cyclin-d1 and Neoplasms

Cyclin D1 (CCND1), a crucial mediator of cell cycle progression, possesses many mutation types with different mutation frequencies in human cancers. The G870A mutation is the most common mutation in CCND1, which produces two isoforms: full-length CCND1a and divergent C-terminal CCND1b. The dysregulation of the CCND1 isoforms is associated with multiple human cancers. Exploring the molecular mechanism of CCND1 isoforms has offer new insight for cancer treatment. On this basis, the alterations of CCND1 gene are described, including amplification, overexpression, and mutation, especially the G870A mutation. Subsequently, we review the characteristics of CCND1 isoforms caused by G870A mutation. Additionally, we summarize cis-regulatory elements, trans-acting factors, and the splice mutation involved in splicing regulation of CCND1. Furthermore, we highlight the function of CCND1 isoforms in cell cycle, invasion, and metastasis in cancers. Importantly, the clinical role of CCND1 isoforms is also discussed, particularly concerning prognosis, chemotherapy, and radiotherapy. Last, emphasis is given to the corrective strategies that modulate the cancerous CCND1 isoforms. Thus, it is highlighting significance of aberrant isoforms of CCND1 as targets for cancer therapy.

Topics: Cyclin D1; Genetic Predisposition to Disease; Humans; Neoplasms; Polymorphism, Single Nucleotide; RNA Splicing

Eugenol (1-allyl-4-hydroxy-3-methoxybenzene) is an important simple phenolic compound mainly derived from Syzygium aromaticum and many other plants. It is traditionally used in ayurveda and aromatherapy for the healing of many health problems. It also has significant applications in dentistry, agriculture, and flavour industry. This simple phenol has an eclectic range of pharmacological properties, such as antioxidant, anti-inflammatory, and anticancer activities. It is regarded as safe by the Food and Agricultural Organization of the United Nations due to its non-carcinogenic and non-mutagenic properties.. The aim of this comprehensive review is to present a critical and systematic assessment of the antitumor ability of eugenol and its associated molecular targets in various cancers.. It was carried out following the preferred reporting items for systematic reviews and meta-analysis guidelines. Risk of bias assessment was performed using the SYstematic review centre for laboratory animal experimentation guidelines. The literature search was performed in standard databases such as Science Direct, PubMed, Google Scholar, Scopus, and Web of Science using the keywords 'eugenol' or 'eugenol essential oil' and 'anti-cancer properties of eugenol'.. The scientific information from fifty-three studies was encompassed in the present review work. Eugenol exhibits significant anticancer effects in a variety of biological pathways, namely apoptosis, autophagy, cell cycle progression, inflammation, invasion, and metastasis. Eugenol-induced apoptosis has been noticed in osteosarcoma, skin tumors, melanoma, leukemia, gastric and mast cells. It decreases the expression of cyclin D1, cyclin B, proliferating cell nuclear antigen, nuclear factor-ƙB, inhibitor of nuclear factor ƙB, and B-cell lymphoma-2. Eugenol increases the expression of B-cell lymphoma-2 (BCL-2) associated X, BH3-interacting domain death agonist, BCL-2 associated agonist of cell death, apoptotic protease activating factor 1, cytochrome c, p21, and p53.. The anticancer potential exhibited by eugenol is mainly attributed to its anti-metastatic, anti-proliferative, anti-angiogenic, anti-inflammatory, cell cycle arrest, apoptotic, and autophagic effects. Hence, the use of eugenol alone or along with other chemotherapeutic anticancer agents is found to be very effective in cancer therapy.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Antioxidants; Apoptotic Protease-Activating Factor 1; Cyclin B; Cyclin D1; Cytochromes c; Eugenol; Neoplasms; Oils, Volatile; Phenols; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Topics: bcl-2-Associated X Protein; Cyclin D1; GTP Phosphohydrolases; Humans; Mechanistic Target of Rapamycin Complex 2; Mitochondrial Proteins; Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt

Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.

Topics: Biomarkers, Tumor; CD8 Antigens; Chemokine CXCL13; Chromosomes, Human, Pair 9; Cohort Studies; Cyclin D1; DNA Copy Number Variations; Exome; Gene Amplification; Humans; Immune Checkpoint Inhibitors; Immune Evasion; Multivariate Analysis; Mutation; Neoplasms; Polymorphism, Single Nucleotide; Receptors, CCR5; T-Lymphocytes; Tumor Burden

The expanding clinical application of CDK4- and CDK6-inhibiting drugs in the managements of breast cancer has raised a great interest in testing these drugs in other neoplasms. The potential of combining these drugs with other therapeutic approaches seems to be an interesting work-ground to explore. Even though a potential integration of CDK4 and CDK6 inhibitors with radiotherapy (RT) has been hypothesized, this kind of approach has not been sufficiently pursued, neither in preclinical nor in clinical studies. Similarly, the most recent discoveries focusing on autophagy, as a possible target pathway able to enhance the antitumor efficacy of CDK4 and CDK6 inhibitors is promising but needs more investigations. The aim of this review is to discuss the recent literature on the field in order to infer a rational combination strategy including cyclin-D1/CDK4-CDK6 inhibitors, RT, and/or other anticancer agents targeting G1-S phase cell cycle transition.

Topics: Animals; Antineoplastic Agents; Autophagy; Cell Cycle; Chemoradiotherapy; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Humans; Neoplasms; Protein Kinase Inhibitors

The relationship between the expression of cyclin D1 and cancer prognosis and outcomes in different malignancies has not been fully elucidated.. In the presented meta-analysis, we assessed the association between the expression level of cyclin D1 with overall survival (OS) in several cancers.. Eligible studies were identified using PubMed, EMBase, Scopus, Web of Sciences and Cochrane Library databases. For the prognostic meta-analysis, study-specific hazard ratios (HRs) of tissue cyclin D1 for survival were obtained. Finally we pooled data derived from one hundred and eight studies comprising 19,224 patients with 10 different cancer types.. In the pooled analysis, high expression of cyclin D1 was significantly related to a poor OS with a pooled HR of 1.11 (95% CI: 1.02-1.20, P = 0.015; random-effects). Sub-group analysis revealed that high expression of cyclin D1 was related to worse OS of head and neck cancers (HR = 2.08, 95% CI: 1.75-2.47; P < 0.001), but not in breast (HR = 1.033, 95% CI: 0.873-1.223, P = 0.702), gastrointestinal (HR = 1.025, 95% CI:0.824-1.275; P = 0.825), bladder (HR = 0.937, CI: 0.844-1.041; P = 0.225) and in lung cancer patients (HR = 1.092, CI: 0.819-1.455; P = 0.549).. Further large, prospective, and well-designed trials are warranted to elucidate the precise clinical importance of cyclin D1 overexpression in the prognosis of cancer patients receiving different treatment regimens.

Topics: Biomarkers, Tumor; Cyclin D1; Humans; Neoplasms; Prognosis; Survival Rate

Topics: Animals; Carcinogenesis; Cell Nucleus; Cyclin D1; DNA Damage; Humans; Neoplasm Invasiveness; Neoplasms

Topics: Cell Adhesion; Cell Cycle Checkpoints; Cyclin D1; Cyclin-Dependent Kinases; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Neoplasm Invasiveness; Neoplasms; Tumor Microenvironment

Topics: Adult; Alleles; Asian People; Cadaver; Correlation of Data; Cyclin D1; Donor Selection; Female; Genetic Association Studies; Genetic Predisposition to Disease; Humans; India; Kidney; Kidney Transplantation; Male; Middle Aged; Neoplasms; Polymorphism, Single Nucleotide; Prognosis; Retrospective Studies; Risk Factors; Tissue and Organ Procurement; Tissue Donors

A growing body of studies has documented the pathological influence of impaired alternative splicing (AS) events on numerous diseases, including cancer. In addition, the generation of alternatively spliced isoforms is frequently noted to result in drug resistance in many cancer therapies. To gain comprehensive insights into the impacts of AS events on cancer biology and therapeutic developments, this paper highlights recent findings regarding the therapeutic routes of targeting alternative-spliced isoforms and splicing regulators to treatment strategies for distinct cancers.

Topics: Adaptor Proteins, Signal Transducing; Alternative Splicing; Antineoplastic Agents; Carcinogenesis; Caspase 9; Cyclin D1; Cyclohexylamines; Epoxy Compounds; Humans; Macrolides; Neoplasm Proteins; Neoplasms; Nuclear Proteins; Oligonucleotides; Pyrans; RNA Splicing Factors; RNA, Messenger; Spiro Compounds; Spliceosomes; Tumor Suppressor Proteins

Radiotherapy (RT) is a powerful tool in the treatment of cancer, having the advantage of preserving normal tissues. Clinical outcomes of RT are significantly improved by technological advances, enabling increased radiation doses directed very specifically to a tumor. However, tumor radioresistance remains a major impediment to effective RT. We have shown that human tumor cells surviving after repeated exposure to fractionated radiation (FR) of X-rays for 1 month have acquired radioresistance through constitutive activation of AKT and downstream cyclin D1 nuclear retention. Tumor radioresistance is also proposed to be an intrinsic characteristic of cancer stem cells (CSC), whose efficient DNA repair is thought to confer this phenotype. We have isolated radioresistant CD133-positive cells following exposure to long-term FR. These cells exhibited the CSC phenotype with activation of the AKT/cyclin D1 pathway. In this review, I summarize our current understanding of the molecular mechanisms underlying tumor radioresistance and propose a strategy for overcoming radioresistance by targeting the AKT/cyclin D1 pathway.. Two different mechanisms: acquired radioresistance of surviving tumor cells after RT and intrinsic radioresistance of CSC are associated with tumor radioresistance. Inhibition of the AKT pathway results in radiosensitization of both types of tumor radioresistance.

Topics: Animals; Antineoplastic Agents; Chemoradiotherapy; Cyclin D1; Humans; Molecular Targeted Therapy; Neoplasms; Proto-Oncogene Proteins c-akt; Radiation Tolerance; Signal Transduction

The translation initiation factor eIF4E mediates a rate-limiting process that drives selective translation of many oncongenic proteins such as cyclin D1, survivin and VEGF, thereby contributing to tumour growth, metastasis and therapy resistance. As an essential regulatory hub in cancer signalling network, many oncogenic signalling pathways appear to converge on eIF4E. Therefore, targeting eIF4E-mediated cap-dependent translation is considered a promising anticancer strategy. This paper reviews the strategies that can be used to target eIF4E, highlighting agents that target eIF4E activity at each distinct level.

Topics: Animals; Antineoplastic Agents; Cyclin D1; Epoxy Compounds; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Macrolides; Neoplasms; Oligonucleotides, Antisense; Protein Biosynthesis; Ribavirin; RNA, Small Interfering; Signal Transduction; Sirolimus; Survivin; Thiazoles; Triterpenes; Vascular Endothelial Growth Factor A

To assess the association between polymorphism rs678653 in human Cyclin D1 gene (CCND1) and the risk of cancer.. Multiple biomedical databases were systematically searched. Pooled odds ratios (OR) and 95% confidence intervals (95% CIs) were calculated in the appropriate model.. In total, 17 case-control studies from 14 articles were included. When combing all available data, no significant association of rs678653 with cancer risk was observed under different genetic models. Stratification by ethnicity also indicated that rs678653 was not correlated with cancer risk in Taiwanese or Indian populations. When stratified by cancer type, no significant association was found between polymorphism rs678653 and digestive tract cancer, head and neck cancer, and gynecological cancer risk.. Our comprehensive meta-analysis suggests that the polymorphism rs678653 in CCND1 has no association with cancer risk in different population and disease contexts, indicating that CCND1 rs678653 does not serve a significant biological function in predicting cancer risk.

Topics: Cyclin D1; Female; Genes, Recessive; Genetic Association Studies; Genetic Predisposition to Disease; Humans; India; Models, Genetic; Neoplasms; Polymorphism, Single Nucleotide; Taiwan

Uncontrolled cellular proliferation, mediated by dysregulation of the cell-cycle machinery and activation of cyclin-dependent kinases (CDKs) to promote cell-cycle progression, lies at the heart of cancer as a pathological process. Clinical implementation of first-generation, nonselective CDK inhibitors, designed to inhibit this proliferation, was originally hampered by the high risk of toxicity and lack of efficacy noted with these agents. The emergence of a new generation of selective CDK4/6 inhibitors, including ribociclib, abemaciclib and palbociclib, has enabled tumour types in which CDK4/6 has a pivotal role in the G1-to-S-phase cell-cycle transition to be targeted with improved effectiveness, and fewer adverse effects. Results of pivotal phase III trials investigating palbociclib in patients with advanced-stage oestrogen receptor (ER)-positive breast cancer have demonstrated a substantial improvement in progression-free survival, with a well-tolerated toxicity profile. Mechanisms of acquired resistance to CDK4/6 inhibitors are beginning to emerge that, although unwelcome, might enable rational post-CDK4/6 inhibitor therapeutic strategies to be identified. Extending the use of CDK4/6 inhibitors beyond ER-positive breast cancer is challenging, and will likely require biomarkers that are predictive of a response, and the use of combination therapies in order to optimize CDK4/6 targeting.

Topics: Aminopyridines; Antineoplastic Combined Chemotherapy Protocols; Benzimidazoles; Breast Neoplasms; Cell Cycle; Clinical Trials as Topic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Female; Forecasting; Humans; Molecular Targeted Therapy; Neoplasms; Piperazines; Purines; Pyridines

Mammalian cells encode three D cyclins (D1, D2, and D3) that coordinately function as allosteric regulators of cyclin-dependent kinase 4 (CDK4) and CDK6 to regulate cell cycle transition from G1 to S phase. Cyclin expression, accumulation, and degradation, as well as assembly and activation of CDK4/CDK6 are governed by growth factor stimulation. Cyclin D1 is more frequently dysregulated than cyclin D2 or D3 in human cancers, and as such, it has been more extensively characterized. Overexpression of cyclin D1 results in dysregulated CDK activity, rapid cell growth under conditions of restricted mitogenic signaling, bypass of key cellular checkpoints, and ultimately, neoplastic growth. This review discusses cyclin D1 transcriptional, translational, and post-translational regulations and its biological function with a particular focus on the mechanisms that result in its dysregulation in human cancers.

Topics: Animals; Antineoplastic Agents; beta Catenin; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 4; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Phosphatidylinositol 3-Kinase; Protein Biosynthesis; Signal Transduction; Transcription, Genetic

While targeting experiments carried out on the genes encoding many cell cycle regulators have challenged our views of cell cycle control, they also suggest that redundancy might not be the only explanation for the observed perplexing phenotypes. Indeed, several observations hint at functions of cyclins and CDK inhibitors that cannot be accounted for by their sole role as kinase regulators. They are found involved in many cellular transactions, depending or not on CDKs that are not directly linked to cell cycle control, but participating to general mechanisms such as transcription, DNA repair or cytoskeleton dynamics. In this review we discuss the roles that these alternative functions might have in cancer cell proliferation and migration that sometime even challenge their definition as proliferation markers.

Topics: Animals; Cell Cycle; Cell Nucleus; Cyclin D1; Cyclin E; Cyclin-Dependent Kinases; Cyclins; Cytoplasm; Cytoskeleton; DNA Damage; DNA Repair; Epithelial-Mesenchymal Transition; Female; Humans; Interphase; Male; Mice; Mitosis; Neoplasm Invasiveness; Neoplasms; Phenotype; Transcription, Genetic

Intensive efforts, over the last decade, have been made to inhibit the kinase activity of cyclins that act as mediators during cell-cycle progression. Activation of the cyclin D1 oncogene, often by amplification or rearrangement, is a major driver of multiple types of human tumors including breast and squamous cell cancers, B-cell lymphoma, myeloma and parathyroid adenoma.. In this review, the authors summarize the activity of cyclins and cyclin-dependent kinases in cell-cycle progression and transcription. They focus on cyclin D1/CDK4/CDK6, a central mediator in the transition from G1 to S phase. Furthermore, the authors discuss the first generation of pan-cyclin-dependent kinase inhibitors that failed to meet expectation and discuss, in detail, the second generation of highly specific cyclin D1/CDK4/CDK6 inhibitors that are proving to be more efficacious.. The mechanism by which cyclin D1 drives tumorigenesis may be dependent on kinase and kinase-independent functions. Further evidence is necessary to delineate the roles of cyclin D1 in early pre-neoplastic lesions where its overexpression may promote genomic instability in a kinase-independent manner.

Topics: Animals; Antineoplastic Agents; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor Proteins; Humans; Neoplasms; Protein Kinase Inhibitors

Although cancer-regulatory genes are dichotomized to oncogenes and tumor-suppressor gene s, in reality they can be oncogenic in one situation but tumor-suppressive in another. This dual-function nature, which sometimes hampers our understanding of tumor biology, has several manifestations: (1) Most canonically defined genes have multiple mRNAs, regulatory RNAs, protein isoforms, and posttranslational modifications; (2) Genes may interact at different levels, such as by forming chimeric RNAs or by forming different protein complexes; (3) Increased levels of tumor-suppressive genes in normal cells drive proliferation of cancer progenitor cells in the same organ or tissue by imposing compensatory proliferation pressure, which presents the dual-function nature as a cell-cell interaction. All these manifestations of dual functions can find examples in the genes along the CCND-CDK4/6-RB axis. The dual-function nature also underlies the heterogeneity of cancer cells. Gene-targeting chemotherapies, including that targets CDK4, are effective to some cancer cells but in the meantime may promote growth or progression of some others in the same patient. Redefining "gene" by considering each mRNA, regulatory RNA, protein isoform, and posttranslational modification from the same genomic locus as a "gene" may help in better understanding tumor biology and better selecting targets for different sub-populations of cancer cells in individual patients for personalized therapy.

Topics: Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Drug Delivery Systems; Genes, Tumor Suppressor; Neoplasms; Oncogenes; Protein Isoforms; Protein Processing, Post-Translational; Regulatory Sequences, Ribonucleic Acid; Retinoblastoma Protein; RNA, Messenger

The acquisition of resistance to chemotherapy is a significant problem in the treatment of cancer, greatly increasing patient morbidity and mortality. Tumors are often sensitive to chemotherapy upon initial treatment, but repeated treatments can select for those cells that were able to survive initial therapy and have acquired cellular mechanisms to enhance their resistance to subsequent chemotherapy treatment. Many cellular mechanisms of drug resistance have been identified, most of which result from changes in gene and protein expression. While changes at the transcriptional level have been duly noted, it is primarily the post-transcriptional processing of pre-mRNA into mature mRNA that regulates the composition of the proteome and it is the proteome that actually regulates the cell's response to chemotherapeutic insult, inducing cell survival or death. During pre-mRNA processing, intronic non-protein-coding sequences are removed and protein-coding exons are spliced to form a continuous template for protein translation. Alternative splicing involves the differential inclusion or exclusion of exonic sequences into the mature transcript, generating different mRNA templates for protein production. This regulatory mechanism enables the potential to produce many different protein isoforms from the same gene. In this review I will explain the mechanism of alternative pre-mRNA splicing and look at some specific examples of how splicing factors, splicing factor kinases and alternative splicing of specific pre-mRNAs from genes have been shown to contribute to acquisition of the drug resistant phenotype.

Topics: Alternative Splicing; Cyclin D1; Drug Resistance, Neoplasm; Humans; Neoplasms; Protein Serine-Threonine Kinases; Receptors, Androgen; RNA Precursors; RNA Splicing Factors; RNA-Binding Proteins; Spliceosomes; STAT2 Transcription Factor

The human CCND1 gene, which encodes the cell-cycle protein cyclin D1, is one of the most frequently amplified genes in human cancers. Cyclin D1 activates the cyclin-dependent kinases CDK4 and CDK6 and drives cell proliferation. Beyond the cell-cycle role, the full repertoire of cyclin D1 functions in cancer cells is still unclear. Emerging evidence indicates that cyclin D1 may play a role in DNA damage response. In this review, we discuss observations linking cyclin D1 to DNA damage repair and summarize our recent findings, which show a cyclin D1 function in homologous recombination-mediated DNA repair.

Topics: Cyclin D1; DNA Damage; DNA Repair; Homologous Recombination; Humans; Molecular Targeted Therapy; Neoplasms; Proteolysis; Proteome; Proteomics

The c-myc is a proto-oncogene that manifests aberrant expression at high frequencies in most types of human cancer. C-myc gene amplifications are often observed in various cancers as well. Ample studies have also proved that c-myc has a potent oncogenicity, which can be further enhanced by collaborations with other oncogenes such as Bcl-2 and activated Ras. Studies on the collaborations of c-myc with Ras or other genes in oncogenicity have established several basic concepts and have disclosed their underlying mechanisms of tumor biology, including "immortalization" and "transformation". In many cases, these collaborations may converge at the cyclin D1-CDK4 complex. In the meantime, however, many results from studies on the c-myc, Ras and cyclin D1-CDK4 also challenge these basic concepts of tumor biology and suggest to us that the immortalized status of cells should be emphasized. Stricter criteria and definitions for a malignantly transformed status and a benign status of cells in culture also need to be established to facilitate our study of the mechanisms for tumor formation and to better link up in vitro data with animal results and eventually with human cancer pathology.

Topics: Animals; Cell Line, Transformed; Cyclin D1; Cyclin-Dependent Kinase 4; Humans; Neoplasm Proteins; Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; ras Proteins

Fractionated radiotherapy (RT) is widely used in cancer therapy for its advantages in the preservation of normal tissues. However, repopulation of surviving tumor cells during fractionated RT limits the efficacy of RT. In fact, repopulating tumors often acquire radioresistance and this is the major cause of failure of RT. We have recently demonstrated that human tumor cells acquire radioresistance when exposed to fractionated radiation (FR) of X-rays every 12 hours for 1 month. The acquired radioresistance was associated with overexpression of cyclin D1, a result of a series of molecular changes; constitutive activation of DNA-PK and AKT with concomitant down-regulation of glycogen synthase kinase-3β (GSK3β) which results in suppression of cyclin D1 proteolysis. Aberrant cyclin D1 overexpression in S-phase induced DNA double strand breaks which activated DNA-PK and established the vicious cycle of cycling D1 overexpression. This overexpression of cyclin D1 is responsible for the radioresistance phenotype of long-term FR cells, since this phenotype was completely abrogated by treatment of FR cells by the API-2, an AKT inhibitor or by a Cdk4 inhibitor. Thus, targeting the AKT/GSK3β/cyclin D1/Cdk4 pathway can be an efficient modality to suppress acquired radioresistance of tumor cells. In this article, I overview the newly discovered molecular mechanisms underlying acquired radioresistance of tumor cells induced by FR, and propose a strategy for eradication of tumors using fractionated RT by overcoming tumor radioresistance.

Topics: Awards and Prizes; Cyclin D1; Cyclin-Dependent Kinase 4; Dose Fractionation, Radiation; Feedback, Physiological; G1 Phase Cell Cycle Checkpoints; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Neoplasms; Nucleosides; Oncogene Protein v-akt; Protein Kinase Inhibitors; Pyridazines; Radiation Tolerance; Signal Transduction; Treatment Failure

The Ser/Thr kinase family, RSK, has been implicated in numerous types of hormone-dependent and -independent cancers. However, there has been little consideration of RSKs as downstream mediators of steroid hormone non-genomic effects or of their ability to facilitate steroid receptor-mediated gene expression. Steroid hormone signaling can directly stimulate the MEK/ERK/RSK pathway to regulate cellular proliferation and survival in transformed cells. To date, multiple mechanisms of RSK and steroid hormone receptor-mediated proliferation/survival have been elucidated. For example, RSK enhances proliferation of breast and prostate cancer cells via its ability to control the levels of the estrogen receptor co-activator, cyclin D1. While in lung and other tumors RSK may control apoptosis via estrogen-mediated regulation of mitochondrial integrity. Thus the RSKs could be important anti-cancer therapeutic targets in many different transformed tissues. The recent discovery of RSK-specific inhibitors will advance our current understanding of RSK in transformation and drive these studies into animal and clinical models. In this review we explore the mechanisms associated with RSK in tumorigenesis and their relationship to steroid hormone signaling.

Topics: Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Neoplasms; Ribosomal Protein S6 Kinases; Signal Transduction; Steroids

Cancer is the result of sequential genetic changes over time that transform a cell into a malignant and ultimately invasive entity. The insight that cancerous cells arise from a series of mutations in oncogenes and tumor suppressors, commonly known as multistep carcinogenesis, has been conceptually elaborated and proven in the last 20 years. Although knowledge about late steps of cancerogenesis and disease progression has greatly advanced, the initial molecular events remain largely unknown. Basic research in Drosophila has started the quest to find early markers that detect initial clonal expansion of precancerous cells. These efforts were spurred by novel findings demonstrating that certain mutations transform cells into super-competitors that expand at the expense of the surrounding epithelial cells without inducing histological changes. This mechanism, discovered as super competition in the fly, might also lie at the heart of a clinical observation termed 'field cancerization'. This review aims to bring together current understanding from basic research on cell competition and clinical studies that have analyzed field characteristics to highlight parallels and possible connections.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Cyclin D1; Drosophila; Genes, Tumor Suppressor; Genetic Markers; Head and Neck Neoplasms; Humans; Loss of Heterozygosity; Models, Biological; Neoplasms; Oncogenes; Precancerous Conditions

Topics: Animals; Chemoprevention; Clinical Trials as Topic; Cyclin D1; Humans; Neoplasms; Retinoids

Perturbations in the regulation of the core cell cycle machinery are frequently observed in human cancers. Cyclin D1 which functions as a mitogenic sensor and allosteric activator of CDK4/6, is one of the more frequently altered cell cycle regulators in cancers. Cyclin D1 is frequently overexpressed in cancers and its overexpression can be attributed to many factors including increased transcription, translation, and protein stability. Although cyclin D1 overexpression is clearly implicated in the affected cancers, overexpression of cyclin D1 is not sufficient to drive oncogenic transformation. Rather, emerging evidence suggests that nuclear retention of cyclin D1 resulting from altered nuclear trafficking and proteolysis is critical for the manifestation of its oncogenicity. This review provides a brief overview of current data documenting various mechanisms underlying aberrant cyclin D1 regulation in human cancers and their impact on neoplastic transformation.

Topics: Animals; Carcinogens; Cell Cycle; Cell Nucleus; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 4; Gene Expression Regulation, Neoplastic; Genomic Instability; Humans; Neoplasms; Protein Kinase Inhibitors; Signal Transduction

In cancer cells, gene expression is altered at the levels of transcription and mRNA maturation, with many splice variants being associated with cancer. Splicing is tightly connected to transcription and can be affected by transcription elongation dynamics. Moreover, various transcriptional coregulators that are altered in cancer, such as the proto-oncogene EWS, are thought to play a role in splicing. A recent study shows that an alteration of EWS in Ewing sarcoma alters the dynamics of RNA polymerase II over the CCND1 proto-oncogene encoding cyclin D1, leading to an increase in its transcription and to an alteration of splicing that results in high levels of the oncogenic cyclin D1b splice isoform. The cyclin D1b isoform is highly expressed in Ewing sarcoma cells and tumors and stimulates Ewing sarcoma cell growth. Thus, alterations of transcriptional regulators in disease may lead to splicing alterations. We review these data and discuss how this concept may apply to various factors that are altered in cancer.

Topics: Animals; Bone Neoplasms; Cyclin D1; Humans; Models, Biological; Neoplasms; Oncogene Proteins, Fusion; Oncogenes; Proto-Oncogene Mas; Proto-Oncogene Protein c-fli-1; RNA Splicing; RNA-Binding Protein EWS; Sarcoma, Ewing; Trans-Activators; Transcription, Genetic

Cell cycle progression is determined by the balance of positive regulators, cyclin-dependent-protein kinases (cdks) relative to negative regulators, cyclin-dependent kinase inhibitors (ckis). D-type cyclins, (D1, D2, D3) are expressed in a tissue-specific manner and are the first cyclins to be expressed during the cell cycle. Of the three D-type cyclins, cyclin D1 is most frequently overexpressed in human cancer. The mechanisms of cyclin D1 overexpression can be attributed to gene amplification, transcriptional activation and altered protein degradation; of these, inhibition of ubiquitin-dependent proteolysis of cyclin D1 is thought to be a primary mechanism of cyclin D1 overexpression in human tumors. Because the identity of the regulators of cyclin D1 proteolysis were largely undefined until recently, it had not been possible to determine whether this regulatory network was directly targeted in primary cancer. Cyclin D1 proteolysis requires phosphorylation by GSK3beta at Thr-286; additional work recently established that p286-D1 is a substrate for the SCF(Fbx4/alphaB-crystallin) E3 ligase. This discovery has facilitated an analysis of SCF(Fbx4/alphaB-crystallin) ligase in human cancers. This recent work revealed that Fbx4 is subject to mutational inactivation in human cancer, resulting in the accumulation of cyclin D1. Molecular analysis of this ligase has revealed striking regulatory features that contribute to regulated cyclin D1 accumulation and support the idea that Fbx4 is a bona fide tumor suppressor.

Topics: alpha-Crystallin B Chain; Animals; Cell Cycle; Cyclin D1; F-Box Proteins; Humans; Models, Biological; Neoplasms; Phosphorylation; SKP Cullin F-Box Protein Ligases; Ubiquitin-Protein Ligases

The G870A polymorphism in the CCND1 gene may influence cancer risk. However, data from published studies with individual low statistical power have been controversial. To evaluate whether combined evidence shows an association between this polymorphism and cancer, we considered all available studies in a meta-analysis. Sixty studies were combined representing data for 18,411 cases and 22,209 controls. In our meta-analysis, we investigated overall sample and two ethnic populations (Caucasians and Asians) as well as nine cancer subtypes. Individuals who are homozygous for A allele (AA) were found to be associated with significantly increased cancer risk in overall sample [odds ratio (OR), 1.23; 95% confidence interval (95% CI), 1.13-1.33; P

Topics: Alleles; Asian People; Chi-Square Distribution; Cyclin D1; Genotype; Humans; Neoplasms; Polymorphism, Genetic; Risk Factors; White People

Cyclin D1 is known as a proto-oncogene whose gene amplification and protein overexpression are frequently observed in tumor cells. It acts as a mitogenic signal sensor and is expressed as a delayed-early response to many mitogenic signals. Cyclin-dependent kinases (CDKs) 4 and 6 are cyclin D1 binding partners, and activated cyclin D1/CDK4 and cyclin D1/CDK6 complex phosphorylate the retinoblastoma protein to induce the expression of target genes essential for S phase entry, resulting in facilitation of the progression from G1 to S phase. As well as acting as a positive regulator of the cell cycle, cyclin D1 is known to bind and modulate the actions of several transcription factors. Since the protein level of cyclin D1 reflects cell cycle progression, the rates of protein production and degradation are strictly regulated. Glycogen synthase kinase-3beta (GSK-3beta), a serine/threonine protein kinase, has been shown to play an important role in the determination of cyclin D1 expression level by regulating mRNA transcription and protein degradation. This review highlights the regulatory mechanisms of cyclin D1 expression level, with special attention to the involvement of GSK-3beta.

Topics: Animals; Antineoplastic Agents; beta Catenin; Celecoxib; Cell Cycle; Cell Differentiation; Curcumin; Cyclin D1; Dyrk Kinases; Enzyme Activation; Enzyme Activators; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Mas; Pyrazoles; Retinoids; Signal Transduction; Sulfonamides; Transcription, Genetic; Ubiquitins; Wnt Proteins

Tobacco carcinogen treatment of immortalized human bronchial epithelial (HBE) cells has uncovered novel targets for cancer chemoprevention. Experiments were conducted with HBE cells and independent treatments with tobacco carcinogens along with the chemopreventive agent all-trans-retinoic acid (RA). That work highlighted D-type and E-type cyclins as novel molecular pharmacologic targets of several chemopreventive agents. G1 cyclins are often aberrantly expressed in bronchial preneoplasia and lung cancers. This implicated these species as targets for clinical cancer chemoprevention. Retinoid regulation mechanisms of D-type cyclins in lung cancer chemoprevention have been comprehensively explored. Retinoid chemoprevention has been mechanistically linked to proteasomal degradation of cyclin D1 and cyclin D3. Threonine 286 mutation stabilized cyclin D1, implicating phosphorylation in this retinoid chemoprevention. Studies with a phospho-specific anti-cyclin D1 antibody confirmed this hypothesis. Glycogen synthase kinase (GSK) inhibitors established a role for this kinase in the retinoid regulation of cyclin D1, but not cyclin D3. Involvement of D-type cyclins in this chemoprevention was shown using small interfering RNAs (siRNAs). Gene profiling experiments highlighted the E1-like ubiquitin-activating enzyme (UBE1L) in the retinoid regulation of cyclin D1. Proof of principle trials have translated these studies into the clinic and established that chemopreventive agents can target D-type cyclins. These findings have been built upon with a targeted combination regimen that cooperatively affects D-type cyclins. Taken together, these preclinical and clinical findings strongly implicate these cyclins as novel molecular pharmacological targets for cancer chemoprevention.

Topics: Animals; Cell Transformation, Neoplastic; Chemoprevention; Clinical Trials as Topic; Cyclin D1; Humans; Neoplasms

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/phosphatidylinositol 3-kinase (PI3K)/AKT constitute an important pathway regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth. PTEN is a dual protein/lipid phosphatase and its main substrate phosphatidyl-inositol 3,4,5 triphosphate (PIP3) is the product of PI3K. Increase in PIP3 recruits AKT to the membrane where is activated by other kinases also dependent on PIP3. Many components of this pathway have been described as causal forces in cancer. PTEN activity is lost by mutations, deletions or promoter methylation silencing at high frequency in many primary and metastatic human cancers. Germ line mutations of PTEN are found in several familial cancer predisposition syndromes. Recently, many activating mutations in the PI3KCA gene (coding for the p110alpha catalytic subunit of PI3K) have been described in human tumors. Activation of PI3K and AKT are reported to occur in breast, ovarian, pancreatic, esophageal and other cancers. Genetically modified mice confirm these PTEN activities. Tissue-specific deletions of PTEN usually provoke cancer. Moreover, an absence of PTEN cooperates with an absence of p53 to promote cancer. However, we have observed very different results with the expression of activated versions of AKT in several tissues. Activated AKT transgenic lines do not develop tumors in breast or prostate tissues and do not cooperate with an absence of p53. This data suggest that an AKT-independent mechanism contributes to PTEN tumorigenesis. Crosses with transgenic mice expressing possible PTEN targets indicate that neither cyclin D1 nor p53 are these AKT-independent targets. However, AKT is more than a passive bridge toward PTEN tumorigenesis, since its expression not only allows but also enforces and accelerates the tumorigenic process in combination with other oncogenes.

Topics: Animals; Apoptosis; Cell Transformation, Neoplastic; Cyclin D1; Enzyme Activation; Female; Humans; Male; Metabolic Networks and Pathways; Mice; Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Tumor Suppressor Protein p53

Cyclin D1 binds to the Cdk4 and Cdk6 to form a pRB kinase. Upon phosphorylation, pRB loses its repressive activity for the E2F transcription factor, which then activates transcription of several genes required for the transition from the G1- to S-phase and for DNA replication. The cyclin D1 gene is rearranged and overexpressed in centrocytic lymphomas and parathyroid tumors and it is amplified and/or overexpressed in a major fraction of human tumors of various types of cancer. Ectopic overexpression of cyclin D1 in fibroblast cultures shortens the G1 phase of the cell cycle. Furthermore, it has been demonstrated that introduction of an antisense cyclin D1 into a human carcinoma cell line, in which the cyclin D1 gene is amplified and overexpressed, causes reversion of the malignant phenotype. Thus, increased expression of cyclin D1 can play a critical role in tumor development and in maintenance of the malignant phenotype. However, it is insufficient to confer transformed properties on primary or established fibroblasts. In this review, we summarize the role of cyclin D1 on tumor development and malignant transformation. In addition, our chemical biology study to understand the regulatory mechanism of cyclin D1 transcription is also reviewed.

Topics: Amino Acid Sequence; Cell Cycle; Cell Transformation, Neoplastic; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Models, Biological; Molecular Sequence Data; Neoplasms; Oncogenes

Cyclin D1 is an important regulator of cell cycle progression and can function as a transcriptionl co-regulator. The overexpression of cyclin D1 has been linked to the development and progression of cancer. Deregulated cyclin D1 degradation appears to be responsible for the increased levels of cyclin D1 in several cancers. Recent findings have identified novel mechanisms involved in the regulation of cyclin D1 stability. A number of therapeutic agents have been shown to induce cyclin D1 degradation. The therapeutic ablation of cyclin D1 may be useful for the prevention and treatment of cancer. In this review, current knowledge on the regulation of cyclin D1 degradation is discussed. Novel insights into cyclin D1 degradation are also discussed in the context of ablative therapy. A number of unresolved questions regarding the regulation of cellular cyclin D1 levels are also addressed.

Topics: Animals; Cell Cycle; Cyclin D1; Disease Progression; Humans; Neoplasms; Peptide Hydrolases; Protein Binding

Endogenous inhibitors of angiogenesis are proved to be a major factor preventing the emergence of clinically manifested stages of human cancer. The protein endostatin, a 20-kD proteolytic fragment of type XVIII collagen, is one of the most active natural inhibitors of angiogenesis. Endostatin specifically inhibits the in vitro and in vivo proliferation of endothelial cells, inducing their apoptosis through inhibition of cyclin D1. On the surface of endothelial cells, endostatin binds with the integrin alpha(5)beta(1) that activates the Src-kinase pathway. The binding of endostatin with integrins also down-regulates the activity of RhoA GTPase and inhibits signaling pathways mediated by small kinases of the Ras and Raf families. All these events promote disassembly of the actin cytoskeleton, disorders in cell-matrix interactions, and decrease in endotheliocyte mobility, i.e., promote the suppression of angiogenesis. Endostatin displays a high antitumor activity in vivo: it inhibits the progression of more than 60 types of tumors. This review summarizes results of numerous studies concerning the biological activity and action mechanism of endostatin.

Topics: Animals; Cyclin D1; Endostatins; Humans; Integrins; Neoplasms; Neovascularization, Pathologic; Protein Binding; rhoA GTP-Binding Protein; Signal Transduction

The ubiquitin-proteasome pathway plays a central role in the degradation of proteins involved in several pathways including the cell cycle, cellular proliferation and apoptosis. Bortezomib is the first proteasome inhibitor to enter clinical use, and received approval by the Food and Drug Administration (FDA) for the treatment of patients with multiple myeloma, therefore validating inhibition of the proteasome as an anticancer target. The approval of Bortezomib was based on a large, international, multicenter phase III trial showing its efficacy and safety compared with conventional therapy. Preclinical data also demonstrates the synergistic effect of bortezomib with other chemotherapeutic agents and its ability to overcome drug resistance. Since then several other proteasome inhibitors have been developed. The anti-tumor activities of bortezomib have been attributed to its effect on pro-apoptotic pathways including the inhibition of NF-kappaB and induction of endoplasmic reticulum stress. However, the molecular mechanisms are not fully understood. In this review, we will summarize the molecular mechanism of apoptosis by bortezomib.

Topics: Animals; Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Cyclin D1; Humans; Multiple Myeloma; Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Ubiquitin

Galectin-3 (Gal-3), a member of the beta-galactoside-binding gene family, distributes inside and outside the cell and has pleiotropic biological functions such as cell growth, cell adhesion, cell-cell interaction, and mRNA processing in a specific situation. In particular, Gal-3 in the nucleus plays a pivotal role in the regulation of cancer-related gene expression, including cyclin D1, TTF-1 and MUC2, presumably associated with tumor progression. Therefore, to understand the mechanism of nuclear import of Gal-3 is very significant and might be developed to the new approach for the cancer treatment. In this review, we focus on the role of Gal-3 in the nucleus and the molecular mechanism of nuclear import pathways of Gal-3, providing the hints for the inhibition of Gal-3 function.

Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Cyclin D1; DNA-Binding Proteins; Galectin 3; Gene Expression Regulation; Humans; Mucin-2; Mucins; Neoplasms; Phosphorylation; Protein Transport; Transcription Factors

Cancer is characterized by uncontrolled cell division resulting from multiple mutagenic events. Cancer chemoprevention strategies aim to inhibit or reverse these events using natural or synthetic pharmacologic agents. Ideally, this restores normal growth control mechanisms. Diverse classes of compounds have been identified with chemopreventive activity. What unites many of them is an ability to inhibit the cell cycle by specifically modulating key components. This delays division long enough for cells to respond to mutagenic damage. In some cases, damage is repaired and in others cellular damage is sufficient to trigger apoptosis. It is now known that pathways responsible for targeting G1 cyclins for proteasomal degradation can be engaged pharmacologically. Emergence of induced cyclin degradation as a target for cancer therapy and chemoprevention in pre-clinical models is discussed in this article. Evidence for cyclin D1 as a molecular pharmacologic target and biological marker for clinical response is based on experience of proof of principle trials.

Topics: Cell Cycle; Cyclin D1; Cyclin G; Cyclin G1; Cyclins; Drug Delivery Systems; Humans; Neoplasms; Proteasome Endopeptidase Complex

Signal transducers and activators of transcription (STAT) are a highly conserved family of transcription factors that are activated by phosphorylation in the cytoplasm, after which they translocate to the nucleus to regulate gene expression. Among the seven STATs, STAT3 is of particular interest due to its constitutive phosphorylation in a large proportion of human cancers and its ability to induce neoplastic transformation. Inhibition of STAT3 can reverse tumor growth in experimental systems while having few effects in normal cells. These findings have implicated STAT3 as a potentially important target for therapeutic intervention. In addition to its well-described role as a transcription factor, STAT3 has been found recently to have important effects in the cytoplasm. Collectively, these functions of STAT3 directly contribute to tumorigenesis, invasion, and metastasis. Given the potential importance of STAT3 as a target for cancer therapy, molecules have been developed that can block STAT3 function at a variety of steps. These drugs show promise as anticancer agents in model systems of a variety of common human cancers. Thus, elucidating the functions of STAT3 and developing agents to inhibit this protein remain important scientific and clinical challenges.

Topics: Animals; Antineoplastic Agents; Cell Nucleus; Cell Transformation, Neoplastic; Cyclin D1; Cytoplasm; Gene Expression Regulation, Neoplastic; Humans; Models, Biological; Neoplasm Metastasis; Neoplasms; Signal Transduction; STAT3 Transcription Factor

C-myc is an oncogene that functions both in the stimulation of cell proliferation and in and apoptosis. C-myc elicits its oncogenic activity by causing immortalization, and to a lesser extent the transformation of cells, in addition to several other mechanisms. C-myc may also enhance or reduce the sensitivity of cancer cells to chemotherapy, but how this dual function is controlled is largely unclear. Cyclin D1 (D1) is another oncogene that drives cell cycle progression; it acts as a growth factor sensor to integrate extracellular signals with the cell cycle machinery, though it may also promote apoptosis. C-Myc collaborates with TGFalpha, epidermal growth factor receptor, Ras, PI3K/Akt, and NF-kappaB. in part via coordination in regulation of D1 expression, because D1 is a common downstream effector of these growth pathways. Coordination of c-Myc with D1 or its upstream activators not only accelerates tumor formation, but also may drive tumor progression to a more aggressive phenotype. Because c-Myc may effect immortalization while D1 or its upstream activators elicit transformation, targeting c-myc and D1 may be a good strategy for cancer prevention. Moreover, since D1 imposes chemoresistance on cancer cells, targeting D1 may also be a good strategy for cancer chemotherapy, whereas practicioners should be cautious to downregulate c-myc for chemotherapy, since c-Myc may elicit apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Cell Transformation, Neoplastic; Cyclin D1; Disease Progression; Genes, myc; Humans; Neoplasms

The cyclin D1 proto-oncogene exercises powerful control over the mechanisms that regulate the mitotic cell cycle, and excessive cyclin D1 expression and/or activity is common in human cancers. Although somatic mutations of the cyclin D1 locus are rarely observed, mounting evidence demonstrates that a specific polymorphism of cyclin D1 (G/A870) and a protein product of a potentially related alternate splicing event (cyclin D1b) may influence cancer risk and outcome. Herein, we review the epidemiological and functional literatures that link these alterations of cyclin D1 to human tumor development and progression.

Topics: Alternative Splicing; Cell Cycle; Cell Transformation, Neoplastic; Cyclin D1; Disease Progression; Epidemiologic Studies; Humans; Neoplasms; Polymorphism, Genetic; Proto-Oncogene Mas

Certain members of the thiazolidinedione (TZD) family of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, such as troglitazone and ciglitazone, exhibit antitumor activities; however, the underlying mechanism remains inconclusive. Substantial evidence suggests that the antiproliferative effect of these TZD members in cancer cells is independent of PPARgamma activation. To discern the role of PPARgamma in the antitumor effects of TZDs, we have synthesized PPARgamma-inactive TZD analogs which, although devoid of PPARgamma activity, retain the ability to induce apoptosis with a potency equal to that of their parental TZDs in cancer cell lines with varying PPARgamma expression status. Mechanistic studies from this and other laboratories have further suggested that troglitazone and ciglitazone mediate antiproliferative effects through a complexity of PPARgamma-independent mechanisms. Evidence indicates that troglitazone and ciglitazone block BH3 domain-mediated interactions between the anti apoptotic Bcl-2 (B-cell leukemia/lymphoma 2) members Bcl-2/Bcl-xL and proapoptotic Bcl-2 members. Moreover, these TZDs facilitate the degradation of cyclin D1 and caspase-8-related FADD-like IL-l-converting enzyme (FLICE)-inhibitory protein through proteasome-mediated proteolysis, and down-regulate the gene expression of prostate-specific antigen gene expression by inhibiting androgen activation of the androgen response elements in the promoter region. More importantly, dissociation of the effects of TZDs on apoptosis from their original pharmacological activity (i.e. PPARgamma activation) provides a molecular basis for the exploitation of these compounds to develop different types of molecularly targeted anticancer agents. These TZD-derived novel therapeutic agents, alone or in combination with other anticancer drugs, have translational relevance in fostering effective strategies for cancer treatment.

Topics: Antineoplastic Agents; Cyclin D1; Humans; Neoplasms; PPAR gamma; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Thiazolidinediones

Cell migration is essential for developmental morphogenesis, tissue repair, and tumor metastasis. A recent study reveals that cyclin D1 acts to promote cell migration by inhibiting Rho/ROCK signaling and expression of thrombospondin-1 (TSP-1), an extracellular matrix protein that regulates cell migration in many settings including cancer. Given the frequent overexpression of cyclin D1 in cancer cells, due to its upregulation by Ras, Rho, Src, and other genes that drive malignant development, the new findings suggest that cyclin D1 may have a central role in mediating invasion and metastasis of cancer cells by controlling Rho/ROCK signaling and matrix deposition of TSP-1.

Topics: Animals; Cell Cycle; Cell Movement; Cyclin D1; Extracellular Matrix; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Neoplasms; Protein Serine-Threonine Kinases; Retinoblastoma Protein; rho-Associated Kinases; Signal Transduction; Thrombospondin 1; Transcription, Genetic

The control of cell proliferation is crucial in maintaining cellular homeostasis and loss of this mechanism is a principle hallmark of cancer cells. A primary target of growth factor signaling is the cyclin D1-dependent kinase (D1-CDK4/6) whose activity promotes G1 phase progression by phosphorylating the retinoblastoma protein (Rb) along with related pocket proteins 107 and p130, relieving inhibition of E2F family transcription factors. Cyclin D1 accumulation is regulated at multiple levels including transcription, post-translational activation and cellular localization throughout the cell cycle. While overexpression of cyclin D1 has been observed in a number of human cancers, mouse cancer models overexpressing D1 have fallen short of establishing a role for cyclin D1 in the initiation of malignant phenotypes suggesting an additional regulatory mechanism exists that prevents cyclin D1-driven cancer. This article will present an overview of current data investigating the regulation of cyclin D1 nuclear localization and the prevalence of these aberrations in cancer. Finally, future avenues of research involving cyclin D1 cellular localization and its regulation in cancer will be addressed.

Topics: Active Transport, Cell Nucleus; Animals; Cell Cycle Proteins; Cell Nucleus; Cell Proliferation; Crk-Associated Substrate Protein; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; G1 Phase; Gene Expression Regulation; Humans; Mice; Models, Biological; Neoplasms; Phosphorylation; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; RNA, Messenger; Signal Transduction; Transcription, Genetic; Ubiquitin-Protein Ligases

In 2005, breast cancer will kill approximately 40,410 women in the U.S., and pancreatic cancer will kill approximately 31,800 men and women in the U.S. Clinical examination and mammography, the currently accepted breast cancer screening methods, miss almost half of breast cancers in women younger than 40 years, approximately one-quarter of cancers in women aged 40-49 years, and one-fifth of cancers in women over 50 years old. Pancreatic cancer progresses rapidly, with only 1% of patients surviving more than 5 years after diagnosis. However, if the disease is diagnosed when it is localized, the 5-year survival is approximately 20%. It would be beneficial to detect breast cancer and pancreatic cancer at the earliest possible stage, when multimodal therapy with surgery, radiotherapy, and chemotherapy have the greatest chance of prolonging survival. Human estrogen receptor-positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, elevated cyclin D1 protein due to overexpression of CCND1 mRNA, and elevated insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC or CCND1 peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C-terminus, and a Tc-99m-chelator peptide on the N-terminus, could measure levels of MYC or CCND1 mRNA noninvasively in human IGF1R-overexpressing MCF7 breast cancer xenografts in immunocompromised mice. Similarly, human pancreatic cancer cells typically display elevated levels of KRAS mRNA and elevated IGF1R. Hence, we also hypothesized that a KRAS Tc-99m-chelator PNA-peptide probe could detect overexpression of KRAS mRNA in pancreatic cancer xenografts by scintigraphic imaging, or by positron emission tomography (PET) with a KRAS Cu-64-chelator PNA-peptide. Human MCF7 breast cancer xenografts in immunocompromised mice were imaged scintigraphically 4-24 h after tail-vein administration of MYC or CCND1 Tc-99m-chelator PNA-peptides, but not after administration of mismatch controls. Similarly, human Panc-1 pancreatic cancer cells xenografts were imaged scintigraphically 4 and 24 h after tail-vein administration of a KRAS Tc-99m-chelator PNA-peptide, and AsPC1 xenografts were imaged by PET 4 and 24 h after tail-vein adminstration of a KRAS Cu-64-chelator PNA-peptide. The radioprobes distributed normally to the kidneys, livers, tumors, and other tissues. External molecular imaging of oncogene mRNAs i

Topics: Animals; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasm Transplantation; Neoplasms; Oncogene Protein p21(ras); Peptide Nucleic Acids; Peptides; Proto-Oncogene Proteins c-myc; Recombinant Fusion Proteins; RNA, Messenger

The retinoblastoma protein pathway (pRb1-cyclin D1-cdk4/6-p16(INK4A)) participates in the regulation of the cellular processes at the transition of G1/S phases of the cell-cycle. Derailments of this pathway, caused either by lack of pRb1 or p16(INK4A) expression or overexpression of cyclin D1 and/or cdk4/6, are implicated in the deregulation of the cell-cycle machinery, resulting in uncontrolled cell proliferation, tumor heterogeneity, invasion and metastasis. Several studies conducted so far have assessed the deregulation of the pRb1-pathway components in various human tumors and cell-lines, provided these pathway alterations play an obligatory role in tumorigenesis. This review briefly summarizes the current information on the pRb1-cyclin D1-cdk4/6-p16(INK4A) alterations in sporadic uterine cancer, placing emphasis on the influence on the dualistic model of endometrial carcinogenesis.

Topics: Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Female; Humans; Neoplasms; Proto-Oncogene Proteins; Retinoblastoma Protein; Tumor Cells, Cultured

SV40 LT and ST antigens cooperate to induce the proliferation and eventual transformation of several human cell types. In natural virus infections, ST often enhances the function of LT when both proteins are present, and it can be difficult to completely separate the roles of the individual proteins. By studying ST in the absence of LT or by replacing ST function with combinations of cellular proteins, several themes have emerged which help define the requirement for ST in human cell transformation. These include the activation of transcription of two cyclins, D and A, along with downregulation of the cyclin kinase inhibitor p27. Modification of these key cell cycle regulators may be influenced by the activation of key downstream targets in the PI3K pathway.

Topics: Animals; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Cell Transformation, Neoplastic; Cyclin A; Cyclin D1; Gene Expression Regulation; Humans; Neoplasms; Phosphatidylinositol 3-Kinases

Topics: Cell Cycle Proteins; Cell Division; Cyclin D1; Enzyme Inhibitors; Gene Expression Regulation; Humans; Mitogens; Neoplasms; Signal Transduction

Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein and promotes progression through the G1-S phase of the cell cycle. Amplification or overexpression of cyclin D1 plays pivotal roles in the development of a subset of human cancers including parathyroid adenoma, breast cancer, colon cancer, lymphoma, melanoma, and prostate cancer. Of the three D-type cyclins, each of which binds cyclin-dependent kinase (CDK), it is cyclin D1 overexpression that is predominantly associated with human tumorigenesis and cellular metastases. In recent years accumulating evidence suggests that in addition to its original description as a CDK-dependent regulator of the cell cycle, cyclin D1 also conveys cell cycle or CDK-independent functions. Cyclin D1 associates with, and regulates activity of, transcription factors, coactivators and corepressors that govern histone acetylation and chromatin remodeling proteins. The recent findings that cyclin D1 regulates cellular metabolism, fat cell differentiation and cellular migration have refocused attention on novel functions of cyclin D1 and their possible role in tumorigenesis. In this review, both the classic and novel functions of cyclin D1 are discussed with emphasis on the CDK-independent functions of cyclin D1.

Topics: Animals; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Transcription, Genetic

The availability of murine models with precisely defined genetic lesions has greatly increased our understanding of the genetic control of cell death, with functional dependence established for a wide range of genes including (amongst others) the p53 and Bcl-2 gene family members, the mismatch repair (MMR) genes and the methyl binding domain family member Mbd4. These studies raised the attractive hypotheses that tumour predisposition may be explained in terms of failed cell death, and also that tumour regression may be initiated through activation of an apoptotic programme. The studies that have addressed these notions have revealed complex consequences of a failed death programme, such that these simple hypotheses have not always been supported. Remarkably, however, some tissues show more predictable responses than others, most apparent in the contrast between the intestine and the haematopoietic system. This review will focus upon a discussion of these relationships, and will also consider the relevance of some of these findings to tumour predisposition and regression.

Topics: Animals; Apoptosis; Base Pair Mismatch; Cell Transformation, Neoplastic; Cyclin D1; DNA Damage; DNA Repair; Endodeoxyribonucleases; Genes, p53; Humans; Mice; Models, Animal; Neoplasms; Signal Transduction

During the past two years, new molecular targets have been discovered which link cell cycle, cell proliferation and cellular growth. It has become more and more evident that whereas gain-of-function mutations in specific genes can lead to cancer, genomic instability plays also an important role in tumour progression. With examples taken from the recent literature, we describe in this short review crucial findings on the molecular mechanisms controlling cell cycle and proliferation. We illustrate how specific combinations of proto-oncogenes alterations can result in tissue-specific tumours. Finally, impairment of the interactions of a cancer cell with its surrounding neighbours is also shown to participate in the progression toward aggressive phenotypes.

Topics: Animals; Antineoplastic Agents, Phytogenic; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Disease Progression; Drug Resistance; Fusion Proteins, bcr-abl; Humans; Mice; Mutation; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Neovascularization, Pathologic; Neural Cell Adhesion Molecules; Paclitaxel; Protein Serine-Threonine Kinases; Radiation Tolerance; Rats; Thrombospondins; Transforming Growth Factor beta; Tuberous Sclerosis

The Cyclin D-Cdk4,6/INK4/Rb/E2F pathway plays a key role in controlling cell growth by integrating multiple mitogenic and antimitogenic stimuli. The components of this pathway are gene families with a high level of structural and functional redundancy and are expressed in an overlapping fashion in most tissues and cell types. Using classical transgenic technology as well as gene-targeting in ES cells, a series of mouse models have been developed to study the in vivo function of individual components of this pathway in both normal homeostasis and tumor development. These models have proven to be useful to define specific as well as redundant roles among members of these cell cycle regulatory gene families. This pathway is deregulated in the vast majority of human tumors by genetic and epigenetic alterations that target at least some of its key members such as Cyclin D1, Cdk4, INK4a and INK4b, pRb etc. As a consequence, some of these molecules are currently being considered as targets for cancer therapy, and several novel molecules, such as Cdk inhibitors, are under development as potential anti-cancer drugs.

Topics: Animals; Cells, Cultured; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Drug Design; Enzyme Inhibitors; G1 Phase; Humans; Neoplasms; Proto-Oncogene Proteins

Cell proliferation is controlled not only by soluble mitogens but also by components of the extracellular matrix (ECM) such as fibronectin, to which cells adhere via the integrin family of transmembrane receptors. Input from both growth factor receptors and integrins is required to stimulate progression through the G1 phase of the cell cycle, via induction of G1 cyclins and suppression of inhibitors of the G1 cyclin-dependent kinases. Extensive crosstalk takes place between integrin and growth factor receptor signaling pathways, and mitogenic signaling is weak and transient in the absence of integrin-mediated cell adhesion. In normal untransformed cells, all of the important mitogenic signal transduction cascades, namely those downstream of the Ras and Rho family small GTPases and the phosphoinositide 3-OH kinase-PKB/Akt pathway, are regulated by integrin-mediated cell adhesion. As a result, these cells are anchorage-dependent for growth. In contrast, constitutive activity of each of these pathways has been reported in cancer cells, which not only reduces their mitogen dependence but also allows these cells to grow in an anchorage-independent fashion.

Topics: Animals; Cell Adhesion; Cell Cycle; Cell Division; Cyclin D1; Fibronectins; Humans; Integrins; Mitogens; Neoplasms; Receptors, Growth Factor; rho GTP-Binding Proteins; Signal Transduction; Transcription, Genetic

A central issue in the study of neoplastic transformation is to understand how proto-oncogene products deregulate normal processes of cell growth and differentiation: an intrinsic aspect of this is to probe the sequence of events leading to altered expression of proto-oncogenes. In the past few years, studies aimed at understanding the regulation and function of protein synthesis initiation factors, eIF4E initially, culminated in the unexpected finding that a moderate overexpression of this factor results in dramatic phenotypic changes, including rapid proliferation and malignant transformation. Conversely, the tumorigenic properties of cancer cells can be strongly inhibited by antisense-RNA against eIF4E, or overexpression of the inhibitory proteins: 4E-BPs. Furthermore, eIF4E is elevated in carcinomas of the breast, head and neck (HNSCC) and prostate, but not in typical benign lesions. This is a strong indication that elevated eIF4E expression may mark a critical transition in cancer progression. Establishing a greater protein synthesis output may be a necessary step for cancer cells in order to sustain their rapid proliferation. However, analysis of cells transformed by eIF4E revealed that the synthesis of only a few proteins was greatly enhanced, while synthesis of most was minimally increased. One possible explanation is that eIF4E causes these effects by specifically increasing the translational efficiency of several oncogene transcripts, leading to overexpression of their products. The feasibility of this hypothesis was confirmed experimentally with the identification of several important products that are specifically upregulated in eIF4E-overexpressing cells. These include: c-Myc, cyclin DI and ODC, which control cycle progression and tumorigenesis; basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF), which are powerful promoters of cell growth and angiogenesis. A deeper understanding of the mRNAs that are strongly dependent on excess eIF4E/F for efficient translation will eventually result in fuller understanding of the fundamental role of translational control in different pathophysiological conditions, including malignancy.

Topics: Animals; Apoptosis; Breast Neoplasms; Carcinoma; Cell Division; Cyclin D1; Disease Progression; Endothelial Growth Factors; Eukaryotic Initiation Factor-4E; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Genes, myc; Head and Neck Neoplasms; Humans; Lymphokines; Metalloendopeptidases; Neoplasm Proteins; Neoplasms; Peptide Initiation Factors; Proto-Oncogene Mas; RNA, Messenger; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Resveratrol (trans-3,4',5-trihydroxystibene) is a phytopolyphenol isolated from the seeds and skins of grapes. Recent studies indicate that resveratrol can block the process of multistep carcinogenesis, namely, tumor initiation, promotion and progression. Resveratrol can also reduce the risk of cardiovascular disease in man. The molecular mechanisms of resveratrol in chemoprevention of cancer and cardiovascular disease are interesting and under intensive investigation. Resveratrol was found to strongly inhibit nitric oxide (NO) generation in activated macrophages, as measured by the amount of nitrite released into the culture medium, and resveratrol strongly reduced the amount of cytosolic inducible nitric oxide synthase (iNOS) protein. The activation of nuclear factor kappa B (NF kappa B) induced by lipopolysaccharide (LPS) was inhibited by resveratrol. The phosphorylation and degradation of nuclear factor inhibitor kappa B alpha (I kappa B alpha) were inhibited by resveratrol simultaneously. Reactive oxygen species (ROS) are regarded as having carcinogenic potential and have been associated with tumor promotion. Resveratrol may act as a reactive oxygen species scavenger to suppress tumor development. In addition, resveratrol may block multistep carcinogenesis through mitotic signal transduction blockade. Reactive oxygen species are pivotal factors in the genesis of heart disease. Meanwhile, efficient endogenous antioxidants, including superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase, are present in tissues. A fine balance between reactive oxygen species and endogenous antioxidants is believed to exist. Any disturbance of this balance in favor of reactive oxygen species causes an increase in oxidative stress and initiates subcellular changes, leading to cardiomyopathy and heart failure. The experimental results indicate that exogenous antioxidant resveratrol is of value in chemopreventing the development of heart disease. It is urgent that more efforts be made to investigate newer therapies employing antioxidants for the chemoprevention of cardiovascular disease and cancer.

Topics: Anticarcinogenic Agents; Carcinogens; Cardiovascular Diseases; Cell Cycle; Cyclin D1; Enzyme Inhibitors; Gene Expression Regulation; Growth Substances; Humans; Lipid Peroxidation; Neoplasms; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Phosphorylation; Protein Processing, Post-Translational; Reactive Oxygen Species; Receptor Protein-Tyrosine Kinases; Resveratrol; Risk Factors; Rosales; Signal Transduction; Stilbenes; Transcription, Genetic; Wine

Topics: Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Genes, Retinoblastoma; Humans; Microtubule-Associated Proteins; Mutation; Neoplasms; Proto-Oncogene Proteins; Tumor Suppressor Proteins

Neoplasia is characterised by abnormal regulation of the cell cycle. Cyclin D1 is a protein derived from the PRAD1, CCND1 or bcl-1 gene on chromosome 11q13, which is involved in both normal regulation of the cell cycle and neoplasia. In the G1 (resting) phase of the cell cycle, cyclin D1 together with its cyclin dependent kinase (cdk) partner, is responsible for transition to the S (DNA synthesis) phase by phosphorylating the product of the retinoblastoma gene (pRB), which then releases transcription factors important in the initiation of DNA replication. Amplification of the CCND1 gene or overexpression of the cyclin D1 protein releases a cell from its normal controls and causes transformation to a malignant phenotype. Analysis of these changes provides important diagnostic information in mantle cell (and related) lymphomas, and is of prognostic value in many cancers. Knowledge of cyclin D1's role in malignancy at the various sites, provides a basis on which future treatment directed against this molecule can proceed.

Topics: Biomarkers, Tumor; Cell Cycle; Cyclin D1; Genes, bcl-1; Humans; Neoplasm Proteins; Neoplasms; Retinoblastoma Protein

Tumour formation relies on a complex combination of genetic and environmental factors. In particular, the contributions from inherited predisposition genes as well as carcinogens, for example from cigarettes or in the diet, are amongst the major contributors to tumorigenesis. Since the study of such processes in particularly difficult in human cancers, the availability of a well-defined model system is of obvious benefit. The mouse skin model of multistage carcinogenesis offers an excellent tool for the study of the target cells, the target genes and the biological events associated with neoplasia. In this system, tumorigenesis occurs in a series of defined stages, each of which is characterized by specific and reproducible alterations in genes such as H-ras, cyclin D1, p53 and p16INK4A. Additional changes occur in the production of, or response to, factors such as transforming growth factor beta (TGF beta). These genetic and biological alterations are mirrored in human tumours of epithelial origin. Hence, research into the general principles of tumour initiation, promotion and progression in the context of the mouse skin model is likely to prove valuable in the continual search for new methods for the diagnosis, prevention, and therapeutic treatment of human cancers.

Topics: Animals; Cell Cycle; Cell Transformation, Neoplastic; Cyclin D1; DNA Damage; Epithelial Cells; Genes, p16; Genes, p53; Genes, ras; Humans; Mice; Models, Biological; Neoplasms; Neoplasms, Experimental

Cell production within an organ is determined by the rate of immigration, proliferation, differentiation, emigration and death of cells. Abnormalities in any one of these processes will disturb normal control of cell production, thereby eliciting hyperplasia can be an early event in neoplasia. Cell death, apoptosis, is a physiological process responsible for removing unwanted cells. It is used in multi-cellular organisms for tissue remodelling during embryogenesis, regulation of cell turnover and as a defence strategy against invading pathogens. In this review article we describe the role of the bcl-2/ced-9 gene family in cancer and discuss the general implications of defects in the apoptosis program for tumourigenesis and resistance of cancer cells to chemotherapy in light of current knowledge of the molecular mechanisms of cell death.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Caenorhabditis elegans Proteins; Cell Transformation, Neoplastic; Cyclin D1; Drug Resistance, Neoplasm; Genes, bcl-2; Genes, p53; Helminth Proteins; Humans; Mutation; Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2

Topics: Cell Cycle; Cyclin D1; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Humans; Mutation; Neoplasms; Oncogene Proteins; Translocation, Genetic

Until recently studies on mutations in cellular genes implicated in multistage carcinogenesis have concentrated mainly on dominant acting mutations in cellular proto-oncogenes, genes that normally mediate agonist-induced signal transduction pathways, and recessive mutations in cellular tumor suppressor genes, whose normal products appear to inhibit cell growth and/or control differentiation and cell-cell interactions. It seems likely, however, that a third category of cellular genes, the cyclins and cyclin-related genes, may also be critical targets during multistage carcinogenesis because of the central role that they play in controlling cell cycle progression. These proteins could, therefore, provide biomarkers for identifying individuals at high risk of developing cancer and also serve as novel targets for chemopreventive agents. This paper reviews evidence that the gene cyclin D1 is amplified and/or overexpressed in a major fraction of human tumors, and that this can occur relatively early in the carcinogenic process. Mechanistic studies indicates that this overexpression plays a critical role in tumor progression as well as the maintenance of the tumorigenic phenotype. Thus, increased cyclin D1 expression can enhance gene amplification and cell transformation and antisense to cyclin D1 can revert malignant cells. The latter findings provide direct evidence that cyclin D and related proteins might be useful markers and also targets for cancer chemoprevention.

Topics: Anticarcinogenic Agents; Biomarkers, Tumor; Cell Cycle; Cyclin D1; Cyclins; Humans; Neoplasms; Oligonucleotides, Antisense; Oncogene Proteins; Risk Factors

Topics: Adenoma; Animals; Chromosomes, Human, Pair 11; Cyclin D1; Cyclins; Gene Amplification; Gene Expression Regulation; Humans; Lymphoma, B-Cell; Neoplasms; Oncogene Proteins; Parathyroid Neoplasms

The cell cycle clock is the central controller of cell proliferation that governs the progress of the cell through its growth cycle, its exit from the active cycle, and its decision to differentiate. Components of the clock are found to be functioning in an aberrant fashion in many types of malignancies. Notable among these is the retinoblastoma protein, pRB, which acts to restrain proliferation in normal cells and suffers inactivation in many types of tumour cells. Its activity is controlled by D-type cyclins in various cell types. We have deleted one of these cyclins--cyclin D1--from the mouse germline and find that its absence leads to a limited range of defects including hypoplastic retinae and the inability of the mammary epithelium to respond to pregnancy-associated hormonal stimulation. Cyclin D1 is overexpressed in many human breast cancers, pointing to a highly specific association of this cell cycle clock component with mammary cell proliferation.

Topics: Activity Cycles; Animals; Cell Cycle; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinases; Cyclins; Genes, Retinoblastoma; Humans; Mice; Neoplasms; Retinoblastoma Protein

Amplification of oncogenes has been observed frequently in various human malignancies and might be of clinical relevance. In the last decade, the exploration of oncogene activation due to DNA amplification in cancer research has mainly focussed on three aspects: (i) the assessment of oncogene amplification as a prognostic marker for survival of cancer patients, (ii) the development of reliable methods for detection of tumors which harbor DNA amplification of oncogenes and (iii) the identification of the gene or genes responsible for the biological (prognostic) significance in tumors with DNA amplification and the characterization of these candidate proto-oncogenes that might help to elucidate their normal function and the role in tumor development. In this review, these three aspects will be highlighted with regard to DNA amplification of the chromosome 11q13 region. Chromosome 11q13 amplification has been found frequently in certain human malignancies; in cancer of the breast and of the head and neck region, amplification of this region is observed in 13 and 29% of tumors, respectively. The 11q13 amplification has been reported to be of clinical relevance in these cancers, since patients with this amplification show a poor clinical course of disease. The amplified 11q13 region is estimated to be 3-5 Mb in size and to harbor many (putative) genes. Recently, two candidate genes, CCND1 and EMS1, were identified which were both over-expressed in all carcinomas with an 11q13 amplification. Therefore, the activation of these genes might confer the selective advantage to these tumors. In addition, the characterization of these two novel genes sustained their potential role in carcinomas with 11q13 amplification.

Topics: Chromosomes, Human, Pair 11; Cortactin; Cyclin D1; Cyclins; Gene Amplification; Genetic Markers; Humans; Microfilament Proteins; Neoplasm Proteins; Neoplasms; Oncogene Proteins; Oncogenes; Prognosis

The D-type cyclins are expressed during the progression from G0/G1 to S phase in the mammalian cell cycle. There is considerable evidence that they contribute to the development of specific cancers, both in humans and in mouse models. For example, cyclin D1 can be activated by chromosomal translocation, DNA amplification and retroviral integration. Cyclins D1, D2 and D3 preferentially associate with two closely related members of the cyclin-dependent kinase family, Cdk4 and Cdk6 and the various complexes are each capable of phosphorylating the retinoblastoma gene product (pRb), at least in vitro. This suggests that the growth promoting effects of the D-cyclins may be manifest via their interactions with tumour suppressor genes.

Topics: Amino Acid Sequence; Animals; Cell Cycle; Cyclin D1; Cyclin-Dependent Kinases; Cyclins; Humans; Molecular Sequence Data; Neoplasms; Oncogene Proteins; Retinoblastoma Protein

Topics: Animals; Apoptosis; Cell Cycle; Cell Division; Cyclin D1; Cyclin-Dependent Kinases; Cyclins; DNA Damage; Gene Expression; Homeostasis; Humans; Mitosis; Models, Biological; Mutation; Neoplasms; Oncogene Proteins; Proto-Oncogenes; S Phase

Bardoxolone methyl, a novel synthetic triterpenoid and antioxidant inflammation modulator, potently induces Nrf2 and inhibits NF-κB and Janus-activated kinase/STAT signaling. This first-in-human phase I clinical trial aimed to determine the dose-limiting toxicities (DLT), maximum tolerated dose (MTD), and appropriate dose for phase II studies; characterize pharmacokinetic and pharmacodynamic parameters; and assess antitumor activity.. Bardoxolone methyl was administered orally once daily for 21 days of a 28-day cycle. An accelerated titration design was employed until a grade 2-related adverse event occurred. A standard 3 + 3 dose escalation was then employed until the MTD was reached. Single dose and steady-state plasma pharmacokinetics of the drug were characterized. Assessment of Nrf2 activation was examined in peripheral blood mononuclear cells (PBMC) by measuring NAD(P)H:quinone oxidoreductase (NQO1) mRNA levels. Immunohistochemical assessment of markers of inflammation, cell cycle, and apoptosis was carried out on tumor biopsies.. The DLTs were grade 3 reversible liver transaminase elevations. The MTD was established as 900 mg/d. A complete tumor response occurred in a mantle cell lymphoma patient, and a partial response was observed in an anaplastic thyroid carcinoma patient. NQO1 mRNA levels increased in PBMCs, and NF-κB and cyclin D1 levels decreased in tumor biopsies. Estimated glomerular filtration rate (eGFR) was also increased.. Bardoxolone methyl was well tolerated with an MTD of 900 mg/d. The increase in eGFR suggests that bardoxolone methyl might be beneficial in chronic kidney disease. Objective tumor responses and pharmacodynamic effects were observed, supporting continued development of other synthetic triterpenoids in cancer.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Renal Cell; Colorectal Neoplasms; Cyclin D1; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Janus Kinases; Kidney Neoplasms; Lymphoma; Male; Maximum Tolerated Dose; Melanoma; Middle Aged; NAD(P)H Dehydrogenase (Quinone); Neoplasms; NF-E2-Related Factor 2; NF-kappa B; Oleanolic Acid; RNA, Messenger; STAT Transcription Factors; Thyroid Neoplasms; Young Adult

The small non-coding RNA miR-34a is a p53-regulated miRNA that acts as a tumour suppressor of colorectal cancer (CRC). Oncogenesis is also negatively influenced by deregulation of the circadian system in many types of tumours with various genetic backgrounds. As the clock gene per2 was recently recognized as one of the target genes of miR-34a, we focused on the miR-34a-mediated influence on the circadian oscillator in CRC cell lines DLD1 and LoVo, which differ in their p53 status. Previously, a sex-dependent association between the expression of per2 and that of miR-34a was demonstrated in CRC patients. Therefore, we also investigated the effect of 17β-estradiol (E2) on miR-34a oncostatic functions. miR-34a mimic caused a pronounced inhibition of per2 expression in both cell lines. Moreover, miR-34a mimic significantly inhibited bmal1 expression in LoVo and rev-erbα expression in DLD1 cells and induced clock gene expression in both cell lines. miR-34a mimic caused a pronounced decrease in sirt1 and cyclin D1 expression, which may be related to the inhibition of proliferation observed after mir-34a administration in DLD1 cells. E2 administration inhibited the migration and proliferation of DLD1 cells. E2 and miR-34a, when administered simultaneously, did not potentiate each other's effects. To conclude, miR-34a strongly influences the expression of components of the circadian oscillator without respect to p53 status and exerts its oncostatic effects via inhibition of sirt1 and cyclin D1 mRNA expression. E2 administration inhibits the growth of DLD1 cells; however, this effect seems to be independent of miR-34a-mediated action. With respect to the possible use of miR-34a in cancer treatment, clock genes can be considered as off-target genes, as changes in their expression induced by miR-34a treatment do not contribute to the oncostatic functions of miR-34a. Possible ambiguous oncogenic characteristics should be taken into consideration in future clinical studies focused on miR-34a.

Topics: Cell Line, Tumor; Cyclin D1; Estradiol; Humans; MicroRNAs; Neoplasms; Sirtuin 1; Tumor Suppressor Protein p53

The study aimed to investigate the in vitro inhibitory activities of spiro N-propargylic β-enaminones, SPEs 1-31, against BCa cells, to perform in silico molecular docking studies to understand the nature of the interaction between the compounds and the ERα, PR, EGFR, and Her2, and to determine the ADMET and drug-likeness properties. Cytotoxic activity was investigated via MTT assay. DNA fragmentation was evaluated via ELISA assay. Cell cycle distributions were investigated by flow cytometry. Expression levels of Bcl-2, Bax, p21 and Cyclin D1 were measured by qRT-PCR and western blot analysis. Molecular docking was done using Autodock/vina software. ADMET analysis was calculated using the ADMETlab 2.0 tool. SPEs 1, 22, and 28 showed selective cytotoxic activity against all BCa cells with SI values >2. SPEs induced apoptosis and caused significant changes in Bcl-2 and Bax levels. The cell cycle was arrested at the S phase and levels of p21 and Cyclin D1 were induced in all BCa cells. Molecular docking analysis revealed that SPE1, SPE22, and SPE28 showed high binding affinities with ERα, PR, EGFR, and Her2. ADMET analysis revealed that SPEs are drug-like compounds as they obey the five rules of Lipinsky and are not toxic. Therefore, these potential anticancer compounds should be further validated by in vivo studies for their appropriate function in human health with a safety profile, and a comprehensive drug interaction study should be performed.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Estrogen Receptor alpha; Humans; Molecular Docking Simulation; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Structure-Activity Relationship

Cyclin-dependent kinase 4 (CDK4) is a member of the cyclin-dependent kinases, a family of protein kinases with outstanding roles in signaling pathways, transcription regulation, and cell division. Defective or overactivated CDK4/cyclin D1 pathway leads to enhanced cellular proliferation, thus being implicated in human cancers. Although the biological role of CDK4 has been extensively studied, its pre-mRNA processing mechanism under normal or pathological conditions is neglected. Thus, the identification of novel CDK4 mRNA transcripts, especially protein-coding ones, could lead to the identification of new diagnostic and/or prognostic biomarkers or new therapeutic targets. In the present study, instead of using the 'gold standard' direct RNA sequencing application, we designed and employed a targeted nanopore sequencing approach, which offers higher sequencing depth and enables the thorough investigation of new mRNAs of any target gene. Our study elucidates for the first time the complex transcriptional landscape of the human CDK4 gene, highlighting the existence of previously unknown CDK4 transcripts with new alternative splicing events and protein-coding capacities. The relative expression levels of each novel CDK4 transcript in human malignancies were elucidated with custom qPCR-based assays. The presented wide spectrum of CDK4 transcripts (CDK4 v.2-v.42) is only the first step to distinguish and assemble the missing pieces regarding the exact functions and implications of this fundamental kinase in cellular homeostasis and pathophysiology.

Topics: Alternative Splicing; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Gene Expression Regulation, Neoplastic; Humans; Nanopore Sequencing; Neoplasms; RNA-Seq; RNA, Messenger; Signal Transduction; Transcription, Genetic

USP2 contributes to the quality control of multiple oncogenic proteins including cyclin D1, Mdm2, Aurora-A, etc., and it is a potential target for anti-cancer drug development. However, currently only a few inhibitors with moderate inhibition activities against USP2 have been discovered. USP2-targeted active compounds with either new scaffolds or enhanced activities are in need. Here in this study, Ub-AMC hydrolysis assay-based screening against ~4000 commercially available drugs and drug candidates was performed to identify USP2-targeted inhibitors. COH29, which was originally developed as an anti-cancer agent by blocking the function of human ribonucleotide reductase (RNR, IC

Topics: Benzamides; Cyclin D1; Down-Regulation; Enzyme Inhibitors; Holoenzymes; Humans; Neoplasms; Ribonucleotide Reductases; Thiazoles; Ubiquitin Thiolesterase; Ubiquitin-Specific Proteases

Frequent activating mutations in the mitogen-activated protein kinase (MAPK) pathway genes have been identified in histiocytoses. MAPK signaling consistently upregulates cyclin D1. The goal of this study was to determine whether cyclin D1 expression by immunohistochemistry is a useful diagnostic marker for periocular histiocytoses and to further characterize their genetic basis.. Retrospective observational case series.. Pathology records were searched for all patients with histiocytoses diagnosed between 1995 and 2020. Eleven histiocyte-rich inflammatory lesions and 10 xanthelasma served as controls. Cyclin D1 immunohistochemistry was performed on all tissues. A subset of histiocytoses was evaluated by next-generation sequencing (NGS) and droplet digital PCR (ddPCR).. There were 36 patients, 15 males (42%) and 21 females (58%), with histiocytoses: 9 juvenile xanthogranuloma (25%), 8 adult-onset asthma and periocular xanthogranuloma (22%), 7 Langerhans cell histiocytosis (19%), 5 Rosai-Dorfman disease (14%), 5 xanthogranuloma-not otherwise specified (14%), 1 Erdheim-Chester disease (3%), and 1 histiocytic sarcoma (3%). Moderate to strong nuclear cyclin D1 expression was present in ≥50% of lesional cells in histiocytoses (23/36, 64%), significantly more when compared to histiocyte-rich inflammatory lesions (0/11, 0%, P<.001) and xanthelasma (0/10, 0%, P<.001). Cyclin D1 was expressed in <10% of lesional cells in all 11 histiocyte-rich inflammatory lesions (P<.001) and all 10 xanthelasma lesions (P<.001). MAPK pathway gene mutations were detected in 12 of 14 (86%) histiocytoses successfully assayed by NGS and/or ddPCR.. Our study confirms that the cyclin D1 immunohistochemical stain is a useful diagnostic marker for periocular histiocytoses, correlating with underlying mutations in MAPK pathway genes.

Topics: Adult; Cyclin D1; Dendritic Cells; Female; Histiocytosis, Langerhans-Cell; Humans; Macrophages; Male; Mitogen-Activated Protein Kinases; Molecular Biology; Neoplasms; Retrospective Studies

The abnormal activation of the Wnt/β-catenin signaling pathway and epithelial-mesenchymal transition (EMT) in drug-resistant tumor cells and cancer stem cells (CSCs) stimulate tumor metastasis and recurrence. Here, a promising combined chemotherapeutic strategy of salinomycin (SL) and doxorubicin (DOX) with specific inhibition of tumor stemness by a targeted co-delivery nanosystem was developed to overcome this abnormal progression. This strategy could be benefit drugs to effectively penetrate and infiltrate into spheres of 3D-cultured breast cancer stem cells (BCSCs). The expression of the Wnt/β-catenin signaling pathway-related genes (β-catenin, LRP6, LEF1, and TCF12) and target genes (Cyclin D1, Cmyc, and Fibronectin) as well as CSC stemness-related genes (Oct4, Nanog, and Hes1) was downregulated by redox-sensitive co-delivery micelles decorated with oligohyaluronic acid as the active targeting moiety. The changes in EMT-associated gene expression (E-cadherin and Vimentin) in vitro showed that the EMT process was also effectively inverted. This strategy achieved a strong inhibitory effect on solid tumor growth and an effective reduction in the risk of tumor metastasis in 4T1 tumor-bearing mice in vivo and effectively alleviated splenomegaly caused by the malignant tumor. Immunohistochemical staining analysis of E-cadherin, Vimentin, and β-catenin confirmed that the inversion of the EMT was also achieved in solid tumors. These results highlight the potential of SL and DOX combined chemotherapeutic strategy for eliminating breast carcinoma. STATEMENT OF SIGNIFICANCE: Cancer stem cells (CSCs), as an important part of tumor heterogeneity, can survive against conventional chemotherapy and initiate tumorigenesis, recurrence, and metastasis. Moreover, non-CSCs can convert into the CSC state through the abnormal Wnt/β-catenin pathway, which is closely related to the epithelial-mesenchymal transition (EMT) process. Here, redox-degradable binary drug-loaded micelles (PPH/DOX+SL) were designed to target CSCs and overcome drug resistance of breast cancer cells. The combined chemotherapy of salinomycin (SL) and doxorubicin (DOX) reversed drug resistance, while the PPH/DOX+SL micelles enhanced the intracellular accumulation and drug penetration of BCSC spheres. The introduction of SL downregulated the expression of tumor stemness genes and the Wnt/β-catenin pathway-related genes and inverted the EMT process. PPH/DOX+SL continuously inhibited tumor growth and invasio

Topics: Animals; beta Catenin; Cadherins; Cell Line, Tumor; Cyclin D1; Doxorubicin; Epithelial-Mesenchymal Transition; Fibronectins; Mice; Micelles; Neoplasms; Neoplastic Stem Cells; Vimentin; Wnt Signaling Pathway

Topics: Antineoplastic Agents; Apoptosis; beta Catenin; Caco-2 Cells; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Matrix Metalloproteinase 7; Molecular Docking Simulation; Neoplasms

to explore proliferative potential of peripheral blood lymphocytes of Chornobyl clean-up workers and persons with malignant neoplasms of the oral cavity, oropharynx and laryngopharynx by level of expression of cyclin D1 and quantitative parameters of cell cycle.. A total of 294 men aged (58.47 ± 7.32) were surveyed, 215 of them were Chornobyl clean"up workers (1986-1987), exposed at the dose range 10.43-3623.31 mSv; 49 persons of the control group and 30persons with malignant neoplasms of the oral cavity, oropharynx and laryngopharynx at III, IVА and IVВ stages ofthe disease. The analysis of parameters of cell cycle and proliferative activity of peripheral blood (PB) lymphocyteswas performed using the flow cytometry. The evaluation of distribution of cells by G0/G1, S, G2/M cell cycle phaseswas done in vivo and in in vitro. Proliferative potential was analyzed by level of expression of cytoplasmic protein ofcyclin D1.. Proliferative potential of PB lymphocytes of Chornobyl clean"up workers and persons with malignant neo"plasms of the oral cavity, oropharynx and laryngopharynx was assessed. An increase in the level of spontaneousсyclin D1 expression and disturbance of сyclin D1-dependent regulation of cell cycle of PB lymphocytes after mito"gen activation were determined in the Chornobyl clean-up workers. An increase in pool of cells in the S" and G2/M"phases of cell cycle was detected, which characterizes high proliferative potential of PB lymphocytes. These changesare most pronounced in the subgroup of persons with a radiation dose of D > 500 mSv, and in persons with oncolo"gical pathology.. A positive linear dependence has been established between the radiation dose and the number of cellsin the S"phase of cell cycle in the subgroup of Chornobyl clean"up workers with a radiation dose of D > 500 mSv. The detected changes of cyclin D1-dependent regulation of cell cycle and proliferative status of lymphocytes depend on the radiation dose, can be a manifestation of genome instability and be a cause for risks of oncogenesis, in a remote period after radiation exposure.. Meta. Doslidyty osoblyvosti proliferatyvnogo potentsialu limfotsytiv peryferychnoï krovi uchasnykivlikvidatsiï naslidkiv avariï na ChAES ta osib, khvorykh na zloiakisni novoutvorennia porozhnyny rota, rotovoï ta gor"tannoï chastyn glotky, za rivnem ekspresiï tsyklinu D1 ta kil'kisnymy pokaznykamy klitynnogo tsyklu.Materialy i metody. Obstezheno 294 cholovikiv u vitsi (58,47 ± 7,32) rokiv, sered nykh: 215 uchasnykiv likvidatsiïnaslidkiv avariï (LNA) na ChAES 1986–1987 rr., dozy zovnishn'ogo oprominennia 10,43–3623,31 mZv; 49 osib kont"rol'noï grupy ta 30 khvorykh na zloiakisni novoutvorennia porozhnyny rota, rotovoï ta gortannoï chastyn glotky III,IVA ta IVV stadiĭ zakhvoriuvannia. Analiz parametriv klitynnogo tsyklu i proliferatyvnoï aktyvnosti limfotsytivperyferychnoï krovi (PK) provodyly metodom protochnoï tsytometriï. Otsiniuvaly rozpodil klityn po G0/G1", S" iG2/M"fazakh klitynnogo tsyklu v umovakh in vivo ta in vitro. Proliferatyvnyĭ potentsial analizuvaly za rivnemekspresiï tsytoplazmatychnogo bilka tsyklinu D1.Rezul'taty. Provedeno otsinku proliferatyvnogo potentsialu limfotsytiv PK uchasnykiv likvidatsiï naslidkivavariï na ChAES ta osib, khvorykh na zloiakisni novoutvorennia porozhnyny rota. V uchasnykiv LNA na ChAES vyznache"no pidvyshchennia rivnia spontannoï ekspresiï tsyklinu D1 ta porushennia tsyklin D1"zalezhnoï reguliatsiï klitynnogotsyklu limfotsytiv PK pislia aktyvatsiï mitogenom. Vyznacheno zbil'shennia pulu klityn u S" ta G2/M"fazakh klityn"nogo tsyklu, shcho kharakteryzuie vysokyĭ proliferatyvnyĭ potentsial limfotsytiv PK. Dani zminy naĭbil'sh vyra"zheni u pidgrupi osib, oprominenykh v dozi D > 500 mZv, ta u patsiientiv z onkologichnoiu patologiieiu. Vysnovky. Vstanovleno pozytyvnu liniĭnu zalezhnist' mizh dozoiu oprominennia ta kil'kistiu klityn S"fazyklitynnogo tsyklu u pidgrupi uchasnykiv LNA na ChAES, oprominenykh u dozakh D > 500 mZv. Vyiavleni zminy tsyklin D1"zalezhnoï reguliatsiï klitynnogo tsyklu ta proliferatyvnogo statusu limfotsytiv zalezhat' vid dozy oprominennia,mozhut' buty proiavom nestabil'nosti genomu i sprychyniaty ryzyky onkogenezu u viddalenomu periodi pislia op"rominennia.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Cell Cycle; Chernobyl Nuclear Accident; Cyclin D1; Emergency Responders; Humans; Lymphocytes; Male; Middle Aged; Mouth; Neoplasms; Occupational Exposure; Pharyngeal Neoplasms; Radiation Dosage; Radiation Exposure; Radiation Injuries; Ukraine

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Division; CRISPR-Cas Systems; Cyclin D1; Cyclin D2; Cyclin D3; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Female; Gene Knockout Techniques; Genes, Tumor Suppressor; HCT116 Cells; HEK293 Cells; Humans; Male; Mice; Neoplasms; Ubiquitin

RNA interference offers therapeutic opportunities for the clinical targeting of otherwise undruggable oncogenes. However RNAi can have off-target effects that considerably increase treatment risks. To manage these side effects and allow an easy subtraction of their activity in healthy tissues, we present here the TAG-RNAi approach where cells that are not designated targets do not have the mRNA tag. Using TAG-RNAi we first established the off-target signatures of three different siRNAs specific to the Cyclin D1 oncogene by RNA-sequencing of cultured cancer cells expressing a FLAG-HA-tagged-Cyclin D1. Then, by symmetrical allografts of tagged-cancer cells and untagged controls on the left and right flanks of model mice, we demonstrate that TAG-RNAi is a reliable approach to study the functional impact of any oncogene without off-target bias. Finally we show, as examples, that mutation-specific TAG-RNAi can be applied to downregulate two oncogenic mutants, KRAS-G12V or BRAF-V600E, while sparing the expression of the wild-type proteins. TAG-RNAi will thus avoid the traditional off-target limitations of RNAi in future experimental approaches.

Topics: Animals; Cell Line, Tumor; Cyclin D1; Humans; Mice; Mice, Nude; Mutation; Neoplasms; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); RNA Interference; RNA, Small Interfering

Binding of transcription factors to mutated DNA sequences is a likely regulator of cancer progression. Noncoding regulatory mutations such as those on the core promoter of the gene encoding human telomerase reverse transcriptase have been shown to affect gene expression in cancer. Using a protein microarray of 667 transcription factor DNA-binding domains and subsequent functional assays, we looked for transcription factors that preferentially bind the mutant hTERT promoter and characterized their downstream effects. One of them, friend leukemia integration 1 (FLI1), which belongs to the E26 transforming-specific family of transcription factors, exhibited particularly strong effects with respect to regulating hTERT expression, while the even better binding ELK3 did not. Depletion of FLI1 decreased expression of the genes for cyclin D1 (CCND1) and E2F transcription factor 2 (E2F2) resulting in a G1/S cell cycle arrest and in consequence a reduction of cell proliferation. FLI1 also affected CMTM7, another gene involved in G1/S transition, although by another process that suggests a balanced regulation of the tumor suppressor gene's activity via opposing regulation processes. FLI1 expression was found upregulated and correlated with an increase in CCND1 expression in pancreatic cancer and brain tumors. In non-neoplastic lung cells, however, FLI1 depletion led to rapid progression through the cell cycle. This coincides with the fact that FLI1 is downregulated in lung tumors. Taken together, our data indicate a cell cycle regulatory hub involving FLI1, hTERT, CCND1 and E2F2 in a tissue- and context-dependent manner.

Topics: Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cyclin D1; Disease Progression; E2F2 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Mutation; Neoplasms; Promoter Regions, Genetic; Protein Array Analysis; Proto-Oncogene Protein c-fli-1; Telomerase

Several experimental strategies in the treatment of cancer include drug alteration of cell cycle regulatory pathways as a useful strategy. Extra-ribosomal functions of human ribosomal protein L3 (uL3) may affect DNA repair, cell cycle arrest and apoptosis. In the present study, we demonstrated that uL3 is required for the activation of G1/S transition genes. Luciferase assays established that uL3 negatively regulates the activity of E2F1 promoter. Induced ribosome-free uL3 reduces Cyclin D1 mRNA and protein levels. Using protein/protein immunoprecipitation methods, we demonstrated that uL3 physically interacts with PARP-1 affecting E2F1 transcriptional activity. Our findings led to the identification of a new pathway mediated by uL3 involving E2F1 and Cyclin D1 in the regulation of cell cycle progression.

Topics: Cell Nucleolus; Cyclin D1; E2F1 Transcription Factor; G1 Phase; HCT116 Cells; Humans; Neoplasms; Ribosomal Protein L3; Ribosomal Proteins; S Phase; Stress, Physiological

The most prominent cancer-associated fibroblasts (CAFs) in tumor stroma is known to form a protective structure to support tumor growth. Anterior gradient-2 (AGR2), a tumor secretory protein is believed to play a pivotal role during tumor microenvironment (TME) development. Here, we report that extracellular AGR2 enhances fibroblasts elongation and migration significantly. The early stimulation of RhoA showed the association of AGR2 by upregulation of G1-S phase-regulatory protein cyclin D1 and FAK phosphorylation through fibroblasts growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR). Our finding indicates that secretory AGR2 alters fibroblasts elongation, migration, and organization suggesting the secretory AGR2 as a potential molecular target that might be responsible to alter fibroblasts infiltration to support tumor growth.

Topics: 3T3 Cells; Animals; Cancer-Associated Fibroblasts; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Culture Media, Conditioned; Cyclin D1; Focal Adhesion Kinase 1; Humans; MCF-7 Cells; Mice; Mucoproteins; Neoplasms; Oncogene Proteins; Phosphorylation; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Vascular Endothelial Growth Factor; rhoA GTP-Binding Protein; Tumor Microenvironment

Angiogenesis is critical to cancer development and metastasis. However, anti-angiogenic agents have only had modest therapeutic success, partly due to an incomplete understanding of tumor endothelial cell (EC) biology. We previously reported that the microRNA (miR)-200 family inhibits metastasis through regulation of tumor angiogenesis, but the underlying molecular mechanisms are poorly characterized. Here, using integrated bioinformatics approaches, we identified the RNA-binding protein (RBP) quaking (QKI) as a leading miR-200b endothelial target with previously unappreciated roles in the tumor microenvironment in lung cancer. In lung cancer samples, both miR-200b suppression and QKI overexpression corresponded with tumor ECs relative to normal ECs, and QKI silencing phenocopied miR-200b-mediated inhibition of sprouting. Additionally, both cancer cell and endothelial QKI expression in patient samples significantly corresponded with poor survival and correlated with angiogenic indices. QKI supported EC function by stabilizing cyclin D1 (CCND1) mRNA to promote EC G1/S cell cycle transition and proliferation. Both nanoparticle-mediated RNA interference of endothelial QKI expression and palbociclib blockade of CCND1 function potently inhibited metastasis in concert with significant effects on tumor vasculature. Altogether, this work demonstrates the clinical relevance and therapeutic potential of a novel, actionable miR/RBP axis in tumor angiogenesis and metastasis.

Topics: Animals; Cell Cycle; Cell Movement; Cell Proliferation; Cells, Cultured; Cyclin D1; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; RNA Interference; RNA-Binding Proteins

It is well known that chromosomal aberrations of tumors are associated with the initiation and progression of malignancy. Fluorescence in situ hybridization (FISH) is a powerful, rapid method to detect chromosome copy number and structural alterations in tissue sections, chromosome, or interphase cellular preparations via hybridization of complementary probe sequences. The technique is based on the complementary nature of DNA double strands, which allows fluorescently labeled DNA probes to be used as probes to label the complementary sequences of target cells, chromosomes, and tissues. FISH technique has many applications, including basic gene mapping, used in pathological diagnosis to detect chromosome and gene copy number aberrations, translocations, microdeletions, and duplications. For the recognition of gene amplifications and deletions, locus-specific probes that are collections of one or a few cloned DNA sequences are routinely used. Multiplex-FISH (M-FISH) technique visualizes all chromosomes with different colors using spectrally distinct fluorophores for each chromosome in one experiment to detect numerical and structural alterations of chromosomes obtained from tumor cells. Recently many of the gene-specific probes are commercially available.

Topics: Chromosome Aberrations; Cyclin D1; DNA Probes; Gene Amplification; Humans; In Situ Hybridization, Fluorescence; Neoplasms

The CCND1 locus is located in 11q13 and encodes the G1-S regulatory protein, cyclin D1. Cyclin D1 is frequently amplified in various types of cancers, and is an attractive potential therapeutic target. Multiplex ligation-dependent probe amplification (MLPA) is a new, high-resolution method for the detection of amplification of numerous genes including CCND1 in small amounts of DNA fragments derived from formalin-fixed, paraffin-embedded material in a single reaction. This approach is, however, based on PCR and averages many different cells, so validation by morphological methods such as fluorescence in situ hybridization (FISH) is theoretically mandatory. Here we describe detection of CCND1 gene copy number variations by commercially available MLPA kits and FISH using a bacterial artificial chromosome (BAC) probe.

Topics: Cyclin D1; DNA; DNA Copy Number Variations; Fluorescence; Humans; In Situ Hybridization, Fluorescence; Multiplex Polymerase Chain Reaction; Neoplasms; Point Mutation

SALL2 is a poorly characterized transcription factor that belongs to the Spalt-like family involved in development. Mutations on SALL2 have been associated with ocular coloboma and cancer. In cancers, SALL2 is deregulated and is proposed as a tumor suppressor in ovarian cancer. SALL2 has been implicated in stemness, cell death, proliferation, and quiescence. However, mechanisms underlying roles of SALL2 related to cancer remain largely unknown. Here, we investigated the role of SALL2 in cell proliferation using mouse embryo fibroblasts (MEFs) derived from Sall2

Topics: Animals; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin E; DNA-Binding Proteins; Fibroblasts; G1 Phase; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Mice, Knockout; Models, Biological; Neoplasms; Phenotype; Promoter Regions, Genetic; Repressor Proteins; RNA, Messenger; S Phase; Transcription Factors; Transcription, Genetic

Expression of cyclin D1 (

Topics: Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Cyclin D1; Humans; MCF-7 Cells; Neoplasms; Oxidative Stress; Retinoblastoma Protein

Previously, we reported that IFN-γ and poly I:C, a TLR3 Pattern Recognition Receptor (PRR) agonist, reduces growth of and induces Cleaved-Caspase-3, ISG54 and p27Kip in B16 melanoma cells. Here, analysis of IFN-γ/PRR synergism was expanded with UM-SCC1 and UM-SCC38 human squamous carcinoma cells and other PRR agonists. As in B16 cells, poly I:C plus IFN-γ synergism reduced UM-SCC1 and UM-SCC38 growth, and no more than 24 h was needed for significant growth reduction. IFN-γ synergism to stem B16 growth also occurred with TLR7, TLR9, TLR4, and STING agonists, but not TLR2 agonist. IFN-γ synergized with TLR3 and TLR4 agonists reducing UM-SCC1 growth, and with TLR7 and TLR3 agonists reducing UM-SCC38 growth. IFN-γ plus poly I:C, which had the most pronounced effect, decreased cyclin-D1, increased G1 cell cycle arrest, and increased Cleaved caspase-3 in B16 cells, as well as RAW264.7, a virus-transformed murine macrophage cell line. Finally, IFN-γ plus poly I:C modulated total but not cell surface expression of immune checkpoint protein PD-L1, as well as cell cycle checkpoint proteins in B16 cells. Thus IFN-γ plus poly I:C, and other PRR agonists, may well be effective adjuvants to cancer immunotherapy against several tumor cell types.

Topics: Animals; B7-H1 Antigen; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Synergism; G1 Phase Cell Cycle Checkpoints; Humans; Interferon-gamma; Mice; Neoplasms; Poly I-C; RAW 264.7 Cells; Toll-Like Receptors

p27 plays critical roles in cell proliferation, differentiation, and apoptosis, which have been well studied in mammals and Drosophila. However, the mechanisms underlying p27 regulation of the cell cycle have not been thoroughly researched. In this study, Genevestigator, Kaplan-Meier Plotter, and the Human Protein Atlas databases were used to analyze the expression of p27, cell division protein kinase 6 (CDK6), and cyclin D1 (CCND1), as well as its prognostic value in different tumor tissues and corresponding normal tissues. Quantitative PCR and immunohistochemistry were used to detect the expression of p27, CDK6, and CCND1 in the tissues of cancer patients. The effects of p27, CDK6, and CCND1 on the proliferation of lung cancer cells were examined by the MTT assay, and flow cytometry was used to investigate the mechanism by which p27 affected cell proliferation. Immunofluorescence, co-immunoprecipitation, and Western blotting were used to determine if p27 interacted with CDK and CCND1 to regulate the cell cycle. The results showed that p27, CDK6, and CCND1 played different roles in tumorigenesis and development, which are in accordance with CDK6 and CCND1 in affecting the cell cycle and cell proliferation. p27 regulated the cell cycle and inhibited cell proliferation by affecting formation of the cell cycle-dependent complex CDK6/CCND1, but did not directly affect the expression of CDK6 and CCND1. Moreover, CCND1 did not regulate the cell cycle alone, but rather, functioned together with CDK6. This study provides insights into the effects of p27 on tumor formation and development, and the underlying regulatory mechanisms.

Topics: A549 Cells; Animals; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p27; Drosophila; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; Neoplasms; Protein Binding

Topics: Activating Transcription Factor 1; Adenocarcinoma; Colonic Neoplasms; Computational Biology; Cyclic AMP Response Element-Binding Protein; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Intracellular Signaling Peptides and Proteins; Kidney Neoplasms; Kruppel-Like Transcription Factors; MicroRNAs; Neoplasms; Rectal Neoplasms; RNA, Long Noncoding; Sp1 Transcription Factor; STAT3 Transcription Factor; Transcription Factors

In spite of the Betulinic acid (BA) being recognized as anticancerous source; its further use in clinical development is greatly hampered because of its poor pharmacokinetic properties. To circumvent these limitations, we synthesized a PI3K target based library of 18 triazole based derivatives and we identified a C-3 cyano analog of betulinic acid (CBA) with significant cell death effects with 5-7 fold higher potency than BA in various cancers. Importantly, no such report is available demonstrating the involvement of BA or its structural analogs in the modulation of PI3K pathway. Using, human leukemia HL-60 cells as a model, we for the first time report that CBA decreased expression of PI3K p110α, p85α, and pAKT in HL-60. Furthermore, we could find significant depletion of pGSK3β, cyclin D1 and increased expression of p21/cip, p27/Kip proteins. CBA induced G0/G1 cell cycle arrest, increased sub-G0 DNA fraction and annexin V binding of the cells besides imparting the typical surface features of cell death. Also, this target specific inhibition was associated with mitochondrial apoptosis as was reflected by expression studies of various proteins together with reactive oxygen species generation and decline in mitochondrial trans membrane potential. The apoptotic effectors i.e., caspase 8 and caspase 9 were found to get upregulated besides PI3K associated DNA repair enzyme i.e., PARP cleavage was observed. Thus, our results elucidated that CBA or other BA based small molecules inhibit PI3K/AKT pathway with induction of subsequent cancer cell death which may be useful therapeutic strategy against leukemias and possibly other cancers.

Topics: Betulinic Acid; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HL-60 Cells; Humans; MCF-7 Cells; Neoplasms; Pentacyclic Triterpenes; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Signal Transduction; Triazoles; Triterpenes

Cyclin D1 (Ccnd1) is a proto-oncogen amplified in many different cancers and nuclear accumulation of Ccnd1 is a characteristic of tumor cells. Ccnd1 activates the transcription of a large set of genes involved in cell cycle progress and proliferation. However, Ccnd1 also targets cytoplasmic proteins involved in the regulation of cell migration and invasion. In this work, we have analyzed by immunohistochemistry the localization of Ccnd1 in endometrial, breast, prostate and colon carcinomas with different types of invasion. The number of cells displaying membranous or cytoplasmic Ccnd1 was significantly higher in peripheral cells than in inner cells in both collective and pushing invasion patterns of endometrial carcinoma, and in collective invasion pattern of colon carcinoma. Also, the cytoplasmic localization of Ccnd1 was higher when tumors infiltrated as single cells, budding or small clusters of cells. To evaluate cytoplasmic function of cyclin D1, we have built a variant (Ccnd1-CAAX) that remains attached to the cell membrane therefore sequestering this cyclin in the cytoplasm. Tumor cells harboring Ccnd1-CAAX showed high levels of invasiveness and metastatic potential compared to those containing the wild type allele of Ccnd1. However, Ccnd1-CAAX expression did not alter proliferative rates of tumor cells. We hypothesize that the role of Ccnd1 in the cytoplasm is mainly associated with the invasive capability of tumor cells. Moreover, we propose that subcellular localization of Ccnd1 is an interesting guideline to measure cancer outcome.

Topics: Amino Acid Motifs; Animals; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cells, Cultured; Colonic Neoplasms; Cyclin D1; Cytoplasm; Endometrial Neoplasms; Female; Humans; Immunohistochemistry; Male; Mice, Nude; Mice, SCID; Microscopy, Confocal; Neoplasm Invasiveness; Neoplasms; Prostatic Neoplasms

The MDM2-p53 pathway has a prominent oncogenic function in the pathogenesis of various cancers. Nutlin-3, a small-molecule antagonist of MDM2-p53 interaction, inhibits proliferation in cancer cells with wild-type p53. Herein, we evaluate the expression of MDM2, both the full length and a splicing variant MDM2-A, and the sensitivity of Nutlin-3 in different cancer cell lines. Included are seven cell lines with wild-type p53 (four mesothelioma, one breast cancer, one chondrosarcoma, and one leiomyosarcoma), two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one mesothelioma cell line harboring a p53 point mutation. Nutlin-3 treatment increased expression of cyclin D1, MDM2, and p53 in cell lines with wild-type p53. Additive effects were observed in cells containing wild-type p53 through coordinated attack on MDM2-p53 binding and cyclin D1 by lentivirual shRNA knockdown or small molecule inhibition, as demonstrated by immunoblots and cell viability analyses. Further results demonstrate that MDM2 binds to cyclin D1, and that an increase in cyclin D1 expression after Nutlin-3 treatment is correlated with expression and ubiquitin E3-ligase activity of MDM2. MDM2 and p53 knockdown experiments demonstrated inhibition of cyclin D1 by MDM2 but not p53. These results indicate that combination inhibition of cyclin D1 and MDM2-p53 binding warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53.

Topics: Cell Line, Tumor; Cyclin D1; Down-Regulation; Gene Knockdown Techniques; HEK293 Cells; Humans; Imidazoles; MCF-7 Cells; Neoplasms; Piperazines; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Cyclin D1 (Ccnd1) together with its binding partner Cdk4 act as a transcriptional regulator to control cell proliferation and migration, and abnormal Ccnd1·Cdk4 expression promotes tumour growth and metastasis. While different nuclear Ccnd1·Cdk4 targets participating in cell proliferation and tissue development have been identified, little is known about how Ccnd1·Cdk4 controls cell adherence and invasion. Here, we show that the focal adhesion component paxillin is a cytoplasmic substrate of Ccnd1·Cdk4. This complex phosphorylates a fraction of paxillin specifically associated to the cell membrane, and promotes Rac1 activation, thereby triggering membrane ruffling and cell invasion in both normal fibroblasts and tumour cells. Our results demonstrate that localization of Ccnd1·Cdk4 to the cytoplasm does not simply act to restrain cell proliferation, but constitutes a functionally relevant mechanism operating under normal and pathological conditions to control cell adhesion, migration and metastasis through activation of a Ccnd1·Cdk4-paxillin-Rac1 axis.

Topics: Animals; Cell Line, Tumor; Cell Membrane; Cyclin D1; Cyclin-Dependent Kinase 4; Cytoplasm; Down-Regulation; Fibroblasts; Gene Knockdown Techniques; HEK293 Cells; Humans; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Paxillin; Phosphorylation; Phosphoserine; Protein Binding; rac1 GTP-Binding Protein; Rats; Substrate Specificity

Synthetic gene circuits are emerging as a versatile means to target cancer with enhanced specificity by combinatorial integration of multiple expression markers. Such circuits must also be tuned to be highly sensitive because escape of even a few cells might be detrimental. However, the error rates of decision-making circuits in light of cellular variability in gene expression have so far remained unexplored. Here, we measure the single-cell response function of a tunable logic AND gate acting on two promoters in heterogeneous cell populations. Our analysis reveals an inherent tradeoff between specificity and sensitivity that is controlled by the AND gate amplification gain and activation threshold. We implement a tumor-mimicking cell-culture model of cancer cells emerging in a background of normal ones, and show that molecular parameters of the synthetic circuits control specificity and sensitivity in a killing assay. This suggests that, beyond the inherent tradeoff, synthetic circuits operating in a heterogeneous environment could be optimized to efficiently target malignant state with minimal loss of specificity.

Topics: Cell Death; Cell Line; Cyclin D1; Fibroblasts; Gene Regulatory Networks; HCT116 Cells; Histones; Humans; Neoplasm Proteins; Neoplasms; Promoter Regions, Genetic; Repressor Proteins; Sensitivity and Specificity; Synthetic Biology

Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB) protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3β (GSK-3β) induces the proteasomal degradation of cyclin D1. The addition of GSK-3β inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3β.

Topics: Annexin A5; Apoptosis; Bufanolides; Cardiotonic Agents; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cellular Senescence; Cyclin D1; G1 Phase; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Maleimides; Medicine, Chinese Traditional; Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Retinoblastoma Protein

Topics: Animals; Breast Neoplasms; Cyclin D1; Female; Humans; Mice; Neoplasms; Protein Kinases; Receptor, ErbB-2

Cyclin D1 immunostaining of non-neoplastic cells has been a source of diagnostic confusion especially in lymphoproliferative lesions. This study has reviewed these in two hundred and thirty-one haematopathological samples stained for cyclin D1. Most cases were formalin-fixed except for a few bone marrow trephines, which were B-5 fixed, and EDTA decalcified. Overall, 94% (216/231) of cases showed one or more types of non-neoplastic cells expressing Cyclin D1 of variable intensity. Endothelial cells and histiocytes were the most commonly identified Cyclin D1 positive cells being positive in 92% (214/231) of cases. Other normal cell types identified included fat cells, stromal fibroblasts, glial cells, spermatocytes, smooth muscle cells, osteoblasts and where present epithelial cells. Many normal cell types can express cyclinD1. Knowledge of these is useful to prevent misinterpretation of cyclin D1 positive tumours.

Topics: Adipocytes; Bone Marrow; Cyclin D1; Endothelial Cells; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Lymphocytes; Neoplasms; RNA, Messenger

RNA-binding proteins of the IMP family (insulin-like growth factor 2 (IGF2) mRNA-binding proteins 1-3) are important post-transcriptional regulators of gene expression. Multiple studies have linked high expression of IMP proteins, and especially of IMP-3, to an unfavorable prognosis in numerous types of cancer. The specific importance of IMP-3 for cancer transformation remains poorly understood. We here show that all three IMPs can directly bind the mRNAs of cyclins D1, D3 and G1 (CCND1, D3 and G1) in vivo and in vitro, and yet only IMP-3 regulates the expression of these cyclins in a significant manner in six human cancer cell lines of different origins. In the absence of IMP-3, the levels of CCND1, D3 and G1 proteins fall dramatically, and the cells accumulate in the G1 phase of the cell cycle, leading to almost complete proliferation arrest. Our results show that, compared with IMP-1 and IMP-2, IMP-3 is enriched in the nucleus, where it binds the transcripts of CCND1, D3 and G1. The nuclear localization of IMP-3 depends on its protein partner HNRNPM and is indispensable for the post-transcriptional regulation of expression of the cyclins. Cytoplasmic retention of IMP-3 and HNRNPM in human cancer cells leads to significant drop in proliferation. In conclusion, a nuclear IMP-3-HNRNPM complex is important for the efficient synthesis of CCND1, D3 and G1 and for the proliferation of human cancer cells.

Topics: Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Cyclin D3; Cyclin G1; Gene Expression Regulation; Heterogeneous-Nuclear Ribonucleoprotein Group M; Humans; Neoplasms; Protein Binding; Protein Transport; RNA Processing, Post-Transcriptional; RNA-Binding Proteins; RNA, Messenger

Selective activation of Rho GTPase cascade requires the release of Rho from RhoGDI (GDP-dissociation inhibitors) complexes. Our previous studies identified RhoGDIα SUMOylation at Lys-138 and its function in the regulation of cancer cell invasion. In the current study, we demonstrate that RhoGDIα SUMOylation has a crucial role in the suppression of cancer cell anchorage-independent growth as well as the molecular mechanisms underlying this suppression. We found that ectopic expression of RhoGDIα resulted in marked inhibition of an anchorage-independent growth with induction of G0/G1 cell cycle arrest, while point mutation of RhoGDIα SUMOylation at residue Lys-138 (K138R) abrogated this growth suppression and G0/G1 cell cycle arrest in cancer cells. Further studies showed that SUMOylation at Lys-138 was critical for RhoGDIα down-regulation of cyclin D1 protein expression and that MEK1/2-Erk was a specific downstream target of SUMOylated RhoGDIα for its inhibition of C-Jun/AP-1 cascade, cyclin d1 transcription, and cell cycle progression. These results strongly demonstrate that SUMOylated RhoGDIα suppressed C-Jun/AP-1-dependent transactivation specifically via targeting MEK1/2-Erk, subsequently leading to the down-regulation of cyclin D1 expression and anti-cancer activity. Our results provide new mechanistic insights into the understanding of essential role of SUMOylation at Lys-138 in RhoGDIα's biological function.

Topics: Amino Acid Substitution; Cell Line, Tumor; Cyclin D1; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Neoplasms; Point Mutation; Proto-Oncogene Proteins c-jun; rho Guanine Nucleotide Dissociation Inhibitor alpha; Sumoylation; Transcription, Genetic

Kruppel-like-factor 17 (KLF17) is a negative regulator of metastasis and epithelial-mesenchymal-transition (EMT). However, its expression is downregulated in metastatic breast cancer that contains p53 mutations. Here, we show that mutant-p53 plays a key role to suppress KLF17 and thereby enhances cancer progression, which defines novel gain-of-function (GOF) of mutant-p53. Mutant-p53 interacts with KLF17 and antagonizes KLF17 mediated EMT genes transcription. Depletion of KLF17 promotes cell viability, decreases apoptosis and induces drug resistance in metastatic breast cancer cells. KLF17 suppresses cell migration and invasion by decreasing CD44, PAI-1 and Cyclin-D1 expressions. Taken together, our results show that KLF17 is important for the suppression of metastasis and could be a potential therapeutic target during chemotherapy.

Topics: Apoptosis; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Survival; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Hyaluronan Receptors; MCF-7 Cells; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Plasminogen Activator Inhibitor 1; RNA Interference; Transcription Factors; Tumor Suppressor Protein p53

This study is to explore new lead compounds by inhibition of Pin1 for anticancer therapy using temperature sensitive mutants. As Pin1 is conserved from yeast to human, we established a high-throughput screening method for Pin1 inhibitors, which employed yeast assay. This method led to the identification of one potent hits, 8-11. In vitro, 8-11 inhibited purified Pin1 enzyme activity with IC50 of (10.40 +/- 1.68) micromol x L(-1), induced G1 phase arrest and apoptosis, showed inhibitory effects on a series of cancer cell proliferation, reduced Cyclin D1 expression, was defined as reciprocally matched for protein-ligand complex in virtual docking analysis and reduced cell migration ability. In vivo, we could observe reduction of tumor volume after treatment with 8-11 in xenograft mice compared with vehicle DMSO treatment. Altogether, these results provide for the first time the involvement of 8-11 in the anticancer activity against Pin1.

Topics: Animals; Apoptosis; Cell Proliferation; Cyclin D1; Drug Screening Assays, Antitumor; G1 Phase; High-Throughput Screening Assays; Humans; Mice; Neoplasms; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Temperature; Xenograft Model Antitumor Assays; Yeasts

Topics: Cell Cycle Proteins; Cyclin D1; Cyclin E; G1 Phase Cell Cycle Checkpoints; Humans; Neoplasms; Oncogene Proteins; Protein Stability

In the present study, the expression of human γ-aminobutyrate type A (GABAA) receptor-binding protein (GABARBP) is downregulated in ovarian cancer cell lines and tissues. We also found that the specific function of GABAPBP was that of a novel pro-apoptotic protein. Both GABARBP and cisplatin suppressed cancer cell proliferation in a concentration-dependent manner. The combined treatment of GABARBP and cisplatin was more effective in inhibiting cell growth, as well as cell migration, than with either drug treatment alone. At the same time, the treatment combination is correlated with the downregulation of cyclin D1 and CDK4, arrested cell cycle progression in the G₀-G₁ phase and enhancing p53 expression, while also reducing Bcl-2 and Bcl-xL expression. The p53 and p21 promoter luciferase activities were induced by GABARBP, whereas there was no effect on the p53-/- and p21-/- system. In addition, p53 activity was validated with UV irradiation and siGABARBP. Taken together, our results indicate that GABARBP can regulate the pro-apoptotic activity of cisplatin via the upregulation of p53 expression.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin D1; Cyclin-Dependent Kinase 4; Gene Expression Regulation, Neoplastic; Humans; Microtubule-Associated Proteins; Neoplasms

Insulin is a secreted peptide hormone identified in human pancreas to promote glucose utilization. Insulin has been observed to induce cell proliferation and myogenesis in C2C12 cells. The precise mechanisms underlying the proliferation of C2C12 cells induced by insulin remain unclear. In this study, we observed for the first time that 10 nM insulin treatment promotes C2C12 cell proliferation. Additionally, 50 and 100 nM insulin treatment induces C2C12 cell apoptosis. By utilizing real-time PCR and Western blotting analysis, we found that the mRNA levels of cyclinD1 and BAD are induced upon 10 and 50 nM/100 nM insulin treatment, respectively. The similar results were observed in C2C12 cells expressing GATA-6 or PPARα. Our results identify for the first time the downstream targets of insulin, cyclin D1, and BAD, elucidate a new molecular mechanism of insulin in promoting cell proliferation and apoptosis.

Topics: Apoptosis; bcl-Associated Death Protein; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Flow Cytometry; GATA6 Transcription Factor; Gene Expression Regulation, Neoplastic; Humans; Insulin; Neoplasms; PPAR alpha; Signal Transduction

Our findings show that upregulation of a wild-type Trop-2 has a key controlling role in human cancer growth, and that tumour development is quantitatively driven by Trop-2 expression levels. However, little is known about the regulation of expression of the TROP2 gene. Hence, we investigated the TROP2 transcription control network. TROP2 expression was shown to depend on a highly interconnected web of transcription factors: TP63/TP53L, ERG, GRHL1/Get-1 (grainyhead-like epithelial transactivator), HNF1A/TCF-1 (T-cell factor), SPI1/PU.1, WT (Wilms' tumour)1, GLIS2, AIRE (autoimmune regulator), FOXM1 (forkhead box M1) and FOXP3, with HNF4A as the major network hub. TROP2 upregulation was shown to subsequently drive the expression and activation of CREB1 (cyclic AMP-responsive-element binding protein), Jun, NF-κB, Rb, STAT1 and STAT3 through induction of the cyclin D1 and ERK (extracellular signal regulated kinase)/MEK (MAPK/ERK kinase) pathways. Growth-stimulatory signalling through NF-κB, cyclin D1 and ERK was shown to require an intact Trop-2 cytoplasmic tail. Network hubs and interacting partners are co-expressed with Trop-2 in primary human tumours, supporting a role of this signalling network in cancer growth.

Topics: Antigens, Neoplasm; Cell Adhesion Molecules; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Membrane Proteins; Neoplasms; NF-kappa B; Signal Transduction

The RPS6KA6 gene encodes the p90 ribosomal S6 kinase-4 (RSK4) that is still largely uncharacterized. In this study we identified a new RSK4 transcription initiation site and several alternative splice sites with a 5'-RACE approach. The resulting mRNA variants encompass four possible first start codons. The first 15 nucleotides (nt) of exon 22 in mouse and the penultimate exon in both human (exon 21) and mouse (exon 24) RSK4 underwent alternative splicing, although the penultimate exon deleted variant appeared mainly in cell clines, but not in most normal tissues. Demethylation agent 5-azacytidine inhibited the deletion of the penultimate exon, whereas two indolocarbazole-derived inhibitors of cyclin-dependent kinase 4 or 6 induced deletion of the first 39 nt from exon 21 of human RSK4. In all human cancer cell lines studied, the 90-kDa wild-type RSK4 was sparse but, surprisingly, several isoforms at or smaller than 72 kDa were expressed as detected by seven different antibodies. On immunoblots, each of these smaller isoforms often appeared as a duplet or triplet and the levels of these isoforms varied greatly among different cell lines and culture conditions. Cyclin D1 inhibited RSK4 expression and serum starvation enhanced the inhibition, whereas c-Myc and RSK4 inhibited cyclin D1. The effects of RSK4 on cell growth, cell death and chemoresponse depended on the mRNA variant or the protein isoform expressed, on the specificity of the cell lines, as well as on the anchorage-dependent or -independent growth conditions and the in vivo situation. Moreover, we also observed that even a given cDNA might be expressed to multiple proteins; therefore, when using a cDNA, one needs to exclude this possibility before attribution of the biological results from the cDNA to the anticipated protein. Collectively, our results suggest that whether RSK4 is oncogenic or tumor suppressive depends on many factors.

Topics: Alternative Splicing; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; Azacitidine; Base Sequence; Blotting, Western; Cell Adhesion; Cell Cycle; Cell Proliferation; Cyclin D1; DNA, Complementary; Exons; Female; Humans; Immunoenzyme Techniques; Immunoprecipitation; Mice; Mice, SCID; Molecular Sequence Data; Neoplasms; Protein Isoforms; Proto-Oncogene Proteins c-myc; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Protein S6 Kinases, 90-kDa; RNA, Messenger; RNA, Small Interfering; Sequence Homology, Nucleic Acid; Transcription Initiation Site; Tumor Cells, Cultured

CyclinD1/CDK4 and cyclinD3/CDK4 complexes are key regulators of the cell progression and therefore constitute promising targets for the design of anticancer agents. In the present study, the key peptide motifs were selected from these two complexes. Chimeric peptides with these peptides conjugated to the protein transduction domain 4 (PTD4) were designed and synthesized. The chimeric peptides, PTD4-D1, PTD4-D3, PTD4-K4 exhibited significant anti-proliferation effects on cancer cell lines. These peptides could compete with the cyclinD/CDK4 complex and induce the G1/S phase arrest and apoptosis of cancer cells. In the tumor challenge experiment, these peptides showed potent antitumor effects with no significant side effects. Our results suggested that these peptides could be served as novel leading compounds with potent antitumor activity.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Cycle; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase 4; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms; Peptide Fragments; Peptides; tat Gene Products, Human Immunodeficiency Virus

Interleukin (IL)-35 is a novel heterodimeric cytokine in the IL-12 family and is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 is expressed in T regulatory (Treg) cells and contributes to the immune suppression function of these cells. In contrast, we found that both IL-35 subunits were expressed concurrently in most human cancer cell lines compared to normal cell lines. In addition, we found that TNF-α and IFN-γ stimulation led to increased IL-35 expression in human cancer cells. Furthermore, over-expression of IL-35 in human cancer cells suppressed cell growth in vitro, induced cell cycle arrest at the G1 phase, and mediated robust apoptosis induced by serum starvation, TNF-α, and IFN-γ stimulation through the up-regulation of Fas and concurrent down-regulation of cyclinD1, survivin, and Bcl-2 expression. In conclusion, our results reveal a novel functional role for IL-35 in suppressing cancer activity, inhibiting cancer cell growth, and increasing the apoptosis sensitivity of human cancer cells through the regulation of genes related to the cell cycle and apoptosis. Thus, this research provides new insights into IL-35 function and presents a possible target for the development of novel cancer therapies.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; fas Receptor; Gene Expression Regulation, Neoplastic; Humans; Inhibitor of Apoptosis Proteins; Interferon-gamma; Interleukin-12 Subunit p35; Interleukins; Minor Histocompatibility Antigens; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Survivin; Tumor Necrosis Factor-alpha; Tumor Suppressor Proteins; Up-Regulation

Justicia spicigera is used for the empirical treatment of cervical cancer in Mexico. Recently, we showed that Justicia spicigera extracts exerted cytotoxic and antitumoral effects and the major component of this extract was kaempferitrin (KM).. The cytotoxic and apoptotic effect of KM on human cancer cells and human nontumorigenic cells were evaluated using MTT and TUNEL assays, and Annexin V/Propidium iodide detection by flow cytometry. The effect of KM on cell cycle was analyzed by flow cytometry with propidium iodide. The apoptotic and cell cycle effects were also evaluated by western blot analysis. Also, different doses of KM were injected intraperitoneally daily into athymic mice bearing tumors of HeLa cells during 32 days. The growth and weight of tumors were measured.. KM induces high cytotoxic effects in vitro and in vivo against HeLa cells. The general mechanisms by which KM induces cytotoxic effects include: cell cycle arrest in G1 phase and apoptosis via intrinsic pathway in a caspase dependent pathway. Also, KM exerts chemopreventive and antitumor effects.. KM exerts cytotoxic and antitumor effects against HeLa cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Survival; Cyclin D1; Humans; In Situ Nick-End Labeling; Kaempferols; Male; Mice; Mice, Nude; Neoplasms; Phytotherapy; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Xenograft Model Antitumor Assays

The constitutive activation of NF-κB is a major event leading to the initiation, development, and progression of cancer. Recently, we showed that the size of preestablished tumors was reduced after the depletion of Kir2.2, an inwardly rectifying potassium channel. To determine the precise mechanism of action of Kir2.2 in the control of tumor growth, we searched for interacting proteins. Notably, NF-κB p65/RelA was identified as a binding partner of Kir2.2 in a yeast two-hybrid analysis. Further analyses revealed that Kir2.2 directly interacted with RelA in vitro and coimmunoprecipitated with RelA from cell lysates. Kir2.2 increased RelA phosphorylation at S536 and facilitated its translocation from the cytoplasm to the nucleus, thereby activating the transcription factor and increasing the expression level of NF-κB targets, including cyclin D1, matrix metalloproteinase (MMP)9, and VEGF. Kir2.2 was overexpressed in human cancer and the expression level was correlated with increased colony formation and tumor growth in mouse tumor models. On the basis of these findings, we propose an unconventional role for Kir2.2 as a constitutive RelA-activating protein, which is likely to contribute to tumor progression in vivo.

Topics: Animals; Cyclin D1; Female; HEK293 Cells; Humans; Mice; Mice, Inbred BALB C; Neoplasms; NF-kappa B; Potassium Channels, Inwardly Rectifying; Transcription Factor RelA

Sulfuretin (3',4',6'-trihydroxyaurone), one of the key flavonoids isolated from Rhus verniciflua, is known to suppress inflammation and oxidative stress. However, the anti-cancer properties of sulfuretin as well as its mechanism of action remain poorly understood. Here, we show that the expression of miR-30C is markedly enhanced in sulfuretin-stimulated cells, consequently promoting apoptosis and cell cycle arrest in human cancer cell lines. The transient transfection of pre-miR-30C resulted in greater than 70% growth inhibition in PC-3 cells and provided strong evidence that miR-30C selectively suppresses the expression of cyclin D1 and D2, but not cyclin D3. Target validation analysis revealed that 3'-UTR of cyclin D2 is a direct target of miR-30C, whereas suppression by miR-30C of cyclin D1 may occur through indirect mRNA regulation. In addition, silencing miR-30C expression partially reversed sulfuretin-induced cell death. Taken together, our data suggest that miR-30C, a tumor suppressor miRNA, contributes to anti-cancer properties of sulfuretin by negatively regulating cyclin D1 and D2, providing important implications of sulfuretin and miR-30C for the therapeutic intervention of human cancers.

Topics: Antineoplastic Agents; Apoptosis; Benzofurans; Cell Line, Tumor; Cyclin D1; Cyclin D2; Down-Regulation; Flavonoids; Humans; MicroRNAs; Neoplasms

Pyruvate kinase catalyzes the rate-limiting final step of glycolysis, generating adenosine triphosphate (ATP) and pyruvate. The M2 tumor-specific isoform of pyruvate kinase (PKM2) promotes glucose uptake and lactate production in the presence of oxygen, known as aerobic glycolysis or the Warburg effect. As recently reported in Nature, PKM2, besides its metabolic function, has a nonmetabolic function in the direct control of cell cycle progression by activating β-catenin and inducing expression of the β-catenin downstream gene CCND1(encoding for cyclin D1). This nonmetabolic function of PKM2 is essential for epidermal growth factor receptor (EGFR) activation-induced tumorigenesis.

Topics: Animals; beta Catenin; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; ErbB Receptors; Glycolysis; Humans; Isoenzymes; Neoplasms; Pyruvate Kinase

The prevalence of ErbB2 amplification in breast cancer has resulted in the heavy pursuit of ErbB2 as a therapeutic target. Although both the ErbB2 monoclonal antibody trastuzumab and ErbB1/ErbB2 dual kinase inhibitor lapatinib have met with success in the clinic, many patients fail to benefit. In addition, the majority of patients who initially respond will unfortunately ultimately progress on these therapies. Activation of ErbB3, the preferred dimerization partner of ErbB2, plays a key role in driving ErbB2-amplified tumor growth, but we have found that current ErbB2-directed therapies are poor inhibitors of ligand-induced activation. By simulating ErbB3 inhibition in a computational model of ErbB2/ErbB3 receptor signaling, we predicted that a bispecific antibody that docks onto ErbB2 and subsequently binds to ErbB3 and blocks ligand-induced receptor activation would be highly effective in ErbB2-amplified tumors, with superior activity to a monospecific ErbB3 inhibitor. We have developed a bispecific antibody suitable for both large scale production and systemic therapy by generating a single polypeptide fusion protein of two human scFv antibodies linked to modified human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with ErbB2 and ErbB3, effectively inhibiting ErbB3 signaling and showing antitumor activity in preclinical models that is dependent on ErbB2 overexpression. MM-111 can be rationally combined with trastuzumab or lapatinib for increased antitumor activity and may in the future complement existing ErbB2-directed therapies to treat resistant tumors or deter relapse.

Topics: Animals; Antibodies, Bispecific; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Design; Female; Humans; Inhibitory Concentration 50; Mice; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Multiprotein Complexes; Neoplasms; Neuregulin-1; Phosphatidylinositol 3-Kinases; Protein Binding; Receptor, ErbB-2; Receptor, ErbB-3; Signal Transduction; Xenograft Model Antitumor Assays

In HCT116 colorectal cancer cells, HeLa cervical cancer cells and HuH-7 hepatoma cells, miR-223 is expressed at a low level. Through infection with lentivirus containing miR-223 precursor, miR-223 [corrected] was overexpressed in all these cells. Interestingly, the expression levels of FOXO1 mRNA and protein, and phosphorylation levels became significantly lower than those of their control. FOXO1 was down-regulated mainly in the cytoplasm, while the nuclear FOXO1 level became relatively high compared to the cytoplasm. As the unphosphorylated active form of FOXO1 increased in the cells, cyclin D1/p21/p27 were up-regulated at either mRNA or protein level. Proliferation of the cells was also greatly inhibited when miR-223 was over-expressed. Therein, our data suggest that miR-223 regulates FOXO1 expression and cell proliferation.

Topics: Cell Line, Transformed; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor Proteins; Cytoplasm; Female; Forkhead Box Protein O1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Liver Neoplasms; Male; MicroRNAs; Neoplasm Proteins; Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Protein Transport; RNA, Messenger; Uterine Cervical Neoplasms

Tumor cells in vivo encounter diverse types of microenvironments both at the site of the primary tumor and at sites of distant metastases. Understanding how the various mechanical properties of these microenvironments affect the biology of tumor cells during disease progression is critical in identifying molecular targets for cancer therapy.. This study uses flexible polyacrylamide gels as substrates for cell growth in conjunction with a novel proteomic approach to identify the properties of rigidity-dependent cancer cell lines that contribute to their differential growth on soft and rigid substrates. Compared to cells growing on more rigid/stiff substrates (>10,000 Pa), cells on soft substrates (150-300 Pa) exhibited a longer cell cycle, due predominantly to an extension of the G1 phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis. Using stable isotope labeling of amino acids in culture (SILAC) and mass spectrometry, we measured the rates of protein synthesis of over 1200 cellular proteins under growth conditions on soft and rigid/stiff substrates. We identified cellular proteins whose syntheses were either preferentially inhibited or preserved on soft matrices. The former category included proteins that regulate cytoskeletal structures (e.g., tubulins) and glycolysis (e.g., phosphofructokinase-1), whereas the latter category included proteins that regulate key metabolic pathways required for survival, e.g., nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway.. The cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of dormant cancer cells, e.g., slow growth rate and reduced metabolism. We suggest that the use of relatively soft gels as cell culture substrates would allow molecular pathways to be studied under conditions that reflect the different mechanical environments encountered by cancer cells upon metastasis to distant sites.

Topics: Acrylic Resins; Adenosine Triphosphate; Biomechanical Phenomena; Bromodeoxyuridine; Cell Line, Tumor; Cellular Microenvironment; Cyclin D1; Extracellular Matrix; Humans; Isotope Labeling; Mass Spectrometry; Neoplasms; Protein Biosynthesis; Proteomics

This paper aims to evaluate the anti-tumour properties of elatol, a compound (sesquiterpene) isolated from algae Laurencia microcladia.. In-vitro and in-vivo anti-tumour properties of elatol were investigated using: MTT assays to assess the cytotoxic effects; flow cytometry analysis to examine the cell cycle and apoptosis; Western blot analysis for determination of the expression of cell cycle and apoptosis proteins; and study of in-vivo tumour growth in mice (C57Bl6 mice bearing B16F10 cells).. Elatol exhibited a cytotoxic effect, at least in part, by inducing cell cycle arrest in the G(1) and the sub-G(1) phases, leading cells to apoptosis. Western blot analysis demonstrated that elatol reduced the expression of cyclin-D1, cyclin-E, cyclin-dependent kinase (cdk)2 and cdk4. A decrease in bcl-xl and an increase in bak, caspase-9 and p53 expression was also observed. In the in-vivo experiment, treatment with elatol was able to reduce tumour growth in C57Bl6 mice.. Elatol promotes a delay in the cell cycle, probably in the G(1)/S transition, activating the apoptotic process and this could be responsible, at least in part, for the in-vivo effects observed. Taken together, the in-vitro and in-vivo experiments suggested that elatol has anti-tumour properties. Further studies should be conducted to clarify the mechanism of action.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Caspase 9; Cell Cycle Checkpoints; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Female; Humans; Laurencia; Male; Melanoma; Mice; Mice, Inbred C57BL; Neoplasms; Phytotherapy; Plant Extracts; Spiro Compounds

We here identify protein kinase D1 (PKD1) as a major regulator of anchorage-dependent and -independent growth of cancer cells controlled via the transcription factor Snail1. Using FRET, we demonstrate that PKD1, but not PKD2, efficiently interacts with Snail1 in nuclei. PKD1 phosphorylates Snail1 at Ser-11. There was no change in the nucleocytoplasmic distribution of Snail1 using wild type Snail1 and Ser-11 phosphosite mutants in different tumor cells. Regardless of its phosphorylation status or following co-expression of constitutively active PKD, Snail1 was predominantly localized to cell nuclei. We also identify a novel mechanism of PKD1-mediated regulation of Snail1 transcriptional activity in tumor cells. The interaction of the co-repressors histone deacetylases 1 and 2 as well as lysyl oxidase-like protein 3 with Snail1 was impaired when Snail1 was not phosphorylated at Ser-11, which led to reduced Snail1-associated histone deacetylase activity. Additionally, lysyl oxidase-like protein 3 expression was up-regulated by ectopic PKD1 expression, implying a synergistic regulation of Snail1-driven transcription. Ectopic expression of PKD1 also up-regulated proliferation markers such as Cyclin D1 and Ajuba. Accordingly, Snail1 and its phosphorylation at Ser-11 were required and sufficient to control PKD1-mediated anchorage-independent growth and anchorage-dependent proliferation of different tumor cells. In conclusion, our data show that PKD1 is crucial to support growth of tumor cells via Snail1.

Topics: Active Transport, Cell Nucleus; Amino Acid Oxidoreductases; Cell Nucleus; Cell Proliferation; Cyclin D1; Cytoplasm; HEK293 Cells; HeLa Cells; Histone Deacetylase 1; Histone Deacetylase 2; Humans; LIM Domain Proteins; Neoplasms; Protein Kinase C; Snail Family Transcription Factors; Transcription Factors; Transcription, Genetic

Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown.. To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC) or caffeic acid (CA) using whole-genome microarrays.. ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were downregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells.. Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2.

Topics: Activating Transcription Factor 2; Caffeic Acids; Caffeine; Cell Line, Tumor; Coffee; Cyclin D1; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Genes, Neoplasm; Humans; Neoplasms; Polyphenols; Reproducibility of Results; RNA, Messenger; STAT5 Transcription Factor

In this study, we synthesized a novel carbazole derivative, MHY407, as a sensitizer of cancer cells to increase DNA damage. We then evaluated the anticancer effects of MHY407 and identified the molecular mechanism for the sensitization of breast cancer cell lines. MHY407 significantly increased DNA damage as determined by DNA breakage, levels of damage-responsive proteins, and DNA foci. In addition, MHY407 increased p21 and decreased cyclin D1 protein levels. MHY407 also involved increased cell cycle arrest at the S phase. Furthermore, in doxorubicin and etoposide-treated breast cancer cells, co-treatment with MHY407 reduced cell viability and increased apoptosis. Co-treatment of MHY407 with doxorubicin or etoposide increased DNA damage-related proteins and foci formation, suggesting that increased DNA damage by MHY407 plays an important role in the sensitization. In addition, MHY407 also sensitized the cancer cells to DNA damaging radiation treatment. These results may contribute to the development of MHY407-based treatments for cancer patients receiving DNA-damage therapy.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carbazoles; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Doxorubicin; Epoxy Compounds; Etoposide; Female; G1 Phase Cell Cycle Checkpoints; Humans; MCF-7 Cells; Neoplasms; Radiation-Sensitizing Agents; S Phase Cell Cycle Checkpoints; Time Factors

The endogenous phospholipid lysophosphatidic acid (LPA) regulates fundamental cellular processes such as proliferation, survival, motility, and invasion implicated in homeostatic and pathological conditions. Hence, delineation of the full range of molecular mechanisms by which LPA exerts its broad effects is essential. We report avid binding of LPA to the receptor for advanced glycation end products (RAGE), a member of the immunoglobulin superfamily, and mapping of the LPA binding site on this receptor. In vitro, RAGE was required for LPA-mediated signal transduction in vascular smooth muscle cells and C6 glioma cells, as well as proliferation and migration. In vivo, the administration of soluble RAGE or genetic deletion of RAGE mitigated LPA-stimulated vascular Akt signaling, autotaxin/LPA-driven phosphorylation of Akt and cyclin D1 in the mammary tissue of transgenic mice vulnerable to carcinogenesis, and ovarian tumor implantation and development. These findings identify novel roles for RAGE as a conduit for LPA signaling and suggest targeting LPA-RAGE interaction as a therapeutic strategy to modify the pathological actions of LPA.

Topics: Animals; Cell Line, Tumor; Cyclin D1; Female; Humans; Lysophospholipids; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Muscle, Smooth, Vascular; Neoplasms; Proto-Oncogene Proteins c-akt; Rats; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Recombinant Proteins; Signal Transduction

Dietary intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) like eicosapentaenoic acid (EPA) decreases cancer risk, while arachidonic acid and other ω-6 PUFAs increase risk, but the underlying mechanisms are unclear. Cytochrome P450 (CYP)-derived epoxides contribute to enhanced tumourigenesis due to ω-6 PUFA intake. Thus, ω-6 arachidonic acid epoxides (EETs) inhibit apoptosis and stimulate proliferation by up-regulating cyclin D1 expression in cells. The present study evaluated the corresponding ω-3 PUFA epoxides and assessed their role in the regulation of cell proliferation.. Four chemically stable EPA epoxides (formed at the 8,9-, 11,12-, 14,15- and 17,18-olefinic bonds) were synthesized and tested against growth-related signalling pathways in brain microvascular endothelial bEND.3 cells. Cell cycle distribution was determined by flow cytometry and cyclin gene expression by immunoblotting and real-time PCR. The role of the p38 mitogen-activated protein (MAP) kinase in cyclin D1 dysregulation was assessed using specific inhibitors and dominant-negative expression plasmids.. The ω-3 17,18-epoxide of EPA decreased cell proliferation, interrupted the cell cycle in S-phase and down-regulated the cyclin D1/cyclin-dependent kinase (CDK)-4 complex, whereas the 8,9-, 11,12- and 14,15-epoxides were either inactive or enhanced proliferation. Cyclin D1 down-regulation by 17,18-epoxy-EPA was mediated by activation of the growth-suppressing p38 MAP kinase, but the alternate EPA-epoxides were inactive.. The present findings suggest that the epoxide formed by CYP enzymes at the ω-3 olefinic bond may contribute to the beneficial effects of ω-3 PUFA by down-regulating cyclin D1 and suppressing cell proliferation.

Topics: Animals; Cell Cycle; Cell Line; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Down-Regulation; Eicosapentaenoic Acid; Endothelial Cells; Enzyme Activation; Epoxy Compounds; Mice; Neoplasms; p38 Mitogen-Activated Protein Kinases; RNA, Messenger; Structure-Activity Relationship; Transfection

Deregulation of the expression of p53R2, a p53-inducible homologue of the R2 subunit of ribonucleotide reductase, has been found in various human cancer tissues; however, the roles p53R2 plays in cancer progression and malignancy remain controversial. In the present study, we examined changes in gene expression profiles associated with p53R2 in cancer cells, using the analysis of cDNA microarray. Gene set enrichment analysis identified that the gene set regulating cell-cycle progression was significantly enriched in p53R2-silencing human oropharyngeal carcinoma KB cells. Attenuation of p53R2 expression significantly reduced p21 expression and moderately increased cyclin D1 expression in both wild-type p53 cancer cells (KB and MCF-7) and mutant p53 cancer cells (PC3 and MDA-MB-231). Conversely, overexpression of p53R2-GFP resulted in an increase in the expression of p21 and decrease in the expression of cyclin D1, which correlated with reduced cell population in S-phase in vitro and suppressed growth in vivo. Furthermore, the MAP/ERK kinase inhibitor PD98059 partially abolished modulation of p21 and cyclin D1 expression by p53R2. Moreover, under the conditions of nonstress and adriamycin-induced genotoxic stress, attenuation of p53R2 in KB cells significantly increased phosphorylated H2AX, which indicates that attenuation of p53R2 may enhance DNA damage induced by adriamycin. Overall, our study shows that p53R2 may suppress cancer cell proliferation partially by upregulation of p21 and downregulation of cyclin D1; p53R2 plays critical roles not only in DNA damage repair but also in proliferation of cancer cells.

Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; KB Cells; Mice; Mice, SCID; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasms; Phosphorylation; Ribonucleotide Reductases; Xenograft Model Antitumor Assays

The retinoblastoma product (RB1) is frequently deregulated in various types of tumors by mutation, deletion, or inactivation through association with viral oncoproteins. The functional loss of RB1 is recognized to be one of the hallmarks that differentiate cancer cells from normal cells. Many researchers are attempting to develop anti-tumor agents that are preferentially effective against RB1-negative tumors. However, to identify patients with RB1-negative cancers, it is imperative to develop predictive biomarkers to classify RB1-positive and -negative tumors.. Expression profiling of 30 cancer cell lines composed of 16 RB1-positive and 14 RB1-negative cancers was performed to find genes that are differentially expressed between the two groups, resulting in the identification of an RB1 signature with 194 genes. Among them, critical RB1 pathway components CDKN2A and CCND1 were included. We found that microarray data of the expression ratio of CCND1 and CDKN2A clearly distinguished the RB1 status of 30 cells lines. Measurement of the CCND1/CDKN2A mRNA expression ratio in additional cell lines by RT-PCR accurately predicted RB1 status (12/12 cells lines). The expression of CCND1/CDKN2A also correlated with RB1 status in xenograft tumors in vivo. Lastly, a CCND1/CDKN2A assay with clinical samples showed that uterine cervical and small cell lung cancers known to have a high prevalence of RB1-decifiency were predicted to be 100% RB1-negative, while uterine endometrial or gastric cancers were predicted to be 5-22% negative. All clinically normal tissues were 100% RB1-positive.. We report here that the CCND1/CDKN2A mRNA expression ratio predicts the RB1 status of cell lines in vitro and xenograft tumors and clinical tumor samples in vivo. Given the high predictive accuracy and quantitative nature of the CCND1/CDKN2A expression assay, the assay could be utilized to stratify patients for anti-tumor agents with preferential effects on either RB1-positive or -negative tumors.

Topics: Animals; Cell Line, Tumor; Cluster Analysis; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HCT116 Cells; HeLa Cells; Hep G2 Cells; Humans; Neoplasm Transplantation; Neoplasms; Neoplasms, Experimental; Oligonucleotide Array Sequence Analysis; Rats; Rats, Inbred F344; Rats, Nude; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The acquisition of proliferative and invasive phenotypes is considered a hallmark of neoplastic transformation; however, the underlying mechanisms are less well known. Lipid phosphate phosphatase-3 (LPP3) not only catalyzes the dephosphorylation of the bioactive lipid sphingosine-1-phosphate (S1P) to generate sphingosine but also may regulate embryonic development and angiogenesis via the Wnt pathway. The goal of this study was to determine the role of LPP3 in tumor cells.. We observed increased expression of LPP3 in glioblastoma primary tumors and in U87 and U118 glioblastoma cell lines. We demonstrate that LPP3-knockdown inhibited both U87 and U118 glioblastoma cell proliferation in culture and tumor growth in xenograft assays. Biochemical experiments provided evidence that LPP3-knockdown reduced β-catenin, CYCLIN-D1, and CD133 expression, with a concomitant increase in phosphorylated β-catenin. In a converse experiment, the forced expression of LPP3 in human colon tumor (SW480) cells potentiated tumor growth via increased β-catenin stability and CYCLIN-D1 synthesis. In contrast, elevated expression of LPP3 had no tumorigenic effects on primary cells.. These results demonstrate for the first time an unexpected role of LPP3 in regulating glioblastoma progression by amplifying β-catenin and CYCLIN-D1 activities.

Topics: Animals; beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Female; Gene Knockdown Techniques; HEK293 Cells; Humans; Mice; Mice, Nude; Neoplasms; Phosphatidate Phosphatase; Signal Transduction; Tumor Burden; Xenograft Model Antitumor Assays

Topics: Cyclin D1; DNA Damage; DNA Repair; Humans; Neoplasms; Protein Binding; Protein Interaction Mapping; Rad51 Recombinase; Recombination, Genetic; Retinoblastoma Protein

Cyclin D1 is a component of the core cell cycle machinery. Abnormally high levels of cyclin D1 are detected in many human cancer types. To elucidate the molecular functions of cyclin D1 in human cancers, we performed a proteomic screen for cyclin D1 protein partners in several types of human tumours. Analyses of cyclin D1 interactors revealed a network of DNA repair proteins, including RAD51, a recombinase that drives the homologous recombination process. We found that cyclin D1 directly binds RAD51, and that cyclin D1-RAD51 interaction is induced by radiation. Like RAD51, cyclin D1 is recruited to DNA damage sites in a BRCA2-dependent fashion. Reduction of cyclin D1 levels in human cancer cells impaired recruitment of RAD51 to damaged DNA, impeded the homologous recombination-mediated DNA repair, and increased sensitivity of cells to radiation in vitro and in vivo. This effect was seen in cancer cells lacking the retinoblastoma protein, which do not require D-cyclins for proliferation. These findings reveal an unexpected function of a core cell cycle protein in DNA repair and suggest that targeting cyclin D1 may be beneficial also in retinoblastoma-negative cancers which are currently thought to be unaffected by cyclin D1 inhibition.

Topics: Animals; Cell Line, Tumor; Comet Assay; Cyclin D1; DNA Damage; DNA Repair; HeLa Cells; Humans; Mice; Neoplasms; Protein Binding; Protein Interaction Mapping; Rad51 Recombinase; Radiation, Ionizing; Recombination, Genetic; Retinoblastoma Protein

High doses of niacin (nicotinic acid) used to treat dyslipidemias cause flushing, due to high levels of prostaglandin D(2) (PGD(2)). GPR109A, a G-protein coupled receptor, triggers the flushing in the skin. In addition to boosting PGD(2), niacin binding to GPR109A activates the entire prostanoid cascade. We found that GPR109A occurs throughout the gastrointestinal tract. Mice that alternated between a 1% niacin diet and a control diet had higher urinary prostaglandin E(2) (PGE(2)) metabolite levels when on niacin (2.8-fold increase; 95% confidence interval, 1.8-3.9). PGE(2) promotes tumors in the intestines, whereas PGD(2) may have an opposite effect, on the basis of our report showing that transgenic hematopoietic prostaglandin D synthase suppresses intestinal adenomas in Apc(Min/+) mice. To determine if either tumor growth or tumor suppression prevails, we fed Apc(Min/+) mice a 1% niacin diet and assessed tumor development. A 1% niacin diet did not affect the number of tumors scored histologically in Apc(Min/+) mice at 14 wk (33 mice on niacin, 33 controls). Although niacin stimulates production of various prostaglandins, our results support an interpretation that very high intakes of niacin are safe in relation to intestinal tumors in this model.

Topics: Adenoma; Animals; Cyclin D1; Diet; Dose-Response Relationship, Drug; Female; Flushing; Humans; Intestines; Intramolecular Oxidoreductases; Isomerases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasms; Neoplasms, Experimental; Niacin; Phospholipases A2, Cytosolic; Prostaglandins; Receptors, G-Protein-Coupled; Receptors, Nicotinic; RNA, Messenger; Vascular Endothelial Growth Factor A

Skp1-Cul1-F-box (SCF) E3 ubiquitin ligase complexes modulate the accumulation of key cell cycle regulatory proteins. Following the G(1)/S transition, SCF(Fbx4) targets cyclin D1 for proteasomal degradation, a critical event necessary for DNA replication fidelity. Deregulated cyclin D1 drives tumorigenesis, and inactivating mutations in Fbx4 have been identified in human cancer, suggesting that Fbx4 may function as a tumor suppressor. Fbx4(+/-) and Fbx4(-/-) mice succumb to multiple tumor phenotypes, including lymphomas, histiocytic sarcomas and, less frequently, mammary and hepatocellular carcinomas. Tumors and premalignant tissue from Fbx4(+/-) and Fbx4(-/-) mice exhibit elevated cyclin D1, an observation consistent with cyclin D1 as a target of Fbx4. Molecular dissection of the Fbx4 regulatory network in murine embryonic fibroblasts (MEFs) revealed that loss of Fbx4 results in cyclin D1 stabilization and nuclear accumulation throughout cell division. Increased proliferation in early passage primary MEFs is antagonized by DNA damage checkpoint activation, consistent with nuclear cyclin D1-driven genomic instability. Furthermore, Fbx4(-/-) MEFs exhibited increased susceptibility to Ras-dependent transformation in vitro, analogous to tumorigenesis observed in mice. Collectively, these data reveal a requisite role for the SCF(Fbx4) E3 ubiquitin ligase in regulating cyclin D1 accumulation, consistent with tumor suppressive function in vivo.

Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Nucleus; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Cyclin D1; DNA Damage; F-Box Proteins; Fibroblasts; Gene Knockout Techniques; Mice; Mice, Transgenic; Neoplasms; Proto-Oncogene Proteins p21(ras); SKP Cullin F-Box Protein Ligases

In the present research we analyze the mechanism of human beta-defensin-2 (hBD-2) influence on cultured malignant epithelial cell growth.. The analysis of a concentration-dependent effect of recombinant hBD-2 (rec-hBD-2) on cell growth patterns and cell cycle distribution has been performed in vitro with 2 cell lines (human lung adenocarcinoma A549 cells and human epidermoid carcinoma A431 cells) using MTT test, flow cytometry and direct cell counting. To study intracellular localization of hBD-2 immunocytofluorescent and immunocytochemical analyses were applied, and effect of hBD-2 on signal cascades involved in cell cycle regulation has been studied by Western blotting.. According to our data, rec-hBD-2 exerts a concentration-dependent effect on the viability of cultured A549 and A431 cells. It causes proproliferative effect at concentrations below 1 nM, significant suppression of cell proliferation at concentration range from 10 nM to 1 μM (p<0.05), and cell death at higher concentrations. Using flow cytometry we have demonstrated that hBD-2 dependent growth suppression is realized via cell cycle arrest at G1/S phase (p<0.05). Also, we have registered significant activation of pRB and decreased expression of Cyclin D1 in cells treated with the defensin compared to untreated control cells, while the expression of p53 remains unaffected. The study of intracellular localization of hBD-2 in these cells has revealed that exogeneously added defensin molecules enter the cells, are distributed throughout the cytoplasm and could be detected in cell nuclei. The model study using A549 cells treated with 1,25-(OH)(2)D(3) has shown similar cell growth suppression effect of native endogenously produced hBD-2.. The results of our study suggest that in malignant epithelial cells hBD-2 may control cell growth via arrest of G1/S transition and activation of pRB.

Topics: Apoptosis; beta-Defensins; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Epithelial Cells; G1 Phase; Humans; Neoplasms; Retinoblastoma Protein; Signal Transduction; Tumor Suppressor Protein p53

A series of anilino substituted pyrimidine sulfonamides were prepared and evaluated for their anticancer activity. These sulfonamides showed promising activity with IC(50) values ranging from 5.6 to 12.3 μM. The detailed biological aspects of some of the promising compounds (3d, 3e and 3g) on the K562 cell line were studied. Interestingly, compounds induced G1 cell cycle arrest and down regulation of G1 phase cell cycle regulatory proteins such as cyclin D1, CDK4. These compounds also exhibited inhibition of NF-κB as well as its downstream target gene Akt1 and the phosphorylated form of AKt ser 474 proteins. One of the representative compound 3e could be considered as the potential lead for its development as a new anticancer agent.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cyclin D1; Down-Regulation; G1 Phase; Humans; Neoplasms; NF-kappa B; Pyrimidines; Sulfonamides

The embryonic pyruvate kinase M2 (PKM2) isoform is highly expressed in human cancer. In contrast to the established role of PKM2 in aerobic glycolysis or the Warburg effect, its non-metabolic functions remain elusive. Here we demonstrate, in human cancer cells, that epidermal growth factor receptor (EGFR) activation induces translocation of PKM2, but not PKM1, into the nucleus, where K433 of PKM2 binds to c-Src-phosphorylated Y333 of β-catenin. This interaction is required for both proteins to be recruited to the CCND1 promoter, leading to HDAC3 removal from the promoter, histone H3 acetylation and cyclin D1 expression. PKM2-dependent β-catenin transactivation is instrumental in EGFR-promoted tumour cell proliferation and brain tumour development. In addition, positive correlations have been identified between c-Src activity, β-catenin Y333 phosphorylation and PKM2 nuclear accumulation in human glioblastoma specimens. Furthermore, levels of β-catenin phosphorylation and nuclear PKM2 have been correlated with grades of glioma malignancy and prognosis. These findings reveal that EGF induces β-catenin transactivation via a mechanism distinct from that induced by Wnt/Wingless and highlight the essential non-metabolic functions of PKM2 in EGFR-promoted β-catenin transactivation, cell proliferation and tumorigenesis.

Topics: Animals; beta Catenin; Cell Line, Tumor; CSK Tyrosine-Protein Kinase; Cyclin D1; ErbB Receptors; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Mice; Neoplasms; NIH 3T3 Cells; Nuclear Proteins; Phosphorylation; Protein Binding; Protein Transport; Protein-Tyrosine Kinases; Pyruvate Kinase; src-Family Kinases

Aberrant activation of Akt plays a pivotal role in cancer development. ATM, a protein deficient in patients with ataxia-telangiectasia disease, is traditionally considered as a nuclear protein kinase that functions as a signal transducer in response to DNA damage. It has recently been shown that ATM is also a cytoplasmic protein that mediates the full activation of Akt in response to insulin. Our study shows that a specific ATM inhibitor, KU-55933, blocks the phosphorylation of Akt induced by insulin and insulin-like growth factor I in cancer cells that exhibit abnormal Akt activity. Moreover, KU-55933 inhibits cancer cell proliferation by inducing G(1) cell cycle arrest. It does so through the downregulation of the synthesis of cyclin D1, a protein known to be elevated in a variety of tumors. In addition, KU-55933 treatment during serum starvation triggers apoptosis in these cancer cells. Our results suggest that KU-55933 may be a novel chemotherapeutic agent targeting cancer resistant to traditional chemotherapy or immunotherapy due to aberrant activation of Akt. Furthermore, KU-55933 completely abrogates rapamycin-induced feedback activation of Akt. Combination of KU-55933 and rapamycin not only induces apoptosis, which is not seen in cancer cells treated only with rapamycin, but also shows better efficacy in inhibiting cancer cell proliferation than each drug alone. Therefore, combining KU-55933 with rapamycin may provide a highly effective approach for improving mammalian target of rapamycin-targeted anticancer therapy that is currently hindered by rapamycin-induced feedback activation of Akt.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Down-Regulation; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Fibroblasts; G1 Phase; Humans; Insulin; Intercellular Signaling Peptides and Proteins; Mice; Morpholines; Neoplasms; Peptide Chain Initiation, Translational; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrones; Sirolimus; Transcription, Genetic; Tumor Suppressor Proteins

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 microM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.

Topics: Animals; Anthraquinones; Antineoplastic Agents; Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 3; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Glioblastoma; Humans; Inhibitor of Apoptosis Proteins; Male; Mice; Mice, Nude; Microtubule-Associated Proteins; Neoplasms; Pancreatic Neoplasms; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; STAT3 Transcription Factor; Sulfonamides; Survivin; Xenograft Model Antitumor Assays

Topics: Animals; Cyclin D1; Endopeptidases; G1 Phase; Mice; Neoplasms; Oncogenes; S Phase; Ubiquitin-Specific Proteases; Ubiquitination

Statins are potent inhibitors of hydroxyl-3-methylglutaryl co-enzyme A (HMG-CoA) reductase, and have emerged as potential anti-cancer agents based on preclinical evidence. In particular, compelling evidence suggests that statins have a wide range of immunomodulatory properties. However, little is known about the role of statins in tumour immune tolerance. Tumour immune tolerance involves the production of immunosuppressive molecules, such as interleukin (IL)-10, transforming growth factor (TGF)-beta and indoleamine-2,3-dioxygenase (IDO) by tumours, which induce a regulatory T cell (T(reg)) response. In this study, we investigated the effect of simvastatin on the production of IL-10, TGF-beta and IDO production and the proliferation of T(regs) using several cancer cell lines, and Lewis lung cancer (3LL) cells-inoculated mouse tumour model. Simvastatin treatment resulted in a decrease in the number of cancer cells (3LL, A549 and NCI-H292). The production of the immune regulatory markers IL-10, TGF-beta in 3LL and NCI-H292 cells increased after treatment with simvastatin. The expression of IDO and forkhead box P3 (FoxP3) transcription factor was also increased in the presence of simvastatin. In a murine 3LL model, there were no significant differences in tumour growth rate between untreated and simvastatin-treated mice groups. Therefore, while simvastatin had an anti-proliferative effect, it also exhibited immune tolerance-promoting properties during tumour development. Thus, due to these opposing actions, simvastatin had no net effect on tumour growth.

Topics: Animals; Cell Count; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cytostatic Agents; Forkhead Transcription Factors; Gene Expression; Humans; Immune Tolerance; Immunosuppressive Agents; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Mice; Mice, Inbred C57BL; Neoplasms; Simvastatin; Spleen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The N-acetyltransferase arrest defective 1 (ARD1) is an important regulator of cell growth and differentiation that has emerged recently as a critical molecule in cancer progression. However, the regulation of the enzymatic and biological activities of human ARD1 (hARD1) in cancer is presently poorly understood. Here, we report that hARD1 undergoes autoacetylation and that this modification is essential for its functional activation. Using liquid chromatography-tandem mass spectrometry and site-directed mutational analyses, we identified K136 residue as an autoacetylation target site. K136R mutation abolished the ability of hARD1 to promote cancer cell growth in vitro and tumor xenograft growth in vivo. Mechanistic investigations revealed that hARD1 autoacetylation stimulated cyclin D1 expression through activation of the transcription factors beta-catenin and activator protein-1. Our results show that hARD1 autoacetylation is critical for its activation and its ability to stimulate cancer cell proliferation and tumorigenesis.

Topics: Acetyl Coenzyme A; Acetylation; Acetyltransferases; Animals; beta Catenin; Cell Growth Processes; Cyclin D1; HeLa Cells; Humans; Mice; N-Terminal Acetyltransferase A; N-Terminal Acetyltransferase E; Neoplasms; Transcription Factor AP-1; Up-Regulation

GRIM-19 (Gene associated with Retinoid-IFN-induced Mortality-19) was originally isolated as a growth suppressor in a genome-wide knockdown screen with antisense libraries. Like classical tumor suppressors, mutations, and/or loss of GRIM-19 expression occur in primary human tumors; and it is inactivated by viral gene products. Our search for potential GRIM-19-binding proteins, using mass spectrometry, that permit its antitumor actions led to the inhibitor of cyclin-dependent kinase 4, CDKN2A. The GRIM-19/CDKN2A synergistically suppressed cell cycle progression via inhibiting E2F1-driven gene expression. The N terminus of GRIM-19 and the fourth ankyrin repeat of CDKN2A are crucial for their interaction. The biological relevance of these interactions is underscored by observations that GRIM-19 promotes the inhibitory effect of CDKN2A on CDK4; and mutations from primary tumors disrupt its ability to interact with GRIM-19 and suppress E2F1-driven gene expression.

Topics: Amino Acid Sequence; Animals; Ankyrin Repeat; Apoptosis Regulatory Proteins; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p16; E2F1 Transcription Factor; G1 Phase; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Mice; Molecular Sequence Data; Mutation; NADH, NADPH Oxidoreductases; Neoplasms; Protein Structure, Tertiary

Recurrence is frequently associated with the acquisition of radioresistance by tumors and resulting failures in radiotherapy. We report, in this study, that long-term fractionated radiation (FR) exposures conferred radioresistance to the human tumor cells, HepG2 and HeLa with cyclin D1 overexpression. A positive feedback loop was responsible for the cyclin D1 overexpression in which constitutively active AKT was involved. AKT is known to inactivate glycogen synthase kinase-3beta (GSK3beta), which is essential for the proteasomal degradation of cyclin D1. The resulting cyclin D1 overexpression led to the forced progression of S-phase with the induction of DNA double strand breaks. Cyclin D1-dependent DNA damage activated DNA-dependent protein kinase (DNA-PK), which in turn activated AKT and inactivated GSK3beta, thus completing a positive feedback loop of cyclin D1 overproduction. Cyclin D1 overexpression led to the activation of DNA damage response (DDR) consisted of ataxia telangiectasia mutated (ATM)- and Chk1-dependent DNA damage checkpoint and homologous recombination repair (HRR). Long-term FR cells repaired radiation-induced DNA damage faster than non-FR cells. Thus, acquired radioresistance of long-term FR cells was the result of alterations in DDR mediated by cyclin D1 overexpression. Inhibition of the AKT/GSK3beta/cyclin D1/Cdk4 pathway by the AKT inhibitor, Cdk4 inhibitor or cyclin D1 targeting small interfering RNA (siRNA) suppressed the radioresistance. Present observations give a mechanistic insight for acquired radioresistance of tumor cells by cyclin D1 overexpression, and provide novel therapeutic targets for recurrent radioresistant tumors.

Topics: Cyclin D1; Cyclin-Dependent Kinase 4; DNA Damage; DNA Repair; DNA-Activated Protein Kinase; Down-Regulation; G1 Phase; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HeLa Cells; Hep G2 Cells; Humans; Neoplasms; Oncogene Protein v-akt; Radiation Tolerance; S Phase; Signal Transduction

Cryptotanshinone (CPT), a natural compound isolated from the plant Salvia miltiorrhiza Bunge, is a potential anticancer agent. However, little is known about its anticancer mechanism. Here, we show that CPT inhibited cancer cell proliferation by arresting cells in G(1)-G(0) phase of the cell cycle. This is associated with the inhibition of cyclin D1 expression and retinoblastoma (Rb) protein phosphorylation. Furthermore, we found that CPT inhibited the signaling pathway of the mammalian target of rapamycin (mTOR), a central regulator of cell proliferation. This is evidenced by the findings that CPT inhibited type I insulin-like growth factor I- or 10% fetal bovine serum-stimulated phosphorylation of mTOR, p70 S6 kinase 1, and eukaryotic initiation factor 4E binding protein 1 in a concentration- and time-dependent manner. Expression of constitutively active mTOR conferred resistance to CPT inhibition of cyclin D1 expression and Rb phosphorylation, as well as cell growth. The results suggest that CPT is a novel antiproliferative agent.

Topics: Antineoplastic Agents; Cell Proliferation; Cyclin D1; Down-Regulation; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Phenanthrenes; Phosphorylation; Protein Kinases; Retinoblastoma Protein; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Clinical Trials as Topic; Cyclin D1; ErbB Receptors; Female; Humans; Lung Neoplasms; Mutation; Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); ras Proteins; Research Design; Retinoid X Receptors; Vascular Endothelial Growth Factor A

Bcl-3 is an atypical member of the inhibitor of NF-kappa B family of proteins since it can function as a coactivator of transcription. Although this oncogene was described in leukemia, it is overexpressed in a number of solid tumors as well. The oncogenic potential of Bcl-3 has been associated with its capacity to increase proliferation by means of activating the cyclin D1 promoter and to its antiapoptotic role mediated by the inhibiton of p53 activity. In the course of dissecting these properties, we found that depleting Bcl-3 protein using shRNAs induce a decrease of proliferation and clonogenic survival associated with the induction of multinucleation and increased ploidy. These effects were associated with a DNA damage response, a delay in G2/M checkpoint and the induction of centrosome amplification.

Topics: Aneuploidy; B-Cell Lymphoma 3 Protein; Cell Cycle; Centrosome; Cyclin D1; Gene Knockdown Techniques; HeLa Cells; Humans; Neoplasms; Promoter Regions, Genetic; Proto-Oncogene Proteins; Transcription Factors

Transcriptional function of cyclin D1, whose deregulation is frequently observed in human cancers, has been suggested to contribute to cancer formation. In the present study, we show that cyclin D1 protein inhibits RUNX3 activity by directly binding to it and interfering with its interaction with p300 interaction in lung cancer cells. Cyclin D1 inhibits p300-dependent RUNX3 acetylation and negatively regulates cyclin-dependent kinase (cdk) inhibitor p21 expression. These transcriptional effects of cyclin D1 do not require cdk4/6 kinase activation. We propose that cyclin D1 provides a transcriptional switch that allows the tumor suppressor activity of RUNX3 to be repressed in cancer cells. Since RUNX3 plays tumor suppressive roles in a wide range of cancers, a non-canonical cyclin D1 function may be critical for neoplastic transformation of the epithelial cells in which RUNX3 regulates proliferation.

Topics: Cell Line, Tumor; Cell Proliferation; Core Binding Factor Alpha 3 Subunit; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; p300-CBP Transcription Factors; Transcription, Genetic; Up-Regulation

Reverse pharmacology, also called the "bedside to bench" approach, that deals with new uses for a well known molecular entity has been used extensively in cancer drug development to identify novel compounds and delineate their mechanisms of action. Here, we show that nimbolide, a triterpenoid isolated from Azadirachta indica, enhanced the apoptosis induced by inflammatory cytokines and chemotherapeutic agents in tumor cells. This limonoid abrogated the expression of proteins associated with cell survival (Bcl-2, Bcl-xL, IAP-1, and IAP-2), proliferation (cyclin D1), invasion (MMP-9), and angiogenesis (VEGF), all regulated by nuclear factor (NF)-κB. Nimbolide inhibited the activation of NF-κB induced by carcinogens and inflammatory stimuli. Constitutively active NF-κB found in most tumor cells was also inhibited. We found that suppression of NF-κB activation by nimbolide was caused by inhibition of IκB kinase (IKK), which led to suppression of IκBα phosphorylation and degradation, nuclear translocation, DNA binding, and gene transcription. Reducing agent reversed the action of the limonoid, suggesting the involvement of a cysteine residue. Replacement of Cys(179) of IKK-β with alanine abolished the effect of nimbolide, suggesting that Cys(179) plays a critical role in inhibiting the NF-κB activation. Overall, our results indicate that nimbolide can sensitize tumor cells to chemotherapeutic agents through interaction with IKK, leading to inhibition of NF-κB-regulated proteins.

Topics: Amino Acid Substitution; Apoptosis; Azadirachta; Blotting, Western; Caspases; Cell Proliferation; Cell Survival; Cyclin D1; Cysteine; Dose-Response Relationship, Drug; Down-Regulation; HEK293 Cells; HL-60 Cells; Humans; I-kappa B Kinase; Jurkat Cells; K562 Cells; Limonins; Molecular Structure; Neoplasms; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Tumor Necrosis Factor-alpha; U937 Cells; Vascular Endothelial Growth Factor A

Trop2 is a cell-surface glycoprotein overexpressed by a variety of epithelial carcinomas with reported low to restricted expression in normal tissues. Expression of Trop2 has been associated with increased tumor aggressiveness, metastasis and decreased patient survival, but the signaling mechanisms mediated by Trop2 are still unknown. Here, we studied the effects murine Trop2 (mTrop2) exerted on tumor cellular functions and some of the signaling mechanisms activated by this oncogene.. mTrop2 expression significantly increased tumor cell proliferation at low serum concentration, migration, foci formation and anchorage-independent growth. These in vitro characteristics translated to increased tumor growth in both subcutaneous and orthotopic pancreatic cancer murine models and also led to increased liver metastasis. mTrop2 expression also increased the levels of phosphorylated ERK1/2 mediating cell cycle progression by increasing the levels of cyclin D1 and cyclin E as well as downregulating p27. The activation of ERK was also observed in human pancreatic ductal epithelial cells and colorectal adenocarcinoma cells overexpressing human Trop2.. These findings demonstrate some of the pathogenic effects mediated by mTrop2 expression on cancer cells and the importance of targeting this cell surface glycoprotein. This study also provides the first indication of a molecular signaling pathway activated by Trop2 which has important implications for cancer cell growth and survival.

Topics: Animals; Antigens, Neoplasm; Cell Adhesion Molecules; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Cyclin D1; Cyclin E; Extracellular Signal-Regulated MAP Kinases; Female; HCT116 Cells; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplasms; Pancreatic Neoplasms; Proteins; Signal Transduction

Cyclin D1 elicits transcriptional effects through inactivation of the retinoblastoma protein and direct association with transcriptional regulators. The current work reveals a molecular relationship between cyclin D1/CDK4 kinase and protein arginine methyltransferase 5 (PRMT5), an enzyme associated with histone methylation and transcriptional repression. Primary tumors of a mouse lymphoma model exhibit increased PRMT5 methyltransferase activity and histone arginine methylation. Analyses demonstrate that MEP50, a PRMT5 coregulatory factor, is a CDK4 substrate, and phosphorylation increases PRMT5/MEP50 activity. Increased PRMT5 activity mediates key events associated with cyclin D1-dependent neoplastic growth, including CUL4 repression, CDT1 overexpression, and DNA rereplication. Importantly, human cancers harboring mutations in Fbx4, the cyclin D1 E3 ligase, exhibit nuclear cyclin D1 accumulation and increased PRMT5 activity.

Topics: Adaptor Proteins, Signal Transducing; Animals; Cell Cycle Proteins; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Cullin Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; DNA Methylation; DNA Replication; Enzyme Activation; F-Box Proteins; Gene Expression Regulation, Neoplastic; Histones; Humans; Lymphoma; Mice; Neoplasms; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Methyltransferases; Protein Stability

Eukaryotic translation initiation factor 4E (eIF4E) is encoded by a potent oncogene which is highly elevated in many human cancers. Few studies have investigated how the level, and thus activity, of eIF4E is regulated in healthy (noncancerous) cells and how they become elevated in malignant cells. Here, our studies reveal a novel mechanism by which eIF4E levels are regulated at the level of mRNA stability. Two factors known to modulate transcript stability, HuR and the p42 isoform of AUF1, compete for binding to the 3' untranslated regions (3'UTRs) of eIF4E mRNAs. We identified a distinct AU-rich element in the 3'UTR of eIF4E which is responsible for HuR-mediated binding and stabilization. Our studies show that HuR is upregulated in malignant cancer specimens characterized by high eIF4E levels and that its depletion leads to reduction in eIF4E levels. Further, HuR and eIF4E regulate a common set of transcripts involved in cellular proliferation (cyclin D1 and c-myc) and neoangiogenesis (vascular endothelial growth factor), which suggests a functional connection between HuR and eIF4E in the regulation of these important processes. In summary, we present a novel model for the regulation of eIF4E expression and show that this model is relevant to elevation of eIF4E levels in malignant cells.

Topics: 3' Untranslated Regions; Antigens, Surface; Binding Sites; Cell Line; Cyclin D1; ELAV Proteins; ELAV-Like Protein 1; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Proto-Oncogene Proteins c-myc; RNA Stability; RNA-Binding Proteins; RNA, Messenger; Up-Regulation; Vascular Endothelial Growth Factor A

Retinoids have significant clinical activity in several human cancers, yet the factors determining retinoid sensitivity in cancer cells are still unclear. Retinoid-induced expression of retinoic acid receptor (RAR) beta(2) is a necessary component of the retinoid anticancer signal in cancer cells. We have previously identified the Estrogen-responsive B Box Protein (EBBP), a member of the Tripartite Motif (TRIM) protein family, as a novel RARbeta2 transcriptional regulator in the retinoid signal. Here we examined the mechanism of the EBBP effect on the retinoid anticancer signal. We assessed retinoid-responsive RARbeta2 transcription in retinoid-resistant breast and lung cancer cells in the presence of chromatin modifying agents. A histone deacetylase (HDAC) inhibitor alone, or in combination with retinoid, was more effective than a demethylating agent in restoring RARbeta2 transcription in resistant cells. Overexpression of EBBP alone markedly increased histone acetylation. The effect of EBBP on retinoid-responsive transcription appeared to be limited to genes with the retinoic acid response element (betaRARE) regulatory sequence, such as CYP26A1. EBBP inhibited cell growth by effects on cyclin D1 and Phospho-Rb, and, reduced cell viability in retinoid-resistant cancer cells. The viability of non-cancer cells was unaffected by EBBP overexpression. Taken together our data suggests that EBBP acts to de-repress transcription of RARbeta2 and CYP26A1, by modifying histone acetylation in retinoid-resistant cancer cells, and, is an important target for drug discovery in retinoid-resistant cancers.

Topics: Acetylation; Apoptosis; Cell Line, Tumor; Cell Survival; Cyclin D1; Cytochrome P-450 Enzyme System; DNA-Binding Proteins; Drug Resistance, Neoplasm; Histones; Humans; Neoplasms; Phosphorylation; Receptors, Retinoic Acid; Retinoblastoma Protein; Retinoic Acid 4-Hydroxylase; Transcription Factors; Tretinoin; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

Asian ginseng (Panax ginseng C. A. Meyer) has been used in Chinese medicine for two thousand years. The root of ginseng contains several saponins (ginsenosides) which are biologically active compounds. Individual ginsenosides suppress tumor cell growth, induce cell differentiation, regulate apoptosis and inhibit metastasis formation. The aim of this study was to evaluate its chemo-preventive effects in an animal test model, through its regulatory effects on apoptosis and the cell cycle.The expression of genes (Bcl-2, Bcl-x and Cyclin D1) which affect apoptosis were examined, in different organs of animals which had consumed a ginseng-containing diet in the presence of a known carcinogen (DMBA). The pattern of gene expression was determined by Q-RT-PCR. The increase of antiapoptotic gene expression after carcinogenic exposure was suppressed by consumption of ginseng which promoted apoptosis.The population is exposed to numerous physical and chemical insults in the modern environment and these include compounds which are known carcinogens. Research has shown that it is possible to interfere with the multi-step process of carcinogenesis through the use of compounds with chemo-preventive effects, such as the inhibition of the activation of antiapoptotic genes.These results support the efficacy of ginseng-containing diets and dietary supplements in the prevention of cancerous diseases.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-X Protein; Carcinogens; Cyclin D1; Disease Models, Animal; Female; Gene Expression; Mice; Mice, Inbred AKR; Neoplasms; Panax; Phytotherapy; Plant Preparations; Plant Roots; Powders; Proto-Oncogene Proteins c-bcl-2

Topics: Animals; Antineoplastic Agents; Catechin; Clinical Trials as Topic; Cyclin D1; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; National Cancer Institute (U.S.); Neoplasms; Signal Transduction; United States; United States Food and Drug Administration

The microRNAs miR-15a and miR-16-1 are downregulated in multiple tumor types and are frequently deleted in chronic lymphocytic leukemia (CLL), myeloma and mantle cell lymphoma. Despite their abundance in most cells the transcriptional regulation of miR-15a/16-1 remains unclear. Here we demonstrate that the putative tumor suppressor DLEU2 acts as a host gene of these microRNAs. Mature miR-15a/miR-16-1 are produced in a Drosha-dependent process from DLEU2 and binding of the Myc oncoprotein to two alterative DLEU2 promoters represses both the host gene transcript and levels of mature miR-15a/miR-16-1. In line with a functional role for DLEU2 in the expression of the microRNAs, the miR-15a/miR-16-1 locus is retained in four CLL cases that delete both promoters of this gene and expression analysis indicates that this leads to functional loss of mature miR-15a/16-1. We additionally show that DLEU2 negatively regulates the G1 Cyclins E1 and D1 through miR-15a/miR-16-1 and provide evidence that these oncoproteins are subject to miR-15a/miR-16-1-mediated repression under normal conditions. We also demonstrate that DLEU2 overexpression blocks cellular proliferation and inhibits the colony-forming ability of tumor cell lines in a miR-15a/miR-16-1-dependent way. Together the data illuminate how inactivation of DLEU2 promotes cell proliferation and tumor progression through functional loss of miR-15a/miR-16-1.

Topics: Bone Marrow; Cell Cycle; Cell Line; Chromatin; Colony-Forming Units Assay; Cyclin D1; Cyclin E; Cyclins; DNA; DNA Primers; Flow Cytometry; Gene Deletion; Humans; Kidney; MicroRNAs; Neoplasms; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Long Noncoding; RNA, Small Interfering; Transferases; Tumor Suppressor Proteins

NF-kappaB activity is tightly regulated by IkappaB class of proteins. IkappaB proteins possess ankyrin repeats for binding to and inhibiting NF-kappaB. The regulatory protein, NPR1 from Brassica juncea possesses ankyrin repeats with sequence similarity to IkappaBalpha subgroup. Therefore, we examined whether stably expressed BjNPR1 could function as IkappaB in inhibiting NF-kappaB in human glioblastoma cell lines. We observed that BjNPR1 bound to NF-kappaB and inhibited its nuclear translocation. Further, BjNPR1 expression down-regulated the NF-kappaB target genes iNOS, Cox-2, c-Myc and cyclin D1 and reduced the proliferation rate of U373 cells. Finally, BjNPR1 decreased the levels of pERK, pJNK and PKCalpha and increased the Caspase-3 and Caspase-8 activities. These results suggested that inhibition of NF-kappaB activation by BjNPR1 can be a promising therapy in NF-kappaB dependent pathologies.

Topics: Active Transport, Cell Nucleus; Amino Acid Sequence; Ankyrin Repeat; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclooxygenase 2; Down-Regulation; eIF-2 Kinase; Gene Expression; Gene Expression Regulation; Genes, Reporter; Humans; I-kappa B Proteins; Inflammation; Molecular Sequence Data; Mustard Plant; Neoplasms; NF-kappa B; Nitric Oxide Synthase Type II; Plant Proteins; Protein Kinase C-alpha; Proto-Oncogene Proteins c-myc

The cyclin D1 proto-oncoprotein is a crucial regulator in cell-cycle progression, and aberrant overexpression of cyclin D1 is linked to tumorigenesis of many different cancer types. By screening ubiquitinated cyclin D1 as a substrate with a deubiquitinase library, we have identified USP2 as a specific deubiquitinase for cyclin D1. USP2 directly interacts with cyclin D1 and promotes its stabilization by antagonizing ubiquitin-dependent degradation. Conversely, USP2 knockdown destabilizes cyclin D1 and induces growth arrest in the human cancer lines where cell growth is dependent on cyclin D1 expression. Of note, cyclin D1 is not universally required for cell-cycle progression. Inactivation of USP2 has either very mild effects on cell growth in normal human fibroblasts or no effect in the cancer cells that do not express cyclin D1. These findings suggest that targeting USP2 is an effective approach to induce growth suppression in the cancer cells addicted to cyclin D1 expression.

Topics: Blotting, Western; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Endopeptidases; Flow Cytometry; HCT116 Cells; Humans; Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transfection; Tumor Suppressor Protein p53; Ubiquitin; Ubiquitin Thiolesterase; Ubiquitination

The small heterodimer partner (SHP; NROB2), a member of the nuclear receptor superfamily, contributes to the biological regulation of several major functions of the liver. However, the role of SHP in cellular proliferation and tumorigenesis has not been investigated before. Here we report that SHP negatively regulates tumorigenesis both in vivo and in vitro. SHP-/- mice aged 12 to 15 months old developed spontaneous hepatocellular carcinoma, which was found to be strongly associated with enhanced hepatocyte proliferation and increased cyclin D1 expression. In contrast, overexpressing SHP in hepatocytes of SHP-transgenic mice reversed this effect. Embryonic fibroblasts lacking SHP showed enhanced proliferation and produced increased cyclin D1 messenger RNA and protein, and SHP was shown to be a direct negative regulator of cyclin D1 gene transcription. The immortal SHP-/- fibroblasts displayed characteristics of malignant transformed cells and formed tumors in nude mice.. These results provide first evidence that SHP plays tumor suppressor function by negatively regulating cellular growth.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Cells, Cultured; Cyclin D1; Embryo, Mammalian; Fibroblasts; Hepatocytes; Liver Neoplasms; Mice; Mice, Knockout; Mice, Nude; Mice, Transgenic; Neoplasms; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Transcription, Genetic; Tumor Suppressor Proteins; Up-Regulation

The epidermal growth factor receptor (EGFR) is frequently overexpressed in various tumours of epidermal origin and is held responsible for tumourigenicity and tumour persistence. Increased nuclear factor (NF)-kappaB activity has been suggested to be involved in the malignant behaviour of EGFR-overexpressing cells. However, the mechanisms that regulate EGF-induced NF-kappaB activation are still largely unknown. Here we show that EGF can induce NF-kappaB-dependent gene expression independently from IkappaBalpha degradation or p100 processing in EGFR-overexpressing HEK293T cells. Moreover, EGF-induced NF-kappaB activation could be inhibited by overexpression of ABINs, which were previously identified as intracellular inhibitors of tumour necrosis factor, interleukin-1 and lipopolysaccharide-induced NF-kappaB activation. Knockdown of ABIN-1 by RNA interference boosted the NF-kappaB response upon EGF stimulation. The C-terminal ubiquitin-binding domain containing region of ABINs was crucial and sufficient for NF-kappaB inhibition. Adenoviral gene transfer of ABINs reduced constitutive NF-kappaB activity as well as the proliferation of EGFR-overexpressing A431 and DU145 human carcinoma cells. Altogether, these results demonstrate an important role for an ABIN-sensitive non-classical NF-kappaB signalling pathway in the proliferation of EGFR-overexpressing tumour cells, and indicate a potential use for ABIN gene therapy in the treatment of cancer.

Topics: Adaptor Proteins, Signal Transducing; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Genetic Therapy; Humans; I-kappa B Proteins; Neoplasms; NF-kappa B; NF-kappa B p52 Subunit; NF-KappaB Inhibitor alpha; Phosphorylation; Protein Structure, Tertiary; RNA Interference; Signal Transduction

There is a growing appreciation of the role that epigenetic alterations can play in oncogenesis. However, given the large number of genetic anomalies present in most cancers, it has been difficult to evaluate the extent to which epigenetic changes contribute to cancer. SNF5 (INI1/SMARCB1/BAF47) is a tumor suppressor that regulates the epigenome as a core member of the SWI/SNF chromatin remodeling complex. While the SWI/SNF complex displays potent tumor suppressor activity, it is unknown whether this activity is exerted genetically via maintenance of genome integrity or epigenetically via transcriptional regulation. Here we show that Snf5-deficient primary cells do not show altered sensitivity to DNA damaging agents, defects in gamma-H2AX induction, or an abrogated DNA damage checkpoint. Further, the aggressive malignancies that arise following SNF5 loss are diploid and genomically stable. Remarkably, we demonstrate that most human SNF5-deficient cancers lack genomic amplifications/deletions and, aside from SNF5 loss, are indistinguishable from normal cells on single-nucleotide polymorphism arrays. Finally, we show that epigenetically based changes in transcription that occur following SNF5 loss correlate with the tumor phenotype. Collectively, our results provide novel insight into the mechanisms of oncogenesis by demonstrating that disruption of a chromatin remodeling complex can largely, if not completely, substitute for genomic instability in the genesis of aggressive cancer.

Topics: Animals; Cell Line, Tumor; Chromosomal Proteins, Non-Histone; Cisplatin; Cyclin D1; Diploidy; DNA Damage; DNA Repair; DNA-Binding Proteins; Epigenesis, Genetic; Etoposide; G2 Phase; Genomic Instability; Histones; Humans; Mice; Neoplasms; Phenotype; Polymorphism, Single Nucleotide; Protein Transport; Rhabdoid Tumor; SMARCB1 Protein; Transcription Factors

Deregulated signaling through the epidermal growth factor receptor (EGFR) is involved in chemoresistance. To identify the molecular determinants of sensitivity to the EGFR inhibitor gefitinib (Iressa, ZD1839) in chemoresistance, we compared the response of matched chemosensitive and chemoresistant glioma and ovarian cancer cell lines. We found that chemoresistant cell lines were 2- to 3-fold more sensitive to gefitinib growth-inhibitory effects, because of decreased proliferation rather than survival. Sensitivity to gefitinib correlated with overexpression and constitutive phosphorylation of HER2 and HER3, but not EGFR, altered HER ligand expression, and enhanced activation of EGF-triggered EGFR pathway. No activating mutations were found in EGFR. Gefitinib fully inhibited EGF-induced and constitutive Akt activation only in chemoresistant cells. In parallel, gefitinib downregulated constitutively phosphorylated HER2 and HER3, and activated GSK3beta with a concomitant degradation of cyclin D1. Ectopically overexpressed HER2 on its own was insufficient to sensitize chemonaive cells to gefitinib. pHER3 coimmunoprecipitated with p85-PI3K in chemoresistant cells and gefitinib dissociated these complexes. siRNA-mediated inhibition of HER3 decreased constitutive activation of Akt and sensitivity to gefitinib in chemoresistant cells. Our study indicates that in chemoresistant cells gefitinib inhibits both an enhanced EGF-triggered pathway and a constitutive HER3-mediated Akt activation, indicating that inhibition of HER3 together with that of EGFR could be relevant in chemorefractory tumors. Furthermore, in combination experiments gefitinib enhanced the effects of coadministered drugs more in chemoresistant than chemosensitive ovarian cancer cells. Combined treatment might be therapeutically beneficial in chemoresistant tumors from ovary and likely from other tissues.

Topics: Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cisplatin; Cyclin D1; DNA, Complementary; Down-Regulation; Drug Resistance, Neoplasm; ErbB Receptors; Female; Gefitinib; Gene Expression Regulation, Neoplastic; Glioma; Humans; Immunoblotting; Immunoprecipitation; Neoplasms; Ovarian Neoplasms; Phosphorylation; Polymerase Chain Reaction; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-2; Receptor, ErbB-3; Repressor Proteins; RNA, Small Interfering; Sequence Analysis, DNA; Signal Transduction

We investigated the differential role of diphenyleneiodonium (DPI), which is widely used as an inhibitor of NADPH oxidase, on the activation of cell cycle regulators in the cell cycle progression. DPI efficiently blocked the transition from G0/G1 to S phase by serum stimulation in quiescent HCT-116 (wild-type p53) and HL-60 (null p53) cells. Concomitant with G0/G1 arrest, HCT-116 cells treated with DPI resulted in strong and sustained upregulation of p53 and p21. p53- or p21-deficient HCT-116 cells using a small interfering RNA (siRNA) significantly increased the progression into S phase by stimulation of DPI, compared with DPI alone. However, the silencing of p53 resulted in more efficient transition into S phase than the silencing of p21 siRNA and significantly inhibited p21 upregulation by DPI stimulation. Interestingly, brief exposure to DPI did not change p53 expression, but showed transient upregulation of p21 and G0/G1 arrest. These results suggest that p53 upregulation sustains G0/G1 cell cycle arrest and p21 upregulation by DPI stimulation in HCT-116 cells. In HL-60 cells, DPI also induced p21 upregulation in a p53-independent manner and the increase of p21 expression seems to be regulated by DPI-mediated ERK activation. Cyclin D1 expression was not significantly affected by DPI treatment in HCT-116 cells. However, in HL-60 cells, DPI irreversibly impaired cyclin D1 upregulation by serum stimulation and a much greater fraction of cells arrested in G0/G1 was observed in HL-60 cells than in HCT-116 cells at 24 h after brief DPI treatment. These results suggest that cyclin D1 is an important regulatory factor in the inhibition of cell cycle progression by DPI in HL-60 cells.

Topics: Cell Cycle; Cell Proliferation; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; HCT116 Cells; HL-60 Cells; Humans; NADPH Oxidases; Neoplasms; Onium Compounds; RNA Interference; RNA, Small Interfering; Signal Transduction; Time Factors; Transfection; Tumor Suppressor Protein p53; Up-Regulation

Tumour heterogeneity is becoming increasingly important as an obstacle to genomic and proteomic technologies designed to improve the diagnosis and treatment of human cancer. In a panel of 19 human in-vitro cancer cell lines, we show marked heterogeneity of proteomic expression of key genes responsible for the control of cell division and death. Patterns of expression of these proteins were unique for each cell line. In addition, dynamic heterogeneity of proteomic expression of Cyclin D1, Cdk1, Cdk4 and even actin was detected. The relative levels of each protein fluctuated independently from experiment to experiment separated only by short passages in tissue culture. Cdk1 and Cdk4 proteomic co-expression (Seabra, 2007) was not, however, affected by dynamic heterogeneity, or, in 4 cell lines, by treatment with D0.1 doses of CDDP. Cdk1/Cdk4 may thus provide a complex molecular target for anti-cancer drug development which is unaffected by tumour heterogeneity and is not disrupted by conventional chemotherapy.

Topics: Antineoplastic Agents; Blotting, Western; CDC2 Protein Kinase; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Tumor Cells, Cultured; Tumor Stem Cell Assay

Cyclin D1 is an important cell cycle regulator, but in cancer its overexpression also increases cellular migration mediated by p27 KIP1 stabilization and RhoA inhibition. Recently, a common polymorphism at the exon 4-intron 4 boundary of the human cyclin D1 gene within a splice donor region was associated with an altered risk of developing cancer. Altered RNA splicing caused by this polymorphism gives rise to a variant cyclin D1 isoform termed cyclin D1b, which has the same N terminus as the canonical cyclin D1a isoform but a distinct C terminus. In this study we show that these different isoforms have unique properties with regard to the cellular migration function of cyclin D1. Although they displayed little difference in transcriptional co-repression assays on idealized reporter genes, microarray cDNA expression analysis revealed differential regulation of genes, including those that influence cellular migration. Additionally, whereas cyclin D1a stabilized p27 KIP1 and inhibited RhoA-induced ROCK kinase activity, promoting cellular migration, cyclin D1b failed to stabilize p27 KIP1 or inhibit ROCK kinase activity and had no effect on migration. Our findings argue that alternate splicing is an important determinant of the function of cyclin D1 in cellular migration.

Topics: Alternative Splicing; Animals; Cell Line, Tumor; Cell Movement; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Gene Expression Regulation; Humans; Mice; Neoplasms; NIH 3T3 Cells; Polymorphism, Genetic; rhoA GTP-Binding Protein; RNA; RNA, Messenger

Arctiin is a major lignan constituent of Arctium lappa and has anti-cancer properties in animal models. It was recently reported that arctiin induces growth inhibition in human prostate cancer PC-3 cells. However, the growth inhibitory mechanism of arctiin remains unknown. Herein we report that arctiin induces growth inhibition and dephosphorylates the tumor-suppressor retinoblastoma protein in human immortalized keratinocyte HaCaT cells. We also show that the growth inhibition caused by arctiin is associated with the down-regulation of cyclin D1 protein expression. Furthermore, the arctiin-induced suppression of cyclin D1 protein expression occurs in various types of human tumor cells, including osteosarcoma, lung, colorectal, cervical and breast cancer, melanoma, transformed renal cells and prostate cancer. Depletion of the cyclin D1 protein using small interfering RNA-rendered human breast cancer MCF-7 cells insensitive to the growth inhibitory effects of arctiin, implicates cyclin D1 as an important target of arctiin. Taken together, these results suggest that arctiin down-regulates cyclin D1 protein expression and that this at least partially contributes to the anti-proliferative effect of arctiin.

Topics: Antineoplastic Agents, Phytogenic; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Furans; Glucosides; Humans; Neoplasms; Retinoblastoma Protein

Thiazolidinediones are oral antidiabetic agents that activate peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and exert potent antioxidant and anti-inflammatory properties. It has also been shown that PPAR-gamma agonists induce G0/G1 arrest and apoptosis of malignant cells. Some of these effects have been suggested to result from inhibition of proteasome activity in target cells. The aim of our studies was to critically evaluate the cytostatic/cytotoxic effects of one of thiazolidinediones (pioglitazone) and its influence on proteasome activity. Pioglitazone exerted dose-dependent cytostatic/cytotoxic effects in MIA PaCa-2 cells. Incubation of tumor cells with pioglitazone resulted in increased levels of p53 and p27 and decreased levels of cyclin D1. Accumulation of polyubiquitinated proteins within cells incubated with pioglitazone suggested dysfunction of proteasome activity. However, we did not observe any influence of pioglitazone on the activity of isolated proteasome and on the proteolytic activity in lysates of pioglitazone-treated MIA PaCa-2 cells. Further, treatment with pioglitazone did not cause an accumulation of fluorescent proteasome substrates in transfected HeLa cells expressing unstable GFP variants. Our results indicate that pioglitazone does not act as a direct or indirect proteasome inhibitor.

Topics: Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cytostatic Agents; Dose-Response Relationship, Drug; HeLa Cells; Humans; Hypoglycemic Agents; Neoplasms; Pioglitazone; PPAR gamma; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Thiazolidinediones; Tumor Suppressor Protein p53

Pre-mRNA splicing and polyadenylation are tightly connected to transcription, and transcriptional stimuli and elongation dynamics can affect mRNA maturation. However, whether this regulatory mechanism has a physio/pathological impact is not known. In cancer, where splice variant expression is often deregulated, many mutated oncogenes are transcriptional regulators. In particular, the Ewing sarcoma (EwSa) oncogene, resulting from a fusion of the EWS and FLI1 genes, encodes a well characterized transcription factor. EWS-FLI1 directly stimulates transcription of the CCND1 protooncogene encoding cyclin D1a and a less abundant but more oncogenic splice isoform, D1b. We show that, although both EWS and EWS-FLI1 enhance cyclin D1 gene expression, they regulate the D1b/D1a transcript ratio in an opposite manner. Detailed analyses of RNA polymerase dynamics along the gene and of the effects of an inhibitor of elongation show that EWS-FLI1 favors D1b isoform expression by decreasing the elongation rate, whereas EWS has opposite effects. As a result, the D1b/D1a ratio is elevated in EwSa cell lines and tumors. The endogenous D1b protein is enriched in nuclei, where the oncogenic activity of cyclin D1 is known to occur, and depleting D1b in addition to D1a results in a stronger reduction of EwSa cell growth than depleting D1a only. These data show that elevated expression of a splice isoform in cancer can be due to an alteration of the transcription process by a mutated transcriptional regulator and provide evidence for a physio/pathological impact of the coupling between transcription and mRNA maturation.

Topics: Alternative Splicing; Bone Neoplasms; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Oncogene Proteins, Fusion; Protein Isoforms; Proto-Oncogene Protein c-fli-1; RNA-Binding Protein EWS; RNA, Messenger; Sarcoma, Ewing; Transcription, Genetic; Up-Regulation

The mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), is a rate-limiting factor of cap-dependent translation initiation. When elevated, eIF4E greatly facilitates translation of a selected spectrum of mRNAs coding for proteins critical to angiogenesis and growth such as vascular endothelial growth factor (VEGF) and cyclin D1. Expression levels of eIF4E, VEGF, and cyclin D1 were examined in multi-tumor tissue microarray by immunohistochemistry and analyzed quantitatively. eIF4E, VEGF and cyclin D1 protein were elevated in tumors of the breast (62, 78, or 40%), colon (72, 77, or 12%), glioblastoma multiforme (48, 68, or 52%), lymphoma (66, 74, or 38%), melanoma (59, 73, or 58%), NSCLC (81, 82, or 29%), ovary (50, 39, or 13%), and prostate (78, 97, or 21%), respectively. eIF4E levels were strongly correlated with VEGF and cyclin D1 in melanoma (Spearman's r=0.97 and 0.77; all P<0.0001); moderately in tumors of the breast (r=0.55 and 0.41; all P<0.0005), colon (0.63 and 0.56; all P<0.0001), lung (0.53 and 0.53; all P<0.005), lymphoma (0.50 and 0.61; all P<0.0005), prostate (0.46 and 0.54; all P<0.005), or ovary (0.56 and 0.46; all P<0.005); and weakly in tumors of glioblastoma multiforme (r=0.20 and 0.31; all P>0.15). The significant association of eIF4E with VEGF and cyclin D1 in multiple tumors supports a role for eIF4E in translational regulation of proteins related to angiogenesis and growth.

Topics: Automation; Cell Line, Tumor; Cyclin D1; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Neoplasms; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Protein Biosynthesis; Vascular Endothelial Growth Factor A

Mutations of the neurofibromatosis 2 (NF2) tumor suppressor gene have frequently been detected not only in schwannomas and other central nervous system tumors of NF2 patients but also in their sporadic counterparts and malignant tumors unrelated to the NF2 syndrome such as malignant mesothelioma, indicating a broader role for the NF2 gene in human tumorigenesis. However, the mechanisms by which the NF2 product, merlin or schwannomin, is regulated and controls cell proliferation remain elusive. Here, we identify a novel GTP-binding protein, dubbed NGB (referring to NF2-associated GTP binding protein), which binds to merlin. NGB is highly conserved between Saccharomyces cerevisiae, Caenorhabditis elegans, and human cells, and its GTP-binding region is very similar to those found in R-ras and Rap2. However, ectopic expression of NGB inhibits cell growth, cell aggregation, and tumorigenicity in tumorigenic schwanomma cells. Down-regulation and infrequent mutation of NGB were detected in human glioma cell lines and primary tumors. The interaction of NGB with merlin impairs the turnover of merlin, yet merlin does not affect the GTPase nor GTP-binding activity of NGB. Finally, the tumor suppressor functions of NGB require merlin and are linked to its ability to suppress cyclin D1 expression. Collectively, these findings indicate that NGB is a tumor suppressor that regulates and requires merlin to suppress cell proliferation.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Adhesion; Cell Movement; Cell Proliferation; Conserved Sequence; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; Mice; Molecular Sequence Data; Neoplasms; Neurofibromin 2; Protein Binding; Rats; Sequence Alignment; Tumor Suppressor Proteins; Two-Hybrid System Techniques; Ubiquitin

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Benzamides; Cyclin D1; Erlotinib Hydrochloride; Gefitinib; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, bcl-1; Humans; Imatinib Mesylate; Mutation; Neoplasms; Oncogenes; Piperazines; Protein Kinase Inhibitors; Pyrimidines; Quinazolines; Signal Transduction; Trastuzumab

Recent evidence indicates that growth hormone-releasing hormone (GHRH) functions as an autocrine/paracrine growth factor for various human cancers. A splice variant (SV) of the full-length receptor for GHRH (GHRHR) is widely expressed in various primary human cancers and established cancer cell lines and appears to mediate the proliferative effects of GHRH. To investigate in greater detail the role of SV1 in tumorigenesis, we have expressed the full-length GHRHR and its SV1 in MCF-7 human breast cancer cells that do not possess either GHRHR or SV1. In accordance with previous findings, the expression of both GHRHR and SV1 restored the sensitivity to GHRH-induced stimulation of cell proliferation, with SV1 being more potent than the GHRHR. Furthermore, MCF-7 cells transfected with SV1 proliferated more quickly than the controls, even in the absence of exogenously added GHRH, suggesting the existence of intrinsic, ligand-independent activity of SV1 after its transfection. In agreement with the stimulation of cell proliferation, the levels of proliferation markers cyclin D1, cyclin E, and proliferating cell nuclear antigen were elevated in MCF-7 cells treated with GHRH, cultured in both serum-free and serum-containing media. In addition, SV1 caused a considerable stimulation of the ability of MCF-7 cells to grow in semisolid medium, an assay considered diagnostic for cell transformation. Collectively, our findings show that the expression of SV1 confers oncogenic activity and provide further evidence that GHRH operates as a growth factor in breast cancer and probably other cancers as well.

Topics: Agar; Alternative Splicing; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Neoplasms; Receptors, Neuropeptide; Receptors, Pituitary Hormone-Regulating Hormone; Transfection

It is widely accepted that increased levels of reactive oxygen species (ROS) contribute to carcinogenesis. However, this claim has not been confirmed by experiments. On the contrary, a growing number of studies clearly demonstrate that ROS are normal cellular signals and induce cell differentiation and apoptosis, the opposite processes to cancer, which is dedifferentiated. Thus, it is hypothesized here that decreased levels of ROS may lead to cancer development, which is supported by following observations: (1) the fast-growing tumor produces ROS at a rate only one-third of the rate found with the control liver mitochondria; (2) the reduction in tumor mitochondrial content indicates low level of ROS production; (3) the low levels of manganese superoxide dismutase in tumor mitochondria also indicate decreased production of ROS, because the enzyme activity is induced by ROS; (4) lipid peroxidation capacity was decreased in human colon carcinomas and Yoshida hepatomas; (5) low levels of lipid peroxidation de-inhibit glucose-6-phosphate dehydrogenase, whose activity is always increased in a variety of cancers without exception. Clarification of real role of ROS in cancer may shed light on the understanding of how impairment of mitochondria leads to malignant transformation of normal cells, and offer new types of strategies for cancer prevention and therapy.

Topics: Colonic Neoplasms; Cyclin D1; Glucosephosphate Dehydrogenase; Humans; Lipid Peroxidation; Mitochondria; Models, Biological; Models, Theoretical; Neoplasms; Oxidation-Reduction; Reactive Oxygen Species; Superoxide Dismutase

Factors that mediate p53 tumor suppressor activity remain largely unknown. In this study we describe a systematic approach to identify downstream mediators of tumor suppressor function of p53, consisting of global gene expression profiling, focused short hairpin RNA (shRNA) library creation, and functional selection of genetic elements cooperating with oncogenic Ras in cell transformation. This approach is based on our finding that repression of gene expression is a major event, occurring in response to p53 inactivation during transformation and immortalization of primary cells. Functional analysis of the subset of genes universally down-regulated in the cells that lacked functional p53 revealed BTG2 as a major downstream effector of p53-dependent proliferation arrest of mouse and human fibroblasts transduced with oncogenic Ras. shRNA-mediated knockdown of BTG2 cooperates with oncogenic Ras to transform primary mouse fibroblasts containing wild-type transcriptionally active p53. Repression of BTG2 results in up-regulation of cyclins D1 and E1 and phosphorylation of Rb and, in cooperation with other oncogenic elements, induces neoplastic transformation of primary human fibroblasts. BTG2 expression was found to be significantly reduced in a large proportion of human kidney and breast carcinomas, suggesting that BTG2 is a tumor suppressor that links p53 and Rb pathways in human tumorigenesis.

Topics: Animals; Cell Transformation, Neoplastic; Cyclin D1; Cyclin E; Female; Fibroblasts; Gene Library; Genes, ras; Genes, Retinoblastoma; Genes, Tumor Suppressor; Humans; Immediate-Early Proteins; Mice; Models, Biological; Neoplasms; Oncogene Proteins; Phosphorylation; Protein Array Analysis; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Antibodies, especially monoclonal antibodies, are highly specific for their target antigens and have found extensive clinical application in the treatment of infectious diseases and neoplasia. However, they have a major shortcoming which, if overcome, would greatly expand their utility: an inability to penetrate the outer membrane of cells and act on intracellular targets. We demonstrated previously that this deficiency could be overcome by covalent linkage of an oligoarginine sequence to the conserved carbohydrate moiety present in the CH2 region of immunoglobulins. Immune specificity was maintained but no attempt was made to test for biological activity related to specificity. Here, we report that a polyarginated monoclonal anti-cyclin D1 enters cells and inhibits cell cycle progression. We demonstrate this with NIH 3T3 cells and with two tumor cell lines, HT29 and SW480. As many tumors overexpress cyclin D1, an intracellular anti-cyclin D1, properly targeted, has the potential to be a novel broad range inhibitor of tumor cell multiplication. Moreover, success with intracellular anti-cyclin D1 suggests that polyarginated antibodies, in general, could be a new, widely applicable experimental tool to investigate and influence intracellular processes, whether native to cells or introduced into cells by outside entities such as viruses.

Topics: Animals; Antibodies, Monoclonal; Cell Cycle; Cell Proliferation; Cyclin D1; Drug Delivery Systems; HT29 Cells; Humans; Immunoenzyme Techniques; Mice; Neoplasms; NIH 3T3 Cells; Peptides; Tumor Cells, Cultured

ATF3 is a stress-inducible gene that encodes a member of the ATF/CREB family of transcription factors. Current literature indicates that ATF3 affects cell death and cell cycle progression. However, controversies exist, because it has been demonstrated to be a negative or positive regulator of these processes. We sought to study the roles of ATF3 in both cell death and cell cycle regulation in the same cell type using mouse fibroblasts. We show that ATF3 promotes apoptosis and cell cycle arrest. Fibroblasts deficient in ATF3 (ATF3(-/-)) were partially protected from UV-induced apoptosis, and fibroblasts ectopically expressing ATF3(-/-) under the tet-off system exhibited features characteristic of apoptosis upon ATF3 induction. Furthermore, ATF3(-/-) fibroblasts transitioned from G(2) to S phase more efficiently than the ATF3(+/+) fibroblasts, suggesting a growth arrest role of ATF3. Consistent with the growth arrest and pro-apoptotic roles of ATF3, ATF3(-) fibroblasts upon Ras transformation exhibited higher growth rate, produced more colonies in soft agar, and formed larger tumor upon xenograft injection than the ATF3(+/+) counterparts. ATF3(-/-) cells, either with or without Ras transformation, had increased Rb phosphorylation and higher levels of various cyclins. Significantly, ATF3 bound to the cyclin D1 promoter as shown by chromatin immunoprecipitation (ChIP) assay and repressed its transcription by a transcription assay. Taken together, our results indicate that ATF3 promotes cell death and cell arrest, and suppresses Ras-mediated tumorigenesis. Potential explanations for the controversy about the roles of ATF3 in cell cycle and cell death are discussed.

Topics: Activating Transcription Factor 3; Animals; Apoptosis; Bromodeoxyuridine; Cell Cycle; Cell Death; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; Chromatin Immunoprecipitation; Cyclin D1; Dose-Response Relationship, Drug; Fibroblasts; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Immunohistochemistry; MAP Kinase Kinase 4; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Transplantation; Neoplasms; Phosphorylation; Plasmids; ras Proteins; Retinoblastoma Protein; Retroviridae; S Phase; Time Factors; Transcription, Genetic; Ultraviolet Rays

alpha-Tocopherol succinate (TS), an analogue of vitamin E, has growth-inhibitory activity in a wide spectrum of in vitro and in vivo cancer models. Here, we report that modulation of oncogenic Ras is associated with TS activity. TS inhibits the proliferation and induces apoptosis of NIH3T3 cells stably transfected with oncogenic K-Ras and H-Ras, but not NIH3T3 cells expressing empty vector. TS treatment resulted in decreased Ras protein levels in oncogenic Ras expressing NIH3T3 cells but not in parental NIH3T3 cells. Treatment with TS suppressed the levels of phospho-Akt and phospho-Erk1/2 in oncogenic Ras expressing NIH3T3 cells. Overexpression of constitutively active phosphoinositide-3-kinase, Akt, and Mek1/2 significantly attenuated TS growth inhibition of oncogenic Ras-transformed NIH3T3 mouse fibroblast cell lines. In addition, transcriptional targets of oncogenic Ras such as c-Myc, cyclin D1, and E2F1 were down-regulated by TS in oncogenic Ras-expressing cells. The above TS effects on oncogenic Ras signaling were also observed in endogenous oncogenic K-Ras expressing HCT 116 (human colon cancer) and MDA-MB-231 (human breast cancer) cells. Taken together, these data show that TS down-regulation of the Ras signaling pathways that are mediated by Mek/Erk and phosphoinositide-3-kinase/Akt plays, at least in part, a critical role in TS inhibition of proliferation and survival of transformed cells. This data supports further investigation of the chemopreventive and therapeutic potential of TS in tumors that are dependent on activated Ras signaling and identifies phosphor-Erk and phosphor-Akt as potential biomarkers of TS activity.

Topics: Animals; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; E2F Transcription Factors; Humans; Mice; Neoplasms; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; ras Proteins; Signal Transduction; Tocopherols; Transcriptional Activation; Vitamin E

Resveratrol is a naturally occurring phytoalexin with antioxidant and antiinflammatory properties. Recent studies suggest that resveratrol possesses anticancer effects, although its mechanism of action is not well understood. We now show that resveratrol inhibits Src tyrosine kinase activity and thereby blocks constitutive signal transducer and activator of transcription 3 (Stat3) protein activation in malignant cells. Analyses of resveratrol-treated malignant cells harboring constitutively-active Stat3 reveal irreversible cell cycle arrest of v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), human breast (MDA-MB-231), pancreatic (Panc-1), and prostate carcinoma (DU145) cell lines at the G0-G1 phase or at the S phase of human breast cancer (MDA-MB-468) and pancreatic cancer (Colo-357) cells, and loss of viability due to apoptosis. By contrast, cells treated with resveratrol, but lacking aberrant Stat3 activity, show reversible growth arrest and minimal loss of viability. Moreover, in malignant cells harboring constitutively-active Stat3, including human prostate cancer DU145 cells and v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), resveratrol treatment represses Stat3-regulated cyclin D1 as well as Bcl-xL and Mcl-1 genes, suggesting that the antitumor cell activity of resveratrol is in part due to the blockade of Stat3-mediated dysregulation of growth and survival pathways. Our study is among the first to identify Src-Stat3 signaling as a target of resveratrol, further defining the mechanism of antitumor cell activity of resveratrol and raising its potential application in tumors with an activated Stat3 profile.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-X Protein; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Female; Gene Expression; Humans; Male; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Resveratrol; Signal Transduction; src-Family Kinases; STAT3 Transcription Factor; Stilbenes

Betulinic acid (BA) is a pentacyclic triterpene found in many plant species, among others in the bark of white birch Betula alba. BA was reported to display a wide range of biological effects, including antiviral, antiparasitic, antibacterial and anti-inflammatory activities, and in particular to inhibit growth of cancer cells. The aim of the study was further in vitro characterization of BA anticancer activity. In this study, we demonstrated a remarkable antiproliferative effect of BA in all tested tumor cell cultures including neuroblastoma, rabdomyosarcoma-medulloblastoma, glioma, thyroid, breast, lung and colon carcinoma, leukemia and multiple myeloma, as well as in primary cultures isolated from ovarian carcinoma, cervical carcinoma and glioblastoma multiforme. Furthermore, we have shown that BA decreased cancer cell motility and induced apoptotic cell death. We also observed decrease of bcl2 and cyclin D1 genes expression, and increase of bax gene expression after betulinic acid treatment. These findings demonstrate the anticancer potential of betulinic acid and suggest that it may be taken into account as a supportive agent in the treatment of cancers with different tissue origin.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Betulinic Acid; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Dose-Response Relationship, Drug; Humans; Neoplasms; Pentacyclic Triterpenes; Proto-Oncogene Proteins c-bcl-2; Triterpenes

Loss of G(1)-S control and aberrations of the p16(Ink4a)-cyclin D1/cyclin-dependent kinase (CDK) 4(6)-pRb-E2F-cyclin E/CDK2 pathway are common in human cancer. Previous studies showed that oncogene-induced aberrant proliferation, such as on cyclin E overexpression, causes DNA damage and checkpoint activation. Here, we show that, in a series of human colorectal adenomas, those with deregulation of cyclin D1 and/or p16(Ink4a) showed little evidence of constitutive DNA damage response (DDR), contrary to cyclin E-overexpressing higher-grade cases. These observations were consistent with diverse cell culture models with differential defects of retinoblastoma pathway components, as overexpression of cyclin D1 or lack of p16(Ink4a), either alone or combined, did not elicit detectable DDR. In contrast, inactivation of pRb, the key component of the pathway, activated the DDR in cultured human or mouse cells, analogous to elevated cyclin E. These results highlight differential effect of diverse oncogenic events on driving the 'cancer cell cycles' and their ability to deregulate the replication-driving CDK2 kinase and to alarm the DDR as a potential anticancer barrier in accordance with their hierarchical positions along the retinoblastoma pathway. Our data provide new insights into oncogene-evoked DDR in human tumorigenesis, with potential implications for individualized management of tumors with elevated cyclin D1 versus cyclin E, due to their distinct clinical variables and biological behavior.

Topics: Cell Line, Tumor; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p16; DNA Damage; E2F1 Transcription Factor; Genes, p53; Humans; Neoplasms; Retinoblastoma Protein

Growth factor-dependent accumulation of the cyclin D1 proto-oncogene is balanced by its rapid phosphorylation-dependent proteolysis. Degradation is triggered by threonine 286 phosphorylation, which promotes its ubiquitination by an unknown E3 ligase. We demonstrate that Thr286-phosphorylated cyclin D1 is recognized by a Skp1-Cul1-F box (SCF) ubiquitin ligase where FBX4 and alphaB crystallin govern substrate specificity. Overexpression of FBX4 and alphaB crystallin triggered cyclin D1 ubiquitination and increased cyclin D1 turnover. Impairment of SCF(FBX4-alphaB crystallin) function attenuated cyclin D1 ubiquitination, promoting cyclin D1 overexpression and accelerated cell-cycle progression. Purified SCF(FBX4-alphaB crystallin) catalyzed polyubiquitination of cyclin D1 in vitro. Consistent with a putative role for a cyclin D1 E3 ligase in tumorigenesis, FBX4 and alphaB crystallin expression was reduced in tumor-derived cell lines and a subset of primary human cancers that overexpress cyclin D1. We conclude that SCF(FBX4-alphaB crystallin) is an E3 ubiquitin ligase that promotes ubiquitin-dependent degradation of Thr286-phosphorylated cyclin D1.

Topics: alpha-Crystallin B Chain; Animals; Catalysis; Cyclin D1; Cytoplasm; F-Box Proteins; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasms; NIH 3T3 Cells; Phosphorylation; Phosphothreonine; Protein Binding; Protein Transport; Proto-Oncogene Mas; RNA, Messenger; SKP Cullin F-Box Protein Ligases; Thermodynamics; Ubiquitin

Splicing of human cyclin D1 (CCND1) mRNA producing transcripts a and b is modulated by a common polymorphism (A --> G) located in a conserved splice donor region at nucleotide 870. CCND1 A/G(870) genotype is associated with tumour progression and clinical outcome in a variety of cancers. Although in vitro expression of cyclin D1 transcript a (CCND1(tra)) has been widely investigated, few studies have examined the expression of CCND1 transcript b (CCND1(trb)). We have studied the effects of inducible expression of human CCND1(trb) in comparison with human CCND1(tra) in a mouse fibroblast knock-out for cyclin D1 (MEF(Cyl-1-/-)). Inducible expression was in stable clones isolated from MEF(Cyl-1-/-) transfectants. Induction of CCND1(tra) produced a 36-kDa protein, which led to a significant increase in the proportion of cells in S-phase, as detected by BrdU incorporation after 32 hr, compared to non-induced cells (p = 0.012). Clones induced to express CCND1(tra) exhibited a significantly increased ability to grow in serum depleted (2% FCS) medium compared to non-induced clones (p = 0.0004). Induced expression of CCND1(trb) in MEF(Cyl-1-/-) transfectants produced a 31-kDa protein and resulted in no significant difference in DNA synthesis, neither did the cells acquire the ability to grow in serum-depleted conditions compared to non-induced cells. Induction of CCND1(trb) significantly enhanced the ability of MEF(Cyl-1-/-) transfectants to form colonies in soft agar, (average 30-fold increase) compared to non-induced clones or those induced to express CCND1(tra). Our data supports the emerging view that CCND1 alternate transcripts encode proteins with differing independent biological functions. We suggest that CCND1(tra) encodes a protein involved in regulating mitogen responsive, anchorage-dependent G(1) progression, whereas CCND1(trb) modulates the ability of the cell to grow in an anchorage-independent manner.

Topics: Animals; Cell Cycle; Cyclin D1; Disease Progression; DNA; Fibroblasts; Genotype; Humans; Mice; Mice, Knockout; Mitogens; Neoplasms; Polymorphism, Genetic; Transfection

The beta-catenin pathway plays a critical role in the pathogenesis of certain types of cancers. To gain insight into mechanisms by which altered receptor tyrosine kinases regulate cytoplasmic beta-catenin accumulation, the effect of an oncogenic receptor originated from Nantes (RON) variant on beta-catenin accumulation and the role of beta-catenin in RON-mediated tumorigenic activities were studied. In NIH3T3 cells harboring oncogenic variant RONDelta160, increased beta-catenin accumulation with tyrosine phosphorylation and nuclear translocation was observed. Overexpression of RONDelta160 also resulted in increased expression of beta-catenin target genes c-myc and cyclin D1. By analyzing cellular proteins that regulate beta-catenin stabilities, it was found that RONDelta160 activates the protein disheveled (DVL) and inactivates glycogen synthase kinase-3beta by Ser-9 residue phosphorylation. These effects were channeled by RONDelta160-activated PI 3-kinase-AKT pathways that are sensitive to specific inhibitors, such as wortmannin, but not to other chemical inhibitors. Silencing RONDelta160 expression by specific small interfering RNA blocked not only beta-catenin expression but also c-myc and cyclin D1 expression, suggesting that RON expression is required for the activation of the beta-catenin signaling pathway. Moreover, it was found that knockdown of the beta-catenin gene expression by small interfering RNA techniques reduces significantly the RONDelta160-mediated NIH3T3 cell proliferation, focus-forming activities and anchorage-independent growth. Thus, the oncogenic RON variant regulates beta-catenin stabilities through activation of DVL and inactivation of glycogen synthase kinase-3beta. The activated beta-catenin cascade is one of the pathways involved in tumorigenic activities mediated by the oncogenic RON variant.

Topics: Active Transport, Cell Nucleus; Alternative Splicing; Androstadienes; Animals; beta Catenin; Blotting, Western; Cell Nucleus; Cyclin D1; Cytoplasm; Cytoskeletal Proteins; Enzyme Inhibitors; Gene Silencing; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Mice; Models, Biological; Neoplasms; NIH 3T3 Cells; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-myc; Receptor Protein-Tyrosine Kinases; RNA, Messenger; RNA, Small Interfering; Serine; Trans-Activators; Tyrosine; Wortmannin

Rabbit monoclonal antibodies (RabMAbs) represent a novel category of immunoreagents that may combine the best properties of both mouse monoclonal antibodies (MMAs) and of rabbit antisera. In the attempt to verify the performance of this new class of antibodies on paraffin-embedded tissue, RabMAbs against estrogen receptor, progesterone receptor, Ki-67, cyclin D1, CD3, CD5, CD23, and synaptophysin were tested on several tumor types as well as normal tissues. The results were compared with those obtained with classic MMAs against the same antigens. RabMAbs appear to offer increased sensitivity with no apparent loss of specificity. On routine use they permit higher working dilutions (5 to 10 times on average), allowing significant improvement in terms of laboratory efficiency. The robustness of RabMAbs is further proved by the fact that in some instances optimal staining can be obtained even without antigen retrieval. In consideration of the high performance observed, routine use of RabMAbs may contribute significantly to standardize diagnostic immunohistochemical procedures.

Topics: Animals; Antibodies, Monoclonal; Biomarkers, Tumor; CD3 Complex; CD5 Antigens; Cyclin D1; Immunohistochemistry; Ki-67 Antigen; Mice; Neoplasms; Rabbits; Receptors, Estrogen; Receptors, IgE; Receptors, Progesterone; Sensitivity and Specificity; Synaptophysin

Previous studies have established constitutive activation of Stat3 protein as one of the molecular changes required for tumorigenesis. To develop novel therapeutics for tumors harboring constitutively active Stat3, compounds from the NCI 2000 diversity set were evaluated for inhibition of Stat3 DNA-binding activity in vitro. Of these, a novel platinum (IV) compound, IS3 295, interacted with Stat3 and inhibited its binding to specific DNA-response elements. Further analysis suggested noncompetitive-type kinetics for the inhibition of Stat3 binding to DNA. In human and mouse tumor cell lines with constitutively active Stat3, IS3 295 selectively attenuated Stat3 signaling, thereby inducing cell growth arrest at G0/G1 phase and apoptosis. Moreover, in transformed cells, IS3 295 repressed expression of cyclin D1 and bcl-xL, two of the known Stat3-regulated genes that are overexpressed in malignant cells, suggesting that IS3 295 mediates anti-tumor cell activity in part by blocking Stat3-mediated sub-version of cell growth and apoptotic signals. Together, our findings provide evidence for the inhibition of Stat3 activity and biological functions by IS3 295 through interaction with Stat3 protein. This study represents a significant advance in small molecule-based approaches to target Stat3 and suggests potential new applications for platinum (IV) complexes as modulators of the Stat3 pathway for cancer therapy.

Topics: Agar; Animals; Apoptosis; Blotting, Western; Bromodeoxyuridine; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cyclin D1; Cytosol; DNA; Fibroblasts; Flow Cytometry; G1 Phase; Gene Expression Regulation; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Ki-67 Antigen; Kinetics; Luciferases; Mice; Neoplasms; NIH 3T3 Cells; Oligonucleotides; Oncogene Protein pp60(v-src); Piperazines; Plasmids; Platinum; Platinum Compounds; Protein Binding; Resting Phase, Cell Cycle; Signal Transduction; Time Factors

A substantial number of genes mutated in human cancers encode components of the cell cycle processes. As the G1/S transition in the cell cycle is a finely regulated biological process, we hypothesized that sequence variations in the promoter region of the related genes might indeed lead to abnormal expression, thus predisposing the individuals carrying these genetic variants to cancer. In this report, we screened the promoter regions of 16 cell cycle checkpoint genes for DNA variants and assessed the functional impact of these promoter region single nucleotide polymorphisms (pSNPs) by combining in silico analysis and in vitro functional assays. We identified 127 pSNPs including 90 with predicted impact on putative binding sites of known transcription factors. Eleven pSNPs were selected for electrophoresis mobility shift assays because of their association with predicted gains of binding sites, and nine pSNPs showed differential allelic shifts in at least one cell line tested. Following the subcloning of the promoter regions into a gene reporter system, we found that at least four promoter haplotypes associated with CCND1, E2F1, HDAC1 and RB1 significantly influenced transcriptional activity in an allele-specific manner. Although the biological significance of these observations still remains to be demonstrated, the expected variability of expression levels in key cell cycle components might influence individual's risk of cancer.

Topics: Binding Sites; Cloning, Molecular; Cyclin D1; E2F1 Transcription Factor; Electrophoretic Mobility Shift Assay; Gene Expression Regulation, Neoplastic; Genes, cdc; Haplotypes; Histone Deacetylase 1; Histone Deacetylases; Humans; Neoplasms; Oligonucleotides; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Retinoblastoma Protein; Transcription Factors

The development and progression of epithelial cancers are the result of an imbalance in signals promoting and inhibiting cellular proliferation and apoptosis. The aim of this study is to evaluate the expression of cell-cycle and apoptosis regulators and correlate them with clinical outcome in the most frequent carcinomas, in order to establish common prognostic biomarkers independent of cancer origin. Using tissue microarrays (TMAs), we have analysed the immuno-expression of Ki-67, Bcl-2, Bax, cyclin D1, cyclin D3, CDK1, CDK2, CDK6, p16, p21, and p27 in a series of 205 carcinomas of the large bowel, breast, lung and prostate (80, 73, 37 and 15 cases, respectively). By univariate analysis, positivity for p27, p16 and Bcl-2 was associated with better overall survival (P<0.0135, P<0.0442 and P<0.0001, respectively). The risk of mortality was 2.3-fold greater in patients without Bcl-2 expression. TMA immunohistochemical analysis identified a subset of epithelial cancers with overlapping alterations in cell-cycle checkpoints, apoptosis regulators and tumour suppressor pathways. We found that in most common epithelial cancers, regardless of origin, Bcl-2 appears to be the key biological factor influencing clinical behaviour.

Topics: Adult; Analysis of Variance; bcl-2-Associated X Protein; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle Proteins; Cyclin D1; Cyclin D3; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Female; Humans; Immunohistochemistry; Intestinal Neoplasms; Ki-67 Antigen; Lung Neoplasms; Male; Middle Aged; Neoplasms; Prognosis; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Survival Analysis; Tissue Array Analysis

Vascular endothelial growth factor (VEGF) and semaphorin-3A (Sema-3A) play important roles in the transduction of promitotic and antimitotic signals, respectively. Here, we report that these conflicting signals are integrated via negative feedback between VEGF and Sema-3A pathways in several primary normal, but not malignant, mesothelial cells. Unlike malignant mesothelial (MM) cells, in which VEGF induces cell proliferation, normal mesothelial (NM) cell growth was repressed by VEGF. Although both cell-types expressed an overlapping set of VEGF tyrosine-kinase receptors, only in NM cells VEGF exposure entails a p38 mitogen-activated protein kinase (MAPK)-dependent increased of Sema-3A production. Inhibition of p38 MAPK (by SB202190 and SB203580) or a dominant-negative mutant of Sema-3A receptor plexin-A1 reversed the inhibitory effects of VEGF in NM cells, increasing cyclin D1 synthesis and cell growth. Conversely, sustained activation of p38 MAPK by the p38 MAPK-activating kinases MKK3 and MKK6 or transfection with Sema-3A inhibited VEGF-induced cyclin D1 up-regulation and MM cell proliferation. Therefore, these results delineate a new role of Sema-3A in VEGF function mediated by p38 MAPK and suggest that the abrogation of regulated Sema-3A expression is responsible for VEGF-driven growth of tumor cells.

Topics: Cell Division; Cell Line; Cell Line, Tumor; Cyclin D1; Enzyme Activation; Epithelial Cells; Feedback, Physiological; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Models, Biological; Neoplasms; p38 Mitogen-Activated Protein Kinases; Semaphorin-3A; Up-Regulation; Vascular Endothelial Growth Factor A

The role of RASSF1A has been elucidated recently in regulating apoptosis and cell cycle progression by inhibiting cyclin D1 accumulation. Aberrant RASSF1A promoter methylation has been found frequently in multiple adult cancer types. Using methylation-specific PCR and reverse transcription-PCR, we investigated epigenetic deregulation of RASSF1A in primary tumors, adjacent nontumor tissues, secondary metastases, peripheral blood cells, and plasma samples from children with 18 different cancer types, in association with their clinicopathologic features.. Regardless of the tumor size, ubiquitous RASSF1A promoter methylation was found in 67% (16 of 24) of pediatric tumors, including neuroblastoma, thyroid carcinoma, hepatocellular carcinoma, pancreatoblastoma, adrenocortical carcinoma, Wilms' tumor, Burkitt's lymphoma, and T-cell lymphoma. A majority (75%) of pediatric cancer patients with tumoral RASSF1A methylation was male. Methylated RASSF1A alleles were also detected in 4 of 13 adjacent nontumor tissues, suggesting that this epigenetic change is potentially an early and critical event in childhood neoplasia. RASSF1A promoter methylation found in 92% (11 of 12) of cell lines largely derived from pediatric cancer patients was significantly associated with transcriptional silencing/repression. After demethylation treatment with 5-aza-2'-deoxycytidine, transcriptional reactivation was shown in KELLY, RD, and Namalwa cell lines as analyzed by reverse transcription-PCR. For the first time, RASSF1A methylation was detected in 54% (7 of 13), 40% (4 of 10), and 9% (1 of 11) of buffy coat samples collected before, during, and after treatment, correspondingly, from pediatric patients with neuroblastoma, thyroid carcinoma, hepatocellular carcinoma, rhabdomyosarcoma, Burkitt's lymphoma, T-cell lymphoma, or acute lymphoblastic leukemia. Concordantly, RASSF1A methylation was found during treatment in plasma of the same patients, suggesting cell death and good response to chemotherapy.. RASSF1A methylation in tumor or buffy coat did not correlate strongly with age, tumor size, recurrence/metastasis, or overall survival in this cohort of pediatric cancer patients. Of importance, epigenetic inactivation of RASSF1A may potentially be crucial in pediatric tumor initiation.

Topics: Adolescent; Alleles; Cell Line; Cell Line, Tumor; Child; Child, Preschool; Cyclin D1; DNA Methylation; Female; Humans; Infant; Infant, Newborn; Male; Neoplasm Metastasis; Neoplasms; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA; Tumor Suppressor Proteins

The proto-oncogene cyclin D1 has been implicated in the genesis of a large proportion of human tumors from diverse histological origins. It has long been assumed that the action of cyclin D1, as an activator of cdk4 and cdk6 and leading to progression through the G1 phase of the cell cycle, underlies its pathological activity. But, more recently, analyses of the patterns of gene expression in human cancer have revealed a previously unappreciated mechanism of action for cyclin D1, suggesting that both cdk-dependent and cdk-independent activities might contribute to tumorigenesis. The development of therapeutics designed to target the aberrant activity of cyclin D1 in human cancers will rely upon an intimate molecular understanding of these distinct mechanisms of actions and their relative importance. Here, we describe the known functions of the cyclin D1 oncogene and delineate the evidence that cdk-independent actions are important for cyclin D1-mediated oncogenesis.

Topics: Animals; Cell Culture Techniques; Cell Cycle; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinases; G1 Phase; Humans; Mice; Models, Biological; Neoplasms; Proto-Oncogene Mas; Proto-Oncogene Proteins

Topics: Anticarcinogenic Agents; Cyclin D1; Cyclin E; Humans; Models, Biological; Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Receptors, Retinoic Acid; Retinoblastoma Protein; Ubiquitin

Estrogen receptor alpha (ERalpha) mediates 17beta-estradiol (E2) actions through the transcription of E2-sensitive target genes. In addition, rapid non-genomic signaling (e.g., MAPK/ERK) occurs. It is now well accepted that these rapid membrane-initiated responses account for E2-related cancer. Beside many beneficial effects on human health, nutritional flavonoids exert protective and anticarcinogenic effects on E2-related cancer. The mechanism underlying these effects seems to be related to flavonoids antioxidant properties and/or to their ability to alter signal transduction protein kinases. In addition, an antiestrogenic activity has been proposed but not yet defined. However, the identification and characterization of the responsible mechanisms for flavonoid antitumoral effects is poorly understood. Here, we investigated the possibility that the antimitogenic effects of flavonoids are transduced by modulating ERalpha-mediated rapid signaling. The ability of two flavonoids, the flavanone naringenin and the flavanol quercetin, with respect of E2, to induce ERalpha activities has been studied in the human cervix epitheloid carcinoma cell line (HeLa) devoid of any estrogen receptors and rendered E2-sensitive by transient transfection with a human ERalpha expression vector. Our results indicate that flavonoids act as E2 mimetic on ERalpha transcriptional activity, whereas they impair the activation of rapid signaling pathways committed to E2-induced proliferation. The resulting decoupling of ERalpha signal transduction could be proposed as a new mechanism in the protective effects of flavonoids against E2-related cancer.

Topics: Cyclin D1; Dose-Response Relationship, Drug; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Flavanones; HeLa Cells; Humans; Neoplasms; Promoter Regions, Genetic; Quercetin; Time Factors

A(3) adenosine receptor (A(3)AR) activation was shown to inhibit the growth of various tumor cells via the down-regulation of nuclear factor kappaB and cyclin D1. To additionally elucidate whether A(3)AR is a specific target, a survey of its expression in tumor versus adjacent normal cells was conducted.. A(3)AR mRNA expression in various tumor tissues was tested in paraffin-embedded slides using reverse transcription-PCR analysis. A comparison with A(3)AR expression in the relevant adjacent normal tissue or regional lymph node metastasis was performed. In addition, A(3)AR protein expression was studied in fresh tumors and was correlated with that of the adjacent normal tissue.. Reverse transcription-PCR analysis of colon and breast carcinoma tissues showed higher A(3)AR expression in the tumor versus adjacent non-neoplastic tissue or normal tissue. Additional analysis revealed that the lymph node metastasis expressed even more A(3)AR mRNA than the primary tumor tissue. Protein analysis of A(3)AR expression in fresh tumors derived from colon (n = 40) or breast (n = 17) revealed that 61% and 78% had higher A(3)AR expression in the tumor versus normal adjacent tissue, respectively. The high A(3)AR expression level in the tumor tissues was associated with elevated nuclear factor kappaB and cyclin D1 levels. High A(3)AR mRNA expression was also demonstrated in other solid tumor types.. Primary and metastatic tumor tissues highly express A(3)AR indicating that high receptor expression is a characteristic of solid tumors. These findings and our previous data suggest A(3)AR as a potential target for tumor growth inhibition.

Topics: Blotting, Western; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Colonic Neoplasms; Cyclin D1; Down-Regulation; Humans; Lung Neoplasms; Lymphatic Metastasis; Melanoma; Neoplasm Metastasis; Neoplasms; NF-kappa B; Receptor, Adenosine A3; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase.

Topics: Animals; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Flow Cytometry; G1 Phase; Genes, Reporter; GTP-Binding Proteins; Humans; Immunoblotting; Mice; Microfilament Proteins; Microscopy, Fluorescence; Mitogen-Activated Protein Kinases; Models, Biological; Muscle Proteins; Neoplasm Proteins; Neoplasms; NIH 3T3 Cells; Oncogene Proteins; Plasmids; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Proto-Oncogene Proteins c-vav; Receptors for Activated C Kinase; Receptors, Cell Surface; Retinoblastoma Protein; S Phase; Signal Transduction; src-Family Kinases; Transfection

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Culture Techniques; Chemokine CXCL12; Chemokines, CXC; Cyclin D1; Drug Screening Assays, Antitumor; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Oncogenes; Stromal Cells

GSK-3beta-dependent phosphorylation of cyclin D1 at a conserved C-terminal residue, Thr-286, promotes CRM1-dependent cyclin D1 nuclear export. Herein, we have identified a short stretch of residues adjacent to Thr-286 that mediates CRM1 association and thus cyclin D1 nuclear export. We found that disruption of this hydrophobic patch, stretching from amino acids 290 to 295 within cyclin D1, results in constitutively nuclear cyclin D1-CDK4 complexes with an increased propensity to potentiate transformation of murine fibroblasts. Our data support a model wherein deregulation of cyclin D1 nuclear export might contribute to human neoplastic growth.

Topics: Active Transport, Cell Nucleus; Amino Acid Sequence; Animals; Cell Proliferation; Cyclin D1; Epitopes; Exportin 1 Protein; Fibroblasts; Humans; Immunoblotting; Karyopherins; Mice; Microscopy, Fluorescence; Models, Biological; Molecular Sequence Data; Neoplasms; NIH 3T3 Cells; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear; Threonine; Time Factors

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are active in cancer therapy. Mechanisms engaged during these clinical responses need to be determined. We reported previously that epidermal growth factor stimulation markedly increased cyclin D1 protein expression in human bronchial epithelial (HBE) cells, and this was opposed by chemoprevention with all-trans-retinoic acid. The current study sought to determine whether the EGFR TKI erlotinib repressed cyclin D1 protein expression in immortalized HBE cells, lung cancer cell lines, and clinical aerodigestive tract cancers.. The BEAS-2B immortalized HBE cell line was exposed to varying concentrations of erlotinib, and effects on proliferation, cell cycle distribution, G1 cyclin expression, and cyclin D1 reporter activity were measured. Non-small-cell lung cancer cell lines were also evaluated for changes in proliferation and cyclin protein expression after erlotinib treatments. A proof of principle clinical trial was conducted. During this study, patients underwent a 9-day course of erlotinib treatment. Pretreatment and posttreatment tumor biopsies were obtained, and changes in candidate biomarkers were determined by immunostaining. Plasma pharmacokinetics and tumor tissue erlotinib concentrations were measured.. Erlotinib, at clinically achievable dosages, repressed BEAS-2B cell growth, triggered G1 arrest, and preferentially reduced cyclin D1 protein expression and transcriptional activation. Erlotinib also preferentially repressed proliferation and cyclin D1 protein expression in responsive, but not resistant, non-small-cell lung cancer cell lines. This occurred in the presence of wild-type EGFR sequence at exons 18, 19, and 21. Five patients were enrolled onto an erlotinib proof of principle clinical trial, and four cases were evaluable. Pharmacokinetic studies established therapeutic erlotinib plasma levels in all patients, but tissue levels exceeding 2 micromol/L were detected in only two cases. Notably, these cases had pathological evidence of response (necrosis) in posttreatment biopsies as compared with pretreatment biopsies. In these cases, marked repression of cyclin D1 and the proliferation marker Ki-67 was detected by immunohistochemical assays. Cases without pathological response to erlotinib did not exhibit changes in cyclin D1 or Ki-67 immunohistochemical expression and had much lower erlotinib tissue levels than did responding cases.. Taken together, these in vitro and in vivo findings provide direct evidence for repression of cyclin D1 protein as a surrogate marker of response in aerodigestive tract cancers to erlotinib treatment. These findings also provide a rationale for combining an EGFR TKI with an agent that would cooperatively repress cyclin D1 expression in clinical trials for aerodigestive tract cancer therapy or chemoprevention.

Topics: Biomarkers, Tumor; Bronchi; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Clinical Trials as Topic; Cyclin D1; DNA; Dose-Response Relationship, Drug; Epithelial Cells; ErbB Receptors; Erlotinib Hydrochloride; Exons; G1 Phase; Gastrointestinal Neoplasms; Gastrointestinal Tract; Humans; Immunoblotting; Immunohistochemistry; Ki-67 Antigen; Kinetics; Luciferases; Necrosis; Neoplasms; Quinazolines; Sequence Analysis, DNA; Time Factors; Transcriptional Activation

Phosphorylation of proteins on serine or threonine residues preceding proline (Ser/Thr-Pro) is a major intracellular signaling mechanism. The phosphorylated Ser/Thr-Pro motifs in a certain subset of phosphoproteins are isomerized specifically by the peptidyl-prolyl cis-trans isomerase Pin1. This post-phosphorylation isomerization can lead to conformational changes in the substrate proteins and modulate their functions. Pin1 interacts with a number of mitotic phosphoproteins, and plays a critical role in mitotic regulation. Recent work indicates that Pin1 is overexpressed in many human cancers and plays an important role in oncogenesis. Pin1 regulates the expression of cyclin D1 by cooperating with Ras signaling and inhibiting the interaction of beta-catenin with the tumor suppressor APC and also directly stabilizing cyclin D1 protein. Furthermore, PIN1 is an E2F target gene essential for the Neu/Ras-induced transformation of mammary epithelial cells. Pin1 is also a critical regulator of the tumor suppressor p53 during DNA damage response. Given its role in cell growth control and oncogenesis, Pin1 could represent a new anti-cancer target.

Topics: Animals; Antineoplastic Agents; beta Catenin; Catalysis; Cyclin D1; Cytoskeletal Proteins; DNA Damage; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; NIMA-Interacting Peptidylprolyl Isomerase; Peptidylprolyl Isomerase; Signal Transduction; Trans-Activators; Tumor Suppressor Protein p53

Cyclin D1 is a proto-oncogene that functions by inactivation of the retinoblastoma tumor suppressor protein, RB. A common polymorphism in the cyclin D1 gene is associated with the production of an alternate transcript of cyclin D1, termed cyclin D1b. Both the polymorphism and the variant transcript are associated with increased risk for multiple cancers and the severity of a given cancer; however, the underlying activities of cyclin D1b have not been elucidated relative to the canonical cyclin D1a. Because cyclin D1b does not possess the threonine 286 phosphorylation site required for nuclear export and regulated degradation, it has been hypothesized to encode a stable nuclear protein that would constitutively inactivate the RB pathway. Surprisingly, we find that cyclin D1b protein does not inappropriately accumulate in cells and exhibits stability comparable to cyclin D1a. As expected, the cyclin D1b protein was constitutively localized in the nucleus, whereas cyclin D1a was exported to the cytoplasm in S-phase. Despite enhanced nuclear localization, we find that cyclin D1b is a poor catalyst of RB phosphorylation/inactivation. However, cyclin D1b potently induced cellular transformation in contrast to cyclin D1a. In summary, we demonstrate that cyclin D1b specifically disrupts contact inhibition in a manner distinct from cyclin D1a. These data reveal novel roles for d-type cyclins in tumorigenesis.

Topics: 3T3 Cells; Alleles; Alternative Splicing; Animals; Binding Sites; Cell Cycle; Cell Nucleus; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cytoplasm; DNA; Flow Cytometry; Green Fluorescent Proteins; Humans; Immunoblotting; Luminescent Proteins; Mice; Microscopy, Fluorescence; Models, Genetic; Neoplasms; Phosphorylation; Plasmids; Polymorphism, Genetic; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Mas; Proto-Oncogene Proteins; Retinoblastoma Protein; Threonine; Time Factors; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Here we describe how patterns of gene expression in human tumors have been deconvoluted to reveal a mechanism of action for the cyclin D1 oncogene. Computational analysis of the expression patterns of thousands of genes across hundreds of tumor specimens suggested that a transcription factor, C/EBPbeta/Nf-Il6, participates in the consequences of cyclin D1 overexpression. Functional analyses confirmed the involvement of C/EBPbeta in the regulation of genes affected by cyclin D1 and established this protein as an indispensable effector of a potentially important facet of cyclin D1 biology. This work demonstrates that tumor gene expression databases can be used to study the function of a human oncogene in situ.

Topics: CCAAT-Enhancer-Binding Protein-beta; Cyclin D1; Databases, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Neoplasms; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Statistics, Nonparametric; Tumor Cells, Cultured

Topics: Animals; Cell Cycle; Cyclin D1; Gene Expression Profiling; Humans; Neoplasms

Effects of gamma-tocopherol on the cell cycle and proliferation were examined in human prostate carcinoma, colorectal adenocarcinoma, and osteosarcoma cells. Many epidemiological studies have suggested an anticancer activity of vitamin E, yet mechanistic studies are sparse to date. Vitamin E consists of four tocopherols (alpha-, beta-, gamma-, delta-) and the corresponding tocotrienols. Because gamma-tocopherol is the predominant form of tocopherol found in the U.S. diet, while alpha-tocopherol is the form of vitamin E most readily found in dietary supplements, we compared physiologically relevant concentrations of these tocopherols and found a more significant growth inhibition effect for gamma- than for alpha-tocopherol. Flow cytometry analysis of gamma-tocopherol treated prostate carcinoma DU-145 cells showed decreased progression into the S-phase. This effect was associated with reduced DNA synthesis as measured by 5-bromo-2'-deoxy-uridine incorporation. Furthermore, Western-blot analysis of gamma-tocopherol treated cells showed decreased levels of cyclin D1 and cyclin E. Taken together, the results indicate that gamma-tocopherol inhibits cell cycle progression via reduction of cyclin D1 and cyclin E levels. Because gamma-tocopherol has a weaker antioxidant capacity than a-tocopherol and gamma-tocopherol more significantly inhibited cell proliferation as well as DNA synthesis than alpha-tocopherol, we suggest a non-antioxidant mechanism to be at the basis of this effect.

Topics: alpha-Tocopherol; Antineoplastic Agents; Caco-2 Cells; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; DNA, Neoplasm; Down-Regulation; gamma-Tocopherol; Humans; Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Proteins

Clinical responses to the HER1 (EGF receptor) inhibitors and HER2/neu/ErbB2 inhibitors correlate with high levels of receptor expression. However, a significant subset of patients with high receptor levels appear to be refractory to treatment. We have observed similar results in the 60 cell lines of the NCI Anti-Cancer Drug Screen using a panel of 11 selective HER1 inhibitors. As expected, low HER1-expressing cell lines were insensitive to HER1 inhibitors. In cell lines with high HER1 expression, low concentrations of HER1 inhibitors potently inhibit both HER1 phosphorylation and the mitogen-activated protein kinase (MAPK) pathway. However, this inhibition did not always correlate with cellular arrest. High HER1-expressing cell lines can be subdivided into two groups based on their sensitivity to HER1 inhibitors. In the sensitive group, receptor and growth inhibition was concordant and occurred at sub-micromolar concentrations of HER1 inhibitors. In the insensitive group, receptor inhibition occurred at a low concentration (< 1 microM) but concentrations that were ten times or higher were required for growth inhibition. Also, neither induction of p21 and cyclin D1 nor p53 status could explain the difference between sensitive and insensitive cells. Although EGF activated the MAPK pathway in all cell lines, only drug-sensitive cell lines responded to EGF (accelerated entry from G1 to S) and to HER1 inhibitors (G1 arrest) by changes in cell cycling. Furthermore, an EGF-dependent immortalized mammary epithelial cell line was extremely sensitive to a panel of HER1 inhibitors. We infer that independence from mitogen-mediated signaling confers insensitivity to HER1 inhibitors in a large subset of cancer cell lines.

Topics: Cell Cycle; Cell Line, Transformed; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Drug Resistance, Neoplasm; Enzyme Induction; Enzyme Inhibitors; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Signaling System; Neoplasm Proteins; Neoplasms; Pyrimidines; Quinazolines; Receptor, ErbB-2; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tyrphostins

We recently reported that overexpression of epidermal growth factor receptor (EGFR) positively correlated with radioresistance of murine carcinomas. Because cyclin D1 is a downstream sensor of EGFR activation, the present study investigated whether a relationship exists between the extent of cyclin D1 expression and in vivo radiocurability of murine tumors. We further investigated the influence of radiation on cyclin D1 expression and the expression of p27, an inhibitor of the cyclin D1 downstream pathway, as well as the relationship of these molecular determinants to cell proliferation and induced apoptosis in tumors exposed to radiation.. Cyclin D1 expression was assayed in nine carcinomas syngeneic to C3Hf/Kam mice using Western blot analysis. These tumors greatly differed in their radioresponse as assessed by TCD(50). The expression of cyclin D1 and p27 proteins was determined by Western blotting. Cell proliferative activity in tumors was determined by proliferating cell nuclear antigen (PCNA) immunochemistry. The effect of irradiation on the expression of cyclin D1 or p27 proteins and on PCNA positivity was determined in the radiosensitive OCa-I and in the radioresistant SCC-VII tumors.. Cyclin D1 expression varied among tumors by 40-fold, and its magnitude positively correlated with poorer tumor radioresponse (higher TCD(50) values). The level of cyclin D1 expression paralleled that of EGFR. A 15-Gy dose reduced constitutive expression of cyclin D1 in the radiosensitive OCa-I tumors, but had no influence on expression of cyclin D1 in the radioresistant SCC-VII tumors. In contrast, 15 Gy increased the expression of p27 in radiosensitive tumors and reduced it in radioresistant tumors. Radiation induced no significant apoptosis or change in the percentage of PCNA-positive (proliferating) cells in SCC-VII tumors with high cyclin D1 levels, but it induced significant apoptosis and a decrease in the percentage of proliferating cells in OCa-I tumors with low cyclin D1 expression.. Our findings show a positive correlation between cyclin D1 expression and tumor radioresistance. The expression of cyclin D1 and p27 was modified by radiation and was associated with cellular response to radiation, but this depended on the pretreatment level of cyclin D1 expression. These findings may have important clinical implications: The pretreatment assessment of cyclin D1 expression could serve as a useful predictor of radiotherapy outcome and assist in selecting an effective treatment modality.

Topics: Animals; Apoptosis; Biomarkers; Blotting, Western; Cell Division; Cyclin D1; ErbB Receptors; Humans; Mice; Mice, Inbred C3H; Microfilament Proteins; Muscle Proteins; Neoplasm Proteins; Neoplasms; Proliferating Cell Nuclear Antigen; Radiation Tolerance; Radiobiology

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Differentiation; Cyclin D1; Gene Expression; Genes, myc; Genes, ras; Genes, Tumor Suppressor; Humans; Mice; Mice, Transgenic; Mutation; Neoplasms; Oncogene Proteins; Oncogenes; Osteosarcoma; Phenotype; Tumor Cells, Cultured

Mot-2 protein is shown to interact with p53 and inhibit its transcriptional activation function. Mot-2 overexpressing stable clones of NIH 3T3 cells were malignantly transformed, however, they had a high level of expression of a p53 downstream gene, p21WAF1. The present study was undertaken to elucidate possible molecular mechanism(s) of such upregulation. An increased level of p21WAF1 expression was detected in stable transfectants although an exogenous reporter gene driven by p21WAF1 promoter exhibited lower activity in these cells suggesting that some post-transcriptional mechanism contributes to upregulation. Western analyses of transient and stable clones revealed that upregulation of p21WAF1 in stable NIH 3T3/mot-2 cells may be mediated by cyclin D1 and cdk-2.

Topics: 3T3 Cells; Animals; CDC2-CDC28 Kinases; Cell Cycle; Cell Line, Transformed; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Gene Expression Regulation, Neoplastic; HSP70 Heat-Shock Proteins; Mice; Neoplasms; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Transcription, Genetic; Tumor Suppressor Protein p53; Up-Regulation

Overexpression of the cyclin D1 oncogene and inactivation of the p53 tumor suppressor have both been implicated in substantial proportions of sporadic human breast cancers. Transgenic mice with cyclin D1 overexpression targeted to mammary tissue by the MMTV enhancer-promoter have been shown to develop mammary cancers. To investigate the relationship between pathways driven by cyclin D1 overexpression and p53 loss during the development of breast cancers, we crossed MMTV-cyclin D1 mice with p53 heterozygous null (p53+/-) mice. In such crossed mice, cyclin D1-driven mammary neoplasia would need to be substantially accelerated by p53 loss in order for mammary tumors to develop prior to the expected onset of non-mammary tumors characteristic of the p53-deficient background alone. Instead, in mice heterozygous or homozygous for p53 deficiency and simultaneously carrying the MMTV-cyclin D1 transgene, only tumors typically found in p53-deficient mice developed and mammary tumors were not observed. Interestingly, MMTV-cyclin D1/p53+/- mice appeared to develop these non-mammary tumors more rapidly than p53+/- mice, and a majority of the sampled non-mammary tumors from MMTV-cyclin D1/p53+/- mice showed 'ectopic' expression of the MMTV-driven cyclin D1 transgene. Within the constraints of possible genetic background effects and limited sensitivity due to the early emergence of non-mammary tumors, these observations provide no evidence that inactivation of p53 confers a major additional selective advantage to mammary cells overexpressing cyclin D1 in this animal model of human breast cancer. Interestingly, the results do raise the possibility that p53 inactivation might complement or cooperate with cyclin D1 deregulation during the development of some types of non-mammary tumors.

Topics: Animals; Cyclin D1; Female; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mammary Tumor Virus, Mouse; Mice; Mice, Knockout; Mice, Transgenic; Neoplasms; Tumor Suppressor Protein p53

Topics: Adult; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Female; Humans; Incidence; Japan; Kidney Transplantation; Male; Middle Aged; Neoplasm Staging; Neoplasms; Postoperative Complications; Rectal Neoplasms; Retrospective Studies; Tumor Suppressor Protein p53

Gene amplifications are common in many different tumor types and may confer diagnostic, prognostic, or therapeutic information for patient management. Tedious experiments are often required to determine which tumor types have amplifications of a specific oncogene. To facilitate rapid screening for molecular alterations in many different malignancies, a tissue microarray consisting of samples from 17 different tumor types was generated. Altogether, 397 individual tumors were arrayed in a single paraffin block. To determine whether results from the literature can be reproduced on minute tissue samples (diameter, 0.6 mm), amplification of three extensively studied oncogenes (CCND1, CMYC, and ERBB2) was analyzed in three fluorescence in situ hybridization experiments from consecutive sections cut from the tissue microarray. Amplification of CCND1 was found in breast, lung, head and neck, and bladder cancer, as well as in melanoma. ERBB2 was amplified in bladder, breast, colon, stomach, testis, and lung cancer. CMYC was amplified in breast, colon, kidney, lung, ovary, bladder, head and neck, and endometrial cancer. These results confirm and even extend existing data in the literature on such amplifications. In summary, we applied three fluorescence in situ hybridization experiments to analyze amplifications of three oncogenes in three x 397 tumors within a week. This demonstrates the power of using minute arrayed tissue specimens for tumor screening.

Topics: Cyclin D1; Gene Amplification; Genes, erbB-2; Genetic Testing; Humans; In Situ Hybridization, Fluorescence; Neoplasms; Proto-Oncogene Proteins c-myc; Sensitivity and Specificity; Time Factors

To investigate the expression of the genes encoding cyclin D1 and p21 in proliferative and non-proliferative cells, as demonstrated by the Ki67 antibody, and to correlate these findings with differentiation.. Immunohistochemistry and immunofluorescence double staining were performed on three breast cancers, two squamous cell cancers of the head and neck, and one ovarium cystadenocarcinoma. In addition, the in vitro effect of cyclin D1 on p21 gene expression in MCF7 breast cancer cells was evaluated.. Immunofluorescence double staining showed a differentiation related gradient in the detection of the Ki67 antigen, cyclin D1, and p21 in squamous cell cancers of the head and neck: Ki67 was detected in the basal layers of the tumour and the cyclin D1 and p21 genes were coexpressed in the higher, more differentiated layers of the tumour. The breast and ovarian cancers often had cells that coexpressed the p21 and cyclin D1 genes, whereas coexpression of cyclin D1 and Ki67 did not occur. Western blot analysis of the MCF7 breast cancer cells showed an upregulation of p21 production when cyclin D1 gene expression was induced.. Overexpression of the cyclin D1 gene seems to lead to growth arrest in a variety of human cancers, possibly through the induction of p21 by cyclin D1. In squamous cell cancer, concerted overexpression of the genes encoding cyclin D1 and p21 might also induce differentiation.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Female; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Neoplasm Proteins; Neoplasms; Tumor Cells, Cultured; Up-Regulation

The development of a malignant tumor involves the progressive acquisition of mutations and epigenetic abnormalities in multiple genes that have highly diverse functions. Some of these genes code for pathways of signal transduction that mediate the action of growth factors. The enzyme protein kinase C plays an important role in these events and in the process of tumor promotion. Therefore, we examined the effects of three inhibitors of protein kinase C, CGP 41251, RO 31-8220, and calphostin C, on human glioblastoma cells. These compounds inhibited growth and induced apoptosis; these activities were associated with a decrease in the level of CDC2 and cyclin B1/CDC2-associated kinase activity. This may explain why the treated cells accumulated in G2-M. In a separate series of studies, we examined abnormalities in cell cycle control genes in human cancer. We have found that cyclin D1 is frequently overexpressed in a variety of human cancers. Mechanistic studies indicate that cyclin D1 can play a critical role in carcinogenesis because: overexpression enhances cell transformation and tumorigenesis; introduction of an antisense cyclin D1 cDNA into either human esophageal or colon cancer cells reverts their malignant phenotype; and overexpression of cyclin D1 can enhance the amplification of other genes. The latter finding suggests that cyclin D1 can enhance genomic instability and, thereby, the process of tumor progression. Therefore, inhibitors of the function of cyclin D1 may be useful in both cancer chemoprevention and therapy. We obtained evidence for the existence of homeostatic feedback loops between cyclins D1 or E and the cell cycle inhibitory protein p27Kip1. On the basis of these and other findings, we hypothesize that, because of their disordered circuitry, cancer cells suffer from "gene addiction" and "gene hypersensitivity," disorders that might be exploited in both cancer prevention and therapy.

Topics: Apoptosis; Cell Cycle; Chemoprevention; Cyclin D1; Drug Design; Genetic Therapy; Humans; Models, Biological; Neoplasms; Oligonucleotides, Antisense

Although several oncogenes, including c-myc, ras and c-raf-1, have been implicated in cellular resistance to ionising radiation, there is less information relating oncogene expression to cis-diamminedichloroplatinum (CDDP) resistance. However, transfection of c-myc or v-H-ras and activation of protein kinase C (PKC), which contributes to the RAF-1, MAP kinase signal transduction pathway, can influence therapeutic response to CDDP. Activation of PKC increases CDDP sensitivity, whilst transfected c-myc or v-H-ras induce CDDP resistance. We have previously reported that human in vitro cell lines show different patterns of sensitivity to CDDP and 4 MeV X-irradiation. In these cells radiation sensitivity is related to high levels of expression of the C-raf-1 proto-oncogene. We thus predicted that cells sensitive to CDDP might show a different relationship to c-raf-1 expression. In addition, because cyclin D1 expression can be upregulated by the myc or ras oncogenes, we also chose to study putative relationships between cyclin D1 protein levels and intrinsic cellular sensitivity to CDDP and gamma-irradiation. We report that in the 16 human cell lines which we have studied, high cyclin D1 expression is related to CDDP resistance but has no relationship with radiation responsiveness, whereas high c-raf-1 expression, although related to radiosensitivity has no relationship with CDDP responsiveness.

Topics: Antineoplastic Agents; Cell Cycle; Cisplatin; Cyclin D1; Cyclins; Drug Resistance, Neoplasm; Gene Expression; Humans; Neoplasms; Oncogene Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Radiation-Sensitizing Agents; RNA, Messenger; Tumor Cells, Cultured

Topics: Animals; Antineoplastic Agents; Apoptosis; Carrier Proteins; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclins; DNA-Binding Proteins; E2F Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Oncogene Proteins; Retinoblastoma-Binding Protein 1; Tetrahydrofolate Dehydrogenase; Transcription Factor DP1; Transcription Factors; Up-Regulation

The p16INK4/CDKN2, D-type cyclins, their partner cyclin-dependent kinases, and retinoblastoma protein constitute a G1 regulatory pathway commonly targeted in oncogenesis. We show that, unexpectedly, abnormalities of p16INK4/CDKN2 occur concomitantly in two-thirds of cancer cell lines harboring aberrations of cyclin D1. Gene and protein transfer experiments demonstrated that concurrent alterations of cyclin D1 and p16 levels cooperate to (de)regulate G1 control in diploid fibroblasts, and that both events influence growth of retinoblastoma (RB)-positive, but not RB-deficient cancer cells. These results show that biological consequences of deregulating individual components along the pathway are unequal, reflecting their hierarchical roles in the G1 checkpoint control. Whereas RB defects eliminate the checkpoint completely, aberrations of the upstream components, such as cyclin D1 and p16INK4/CDKN2, can cooperate in multistep tumorigenesis.

Topics: Base Sequence; Carrier Proteins; Cell Division; Chromosome Aberrations; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclins; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Neoplasms; Oncogene Proteins; Tumor Cells, Cultured

Complexes of cyclin-dependent kinases (cdk) and their partner cyclins drive the cell through the cell cycle, each such complex phosphorylating a distinct set of proteins at a particular check-point or phase of the cycle. Immunocytochemical detection of cyclins combined with measurement of cellular DNA content by flow cytometry makes it possible to relate expression of each of these proteins with the actual cell cycle position, without the necessity of cell synchronization. In the present study, we have investigated expression of E and D type cyclins in G1 cells and in cells entering S phase, in eight different human hematopoietic and solid tumour cell lines (two leukaemias, a lymphoma, three breast carcinomas, a colon carcinoma and a bladder transitional cell carcinoma) during their exponential phase of growth, as well as in normal mitogen stimulated lymphocytes. In all the cell types studied, the average level of D type cyclin expression was invariable throughout the cell cycle. A great intercellular variability, in particular of the G1 cell subpopulations, and the presence of a large fraction of G1, S and G2 + M cells that were cyclin D negative (20-40% in tumour cell lines and about 80% among lymphocytes), were other characteristic features of D type cyclin expression. In contrast to D type cyclins, the expression of cyclin E was discontinuous during the cycle, peaking at the time of cell entrance to S. Also, a well defined threshold in expression of cyclin E characterized cells that were entering S phase, and virtually no cyclin E negative cells were seen during the early portion of S phase. The data indicate that while cell entrance to S phase is unrelated to expression of D type cyclins (at the time of entrance), accumulation of cyclin E up to critical level is a prerequisite for initiation of DNA replication. The great intercellular variability in expression of D type cyclins and their invariant average level across the cell cycle suggest that these cyclins, in addition to their acknowledged function in promoting cell progression through mid- to late-G1 may have other role(s), related or unrelated to the cell cycle progression. The presence of a large number of D type cyclin negative cells in all phases of the cycle suggests that during exponential growth the cells may not express this protein and yet may traverse the cycle, including G1 phase.

Topics: Blotting, Western; Cells, Cultured; Cyclin D1; Cyclin D3; Cyclins; Humans; In Vitro Techniques; Lymphocytes; Neoplasms; Oncogene Proteins; S Phase

Cyclin D1 is a cell cycle regulator essential for G1 phase progression and a candidate proto-oncogene whose deregulated expression has been implicated in pathogenesis of several types of cancer. We have examined expression of cyclin D1 in 212 primary tumours of five histogenetically distinct types by immunohistochemistry and found strong aberrant accumulation of the protein in 21%, and a moderate overabundance in further 25% of cases. While the abnormalities were more frequent in carcinomas of the breast, i.e. the cancer type known for cyclin D1 gene amplification, aberrant expression was also seen in significant subsets of colorectal cancers, soft tissue sarcomas, uterine carcinomas and malignant melanomas. Comparison of distinct stages of tumour progression showed concordant cyclin D1 patterns in the in situ vs invasive breast carcinoma components (n = 37) and between primary and metastatic lesions (n = 51) of several tumour types. The specificity of the immunohistochemical data was supported by immunoblotting analysis of tissue and tumour lysates, and the tumour-specific over-expression was confirmed by computer-assisted image analysis. These observations suggest that alterations of cyclin D1 expression represent a common feature of malignancies of diverse histogenesis and indicate that both the spectrum of tumour types and the frequency of cyclin D1 aberrations significantly exceed previous estimations based on genetic analyses.

Topics: Blotting, Western; Cell Cycle; Cyclin D1; Cyclins; Humans; Image Processing, Computer-Assisted; Immunoenzyme Techniques; Neoplasms; Oncogene Proteins; Proto-Oncogene Mas

Topics: Base Sequence; Carrier Proteins; Cell Line; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Cyclins; DNA Primers; Enzyme Inhibitors; Female; Gene Deletion; Gene Expression Regulation, Neoplastic; Genes, Retinoblastoma; Genes, Tumor Suppressor; Homozygote; Humans; Molecular Sequence Data; Neoplasms; Oncogene Proteins

Cell cycle arrest at the G1 checkpoint allows completion of critical macromolecular events prior to S phase. Regulators of the G1 checkpoint include an inhibitor of cyclin-dependent kinase, p16INK4; two tumor-suppressor proteins, p53 and RB (the product of the retinoblastoma-susceptibility gene); and cyclin D1. Neither p16INK4 nor the RB protein was detected in 28 of 29 tumor cell lines from human lung, esophagus, liver, colon, and pancreas. The presence of p16INK4 protein is inversely correlated with detectable RB or cyclin D1 proteins and is not correlated with p53 mutations. Homozygous deletions of p16INK4 were detected in several cell lines, but intragenic mutations of this gene were unusual in either cell lines or primary tumors. Transfection of the p16INK4 cDNA expression vector into carcinoma cells inhibits their colony-forming efficiency and the p16INK4 expressing cells are selected against with continued passage in vitro. These results are consistent with the hypothesis that p16INK4 is a tumor-suppressor protein and that genetic and epigenetic abnormalities in genes controlling the G1 checkpoint can lead to both escape from senescence and cancer formation.

Topics: Base Sequence; Carrier Proteins; Cell Cycle; Chromosomes, Human, Pair 9; Cloning, Molecular; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Cyclins; DNA Primers; DNA, Neoplasm; Gene Deletion; Genes; Genes, Retinoblastoma; Humans; In Situ Hybridization, Fluorescence; Molecular Sequence Data; Neoplasms; Oncogene Proteins; Polymorphism, Single-Stranded Conformational

Topics: Animals; Cell Cycle; Cell Differentiation; Cell Transformation, Neoplastic; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Cyclins; G1 Phase; Genes, Tumor Suppressor; Humans; Neoplasms; Oncogene Proteins; Oncogenes; Protein Kinases; Proto-Oncogene Proteins

Accumulating evidence indicates that the activation of cellular oncogenes is a cause of some human cancers. ErbB-1, erbB-2 and abl oncogenes encoding tyrosine kinases, ras oncogenes encoding GTP binding proteins and myc oncogenes whose functions are not well understood are some examples. Therefore, agents which inhibit the activity of these oncogene products may provide new means to overcome certain human tumors. Herbimycin A and tyrphostins have been found and developed as inhibitors of tyrosine kinases and the effectiveness of these agents against tumors of Ph1-positive leukemia (CML, ALL) or squamous cell carcinomas has been reported. Although specific inhibitors of ras or myc oncogene products have not yet been described, recent studies on the processing of Ras proteins toward the cell membrane provide a strategy to search for inhibitors of ras functions.

Topics: Antibiotics, Antineoplastic; Benzoquinones; Carcinoma, Squamous Cell; Catechols; Cyclin D1; Female; Genes, ras; Humans; Lactams, Macrocyclic; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Neoplasms; Nitriles; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Quinones; Rifabutin; Tyrphostins