cyclin-d1 and Multiple-Myeloma

Multiple myeloma (MM) is a hematological malignancy caused by the clonal expansion of plasma cells. The incidence of MM worldwide is increasing with greater than 140 000 people being diagnosed with MM per year. Whereas 5-year survival after a diagnosis of MM has improved from 28% in 1975 to 56% in 2012, the disease remains essentially incurable. In this review, we summarize our current understanding of MM including its epidemiology, genetics and biology. We will also provide an overview of MM management that has led to improvements in survival, including recent changes to diagnosis and therapies. Areas of unmet need include the management of patients with high-risk MM, those with reduced performance status and those refractory to standard therapies. Ongoing research into the biology and early detection of MM as well as the development of novel therapies, such as immunotherapies, has the potential to influence MM practice in the future.

Topics: Biomarkers, Tumor; Cyclin D1; Exosome Multienzyme Ribonuclease Complex; Genetic Predisposition to Disease; Histone Demethylases; Humans; Immunotherapy; Multiple Myeloma; Mutation; Plasma Cells; Repressor Proteins; Risk Factors; Survival Rate; Transcriptional Elongation Factors

Recently, large sequencing studies have provided insights into the mutational landscape of multiple myeloma (MM), identifying actionable mutations and giving a precious opportunity for exploring new targeted therapies. The main goal of precision medicine, matching patients with the right drug, seems to be closer than ever. However, no targeted therapies in MM are approved yet. Several clinical trials testing targeted drugs and enrolling patients with MM are currently ongoing and will provide predictive biomarkers that might support clinical decision making. In this review, we evaluate the evidence supporting the implementation of precision medicine in MM and we discuss the challenges that should be dealt with in this imminent and promising new era.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Cyclin D1; DNA Repair; Gene Expression Regulation, Neoplastic; Genes, myc; Humans; Immunotherapy; Mitogen-Activated Protein Kinases; Molecular Targeted Therapy; Multiple Myeloma; Phosphatidylinositol 3-Kinases; Precision Medicine; Prognosis; Proto-Oncogene Proteins c-akt; ras Proteins; Receptor, Fibroblast Growth Factor, Type 3; Signal Transduction; TOR Serine-Threonine Kinases; Translocation, Genetic

A 71-year-old woman had been diagnosed as having osteosclerotic myeloma (BJP-λ type) three years prior to the current presentation, based on tumor biopsy from the forehead showing plasmacytoma with systemic osteosclerotic lesions. At 71 years of age, she underwent transverse colectomy for a tumor in the hepatic flexure of the large intestine, and it was diagnosed as IgH/CCND-1-positive plasmacytoma of the large intestine. Although serum vascular endothelial cell growth factor (VEGF) was not elevated, the plasmacytoma was largely positive for VEGF staining. She subsequently experienced transformation to aggressive myeloma over a short period of time. Osteosclerotic myeloma is a rare disease that accounts for less than 3% of all myelomas, and requires differentiation from POEMS syndrome. In this case, peripheral nerve symptoms, which are necessary for the diagnosis of POEMS syndrome, were not confirmed. Thus, this case was diagnosed as having osteosclerotic myeloma. By contrast, abnormal IgH/CCND-1 is confirmed in 15% of patients with myeloma, and 25% of those with POEMS syndrome. While it is unclear whether this genetic abnormality is involved in the development of an osteosclerotic lesion, it is expected that data from patients with osteosclerotic myeloma and POEMS syndrome will be accumulated in the future, allowing clarification of the relationship between the genetic abnormality and osteosclerosis.

Topics: Aged; Cyclin D1; Fatal Outcome; Female; Humans; Immunoglobulin Heavy Chains; Multiple Myeloma; Translocation, Genetic

Multiple myeloma is a monoclonal tumor of plasma cells, and its development is preceded by a premalignant tumor with which it shares genetic abnormalities, including universal dysregulation of the cyclin D/retinoblastoma (cyclin D/RB) pathway. A complex interaction with the BM microenvironment, characterized by activation of osteoclasts and suppression of osteoblasts, leads to lytic bone disease. Intratumor genetic heterogeneity, which occurs in addition to intertumor heterogeneity, contributes to the rapid emergence of drug resistance in high-risk disease. Despite recent therapeutic advances, which have doubled the median survival time, myeloma continues to be a mostly incurable disease. Here we review the current understanding of myeloma pathogenesis and insight into new therapeutic strategies provided by animal models and genetic screens.

Topics: Bone Remodeling; Cell Transformation, Neoplastic; Cyclin D1; Disease Progression; Drug Resistance, Neoplasm; Genes, bcl-1; Genes, Retinoblastoma; Humans; Immunoglobulin Heavy Chains; Immunophenotyping; Models, Biological; Monoclonal Gammopathy of Undetermined Significance; Multiple Myeloma; Neoplasm Proteins; Neoplastic Stem Cells; Oncogene Proteins, Fusion; Osteoclasts; Precancerous Conditions; Retinoblastoma Protein; Signal Transduction; Stem Cell Niche; Translocation, Genetic; Tumor Microenvironment

Plasma cell myeloma is a heterogenous disease with variable clinical presentation and outcome. The prognosis is largely determined by tumor biology. Newer therapeutic agents are rapidly changing the survival outlook of myeloma patients.

Topics: Cyclin D1; Flow Cytometry; Histone-Lysine N-Methyltransferase; Humans; Immunoglobulin Heavy Chains; Immunophenotyping; Multiple Myeloma; Prognosis; Receptor, Fibroblast Growth Factor, Type 3; Repressor Proteins; Translocation, Genetic

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

Topics: Animals; Cell Transformation, Neoplastic; Cyclin D1; Humans; Multiple Myeloma; Translocation, Genetic

There appear to be two pathways involved in the pathogenesis of premalignant non-immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). Nearly half of tumors are nonhyperdiploid, and mostly have one of five recurrent IgH translocations: 16% 11q13 (CCN D1), 3% 6p21 (CCN D3), 5% 16q23 (MAF), 2% 20q12 (MAFB), and 15% 4p16 (FGFR3 and MMSET). The remaining hyperdiploid tumors have multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21, and infrequently one of these five translocations. Although cyclin D1 is not expressed by healthy lymphoid cells, it is bi-allelically dysregulated in a majority of hyperdiploid tumors. Virtually all MM and MGUS tumors have dysregulated and/or increased expression of cyclin D1, D2, or D3, providing an apparent early, unifying event in pathogenesis. The patterns of translocations and cyclin D expression (TC) define a novel classification that includes eight groups: 11q; 6p; MAF; 4p; D1 (34%); D1+D2 (6%); D2 (17%); and none (2%). The hyperdiploid D1 group is virtually absent in extramedullary MM and MM cell lines, suggesting a particularly strong dependence on interaction with the bone marrow microenvironment. Despite shared progression events (RAS mutations, MYC dysregulation, p53 mutations, and additional disruption of the retinoblastoma pathway), the phenotypes of MGUS and MM tumors in the eight TC groups is determined mainly by early oncogenic events. Similar to acute lymphocytic leukemia, MM seems to include several diseases (groups) that have differences in early or initiating events, global gene expression patterns, bone marrow dependence, clinical features, prognosis, and response to therapy.

Topics: Biopsy, Needle; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; Male; Molecular Biology; Multiple Myeloma; Neoplasm Staging; Sensitivity and Specificity; Translocation, Genetic

We used a sensitive real-time reverse transcription-polymerase chain reaction assay to quantify cyclin D1 mRNA levels in bone marrow samples collected at diagnosis from 74 newly diagnosed multiple myeloma (MM) patients who were randomized to undergo either single or double autologous peripheral blood stem cell transplantation as part of first-line therapy for their malignancy. In 46 cases, fluorescence in situ hybridization (FISH) analysis and/or conventional cytogenetics were performed to detect chromosome 11 abnormalities. Patients with the t(11;14) or trisomy 11 significantly overexpressed cyclin D1 (P <.0001) in comparison with patients without 11q abnormalities, who had cyclin D1 mRNA levels similar to healthy donors. Overall, 32 (43%) of 74 patients showed cyclin D1 overexpression. No difference was found between cyclin D1-positive (group A) and cyclin D1-negative (group B) patients with respect to presenting clinical and laboratory characteristics, including chromosome 13 abnormalities, as well as to response to therapy and overall survival, both of which were calculated on an intent-to-treat basis. Patients who overexpressed cyclin D1 had significantly longer duration of remission in comparison with patients who did not (41 vs 26 months, respectively; P =.02). As a result, median event-free survival (EFS) was longer in group A than in group B (33 vs 24 months, respectively; P =.055). We concluded that cyclin D1 overexpression is closely associated with 11q abnormalities and identifies a subset of MM patients who are more likely to have prolonged duration of remission and EFS following autologous transplantation.

Topics: Adult; Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Chromosome Aberrations; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 13; Combined Modality Therapy; Cyclin D1; Cyclophosphamide; Dexamethasone; Disease-Free Survival; Doxorubicin; Female; Gene Expression; Hematopoietic Stem Cell Transplantation; Humans; Male; Melphalan; Middle Aged; Multiple Myeloma; Predictive Value of Tests; Prognosis; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Sensitivity and Specificity; Vincristine

To demonstrate the clinical features and prognostic impact of cyclin D1 positivity in patients with amyloid light chain amyloidosis (AL).. We consecutively included 71 patients diagnosed with AL with cyclin D1 positivity between February 2008 and January 2022. t(11;14) was examined through interphase fluorescence in situ hybridization using bone marrow cells.. The median age of the patients was 73 years, and 53.5% were male. The underlying diseases included symptomatic multiple myeloma, smoldering multiple myeloma, Waldenström macroglobulinemia, and monoclonal gammopathy of undetermined significance, representing 33.8%, 26.8%, 2.8%, and 36.6%, respectively. The prevalence of cyclin D1 and t(11;14) was 38.0% and 34.7%, respectively. Higher frequency of light chain paraprotein type was seen in cyclin D1-positive patients with AL than in cyclin D1-negative patients (70.4% vs 18.2%). The median overall survival (OS) of patients with AL with and without cyclin D1 expression was 18.9 months and 73.1 months, respectively (P = .019). Early death occurred in 44.4% of cyclin D1-positive patients and 31.8% of cyclin D1-negative patients. Moreover, 83.3% of cyclin D1-positive patients and 21.4% of cyclin D1-negative patients died of cardiac causes.. Cyclin D1 immunohistochemistry accurately identified patients with t(11;14). Cyclin D1-positive patients had significantly inferior OS compared with cyclin D1-negative patients.

Topics: Aged; Cyclin D1; Female; Humans; Immunoglobulin Light-chain Amyloidosis; In Situ Hybridization, Fluorescence; Male; Multiple Myeloma; Paraproteinemias

Although long non-coding RNA (lncRNA) HCP plays essential roles in human cancers, its function and mechanism in multiple myeloma (MM) have not crystallized.. HCP5 level in MM was assessed through qRT-PCR. A series of functional investigations were conducted to evaluate the influences of HCP5 on proliferation and apoptosis. Bioinformatics analysis and RIP/RNA pull-down assays were carried out to determine the relationships among HCP5, miR-128-3p, and PLAGL2. Relative protein level was determined through Western blot. A xenograft tumor model was applied for validating the roles of HCP5/miR-128-3p/PLAGL2 axis in vivo.. HCP5 was significantly increased in MM. HCP5 knockdown effectively thwarted the proliferative rate and cell cycle of MM cell lines and suppressed tumor growth. HCP5 regulated PLAGL2 expression by sponging miR-128-3p. PLAGL2 overexpression effectively rescued cells from influences by sh-HCP5 on cell proliferative and apoptotic rates. Additionally, HCP5 knockdown significantly inhibited Wnt/β-catenin/cyclin D1 signaling, and these effects were eliminated by PLAGL2 overexpression.. Our study revealed that HCP5/miR-128-3p/PLAGL2 is closely correlated to MM development by modulating Wnt/β-catenin/cyclin D1 signaling. HCP5 promoted cell proliferation and tumor formation of MM cells by activating the Wnt/β-catenin/CCND1 signaling pathway by sponging miR-128-3p to increase PLAGL2 expression.

Topics: beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Multiple Myeloma; RNA-Binding Proteins; RNA, Long Noncoding; Transcription Factors; Wnt Signaling Pathway

Multiple myeloma is a genetically heterogeneous cancer of the bone marrow plasma cells (PC). Distinct myeloma transcriptome profiles are primarily driven by myeloma initiating events (MIE) and converge into a mutually exclusive overexpression of the CCND1 and CCND2 oncogenes. Here, with reference to their normal counterparts, we find that myeloma PC enhanced chromatin accessibility combined with paired transcriptome profiling can classify MIE-defined genetic subgroups. Across and within different MM genetic subgroups, we ascribe regulation of genes and pathways critical for myeloma biology to unique or shared, developmentally activated or de novo formed candidate enhancers. Such enhancers co-opt recruitment of existing transcription factors, which although not transcriptionally deregulated per se, organise aberrant gene regulatory networks that help identify myeloma cell dependencies with prognostic impact. Finally, we identify and validate the critical super-enhancer that regulates ectopic expression of CCND2 in a subset of patients with MM and in chronic lymphocytic leukemia.

Topics: Bone Marrow Cells; Carcinogenesis; Case-Control Studies; Cell Line, Tumor; Chromatin; Cyclin D1; Cyclin D2; Enhancer Elements, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Histone-Lysine N-Methyltransferase; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Multiple Myeloma; Plasma Cells; Proto-Oncogene Proteins c-maf; Repressor Proteins; Survival Analysis; Transcriptome

Topics: Antineoplastic Agents, Phytogenic; beta Catenin; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Humans; Morus; Multiple Myeloma; Plant Bark; Plant Roots; Proto-Oncogene Proteins c-myc; Triterpenes; Ursolic Acid; Wnt Signaling Pathway

Plasma cell myeloma (PCM) involving skin is rare and occurs in 1% to 4% of patients with PCM. We evaluated the clinicopathologic features, cytogenetic findings and clinical follow-up in a series of PCM cases with cutaneous involvement.. Cases of PCM with cutaneous involvement were retrospectively reviewed with clinical data.. Skin involvement in PCM occurred in older individuals (mean, 75 years) and was more frequent in men (7/10 patients). All cases showed bone marrow involvement preceding the cutaneous lesions. Histopathologically, the infiltrate was plasmacytic (n = 5) or primitive or plasmablastic (n = 4), and 1 case showed predominantly lymphoplasmacytic features with cyclin D1 immunoreactivity and CCND1 gene rearrangement. Concurrent amyloid deposition was seen in one biopsy, and another case demonstrated coexisting squamous cell carcinoma. The most common immunophenotype was CD138+, CD20-, and CD56+ with light chain restriction. Cytogenetic analysis (available for 7 cases) showed multiple hyperdiploid abnormalities. Follow-up was available for 8 cases (mean, 42 months; range, 11-156 months) and showed short-term disease-related death in 7 of 8 patients.. Cutaneous involvement in PCM demonstrates a diverse cytomorphologic spectrum with plasmacytic, plasmablastic, or lymphoplasmacytic features and may show concurrent amyloid deposition or neoplasms such as squamous cell carcinoma. Cutaneous involvement typically occurs late in the course of the disease and likely portends poor outcome.

Topics: Aged; Aged, 80 and over; Bone Marrow; Cyclin D1; Female; Gene Rearrangement; Humans; Immunophenotyping; Male; Middle Aged; Multiple Myeloma; Plasma Cells; Retrospective Studies; Skin

Structural chromosomal changes including copy number aberrations (CNAs) are a major feature of multiple myeloma (MM), however their evolution in context of modern biological therapy is not well characterized. To investigate acquisition of CNAs and their prognostic relevance in context of first-line therapy, we profiled tumor diagnosis-relapse pairs from 178 NCRI Myeloma XI (ISRCTN49407852) trial patients using digital multiplex ligation-dependent probe amplification. CNA profiles acquired at relapse differed substantially between MM subtypes: hyperdiploid (HRD) tumors evolved predominantly in branching pattern vs. linear pattern in t(4;14) vs. stable pattern in t(11;14). CNA acquisition also differed between subtypes based on CCND expression, with a marked enrichment of acquired del(17p) in CCND2 over CCND1 tumors. Acquired CNAs were not influenced by high-dose melphalan or lenalidomide maintenance randomization. A branching evolution pattern was significantly associated with inferior overall survival (OS; hazard ratio (HR) 2.61, P = 0.0048). As an individual lesion, acquisition of gain(1q) at relapse was associated with shorter OS, independent of other risk markers or time of relapse (HR = 2.00; P = 0.021). There is an increasing need for rational therapy sequencing in MM. Our data supports the value of repeat molecular profiling to characterize disease evolution and inform management of MM relapse.

Topics: Cyclin D1; DNA Copy Number Variations; Humans; Lenalidomide; Melphalan; Multiple Myeloma; Nerve Tissue Proteins; Prognosis; Recurrence

Interaction with bone marrow stromal cells (BMSCs) has been suggested as an important mechanism for the progression of multiple myeloma (MM) cells, while exosomes are crucial mediators for cell-to-cell communication. The study was to investigate the miRNA profile changes in exosomes released by BMSCs of MM patients and explore their possible function roles.The microarray datasets of exosomal miRNAs in BMSCs were downloaded from the Gene Expression Omnibus database (GSE110271: 6 MM patients, 2 healthy donors; GSE78865: 4 donors and 2 MM patients; GSE39571: 7 MM patients and 4 controls). The differentially expressed miRNAs (DEMs) were identified using the LIMMA method. The target genes of DEMs were predicted by the miRwalk 2.0 database and the hub genes were screened by constructing the protein-protein interaction (PPI) network, module analysis and overlapping with the differentially expressed genes (DEGs) after overexpression or knockout of miRNAs.Three downregulated DEMs were found to distinguish MM from normal and MM-MGUS controls in the GSE39571 dataset; one downregulated and one upregulated DEMs (hsa-miR-10a) could differentiate MM from normal and MM-MGUS controls in the GSE110271-GSE78865 merged dataset. Furthermore, 11 downregulated (hsa-miR-16) and 1 upregulated DEMs were shared between GSE39571 and merged dataset when comparing MM with normal samples. The target genes were predicted for these 17 DEMs. PPI with module analysis showed IGF1R and CCND1 were hub genes and regulated by hsa-miR-16. Furthermore, EPHA8 was identified as a DEG that was downregulated in MM cells when the use of has-miR-10a mimics; while IGF1R, CCND1, CUL3, and ELAVL1 were also screened as DEGs that were upregulated in MM cells when silencing of hsa-miR-16.BMSCs-derived exosomal miR-10a and miR-16 may be involved in MM progression by regulating EPHA8 or IGF1R/CCND1/CUL3/ELAVL1, respectively. These exosomal miRNAs or genes may represent potential biomarkers for diagnosis of MM and prediction of progression and targets for developing therapeutic drugs.

Topics: Biomarkers, Tumor; Case-Control Studies; Cell Line, Tumor; Cullin Proteins; Cyclin D1; Databases, Genetic; Disease Progression; Down-Regulation; ELAV-Like Protein 1; Exosomes; Gene Expression Regulation, Neoplastic; Humans; Linear Models; Mesenchymal Stem Cells; Microarray Analysis; MicroRNAs; Multiple Myeloma; Protein Interaction Maps; Receptor, EphA8; Receptor, IGF Type 1; Up-Regulation

Venetoclax is a highly potent, selective BCL2 inhibitor capable of inducing apoptosis in cells dependent on BCL2 for survival. Most myeloma is MCL1-dependent; however, a subset of myeloma enriched for translocation t(11;14) is codependent on BCL2 and thus sensitive to venetoclax. The biology underlying this heterogeneity remains poorly understood. We show that knockdown of cyclin D1 does not induce resistance to venetoclax, arguing against a direct role for cyclin D1 in venetoclax sensitivity. To identify other factors contributing to venetoclax response, we studied a panel of 31 myeloma cell lines and 25 patient samples tested for venetoclax sensitivity. In cell lines, we corroborated our previous observation that BIM binding to BCL2 correlates with venetoclax response and further showed that knockout of BIM results in decreased venetoclax sensitivity. RNA-sequencing analysis identified expression of B-cell genes as enriched in venetoclax-sensitive myeloma, although no single gene consistently delineated sensitive and resistant cells. However, a panel of cell surface makers correlated well with ex vivo prediction of venetoclax response in 21 patient samples and may serve as a biomarker independent of t(11;14). Assay for transposase-accessible chromatin sequencing of myeloma cell lines also identified an epigenetic program in venetoclax-sensitive cells that was more similar to B cells than that of venetoclax-resistant cells, as well as enrichment for basic leucine zipper domain-binding motifs such as BATF. Together, these data indicate that remnants of B-cell biology are associated with BCL2 dependency and point to novel biomarkers of venetoclax-sensitive myeloma independent of t(11;14).

Topics: B-Lymphocytes; Basic-Leucine Zipper Transcription Factors; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Multiple Myeloma; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Translocation, Genetic

Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain potential to transform by reprograming, and then chromosomal aberrations cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we have already established normal B cell-derived induced pluripotent stem cells (BiPSCs). Here we established two BiPSCs with reciprocal cTr t(11;14) using the CRISPR/Cas9 system; the cleavage site were located in the IgH Eμ region of either the VDJ rearranged allele or non-rearranged allele of IgH and the 5'-upsteam region of the CCND1 (two types of BiPSC13 with t(11;14) and MIB2-6 with t(11;14)). Furthermore, p53 was deleted using the CRISPR/Cas9 system in BiPSC13 with t(11;14). These BiPSCs differentiated into hematopoietic progenitor cells (HPCs). However, unlike cord blood, those HPCs did not differentiated into B lymphocytes by co-culture with BM stromal cell. Therefore, further ingenuity is required to differentiate those BiPSCs-derived HPCs into B lymphocytes.

Topics: B-Lymphocytes; Cell Differentiation; Cell Line, Tumor; Chromosome Aberrations; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; CRISPR-Cas Systems; Cyclin D1; Hematopoietic Stem Cells; Humans; Immunoglobulin Heavy Chains; In Situ Hybridization, Fluorescence; Induced Pluripotent Stem Cells; Multiple Myeloma; Translocation, Genetic; Tumor Suppressor Protein p53; VDJ Exons

Myeloma-related bone disease (MBD) is a common complication of multiple myeloma (MM), which can both decrease life quality and influence the prognosis of the patients. We have found that CCN1 stimulated proliferation and differentiation of osteoblasts in MM in vitro and in vivo, while its mechanism still remains unknown.. Bone marrow mononuclear cells were collected from MM patients and differentiated into the osteoblasts. After co-culture with CCN1 in vitro, the intracellular signaling antibody array and western blot were performed to explore the signaling pathway. Furthermore, GSK3β inhibitor TWS119 was used to check the pathway of CCN1 might have on osteoblasts in vitro.. For the protein array kit, the expressions of GSK3β, 4E-BP1, and PTEN are decreased in CCN1 group. For western blots, the CCN1 group also has lower expression comparing to the control group in PTEN (P = .031). Meanwhile p-AKT and cyclinD1 levels have increased in the CCN1 group (P = .002, P = .039). After adding TWS119 as another group, western blot was performed again to verify the pathway. For upstream proteins PTEN and p-AKT, TWS119 group has higher expression level compared to that in CCN1 group (P = .003, P = .001). And for downstream protein cyclinD1, TWS119 group also presented higher level than the control group (P = .02). CCN1 could have almost the same effect on GSK3β as the specific inhibitor TWS119 had.. CCN1 can stimulate osteoblasts through PTEN/AKT/GSK3β/cyclinD1 pathway in MBD, which has the potential to be a novel therapy of MBD.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Bone Diseases; Case-Control Studies; Cells, Cultured; Cyclin D1; Cysteine-Rich Protein 61; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3 beta; Humans; Male; Middle Aged; Multiple Myeloma; Osteoblasts; Prognosis; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase

Cyclin D1 (CCND1) overexpression is an early and unifying oncogenic event in mantle cell lymphoma (MCL) and multiple myeloma (MM) with chromosome 11q13 abnormalities. Herein, we report newly discovered transcript variants of the CCND1 gene in MCL and MM cells with chromosome 11q13 abnormalities. These transcript variants, designated CCND1.tv., covered the full-length coding region of CCND1 with longer 5'-untranslated regions (5'-UTRs) of CCND1 and occasionally contained a novel exon. CCND1.tv. was specifically detectable in patient-derived primary MCL or MM cells with chromosomal translocation t(11;14)(q13;q32), but not in t(11;14)-negative cells. The lengths of the 5'-UTR sequences of CCND1.tv. differed among patients and cell lines. Introduction of CCND1.tv. led to increased expression of normal-sized CCND1 protein in HEK293 cells. Furthermore, mTOR inhibition by rapamycin or serum starvation reduced ectopic expression of CCND1.tv.-derived CCND1 protein, but not 5'-UTR less CCND1-derived CCND1 protein in HEK293 cells, suggesting that the protein expression of CCND1.tv. is regulated by the mTOR pathway. Our results suggest that the aberrant expression of CCND1.tv. may contribute to the understanding of the pathogenesis of MCL and MM with 11q13 abnormalities.

Topics: 5' Untranslated Regions; Cell Line, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Exons; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lymphoma, Mantle-Cell; Multiple Myeloma; Signal Transduction; TOR Serine-Threonine Kinases; Transcription, Genetic; Translocation, Genetic

The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.. Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.. LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low.. These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

Topics: Bone Marrow; Cyclin D1; Gene Rearrangement; High-Throughput Nucleotide Sequencing; Humans; Immunoglobulin Heavy Chains; Immunoglobulin lambda-Chains; Immunoglobulins; Leukemia, Lymphocytic, Chronic, B-Cell; Limit of Detection; Multiple Myeloma; Neoplasm, Residual; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proto-Oncogene Proteins c-bcl-2; Reagent Kits, Diagnostic; Translocation, Genetic

Multiple Myeloma (MM) is a hematological malignancy with genomic heterogeneity and poor survival outcome. Apart from the central role of genetic lesions, epigenetic anomalies have been identified as drivers in the development of the disease.. Alterations in the DNA methylome were mapped in 52 newly diagnosed MM (NDMM) patients of six molecular subgroups and matched with loci-specific chromatin marks to define their impact on gene expression. Differential DNA methylation analysis was performed using DMAP with a ≥10% increase (hypermethylation) or decrease (hypomethylation) in NDMM subgroups, compared to control samples, considered significant for all the subsequent analyses with p<0.05 after adjusting for a false discovery rate.. We identified differentially methylated regions (DMRs) within the etiological cytogenetic subgroups of myeloma, compared to control plasma cells. Using gene expression data we identified genes that are dysregulated and correlate with DNA methylation levels, indicating a role for DNA methylation in their transcriptional control. We demonstrated that 70% of DMRs in the MM epigenome were hypomethylated and overlapped with repressive H3K27me3. In contrast, differentially expressed genes containing hypermethylated DMRs within the gene body or hypomethylated DMRs at the promoters overlapped with H3K4me1, H3K4me3, or H3K36me3 marks. Additionally, enrichment of BRD4 or MED1 at the H3K27ac enriched DMRs functioned as super-enhancers (SE), controlling the overexpression of genes or gene-cassettes.. Therefore, this study presents the underlying epigenetic regulatory networks of gene expression dysregulation in NDMM patients and identifies potential targets for future therapies.

Topics: Aneuploidy; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 4; Cyclin D1; Cyclin D2; DNA Methylation; DNA, Neoplasm; Epigenesis, Genetic; Epigenome; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Ontology; Gene Regulatory Networks; Histone Code; Histones; Humans; Multiple Myeloma; Neoplasm Proteins; Plasma Cells; Promoter Regions, Genetic; Proto-Oncogene Proteins c-maf; Translocation, Genetic

Multiple myeloma (MM) is a heterogeneous plasma cell malignancy that remains challenging to cure. Global hypomethylation correlates with an aggressive phenotype of the disease, while hypermethylation is observed at particular regions of myeloma such as B cell-specific enhancers. The recently discovered active epigenetic mark 5-hydroxymethylCytosine (5hmC) may also play a role in tumor biology; however, little is known about its level and distribution in myeloma. In this study, we investigated the global level and the genomic localization of 5hmC in myeloma cells from 40 newly diagnosed patients, including paired relapses, and of control individuals.. Compared to normal plasma cells, we found global 5hmC levels to be lower in myeloma (P < 0.001). Higher levels of 5hmC were found in lower grades of the International Staging System prognostic index (P < 0.05) and tend to associate with a longer overall survival (P < 0.1). From the hydroxymethylome data, we observed that the remaining 5hmC is organized in large domains overlapping with active chromatin marks and chromatin opening. We discovered that 5hmC strongly persists at key oncogenic genes such as CCND1, CCND2 and MMSET and characterized domains that are specifically hydroxymethylated in myeloma subgroups. Novel 5hmC-enriched domains were found at putative enhancers of CCND2 and MYC in newly diagnosed patients.. 5hmC level is associated with clinical aspects of MM. Mapping 5hmC at a genome-wide level provides insights into the disease biology directly from genomic DNA, which makes it a potent mark to study epigenetics on large patient cohorts.

Topics: 5-Methylcytosine; Chromatin; Cyclin D1; Cyclin D2; DNA Methylation; Epigenesis, Genetic; Epigenomics; Female; Gene Expression Regulation, Neoplastic; Genome; Histone-Lysine N-Methyltransferase; Humans; Male; Middle Aged; Multiple Myeloma; Phenotype; Proto-Oncogene Proteins c-myc; Regulatory Sequences, Nucleic Acid; Repressor Proteins; Severity of Illness Index

We attempted to clarify the regulatory mechanism of UCA1/miR-331-3p/IL6R on cell progression in multiple myeloma (MM).. The expression of UCA1, miR-331-3p, and IL6R in tumor tissues and cells was measured by qRT-PCR. Cell Counting Kit-8 (CCK-8) was conducted to detect cell proliferation, and flow cytometry assay was applied to examine cell apoptosis. Protein expression of L6R, p-JAK2, p-STAT3, c-Myc, CyclinD1, Bcl-2, and Bax was detected by Western blot assay. The interaction among miR-331-3p, UCA1, and IL6R was determined by Luciferase reporter system. Murine xenograft assay was performed to confirm the biological function of UCA1 in vivo.. The expression of UCA1 and IL6R was up-regulated, while miR-331-3p was down-regulated in MM tumors and cell lines compared with normal tissues and cells. By calculation, miR-331-3p was correlated with UCA1 or IL6R inversely. In addition, UCA1 knockdown suppressed cell proliferation and promoted apoptosis in vitro and in vivo. Luciferase reporter system confirmed the interaction between miR-331-3p and UCA1 or IL6R. More importantly, UCA1 restored miR-331-3p mediated inhibition of proliferation and promotion on apoptosis of MM cells. Consistently, IL6R rescued UCA1 knockdown caused repression on MM cell growth and elevation on apoptosis. Besides, UCA1 facilitated the activation of the JAK2/STAT3 signaling pathway by enhancing IL6R expression via targeting miR-331-3p.. UCA1 accelerates proliferation and suppresses apoptosis in MM by targeting miR-331-3p/IL6R axis to activate JAK2/STAT3 pathway, providing potential targets for the diagnosis and therapy of MM.

Topics: Apoptosis; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 2; MicroRNAs; Multiple Myeloma; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Receptors, Interleukin-6; RNA, Long Noncoding; Signal Transduction; STAT3 Transcription Factor; Transfection; Up-Regulation; Xenograft Model Antitumor Assays

Plasma cell neoplasms may exhibit variations in morphology and immunophenotype, which can mimic mature B-cell lymphoproliferative disorders and pose diagnostic challenges. This case illustrates a rare entity of plasma cell myeloma, where the entire plasma cell population exhibited lymphoid morphology, negativity for CD138, positivity for CD20 and cyclin D1, and positive fluorescence in situ hybridisation for t(11;14) and del(17 p), mimicking a mature B-cell lymphoproliferative disorder, in particular mantle cell lymphoma. In this case, a careful analysis of flow cytometry gating strategies and use of other ancillary tests were keys for correct diagnosis. In addition to the diagnostic implications due to its rarity, CD138-negative plasma cell myeloma may represent a unique entity, which is associated with 'stem cell'-like clonogenic properties, more aggressive clinical behaviour and resistance to chemotherapy.

Topics: Aged; Antigens, CD20; Cyclin D1; Diagnosis, Differential; Female; Humans; Lymphoma, Mantle-Cell; Multiple Myeloma; Syndecan-1

The t(11;14)/CCND1-IGH, t(4;14)/NSD2(MMSET)-IGH, and t(14;16)/IGH-MAF gene rearrangements detected by fluorescence in situ hybridization (FISH) are used for risk stratification in patients with multiple myeloma (MM). Compared with conventional FISH techniques using fresh cells, immunohistochemistry (IHC) is much more cost- and time-efficient, and can be readily applied to routinely prepared formalin-fixed, paraffin-embedded (FFPE) materials. In this study, we performed tissue FISH and IHC employing FFPE specimens, and examined the usefulness of IHC as a tool for detecting CCND1, NSD2, and MAF gene rearrangements. CD138 signals were used to identify plasma cells in tissue FISH and IHC analyses. With cohort 1 (n = 70), we performed tissue FISH and subsequently IHC, and determined IHC cut-off points. In this cohort, the sensitivity and specificity for the 3 molecules were ≥.90 and ≥.96, respectively. With cohort 2, using MM cases with an unknown gene status (n = 120), we performed IHC, and the gene status was estimated using the cut-off points determined with cohort 1. The subsequent FISH analysis showed that the sensitivity and specificity for the 3 molecules were ≥.92 and ≥.98, respectively. CCND1, NSD2, and MAF gene rearrangements were estimated accurately by IHC, suggesting that conventional FISH assays can be replaced by IHC.

Topics: Aged; Aged, 80 and over; Cohort Studies; Cyclin D1; Female; Gene Rearrangement; Histone-Lysine N-Methyltransferase; Humans; Immunohistochemistry; Male; Middle Aged; Multiple Myeloma; Proto-Oncogene Proteins c-maf; Repressor Proteins; Sensitivity and Specificity

Topics: Animals; Cell Line, Tumor; Cell Survival; Cyclin D1; Drug Design; Gene Silencing; Heterografts; Humans; Mice; Multiple Myeloma; Proto-Oncogene Proteins c-bcl-2; RNA, Guide, Kinetoplastida; RNA, Messenger; Survival Analysis

Interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment promotes the proliferation, survival and chemoresistance of MM. The mTOR pathway plays a key role in these undesirable BM microenvironment-mediated events. We synthesized a novel alkaloid compound, DCZ0358, that effectively inhibits mTOR signaling via dual mTORC1/2 inhibition and exhibits potent anti-MM activity in cultured and primary MM cells, as well as a MM xenograft model but has little effect on normal cells. Importantly, we show that this compound can block the BM stromal cell-mediated activation of mTOR/Akt signaling and antagonizes the protective effect of the BM microenvironment. Moreover, DCZ0358 abrogates the bortezomib-triggered activation of Akt, leading to the synergism of DCZ0358 and bortezomib in MM cells. Taken together, our results provide the proof-of-concept for clinical evaluation of DCZ0358, alone or in combination, as an anti-MM agent in MM therapy.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Marrow; Bortezomib; Cell Line, Tumor; Cell Survival; Cyclin D1; Cyclin-Dependent Kinases; Drug Synergism; Enzyme Activation; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Mice, Inbred BALB C; Mice, Nude; Multiple Myeloma; Proto-Oncogene Proteins c-akt; Tumor Microenvironment

We examined a set of 805 cases that underwent DNA sequencing using the FoundationOne Heme (F1H) targeted sequencing panel and gene expression profiling. Known and likely variant calls from the mutational data were analyzed for significant associations with gene expression defined translocation cyclin D (TC) molecular subgroups. The spectrum of KRAS, NRAS, and BRAF codon mutations varied across subgroups with NRAS mutations at Q61 codon being common in hyperdiploid (HRD) and t(11;14) myeloma while being rare in MMSET and MAF. In addition, the presence of RAS-RAF mutations was inversely associated with NFκB pathway activation in all subgroups excluding MAF. In the MMSET subgroup, cases with low FGFR3 expression frequently had RAS-RAF mutations. Conditional inference tree analysis determined that mutation and homozygous deletion of TP53, CDKN2C, and RB1 were key prognostic factors associated with adverse outcome in a non-relapse clinical setting. In conclusion, this study highlights the heterogeneity in the distribution and clinical outcomes of RAS codon and other mutations in multiple myeloma dependent upon primary molecular subgroup.

Topics: Codon; Colorectal Neoplasms; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p18; DNA; DNA Mutational Analysis; Gene Expression Profiling; GTP Phosphohydrolases; Humans; Membrane Proteins; Multiple Myeloma; Mutation; NF-kappa B; Prognosis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Receptor, Fibroblast Growth Factor, Type 3; Retinoblastoma Binding Proteins; Sequence Analysis, DNA; Signal Transduction; Translocation, Genetic; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

Multiple myeloma (MM) is a frequently incurable hematological cancer in which overactivity of MYC plays a central role, notably through up-regulation of ribosome biogenesis and translation. To better understand the oncogenic program driven by MYC and investigate its potential as a therapeutic target, we screened a chemically diverse small-molecule library for anti-MM activity. The most potent hits identified were rocaglate scaffold inhibitors of translation initiation. Expression profiling of MM cells revealed reversion of the oncogenic MYC-driven transcriptional program by CMLD010509, the most promising rocaglate. Proteome-wide reversion correlated with selective depletion of short-lived proteins that are key to MM growth and survival, most notably MYC, MDM2, CCND1, MAF, and MCL-1. The efficacy of CMLD010509 in mouse models of MM confirmed the therapeutic relevance of these findings in vivo and supports the feasibility of targeting the oncogenic MYC-driven translation program in MM with rocaglates.

Topics: Animals; Cell Line, Tumor; Cyclin D1; Humans; Mice; Multiple Myeloma; Proto-Oncogene Proteins c-maf; Proto-Oncogene Proteins c-myc; Xenograft Model Antitumor Assays

Immunoglobulin light chain (AL) amyloidosis is characterized by tissue deposition of amyloid fibers derived from immunoglobulin light chain. AL amyloidosis and multiple myeloma (MM) originate from monoclonal gammopathy of undetermined significance. We wanted to characterize germline susceptibility to AL amyloidosis using a genome-wide association study (GWAS) on 1229 AL amyloidosis patients from Germany, UK and Italy, and 7526 healthy local controls. For comparison with MM, recent GWAS data on 3790 cases were used. For AL amyloidosis, single nucleotide polymorphisms (SNPs) at 10 loci showed evidence of an association at P<10

Topics: Adult; Aged; Aged, 80 and over; Amyloidosis; Cyclin D1; Female; Genetic Predisposition to Disease; Genome-Wide Association Study; Humans; Immunoglobulin Light Chains; Male; Middle Aged; Multiple Myeloma; Polymorphism, Single Nucleotide

Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3'UTR in CCND2 overexpression in multiple myeloma.. Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3'UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3' rapid amplification of cDNA ends (RACE)-PCR.. We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3'RACE experiments revealed that changes in CCND2 3'UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3'UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction.. Overall, these results highlight the impact of CCND2 3'UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma.

Topics: 3' Untranslated Regions; Alternative Splicing; Cell Line, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Cyclin D2; Cyclin D3; DNA Methylation; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Multiple Myeloma; Promoter Regions, Genetic; RNA Processing, Post-Transcriptional; RNA, Messenger; Translocation, Genetic; Up-Regulation

Topics: Cyclin D1; Erythroid Cells; Genes, myc; Humans; Karyotype; Male; Middle Aged; Multiple Myeloma; Oncogene Proteins, Fusion; Phagocytosis; Tetraploidy

Multiple myeloma (MM) is characterized by over-expression of cyclin D1 (CCND1) or D2 (CCND2), which control G1 phase cell-cycle progression. Proteolytic degradation of CCND1 (but not CCND2), resulting in G1 arrest, is reported in non-MM cells post-DNA damage, affecting DNA repair and survival. We examined the effect of ionizing radiation (IR) on D-cyclin levels and cell-cycle kinetics of MM cells, exploring differences based on D-cyclin expression. We showed that CCND1 is downregulated, whereas CCND2 is not, following IR. This did not lead to hypo-phosphorylation of retinoblastoma protein or G1 arrest. Both CCND1- and CCND2-expressing MM cells arrested in S/G2/M, and did not differ in other cell-cycle proteins or sensitivity to IR. When treated with a CDK4/6 inhibitor, both CCND1 and CCND2 MM cells arrested in G1 and therefore are subject to physiological regulation at this checkpoint. Immunoprecipitation showed that, despite CCND1 degradation following IR, sufficient protein remains bound to CDK4/6 to prevent G1 arrest. Aberrant expression of CCND1 driven from the IGH promoter in t(11;14) MM cells maintains progression through G1 to arrest in S/G2/M. Differential expression of D-cyclin does not appear to affect cell-cycle response to IR, and is unlikely to underlie differential sensitivity to DNA damage.

Topics: Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cells, Cultured; Cyclin D; Cyclin D1; Cyclin D2; DNA Damage; Humans; Multiple Myeloma; Radiation, Ionizing

The interactions of multiple myeloma (MM) cells with their microenvironment are crucial for pathogenesis. MM cells could interact differentially with their microenvironment depending on the type of cyclin D they express. We established several clones that constitutively express cyclin D1 from the parental RPMI8226 MM cell line and analyzed the impact of cyclin D1 expression on cell behavior. We performed a gene expression profiling study on cyclin D1-expressing vs. control cells and validated the results by semi-quantitative RT-PCR. The expression of cyclin D1 altered the transcription of genes that control adhesion and migration. We confirmed that cyclin D1 increases cell adhesion to stromal cells and fibronectin, stabilizes F-actin fibers, and enhances chemotaxis and inflammatory chemokine secretion. Both control and cyclin D1-expressing cells were more resistant to acute carfilzomib treatment when cultured on stromal cells than in suspension. However, this resistance was specifically reduced in cyclin D1-expressing cells after pomalidomide pre-treatment that modifies tumor cell/microenvironment interactions. Transcriptomic analysis revealed that cyclin D1 expression was also associated with changes in the expression of genes controlling metabolism. We also found that cyclin D1 expression disrupted the redox balance by producing reactive oxygen species. The resulting oxidative stress activated the p44/42 mitogen-activated protein kinase (or ERK1/2) signaling pathway, increased cell adhesion to fibronectin or stromal cells, and controlled drug sensitivity.Our results have uncovered a new function for cyclin D1 in the control of redox metabolism and interactions of cyclin D1-expressing MM cells with their bone marrow microenvironment.

Topics: Cell Adhesion; Cell Line, Tumor; Cell Movement; Chemokines; Cyclin D1; Drug Resistance, Neoplasm; Extracellular Signal-Regulated MAP Kinases; Humans; Multiple Myeloma; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Thalidomide; Tumor Microenvironment

Topics: Aged; Cyclin D1; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; Multiple Myeloma; Plasma Cells; Translocation, Genetic

Intensive protein synthesis is a unique and differential trait of the multiple myeloma (MM) cells. Previously we showed that tetraspanin overexpression in MM cell lines attenuated mTOR and PI3K cascades, induced protein synthesis, activated unfolded protein response (UPR), and caused autophagic death, all suggesting breach of proteostasis. Here we assessed the role of translation initiation in the tetraspanin‑induced MM cell death with emphasis on eIF4E translation initiation factor. We showed tetraspanins attenuated peIF4E and its targets [c‑Myc, cyclin D1 (cycD1)]; eIF4E attenuation was Akt-dependent. eIF4E inhibition in MM cells [bone marrow (BM), lines] by siRNA and/or the anti‑viral drug and competitive eIF4E inhibitor ribavirin (RBV) deleteriously affected MM cells in a similar manner to the overexpression of tetraspanins. Furthermore, combined application of RBV and velcade had a synergistic anti‑MM effect. Our results demonstrate that breach of proteostasis via eIF4E inhibition is an attractive therapeutic approach that may be relatively easily achieved by employing RBV, making this strategy readily translatable into the clinic.

Topics: Autophagy; Cell Line, Tumor; Cyclin D1; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-myc; Ribavirin; RNA, Small Interfering; Tetraspanins; TOR Serine-Threonine Kinases; Unfolded Protein Response

We describe herein the case of a 64-year-old man with a diagnosis of plasma cell myeloma (PCM). A chromosome analysis based on G-banding and spectral karyotyping revealed the following complex karyotype: 46,XY,del(3)(p?), t(4;15)(q31;q24),t(9;14;11)(p13;q32;q13),add(15)(q24),add(18)(q21). Fluorescence in situ hybridization (FISH) detected one signal each for the immunoglobulin heavy chain (IGH) and cyclin D1 (CCND1) genes, and three fusion signals of IGH and CCND1. FISH analysis of metaphase spreads revealed fusion signals on the derivative chromosomes 9, 11, and 14. Immunohistochemical analysis identified abnormal expression of CCND1 and PAX5. PAX5-positive PCM is rare because the down-regulation of PAX5 is essential for the terminal differentiation of B cells into plasma cells. To the best of our knowledge, this is the first reported case of a novel complex variant translocation of t(11;14)(q13;q32) and t(9;14)(p13;q32).

Topics: Chromosome Banding; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 9; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immunoglobulin Heavy Chains; In Situ Hybridization, Fluorescence; Karyotyping; Male; Middle Aged; Multiple Myeloma; PAX5 Transcription Factor; Plasma Cells; Translocation, Genetic

Topics: Adult; Aged; Aged, 80 and over; CD56 Antigen; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 16; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Multiple Myeloma; Neoplasm Proteins; Survival Analysis; Translocation, Genetic; Treatment Outcome

Cyclin D1 and its kinase partners control cell cycle progression. Cyclin D1 is frequently deregulated in various cancers, including malignant hemopathies, and tumor cells display uncontrolled cell proliferation. Cyclin D1 is not expressed in the B-cell lineage but is found in multiple myeloma (MM) cells in almost 50% of patients with this condition. Paradoxically, cyclin D1 expression is associated with a good prognosis and longer overall survival in MM patients.. We used two independent MM cell lines (RPMI 8226 and LP1) to generate several clones stably expressing either the green fluorescent protein (GFP) or a GFP-cyclin D1 fusion protein, and we analyzed the properties acquired following cyclin D1 expression.. Whole-genome expression analysis in the cell clones indicated that cyclin D1 profoundly modified several cellular functions, including the regulation of apoptotic cell death. We studied the apoptotic response of GFP- and GFP-cyclin D1-expressing clones to bortezomib-treatment. We found that the apoptotic response occurred faster and was of a greater amplitude in cyclin D1-expressing cells. Cyclin D1 expression enhanced the caspase-dependent apoptosis mediated by the intrinsic mitochondrial pathway. More importantly, cyclin D1 also activated the unfolded protein response (UPR) and induced endoplasmic reticulum (ER) stress-mediated apoptosis.. The ER is well known to be a crucial regulator of plasma cell death and it plays the same role in their malignant counterparts, myeloma cells. This role involves activation of the UPR controlled at least in part by cyclin D1.

Topics: Apoptosis; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Cyclin D1; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Genome, Human; Humans; Multiple Myeloma; Neoplasm Proteins; Prognosis; Signal Transduction; Unfolded Protein Response

Cyclin D1 is an important cell cycle regulator implicated in the pathogenesis of many cancer types. In particular, translocation and overexpression of cyclin D1 are common events in multiple myeloma (MM), suggesting that it may drive the initiation and progression of this malignancy. However, the association between genetic polymorphisms of cyclin D1 and the risk for developing MM remains poorly characterized. We performed a case-control study with 67 patients with MM and 66 healthy controls in a Chinese population. The cancer-associated G870A single nucleotide polymorphism (SNP) of CCND1, the gene encoding cyclin D1, was determined by polymerase chain reaction and restriction fragment length polymorphism. We found that there was a strong association between the homozygous variant allele  (GG) and MM susceptibility, with an odds ratio (OR) of 4.679 [95% confidence interval (CI): 1.081-5.647]. When further stratified analysis was performed, the increased risk was only evident in individuals over 60 years old (OR=3.297; 95%CI: 1.058-10.276). Our results suggest that the CCND1 G870A SNP may be critically involved in MM development.

Topics: Adult; Aged; Alleles; Cyclin D1; Female; Genetic Association Studies; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Multiple Myeloma; Polymorphism, Single Nucleotide; Risk Factors

Topics: Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 9; Cyclin D1; Forkhead Transcription Factors; Humans; Multiple Myeloma; Phosphoric Diester Hydrolases; Polymorphism, Single Nucleotide

Due to its cytotoxic effect in lymphoid cells, dexamethasone is widely used in the treatment of multiple myeloma (MM). However, only a subset of myeloma patients responds to high-dose dexamethasone. Despite the undeniable anti-myeloma benefits of dexamethasone, significant adverse effects have been reported. We re-evaluate the anti-tumor effect of dexamethasone according to the molecular heterogeneity of MM. We demonstrated that the pro-death effect of dexamethasone is related to the genetic heterogeneity of MM because sensitive cell lines were restricted to MAF and MMSET signature subgroups, whereas all CCND1 cell lines (n = 10) were resistant to dexamethasone. We demonstrated that the glucocorticoid receptor expression was an important limiting factor for dexamethasone-induced cell death and we found a correlation between glucocorticoid receptor levels and the induction of glucocorticoid-induced leucine zipper (GILZ) under dexamethasone treatment. By silencing GILZ, we next demonstrated that GILZ is necessary for Dex induced apoptosis while triggering an imbalance between anti- and pro-apoptotic Bcl-2 proteins. Finally, the heterogeneity of the dexamethasone response was further confirmed in vivo using myeloma xenograft models. Our findings suggested that the effect of dexamethasone should be re-evaluated within molecular subgroups of myeloma patients to improve its efficacy and reduce its adverse effects.

Topics: Animals; Antineoplastic Agents, Hormonal; Apoptosis; Cell Death; Cyclin D1; Dexamethasone; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Glucocorticoids; Humans; Mice; Mice, SCID; Multiple Myeloma; Neoplasm Transplantation; Oligonucleotide Array Sequence Analysis; Receptors, Glucocorticoid; RNA Interference

To study the clinical and pathologic features of multiple myeloma(MM) with CCND1.. Retrospectively analyzed the clinical and pathologic profiles of 158 patients with MM from 2010 to 2013. The clinical and morphologic features of bone marrow aspiration, biopsy and immunophenotypic analysis which was carried out by flow cytometry and immunohistochemistry were analyzed in all patients with MM respectively. CCND1 translocation was studied by FISH method in all cases. Classical cytogenetic studies of bone marrow were performed in 24 cases whose CCND1 was positive.. In the 158 patients with MM, CCND1 was detected in 31 patients (19.6%）. In 31 patients, type IgA, IgD, IgG, IgM, light-chain only and nonsecretory MM were 4 cases,4 cases,11 cases,1 case, 6 cases and 5 cases respectively. A high incidence of CCND1 was observed in IgD and nonsecretory MM comparied with IgA and IgG respectively (P<0.05). but no statistical significance was reached between κ and λ type patients (P=0.627). The morphology of plasma cell in bone marrow biopsies were small Lymphocyte- Like 24 cases,mature plasma cell 6 cases and immature plasma cell 1 case. Immunophenotype of all 31 cases was CD38⁺CD138⁺CD19⁻CD45⁻, (CD56⁺ in 11 cases, CD20⁺ in 9 cases, CD117⁺ in 3 cases. MM with CCND1 showed a strong association with CD20 expression, the lack of CD56 expression. Immunohistochemistry showed positive for cyclinD1 in 22 cases.. A high incidence of CCND1 was detected in the IgD and nonsecretory MM, and correlated with Small Lymphocyte- Like, higher positive rate of CD20, cyclinD1 and the lack of CD56 expression. MM with CCND1 must be distinguished from LPL and other mature B cell lymphomas which have plasmacytoid differentiation.

Topics: Biopsy; Bone Marrow; Cyclin D1; Flow Cytometry; Humans; Immunohistochemistry; Immunophenotyping; In Situ Hybridization, Fluorescence; Multiple Myeloma; Plasma Cells; Retrospective Studies; Translocation, Genetic

Almost all patients with multiple myeloma (MM) have chromosomal translocation which can result in genetic variation. There are mainly five types of chromosomal translocations, involving the IGH gene translocation to 11q13 (CCND1), 4p16 (FGFR/MMSET), 16q23 (MAF), 6p21 (CCND3) and 20q11 (MAFB). It is possible that all IGH translocations converge on a common cell cycle signal pathway. Some MM develops through a multistep transformation from monoclonal gammopathy of undetermined significance (MGUS) to smoldering MM (SMM) and eventually to MM and plasma cell leukemia (PCL). Similarly to what Darwin proposed in the mid-19th century-random genetic variation and natural selection in the context of limited resources, MM clonal evolution follow branching and nonlinear mode. The failure of MM treatment is usually related with the minimal subclone which is hardly found at newlydiagnosed.

Topics: Clonal Evolution; Cyclin D1; Genes, Immunoglobulin Heavy Chain; Humans; Multiple Myeloma; Translocation, Genetic

Solitary plasmacytoma of bone (SPB) is a rare tumor that represents a minority of patients with plasma cell localized malignancy characterized by a single osteolytic bone lesion. The molecular mechanism underlying the genesis of SPB has remained enigmatic. Signal transducers and activators of transcription-3 (STAT3) is often activated in human cancers and is implicated in tumorigenesis. In the present work, the immunohistochemical expression of pSTAT3 (the active isoform of STAT3) and its potential downstream mediators (Bcl-2, Bcl-xL, c-Myc, cyclin D1, VEGF, cIAP-2, Mcl-1, and survivin) were examined, clinical features were investigated, and relative prognostic factors were analyzed. Positive expression of Bcl-2 was observed in 46.7 % (14/30) of patients, c-Myc in 36.7 % (11/30), and cyclin D1 in 23.3 % (7/30). By univariate analysis, Bcl-2 expression was found to be closely associated with shorter overall survival (OS) and progression-free survival. Bcl-2 and c-Myc positive expression were also found to be a factor that affect time to progression to multiple myeloma. In conclusion, results showed Bcl-2 expression to be a clinically significant prognostic indicator for SPB patients and constitutive activated STAT3 may not be the sole primary regulatory mechanism.

Topics: Adult; Aged; Biomarkers, Tumor; Bone and Bones; Bone Neoplasms; Cohort Studies; Cyclin D1; Disease Progression; Female; Follow-Up Studies; Humans; Male; Middle Aged; Multiple Myeloma; Neoplasm Proteins; Plasmacytoma; Prognosis; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Retrospective Studies; Survival Analysis; Young Adult

Topics: Algorithms; Antigens, CD20; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cluster Analysis; Cohort Studies; Cyclin D1; Gene Expression Profiling; Humans; Kaplan-Meier Estimate; Multiple Myeloma; Prognosis; Translocation, Genetic; Treatment Outcome; Wnt Proteins; Wnt Signaling Pathway

Cyclin D1 is involved in normal regulation of the cell cycle and in neoplasia. Inhibition of cyclin D1 function markedly attenuates the proliferation of fibroblasts of colon, esophageal, lung, and pancreatic cancer. However, the prognostic value of overexpression of cyclin D1 in multiple myeloma is still a point of debate. This study aimed at evaluating the effect of cyclin D1 gene amplification in multiple myeloma on overall survival and response to therapy.. Fifty patients with multiple myeloma were retrospectively studied. Cyclin D1 gene amplification was studied in bone marrow biopsies of these patients using FISH. An immunohistochemical study of the bone marrow biopsies was done to detect MDR1 protein expression. The correlations between the cyclin D1 gene amplification and overall survival and MDR1 expression were studied and analyzed statistically.. Cyclin D1 gene amplification was found in 20% of myeloma patients and was associated with higher percentage of plasma cell infiltration of the bone marrow and increased liability for multiple osteolytic lesions. Cyclin D1-positive patients had a significantly lower progression-free and overall survival and higher levels of MDR1 compared with cyclin D1-negative patients. Cyclin D1 levels showed a highly statistically significant positive correlation with MDR1 levels (R, 0.8 and P < .0001).. We suggest that there is an association between cyclin D1 gene amplification and disease severity, unfavorable prognosis, and increased expression of MDR1 in multiple myeloma patients.

Topics: Aged; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bone Neoplasms; Cyclin D1; Gene Amplification; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Middle Aged; Multiple Myeloma; Neoplasm Staging; Odds Ratio; Prognosis

The identification of the active compounds of herbal medicines and the molecular targets of those compounds is an attractive therapeutic objective. Reynoutria elliptica has been used for the treatment of various inflammatory diseases as a Korean folk remedy. Based on the evidence that anti-inflammatory agents frequently exert antiproliferative activity, we tested two sesquiterpene derivatives, 8-hydrocalamenene (HC) and 8,14-dihydrocalamenene (DHC), for their ability to induce apoptosis and suppress signal transducer and activator of transcription 3 (STAT3) activation in multiple myeloma (MM) U266 cells. We found that HC inhibited cell viability in U266, but not in peripheral blood mononuclear cells. HC exerted significant cytotoxicity and induced substantial subG1-phase arrest and apoptosis as compared with DHC. HC inhibited the expression of gene products involved in antiapoptosis (Bcl-2 and Bcl-xL), proliferation (cyclin D1), and invasion (MMP-9), all of which are known to be regulated by STAT3. Furthermore, HC up-regulated cyclin-dependent kinase inhibitor p21 and induced apoptosis through the activation of caspase-8, -9, and -3 in U266 cells. Interestingly, HC blocked constitutive STAT3 activation through the inhibition of activation of upstream kinases Janus-like kinase 1 (JAK1), JAK2, and c-Src and up-regulated PIAS3. Deletion of STAT3 reversed cytotoxic effects and the down-regulation of cyclin D1 and c-myc by HC in MM cells. Finally, this sesquiterpene significantly synergized the cytotoxic and apoptotic effects of bortezomib in U266 cells. Taken together, these results suggest that HC is a novel blocker of STAT3 activation which may have a potential in the prevention and treatment of MM.

Topics: Antineoplastic Agents, Phytogenic; Caspase 8; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Humans; Janus Kinase 2; Multiple Myeloma; Plant Extracts; Polycyclic Sesquiterpenes; Polygonum; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Terpenes

Mice susceptible to plasma cell tumors provide a useful model for human multiple myeloma. We previously showed that mice expressing an Eµ-v-abl oncogene solely develop plasmacytomas. Here we show that loss of the proapoptotic BH3-only protein Bim or, to a lesser extent, overexpression of antiapoptotic Bcl-2 or Mcl-1, significantly accelerated the development of plasmacytomas and increased their incidence. Disease was preceded by an increased abundance of plasma cells, presumably reflecting their enhanced survival capacity in vivo. Plasmacytomas of each genotype expressed high levels of v-abl and frequently harbored a rearranged c-myc gene, probably as a result of chromosome translocation. As in human multiple myelomas, elevated expression of cyclin D genes was common, and p53 deregulation was rare. Our results for plasmacytomas highlight the significance of antiapoptotic changes in multiple myeloma, which include elevated expression of Mcl-1 and, less frequently, Bcl-2, and suggest that closer attention to defects in Bim expression is warranted.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Blotting, Western; Cells, Cultured; Cyclin D1; Female; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Multiple Myeloma; Mutation; Myeloid Cell Leukemia Sequence 1 Protein; Plasmacytoma; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-abl; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Protein p53

A 69-year-old male was referred to our hospital because of anemia, renal insufficiency, and a positive urine test for Bence-Jones protein. A bone marrow examination showed 73.7% of myeloma cells with lymphoplasmacytic morphology, the strong expressions of CD20 and CD23 by flow cytometry, and the chromosomal aberration of CCND1/IGH by FISH analysis. He was diagnosed with multiple myeloma, IgG-λ type. The initial treatment with bortezomib plus dexamethasone (BD) provided a rapid decrease in the level of IgG; however, he developed bortezomib-induced recurrent paralytic ileus accompanied by aspiration pneumonia during the second course. Interestingly, CD23 expression on myeloma cells decreased from 87.7% to 2.2% after 2 courses of BD. Negative CD23 expression was maintained following lenalidomide plus dexamethasone therapy. There are extremely few reports on CD23 expression on myeloma cells, and this is the first case report of multiple myeloma in which CD23 expression was lost after BD therapy.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Bortezomib; Cyclin D1; Dexamethasone; Humans; Male; Multiple Myeloma; Pyrazines; Receptors, IgE

A number of specific chromosomal abnormalities define the subgroups of multiple myeloma. In a meta-analysis of two genome-wide association studies of multiple myeloma including a total of 1,661 affected individuals, we investigated risk for developing a specific tumor karyotype. The t(11;14)(q13;q32) translocation in which CCND1 is placed under the control of the immunoglobulin heavy chain enhancer was strongly associated with the CCND1 c.870G>A polymorphism (P = 7.96 × 10(-11)). These results provide a model in which a constitutive genetic factor is associated with risk of a specific chromosomal translocation.

Topics: Case-Control Studies; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Genome-Wide Association Study; Genome, Human; Genotype; Humans; In Situ Hybridization, Fluorescence; Karyotyping; Multiple Myeloma; Phenotype; Polymorphism, Single Nucleotide; Risk Factors; Translocation, Genetic

To delineate the pathology of immunoglobulin M-producing multiple myeloma (IgM MM).. Clinicopathologic data were reviewed for 15 cases meeting World Health Organization criteria for MM and having a serum IgM paraprotein. Immunohistochemistry was performed on diagnostic bone marrow specimens for common B-cell and plasma cell markers.. Of the 15 IgM MM bone marrows reviewed, 6 (40%) had lymphoplasmacytoid cytology, and 12 (80%) expressed CD19, CD20, and/or CD45. Cyclin D1 expression was common (11 cases, 73%) and usually associated with t(11;14). No cases expressed CD5 or had an associated CD5-positive B-cell population. CD117 was positive in 20% of cases.. The frequent B-cell-associated antigen expression by IgM MM distinguishes it from other MM types, causing significant pathologic overlap with lymphoplasmacytic lymphoma (LPL). However, IgM MM is usually distinguished from LPL by aberrant cyclin D1 expression or t(11;14). Therefore, assessing for these abnormalities is recommended when evaluating bone marrow involvement by IgM-associated lymphoplasmacytoid disorders.

Topics: Aged; B-Lymphocytes; Biomarkers, Tumor; Bone Marrow; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Female; Humans; Immunoglobulin M; Male; Middle Aged; Multiple Myeloma; Paraproteinemias; Rare Diseases; Translocation, Genetic

Accumulating evidence suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. It is not known, however, whether genome organization differs in nonmalignant cells from patients as compared to their cellular counterparts from healthy donors. This could contribute to translocation potential causing cancer. Multiple myeloma is a hematopoietic cancer of the B-lineage, characterized by karyotypic instability, including chromosomal translocations involving the IGH locus and several translocation partners. Utilizing 3-D FISH and confocal imaging, we investigate whether nuclear spatial positioning of the translocation-prone gene loci, IGH, FGFR3, and CCND1 differs in nonmalignant cell subsets from multiple myeloma patients as compared to positioning in their corresponding healthy donor cell subsets. 3-D analysis software was used to determine the spatial proximity of potential translocation pairs and the radial distribution of each gene. We observed that in all cell subsets, the translocation-prone gene loci are intermediately located in the nucleus, while a control locus occupies a more peripheral position. In nonmalignant B-cells from multiple myeloma patients, however, the translocation-prone gene loci display a more central nuclear position and close spatial proximity. Our results demonstrate that gene positioning in nonmalignant B-cells from multiple myeloma patients differs from that in healthy donors, potentially contributing to translocation probability in patient cells. We speculate that genome reorganization in patient B-cells may closely reflect gene positioning at the time the multiple myeloma-specific translocation initially formed, thus influencing translocation probability between proximal loci in the B-cell population from which the malignancy emerged.

Topics: B-Lymphocytes; Bone Marrow Cells; Case-Control Studies; Cell Nucleus; Cyclin D1; DNA, Intergenic; Genetic Loci; Hematopoietic Stem Cells; Humans; Immunoglobulin Heavy Chains; In Situ Hybridization, Fluorescence; Multiple Myeloma; Receptor, Fibroblast Growth Factor, Type 3; Translocation, Genetic

Multiple Myeloma (MM) is a lymphatic neoplasm characterized by clonal proliferation of malignant plasma cell that eventually develops resistance to chemotherapy. Drug resistance, differentiation block and increased survival of the MM tumor cells result from high genomic instability. Chromosomal translocations, the most common genomic alterations in MM, lead to dysregulation of cyclin D, a regulatory protein that governs the activation of key cell cycle regulator--cyclin dependent kinase (CDK). Genomic instability was reported to be affected by over expression of another CDK regulator--cyclin E (CCNE). This occurs early in tumorigenesis in various lymphatic malignancies including CLL, NHL and HL. We therefore sought to investigate the role of cyclin E in MM. CCNE1 expression was found to be heterogeneous in various MM cell lines (hMMCLs). Incubation of hMMCLs with seliciclib, a selective CDK-inhibitor, results in apoptosis which is accompanied by down regulation of MCL1 and p27. Ectopic over expression of CCNE1 resulted in reduced sensitivity of the MM tumor cells in comparison to the paternal cell line, whereas CCNE1 silencing with siRNA increased the cell sensitivity to seliciclib. Adhesion to FN of hMMCLs was prevented by seliciclib, eliminating adhesion-mediated drug resistance of MM cells. Combination of seliciclib with flavopiridol effectively reduced CCNE1 and CCND1 protein levels, increased subG1 apoptotic fraction and promoted MM cell death in BMSCs co-culture conditions, therefore over-coming stroma-mediated protection. We suggest that seliciclib may be considered as essential component of modern anti MM drug combination therapy.

Topics: Apoptosis; Cell Line, Tumor; Cyclin D1; Cyclin E; Flavonoids; Gene Expression Profiling; Gene Expression Regulation; Gene Silencing; Genomic Instability; Humans; Multiple Myeloma; Oncogene Proteins; Piperidines; Protein Kinase Inhibitors; Purines; RNA, Small Interfering; Roscovitine

Multiple myeloma (MM) comprises 1% of all malignancies and 13% of hematological malignancies in the Caucasian population. Yearly incidence is 4/100,000 in the US and is higher in blacks and males [1]. The pathogenesis of the disease is relatively unknown; several chromosomal abnormalities have been related to the development of the disease,but none is characteristic of MM. Cyclin-D1 is a protein encoded by the CCND1 (bcl-1) gene on chromosome 11q13, and is an important regulator of G1 to S phase progression.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; Bone Marrow; Cohort Studies; Cyclin D1; Female; Follow-Up Studies; Greece; Humans; Male; Middle Aged; Multiple Myeloma; Neoplasm Staging; Prognosis; RNA, Messenger; Survival Analysis; Up-Regulation

To investigate the effect of baicalein on proliferation and migration of multiple myeloma (MM) cell lines and its molecular mechanism.. The MM cell line RPMI-8226 and U266 cells were used as the model, and treated with different concentration and time of baicalein the effect of baicalein on the MM cells proliferation was assessed by MTT assay. With or without baicalein or Interleukin-6 (IL-6) treatment, the β-catenin protein level was analyzed by immunofluorescence assay and western blot assay and mRNA levels of β-catenin, c-myc, cyclin D1 and integrin 7 gene by RT-PCR. Transwell chamber migration assay was used to detect the cells migration ability with different concentration of baicalein cultured.. Baicalein inhibited the MM cell line RPMI 8226 and U266 cell proliferation in a dose- and time-dependent manner. It simultaneously inhibited β-catenin protein level to resist the effect of IL-6 on inducing MM cell proliferation, and resulted in decrease of β-catenin, c-myc, cyclinD1 and integrin β7 mRNA levels. Baicalein also decreased migration ability of MM cells in a dose-dependent manner by SDF-1.. Baicalein can inhibit MM cells proliferation and migration, and its molecular mechanisms are associated with inhibition of proliferation related genes β-catenin, c-myc, cyclin D1 and integrin β7 expression.

Topics: Antigens, CD; beta Catenin; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Flavanones; Humans; Integrin alpha Chains; Interleukin-6; Multiple Myeloma; Proto-Oncogene Proteins c-myc; RNA, Messenger

Recent reports have indicated that decursin can induce apoptosis, suppress tumor growth, and inhibit angiogenesis. In this experiment, we investigated how decursin could potentiate the cytotoxic effects of bortezomib in human multiple myeloma cells. We found that decursin inhibited cell viability in U266, MM.1S and ARH77 cells, but not in peripheral blood mononuclear cells (PBMC). Decursin-induced apoptosis through the activation of caspase-8, -9, and -3 in U266 cells. This correlated with the down-regulating of cyclin D1, bcl-2, bcl-xL, survivin, and the vascular endothelial growth factor (VEGF), which are all regulated by the activation of signal transducers and the activator of transcription 3 (STAT3). Indeed, decursin inhibited constitutive STAT3 activation through inhibition of the activation of Janus-activated kinase 2 (JAK2) in U266 cells. In addition, decursin inhibited interleukin-6-inducible STAT3 activation in a time-dependent manner in MM.1S cells. Interestingly, decursin significantly potentiated the apoptotic effects of bortezomib in U266 cells. These effects of decursin were correlated with the suppression of constitutive STAT3 activation in U266 cells. Overall, these results suggest that decursin is a novel blocker of STAT3 activation and it may be a potential candidate for overcoming chemo-resistance through suppression of this signaling.

Topics: Apoptosis; Benzopyrans; Boronic Acids; Bortezomib; Butyrates; Caspase 3; Cell Line, Tumor; Cyclin D1; DNA; G1 Phase; Humans; Interleukin-6; Janus Kinase 2; Multiple Myeloma; Phosphorylation; Pyrazines; Signal Transduction; STAT3 Transcription Factor

Lenalidomide plays an important role in our chemotherapeutic armamentarium against multiple myeloma, in part by exerting direct anti-proliferative and pro-apoptotic effects. Unfortunately, long-term exposure leads to the development of drug resistance through unknown mechanisms, and we therefore sought to identify pathways that could be responsible for this phenotype. Chronic drug exposure produced myeloma cell lines that were tolerant of the direct effects of lenalidomide, with a degree of resistance of up to 2,500-fold. Gene expression profiling and pathway analysis identified dysregulation of the Wnt/β-catenin pathway as a consistent change across four independent cell isolates, and a pair of primary plasma cell samples. Acute drug treatment also increased β-catenin transcription by 3-fold or more, and both acute and chronic exposure resulted in enhanced accumulation of β-catenin protein by up to 20-fold or more. This produced Wnt/β-catenin pathway activation, as judged by increased activity of a lymphoid enhancer factor/T-cell factor promoter reporter, and enhanced accumulation of the downstream targets cyclin D1 and c-Myc. Components of the β-catenin destruction complex were also impacted by lenalidomide, which suppressed casein kinase 1α expression while augmenting glycogen synthase kinase 3α/β phosphorylation. Stimulation of Wnt/β-catenin signaling with recombinant Wnt-3a, or by overexpression of β-catenin, reduced the anti-proliferative activity of lenalidomide. Conversely, suppression of β-catenin with small hairpin RNAs restored plasma cell sensitivity to lenalidomide. Together, these findings support the hypothesis that lenalidomide mediates activation of Wnt/β-catenin signaling in plasma cells as a mechanism of inducible chemoresistance through effects at the transcriptional and post-translational levels.

Topics: Antineoplastic Agents; beta Catenin; Casein Kinase Ialpha; Cell Line, Tumor; Cyclin D1; Drug Resistance, Neoplasm; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lenalidomide; Multiple Myeloma; Plasma Cells; Proto-Oncogene Proteins c-myc; Signal Transduction; Thalidomide; Transcription, Genetic; Wnt Proteins; Wnt3 Protein; Wnt3A Protein

Interphase fluorescence in situ hybridization (FISH) can identify submicroscopic deletions adjacent to the breakpoints of rearrangements undetected by conventional cytogenetics. In this study, the characteristics and frequency of the IgH deletion identified by interphase FISH were investigated in patients with multiple myeloma (MM) and chronic lymphocytic leukemia (CLL).. The study group included 29 patients with MM and eight patients with CLL. Interphase FISH was performed with the IgH dual color, break-apart rearrangement probe and the IgH/CCND1 dual color, dual fusion translocation probe.. The IgH deletion was found in 14% (4/29) of patients with MM and 13% (1/8) of the patients with CLL. Four patients had deletions of the whole or variable region of IgH on the native chromosome 14, whereas one patient had a deletion of the IgH variable region on a der(11)t(11;14). In two patients, the IgH break-apart FISH showed both patterns with and without IgH deletions. In cases showing the same pattern by IgH break-apart FISH, the IgH/CCND1 FISH showed different patterns, and vice versa.. A variety of patterns of the IgH deletion were identified by interphase FISH using IgH break-apart and IgH/CCND1 probes in patients with MM and CLL. The results of this study suggest that the integrated information obtained with IgH break-apart and IgH/CCND1 FISH was needed to interpret FISH results unambiguously.

Topics: Aged; Chromosomes, Human, Pair 14; Cyclin D1; Female; Gene Deletion; Humans; Immunoglobulin Heavy Chains; In Situ Hybridization, Fluorescence; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Multiple Myeloma; Translocation, Genetic

Multiple myeloma (MM) is a malignancy of the plasmocyte and is associated with various symptoms such as anemia, immunodeficiency, bone lesions and kidney insufficiency. Although prognosis was poor until some years ago, recent advances that introduced newer molecular targeting agents such as bortezomib and thalidomide have resulted in a better prognosis for MM. However, clinical manifestations and the relationship between cellular and molecular findings, including chromosomal translocation and the related overexpression of oncogenes such as CCND1 (cyclin D1) and FGFR3 (fibroblast growth factor receptor 3), remain unclear. It has been reported that a specific translocation may influence the prognosis of MM. Although translocations and overexpressed genes should be examined in ordinary clinical investigations, limited definitive assays for translocation involve the use of FISH (fluorescent in situ hybridization) or SKY (special karyotypic) methods. We therefore, attempted to establish a quick detection method for major translocated genes such as FGFR3, CCND1, CCND3 and MAF using multiplex RT-PCR (MP-RT-PCR). MP-RT-PCR can be performed within several to 24 h after bone marrow samples are taken. Two of 21 bone marrow blood samples from MM patients were analyzed using MP-RT-PCR and double-color FISH, and the results of both methods were compatible. Future utilization and elaboration of this method may help our understanding of the cell biology and clinical features of MM.

Topics: Aged; Aged, 80 and over; Cyclin D1; Cyclin D3; Female; Gene Expression; Humans; In Situ Hybridization, Fluorescence; Male; Middle Aged; Multiple Myeloma; Proto-Oncogene Proteins c-maf; Receptor, Fibroblast Growth Factor, Type 3; Reverse Transcriptase Polymerase Chain Reaction; Translocation, Genetic

This study was aimed to investigate the effect of curcumin in combination with bortezomib on the proliferation and apoptosis of human MM cell line H929 in vitro, and to explore its mechanisms. MTT assay was applied to detect the inhibitory effects of curcumin and bortezomib either alone or combined at different concentrations on H929 cells, and flow cytometry was employed to assay the apoptosis rate. In addition, RT-PCR was used to analyze the mRNA expression of gene BCL-2, BAX, cyclin D1. Immunofluorescence technique was performed to study the location changes of NF-κB P65 in different groups. The results showed that both curcumin and bortezomib inhibited the proliferation of MM cell line H929 in dose-dependent manner, and combination of these two drugs displayed synergistical effect. A much higher apoptosis rate was determined by flow cytometry in combinative groups than that in single or control group. And RT-PCR showed, as compared with curcumin or bortezomib group, there was mRNA expression decrease of BCL-2, cyclin D1 but increase of BAX in combined group. The expression of NF-κB P65 in nucleus was downregulated in either the curcumin or bortezomib group, however, distribution of NF-κB P65 in cytoplasm was observed in combined group. It is concluded that the combination of curcumin and bortezomib is much more effective for the inhibiting proliferation and promoting apoptosis of H929 cell line, which may function by inhibiting the transcription of NF-κB and apoptosis-related genes.

Topics: Apoptosis; bcl-2-Associated X Protein; Boronic Acids; Bortezomib; Cell Line, Tumor; Cell Proliferation; Curcumin; Cyclin D1; Drug Therapy, Combination; Humans; Multiple Myeloma; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Transcription Factor RelA

Multiple myeloma is a plasma cell malignancy that is heterogeneous with respect to its causative molecular abnormalities and the treatment response of patients. The Bcl-2 protein family is critical for myeloma cell survival. ABT-737 is a cell-permeant compound that binds to Bcl-2 and Bcl-x(L) but not to Mcl-1. Using a myeloma cell line collection (n = 25) representative of different molecular translocations, we showed that ABT-737 effectively kills a subset of cell lines (n = 6), with a median lethal dose ranging from 7 ± 0.4 nM to 150 ± 7.5 nM. Of interest, all sensitive cell lines harbored a t(11;14). We demonstrated that ABT-737-sensitive and ABT-737-resistant cell lines could be differentiated by the BCL2/MCL1 expression ratio. A screen of a public expression database of myeloma patients indicates that the BCL2/MCL1 ratio of t(11;14) and hyperdiploid patients was significantly higher than in all other groups (P < .001). ABT-737 first induced the disruption of Bcl-2/Bax, Bcl-2/Bik, or Bcl-2/Puma complexes, followed by the disruption of Bcl-2 heterodimers with Bak and Bim. Altogether, the identification of a subset of cell lines and primary cells effectively killed by ABT-737 alone supported the evaluation of ABT-263, an orally active counterpart to ABT-737, for the treatment of t(11;14) and hyperdiploid groups of myeloma harboring a Bcl-2(high)/Mcl-1(low) profile.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Biphenyl Compounds; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Tumor Cells, Cultured

The combined use of microarray technologies and bioinformatics analysis has improved our understanding of biological complexity of multiple myeloma (MM). In contrast, the application of the same technology in the attempt to predict clinical outcome has been less successful with the identification of heterogeneous molecular signatures. Herein, we have reconstructed gene regulatory networks in a panel of 1,883 samples from MM patients derived from publicly available gene expression sets, to allow the identification of robust and reproducible signatures associated with poor prognosis across independent data sets.. Gene regulatory networks were reconstructed by using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) and microarray data from seven MM data sets. Critical analysis of network components was applied to identify genes playing an essential role in transcriptional networks, which are conserved between data sets.. Network critical analysis revealed that (i) CCND1 and CCND2 were the most critical genes; (ii) CCND2, AIF1, and BLNK had the largest number of connections shared among the data sets; (iii) robust gene signatures with prognostic power were derived from the most critical transcripts and from shared primary neighbors of the most connected nodes. Specifically, a critical-gene model, comprising FAM53B, KIF21B, WHSC1, and TMPO, and a neighbor-gene model, comprising BLNK shared neighbors CSGALNACT1 and SLC7A7, predicted survival in all data sets with follow-up information.. The reconstruction of gene regulatory networks in a large panel of MM tumors defined robust and reproducible signatures with prognostic importance, and may lead to identify novel molecular mechanisms central to MM biology.

Topics: Adaptor Proteins, Signal Transducing; Algorithms; Calcium-Binding Proteins; Cyclin D1; Cyclin D2; DNA-Binding Proteins; Gene Expression Profiling; Gene Regulatory Networks; Humans; Microfilament Proteins; Models, Genetic; Multiple Myeloma; Oligonucleotide Array Sequence Analysis; Treatment Outcome

Topics: Bone Marrow; Cell Size; Cyclin D1; Female; Humans; Lymphocytes; Middle Aged; Multiple Myeloma; Oncogene Proteins, Fusion; Plasma Cells; Translocation, Genetic

Multiple myeloma (MM) is a malignancy of the plasma cells (PCs) characterized by a wide variety of genetic and chromosomal abnormalities. In recent years, major attention was drawn to the significance of chromosomal aberrations involving chromosome arm 13q and the IGH region on chromosome band 14q32 as a prognostic indicator in MM. In this study we applied a combined cell morphology and FISH method for the analysis of coexistence of t(11;14)(q13;q32) with deletions of the long arm of chromosome 13 (Delta13) in PCs from 51 MM patients using several probes for the 13q14, 11q13, and IGH regions. We found 15 different variants of the t(11;14) that are the consequence of different 11q13 breakpoints and various deletions of Variable (del IGH Var) or Constant (del IGH Const) IGH segments and also duplications and losses of the IGH gene on the normal nontranslocated chromosome 14 as well as IGH/Cyclin D1 (CCND1) fusion on der(14) and CCND1/IGH fusions on der(11). A strong association between Delta13 and specific variants of t(11;14) was found: variants with deletion of the IGH gene or its segments were found only in MM cases with deleted chromosome 13, while the common translocation t(11;14) was found only in the MM cases with normal chromosome arm 13q. In contrast, we did not find any association between Delta13 and deletions of the IGH gene or its segments in the MM patients with t(4;14)(p16;q32).

Topics: Cell Shape; Chromosome Aberrations; Chromosome Breakage; Chromosome Deletion; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 14; Cyclin D1; Humans; Immunoglobulin Heavy Chains; In Situ Hybridization, Fluorescence; Multiple Myeloma; Prognosis; Translocation, Genetic

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD20; Biomarkers, Tumor; Cyclin D1; Cyclin D2; Female; Humans; Male; Middle Aged; Multiple Myeloma; Neoplasm Proteins; Survival Analysis

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Combined Chemotherapy Protocols; Boronic Acids; Bortezomib; Combined Modality Therapy; Cyclin D1; Dexamethasone; Hematopoietic Stem Cell Transplantation; Humans; Immunocompromised Host; Immunosuppression Therapy; Lenalidomide; Lymphoma, Mantle-Cell; Male; Melphalan; Middle Aged; Multiple Myeloma; Neoplasm Proteins; Neoplasms, Second Primary; Pyrazines; Rituximab; Thalidomide; Transplantation, Autologous

Aberrant expression of cyclin D1 is a common feature in multiple myeloma (MM) and always associated with mantle cell lymphoma (MCL). CCND1 gene is alternatively spliced to produce two cyclin D1 mRNA isoforms which are translated in two proteins: cyclin D1a and cyclin D1b. Both isoforms are present in MM cell lines and primary cells but their relative role in the tumorigenic process is still elusive.. To test the tumorigenic potential of cyclin D1b in vivo, we generated cell clones derived from the non-CCND1 expressing MM LP-1 cell line, synthesizing either cyclin D1b or cyclin K, a structural homolog and viral oncogenic form of cyclin D1a. Immunocompromised mice injected s.c. with LP-1K or LP-1D1b cells develop tumors at the site of injection. Genome-wide analysis of LP-1-derived cells indicated that several cellular processes were altered by cyclin D1b and/or cyclin K expression such as cell metabolism, signal transduction, regulation of transcription and translation. Importantly, cyclin K and cyclin D1b have no major action on cell cycle or apoptosis regulatory genes. Moreover, they impact differently cell functions. Cyclin K-expressing cells have lost their migration properties and display enhanced clonogenic capacities. Cyclin D1b promotes tumorigenesis through the stimulation of angiogenesis.. Our study indicates that cyclin D1b participates into MM pathogenesis via previously unrevealed actions.

Topics: Animals; Cell Cycle; Cell Line; Cell Movement; Cell Separation; Chick Embryo; Cyclin D1; Cyclins; Female; Flow Cytometry; Humans; Immunoblotting; Immunohistochemistry; Mice; Mice, Nude; Multiple Myeloma; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction

Cyclins D1, D2, and D3 (CCND1, 2, 3) are regulated by proteasomal degradation. Their overexpression in multiple myeloma (MM) has prognostic value. We performed this pilot study to analyze a possible association between CCND1-3 overexpression and response to treatment with the proteasome inhibitor bortezomib, since a specific prognostic marker for bortezomib response has not been reported, but would be ideal to predict who benefits most from bortezomib in times of several potentially efficient therapeutic options. Bone marrow (BM) specimens of 20/47 consecutive patients were available for reliable CCND1-3 analyses by real-time PCR. With CCND1 overexpression in 9/20 patients, the risk for progression after bortezomib treatment was significantly decreased (HR 0.102, 95% CI 0.021-0.498, p = 0.0048) and progression-free survival substantially prolonged (p = 0.0011). Our study is the first to suggest that overexpressed CCND1 in MM is an independent prognostic marker associated with a more durable response to bortezomib. These preliminary results warrant a larger study.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Bone Marrow; Boronic Acids; Bortezomib; Cyclin D1; Cyclin D2; Cyclin D3; DNA; Drug Resistance, Neoplasm; Female; Humans; Male; Middle Aged; Multiple Myeloma; Neoplasm Recurrence, Local; Pilot Projects; Polymerase Chain Reaction; Pyrazines; Salvage Therapy; Survival Rate; Treatment Outcome

Topics: Antineoplastic Agents; Boronic Acids; Bortezomib; Cyclin D1; Humans; Multiple Myeloma; Precision Medicine; Pyrazines; Treatment Outcome

We histologically and immunohistochemically studied 95 bone marrow (BM) reactive plasmacytoses. Ten biopsies from plasma cell myeloma (PCM) patients served as a control group. In addition, we studied 10 monoclonal gammopathy of undetermined significance (MGUS) cases. Histologically, plasmacytosis varied between 5% and 25% with an interstitial pattern of plasma cell (PC) distribution being characteristically displayed. Immunohistochemically, we did not find any CD56/NCAM nor cyclin D1 expression in all biopsies (95 of 95, 100%), not even a weak, doubtful one; PCs were all polyclonal and CD138 positive. On the contrary, myeloma-associated PCs showed monoclonality for kappa- or lambda- light chain and strong CD56/NCAM immunoreactivity (8 of 10, 80%); four of them were cyclin D1 positive. Osteoblasts exhibited similar CD56/NCAM expression in both groups. Our data confirm the diagnostic utility of CD56/NCAM in the phenotypic characterization of polyclonal plasma cells, suggesting an important role of this particular immunomarker in the BM trephine study of polyclonal versus neoplastic plasmacytic infiltrations.

Topics: Bone Marrow; CD56 Antigen; Cyclin D1; Humans; Immunoenzyme Techniques; Immunoglobulin lambda-Chains; Multiple Myeloma; Neural Cell Adhesion Molecules; Plasma Cells; Retrospective Studies

The Karpas-620 human myeloma cell line (HMCL) expresses high levels of Cyclin D1 (CCND1), but has a der(8)t(8;11) and a der(14)t(8;14), and not a conventional t(11;14). Fluorescent in situ hybridization (FISH) and array comparative genomic hybridization (aCGH) studies suggest that der(14)t(11;14) from a primary translocation underwent a secondary translocation with chromosome 8 to generate der(8)t(8;[14];11) and der(14)t(8;[11];14). Both secondary derivatives share extensive identical sequences from chromosomes 8, 11, and 14, including MYC and the 3' IgH enhancers. Der(14), with MYC located approximately 700 kb telomeric to the 3'IGH enhancer, expresses MYC. By contrast, der(8), with both CCND1 and MYC repositioned near a 3'IGH enhancer, expresses CCND1, which is telomeric of the enhancer, but not MYC, which is centromeric to the enhancer. The secondary translocation that dysregulated MYC resulted in extensive regions from both donor chromosomes being transmitted to both derivative chromosomes, suggesting a defect in DNA recombination or repair in the myeloma tumor cell.

Topics: Animals; Cell Line, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 8; Comparative Genomic Hybridization; Cyclin D1; Enhancer Elements, Genetic; Gene Dosage; Humans; In Situ Hybridization, Fluorescence; Mice; Multiple Myeloma; Proto-Oncogene Proteins c-myc; RNA; Translocation, Genetic

Prior studies of myeloma treated with conventional chemotherapy or autologous stem cell transplantation have shown immunohistochemistry for cyclin D1, fibroblast growth factor receptor 3, and p53 to be prognostically significant. The clinical significance of these phenotypic abnormalities in thalidomide-based regimens is currently unknown. We examined the clinical significance of immunohistochemistry for cyclin D1, fibroblast growth factor receptor 3, p53, and cyclin D3 in 94 patients treated with pegylated doxorubicin, vincristine, dexamethasone, and thalidomide, including 49 newly diagnosed and 45 relapsed/refractory patients. The incidence of positivity for cyclin D1, fibroblast growth factor receptor 3, p53, and cyclin D3 was similar in newly diagnosed versus relapsed/refractory groups (37%, 8%, 4%, and 2% versus 42%, 7%, 11%, and 2%, respectively). In contrast to prior studies of other therapeutic regimens, cyclin D1 negativity or fibroblast growth factor receptor 3 positivity did not convey an adverse progression-free or overall survival. p53 positivity, although uncommon, was associated with shorter progression-free and overall survival in newly diagnosed cases. The findings suggest that a thalidomide-based regimen may overcome the poor prognosis associated with a cyclin D1-negative or fibroblast growth factor receptor 3-positive phenotype.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Cell Nucleus; Clinical Trials, Phase II as Topic; Cyclin D1; Dexamethasone; Doxorubicin; Female; Fibroblast Growth Factor 3; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; Middle Aged; Multiple Myeloma; Ohio; Polyethylene Glycols; Survival Rate; Thalidomide; Tumor Suppressor Protein p53; Vincristine

We have shown that heightened AKT activity sensitized multiple myeloma cells to the antitumor effects of the mammalian target of rapamycin inhibitor CCI-779. To test the mechanism of the AKT regulatory role, we stably transfected U266 multiple myeloma cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analogue, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mammalian target of rapamycin inhibitors to down-regulate D-cyclin expression was significantly greater in AKT-transfected multiple myeloma cells due, in part, to the ability of AKT to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was shown in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild-type PTEN. Because extracellular signal-regulated kinase (ERK)/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells showed significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant "low-AKT" myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down-regulated D-cyclin protein expression and G1 arrest. However, ectopic overexpression of an activated MEK gene did not increase cap-independent translation of D-cyclin in "high-AKT" myeloma cells, indicating that mitogen-activated protein kinase/ERK kinase/ERK activity was required, but not sufficient, for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in multiple myeloma cells and ERK facilitates activity.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Mice; Multiple Myeloma; Phosphorylation; Protein Biosynthesis; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Retinoblastoma; Ribosomes; Sirolimus; TOR Serine-Threonine Kinases

Multiple myeloma is an incurable disease with heterogeneous clinical behavior. Bortezomib has offered some patients with relapsed and refractory disease an opportunity for prolonged survival. However, there remains a paucity of data in patients treated with bortezomib that accurately delineates and identifies such patients. This information is crucial to guide management.. In this study, we aimed to identify the patients most likely to respond to bortezomib salvage therapy. We analyzed the baseline clinical variables and profiled the baseline expression of a broad range of immunohistochemical markers of cell cycle activity, apoptosis, and angiogenesis in a large cohort of multiply relapsed myeloma patients recruited to one of two prospective multicentre trials assessing the efficacy of bortezomib salvage therapy.. Using the European Group for Bone Marrow Transplantation criteria, response (complete or partial) to bortezomib salvage therapy was associated with a previous history of complete response to alternative antimyeloma treatment. Patients who expressed cyclin D1 were more likely to achieve a response. In contrast, patients who expressed p16(INK4A), cytoplasmic p53, and the highest intensity of Bcl-2 staining had a poor response. Patients who achieved a response to bortezomib and those patients who expressed cyclin D1 at baseline showed a significant survival advantage. Patients who expressed FGFR3, a poor prognostic marker, responded equally well and had similar outcomes with bortezomib compared with FGFR3-negative patients.. Baseline clinical variables and selective immunohistochemical markers expressed by patients may be used effectively to identify patients that are most likely to achieve a meaningful clinical response to bortezomib salvage therapy.

Topics: Adult; Aged; Antineoplastic Agents; Boronic Acids; Bortezomib; Cell Cycle; Cohort Studies; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Humans; Immunohistochemistry; Male; Middle Aged; Multiple Myeloma; Prognosis; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Treatment Outcome; Tumor Suppressor Protein p53

Cyclin D dysregulation and overexpression is noted in the majority of multiple myeloma (MM) patients, suggesting its critical role in MM pathogenesis. Here, we sought to identify the effects of targeting cyclin D in MM. We first confirmed cyclin D mRNA overexpression in 42 of 64 (65%) patient plasma cells. Silencing cyclin D1 resulted in >50% apoptotic cell death suggesting its validity as a potential therapeutic target. We next evaluated P276-00, a clinical-grade small-molecule cyclin-dependent kinase inhibitor as a way to target the cyclins. P276-00 resulted in dose-dependent cytotoxicity in MM cells. Cell-cycle analysis confirmed either growth arrest or caspase-dependent apoptosis; this was preceded by inhibition of Rb-1 phosphorylation with associated downregulation of a range of cyclins suggesting a regulatory role of P276-00 in cell-cycle progression through broad activity. Proliferative stimuli such as interleukin-6, insulin-like growth factor-1 and bone-marrow stromal cell adherence induced cyclins; P276-00 overcame these growth, survival and drug resistance signals. Because the cyclins are substrates of proteasome degradation, combination studies with bortezomib resulted in synergism. Finally, in vivo efficacy of P276-00 was confirmed in an MM xenograft model. These studies form the basis of an ongoing phase I study in the treatment of relapsed/refractory MM.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Bone Marrow; Boronic Acids; Bortezomib; Caspases; Cell Adhesion; Cell Cycle; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor Proteins; Down-Regulation; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Drug Synergism; Flavones; Gene Expression Profiling; Humans; Insulin-Like Growth Factor I; Interleukin-6; Male; Mice; Mice, SCID; Multiple Myeloma; Oligonucleotide Array Sequence Analysis; Phosphorylation; Pyrazines; Retinoblastoma Protein; Stromal Cells; Transplantation, Heterologous; Tumor Cells, Cultured

Multiple myeloma (MM) is an incurable B-cell malignancy characterised by uncontrolled growth and accumulation of malignant plasma cells in the bone marrow. Aberrant expression of CD56 in patients with MM is thought to contribute to a worsened disease course and metastasis. We therefore investigated the regulation of the CD56 promoter in relation to typical clinical factors. We used qPCR and FACS to measure the expression levels of CD56, and potential regulatory factors in patients with MM and related these with MM progression/prognosis. The transcription factors BTBD3, Pax5, RUNX1 and MMSET were positively associated with CD56 expression, as was CYCLIN D1, which is involved in disease progression, anti-apoptosis and proliferation. RUNX1 was negatively associated with the survival of stem-cell transplanted patients. Our findings propose four potential activators of the CD56 promoter and for CD56 to be involved in proliferation and anti-apoptosis, leading to disease progression in MM.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; CD56 Antigen; Cell Proliferation; Cyclin D1; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Multiple Myeloma; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Messenger; Survival Rate; Transcription Factors

Genetic and epigenetic changes in multiple myeloma (MM) correlate with the stage of the disease. Therefore, we investigated how cytostatics and gamma radiation influence MM-associated histone modifications. ChIP-PCR and ChIP-on-chip technologies were used to quantify H3K9 acetylation and H3K9 dimethylation at select loci in MM patients, lymphoblastoid ARH-77, and myeloma MOLP-8 cells. Genome-wide analysis revealed that the cytostatic, melphalan, increased H3K9 acetylation at multiple gene promoters in ARH-77 cells. Melphalan and gamma radiation also influenced histone modification of prognostically important c-myc and CCND1 genes in ARH-77 and MOLP-8 cells. Moreover, H3K9 acetylation at c-myc and CCND1 promoters was increased in individual MM patients after melphalan treatment. Western blotting revealed that these effects were accompanied by changes in c-MYC and cyclin D1 protein levels. Taken together, we showed that cytostatics significantly alter histone modification of tumor-related genes which is indispensable for understanding cancer therapies.

Topics: Antineoplastic Agents; Apoptosis; Base Sequence; Cell Proliferation; Chromatin Immunoprecipitation; Combined Modality Therapy; Cyclin D1; DNA Primers; Epigenesis, Genetic; Flow Cytometry; Gamma Rays; Genes, myc; Humans; Multiple Myeloma; Polymerase Chain Reaction

The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. We identify DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. Loss of DEPTOR activates S6K1, Akt, and SGK1, promotes cell growth and survival, and activates mTORC1 and mTORC2 kinase activities. DEPTOR overexpression suppresses S6K1 but, by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt. Consistent with many human cancers having activated mTORC1 and mTORC2 pathways, DEPTOR expression is low in most cancers. Surprisingly, DEPTOR is highly overexpressed in a subset of multiple myelomas harboring cyclin D1/D3 or c-MAF/MAFB translocations. In these cells, high DEPTOR expression is necessary to maintain PI3K and Akt activation and a reduction in DEPTOR levels leads to apoptosis. Thus, we identify a novel mTOR-interacting protein whose deregulated overexpression in multiple myeloma cells represents a mechanism for activating PI3K/Akt signaling and promoting cell survival.

Topics: Cell Line; Cell Survival; Cyclin D1; Cyclin D3; Cyclins; Humans; Intracellular Signaling Peptides and Proteins; Multiple Myeloma; Phosphatidylinositol 3-Kinases; Protein Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases

Chromosomal rearrangements and copy number variation are frequently observed in cancer cells, including multiple myeloma (MM). Karyotypic abnormalities seen in MM cells correlate with the disease stage and drug responses. Here, we investigate the nuclear arrangement of the 1q21 region; amplification of this region is an important diagnostic and prognostic marker of MM. We examined the lymphoblastoid cell line CD138- ARH-77, multiple myeloma CD138+ MOLP-8 cells, and the CD138+ bone marrow fraction of patients diagnosed with MM. In this experimental system, we observed that gamma-radiation and selected cytostatic drugs such as melphalan and dexamethasone did not significantly alter the nuclear radial arrangement of the 1q21 region and other relevant regions of chromosome 1. Similarly, conserved nuclear radial positioning after cytostatic treatment was observed for the c-myc, TP53, CCND1, and IgH loci. When analyzed Mcl-1, a protein encoded by a gene mapped to the 1q21 region, we found that the variant Mcl1S is highly expressed in multiple myeloma MOLP-8 cells, but not in peripheral blood lymphocytes of healthy donors or lymphoblastoid ARH-77 cells; this is in contrast to the expression pattern of the Mcl-1L variant. On the basis of these observations we suggest that the 1q21 region is an important diagnostic marker of MM, particularly the gene encoding the Mcl-1S variant, which can be easily detected by western analysis.

Topics: Biomarkers, Tumor; Cell Line, Tumor; Cell Nucleus; Chromosome Mapping; Chromosomes, Human, Pair 1; Cyclin D1; Humans; Immunoglobulin Heavy Chains; Interphase; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2

Topics: Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; G1 Phase; Humans; Lymphoma, Mantle-Cell; Multiple Myeloma; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; S Phase

Plasma cell myeloma is a frequent hematogeneous disorder that occurs mainly in older people. Not only bone marrow smears but also clots and/or biopsied specimens are often taken for confirmation of pathological diagnosis. Some specimens show sheet-like plasma cell proliferation associated with immunoglobulin monotype on immunohistology, which readily leads to diagnosis, but many samples do not clearly show light-chain restriction. The aim of the present study was therefore to examine CD79a expression because some samples had reduced expression or none at all. The immunoreactivity of CD79a was categorized into three groups: positive, weakly positive and negative, compared with scattering non-neoplastic plasma cells in the same specimen. Out of 100 specimens of plasma cell myeloma, 48% were positive for CD79a, 15% were weakly positive, and 37% were negative. In contrast, overexpression of cyclinD1 was detected in 26% of examined samples. CD79a-negative cases had a significantly lower percentage of positive staining for cyclinD1 than CD79a-positive or weakly positive cases. Clinicopathological data showed that CD79a-negative expression was associated with decreased platelet numbers in patients. The present study indicates that downregulation or loss of CD79a and/or overexpression of cyclin D1, observed in 59% of neoplastic plasma cell samples, could provide a strong diagnostic clue without regard to the results of immunoglobulin light-chain restriction.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD20; CD79 Antigens; Cyclin D1; Down-Regulation; Female; Humans; Immunohistochemistry; Male; Middle Aged; Multiple Myeloma; Proto-Oncogene Proteins c-maf

We established a myeloma cell line (RPMI8226) with cyclin D1 overexpression in which the transfected cyclin D1 gene was stably expressed. D1 transfectants showed down-regulation of cyclin D2. Cell proliferation analysis did not show any differences among RPMI8226, mock control, and D1 transfectants. The number of S-phase cells increased while the number of G0/G1- and G2/M-phase cells decreased in D1 transfectants, which indicates a prolonged S-phase caused by cyclin D1 transfection. A decreased number of G2/M-phase cells was also detected in myeloma cells of patients with translocation t(11;14)(q13;q32). Western blot analysis revealed an increase in the hyperphosphorylated form of retinoblastoma (Rb) protein in D1 transfectants; however, the expression of p53, p16, Bax, Bad, Bcl-2, and Mcl-1 did not significantly change. Treatment with anti-myeloma drugs (melphalan, dexamethasone, bortezomib and immunomodulatory compounds) induced apoptosis earlier in D1 transfectants compared with RPMI8226 and mock control via the activation of both caspase-8 and -9. However, we could not detect a relationship between cyclin D1 expression and the response to treatment with VAD and bortezomib. Therefore, we assume that high sensitivity to anti-myeloma drugs depends on the duration of the S-phase, but a clinical response might depend on the number of myeloma cells with cyclin D1 overexpression.

Topics: Aged; Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Dexamethasone; Female; Humans; Intracellular Signaling Peptides and Proteins; Male; Melphalan; Middle Aged; Multiple Myeloma; Phosphorylation; Pyrazines; Retinoblastoma Protein; Thalidomide; Time Factors; Transfection; Up-Regulation; Vincristine

Several studies have confirmed that CD20 is expressed in about 20% of multiple myeloma (MM) cases. This is closely related to a chromosomal translocation between chromosome 11 and chromosome 14, which results in the expression of CyclinD1. The use of rituximab (RIT) in the treatment of CD20-positive MM has been reported, however its effectiveness is still not well established. We encountered a case of CD20-positive/CyclinD1-positive MM; interestingly, CD20 expression could not be detected in MM cells following RIT-combined chemotherapy, while it gradually recovered when RIT therapy was discontinued. This is the first report in which the transition of CD20 expression was accurately analyzed by flow cytometry and immunohistochemical staining before, during and after RIT treatment. Consequently, this case provides insight regarding the mechanism through which CD20 expression is lost following RIT therapy for CD20-positive lymphoid neoplasm, as well as the efficacy of RIT in CD20-positive MM.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Antineoplastic Agents; Biomarkers, Tumor; Cyclin D1; Female; Gene Expression; Humans; Middle Aged; Multiple Myeloma; Rituximab; Treatment Outcome

Overlapping of essential thrombocythemia (ET) and multiple myeloma (MM) has been extremely rare. Our report concerns a case with concomitant ET and MM, where JAK2V617F was present in non-myeloma peripheral blood leukocytes and bone marrow (BM) hematopoietic cells, but not in BM-derived CD138-positive myeloma cells. In contrast, double-color fluorescence in situ hybridization analysis showed that BM-derived CD138-positive myeloma cells possessed the gene translocation between the immunoglobulin heavy chain gene and the cyclin D1 gene, which was not involved in non-myeloma hematopoietic cells. This is the first case with concomitant ET and MM in which the 2 hematologic neoplasms were shown to have originated from separate malignant clones at hierarchically different differentiation levels resulting from independent acquisition of different molecular aberrations. Among the 10 reported cases, including ours, ET preceded MM in 8 cases, but MM never preceded ET. We suggest that MM clones may have greater proliferative potency compared with ET clones, and that the treatment modification from ET to MM did not seem to exacerbate ET in most reported cases, perhaps because of the suppression of the ET clone by both the cytotoxic effect of anti-myeloma therapy and the clonal repression by MM progression.

Topics: Aged; Amino Acid Substitution; Cyclin D1; Female; Hematopoietic Stem Cells; Humans; Immunoglobulin Heavy Chains; Janus Kinase 2; Leukocytes; Multiple Myeloma; Mutation, Missense; Syndecan-1; Thrombocytopenia; Translocation, Genetic

Multiple myeloma (MM) frequently shows overexpression of cyclin D1, either due to a t(11;14)(q13;q32) translocation, or in association with polysomy 11. The predominant expression of a cyclin D1 mRNA isoform lacking the 3'-untranslated region (Delta3'UTR) is associated with higher total cyclin D1 mRNA levels, increased proliferation and poor prognosis in mantle cell lymphoma, and can be caused by genetic alterations of the 3'UTR region. The role of this cyclin D1 isoform in MM is unknown. We therefore quantified levels of total and Delta3'UTR cyclin D1 mRNA by real-time RT-PCR in cytogenetically characterized cyclin D1+MM primary cases, and cyclin D1+cell lines. Both long and Delta3'UTR cyclin D1 transcripts were expressed in 35/41 MM cases, but none of the samples showed complete loss of the long transcript or genomic alterations of the 3'UTR. Predominance of the Delta3'UTR mRNA was associated with higher cyclin D1 levels in cases with t(11;14), but did not correlate with the proliferation rate, suggesting a different role of this isoform in MM.

Topics: 3' Untranslated Regions; Adult; Aged; Aged, 80 and over; Cell Line, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Female; Humans; Male; Middle Aged; Multiple Myeloma; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Deletion; Translocation, Genetic

D-cyclins are regulators of cell division that act in a complex with cyclin-dependent kinases to commit cells to a program of DNA replication. D-cyclins are overexpressed in many tumors, including multiple myeloma and leukemia, and contribute to disease progression and chemoresistance. To better understand the role and impact of D-cyclins in hematologic malignancies, we conducted a high throughput screen for inhibitors of the cyclin D2 promoter and identified the drug cyproheptadine. In myeloma and leukemia cells, cyproheptadine decreased expression of cyclins D1, D2, and D3 and arrested these cells in the G(0)/G(1) phase. After D-cyclin suppression, cyproheptadine induced apoptosis in myeloma and leukemia cell lines and primary patient samples preferentially over normal hematopoietic cells. In mouse models of myeloma and leukemia, cyproheptadine inhibited tumor growth without significant toxicity. Cyproheptadine-induced apoptosis was preceded by activation of the mitochondrial pathway of caspase activation and was independent of the drug's known activity as an H1 histamine and serotonin receptor antagonist. Thus, cyproheptadine represents a lead for a novel therapeutic agent for the treatment of malignancy. Because the drug is well tolerated and already approved in multiple countries for clinical use as an antihistamine and appetite stimulant, it could be moved directly into clinical trials for cancer.

Topics: Animals; Apoptosis; Cell Line; Cell Line, Tumor; Cyclin D1; Cyclin D2; Cyclin D3; Cyclins; Cyproheptadine; Drug Screening Assays, Antitumor; Gene Expression Regulation; Humans; Leukemia, Myeloid, Acute; Mice; Multiple Myeloma

Lycorine is a natural anti-tumor alkaloid extracted from Amaryllidaceae and has various biological effects on malignant cells. The present study explores the effects of lycorine on the human multiple meyloma cell line, KM3, and the possible mechanisms of these effects. An MTT assay showed that lycorine had significant inhibitory activity on KM3 cells. The growth rates of the KM3 cells exposed to lycorine evidently slowed down. Cell fluorescent apoptotic morphological changes, DNA degradation fragments, and a sub-G1 peak were detected, indicating the occurrence of cell apoptosis after lycorine treatment. Furthermore, the release of mitochondrial cytochrome c, the augmentation of Bax with the attenuation of Bcl-2, and the activation of caspase-9, -8, and -3 were also detected, suggesting that the mitochondrial pathway and the death acceptor pathway were also involved. The results also showed that lycorine was able to block the cell cycle at the G0/G1 phase through the downregulation of both cyclin D1 and CDK4. In summary, lycorine can suppress the proliferation of KM3 cells and reduce cell survival by arresting cell cycle progression as well as inducing cell apoptosis.

Topics: Amaryllidaceae Alkaloids; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Cyclin-Dependent Kinase 4; Cytochromes c; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; Phenanthridines; Plant Extracts; Proto-Oncogene Proteins c-bcl-2

Plasma cell myelomas (PMs) exhibit clinical and molecular heterogeneity. To date, morphology and immunohistochemistry on bone marrow trephines are of limited value to stratify patients into different prognostic categories. However, some chromosomal translocations are of prognostic and/or of predictive importance in PMs. In this study, the prognostic significance of morphology, CyclinD1 expression, proliferation index (Mib1) and presence of the translocations FGFR3/IgH [t(4;14)] and CCND1/IgH [t(11;14)] are compared in 119 patients with PM. Immunohistochemistry and fluorescence in situ hybridization analysis were carried out on a tissue microarray containing bone marrow trephines. Hundred and one PMs showed a mature morphology whereas 10 were immature. All but one PM carrying a translocation showed a mature morphology. Patients with a t(4;14) (12%) had a statistically significant shorter 1-year survival (P=0.004), whereas those with a t(11;14) (21%) had a trend towards a better clinical outcome. CyclinD1 protein expression was not significantly associated with survival. Besides the t(4;14), an immature morphology (P<0.001) and a proliferation index (Mib1) of more than 10% (P=0.002) were associated with a significantly worse outcome. A high occurrence of strong CyclinD1 protein expression in the tumor cells was predictive of either a t(11;14) or of a low level amplification of the CCND1 gene, suggesting that different molecular mechanisms may have lead to an over-expression of the CyclinD1 protein in PMs. These findings demonstrate that a high proliferation rate and translocations involving the IgH locus can stratify mature PMs into groups with distinct survival probabilities.

Topics: Biomarkers, Tumor; Bone Marrow Cells; Cell Proliferation; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 4; Cyclin D1; Female; Humans; In Situ Hybridization, Fluorescence; Ki-67 Antigen; Male; Middle Aged; Molecular Epidemiology; Multiple Myeloma; Prognosis; Retrospective Studies; Survival Rate; Switzerland; Tissue Array Analysis; Translocation, Genetic

Capsaicin, a constituent of green and red peppers, has been linked with suppression of tumorigenesis through a mechanism that is not well understood. Because the transcription factor signal transducer and activator of transcription 3 (STAT3) has been closely linked with tumorigenesis, we investigated the effect of this vanilloid on the STAT3 pathway in human multiple myeloma cells.. The effect of capsaicin on both constitutive and interleukin-6-induced STAT3 activation, associated protein kinases, and STAT3-regulated gene products involved in proliferation, survival and angiogenesis, cellular proliferation, and apoptosis in multiple myeloma cells was investigated.. We found that capsaicin inhibited constitutive activation of STAT3 in multiple myeloma cells in a dose- and time-dependent manner, with minimum effect on STAT5. Capsaicin also inhibited the interleukin-6-induced STAT3 activation. The activation of Janus-activated kinase 1 and c-Src, implicated in STAT3 activation, was also inhibited by the vanilloid, with no effect on extracellular signal-regulated kinase 1/2 activation. Pervanadate reversed the capsaicin-induced down-regulation of STAT3, suggesting the involvement of a protein tyrosine phosphatase. Capsaicin down-regulated the expression of the STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin, and vascular endothelial growth factor. Finally, capsaicin induced the accumulation of cells in G(1) phase, inhibited proliferation, and induced apoptosis, as indicated by caspase activation. Capsaicin also significantly potentiated the apoptotic effects of Velcade and thalidomide in multiple myeloma cells. When administered i.p., capsaicin inhibited the growth of human multiple myeloma xenograft tumors in male athymic nu/nu mice.. Overall, these results suggest that capsaicin is a novel blocker of the STAT3 activation pathway, with a potential role in the prevention and treatment of multiple myeloma and other cancers.

Topics: Antineoplastic Agents; Apoptosis; bcl-X Protein; Boronic Acids; Bortezomib; Capsaicin; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cyclin D1; DNA; Extracellular Signal-Regulated MAP Kinases; G1 Phase; Humans; Inhibitor of Apoptosis Proteins; Interleukin-6; Janus Kinase 1; Microtubule-Associated Proteins; Multiple Myeloma; Neoplasm Proteins; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-bcl-2; Pyrazines; STAT3 Transcription Factor; Survivin; Thalidomide; Vascular Endothelial Growth Factor A

Plasma cell myelomas (PCMs) are traditionally diagnosed by the percentage (%) of plasma cells (PCs) in the bone marrow aspirate differential combined with clinical parameters and radiographic findings. PCs are most reliably quantitated in bone marrow (BM) tissues by CD138 immunohistochemistry (IHC). However, there are no correlations of % CD138+ cells with clinical parameters or overall survival (OS). The presence of cyclin D1 has correlated with worst prognosis, but cyclin D1 has not been correlated with routine cytogenetics. CD56+, although not significantly reported in reactive plasmacytoses, monoclonal gammopathy of undetermined significance (MGUS), nor in lymphoplasmacytic lymphomas (LPLs), has not been evaluated in borderline diagnostic (borderline) cases.. It includes: (1) correlating the percentages of PCs by CD138 IHC, cyclin D1 status, and CD56 expression with clinical parameters and OS in PCMs, (2) correlating cyclin D1 status with routine cytogenetics in PCMs, borderline cases, and MGUSs, and (3) analyzing CD56 expression in PCMs, borderline cases, MGUSs, and LPLs.. Bone marrow aspirates, BM touch preparations, and BM clot and/or biopsy sections with CD138/kappa/lambda IHC (44-PCMs, 9-MGUSs, 17-borderline cases, 3-LPLs, and 3-reactive plasmacytoses) were reviewed and stained with CD56 and cyclin D1.. Increased CD138+ cells did not correlate significantly with clinical parameters or OS. Cyclin D1+ did not correlate with the presence of a t(11;14) by routine cytogenetics [although detected in all t(11;14)+ cases], clinical parameters, nor OS. CD56 expression was identified in PCMs, MGUSs, and LPL but not in reactive plasmacytoses. CD56+ did not distinguish PCMs, MGUS, and LPLs, and did not correlate with clinical parameters or OS. CD56 and cyclin D1 IHC were better evaluated in BM clot than biopsy sections.

Topics: CD56 Antigen; Cyclin D1; Female; Humans; Immunohistochemistry; Lymphocyte Count; Male; Multiple Myeloma; Plasma Cells; Syndecan-1

Dysregulation of cyclin D1 expression is one of the most common genetic aberrations found in hematopoietic malignancies, including multiple myeloma. To address the effects of cyclin D1 overexpression might have on the response of malignant hematopoietic cells to CDK inhibitors, the impact of ectopic cyclin D1 overexpression on the response of human multiple myeloma U266 cells to various cyclin-dependent kinase (CDK) inhibitors was examined. Cyclin D1 overexpression markedly increased the apoptotic response of cells to the CDK inhibitors flavopiridol, roscovitine, and R-roscovitine. Ectopic expression of cyclin D1 resulted in p21(CIP1) accumulation, an effect that was diminished by CDK inhibitor exposure. In pRb-null U266 cells, enforced overexpression of cyclin D1 diminished CDK inhibitor-mediated dephosphorylation of the pocket proteins p130 and p107, reduced binding of E2F1 and E2F4 to p130 and p107, and attenuated inhibition of E2F activity. Notably, CDK inhibitors failed to reduce the S phase fraction in cyclin D1/U266 cells in contrast to effects in their wild-type counterparts. Finally, cyclin D1/U266 cells exhibited diminished basal NF-kappaB activity compared to controls, which was essentially completely abrogated by CDK inhibitor exposure. Together, these findings suggest that dysregulation of cyclin D1 sensitizes human myeloma cells to the actions of CDK inhibitors through mechanisms involving interference with p21(CIP1) expression, dephosphorylation of pocket proteins and inactivation of E2Fs culminating in S phase entry, as well as inactivation of NF-kappaB, leading to apoptosis rather than growth arrest.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Drug Screening Assays, Antitumor; E2F Transcription Factors; Gene Expression; Humans; Multiple Myeloma; NF-kappa B; Phosphorylation; Protein Binding; Protein Kinase Inhibitors; Retinoblastoma-Like Protein p107; Retinoblastoma-Like Protein p130; S Phase; STAT3 Transcription Factor; Up-Regulation

The significance of cyclin D1 expression in plasma cell myeloma was examined by immunohistochemical analysis using a newly available rabbit monoclonal antibody with superior staining properties. Two patterns of positive staining were observed, each associated with distinct pathologic features. Strong cyclin D1 staining was associated with increased plasma cells at diagnosis (P=.026), lymphoplasmacytic morphologic features (P=.029), CD20 expression (P=.040), and t(11;14)(q13;q32) as detected by interphase fluorescence in situ hybridization with simultaneous CD138 immunofluorescence (P<.001). In contrast, weak staining was associated with hyperdiploidy (P=.02) and gains of the CCND1 locus (P=.01). Overall survival was longer in the cyclin D1+ cases than in cyclin D1- cases (estimated 3-year survival, 73% vs 27%; P=.005). Improved survival was seen in the strongly positive and weakly positive groups compared with cyclin D1- cases (P=.005). This report shows for the first time that cyclin D1 immunohistochemical analysis can provide prognostic information in plasma cell myeloma.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD20; Biomarkers, Tumor; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Clinical Trials, Phase II as Topic; Cyclin D1; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Membrane Glycoproteins; Multiple Myeloma; Prognosis; Proteoglycans; Rabbits; Survival Analysis; Syndecan-1; Syndecans; Translocation, Genetic

Several studies including ours have suggested that lack of CD56 in multiple myeloma (MM) defines a unique patient subset with poorer prognosis. However, the mechanism underlying this aggressive behavior of CD56(-) MM has not been well elucidated. Interleukin-6 (IL-6) or insulin-like growth factor I (IGF-I) induce proliferation of MM cells. In this study, we report about the relationship between CD56 expression and responsiveness to these cytokines.. We sorted out both CD56(-) and CD56(+) fractions from MM cell lines such as KMS-21-BM and U-266, and investigated their different responsiveness to IL-6 or IGF-I. Furthermore, we compared the effects of these cytokines on the regulation of cell-cycle distribution between CD56(-) and CD56(+) cells.. Although CD56(-) cells in both KMS-21-BM and U-266 cells responded significantly to IL-6, CD56(+) cells did not. Ki-67(+) cells in the CD56(-) cells were significantly increased by IL-6. Western blotting showed that IL-6 phosphorylated Akt, and upregulated and downregulated the level of cyclin D1 and p27 protein in the CD56(-) KMS-21-BM cells, respectively. LY-294002 completely blocked these effects of IL-6. On the other hand, Ki-67(+) cells in the CD56(+) cells did not respond to IL-6. Anti-IGF-I mAb significantly reduced Ki-67(+) cells only in the CD56(+) cells. IGF-I phosphorylated Akt and upregulated cyclin D1 in the CD56(+) KMS-21-BM cells, which was completely blocked by LY294002.. These results suggest that CD56(-) and CD56(+) MM cells could be stimulated by IL-6 and IGF-I, respectively, via PI3-K/Akt pathway, and provide useful information for anticytokine therapies.

Topics: Antibodies, Monoclonal; CD56 Antigen; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromones; Cyclin D1; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Interleukin-6; Ki-67 Antigen; Morpholines; Multiple Myeloma; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Processing, Post-Translational; Signal Transduction

Rice protein isolate (RPI) has been reported to reduce the incidence of 7,12-dimethylbenz[a]anthracene-induced mammary tumors in rats. To determine the potential role of phytochemicals associated with the RPI, we studied in vitro antitumor activities of an ether fraction from RPI using human tumor cell lines, including two human breast carcinoma cell lines (MDA-MB-453 and MCF-7) and two myeloma cell lines (RPMI-8226 and IM-9). Concentration-dependent antiproliferative effects of the ether fraction were observed in all cell lines using the standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Fraction-induced apoptosis (P < 0.05) was detected in all cell lines, and this was associated with the induction of proapoptotic bax protein and cdk inhibitors (p21) and the suppression of cdk4 and cyclin D1 activity. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) with both positive and negative modes was used to analyze the phytochemicals in the ether fraction from RPI. Fifty-seven phytochemicals were identified or characterized by their diagnostic fragmentation patterns and direct comparison with the authentic standards on the basis of electrospray ionization-MS/MS data. The major components bound to RPI were lysoglycerophospholipids, fatty acids, and fatty acid 3-[2-(2,3-dihydroxy-propoxycarbonyl)-2-hydroxy-ethoxy]-2-hydroxy-propyl esters.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Cell Division; Cell Line, Tumor; Chromatography, Liquid; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p21; Humans; Mass Spectrometry; Multiple Myeloma; Oryza; Plant Proteins

To assess a possible role in tumor progression, the occurrence and type of K- and N-RAS mutations were determined in purified tumor cells, including samples from patients with premalignant monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM), and extramedullary plasma cell (PC) tumors (ExPCTs). Immunophenotypic aberrant PCs were flow sorted from 20 MGUS, 58 MM, and 13 ExPCT patients. One RAS mutation was identified in 20 MGUS tumors (5%), in contrast to a much higher prevalence of RAS mutations in all stages of MM (about 31%). Further, oncogene analyses showed that RAS mutations are not evenly distributed among different molecular subclasses of MM, with the prevalence being increased in MM-expressing cyclin D1 (P = .015) and decreased in MM with t(4;14) (P = .055). We conclude that RAS mutations often provide a genetic marker if not a causal event in the evolution of MGUS to MM. Surprisingly, RAS mutations were absent in bone marrow tumor cells from all patients with ExPCT, a result significantly different from intramedullary MM (P = .001). From 3 of 6 patients with paired intramedullary and extramedullary PCs and identical immunoglobulin heavy chain gene (IgH) sequences, RAS mutations were identified only in extramedullary PCs, suggesting a role for RAS mutations in the transition from intramedullary to extramedullary tumor.

Topics: Base Sequence; CD56 Antigen; Cell Line, Tumor; Cell Transformation, Neoplastic; Cyclin D1; Humans; Monoclonal Gammopathy of Undetermined Significance; Multiple Myeloma; Mutation; ras Proteins

Immunoglobulin light chain amyloidosis (AL) is characterized by a clonal expansion of plasma cells within the bone marrow. Gene expression analysis was used to identify a unique molecular profile for AL using enriched plasma cells (CD138+) from the bone marrow of 24 patients with AL and 28 patients with multiple myeloma (MM) and 6 healthy controls. Class prediction analysis (PAM) revealed a subset of 12 genes, which included TNFRSF7 (CD27), SDF-1, and PSMA2, that distinguished between these 2 groups with an estimated and observed accuracy of classification of 92%. This model was validated with an independent dataset of 11 patients with AL and 12 patients with MM with 87% accuracy. Differential expression for the most discriminant genes in the 12-gene subset was validated using quantitative real-time polymerase chain reaction and protein expression analysis, which upheld the observations from the micro-array expression data. Functional analyses using a novel network mapping software revealed a number of potentially significant pathways that were dysregulated in patients with AL, with those regulating proliferation, apoptosis, cell signaling, chemotaxis, and migration being substantially represented. This study provides new insight into the molecular profile of clonal plasma cells and its functional relevance in the pathogenesis of light chain amyloidosis.

Topics: Aged; Amyloidosis; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Clone Cells; Cyclin D1; Gene Expression; Gene Expression Profiling; Humans; Immunoglobulin Light Chains; Middle Aged; Multiple Myeloma; Plasma Cells; Polymerase Chain Reaction; Predictive Value of Tests; Reproducibility of Results; Retinoblastoma Protein; Translocation, Genetic

Multiple myeloma (MM) is a fatal lymphoid malignancy that is incurable with conventional modalities of chemotherapy. Strong and constitutive activation of nuclear factor kappa B (NF-kappaB) is a common characteristic of MM cells. In our study we successfully target NF-kappaB with a novel NF-kappaB inhibitor dehydroxymethylepoxyquinomycin (DHMEQ). DHMEQ completely abrogates constitutive NF-kappaB activity and induces apoptosis of MM cells, whereas control peripheral blood mononuclear cells (PBMC) are resistant to NF-kappaB inhibition and apoptosis by DHMEQ treatment. DHMEQ inhibition of NF-kappaB triggers activation of caspases 8 and 9, as well as G0/G1 cell cycle arrest accompanied by downregulation of antiapoptotic genes Bcl-XL and c-FLIP and cell cycle progression gene cyclins D1 and D2. DHMEQ-mediated inhibition of vascular endothelial growth factor (VEGF) production in MM cells raises the possibility that DHMEQ abrogates the autocrine VEGF loop and enhances its antitumor effects by inhibiting neovascularization in the bone marrow. Using an in vivo NOD/SCID/gammac(null) (NOG) mice model, we show that DHMEQ has a potent inhibitory effect on the growth of MM cells. Compared to other compounds having the potential to inhibit NF-kappaB, DHMEQ is a unique compound that blocks the translocation of NF-kappaB p65 into the nucleus and selectively targets NF-kappaB activated in tumor cells. Therefore, our study presents a new molecular target therapy in MM.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Benzamides; Calcium-Binding Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Cyclin D; Cyclin D1; Cyclins; Cyclohexanones; Disease Models, Animal; Down-Regulation; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mice; Mice, Inbred NOD; Mice, SCID; Multiple Myeloma; Nerve Tissue Proteins; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Synaptotagmin I; Synaptotagmins; Translocation, Genetic; Vascular Endothelial Growth Factor A

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus and various partner loci frequently are associated with multiple myeloma (MM). We investigated the expression profiles of the FGFR3/MMSET, CCND1, CCND3, MAF, and MAFB genes, which are involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), t(14;16)(q32;q23), and t(14;20)(q32;q12), respectively, in purified plasma cell populations from 39 MMs and six plasma cell leukemias (PCL) by DNA microarray analysis and compared the results with the presence of translocations as assessed by dual-color FISH or RT-PCR. A t(4;14) was found in 6 MMs, t(11;14) in 9 MMs and 1 PCL, t(6;14) in 1 MM, t(14;16) in 2 MMs and 1 PCL, and t(14;20) in 1 PCL. In all cases, the translocations were associated with the spiked expression of target genes. Furthermore, gene expression profiling enabled the identification of putative translocations causing dysregulation of CCND1 (1 MM and 1 PCL) and MAFB (1 MM and 1 PCL) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. Markedly increased MMSET expression was found in 1 MM showing associated FGFR3 and MMSET signals on an unidentified chromosome. Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations in MM.

Topics: Adult; Aged; Aged, 80 and over; Carrier Proteins; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 16; Chromosomes, Human, Pair 20; Chromosomes, Human, Pair 4; Chromosomes, Human, Pair 6; Cyclin D1; Cyclin D3; Cyclins; DNA-Binding Proteins; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone-Lysine N-Methyltransferase; Humans; In Situ Hybridization, Fluorescence; Macrophage-Activating Factors; MafB Transcription Factor; Male; Microarray Analysis; Middle Aged; Multiple Myeloma; Oncogene Proteins; Oncogene Proteins, Fusion; Oncogenes; Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 3; Receptors, Fibroblast Growth Factor; Repressor Proteins; Transcription Factors; Translocation, Genetic

Primary extramedullary plasmacytomas are infrequent, typically solitary, plasma cell neoplasms that generally pursue an indolent clinical course but may, rarely, convert to multiple myeloma. Phenotypic differences between these two entities are not well defined. Twenty-eight cases of primary extramedullary plasmacytoma and 26 cases of both medullary (n = 17) and extramedullary (n = 9) multiple myeloma were analysed for the expression of proteins known to play a role in the biology of multiple myeloma. Immunohistochemistry was performed on paraffin wax sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, p27, p21, p53, MIB1, CD20, and CD56. Twenty-three extramedullary plasmacytomas were localized in the upper aerodigestive tract, four in the lymph nodes, and one in the testis. There was a strong male predominance (M : F = 6 : 1). None of the patients died from the disease or progressed to multiple myeloma (mean follow-up 50 months). Nine patients developed local relapse and one patient's tumour evolved into a B-cell non-Hodgkin's lymphoma. In contrast to both intra- and extra-medullary multiple myeloma, extramedullary plasmacytoma showed absence of cyclin D1 (p < 0.001) and infrequent expression of CD56 (p < 0.001). Furthermore, extramedullary plasmacytomas were characterized by weaker staining for Bcl-2 protein and rare overexpression of p21 and p53. In comparison to extramedullary multiple myeloma, extramedullary plasmacytoma showed a more mature morphology and lower proliferation indices (p = 0.008). There was no association between the phenotypic parameters investigated and clinical outcome in extramedullary plasmacytoma. In summary, extramedullary plasmacytoma and multiple myeloma show significant immunophenotypic differences, some of which may be of both diagnostic utility and biological relevance.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; CD56 Antigen; Cyclin D1; Diagnosis, Differential; Female; Follow-Up Studies; Humans; Immunophenotyping; Male; Middle Aged; Multiple Myeloma; Neoplasm Proteins; Neoplasm Staging; Plasmacytoma; Retrospective Studies; RNA, Messenger; RNA, Neoplasm

Two oncogenic pathways have been hypothesized for multiple myeloma (MM) and premalignant monoclonal gammopathy of undetermined significance (MGUS) tumors: a nonhyperdiploid pathway associated with a high prevalence of IgH translocations and a hyperdiploid pathway associated with multiple trisomies of 8 chromosomes. Cyclin D1, D2, or D3 expression appears to be increased and/or dysregulated in virtually all MM tumors despite their low proliferative capacity. Translocations can directly dysregulate CCND1 (11q13) or CCND3 (6p21), or MAF (16q23) or MAFB (20q11) transcription factors that target CCND2. Biallelic dysregulation of CCND1 occurs in nearly 40% of tumors, most of which are hyperdiploid. Other tumors express increased CCND2, either with or without a t(4;14) translocation. Using gene expression profiling to identify 5 recurrent translocations, specific trisomies, and expression of cyclin D genes, MM tumors can be divided into 8 TC (translocation/cyclin D) groups (11q13, 6p21, 4p16, maf, D1, D1+D2, D2, and none) that appear to be defined by early, and perhaps initiating, oncogenic events. However, despite subsequent progression events, these groups have differing gene expression profiles and also significant differences in the prevalence of bone disease, frequency at relapse, and progression to extramedullary tumor.

Topics: Bone Diseases; Cyclin D1; Cyclin D2; Cyclin D3; Cyclins; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; Phenotype; Reproducibility of Results; RNA, Messenger; Translocation, Genetic; Trisomy

In this study we report that R-etodolac (SDX-101), at clinically relevant concentrations, induces potent cytotoxicity in drug-sensitive multiple myeloma (MM) cell lines, as well as in dexamethasone (MM.1R)-, doxorubicin (Dox40/RPMI8226)-, and bortezomib (DHL4)-resistant cell lines. Immunoblot analysis demonstrates that R-etodolac induces apoptosis characterized by caspase-8, -9, and -3 and PARP (poly-ADP [adenosine diphosphate]-ribose polymerase) cleavage and down-regulation of cyclin D1 expression. Subcytotoxic doses of R-etodolac up-regulate myeloid cell leukemia-1 proapoptotic variant (Mcl-1S), while enhancing dexamethasone (Dex)-induced caspase activation and apoptosis. The combination of R-etodolac with Dex results in a highly synergistic cytotoxic effect. R-etodolac also induces apoptosis against primary cells isolated from patients with MM refractory to chemotherapy. Although interleukin 6 (IL-6) and insulin-like growth factor-1 (IGF-1) abrogate Dex-induced MM cell cytotoxicity, neither IL-6 nor IGF-1 protects against R-etodolac-induced cytotoxicity in MM cells. R-etodolac also inhibits viability of MM cells adherent to bone marrow stromal cells (BMSCs), thereby overcoming a mechanism of drug resistance commonly observed with other conventional chemotherapeutic agents. Our data, therefore, indicate that R-etodolac circumvents drug resistance in MM cells at clinically relevant concentrations, targets Mcl-1, and can be synergistically combined with Dex.

Topics: Antineoplastic Agents; Apoptosis; Bone Marrow Cells; Caspases; Cell Adhesion; Cell Line, Tumor; Cyclin D1; Dexamethasone; Drug Resistance, Neoplasm; Drug Synergism; Etodolac; Humans; Insulin-Like Growth Factor I; Interleukin-6; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Stereoisomerism

Overexpression of cyclin D1 is a common event in solid and hematological cancers. In multiple myeloma (MM), a B-cell hemopathy, one mechanism responsible for cyclin D1 expression is the translocation t(11;14)(q13;q32). But this translocation does not account for cyclin D1 gene activation in all MM. We have hypothesized that epigenetic events could regulate cyclin D1 gene transcription.. Using 6 MM cell lines representative of different cyclin D1 expression levels and exhibiting various chromosome 11 abnormalities, as well as normal B cells, we studied DNA methylation and histone acetylation of the cyclin D1 promoter.. With the bisulfite sequencing technique, we have studied the DNA methylation status of the core minimal cyclin D1 promoter containing Sp1- and CRE-binding sites. Our results show that this region is not methylated in 6 human MM cell lines as well as in normal B lymphocytes, independently of cyclin D1 expression. Treatment with the DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-Aza) had no effect on cyclin D1 gene transcription. Chromatin immunoprecipitation (ChIP) experiments indicated that acetylated histones H4 are located at both the active and inactive cyclin D1 promoter. Treatment with a histone deacetylase inhibitor, trichostatin A (TSA), had no effect on gene transcription, nor had combined TSA plus 5-Aza treatment.. The cyclin D1 gene is silenced within the B lineage by a mechanism different from DNA methylation or histone deacetylation.

Topics: Acetylation; Base Sequence; Cell Line, Tumor; Cyclin D1; DNA Methylation; DNA Primers; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Multiple Myeloma; Promoter Regions, Genetic; Transcriptional Activation

Nuclear factor-kappaB (NF-kappaB) is constitutively activated in multiple myeloma cells. Several proteasome inhibitors have been shown to be effective against multiple myeloma and may act by inhibiting degradation of IkappaBalpha. Here, we examined the biological effects of a new type of NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), which is reported to directly inhibit the cytoplasm-to-nucleus translocation of NF-kappaB. A multiple myeloma cell line, 12PE, which is defective for IkappaBalpha protein, was utilized to determine if IkappaBalpha is concerned with the action of DHMEQ. Meanwhile, U266 was used as a multiple myeloma cell line with normal IkappaBalpha. A proteasome inhibitor, gliotoxin, which is an inhibitor of degradation of phosphorylated IkappaBalpha, failed to inhibit translocation of NF-kappaB in 12PE. In contrast, DHMEQ equally inhibited translocation of NF-kappaB to the nucleus and induced apoptosis to both multiple myeloma cell lines, suggesting that apoptosis resulting from DHMEQ is IkappaBalpha independent. DHMEQ also induced apoptosis in freshly isolated multiple myeloma cells. After DHMEQ treatment, cleavage of caspase-3 and down-regulation of cyclin D1 were observed in both cell lines. In addition, administration of DHMEQ resulted in a significant reduction in tumor volume in a plasmacytoma mice model compared with control mice. Our results show that DHMEQ could potentially be a new type of molecular target agent for multiple myeloma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzamides; Caspase 3; Caspases; Cyclin D1; Cyclohexanones; Down-Regulation; Drug Screening Assays, Antitumor; Enzyme Activation; Female; Humans; I-kappa B Proteins; Mice; Mice, Inbred ICR; Mice, SCID; Minor Histocompatibility Antigens; Multiple Myeloma; NF-kappa B; NF-KappaB Inhibitor alpha; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM). A recently proposed classification grouped MM patients into five classes on the basis of their cyclin D expression profiles and the presence of the main translocations involving the immunoglobulin heavy chain locus (IGH) at 14q32. In this study, we provide a molecular characterization of the identified translocations/cyclins (TC) groups.. The gene expression profiles of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes and identify their transcriptional fingerprints. The cyclin D expression data were validated by means of real-time quantitative polymerase chain reaction analysis; fluorescence in situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion.. Class-prediction analysis identified 112 probe sets as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. The TC2 group, which showed extra copies of the CCND1 locus and no IGH translocations or the chromosome 13q deletion, was characterized by the overexpression of genes involved in protein biosynthesis at the translational level. A meta-analysis of published data sets validated the identified gene expression signatures.. Our data contribute to the understanding of the molecular and biologic features of distinct MM subtypes. The identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.

Topics: Adult; Aged; Aged, 80 and over; Cyclin D1; Female; Gene Expression; Gene Expression Profiling; Humans; In Situ Hybridization, Fluorescence; Male; Middle Aged; Multiple Myeloma; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Translocation, Genetic

Multiple myeloma is a tumor of somatically mutated, isotype-switched plasma cells that accumulate in the bone marrow leading to bone destruction and bone marrow failure. The germinal center processes of somatic hypermutation and switch recombination are implicated in the development of recurrent immunoglobulin gene translocations in 40% of patients. These affect five loci: 11q13, 6p21, 4p16, 16q23 and 20q11, leading to dysregulation of CCND1, CCND2, FGFR3/MMSET, c-MAF and MAFB respectively. The remaining 60% of patients can be divided into four groups based on their expression of CCND1 and CCND2. The largest group (40%) ectopically express CCND1 bi-allelically and have hyperdiploidy with multiple trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21. The translocation and cyclin D (TC) groups identify patients with different genetics, biology, clinical features, prognosis and response to therapy.

Topics: B-Lymphocytes; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 13; Cyclin D1; Cyclin D2; Cyclins; Diploidy; Humans; Immunoglobulin Heavy Chains; MafB Transcription Factor; Middle Aged; Monoclonal Gammopathy of Undetermined Significance; Multiple Myeloma; Paraproteinemias; Proto-Oncogene Proteins c-maf; Receptor, Fibroblast Growth Factor, Type 3; Recurrence; Translocation, Genetic; Trisomy

Multiple myeloma, the second most common hematopoietic cancer, ultimately becomes refractory to treatment when self-renewing multiple myeloma cells begin unrestrained proliferation by unknown mechanisms. Here, we show that one, but not more than one, of the three early G(1) D cyclins is elevated in each case of multiple myeloma. Cyclin D1 or D3 expression does not vary in the clinical course, but that alone is insufficient to promote cell cycle progression unless cyclin-dependent kinase 4 (cdk4) is also elevated, in the absence of cdk6, to phosphorylate the retinoblastoma protein (Rb). By contrast, cyclin D2 and cdk6 are coordinately increased, thereby overriding the inhibition by cdk inhibitors p18(INK4c) and p27(Kip1) and phosphorylating Rb in conjunction with the existing cdk4. Thus, cyclin D1 pairs exclusively with cdk4 and cdk6 pairs only with cyclin D2, although cyclin D2 can also pair with cdk4 in multiple myeloma cells. The basis for this novel and specific cdk/D cyclin pairing lies in differential transcriptional activation. In addition, cyclin D1- or cyclin D3-expressing multiple myeloma cells are uniformly distributed in the bone marrow, whereas cdk6-specific phosphorylation of Rb occurs in discrete foci of bone marrow multiple myeloma cells before proliferation early in the clinical course and is then heightened with proliferation and disease progression. Mutually exclusive cdk4/cyclin D1 and cdk6/cyclin D2 pairing, therefore, is likely to be a critical determinant for cell cycle reentry and progression and may play a pivotal role in the expansion of self-renewing multiple myeloma cells.

Topics: Adult; Aged; Aged, 80 and over; Cyclin D1; Cyclin D2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Female; G1 Phase; Humans; Male; Middle Aged; Multiple Myeloma; Phosphorylation; Retinoblastoma Protein

Stratification of patients with multiple myeloma (MM) may be important. We investigated 138 MM patients, focusing on correlations between CD20 expression, 11 ; 14 translocation, morphology of MM cells, cyclin D1 immunostaining, and the prognosis. About 15% of patients (7/47cases) were CD20-positive, small mature MM cells, with positive cyclin D1 in the nucleus and 11; 14 translocation. Two color analysis of CD38 x CD20 antigens may be necessary to investigate CD20 expression on MM cells. Rituximab may be effective for the treatment of CD20-positive MM.

Topics: Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Antineoplastic Agents; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Female; Gene Expression; Humans; Male; Middle Aged; Multiple Myeloma; Plasma Cells; Prognosis; Rituximab; Translocation, Genetic

Conflicting data are reported on the clinical significance of cyclin D1 deregulation in multiple myeloma. The aim of this study was to evaluate the incidence and prognostic significance of cyclin D1 expression and p53 mutations in multiple myeloma, as well as the relationship of their expression with selected clinical data, histological features, and proliferative activity of myeloma cells. We analyzed bone marrow biopsy specimens obtained from 59 patients with newly diagnosed multiple myeloma. Expression of cyclin D1 and p53 was analyzed using standard immunohistochemical method of B5-fixed and routinely processed paraffin-embedded bone marrow specimens. Cyclin D1 was overexpressed in 14/59 (27%) and p53 in 5/59 (8.5%) specimens. There was no significant correlation between cyclin D1 overexpression and age, gender, clinical stage (Durie-Salmon classification), extent of osteolytic lesions, type of monoclonal protein, hemoglobin concentration, platelet count, serum concentration of creatinine, calcium, C-reactive protein, and beta2-microglobulin. No association was observed between the expression of cyclin D1 and the extent of bone marrow infiltration, histological grade, proliferative activity index (measured with Ki-67 immunoreactivity) and response to therapy. No significant difference was observed regarding overall survival between cyclin D1 positive and cyclin D1 negative patients (29 vs 36 mo, p = 0.76). Results of this study did not revealed prognostic significance of cyclin D1 overexpression in multiple myeloma. Mutations of p53 gene are rare events in myeloma, suggesting their limited role in the pathogenesis of the disease.

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow; Cyclin D1; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Male; Middle Aged; Multiple Myeloma; Prognosis; Survival Analysis; Tumor Suppressor Protein p53

The t(11;14)(q13;q32) is the most common translocation in multiple myeloma (MM), resulting in up-regulation of cyclin D1. We used a segregation fluorescence in situ hybridization (FISH) assay to detect t(11;14) breakpoints in primary MM cases and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify cyclin D1 and MYEOV (myeloma overexpressed) expression, another putative oncogene located on chromosome 11q13. High levels of cyclin D1 mRNA (cyclin D1/TBP [TATA box binding protein] ratio > 95) were found exclusively in the presence of a t(11;14) translocation (11/48 cases; P <.00001). In addition, a subgroup of MM cases (15/48) with intermediate to low cyclin D1 mRNA (cyclin D1/TBP ratio between 2.3 and 20) was identified. FISH analysis ruled out a t(11; 14) translocation and 11q13 amplification in these cases; however, in 13 of 15 patients a chromosome 11 polysomy was demonstrated (P <.0001). These results indicate an effect of gene dosage as an alternative mechanism of cyclin D1 deregulation in MM. The absence of chromosome 11 abnormalities in 2 of 15 patients with intermediate cyclin D1 expression supports that there are presumably other mechanism(s) of cyclin D1 deregulation in MM patients. Our data indicate that deregulation of MYEOV is not favored in MM and further strengthens the role of cyclin D1 overexpression in lymphoid malignancies with a t(11;14)(q13;q32) translocation.

Topics: Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Gene Dosage; Humans; In Situ Hybridization, Fluorescence; Multiple Myeloma; Oncogene Proteins; Osteolysis; Proto-Oncogene Proteins; RNA, Messenger; Translocation, Genetic

The purpose of this study was to examine interactions between the proteasome inhibitor bortezomib (Velcade) and the histone deacetylase (HDAC) inhibitors sodium butyrate and suberoylanilide hydroxamic acid in human multiple myeloma (MM) cells that are sensitive and resistant to conventional agents.. MM cells were exposed to bortezomib for 6 h before the addition of HDAC inhibitors (total, 26 h), after which reactive oxygen species (ROS), mitochondrial dysfunction, signaling and cell cycle pathways, and apoptosis were monitored. The functional role of ROS generation was assessed using the free radical scavenger N-acetyl-l-cysteine.. Preincubation with a subtoxic concentration of bortezomib markedly sensitized U266 and MM.1S cells to sodium butyrate- and suberoylanilide hydroxamic acid-induced mitochondrial dysfunction; caspase 9, 8, and 3 activation; and poly(ADP-ribose) polymerase degradation; resulting in synergistic apoptosis induction. These events were associated with nuclear factor kappaB inactivation, c-Jun NH(2)-terminal kinase activation, p53 induction, and caspase-dependent cleavage of p21(CIP1), p27(KIP1), and Bcl-2, as well as Mcl-1, X-linked inhibitor of apoptosis, and cyclin D1 down-regulation. The bortezomib/HDAC inhibitor regimen markedly induced ROS generation; moreover, apoptosis and c-Jun NH(2)-terminal kinase activation were attenuated by N-acetyl-l-cysteine. Dexamethasone- or doxorubicin-resistant MM cells failed to exhibit cross-resistance to the bortezomib/HDAC inhibitor regimen, nor did exogenous interleukin 6 or insulin-like growth factor I block apoptosis induced by this drug combination. Finally, bortezomib/HDAC inhibitors induced pronounced lethality in primary CD138(+) bone marrow cells from MM patients, but not in the CD138(-) cell population.. Sequential exposure to bortezomib in conjunction with clinically relevant HDAC inhibitors potently induces mitochondrial dysfunction and apoptosis in human MM cells through a ROS-dependent mechanism, suggesting that a strategy combining these agents warrants further investigation in MM.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Survival; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radical Scavengers; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intracellular Membranes; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Oxidative Stress; Oxygen; Protease Inhibitors; Proteins; Proteoglycans; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Reactive Oxygen Species; Sodium Oxybate; Syndecan-1; Syndecans; Tumor Suppressor Proteins; Vorinostat; X-Linked Inhibitor of Apoptosis Protein

Topics: Bone Marrow; Cyclin D1; Humans; Male; Middle Aged; Multiple Myeloma

Topics: Cyclin D1; Gene Expression; Humans; Intercellular Signaling Peptides and Proteins; Multiple Myeloma; Oligonucleotide Array Sequence Analysis; Osteolysis; Proteins; RNA, Messenger; Translocation, Genetic

Cyclin D1 is a positive-regulator of the cell cycle and is overexpressed in myeloma cells with t(11;14)(q13;q32). First, we analyzed whether there was a correlation between cyclin D1 overexpression and the presence of Ki67-positive myeloma cells in multiple myeloma (MM). Cyclin D1 overexpression was examined by competitive RT-PCR. Then we found these two markers were present independently in a given case. FISH analysis revealed that cyclin D1 over-expression was caused by t(11;14)(q13;q32) or extra copies of B-cell leukemia/lymphoma-1 (BCL-1/CCND1), and unknown mechanism without them. We compared the gene expression between myeloma cells with cyclin D1 overexpression and those without it using cDNA microarray analysis. Analysis of the expression profiles showed that the significantly up-regulated genes included cyclin D1, cell division cycle 37 (CDC37) and B-cell leukemia/lymphoma-2 (BCL-2), while the down-regulated genes included cyclin D2 and CD9 antigen (p24) in MM cases with cyclin D1 overexpression. However, hierarchical clustering analysis of the data showed that myeloma cells of MM cases with cyclin D1 overexpression could not be distinguished clearly from those without it. Real-time RT-PCR showed that the expression of CDC37 gene was significantly up-regulated in MM patients with cyclin D1 overexpression compared with those without it (p=0.0418). However, there was no significant difference in BCL-2 gene (p=0.5748). These results suggested that MM cases with cyclin D1 overexpression do not constitute a specific group, and cyclin D1 overexpression may not be caused only by abnormality of the BCL-1/CCND1 gene. The CDC37 may collaborate with cyclin D1 in progression of MM.

Topics: Biomarkers, Tumor; Cell Cycle Proteins; Chaperonins; Chromosomes, Human, Pair 13; Cyclin D1; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; Multiple Myeloma; Oligonucleotide Array Sequence Analysis; Proto-Oncogene Proteins c-bcl-2; Translocation, Genetic; Up-Regulation

A large number of chromosomal abnormalities have been detected in multiple myeloma (MM). The most frequent are chromosome 13q deletions and translocations affecting the immunoglobulin heavy chain gene (IGH). Recent studies using comparative genomic hybridization (CGH) have shown that gains of 11q represent one of the most frequent genomic changes in MM. However CGH is not generally used in routine clinical laboratories.. In the present study, efficiency of fluorescent in situ analysis (FIS)H analysis in the detection of 11q abnormalities in MM patients was investigated. Cytogenetic and FISH studies with three different specific probes for the regions containing the genes BCL1 (11q13), ATM (11q22) and MLL (11q23) were simultaneously performed in 52 patients: 9 cases with 11q abnormalities detected by conventional cytogenetics and 43 cases without 11q abnormalities. FISH analysis identified 11q aberrations that were undetected by cytogenetics in 16 out the 43 cases (37%).. Gains on 11q were present in 13 cases (30%) while rearrangements on 11q were observed in the remaining 3 cases. No losses were found. All 11q gains involved the three regions analyzed (BCL1, ATM and MLL genes) while only rearrangements of BCL1 were observed. In all control cases the 11q alterations were confirmed by FISH. A good overall correlation between CGH and FISH was observed. Nevertheless gains on BCL1, ATM and MLL genes were observed in 3 cases displaying a normal CGH.. In summary, chromosomal abnormalities on 11q are frequent in MM. FISH studies demonstrate a high sensitivity at detecting this abnormality and should be used in the routine evaluation of MM.

Topics: Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Chromosome Aberrations; Chromosome Banding; Chromosomes, Human, Pair 11; Cohort Studies; Cyclin D1; DNA-Binding Proteins; Female; Genetic Markers; Histone-Lysine N-Methyltransferase; Humans; In Situ Hybridization, Fluorescence; Karyotyping; Male; Multiple Myeloma; Myeloid-Lymphoid Leukemia Protein; Nucleic Acid Hybridization; Protein Serine-Threonine Kinases; Sensitivity and Specificity; Tumor Suppressor Proteins

Interactions between the cyclin-dependent kinase inhibitor flavopiridol and the small-molecule Bcl-2 antagonist HA14-1 were examined in human multiple myeloma cells. Whereas individual treatment of U266 myeloma cells with 10 micromol/L HA14-1 or 100 nmol/L flavopiridol had little effect, exposure of cells to flavopiridol (6 hours) followed by HA14-1 (18 hours) resulted in a striking increase in mitochondrial dysfunction (cytochrome c and Smac/DIABLO release; loss of mitochondrial membrane potential), activation of the caspase cascade, apoptosis, and diminished clonogenic survival. Similar findings were noted in other myeloma cell lines (e.g., MM.1S, RPMI8226, and NCI-H929) as well as in those resistant to dexamethasone and cytotoxic agents (e.g., MM.1R, 8226/Dox40, and 8226/LR5). Combined exposure to flavopiridol and HA14-1 was associated with down-regulation of Mcl-1 and Bcl-xL, Bid cleavage, and mitochondrial translocation of Bax. Flavopiridol/HA14-1-treated cells also exhibited a pronounced activation of Jun NH2-terminal kinase, a modest activation of p38 mitogen-activated protein kinase, and down-regulation of cyclin D1. Flavopiridol/HA14-1-induced apoptosis was associated with a marked increase in reactive oxygen species generation; moreover,both events were attenuated by the antioxidant N-acetyl-l-cysteine. Finally, in contrast to dexamethasone, flavopiridol/HA14-1-induced lethality was unaffected by exogenous interleukin-6 or insulin-like growth factor-I. Together, these findings indicate that flavopiridol and the small-molecule Bcl-2 antagonist HA14-1 cooperate to trigger oxidant injury, mitochondrial dysfunction, caspase activation, and apoptosis in human multiple myeloma cells and suggest that this approach may warrant further evaluation as an antimyeloma strategy.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Benzopyrans; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Cyclin D1; Cyclin-Dependent Kinases; Cytochromes c; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Free Radicals; Humans; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Nitriles; p38 Mitogen-Activated Protein Kinases; Piperidines; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

The signal transducer and activator of transcription molecules (Stats) play key roles in cytokine-induced signal transduction. Recently, it was proposed that constitutively activated Stat 3 (Stat 3 phosphorylated) contributes to the pathogenesis of multiple myeloma (MM) by preventing apoptosis and inducing proliferation. The study aim was to investigate Stat 3 activation in a series of multiple myeloma (MM) cases and its effect on downstream targets such as the anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2, and the cell-cycle protein cyclin D1. Forty-eight cases of MM were analyzed. Immunohistochemistry was performed on paraffin sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, Mcl-1, p21, Stat 3, and Stat 3 phosphorylated (P). Their specificity was corroborated by Western blot analysis using eight human MM cell lines as control. The proliferation rate was assessed with the antibody MiB1. In addition, the mRNA levels of cyclin D1 and Stat 3 were determined by quantitative real-time reverse transcriptase-polymerase chain reaction of paraffin-embedded microdissected tissue. Three different groups determined by the expression of Stat 3P and cyclin D1 (protein and mRNA) were identified: group 1, Stat 3-activated (23 cases, 48%). All cases revealed nuclear expression of Stat 3P. No elevation of Stat 3 mRNA was identified in any of the cases. Three cases in this group showed intermediate to low cyclin D1 protein and mRNA expression. Group 2 included 15 (31%) cases with cyclin D1 staining and lack of Stat 3P. All cases showed intermediate to high levels of cyclin D1 mRNA expression. Group 3 included 10 (21%) cases with no expression of either cyclin D1 or Stat 3P. High levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were identified in 89% and 100% of all cases, respectively. In contrast to Bcl-xL and Mcl-1, the expression of Bcl-2 showed an inverse correlation with proliferation rate (P: 0.0003). No significant differences were found between the three groups in terms of proliferation rate or expression of anti-apoptotic proteins. However, cyclin D1+ cases were always well differentiated and were more likely to show a lymphoplasmocytoid differentiation (chi-square = 9.55). Overall, constitutive activation of Stat 3 was found in almost half (48%) of the investigated MM cases. However, this does not seem to have a major impact on the expression of anti-apoptotic proteins and proliferation. We showed that cyclin D1 overexpression and Stat 3 activatio

Topics: Aged; Apoptosis; Base Sequence; Cyclin D1; DNA Primers; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-6; Male; Middle Aged; Multiple Myeloma; Neoplasm Staging; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor; Trans-Activators

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Cyclin D1; Diagnosis, Differential; Female; Groin; Humans; Multiple Myeloma; Osteolysis; Pain; Radiography; Reverse Transcriptase Polymerase Chain Reaction; Skull

Previously, we showed that monensin, Na+ ionophore, potently inhibited the growth of acute myelogenous leukemia and lymphoma cells. Here, we investigated the antiproliferative effect of monensin on human myeloma cell lines. Monensin significantly inhibited the proliferation of myeloma cell lines examined with IC50 of about 1 micro M. Cell cycle analysis indicated that monensin induced a G1 and/or a G2-M phase arrest in these cell lines. To address the mechanism of the antiproliferative effect of monensin, we examined the effect of this drug on cell cycle-related proteins in NCI-H929 cells. Monensin decreased the levels of CDK2, CDK6, cdc2, cyclin A, cyclin B1, cyclin D1 and cyclin E proteins but did not alter CDK4 protein. While p21 was increased by monensin, p27 was not. In addition, monensin markedly enhanced the binding of p21 with CDK6 and cdc2. Furthermore, the activities of CDK2- and CDK6-associated kinases were reduced in association with hypophosphorylation of Rb protein. The activity of cdc2-associated kinase was decreased, which was accompanied by reduction of cdc25C phosphatase. Also, monensin induced apoptosis in myeloma cells, as evidenced by annexin V binding assay and flow cytometric detection of sub-G1 DNA content. This apoptotic process was associated with down-regulation of Bcl-2, loss of mitochondria transmembrane potential (Deltapsim) and an increase of caspase-3 activity. In addition, monensin caused the up-regulation of ERK and p38 kinase activities. Taken together, these results have demonstrated for the first time that monensin potently inhibited the proliferation of human myeloma cell lines, especially NCI-H929 cells, via cell cycle arrest in association with p21 and apoptosis.

Topics: Antifungal Agents; Apoptosis; Blotting, Western; CDC2 Protein Kinase; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Cell Separation; Cyclin D1; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Dose-Response Relationship, Drug; Down-Regulation; Electrophoresis, Polyacrylamide Gel; Flow Cytometry; G2 Phase; Humans; Inhibitory Concentration 50; Membrane Potentials; Microfilament Proteins; Mitochondria; Mitosis; Models, Chemical; Monensin; Multiple Myeloma; Muscle Proteins; Precipitin Tests; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Time Factors

Chromosomal translocations involving the immunoglobulin heavy chain gene (IgH) and nonrandom protooncogene loci are the hallmark of genetic alterations found not only in multiple myeloma (MM), but also in premalignant stages of MM, including monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM). We studied the frequency of IgH (14q32) rearrangements and their partner chromosomes in 16 Japanese patients with MGUS (13 cases), and SMM (3 cases) by means of interphase double-color fluorescence in situ hybridization (DCFISH) applied to purified plasma cells and using CD138-bead selection. IgH rearrangement was recognized in nine of the patients (56.3%). Protooncogene loci juxtaposed to IgH were identified in seven cases including CCND1 (11q13) in six cases and FGFR3 (4p16) in one. Four out of the six t(11;14)-positive cases showed nuclear staining of the cyclin D1 protein, whereas none of the seven t(11;14)-negative cases did. Moreover, neither MUM1(6p25)-IgH nor MAFB(20q11)-IgH fusion signals were observed. This suggests to us that cyclin D1 deregulation due to the presence of t(11;14) is involved in the early development of plasma cell neoplasms, and that this event alone is not enough for the development of symptomatic myeloma.

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow; Chromosome Aberrations; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Female; Gene Rearrangement; Humans; Immunoenzyme Techniques; Immunoglobulin Heavy Chains; In Situ Hybridization, Fluorescence; Male; Middle Aged; Multiple Myeloma; Paraproteinemias; Translocation, Genetic

This study investigated the expression pattern in primary plasma cells (PCs) of putative oncogenes suggested to be involved in multiple myeloma (MM) development. cDNA archives were generated by global reverse transcription polymerase chain reaction from CD38++/CD19-/CD56-/++ aberrant PCs of a prospective cohort of 96 subjects, including healthy individuals, patients with monoclonal gammopathies of undetermined significance (MGUS), MM and MM with extramedullary manifestations (ExMM). The cDNA archives were analysed quantitatively for expression of the cyclin D1, fibroblast growth factor receptor 3 (FGFR3), C-MYC, C-MAF and cyclin D3 oncogenes. In addition, all patients were screened for IGH-MMSET hybrid transcripts. None of the analysed oncogenes was randomly distributed. C-MYC and cyclin D3 expression increased at the extramedullary transformation stage. Furthermore, C-MYC and cyclin D3 expression in CD56+ MM was similar to MGUS, whereas CD56- MM was similar to ExMM. FGFR3/IGH-MMSET was only observed among CD56+ MM patients, whereas an increased frequency of C-MAF dysregulation was seen among CD56- MM. High cyclin D1 expression levels were identified at similar frequencies at all stages, whereas the frequency of patients with low cyclin D1 levels increased during MM development. These data support the stepwise transformation model accumulating genetic alterations and proliferative capacity during MM initiation and development resulting in different clinical entities.

Topics: Cell Transformation, Neoplastic; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 4; Cyclin D1; Disease Progression; DNA, Complementary; DNA, Neoplasm; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; Oncogene Proteins; Oncogenes; Paraproteinemias; Plasma Cells; Prospective Studies; Reverse Transcriptase Polymerase Chain Reaction; Translocation, Genetic

Topics: Chromosome Aberrations; Chromosomes, Human, Pair 11; Cyclin D1; Humans; Multiple Myeloma; Prognosis

Translocations involving immunoglobulin (Ig) loci and chromosome 13 monosomy (Delta 13) are frequent cytogenetic findings in multiple myeloma (MM). Similar chromosomal aberrations have been identified in the monoclonal gammopathy of undetermined significance (MGUS), but their prevalence and significance remain uncertain. Bone marrow from 72 patients with MGUS (n = 62) and smoldering MM (n = 10) was evaluated for translocations between the Ig heavy chain (IgH) and chromosomes 4, 11, and 16, translocations involving Ig light chain-lambda (IgL-lambda, and Delta 13. Fluorescence in situ hybridization (FISH) analysis was done on clonal plasma cells (PCs) detected by immunofluorescence (cIg-FISH) of the cytoplasmic light chain. We also studied cells for cyclin D1 and FGFR3 up-regulation by immunohistochemistry and immunofluorescence, respectively. Twenty-seven (46%) of 59 patients had IgH translocations, and 4 (11%) of 37 had an IgL-lambda translocation. A t(11;14)(q13;q32) was found in 15 (25%) of 59 patients, a t(4;14)(p16.3;q32) in 9% of patients, and a t(14;16)(q32;q23) in 5% of patients. All patients with t(4;14)(p16.3;q32) tested (n = 3) had intense cytoplasmic fluorescence with an anti-FGFR3 antibody. PC nuclear staining of cyclin D1 was only observed in patients with t(11;14)(q13;q32); Delta 13 was detected in the clonal PCs in 50% of patients. The percentage of abnormal PCs varied with any given abnormality. No obvious clinical or biologic correlations were associated with these chromosome abnormalities. Similar translocations are found in both MGUS and MM, including t(4;14)(p16.3;q32) and t(14;16)(q32;q23). Moreover, Delta 13 is common in MGUS and unlikely to play a predominant role in the evolution of MGUS to MM.

Topics: Aneuploidy; Chromosome Aberrations; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 16; Chromosomes, Human, Pair 4; Cyclin D1; Fluorescent Antibody Technique; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunohistochemistry; In Situ Hybridization, Fluorescence; Multiple Myeloma; Paraproteinemias; Prognosis; Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 3; Receptors, Fibroblast Growth Factor; Translocation, Genetic

The retinoblastoma protein (pRb), p16(INK4A), D-type cyclins, and their partners cyclin-dependent kinase (CDK) 4 and 6 constitute a G(1) regulatory pathway commonly targeted in tumorigenesis. Several malignancies show a reciprocal correlation between genetic alterations of single members of the pRb pathway. Therefore, we determined the frequency of Rb deletions and cyclin D1 alterations by fluorescence in situ hybridization as well as 5' CpG island hypermethylation of the p16(INK4A)gene using methylation-specific polymerase chain reaction in bone marrow mononuclear cells from 82 individuals with plasma cell disorders. Alterations in at least one of the components of the pathway were found in 75%. Cyclin D1 translocations or amplifications were detected in 14/82 (17.1%), Rb deletions at 13q14 in 23/82 (28%) of the cases, including three (3.6%) homozygous deletions. p16(INK4A) was hypermethylated in 33/57 (57.9%) of the samples. Further analysis revealed a highly significant correlation between cyclin D1 alterations and extramedullar or leukemic myeloma manifestations (P = 0.014; Fisher's test). Whereas Rb deletions seemed to occur alternatively to cyclin D1 alterations, no reciprocal correlation was found between p16(INK4A) hypermethylations and cyclin D1 or Rb locus aberrations. Cyclin D1 locus alterations and Rb deletions were associated with a significantly worse prognosis whereas p16(INK4A) hypermethylation had no impact on survival. We conclude that cyclin D1 and Rb aberrations seem to occur as alternative events in plasma cell malignancies and contribute to clinical course and prognosis. In contrast, although p16(INK4A) hypermethylation is frequent, inactivation of p16(INK4A) seems not to be involved in the pathogenesis of plasma cell disorders.

Topics: Chromosome Aberrations; Chromosome Mapping; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 13; Chromosomes, Human, Pair 14; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Female; Gene Deletion; Humans; Immunoglobulins; Leukemia, Plasma Cell; Male; Middle Aged; Multiple Myeloma; Neoplasm Staging; Retinoblastoma Protein; Survival Rate; Translocation, Genetic

Recent work identifies the AKT kinase as a potential mediator of tumor expansion in multiple myeloma. The finding of PTEN mutations in several myeloma cell lines suggests that loss of PTEN function may be one mechanism by which AKT activity is increased in this disease. Because PTEN-deficient myeloma cells may have up-regulated activity of the mammalian target of rapamycin (mTOR), downstream of AKT, they may be particularly sensitive to mTOR inhibition. To test this hypothesis, we challenged myeloma cell lines with CCI-779, a newly developed analogue of rapamycin and an efficient inhibitor of mTOR. Three of four PTEN-deficient cell lines with constitutively active AKT were remarkably sensitive to cytoreduction and G(1) arrest induced by CCI-779 with ID(50) concentrations of <1 nM. In contrast, myeloma cells expressing wild-type PTEN were >1000-fold more resistant. Acute expression of a constitutively active AKT gene in CCI-779-resistant myeloma cells containing wild-type PTEN and quiescent AKT did not convert them to the CCI-779-sensitive phenotype. Conversely, expression of wild-type PTEN in CCI-779-sensitive, PTEN-deficient myeloma cells did not induce resistance. Differential sensitivity did not appear to be due to differences in the ability of CCI-779 to inhibit mTOR and induce dephosphorylation of p70S6kinase or 4E-BP1. However, CCI-779 inhibited expression of c-myc in CCI-sensitive PTEN-null myeloma cells but had no effect on expression in CCI-resistant cells. In contrast, cyclin D1 expression was not altered in either sensitive or resistant cells. These results indicate that PTEN-deficient myeloma cells are remarkably sensitive to mTOR inhibition. Although the results of transfection studies suggest that the level of PTEN and AKT function per se does not regulate sensitivity, PTEN/AKT status may be a good predictive marker of sensitivity.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Carrier Proteins; Cell Cycle Proteins; Cyclin D1; Enzyme Activation; Humans; Multiple Myeloma; Mutation; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Kinase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; PTEN Phosphohydrolase; Ribosomal Protein S6 Kinases; Sirolimus; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins

Overexpression of cyclin D1 mRNA and protein as a result of the chromosomal translocation t(11;14)(q13;q32) is a highly specific molecular marker of mantle cell lymphoma, but cyclin D1 dysregulation can also be found in other B-cell neoplasias. The aim of the study was to develop a precise and reliable tool for quantitation of cyclin D1 mRNA suitable for archival clinical specimens.. A real-time reverse transcription-PCR (RT-PCR) assay was used to quantitate cyclin D1 mRNA copy numbers. Using 2000 microdissected cells as template, 104 formalin-fixed, paraffin-embedded lymph node, spleen, and decalcified bone marrow biopsies from a panel of 95 cases of B-cell non-Hodgkin's lymphomas (B-NHLs) were analyzed. In addition, cyclin D1 protein expression was assessed by immunohistochemistry.. Strong cyclin D1 mRNA overexpression was detected in mantle cell lymphomas (23 of 23), hairy cell leukemias (5 of 19), and multiple myelomas (7 of 23) with particularly high levels in 2 of the latter cases. Intermediate transcript levels were found in 5 of 23 multiple myelomas and 7 of 19 hairy cell leukemias. B-cell chronic lymphocytic leukemias (10 of 10), follicular lymphomas (9 of 9), mucosa-associated lymphoid tissue lymphomas (5 of 5) and reactive lymphoid tissues with the exception of normal spleen had no or very low cyclin D1 expression. In comparison with real-time RT-PCR, immunohistochemistry showed a lower level of sensitivity, more variability, and did not allow accurate quantitation.. Real-time RT-PCR for cyclin D1 mRNA is an excellent tool for the differential diagnosis of B-NHLs and, in combination with microdissection, a powerful approach for retrospective trials using archival clinical specimens as tissue source. Furthermore, real-time RT-PCR may help to identify subgroups of B-NHLs according to cyclin D1 mRNA copy numbers and to investigate the possible influence of different chromosomal breakpoints on cyclin D1 expression.

Topics: Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Computer Systems; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Leukemia, B-Cell; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoid Tissue; Lymphoma, B-Cell; Lymphoma, B-Cell, Marginal Zone; Lymphoma, Follicular; Lymphoma, Mantle-Cell; Multiple Myeloma; Neoplasm Proteins; Paraffin Embedding; Pseudolymphoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Translocation, Genetic

Multiple myeloma (MM) is a plasma cell malignancy preliminary localized in the bone marrow and characterized by its capacity to disseminate. IL-6 and IGF-1 have been shown to mediate proliferative and anti-apoptotic signals in plasmocytes. However, in primary plasma-cell leukemia (PCL) and in end-stage aggressive extramedullar disease, the cytokine requirement for both effects may be not mandatory. This suggests that constitutive activation of signaling pathways occurs. One of the signaling pathways whose deregulation may play an oncogenic role in MM is the phosphatidylinositol 3-kinase (PI 3-K) pathway. In human growth factor-independent MM cell lines OPM2 and RPMI8226, we show that the PI 3-K inhibitors LY294002 and Wortmannin strongly inhibited cell proliferation, whereas inhibition of the mammalian Target Of Rapamycin (mTOR)/P70-S6-kinase (P70(S6K)) pathway with rapamycin or of the Mitogen-Activated Protein Kinase (MAPK) pathway with PD98059 had minimal effect on proliferation. In both cell lines, constitutive activation of the PI 3-K/Akt/FKHRL-1, mTOR/P70(S6K) and MAPK pathways was detected. LY294002 inhibited phosphorylation of Akt, FKHRL-1 and P70(S6K) but had no effect on ERK1/2 phosphorylation, indicating that the PI 3-K and MAPK pathways are independent. IGF-1 but not IL-6 increased phosphorylation of Akt, FKHRL-1 and P70(S6K). Purified plasmocytes from four patients with MM and two patients with primary PCL were studied. In three of them including the two patients with PCL, constitutive phosphorylation of Akt, FKHRL-1 and P70(S6K) was present, inhibited by LY294002 and enhanced by IGF-1. In these patients with constitutive Akt activation, normal PTEN expression was detected. PI 3-K inhibition induced caspase-dependent apoptosis as confirmed by inhibition with the large spectrum caspase inhibitor Z-VAD-FMK and cleavage of pro-caspase-3. Both cell lines spontaneously expressed Skp2 and cyclin D1 proteins at high levels but no p27(Kip1) protein. In the presence of LY294002, cell-cycle arrest in G0/G1 was observed, p27(Kip1) protein expression was up-regulated whereas the expression of both Skp2 and cyclin D1 dramatically diminished. PI 3-K-dependent GSK-3alpha/beta constitutive phosphorylation was also detected in OPM2 cells that may contribute to high cyclin D1 expression. Overall, our results suggest that PI 3-K has a major role in the control of proliferation and apoptosis of growth factor-independent MM cell lines. Most of the biological effec

Topics: Apoptosis; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line; Cyclin D1; Fas Ligand Protein; fas Receptor; Humans; Interleukin-6; Membrane Glycoproteins; Microfilament Proteins; Mitogen-Activated Protein Kinases; Multiple Myeloma; Muscle Proteins; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Ribosomal Protein S6 Kinases; S-Phase Kinase-Associated Proteins; TOR Serine-Threonine Kinases; Tumor Suppressor Proteins

The DNA microarray technology enables the identification of the large number of genes involved in the complex deregulation of cell homeostasis taking place in cancer. Using Affymetrix microarrays, we have compared the gene expression profiles of highly purified malignant plasma cells from nine patients with multiple myeloma (MM) and eight myeloma cell lines to those of highly purified nonmalignant plasma cells (eight samples) obtained by in vitro differentiation of peripheral blood B cells. Two unsupervised clustering algorithms classified these 25 samples into two distinct clusters: a malignant plasma cell cluster and a normal plasma cell cluster. Two hundred and fifty genes were significantly up-regulated and 159 down-regulated in malignant plasma samples compared to normal plasma samples. For some of these genes, an overexpression or downregulation of the encoded protein was confirmed (cyclin D1, c-myc, BMI-1, cystatin c, SPARC, RB). Two genes overexpressed in myeloma cells (ABL and cystathionine beta synthase) code for enzymes that could be a therapeutic target with specific drugs. These data provide a new insight into the understanding of myeloma disease and prefigure that the development of DNA microarray could help to develop an 'à la carte' treatment in cancer disease.

Topics: Adult; Aged; Anaphase-Promoting Complex-Cyclosome; Apoptosis; Bone Remodeling; Cell Line; Cyclin D1; Cystathionine beta-Synthase; Female; Gene Expression Profiling; Genes, abl; Genes, myc; Humans; Ligases; Male; Middle Aged; Multiple Myeloma; Oligonucleotide Array Sequence Analysis; Plasma Cells; Signal Transduction; Ubiquitin-Protein Ligase Complexes

Multiple myeloma (MM) is a clonal neoplasm of plasma cells which offers an excellent model to study multistep molecular oncogenesis. In 20-25% of primary tumors and cell lines examined, cyclin D1 is overexpressed due to the translocation t(11;14)(q13;q32). We have characterized cyclin-dependent kinase inhibitor p15 (CDKN2B), p16 (CDKN2A) and p18 (CDKN2C) deletions in cyclin D1-expressing and non-expressing MM cell lines. p18 was found to be frequently deleted (38%); in some cases p18 deletions coexisted with hemizygous p16 deletion. To examine the function of p18 as a putative tumor suppressor in myeloma cells, a zinc-inducible p18 construct was stably transfected into KMS12, a MM cell line with biallelic p18 and monoallelic p16 deletions as well as cyclin D1 overexpression. Ectopic expression of p18 caused 40-45% growth suppression as determined by trypan blue exclusion and MTS assays. p18 induction also resulted in apoptosis, suggesting that inhibition of the cyclin D1/CDK/pRb pathway in these tumor cells could be a crucial step toward the induction of tumor regression via apoptotic cell death. This cell cycle pathway is thus frequently mutated and provides a potentially novel target for gene therapeutic or pharmacologic approaches to human myeloma.

Topics: Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin D1; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p18; Cyclin-Dependent Kinases; Enzyme Inhibitors; Gene Deletion; Genes, Tumor Suppressor; Genotype; Humans; Lymphoma, Mantle-Cell; Multiple Myeloma; Neoplasm Proteins; Protein Serine-Threonine Kinases; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

Cyclin D1, encoded by the CCND1 gene, is immunohistochemically detectable in up to one-third of cases of multiple myeloma (MM). To examine the mechanism of cyclin D1 overexpression, we compared cyclin D1 immunoreactivity with the results of conventional cytogenetics to determine if the t(11;14)(q13;q32) or other abnormalities of 11q11-14 explained cyclin D1 overexpression. Karyotypic abnormalities were found in 45 out of 67 (67%) MM cases; the t(11;14) was present in seven cases (10%). Additional 11q11-14 abnormalities were not identified. The t(11;14) correlated with cyclin D1 upregulation in low to intermediately proliferative MM, but was not present in highly proliferative tumours (assessed using bromodeoxyuridine labelling index). Cyclin D1 indirectly activates the transcription factor E2F-1. In the bone marrow biopsy specimens of MM cases, E2F-1 was concurrently expressed with cyclin D1 (P = 0.001), indicating that cyclin D1 is functional. However, as neither E2F-1 nor cyclin D1 expression correlated with proliferative activity, the speculation that t(11;14) upregulates the CCND1 gene to induce higher proliferation and possibly more aggressive disease is not supported. We conclude that in low to intermediately proliferative MM cases, cyclin D1 is probably upregulated by t(11;14), but an alternative mechanism is more probable in highly proliferative MM.

Topics: Adult; Aged; Bone Marrow Cells; Bromodeoxyuridine; Carrier Proteins; Cell Cycle Proteins; Cell Division; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Cytogenetic Analysis; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Female; Flow Cytometry; Gene Amplification; Humans; Immunohistochemistry; Male; Middle Aged; Multiple Myeloma; Retinoblastoma-Binding Protein 1; Transcription Factor DP1; Transcription Factors; Translocation, Genetic

Among the recently discovered myeloma-specific gene alterations associated with chromosomal translocations, cyclin D1/PRAD1/Bcl-1 overexpression caused by t(11;14)(q13;q32) is considered to be the most frequent in myeloma patients and cell lines, and may be a prognostic factor clinically. To elucidate the cellular biological role of overexpressed cyclin D1 in myeloma cells, we examined the mRNA expression levels of cell cycle regulators including three cyclin Ds, cyclin-dependent kinase inhibitors (CDK-Is) and accelerators. Cyclin D1 overexpression was clearly demonstrated in the lines with abnormal 11q13 and associated with overexpression of S and G2 accelerator genes. The cyclin D1-overexpressing lines tended to have a shortened G1 phase compared with the non-expressing lines. In addition, artificial silencing using antisense oligonucleotides for cyclin D1 suppressed the growth rate of some but not all cyclin D1-overexpressing cells. These results indicate that overexpression of cyclin D1 caused by cytogenetic abnormalities may make cells progress through the cell cycle rapidly, but it seems that other factors such as cyclin D2 and translocation-related genes affect the cell cycle progression in myeloma cells.

Topics: Cell Cycle; Cell Cycle Proteins; Cell Division; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; DNA Primers; Gene Expression; Humans; Immunohistochemistry; Multiple Myeloma; Oligonucleotides, Antisense; Reverse Transcriptase Polymerase Chain Reaction; Translocation, Genetic; Tumor Cells, Cultured

Cyclin D1 expression was evaluated by immunohistochemical analysis and biotin-labeled in situ hybridization (ISH) in a series of 71 decalcified, paraffin-embedded bone marrow biopsy specimens from patients with multiple myeloma (MM). Cyclin D1 messenger RNA (mRNA) overexpression was detected by ISH in 23 (32%) of 71 cases, whereas cyclin D1 protein was identified by immunohistochemical analysis in 17 (24%) of 71 specimens. All cases that were positive by immunohistochemical analysis also were positive by ISH. Statistically significant associations were found between cyclin D1 overexpression and grade of plasma cell differentiation and between cyclin D1 overexpression and extent of bone marrow infiltration. Our findings demonstrate the following: (1) ISH for cyclin D1 mRNA is a sensitive method for the evaluation of cyclin D1 overexpression in paraffin-embedded bone marrow biopsy specimens with MM. (2) ISH is more sensitive than immunohistochemical analysis in the assessment of cyclin D1 expression. (3) Cyclin D1 overexpression in MM is correlated positively with higher histologic grade and stage.

Topics: Antigens, CD20; Biopsy; Biotinylation; Bone Marrow; Cell Differentiation; Cyclin D1; Female; Gene Expression; Humans; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Immunohistochemistry; In Situ Hybridization; Male; Middle Aged; Multiple Myeloma; Neoplasm Staging; Paraffin; Plasma Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Embedding

Two new human myeloma cell lines were established from pleural effusion and bone marrow malignant cells derived from a single patient, who manifested hyperammonemia associated with multiple myeloma, and these were characterized. Both lines possess t(11;14)(q13;q32) and t(8;14)(q24;q32) reciprocal translocations and overexpress cyclin D1, but not c-myc. Human myeloma lines including these new lines produced and secreted excess ammonia into culture medium more than non-myelomatous hematological cell lines. In addition, these two lines were revealed to have high surface CD7 expression correlated with relatively high mRNA expression by MP-RT-PCR. Among 8 human myeloma lines, half of them revealed significant surface expression of CD7 and a positive correlation between expression levels of protein and message. CD7 message was also detected in surface negative lines. Consequently, there may be posttranslational regulation of the CD7 molecule, whose cellular biological role in expressing cells has not been elucidated.

Topics: Adult; Ammonia; Antigens, CD7; Bone Marrow Cells; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 8; Cyclin D1; Humans; Hyperammonemia; Male; Multiple Myeloma; Pleural Effusion, Malignant; Proto-Oncogene Proteins c-myc; Translocation, Genetic; Tumor Cells, Cultured

The rearrangement of Bcl-1 gene (Bcl-1/IgH rearrangement) and expression of cyclin D1 in multiple myeloma (MM) precursor cells were studied and the role of cyclin D1 in the pathogenesis of MM was investigated. The BCL-1 rearrangement and cyclin D1 protein expression in 15 cases of MM were detected. By using hemi-nested polymerase chain reaction (PCR) the genomic DNA from fresh peripheral blood and bone marrow was amplified and the expression of cyclin D1 in the smears was detected by using immunohistochemical method. Ten volunteer with normal bone marrow served as control group. The results showed Bcl-1 rearrangement was detectable in 3/15 (20%) MM patients and cyclin D, expression in 4/15 (27%) MM patients. Bcl-1 rearrangement and cyclin D1 protein expression were also detected in MM precursor cells. No overexpression of cyclin D1 or the rearrangement of the Bcl-1 gene was found in the 10 volunteers. It was concluded that Bcl-1 rearrangement and cyclin D1 protein overexpression were detected in MM precursor cells, speculating that overexpression of cyclin D1 protein may play an initial (critical) role in the pathogenesis of MM.

Topics: Adult; Aged; Cyclin D1; Erythroid Precursor Cells; Female; Gene Rearrangement; Genes, bcl-1; Humans; Male; Middle Aged; Multiple Myeloma; Myosin Light Chains

EB1089, a 1,25-dihydroxyvitamin D(3) analog, has been known to have potent antiproliferative properties in a variety of malignant cells in vitro and in vivo. In the present study, we analyzed the effect of EB1089 on human myeloma cell lines. EB1089 inhibited the proliferation of NCI-H929 cells and RPMI8226 cells in a dose-dependent manner among three myeloma cell lines tested. The antiproliferative effect of EB1089 on myeloma cells was related to the expression level of vitamin D receptor. To investigate the mechanism of the antiproliferative effect of EB1089, cell cycle analysis was attempted in EB1089-sensitive NCI-H929 cells. EB1089 (1 x 10(-8) M) efficiently induced G(1) arrest of the cell cycle. Analysis of G(1) regulatory proteins demonstrated that protein levels of CDK2, CDK4, cyclin D1, and cyclin A were decreased in a time-dependent manner, but not those of CDK6 and cyclin E, by EB1089. In addition, EB1089 (1 x 10(-8) M, 72 h) increased the protein level of the CDKI p27 and markedly enhanced the binding of p27 with CDK2 compared to EB1089-untreated cells. Furthermore, the activity of CDK2-associated cyclin kinase was decreased, which was accompanied by the reduction of cyclin-D1-, cyclin-E-, and cyclin-A-associated kinase activities, resulting in the hypophosphorylation of Rb protein. These results suggest that EB1089 can inhibit the proliferation of human myeloma cells, especially NCI-H929 cells, via a G(1) block in association with the induction of p27 and the reduction of CDK2 activity.

Topics: Antineoplastic Agents; Calcitriol; CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin A; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; G1 Phase; Humans; Kinetics; Microtubule-Associated Proteins; Multiple Myeloma; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptors, Calcitriol; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Proteins

Through the application of the NIH/3T3 tumorigenicity assay to DNA from a gastric carcinoma, we have identified a novel transforming gene, designated myeov (myeloma overexpressed gene in a subset of t[11;14]-positive multiple myelomas). Sequence analyses did not reveal any homology with sequences present in the GenBank, except the deduced protein structure predicts a transmembrane localization. Myeov was mapped to chromosome 11q13 and localized by DNA fiber fluorescence in situ hybridization (FISH) 360-kilobase (kb) centromeric of cyclin D1. In 3 of 7 multiple myeloma (MM) cell lines with a t(11;14)(q13;q32) and cyclin-D1 overexpression, Northern blot analysis revealed overexpression of myeov as well. In all 7 cell lines, the translocation breakpoint was mapped within the 360-kb region between myeov and cyclin D1. DNA fiber FISH with a contig of probes covering the constant region of the immunoglobulin heavy chain (IgH) revealed that exclusively in the 3 myeov-overexpressing cell lines (KMS-12, KMS-21, and XG-5), either the 5' E(mu) enhancer or the most telomeric 3' Ealpha enhancer was juxtaposed to myeov. Although cyclin D1 overexpression represents a characteristic feature of all MM cell lines with t(11;14), our results demonstrate aberrant expression of a second putative oncogene in a subset of these cases, due to juxtaposition to IgH enhancers. The clinical relevance of this dual activation remains to be elucidated. (Blood. 2000;95:2691-2698)

Topics: Amino Acid Sequence; Chromosome Mapping; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; DNA, Neoplasm; Humans; Molecular Sequence Data; Multiple Myeloma; Neoplasm Proteins; Translocation, Genetic; Tumor Cells, Cultured

The t(11;14)(q13;q32) chromosomal translocation, the hallmark of mantle cell lymphoma (MCL), is recurrently found in multiple myelomas (MM) by means of conventional cytogenetics. Unlike MCL, recent molecular studies of MM-derived cell lines with t(11;14) have indicated that the breakpoints are highly dispersed over the 11q13 region; however, the fact that cyclin D1 is generally overexpressed in these cell lines suggests that this gene is the target of the translocation. To evaluate further the involvement of cyclin D1 in MM, we used immunohistochemistry and fluorescence in situ hybridization to investigate cyclin D1 expression and the presence of chromosome 11 abnormalities in a representative panel of 48 MM patients (40 at diagnosis and 8 at relapse). Cyclin D1 overexpression occurred in 12/48 (25%) of cases; combined immunohistochemistry and fluorescence in situ hybridization analyses in 39 patients showed cyclin D1 positivity in all of the cases (7/7) bearing the t(11;14), in two of the 13 cases with trisomy 11, and in one of the 19 cases with no apparent abnormalities of chromosome 11. Our data indicate that the t(11;14) translocation in MM leads to cyclin D1 overexpression and that immunohistochemical analysis may represent a reliable means of identifying this lesion in MM.

Topics: Adult; Aged; Aged, 80 and over; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Female; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; Middle Aged; Multiple Myeloma; Neoplasm Staging; Translocation, Genetic

Approximately 30% of myeloma patients express cyclin D1 RNA and protein. The low incidence of translocation t(11; 14) detected by conventional cytogenetics suggests that the up-regulation of cyclin D1 protein might result from other mechanisms as well as from gene amplification. Therefore, the frequency and the clinical and prognostic implications of cyclin D1 amplification were examined. We highly purified myeloma cells from bone marrow by magnetic cell sorting and analysed 50 myelomas by fluorescence in situ hybridization (FISH) using probes specific for cyclin D1 and 20 samples by immunoblotting to detect cyclin D1 expression. The amplification of cyclin D1 gene was found in 19 of 50 analysed patients and was associated with expression of cyclin D1 protein. The amplification correlated significantly with the bone marrow infiltration, plasma cell morphology and labelling index as well as serum beta2-microglobulin, C-reactive protein (CRP) and creatinine levels. In univariate analysis, the amplification of the cyclin D1 gene was a significantly unfavourable parameter with regard to overall survival (P = 0.0064) and progression-free survival (P = 0. 0005). In multivariate analysis, cyclin D1 amplification and serum beta2-microglobulin were independent and well-suited parameters for predicting survival. The detection of cyclin D1 amplification seems to be of promising prognostic value in multiple myeloma.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Analysis of Variance; beta 2-Microglobulin; Biomarkers; Case-Control Studies; Child; Child, Preschool; Cyclin D1; Disease-Free Survival; Gene Amplification; Genes, bcl-1; Humans; In Situ Hybridization, Fluorescence; Infant; Infant, Newborn; Middle Aged; Multiple Myeloma; Prognosis; Proportional Hazards Models

We identified 24 cases of multiple myeloma with the t(11;14)(q13;q32). In 22 cases, the t(11;14)(q13;q32) was part of a complex karyotype, and in 2 cases it was an isolated abnormality. All patients had clinical and laboratory features consistent with multiple myeloma. The median degree of plasma cell involvement in the bone marrow was 60%, and in 10 cases, the plasma cells had a lymphoplasmacytoid appearance. Of the 24 cases, 21 had intermediate or high proliferative rates based on labeling index studies. Immunohistochemical studies performed on all bone marrow biopsy specimens showed strong cyclin D1 nuclear positivity in 19 cases. There also was strong cyclin D1 nuclear positivity found in 6 of 30 additional cases without the t(11;14)(q13;q32) demonstrated by routine cytogenetics. The t(11;14)(q13;q32) in multiple myeloma results in overexpression of the cyclin D1 protein, which can be demonstrated by immunohistochemical stain. The cyclin D1 stain results in the additional cases of multiple myeloma suggest that the t(11;14)(q13;q32) may be more common than previously thought and may be missed by routine cytogenetics, particularly if the proliferative rate is low.

Topics: Adult; Aged; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Multiple Myeloma; Translocation, Genetic

Topics: Cyclin D1; Cyclin D2; Cyclin D3; Cyclins; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Hairy Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoproliferative Disorders; Multiple Myeloma; Neoplasm Proteins; RNA, Messenger; RNA, Neoplasm; Splenic Neoplasms

Chromosomal abnormalities are detected in 50 to 70% of patients with multiple myeloma (MM). By conventional cytogenetic analysis, a t(11;14)(q13;q32) is observed at a frequency of 3 to 14%.. To demonstrate a cyclin D1 expression in MM patients or MM cell lines, 14 patients with multiple myeloma (MM) and nine human multiple myeloma cell lines (HMCL) were screened by a competitive RT-PCR and/or Northern blot analysis for cyclin D1 expression. Furthermore, we screened 10 MM patients with FISH to demonstrate a relationship between the cyclin D1 expression and the presence of the t(11;14).. Five HMCL had a cyclin D1 overexpression: three of them had a t(11;14)(q13;q32) and two had extra copies of chromosome 11. A cyclin D1 expression was found at diagnosis in seven out of 14 untreated MM patients (50%). Out of 14 MM patients, FISH studies were performed in 10 patients. A t(11;14) was detected in three out of 10 patients and extra copies of chromosome 11 were found in two additional patients.. Cyclin D1 expression is a common event in MM patients (50%) and is associated either with a t(11;14)(q13;q32) or extra copies of chromosome 11. The prognostic role of the cyclin D1 expression and the level of this expression, as compared to other B-cell chronic lymphoproliferative disorders such as mantle cell lymphoma or hairy cell leukemia, remains to be determined in the pathogenesis of multiple myeloma.

Topics: Bone Marrow; Chromosome Mapping; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; DNA Primers; Female; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; Male; Multiple Myeloma; Neoplasm Staging; Plasma Cells; Reverse Transcriptase Polymerase Chain Reaction; Translocation, Genetic; Tumor Cells, Cultured

Oncogenes are often dysregulated in B cell tumors as a result of a reciprocal translocation involving an immunoglobulin locus. The translocations are caused by errors in two developmentally regulated DNA recombination processes: V(D)J and IgH switch recombination. Both processes share the property of joining discontinuous sequences from one chromosome and releasing intervening sequences as circles that are lost from progeny cells. Here we show that these intervening sequences may instead insert in the genome and that during productive IgH mu-epsilon switch recombination in U266 myeloma tumor cells, a portion of the excised IgH switch intervening sequences containing the 3' alpha-1 enhancer has inserted on chromosome 11q13, resulting in overexpression of the adjacent cyclin D1 oncogene.

Topics: Base Sequence; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Switch Region; In Situ Hybridization, Fluorescence; Molecular Sequence Data; Multiple Myeloma; Oncogenes; Recombination, Genetic; Sequence Alignment; Translocation, Genetic; Tumor Cells, Cultured

We analysed PRAD1/cyclin D1 expression in 20 patients with plasma cell malignancy by Northern analysis. 6/17 multiple myeloma patients and 3/3 plasma cell leukaemia patients showed PRAD1/cyclin D1 expression. This incidence appeared to be higher than the expected incidence based on previous studies. Southern analysis did not show rearrangement of the bcl-1 region. Although there was no statistical difference, the PRAD1/cyclin D1 negative group showed a 1-year survival of 81.8%, 3-year survival of 45.5% and 5-year survival of 22.7%, and those for the PRAD1/cyclin D1 positive group were 63.5%, 16.9% and 16.9%, respectively. Further study is required to determine whether PRAD1/cyclin D1 expression is a prognostic factor.

Topics: Aged; Blotting, Southern; Cyclin D1; Female; Humans; Leukemia, Plasma Cell; Male; Middle Aged; Multiple Myeloma; Prognosis; Survival Analysis

The t(11;14)(q13;q32) translocation with cyclin D1 overexpression commonly is found in multiple myeloma (MM) and in mantle cell lymphoma (MCL). Several reports have shown that p53 mutations in MCL lead to blastoid transformation and a worse prognosis; however, the role of p53 mutations in MM with t(11;14) is unclear.. In this study the authors describe a patient with MM with t(11;14) and a p53 mutation at presentation and characterized a cell line, MEF-1, established from this patient. Immunohistochemical analysis of p53 and cyclin D1 proteins was performed. The p53 gene was analyzed by polymerase chain reaction-single strand conformation polymorphism and direct sequencing. The expression of cyclin D1 mRNA was examined by Northern blot analysis.. MEF-1 had t(11;14) with overexpression of cyclin D1 mRNA and produced immunoglobulin kappa-light chain. MEF-1 had a mutation in exon 7 (codon 255-257) of the p53 gene, which was noted in the patient's myeloma cells.. p53 mutations may be important genetic events in disease progression of MM with t(11;14). The MEF-1 cell line may be a useful tool to study mechanisms of progression in MM based on abnormalities of the cyclin D1 gene.

Topics: Aged; Bone Marrow; Bone Neoplasms; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Epstein-Barr Virus Infections; Fatal Outcome; Female; Forearm; Gene Expression Regulation, Neoplastic; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genes, p53; Herpesvirus 4, Human; Humans; Immunophenotyping; Karyotyping; Multiple Myeloma; Myeloma Proteins; Neoplasm Proteins; Polymorphism, Single-Stranded Conformational; Translocation, Genetic; Tumor Cells, Cultured

t(11;14)(q13;q32) observed in B-cell malignancies is associated with cyclin D1 (bcl-1, PRAD1, CCND1) overexpression. We devised a simple competitive reverse transcription-polymerase chain reaction (RT-PCR) assay for rapid detection of cyclin D1 overexpression. Sharing a single upstream primer derived from a homologous sequence in cyclins D1, D2 and D3, each PCR product serves as a competitor and cyclin D1 overexpression is determined by comparing the intensities of the three amplified products. We analyzed cyclin D1 in clinical specimens from 104 patients with lymphoid malignancies. Cyclin D1 overexpression was evident in 13 of 104 (7/72 non-Hodgkin's lymphomas, 0/6 adult T-cell lymphoma/leukemias, 0/4 Hodgkin's diseases, 0/11 acute lymphoblastic leukemias, 3/4 multiple myelomas, 1/2 Waldenström's macroglobulinemias, 1/2 prolymphocytic leukemias and 1/3 chronic lymphocytic leukemias). Among 72 patients for whom cytogenetic studies had been done, all 7 patients with t(11;14) were positive. The relative expression levels of D-type cyclins altered dramatically in the presence of t(11;14). Thus, this RT-PCR assay can identify tumors with cyclin D1 overexpression. Cyclin D1 overexpression was frequent in extranodal specimens (11 out of 32 vs. 2 of 72 lymph nodes) and was restricted to specific types of lymphoid malignancies, as observed using other methods. This reliable assay should be suitable to provide clinical guidance for the diagnosis and management of lymphoid malignancies, especially in the case of extranodal involvement.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bone Marrow; Cyclin D1; Female; Hodgkin Disease; Humans; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, Non-Hodgkin; Lymphoproliferative Disorders; Male; Middle Aged; Multiple Myeloma; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Transcription, Genetic; Waldenstrom Macroglobulinemia

Approximately 30% of multiple myelomas (MMs) express cyclin D1 when assessed using immunohistochemical techniques. Cyclin D1 expression correlates with greater tumor burden in MM, because cyclin D1-positive cases are more frequently associated with extensive bone marrow involvement, i.e., high pathologic stage, than are cyclin D1-negative cases. The mechanisms that explain this association are unknown. To explore other differences between cyclin D1-positive and cyclin D1-negative MMs, we assessed 59 MMs immunohistochemically for several G1 cell-cycle regulatory proteins, including cyclin D1, E2F-1, p53, mdm-2, and p21waf-1, using routinely fixed and processed, paraffin-embedded bone marrow specimens. Twenty MMs (34%) were cyclin D1 positive, and 39 (66%) were cyclin D1 negative. Eighteen (90%) of 20 cyclin D1-positive MMs were Stage III, in contrast to 19 (49%) of 39 cyclin D1-negative MMs (P = .003). Cyclin D1-positive MMs were more likely to express E2F-1 (16/20 vs. 4/39, P < .001), p53 (11/20 vs. 10/39, P = .041), and p21waf-1 (12/20 vs. 7/39, P = .003). There was no significant difference in mdm-2 expression between these groups. We also assessed proliferation rate using an antibody specific for the Ki-67 antigen. A relatively high percentage (> 20%) of Ki-67-positive cells was found in cyclin D1-positive MMs compared with cyclin D1-negative MMs (13/20 vs. 3/39, P < 0.001). These results suggest that cyclin D1-positive MMs are more likely to possess additional derangements involving other G1 cell-cycle regulatory proteins. We speculate that these abnormalities might result in increased proliferation, thereby explaining the correlation between cyclin D1 expression and greater tumor burden.

Topics: Bone Marrow Cells; Carrier Proteins; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Enzyme Inhibitors; Humans; Immunoenzyme Techniques; Ki-67 Antigen; Multiple Myeloma; Neoplasm Proteins; Nuclear Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Retinoblastoma-Binding Protein 1; Transcription Factor DP1; Transcription Factors; Tumor Suppressor Protein p53

Topics: Chromosome Mapping; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Cyclins; Humans; Immunoglobulin Constant Regions; Immunoglobulin Heavy Chains; In Situ Hybridization, Fluorescence; Multiple Myeloma; Oncogene Proteins; Translocation, Genetic; Tumor Cells, Cultured

In this study, we analyzed 69 plasma cell neoplasms, including 54 multiple myelomas (MMs), 3 cases of plasma cell leukemia, and 12 plasmacytomas, for expression of cyclin D1 protein using an immuno-histochemical method applied to routinely fixed, paraffin-embedded tissue sections. Cyclin D1 was expressed in 18 (26%) of 69 plasma cell neoplasms, including 16 (30%) of 54 MMs and 2 (17%) of 12 plasmacytomas. Three cases of plasma cell leukemia were negative. Cyclin D1 expression correlated with the cytologic differentiation and histologic stage of MM. Twelve (75%) of 16 MMs had intermediate or immature cytologic features and were histologic Stage III. With use of a polymerase chain reaction assay, we also analyzed six cases (three cyclin D1 positive, three cyclin D1 negative) for the t(11;14); one MM carried the t(11;14) and expressed cyclin D1 protein. We conclude that cyclin D1 expression occurs in approximately one-quarter of plasma cell neoplasms and correlates with the degree of cytologic differentiation and histologic stage. The relatively high frequency of cyclin D1 expression, compared with the less than 5% incidence of the t(11;14) detected by conventional cytogenetics reported in the literature, suggests that upregulation of cyclin D1 protein might be the result of mechanisms other than the t(11;14).

Topics: Adult; Aged; Aged, 80 and over; Cyclin D1; Female; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunohistochemistry; Leukemia, Plasma Cell; Male; Middle Aged; Multiple Myeloma; Paraffin Embedding; Plasmacytoma; Polymerase Chain Reaction; Survival Rate

Myeloma cells consist of immature, intermediate and mature cells with respect to expression of VLA-5 (CD49e) and MPC-1 adhesion molecules. VLA-5(-)MPC-1(-) immature myeloma cells respond to interleukin 6 (IL-6) to proliferate in vitro. but VLA-5+MPC-1+ mature myeloma cells have almost no proliferative activity with higher secretory activity of M-protein in vitro. In order to further clarify the biological differences between these immature and mature myeloma cells, we examined survival of these cells with or without IL-6 in vitro, and investigated the underlying mechanism of the proliferative or non-proliferative character of these cells by examining expression of cell cycle regulators such as cyclin D1 and inhibitors for cyclin-dependent kinase (Cdk), p16INK4A, p21CIP1 and p27KIP1 by RT-PCR and immunohistochemistry. In vitro survival of these myeloma cells was examined by flow cytometric quantification of fluorescein diacetate (FDA) and propidium iodide (PI) staining. Immature myeloma cells rapidly entered apoptosis without IL-6, but mature myeloma cells could survive without IL-6 as well as normal mature plasma cells. Immature myeloma cells as well as myeloma cell lines expressed cyclin D1 mRNA and protein, but not any Cdk inhibitors. On the other hand, mature myeloma cells did not express cyclin D1 but expressed p16, not p21 or p27, as well as normal mature plasma cells. Therefore these results show that immature myeloma cells constitutively express cyclin D1 and can proliferate, and mature myeloma cells as well as normal mature plasma cells preferentially express p16 and can survive for a long time without proliferation.

Topics: Base Sequence; Cell Separation; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p16; Humans; Immunohistochemistry; Molecular Sequence Data; Multiple Myeloma; Polymerase Chain Reaction; Tumor Cells, Cultured

Translocations involving the IgH locus at chromosomal locus 14q32.3 are a common event in many B-cell malignancies. The translocations, which generally occur into JH and switch regions, are mediated by errors in the two developmentally regulated, lymphocyte-specific pathways: VDJ-and switch-mediated recombination. Dysregulation of cyclin D1 by a t(11;14)(q13;q32) translocation occurs in most cases of mantle-cell lymphoma and in approximately 30% of multiple myeloma (MM) tumors in which a 14q32 translocation can be detected. We show here that in two of three myeloma lines that overexpress cyclin D1, there is an 11;14 translocation into a gamma switch region, suggesting an error in switch recombination. By contrast, 11;14 translocations in mantlecell lymphoma are invariably into or near a JH segment, suggesting an error in VDJ recombination. This is consistent with the fact that myeloma cells have undergone lgH switch recombination, whereas mantle-cell lymphoma cells generally have not.

Topics: Base Sequence; Blotting, Southern; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Cyclins; DNA Nucleotidyltransferases; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genes, Switch; Humans; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin Heavy Chains; Lymphoma, B-Cell; Molecular Sequence Data; Multiple Myeloma; Myeloma Proteins; Oncogene Proteins; Recombination, Genetic; Translocation, Genetic; VDJ Recombinases

Cyclin D1/PRAD1 (bcl-1) is a recently discovered proto-oncogene that is overexpressed in mantle cell lymphomas and several other human tumors. In a previous study, the authors demonstrated expression of cyclin D1 in 15 of 15 cases of mantle cell lymphoma and 1 of 8 cases of B-chronic lymphocytic leukemia (B-CLL) using a polyclonal antibody and microwave enhanced immunohistochemical staining method on paraffin-embedded tissue sections. In this study, 107 additional B- and T-cell neoplasms were studied, including 47 cases of high grade lymphoma (33 diffuse large B-cell type, 9 Burkitt and Burkitt-like, 4 precursor T-lymphoblastic lymphoma, and 1 adult T-cell lymphoma/leukemia), 38 additional cases of low grade B-cell lymphoma (18 CLL, 15 hairy cell leukemia and 5 mantle cell lymphoma), and 22 plasmacytomas for expression of cyclin D1 using the same immunohistochemical staining technique. All cases of mantle cell lymphoma showed diffuse nuclear staining. No additional cases of CLL showed cyclin D1 expression. In contrast, 1 of 15 hairy cell leukemias and 1 of 22 plasmacytomas showed cyclin D1 staining. None of the high grade lymphomas demonstrated expression of cyclin D1 protein by immunostaining, including three cases of large B-cell lymphoma that coexpressed CD5. The authors conclude that cyclin D1 is expressed in all cases of mantle cell lymphoma, and only in very rare cases of B-CLL, hairy cell leukemia and plasmacytoma/myeloma. Cyclin D1 does not appear to play an important role in high grade lymphomas. In addition, most CD5 positive high grade B-cell lymphomas do not express cyclin D1, and are not likely to be derived from mantle cell lymphoma or other lymphomas with t(11;14).

Topics: Cyclin D1; Cyclins; Humans; Immunohistochemistry; Leukemia, Hairy Cell; Lymphoma; Lymphoma, B-Cell; Lymphoma, Non-Hodgkin; Multiple Myeloma; Oncogene Proteins; Plasmacytoma; Proto-Oncogene Mas; Staining and Labeling

The t(11;14)(q13;q32) chromosomal translocation is associated with several B-cell lymphoproliferative disorders and is thought to result in upregulation of expression of PRAD1/cyclin D1 proto-oncogene. A patient with multiple myeloma of IgG kappa-type with t(11;14)(q13;q32) is now shown to overexpress PRAD1. The clinical stage of the disease was advanced (IIIA), with a myeloma cell count of 94.6% in the bone marrow. Chromosomal analysis of bone marrow cells showed t(11;14)(q13;q32) in five of 20 metaphases as well as other karyotypic features. Northern blot analysis of RNA prepared from myeloma cells revealed overexpression of PRAD1. Multiple myeloma with t(11;14)(q13;q32) has been associated with an aggressive clinical course. Although neither myeloma cells in the peripheral blood nor extramedullary lesions were apparent in the present patient, the myeloma was refractory to several chemotherapeutic regimens from the beginning. Detection of PRAD1 expression may offer an easier alternative to cytogenetic analysis in myeloma and is a potentially useful indicator of a poor prognosis.

Topics: Blotting, Northern; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Cyclins; Female; Gene Expression; Humans; Middle Aged; Multiple Myeloma; Oncogene Proteins; Prognosis; Proto-Oncogene Mas; RNA, Neoplasm; Translocation, Genetic

To identify genes activated by chromosome translocation t(11;14)(q13;q32), mRNA levels of five genes (cyclin D1, EXP1, MB38, HST1, and INT2) at chromosome 11q13 were investigated. The cyclin D1 mRNA increased in BCL-1-rearranged B-cell tumor cell lines SP-49, NOP-2, FLAM-76, KMS-12-PE, and KMS-12-BM cells, while it was not detected in cell lines without the translocation, Raji, U266, and HEL cells. A significant amount of the MB38 mRNA was detected irrelevantly to the translocation in all of these cell lines. The mRNAs of EXP1, HST1, and INT2 were undetectable in these cells. The results suggested that the translocation activates cyclin D1 alone, while the mRNA levels of the other four genes are regulated independently of the translocation.

Topics: Blotting, Northern; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Cyclins; Humans; Lymphoma, B-Cell; Multiple Myeloma; Oncogene Proteins; RNA, Messenger; RNA, Neoplasm; Translocation, Genetic

We have analyzed the BCL1 locus in a series of 24 B-cell tumors and cell lines with rearrangements of 11q13 (mostly t(11;14)(q13;q32) translocations). Using Southern hybridization and/or fluorescence in situ hybridization (FISH) on metaphase chromosomes, we have not only confirmed the scattering of the breakpoints between the BCL1 locus and the cyclin D1 gene (CCND1), but also shown that some of the breakpoints could be as far as 500 kb on either side of the latter. Expression of CCND1 was not restricted to cases with t(11;14)(q13;q32), but was also associated with other 11q13 rearrangements, such as a t(8;11)(p22;q13). Whatever the alteration at 11q13, a correlation was observed between the expression of CCND1 and the presence of a breakpoint within 150 kb upstream of the gene. On the contrary, three samples, including a bona fide t(11;14) translocation and two cases with breakpoints located outside the BCL1-CCND1 area, did not exhibit detectable levels of CCND1 transcripts. Our results raise the possibility that several discrete molecular events can take place at 11q13 in B-cell malignancies.

Topics: Base Sequence; Blotting, Northern; Blotting, Southern; Chromosomes, Human, Pair 11; Cosmids; Cyclin D1; Cyclins; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Gene Rearrangement, B-Lymphocyte; Humans; In Situ Hybridization, Fluorescence; Leukemia, B-Cell; Lymphoma, Non-Hodgkin; Molecular Sequence Data; Multiple Myeloma; Oncogene Proteins; Polymorphism, Restriction Fragment Length; Prospective Studies; Restriction Mapping; Retrospective Studies; RNA, Neoplasm; Transcription, Genetic; Translocation, Genetic

Translocation (11;14)(q13;q32) is a recurring chromosome abnormality which is found non-randomly in non-Hodgkin's lymphoma, especially of follicular type, as well as in B-cell chronic lymphocytic leukaemia, Waldenström's macroglobulinaemia and multiple myeloma. To define further the prevalence of this abnormality in multiple myeloma, we studied a series of 17 patients with this disease with concomitant chromosome analysis and Southern blotting, using a probe specific for the major translocation cluster of the BCL-1 oncogene which is located at chromosome 11q13. Karyotype analysis of 14 evaluable patients revealed three cases with abnormalities of chromosome 11q13, two of them with t(11;14)(q13;q32) and one with del (11)(q13). Southern blot analysis showed no rearrangements in BclI and HindIII digests of DNA from 17 patients including the three patients with anomaly of chromosome 11q13, using a BCL-1 specific probe. A possible restriction length polymorphism was detected in EcoRI cut DNA of five out of 11 patients studied. Therefore the chromosomal break point in our cases with abnormality of chromosome 11q13 must lie outside the major translocation cluster of the BCL-1 gene. However, in centrocytic lymphoma rearrangement of the BCL-1 oncogene has been detected in up to 50% of cases. Location of the break point may therefore be dependent on the differentiation of the transformed B-cell.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Southern; Chromosomes; Cyclin D1; DNA; Female; Gene Rearrangement, B-Lymphocyte; Humans; Karyotyping; Male; Middle Aged; Multiple Myeloma; Proto-Oncogene Proteins; Translocation, Genetic

The proto-oncogene PRAD1 (parathyroid adenoma 1) on chromosome 11q13 was found to be overexpressed in all five B-cell lines with t(11;14)(q13;q32) translocation tested. One B-cell lymphoma and four myeloma cell lines with this translocation demonstrated more than 10-fold overexpression as determined by Northern blot analysis, when compared with normal lymphoid tissues such as thymus, spleen and lymph node. Hematopoietic cell lines without the translocation were also examined, but none of these demonstrated the overexpression, confirming that overexpression of the PRAD1 gene is associated with t(11;14) translocation. A truncated form of mRNA was seen in one of five cell lines with the translocation, SP-49. Hybridization with different regions of the PRAD1 cDNA revealed that the truncated form of mRNA retained the coding region but had lost the 3' untranslated region. Southern blot analysis demonstrated a gene rearrangement in this SP-49 cell line. To study the genetic alteration responsible for the truncated form of mRNA in this cell line, the rearranged allele as well as the germline allele were cloned. The restriction map revealed that the rearranged portion was at the 3' end of the PRAD1 gene, eliminating the mRNA-destabilizing signal AUUUA. Human-rodent hybrid cell analysis demonstrated that the region introduced 3' of PRAD1 was derived from chromosome 11, suggesting that the PRAD1 gene region is deleted at the 3' end. Over-expression of the PRAD1 gene in association with t(11;14)(q13;q32) translocation suggested that in these cases the regulation of PRAD1 was altered by the juxtaposed gene, most likely the immunoglobulin heavy-chain gene from chromosome 14.

Topics: Base Sequence; Chromosome Deletion; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 14; Cyclin D1; Cyclins; Gene Expression; Gene Rearrangement, B-Lymphocyte; Humans; Lymphoma, B-Cell; Molecular Sequence Data; Multiple Myeloma; Oncogene Proteins; Proto-Oncogene Mas; RNA, Messenger; RNA, Neoplasm; Translocation, Genetic; Tumor Cells, Cultured