transforming-growth-factor-beta and Adenoma

The pituitary gland is an organ that functionally connects the hypothalamus with the peripheral organs. The pituitary gland is an important regulator of body homeostasis during development, stress, and other processes. Pituitary adenomas are a group of tumors arising from the pituitary gland: they may be subdivided in functional or non-functional, depending on their hormonal activity. Some trophic and neurotrophic factors seem to play a key role in the development and maintenance of the pituitary function and in the regulation of hypothalamo-pituitary-adrenocortical axis activity. Several lines of evidence suggest that trophic and neurotrophic factors may be involved in pituitary function, thus suggesting a possible role of the trophic and neurotrophic factors in the normal development of pituitary gland and in the progression of pituitary adenomas. Additional studies might be necessary to better explain the biological role of these molecules in the development and progression of this type of tumor. In this review, in light of the available literature, data on the following neurotrophic factors are discussed: ciliary neurotrophic factor (CNTF), transforming growth factors β (TGF‑β), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), vascular endothelial growth inhibitor (VEGI), fibroblast growth factors (FGFs) and epidermal growth factor (EGF) which influence the proliferation and growth of pituitary adenomas.

Topics: Adenoma; Animals; Ciliary Neurotrophic Factor; Disease Progression; Epidermal Growth Factor; Fibroblast Growth Factors; Glial Cell Line-Derived Neurotrophic Factor; Humans; Nerve Growth Factor; Nerve Growth Factors; Pituitary Gland; Pituitary Neoplasms; Transforming Growth Factor beta; Tumor Necrosis Factor Ligand Superfamily Member 15; Vascular Endothelial Growth Factor A

It is sometimes difficult but finally possible to distinguish colitis-associated neoplasms from sporadic neoplasms. The frequency of detection of precursor lesions of carcinoma (e.g. dysplasia, intraepithelial neoplasia and adenoma) has increased in recent years, which is most probably due to better endoscopic detection and thus improved histological diagnosis. Carcinogenesis of colitis-associated neoplasms is different from carcinogenesis in sporadic neoplasms because mutations and epigenetic changes are different or may occur at a different point in time. In the present article, these differences will be described and placed in context with carcinogenesis in ulcerative colitis.

Topics: Adenoma; Carcinoma in Situ; Cell Transformation, Neoplastic; Chromosome Aberrations; Colitis, Ulcerative; Colonic Neoplasms; DNA Methylation; DNA Mutational Analysis; Epigenesis, Genetic; Humans; Intestinal Mucosa; Microsatellite Instability; Neoplasm Invasiveness; Neoplasm Staging; Oxidative Stress; Precancerous Conditions; Reactive Oxygen Species; Transforming Growth Factor beta

Colon cancer is the third most common cancer and third most common cause of cancer-related death in the USA according to 2008 American Cancer Society statistics. The carcinogenesis of colon cancer has been associated with both genetics and environmental factors. It has been found that several signal pathways, including K-ras, Src/PI3K/Akt, beta-catenin, TGFbeta and p53 play critical roles in its pathogenesis. The 5 y survival rate of metastatic colon cancer is below 10%. Thus, it is necessary to further understand its biology and search for effective therapy. Azoxymethane (AOM) is a common model for colon cancer. It can specifically induce colon cancer similar to the pathogenesis of human sporadic colon cancer. Thus, it has been extensively used in the study of the molecular biology, prevention and treatment of colon cancer. After administration, AOM is metabolised into methylazoxymethanol by CYP2E1, which causes DNA mutations. Mutation of K-ras activates this pathway and its downstream PI3K/Akt pathway and MAPK pathway. Mutation of beta-catenin also prevents it from being degraded by GSK-3 and accumulation of beta-catenin leads to cell proliferation. TGFbeta, a pro-apoptotic protein, is inhibited. All of these changes form the basis of AOM carcinogenesis. This model has been used in the study of the genetic deficiencies of colon cancer and in the prevention and treatment of the disease. For example, TGF-betaR2 and adiponectin knockout mice are more susceptible to AOM, while high amylose cornstarch, green tea and artemisia have protective effects.

Topics: Adenocarcinoma; Adenoma; Adiponectin; Animals; Anticarcinogenic Agents; Apoptosis; Azoxymethane; Carcinogens; Colonic Neoplasms; Cytochrome P-450 CYP2E1; Diet; DNA Damage; Genes, ras; Humans; MAP Kinase Signaling System; Methylazoxymethanol Acetate; Mice; Mice, Knockout; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta2

Pituitary adenomas are common monoclonal neoplasms accounting for approximately one-fifth of primary intracranial tumors. Prolactin-secreting pituitary adenomas (prolactinomas) are the most common form of pituitary tumors in humans. They are associated with excessive release of the hormone prolactin and increased tumor growth, giving rise to severe endocrine disorders and serious clinical concerns for the patients. Recent studies indicated that the activin/TGF-beta family of growth factors plays a prominent role in regulating pituitary tumor growth and prolactin secretion from anterior pituitary lactotrope cells. Furthermore, these studies highlighted the tumor suppressor menin and the protein Smads as central regulators of these biological processes in the pituitary. Alterations in the activin/TGF-beta downstream signaling pathways are critical steps towards tumor formation and progression. This chapter will review the role and intracellular molecular mechanisms of action by which activin, TGF-beta, Smads and menin act in concert to prevent pituitary tumor cell growth and control hormonal synthesis by the anterior pituitary.

Topics: Activins; Adenoma; Gene Expression Regulation, Neoplastic; Humans; Pituitary Neoplasms; Prolactin; Proto-Oncogene Proteins; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) signaling occurring during human colorectal carcinogenesis involves a shift in TGF-beta function, reducing the cytokine's antiproliferative effect, while increasing actions that promote invasion and metastasis. TGF-beta signaling involves phosphorylation of Smad3 at serine residues 208 and 213 in the linker region and serine residues 423 and 425 in the C-terminal region. Exogenous TGF-beta activates not only TGF-beta type I receptor (TbetaRI) but also c-Jun N-terminal kinase (JNK), changing unphosphorylated Smad3 to its phosphoisoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker phosphorylated Smad3 (pSmad3L). Either pSmad3C or pSmad3L oligomerizes with Smad4, and translocates into nuclei. While the TbetaRI/pSmad3C pathway inhibits growth of normal epithelial cells in vivo, JNK/pSmad3L-mediated signaling promotes tumor cell invasion and extracellular matrix synthesis by activated mesenchymal cells. Furthermore, hepatocyte growth factor signaling interacts with TGF-beta to activate the JNK/pSmad3L pathway, accelerating nuclear transport of cytoplasmic pSmad3L. This reduces accessibility of unphosphorylated Smad3 to membrane-anchored TbetaRI, preventing Smad3C phosphorylation, pSmad3C-mediated transcription, and antiproliferative effects of TGF-beta on epithelial cells. As neoplasia progresses from normal colorectal epithelium through adenoma to invasive adenocarcinoma with distant metastasis, nuclear pSmad3L gradually increases while pSmad3C decreases. The shift from TbetaRI/pSmad3C-mediated to JNK/pSmad3L-mediated signaling is a major mechanism orchestrating a complex transition of TGF-beta signaling during sporadic human colorectal carcinogenesis. This review summarizes the recent understanding of Smad3 phosphoisoform-mediated signaling, particularly 'cross-talk' between Smad3 and JNK pathways that cooperatively promote oncogenic activities. Understanding of these actions should help to develop more effective therapy against human colorectal cancer, involving inhibition of JNK/pSmad3L pathway.

Topics: Adenocarcinoma; Adenoma; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Colorectal Neoplasms; Disease Progression; Humans; MAP Kinase Kinase 4; Neoplasm Invasiveness; Phosphorylation; Protein Isoforms; Serine; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

There is substantial evidence to support the contention that the Smad portion of the TGF-beta signal transduction pathway provides an important tumor-suppressor function. Mutational loss of function of Smad pathway members have been associated with the development of human cancers and appear to be causative in selected rodent carcinogenesis models. TGF-beta also has multiple other actions that appear to be independent of the growth-inhibitory/tumor suppressor effects. The predominant effect of TGF-beta appears to be dependent on the context of the responding cell. Once transformation has occurred, TGF-beta effects may be detrimental and may actually promote tumor cell survival, invasion, and metastasis. Recent work suggests that these effects may involve TGF-beta regulation of COX-2 and other pathways that may contribute to tumor cell aggressiveness. In gaining a better understanding of the mechanisms by which TGF-beta may promote tumor progression, it is likely that new therapeutic strategies may be developed that preserve tumor-suppressor function of TGF-beta while inhibiting the tumor-promoting effects.

Topics: Adenoma; Animals; Colorectal Neoplasms; Humans; Mice; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Topics: Adenoma; Animals; Cell Division; Cell Transformation, Neoplastic; Colorectal Neoplasms; DNA-Binding Proteins; Gene Expression Regulation; Humans; Intestinal Polyps; Mice; Mutation; Signal Transduction; Smad3 Protein; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

HGF (Hepatocyte Growth Factor), TGFbeta1 (Transforming Growth Factor beta1) and IGF-I (Insulin Like Growth Factor I) are cytokines that are involved in the parathyroid tumors formation and growth. We tried to determine, if there are changes and relationships in the production of these cytokines by tumor cells of parathyroid tumors.. We determined concentrations of HGF, TGFbeta1 and IGF-I in serum from peripheral blood of 16 patients with parathyroid adenoma and of 8 patients with parathyroid secondary hyperplasia before and after parathyroidectomy. Results were compared with serum levels in healthy people.. Both preoperative and postoperative HGF serum levels in patients with parathyroid adenoma and secondary hyperplasia are significantly higher than in healthy people. Preoperative and postoperative serum levels of TGFbeta1 in parathyroid adenoma and postoperative TGFb1 serum levels in parathyroid secondary hyperplasia are higher, compared with those in the healthy population and in parathyroid secondary hyperplasia preoperatively. There are no significant differences of IGF-I serum levels among the all investigated groups of patients.. Changes in the growth factors production by parathyroid tumor cells are reflected by their concentrations in peripheral blood. The elevation of HGF serum levels in patients with parathyroid adenoma and hyperplasia can be explained by very high HGF production by tumor cells. Nevertheless, there is no decrease of HGF serum levels after the parathyroidectomy. That may be the result of the extratumoral production of this cytokine. Also TGFbeta1 and IGF-I serum levels indicate high possibility of the extratumoral production of these cytokines. Higher postoperative IGF-I serum levels (but not significantly) in parathyroid secondary hyperplasia are in accordance with its bone production.

Topics: Adenoma; Cytokines; Hepatocyte Growth Factor; Humans; Hyperplasia; Insulin-Like Growth Factor I; Parathyroid Glands; Parathyroid Neoplasms; Reference Values; Transforming Growth Factor beta

Microsatellite instability (MSI) and frameshift mutations in genes containing nucleotide repeats have been reported in a subset of gastric carcinomas, but the mutational profiles in precancerous lesions have not been characterized. To characterize the genetic events during gastric carcinogenesis, we analyzed DNA from 56 gastric adenomas and 167 gastric carcinomas for MSI using five microsatellite markers and for frameshift mutations at coding nucleotide repeats of the type II transforming growth factor beta receptor, BAX, hMSH3, hMSH6, IGF II receptor, and E2F-4 genes. On the basis of the number of markers displaying instability per tumor, the tumors were divided into three groups: those with two or more of the five markers showing instability (high MSI [MSI-H]), those with one of the five markers showing instability (low MSI [MSI-L]), and those with no instability. MSI-H was found in 8 adenomas (14%) and 19 carcinomas (11%), and MSI-L was found in 8 adenomas (14%) and 9 carcinomas (5%). These groups were tested for correlations with several clinicopathologic parameters. MSI-H gastric adenomas were related to the high histologic grade of composing dysplastic glands (p = 0.004), and MSI-H gastric carcinomas were associated with exophytic tumor growth (p = 0.005). We found 48 frameshift mutations at coding nucleotide repeats of the six genes, and all mutations except one were found in MSI-H gastric tumors. Only one of the 17 MSI-L tumors showed frameshift mutations at coding nucleotide repeats of the transforming growth factor beta receptor II gene. Compared with MSI-H gastric carcinomas, MSI-H adenomas had no mutations in the hMSH6 and the IGF II receptor genes, less frequent mutations in the transforming growth factor beta receptor II (38% versus 63%), BAX (13% versus 37%), and hMSH3 (13% versus 37%) genes, and more frequent mutations in the E2F-4 (50% versus 37%) gene. Our findings suggest that MSI and E2F-4 mutations are early genetic events and that mutations of the other five genes are accumulated during the progression of gastric carcinomas with MSI.

Topics: Adenoma; bcl-2-Associated X Protein; Carcinoma; Disease Progression; DNA-Binding Proteins; E2F4 Transcription Factor; Female; Frameshift Mutation; Genetic Code; Humans; Male; Melanocyte-Stimulating Hormones; Microsatellite Repeats; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptor, IGF Type 2; Stomach Neoplasms; Transcription Factors; Transforming Growth Factor beta

RAC1B is a tumour-related alternative splice isoform of the small GTPase RAC1, found overexpressed in a large number of tumour types. Building evidence suggests it promotes tumour progression but compelling in vivo evidence, demonstrating a role in driving tumour invasion, is currently lacking. In the present study, we have overexpressed RAC1B in a colorectal cancer mouse model with potential invasive properties. Interestingly, RAC1B overexpression did not trigger tumour invasion, rather it led to an acceleration of tumour initiation and reduced mouse survival. By modelling early stages of adenoma initiation we observed a reduced apoptotic rate in RAC1B overexpressing tumours, suggesting protection from apoptosis as a mediator of this phenotype. RAC1B overexpressing tumours displayed attenuated TGFβ signalling and functional analysis in ex vivo organoid cultures demonstrated that RAC1B negatively modulates TGFβ signalling and confers resistance to TGFβ-driven cell death. This work defines a novel mechanism by which early adenoma cells can overcome the cytostatic and cytotoxic effects of TGFβ signalling and characterises a new oncogenic function of RAC1B in vivo.

Topics: Adenoma; Adenomatous Polyposis Coli; Animals; Apoptosis; Carcinogenesis; Disease Models, Animal; Down-Regulation; Gene Expression Regulation, Neoplastic; Intestines; Mice; Models, Biological; rac1 GTP-Binding Protein; Signal Transduction; Survival Analysis; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Mutations in the APC gene and other genes in the Wnt signaling pathway contribute to development of colorectal carcinomas. R-spondins (RSPOs) are secreted proteins that amplify Wnt signaling in intestinal stem cells. Alterations in RSPO genes have been identified in human colorectal tumors. We studied the effects of RSPO1 overexpression in Apc. An adeno associated viral vector encoding RSPO1-Fc fusion protein, or control vector, was injected into Apc. Intestines from Apc. Expression of RSPO1 in Apc

Topics: Adenoma; Animals; Disease Models, Animal; Intestinal Neoplasms; Mice; Organoids; Thrombospondins; Transforming Growth Factor beta; Wnt Signaling Pathway

Nonfunctioning pituitary adenoma is a common intracranial tumor. Though benign in the majority of cases, complications can be excruciating to the affected individual, and recurrences after tumor removal may happen with more aggressive clinical features. T regulatory (Treg) cells are generally considered a tumor-promoting immune cell type in malignant cancers with currently unclear roles in pituitary adenoma patients. Therefore, we investigated the frequency and functional characteristics of Treg cells in nonfunctioning pituitary adenoma patients before and after tumor removal. Compared to healthy controls, untreated patients with nonfunctioning pituitary adenomas presented an overrepresentation of highly functional circulating FOXP3

Topics: Adenoma; Adult; Cell Proliferation; Female; Forkhead Transcription Factors; Hepatitis A Virus Cellular Receptor 2; Humans; Immune Tolerance; Interleukin-10; Lymphocyte Activation; Male; Middle Aged; Pituitary Neoplasms; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

In pituitary adenomas, early recurrences and resistance to conventional pharmacotherapies are common, but the mechanisms involved are still not understood. The high expression of epidermal growth factor receptor 2 (HER2)/extracellular signal-regulated kinase (ERK1/2) signal observed in human pituitary adenomas, together with the low levels of the antimitogenic transforming growth factor beta receptor 2 (TBR2), encouraged us to evaluate the effect of the specific HER2 inhibition with trastuzumab on experimental pituitary tumor cell growth and its effect on the antiproliferative response to TGFB1. Trastuzumab decreased the pituitary tumor growth as well as the expression of ERK1/2 and the cell cycle regulators CCND1 and CDK4. The HER2/ERK1/2 pathway is an attractive therapeutic target, but its intricate relations with other signaling modulators still need to be unraveled. Thus, we investigated possible cross-talk with TGFB signaling, which has not yet been studied in pituitary tumors. In tumoral GH3 cells, co-incubation with trastuzumab and TGFB1 significantly decreased cell proliferation, an effect accompanied by a reduction in ERK1/2 phosphorylation, an increase of SMAD2/3 activation. In addition, through immunoprecipitation assays, a diminution of SMAD2/3-ERK1/2 and an increase SMAD2/3-TGFBR1 interactions were observed when cells were co-incubated with trastuzumab and TGFB1. These findings indicate that blocking HER2 by trastuzumab inhibited pituitary tumor growth and modulated HER2/ERK1/2 signaling and consequently the anti-mitogenic TGFB1/TBRs/SMADs cascade. The imbalance between HER2 and TGFBRs expression observed in human adenomas and the response to trastuzumab on experimental tumor growth may make the HER2/ERK1/2 pathway an attractive target for future pituitary adenoma therapy.

Topics: Adenoma; Adult; Cell Cycle; Cell Proliferation; Female; Humans; Male; Middle Aged; Phosphorylation; Pituitary Neoplasms; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Trastuzumab; Young Adult

Colorectal cancer (CRC) results from the accumulation of gene mutations and epigenetic alterations in colon epithelial cells, which promotes CRC formation through deregulating signaling pathways. One of the most commonly deregulated signaling pathways in CRC is the transforming growth factor β (TGF-β) pathway. Importantly, the effects of TGF-β signaling inactivation in CRC are modified by concurrent mutations in the tumor cell, and these concurrent mutations determine the ultimate biological effects of impaired TGF-β signaling in the tumor. However, many of the mutations that cooperate with the deregulated TGF-β signaling pathway in CRC remain unknown. Therefore, we sought to identify candidate driver genes that promote the formation of CRC in the setting of TGF-β signaling inactivation. We performed a forward genetic screen in mice carrying conditionally inactivated alleles of the TGF-β receptor, type II (Tgfbr2) using Sleeping Beauty (SB) transposon mediated mutagenesis. We used TAPDANCE and Gene-centric statistical methods to identify common insertion sites (CIS) and, thus, candidate tumor suppressor genes and oncogenes within the tumor genome. CIS analysis of multiple neoplasms from these mice identified many candidate Tgfbr2 cooperating genes and the Wnt/β-catenin, Hippo and MAPK pathways as the most commonly affected pathways. Importantly, the majority of candidate genes were also found to be mutated in human CRC. The SB transposon system provides an unbiased method to identify Tgfbr2 cooperating genes in mouse CRC that are functionally relevant and that may provide further insight into the pathogenesis of human CRC.

Topics: Adenocarcinoma; Adenoma; Animals; Colorectal Neoplasms; DNA Transposable Elements; Genes, Neoplasm; Genes, Tumor Suppressor; Genetic Association Studies; Humans; Mice; Mice, Knockout; Mice, Transgenic; Mutagenesis, Insertional; Neoplasm Proteins; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Sequence Analysis, DNA; Signal Transduction; Species Specificity; Transforming Growth Factor beta

Mutational processes and signatures that drive early tumorigenesis are centrally important for early cancer prevention. Yet, to date, biomarkers and risk factors for polyps (adenomas) that inordinately and rapidly develop into colon cancer remain poorly defined. Here, we describe surprisingly high mutational profiles through whole-genome sequence (WGS) analysis in 2 of 4 pairs of benign colorectal adenoma tissue samples. Unsupervised hierarchical clustered transcriptomic analysis of a further 7 pairs of adenomas reveals distinct mutational signatures regardless of adenoma size. Transitional single nucleotide substitutions of C:G>T:A predominate in the adenoma mutational spectrum. Strikingly, we observe mutations in the TGF-β pathway and CEA-associated genes in 4 out of 11 adenomas, overlapping with the Wnt pathway. Immunohistochemical labeling reveals a nearly 5-fold increase in CEA levels in 23% of adenoma samples with a concomitant loss of TGF-β signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-β signaling. Our study uncovers diverse mutational processes underlying the transition from early adenoma to cancer. This has broad implications for biomarker-driven targeting of CEA/TGF-β in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression.

Topics: Adenoma; Apoptosis; Biomarkers, Tumor; Blotting, Western; Carcinoembryonic Antigen; Cell Movement; Cell Proliferation; Cells, Cultured; Colon; Colonic Neoplasms; Disease Progression; Gene Expression Regulation, Neoplastic; High-Throughput Nucleotide Sequencing; Humans; Immunoenzyme Techniques; Immunoprecipitation; Mutation; Signal Transduction; Transforming Growth Factor beta

The heterogeneous nature of colorectal cancer (CRC) complicates prognosis and is suggested to be a determining factor in the efficacy of adjuvant therapy for individual patients. Based on gene expression profiling, CRC is currently classified into four consensus molecular subtypes (CMSs), characterized by specific biological programs, thus suggesting the existence of unifying developmental drivers for each CMS Using human organoid cultures, we investigated the role of such developmental drivers at the premalignant stage of distinct CRC subtypes and found that TGFβ plays an important role in the development of the mesenchymal CMS4, which is of special interest due to its association with dismal prognosis. We show that in tubular adenomas (TAs), which progress to classical CRCs, the dominating response to TGFβ is death by apoptosis. By contrast, induction of a mesenchymal phenotype upon TGFβ treatment prevails in a genetically engineered organoid culture carrying a BRAF(V) (600E) mutation, constituting a model system for sessile serrated adenomas (SSAs). Our data indicate that TGFβ signaling is already active in SSA precursor lesions and that TGFβ is a critical cue for directing SSAs to the mesenchymal, poor-prognosis CMS4 of CRC.

Topics: Adenoma; Carcinogenesis; Colorectal Neoplasms; Humans; Organoids; Signal Transduction; Transforming Growth Factor beta

Stepwise acquisition of oncogene mutations and deletion/inactivation of tumor suppressor genes characterize the development of colorectal cancer (CRC). These genetic events interact with discrete morphologic transitions from hyperplastic mucosa to adenomatous areas, followed by in situ malignant transformation and finally invasive carcinoma. The goal of this study was to identify tissue markers of the adenoma-carcinoma morphogenetic transitions in CRC.. We analyzed the patterns of expression of growth regulatory and stem cell markers across these distinct morphologic transition zones in 735 primary CRC tumors. In 202 cases with preserved adenoma-adenocarcinoma transition, we identified, in 37.1% of cases, a zone of adenomatous epithelium, located immediately adjacent to the invasive component, that showed rapidly alternating intraglandular stretches of PTEN+ and PTEN- epithelium. This zone exactly overlapped with similar alternating expression of Ki-67 and inversely with the transforming growth factor-beta (TGF-β) growth regulator SMAD4. These zones also show parallel alternating levels and/or subcellular localization of multiple cancer stem/progenitor cell (CSC) markers, including β-catenin/CTNNB1, ALDH1, and CD44. PTEN was always re-expressed in the invasive tumor in these cases, unlike those with complete loss of PTEN expression. Genomic microarray analysis of CRC with prominent CSC-like expansions demonstrated a high frequency of PTEN genomic deletion/haploinsufficiency in tumors with CSC-like transition zones (62.5%) but not in tumors with downregulated but non-alternating PTEN expression (14.3%). There were no significant differences in the levels of KRAS mutation or CTNNB1 mutation in CSC-like tumors as compared to unselected CRC cases.. In conclusion, we have identified a distinctive CSC-like pre-invasive transition zone in PTEN-haploinsufficient CRC that shows convergent on-off regulation of the PTEN/AKT, TGF-β/SMAD and Wnt/β-catenin pathways. This bottleneck-like zone is usually followed by the emergence of invasive tumors with intact PTEN expression but dysregulated TP53 and uniformly high proliferation rates.

Topics: Adenocarcinoma; Adenoma; beta Catenin; Biomarkers, Tumor; Cell Proliferation; Colorectal Neoplasms; Disease Progression; Haploinsufficiency; Humans; Mutation; Neoplasm Invasiveness; Neoplastic Stem Cells; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Tumor Suppressor Proteins; Wnt Signaling Pathway

In the majority of microsatellite-stable colorectal cancers (CRCs), an initiating mutation occurs in the adenomatous polyposis coli (APC) or β-catenin gene, activating the β-catenin/TCF pathway. The progression of resulting adenomas is associated with oncogenic activation of KRas and inactivation of the p53 and TGF-β/Smad functions. Most established CRC cell lines contain mutations in the TGF-β/Smad pathway, but little is known about the function of TGF-β in the early phases of intestinal tumorigenesis. We used mouse and human ex vivo 3D intestinal organoid cultures and in vivo mouse models to study the effect of TGF-β on the Lgr5(+) intestinal stem cells and their progeny in intestinal adenomas. We found that the TGF-β-induced apoptosis in Apc-mutant organoids, including the Lgr5(+) stem cells, was mediated by up-regulation of the BH3-only proapoptotic protein Bcl-2-like protein 11 (Bim). BH3-mimetic compounds recapitulated the effect of Bim not only in the adenomas but also in human CRC organoids that had lost responsiveness to TGF-β-induced apoptosis. However, wild-type intestinal crypts were markedly less sensitive to TGF-β than Apc-mutant adenomas, whereas the KRas oncogene increased resistance to TGF-β via the activation of the Erk1/2 kinase pathway, leading to Bim down-regulation. Our studies identify Bim as a critical mediator of TGF-β-induced apoptosis in intestinal adenomas and show that the common progression mutations modify Bim levels and sensitivity to TGF-β during intestinal adenoma development.

Topics: Adenoma; Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Blotting, Western; Cells, Cultured; Chromatography, Gel; DNA Primers; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Intestinal Neoplasms; Membrane Proteins; Mice; Microarray Analysis; Organoids; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Transforming Growth Factor beta

Interleukin (IL)-17A is an important pro-inflammatory cytokine and involved in the colorectal carcinogenesis. In this study, the authors evaluated the dynamic change of IL-17A expression in the tumor microenvironment throughout the colorectal adenoma-carcinoma sequence.. Using quantitative real-time PCR (polymerase chain reaction) and semi-quantitative immunohistochemistry, the authors examined the expression level of IL-17A in 50 of human colorectal adenoma tissues, 50 of colorectal cancer (CRC) tissues and 15 controls. The relationship between IL-17A expression and clinicopathological parameters throughout the sequence was also evaluated.. The results revealed a step-up increased IL-17A mRNA level throughout the colorectal adenoma-carcinoma sequence, which began to increase in the adenomas and became even higher in the CRCs; notably, the increase of IL-17A mRNA level in the adenomatous tissues was associated with the severity of dysplasia. Immunohistochemical analysis confirmed the real-time PCR results and revealed gradually increasing IL-17A cells in both the stroma and adenomatous/cancerous epithelium. In addition, the quantitative real-time PCR result has also revealed an increased expression of TH17-stimulating factors throughout the sequence.. IL-17A and TH17 are highly activated throughout the colorectal adenoma-carcinoma sequence.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Analysis of Variance; Carcinoma; Case-Control Studies; Cell Transformation, Neoplastic; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Interleukin-17; Interleukin-1beta; Interleukin-23; Interleukin-6; Male; Middle Aged; Real-Time Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Th17 Cells; Transforming Growth Factor beta

MicroRNAs (miRs) are small, 16-29 nucleotide long, non-coding RNA molecules which regulate the stability or translational efficiency of targeted mRNAs via RNA interference. MiRs participate in the control of cell proliferation, cell differentiation, signal transduction, cell death, and they play a role in carcinogenesis. The aims of our study were to analyse the expression profile of miRs in sporadic clinically non-functioning pituitary adenomas (NFPA) and in normal pituitary tissues, and to identify biological pathways altered in these pituitary tumors. MiR expression profiles of 12 pituitary tissue specimens (8 NFPA and 4 normal pituitary tissues) were determined using miR array based on quantitative real-time PCR with 678 different primers. Five overexpressed miRs and mRNA expression of Smads (Smad1-9), MEG and DLK1 genes were evaluated with individual Taqman assays in 10 NFPA and 10 normal pituitary tissues. Pathway analysis was performed by the DIANA-mirPath tool. Complex bioinformatical analysis by multiple algorithms and association studies between miRs, Smad3 and tumor size was performed. Of the 457 miRs expressed in both NFPA and normal tissues, 162 were significantly under- or overexpressed in NFPA compared to normal pituitary tissues Expression of Smad3, Smad6, Smad9, MEG and DLK1 was significantly lower in NFPA than in normal tissues. Pathway analysis together with in silico target prediction analysis indicated possible downregulation of the TGFβ signaling pathway in NFPA by a specific subset of miRs. Five miRs predicted to target Smad3 (miR-135a, miR-140-5p, miR-582-3p, miR-582-5p and miR-938) were overexpressed. Correlation was observed between the expression of seven overexpressed miRs and tumor size. Downregulation of the TGFβ signaling through Smad3 via miRs may have a possible role in the complex regulation of signaling pathways involved in the tumorigenesis process of NFPA.

Topics: Adenoma; Adult; Aged; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Male; MicroRNAs; Middle Aged; Oligonucleotide Array Sequence Analysis; Pituitary Neoplasms; Signal Transduction; Transforming Growth Factor beta; Validation Studies as Topic

Functional loss of the tumor suppressor Smad4 is involved in pancreatic and colorectal carcinogenesis and has been associated with the acquisition of invasiveness. We have previously demonstrated that the heterotrimeric basement membrane protein laminin-332 is a Smad4 target. Namely, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-332; its loss is thus implicated in the reduced or discontinuous deposition of the heterotrimeric basement membrane molecule as evident in carcinomas. Uncoupled expression of laminin genes, on the other hand, namely overexpression of the laminin-gamma2 chain is an impressive marker at invasive edges of carcinomas where tumor cells are maximally exposed to signals from stromal cell types like macrophages. As Smad4 is characterized as an integrator of multiple extracellular stimuli in a strongly contextual manner, we asked if loss of Smad4 may also be involved in uncoupled expression of laminin genes in response to altered environmental stimuli. Here, we address Smad4 dependent effects of the prominent inflammatory cytokine TNFalpha on tumor cells.. Smad4-reconstituted colon carcinoma cells like adenoma cells respond to TNFalpha with an increased expression of all three chains encoding laminin-332; coincubation with TGFbeta and TNFalpha leads to synergistic induction and to the secretion of large amounts of the heterotrimer. In contrast, in Smad4-deficient cells TNFalpha can induce expression of the gamma2 and beta3 but not the alpha3 chain. Surprisingly, this uncoupled induction of laminin-332 chains in Smad4-negative cells rather than causing intracellular accumulation is followed by the release of gamma2 into the medium, either in a monomeric form or in complexes with as yet unknown proteins. Soluble gamma2 is associated with increased cell migration.. Loss of Smad4 may lead to uncoupled induction of laminin-gamma2 in response to TNFalpha and may therefore represent one of the mechanisms which underlie accumulation of laminin-gamma2 at the invasive margin of a tumor. The finding, that gamma2 is secreted from tumor cells in significant amounts and is associated with increased cell migration may pave the way for further investigation to better understand its functional relevance for tumor progression.

Topics: Adenoma; Amino Acid Sequence; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Drug Synergism; Gene Knockdown Techniques; Humans; Kalinin; Laminin; Mass Spectrometry; Molecular Sequence Data; NF-kappa B; Promoter Regions, Genetic; Protein Conformation; Smad4 Protein; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-beta/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-beta and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-beta and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins.. The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry.. The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions.. The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-beta/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.

Topics: Activins; Adenoma; Carcinoma, Papillary, Follicular; Goiter, Nodular; Humans; Signal Transduction; Smad Proteins, Receptor-Regulated; Smad2 Protein; Smad3 Protein; Smad4 Protein; Smad7 Protein; Thyroid Neoplasms; Transforming Growth Factor beta

The epithelial-mesenchymal transition (EMT) occurs commonly during carcinoma invasion and metastasis, but not during early tumorigenesis. Microarray data demonstrated elevation of vimentin, a mesenchymal marker, in intestinal adenomas from Apc Min/+ (Min) mice. We have tested the involvement of EMT in early tumorigenesis in mammalian intestines by following EMT-associated markers. Elevated vimentin RNA expression and protein production were detected within neoplastic cells in murine intestinal adenomas. Similarly, vimentin protein was detected in both adenomas and invasive adenocarcinomas of the human colon, but not in the normal colonic epithelium or in hyperplastic polyps. Expression of E-cadherin varied inversely with vimentin. In addition, the expression of fibronectin was elevated while that of E-cadherin decreased. Canonical E-cadherin suppressors, such as Snail, were not elevated in the same tumor. Elevated vimentin expression in the adenoma was not correlated with persistent Ras signaling, but was strongly correlated with reduced proliferation indices, active Wnt signaling, and TGF-beta signaling, as demonstrated by its dependence on Smad3. We designate our observations of expression of only some of the canonical features of EMT as "truncated EMT". These unexpected observations are interpreted as reflecting the involvement of a core of the EMT system during the tissue remodeling of early tumorigenesis.

Topics: Adenoma; Adenomatous Polyposis Coli Protein; Animals; Azoxymethane; Cadherins; Cell Proliferation; Epithelium; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Intestinal Neoplasms; Intestinal Polyps; Mesoderm; Mice; Mutation; Neoplasm Invasiveness; Phenotype; ras Proteins; Signal Transduction; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta; Vimentin; Wnt Proteins

Clinical evidence suggests that estradiol replacement therapy reduces colon cancer risk in 'post'menopausal women. In colon epithelial cells, the estrogen receptor beta (ERbeta) is the predominant ER subtype and is thought to mediate the genomic effect of estrogens. The first aim of this study was to investigate the consequence of ERbeta deficiency on intestinal tumorigenesis in the Apc(Min/+) mouse model. Furthermore, to explore the biological mechanisms by which estrogens may influence the pathogenesis of colorectal cancer, we performed gene expression profiles in colonocytes from ovariectomized wild-type (WT) vs. ERbeta(-/-) mice, treated with estradiol (E(2)) or vehicle. Specifically in female, ERbeta deficiency was found to be associated with higher adenoma multiplicity in the small intestine, but not in the colon. Furthermore, tumors from ERbeta(-/-)Apc(Min/+) female mice were on average significantly larger than those from control Apc(Min/+) mice. Higher steady-state proliferation in epithelial cells of the jejunum and colon from ERbeta(-/-)Apc(Min/+) vs. Apc(Min/+) female mice was confirmed by BrdU incorporation assay. Interestingly, functional categorization of microarray results revealed the TGFbeta signaling pathway to be modulated in colonocytes, especially for the WT + E(2) vs. WT + Vehicle and the ERbeta(-/-) + E(2) vs. WT + E(2) comparisons. Using quantitative PCR analysis, we observed transcripts from ligands of the TGFbeta pathway to be upregulated in colonocytes from E(2)-treated WT and ERbeta(-/-) mice and downregulated in ERbeta-deficient mice, mostly in an E(2)-independent manner. Therefore, our results demonstrate that ERbeta deficiency enhances small intestinal tumorigenesis and suggest that modulation of the TGFbeta signaling pathway could contribute to the protective role of estrogens on intestinal tumorigenesis.

Topics: Adenoma; Animals; Bromodeoxyuridine; Colonic Neoplasms; Disease Models, Animal; Estrogen Receptor beta; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Intestinal Neoplasms; Intestine, Small; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Oligonucleotide Array Sequence Analysis; Ovariectomy; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

The tumor suppressor Smad4 is involved in carcinogenesis mainly of the pancreas and colon. Functional inactivation of Smad4 is a genetically late event that occurs upon transition from premalignant stages to invasive and metastatic growth. Smad4 encodes an intracellular messenger common to all signalling cascades induced by members of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. Despite extensive knowledge about the mechanisms of TGF-beta/Smad signal transduction, little is known about Smad4 targets involved in the transition to malignancy. The hallmark of invasive growth is a breakdown of the basement membrane (BM), a specialized sheet of extracellular matrix produced through cooperation of epithelial and stromal cells. Laminin-5, a heterotrimeric epithelial-derived BM component, is commonly lost in carcinomas but not in premalignant tumors. Herein, we report that in human colon and pancreatic tumor cells, Smad4 functions as a positive transcriptional regulator of all three genes encoding laminin-5. Coordinate re-expression of the three laminin-5 chains induced by reconstitution of Smad4 leads to secretion and deposition of the heterotrimeric molecule in BM-like structures. These data define the expression control of an essential BM component as a novel function for the tumor suppressor Smad4.

Topics: Adenocarcinoma; Adenoma; Basement Membrane; Cell Adhesion Molecules; Cell Line, Tumor; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Kalinin; Pancreatic Neoplasms; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) and Wnt ligands function in numerous developmental processes, and alterations of both signaling pathways are associated with common pathologic conditions, including cancer. To obtain insight into the extent of interdependence of the two signaling cascades in regulating biological responses, we used an oligonucleotide microarray approach to identify Wnt and TGF-beta target genes using normal murine mammary gland epithelial cells as a model. Combination treatment of TGF-beta and Wnt revealed a novel transcriptional program that could not have been predicted from single ligand treatments and included a cohort of genes that were cooperatively induced by both pathways. These included both novel and known components or modulators of TGF-beta and Wnt pathways, suggesting that mutual feedback is a feature of the coordinated activities of the ligands. The majority of the cooperative targets display increased expression in tumors derived from either Min (many intestinal neoplasia) or mouse mammary tumor virus (MMTV)-Wnt1 mice, two models of Wnt-induced tumors, with nine of these genes (Ankrd1, Ccnd1, Ctgf, Gpc1, Hs6st2, IL11, Inhba, Mmp14, and Robo1) showing increases in both. Reduction of TGF-beta signaling by expression of a dominant-negative TGF-beta type II receptor in bigenic MMTV-Wnt1/DNIIR mice increased mammary tumor latency and was correlated with a decrease in expression of Gpc1, Inhba, and Robo1, three of the TGF-beta/Wnt cooperative targets. Our results indicate that the TGF-beta and Wnt/beta-catenin pathways are firmly intertwined and generate a unique gene expression pattern that can contribute to tumor progression.

Topics: Adenoma; Animals; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Humans; Intestinal Neoplasms; L Cells; Mammary Neoplasms, Experimental; Mice; Mice, Inbred C57BL; Mice, Transgenic; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Wnt Proteins; Wnt3 Protein

Smads are signal transducers for the members of the TGF-beta superfamily. Of these Smads, Smad4 is essential for TGF-beta signaling. The purpose of this study was to elucidate Smad4 expression and localization in 65 gastric adenomas, 49 intestinal-type and 39 diffuse type of gastric adenocarcinomas (including 12 cases of fresh frozen tissue) using Real-time RT-PCR and immunohistochemistry. Real-time RT-PCR showed that intestinal type gastric adenocarcinomas have higher Smad4 mRNA expression than diffuse type gastric adenocarcinomas. Immunohistochemical stain for Smad4 revealed that expression of Smad4 was significantly lower in diffuse-type gastric adenocarcinoma than intestinal-type gastric adenocarcinomas. Also, higher Smad4 protein expression in intestinal type gastric adenocarcinomas than overall gastric adenoma was noted. The rate of reduced Smad4 expression was higher in advanced gastric cancer than early gastric cancer. These results suggest that Smad4 might play different roles in human gastric carcinogenesis, especially between intestinal type and diffuse type of gastric adenocarcinoma.

Topics: Adenocarcinoma; Adenoma; Adult; Aged; Base Sequence; DNA-Binding Proteins; Female; Gastric Mucosa; Gene Expression; Humans; Immunohistochemistry; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Smad4 Protein; Stomach Neoplasms; Trans-Activators; Transforming Growth Factor beta

The latent transcription factor Stat3 is activated by gp130, the common receptor for the interleukin (IL)-6 cytokine family and other growth factor and cytokine receptors. Ligand-induced dimerization of gp130 leads to activation of the Stat1, Stat3 and Shp2-Ras-Erk signaling pathways. Here we assess genetically the contribution of exaggerated Stat3 activation to the phenotype of gp130 (Y757F/Y757F) mice, in which a knock-in mutation disrupts the negative feedback mechanism on gp130-dependent Stat signaling. Compared to gp130 (Y757F/Y757F) mice, reduced Stat3 activation in gp130 (Y757F/Y757F) Stat3(+/-) mice increased their lifespan, prevented splenomegaly, normalized exaggerated hepatic acute-phase response and lymphocyte trafficking, and suppressed the growth of spontaneously arising gastric adenomas in young mice. These lesions share histological features of gastric polyps in aging mice with monoallelic null mutations in Smad4, which encodes the common transducer for transforming growth factor (TGF)-beta signaling. Indeed, hyperactivation of Stat3 desensitizes gp130 (Y757F/Y757F) cells to the cytostatic effect of TGF-beta through transcriptional induction of inhibitory Smad7, thereby providing a novel link for cross-talk between Stat and Smad signaling in gastric homeostasis.

Topics: Adenoma; Animals; Cytokine Receptor gp130; Histological Techniques; Immunoblotting; Mice; Mice, Mutant Strains; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad7 Protein; STAT3 Transcription Factor; Stomach Neoplasms; Transcriptional Activation; Transforming Growth Factor beta

IGF-I, HGF, TGFbeta1, bFGF and VEGF are involved in the pathogenesis of thyroid gland tumors and their growth. We decided to find whether changes in the production of these cytokines by thyroid tumor cells are reflected by changes of their peripheral blood. Using ELISA kits, we measured the concentrations of growth factors in the peripheral blood serum in 28 patients with thyroid gland tumors (14 adenomas, 14 papillary carcinomas) and compared these concentrations with those in healthy people. We found significantly lower serum levels of IGF-I in patients with thyroid adenoma compared to the healthy population. Serum levels of HGF and bFGF were significantly higher in patients with thyroid adenoma and papillary carcinoma compared with those in healthy subjects. Serum concentrations of TGFbeta1 and VEGF were not significantly different in any groups of investigated subjects. Changes in the production of these cytokines by thyroid gland tumor cells are reflected in their peripheral blood levels, but these levels also depend on a number of other physiological and pathological processes in the organism. However, significant differences of HGF and bFGF serum levels can be explained by their very high production by thyroid tumor cells and by their strong effect on the follicular and endothelial cell proliferation.

Topics: Adenoma; Biomarkers, Tumor; Carcinoma, Papillary; Female; Fibroblast Growth Factor 2; Growth Substances; Hepatocyte Growth Factor; Humans; Insulin-Like Growth Factor I; Male; Neovascularization, Pathologic; Thyroid Neoplasms; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Topics: Activin Receptors, Type I; Adenoma; Adolescent; Adult; Aged; Bone Morphogenetic Protein Receptors, Type I; Colorectal Neoplasms; DNA Glycosylases; DNA Mutational Analysis; DNA-Binding Proteins; DNA, Neoplasm; Female; Germ-Line Mutation; Humans; Loss of Heterozygosity; Male; Middle Aged; N-Glycosyl Hydrolases; Neoplasms, Multiple Primary; Polymorphism, Single-Stranded Conformational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Receptors, Growth Factor; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Wnt Proteins; Zebrafish Proteins

Galectin-3 (Gal-3), a beta-galactoside-binding protein, has been implicated in a variety of biological functions, including cell proliferation and differentiation, tumor cell adhesion, angiogenesis, apoptosis, tumor progression, and metastasis. We investigated the role of Gal-3 in the development and progression of pituitary tumors. Immunohistochemical and Western blot analysis of normal and neoplastic human pituitaries showed that only lactotroph (PRL) and corticotroph (ACTH) hormone-producing cells and tumors expressed Gal-3. Gal-3 was present in 24 of 38 (63.2%) PRL adenomas, 5 of 6 (83.3%) PRL carcinomas, 19 of 41 (46.3) ACTH adenomas, and 7 of 8 (87.5%) ACTH carcinomas, but not in 112 other pituitary adenomas and carcinomas. Pituitary folliculo-stellate cells, which have macrophage-type functions in the anterior pituitary, also expressed Gal-3. Hyperplastic and neoplastic pituitaries from p27(Kip1) (p27)-null mice, which produce mainly ACTH, showed increased Gal-3 expression levels compared with control mice. Treatment with transforming growth factor beta1, which regulates pituitary cell proliferation, reduced Gal-3 as well as p27 expression levels in cultured HP75 pituitary cells and Gal-3 in cultured pituitary cells from p27-null mice, suggesting that p27 is not necessary for the inhibitory effects of transforming growth factor beta1 on the cell cycle in the pituitary. The role of Gal-3 in pituitary cell function was examined by RNA interference experiments. Inhibition of Gal-3 gene expression by RNA interference decreased HP75 cell proliferation and increased apoptosis. These results indicate that Gal-3 has an important role in pituitary cell proliferation and tumor progression.

Topics: Adenoma; Animals; Blotting, Western; Carcinoma; Cell Cycle Proteins; Cell Division; Cyclin-Dependent Kinase Inhibitor p27; Disease Progression; DNA, Antisense; Galectin 3; Humans; Immunohistochemistry; In Situ Hybridization; Mice; Pituitary Neoplasms; RNA Interference; RNA, Small Interfering; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tumor Suppressor Proteins

Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the tumor growth factor beta (TGF-beta) superfamily. Several observations suggest that it plays a role in colorectal carcinoma (CRC). In particular, MIC-1 is markedly up-regulated in colorectal cancers as well as in premalignant adenomas. This study examines the relationship of serum MIC-1 levels and genotypes to clinical and pathologic features of colonic neoplasia.. We confirmed the presence of MIC-1 in CRC tissue and the cell line CaCo-2. The normal range for serum MIC-1 levels was defined in 260 healthy blood donors, and the differences between normal subjects and 193 patients having adenomatous polyps or CRC were then determined. In a separate cohort of 224 patients, we evaluated the relationship of MIC-1 serum level and genotype to standard tumor parameters and outcome measures.. MIC-1 was expressed in CRC tissue and the cancer cell line CaCo-2. There was a progressive increase in serum MIC-1 levels between normal individuals [mean (M) = 495 pg/ml, SD = 210), those with adenomatous polyps (M = 681 pg/ml, SD = 410), and those with CRC (M = 783 pg/ml, SD = 491)]. Serum MIC-1 level was correlated with the extent of disease so that the levels were higher in patients with higher Tumor-Node-Metastasis stage. There were significant differences in time to relapse and overall survival between subjects with different MIC-1 levels and genotypes.. This study identifies a strong association between MIC-1 serum levels and neoplastic progression within the large bowel. We suggest that the measurement of serum MIC-1 levels and determination of MIC-1 genotype may have clinical use in the management of patients with CRC.

Topics: Adenoma; Adenomatous Polyps; Alleles; Carcinoma; Cell Line, Tumor; Cohort Studies; Colorectal Neoplasms; Cytokines; Disease-Free Survival; Female; Genotype; Growth Differentiation Factor 15; Humans; Immunohistochemistry; Logistic Models; Lymphocytes; Male; Neoplasm Metastasis; Prognosis; Time Factors; Transforming Growth Factor beta; Treatment Outcome; Up-Regulation

In the liver, transforming growth factor beta-1 (TGF-beta1) constitutes a major negative growth regulating factor involved in the control of cell numbers; failure of this control mechanism has been associated with the development of liver cancer. Since no reports on the in vivo effects of exogenously administered TGF-beta1 on apoptosis in liver tumors have been published yet, we studied hepatocyte sensitivity to the proapoptotic action of TGF-beta1 in stages of chemically induced mouse liver carcinogenesis.. Mouse liver carcinogenesis was initiated by a single dose of N-nitrosodiethylamine (NDEA, 90 mg/kg b.w., i.p.) to 5-week-old B6C3F1 mice. After 2 weeks, mice received either standard diet or a diet containing phenobarbital (PB, 90 mg/kg b.w) for 85 weeks. Four hours before being killed mice received a single dose of TGF-beta1 (56 microg or 200 microg TGF-beta1/kg of b.w., injected into the tail vein). Quantitative histological analysis of mitosis and apoptosis in normal liver tissue (NL), putative preneoplastic foci (PPF), hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC) was performed on H&E-stained liver sections.. In NDEA and NDEA + PB-treated mice, NL exhibited a very low incidence of apoptosis and mitosis, which increased in HCA and HCC. In the lesions apoptoses ranged between 0.03 and 0.6%. Two hundred micrograms of TGF-beta1/kg stimulated apoptoses in NL as well as in neoplastic lesions (significant increase in NL, HCA, and HCC); the most pronounced proapoptotic action of TGF-ss1 was observed in lesions of NDEA+PB pretreated mice (about 1.7%). Fifty-six microg TGF-beta1/kg had no detectable effect on apoptosis.. These observations indicate that during chemically induced liver carcinogenesis in B6C3F1 mice basal rates of apoptoses in adenoma and carcinoma are higher than in normal liver and can be further increased by a proapoptotic cytokine.

Topics: Adenoma; Animals; Antineoplastic Agents; Apoptosis; Carcinoma; Liver Neoplasms, Experimental; Mice; Transforming Growth Factor beta; Transforming Growth Factor beta1

Based upon molecular allelotyping and comparative genomic hybridization studies, chromosome 15q is the likely location of a tumor suppressor gene important in the pathogeneses of sporadic enteropancreatic endocrine tumors and parathyroid adenomas. Interest has focused on Smad3 as a candidate endocrine tumor suppressor gene because 1) it is localized to 15q and 2) it encodes a TGF beta signaling molecule that has been identified as a binding partner of the multiple endocrine neoplasm type 1 gene product menin, itself involved in enteropancreatic and parathyroid neoplasia. To determine whether Smad3 plays a primary role in development of these tumors, 20 enteropancreatic tumors and 67 parathyroid adenomas were investigated for loss of heterozygosity at DNA markers surrounding Smad3. Twenty percent of enteropancreatic tumors and 24% of parathyroid adenomas showed loss. All 9 coding exons and intron-exon boundaries of the Smad3 gene were then sequenced in genomic DNA from all 20 enteropancreatic and 25 parathyroid tumors, including every case with loss of heterozygosity. No acquired clonal mutations, insertions, or microdeletions in Smad3 were detected in any tumors. Because inactivating somatic mutation is the hallmark of an authentic tumor suppressor, Smad3 is unlikely to function as a classical tumor suppressor gene in the pathogenesis of sporadic parathyroid or enteropancreatic endocrine tumors.

Topics: Adenoma; DNA Mutational Analysis; DNA-Binding Proteins; Genes, Tumor Suppressor; Humans; Islets of Langerhans; Neoplasm Proteins; Pancreatic Neoplasms; Parathyroid Neoplasms; Polymorphism, Genetic; Proto-Oncogene Proteins; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

In the normal liver, the transforming growth factor beta (TGF-beta) signaling pathway plays an important role in inhibiting hepatocyte growth. This effect is mediated through Smad4 (or Dpc4), a tumor-suppressor gene that affects gene transcription and controls cell growth. A loss of Smad4 is associated with carcinoma in a number of other organs, including the pancreas and colon. Despite these facts, several recent studies using cDNA microarrays have surprisingly shown overexpression of Smad4 in hepatocellular carcinoma (HCC). Because Smad4 plays a central role in the TGF-beta signaling pathway, we hypothesized that activation of the TGF-beta signaling pathway may explain Smad4 overexpression. To investigate this, 21 surgically resected HCCs were immunostained with antibodies to Smad4 and TGF-beta receptor II. Tumor and normal liver tissues were stained in all cases, and expression in the tumor was scored in comparison to the nonneoplastic liver. Thirteen hepatic adenomas were also immunostained as a control group. The average age at resection was 58 +/- 16 years for the 17 men and 4 women with HCC. TGF-beta receptor II was weakly expressed in the hepatocyte cytoplasm of all normal livers and was overexpressed in 10 of 21 HCCs. Of these 10 HCCs increased Smad4 immunolabeling was also present in 10 of 10 cases. In contrast, of the 11 of HCCs that did not show TGF-beta overexpression, only 1 showed increased Smad4 immunolabeling. Increased TGF-beta receptor II and Smad4 labeling was associated with a worse nuclear grade and increased mitotic activity. For the hepatic adenomas, the 13 women had an average age at resection of 36 +/- 10 years. Whereas 2 adenomas showed over expression of TGF-beta receptor II, there was no Smad4 overexpression in any case. In conclusion, increased Smad4 protein expression in HCC is tightly linked to overexpression of TGF-beta II receptors and is associated with increased mitoses and a worse nuclear grade. Hepatic adenomas only rarely show overexpression of TGF-beta II receptors and did not show increased Smad4 labeling. The results from this study indicate that Smad4 protein overexpression is present in a subset of HCCs and is strongly correlated with immunostaining for TGF-beta II receptor, findings that may represent activation or dysregulation of the TGF-beta signaling pathway.

Topics: Adenoma; Biomarkers, Tumor; Carcinoma, Hepatocellular; DNA-Binding Proteins; Female; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Liver Neoplasms; Male; Middle Aged; Mitotic Index; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Retrospective Studies; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) inhibits growth and induces apoptosis of colon epithelial cells. Binding of TGF-beta to its receptor induces phosphorylation of the Smad proteins Smad2 and Smad3, which then form heteromeric complexes with Smad4, translocate to the nucleus, and activate gene transcription. Smad4 function has been considered an obligate requirement for TGF-beta signaling, and Smad4 mutations present in some cancers have been considered sufficient to inactivate TGF-beta signaling. In this work, we describe studies with a nontransformed human colon epithelial cell line that is mutant for Smad4 but remains growth-inhibited by TGF-beta. The colon cell line VACO-235 has lost one of its Smad4 alleles via a chromosome 18q deletion. The remaining allele bears two missense point mutations located in regions important for Smad4 trimer formation, which is thought necessary for Smad4 function. As expected, pSBE4-BV/Luc, a Smad4-activated transcriptional reporter, was inactive in VACO-235. Nonetheless, VACO-235 demonstrated 80% growth inhibition in response to TGF-beta, as well as retention of some TGF-beta-mediated activation of the p3TP-Lux transcriptional reporter. Transient transfection of the VACO-235 Smad4 mutant allele into a Smad4-null cell line confirmed that this allele is functionally inactive as assayed by both the pSBE4-BV and p3TP-Lux reporters. The simplest explanation of these results is that there is a non-Smad4-dependent pathway for TGF-beta-mediated signaling and growth inhibition in VACO-235 cells.

Topics: Adenoma; Cell Division; Colonic Neoplasms; DNA-Binding Proteins; Genes, Reporter; Growth Inhibitors; Humans; Luciferases; Mutation; Smad4 Protein; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor (TGF) beta1 is a growth factor with wide-ranging effects on proliferation, differentiation, immunosuppression, apoptosis and matrix remodelling. TGFbeta1 seems to have an antitumorigenic role in the gastrointestinal tract but may also be associated with the development of colorectal cancer. Initially, TGFbeta1 is produced in a latent (precursor) form in epithelial cells and then is activated by a not clearly understood multistep process. In this study, we analysed precursor TGFbeta1 protein expression (n=40) and TGFbeta1 gene expression (n=49) in human colorectal adenocarcinomas and 49 normal adjacent tissue. Out of these 49 normal tissues 40 were matched. Western blot analysis revealed that the precursor TGFbeta1 protein levels were generally lower in colorectal cancerous tissue compared to adjacent non-cancerous tissue (P<0.001). Furthermore, with real-time PCR our results cannot reflect a statistically significant difference in TGFbeta1 gene expression between the tumour tissue and normal tissue. These finds indicate that it is likely that there are mechanisms which control precursor TGFbeta1 protein expression by factor(s) at the level of pre-translation of the TGFbeta1 transcript and/or at the level of post-translation of the TGFbeta1 protein in the tumours. This process may be related to carcinogenesis and poses the question whether the suppression of the precursor TGFbeta1 is an early event, in vivo, in the human colorectal adenoma-carcinoma sequence.

Topics: Adenoma; Aged; Aged, 80 and over; Blotting, Western; Carcinoma; Colon; Colorectal Neoplasms; DNA, Complementary; Female; Humans; Immunohistochemistry; Male; Middle Aged; Polymerase Chain Reaction; Protein Biosynthesis; Protein Precursors; Transforming Growth Factor beta; Transforming Growth Factor beta1

We tried to elucidate the effects of sulindac on human colorectal carcinoma.. Sulindac (300 mg/day) was administered for two weeks before operation to 33 patients with sporadic colorectal carcinoma (Sulindac Group). Resected specimens were used to detect apoptosis by terminal dUTP nick end labeling and transforming growth factor (TGF)-beta1 expression by immunohistochemistry. The results were compared with those from the historical Control Group. Twenty-nine available preoperative biopsies taken from carcinomas before sulindac prescription and 22 concurrent colorectal adenomas (9 and 13 in Sulindac and Control Groups, respectively) in the resected specimen were also examined regarding TGF-beta1 expression.. In the resected carcinomas and adenomas, more frequent apoptosis and higher TGF-beta1 scores were observed in the Sulindac Group than in the Control Group. Overexpression of TGF-beta1 and apoptosis occurred in the same region in adenomas but not in carcinomas. A positive correlation between TGF-beta1 scores and apoptotic frequency was found in adenomas (P = 0.01, rho = 0.91) but not in carcinomas (P = 0.89, rho = 0.03).. We conclude that sulindac induces apoptosis in human colorectal carcinomas as well as in adenomas. Also, one of the antineoplastic effects of sulindac might be mediated by upregulating TGF-beta1 expression, particularly in colorectal adenomas.

Topics: Adenoma; Aged; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Carcinoma; Colorectal Neoplasms; Female; Humans; Male; Middle Aged; Preoperative Care; Sulindac; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Alterations in the transforming growth factor-beta (TGF-beta) pathway are implicated in the pathogenesis of colorectal cancer. We hypothesize that alterations in the TGF-beta pathway contribute to differential sensitivity of mice to the colon carcinogen azoxymethane (AOM). A/J (sensitive) and AKR/J (resistant) mice were injected intraperitoneally with AOM (10 mg/kg of body weight once a week for 6 wk). Twenty-four weeks after AOM exposure, mutational analysis of TGF-beta type II receptor (TbetaR-II) from normal colons and from tumors showed no AOM-induced alterations. A significant decrease (1.5-fold, P < 0.05) in TbetaR-II mRNA levels, however, was found in A/J tumors with the RNase protection assay. Immunofluorescence of TbetaR-II showed marked loss of staining in A/J tumors. The RNase protection assay and sequence analysis of the downstream signaling molecule Smad3 revealed no carcinogen-induced alterations in either strain. To gain further insight into the functionality of the pathway, expression of TGF-beta, TGF-beta type I receptor (TbetaR-I), and several downstream targets of TGF-beta signaling, including Smad7, c-myc, and p15, was examined. Although no alterations in TGF-beta, TbetaR-I, or Smad7 were found in tumors, a significant increase in c-myc expression (2.5-fold, P < 0.05 ) and a significant decrease in p15 expression (4.5-fold, P < 0.05 ) were noted. Concomitant repression of TbetaR-II and overexpression of c-myc may render epithelial cells insensitive to TGF-beta-mediated growth arrest, a possibility that also is suggested by this model. The significant decrease in p15 expression in tumors provides additional evidence that TGF-beta signaling may be markedly attenuated during colon tumorigenesis.

Topics: Adenoma; Animals; Azoxymethane; Carcinogens; Cell Cycle Proteins; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p15; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation, Neoplastic; Male; Mice; Mice, Inbred A; Mice, Inbred AKR; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Smad3 Protein; Smad7 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Tumor Suppressor Proteins

Mice heterozygous for deletion of the transforming growth factor beta1 (TGF-beta1) gene show an enhanced rate of lung tumorigenesis following carcinogen treatment. Since the growth inhibitory activity of TGF-beta1 in epithelial cells is associated with K-ras p21, and K-ras mutations commonly occur in chemically-induced mouse lung tumors, we postulated that tumors in heterozygous TGF-beta1 mice might be more likely to have K-ras mutations compared with tumors in wildtype TGF-beta1 mice. Urethane-induced lung tumors in AJBL6 TGF-beta1 +/- and +/+ mice were examined for K-ras mutations by polymerase chain reaction/single strand conformation polymorphism analysis and sequencing. Mutation frequencies were similar in both genotypes: 12/18 +/- tumors (67%) and 10/16 +/+ tumors (62%). Mutations occurred in 80% +/- and 75% +/+ carcinomas, but in only 50% of the adenomas of both TGF-beta1 genotypes. Codon 61 A-->G transition mutations were predominant, occurring in 61% +/- and 44% +/+ tumors. Three +/- (17%) and three +/+ (19%) tumors showed codon 12 mutations, mostly G-->A transitions. Two +/- tumors had both codon 61 and codon 12 mutations. Interestingly, carcinomas with mutations in codon 61 were larger than those with codon 12 changes. It appears that the mechanism of enhanced susceptibility of TGF-beta1+/- mice to urethane-induced lung carcinogenesis does not involve selective development of tumors with K-ras mutations.

Topics: Adenoma; Animals; Carcinogenicity Tests; Carcinogens; Carcinoma; Crosses, Genetic; DNA Mutational Analysis; DNA, Neoplasm; Female; Genes, ras; Genetic Predisposition to Disease; Genotype; Heterozygote; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mutagenicity Tests; Mutation; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Transforming Growth Factor beta; Transforming Growth Factor beta1; Urethane

Transforming growth factor (TGF) beta1 has an antitumorigenic role in the gastrointestinal tract and may be associated with the development of colon neoplasia. In the present study we investigated whether TGF-beta1 production in mucosa is lower in the distal colon, which is where clinical evidence shows that the incidence of colon neoplasia is higher, and whether TGF-beta1 levels were lower in the mucosa of patients with colon adenoma. Production of colon mucosa TGF-beta1 was investigated by means of a 24-hour organ culture with biopsy specimens taken from different segments of the colon of 58 normal subjects by using an enzyme immunoassay. TGF-beta1 production in colon mucosa from locations near the site of sporadic adenoma was also investigated in 46 patients. TGF-beta1 production gradually increased from the rectum to the ascending colon in a statistically significant manner in both normal (r = 0.77, P < .0001) and adenoma-bearing (r = 0.8, P < .0001) mucosa. When TGF-beta1 production was compared between normal and adenoma-bearing mucosa, levels were lower in the latter, although statistically significant results were seen only in the transverse colon (P < .05). TGF-beta1 production has clear site dependency, being lowest in the rectum and highest in the ascending colon. Furthermore, low levels of TGF-beta1 are associated with the development of adenoma. Our results suggest the possibility that this site dependency is associated with the higher epidemiologic incidence of colon neoplasia in the distal colon.

Topics: Actins; Adenoma; Adult; Aged; Biopsy; Colon; Colonic Neoplasms; Female; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Male; Middle Aged; Organ Culture Techniques; Rectum; RNA, Messenger; Tissue Distribution; Transforming Growth Factor beta

The transforming growth factor-betas (TGF-beta s) are multifunctional proteins that inhibit the proliferation of many epithelial cells through a set of cell protein receptors that includes the TGF-beta type I (RI) and type II (RII) receptors. Loss of growth inhibition by TGF-beta is thought to contribute to the development of many types of tumors. In the present study, we have examined expression of the proteins and mRNAs for TGF-beta 1, TGF-beta RI, and TGF-beta RII in normal human lung, well-characterized non-small cell lung cancer (NSCLC) cell lines, and primary NSCLC specimens. Immunohistochemical staining for TGF-beta 1, TGF-beta RI, and TGF-beta RII using specific antibodies in normal human lung showed expression of the 3 proteins in the epithelium of bronchi and bronchioles as well as in alveoli. Differential expression of TGF-beta RI and TGF-beta RII proteins was detected in 5 NSCLC cell lines using Western blot analysis, with reduced levels in 3 cell lines. A panel of 45 formalin-fixed and paraffin-embedded NSCLC specimens showed positive immunostaining for TGF-beta 1, TGF-beta RI, and TGF-beta RII, with reduced TGF-beta RII in poorly differentiated adenocarcinomas and squamous cell carcinomas and some moderately differentiated adenocarcinomas. In situ hybridization studies conducted with specific riboprobes for TGF-beta 1, TGF-beta RI, and TGF-beta RII showed corresponding localization of expression of the mRNAs in the specimens that showed positive immunostaining for the proteins. To investigate the roles of TGF-beta 1, TGF-beta RI, and TGF-beta RII in chemically induced mouse lung tumorigenesis, we examined the expression of their proteins and mRNAs in 2 mouse model systems. Whereas expression of the proteins and mRNAs for TGF-beta 1 and TGF-beta RI was comparable in lung adenomas and bronchioles of A/J mice treated with benzo(alpha)pyrene, decreased immunostaining and hybridization for TGF-beta RII protein and mRNA was detected in 50% of lung adenomas in these mice. Interestingly, expression of TGF-beta 1 and the TGF-beta receptor proteins was similar to that of bronchioles in C57B1/6 mice and their littermates heterozygous for deletion of the TGF-beta 1 gene treated with diethylnitrosamine. These data show that reduced levels of expression of TGF-beta RII occur in some, but not all, human and mouse lung tumors. This suggests that different mechanisms of action, some of which may involve the TGF-beta signaling pathway, may contribute to the p

Topics: Adenoma; Animals; Blotting, Western; Bronchi; Carcinogens; Carcinoma, Non-Small-Cell Lung; Disease Models, Animal; DNA Primers; Female; Humans; Immunohistochemistry; In Situ Hybridization; Lung Neoplasms; Mice; Mice, Inbred A; Mice, Inbred C57BL; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

TGFbeta1, a multifunctional growth modulator, inhibits the proliferation of epithelial cells. TGFbeta1 signaling is dependent on the heterodimerization of the TGFbeta1 receptor II (TGFbeta1RII) with the TGFbeta1 receptor I (TGFbeta1RI). The cytoplasmic proteins Smads are the mediators of the TGFbeta1 signal. TGFbeta1 regulates adult and fetal adrenal growth and function. Previously we have shown by Northern analysis that TGFbeta1mRNA was well expressed in normal adrenal and in adrenocortical adenomas but reduced in carcinomas. To investigate whether TGFbeta1 receptors may act as tumor suppressors of adrenal tumorigenesis, 16 adenomas and 12 carcinomas were studied. We have used SSCP analysis to scan for inactivating mutations in carcinomas. All tumor samples were negative for somatic alterations of both genes. A competitive RT-PCR system was developed to compare the levels of expression of TGFbeta1, TGFbeta1R-I and TGFbeta1R-II, Smad-2 and Smad-4 genes in all tumors. In our study, we confirmed the presence of reduced levels of TGFbeta1 in carcinomas. On the contrary, Smad-4 gene levels were elevated in carcinomas when compared to that of adenomas. No significant differences were observed in gene expression of TGFbeta1RI and Smad-2. Our results suggest that mutations of TGFbeta1 receptors appear not to be involved in adrenal tumorigenesis. Adrenal carcinomas showed a significant reduction of the TGFbeta1 mRNA levels but on the contrary Smad 4 mRNA levels were significantly increased.

Topics: Adenoma; Adrenal Cortex Neoplasms; Carcinoma; DNA-Binding Proteins; Humans; Mutation; Receptors, Transforming Growth Factor beta; RNA, Messenger; Signal Transduction; Smad4 Protein; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1

Many colorectal cancer cells are resistant to the anti-proliferative effects of transforming growth factor-beta (TGF-beta). TGF-beta also acts as paracrine factor from cancer cells on their mesenchymal cells. The aim of this study was to examine the expression of TGF-beta and its receptors in human colorectal cancer tissue and determine any relationship with cancer growth. In situ hybridization and Northern blot hybridization detection of TGF-beta1, type I and type II receptor mRNA and immunohistochemical staining of TGF-beta1 were performed using 11 human colorectal adenomas, 22 colorectal cancers and ten normal colorectal mucosas as control. TGF-beta receptor mRNAs were expressed mainly by normal colorectal epithelial cells and adenoma. However, mRNAs for TGF-beta receptors were only faintly, if at all, expressed in eight of 22 human colorectal cancers. In addition, intense signals of TGF-beta1 mRNA and the protein were detected in all colorectal cancers. TGF-beta receptor mRNAs and TGF-beta1 protein were also distributed in fibroblasts and endothelial cells in the interstitium. Moreover, Smad 4 protein was translocated to nucleus in primarily cultured adenoma cells, but not in cancer cells after TGF-beta stimulation. The escape of human colon cancer from TGF-beta-mediated growth inhibition by down-regulation of TGF-beta receptors as well as the effects of TGF-beta on stroma formation and angiogenesis indicate a possible role for TGF-beta in the progression of colon cancer in an intact host.

Topics: Adenoma; Adult; Aged; Blotting, Northern; Colorectal Neoplasms; Down-Regulation; Female; Humans; Immunohistochemistry; In Situ Hybridization; Male; Middle Aged; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta

The transforming growth factor beta (TGF-beta) pathway is known to play an important role in both human and urine colon cancer. However, the staging, ligand specificity, and mechanism underlying the tumor suppressive activity of this pathway are unknown. We developed a mouse model for colon cancer that identifies an early role for TGF-beta1 in tumor suppression and implicates TGF-beta2 or TGF-beta3 in the prevention of metastasis. Analysis of the development of colon cancer in TGF-beta1 knockout mice pinpoints the defect to the hyperplasty/adenoma transition and reveals that the mechanism involves an inability to maintain epithelial tissue organization and not a loss of growth control, increased inflammatory activity, or increased genetic instability. These mice provide a unique opportunity to investigate the specific role of TGF-beta1 at this critical transition in the development of colon cancer.

Topics: Adenocarcinoma; Adenoma; Adenomatous Polyposis Coli Protein; Animals; Apoptosis; beta Catenin; Biomarkers; Cecum; Cell Division; Cell Transformation, Neoplastic; Colon; Colonic Neoplasms; Crosses, Genetic; Cytoskeletal Proteins; Disease Progression; DNA; DNA-Binding Proteins; DNA, Neoplasm; Genes, APC; Genetic Predisposition to Disease; Humans; Hyperplasia; Inflammation; Intestinal Mucosa; Mice; Mice, Knockout; Microsatellite Repeats; Neoplasm Metastasis; Nuclear Proteins; Specific Pathogen-Free Organisms; Trans-Activators; Transforming Growth Factor beta

Transforming growth-factor beta1 (TGF-beta1) influences a number of specific functions of adrenocortical cells in several animal species. The aim of our study was to evaluate by immunohistochemical analysis the presence and distribution of TGF-beta1 in normal adrenal tissue and in different adrenal tumours.. We analysed 8 functioning (5 adenomas and 3 carcinomas) and 15 non functioning (6 adenomas and 9 carcinomas) adrenal tumours and 6 normal adrenal glands.. In normal adrenal glands, the glomerulosa and the reticularis zones displayed diffuse cytoplasmic staining, while the fasciculata zone was almost completely negative. Functioning adenomas displayed cytoplasmic staining restricted to compact cells while in nonfunctioning adenomas, prevalently composed by clear cells, no staining was observed. Overall, adrenal carcinomas were characterized by the lack of cytoplasmic positivity and by sporadic positive cells around vessels both in functioning and in nonfunctioning tumours.. TGF-beta1 expression is associated with active steroid secretion in normal adrenal tissue, as well as in benign cortical adenomas, while this relationship is lost in primary adrenal malignancies. These data provide indirect evidence for a regulatory role played by TGF-beta1 on steroid secretory pathways.

Topics: Adenoma; Adolescent; Adrenal Cortex; Adrenal Cortex Neoplasms; Adult; Carcinoma; Cytoplasm; Female; Humans; Immunohistochemistry; Male; Middle Aged; Transforming Growth Factor beta; Zona Glomerulosa; Zona Reticularis

Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from hypothalamic tissues based on its ability to stimulate cAMP production in cultured anterior pituitary cells. Recent studies have suggested a functional role for PACAP in the apoptosis of brain cells. However, the role of PACAP in regulating apoptosis in human pituitary adenomas has not previously been examined. Analysis of the cultured human pituitary adenoma cell line HP75, which expresses all three major PACAP receptors, showed that both PACAP-38 and PACAP-27 inhibited TGF-beta1-induced apoptosis. Treatment with the PACAP receptor antagonists PACAP 6-38 (PACAP type I receptor antagonist) and (p-chloro-D-Phe(6), Leu(17))-VIP (PACAP type II receptor antagonist) blocked the effects of PACAP-38 on the inhibition of transforming growth factor-beta1 (TGF-beta1)-induced apoptosis, confirming the specificity of the role of PACAP. Treatment with forskolin but not phorbol 12-myristate 13-acetate (PMA) also inhibited TGF-beta1-induced apoptosis. TGF-beta1 treatment was associated with an increase in mitogen-activated protein kinase (MAP kinase) when analyzed by Western blotting, but PACAP inhibition of TGF-beta1-induced apoptosis was not associated with activation of MAP kinase. Immunocytochemical analysis of the cell cycle cyclin-dependent kinase inhibitor p27 showed that treatment with TGF-beta1, forskolin, PMA, and PACAP increased p27 expression in cultured HP75 cells. These results indicate that PACAP is a highly specific inhibitor of TGF-beta1-induced apoptosis in the HP75 human pituitary adenoma cell line and that PACAP, TGF-beta1, forskolin, and PMA all stimulate expression of the TGF-beta-regulated cell cycle protein p27 in the HP75 human pituitary adenoma cell line. The HP75 cell line can be used as a model to study the regulation of apoptosis in human pituitary cells.

Topics: Adenoma; Apoptosis; Cell Cycle; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Humans; Immunohistochemistry; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Neuropeptides; Nucleic Acid Hybridization; Pituitary Adenylate Cyclase-Activating Polypeptide; Pituitary Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins

Immunohistochemical investigation of bone morphogenetic protein-2 (BMP-2) and type II collagen, two cartilage-associated proteins, was undertaken using monoclonal antibodies in 20 cases of salivary pleomorphic adenoma (PA) in order to explore their possible roles in chondroid differentiation of this tumor. Other salivary gland tumors, including adenoid cystic carcinoma (17 cases), polymorphous low-grade adenocarcinoma (10 cases), basal cell adenoma (3 cases), basal cell adenocarcinoma (1 case), and epithelial-myoepithelial carcinoma (2 cases), were also examined for comparison. In PA, BMP-2 immunoreactivity was detected in the luminal and non-luminal cells of the tubulo-ductal structures, plasmacytoid cells, and other scattered tumor cells in solid areas. In addition, tumor cells in chondroid areas in most cases (14/15), and stellate cells in myxoid areas in many cases (7/19), were also intensely labeled for BMP-2. Furthermore, BMP-2 was also detected in the non-neoplastic ductal cells in salivary glands, whereas no other salivary gland tumors were positively stained for this protein. Type II collagen was localized in the intercellular matrix of chondroid areas and in a few chondroid differentiating cells in myxoid areas, confirming its cartilage-specificity. A proportional relationship was observed between BMP-2 expression and chondroid formation, although BMP-2 was also stained in occasional PAs without chondroid formation. It is speculated that BMP-2 might be secreted by tumor cells and play a role in chondroid formation in PA by inducing some tumor cells to produce type II collagen and other chondroid matrical substances, like glycosaminoglycans. The expression of BMP-2 is specific to PA and may possibly be used as a useful marker in differentiating PA from other salivary gland tumors.

Topics: Adenocarcinoma; Adenoma; Adenoma, Pleomorphic; Biomarkers, Tumor; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Carcinoma; Collagen; Humans; Immunohistochemistry; Salivary Gland Neoplasms; Transforming Growth Factor beta

Bcl-2 expression is confined to the base of the colonic crypt, whereas transforming growth factor beta (TGFbeta) is expressed in the upper crypt, as are the apoptotic death promoters, Bak and Bax. In colonic adenoma cells, TGFbeta induces a growth arrest. In some adenoma cell lines, this is accompanied by apoptosis and in others it is not. In this study, we used two human colonic adenoma cell lines: RG/C2, in which TGFbeta induces a G1 arrest without apoptosis, and BH/C1, in which TGFbeta induces both a G1 arrest and apoptosis. TGFbeta does not induce apoptosis in RG/C2 cells even if hydrocortisone and insulin are removed from the culture medium. In BH/C1 cells, TGFbeta induces apoptosis in the presence of insulin and hydrocortisone. Apoptosis induced by TGFbeta is preceded by a reduction in p26-Bcl-2 protein levels. There was no change in the levels of the p30 phosphorylated form of Bcl-2 or in levels of the proapoptotic proteins Bax or Bak. RG/C2 cells did not show decreased Bcl-2 levels in response to TGFbeta-induced growth inhibition. Therefore, TGFbeta regulates Bcl-2 expression in colonic adenoma cells which undergo apoptosis in response to TGFbeta, but not in those which are growth inhibited, but resistant to TGFbeta-induced apoptosis. TGFbeta may play an important role in the colonic epithelium, not only in the inhibition of cell proliferation, but also in the regulation of apoptosis.

Topics: Adenoma; Apoptosis; Colorectal Neoplasms; Humans; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor (TGF)-beta has been implicated in the regulation of normal and neoplastic anterior pituitary cell function. TGF-beta regulates the expression of various proteins, including p27Kip1 (p27), a cell cycle inhibitory protein. We examined TGF-beta, TGF-beta type II receptor (TGF-beta-RII), and p27 expression in normal pituitaries, pituitary adenomas, and carcinomas to analyze the possible roles of these proteins in pituitary tumorigenesis. Normal pituitary, pituitary adenomas, and pituitary carcinomas all expressed TGF-beta and TGF-beta-RII immunoreactivity. Reverse transcription polymerase chain reaction analysis showed TGF-beta 1, -beta 2, and -beta 3 isoforms and TGF-beta-RII in normal pituitaries and pituitary adenomas. Pituitary adenomas cells cultured for 7 days in defined media showed a biphasic response to TGF-beta with significant inhibition of follicle-stimulating hormone secretion at higher concentrations (10(-9) mol/L) and stimulation of follicle-stimulating hormone secretion at lower concentrations (10(-13) mol/L) of TGF-beta 1 in gonadotroph adenomas. Immunohistochemical analysis for p27 protein expression showed the highest levels in nontumorous pituitaries with decreased immunoreactivity in adenomas and carcinomas. When nontumorous pituitaries and various adenomas were analyzed for p27 and specific hormone production, growth hormone, luteinizing hormone, and thyroid-stimulating hormone cells and tumors had the highest percentages of cells expressing p27, whereas adrenocorticotrophic hormone cells and tumors had the lowest percentages. Immunoblotting analysis showed that adrenocorticotrophic hormone adenomas also had the lowest levels of p27 protein. Semiquantitative reverse transcription polymerase chain reaction and Northern hybridization analysis did not show significant differences in p27 mRNA expression in the various types of adenomas or in nontumorous pituitaries. In situ hybridization for p27 mRNA showed similar distributions of the gene product in nontumorous pituitaries, pituitary adenomas, and carcinomas. These results indicate that TGF-beta and TGF-beta-RII are widely expressed in nontumorous pituitaries and in pituitary neoplasms and that TGF-beta 1 regulates pituitary hormone secretion. The levels of the TGF-beta-regulated protein p27 decreases in the progression of normal to neoplastic pituitaries. In contrast, the mRNA levels of p27 remained relatively constant in nontumorous pituitaries, pituitary adeno

Topics: Adenoma; Carcinoma; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Immunohistochemistry; In Situ Hybridization; Microtubule-Associated Proteins; Pituitary Gland; Pituitary Neoplasms; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Suppressor Proteins

Transforming growth factor-beta proteins are key regulators of cellular growth and differentiation. To understand the role of TGF-beta in colonic tumour progression, 47 paraffin embedded samples from colonic tumours (13 adenomas, and 34 adenocarcinomas) were studied. Gene mutations in the region coding for the active protein were studied by PCR SSCP analysis of exons 5, 6, and 7. TGF-beta1 mRNA expression and localization were studied by NISH using cDNA probes generated by RT-PCR. Protein distribution was investigated by immunohistochemistry using antibodies against both intracellular and extracellular forms. Three mutations were found: one in exon 5 (Dukes C) and two in exon 7 (Dukes C and A). TGF-beta1 mRNA was observed in 9 (69%) of 13 adenomas and in 30 (88%) of 34 adenocarcinomas. Levels of TGF-beta1 mRNA and proteins were higher in colorectal carcinomas than in colorectal adenomas. Tubular adenomas associated with dysplasia showed greater expression of TGF-beta1 than adenomas without dysplasia and than non-neoplastic colonic mucosa. In dysplasia and cancer, epithelial neoplastic cells and stromal cells were positive for TGF-beta1. In normal tissue endothelial cells and granulocytes sporadically showed immunoreactivity for TGF-beta1, whereas epithelial cells were all negative. The three mutations in TGF-beta1 exons 5, 6 and 7 found in colorectal adenocarcinomas suggest that TGF-beta1 mutation may play a role in colorectal carcinogenesis and that the presence of the mutant form contributes to the transformed state. Our two other findings, the loss of staining gradient in normal colonic mucosa and the higher levels of TGF-beta1 mRNA and proteins in colorectal carcinomas than in colorectal adenomas, indicate that the de-regulation of TGF-beta1 expression may occur as an early event in colorectal carcinogenesis. Although TGF-beta1 expression is generally a reflection of cellular differentiation, tumour grade is not associated with TGF-beta1 mRNA expression. Beside being present in the epithelial cells of the colonic tumours, TGF-beta1 mRNA also occurred in the stroma: its localization in the macrophages adjacent to tumour strongly suggests that TGF-beta1 could be secreted by macrophages. This hypothesis should lead to new therapeutic strategies for colorectal carcinoma.

Topics: Adenocarcinoma; Adenoma; Colonic Neoplasms; DNA Probes; DNA, Neoplasm; Exons; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Mutation; Paraffin Embedding; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; RNA, Messenger; Transforming Growth Factor beta

The high incidence of colorectal cancer in Western society is believed to be strongly related to diet. Mutation of the p53 gene is a late event in colorectal carcinogenesis, and thus, the majority of pre-malignant adenomas express wild-type p53. As loss of p53 protein function is an important step in colorectal carcinogenesis, we investigated whether naturally occurring lumenal factors can modulate the expression of p53 in non-tumorigenic human colonic adenoma cell lines. Levels of p53 protein and mRNA were measured in adherent cells which had been incubated with growth-inhibitory concentrations of sodium butyrate (a by-product of dietary fibre fermentation) or sodium deoxycholate (a bile acid) for up to 48 hr. We report that both butyrate and deoxycholate can down-regulate the expression of wild-type and mutant p53. In contrast, incubation for 48 hr with the endogenous inhibitory growth factor TGFbeta1 did not alter p53 protein expression. Thus, in addition to cellular mechanisms which regulate p53 function, such as post-translational stabilisation, nuclear exclusion, negative feedback inhibition of p53 mRNA translation or binding of p53 by cellular proteins, p53 protein levels also may be regulated by changes in the level of p53 gene transcription. Furthermore, we show that lumenal factors are able to affect directly the expression of p53 protein in colonic epithelial cells.

Topics: Adenoma; Butyrates; Butyric Acid; Cell Division; Cholagogues and Choleretics; Colonic Neoplasms; Deoxycholic Acid; Down-Regulation; Gene Expression Regulation, Neoplastic; Histamine Antagonists; Humans; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Activin, a member of the transforming growth factor-beta (TGF beta) cytokine family, acts as a pituitary cell mitogen via a novel family of receptor-linked serine/threonine (Ser/Thr) kinases. Pituitary tumors synthesize activin subunits, and the autocrine action of these growth factors may modulate tumor proliferation. We, therefore, investigated the expression of activin/TGF beta type I receptor messenger ribonucleic acids (mRNAs), designated ALK1 through ALK5 (ALK = activin receptor-like kinase), and type II receptor mRNAs using RT-PCR in 34 human pituitary adenomas of all phenotypes and normal pituitary tissue. ALK2 and ALK5, specific mediators of activin and TGF beta signals, respectively, were found to be expressed only in tumor and not in normal pituitary cells, and ALK2 expression was found only in tumors of a mammosomatotroph cell lineage. ALK1, ALK3, and ALK4 mRNAs were found in both normal and neoplastic pituitary cells. The alternatively spliced cytoplasmic domain of ALK4 consists of 11 kinase subdomains, that are critical for modulating receptor function and intracellular signaling. Truncated forms of the ALK4 cytoplasmic domain lacking these subdomains may attenuate activin signal transduction and affect both tumor phenotype and proliferation via the formation of inactive type I/type II complexes. Three truncated ALK4 receptor mRNAs generated by alternate splicing of the cytoplasmic Ser/Thr kinase domain were found to be tumor specific. One of these truncated receptor mRNAs, ALK4-5, is a novel splice variant that has not been previously described. Expression of the ActRII and T beta RII type II receptor mRNAs, which specifically bind activin and TGF beta, respectively, was highly prevalent among all tumor subtypes and normal pituitary tissue. However, ActRIIB, an activin-specific type II receptor that displays a 3- to 4-fold higher affinity for ligand than ActRII, was expressed in 94% of tumors, but was not prevalent in normal tissue. These data are the first to demonstrate tumor-specific expression of Ser/Thr kinase receptors mRNAs and their splice variants in human pituitary adenomas.

Topics: Activin Receptors; Activins; Adenoma; Adolescent; Adult; Alternative Splicing; Amino Acid Sequence; Base Sequence; Gene Expression; Humans; Inhibins; Middle Aged; Molecular Sequence Data; Pituitary Neoplasms; Polymerase Chain Reaction; Receptors, Growth Factor; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta

Topics: Adenoma; Adenomatous Polyposis Coli; Adenomatous Polyposis Coli Protein; Animals; Biological Evolution; Colonic Neoplasms; Cytoskeletal Proteins; DNA Repair; DNA-Binding Proteins; DNA, Satellite; Fungal Proteins; Humans; Mice; Microsatellite Repeats; Models, Genetic; Mutation; MutS Homolog 2 Protein; Neoplasms; Transforming Growth Factor beta

There is increasing evidence for the role of cytokines in pituitary differentiated function and tumorigenesis, but the spectrum of cytokines found in the pituitary is unknown. Therefore profiles of cytokine expression were determined in different human anterior pituitary adenoma sub-types.. The reverse transcriptase-linked polymerase chain reaction (PCR) was used to identify the presence of cytokine mRNA within human pituitary adenomas.. Seventeen pituitary adenoma biopsies removed at transsphenoidal surgery were examined: 4 somatotrophinomas, 7 non-functional adenomas, 4 prolactinomas, one case of Cushing's disease and one case of Nelson's syndrome.. RNA was extracted from each adenoma biopsy and reverse transcribed into cDNA. This was specifically amplified in a PCR using oligonucleotide primers complementary to each cytokine. The cytokines investigated were interleukin (IL)-I alpha, IL-I beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, tumour necrosis factor (TNF)-alpha, TNF-beta and transforming growth factor (TGF)-beta 1, beta 2 and beta 3. The products of each PCR were visualized using agarose gel electrophoresis.. All 17 adenomas expressed IL-8 transcripts, but no expression of IL-2, IL-5 or IL-7 was found. IL-6 was expressed in all 4 somatotrophinomas, 3 of 7 non-functional tumours, 2 of 4 prolactinomas and in the single case of Nelson's syndrome. At least one of the 3 isoforms of TGF-beta was found in all but 2 tumours; one prolactinoma and one non-functional adenoma. IL-1 alpha, IL-beta, IL-4, TNF-alpha and TNF-beta were expressed sporadically by individual adenomas.. These data suggest that whilst IL-8 may be important, the local expression of the cytokines IL-2, IL-5 and IL-7 is not important in human anterior pituitary tumorigenesis.

Topics: Adenoma; Adult; Aged; Base Sequence; Cushing Syndrome; Cytokines; DNA Primers; Female; Growth Hormone; Humans; Interleukin-6; Interleukin-8; Male; Middle Aged; Molecular Sequence Data; Nelson Syndrome; Pituitary Gland, Anterior; Pituitary Neoplasms; Polymerase Chain Reaction; Prolactinoma; RNA, Messenger; Transforming Growth Factor beta

Transforming growth factor-beta proteins are key regulators of cellular growth and differentiation. To understand the role of TGF-beta in colonic tumour progression, 47 paraffin-embedded samples from colonic tumours (13 adenomas, and 34 adenocarcinomas) were studied. Gene mutations in the region coding for the active protein were studied by PCRSSCP analysis of exons 5, 6, and 7. TGF-beta 1 mRNA expression and localization were studied by NISH using cDNA probes generated by RT-PCR. Protein distribution was investigated by immunohistochemistry using antibodies against both intracellular and extracellular forms. Three mutations were found: one in exon 5 (Dukes C) and two in exon 7 (Dukes C and A). mRNA TGF-beta 1 was observed in 9 (69%) of 13 adenomas and in 30 (88%) of 34 adenocarcinomas. Levels of TGF-beta 1 mRNA and proteins were higher in colorectal carcinomas than in colorectal adenomas. Tubular adenomas associated with dysplasia showed greater expression of TGF-beta 1 than adenomas without dysplasia and than non neoplastic colonic mucosa. In dysplasia and cancer, epithelial neoplastic cells and stromal cells were positive for TGF-beta 1. In normal tissue endothelial cells and granulocytes sporadically showed immunoreactivity for TGF-beta 1, whereas epithelial cells were all negative. The three mutations in TGF-beta 1 exons 5, 6 and 7 found in colorectal adenocarcinomas suggest that TGF-beta 1 mutation may play a role in colorectal carcinogenesis and that the presence of the mutant form contributes to the transformed state. Our two other findings, the loss of staining gradient in normal colonic mucosa and the higher levels of TGF-beta 1 mRNA and proteins in colorectal carcinomas than in colorectal adenomas, indicate that the deregulation of TGF-beta 1 expression may occur as an early event in colorectal carcinogenesis. Although TGF-beta 1 expression is generally a reflection of cellular differentiation, tumour grade is not associated with mRNA TGF-beta 1 expression. As well as being present in the epithelial cells of the colonic tumours, mRNA TGF-beta 1 also occurred in the stroma. Its localization in the macrophages adjacent to tumour strongly suggests that TGF-beta 1 could be secreted by macrophages. This hypothesis should lead to new therapeutic strategies for colorectal carcinoma.

Topics: Adenocarcinoma; Adenoma; Colonic Neoplasms; Exons; Humans; Immunohistochemistry; Mutation; RNA, Messenger; Transforming Growth Factor beta

VACO-330, a nontransformed cell line established from a human colon adenoma, undergoes spontaneous apoptosis and shedding of cells into the culture medium. Shed cells were shown to be apoptotic, both by nuclear morphology and by generation of a typical "laddered" pattern of degraded DNA. Quantitation of DNA released into the medium, compared with the amount retained on the plate, demonstrated that 6.2 +/- 1.1% of the total cell mass underwent apoptotic death daily. The addition of transforming growth factor beta (20 ng/ml) accelerated this spontaneous apoptotic rate 3.2-fold. Moreover, apoptosis could be rapidly induced in up to 45% of the VACO-330 cells by using brief exposure to a calcium chelating medium to release the cells from the substratum. We suggest that transforming growth factor beta is a likely physiological regulator of apoptosis during maturation of the colonic epithelial cells. We additionally suggest the existence of an alternate pathway, which at the time of shedding from the crypt induces apoptosis in colonic epithelial cells that have escaped earlier apoptotic signals.

Topics: Adenoma; Apoptosis; Cell Adhesion; Cell Differentiation; Collagen; Colonic Neoplasms; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured

To determine whether a single mutational event in one p53 gene is sufficient to confer a significant growth advantage on a colonic epithelial cell, the 143(Ala) p53 mutation was previously expressed in the human colonic adenoma derived cell line AA/C1 (which is wild type for p53) and shown to have no effect on it's in vitro or in vivo growth characteristics. In this investigation, by expressing the 175(His), 248(Trp) or 273(His) mutations in the same AA/C1 cell line, we have shown that this failure to affect the growth of the cells was not mutant specific. We have also demonstrated, using induction of MDM2 protein and the ability of the cells to undergo a p53 dependent G1 arrest, that the 143(Ala), 175(His) or 248(Trp) transfected cells retain functional endogenous wild type p53 activity, and suggest that these p53 mutations would not have a fully dominant negative mode of action in vivo. In contrast, one of the two AA/C1 cell lines transfected with the 273(His) mutation did fail to cell cycle arrest after gamma irradiation, indicating that this mutation can act as a dominant negative. However even loss of wild type p53 function in this cell line was insufficient to directly effect the growth rate of the AA/C1 cells, suggesting that acquisition of the 273(His) mutation may contribute to malignant progression through genomic instability (by inhibiting the G1 arrest) and that other mutations are required before outgrowth of the cell population containing the p53 mutation.

Topics: Adenoma; Base Sequence; Cell Division; Colorectal Neoplasms; DNA Primers; G1 Phase; Gamma Rays; Genes, Dominant; Genes, p53; Humans; Molecular Sequence Data; Mutation; Neoplasm Proteins; Nuclear Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

In cell culture, type alpha transforming growth factor (TGF-alpha) stimulates epithelial cell growth, whereas TGF-beta 1 overrides this stimulatory effect and is growth inhibitory. Transgenic mice that overexpress TGF-alpha under control of the mouse mammary tumor virus (MMTV) promoter/enhancer exhibit mammary ductal hyperplasia and stochastic development of mammary carcinomas, a process that can be accelerated by administration of the chemical carcinogen 7,12-dimethylbenz[a]anthracene. MMTV-TGF-beta 1 transgenic mice display mammary ductal hypoplasia and do not develop mammary tumors. We report that in crossbreeding experiments involving the production of mice carrying both the MMTV-TGF-beta 1 and MMTV-TGF-alpha transgenes, there is marked suppression of mammary tumor formation and that MMTV-TGF-beta 1 transgenic mice are resistant to 7,12-dimethylbenz[a]anthracene-induced mammary tumor formation. These data demonstrate that overexpression of TGF-beta 1 in vivo can markedly suppress mammary tumor development.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Adenocarcinoma; Adenoma; Aging; Animals; Crosses, Genetic; Enhancer Elements, Genetic; Exons; Female; Globins; Male; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Transgenic; Polymerase Chain Reaction; Promoter Regions, Genetic; Rabbits; Transforming Growth Factor beta

Transforming growth factor beta 1 (TGF beta 1) is a multifunctional cytokine. The role of TGF beta 1 in hepatocarcinogenesis is still a matter of discussion. To assess the expression of TGF beta 1 in human liver tumors, the following study was conducted.. Formalin fixed, paraffin embedded sections of 50 hepatocellular carcinomas (HCC), 30 cholangiocellular carcinomas (CCC), 15 hepatocellular adenomas (HCA), 15 focal nodular hyperplasias (FNH), and 20 normal livers (NL) were immunostained with a monoclonal anti-TGF beta 1 antibody (clone TB-21). TGF beta 1 mRNA was measured in snap frozen tissue of 2 HCC, 3 HCA, 2 FNH, and 3 NL of the immunohistochemistry group using slot blot hybridizations. Total RNA was extracted, and slot blots were hybridized with 32P endlabeled TGF beta 1 oligonucleotides. TGF beta 1 mRNA was measured in cpm with a scintillation counter.. TGF beta 1 protein was strongly expressed in 14/15 FNH and 13/15 HCA, whereas it was scarcely detectable in 46/50 HCC and 25/30 CCC. All NL were moderately positive. The 2 HCC cases contained far less TGF beta 1 mRNA than the HCA, FNH, and NL examined here.. TGF beta 1 expression is markedly lower in malignant liver tumors than in benign tumors or NL. Therefore, TGF beta 1 down-regulation may be an important step in hepatocarcinogenesis. Additionally, TGF beta 1 immunostaining may be useful in distinguishing well-differentiated HCC from adenomas.

Topics: Adenoma; Carcinoma, Hepatocellular; Cholangiocarcinoma; Humans; Hyperplasia; Immunohistochemistry; In Situ Hybridization; Liver; Liver Neoplasms; Precancerous Conditions; RNA, Messenger; Transforming Growth Factor beta

It is well known that TSH is the main factor responsible for thyrocyte proliferation and growth. Recent studies have shown that other growth factors, including transforming growth factor-beta 1 (TGF-beta 1), have an important role in the control of thyrocyte proliferation and differentiation. The aim of the study was to evaluate the expression of the TGF-beta 1 gene in thyroid follicular adenoma (FA) by Northern analysis, and its protein localization by immunohistochemistry. Surgically removed thyroid tissue from 56 patients with thyroid FA was screened for the study. Normal thyroid tissue from 4 patients with papillary carcinoma was used as a control. Sixteen FA (8 with a "cold" and 8 with a "hot" scintiscan pattern) having homogeneous histological characteristics were subsequently selected. FA showed greater TGF-beta 1 gene expression than control tissue. There was not a statistically significant difference between "cold" and "hot" FA. Immunohistochemistry analysis showed that TGF-beta 1 was located in various histological structures of the adenomas (thyrocytes, endothelium, perinervium and connective tissue); on the other hand, perinodular and control tissue did not show appreciable TGF-beta 1 protein. Our data suggest that TGF-beta 1 may be involved in the pathogenesis of FA. The different TGF-beta 1 distribution in thyrocytes, endothelium, perinervium and connective tissue in FA suggests that TGF-beta 1 may be variably expressed during the natural history of FA. Since no significant difference in TGF-beta 1 gene expression between "hot" and "cold" adenomas was found, it appears that other factors are involved in their functional differentiation.

Topics: Adenoma; Adult; Female; Humans; Immunohistochemistry; Male; Middle Aged; RNA, Neoplasm; Thyroid Neoplasms; Transforming Growth Factor beta

We describe the spontaneous progression of a colon adenoma cell line to tumorigenicity and growth factor independence. This system allows direct comparison of biologic stages of malignant progression with alterations of colon cancer suppressor genes and oncogenes. VACO-235, a human colon adenoma cell line, is at early passages nontumorigenic in the nude mouse, unable to grow in soft agar, growth stimulated by serum and EGF, and growth inhibited by TGF-beta. VACO-235 daughter passages 93 and higher have in culture spontaneously progressed to being weakly tumorigenic, but retain all other growth characteristics of VACO-235 early passages. A mouse xenograft from late passage VACO-235 was reestablished in culture as the granddaughter cell line, VACO-411. VACO-411 is highly tumorigenic, clones in soft agar, and is unresponsive to serum, EGF, and TGF-beta. Early passage VACO-235 bears a mutant K-ras allele, bears only mutant APC alleles, expresses no DCC transcripts, and expresses only wild type p53 transcripts. VACO-411 retains the identical genotype, still expressing only wild type p53. Colonic cells after ras mutation, APC mutation, and DCC inactivation remain nontumorigenic and growth factor dependent. Malignant progression involves at least two additional steps, and in VACO-411 can proceed by a novel pathway not requiring p53 inactivation.

Topics: Adenoma; Animals; Base Sequence; Cell Division; Chromosome Aberrations; Colonic Neoplasms; Female; Genes, p53; Humans; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Mutation; Oncogenes; Transforming Growth Factor beta; Tumor Cells, Cultured

Mutation of the p53 gene is thought to be a late event in human colorectal carcinogenesis, involved in the malignant conversion of the adenoma to the carcinoma. One of the questions that we hoped to address was whether, in vivo, a single mutational event in one p53 gene is sufficient to confer a significant growth advantage on a colonic epithelial cell. Such a growth advantage could result either from an increase in growth rate and/or loss of response to inhibitory growth signals naturally present in the colonic crypt. We therefore introduced the pC53-SCX3 143 (Val-Ala) p53 mutation into a non tumorigenic adenoma derived cell line, AA/C1, which contained a truncating APC mutation, activating K-ras mutation but was wild-type for the p53 protein. High levels of mutant p53 protein were detected in the pC53-SCX3 transfected AA/C1 cell lines but was found not to affect either the in vitro (colony forming efficiency, anchorage independence) or in vivo (tumorigenicity in nude mice) growth, when compared to vector control or the parental AA/C1 cell line. In addition, to test whether the cells become less sensitive to inhibitory growth factors, the response of the cell lines to the naturally occurring growth inhibitor TGF beta was also investigated. Even though TGF beta had previously been implicated in the control of growth of intestinal epithelium, expression of the mutant p53 protein did not affect the sensitivity of the parental AA/C1 cell line to TGF beta. Under the experimental conditions tested expression of the 143 (Val-Ala) p53 protein was unable to affect the in vitro or in vivo growth characteristics of the adenoma derived AA/C1 cell line. When compared to other studies, these results suggest that the genetic background of the individual recipient cell may greatly influence the effect of expression of a particular p53 mutation.

Topics: Adenoma; Animals; Cell Division; Colonic Neoplasms; DNA, Neoplasm; Humans; Mice; Mice, Nude; Mutation; Neoplasm Transplantation; Phenotype; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Samples of colorectal carcinoma, adenoma and normal colorectal mucosa were examined for the expression of TGF-beta by immunohistochemistry. Immunoreactivity for TGF-beta was present in 52 out of a total of 58 samples of normal mucosa examined. In adenomas and carcinomas TGF-beta expression was observed in eight out of ten and 46 out of 48 samples respectively and was largely restricted to epithelial cells. In normal mucosa differential expression of TGF-beta was present within epithelial cells, those in the upper parts of the crypts showing enhanced immunoreactivity compared to cells in the proliferative compartment. This pattern of differential staining is consistent with TGF-beta having an important role in the control of growth and differentiation in colonic mucosa.

Topics: Adenoma; Cell Differentiation; Colon; Colonic Neoplasms; Epithelial Cells; Epithelium; Humans; Immunohistochemistry; Intestinal Mucosa; Transforming Growth Factor beta

Immunohistochemical localization of transforming growth factor-beta 1 (TGF-beta 1) was studied in the lungs of rats given crystalline silica or ferric oxide by single intratracheal instillation. Ferric oxide elicited no progressive granulomatous reaction, no epithelial hyperplasia, and no lung tumors; no demonstrable reactivity to TGF-beta 1 was observed. Silica induced a granulomatous reaction with progressive fibrosis, adjacent alveolar type II hyperplasia, and alveolar carcinomas. Rabbit polyclonal antibodies to synthetic peptides corresponding to the first 30 amino acids of mature TGF-beta 1, anti-LC (1-30), and anti-CC (1-30) were used for the localization of intracellular and extracellular TGF-beta 1. An antibody to a peptide corresponding to amino acids 266-278 of the TGF-beta 1 precursor sequence, anti-Pre (266-278), was used to detect the TGF-beta precursor and the latency-associated peptide. Intracellular mature TGF-beta (anti-LC) was demonstrated in fibroblasts and macrophages located at the periphery of silicotic granulomas and in fibroblasts adjacent to hyperplastic type II cells. Extracellular mature TGF-beta 1 was localized in the connective tissue matrix of the granulomas and in the stroma of both hyperplastic type II cells and well-differentiated adenocarcinomas. Immunoreactivity to anti-Pre was localized, intracellularly, in hyperplastic alveolar type II cells and their proliferative lesions adjacent to granulomas, in adenomas, but not in adenocarcinomas. The hyperplastic type II cells appear to be the sites of production and secretion of TGF-beta 1, which may regulate their own growth and differentiation and mediate the production of extracellular TGF-beta 1-associated matrix. The lack of reactivity to TGF-beta 1 precursor in the adenocarcinomas is consistent with the loss of normal cellular differentiation and function. TGF-beta 1 appears to have a pathogenetic role in silica-induced mesenchymal and epithelial lesions. The role of TGF-beta 1 and other cytokines in silica-induced carcinogenesis requires further investigation.

Topics: Adenocarcinoma; Adenoma; Animals; Disease Models, Animal; Female; Ferric Compounds; Hyperplasia; Lung; Lung Neoplasms; Male; Protein Precursors; Pulmonary Alveoli; Rats; Rats, Inbred F344; Silicon Dioxide; Silicosis; Transforming Growth Factor beta

The purpose of this study was to determine whether cultured colonic adenoma and carcinoma cells undergo apoptosis (programmed cell death) in vitro and whether specific growth and dietary factors, thought to be involved in the control of growth and differentiation of human colonic cells, could induce cell death through apoptosis. In cell lines originating from 6 colorectal adenomas and 7 carcinomas, spontaneous apoptosis was observed. Sodium butyrate, a naturally occurring fatty acid, is present in the human large bowel in millimolar amounts as a result of bacterial fermentation of dietary fibre. Sodium butyrate, at physiological concentrations, induced apoptosis in 2 adenoma cell lines, RG/C2 and AA/Cl, and in the carcinoma cell line PC/JW/FI. In contrast, transforming growth factor beta 1, which is thought to have an important role in the control of growth in colonic epithelium, did not induce apoptosis. Neither RG/C2 nor PC/JW/FI contain wild-type p53, therefore this tumour-suppressor gene is not required to mediate signals for the induction of apoptosis in colonic tumour cells. Our studies report the induction of apoptosis in colonic tumour cells by the naturally occurring fatty acid sodium butyrate. Since sodium butyrate is produced by bacterial fermentation of dietary fibre, the observation that this fatty acid can induce apoptosis could, in part, explain why a high-fibre diet appears to be protective against colon cancer. Escape from the induction of programmed cell death may be an important event in colorectal carcinogenesis.

Topics: Adenoma; Apoptosis; Butyrates; Butyric Acid; Carcinoma; Colonic Neoplasms; Dietary Fiber; DNA, Neoplasm; Genes, p53; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured

Measurable endothelin (ET) was found in serum-free medium of cultured primary thyroid cells derived from human thyroid tissues after 3 days incubation at levels ranging from undetectable to 35 fmol/100,000 cells. Out of 12 thyroid glands studied, 2 responded to transforming growth factor-beta (TGF-beta) treatment with increased ET secretion into medium. Detailed study of cells derived from one of these thyroids showed TGF-beta at 1 ng/ml stimulated ET secretion from 13.7 to 33.3 fmol/100,000 cells after 3 days incubation. Although TSH alone did not significantly modulate ET release into medium, TSH enhanced the stimulatory effect of TGF-beta. A combination of TSH at 1 mU/ml and TGF-beta at 1 ng/ml stimulated ET secretion to 63.8 fmol/100,000 cells after 3 days incubation. Immunostaining studies demonstrated the presence of intracellular immunoreactive ET, largely localized in perinuclear regions, which was greatly stimulated by TSH but not by TGF-beta. These observations suggest that TSH may stimulate only ET synthesis, whereas TGF-beta may stimulate both synthesis and secretion. Binding of [125I]ET-1 to receptor on thyroid cells was dose-dependently stimulated by TGF-beta (0-10 ng/ml) pretreatment for 3 days. Scatchard analysis of competitive binding data from TGF-beta (1 ng/ml)-treated cells indicated that increased binding was the result of increased receptor number and not increased receptor affinity. TSH (0-10 mU/ml), though not as potent as TGF-beta, also dose-dependently stimulated ET binding. Treatment of thyrocytes with 1 mU/ml TSH for 3 days did not significantly affect ET receptor number or binding affinity. These results illuminate aspects of a possible autocrine regulation of ET in the thyroid gland.

Topics: Adenoma; Cells, Cultured; Endothelins; Endothelium, Vascular; Goiter; Goiter, Nodular; Graves Disease; Humans; Iodine Radioisotopes; Kinetics; Receptors, Endothelin; Reference Values; Thyroglobulin; Thyroid Diseases; Thyroid Gland; Thyroid Neoplasms; Thyrotropin; Transforming Growth Factor beta

Activating mutations of the ras oncogene family occur at high frequency in all stages of thyroid tumorigenesis, both human and experimental. To test the causal nature of this association, and to investigate the biological role of ras mutation, we introduced a mutant c-Ha-ras gene into normal rat thyroid follicular cells using an ecotropic retroviral vector. The major immediate effect was to greatly extend the proliferative lifespan of these cells in culture from less than 3 to more than 15 doublings, without any observable loss of growth-factor dependence or differentiated functions. This in vitro phenotype strongly supports an initiating role for ras mutation in the genesis of benign thyroid tumors (adenomas) in vivo. Spontaneous transformation was observed at low frequency on continuous culture of mutant ras-expressing cells, giving rise to fully immortalized, growth factor-independent, highly tumorigenic lines. Transformation was associated with (i) loss of responsiveness to the growth inhibitor TGF-beta 1, and (ii) greatly increased nuclear levels of p53 protein, which unexpectedly was not due to point mutation in the conserved regions of the p53-coding sequence. We postulate that these two phenomena are causally related to each other and to the transformed phenotype.

Topics: Adenoma; Animals; Base Sequence; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; DNA Probes; Dose-Response Relationship, Drug; Genes, ras; In Vitro Techniques; Male; Molecular Sequence Data; Mutation; Proto-Oncogene Proteins p21(ras); Rats; Rats, Inbred Strains; Serum Albumin, Bovine; Thyroglobulin; Thyroid Neoplasms; Thyrotropin; Transduction, Genetic; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Using the thyroid follicular cell as a model for multi-stage carcinogenesis, we have investigated the role of two potential negative growth regulators ('anti-oncogenes') in epithelial tumour progression--transforming growth factor-beta 1 (TGF beta 1) and p53. Normal follicular cells, as expected, showed marked growth inhibition in response to TGF beta 1. Adenoma cells were equally inhibited. In contrast, spontaneously and SV40-immortalised follicular cell lines showing features of malignant transformation (notably loss of growth factor dependence) had lost all responsiveness to TGF beta 1, accompanied by a partial loss of its receptors. p53 protein was below detectable limits in normal and in adenoma cells but in contrast very high levels were observed in all three transformed lines. In the SV40-immortalised cells, this was expected in view of the known stabilising effect of the viral large T protein. In the spontaneous line we found strong evidence for point mutation of p53, which is known to have the same effect. Both mechanisms result in loss of p53 tumour suppressor function despite increased protein content. We conclude that loss of inhibition by TGF beta and inactivation of p53 are important steps in in vitro immortalisation and/or in vivo tumour progression in human thyroid follicular cells, and speculate that p53 may mediate or be required for the inhibitory signal normally induced by TGF beta 1.

Topics: Adenoma; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Epithelium; Humans; Immunohistochemistry; Precipitin Tests; Thyroid Gland; Thyroid Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The growth of three non-tumorigenic human colonic adenoma cell lines, designated AA/C1, RG/C2 and RR/C1, was inhibited by low concentrations of transforming growth factor beta (TGF-beta) (0.05-0.5 ng ml-1). However, the growth of five human colon cancer cell lines under identical conditions was resistant to high concentrations of TGF-beta (2-10 ng ml-1). This is the first report of well-characterized premalignant human colonic cells showing sensitivity to TGF-beta. The TGF-beta-sensitive adenoma cell line AA/C1 was derived from a relatively large adenoma with a K-ras gene mutation and represents a relatively late-stage adenoma, indicating that loss of response to TGF-beta occurs at a relatively late stage in colorectal carcinogenesis and that the presence of a ras gene mutation does not necessarily confer resistance to TGF-beta. Of further interest, the RG/CZ cell line has a p53 mutation showing that p53 mutations do not necessarily lead to TGF-B insensitivity. Furthermore, in this paper we show that the conversion of the AA/C1 adenoma cell line to a tumorigenic phenotype [Williams et al., (1990) Cancer Res., 50, 4724] is accompanied by a reduced response to the growth-inhibitory effects of TGF-beta up to 10 ng ml-1. Reduced responsiveness to the inhibitory effects of TGF-beta may be an important event in the loss of growth control in colorectal carcinogenesis.

Topics: Adenoma; Carcinoma; Cell Line, Transformed; Cell Transformation, Neoplastic; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Resistance; Genes, ras; Humans; Mutation; Phenotype; Precancerous Conditions; Transforming Growth Factor beta; Tumor Cells, Cultured