Growth inhibition of transforming growth factor beta on human gastric carcinoma cells: receptor and postreceptor signaling.

The effect of transforming growth factor beta (TGF-beta) on human gastric carcinoma cell lines was examined. Cell growth and DNA synthesis of TMK-1 were inhibited by TGF-beta, whereas MKN-28 presented no response to TGF-beta. Scatchard plot analysis of TGF-beta binding showed that TMK-1 had a relatively small number of high-affinity receptors, whereas MKN-28 had a large number of low-affinity receptors. By affinity labeling, only the type I receptor (Mr 65,000) for TGF-beta was detected in TMK-1, while three types of receptors, type I, type II (Mr 85,000-95,000), and type III (Mr 250,000-350,000), for TGF-beta were present in MKN-28. TGF-beta treatment reduced p34cdc-2 kinase activity and the level of phosphorylation of retinoblastoma protein in TMK-1, whereas it did not affect them in MKN-28. mRNAs for MYC and platelet-derived growth factor B chain were increased by treatment of TGF-beta on TMK-1. cAMP-responsive element binding activity was decreased by TGF-beta treatment in MKN-28 but not in TMK-1. This was closely correlated with protein kinase C activity. These results suggest that the type I receptor for TGF-beta in human gastric carcinoma cells may be mainly linked with the growth inhibition of TGF-beta by a decrease in retinoblastoma protein phosphorylation by p34cdc-2 without suppression of MYC expression. Conversely, TGF-beta may reduce protein kinase C activity and cAMP-responsive element binding activity in TGF-beta-resistant gastric carcinoma cells.     

Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines.

In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II mRNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF-beta receptor proteins beta-glycan mRNA was rapidly down-regulated and this effect was sustained throughout the 24 h observation period. RI and RII mRNAs were slightly increased 24 h after treatment. In one cell line sensitive to growth inhibition by TGF-beta, 1 but lacking beta-glycan expression, and one cell line expressing only beta-glycan and thus TGF-beta 1 -resistant, no autoregulation of mRNA of either TGF-beta receptor was demonstrated. The results suggest that TGF-beta 1 regulates the expression of its receptors, in particular beta-glycan, and that this effect is dependent on co-expression of beta-glycan, RI and RII.     

Transcriptional repression of the transforming growth factor-beta type I receptor gene by DNA methylation results in the development of TGF-beta resistance in human gastric cancer

The transforming growth factor-beta (TGF-beta) signaling pathway subserves an essential tumor suppressor function in various cell types. A heteromeric complex composed of TGF-beta type I (RI) and type II (RII) receptors is required for TGF-beta signaling. We have identified a subset of human gastric cancer cell lines which are insensitive to TGF-beta and which express a low level of TGF-beta type I receptor mRNA relative to a gastric cancer cell line which is highly responsive to TGF-beta. Using these cells, we show that hypermethylation of a CpG island in the 5' region of the TGF-beta RI gene provides another potentially important mechanism of escape from negative growth control by TGF-beta. This hypermethylation was found in four of five human gastric cancer cell lines and five out of 40 (12.5%) primary tumors examined. In human gastric cancer cell lines, treatment with the demethylating agent, 5-aza-2'-deoxycytidine, resulted in increased expression of the TGF-beta RI gene, but not the RII gene. Transient transfection of an RI expression vector into the TGF-beta resistant SNU-601 cell line restores TGF-beta responsiveness. These findings suggest that one of the mechanisms of escape from autocrine or paracrine growth control by TGF-beta during carcinogenesis could involve aberrant methylation of CpG islands in the 5' region of the TGF-beta RI gene.     

The loss of TGF-beta signaling promotes prostate cancer metastasis

In breast and colon cancers, transforming growth factor (TGF)-beta signaling initially has an antineoplastic effect, inhibiting tumor growth, but eventually exerts a proneoplastic effect, increasing motility and cancer spread. In prostate cancer, studies using human samples have correlated the loss of the TGF-beta type II receptor (T beta R II) with higher tumor grade. To determine the effect of an inhibited TGF-beta pathway on prostate cancer, we bred transgenic mice expressing the tumorigenic SV40 large T antigen in the prostate with transgenic mice expressing a dominant negative T beta R II mutant (DN II R) in the prostate. Transgene(s) and TGF-beta 1 expression were identified in the prostate and decreased protein levels of plasminogen activator inhibitor type I, as a marker for TGF-beta signaling, correlated with expression of the DN II R. Although the sizes of the neoplastic prostates were not enlarged, increased amounts of metastasis were observed in mice expressing both transgenes compared to age-matched control mice expressing only the large T antigen transgene. Our study demonstrates for the first time that a disruption of TGF-beta signaling in prostate cancer plays a causal role in promoting tumor metastasis.     

Autocrine Transforming Growth Factor-beta Growth Pathway in Murine Osteosarcoma Cell Lines Associated with Inability to Affect Phosphorylation of Retinoblastoma Protein

Purpose. Production of active transforming growth factor-beta (TGF-beta ) by human osteosarcoma may contribute to malignant progression through mechanisms that include induction of angiogenesis, immune suppression and autocrine growth stimulation of tumor cell growth.To study events associated with induction of cell proliferation by TGF-beta , we have evaluated the TGF-beta pathway in two murine osteosarcoma cell lines, K7 and K12.Results. Northern and immunohistochemical analyses show that each cell line expressesTGF-beta1 and TGF-beta3 mRNA and protein. Both cell lines secrete activeTGF-beta 1 and display a 30-50% reduction in growth when cultured in the presence of a TGF-beta blocking antibody. Expression of TGF-beta receptors TbetaRI, TbetaRII and TbetaRIII is demonstrated by affinity labeling with (125) -TGF-beta 1, and the intermediates, Smads 2, 3 and 4, are uniformly expressed. Smads 2 and 3 are phosphorylated in response toTGF-beta , while pRb phosphorylation in each osteosarcoma cell line is not affected by either exogenousTGF-beta or TGF-beta antibody.Conclusions. The data implicate events downstream of Smad activation, including impaired regulation of pRb, in the lack of a growth inhibitory response toTGF-beta , and indicate that this murine model of osteosarcoma is valid for investigating the roles of autocrineTGF-beta in vivo.     

Transforming growth factor beta 1 (TGF-beta 1) inhibits retinoblastoma gene expression but not pRB phosphorylation in TGF-beta 1-growth stimulated colon carcinoma cells.

The response of the retinoblastoma (RB) gene and its product (pRB) to transforming growth factor beta 1 (TGF-beta 1) was studied in three types of colon carcinoma cells derived from the same parental line. TGF-beta 1 was a growth inhibitor for two enterocytic-differentiated lines, a growth stimulator for two undifferentiated lines, and had no effect on two goblet cell-differentiated lines. TGF-beta 1 treatment for 3 days decreased RB gene expression and pRB level two- to threefold in each responsive line. When treated with TGF-beta 1 beginning in early G1, enterocytic cells were arrested in G1 and pRB remained under-phosphorylated and in low abundance. Neither goblet cell line exhibited these responses to TGF-beta 1 because they were shown to lack TGF-beta 1 type I and II receptors. Thus during colonocyte differentiation goblet cells lose responsiveness to TGF-beta 1 by down-regulating TGF-beta 1 receptors, while enterocytic cells retain and exhibit responsiveness to TGF-beta 1 through modulations of pRB. Both of the undifferentiated lines exhibited mixed responses to TGF-beta 1: a decrease in total amount of RB mRNA and pRB protein yet an increase in pRB phosphorylation consistent with increased cell cycling. Therefore, TGF-beta 1 controls RB function by two separable mechanisms, the regulation of pRB phosphorylation and the control of RB mRNA and protein level.     

Alteration of cell growth and morphology by overexpression of transforming growth factor beta type II receptor in human lung adenocarcinoma cells

TGF-beta is a potent inhibitory regulator of cell growth, which is transduced through interaction between type I (RI) and type II (RII) receptors that form heteromeric kinase complexes. Abnormal expression of these receptors has been identified in several human epithelial cancers and has been shown to be highly associated with resistance to TGF-beta. In this study, we investigated the expression of RI and RII in 13 human non-small cell lung cancer cell lines (NSCLCs) and demonstrated decreased or loss of RII expression in five lung cancer cell lines, but not of RI. Of these cell lines, the role of RII in NCI-H358 adenocarcinoma, which lacks RII and is insensitive to TGF-beta, was investigated by transducing this cell line with a recombinant retrovirus expressing full-length TGF-beta RII. Stably transfected cells showed significant increase in RII mRNA and protein expression. These cells responded to exogenous TGF-beta1 with suppressed proliferation in a dose-dependent manner and G1 arrest accompanied by morphological change distinct from control cells. We also investigated whether overexpression of dominant-negative RII (dnRII) in NCI-H441 adenocarcinoma, which is sensitive but expresses low levels of RII, could block signaling through the receptor complex. The overexpression of this kinase-domain-truncated RII by expressing the retroviral dnRII construct led to loss of the ability to respond to TGF-beta1 and an exhibition of uncontrolled growth. These results suggest a close association between the loss of the expression of wild-type TGF-beta RII and carcinogenesis in human lung cancer cells.     

Expression of transforming growth factor beta 1 by human endometrial carcinoma cell lines: inverse correlation with effects on growth rate and morphology

We examined the effects of transforming growth factor beta 1 (TGF-beta 1) on various aspects of the cell biology of human endometrial carcinoma (HEC) cell lines in vitro, as well as the expression of TGF-beta 1 mRNA by these cell lines. Cell lines from eight HEC tumors, representing a variety of histological subtypes, were studied in order to test the generality of conclusions regarding the effects of TGF-beta 1 on this particular tumor cell type. The growth of five HEC cell lines was inhibited by TGF-beta 1 (10 ng/ml), while growth of three cell lines was not inhibited. The effects on growth correlated with morphological alterations induced by TGF-beta 1; the cell lines with inhibited growth displayed a larger, flatter, more contact-inhibited phenotype, while the cell lines whose growth ws not inhibited showed few discernible morphological alterations in response to TGF-beta 1. Northern analysis of TGF-beta 1 mRNA levels revealed that the three HEC cell lines unresponsive to TGF-beta 1 treatment expressed relatively large amounts of TGF-beta 1. Correspondingly, the five HEC cell lines which responded to TGF-beta 1 with growth and morphological changes expressed much lower levels of TGF-beta 1 mRNA. These results suggest that the sensitivity of human HEC cell lines to TGF-beta 1 is variable and that this sensitivity is inversely correlated with the level of expression of TGF-beta 1.     

Overexpression of H-ras oncogene induces resistance to the growth-inhibitory action of transforming growth factor beta-1 (TGF-beta 1) and alters the number and type of TGF-beta 1 receptors in rat intestinal epithelial cell clones.

In this report, we utilize rat intestinal cell (IEC-18) clones expressing an activated human H-ras gene to investigate the relationship between malignant transformation and growth control by transforming growth factor beta (TGF-beta). We demonstrate that clones expressing high levels of H-ras oncogene lose sensitivity to the growth inhibitory action of TGF-beta. The loss of sensitivity is related to the degree of H-ras expression and is shown to be a direct consequence of H-ras expression through the use of a clonal cell line with inducible expression of activated H-ras. Co-incident with the loss of growth inhibition, ras-expressing clones display an altered expression of TGF-beta-binding proteins as detectable by [125I]TGF-beta cross-linking. While IEC-18 cells express type II (92 kDa) binding protein predominantly, H-ras expression induces a shift to predominantly type I (69 kDa) binding protein expression.     

Reduced levels of transforming growth factor-beta type I receptor in human gastric carcinomas.

The expressions of transforming growth factor beta (TGF-beta) and its receptor and TGF-beta inhibitory element (TIE)-binding protein were examined on human gastric carcinomas by Northern blot hybridization, immunohistochemistry, affinity labeling and gel retardation analysis. TGF-beta mRNA was expressed in tumor and normal tissues at various levels. Immunohistochemically, TGF-beta expression was confirmed to be present within tumor cells. Out of the 17 human gastric carcinoma tissues, 14 (82%) showed a reduction in the level of type I receptor (65 kDa) for TGF-beta when compared to corresponding normal mucosas. Interestingly, in seven of the 14 tumors the level of TIE-binding protein in the tumor tissue was lower than that in normal mucosa. Human gastric carcinoma cell line TMK-1, whose growth was inhibited by TGF-beta, had only type I receptor for TGF-beta and showed a high level of TIE-binding protein. Conversely, MKN-1, a TGF-beta-resistant cell line, exhibited an extremely low level of TGF-beta receptor and had no TIE-binding protein. These results overall indicate that although human gastric carcinoma cells produced TGF-beta, they showed a reduction in TGF-beta type I receptor and a low level of TIE-binding protein, resulting in escape from growth inhibition by TGF-beta.     

Soluble type II transforming growth factor-beta (TGF-beta) receptor inhibits TGF-beta signaling in COLO-357 pancreatic cancer cells in vitro and attenuates tumor formation.

Human pancreatic ductal adenocarcinomas overexpress transforming growth factor-betas (TGF-betas). This overexpression has been correlated with decreased patient survival. TGF-betas bind to a type II TGF-beta receptor (TbetaRII) dimer, which heterotetramerizes with a type I TGF-beta receptor (TbetaRI) dimer, thereby activating downstream signaling. PURPOSE AND EXPERIMENTAL DESIGN: To determine whether blocking TGF-beta actions would suppress pancreatic cancer cell growth in vivo, we expressed a soluble TbetaRII, encoding amino acids 1-159 of the extracellular domain in COLO-357 human pancreatic cancer cells. This cell line expresses all of the three mammalian TGF-beta isoforms and is growth inhibited by TGF-beta in vitro. RESULTS: COLO-357 clones expressing soluble TbetaRII were no longer growth inhibited by exogenous TGF-beta1 and exhibited a marked decrease in their invasive capacity in vitro. When injected s.c. into athymic mice, these clones exhibited attenuated growth rates and angiogenesis and decreased levels of plasminogen activator inhibitor-1 mRNA as compared with tumors formed by sham-transfected cells. CONCLUSIONS: These results indicate that endogenous TGF-betas can confer a growth advantage in vivo to a pancreatic cancer cell line that is growth inhibited in vitro and suggest that a soluble receptor approach can be used to block these tumorigenic effects of TGF-betas.     

Isolation and characterization of Kirsten murine sarcoma virus-transformed mouse keratinocytes resistant to transforming growth factor beta

BALB/MK (MK) is a continuous murine keratinocyte line whose cells are strictly dependent on exogenous epidermal growth factor (EGF) for growth in culture. A derivative cell, KC, resulted from Kirsten murine sarcoma virus transformation, and these cells no longer require EGF for their growth. Despite differences in MK and KC growth conditions, both cell lines are growth inhibited by picomolar concentrations of transforming growth factor-beta (TGF-beta). When MK and KC cells were maintained in the presence of TGF-beta, resistant variants eventually proliferated only from the KC population. In an attempt to determine the mechanism of development of TGF-beta resistance, the TGF-beta-resistant cells (KCR cells) were compared with TGF-beta-sensitive KC cells with regard to growth properties, TGF-beta 1 binding characteristics, and gene expression. KCR cells continued to synthesize DNA and proliferated in the presence of TGF-beta 1 concentrations up to 2 nM, which was 500-fold greater than the ED50 for the sensitive cells. Although the KCR cells possess similar receptor numbers and affinity for TGF-beta 1, we observed differences in affinity cross-linking studies. The KCR cells expressed more of the type III, high molecular weight cell surface binding protein and less of the type II than the KC cells. The type I moiety was clearly altered to a smaller size in some, but not all, KCR cells. In gene regulation studies, there was no apparent difference in c-Ki-ras and v-Ki-ras mRNA levels in the KC and KCR cells. Additionally, expression of TGF-alpha and TGF-beta 1 mRNA was similar in MK, KC, and KCR cells. The expression of proliferation-associated genes, such as c-myc and MGSA/c-gro/kc, which were markedly decreased by TGF-beta 1 in the MK and KC cells, was not altered by TGF-beta 1 in the KCR cells. The data suggest that the loss of TGF-beta 1 responsiveness in the KCR cells was due to an alteration in the TGF-beta receptor that did not permit signal transduction, although the existence of postreceptor alterations cannot be excluded.     

A dominant negative mutation of transforming growth factor-beta receptor type II gene in microsatellite stable oesophageal carcinoma

Recent investigations revealed microsatellite instability in colon cancers are associated with mutations of the transforming growth factor-beta receptor type II gene (TGF-beta RII) that encodes a transmembrane protein containing an intracellular serine/threonine kinase domain. Activation of TGF-beta receptor type I (RI) and RII by TGF-beta induces nuclear translocation of Smad proteins including Smad2 and Smad4 that have been originally identified as tumour suppressor genes. We have previously reported six cases with microsatellite instability in 32 oesophageal carcinomas. In this study, we analysed genetic mutations of TGF-beta RII, Smad2 and Smad4 in these oesophageal carcinoma tissues and established 16 cell lines. No genetic mutation was detected in any tissues or cell lines except one tissue sample of microsatellite stable oesophageal carcinoma, that is, a mis-sense mutation of glutamic acid to glutamine at codon 526 (E526Q) in the TGF-beta RII serine/threonine kinase domain. Interestingly, the mutant TGF-beta RII E526Q can completely inhibit TGF-beta-induction of nuclear translocation of Smad4 protein in oesophageal carcinoma cells. This mutation of TGF-beta RII that is not associated with microsatellite instability might make a dominant negative effect on TGF-beta signal transduction in oesophageal carcinoma.     

Characterization of a newly established human pancreatic carcinoma cell line, UK Pan-1

BACKGROUND: A highly tumorigenic cell line designated as UK Pan-1 was established in a surgically removed human pancreatic adenocarcinoma and characterized as having many of the genotypic and phenotypic alterations commonly found in pancreatic tumors. METHODS: The cell line was characterized by its morphology, growth rate in monolayer culture and soft agar, tumorigenicity in nude mice, and chromosomal analysis. Furthermore, the status of p53, Ki-ras mutation and transforming growth factor (TGF)-/receptor expression were determined. The characteristics of UK Pan-1 were compared with those of other commonly used pancreatic carcinoma cell lines. RESULTS: Quiescent UK Pan-1 cells could be stimulated to proliferate in growth factor free nutrient media, indicating a growth factor independent phenotype. UK Pan- 1 cells grew in soft agar and rapidly formed tumors in nude mice. This cell line possesses a mutation at codon 12 of the c-Ki-ras-2 gene that is commonly found in pancreatic carcinoma. Fluorescence in situ hybridization showed that two alleles of p53 tumor suppressor gene were present in UK Pan-1. However, sequencing analysis revealed a mutation in one allele at exon 8, codon 273 (G to A; Arg to His). Additional growth assays indicated that the cell line was insensitive to negative growth regulation induced by exogenous TGF-beta. Molecular analysis of the TGF-beta signaling pathway showed that UK Pan-1 did not express appreciable levels of the TGF-beta receptor type I, II, or III mRNAs, but did express DPC4 mRNA. Karyotype analysis revealed an 18q21 deletion indicating a possible loss of heterozygosity for DPC4, as well as other chromosomal deletions and rearrangements. CONCLUSIONS: This study indicates that UK Pan-1 is a highly tumorigenic cell line possessing a molecularly complex pattern of mutations that may be used as a model to further the understanding of the mechanisms responsible for the development of pancreatic carcinoma.     

Effects of transforming growth factor beta-1 on growth-regulatory genes in tumour-derived human oral keratinocytes.

This study examined the effect of transforming growth factor beta-1 (TGF-beta 1) on c-myc, RB1, junB and p53 expression together with pRb phosphorylation, in carcinoma-derived and normal human oral keratinocytes with a range of inhibitory responses to this ligand. Amplification of c-myc was observed in eight of eight tumour-derived cell lines and resulted in corresponding mRNA expression. The down-regulation of c-myc expression by TGF-beta 1 predominantly reflected growth inhibition by TGF-beta 1, but in two of eight tumour-derived cell lines which were partially responsive to TGF-beta 1 c-myc expression was unaltered by this ligand. While RB1 mRNA levels were unaltered by TGF-beta 1, the ligand caused the accumulation of the underphosphorylated form of the Rb protein in all cells irrespective of TGF-beta 1-induced growth arrest. junB expression was up-regulated by TGF-beta 1 in cells with a range of growth inhibitory responses. All cells contained mutant p53. TGF-beta 1 did not affect p53 mRNA expression in both tumour-derived and normal keratinocytes and there was no alteration in p53 protein levels in keratinocytes expressing stable p53 protein following TGF-beta 1 treatment. The data indicate that TGF-beta-induced growth control can exist independently of the presence of mutant p53 and the control of Rb phosphorylation and c-myc down-regulation. It may be that TGF-beta growth inhibition occurs via multiple mechanisms and that the loss of one pathway during tumour progression does not necessarily result in the abrogation of TGF-beta-induced growth control.