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Transforming growth factor beta1 increases the expressions of high molecular weight tropomyosin isoforms and vinculin and suppresses the transformed phenotypes in human lung carcinoma cells.

Exposure of the human lung carcinoma cell line, A549 cells, to transforming growth factor beta1 (TGFbeta1) resulted in an alteration in the morphology from epithelial-like to fibroblastic flat cells. Immunofluorescent study revealed that microfilament organization was partially restored in the TGFbeta1-treated cells. We also investigated the effects of TGFbeta1 on the transformed phenotypes including the anchorage-independent growth and invasive ability of A549 cells and found that the phenotypes were strikingly suppressed in TGFbeta1-treated A549 cells. Accompanying these changes, the levels of the expression of high molecular weight tropomyosin (TM) isoforms, especially TM1 and TM2, were significantly increased in the TGFbeta1-treated cells. The expression level of vinculin was also increased in these cells. These results suggest that the increased tropomyosin and vinculin syntheses may be involved in the suppression of the transformed phenotypes of A549 cells.

Actins↗

Biologically active precursor for transforming growth factor type alpha, released by retrovirally transformed cells.

Retrovirus-transformed rat cells that actively express transforming growth factor type alpha (TGF-alpha) release into the medium a soluble protein of 17-19 kDa that has been identified as a precursor for TGF-alpha. The identification of this protein as a TGF-alpha precursor is based on its recognition by specific antibodies and by the ability of elastase to convert this protein into a 6-kDa polypeptide with the properties of mature TGF-alpha. This TGF-alpha precursor binds to epidermal growth factor/TGF-alpha receptors and activates the receptor-associated tyrosine kinase activity in intact cells. The biological potency of this precursor is not markedly increased by conversion into mature TGF-alpha in vitro. These studies demonstrate the ability of transformed cells to release a TGF-alpha precursor capable of strong mitogenic action in vivo.

Animals↗

Transformation of normal rat kidney (NRK) cells by an infectious retrovirus carrying a synthetic rat type alpha transforming growth factor gene.

We synthesized a gene for rat type alpha transforming growth factor (TGF-alpha) consisting of the leader sequence and the sequence coding for the mature 50-amino acid peptide without the C-terminal processed region. This gene was inserted into the retrovirus vector pSW272, which is derived from spleen necrosis virus, to obtain an infectious recombinant virus carrying the rat TGF-alpha gene. This recombinant virus can infect normal rat kidney (NRK) cells and allow these cells to grow in soft agar in the presence of TGF-beta. Transformed cells isolated from colonies grown in soft agar contain an integrated form of the recombinant virus and secrete biologically active TGF-alpha into their medium. These results show that a biologically active TGF-alpha can be produced by a gene synthesized from only part of the coding region and that the infectious retrovirus carrying the TGF-alpha gene (SW355) can function as a transforming agent in NRK cells in the presence of TGF-beta.

Animals↗

Differential Taxol-dependent arrest of transformed and nontransformed cells in the G1 phase of the cell cycle, and specific-related mortality of transformed cells.

Taxol (paclitaxel) induces a microtubule hyperassembled state, and effectively blocks cells in mitosis. Here we report that Taxol also induces a stable late-G1 block in nontransformed REF-52 and WI-38 mammalian fibroblast cells, but not in T antigen-transformed cells of the same parental lineage. G1 arrest is characterized by partially dephosphorylated pRb, and inactive cdk2 kinase. Nontransformed cells recover normally from Taxol arrest. In contrast, T antigen transformed cells continue inappropriately past both G1 and G2-M in the presence of Taxol, and undergo a rapid death upon release. These results demonstrate a microtubule sensitive step in G1 regulation of nontransformed fibroblast cells. Also, Taxol selectively induces death of transformed cells, possibly because they slip the Taxol-dependent G1 arrest, as well as G2/M arrest, which are both specific to nontransformed cells.

Animals↗

SV40-transformed human diploid cells that remain transformed throughout their limited lifespan.

Of ninety-one subcultured foci of SV40-transformed WI-38 human diploid fibroblasts, none yielded cells that grew indefinitely, but all cells in each subculture continued to produce T antigen and to look morphologically transformed throughout their lifespan. These results are consistent with the commitment theory of fibroblast senescence, but predict that a special transformation event is necessary to accout for the rare survivors.

Antigens, Neoplasm↗

Virocrine transformation: the intersection between viral transforming proteins and cellular signal transduction pathways.

This review describes a mechanism of viral transformation involving activation of cellular signaling pathways. We focus on four viral oncoproteins: the E5 protein of bovine papillomavirus, which activates the platelet-derived growth factor beta receptor; gp55 of spleen focus forming virus, which activates the erythropoietin receptor; polyoma virus middle T antigen, which resembles an activated receptor tyrosine kinase; and LMP-1 of Epstein-Barr virus, which mimics an activated tumor necrosis factor receptor. These examples indicate that diverse viruses induce cell transformation by activating cellular signal transduction pathways. Study of this mechanism of viral transformation will provide new insights into viral tumorigenesis and cellular signal transduction.

Animals↗

Transforming acidic coiled coil 1 promotes transformation and mammary tumorigenesis.

Transforming acidic coiled coil 1 (TACC1) is a putative oncogene located within a breast cancer amplicon found on human chromosome 8p11. Although TACC1 has been reported to transform fibroblasts, it is also down-regulated in a subset of mammary tumors treated with anthracyclin. Here, we show that ectopic TACC1 overexpression can cooperate with Ras to induce focus formation in murine fibroblast cultures and prevent death caused by overexpression of Pten or a dominant-negative form of protein kinase B (PKB)/Akt. In transgenic mice carrying TACC1 under the control of the mouse mammary tumor virus promoter, TACC1 expression reduced apoptosis during mammary gland involution, increased the penetrance of mammary tumors in a pten+/- background, and decreased the average age of mammary tumor onset in a mouse model based on a phosphatidylinositol 3'-kinase (PI3K)-decoupled mutant of polyoma middle T. Elevated levels of both phospho-PKB and phospho-extracellular signal-regulated kinase were found in mammary tissue containing the TACC1 transgene. Thus, TACC1 positively regulates the Ras and PI3K pathways, promotes Ras-mediated transformation, and prevents apoptosis induced by PI3K pathway inhibition. TACC1 also cooperates with tumorigenic mutations in the PI3K pathway and thereby plays an oncogenic role in tumor formation in the murine mammary gland.

Animals↗

The use of cystatin C to inhibit epithelial-mesenchymal transition and morphological transformation stimulated by transforming growth factor-beta.

INTRODUCTION: Transforming growth factor-beta (TGF-beta) is a potent suppressor of mammary epithelial cell (MEC) proliferation and is thus an inhibitor of mammary tumor formation. Malignant MECs typically evolve resistance to TGF-beta-mediated growth arrest, enhancing their proliferation, invasion, and metastasis when stimulated by TGF-beta. Recent findings suggest that therapeutics designed to antagonize TGF-beta signaling may alleviate breast cancer progression, thereby improving the prognosis and treatment of breast cancer patients. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-beta type II receptor antagonist that inhibits TGF-beta binding and signaling in normal and cancer cells. We hypothesized that the oncogenic activities of TGF-beta, particularly its stimulation of mammary epithelial-mesenchymal transition (EMT), can be prevented by CystC. METHOD: Retroviral infection was used to constitutively express CystC or a CystC mutant impaired in its ability to inhibit cathepsin protease activity (namely Delta14CystC) in murine NMuMG MECs and in normal rat kidney (NRK) fibroblasts. The effect of recombinant CystC administration or CystC expression on TGF-beta stimulation of NMuMG cell EMT in vitro was determined with immunofluorescence to monitor rearrangements of actin cytoskeletal architecture and E-cadherin expression. Soft-agar growth assays were performed to determine the effectiveness of CystC in preventing TGF-beta stimulation of morphological transformation and anchorage-independent growth in NRK fibroblasts. Matrigel invasion assays were performed to determine the ability of CystC to inhibit NMuMG and NRK motility stimulated by TGF-beta. RESULTS: CystC and Delta14CystC both inhibited NMuMG cell EMT and invasion stimulated by TGF-beta by preventing actin cytoskeletal rearrangements and E-cadherin downregulation. Moreover, both CystC molecules completely antagonized TGF-beta-mediated morphological transformation and anchorage-independent growth of NRK cells, and inhibited their invasion through synthetic basement membranes. Both CystC and Delta14CystC also inhibited TGF-beta signaling in two tumorigenic human breast cancer cell lines. CONCLUSION: Our findings show that TGF-beta stimulation of initiating metastatic events, including decreased cell polarization, reduced cell-cell contact, and elevated cell invasion and migration, are prevented by CystC treatment. Our findings also suggest that the future development of CystC or its peptide mimetics hold the potential to improve the therapeutic response of human breast cancers regulated by TGF-beta.

Animals↗

Characterization and purification of a secreted plasminogen activator inhibitor (PAI-1) induced by transforming growth factor-beta 1 in normal rat kidney (NRK) cells: decreased PAI-1 expression in transformed NRK cells.

Type 1 transforming growth factor-beta (TGF beta 1) was found to be a potent inducer of the secretion of a 49,000 mol wt protein by normal rat kidney (NRK) cells. This protein was related to type 1 plasminogen activator inhibitor (PAI-1) on the basis of molecular mass, activity in the presence of sodium dodecyl sulfate, immunoprecipitation by antibodies to PAI-1, and N-terminal sequence analysis. PAI-1 levels in the conditioned medium of NRK cells were increased 5- to 11-fold when cells were incubated with picomolar concentrations of TGF beta 1 for 24 h, reaching a concentration of approximately 0.3 microgram/ml. The secreted PAI-1 was deposited in the NRK extracellular matrix as well as released into the culture medium. A spontaneously transformed NRK cell line was found to secrete 3-4 times less PAI-1, in the absence or presence of TGF beta 1, compared to the parent cell line, while PAI-1 secretion in Kirsten sarcoma virus-transformed NRK cells was almost completely abrogated. A novel purification procedure was established, which results in the isolation of highly active and detergent-free TGF beta 1-induced PAI-1.

Amino Acid Sequence↗

Induction of transforming growth factor alpha expression in mouse mammary epithelial cells after transformation with a point-mutated c-Ha-ras protooncogene.

NOG-8 ras cells are a normal mouse mammary epithelial cell line transfected with a plasmid containing a glucocorticoid-inducible mouse mammary tumor virus long terminal repeat linked to the activated c-Ha-ras protooncogene. After addition of dexamethasone, there is a rapid induction (within 1-3 h) of p21ras protein that is concomitant with a parallel induction of the c-Ha-ras specific mRNA. After 4-6 days of dexamethasone treatment, NOG-8 ras cells are able to grow as colonies in semisolid medium. Between 9 and 12 days of dexamethasone treatment, there is a 5- to 6-fold increase of transforming growth factor alpha (TGF alpha) activity in the conditioned medium from NOG-8 ras cells. A 60-65% reduction in epidermal growth factor cell surface receptors on NOG-8 ras cells also occurs during this time interval. A 3- to 4-fold increase of the expression of a specific TGF alpha mRNA can be detected within 2 days of dexamethasone treatment, preceding the increase in TGF alpha protein found in the conditioned medium. Exogenous TGF alpha is able to stimulate in a dose-dependent fashion the anchorage-dependent and anchorage-independent growth of NOG-8 ras cells to a level comparable to that observed in dexamethasone treated ras-transformed NOG-8 ras cells. These results suggest that the enhanced expression of TGF alpha after induction of an activated ras protooncogene may be necessary for the anchorage-independent growth and subsequent morphological changes and the enhanced growth rate observed in ras-transformed mammary epithelial cells.

Animals↗

Transforming growth factor-beta expression in fibropapillomas induced by bovine papillomavirus type 1, in normal bovine skin, and in BPV-1-transformed cells.

There is substantial evidence to suggest that transforming growth factor-beta (TGF-beta) plays an important role in wound healing and tissue repair as well as in carcinogenesis. It has also been observed that naturally occurring bovine papillomavirus type 1 (BPV-1)-induced bovine fibropapillomas occur predominantly at traumatized sites of the body, suggesting that humoral factors released in wounds might be important for papillomavirus infection. We have therefore investigated the possible role of TGF-beta 1 in BPV-1 infections. Two antipeptide antibodies which recognize different epitopes in the N-terminus of TGF-beta 1 were used to localize TGF-beta 1 in bovine fibropapillomas and normal bovine skin using immunohistochemical methods. Staining by anti-LC(1-30) is intracellular in suprabasal keratinocytes of the epidermis as well as the hair follicles and sebaceous glands and correlates with known sites of TGF-beta 1 mRNA synthesis. Anti-CC(1-30) staining is extracellular in the immediately underlying dermis. Neither the pattern nor intensity of TGF-beta 1 staining was affected by BPV-1 infection. C127 cells and BPV-1-transformed C127 cells were compared for TGF-beta 1 mRNA expression and secretion of TGF-beta 1 peptide. Although the levels of messenger RNA and secreted TGF-beta 1 peptide were similar in both cell types, five- to six-fold greater amounts of TGF-beta-like activity per cell was detected in media conditioned by the uninfected cells. TGF-beta 1 treatment had no effect on the growth rate of either cell type or on BPV-1 gene expression in the transformed cells.

Animals↗

Dominant suppression of adenovirus mediated transformation and insufficiency of p105Rb binding as a condition for oncogenic transformation.

An adenovirus-specific transformation resistant cell line (G2) expressing biologically active E1a proteins and originally isolated as a revertant from Ad2-transformed rat cells (F4), was shown to form stable Rb-E1a and 300K-E1a complexes in immunoprecipitation experiments. Consistent with the transformation resistant phenotype, cell hybrids between G2 and F4 were all nontumorigenic. Retrovirus insertion mutagenesis resulted in tumorigenic cell lines and identified a common locus responsible for the E1a-specific dominant tumor suppressor phenotype of G2 cells.

Adenoviruses, Human↗

Secretion, glycosylation, and phosphorylation of rat-specific, transformation-associated proteins in Moloney murine sarcoma virus-transformed rat cells.

Previously, we detected, by monoclonal antibody, three Moloney murine sarcoma virus (Mo-MSV)-activated intracellular transformation-associated proteins (TAP), (P66, P63, and P60) in several Mo-MSV-transformed rat cell lines (Chan et al., Biochem. Biophys. Res. Commun., 134: 1223-1230, 1986) and found the release of TAP into the extracellular medium with a change from three intracellular polypeptides to two extracellular polypeptides (P68 and P64) (Li et al., Virology, 156: 91-100, 1987). Since then, we have further analyzed TAP in terms of their secretion, glycosylation, and phosphorylation in a temperature-sensitive Mo-MSV-transformed normal rat kidney (NRK) cell line, the 6M2 line, and found these results. Extracellular TAP were detected by immunoblotting and immunoprecipitation techniques as two polypeptides (P68 and P64). The secretion of TAP was rapid, with a 50% secretion rate of 78 min. Both intracellular (except for P63) and extracellular TAP were glycosylated. As a result of inhibition of glycosylation by the antibiotic tunicamycin, a fourth intracellular TAP (P58) and a third extracellular TAP (P60) were found. The new results suggest that the intracellular TAP were probably changed from four polypeptides (P66, P63, P60, and P58) to three (P66, P63, and P60) during the glycosylation process. Likewise, the three extracellular TAP were changed to two (P68 and P64) as a result of further glycosylation and subsequent secretion into the extracellular medium. Inhibition of glycosylation by tunicamycin (0.5 microgram/ml) reduced the TAP secretion rate by about 39%. Extracellular TAP (P68 and P64) as well as P85gag-mos and P58gag were found to be phosphoproteins.

Animals↗

A high-molecular-weight PDGF-like factor secreted by v-sis transformed cells leads to growth stimulation and transformation.

Transformation by the v-sis containing simian sarcoma virus (SSV) is believed to be mediated by constitutive production of a platelet-derived growth factor (PDGF)-like molecule and its interaction with the PDGF receptor. SSV-transformed nonproducer NRK cells release into their tissue culture supernatant a high-molecular-weight factor (150-300 kDa) with biological activities closely resembling those of human PDGF. The partially purified high-molecular-weight factor competes with 125I-PDGF for receptor binding and leads to growth stimulation and anchorage-independent growth of normal cells. All these activities can be blocked by antibodies against PDGF and SSV-NP cells. The formerly described SSV transformation-specific glycopeptide (Thiel and Hafenrichter, 1984), now termed gp200sis, is recognized by the same specific antibodies and shows a striking resemblance in size. We conclude that gp200sis is responsible for the described PDGF-like activities.

Animals↗

Transforming activity of crude extract of pseudorabies virus-transformed cells.

Crude extract of pseudorabies virus (PRV)-transformed human (H-PR-1) cells induced transformation in human embryonic lung (HEL) cells. When the extract was removed, the acquired cell morphology remained unchanged, but the saturation density of cells was decreased. The transforming effect of the extract was neutralized with anti-PRV IgG.

Cell Transformation, Viral↗

[Genetic transformation of somatic cells. I. Clone of Chinese hamster cells defective in thymidine kinase and characterized by high transformation efficiency].

A characteristics is given of clone A238 of the Chinese hamster cells deficient in thymidine kinase (TK). The isolation procedure is described. Upon transformation with the aid of DNA of plasmids, containing thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1) clone A238 cells show frequency (7.10(-5) and efficiency (130 TK+ colonies per 1 microgram of plasmid DNA) compatible with those of mouse line LMtk- cells. Modified transformation and selection conditions of clone A238 cells expressing TK-gene of HSV1 are demonstrated. A simple method is described for discriminating somatic cells, expressing either their proper or a virus TK-gene according to the cloning efficiency of cells on the HAT medium containing thymidine in concentration 100 micrograms/ml. It is shown that at the fixed total DNA concentrations a complete replacement of the eukaryotic carrier DNA for the plasmid DNA, containing no tg gene of HSV1, decreases but only insignificantly the frequency and efficiency of transformation.

Animals↗

Induction of anchorage-independent growth by epidermal growth factor and altered sensitivity to type beta transforming growth factor in partially transformed rat kidney cells.

A partially transformed cell line (NRK-PT14) was isolated from normal rat kidney (NRK) cells. Like NRK cells, NRK-PT14 cells required epidermal growth factor for anchorage-independent growth, but lost the additional requirement for exogenous type beta transforming growth factor (TGF-beta). Compared to NRK cells, NRK-PT14 cells did not secrete elevated levels of TGF-beta, but exhibited an altered response to this growth factor. Monolayer growth of NRK cells in a serum-free medium was inhibited by TGF-beta, whereas growth of NRK-PT14 cells was stimulated by TGF-beta. In addition, TGF-beta stimulated epidermal growth factor binding to high affinity sites in NRK cells, but decreased epidermal growth factor binding to NRK-PT14 cells during growth of the cells in serum-free medium. These qualitative changes in the response to TGF-beta may be representative of an intermediate stage in the spontaneous transformation of NRK cells.

Animals↗

Purification and biological properties of type beta transforming growth factor from mouse transformed cells.

Transforming growth factor type beta (TGF beta) has been purified from serum-free culture fluids of transformed mouse L-929 cells which are capable of continual growth in serum-free medium in the absence of any exogenously added polypeptide growth factors. TGF beta has been purified to homogeneity as judged by NH2-terminal amino acid sequence analysis. Analysis of the purified polypeptide by gel electrophoresis indicates that TGF beta is composed of two polypeptide chains of Mr 12,500 cross-linked by disulfide bonds. TGF beta was characterized by its ability to induce anchorage-dependent normal rat kidney (NRK) cells to grow in soft agar in the presence of epidermal growth factor (EGF). TGF beta was also able to enhance both EGF-induced DNA synthesis and cell proliferation on growth-arrested NRK cells in monolayer cultures under serum-free conditions. We also show that in mouse melanoma B-16 cells under serum-free conditions TGF beta stimulates both DNA synthesis in monolayer cultures and anchorage-independent growth in soft agar. Paradoxically, the anchorage-independent growth in the presence of serum of many human cell lines, including melanomas, and mammary, prostatic, vulvar, and lung carcinomas is inhibited by TGF beta at saturating concentrations similar to those that stimulate colony formation of the rodent cell lines L-929 and B-16 under serum-free conditions. The peculiar action of TGF beta is further revealed by the observations that while EGF and TGF beta synergize to induce inhibition of anchorage-independent growth of A-431 human vulvar carcinoma cells, their effects on the anchorage-independent growth of one human lung carcinoma cell line (A-549) and two human prostatic carcinoma cell lines (PC-3 and DU-145) are antagonistic. Moreover, we show that in the rodent and human cell lines TGF beta interacts with specific cellular receptors which may mediate the actions of TGF beta. We conclude that the expression of both TGF beta and TGF beta receptors by L-929 cells and the stimulation of growth of L-929 cells in serum-free medium by TGF beta suggests that TGF beta may be important for maintaining the transformed state of this tumor cell line, and the way in which a cell responds to TGF beta is dependent on the presence or absence of growth factors contained in the serum.

Amino Acid Sequence↗