Direct tumorigenic conversion of human gallbladder carcinoma cells by v-src but not by activated c-H-ras oncogene

The roles of activated ras and src oncogene products in the acquisition of fully neoplastic phenotype by human gallbladder adenocarcinoma cells were investigated by co-transfecting non-tumorigenic HAG-1 human gallbladder carcinoma cells with the pSV2neo plasmid and a plasmid carrying either activated c-H-ras or v-src oncogene. G-418-resistant clones were isolated and assessed for the acquisition of anchorage-independent growth potential. Neither the 10 established clones transfected with pSV2neo alone nor the 17 clones transfected with activated c-H-ras, including 4 clones expressing the mutated p21^H-ras protein, could form colonies in soft agar. By contrast, out of 10 clones transfected with v-src, 2 formed colonies in soft agar and produced tumors in athymic nude mice, the resulting progressive neoplasms being poorly differentiated adenocarcinomas. These tumorigenic clones were shown to have v-src DNA and mRNA levels with p60^v-src protein, but there were no significant chromosomal alterations following tumorigenic conversion. Moreover, herbimycin A, a selective src-kinase inhibitor, markedly reduced clonogenic growth of these cells in soft agar rather than monolayer growth, suggesting that anchorage-independent growth of the v-src-transformed HAG-1 cells might be driven directly by p60^v-src kinase activity. Taken together, our data suggest that the fully neoplastic conversion of HAG-1 cells depends on src-related tyrosine-kinase activity, but not solely on the function mediated by activated ras, thus providing evidence of an src-related signaling pathway for the acquisition of tumorigenic potential by human gallbladder adenocarcinoma cells. © 1995 Wiley-Liss, Inc.     

Inhibitors of anchorage-independent growth affect the growth of transformed cells on poly(2-hydroxyethyl methacrylate)-coated surfaces

We have described a microplate colorimetric assay to quantitate anchorage-independent cell growth, using plates coated with an anti-adhesive polymer poly (2-hydroxyethyl methacrylate) (polyHEMA). We investigated whether this method could be applied to human cancer cells of epithelial origin. HAG-I, a non-tumorigenic human gall-bladder carcinoma cell line, and its pSVneo and c-H-ras transfectants, which are also non-tumorigenic, did not grow on a polyHEMA-surface. In contrast, the v-src transformant which produced tumors in nude mice and formed colonies in soft agar, was able to proliferate on the coated surface, suggesting that tumorigenicity of human cancer cells correlates with the ability to grow on a polyHEMA-coated surface. We report on the feasibility of this method as a screening system for inhibitors of oncogenic transformation. Herbimycin A and radicicol, which have been reported to block Src function, suppressed the growth of v-src-transformed NRK and HAG-I cells on the non-adhesive polyHEMA-surface at concentrations significantly lower than on plastic. Differences in the inhibitory concentrations were not observed with KB cells, and cytotoxic agents such as adriamycin did not show any selectivity between the 2 surfaces. Growth of ras-transformed cells on the coated surface was selectively blocked by L-739,749, a farnesyltransferase inhibitor. The results demonstrate that compounds causing reversion of transformed cells to normal, hence, selectively inhibiting cell growth in anchorage-independent conditions, can be screened using this microtiter plate assay. © 1996 Wiley-Liss, Inc.     

Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidermal growth factor receptor in human gallbladder adenocarcinoma cells

Purpose: Although gefitinib, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, has been demonstrated to exhibit its antitumor activity by the blockade of EGF receptor, the role of signaling pathways downstream of EGFR in gefitinib sensitivity remains unknown. In this study, we investigated the mechanistic role of Src and Ras, major oncogene products implicated in the pathogenesis of many human cancers in gefitinib sensitivity. Methods: Using parental and v-src- or c-H-ras-transfected HAG-1 human gallbladder adenocarcinoma cell lines, effects of gefitinib on cytotoxicity, cell cycle purtubation and apoptosis, and tyrosine phosphorylation of EGFR, Akt, and Erk were determined by WST-1 assay, flow cytometry, and Western blots, respectively. Results: Activated Ras and Src conferred a strong resistance to gefitinib by nearly 30-fold and 200-fold, respectively. Gefitinib induced accumulation of cells in the G0/G1 phase of the cell cycle at 24-h, with progressive expansion of apoptotic cell population in parental HAG-1 cells, but these effects were completely abolished in v-src- or c-H-ras-transfected cell line. Upon gefitinib treatment, EGFR activation and subsequent downstream activation through Erk and Akt were significantly inhibited in HAG-1 cells. By contrast, gefinitib failed to inhibit the activation of both Akt and Erk in v-src-transfected cells and Erk, but not Akt in c-H-ras-transfected cells, despite the blockade of EGFR activation in these respective cell lines. Treatment of v-src-transfected cells with herbimycin A, a Src tyrosine kinase inhibitor, partially reversed the gefitinib resistance, with concomitant inhibition of Akt and Erk. Conclusion: Our results suggest that activated Ras and Src could induce gefitinib resistance by activating either or both of Akt and Erk signaling pathways, thus providing a strategic rationale for assessment of these specific signaling molecules downstream of EGFR to customize treatment.Baoli Qin and Hiroshi Ariyama contributed equally to this work.     

Association of viral oncogene-induced changes in gap junctional intercellular communication and morphological transformation in BALB/c3T3 cells

In order to study the relationship between altered gap junctionalintercellular communication (GJIC) and induction of cell transformationby oncogenes, we transfected six viral oncogenes into BALB/c3T3A31-1-1 cells. BALB/c3T3 cells with v-src, v-ras or polyomamiddle T (PyMT) genes grew in soft agar and formed distincttransformed foci in the absence or presence of a vast excessof non-transfected cells. On the other hand, those with v-myc,v-fos or polyoma large T (PyLT) genes expressed less distinctlytransformed phenotypes (less transformed morphology, highersaturation density than non-transfected counterparts and lessgrowth in soft agar), and did not form distinct foci in coculturewith non-transformed cells. When their homologous GJIC capacitieswere examined by the microinjection/dye transfer assay, no decreasein GJIC was observed in any of the v-onc-transformed cells.Non-transformed and all v-onc-transformed cell lines expressedsimilar levels of connexin 43 mRNA. v-myc-, v-fos- and PyLT-transformedcells, but not v-ras-, v-src- and PyMT-transformed cells wereable to communicate heterologously with non-transformed cells.Tumor promoting phorbol esters strongly inhibited GJIC of non-transformedand all v-onc-transformed BALB/c3T3 cell lines. In coculturesof v-myc-, v-fos- or PyLT-transformed cells with non-transformedBALB/c3T3 A31-1-1 cells, 12-O-tetradecanoylphorbol-13-acetate(TPA) increased the number of transformed foci. However, whenthese v-onc-transformed cells were co-cultured with non-transformedBALB/c3T3 A31-1-13 cells (which lose GJIC at growth confluence,as if TPA had been added), no morphologically transformed fociappeared. These results suggest that factors other than GJICare involved in the suppression of oncogene-transformed cellsby surrounding normal counterparts.     

Comparison of three recombinant murine leukemia viruses carrying the v-src oncogene of avian sarcoma virus: Differences in in vitro transformation and in vivo pathogenicity

We previously described a recombinant Moloney murine leukemia virus (Mo-MuLV) carrying the v-src oncogene, Mo-MuLV(src). Mo-MuLV(src) encodes a gag-src fusion protein, transforms cells in culture, and induces fibrosarcomas in vivo. To compare transforming properties of the gag-src fusion protein to pp60^src encoded by Rous sarcoma virus, we constructed a new recombinant virus, Mo-MuLV(+src). Mo-MuLV(+src) encodes pp60^src in the context of Mo-MuLV. Cells transformed by Mo-MuLV(+src) were round and formed colonies in soft agar, whereas Mo-MuLV(src)-infected cells were fusiform and did not grow in suspension. Thus, the extent of transformation induced by Mo-MuLV(+src) was greater than that induced by Mo-MuLV(src). Subcutaneous inoculation of either virus into neonatal NIH Swiss mice resulted in fibrosarcomas at the site of injection. Further studies indicated that tumors induced by Mo-MuLV(+src) grew rapidly but rarely metastasized. In contrast, tumors induced by Mo-MuLV(src) grew somewhat more slowly but metastasized with a high frequency (60%). These viruses may provide a useful model system for tumor metastasis. Another src-containing virus was also studied, MRSV (constructed by Anderson and Scolnick). MRSV also encodes pp60^src but in the context of amphotropic MuLV. When injected intravenously into six-week-old mice, MRSV induced splenomegaly and spleen foci but no solid tumors, as reported previously. In contrast, Mo-MuLV(src)-induced fibrosarcomas mostly in the spleen under the same inoculation protocol. These results suggest that the v-src oncogene was the major pathogenic determinant in neonatal mice for all three src-containing viruses; however, variations in the nature of the transforming protein modulated the behavior of the induced tumors. In adult mice, greater differences in pathogenicity were observed.     

Mechanisms of unusually high antioxidant activity of RSV-SR-transformed cells and of its suppression by activated p21ras

We have previously demonstrated that hamster embryo fibroblasts (HEFs) transformed by Rous Sarcoma virus, Schmidt-Ruppin strain (RSV-SR) are highly resistant to damage by H₂O₂ (H₂O₂^R), (in contrast to HEFs transformed spontaneously, or by bovine adenovirus and SV40), while N-ras transfection of RSV-SR transformants leads to suppression of pp60^v-src and of H₂O₂^R. In this study we have examined (1) mechanisms of antioxidant activity (AOA) of HEFs transformed by these agents and (2) the possible role of the v-src gene in unusually high AOA of RSV-SR transformants and of activated ras oncogenes in its suppression. All transformants exhibit increased catalase and glutathione peroxidase (GP) activities, while SOD, glutathione and glutathione reductase (GR) were reduced. As compared with other transformants, the significantly higher catalase and the low SOD activities were characteristic of RSV-SR-transformants, while an increase in GP was observed in all types of transformants. Correspondingly, RSV-SR-transformants showed an extremely high H₂O₂-catabolizing activity (H₂O₂^CA) and no lipid peroxidation chain reaction (LPCR). N-ras-induced suppression of pp60^v-src of RSV-SR-transformed HEFs coincided with the suppression of catalase, GP, H₂O₂^R and H₂O₂^CA. However, suppression of catalase and GP was also observed in N-ras- and Ha-ras-transfected, spontaneously transformed HEFs. Thus, extremely high catalase activity and suppression of LPCR are apparently the main mechanisms of the unusually high H₂O₂^R of RSV-SR transformants, while its suppression by activated ras oncogenes may also take place in some transformants, free of v-src activity. © 1996 Wiley-Liss, Inc.     

A connexin 43 antisense vector reduces the ability of normal cells to inhibit the foci formation of transformed cells

Antisense gene constructs have been very useful in the functional analysis of genes and their products. In this report we used a connexin 43 (Cx43) antisense gene construct to study the role that heterologous gap-junctional intracellular communication (GJIC) plays in the ability of untransformed fibroblasts to suppress the foci-forming ability of src oncogene-transformed cells. Untransformed Rat-1 fibroblasts transfected with the Cx43 antisenese DNA construct showed marked decreases in Cx43 RNA and protein, which were accompanied by a corresponding decrease in GJIC. These Cx43 antisense-transfected cells maintained normal cell morphology, growth rates, and saturation densities and did not grow in soft-agar suspension. However, in coculture experiments, the Cx43 antisense cells were less effective than vector-alone-transfected, sense-trans-fected, and untransfected cells at in hibiting foci formation of pp60^v-src-transformed cells. These effects of junctionally competent, normal cells were associated with the existence of heterologous GJIC with the transformed cells and did not appear to result from the elaboration of a stable, diffusible inhibitory factor. Thus, gap-junction-mediated transfer of putative regulatory molecules may play a role in the ability of untransformed cells to suppress the expression of certain properties of transformed cells. ©1994 Wiley-Liss, Inc.     

Effects of tyrosine kinase inhibitors on the proliferation of human breast cancer cell lines and proteins important in the RAS signaling pathway

Breast cancers frequently over-express a number of growth factor receptors. In addition, elevated src family kinase activity is present in a percentage of these neoplasms and has been implicated in signal transduction in these cells. Therefore, inhibiting tyrosine kinase activity is a potential approach for treating these tumors. Utilizing the SKBR3 and MCF-7 breast cancer cell lines, we evaluated the effects of broadly targeting growth factor receptor and cytoplasmic tyrosine kinases with tyrosine kinase inhibitors (herbimycin A and genistein) to inhibit proliferation. We also evaluated these inhibitors' effects on proteins that regulate ras function, which is a convergence point for signaling through both src family kinases and a number of growth factor receptors with tyrosine kinase activity (e.g., epidermal growth factor and erbB-2 receptors). We specifically evaluated whether these compounds affected 2 recently discovered proteins involved in controlling ras function: Shc, which is tyrosine-phosphorylated by src and activated growth factor receptors, and Grb-2, which mediates signal transduction from activated growth factor receptors through ras. We evaluated their effects on tyrosine phosphorylation of Shc, binding of Grb-2 to Shc and MAP kinase activity. Both cell lines were inhibited in a dose-dependent manner by each compound. This was accompanied by decreased Shc tyrosine phosphorylation, Shc's association with Grb-2 and MAP kinase activity. Thus, tyrosine kinase inhibitors can inhibit proliferation of breast cancer cells, accompanied by inhibition of signal transduction steps potentially mediated through ras. Tyrosine kinase inhibitors might, therefore, be useful for the treatment of breast cancer. © 1996 Wiley-Liss, Inc.     

IFNγ induction of p21WAF1 in prostate cancer cells: Role in cell cycle,alteration of phenotype and invasive potential

Type I and type II interferons (IFNs) are known to exert antitumor effects on a variety of tissues and cell types. We have previously shown that the type I IFN IFNα induces the expression of the cyclin-dependent kinase inhibitor p21^WAF1 and inhibits the cell cycle of the human prostate adenocarcinoma cell line, DU145, that carries mutations in the tumor suppressor gene products p53 and pRB. We now show that the type II IFN IFNγ similarly induces the expression of p21^WAF1 and inhibits the cell cycle of DU145 cells. In addition, we show that while both IFNs exert antiproliferative activity, only IFNγ induced phenotypic changes in these cells that accompanied the antiproliferative effect. For example, IFNγ, but not IFNα, caused a significant reduction in epidermal growth factor receptor expression as well as an increase in the adhesion molecules intercellular adhesion molecule-1 and integrin α3. These phenotypic changes in DU145 cells are suggestive of the acquisition of a non-tumorigenic state. Consistent with these findings, IFNγ showed a significantly lower invasive ability in in vitro assays using invasion chambers. Thus, IFNγ inhibits both the cell cycle and the metastatic potential of DU145 cells independent of the p53 and RB status, and our data describe a mechanism for mediating the antitumor capabilities of IFNγ that bypasses tumor suppressor genes like p53. Int. J. Cancer77:138–145, 1998.© 1998 Wiley-Liss, Inc.     

c-myc Gene activation as a permanent trait of RSV-infected quail cells

A high level of c-myc gene expression was found to be a constant feature of v-src transformation. The c-myc gene product was analyzed in quail embryo cells transformed by different mutants of Rous sarcoma virus (RSV) that were temperature-sensitive with respect to various parameters of v-src function. The high-level expression of c-myc proved not to be temperature-sensitive: At both permissive (35°C) and non-permissive (41°C) temperatures, the same high levels of c-myc gene product were found for all RSV mutants tested. This result, in agreement with earlier evidence for a v-src-induced proliferative stimulus which was unaffected by ts mutants at the non-permissive temperature, shows that certain v-src functions have not yet been fully characterized.     

Different metastatic potentials of ras- and src-transformed BALB/c 3T3 A31 variant cells

The metastatic phenotype of tumor cells is thought to be induced by an aberrant signaling cascade or cascades that are different from those required for tumorigenicity. Oncogene-transfected cells with different tumorigenicities and metastatic potentials have been used to identify such pathways and responsible molecules. However, oncogenes that can induce tumorigenicity in recipient cells also frequently induce the metastatic phenotype at the same time. The difficulty in obtaining cell lines that are tumorigenic but not metastatic has hampered such studies. In this report, we transfected the activated c-Ha-ras oncogene into BALB/c 3T3 A31 variant cells and found that the transfectants were tumorigenic but they did not form metastatic lung nodules in the experimental metastasis assay. The phenotype was very stable and was maintained during cultivation. On the other hand, the metastatic potentials of either the transfected cells or the original variant cells could be induced by transfection of the v-src oncogene. The src transfectants formed extensive nodules in lung when injected into the tail veins of congeneric mice. The cell motility of the metastatic src transfectants on Matrigel-coated dishes was greater than that of the ras transfectants. The src transfectants were also invasive in Matrigel when analyzed on a filter. These variant cells transformed by the ras and src oncogenes will be a useful system for identifying the signaling cascades responsible for the metastatic potential of tumors. © 1996 Wiley-Liss, Inc.     

Sequence variation in the SRC gene product affects metastasis formation: The central,but not exclusive,role of the tumor immune response

Sequence variation in the src gene product could, in principle, influence metastasis formation through either of 2 effects: an alteration in tumor antigenicity or a non-immune-mediated change in one or more src-associated functions. Our present results establish that both mechanisms underlie the difference in relative levels of metastasis formation induced by the v-src vs. the c-src(527) oncogene. A point that emerges from this analysis is the segregation, within a chicken line genotypically uniform at the major histocompatibility (B) complex (MHC), of a phenotype defined by strong resistance to secondary v-src-induced tumor challenge. The pattern of segregation is consonant with the possibility that a gene unlinked to the MHC governs immune response levels to v-src-encoded tumor antigen. © 1996 Wiley-Liss, Inc.     

Cellular pharmacology ofd-3-azido-3-deoxy-myo-inositol,an inhibitor of phosphatidylinositol signaling having antiproliferative activity

d-3-Azido-3-deoxy-myo-inositol (3AMI) is an inhibitor of the growth of v-sis-transformed NIH 3T3 cells but not of wild-type NIH 3T3 cells, whose effects may be mediated through the phosphatidylinositol-3-kinase pathway. We studied some properties of the cellular pharmacology of 3AMI using high-specific-activity [³H]-3AMI. The uptake of [³H]-3AMI by wild-type NIH 3T3 and v-sis NIH 3T3 cells was similar. [³H]-3AMI was a substrate for phosphatidylinositol synthetase, with the maximal velocity (V_max) being 1.0 nmol min^–1 mg^–1 and the Michaelis constant (K _m) being 23 mM. Corresponding values obtained for [³H]-myo-inositol as a substrate were 5.5 nmol min^–1 mg^–1 and 3.2 mM. [³H]-3AMI was incorporated into the cellular inositol lipids of v-sis NIH 3T3 cells to a similar extent as that observed for [³H]-myo-inositol but was not incorporated into the inositol lipids of wild-type NIH 3T3 cells. The [³H]-3AMI incorporated by the v-sis NIH 3T3 cells was present in the phosphatidylinositol and phosphatidylinositol phosphate fractions but not in bisphosphorylated phosphatidylinositol.myo-Inositol antagonized the growth-inhibitory effects of 3AMI. The v-sis NIH 3T3 cells were found to be more sensitive than the wild-type NIH 3T3 cells to growth inhibition (without 3AMI) caused by the removal ofmyo-inositol from the medium. The results of the study suggest that 3AMI is an antimetabolite ofmyo-inositol. The relative sensitivity of v-sis NIH 3T3 and some other cells to 3AMI may be a reflection of increasedmyo-inositol requirements for the growth of these cells as compared with wild-type NIH 3T3 cells.Abbreviations 3AMI d-3-azido-3-deoxy-myo-inositol - 3AmMI d-3-amino-3-deoxy-myo-inositol - PIPLC phosphoinositide-selective phospholipase - C PtdIns(4,5)P₂, phosphatidylinositol(4,5)bisphosphate - Ins(1,4,5)P₃ myo-inositol(1,4,5)trisphosphate - Ins(1,3,4,5)P₄ myo-inositol(1,3,4,5)tetrakisphosphate - PKC protein kinase C - PtdIns3 K phosphatidylinositol-3-kinase - PDGF plateletderived growth factor - EGF epidermal growth factor - CDP diglyceride, cytidine dipalmitoyl diphosphodiglyceride - DMEM Dulbecco's modified Eagle's medium - PBS Dulbecco's phosphate-buffered saline - HCS heat-inactivated calf serum - IC₅₀ concentration required to cause 50% inhibition - PtdIns synthetase; phosphatidylinositol synthetase - DAG diacylglycerol     

Synergistic anti-tumor effects with co-expression of GM-CSF and IFN-γ in murine tumors

We explored the potential therapeutic benefit of introducing GM-CSF, IFN-γ or a combination of both factors into CT26 tumor cells. CT26 cells secreting either GM-CSF or IFN-γ exhibited delayed tumorigenicity; however, cells expressing both GM-CSF and IFN-γ did not form tumors. Even when wild type CT26 cells were introduced into a distant site of mice that had been inoculated with CT26/GM-CSF/IFN-γ cells, no tumors were generated. Furthermore, when we injected GM-CSF + IFN-γ cells into animals bearing established tumors, the tumors were either rejected or their development was delayed, suggesting that synergistic effects were induced against these tumors via a systemic immune response. Histopathological examination of the tumors injected with cells expressing GM-CSF and IFN-γ combined showed necrosis and few signs of malignancy. The growth of tumors from mice treated with CT26/GM-CSF/IFN-γ cells exhibited a delay in tumor formation and no effects were seen in athymic nude mice, which are deficient in T lymphocytes, or in splenectomized nude mice, which are deficient in natural killer (NK) cells, respectively. Our data indicate a dual role for T and NK cells in mediating the anti-tumor activity of this therapy. Our results suggest that transduction of tumor cells with both GM-CSF + IFN-γ results in a powerful synergistic effect of the 2 cytokines that is of greater therapeutic benefit than transduction with either cytokine alone. Int. J. Cancer 77:907–912, 1998.© 1998 Wiley-Liss, Inc.     

D-3-Deoxy-3-substitutedmyo-inositol analogues as inhibitors of cell growth

Summary A number of unnaturald-3-deoxy-3-substitutedmyo-inositols were synthesized and their effects on the growth of wild-type NIH 3T3 cells and oncogenetransformed NIH 3T3 cells were studied. The compounds were found to exhibit a diversity of growth-inhibitory activities and showed selectivity in inhibiting the growth of some transformed cells as compared with wild-type cells. Remarkably,d-3-deoxy-3-azido-myo-inositol exhibited potent growth-inhibitory effects toward v-sis-transformed NIH 3T3 cells but had little effect on the growth of wildtype cells. The growth-inhibitory effects of themyo-inositol analogues were antagonized bymyo-inositol. Since [³H]-3-deoxy-3-fluoro-myo-inositol was shown to be taken up by cells and incorporated into cellular phospholipids, we suggest that these unnaturalmyo-inositol analogues may act as antimetabolites in the phosphatidylinositol intracellular signalling pathways. Because cells transformed by oncogenes often exhibit elevated phosphatidylinositol turnover, the inhibition of signalling pathways that mediate oncogene action could offer new opportunities for controlling the growth of cancer cells.     

Effects of interferon-α on human b cells: Repression of apoptosis and prevention of cell growth are independent responses of burkitt lymphoma lines

We have previously shown that interferon-α (IFN-α) can repress apoptosis in Burkitt lymphoma (BL) cells. In this study, we have compared this protective response with a further, well-established effect of IFN-α on BL cells, that of growth arrest. Of a panel of BL lines comprising (i) EBV-positive and -negative lines that retain the phenotype of the parental tumour cells and (ii) the prototype IFN-α-growth-inhibited line, Daudi, only Daudi cells were found to undergo substantial growth inhibition in response to the cytokine. By contrast, all lines, with the notable exception of Daudi, were protected by IFN-α from high-rate apoptosis initiated by the Ca²⁺ ionophore ionomycin. lonomycin failed to elicit an IFN-α-repressible apoptotic response in either wild-type Daudi cells or IFN-resistant sublines that were refractory to the growth-arresting effects of the cytokine. Analysis of c-myc protein levels confirmed previous observations that repression of apoptosis in IFN-α-rescuable BL cells was associated with an early inhibition of myc that was followed by a return to high-level expression. Significantly, ionomycin alone induced a comparable transient inhibition of myc protein in Daudi cells. In Daudi cells, but not in IFN-α-rescuable BL cells, renewed expression of myc observed after the early, transient down-regulation was followed by sustained down-regulation of the protein, which paralleled growth arrest. Our results indicate that long-term growth arrest and repression of apoptosis in BL are distinct cellular responses to IFN-α. © 1995 Wiley-Liss, Inc.     

Growth suppression of transformed BALB/c 3T3 cells by transforming growth factor {beta}1 occurs only in the presence of their normal counterparts

Transforming growth factor ß1 (TGF-ß1) enhancesthe yield of transformed foci of BALB/c 3T3 cells, but the continuouspresence of TGF-ß1 after foci formation inhibits thegrowth of transformed foci. The focus-forming ability of Ha-ras-,v-src-and PyMT-transformed cells growing on a monolayer of non-transformedcells was completely suppressed by TGF ß1, whereasgrowth of the transformed cells was little inhibited by TGF-ß1in the absence of their normal counterparts. The inhibitionby TGF-ß1 of focus formation by transformed BALB/c3T3 cells on a normal cell monolayer remained when TGF-ß1was removed from the culture medium after 2 weeks. However,the transformed cells were not killed, since they grew in cultureconditions under which only transformed cells are able to grow(soft agar). These results suggest that TGF-ß1 suppressesgrowth of transformed cells in the presence of normal cells.Furthermore, when non-transformed cells were treated with TGF-ß1before co-culture with Ha-ras-transformed cells, formation oftransformed foci was inhibited. When normal and transformedcells were cultured in the same dish but separated physically,focus formation was still Inhibited. On the other hand, TGF-ß1enhanced the growth and changed the morphology of non-transformedcells only in the presence of transformed counterparts. Thegrowth inhibitory effect of TGF-ß1 on transformedcells and its growth stlmulatory effect on non-transformed cellsin co-culture conditions suggest the induction of reciprocalparacrine growth regulatory factors. As TGF-ß1 inhibitsthe growth of transformed BALB/c 3T3 cells only in the presenceof their normal counterparts, a paracrine negative growth controlmechanism appears to be operating.     

No evidence of correlation between mutation at codon 531 of src and the risk of colon cancer in Chinese

The protein tyrosine kinase activity of c-src proto-oncogene product, pp60^c-src, is elevated in a number of human cancers, including colon cancer. Phosphorylation of human pp60^c-src carboxy-terminal tyrosine 530 suppresses its kinase activity. A recent report suggested that the risk of colon cancer is higher for those who carry a C→T transition mutation on codon 531 (Gln-531→Amber-531) of src gene. This mutation caused a prematured translation termination and up-regulated the kinase activity. To examine whether this mutation could be a risk factor for colon carcinoma in the Chinese population, we used the same PCR-based assay to analyze src genotypes of 131 colon cancers and other various types of carcinoma. No mutation was detected in all specimens that were screened in this study. Thus, mutation at Gln-531 of src gene does not seem to be involved in the development of colon cancer in Chinese ethnicity.