Cooperative transforming activities of ras, myc, and src viral oncogenes in nonestablished rat adrenocortical cells.

Early-passage rat adrenocortical cells were infected with Kirsten murine sarcoma virus and MMCV mouse myc virus, two retroviruses carrying the v-Ki-ras and v-myc oncogenes, respectively. Efficient morphological transformation required coinfection with the two viruses, was dependent on the presence of high serum concentrations, and was not immediately accompanied by growth in soft agar. The doubly infected cells coordinately acquired the capacity for anchorage- and serum-independent growth during passage in culture. The appearance of such highly transformed cells was correlated with the emergence of a dominant clone, as suggested by an analysis of retrovirus integration sites. These results indicate that the concerted expression of v-Ki-ras and v-myc could induce rapid morphological transformation of nonestablished adrenocortical cells but that an additional genetic or epigenetic event was required to permit full transformation by these two oncogenes. In contrast, v-src, introduced by retrovirus infection in conjunction with v-myc, rapidly induced serum- and anchorage-independent growth. Therefore, the p60v-src protein-tyrosine kinase, unlike p21v-ras, is apparently not restricted in the induction of a highly transformed phenotype in adrenocortical cells. This system provides an in vitro model for the progressive transformation of epithelial cells by dominantly acting oncogenes.     

Effects of cellular transformation on expression of plasminogen activator inhibitors 1 and 2. Evidence for independent regulation

Expression of plasminogen activators (PA) has been reported to be associated with invasive tumor growth and increased metastatic ability. In order to delineate changes in PA and PA inhibitor (PAI) expression that accompany cellular transformation, we studied oncogene-containing variants of the Rat-1 cell line. We report here that transfection of the oncogenes v-src, erbB, c-myc, v-myc, N-myc, and EJras into these cells does not result in detectable PA activity in conditioned media or cell extracts. In addition, Northern blot analysis fails to demonstrate urokinase mRNA in Rat-1 cells or transfectants. Moreover, cells transformed by EJras and v-src but not other oncogenes secrete an active placental-type PAI, PAI-2. Using inducible EJras constructs, we find that increased PAI-2 gene expression is detectable within 6-12 h after treatment with the inducing agent. Peak expression of PAI-2 mRNA is increased 10-15-fold over base line, and high levels are maintained for at least 72 h. In contrast to the results with PAI-2, secretion of endothelial-type PAI-1 into conditioned media is sharply down-regulated by several oncogenes. Thus, we have found that PAI-1 and PAI-2 are independently regulated in transformed variants of Rat-1 cells. The specific induction of PAI-2 in cells transformed by oncogenic ras and src suggests that this protease inhibitor may have a previously unsuspected role in malignancy.     

Lymphocytes infiltrating human ovarian tumors. I. Role of Leu-19 (NKH1)-positive recombinant IL-2-activated cultures of lymphocytes infiltrating human ovarian tumors

Tumor-infiltrating lymphocytes (TIL) were obtained from human ovarian tumors, expanded in the presence of IL-2 in culture and studied for cytotoxicity against fresh autologous and allogeneic ovarian carcinoma (CA) targets. TIL from ovarian tumors grew well in long term cultures, achieving from 8- to 682-fold expansion. TIL cultured with IL-2 were cytotoxic against both autologous and allogeneic fresh ovarian CA targets, and no specificity for autologous tumor could be demonstrated in any of the cultures. In all fresh TIL preparations, CD3+ lymphocytes were the major cell type and contained a high proportion (up to 51%) of activated (IL-2R+) cells as determined by two-color flow cytometry. Sorting of bulk TIL cultures followed by cytotoxicity assays identified the Leu-19+ cells, both CD3+ and CD3-, as effectors of cytotoxicity against autologous and allogeneic tumor cell targets. Cold target inhibition assays showed that allogeneic targets (both ovarian CA and a sarcoma) competed effectively with autologous ovarian CA targets for Leu-19+ effectors in TIL cultures. mAb to Leu-19 or Leu-2a did not block lysis of autologous targets by sorted effectors. OKT3 antibody augmented lysis of autologous targets by CD3+Leu-19- effectors only. These results show that non-MHC-restricted Leu-19+ effectors in cultures of TIL with 1000 U/ml of rIL-2 mediate lysis of autologous and allogeneic tumor cells. The CD3+Leu-19- cells, the main population in these cultures, do not mediate tumor lysis. To determine the phenotype of antitumor effectors in IL-2 cultures of TIL, cell sorting followed by functional assays are necessary.     

C127 cells resistant to transformation by tyrosine protein kinase oncogenes

C127 is a nontumorigenic mouse cell line widely used in in vitro transformation assays due to its normal morphological appearance and its very low levels of spontaneous transformation. We now report that C127 cells are resistant to transformation by tyrosine protein kinase oncogenes derived from growth factor receptors such as the retroviral v-fms and the human trk transforming genes. In contrast, these cells could be efficiently transformed by members of the ras oncogene family and by serine/threonine kinase oncogenes such as v-mos and v-raf. C127 cells were also found to be resistant to transformation by v-src, the prototype of a large family of tyrosine protein kinase oncogenes whose products are associated with the inner side of the plasma membrane. However, morphologically normal C127 cells expressing pp60v-src acquired a transformed phenotype upon continuous passage in vitro. Somatic cell hybrids (neoR, hygroR) obtained by fusion of G418-resistant C127 cells expressing p70trk (neoR) and hygromycin-resistant NIH3T3 cells (hygroR) exhibited transformed properties as determined by their ability to grow in semisolid agar. In contrast, no such growth was observed when these neoR p70trk-containing C127 cells were fused to control hygroR C127 cells. These results indicate that C127 cells may either lack or express insufficient levels of certain critical substrate(s) necessary for the onset of transformation by tyrosine protein kinase oncogenes.     

Malignant transformation of an epithelial cell by v-Src via tv-a-mediated retroviral infection: a new cell model for studying carcinogenesis

Most human cancers are of epithelial origin, but many cell culture models for the study of cancer-causing genes use fibroblasts. In addition, efficient delivery and stable expression of foreign genes into non-transformed cell lines are often difficult. To address both questions, we here established a non-transformed rat kidney epithelial RK3E cell line that constitutively expresses tv-a (receptor for subgroup A avian leukosis virus, ALV) for delivery of foreign genes via avian retroviral infection. This cell line (RK3E/tv-a) allows efficient and stable expression of either single or multiple foreign genes. Furthermore, tv-a-mediated delivery of various oncogenes (v-src, H-ras, myc or akt) leads to malignant transformation. v-src-transformed cells exhibited classical cancerous phenotypes in vitro, and induced tumor formation and lung metastasis upon injecting into immunodeficient mice. Expression profiles of downstream molecular effectors (E-cadherin, beta-catenin, cyclin D1, Myc, VEGF, MMP-2, and MMP-9) in these cells correlate with characteristics of cancerous phenotypes. This new cell model serves as a useful tool to study cancer-causing genes in epithelial cell type.     

Differential response to retinoic acid of Syrian hamster embryo fibroblasts expressing v-src or v-Ha-ras oncogenes.

It has been shown that treatment of many but not all tumor cell lines with retinoids affects cell proliferation and expression of the transformed phenotype. To determine whether the response of the tumor cell to retinoids is influenced by specific oncogenes activated in the cell, we studied the action of these agents in the immortal, nontumorigenic Syrian hamster embryo cell lines DES-4 and 10W transfected with either v-Ha-ras or v-src oncogenes. In this paper we show that in transformed DES-4 cells expressing v-src, retinoic acid inhibited anchorage-independent growth, reduced saturation density, and inhibited the induction of ornithine decarboxylase by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, retinoic acid enhances the expression of the transformed phenotype in DES-4-derived cells that express v-Ha-ras. In these cells retinoic acid increases the number and the average size of colonies formed in soft agar. Moreover, retinoic acid enhances ornithine decarboxylase activity and acts in a synergistic fashion with 12-O-tetradecanoylphorbol-13-acetate. These results indicate that oncogenes activated in cells can indeed influence the response of cells to retinoids. Retinoic acid does not appear to alter the levels of pp60src or p21ras proteins in these cells, suggesting that retinoic acid does not affect the synthesis of these oncogene products. Furthermore, retinoic acid does not affect the protein kinase activity of pp60src. Transformed cell lines derived from 10W cells responded differently, indicating that the presence of a specific oncogene is not the only factor determining the response to retinoids. Possible mechanisms by which retinoic acid may interfere with the expression of the oncogene products are discussed.     

Neoplastic transformation of normal and carcinogen-induced preneoplastic Syrian hamster embryo cells by the v-src oncogene.

The ability of cloned Rous sarcoma virus (RSV) DNA encoding the v-src oncogene to neoplastically transform normal, diploid Syrian hamster embryo (SHE) cells was examined. Transfection of RSV DNA into early passage SHE cells resulted in a low but significant number of tumors when treated cells were injected into nude mice. Tumors formed with a low frequency (two tumors out of ten sites injected) and only after a long latency period (14 weeks). In contrast to the normal SHE cells, several different carcinogen-induced preneoplastic immortal SHE cell lines were highly susceptible to transformation by the v-src oncogene to the neoplastic phenotype. Tumors formed with high efficiency and a short latency period (less than 3 weeks). Further studies were performed to determine the basis for the inefficient transformation of the normal SHE cells. NeoR clones isolated after cotransfection of SHE cells with pSV2-neo and RSV DNAs were neither morphologically altered nor immortal and did not contain detectable levels of the v-src gene product. These results suggest that neoplastic transformation by v-src DNA in the normal cells is initially suppressed. However, cells from a v-src-induced tumor expressed v-src RNA, and antibody to v-src protein precipitated from the tumor cells a 60,000-molecular-weight protein which displayed protein kinase activity. Karyotypic analyses confirmed that the tumor was derived from Syrian hamster cells and suggested that it was clonal in nature. These results indicate that the v-src oncogene was primarily responsible for neoplastic transformation of SHE cells. In contrast to the results with the v-src oncogene, our previous studies showed that v-Ha-ras oncogene alone is unable to induce neoplastic transformation of SHE cells. Furthermore, the v-myc oncogene was able to compliment v-Ha-ras to neoplastically transform SHE cells, while cotransfection with v-src plus v-myc did not increase the incidence of tumors.     

Studies on the changes in calcium fluxes in ts-RSV LA 90 cells transformation

To study the role of Ca2+ fluxes and [Ca2+]i in cell transformation by the v-src gene, ts-RSV LA 90 cells was used in this experiment. Ca2+ fluxes across the plasma membrane was measured with radioisotopes. The relative [Ca2+]i in LA 90 cells loaded Indo-1AM was measured by computer-based Optical Multichannel Analyzer connected with fluorescence microscopy. It was observed that changes in rate of Ca2+ fluxes across the plasma membrane are one of the earliest detectable changes of LA 90 cells transformation. Rates of Ca2+ fluxes in transformed LA 90 cells (40 degrees C) is higher than that in normal LA 90 cells (33 degrees C) and rates of Ca2+ fluxes increased in 25 minutes when LA 90 cells shifted from nonpermissive (40 degrees C) to permissive (33 degrees C) temperature. TMB-8 inhibited increases in rate of Ca2+ efflux induced by pp 60 v-src, and increase in rate of Ca2+ efflux in normal LA 90 cells was stimulated by calf serum. The rate of Ca2+ efflux was related to the changes in temperature. The increase in rate of Ca2+ influx induced by pp 60 v-src could be blocked by verapamil. The rate of Ca2+ influx was not affected by the changes in temperature. The increase in relative [Ca2+]i induced by pp 60 v-src is one of the early events in the transformation process. The level of [Ca2+]i in transformed LA 90 cells was about 2-3 times as much as that in normal LA 90 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of herbimycin A and various SH-reagents on p60v-src kinase activity in vitro

Herbimycin A is an antiobiotic which reverses transformation caused by src family oncogenes. It inactivates p60v-src in vitro, possibly by binding to reactive SH-group(s) of the kinase. We examined effects of various SH-reagents on p60v-src and observed that N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM) or N-(9-acridinyl)maleimide (NAM) were potent inactivators of the kinase, whereas N-ethylmaleimide (NEM) required high concentrations, and iodoacetamide was totally ineffective in reducing the kinase activity. Pretreatment of p60v-src immune-complex with NEM and iodoacetamide, however, protected the kinase from inactivation by herbimycin A, BIPM, and NAM. The results suggest that SH-group(s) to which herbimycin A binds is not essential for the kinase activity, but is positioned in the vicinity of the active center.     

Transformation by viral and cellular oncogenes of a mouse BALB/3T3 cell mutant resistant to transformation by chemical carcinogens.

The mouse cell line MO-5 is resistant to transformation by various chemical carcinogens and also by UV irradiation (C. Yasutake, Y. Kuratomi, M. Ono, S. Masumi, and M. Kuwano, Cancer Res. 47:4894-4899, 1987). Northern (RNA) blot analysis showed active expression of ras and myc genes in MO-5 and BALB/3T3 cells. The effect of transfection of various oncogenes on transformation was compared in MO-5 cells and parental BALB/3T3 cells. Activated c-H-ras, c-N-ras, and v-mos gene induced transformation foci of MO-5 and BALB/3T3. Introduction of the polyomavirus middle T-antigen (mTag) or the Rous sarcoma virus-related oncogene v-src, however, efficiently transformed BALB/3T3 but not MO-5 cells. Expression and phosphorylation of mTag and the associated c-src proteins were observed in mTag-transfected clones of MO-5 as in BALB/3T3 and phosphorylation of the src protein was observed in v-src-transfected BALB/3T3 and MO-5 clones. Hybrids between mTag- or v-src-induced transformants of BALB/3T3 and untransformed MO-5 maintained the transformation phenotype, suggesting that no dominant suppressor of transformation exists in MO-5. A hybrid clone between BALB/3T3 and MO-5 induced efficient transformation foci after transfection with the mTag gene, suggesting that the deficient transformation phenotype of MO-5 was recessive. Instead, some other alteration of MO-5, plausibly membrane function, might lead to abortive transformation by chemical carcinogens and also by mTag and the v-src gene product.     

Blockage of morphological transformation of chick embryo fibroblasts infected with Rous sarcoma virus by N-myristoyl glycinal diethylacetal in vitro

N-Myristoyl glycinal diethylacetal strongly inhibited morphological transformation of chick embryo fibroblasts infected with a temperature-sensitive mutant (tsNY68) of Rous sarcoma virus. Myristoylated or nonmyristoylated pp60v-src, which were expressed in tsNY68-infected cells in the absence or presence of the compound, were identified separately by fluorography or immunoblotting analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]myristate-labeled cell lysate. The results taken together suggest that the blockage of morphological transformation was caused by prevention of protein myristoylation of the transforming protein pp60v-src.     

Coinfection with viruses carrying the v-Ha-ras and v-myc oncogenes leads to growth factor independence by an indirect mechanism.

The concomitant expression of certain oncogenes can transform normal diploid rodent cells into transplantable tumorigenic cells. The mechanism by which these oncogenes collaborate is unclear. Recent findings (M. Oshimura, T. M. Gilmer, and J. C. Barrett, Nature [London] 316:636-639, 1985) raise the possibility that karyotypic changes, including monosomy for chromosome 15, are required to induce tumorigenicity in Syrian hamster embryo cells transfected in vitro with v-Ha-ras and v-myc DNAs. We studied the effect of the oncogenes v-Ha-ras and v-myc, introduced by viral infection, on murine hematopoietic cells. The induction of growth factor independence by the two oncogenes was used as an in vitro correlate of tumorigenicity. After a period of reduced growth rate reminiscent of the growth rate of cells in crisis, the doubly infected cells became growth factor independent. These cells showed a great variability in their karyotypes.     

Suppression of the neoplastic phenotype by transfection of phospholipase C beta 3 to neuroendocrine tumor cells.

The expression of phospholipase C beta 3 (PLCB3) is low or absent in several neuroendocrine neoplasias. To investigate the role of PLCB3 in the neuroendocrine tumorigenesis, we transfected a PLCB3 construct to three neuroendocrine tumor cell lines with a low PLCB3 expression. The growth rate and tumorigenicity were assessed in vitro by [3H]thymidine incorporation and cell counting, in vivo, by xenografting to nude mice. In vitro, PLCB3 expressing clones showed a significant growth inhibition. The tumor weight was reduced for one of the two xenografted PLCB3-transfected cell lines and in both, a reduced number of proliferating (Ki-67 positive) cells was observed. This study implies an essential role for PLCB3 in the neuroendocrine tumorigenesis.     

The amino-terminal 14 amino acids of v-src can functionally replace the extracellular and transmembrane domains of v-erbB.

The retroviral oncogene v-erbB encodes a truncated form of the receptor for epidermal growth factor, an integral membrane protein-tyrosine kinase. By contrast, the oncogene v-src encodes a protein-tyrosine kinase that is a peripheral membrane protein. The morphologies and spectra of cells transformed by these two oncogenes differ. In an effort to identify the functional determinant(s) of these differences, we constructed and tested first deletion mutants of v-erbB and then chimeras between v-src and v-erbB. As reported previously, the absence of any membrane anchorage eliminated transformation by v-erbB. Anchorage of the cytoplasmic kinase domain of v-erbB to membranes with amino-terminal portions of the v-src protein permitted transformation. The phenotype and spectrum of transformation were those expected for v-erbB rather than for v-src. The transforming chimeras lost their biological activity if the signal for myristylation at the amino terminus of v-src was compromised by mutation. Biochemical fractionations revealed a correlation between transforming activity and the association of chimeric gene products with the membrane fraction of the cell. For reasons not yet apparent, the combined presence of membrane anchorage domains of v-src, and the transmembrane domain of v-erbB in the same chimera typically (but not inevitably) impeded transformation. Our results suggest that the specificity of transformation by v-erbB resides in the selection of substrates by the cytoplasmic domain of the gene product. The protein retains access to those substrates even when anchored to the membrane in the manner of a peripheral rather than a transmembrane protein.     

Transformation of human and murine fibroblasts without viral oncoproteins

Murine embryo fibroblasts are readily transformed by the introduction of specific combinations of oncogenes; however, the expression of those same oncogenes in human cells fails to convert such cells to tumorigenicity. Using normal human and murine embryonic fibroblasts, we show that the transformation of human cells requires several additional alterations beyond those required to transform comparable murine cells. The introduction of the c-Myc and H-RAS oncogenes in the setting of loss of p53 function efficiently transforms murine embryo fibroblasts but fails to transform human cells constitutively expressing hTERT, the catalytic subunit of telomerase. In contrast, transformation of multiple strains of human fibroblasts requires the constitutive expression of c-Myc, H-RAS, and hTERT, together with loss of function of the p53, RB, and PTEN tumor suppressor genes. These manipulations permit the development of transformed human fibroblasts with genetic alterations similar to those found associated with human cancers and define specific differences in the susceptibility of human and murine fibroblasts to experimental transformation.     

Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog.

The lipid bound to p60src, the transforming protein of Rous sarcoma virus, has been identified by gas and thin-layer chromatography as the 14-carbon saturated fatty acid, myristic acid. The protein can be labeled biosynthetically with either [3H]myristic acid or [3H]palmitic acid. Incorporation of [3H]myristic acid was noticeably greater than incorporation of [3H]palmitic acid. All of the [3H]myristic acid-derived label in p60src was present as myristic acid. In contrast, none of the radioactivity derived from [3H]palmitic acid was recovered as palmitic acid. Instead, all 3H incorporated into p60src from [3H]palmitic acid arose by metabolism to myristic acid. The cellular tyrosine kinase, p60c-src also contains myristic acid. By comparison of the extent of myristylation of p60v-src with that of the Moloney murine leukemia virus structural protein precursor, Pr65gag, we estimate that greater than 80% of the molecules of p60v-src contain one molecule of this fatty acid. Myristylation is a rare form of protein modification. p60v-src contains 10 to 40% of the myristic acid bound to protein in cells transformed by Rous sarcoma virus and is easily identified in total cell lysates when [3H]myristic acid-labeled proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the amount of [3H]myristic acid-labeled p60src in total cell lysates and in immunoprecipitates suggests that immunoprecipitation with rabbit anti-Rous sarcoma virus tumor sera detects ca. 25% of the p60src present in cells.     

Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin

A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.     

Differentiation of retrovirus-infected avian neuroretina cells

The effects of oncogenic retroviruses on the expression of differentiation markers were studied in monolayer cultures of chick and quail embryo neuroretinas. Transformation by Rous sarcoma virus (RSV) did not affect the appearance of synapses, and the expression of glutamic acid decarboxylase was stimulated by pp60v-src, the product of the src gene. Quail embryo neuroretina cells transformed by Mill Hill 2 (which contains the two oncogenes v-mil and v-myc) were induced to proliferate into permanent cultures that synthesized crystallins and produced lentoid bodies. In contrast, transformation with a temperature-sensitive mutant of RSV reversibly blocked the production of crystallins and lentoid bodies. These data show that given cellular genes can respond differently to distinct oncogenes.