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.     

Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: temperature sensitivity of the transformed phenotype and retransofrmation by wild-type virus.

The function of the A gene of simian virus 40 (SV40) in transformation of BALB/c-3T3 cells was investigated by infecting at the permissive temperature with wild-type SV40 and with six tsA mutants whose mutation sites map at different positions in the early region of the SV40 genome. Cloned transformants were then characterized as to the temperature sensitivity of the transformed phenotype. Of 16 tsA transformants, 15 were temperature sensitive for the ability to overgrow a monolayer of normal cells, whereas three of three wild-type transformants were not. This pattern of temperature sensitivity of the transformed phenotype was also observed when selected clones were assessed for the ability to grow in soft agar and in medium containing low concentration of serum. The temperature resistance of the one exceptional tsA transformant could be attributed neither to the location of the mutation site in the transforming virus nor to transformation by a revertant virus. This temperature-resistant tsA transformant, however, was demonstrated to contain a higher intracellular concentration of SV40 T antigen than a temperature-sensitive line transformed by the same tsA mutant. A tsA transformant displaying the untransformed phenotype at the nonpermissive temperature was found to be susceptible to retransformation by wild-type virus at this temperature, demonstrating that the temperature sensitivity of the tsA transformants is due to the viral mutation and not to a cellular defect. These results indicate that continuous expression of the product of the SV40 A gene is required to maintain the transformed phenotype in BALB/c-3T3 cells.     

Malignant transformation of mouse BALB/3T3 cells by polyoma middle T antigen requires epidermal growth factor receptor expression.

The mouse cell line MO-5, which is defective in receptor-binding activity of epidermal growth factor (EGF), is very poorly transformed by polyoma middle T antigen or v-src gene, but activated c-H-ras and v-mos gene can induce the transformation (M. Ono, M. Yakushinji, K. Segawa, and M. Kuwano, Mol. Cell. Biol., 8: 4190-4196, 1988). We established clones of MO-5 expressing a functional EGF receptor (EGF-R) after introduction of the human EGF-R complementary DNA into MO-5 (MNER23 and MNER31), and we also established a clone (BNER4) expressing human EGF-R from the parental cell line, BALB/3T3. MNER23, MNER31, and BNER4 expressed EGF-R activity at about 2- to 6-fold higher levels than did control BALB/3T3 cells. A marked increase in DNA synthesis in response to EGF was observed in these BNER4, MNER23, and MNER31 cell lines compared to BALB/3T3 cells; however, there was little if any increase in DNA synthesis of MO-5 in the presence of EGF. Introduction of the polyoma middle T antigen gene into BALB/3T3, BNER4, MNER23, and MNER31 resulted in the appearance of transformation foci, but MO-5 again showed little response. We purified clones B4-mT-2, M23-mT-1, M23-mT-2, M23-mT-3, and M31-mT-13 from transformation foci of BNER4, MNER23, and MNER31 cells, which were respectively transfected with the middle T antigen. All of the middle T antigen-positive transfectants demonstrated abilities to form both colonies in soft agar and tumors in nude mice. The presence of EGF-R appears to be indispensable for malignant transformation by polyoma middle T antigen.     

Distribution and functional analysis of a 120- to 130-kDa T-cell surface antigen

A monoclonal antibody (mAb), SPV-L14, was raised that detected a human T-cell surface antigen with a molecular weight (MW) of 120 kDa on resting and phytohemagglutinin-activated peripheral blood T lymphocytes (PBL). An additional band with a MW of 130 kDa could be precipitated with variable intensities from thymocytes, neoplastic T cells, and CD4+- or CD8+ T-cell clones. Based on their reactivity with SPV-L14 and a mAb directed against CD3, four subpopulations of CD2+ lymphocytes could be detected and their existence was confirmed at the clonal level. The majority (95%) of the CD3+ cells were SPV-L14+, whereas 5% were CD3+, SPV-L14-. Among cloned cell lines CD3-,SPV-L14- and CD3-,SPV-L14+ cells were found to exist. The CD3-,SPV-L14- and CD3-,SPV-L14+ clones were shown to have NK cell activity, indicating that the 120- to 130-kDa antigen is expressed heterogeneously on CD3- NK cell clones. In addition, neoplastic T cells representing these four subpopulations were shown to exist. Although the tissue distribution and the MW of the SPV-L14 target antigen strongly suggest that SPV-L14 reacts with an epitope on CD6, the SPV-L14 mAb did not react with resting or activated B cells or with malignant B cells. Blocking studies showed that SPV-L14 inhibited the proliferative response of PBL, induced by anti-CD3 mAb, but that SPV-L14 did not affect the proliferation induced by phytohemagglutinin. These results suggest that the 120- to 130-kDa MW antigen is associated with T-cell proliferation, depending on the mode of activation.     

Responses of mouse cell lines transformed by various means to histidinol/cytosine arabinoside treatment

The responses of the phenotypically-normal BALB/c 3T3 mouse cell line A31 and three of its transformed derivatives, BP-A31 (a benzo(a)pyrene transformant), M-A31 and SV-A31 (Moloney murine sarcomavirus and SV-40 virus transformants, respectively) to L-histidinol and to L-histidinol/cytosine arabinoside(AraC) combinations were investigated. As observed in comparable studies with other normal cell lines, prior treatment of the A31 parental line with L-histidinol led to significant protection from AraC-mediated toxicity. However, in contrast to previous analyses of a number of other transformed cell lines which showed enhanced AraC-mediated toxicity upon prior exposure to L-histidinol, the three types of A31 transformants were afforded varying degrees of protection from AraC when treated with histidinol. These findings support the concept that transformed, tumorigenic cells may retain certain growth properties of their nontumorigenic parental cells.     

Transfer and co-amplification of a gene encoding a 96-kDa immune IFN-inducible human melanoma-associated antigen. Preferential expression by mouse melanoma host cells

An immune IFN-inducible human melanoma-associated glycoprotein Ag, 96-kDa MAA, having preferential distribution on metastases has been defined by mouse mAb CL203.4. To initiate molecular genetic analysis of 96-kDa MAA, the gene encoding the Ag was transfected into mouse B16 melanoma cell clone B78H1. Formation of B78H1-transfectant colonies expressing a surface Ag reactive with mAb CL203.4 in an immunorosetting assay was dependent on addition of chromosomal DNA from human melanoma cells [primary (1 degree) transfer] or from Ag-expressing transfectant cells (2 degrees, 3 degrees, 4 degrees transfer). The mAb CL203.4-reactive species expressed by the transfectant cells is a glycoprotein with a molecular mass 93-kDa, within the range of 93 to 96-kDa observed for the endogenous human Ag. In the presence of tunicamycin, an inhibitor of N-linked glycosylation, both mouse melanoma transfectants and human melanoma cells express a 50- to 51-kDa antigenic species. Human alu family repeat sequences (h-alu) are present in the genomes of 3 degrees transfectant cells. Continued presence of these h-alu after dilution of extraneous human DNA by three cycles of transfection suggests their association with the transferred 96-kDa MAA gene. Use of a selective co-amplification procedure led to transfectant cells' increased expression of 96-kDa MAA and to commensurate increases in their content of presumed 96-kDa MAA gene-associated h-alu. Preferential DNA-mediated transferability of the 96-kDa MAA+ phenotype into B78H1 cells as compared with LMTK- mouse fibroblasts suggests host cell specificity of 96-kDa MAA gene expression.     

Transforming growth factor-beta (TGF-beta)-resistant helper T lymphocyte clones show a concomitant loss of all three types of TGF-beta receptors.

Three ovalbumin-specific T helper cell lines (OVA-7T cells) that differ in their susceptibility to the immunosuppressive effects of transforming growth factor-beta (TGF-beta) were cloned. The frequency of TGF-beta-resistant OVA-7T cell clones correlated with the decline of TGF-beta sensitivity in the original OVA-7T parental cell lines. In TGF-beta-resistant OVA-7T cell clones, TGF-beta inhibited neither the growth of the T cells nor their secretion of granulocyte macrophage CSF. TGF-beta suppressed the expression of c-myc mRNA in OVA-7T-responder but not in OVA-7T-nonresponder cells. TGF-beta resistance was found to be due to a loss of TGF-beta high-affinity binding sites, with an absence of expression of the distinct 54-, 70-, 110-, and 250-kDa surface proteins that bind TGF-beta on TGF-beta-susceptible T cells. Loss of TGF-beta R may enable T cells to escape the negative feedback control provided by TGF-beta secreted from activated T cells during an immune response.     

Activated c-raf gene in a rat hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline

A rat hepatocellular carcinoma, IQ7, induced by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) gave two transformants of NIH 3T3 cells on DNA mediated gene transfer. One of these transformants was examined further and secondary and tertiary transformants were obtained. The secondary transformant was tumorigenic in nude mice. The activated oncogene in this primary transformant was identified as rat c-raf by Southern blot analysis.     

A Swiss 3T3 variant cell line resistant to the effects of tumor promoters cannot be transformed by src.

To study the relationship between oncogenesis by v-src and normal cellular signalling pathways, we determined the effects of v-src on 3T3-TNR9 cells, a Swiss 3T3 variant which does not respond mitogenically to tumor promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We found that src was unable to transform these variant cells, whether the oncogene was introduced by infection with a murine retrovirus vector or by transfection with plasmid DNA. 3T3-TNR9 cells were not inherently resistant to transformation, since infection with similar recombinant retroviruses containing either v-ras or v-abl did induce transformation. Further analysis of Swiss 3T3 and 3T3-TNR9 cell populations infected with the v-src-containing retrovirus revealed that although the amount of v-src DNA in each was approximately the same, the level of the v-src message and protein and the overall level of phosphotyrosine expressed in the infected variants was much less than in infected parental cells. Cotransfection experiments using separate v-src and neo plasmids revealed a decrease in the number of G418-resistant colonies when transfections of TNR9 cells occurred in the presence of the src-containing plasmid, suggesting a growth inhibitory effect of v-src on 3T3-TNR9 cells, as has also been found for TPA itself. Since v-src cannot transform this variant cell line, which does not respond mitogenically to the protein kinase C agonist TPA, we suggest that src makes use of the protein kinase C pathway as part of its signalling activities.     

Activated H-ras transforms rat intestinal epithelial cells with expression of alpha-TGF

We describe malignant transformation of cultured rat intestinal epithelial cells (IEC-18) by transfection with the activated human H-ras gene cloned from the EJ bladder carcinoma. Transformed cells showed a marked morphological change, expressed high levels of the transfected H-ras gene, were able to grow in agar and expressed antigenic markers identical with parental IEC-18 cells. When injected into syngeneic rats these cells formed rapidly growing tumors expressing the same intestinal-specific antigenic markers as the injected cells. Parallel to the high expression of H-ras mRNA in the transformants we document overexpression of rat alpha-TGF mRNA.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

c-myc gene expression and interleukin-2 receptor levels in cloned human CD2+,CD3+ and CD2+,CD3- lymphocytes

The levels of c-myc mRNA and interleukin-2 receptors (IL-2 Rec) were studied in human peripheral blood lymphocytes (PBL); mature CD2+,CD3+ T cell clones and CD2+,CD3- natural killer (NK) cell clones, and CD2+,CD3+ and CD2-,CD3- T lymphoma cell lines. A transient induction of the expression of c-myc and IL-2 Rec was observed in PBL after activation with phytohemagglutinin (PHA). Expression of c-myc and IL-2 Rec was also found in the CD2+,CD3+ and CD2+,CD3- clones. The CD2+,CD3+ showed higher levels of c-myc mRNA and IL-2 Rec than the CD2+,CD3- clones. In three T lymphoma cell lines constitutively high levels of c-myc mRNA but no IL-2 Rec were found. Only in JURKAT (CD2+,CD3+), c-myc mRNA levels could be further enhanced by PHA. These results suggest that in the presence of PHA, expression of c-myc and IL-2 Rec is induced via the CD3 receptor, and in the absence of PHA and/or the CD3 receptor alternative routes of induction are involved.     

Immortalization by c-myc, H-ras, and Ela oncogenes induces differential cellular gene expression and growth factor responses.

Early-passage rat kidney cells were immortalized or rescued from senescence with three different oncogenes: viral promoter-driven c-myc, H-ras (Val-12), and adenovirus type 5 E1a. The normal c-myc and H-ras (Gly-12) were unable to immortalize cells under similar conditions. Quantitation of RNA in the ras-immortalized lines demonstrated that the H-ras oncogene was expressed at a level equivalent to that of the normal H-ras gene in established human or rat cell lines. Cell lines immortalized by different oncogenes were found to have distinct growth responses to individual growth factors in a short-term assay. E1a-immortalized cells were largely independent of serum growth factors, whereas c-myc-immortalized cells responded to serum better than to epidermal growth factor and insulin. H-ras-immortalized cells responded significantly to insulin alone and gave a maximal response to epidermal growth factor and insulin. Several cellular genes associated with platelet-derived growth factor stimulation, including c-myc, were expressed at high levels in the H-ras-immortalized cells, and c-myc expression was deregulated, suggesting that the H-ras oncogene has provided a "competence" function. H-ras-immortalized cells could not be morphologically transformed by secondary transfection with a long terminal repeat-c-myc oncogene, but secondary transfection of the same cells with H-ras (Val-12) produced morphologically transformed colonies that had 20- to 40-fold higher levels of H-ras oncogene expression. Thus, transformation in this system is dependent on high levels of H-ras oncogene expression rather than on the presence of activated H-ras and c-myc oncogenes in the same cell.     

Induction of high-grade anti-tumor immunity by use of a recombinant H-2Kb/avian erythroblastosis virus erbB gene transfectant

Summary The recombinant H-2K^b-erbB gene, encoding for a part of the H-2 class I antigen and the kinase domain of the V-erbB peptide, was successfully introduced into murine mastocytoma P815 variant P1.HTR cells, which resulted in low but significant cell-surface expression of the hybrid gene product. When the chimeric gene transfectant was inoculated into the CDF₁ mice, it soon grew but regressed thereafter. The tumorigenicity of this transfectant was lower than the H-2K^b gene transfectant that expressed the H-2K^b antigen at a comparable level. These CDF₁ mice that had received the chimeric gene transfectant obtained a high-grade anti-tumor immunity against the challenge of a high dose of parental tumor. Corresponding to these observations, anti-tumor cytotoxic T lymphocytes, which lyse parental P1.HTR cells but not syngeneic L1210 or NS-1 tumor cells, were developed in the peritoneal cavity of mice that had been inoculated with the transfectant and parental tumor. Definite antibody activity binding to parental P1.HTR tumor cells was also demonstrated in the sera of these mice, precipitating 40-kDa, 74-kDa and 98-kDa molecules from the surface of the radiolabeled P1-HTR tumor cells. The results suggested that the chimeric H-2-erbB gene transfectant efficiently triggers both cellular and humoral anti-tumor immune responses.     

Modulation of transforming growth factor type beta action by activated ras and c-myc.

Transfection of C3H/10T1/2 cells with a c-myc gene resulted in the acquisition of responsiveness to transforming growth factor type beta. Cells transfected with an activated H-ras gene or an H-ras and c-myc gene, however, exhibited a transformed morphology and spontaneous soft-agar growth, a phenotype induced reversibly by transforming growth factor type beta in responsive fibroblasts.     

Growth factor-deprived BALB/c 3T3 murine fibroblasts can enter the S phase after induction of c-myc gene expression.

Induction of quiescent BALB/c 3T3 murine fibroblasts by platelet-derived growth factor (PDGF) or fibroblast growth factor (FGFs) is accompanied by induction of c-myc gene expression. To study the role of c-myc in cell growth, we transfected BALB/c 3T3 cells with a plasmid construct containing a glucocorticoid-inducible c-myc gene. When these transfected cells were growth arrested in PDGF-FGF-freedefined medium, glucocorticoid treatment induced S-phase DNA synthesis. This induction of DNA synthesis was inefficient, and cell proliferation was not evident, suggesting that growth factors act through stimulation of c-myc expression together with other intracellular events.     

Serine/threonine-specific protein kinase activity associated with viral pp60src protein

Three different types of experiments are presented in this paper, the results of which converge to indicate that the viral src protein associates with and modulates the activity and/or the specificity of a serine/threonine protein kinase. Firstly, a 60-kDa protein from extracts of FR3T3 rat fibroblasts transformed by wild-type Rous sarcoma virus (SRD-FR3T3) is shown to be immunoprecipitated with a monoclonal antibody (mAb) raised against bacterially produced pp60v-src, the mAb327 [Lipsich, L. A., Lewis, A. J. & Brugge, J. S. (1983) J. Virol. 48, 352-360] and to be phosphorylated in vitro at serine/threonine/tyrosine residues, in the ratio 25:53:22. Under the same experimental conditions, the pp60c-src protein immunoprecipitated with mAb327 from extracts of NIH c-src overexpresser cells is phosphorylated exclusively on tyrosine residues. Secondly, the results of immunoprecipitation experiments using a tumor-bearing rabbit (TBR) serum and reported in an earlier work [David-Pfeuty, T. & Hovanessian, A. (1984) Eur. J. Biochem. 140, 325-342], together with those reported here, suggest that the TBR-immunoprecipitated pp60v-src coprecipitates with a cellular protein related to the 60-kDa subunit of the Ca2+/calmodulin protein kinase II from brain. Finally, partially purified preparations of pp60v-src, but not of pp60c-src, are shown to contain a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates a 52-kDa protein substrate.