Growth Of NIH3T3 Cells Research Articles

Apobec-T is widely expressed in normal and cancerous gastrointestinal tissues and its inducible overexpression alters growth in NIH3T3 cells

Background: Apobec-1 is an RNA-specific cytidine deaminase which mediates a single C to U change in the nuclear apolipoproteinB mRNA in mammalian small intestine. Human apobec-1 gene expression is regulated through differential altemative splicing, whereby an alternate splice acceptor site results in tissue and developmental stage specific exclusion of exon 2. This splice variant results in a frame shift in the processed mRNA which generates a novel 36 amino acid polypeptide, apobec-T, mRNA encoding apobec-T predominates during intestinal ontogenesis and was found to be overexpressed in human colorectal tumors when compared to matched normal tissue. Our aims in this study were to further examine the distribution of apobec-T expression in normal and cancerous gastrointestinal tissues and to evaluate the in vivo effects of apobec-T overexpression on cell proliferation by means of inducible expression. Results: Immunocytochemical staining of paraffin sections of normal stomach, small intestine and colon using an antipeptide antibody confirms the presence of apobec-T throughout the human gastrointestinal tract. Immunocytochemical analysis of 3 gastric adenocarcinomas, 5 small intestinal adenocarcinomas and 6 colonic adenocarcinomas revealed the presence of apobec-T in a diffuse pattern within epithelial cells throughout the tumors. 7 tubular adenomas also demonstrated the presence of apobec-T staining. Interestingly, 5 of 5 hepatic metastases from colon carcinomas demonstrated apobec-T staining. None of 5 primary hepatocellular carcinomas examined demonstrated apobec-T mRNA or protein, suggesting that the presence of apobec-T may be a useful surrogate marker of luminal GI tract malignancy. In order to determine the effects of apobec-T expression on cell proliferation, the splice variant mRNA was reverse transcribed and cloned downstream of a tetracycline-response element and transfected into NIH 3T3 cells expressing the tetR/VP16 fusion. One clone was identified in which expression of apobec-T could be induced 17-fold following withdrawal of tetracycline. Maximal induction of apobec-T in this cell line represents I l-fold higher levels of apobec-T mRNA than is expressed in normal adult jejunum. These cells were plated in 96 well dishes, +/tetracycline, and an XTT assay was performed daily on replicate plates to quantitate cell number. On days 4-6 after induction the cells expressing apobec-T were significantly fewer in number when compared to matched uninduced cells (paired t-test, p<0.01). Conclusions:Apobec-T peptide is detectable in normal and cancerous gastrointestinal tissues. High level overexpression of apobec-T in NIH3T3 cells utilizing a tetracycline-inducible system results in a significant decrease in cell number after 4 days of growth. Apobec-T may function as a modulator of cellular proliferation or apoptosis.

Both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-Ras prenylation but each alone is sufficient to suppress human tumor growth in nude mouse xenografts.

The ability of Ras oncoproteins to cause malignant transformation requires their post-translational modifications by prenyl groups. Because K-Ras can be both farnesylated and geranylgeranylated it is not known whether both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for suppressing human tumor growth in whole animals. In this paper we report that oncogenic Ras processing, MAP kinase activation and growth in nude mice are inhibited by the farnesyltransferase inhibitor FTI-276 in H- and N-Ras transformed NIH3T3 cells; whereas in KB-Ras transformed NIH3T3 cells both FTI-276 and the geranylgeranyltransferase I inhibitor GGTI-297 are required for inhibition. Furthermore, human lung A-549 and Calu-1 carcinoma cell lines were found to co-express H-, N- and K-Ras. In Calu-1 cells, the processing of H- and N-Ras is inhibited greatly by FTI-276 but only partially by GGTI-297 whereas K-Ras processing inhibition requires both FTI-276 and GGTI-297. In contrast, in A-549 cells the processing of H- and N-Ras is inhibited only by FTI-276 and K-Ras processing is resistant to co-treatment with FTI-276 and GGTI-297. Yet, the growth in nude mice of A-549 and Calu-1 xenografts, both of which express K-Ras mutations, is inhibited by FTI-276 (80% inhibition) and GGTI-297 (60%). Furthermore, FTI-276 inhibits tumor growth of NIH3T3 cells transformed by a form of oncogenic H-Ras that is exclusively geranylgeranylated and whose processing is resistant to this inhibitor. Taken together, the results demonstrate that both FTase and GGTase I inhibitors are required for inhibition of K-Ras processing but that each alone is sufficient to suppress human tumor growth in nude mice.

Mutation of C-Terminal Tyrosine Residues Y497/Y504 of the Src-Family Member Bsk/Iyk Decreases NIH3T3 Cell Proliferation

To elucidate the properties of the Src-family member Bsk/Iyk, NIH3T3 cells were transfected with wild-type Bsk/Iyk or Bsk/Iyk carrying Y497F, Y504F or Y497/504F mutations. These positions are putatively homologous to tyr-527 in Src. The Bsk/IykY497/504F cells displayed a decreased cell growth rate, parallelled by an augmentation of the fraction of cells in G1-phase. The Bsk/IykY497/504F double-mutation decreased the [3H]thymidine incorporation. No effects on NIH3T3 cell growth could be seen in cells expressing wild-type Bsk/Iyk or the other Bsk/Iyk mutants. In vitro kinase reactions performed on immuno-precipitates from NIH3T3 cells expressing wild-type or mutated Bsk/Iyk revealed increased relative [32P]-incorporation into Bsk/Iyk isoforms containing the Y504F and Y497/504F mutations compared with wild-type Bsk/Iyk. The Y497F and Y497/504F mutations elevated the proportion of [32P]-incorporation into a 57 kDa Bsk/Iyk product relative to that into the 60 kDa isoform. The Y497F Bsk/Iyk mutant not only increased the relative amount of p57 Bsk/Iyk but also transferred this isoform to the nuclear subcellular fraction. The results suggest that Bsk/Iyk has unique regulatory properties, and that this kinase might serve a role in inhibiting cell replication.

Decreased Retinoylation in NIH 3T3 Cells Transformed with Activated Ha-ras

Retinoylation (retinoic acid acylation) is a posttranslation modification of proteins occurring in a variety of mammalian cell lines andin vivo.To gain further knowledge of the role of retinoylation we studied it in NIH 3T3 cells and NIH 3T3 cells transformed by an activated Ha-rasoncogene (NIH Ha-ras-3T3 cells). In serum-free medium retinoic acid (RA) inhibited growth of NIH 3T3 cells but did not inhibit growth of NIH Ha-ras-3T3 cells. After incubation with [3H]RA, the level of retinoylated protein in NIH 3T3 cells was about 1.5-fold greater than in NIH Ha-ras-3T3 cells. On one-dimensional polyacrylamide gel electrophoresis, both the rate and the extent of retinoylation were greater in NIH 3T3 cells. We detected about 40 retinoylated proteins in NIH 3T3 cells by two-dimensional polyacrylamide gel electrophoresis. Only about 15 proteins were retinoylated, but at reduced levels, in NIH Ha-ras-3T3 cells. These results suggest that the activatedrasoncogene inhibits retinoylation. This inhibition may in turn be related to the loss of other RA responses of NIH 3T3 cells, including growth inhibition, retinoic acid catabolism, down-regulation of fibronectin biosynthesis, and induction of tissue-type transglutaminase, which are not seen to the same extent in NIH Ha-ras-3T3 cells.

Turnover of the Acyl Phosphates of Human and Murine Prothymosin α in Vivo

Prothymosin alpha is a small, highly acidic, abundant, nuclear, mammalian protein which is essential for cell growth. Our laboratory has recently shown that primate prothymosin alpha contains stoichiometric amounts of phosphate on the glutamyl groups of the protein and that in vitro the phosphate undergoes rapid hydrolysis or transfer to a nearby serine residue. Here an assay for the presence of acyl phosphates in vivo has been developed by measuring stable phosphoserine and phosphothreonine in vitro. The assay was used to determine the half-life of the acyl phosphates on prothymosin alpha in vivo by pulse-labeling HeLa cells with [32P]orthophosphate and chasing using three different techniques: permeabilization with digitonin to allow extracellular ATP to equilibrate with the intracellular pool; electroporation in the presence of ATP to reduce the specific activity of [32P]ATP by expansion of the pool; and incubation with inorganic phosphate. Regardless of the method, the phosphate turned over with a half-life of 75-90 min. The ability of cells to phosphorylate old prothymosin alpha molecules was established by demonstrating equivalent labeling of the protein with [32P]orthophosphate in the presence and absence of cycloheximide. The half-life of the acyl phosphates was also studied in resting and growing NIH3T3 cells, with measured values of 30-35 and 70 min, respectively. Our data suggest that the "activity" of prothymosin alpha involves the turnover of its acyl phosphates and that it participates in a function common to all nucleated mammalian cells regardless of whether they are quiescent or undergoing rapid proliferation. This is the first measurement of the stability of protein-bound acyl phosphates in vivo.

High-level expression of human c-Src can cause a spherical morphology without loss of anchorage-dependent growth of NIH 3T3 cells

To investigate whether overexpression of human c-Src leads to cell rounding in anchorage-dependent NIH 3T3 fibroblasts, we have established c-Src-inducible cell lines using a lac repressor–operator system. RN1 cells, which expressed c-Src at a high level after induction, exhibited a spherical morphology and ceased to grow in monolayer culture. RN1 cells, however, exhibited neither focus-forming ability nor anchorage-independent growth potential with or without induction. Induced RN1 c-Src was phosphorylated at Ser 75, a previously reported spherical cell-associated site, and at Tyr 419. These data demonstrated for the first time that highly elevated human c-Src tyrosine kinase activity can cause NIH 3T3 cells to have a spherical morphology without loss of anchorage-dependent growth. The inducible cell line should be useful to study the mechanism for cell rounding by c-Src.

Human Immunodeficiency Virus Type 1VprGene Product Prevents Cell Proliferation on Mouse NIH3T3 Cells without the G2Arrest of the Cell Cycle

Human immunodeficiency virus type 1 Vpr is a 96-amino-acid virion-associated protein that arrests cells in the G2phase of the cell cycle in peripheral blood lymphocytes, HeLa, 293, 293T, A549, Jurkat, CEM, SupT1, CV-1 and COS1 cells. When we transfected Vpr expression vector into mouse NIH3T3 and then cultured it in the presence of G418, NIH3T3 cells were the drug resistant cells yielded. The surviving colonies, however, exhibited a degenerating morphology up to 8∼20-fold smaller than the control vector colonies. In addition, the growth of NIH3T3 cells transiently transfected with Vpr expression vector declined dramatically compared with that of transfectants with control vector, suggesting that Vpr significantly interferes with cell proliferation of NIH3T3 cells. Cell cycle characterization by flow cytometry indicated that expression of Vpr did not induce G2cessation in NIH3T3. These findings strongly suggest that Vpr has a novel pathway to retard cell growth independently and arrests the G2phase of the cell cycle.

Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth.

Eps8, a substrate of receptor tyrosine kinases, is an SH3 domain containing protein that plays an important role in mitogenic signaling. To determine the cellular function of eps8, we used the SH3 domain of eps8 to screen a human fibroblast M426 expression library and identified, a full-length cDNA clone of 3.2 kb. We designated this clone e3B1 for eps8 SH3 domain binding protein 1. Northern analysis revealed that expression of e3B1 mRNA was ubiquitous in human tissues. The e3B1 gene encodes a SH3 domain containing protein. We show that anti-e3B1 antibodies detect three cytosolic protein species of 65, 68 and 72 kDa in cell lysate isolated from asynchronously growing NIH3T3 cells. E3B1 binds to the SH3 domain of eps8 and Abl in vitro. We also demonstrated that e3B1 associates with eps8 in vivo. Phosphatase digestion and phosphoamino acid analysis revealed that p65e3B1 is a phosphoserine containing protein and p72e3B1 and p68e3B1 are hyperserine-phosphorylated form of p65e3B1. We further determined that the p65e3B1 was the most abundant in serum-starved NIH/EGFR cells. Time course studies initiated by the addition of epidermal growth factor (EGF) revealed that the p72e3B1 started to accumulate at 4 h, peaked at 8 h and remained high until 24 h. Finally, we demonstrate that NIH/EGFR fibroblasts overexpressing e3B1 grow more slowly relative to matched controls.

In the Absence of Streptomycin, Minoxidil Potentiates the Mitogenic Effects of Fetal Calf Serum, Insulin-Like Growth Factor 1, and Platelet-Derived Growth Factor on NIH 3T3 Fibroblasts in a K + Channel-Dependent Fashion

There is considerable evidence to suggest that the opening of K+ channels plays an important role in stimulating mitogenesis. K+ channel blockers have been shown to inhibit mitogenesis in vitro, mitogens increase cytosolic membrane K+ channel permeability, K+ channel openers stimulate hair growth in vivo, and the Ras/Raf signal transduction pathway induces K+ channel activity. Paradoxically, however, K+ channel openers such as minoxidil have been reported in vitro not to modulate, or even to inhibit, mitogenesis in a range of cell types. Only untherapeutic concentrations have stimulated mitogenesis. These experiments, however, appear to have been carried out in the presence of aminoglycoside antibiotics, which inhibit potassium channel activity. We now report that in the absence of aminoglycoside antibiotics, minoxidil at 10 microg/ml (0.05 mM) causes a significant stimulation of proliferation of NIH 3T3 fibroblasts maintained over a 10-d period in 5% fetal calf serum-supplemented medium. Further, we show that in the presence of 100 microg streptomycin per ml, minoxidil at 10 microg/ml produces an initial inhibition of proliferation, which apparently confirms, in NIH 3T3 fibroblasts, that the inhibition of mitogenesis by minoxidil in the presence of streptomycin is an artifact. The potentiation of NIH 3T3 cell growth by minoxidil can be attributed to the opening of potassium channels, because the potassium channel blocker tolbutamide (5 mM) or combinations of the blockers tolbutamide (1 mM)/tetraethylammonium (2 mM) or glibenclamide (1 microM)/apamin (10 nM) block the minoxidil-induced stimulation of growth. We also demonstrate that minoxidil is able to significantly potentiate the mitogenic effects of both platelet-derived growth factor and insulin-like growth factor 1 on NIH 3T3 fibroblasts in the presence of CPSR-2 (a cytokine free serum substitute). Thus we have shown that minoxidil potentiates the mitogenic effects of fetal calf serum in vitro on NIH 3T3 fibroblasts by opening potassium channels and is also able to potentiate the mitogenic effects of the growth factors platelet-derived growth factor and insulin-like growth factor 1.

Processing of the Nedd2 precursor by ICE‐like proteases and granzyme B

The Nedd2/Ich-1 protein belongs to a growing family of mammalian cysteine proteases similar to interleukin-1 beta converting enzyme (ICE). Because of their similarity to the Cacnorhabditis elegans cell death protein CED-3, the ICE-like proteins are thought to play a key role in the execution of apoptosis. The active form of ICE is a tetramer consisting of two heterodimers (p20 + p10)2 derived from the cleavage of the pro-enzyme. In the present communication we show that the p51 Nedd2 precursor (pro-Nedd2) is also cleaved into p20-like (p19) and p10-like (p12) subunits by extracts prepared from cultured cell lines. Extracts from apoptotic NIH-3T3 cells but not normal growing NIH-3T3 cells also contained pro-Nedd2 cleaving activity. The processing of pro-Nedd2 by cell extracts was inhibited by characteristic inhibitors of ICE-like proteases. Additionally we show that pro-Nedd2 (p51) can be processed in vitro by active CPP32 and ICE, and to a lesser extent by Mch2 and Nedd2. Granzyme B, a serine protease required for cytotoxic T lymphocyte (CTL) mediated killing of target cells, also cleaved pro-Nedd2 to p19 + p12 subunits. Our observations suggest that Nedd2 activation requires cleavage by one or more ICE-like proteases that lie upstream in the proteolytic cascade. Cleavage of pro-Nedd2 by granzyme B indicates that Nedd2 may be one of the downstream effectors in the CTL-mediated killing of target cells.

TGF-beta 1 perturbation of the fibroblast cell cycle during exponential growth: switching between negative and positive regulation.

We have demonstrated previously that SV40 T antigen and serum regulate the length of G1 in exponentially growing NIH-3T3 cells in part by inhibiting density dependent negative cell cycle regulation. In these studies it was suggested that T antigen positively regulated G1 in a density independent manner as well. In this report we show that, 24 h after treatment, TGF-beta 1 perturbs the cell cycle of exponentially growing fibroblasts in a manner similar to T antigen. However, prior to 24 h, TGF-beta 1 produced a negative response, elongating the G1 phase of the cell cycle that was followed by a positive response, both of which were density independent. This biphasic response was measured between 0 and 12 h post-treatment and was relative to responses from serum. This switch from an early inhibitory effect to a late stimulatory effect was associated with changes in Rb phosphorylation, the timing and magnitude of which indicated that Rb may be directly regulating TG1 rather than reporting changes in the population. This is further substantiated by abrogation of the inhibitory effect by expression of wild-type SV40 T antigen and retention of the effect in cells that express an Rb-binding mutant of T antigen (K1). The biphasic regulatory effects of TGF-beta 1 were also displayed in WI-38 and IMR-90 human fibroblasts. This suggests that this biphasic effect is a property of fibroblasts.

The Multiple Endocrine Neoplasia Type 2B Point Mutation Alters Long-term Regulation and Enhances the Transforming Capacity of the Epidermal Growth Factor Receptor

The RET proto-oncogene encodes a member of the receptor tyrosine kinase family. Multiple endocrine neoplasia type 2B (MEN 2B) is caused by the mutation of a conserved methionine to a threonine in the catalytic domain of the RET kinase. When the MEN 2B point mutation was introduced into the epidermal growth factor (EGF) receptor (M857T EGFR), the intrinsic tyrosine kinase activity of the mutant receptor was similar to that of wild-type EGF receptor and remained ligand-dependent. However, the mutant receptor showed an enhanced transforming capacity compared to the wild-type receptor as judged by its ability to mediate the growth of NIH 3T3 cells in soft agar. Using the oriented peptide library approach to examine substrate specificity, the M857T mutation was found to be associated with a decrease in the selectivity of the receptor for Phe and an increase in the selectivity for acidic residues at the P + 1 position as compared to wild-type EGF receptor. Short-term responses to EGF were similar in cells expressing wild-type and M857T EGF receptors. However, significant differences in receptor down-regulation were observed between the two receptors. These data demonstrate that the MEN 2B point mutation alters the substrate specificity of receptor tyrosine kinases and suggest that the enhanced oncogenesis associated with the MEN 2B mutation may be due in part to alterations in receptor regulation.

Activation of bruton's tyrosine kinase (BTK) by a point mutation in its pleckstrin homology (PH) domain

Bruton's tyrosine kinase (BTK) is a nonreceptor tyrosine kinase critical for B cell development and function. Mutations in BTK result in X-linked agammaglobulinemia (XLA) in humans and X-linked Immunodeficiency (xid) in mice. Using a random mutagenesis scheme, we isolated a gain-of-function mutant called BTK* whose expression drives growth of NIH 3T3 cells In soft agar. BTK* results from a single point mutation In the pleckstrin homology (PH) domain, where a Glu is replaced by Lys at residue 41. BTK* shows an increase In phosphorylation on tyrosine residues and an increase in membrane targeting. Transforming activity requires kinase activity, a putative autophosphorylation site, and a functional PH domain. Mutation of the SH2 or SH3 domains did not affect the activity of BTK*. Expression of BTK* could also relieve IL-5 dependence of a B lineage cell line. These results show that transformation activation and regulation of BTK are critically dependent on the PH domain.

Vacuolar H+-ATPase Mutants Transform Cells and Define a Binding Site for the Papillomavirus E5 Oncoprotein

The 16K subunit of the vacuolar H(+)-ATPase binds specifically to the bovine (BPV) and human (HPV) papillomavirus E5 oncoproteins, and it has been suggested that this interaction may contribute to cell transformation (Goldstein, D. J., and Schlegel, R. (1990) EMBO J. 9, 137-146; Goldstein, D. J., Finbow, M. E., Andresson, T., McLean, P., Smith, K., Bubb, V. J., and Schlegel, R. (1991) Nature 352, 347-349; Conrad, M., Bubb, V. J., and Schlegel, R. (1993) J. Virol. 67, 6170-6178; Goldstein, D. J., Toyama, R., Schlegel, R., and Dhar, R. (1992) Virology 190, 889-893). We generated mutations within the 16K protein to define binding domains for BPV-1 E5 as well as to characterize the role of 16K in cell transformation. 16K consists predominantly of 4 transmembrane (TM) domains. We showed that mutations within the TM4 domain severely inhibited E5 binding. More specifically, conversion of glutamic acid 143 to arginine within TM4 severely reduced 16K/E5 binding, suggesting that charged interactions facilitated efficient binding. This hypothesis was confirmed by demonstrating that binding to the defective 16K arginine mutant could be restored by complementary charge mutations in E5; conversion of E5 glutamine 17 to glutamic acid or aspartic acid enhanced interactions with the 16K arginine mutant. Surprisingly, mutants in TM4 not only bound poorly to wild-type E5 but were converted into an oncoprotein and induced anchorage-independent growth of NIH 3T3 cells. These data define glutamic acid 143 in the 16K TM4 domain and glutamine 17 within E5 as important contributors to E5/16K binding and suggest a role for the 16K protein in the regulation of cell proliferation.

Enhancer-mediated role for polyomavirus middle T/small T in DNA replication

A major role for polyomavirus middle T/small T antigens in viral DNA synthesis was uncovered by examining the replication of middle T/small T-deficient mutants (hr-t mutants). hr-t mutants in the A2 genetic background showed a 16- to 100-fold defect in genome accumulation relative to the wild type when infections were carried out in exponentially growing NIH 3T3 cells in medium supplemented with low levels of serum (< 2.0%). A proportional decrease in the level of viral early transcripts was also seen. The replication defect of the hr-t mutants was partially overcome in the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. The defect was also alleviated by a duplication encompassing the alpha core enhancer domain that contains binding sites for the transcriptional activators PEA1/AP-1 and PEA3/c-ets. Such a duplication is present in all naturally occurring hr-t mutants and absent in the A2 strain. The effects of 12-O-tetradecanoylphorbol-13-acetate and alpha core duplication were additive but did not fully complement the absence of middle T/small T. In mixed infection competition experiments with two hr-t mutants, a genome that carried an alpha core duplication had a replication advantage (up to 17-fold) over a genome without duplication. This result demonstrates that one effect of the duplication is exerted directly at the level of DNA replication. The advantage of the duplication-bearing genome was established during the earliest stages of replication and was not further amplified in later rounds of replication. In the presence of middle T/small T, both genomes replicated to high levels and the advantage of the duplication-bearing genome was eliminated. On the basis of these results, we propose that factors that bind the alpha core domain (presumably PEA1 and PEA3) are present in limiting amounts in exponentially growing NIH 3T3 cells and play a crucial role in polyomavirus DNA replication. We further suggest that middle T and/or small T stimulates viral DNA replication by activating these factors. The fact that all middle T-/small T-defective hr-t mutants have evolved to contain enhancer duplications that encompass the PEA1 and PEA3 binding sites in the alpha core domain and partially restore their replication defect (A. Amalfitano, M. C. Chen, and M. Fluck, unpublished data) provides an adequate explanation for the fact that the importance of the role of the middle T and/or small T function in DNA replication has not been recognized previously. Much evidence is available in support of separate elements of this model.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet-derived growth factor (PDGF) B and A homodimers transform murine fibroblasts depending on the genetic background of the cell.

The endogenously overexpressed c-sis (platelet-derived growth factor (PDGF) B-chain) proto-oncogene and its viral counterpart v-sis effectively transform NIH 3T3 cells by an autocrine mechanism, whereas the PDGF A-chain gene does not. However, PDGF B continuously cultured with confluent NIH 3T3 cells fails to induce phenotypic transformation as would be predicted by the autocrine model. We now have established conditions in which subconfluent, logarithmically growing NIH 3T3 cells are efficiently transformed by exogenous PDGF B but not PDGF A. Clonally selected cells from transformed foci remain transformed when continuously cultured with PDGF B but revert to the non-transformed phenotype without PDGF B or if PDGF A is substituted for PDGF B, suggesting that PDGF B complements a genetically stable NIH 3T3 cell phenotype to induce transformation and initiates a signal different from that of PDGF A required to induce transformation. Neither PDGF A nor PDGF B transform normal rat kidney (NRK) cells under identical conditions of culture. However, we now demonstrate that exogenous PDGF A and PDGF B independently transform NRK cells that endogenously overexpress the anti-apoptotic bcl-2 gene. These results suggest that both PDGF A and PDGF B effectively transform murine fibroblasts with a compatible genotype, that transformation by the PDGF isoforms requires complementation with a compatible genotype in a multistep transformational process, and that the autocrine mechanism is required but not sufficient for transformation of murine fibroblasts by PDGF.

Synthesis and functional studies on nucleic acids. Problems related to synthetic ras genes

Synthesis of a gene for an oncoprotein, ras p21 and its functions are described. Point mutations in hot spots of ras genes have been found in human cancer cells and produce activated p21 which result in transformation of cultured NIH3T3 cells. To produce normal and activated p21 in quantity for biochemical and structural studies, genes encoding these proteins were synthesized and expressed in E. coli. Normal and activated RAS proteins were tested for their GTPase activity and three dimensional structures were determined by X-ray crystallography. Transforming activities of the synthetic genes have been tested by transfecting their expression vectors to NIH3T3 cells and the synthetic activated genes were found to transform these cells indicating that the product of the activated gene is responsible for these malignant growth of the cells. These activities were proved to be inhibited by transfecting designed ribozyme genes. These synthetic genes were used to investigate mutagenesis of damaged bases such as 7,8-dihydro-8-oxoguanine and thymine photodimers, by introducing the damaged base in hot spots of the oncogene. These unnatural bases in the ras gene were found to be mutagenic and cause malignant growth of NIH3T3 cells.

Pleiotrophin transforms NIH 3T3 cells and induces tumors in nude mice.

The pleiotrophin (PTN) gene (Ptn) encodes an 18-kDa protein that is highly conserved among mammalian species and that functions as a weak mitogen and promotes neurite-outgrowth activity in vitro. To further investigate the role PTN plays in regulating cell growth, we overexpressed the bovine PTN cDNA and now show that PTN phenotypically transforms NIH 3T3 cells, as evidenced by increased cell number at confluence, focus formation, anchorage-independent growth, and tumor formation in the nude mouse. The results demonstrate that the Ptn gene has the potential to regulate NIH 3T3 cell growth and suggest that PTN may influence abnormal cell growth in vivo.

Activating and inactivating mutations of the alpha subunit of Gi2 protein have opposite effects on proliferation of NIH 3T3 cells.

Previous studies have demonstrated that mutations of highly conserved residues in the alpha subunit of Gs (alpha s) can inhibit either the intrinsic GTPase activity (glutamine-227 to leucine, Q227L) or the ability of the protein to be activated by GTP (glycine-226 to alanine, G226A). We stably transfected NIH 3T3 cells with cDNAs encoding Gi2 alpha subunit (alpha i2) containing either wild-type sequence or the homologous mutations Q205L and G204A. High expression of wild-type alpha i2, Q205L alpha i2, and G204A alpha i2 was confirmed in transfected cells by immunoblot analysis. The overexpression of all three alpha i2 proteins was accompanied by an increase in beta-subunit expression. Q205L alpha i2 was a poor substrate for ADP-ribosylation by pertussis toxin as compared with wild-type alpha i2. Expression of Q205L alpha i2 markedly decreased forskolin- or cholera toxin-stimulated intracellular cAMP levels in intact cells, confirming the constitutively activated state of the protein. In contrast, G204A alpha i2 increased intracellular cAMP and was resistant to guanosine 5'-[gamma-thio]triphosphate-induced inhibition of ADP-ribosylation by pertussis toxin, as expected for an inactive alpha i2. Transfection of wild-type, Q205L, or G204A alpha i2 cDNA did not induce focus formation of NIH 3T3 cells. However, overexpression of Q205L alpha i2 induced a decreased serum requirement, a reduced doubling time, and an 8- to 10-fold increase in [3H]thymidine incorporation. Q205L alpha i2 cells formed small colonies in soft agar, demonstrating some degree of anchorage-independent proliferation. Expression of G204A alpha i2 slowed the growth of NIH 3T3 cells. We conclude that alpha i2 plays an important role in regulation of fibroblast growth.

The full-length E6 protein of human papillomavirus type 16 has transforming and trans-activating activities and cooperates with E7 to immortalize keratinocytes in culture.

The wild-type E6 and E7 genes of human papillomavirus type 16 (HPV16) can cooperate to immortalize normal human keratinocytes in culture. The E6 open reading frame of HPV16 and other HPV types highly associated with cervical cancer has the potential of encoding both full-length E6 and two truncated E6* proteins, the latter being generated via splicing within the E6 open reading frame portion of the E6-E7 polycistronic transcript. Those types, such as HPV6, that are infrequently associated with cervical carcinoma lack the splice site and encode only a full-length E6. We have now found that, in addition to cooperating with E7 to immortalize keratinocytes, HPV16 E6 can induce anchorage-independent growth in NIH 3T3 cells and trans-activate the adenovirus E2 promoter. HPV6 E6 was also able to trans-activate the adenovirus E2 promoter, although it was inactive in both cell transformation assays. An HPV16 splice site mutant which expressed only the full-length HPV16 E6 was active in all three assays, indicating that the E6* proteins are not required for these activities. The plasmid which encodes the E6* proteins was inactive and did not potentiate the activity of the HPV16 splice site mutant. The mutation that prevented splicing in E6-E7 mRNA severely reduced the level of E7 protein and increased E6 protein. Taken together, the results suggest that the primary function of the splice within E6 is to facilitate the translation of E7 and reduce translation of full-length E6, rather than to generate biologically active E6* proteins.