Altered expression and distribution of the cytoskeletal-associated p35 protein in NIH 3T3 cells transformed with the Harvey sarcoma virus v-ras oncogene

1. Cytoskeletal events associated with retroviral oncogene (v-ras)-mediated transformation were studied in NIH 3T3 fibroblasts and their v-ras-transfected counterparts (3T3/H-1 cells). 2. Abnormal microfilament networks seen in 3T3/H-1 cells reflected significant decreases (approximately 90%) in two cytoskeletal-associated proteins (tropomyosin-1, p35). Neither actin content nor actin mRNA levels were altered, however, v-ras transfectants. 3. p35 mRNA activity in both NIH 3T3 and 3T3/H-1 cells was similar although differential compartmentalization of p35 to the detergent-resistant cytoskeletal fraction was evident only in normal fibroblasts. 4. Proper cytoskeletal organization may be a factor in the regulation of p35 mRNA translation in situ or influence the stability of p35 independent of translational rate.     

NIH 3T3 cells transfected with human tumor DNA lose the transformed phenotype when treated with swainsonine

Phenotypic expression of transformation was inhibited by swainsonine at concentrations which affect the late stages of glycoprotein processing but not growth of cells. In the presence of swainsonine, NIH 3T3 fibroblasts transfected with human tumor DNA (al-l) no longer grew in soft agar or expressed complex type oligosaccharides characteristic of transformed cells. Thus, it appears that glycoproteins with fully processed oligosaccharides are necessary for the maintenance of the transformed phenotype in these cells.     

Reverse transformation of Harvey murine sarcoma virus-transformed NIH/3T3 cells by site-selective cyclic AMP analogs

Eighteen site-selective cAMP analogs modified at either the C-8 position or the C-6 position were tested for their growth regulatory effects on the Harvey murine sarcoma virus-transformed NIH/3T3 clone 13-3B-4 cells grown in a serum-free defined medium. All 18 analogs, when tested individually, exhibited an appreciable growth inhibitory effect at micromolar concentrations. The most potent growth inhibitory analogs contained a thio moiety at the C-8 position. In general, C-6 analogs required 5-10-fold greater concentrations than C-8 analogs to produce the same degree of growth inhibition. The growth inhibition induced by these analogs was accompanied by a change in cell morphology; cells treated with the analogs exhibited the morphology characteristic of untransformed fibroblasts, while untreated cells retained a transformed phenotype. The regulatory subunit of cAMP-dependent protein kinase, the cAMP receptor protein, has two different intrachain cAMP binding sites, and cAMP analogs modified at the C-8 position (C-8 analogs) are generally selective for Site 1, while analogs modified at the C-6 position (C-6 analogs) are generally selective for Site 2. Thus, C-8 and C-6 analogs were tested in combination to enhance the growth regulatory effect. Both growth inhibition and morphological change were enhanced synergistically by a combination of the C-6 and C-8 analogs. Two C-6 analogs or two C-8 analogs added together did not cause synergism. For both growth inhibition and phenotypic change, C-8 thio analogs acted far more synergistically than C-8 amino analogs when cells were treated in combination with C-6 analogs, suggesting a response of the RII rather than the RI cAMP receptor protein. DEAE-cellulose chromatography revealed that the growth inhibition, in fact, correlates with an increase of the RII cAMP receptor protein and a decrease of the RI receptor protein. The growth inhibitory effect of the site-selective analogs was not due to the cytotoxic effect of adenosine metabolites as shown by the different behavior of 8-Cl-cAMP compared with 8-Cl-adenosine in 1) cell cycle effects and 2) release from growth inhibition. It is concluded that the observed growth inhibition and phenotypic reversion of 13-3B-4 cells is most likely mediated through the cellular effector, the RII cAMP receptor protein.     

Helper-independent transformation by unintegrated Harvey sarcoma virus DNA.

We have studied the unintegrated infectious DNA of Harvey sarcoma virus (Ha-SV) and Moloney leukemia virus (Mo-MuLV). The source of infectious viral DNA was the Hirt supernatant fraction from cells acutely infected with Ha-SV and Mo-MuLV. To obtain a direct quantitative assay for infectious viral DNA, recipient mouse cells were first exposed to calcium phosphate-precipitated viral DNA and then treated with dimethyl sulfoxide. Infectivity was monitored by focus formation for Ha-SV and XC plaque formation for Mo-MuLV. The viral DNA titration pattern followed single-hit kinetics for both foci and plaques, indicating that a single molecule carried information for each function. Focus-forming and plaque-forming activity were present in different molecules, since these two biological activities could be separated from each other by agarose gel electrophoresis. The focus-forming molecule was linear DNA with a molecular weight of about 4 x 10(6) daltons. The focus-forming activity of the viral DNA was sensitive to EcoRI and resistant to XhoI restriction endonucleases, whereas the plaque-forming activity was resistant to EcoRI and sensitive to XhoI. The generation of helper-independent foci indicates that Ha-SV DNA can transform mouse cells in the absence of helper virus or its proteins.     

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

Internalization and recycling of CD4 transfected into HeLa and NIH3T3 cells.

The internalization of CD4, a T cell differentiation antigen and the receptor for the human immunodeficiency viruses (HIV-1 and -2), has been examined in HeLa and murine 3T3 cells transfected with CD4 cDNA. Fab' fragments of the anti-CD4 monoclonal antibody Leu3a were generated by pepsin digestion and used as a specific monovalent, non-crosslinking ligand for CD4. These Fab' fragments were shown to bind to CD4 on the transfected cells with an affinity similar to that of HIV gp120, and inhibited HIV infection of lymphocytic cells. The Fab' fragments were radioiodinated and used in an acid-stripping endocytosis assay to demonstrate that the CD4 expressed on transfected HeLa and NIH3T3 cells was internalized. Approximately 1.5-2% of the total cell-bound [125I]Fab' fragments were internalized per minute. Furthermore, the internalized [125I]Fab' fragments could be shown to recycle to the cell surface. After 30-60 min a steady state was reached between internalization and recycling, with approximately 30-40% of the total cellular CD4 pool residing inside the cell. Similar results were obtained in studies with the intact divalent radiolabelled Leu3a antibody. These data demonstrate that CD4 expressed on transfected non-lymphoid cells is constitutively endocytosed and recycled.     

Physical map of biologically active Harvey sarcoma virus unintegrated linear DNA.

BALB/c JLS V9 cells recently infected with Harvey sarcoma virus-murine leukemia virus (HSV-MuLV) complex contained unintegrated HSV linear DNA of 6.0-kilobase pair mass. The cells also contained two HSV closed circular DNA species along with MuLV-encoded linear and closed circular DNA species. HSV 6.0-kilobase pair linear DNA induced focal transformation upon transfection of NIH 3T3 mouse fibroblasts, and the biological activity of HSV DNA did not require helper MuLV functions. A physical map of restriction endonuclease cleavage sites along HSV 6.0-kilobase pair linear DNA was derived. Comparison of this map with one for Moloney MuLV DNA showed that the HSV and Moloney MuLV genomes are identical near their viral RNA 3' ends.     

Cell surface glycolipids of transformed NIH 3T3 cells transfected with DNAs of human bladder and lung carcinomas.

Neutral glycolipids and gangliosides of NIH 3T3 cells oncogenically transformed by transfection of DNAs from human lung carcinoma (Lx-1) and human bladder carcinoma (Ej) have been investigated. The chemical quantity and the degree of cell surface exposure of gangliotriaosylceramide (Gg3) were greatly enhanced in NIH 3T3 cells transformed by transfection of DNAs of either Lx-1 or Ej carcinoma cells. An identical but more conspicuous change in cell surface exposure of Gg3 was observed in BALB/c 3T3 cells transformed by murine sarcoma virus Kirsten strain, but the same glycolipid was absent in the original Lx-1 or Ej human carcinomas. The mechanism that defines the chemical quantity and the organization of glycolipids is controlled by multiple factors. These include not only the quantity but also the organization of glycosyl transferases and hydrolases in membranes. This also involves membrane dynamics regulated through a cytoskeletal-membrane conjunction which may determine the degree of glycolipid exposure at the cell surface. The similarity of the chemical and organizational change of a single glycolipid, Gg3, between 3T3 transformants by Kirsten murine sarcoma virus and those by transfection of human cancer DNAs may indicate a common biochemical basis triggered by activation of the oncogene.     

Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells.

Clinical evidence suggests that hepatitis C virus (HCV) is etiologically involved in hepatic cancer and liver cirrhosis. To investigate whether the HCV nonstructural protein NS3 has oncogenic activity, NIH 3T3 cells were transfected with an expression vector containing cDNA for the 5'- or 3'-half sequence of the HCV genome segment encoding NS3. Only cells transfected with the 5'-half cDNA rapidly proliferated, lost contact inhibition, grew anchorage independently in soft agar, and formed tumors in nude mice. PCR analysis confirmed the presence of the 5'-half DNA in the transfectants. These results suggest that the 5' region of the HCV genome segment encoding NS3 is involved in cell transformation.     

An altered DNA sequence encompassing the ras gene of Harvey murine sarcoma virus.

The DNA fragment encompassing the ras gene of Harvey murine sarcoma virus was sequenced and assigned the coding region of a transforming protein, p21, to the sequence. Examination of nucleotide sequence, taken together with the result of analysis of the ras mRNAs (1), has revealed that p21 is encoded from a continuous coding region starting with the 5' proximal initiation codon but not a processed protein. However, there were found several differences between the sequence published by Dhar et. al. (2) and ours, including 9 deletions, 7 substitutions and 2 insertions of nucleotides in the published sequence of 997 nucleotides in length. Among these, one of the substitutions occurring in the coding region resulted in amino acid replacement of glycine by alanine at position 122 of p21. The evidences are presented with some of actual gel autoradiographs.     

Transformation of NIH 3T3 cells by DNA of the MCF-7 human mammary carcinoma cell line induces expression of an endogenous murine leukemia provirus.

Previous studies identified two glycoproteins of 86 (gp86) and 72 (gp72) kilodaltons and two nonglycosylated proteins of 70 (p70) and 19 (p19) kilodaltons which were specifically expressed in NIH cells transformed by DNA of the MCF-7 human mammary carcinoma cell line. Pulse-chase experiments and the use of tunicamycin to inhibit glycosylation suggested that gp86, gp72, and p19 were related as precursor products. Characteristics of the four transformation-associated proteins resembled those of murine leukemia virus (MuLV) proteins. Sera raised against disrupted MuLV immunoprecipitated the same four proteins in extracts of NIH(MCF-7) cells and MuLV-infected NIH 3T3 cells. In addition, a monoclonal antibody against MuLV gp70 immunoprecipitated proteins gp86 and gp72, whereas a monoclonal antibody against MuLV p15(E) immunoprecipitated gp86 and p19. These results indicate that proteins gp86, gp72, and p19 expressed in NIH(MCF-7) transformants correspond to MuLV envelope proteins gp80env, gp70, and p15(E), respectively. The transformation-associated protein p70 appears to be a non-envelope MuLV protein, most likely p65gag. Northern blot analysis confirmed that transformation of NIH cells by MCF-7 mammary carcinoma DNA led to the induction of an endogenous MuLV provirus.     

Uptake of cadmium is diminished in transfected mouse NIH/3T3 cells enriched for metallothionein.

To determine the relationship between cellular uptake of cadmium and content of metallothionein, we measured uptake of 109Cd in cells that differed in content of metallothionein (MT). MT cells were derived from NIH/3T3 cells by transfection with a plasmid containing the genome of bovine papilloma virus and the mouse metallothionein-I gene, driven by the promotor for the glucose-regulated protein of 78 kDa. Control cells were similarly transfected with bovine papilloma virus-based plasmids with the gene for metallothionein inverted and thus separated from the promoter (TM), or deleted, along with the promoter (BPA). The number of copies of bovine papilloma virus-based plasmids was similar in MT, TM, and BPA cells, approximately 100 per cell. MT cells were more than 10 times more resistant to the lethal effect of cadmium than were the control cells. Synthesis of metallothionein was 15-fold greater in the MT cells than in the TM or BPA cells. The uptake of 109Cd by the cells enriched in metallothionein was 4-fold less than by the control cells. These data suggest that an increased content of metallothionein may protect some cells from the toxic effects of cadmium, in part, by diminishing uptake of the metal.     

Effect of mycoplasma on polyamine synthesis and levels in NIH 3T3 cells and in cells transformed by Rous sarcoma virus

Abstract Infecting NIH 3T3 cells with different species of mycoplasmas resulted only in a slight decrease in ornithine decarboxylase (ODC) activity and in the appearance of cadaverine in the infected cells. Similarly, the presence of mycoplasma in NIH 3T3 cells infected with a temperature-sensitive mutant of Rous Sarcoma virus did not bring about any significant changes either in the pattern of ODC activity or in putrescine levels, when transferred to the permissive temperature. This indicates that mycoplasmal contamination of cultures may not significantly change the putrescine metabolism in host cells. On the other hand, the presence of cadaverine in cultured cells may be attributed to contamination by mycoplasma.     

Resistance to anticancer drugs in NIH3T3 cells transfected with c-myc and/or c-H-ras genes

NIH3T3 cells transfected with c-H-ras and/or c-myc genes were examined for differences in drug sensitivity. The two transfectants used were NIH3T3-nm-1 (nm-1), pT22-3-nm-2. They were transfected with c-myc, c-myc plus activated c-H-ras, respectively. The relative resistances (IC50 values of transfectants/those of NIH3T3 cells) to cisplatin, adriamycin, 4-hydroperoxycyclophosphamide, melphalan, and CPT-11 were 2.1, 1.6, 4.7, 4.9, 1.6, respectively for nm-1 and 1.6, 2.2, 3.3, 9.1 and 2.2, respectively for nm-2. These results strongly suggest that the expression of the c-myc gene plays a role for the acquisition of drug resistance. The c-myc gene is believed to provide us an important clue for determining the mechanism of drug resistance.     

NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype.

In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.     

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.     

Functional organization of the Harvey murine sarcoma virus genome.

The comparative infectivity of Harvey murine sarcoma virus (Ha-MuSV) DNA for NIH 3T3 cells was determined for supercoiled Ha-MuSV DNA molecularly cloned in lambda phage and pBR322 at its unique EcoRI site (which is located near the middle of the 6-kilobase pair [kbp] unintegrated linear viral DNA) and for two cloned subgenomic fragments: one was 3.8 kbp and lacked about 1 kbp from each side of the EcoRI site, and the second did not contain the 3 kbp of the unintegrated linear viral DNA located on the 3' side of the EcoRI site. Each subgenomic DNA induced foci of transformed cells, but with a lower relative efficiency then genomic DNA. Transfection with intact vector Ha-MuSV DNA yielded results similar to those obtained after separation of Ha-MuSV DNA from vector DNA. Cells lines were then derived from individual foci transformed with each type of viral DNA. Focus-forming virus was recovered from transformed cells after superinfection with a helper-independent virus, but the efficiency varied by several orders of magnitude. For several transformed lines, the efficiency of recovery of focus-forming virus was correlated with the structure of the Ha-MuSV DNA in the cells before superinfection. When 32P-labeled Ha-MuSV DNA probes specific for sequences on either the 3' or 5' side of the EcoRI site were used to analyze the viral RNA in the transformed cell lines, all lines were found to hybridize with the 5' probe, but some lines did not hybridize with the 3' probe. The transformed lines contained high levels of the Ha-MuSV-coded p21 or its associated GDP-binding activity. We conclude that the transforming region and the sequences that code for the viral p21 protein are both located within the 2 kilobases closest to the 5' end of the Ha-MuSV genome.     

Mitochondrial DNA from colorectal cancer cells promotes the malignant phenotype of NIH3T3 cells

We investigated the potential role of mitochondrial DNA (mtDNA) in colorectal carcinogenesis by constructing a eukaryotic expression vector of the mitochondrial D-loop gene from colorectal cancer cell SW480 and transfected NIH3T3 cells. The NIH3T3/SW480 cells exhibited a significantly increased growth rate and colony formation rate, and also had a decreased apoptotic rate. Polyploidy and aberrant chromosomes were detected in the NIH3T3/SW480 cells by chromosome karyotype analysis. Our results suggested that mtDNA from colorectal cancer cells promotes the malignant phenotype of NIH3T3 cells. Further study of the biological functions of NIH3T3/SW480 cells might be helpful in understanding the role of mtDNA in colorectal carcinogenesis.     

Expression of anti-sense mRNA in H-ras transfected NIH/3T3 cells does not suppress the transformed phenotype

We have attempted to reverse the transformed phenotype of cells expressing the H-ras oncogene. A plasmid in which the first exon of the H-ras oncogene was coupled to the SV40 early promoter in an anti-sense orientation was constructed. This construct was introduced into a clone of H-ras-transformed NIH/3T3 cells. Simultaneous expression of both the SV40 anti-sense construct and H-ras was observed. Anti-sense RNA was present in a 10-20-fold excess over sense H-ras RNA. Only a small fraction of the cytoplasmic RNA was present in a sense: anti-sense duplexed form. The expression of anti-sense H-ras RNA was not accompanied by a phenotypic reversion of transformed cells. The only phenotypic reversion we observed was accompanied by a loss of transfected H-ras sequences. The loss of transfected H-ras sequences occurs with a high frequency in cells supertransfected with the SV40 anti-sense construct.