Temperature dependency for maintenance of transformation in mouse cells transformed by simian virus 40 tsA mutants.

Mouse embryo fibroblasts and 3T3 cells were transformed by wild-type, tsB4, tsA7, tsA58, and tsA209 simian virus 40. Clones of transformants were generated both in soft agar and in liquid medium by focus formation and at both high and relatively low multiplicities of infection. All transformants were assayed for three phenotypes of transformation: (i) the ability to form highly multinucleated cells in cytochalasin B-supplemented medium, i.e., uncontrolled nuclear division; (ii) the capacity to continue DNA synthesis at increasing cell density; and (iii) the ability to form colonies in soft agar. The great majority of mouse embryo fibroblast transformants generated with tsA mutant virus were temperature sensitive for transformation in all three assays, regardless of the input multiplicity or whether they were generated in liquid medium or soft agar. These transformants exhibited a normal or near-normal phenotype at the nonpermissive temperature of 40 degrees C. All but one of the transformants which appeared transformed at both temperatures were in the A209 group. In contrast to mouse embryo fibroblasts, transformants generated with 3T3 cells and tsA virus were often not temperature sensitive, exhibiting the transformation phenotypes at both temperatures. This phenomenon was more often observed when 3T3 transformants were generated in soft agar. These results, along with other published data, suggest that uncontrolled nuclear division and uncontrolled DNA synthesis are a function of the simian virus 40 A gene. Finally, with the 3T3 transformants, there was often discordance in the expression of transformation among the three phenotypes. Some tsA transformants were temperature sensitive in one of two assays but were transformed at both 33 and 40 degrees C in the remaining assay(s). Other transformants exhibited a normal cytochalasin B response at either temperature but were temperature sensitive in the other assays.     

Temperature-sensitive growth regulation in one type of transformed rat cells induced by the tsa mutant of polyoma virus.

A fibroblast line of the 3T3 type with a low saturation density was established from Fisher rat embryo cells. After infection with either wild-type or tsa mutant polyoma virus, transformants were isolated and cloned at 33 degrees C on the basis of their ability either to grow as dense foci on plastic in liquid medium (type N) or to form colonies in soft agar (type A). Polyoma T antigen was detected in all of the transformed lines. The following growth characteristics were studied for both types at 33 and 41 degrees C: saturation density, growth in soft agar and at a low serum concentration, colony-forming ability, and generation time. tsa-N transformants behaved at 33 degrees C similarly to transformed cells, but reverted at 41 degrees C to the nontransformed phenotype for all of these characters. tsa-A transformants and all of the wild-type transformants exhibited the transformed phenotype at both low and high temperatures. These results led us to distinguish at least two types of virus-induced transformants. In one of them, the activity of the protein affected by the tsa mutation appears to be necessary for the expression of several of the characters defining the transformed state.     

Transformation of BALB/c-3T3 cells by tsA mutants of simian virus 40: effect of transformation technique on the transformed phenotype.

Simian virus 40 tsA-transformed BALB/c-3T3 cells isolated as foci of overgrowth in liquid medium were compared with those isolated as colonies in soft agar. Efficiencies of transformation were equivalent in the two procedures. Cells isolated as foci were able to grow in agar and vice versa. No difference in temperature sensitivity of the transformed phenotype was detected when tsA transformants selected in agar were compared with those selected as foci. The use of the two different transformation procedures, then, did not form the basis for generation of different transformed phenotypes, and transformants generated in both ways were dependent upon expression of the A gene for maintenance of the transformed state.     

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.     

trans-Dominant suppressor mutations of the H-ras oncogene

Site-directed mutagenesis of the conserved sequence motifs of p21 generated a group of mutant p21s defective in GTP binding. Some of these mutants were highly transforming, whereas others were transformation defective. Among the latter group, we found two mutants, derived from the v-H-ras oncogene by substituting the asparagine-116 with tyrosine and isoleucine, that exhibited a trans-dominant activity of suppressing the transformed phenotype of NIH3T3 cells induced by a long terminal repeat-linked c-H-ras and a wild-type v-H-ras. They caused reduction of the colony-forming efficiency in soft agar (78% in c-ras-transformed cells; 55% in v-ras cells) and morphological reversion of ras transformants. Subclones of revertants expressed a great excess of mutant p21 relative to the c-ras p21 present in these cells. These mutants were not lethal to NIH3T3 cells. Apparently, defective proteins encoded by suppressor mutants sequestered vital targets for ras function. Suppressor mutants also induced morphological reversion of NIH3T3 cells transformed by src, fes/flp, sis, and fms oncogenes, suggesting that these oncogenes function upstream to ras in the signaling pathways. Cells transformed by mos and a chemical carcinogen were unaffected.     

Rat cells transformed by simian virus 40 give rise to tumor cells which contain no viral proteins and often no viral DNA.

Rat 3T3 cells transformed by simian virus 40 were injected into rats to examine their capacity to develop into tumors. Both large T-dependent (N) transformants and large T-independent (A) transformants were used. All the transformed cell lines contained large T and small t and could multiply efficiently in agar. Only some transformants could develop into tumors. All tumor cells examined had lost both large T and small t. Tumor cells in which the viral genome could still be detected were found together with tumor cells in which the simian virus 40 DNA could no longer be detected. N transformants which displayed the transformed phenotype in a temperature-sensitive manner became temperature insensitive during tumor formation.     

Unstable transformation of mouse 3T3 cells by transfection with DNA from normal human lymphocytes.

DNA fragments (0.5-4.5 kb) of normal human lymphocytes induced pre-neoplastic mouse NIH/3T3 cells after transfection to grow in soft agar medium at low efficiency (0.0007 colonies/micrograms DNA/10(6) cells). In secondary transfections high mol. wt. DNA (greater than 20 kb) of cells transformed by DNA fragments induced neoplastic transformation with high efficiency (0.16-1.1 soft agar colonies/micrograms DNA/10(6) cells). These results confirm previous data obtained by others with chicken and mouse donor DNA. We describe here that independent secondary transformants harbored human Alu repetitive DNA sequences on similar restriction fragments and formed progressively growing tumors in BALB/c mice or nude mice. The corresponding primary transformants were not tumorigenic, however, and the ability to proliferate in semi-solid agar medium was gradually lost when the cells were grown as non-confluent monolayers. Furthermore, in contrast to secondary transformants, DNA from primary transformants showed only relatively weak hybridization to a human Alu repetitive DNA probe. We conclude that in primary transformants the transformed phenotype is expressed in an unstable fashion whereas secondary transformants appear to be stably transformed.     

Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.     

Conditions leading to the establishment of the N (a gene dependent) and A (a gene independent) transformed states after polyoma virus infection of rat fibroblasts.

Infection of normal rat fibroblasts (FR 3T3) with the early tsa mutant of polyoma virus may lead to either the A or the N phenotype, tsa-A transformants, originally derived by agar selection, are not temperature dependent for maintenance of the transformed phenotype, whereas tsa-N transormants revert at high temperature to normal growth control. A transformants did not result from an independent cellular mutation selected in agar medium, but rather from a transformation process distinct from that leading to the N state. It occurred in both liquid and agar media when the infected cells were maintained under growth-restricting conditions, such as absence of anchorage and contact inhibition at confluency. N transformation occurred in cells maintained in active growth after virus infection (sparse cultures on a solid substratum). Physiological conditions during a critical period after virus infection thus appear to be a crucial parameter of the transformation process.     

Adenovirus type 12 gene 401 function and maintenance of transformation.

Rat (3Y1) and hamster embryo brain cells were transformed by wild-type adenovirus type 12 or the DNA-minus temperature-sensitive mutant ts401. The ts401-transformed 3Y1 cells, but not the wild-type transformants, displayed a temperature-sensitive response with respect to the following characteristics of the transformed phenotype: morphology, saturation density, growth rate, cloning in soft agar, colony formation on plastic at low cell densities in 1% serum medium, and the T antigen(s). Temperature shift-down experiments showed that the density-dependent inhibition of growth of the ts401-transformed cells was reversible, as was, to some extent, the low efficiency of colony formation at low cell densities in 1% serum. Examination of hamster transformants for their ability to clone in soft agar at permissive and nonpermissive temperatures showed that this property was temperature dependent, again only in the ts401 transformants and not in the wild-type transformants. Alteration in uptake of 2-deoxyglucose or in intracellular cyclic AMP content was not a characteristic of the adenovirus-transformed phenotype in the 3Y1 cells. The findings suggest that an active 401 function is required for maintenance of the adenovirus-transformed cell pheno-type.     

Murine gamma interferon inhibits v-mos-induced fibroblast transformation via down regulation of retroviral gene expression.

Expression of the retroviral vector Neor myeloproliferative sarcoma virus (MPSV), which contains the v-mos oncogene and the neomycin resistance gene, leads to neoplastic transformation of mouse fibroblasts. Murine recombinant gamma interferon (IFN-gamma) could revert the neoplastic properties of established Neor MPSV-transformed cell lines to an apparently untransformed phenotype. In the presence of IFN-gamma, the Neor MPSV transformants showed a greater than 97% reduction of cloning efficiency in soft agar, strongly reduced proliferative capacity, and morphological changes. The IFN-gamma-induced phenotypic reversion was preceded by a rapid and selective reduction of all retroviral RNA species, apparently due to IFN-gamma action on the long terminal repeat of Neor MPSV. The mRNA levels of cellular genes either remained unaffected (beta-actin) or were even enhanced (H-2) in IFN-gamma-treated Neor MPSV-transformed cells. Upon removal of IFN-gamma, retroviral gene expression was fully recovered and a gradual reappearance of the transformed phenotype of these cells within 3 weeks was noted. These data show that IFN-gamma can cause a virtually complete, but reversible, inhibition of v-mos-induced neoplastic properties in transformed fibroblasts by selective down regulation of retroviral RNA levels.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

Biological and biochemical studies of cells transformed by simian virus 40 temperature-sensitive gene A mutants and A mutant revertants.

The growth properties of hamster cells transformed by wild-type Simian virus 40 (SV40), by early SV40 temperature-sensitive mutants of the A complementation group, and by spontaneous revertants of these mutants were studied. All of the tsA mutant-transformed cells were temperature sensitive in their ability to form clones in soft agar and on monolayers of normal cells except for CHLA-30L1, which was not temperature sensitive in the latter property. All cells transformed by stable revertants of well-characterized tsA mutants possessed certain growth properties in common with wild-type-transformed cells at both temperatures. Virus rescued from tsA transformants including CHLA30L1 was temperature sensitive for viral DNA replication, whereas that rescued from revertant and wild-type transformants was not thermolabile in this regard. T antigen present in crude extracts of tsA-transformed cells including CHLA30L1, grown at 33 degreeC, was temperature sensitive by in vitro immunoassay, whereas that from wild-type-transformed cells was relatively stable. T antigen from revertant transformants was more stable than the tsA protein. Partially purified T antigen from revertant-transformed cells was nearly as stable as wild-type antigen in its ability to bind DNA after heating at 44 degrees C, whereas T antigen from tsA30 mutant-transformed cells was relatively thermolabile. These results further indicate that T antigen is a product of the SV40 A gene. Significantly more T antigen was found in extracts of CHLA30L1 grown to high density at the nonpermissive temperature than in any other tsA-transformed cell similarly grown. This is consistent with the suggestion that the amount of T antigen synthesized in CHLA30L1 is large enoughto allow partial expression of the transformed phenotype at the restrictive temperature. Alternatively, the increase in T antigen concentration may be secondary to one or more genetic alterations that independently affect the transformed phenotype of these cells.     

Polyoma virus middle t antigen: a tumor progression factor.

Polyoma virus (PyV) deletion mutant dl23 (affecting both large T and middle t but not small t antigens) was used to study transformation of 3T3 rat cells. This mutant generated stable transformants in the agar assay at a frequency similar to that of wild-type virus (WT). However, WT-induced transformants were detected 3 weeks after infection, whereas those induced by the mutant could not be detected until 6 to 8 weeks after infection. In this respect, dl23 PyV behaved like WT simian virus 40 (SV40). Cells transformed by WT SV40 or by dl23 PyV were similar in all their transformed properties. Those transformed by WT PyV were different from the others on the basis of morphology, cell adhesion to the substrate, release of protease activity, efficiency of doubling in agar, growth rate, and time required for tumor formation. Saturation density, the ability to grow in agar, the serum requirement for cloning, and the ability to grow on a cell monolayer were similar for all transformants. Middle t antigen enhanced membrane alterations and growth rate of the transformed cells, shortening the time required for tumor formation in rats.     

Unstable expression and amplification of a transfected oncogene in confluent and subconfluent cells.

NIH 3T3 cells were transfected with a plasmid containing the transforming gene, v-src, from Rous sarcoma virus. One of the transformed cell lines isolated reverted to a flat, nontransformed morphology after cloning through soft agar. This cell line did not express the src gene and could no longer grow in soft agar. When these cells were held at confluence, spontaneous foci appeared which eventually covered the dish. The appearance of foci correlated with an increase in v-src gene expression, ability to grow in soft agar, and tumorigenicity in mice. When these transformed cells were trypsinized and held at subconfluence, both v-src expression and the transformed phenotype were progressively lost. Whereas rearrangement of the transfected gene was not detected, the gene copy number in the transformed cells was markedly increased (greater than 50-fold). Confluence-dependent gene amplification and deamplification have been retained after several cycles of growth alternately at high and low density, in cells recloned through soft agar, and after cells had been maintained continuously at high or low density. The results suggest that, in this cell line, reversible gene amplification plays a central role in expression of the transfected gene.     

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.     

The genetic stability of Penicillium chrysogenum transformants in a fermentor

Summary A number of transformants of Penicillium chrysogenum have been obtained with the plasmid vector p3SR2. Southern analysis showed that transformation had occurred by integration of vector sequences into the nuclear DNA of the fungus. A number of transformants were tested for stability of the transformed phenotype in agar medium and some were found to be unstable. Two transformants, shown to be stable in agar culture, were grown in 5-l batch fermentors for further stability tests. Over periods of up to 312 h in the fermentor both transformants were 100% stable with respect to the transformed phenotype. In addition Southern analysis of DNA extracted from the spent mycelium showed that no change had occurred in the position of the integrated vector sequences within the transformant nuclear DNA.     

The propeptide domain of lysyl oxidase induces phenotypic reversion of ras-transformed cells

Lysyl oxidase is an extracellular enzyme critical for the normal biosynthesis of collagens and elastin. In addition, lysyl oxidase reverts ras-mediated transformation, and lysyl oxidase expression is down-regulated in human cancers. Since suramin inhibits growth factor signaling pathways and induces lysyl oxidase in ras-transformed NIH3T3 cells (RS485 cells), we sought to investigate the effects of suramin on the phenotype of transformed cells and the role of lysyl oxidase in mediating these effects. Suramin treatment resulted in a more normal phenotype as judged by growth rate, cell cycle parameters, and morphology. beta-aminopropionitrile, the selective inhibitor of lysyl oxidase enzyme activity, was remarkably unable to block suramin-induced reversion. By contrast, ectopic antisense lysyl oxidase demonstrated that lysyl oxidase gene expression mediated phenotypic reversion. Since lysyl oxidase is synthesized as a 50 kDa precursor and processed to a 30 kDa active enzyme and 18 kDa propeptide, the effects of these two products on the transformed phenotype of RS485 cells were then directly assessed in the absence of suramin. Here we report, for the first time, that the lysyl oxidase propeptide, and not the lysyl oxidase enzyme, inhibits ras-dependent transformation as determined by effects on cell proliferation assays, growth in soft agar, and Akt-dependent induction of NF-kappaB activity. Thus, the lysyl oxidase propeptide, which is released during extracellular proteolytic processing of pro-lysyl oxidase, functions to inhibit ras-dependent cell transformation.     

Retention of herpes simplex virus type II sequences in bglII n-transformed cells after co-transfection with a selectable marker.

Transformation of mammalian cells by total u.v.-inactivated herpes simplex virus II (HSVII) or cloned fragments thereof (BglII n, BglII C) has been complicated both by a low efficiency of oncogenic transformation and the disappearance of viral DNA and/or viral products initially detected in the transformed cell lines. In an attempt to effect a stable integration of BglII n and to elucidate the role of HSVII in oncogenic transformation, we have co-transfected NIH 3T3 cells with pAG60, a plasmid which confers resistance to the G418 antibiotic, and plasmids containing either BglII n in its entirety (pNB2) or one of five subfragments of BglII n. Several isolated clones exhibit a transformed phenotype as expressed by rapid growth in low serum concentrations and colony formation in soft agar. We have obtained a markedly reduced frequency of biochemical transformants when co-transfecting pNB2 in comparison with the numbers obtained when cotransfecting the five subfragments. Furthermore, a greater proportion of subfragment-transfected colonies contain viral DNA, and in higher copy number, than observed in the pAG60/pNB2 clones. We have also found viral DNA to be more stably integrated in the subfragment-transfected clones than in the pNB2-transfected clones.     

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.