The majority of independently transformed BHK cell clones share a single functional lesion which determines anchorage independence and influences tumorigenicity

Summary Expression of the anchorage-independent transformed phenotype in BHK 21/13 cells generally behaves as a recessive trait. When chemically induced and spontaneously arising transformants are fused to the nontransformed parent line, transformation is initially suppressed, reappearing after extended growth of the hybrids. In this paper, complementation for the expression of anchorage independence was sought among a large group of such transformants, all independently derived from BHK 21/13 cells. Tumorigenicity studies on selected hybrids and parental lines indicated that the in vitro trait of anchorage independence is an accurate indicator of in vivo neoplasia for these cells. Seventeen of the 18 clones tested did not complement one or more of three tester strains. This result indicates that anchorage independence arose in these clones as a result of lesions in the same genetic function and suggests that the final step in the progressive changes of carcinogenesis may frequently be restricted to lesions at a single locus. This investigation was supported by National Institutes of Health grant CA27306.     

Induction of a step in carcinogenesis that is normally associated with mutagenesis by nonmutagenic concentrations of 5-azacytidine.

The permanent cell line BHK-21/cl 13 can be transformed by mutagenic carcinogens as the result of the induction of a recessive somatic mutation. Yet when these cells were treated with 5-azacytidine under conditions in which no mutants resistant to either ouabain or 6-thioguanine could be detected, they were transformed efficiently. These transformants were induced, not selected. 6-Azacytidine was ineffective at transforming BHK cells; 2'-deoxy-5-azacytidine was exceptionally effective. When tested by cell fusion, transformants induced by 5-azacytidine fell into the same complementation group as those induced by highly mutagenic carcinogens, but they were phenotypically distinct in that they were unstable during prolonged passage and rarely displayed the temperature-limited phenotypes so common among BHK transformants induced by strongly mutagenic carcinogens. These results raise the possibility that a cell can be induced by either genetic or epigenetic means to traverse the same single step in carcinogenesis.     

Suppression of the transformed phenotype of hepatoma cells after hybridization with normal diploid fibroblasts

Hybrids were generated between mouse hepatoma cells which exhibit a transformed phenotype, and rat normal diploid fibroblasts. Most isolated hybrid clones contain a single set of chromosomes from each parent. Such clones grow to low saturation densities and are unable to grow or to form colonies in soft agar. The transformed phenotype of the parental hepatoma cells is thus suppressed in these hybrids. Suppression is very stable; however, subclones which have regained a transformed phenotype could be selected; these subclones show a significant reduction of their chromosome number. Amongst the hybrid clones isolated after fusion, a few are characterized by an excess of mouse chromosomes and a reduced number of rat chromosomes. Such clones exhibit a transformed phenotype. Our results show that, provided the hybrids contain an almost complete single set of chromosomes of each parent, spontaneous transformation behaves as a recessive trait in hybrids formed with normal diploid cells.     

Suppression of the chemically transformed phenotype of BHK cells by a human cDNA.

Transformation of the baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis (A. Stoler and N. P. Bouck, Proc. Natl. Acad. Sci. USA 82:570-574, 1985). A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent normal cellular phenotype after transfection with human cDNA. The human cDNA capable of inducing stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.     

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.     

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.     

Expression of the ASV src gene in hybrids between normal and virally transformed cells: Specific suppression occurs in some hybrids but not others

Somatic cell hybrids have been made between clones of rat cells transformed by avian sarcoma virus and rat or mouse cells that are untransformed. Intraspecies hybrids were either predominantly morphologically normal or predominantly transformed, some clones that formed transformed intraspecies hybrids yielding normal interspecies hybrids. Untransformed hybrids usually showed no detectable alteration in the structure or location of the integrated provirus, but viral RNA and pp60^src kinase activities were much reduced. No decrease in viral gene expression was seen in transformed hybrids. Thus hybrid suppression of viral transformation, mediated in trans by the untransformed parent, is a specific event that depends on both untransformed and transformed parental parameters.     

Collagen synthesis in normal BHK cells and temperature-sensitive chemically transformed BHK cells.

Collagen synthesis in normal BHK 21/cl 13 and chemically transformed temperature sensitive BHK 21/cl 13 cells (Me2N4) was assessed by examination of hydroxyproline formation and collagenase-susceptible protein. The Me2N4 cells lost their ability to synthesize collagen at both permissive and nonpermissive temperatures for transformation. These conclusions were confirmed by polyacrylamide-gel eletrophoresis and CM-cellulose chromatography. Prolyl hydroxylase activity was present in both normal and transformed cells even when no collagen could be demonstrated. The production of noncollagen protein, although decreased in the transformed cell, did not change as drastically as the collagen synthesis.     

Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803

The facultative heterotrophic cyanobacterium Synechocystis sp. strain PCC 6803 was transformed by HaeII Cmr fragments ligated at random to HaeII DNA fragments of the host genome. A similar transformation was done with an AvaII Kmr marker ligated to AvaII host DNA fragments. Integration of the resistance markers into the host genome led to a high frequency of stable Kmr and Cmr transformants. Physical analysis of individual transformants indicated that this result was due to homologous recombination by conversionlike events leading to insertion of the Cmr (or Kmr) gene between two HaeII (or AvaII) sites of the host genome, with precise deletion of the host DNA between these sites. In contrast, integrative crossover of circular DNA molecules with homology to the host DNA is very rare in this cyanobacterium. Strain PCC 6803 was shown to have about 12 genomic copies per cell in standard growth conditions, which complicates the detection of recessive mutations induced by chemical or UV mutagenesis. Random disruption of the host DNA by insertional transformation provides a convenient alternative to transposon mutagenesis in cyanobacteria and may help to overcome the difficulties encountered in generating recessive mutants by classical mutagenesis.     

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.     

Expression of the SV40-transformed phenotype in hybrids between conditional and wild-type transformants

The nature of two different SV40-transformed cell lines which are temperature sensitive (ts) for the expression of the transformed phenotype has been analysed by somatic cell hybridization. Ts 23A cells are temperature sensitive in their ability to grow in medium containing low serum; in hybrids between these cells and a standard SV40-transformed BALB/c 3T3 cell line, the mutation seems to be dominant, at least for some parameters. One hundred percent of the hybrid clones have a very low efficiency of plating in medium containing 1% serum, but only 50% of them show growth inhibition in mass culture under the same conditions. Ts H615 cells revert to a normal phenotype at 39 °C for most parameters of transformation. The mutation appears to be recessive, as the hybrids generally behave like transformed cells at 39 °C. However, the expression of this mutation is not completely suppressed, since DNA synthesis after confluence shows some degree of inhibition in the hybrids at 39 °C. A considerable variation in the behavior of individual hybrid clones and in the degree of inhibition of some parameters has been observed and is discussed in terms of possible mechanism(s) of transformation.     

Revertants of v-fos-transformed rat fibroblasts: suppression of transformation is dominant.

Phenotypic revertants of Finkel-Biskis-Riley (FBR)-murine sarcoma virus-transformed rat fibroblasts were isolated on the basis of their adherence to plastic tissue culture dishes in the absence of divalent cations. Some revertants had sustained deletions or inactivating mutations of the v-fos gene. However, two revertants expressed a functional v-fos gene at levels equal to that in the transformed parental cells, and therefore phenotypic reversion was due to mutations in nonviral genes. These revertants were considered nontransformed according to four criteria: (i) they were flat and had a nontransformed morphology, (ii) they were contact inhibited when grown to confluence, (iii) they did not display anchorage-independent growth in soft agar, and (iv) they did not form tumors in nude mice. Somatic-cell hybrids between the revertants and the transformed parental cells were nontransformed, suggesting that the revertants had sustained an activating mutation of a gene capable of suppressing transformation. The expression of c-jun, junB, and junD was not altered in the revertants, and they could not be transformed by transfection with a c-jun expression vector. The revertants were resistant to transformation by an activated c-Ha-ras gene but were susceptible to transformation by simian virus 40. Our results demonstrate the existence of a class of revertants that harbor genes capable of suppressing transformation by v-fos and some other oncogenes. This contrasts with previously described revertants of transformation by v-fos that contain recessive mutations.     

Suppression of the transformed phenotype with retention of the viral ‘src’ gene in cell hybrids between rous sarcoma virus-transformed rat cells and untransformed mouse cells

Hybrid cell lines between untransformed mouse 3T3TK-cells and normal rat kidney (NRK) cells transformed by the B77 strain of Rous Sarcoma Virus (RSV) express a non-transformed phenotype, as determined by anchorage-dependent growth and organization of microfilament bundles. Virus rescue experiments and genetic experiments using an RSV mutant temperature-sensitive for maintenance of the transformed phenotype demonstrate that RSV is retained in the non-transformed hybrids. The action of the viral transformation gene ‘src’ therefore appears to be ‘suppressed’ in these hybrids. The suppressed hybrids generate variants in which the expression of the transformed phenotype and the ‘src’ gene is regained. This system should prove to be of value in identifying cellular genes involved in the expression of virally induced transformation.     

Transformed BHK cells exhibiting normal or subnormal sugar uptake

The uptake of deoxyglucose was compared in BHK cells and in DMN4B cells, a conditionally transformed line of BHK cells which exhibits transformed behavior at 38.5° but not at 32°. At 32°, DMN4B cells took up deoxyglucose more slowly than BHK cells, reflecting a higher Km for uptake of this sugar. When both cell lines were grown at 38.5°, the Km for DMN4B cells was reduced to a level only slightly greater than for BHK cells, and deoxyglucose uptake became similar in the two cell lines. Growth in glucose-free medium for 22 hours stimulated deoxyglucose uptake in both BHK and DMN4B cells; under these conditions, uptake was equal in the two cells lines, both at 32° and 38.5°. Glycolysis, as measured by lactic acid production, was slower in DMN4B than BHK cells, but in contrast to deoxyglucose uptake, this difference was observed at 38.5° rather than 32°. The observation that the subnormal deoxyglucose uptake of DMN4B cells in the untransformed state (32°) can be normalized by growth at 38.5°, a temperature permissive for transformation, suggests that membrane changes facilitating sugar uptake, which have been found in other transformed cells, are associated with transformation in DMN4B cells as well. However, the failure of uptake to exceed normal in these cells indicates that their transformed behavior is not attributable to excessive sugar uptake per se.     

A biological-based model that links genomic instability, bystander effects, and adaptive response

This paper links genomic instability, bystander effects, and adaptive response in mammalian cell communities via a novel biological-based, dose-response model called NEOTRANS3. The model is an extension of the NEOTRANS2 model that addressed stochastic effects (genomic instability, mutations, and neoplastic transformation) associated with brief exposure to low radiation doses. With both models, ionizing radiation produces DNA damage in cells that can be associated with varying degrees of genomic instability. Cells with persistent problematic instability (PPI) are mutants that arise via misrepair of DNA damage. Progeny of PPI cells also have PPI and can undergo spontaneous neoplastic transformation. Unlike NEOTRANS2, with NEOTRANS3 newly induced mutant PPI cells and their neoplastically transformed progeny can be suppressed via our previously introduced protective apoptosis-mediated (PAM) process, which can be activated by low linear energy transfer (LET) radiation. However, with NEOTRANS3 (which like NEOTRANS2 involves cross-talk between nongenomically compromised [e.g., nontransformed, nonmutants] and genomically compromised [e.g., mutants, transformants, etc.] cells), it is assumed that PAM is only activated over a relatively narrow, dose-rate-dependent interval (D(PAM),D(off)); where D(PAM) is a small stochastic activation threshold, and D(off) is the stochastic dose above which PAM does not occur. PAM cooperates with activated normal DNA repair and with activated normal apoptosis in guarding against genomic instability. Normal repair involves both error-free repair and misrepair components. Normal apoptosis and the error-free component of normal repair protect mammals by preventing the occurrence of mutant cells. PAM selectively removes mutant cells arising via the misrepair component of normal repair, selectively removes existing neoplastically transformed cells, and probably selectively removes other genomically compromised cells when it is activated. PAM likely involves multiple pathways to apoptosis, with the selected pathway depending on the type of cell to be removed, its cellular environment, and on the nature of the genomic damage.     

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.     

Altered low density lipoprotein receptor regulation is associated with cholesteryl ester accumulation in simian virus 40 transformed rodent fibroblast cell lines

Summary Several SV40 transformed REF52 cell lines were found to accumulate 5 to 10 times more cholesteryl esters compared to their parent line REF52 when cultured in 10% serum. Under this culture condition, the cholesteryl ester to phospholipid ratio was 0.4∶1 and 2.0∶1 to 3.8∶1 for normal and SV40 tranformed cells, respectively. The mechanism underlying cholesteryl ester accumulation in SV40 transformed lines was investigated. We found that 1) the rate of thede novo cholesterol and cholesteryl ester synthesis was roughly equal in normal and transformed derivatives; 2) the accumulation of cholesteryl esters in the transformants would not occur when cultured in lipoprotein-deficient medium and reappeared upon their return to low density lipoprotein-containing medium; 3) the transformants expressed twice as many low density lipoprotein receptors and were less sensitive to LDL-induced receptor down regulation compared to their nontransformed counterparts. The results indicate that SV40 transformed lines exhibited an accelerated lipid uptake from the culture medium due to an altered regulation of low density lipoprotein receptor activity. Supported by NCI grant CA 38016 Editor's Statement This article reports alteration of cholesterol metabolism in rat cells by viral transformation and provides an explanation for the phenomenon at a molecular level. The results may be widely applicable to other instances of viral transformation or virus infection.     

Transformation of murine melanocytes by basic fibroblast growth factor cDNA and oncogenes and selective suppression of the transformed phenotype in a reconstituted cutaneous environment

Constitutive expression of basic fibroblast growth factor (bFGF), a common characteristic of metastatic melanomas, was reproduced in vitro by infection of normal murine melanocytes with a recombinant retrovirus carrying a cDNA for bFGF. Expression of bFGF in these cells conferred autonomous growth in culture and extinguished differentiated functions, such as the synthesis of melanin and formation of dendrites. Independence from exogenous bFGF and loss of differentiated functions in vitro were induced also by transformation of melanocytes with the oncogenes myc, Ela, ras, and neu, although bFGF was not expressed by the respective transformants. As shown in skin reconstitution experiments onto syngeneic mice and subcutaneous injections into nude mice, the various transformants differed in their behavior in vivo. The bFGF transformants did not form tumors. They reverted to having a normal, melanotic phenotype and restricted growth. Myc and Ela transformants grew as tumors in nude mice but not in syngeneic, immunocompetent animals. Ras-transformed melanocytes were always tumorigenic, whereas the formation of tumors by neu transformants was suppressed by the concomitant grafting of keratinocytes in reconstituted skin of syngeneic mice. These data show that melanocytes genetically manipulated to produce bFGF acquire properties in vitro similar to those of metastatic melanoma cells or those induced by various oncogenes but that constitutive production of bFGF by itself is insufficient to make melanocytes tumorigenic. The experiments also show that melanocytes transformed by the selected oncogenes respond differentially to various environments in vivo.     

Fibroblast Growth Factor stimulates anchorage independent growth in agar of BHK21/13 and SV3T3 cells

The effect of Fibroblast Growth Factor (FGF) on the growth in suspension in soft agar of secondary baby hamster kidney fibroblasts, the partially transformed cell line BHK21/13 and its virally transformed derivative SV28 was studied. Only BHK21/13 cells could be induced by FGF to form colonies in soft agar. Secondary baby hamster kidney fibroblasts did not grow in suspension even with FGF, whereas SV28 grew independently of the presence of FGF. Thus viral transformation of BHK21/13 was not a prerequisite for growth in agar if FGF was present. Epidermal Growth Factor (EGF) was ineffective at promoting growth in agar of secondary baby hamster kidney fibroblasts or BHK21/13 cells. Balb/c 3T3 behaves like the secondary baby hamster kidney and SV3T3 like BHK21/13. We conclude there are three degrees of transformation with different potentials for growth in agar.     

Collagen synthesis in normal BHK cells and temperature-sensitive chemically transformed BHK cells

Summary Collagen synthesis in normal BHK 21/cl 13 and chemically transformed temperature-sensitive BHK 21/cl 13 cells (Me₂N4) was assessed by examination of hydroxyproline formation and collagenase-susceptible protein. The Me₂N4 cells lost their ability to synthesize collagen at both permissive and nonpermissive temperatures for transformation. These conclusions were confirmed by polyacrylamide-gel electrophoresis and CM-cellulose chromotography. Prolyl hydroxylase activity was present in both normal and transformed cells even when no collagen could be demonstrated. The production of noncollagen protein, although decreased in the transformed cell, did not change as drastically as the collagen synthesis. This paper was supported in part by a grant from the Public Health Service (AG00001), and by the Medical Research Service of the Veterans Administration.