Analysis of type 5 adenovirus transformation with a cloned rat embryo cell line (CREF).

A recently isolated cloned cell line of Fischer rat embryo fibroblasts (CREF) can be transformed at high frequency by adenovirus serotype 5 (Ad5). The CREF cells display a near diploid karyotype, do not crisscross at confluency, can be maintained at confluency for greater than 7 wk at 36 degrees C, and do not form macroscopic colonies when seeded in agar. Transformed cells are identified by their altered morphology and the presence of adenovirus DNA sequences in the transformants, which can be demonstrated by lysing cells directly on nitrocellulose filters and hybridizing with 32P-labeled Ad5 DNA (spot hybridization). The frequency of transformation at 36 degrees C is approximately equal to 2 x 10(-4) with wild-type Ad5 and approximately equal to 2 x 10(-3) with the temperature-sensitive mutant H5ts125. Southern blot hybridization analysis indicates that five out of six wild-type Ad5- and six out of six H5ts125-transformed CREF clones isolated at 36 degrees C contain the complete integrated Ad5 genome. Preliminary characterization of four transformed clones (two wild-type and two H5ts125) indicates that, even though transformation was done in CREF cells (a clonal cell line), they differ in their biological properties, such as saturation density and anchorage dependence.     

Temperature-Dependent Properties of Cells Transformed by a Thermosensitive Mutant of Polyoma Virus

In cells transformed by ts-3, a thermosensitive mutant of polyoma virus, the loss of inhibition of DNA synthesis by topographical factors (topo-inhibition), is rendered temperature-dependent, providing evidence that the viral genome controls this essential aspect of transformation. The expression of two other attributes of transformed cells, growth in agar and serum requirement for initiation of DNA synthesis in a wound in culture, is not made temperature-dependent. In productive infection of BALB-3T3 cells by ts-3, virus-induced stimulation of cellular DNA synthesis and movement is rendered temperature-dependent.     

Interactions between adenovirus, a tumor promoter, and chemical carcinogens in transformation of rat embryo cell cultures.

The tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA) caused a 2- to 3-fold enhancement of transformation of secondary rat embryo cells that had been injected with a temperature-sensitive mutant of adenovirus type 5(H5ts 125). In addition, transformed foci appeared earlier and were larger in cultures grown in the presence of TPA. The addition of TPA could be delayed until up to 7 days after viral injection and still enhancement was observed. Exposure of the cells to 7,12-dimethylbenz[a]pyrene prior to H5ts125 infection also resulted in a 2- to 4-fold enhancement of transformation, and this enhancement was further augmented 2- to 3-fold when cells were grown in TPA after virus infection. Whereas TPA did not enhance the cloning efficiency of normal rat embryo cells, it did enhance the cloning efficiency of isolated colonies of adenovirus-transformed cells when these were grown alone or cocultured with a 100-fold excess of normal rat embryo cells. The enhancement of adenovirus transformation by TPA appears to be due to its ability to facilitate expression of the transformed state rather than an effect on virus uptake or integration.     

Mutations in the E1a gene of adenovirus type 5 alter the tumorigenic properties of transformed cloned rat embryo fibroblast cells.

The adenovirus type 5 mutants H5hr1 and H5dl101 contain modifications in the E1a gene affecting the 13S mRNA-encoded 289-amino acid polypeptide and exhibit a cold-sensitive transformation phenotype upon infection of cloned rat embryo fibroblast (CREF) cells. Transformed cell lines expressing solely E1a or E1a and E1b gene products derived from these viruses display enhanced anchorage-independent growth at 37 degrees C versus 32 degrees C and display a cytoskeletal architecture resembling untransformed fibroblastic CREF cells. In contrast, CREF cells transformed by H5wt or the E1a and E1b region of H5wt grow with similar efficiency in agar at 37 degrees C or 32 degrees C and exhibit an epithelioid morphology that is associated with an altered cytoskeleton. Regardless of the expression or presence of other viral early regions, including E1b, E2a, and E4 genes, specific CREF cell lines expressing an altered 289-amino acid protein and a wild-type 12S mRNA-encoded 243-amino acid protein were capable of inducing tumors in nude mice and in immunocompetent syngeneic Fischer rats. In sharp contrast, cells expressing a wild-type 289-amino acid protein were unable to induce tumors in either nude mice or syngeneic rats. The ability to induce tumors did not correlate with alterations in the pattern of viral DNA integration or differential expression of the E1a and E1b genes, nor was the tumor induction a consequence of unique properties of the immortal parental CREF cell line.     

Cold-sensitive expression of transformation by a host range mutant of type 5 adenovirus.

hr1, an E1a (0-4.5 map units) host range mutant of type 5 adenovirus (Ad5), transformed a cloned rat embryo fibroblast (CREF) cell line at about a 5-fold higher frequency than wild-type (wt) Ad5 when cells were cultured at 37 degrees C. However, if the cells were infected with hr1 and maintained at 32 degrees C morphological transformation did not occur. When infected cells were shifted from 32 degrees C to 37 degrees C 2 wk postinfection, the frequency of transformation by 6 wk was identical to that of cells grown continuously at 37 degrees C, whereas cultures shifted from 37 degrees C to 32 degrees C 2 wk postinfection displayed a greater than 96% reduction in morphological transformation. hr1-transformed cells had a fibroblastic morphology as contrasted with the typical epithelioid morphology of wt Ad5-transformed cells, but hr1- and wt Ad5-transformed cells had similar saturation densities, growth rates, and agar cloning efficiencies when assayed at 37 degrees C. However, when cells transformed by hr1 at 37 degrees C were grown at 32 degrees C, they had a saturation density close to that of normal CREF cells and grew at a lower efficiency in agar than wt-transformed cells. DNA transfer/hybridization analysis of two hr1-transformed cloned cell lines, A2 and B3, indicated that A2 cells contained a complete integrated copy of the Ad5 genome, whereas in B3 cells only part of the Ad5 genome was integrated. RNA transfer and RNA/DNA filter hybridization analyses indicated that the types of viral messenger RNAs and the relative amounts of RNA transcribed were similar in the A2 and B3 cell lines when they were grown at 32 degrees C and 37 degrees C. Indirect immunofluorescence, with antisera from hamsters bearing Ad-induced tumors, indicated a temperature dependence in staining--i.e., cells grown at 37 degrees C or shifted from 32 degrees C to 37 degrees C contained intense, particulate staining in the nuclear region, whereas the staining was decreased significantly in cells cultured at 32 degrees C and in cells shifted from 37 degrees C to 32 degrees C. These findings indicate that the gene product affected by the hr1 mutation is cold sensitive and is essential for the expression of the characteristics that identify the transformed cell.     

Neoplastic transformation of chimpanzee cells induced by adenovirus type 12--simian virus 40 hybrid virus.

The adenovirus 12--simian virus 40 hybrid virus produced neoplastic transformation of chimpanzee skin fibroblasts in vitro. The transformed fibroblasts showed morphological alteration and became permanent lines. The transformed cells contained both adenovirus 12 and simian virus 40 large tumor antigens and were virus producers. However at passage 9, one line (WES) was found to be a nonproducer, producing neither infectious virus nor virus-specific antigen detectable by the complement fixation test. Virus particles were not detected nor could infectious hybrid virus be rescued from this line by cocultivation with Vero cells. The transformed cells formed large cell aggregates and grew in liquid growth medium above an agar base, formed colonies in soft agar, and grew to high saturation densities; the normal chimpanzee skin fibroblasts did not. One transformed WES line produced tumors when transplanted subcutaneously into newborn nude mice, thus providing an important tool for studying tumor immunity in the chimpanzee.     

Astrocyte elevated gene-1 (AEG-1) functions as an oncogene and regulates angiogenesis

Astrocyte-elevated gene-1 (AEG-1) expression is increased in multiple cancers and plays a central role in Ha-ras-mediated oncogenesis through the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Additionally, overexpression of AEG-1 protects primary and transformed human and rat cells from serum starvation-induced apoptosis through activation of PI3K/Akt signaling. These findings suggest, but do not prove, that AEG-1 may function as an oncogene. We now provide definitive evidence that AEG-1 is indeed a transforming oncogene and show that stable expression of AEG-1 in normal immortal cloned rat embryo fibroblast (CREF) cells induces morphological transformation and enhances invasion and anchorage-independent growth in soft agar, two fundamental biological events associated with cellular transformation. Additionally, AEG-1-expressing CREF clones form aggressive tumors in nude mice. Immunohistochemistry analysis of tumor sections demonstrates that AEG-1-expressing tumors have increased microvessel density throughout the entire tumor sections. Overexpression of AEG-1 increases expression of molecular markers of angiogenesis, including angiopoietin-1, matrix metalloprotease-2, and hypoxia-inducible factor 1-α. In vitro angiogenesis studies further demonstrate that AEG-1 promotes tube formation in Matrigel and increases invasion of human umbilical vein endothelial cells via the PI3K/Akt signaling pathway. Tube formation induced by AEG-1 correlates with increased expression of angiogenesis markers, including Tie2 and hypoxia-inducible factor-α, and blocking AEG-1-induced Tie2 with Tie2 siRNA significantly inhibits AEG-1-induced tube formation in Matrigel. Overall, our findings demonstrate that aberrant AEG-1 expression plays a dominant positive role in regulating oncogenic transformation and angiogenesis. These findings suggest that AEG-1 may provide a viable target for directly suppressing the cancer phenotype.     

Detection and Quantitation of Simian Virus 40 Genetic Material in Abortively Transformed BALB/3T3 Clones

Infection with simian virus 40 is known to induce many cells to synthesize DNA and to divide in a medium lacking serum protein growth factor(s) that is essential for growth of uninfected cells (factor-free medium). Cells infected under these conditions then go through several rounds of division, since colonies containing more than 100 cells are formed. Many of these colonies are abortively transformed since, upon subsequent passage of the cells in standard medium, they can no longer grow in factor-free medium and show no other properties of viral transformation. We have examined these abortively transformed cells for the presence of simian virus 40 DNA sequences. Of the three clones tested, two were found to contain viral genetic material despite the fact that they were phenotypically normal.The number of simian virus 40 genome equivalents present was determined by measurement of DNA reassociation kinetics on hydroxyapatite. Two of the abortively transformed lines contained approximately five viral genome equivalents per diploid cell, while the DNA from a third abortive transformant was indistinguishable from that of uninfected BALB/3T3 cells. A standard simian virus 40 transformant, isolated under similar conditions, contained two copies of the viral genome per cell. The abortive transformants also appear to contain the entire viral genome rather than multiple partial copies. Subclones of one abortively transformed line containing five copies per cell had 2.7-10 copies of viral genetic material per diploid cell.     

Persistence of type 5 adenovirus DNA in cells transformed by temperature-sensitive mutant, H5ts125.

The characteristic of H5ts125, a temperature-sensitive DNA-minus mutant, to transform 3 to 8 times more rat embryo cells than wild-type 5 adenovirus was correlated with the persistence of an increased proportion of the viral genome in cells independently transformed at the nonpermissive (39.5 degrees) or semipermissive (36 degrees) temperature. Reassociation kinetics of the hybridization of 32P-labeled,HindIII restriction fragments of the viral genome and excess unlabeled, transformed cell DNA was used to measure the quantity of the viral genome in transformed cells. Three of four cell lines independently transformed and maintained at 36 degrees contained all regions of the viral genome; one line transformed at 39.5 degrees contained multiple copies representing all of the viral DNA; and one line contained a large proportion of the viral genome. The cell line transformed and maintained at 32 degrees contained a quantity of viral genome consistent with that usually found in cells transformed by wild-type 5 adenovirus.     

Initiation and maintenance of cell transformation by simian virus 40: a viral genetic property.

The transforming ability in 10% serum medium of the temperature-sensitive mutants of simian virus 40 in the complementation group III (ts640 type mutants) was greatly reduced when the infected rat 3Y1 cells were incubated at the restrictive temperature of 40 degrees or incubated first at 40 degrees for 3 days and then shifted to the permissive temperature of 33 degrees. Transformation did occur efficiently after incubation at 33 degrees or after an initial incubation at 33 degrees for 5 days followed by a shift to 40 degrees. When growth properties of 3Y1 cells transformed at 33 degrees by the group III mutants were examined at 40 degrees, several aspects of the transformed state were rendered temperature-sensitive. These aspects were the ability of cells to grow in low serum (1.5%) medium and to make colonies, in 10% serum medium, on monolayers of untransformed 3Y1 cells and in soft agar. It is concluded that a simian virus 40 gene (cistron III) controls the initiation, as well as at least some aspects of the maintenance, of transformation and that the initiation reaction is a more heat-labile event than the maintenance reaction(s) under the experimental conditions.     

Irreversibility of cellular aging and neoplastic transformation: a clonal analysis.

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in a persistent impairment of proliferation when the cells are subcultured at low density and a greatly increased probability of neoplastic transformation in assays for transformation. These properties, along with the large accumulation of age pigment bodies in the confluent cells, are cardinal cellular characteristics of aging in organisms and validate the system as a model of cellular aging. Two cultures labeled alpha and beta were obtained after prolonged confluence; both were dominated by cells that were both slowed in growth at low population density and enhanced in growth capacity at high density, a marker of neoplastic transformation. An experiment was designed to study the reversibility of these age-related properties by serial subculture at low density of the two uncloned cultures and their progeny clones derived from assuredly single cells. Both uncloned cultures had many transformed cells and a reduced growth rate on subculture. Serial subculture resulted in a gradual increase in growth rates of both populations, but a reversal of transformation only in the alpha population. The clones originating from both populations varied in the degree of growth impairment and neoplastic transformation. None of the alpha clones increased in growth rate on low density passage nor did the transformed clones among them revert to normal growth behavior. The fastest growing beta clone was originally slower than the control clone, but caught up to it after four weekly subcultures. The other beta clones retained their reduced growth rates. Four of the five beta clones, including the fastest grower, were transformed, and none reverted on subculture. We conclude that the apparent reversal of impaired growth and transformation in the uncloned parental alpha population resulted from the selective growth at low density of fast growing nontransformed clones. The reversal of impaired growth in the uncloned parental beta population was also the result of selective growth of fast growing clones, but in this case they were highly transformed so no apparent reversal of transformation occurred. The clonal results indicate that neither the impaired growth nor the neoplastic transformation found in aging cells is reversible. We discuss the possible contribution of epigenetic and genetic processes to these irreversible changes.     

Critical role played by thyroid hormone in induction of neoplastic transformation by chemical carcinogens in tissue culture.

Incubation of primary cultures of hamster embryo cells (HEC) or mouse fibroblasts (C3H/10T1/2 cells) in media depleted of thyroid hormones does not alter cell growth or survival but renders the cells resistant to neoplastic transformation by benzo[a]pyrene (B[a]P) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), carcinogens which yield transformation rates of 10(-4)-10(-2) in media supplemented with triiodothyronine (T3). In C3H/10T1/2 cells, the times of addition or removal of the hormone indicate that T3 exerts maximum effect when added 12 hr prior to treatment with B[a]P and that the progression of transformation from the time of initiation by the carcinogen to full expression and the appearance of transformed foci was independent of the presence or absence of the hormone in the medium. Dependence of transformation on T3 concentration in the medium was observed over the physiological range of 1 pM to 100 nM in C3H/10T1/2 cells treated with B[a]P. These results were similar to our previous findings on the T3 dose-related induction of radiogenic transformation and of Na+,K+-ATPase activity. The latter effect was used as a measure of T3 induction of protein synthesis. A further indication of the potential involvement of protein synthesis in T3 action is the suppression of T3- and B[a]P-dependent transformation by cycloheximide at concentrations that inhibit protein synthesis by approximately equal to 50% in the C3H/10T1/2 cells. We suggest that thyroid hormone induces the synthesis of a host protein that plays a key role in neoplastic transformation by direct-acting chemical carcinogens and by those requiring metabolic activation. In our previous studies, similar T3-dependent mechanisms were implicated in radiogenic transformations.     

In vitro complementation as an assay for purification of adenovirus DNA replication proteins.

As an approach to the purification of adenovirus-encoded DNA replication proteins, we have developed in vitro complementation assays that make use of viral mutants defective in DNA replication in vivo. Nuclear extracts prepared from cells infected with H5ts36 or H5ts125, two such mutants belonging to different complementation groups, were found to be defective in viral DNA replication in vitro. However, replication activity could be restored by mixing the two extracts. Replication activity in either extract also could be restored by addition of appropriate replication-deficient fractions purified from cells infected with wild-type adenovirus. By using such assays, H5ts36- and H5ts125-complementing activities were extensively purified. As expected, purified H5ts125-complementing activity consisted of a single major polypeptide, the 72-kilodalton (kDal) adenovirus DNA binding protein. The purified H5ts36-complementing activity consisted of the 80-kDal adenovirus terminal protein precursor and two other major polypeptides with apparent molecular masses of 140 and 65 kDal. Formation of the 80-kDal terminal protein-dCMP complexes, the proposed initial step in adenovirus DNA replication, required components in the purified H5ts36-complementing fraction and a cellular factor(s) but did not require the adenovirus DNA binding protein. The complete in vitro adenovirus DNA replication reaction was reconstituted from the purified H5ts36-complementing activity, the adenovirus DNA binding protein, and an extract from uninfected cells.     

Reversion of the Jun-induced oncogenic phenotype by enhanced synthesis of sialosyllactosylceramide (GM3 ganglioside)

In the mouse fibroblast cell line C3H 10T1/2 and the chicken fibroblast cell line DF1, the ganglioside GM3 is the major glycosphingolipid component of the plasma membrane. Expression of the viral oncoprotein Jun (v-Jun) induces transformed cell clones with greatly reduced levels of GM3 and GM3 synthase (lactosylceramide α2,3-sialyltransferase) mRNA in both 10T1/2 and DF1 cell cultures. Compared with nontransformed controls, v-Jun transfectants show enhanced ability of anchorage-independent growth, and their growth rates as adherent cells are increased. When the mouse GM3 synthase gene is transfected with the pcDNA vector into v-Jun-transformed 10T1/2 cells, the levels of GM3 synthase and corresponding mRNA are restored to those of control cells. Reexpression of GM3 correlates with a reduced ability of the cells to form colonies in nutrient agar. Similarly, when the newly cloned chicken GM3 synthase gene is transfected into v-Jun-transformed DF1 with the pcDNA vector, the GM3 synthase level is restored to that of control cells, and the ability of the cells to form agar colonies is reduced. The levels of GM3 in the cell also affect membrane microdomains. The complex of GM3 with tetraspanin CD9 and integrin α5β1 inhibits motility and invasiveness. The amounts of this complex are greatly reduced in transformed cells. Expression of GM3 and consequent reversion of the transformed phenotype results in increased levels of that microdomain complex.     

Production of platelet-derived growth factor-like molecules and reduced expression of platelet-derived growth factor receptors accompany transformation by a wide spectrum of agents.

A series of nontransformed human and murine cells and derivative cell lines transformed by methylcholanthrene; by simian virus 40, Kirsten and Moloney murine sarcoma viruses, simian sarcoma virus, and adenovirus; and by a "spontaneous" event in culture were examined for the expression of receptors for the platelet-derived growth factor (PDGF) and for production of substances able to compete with 125I-labeled PDGF for binding to the cell-surface PDGF receptor. In each case, transformation resulted in a 50-100% decrease in available PDGF receptors. All transformed cells except the methylcholanthrene-transformed mouse cells produce a PDGF competitor into the conditioned medium. Levels of PDGF competitor in conditioned medium at the end of a 48-hr collection were as high as 2 ng/ml--high enough to be measured by radioreceptor assay diluted 1:30 and to maximally stimulate [3H]thymidine incorporation by human fibroblasts. The PDGF competitor activity detected in a radioreceptor assay does not reflect irreversible (e.g., proteolytic) damage to the receptor of test cells since its effects are reversed by acetic acid dissociation. Antiserum against human PDGF neutralizes 20-80% of the PDGF competitor found in conditioned medium from different transformed human cells and 100% of the activity from normal human endothelial cells. The possibility that induction of expression of the cellular PDGF gene may be involved in the mechanism of transformation of PDGF-responsive mesenchymal cells is discussed.     

The human transforming growth factor type alpha coding sequence is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.

A peptide secreted by some tumor cells in vitro imparts anchorage-independent growth to normal rat kidney (NRK) cells and has been termed transforming growth factor type alpha (TGF-alpha). To directly investigate the transforming properties of this factor, the human sequence coding for TGF-alpha was placed under the control of either a metallothionein promoter or a retroviral long terminal repeat. These constructs failed to induce morphological transformation upon transfection of NIH 3T3 cells, whereas viral oncogenes encoding a truncated form of its cognate receptor, the EGF receptor, or another growth factor, sis/platelet-derived growth factor 2, efficiently induced transformed foci. When NIH 3T3 clonal sublines were selected by transfection of TGF-alpha expression vectors in the presence of a dominant selectable marker, they were shown to secrete large amounts of TGF-alpha into the medium, to have downregulated EGF receptors, and to be inhibited in growth by TGF-alpha monoclonal antibody. These results indicated that secreted TGF-alpha interacts with its receptor at a cell surface location. Single cell-derived TGF-alpha-expressing sublines grew to high saturation density in culture. However, when plated as single cells on contact-inhibited monolayers of NIH 3T3 cells, they failed to form colonies, whereas v-sis- and v-erbB-transfected cells formed transformed colonies under the same conditions. Moreover, TGF-alpha-expressing sublines were not tumorigenic in nude mice. These and other results imply that TGF-alpha exerts a growth-promoting effect on the entire NIH 3T3 cell population after secretion into the medium but little, if any, effect on the individual cell synthesizing this factor. It is concluded that the normal coding sequence for TGF-alpha is not a direct-acting oncogene when overexpressed in NIH 3T3 cells.     

Potential role of the src gene product in inhibition of gap-junctional communication in NIH/3T3 cells.

The effects of the src gene on the activity of protein kinase C and intercellular communication have been studied in transformed NIH/3T3 clones isolated from soft agar following transfection with the plasmid carrying the v-src gene (psrc-11). Six transformed clones that were studied contained newly incorporated v-src genes in the genome, had an increased amount of pp60src, and showed enhanced activities of protein kinase C. Intercellular communication, studied by observing with autoradiography the transfer of [3H]uridine nucleotide from prelabeled donor cells to recipient cells in contact, was found to be reduced in transformed clones as compared to parental NIH/3T3 cells. Treatment with phorbol 12-myristate 13-acetate was also found to increase protein kinase C activity and to reduce intercellular communication in normal NIH/3T3 cells. These results suggest that the v-src gene product, in a manner similar to some of the powerful tumor promoters, may directly or indirectly affect cell-cell communication.     

Autocrine transformation by chimeric signal peptide-basic fibroblast growth factor: reversal by suramin.

NIH 3T3 cells transfected with basic fibroblast growth factor (bFGF) fused to an immunoglobulin signal peptide sequence are transformed in vitro and tumorigenic in vivo. The transformed phenotype of chimeric signal peptide-bFGF (spbFGF) cells is characterized by an enhanced proliferation rate compared to parental NIH 3T3 cells, density- and anchorage-independent growth, a transformed morphology, and lack of cell adhesion. The rate of spbFGF cell proliferation is not diminished by anti-bFGF neutralizing antibodies. 125I-labeled bFGF receptor cross-linking and binding studies suggest that surface FGF receptors in spbFGF cells are unavailable and down-regulated. The FGF receptors are also down-regulated in K-fgf-transformed cells but not in parental 3T3, native bFGF-transfected, and ras-transformed NIH 3T3 cells. The addition of suramin to spbFGF and K-fgf cells rapidly promotes the up-regulation of FGF receptors. Suramin also induces lowering of the proliferation rate to that of parental cells, anchorage-dependent growth, assembly of cytoskeletal filaments, cellular adhesion, and spreading. These results suggest that spbFGF cells undergo autocrine transformation, possibly by an internal autocrine loop, in which there is constitutive activation of the FGF receptor. Suramin inhibits autocrine transformation, leading to a normal untransformed phenotype.     

High rate of diversification and reversal among subclones of neoplastically transformed NIH 3T3 clones.

NIH 3T3 cells undergo neoplastic transformation when exposed to conditions of moderate physiological growth constraint. One of several characteristics of this transformation that indicates its adaptational nature is its gradual reversibility under conditions of unconstrained growth. We explored the origins of reversibility by isolating cells from each of three highly transformed foci and comparing their focus-forming capacity with that of derivative clones and subclones. A high proportion of the parental cells made dense foci. Six of the nine clones obtained from the three foci produced foci, though the percentage varied widely. The other three clones produced no foci at all. The transformed clones were subcloned and analyzed to evaluate the possibility that the negative clones were genuine revertants, rather than being derived from a small minority of nontransformed cells surrounding or underlying the original foci. In each case the subclones varied widely in the percentage of focus-forming cells and the average was much lower than the parental clone from which they were derived. Indeed, 15 of the 53 subclonal populations produced no foci. The high degree of heterogeneity, including complete reversal of focus-forming capacity, provides additional support for the hypothesis that "spontaneous" transformation is driven by an adaptive response to moderate growth constraint rather than by one or more effectively irreversible mutations.