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Cells transformed by the simian virus 40 (SV40) large T antigen (Tag) contain elevated levels of cellular p53 protein. To quantify this relationship, levels of p53 were measured in NIH 3T3 cells that expressed different concentrations of Tag. Using immunoblotting, average p53 levels were shown to increase linearly with Tag concentrations in these cell lines. Single-cell measurements were also performed using flow cytometry to measure p53 immunofluorescence. Surprisingly, the flow cytometry experiments showed that two distinct cell populations, based on p53 content, were present in cells expressing high levels of Tag. One cell population contained elevated p53 levels. A second population did not contain elevated p53, even though high concentrations of Tag were present in the cells. This latter cell population did not appear to arise because of mutations in either Tag or p53. The two cell populations also had phenotypic differences. In exponentially growing cells, Tag alters the cell cycle distribution (decreases the percentage of G1 phase cells and increases the percentages of S and G2 + M phase cells). This phenotype was maximum in the cell population containing elevated p53. A lesser phenotype was found in the cell population that did not contain elevated p53. These data show, firstly, that cells can express significant levels of Tag and not contain elevated levels of p53 and, secondly, that elevated p53 correlates with the altered cell cycle distribution produced by Tag in growing cells.

Overexpression of genes encoding E2F transcription factors can transform some cultured cell lines and cause apoptosis of others. Apoptosis due to E2F overexpression requires the presence of wild-type p53. Cell lines in which stable E2F overexpression is possible might be expected, therefore, to contain mutant p53. In this report, it was asked whether endogenous p53 was mutant or wild-type in four established fibroblast cell lines that this laboratory previously showed stably overexpressed and were transformed by exogenous E2F-1. Unexpectedly, it was found that the p53 in these cells was wild-type by the criteria of immunoprecipitation with conformation-specific, p53 monoclonal antibodies and by transactivation of a p53-dependent reporter gene construct in transient transfection assays. These data indicate that stable overexpression of E2F-1 is possible in the presence of wild-type p53 and may result in cell transformation.

A set of cell lines was constructed by infection of established murine fibroblasts with recombinant retroviruses encoding the simian virus 40 large T antigen (Tag) gene. By immunofluorescence flow cytometry, it was shown that these cell lines expressed Tag over at least a 20-fold concentration range. Using these cells, the dose-response relationship between Tag concentration and a phenotype detected by flow cytometry that measures the rate at which proliferating cells transit the cell cycle (i.e. cell-cycling phenotype) was determined. This relationship between Tag concentration and phenotype was not linear. Instead, the cell-cycling phenotype became saturated at relatively low Tag concentrations, i.e. a further increase in Tag concentration did not change the phenotype. The dose-response relationship between Tag and a second phenotype, colony formation in soft agar, was also determined. Colony formation in soft agar is a measurement of cell transformation. In contrast to the cell-cycling phenotype, transformation was linearly related to Tag over the entire 20-fold Tag concentration range. This phenotype did not saturate at high Tag concentrations. Therefore, the dose-response relationship between Tag concentration and the cell-cycling phenotype was different from that between Tag concentration and cellular transformation. Since the Tag gene is comprised of multiple genetic domains that independently affect cellular proliferation, one possibility is that the differences in dose-response of the two phenotypes indicate that different genetic domains of the gene are necessary for production of each phenotype.

MCF-7, a breast cancer-derived cell line, is deficient of caspase 3 and relatively insensitive to many chemotherapeutic agents. To study the association of caspase 3 deficiency and chemotherapeutic resistance, we reconstituted caspase 3 in MCF-7 cells and characterized their apoptotic response to doxorubicin and etoposide. Western blots demonstrated that caspase 3 was constitutively expressed in the reconstituted MCF-7 cells. Both morphological observation and survival assays showed that caspase 3 reconstitution significantly sensitized MCF-7 cells to both drugs. Remarkably increased activation of caspases 3, 6, and 7, cleavage of cellular death substrates, and DNA fragmentation were detected in the reconstituted MCF-7 cells after drug treatment. Together, these data demonstrated a specific role for caspase 3 in chemotherapy-induced apoptosis and in activation of caspases 6 and 7. Our results also suggest that caspase 3 deficiency may contribute to chemotherapeutic resistance in breast cancer.

Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G(1)/S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G(1) arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells.

To examine the pattern of retroviral vector gene expression during early stages of infection, a Moloney murine leukemia virus (M-MuLV)-based vector that transcribes the simian virus 40 (SV40) large T antigen (Tag) gene from the viral long terminal repeat (LTR) was used to infect proliferating rodent fibroblasts. At various times after infection, cells were fixed and stained for Tag by indirect immunofluorescence, and for DNA using propidium iodide. Tag immunofluorescence and DNA content were both quantified by flow cytometry. The results showed that Tag expression was first detected exclusively in the late G1 and early S phases of the cell cycle, approximately 12 h postinfection. The infection was synchronous in that Tag-expressing cells detected at 12 h, in late G1 and early S, moved as a discrete population through S phase, into the G2 + M phases of the cell cycle and then back into G1 during the next 8-10 h. The presence of a synchronous Tag-expressing cell population suggests that, at time of infection, cells in certain phases of the cell cycle were more susceptible to infection than cells in other phases. This may be related to synchronizing events that must occur before viral genes are expressed in infected cells; one such event may be integration of viral DNA into cellular chromosomes (i.e., provirus formation) that requires cells to pass through an M phase.

The E2F transcription factor can regulate expression of numerous cellular genes controlling proliferation, including proto-oncogenes and genes regulating cell cycle progression. Therefore, genes comprising the E2F gene family could potentially contribute to carcinogenesis. To test the potential of E2F to act as a transforming gene, a cDNA encoding E2F-1 was constitutively overexpressed in established rodent cells using a retroviral vector. Overexpressed E2F-1 was functional, as shown by stimulation of a transfected adenovirus E2 promoter driving a chloramphenicol acetyltransferase reporter gene in E2F-1 overexpressing cells. This stimulation was dependent on functional E2F binding sites in the promoter. Examination of phenotype showed that E2F-1 overexpression mediated cell transformation as measured by the ability of cells to form colonies in soft agar medium. In addition, overexpressed E2F-1 shortened the duration of the G1 cell cycle phase in proliferating cells, a property characteristic of other transforming genes. These data provide direct evidence that E2F-1 can act as a transforming gene and a critical regulator of cell cycle progression and suggest the possibility of E2F involvement in carcinogenesis.

Simian virus 40 (SV40) large T antigen (Tag) expression results in reduced percentages of G1-phase cells and increased percentages of S- and G2+M-phase cells in exponentially growing fibroblast populations as compared with identical cell populations not expressing Tag. This effect is the result of reduced G1 and increased G2+M cell cycle phase durations caused by Tag [Sladek, T.L.Jacobberger, J.W. (1992). J. Virol., 66, 1059-1065]. Using recombinant retroviruses to manipulate Tag expression over a 25-fold range, it is shown here that the magnitude of this cell cycle phenotype increases as a function of increasing intracellular Tag concentration. This effect of Tag on the cell cycle is not independent of negative regulation by cellular mechanisms since exponentially growing cell populations producing high and increasing levels of Tag, increase the fraction of cells residing in G1 and decrease the fraction in S and G2+M as a function of cell density. Therefore, the data in this paper show, first, that Tag is a concentration-dependent, positive cell cycle regulator in exponentially proliferating cells and, second, that endogenous cellular mechanisms negatively regulating the cell cycle in response to cell density override the effect of Tag.

Double-stranded DNA from mycoplasma virus L2 can transfect Acholeplasma laidlawii cells in the presence of polyethylene glycol (T. L. Sladek and J. Maniloff, J. Bacteriol. 155:734-741, 1983). We report here that both single-stranded DNA and double-stranded replicative form DNA, from the single-stranded DNA mycoplasma virus L51, are also infectious in this system. For both DNAs transfection frequencies were in the range of 10(-8) transfectants per DNA molecule and 10(-3) transfectants per CFU. An unexpected finding was that both DNAs could transfect A. laidlawii strain REP-, a variant which is a nonpermissive host for single-stranded DNA mycoplasma viruses due to a block in viral DNA replication (Nowak et al., J. Bacteriol. 127:832-836, 1976). The number of viruses produced by transfected REP- cells was comparable to the number produced by both transfected and infected wild-type cells. Therefore, transfected L51 DNAs are able to bypass the replication block in REP- cells that occurs when these cells are infected by L51 virions.

We have found that mycoplasma virus L172 is an enveloped globular virion containing circular, single-stranded DNA of 14.0 kilobases. L172 has been reported by other workers to have a double-stranded DNA genome of 13 to 17 kilobase pairs and has been classified as a plasmavirus, a group for which mycoplasma virus L2 is the type member. Mycoplasma viruses L172 and L2 differ in genome size and structure, DNA base composition, and protein composition, and they have no detectable DNA homology. As the only reported enveloped virion containing single-stranded DNA, L172 represents a new group of viruses.

Transfection of Acholeplasma laidlawii strain JA1 with viral DNAs that have been sequence-specifically methylated in vitro has led us to previously postulate that JA1 cells contain an enzyme which cleaves only DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing that base. In this paper we show that an endonuclease activity is present in extracts from JA1 cells but not in extracts from a restriction-deficient variant of JA1. The partially purified enzyme cleaves not only DNA containing 5-methylcytosine, but also DNA containing no methylated bases. We discuss why this endonuclease activity may not have the same in vitro specificity as would be expected from in vivo experiments.

Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JA1 and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JA1 cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JA1. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JA1 restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In contrast, JA1 cells (which contain no methylated DNA bases) have a newly discovered type of restriction-modification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JA1 cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JA1, which retains the JA1 modification phenotype, was isolated, indicating that JA1 cells have a gene product with restriction specificity for DNA containing 5-methylcytosine.