Ras-dependent cell cycle commitment during G2 phase.

Synchronization used to study cell cycle progression may change the characteristics of rapidly proliferating cells. By combining time-lapse, quantitative fluorescent microscopy and microinjection, we have established a method to analyze the cell cycle progression of individual cells without synchronization. This new approach revealed that rapidly growing NIH3T3 cells make a Ras-dependent commitment for completion of the next cell cycle while they are in G2 phase of the preceding cell cycle. Thus, Ras activity during G2 phase induces cyclin D1 expression. This expression continues through the next G1 phase even in the absence of Ras activity, and drives cells into S phase.

Influence of cell cycle and oncogene activity upon topoisomerase IIalpha expression and drug toxicity.

The cell cycle, oncogenic signaling, and topoisomerase (topo) IIalpha levels all influence sensitivity to anti-topo II drugs. Because the cell cycle and oncogenic signaling influence each other as well as topo IIalpha levels, it is difficult to assess the importance of any one of these factors independently of the others during drug treatment. Such information, however, is vital to an understanding of the cellular basis of drug toxicity. We, therefore, developed a series of analytical procedures to individually assess the role of each of these factors during treatment with the anti-topo II drug etoposide. All studies were performed with asynchronously proliferating cultures by the use of time-lapse and quantitative fluorescence staining procedures. To our surprise, we found that neither oncogene action nor the cell cycle altered topo IIalpha protein levels in actively cycling cells. Only a minor population of slowly cycling cells within these cultures responded to constitutively active oncogenes by elevating topo IIalpha production. Thus, it was possible to study the effects of the cell cycle and oncogene action on drug-treated cells while topo IIalpha levels remained constant. Toxicity analyses were performed with two consecutive time-lapse observations separated by a brief drug treatment. The cell cycle phase was determined from the first observation, and cell fate was determined from the second. Cells were most sensitive to drug treatment from mid-S phase through G(2) phase, with G(1) phase cells nearly threefold less sensitive. In addition, the presence of an oncogenic src gene or microinjected Ras protein increased drug toxicity by approximately threefold in actively cycling cells and by at least this level in the small population of slowly cycling cells. We conclude that both cell cycle phase and oncogenic signaling influence drug toxicity independently of alterations in topo IIalpha levels.

The p38 pathway provides negative feedback for Ras proliferative signaling.

Ras activates three mitogen-activated protein kinases (MAPKs) including ERK, JNK, and p38. Whereas the essential roles of ERK and JNK in Ras signaling has been established, the contribution of p38 remains unclear. Here we demonstrate that the p38 pathway functions as a negative regulator of Ras proliferative signaling via a feedback mechanism. Oncogenic Ras activated p38 and two p38-activated protein kinases, MAPK-activated protein kinase 2 (MK2) and p38-related/activated protein kinase (PRAK). MK2 and PRAK in turn suppressed Ras-induced gene expression and cell proliferation, whereas two mutant PRAKs, unresponsive to Ras, had little effect. Moreover, the constitutive p38 activator MKK6 also suppressed Ras activity in a p38-dependent manner whereas arsenite, a potent chemical inducer of p38, inhibited proliferation only in a tumor cell line that required Ras activity. MEK was required for Ras stimulation of the p38 pathway. The p38 pathway inhibited Ras activity by blocking activation of JNK, without effect upon ERK, as evidenced by the fact that PRAK-mediated suppression of Ras-induced cell proliferation was reversed by coexpression of JNKK2 or JNK1. These studies thus establish a negative feedback mechanism by which Ras proliferative activity is regulated via signaling integrations of MAPK pathways.

Ras stimulates DNA topoisomerase II alpha through MEK: a link between oncogenic signaling and a therapeutic target.

Topoisomerase II alpha (topo II alpha) is a major target of antitumor treatments. In an effort to determine why this protein might be a better target in tumor cells than in normal cells, we attempted to determine if the altered proliferative signaling in a tumor cell might effect the levels of expression of the topo II alpha gene. In support of this idea, it was found that topo II alpha was elevated following microinjection of oncogenic Ras protein. Oncogenic ras was further shown to stimulate the topo II alpha promoter. Stimulation by ras was independent of the normal cell cycle regulation of this promoter. Transactivation of topo II alpha by ras required both the MEK/ERK pathway, and the stress-associated protein kinase (SAPK) signaling pathway. As a direct confirmation that both ERK and SAPK were involved in topo II alpha regulation, a constitutively active MEKK that stimulates these two kinases simultaneously was shown to strongly induce topo II alpha promoter activity. Activation of either pathway alone, on the other hand, only slightly stimulated the topo II alpha promoter. Deletion analyses showed that elements near both the 5' and 3' ends of the promoter were responsible for the ras stimulation. Site-directed mutagenesis further demonstrated that an Ets-like binding site near the 5' end (-480 to -475) was one of the responsive elements. Taken together, these studies demonstrate the direct role of Ras signaling in stimulation of topo II alpha expression, and thereby establish a link between the action of a common tumor mutation and the target of multiple anti-tumor reagents.

Cyclin D1 production in cycling cells depends on ras in a cell-cycle-specific manner.

BACKGROUND: Cellular Ras and cyclin D1 are required at similar times of the cell cycle in quiescent NIH3T3 cells that have been induced to proliferate, but not in the case of cycling NIH3T3 cells. In asynchronous cultures, Ras activity has been found to be required only during G2 phase to promote passage through the entire upcoming cell cycle, whereas cyclin D1 is required through G1 phase until DNA synthesis begins. To explain these results in molecular terms, we propose a model whereby continuous cell cycle progression in NIH3T3 cells requires cellular Ras activity to promote the synthesis of cyclin D1 during G2 phase. Cyclin D1 expression then continues through G1 phase independently of Ras activity, and drives the G1-S phase transition. RESULTS: We found high levels of cyclin D1 expression during the G2, M and G1 phases of the cell cycle in cycling NIH3T3 cells, using quantitative fluorescent antibody measurements of individual cells. By microinjecting anti-Ras antibody, we found that the induction of cyclin D1 expression beginning in G2 phase was dependent on Ras activity. Consistent with our model, cyclin D1 expression during G1 phase was particularly stable following neutralization of cellular Ras. Finally, ectopic expression of cyclin D1 largely overcame the requirement for cellular Ras activity during the continuous proliferation of cycling NIH3T3 cells. CONCLUSIONS: Ras-dependent induction of cyclin D1 expression beginning in G2 phase is critical for continuous cell cycle progression in NIH3T3 cells.

Cell cycle arrest and morphological alterations following microinjection of NIH3T3 cells with Pur alpha.

Levels of Pur alpha, a protein implicated in control of both DNA replication and gene transcription, fluctuate during the cell cycle, being lowest in early S phase and highest just after mitosis. Here we have employed a new video time-lapse technique enabling us to determine the cell cycle position of each cell in an asynchronous culture at a given time and to ask whether introduction of Pur alpha protein at specific times can affect cell cycle progression. Approximately 80% of all NIH3T3 cells injected with Pur alpha were inhibited from passing through mitosis. Cells injected with Pur alpha during S or G2 phases were efficiently blocked with a 4N (G2 phase) DNA level, as determined by quantitative DNA photometry of individual cells. Of the cells injected with Pur alpha during G1 phase, 40% experienced a rapid cell death characterized by extreme cellular fragmentation. Of those G1 injected cells which remained viable, approximately equal numbers were arrested with either 2N or 4N DNA levels. Cells arrested by Pur alpha in G2 phase grew to cover a large surface area. These results link fluctuations in Pur alpha levels to aspects of cell cycle control.

Cellular ras and cyclin D1 are required during different cell cycle periods in cycling NIH 3T3 cells.

Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.

Adenovirus-mediated gene transfer of dominant negative Ha-Ras inhibits proliferation of primary meningioma cells.

OBJECTIVE: Previous studies demonstrated that activation of receptor tyrosine kinases in human meningiomas by an autocrine or paracrine growth-stimulatory loop plays an important role in meningioma proliferation. Although it is well established that the proliferative signal from protein tyrosine kinase receptors is transduced through Ras proteins, the relevance of the Ras pathway in meningioma proliferation, to our knowledge, has not been studied. The purpose of this study was, therefore, to determine whether Ras proteins are functionally important in meningioma proliferation. METHODS: Meningioma cells of nine primary cell cultures were infected with the recombinant adenovirus Ad-rasN17 encoding the dominant negative Ras protein or control adenovirus Ad-pAC. Ras-N17 is a Ras mutant protein with substitution of asparagine for serine at position 17 in the cellular Ha-Ras protein that inhibits function of all endogenous cellular Ras proteins. Proliferation of meningioma cells was measured using [3H]thymidine or 5-bromo-2'-deoxyuridine labeling and detection assays. RESULTS: Infection of meningioma cells with Ad-rasN17 dramatically increased the expression levels of the Ras-N17 mutant protein and inhibited phosphorylation of the mitogen-activated protein kinases, compared with uninfected cells or cells infected with the control adenovirus. Suppression of Ras proteins inhibited proliferation of all exponentially growing and growth-arrested meningioma cells stimulated with serum. CONCLUSION: The obtained results suggest that proliferation of primary meningioma cells is dependent on the presence of functional Ras proteins. Therefore, inhibition of the Ras pathway may be important in preventing growth factor-stimulated meningioma proliferation.

p53 inhibits entry into mitosis when DNA synthesis is blocked.

Human and mouse fibroblasts with normal p53 fail to enter mitosis when DNA synthesis is blocked by aphidicolin or hydroxyurea. Isogenic p53-null fibroblasts do enter mitosis with incompletely replicated DNA, revealing that p53 contributes to a checkpoint that ensures that mitosis does not occur until DNA synthesis is complete. When treated with N-(phosphonacetyl)-L-aspartate (PALA), which inhibits pyrimidine nucleotide synthesis, leading to synthesis of damaged DNA from highly unbalanced dNTP pools, p53-null cells enter mitosis after they have completed DNA replication, but cells with wild-type p53 do not, revealing that p53 also mediates a checkpoint that monitors the quality of newly replicated DNA.

p21Waf1 inhibits the activity of cyclin dependent kinase 2 by preventing its activating phosphorylation.

Prostaglandin A2 (PGA2), a potent inhibitor of the growth of many cell types, inhibits G1 phase cyclin dependent kinases (cdk). Although PGA2 suppresses cyclin D1 and elevates p21Waf1 levels, it was the failure of cdk2 to become activated by phosphorylation which correlated best with growth inhibition. In kinetic studies, cdk2 activation was inhibited efficiently only if p21Waf1 levels increased prior to the activating phosphorylation; suggesting that p21Waf1 had blocked this phosphorylation. This model was confirmed in cells from p21Waf1 knockout mice where PGA2 was completely unable to block the activating phosphorylation of cdk2, or inhibit cdk2 activity. As expected, growth inhibition of p21Waf1(-/-) cells was not observed at PGA2 concentrations which inhibited cdk2 activity and growth of p21Waf1(+/+) cells.

Protection against cyclophosphamide-induced alopecia and inhibition of mammary tumor growth by topical 1,25-dihydroxyvitamin D3 in mice.

Twenty-one-day-old BALB/c mice were shaved on the back to synchronize hair growth. On day 30 or 31, when at least 90% of mice exhibited hair regrowth in the shaved area, 1,25(OH)2D3 was applied topically to the shaved area daily for 5 days. On the 6th day, cyclophosphamide (Cytoxan, CTX) was injected i.p. to induce hair loss in the shaved area. Alopecia was induced in a dose-dependent manner by CTX treatment within 1 to 2 weeks. This effect was reduced significantly if mice were pre-treated with 1,25(OH)2D3, though only slight protection was observed in female mice. Interestingly, this 1,25(OH)2D3-mediated protection against hair loss was attenuated in male mice but became more significant in female mice when they were inoculated with the EMT-6 murine mammary tumor prior to treatment. More importantly, topical treatment with 1,25(OH)2D3 alone was able to inhibit EMT-6 tumor growth in both male and female BALB/c mice. Furthermore, 1,25(OH)2D3 pre-treatment also augmented the anti-tumor effect of CTX. Our results demonstrate that topical application of 1,25(OH)2D3 can protect against CTX-induced alopecia both in tumor-free and in tumor-bearing mice in a sex-dependent manner. Moreover, 1,25(OH)2D3 was shown, either alone or in combination with CTX, to inhibit tumor growth.

Neurofibromin colocalizes with mitochondria in cultured cells.

Mutations in neurofibromatosis type 1 target the gene coding for neurofibromin. While neurofibromin is able to accelerate the rate of GTP hydrolysis by cellular Ras proteins, its biological function is not well understood. To gain information regarding its function, the intracellular localization of neurofibromin was analyzed in cultured cell lines using polyclonal antisera raised against four neurofibromin-specific peptides, three from the carboxyl terminus and one from the amino terminus. In methanol-fixed cells distinct rod-like structures distributed throughout the cytoplasm were recognized by the antisera. Similar structures were seen with each antiserum, including affinity-purified antibodies, and in each of the cultured cell lines tested. Similar structures were seen in paraformaldehyde-fixed cells. Double staining experiments showed that these structures colocalize with mitochondria, but not with actin, beta-tubulin, or endoplasmic reticulum. When actin or tubulin structures within the cell were disrupted by separate antimitotic drugs, these stained structures retained their shape. Neurofibromin association with mitochondria was confirmed biochemically when highly purified mitochondrial fractions from bovine heart tissue were shown in Western analysis to contain neurofibromin. This association might be helpful in predicting identification of some of the cellular proteins with which neurofibromin interacts.

Reduced expression of schwannomin/merlin in human sporadic meningiomas.

OBJECTIVE: The neurofibromatosis type 2 gene is frequently mutated in sporadic meningiomas. The protein product of the neurofibromatosis type 2 gene is called schwannomin or merlin. Its expression in leptomeningeal cells from which meningiomas are derived and the characteristics of mutated forms in meningiomas, to our knowledge, have not been previously studied. METHODS: Immunoblotting and immunoprecipitation experiments with two specific antibodies were used to determine the size and subcellular distribution of schwannomin/merlin in rabbit and human brain tissue and established human leptomeningeal LTAg2B cells. Immunoblotting was used to determine the expression level of schwannomin/merlin in 14 human sporadic meningiomas. RESULTS: Both antibodies detect a protein of approximately 66 kDa, which is predominantly expressed in the Triton X-100-insoluble fraction of the brain and LTAg2B cells. The levels of schwannomin/merlin were severely reduced in eight tumors (57%) when compared with the expression levels in the human brain, LTAg2B cells, and the remaining six meningiomas. All six tumors with the normal schwannomin/merlin expression were of meningotheliomatous type. In contrast, all other histological types and one meningotheliomatous tumor with psammoma bodies were deficient in the 66-kDa schwannomin/merlin. Although nonsense mutations leading to premature stop codons are common in the neurofibromatosis type 2 gene in meningiomas, we found no evidence of truncated schwannomin/merlin forms in the tumors analyzed. CONCLUSION: The absence of complete schwannomin/merlin in almost 60% of primary sporadic meningiomas seems to be an important factor in meningioma tumorigenesis. The development of meningotheliomatous meningiomas is probably linked to alterations in other oncogenes or tumor suppressor genes.

Reduced expression of neurofibromin in human meningiomas.

Meningiomas are common, mostly benign, tumours arising from leptomeningeal cells of the meninges, which frequently contain mutations in the neurofibromatosis type 2 (NF2) gene. In this study, we analysed a protein product of the neurofibromatosis type 1 (NF1) gene, neurofibromin, in human established leptomeningeal cells LTAg2B, in 17 sporadic meningiomas and in a meningioma from a patient affected by NF2. The expression level of neurofibromin was determined by immunoblotting and immunoprecipitation with anti-neurofibromin antibodies. The functional status of neurofibromin was analysed through its ability to stimulate the intrinsic GTPase activity of p21 ras. In the cytosolic extracts of four sporadic meningiomas and in the NF2-related meningioma, the expression level and the GTPase stimulatory activity of neurofibromin were drastically reduced compared with the level present in the human brain, human established leptomeningeal cells LTAg2B and the remaining 13 meningiomas. Our results suggest that neurofibromin is expressed in leptomeningeal cells LTAg2B and in most meningiomas, i.e. tumours derived from these cells. The reduced expression and GTPase stimulatory activity of neurofibromin was found in about 23% of meningiomas and in the single NF2-related meningioma analysed. These results suggest that decreased levels of neurofibromin in these tumours may contribute to their tumorigenesis.

Oncogenic transformation potentiates apoptosis, S-phase arrest and stress-kinase activation by etoposide.

The exact mechanisms for the selective toxicity of chemotherapeutic drugs against tumor cells are not fully understood. We designed a series of experiments to test the possibility that the positive proliferative signal initiated by oncogenes might change the sensitivity for apoptosis induction by the anticancer drug etoposide (VP16), an inhibitor of topoisomerase II (Topo II). Treatment with VP16 induced significantly increased apoptosis in NIH3T3 cells transformed by oncogenic src, ras or raf, compared with the normal 3T3 cells. Apopototic changes involved nuclear DNA fragmentation, morphological alterations and decreased viability. Furthermore it was shown that stress-activated protein kinase (SAPK) was activated much more strongly in all three transformed lines compared to untransformed cells by VP16 treatment, while slight activation of extracellular signal-regulated kinase (ERK1) was observed in all four cell lines. In addition, the transformed cells displayed arrest in mid-S-phase following the treatment, whereas NIH3T3 cells were primarily arrested in late S and G2/M phase. Finally, the cyclin-dependent kinase inhibitor p21 WAF1 was induced in all four cell lines, although induction of p53 was not detected in any of these cell lines. Taken together our results demonstrated that oncogenic transformation can sensitize the cells to apoptosis induction, stress kinase activation and cell cycle arrest in response to VP16 treatment. These results may have important implications for understanding the selective toxicity of anti-cancer drugs in tumor cells.

Antisense oligonucleotides demonstrate a dominant role of c-Ki-RAS proteins in regulating the proliferation of diploid human fibroblasts.

Although members of the RAS protein family (Ha-, Ki-, and N-RAS) are known to play a key role in normal cell proliferation and to be frequently mutated in naturally occurring tumors, it remains unclear which of these proteins functions to regulate growth in normal cells. Gene-specific oligonucleotides (oligos) against c-Ki-RAS (ISIS 6957), c-Ha-RAS (ISIS 2503), and oncogenic Ha-RAS (ISIS 2570) were used to analyze the requirement for individual RAS proteins in the proliferation of diploid human lung fibroblasts (MRC-5), and human bladder carcinoma cell lines with (T24) or without (J-82) a RAS mutation. The oncogenic Ha-RAS oligo substantially inhibited T24 cell proliferation, whereas the c-Ki-RAS and control (ISIS 1966) oligos had little effect. Interestingly, in MRC-5 cells the c-Ki-RAS but not c-Ha-RAS oligo was effective in inhibiting cell proliferation. No inhibition was seen in the J-82 cells with either oligo. In Western analysis, p21 RAS protein was decreased following treatment with the oncogenic Ha-RAS oligo in T24 cells or the c-Ki-RAS oligo in MRC-5 cells, whereas no reductions were observed in J-82 cells with either oligo. The specificity of these oligos was demonstrated in Northern analyses in which both Ha-RAS and Ki-RAS oligo treatment resulted in reduced levels of their respective mRNAs in all three cell lines, whereas the mutant Ha-RAS mRNA in T24 cells was most effectively reduced with the oncogenic Ha-RAS oligo. These results demonstrate that oncogenic Ha-RAS plays an important role in the proliferation of T24 cells, whereas c-Ki-RAS contributes predominantly to the proliferation of normal MRC-5 cells.

Prostaglandin A2 blocks the activation of G1 phase cyclin-dependent kinase without altering mitogen-activated protein kinase stimulation.

Prostaglandin A2 (PGA2) reversibly blocked the cell cycle progression of NIH 3T3 cells at G1 and G2/M phase. When it was applied to cells synchronized in G0 or S phase, cells were blocked at G1 and G2/M, respectively. The G2/M blockage was transient. Microinjected oncogenic leucine 61 Ras protein could not override the PGA2 induced G1 blockage, nor could previous transformation with the v-raf oncogene. The serum-induced activation of mitogen-activated protein kinase was not inhibited by PGA2 treatment. These data suggest that PGA2 blocks cell cycle progression without interfering with the cytosolic proliferative signaling pathway. Combined microinjection of E2F-1 and DP-1 proteins or microinjected adenovirus E1A protein, however, could induce S phase in cells arrested in G1 by PGA2, indicating that PGA2 does not directly inhibit the process of DNA synthesis. In quiescent cells, PGA2 blocked the normal hyperphosphorylation of the retinoblastoma susceptible gene product and the activation of cyclin-dependent kinase (CDK) 2 and CDK4, in response to serum stimulation. PGA2 treatment elevated the p21Waf1/Cip1/Sdi1 protein expression level. These data indicate that PGA2 may arrest the cell cycle in G1 by interfering with the activation of G1 phase CDKs.

Transient blockage of proliferative signalling: a novel strategy for protective chemotherapy.

An intact proliferative signalling pathway is essential to the growth of all normal cells, but is often not required by tumor cells. This fact was used to devise a protective chemotherapeutic protocol potentially applicable to all tissues. Four treatments were chosen to temporarily disrupt proliferative signalling. They acted either upstream, at, or downstream of cellular ras activity. As expected, the cell cycle progression of normal cells was temporarily interrupted, while those cells transformed by tumor genes, or tumor cells themselves often were not affected. During these cell cycle blocking treatments the cells were exposed to the topoisomerase inhibitor m-AMSA. This anti-cancer drug is selectively toxic to cycling cells. In each case the tumor cells were selectively killed as judged either by their ability to incorporate labeled thymidine, replate, or grow. These studies suggest new ways to utilize current drugs or search for new ones.

A conserved region of c-Ha-Ras is required for efficient GTPase stimulation by GTPase activating protein but not neurofibromin.

The effector binding domain and the switch II region of c-Ha-Ras are necessary for p120GAP-stimulated GTP hydrolysis. We report a third region of c-Ha-Ras located within the alpha 3 helix (amino acids 101-103) which is also required for efficient p120GAP, but not neurofibromin-mediated hydrolysis. This highly conserved region of the Ras protein was investigated using an insertion-deletion mutant (Ras-100LIR104) originally characterized by Willumsen et al. (Willumsen, B. M., Adari, H., Zhang, K., Papageorge, A. G., Stone, J. C., McCormick, F., and Lowy, D. R (1989) in The Guanine Nucleotide Binding Proteins; Common Structural and Functional Properties (Bosch, L., Kraal, B., and Parmeggiani, A., eds) pp. 165-178, Plenum Press, New York). The 100LIR104 substitution did not alter the intrinsic hydrolytic rate of the protein. The p120GAP-stimulated hydrolysis of Ras-100LIR104, however, was decreased by 2-3-fold compared to wild type Ras. This decrease in p120GAP-stimulated hydrolysis was not due to its inability to physically associate with Ras-100LIR104. GTP (as determined by competitive binding assays). Surprisingly, neurofibromin-stimulated GTP hydrolysis was unaltered by the mutation. Finally, no differences were observed in the ability of either the p120GAP catalytic domain or the neurofibromin GRD to accelerate Ras-100LIR104 GTPase activity, indicating that the amino-terminal noncatalytic GAP region is critical for p120GAP-stimulated GTP hydrolysis. This is the first report of a Ras mutation which differentiates between p120GAP and neurofibromin activity.

The adenovirus E1A protein overrides the requirement for cellular ras in initiating DNA synthesis.

The adenovirus E1A protein can induce cellular DNA synthesis in growth-arrested cells by interacting with the cellular protein p300 or pRb. In addition, serum- and growth factor-dependent cells require ras activity to initiate DNA synthesis and recently we have shown that Balb/c 3T3 cells can be blocked in either early or late G1 following microinjection of an anti-ras antibody. In this study, the E1A 243 amino acid protein is shown through microinjection not only to shorten the G0 to S phase interval but, what is more important, to override the inhibitory effects exerted by the anti-ras antibody in either early or late G1. Specifically, whether E1A is co-injected with anti-ras into quiescent cells or injected 18 h following a separate injection of anti-ras after serum stimulation, it efficiently induces cellular DNA synthesis in cells that would otherwise be blocked in G0/G1. Moreover, injection of a mutant form of E1A that can no longer associate with p300 is just as efficient as wild-type E1A in stimulating DNA synthesis in cells whose ras activity has been neutralized by anti-ras. The results presented here show that E1A is capable of overriding the requirement of cellular ras activity in promoting the entry of cells into S phase. Moreover, the results suggest the possibility that pRb and/or pRb-related proteins may function in a ras-dependent pathway that enables E1A to achieve this activity.