Inhibition Of Cellular DNA Synthesis Research Articles

Adenovirus-mediated expression of MOAP-1, Bax and RASSF1A antagonizes chemo-drug resistance of human breast cancer cells expressing cancer stem cell markers

Cancer is one of the major leading causes of mortality globally and chemo-drug-resistant cancers pose significant challenges to cancer treatment by reducing patient survival rates and increasing treatment costs. Although the mechanisms of chemoresistance vary among different types of cancer, cancer cells are known to share several hallmarks, such as their resistance to apoptosis as well as the ability of cancer stem cells to produce metastatic daughter cells that are resistant to chemotherapy. To address the issue of chemo-drug resistance in cancer cells, a tetracistronic expression construct, Ad-MBR-GFP, encoding adenovirus-mediated expression of MOAP-1, Bax, RASSSF1A, and GFP, was generated to investigate its potential activity in reducing or inhibiting the chemo-drug resistant activity of the human breast cancer cells, MCF-7-CR and MDA-MB-231. When infected by Ad-MBR-GFP, the cancer cells exhibited round cell morphology and nuclei condensation with positive staining for annexin-V. Furthermore, our results showed that both MCF-7-CR and MDA-MB-231 cells stained positively for CD 44 and negatively for CD 24 (CD44+/CD24-) with high levels of endogenous ALDH activity whereas SNU-1581 breast cancer cells were identified as CD 44-/CD 24- cells with relatively low levels of endogenous ALDH activity and high sensitivity toward chemo-drugs, suggesting that both CD 44 and ALDH activity contribute to chemo-drug resistance. Moreover, both MCF-7-CR and MDA-MB-231 cells showed strong chemo-drug sensitivity to cisplatin when the cells were infected by Ad-MBR-GFP, leading to 9-fold and 2-fold reduction in the IC 50 values when compared to cisplatin treatment alone, respectively. The data were further supported by 3D (soft agar) and spheroid cell models of MCF-7-CR and MDA-MB-231 cells which showed a 2-fold reduction of a number of cell colonies and spheroid size when treated with both Ad-MBR-GFP and cisplatin, and compared to control. Other than chemo-sensitivity, Ad-MBR-GFP-infected cancer cells retarded cell migration. Flow cytometry analysis showed that the mechanism of action of Ad-MBR-GFP involved cell cycle arrest at the G1 phase and inhibition of cellular DNA synthesis. Taken together, our investigation showed that Ad-MBR-GFP mediated chemo-drug sensitization in the infected cancer cells involved the activation of apoptosis signaling, cell cycle arrest, and inhibition of DNA synthesis, suggesting that Ad-MBR-GFP is potentially efficacious for the treatment of chemo-drug resistant cancers.

Activation of E2F-dependent transcription by the mouse cytomegalovirus M117 protein affects the viral host range.

Cytomegaloviruses (CMVs) have a highly restricted host range as they replicate only in cells of their own or closely related species. To date, the molecular mechanisms underlying the CMV host restriction remain poorly understood. However, it has been shown that mouse cytomegalovirus (MCMV) can be adapted to human cells and that adaptation goes along with adaptive mutations in several viral genes. In this study, we identify MCMV M117 as a novel host range determinant. Mutations in this gene enable the virus to cross the species barrier and replicate in human RPE-1 cells. We show that the M117 protein is expressed with early kinetics, localizes to viral replication compartments, and contributes to the inhibition of cellular DNA synthesis. Mechanistically, M117 interacts with members of the E2F transcription factor family and induces E2F target gene expression in murine and human cells. While the N-terminal part of M117 mediates E2F interaction, the C-terminal part mediates self-interaction. Both parts are required for the activation of E2F-dependent transcription. We further show that M117 is dispensable for viral replication in cultured mouse fibroblasts and endothelial cells, but is required for colonization of mouse salivary glands in vivo. Conversely, inactivation of M117 or pharmacological inhibition of E2F facilitates MCMV replication in human RPE-1 cells, whereas replacement of M117 by adenovirus E4orf6/7, a known E2F activator, prevents it. These results indicate that E2F activation is detrimental for MCMV replication in human cells. In summary, this study identifies MCMV M117 as a novel E2F activator that functions as a host range determinant by precluding MCMV replication in human cells.

The Cellular Protein MCM3AP Is Required for Inhibition of Cellular DNA Synthesis by the IE86 Protein of Human Cytomegalovirus

Like all DNA viruses, human cytomegalovirus (HCMV) infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G0-G1 phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86) protein - an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3) associated protein (MCM3AP), which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner.

Depletion of Cellular Pre-Replication Complex Factors Results in Increased Human Cytomegalovirus DNA Replication

Although HCMV encodes many genes required for the replication of its DNA genome, no HCMV-encoded orthologue of the origin binding protein, which has been identified in other herpesviruses, has been identified. This has led to speculation that HCMV may use other viral proteins or possibly cellular factors for the initiation of DNA synthesis. It is also unclear whether cellular replication factors are required for efficient replication of viral DNA during or after viral replication origin recognition. Consequently, we have asked whether cellular pre-replication (pre-RC) factors that are either initially associated with cellular origin of replication (e.g. ORC2), those which recruit other replication factors (e.g. Cdt1 or Cdc6) or those which are subsequently recruited (e.g. MCMs) play any role in the HCMV DNA replication. We show that whilst RNAi-mediated knock-down of these factors in the cell affects cellular DNA replication, as predicted, it results in concomitant increases in viral DNA replication. These data show that cellular factors which initiate cellular DNA synthesis are not required for the initiation of replication of viral DNA and suggest that inhibition of cellular DNA synthesis, in itself, fosters conditions which are conducive to viral DNA replication.

FNC, a novel nucleoside analogue inhibits cell proliferation and tumor growth in a variety of human cancer cells

Inhibition of cellular DNA synthesis is a strategy to block cancer cell division. Nucleoside analogues can incorporate into DNA and terminate DNA strand elongation. So far, several nucleoside analogues have been successfully used as anticancer drugs. FNC, 2′-deoxy-2′-β-fluoro-4′-azidocytidine is a novel cytidine analogue which demonstrated potent activity against hepatitis C virus (HCV). To investigate the therapeutic potential of FNC in human cancers we studied its activity in a number of cancer cells in vitro and in vivo. FNC potently inhibited cell proliferation with an IC 50 of 0.95–4.55 μM in a variety of aggressive human cancer cell lines including B-cell non-Hodgkin's lymphomas, lung adenocarcinoma and acute myeloid leukemia. Cells treated with FNC exhibited G1 and S cell cycle arrest at high and low dose, respectively, which confirms the mechanism of action of nucleoside analogues. Treatment of B-NHL cell lines with FNC induced apoptosis in a dose and time dependent manner. Finally, mouse xenograft models of hepatocarcinoma (H22), sarcoma (S180) and gastric carcinoma (SGC7901) demonstrated that FNC had significant tumor growth inhibition activity in a dose-dependent manner with low toxicity. Together, our results suggest that FNC may be a valuable therapy in cancer patients and warrant early phase clinical trial evaluation.

Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G(2)-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1;E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

Hydroxyurea Associated Leg Ulcer Succesfully Treated with Hyperbaric Oxygen in a Diabetic Patient

Oxygen tension in healing tissues is heterogeneous. Increased oxygen mostly stimulates repair mechanisms and enhances tissue healing. Hyperbaric oxygen therapy increases blood and tissue oxygen content and may help maintain cellular integrity and function. Hydroxyurea (HU) is a cytotoxic agent, which leads to inactivation of ribonucleotide reductase, inhibition of cellular DNA synthesis, and cell death in the S phase. HU induced leg ulcers occur after use of this agent for a long time and at higher cumulative doses. Here we describe a diabetic patient with foot ulcer associated with HU treatment for polycythemia vera, who was treated successfully with hyperbaric oxygen and general wound care after discontinuation of HU. Faster improvement of leg ulcer in our patient compared to literature regarding HU withdrawal as single therapy suggests that hyperbaric oxygen may be helpful in the management of HU associated leg ulcers, especially in diabetic subjects.

Inhibition of Cellular DNA Synthesis by the Human Cytomegalovirus IE86 Protein Is Necessary for Efficient Virus Replication

Human cytomegalovirus (HCMV) expresses several proteins that manipulate normal cellular functions, including cellular transcription, apoptosis, immune response, and cell cycle control. The IE2 gene, which is expressed from the HCMV major immediate-early (MIE) promoter, encodes the IE86 protein. IE86 is a multifunctional protein that is essential for viral replication. The functions of IE86 include transactivation of cellular and viral early genes, negative autoregulation of the MIE promoter, induction of cell cycle progression from G0/G1 to G1/S, and arresting cell cycle progression at the G1/S transition in p53-positive human foreskin fibroblast (HFF) cells. Mutations were introduced into the IE2 gene in the context of the viral genome using bacterial artificial chromosomes (BACs). From these HCMV BACs, a recombinant virus (RV) with a single amino acid substitution in the IE86 protein was isolated that replicates slower and to lower titers than wild-type HCMV. HFF cells infected with the Q548R RV undergo cellular DNA synthesis and do not arrest at any point in the cell cycle. The Q548R RV is able to negatively autoregulate the MIE promoter, transactivate viral early genes, activate cellular E2F-responsive genes, and produce infectious virus. This is the first report of a viable recombinant HCMV that is unable to inhibit cellular DNA synthesis in infected HFF cells.

Ganoderiol F, a ganoderma triterpene, induces senescence in hepatoma HepG2 cells

Ganoderiol F (GolF), a tetracyclic triterpene, was isolated from Ganoderma amboinense and found to induce senescence of cancer cell lines. GolF induced growth arrest of cancer cell lines HepG2, Huh7 and K562, but exerted much less effect in hepatoma Hep3B cells and normal lung fibroblast MRC5 cells, and no effect on peripheral blood mononuclear cells. GolF treatment of the cancer cells, with the exception of Hep3B, resulted in prompt inhibition of DNA synthesis and arrest of cell progression cycle in G1 phase. Short-term exposure of HepG2 cells to GolF temporarily arrested progression of the cell cycle; cell growth was recovered if the drug was withdrawn from the medium after a 24-h exposure. After 18 days of continuous treatment of HepG2 cells with 30 μM GolF, over 50% of cells were found to be enlarged and flattened, and were β-galactosidase positive phenotypes of senescent cells. GolF was found to inhibit activity of topoisomerases in vitro, which may contribute to the inhibition of cellular DNA synthesis. Activation of the mitogen-activated protein kinase EKR and up-regulation of cyclin-dependent kinase inhibitor p16 were found in early stages of GolF treatment and were presumed to cause cell-cycle arrest and trigger premature senescence of HepG2 cells. The growth-arrest and senescence induction capability on cancer cells suggest anticancer potential of GolF.

Autoantibodies to the epidermal growth factor receptor in systemic sclerosis, lupus, and autoimmune mice.

Autoantibodies to the recombinant extracellular domain of epidermal growth factor receptor (exEGFR) were detected by ELISA in the serum of Fas-defective old MRL/MpJ/lpr and C3H/HeJ/gld mice, but not young mice from these strains, or nonautoimmune young and old BALB/c, MRL/MpJ/++, and C3H/HeJ/MMTV mice. Compared with control human subjects without autoimmune disease, the frequency of exEGFR-binding autoantibodies was increased in scleroderma (systemic sclerosis) patients and to a lesser extent in lupus patients. Phage autoantibodies (Fv fragments) isolated from a lupus library by selection on a linear epitope of EGFR (residues 294-310) displayed the ability to bind exEGFR. Treatment of EGFR-expressing A431 cells with autoantibodies purified by affinity chromatography on immobilized exEGFR resulted in specific staining of the cells. Short-lived but strong inhibition of cellular DNA synthesis was observed in the presence of the autoantibodies. We concluded that autoantibody responses to EGFR hold the potential of fulfilling a pathogenic role in autoimmune disease.

Synergistic inhibition of herpesvirus replication by docosanol and antiviral nucleoside analogs

Interactions between docosanol ( n-docosanol, behenyl alcohol) and nucleoside or pyrophosphate analogs were investigated in vitro. The anti-HSV activity of acyclovir (ACV) was synergistically enhanced by treatment of cells with docosanol as judged by inhibition of progeny virus production and plaque formation. This drug interaction between ACV and docosanol was observed with laboratory strains of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), oral and genital clinical isolates of HSV, cytomegalovirus (CMV), and varicella zoster virus (VZV). Near optimal concentrations of docosanol plus ACV inhibited HSV replication >99% more than either drug alone, including emergence of ACV-resistant variants. The response was observed with African Green Monkey kidney cells, normal human foreskin cells, and normal human lung cells. Treatment of cells with docosanol also synergistically intensified the inhibition of HSV production by all tested nucleoside analogs, including trifluorothymidine (TFT), adenine arabinoside (Ara-A), and ribavirin. An additive anti-HSV effect was observed with docosanol and phosphonoformate (PFA). No evidence was found for either synergistic inhibition of cellular DNA synthesis or induction of overt cellular toxicity when docosanol was combined with ACV, TFT, Ara-A, ribavirin, PFA, 8-azaguanine, or 5-fluorouracil. The ability of docosanol treatment to increase the antiviral activities of nucleoside analog antiviral drugs, coupled with a lack of toxic interactions, translates to substantial improvements in drug selectivity ratios.

Selective regulation of human immunodeficiency virus-infected CD4(+) lymphocytes by a synthetic immunomodulator leads to potent virus suppression in vitro and in hu-PBL-SCID mice.

We have previously observed that the synthetic immunomodulator Murabutide inhibits human immunodeficiency virus type 1 (HIV-1) replication at multiple levels in macrophages and dendritic cells. The present study was designed to profile the activity of Murabutide on CD8-depleted phytohemagglutinin-activated lymphocytes from HIV-1-infected subjects and on the outcome of HIV-1 infection in severe combined immunodeficiency mice reconstituted with human peripheral blood leukocytes (hu-PBL-SCID mice). Maintaining cultures of CD8-depleted blasts from 36 patients in the presence of Murabutide produced dramatically reduced levels of viral p24 protein in the supernatants. This activity correlated with reduced viral transcripts and proviral DNA, was evident in cultures harboring R5, X4-R5, or X4 HIV-1 isolates, was not linked to inhibition of cellular DNA synthesis, and did not correlate with beta-chemokine release. Moreover, c-myc mRNA expression was down-regulated in Murabutide-treated cells, suggesting potential interference of the immunomodulator with the nuclear transport of viral preintegration complexes. On the other hand, daily treatment of HIV-1-infected hu-PBL-SCID mice with Murabutide significantly reduced the viral loads in plasma and the proviral DNA content in human peritoneal cells. These results are the first to demonstrate that a clinically acceptable synthetic immunomodulator with an ability to enhance the host's nonspecific immune defense mechanisms against infections can directly regulate cellular factors in infected lymphocytes, leading to controlled HIV-1 replication.

Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways.

Most cervical carcinomas express high-risk human papillomaviruses (HPVs) E6 and E7 proteins, which neutralize cellular tumor suppressor function. To determine the consequences of removing the E6 and E7 proteins from cervical cancer cells, we infected HeLa cells, a cervical carcinoma cell line that contains HPV18 DNA, with a recombinant virus that expresses the bovine papillomavirus E2 protein. Expression of the E2 protein resulted in rapid repression of HPV E6 and E7 expression, followed approximately 12 h later by profound inhibition of cellular DNA synthesis. Shortly after E6/E7 repression, there was dramatic posttranscriptional induction of p53. Two p53-responsive genes, mdm2 and p21, were induced with slightly slower kinetics than p53 and appeared to be functional, as assessed by inhibition of cyclin-dependent kinase activity and p53 destabilization. There was also dramatic posttranscriptional induction of p105(Rb) and p107 after E6/E7 repression, followed shortly thereafter by induction of p130. By 24 h after infection, only hypophosphorylated p105(Rb) was detectable and transcription of several Rb/E2F-regulated genes was dramatically repressed. Constitutive expression of the HPV16 E6/E7 genes alleviated E2-induced growth inhibition and impaired activation of the Rb pathway and repression of E2F-responsive genes. This dynamic response strongly suggests that the p53 and Rb tumor suppressor pathways are intact in HeLa cells and that repression of HPV E6 and E7 mobilizes these pathways in an orderly fashion to deliver growth inhibitory signals to the cells. Strikingly, the major alterations in the cell cycle machinery underlying cervical carcinogenesis can be reversed by repression of the endogenous HPV oncogenes.

Linkage between reovirus-induced apoptosis and inhibition of cellular DNA synthesis: role of the S1 and M2 genes.

The mammalian reoviruses are capable of inhibiting cellular DNA synthesis and inducing apoptosis. Reovirus strains type 3 Abney (T3A) and type 3 Dearing (T3D) inhibit cellular DNA synthesis and induce apoptosis to a substantially greater extent than strain type 1 Lang (T1L). We used T1L x T3A and T1L x T3D reassortant viruses to identify viral genes associated with differences in the capacities of reovirus strains to elicit these cellular responses to viral infection. We found that the S1 and M2 genome segments determine differences in the capacities of both T1L x T3A and T1L x T3D reassortant viruses to inhibit cellular DNA synthesis and to induce apoptosis. These genes encode viral outer-capsid proteins that play important roles in viral attachment and disassembly. To extend these findings, we used field isolate strains of reovirus to determine whether the strain-specific differences in inhibition of cellular DNA synthesis and induction of apoptosis are also associated with viral serotype, a property determined by the S1 gene. In these experiments, type 3 field isolate strains were found to inhibit cellular DNA synthesis and to induce apoptosis to a greater extent than type 1 field isolate strains. Statistical analysis of these data indicate a significant correlation between the capacity of T1L x T3A and T1L x T3D reassortant viruses and field isolate strains to inhibit cellular DNA synthesis and to induce apoptosis. These findings suggest that reovirus-induced inhibition of cellular DNA synthesis and induction of apoptosis are linked and that both phenomena are induced by early steps in the viral replication cycle.

Prolonged gene expression and cell survival after infection by a herpes simplex virus mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22.

Very early in infection, herpes simplex virus (HSV) expresses four immediate-early (IE) regulatory proteins, ICP4, ICP0, ICP22, and ICP27. The systematic inactivation of sets of the IE proteins in cis, and the subsequent phenotypic analysis of the resulting mutants, should provide insights into how these proteins function in the HSV life cycle and also into the specific macromolecular events that are altered or perturbed in cells infected with virus strains blocked very early in infection. This approach may also provide a rational basis to assess the efficacy and safety of HSV mutants for use in gene transfer experiments. In this study, we generated and examined the phenotype of an HSV mutant simultaneously mutated in the ICP4, ICP27, and ICP22 genes of HSV. Unlike mutants deficient in ICP4 (d120), ICP4 and ICP27 (d92), and ICP4 and ICP22 (d96), mutants defective in ICP4, ICP27, and ICP22 (d95) were visually much less toxic to Vero and human embryonic lung cells. Cells infected with d95 at a multiplicity of infection of 10 PFU per cell retained a relatively normal morphology and expressed genes from the viral and cellular genomes for at least 3 days postinfection. The other mutant backgrounds were too toxic to allow examination of gene expression past 1 day postinfection. However, when cell survival was measured by the capacity of the infected cells to form colonies, d95 inhibited colony formation similarly to d92. This apparent paradox was reconciled by the observation that host cell DNA synthesis was inhibited in cells infected with d120, d92, d96, and d95. In addition, all of the mutants exhibited pronounced and distinctive alterations in nuclear morphology, as determined by electron microscopy. The appearance of d95-infected cells deviated from that of uninfected cells in that large circular structures formed in the nucleus. d95-infected cells abundantly expressed ICP0, which accumulated in fine punctate structures in the nucleus at early times postinfection and coalesced or grew to the large circular objects that were revealed by electron microscopy. Therefore, while the abundant accumulation of ICPO in the absence of ICP4, ICP22, and ICP27 may allow for prolonged gene expression, cell survival is impaired, in part, as a result of the inhibition of cellular DNA synthesis.

Avian Reovirus σC Protein Contains a Putative Fusion Sequence and Induces Fusion when Expressed in Mammalian Cells

The biological functions of the structural protein σC, from avian reovirus strain RAM-l, were investigated in this study. A putative fusion peptide in σC was recognized in the deduced amino acid sequence by homology with Pneumovirus fusion sequences, and it was thus postulated that this protein may be involved in the formation of syncytia in cells infected with RAM-1. The σC gene was cloned and expressed in mammalian [COS7) cells and the σC protein was found to induce syncytia. It was therefore concluded that this protein is indeed responsible for avian reovirus-induced cell fusion. It was also found that σC caused condensation of the nuclei within a syncytium, as observed in RAM-1-infected cells. On the basis that lids represented condensation of the chromatin, the inhibition of cellular DNA synthesis by the virus and by the σC protein was measured. It was found that the virus caused a 50% reduction in cellular DNA synthesis, but the σC protein did not inhibit DNA synthesis. Therefore pyknosis of the nuclei and inhibition of cellular DNA synthesis by RAM-1 are likely to be separate events.

Inhibition of Cellular and SV40 DNA Replication by the Adeno-Associated Virus Rep Proteins

In order to define the mechanism used by the adeno-associated virus replication ( rep) gene to mediate inhibition of cell proliferation, we have studied its effects on SV40 and cellular DNA replication. SV40 DNA replication was inhibited by the presence of the rep gene in human 293 cells, and the inhibition was not linked to suppression of SV40 early gene expression, Using double-immunofluorescence assays that measured both rep gene expression and bromodeoxyuridine incorporation, we found that the presence of the Rep78 and Rep68 proteins correlated with inhibition of cellular DNA synthesis in NIH3T3 cells. This links the rep gene's anti-proliferative effects to either: ( i) a direct inhibition of DNA synthesis or ( ii) a possible cell cycle block.

INHIBITION OF CELL-PROLIFERATION BY AN ADENOVIRUS VECTOR EXPRESSING THE HUMAN WILD TYPE-P53 PROTEIN

We have developed human adenovirus 5 (Ad5) vectors expressing the wild type human p53 protein or a mutant p53 form under the control of the human cytomegalovirus immediate early gene promoter. Human cells infected with these vectors expressed high levels of p53, accumulating 20-40 fold more protein than found in normal human fibroblasts. The ability of the vectors to affect proliferation of cells in culture was assessed by measuring cell DNA synthesis and colony forming ability after infection with viruses. When the p53 deficient ovarian carcinoma cell line, SKOV-3, was infected with Adp53wt expressing the wild type (wt) p53 protein, a significant inhibition of cellular DNA synthesis was observed, relative to cells infected with Adp53m expressing mutant p53, or a control virus, AdLacZ, expressing bacterial beta-galactosidase. Inhibition was dependent on multiplicity of infection, with no significant effect below 5 pfu/cell, and maximal effect between 25 and 100 PFU/cell which resulted in approximately 95% inhibition of SKOV-3 cell DNA synthesis relative to mock infected cells. Infection of normal human fibroblasts with Adp53wt also inhibited DNA synthesis but to a significantly lesser degree. SKOV-3 cell survival, assayed by ability to form colonies, was reduced at least 10 fold after infection with Adp53wt compared to colony forming ability of cells after infection with either AdLacZ or Adp53m. The results of these studies indicate that p53 expressed by Ad vectors can inhibit proliferation in culture of p53 negative cells by at least 95%, and suggest that such vectors might similarly inhibit the proliferation of tumor cells in vivo.

Expression of the two reovirus S1 gene products in transfected mammalian cells

Reovirus genomic segment S1, which has been implicated in the viral inhibition of cellular DNA synthesis, is transcribed into a single mRNA that encodes two proteins, the ∼ 49-kDa hemagglutinin, σ1, and the apparently nonstructural protein, p14. These two polypeptides have been expressed in mammalian cells, together or independently, in order to assess their role in the shutdown of host DNA replication. Results obtained with transient and stable expression systems demonstrate that production of serotype 3 σ1 and p14 together or individually is not sufficient to change the kinetics of DNA replication in uninfected cells. However, inhibition of DNA synthesis by reovirus type 1 infection was enhanced in cells producing type 3σ1 and p14 but not σ1 by itself. In addition, expression of p14 alone led to increased cytopathic effects following infection by either type 1 or type 3 virus. The results suggest that interactions with other viral components are required to elicit the effects of the S1-specified polypeptides on cellular DNA synthesis.

Comparison of the Mechanism of Toxicity of 3′-Azido-3′-Deoxythymidine and 3′-Fluoro-3′-Deoxythymidine in Human Lymphocytes

The thymidine analogue 3′-azido-3′-deoxythymidine (AZT), whilst a useful drug for the treatment of acquired immunodeficiency syndrome, produces toxic side-effects which can be severe and can interfere with therapy. The toxic mechanism of AZT is unknown. We have investigated the relationship between the phosphorylation and effect on natural dNTP pools of 3′-azido-3′-deoxythymidine and the closely related 3′-fluoro-3′-deoxythymidine and their toxicity in human lymphocytes. We attempted to reduce the toxicity by co-administration of natural nucleosides. The toxicity of 3′-fluoro-3′-deoxythymidine could be reduced with thymidine or deoxyuridine five- and 10-fold, respectively. The toxicity of 3′-azido-3′-deoxythymidine could be reduced twofold with cytidine or uridine but was increased by all other nucleosides, including thymidine. Neither analogue caused significant changes in the dNTP pools at cytotoxic concentrations; the effect of the nucleosides in reducing toxicity was not owing to replacement of a depleted dNTP. Thymidine reduced the phosphorylation of 3′-azido-3′-deoxythymidine and 3′-fluoro-3′-deoxythymidine 6 and 17 times, respectively. 3′-azido-3′-deoxythymidine and 3′-fluoro-3′-deoxythymidine appear to have different mechanisms of toxicity. The toxic mechanism of 3′-fluoro-3′-deoxythymidine is probably inhibition of cellular DNA synthesis by the triphosphate. The toxicity of 3′-azido-3′-deoxythymidine in lymphocytes does not appear to be directly related to the amounts of the phosphorylated forms. The mechanism may be interference with RNA metabolism or precursors, perhaps by the nucleoside.