Inhibitor Of DNA Polymerase Alpha Research Articles

Inhibition of eukaryotic dna polymerase alpha by persimmon (Diospyros kaki) extract and related polyphenols.

The effects of persimmon extract (Diospyros kaki) and related polyphenols on eukaryotic DNA polymerase alpha were examined. It was found that persimmon extract, epigallocatechin gallate, and epicatechin gallate strongly inhibited the activity of DNA polymerase alpha purified from calf thymus. Among these polyphenols, persimmon extract had the most potent effect on DNA polymerase alpha activity and the concentration of persimmon extract producing 50% inhibition of the activity was 0.191 microM. Persimmon extract showed a weaker effect on DNA polymerase beta and slightly inhibited primase and DNA polymerase I. The inhibition of DNA polymerase alpha by persimmon extract was competitive with the template-primer and noncompetitive with dTTP substrate. The Ki value of DNA polymerase alpha for persimmon extract was estimated to be 70 nM. Moreover, persimmon extract inhibited [3H]thymidine incorporation of human peripheral lymphocyte cells stimulated by PHA.

Antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) adenine, a novel deoxyadenosine analog, against human colon tumor xenografts by oral administration.

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) adenine (Cl-F-araA) is a novel deoxyadenosine analog, which inhibits DNA synthesis by inhibiting DNA polymerase alpha and ribonucleotide reductase. Cl-F-araA shows potent antiproliferative activity against several leukemic cell lines including those of human origin and is also effective against murine solid tumors, in particular being curative against colon tumors. We therefore decided to investigate whether Cl-F-araA is effective against human colon tumors, in particular by oral administration, since it has improved stability compared with other deoxyadenosine analogs. Antiproliferative activity in vitro was determined from cell counts. Subcutaneously inoculated xenograft models and a liver micrometastases model were used for assessment of antitumor activity in vivo. Cl-F-araA showed potent antiproliferative activity against four human colon tumor cell lines (HCT116, HT-29, DLD-1, WiDr), with a 50% growth-inhibitory concentration (IC50) of 0.26 microM with a 72-h exposure. This activity was greater than those of fludarabine desphosphate and cladribine, other deoxyadenosine analogs, which showed IC50 values of 19 microM and 0.35 microM, respectively. Cl-F-araA showed potent antitumor activity against four human colon tumor xenograft models (HT-29, WiDr, Co-3, COLO-320DM) in a 5-day daily administration schedule, which was shown to be the most effective of three administration regimens tested (single, twice-weekly, 5-day daily). In particular, oral administration showed significantly superior activity, with a regressive or cytostatic growth curve, compared with intravenous administration. In addition, Cl-F-araA was effective at only one-sixteenth of the maximum dose tested in a 10-day daily administration schedule. Therapeutic efficiency seemed to increase in proportion to the frequency of administration. Cl-F-araA also decreased liver micrometastases created by intrasplenic injection of human colon tumor cells, leading to complete suppression at the maximum dose tested. These results suggest that Cl-F-araA might be clinically effective against human colon cancers using a daily oral administration schedule.

Role of G1 phase in the UV-induced apoptosis of EL-4 mouse lymphoma cells.

Although UV is known to induce apoptotic cell death to various animal cells, relationship between cell cycle and UV-induced apoptosis is still unclear. In this study, we investigated the role of G1 phase in UV-induced apoptosis by using EL-4 mouse lymphoma cells which have wild type p53. After 500 J/m2 UV irradiation, an increase of apoptotic fraction was accompanied by cell cycle accumulation in the G1 phase. Apoptotic fraction after UV-exposure was remarkably augmented by treatment with 2-AP, a G1 checkpoint inhibitor. In contrast, aphidicolin, an inhibitor of DNA polymerase-alpha, suppressed the rate of apoptotic fraction. These results suggest that mandatory cell cycle progression from G1 to S leaves the damaged DNA unrepaired and may increase the apoptotic fraction. To investigate the precise mechanism in the G1 phase, UV was exposed to the G1-synchronized cells and apoptotic fraction was serially observed. Synchronized EL-4 cells passed through the G1 phase in 8 h. Within the G1 phase, late-G1 cells (6 h after M) were more sensitive to UV-induced apoptosis than early-G1 cells (2 h after M) (49.7 +/- 9.0% vs. 41.5 +/- 8.5%, p < 0.05). In HL-60 cells, lacking in p53 expression, such a difference was not observed. Western blot analysis revealed that expression of p53 in synchronized EL-4 cells was increasingly enhanced during G1 phase. After UV-exposure, p53 expression gradually decreased in early-G1 cells, but it was kept at almost the same level in late-G1 cells. In addition, bcl-2 expression in early-G1 cells showed a more rapid and larger increase than that in late-G1 cells. These results suggest that susceptibility of the G1 cells to UV-induced apoptosis depends on their position within the G1 phase, and late-G1 is more sensitive than early-G1. Sensitivity to UV-induced apoptosis is closely related to the expression level of p53 and bcl-2 proteins. Early-G1 cells may be able to take enough time to repair damaged DNA until they reach the G1 checkpoint compared to the late-G1 cells.

Antisense oligonucleotide complementary to the BamHI-H gene family of Marek's disease virus induced growth arrest of MDCC-MSB1 cells in the S-phase.

DNA synthesis was effectively inhibited by antisense oligonucleotide A1 complementary to the BamHI-H gene family in Marek's disease virus (MDV)-derived lymphoblastoid MDCC-MSB1 cells. When a cell cycle distribution of a total cell population was analyzed by flow cytometry, the proportion of S-phase cells increased in the cell populations by treatment with oligonucleotide A1. Approximately 60-70% of the cells appeared in the S phase for 24 and 36 hr of incubation in the presence of oligonucleotide A1 (20-30% in the untreated control cells). The inhibition of cell cycle progression by treatment with oligonucleotide A1 was reversible. When the cells were treated with 5 microM aphidicolin for 12 hr, a similar pattern of cell cycle distribution was observed to that obtained after treatment with oligonucleotide A1. Aphidicolin is an inhibitor of cellular DNA polymerase alpha, and it halts progression of the cell cycle at the G1/S border or early S phase. When the cells were treated with aphidicolin for 12 hr and subsequently incubated with oligonucleotide A1, no significant difference was observed in the cycle phase distribution of cells in the presence and absence of oligonucleotide A1. In contrast, when the cells were treated with oligonucleotide A1 for 12 hr and subsequently incubated with aphidicolin, the cell cycle did not progress from the G1/S border or early S phase to the next phase.

The relationship between the antitumor activity and the ribonucleotide reductase inhibitory activity of (E)-2'-deoxy-2'-(fluoromethylene) cytidine, MDL 101,731.

(E)-2'-deoxy-2'-(fluoromethylene) cytidine (MDL101,731) is a new deoxycytidine analog which shows potent antitumor activity against several human tumor models. We previously showed that MDL101,731 inhibited human ribonucleotide reductase (RNR) in HeLa S3 human cervical carcinoma cells. Recently, it has been reported that another deoxycytidine analog, 2'-deoxy-2'-methylidenecytidine (DMDC) which also inhibits RNR from Escherichia coli, does not inhibit RNR in intact L1210 murine leukemia cells. MDL101,731 was designed as an inhibitor of RNR, so it is important to know the contribution of the RNR inhibitory activity of the drug on its antitumor efficacy in vivo. Therefore, we examined the relationship between the antitumor activity and RNR inhibitory activity of MDL101,731 using LX-1 human lung carcinoma which was highly sensitive to this drug. MDL101,731 showed strong inhibition of RNR activity in LX-1 lung carcinoma by both i.v. and p.o. administration. Administration of 15 mg/kg i.v. and 30 mg/kg p.o. of MDL101,731, doses which showed almost the same degree of antitumor activity against LX-1 lung carcinoma on a daily 5 day schedule, caused a similar degree and similar kinetics of inhibition of RNR in LX-1 lung carcinoma at least for 12 h after administration. On the other hand, DMDC as well as 1-beta-D-arabinofuranosyl-cytosine (ara-C), which is a well-known deoxycytidine analog and inhibits DNA polymerase alpha, did not inhibit RNR in LX-1 lung carcinoma at doses demonstrating antitumor activity. These results indicate that MDL101,731 exhibited antitumor activity through inhibition of RNR activity in tumor cells in vivo and the mechanism of antitumor action of MDL 101,731 might be different from those of DMDC and ara-C, at least in part.

Sulfoquinovosyldiacylglycerol, KM043, a new potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1 from a marine red alga, Gigartina tenella.

A new sulfolipid, KM043, which belongs to the 6-sulfo-alpha-D-quinovopyranosyl-(1-->3')-1',2'-diacylglycerol (SQDG) class of compounds, has been isolated from a marine red alga, Gigartina tenella, as a potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1. Its structure was determined on the basis of spectroscopic and gas chromatographic analyses. The inhibition was dose-dependent, and complete (more than 90%) inhibition of DNA polymerase alpha (pol. alpha), DNA polymerase beta (pol. beta) and HIV-reverse transcriptase type 1 (HIV-RT) was observed at concentrations of 5, 10, and 30 microM, respectively.

A 5'-monophosphate form of bredinin selectively inhibits the activities of mammalian DNA polymerases in vitro.

Bredinin is an immunosuppressive drug which is used clinically in Japan. In this study, we investigated bredinin's molecular mode of action to clarify its immunosuppressive effects. We focused on the DNA polymerases in the somatic DNA synthesis which may be required in the process of lymphocyte differentiation. We found that bredinin-5'-monophosphate (breMP) could be a potent inhibitor of mammalian DNA polymerase alpha(pol.alpha) and (pol.beta) in vitro, although bredinin itself has no such effects. BreMP inhibited the pol. alpha activity at less than 7 micrograms/ml and the pol. activity at 7 micrograms/ml. Neither breMP nor bredinin influenced the activities of a plant DNA polymerase, prokaryotic DNA polymerases such as E. coli DNA polymerase I and Taq DNA polymerase, or DNA-metabolic enzymes such as DNase I, indicating that breMP selectively suppressed the activities of the mammalian DNA polymerases. For pol., beta breMP acted by competing with both the substrate and template-primer. For pol. alpha, it acted by competing only with the substrate, and non-competitively with the template-primer. The ribose of bredinin is quickly and quantitatively converted to its ribose-5'-phosphate form in vivo as soon as it is incorporated into cells. The action mode of bredinin and its use as an immunosuppressive drug are discussed based on these results.

Rat hepatoma Reuber H-35 cells produce factors that promote the hatching of mouse embryos cultured in vitro.

The effects of coculture and conditioned medium of rat hepatoma Reuber H-35 cells on the subsequent in vitro development and hatching of mouse 2-cell embryos were examined. The hatching of embryos obtained from CD-1 mice was accelerated by coculture with Reuber H-35 cells in the presence of 3 mg/ml BSA. The promoting effect on complete hatching from zona pellucida was evident even in cell-conditioned medium containing 60 micrograms/ml BSA. In the presence of 60 micrograms/ml BSA, more than 20% of embryos completely hatched, whereas none hatched in the control culture. The promoting activity was also found in both the M(r) < 10,000 and the M(r) > 10,000 subfractions of the conditioned medium separated by ultrafiltration. The cell number per blastocyst was increased to 1.1- to 1.3 times the control by culturing embryos from the 2-cell stage with the conditioned medium or its subfractions. The effective target of promoting factors for complete hatching was after the morula stage, and blastocysts hatched completely even when incubated in conditioned medium for 6 h. Inhibitors of DNA polymerase alpha, protein synthesis, and protein kinase partially reduced (40-90% inhibition) the promoting effect of the conditioned medium. On the other hand, protease inhibitors showed no effect. In a caseinolytic assay, protease activity was undetectable in the conditioned medium. Incubating the 125I-labeled proteins derived from the M(r) > 10,000 fraction with blastocysts revealed that at least 9 proteins with apparent molecular masses of 76, 60, 49, 38, 34, 31, 24, 22, and 18 kDa specifically bound to or accumulated in the embryos. Moreover, reverse-transcriptase polymerase chain reaction showed that Reuber H-35 cells expressed mRNAs for epidermal growth factor, transforming growth factors alpha and beta 1, and stem cell factor. These results indicated that embryonic development and the process of zona hatching was accelerated by factors synthesized by Reuber H-35 cells. This and other studies demonstrated that Reuber H-35 cells exert positive (later than 2-cell stage) and negative (at 2-cell stage) effects upon the development of mouse embryos at different embryonic stages. These factors will serve as valuable tools to clarify the proliferating and differentiating mechanisms of the preimplantation embryo.

Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase-I: evidence for cell cycle-independent toxicity.

Camptothecin is an S-phase-specific anticancer agent that inhibits the activity of the enzyme DNA topoisomerase-I (topo-I). Irreversible DNA double-strand breaks are produced during DNA synthesis in the presence of camptothecin, suggesting that this agent should not be toxic to nondividing cells, such as neurons. Unexpectedly, camptothecin induced significant, dose-dependent cell death of postmitotic rat cortical neurons in vitro; astrocytes were more resistant. Aphidicolin, an inhibitor of DNA polymerase alpha, did not prevent camptothecin-induced neuronal death, while death was prevented by actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole as well as cycloheximide and anisomycin, inhibitors of RNA and protein synthesis, respectively. Camptothecin-induced neuronal death was apoptotic, as characterized by chromatin condensation, cytoplasmic shrinking, plasma membrane blebbing, and fragmentation of neurites. DNA fragmentation was also confirmed by the use of the in situ DNA end labeling assay. In addition, aurintricarboxylic acid, an inhibitor of the apoptotic endonuclease, partially protected against camptothecin-induced neuronal death. The toxicity of stereoisomers of a camptothecin analogue was stereospecific, demonstrating that toxicity was a result of inhibition of topo-I. The difference in sensitivity to camptothecin between neurons and astrocytes correlated with their transcriptional activity and level of topo-I protein expression. These data indicate important roles for topo-I in postmitotic neurons and suggest that topo-I inhibitors can induce apoptosis independent of DNA synthesis. We suggest a model based on transcriptionally mediated DNA damage, a novel mechanism of action of topo-I poisons.

Enhancement of melphalan activity by inhibition of DNA polymerase-alpha and DNA polymerase-beta.

Our previous studies exploring melphalan resistance in the human rhabdomyosarcoma xenograft TE-671 MR revealed elevation of DNA polymerase-alpha and DNA polymerase-beta. The present study evaluated the alteration of melphalan activity in TE-671 (melphalan-sensitive) and TE-671 MR (melphalan-resistant) subcutaneous xenografts in nude mice after DNA polymerase-alpha was inhibited using aphidicolin glycinate (AG) and DNA polymerase-beta was inhibited using dideoxycytidine (DDC). Administration of AG or DDC did not produce toxicity or demonstrate antineoplastic activity when given alone. AG (90 mg/m2) enhanced the activity of melphalan against TE-671, with growth delays increasing by 8.4, 15.8, and 21.2 days over the regimen with melphalan only. AG (180 mg/m2) only modestly increased melphalan activity against TE-671 MR, with the growth delays increasing from 9.6 and 12.1 days using melphalan alone to 12.1 and 14.5 days using melphalan plus AG. AG (180 mg/m2) plus melphalan (the dose lethal to 10% of animals) produced greater weight loss compared with melphalan alone, whereas DDC plus melphalan produced no additional toxicity. DDC modestly enhanced the activity of melphalan plus AG against TE-671 MR. AG plus O6-benzylguanine did not increase the activity of 1,3-bis(2-chloroethyl)-1-nitrosourea against TE-671 or TE-671 MR. AG (90 mg/m2 and 180 mg/m2) inhibited DNA polymerase-alpha to 80% and 72% of control in TE-671 and 64% and 37% in TE-671 MR, and DDC inhibited DNA polymerase-beta to 59% in TE-671 and 48% in TE-671 MR. These results suggest a role for AG-mediated enhancement of melphalan activity, particularly in the treatment of newly diagnosed, melphalan-sensitive tumors.

Effects of cell-cycle-arrest agents on cleavage and development of mouse embryos.

In mammals, there are no reliable methods for synchronizing cell division of early embryos without reducing their ability to develop into blastocysts and fetuses. The present study was undertaken to examine the in vitro inhibition of cell division of four-cell mouse embryos by cell cycle arrest agents. The reversibility of the agents was also tested by examining the developmental ability of treated embryos. Four-cell mouse embryos obtained at 54 hr post-human chorionic gonadotrophin (post-hCG) were cultured for 4, 8, 12, or 16 hr in media supplemented with either nocodazole, an inhibitor of tubulin polymerization, 6-dimethylaminopurine (6-DMAP), an inhibitor of maturation promoting factor (MPF) activation, or aphidicolin, a specific inhibitor of DNA polymerase alpha. Reversibility and toxicity of these agents were both dose and time dependent. For all three agents, prolonging cleavage arrest for 8 or 16 hr (at the effective concentrations) caused embryo lethality. Although nocodazole treatment was least cytotoxic, 6-DMAP and aphidicolin concentrations which induce cleavage arrest were detrimental to development beyond the blastocyst stage. The results of this study show that the development of embryos treated with these three cell-cycle-arrest agents is dose and incubation time dependent. Toxic effects beyond the blastocyst stage could only be minimized for nocodazole by reducing the exposure time of treatment and concentration of the mitotic inhibitor. However, these results render doubt on the usefulness of 6-DMAP and aphidicolin for synchronization studies leading to embryo transfer procedures.

Fluorodeoxyuridine-mediated cytotoxicity and radiosensitization require S phase progression.

The role of cell cycle redistribution in fluoropyrimidine-mediated radiosensitization remains unresolved. To determine if radiosensitization resulted from the redistribution of cells into a sensitive phase of the cell cycle, we assessed fluorodeoxyuridine (FdUrd)-mediated radiosensitization in flow-sorted mid-S phase HT29 human colon cancer cells. We hypothesized that if FdUrd-mediated radiosensitization were strictly the result of cell cycle redistribution, FdUrd-treated mid-S phase cells would remain as radioresistant as mid-S phase cells cultured in the absence of drug. However, we found that the mid-S phase cells from FdUrd-treated populations were markedly radiosensitized. To assess the role of S phase progression in radiosensitization, we exposed FdUrd-treated cells to aphidicolin, an inhibitor of DNA polymerase alpha, prior to irradiation. We found that aphidicolin blocked the radiosensitizing (and cytotoxic) effects of FdUrd. These results, combined with our previous observations that FdUrd-treated cells at the G1/S boundary are minimally sensitized, appear to disprove the hypothesis that sensitization results strictly from cell cycle distribution. Furthermore, they suggest that a key aspect of both FdUrd-mediated radiosensitization and cytotoxicity is S phase progression on a damaged DNA template.

Human Xeroderma Pigmentosum Group A Protein Interacts with Human Replication Protein A and Inhibits DNA Replication

Human replication protein A (RPA; also known as human single-stranded DNA binding protein, or HSSB) is a multisubunit complex involved in both DNA replication and repair. While the role of RPA in replication has been well studied, its function in repair is less clear, although it is known to be involved in the early stages of the repair process. We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA. We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system. XPAC inhibited SV40 DNA replication in vitro, and this inhibition was reversed by the addition of RPA but not by the addition of DNA polymerase alpha-primase complex, SV40 large tumor antigen, or topoisomerase I. This inhibition did not result from an interaction between XPAC and single-stranded DNA (ssDNA), or from competition between RPA and XPAC for DNA binding, because XPAC does not show any ssDNA binding activity and, in fact, stimulates RPA's ssDNA binding activity. Furthermore, XPAC inhibited DNA polymerase alpha activity in the presence of RPA but not in RPA's absence. These results suggest that the inhibitory effect of XPAC on DNA replication probably occurs through its interaction with RPA.

Effect of microinjection time during postfertilization S-phase on bovine embryonic development.

Microinjection into bovine zygotes was performed to evaluate the effects of the timing of injection during the phase of DNA replication on the subsequent in vitro development of embryos and expression of injected chicken beta-actin promoter-lac Z gene construct. The period of DNA replication of bovine zygotes, determined by 3H-thymidine incorporation, begins between 12 hr and 13 hr postinsemination (hpi) of in vitro matured oocytes, reaches a maximum from 17 hpi to 19 hpi, and is complete by 21-22 hpi. Aphidicolin, an inhibitor of DNA polymerase alpha, was used to synchronize the pronuclei and the zygote population. Treatment with aphidicolin at 9-18 hpi arrested DNA replication without affecting formation of the pronuclei or embryo development. Cycloheximide, an inhibitor of protein synthesis, was used for nucleocytoplasmic resynchronization of the aphidicolin-treated zygotes. Microinjection was performed at 15 (early), 18 (mid), and 21 (late S phase) hpi. Embryonic development was affected following each of the three microinjection times. The development of zygotes injected at 18 hpi was significantly higher (P < 0.01) after 5 days of culture than those injected at 15 hpi or 21 hpi. Expression of the marker gene was observed in the higher stage of development (> 16 cells) only in the zygotes injected at 18 hpi. At the earlier stages of development, the proportions of embryos showing expression of the foreign gene were the same for all microinjection times. In aphidicolin- and cycloheximide-treated zygotes, expression of the marker gene followed the same curve as development, i.e., expression was low when injected early or late and higher (P < 0.005) when injected in the middle of zygotic S phase. The ability of the embryos to survive microinjection and to express the marker gene as a function of hpi seems to be influenced mostly in the cytoplasm processing stage rather than the pronuclei processing stage.

Productive HIV-1 infection of macrophages restricted to the cell fraction with proliferative capacity.

Retroviruses establish productive infection only in proliferating cells. Macrophages are often considered to be non-proliferating in vitro yet are susceptible to HIV-1 infection. This has led to the conclusion that HIV-1 can establish infection independent of host cell proliferation. We here report that a small proportion of macrophages does have proliferative capacity. A comparable small fraction of monocyte derived macrophages (MDM) supported productive HIV-1 infection as demonstrated in limiting dilution culture. Fluorescence activated cell sorting on the basis of incorporation of BrdUrd, a thymidine analog, and subsequent PCR analysis revealed the presence of proviral DNA only in the BrdUrd positive cell fraction with DNA synthesizing activity. To identify which phase of cell cycle is required for establishment of productive infection, growth arrest in G1 or G1/S phase prior to inoculation was performed. gamma-Irradiation, which arrests primary cells in G1, prevented both cell proliferation and establishment of productive infection in MDM. Treatment of MDM with aphidicolin, a specific inhibitor of DNA polymerase alpha and delta which arrests cells in G1/S phase of the cell cycle, also inhibited DNA synthesis but did not prevent establishment of productive infection which is completely analogous to observations in T cells. Our data thus indicate that not cell division itself but cellular conditions that coincide with cell proliferation are apparently indispensable for establishment of productive infection.

Nuclear and Organelle DNA Replication during Spore Germination in Bryophytes and Equisetum

AbstractIn contrast to ferns, the spore nuclei of the moss, Funaria hygrometrica, are in the G2 phase. They do not incorporate the thymidine analogue BUdr before the first mitosis. When spores germinate in the presence of aphidicolin, an inhibitor of the (nuclear) DNA polymerase alpha, they give rise to two‐celled, negatively phototropic protonemata, consisting of relatively long and thin cells. Determinations of nuclear DNA content further confirm the presence of G2‐nuclei in Funaria spores. Spores of Pogonatum urnigerum and of Equisetum hyemale likewise contain G2‐nuclei, as shown by BUdr incorporation or experiments with aphidicolin, respectively. In germinating Funaria spores, the organelle DNA is synthesized very early and independent of nuclear DNA. A gradient in replication of organelle DNA is found only in the long tip cells which are formed in the presence of aphidicolin.

Phi 29 DNA polymerase active site. Residue ASP249 of conserved amino acid motif “Dx2SLYP” is critical for synthetic activities.

phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid sequence similarity and sensitivity to inhibitors of eukaryotic DNA polymerase alpha. In this paper, site-directed mutants in the phi 29 DNA polymerase residues Asp249, Ser252, Leu253, and Pro255 of the conserved amino acid motif "Dx2SLYP" are described. Two mutants, D249E and S252R, were drastically affected in all the synthetic activities, whereas their 3' to 5' exonuclease activity and interaction with the TP primer was normal. Mutant D249E, slightly affected in template-primer binding, was completely inactive in all conditions tested, suggesting that Asp249 could be playing a direct role in catalysis. On the other hand, mutant S252R, strongly affected in template-primer binding, showed some DNA polymerization activity in the presence of Mn2+. Mutants S252G and P255S showed a reduced template-primer binding ability; these mutants, together with mutant L253V, showed metal ion-dependent phenotypes in their synthetic activities and altered sensitivities to the PPi analog phosphonoacetic acid. All these results support the hypothesis that the Dx2SLYP motif forms part of the polymerization active site of the phi 29 DNA polymerase, being the Asp249 residue critical both for protein-primed initiation and DNA polymerization.

Retinal differentiation from multipotential pineal cells of the embryonic quail

Pineal cells of the embryonic quail are multipotent stem cells which are able to differentiate in vitro into pigmented epithelial cells, lens cells and skeletal muscle fibers. Neuronal expression was added in this study in the repertory of differentiating potency of pineal cells. We used immunohistochemical methods to characterize neuronal properties with antibodies against serotonin, GABA, tyrosine hydroxylase and neuron-specific antigen (HPC-1) in addition to the enzyme histochemistry for acetylcholinesterase activity. Cells in the culture were found to be positively stained with these methods, suggesting that embryonic pineal cells are neuropotent to differentiate various types of neuronal cells. We have studied the culture conditions which favor increment of neuronal cells with extension of neuritic processes, and we have found that neuronal cells are maintained for quite a long period under suppressive conditions of DNA synthesis and under the effect of basic fibroblast growth factor (FGF). Suppression of DNA synthesis was achieved by the addition of aphidicolin, an inhibitor of DNA polymerase alpha, in the medium. Time lapse videograph revealed two different cell types participated in neurogenesis; a minor population of small round cells and a major one of flat epithelial cells. Since embryonic quail pineal cells have been shown to differentiate into two types of photoreceptors, the present results show wider retinal potency of cell differentiation by embryonic pineal cells. The cessation of DNA synthesis as well as growth factor(s) may be positively involved in the mechanisms of determination and differentiation of pineal neurons.

Inhibition of DNA repair with aphidicolin enhances sensitivity of targets to tumor necrosis factor.

To test whether DNA injury contributes to TNF-induced cytotoxicity, we attempted to enhance DNA injury by inhibiting its repair and then assessing effects on cytotoxicity. DNA repair, assayed as unscheduled DNA synthesis, was first detected in TNF-sensitive targets by 2-3 h of incubation with TNF. Targets resistant to TNF cytotoxicity did not demonstrate significant repair replication. Repair preceded the detection of TNF-induced DNA injury, which was subsequently demonstrated by a double-stranded DNA fragmentation assay, sedimentation of DNA in neutral and alkaline sucrose gradients, and gel electrophoresis of extracted DNA. This suggested that early during exposure to TNF, DNA repair proceeds more rapidly than strand breakage. To inhibit repair, nontoxic concentrations of aphidicolin (inhibitor of DNA polymerase-alpha) and dideoxythymidine (inhibitor of DNA polymerase-beta and gamma) were used. Aphidicolin inhibited repair and consistently sensitized to TNF cytotoxicity, decreasing the ID50 for TNF at least 10- to 50-fold. In contrast, dideoxythymidine had no effect on repair or cytotoxicity. Deoxycytidine, which competitively inhibits binding of aphidicolin to DNA polymerase, blocked the sensitization in a concentration-dependent fashion. In targets sensitized with aphidicolin, TNF-induced strand breakage was accelerated, being detected by 4 h of culture in the sucrose gradient assay. Sensitization to TNF was not due to a heightened activation of poly (ADP-ribose) polymerase. These results indicate that TNF-induced strand breakage participates in TNF-induced cytotoxicity and that the level of DNA repair plays a role in determining relative sensitivity of targets.

Human immunodeficiency virus type 1 DNA synthesis, integration, and efficient viral replication in growth-arrested T cells.

Human immunodeficiency virus type 1 (HIV-1) replicates efficiently in nonproliferating monocytes and macrophages but not in resting primary T lymphocytes. To determine the contribution of cell division to the HIV-1 replicative cycle in T cells, we evaluated HIV-1 expression, integration of proviral DNA, and production of infectious progeny virus in C8166 T-lymphoid cells blocked in cell division by treatment with either mitomycin, a DNA cross-linker, or aphidicolin, a DNA polymerase alpha inhibitor. The arrest of cell division was confirmed by assay of [3H]thymidine uptake; the nondividing cells remained viable for at least 3 days after treatment. HIV-1 was expressed and replicated equally well in nondividing and dividing C8166 cells, as judged by the comparison of the levels of p24 core antigens in culture supernatants, the proportion of cells expressing HIV-1 specific antigens, the pattern and quantity of HIV-1 DNA present in the extrachromosomal and total cellular DNA fractions, and the biological activity of progeny viruses. A polymerase chain reaction-based viral DNA integration assay indicated that HIV-1 provirus was integrated in C8166 cells treated with either of the two inhibitors of cell division. Similar results were obtained by using growth-arrested Jurkat T-lymphoid cells. We conclude that cell division and cellular DNA synthesis are not required for efficient HIV-1 expression in T cells.