Shunji Izuta

Reactivation of Lytic Replication from B Cells Latently Infected with Epstein-Barr Virus Occurs with High S-Phase Cyclin-Dependent Kinase Activity while Inhibiting Cellular DNA Replication

ABSTRACT Productive infection and replication of herpesviruses usually occurs in growth-arrested cells, but there has been no direct evidence in the case of Epstein-Barr virus (EBV), since an efficient lytic replication system without external stimuli does not exist for the virus. Expression of the EBV lytic-switch transactivator BZLF1 protein in EBV-negative epithelial tumor cell lines, however, is known to arrest the cell cycle in G0/G1 by induction of the tumor suppressor protein p53 and the cyclin-dependent kinase (CDK) inhibitors p21WAF-1/CIP-1 and p27KIP-1, followed by the accumulation of a hypophosphorylated form of the Rb protein. In order to determine the effect of the onset of lytic viral replication on cellular events in latently EBV-infected B LCLs, a tightly controlled induction system of the EBV lytic-replication program by inducible BZLF1 protein expression was established in B95-8 cells. The induction of lytic replication completely arrested cell cycle progression and cellular DNA replication. Surprisingly, the levels of p53, p21WAF-1/CIP-1, and p27KIP-1 were constant before and after induction of the lytic program, indicating that the cell cycle arrest induced by the lytic program is not mediated through p53 and the CDK inhibitors. Furthermore, although cellular DNA replication was blocked, elevation of cyclin E/A expression and accumulation of hyperphosphorylated forms of Rb protein were observed, a post-G1/S phase characteristic of cells. Thus, while the EBV lytic program promoted specific cell cycle-associated activities involved in the progression from G1 to S phase, it inhibited cellular DNA synthesis. Such cellular conditions appear to especially favor viral lytic replication.

Inhibition of DNA primase by sphingosine and its analogues parallels with their growth suppression of cultured human leukemic cells

Sphingosine is a potent inhibitor of a mammalian DNA primase in vitro (Simbulan et al., Biochemistry 33, 9007‐9012, 1994). Here we measured the inhibition of DNA primase in vitro by 9 sphingosine‐analogues with respect to RNA primer synthesis and DNA primase‐dependent DNA synthesis, and their potencies of inhibition in vitro were compared with their in vivo effects on human leukemic cells. Sphingosine, phytosphingosine and N, N‐dimethylsphingosine strongly inhibited the activity of purified calf thymus DNA primase, and also inhibited the growth of human leukemic cell line HL‐60, exerting strong cytotoxicity. Dihydrosphingosine and cis‐sphingosine, which showed more subtle inhibition of DNA primase in vitro, moderately inhibited the cell growth in vivo and caused cell death. In contrast, N‐acyl‐, N‐octyl‐, and N‐acetylsphingosine (ceramides) showing little inhibition of DNA primase suppressed cell growth only slightly. HL 60 cell was arrested at Go/G1 phase by exogenously added sphingosine. From these results, it is suggested that DNA primase is one of targets of sphingosine, an effector molecule in apoptosis.

Poly(ADP-ribose) polymerase stimulates DNA polymerase α by physical association

Abstract The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP-ribose) polymerase on the activity of DNA polymerase alpha was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 micrograms/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase alpha by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase beta, DNA polymerase gamma, and primase, indicating that its effect is specific for DNA polymerase alpha. Apparently, poly(ADP-ribosyl)ation of DNA polymerase alpha was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP-ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the reaction in competition with template DNA, while, at higher DNA doses, it greatly stimulated the reaction by increasing the Vmax of the reaction. By the automodification of poly(ADP-ribose) polymerase, however, both the inhibition at low DNA concentration and the stimulation at high DNA doses were largely lost. Furthermore, stimulation by poly(ADP-ribose) polymerase could not be attributed to its DNA-binding function alone since its fragment, containing only the DNA-binding domain, could not exert full stimulatory effect on DNA polymerase, as of the intact enzyme. Poly(ADP-ribose) polymerase is co-immunoprecipitated with DNA polymerase alpha, using anti-DNA polymerase alpha antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase alpha. In a crude extract of calf thymus, a part of poly(ADP-ribose) polymerase activity existed in a 400-kDa, as well as, a larger 700-kDa complex containing DNA polymerase alpha, suggesting the existence in vivo of a complex of these two enzymes.

Flavocristamides A and B, new DNA polymerase α inhibitors from a marine bacterium Flavobacterium sp.

Abstract Flavocristamides A (1) and B (2), two new sulfonolipids with inhibitory activity against DNA polymerase α, have been isolated from a marine bacterium Flavobacterium sp. and the structures containing absolute stereochemistry were determined by spectroscopic data and chemical means.

Phosphorylated retinoblastoma protein stimulates DNA polymerase α

Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10 – 100-fold the activity of DNA polymerase α from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On the other hand, a hyperphosphorylated Rb protein, obtained from insect cells overexpressing Rb protein, cyclin E and cyclin-dependent kinase 2 simultaneously, stimulated DNA polymerase α more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. Rb protein isolated from human Burkitt lymphoma Raji cells also stimulated DNA polymerase α. In contrast, Rb protein did not affect eukaryotic DNA primase or Klenow fragment of Escherichia coli DNA polymerase I. By immunoprecipitation using anti-DNA polymerase α antibody, Rb protein in nuclear extract of Raji cells was co-precipitated with DNA polymerase α. This result indicates that DNA polymerase α exists as a complex containing phosphorylated Rb protein in cells. DNA polymerase α specifically bound to a purified Rb protein-immobilized Sepharose column. Rb protein also bound to DNA polymerase α trapped to anti-DNA polymerase α antibody-Sepharose column, suggesting the direct association of these two proteins. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication.

Sulfated glycoglycerolipid from Archaebacterium inhibits eukaryotic DNA polymerase α, β and retroviral reverse transcriptase and affects methyl methanesulfonate cytotoxicity

A sulfated glycoglycerolipid, 1‐O‐(6′‐sulfo‐α‐d‐glucopyranosyl)‐2,3‐di‐O‐phytanyl‐sn‐glycerol (KN‐208), a derivative of the polar lipid isolated from an archaebacterium, strongly inhibited DNA polymerase (pol) α and pol β in vitro among 5 eukaryotic DNA polymerases (α, β, γ, δ, and ϵ). It also inhibited Escherichia coli DNA polymerase I Klenow fragment (E. coli pol I) and human immunodeficiency virus reverse transcriptase (HIV RT). The mode of inhibition of these polymerases was competitive with the DNA template primer and was non‐competitive with the substrate dTTP. KN‐208 inhibited pol β most strongly, with a Ki value of 0.05 μM, 10‐fold lower than that for pol α (0.5 μM) and 60‐ or 140‐fold lower than that for HIV RT (3 μM) or for E. coli pol I (7 μM), respectively. The loss of sulfate on the 6′‐position of glucopyranoside of this compound completely abrogated inhibition. However, the hydrophilic part of KN‐208, glucose 6‐sulfate alone, showed no inhibition. Other sulfated compounds containing different hydrophobic structures, such as dodecyl sulfate and cholesterol sulfate, exhibited a much weaker inhibition. Our results suggest that the whole molecular structure of KN‐208 is required for inhibition. KN‐208 was shown to be modestly cytotoxic for the human leukemic cell line K562. Interestingly, a subcytotoxic dose of KN‐208 increased the sensitivity of the human leukemic cells to an alkylating agent, methyl methanesulfonate, while it did not potentiate the effects of ultraviolet light or of cisplatin. Int. J. Cancer 76:512–518, 1998.© 1998 Wiley‐Liss, Inc.

A novel platinum compound inhibits telomerase activity in vitro and reduces telomere length in a human hepatoma cell line

Telomerase activity is detectable in most human tumors but not in most normal somatic cells or tissues. Telomerase inhibition has, therefore, been proposed as a novel and potentially selective strategy for antitumor therapy. In the present study, we found that platinum compounds, including cisplatin [cis‐diamminedichloro‐platinum (II)], strongly inhibited the activity of partially purified rat telomerase. Among the agents tested, 2,3‐dibromosuccinato [2‐(methylaminomethyl)pyridine]platinum (II) (compound E) exhibited the strongest inhibition, with an median inhibitory concentration (IC50) of 0.8 μM. The mode of inhibition was noncompetitive with either dNTPs or TS (first) primer, with Ki values estimated to be 2.3 or 3.9 μM for varied TS primer or dNTPs, respectively. Notably, cisplatin also inhibited the telomerase activity, with an IC50 of 2.0 μM. Again, the mode of inhibition was noncompetitive, with Ki values estimated as 7.3 or 8.1 μM. Preincubation of TS primer with compound E did not affect the telomerase inhibition, whereas preincubation with cisplatin caused remarkable enhancement. Treatment of a human hepatoma cell line HepG2 with a low concentration of compound E gradually reduced the telomere length, indicating that this compound was able to inhibit telomerase in living cells as well as in vitro.

PCNA Mono-Ubiquitination and Activation of Translesion DNA Polymerases by DNA Polymerase α

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

Sequence‐dependent Termination of Mammalian DNA Polymerase Reaction by a New Platinum Compound, (–)‐(R)‐2‐Aminomethylpyrrolidine(1,1‐cyclobutane‐dicarboxylato)‐2‐platinum(II) Monohydrate

We examined the mechanisms of the inhibition of DNA synthesis by a new platinum compound, (‐)‐(R)‐2‐aminomethylpyrrolidine(1,1‐cyclobutane‐dicarboxylato)‐2‐platinum(II) monohydrate (DWA‐2114R), a derivative of the antitumor drug cis‐ diamminedichloroplatinum(II) (CDDP), using prokaryotic and eukaryotic DNA polymerases. Preincubating activated DNA with CDDP or DWA‐2114R reduced its template activity for prokaryotic and eukaryotic DNA polymerases in a dose‐dependent manner. DWA2114R required six times greater drug concentration and two times longer incubation time to show the same decrease of the template activity compared to CDDP. Treatment of primed pUC118 ssDNA templates with the two drugs followed by second‐strand synthesis by prokaryotic and eukaryotic DNA polymerases revealed that DWA2114R bound to DNA in a similar manner to CDDP and these adducts blocked DNA elongation by DNA polymerases of eukaryotes as well as of prokaryotes. With these two drugs, the elongations by E. coli DNA polymerase I (Klenow fragment), T7 DNA polymerase and calf thymus DNA polymerase α were strongly arrested at guanine‐guanine sequences (GG). Stop bands were also observed at adenine‐guanine sequences (AG) guanine‐adenine‐guanine sequences (GAG) and mono‐guanine sequence (G). Calf testis DNA polymerase β was also arrested efficiently at AG, GAG and G, but much more weakly at GG. This pattern was common to DWA2114R and CDDP.

The Antitumor Mechanism of 1‐(2‐Deoxy‐2‐fluoro‐4‐thio‐β‐D‐arabinofuranosyl)‐cytosine: Effects of Its Triphosphate on Mammalian DNA Polymerases

The mechanism of action of the antitumor nucleoside analog l‐(2‐deoxy‐2‐fluoro‐4‐thio‐β‐D‐arabinofuranosyl)cytosine (4′‐thio‐FAC) was investigated. 4′‐Thio‐FAC inhibited cellular DNA synthesis, but not RNA and protein syntheses. We observed potent inhibitory action of the triphosphate of 4′‐thio‐FAC (4′‐thio‐FACTP) against DNA polymerase α, whereas it showed moderate inhibition of DNA polymerase P and little inhibition of DNA polymerase β. The kinetic analysis showed that the inhibition mode of 4′‐thio‐FACTP towards DNA polymerase a was mixed type, implying a chain‐terminating effect of 4′‐thio‐FACTP. The triphosphate of 2′‐deoxy‐2′,2′‐difluorocytidine (gemcitabine), a known antitumor nucleoside, did not show potent inhibition of these three DNA polymerases. Thus, the effect of the diphosphate of gemcitabine on ribonucleotide reductase was suggested to be more important for the antitumor action of gemcitabine. From these findings, the main target enzymes of 4′‐thio‐FAC and gemcitabine appear to be different. We found a synergistic effect of the two drugs in an in vitro model, which supports the above idea.