The effects of dehydroaltenusin, a novel mammalian DNA polymerase alpha inhibitor, on cell proliferation and cell cycle progression.

As described previously, a natural product isolated from fungus (Acremonium sp.), dehydroaltenusin, is an inhibitor of mammalian DNA polymerase alpha in vitro [Y. Mizushina, S. Kamisuki, T. Mizuno, M. Takemura, H. Asahara, S. Linn, T. Yamaguchi, A. Matsukage, F. Hanaoka, S. Yoshida, M. Saneyoshi, F. Sugawara, K. Sakaguchi, Dehydroaltenusin, a mammalian DNA polymerase alpha inhibitor, J. Biol. Chem. 275 (2000) 33957_33961]. In this study, we investigated the interaction of dehydroaltenusin with lipid bilayers using an in vitro liposome system, which is a model of the cell membrane, and found that approximately 4% of dehydroaltenusin was incorporated into liposomes. We also investigated the influence of dehydroaltenusin on cultured cancer cells. Dehydroaltenusin inhibited the growth of HeLa cells with an LD50 value of 38 microM, and as expected, S phase accumulation in the cell cycle. The total DNA polymerase activity of the extract of incubated cells with dehydroaltenusin was 23% lower than that of nontreated cells. Dehydroaltenusin increased cyclin E and cyclin A levels. In the analysis of the cell cycle using G1/S synchronized cells by employing hydroxyurea, the compound delayed both entry into the S phase and S phase progression. In a similar analysis using G2/M synchronized cells by employing nocodazole, the compound accumulated the cells at G1/S and inhibited entry into the S phase. Thus, the pharmacological abrogation of cell proliferation by dehydroaltenusin may prove to be an effective chemotherapeutic agent against tumors.

Fatty acids selectively inhibit eukaryotic DNA polymerase activities in vitro.

The in vitro relationship between eukaryotic DNA polymerases and fatty acids was investigated. Some fatty acids strongly inhibited the activities of DNA polymerase alpha and/or beta in vitro. The kinetics of inhibition by linoleic acid showed that DNA polymerase alpha was non-competitively inhibited with respect to the DNA template and substrate (dTTP), while DNA polymerase beta was inhibited competitively with both DNA and substrate.

Vitamin A-related compounds, all-trans retinal and retinoic acids, selectively inhibit activities of mammalian replicative DNA polymerases.

Retinoic acids, vitamin A-related compounds, are known to be inhibitors of telomerase. We found that fucoxanthin from the sea alga Petalonia bingamiae is a potent inhibitor of mammalian replicative DNA polymerases (i.e., pol alpha, delta and epsilon). Since fucoxanthin is a carotenoid (provitamin A-related) compound, we characterized the biochemical modes of vitamin A-related compounds including vitamin A and provitamin A in this report. Subsequently, we found that fucoxanthin, all-trans retinal (RAL, vitamin A aldehyde) and all-trans retinoic acid (RA, vitamin A acid) inhibited the activities of replicative DNA polymerases with IC(50) values of 18-190, 14-17 and 8-30 microM, respectively. On the other hand, all-trans retinol (vitamin A) did not influence any of the DNA polymerase activities. RA inhibited not only the activities of pol alpha, delta and epsilon with IC(50) values of 30, 28 and 8 microM, respectively, but of pol beta with an IC(50) value of 27 microM. The tested vitamin A-related compounds did not influence the activities of DNA polymerases from a higher plant, cauliflower, prokaryotic DNA polymerases, or DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. RAL and RA should be called selective inhibitors of mammalian DNA polymerases including telomerase, and RAL was a specific inhibitor of mammalian replicative DNA polymerases. As expected from these results in vitro, some of them could prevent the growth of NUGC-3 human gastric cancer cells, and especially RAL was a potent antineoplastic agent with an LD(50) value of 19 microM. The cells were halted at G1 phase in the cell cycle by RAL.

NaF and mononucleotides as inhibitors of 3'-5'-exonuclease activity and stimulators of polymerase activity of E. coli DNA polymerase I Klenow fragment.

It has been shown that, in the absence of dATP in the poly(dT).oligo(dA) template-primer complex, the rate of primer cleavage by the E. coli DNA polymerase I Klenow fragment equals 4% of polymerization rate, while in the presence of dATP it equals as much as 50-60%. NaF and NMP taken separately inhibit exonuclease cleavage of oligo(dA) both with and without dATP. The addition of NaF (5-10 mM) or NMP (5-20 mM) increases the absolute increment of polymerization rate 5-9-fold relative to the absolute decrement of the rate of nuclease hydrolysis of primer. This proves the assumption that not more than 10-20% of primer molecules, interacting with the exonuclease center of polymerase, are cleaved by the enzyme. Presumably, NaF and nucleotides disturb the coupling of the 3'-end of oligonucleotide primer to the exonuclease center of the enzyme. As the primers mostly form complexes with the polymerizing center, the reaction of polymerization is activated.

[Selective inhibition of 3'-5'-exonuclease activity of DNA-polymerase I from Escherichia coli by a fluoride ion]. / Selektivnoe ingibirovanie ftorid-ionom 3'-5'-ékzonukleaznoi aktivnosti DNK-polimerazy I Escherichia coli.

The effect of NaF on the enzymatic activities of the large fragment of E. coli DNA polymerase I (Klenow enzyme-KE) with different DNA-substrates was studied. It was shown that fluoride ion at concentrations of 5-10 mM efficiently inhibits the 3'----5' exonuclease activity of KE but does not affect the polymerase activity of the enzyme. Selective inhibition of the 3'----5' exonuclease activity of KE is Mg-dependent and is observed with double- or single-stranded DNAs. In reaction with the 14-mer oligonucleotide annealed with single-stranded phage M13 DNA the enzyme was found not only to perform the exonucleolytic hydrolysis of the primers but to catalyse also a limited elongation of some primers, adding a few nucleotide residues in the absence of exogenous dNTP. The primer elongation is inhibited by inorganic pyrophosphatase and is stimulated by micromolar concentrations of exogenous pyrophosphate thus suggesting a possible role of PPi contamination in dNTP generation via pyrophosphorolysis. Traces of precursors in DNA preparations obtained by generally employed methods may serve as another source of nucleotides for the primer elongation.

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.

Mode of inhibition of HIV-1 reverse transcriptase by polyacetylenetriol, a novel inhibitor of RNA- and DNA-directed DNA polymerases.

Polyacetylenetriol (PAT), a natural marine product from the Mediterranean sea sponge Petrosia sp., was found to be a novel general potent inhibitor of DNA polymerases. It inhibits equally well the RNA- and DNA-dependent DNA polymerase activities of retroviral reverse transcriptases (RTs) (i.e. of HIV, murine leukaemia virus and mouse mammary tumour virus) as well as cellular DNA polymerases (i.e. DNA polymerases alpha and beta and Escherichia coli polymerase I). A study of the mode and mechanism of the polymerase inhibition by PAT has been conducted with HIV-1 RT. PAT was shown to be a reversible non-competitive inhibitor. PAT binds RT independently and at a site different from that of the primer-template and dNTP substrates with high affinity (K(i)=0.51 microM and K(i)=0.53 microM with dTTP and with dGTP as the variable substrates respectively). Blocking the polar hydroxy groups of PAT has only a marginal effect on the inhibitory capacity, thus hydrophobic interactions are likely to play a major role in inhibiting RT. Preincubation of RT with the primer-template substrate prior to the interaction with PAT reduces substantially the inhibition capacity, probably by preventing these contacts. PAT does not interfere with the first step of polymerization, the binding of RT to DNA, nor does the inhibitor interfere with the binding of dNTP to RT/DNA complex, as evident from the steady-state kinetic study, whereby K(m) remains unchanged. We assume, therefore, that PAT interferes with subsequent catalytic steps of DNA polymerization. The inhibitor may alter the optimal stereochemistry of the polymerase active site relative to the primer terminus, bound dNTP and the metal ions that are crucial for efficient catalysis or, alternatively, may interfere with the thumb sub-domain movement and, thus, with the translocation of the primer-template following nucleotide incorporation.

Injection of poly(beta-L-malate) into the plasmodium of Physarum polycephalum shortens the cell cycle and increases the growth rate.

Poly(beta-L-malate) (PMLA) has been reported as an unconventional, physiologically important biopolymer in plasmodia of myxomycetes, and has been proposed to function in the storage and transport of nuclear proteins by mimicking the phospho(deoxy)ribose backbone of nucleic acids. It is distributed in the cytoplasm and especially in the nuclei of these giant, multinucleate cells. We report here for the first time an increase in growth rate and a shortening of the cell cycle after the injection of purified PMLA. By comparing two strains of Physarum polycephalum that differed in their production levels of PMLA, it was found that growth activation and cell cycle shortening correlated with the relative increases of PMLA levels in the cytoplasm or the nuclei. Growth rates of a low PMLA producer strain (LU897 x LU898) were increased by 40-50% while those of a high producer strain (M(3)CVIII) were increased by only 0-17% in comparison with controls. In both strains, shortening of the cell cycle occurred to a similar extent (7.2-9.5%), and this was associated with similar increases in nuclear PMLA levels. The effects showed saturation dependences with regard to the amount of injected PMLA. A steep rise of intracellular PMLA shortly after injection was followed by the appearance of histone H1 in the cytoplasm. The increase in growth rate, the shortening of the cell cycle duration and the appearance of H1 in the cytoplasm suggest that PMLA competes with nucleic acids in binding to proteins that control translation and/or transcription. Thus, PMLA could play an important role in the coordination of molecular pathways that are responsible for the synchronous functioning of the multinucleate plasmodium.

Antitumor mechanisms of zinostatin stimalamer (YM881).

The mechanism of antitumor action of zinostatin stimalamer (YM881) was studied. YM881 suppressed colony formation of HeLa cells dose-dependently, and showed cytocidal activity. The compound inhibited DNA synthesis, but neither RNA nor protein synthesis in L1210 cells, and induced cellular DNA strand breaks in HeLa cells and DNA cleavage of PM2 phage supercoiled DNA. The compound was also shown to cause typical G2/M phase arrest in the L1210 cell cycle, and inhibited activity of DNA polymerase alpha of HeLa cells, but only at a high concentration. These results suggest that the antitumor and cytotoxic mechanisms of YM881 are identical to those of neocarzinostatin (NCS), and were due to cellular DNA strand breaks induced by direct DNA-cleaving activity and the subsequent inhibition of DNA synthesis.