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Title: Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase {alpha}

Abstract

In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol {alpha} from a fungus (Alternaria tennuis). We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited only mammalian pol {alpha} with IC{sub 50} value of 0.5 {mu}M, and did not influence the activities of other replicative pols such as pols {delta} and {epsilon}, but also showed no effect on pol {alpha} activity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. The compound also inhibited the proliferation of human cancer cells with LD{sub 50} values of 38.0-44.4 {mu}M. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, dehydroaltenusin was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that increased tumor necrosis and decreased mitotic index were apparently detected by the compound in vivo. Therefore, dehydroaltenusin could be of interest as not only a mammalian pol {alpha}-specific inhibitor, but also as a candidate drug for anti-cancer treatment.

Authors:
 [1];  [2];  [2];  [3];  [1];  [4];  [5];  [4];  [4];  [1];  [6];  [7];  [8]
  1. Laboratory of Food and Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180 (Japan)
  2. Biomedical Research Center Laboratory of Biomedical Engineering, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556 (Japan)
  3. Marine Biomedical Institute, Sapporo Medical University School of Medicine, Oshidomari, Rishirifuji, Hokkaido 097-0101 (Japan)
  4. Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510 (Japan)
  5. RIKEN - Institute of Physical and Chemical Research, Wako, Saitama 351-0198 (Japan)
  6. (Japan)
  7. Department of Pathology, Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556 (Japan)
  8. Laboratory of Food and Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180 (Japan) and Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180 (Japan). E-mail: mizushin@nutr.kobegakuin.ac.jp
Publication Date:
OSTI Identifier:
20857953
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 352; Journal Issue: 2; Other Information: DOI: 10.1016/j.bbrc.2006.11.021; PII: S0006-291X(06)02491-0; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BROMIDES; DMSO; DNA; DNA POLYMERASES; DNA REPLICATION; DRUGS; EDTA; ENZYME INHIBITORS; FUNGI; HELA CELLS; IN VIVO; LETHAL DOSES; MICE; MITOTIC INDEX; NECROSIS; NEOPLASMS; PHOSPHATES; SCREENING; TETRAZOLIUM; TOXICITY

Citation Formats

Maeda, Naoki, Kokai, Yasuo, Ohtani, Seiji, Sahara, Hiroeki, Kuriyama, Isoko, Kamisuki, Shinji, Takahashi, Shunya, Sakaguchi, Kengo, Sugawara, Fumio, Yoshida, Hiromi, Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Sato, Noriyuki, and Mizushina, Yoshiyuki. Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase {alpha}. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2006.11.021.
Maeda, Naoki, Kokai, Yasuo, Ohtani, Seiji, Sahara, Hiroeki, Kuriyama, Isoko, Kamisuki, Shinji, Takahashi, Shunya, Sakaguchi, Kengo, Sugawara, Fumio, Yoshida, Hiromi, Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Sato, Noriyuki, & Mizushina, Yoshiyuki. Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase {alpha}. United States. doi:10.1016/j.bbrc.2006.11.021.
Maeda, Naoki, Kokai, Yasuo, Ohtani, Seiji, Sahara, Hiroeki, Kuriyama, Isoko, Kamisuki, Shinji, Takahashi, Shunya, Sakaguchi, Kengo, Sugawara, Fumio, Yoshida, Hiromi, Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Sato, Noriyuki, and Mizushina, Yoshiyuki. Fri . "Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase {alpha}". United States. doi:10.1016/j.bbrc.2006.11.021.
@article{osti_20857953,
title = {Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase {alpha}},
author = {Maeda, Naoki and Kokai, Yasuo and Ohtani, Seiji and Sahara, Hiroeki and Kuriyama, Isoko and Kamisuki, Shinji and Takahashi, Shunya and Sakaguchi, Kengo and Sugawara, Fumio and Yoshida, Hiromi and Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180 and Sato, Noriyuki and Mizushina, Yoshiyuki},
abstractNote = {In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol {alpha} from a fungus (Alternaria tennuis). We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited only mammalian pol {alpha} with IC{sub 50} value of 0.5 {mu}M, and did not influence the activities of other replicative pols such as pols {delta} and {epsilon}, but also showed no effect on pol {alpha} activity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. The compound also inhibited the proliferation of human cancer cells with LD{sub 50} values of 38.0-44.4 {mu}M. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, dehydroaltenusin was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that increased tumor necrosis and decreased mitotic index were apparently detected by the compound in vivo. Therefore, dehydroaltenusin could be of interest as not only a mammalian pol {alpha}-specific inhibitor, but also as a candidate drug for anti-cancer treatment.},
doi = {10.1016/j.bbrc.2006.11.021},
journal = {Biochemical and Biophysical Research Communications},
number = 2,
volume = 352,
place = {United States},
year = {Fri Jan 12 00:00:00 EST 2007},
month = {Fri Jan 12 00:00:00 EST 2007}
}
  • A new polyclonal antibody against the ..cap alpha..-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell..cap alpha..-polymerase. The antibody neutralized ..cap alpha..-polymerase activity and was strong and specific for the ..cap alpha..-polymerase catalytic polypeptide (M/sub r/ 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in lambdagt11. A positive phage was identified and plaque purified. This phage, designated lambdapol..cap alpha..1.2, also was found to be positive with an antibody against Drosophila ..cap alpha..-polymerase. The insert in lambdapol..cap alpha..1.2 (1183 base pairs) containedmore » a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified ..cap alpha..-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating ..cap alpha..-polymerase. This indicated the lambdapol..cap alpha..1.2 insert encoded an ..cap alpha..-polymerase epitope and suggested that the cDNA corresponded to an ..cap alpha..-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding ..cap alpha..-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is approx.5.4 kilobases.« less
  • DNA polymerases delta and ..cap alpha.. were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl) deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase ..cap alpha.. were determined. The effects of these inhibitors on DNA replication, measured with (/sup 3/H)TTP, in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and ..cap alpha.. in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase ..cap alpha.. or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition ofmore » purified polymerase ..cap alpha.. or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase ..cap alpha.. activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase ..cap alpha.. activity reduced, but did not abolish, DNA replications in permeable cells. BuPdGTP, as well as anti-polymerase ..cap alpha.. antibodies, inhibited DNA replication in a nonlinear manner as a function of time. A concentration of BuPdGTP which abolished polymerase ..cap alpha.. activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments. This information suggests that polymerases ..cap alpha.. and delta are involved in both the synthesis and maturation of nascent DNA. This is the first report to present evidence suggesting that both polymerases ..cap alpha.. and delta play a significant role in mammalian DNA replication.« less
  • An inhibitor of poly(ADP-ribose) synthesis, 3-aminobenzamide (3AB), at low concentrations was found to reduce strand-break frequencies and increase repair replication in human lymphoid cells damaged by methyl methanoesulfonate. A concentration of 0.1 mM 3AB was adequate to produce a maximum effect on strand-break frequencies and repair replication. This evidence, together with previous measurements, demonstrates that 3AB cannot be regarded as an inhibitor of DNA repair; rather, it actually accelerates the ligation of DNA repair patches. Previous considerations of 3AB as a repair inhibitor may have derived from the use of excessive concentrations above 1 mM that may have stimulated additionalmore » damage and from the use of ethyl alcohol as a solvent for 3AB. Interpretations of the role of single-strand breaks and poly(ADP-ribose) in DNA repair, differentiation, and gene activity may need reevaluation because they have frequently been based on an erroneous notion of 3AB as a repair inhibitor, when its mode of action is, in fact, more complex.« less
  • We are investigating the molecular mechanisms of how metal ions affect the fidelity of DNA replication. In our DNA replication system primed templates site-specifically modified with a model mutagenic lesion. O{sup 6}-methyldeoxyguanosine (O{sup 6}mG), are replicated in vitro by various purified DNA polymerases. O{sup 6}mG blocks DNA replication by human DNA polymerase {beta} but is less inhibitory to E. coli DNA Polymerase I-Klenow Fragment (KF) and its 3`-5` exonuclease deficient counterpart [KF (exo{sup {minus}})]. All three DNA polymerases exhibit a strong prelesion block and decreased rates of nucleotide extension. Polymerase {beta} exhibits discrimination against the incorporation of the right (dC)more » versus the wrong (dT) base. dT is incorporated in preference to dC opposite O{sup 6}mG-dT. KF (exo{sup {minus}}), on the other hand, extends the O{sup 6}mG-dT base pair more efficiently than O{sup 6}mG-dC. Thus individual polymerases may have opposing preferences for incorporation versus extension. Our previous studies have shown that chromium (III) [Cr(III)] increases DNA polymerase processivity and lowers the fidelity of DNA replication. At low final concentrations (about 0.1 {mu}M) Cr(III) stimulates the rate of nucleotide incorporation opposite O{sup 6}mG by KF(exo{sup {minus}}) and, to a lesser extent, by polymerase {beta}. Cr(III) does not affect incorporation of dT opposite dA, but decreases by 10-fold the K{sub M} for incorporation of dT opposite O{sup 6}mG. This constitutes an important mutagenic effect. Further experiments are underway to determine how Cr(III) affects the DNA binding and kinetic parameters of these exonuclease deficient DNA repair polymerases.« less
  • Treatment of the TsAF8 temperature-sensitive (TS) mutant of Syrian hamster BHK-21 cells, with calcium phosphate precipitates of genomic TS/sup +/ DNAs from a variety of mammalian cell lines permitted the selection of TS/sup +/ colonies at 40/sup 0/C. TS/sup +/ transformation events were distinguished from spontaneous TS/sup +/ reversions in experiments in which ..cap alpha..-amanitin-sensitive (Ama/sup s/) TS/sup +/ DNA was used to transform an AMA/sup R/ derivative of TsAF8 cells and Ama/sup R/ TS/sup +/ DNA was used to transform Ama/sup s/ TsAF8 cells. In each case it was possible to demonstrate the unselected acquisition of the appropriate Ama/supmore » s/ or Ama/sup R/ phenotype with the selected TS/sup +/ allele. Each of these TS/sup +/ transformed cell lines when grown at 40/sup 0/C contained an RNA polymerase II activity with a sensitivity to inhibition by ..cap alpha..-amanitin characteristic of the particular DNA used to transform the TS cells, whereas at 34/sup 0/C the same cells contained a mixture of Ama/sup R/ and Ama/sup s/ polymerase II activities. Together, these data provide convincing evidence that the RNA polymerase II gene determining sensitivity to inhibition by ..cap alpha..-amanitin can be transferred to TsAF8 cells and that the TS defect in TsAF8 is a polymerase II mutation.« less