ABSTRACT
BACKGROUND: Chronic myelogenous leukemia (CML) is characterized by an initial chronic phase that invariably evolves to the more aggressive phase of blast crisis. Although the determinants of this transition are still unknown, it has been shown that the blast crisis is accompanied by genetic instability. MATERIALS AND METHODS: The expression and activity of the error-prone DNA polymerase beta (pol beta) were investigated in blood samples from CML patients, by Western blotting and by an in vitro replication assay, respectively. RESULTS: Pol beta expression and activity were significantly higher in CML samples compared to those of healthy donors. CONCLUSION: Our results suggest that the excess of pol beta in CML could contribute to the genetic instability observed during the evolution of the disease from the chronic phase to blast crisis.
Subject(s)
DNA Polymerase beta/blood , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Blotting, Western , DNA Polymerase beta/biosynthesis , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/blood , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Neutrophils/enzymologyABSTRACT
DNA polymerase beta (pol beta) is a nuclear enzyme that is tightly bound to chromatin. Release of the pol beta activity into serum, therefore, may indicate the occurrence of massive destruction of cell nuclei in organs or tissues. In the present study, we made a liver injury model rat by the intraperitoneal injection of D-galactosamine hydrochloride (GalN, 500 mg/kg) and lipopolysaccharide (LPS, 100 microg/kg). Serum from the GalN/LPS-treated rats showed a high level of pol beta activity up to 118 pmol/0.5 microl serum (4700 cpm) at 12 h after the treatment, while the control rat serum showed the back ground level (3.8 pmol/0. 5 microl, 150+/-70 cpm). The serum pol beta activity was sensitive to inhibition by 2',3'-dideoxyTTP and by an anti-rat pol beta antibody. Among 30 rats treated with GalN/LPS, 10 rats died within 120 h (dead group). Serum pol beta activity in the dead group was as high as 23.0+/-19.5 pmol/0.5 microl (925+/-778 cpm) at 10 h after the treatment, while in alive group (n=20), it was 3.7+/-3.2 pmol. Levels of the serum pol beta activity correlated well with the prognosis of GalN/LPS-treated rats based on an analysis of the receiver-operator characteristic curves.
Subject(s)
DNA Polymerase beta/blood , Galactosamine/toxicity , Hepatic Encephalopathy/enzymology , Lipopolysaccharides/toxicity , Liver/pathology , Alanine Transaminase/blood , Animals , Biomarkers/blood , Dose-Response Relationship, Drug , Hepatic Encephalopathy/blood , Hepatic Encephalopathy/chemically induced , Liver/enzymology , Male , Predictive Value of Tests , Prognosis , Rats , Rats, Wistar , Time FactorsABSTRACT
BACKGROUND: Alterations in the expression levels of genes and proteins involved in oxidative stress and DNA damage response underlie the phenotypic changes associated with aging. We have investigated whether the quality of dietary fat alters postprandial gene expression and protein levels involved in p53-dependent DNA repair and whether the supplementation with Coenzyme Q10 improves this situation in an elderly population. METHODS: Twenty participants were randomized to receive three isocaloric diets each for 4 weeks: Mediterranean diet supplemented with Coenzyme Q10, Mediterranean diet, saturated fatty acid-rich diet. After a 12-hour fast, volunteers consumed a breakfast with a fat composition similar to that consumed in each of the diets. Gadd45a, Gadd45b, OGG1, APE-1/Ref-1, DNApolß, and XPC gene expression and nuclear Gadd45a, APE-1/Ref-1, and DNApolß protein levels were determined in peripheral blood mononuclear cells. RESULTS: Mediterranean diet and Mediterranean diet supplemented with Coenzyme Q10diets downregulated Gadd45a protein levels compared with the saturated fatty acid-rich diet. Moreover, Mediterranean diet supplemented with Coenzyme Q10diet evoked lower postprandial Gadd45a, Gadd45b, XPC, DNApolß and OGG1 gene expression and lower APE-1/Ref-1 and DNApolß protein levels than the saturated fatty acid-rich diet. CONCLUSIONS: Our results support a beneficial effect of Mediterranean diet and Mediterranean diet supplemented with Coenzyme Q10 on DNA damage as compared to the detrimental action of a saturated fatty acid-rich diet, which triggers the p53-dependent DNA repair machinery.
Subject(s)
Aging/metabolism , DNA Repair , Diet, Mediterranean , Ubiquinone/analogs & derivatives , Aged , Aging/genetics , Cell Cycle Proteins/blood , Cell Cycle Proteins/genetics , Cross-Over Studies , DNA Glycosylases/genetics , DNA Polymerase beta/blood , DNA Polymerase beta/genetics , DNA Repair/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/blood , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-Binding Proteins/genetics , Dietary Supplements , Female , Gene Expression , Genes, p53 , Humans , Leukocytes, Mononuclear/metabolism , Male , Nuclear Proteins/blood , Nuclear Proteins/genetics , Postprandial Period/genetics , Postprandial Period/physiology , RNA, Messenger/blood , RNA, Messenger/genetics , Ubiquinone/administration & dosageABSTRACT
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.