8-hydroxyguanine and Leukemia

Mutations and polymorphisms of OGG1, the major mammalian 8-oxoguanine repair activity, are associated with increased risk for several cancers. Decreased 8-oxoguanine repair capacity due to variant forms of the OGG1 gene is a common feature of numerous cancer cell lines. One such cell line, human KG-1 leukemia cells, has previously been demonstrated to be deficient in the excision of 8-oxoguanine from oxidatively damaged DNA. KG-1 cells have a homozygous R229Q amino acid substitution in OGG1 that has been presumed to alter the function of OGG1 and result in elevated levels of genomic 8-oxoG and hypersensitivity to 8-hydroxydeoxyguanosine nucleoside and ionizing radiation observed in KG-1 cells.. We characterized the enzymatic activity of R229Q OGG1 and the effect of the enzyme on cell survival following treatment with DNA damaging agents.. R229Q OGG1 had activity similar to the wild-type enzyme, yet was easily heat inactivated at physiological temperature. R229Q OGG1 expressed in human cells had significantly lower activity than wild-type OGG1 and was also highly thermolabile. Expression of R229Q OGG1 sensitized KG-1 cells to killing by menadione and 8-hydroxydeoxyguanosine, but not ionizing radiation.. These results suggest that decreased 8-oxoguanine repair in KG-1 is due to thermolability of R229Q OGG1 and that the enzyme variant increases cellular susceptibility to killing resulting from oxidative DNA damage. The R229Q OGG1 variant is a validated polymorphism prevalent in world populations and not an isolated mutation in KG-1 cells, thus the R229Q OGG1 allele may be a novel marker for cancer susceptibility.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Alleles; Amino Acid Substitution; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Deoxyguanosine; DNA Damage; DNA Glycosylases; Gene Expression; Guanine; Hot Temperature; Humans; Leukemia; Polymorphism, Genetic; Radiation, Ionizing; Vitamin K 3; Vitamins

We reported previously that KG-1, a human acute leukemia cell line, has mutational loss of 8-oxoguanine (8-hydroxyguanine; oh8Gua) glycosylase 1 (OGG1) activity and undergoes apoptotic death after treatment with 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodeoxyguanosine, 8-hydroxydeoxyguanosine; oh8dG). In our present study, we further characterized the effects of oh8dG in KG-1 cells and found that, in addition to apoptosis, oh8dG induced the arrest of KG-1 at the G1 phase. Simultaneously, oh8dG-treated KG-1 showed an increase in the oh8Gua content of DNA, upregulation of p21 (an inhibitor of cdk), and Ras inactivation. Moreover, the upregulation of p21 was followed by the inactivations of cdk4 and cdk2, the hypophosphorylation of Rb, and a marked decline in the expression of c-myc (a gene regulated by E2F that is a transcription factor whose activity is suppressed when it is bound to hypophosphorylated Rb). Ras inactivation was also followed by the inactivation of ERK kinase (MEK) and the inactivation of AP-1, a downstream target of the Ras signaling pathway. The specific MEK inhibitors, PD98059 and U0126, also induced G1 arrest. These findings suggest that p21 upregulation and Ras inactivation contribute to G1 arrest. An increase of oh8Gua content in DNA does not seem to be a principal contributor to G1 arrest, however, because the kinetics of increases of oh8Gua content in DNA and of G1 cell number did not coincide. We report that oh8dG induces the arrest of KG-1 growth at the G1 phase mainly by upregulating p21 and inactivating Ras.

Topics: Acute Disease; Apoptosis; Cyclin-Dependent Kinase Inhibitor p21; G1 Phase; Guanine; Humans; Leukemia; Proto-Oncogene Proteins p21(ras); Signal Transduction; Tumor Cells, Cultured; Up-Regulation

Our previous study showed that KG-1, a human acute leukemia cell line, has mutational loss of 8-oxoguanine (8-hydroxyguanine; oh(8)Gua) glycosylase 1 (OGG1) activity and that its viability is severely affected by 8-hydroxydeoxyguanosine (8-oxodeoxyguanosine; oh(8)dG). In the present study, the nature of the killing action of oh(8)dG on KG-1 was investigated. Signs observed in oh(8)dG-treated KG-1 cells indicated that death was due to apoptosis, as demonstrated by: increased sub-G(1) hypodiploid (apoptotic) cells, DNA fragmentation, and apoptotic body formation; loss of mitochondrial transmembrane potential, the release of cytochrome c from mitochondria into the cytosol, and the down-regulation of bcl-2; and the activation of caspases 8, 9, and 3, and the efficient inhibition of the apoptotic process by caspases inhibitors. This apoptosis appears not to be associated with Fas/Fas ligand because the expressions of these proteins were unchanged. Apoptotic KG-1 cells showed a high concentration of oh(8)Gua in DNA. Moreover, the increased concentration of oh(8)Gua in DNA, and the apoptotic process were not suppressed by the antioxidant, N-acetylcysteine, and thus the process is independent of reactive oxygen species. Of the 18 cancer cell lines treated with oh(8)dG, 3 cell lines (H9, CEM-CM3, and Molt-4) were found to be committed to apoptosis, and all of these showed very low OGG1 activity and a marked increase in the concentration of oh(8)Gua in DNA. These observations indicate that in addition to its mutagenic action, oh(8)Gua in DNA disturbs cell viability by inducing apoptosis.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Apoptosis; Cell Division; Cell Line, Tumor; Deoxyguanosine; DNA; DNA-Formamidopyrimidine Glycosylase; Escherichia coli Proteins; Fas Ligand Protein; fas Receptor; Guanine; Humans; Leukemia; Membrane Glycoproteins; Reactive Oxygen Species