Potentiation of temozolomide cytotoxicity by inhibition of DNA polymerase beta is accentuated by BRCA2 mutation.

Base excision repair (BER) plays a critical role in the repair of bases damaged by oxidative metabolism or alkylating agents, such as those commonly used in cancer therapy. Incomplete BER generates intermediates that require activation of homology-dependent DNA repair to resolve. We investigated the effects of lithocholic acid (LCA), an inhibitor of the key BER enzyme DNA polymerase beta (pol beta), in cells deficient in expression of the homology-dependent repair factor BRCA2. In vitro studies show that LCA suppresses the DNA polymerase and 5'-deoxyribose phosphate lyase activities of DNA pol beta by preventing the formation of a stable pol beta-DNA complex, reducing BER effectiveness. Cytotoxicity assays based on colony formation revealed that LCA exhibits synergism with the alkylating agent temozolomide, which engages BER through DNA methylation, and that the degree of synergism is increased in cells lacking functional BRCA2. BRCA2-deficient cells also showed heightened susceptibility to both LCA and temozolomide individually. The potentiation of temozolomide cytotoxicity by LCA owes to the conversion of single-stranded DNA breaks generated through incomplete BER of methylated nucleotides into double-stranded breaks during DNA replication, as indicated by gammaH2AX immunofluorescence. Death seems to be induced in cotreated cells through an accumulation of persistent double-stranded DNA breaks. Mutations of the BRCA2 gene have been extensively characterized and are present in various cancers, implying that inhibition of BER may offer a means to augment tumor selectivity in the use of conventional cancer therapies.

Human terminal deoxynucleotidyl transferases as novel targets for anticancer chemotherapy.

Mammalian terminal deoxyribonucleotidyl transferase (TDT) catalyzes the non-template-directed polymerization of deoxyribonucleoside triphosphates and has a key role in V(D)J recombination during lymphocyte and repertoire development. Over 90% of leukemic cells in acute lymphocytic leukemia and approximately 30% of leukemic cells in the chronic myelogenous leukemia crisis show elevated TDT activity. This finding is connected to a poor prognosis and response to chemotherapy and reduced survival time. On the other hand, recent data indicated that TDT is not the only terminal deoxyribonucleotidyl transferase in mammalian cells. Its close relative, DNA polymerase (pol) pol lambda can synthesize DNA both in a template dependent (DNA polymerase) and template-independent (terminal deoxyribonucleotidyl transferase) fashion. Pol lambda might be involved in the nonhomologous end-joining (NHEJ) recombinational repair pathway of DNA double strand breaks (DSBs). Specific inhibitors of these enzymes hold the potential to be developed into a novel class of antitumoral agents. In this review, we will summarize the recent advances in the synthesis and characterization of the first classes of specific inhibitors of mammalian terminal transferases and their potential applications.

Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1.

Molecular interactions among cell cycle and DNA repair proteins have been described, but the impact of many of these interactions on cell cycle control and DNA repair remains unclear. The cyclin-dependent kinase inhibitor, p21, is known to be involved in DNA damage-induced cell cycle arrest and blocking DNA replication and repair. Participation of p21 has been implicated in nucleotide excision repair. However, the role of p21 in the base excision repair (BER) pathway has not been thoroughly studied. In the present investigation, we treated isogenic mouse embryonic fibroblast (MEF) cell lines containing wild-type (MEF-polbeta) or DNA polymerase beta (polbeta) gene-knockout (MEFpolbetaKO) with oxidative DNA-damaging agent, plumbagin, and examined its effect on p21 levels and BER activity. Plumbagin treatment caused a S-G(2)/M phase arrest and cell death of both MEF cell lines, induced p21 levels, and decreased p21-mediated long-patch (LP) BER by blocking DNA ligase activity in the polbeta-dependent pathway and by blocking both FEN1 and DNA ligase activity in polbeta-independent pathway. These findings suggest that plumbagin induced p21 levels play a regulatory role in cell cycle arrest, apoptosis, and polbeta-dependent and -independent LP-BER pathways in MEF cells.

Antitumor activity of 2',3'-dideoxycytidine nucleotide analog against tumors up-regulating DNA polymerase beta.

DNA polymerase beta (Pol beta), an error-prone DNA-synthesizing enzyme tightly down-regulated in healthy somatic cells, has been shown to be overexpressed in many human tumors. In this study, we show that treatment with the 2',3'-dideoxycytidine (ddC) nucleoside analog inhibited in vitro and in vivo the proliferation of Pol beta-transfected B16 melanoma cells, which up-regulate Pol beta compared with control isogenic cells. The administration of ddC also increased specifically the survival of mice bearing Pol beta-overexpressing B16 melanoma. When the phosphorylated form of ddC was electrotransfered into Pol beta-transfected melanoma, the cell growth inhibition was strengthened, strongly suggesting that the cytotoxic effect results from incorporation of the chain terminator into DNA. Using in vitro single- and double-stranded DNA synthesis assays, we demonstrated that excess Pol beta perturbs the replicative machinery, favors ddC-TP incorporation into DNA, and consequently promotes chain termination. Therefore, the use of chain terminator anticancer agents could be suitable for the treatment of tumors with a high level of Pol beta.

Improved immunodetection of nuclear antigens after sodium dodecyl sulfate treatment of formaldehyde-fixed cells.

Immunostaining techniques are commonly employed to determine the temporal and spatial patterns of cellular components in in situ preparations of cells and tissues. Usually, cells are formalin-fixed, permeabilized with nonionic detergents, and probed with specific antibodies. The incorporation of a sodium dodecyl sulfate (SDS) treatment after chemical crosslinking has been shown to improve the immunodetection of some cytosolic and cell surface antigens. By incorporating an SDS treatment after crosslinking, we report a significant improvement in the detection of two nuclear antigens (i.e.) the DNA binding proteins apurinic/apyrimidinic endonuclease and DNA polymerase-beta) and bromodeoxyuridine-tagged DNA by indirect immunofluorescence of whole cells. In bromodeoxyuridine-tagged DNA, the improvement in detection after an SDS treatment was observed only after long incorporation protocols (>48 hr) and, interestingly, it was more pronounced in cultured human foreskin keratinocytes than in bovine aorta endothelial cells. In addition, the SDS treatment proved in these studies to be superior to the standard Triton X-100 permeabilization. SDS thus provides a potential means to visualize previously undetectable or poorly detectable nuclear antigens.

The cellular basis of the efficacy of the trinuclear platinum complex BBR 3464 against cisplatin-resistant cells.

Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional mononuclear platinum-based drugs. In this study we performed a comparative study of cisplatin and of the triplatinum complex BBR 3464 in a human osteosarcoma cell system (U2-OS) including an in vitro selected cisplatin-resistant subline (U2-OS/Pt). BBR 3464 was extremely potent in comparison with cisplatin in U2-OS cells and completely overcame resistance of U2-OS/Pt cells. In both cell lines, BBR 3464 accumulation and DNA-bound platinum were higher than those observed for cisplatin. On the contrary, a low frequency of interstrand cross-links after exposure to BBR 3464 was found. Differently from the increase of DNA lesions induced by cisplatin, kinetics studies indicated a low persistence of interstrand cross-link formation for BBR 3464. Western blot analysis of DNA mismatch repair proteins revealed a marked decrease of expression of PMS2 in U2-OS/Pt cells, which also exhibited microsatellite instability. Studies on DNA mismatch repair deficient and proficient colon carcinoma cells were consistent with a lack of influence of the DNA mismatch repair status on BBR 3464 cytotoxicity. In conclusion, the cytotoxic potency and the ability of the triplatinum complex to overcome cisplatin resistance appear to be related to a different mechanism of DNA interaction (formation of different types of drug-induced DNA lesions) as compared to conventional mononuclear complexes.

Chromium(III) decreases the fidelity of human DNA polymerase beta.

Certain particulate compounds of hexavalent chromium are well-known occupational and environmental human carcinogens. Hexavalent chromium primarily enters the cells and undergoes metabolic reduction; however, the ultimate trivalent oxidation state of chromium, Cr(III), predominates within the cell. DNA-bound Cr(III) has been previously shown to decrease the fidelity of replication in the M13 phage mutation assay. This study was done to understand how Cr(III), in the presence of physiological concentrations of magnesium, affects the kinetic parameters of steady-state DNA synthesis in vitro across site-specific O6-methylguanine (m6dG) residues by DNA polymerase beta (pol beta). Cr(III) binds to the short oligomer templates in a dose-dependent manner and stimulates the activity of pol beta. Cr(III) stimulates the mutagenic incorporation of dTTP opposite m6dG more than the nonmutagenic incorporation of dCTP, and thereby Cr(III) further decreases the fidelity of DNA synthesis across m6dG by pol beta. In contrast, Cr(III) does not affect the fidelity of DNA synthesis across the normal template base, dG. Both the enhanced activity and the mutagenic lesion bypass in the presence of Cr(III) may be associated with Cr(III)-dependent stimulation of pol beta binding to DNA as reported here. This study shows some of the mechanisms by which mutagenic chromium affects DNA synthesis.

Engagement of DNA polymerases during apoptosis.

DNA replicative and repair machinery was investigated by means of different techniques, including in vitro nuclear enzymatic assays, immunoelectron microscopy and confocal microscopy, in apoptotic cell lines such as HL-60 treated with methotrexate, P815 and K562 exposed to low temperatures and Friend cells exposed to ionizing radiation. The results showed a shift of DNA polymerase alpha and beta activities. DNA polymerase alpha, which in controls was found to be the principal replicative enzyme driving DNA synthesis, underwent, upon apoptosis, a large decrease of its activity being replaced by DNA polymerase beta which is believed to be associated with DNA repair. Such a modulation was concomitant with a topographical redistribution of both DNA polymerase alpha and the incorporation of BrdUrd throughout the nucleus. Taken together, these results indicate the occurrence of a dramatic response of the DNA machinery, through a possible common or at least similar behaviour when different cell lines are triggered to apoptosis. Although this possibility requires further investigation, these findings suggest an extreme attempt of the cell undergoing apoptosis to preserve its nuclear environment by switching on a repair/defence mechanism during fragmentation and chromatin margination.