Interactive effects of inhibitors of poly(ADP-ribose) polymerase and DNA-dependent protein kinase on cellular responses to DNA damage

DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase (PARP) are activated by DNA strand breaks and participate in DNA repair. We investigated the interactive effects of inhibitors of these enzymes [wortmannin (WM), which inhibits DNA-PK, and 8-hydroxy-2-methylquinazolin-4-one (NU1025), a PARP inhibitor] on cell survival and DNA double-strand break (DSB) and single-strand break (SSB) rejoining in Chinese hamster ovary-K1 cells following exposure to ionizing radiation (IR) or temozolomide. WM (20 microM) or NU1025 (300 microM) potentiated the cytotoxicity of IR with dose enhancement factors at 10% survival (DEF10) values of 4.5 +/- 0.6 and 1.7 +/- 0.2, respectively. When used in combination, a DEF10 of 7.8 +/- 1.5 was obtained. WM or NU1025 potentiated the cytotoxicity of temozolomide, and an additive effect on the DEF10 value was obtained with the combined inhibitors. Using the same inhibitor concentrations, their single and combined effects on DSB and SSB levels following IR were assessed by neutral and alkaline elution. Cells exposed to IR were post-incubated for 30 min to allow repair to occur. WM or NU1025 increased net DSB levels relative to IR alone (DSB levels of 1.29 +/- 0.04 and 1.20 +/- 0.05, respectively, compared with 1.01 +/- 0.03 for IR alone) and the combination had an additive effect. WM had no effect on SSB levels, either alone or in combination with NU1025. SSB levels were increased to 1.27 +/- 0.05 with NU1025 compared with IR alone, 1.02 +/- 0.04. The dose-dependent effects of the inhibitors on DSB levels showed that they were near maximal by 20 microM WM and 300 microM NU1025. DSB repair kinetics were studied. Both inhibitors increased net DSB levels over a 3 h time period; when they were combined, net DSB levels at 3 h were identical to DSB levels immediately post-IR. The combined use of DNA repair inhibitors may have therapeutic potential.     

Inhibition of PI3 kinases enhances the sensitivity of non-small cell lung cancer cells to ionizing radiation

Non-small cell lung cancer (NSCLC) cells are relatively resistant to ionizing radiation (IR). The phosphatidylinositol 3 (PI3) kinases are members of a family of lipid kinases that mediate cellular functions, including cell growth, proliferation and DNA repair, which may contribute to radioresistance. We studied whether inhibition of PI3 kinases could increase the response of NSCLC cells to γ-irradiation. The results showed that pretreatment of PI3 kinase inhibitor wortmannin dose-dependently radiosensitized NSCLC A549 and H1650 cells by inhibiting colony formation, which was due to enhanced G2/M arrest and apoptosis by wortmannin. The accelerated apoptosis was accompanied by increased loss of mitochondrial membrane potential (MMP) and cytochrome c release to the cytoplasm. In addition, wortmannin pretreatment significantly increased caspase-3 activation, which was associated with the repression of X-linked inhibitor of apoptosis protein (XIAP). The radio-sensitizing effect of wortmannin was correlated with the inhibition of phosphorylated PKB/Akt level. Furthermore, wortmannin down-regulated the expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) which is involved in DNA double stand break (DSB) repair, as a result, leading to the inhibition of DSBs rejoining, as indicated by increased level of γ-H2AX at 24 h after IR. Taken together, our results demonstrate that wortmannin acts as a powerful radiosensitizer in NSCLC cells by inhibiting PI3K/Akt survival signaling and DNA repair protein DNA-PKcs, suggesting that PI3 kinase inhibitors may represent a novel strategy for overcoming resistance to IR-induced apoptosis in NSCLC cells.     

ＤＮＡ双链断裂修复通路与遗传性乳腺癌的关系

遗传性乳腺癌具有家族聚集、早发、双侧等特点,多为易感基因发生胚系突变所致。ＤＮＡ损伤修复是哺乳动物细胞保证遗传物质稳定性的重要机制。双链断裂是最严重的ＤＮＡ损伤之一,修复过程涉及同源重组和非同源末端连接通路。ＤＮＡ双链断裂修复或信号传导相关基因或蛋白功能缺陷可以诱导染色体不稳定而增加乳腺癌的易感性。与ＤＮＡ修复功能相关的链交联剂和ＰＡＲＰ-１抑制剂为ＢＲＣＡ相关遗传性乳腺癌的治疗提供了新的途径。本文就ＤＮＡ双链断裂修复通路相关基因的突变与遗传性乳腺癌发病的关系作一综述。     

Heat exposure enhances radiosensitivity by depressing DNA-PK kinase activity during double strand break repair

Purpose: From the role of double strand DNA dependent protein kinase (DNA-PKcs) activity of non-homologous end joining (NHEJ) repair for DNA double strand breaks (DSBs), we aim to define possible associations between thermo-sensitisation and the enzyme activities in X-ray irradiated cells. Materials and methods: DNA-PKcs deficient mouse, Chinese hamster and human cultured cells were compared to the parental wild-type cells. The radiosensitivities, the number of DSBs and DNA-PKcs activities after heat-treatment were measured. Results: Both DNA-PKcs deficient cells and the wild-type cells showed increased radiosensitivities after heat-treatment. The wild-type cells have two repair processes; fast repair and slow repair. In contrast, DNA-PKcs deficient cells have only the slow repair process. The fast repair component apparently disappeared by heat-treatment in the wild-type cells. In both cell types, additional heat exposure enhanced radiosensitivities. Although DNA-PKcs activity was depressed by heat, the inactivated DNA-PKcs activity recovered during an incubation at 37?°C. DSB repair efficiency was dependent on the reactivation of DNA-PKcs activity. Conclusion: It was suggested that NHEJ is the major process used to repair X-ray-induced DSBs and utilises DNA-PKcs activity, but homologous recombination repair provides additional secondary levels of DSB repair. The thermo-sensitisation in X-ray-irradiated cells depends on the inhibition of NHEJ repair through the depression of DNA-PKcs activities.     

Inhibition of poly(ADP-ribose) polymerase increases ({+/-})-anti-benzo[a]pyrene diolepoxide-induced micronuclei formation and p53 accumulation in isolated human peripheral blood lymphocytes

In response to DNA damage, in particular DNA strand breaks,the proposed roles for normal tumour suppressor protein p53are to increase the period of time available for DNA repairprior to replication, or to direct damaged cells into programmedcell-death. Since treatment of mammalian cells with (±)-anti-benzo[a]pyrenediolepoxide [(±)-anti-BPDE]—a mixture of metabolitescomprising the most reactive (+ )-anti-enantiomer of the fullenvironmental carcinogen benzo[a]pyrene—has been shownto result in induction of DNA repair processes and consequentlyin DNA strand break formation, the aim of the present studywas to investigate whether p53 accumulation is induced in (±)-anti-BPDE-treatedphytohaemagglutinin-stimulated human peripheral blood lymphocytes(PBLs). Both immunocytochemical and immunoblot analysis indicatedthat treatment of PBLs with (±)-anti-BPDE results inp53 accumulation. Optimal accumulation was observed at 2.5 uM,while no increase of p53 levels was observed at concentrations<2.5 µM and >10 µM. Further, (±)-anti-BPDE-inducedp53 accumulation in PBLs was found to be time-dependent withaccumulation up to 24 h after the onset of treatment. Treatmentof PBLs with 2.5 µM of (±)-anti-BPDE and 1 mM of3-aminobenzamide, an inhibitor of the DNA strand break-dependentenzyme poly(ADP-ribose) polymerase, resulted in increased p53levels, in comparison to cells treated with (±)-anti-BPDEalone. This combination also potentiated the frequency of (±)-anti-BPDE-inducedmicronuclei. These findings suggest that (±)-anti-BPDE-inducedDNA strand break formation is responsible for the observed p53accumulation. It is unlikely that poly(ADP-ribose) polymer formationis a prerequisite in the process of p53 accumulation, as triggeredby DNA strand-break inducing agents like (±)-anti-BPDE.It is hypothesized that p53-dependent pathways may be activatedin phytohaemagglutinin-stimulated human peripheral blood lymphocytesexposed ex vivo to (±)-anti-BPDE.     

Association between DNA strand breaks and specific DNA adducts in murine hepatocytes following in vivo and in vitro exposure to N-hydroxy-2-acetylaminofluorene and N-acetoxy-2-acetylaminofluorene

N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-acetoxy-2-acetylaminofluorene(N-OAc-AAF) have previously been shown to induce dose-dependentDNA strand breaks in primary hepatocytes from mice and rats.In an attempt to determine the relationship between the extentof DNA strand breaks and the formation of specific DNA-carcinogenbound adducts in murine liver, the capability of N-OH-AAF andN-OAc-AAF to induce both DNA single strand breaks and adductformation in in vivo and in primary hepatocytes was measured.N-OH-AAF induced a low level of DNA damage in F344 rats (10mg/kg, i.p.) and in B6 mice (40 mg/kg, i.p.) 4 h after treatment.The DNA adducts identified in vivo were N-(guanin-8-yl)-2-acetylaminofluorene(Gua-C8-AAF) 55% versus 11%, N-(guanin-8-yl)-2-aminofluorene(Gua-C8-AF) 34% versus 67% and Mguanin-N²-yl)-2-acetylaminofluorene(Gua-N²-AAF) 11% versus 10%, respectively, for rat and mouseliver. An additional unknown adduct (12%) was detected in mouseliver. Dose dependent DNA binding and formation of individualDNA adducts were observed in rat and mouse primary hepatocytesfollowing 1 h exposure to [ring-³H]-N-OH-AAF (0.1-20 µM)and [ring-³-N-OAc-AAF (5–20 /M). The patterns of DNA adductsin mouse and rat primary hepatocytes exposed to N-OH-AAF andN-OAc-AF were similar to those obtained in liver following invivo treatment with N-OH-AAF. The deacetylase inhibitor, paraoxon(10^–4M) completely inhibited DNA damage induced by N-OH-AAFin mouse and partially in rat hepatocytes while DNA damage causedby N-OAc-AAF was only partially inhibited by paraoxon (10^–4M) in both species. Parallel experiments showed that paraoxon,at low concentration (10^– M), did not alter either thelevel of DNA binding or the pattern of adduct formation in rathepatocytes treated with N-OH-AAF (20 µM). However, at10^–4 M paraoxon partially blocked DNA binding (60%) andthe formation of Gua-C8-AAF (95%) and Gua-N²-AAF (80%) whileGua-C8-AF was increased twofold. In mouse hepatocytes paraoxonpretreatment (10^–4M) inhibited the formation of Gua-C8-AFby 70% following exposure to N-OH-AAF (20 µM). Gua-C8-AAFand Gua-N²-AAF were also inhibited but only at 10^–4M paraoxon.Paraoxon (10^–6 and 10^–4 M) pretreatment induceddosedependent partial inhibition of the covalent binding ofN-OAc-AAF to rat DNA and the formation of all guanine adducts.In the mouse, paraoxon (10^–6 and 10^–4 M) inhibitedthe formation of Gua-C8-AF while it increased Gua-C8-AAF. Theseresults indicate that a positive correlation exists betweenthe extent of DNA strand breaks and the formation of eitherGua-C8-AAF or Gua-C8-AF.     

Links between DNA double strand break repair and breast cancer: accumulating evidence from both familial and nonfamilial cases

DNA double strand break (DSB) repair dysfunction increases the risk of familial and sporadic breast cancer. Advances in the understanding of genetic predisposition to breast cancer have also been made by screening naturally occurring polymorphisms. These studies revealed that subtle defects in DNA repair capacity arising from low-penetrance genes, or combinations thereof, are modified by other genetically determined or environmental risk factors and correlate to breast cancer risk. Overexpression of DSB repair enzymes, absence of surveillance factors and mutation or loss of heterozygosity in any of these genes contributes to the pathogenesis of sporadic breast cancers. The results identifying DSB repair defects as a common denominator for breast cancerogenesis focus attention on functional assays in order to assess DSB repair capacity as a diagnostic tool to detect increased breast cancer risk and to enable therapeutic strategies specifically targeting the tumor.     

DNA double strand break repair inhibition as a cause of heat radiosensitization: re-evaluation considering backup pathways of NHEJ.

Heat shock is one of the most effective radiosensitizers known. As a result, combination of heat with ionizing radiation (IR) is considered a promising strategy in the management of human cancer. The mechanism of heat radiosensitization has been the subject of extensive work but a unifying mechanistic model is presently lacking. To understand the cause of excessive death in irradiated cells after heat exposure, it is necessary to characterize the lesion(s) underlying the effect and to determine which of the pathways processing this lesion are affected by heat. Since DNA double strand breaks (DSBs) are the main cause for IR-induced cell death, inhibition of DSB processing has long been considered a major candidate for heat radiosensitization. However, effective radiosensitization of mutants with defects in homologous recombination repair (HRR), or in DNA-PK dependent non-homologous end joining (D-NHEJ), the two primary pathways of DSB repair, has led to the formulation of models excluding DSBs as a cause for this phenomenon and attributing heat radiosensitization to inhibition of base damage processing. Since direct evidence for a major role of base damage in heat radiosensitization, or in IR-induced killing for that matter, is scarce and new insights in DSB repair allow alternative interpretations of existing data with repair mutants, we attempt here a re-evaluation of the role of DSBs and their repair in heat radiosensitization. First, we reanalyse data obtained with various DSB repair mutants on first principles and in the light of the recent recognition that alternative pathways of NHEJ, operating as backup (B-NHEJ), substantially contribute to DSB repair and thus probably also to heat radiosensitization. Second, we review aspects of combined action of heat and radiation, such as modulation in the cell-cycle-dependent variation in radiosensitivity to killing, as well as heat radiosensitization as a function of LET, and examine whether the observed effects are compatible with DSB repair inhibition. We conclude with a model reclaiming a central role for DSBs in heat radiosensitization.     

Radiosensitization and DNA repair inhibition by the combined use of novel inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase-1

The DNA repair enzymes, DNA-dependent protein kinase (DNA-PK) and poly(ADP-ribose) polymerase-1 (PARP-1), are key determinants of radio- and chemo-resistance. We have developed and evaluated novel specific inhibitors of DNA-PK (NU7026) and PARP-1 (AG14361) for use in anticancer therapy. PARP-1- and DNA-PK-deficient cell lines were 4-fold more sensitive to ionizing radiation (IR) alone, and showed reduced potentially lethal damage recovery (PLDR) in G(0) cells, compared with their proficient counterparts. NU7026 (10 micro M) potentiated IR cytotoxicity [potentiation factor at 90% cell kill (PF(90)) = 1.51 +/- 0.04] in exponentially growing DNA-PK proficient but not deficient cells. Similarly, AG14361 (0.4 micro M) potentiated IR in PARP-1(+/+) (PF(90) = 1.37 +/- 0.03) but not PARP-1(-/-) cells. When NU7026 and AG14361 were used in combination, their potentiating effects were additive (e.g., PF(90) = 2.81 +/- 0.19 in PARP-1(+/+) cells). Both inhibitors alone reduced PLDR approximately 3-fold in the proficient cell lines. Furthermore, the inhibitor combination completely abolished PLDR. IR-induced DNA double strand break (DNA DSB) repair was inhibited by both NU7026 and AG14361, and use of the inhibitor combination prevented 90% of DNA DSB rejoining, even 24-h postirradiation. Thus, there was a correlation between the ability of the inhibitors to prevent IR-induced DNA DSB repair and their ability to potentiate cytotoxicity. Thus, individually, or in combination, the DNA-PK and PARP-1 inhibitors act as potent radiosensitizers and show potential as tools for anticancer therapeutic intervention.     

Double strand break repair components are frequent targets of microsatellite instability in endometrial cancer

AimDNA double strand break (DSB) repair is a central cellular mechanism of the DNA damage response to maintain genomic stability. DSB components are frequently mutated in colorectal cancer with microsatellite instability (MSI). We investigated whether DSB repair is involved in endometrial cancer (EC) with MSI.MethodsMononucleotide microsatellite tracts of 14 genes of the DSB repair system were analysed in a series of 41 EC with MSI. Among these genes, the microcephalin 1 (MCPH1/BRIT1) has never been tested as target of MSI in tumour series.ResultsThe most frequently mutated gene was DNAPKcs (n = 14, 34%) followed by RAD50 (n = 7, 17%), MRE11, ATR and BRCA1 (n = 6, 15%), and by CtIP and MCPH1 (n = 5, 12%). While DSB biallelic mutations were infrequent, a high proportion of tumours (n = 30, 73%) presented mutations at some component of the DSB repair pathway, and almost half of them showed alterations at two or more components. Tumours with mutations in two or more genes were significantly associated with advanced grade (p = 0.03) and vascular invasion (p = 0.02) and marginally associated with advanced stage (p = 0.07).ConclusionsOur results suggest that in EC, the DSB repair is a relatively common mutational target of MSI and might contribute to tumour progression, and also that MCHP1 may be a novel target gene of MSI.     

Double‐strand break repair‐adox: Restoration of suppressed double‐strand break repair during mitosis induces genomic instability

Double‐strand breaks (DSBs) are one of the severest types of DNA damage. Unrepaired DSBs easily induce cell death and chromosome aberrations. To maintain genomic stability, cells have checkpoint and DSB repair systems to respond to DNA damage throughout most of the cell cycle. The failure of this process often results in apoptosis or genomic instability, such as aneuploidy, deletion, or translocation. Therefore, DSB repair is essential for maintenance of genomic stability. During mitosis, however, cells seem to suppress the DNA damage response and proceed to the next G₁ phase, even if there are unrepaired DSBs. The biological significance of this suppression is not known. In this review, we summarize recent studies of mitotic DSB repair and discuss the mechanisms of suppression of DSB repair during mitosis. DSB repair, which maintains genomic integrity in other phases of the cell cycle, is rather toxic to cells during mitosis, often resulting in chromosome missegregation and aberration. Cells have multiple safeguards to prevent genomic instability during mitosis: inhibition of 53BP1 or BRCA1 localization to DSB sites, which is important to promote non‐homologous end joining or homologous recombination, respectively, and also modulation of the non‐homologous end joining core complex to inhibit DSB repair. We discuss how DSBs during mitosis are toxic and the multiple safeguard systems that suppress genomic instability.     

Nontoxic concentration of DNA‐PK inhibitor NU7441 radio‐sensitizes lung tumor cells with little effect on double strand break repair

High‐linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end‐joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non‐toxic concentration and carbon ions. NU7441‐treated non‐small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X‐rays and carbon ions (290 MeV/n, 50 keV/μm). Cell survival was measured by clonogenic assay. DNA DSB repair, cell cycle distribution, DNA fragmentation and cellular senescence induction were studied using a flow cytometer. Senescence‐associated protein p21 was detected by western blotting. In the present study, 0.3 μM of NU7441, nontoxic to both normal and tumor cells, caused a significant radio‐sensitization in tumor cells exposed to X‐rays and carbon ions. This concentration did not seem to cause inhibition of DNA DSB repair but induced a significant G2/M arrest, which was particularly emphasized in p53‐null H1299 cells treated with NU7441 and carbon ions. In addition, the combined treatment induced more DNA fragmentation and a higher degree of senescence in H1299 cells than in A549 cells, indicating that DNA‐PK inhibitor contributes to various modes of cell death in a p53‐dependent manner. In summary, NSCLC cells irradiated with carbon ions were radio‐sensitized by a low concentration of DNA‐PK inhibitor NU7441 through a strong G2/M cell cycle arrest. Our findings may contribute to further effective radiotherapy using heavy ions.     

Lymphoid and fibroblastic cell lineages from radiosensitive cancer patients: molecular analysis of DNA double strand break repair by major non-homologous end-joining sub-pathways

Aims: Radiation therapy (RT) is used in the treatment of approximately half of all cancer patients. Although there have been great improvements in tumor localization and the technical accuracy of RT delivery, some RT patients still have idiosyncratic hypersensitivity to ionizing radiation (IR) in their normal tissues. Although much effort has been expended in the search for assays that could detect radiosensitive individuals prior to treatment and facilitate tailored therapy; a suitable and clinically practical predictive assay has yet to be realized. Since DNA double‐strand breaks (DSB) are a major lesion caused by IR, we hypothesized that radiation hypersensitive individuals might be deficient in the repair of such lesions. Methods: To test this hypothesis we quantitatively and functionally characterized DSB repair of the two major non‐homologous end‐joining (NHEJ) sub‐pathways in a pilot study using a plasmid repair reconstitution assay in lymphoblastoid and fibroblast cell lines from radiosensitive cancer patients and controls. Experiments using well‐characterized mammalian DSB repair mutants demonstrated the ability of the assay to distinguish NHEJ sub‐pathways. The proportion of direct end‐joining repair compared with that of microhomology‐directed repair was used as a functional end‐point of DSB repair competence in the different cell lines. Results: We found that the overall level of NHEJ sub‐pathway repair competency was similar in cell lines from radiosensitive patients and controls. Conclusion: These data suggest that this assay in these cell lineages has limited usefulness as a predictive screen for the endogenous DNA DSB repair competency of radiosensitive cancer patients' cells but can usefully characterize major cellular DSB repair phenotypes.     

Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining

A DNA double strand break (DSB) is a highly toxic lesion, which can generate genetic instability and profound genome rearrangements. However, DSBs are required to generate diversity during physiological processes such as meiosis or the establishment of the immune repertoire. Thus, the precise regulation of a complex network of processes is necessary for the maintenance of genomic stability, allowing genetic diversity but protecting against genetic instability and its consequences on oncogenesis. Two main strategies are employed for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by single-stranded DNA (ssDNA) resection and requires sequence homology with an intact partner, while NHEJ requires neither resection at initiation nor a homologous partner. Thus, resection is an pivotal step at DSB repair initiation, driving the choice of the DSB repair pathway employed. However, an alternative end-joining (A-EJ) pathway, which is highly mutagenic, has recently been described; A-EJ is initiated by ssDNA resection but does not require a homologous partner. The choice of the appropriate DSB repair system, for instance according the cell cycle stage, is essential for genome stability maintenance. In this context, controlling the initial events of DSB repair is thus an essential step that may be irreversible, and the wrong decision should lead to dramatic consequences. Here, we first present the main DSB repair mechanisms and then discuss the importance of the choice of the appropriate DSB repair pathway according to the cell cycle phase. In a third section, we present the early steps of DSB repair i.e., DSB signaling, chromatin remodeling, and the regulation of ssDNA resection. In the last part, we discuss the competition between the different DSB repair mechanisms. Finally, we conclude with the importance of the fine tuning of this network for genome stability maintenance and for tumor protection in fine.     

Synergistic cytotoxicity and DNA strand break formation by bromodeoxyuridine and bleomycin in human tumor cells

5-Bromo-2'-deoxyuridine (BrdUrd) is a thymidine analogue whose cellular effects are related to its incorporation into DNA. BrdUrd is a known radiosensitizing agent that could potentially enhance the activity of chemotherapeutic agents that interact directly with DNA. Therefore we studied the interaction of BrdUrd and bleomycin in a human head and neck squamous carcinoma cell line, SQ20B. Using a colony-forming assay and analyzing results by the median-effect method, we have shown that there is synergistic cytotoxicity between BrdUrd and bleomycin. Synergism is evident when BrdUrd is administered prior to bleomycin or when the two drugs are applied simultaneously and is evident at a variety of BrdUrd:bleomycin concentration ratios. Alkaline elution of DNA from cells exposed to BrdUrd and bleomycin demonstrated greater single strand break formation than expected from the individual single strand break frequencies induced by each drug alone. BrdUrd did not affect the rate of repair of bleomycin-induced single strand breaks or the formation of double strand breaks. Although the mechanism of this interaction at the molecular level is unclear, our studies suggest that a direct interaction of bleomycin with BrdUrd-substituted DNA may be the cause of the synergism of these two agents.     

Induction of DNA double-strand breaks by 8-methoxycaffeine: cell cycle dependence and comparison with topoisomerase II inhibitors

We have studied the ability of 8-methoxycaffeine (8-MOC)—oneof the most effective caffeine derivatives in inducing chromosomalaberrations—to induce DNA double strand breaks (DSB) inpurified human T lymphocytes during the cell cycle. Etoposide-or ellipticine-mediated DNA break frequency was used as a parameterof topoiso merase II activity. DNA-DSB induced by either 8-MOCor VP16 or ellipticine rose co-ordinately with the level ofDNA topoisomerase II and with the onset of DNA replication.At concentrations between 10 and 50 .µM 8-MOC was     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced tumor promotion and ornithine decarboxylase activity by quercetin: possible involvement of lipoxygenase inhibition

Quercetin (30 µmol/mouse) markedly suppressed the effectof 12-O-tetradecanoylphorbol-13-acetate (TPA, 20 nmol/mouse)on skin tumor formation in the CD-1 mice initiated by 7,12-dimethylbenz[a]anthracene(200 nmol/mouse). TPA (20 nmol/mouse)-induced epidermal ornithinedecarboxylase (ODC) activity was also inhibited by quercetin(10–30 µmol/mouse), but it failed to inhibit thestimulation, of epidermal DNA synthesis by TPA. In addition,quercetin potently inhibited lipoxygenase from 105 000 g supernatantof epidermal homogenate of mice. The 50% inhibition of lipoxygenasewas observed by quercetin at 1.3 µM. These results suggestthat the inhibition of lipoxygenase by quercetin is one of themajor actions of the above agent to inhibit tumor promotionand TPA-induced ODC activity.     

Effects of inhibitors of DNA strand break repair on HeLa cell radiosensitivity

The effects of three drugs (hydroxyurea, 1-beta-arabinofuranosylcytosine, and diamide) known to inhibit DNA synthesis on the repair of ionizing radiation-induced DNA single-strand breaks measured by alkaline elution and on cellular radiosensitivity were examined. Inhibition of repair was observed at 10(-2) M hydroxyurea, 10(-4) M 1-beta-D-arabinofuranosylcytosine, and 5 X 10(-5) M diamide, levels causing only 10% cell kill. While the mechanisms by which the drugs inhibit DNA synthesis differ, they are equally effective at inhibiting repair; without drug, cells, after a dose of 10 grays, repair 35% of DNA strand breaks in 3 min and a further 35% in 1 hr; with drug, only 10% is repaired in 3 min, and the deficiency in repair amount remains, even after 60 min. The effect of similar drug treatment on radiation-induced cell killing shows that radiosensitivity is increased; the major effect is reduction in D0 from 1.3 grays to approximately 0.8 grays with smaller effects on Dq. The data are consistent with the hypothesis that radiation produces potential double-strand breaks in DNA which, if not rapidly repaired, are converted into lethal actual double-strand breaks.