Toxicity Of DNA-damaging Agents Research Articles

PARP inhibitor BMN-673 induced apoptosis by trapping PARP-1 and inhibiting base excision repair via modulation of pol-β in chromatin of breast cancer cells

PARP inhibitors emerged as clinically effective anti-tumor agents in combination with DNA damaging agents but the toxicity of DNA damaging agents and their off-target effects caused serious problems in cancer therapy. They confer cytotoxicity in cancer cells both by catalytic inhibition and trapping of PARP-1 at the DNA damage site. There is a lack of direct evidence to quantitatively determine the trapped PARP-1 in cellular DNA. Here, we have precisely evaluated the mechanism of PARP trapping mediated anti-cancer action of Quinacrine (QC), BMN-673, and their combination (QC + BMN-673) in breast cancer cells. We introduced a strategy to measure the cellular PARP trapping potentiality of BMN-673 in QC pretreated cells using a fluorescence-based assay system. It was found that QC+ BMN-673 induced apoptosis by triggering DNA damage in breast cancer cells. Treatment with QC + BMN-673 stimulated the expression of PARP-1 in the chromatin compared to that of PARP-2 and PARP-3. QC + BMN-673 treatment also caused a dose-dependent and time-dependent accumulation of PARP-1 and inhibition of PARylation in the chromatin. Upregulation of BER components (pol-β and FEN-1), an unchanged HR and NHEJ pathway proteins, and reduction of luciferase activity of the cells transfected with R-p21-P (LP-BER) were noted in combined drug-treated cells. Interestingly, silencing of pol-β resulted in unchanged PARP-1 trapping and PAR activity in the chromatin with increasing time after QC + BMN-673 treatment without altering APC and FEN-1 expression. Thus, our data suggested that the QC + BMN-673 augmented breast cancer cell death by pol-β mediated repair inhibition primarily through trapping of PARP-1 besides PARP-1 catalytic inhibition.

Abstract LB-106: Microtubule-targeting agents (MTAs) disrupt intracellular trafficking of DNA repair proteins and augment the toxicity of DNA damaging agents (DDAs)

Abstract MTAs have long been thought to cause cell death primarily by inducing mitotic arrest, a paradigm that applies to rapidly dividing cells in preclinical models. However, mitotic arrest cannot explain the activity of MTAs in much more slowly growing human cancers. In the latter, we have proposed interference with trafficking on interphase microtubules (MTs) as the principal mechanism of cytotoxicity. Satisfying this paradigm requires identification of the proteins whose trafficking on MTs, when disrupted, leads to cytotoxicity. MTAs in combination with DNA-damaging agents (DDAs) have emerged as preferred regimens for the treatment of ovarian, lung, some head and neck cancers, and many lymphomas. We proposed that by interfering with the trafficking of DNA repair proteins, MTAs could synergize with DDAs, augmenting their toxicity and enhancing cell death. To explore this hypothesis, we systematically investigated the effects of either paclitaxel or vincristine on treatment-induced DNA damage and on the distribution and biology of nine different proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, TP53, 53BP1 and p63. In several cell models including A549 cells and four Burkitt's lymphoma models (CA46, DG-75, Ramos and ST486) addition of vincristine increased cytoplasmic retention of the DNA repair proteins, thus excluding a greater fraction from the nucleus. The latter effect was observed only with MTAs and not with an inhibitor of Aurora kinase, despite similar cell cycle effects, confirming the cytoplasmic retention seen with MTAs is not due to alterations in the cell cycle. Increased cytoplasmic retention of DNA repair proteins following MT disruption suggests these proteins traffic on MTs and are vulnerable to MTAs, and this is supported by both confocal microscopy demonstrating co-localization of DNA repair proteins and α- or αβ-tubulin, as well as co-immunoprecipitation of these proteins with antibodies against the MT motor, dynein. In both A549 and MCF cells, the repair of DNA as measured by the level and persistence of γ-H2AX, was prolonged by the addition of paclitaxel to radiation. Moreover, when MCF7 or A549 cells were treated with either adriamycin or etoposide, γ-H2AX was induced to higher levels and for longer times when cells were treated with vincristine prior to and with the DDA and during DDA washout, when vincristine was present. In comparison, γ-H2AX decline was more rapid after DDA washout for cells treated only with the DDA. Together these data demonstrate that many DNA damage repair proteins travel on MTs and that the addition of MTAs promotes their sequestration in the cytoplasm. By interfering with the repair of DNA, cytoplasmic retention results in greater toxicity and likely explains why combinations of MTAs and DDAs have emerged as favored drug combinations for therapy of a diverse group of cancers. Citation Format: Marianne S. Poruchynsky, Edina Komlodi-Pasztor, Julia Wilkerson, Shana Trostel, Mauricio Burroto-Pichun, Tito Fojo. Microtubule-targeting agents (MTAs) disrupt intracellular trafficking of DNA repair proteins and augment the toxicity of DNA damaging agents (DDAs). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-106. doi:10.1158/1538-7445.AM2014-LB-106

Knockdown of Chk1 sensitizes human colon carcinoma HCT116 cells in a p53-dependent manner to lidamycin through abrogation of a G2/M checkpoint and induction of apoptosis

Recent advances in cell cycle regulation have led to a suggestion of therapeutically targeting cell cycle checkpoint pathways in cancer cells to increase the toxicity of DNA-damaging agents. In this study, we investigate whether knockdowns of checkpoint kinases Chk1 and Chk2 by RNA interfering potentiate the cytotoxicity and abrogate G2/M checkpoint induced by DNA- damaging agent lidamycin (LDM) in HCT116 cells with different p53 status. Our results showed that Chk1 knockdown enhanced the cytotoxicity of LDM through abrogating G2/M arrest and increasing apoptosis to a greater extent in HCT116 p53-/- cells than in p53wt cells. Abrogation of LDM-induced G2/M arrest by Chk1 knockdown was associated with reducing the inactivated phosphorylations of Cdc25C and Cdc2. LDM-induced γ-H2AX was increased in cells with Chk1 knockdown, indicating that DNA double-strand breaks (DSBs) were enhanced. Furthermore, knockdown of Chk1 also increased LDM-mediated apoptotic cell death in p53 knockout cells with activation of caspase-2 and caspase-3. On the contrary, knockdown of Chk2 had no impact on G2/M arrest or apoptosis induced by LDM. Moreover, dual knockdown of Chk1 and Chk2 failed to achieve better efficacy than Chk1 alone. Taken together, we suggest that Chk1 is a potential therapeutic target to sensitize human p53 deficient cancer cells to LDM.

P53: an ubiquitous target of anticancer drugs.

The p53 tumor suppressor can induce growth arrest, apoptosis and cell senescence. Not surprisingly, p53 is an appealing target for therapeutic intervention. Although current anticancer agents do not directly interact with p53, these agents (including DNA damaging drugs, antimetabolites, microtubule-active drugs and inhibitors of the proteasome) cause accumulation of wt p53. Depending on the p53 status of cancer cells, diverse therapeutic strategies are under development. These include pharmacological rescue of mutant p53 function and reactivation of wt p53 in E6-expressing cells. For protection of normal cells, strategies range from abrogation of wt p53 induction, thereby decreasing the toxicity of DNA damaging agents, to activation of wt p53-dependent checkpoints, thereby protecting cells against cell cycle-dependent therapeutics.

Cisplatin-DNA adducts are molecular decoys for the ribosomal RNA transcription factor hUBF (human upstream binding factor).

The toxicity of DNA-damaging agents is widely believed to result from the formation of lesions that block polymerases or disrupt the integrity of the genome. A mechanism heretofore not addressed is that DNA damage may titrate essential DNA-binding proteins away from their natural sites of action. This report shows that the ribosomal RNA (rRNA) transcription factor hUBF (human upstream binding factor) binds with striking affinity (Kd(app) approximately 60 pM) to the intrastrand cis-[Pt(NH3)2](2+-d(GpG) crosslink formed by the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin). When protein blots of human cell extracts are probed with cisplatin-modified DNA, 97- and 94-kDa proteins are detected, consistent with the known sites of hUBF species. A similar analysis of blots containing in vitro translated hUBF confirmed that the protein binds cisplatin adducts with high specificity. By contrast, DNA adducts of the clinically ineffective trans isomer of cisplatin, trans-diamminedichloroplatinum(II), are not recognized by hUBF. DNase I inhibition patterns of hUBF bound to a 100-base-pair DNA fragment containing a centrally located cis-[Pt(NH3)2](2+)-d(GpG) crosslink reveal specific protein-DNA interactions in a 14-base-pair region flanking the adduct. The affinity of hUBF for the rRNA promoter is similar (Kd(app) approximately 18 pM) to that measured for the cisplatin adduct. In addition, we observe that the hUBF-promoter interaction is highly sensitive to the antagonistic effects of cisplatin-DNA adducts. These results suggest that a cisplatin-mediated transcription-factor-hijacking mechanisms could disrupt rRNA synthesis, which is stimulated in proliferating cells.

Programmed cell death and adenine deoxynucleotide metabolism in human lymphocytes

Agents that cause the accumulation of DNA strand breaks are directly cytotoxic to non-dividing normal human peripheral blood lymphocytes, and to chronic lymphocytic leukemia (CLL) cells. Activation of poly(ADP-ribose) polymerase (ADPRP), and the resultant consumption of NAD, play an essential role in mediating the toxicity of these agents. Human peripheral blood lymphocytes contain a substantial number of alkali-sensitive DNA sites, reflecting ongoing DNA strand breakage and repair. However, resting lymphocytes have a limited capacity to synthesize NAD. Pulse-chase experiments indicate that approximately 75% of their NAD turnover is due to ADPRP activity. Exposure of the cells in vitro to deoxyadenosine, or to 2-chlorodeoxyadenosine (CdA, an adenosine deaminase resistant deoxyadenosine congener), caused an increase in DNA strand breaks, rapid NAD consumption, ATP depletion and cell death. Supplementation of the medium with inhibitors of poly(ADP-ribose) polymerase blocks the fall in cellular NAD and ATP, and protects the lymphocytes from the toxicity of DNA damaging agents. Slowly dividing malignant lymphocytes from patients with CLL are also susceptible to lethal NAD depletion following DNA damage. 2-chlorodeoxyadenosine (CdA) induced massive DNA strand break formation in CLL cells in vitro and a fall in NAD and ATP pools. In an initial clinical trial, several CLL patients, and two patients with hairy cell leukemia, have responded to treatment with CdA, with minimal toxicity. Thus, the suicidal activation of ADPRP in response to DNA damage has been rationally exploited in the treatment of chronic lymphoid malignancies.

Inhibition of DNA repair by deoxyadenosine in resting human lymphocytes.

Profound lymphopenia is characteristic of immunodeficient children who lack adenosine deaminase (ADA). When ADA is inactive, deoxyadenosine (dAdo) is phosphorylated by immature T lymphoblasts and inhibits cell division. However, dAdo also causes the slow accumulation of DNA strand breaks in nondividing, mature human peripheral blood lymphocytes. To explore the basis for this phenomenon, we have assessed the effects of dAdo and other deoxynucleosides on the repair of gamma-radiation induced DNA strand breaks in resting normal lymphocyte cultures. As measured by a sensitive DNA unwinding assay, most DNA strand breaks were rejoined within 2 hr after exposure of lymphocytes to 500 rad. In medium supplemented with deoxycoformycin, a tight binding ADA inhibitor, dAdo retarded DNA rejoining in a dose and time dependent manner. The inhibition required dAdo phosphorylation. Over an 8-hr period, 10 microM dAdo gradually rendered peripheral blood lymphocytes incompetent for DNA repair. Among several other compounds tested, 2-chlorodeoxyadenosine, an ADA resistant dAdo congener with anti-leukemic and immunosuppressive activity, was the most powerful inhibitor of DNA repair, exerting significant activity at concentrations as low as 100 nM. Both dAdo and 2-chlorodeoxyadenosine blocked unscheduled DNA synthesis in irradiated resting lymphocytes, as measured by [3H]thymidine uptake. On the basis of this and other data, we suggest that quiescent peripheral blood lymphocytes break and rejoin DNA at a slow and balanced rate. The accumulation of dATP progressively retards the DNA repair process and thereby fosters the time-dependent accretion of DNA strand breaks. By inhibiting DNA repair, dAdo, 2-chlorodeoxyadenosine and related compounds may substantially potentiate the toxicity of DNA damaging agents to normal and malignant lymphocytes.

Differential effects of NAD, nicotinamide and related compounds upon growth and nucleoside incorporation in human cells

Two human melanoma cell lines, MM96 and MM127, were found to be highly sensitive to the toxicity of adenosine ( d 50 100–150 μg/ml) compared with other melanoma lines. HeLa cells and a lymphoblastoid line ( d 50 > 500 μg/ml). The MM127 line was also sensitive to NAD ( d 50 41 μg/ml) compared with the other lines ( d 50 > 400 μg/ml), and accumulated three-fold more NAD-derived isotopic label. Nicotinamide exhibited little toxicity in any cell type ( d 50 > 400 μg/ml); 25–100 μg/ml nicotinamide greatly increased the plating efficiency of melanoma cells and fibroblasts when low levels of foetal calf serum were used. The toxicity of DNA-damaging agents (alkylating agents and u.v.) in melanoma cells was not reduced in the presence of NAD, adenosine or nicotinamide. Studies of the effects of the latter compounds upon the incorporation of deoxynucleosides showed that: (a) melanoma cells have lower purine pools than fibroblasts; (b) [ 3H]deoxyguanosine incorporation was inhibited more than [ 3H]deoxyadenosine incorporation; (c) incorporation of [ 3H]deoxyadenosine and [ 3H]deoxyadenosine and [ 3H]deoxyguanosine into RNA was inhibited by adenosine, thus providing a method for determination of guanine-specific DNA repair; and (d) NAD enhanced thymidine incorporation in intact melanoma cells but not in fibroblasts, in a pattern similar to the release from template restriction previously reported for permeabilised tumour cells.