Drug-induced DNA Damage Research Articles

Abstract 1590: Expression and function of Sirt6 in muscle invasive bladder cancer

Abstract Unlike most other solid tumors, there is limited success of targeted agents in treating metastatic bladder cancer (BC), a lethal disease with a median overall survival of merely 13-15 months. No prevalent “drugable” driver mutations have been identified in muscle invasive bladder cancer (MIBC) so far. Several high profile publications in the past few years have put Sirt6, a member of the class III histone deacetylase in the center stage of cancer metabolism and DNA repair. We therefore assessed the expression and function of Sirt6 in MIBC. Inconsistent with its proposed function as a tumor suppressor in colon cancer, the gene expression datasets available in Oncomine did not reveal significant differences in Sirt6 mRNA levels among normal urothelium, superficial BC and MIBC. Compared to normal urothelium, carcinoma in situ, superficial BC, the median Sirt6 mRNA was the lowest in MIBC in the Dyrkjot bladder 3 dataset, but this was not statistically significant. We then studied Sirt6 protein expression with immunohistochemistry (IHC) in commercial tissue microarray as well as radical cystoprostatectomy samples in our total cancer care tumor bank. Sirt6 IHC were reviewed by pathologist and considered positive for Sirt6 if more than 5% of the tumor cells had at least 1+ Sirt6 nuclear staining. Positive Sirt6 IHC was detected in 33/56 (59%) of T2 (grade 1-3), 2/23 T3 (9%) and 0/3 T4 urothelial carcinoma. All the grade 3/high grade T3 urothelial carcinomas were negative for Sirt6 IHC. Negative Sirt6 IHC was also noted in 2 adenocarcinomas, 1 sarcomatoid carcinoma and 1 small cell MIBC. 1/18 (5%) lymph node metastasis had positive Sirt6 IHC. Consistent with its role of inhibiting aerobic glycolysis, bladder cancer cell lines with higher Srit6 have lower glucose uptake and lower lactate production. Knocking down Sirt6 expression in bladder cancer cell lines, however, did not sensitize bladder cancer lines to drug induced DNA damage. Among MIBC, much higher frequency of Sirt6 IHC positivity was noted in T2 urothelial carcinoma than T3, T4 and non-urothelial carcinomas. Data with bladder cancer cell lines suggest the function of Sirt6 in bladder cancer is primarily inhibiting aerobic glycolysis. Citation Format: Minghui Wu, Shohreh Dickinson, Jingsong Zhang. Expression and function of Sirt6 in muscle invasive bladder cancer. [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 1590. doi:10.1158/1538-7445.AM2014-1590

Abstract 419: The role of histone methyltransferase G9a in chemotherapeutic drugs-induced DNA damage

Abstract Head and neck squamous cell carcinoma (HNSCC) is one of the serious life-threatening diseases in the world. In addition to surgery, the conventional treatments for oral cancer includes radiation and chemotherapy, which usually act by induction of DNA damage. However, approximately one-third of treated patients will experience local or regional recurrence. G9a is a histone methyltransferase responsible for mono- or di-methylation of lysine 9 in histone H3. Our recent work has demonstrated that G9a level is highly correlated with aggressiveness and poor prognosis of lung, ovarian and colon cancer patients in Taiwanese population. Here, we found that G9a expression in HNSCC is upregulated and positively correlated with poor prognosis. Moreover, knockdown of G9a resulted in decrease cell survival and cologenic capacity following treating with chemotherapeutic drug, hydroxyurea and etoposide. Futhermore, depletion of G9a caused sustained phosphorylation of γ-H2AX after etoposide treatment. Together, We suggest that G9a may contribute to HNSCC proliferation by improvement of DNA double break repair. Our study paves the way for exploring the blockade of G9a overexpression as a novel approach for the prevention and treatment of HNSCC. Citation Format: Chia-Wen Liu. The role of histone methyltransferase G9a in chemotherapeutic drugs-induced DNA damage. [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 419. doi:10.1158/1538-7445.AM2014-419

Nanodrug delivery in reversing multidrug resistance in cancer cells.

Different mechanisms in cancer cells become resistant to one or more chemotherapeutics is known as multidrug resistance (MDR) which hinders chemotherapy efficacy. Potential factors for MDR includes enhanced drug detoxification, decreased drug uptake, increased intracellular nucleophiles levels, enhanced repair of drug induced DNA damage, overexpression of drug transporter such as P-glycoprotein(P-gp), multidrug resistance-associated proteins (MRP1, MRP2), and breast cancer resistance protein (BCRP). Currently nanoassemblies such as polymeric/solid lipid/inorganic/metal nanoparticles, quantum dots, dendrimers, liposomes, micelles has emerged as an innovative, effective, and promising platforms for treatment of drug resistant cancer cells. Nanocarriers have potential to improve drug therapeutic index, ability for multifunctionality, divert ABC-transporter mediated drug efflux mechanism and selective targeting to tumor cells, cancer stem cells, tumor initiating cells, or cancer microenvironment. Selective nanocarrier targeting to tumor overcomes dose-limiting side effects, lack of selectivity, tissue toxicity, limited drug access to tumor tissues, high drug doses, and emergence of multiple drug resistance with conventional or combination chemotherapy. Current review highlights various nanodrug delivery systems to overcome mechanism of MDR by neutralizing, evading, or exploiting the drug efflux pumps and those independent of drug efflux pump mechanism by silencing Bcl-2 and HIF1α gene expressions by siRNA and miRNA, modulating ceramide levels and targeting NF-κB. “Theragnostics” combining a cytotoxic agent, targeting moiety, chemosensitizing agent, and diagnostic imaging aid are highlighted as effective and innovative systems for tumor localization and overcoming MDR. Physical approaches such as combination of drug with thermal/ultrasound/photodynamic therapies to overcome MDR are focused. The review focuses on newer drug delivery systems developed to overcome MDR in cancer cell.

Loss of urokinase receptor sensitizes cells to DNA damage and delays DNA repair.

DNA damage induced by numerous exogenous or endogenous factors may have irreversible consequences on the cell leading to cell cycle arrest, senescence and cell death. The DNA damage response (DDR) is powerful signaling machinery triggered in response to DNA damage, to provide DNA damage recognition, signaling and repair. Most anticancer drugs induce DNA damage, and DNA repair in turn attenuates therapeutic efficiency of those drugs. Approaches delaying DNA repair are often used to increase efficiency of treatment. Recent data show that ubiquitin-proteasome system is essential for signaling and repair of DNA damage. However, mechanisms providing regulation of proteasome intracellular localization, activity, and recruitment to DNA damage sites are elusive. Even less investigated are the roles of extranuclear signaling proteins in these processes. In this study, we report the involvement of the serine protease urokinase-type plasminogen activator receptor (uPAR) in DDR-associated regulation of proteasome. We show that in vascular smooth muscle cells (VSMC) uPAR activates DNA single strand break repair signaling pathway. We provide evidence that uPAR is essential for functional assembly of the 26S proteasome. We further demonstrate that uPAR mediates DNA damage-induced phosphorylation, nuclear import, and recruitment of the regulatory subunit PSMD6 to proteasome. We found that deficiency of uPAR and PSMD6 delays DNA repair and leads to decreased cell survival. These data may offer new therapeutic approaches for diseases such as cancer, cardiovascular and neurodegenerative disorders.

Effects of Four Chemotherapeutic Agents, Bleomycin, Etoposide, Cisplatin, and Cyclophosphamide, on DNA Damage and Telomeres in a Mouse Spermatogonial Cell Line1

Treatment with chemotherapeutics agents may induce persistent DNA damage in male germ cells with the possibility of long-term consequences on fertility and progeny outcome. Telomeres, specialized structures at the physical ends of chromosomes, play an important role in the maintenance of genetic stability and in the response of somatic cells to anticancer drugs. Our objective was to test the hypothesis that exposure to bleomycin, etoposide, or cisplatin (the drugs used to treat testicular cancer) or cyclophosphamide (an anticancer agent and immunosuppressant) targets telomeres in the male germ line. C18-4 spermatogonial cells were exposed to bleomycin, etoposide, cisplatin, or 4-hydroperoxycyclophosphamide (4OOH-CPA, a preactivated analog of cyclophosphamide). All four anticancer drugs induced a significant increase in DNA damage in C18-4 cells, as assessed by gamma-H2AX immunofluorescence. Interestingly, the gamma-H2AX signal was localized to telomeres after treatment with bleomycin, cisplatin, and 4OOH-CPA, but not etoposide. Mean telomere lengths, the intensity of the telomere fluorescence in situ hybridization signal, telomerase activity, and the expression of the telomerase enzyme mRNA components, Tert and Terc, were reduced by exposure to cisplatin and 4OOH-CPA, but not by bleomycin or etoposide. Thus, although all four anticancer drugs induced DNA damage in this spermatogonial cell line, telomeres were not specifically affected by etoposide and only the two alkylating agents, cisplatin and 4OOH-CPA, induced telomere dysfunction. This telomere dysfunction may contribute to infertility and developmental defects in the offspring.

Dynamic regulation of Rad51 by E2F1 and p53 in prostate cancer cells upon drug-induced DNA damage under hypoxia.

Intratumoral hypoxia has been proposed to create a "mutator" phenotype through downregulation of DNA repair, leading to increased genomic instability and drug resistance. Such downregulation of DNA repair has been proposed to sensitize hypoxic cancer cells to DNA-damaging agents and inhibitors of DNA repair. Here, we showed that prostate cancer cells with mutant p53 were resistant to the poly(ADP-ribose) polymerase inhibitor, veliparib (2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide, dihydrochloride; Abbott Laboratories, Abbott Park, IL), and the DNA-damaging topoisomerase I inhibitor camptothecin-11 (CPT-11) or SN38 (7-ethyl-10-hydroxycamptothecin) under hypoxia. Upregulation of Rad51 by E2F1 upon DNA damage under hypoxia contributed to such resistance, which was reversed by either inhibiting RAD51 transcription with small interfering RNA or by expressing wild-type p53 in the p53 null prostate cancer line. Accumulation of endogenous p53 but not E2F1 and suppressed RAD51 transcription was observed in prostate cancer line with wild-type p53 after DNA damage under hypoxia. Combining veliparib with CPT-11 significantly enhanced DNA damage and apoptosis under both hypoxic and normoxic culture conditions. Such enhanced DNA damage and antitumor activities were seen in the presence of Rad51 upregulation and confirmed in vivo with PC3 mouse xenografts. These data illustrate a dynamic regulation of Rad51 by E2F1 and p53 in prostate cancer cells' response to hypoxia and DNA damage. The veliparib and CPT-11 combination can be further explored as a treatment of metastatic castration-resistant prostate cancers that have frequent p53 mutations and enriched genomic instability.

INTERACTION OF METHOTREXATE WITH DNA USING GOLD NANOPARTICLES AS A PROBE

□ The use of gold nanoparticles as a probe to investigate the interaction of DNA with methotrexate is reported. Methotrexate has widespread use in the treatment of tumors and auto-immune diseases, and is an anti-metabolite of folic acid, having a direct or indirect effect on various molecular targets, which influences DNA replication and cell proliferation by integrating between the spaces between adjacent DNA base pairs. A simple method to monitor the interaction of DNA with methotrexate was developed, using ultraviolet – visible spectroscopy and spectroflurometry with gold nanoparticles as the probe. In a mixture of DNA and methotrexate, as the proportion of free DNA increased, the absorption of gold nanoparticles decreased and the fluorescence intensity increased. This article confirmed that as the interaction between DNA and methotrexate increased, less free DNA was available to bind with gold nanoparticles present in solution, reducing the absorption and enhancing the fluorescence of the nanoparticles. The limit of detection and limit of quantification were 160 ng/mL and 50 ng/mL, respectively. The results demonstrated the use of gold nanoparticles as a probe for the interaction of DNA with methotrexate, which may be an important tool to measure drug-induced DNA damage.

Optimization of the Lactam Side Chain of 7-Azaindenoisoquinoline Topoisomerase I Inhibitors and Mechanism of Action Studies in Cancer Cells

Optimization of the lactam ω-aminoalkyl substituents in a series of 7-azaindenoisoquinolines resulted in new anticancer agents with improved Top1 inhibitory potencies and cancer cell cytotoxicities. The new compounds 14–17 and 19 exhibited mean graph midpoint cytotoxicity (GI50) values of 21–71 nM in the NCI panel of 60 human cancer cell cultures. Ternary 7-azaindenoisoquinoline–DNA–Top1 cleavage complexes that persist for up to 6 h were detected in HCT116 colon cancer cells. Ternary complexes containing 7-azaindenoisoquinolines were significantly more stable than those in which camptothecin was incorporated. DNA content distribution histograms showed S-phase block 3 h after drug removal. Drug-induced DNA damage in HCT116 cells was revealed by induction of the histone γ-H2AX marker. The 7-azaindenoisoquinolines were able to partially overcome resistance in several drug-resistant cell lines, and they were not substrates for the ABCB1 drug efflux transporter. Molecular modeling studies indicate that the 7-azaindenoisoquinolines intercalate at the DNA cleavage site in DNA–Top1 covalent complexes with the lactam side chain projecting into the major groove. Overall, the results indicate that the 7-azaindenoisoquinolines are promising anticancer agents that merit further development.

Reverse engineering of drug induced DNA damage response signalling pathway reveals dual outcomes of ATM kinase inhibition

Reverse engineering of drug induced DNA damage response signalling pathway reveals dual outcomes of ATM kinase inhibition

ATG5 is induced by DNA-damaging agents and promotes mitotic catastrophe independent of autophagy

Anticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.

Critical role of FANCC in JAK2 V617F mutant-induced resistance to DNA cross-linking drugs

A point mutation (V617F) of tyrosine kinase Janus kinase 2 (JAK2) is found in the majority of patients with myeloproliferative neoplasms (MPNs) and an aberrant signaling pathway induced by constitutively active JAK2 V617F mutant is a hallmark of MPNs. Cells transformed by JAK2 V617F mutant exhibited resistance to anti-cancer drugs such as cisplatin (CDDP), mitomycin C (MMC) and bleomycin (BLM). We first found that the expression of FANCC, a member of the Fanconi anemia (FA) proteins, was significantly induced by JAK2 V617F mutant through activation of signal transducers and activators of transcription 5 (STAT5). In addition, monoubiqitination and foci formation of FANCD2, which are critical for activation of the FA pathway, were increased in cells transformed by JAK2 V617F mutant, compared to cells expressing wild-type JAK2. Interestingly, knockdown of FANCC in cells expressing JAK2 V617F mutant induced not only the reduction of monoubiqitination and foci formation of FANCD2 but also the enhancement of sensitivity to DNA damage induced by CDDP and MMC but not BLM. Taken together, FANCC is most likely to be critical for resistance to DNA cross-linking drug-induced DNA damage in cells transformed by JAK2 V617F mutant.

Tempol prevents genotoxicity induced by vorinostat: role of oxidative DNA damage

Vorinostat is a member of histone deacetylase inhibitors, which represents a new class of anticancer agents for the treatment of solid and hematological malignancies. Studies have shown that these drugs induce DNA damage in blood lymphocytes, which is proposed to be due to the generation of oxidative lesions. The increase in DNA damage is sometimes associated with risk of developing secondary cancer. Thus, finding a treatment that limits DNA damage caused by anticancer drugs would be beneficial. Tempol is a potent antioxidant that was shown to prevent DNA damage induced by radiation. In this study, we aimed to investigate the harmful effects of vorinostat on DNA damage, and the possible protective effects of tempol against this damage. For that, the spontaneous frequency of sister chromatid exchanges (SCEs), chromosomal aberrations (CAs), and 8-hydroxy-2-deoxy guanosine (8-OHdG) levels were measured in cultured human lymphocytes treated with vorinostat and/or tempol. The results showed that vorinostat significantly increases the frequency of SCEs, CAs and 8-OHdG levels in human lymphocytes as compared to control. These increases were normalized by the treatment of cells with tempol. In conclusion, vorinostat is genotoxic to lymphocytes, and this toxicity is reduced by tempol. Such results could set the stage for future studies investigating the possible usefulness of antioxidants co-treatment in preventing the genotoxicity of vorinostat when used as anticancer in human.

Regulation of Rad17 Protein Turnover Unveils an Impact of Rad17-APC Cascade in Breast Carcinogenesis and Treatment*

Aberrant regulation of DNA damage checkpoint function leads to genome instability that in turn can predispose cellular tissues to become cancerous. Previous works from us and others demonstrated the role of Rad17 in either activation or termination of DNA damage checkpoint function. In the current study, we have revealed the unexpected accumulation of Rad17 in various types of breast cancer cell lines as well as human breast cancer tissues. We observed that Rad17 protein turnover rate in breast epithelial cells is much faster than in breast cancer cells, where the turnover of Rad17 is regulated by the Cdh1/APC pathway. We further observed that Rad17-mediated checkpoint function is modulated by proteolysis. Stabilization of Rad17 disrupts cellular response to chemotherapeutic drug-induced DNA damage and enhances cellular transformation. In addition, manipulation of Rad17 by RNA interference or stabilization of Rad17 significantly sensitize breast cancer cell to various chemotherapeutic drugs. Our present results indicate the manipulation of Rad17 proteolysis could be a valuable approach to sensitize breast cancer cell to the chemotherapeutic treatment despite of the critical role in governing DNA damage response and cellular recovery from genotoxic stress.

14-3-3ε Boosts Bleomycin-induced DNA Damage Response by Inhibiting the Drug-resistant Activity of MVP

Major vault protein (MVP) is the predominant constituent of the vault particle, the largest known ribonuclear protein complex. Although emerging evidence have been establishing the links between MVP (vault) and multidrug resistance (MDR), little is known regarding exactly how the MDR activity of MVP is modulated during cellular response to drug-induced DNA damage (DDR). Bleomycin (BLM), an anticancer drug, induces DNA double-stranded breaks (DSBs) and consequently triggers the cellular DDR. Due to its physiological implications in hepatocellular carcinoma (HCC) and cell fate decision, 14-3-3ε was chosen as the pathway-specific bait protein to identify the critical target(s) responsible for HCC MDR. By using an LC-MS/MS-based proteomic approach, MVP was first identified in the BLM-induced 14-3-3ε interactome formed in HCC cells. Biological characterization revealed that MVP possesses specific activity to promote the resistance to the BLM-induced DDR. On the other hand, 14-3-3ε enhances BLM-induced DDR by interacting with MVP. Mechanistic investigation further revealed that 14-3-3ε, in a phosphorylation-dependent manner, binds to the phosphorylated sites at both Thr52 and Ser864 of the monomer of MVP. Consequently, the phosphorylation-dependent binding between 14-3-3ε and MVP inhibits the drug-resistant activity of MVP for an enhanced DDR to BLM treatment. Our findings provide an insight into the mechanism underlying how the BLM-induced interaction between 14-3-3ε and MVP modulates MDR, implicating novel strategy to overcome the chemotherapeutic resistance through interfering specific protein-protein interactions.

PARP and CHK inhibitors interact to cause DNA damage and cell death in mammary carcinoma cells

The present studies examined viability and DNA damage levels in mammary carcinoma cells following PARP1 and CHK1 inhibitor drug combination exposure. PARP1 inhibitors [AZD2281 ; ABT888 ; NU1025 ; AG014699] interacted with CHK1 inhibitors [UCN-01 ; AZD7762 ; LY2603618] to kill mammary carcinoma cells. PARP1 and CHK1 inhibitors interacted to increase both single strand and double strand DNA breaks that correlated with increased γH2AX phosphorylation. Treatment of cells with CHK1 inhibitors increased the phosphorylation of CHK1 and ERK1/2. Knock down of ATM suppressed the drug-induced increases in CHK1 and ERK1/2 phosphorylation and enhanced tumor cell killing by PARP1 and CHK1 inhibitors. Expression of dominant negative MEK1 enhanced drug-induced DNA damage whereas expression of activated MEK1 suppressed both the DNA damage response and tumor cell killing. Collectively our data demonstrate that PARP1 and CHK1 inhibitors interact to kill mammary carcinoma cells and that increased DNA damage is a surrogate marker for the response of cells to this drug combination.

Abstract 4063: Histone methyltransferase G9a promotes the oral cancer cells recovery from drug-induced DNA damage.

Abstract Oral cancer is one of the serious life-threatening diseases in the world. In addition to surgery, the conventional treatment for oral cancer includes radiation and anti-cancer drugs, which usually act by induction of DNA damage. However, approximately one-third of treated patients will experience local or regional recurrence. G9a is a histone methyltransferase responsible for mono- or dimethylation of lysine 9 in histone H3. Our recent work has demonstrated that G9a level is highly correlated with aggressiveness and poor prognosis of lung, ovarian and colon cancer patients in Taiwanese population. Here, we report that knockdown of G9a resulted in decrease cell survival and cologenic capacity following DNA damage induced independently by etoposide and ionizing radiation. Depletion of G9a caused sustained phosphorylation of γ-H2AX after etoposide treatment. Together, our data suggest that a novel function of G9a may increase the DNA repair efficiency and promote the oral cancer cells recovery and survival from the DNA damage agents. We suggest that G9a may contribute to oral cancer proliferation by improvement of homologous recombination repair of DNA double break. Our study paves the way for exploring the blockade of G9a overexpression as a novel approach for the prevention and treatment of oral cancer. Citation Format: Chia-Wen Liu, Shih-Ting Cha, Ching-Ting Tan, Min-Liang Kuo. Histone methyltransferase G9a promotes the oral cancer cells recovery from drug-induced DNA damage. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4063. doi:10.1158/1538-7445.AM2013-4063

CDK4 inhibition restores G₁-S arrest in MYCN-amplified neuroblastoma cells in the context of doxorubicin-induced DNA damage

Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the G₁-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19INK4D overexpression partly restored G₁-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19INK4D, but not p16INK4A, in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G₁-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G₁ checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G₁-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.

(PARP)-1 N-Terminal Fragment Down Regulates Endogenous PARP-1 Expression and Activity and Sensitises Cells to Oxidative Stress

Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays a vital role in DNA repair. This makes it an attractive anticancer therapeutic target. It is activated by DNA breaks and catalyses the synthesis of homopolymers of ADP-ribose from NAD+. Competitive inhibitors as well as the non-catalytic, DNA-binding domain of PARP-1 abolish its polymer synthesising function. Such inhibitors of PARP-1 have been used in clinical trials and are known to cause nausea, fatigue and haematological events and with chronic use the risk of drug-induced DNA damage and tumorigenesis. In this study, I have investigated the effect of PARP-1-N-terminal fragment on endogenous PARP-1 activity and expression in mammalian cells. To elicit DNA damage response, different concentrations of H2O2 were used. For visualisation of the effects by live imaging, 750 bp PARP-1-N-terminal fragment was tagged to EGFPN1 vector. My data shows an inverse correlation between the expression of this fragment and poly (ADP-ribose) synthesis at lower concentrations of H2O2 and induction of apoptosis at higher concentrations. My experimental evidence also supports regulation of endogenous PARP-1 expression by the fragment in the absence of DNA damage. This construct has allowed for the visualisation of its functional ability to sensitise cells to oxidative damage and induce apoptosis as observed by the formation of apoptotic precursors and caspase cleavage products in live cells. The data also provides direct evidence for its therapeutic potential in chemotherapy or radiotherapy to avert necrosis induced inflammatory response.

RhoJ Regulates Melanoma Chemoresistance by Suppressing Pathways That Sense DNA Damage

Melanomas resist conventional chemotherapeutics, in part, through intrinsic disrespect of apoptotic checkpoint activation. In this study, using an unbiased genome-wide RNA interference screen, we identified RhoJ and its effector PAK1, as key modulators of melanoma cell sensitivity to DNA damage. We find that RhoJ activates PAK1 in response to drug-induced DNA damage, which then uncouples ATR from its downstream effectors, ultimately resulting in a blunted DNA damage response (DDR). In addition, ATR suppression leads to the decreased phosphorylation of ATF2 and consequent increased expression of the melanocyte survival gene Sox10 resulting in a higher DDR threshold required to engage melanoma cell death. In the setting of normal melanocyte behavior, this regulatory relationship may facilitate appropriate epidermal melanization in response to UV-induced DNA damage. However, pathologic pathway activation during oncogenic transformation produces a tumor that is intrinsically resistant to chemotherapy and has the propensity to accumulate additional mutations. These findings identify DNA damage agents and pharmacologic inhibitors of RhoJ/PAK1 as novel synergistic agents that can be used to treat melanomas that are resistant to conventional chemotherapies.

Development and validation of a PCR‐based assay for the selection of patients more likely to benefit from therapeutic treatment with alkylating drugs

Previous studies have indicated that the levels of DNA damage induced in peripheral blood mononuclear cells by the alkylating drugs melphalan, cisplatin and carboplatin can serve as useful biomarkers predictive of the therapeutic response of cancer patients to these drugs. In the present study we developed a quantitative PCR-based assay, for the measurement of DNA damage. The advantages of this methodology are based on: its far greater sensitivity (about 250 times) than the traditional Southern blot-based method (the detection limit is ~10-20 lesions/10(6) nucleotides from the equivalent DNA of ~8000 cells); its simplicity and speed (results obtained within ~8h); its excellent reproducibility, with a coefficient of variance of 10-15% for different DNA preparations from similarly treated cells; its requirement for only minute amounts of material, and; the avoidance of radioisotope labeling. Moreover, emphasis was given to translate basic research findings into clinical practice through the validation of this assay for prediction of clinical outcome in multiple myeloma patients. In order to develop and validate a simple, sensitive and rapid method for the quantitation of alkylating drug-induced DNA damage. HepG2 cells and blood samples were treated with alkylating drugs (melphalan, cisplatin, carboplatin). Gene-specific damage was examined using Southern blot and a multiplex long quantitative PCR (QPCR) carried out in a 7 kb fragment (part of the p53 gene) and a 0.5 kb fragment (part of the IFN-β1 sequence; internal standard). The extent of PCR amplification of a p53 fragment was inversely proportional to the treatment concentrations of all anticancer drugs examined, indicating a dose-related inhibition by the DNA adducts formed. Parallel analysis of the same samples using both Southern blot and QPCR showed that the DNA adducts measured by QPCR corresponded to the interstrand cross-links in the case of melphalan, and to total drug-induced lesions in the case of the platinum drugs. The detection limit was ~10-20 lesions/10(6) nucleotides using DNA from ~8000 cells. The method is about 250 times more sensitive than the Southern blot-based method and the reproducibility is excellent, with an intraday coefficient of variance (CV) of 5-9% and an interday CV of 4-12%. Application of the QPCR assay to ex vivo melphalan-treated peripheral blood mononuclear cells from multiple myeloma patients, showed that the positive predictive value of this assay for clinical response to melphalan therapy was 92.9%. The PCR-based assay developed in this study can be used for the selection of cancer patients more likely to benefit from therapeutic treatment with alkylating drugs.