Intrastrand DNA Cross-links Research Articles

Can the response to a platinum-based therapy be predicted by the DNA repair status in non-small cell lung cancer?

Preclinical evidence has been accumulating on the impact of the DNA repair status on the sensitivity/resistance to anticancer agents in different tumor types, including lung cancer. The possibility to predict the response to therapy, and specifically to platinum agents, based on tumor specific DNA repair functionality would enable to tailor its use only in those patients with maximum chances to respond, avoiding the burden of toxicity in those ones with lesser chances. We here reviewed the clinical evidence on the prognostic role of DNA repair markers and/or functional assays in predicting the response to a platinum-based chemotherapy in lung cancer patients. Consequently, we focused on those proteins involved in pathways repairing platinum induced DNA inter-strand and intra-strand crosslinks. Most promising clinical trials targeting the nucleotide repair protein ERCC1 in non-small cell lung cancer later on suffered from serious drawbacks. Nevertheless, these results spurred a variety of preclinical studies on a multitude of alternative DNA repair markers. However so far, no one of the analyzed DNA repair markers can be considered a reliable and mature biomarker for selecting patients. We discuss the reasons for such failure which discloses novel strategies for the future.

DNA-crosslinker cisplatin eradicates bacterial persister cells.

For all bacteria, nearly every antimicrobial fails since a subpopulation of the bacteria enter a dormant state known as persistence, in which the antimicrobials are rendered ineffective due to the lack of metabolism. This tolerance to antibiotics makes microbial infections the leading cause of death worldwide and makes treating chronic infections, including those of wounds problematic. Here, we show that the FDA-approved anti-cancer drug cisplatin [cis-diamminodichloroplatinum(II)], which mainly forms intra-strand DNA crosslinks, eradicates Escherichia coli K-12 persister cells through a growth-independent mechanism. Additionally, cisplatin is more effective at killing Pseudomonas aeruginosa persister cells than mitomycin C, which forms inter-strand DNA crosslinks, and cisplatin eradicates the persister cells of several pathogens including enterohemorrhagic E. coli, Staphylococcus aureus, and P. aeruginosa. Cisplatin was also highly effective against clinical isolates of S. aureus and P. aeruginosa. Therefore, cisplatin has broad spectrum activity against persister cells. Biotechnol. Bioeng. 2016;113: 1984-1992. © 2016 Wiley Periodicals, Inc.

Abstract A01: A comparison of the mechanisms and cytotoxic activity of dianhydrogalactitol (VAL-083) to cisplatin in ovarian tumor models harboring wild-type and mutant p53.

Abstract Ovarian cancers are usually treated with platinum-based therapies, which produce a 70% response rate. However, many patients relapse as tumors become resistant to cisplatin and carboplatin, resulting in a 5-year survival rate of about 20%. Onset of drug resistance is a major factor limiting the clinical utility of platinum-based therapeutic regimens drugs and, therefore, new agents are needed to circumvent resistance. Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent, whose cytotoxic activity is due to the formation of DNA cross links at the N7 position of guanine. Unlike cisplatin and carboplatin, which predominantly form intrastrand DNA cross-links, VAL-083 derives its anti-cancer activity interstrand DNA cross-links. More importantly, VAL-083 has demonstrated clinical activity against a range of tumor types, including ovarian cancer in historical NCI-sponsored clinical studies. Platinum drug resistance is normally ascribed to several mechanisms, with mutation in wild-type p53 playing a critical role, particularly in high-grade ovarian serous carcinoma (HGOSC), where the incidence of this mutation can be substantial. However, recent analysis of the TCGA database indicates that the survival rate in wild-type p53 HGOSC is no better or perhaps even worse. Using a 5-day MTT assay and fitting a sigmoidal curve to the dose-response data to determine IC50 values, we have similarly found that resistance to cisplatin in a panel of ovarian tumor models is greater when p53 is wild-type (median IC50: 4-7 µM vs. 1-2 µM for mutant/null p53 models; in comparison, sensitive wild-type p53 A2780 cells have an IC50 of 0.2-0.3 μM). Previous studies in our lab suggest that factors downstream from p53, including MDM4 and p21, may contribute to cisplatin-resistance in ovarian cancer models with high cisplatin-resistance and wild-type 53, like 2780CP-16. We thus sought to investigate the potential of VAL-083 to circumvent cisplatin-resistance in five p53 wild-type ovarian cancer models: one cisplatin-sensitive A2780, and four cisplatin-resistant 2780CP-16, OVCAR-10, Hey and OVCA-433. IC50 values of VAL-083 were generated using the MTT assay and sigmoidal curve fitting of data, as described above. The baseline IC50 for VAL-083 against A2780 cell was about 0.5 μM. The IC50 for VAL-083 in the cisplatin-resistant cell-lines 2780CP-16, OVCAR-10, Hey and OVCA-433 were 4- to 7-fold greater; however, VAL-083 was substantially more potent in comparison to cisplatin in these models where corresponding IC50 values were 10- to over 25-fold greater. These results demonstrate that there is only partial cross-resistance between cisplatin and VAL-083 further suggesting distinct modes of action for the two drugs. In order to examine whether the partial circumvention of cisplatin-resistance was p53-dependent, cytotoxicity was determined in isogenic HCT-116p53-/- and HCT-116p53+/+ models. These studies demonstrated that loss of p53 increased resistance to cisplatin by 2-5-fold whereas loss of p53 only increased resistance to VAL-083 about 1.7-fold. These results suggest that VAL-083 is less dependent on p53 for its cytotoxic activity. Immunoblots confirmed this in 2780CP-16 cells, where VAL-083 was more effective than cisplatin at increasing p53 and p21 levels, and induced relatively greater Ser-15 and Ser-20 phosphorylations, further supporting different modes of action for the two drugs. In contrast, both drugs were equally effective in inducing these markers of DNA damage in A2780 cells. The non-overlapping mechanisms of action suggested a potential therapeutic benefit for combinations of VAL-083 with cisplatin. We have previously reported that the combination of VAL-083 with cisplatin in wild-type p53 NSCLC models H460 and A549, and mutant p53 NSCLC H1975 demonstrated significant super-additivity (p<0.05) and synergy (CI < 1) in all three cell-lines. Taken together these results demonstrate the effectiveness of VAL-083 against refractory cisplatin-resistant ovarian cancers and raise the potential for treatment of platinum-resistant ovarian cancers or a combination regimen with cisplatin. (Supported in part by NCI RO1 CA160687 to ZHS). Citation Format: Jeffrey A. Bacha, Michelle Martinez-Rivera, Guanghan He, Xiaolei Xie, Anne Steino, Sarath Kanekal, Dennis M. Brown, Zahid H. Siddik. A comparison of the mechanisms and cytotoxic activity of dianhydrogalactitol (VAL-083) to cisplatin in ovarian tumor models harboring wild-type and mutant p53. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A01.

Nucleotide excision repair (NER) alterations as evolving biomarkers and therapeutic targets in epithelial cancers.

The nucleotide excision repair (NER) pathway is a highly conserved and remarkably versatile DNA repair pathway that functions to identify and repair bulky intrastrand DNA crosslinks generated by a variety of genotoxic agents including platinum drugs. Platinum analogs are active anticancer agents and constitute the backbone of first-line chemotherapy used in a number of epithelial malignancies. Given the role of the NER pathway in repair of platinum-induced DNA damage, a number of studies have recently investigated the prevalence and of NER pathway alterations in various epithelial tumors as well as their association with response to platinum and other chemotherapy agents. In epithelial ovarian cancer (EOC), analysis of the TCGA data set revealed that nearly half of all EOCs harbor an alteration in the homologous recombination (HR) pathway, which includes BRCA1 and BRCA2 (BRCA1/2). In addition, 8% of high-grade serous EOCs have an alteration (non-synonymous mutation, splice site mutation, promoter hypermethylation, or homozygous deletion) of at least one gene in the NER pathway, and half of these NER alterations (4% of tumors overall) occurred in the absence of a known HR alteration. [1] Patients with tumors harboring NER alterations had improved overall and progression-free survival compared to tumors without NER or BRCA1/2 alterations. Given that nearly all EOC patients receive platinum-based chemotherapy, and because the durability of platinum response is closely associated with survival in these patients, the improved survival of patients with NER-altered tumors likely reflects increased platinum sensitivity in this population. Moreover, survival of patients with NER-altered tumors was similar to patients with tumors harboring BRCA1/2 alterations, suggesting that NER pathway alterations may contribute to EOC platinum sensitivity to an extent similar to the effect of BRCA1/2 loss. Importantly, unlike BRCA1/2 alterations, NER pathway alterations were not associated with sensitivity to poly(ADP-ribose) polymerase (PARP)-inhibitors. Functional analysis of several NER mutations identified in the TCGA cohort revealed that expression of the mutant in a NER-deficient background failed to rescue cisplatin sensitivity, whereas no difference in PARP-inhibitor sensitivity or HR activity was noted. Together, these findings identify an important subset of NER-deficient, HR-proficient EOCs with discordant platinum and PARP-inhibitor profiles, and underscore the potential role of NER pathway alterations as predictive biomarkers of response to specific anticancer therapies. More broadly, NER pathway alterations are also being identified in other epithelial tumor types and may serve as important biomarkers in diverse clinical settings where platinum agents are commonly employed. In urothelial carcinoma, we and others recently identified recurrent ERCC2 mutations, and show that ERCC2- deficient tumors have increased response rates to cisplatin- based chemotherapy regimens. [2, 3] Review of the TCGA dataset using the cBIO portal reveals presence of NER alterations in several other epithelial cancers [4] (Figure ​(Figure1),1), and additional studies may identify other clinical contexts in which NER pathway alterations could be used to inform therapy selection. Figure 1 Frequency of NER pathway gene mutations in several solid tumor types Finally, it is important to note that NER pathway alterations may also render tumors susceptible to novel anticancer therapies such as targeted immunotherapies and cell cycle checkpoint inhibitors. Given that NER- mutated tumors harbor higher mutational loads, they may possess more tumor-specific neoantigens, resulting in increased number of tumor-infiltrating lymphocytes (TILs) [5]. Several studies have shown that hypermutated tumors such as mismatch repair (MMR)-deficient or POLE-mutated tumors are associated with higher neoantigen loads and an elevated number of TILs, which is counterbalanced by overexpression of immune checkpoints such as PD-1/PD-L1 AND CTLA4 [6]. Such hypermutated tumors have been shown to particularly susceptible to immune checkpoint inhibitors targeting the PD-1/PD-L1 or CTLA4 pathways [7]. Finally, NER-deficient tumors are particularly sensitive to cell cycle checkpoint inhibition, in particular inhibitors of the ATR-CHK1-WEE1 pathway. ATR is activated by DNA single-strand–double-strand junctions that arise as intermediates in NER and triggers the intra-S phase and the G2 checkpoints via phosphorylation of CHK1 [8]. Activated Chk1 in turns phosphorylates WEE1 (resulting in its activation) and cell division cycle 25 (CDC25A and CDC25C) phosphatases (resulting in their inhibition) to inhibit cell cycle progression through the coordinated suppression of cyclin-dependent kinase activity. Several approaches to inhibit the ATR/CHK1/WEE1 pathway including ATR inhibitors (such as VX-970 and AZD6738), WEE1 inhibitors (such as AZD1775) and CHK1 inhibitors (GDC-0425 and LY2606368) are currently in early clinical trial evaluation in a number of epithelial cancers, and NER alterations may serve as a useful biomarker to define a subset of tumors that are particularly sensitive to these agents.

Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells

The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury.

The Translesion Polymerase ζ Has Roles Dependent on and Independent of the Nuclease MUS81 and the Helicase RECQ4A in DNA Damage Repair in Arabidopsis.

DNA polymerase zeta catalytic subunit REV3 is known to play an important role in the repair of DNA damage induced by cross-linking and methylating agents. Here, we demonstrate that in Arabidopsis (Arabidopsis thaliana), the basic polymerase activity of REV3 is essential for resistance protection against these different types of damaging agents. Interestingly, its processivity is mainly required for resistance to interstrand and intrastrand cross-linking agents, but not alkylating agents. To better define the role of REV3 in relation to other key factors involved in DNA repair, we perform epistasis analysis and show that REV3-mediated resistance to DNA-damaging agents is independent of the replication damage checkpoint kinase ataxia telangiectasia-mutated and rad3-related homolog. REV3 cooperates with the endonuclease MMS and UV-sensitive protein81 in response to interstrand cross links and alkylated bases, whereas it acts independently of the ATP-dependent DNA helicase RECQ4A. Taken together, our data show that four DNA intrastrand cross-link subpathways exist in Arabidopsis, defined by ATP-dependent DNA Helicase RECQ4A, MMS and UV-sensitive protein81, REV3, and the ATPase Radiation Sensitive Protein 5A.

Abstract 3696: Molecular dynamics simulations of C7/C7′-linked pyrrolobenzodiazepine (PBD) dimers

Abstract The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a group of naturally occurring and synthetic DNA minor-groove binding agents (MGBAs) characterised by their ability to covalently bind to the C2-amino groups of guanine bases. Although natural PBD monomers have low micromolar cytotoxicity, synthetic PBD dimers have garnered significant attention due to their potent cytotoxicity and use in antibody-drug conjugates (ADCs). The cytotoxic nature of PBD dimers is thought to relate to their ability to form inter- and intrastrand cross-links in DNA. The first known PBD dimer had its two PBD units linked through their C7/C7′-positions, and was reported by Suggs and co-workers. This molecule was shown to reversibly cross-link DNA, a feature that may account for its poor cytotoxicity relative to C8-linked dimers which are used as ADC payloads. To investigate this, we have undertaken Molecular Dynamics (MD) studies on the interaction of C7-dimers with DNA and compared results to those for C8-linked PBD dimers. This has led us to propose an alternative mechanism to explain the discrepancy in potencies as described below. MD simulations were conducted over 10ns in explicit solvent. In this study, Suggs’ published C7/C7′-linked dimer and an equivalent C8/C8′ linked structure were first compared, and this was followed by comparison of the known C8-linked PBD dimer SG-2202 and an equivalent C7/C7′-linked version. SG-2202 is known to preferentially bind to the sequence 5′-pu-G-A-A/T-A/T-C-py-3′. As each of the PBD dimers analysed have an equivalent central linker (i.e., five methylenes), each of the structures were simulated in the sequence 5′-GCGATTCTCGC-3′. The results suggested that C8-linked dimers interact with DNA to a far greater extent than C7-linked dimers, a feature attributable to the level of curvature (“isohelicity”) of C8-linked dimers, and this was supported by free energy of binding calculations. Crucially, the simulations showed that the C7-linked dimer family does not form stabilising non-covalent interactions with the DNA minor-groove as previously thought. Furthermore, due to its poor isohelicity, simulations predict that only a single alkylation event takes place initially, and this is followed by the second alkylation which is dependent on DNA breathing to align the second PBD unit with an appropriate guanine. Simulations suggested that the C7-position of any PBD has two potential orientations: (1) bulky groups such as a sugar (i.e., as in sibiromycin) point out of the minor groove, and (2) smaller substituents (e.g., a methylene linker) point along the minor groove floor. The latter explains why it is possible for C7-methylene-linked PBD dimers to cross-link DNA albeit with less efficiency compared to C8-linked dimers. Studies are now underway to analyse the curvature of PBD-type molecules to develop tools (e.g., QSAR) for the correlation of shape and cytotoxicity to facilitate the discovery of MGBAs with more potent cytotoxicity. Citation Format: Paul J. M. Jackson, Khondaker Miraz Rahman, David E. Thurston. Molecular dynamics simulations of C7/C7′-linked pyrrolobenzodiazepine (PBD) dimers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3696. doi:10.1158/1538-7445.AM2015-3696

Abstract 5495: Identification of markers predictive of phosphoramide mustard (PM) cellular sensitivity in lymphoblast cell lines

Abstract Cyclophosphamide (Cy) a DNA alkylating agent, is commonly used for treatment of several malignancies such as breast cancer and lymphoma. It is also widely used in conditioning regimens for bone marrow transplantation. Cy is a prodrug requiring activation to 4-hydroxycyclophosphamide by hepatic cytochrome P450 enzymes. 4-hydroxy-Cy non-enzymatically converts to active phosphoramide mustard (PM), which produces inter and intra-strand DNA crosslinks causing DNA breaks and hence the cytotoxicity. Although of great clinical utility the dose limiting side effects of Cy as myelosuppression, nephrotoxicity, cardiotoxicity, neurotoxicity and bladder toxicity posses clinical challenge. Genetic polymorphisms in P450 enzymes especially CYP2B6 as well as phase II drug metabolizing enzymes has been shown to partially explain the inter-patient variation in response in some studies however role of pharmacodynamics players is still not clear. Thus, we designed the present study to identify genes predictive of phosphoramide mustard response. We utilized EBV transformed lymphoblast cell lines (LCLs) that are part of International HapMap project as the model system and treated these with PM to by pass the CYP mediated activation of Cy. Since most of the Cy metabolizing genes (CYPs) are expressed in liver but not as much in LCLs, use of PM instead of Cy in the present study enriched our ability to identify genes of relevance to the pharmacodynamic significance. Briefly we treated LCLs with six different concentrations of PM (100, 50, 20, 10, 1, and 0.1 μM), due to stability issues PM was prepared fresh for each experiment. Cell viability was determined 48 hr post drug treatment by MTT assays. Genome-wide gene expression and genotype data was obtained from publically available source and was evaluated for association with PM cellular sensitivity. Top 100 SNPs (p< 2×10-5) mapped to 10 genes of biological significance including CREBBP, DPP10, HYAL3, XRCC1, SNAPC3, CCDC21, PSIP1, RASSF1, and SALL4 CREBBP (CREB binding protein) is a major transcriptional regulator for both estrogen dependent breast cancer signaling and Lymphotoxin b receptor signaling pathways. CREBBP has an intrinsic histone acetyltransferase activity that acts as a scaffold to stabilize additional protein interactions with the transcription complex. Gene-expression association analysis identified 34 genes significantly associated with PM cytotoxicity including EIF4E2, MMP16, MUC6, NDUFA2, NDUFA9 and SDHB. Identification of SNPs in XRCC1 further confirmed the impact of altered DNA damage repair/response pathway in compromising PM response. Our results identified genetic markers of pharmacodynamic significance to Cy response thus opening up opportunities for clinical validation of these in patients receiving Cy and moving a step closer to personalized medicine. Citation Format: Lata Chauhan, Brooke L. Fridley, Alice Wang, Jatinder Kaur Lamba. Identification of markers predictive of phosphoramide mustard (PM) cellular sensitivity in lymphoblast cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5495. doi:10.1158/1538-7445.AM2015-5495

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins.

Platinum-resistance and AKT Over-expression in Ovarian Cancer

Ovarian cancer is among the most common cause of cancer death and ranks first in the number of deaths each year in the field of gynaecological malignancies. This is due to its late diagnosis and the development of chemoresistance. Platinum derivates, including cisplatinum and carboplatinum in combination with paclitaxel, are the first-line chemotherapeutic agents. Platinum derivates intercalates irreversibly into the DNA and creates inter- and intra-strand DNA cross-links. During cell division, platinum-DNA-adducts block the replication machinery, inducing DNA damage and apoptosis. Nearly all patients respond to first line chemotherapy before it comes later to recurrence of the disease. At time of recurrence, tumours are usually more aggressive, form metastasis in secondary tissues and acquire resistance to conventional chemotherapeutics. Drug resistance is a common problem in tumour therapy not only restricted to ovarian cancer. This is characterized by gene mutations, increased DNA repair, reduced drug efficacy and enhanced drug clearance and detoxification. Up to now the complex molecular mechanism of chemoresistance is not well understood but increasing evidence points towards AKT over-expression and alteration of the PI3K/AKT/mTOR cascade as a central mechanistic reason for this resistance.

Attenuated XPC expression is not associated with impaired DNA repair in bladder cancer.

Bladder cancer has a high incidence with significant morbidity and mortality. Attenuated expression of the DNA damage response protein Xeroderma Pigmentosum complementation group C (XPC) has been described in bladder cancer. XPC plays an essential role as the main initiator and damage-detector in global genome nucleotide excision repair (NER) of UV-induced lesions, bulky DNA adducts and intrastrand crosslinks, such as those made by the chemotherapeutic agent Cisplatin. Hence, XPC protein might be an informative biomarker to guide personalized therapy strategies in a subset of bladder cancer cases. Therefore, we measured the XPC protein expression level and functional NER activity of 36 bladder tumors in a standardized manner. We optimized conditions for dissociation and in vitro culture of primary bladder cancer cells and confirmed attenuated XPC expression in approximately 40% of the tumors. However, NER activity was similar to co-cultured wild type cells in all but one of 36 bladder tumors. We conclude, that (i) functional NER deficiency is a relatively rare phenomenon in bladder cancer and (ii) XPC protein levels are not useful as biomarker for NER activity in these tumors.

DNA Damage Initiated by the Sulfur Mustard Analog Mechlorethamine (methylbis(2‐chloroethyl)amine) in Mouse Keratinocytes is Associated with an Oxidative Stress Response

Sulfur mustard and nitrogen mustard (HN2, mechlorethamine) are bifunctional alkylating agent that cause dermal inflammation, edema and blistering. Skin keratinocytes are known to be important targets for mustards, which form both monofunctional DNA adducts and inter- and intrastrand DNA cross-links in the cells leading to cytotoxicity. In these studies, we used PAM212 mouse keratinocytes to analyze mechanisms underlying HN2 toxicity. Treatment of PAM212 cells with HN2 (3-30 μM) caused cytostasis; this was associated with a cell cycle block in late S phase and G2M phase. HN2 also caused DNA double strand breaks as shown by increased expression of phospho-H2A.X, a H2A histone variant that binds to double stranded DNA breaks creating a repair recognition domain. HN2-induced phosphorylation of H2A.X was both time- and concentration-dependent. Maximal increases were evident in cells at 24 h post HN2. HN2 induced double strand breaks were correlated with increases in the cellular antioxidants heme oxygenase-1 (HO-1) and heat shock protein27 (hsp27), which peaked 3 h and 6 h post HN2-treatment. These findings are consistent with the idea that HN2 initiates a DNA damage response in mouse keratinocytes that leads to induction of an adaptive stress response. Interfering with DNA strand breaks may be an effective strategy to mitigate mustard-induced damage in keratinocytes. Support: AR055073 and ES005022

Curcumin reverses cisplatin resistance in cisplatin-resistant lung caner cells by inhibiting FA/BRCA pathway.

Cisplatin (DDP) is the most widely used chemotherapy agent for treatment of malignancies including lung cancer. However, the effectiveness of DDP is often weakened by acquired resistance of tumor cells. DDP kills cancer cells primarily by creating intrastrand and interstrand DNA cross-links, which block DNA replication. The Fanconi anemia (FA)/BRCA pathway is a DNA cross-link damage repair pathway, which regulates cellular resistance to DNA cross-link agents, such as DDP. Some study has shown that natural compound curcumin sensitize human ovarian and breast cancer cells to DDP. However, whether curcumin may reverse resistance to DDP in DDP-resistant lung cancer cells has not been understood. In this study, we showed that curcumin enhanced the proliferation inhibitory effect of DDP and promote DDP-induced apoptosis in A549/DDP cells (DDP-resistant lung adenocarcinoma cells). Moreover, we observed that FA/BRCA pathway DNA damage repair processes, such as DDP-induced FANCD2 monoubiquitination and nuclear foci formation were downregulated in the presence of curcumin, suggesting that curcumin enhanced sensitivity to DDP in A549/DDP cells through the inhibition of FA/BRCA pathway. Furthermore, the calculation of q value and apoptosis analyses revealed that curcumin in combination with DDP could exert a synergistic cytotoxic effect in A549/DDP cells, further demonstrating that curcumin can reverse cisplatin resistance of A549/DDP cells. In conclusion, by suppressing the FA/BRCA pathway DNA repair, curcumin potentiates DDP-induced proliferation inhibitory effect and apoptosis in A549/DDP cell, indicating that curcumin may serve as a chemosensitizer to cross-link-inducing anticancer drugs DDP.

Crosstalk between translesion synthesis, Fanconi anemia network, and homologous recombination repair pathways in interstrand DNA crosslink repair and development of chemoresistance.

Bifunctional alkylating and platinum based drugs are chemotherapeutic agents used to treat cancer. These agents induce DNA adducts via formation of intrastrand or interstrand (ICL) DNA crosslinks, and DNA lesions of the ICL type are particularly toxic as they block DNA replication and/or DNA transcription. However, the therapeutic efficacies of these drugs are frequently limited due to the cancer cell's enhanced ability to repair and tolerate these toxic DNA lesions. This ability to tolerate and survive the DNA damage is accomplished by a set of specialized low fidelity DNA polymerases called translesion synthesis (TLS) polymerases since high fidelity DNA polymerases are unable to replicate the damaged DNA template. TLS is a crucial initial step in ICL repair as it synthesizes DNA across the lesion thus preparing the damaged DNA template for repair by the homologous recombination (HR) pathway and Fanconi anemia (FA) network, processes critical for ICL repair. Here we review the molecular features and functional roles of TLS polymerases, discuss the collaborative interactions and cross-regulation of the TLS DNA damage tolerance pathway, the FA network and the BRCA-dependent HRR pathway, and the impact of TLS hyperactivation on development of chemoresistance. Finally, since TLS hyperactivation results from overexpression of Rad6/Rad18 ubiquitinating enzymes (fundamental components of the TLS pathway), increased PCNA ubiquitination, and/or increased recruitment of TLS polymerases, the potential benefits of selectively targeting critical components of the TLS pathway for enhancing anti-cancer therapeutic efficacy and curtailing chemotherapy-induced mutagenesis are also discussed.

The stability of DNA intrastrand cross-links of antitumor transplatin derivative containing non-bulky methylamine ligands.

Oligonucleotides modified by clinically ineffective trans-diamminedichloridoplatinum(II) (transplatin) have been shown to be effective modulators of gene expression. This is so because in some nucleotide sequences the 1,3-GNG intrastrand adducts formed by transplatin in double-helical DNA readily rearrange into interstrand cross-links so that they can cross-link the oligonucleotides to their targets. On the other hand, in a number of other sequences these intrastrand adducts are relatively stable, which represents the major difficulty in the clinical use of the antisense transplatin-modified oligonucleotides. Therefore, we examined in this study, the stability of 1,3-GNG intrastrand adducts in double-helical DNA formed by a new antitumor derivative of transplatin, trans-[Pt(CH3NH2)2Cl2], in the sequence contexts in which transplatin formed relatively stable intrastrand cross-links which did not readily rearranged into interstrand cross-links. We have found that 1,3-GNG intrastrand adducts in double-helical DNA formed by trans-[Pt(CH3NH2)2Cl2] even in such sequences readily rearrange into interstrand cross-links. This work also suggests that an enhanced frequency of intrastrand cross-links yielded by trans-[Pt(CH3NH2)2Cl2] is a consequence of the fact that these DNA lesions considerably distort double-helical DNA in far more sequence contexts than parent transplatin. Our results suggest that trans-[Pt(CH3NH2)2Cl2]-modified oligonucleotides represent promising candidates for new agents in antisense or antigene approach.

Are dinucleoside monophosphates relevant models for the study of DNA intrastrand cross-link lesions? The example of g[8-5m]T.

Oxidatively generated tandem lesions such as G[8-5m]T pose a potent threat to genome integrity. Direct experimental studies of the kinetics and thermodynamics of a specific lesion within DNA are very challenging, mostly due to the variety of products that can be formed in oxidative conditions. Dinucleoside monophosphates (DM) involving only the reactive nucleobases in water represent appealing alternative models on which most physical chemistry and structural techniques can be applied. However, it is not yet clear how relevant these models are. Here, we present QM/MM MD simulations of the cyclization step involved in the formation of G[8-5m]T from the guanine-thymine (GpT) DM in water, with the aim of comparing our results to our previous investigation of the same reaction in DNA ( Garrec , J. , Patel , C. , Rothlisberger , U. , and Dumont , E. ( 2012 ) J. Am. Chem. Soc. 134 , 2111 - 2119 ). We show that, despite the different levels of preorganization of the two systems, the corresponding reactions share many energetic and structural characteristics. The main difference lies in the angle between the G and T bases, which is slightly higher in the transition state (TS) and product of the reaction in water than in the reaction in DNA. This effect is due to the Watson-Crick H-bonds, which are absent in the {GpT+water} system and restrain the relative positioning of the reactive nucleobases in DNA. However, since the lesion is accommodated easily in the DNA macromolecule, the induced energetic penalty is relatively small. The high similarity between the two reactions strongly supports the use of GpT in water as a model of the corresponding reaction in DNA.

Frequencies of mutagenic translesion DNA synthesis over cisplatin-guanine intra-strand crosslinks in lacZ plasmids propagated in human cells

Cisplatin (cis-diamminedichloroplatinum(II)), a widely used anticancer drug, forms inter- and intra-strand DNA crosslinks. The major intra-strand crosslinks are Pt adducts at 1,2-d(GpG) and 1,3-d(GpNpG) (Pt-GG and Pt-GNG, respectively). Although most of the intra-strand crosslinks are removed by the nucleotide excision repair (NER), the remaining crosslinks can cause mutations through the translesion DNA synthesis (TLS) during chromosome replication. To understand the precise mechanism of cisplatin mutagenesis in human cells, the plasmid carrying a single Pt-GG or 1,3-d(GpTpG) crosslink (Pt-GTG) site-specifically in lacZ gene was constructed and propagated in NER-defective xeroderma pigmentosum cells. The plasmids retrieved from the cells were introduced into indicator bacterial cells to access frequencies of TLS and mutations. The experiments revealed that Pt-GTG blocked DNA replication more strongly and caused more mutations (29.1%) than Pt-GG (1.7%). Most mutations were G to A or T base changes at 5′ G residue in the Pt-GTG crosslinks. These results indicate that the Pt-GTG crosslinks become effective obstacles for cancer cell division, and have an important role for cisplatin cancer therapy.

The Expression of Ribonucleotide Reductase M2 in the Carcinogenesis of Uterine Cervix and Its Relationship with Clinicopathological Characteristics and Prognosis of Cancer Patients

BackgroundTo investigate the implication of ribonucleotide reductase M2 (RRM2) in the carcinogenesis of uterine cervix and its relationship with clinicopathological characteristics and prognosis of cancer patients.Methodology and Principal FindingsThe impact of RRM2 on cell viability was investigated in SiHa cervical cancer cells after RRM2 knockdown and the addition of cisplatin, which induces inter- and intra-strand DNA crosslinks. RRM2 immunoreactivity was evaluated by semi-quantitative H score among 29 normal, 30 low-grade dysplasia, 30 high-grade dysplasia and 103 invasive cancer tissue specimens of the uterine cervix, using tissue microarrays. RRM2 was then correlated with the clinicopathological variables of cervical cancer and patient survival. A greater toxic effect on cell viability using cisplatin was reflected by the greater reduction in RRM2 protein expression in SiHa cells. The RRM2 expression in cancer tissues was higher than that in high-grade dysplasia, low-grade dysplasia or normal cervical tissues. RRM2 upregulation was correlated with deep stromal invasion, large tumors and parametrial invasion and predicted poor survival.ConclusionsRRM2 is a new molecular marker for the diagnosis and clinical outcomes of cervical cancer. It is involved in cervical carcinogenesis and predicts poor survival, and may be a potential therapeutic target including in cisplatin treatment.

ERCC1 function in nuclear excision and interstrand crosslink repair pathways is mediated exclusively by the ERCC1-202 isoform

ERCC1 (excision repair cross-complementation group 1) plays essential roles in the removal of DNA intrastrand crosslinks by nucleotide excision repair, and that of DNA interstrand crosslinks by the Fanconi anemia (FA) pathway and homology-directed repair processes (HDR). The function of ERCC1 thus impacts on the DNA damage response (DDR), particularly in anticancer therapy when DNA damaging agents are employed. ERCC1 expression has been proposed as a predictive biomarker of the response to platinum-based therapy. However, the assessment of ERCC1 expression in clinical samples is complicated by the existence of 4 functionally distinct protein isoforms, which differently impact on DDR. Here, we explored the functional competence of each ERCC1 protein isoform and obtained evidence that the 202 isoform is the sole one endowed with ERCC1 activity in DNA repair pathways. The ERCC1 isoform 202 interacts with RPA, XPA, and XPF, and XPF stability requires expression of the ERCC1 202 isoform (but none of the 3 others). ERCC1-deficient non-small cell lung cancer cells show abnormal mitosis, a phenotype reminiscent of the FA phenotype that can be rescued by isoform 202 only. Finally, we could not observe any dominant-negative interaction between ERCC1 isoforms. These data suggest that the selective assessment of the ERCC1 isoform 202 in clinical samples should accurately reflect the DDR-related activity of the gene and hence constitute a useful biomarker for customizing anticancer therapies.

ChemInform Abstract: Mitomycinoid Alkaloids: Mechanism of Action, Biosynthesis, Total Syntheses, and Synthetic Approaches

AbstractReview: 379 refs.