Keisha Milum

Modulation of Nucleotide Excision Repair by Mammalian SWI/SNF Chromatin-remodeling Complex

Accessibility within chromatin is an important factor in the prompt removal of UV-induced DNA damage by nucleotide excision repair (NER). Chromatin remodeling by the SWI/SNF complex has been shown to play an important modulating role in NER in vitro and yeast in vivo. Nevertheless, the molecular basis of cross-talk between SWI/SNF and NER in mammalian cells is not fully understood. Here, we show that knockdown of Brg1, the ATPase subunit of SWI/SNF, negatively affects the elimination of cyclobutane pyrimidine dimers (CPD), but not of pyrimidine (6, 4)pyrimidone photoproducts (6-4PP) following UV irradiation of mammalian cells. Brg1-deficient cells exhibit a lower chromatin relaxation as well as impaired recruitment of downstream NER factors, XPG and PCNA, to UV lesions. However, the assembly of upstream NER factors, DDB2 and XPC, at the damage site was unaffected by Brg1 knockdown. Interestingly, Brg1 interacts with XPC within chromatin and is recruited to UV-damaged sites in a DDB2- and XPC-dependent manner. Also, postirradiation decrease of XPC levels occurred more rapidly in Brg1-deficient than normal cells. Conversely, XPC transcription remained unaltered upon Brg1 knockdown indicating that Brg1 affects the stability of XPC protein following irradiation. Thus, Brg1 facilitates different stages of NER by initially modulating UV-induced chromatin relaxation and stabilizing XPC at the damage sites, and subsequently stimulating the recruitment of XPG and PCNA to successfully culminate the repair.

NER initiation factors, DDB2 and XPC, regulate UV radiation response by recruiting ATR and ATM kinases to DNA damage sites

ATR and ATM kinases are central to the checkpoint activation in response to DNA damage and replication stress. However, the nature of the signal, which initially activates these kinases in response to UV damage, is unclear. Here, we have shown that DDB2 and XPC, two early UV damage recognition factors, are required for the damage-specific ATR and ATM recruitment and phosphorylation. ATR and ATM physically interacted with XPC and promptly localized to the UV damage sites. ATR and ATM recruitment and their phosphorylation were negatively affected in cells defective in DDB2 or XPC functions. Consequently, the phosphorylation of ATR and ATM substrates, Chk1, Chk2, H2AX, and BRCA1 was significantly reduced or abrogated in mutant cells. Furthermore, UV exposure of cells defective in DDB2 or XPC resulted in a marked decrease in BRCA1 and Rad51 recruitment to the damage site. Conversely, ATR- and ATM-deficiency failed to affect the recruitment of DDB2 and XPC to the damage site, and therefore did not influence the NER efficiency. These findings demonstrate a novel function of DDB2 and XPC in maintaining a vital cross-talk with checkpoint proteins, and thereby coordinating subsequent repair and checkpoint activation.

Differential contributory roles of nucleotide excision and homologous recombination repair for enhancing cisplatin sensitivity in human ovarian cancer cells

BackgroundWhile platinum-based chemotherapeutic agents are widely used to treat various solid tumors, the acquired platinum resistance is a major impediment in their successful treatment. Since enhanced DNA repair capacity is a major factor in conferring cisplatin resistance, targeting of DNA repair pathways is an effective stratagem for overcoming cisplatin resistance. This study was designed to delineate the role of nucleotide excision repair (NER), the principal mechanism for the removal of cisplatin-induced DNA intrastrand crosslinks, in cisplatin resistance and reveal the impact of DNA repair interference on cisplatin sensitivity in human ovarian cancer cells.ResultsWe assessed the inherent NER efficiency of multiple matched pairs of cisplatin-sensitive and -resistant ovarian cancer cell lines and their expression of NER-related factors at mRNA and protein levels. Our results showed that only the cisplatin-resistant ovarian cancer cell line PEO4 possessed an increased NER capacity compared to its inherently NER-inefficient parental line PEO1. Several other cisplatin-resistant cell lines, including CP70, CDDP and 2008C13, exhibited a normal and parental cell-comparable NER capacity for removing cisplatin-induced DNA intrastrand cross-links (Pt-GG). Concomitant gene expression analysis revealed discordance in mRNA and protein levels of NER factors in various ovarian cancer cell lines and NER proteins level were unrelated to the cisplatin sensitivity of these cell lines. Although knockdown of NER factors was able to compromise the NER efficiency, it only caused a minimal effect on cisplatin sensitivity. On the contrary, downregulation of BRCA2, a critical protein for homologous recombination repair (HRR), significantly enhanced the efficacy of cisplatin in killing ovarian cancer cell line PEO4.ConclusionOur studies indicate that the level of NER factors in ovarian cancer cell lines is neither a determinant of their NER capacity nor of the sensitivity to cisplatin, and suggest that manipulation of the HRR but not the NER factor expression provides an effective strategy for sensitizing cisplatin-resistant tumors to platinating agents.

Stem cell protein Piwil2 modulates chromatin modifications upon cisplatin treatment

Piwil2 (mili in mouse or hili in humans), a member of the PIWI/Argonaute gene family, plays important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. Recent demonstration of stable Piwil2 expression in pre-cancerous stem cells and in various human and animal tumor cell lines suggests its association in tumorigenesis. Here, we show that cisplatin induces chromatin relaxation in Mili-wild type (WT) mouse embryonic fibroblasts (MEFs), but not in Mili-knockout (KO) MEFs. Moreover, in contrast to Mili-WT MEFs, Mili-KO MEFs showed a discernable H3 hypoacetylation response upon cisplatin treatment. Levels of the histone acetyltransferase (HAT), p300, were dramatically different due to a consistent cisplatin post-treatment decrease in Mili-WT and an increase in Mili-KO MEFs. Concomitant reduction of specific HAT activity of p300 could explain the decrease of H3 acetylation in Mili-KO MEFs. Our data also shows Mili is required for maintaining the euchromatic marks in MEFs upon cisplatin treatment. In addition, Mili-KO MEFs exhibited a significant deficiency in repairing cisplatin-induced DNA damage and displayed higher sensitivity to cisplatin. Further analysis revealed that Piwil2 was also enhanced in two completely different cisplatin-resistant ovarian cancer cell lines. Interestingly, knockdown of Piwil2 expression in these two cell lines also resulted in their enhanced sensitivity to cisplatin and decreased their efficiency for removing cisplatin-induced DNA intrastrand crosslinks (Pt-GG). The overall data showed that Piwil2 is a key factor in regulating chromatin modifications especially in response to cisplatin. To conclude, the overexpression of Piwil2 in some cancers could lead to cellular cisplatin resistance, possibly due to enhanced chromatin condensation affecting normal DNA repair.

Overexpression of DDB2 enhances the sensitivity of human ovarian cancer cells to cisplatin by augmenting cellular apoptosis.

Cisplatin is one of the most widely used anticancer agents, displaying activity against a wide variety of tumors. However, development of drug resistance presents a challenging barrier to successful cancer treatment by cisplatin. To understand the mechanism of cisplatin resistance, we investigated the role of damaged DNA binding protein complex subunit 2 (DDB2) in cisplatin‐induced cytotoxicity and apoptosis. We show that DDB2 is not required for the repair of cisplatin‐induced DNA damage, but can be induced by cisplatin treatment. DDB2‐deficient noncancer cells exhibit enhanced resistance to cell growth inhibition and apoptosis induced by cisplatin than cells with fully restored DDB2 function. Moreover, DDB2 expression in cisplatin‐resistant ovarian cancer cell line CP70 and MCP2 was lower than their cisplatin‐sensitive parental A2780 cells. Overexpression of DDB2 sensitized CP70 cells to cisplatin‐induced cytotoxicity and apoptosis via activation of the caspase pathway and downregulation of antiapoptotic Bcl‐2 protein. Further analysis indicates that the overexpression of DDB2 in CP70 cells downregulates Bcl‐2 expression through decreasing Bcl‐2 mRNA level. These results suggest that ovarian cancer cells containing high level of DDB2 become susceptible to cisplatin by undergoing enhanced apoptosis.

A High-Affinity Near-Infrared Fluorescent Probe to Target Bombesin Receptors

PurposeThis study aimed to create new optical surgical navigation NIRF probes for prostate and breast cancers.ProceduresIR800-linker-QWAVGHLM-NH2 with linker = GSG, GGG, and G-Abz4 were synthesized and characterized. IC50 for bombesin receptors (BBN-R) in PC-3 prostate and T47D breast cancer cells, fluorescence microscopy in PC-3 cells, and NIRF imaging in mice PC-3 tumor xenografts were studied.ResultsGGG, GSG, and G-Abz4 derivatives had IC50 (nM) for BBN-R+ PC-3 cells = 187 ± 31, 56 ± 5, and 2.6 ± 0.2 and T47D cells = 383 ± 1, 57.4 ± 1.2, and 3.1 ± 1.1, respectively. By microscopy the Abz4 derivative showed the highest uptake, was competed with by BBN, and had little to no binding to BBN-R− cells. In NIRF imaging the G-Abz4 probe was brighter than GGG probe in BBN-R+ tissues in vivo and tissues, tumors, and tumor slices ex vivo. Uptake could be partially blocked in BBN-R+ pancreas but not visibly in tumor.ConclusionsLinker choice can dominate peptidic BBN-R binding. The G-Abz4 linker yields a higher affinity and specific BBN-R binder in this series of molecules.

Localization of CaSR antagonists in CaSR-expressing medullary thyroid cancer.

OBJECTIVE Image-based localization of medullary thyroid cancer (MTC) and parathyroid glands would improve the surgical outcomes of these diseases. MTC and parathyroid glands express high levels of calcium-sensing receptor (CaSR). The aim of this study was to prove the concept that CaSR antagonists specifically localize to CaSR-expressing tumors in vivo. DESIGN We synthesized two isomers of a known CaSR calcilytic, Calhex 231, and four new analogs, which have a favorable structure for labeling. Their antagonistic activity was determined using immunoblots demonstrating decreased ERK1/2 phosphorylation after calcium stimulation in human embryonic kidney cells overexpressing CaSR. Compound 9 was further radiolabeled with (125)I and evaluated in nude mice with and without heterotransplanted xenografts of MTC cell lines, TT and MZ-CRC-1, that do and do not express CaSR, respectively. RESULTS Two newly synthesized compounds, 9 and 11, exhibited better antagonistic activity than Calhex 231. The half-life of (125)I-compound 9 in nude mice without xenografts was 9.9 hours. A biodistribution study in nude mice bearing both tumors demonstrated that the uptake of radioactivity in TT tumors was higher than in MZ-CRC-1 tumors at 24 hours: 0.39 ± 0.24 vs 0.18 ± 0.12 percentage of injected dose per gram of tissue (%ID/g) (P = .002), with a ratio of 2.25 ± 0.62. Tumor-to-background ratios for TT tumors, but not MZ-CRC-1 tumors, increased with time. Tumor-to-blood values increased from 2.02 ± 0.52 at 1 hour to 3.29 ± 0.98 at 24 hour (P = .015) for TT tumors, and 1.7 ± 0.56 at 1 hour to 1.48 ± 0.33 at 24 hour (P = .36) for MZ-CRC-1 tumors. CONCLUSIONS Our new CaSR antagonists specifically inhibit CaSR function in vitro, preferentially localize to CaSR-expressing tumors in vivo, and therefore have the potential to serve as scaffolds for further development as imaging pharmaceuticals.

Abstract 186: NER factor XPC enhances DNA damage-induced apoptosis independently of p53 function

Xeroderma Pigmentosum complementation group C (XPC) is a 940-aa protein engaged in the DNA damage recognition during nucleotide excision repair (NER). It was reported that one of the XPC polymorphisms, Lys939Gln, is associated to cancer predisposition, although this variant did not affect NER. Thus, another mechanism must exist to contribute to this cancer susceptibility. In addition, this XPC genetic polymorphism correlates with the failure of imatinib treatment in patients with chronic myeloid leukemia. We reasoned that XPC must be influencing other functions unrelated to DNA repair. Here, we demonstrated that XPC can enhance the apoptosis induced by various DNA damaging agents, including ionizing radiation (IR), ultraviolet light (UV), cisplatin, and etoposide. In addition, XPC-mediated apoptosis is independent of p53, as reflected by the findings that knock-down of XPC expression compromised the UV or IR-induced apoptosis in p53-deficient Li-Fraumeni Syndrome (LFS) MDAH041 and HCT116 cell lines, while having no influence on apoptosis in their p53-proficient counterparts. Also, in response to UV irradiation, XPC deficiency did not affect the mitochondrial cytochrome c release or had only slight effect on the activation of caspase-3. However, XPC deficiency compromised the UV-induced activation of caspase-9. Therefore, XPC seems to function downstream of mitochondrial membrane permeabilization (MMP) and upstream of caspase-9 activation during DNA damage-induced apoptosis. We further demonstrated that XPC deficiency impaired the UV-induced caspase-2 activation, as reflected by reduced cleavage of long isoform of caspase-2 (CASP-2L) in XP-C cells compared to XPC-restored XP-C cells following UV irradiation. Meanwhile, the short isoform of caspase-2 (CASP-2S) was found to be induced in XP-C cells at early times following UV irradiation, while it remained unchanged in XPC-restored XP-C cells. Given that caspase 2 gene generates both proapoptotic (CASP-2L) and antiapoptotic (CASP-2S) isoforms by alternative splicing, our data suggest that XPC enhances DNA damage-induced apoptosis by inhibiting the production of anti-apoptotic CASP-2S via regulation of the alternative splicing of caspase-2. (Supported by NIH grants CA93413, ES2388 and ES12991). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 186. doi:10.1158/1538-7445.AM2011-186

Abstract 3946: Stem cell protein piwil2 enhances nucleotide excision repair through regulating chromatin remodeling following DNA damage

The piwi family gene, which are defined by conserved PAZ and Piwi domains, play important roles in stem cell self-renewal, RNA silencing, and translational regulation in various organisms. Three piwi homologs have been identified in the mouse genome. Amongst these, mili (piwil2) protein expression appears to be associated with tumorigenesis as evidenced by the facts that piwil2 stably expresses in pre-cancerous stem cells and also appears at variable levels in different human and animal tumor cell lines. Based on our preliminary data showing that piwil2 is induced in normal cultured human fibroblasts in vitro by physical and chemical genotoxic agents, we hypothesized that piwil2 might function in DNA damage response. Here, we demonstrated that mammalian cells lacking piwil2 are hypersensitive to cisplatin and UV irradiation. In addition, nucleotide excision repair (NER) efficiency is significantly decreased in the absence of piwil2 as assessed by the removal of cisplatin-induced intra-strand cross-links (Pt-GG) and UV-induced cyclobutane pyrimidine dimers (CPDs) from genomic DNA. Although quantitative real-time PCR analysis indicated higher transcript levels of various NER proteins in piwil2 knockout mouse embryo fibroblasts (MEFs), the Western blotting did not show an obvious difference in the NER protein levels between mili+/+ and mili−/− MEFs. Since piwil2 is believed to orchestrate chromatin remodeling, we reasoned that low NER efficiency in mili−/− MEFs may result from disrupted chromatin remodeling in response to UV or cisplatin induced DNA damage. Thus, chromatin relaxation and histone acetylation was analyzed in these MEFs following treatment with UV or cisplatin. MEFs lacking piwil2 showed less sensitivity to micrococcal nuclease (MNase) digestion compared with piwil2-proficient MEFs, indicating a compact chromatin structure in these cells. Moreover, in contrast to mili+/+ MEFs, mili−/− MEFs exhibited decreased acetylated Histone H3 upon both UV and cisplatin treatment. This decrease could be blocked by histone deacetylase (HDAC) inhibition indicating that piwil2 may function in damage-induced chromatin remodeling via inhibiting the HDAC activity. In summary, our data suggests that piwli2 can participate in the NER through regulating chromatin relaxation which could be a pivotal factor in genotoxin-induced tumorigenesis as well as efficacy of cancer therapeutics. (Supported by NIH grants CA93413, ES2388 and ES12991 to AAW). Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3946.

Abstract 3935: H3K56 is deacetylated in response to UV-irradiation and recovery of acetylation is regulated by ATR and ATM checkpoint kinases

DNA damage results in cell cycle arrest by checkpoint activation until successful completion of DNA repair. Cell cycle arrest and DNA repair processes involve changes in chromatin architecture, which help access of proteins involved in these processes. After DNA repair, the chromatin structure and cell cycle progression are restored, but the mechanism is largely unknown. Recent studies implicated a role of histone chaperones, CAF1 and ASF1 in DNA damage repair through histone modification as well as histone assembly and disassembly. CAF1 depletion can lead to a G1 and S phase checkpoint response, in an ATR-dependent manner. In yeast, ASF1 chaperone associates with Rtt109 HAT complex which is required for H3K56 acetylation and is important for checkpoint recovery. Recently, Das et. al. showed that mammalian ASF1 interacts with p300/CBP histone acetylase and acetylates H3K56, which is deposited at the damage site in a CAF1-dependent manner. In contrast, Tjeertes et. al. showed that H3K56Ac is present in undamaged cells and is reduced in response to DNA damage. Due to the contradictory nature of H3K56 acetylation from these studies, we wanted to establish a solid foundation related to the nature of this important UV damage response event in human cells. For this, cells were irradiated and the status of H3K56 acetylation was determined immediately and up to 48 hr after UV-irradiation. Our data unambiguously show that in comparison to the steady levels of control actin, H3K56 acetylation decreased gradually and was completely eliminated at 24 hr following 20 J/m2 UV dose. However, H3K56 acetylation exhibited full recovery after 48 hr at 2.5, 5 and 10 J/m2 UV doses. Thus, UV-irradiation causes H3K56 deacetylation, which is promptly restored possibly due to the successful repair and checkpoint recovery. We observed a similar effect on H3K56 deacetylation in response to UV damage using NHF cells. Since, H3K56 acetylation has been implicated in checkpoint recovery after completion of DNA repair, we tested whether H3K56 acetylation/deacetylation is regulated by ATR and ATM checkpoint kinases. Interestingly, in Seckel cells (lower ATR level) H3K56 acetylation is dramatically reduced even in the absence of UV-irradiation suggesting that H3K56 acetylation is regulated by ATR. In contrast, in A-T cells (ATM-deficient), H3K56 acetylation did not change in the absence of UV-irradiation. In A-T cells, H3K56 is deacetylated in response to UV-irradiation as in NHF cells, but the acetylation did not recover after 48 hr post-repair. These data clearly indicate a role of ATR/ATM in recovery of H3K56 acetylation. We propose that H3K56 acetylation presents a histone mark needed for ATR/ATM-mediated checkpoint recovery. (This work was supported by NIH grants CA93413, ES2388 and ES12991) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3935.