Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UVlesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

For patients with inoperable neuroendocrine tumors (NETs) expressing somatostatin receptors, peptide receptor radionuclide therapy (PRRT) with 177Lu-[DOTA0-Tyr3]-octreotate (177Lu-octreotate) is one of the most promising targeted therapeutic options but it rarely achieves cure. Therefore, different approaches are being tested to increase the efficacy of 177Lu-octreotate PRRT in NET patients. Using the gastroenteropancreatic BON-1 and the bronchopulmonary NCI-H727 as NET cell models, here we report that pharmacological inhibitors of DNA repair-associated enzyme poly(ADP-ribose) polymerase-1 (PARPi) potentiate the cytotoxic effect of 177Lu-octreotate on 2D monolayer and 3D spheroid models of these two types of NET cells. PARPi mediates this effect by enhancing 177Lu-octreotate-induced cell cycle arrest and cell death. Thus, the use of PARPi may offer a novel option for improving the therapeutic efficacy of 177Lu-octreotate PRRT of NETs.

The existing methodologies for studying robust responses of poly (ADP-ribose) polymerase-1 (PARP-1) to DNA damage with strand breaks are often not suitable for examining its subtle responses to altered DNA without strand breaks, such as UV-damaged DNA. Here we describe two novel assays with which we characterized the interaction of PARP-1 with UV-damaged DNA in vivo and in vitro. Using an in situ fractionation technique to selectively remove free PARP-1 while retaining the DNA-bound PARP-1, we demonstrate a direct recruitment of the endogenous or exogenous PARP-1 to the UV-lesion site in vivo after local irradiation. In addition, using the model oligonucleotides with single UV lesion surrounded by multiple restriction enzyme sites, we demonstrate in vitro that DDB2 and PARP-1 can simultaneously bind to UV-damaged DNA and that PARP-1 casts a bilateral asymmetric footprint from −12 to +9 nucleotides on either side of the UV-lesion. These techniques will permit characterization of different roles of PARP-1 in the repair of UV-damaged DNA and also allow the study of normal housekeeping roles of PARP-1 with undamaged DNA.The abundance of poly (ADP-ribose) polymerase-1 (PARP-1) in mammalian cells and its rapid catalytic activation to form polymers of ADP-ribose (PAR) in the presence of various types of DNA damages with or without strand breaks has made it an ideal first responder at the lesion site to influence downstream events 1,2 . Apart from DNA damages, PARP-1 is also recruited to DNA during normal physiological processes such as transcription and chromatin remodeling 3 , which do not involve overt DNA damage but just altered DNA structures. While we know much more about how PARP-1 rapidly recognizes and binds to single or double strand breaks in DNA, we know very little about how PARP-1 interacts with DNA damages or altered DNA structures without strand breaks. The key reason is that the existing methodologies that readily identify interactions of PARP-1 with DNA strand breaks are not sufficiently sensitive to study the relatively weaker responses of PARP-1 to DNA damage without strand breaks. The response of PARP-1 to UVC-induced direct photolesions, such as cyclobutane pyrimidine dimers (CPD) that are formed without any DNA strand breaks exemplifies this problem.Recent studies from others and our team have shown the involvement of PARP-1 in the host cell reactivation 4 and specifically in the nucleotide excision repair (NER) of UV-damaged DNA through its interaction with early NER protein DDB2 [5][6][7] . Additional studies have shown that downstream NER proteins XPA 8,9 and XPC 10 are PARylated. Thus, PARP-1 possibly has multiple roles in NER, but we do not yet fully understand its interactions with UV-damaged DNA or other NER proteins due to two major challenges. The first challenge is that unlike for many NER proteins, the abundance of endogenous PARP-1 in the nucleus makes it nearly impossible to visualize its dynamics of recruitment to UV-damaged DNA in situ using conventional immunocytological methods. T...

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Changes in lifestyle lead to insulin resistance (IR) in females ultimately predisposing them towards infertility. In addition, cadmium (Cd), an environmental endocrine disruptor, is reported for detrimental effects on granulosa cells, thus leading to ovarian dysfunction. A combination of these factors, lifestyle and environment, seems to play a role in etiology of idiopathic infertility that accounts for 50% amongst the total infertility cases. To address this issue, we made an attempt to investigate the extent of Cd impact on insulin-resistant (IR) granulosa cells. We exposed adult female Charles Foster rats to dexamethasone and confirmed IR condition by fasting insulin resistance index (FIRI). On treatment of IR rats with Cd, the preliminary studies demonstrated prolonged estrous cyclicity, decrease in serum estradiol concentrations, abnormal histology of ovary, and increased granulosa cell death. Further gene and protein expression studies of steroidogenic acute regulatory (StAR) protein, 17β-hydroxysteroid dehydrogenase (17β-HSD), and cytochrome P450 aromatase (CYP19A1) were performed. Protein expression studies demonstrated significant decrease in treated groups when compared with control. Study revealed that, in spite of the molecular parameters being affected at varied level, overall ovarian physiology is maximally affected in IR and Cd coexposed group, thus mimicking the condition similar to those prevailing in infertile females.

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The vernier caliper has been used as a gold standard to measure the length, width and height of skin tumors to calculate their total area and volume. It is a simple method for collecting data on a few tumors at a time, but becomes tedious, time-consuming and stressful for the animals and the operator when used for measuring multiple tumors in a large number of animals in protocols such as UVB-induced non-melanoma skin cancer (NMSC) in SKH-1 mice. Here, we show that photographic images of these mice taken within a few minutes under optimized conditions can be subjected to computerized analyses to determine tumor volume and area as accurately and precisely as the caliper method. Unlike the caliper method, the photographic method also records the incidence and multiplicity of tumors, thus permitting comprehensive measurement of tumor burden in the animal. The simplicity and ease of this method will permit more frequent monitoring of tumor burden in long protocols, resulting in the creation of additional data about dynamic changes in progression of cancer or the efficacy of therapeutic intervention. The photographic method can broadly substitute the caliper method for quantifying other skin pathologies.