PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining.

The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.

Poly (ADP-ribose) Polymerase-1 modulations in the genesis of thrombosis.

Thrombosis, a coagulation disorder, occurs due to altered levels of coagulation, fibrinolytic and immune factors, which are otherwise known to maintain hemostasis in normal physiological conditions. Here, we review the direct and indirect participation of a multifunctional nuclear enzyme poly (ADP-ribose) polymerase-1 (PARP1) in the expression of key genes and cellular processes involved in thrombotic pathogenesis. PARP1 biological activities range from maintenance of genomic integrity, chromatin remodeling, base excision DNA repair, stress responses to cell death, angiogenesis and cell cycle pathways. However, under homeostatic imbalances, PARP1 activities are linked with the pathogenesis of diseases, including cancer, aging, neurological disorders, and cardiovascular diseases. Disease-associated distressed cells employ a variety of PARP-1 functions such as oxidative damage exacerbations, cellular energetics and apoptosis pathways, regulation of inflammatory mediators, promotion of endothelial dysfunction, and ERK-mediated signaling in pathogenesis. Thrombosis is one such pathogenesis that comprises exacerbation of coagulation cascade due to biochemical alterations in endothelial cells, platelet activation, overexpression of adhesion molecules, cytokines release, and leukocyte adherence. Thus, the activation of endothelial and inflammatory cells in thrombosis implicates a potential role of PARP1 activation in thrombogenesis. This review article explores the direct impact of PARP1 activation in the etiology of thrombosis and discusses PARP1-mediated endothelial dysfunction, inflammation, and epigenetic regulations in the disease manifestation. Understanding PARP1 functions associated with thrombosis may elucidate novel pathogenetic mechanisms and help in better disease management through newer therapeutic interventions targeting PARP1 activity.

Poly(ADP-ribosyl)ation temporally confines SUMO-dependent ataxin-3 recruitment to control DNA double-strand break repair.

DNA damage-induced SUMOylation serves as a signal for two antagonizing proteins that both stimulate repair of DNA double-strand breaks (DSBs). Here, we demonstrate that the SUMO-dependent recruitment of the deubiquitylating enzyme ataxin-3 to DSBs, unlike recruitment of the ubiquitin ligase RNF4, additionally depends on poly [ADP-ribose] polymerase 1 (PARP1)-mediated poly(ADP-ribosyl)ation (PARylation). The co-dependence of ataxin-3 recruitment on PARylation and SUMOylation temporally confines ataxin-3 to DSBs immediately after occurrence of DNA damage. We propose that this mechanism ensures that ataxin-3 prevents the premature removal of DNA repair proteins only during the early phase of the DSB response and does not interfere with the subsequent timely displacement of DNA repair proteins by RNF4. Thus, our data show that PARylation differentially regulates SUMO-dependent recruitment of ataxin-3 and RNF4 to DSBs, explaining how both proteins can play a stimulatory role at DSBs despite their opposing activities.

Combination treatments to enhance peptide receptor radionuclide therapy of neuroendocrine tumours.

The incidence of neuroendocrine tumours (NETs) is increasing, but curative therapeutic options are limited because diagnosis is often delayed until the tumour has metastasized. Peptide receptor radionuclide therapy (PRRT) is among the most effective therapeutic options for metastatic NETs because of targeted delivery of radioactivity to the tumour via the somatostatin receptor (SSTR) and relatively low systemic toxicity. However, current PRRT regimes result in palliation rather than cure, and higher doses of PRRT that might achieve remission would also be too toxic to the patients. Therefore, there is a need to improve PRRT of NETs by combining it with other agents to achieve maximum benefits from the internal radiation therapy, while sparing non-target organs from radiation toxicity. Here we review various current and potential combination strategies to improve 177Lu-octreotate-based PRRT of NET, some of which could also apply to other radionuclide therapies. These strategies include co-administered drugs that improve delivery of the radiopharmaceutical via increased tumour perfusion or through increased SSTR density at tumour surface. Other combinations are aimed at enhancing the biological effects of the radiation-induced DNA damage in tumour cells or generating additional DNA damage burden to effectively increase the cytotoxicity of PRRT. We also propose an algorithm for stratifying NET patients to receive or not combination therapies with PRRT. Considering that PRRT and many of these combination agents are already used for treating patients with NET and other cancers, the proposed strategies to improve the efficacy of PRRT could be rapidly translated into the clinic.

Enhanced Dark-Field Hyperspectral Imaging and Spectral Angle Mapping for Nanomaterial Detection in Consumer Care Products and in Skin Following Dermal Exposure.

Consumer personal care products, and cosmetics containing nanomaterials (NM), are increasingly available in the Canadian market. Current Canadian regulations do not require product labeling for ingredients that are present in the nanoscale. As a result, unless voluntarily disclosed, it is unclear which products contain NM. The enhanced dark-field hyperspectral imaging (EDF-HSI) coupled with spectral angle mapping (SAM) is a recent technique that has shown much promise for detection of NM in complex matrices. In the present study, EDF-HSI was used to screen cosmetic inventories for the presence of nano silver (nAg), nano gold (nAu), and nano titanium dioxide (nTiO2). In addition, we also assessed the potential of EDF-HSI as a tool to detect NM in skin layers following application of NM products in vitro on commercially available artificial skin constructs (ASCs) and in vivo on albino hairless SKH-1 mouse skin. Spectroscopic analysis positively detected nAu (4/9 products) and nTiO2 (7/13 products), but no nAg (0/6 products) in a subset of the cosmetics. The exposure of ASCs for 24 h in a Franz diffusion cell system to a diluted cosmetic containing nTiO2 revealed penetrance of nTiO2 through the epidermal layers and was detectable in the receptor fluid. Moreover, both single and multiple applications of nTiO2 containing cosmetics on the dorsal surface of SKH-1 mice resulted in detectable levels of trace nTiO2 in the layers of the skin indicating that penetrance of NM was occurring after each application of the product. The current study demonstrates the sensitivity of EDF-HSI with SAM mapping for qualitative detection of NM present in cosmetic products per se and very low levels in complex biological matrices on which these products are applied.

Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

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 UV-lesion 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.

Role of poly(ADP-ribose) polymerase-1 in the removal of UV-induced DNA lesions by nucleotide excision repair.

Among the earliest responses of mammalian cells to DNA damage is catalytic activation of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Activated PARP-1 forms the polymers of ADP-ribose (pADPr or PAR) that posttranslationally modify its target proteins, such as PARP-1 and DNA repair-related proteins. Although this metabolism is known to be implicated in other repair pathways, here we show its role in the versatile nucleotide excision repair pathway (NER) that removes a variety of DNA damages including those induced by UV. We show that PARP inhibition or specific depletion of PARP-1 decreases the efficiency of removal of UV-induced DNA damage from human skin fibroblasts or mouse epidermis. Using NER-proficient and -deficient cells and in vitro PARP-1 assays, we show that damaged DNA-binding protein 2 (DDB2), a key lesion recognition protein of the global genomic subpathway of NER (GG-NER), associates with PARP-1 in the vicinity of UV-damaged chromatin, stimulates its catalytic activity, and is modified by pADPr. PARP inhibition abolishes UV-induced interaction of DDB2 with PARP-1 or xeroderma pigmentosum group C (XPC) and also decreases localization of XPC to UV-damaged DNA, which is a key step that leads to downstream events in GG-NER. Thus, PARP-1 collaborates with DDB2 to increase the efficiency of the lesion recognition step of GG-NER.

Role of poly(ADP-ribose) polymerase-1 in regulating human islet cell differentiation.

Poly(ADP-ribose) polymerase-1 (PARP1), a fundamental DNA repair enzyme, is known to regulate ß cell death, replication, and insulin secretion. PARP1 knockout (KO) mice are resistant to diabetes, while PARP1 overactivation contributes to ß cell death. Additionally, PARP1 inhibition (PARPi) improves diabetes complications in patients with type-2 diabetes. Despite these beneficial effects, the use of PARP1 modulating agents in diabetes treatment is largely neglected, primarily due to the poorly studied mechanistic action of PARP1 catalytic function in human ß cell development. In the present study, we evaluated PARP1 regulatory action in human ß cell differentiation using the human pancreatic progenitor cell line, PANC-1. We surveyed islet census and histology from PARP1 wild-type versus KO mice pancreas in a head-to-head comparison with PARP1 regulatory action for in-vitro ß cell differentiation following either PARP1 depletion or its pharmacological inhibition in PANC-1-differentiated islet cells. shRNA mediated PARP1 depleted (SiP) and shRNA control (U6) PANC-1 cells were differentiated into islet-like clusters using established protocols. We observed complete abrogation of new ß cell formation with absolute PARP1 depletion while its inhibition using the potent inhibitor, PJ34, promoted the endocrine ß cell differentiation and maturation. Immunohistochemistry and immunoblotting for key endocrine differentiation players along with ß cell maturation markers highlighted the potential regulatory action of PARP1 and augmented ß cell differentiation due to direct interaction of unmodified PARP1 protein elicited p38 MAPK phosphorylation and Neurogenin-3 (Ngn3) re-activation. In summary, our study suggests that PARP1 is required for the proper development and differentiation of human islets. Selective inhibition with PARPi can be an advantage in pushing more insulin-producing cells under pathological conditions and delivers a potential for pilot clinical testing for ß cell replacement cell therapies for diabetes.

Deubiquitinating enzymes and the proteasome regulate preferential sets of ubiquitin substrates.

The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely unknown. Here, we compare the contributions of the proteasome and DUBs on the global ubiquitinome, using UbiSite technology, inhibitors and mass spectrometry. We uncover large dynamic ubiquitin signalling networks with substrates and sites preferentially regulated by DUBs or by the proteasome, highlighting the role of DUBs in degradation-independent ubiquitination. DUBs regulate substrates via at least 40,000 unique sites. Regulated networks of ubiquitin substrates are involved in autophagy, apoptosis, genome integrity, telomere integrity, cell cycle progression, mitochondrial function, vesicle transport, signal transduction, transcription, pre-mRNA splicing and many other cellular processes. Moreover, we show that ubiquitin conjugated to SUMO2/3 forms a strong proteasomal degradation signal. Interestingly, PARP1 is hyper-ubiquitinated in response to DUB inhibition, which increases its enzymatic activity. Our study uncovers key regulatory roles of DUBs and provides a resource of endogenous ubiquitination sites to aid the analysis of substrate specific ubiquitin signalling.

XPC-PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair.

Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.

Common fragile sites in colon cancer cell lines: role of mismatch repair, RAD51 and poly(ADP-ribose) polymerase-1.

Common fragile sites (CFS) are specific chromosomal areas prone to form gaps and breaks when cells are exposed to stresses that affect DNA synthesis, such as exposure to aphidicolin (APC), an inhibitor of DNA polymerases. The APC-induced DNA damage is repaired primarily by homologous recombination (HR), and RAD51, one of the key players in HR, participates to CFS stability. Since another DNA repair pathway, the mismatch repair (MMR), is known to control HR, we examined the influence of both the MMR and HR DNA repair pathways on the extent of chromosomal damage and distribution of CFS provoked by APC and/or by RAD51 silencing in MMR-deficient and -proficient colon cancer cell lines (i.e., HCT-15 and HCT-15 transfected with hMSH6, or HCT-116 and HCT-116/3+6, in which a part of a chromosome 3 containing the wild-type hMLH1 allele was inserted). Here, we show that MMR-deficient cells are more sensitive to APC-induced chromosomal damage particularly at the CFS as compared to MMR-proficient cells, indicating an involvement of MMR in the control of CFS stability. The most expressed CFS is FRA16D in 16q23, an area containing the tumour suppressor gene WWOX often mutated in colon cancer. We also show that silencing of RAD51 provokes a higher number of breaks in MMR-proficient cells with respect to their MMR-deficient counterparts, likely as a consequence of the combined inhibitory effects of RAD51 silencing on HR and MMR-mediated suppression of HR. The RAD51 silencing causes a broader distribution of breaks at CFS than that observed with APC. Treatment with APC of RAD51-silenced cells further increases DNA breaks in MMR-proficient cells. The RNAi-mediated silencing of PARP-1 does not cause chromosomal breaks or affect the expression/distribution of CFS induced by APC. Our results indicate that MMR modulates colon cancer sensitivity to chromosomal breaks and CFS induced by APC and RAD51 silencing.

Poly (ADP-ribose) polymerase (PARP) inhibition in cancer: Potential impact in cancer stem cells and therapeutic implications.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are used in mono- or combination therapies for several malignancies. They are also used as maintenance therapy for some cancers after initial treatment. While the focus of this therapeutic approach is on the effect of PARP inhibition on the bulk tumour cells, in this review, we discuss their effect on the cancer stem cells. We identify key mediators and pathways in cancer stem cells whose response to PARP inhibition is not necessarily the same as the rest of the tumour cells. Since the cancer stem cells are known drivers of growth of tumours and their resistance to therapy, the clinical outcome might be drastically different than what is expected, if the effect of PARP inhibition on the cancer stem cells is not taken into account.

Chemotherapy-Induced Upregulation of Somatostatin Receptor-2 Increases the Uptake and Efficacy of 177Lu-DOTA-Octreotate in Neuroendocrine Tumor Cells.

The peptide receptor radionuclide therapy (PRRT) with 177Lu-DOTA-octreotate (LuTate) is recommended for different types of neuroendocrine tumors (NETs) which overexpress somatostatin receptors (SSTR). A combination with chemotherapy improves objective response to LuTate in NET patients and here we characterized chemotherapy-induced upregulation of SSTR2 receptors as a cause for this improved response to LuTate. The NET cell lines with low (BON-1) or relatively high (NCI-H727) SSTR2-expression levels, and non-NET cancer and normal cells were treated with chemotherapeutic drugs and assessed for upregulation of SSTR2. We report that an exposure to low or high doses of drugs, such as temozolomide for 24 h or 5 day results in upregulation of SSTR2 between 3-7 days, increased LuTate uptake and decreased rate of cell proliferation. This effect is at the level of SSTR2-mRNA and is more pronounced in low SSTR2 expressing BON-1 than in high SSTR2 expressing NCI-H727 or non-NET cancer or normal cells. Thus, a properly timed pre-treatment with low-dose chemotherapy could not only improve therapeutic efficacy of LuTate in NET patients who are presently eligible for PRRT, but also allow PRRT to be administered to patients with low SSTR-expressing NETs, who would otherwise not respond to this modality because of insufficient radiation delivery.

Methods to Study Intracellular Movement and Localization of the Nucleotide Excision Repair Proteins at the DNA Lesions in Mammalian Cells.

Nucleotide excision repair (NER) is the most versatile DNA repair pathway that removes a wide variety of DNA lesions caused by different types of physical and chemical agents, such as ultraviolet radiation (UV), environmental carcinogen benzo[a]pyrene and anti-cancer drug carboplatin. The mammalian NER utilizes more than 30 proteins, in a multi-step process that begins with the lesion recognition within seconds of DNA damage to completion of repair after few hours to several days. The core proteins and their biochemical reactions are known from in vitro DNA repair assays using purified proteins, but challenge was to understand the dynamics of their rapid recruitment and departure from the lesion site and their coordination with other proteins and post-translational modifications to execute the sequential steps of repair. Here, we provide a brief overview of various techniques developed by different groups over last 20 years to overcome these challenges. However, more work is needed for a comprehensive knowledge of all aspects of mammalian NER. With this aim, here we provide detailed protocols of three simple yet innovative methods developed by many teams that range from local UVC irradiation to in situ extraction and sub-cellular fractionation that will permit study of endogenous as well as exogenous NER proteins in any cellular model. These methods do not require unique reagents or specialized instruments, and will allow many more laboratories to explore this repair pathway in different models. These techniques would reveal intracellular movement of these proteins to the DNA lesion site, their interactions with other proteins during repair and the effect of post-translational modifications on their functions. We also describe how these methods led us to identify hitherto unexpected role of poly(ADP-ribose) polymerase-1 (PARP1) in NER. Collectively these three simple techniques can provide an initial assessment of the functions of known and unknown proteins in the core or auxiliary events associated with mammalian NER. The results from these techniques could serve as a solid foundation and a justification for more detailed studies in NER using specialized reagents and more sophisticated tools. They can also be suitably modified to study other cellular processes beyond DNA repair.

A panel of criteria for comprehensive assessment of severity of ultraviolet B radiation-induced non-melanoma skin cancers in SKH-1 mice.

The study of causes and cures for ultraviolet B radiation (UVB)-induced non-melanoma skin cancers (NMSC) has been greatly facilitated by use of the albino SKH-1 hairless mice. These mice develop multiple tumors of different sizes and the severity of cancer is often measured by one or more of the four criteria, namely the prevalence, multiplicity, area and volume of tumors. However, there are inherent limitations of each criterion: the prevalence and number do not account for size differences among tumors, area measurement ignores the tumor height, and volume measurement overcompensates for the height at the cost of planar dimensions. Here, using our dataset from an ongoing NMSC study, we discuss the limitations of these four criteria, and suggest refinements in measuring prevalence. We recommend the use of three more criteria, namely the Knud Thomsen tridimensional surface that apportions optimal weightage to three tumor dimensions, weekly occurrence of new tumors and tumor growth-rate to reveal initiation and growth of tumors in early and late phase of NMSC development, respectively. Together, use of this comprehensive panel of seven criteria can provide an accurate assessment of severity of NMSC and lead to a testable hypothesis whether the experimental manipulation of mice has affected the early initiation or growth phase of NMSC tumors.

Loss of ZBTB24 impairs nonhomologous end-joining and class-switch recombination in patients with ICF syndrome.

The autosomal recessive immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is a genetically heterogeneous disorder. Despite the identification of the underlying gene defects, it is unclear how mutations in any of the four known ICF genes cause a primary immunodeficiency. Here we demonstrate that loss of ZBTB24 in B cells from mice and ICF2 patients affects nonhomologous end-joining (NHEJ) during immunoglobulin class-switch recombination and consequently impairs immunoglobulin production and isotype balance. Mechanistically, we found that ZBTB24 associates with poly(ADP-ribose) polymerase 1 (PARP1) and stimulates its auto-poly(ADP-ribosyl)ation. The zinc-finger in ZBTB24 binds PARP1-associated poly(ADP-ribose) chains and mediates the PARP1-dependent recruitment of ZBTB24 to DNA breaks. Moreover, through its association with poly(ADP-ribose) chains, ZBTB24 protects them from degradation by poly(ADP-ribose) glycohydrolase (PARG). This facilitates the poly(ADP-ribose)-dependent assembly of the LIG4/XRCC4 complex at DNA breaks, thereby promoting error-free NHEJ. Thus, we uncover ZBTB24 as a regulator of PARP1-dependent NHEJ and class-switch recombination, providing a molecular basis for the immunodeficiency in ICF2 syndrome.

Inhibition of homologous recombination by treatment with BVDU (brivudin) or by RAD51 silencing increases chromosomal damage induced by bleomycin in mismatch repair-deficient tumour cells.

Mismatch repair (MMR) has been shown to control homologous recombination (HR) by aborting strand exchange between divergent sequences. We previously demonstrated that MMR-deficient tumour cells are more resistant to chromosomal damage induced by bleomycin (BLM) during the G(2) phase, likely due to the lack of the MMR inhibitory effect on HR. Aim of this study was to investigate whether inhibition of HR by the nucleoside analogue BVDU [(E)-5(2-bromovinyl)-2'-deoxyuridine, brivudin], or silencing of genes involved in HR function, might affect sensitivity of MMR-deficient tumour cells to DNA damage induced by BLM in G(2). The results indicated that BVDU increased chromatid damage and DNA double strand breaks induced by BLM only in MMR-deficient MT-1, HL-60R, HCT116 cells, which are more resistant to BLM with respect to MMR-proficient TK-6, HL-60S and HCT116/3-6 lines. Silencing of RAD51, a key component of HR, increased sensitivity of MMR-deficient HCT-15 cells to BLM clastogenicity; in this case combined treatment with BVDU had no additional effect. Similarly, treatment with BVDU did not affect BLM clastogenicity in CAPAN-1 cells, characterized by a defective HR due to BRCA2 mutations. Conversely, BVDU increased chromatid breaks induced by BLM in HCT-15 cells transiently silenced for DNA-PK catalytic subunit, which plays a key role in non-homologous end joining. The BVDU-mediated increase of chromatid breaks in MMR-deficient cells did not depend on its previously reported inhibitory effect on poly(ADP-ribose) polymerase (PARP). In fact, it was observed also in cells stably silenced for PARP-1, which is responsible for most of cellular PARP activity. These data support the suggestion that the higher sensitivity of MMR-proficient versus MMR-deficient cells to BLM-induced chromatid breaks in the G(2) phase is a consequence of the inhibition of HR by MMR. In MMR-deficient cells, BVDU attenuates the repair of BLM-induced DSBs and this is likely to occur via inhibition of HR.

Stable depletion of poly (ADP-ribose) polymerase-1 reduces in vivo melanoma growth and increases chemosensitivity.

Poly(ADP-ribose) polymerase (PARP)-1, which plays a key role in DNA repair, inflammation and transcription, has recently been shown to be involved in angiogenesis. The aim of this study was to investigate PARP-1 role in melanoma aggressiveness and chemoresistance in vivo using clones stably silenced for PARP-1 expression. Whilst the growth characteristics of PARP-1-deficient melanoma cells were comparable to those of PARP-1-proficient cells in vitro, their tumourigenic potential in vivo was significantly compromised. In fact, mice challenged intra-muscle with PARP-1-deficient cells showed a delayed development of measurable tumour nodules, which were also significantly reduced in size with respect to those of mice inoculated with PARP-1-proficient cells. Moreover, animals challenged intra-cranially with PARP-1-deficient cells, a model that mimics CNS localisation of melanoma, showed an increased survival. Immunohistochemical analyses of PARP-1-depleted melanoma grafts indicated a reduced expression of the angiogenesis marker PECAM-1/CD31 and of the pro-inflammatory mediators TNF-alpha and GITR. Notably, PARP-1-silenced melanoma was extremely sensitive to temozolomide, an anticancer agent used for the treatment of metastatic melanoma. These results provide novel evidence for a direct role of PARP-1 in tumour aggressiveness and chemoresistance.

Potentiation of 177Lu-octreotate peptide receptor radionuclide therapy of human neuroendocrine tumor cells by PARP inhibitor.

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.

Comprehensive measurement of UVB-induced non-melanoma skin cancer burden in mice using photographic images as a substitute for the caliper method.

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.