Girish M. Shah

New paradigm for lymphocyte granule-mediated cytotoxicity: Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis

Lymphocyte granule-mediated apoptosis is postulated to entail the formation of membrane pores by perforin. Then soluble granzyme reaches the cytosol either through these pores or by reparative pinocytosis. We demonstrate here that Jurkat cells bind and internalize granzyme B via high affinity binding sites without toxic consequence. Apoptosis occurs, however, if sublytic perforin is added to targets washed free of soluble granzyme B. We suggest that granule-mediated apoptosis mimics viral strategies for cellular entry. Accordingly, co-internalization of granzyme B with adenovirus, a virus that escapes endosomes to reach the cytosol, also induced apoptosis. Poly(ADP-ribose) polymerase cleavage and processing of CPP32, ICE-LAP3, and Mch2 were detected at 30 min, while cytosolic acidification and DNA fragmentation occurred at 60 min. Annexin V binding and membrane permeabilization arose at 4 h. The concurrent activation of the Ced-3 proteases differed from the rate at which each cysteine protease is cleaved in vitro by granzyme B. Thus, granzyme B may not directly process these proteases in whole cells but rather may function by activating a more proximal enzyme. These results indicate that adenovirus-mediated delivery of granzyme B is suitable for elucidating biochemical events that accompany granule-mediated apoptosis.

Mode of action of poly(ADP-ribose) glycohydrolase.

The turnover of the homopolymer of ADP-ribose, which is known to be involved in many DNA-related functions, is controlled by 2 principal enzymes. Poly(ADP-ribose) polymerase (EC 2.4.2.30) synthesizes the polymer from NAD, and poly(ADP-ribose) glycohydrolase (PARG) is the major enzyme responsible for its catabolism (Thomassin et al. (1992) Biochim. Biophys. Acta 1137, 171-181). In vivo, poly(ADP-ribose) polymers constitute a heterogeneous population of branched polymers attaining sizes of 200-400 residues. They are rapidly degraded by PARG, displaying variable kinetic parameters as a function of polymer size. Several studies have suggested that PARG acts exoglycosidically on its substrate but others observed that it could act endo/exo-glycosidically. We analysed the mode of action of PARG under conditions most suitable for expression of all the activities of PARG, using HPLC purified long free polymer and very pure PARG. We conclusively show that on large free polymers, PARG exhibits endoglycosidic activity along with exoglycosidic activity. This endoglycosidic activity could have a significant role during cellular response to DNA damage.

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.

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

Summary 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.

Mechanism of early biphasic activation of poly(ADP-ribose) polymerase-1 in response to ultraviolet B radiation.

The damage to DNA caused by ultraviolet B radiation (280-320 nm) contributes significantly to development of sunlight-induced skin cancers. The susceptibility of mice to ultraviolet B-induced skin carcinogenesis is increased by an inhibitor of the DNA damage-activated nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP), hence PARP activation is likely to be associated with cellular responses that suppress carcinogenesis. To understand the role of activated PARP in these cellular functions, we need to first clearly identify the cause of PARP activation in ultraviolet B-irradiated cells. Ultraviolet B, like ultraviolet C, causes direct DNA damage of cyclobutane pyrimidine dimer and 6, 4-photoproduct types, which are subjected to the nucleotide excision repair. Moreover, ultraviolet B also causes oxidative DNA damage, which is subjected to base excision repair. To identify which of these two types of DNA damage activates PARP, we examined mechanism of early PARP activation in mouse fibroblasts exposed to ultraviolet B and C radiations. The ultraviolet B-irradiated cells rapidly activated PARP in two distinct phases, initially within the first 5 minutes and later between 60-120 minutes, whereas ultraviolet C-irradiated cells showed only the immediate PARP activation. Using antioxidants, local irradiation, chromatin immunoprecipitation and in vitro PARP assays, we identified that ultraviolet radiation-induced direct DNA damage, such as thymine dimers, cause the initial PARP activation, whereas ultraviolet B-induced oxidative damage cause the second PARP activation. Our results suggest that cells can selectively activate PARP for participation in different cellular responses associated with different DNA lesions.

CHARACTERIZATION OF ANTI-PEPTIDE ANTIBODIES DIRECTED TOWARDS THE AUTOMODIFICATION DOMAIN AND APOPTOTIC FRAGMENT OF POLY(ADP-RIBOSE) POLYMERASE

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a highly conserved nuclear enzyme present in higher eukaryotes. PARP is activated following DNA damage, is implicated in DNA repair, and its proteolysis has been shown to be an early marker of programmed cell death or apoptosis. In order to better understand the role of PARP in apoptosis and DNA repair and also to study PARP automodification, we have developed anti-peptide sera directed against four peptides from the conserved automodification domain of PARP. Four peptides were synthesized according to the four branched Multiple Antigenic Peptide (MAP) system and injected into rabbits. Immune sera were titrated by ELISA and analysed in Western blotting experiments on cell lines. The sera were also analysed for their capacity to inhibit PARP activity in an in vitro assay. Of the eight sera developed (two for each peptide), a serum directed against a peptide localized at the C-terminal part of the automodification domain of PARP (#422) appeared to be the best antibody to detect PARP from different species. All antipeptide antibodies were efficient in detecting the apoptotic fragment of PARP during programmed cell death in HL-60 apoptotic cells. None of the serum alone was able to completely inhibit PARP activity but combinations of the sera could significantly reduce automodification of PARP consistent with the localization of half of the automodification sites on bovine PARP. Sera were also used to map proteolysed purified PARP and to immunoprecipitate purified bovine PARP.

Biochemical Assessment of Niacin Deficiency Among Carcinoid Cancer Patients

OBJECTIVE:Carcinoid cancer patients often have elevated levels of serotonin or its precursor 5-hydroxytryptophan. Normally, serotonin synthesis accounts for a small fraction of tryptophan catabolism, which should be directed along a pathway that allows partial conversion to niacin; hence, increased diversion of tryptophan toward serotonin could cause variable degrees of niacin deficiency in carcinoid patients. Therefore, the prevalence of niacin deficiency among carcinoid patients was investigated by clinical assessment of pellagra and biochemical assessment of whole blood niacin number, a ratio derived from two biologically active forms of niacin (NAD/NADP × 100).METHODS:Clinical and biochemical niacin status were assessed in a cohort of newly diagnosed carcinoid patients with carcinoid syndrome (CCS, n = 36), carcinoid patients without carcinoid syndrome (CWCS, n = 32) and noncarcinoid controls (n = 24) recruited at two primary care clinics. Other aspects of serotonin metabolism were measured by analyses of plasma serotonin and tryptophan and urinary excretion of 5-hydroxyindoleacetic acid.RESULTS:Biochemical niacin deficiency (niacin number < 130) was significantly more common in CCS patients (10 out of 36) compared to controls (p < 0.05, Fishers exact test), while CWCS patients displayed an incidence that was not significantly elevated (4 out of 32). Only one CCS patient, who was also identified biochemically as niacin deficient, was clinically diagnosed with pellagra.CONCLUSION:Biochemical niacin deficiency is more prevalent among newly diagnosed CCS patients than in controls. Manifestation of pellagra is a less sensitive indicator, and dependence on this endpoint could lead to a lack of appropriate nutritional support for this group of patients.

Resistance to PARP-Inhibitors in Cancer Therapy

The pharmacological inhibitors of poly(ADP-ribose) polymerase (PARP) family of proteins have shown promising results in preclinical studies and clinical trials as a monotherapy or in combination therapy for some cancers. Thus, usage of PARP-inhibitors (PARPi) in cancer therapy is bound to increase with time, but resistance of cancer cells to PARPi is also beginning to be observed. Here we review different known and potential mechanisms by which: (i) PARPi kill cancer cells; and (ii) cancer cells develop resistance to PARPi. Understanding the lethality caused by PARPi and the countermeasures deployed by cancers cells to survive PARPi will help us rationalize the use of this new class of drugs in cancer therapy.

Role of poly(ADP-ribose) polymerase in rapid intracellular acidification induced by alkylating DNA damage

In response to high levels of DNA damage, catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) triggers necrotic death because of rapid consumption of its substrate β-nicotinamide adenine dinucleotide and consequent depletion of ATP. We examined whether there are other consequences of PARP activation that could contribute to cell death. Here, we show that PARP activation reaction in vitro becomes acidic with release of protons during hydrolysis of β-nicotinamide adenine dinucleotide. In the cellular context, we show that Molt 3 cells respond to DNA damage by the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) with a dose-dependent acidification within 30 min. Whereas acidification by 0.15 pH units induced by 10 μM MNNG is reversed within 1 h, 100 μM MNNG-induced acidification by 0.5–0.6 pH units is persistent up to 7 h. Acidification is a general DNA damage response because H2O2 exposure also acidifies Molt 3 cells, and MNNG causes acidification in Jurkat, U937, or HL-60 leukemia cells and in PARP+/+ fibroblasts. Acidification is significantly decreased in the presence of PARP inhibitors or in PARP−/− fibroblasts, suggesting a major role for PARP activation in acidification. Inhibition of proton export through ATP-dependent Na+/H+ exchanger is another major cause of acidification. Using the pH clamp method to either suppress or introduce changes in cellular pH, we show that brief acidification by 0.5–0.6 pH units may be a negative regulator of apoptosis while permitting necrotic death of cells with extensively damaged DNA.

Calcitonin Stimulates Multiple Stages of Angiogenesis by Directly Acting on Endothelial Cells

Although a strong correlation between neuroendocrine differentiation and angiogenesis of prostate cancer has been reported, no mechanistic link between the two events has been established. Because neuropeptide calcitonin is secreted by prostate tumors and endothelial cells are known to express calcitonin receptor-like receptor, we examined the potential action of calcitonin on endothelial cells. The presence of calcitonin receptor, calcitonin receptor-like receptor, and receptor activity-modifying proteins in human microvessel endothelial-1 cells was tested by reverse transcriptase-PCR (RT-PCR). The proangiogenic action of calcitonin was examined in several in vitro models of angiogenesis using HMEC-1 cells and also in vivo using dorsal skinfold assays. Calcitonin expression of PC-3M cells was modulated, and its effect on angiogenesis was examined in in vitro as well as in vivo models. The results of RT-PCR and radioligand receptor assays showed the presence of functional calcitonin receptor in HMEC-1 cells. Calcitonin stimulated all phases of angiogenesis through the calcitonin receptor, but its effect on tube morphogenesis by endothelial cells occurred at the concentration of the Kd of calcitonin receptor. Silencing of calcitonin receptor expression in HMEC-1 cells abolished calcitonin-induced tube formation. Vascular endothelial growth factor antibodies attenuated but did not abolish calcitonin-induced tube morphogenesis. PC-3M prostate cancer cells induced angiogenesis in in vivo and in vitro models. Overexpression of calcitonin in PC-3M cells increased their angiogenic activity, whereas the silencing of calcitonin expression abolished it. These results show that prostate tumor-derived calcitonin may play an important role in prostate tumor growth by regulating intratumoral vascularization.