Roles of poly(ADP-ribosyl)ation and PARP in apoptosis, DNA repair, genomic stability and functions of p53 and E2F-1.
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3T3 Cells↗
Biomedical subjects
D S Rosenthal
Publications and source records attributed to D S Rosenthal.
Chromosomal aberrations in PARP(-/-) mice: genome stabilization in immortalized cells by reintroduction of poly(ADP-ribose) polymerase cDNA.
Depletion of poly(ADP-ribose) polymerase (PARP) increases the frequency of recombination, gene amplification, sister chromatid exchanges, and micronuclei formation in cells exposed to genotoxic agents, implicating PARP in the maintenance of genomic stability. Flow cytometric analysis now has revealed an unstable tetraploid population in immortalized fibroblasts derived from PARP(-/-) mice. Comparative genomic hybridization detected partial chromosomal gains in 4C5-ter, 5F-ter, and 14A1-C1 in PARP(-/-)mice and immortalized PARP(-/-)fibroblasts. Neither the chromosomal gains nor the tetraploid population were apparent in PARP(-/-) cells stably transfected with PARP cDNA [PARP(-/-)(+PARP)], indicating negative selection of cells with these genetic aberrations after reintroduction of PARP cDNA. Although the tumor suppressor p53 was not detectable in PARP(-/-) cells, p53 expression was partially restored in PARP(-/-) (+PARP) cells. Loss of 14D3-ter that encompasses the tumor suppressor gene Rb-1 in PARP(-/-) mice was associated with a reduction in retinoblastoma(Rb) expression; increased expression of the oncogene Jun was correlated with a gain in 4C5-ter that harbors this oncogene. These results further implicate PARP in the maintenance of genomic stability and suggest that altered expression of p53, Rb, and Jun, as well as undoubtedly many other proteins may be a result of genomic instability associated with PARP deficiency.
Animals↗
Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol alpha expression during early S phase.
E2F-1, a transcription factor implicated in the activation of genes required for S phase such as DNA pol alpha, is regulated by interactions with Rb and by cell-cycle dependent alterations in E2F-1 abundance. We have shown that depletion of poly(ADP-ribose) polymerase (PARP) by antisense RNA expression downregulates pol alpha and E2F-1 expression during early S phase. To examine the role of PARP in the regulation of pol alpha and E2F-1 gene expression, we utilized immortalized mouse fibroblasts derived from wild-type and PARP knockout (PARP-/-) mice as well as PARP-/- cells stably transfected with PARP cDNA [PARP-/-(+PARP)]. After release from serum deprivation, wild-type and PARP-/-(+PARP) cells, but not PARP-/- cells, exhibited a peak of cells in S phase by 16 h and had progressed through the cell cycle by 22 h. Whereas [3H]thymidine incorporation remained negligible in PARP-/- cells, in vivo DNA replication maximized after 18 h in wild-type and PARP-/-(+PARP) cells. To investigate the effect of PARP on E2F-1 promoter activity, a construct containing the E2F-1 gene promoter fused to a luciferase reporter gene was transiently transfected into these cells. E2F-1 promoter activity in control and PARP-/-(+PARP) cells increased eightfold after 9 h, but not in PARP-/- cells. PARP-/- cells did not show the marked induction of E2F-1 expression during early S phase apparent in control and PARP-/-(+PARP) cells. RT - PCR analysis and pol alpha activity assays revealed the presence of pol alpha transcripts and a sixfold increase in activity in both wild-type and PARP-/-(+PARP) cells after 20 h, but not in PARP-/- cells. These results suggest that PARP plays a role in the induction of E2F-1 promoter activity, which then positively regulates both E2F-1 and pol alpha expression, when quiescent cells reenter the cell cycle upon recovery from aphidicolin exposure or removal of serum.
Animals↗
Poly(ADP-ribosyl)ation of p53 during apoptosis in human osteosarcoma cells.
Spontaneous apoptosis in human osteosarcoma cells was observed to be associated with a marked increase in the intracellular abundance of p53. Immunoprecipitation and immunoblot analysis revealed that, together with a variety of other nuclear proteins, p53 undergoes extensive poly(ADP-ribosyl)ation early during the apoptotic program in these cells. Subsequent degradation of poly(ADP-ribose) (PAR), attached to p53 presumably by PAR glycohydrolase, the only reported enzyme to degrade PAR, was apparent concomitant with the onset of proteolytic processing and activation of caspase-3, caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP), and internucleosomal DNA fragmentation during the later stages of cell death. The decrease in PAR covalently bound to p53 also coincided with the marked induction of expression of the p53-responsive genes bax and Fas. These results suggest that poly(ADP-ribosyl)ation may play a role in the regulation of p53 function and implies a regulatory role for PARP and/or PAR early in apoptosis.
Apoptosis↗
Talking with patients about screening for prostate cancer.
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Colorectal Neoplasms↗
Involvement of PARP and poly(ADP-ribosyl)ation in the early stages of apoptosis and DNA replication.
We have focused on the roles of PARP and poly(ADP-ribosyl)ation early in apoptosis, as well as during the early stages of differentiation-linked DNA replication. In both nuclear processes, a transient burst of PAR synthesis and PARP expression occurs early, prior to internucleosomal DNA cleavage before commitment to apoptosis as well as at the round of DNA replication prior to the onset of terminal differentiation. In intact human osteosarcoma cells undergoing spontaneous apoptosis, both PARP and PAR decreased after this early peak, concomitant with the inactivation and cleavage of PARP by caspase-3 and the onset of substantial DNA and nuclear fragmentation. Whereas 3T3-L1, osteosarcoma cells, and immortalized PARP +/+ fibroblasts exhibited this early burst of PAR synthesis during Fas-mediated apoptosis, neither PARP-depleted 3T3-L1 PARP-antisense cells nor PARP -/- fibroblasts showed this response. Consequently, whereas control cells progressed into apoptosis, as indicated by induction of caspase-3-like PARP-cleavage activity, PARP-antisense cells and PARP -/- fibroblasts did not, indicating a requirement for PARP and poly(ADP-ribosyl)ation of nuclear proteins at an early reversible stage of apoptosis. In parallel experiments, a transient increase in PARP expression and activity were also noted in 3T3-L1 preadipocytes 24 h after induction of differentiation, a stage at which approximately 95% of the cells were in S-phase, but not in PARP-depleted antisense cells, which were consequently unable to complete the round of DNA replication required for differentiation. PARP, a component of the multiprotein DNA replication complex (MRC) that catalyzes viral DNA replication in vitro, poly(ADP-ribosyl)ates 15 of approximately 40 MRC proteins, including DNA pol alpha, DNA topo I, and PCNA. Depletion of endogenous PARP by antisense RNA expression in 3T3-L1 cells results in MRCs devoid of any DNA pol alpha and DNA pol delta activities. Surprisingly, there was no new expression of PCNA and DNA pol alpha, as well as the transcription factor E2F-1 in PARP-antisense cells during entry into S-phase, suggesting that PARP may play a role in the expression of these proteins, perhaps by interacting with a site in the promoters for these genes.
3T3 Cells↗
Prolongation of the p53 response to DNA strand breaks in cells depleted of PARP by antisense RNA expression.
The observation that 3-aminobenzamide, which inhibits a variety of ADP-ribose transferases, prolongs the gamma-irradiation-induced increase in intracellular p53 concentration suggested that one or more of such enzymes may determine the duration of the p53 response during G1 arrest. The role of poly(ADP-ribose) polymerase (PARP), an abundant nuclear enzyme activated by DNA strand breaks, in the p53 response to y-irradiation was investigated in Burkitt's lymphoma AG876 cells stably transfected with an inducible PARP antisense construct. Immunoblot analysis revealed that the cellular content of PARP was reduced to virtually undetectable levels after incubation of transfected cells for 72 h with the inducer dexamethasone. In noninduced antisense cells, the p53 concentration reached a maximum 2 h after exposure to 6.3 Gy of gamma-radiation and returned to control values by 4 h. In contrast, the p53 response in PARP-depleted antisense cells peaked at 4 h, with the levels of p53 remaining elevated for up to 12 h after y-irradiation. The maximal increase in p53 concentration was similar in both induced and noninduced cells. These results thus indicate that PARP activity, in part, determines the duration, but not the magnitude, of the p53 response to DNA damage.
Burkitt Lymphoma↗
The National Cancer Data Base report on prostate carcinoma after the peak in incidence rates in the U.S. The American College of Surgeons Commission on Cancer and the American Cancer Society.
BACKGROUND: Prostate carcinoma incidence has been declining since 1992 after a period of marked increase. Recent data from the National Cancer Data Base (NCDB) were examined to assess changes in prostate carcinoma patient characteristics and disease and treatment patterns coincidental to this decline. The NCDB is a program of the American College of Surgeons Commission on Cancer and the American Cancer Society that collects timely data from institutions representing every level of cancer care. METHODS: Data from 1114 hospitals regarding 103,979 patients diagnosed with prostate carcinoma in 1992 and from 1144 hospitals regarding 72,337 patients diagnosed in 1995 were studied. Patient and disease characteristics studied included age, race, American Joint Committee on Cancer stage, and tumor histologic grade. Trends in treatment and variations in treatment by region also were examined. RESULTS: The average age at diagnosis declined by 2 years over the period studied and the proportion of patients identified as African-American increased from 8.8% to 11.8%. The proportion of patients diagnosed with localized disease and the proportion of tumors of moderately differentiated histologic grade increased. The overall proportion of patients treated by radical prostatectomy increased and the use of external beam radiation treatment decreased. Substantial regional variation in treatment patterns was observed. The Pacific and Mountain state regions of the U. S. had the highest proportions of patients treated by radical prostatectomy although increases were occurring in other regions. Use of external beam radiation treatment was highest in the Northeast and Southeast states and was declining in the majority of regions. Use of brachytherapy radiation treatment by implantation of radioactive seeds was infrequent but increasing. CONCLUSIONS: Comparison of the basic characteristics of prostate carcinoma data from 1992 and 1995 shows that patients are being diagnosed at younger ages and earlier stages, the proportion of African-Americans has increased, and more patients are being treated for cure. Regional differences in patterns of care persist but may be moderating. The annual prostate carcinoma death rate in the U.S. has declined an average of 1% per year since 1990.
Age Factors↗
Induction of apoptosis in proliferating human endothelial cells by the tumor-specific antiangiogenesis agent combretastatin A-4.
The antiangiogenic, tubulin-binding drug combretastatin A-4 exhibits a selective toxicity for proliferating endothelial cells in vitro and induces vascular shutdown in tumor models in vivo. The mechanism of combretastatin A-4 cytotoxicity has now been investigated with cultured proliferating human umbilical vein endothelial cells by examining various markers of apoptosis. Incubation of cells with 0.1 mM combretastatin A-4 induced the conversion (first detected after 6 h) of the CPP32 proenzyme to active caspase-3, a cysteine protease that plays an important role in apoptosis in many cell types; the drug also increased caspase-3 activity. Another early event observed was the binding of annexin V to 50% of the cells 8 h after drug treatment. Internucleosomal DNA fragmentation, another hallmark of apoptosis, was detected in cells incubated with 0.1 mM combretastatin A-4 for 24 h. Staining with Hoechst 33258 revealed that about 75% of cells exhibited a nuclear morphology characteristic of apoptosis after incubation with drug for 24 h. Incubation of cells for up to 8 h with combretastatin A-4 did not induce the release of lactate dehydrogenase or increase the uptake of propidium iodide, both indicators of membrane integrity. These results indicate that the selective cytotoxic effect of combretastatin A-4 is mediated by the induction of apoptosis rather than by necrosis and may provide an enhanced clinical strategy in cancer chemotherapy with this new agent.
Animals↗
The E7 protein of human papillomavirus type 16 sensitizes primary human keratinocytes to apoptosis.
The 'high risk' human papillomaviruses are associated with the development of anogenital carcinomas and their E6 and E7 genes possess immortalizing and transforming functions in several in vitro culture systems. Recently the E6 gene has also been shown to enhance the apoptosis of human mammary epithelial cells. To determine the apoptotic activity of these oncogenes in the natural host cell, we infected genital keratinocytes with retroviruses expressing either HPV-16 E6, E7, or both the E6 and E7 (E6/7) genes. Apoptosis was quantitated under normal growth conditions or when induced by tumor necrosis factor alpha/cycloheximide or sulfur mustard. In contrast to previous findings with mammary epithelial cells, the E6 gene did not significantly augment either spontaneous or induced apoptosis. E6 also did not suppress apoptosis in normal keratinocytes (despite dramatically reducing their p53 levels), suggesting that p53-independent events mediated this effect. In contrast, E7 increased both spontaneous and induced apoptosis as well as the cellular levels of p53 and p21 protein. Interestingly, co-expression of E6 abrogated E7-facilitated apoptosis by tumor necrosis factor alpha nearly completely, but had only a minor protective effect on sulfur mustard induced apoptosis in these cells, demonstrating at least in part the p53-dependence and -independence of these two apoptotic pathways. Finally, our results indicate that the apoptosis of normal and E7-expressing keratinocytes is differentially affected by E6 expression and that E7, when unaccompanied by E6, sensitizes keratinocytes to apoptosis.
Apoptosis↗
Regulation of the expression or recruitment of components of the DNA synthesome by poly(ADP-ribose) polymerase.
Poly(ADP-ribose) polymerase (PARP) is a component of the multiprotein DNA replication complex (MRC, DNA synthesome) that catalyzes replication of viral DNA in vitro. PARP poly(ADP-ribosyl)ates 15 of the approximately 40 proteins of the MRC, including DNA polymerase alpha (DNA pol alpha), DNA topoisomerase I (topo I), and proliferating-cell nuclear antigen (PCNA). Although about equal amounts of MRC-complexed and free forms of PCNA were detected by immunoblot analysis of HeLa cell extracts, only the complexed form was poly(ADP-ribosyl)ated, suggesting that poly(ADP-ribosyl)ation of PCNA may regulate its function within the MRC. NAD inhibited the activity of DNA pol delta in the MRC in a dose-dependent manner, whereas the PARP inhibitor, 3-AB, reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by characterizing the complex purified from 3T3-L1 cells before and 24 h after induction of a round of DNA replication required for differentiation of these cells; at the latter time point, approximately 95% of the cells are in S phase and exhibit a transient peak of PARP expression. The MRC was also purified from similarly treated 3T3-L1 cells depleted of PARP by antisense RNA expression; these cells do not undergo DNA replication nor terminal differentiation. Both PARP protein and activity and essentially all of the DNA pol alpha and delta activities exclusively cosedimented with the MRC fractions from S phase control cells, and were not detected in the MRC fractions from PARP-antisense or uninduced control cells. Immunoblot analysis further revealed that, although PCNA and topo I were present in total extracts from both control and PARP-antisense cells, they were present in the MRC fraction only from induced control cells, indicating that PARP may play a role in their assembly into an active DNA synthesome. In contrast, expression of DNA pol alpha, DNA primase, and RPA was down-regulated in PARP-antisense cells, suggesting that PARP may be involved in the expression of these proteins. Depletion of PARP also prevented induction of the expression of the transcription factor E2F-1, which positively regulates transcription of the DNA pol alpha and PCNA genes; thus, PARP may be necessary for expression of these genes when quiescent cells are stimulated to proliferate.
3T3 Cells↗
Transient poly(ADP-ribosyl)ation of nuclear proteins and role of poly(ADP-ribose) polymerase in the early stages of apoptosis.
A transient burst of poly(ADP-ribosyl)ation of nuclear proteins occurs early, prior to commitment to death, in human osteosarcoma cells undergoing apoptosis, followed by caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP). The generality of this early burst of poly(ADP-ribosyl)ation has now been investigated with human HL-60 cells, mouse 3T3-L1, and immortalized fibroblasts derived from wild-type mice. The effects of eliminating this early transient modification of nuclear proteins by depletion of PARP protein either by antisense RNA expression or by gene disruption on various morphological and biochemical markers of apoptosis were then examined. Marked caspase-3-like PARP cleavage activity, proteolytic processing of CPP32 to its active form, internucleosomal DNA fragmentation, and nuclear morphological changes associated with apoptosis were induced in control 3T3-L1 cells treated for 24 h with anti-Fas and cycloheximide but not in PARP-depleted 3T3-L1 antisense cells exposed to these inducers. Similar results were obtained with control and PARP-depleted human Jurkat T cells. Whereas immortalized PARP +/+ fibroblasts showed the early burst of poly(ADP-ribosyl)ation and a rapid apoptotic response when exposed to anti-Fas and cycloheximide, PARP -/- fibroblasts exhibited neither the early poly (ADP-ribosyl)ation nor any of the biochemical or morphological changes characteristic of apoptosis when similarly treated. Stable transfection of PARP -/- fibroblasts with wild-type PARP rendered the cells sensitive to Fas-mediated apoptosis. These results suggest that PARP and poly(ADP-ribosyl)ation may trigger key steps in the apoptotic program. Subsequent degradation of PARP by caspase-3-like proteases may prevent depletion of NAD and ATP or release certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition, both of which might be required for late stages of apoptosis.
3T3 Cells↗
Collaborative approaches to purchasing and managing oncology services for a prepaid population.
BACKGROUND: Change created by managed care plans is producing tensions among oncology care providers, health plans, patients, and employers. Managed care plans, which now are dominant, are engendering concern among some patients and providers because those plans limit provider choice and reimbursement, as well as inject themselves in clinical decision-making. Collaborative approaches to purchasing and managing oncology service for a prepaid population should help reduce these tensions. METHODS: This article provides a case history of how Harvard University Health Services, a managed care delivery and insurance program serving the university collaborates with contracted oncology providers. The described approach defines mutually beneficial payment and shared responsibility for care in the context of patient centered values. As a part of this collaborative effort, liberal experimental treatment coverage is offered. RESULTS: Patient care is improved because the flow of clinical information among caregivers is improved. When communication is more open, nurse case managers who work for the managed care plan also can arrange for covered services in a more expeditious manner. CONCLUSIONS: Collaboration among health plans, oncologists, and other health care providers to provide patients with high quality, cost-effective care on a fully informed basis is challenging, but not impossible. The key is to define common ground and commitment to the needs of patients among health plans and providers.
Cooperative Behavior↗
Sphingosine 1-phosphate inhibits activation of caspases that cleave poly(ADP-ribose) polymerase and lamins during Fas- and ceramide-mediated apoptosis in Jurkat T lymphocytes.
Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7.
Apoptosis↗
Sulfur mustard induces markers of terminal differentiation and apoptosis in keratinocytes via a Ca2+-calmodulin and caspase-dependent pathway.
Sulfur mustard (SM) induces vesication via poorly understood pathways. The blisters that are formed result primarily from the detachment of the epidermis from the dermis at the level of the basement membrane. In addition, there is toxicity to the basal cells, although no careful study has been performed to determine the precise mode of cell death biochemically. We describe here two potential mechanisms by which SM causes basal cell death and detachment: namely, induction of terminal differentiation and apoptosis. In the presence of 100 microM SM, terminal differentiation was rapidly induced in primary human keratinocytes that included the expression of the differentiation-specific markers K1 and K10 and the cross-linking of the cornified envelope precursor protein involucrin. The expression of the attachment protein, fibronectin, was also reduced in a time- and dose-dependent fashion. Features common to both differentiation and apoptosis were also induced in 100 microM SM, including the rapid induction of p53 and the reduction of Bcl-2. At higher concentrations of SM (i.e., 300 microM), formation of the characteristic nucleosome-sized DNA ladders, TUNEL-positive staining of cells, activation of the cysteine protease caspase-3/apopain, and cleavage of the death substrate poly(ADP-ribose) polymerase, were observed both in vivo and in vitro. Both the differentiation and the apoptotic processes appeared to be calmodulin dependent, because the calmodulin inhibitor W-7 blocked the expression of the differentiation-specific markers, as well as the apoptotic response, in a concentration-dependent fashion. In addition, the intracellular Ca2+ chelator, BAPTA-AM, blocked the differentiation response and attenuated the apoptotic response. These results suggest a strategy for designing inhibitors of SM vesication via the Ca2+-calmodulin or caspase-3/PARP pathway.
Apoptosis↗
Build consensus and develop collaborative practice guidelines.
With differing treatment guidelines available from many sources, collaborating advanced practice nurses and physicians at this facility recognized the need for one clear, acceptable set. Survey results show that clinicians incorporate all or most of the guidelines into practice (91%).
Ambulatory Care↗
Intact cell evidence for the early synthesis, and subsequent late apopain-mediated suppression, of poly(ADP-ribose) during apoptosis.
Poly(ADP-ribose) polymerase (PARP), which is catalytically activated by DNA strand breaks, has been implicated in apoptosis, or programmed cell death. A protease (CPP32) responsible for the cleavage of PARP and necessary for apoptosis was recently purified and characterized. The coordinated sequence of events related to PARP activation and cleavage in apoptosis has now been examined in individual cells. Apoptosis was studied in a human osteosarcoma cell line that undergoes a slow (8 to 10 days), spontaneous, and reproducible death program in culture. Changes in the abundance of intact PARP, poly(ADP-ribose) (PAR), and a proteolytic cleavage product of PARP that contains the DNA-binding domain were examined during apoptosis in the context of individual, whole cells by immunofluorescence with specific antibodies. The synthesis of PAR from NAD increased early, within 2 days of cell plating for apoptosis, prior to the appearance of internucleosomal DNA cleavage and before the cells become irreversibly committed to apoptosis, since replating yields viable, nonapoptotic cells. Strong expression of full-length PARP was also detected, by immunofluorescence as well as by Western analysis, during this same time period. However, after approximately 4 days in culture, the abundance of both full-length PARP and PAR decreased markedly. After 6 days, a proteolytic cleavage product containing the DNA-binding domain of PARP was detected immunocytochemically and confirmed by Western analysis, both in the nuclei and in the cytoplasm of cells. A recombinant peptide spanning the DNA-binding domain of PARP was expressed, purified, and biotinylated, and was then used as a probe for DNA strand breaks. Fluorescence microscopy with this probe revealed extensive DNA fragmentation during the later stages of apoptosis. This is the first report, using individual, intact cells, demonstrating that poly(ADP-ribosyl)ation of nuclear proteins occurs prior to the commitment to apoptosis, that inactivation and cleavage of PARP begin shortly thereafter, and that very little PAR per se is present during the later stages of apoptosis, despite the presence of a very large number of DNA strand breaks. These results suggest a negative regulatory role for PARP during apoptosis, which in turn may reflect the requirement for adequate NAD and ATP during the later stages of programmed cell death.
Apoptosis↗
Detection of DNA breaks in apoptotic cells utilizing the DNA binding domain of poly(ADP-ribose) polymerase with fluorescence microscopy.
The DNA binding domain (DBD) of poly(ADP-ribose) polymerase (PARP) has proved to be a novel, highly sensitive probe for detecting DNA breaks in intact cells undergoing apoptosis. A recombinant peptide spanning the DNA binding domain of PARP was expressed, purified and used to detect DNA strand breaks in fixed cells. Fluorescence microscopy with this probe followed by detection with anti-PARP antisera initially revealed an increased binding following treatment of cells with DNA strand-breaking agents (such asN-methyl-N'-nitro-N-nitrosoguanidine) and, subsequently, using biotinylated PARP DBD, during the later stages of apoptosis in several cell systems, when internucleosomal strand breaks became evident. This procedure was found to be at least as sensitive and required fewer steps to detect DNA strand breaks than those utilizing Klenow incorporation of biotinylated nucleotides.
Apoptosis↗