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HL-60 cells undergo apoptotic DNA fragmentation following X-irradiation. HL-60 (lanes 1 and 2) and HCW-2 (lanes 3 and 4) cells were either left untreated (lanes 1 and 3) or exposed to 10 Gy of X rays (lanes 2 and 4), and at 72 h postirradiation the status of chromosomal DNA was assessed by agarose gel analysis. Lane M, 123-bp DNA markers.
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The p53 tumor suppressor gene is thought to be required for the induction of programmed cell death (apoptosis) initiated by
DNA damage. We show here, however, that the human promyelocytic leukemia cell line HL-60, which is known to be deficient in
p53 because of large deletions in the p53 gene, can be induced to undergo apoptosis following X-irradi...
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... and analyzed by agarose gel electrophoresis. In HL-60 cells, there was a large amount of fragmented DNA (Fig. 5, compare lanes 1 and 2). The observed nucleosomal, ladder-like pattern of fragmented DNA is a hallmark of apoptosis (65). As expected, there was little DNA degradation in X-irradiated HCW-2 cells even at 72 h postirradiation (Fig. 5, compare lanes 3 and 4). Thus, X-irradiated HL-60 cells died by apoptosis, whereas HCW-2 cells were resistant to apoptosis induction by X rays. G 1 -arrested HL-60 cells are resistant to apoptotic induction by X rays. The above results strongly indicated that following DNA damage, HL-60 cells progressed to the G 2 phase of the cell cycle, whereupon they ...
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... the status of bax in these two cell lines. Northern analysis showed that bax mRNA was approximately equally expressed in the two cell lines (Fig. 8A, compare lanes 1 and 4). X-irradiation resulted in a slight down regulation of bax mRNA in HL-60 cells at 24 and 48 h (Fig. 8A, lanes 2 and 3), while bax expression remained unchanged in HCW-2 cells (Fig. 8A, lanes 5 and 6). Interestingly, Western analysis showed that, in comparison with HL-60 cells, bax was highly overex- pressed in HCW-2 cells (Fig. 8B, compare lanes 1 and 4). This observation suggests that significant regulation of bax may oc- cur at the posttranscriptional level (13). In HL-60 cells, bax protein increased slightly following ...
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... cells were exposed to 10 Gy of X rays, and then at 0 (lanes 1 and 4), 10 (lanes 2 and 5), or 25 (lanes 3 and 6) h, cell extracts were prepared and subjected to a Western analysis using an antibody specific for bcl-2. these cells (Fig. 8B, lanes 2 and 3). However, in HCW-2 cells, bax expression was unaffected by X-irradiation at subsequent times (Fig. 8B, lanes 5 and 6). Thus, again paradoxically, HCW-2 cells are more radioresistant and apoptosis resistant than the parental HL-60 cells, even though they overexpress ...
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... to that observed in large variety of p53 wild-type cell lines (1, 2, 16). In addition, no DNA repair defect was observed following UV irradiation of a p53-deficient mouse cell line (24). Thus, a direct role for p53 in DNA repair seems to be ruled out. We have also demonstrated that X rays can induce apoptosis in the p53- deficient HL-60 cell line (Fig. 4 and 5). This agrees with the observation that apoptosis can still be stimulated by other factors, such as glucocorticoids, in p53-knockout animals (8,39). Thus, it also seems highly unlikely that p53 actively par- ticipates in apoptotic DNA ...
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Citations
... 4 Disruption of the checkpoint systems may limit or prevent DNA repair, leading to mitotic defects such as chromosomal abnormalities, apoptosis and/or programmed cell death. 5,6 In particular, the G2/M checkpoint helps to maintain genetic stability by blocking damaged or incompletely replicated DNA from entering mitosis until repair is complete. 7 Cell cycle regulators such as checkpoint kinases, p53, polo-like kinases, Aurora kinases and never in mitosis gene A (NIMA)-related kinases are involved in controlling the G2/M checkpoint and regulating centrosome segregation. ...
NIMA‐related kinase 2 (NEK2) is a serine/threonine protein kinase that regulates mitosis and plays pivotal roles in cell cycle regulation and DNA damage repair. However, its function in porcine embryonic development is unknown. In this study, we used an NEK2‐specific inhibitor, JH295 (JH), to investigate the role of NEK2 in embryonic development and the underlying regulatory mechanisms. Inhibition of NEK2 after parthenogenesis activation or in vitro fertilization significantly reduced the rates of cleavage and blastocyst formation, the numbers of trophectoderm and total cells and the cellular survival rate compared with the control condition. NEK2 inhibition delayed cell cycle progression at all stages from interphase to cytokinesis during the first mitotic division; it caused abnormal nuclear morphology in two‐ and four‐cell stage embryos. Additionally, NEK2 inhibition significantly increased DNA damage and apoptosis, and it altered the expression levels of DNA damage repair‐ and apoptosis‐related genes. Intriguingly, NEK2 inhibition downregulated the expression of β‐catenin and its downstream target genes. To validate the relationship between Wnt/β‐catenin signalling and NEK2 during porcine embryonic development, we cultured porcine embryos in JH‐treated medium with or without CHIR99021, a Wnt activator. CHIR99021 co‐treatment strongly restored the developmental parameters reduced by NEK2 inhibition to control levels. Our findings suggest that NEK2 plays an essential role in porcine embryonic development by regulating DNA damage repair and normal mitotic division via the Wnt/β‐catenin signalling pathway.
... However, the onset of carcinoma and indeed the immortalisation process of cells can alter the normal control of the cell cycle (Stacey et al. 2009). There are three important checkpoints during cell cycle, the first, G1 checkpoint between the G1/S phase, the second, G2 checkpoint between the G2/M phase and the spindle checkpoint in the mitotic phase between metaphase and anaphase (Han et al. 1995;Gorbsky 2001;Seluanov et al. 2009). Interestingly, statistically significant (P C 0.05) differences were noted in the cell cycle assay, which were seen to be dependent on the working concentration of the Collagen concentration, cell population numbers in the G0/G1 phase decreasing and S-Phase population numbers increasing with decreasing Collagen working concentration, indicating that the presence of the Collagen substrate most likely altered the cycle of the HeLa cells (Fig. 6) by arresting cells in the G0/G1 phase. ...
In this study, the cellular viability and function of immortalized human cervical and dermal cells are monitored and compared in conventional 2D and two commercial 3D membranes, Collagen and Geltrex, of varying working concentration and volume. Viability was monitored with the aid of the Alamar Blue assay, cellular morphology was monitored with confocal microscopy, and cell cycle studies and cell death mechanism studies were performed with flow cytometry. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to 3D environment causing alterations to effective resazurin concentration, uptake and conversion rates, which was dependent on exposure time, but also due to the effect of the membrane itself on cellular function. These effects were verified by flow cytometry, in which no significant differences in viable cell numbers between 2D and 3D systems were observed after 24 h culture. The results showed the observed effect was different after shorter exposure periods, was also dependent on working concentration of the 3D system and could be mediated by altering the culture vessel size. Cell cycle analysis revealed cellular function could be altered by growth on the 3D substrates and the alterations were noted to be dependent on 3D membrane concentration. The use of 3D culture matrices has been widely interpreted to result in “improved viability levels” or “reduced” toxicity or cellular “resistance” compared to cells cultured on traditional 2D systems. The results of this study show that cellular health and viability levels are not altered by culture in 3D environments, but their normal cycle can be altered as indicated in the cell cycle studies performed and such variations must be accounted for in studies employing 3D membranes for in vitro cellular screening.
... Tumor protein p53 (p53) is a common tumor suppressor protein that is frequently activated when the cell is damaged, and which serves a role in various signaling pathways to regulate cell survival and death. p53 can affect DNA repair and cell cycle progression, and serves an important role in the inhibition of tumorigenesis (22)(23)(24)(25). If a mutation of the gene encoding p53 occurs, the mutated p53 protein produced cannot trigger apoptosis, resulting in tumorigenesis (26)(27)(28). ...
Hemangioma is one of the most common types of infantile vascular benign tumor. The aim of the present study was to investigate the role of B-cell lymphoma 2 (Bcl-2) and tumor protein p53 (p53) in the proliferation and apoptosis of hemangioma cells. A total of 38 paraffin-embedded hemangioma specimens (16 males and 22 females) and another 5 paraffin-embedded healthy surrounding tissue samples, collected between January 2007 and December 2010, were obtained from the Department of Pathology at Renmin Hospital of Wuhan University (Wuhan, China). Immunohistochemistry, hematoxylin and eosin staining, and quantum dot double staining were used to detect the expression of proliferating cell nuclear antigen (PCNA), Bcl-2 and p53 in hemangioma and healthy surrounding skin tissue samples. All hemangioma specimens were classified into proliferative or the involuting stage hemangioma according to Mulliken's criteria and their expression of PCNA. The results of the quantum dot double staining were analyzed using a multi-spectral imaging system. One-way analysis of the variance and the Student-Newman-Keuls q test were performed to statistically analyze the data. There were 24 cases of proliferative stage and 14 cases of involuting stage hemangioma among the specimens. Immunohistochemical analysis results indicated a high expression of Bcl-2 and p53 in proliferative stage hemangioma tissue samples, and low expression in involuting stage hemangioma and healthy tissue samples. Statistical analysis of the results from quantum dot double staining demonstrated that the expression of Bcl-2 and p53 in proliferative hemangioma was significantly increased compared with that in involuting stage specimens (P<0.05) and healthy tissue samples (P<0.05). No significant difference in Bcl-2 and p53 expression was identified between the involuting hemangioma and healthy surrounding tissue samples. The higher expression of Bcl-2 and p53 in proliferative hemangioma suggests that Bcl-2 may cause an imbalance between endothelial cell proliferation and apoptosis through the inhibition of endothelial cell apoptosis. Furthermore, p53 may promote the proliferation of endothelial cells in proliferative hemangioma.
... Accordingly, in this study, the effect of HAs on cell cycle and proteins involved in the cell cycle regulation in human promyelocytic leukemia (HL-60) cell line was investigated. The HL-60, which lack of p53 expression was attributed to p53 gene deletion (Wolf and Rotter, 1985), can be induced to undergo apoptosis following chemicals or radiation treatments (Brodska et al., 2014;Han et al., 1995). The tumor suppressor p53 is also the key protein in checkpoint pathways, which activation may lead to growth arrest at both G1 and G2/M phases in several cancer cells (Stark and Taylor, 2006). ...
Cell cycle regulation is an important issue in cancer therapy. Delphinidin and cyanidin are two major anthocyanins of the roselle plant (Hibiscus sabdariffa). In the present study, we investigated the effect of Hibiscus anthocyanins (HAs) on cell cycle arrest in human leukemia cell line HL-60 and the analyzed the underlying molecular mechanisms. HAs extracted from roselle calyces (purity 90%) markedly induced G2/M arrest evaluated with flow cytometry analysis. Western blot analyses revealed that HAs (0.1–0.7 mg mL−1) induced G2/M arrest via increasing Tyr15 phosphorylation of Cdc2, and inducing Cdk inhibitors p27 and p21. HAs also induced phosphorylation of upstream signals related to G2/M arrest such as phosphorylation of Cdc25C tyrosine phosphatase at Ser216, increasing the binding of pCdc25C with 14-3-3 protein. HAs-induced phosphorylation of Cdc25C could be activated by ATM checkpoint kinases, Chk1, and Chk2. We first time confirmed that ATM-Chk1/2-Cdc25C pathway as a critical mechanism for G2/M arrest in HAs-induced leukemia cell cycle arrest, indicating that this compound could be a promising anticancer candidate or chemopreventive agents for further investigation. © 2016 Wiley Periodicals, Inc. Environ Toxicol, 2016.
... Although the tumour suppressor gene p53 is known to play a key role in the translocation of Bax or Bak onto mitochondria to mediate in apoptotic cell death, its functionality and wild-type status are not required for cells to undergo Bax-or Bak-mediated apoptosis [6,7]. In fact, the translocation of Bax to mitochondria in response to exposure to cytotoxic agents has been demonstrated in cells lacking functional p53 [8,9]. ...
... The p53 gene is known to exhibit point mutations and is dysfunctional in DU145 cells [16]. However, wild-type or functional p53 is not always required for cells to undergo mitochondria-mediated apoptosis [6,7]. In fact, the translocation of Bax or Bak to mitochondria in response to exposure to cytotoxic agents has been demonstrated in cells lacking functional p53 [8,9]. ...
... Since HL-60 cells are p53-deficient, apoptosis after irradiation can be induced by a p53-independent mechanism at the G2 checkpoint [31]. Those cells also exhibit a markedly longer G2 arrest for DNA damage repair in coordination with their higher radioresistance [33]. Our present work describes a dose-dependent effect of low-dose irradiation on apoptosis of HL-60 cells. ...
The biological effects of low-dose radiation have attracted attention, but data are currently insufficient to fully understand
the beneficial role of the phenomenon. In the present study, we have investigated the effects of low doses of gamma-irradiation
alone and in combination with all-trans-retinoic acid (RA) on proliferation, apoptosis and differentiation of the human promyelocytic leukemia HL-60 cells. Changes
in cell behavior and protein expression were determined with the use of light and fluorescent microscopy, immunocytochemical
and Western blot analysis. Low-dose irradiation with 1–100 cGy caused a dose-dependent inhibition of HL-60 cell proliferation,
and induced apoptosis and differentiation to granulocytes with an increase in the number of CD15-positive cells. Pre-irradiation
with 1–100 cGy for 24 h before treatment with RA promoted apoptosis but did not impair RA-induced differentiation. Both processes
were associated with a decrease in the expression of the proliferating cell nuclear antigen (PCNA), BCL-2, c-MYC, and changes
in both cytosolic and nuclear levels of protein tyrosine-phosphorylation as well as protein kinase C alpha or beta isoforms.
These results demonstrate the beneficial role of low-dose irradiation in modulating leukemia cell proliferation, differentiation
and apoptosis.
... p53 führt unter anderem zur Aktivierung von Proteinen der bcl-2-Familie und der BAX-Subfamilie, die sich daraufhin an die äußere Mitochondrienmembran heften und zur Freisetzung von Cytochrom C führen [Adams und Cory, 1998]. Dies bewirkt die Ausbildung des so genannten Apoptosoms -einem Komplex aus Cytochrom C, Apaf-1 und Caspase 9 [Cain et al., 1999] Auch p53-unabhängige Apoptose ist beschrieben worden [Han et al., 1995;Seki et al., 1994]. So können Nur77 [Li et al., 2000], Caspase 2 [Tinel und Tschopp, 2004], p73 [Melino et al., 2004] oder Chk2 [Yang et al., 2002] Der Ausfall eines Gens kann von anderen kompensiert werden. ...
Das Nijmegen-Breakage-Syndrom (NBS) ist ein autosomal-rezessiv vererbtes Chromosomenbruchsyndrom, dem in > 90% der Patienten eine 5bp-Deletion im Nbs1-Gen zugrundeliegt. Klinisches Hauptmerkmal ist ein stark erhöhtes Krebsrisiko, insbesondere für B-Zell-Lymphome. Bereits bekannt ist die Funktion des entsprechenden Genprodukts, Nibrin, bei den für die Krebsprävention wichtigen Mechanismen der DNA-Reparatur und der Zellzykluskontrolle. Daneben spielt die Apoptose eine essentielle Rolle bei der Krebsentstehung. Zu untersuchen ob Nibrin auch hier Funktionen übernimmt war Gegenstand dieser Arbeit. Eine Störung der Apoptose könnte dabei mitverantwortlich für das hohe Krebsrisiko der NBS-Patienten sein. Kern der Arbeit war die Untersuchung von NBS-B-Lymphozyten hinsichtlich ihrer Fähigkeit, nach einer DNA-Schädigung die Apoptose zu induzieren. Hierzu wurde in den entsprechenden Zellen mittels Bleomycin der Apoptoseprozess ausgelöst und die prozentualen Apoptoseraten durchflusszytometrisch bestimmt. Die Mehrheit der NBS-Zelllinien zeigte eine Störung in der Apoptoseinduktion im Sinne signifikant verminderter Apoptoseraten. Dies weist auf eine Funktion des Nibrins bei der Induktion der Apoptose hin. Andere NBS-Zelllinien zeigten normale Apoptoseindices. Dies könnte auf dem individuellen genetischen Hintergrund der Zellen beruhen, der auch für die erhebliche klinische Variabilität des Krankheitsbildes verantwortlich ist. Eine Korrelation der Apoptoseraten mit der Krebsinzidenz zeigte, dass alle Patienten mit reduzierten Apoptoseraten bereits Lymphome entwickelt hatten, während Patienten mit normalen Apoptoseindices bisher keine Lymphome aufwiesen. Möglicherweise gibt es also generell zwei Gruppen von NBS-Patienten - Patienten mit höherem und mit niedrigerem Entartungsrisiko, wobei eine verminderte Apoptoseinduktion als Risikofaktor für Krebs angesehen werden könnte.
... A toxic effect was seen at concentrations >100 M and the IC 50 differed slightly for the different cell types tested, HL-60 cells being more sensitive than Raji cells. HL60 cells are p53-deficient due to deletions in the gene coding for this protein [24], but nevertheless they undergo apoptosis readily and show a G2 checkpoint [25]. They have been found to be more sensitive than other cell lines in studies of anticancer drugs [26,27]. ...
Compounds of the 1,4-dihydropyridine (1,4-DHP) series have been shown to reduce spontaneous, alkylation- and radiation-induced mutation rates in animal test systems. Here we report studies using AV-153, the 1,4-DHP derivative that showed the highest antimutagenic activity in those tests, to examine if it modulates DNA repair in human peripheral blood lymphocytes and in two human lymphoblastoid cell lines, Raji and HL-60. AV-153 caused a 50% inhibition of growth (IC50) of Raji and HL-60 cells at 14.9+/-1.2 and 10.3+/-0.8mM, respectively, but did not show a cytotoxic effect at concentrations <100 microM. Alkaline single-cell gel electrophoresis (comet) assays showed that AV-153 reduced the number of DNA strand breaks in untreated cells and also in cells exposed to 2 Gy of gamma-radiation, 100 microM ethylmethane sulfonate (EMS), or 100 microM H2O2. DNA damage was reduced by up to 87% at AV-153 concentrations between 1 and 10nM, and a positive dose-effect relationship was seen between 0.01 and 1 nM. Comparison of the kinetics of DNA strand-break rejoining in the presence and absence of AV-153 revealed a considerable influence on the rate of repair. In view of the resemblance of this compound's structure to that of dihydronicotinamide, a substrate for poly(ADP-rybose)polymerase, the modulation of DNA repair by AV-153 could involve an influence on poly(ADP)ribosylation.
... The ubiquitous distribution of nicotinic acetylcholine receptors and their possible involvements in many medical fields, especially in cancer, prompt us to initiate investigations on apoptosis. Our model is based on the use of a radiomimetic agent bleomycin on HL-60 cell line known to be from hematological origin and likely to express nicotinic acetylcholine receptors (Richman, 1979;Lebargy et al., 1996;Benhammou et al., 2000;Villiger et al., 2002) together with the bcl-2 anti-apoptotic gene (Han et al., 1995;Wen et al., 2000). The aim of the study was (i) to observe the nicotine effects on bleomycin-induced apoptosis in HL-60 cell line, (ii) to determine whether nicotine acted through nicotinic acetylcholine receptors, and (iii) if so, to determine pharmacologically the subunit of the nicotinic acetylcholine receptors. ...
The subunit composition of nicotinic acetylcholine receptors involved in apoptosis is an ongoing question. HL-60 cells were used in order to investigate the implication of nicotinic acetylcholine receptors in bleomycin-induced apoptosis. We found that bleomycin-induced apoptosis was significantly enhanced by nicotine and was blocked by nicotinic acetylcholine receptor antagonists, including alpha-bungarotoxin, a competitive antagonist of alpha 7 nicotinic receptor. Among the other agonists tested, 3-[2,4-dimethoxybenzylidene]anabaseine (GTS-21)-selective agonist for alpha 7-nicotinic acetylcholine receptor-, but not epibatidine or cytisine, enhanced bleomycin-induced apoptosis. In addition to these results, the detectable presence of alpha 7-mRNA supports a key role of alpha 7-nicotinic acetylcholine receptors in the modulation of the induced apoptosis by nicotine.
... G1-and G2 checkpoints are crucial for the detection of DNA damage, DNA repair and induction of apoptosis. Prior to apoptosis induction, p53-deficient cells arrest in the G2-phase of the cell cycle probably due to the lack of an intact G1 checkpoint (Han et al., 1995). This G2 arrest is then required for the repair of DNA damage induced by radiation. ...
... The decrease of survivin that is expressed in G2/M and protects cells from apoptosis during G2/M transitions by inhibiting the terminal effector caspases À3 and À7 may contribute to the toxicity of GT. For p53-deficient HL-60 cells lacking the G1 arrest, the G2/M checkpoint is crucially important for the decision to repair DNA damage induced by radiation or undergo apoptosis (Han et al., 1995). As shown in HL-60 cells and other cell lines, that is, fibroblasts (Powell et al., 1995) or human lung cancer cells (Russell et al., 1995), the length of G2/M arrest is correlated with radioresistance. ...
Radioresistance markedly impairs the efficacy of tumor radiotherapy and may involve antiapoptotic signal transduction pathways that prevent radiation-induced cell death. A common cellular response to genotoxic stress induced by radiation is the activation of the nuclear factor kappa B (NF-kappaB). NF-kappaB activation in turn can lead to an inhibition of radiation-induced apoptotic cell death. Thus, inhibition of NF-kappaB activation is commonly regarded as an important strategy to abolish radioresistance. Among other compounds, the fungal metabolite gliotoxin (GT) has been reported to be a highly selective inhibitor of NF-kappaB activation. Indeed, low doses of GT were sufficient to significantly enhance radiation-induced apoptosis in HL-60 cells. However, this effect turned out to be largely independent of NF-kappaB activation since radiation of HL-60 cells with clinically relevant doses of radiation induced only a marginal increase in NF-kappaB activity, and selective inhibition of NF-kappaB by SN50 did not result in a marked enhancement of GT-induced apoptosis. GT induced activation of JNKs, cytochrome c release from the mitochondria and potently stimulated the caspase cascade inducing cleavage of caspases -9, -8, -7 and -3. Furthermore, cleavage of the antiapoptotic protein X-linked IAP and downregulation of the G2/M-specific IAP-family member survivin were observed during GT-induced apoptosis. Finally, the radiation-induced G2/M arrest was markedly reduced in GT-treated cells most likely due to the rapid induction of apoptosis. Our data demonstrate that various other pathways apart from the NF-kappaB signaling complex can sensitize tumor cells to radiation and propose a novel mechanism for radiosensitization by GT, the interference with the G2/M checkpoint that is important for repair of radiation-induced DNA damage in p53-deficient tumor cells.
