Relationship of DNA damage signaling to DNA replication following treatment with DNA topoisomerase inhibitors camptothecin/topotecan, mitoxantrone, or etoposide.

DNA topoisomerase I (Top1) and topoisomerase II (Top2) inhibitors are widely used to treat a variety of cancers. Their mechanism of action involves stabilization of otherwise transient ("cleavable") complexes between Top1 or Top2 and DNA; collisions of DNA replication forks with such stabilized complexes lead to formation of DNA double-strand breaks (DSBs). In this study, using 5-ethynyl-2'deoxyuridine (EdU) as a DNA precursor, we directly assessed the relationship between DNA replication and induction of DSBs revealed as γH2AX foci in A549 cells treated with Top1 inhibitors topotecan (Tpt) or camptothecin (Cpt) and Top2 inhibitors mitoxantrone (Mxt) and etoposide (Etp). Analysis of cells by multiparameter laser scanning cytometry following treatment with Tpt or Cpt revealed that only DNA replicating cells showed induction of γH2AX and a strong correlation between DNA replication and formation of DSBs (r = 0.86). In cells treated with Mxt or Etp, the correlation was weaker (r = 0.52 and 0.64). In addition, both Mtx and Etp caused induction of γH2AX in cells not replicating DNA. Confocal imaging of nuclei of cells treated with Tpt revealed the presence of γH2AX foci predominantly in DNA replicating cells and close association and co-localization of γH2AX foci with DNA replication sites. In cells treated with Mxt or Etp, the γH2AX foci were induced in DNA replicating as well as non-replicating cells but the close association between a large proportion of γH2AX foci and DNA replication sites was also apparent. The data are consistent with the view that collision of DNA replication forks with cleavable Top1-DNA complexes stabilized by Tpt/Cpt is the sole cause of induction of DSBs. Additional mechanisms such as involvement of transcription and/or generation of oxidative stress may contribute to DSBs induction by Mxt and Etp. The confocal analysis of the association between DNA replication sites and the sites of DSBs (γH2AX foci) opens a new approach for mechanistic studies of the involvement of DNA replication in induction of DNA damage.

Cytometry of DNA replication and RNA synthesis: Historical perspective and recent advances based on "click chemistry".

This review covers progress in the development of cytometric methodologies designed to assess DNA replication and RNA synthesis. The early approaches utilizing autoradiography to detect incorporation of (3) H- or (14) C-labeled thymidine were able to identify the four fundamental phases of the cell cycle G(1) , S, G(2) , and M, and by analysis of the fraction of labeled mitosis (FLM), to precisely define the kinetics of cell progression through these phases. Analysis of (3) H-uridine incorporation and RNA content provided the means to distinguish quiescent G(0) from cycling G(1) cells. Subsequent progress in analysis of DNA replication was based on the use of BrdU as a DNA precursor and its detection by the quenching of the fluorescence intensity of DNA-bound fluorochromes such as Hoechst 33358 or acridine orange as measured by flow cytometry. Several variants of this methodology have been designed and used in studies to detect anticancer drug-induced perturbations of cell cycle kinetics. The next phase of method development, which was particularly useful in studies of the cell cycle in vivo, including clinical applications, relied on immunocytochemical detection of incorporated halogenated DNA or RNA precursors. This approach however was hampered by the need for DNA denaturation, which made it difficult to concurrently detect other cell constituents for multiparametric analysis. The recently introduced "click chemistry" approach has no such limitation and is the method of choice for analysis of DNA replication and RNA synthesis. This method is based on the use of 5-ethynyl-2'deoxyuridine (EdU) as a DNA precursor or 5-ethynyluridine (EU) as an RNA precursor and their detection with fluorochrome-tagged azides utilizing a copper (I) catalyzed [3+2] cycloaddition. Several examples are presented that illustrate incorporation of EdU or EU in cells subjected to DNA damage detected as histone H2AX phosphorylation that have been analyzed by flow or laser scanning cytometry.

Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho-specific antibodies.

By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway. DNA damage in human pulmonary adenocarcinoma A549 cells was induced by treatment with the DNA topoisomerase I inhibitor camptothecin and phosphorylation of histone H2AX on Ser139 (γH2AX) and of ATM on Ser1981 was detected with phospho-specific Abs; cellular fluorescence was measured by laser scanning cytometry (LSC). The sensitivity and accuracy of detection of H2AX and ATM phosphorylation concurrent with the detection of DNA replication by EdU incorporation and "click chemistry" was found in ZBF fixed cells to be comparable to that of cell fixed in formaldehyde. The accuracy of DNA content measurement as evident from the resolution of DNA content frequency histograms of cells stained with DAPI was somewhat better in ZBF- than in formaldehyde-fixed cells. The pattern of chromatin condensation revealed by the intensity of maximal pixel of DAPI that allows one to identify mitotic and immediately post-mitotic cells by LSC was preserved after ZBF fixation. ZBF fixation was also compatible with the detection of γH2AX foci considered to be the hallmarks of induction of DNA double-strand breaks. Analysis of cells by flow cytometry revealed that ZBF fixation of lymphoblastoid TK6 cells led to about 60 and 33% higher intensity of the side and forward light scatter, respectively, compared to formaldehyde fixed cells.

Induction of DNA damage signaling by oxidative stress in relation to DNA replication as detected using "click chemistry".

Induction of DNA damage by oxidants such as H(2) O(2) activates the complex network of DNA damage response (DDR) pathways present in cells to initiate DNA repair, halt cell cycle progression, and prepare an apoptotic reaction. We have previously reported that activation of Ataxia Telangiectasia Mutated protein kinase (ATM) and induction of γH2AX are among the early events of the DDR induced by exposure of cells to H(2) O(2) , and in human pulmonary carcinoma A549 cells, both events were expressed predominantly during S-phase. This study was designed to further explore a correlation between these events and DNA replication. Toward this end, we utilized 5-ethynyl-2'deoxyuridine (EdU) and the "click chemistry" approach to label DNA during replication, followed by exposure of A549 cells to H(2) O(2) . Multiparameter laser scanning cytometric analysis of these cells made it possible to identify DNA replicating cells and directly correlate H(2) O(2) -induced ATM activation and induction of γH2AX with DNA replication on a cell by cell basis. After pulse-labeling with EdU and exposure to H(2) O(2) , confocal microscopy was also used to examine the localization of DNA replication sites ("replication factories") versus the H2AX phosphorylation sites (γH2AX foci) in nuclear chromatin in an attempt to observe the absence or presence of colocalization. The data indicate a close association between DNA replication and H2AX phosphorylation in A549 cells, suggesting that these DNA damage response events may be triggered by stalled replication forks and perhaps also by induction of DNA double-strand breaks at the primary DNA lesions induced by H(2) O(2) .

Kinetics of the UV-induced DNA damage response in relation to cell cycle phase. Correlation with DNA replication.

It has been reported that exposure to UV light triggers DNA damage response (DDR) seen as induction of gammaH2AX not only in S- but also in G(1)-phase cells. In the present study, in addition to gammaH2AX, we assessed other markers of DDR, namely phosphorylation of ATM on Ser1981, of ATM/ATR substrate on Ser/Thr at SQ/TQ cluster domains and of the tumor suppressor p53 on Ser15, in human pulmonary carcinoma A549 cells irradiated with 50 J/m(2) of UV-B. Phosphorylation of these proteins detected with phospho-specific Abs and measured by laser scanning cytometry in relation the cell cycle phase was found to be selective to S-phase cells. The kinetics of phosphorylation of ATM was strikingly similar to that of ATM/ATR substrate, peaking at 30 min after UV irradiation and followed by rapid dephosphorylation. The peak of H2AX phosphorylation was seen at 2 h and the peak of p53 phosphorylation at 4 h after exposure to UV light. Local high spatial density of these phospho-proteins reported by intensity of maximal pixel of immunofluorescence in the DDR nuclear foci was distinctly more pronounced in the early compared to late portion of S-phase. Exposure of cells to UV following 1 h pulse-labeling of their DNA with 5-ethynyl-2'deoxyuridine (EdU) made it possible to correlate the extent of DNA replication during the pulse with the extent of the UV-induced H2AX phosphorylation within the same cells. This correlation was very strong (R(2) = 0.98) and the cells that did not incorporate EdU showed no evidence of H2AX phosphorylation. The data are consistent with the mechanism in which stalling of DNA replication forks upon collision with the primary UV-induced DNA lesions and likely formation of double-strand DNA breaks triggers DDR. The prior reports (including our own) on induction of gammaH2AX in G(1) cells by UV may have erroneously identified cells initiating DNA replication following UV exposure as G(1) cells due to the fact that their DNA content did not significantly differ from that of G(1) cells that had not initiated DNA replication.

New biomarkers probing depth of cell senescence assessed by laser scanning cytometry.

The imaging analytical capabilities of laser scanning cytometer (LSC) have been used to assess morphological features considered to be typical of the senescent phenotype. The characteristic "flattening" of senescent cells was reflected by the decline in the density of staining (intensity of maximal pixel) of DNA-associated fluorescence [4,6-diamidino-2-phenylindole (DAPI)] paralleled by an increase in nuclear size (area). The decrease in ratio of maximal pixel to nuclear area was even more sensitive senescence biomarker than the change in maximal pixel or nuclear area, each alone. The saturation cell density at plateau phase of growth recorded by LSC was found to be dramatically decreased in cultures of senescent cells, thereby also serving as an additional marker. The induction of cyclin dependent kinase inhibitors p21(WAF1) and p27(KIP1) and γH2AX and activation of ATM markers of DNA damage response were measured in parallel with DNA/DAPI maximal pixel and nuclear area. These biomarker indices were expressed in quantitative terms by reporting them as a fraction of the respective controls. The effect of treatment of A549 and WI-38 cells with different concentrations of mitoxantrone (Mxt) and trichostatin A for various time periods was studied to assess the degree (depth) of cell senescence. Also assessed was the effect of 2-deoxy-D-glucose, the agent attenuating metabolic cell activity, on the depth of senescence induced by Mxt. A relationship between the ability of cells to synthesize RNA (incorporate 5-ethynyluridine) that leads to growth imbalance and induction of cell senescence was also studied. The data show that morphometric analysis of cellular attributes by LSC offers an attractive tool to detect cell senescence and measure its degree particularly in assessing effects of the factors that enhance or attenuate this process. This methodology is of importance in light of the evidence that cellular senescence is not only a biological process that is fundamental for organismal aging but also impedes formation of induced-pluripotent stem cells providing the barrier for neoplastic transformation and is the major mechanism of induction of reproductive cell death during treatment of solid tumors.

Induction of DNA damage response by the supravital probes of nucleic acids.

The aim of this study was to assess the potential DNA damage response (DDR) to four supravitally used biomarkers Hoechst 33342 (Ho 42), DRAQ5, DyeCycle Violet (DCV), and SYTO 17. A549 cells were exposed to these biomarkers at concentrations generally applied to live cells and their effect on histone H2AX (Ser 139), p53 (Ser15), ATM (Ser1981), and Chk2 (Thr68) phosphorylation was assessed using phospho-specific Abs. Short-term treatment with Ho 42 led to modest degree of ATM activation with no evidence of H2AX, Chk2, or p53 phosphorylation. However, pronounced ATM, Chk2, and p53 phosphorylation and perturbed G(2) progression were seen after 18 h. While short-term treatment with DRAQ5 induced ATM activation with no effect on H2AX, Chk2, and p53, dramatic changes marked by a high degree of H2AX, ATM, Chk2, and p53 phosphorylation, all occurring predominantly in S phase cells, and a block in cell cycle progression, were seen after 18 h exposure. These changes suggest that the DRAQ5-induced DNA lesions may become converted into double-strand DNA breaks during replication. Exposure to DCV also led to an increase in the level of activated ATM and Chk2 as well as of phosphorylated p53 and accumulation of cells in G(2)M and S phase. Exposure to SYTO 17 had no significant effect on any of the measured parameters. The data indicate that supravital use of Ho 42, DRAQ5, and DCV induces various degrees of DDR, including activation of ATM, Chk2 and p53, which may have significant consequences on regulatory cell cycle pathways and apoptosis.

DNA damage response induced by tobacco smoke in normal human bronchial epithelial and A549 pulmonary adenocarcinoma cells assessed by laser scanning cytometry.

Cigarette smoke (CS) is a major cause of lung cancer and a contributor to the development of a wide range of other malignancies. There is an acute need to develop a methodology that can rapidly assess the potential carcinogenic properties of the genotoxic agents present in CS. We recently reported that exposure of normal human bronchial epithelial cells (NHBEs) or A549 pulmonary carcinoma cells to CS induces the activation of ATM through its phosphorylation on Ser1981 and phosphorylation of histone H2AX on Ser139 (gammaH2AX) most likely in response to the formation of potentially carcinogenic DNA double-strand breaks (DSBs). To obtain a more complete view of the DNA damage response (DDR) we explored the correlation between ATM activation, H2AX phosphorylation, activation of Chk2 through its phosphorylation on Thr68, and phosphorylation of p53 on Ser15 in NHBE and A549 cell exposed to CS. Multiparameter analysis by laser scanning cytometry made it possible to relate these DDR events, detected immunocytochemically, with cell cycle phase. The CS-dose-dependent induction and increase in the extent of phosphorylation of ATM, Chk2, H2AX, and p53 were seen in both cell types. ATM and Chk2 were phosphorylated approximately 1 h prior to phosphorylation of H2AX and p53. The dephosphorylation of ATM, Chk2, and H2AX was seen after 2 h following CS exposure. The dose-dependency and kinetics of DDR were essentially similar in both cell types, which provide justification for the use of A549 cells in the assessment of genotoxicity of CS in lieu of normal bronchial epithelial cells. The observation that DDR was more pronounced in S-phase cells is consistent with the mechanism of induction of DSBs occurring as a result of collision of replication forks with primary lesions such as DNA adducts that can be caused by CS-generated oxidants. The cytometric assessment of CS-induced DDR provides a means to estimate the genotoxicity of CS and to explore the mechanisms of the response as a function of cell cycle phase and cell type.

Cytometric analysis of DNA damage: phosphorylation of histone H2AX as a marker of DNA double-strand breaks (DSBs).

Phosphorylation of histone H2AX on Ser 139 is a sensitive reporter of DNA damage, particularly if the damage involves induction of DNA double-strand breaks (DSBs). Phosphorylated H2AX has been named gammaH2AX and its presence in the nucleus can be detected immunocytochemically. Multiparameter analysis of gammaH2AX immunofluorescence by flow or laser-scanning cytometry allows one to measure extent of DNA damage in individual cells and to correlate it with their position in the cell cycle and induction of apoptosis. This chapter presents the protocols and outlines applications of multiparameter cytometry in analysis of H2AX phosphorylation as a reporter of the presence of DSBs.

gammaH2AX: A potential DNA damage response biomarker for assessing toxicological risk of tobacco products.

Differentiation among American cigarettes relies primarily on the use of proprietary tobacco blends, menthol, tobacco substitutes, paper porosity, paper additives, and filter ventilation. These characteristics substantially alter per cigarette yields of tar and nicotine in standardized protocols promulgated by government agencies. However, due to compensatory alterations in smoking behavior to sustain a preferred nicotine dose (e.g., by increasing puff frequency, inhaling more deeply, smoking more cigarettes per day, or blocking filter ventilation holes), smokers actually inhale similar amounts of tar and nicotine regardless of any cigarette variable, supporting epidemiological evidence that all brands have comparable disease risk. Consequently, it would be advantageous to develop assays that realistically compare cigarette smoke (CS)-induced genotoxicity regardless of differences in cigarette construction or smoking behavior. One significant indicator of potentially carcinogenic DNA damage is double strand breaks (DSBs), which can be monitored by measuring Ser 139 phosphorylation on histone H2AX. Previously we showed that phosphorylation of H2AX (defined as gammaH2AX) in exposed lung cells is proportional to CS dose. Thus, we proposed that gammaH2AX may be a viable biomarker for evaluating genotoxic risk of cigarettes in relation to actual nicotine/tar delivery. Here we tested this hypothesis by measuring gammaH2AX levels in A549 human lung cells exposed to CS from a range of commercial cigarettes using various smoking regimens. Results show that gammaH2AX induction, a critical event of the mammalian DNA damage response, provides an assessment of CS-induced DNA damage independent of smoking topography or cigarette type. We conclude that gammaH2AX induction shows promise as a genotoxic bioassay offering specific advantages over the traditional assays for the evaluation of conventional and nonconventional tobacco products.

Kinetics of histone H2AX phosphorylation and Chk2 activation in A549 cells treated with topotecan and mitoxantrone in relation to the cell cycle phase.

The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitors doxorubicin, etoposide and mitoxantrone (MXT) are widely used antitumor drugs. They stabilize otherwise transient ("cleavable") complexes of topo1 or topo2 with DNA, respectively. Collisions of DNA replication forks (during replication) or progressing RNA polymerase molecules (during transcription) with these complexes convert them into double-strand DNA breaks (DSBs). Formation of DSBs triggers activation of ATM and phosphorylation of histone H2AX, the markers that have been used to correlate DNA damage with cell cycle phase or induction of apoptosis. In the present study we explored a relationship between H2AX phosphorylation and activation of checkpoint kinase 2 (Chk2) in human lung carcinoma A549 cells treated with TPT or with MXT. Activation of Chk2 was detected immunocytochemically using a phospho-specific (Thr68) Ab and measuring Chk2-Thr68(P)immunofluorescence (IF), concurrently with DNA content, by laser scanning cytometry. In the untreated cells, activated Chk2 was present predominantly in centrosomes. Upon treatment with TPT or MTX, the activated Chk2 presented itself in form of either minute or large IF foci in the cell's nucleoplasm. H2AX phosphorylation whether induced by TPT or MXT was rapid, with the maximal rate occurring during the initial 2 h and peaking at 2 h of treatment. TPT or MXT induced Chk2 activation occurred at a distinctly slower pace, peaking at 4 h. While TPT-induced H2AX phosphorylation and Chk2 activation were maximal in S-phase cells, Chk2 activation was also much pronounced in G(2)M cells; the least affected by TPT were G(1) cells. MTX-induced H2AX phosphorylation was maximal in G(1) cells while Chk2 activation was maximal in G(2)M and minimal in G(1) cells. The pattern of cell-cycle phase specific response to TPT or MXT by H2AX phosphorylation and Chk2 activation was different when measured either as integrated or maximal pixel of gammaH2AX or Chk2-Thr68(P) IF, the former reflecting total IF per nucleus the latter stressing the punctate (foci) character of expression of these phospho-modified proteins.

Down-regulation of androgen-receptor and PSA by phytochemicals.

The androgen receptor (AR) signaling pathway continues to be active in hormone resistant prostate cancer (HRPC) and can inappropriately activate transcription. Consequently the AR is a therapeutic target for HRPC. We reported that PC-SPES is active against HRPC, partly due to its actions in down-regulating AR protein expression and modulating cell cycle. Further investigation has identified five active anticancer compounds. This study describes the effects of three of these compounds (oridonin, isoliquiritigenin and wogonin) on cell proliferation, cell apoptosis, cell cycle parameters, AR and PSA protein expression. In each case, these compounds have independent activities which may partly contribute to the biological activity of PC-SPES.

Assessment of DNA double-strand breaks and gammaH2AX induced by the topoisomerase II poisons etoposide and mitoxantrone.

Double-strand breaks (DSBs) are highly deleterious DNA lesions as they lead to chromosome aberrations and/or apoptosis. The formation of nuclear DSBs triggers phosphorylation of histone H2AX on Ser-139 (defined as gammaH2AX), which participates in the repair of such DNA damage. Our aim was to compare the induction of gammaH2AX in relation to DSBs induced by topoisomerase II (TOPO II) poisons, etoposide (ETOP) and mitoxantrone (MXT), in V79 cells. DSBs were measured by the neutral comet assay, while gammaH2AX was quantified using immunocytochemistry and flow cytometry. Stabilized cleavage complexes (SCCs), lesions thought to be responsible for TOPO II poison-induced genotoxicity, were measured using a complex of enzyme-DNA assay. In the case of ETOP, a no observed adverse effect level (NOAEL) and lowest observed effect level (LOEL) for genotoxicity was determined; gammaH2AX levels paralleled DSBs at all concentrations but significant DNA damage was not detected below 0.5 microg/ml. Furthermore, DNA damage was dependent on the formation of SCCs. In contrast, at low MXT concentrations (0.0001-0.001 microg/ml), induction of gammaH2AX was not accompanied by increases in DSBs. Rather, DSBs were only significantly increased when SCCs were detected. These findings suggest MXT-induced genotoxicity occurred via at least two mechanisms, possibly related to DNA intercalation and/or redox cycling as well as TOPO II inhibition. Our findings also indicate that gammaH2AX can be induced by DNA lesions other than DSBs. In conclusion, gammaH2AX, when measured using immunocytochemical and flow cytometric methods, is a sensitive indicator of DNA damage and may be a useful tool in genetic toxicology screens. ETOP data are consistent with the threshold concept for TOPO II poison-induced genotoxicity and this should be considered in the safety assessment of chemicals displaying an affinity for TOPO II and genotoxic/clastogenic effects.

ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke.

BACKGROUND: In response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on Ser139 by phosphoinositide 3-kinase related protein kinases (PIKKs) such as ataxia telangiectasia mutated (ATM), ATM-and Rad-3 related (ATR) kinase, or by DNA dependent protein kinase (DNA-PKcs). When DNA damage primarily involves formation of DNA double-strand breaks (DSBs), H2AX is preferentially phosphorylated by ATM rather than by the other PIKKs. We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE) to cigarette smoke (CS) induced phosphorylation of H2AX. RESULTS: We report here that H2AX phosphorylation in A549 cells induced by CS was accompanied by activation of ATM, as revealed by ATM phosphorylation on Ser1981 (ATM-S1981P) detected immunocytochemically and by Western blotting. No cell cycle-phase specific differences in kinetics of ATM activation and H2AX phosphorylation were observed. When cells were exposed to CS from cigarettes with different tobacco and filter combinations, the expression levels of ATM-S1981P correlated well with the increase in expression of phosphorylated H2AX (gammaH2AX) (R = 0.89). In addition, we note that while CS-induced gammaH2AX expression was localized within discrete foci, the activated ATM was distributed throughout the nucleoplasm. CONCLUSION: These data implicate ATM as the PIKK that phosphorylates H2AX in response to DNA damage caused by CS. Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations. As checkpoint kinase (Chk) 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences related to the halt in cell cycle progression and increased propensity to undergo apoptosis. Defining the nature and temporal sequence of molecular events that are disrupted by CS through activation and eventual dysregulation of normal defense mechanisms such as ATM and its downstream effectors may allow a more precise understanding of how CS promotes cancer development.

The 2,6-disubstituted purine reversine induces growth arrest and polyploidy in human cancer cells.

Reversine (RV) is the synthetic purine identified from a protein kinase-based screen of purine mimetics and it has been shown to induce muscle myoblast differentiation into progenitor cells that can be further converted into other cell lineages. Since protein kinases play a pivotal role in cell cycle control, we hypothesize that RV might affect the proliferation of cancer cells. Herein we report that RV inhibited growth of cultured human tumor cells, respectively, PC-3, HeLa, CWR22Rv1, and DU-145 cells, and induced accumulation of polyploidal cells with > or =4N DNA content. However, RV was without effect on growth of normal prostate epithelial cells. RV-treated PC-3 cells showed enlarged nuclei and an estimated 100-fold increase in cell size. Moreover, PC-3 cells treated with RV for 2-4 days were accompanied by a marked increase in the expression of p21(WAF1), a modest elevation in the levels of cyclin D3 and CDK6 and concomitantly, also a substantial reduction in cyclin B and CDK1. These results suggest that RV may induce polyploidy and increase in cell size by up-regulating p21(WAF1) and cyclin D3/CDK6, while simultaneously suppressing the expression of cyclin B and CDK1.

Noncatalytic requirement for cyclin A-cdk2 in p27 turnover.

Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27(kip1) is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G(1) cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCF(skp2) complex.

Cell cycle arrest and apoptosis induced by human Polo-like kinase 3 is mediated through perturbation of microtubule integrity.

Human Polo-like kinase 3 (Plk3, previously termed Prk or Fnk) is involved in regulation of cell cycle progression through the M phase (B. Ouyang, H. Pan, L. Lu, J. Li, P. Stambrook, B. Li, and W. Dai, J. Biol. Chem. 272:28646-28651, 1997). Here we report that in most interphase cells endogenous Plk3 was predominantly localized around the nuclear membrane. Double labeling with Plk3 and gamma-tubulin, the latter a major component of pericentriole materials, revealed that Plk3 was closely associated with centrosomes and that its localization to centrosomes was dependent on the integrity of microtubules. Throughout mitosis, Plk3 appeared to be localized to mitotic apparatus such as spindle poles and mitotic spindles. During telophase, a significant amount of Plk3 was also detected in the midbody. Ectopic expression of Plk3 mutants dramatically changed cell morphology primarily due to their effects on microtubule dynamics. Expression of a constitutively active Plk3 (Plk3-A) resulted in rapid cell shrinkage, which led to formation of cells with an elongated, unsevered, and taxol-sensitive midbody. In contrast, cells expressing a kinase-defective Plk3 (Plk3(K52R)) mutant exhibited extended, deformed cytoplasmic structures, the phenotype of which was somewhat refractory to taxol treatment. Expression of both Plk3-A and Plk3(K52R) induced apparent G(2)/M arrest followed by apoptosis, although the kinase-defective mutant was less effective. Taken together, our studies strongly suggest that Plk3 plays an important role in the regulation of microtubule dynamics and centrosomal function in the cell and that deregulated expression of Plk3 results in cell cycle arrest and apoptosis.

N-acetylcysteine conjugate of phenethyl isothiocyanate enhances apoptosis in growth-stimulated human lung cells.

We previously showed that dietary treatment with the N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) inhibited benzo(a)pyrene-induced lung tumorigenesis in A/J mice, and that tumor inhibition was associated with induction of activator protein-1 (AP-1) activity and stimulation of apoptosis in the lungs of mice. In the present study, we show that PEITC-NAC also induces apoptosis and AP-1 activity in human lung adenocarcinoma A549 cells, and that activation of AP-1 is important in PEITC-NAC induced apoptosis in these cells. PEITC-NAC induced AP-1 binding activity in A549 cells in a dose- and time-dependent manner; peak activity appeared at 10 micromol/L after 24 hours. At that time, flow cytometric analysis showed a sub-G1 peak, indicating that approximately 4.5% of the cells had undergone apoptosis. When wild-type c-jun cDNA was transfected into A549 cells, PEITC-NAC-mediated apoptosis was greatly increased in the c-jun-transfected cells compared with the control vector-transfected cells, based on cell morphology and analysis of DNA fragmentation. Furthermore, cells that were pretreated with 100 nmol/L 12-O-tetradecanoyl phorbol-13-acetate, and then treated with 25 micromol/L PEITC-NAC, underwent enhanced apoptosis compared with cells that were treated with PEITC-NAC alone; cells treated with 12-O-tetradecanoyl phorbol-13-acetate alone showed active cell growth without apoptosis. Bivariate flow cytometric analysis of DNA strand breaks versus DNA content showed that apoptosis induced by PEITC-NAC occurred predominantly in the G2-M phase. These findings suggest that growth-stimulated cells with an elevated basal AP-1 activity, i.e., A549 cells transfected with wild-type c-jun or treated with a tumor promoter, were more sensitive to PEITC-NAC-mediated apoptosis. The observation that PEITC-NAC induces apoptosis predominantly in growth-promoted cells, such as neoplastic cells, suggests a selective mechanism by which PEITC-NAC inhibits lung carcinogenesis.

Activation of ATM and histone H2AX phosphorylation induced by mitoxantrone but not by topotecan is prevented by the antioxidant N-acetyl-L-cysteine.

The DNA topoisomerase II (topo2) inhibitor mitoxantrone (MXT) and topo1 inhibitor topotecan (TP) are antitumor drugs widely used to treat different types of cancer. Their mechanism of action is thought to stabilize otherwise transient ("cleavable") complexes between topo2 or topo1 and DNA; the collisions of the DNA replication fork during replication, or RNA polymerase during transcription, with these complexes convert them into double-strand DNA breaks (DSBs), potentially lethal lesions that may trigger apoptosis. In the present study we observed that treatment of human lung carcinoma A549 or promyelocytic leukemic HL-60 cells with MXT led to ATM activation and phosphorylation of histone H2AX on Ser-139, the reporters of induction of DSBs, in all phases of the cell-cycle. Only S-phase cells, however, underwent apoptosis after treatment with MXT, which implied that DSBs in the cells replicating DNA were more effective in triggering apoptosis than DSBs in G(1) or G(2)M phase cells. Unlike MXT, the treatment with TP induced ATM activation and H2AX phosphorylation almost exclusively in S-phase cells and only S phase cells underwent apoptosis. The induction of both ATM activation and H2AX phosphorylation by MXT was prevented to a large extent by N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS). The protective effect of NAC was observed for cells in all phases of the cell cycle. NAC offered no protection at all against TP. The induction of DSBs by MXT, thus, appears to be predominantly mediated through ROS, while DSBs generated during treatment with TP most likely are a consequence of collisions of replication forks with the "cleavable" complexes.

Constitutive histone H2AX phosphorylation on Ser-139 in cells untreated by genotoxic agents is cell-cycle phase specific and attenuated by scavenging reactive oxygen species.

DNA damage, particularly when it involves formation of double-strand breaks (DSBs), triggers phosphorylation of histone H2AX on Ser-139. Phosphorylated H2AX has been named gammaH2AX, and induction of gammaH2AX in cells exposed to genotoxic agents is considered a sensitive and specific reporter of DNA damage. However, in untreated normal cells as well in the cells of various tumor lines cells, a fraction of histone H2AX molecules remain phosphorylated. In the present study, we observed that the extent of this constitutive H2AX phosphorylation varies depending on the cell type (line) and on cell cycle phase and, in most cell types, S and G(2)/M phase cells exhibit greater levels of H2AX phosphorylation than do cells in the G(1) phase. Furthermore, constitutive H2AX phosphorylation in human pulmonary carcinoma A549, lymphoblastoid TK6, and in normal bronchial epithelial cells was reduced following cell exposure to N-acetyl-L-cysteine, a scavenger of reactive oxygen intermediates; the reduction was most pronounced for G(2)M cells. Growth of A549 cells in the presence of buthionine sulfoximine, an inhibitor of glutathione synthetase, amplified the level of constitutive H2AX phosphorylation in A549 cells. The observed constitutive H2AX phosphorylation may be a reflection of the ongoing DNA damage mediated by reactive oxygen species (ROS) generated by metabolic activity during progression through the cell cycle, leading to formation of DSBs during the S phase. Because cumulative DNA damage in proliferating cells mediated by ROS is considered the key mechanism for cell ageing, the present approach to estimate the degree of attenuation of constitutive H2AX phosphorylation by antioxidants may provide a convenient tool to assess the DNA-protective and possible anti-ageing properties of other agents.