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It has been reported that exposure to UV light triggers DNA damage response DDR seen as induction of γH2AX not only in S but also in G1phase cells. In the present study, in addition to γH2AX, we assessed other markers of DDR, namely phosphorylation of ATM on Ser1981, of ATMATR substrate on SerThr at SQTQ cluster domains and of the tumor suppressor p53 on Ser15, in human pulmonary carcinoma A549 cells irradiated with 50 Jm2of UVB. Phosphorylation of these proteins detected with phosphospecific Abs and measured by laser scanning cytometry in relation the cell cycle phase was found to be selective to Sphase cells. The kinetics of phosphorylation of ATM was strikingly similar to that of ATMATR 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 phosphoproteins 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 Sphase. Exposure of cells to UV following 1 h pulselabeling of their DNA with 5ethynyl2′deoxyuridine EdU made it possible to correlate the extent of DNA replication during the pulse with the extent of the UVinduced H2AX phosphorylation within the same cells. This correlation was very strong R2 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 UVinduced DNA lesions and likely formation of doublestrand DNA breaks triggers DDR. The prior reports including our own on induction of γH2AX in G1cells by UV may have erroneously identified cells initiating DNA replication following UV exposure as G1cells due to the fact that their DNA content did not significantly differ from that of G1cells that had not initiated DNA replication. © 2009 International Society for Advancement of Cytometry

Accumulation of reactive oxygen species (ROS)-induced damage and mutations in the genomic DNA is considered the primary etiology of aging and age-related pathologies including cancer. Strategies aimed at slowing these conditions often involve protecting against oxidative DNA damage viamodulation of the intracellular redox state. Recently, a biomarker of DNA double-strand breaks (DSBs), serine 139-phosphorylated histone H2AX (?H2AX), and its upstream mediator, activated PI-3-related kinase, ATM (ATMP1981), were shown to be constitutively expressed in cells and modulated by antioxidant treatment. Thus, both constitutive histone H2AX phosphorylation (CHP) and constitutive ATM activation (CAA) are thought to reflect a cell’s response to endogenous ROS-induced DSBs. In the present study, we investigated the effects of a battery of fluoroquinolone (FQ) compounds, namely ciprofloxacin, enrofloxacin, gatifloxacin, lomefloxacin and ofloxacin, on CHP and CAA in human TK6 lymphoblastoid cells. All FQs tested reduced CHP and CAA compared to controls following 6 and 24h treatment with CAA being more sensitive to their effects at both time points. In addition, intracellular ROS levels and mitochondrial activities were also lowered in FQ-treated cells at 6 and 24h. We presume that FQs mediate this effect viaa combination of ROS-scavenging and mitochondrial suppression and therefore may protect against the onset or may slow the progression of numerous oxidative pathophysiological conditions.

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 G1 or G2M 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.

Histone H1 and H3 phosphorylation associated with chromatin condensation during mitosis has been studied extensively. Less is known on histone modifications that occur during premature chromosome condensation (PCC). The aim of the present study was to reveal the status of histone H3 and H2AX phosphorylation on Ser‐10 and Ser‐139, respectively, as well as ATM activation through phosphorylation on Ser‐1981, during PCC, and relate these events to cell‐cycle phase and to initiation of apoptosis.To induce PCC, A549 and HL‐60 cells were exposed to the phosphatase inhibitor calyculin A (Cal A). Phosphorylation of histone H3 and H2AX as well as ATM activation were detected immunocytochemically concurrent with analysis of cellular DNA content and activation of caspase‐3, a marker of apoptosis. The intensity of cellular fluorescence was measured by flow‐ or laser scanning cytometry.Induction of PCC led to rapid histone H3 phosphorylation, followed by activation of ATM and then H2AX phosphorylation in both, HL‐60 and A549 cells. All these events occurred sequentially, prior to caspase‐3 activation, and affected cells in all phases of the cell cycle. ATM activation and H2AX phosphorylation was seen during mitosis of A549 but not HL‐60 cells.Because the Cal A‐induced phosphorylation of histone H3 and H2AX, and of ATM, precede caspase‐3 activation these modifications are pertinent to PCC and not to apoptosis‐associated chromatin condensation. The sequence of histone H3 and H2AX phosphorylation and ATM activation during PCC is compatible with a role of ATM in mediating phosphorylation of H2AX but not H3. Mitosis in some cell types may proceed without ATM activation and H2AX phosphorylation. © 2006 International Society for Analytical Cytology

DNA replication stress often induces DNA damage. The antitumor drug hydroxyurea (HU), a potent inhibitor of ribonucleotide reductase that halts DNA replication through its effects on cellular deoxynucleotide pools, was shown to damage DNA inducing double‐strand breaks (DSBs). Aphidicolin (APH), an inhibitor of α‐like DNA polymerases, was also reported to cause DNA damage, but the evidence for induction of DSBs by APH is not straightforward. Histone H2AX is phosphorylated on Ser 139 in response to DSBs and one of the protein kinases that phosphorylate H2AX is ataxia telangiectasia mutated (ATM); activation of ATM is through its phosphorylation of Ser 1981. The present study was undertaken to reveal whether H2AX is phosphorylated in cells exposed to HU or APH and whether its phosphorylation is mediated by ATM.HL‐60 cells were treated in cultures with 0.1–5.0 mM HU or 1–4 μM APH for up to 5 h. Activation of ATM and H2AX phosphorylation was detected immunocytochemically using Ab specific to Ser1981‐ATM or Ser 139‐H2AX epitopes, respectively, concurrent with measurement of cellular DNA content.While exposure of cells to HU led to H2AX phosphorylation selectively during S phase and the cells progressing through the early portion of S (DI = 1.1–1.4) were more affected than late‐S phase (DI = 1.6–1.9) cells, ATM was not activated by HU. In fact, the level of constitutive (“programmed”) ATM phosphorylation was distinctly suppressed, in all phases of the cell cycle, at 0.1–5.0 mM HU. Cells' exposure to APH also resulted in H2AX phosphorylation at Ser139 with no evidence of ATM activation, and as in the case of HU, the early‐S cells were more affected than the late‐S phase cells. The rise in frequency of apoptotic cells became apparent after 2 h of exposure to HU or APH, and all apoptotic cells had markedly elevated levels of both H2AX‐Ser139 and ATM‐Ser1981 phosphorylation.The lack of correlation between H2AX phosphorylation and ATM activation indicates that protein kinase(s) other than ATM (ATR and/or DNA‐dependent protein kinase) are activated by DSBs induced by replication stress. Interestingly, HU inhibits the constitutive (“programmed”) level of ATM phosphorylation in untreated cells. However, DNA fragmentation during apoptosis activates ATM and dramatically increases level of H2AX phosphorylation. © 2006 International Society for Analytical Cytology

Removal of the nucleic acid-bound fluorochrome is desirable when stained cells have to be reanalyzed using other fluorochromes. It is also often desirable to remove DNA-bound antitumor drugs from drug-treated cells, to improve cell staining. We have previously observed that in aqueous solutions, the methylxanthine caffeine (CFN) decreases interactions between planar aromatic molecules such as intercalating dyes or antitumor drugs and nucleic acids. The aim of this study was to explore whether this property of CFN can be utilized to remove the DNA-bound intercalating dyes propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) from the cells and whether the bleached cells can be restained and reanalyzed. HL-60 cells were fixed in 70% ethanol and their DNA was stained with PI or 7-AAD. The cells were then rinsed with a 0.05 M solution of CFN in phosphate-buffered saline (PBS) or with PBS alone. The decrease in intensity of cell fluorescence during rinsing was measured by laser scanning cytometry (LSC) to obtain the bleaching kinetics of individual cells. The bleached cells were then restained with PI, 7-AAD, or the protein-specific fluorochrome sulforhodamine 101(S101). Their fluorescence was measured again by LSC. In addition, free DNA was subjected to gel electrophoresis, DNA bands in the gels were stained with ethidium bromide (EB), and the gels were rinsed with a solution of CFN or PBS to bleach the DNA band's fluorescence. Rinsing the PI or 7-AAD–stained cells with solutions of CFN led to nearly complete removal of PI and a more than 75% decrease in 7-AAD fluorescence after 10 min. The rinse with PBS decreased the PI cell fluorescence intensity by less than 30% and the 7-AAD fluorescence by about 50%. The differences in kinetics of PI or 7-AAD removal by CFN from G2/M versus G1 cells suggest that these intercalators bind more strongly to DNA in chromatin of G2/M than G1 cells. The CFN-bleached cells were then successfully stained with S101 and again with PI or 7-AAD. The bivariate analysis of the LSC merged files of the cells sequentially stained with PI and S101 revealed typical DNA/protein distributions. The fluorescence of EB-stained DNA bands in gels was also nearly completely removed by rinsing gels in 0.05 M CFN; PBS alone had a distinctly lesser effect. Solutions of CFN can dissociate the DNA-bound PI, 7-AAD, EB, and possibly other intercalating fluorochromes. The bleached cells can be restained and reanalyzed by LSC. This approach can also be used to remove such fluorochromes from nucleic acids immobilized in gels and perhaps in other solid matrices. Analysis of the kinetics of fluorochrome removal from cells can possibly be used to study their binding affinities to nucleic acids in situ. Cytometry 43:38–45, 2001. © 2001 Wiley-Liss, Inc.

The presence of sperm cells characterized by increased sensitivity of DNA in situto denaturation and by abnormal morphology (shape) in semen is associated with low fertility. Such cells often appear in increasing numbers following exposure to toxic or mutagenic agents. In the present study, the sensitivity of DNA in situto acid-induced denaturation was assayed in sperm cells of 25 patients attending a human fertility center. The same samples were also subjected to analysis of DNA strand breaks. A new method for detecting DNA strand breaks in situby labeling 3′-OH termini with biotinylated dUTP in a reaction employing exogenous terminal deoxynucleotidyl transferase has been applied. Both methods utilized flow cytometry. A good correlation was observed in all samples between the percentage of sperm cells with DNA strand breaks and the percentage of cells characterized by an increased sensitivity of DNA to denaturation (r= 0.87; P≤ -0.05). It is likely, thus, that the sperm cells characterized by increased DNA sensitivity to denaturation have extensive DNA strand breakage. The above properties of abnormal sperm cells, which in all probability are dead in terms of their reproductive capacity, resemble properties of apoptotic somatic cells. Activation of the endogenous endonuclease which causes extensive DNA breakage, thus, is characteristic to both the programmed death of somatic cells (apoptosis) and functional elimination (of possibly defective) germ cells from the reproductive pool.

A new technique for simultaneous multiparameter deoxyribonucleic acid, ribonucleic acid and nuclear size measurements by flow cytometry was applied to the examination of bladder irrigation cytology specimens from 107 urologic patients. The cell samples from patients with bladder carcinoma could be distinguished from normal by 2 features: 1) an increase in the proportion of bladder epithelial cells with more than diploid deoxyribonucleic acid and 2) aneuploid cell peaks. These criteria identified 12 of 13 cases of invasive carcinoma, 24 of 28 cases of carcinoma in situ and 11 of 13 cases of papillary carcinoma. An increased proportion of cells with more than diploid deoxyribonucleic acid or aneuploidy was found in 9 of 14 patients with papilloma and 6 of 19 patients with a history of bladder tumors but no evident disease at present—these were believed owing to increased epithelial proliferative rates or nuclear chromatin abnormalities not visible by light microscopy. None of the 20 patients who had never had bladder tumors was abnormal. While the results in this small clinical trial have been most encouraging an additional descriptor of nuclear chromatin structure is believed necessary to discriminate benign, reactive proliferative epithelium from neoplasm when the latter is near diploid or shedding few cells. Studies to develop such a parameter presently are under way.

The effects of 1,4-bis(2'-chloroethyl)-1,4-diazabicyclo-[2.2.1] heptane diperchlorate (CBH; NSC 57198) on cell viability, growth, progression through the cell cycle, survival, and differentiation were investigated in suspension cultures of murine lymphocytic leukemia (L1210) and erythroleukemic (FL) cells and normal human lymphocytes stimulated with phytohemagglutinin (PHA) and in adherent cultures of Chinese hamster ovary (CHO) cells. CBH was equally cytotoxic toward stationary and exponentially growing CHO cells. Cell viability was diminished by 50% following 24 hr exposure to approximately 50 μg CBH per ml. Treatment of quiescent human lymphocytes for 24 hr with up to 100 μg CBH per ml did not appreciably diminish cell viability though the subsequent stimulation of such lymphocytes with PHA was inhibited in a dose dependent fashion. L1210, FL cells, and PHA stimulated human lymphocytes were equally sensitive to CBH, 50% inhibition of growth was obtained following 24 hr treatment with 25 μg CBH per ml. Incubation for up to 48 hr with CBH did not result in differentiation of FL cells to mature hemoglobin containing cells. Constant exposure of L1210 cells and PHA-stimulated human lymphocytes to 10-50 μg CBH per ml resulted in accumulation of cells in G2 + M phase; higher drug concentrations resulted in cell arrest in mid to late S phase and G2 phase. A short 1-hr pulse of the drug resulted in a transient accumulation of L1210 cells in S and G2 phases. However, cells recovered from a short pulse of drug and by 48 hr, both cell proliferation and the cell cycle distribution appeared normal. A detailed analysis of cell cycle progression of L1210 cells in the presence of the drug indicated that the duration of G2 phase was extended at low concentrations (10 μg/ml) while the transit of cells through S was retarded with subsequent accumulation in late S and G2 phase at higher (50 μg/ml) concentrations. Concomitant with cell arrest in S and G2 phase an increase in cellular RNA content indicating unbalanced growth was observed. This state of unbalanced growth was reversible in cultures exposed to a 1-hr pulse of up to 100 μg CBH per ml; cellular RNA content returned to control values by 48 hr. No effect on nuclear chromatin as assayed by acid denaturation was observed. Though the exact mechanism of drug action is not known, the data are not incompatible with the drug acting as an alkylating agent.

Cyclins, the regulatory subunits of their respective cyclin-dependent kinases, are the key components of the cell-cycle progression machinery. Some cyclins are expressed discontinuously during the cell cycle, their synthesis and degradation being strictly scheduled. The presence of these cyclins in the cell, therefore, provides landmarks of the cell cycle, in addition to DNA replication and mitosis. Cyclin A is expressed in late S and G2phase and degraded during mitosis just prior to metaphase. Degradation of another "mitotic" cyclin, cyclin B1, occurs later, at the transition from metaphase to anaphase. Based on the difference in time of degradation of cyclin A versus cyclin B1 it was possible, in the present study, to discriminate between G2and mitotic (postprophase) MOLT-4 leukemic cells, by multiparameter (cellular DNA content versus cyclin expression) flow cytometry. The cells arrested in metaphase by Vinblastine were cyclin A negative and had an elevated level of cyclin B1. The cells arrested in G2by the DNA topoisomerase II inhibitor m-AMSA had a very high level of cyclin B1 expression and unchanged expression of cyclin A. During stathmokinesis induced by Vinblastine the percentage of mitotic cells estimated by analysis of cellular DNA content and cyclin A expression was identical to that estimated by the alternative method based on in situDNA denaturation followed by staining with acridine orange. Thus, differences in expression of cyclins A and B1 make it possible to discriminate cells that have the same DNA content but reside in different phases of the cycle, such as DNA diploid cells in G2versus tetraploid G1cells or mitotic versus G2cells.

The term cell necrobiology is introduced to comprise the life processes associated with morphological, biochemical, and molecular changes which predispose, precede, and accompany cell death, as well as the consequences and tissue response to cell death. Two alternative modes of cell death can be distinguished, apoptosis and accidental cell death, generally defined as necrosis. The wide interest in necrobiology in many disciplines stems from the realization that apoptosis, whether it occurs physiologically or as a manifestation of a pathological state, is an active mode of cell death and a subject of complex regulatory processes. A possibility exists, therefore, to interact with the regulatory machinery and thereby modulate the cell's propensity to die in response to intrinsic or exogenous signals. Flow cytometry appears to be the methodology of choice to study various aspects of necrobiology. It offers all the advantages of rapid, multiparameter analysis of large populations of individual cells to investigate the biological processes associated with cell death. Numerous methods have been developed to identify apoptotic and necrotic cells and are widely used in various disciplines, in particular in oncology and immunology. The methods based on changes in cell morphology, plasma membrane structure and transport function, function of cell organelles, DNA stability to denaturation, and endonucleolytic DNA degradation are reviewed and their applicability in the research laboratory and in the clinical setting is discussed. Improper use of flow cytometry in analysis of cell death and in data interpretation also is discussed. The most severe errors are due to i) misclassification of nuclear fragments and individual apoptotic bodies as single apoptotic cells, ii) assumption that the apoptotic index represents the rate of cell death, and iii) failure to confirm by microscopy that the cells classified by flow cytometry as apoptotic or necrotic do indeed show morphology consistent with this classification. It is expected that flow cytometry will be the dominant methodology for necrobiology. Cytometry 27:1–20, 1997. © 1997 Wiley-Liss, Inc.

A mathematical model is used to estimate the cell cycle phase specific action of a new anticancer drug, CI-921, based on results of a short term (8 hr) stathmokinetic (blocked divisions) study on Friend leukemia cells. The estimate obtained is in the form of a sequence of fractions of the cell flow blocked in successive subcompartments of the cell cycle. At specific drug concentrations, the information contained in this estimate is sufficient to correctly predict the results of long term (24 hr) continuous drug exposure. The result obtained seems to be of interest, since the ability to predict the cell cycle phase specific effects of anticancer drugs is crucial for any attempt to improve the existing chemotherapeutic treatment schedules for cancer. This study required developing new mathematical techniques, among them a nonparametric method for estimation of the distributions of cell residence times in the G1phase of the cell cycle (Appendix). The methods developed are general enough to be applicable, in principle, to any chemotherapeutic agent the action of which is distributed throughout the cell cycle.