Concurrent detection of lysosome and tissue transglutaminase activation in relation to cell cycle position during apoptosis induced by different anticancer drugs.

Described is the new cytometric approach do detect either stimulation or a collapse of lysosomal proton pump (lysosomes rupture) combined with activation of transglutaminase 2 (TG2) during induction of apoptosis. Apoptosis of human lymphoblastoid TK6 cells was induced by combination of 2-deoxyglucose with the isoquinoline alkaloid berberine, by DNA topoisomerase I inhibitor camptothecin, its analog topotecan, topoisomerase II inhibitors etoposide or mitoxantrone, as well as by the cytotoxic anticancer ribonuclease ranpirnase (onconase). Activity of the proton pump of lysosomes was assessed by measuring entrapment and accumulation of the basic fluorochrome acridine orange (AO) resulting in its metachromatic red luminescence (F>640 ) within these organelles. Activation of TG2 was detected in the same cell subpopulation by the evidence of crosslinking of cytoplasmic proteins revealed by the increased intensity of the side light scatter (SSC) as well as following cell lysis by detergent, by its red fluorescence after staining by sulforhodamine 101. Because at low AO concentration nuclear DNA of the lysed cells was stoichiometrically stained green (F530 ) its quantity provided information on effects of the drug treatments on cell cycle in relation to activation of TG2. The data reveal that activation of lysosomal proton pump was evident in subpopulations of cells treated with 2-deoxyglucose plus berberine, topotecan, etoposide and mitoxantrone but not with ranpirnase. The collapse of lysosomal proton pump possibly reporting rupture of these organelles was observed in definite cell subpopulations after treatment with each of the studied drugs. Because regardless of the inducer of apoptosis TG2 activation invariably was correlated with lysosomes rupture it is likely that it was triggered by calcium ions or protons released from the ruptured lysosomes. This new methodological approach offers the means to investigate mechanisms and factors affecting autophagic lysosomes proton pump activity vis-à-vis TG2 activation that are common in several pathological states. © 2019 International Society for Advancement of Cytometry.

Synergy of 2-deoxy-D-glucose combined with berberine in inducing the lysosome/autophagy and transglutaminase activation-facilitated apoptosis.

Utilizing a variety of flow cytometric methods evidence was obtained indicating that a combination of the glucose analog 2-deoxy-D-glucose (2-dG) and the plant alkaloid berberine (BRB) produces synergistic effect in the induction of apoptosis in human lymphoblastoid TK6 cells. The synergistic effect is seen at concentrations of the drugs at which each of them alone shows no cytotoxicity at all. The data suggest that the combination of these drugs, which are known in terms of their overall toxicity, side effects and pharmacokinetics may be considered for further studies as chemopreventive and cancer treatment modalities. Of interest are results indicating that rapamycin, which similarly to BRB, suppresses mTOR signaling, when combined with 2-dG shows no synergistic properties. Metformin, on other hand, requires much higher concentration to show the synergy with 2-dG. Also of interest are the findings pertaining to the methodology of the present study. Specifically, dynamic assessment of cellular viability was performed by using the DRAQ7 cell exclusion fluorochrome present in cultures from 0 to 72 h. Concurrent measurement of lysosomal proton pump using acridine orange as the probe shows activation of lysosomes in the cells treated with 2-dG or BRB alone as well as with the drugs combined. Apoptosis was assessed by measuring DNA fragmentation, cell cycle, activation of caspase-3 and tissue transglutaminase (Tgase). A novel cytometric method was developed based on analysis of lysosomal (acidic vesicles) proton pump in live cells followed by cell lysis with detergent and fluorochrome labeling of proteins and DNA to analyze Tgase activation concurrently with cell cycle, in same population of cells. The data show that the cell subpopulation undergoing apoptosis has increased side (right-angle) light scatter likely due to the presence of the crosslinked (solid state) proteins, the consequence Tgase activation.

In search of antiaging modalities: evaluation of mTOR- and ROS/DNA damage-signaling by cytometry.

This review presents the evidence in support of the IGF-1/mTOR/S6K1 signaling as the primary factor contributing to aging and cellular senescence. Reviewed are also specific interactions between mTOR/S6K1 and ROS-DNA damage signaling pathways. Outlined are critical sites along these pathways, including autophagy, as targets for potential antiaging (gero-suppressive) and/or chemopreventive agents. Presented are applications of flow- and laser scanning- cytometry utilizing phospho-specific Abs, to monitor activation along these pathways in response to the reported antiaging drugs rapamycin, metformin, berberine, resveratrol, vitamin D3, 2-deoxyglucose, and acetylsalicylic acid. Specifically, effectiveness of these agents to attenuate the level of constitutive mTOR signaling was tested by cytometry and confirmed by Western blotting through measuring phosphorylation of the mTOR-downstream targets including ribosomal protein S6. The ratiometric analysis of phosphorylated to total protein along the mTOR pathway offers a useful parameter reporting the effects of gero-suppressive agents. In parallel, their ability to suppress the level of constitutive DNA damage signaling induced by endogenous ROS was measured. While the primary target of each of these agents may be different the data obtained on several human cancer cell lines, WI-38 fibroblasts and normal lymphocytes suggest common downstream mechanism in which the decline in mTOR/S6K1 signaling and translation rate is coupled with a reduction of oxidative phosphorylation and ROS that leads to decreased oxidative DNA damage. The combined assessment of constitutive γH2AX expression, mitochondrial activity (ROS, ΔΨm), and mTOR signaling provides an adequate gamut of cell responses to test effectiveness of gero-suppressive agents. Described is also an in vitro model of induction of cellular senescence by persistent replication stress, its quantitative analysis by laser scanning cytometry, and application to detect the property of the studied agents to attenuate the induction of senescence. Discussed is cytometric analysis of cell size and heterogeneity of size as a potential biomarker used to asses gero-suppressive agents and longevity.

DNA damage signaling, impairment of cell cycle progression, and apoptosis triggered by 5-ethynyl-2'-deoxyuridine incorporated into DNA.

The "click chemistry" approach utilizing 5-ethynyl-2'-deoxyuridine (EdU) as a DNA precursor was recently introduced to assess DNA replication and adapted to flow- and imaging-cytometry. In the present study, we observed that EdU, once incorporated into DNA, induces DNA damage signaling (DDS) such as phosphorylation of ATM on Ser1981, of histone H2AX on Ser139, of p53 on Ser15, and of Chk2 on Thr68. It also perturbs progression of cells through the cell cycle and subsequently induces apoptosis. These effects were observed in non-small cell lung adenocarcinoma A549 as well as in B-cell human lymphoblastoid TK6 and WTK1 cells, differing in the status of p53 (wt versus mutated). After 1 h EdU pulse-labeling, the most affected was cells progression through the S phase subsequent to that at which they had incorporated EdU. This indicates that DNA replication using the template containing incorporated EdU is protracted and triggers DDS. Furthermore, progression of cells having DNA pulse-labeled with EdU led to accumulation of cells in G2 , likely by activating G2 checkpoint. Consistent with the latter was activation of p53 and Chk2. Although a correlation was observed in A549 cells between the degree of EdU incorporation and the extent of γH2AX induction, such correlation was weak in TK6 and WTK1 cells. The degree of perturbation of the cell cycle kinetics by the incorporated EdU was different in the wt p53 TK6 cells as compared to their sister WTK1 cell line having mutated p53. The data are thus consistent with the role of p53 in modulating activation of cell cycle checkpoints in response to impaired DNA replication. The confocal microscopy analysis of the 3D images of cells exposed to EdU for 1 h pulse and then grown for 24 or 48 h revealed an increased number of colocalized γH2AX and p53BP1 foci considered to be markers of DNA double-strand breaks and enlarged nuclei.

DNA damage signaling assessed in individual cells in relation to the cell cycle phase and induction of apoptosis.

Reviewed are the phosphorylation events reporting activation of protein kinases and the key substrates critical for the DNA damage signaling (DDS). These DDS events are detected immunocytochemically using phospho-specific Abs; flow cytometry or image-assisted cytometry provide the means to quantitatively assess them on a cell by cell basis. The multiparameter analysis of the data is used to correlate these events with each other and relate to the cell cycle phase, DNA replication and induction of apoptosis. Expression of γH2AX as a possible marker of induction of DNA double strand breaks is the most widely studied event of DDS. Reviewed are applications of this multiparameter approach to investigate constitutive DDS reporting DNA damage by endogenous oxidants byproducts of oxidative phosphorylation. Also reviewed are its applications to detect and explore mechanisms of DDS induced by variety of exogenous agents targeting DNA such as exogenous oxidants, ionizing radiation, radiomimetic drugs, UV light, DNA topoisomerase I and II inhibitors, DNA crosslinking drugs and variety of environmental genotoxins. Analysis of DDS induced by these agents provides often a wealth of information about mechanism of induction and the type of DNA damage (lesion) and is reviewed in the context of cell cycle phase specificity, DNA replication, and induction of apoptosis or cell senescence. Critically assessed is interpretation of the data as to whether the observed DDS events report induction of a particular type of DNA lesion.

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.

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.

Analysis of cellular DNA content by flow and laser scanning cytometry.

This chapter covers several aspects of methodology of DNA content analysis in individual cells that is most commonly used for assessment of DNA ploidy and for enumeration of cells in particular phases of the cell cycle. Briefly presented are general principles of instrumentation and cell analysis by flow- and laser scanning- cytometry. Described are major methods designed to stain DNA with fluorochromes in live cells, in detergent-permeabilized cells, in cells fixed prior to DNA staining as well as in nuclei of cells isolated from paraffin-embedded tissues. Briefly addressed are approaches to estimate cellular DNA content in conjunction with cellular immunophenotype. Discussed are factors that affect accuracy of DNA content measurement such as: (i) differences in chromatin structure of the analyzed cells that restrict DNA accessibility to fluorochromes, (ii) stoichiometry of interaction between fluorochromes and DNA in chromatin and (iii) chemical mass action law defining dependency of fluorochrome binding to DNA in relation to fluorochrome concentration and number of potential binding sites in a sample. Described also are controls used to ensure accuracy of DNA ploidy determination, the principles in ploidy assessment and possible pitfalls in analysis.

Attenuation of the oxidative burst-induced DNA damage in human leukocytes by hyaluronan.

Oxidative burst provides the mechanism for specialized phagocytes, such as granulocytes or monocytes, to kill invading microorganisms through generation of superoxide anions. However, the oxidants generated during the burst damage DNA of the phagocytes and neighboring cells. Human blood leukocytes treated with phorbol myristate acetate (PMA) are considered to represent the experimental model of induction of oxidative burst. We recently reported that DNA damage in PMA-treated leukocytes is assessed by cytometric analysis of the induction of histone H2AX phosphorylation and Ataxia Telangiectasia Mutated (ATM) activation. In the present study we observed that hyaluronic acid (HA) of average molecular weight (MW) 5.4x10(6) and 2x10(6) at 0.1% (w/v) concentration significantly attenuated H2AX phosphorylation and ATM activation induced in leukocytes during oxidative burst. HA also reduced the intracellular level of PMA-induced reactive oxidants as measured by the ability of cells to oxidize 2',7'-dihydro-dichlorofluorescein-diacetate. No such effect was seen with HA of 6x10(4) MW. The data are consistent with earlier observations that HA of high MW protects DNA from oxidative damage induced by endo- or exogenous oxidants. The anti-oxidant effect of HA seen during oxidative burst also explains its anti-inflammatory effect when used to treat arthritic joints.

Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage.

This unit describes immunocytochemical detection of histone H2AX phosphorylated on Ser-139 (γH2AX) to reveal DNA damage, particularly when the damage involves the presence of DNA double-strand breaks (DSBs). These breaks often result from DNA damage induced by ionizing radiation or by treatment with anticancer drugs such as DNA topoisomerase inhibitors. Furthermore, DSBs are generated in the course of DNA fragmentation during apoptosis. The unit presents strategies to distinguish radiation- or drug-induced DNA breaks from those intrinsically formed in untreated cells or associated with apoptosis. The protocol describes immunocytochemical detection of γH2AX combined with measurement of DNA content to identify cells that have DNA damage and concurrently to assess their cell-cycle phase. The detection is based on indirect immunofluorescence using FITC- or Alexa Fluor 488-labeled antibody, with DNA counterstained with propidium iodide and cellular RNA removed with RNase A. © 2019 by John Wiley & Sons, Inc.

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.

Attenuation of acridine mutagen ICR-191--DNA interactions and DNA damage by the mutagen interceptor chlorophyllin.

We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.

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.

Upregulation of PD­L1 expression by resveratrol and piceatannol in breast and colorectal cancer cells occurs via HDAC3/p300­mediated NF­κB signaling.

Programmed cell death ligand 1 (PD­L1) expressed in cancer cells interacting with its receptor programmed cell death 1 (PD­1) expressed in immune cells represents a regulatory axis linked to the suppression and evasion of host immune functions. The blockade of PD­1/PD­L1 interaction using monoclonal antibodies has emerged as an effective therapy for several solid tumors; however, durable response has been observed in a subset of patients with PD­L1-positive tumors. Thus, the understanding of the mechanisms responsible for the expression of PD­L1 in tumor cells may help to improve the response to PD­L1 blockade therapies. In this study, we investigated whether resveratrol, a grape-derived stilbenoid with immunoregulatory activity, modulates the expression of PD­L1 in breast and colorectal cancer cells. The surface expression of PD­L1 was determined by flow cytometry in cancer cells treated with resveratrol and/or piceatannol. Each stilbenoid alone induced PD­L1 and when used in combination, elicited a synergistic upregulation of PD­L1 in some cell lines. The induction of PD­L1 by the combined use of stilbenoids was most pronounced in the Cal51 triple-negative breast cancer (TNBC) and SW620 colon cancer cells. The observed induction of PD­L1 was transcriptionally mediated by nuclear factor (NF)-κB, as shown by NF­κB reporter assays, the nuclear accumulation of the p65 subunit of NF­κB, inhibition by the IKK inhibitor, BMS­345541, and histone the modification inhibitors, resminostat, entinostat or anacardic acid. Combined treatment with resveratrol and piceatannol also decreased tumor cell survival as indicated by the upregulation of the DNA damaging marker, γH2AX, the cleavage of caspase 3, the downregulation of the survival markers, p38-MAPK/c­Myc, and G1-to-S cell cycle arrest.

Mild hyperthermia predisposes tumor cells to undergo apoptosis upon treatment with onconase.

Onconase (ONC), (ranpirnase) a cytotoxic ribonuclease isolated from amphibian oocytes and early embryos targeting tumor cells in vitro and in vivo, is currently in a confirmatory Phase IIIb clinical trial for unresectable malignant mesothelioma where it demonstrates antitumor activity with relatively minor overall toxicity to patients. Since hyperthermia has been shown to be synergistic with certain antitumor modalities, the aim of the present study was to explore whether the cytotoxic effects of ONC can be enhanced under conditions of mild hyperthermia. Treatment of human lymphoblastoid TK6 cells with 2 or 5 microg/ml of ONC at 40 degrees C for 24 or 48 h led to 64-200% enhancement in incidence of apoptosis assessed by frequency of cells showing the presence of activated (cleaved) caspase-3 or activated serine proteases, compared to treatment at 37.5 degrees C. The incidence of apoptosis at 40 degrees C in the absence of ONC was unchanged compared to 37.5 degrees C, for up to 48 h. Although at 41 degrees C in absence of ONC the incidence of apoptosis was elevated compared to 37 degrees C the cytotoxicity of ONC was further enhanced and the overall pro-apoptotic effect was above the level of additive effects of ONC plus that of 41 degrees C-hyperthermia. While the mechanism of the observed enhancement of ONC cytotoxicity is currently under investigation, the findings suggest that a combination of ONC and mild hyperthermia should be explored to increase effectiveness of ONC in cancer treatment.

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.

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.

ATM Activation and H2AX Phosphorylation Induced by Genotoxic Agents Assessed by Flow- and Laser Scanning Cytometry.

Activation of Ataxia Telangiectasia Mediated protein kinase (ATM) by its phosphorylation on serine 1981 and phosphorylation of histone H2AX on serine 139 (γH2AX) are the key events reporting DNA damage, primarily formation of DNA double strand breaks. These events are detected immunocytochemically in individual cells using phospho-specific Abs. The protocols are presented that describe the methodology of immunofluorescent labeling of cells in conjunction with specific staining of cellular DNA. Flow- and imaging-cytometry, the latter exemplified as laser scanning cytometry, is used to quantify intensity of cellular fluorescence reporting activation of ATM and induction of γH2AX with respect to cellular DNA content, which in turn reports the cell cycle phase. Different protocols are presented for analysis of cells either grown in suspension or attached to surface of culture vessels. Examples of ATM activation and H2AX phosphorylation in response to DNA damage in leukemic HL-60 cells by DNA topoisomerase I inhibitor topotecan, and in lung carcinoma A549 cells by hydrogen peroxide, are presented.