X Li

New York Medical College, New York City, NY, United States

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Publications (25)98.03 Total impact

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    ABSTRACT: Onconase (ONC), an antitumor ribonuclease from oocytes of a frog Rana pipiens, capable of inducing apoptosis in many cell lines is synergistic with several other anticancer drugs. Since cytotoxic effects of numerous drugs are modulated by reactive oxygen intermediates (ROI), we have studied effects of ONC on the intracellular level of oxidants in several normal cell types as well as tumor cell lines. It is demonstrated for the first time that ONC substantially decreases the content of ROI in all cell lines studied. This effect depends on the ribonucleolytic activity of the enzyme and is due to both, decreased rate of ROI generation and accelerated rate of their degradation. Onconase decreases the mitochondrial transmembrane potential and consequently, generation of ATP. Simultaneously the enzyme decreases the expression of an antiapoptotic protein Bcl-2, and upregulates the proapoptotic Bax protein. These finding are consistent with the enzyme propensity to induce apoptosis. The observed antioxidant activity of ONC may be an important element of its cytotoxicity towards cancer cells. The enzyme seems to exert its biological activities by interfering with the redox system of cellular regulation.
    International Journal of Oncology 10/2007; 31(3):663-9. · 2.66 Impact Factor
  • Methods in cell biology 02/2001; 66:69-109. · 1.44 Impact Factor
  • Methods in cell biology 02/2001; 64:421-43. · 1.44 Impact Factor
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    ABSTRACT: During induction of apoptosis, the pro-apoptotic member of the Bcl-2 protein family (Bax) undergoes translocation to the mitochondria. The translocation, which leads to accumulation of Bax in the mitochondrial intermembrane space, appears to be the critical event determining release of cytochrome c to cytosol: the latter event triggers the irreversible steps of apoptosis, namely, the activation of caspases and the initiation of the degradation of many proteins. The aim of this study was to utilize the morphometric capabilities of the laser scanning cytometer (LSC) and adapt this instrument to detect and measure in situ the process of translocation of Bax to mitochondria. Human breast carcinoma MCF-7 cells growing on microscope slides were treated with the DNA topoisomerase I inhibitor, camptothecin (CPT). CPT is known to induce apoptosis preferentially of S-phase cells. The cells were fixed and permeabilized on the slides, their DNA was stained with propidium iodide (PI), Bax was detected immunocytochemically with the fluoresceinated antibody, and red and green fluorescence emission was measured by the LSC. Prior to induction of apoptosis, Bax was uniformly and diffusely dispersed in the cell nucleus and cytoplasm. Its translocation and accumulation in mitochondria in cells undergoing apoptosis were detected and measured by the LSC as the increase in intensity of maximal pixel of Bax immunofluorescence. Bivariate analysis of DNA content versus maximal pixel of Bax fluorescence revealed that the CPT-induced Bax translocation into mitochondria was preferential to S-phase cells. Total cellular Bax immunofluorescence measured by flow cytometry, however, was increased in all phases of the cycle without a preference to S-phase cells. Changes in abundance and localization of particular proteins that undergo translocation within the cell, leading to their altered local density, may be conveniently detected by the LSC by taking advantage of its morphometric capabilities. Measurement of total cellular Bax immunofluorescence by flow cytometry combined with analysis of its translocation by LSC revealed that apoptosis of S-phase cells induced by CPT was unrelated to overall Bax abundance per cell but correlated with its accumulation in mitochondria.
    Cytometry 11/2000; 41(2):83-8.
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    ABSTRACT: The DNA topoisomerase I (topI) inhibitor camptothecin (CPT), stabilizes so-called cleavable complexes which consist of topI covalently attached to 3' OH ends of DNA nicks. Collisions between the progressing DNA replication forks (occurring in S phase cells) or between the transcription driven RNA polymerase molecules (occurring in G1, S and G2 cells) and these complexes convert the latter into secondary DNA lesions which are unrepairable and lethal to the cell. Changes induced by CPT in the level of the tumor suppressor p53, cyclin-dependent kinase inhibitor p21WAF1 and proapoptotic protein Bax (all detected immunocytochemically), were measured separately in the nucleus and cytoplasm of individual human breast carcinoma MCF-7 cells by laser scanning cytometry (LSC) in relation to cell cycle position and induction of apoptosis. The initial transient cell arrest at the G1 checkpoint seen at 8-16 h of treatment with 0.15 microM CPT was accompanied by the rapid accumulation of p53 (preventable by cycloheximide) in the nucleus; the rise (>20-fold) in p53 was maximal for S phase cells. The magnitude of the nuclear p53 increase induced by CPT, at maximum, was 2-fold higher than that induced by the proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (LLnL). While the accumulation of p53 was seen in all phases of the cycle, only G1 cells responded by induction ( approximately 60-fold increase) of p21WAF1. Inhibition of DNA replication by aphidicolin prevented the accumulation of p53 in S and G2/M but had no effect on its induction in G1 cells. Perturbation of cell progression through S phase was seen between 24-72 h of treatment, and it coincided with induction of Bax and apoptosis (both maximal in S phase cells). Thus, the changes observed in S phase cells (nuclear accumulation of p53 preventable by aphidicolin, induction of Bax, apoptosis), triggered by the collisions of DNA replication forks with the CPT-induced lesions, were distinct from the changes in G1 (nuclear p53 accumulation unaffected by aphidicolin, induction of p21WAF1) presumably triggered by collisions of RNA polymerase with the CPT-lesions. Great heterogeneity in expression of p53 and p21WAF1 of the G1 cell population in response to CPT was observed, which may reflect the intercellular variability in the rate of transcription (i.e., frequencies of collisions of RNA polymerase with the lesions). Thus, differences in the transcriptional activity of G1 cells may play a role in their sensitivity to CPT and similar topI inhibitors.
    International Journal of Oncology 12/1999; 15(5):861-71. · 2.66 Impact Factor
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    ABSTRACT: Flow cytometry techniques that are widely used in studies of cell death, and particularly in the identification of apoptotic cells, generally rely on the measurement of a single characteristic biochemical or molecular attribute. These methods fail to recognize cell death lacking that attribute, as in some examples of atypical apoptosis. Since apoptosis was originally defined by morphologic criteria, we suggest that for any new cell system the cytometry-defined apoptosis be confirmed by morphologic examination. This quality assurance measure is now provided by laser scanning cytometry (LSC). LSC measurements of cell fluorescence are precise and highly sensitive, comparable to flow cytometry (FCM), and can be carried out on cells on slides, permitting cell by cell correlation of fluorescence cytometry with visual microscopic morphology. In this report we describe adaptations of various flow cytometry techniques for detection of apoptosis by laser scanning cytometry. We also describe features unique to LSC that are useful in recognizing apoptosis. Hyperchromicity of DNA, reflecting chromatin condensation, is evidenced by high maximal pixel values for fluorescence of the DNA-bound fluorochrome. Mitochondrial probes that have been adapted to LSC to measure the drop in mitochondrial transmembrane potential that occurs early in apoptosis include rhodamine 123, 3,3'-dihexiloxadicarbocyanine [DiOC6(3)], and the aggregate dye 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). The changes in plasma membrane phospholipids and transport function, also early in apoptosis, are probed by a combination of the fluoresceinated annexin V and DNA fluorochromes such as propidium or 7-aminoactinomycin D. We also review methods of detection of apoptosis based on analysis of DNA fragmentation and their application to clinical oncology. Visual examination of the presumed apoptotic cells detected by cytometry makes it possible to discriminate those that are genuine from monocytes/macrophages that have ingested nuclear fragments via apoptotic bodies. Applications of flow cytometry and laser scanning cytometry in analysis of cell death are discussed and their respective advantages and disadvantages compared.
    Cytometry 04/1999; 35(3):181-95.
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    ABSTRACT: The presence of DNA strand breaks resulting from the cleavage of nuclear DNA by the apoptosis-associated endonuclease(s) is one of the most characteristic features of apoptotic cells (1,2). A widely used methodology to detect apoptotic cells thus relies on labeling DNA strand breaks in situ either with fluorochromes (3,4) or absorption dyes (5–9). The advantage of strand break labeling with fluorochromes is that such cells can be rapidly analyzed by flow cytometry. When cellular DNA content is also measured in these cells, the bivariate analysis of such data provides information about the DNA ploidy and cell-cycle phase specificity of apoptosis (4,10).
    Methods in molecular biology (Clifton, N.J.) 02/1999; 113:607-19.
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    ABSTRACT: Onconase is a 12 kDa protein homologous to pancreatic RNase A isolated from amphibian oocytes which shows cytostatic and cytotoxic activity in vitro, inhibits growth of tumors in mice and is in phase III clinical trials. The present study was aimed to reveal mechanisms by which onconase perturbs the cell cycle progression. Human histiocytic lymphoma U937 cells were treated with onconase and expression of cyclins D3 and E, as well as of the cyclin-dependent kinase inhibitors (CKIs) p16INK4A, p21WAF1/CIP1 and p27KIP1 (all detected immunocytochemically) was measured by multiparameter flow cytometry, in relation to the cell cycle position. Also monitored was the status of phosphorylation of retinoblastoma protein (pRb) by a novel method utilizing mAb which specifically detects underphosphorylated pRb in individual cells. Cell incubation with 170 nM onconase for 24 h and longer led to their arrest in G1 which was accompanied by a decrease in expression of cyclin D3, no change in cyclin E, and enhanced expression of all three CKIs. pRb was underphosphorylated in the onconase arrested G1 cells but was phosphorylated in the cells that were still progressing through S and G2/M in the presence of onconase. The cytostatic effect of onconase thus appears to be mediated by downregulation of cyclin D3 combined with upregulation of p27KIP1, p16INK4A and p21WAF1/CIP1, the events which may prevent phosphorylation of pRb during G0/1 and result in cell arrest at the restriction point controlled by Cdk4/6 and D type cyclins.
    Leukemia 09/1998; 12(8):1241-8. · 10.16 Impact Factor
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    ABSTRACT: Cyclins A and B1 activate cyclin-dependent kinases CDK2 and CDC2, which regulate cell progression through S and G2. Expression of these cyclins is generally measured in populations of synchronized cells, by immunoblotting. Such studies neither provide information regarding intercellular variability in cyclin expression nor yield precise data on a time relationship between initiation and termination of DNA replication in relation to cyclin expression. Furthermore, cell synchronization by DNA polymerase inhibitors or excess of thymidine induces cell growth imbalance and alters expression of cyclins, thereby introducing an experimental bias. Using a novel flow cytometric method of detection of incorporated bromodeoxyuridine (BrdUrd) in the present study, we have been able to correlate expression of immunocytochemically discerned cyclins A and B1 with incorporation of BrdUrd and the cell cycle position of individual MOLT-4 cells. On the basis of differences in amount of incorporated BrdUrd and DNA content, the following cohorts of cells in narrow windows of the cell cycle were identified: (a) cells initiating and (b) terminating DNA replication during a 1-h pulse of BrdUrd; (c) cells replicating DNA throughout the duration of BrdUrd pulse; (d) G1 cells; and (e) G2 cells that remained in G2 for at least 1 h after exiting S phase. These populations were characterized with respect to expression of cyclins A and B1. Expression of cyclin A was an early event of S phase, and 84% of cells entering S phase during 1 h of exposure to BrdUrd were already cyclin A positive. More than 95% of S-phase cells, as well as the cells exiting S during BrdUrd pulse, were also cyclin A positive. The maximal rate of accumulation of cyclin A was seen during the first hour of progression through S phase. In contrast, the maximal accumulation rate of cyclin B1 showed cells during the first hour of progression through G2. A strong correlation between expression of cyclin A and the rate of DNA replication, estimated by the degree of BrdUrd incorporation (r = 0.99), was observed.
    Cancer Research 04/1997; 57(5):803-7. · 8.65 Impact Factor
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    ABSTRACT: 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 02/1997; 27(1):1-20.
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    ABSTRACT: Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated nucleotides has been recently proposed as a means of analyzing DNA replication and detecting proliferating cells. The presence of numerous in situ DNA strand breaks is also an inherent feature of apoptotic cells. The aim of the present study was to differentially label DNA strand breaks in apoptotic cells vs photolysis-induced breaks in BrdUrd incorporating cells. This would allow one, by multicolor staining, to identify these respective cells in the same sample preparation. Toward this end, exponentially growing HL-60 cells were pulse labeled with BrdUrd and then were subjected to hyperthermia or treated with DNA topoisomerase I inhibitor camptothecin to induce apoptosis. DNA strand breaks in apoptotic cells were first labeled directly with fluorochrome-conjugated dUTP or dCTP, followed by dideoxynucleotide (to terminate chain elongation), in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The cells were subsequently exposed to UV light to photolyze DNA containing the incorporated BrdUrd. The photolysis-induced DNA strand breaks were, in turn, labeled with digoxygenin- or biotin-conjugated dUTP followed by digoxygenin antibody or avidin, respectively, conjugated with fluorochrome of another color. Alternatively, DNA strand breaks were labeled with BrdUTP which was then detected by FITC-conjugated anti-BrdUrd MoAb. Following counterstaining of cellular DNA with a fluorochrome of a third color it was possible to identify apoptotic cells, cells incorporating BrdUrd, and cells having no DNA strand breaks. Cell fluorescence was measured either by flow cytometry or with the microscope-based laser scanning cytometer. The SBIP approach also offers a possibility to study a colocalization of the immunocytochemically detectable cell constituents at the DNA replication points by microscopy. Using this approach the presence of the proliferating cell nuclear antigen at the DNA replication sites was revealed in MCF-7 breast carcinoma cells.
    Experimental Cell Research 02/1996; 222(1):28-37. · 3.56 Impact Factor
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    ABSTRACT: In situ presence of numerous DNA strand breaks is a typical feature of apoptotic cells. Selective DNA strand break induction by photolysis (SBIP) at sites that contain incorporated halogenated DNA precursors has recently been proposed as a method of analysing DNA replication. Detection of DNA strand breaks, thus, enables one to identify apoptotic and/or DNA replicating cells. The current methods for DNA strand break labelling rely on the use of exogenous terminal deoxynucleotidyl transferase which either directly attaches the fluorochrome conjugated triphosphodeoxynucleotides to 3'OH ends in the breaks, or indirectly labels 3'OH ends with digoxygenin or biotin conjugated triphosphodeoxynucleotides. A limitation of these methodologies, especially restricting their routine application in the clinic, is high cost of reagents. In the present study we have tested whether relatively simple compound BrdUTP, which is approximately three orders of magnitude less expensive than dUTP conjugated to digoxygenin, can be used as marker of DNA strand breaks. Apoptosis of HL-60 cells was induced by DNA topoisomerase I inhibitor camptothecin. The incorporated BrdUTP was detected by fluoresceinated anti-BrdUrd MoAb. Cellular fluorescence was measured by flow cytometry as well as by Laser Scanning Cytometer (LSC). The data show that intensity of DNA strand break labelling with BrdUTP was nearly four- and two-fold higher than that obtained with the indirect labelling using biotin- or digoxygenin-conjugated dUTP, respectively, and over eight-fold higher than in the case of direct labelling with the fluorochrome (fluorescein or BODIPY)-conjugated deoxynucleotides. The increased labelling of DNA strand breaks with BrdUTP may reflect more efficient incorporation of this precursor by terminal transferase, compared to the nucleotides with bulky fluorochrome conjugates. DNA strand break labelling with BrdUTP, thus, offers a possibility of more sensitive (and at lower cost) detection of apoptotic or DNA replicating cells, compared to the alternative methods of DNA strand break labelling.
    Cell Proliferation 12/1995; 28(11):571-9. · 2.27 Impact Factor
  • Leukemia 12/1995; 9(11):1961-6. · 10.16 Impact Factor
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    ABSTRACT: A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deoxynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20-30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluoresceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.
    Biotechnic and Histochemistry 10/1995; 70(5):234-42. · 1.06 Impact Factor
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    ABSTRACT: There are conflicting data in the literature as to whether cytotoxicity of the cytidine antimetabolite 5'-azacytidine (AZC) is a consequence of its incorporation into RNA, DNA, or both. Because apoptosis appears to be the predominant mode of tumor cell death after treatment with most antitumor drugs, and in the case of some drugs, the proclivity of the cell to undergo apoptosis varies depending on the cell cycle position, this study was aimed toward elucidating whether induction of apoptosis by AZC is cell cycle phase specific. Human promyelocytic leukemic HL-60 cells were treated with varying concentrations of AZC, and flow cytometric methods that identify apoptotic cels and provide information about the cell cycle distribution of the apoptotic and nonapoptotic cell populations were used. At 2-6 microM concentrations of AZC, the cells in the G1 phase preferentially underwent apoptosis, whereas the cells in G2-M were particularly resistant. Although incorporation of bromouridine into RNA was suppressed at that low AZC concentration, the rate of 5'-bromo-2-deoxyuridine incorporation into DNA was not significantly affected. At an AZC concentration of 8-40 microM, no cell cycle phase specificity in induction of apoptosis was apparent, but both the rate of 5'-bromo-2-deoxyuridine incorporation into DNA and bromouridine into RNA were reduced in proportion to drug concentration. The data suggest that the mechanism of cell killing by AZC may be different, depending on its concentration. Namely, whereas incorporation of AZC into RNA may play a predominant role in the induction of cytotoxicity of G1 cells at low drug concentrations, the perturbation of both RNA and DNA metabolism may be responsible for triggering cell death in the G1 and S phases, as is seen at higher concentrations of this antimetabolite.
    Cancer Research 08/1995; 55(14):3093-8. · 8.65 Impact Factor
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    ABSTRACT: The methods of in situ labeling of DNA strand breaks have been used to identify apoptotic cells and/or DNA replicating cells. While discrimination of apoptotic cells is based on the inherent presence of numerous DNA strand breaks in their chromatin, DNA proliferating cells can be discriminated by the selective DNA strand break induction by photolysis (SBIP) methodology at the sites that contain incorporated bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd). In both instances, DNA strand breaks are labeled with biotin- or digoxygenin-conjugated deoxynucleotides, preferably in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase; fluorescein tagged avidin (streptavidin) or digoxygenin antibody is used in the second step of the reaction. In the present study, DNA strand break labeling was simplified by using directly labeled deoxynucleotides, in a single-step reaction. Cell fluorescence was measured by flow cytometry as well as by a microscope-based laser scanning multiparameter cytometer. Apoptotic cells in HL-60 cultures treated with camptothecin or in primary cultures of non-Hodgkin's lymphoma cells treated with prednisolone were easily identified utilizing BODIPY-conjugated dUTP (B-dUTP). Apoptotic cells were also recognized using fluorescein-conjugated dUTP or dATP, although the discrimination was more pronounced with B-dUTP. The single-step procedure, requiring fewer centrifugation steps, resulted in less cell loss compared to the two-step cell labeling technique.(ABSTRACT TRUNCATED AT 250 WORDS)
    Cytometry 07/1995; 20(2):172-80.
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    ABSTRACT: It has been reported that the cytotoxic effect of tumor necrosis factor (TNF) on cells of several tumor cell lines was potentiated in culture media lacking glucose. Also, the antitumor effect of TNF was shown to be enhanced in vivo in mice treated with insulin to reduce their blood glucose level. The present study was aimed to reveal whether (a) the administration of the glucose antimetabolite 2-deoxy-D-glucose (2DG) has an effect similar to that of reduction of the extracellular glucose concentration; (b) the combined treatment with TNF and 2DG, similar to TNF alone, leads to apoptosis; and (c) there is a preference of cells in a particular phase of the cell cycle to undergo apoptosis in the presence of these agents. Exponentially growing human histiocytic lymphoma U937 cells were exposed to 0.1-0.5 nM of recombinant human TNF-alpha in the absence and presence of 1.0-5.0 mM 2DG. Analysis of the cell proliferation rates and their viability revealed that cytotoxicity of TNF was markedly potentiated by 2DG. Thus, administration of 1.0 mM 2DG to the cultures treated with 0.3 nM recombinant human TNF-alpha increased by 2-3-fold the percentage of dead cells after 24-72 h. The antimetabolite alone, at that low concentration, showed minimal cytotoxicity. More than additive cytotoxic effects also were seen at 2.5 and 5.0 mM concentrations of 2DG. Apoptosis was identified by typical changes in cell morphology, preferential degradation of internucleosomal DNA, and in situ extensive DNA strand breakage. The number of cells with DNA strand breaks after 24-h incubation was increased from 13% (0.1 nM TNF alone) to 20 or 45% in the presence of 2.5 or 5.0 mM 2DG, respectively. There was no evidence of a significant cell cycle phase preference in induction of apoptosis by combined treatment with recombinant human TNF-alpha and 2DG, although 2DG alone reduced the percentage of cells in S and G2 + M, apparently by arresting cells in G1. These data, along with observations in other cell systems, suggest that simultaneous stimulatory signals for growth induction, presumed to be provided by TNF, and growth suppression (inhibition of glycolysis) may preferentially trigger apoptosis of transformed cells. The data also suggest that 2DG may be an effective adjunct to TNF in the clinic, increasing the antitumor potency of this cytokine.
    Cancer Research 02/1995; 55(2):444-9. · 8.65 Impact Factor
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    ABSTRACT: A new method is described which combines the identification of DNA replicating and apoptotic cells in a single measurement by flow cytometry. The detection of DNA replicating cells is based on incorporation of 5-bromo-2'-deoxyuridine or 5-iodo-2'-deoxyuridine, followed by selective photolysis at the site of incorporation of the halogenated DNA precursors. Single-strand breaks in DNA resulting from the photolysis are subsequently labeled with digoxygenin or biotin-conjugated dUTP in a reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. The double-stranded DNA breaks in apoptotic cells resulting from activation of the endonuclease can be labeled in this reaction as well. However, in contrast to the photolysed DNA, the low molecular weight fraction of DNA of apoptotic cells is extractable from the cells, and the degree of DNA elution can be modulated by cross-linking with formaldehyde. Thus, apoptotic cells can be distinguished and quantified by virtue of their fractional DNA content. Replication of less than 1% of a genome of a cell in the presence of 5-bromo-2'-deoxyuridine (equivalent of a 5-min 10 microM 5-bromo-2'-deoxyuridine pulse) can be detected by the selective photolysis method. The method was applied to study apoptosis and proliferation of human leukemic HL-60 cells and normal, mitogen-stimulated lymphocytes. Whereas apoptosis of HL-60 cells induced by the DNA topoisomerase I inhibitor camptothecin was selective to DNA replicating cells, apoptosis induced by hyperthermia showed no such selectivity. Lymphocytes that preferentially underwent apoptosis in cultures stimulated by phytohemagglutinin did not initiate DNA replication. By offering the possibility for identification of both DNA replicating and apoptotic cells in a single measurement, the method may find an application in studies of the prognostic value of both cell proliferation and death in human tumors and the apoptotic response of DNA replicating vs. nonreplicating cells to different treatments.
    Cancer Research 09/1994; 54(16):4289-93. · 8.65 Impact Factor
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    ABSTRACT: CD4 is a T lymphocyte receptor for major histocompatibility complex class II antigens. It is referred to as coreceptor because it synergizes with the T cell receptor for antigen when both receptors become engaged simultaneously. We show here in mice that when engaged by antibody independently of the T cell antigen receptor, CD4 induces T cells to undergo apoptosis. Several features of this process were identified. The expression of an intact Fas protein is a requirement for CD4-mediated T cell death. Mice homozygous for the lpr mutation which are defective in the expression of Fas and in their ability to delete lymphocytes apoptotically fail to delete anti-CD4-reactive T cells. Sessile anti-CD4-reactive T cells leave their homing environment in lymphoid organs and modulate their cell surface molecules, e.g. CD2, CD3, CD4. A massive influx of lymphoid cells with null-cell phenotype occurs in the blood where they begin to reexpress cell surface markers. With their arrival in the circulation, anti-CD4-reactive T cells develop features of DNA degradation typical of apoptosis. More than one third of the circulating lymphoid cells show apoptotic features 7-8 h after anti-CD4 injection. Their frequency declines subsequently presumably due to their physical disintegration via shedding of apoptotic bodies and phagocytosis. Our data show that when not obliged to the activation process by the antigen receptor, CD4 can mediate deletion signals. Thus, besides functioning as coreceptor with the antigen receptor, CD4 has a function of its own in facilitating the induction of apoptosis.
    European Journal of Immunology 08/1994; 24(7):1549-52. · 4.97 Impact Factor
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    ABSTRACT: 5-Bromouridine (BrUrd) and several analogues of this antimetabolite exhibit antiviral activity and are potent inhibitors of the human immunodeficiency virus. The antitumour activity of BrUrd, however, in comparison with 5-fluorouridine or 5-fluorouracil, is less pronounced. Because BrUrd is incorporated into RNA and can be detected immunocytochemically and analysed by cytometry, it may prove useful as an RNA precursor to assay cell transcriptional activity. The aim of the present study was to evaluate the cell cycle effects of BrUrd incorporation in human lymphocytic leukaemic MOLT-4 and HL-60 cells and mitogenically stimulated normal peripheral blood lymphocytes. BrUrd suppressed cell proliferation and induced cell death; when measured after 72 h of treatment, the LD50 was 10 and 20 microM for HL-60 and MOLT-4, respectively, and LD90 was 100 microM, for both cell lines. BrUrd was maximally incorporated by the cells progressing through S phase of the cycle and the S phase cells were most severely perturbed by the drug, which was detected in RNA but not in DNA. Suppression of the S phase traverse of MOLT-4, HL-60 and normal lymphocytes was seen at > or = 30 microM BrUrd concentration. Also sensitive was the cell traverse through G2+M which, in the case of lymphocytes, HL-60 and MOLT-4 cells, was perturbed at > or = 30, > or = 200 and 500 microM BrUrd concentrations, respectively. Transition of lymphocytes from G0 to G1 was little affected at < 100 microM, and, although suppressed, was still observed even at 500 microM BrUrd concentration. Apoptosis of HL-60 cells and lymphocytes was observed at > or = 50 microM BrUrd after 24 h of incubation; death of MOLT-4 cells had mixed features of apoptosis and necrosis and resembled the 'mitotic' or 'reproductive cell death' as described in other cell systems. The cytostatic and cytotoxic effects of BrUrd should be taken into consideration when using this antimetabolite as an antiviral agent in the clinic or as an RNA precursor in assays of cell transcriptional activity.
    Cell Proliferation 07/1994; 27(6):307-19. · 2.27 Impact Factor

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Institutions

  • 1993–2001
    • New York Medical College
      • • Department of Pathology
      • • Brander Cancer Research Institute
      New York City, NY, United States
  • 1997–2000
    • Cancer Research Institute
      New York City, New York, United States
  • 1994
    • Université Libre de Bruxelles
      • Faculty of Medicine
      Brussels, BRU, Belgium