The cell cycle effects of camptothecin.
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Animals↗
Biomedical subjects
Z Darzynkiewicz
Publications and source records attributed to Z Darzynkiewicz.
Induction of apoptosis by camptothecin and topotecan.
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Antineoplastic Agents, Phytogenic↗
DNA segments sensitive to single-strand-specific nucleases are present in chromatin of mitotic cells.
It was observed before that DNA in situ in chromatin of mitotic cells is more sensitive to denaturation than DNA in chromatin of interphase cells. DNA sensitivity to denaturation, in these studies, was analyzed by exposing cells to heat or acid and using acridine orange (AO), the metachromatic fluorochrome which can differentially stain double-stranded (ds) vs single-stranded (ss) nucleic acids, as a marker of the degree of DNA denaturation. However, without prior cell treatment with heat or acid no presence of single-stranded DNA in either mitotic or interphase cells was detected by this assay. In the present experiments we demonstrate that DNA in situ in mitotic cells, without any prior treatment that can induce DNA denaturation, is sensitive to ss-specific S1 and mung bean nucleases. Incubation of permeabilized human T cell leukemic MOLT-4, promyelocytic HL-60, histiomonocytic lymphoma U937 cells, or normal PHA-stimulated lymphocytes with S1 or mung bean nucleases generated extensive DNA breakage in mitotic cells. DNA strand breaks were detected using fluorochrome-labeled triphosphonucleotides in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. Under identical conditions of the cells' exposure to ss-specific nucleases, DNA breakage in interphase cells was of an order of magnitude less extensive compared to mitotic cells. The data indicate that segments of DNA in mitotic chromosomes, in contrast to interphase cells, may be in a conformation which is sensitive to ss nucleases. This may be a reflection of the differences in the torsional stress of DNA loops between interphase and mitotic chromatin. Namely, greater stress in mitotic loops may lead to formation of the hairpin-loop structures by inverted repeats; such structures are sensitive to ss nucleases. The present method of detection of such segments appears to be more sensitive than the use of AO. The identification of mitotic cells based on sensitivity of their DNA to ss nucleases provides an additional method for their quantification by flow cytometry.
Bromodeoxyuridine↗
Cytometry of cyclin proteins.
Cyclins are key components of the cell cycle progression machinery. They activate their partner cyclin-dependent kinases (CDKs) and possibly target them to respective substrate proteins within the cell. CDK-mediated phosphorylation of specific sets of proteins drives the cell through particular phases or checkpoints of the cell cycle. During unperturbed growth of normal cells, the timing of expression of several cyclins is discontinuous, occurring at discrete and well-defined periods of the cell cycle. Immunocytochemical detection of cyclins in relation to cell cycle position (DNA content) by multiparameter flow cytometry has provided a new approach to cell cycle studies. This approach, like no other method, can be used to detect the unscheduled expression of cyclins, namely, the presentation of G1 cyclins by cells in G2/M and of G2/M cyclins by G1 cells, without the need for cell synchronization. Such unscheduled expression of cyclins B1 and A was seen when cell cycle progression was halted, e.g., after synchronization at the G1/S boundary by inhibitors of DNA replication. The unscheduled expression of cyclins B1 or E, but not of A, was also observed in some tumor cell lines even when their growth was unperturbed. Likewise, whereas the expression of cyclins D1 or D3 in nontumor cells was restricted to an early section of G1, the presentation of these proteins in many tumor cell lines also was seen during S and G2/M. This suggests that the partner kinase CDK4 (which upon activation by D-type cyclins phosphorylates pRB committing the cell to enter S) is perpetually active throughout the cell cycle in these tumor lines. Expression of cyclin D also may serve to discriminate G0 vs. G1 cells and, as an activation marker, to identify the mitogenically stimulated cells entering the cell cycle. Differences in cyclin expression make it possible to discriminate between cells having the same DNA content but residing at different phases such as in G2 vs. M or G2/M of a lower DNA ploidy vs. G1 cells of a higher ploidy. The expression of cyclins D, E, A and B1 provides new cell cycle landmarks that can be used to subdivide the cell cycle into several distinct subcompartments. The point of cell cycle arrest by many antitumor agents can be estimated with better accuracy in relation to these compartments compared to the traditional subdivision into four cell cycle phases. The latter applications, however, pertain only to normal cells or to tumor cells whose phenotype is characterized by scheduled expression of cyclins. As sensitive and specific indicators of the cell's proliferative potential, the cyclins, in particular D-type cyclins, are expected to be key prognostic markers in neoplasia.
Animals↗
Effect of protease inhibitors on early events of apoptosis.
Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization of in situ DNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence of in situ DNA strand breaks. DNA stability was estimated by the measure of its sensitivity in situ to denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4',6-diamidino-2- phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of >=50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNA in situ, and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to >=50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.
Animals↗
Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color.
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.
Antibodies, Monoclonal↗
Unscheduled expression of cyclins D1 and D3 in human tumour cell lines.
D-type cyclins are involved in regulation of cell traverse through G1 primarily by activating the cyclin-dependent kinase 4 (CDK4) and targeting it to the retinoblastoma tumour suppressor protein. There is a vast body of evidence that defective expression of D-type cyclins is associated with tumour development and/or progression. Immunocytochemical detection of D cyclins combined with multiparameter flow cytometry makes it possible to measure the expression of these proteins in individual cells in relation to their cell cycle position without the need for cell synchronization. This approach was used in the present study to compare the cell cycle phase specific expression of cyclins D3 and D1 in human normal proliferating lymphocytes and fibroblasts, respectively, with nine tumour cell lines of different lineage. During exponential, unperturbed growth, expression of cyclin D1 in fibroblasts from donors of different age, or cyclin D3 in lymphocytes, was limited to mid-G1 cells: Less than 7% of the cells entering S phase or progressing through S and G2 were cyclin D positive. In contrast, expression of either cyclin D1 or cyclin D3 in tumour cell lines of different lineage was not limited to G1 phase. Namely, over 80% of the cells in S and G2+M were cyclin D positive in eight of the nine cell lines studied. The data indicate that while expression of cyclin D1 or D3 in normal cells is discontinuous, occurring transiently in G1, these proteins are expressed in some tumour lines persistently throughout the cell cycle. This suggests that the partner kinase CDK4 is perpetually active throughout the cell cycle in these tumour lines.
Cell Cycle↗
Laser scanning cytometer (LSC) analysis of fraction of labelled mitoses (FLM).
In this report we describe the successful application of a novel microscope-based multiparameter laser scanning cytometer (LSC) to measure duration of different phases of cell cycle in HL-60 human leukaemic cell lines by the fraction of labelled mitoses (FLM) method. Exponentially growing cells were harvested after various time intervals following pulse-labelling with 5'-bromo-2'-deoxyuridine (BrdUrd), cytocentrifuged, fixed in ethanol, and then exposed to UV light to induce DNA strand breaks at the sites of incorporated BrdUrd. The 3' OH termini of the photolytically generated DNA strand breaks were labelled with BrdUTP in the reaction catalysed by exogenous terminal deoxynucleotidyl transferase (TdT), followed by FITC-labelled BrdUrd antibodies. DNA was counterstained with propidium iodide (PI). Due to differences in chromatin structure between the interphase and mitotic cells, the LSC identified the latter by virtue of their higher red (PI) fluorescence intensity values among all pixels over the measured cell. To confirm that the cells selected were indeed cells in mitosis, predominantly in metaphase, the recorded X-Y coordinates of selected cells were used to re-position the cell for their visual examination. From the time lapse analysis of percentage BrdUrd-labelled cells progressing through mitosis it was possible to calculate the duration of individual phases of the cell cycle. The duration of S (Ts) and G2 + M (TG2 + M) was 8 and 3 h, respectively, and the minimal duration of G2 (TG2) was 2 h. The cell cycle time (Tc) estimated for the cohort of the most rapidly progressing cells was 13 h. The ability to automatically and rapidly discriminate mitotic cells combined with the possibility of their subsequent identification by image analysis makes LSC the instrument of choice for the FLM analysis.
Bromodeoxyuridine↗
Leukemia treatment in severe combined immunodeficiency mice by antisense oligodeoxynucleotides targeting cooperating oncogenes.
Transformation of hematopoietic cells by the p210bcr/abl tyrosine kinase appears to require the expression of a functional MYC protein, suggesting that simultaneous targeting of BCR-ABL and c-myc might be a rational strategy for attempting treatment of Phil-adelphia leukemia. To test this hypothesis, severe combined immunodeficiency mice injected with Philadelphia leukemic cells were treated systemically with equal doses of bcr-abl or c-myc antisense oligodeoxynucleotides (ODNs) or with both ODNs in combination. Compared with the mice treated with individual agents, the disease process was much slower in the group treated with both ODNs, as revealed by flow cytometry, clonogenic assay, and reverse transcriptase-polymerase chain reaction analysis to detect leukemic cells in mouse tissue cell suspensions, and by enumeration of liver metastases. The retardation of the disease process was positively correlated with a markedly increased survival of leukemic mice treated with both ODNs. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.
Animals↗
Effects of organic and inorganic selenium compounds on rat mammary tumor cells.
To explore cellular effects of potent organoselenium chemopreventive agents we have used a rat mammary tumor cell line. We demonstrate that 1,4-phenylenebis(methylene) selenocyanate (p-XSC) at a dose of 5 microM is a more potent inhibitor of DNA, RNA and protein synthesis as well as of mitochondrial transmembrane potential than its chemopreventive counterparts benzyl selenocyanate (BSC) and sodium selenite. These differences were also reflected in reduced growth rate by 24 and 48 hr. Cell-cycle and cell-morphology analysis revealed that higher doses of p-XSC (10 microM) caused DNA fragmentation which was accompanied with partial loss of nuclear stainability, whereas BSC caused a noticeable change in cell-cycle distribution and extensive micronucleation. Overall, our results point to cellular targets of selenium compounds which may mediate their chemopreventive activities in mammary tissues.
Animals↗
Overexpression of DR-nm23, a protein encoded by a member of the nm23 gene family, inhibits granulocyte differentiation and induces apoptosis in 32Dc13 myeloid cells.
Chronic myelogenous leukemia evolves in two clinically distinct stages: a chronic and a blast crisis phase. The molecular changes associated with chronic phase to blast crisis transition are largely unknown. We have identified a cDNA clone, DR-nm23, differentially expressed in a blast-crisis cDNA library, which has approximately 70% sequence similarity to the putative metastatic suppressor genes, nm23-H1 and nm23-H2. The deduced amino acid sequence similarity to the proteins encoded by these two latter genes is approximately 65% and includes domains and amino acid residues (the leucine zipper-like and the RGD domain, a serine and a histidine residue in the NH2- and in the COOH-terminal portion of the protein, respectively) postulated to be important for nm23 function. DR-nm23 mRNA is preferentially expressed at early stages of myeloid differentiation of highly purified CD34+ cells. Its constitutive expression in the myeloid precursor 32Dc13 cell line, which is growth-factor dependent for both proliferation and differentiation, results in inhibition of granulocytic differentiation induced by granulocyte colony-stimulating factor and causes apoptotic cell death. These results are consistent with a role for DR-nm23 in normal hematopoiesis and raise the possibility that its overexpression contributes to differentiation arrest, a feature of blastic transformation in chronic myelogenous leukemia.
Amino Acid Sequence↗
Induction of apoptosis by 5-azacytidine: drug concentration-dependent differences in cell cycle specificity.
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.
Apoptosis↗
Single-step procedure for labeling DNA strand breaks with fluorescein- or BODIPY-conjugated deoxynucleotides: detection of apoptosis and bromodeoxyuridine incorporation.
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)
Apoptosis↗
2-Deoxy-D-glucose enhances sensitivity of human histiocytic lymphoma U937 cells to apoptosis induced by tumor necrosis factor.
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.
Apoptosis↗
Apoptosis in antitumor strategies: modulation of cell cycle or differentiation.
There is a strong evidence that administration of antitumor drugs triggers apoptotic death of target cells. A characteristic feature of apoptosis is active participation of the affected cell in its demise. Attempts have been made, therefore, to potentiate the cytotoxicity of a variety of agents by modulating the propensity of cells to respond by apoptosis. Several strategies to enhance apoptosis that involve modulation of the cell cycle or differentiation are discussed. Loss of control of the G1 checkpoint in tumor cells allows one to design treatments that arrest normal cells at the checkpoint and attempt to selectively kill tumor cells with S phase specific drugs. The possibility of a restoration of the apoptosis triggering function of the tumor suppressor gene p53 when the G1 checkpoint function is abolished is expected to increase tumor cells' sensitivity to S phase poisons. Because induction of apoptosis by many antitumor drugs is cell cycle phase specific, drug combinations that preferentially trigger apoptosis at different phases of the cycle, or recruitment of cells to the sensitive phase, offer another antitumor strategy. There is also evidence that apoptosis is potentiated when cell differentiation is triggered following DNA damage. This observation suggests that strategies which combine DNA damaging and differentiating drugs, under conditions where the latter are administered following DNA damage caused by the former, may be successful.
Animals↗
Discrimination of G2 and mitotic cells by flow cytometry based on different expression of cyclins A and B1.
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 G2 phase 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 G2 and 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 G2 by 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 situ DNA 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 G2 versus tetraploid G1 cells or mitotic versus G2 cells.
Cell Cycle↗
Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation.
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.
Apoptosis↗
Threshold expression of cyclin E but not D type cyclins characterizes normal and tumour cells entering S phase.
Complexes of cyclin-dependent kinases (cdk) and their partner cyclins drive the cell through the cell cycle, each such complex phosphorylating a distinct set of proteins at a particular check-point or phase of the cycle. Immunocytochemical detection of cyclins combined with measurement of cellular DNA content by flow cytometry makes it possible to relate expression of each of these proteins with the actual cell cycle position, without the necessity of cell synchronization. In the present study, we have investigated expression of E and D type cyclins in G1 cells and in cells entering S phase, in eight different human hematopoietic and solid tumour cell lines (two leukaemias, a lymphoma, three breast carcinomas, a colon carcinoma and a bladder transitional cell carcinoma) during their exponential phase of growth, as well as in normal mitogen stimulated lymphocytes. In all the cell types studied, the average level of D type cyclin expression was invariable throughout the cell cycle. A great intercellular variability, in particular of the G1 cell subpopulations, and the presence of a large fraction of G1, S and G2 + M cells that were cyclin D negative (20-40% in tumour cell lines and about 80% among lymphocytes), were other characteristic features of D type cyclin expression. In contrast to D type cyclins, the expression of cyclin E was discontinuous during the cycle, peaking at the time of cell entrance to S. Also, a well defined threshold in expression of cyclin E characterized cells that were entering S phase, and virtually no cyclin E negative cells were seen during the early portion of S phase. The data indicate that while cell entrance to S phase is unrelated to expression of D type cyclins (at the time of entrance), accumulation of cyclin E up to critical level is a prerequisite for initiation of DNA replication. The great intercellular variability in expression of D type cyclins and their invariant average level across the cell cycle suggest that these cyclins, in addition to their acknowledged function in promoting cell progression through mid- to late-G1 may have other role(s), related or unrelated to the cell cycle progression. The presence of a large number of D type cyclin negative cells in all phases of the cycle suggests that during exponential growth the cells may not express this protein and yet may traverse the cycle, including G1 phase.
Blotting, Western↗