Cells have been cultured from the brains of 60-day-old rats and propagated through 12 passages. The cells contain the high and middle, but not low, molecular weight neurofilament subunits and neuron-specific enolase, demonstrated by immunoblotting and immunocytochemistry with redundant antibodies. The cells did not have the morphology of neurons when cultured in medium containing fetal calf serum and growth factors. In low serum medium containing the same growth factors with the addition of dibutyryl cyclic AMP, the cells became smaller and developed long processes. Three clonal lines derived from these cultures had the same properties. These observations are in agreement with recent observations using mouse and human brain tissue and demonstrate that proteins normally associated with neurons can be found in dividing cells cultured from the brains of young adult rats.

Animals↗

PubMed1994

The cell cycle effects and induction of apoptosis by 5-bromouridine in cultures of human leukaemic MOLT-4 and HL-60 cell lines and mitogen-stimulated normal lymphocytes.

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.

Antimetabolites↗

PubMed1994

Myocyte cellular hyperplasia and myocyte cellular hypertrophy contribute to chronic ventricular remodeling in coronary artery narrowing-induced cardiomyopathy in rats.

To determine whether cardiac failure produced by chronic coronary artery stenosis was associated with the activation of myocyte cellular hyperplasia in the myocardium, the changes in number and size of left ventricular myocytes were measured in rats 3 months after surgery. The hypertrophied left ventricle was found to possess 44%, 32%, 49%, and 48% fewer mononucleated, binucleated, trinucleated, and tetranucleated myocytes, respectively. In contrast, the hypertrophied right ventricle contained 1.49 x 10(6) more myocytes as a result of a 2.1-fold, 1.4-fold, and 1.4-fold increase in mononucleated, binucleated, and tetranucleated myocytes. Myocyte cell volume was seen to increase 49% and 21% in left and right ventricular myocytes, respectively. The process of myocyte cellular hyperplasia in the right ventricular myocardium was accompanied by capillary proliferation, and these events were responsible for the parallel addition of newly formed cells and capillaries within the wall and mural thickening. Moreover, the in-series insertion of new myocytes contributed to right ventricular dilatation after coronary artery stenosis. In view of the fact that extensive myocardial damage and cell loss may have masked the phenomenon of myocyte cellular hyperplasia in the left ventricle, the presence of DNA synthesis in myocyte nuclei was evaluated at 3 days, 1 week, 2 weeks, 1 month, and 3 months after coronary artery stenosis. Bromodeoxyuridine (BrdU) labeling markedly increased in myocyte nuclei of both ventricles, reaching its peak at 1 and 2 weeks. BrdU labeling of nonmyocyte nuclei also increased but mostly at 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Apoptotic cell death during treatment of leukemias.

The apoptosis-associated DNA strand breaks were detected in situ, in individual leukemic cells in peripheral blood and bone marrow of over 110 patients with different types of leukemia (ALL, AML, CML in blastic crisis, APL), prior to and during routine chemotherapy. The DNA strand breaks were labeled with digoxigenin- or biotin-conjugated dUTP in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase, and the cells, counterstained for DNA, were analyzed by bivariate flow cytometry. The proportion of cells with DNA strand breaks prior to therapy, most likely reflecting spontaneous apoptosis, varied from 0.1 to 16%, but in the large majority of cases was below 3%. Administration of drugs of different classes, which included DNA topoisomerase I (Topotecan) and II (mitoxantrone, VP-16) inhibitors, antimetabolite (ara-C) or microtubule poison (Taxol), all triggered the appearance of cells with extensive DNA breakage, typical of apoptosis, to up to 80%. The peak of the response, measured as maximal percent of cells with DNA strand breaks, which varied between individual patients by as much as factor 10, was generally seen between 8 to 24 h after the initial administration of DNA topoisomerase inhibitors, and somewhat later (48-72 h) during the response to Taxol or ara-C. Thus, the data show that the response to treatment with a variety of drugs, in terms of induction of apoptosis, can be conveniently measured by the present method. The prognostic value of the apoptotic index, before, as well as during treatment, is being estimated for each type of leukemia, in the ongoing prospective studies.

Antineoplastic Agents↗

PubMed1994

Apoptotic death of lymphocytes upon treatment with 2-chloro-2'-deoxyadenosine (2-CdA).

In this work we addressed the question if the difference in the mechanism by which 2-CdA kills resting and proliferating cells could be responsible for the therapeutic window of the drug. We show that 2-CdA triggers programmed cell death in proliferating human promyelocytic cell line, HL-60, human lymphocytic cell line, MOLT-4, and human peripheral blood lymphocytes stimulated to proliferation by PHA. Under our experimental conditions 2-CdA failed to induce apoptosis in the resting human peripheral blood lymphocytes despite induction of massive apoptosis in the same lymphocytes stimulated to proliferation by PHA. We also show that 2-CdA-induced apoptosis in HL-60 and MOLT-4 cells can not be prevented by addition of nicotinamide or inhibiting poly(ADP-ribose) synthetase by 3-aminobenzamide. In the case of HL-60 cells apoptosis is specific to the S phase of the cell cycle. Taking together these data suggest that selective induction of apoptosis in proliferating cells may be responsible for the therapeutic value of 2-CdA.

Antimetabolites, Antineoplastic↗

PubMed1994

Altered susceptibility of differentiating HL-60 cells to apoptosis induced by antitumor drugs.

It has been reported that human promyelocytic leukemic HL-60 cells which undergo differentiation fail to respond by apoptosis when treated with antitumor drugs, predominantly DNA topoisomerase inhibitors. Because S phase cells are selectively sensitive to these drugs, and during differentiation there is a reduction in the proportion of cells in S phase, the reported decrease in the number of apoptotic cells could simply be a reflection of the paucity of sensitive cells in these cultures. Using cytometric methods which allow apoptosis to be related to cell cycle position, we have compared the apoptotic response of HL-60 cells growing exponentially and induced to myeloid differentiation by dimethyl sulfoxide (DMSO). The cells were treated with: (i) the DNA topoisomerase I inhibitor camptothecin (CAM), which selectively triggers apoptosis or S phase cells; (ii) the nucleoside antimetabolite 5-azacytidine (AZC) and hyperthermia, both of which preferentially affects G1 cells; and (iii) gamma radiation, which causes apoptosis predominantly of G2 + M cells. The cells exposed to 1.4% DMSO for 24 or 48 h were significantly more resistant to response by apoptosis, regardless of the nature of the agent and regardless of their position in the cell cycle. Thus, induction of differentiation lowers the cell's ability to respond to a variety of damaging agents by apoptosis and this effect is not correlated with cell cycle position. In addition, the difference in response was unrelated to expression of the apoptosis-modulating protein bcl-2, which appeared unchanged following 48 h exposure to DMSO. On the other hand, when the cells were pretreated with low concentrations of CAM or AZC, washed free of drug, and then treated with DMSO, the proportion of cells undergoing apoptosis was markedly increased, relative to drug-treated cells returned to DMSO-free medium. The present data may indicate that while the drug-induced damage screening mechanisms, which are linked to triggering apoptosis, may be more proficient in proliferating cells, the effectors of apoptosis are more expressed in cells undergoing differentiation. The data also suggest that the efficiency of chemotherapeutic agents or radiation may be reduced if a differentiating agent is used in combination therapy and is administered first. An enhancement of apoptosis, however, may be expected if the differentiating drug is administered in the reverse sequence.

Antineoplastic Agents↗

PubMed1993

Simultaneous analysis of cell cycle kinetics at two different DNA ploidy levels based on DNA content and cyclin B measurements.

The methods of cell cycle analysis that rely on DNA content measurements cannot discriminate between cells at different phases of the cycle if these cells have similar DNA content. This limitation can be circumvented by measurement of another cell cycle phase-specific cell constituent in addition to DNA content, followed by bivariate analysis of the correlated data. The aim of the present study was to explore the utility of a monoclonal antibody against the G2- and M phase-specific regulatory protein cyclin B for discrimination of cell populations with overlapping DNA content. This analysis, which was based on correlated DNA/cyclin B content measurements by flow cytometry, was applied to human lymphocytic leukemic MOLT-4 cells. The onset of cyclin B synthesis was observed in the last one third of S phase with its maximum accumulation in G2 and M phases; cells in G1 and early- and mid-S phases were negative. Cells arrested in metaphase by vinblastine expressed high levels of this protein, although not as high as in cells arrested in G2 by the DNA topoisomerase II inhibitor m-AMSA. Disruption of cytokinesis by the protein kinase inhibitor staurosporine led to DNA rereplication, cell progression through the chromatin cycle at higher DNA ploidy, and induction of polyploidy. It was possible, utilizing the cyclin B antibody, to discriminate between G2 + M cells with a 2C level of DNA and G1 cells with 4C DNA, as well as to distinguish doublets of G1 cells with a 2C DNA level. Thus, the rate of cell entrance to G1 at the 4C DNA level and the rates of progression through the cycle at both the 2C and 4C DNA levels could be simultaneously estimated. The data indicate that, in the presence of 0.1 microM staurosporine, cytokinesis of all MOLT-4 cells is impaired and the cells enter to and progress through the chromosome cycle at 4C DNA at the same rates as at 2C DNA. This approach can be helpful in the analysis of DNA ploidy and the cell cycle of human tumors when there is an overlap in DNA content values between normal stromal or infiltrating cells and aneuploid tumor cell population and may be the method of choice to investigate the activity of antitumor drugs which impair cytokinesis but do not interfere with progression of cells through the chromatin cycle.

Alkaloids↗

PubMed1993

Caffeine prevents apoptosis and cell cycle effects induced by camptothecin or topotecan in HL-60 cells.

Caffeine (3,7-dihydro-1,3,7,-trimethyl-1H-purine-6,6-dione; CAF) is known to potentiate the cytotoxic effects of DNA damaging agents such as ionizing radiation and alkylating agents. In contrast, however, the cytotoxic and cytostatic activity of aromatic, DNA-intercalating, DNA topoisomerase II inhibitors such as Adriamycin, ellipticine, or mitoxantrone are diminished in the presence of CAF. To resolve whether the protective effect of CAF is associated with a particular mechanism of drug interaction (e.g., intercalation into DNA, inhibition of DNA topoisomerase II), or the aromatic nature of the drug structure, per se, we have presently studied the effects of CAF on the cytostatic and cytotoxic action of camptothecin (CAM) and its less toxic but more water soluble derivative topotecan (TPT) on HL-60 human myelogenous leukemia cells: both drugs have aromatic structures but are nonintercalating inhibitors of DNA topoisomerase I. By using spectroscopy and titration microcalorimetry, we have also studied the direct interaction between CAF and TPT in solution. Low (20 nM) concentrations of CAM or TPT perturbed progression of HL-60 cells through S-phase, whereas higher concentrations (0.15 microM) of these drugs induced apoptosis; both effects were easily demonstrable after 4 h of treatment. When added simultaneously with CAM or TPT, CAF prevented both effects. The protective effect of CAF was concentration dependent and evident within the concentration range of 1-5 mM; nearly total protection was seen at a CAF concentration of 5 mM. The bathochromic and hypochromic shift in the absorption spectrum of the water soluble compound TPT upon addition of CAF indicated that CAF and TPT interact (stack) in a fashion similar to that previously observed for CAF and DNA intercalators. Microcalorimetric measurements of TPT titration with CAF indicate an exothermic reaction between these compounds (the enthalpy change was delta H degree = -4.2 kcal/mol), which is consistent with a stacking model of CAF-TPT interaction. Thus, the ability of CAF to protect HL-60 cells against the cell kinetic effects of CAM or TPT, as in the case of DNA intercalating topoisomerase II inhibitors, is most likely due to formation of complexes between CAF and these aromatic molecules, which result in reducing the effective concentration of the free form of these drugs available to the cells.

Antineoplastic Agents↗

PubMed1993

The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents.

The studies were aimed to detect the cell cycle-associated differences in the susceptibility of HL-60 cells to apoptosis induced by diverse agents. Exponentially growing HL-60 cells were treated with the DNA topoisomerase I inhibitor camptothecin; the DNA topoisomerase II inhibitors teniposide, m-AMSA, Mitoxantrone, or Fostriecin; the presumed tyrosine kinase inhibitor genistein; a serine/threonine kinase inhibitor H7; the protein synthesis inhibitor cycloheximide; the DNA replication inhibitor hydroxyurea; the nucleoside antimetabolites 1-beta-D-arabinofuranosylcytosine and 5-azacytidine; and the alkylating agent nitrogen mustard, cisplatin, hyperthermia, and gamma irradiation. Endonucleolysis, which accompanied apoptosis induced by these agents, was assessed by two different flow cytometric methods, one based on DNA content measurements following extraction of low molecular weight DNA, and another using exogenous terminal deoxynucleotidyl transferase to label in situ DNA strand breaks. Each method allowed for both identification of apoptotic cells and analysis of the cell cycle distribution of the unaffected cell population; the method using terminal transferase also allowed for identification of the cell cycle position of apoptotic cells. Confirmed by analysis of DNA degradation by gel electrophoresis and changes in cell morphology, apoptosis was observed as early as 3 h after administration of most drugs and for some drugs was cell cycle phase specific. Cells progressing through S phase were selectively susceptible when treated with camptothecin, teniposide, m-AMSA, Mitoxantrone, H7, hydroxyurea, and 1-beta-D-arabinofuranosylcytosine. Cells in G2-M preferentially underwent apoptosis in cultures treated with H7 or with gamma-irradiation. Cells in G1 phase were preferentially affected by 5-azacytidine, nitrogen mustard, and hyperthermia. No significant cell cycle specificity was observed in the case of Fostriecin, genistein, cycloheximide, or cisplatin. The cell cycle related difference in susceptibility to apoptosis may be a reflection of both the severity of the lesion induced by a given drug and the ability of the cells to repair that lesion; both can vary depending on the cell cycle phase.

Antineoplastic Agents↗

PubMed1993

Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays.

DNA strand breaks which occur in HL-60 cells as a result of activation of endonuclease during apoptosis induced by cell treatment with the DNA topoisomerase I inhibitor camptothecin and topoisomerase II inhibitors teniposide, 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide, and fostriecin were labeled in situ, in individual fixed and permeabilized cells, with biotinylated dUTP (detected by fluoresceinated avidin), using the terminal deoxynucleotidyl transferase or nick translation assays. During the early stage of apoptosis, prior to nuclear fragmentation, the breaks were predominantly localized at the nuclear periphery, close to the nuclear envelope. In more advanced stages, all cellular DNA, then localized within the cell as dense, homogeneous granules of a variety of sizes, was strongly labeled, indicating extensive and more uniform distribution of breaks throughout genomic DNA. Bivariate analysis of the incorporated biotinylated dUTP and cellular DNA content by flow cytometry made it possible to estimate the kinetics of the labeling reaction and relate DNA breaks to cell position in the cycle. The kinetics of biotinylated dUTP incorporation was faster, and the distinction of cells with DNA breaks was more pronounced, using the terminal transferase rather than the nick translation assay. Camptothecin, teniposide, and 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide induced DNA breaks preferentially in S-phase cells, having little effect on cells in the G1 phase of the cycle. In contrast, fostriecin affected cells indiscriminately, in all phases of the cell cycle. The method of detection of DNA strand breaks (3'-hydroxyl termini) in individual cells offers several advantages and can be applied to clinical material (tumor biopsies) to study the induction of apoptosis in tumors during treatment, as a possible prognostic marker. The protein-associated DNA breaks in the "cleavable" DNA-topoisomerase complexes, which are the primary lesions induced by the inhibitors and precede apoptosis, were not detectable by the present methods.

Apoptosis↗

PubMed1993

Altered sensitivity of DNA in situ to denaturation in apoptotic cells.

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Apoptosis↗

PubMed1993

Characterization of lineage-negative blast subpopulations derived from normal and chronic myelogenous leukemia bone marrows and determination of their responsiveness to human c-kit ligand.

Lineage-negative (lin-) normal and chronic myelogenous leukemia (CML) marrow blast populations were obtained by negative selection and subsequently separated on the basis of size by velocity sedimentation. The three subpopulations of lin- blasts obtained were enriched for F8 (the more primitive small blasts), F11 (blasts intermediate in size), and F13 (the more mature large blasts). We examined the morphological and phenotypic characteristics and cell cycle status of the subpopulations and determined the responsiveness of granulocyte-monocyte progenitors (colony-forming units/granulocyte-macrophage) derived from each subpopulation to mast cell growth factor in combination with granulocyte (G-CSF) or granulocyte-macrophage (GM-CSF) colony-stimulating factors alone and in combination. Morphological assessment revealed that an increased proportion of CML lin- blasts exhibited early cytoplasmic maturation as evidenced by the appearance of azurophilic (nonspecific) granules in the cytoplasm. Although the percentages of CML and normal small blasts expressing CD34 were similar, the proportion of CML lin- blasts expressing CD34 declined in the intermediate and more mature large lin- blast subpopulations by about 50%, whereas the percentage of CD34+ normal blasts remained essentially the same, indicating an earlier loss of CD34 expression by CML lin- blasts. In addition, the percentages of CML small blasts expressing CD33 were higher than normal (26-61% versus 0-16%, respectively), indicating that a higher proportion of CML small lin- blasts had a more mature phenotype. Mast cell growth factor addition to cultures stimulated by G-CSF, GM-CSF, or G-CSF plus GM-CSF, exerted the greatest synergistic effect (increased colony number and size) in the normal small and intermediate lin- blast cultures, but mast cell growth factor had considerably less effect, or no effect, in cultures of comparable CML subpopulations, indicating that CML lin- progenitors had a somewhat lower requirement for multiple growth factors. The findings suggest that the differences observed between normal and CML marrow subpopulations are proportional differences and that a greater proportion of CML lin- blast subpopulations exhibit characteristics associated with a more advanced stage of maturation than comparable normal lin- blast subpopulations.

Bone Marrow↗

PubMed1993

Myocyte DNA synthesis with aging: correlation with ventricular loading in rats.

To determine whether the detrimental mechanical and anatomical changes that occur biventricularly with aging are associated with activation of DNA synthesis, flow cytometric analysis was performed on myocyte nuclei prepared from the left and right ventricles of rats at 4, 12, 20, and 29 months of age. Heart weight increased significantly with age, and this growth adaptation was associated with the development of left ventricular failure and right ventricular dysfunction. These phenomena were coupled with marked elevations in diastolic wall stress and increases in the percentage of myocyte nuclei in S+G2M in both ventricles. Linear regression analyses revealed a direct correlation between the fraction of myocytes that entered the cell cycle and diastolic pressure and wall stress. An inverse relation was found between the percentage of myocyte nuclei in S+G2M and +dP/dt and systolic wall stress. Thus the depression of hemodynamic performance coupled with alterations in the loading conditions contributes, at least in part, to increased DNA synthesis in cardiac myocytes with age.

Aging↗

PubMed1993

Different patterns of apoptosis of HL-60 cells induced by cycloheximide and camptothecin.

Cells of the human promyelocytic HL-60 line, when treated with a variety of antitumor agents in the presence of the protein synthesis inhibitor cycloheximide (CHX), or with CHX alone, rapidly undergo apoptosis ("active cell death"). It is presumed, therefore, that such cells are "primed" to apoptosis in that no new protein synthesis is required for induction of their death. We have studied apoptosis of HL-60 cells triggered by the DNA topoisomerase I inhibitor camptothecin (CAM) in the absence and presence of CHX and apoptosis induced by CHX alone. Two different flow cytometric methods were used, each allowing us to relate the apoptosis-associated DNA degradation to the cell cycle position. Apoptosis induced by CAM was limited to S phase cells, e.g., at a CAM concentration of 0.15 microM, nearly 90% of the S phase cells underwent apoptosis after 4 h. In contrast, apoptosis triggered by CHX was indiscriminate, affecting all phases of the cycle: approximately 40% of the cells from each phase the cycle underwent apoptosis at 5 microM CHX concentration. When CAM and CHX were added together, the pattern of apoptosis resembled that of cycloheximide alone, namely, cells in all phases of the cycle in similar proportion were affected. Thus, CHX, while itself inducing apoptosis of a fraction of cells, protected the S phase cells against apoptosis triggered by CAM. Because CHX (5 microM) did not significantly affect the rate of cell progression through S phase, the observed protective effect was most likely directly related to inhibition of protein synthesis, rather than to its possible indirect effect on DNA replication. Furthermore, whereas apoptosis (DNA degradation) triggered by CAM was prevented by the serine protease inhibitor N-tosyl-L-lysylchloromethyl ketone (TLCK), this process was actually potentiated by this inhibitor when induced by CHX. The present data indicate differences in mechanism of apoptosis triggered by CAM (and perhaps other antitumor drugs) as compared with CHX. Apoptosis caused by CHX may be unique in that it may not involve new protein synthesis. These data are compatible with the assumption that the loss of a hypothetical, rapidly turning over suppressor of apoptosis may be the trigger of apoptosis of HL-60 cells treated with CHX, whereas de novo protein synthesis is required when apoptosis is triggered by other agents.

Apoptosis↗