Effects of ellipticine on cell survival and cell cycle progression in cultured mammalian cells

The effects of ellipticine [5,11-dimethyl-6H-pyrido(4,3-b)carbazole; NSC 71795] on cell viability, growth, and colony formation were investigated in suspension (Friend leukemia and L1210) and adherent [Chinese hamster ovary (CHO)]tumor cell systems as well as in mitogen-stimulated human peripheral blood lymphocyte cultures. Cell cycle progression and the terminal point of action of the drug were monitored by flow cytometry. Ellipticine was cytostatic for all cell lines tested, blocking cells in G2 phase following 24 hr constant exposure at concentrations in the range of 1.0 microgram/ml. A 10 times higher drug concentration was required to block cells in G2 if the cells were exposed for only 30 min to the drug followed by 23.5 hr culture in drug-free medium. Formation of CHO cell colonies was inhibited by 50% following exposure to ellipticine for 2 hr at 6.0 microgram/ml or for 24 hr at 0.3 microgram/ml. Fifty % cell kill in asynchronously growing Friend leukemia and L1210 cells was obtained following exposure to ellipticine for 24 hr at 2.0 microgram/ml and 1.15 microgram/ml, respectively, whereas human peripheral blood lymphocytes required 66 hr exposure to 1.0 microgram/ml to kill 50% of the cells. Phytohemagglutinin-stimulated lymphocytes were remarkably resistant to the cytotoxic effect of ellipticine but did display a dose-dependent inhibition of stimulation and accumulation in G2 whether the drug was added prior to our during active cell proliferation. Ellipticine, at cytostatic concentrations, had a marked effect on cellular RNA content. Friend leukemia cells, blocked in G2 by the drug, doubled their RNA content compared to control cells. L1210 and CHO cells, but not lymphocytes, also increased in RNA content following ellipticine treatment. Drug concentrations which blocked cells in G2 also led in the case of Friend leukemia and L1210 but not CHO cells to an increase in the proportion of cells with greater than 4C amounts of DNA.     

Effects of chartreusin on cell survival and cell cycle progression

Chartreusin was lethal to both L1210 and P388 cells in culture with 90% of the cells being killed after a 24-hr exposure to 1.1 and 2.6 microgram/ml, respectively. The lethality of the drug increased in direct proportion to dose and exposure time. Both L1210 and Chinese hamster ovary cells in S phase were more sensitive to the lethality of the drug than were their corresponding non-S-phase cells. L1210 cells were partially synchronized by exposing an asynchronous culture to [methyl-3H]thymidine (20 Ci/mmol) and Colcemid for 3 hr. Synchronous culture of Chinese hamster ovary cells was established by planting mitotic cells. The progression of cells through the cell cycle was studied with flow microfluorometry both in the presence of the drug and after the drug had been washed off. In the presence of chartreusin the progression of mitotic cells into G1 was not affected. The movement of G1 cells into S was slower, and the movement of G2 cells into mitosis was blocked. When the drug was removed, the G2 to M block persisted for at least 4 hr but the progression of G1 cells to S was no longer inhibited.     

Effects of etoposide (VP-16) on the survival and progression of cultured HeLa S3 cells through the cell cycle

The survival of cultured HeLa S3 cells and their progression through the cell cycle after exposure to Etoposide (VP-16) were studied. The dose survival curve of exponentially growing cells was of a biphasic nature; the curve had an exponential slope starting from a small shoulder, and finally attained a plateau against increasing concentrations of the drug. Throughout the cell cycle, VP-16 exerted its main killing activity on cells in the late S and G2 phases, whereas cells in the G1 phase were resistant to the drug. By using a low concentration of VP-16, which allowed 85% of the treated cells to survive, it was found that the inhibition of the cell progression by the drug was greatest in the late S and G2 phases.     

Effects of 5-aza-2'-deoxycytidine on survival and cell cycle progression of L1210 leukemia cells

The in-vitro effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-dCyd), on DNA synthesis, growth, cloning in agar, and cell cycle traverse of L1210 leukemia cells were studied. 5-Aza-dCyd at 0.1 microgram/ml for 10 hr (cytotoxic concentration) did not inhibit DNA synthesis but produced a very potent growth inhibition, and changed markedly the DNA flow cytometric histograms. A 5-h continuous exposure to the drug at concentrations ranging from 0.1 to 10 micrograms/ml caused an accumulation of cells in the S portion of the DNA histograms indicating a slowing of the progression of cells in the S phase. A longer exposure time (10 h) at the same concentrations led to a bimodal DNA distribution (peaks at G1 and G2-M) and a depletion of the S phase. When the exposure time to 5-aza-dCyd (0.1 microgram/ml) was extended to 15 and 20 h, there was a decrease in the G2-M peak and an augmentation of the G1 peak. To determine if 5-aza-dCyd produced a block in cell cycle progression, L1210 cells were treated for 10 h with colcemid and 5-aza-dCyd simultaneously for 10 h. Colcemid alone, or colcemid in combination with 5-aza-dCyd produced an accumulation of cells under a single G2-M peak. This indicates that 5-aza-dCyd did not block the progression of L1210 cells through S phase, but only produced a slowing down of this event. These results, indicating that 5-aza-dCyd does not block cell cycle progression and that its cytotoxic action is not self-limiting, are of importance for designing future clinical trials.     

Effect of gossypol on DNA synthesis and cell cycle progression of mammalian cells in vitro

Gossypol, a yellow phenolic compound extracted from cotton plants that is being used as a male antifertility drug, has been found to have cytotoxic effects. The objective of the present study was to determine the cause of these cytotoxic effects in cultured cells and to ascertain whether cells in certain phases of the cell cycle were more sensitive to the drug than were cells in other phases. HeLa or Chinese hamster ovary cells were exposed to various concentrations of gossypol for varying periods of time, and the effects of the drug on the growth rate, cell cycle traverse, plating efficiency, and macromolecular synthesis were measured. The results of this study indicate that gossypol is a specific inhibitor of DNA synthesis; it has no effect on RNA and protein synthesis at a concentration of 10 micrograms/ml and hence has no effect on the cell cycle traverse of cells from G2 to the beginning of S phase. In the presence of the drug, cells can enter S phase but fail to complete replication. The effects of the drug on growth rate and plating efficiency indicate that there is a threshold concentration at which gossypol becomes effective as a cytotoxic agent. Gossypol did not increase chromosome aberrations in the treated cells. Since gossypol irreversibly blocks cells in S phase, it could be useful as an antitumor drug.     

Effects of 9-OH-ellipticine on cell survival, macromolecular syntheses, and cell cycle progression in sensitive and resistant Chinese hamster lung cells

In an effort to understand the mechanism of action of the DNA-intercalating antitumor agent 9-hydroxyellipticine (9-OH-E), we have examined the effects of this drug on the cell survival, macromolecular syntheses, and cell cycle progression in sensitive and resistant cells. Our results show that 9-OH-E toxicity on sensitive and resistant cells involves different mechanisms of action: the drug toxicity in the sensitive cells appears to result from lethal lesions mediated through the interaction of the drug with an intracellular protein, independently of any effect of the drug on the macromolecular syntheses; in the resistant cells, the cell death occurs concomitantly with the inhibition of these syntheses. Cell cycle progression analysis after 9-OH-E treatment showed that, in the sensitive cells, the drug is inducing a G1 and a G2 block, which are both released in the presence of 1 mM caffeine, without any effect on the 9-OH-E toxicity. In the resistant cells, a G2 block was also observed but only when the cells were resuming their growth after about a 30- to 40-h growth arrest. Caffeine release of this block, which again had no effect on 9-OH-E toxicity, was only observed when it was added from 40 to 60 h after 9-OH-E treatment, when the cells resumed their growth. Finally in the sensitive cells, cycloheximide exerted an inhibitory effect on 9-OH-E toxicity when it was added before and during the cell exposure to the drug. This effect was interpreted as indicating that 9-OH-E toxicity in the sensitive cells relies on a protein which is not induced by the drug but has to be present in the cells when the drug is added. The possible implication of DNA topoisomerases in 9-OH-E toxicity mechanism is discussed.     

Effects of ARHI on cell cycle progression and apoptosis levels of breast cancer cells

The purposes of this study were to investigate the role of Aplysia Ras Homolog I (ARHI) on cell growth, proliferation, apoptosis, and other biological characteristics of HER2-positive breast cancer cells. Our goal was to provide experimental evidence for the development of future effective treatments of HER2-positive breast cancer. A pcDNA3.1-ARHI eukaryotic expression vector was constructed and transfected into the human HER2-positive breast cancer cell lines SK-BR-3 and JIMT-1. Then, various experimental methods were utilized to analyze the biological characteristics of ARHI-expressing breast cancer cells and to examine the impact of expression of the ARHI gene on cyclin D1, p27^Kip1, and calpain1 expression. We further analyzed the cells in each group after treatment with trastuzumab to examine the effects of this drug on various cellular characteristics. When we compared pcDNA3.1-ARHI-expressing SK-BR-3 and JIMT-1 cells to their respective empty vector and control groups, we found that cell viability was significantly lower (p?<?0.05) in the ARHI-expressing cells, and the proportions of G1 phase cells and apoptotic cells were significantly higher in the ARHI-expressing cells (p?<?0.05). In all groups of SK-BR-3 cells, trastuzumab treatment significantly decreased cell growth (p?<?0.05). The proportion of cells in G1 phase and the number of apoptotic cells in the pcDNA3.1-ARHI-expressing group were significantly higher than that in the empty vector group and the control group (p?<?0.05). The growth of pcDNA3.1-ARHI-transfected JIMT-1 cells was significantly decreased (p?<?0.05), while the proportion of apoptotic cells was significantly increased (p?<?0.05). Cell growth, viability, and the percentage of apoptotic cells were similar between the JIMT-1 empty vector and control groups. ARHI expression inhibited cyclin D1 expression in SK-BR-3 cells and JIMT-1 cells, while it promoted p27^Kip1 and calpain1 expression in these cells. ARHI expression inhibits the growth and proliferation of HER2-positive breast cancer cells, while it also promotes apoptosis in these cells. ARHI expression also improves the sensitivity of JIMT-1 cells to trastuzumab by inducing apoptosis.     

Comparative effects of three nitrosourea derivatives on mammalian cell cycle progression.

Three nitrosourea analogs, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and 1-trans-(2-chloroethyl)-3-cyclohexyl-1-(4-methylcyclohexyl)-1-nitrosourea, were examined for effects on survival and cell cycle traverse capacity in exponentially growing (cycling) populations of line CHO Chinese hamster cells and in cultures arrested in G1 by isoleucine deprivation during treatment with drugs, then returned to the cycling mode by restoration of isoleucine (noncycling cells). Among parameters studied were survival, cell division, DNA initiation capacities, cell cycle distributions, and rates of cell cycle traverse in drug-treated cycling and noncycling cells utilizing a protocol combining autoradiography, cell number enumeration, and flow microfluorometry. The results obtained were in generally good agreement with results obtained in vivo in other studies and included the following. Cells treated with any of these agents accumulated preferentially in late S and G2, primarily the result of a gross increase in duration of these phases of the cell cycle. There was also a prolongation of doubling time during the early stages following drug treatment and return to the proliferating mode of cells which ultimately survived. All three drugs induced mitotic nondisjunction in cells capable of dividing and also induced polyploidy by allowing multiple rounds of progression through the cell cycle in the absence of an intervening cell division. In treated populations, the G2-arrested and polyploid cells were among the first cells to die. Treated, noncycling cells that were returned to cycle exhibited a lower survival capacity than did treated, cycling cells. Finally, 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea and 1-trans(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea induced a dramatic alteration in clonal morphology and growth patterns in surviving cells that persisted for at least a week after drug removal. The results obtained suggest that our model system may be useful as a predictive guide for determining response of susceptible tumor cells to treatment with chemotherapeutic agents.     

Effects of tamoxifen on cell cycle progression of synchronous MCF-7 human mammary carcinoma cells

The effects of tamoxifen on cell cycle progression and clonogenic survival have been examined using synchronized cultures of MCF-7 human mammary carcinoma cells. Cell synchrony was induced by mitotic selection. Subsequent cell cycle analyses, using DNA flow cytometry, showed that 85% of synchronized cells had a mean cell cycle time of 21.3 hr with mean phase durations of 9 hr for G0-G1, 9.3 hr for S, and 3 hr for G2 + M. A slowly cycling or noncycling subpopulation comprising 15% of the total population was also observed. Exposure to tamoxifen (5 to 12.5 microM) resulted in a dose-dependent reduction in the number of cells progressing through G0-G1 and entering S phase. Those cells which were not retained in G0-G1, however, appeared to traverse G0-G1 and the remainder of the cell cycle at a rate only slightly less than that of untreated controls. Further experiments demonstrated that the major sensitivity to tamoxifen in terms of both inhibition of cell cycle progression and drug cytotoxicity was restricted to a short interval in the middle of G0-G1. This 2- to 4-hr period of maximum drug sensitivity began approximately 4 hr after mitotic selection, with drug exposures outside this time frame having markedly fewer effects. The significance of these observations in the light of previous studies with asynchronous populations of MCF-7 cells is discussed.     

Cellular pharmacokinetics of aclacinomycin A in cultured L1210 cells

Summary Aclacinomycin, which is more lipophilic than daunorubicin and doxorubicin, is taken up and released more rapidly and extensively by L1210 cells. After 5 h of incubation 91% of aclacinomycin is found in the nuclei of L1210 cells and the drug present in the post-nuclear fraction is distributed between the lysosomes and the cytosol. After an incubation of 5 h aclacinomycin decreases the density of the lysosomes. This effect is not observed either with doxorubicin or daunorubicin or when the cells are incubated with aclacinomycin for only 30 min.     

Effect of ACNU, a water-soluble nitrosourea derivative, on survival and cell progression of cultured HeLa S3 cells

Effects of a water-soluble nitrosourea derivative, 1-(4-amino-2-methylpyrimidin-5-yl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride [ACNU]. on survival and cell progression of HaLe S3 cells was investigated. The survival of exponentially growing cells exposed to increasing concentrations of the drug was characterized by a threshold-type survival curve (D0 = 7.0 micrograms/ml X 1 hr, Dq = 3.5 micrograms/ml X 1 hr). ACNU exerted its main killing effect on cells in G1 and G2 + M phases, whereas cells in S phase were resistant to the drug. Changes in survival response as a function of cell cycle were mainly dependent upon the extent of the exponential slope of the survival curve. Cell progression effects were examined by using a low concentration of ACNU in which 80% of treated cells could survive. Cells in G1 and early S phases at the time of treatment were not prevented from entering S phase but prolonged in duration of S phase followed by a marked delay in progression through G2 phase. However, such a delay in cell progression time was reduced in cells treated in mid S phase as compared with G1 and early S phases. Cells treated in late S and G2 phases could normally progress into mitosis.     

Connecting signaling and cell cycle progression in growth factor-stimulated cells

A widely used model system to investigate cell proliferation is stimulation of serum-arrested cells with growth factors. Recent data suggest that there are two waves of growth factor-dependent signaling events required for a proliferative response. One is an acute burst of signaling, which occurs immediately after growth factor stimulation and lasts for 30 - 60 min. The other occurs in a different time frame (8 - 12 h post stimulation), and involves activation of cyclin dependent kinases (Cdks). In addition to a general overview of growth factor-dependent signaling, we present our 'two wave' hypothesis for how signaling and cell cycle progression are linked.     

Resveratrol inhibits cell cycle progression in U937 cells

Resveratrol, a naturally occurring stylbene present in grapes, is proposed to be responsible for the positive effects of red wine consumption on cardiovascular diseases (French paradox). In recent years this molecule has also been proposed as a cancer chemopreventive agent. The aim of the present study was to investigate the mechanisms by which resveratrol inhibits tumor growth. For this purpose, U937 cells were exposed to resveratrol at concentrations usually present in red wine, and the effects on proliferation, death and cell cycle machinery were assessed. The U937 cell growth was impaired due to reduced cell proliferation, without significant induction of apoptosis. This anti-proliferative effect is associated with modulations in the pattern of DNA distribution, with a reduction of G0/G1, particularly G2-M peaks and accumulation in the S phase of the cell cycle. This result was confirmed by the observation that the rate of [3H]-thymidine incorporation into DNA was significantly reduced in resveratrol-treated U937 cells. Consistent with this observation, in the same experimental conditions, the activity of ribonucleotide reductase, an enzyme critically involved in the process of DNA duplication, decreased. The altered progression in the cell cycle could depend on modulations in the activity of cyclin dependent kinases and their inhibitors. After exposure of U937 cultures to resveratrol, the expression of cyclins A and E, as well as that of CDK2 increased, while that of p21CIP was significantly reduced. The data shown in this report suggests that the prevention of cancer development exerted by resveratrol may result from modulations of molecules involved in the regulation of cell cycle progression, which block the cells at the S phase checkpoint.     

Effects of ErbB-2 overexpression on mitogenic signalling and cell cycle progression in human breast luminal epithelial cells

Most breast cancers arise from luminal epithelial cells and 25-30% of these tumours overexpress the ErbB-2 receptor. Herein, a non-transformed, immortalized cell system was used to investigate the effects of ErbB-2 overexpression in luminal epithelial cells. The phenotypic consequence of ErbB-2 overexpression is a shortening of the G1 phase of the cell cycle and early S phase entry, which leads to hyperproliferation. We show that this effect was mediated through the up-regulation of cdk6 and cyclins D1 and E, and enhanced degradation and relocalization of p27(Kip1). These changes were effected predominantly through enhanced MAPK signalling, resulting in cdk2 hyperactivation. PI3K signalling also participated in cell cycle progression, since PI3K and MAPK coordinately regulated changes in cyclin D1 and cdk6 expression. Cdk4 activity was not required for cell cycle progression in these cells, and was constitutively inhibited through its association with p16(INK4A). MAPK-dependent induction of p21(Cip1) was also necessary for G1 phase progression, although its degradation by the proteasome was required for S phase entry. These data provide new insights into the complex molecular mechanisms underlying mitogenic cell cycle control in luminal epithelial cells, the cell type relevant to primary breast cancer, and show how ErbB-2 overexpression subverts this normal control.     

Effect of flavonoids on cell cycle progression in prostate cancer cells.

The effect of some flavonoids, which are components of fruits, vegetables, and peas, on the cell cycle progression of human LNCaP prostate cancer cells has been investigated in this study. Genistein arrested the cell cycle at the G2/M phases, which is attributed to the suppression of cyclin B expression. In addition, genistein induced the cyclin-dependent kinase inhibitor p21, which does not depend on p53 activation. Apigenin and luteolin also increased p21 levels, but quercetin did not. Apigenin induced p21 production through a p53-dependent pathway, but luteolin did so in a p53-independent manner. These results suggest that flavonoids are potent regulators of cyclin B and p21 for cell cycle progression, which may play some roles in prevention of carcinogenesis.