The induction of apoptosis by chemotherapeutic agents occurs in all phases of the cell cycle.

A1.1 T-cell hybridoma cells exposed to either actinomycin D (1 microgram/ml), camptothecin (200 ng/ml) or aphidicolin (10 micrograms/ml) for 16 hrs at 37 degrees C die via apoptosis. The cell death was independent of RNA synthesis, in contrast to previous data reported for other forms of apoptosis in murine lymphocyte cells and their derived lines. Each of the three agents described appeared to induce death in all phases of the cell cycle in asynchronously proliferating cells. G1 cells appeared to be more susceptible to the effects of camptothecin and contrasts with other reports which detail its selectivity for S and G2 phase cells. This might indicate that cells are progressing into S phase before dying or, alternatively, cells may indeed be dying in G1. When elutriated synchronised cells were exposed to each of the three cytotoxic agents cell death occurred in all phases of the cell cycle. In view of the fact that G1 and S phase cells did not cycle to any appreciable extent during drug exposure, it was likely that ensuing death, occurred specifically from these phases. G2/M cells, however, moved rapidly into G1 in the presence of each drug, thus making it difficult to determine whether G2/M cells were capable of undergoing drug-induced apoptosis. To overcome this problem, nocodazole (50 ng/ml) was used to block asynchronous cells in M phase. When these cells were exposed to actinomycin D, aphidicolin or camptothecin, cell death ensued via apoptosis.     

Effects of gossypol on the cell cycle phases in T-47D human breast cancer cells.

Since gossypol, a naturally occurring component of cottonseed oil, exhibits a broad spectrum of activities, we have examined it as an antitumor agent on breast cancer. The effects of different concentrations of gossypol on the T-47D human breast cancer cell cycle phases were studied using cytometric image processing on Feulgen stained nuclei. The proportion of cells at different cell cycle phases was determined by discriminate analysis of the image parameters and gave good classification ranging from 86 to 100%. Gossypol was found to increase the G0/G1 fraction of the T-47D cells. This cell kinetic alteration by gossypol was shown to be dose dependent and reversible. Complete reversal of the effect of gossypol was observed after four days with a simple change to gossypol-free medium. The cells then progressed into S and G2/M phase, thus indicating that gossypol-treated cells remain viable. Gossypol was shown to have a strong inhibitory effect on cellular proliferation in T-47D cells. It was also found that this agent is only toxic to cells at the highest dose tested (10 microM). The results of this study may be of clinical significance in the treatment of breast cancer, since gossypol shows strong antiproliferative properties.     

Effect of normal and tumor factors on different phases of cell populations cycle

In the present experiments we studied the effect of extracts from intact liver (LE), ES2 tumor extract (TE), plasmas from intact mice (PI), and from tumor bearing animals (PT) on different phases of hepatocytes and renocytes cell cycles. C3HS 28-day-old male mice, standardized for periodicity analysis, were injected at 16:00 hours and killed every 4 hours during a circadian cycle at 20:00/04; 00:00/08; 04:00/12; 08:00/16; 12:00/20 and 16:00/24 (time of day/hours post treatment). Colchicine (2 microg/g) was injected 4 hours before killing them. Samples of livers and kidneys were processed for histology and mitotic index determinations. The results were expressed as colchicine arrested metaphases per 1000 nuclei. The TE, LE and PI had a promoting effect on the mitotic activity of hepatocytes during the first 12 hours post treatment. During the subsequent 12 hours, not only these treatments but also the PI had an inhibiting effect on the mitotic activity of the same cell population. Also the TE and the PT had a promoting effect on the mitotic activity of the renocytes during the first 12 hours while the effect of all treatments showed a clear inhibition of the mitotic activity studied during the last 12 hours. Taking into account the time elapsed between the injections and the measurements made in these light-dark synchronized animals, we conclude that the increase in mitotic index observed in those tissues stemmed from a reinitiation of cell-cycle traverse in a subpopulation of G2-arrested, noncycling cells.     

Glutathione contents in human and rodent tumor cells in various phases of the cell cycle

Experiments were carried out to investigate whether or not the cell cycle dependent cytotoxicity of adriamycin (ADR) was a consequence of variations in cellular glutathione (GSH) levels in different phases of the cell cycle. The GSH content of a range of rodent and human tumor cell lines, grown both in vivo and in vitro, were measured by high performance liquid chromatography. Enrichment of cells in various cell cycle phases was accomplished by centrifugal elutriation. The GSH content of cells in the different phases of the cell cycle increased in proportion to the increases in cell volume from G1 to S phase to G2-M. However, the apparent differences in GSH content across the cell cycle were eliminated when GSH content was normalized according to cell volume. This observation held true for all cell lines studied. The cell cycle dependent cytotoxicity of ADR therefore was not related to cell cycle dependent variations in GSH content. Buthionine sulfoximine, a specific inhibitor of GSH synthesis, depleted the GSH of cells in G1, S, and G2-M of the cell cycle by similar rates and enhanced the cytotoxicity of ADR to similar extents. These results suggest that although GSH does confer a significant degree of protection against the toxic effects of ADR in general, the more specific differences in response to ADR across the cell cycle probably were not the consequence of thiol variations but rather the result of other as yet unidentified factors.     

Radiosensitization by pemetrexed of human colon carcinoma cells in different cell cycle phases

PURPOSE: The novel folate antimetabolite Alimta (pemetrexed disodium, LY231514) exhibits antitumor activity in a broad array of human malignancies and was recently found to enhance radiation-induced cell killing in vitro. In the present study, a possible cell cycle phase-specific radiosensitization by pemetrexed was assessed. METHODS AND MATERIALS: Widr human colon carcinoma cells were synchronized by serum withdrawal/stimulation that yielded about 80% cells with G1 DNA content 6 h after replating and more than 60% S-phase cells after 22 h, as assessed by flow cytometry. The respective cultures were irradiated with doses up to 12 Gy in combination with a subtoxic pemetrexed exposure (1.06 microM for 2 h: about 80% survival), or after mock treatment. Survival curves were generated by the clonogenic assay; apoptosis was measured by sub-G1 DNA flow cytometry. RESULTS: The combination treatment of the G1 cells and of the more radioresistant S-phase cell preparations yielded survival rates that were lower than expected for independent cell killing. Radiosensitization, calculated as the ratio of the mean inactivation doses without or with drug exposure (enhancement ratio), was not significantly different for the two cell preparations (enhancement ratio of 2.1 and 2.3, respectively) and was similar to the previously reported value for log-phase cells. Pemetrexed exposure was unable to stimulate an apoptotic response of these cells to radiation. CONCLUSIONS: Radiosensitization by pemetrexed is not cell cycle phase-specific, and the relative radioresistance of S-phase cells is retained. Apoptosis seems to have no influence on radiosensitization in this cell line.     

Flow cytometric immunofluorescence assay for quantification of cyclobutyldithymine dimers in separate phases of the cell cycle

Quantitative immunofluorescence assays for the measurement ofcyclobutyldithymine dimers (T < > T) based on computer-assistedimmunofluorescence microscopy have recently been described.Here we present a modified assay for T < > T based onflow cytometry. This method has the advantage that T < >T can be quantified in separate phases of the cell cycle bythe fluorescent counterstaining of nuclear DNA and subsequentselection on DNA content. The H3 monoclonal antibody directedat T < > T binds to partially denatured DNA in situ. Theantibody is labeled with fluorescein isothiocyanate (FITC) andDNA is stained with the intercalating dye 7-amino-actinomycinD. FITC fluorescence increases linearly with dose of UV-C radiation(up to 45 J/m²) of cultured human fibroblasts. A linear fluorescence—doserelationship was also found for epidermal cells of SKH:HR1 hairlessmice after in vivo irradiation with UV-B (FS40 sunlamp, up to3750 J/m²). This technique allows a quick assessment of UV damagelevels in 10 000s of cells and makes immunofluorescence of DNAdamage more accessible to other research groups.     

Expression of surface antigens during the cell cycle in different growth phases of American and African Burkitt's lymphoma cell lines

We have studied the expression of five surface antigens in eight Burkitt's lymphoma cell lines during different phases of the cell cycle and in different growth phases (logarithmic and stationary). Cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and DNA content (propidium iodide), and dual parameter measurements were performed with a flow cytometer. Analysis of cells in specific cell cycle phases during log-phase growth revealed a 1.6-fold increase in surface antigen expression as cells passaged from G1 to G2/M. This is almost identical to the measured increase in cell surface area which occurs during passage of cells through the cell cycle and indicates that under optimal conditions surface antigen density is maintained during cell doubling. We also observed a consistent reduction, by about 50%, in the expression of surface IgM (mu), k-light chain, and B1 on the cell lines during a 5-day culture period. Cell lines that only weakly expressed surface IgM were found to have a more rapid decrease, and in such cell lines IgM was ultimately completely lost from the cell surface. In contrast, the expression of beta 2-microglobulin and HLA-ABC increased in some cell lines, whereas in others a significant decrease of both beta 2-microglobulin and HLA expression was demonstrated as the cells entered stationary growth phase. Decreased cell volume (and therefore surface area) associated with declining growth rate and fewer late S or G2/M cells could account for 20-30% of the observed reduction in surface IgM, k-light chain, and B1 expression, but the major decrement in fluorescence intensity was due to a reduction in the density of these surface antigens. Thus, the ability to maintain surface antigen densities is frequently lost in suboptimal culture conditions.     

Hypoxia induces p53 accumulation in the S-phase and accumulation of hypophosphorylated retinoblastoma protein in all cell cycle phases of human melanoma cells.

Hypoxia has been shown to induce accumulation of p53 and of hypophosphorylated retinoblastoma protein (pRb) in tumour cells. In this study, the cell cycle dependence of p53 accumulation and pRb hypophosphorylation in four human melanoma cell lines that are wild type for p53 was investigated using two-parameter flow cytometry measurements of p53 or pRb protein content and DNA content. The hypoxia-induced increase in p53 protein was higher in S-phase than in G1 and G2 phases in all cell lines. The accumulation of p53 in S-phase during hypoxia was not related to hypoxia-induced apoptosis or substantial cell cycle specific cell inactivation during the first 24 h of reoxygenation. pRb was hypophosphorylated in all cell cycle phases by hypoxia treatment. The results did not support a direct link between p53 and pRb during hypoxia because p53 was induced in a cell cycle-specific manner, whereas no cell cycle-dependent differences in pRb hypophosphorylation were detected. Only a fraction of the cell populations (0.60+/-0.10) showed hypophosphorylated pRb. Thus, pRb is probably not the only mediator of the hypoxia-induced cell cycle block seen in all cells and all cell cycle phases. Moreover, the cell cycle-dependent induction of p53 by hypoxia suggests that the primary function of p53 accumulation during hypoxia is other than to arrest the cells.     

Control of the cell cycle

Cell biology has made major progress in identifying the molecules that drive the cell cycle. The evidence accumulating from these studies indicates that derangements in the cell cycle machinery contribute to the uncontrolled cell growth of tumours. The cell cycle machinery has been found to be substantially altered in tumour cells and also may be crucial for carcinogenesis. In this context, various aspects of tumour cell growth have been studied in an effort to understand 1) why tumour cells display uncontrolled growth, 2) why radiation selectively affects growing cells, and 3) whether aspects of the cell cycle and tumour cell growth may be used in tumour diagnosis and prognosis.     

Human papillomavirus life cycle: active and latent phases

Productive infection by human papillomaviruses (HPV) is dependent upon the differentiation of the host cell. Following entry into basal epithelial cells, HPV genomes are established as autonomous replicating extrachromosomal elements and a low level of HPV expression occurs. Upon differentiation of infected cells, productive replication and expression of capsid genes is induced resulting in the synthesis of progeny virions. Evidence from immunosuppressed patients as well as individuals with recurring laryngeal papillomatosis suggest that certain HPV types can exist in a latent state. In latently infected cells, HPV DNA may be present but no differentiation-dependent synthesis of virions occurs. The presence of a latent state for HPVs can be a determining factor in the effectiveness of therapeutic methods for treatment of infections.     

Effects of ketoconazole on the proliferation and cell cycle of human cancer cell lines.

The growth-inhibitory effects of ketoconazole, an antifungal agent which inhibits arachidonic acid lipoxygenases and cytochrome P-450 enzymes, were tested in human colon and breast cancer cell lines. In the serum independent HT29-S-B6 colon cell clone, ketoconazole reduced cell proliferation and [3H]thymidine incorporation in a dose-dependent fashion, with a 50% inhibitory concentration of approximately 2.5 microM. Flow cytometry showed an accumulation of cells in the G0-G1 phase of the cell cycle and a concomitant decrease of the percentage of cells in S phase. Ketoconazole also inhibited [3H]thymidine incorporation in the hormone-independent breast cancer cells MDA-MB-231 and Evsa-T, with respective 50% inhibitory concentration of approximately 13 and 2 microM. The mechanism of action of ketoconazole is unknown. However, another lipoxygenase inhibitor, BW755C, inhibited only weakly [3H]-thymidine incorporation and accumulated the cells in S and G2. Conversely, clotrimazole and SKF525A, inhibitors of cytochrome P-450 enzymes, had effects similar to those of ketoconazole on HT29-S-B6 cells whereas metronidazole and secnidazole, other azole derivatives which do not inhibit cytochrome P-450 enzymes, had no effect. The results suggest that cytochrome P-450 enzyme(s) activity(ies) could be implicated in the antiproliferative effects of ketoconazole.     

Deoxycytidine kinase, thymidine kinase and cytidine deaminase and the formation of Ara-CTP in leukemic cells in different phases of the cell cycle

In this study we investigated the Ara-CTP-forming capacity of leukemic cells in different phases of the cell cycle. Cells from two leukemic cell lines and leukemic bone marrow cells from patients and rats (BNML model) with acute myelocytic leukemia were separated according to cell cycle phase by means of an albumin density gradient in a specially designed sedimentation chamber. We found that the activity of CdR kinase and Cyt deaminase is much less influenced by cell-cycle phase progression than TdR kinase activity. For the leukemic cell lines HL-60 and BNML-CL/O CdR kinase activity is even independent of cell-cycle phase. In addition, Ara-CTP formation is not restricted to cells in S-phase. Cell cycle phase-independent Ara-CTP formation creates a situation in which cells which are not in S-phase during exposure to Ara-C might undergo the cytotoxic effects of Ara-C as soon as they enter S-phase.