Cell-cycle inhibition by misonidazole of human cells cultivated in vitro under aerobic conditions.

By means of flow cytometric recording of DNA histograms and counting of cells in synchronized populations, we have found that misonidazole (MIS) in clinically relevant concentrations induces cell-kinetic changes in human cells (NHIK 3025) cultivated in vitro under aerobic conditions. The effect seems to be a general lengthening of the cell cycle, affecting all phases. However, induction of this effect is phase-dependent, since only cells exposed to MIS during mitosis and/or early G1 will suffer significant cell-cycle prolongation. In exponentially growing populations this effect of MIS leads to a transient increase in the fraction of G1 cells and a corresponding decrease in the fraction of S cells. The possible significance of this effect for the clinical use of MIS is discussed

Inhibition of cell-cycle progression by acute treatment with various degrees of hypoxia: modifications induced by low concentrations of misonidazole present during hypoxia.

The effect on cell-cycle progression in various phases of the cell cycle caused by an acute exposure to hypoxia in absence and presence of misonidazole (MISO) was investigated. Exponentially growing and synchronized cells of the human line NHIK 3025 were exposed to different degrees of hypoxia for a short period (1.5 or 3 h). The cell-cycle progression was studied both during and after hypoxia by flow-cytometric recording of DNA-histograms from treated and untreated cells. The rate of cell-cycle progression was reduced during hypoxia only if the O2-concentration was below 1000 ppm. The inhibition was phase specific with a strong effect in S (reduced DNA-synthesis), and a specific cell-cycle inhibition in late G1, probably at the G1/S-border. For cells inhibited (or arrested for extreme hypoxia) at the G1/S-border, the cell-cycle progression changed back to normal shortly after aerobic conditions were re-established. For cells rendered hypoxic and inhibited during S, hypoxia exerted a lasting effect expressed by a low cell-cycle progression rate even after aerobic conditions were re-established. This effect was strongly dependent on both the degree and the duration of the hypoxic treatment. The presence of a low concentration of MISO (0.05 mM) during hypoxia did not affect the cell-cycle progression during hypoxia at any O2-concentration. For cells rendered hypoxic during S, however, MISO (0.05 mM) counteracted the lasting effect of hypoxia for all concentrations of O2 where this lasting effect was observed

Low concentrations of misonidazole counteract effects of extreme hypoxia on cells in S.

Populations of NHIK 3025 cells synchronized by mitotic selection were exposed at 37 degrees C to extreme hypoxia in absence and presence of misonidazole (MISO). Cells in G1, S or G2 and mitosis were treated for 3 h. Inhibition of cell-cycle progression by this treatment was measured by flow cytometry of DNA histograms and cell inactivation was measured by colony formation. The exposure to hypoxia alone of cells in G1 or in G2 and mitosis led to only minor cell-cycle inhibition, and hardly reduced cell survival. However, the exposure of cells in S to hypoxia alone had a strong inhibitory effect on cell-cycle progression, and cell survival was only 40% of untreated cells. Low concentrations of MISO (0.05-0.4 mM) during exposure of cells in S to hypoxia, produced less cell-cycle inhibition than after hypoxia alone, and cell survival was restored to 100%. The presence of MISO during the 3h exposure to hypoxia of cells in G1 or in G2 and mitosis only increased the effects of hypoxia alone. MISO at concentrations greater than 0.8 mM during hypoxia produced cell inactivation, for all phases of the cell cycle, comparable to that already known from the literature

Regulation of cell proliferation under extreme and moderate hypoxia: the role of pyrimidine (deoxy)nucleotides.

In the present study we have used flow cytometric DNA measurements on synchronised human NHIK 3025 cells to measure cell cycle progression under various conditions of reduced oxygenation. Our data indicate that addition of 0.1 mM deoxycytidine or uridine has no effect on the oxygen-dependent arrest in late G1 or on the inhibition of cell proliferation through S-phase under extremely hypoxic conditions. Following reoxygenation of cells exposed to extremely hypoxic conditions in G2 initiation of DNA synthesis in the subsequent cell cycle is delayed by several hours. This G2-induced delay is completely abolished for approximately 60% of the cell population by addition of deoxycytidine to hypoxic G2 cells. This finding supports our previous proposal that important steps in the preparation for DNA synthesis occur during G2 of the previous cell cycle, and it indicates that this preparation is connected to the de novo synthesis of pyrimidine deoxynucleotide precursors. The results show that cells are able to enter S-phase in the presence of 100-1,300 p.p.m. (0.01-0.13%) oxygen (here denoted 'moderate hypoxia'), but they are not able to complete DNA synthesis under such conditions. However, the cell cycle inhibition induced under moderate hypoxia is partially reversed in the presence of exogenously added deoxycytidine and uridine, while no such reversal is seen in the presence of purine deoxynucleosides (deoxyadenosine and deoxyguanosine). Thus, both deoxycytidine and uridine could replace reoxygenation under these conditions. These results indicate that the reduction of CDP to dCTP by ribonucleotide reductase, an enzyme which requires oxygen as an essential factor for the formation of tyrosyl radicals for its catalytic activity, does not seem to be the limiting step responsible for the reduced dCTP pool observed under moderate hypoxia. We conclude that the oxygen-dependent catalytic activity of the M2 subunit of ribonucleotide reductase is still intact and functional in NHIK 3025 cells even at oxygen concentration as low as 100 p.p.m. Therefore the cell cycle inhibition observed is probably due to inhibition of the respiratory chain-dependent UMP synthesis at the stage of dihydroorotate dehydrogenase

The mechanism of photodynamic inactivation of human cells in vitro in the presence of haematoporphyrin.

The photosensitizing effect of haematoporphyrin (HP) on human cells of the established line NHIK 3025 has been studied. Fluorescence measurements show that HP is bound to these cells. Serum proteins also bind HP, and the presence of 10% human serum during incubation with HP (3 X 10(-4)M) reduces the cellular uptake of HP by 75% or more. The photosensitized inactivation is enhanced when the cells are suspended in D2O-buffer during irradiation. This indicates that singlet oxygen is involved in the inactivation. Two findings indicate that the photoinduced damage is repairable: firstly, the fraction of cells surviving a given light dose decreases with decreasing irradiation temperature, and secondly, the survival curves have a shoulder at low exposures of light

The binding of cis-dichlorodiammineplatinum(II) to extracellular and intracellular compounds in relation to drug uptake and cytotoxicity in vitro.

The biological consequence of the binding of cis-dichlorodiammineplatinum(II) (cis-DDP) to serum protein as well as to cellular components in general, was studied on human NHIK 3025 cells in vitro. As expected, we found that the cytotoxicity of cis-DDP was lost by binding to serum protein, and that protein-bound platinum was impermeable to the cells. As we have previously shown that electropermeabilisation may transiently increase the influx of cis-DDP, we applied this technique in an attempt to increase the efflux of cis-DDP or any other cytotoxic intermediates. Our data demonstrate that if cells are electropermeabilised shortly after treatment with cis-DDP, cell survival increased. This indicates that cis-DDP in an active form is released from the cells; furthermore, the plasma membrane represents a barrier against efflux, as it has also been shown to be against influx of active cis-DDP. Thus, our data are consistent with the idea that there must be an intracellular pool of either cis-DDP, or some biologically active intermediates, in cells treated with this drug. Additionally, our data indicate that the binding rate of cis-DDP to biological molecules is much quicker intracellularly than in the extracellular environment: We found the biological half-life at 37 degrees C to be about 2.1 h in human serum and about 11 min inside our cells

The retinoblastoma protein-associated cell cycle arrest in S-phase under moderate hypoxia is disrupted in cells expressing HPV18 E7 oncoprotein.

We have studied the role of the oxygen-dependent pyrimidine metabolism in the regulation of cell cycle progression under moderate hypoxia in human cell lines containing functional (T-47D) or non-functional (NHIK 3025, SAOS-2) retinoblastoma gene product (pRB). Under aerobic conditions, pRB exerts its growth-regulatory effects during early G1 phase of the cell cycle, when all pRB present has been assumed to be in the underphosphorylated form and bound in the nucleus. We demonstrate that pRB is dephosphorylated and re-bound in the nucleus in approximately 90% of T-47D cells located in S and G2 phases under moderately hypoxic conditions. Under these conditions, no T-47D cells entered S-phase, and no progression through S-phase was observed. Progression of cells through G2 and mitosis seems independent of their functional pRB status. The p21WAF1/CIP1 protein level was significantly reduced by moderate hypoxia in p53-deficient T-47D cells, whereas p16(INK4a) was not expressed in these cells, suggesting that the hypoxia-induced cell cycle arrest is independent of these cyclin-dependent kinase inhibitors. The addition of pyrimidine deoxynucleosides did not release T-47D cells, containing mainly underphosphorylated pRB, from the cell cycle arrest induced by moderate hypoxia. However, NHIK 3025 cells, in which pRB is abrogated by expression of the HPV18 E7 oncoprotein, and SAOS-2 cells, which lack pRB expression, continued cell cycle progression under moderate hypoxia provided that excess pyrimidine deoxynucleosides were present. NHIK 3025 cells express high levels of p16INK4a under both aerobic and moderately hypoxic conditions, suggesting that the inhibitory function of p16(INK4a) would not be manifested in such pRB-deficient cells. Thus, pRB, a key member of the cell cycle checkpoint network, seems to play a major role by inducing growth arrest under moderate hypoxia, and it gradually overrides hypoxia-induced suppression of pyrimidine metabolism in the regulation of progression through S-phase under such conditions

Synergistic cell inactivation by cis-dichlorodiammineplatinum in combination with 1-propargyl-5-chloropyrimidin-2-one.

A synergistic effect with respect to inactivation of human NHIK 3025 cells cultured in vitro was displayed when treatment with cis-dichlorodiammineplatinum(II) (cis-DDP) and the mitotic inhibitor 1-propargyl-5-chloropyrimidin-2-one (NY 3170) were given in simultaneous combination. Cell inactivation was measured by loss of colony-forming ability. Treatment with NY 3170 alone produced no significant inactivation at concentrations up to 2 mM. However, treatment with NY 3170 in combination with cis-DDP induced increased cell inactivating effects equal to a doubling of either the concentration of cis-DDP or treatment time. Scheduling of NY 3170 treatment in relationship to a 1 h cis-DDP pulse revealed that synergism occurred only when the two drugs were present simultaneously. The inactivating effect of 10 microM cis-DDP in combination with 2 mM NY 3170 given to synchronized NHIK 3025 cells at various stages of the cell cycle was also determined. For cells treated in S or in G2 + M cell survival was reduced by a factor of 5 after a 1 h treatment with the drug combination as compared to similar treatment with cis-DDP alone. The cells appeared to be most sensitive at the time of initiation of DNA synthesis. Here cell survival was reduced by a factor of 100 following treatment with the drug combination than following treatment with cis-DDP alone. Measurement of cell-associated platinum by atomic absorption spectroscopy indicated that cellular uptake of cis-DDP was increased when NY 3170 was simultaneously present during drug treatment

Cell-cycle inhibitory effects of the mitotic inhibitor NY 3170 on human cells in vitro.

Effects of the mitotic inhibitor NY 3170 (1-propargyl-5-chloropyrimidin-2-one) on cell-cycle kinetics of NHIK 3025 cells were studied by means of time-lapse microcinematography, pulsed incorporation of [3H] thymidine, flow cytometry, and mitotic index. All the experiments were performed with cells synchronized by mitotic selection. Mitotic inhibition as well as inhibition in interphase was examined. The small fraction of cells able to escape mitotic arrest at 0.2mM NY 3170 had spent about 12 h in metaphase. The metaphase block was complete at 0.3 mM. For comparison, complete metaphase arrest of NHIK 3025 cells was reached at 8 mM after treatment with the parent substance NY 3000 (5-chloropyrimidin-2-one, previously reported). At 0.3mM NY 3170 interphase was also considerably prolonged. All stages of interphase were prolonged, in contrast to the interphase prolongation after treatment with high concentrations of the mitotic inhibitors vincristine and vinblastine, which occurs in G2. It was shown that the presence of NY 3170 during mitosis is a necessary and sufficient condition for metaphase arrest, thus demonstrating that metaphase arrest is not dependent on some preceding event in interphase

Selection of tumour cell subpopulations occurs during cultivation of human tumours in soft agar. A DNA flow cytometric study.

To examine whether selection of tumour cell subpopulations occurs during cultivation in soft agar, we compared in 23 human tumours of different histological types the DNA content of cells from colonies formed in soft agar (method of Courtenay and Mills, 1978) with that of the original tumour cells. The ploidy as well as the fraction of cells in S phase were determined from DNA histograms after staining of the nuclei with a propidium-iodide procedure and flow cytometric recordings. In 8 of 17 aneuploid tumours analysed, specific aneuploid subpopulations disappeared during cultivation or new aneuploid populations, not demonstrable in the original cell suspensions, appeared in the colonies. In 9 cases identical aneuploid populations were found in the colonies and the tumours. In one of 6 diploid tumours examined, aneuploid cell populations not revealed in the original cell suspension, were found in addition to diploid cells, whereas 5 tumours gave rise to colonies containing a purely diploid population. The results show that in a variety of human malignant tumours cultivation in soft agar may select specific aneuploid tumour cell populations