Reidar Oftebro

Cell cycle characteristics of synchronized and asynchronous populations of human cells and effect of cooling of selected mitotic cells.

The method of synchronizing cells by means of mitotic selection has been adapted to the human line NHIK 3025. Increase in cell number as a function of time in asynchronous and synchronous populations was studied as well as mitotic index as a function of time after selection of synchronized populations. Phase durations of the cell cycle of synchronous populations were determined by 3 H‐thymidine incorporation and scintillation counting. The relative phase durations of exponentially growing asynchronous populations were determined by mathematical analysis of DNA‐histograms recorded by flow cytofluorimetry. Both the generation time and the various phase durations of the cell cycle were found to be the same in asynchronous and synchronous populations. It was found that NHIK 3025 cells are damaged by cooling to 4 and 0°C so that cooling of selected cells in order to increase the yield would reduce the quality of the synchronized populations.

X-ray inactivation of human cells in tissue culture under aerobic and extremely hypoxic conditions in the presence and absence of TMPN.

SummaryA technique is described which permits irradiation of suspensions of human cells in tissue culture either aerobically (equilibrated with air) or under extremely hypoxic conditions (flushed with a mixture of 97 per cent N2 and 3 per cent CO2, the oxygen content of which was less than 15 p.p.m.).An established cell-line, NHIK 3025, originally derived from an early stage of human cancer of the cervix, was used. The D0 value of these cells under aerobic conditions was 130 rads and the extrapolation number about 3. Under extremely hypoxic conditions, the dose-effect curve was exponential with a D0 value of 600 rads. The o.e.r. derived from the ratio of the D0 values was therefore 4·6.2,2,6,6tetramethyl-4-piperidinol-N-oxyl (TMPN) at a concentration of 10−2 M exerted a pronounced sensitizing effect under extremely hypoxic conditions (TMPN e.r. = 2·5), and yielded dose-effect curves with an extrapolation number of 3. The high radio-resistance of the cell under extremely hypoxic conditions in the absence o...

Further Studies on Mitosis of Bi- and Multinucleate HeLa Cells

Mitosis of 37 binucleate, 15 trinucleate, 5 tetranucleate, 1 pentanucleate, 1 hexanucleate and 2 9-nucleate HeLa cells was studied by means of time lapse phase contrast microcinematography of living material. When 37 cases of mitosis of binucleate cells were grouped according to the number and nuclearity of daughter cells, 12 different groups were found. The number of nuclei produced in most cases was 4 (21 cases), but examples of 3 nuclei (8 cases), 2 (3 cases), 5 (3 cases), and 6 (1 case) were also found. The number of cells produced was 3 in 16 cases, 2 in 14 cases, 4 in 5 cases, and 1 in 1 case. One cell burst in metaphase. In 25 of the 37 experiments at least 1 daughter cell had more than one nucleus. When the 15 cases of mitosis of trinucleate cells were grouped according to the number and nuclearity of daughter cells, 10 different groups were found. The number of nuclei produced was 3 in 3 cases, 4 in 3 cases, 5 in 1 case, and 6 in 2 cases. The number of cells produced was 4 in 5 cases, 3 in 2 case...

Effects of benzaldehyde on survival and cell-cycle kinetics of human cells cultivated in vitro

Synchronized cells of the human line NHIK 3025 were used to study inactivating and cell-cycle inhibitory effects induced by benzaldehyde. Inactivation was measured as loss of colony-forming ability after treatment of exponentially growing or synchronized cells. Cell-cycle inhibition was measured by flow cytometric recordings of DNA-histograms and microscopic recordings of cell division in synchronized cells. Treatment with benzaldehyde for 4 or 24 hr showed that a marked decrease in survival took place for concentrations above 6.4 mM. Cell-cycle inhibition was observed at concentrations as low as 0.8 mM. Synchronized cells were treated with 3.2 and 6.4 mM benzaldehyde for 8 hr starting at various stages of the cell-cycle. Both the colony-forming ability and the rate of cell-cycle traverse was measured. No difference in sensitivity was found whether the treatment was given in G1, S or in G2. Thus the results show that there is no specific part of interphase where the cells are particularly sensitive with respect to either the inactivating or the cell-cycle inhibitory effects of benzaldehyde in concentrations up to 6.4 mM. When benzaldehyde was present during mitosis both the inactivating and the cell-cycle inhibitory effects were markedly enhanced as compared to the corresponding effects during interphase. It is concluded that benzaldehyde must affect some process within the cell which represents a general requirement for cell-cycle progression. In addition, there are effects on processes that take place only during the last few minutes before and/or during mitosis.

Effects of benzaldehyde on protein metabolism of human cells cultivated in vitro

The mechanism by which the antitumour agent benzaldehyde inhibits cell growth has been investigated. Human NHIK 3025 cells were synchronized by selection of mitotic cells and the protein content at various stages of the cell cycle was recorded by use of flow cytometry. In the presence of benzaldehyde (concentrations above 0.5 mM, approximately 50 micrograms/ml) the rate of protein accumulation was reduced to the same extent throughout the cell cycle. The rates of protein synthesis and protein degradation were measured by incorporation and release, respectively, of radioactively labelled valine in exponentially growing cells. It was found that benzaldehyde primarily reduced the rate of protein synthesis, while it induced only a very small effect (reduction) on the rate of protein degradation. When comparing the rate of cell-cycle progression with the rate of protein accumulation, it was found that the median interphase duration was equal to the protein doubling time even for concentrations of benzaldehyde giving a marked reduction in the rate of protein accumulation. Similar results have been observed on these cells using the specific protein synthesis inhibitor cycloheximide. However, the two drugs have different effects during mitosis, since benzaldehyde but not cycloheximide induces a specific mitotic inhibition. It is, therefore, possible that benzaldehyde inhibits the protein synthesis by a mechanism different from that of cycloheximide, a mechanism which simultaneously results in a specific mitotic inhibition. A hypothesis is proposed on the mechanism of action of benzaldehyde: that the drug might inhibit a process in the cells which activates enzymes. Such an effect might possibly entail a reduced protein synthesis as well as a prolonged mitosis. In addition, it might also count for the reported de-transforming activity of benzaldehyde on malignant cells.

Metahalones, a new class of metaphase inhibitors.

We have now extended our work on this class of substances and have attempted to establish the structure-activity relationship. The results suggest that the metaphase-inhibitory activity is associated with the 2-0~0 group and the 5-halogen, and that certain substituents in other positions are permissable for the retainment of activity. We should like to suggest the name Metahalones for this class of inhibitors. Meta from metaphase, ha2 from halogen and one because the activity is associated with the one-form of the 2-hydroxy group; O-substituted derivatives as shown below are inactive whereas N-substituted derivates are active.

Reduction of cis-dichlorodiammineplatinum-induced cell inactivation by benzaldehyde

The inactivating effect of a combined treatment of human cells (NHIK 3025) in culture with cis-dichlorodiammineplatinum(II) (cis-DDP) and the protein synthesis inhibitor benzaldehyde was tested. Cell inactivation was measured as loss of colony-forming ability following drug treatment. While 3.2 mM benzaldehyde had no effect on the cell survival when given alone, it reduced the effect of 10 microM cis-DDP significantly when the two drugs were added simultaneously. Scheduling experiments indicate that benzaldehyde must be present immediately before addition of, or simultaneously with, cis-DDP for optimal reduction of cell inactivation. Benzoic acid, benzyl alcohol or the protein synthesis inhibitor cycloheximide did not reduce the inactivating effect of cis-DDP. Cells synchronized by mitotic selection were used to determine the variation in the responses throughout the cell cycle. It was found that concomitant 2-hr treatment of synchronized cells with 3.2 mM benzaldehyde and 10 microM cis-DDP at various times during the cell cycle resulted in a consistently greater surviving fraction of cells than 10 microM cis-DDP alone. Benzaldehyde thus reduced the inactivating effect of cis-DDP in all phases of the cell cycle. The effect of benzaldehyde in combination with two alkylating agents, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and nitrogen mustard (HN2), was also studied. Benzaldehyde was not found to influence the effects on cell survival induced by these drugs.

Pyrimidinones as reversible metaphase arresting agents

Abstract 5-Halo- N (1)-substituted 2(1 H )-pyrimidinones have the ability to cause reversible arrest of mitosis during metaphase. Highly active compounds have a heteroatom ( O , S or N ) in the β-position of the N (1)-carbon chain which is further substituted by an aryl group. In vitro data have been provided. It is suggested that reversible metaphase inhibitors can be used as synchronizing agents of cell-cycles by applying them in a sequential manner when a phase-specific cytotoxic drug is used in the treatment of diseases caused by uncontrolled rapidly proliferating cells. The active compounds are prepared from 2-pyrimidinones by alkylation reactions. The key reactants are α-chloroalkyl ethers, sulfides and amides; methods for their syntheses have been described.

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.

The Radiosensitizing and Radioprotective Effect of the Organic Nitroxyl-free Radical TMPN on Extremely Hypoxic Human Cells of Line NHIK 3025, Cultivated in Vitro

SummaryHuman cells from the established line NHIK 3025 have been irradiated in suspension under extremely hypoxic conditions ( 400–800 rads, dependent on TMPN-concentration) and increases with increasing TMPN-concentrations.At low doses (<400–800 rads) TMPN was found to exert a protective effect. Below 300 rads, the protection was found to be optimal for a TMPN-concentration between 0·5 and 3 p.p.m. Within the concentration limits here studied it was found that the dose-range in which TMPN exerts a protection increases with decreasing TMPN-concentration.The slope of the survival curves was found to increase when the dose exceeded about 2000 rads, irrespective of whether the cells were in contact with 1 mM, 0·5 mM or no T...