Øystein W. Rønning

Protein synthesis and protein degradation through the cell cycle of human NHIK 3025 cells in vitro

Abstract The rates of protein synthesis and protein degradation through the cell cycle of human NHIK 3025 cells in balanced growth were studied in cells synchronized by mitotic selection. The rates of protein synthesis and protein degradation per cell were found to increase smoothly from the first measurements after selection (mitosis) and until the beginning of the next mitosis. By the end of the cell cycle, both the rate of protein synthesis per cell and the rate of protein degradation per cell had doubled their values relative to the extrapolated values at the beginning of the cell cycle. The increase in the rates of protein synthesis and protein degradation seemed to follow the increase in total protein content closely, i.e. the rates of protein synthesis and protein degradation given as percentage of total protein were constant throughout the cell cycle of NHIK 3025 cells in balanced growth. The rate of protein accumulation, either measured directly or calculated as the difference between the rates of protein synthesis and protein degradation, allowed for a doubling of the protein content during the median time period required to complete the cell cycle.

Progress through G1 and S in relation to net protein accumulation in human NHIK 3025 cells

We have investigated whether human NHIK 3025 cells are dependent upon a net increase in cellular protein content in order to traverse G1 and S. The increase in DNA and protein content was studied by means of two-parameter flow cytometry using populations of cells synchronized by mitotic selection. By adding 1 microM cycloheximide to the medium protein synthesis was partially inhibited, resulting in negligible net accumulation of protein. The cells were able to enter S and progress through S under such conditions. The latter was the case whether the cells had been accumulating protein during G1 or not. The results further indicate that the larger cells enter S earlier and traverse S at a higher rate than the smaller cells. Our conclusion is that net accumulation of protein does not seem to be a prerequisite for traverse through G1 and S, i.e. DNA replication may be dissociated from the general growth of cell mass.

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.

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.

The origin of variability in cell cycle durations of NHIK 3025 cells

Abstract The origin of cell cycle variability was investigated in NHIK 3025 cells synchronized by mitotic selection from an exponentially growing population. The variability in G1 durations was measured by flow cytometric analysis of the fraction of cells in G1 as a function of time after mitotic selection. Immediately before the first cells entered S, medium containing 2.0 mM thymidine was added to the cells, and removed when all the cells had reached S. Since the cells had approximately the same DNA content upon removal of the thymidine, the variability in the durations of S+G2+M was measured by counting the fraction of undivided cells as a function of time after removing the thymidine. Such a thymidine treatment did not affect the naturally occurring variability in cell cycle durations generated after the start of S. The results indicate that the cell cycle variability of NHIK 3025 cells can be adequately described by a cell cycle model consisting of at least two compartments, which the cells leave according to first order kinetics. The model accounts for the initial shoulder of the curve representing the fraction of undivided cells as a function of time after mitotic selection. Furthermore, it accounts for the reduction in the rate of entry into the subsequent cell cycle compared to the rate of entry into S. Both rate constants were equally reduced after serum stepdown.

Effect of different growth factors on cell cycle traverse and protein growth of human cells in culture

Human epithelioid cells (NHIK 3025) are able to traverse at least one cell cycle, if the serum concentration in the medium is reduced to only 0.3% when the cells are in mitosis. The cell cycle and protein doubling times were prolonged considerably under such conditions. The protein doubling time was, however, prolonged much more than the cell cycle. The addition of purified growth factors (insulin, MSA, EGF, FGF or PDGF) did not shorten G1 or the cell cycle, although insulin, EGF and PDGF increased the rate of protein synthesis. When the growth factors were added to the medium two and two in combination, it was found that four combinations (out of ten possible) increased the rate of progress through G1, whereas three combinations increased the rate of progress through the total cell cycle. Only two combinations increased the rate of protein accumulation to such an extent that the protein content doubled during the cell cycle. Both of these combinations also shortened the cell cycle. Since different combinations of growth factors were found to regulate the various events related to growth of NHIK 3025 cells, it is suggested that these events are under separate control.