Nucleic acids methylation of synchronized BHK 21 HS 5 fibroblasts during the mitotic phase

The methylation of nucleic acids has been investigated during the cell cycle of an asparagine dependent strain of transformed fibroblasts (BHK 21 HS 5). The synchrony was carried out by a partial asparagine starvation of cells for 24 hours. The amino acid supply induced all cells to enter synchronously the G₁ phase. Methylation and DNA synthesis were respectively measured by pulsed [methyl-¹⁴C] methionine and [methyl-³H] thymidine incorporation. DNA methylation followed a biphasic pattern with maximal methyl incorporations during both S phase and mitosis. A partial desynchronisation induced the S phase of the second cycle to proceed before all the cells have achieved their division. Hydroxyurea was used in order to inhibit the DNA synthesis of cells entering the second cell cycle, which might interfer with the mitosis of the first one. The inhibitor was added either at the first beginning of cell division or during all the G₁ phase. In both conditions it suppressed ³H thymidine incorporation of the second cycle. However, mitosis took place and methylations occurred as in previous experiments. The DNA methylation of the mitotic phase in the first cell cycle could thus be dissociated from the classical post-synthetic DNA maturation and did not correspond to any DNA methylation appearing in the course of the second cell cycle.     

Cell cycle kinetics, tissue transglutaminase and programmed cell death (apoptosis).

Studies were undertaken on a highly metastatic hamster fibrosarcoma cell line with a view to assessing whether cells entering into apoptosis, measured by counting the number of transglutaminase mediated detergent insoluble envelopes, has any synchrony with a particular phase of the cell cycle. A double exposure of thymidine was used to block cells in early S-phase. Flow cytometry in combination with [3H]thymidine incorporation into DNA was used to assess the degree of synchrony and progression through the different phases of cell cycle. The apoptotic index was found to be at its maximum in mid-S-phase. Measurement of transglutaminase activity in each phase of the cell cycle indicated that the specific activity was also at its greatest during mid S-phase. The level of enzyme was relatively unchanged throughout the cell cycle indicating that the regulation of transglutaminase activity occurs primarily through effects on catalytic activity rather than enzyme synthesis.     

The activities of thymidine metabolising enzymes during the cell cycle of a human lymphocyte cell line LAZ-007 synchronised by centrifugal elutriation

The activities throughout the cell cycle of thymidine kinase (EC 2.7.1.21), dihydrothymine dehydrogenase (EC 1.3.1.2), thymidine phosphorylase (EC 2.4.2.4) and dTMP phosphatase (EC 3.1.3.35) were measured in the Epstein-Barr virally transformed human B lymphocyte line LAZ-007. Cells were synchronised at different stages of the cell cycle using the technique of centrifugal elutriation. The degree of synchrony in each cycle-stage cell population was determined by flow microfluorimetric analysis of DNA content and by measurement of thymidine incorporation into DNA. The activity of the anabolic enzyme thymidine kinase was low in the G₁ phase cells, but increased many-fold during the S and G₂ phases, reaching a maximum after the peak of DNA synthesis, then decreasing in late G₂ + M phase. By contrast, the specific activities of the enzymes involved in thymidine and thymidylate catabolism, dihydrothymine dehydrogenase, thymidine phosphorylase and dTMP phosphatase remained essentially constant throughout the cell cycle, indicating that the fate of thymidine at different stages of the cell cycle is governed primarily by regulation of the level of the anabolic enzyme thymidine kinase and not by regulation of the levels of thymidine catabolising enzymes.     

Membrane fatty acid changes during the cell cycle of CV-1 cells

Monolayers of CV-1 cells were synchronized at the G1/S boundary of the cell cycle by a 24-h 2 mM thymidine blockade. Uptake of tritiated thymidine indicated that the peak DNA synthesis occurred 6-8 h after release from the block and that cell cycle time was 18-20 h. The fatty acid composition of phospholipids extracted from cells at 0, 7, and 18 h postblockade was measured by gas chromatography. The results indicate cyclic changes in membrane fatty acids with a significant increase in long-chain polyunsaturated fatty acids during the DNA synthesis phase (S phase) of the cell cycle.     

Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan

The effects of cell surface heparan sulfate proteoglycan (HSPG) prepared from log and confluent monolayers of a rat hepatoma cell line on hepatoma cell growth were studied. When HSPG isolated from confluent cells was added exogenously to log phase cells, it was internalized and free heparan sulfate (HS) chains appeared transiently in the nucleus. Concurrently, the growth of the treated cells was inhibited, but the cells resumed logarithmic growth as the level of nuclear HS fell, and the cells grew to confluence and became contact inhibited. When HSPG prepared from log-phase hepatoma cells was added exogenously to log phase cells, it was internalized but very little of the internalized HS appeared in the nucleus, and there was no change in the rate of cell growth. However, when the rate of cell growth was reduced by culture of the cells in serum- and insulin-deficient medium, HSPG prepared from log-phase cells stimulated the growth rate of these slow-growing cells. The cell cycle dependency of HSPG uptake and growth inhibition was studied in cultures synchronized by a thymidine/aphidicolin double block. When [35SO4]HSPG from confluent cells was added to synchronized cells just as they were released from the second block, a portion of the [35SO4]HSPG was internalized and [35SO4]HS appeared in the nucleus. However, at mitosis the [35SO4]HS disappeared almost completely from all of the cellular pools, and after mitosis, more of the [35SO4]HSPG was taken up and [35SO4]HS reappeared in the nucleus and remained in the nucleus until the cells divided again. When cultures were released from the aphidicolin block, both control and HSPG-treated cells progressed through the S, the G2, and the M phases of the cell cycle. However, the length of the G1 phase of the cycle was increased in the HSPG-treated cells. The treated cultures then progressed through the second S, G2, and M phases. Thus, the inhibition of cell division occurred in the G1 phase of the cell cycle, prior to the G1/S boundary. Addition of the HSPG to the synchronized cultures just after the first mitosis resulted in an immediate arrest of the cell cycle in G1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of thymidine kinase and dihydrofolate reductase genes in mammalian ts mutants of the cell cycle

Thymidine kinase and dihydrofolate reductase mRNA levels and enzyme activities were determined in two temperature-sensitive cell lines, tsAF8 and ts13, that growth arrest in the G1 phase of the cell cycle at the restrictive temperature. The levels of thymidine kinase mRNA and enzyme activity increased markedly in both cell lines serum stimulated from quiescence at the permissive temperature. At the nonpermissive temperature, the levels of thymidine kinase mRNA and enzyme activity remain at the low levels of quiescent G0 cells. The levels of dihydrofolate reductase mRNA as well as the enzyme activity also increase when both cell lines are serum stimulated at the permissive temperature. When ts13 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity declines rapidly and dihydrofolate reductase mRNA is below detectable levels. On the contrary, when tsAF8 cells are serum stimulated at the nonpermissive temperature dihydrofolate reductase enzyme activity increases and mRNA levels are detectable slightly above G0 levels, even though the cells are blocked in the G1 phase. Studies with 2 other cDNA clones (one with an insert whose expression is cell cycle dependent and the other with an insert whose expression is not cell cycle dependent) indicate that the results are not due to aspecific toxicity or the effect of temperature. We conclude that the expression of different genes is affected differently by the ts block in G1, even when these genes are all growth-related.     

Fluctuation of alkaline phosphatase activity in synchronized heteroploid cell cultures: effects of prednisolone

In two heteroploid cell lines synchronized with thymidine double block, activity of alkaline phosphatase decreased during the 12 hour period preceding mitotic peak. A return to high values was observed during the next 12 hours of synchronous cycle. Prednisolone (11β, 17α, 21-trihydroxy-1,4-pregnadiene-3,20-dione), when added to such cell cultures increased alkaline phosphatase activity in one of the cell lines (Henle embryonic intestine) but had the opposite effect on another line (HeLa-S3) in which the enzyme activity was decreased. Neither effect could be demonstrated if the hormone was added at the end of S phase or if cells were arrested in metaphase by vinblastine sulfate.     

Replicatin of the resident marek''s disease virus genome in synchronized nonproducer MKT-1 cells

MKT-1, a virus nonproducer lymphoblastoid cell line established from a Marek's disease tumor, was synchronized by double thymidine block to determine the sequence of events in the synthesis of cellular and latent marek's disease virus DNA. Cellular DNA synthesis was measured by incorporation of [3H]thymidine, whereas viral DNA synthesis was determined by DNA-DNA reassociation kinetics. The results of these studies indicate that the resident Marek's disease viral DNA in MKT-1 cells replicates during the early S phase of the cell cycle, before the onset of active cellular DNA synthesis. This observation is similar to that seen in the replication of resident Epstein-Barr virus DNA in synchronized Raji cells.     

Thymidine block does not synchronize L1210 mouse leukaemic cells: implications for cell cycle control, cell cycle analysis and whole-culture synchronization

Abstract.   It has been predicted that whole-culture methods of synchronization cannot synchronize cells. We have tested whether thymidine block, one type of whole-culture synchronization, can synchronize L1210 cells. We demonstrate experimentally that the thymidine block method cannot produce a synchronized culture. Although thymidine-treated cells are arrested primarily with an S-phase amount of DNA, there is no narrowing of the cell size distribution and there is no synchronized division pattern following release from the thymidine block. In contrast to a whole-culture synchronization method, cells produced by a selective (i.e. non-whole-culture) method not only have a specific DNA content, but also have a narrow size distribution and divide synchronously. Generalizing the results to other cell lines, we suggest that these conclusions call into question experimental measurements of gene expression during the division cycle based on thymidine inhibition synchronization.     

Cytofluorometry of lymphocytes infected with Epstein-Barr virus: effect of phosphonoacetic acid on nucleic acid.

DNA synthesis in Epstein-Barr virus (EBV)-infected lymphocytes was inhibited by phosphonoacetic acid (PAA) as measured by [3H]thymidine incorporation. PAA, at a concentration of 200 microgram/ml, inhibited [3H]thymidine incorporation by human umbilical cord lymphocytes infected with EBV strain P94 but had little effect on DNA synthesis in mitogen-stimulated cells. Transformed cell lines did not develop from infected cord cell cultures treated with 100 microgram of PAA per ml. Cytofluorometric analysis showed marked increases in cellular nucleic acid content (RNA plus DNA) as early as 9 days after infection of cord cells in the absence of PAA and before significant enhancement of [3H]thymidine incorporation became apparent. Moreover, EBV led to increases in cellular nucleic acid even when 200 microgram of PAA per ml was added to cell cultures before infection. The apparent discrepancy between results obtained by [3H]thymidine incorporation and cytofluorometry is explained either by significant inhibition of cellular DNA polymerases by PAA or by a block at the G2 + M phase of the cell cycle. The data suggest that EBV initiates alterations in cellular nucleic acid synthesis or cell division without prior replication of viral DNA by virus-induced DNA polymerases.     

Nucleoside phosphotransferase activity through the growth and cell cycle of Tetrahymena pyriformis GL-I

Tetrahymena pyriformis GL-I were synchronized by three different techniques and nucleoside phosphotransferase activity measured through the different cell cycles obtained. In cells that were starved and then refed, activity did not increase until 75 min after refeeding. This increase in activity occurred well before nuclear DNA synthesis and was not blocked by hydroxyurea. In cells synchronized by the induction technique of one heat shock per generation and the selection technique of differential density labelling, enzyme activity increased continuously over the cell cycle but did not double. However, during early logarithmic growth nucleoside phosphotransferase activity more than doubled over one cell cycle time while late in log growth phase less than a doubling was observed. Cycloheximide and mixed extract experiments suggest that the patterns of activity observed reflect the patterns of enzyme synthesis. These results are discussed with respect to the pattern of activity observed for thymidine kinase in other organisms.     

Control of B cell proliferation: arrest of B cells in late G1 underlies immunosuppression induced by plasma cell tumors

Immunosuppression in mice bearing plasma cell tumors (PC-mice) provides a model system for the study of negative B cell regulation. Our previous studies demonstrated that B cell proliferation is suppressed in these mice by a cascade of interactions involving macrophages and soluble factors. The present report pinpoints the G1 phase of the cell cycle as the stage of B cell proliferation inhibited in PC-mice. Modulation of surface immunoglobulin (sIg) with anti-mu, an early membrane activation event, occurred normally on B cells from the spleens of PC-mice. However, examination of the size profile and the expression of sIgD and sIgM on B cells from the spleens of PC-mice showed an accumulation of large-sized, low intensity sIgD+ cells, suggesting a block in B cell activation in the late G1 phase of the cell cycle. This was confirmed by experiments in vitro that demonstrated that although LPS-stimulated B cells from the spleens of PC-mice enlarged to a size characteristic of G1 phase, most did not additionally enlarge into S phase even after 3 days of culture, nor did they incorporate significant amounts of [3H]thymidine. Additional confirmation of a block in late G1 was obtained by using analysis of [3H]thymidine incorporation, cell size, and cell cycle after normal cells were cultured in supernatants from cloned PC lines containing the factor(s) that initiates the cascade of events leading to suppression of B cell proliferation. The relevance of these findings to PC-induced immunosuppression and to the regulation of normal B cell proliferation during the G1 phase of the cell cycle is discussed.     

Thymidine as synchronizing agent. 3. Persistence of cell cycle patterns of phosphatase activities and elevation of nuclease activity during inhibition of DNA synthesis

Synchronous cultures of HeLa cells were obtained by selective detachment of cells in mitosis and fluctuations in enzyme activity were followed during the subsequent cell cycle. The enzymes measured were alkaline and acid phosphatases and a nuclease active on denatured DNA at alkaline pH (alkaline DNase). Each of these enzymes showed a different pattern of activity in the cell cycle, but a temporal relationship to the DNA synthetic phase was apparent in each case. Treatment of the cultures at the beginning of the cell cycle with 15 mM thymidine did not alter the subsequent pattern of fluctuations in activity of alkaline phosphatase or of acid phosphatase, although DNA synthesis was fully inhibited by this treatment. This indicates that the pattern of activity of some enzymes is not linked to DNA replication. On the other hand, the pattern of fluctuations in the activity of alkaline DNase was abolished by thymidine treatment, and elevation of the activity of this enzyme was observed. These results suggest complex and variable relationships between phases of the cell cycle and enzyme activity, and show that inhibition of DNA synthesis is not a suitable procedure for induction of culture synchrony if enzyme activities are to be studied.     

Synthesis and secretion of albumin by a synchronized rat hepatoma cell line.

The rat hepatoma cell H4-12 which synthesizes and secretes albumin was synchronized by growth in isoleucine-deficient medium followed by a second block with excess thymidine. Albumin synthesis and secretion was measured in the synchronized cells at different time intervals representative of early S, late S, G2, mitosis, early G1 and late G1 phases of the cell cycle. Maximal albumin synthesis occurred during G1 although significant synthesis also occurred during the other cell cyle phases. Most (75--80%) of the radioactive albumin produced during a 15 min pulse incubation with L-[4,5-3H] leucine was found in the microsomal cell fraction and this nascent albumin was secreted into the incubation medium during a 160 min chase period. Fifty percent of the nascent albumin was secreted by 50--55 min and this pattern of secretion did not change during the cell cycle. These data indicate that albumin synthesis occurs throughout the cell cycle but that it is preferred during G1. The rate of intracellular transport and secretion of albumin does not vary during the different phase of the cell cycle.     

Partial purification and characterization of an endothelial cell growth regulator from the bovine ovary

An inhibitor of endothelial cell thymidine incorporation in vitro was partly purified from cow ovaries using ammonium sulphate (AS) precipitation. Supernatant fluid from the 100,000 g pellet of freshly homogenized ovaries was subjected to stepwise AS precipitation. Precipitates were collected sequentially at 40%, 60%, 80% and 95% saturation, and then each was dissolved, dialysed (Mr 8000 cutoff) and examined in tissue culture for effects on cellular thymidine incorporation by cow pulmonary artery endothelial cells (CPAE) and mouse fibroblasts (L929 and 3T3). The 80% AS precipitate (ppt.) inhibited the in-vitro uptake of [3H]thymidine by CPAE and L929 cells, but not 3T3 cells. Heparin-Sepharose (HS) chromatography of the 80% AS ppt. revealed that the inhibitory activity on CPAE and L929 cells did not bind to HS; the inhibitory fraction was found in the HS column breakthrough (80% BT). The 80% BT fraction reduced CPAE[3H]thymidine uptake as determined by autoradiography and increased cellular uptake of trypan blue. Serial fractions from Sephacryl S-200 exclusion chromatography of the 80% BT contained CPAE inhibitory activity in the Mr range 30,000-50,000. The inhibitory activity on endothelial cells and L929 fibroblasts and the non-reduced molecular weight range of that fraction are similar to those of tumour necrosis factor alpha (TNF alpha). The results indicate that the cow ovary contains a fraction that inhibits endothelial cell growth in vitro and may have important roles in follicular atresia and luteal regression.     

Lack of correlation between thymidine kinase activity and changes of DNA synthesis with tumour age: an in vivo study in Ehrlich ascites tumour

Thymidine kinase (TK) and its isoenzymes were studied in relation to age of Ehrlich ascites tumour cells growing in vivo. Various steps of the pathway of thymidine through deoxynucleotide metabolism were studied: [3H]-thymidine cellular uptake and incorporation into DNA; the cellular nucleotide pools; and the concentration of thymidine in ascites. In addition, the proportion of cells in the various parts of the cell cycle and the bromodeoxyuridine labelling index were determined. Four isoenzymes at pI 4.1, 5.3, 6.9 and 8.3 were identified using isoelectric focusing. The TK activity declined with age of the tumour by about 90%, mostly due to a decrease of the isoenzyme at pI 8.3. However, this decline was neither related to the changes in DNA synthesis rate of the cells with tumour age, nor to the proportion of cells in S-phase or the bromodeoxyuridine (BrdU) labelling index. In contrast, the contribution of DNA synthesis via the thymidine salvage pathway relative to the total DNA synthesis increased from less than 1% at exponential growth to about 15% at plateau phase of growth. Blocking of DNA synthesis by aphidicolin did not change the TK activity. We therefore conclude that changes in TK activity and changes in cell growth are epiphenomena rather than causally related to each other. All nucleotide pools decreased with tumour age. The inhibition of TK by an increase in the deoxythymidine triphosphate pool could therefore be excluded. With a decrease of the TK activity during tumour growth, increasing amounts of TdR were excreted by the cells and accumulated in the ascites fluid. To explain our results on TK activity we propose a substrate cycle in which thymidine monophosphate supplied by de novo synthesis is dephosphorylated and is then either phosphorylated by TK to thymidine monophosphate or excreted by the cell.     

The association between prolyl hydroxylase metabolism and cell growth in cultured L-929 fibroblasts

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis, its active form is a tetramer (alpha 2 beta 2). In L-929 fibroblasts in the log phase of culture there is a low level of active enzyme. When the cell culture reaches confluency, prolyl hydroxylase activity in cells increases by a process that requires de novo RNA and protein synthesis. The same result may be achieved by crowding the cells (replating log phase cells at the density of stationary phase cells). In the work reported here we further examined induction of the enzyme. RNA synthesis necessary for enzyme induction is complete 6 h after "crowding" while protein synthesis requires 12 h. Thymidine (0.2-0.5 mM) added to log phase cells will also cause enzyme induction to the level found in "crowded" or resting cells. We also looked at the decay of the enzyme activity after subculture. This occurs rapidly (enzyme half-life is 1-2 h) and is concurrent with the re-entry of resting cells into cell cycle; however, thymidine added at the time of subculture to block DNA synthesis does not prevent the loss of prolyl hydroxylase activity. These results suggest that when cells are not engaged in propagation, they begin to synthesize luxury proteins such as prolyl hydroxylase. However, the loss of prolyl hydroxylase during subculture is probably not a direct consequence of DNA synthesis.     

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts: I. Temporal relationships between HMG-CoA reductase activity,sterol biosynthesis and thymidine incorporation into DNA

Temporal relationships between hydroxymethylglutaryl-CoA reductase activity, biosynthesis of C₂₇ sterols, and [³H]thymidine incorporation into DNA were studied in a rat embryo fibroblast cell line synchronized by double thymidine block and cultured in cholesterol-containing medium. Cyclic variations of HMG-CoA reductase activity and C₂₇ sterols occurred, with two maxima in S and G₂M phases; the relative shortness of the G₁ phase (3 h) in these cells could be responsible for the shift of sterol synthesis in the S phase. No noticeable variation of the individual C₂₇ sterols was observed during the entire cell cycle. In each experiment, there was a good linear correlation between HMG-CoA reductase activity and C₂₇ sterol synthesis, but from one experiment to another, a given level of enzymatic activity led to varying levels of [2-¹⁴C]acetate incorporation into sterols. In our experimental conditions, total HMG-CoA reductase activity is measured, and the preceding observation could be explained by a varying degree of phosphorylation of the enzyme depending on the metabolic state of the cells at the start of the experiment. The cyclic variations of the enzyme activity seem to be due more to increased synthesis at given times of the cycle than to periodic dephosphorylation. We question the existence of a relationship between cell division and cyclic sterol synthesis occurring in cells cultured in cholesterol-containing medium.