Deoxyribonuclease II activity in relation to cell cycle in synchronized HeLa S3 cells

Studying the activity of DNase II in relation to cell cycle in synchronized HeLa S3 cells show a two to seven fold increase in DNase II activity at those times when DNA synthesis is taking place. The peaks of DNase II activity coincide with the peaks of DNA synthesis. The increased DNase II activity could be prevented by puromycin, suggesting that the enzyme activity increased at the S phase was caused by synthesis of new molecules rather than the activation of existing molecules. Acid phosphatase (as a marker for lysosomal enzymes) does not show an induction similar to that observed for DNase II in relation to cell cycle.     

Enzyme and Nucleic Acid Formation During Synchronous Growth of Rhodopseudomonas spheroides

The synthesis of various cell components was examined during the anaerobic photosynthetic growth of synchronous populations of Rhodopseudomonas spheroides. Net deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein increased continuously as did the rate of incorporation of radioactive precursors into protein. The rates of incorporation of radioactive precursors into RNA and DNA were marked by abrupt discontinuities. It is not clear whether these discontinuities represent changes in rates of synthesis or fluctuations in precursor pools. Although the synthesis of bacteriochlorophyll occurred in a continuous manner, those enzymes examined which are involved in the synthesis of tetrapyrroles, i.e., succinyl CoA thiokinase, delta-aminolevulinic acid synthetase, and delta-aminolevulinic acid dehydrase, increased discontinuously. Two other enzymes not involved in tetrapyrrole biosynthesis were examined. Alkaline phosphatase increased in a stepwise manner during the division cycle, whereas the synthesis of ornithine transcarbamylase increased rapidly before leveling off for a period of time until synthesis began again. In each instance of discontinuous enzyme synthesis, increases occurred at regular and characteristic times during the division cycle. Ammonium sulfate precipitation was employed to remove low molecular weight end product inhibitors from enzyme preparations. These studies suggested that the stepwise increases in enzyme activity observed in the present investigation were not affected by periodic end product inhibition. A temporal map of enzyme synthesis during the division cycle was constructed. Both delta-aminolevulinic acid synthetase and delta-aminolevulinic acid dehydrase appeared early in the division cycle, whereas alkaline phosphatase and succinyl CoA thiokinase appeared later on.     

Activity of lysosomal enzymes in the various cell cycle phases of synchronized HeLa cells.

The activities of 5 lysosomal enzymes (acid DNase, β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase and cathepsin D) were measured in HeLa cells in various cell cycle phases. The cells were synchronized either by shake-off of mitotic cells followed by resuspension in fresh medium, or by addition of amethopterin and adenosine for 16 h and reversal with thymidine. Metaphase arrest was obtained with colcemid in cells previously synchronized by means of amethopterin/thymidine. The specific activities (activity/mg protein) of the different enzymes were found to be constant following synchronization both with the shake-off technique and with the amethopterin/thymidine treatment. Furthermore, the specific enzyme activities were unaltered by metaphase arrest by colcemid. Our data indicate that lysosomal enzyme synthesis is continuous during the cell cycle of HeLa cells. The specific activity of β-glucuronidase was found to be about 3 times higher in HeLa cells grown in suspension cultures than in cells grown on solid surface. The activities of the other enzymes measured were approximately equal in suspension cells and surface cells.     

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.     

Studies on the synthesis and degradation of DNA in developing and old chick cerebellum

The levels of DNA, RNA, protein and acid and alkaline DNase were studied in developing and old chick cerebellum. The in vitro synthesis of DNA, by both chick cerebrum and cerebellum was also studied, by following the incorporation of [3H]thymidine into DNA. It was observed that the increase in DNA content of chick cerebellum continued well beyond adult stages of life span. Maximal DNA synthesis, as judged by the [3H]thymidine incorporation, was noticed during the early embryonic development but decreased with advancement of age. There was, however, another peak of activity, although smaller, at about 9 months of age. Both cerebrum and cerebellum showed similar patterns. The highest specific activity of acid DNase was also found during the early period of cerebellar development, that is at a time when rapid cellular proliferation was occurring. The activity steadily declined with the aging and in 2-year-old cerebellum very little activity could be detected. Alkaline DNase, on the other hand, not only exhibited high activity during the early development but also remained at a significant level even in old cerebellum. It is concluded that acid DNase shows a positive correlation to the early embryonic DNA synthesis but not to the cell increase occurring in old age.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.     

Flow cytometric determination of cell cycle phase-specific changes in cellular phosphatase and glycosidase activities

The activities of two phosphatases (E.C. 3.1.3.1 and 3.1.4.1) and four glycosidases (E.C. 3.2.1.21, 3.2.1.30, 3.2.1.31 and 3.2.1.51) were measured by fluorescence spectrophotometry, and flow cytometry, in mitogen-stimulated lymphocytes, and in cultures of Molt-4-F and F-89 cell lines, synchronized by hydroxyurea or thymidine. All enzymes were active throughout the cycle but the activities of three enzymes were elevated at specific points in the cycle, alkaline phosphatase activity increased at G2 + M/G1 boundary and in early S-phase, the activity of beta-L fucosidase was elevated in G1 and late S-phase. Orthophosphate diesterase activity was elevated at the G1/S boundary, and during G2 + M. The increase in beta-L fucosidase activity was due to an increased number of cells showing activity, whilst the increase in orthophosphate diesterase activity was attributable to an increase in cellular enzyme activity. Only the activities of orthophosphate diesterase and beta-L fucosidase were measurable by flow cytometry, alkaline phosphatase activity was mainly extracellular, and therefore not detectable by flow cytometric methods employed.     

Inhibitory effects of somatostatin on growth and differentiation in cultured intestinal mucosa

The effect of somatostatin on mucosal DNA, protein and brush border enzymes was studied in organ cultured rabbit jejunum and ileum. Compared to control cultures, somatostatin reduced the biopsy DNA and protein content in parallel in the jejunum, but was ineffective in the ileum. This was probably due to a direct growth inhibition, since DNA and brush border enzyme activity from desquamated cells in the postculture medium were unaffected. In addition, a direct inhibition of jejunal villous cell differentiation by somatostatin was reflected in a significant decrease of sucrase, maltase and alkaline phosphatase activity. In the ileum, only the specific activity of alkaline phosphatase was reduced. The key enzyme of cholesterol synthesis, HMG-CoA-reductase, was measured as an intracellular enzyme control and was not influenced by the hormone. The high somatostatin concentrations necessary to achieve the effects are not an artefact of hormone degradation during culture, as shown by radioimmunoassay, and suggest a local or "paracrine" rather than systemic, inhibitory action of somatostatin on intestinal growth and differentiation.     

Enhanced Deoxyribonucleic Acid Polymerase Activity in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12

Deoxyribonucleic acid (DNA) polymerase activity was induced at approximately 18 to 20 hr after infection of secondary cultures of human embryonic kidney cells with adenovirus type 2 or type 12, and, at 30 to 50 hr after infection, the activity of this enzyme increased two- to threefold. The activity of thymidine kinase was also induced, but the activity of deoxycytidylic deaminase was not. The DNA content per cell at 71 hr after infection was 1.6-fold greater in adenovirus 2-infected cultures, and approximately 2.4-fold greater in adenovirus 12-infected cultures, than in the noninfected cultures. Several properties of DNA polymerase were studied. The enzymes in normal and adenovirus 2- or 12-infected cell extracts were saturated by approximately the same concentration of heat-denatured salmon sperm DNA primer (160 mug/ml); the enzyme activities had a similar broad pH optimum between 7.5 and 9. Extracts prepared from cells infected by either adenovirus did not activate DNA polymerase from noninfected cells, nor did the noninfected cell extracts inhibit enzyme activity of infected cell extracts. DNA polymerase in both normal and adenovirus 2- or 12-infected cells was located predominantly in the nucleus. In each case, the cytoplasm had only 30% of the enzyme activity of the nucleus. At 40 hr after infection with adenovirus 2 or 12, the activities of the enzyme in the nuclear and cytoplasmic fractions increased two- to threefold. Puromycin, an inhibitor of protein synthesis, prevented DNA polymerase induction when added to cultures during the 18- to 30-hr postinfection period, and it arrested the additional increase in enzyme activity when added after enzyme induction began. However, the increases in both DNA polymerase and thymidine kinase activities took place after treatment of infected cultures with 1-beta-d-arabinofuranosylcytosine, an inhibitor of DNA synthesis and adenovirus growth.     

Effects of recombinant human interleukin 1 alpha and interleukin 1 beta on cell growth and alkaline phosphatase of the mouse osteoblastic cell line MC3T3-E1

Recombinant human interleukin 1 (rhIL-1)alpha and rhIL-1 beta were examined for their effects on DNA synthesis, cell growth and alkaline phosphatase activity of the mouse osteoblastic cell line MC3T3-E1. The relative activity of rhIL-1 alpha and rhIL-1 beta was compared in terms of the units which induced half-maximal [3H]thymidine uptake into mouse thymocyte cultures exposed to IL-1. Both rhIL-1 alpha and rhIL-1 beta significantly inhibited DNA synthesis and division of the cells in a concentration- and cultivation time-dependent fashion. In contrast, rhIL-1 alpha and rhIL-1 beta markedly increased alkaline phosphatase activity, which is a marker of osteoblastic differentiation. This activity in cells treated with rhIL-1 alpha and rhIL-1 beta increased about 2.0- and 1.7-fold, respectively, compared with that of control cultures. Inhibition of the DNA synthesis and stimulation of alkaline phosphatase activity by both types of rhIL-1 were completely neutralized by treatment with their respective polyclonal antisera. Also, inhibition of DNA synthesis was unaffected by the addition of cyclooxygenase and lipoxygenase inhibitors, and stimulation of alkaline phosphatase activity was unaffected by the addition of indomethacin. These results indicate that both rhIL-1 alpha and rhIL-1 beta have qualitatively similar biological effects on osteoblastic cells. They also suggest that IL-1 is an important modulator of the growth and differentiation of osteoblasts.     

Inhibition of Thymidine Kinase Activity and Deoxyribonucleic Acid Synthesis in L Cells Infected with the Meningopneumonitis Agent

The activities of enzymes related to deoxyribonucleic acid (DNA) synthesis were studied in uninfected L cells and in L cells infected with Chlamydia psittaci (strain meningopneumonitis). The meningopneumonitis agent multiplied normally but failed to induce the synthesis of thymidine kinase in LM (TK(-)) cells which contain no thymidine kinase in the uninfected state. It was concluded that this microorganism has no thymidine kinase of its own and that it does not depend on the functioning of the host enzyme for synthesizing its DNA. Exposure of clone 5b L cells to the meningopneumonitis agent was followed by a decline in their thymidine kinase activity to nearly zero levels, whereas the levels of uridine kinase and thymidylate synthetase remained unchanged. Inhibition of thymidine kinase activity in L cells occurred soon after infection and required new protein synthesis by the meningopneumonitis agent. This inhibition occurred before inhibition of host DNA synthesis, but it was not an essential prelude to the latter inhibition. On the basis of this and previous investigations and in light of present knowledge of the mammalian cell cycle, it was postulated that the meningopneumonitis agent inhibits macromolecular synthesis in L cells by preventing the initiation of a new cell cycle.     

Effects of recombinant human interleukin 1a and interleukin 1β on cell growth and alkaline phosphatase of the mouse osteoblastic cell line MC3T3-E1

Recombinant human interleukin 1 (rhIL-1)α and rhIL-1β were examined for their effects on DNA synthesis, cell growth and alkaline phosphatase activity of the mouse osteoblastic cell line MC3T3-E1. The relative activity of rhIL-1α and rhIL-1β was compared in terms of the units which induced half-maximal [³H]thymidine uptake into mouse thymocyte cultures exposed to IL-1. Both rhIL-1α and rhIL-1β significantly inhibited DNA synthesis and division of the cells in a concentration- and cultivation time-dependent fashion. In contrast, rhIL-lα and rhIL-1β markedly increased alkaline phosphatase activity, which is a marker of osteoblastic differentiation. This activity in cells treated with rhIL-1α and rhIL-1β increased about 2.0- and 1.7-fold, respectively, compared with that of control cultures. Inhibition of the DNA synthesis and stimulation of alkaline phosphatase activity by both types of rhIL-1 were completely neutralized by treatment with their respective polyclonal antisera. Also, inhibition of DNA synthesis was unaffected by the addition of cyclooxygenase and lipoxygenase inhibitors, and stimulation of alkaline phosphatase activity was unaffected by the addition of indomethacin. These results indicate that both rhIL-1α and rhIL-1β have qualitatively similar biological effects on osteoblastic cells. They also suggest that IL-1 is an important modulator of the growth and differentiation of osteoblasts.     

Physarum thymidine kinase. A step or a peak enzyme depending upon temperature of growth.

The variations of thymidine kinase or ATP:thymidine 5'-phosphotransferase (EC 2.7.1.21) during the cell cycle of Physarum polycephalum plasmodia have been studied at two extreme physiological temperatures: 22 degrees C and 32 degrees C. At 22 degrees C the enzyme activity increases near mitosis and stays constant during late S and G2 phases, exhibiting the typical pattern of a 'step enzyme'. But at 32 degrees C thymidine kinase activity goes through a maximum 1 h 30 min after mitosis and decreases during the subsequent phases as expected for a 'peak enzyme'. The rate of enzyme degradation and/or inactivation, measured in the presence of metabolic poisons (cycloheximide or dinitrophenol), appears to follow a simple exponential function with a half-life of approximately 3 h and 1 h at 22 degrees C and 32 degrees C respectively. The effect of growth temperature on the decrease of thymidine kinase activity can account entirely for the differences in the pattern of enzyme activity at the two extreme temperatures. Tentative calculations indicate that the rate of enzyme synthesis is nearly constant during the cell cycle except near mitosis, where it is temporarily increased. The results suggest the existence of a regulatory mechanism able to modulate the rate of synthesis of thymidine kinase during the cell cycle.     

Brain DNA metabolism in myelin deficient mutant jp,jpmsd and qk mice

We studied metabolism of brain DNA in three myelin deficient mutants qk, jp and jp^msd mice. The DNA content, the in vivo incorporation of [¹⁴C]thymidine in DNA and the activity of acid DNase in tissues (cerebellum and cerebrum) from normal littermates and affected mice were compared. The results showed that neither the DNA content, the incorporation of [¹⁴C]thymidine in DNA nor the activity of acid DNase in brain were altered in qk affected mice. In jp^msd mice, however, the DNA content as well as the incorpation of thymidine in DNA were reduced in both cerebellum and cerebrum, but the activity of acid DNase was reduced in cerebrum only. In jp mice, although the DNA content was reduced in both cerebellum and cerebrum, the incorporation of thymidine in DNA and the activity of acid DNase were reduced in cerebrum only. The data suggest a) that in qk mutants DNA metabolism and hence cell (glial) proliferation is not affected; b) that in jp^msd mutants DNA synthesis, and thus the cell proliferation is reduced in cerebellum as well as in cerebrum of the affected mice and c) that in jp mutants the synthesis of DNA and the cell proliferation is reduced in cerebrum but not in cerebellum.     

Glucose metabolism and template synthesis in the mitotic cycle of human diploid fibroblasts

The correlation between the rates of protein and nucleic acid synthesis and the activity of the key enzymes of glycolysis (hexokinase, phosphofructokinase) and pentose phosphate cycle (glucose-6-phosphate dehydrogenase) in the mitotic cycle of human diploid fibroblasts synchronized by double thymidine block was studied. It was found that the removal of the thymidine block is followed by short-term (presumably, non-specific) simultaneous stimulation of matrix syntheses, as well as by glycolytic and pentose phosphate cycle enzyme syntheses. By the beginning of the S-phase, all the processes appear to be inhibited, followed by gradual activation of glycolysis and pentose phosphate cycle reactions. The implementation of the cell cycle is concomitant with stepwise transitions of protein and hexokinase synthesis rates and ATP content to one of the following levels--basal, intermediate or maximal. Changes in the activity of glucose-6-phosphate dehydrogenase in the course of the cell cycle appear as oscillations, those in phosphofructokinase as alternative states. At stage M, the oscillatory processes are temporarily quenched, whereas the ATP content occupies an intermediate level. In contrast with diploid fibroblasts, in transformed T9 cells the enzyme activity is much higher, and the fluctuations in activity throughout the cell cycle are less noticeable. Presumably, in transformed cells the enzyme activity is at the maximum level and is not prone to effector regulation.     

Alkaline phosphatase activity of normal and transformed cultured human cells

A study was made of the relationship between the activity of alkaline phosphatase and the proliferation of cultured human cells with different replicative potentials. It is shown that alkaline phosphatase plays a role as one of endogenic stimulators of cellular proliferation. The ageing of diploid cells is accompanied by a decrease in the enzyme activity. Maximum activity was observed during a period of logarithmic cell growth. Addition of placental alkaline phosphatase to the synchronized diploid cells stimulated DNA synthesis in the S-phase of the cell cycle. Heteroploid cells with a high growth rate possessed a 30-100 times higher alkaline phosphatase activity than in the diploid cells. Under certain conditions alkaline phosphatase may presumably function as a proteinkinase.     

Macromolecular Synthesis During Microcycle Sporogenesis of Bacillus cereus T

Microcycle sporogenesis induced in Bacillus cereus T by phosphate limitation occurs over a narrow range of phosphate to spore inoculum ratios. Sufficient phosphate is required to satisfy the demands for a twofold increase in deoxyribonucleic acid; net ribonucleic acid synthesis is not required. The total ribonucleic acid content of the culture was variable, and deoxyribonucleic acid synthesis was restricted to a twofold increase. Developmental changes during outgrowth occurred synchronously, whereas enzyme synthesis was periodic. The timing of the synthesis of tricarboxylic cycle enzymes, extracellular protease, arginase, histidase, and alkaline phosphatase was measured. Histidase could be induced after 2.5 hr throughout microcycle sporogenesis. Several other features of macromolecular synthesis during microcycle sporogenesis are described. Differences between this pattern and those observed during outgrowth leading to cell division are discussed. A technique for accurately estimating the levels and time of synthesis of incompletely extractable, labile enzymes is also presented.     

Changes in enzyme activity during differentiation in Chlamydomonas reinhardtii

The patterns of alanine dehydrogenase, glutamate dehydrogenase and malate dehydrogenase activity were studied during the normal vegetative cell cycle and during the process of gametic differentiation and dedifferentiation in synchronized cultures of Chlamydomonas reinhardtii. During all three phases of growth and differentiation the synthesis of DNA was also measured. During gametic differentiation all three enzyme levels were suppressed compared to vegetative cells although DNA and cell number were comparable. During gametic dedifferentiation no DNA synthesis occurred during the first 24 h cycle and only a doubling during the second. It was not until the third cycle that a normal 4-fold increase in DNA was observed. Cell number followed a similar pattern. Athough the levels of alanine dehydrogenase and malate dehydrogenase were uniformly low during the first cycle when glutamate dehydrogenase increased 4-fold, during the second cycle the patterns of these enzymes changed markedly. The enzymes did not attain levels characteristic of vegetative cells until the third cycle.     

Enzymic activity in rat uterus during early pregnancy.

Total protein, RNA and DNA content and the activity of acid and alkaline phosphatases, 5'-nucleotidase and isocitrate dehydrogenase were studied in rat uterus during the first 8 days of pregnancy. Isocitrate dehydrogenase activity showed marked fluctuations from day to day. Nucleotidase and acid phosphatase activities showed a significant increase on day 8. The most marked change in activity was that of alkaline phosphatase which showed a 10-fold increase between days 6 and 8, due largely to an increase in the activity of this enzyme in the decidual nodule. The rise in alkaline phosphatase activity did not occur in rats ovariectomized on days 1, 2 or 4 of pregnancy and was markedly decreased in those ovariectomized on day 6. [3H]-uridine incorporation into RNA showed a significant increase between days 2 and 6 whereas [3H]-thymidine incorporation into DNA showed a significant increase on day 6.