Ribosomal RNA metabolism in synchronized plasmacytoma cells

Ribosome synthesis and metabolism has been studied in a plasmacytoma cell line synchronized by isoleucine deprivation. Ribosomal RNA (rRNA) was characterized by gel electrophoresis. The rate of ribosome synthesis (as measured by the appearance of labelled rRNA in the cytoplasm) varied greatly during the cell cycle. It was low during the G l phase, increased rapidly during the S phase, remained high during part of the G 2 phase, and dropped to a minimum during mitosis. A slowdown in the increasing rate of RNA synthesis was observed during the middle of the S phase.No significant decrease in the total nucleotide pool per cell could be observed during the S phase. The accumulation of RNA (as determined by absorbance measurements) was highest during the S and G 2 phases.Pulse labelling of rRNA and pulse chase experiments demonstrated that newly synthesized ribosomal subunits entered into free polysomes to the highest extent during the S phase. The percentage of membrane-bound polysomes of total polysomes increased during the G 1 phase, as did the percentage of labelled rRNA in the membrane-bound fraction.     

Synthesis of poly(adenosine diphosphate ribose) in synchronized Chinese hamster cells.

Chinese hamster ovary cells were synchronized by mitotic selection and used to study the relation of poly(adenosine diphosphate ribose) synthesis to DNA synthesis and the different phases of the cell cycle. DNA synthesis was measured in cells rendered permeable to exogenously supplied nucleotides. Poly(ADPR) synthesis was also measured in permeable cells in the presence of both minimum and maximum DNA damage. The maximum DNA damage was produced by treating the cells with saturating concentrations of DNase. As anticipated, the DNA synthesis complex showed its maximum activity during S phase and showed 4–5-fold less activity during the other phases of the cell cycle. The basal level of poly(ADPR) synthesis was elevated during G1, fell to its lowest level during S phase, then increased during G2 and rose to its highest level during G1. The DNase responsive activity of poly(ADPR) synthesis was relatively constant thru the cell cycle but showed a peak at the end of S phase; then the activity decreased during the subsequent G2-M period.     

Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein g mRNA into membrane-bound polyribosome complexes

Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.     

Changes in cell-cycle duration and growth fraction in the shoot meristem of Sinapis during floral transition

The cell-cycle duration and the growth fraction were estimated in the shoot meristem of Sinapis alba L. during the transition from the vegetative to the floral condition. Compared with the vegetative meristem, the cell-cycle length was reduced from 86 to 32 h and the growth fraction, i.e. the proportion of rapidly cycling cells, was increased from 30–40% to 50–60%. These changes were detectable as early as 30 h after the start of the single inductive long day. The faster cell cycle in the evoked meristem was achieved by a shortening of the G1 (pre-DNA synthesis), S (DNA synthesis) and G2 (post-DNA synthesis) phases of the cycle. In both vegetative and evoked meristems, both-the central and peripheral zones were mosaics of rapidly cycling and non-cycling cells, but the growth fraction was always higher in the peripheral zone.Abbreviations G1 pre-DNA synthesis phase - G2 post-DNA synthesis phase - GF growth fraction - M mitosis phase - PLM percentage-labelled-mitoses method - S DNA synthesis phase - TdR thymidine     

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.     

Alterations of cell cycle kinetics and vimentin expression in TPA-treated, asynchronous MPC-11 mouse plasmacytoma cells

Vimentin expression throughout the cell cycle has been analyzed at the single-cell level in asynchronously growing MPC-11 cells using multiparameter flow cytometry. We have previously shown that these cells normally lack detectable amounts of intermediate filament proteins. In the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), cell proliferation ceases and large quantities of the intermediate filament protein vimentin are synthesized and accumulate in most of the cells. In the present study, the short-term effect of TPA on distribution of cells within the cell cycle was a depletion in early S phase followed by a depletion in mid- and late S phase. In parallel, the G1-phase fraction increased significantly. In addition, a delay in progression through G2/M phase was observed. These data strongly suggest an inhibition of progression of cells through the cell cycle in G1 phase as the primary event on cell cycle kinetics elicited by TPA. Vimentin accumulation could be detected by flow cytometry as early as 2 h after TPA addition; at this time, the percentage of vimentin-positive cells was highest in G2/M phase. Prolonged TPA treatment induced vimentin accumulation in cells of all cell cycle phases. However, even at later times, the G1-phase population consisted of two subpopulations with low and high vimentin content, respectively. The fraction of cells which displayed a higher level of vimentin probably represents those G1-phase cells which previously had undergone cell division in the presence of TPA. Our data indicate that TPA-induced vimentin synthesis is regulated in a cell cycle-dependent manner and is maximally induced in cells which have passed a putative cell cycle restriction point in G1 phase.     

Energy metabolism and ATP turnover time during the cell cycle of Ehrlich ascites tumour cells

The energy production in different parts of the cell cycle due to aerobic and aerobic glycolytic metabolism and ATP turnover time was estimated by measuring the oxygen consumption, lactate-pyruvate and ATP content of Ehrlich ascites tumour cells growing in vivo. Cell fractions of high purity from the various parts of the cell cycle were obtained by means of elutriator centrifuging. The total energy production for one cell cycle was estimated to be 19 × 10^?12 mol ATP, 60% of which was due to the aerobic metabolism. Whereas the total ATP production is unchanged during G1 a fairly exponential increase is found during the S and G2 + M phases. The total cellular ATP content increases from 12 fmol ATP at early G1 to 28 fmol ATP at G2 + M; this increase, however, is discontinuous and is most pronounced during G1 and during late S phase S phase/G2 + M. The ATP turnover time, as defined as the ratio between ATP content and ATP production, was found to increase significantly from 75 sec in early G1 to 120 sec in late G1 but was constantly 100 sec during the early, middle and late S phase as well as G2 + M. These variations indicate maximum energy-requiring processes during early G1 period of the cell cycle and are discussed in relation to K⁺Na⁺ flux and macromolecule synthesis.     

The simulation of thymidine kinase enzyme kinetics in cultured cells using the computer language cellsim

Thymidine kinase is an enzyme that occurs in cells actively synthesizing DNA. In studies of synchronized cell populations, it has been shown that the enzyme activity disappears during the G1 phase of the cell cycle and reappears during the S and G2 phases. Its reappearance is consistent with the synthesis of the mRNA for this enzyme during the S and G2 phases and its immediate translation into active enzyme by the protein synthesis machinery within the cell. The disappearance of the enzyme is consistent with the cessation of mRNA synthesis by mitotic cells. We have now tested this concept by computer simulation of a growing cell population in which a specific mRNA is generated while cells are in the S and G2 phases of the cell cycle. The computer simulation was done using the simulation language Cellsim designed for modeling populations of cells. The Cellsim program which we developed allowed each cell to make about 1 mRNA molecule per min during the S and G2 phases. Every 3 min each mRNA molecule generated a protein enzyme molecule. The mRNA had a half-life of about 9 min, and the enzyme had a half-life of about 150 min. When these molecular parameters were coupled to the cell cycle parameters for Chinese hamster fibroblasts, the resulting curve of enzyme production with time closely matched the observed kinetics of enzyme activity seen in synchronized cells. The only part of the curve that did not fit was the rapid drop in enzyme activity which was seen as the population of mitotic cells was permitted to enter G1. This drop in activity was not seen in mitotic cells blocked with Colcemid where mRNA synthesis must be lacking. Earlier studies have shown that the Gl cells do not contain any inhibitor of enzyme activity. It therefore appears that the enzyme molecule is more unstable during the G1 phase than in any of the other phases of the cell cycle.     

The difference in murine CDC2 kinase activity between cytoplasmic and nuclear fractions during the cell cycle

The mouse analog of yeast CDC2+ kinase was detected in the cytoplasmic and nuclear fractions of cultured mouse FM3A cells. Its activity in the nuclear fraction increased in the G2/M phase became seven times higher than that in the G1/S phase, while the activity in the cytoplasmic fraction remained was almost constant from the G1/S to G1 phases. The activity in the cytoplasmic fraction was similar to that in the nuclear fraction in the G2/M phase. The amount of the enzyme remained almost constant during the cell cycle in both the nuclear and cytoplasmic fractions. These findings suggest that the cytoplasmic enzyme might play an independent role in the cell cycle.     

Induction of gene mutations and the lethal action of ultraviolet rays in synchronized Chinese hamster cells

Lethal and mutagenic effects of UV light were studied in two synchronized UV-sensitive Chinese hamster cell clones differing in the degree of sensitivity (CHS1, CHS2). It is shown that the phase of mitosis is most resistant to the lethal effect of UV. The sensitivity of both cell clones increases in the pre-synthetic phase and reaches its maximum during the phase of DNA synthesis. Positive correlation of cell sensitivity to mutagenic and lethal action of UV was observed when studying induced mutability in both cell clones during the phase of DNA synthesis. However, the study of the mutagenic effect of UV on different phases of the synthesis. However, the study of the mutagenic effect of UV on different phases of the cell cycle (M, G1, S) in the less UV-sensitive cell clone has revealed that the maximal mutation yield takes place when cells are irradiated at G1 (CHS1). The discrepancy observed may be due to different probability of the phenotypic detection of pre-mutational lesions, arising at different phases of the cell cycle. It is shown that only one cell generation is necessary for the expression of pre-mutational changes. These data allow to conclude that the increased mutation rate observed at G1 (as compared with S) reveals rather a probability of the expression but not of the occurrence of pre-mutational lesions. It is suggested that the fixation of mutations in the cells studied proceeds during the post-replication repair synthesis.     

Simian virus 40 induces multiple S phases with the majority of viral DNA replication in the G2 and second S phase in CV-1 cells

The infection of permissive monkey kidney cells (CV-1) with simian virus 40 induces G1 growth-arrested cells into the cell cycle. After completion of the first S phase and movement into G2, mitosis was blocked and the cells entered another DNA synthesis cycle (second S phase). Growth-arrested CV-1 cells replicated significant amounts of viral DNA in the G2 phase with the majority of synthesis occurring during the second S phase. When mimosine-blocked (G1/S) infected cells were released into the cell cycle, a major portion of the viral DNA was detected in G2 with the largest accumulation in the second S phase. The total DNA produced per infected cell was 10-12C with approximately 0.5-2C of viral DNA replicated per cell. Therefore the majority of the DNA per cell was cellular, 4C from the first S phase and approximately 4-6C from the second cellular synthesis phase.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

Treatment of cells with the polyamine analog N, N11-diethylnorspermine retards S phase progression within one cell cycle.

When Chinese hamster ovary cells were seeded in the presence of the spermine analog N1,N11-diethylnorspermine (DENSPM), cell proliferation ceased; this was clearly apparent by cell counting 2 days after seeding the cells. However, 1 day after seeding there was a slight difference in cell number between control and DENSPM-treated cultures. To investigate the reason for this easily surpassed slight difference, we used a sensitive bromodeoxyuridine/flow cytometry method. Cell cycle kinetics were studied during the first cell cycle after seeding cells in the absence or presence of DENSPM. Our results show that DENSPM treatment did not affect the progression of the cells through G1 or the first G1/S transition that took place after seeding the cells. The first cell cycle effect was a delay in S phase as shown by an increase in the DNA synthesis time. The following G2/M transition was not affected by DENSPM treatment. DENSPM treatment inhibited the transient increases in putrescine, spermidine, and spermine pools that took place within 24 h after seeding. Thus, in conclusion, the first cell cycle phase affected by the inhibition of polyamine biosynthesis caused by DENSPM was the S phase. Prolongation of the other cell cycle phases occurred at later time points, and the G1 phase was affected before the G2/M phase.     

RNA metabolism and membrane-bound polysomes in relation to globulin biosynthesis in cotyledons of developing field beans (Vicia faba L.).

In cotyledon cells of developing field beans the RNA content per cell does not change in the second half of developmental period 2, whereas globulin biosynthesis continues. The constant RNA content per cell results from an equilibrium between RNA synthesis and degradation. All types of RNA are synthesized until the end of globulin biosynthesis, but poly(A)-containing RNA was preferentially labelled during maximum globulin formation. During stage 2 of seed development of poly(A)-containing RNA fraction represents a discrete peak in the 12--18-S region on agarose gels and corresponds to the peak of poly(A)-containing RNA isolated from polysomes. alpha-Amanitin inhibits selectively the labelling of poly(A)-containing RNA and concomitantly globulin formation. Translation of total poly(A)-containing RNA, free and membrane-bound polysomes in a cell-free wheat germs demonstrates that the globulins are preferentially produced on membrane-bound polysomes and that poly(A)-containing RNA includes the mRNA for both vicilin and legumin.     

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity.     

Cell-Cycle Dependent Appearance of Murine Leukemia-Sarcoma Virus Antigens

Normal rat kidney (NRK) cells, NRK cells infected with Rauscher murine leukemia virus, and NRK cells infected with Kirsten murine sarcoma-leukemia virus (NRK-K) were synchronized by a double thymidine block. At intervals after release from thymidine blockage, the cells were examined for the presence of viral antigens in the cytoplasm and on the cell surface by immunofluorescent microscopy by using goat anti-Rauscher murine leukemia virus and goat anti-Moloney leukemia virus (Tween-ether disrupted) sera. Detection of viral antigens in the cytoplasm was periodic during the cell cycle. Antigens were detected first during the S phase, increased during the G2 phase, and disappeared during the M and G1 phases. A similar pattern of surface immunofluorescence was observed. Infectious virus was detected in culture fluids from synchronized cells during the M phase. Surface immunofluorescence was detected in NRK-K cells with anti-Rauscher murine leukemia virus and may represent the presence of group-specific antigens on the cell surface. Control, uninfected NRK cells, which did not normally fluoresce, showed weak immunofluorescence during the S and G2 phases after synchronization. Synchronization can be used to amplify latent oncornavirus expression.     

Cell cycle kinetics and monoclonal antibody productivity of hybridoma cells during perfusion culture

The flow-cytometric (FCM) analysis of bivariate DNA/lgG distributions has been conducted to study the cell cycle kinetics and monoclonal antibody (MAb) production during perfusion culture of hybridoma cells. Three different perfusion rates were employed to demonstrate the dependency of MAb synthesis and secretion on cell cycle and growth rate. The results showed that, during the rapid growth period of perfusion culture, the level of intracellular igG contents of hybridoma cells changed significantly at each perfusion rate, while the DNA histograms showing cell cycle phases were almost constant. Meanwhile, during the reduced growth period of perfusion culture, the fraction of cells in the S phase decreased, and the fraction cells in the G1/G0 phase increased with decreasing growth rate. The fraction of cells in the G2/M phase was relatively constant during the whole period of perfusion culture. Positive correlation was found between mean intracellular IgG contents and the specific MAb production rate, suggesting that the deletion of intracellular IgG contents by a flow cytometer could be used as a good indicator for the prediction of changes in specific MAb productivity following manipulation of the culture condition. (c) 1994 John Wiley & Sons, Inc.     

Flow cytometric study of cultured mammalian cells.

Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of flow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics. The maximum proportion of S-cells was reached early in the growth phase while a population of low fluorescence cells with lower polidy than G1, dead cells and fragmented nuclei emerged during the death phase. Supplementation with amino acids during the exponential phase prolonged the growth cycle by enhancing cell proliferation. The fraction of S/G2 cells was much reduced by a reduction in serum concentration but was maintained during the prolonged non-proliferating "stationary" phase. The magnitude of Rhodamine 123 staining showed a consistent and general decrease during late exponential and decline phases. This trend was accompanied by an increase in the fraction of the Propidium Iodide-stained population which reflected the deteriorating metabolic and membrane integrity. Decrease in mean fluorescence intensity for DNA, RNA, protein and intracellular IgG was noted at the decline phase. Intracellular immunofluorescence was a more reliable indicator of antibody productivity than surface immunofluorescence.     

Level of mIa expression on mitogen-stimulated murine B lymphocytes is dependent on position in cell cycle

Using correlated flow cytometric analysis of cell surface Ia antigen expression (immunofluorescence) and cell cycle phase (pulse-width of axial light extinction), we have quantitated changes in expression of mIa antigen on murine B cells during progression through cell cycle. Our results indicate that density of mIa expressed on mitogen-stimulated B cells increases fourfold to fivefold during the transition from G0 to G1. By early S phase, mIa density has decreased by fourfold to fivefold relative to peak expression. This decrease becomes more evident by late S, G2, and M phases, when an eightfold decrease in mIa antigen density is observed relative to peak levels. This decrease results in mIa antigen expression lower than that of resting, unstimulated B cells. Therefore, maximum mIa antigen expression occurs during G0 to G1 transition and in early G1, when a requirement for I region-restricted, antigen-driven T cell help for thymus-dependent, antigen-driven B cell activation has been demonstrated.     

Phospholipids are synthesized in the G2/M phase of the cell cycle

Very little is known about the metabolism of phospholipids in the G2 and M phases of the cell cycle, but limited studies have led to the postulation that phospholipid synthesis ceases during this period. To investigate whether phospholipids are synthesized in the G2/M phase of the cell cycle, protocols were developed to produce synchronized MCF-7 cell populations with greater than 80% of the cells in G1/S or G2/M phases that moved in synchrony following removal of the blocking agent. Analysis of the activities of key phosphatidylcholine and phosphatidylethanolamine biosynthetic enzymes in subcellular fractions obtained from MCF-7 cells at different cell cycle phases revealed that there was robust activity of key enzymes in the fractions prepared from MCF-7 cells in G2/M phase. Radiolabeled choline and ethanolamine were rapidly incorporated into cells maintained at G2/M phase with nocodazole, and the rates of incorporation were similar to those obtained in cells allowed to progress into the G1 phase. Furthermore, radiolabeled glycerol was incorporated into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid in MCF-7 cells maintained at G2/M phase with nocodazole. Similar results were obtained in CHO cells. These results demonstrate that glycerophospholipid synthesis is very active in the G2/M phase of these cells. Therefore, the postulated cessation of phospholipid synthesis in G2/M phases is not applicable to all cell types.