Cell Cycle Of Fibroblasts Research Articles

Changes in distribution of nuclear matrix antigens during the mitotic cell cycle.

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.

Regulation of growth of human diploid fibrobalasts by factors elaborated by activated lymphoid cells

Fibroblasts growth synthesis activities appear to be under exquisite control. This control is mediated in part by substances present in blood plasma or released by other cells. We have studied the role of peripheral blood mononuclear cells (PBM) activated with phytohemagglutinin-P (PHA) on DNA synthesis, proliferation, and the cell cycle of human diploid fibroblasts. Culture medium from activated but not from unactivated PBM cultures inhibited fibroblast DNA synthesis and growth in a dose-dependent manner. The activity, which was designated as lymphocyte factor (LF), was very potent; it inhibited 50% of the DNA synthesis and cell growth at a dilution of 1:160. It has a molecular weight between 50,000 and 100,000 daltons and it is destroyed by trypsin digestion or by heating at 80 degrees C for 30 minutes. The activity was not due to the presence of prostaglandin. Furthermore, using immunoprecipitation and affinity chromatography, it was shown conclusively to to be distinctly different from alpha lymphotoxin (alpha-LT). It was not cytotoxic, as shown by the 51chromium release technique. Using flow microfluorimetry it was shown that the activity regulates fibroblast growth by preventing quiescent cells in the G0 or G1 stage of the cell cycle from entering the S phase. Cells already in S at the time of exposure complete DNA synthesis but cannot divide, and they accumulate in G2. The activity also has marked effects on protein synthesis. Activated mononuclear cells may play a major role in regulating fibroblast growth and synthesis in normally healing wounds and in acute and chronic inflammatory processes.

Prolongation of S phase and whole cell cycle in Werner's syndrome fibroblasts

The cell cycle was determined in early passage fibroblasts from Werner's syndrome and normal subjects. The average cell cycle time was prolonged in Werner's syndrome cells due to changes in the duration of S phase. Using alkaline sucrose gradient sedimentation, the rate of DNA elongation was examined, and no difference was observed between Werner's syndrome cells and normal cells.

Role of src gene in growth regulation of Rous sarcoma virus-infected chicken embryo fibroblasts.

We report here a study of the mechanisms leading to loss of growth control in chicken embryo fibroblasts transformed by Rous sarcoma virus (RSV). We have been particularly concerned with the role of the src gene in this process, and have used RSV mutants temperature sensitive (ts) for transformation to investigate the nature of the growth regulatory lesion. The two principal findings were (1) the stationary phase of the cell cycle (G1) in chick embryo fibroblasts seems to have two distinct regulatory compartments (using the terminology of Brooks et al. we refer to these as 'Q' and 'A' states). When rendered stationary at 41.5 degrees C by serum deprivation, normal cells enter a Q state, but cells infected with the ts-mutant occupy an A state. (2) Whereas normal cells can occupy either state depending on culture conditions, the ts-infected cells, at 41.5 degrees C, do not seem to enter Q even though a known src gene product, a kinase, is reported to be inactive at this temperature. We discuss the possibility that viral factors other than the active src protein kinase influence growth control in infected cultures.

Histone mRNA metabolism during the mouse fibroblast cell cycle

The synthesis of histone mRNA in the nucleus and the eventual appearance of histone mRNA in the cytoplasm of 3T6 Swiss mouse embryo fibroblasts was investigated with a highly labelled cDNA probe complementary to histone mRNA. Very low levels of histone mRNA were found in G 1 phase nuclei, with even lower levels in the cytoplasm. At the onset of DNA synthesis, there is a rapid increase in histone mRNA synthesis, with a concomitant increase of histone mRNA in the cytoplasm followed by its decline in both nucleus and cytoplasm on entering the G 1 phase of the cell cycle. The decline in the nucleus is much more rapid than in the cytoplasm.

Procollagen mRNA metabolism during the fibroblast cell cycle and its synthesis in transformed cells

Procollagen mRNA was isolated from mouse embryos and used for the synthesis of a highly labelled cDNA probe complementary to collagen mRNA. This probe was used for the investigation of procollagen mRNA metabolism during the cell cycle of 3T6 mouse embryo fibroblasts in culture. Titration hybridization experiments revealed that procollagen mRNA was present throughout the cell cycle following stumulation of confluent monolayers. Procollagen mRNA levels of sparse cultures appeared similar to those of unstimulated monolayers. The fluctuating levels of collagen synthesis during the cell cycle can be ascribed to changes in the amount of collagen mRNA present. In mouse sarcoma virus transformed 3T3 cells only 20--30% of the amount of procollagen mRNA in 3T3 cells is present indicating that the decline in collagen synthesis is due to mRNA availability.

Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle

Platelet-derived growth factor and the regulation of the mammalian fibroblast cell cycle

Effects of Leukocyte and Fibroblast Interferon on Events in the Fibroblast Cell Cycle

Serum-depleted human foetal skin fibroblasts were stimulated by addition of 10% foetal calf serum to proliferate synchronously for at least one cell cycle. This proliferation was suppressed by leukocyte or fibroblast interferon (IF), which prolonged the G1 phase and diminished the rate of DNA synthesis during the S phase in a dose-dependent manner. When used in identical concentration, as judged in terms of units of antiviral activity, fibroblast IF had more pronounced effects on cell cycle events than leukocyte IF. Interferon exerted its effect in early G1, before the cells were irreversibly committed to DNA synthesis.

Sister chromatid exchange and cell cycle in fibroblasts of Bloom's syndrome

Sister chromatid exchange (SCE) has been studied in the fibroblasts of five Bloom's syndrome patients, one heterozygote, and two normal individuals. The high frequency of SCE already known in the lymphocytes of Bloom's syndrome was also found in the fibroblasts of all five patients. However, populations with low and high frequency of SCE were not found. In addition, chromosome aberrations appeared with a lower frequency. The cell cycle duration in the Bloom's fibroblasts appeared to be similar to that in the normal cell line, and the difference in the growth pattern appeared to be due to the variation in the mitotic index. The cell cycle lasted about 24 h in at least four of the Bloom's lines studied during the present experiments.

Cell size, cell cycle and transition probability in mouse fibroblasts

This paper describes the relationship between cell size and cell division in two situations. In the first, quiescent cells were sorted on the basis of cell size using a fluorescence-activated cell sorter and returned to culture. The results of this type of experiment are compatible with the idea that once cells have completed a size-dependent lag, the rate of entry of cells into S phase is controlled by a rate-limiting random event (or transition). The second kind of experiment follows the kinetics of complete cell cycles in rapidly proliferating cells whose mothers had been sorted on the basis of cell size. The cells born of small mother cells have longer cycle times than cells derived from large mothers. The difference in the cycle time of these two classes was due to differences in the B phase of the cell cycle [containing S, G2, M and part of G1 (G1B)], transition probability being the same in both size classes. Our results show that S, G2 and M are unaffected by size, thus confining the effect of size to G1B. It seems probable that the variability of B phase in cloned cell populations is partly due to variations of cell size at division, and correlations between the cycle times of sister cells result because sibling cells are more similar in size than unrelated cells. The major factor controlling cell division in mouse fibroblasts is shown, however, to be the transition probability; size has a more minor role.

The cell cycle of low passage and high passage human diploid fibroblasts

Using mitotic selection, synchronous populations of passage 25 and passage 50 WI38 cells were obtained. Thymidine incorporation, mitotic index, and labeling index were determined. No great differences could be seen in the organization of the cell cycle of low passage and high passage cells. Although labeling indices for passage 25 and passage 50 exponential cells were 83% and 37%, respectively, 75% of all mitotically selected cells were found to enter S phase, regardless of passage number. Thus, the loss of late passage diploid cell cultures does not occur from alteration of the cell cycle in the majority of rapidly proliferating cells.

Phosphorylation of nonhistone chromosomal proteins early during the prereplicative phase of the cell cycle of WI-38 human diploid fibroblasts

Stimulation of confluent monolayers of nondividing WI-38 cells to proliferate is accompanied by a doubling in the rate of phosphorylation of nonhistone chromosomal proteins during the first hour. The use of cycloheximide to inhibit protein synthesis does not appear to affect phosphorylation of the nonhistones. Nondividing and serum-stimulated cells exhibit qualitative differences in the phosphorylation and dephosphorylation of individual polypeptide peaks resolved by polyacrylamide gel electrophoresis. These results are consistent with other evidence implicating nonhistone protein phosphorylation as an early event involved in the activation of transcription occurring during cell proliferation.

Independent fluctuations of cytidine and adenosine diphosphate reductase activities in cultured Chinese hamster fibroblasts

The activities of CDP and ADP reductases were determined throughout the cell cycle of Chinese hamster fibroblasts synchronized by partial deprivation of isoleucine. Both enzyme activities were increased in S phase as compared to G 1 phase. CDP reductase increased about 8-fold while ADP reductase increased about 2.5-fold. The ratio of CDP to ADP reductase was 0.26 at the G 1 and early S phases of the cell cycle; the ratio was increased to 0.83 by late S phase. Addition of actinomycin D or cycloheximide to cell cultures in G 1 phase prevented the increase of both CDP and ADP reductase activities in the latter part of the cell cycle, but the ratio of the two activities was not affected. The ratio of CDP to ADP reductase activities varied from 0.8 to 3 in different populations of exponentially growing DON cells. These results show that CDP and ADP reductase activities vary independently in growing cells. The independent variation with cell growth of CDP and ADP reductases suggests important individual functions of the deoxynucleotides during the cell cycle apart from their common role as precursors of DNA synthesis.

Regulation of fibroblast cell cycle by serum.

WHEN “normal” fibroblasts are deprived of serum they cease growing and accumulate in the G1 or G0 stage of the cell cycle1. On restoration of serum, the cells begin to enter S phase quasi-synchronously after a well defined lag period which seems to be characteristic of the cell type. Following the studies of Todaro et al.2, Burk3, and Temin4, it is widely believed that exposure to serum for only a portion of this lag period is sufficient to commit the cells irreversibly to initiate a round of DNA replication. Thus, removal of the serum after only a few hours, and before the end of the lag period, does not affect subsequent DNA synthesis in a significant proportion of the population. It has been apparent, nevertheless, that the proportion of such “committed” cells depends on the duration of the serum pulse2–5. Indeed, the relationship between the length of exposure to serum and the proportion of committed cells is approximately linear with time and (with 3T3 cells) extends beyond the end of the lag phase itself (Fig. 1). Clearly, some cells which are stimulated to initiate DNA synthesis during continuous exposure to serum are not committed by the end of the lag period, since removal of the serum at this point reduced the proportion of labelled cells, at 24 h, from 76 to 53% (Fig. 1b).

Gene activation in human diploid cells: Age-dependent modifications in the stability of messenger RNAs for nonhistone chromosomal proteins

Serum stimulation of early as well as late passages of nondividing WI-38 human diploid fibroblasts to proliferate, results in DNA synthesis beginning at 12 hours and mitosis at 20 hours. A 2-fold increase in the transcriptional activity of chromatin isolated from early and late passage WI-38 cells is evident one hour following serum stimulation. An increased synthesis and association with the genome of two defined molecular weight classes of nonhistone chromosomal proteins one hour following serum stimulation of early and late passage cells is also observed. The increased chromatin template activity and nonhistone chromosomal protein synthesis occur in early passage cells stimulated to proliferate in the presence of actinomycin D. However, when late passage WI-38 cells are stimulated in the presence of actinomycin D, increases in chromatin template activity and nonhistone chromosomal protein synthesis are not observed one hour following serum stimulation. The possibility that nonhistone chromosomal protein synthesis and activation of transcription early during the prereplicative phase of the cell cycle in early passage human diploid fibroblasts may be regulated at the translational level is discussed. Consideration is also given to the possibility that there may be an age-dependent modification in such a regulatory mechanism.

Defects in the cell cycle of fibroblasts obtained from patients with cystic fibrosis.

Some years ago, Danes and Beam (1) discovered that the fibroblasts obtained from the cutaneous biopsy of patients diagnosed as cystic fibrotic were more metachromatic than similar cells derived from patients with conditions other than cystic fibrosis. In our studies of the population doubling times of diploid human fibroblasts in culture (2), we reported that the fibroblasts derived from cutaneous biopsies of three different patients with cystic fibrosis had significantly longer population doubling times than did either normal diploid human fibroblasts or cells derived from patients with acid mucopolysac-charidoses (Hunter's and Hurler's disease). Further, we have reported (3) that cystic fibrosis-derived cutaneous fibroblasts do not make normal amounts of collagen hydroxypro-line when exposed in vitro to ascorbic acid and radioactive proline.The findings, then, of metachromasia, prolonged population doubling time and failureof collagen synthesis on the part of fibroblasts derived from the skin of patient...

Temporal variation of adenine ribonucleotides during the cell cycle of Chinese hamster fibroblasts in culture

Chinese hamster fibroblasts in monolayer culture (Don-C cell line) were synchronized by selective detachment of metaphase cells after brief treatment with colcemid. Replicate monolayer cultures were harvested at intervals after synchronization and ethanolic extracts were prepared for the determination of adenine ribonucleotides with the luciferin-luciferase assay. The level of ATP increased approx. 145% during the cell cycle, with the most rapid increase occurring during the G1 phase. One hour after synchronization (early G 1 phase), 1.3 nmoles of ATP 10 6 cells were observed; a maximum of 3.2 nmoles of ATP 10 6 cells was reached at 12 h (G 2 phase). The adenylate energy charge, ( ATP + 1 2 ADP)/( ATP + ADP + AMP) was lowest during the G 1 phase (0.7) and increased to 0.9 during the late S and G 2 phase. A slight decrease of energy charge was observed during the second mitosis.

Selective inhibition of the cell cycle of cultured human diploid fibroblasts by aminonucleoside of puromycin.

AbstractAminonucleoside (AMS) inhibited the cell cycle of human lung fibroblasts at a point in G1 phase and at another point in G2 phase. Even when this inhibition was fully established, DNA synthesis and mitosis which were in progress proceeded normally. Inhibition of RNA synthesis in the cultures preceded the effects on DNA synthesis and mitosis, but inhibition of protein synthesis could not be detected.These points of potential inhibition do not exist in the cell cycle of HeLa cell, or are not affected by aminonucleoside. Here inhibition of cell proliferation by AMS was less marked, and when inhibition eventually occurred it was not specific for any point of the cell cycle. The rate of entry of the inhibitor was similar in both types of cell.

RNA SYNTHESIS IN THE DIFFERENT PHASES OF CELL CYCLE OF CHICK EMBRYO CELLS.

RNA synthesis was studied at different phases of the cell cycle of chick embryo fibroblasts, which were synchronized by medium replacement in the confluent phase. The synthesis of DNA started at 4 hr and continued for 8 hr. RNA synthesis increased with time after medium change. The ratio of total amount of radioactivity in nuclear RNA prepared at 0, 2 and 8 hr was 1.0:1.03:5.05. The distribution of radioactive RNA in the sedimentation pattern was similar, showing remarkable incorporation in 45S region of ribosomal precursor RNA. The base composition of newly synthesized RNA, however, varied at different time intervals after medium replacement. Even within the G1 phase, the molar percentage of G and C was quite different. Treatment with actinomycin D at a concentration of 0.02 μg/ml for 1 hr specifically inhibited ribosomal RNA synthesis. At 2 hr after medium change, ribosomal and AU-rich RNA including larger than 28S were synthesized in about equal amounts.