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PubMed1996

Translocation of the retinoblastoma gene product during mitosis.

This study reports the immunocytochemical localization of the retinoblastoma gene product within synchronized normal human keratinocytes. Data suggest that mitotic spindles function in the transport of the retinoblastoma tumor suppressor gene product during cell division. A diffuse anti-pRB reactivity was detected within the nuclei of G1-phase keratinocytes, although staining was not evident within the nucleoli. During S-phase and G2-phase the anti-pRB reactivity was localized to discrete regions within the nuclear compartment. The anti-pRB reactivity of M-phase cells was localized to the mitotic spindles and microtubule nucleation centers. Immunoprecipitation and Western blotting of the pRB antigen from synchronized keratinocytes showed that the apparent polypeptide molecular weight of pRB increased from 105 kDa during G1-phase to 115 kDa during M-phase. Immunoprecipitation of the pRB antigen from mitotic spindles resulted in the coprecipitation of two polypeptides with apparent polypeptide molecular weights of 115 and 50 kDa. Western blotting of the immunoprecipitates from purified keratinocyte mitotic spindles showed that beta-tubulin was the 50-kDa polypeptide associated with hyperphosphorylated pRB.

Biological Transport↗
PubMed1998

On the interpretation of the shortening of the G1-phase by overexpression of cyclins in mammalian cells.

Increasing the concentration of cyclins in mammalian cells leads to a shortening of the G1-phase of the division cycle. This observation has been interpreted as indicating that these cyclins act during, and are rate limiting for, passage through the G1-phase. Here it is argued that it is not possible to interpret experiments involving cyclin overexpression-induced changes in the lengths of individual cell cycle phases without considering changes in the overall cellular growth rate. A rigorous reanalysis of these experiments demonstrates that the results are consistent with the proposal that the shortening of the G1-phase is merely due to an increase in the rate of mass synthesis in all phases of the cell cycle. Increased cyclin concentrations leads to a faster rate of mass synthesis and a concomitant shortening of the G1-phase. Cyclins can also affect the length of the S- and G2-phases, which leads to the observed shortening of the G1-phase. Thus, the experiments on cyclin overexpression and their effect on G1-phase length cannot be used to support the proposal that cyclins act specifically during the G1-phase of the division cycle.

Animals↗
PubMed1998

Replication of nuclei from cycling and quiescent mammalian cells in 6-DMAP-treated Xenopus egg extract.

Nuclear membrane permeabilization is required for replication of quiescent (G0) cell nuclei in Xenopus egg extract. We now demonstrate that establishment of replication competence in G0 nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesis is that G0 nuclei lose the license to replicate following growth arrest and that this positive activity is required for relicensing DNA for replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prepare egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), G2-phase (unlicensed), and G0 were permeabilized and assayed for replication in 6-DMAP-treated and untreated extracts supplemented with [alpha-32P]dATP or biotinylated-dUTP. Very little radioactivity was incorporated into nascent DNA in each nuclear population; however, nearly all nuclei in each population incorporated biotin in 6-DMAP extract. The pattern of biotin incorporation within these nuclei was strikingly similar to the punctate pattern observed within nuclei incubated in aphidicolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substitution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins which escape 6-DMAP inhibition. We conclude that 6-DMAP-treated egg extract cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the "licensed state" of G0 nuclei using the assays described here.

3T3 Cells↗
PubMed1975

Heparin effects on cultured mammalian cells.

Plasmacytoma cells exposed to heparin exhibited zeiotic blebs in the G1 phase, S phase, and early G2 phase. Zeiosis was not seen in mitotic cells. This heparin effect was reversible. Also fibroblasts were sensitive to heparin. After trypsinization of fibroblasts, heparin produced large non-reversible zeiotic blebs in the cells, except in those in mitosis. The primary target for heparin is apparently to be sought among components of the cellular periphery.

Animals↗
PubMed1992

DNA synthesis in the early embryo of the nematode Ascaris suum.

We have used microspectrofluorimetry to measure the rate of DNA synthesis in the first two embryonic cell cycles of the parasitic nematode Ascaris suum. The S-phase of the early Ascaris cell cycles occupies at most 1 hr; G2 phase is prominent and occupies approximately 11 hr; no G1 phase could be detected. These results contrast with our previous measurements made with embryos of the free-living nematode Caenorhabditis elegans, in which the earliest cell cycles consist of simple alternations between S and M phases.

Animals↗
PubMed1987

Increased density of class I major histocompatibility complex antigens and decreased density of T-cell differentiation antigens in the early stages of T-cell activation.

Major histocompatibility complex (MHC) antigens and T-cell differentiation antigens on activated T cells play a central role in T-cell interactions. In the present study, we have analyzed time courses of both quantity and density of the T-cell differentiation antigens, CD3 (T3), CD4 (T4), and CD8 (T8), as well as MHC antigens, on the cell surface of T cells, and made correlated measurements of DNA content with the surface antigen quantity as well with RNA content and cell size following activation of T cells by phytohemagglutinin. We found that the quantity and density of class I MHC antigens increase within 24 hr following activation and then decrease, while the quantity and density of the T-cell differentiation antigens decrease within 24 hr following activation, which suggests that T-cell recognition involving class I MHC gene products occurs at an early stage of T-cell activation. Class II MHC antigens can be detected on more than 40% of T cells as the expression of the T-cell differentiation antigens increases much later in the response. Cell cycle studies demonstrated that the density of class I MHC, CD3, CD4, and CD8 antigens was greater in G0/G1 phase cells than G2 phase cells at all times tested during T-cell activation. Our findings suggest that T cells demonstrate a differential regulation in expression of MHC and T-cell differentiation antigens following activation which may reflect their role in cellular interactions.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1999

Circadian variation in the expression of cell-cycle proteins in human oral epithelium.

At the tissue level, there is experimental and clinical data to suggest a cytokinetic coordination of the cell cycle with a greater proportion of cycling cells entering S-phase and mitosis at specific times of the day. The association of certain cell-cycle proteins with defined events in the cell cycle is well established and may be used to study the timing of cell-cycle phases over 24 hours. In this study oral mucosal biopsies were obtained from six normal human volunteers at 4-hour intervals, six times over 24 hours. Using immunohistochemistry, the number of positive cells expressing the proteins p53, cyclin-E, cyclin-A, cyclin-B1, and Ki-67 was determined for each biopsy and expressed as the number of positive cells per mm of basement membrane. We found a statistically significant circadian variation in the nuclear expression of all of these proteins with the high point of expression for p53 at 10:56 hours, cyclin-E at 14:59 hours, cyclin-A at 16:09 hours, cyclin-B1 at 21:13 hours, and Ki-67 at 02:50 hours. The circadian variation in the nuclear expression of cyclins-E (G1/S phase), -A (G2-phase), and -B1 (M-phase) with a normal physiological progression over time suggests a statistically significant circadian variation in oral epithelial cell proliferation. The finding of a circadian variation in the nuclear expression of p53 protein corresponding to late G1 is novel. This information has clinical implications regarding the timing of chemotherapy and radiotherapy.

Adult↗
PubMed2000

Cell cycle regulation in Schizosaccharomyces pombe.

Cdc2, a cyclin-dependent kinase, controls cell cycle progression in fission yeast. New details of Cdc2 regulation and function have been uncovered in recent studies. These studies involve cyclins that associate with Cdc2 in G1-phase and the proteins that regulate inhibitory phosphorylation of Cdc2 during S-phase and G2-phase. Recent investigations have also provided a better understanding of proteins that regulate DNA replication and that are directly or indirectly controlled by Cdc2.

CDC2 Protein Kinase↗
PubMed2004

A novel immunohistochemical method for estimating cell cycle phase distribution in ovarian serous neoplasms: implications for the histopathological assessment of paraffin-embedded specimens.

We have investigated whether immunohistochemical markers can identify differences in cell cycle phase distribution in ovarian serous neoplasms, including borderline tumours of different grades. Sections of normal ovary (n=18), serous cystadenoma (n=21), borderline serous tumours (n=21) and serous cystadenocarcinoma (n=15) were analysed by immunohistochemistry using markers of cell cycle entry (Mcm-2) and cell cycle phase, including cyclin D1 (mid-to-late G1), cyclin A (S phase), cyclin B1 (G2 phase) and phosphohistone H3 (mitosis). Double-labelling confocal microscopy confirmed marker phase specificity and phase estimations were corroborated by flow cytometry. On progression from normal ovary through serous cystadenoma and borderline tumours to cystadenocarcinomas, expression of Mcm-2 (P<0.0001), cyclin D1 (P=0.002), cyclin A (P<0.0001), cyclin B1 (P<0.0001) and phosphohistone H3 (P<0.0001) increased, paralleled by an increase in the S-phase fraction (cyclin A : Mcm-2 ratio; P=0.002). Borderline tumours of increasing grade also showed increased Mcm-2 and cyclin A expression, together with an increase in the S-phase fraction. Immunohistochemistry can be used to estimate cell cycle phase distribution in ovarian serous neoplasms, giving results similar to flow cytometric analysis and enabling direct assessment of tumour heterogeneity. Immunohistochemical estimates of the S-phase fraction may identify serous borderline tumours likely to exhibit malignant progression and/or select serous cystadenocarcinomas likely to respond to adjuvant therapy.

Biomarkers, Tumor↗
PubMed1996

Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase.

Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the G2 phase and mitosis to highest abundance in the G1 phase. Since this was rather surprising, we verified it by quantifying phase-specific mRNA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the G1 phase that may be related to cell cycle control.

Cell Cycle↗
PubMed1979

Endocytosis and chloroquine accumulation during the cell cycle of hepatoma cells in culture.

Variations of endocytic and of lysosomal functions during the cell cycle have been investigated in synchronized hepatoma cells (derived from Morris hepatoma 7288c) by following the cellular uptake of horseradish peroxidase, dextran (mol wt. 70,000), and chloroquine. Cell fractionation and cytochemistry show that in asynchronously growing cells exposed for 1 h to 5 mg/ml peroxidase, the bulk of the enzyme taken up by the cells is found in phagosomes. By using the same experimental system with synchronized HTC cells, large variations of endocytosis are observed during the cell cycle. Peroxidase uptake is lowest during mitosis, increases 5--10 times during G1 phase, reaches a plateau, and finally decreases at the end of S phase and during G2 phase. A similar evolution is observed for the uptake of dextran (0.5 or 1 mg/ml), but it is likely that a significant part of the polysaccharide is still associated with the pericellular surface after 1 h. Moreover, dextran is transferred more slowly than peroxidase to lysosomes. Cellular accumulation of chloroquine is related to intralysosomal pH or to the buffering capacity of lysosomes. Our results show that this drug is taken up more rapidly during G1 and S phases while the rate of accumulation is lowest in mitotic cells. The results are discussed in relation to the modifications of the physical properties of lysosomes during the cell cycle observed previously by cell fractionation and electron microsocopy, and to the possible role of lysosomes in the initiation of mitosis. Cyclic changes of endocytosis in actively dividing cells are demonstrated by our observations and may induce large differences in the uptake rate of extracellular substances.

Animals↗
PubMed1996

Antitumor effect of hydrolyzed anthocyanin from grape rinds and red rice.

When Balb/C mice that were fed with red glutinous rice, white ordinary rice, or commercially available standard food were inoculated with syngeneic Meth/A lymphoma cells i.p., the group fed the red rice survived longer than the other two groups. In order to determine if the anthocyanins contained in red-coloured seeds and fruit rinds were responsible for the tumor suppressive effect, we added anthocyanins extracted from grape rinds and glutinous red rice to petri dishes that had been seeded with HCT-15 cells. After 4 days of culture, cell counts were made. These anthocyanins were not effective in suppressing the tumor growth. However, anthocyanidins, which were generated by keeping anthocyanins in 5 to 6% HC1 methanol for 5 to 6 hours, were effective in the suppression of tumor growth. Flowcytometric histograms were made after 2 days of culture with these bioflavonoids. The histogram of cells treated with crude anthocyanin was similar to that of the control. Hydrolyzed anthocyanidins gave the elevation of S phase, suggesting a block in the step from S-phase to G2-phase. It seemed that the anthocyanidins contained in the grape rinds and red rice were effective on the suppression of cell growth as observed previously for anthocyanins extracted from the petals of higher plants.

Animals↗
PubMed1998

Antitumor effect of gold as revealed by growth suppression of cultured cancer cells.

Gold agents have been widely used for the treatment of rheumatoid arthritis. We studied the growth inhibiting effect of such an agent on malignant cells in vitro. HCT-15, AGS cells derived from a human malignancy, and Meth/A cells from a malignant lymphoma of Balb/C mice were cultured separately with gold agent at concentrations of 2 micrograms/ml. Four days after the cultures had been incubated in a 5% CO2 incubator at 37 degrees C, cell counts were made; and significance of differences was analyzed by Student's t test. Additionally, HCT-15 cells were cultured with gold for two days, and then the cells were analyzed by flow cytometry. The growth of HCT-15, AGS, and Meth/A cells was suppressed by gold. Fifty percent suppression was observed at a concentration between 50 micrograms/ml and 10 micrograms/ml for HCT-15 cells, between 125 micrograms/ml and 50 micrograms/ml for AGS cells, and between 125 micrograms/ml and 50 micrograms/ml for Meth/A cells. Fifty percent suppression of HCT-15 cell growth by cisplatinum was found between 50 micrograms/ml and 10 micrograms/ml. Flow cytometric findings showed a significant rise in the tetraploid peak, a mild rise in the resion between diploid and tetraploid peaks, and an increase in cells with a ploidy greater than four. These data suggest that gold blocks the S phase, G2 to M phase, and M phase as well. To observe the cytotoxicity of gold, each of 10 of 4 week-old Balb/C mice was injected s.c. at a dose of 10 mg/kg or 2 mg/kg every other day for a total of 3 injections, or was administered the gold at 30 mg/kg/day p.o. for 10 days. All mice were still alive after 20 days of observation. Cisplatinum at a dose of 10 mg/kg was also injected s.c. one time into each of 10 mice, and 60% of the animals died within 10 days after the injection.

Animals↗
PubMed1982

Effect of folate deficiency on the reproductive organs of female rhesus monkeys: a cytomorphological and cytokinetic study.

The effect of folate deficiency was studied on the cytomorphology and the kinetics of proliferation of ovarian granulosa cells, and the epithelial cells lining the uterus, cervix and vagina of six sexually mature female rhesus monkeys. The cellular kinetics were study by in vivo labeling with [3H]thymidine, followed by autoradiography of the histological sections of tissue biopsies taken at various time intervals and labeled mitosis curves. The folate-deficient monkeys showed an increased number of atresic and cystic ovarian follicles with depletion of granulosa cells. There was also a significant impairment of the proliferation kinetics of granulosa cells as indicated by decreased [3H]thymidine labeling index (L.I.), and marked prolongation of the pre- DNA-synthetic time (G1 phase); the DNA-synthetic (S phase) and postsynthetic (G2 phase) periods were only mildly prolonged. These changes may have impaired the ovarian synthesis of the steroidal hormones, estrogen and progesterone. The cervico-vaginal epithelium showed megaloblastosis, multinucleation and impairment of orderly proliferation and maturation. Less pronounced changes were also seen in the uterus. The cytomorphological and functional disturbances in the uterus, cervix and vagina may be partly secondary to those in the ovaries, and partly due to the depletion of their essential folate coenzymes.

Animals↗
PubMed1976

Susceptibility of fetal lung to transplacental 1-ethyl-1-nitrosourea: its relation to epithelial proliferation.

The relationship between the age-dependence of the number of lung adenomas produced by exposure of fetal mouse lung to 1-ethyl-1-nitrosourea (ENU) and changes in the number of epithelial cells within the population that are in the cell cycle on different gestation days was studied. The epithelial cells lining peripheral tubules of growing lung were counted by morphometric methods, and, based on previous studies, their distribution in various phases of the cell cycle was estimated. The curve of multiplicity of adenomas was biphasic, with the maximum number in fetuses exposed on days 16 and 17 and the minimum in those exposed on day 19. The number of epithelial cells in the proliferative cycle followed a similar pattern. A linear regression curve relating the number of cells in the cell cycle to the number of adenomas produced 16 weeks after exposure to ENU showed a correlation coefficient of 0.82. The number of adenomas was more highly correlated with the number of cells in the G1 phase at carcinogen exposure than with the number of cells in either the S phase or the G2 phase and mitosis. The incidence of adenomas per 10(6) cells in cycle was greater when fetuses were exposed to ENU on day 15, and tumors induced on this day grew to a significantly larger size by 16 weeks after birth than did those induced on days 17, 18, or 19. Factors of possible significance in this age-related response may have been the high growth fraction in fetal lung epithelium on day 15, the short cell-cycle time, and the longer period available for cell proliferation between tumor initiation and birth.

Adenoma↗
PubMed1985

Purification and characterization of S-adenosylhomocysteine hydrolase from mouse mastocytoma P-815 cells. Evidence for cell cycle-specific fluctuation of the enzyme activity.

S-Adenosylhomocysteine hydrolase [EC 3.3.1.1] was purified to electrophoretic homogeneity from mastocytoma P-815 cells. The purified enzyme had a molecular weight of 190,000, as estimated by Sephadex G-200 chromatography, and a monomer molecular weight of 45,000, as determined by polyacrylamide gel electrophoresis in the presence of SDS. The Km value for adenosine was 0.29 microM and the Vmax value 4.5 mumol S-adenosylhomocysteine X min-1 X mg-1 in the synthetic reaction, while the Km value for S-adenosylhomocysteine was 0.77 microM and the Vmax 0.48 mumol adenosine X min-1 X mg-1 in the hydrolytic reaction. The purified enzyme also had one binding site for adenosine (KD = 2.61 X 10(-7) M) and one for cAMP (KD = 1.6 X 10(-7) M). Using rabbit antiserum raised against the purified enzyme, it was shown that the enzyme activity and enzyme synthesis fluctuated during the cell cycle of mastocytoma cells, reaching the maximum levels as the cells changed from the G1/S phase to the G2 phase.

Adenosine↗
PubMed1999

Molecular markers and cell cycle inhibitors show the importance of cell cycle progression in nematode-induced galls and syncytia.

Root knot and cyst nematodes induce large multinucleated cells, designated giant cells and syncytia, respectively, in plant roots. We have used molecular markers to study cell cycle progression in these specialized feeding cells. In situ hybridization with two cyclin-dependent kinases and two cyclins showed that these genes were induced very early in galls and syncytia and that the feeding cells progressed through the G2 phase. By using cell cycle blockers, DNA synthesis and progression through the G2 phase, or mitosis, were shown to be essential for gall and syncytium establishment. When mitosis was blocked, further gall development was arrested. This result demonstrates that cycles of endoreduplication or other methods of DNA amplification are insufficient to drive giant cell expansion. On the other hand, syncytium development was much less affected by a mitotic block; however, syncytium expansion was inhibited.

Animals↗
PubMed1975

Existence of two chalone-like substances in intestinal extract from the adult newt, inhibiting embryonic intestinal cell proliferation.

The inhibiting effect of tissue extract from fully differentiated intestinal mucosa of adult animals on proliferation kinetics of exponentially growing embryonic epithelial gut cell populations was studied in the newt Pleurodeles waltlii. Crude extract was fractionated by G-200 Sephadex chromatography and the effect of fractions on cell proliferation was studied using both mitotic index and 3-H-thymidine incorporation methods. The inhibitions we obtained were then displayed by means of cytophotometric study of age distribution of intestinal gut cells around the cell cycle, measuring the Feulgen-DNA content. The results revealed the presence of two chalone-like substances in the intestine of adults. One (factor 1) is characterized by a molecular weight of between 120,000 and 150,000 and inhibits the cell cycle at the end of the G1 phase, the other (factor 2) is characterized by a molecular weight lower than 2000 and inhibits the cell cycle in the course of the G2 phase. The cells delayed in the G2 phase escape from inhibition but the cells delayed in the G1 phase do not, although availability time of both factor 1 and factor 2 is about 12 hr. It is thus thought that cells prevented from dividing in G1 phase are indefinitely delayed in this phase and possibly differentiate.

Animals↗