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PubMed1991

A comparison between the epidermal regenerative responses provoked by a skin irritant and a tumor promoter using anti-BrdUrd/DNA flow cytometry.

The proliferative responses induced in hairless mouse epidermis after application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the skin irritant cantharidin were investigated. Doses known to give the same degree of hyperplasia were chosen. Mice were pulse-labeled with bromodeoxyuridine (BrdUrd) 30 min prior to or 24 h after a single application of either cantharidin or TPA, and thereafter killed at various time intervals. The basal cells were isolated from epidermis, fixed in 70% ethanol and prepared for bivariate BrdUrd/DNA flow cytometric analysis. Cells pulse-labeled in S phase 30 min prior to treatment with cantharidin or TPA were slightly delayed in their progression through S phase and temporarily blocked in G2 phase. However, they were still able to re-enter S phase 18 h later, indicating a shortening of the G1 phase. Cells pulse-labeled 24 h after treatment had a considerably reduced cell cycle time, i.e. reduced G1 transit time. Hence, the wave of cells entering S phase from 16 h after injury could be explained by an immediate reduction in G1 transit time, without assuming recruitment of temporarily resting G0/G1 cells. Although cantharidin caused the longest initial delay in cell cycle progression, the subsequent proliferative response was less pronounced than that provoked by TPA. Rapid proliferation may allow for clonal expansion of initiated cells. The higher ability of TPA to induce rapid proliferation, apparently without causing any severe initial cell damage, may thus be related to its higher tumor promoting ability.

Animals↗
PubMed1983

Density-dependent growth control of adult rat hepatocytes in primary culture.

Adult rat hepatocytes in primary culture, which show various liver functions, did not show any mitosis at confluent cell density, although they entered the S phase and remained in the G2 phase, judging by cytofluorometry, when insulin and epidermal growth factor (EGF) were added to 2-day cultures (Tomita, Y., Nakamura, T., & Ichihara, A. (1981) Exp. Cell Res. 135, 363-371). However, when the cell density was decreased by half or one third, the number of nuclei and cell number increased to 1.5-2.0 times that after culture for 35 h with insulin and EGF. Moreover, at these lower densities, DNA synthesis started much earlier, although at the usual high density DNA synthesis with these two hormones did not start until the hepatocytes had been cultured for over 40 h. These results suggest that proliferation of mature rat hepatocytes is regulated by the cell density. First, cells in G0 enter the G1 phase density-dependently; then cells in the G1 phase seem to be stimulated to enter the S phase by insulin and EGF, and a low cell density may permit cells after DNA synthesis to enter the M phase. DNA synthesis of rat hepatocyte cultures at low cell density was strongly inhibited by co-culture with a dense culture. Therefore, the density-dependent mechanism of hepatocyte proliferation seems to involve regulation by a soluble inhibitor(s) secreted by the hepatocytes into the culture medium.

Animals↗
PubMed1986

Acceptor proteins for (ADP-ribose)n in the HeLa S3 cell cycle.

The acceptor proteins for (ADP-ribose)n were investigated by using nuclei or chromosomes isolated from specific phases of the cell cycle of HeLa S3 cells. Analysis of HMG proteins and histone H1 by acetic acid/urea polyacrylamide gel electrophoresis demonstrated that the (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 increased by 12- and 5-fold, respectively, in the metaphase chromosomes as compared with that in the G1 phase cell nuclei. The degree of (ADP-ribosyl)n-ation of these proteins in the S phase cell nuclei was as low as that in G1 phase cell nuclei. In the G2 phase cell nuclei, the degrees of (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 were about 5- and 2-fold greater, respectively, as compared with that in the G1 phase cell nuclei. The (ADP-ribosyl)n-ation of HMG 1 and 2 was constant through the cell cycle except for a slight decrease in the S phase. The data may imply that the (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 is linked to chromatin structural changes in mitosis.

Cell Cycle↗
PubMed1997

Effects of olomoucine, a selective inhibitor of cyclin-dependent kinases, on cell cycle progression in human cancer cell lines.

We have studied the effects of olomoucine, a selective inhibitor of cdk2, cdc2 and MAP kinase, on the rate of proliferation and the cell cycle progression in human cancer cells in culture. Olomoucine inhibited the growth of the KB 3-1, MDA-MB-231 and Evsa-T cell lines in a concentration-dependent manner, with EC50 values of 45, 75 and 85 microM, respectively. Incubation of exponentially growing KB 3-1 cells in the presence of olomoucine led to an increased proportion of cells in G1 phase after 24 h or more of incubation. Olomoucine failed to rapidly affect the phosphorylation of the Rb tumor-supressor gene product. However, [3H]thymidine incorporation into the cell DNA was rapidly inhibited. We show that this inhibition is due, at least in part, to the diminution of thymidine entry into the cells. Surprisingly, all these cell lines, when synchronized at the G1/S interface and relaxed in the presence of olomoucine, progressed unhindered through the S phase. Under these conditions, the G2 phase transit was markedly retarded but not prevented. Insufficient permeability of the cell membrane to olomoucine may explain the low activity of the drug.

Cell Cycle↗
PubMed1987

Cytostatic effects of radiographic contrast media in synchronized cell cultures.

The cytostatic effects of conventional high osmolal ionic contrast media (meglumine-calcium metrizoate and Na-metrizoate) and new low osmolal nonionic contrast media (iohexol and iopamidol) in synchronized cell cultures were tested. The cell-cycle prolongation was most pronounced when the contrast media were added in the G1 phase, but there was also a marked effect when the contrast media were added in the S phase or late in the G2 phase. The cytostatic effect even persisted into the first cell cycle following the termination of the exposure. All four contrast media exerted effects stronger than that of equiosmolal saline. Iohexol and iopamidol produced a more severe effect than meglumine-calcium metrizoate and Nametrizoate at equal osmolality. Thus, the cytostatic effect of contrast media cannot be explained only by hypertonicity; the contrast media must have an additional specific cytostatic effect. When the cytostatic effect was related to iodine concentration, the new low osmolal nonionic contrast media influenced the cell cycle less than the conventional high osmolal ionic contrast media.

Cell Cycle↗
PubMed1983

A model for human medulloblastoma. Growth, morphology, and chromosomal analysis in vitro and in athymic mice.

A model was developed for the in vitro and in vivo study of the continuous cell line, TE-671, derived from a human medulloblastoma. TE-671 grew in vitro as sheets of uniform triangular- to spindle-shaped cells with round to oval nuclei and sparse cytoplasm. The in vitro population doubling time was 31.48 hours (h). Mean colony forming efficiency in an agarose medium was 9.0 +/- 3.0%. TE-671 grew subcutaneously in athymic mice as lobulated masses composed of sheets of small uniform cells with round hyperchromatic nuclei and scanty cytoplasm. Perivascular pseudorosettes were commonly seen in early in vivo passage. The in vivo doubling time of subcutaneous tumors was 3.17 +/- 1.02 days, with a latency to 500 mm3 of 20.66 +/- 1.48 days. The cell cycle time (Tc) was 24 h, S phase was 14 h, G1 phase was seven h, and G2 phase was three h. Intracerebral tumors grew as discrete masses composed of diffuse sheets of small anaplastic cells with frequent mitoses and perivascular pseudorosettes. Both in vitro and in vivo chromosome studies revealed modal chromosome counts in the near tetraploid range with the same marker chromosomes as described in the original report of this cell line. The in vitro and in vivo growth of TE-671 provides the best available experimental model for the analysis of the biological properties and chemosensitivity of human medulloblastoma.

Animals↗
PubMed1980

Cell-production rates estimated by the use of vincristine sulphate and flow cytometry. II. Correlation between the cell-production rates of ageing ascites tumours and the number of S phase tumour cells.

A new method for the evaluation of cell production rates combining flow cytometry (FCM) and the stathmokinetic method using vincristine sulphate (VS) has been used for the analysis of three aneuploid ascites tumours at different stages of growth. Using this technique it was possible to estimate the well-known decrease in cell production rates of ageing ascites tumours. The percentage of normal host cells in the aneuploid tumours studied was easily determined by FCM prior to the calculation of the tumour cell-production rates. A correlation was found between the percentage of tumour cells in the S phase and the tumour cell-production rate. This correlation is probably explained by the gradual transfer of proliferating cells in S phase to resting G1 and G2 phases with increasing tumour age.

Animals↗
PubMed2005

Molecular cloning of a novel mouse testis-specific spermatogenic cell apoptosis inhibitor gene mTSARG7 as a candidate oncogene.

A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and 11 introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFP-tagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(-)/mTSARG7 plasmid was constructed and introduced into GC-1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.

Amino Acid Sequence↗
PubMed1980

Tumor promoter TPA mimics irradiation effects on the cell cycle of HeLa cells.

When asynchronous and synchronous HeLa cells were incubated with small doses (10(-7) M) of tumor promoter 12 O-tetradecanoylphorbol-13-acetate (TPA), a variety of transient alterations in the replication cycle were detected within 24 hours by the use of independent methods. Especially, a delayed passage through the S phase and influences on the G2 phase resemble x-ray irradiation effects on cell cultures. None of these alterations was observed with the hyperplasiogenic but nonpromoting 4-O-methyl-TPA.

Cell Cycle↗
PubMed1997

TKRP125, a kinesin-related protein involved in the centrosome-independent organization of the cytokinetic apparatus in tobacco BY-2 cells.

Analysis of a cDNA for a 125 kDa polypeptide, previously isolated from phragmoplasts of tobacco BY-2 cells as a candidate for a plus end-directed microtubule motor, revealed this polypeptide to be a novel member of the kinesin superfamily. We named this protein TKRP125 (tobacco kinesin-related polypeptide of 125 kDa). The strong similarity between TKRP125 and members of the bimC subfamily in terms of the amino acid sequence of the amino-terminal motor domain indicated that TKRP125 belonged to the bimC subfamily. An antibody against a short peptide from the motor domain of TKRP125 inhibited the GTP- or ATP-dependent translocation of phragmoplast microtubules in membrane-permeabilized BY-2 cells, suggesting a role for TKRP125 in microtubule translocation, which is considered to be involved in the formation and/or maintenance of the bipolar structure of the phragmoplast. The expression of TKRP125 was found to be cell cycle-dependent. TKRP125 was not present in cells at the G1 phase. It began to appear at the S phase and accumulated during the G2 phase. The distribution of TKRP125 changed as the arrangement of microtubules changed with the progression of the cell cycle. TKRP125 was distributed along cortical microtubules during the S phase and along microtubules in the preprophase band and perinuclear microtubules in premitotic cells. It was also present in the nucleus in premitotic cells. In cells in M phase, TKRP125 was distributed along spindle microtubules. It accumulated at the equatorial plane of the spindle as the spindle elongated. In cytokinetic cells, TKRP125 was colocalized with phragmoplast microtubules. These observations suggest the possible involvement of TKRP125 in the cell cycle-dependent changes in arrays of microtubules, including the organization of the phragmoplast, and in the movement of chromosomes in anaphase cells.

Amino Acid Sequence↗
PubMed1975

Some evidence for replication-transcription coupling in Physarum polycephalum.

Hydroxyurea, at concentrations of 40-60 mM, selectively and effectively blocked incorporation of thymidine into DNA. Inhibition occurred within 5-10 min of application of the agent when DNA synthesis was in progress, while the onset of replication at the beginning of S-phase and DNA synthesis in G2 phase were not affected. Uridine incorporation into TCA-precipitable material, in the presence of hydroxyurea, was significantly (up to 70%) inhibited in early S-phase of the cell cycle. Selective inhibition of RNA synthesis was confirmed for RNA separated into rRNA-rich and poly(A)-rich RNA fractions and analysed by the 2 kinds of DNA-RNA hybridization reactions. Uridine incorporation into poly (A) RNA was also inhibited under conditions where cycloheximide prevented maturation of nascent DNA molecules in early S-phase. We assume that chromatin which is replicating early DNA sequences may be a more competent template for transcription.

Chromatin↗
PubMed2005

The effect of wortmannin on radiation-induced chromosome aberration formation in the radioresistant tumor cell line WiDr.

We analyzed the formation of radiation-induced chromosome aberrations in the cells of the radioresistant colon carcinoma cell line WiDr after treatment with wortmannin, an inhibitor of PI-3 kinases, including DNA-PK. Cells irradiated in G0/G1 phase with 200 kV X rays were treated with wortmannin before or after irradiation. Chromosome-type and chromatid-type aberrations were scored in metaphase cells by either Giemsa staining or FISH. Moreover, DNA-PK activity was measured in the absence and presence of wortmannin. In irradiated G0/G1-phase WiDr cells, only chromosome-type aberrations, including simple and complex exchanges and excess acentrics, were observed. After addition of 1 to 20 microM wortmannin, the formation of chromosome-type exchange aberrations was completely suppressed. The irradiated cells displayed exclusively chromatid-type aberrations including simple and complex chromatid exchanges and chromatid/isochromatid breaks. Whether the chromatid-type aberrations arise during G0/G1 as a result of homologous recombination processes coping with damaged DNA or whether DNA damage induced during G0/G1 phase persists until S and G2 phase and is then processed by homologous recombination pathways must be investigated further.

Androstadienes↗
PubMed2002

[Expression and relocation of B23 in the process of rat liver cell hyperplasia and liver regeneration].

OBJECTIVE: To study the B23 expression and relocation in the process of liver regeneration at different time periods after partial hepatectomy (PH) on healthy rats, and to study the feasibility of using B23 as a proliferation marker. METHODS: Eighteen rats underwent partial hepatectomy (PH). The regenerative liver tissues were obtained at day 1, 2, 3, 4,and 7 after PH. The expression of B23 in hepatocytes of the regenerative tissues was measured using monoclonal antibody against nucleophosmin/B23 by Western blot and immunohistochemistry. The expression of proliferate cell nuclear antigen (PCNA) in these cells were measured using monoclonal antibody against PCNA by both methods for comparison with that of B23. RESULTS: Expression of B23 in the steady state of liver cells before PH was hardly detected by both methods. The increased expression of B23 in the regenerative liver cells was quantitatively changed like a parabolic curve at day 1 to days 7 after PH, with the peak expression of B23 at day 3 after PH. This quantitative variation of B23 expression was similar to that of PCNA expression. The variation of location of B23 in the regenerative liver cells was observed from nucleolus (G1-S phase) to nucleoplasm (S-G2 phase) and to cytoplasm and mitotic chromosome (M phase). CONCLUSION: B23 expression is quantitatively increased and moves in location in the process of liver regeneration after PH. B23 may be regarded as a proliferation marker in liver regeneration and liver cell hyperplasia.

Animals↗
PubMed1992

[Synthesis and supramolecular organization of components of the extracellular matrix by fibroblasts cultured in a collagen lattice].

Fibroblasts cultured in a collagen gel contract and organize the gel into a three-dimensional matrix of collagen fibers. Within this matrix, the fibroblast cell cycle is blocked at the G1 phase but also at the G2 phase. The fibroblasts produce the main extracellular matrix components (collagen, noncollagen proteins, glycosaminoglycans), although in small amounts. Studies using this in vitro model with radiolabeled precursor substances (14C proline, 3H glucosamine) demonstrated production of supermolecular complexes which resisted to proteolysis by pepsin and collagenase and could not be isolated by saline precipitation. Polyclonal antibodies identified type I collagen, type VI collagen and fibronectin in this coherent supermolecular structure. The presence of glycosaminoglycans was also demonstrated by alcian blue precipitation.

Cells, Cultured↗
PubMed2005

Molecular cloning and primary functional analysis of a novel human testis-specific gene.

In this study, a new data mining tool called Digital Differential Display (DDD) from the NCBI was used to predict testis-specific expressed genes from the expressed sequence tag (EST) database. DDD (digital differential display) was performed between nine testis libraries and seventy libraries derived from other tissues. We identified a new contig of ESTs (HS. 326528) which was from testis libraries. To validate the use of bioinformatic approaches in gene discovery, the ESTs (HS. 326528), which were predicted to be testis-specific, were chosen for further study. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis of matched sets of cDNAs from testis and other tissues indicated that the ESTs were specifically expressed in testis. This result was further validated by multi-tissue Northern blot. The full-length cDNA encompassing the entire open reading frame was cloned and, in view of its apparent specificity to testis, the gene was termed homo sapiens spermatogenesis-related gene 8---SRG8 (GenBank accession number: AY489187). The gene whose full cDNA length is 1 044 bp containing 3 exons and 2 introns is located in human chromosome 15q26.2, the cDNA encodes a novel protein of 105 amino acides with a theoretical molecular weight of 11.7 kD and isoelectric point of 10.09 which shares no significant homology with any known proteins in database. Real time PCR analysis of testis of different developmental periods revealed that SRG8 gene is significantly expressed in adult testis. The green fluorescent protein produced by pEGFP-C3/SRG8 was detected in the nucleus of HeLa cells after 24 h post-transfection. Cell cycle analysis showed that SRG8 can accelerate HeLa cells to traverse the S-phase and enter the G2-phase compared with the control without transfection of SRG8, which suggested that this gene plays an important role in the development of testis. The discovery of SRG8 showed that DDD combined with experiments is a feasible, time-saving strategy to identify new candidate genes for testis-specific development

Adult↗
PubMed1991

Cell cycle-regulated and proliferation stimulus-responsive genes.

We have reviewed here genes whose expression may vary during the "cell cycle" and we discuss the underlying regulatory mechanisms. Given a correlation between the cell cycle and expression of a particular gene, the question arises whether that gene regulates the cycle, whether the cycle regulates that gene, or whether the correlation is simply the consequence that both the cell cycle and that gene respond to the same signal(s). Gene expression is regulated at diverse levels, and the relative importance of regulation at these different levels depends on which version of the cell cycle one has in mind; depending upon the context, the concept of the (higher eukaryote) cell cycle has a number of different operational meanings. Thus the first few divisions of the fertilized egg consist of successive S and M phases, with insignificant G1 and G2 phases, regulated entirely at the translational and post-translational level by the phosphorylation/dephosphorylation of p34cdc2 and the synthesis/degradation of one or more cyclins-keyed perhaps to the cytoplasm/nucleoplasm ratio and the completion of DNA replication. In contrast, cells stimulated to exit quiescence, (G0), require new gene transcription and changes in the post-transcriptional control of gene expression. Cells proliferating in a constant environment proceed directly from mitosis into G1 and are less dependent on (but not independent of) new transcription; here controls at the post-transcriptional and post-translational levels are more pronounced. In addition to regulation by p34cdc2, input from cell-specific growth factors or other extracellular signals is essential for most untransformed cells to continue through the cycle. Many transformed cells in contrast do not require exogenous signals and are altered in the way that key regulatory genes (e.g., p53, RB) are controlled. While cells of many lower eukaryotes appear capable of an indefinite number of cell cycles, the typical higher eukaryotic cell appears to have a limit on this number--untransformed, nonestablished vertebrate cells are usually mortal. For unknown reasons, established cell lines and certain embryonic or stem cells under the right conditions, are immortal and capable of indefinite proliferation. Apparently, the price paid to construct a differentiated multicellular organism is a limit on the number of cell divisions that the constituent somatic cells are capable of undergoing.

Amino Acid Sequence↗
PubMed1989

Differential [32P]-labeling of chemically induced stress proteins in S-phase mouse lymphoma cells.

A portion of our research involves the investigation of biomarkers as preclinical indicators of toxic stress. Stemming from this effort, we report three unique proteins that phosphorylate and synthesize during the S-phase of lymphoma cells following chemical insult. Mouse lymphoma cell nuclei were physically sorted (using a fluorescence activated cell sorter) from partitions of the cell cycle and specific nuclear proteins in each partition were examined by gel microelectrophoresis. The changes in [32P]-incorporation by stress proteins (SPs) were examined in each of seven partitions following administration of sodium arsenite. Four SPs [80,000-84,000 relative molecular mass (Mr)], designated 'c','b','x', and 'y', underwent significant alterations in [32P]-labeling and each exhibited varying degrees of differential [32P]-incorporation in partition 3 of G1 phase, all five partitions of S-phase, and partition 1 of G2 phase of the cell cycle. Three other predominantly S-phase SPs (designated S1, S2 and S3) were phosphorylated after sodium arsenite treatment. Stress protein S1 was labeled exclusively in S-phase, while proteins S2 and S3 were labeled only in partition 3 of G1 and S-phase. Stress protein S1 possessed an identical isoelectric point, molecular mass, distribution of polypeptide fragments and immunochemical determinants as S-phase SPs found previously in mouse spleen (X') and mouse liver (LP-S). The identical biochemical characteristics of these three S-phase (SPs), found in diverse tissue types, suggest they are homologous.

Animals↗
PubMed1989

Energy supply of the mitotic cell cycle and the Na+/H+-antiport in ascites tumors.

The activation of Na+ transport is due to the exchange of protons formed via glucose conversion into lactate for Na+, i.e., to the stimulation of the Na+/H+-antiport. Experimental results and theoretical calculations suggest that in glucose-containing medium the Na+ transport increases from 0.75 to 1.78 pmol/hour per cell. The permeability of plasma membranes for K+ increases 2.75 fold, while the passive flux of Na+ diminishes. The intensity of O2 adsorption by ascites tumor cells does not practically depend on the monovalent cation concentration gradient between the cells and the culture medium, whereas the rate of glycolysis decreases simultaneously with the diminution of the concentration gradient. In synchronized cultures at the beginning of the mitotic cycle, the bulk of ATP resynthesized via glycolysis is utilized for the synthesis of biopolymers, whereas that at the end of the S-phase and in the G2-phase is utilized for cation transport across plasma membranes. From 35 to 100% of the whole amount of ATP resynthesized via glycolysis is utilized for transport purposes. It is concluded that the observed increase in the Na+/K+ ratio in ascites tumor cells is connected with their enhanced ability to synthesize lactic acid. Presumably, glycolysis is one of the regulatory mechanisms of intracellular ratios of monovalent cations.

Animals↗