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PubMed1975

In vitro epidermal cell proliferation in rat skin plugs.

Full-thickness skin plugs from immature and adult rats have been shown to incorporate -3H-thymidine in vitro in a semisynchronous fashion for up to 54 hr. DNA synthesis is minimal at 16 hr post sacrifice but increases again at 24 and 48 hr, and cells in S phase at 48 hr have passed through at least one mitosis in vitro (between 32 and 39 hr). Advantage can be taken of this semisynchronous burst of DNA synthetic activity to test the effects of potential inhibitors of skin cell proliferation, and to determine at which phase of the cell cyclic these inhibitors act. High concentrations of epinephrine or isoproterenol (10--5 M) are required to cause inhibition but the effect is specific for the G2 phase of the cell cycle. Propranolol does not demonstrate beta-antagonism in this system, since it is a very potent inhibitor acting in G1 phase. Dibutyryl cycle AMP and theophylline have two effects: one is a specific inhibitory action on cells in the G2 phase and the second is a short-term action limiting thymidine uptake by cells in S phase without affecting the transition of cells from G1 to S.

Aminophylline↗
PubMed1982

Ultrastructural changes in HeLa cells following treatment with the antineoplastic agent cis-acid.

The ultrastructure of HeLa cells arrested in the G2 phase of the cell cycle by the antineoplastic agent cis-acid was studied and compared with that of normal G2-phase-synchronized cells. The results demonstrate that the chromatin of drug-treated cells was fragmented and clumped, the membranous structures had degenerated or were destroyed and the polyribosomes were decreased. The mitochondria were swollen and vacuolated, suggesting that energy production might have been impaired.

Cell Cycle↗
PubMed1975

[Effect of methotrexate on the leukemic cell cycle in mouse ascitic leukemia L1210].

In cells of L1210 ascite leukemia cells, methotrexate inhibited H3-thymidine incorporation, blocked shortly (during 4 hours) the G1 leads to S transition, and did not affect cells in G2-phase or in the late S phase. Almost half a cell population was degenerated and cells in S- and G1-phases were affected in equal proportion. This may suggest that methotrexate is not S-phase specific for cells of leukemia L1210. A simultaneous administration of vinblastine increases the antitumour effect of methotrexate. Cells in G2-phase constitute, presumably, a significant proportion of cells recovered after methotrexate administration. A comparison of the data obtained with literature evidence shows that in the sensitive (leukemia L1210) and resistant (acute mieloid leukemia of man) forms of leukemia, methotrexate affects cells that are in S-phase, whereas cells being in G1-phase are affected only when the sensitive tumours are treated.

Animals↗
PubMedDate not supplied

Malignant melanoma in children and congenital melanocytic nevi: DNA content and cell cycle analysis by flow cytometry.

Malignant melanoma (MM) in children, although a rare neoplasm, can occur within a preexisting congenital melanocytic nevus (CMN). All the potential risk factors for this phenomenon are not well known, but increases in S phase and G2 + M phase of cell cycle, DNA aneuploidy, and cell cycle abnormalities in precursor lesions might be among the risk factors. Using paraffin-embedded tissue, we performed a retrospective analysis of DNA content, aneuploidy, and cell cycle by flow cytometry. Two groups of patients were analyzed: 28 children with CMN who did not developed MM, and 6 patients who further developed MM. In this second group, three patients had four biopsies done before the appearance of MM and in two patients biopsies were done after the appearance of MM. All CMN not associated with MM exhibited diploid cells only, their S phase was 11.5% (+/- 3.8), and their G2 + M phase was 2.5% (+/- 2.2). Among those patients who developed MM, 3/6 had an S phase > 15.5 and a G2 + M phase > 2.3 prior to the appearance of MM. Two out of six patients had a tetraploid DNA when MM developed and died with a disseminated MM. They had an S phase > 15.5 and their G2 + M phase was > 2.5. We propose that evaluation of DNA content and cell cycle by flow cytometry is a useful method to supplement biopsy findings in children with CMN who have lesions suspicious of developing a MM.

Adolescent↗
PubMedDate not supplied

Cell kinetics (SAMBA 200) of estradiol stimulated long-term phenol red withdrawn cultured breast cancer cells.

When estradiol stimulation was performed on hormone-responsive MCF-7 human breast cancer cells maintained in phenol red-containing medium until 24h before experiments, the cell number and the cell surface transferrin receptor level, an early marker of estradiol stimulation, were strongly increased. In contrast, similar treatment performed on MCF-7 cells grown in phenol red-free medium up to 12 months before experiments induced no stimulating effect on the cell number, although transferrin receptors were still enhanced. Since the appearance of transferrin receptors occurs before the cells begin DNA synthesis in late G1 phase, we assumed that the discrepancy between cell counts and cell surface transferrin receptors might involve cell kinetic perturbations. The proportion of cells in the (S + G2) phases and the duration of the cell cycle phases were analysed using the SAMBA 200 cell image processor. The data presented in this study failed to indicate any block in the cell cycle and the duration of the S and G2 phases were found to be unchanged. The lack of effect of estradiol stimulation on the growth of MCF-7 cells maintained several months without phenol red is not thus a consequence of cell cycle perturbations.

Breast Neoplasms↗
PubMed1996

Temporal patterns of gene expression of G1-S cyclins and cdks during the first and second mitotic cell cycles in mouse embryos.

Cell-cycle progression in somatic cells is regulated by a family of cyclins and cyclindependent kinases (cdks) that form specific complexes as a function of cell-cycle progression. However, the transcript abundance of G1-S cyclins and cdks during the meiotic and mitotic cell cycles of mammalian embryos has not been previously reported. Using a reverse transcription-polymerase chain reaction (PCR) assay that detects changes in either mRNA abundance or polyadenylation state, we examined the relative levels of gene expression for the G1-S cyclins and cdks, as well as for p21, p27, and the retinoblastoma (Rb) gene in mouse oocytes, metaphase II-arrested eggs, and 1-2-cell embryos. The PCR products for cyclins D1, D3, and A, as well as cdk4, p21, and Rb, displayed similar levels in meiotically incompetent and competent oocytes, as well as in metaphase II-arrested eggs. The levels of PCR products for cyclin D2, p27, and two forms of cdk2 were similar in meiotically incompetent and competent oocytes but decreased during oocyte maturation. Finally, the level of PCR products for cyclin E and cdk2 gradually decreased during the progression from meiotically incompetent oocytes to metaphase II-arrested eggs. When the levels of PCR products for the G1-S regulatory genes were evaluated during the first and second mitotic cell cycles, four main patterns were found: 1) steady levels for cyclin A; 2) steady levels followed by a 2-3-fold increase during the G2 phase of the second mitotic cell cycle for cyclins D1, E, cdk2, and p21; 3) a transient increase during the S and/or G2 phases of the first mitotic cell cycle for p27, cyclin D3, and the two forms of cdk2; and 4) higher levels during the first cell cycle and then a decrease with lower levels during the second mitotic cell cycle for cyclin D2 and Rb. cdk4 expression displayed a combination of patterns 2 and 3. The increase in the amount of PCR product for the cdk4 gene during the first mitotic cell cycle was due to polyadenylation, whereas the increase in the amount of PCR product for cdk4, cdk2, and cyclins D1 and E in the second mitotic cell cycle was a product of activation of the embryonic genome.

Amanitins↗
PubMed2002

Aphidicolin induces 6-thioguanine resistant mutants in human diploid fibroblasts.

Cytotoxic and mutagenic effects of aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta, were studied in human diploid VH-10 fibroblasts. The cells were treated (2 or 4h) with APC at concentration ranges of 10-40 microM. The effect of APC on cell survival after 4 h treatment was significantly higher than after 2 h treatment. The mutagenicity of APC was investigated at the HPRT locus, and the frequency of HPRT mutants was estimated by selection in medium containing 6-thioguanine (6-TG). Treatment of fibroblast cells with 20 microM of APC for 2 or 4 h resulted approximately in 5 or 10 times increase of 6-TG resistant mutant frequencies, respectively, compared to untreated control cells. The cell cycle analyses performed during the expression time (9-12 days) have shown that after 2 and 4h treatment with APC the cells were blocked in G2 phase during the majority of the expression period, compared to control cells. Four days after the treatment, the amount of cells in G2 phase increased about two-fold (28.6-31.8% compared to 13.5% in the untreated cells). The mode of cell death during the expression time was via necrosis, rather than apoptosis, which was demonstrated by fluorescein-diacetate (FDA)-staining and terminal dUTP nick end labeling (TUNEL)-method.

Antimetabolites, Antineoplastic↗
PubMed2003

[Screening of new binding partners of CIKS with yeast two-hybrid system].

The NF-kappaB transcription factor plays important regulatory roles in inflammation, apoptosis, immune and stress responses. IkappaB kinase (IKK) composed of two catalytic subunits and a regulator subunit, acts as a key component of NF-kappaB activation pathway through phosphorylation of IkappaB, the inhibitor of NF-kappaB. CIKS (connection to IKK and SAPK), a newly identified cellular protein, is involved in NF-kappaB and JNK activation. Although it has been known that CIKS interacts with IKK complex, and activates both IKK and SAPK when overexpressed; the underling mechanisms are poorly understood. To better understand the physiological roles of CIKS, we have screened human HeLa MATCHMAKER cDNA library for new binding partners of CIKS by using the yeast two-hybrid system with truncated CIKS (151-574) as the bait. The yeast strain AH109 was sequentially transformed with the bait plasmid and the library. The transformants were screened on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal)selective plates. Positive clones were restreaked on SD(-Leu/-Trp / + X-alpha-gal)plates three times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. After 3 screenings on SD(-Leu/-Trp / + X-alpha-gal), the positive clones were further verified on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal) plates. The inserts in AD/library plasmids were amplified by PCR and PCR products were characterized by Hae III digestion to eliminate the duplicates. After screening in selective plates, the positive AD/library plasmids were rescued via transformation of E. coli HB101 and the interactions of CIKS (151-574) with positive AD/library plasmids were further assessed by yeast two-hybrid analysis. Finally, the DNA sequences of the positive AD/library plasmids were determined and BLAST analysis against the databases was performed. The BLAST results indicate that the inserts in the positive plasmids encode RIKEN cDNA 473340F03, PLAC8, CD27BP (Siva-1), CDC5L, SnRNP smB, and DVL2. CDC5L is a key component of the multi-protein complex essential for the formation of pre-mRNA splicing product and is not required for spliceosome assembly. A role for CDC5L in the cell division cycle has been precious suggested as its overexpression of this protein in mammalian cells leads to a shortening G2 phase of the cell cycle, and a negative-dominant mutant of CDC5L lacking the N-terminal activation domain delays the G2 phase cell's entry into the mitosis. It has been reported that SnRNP smB participates in pre-mRNA splicing and CD27BP (Siva-1) binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis and regulates T cell homeostasis. The functions of RIKEN cDNA 473340F03, PLAC8 and DVL2 are unknown. It has been suggested that CIKS functions as a critical component for cross-talk between NF-kappaB and JNK signaling pathways. IKK subunits, which have been demonstrated to interact with CIKS, were not shown up in this experiment. We speculate that the truncated CIKS (151-574) bait may not contain the binding domain that mediates the interaction of IKK subunits with CIKS. Taken together, the above results suggest that CIKS may play a role in cell regulation through interacting with various cellular proteins. Further investigations are required to characterize these interactions.

Adaptor Proteins, Signal Transducing↗
PubMed1983

The cell cycle dependence of thermotolerance. I. CHO cells heated at 42 degrees C.

We examined the dependence of heat killing and thermotolerance on the position and progression of Chinese hamster ovary (CHO) cells in the cell cycle. We measured cell cycle perturbations and survival of asynchronous and synchronized G1-, S-, and G2-phase cells resulting from continuous heating at 42.0 degrees C for up to 80 hr. Thermotolerance under these conditions was transient in nature, was dependent on the position of cells in the cell cycle, and occurred concurrently with a heat-induced delay of progression of G1- and G2-phase cells. When G1 cells were heated, survival decreased to 25% after 4 hr, at which time the thermotolerance was expressed. For G2 cells survival decreased initially at the same rate (T0 congruent to 3 hr) but thermotolerance was not expressed until approximately 12 hr, at which time the survival was 4%. The rate of decrease in survival was much more rapid for cells heated in mid-S phase (T0 congruent to 0.5 hr), and these cells did not express thermotolerance at a measurable level. Concurrent with the expression of thermotolerance, the progression of cells heated in G1 and G2 was delayed. Following the expression of tolerance, progression resumed at a rate approximately equal to the rate of decrease in survival of the G1 population. Cells heated in mid-S phase continued to progress through the cell cycle until they reached G2, where they were also delayed.

Animals↗
PubMed1994

Comparison of the effects of genistein and amsacrine on leukemia cell proliferation.

Genistein is an inhibitor of the enzymes protein tyrosine kinase and topoisomerase-II. It induces G2-phase arrest in human Jurkat and murine P388 leukemia cells at concentrations at which it is also cytotoxic. The effects of genistein have been investigated on Jurkat and P388 leukemia sublines that manifest multidrug resistance. Cells that possess altered topoisomerase-II activity ("atypical" multidrug resistance) are resistant to both the G2 phase-arresting and cytotoxic effects of genistein. The ability of genistein to impede progression through the cell cycle and kill cells is similar to that of amsacrine, a classical topoisomerase-II poison. This result identifies topoisomerase-II rather than tyrosine kinase activity as the target of genistein-mediated cytotoxicity and G2-phase arrest.

Amsacrine↗
PubMed1975

Proliferation of ineffective erythropoiesis with nuclear abnormalities and megaloblastoid appearance in preleukaemia.

Erythropoiesis of two patients in the early stage of acute leukaemia and two patients with refractory anaemia and hypercellular bone marrow (preleukaemia?) was studied with the cytophotometric-autoradiographic method. Megaloblastoid erythroblasts show a decreased proliferative activity in comparison to the morphologically normal cells and are in the maturation stage of E4 (early polychromatic normoblasts) mainly in the G1-phase; they are therefore largely comparable to the megaloblastoid erythroblasts in erythroleukaemia. Erythroblasts in preleukaemia with nuclear abnormalities are found in a high percentage in the G2-phase or are unlabelled with a DNA content of between diploid and tetraploid value. They show a similar proliferative behaviour to the megaloblasts in pernicious anaemia. Early polychromatic erythroblasts arrested in G2-phase can differentiate without mitosis into tetraploid mature erythroblasts (E5). They can divide elsewhere endomitotically, produce binucleated E5 or take up the DNA synthesis and become polyploid. E4 with nuclear abnormalities do not proliferate and are mainly found in the premitotic phase.

Acute Disease↗
PubMed1995

Repression of cell cycle progression by antisense HMG2 RNA.

The expression level of the HMG2 gene during the cell cycle of rat fibroblast cell lines was analyzed. Northern analysis demonstrated that the level of HMG2 mRNA was markedly enhanced in the post-S phase and reached a maximum at G2 phase, suggesting that expression of the HMG2 gene is not coupled with DNA synthesis. Progression of the cell cycle of COS-1 cells was repressed during G1 to S phase by expression of the antisense RNA for HMG2, resulting in a decrease of cell growth. These results suggest that the fluctuation of the HMG2 message during the cell cycle is not a consequence of but a prerequisite for cell proliferation.

Animals↗
PubMed1993

Characterization of the first cell cycle in human zygotes: implications for cryopreservation.

OBJECTIVE: To study the temporal pattern of deoxyribonucleic acid (DNA) synthesis leading up to the first mitotic division in human one-cell stage zygotes. SETTING: In vitro fertilization program of a university hospital. PATIENTS: Couples donating spare embryos before the existence of an embryo freezing program. DESIGN: Incorporation of 3H-thymidine was examined between 9 to 27 hours after insemination in 72 untransferred human zygotes containing two pronuclei. Microscopic observations on an additional 978 transferred zygotes extended the 3H-thymidine incorporation data. RESULTS: The first thymidine incorporation was seen 9 to 10 hours after insemination, and the heaviest incorporation occurred between 11 and 13 hours. Subsequently, thymidine labeling declined, and chromosomal condensation and cell division first occurred approximately 19 to 20 hours. At 20 hours after insemination, 89% of the zygotes had two visible pronuclei (PN). In contrast, by 24 hours, 41% had no visible PN, whereas 5% had cleaved to the two-cell stage. By 27 hours, 38% had cleaved to the two-cell stage, and only 25% still had two visible PN. CONCLUSIONS: These results suggest that the DNA-synthetic, S-phase of the human zygote is initiated by approximately 9 to 10 hours after insemination and is completed approximately 3 to 5 hours later. The duration of G2 phase and mitosis is in the range of 4 to 6 and 3 to 3.5 hours, respectively. Because zygotes may be particularly susceptible to damage during the S phase of the cell cycle, these findings suggest that the optimal time for freezing of human zygotes may be approximately 20 to 22 hours after insemination when the majority of zygotes should have entered the G2 phase, before pronuclear dissolution and chromosome condensation.

Adult↗
PubMed2002

Vincristine induces somatic segregation, via mitotic crossing-over, in diploid cells of Aspergillus nidulans.

Vincristine is an alkaloid widely used as an antineoplastic agent. In eukaryotic cells the drug causes blockage in the G2 phase of the cell cycle and an increase in the frequency of sister chromatid exchanges. Due to the fact that germinating Aspergillus nidulans cells spend most of their cycle in G2 phase, they provide an excellent system for the study of mitotic crossing-over. Taking into account that mitotic crossing-over occurs during G2 period, the evaluation of recombinagenic and aneugenic potential of vincristine is provided with regard to two diploid strains of A. nidulans: a wild strain (uvsH+//uvsH+) and a defective one in DNA repair (uvsH//uvsH). Drug toxicity and its effect on the asexual cycle of A. nidulans has been evaluated as well. Treatment of both strains with vincristine did not change colony growth in the culture, however cytological analyses showed aberrant conidiophores. Recombinagenic potential of vincristine was evaluated by induction of gene homozygosis originally present in heterozygosity diploid strains (Homozygotization Index). Results show that vincristine induces mitotic crossing-over and higher frequency of aneuploid mitotic segregants. The results also show the recombinagenic and aneuploidogenic potential of vincristine and suggest its participation in the induction of secondary malignancies.

Aneuploidy↗
PubMed1992

[Flow cytometric BrdU/DNA assay for anticancer agent sensitivity test].

The significance of BrdU/DNA double staining method using flow cytometry as a chemosensitivity test of anticancer agents is herein reported. Experimentally, CDDP, ADM and MMC yielded a significant increase of G2 phase fraction at 24, 48 and 72 hours, and caused a significant decrease of BrdU labeling index at 48 hours. The changes of G2 phase fraction and BrdU labeling index were correlated well with the result of colony forming test, and, therefore, were considered as a useful index of lethal effect of anticancer agents. For the clinical application, enzymatic preparation and discontinuous Ficoll density gradient method might be advantageous to collect viable tumor cells from solid specimen. Flow cytometric BrdU/DNA assay can be clinically applied as a chemosensitivity test in selecting effective anticancer drugs.

Antineoplastic Agents↗
PubMed1990

[An experimental study of anticancer agent sensitivity test in human gastric cancer cell lines by flow cytometry].

The purpose of this study is to assess the lethal and kinetic effects of CDDP, ADM, MMC and 5FU on human gastric cancer cell lines, MKN28, MKN45 and KATO III. The lethal effect was examined by growth inhibition test and colony forming test. The DNA content and DNA synthesis rate of individual cells were simultaneously measured by DNA/BrdU double staining method. In growth inhibition test, MKN45 was sensitive to CDDP, and all cell lines were sensitive to ADM, MMC and 5FU. On the other hand, in colony forming test, these cell lines were sensitive to all drugs. In the cell kinetics, CDDP, ADM and MMC yielded a significant increase of G2 phase fraction at 24, 48 and 72 hours, and caused a significant decrease of BrdU labeling index at 48 hours. The changes of G2 phase fraction and BrdU labeling index were correlated well to the lethal effect of CDDP, ADM and MMC. However, 5FU did not cause these changes to the cell lines employed in the cell kinetic study. Therefore, it was suggested that these results of the cell kinetics might be applied to anticancer agent sensitivity test by selecting adequate anticancer drugs.

Antibodies, Monoclonal↗
PubMedDate not supplied

Epidermal cell kinetics in hairless mice after bleomycin. II. Perturbations after multiple doses.

Bleomycin (BLM) at doses of 0.1 mg/animal was injected into hairless mice every 4 hours for 4 days. Epidermal cell cycle phase distributions were determined by flow cytofluorometric DNA measurements. Labeling indices and mean grain counts were determined by means of autoradiography, and mitotic rates were determined by demecolcine arrest of metaphases. A small, partially reversible accumulation of cells with G2 phase DNA content was observed subsequent to BLM administration. Most of the cells accumulated in G2 phase, however, seemed to stop proliferation and die in this phase. Thus, BLM does not fulfill the criteria for a good synchronizing agent in the hairless mouse epidermis. Flow cytofluorometry alone cannot determine whether piling up of cells in a particular cell cycle phase is true synchrony or an accumulation of lethally damaged cells. Several cell kinetic methods should be used to demonstrate real synchrony.

Animals↗
PubMed2001

Transcription activating property of autoantigen SG2NA and modulating effect of WD-40 repeats.

Autoantibodies to intracellular proteins have been detected in sera of patients with various forms of cancer. Nuclear autoantigen SG2NA (S, G2 phase nuclear antigen) was isolated using autoantibodies from a patient with bladder and lung cancers and its expression is enhanced in the S and G2 phases of the cell cycle. Molecular cloning revealed that the C-terminal region of SG2NA contains six WD-40 repeats, motifs that are present in a large family of proteins with diverse functions. We show that the N-terminal region of SG2NA (aa 1-391) acted as a strong transcriptional activator in both yeast and mammalian cells. In contrast, the C-terminal WD-40 repeats had an inhibitory effect on transcription activation. We performed molecular swapping experiments by substituting the WD-40 repeats of SG2NA with those of yeast Met30 and Cdc4 and showed that the WD-40 regions from either Met30 or Cdc4 were capable of reproducing transcription repression function. The SG2NA WD-40 repeats were also able to repress basal level transcription and transactivation function of a GAL4-VP16 chimera. These observations suggest that some WD-40 repeats may have, as one of their functions, a negative regulatory role in the biological activities of their own and perhaps other proteins.

Animals↗