Effects of ionizing- and UV B-radiation on proteins controlling cell cycle progression in human cells: comparison of the MCF-7 adenocarcinoma and the SCL-2 squamous cell carcinoma cell line.

MCF-7 and SCL-2 cells were irradiated with UV B-radiation or with 137Cs gamma-radiation, in order to investigate cell cycle checkpoint control mechanisms. Effects of both qualities of radiation were investigated for the two cell lines in regard to p53 protein levels, and alterations in Cdk1 (cyclin dependent kinase 1) and Cdk2 phosphorylation were monitored. SCL-2 cells constitutively overexpressed a form of p53 protein whose abundance remained unchanged after irradiation, whereas MCF-7 cells expressed wild type p53 whose abundance increased after irradiation. Accordingly, MCF-7 cells showed a strong G1 phase arrest, whereas SCL-2 cells were only delayed in S phase (after UV B-irradiation) and arrested in G2 phase (after gamma-irradiation and UV B-irradiation), as monitored by flow cytometry. In MCF-7 cells increased p53 levels were observed for up to 30 h after gamma-irradiation and up to 20 h after UV B-irradiation. Only in SCL-2 cells was there a significant radiation induced inactivation of Cdk1 by hyperphosphorylation. This effect was prevented by culturing cells in the presence of caffeine after irradiation. After UV B-irradiation the inactivation of Cdk1 was less pronounced and only partially diminished in the presence of caffeine. No alteration in Cdk2 phosphorylation was observed after irradiation in either cell line.

UV-B-induced cell cycle perturbations, micronucleus induction, and modulation by caffeine in human keratinocytes.

UV-B-induced perturbations of cell cycle progression in asynchronous human keratinocytes were analysed during two cell cycles with respect to their cell cycle stage at the time of irradiation using BrdUrd/Hoechst flow cytometry. Exponentially growing SCL-2-keratinocytes exposed to UV-B radiation showed a short delay in G1-phase exit and were blocked in the S and G2/M phases of the first cell cycle. UV-A wavelengths did not show any detectable effect on cell cycle progression. In contrast, 137Cs-irradiation of these cells induced a temporary G2 block only. Micronucleus frequency increased in gamma-irradiated cells as soon as the cells started to divide and reached a plateau when most of the cells had divided. Continuous treatment with caffeine starting immediately after 137Cs gamma-irradiation prevented accumulation of cells in G2 phase, but did not influence the frequency of micronuclei. In UV-B-irradiated keratinocytes, however, the damage-induced cell cycle perturbations were merely reduced by caffeine, but not eliminated. Compared with gamma-irradiation a moderate induction of micronuclei was observed in UV-B-irradiated cells. Caffeine, however, potentiated the induction of micronuclei by UV-B. These different effects on cell cycle kinetics and micronucleus induction indicate different mechanisms of DNA damage caused by UV-B- and gamma-irradiation that may be repaired through different pathways.

Induction of cell cycle perturbations by the tear gas 2-chlorobenzylidene malonitrile in synchronously and asynchronously proliferating mammalian cells.

The effects of the tear gas 2-chlorobenzylidene malonitrile (CS) on mammalian cell proliferation were studied in detail using bromodeoxyuridine/Hoechst flow cytometry. In synchronized (G0/G1-phase) Chinese hamster embryo (CHE) cells, exposure to CS (60 microM) caused a permanent arrest in the G0/G1 phase in 50% of the cells and a delayed G0/G1 phase exit. In asynchronously growing CHE cells, the CS-induced cell kinetic perturbations varied with the cell cycle stage during treatment. While G1-phase cells showed a delayed progression through S and G2/M phases, S-phase cells were mainly inhibited in the G2/M compartment of the first cell cycle. In contrast, CS-treated, asynchronous, amniotic fluid-derived, fibroblast-like (AFFL) cells exhibited a prolonged transit through the G2/M phase of the first cell cycle regardless of the cell cycle stage during treatment. This indicates that the induced cytotoxicity of CS is a function of both the cell cycle phase and the particular type of cells.

Flow cytometric analysis of bromodeoxyuridine-induced micronuclei.

The effects of DNA substitution by the thymidine analogue 5-bromodeoxyuridine (BrdU) on cell cycle progression and micronucleus induction were studied in different mammalian cell cultures. Simultaneous flow cytometric measurements of DNA content and side scatter of nuclei in Chinese hamster embryo (CHE) cells revealed a concentration-dependent temporary block in the G2/M phase of the first cell cycle. NIH 3T3 cells and human amniotic fluid fibroblast-like cells, on the contrary, did not show any cell cycle disturbances in the presence of BrdU. Micronucleus frequency increased as soon as CHE cells started to divide and reached a plateau when all cells have divided. The height of this plateau was almost equal for 60 and 100 microM BrdU. This saturation of micronucleus induction was due to a saturation of BrdU incorporation into DNA already at a doses of 60 microM as shown by the BrdU/Hoechst quenching technique. Indirect immunofluorescent staining of kinetochores with CREST antibodies revealed that nearly all BrdU-induced micronuclei were kinetochore-negative suggesting the presence of acentric chromosome fragments in these micronuclei. DNA distributions of micronuclei measured by flow cytometry showed several peaks representing micronuclei which contain DNA fragments of defined sizes induced by non-random breakage of chromosomes 1 and X as verified by flow karyotyping and C-banding.

Induction of replicative senescence by 5-azacytidine: fundamental cell kinetic differences between human diploid fibroblasts and NIH-3T3 cells.

To analyse the putative role of methylation of cytosine residues in the nuclear DNA as a regulatory step during cellular ageing, we incubated ageing human amniotic fluid derived fibroblast-like cells and non-ageing NIH-3T3 cells with 5-azacytidine. BrdUrd/Hoechst and acridine orange (AO) flow cytometry was used to compare the effects of the base analogue on cell proliferation and cell differentiation. In NIH-3T3 cultures, 96h exposures to 4 microM 5-azacytidine caused diminished cell proliferation due to cell arrest in the G1 compartments of the second and third cell cycles of serum stimulated cells. The exit from the G0/G1 compartment was not affected. The 5-azacytidine induced cell kinetic disturbances were unstable in NIH-3T3 cultures, such that pre-treated cells reverted to normal cell cycle transit within 2-3 days after termination of treatment. In contrast, 5-azacytidine pre-treated amniotic fluid derived fibroblast-like cell cultures showed persistently elevated G2 phase arrests and delayed G0/G1 phase exit kinetics, which explain the premature cessation of proliferation observed in these primary cultures. In both cell systems, 5-azacytidine exposed cultures showed elevated numbers of G1 phase cells with increased RNA content as revealed by AO flow cytometry. Again, this effect was reversible in NIH-3T3 cells but not in amniotic fluid derived fibroblast-like cells. These contrasting responses to 5-azacytidine are likely to reflect intrinsic differences in methylation patterns or de novo methylase activity between ageing cell strains and non-ageing cell lines.

Characterization of embryonic antigens in the plasma of developing chick embryos.

Two prominent alpha-fetoproteins are found in chick embryo plasma up to and slightly beyond hatching. Only antisera against chick embryo plasma resolve them. alpha3-fetoprotein is a lipoprotein, the first fetoprotein thus described. It probably functions to transport lipid from the yolk to the embryo. The other fetoprotein (alpha4) is a major glycoprotein. Both are made by the liver and yolk sac. From 9 days onward, albumin is the principal export protein of the liver; at 7 days, when it first appears in the plasma, the yolk sac, but not the liver, makes it. The reverse situation occurs at 9 days. These facts suggest a humoral relationship between the yolk sac and liver. The embryo synthesizes every plasma protein including fetoproteins and "adult" proteins (prealbumin, albumin, and transferrin). No "cold" or unlabeled proteins are seen.