Effect of ultraviolet (UV) A, UVB or ionizing radiation on the cell cycle of human melanoma cells.

BACKGROUND: One important component of the cellular response to irradiation is the activation of cell cycle checkpoints. It is known that both ultraviolet (UV) radiation and ionizing radiation (IR) can activate checkpoints at transitions from G(1) to S phase, from G(2) phase to mitosis and during DNA replication. OBJECTIVES: To evaluate the effects of irradiation with different wavelengths on cell cycle alterations. METHODS: p53-deficient IPC-298 melanoma cells were irradiated with 10 J cm(-2) UVA, 40 mJ cm(-2) UVB, or with 7.5 Gy IR. Cell cycle effects were then determined by DNA/5-bromodeoxyuridine dual-parameter flow cytometry. RESULTS: IPC-298 cells irradiated in G(1) with UVA were not arrested at the G(1)/S transition, but at the G(2)/M transition. Despite p53 deficiency, the cells showed a G(1) arrest after UVB exposure. Furthermore, IR did not affect G(1) or S phase, but induced G(2) phase arrest. Hence, the effects of UVA, but not of UVB, on the cell cycle in p53-deficient melanoma cells are comparable with those of IR. CONCLUSIONS: UVA and IR induce radical-mediated strand breaks and DNA lesions, and UVB essentially induces thymine dimers that lead to excision repair-related strand breaks. Different cell cycle effects may be a consequence of different types of DNA damage. The results showed that UVB-irradiated p53-deficient cells are arrested in G(1). Irradiation with the solar radiation component UVB can therefore result in a beneficial retardation of tumour promotion in human skin carrying p53-mutated cell clones.

Protein kinase inhibitors modulate time-dependent effects of UV and ionizing irradiation on ICAM-1 expression on human hepatoma cells.

PURPOSE: To investigate modulation of the expression of the adhesion protein ICAM-1 by UV and ionizing irradiation. MATERIALS AND METHODS: HepG2 hepatoma cells were irradiated in vitro with UVB (20 mJ cm(-2)) or X-rays (5 Gy), respectively. Gene expression of ICAM-1 after irradiation was quantified by RT-PCR. Cell surface density of ICAM-1 was determined by flow cytometry. Protein or lipid kinase inhibitors were used to clarify radiation-induced transduction pathways that control ICAM-1 expression. Immuno-electron microscopy and dot-blot analysis were used to examine localization of ICAM-1. RESULTS: The study showed time-dependent effects of ionizing and UV irradiation on ICAM-1 expression of HepG2 cells. After an immediate transient decrease of ICAM-1 cell surface expression within minutes to hours, ICAM-1 expression increased up to 1.35-fold over normal level at 48 h post-irradiation. Irradiation caused ICAM-1 to become internalized into lysosomes. Additionally, ICAM-1 together with parts of the cell were pinched off. Finally, ICAM-1 levels were down- and up-regulated by decreased or increased gene expression. The early decrease of ICAM-1 expression could be blocked by a potent PKC inhibitor (BisX), whereas the increase of ICAM-1 after 24 h was prevented by addition of the p38 MAP kinase inhibitor SB 203580. CONCLUSION: The data suggest that ICAM-1 expression is modulated by UV, as well as ionizing radiation, in a time-dependent way involving PKC and p38 MAP kinase pathways.

Proliferation kinetics and prognosis in gastric cancer after resection.

The influence of proliferation and proliferation kinetics on prognosis in gastric cancer after complete resection are controversial. In a prospective study we investigated the tumour specimens of 111 patients after resection of gastric cancer, who received 200 mg intravenous (i.v.) bromodeoxyuridine (BrdU) pre-operatively. The following biological parameters were analysed in the tumour tissue using flow-cytometry: DNA ploidy, proportion of S-phase cells, BrdU labelling index (LI), DNA synthesis time (T(s)), potential tumour doubling time (T(pot)), proliferating cell nuclear antigen (PCNA) and Ki-67 LI. The median follow-up time was 40 months (range 19-62 months). Besides the established pathohistological prognostic factors, univariate analysis revealed a prognostic influence on survival for BrdU LI, T(pot) and the proportion of S-phase cells. By multivariate Cox analysis of the completely resected cases, only tumour stage and T(pot) had a significant, independent influence on survival. By classification and regression trees (CART) analysis, resection status, tumour stage and T(pot) defined risk groups with significantly different outcomes. A short T(pot) was a predictor of better survival in stage I, II and IIIA tumours. Ploidy and the other investigated proliferation-related parameters failed to demonstrate any influence on prognosis after resection of gastric cancer.

Early changes in cell cycle kinetics after ionizing irradiation below 1 GY.

The present study describes a procedure for quantifying cell cycle alterations within 15 h after radiation with doses below 1 Gy. For detection and assessment of the relevant changes, 5-bromo-2'-deoxyuridine (BrdUrd)-labelling and flow cytometry were used. Using this approach, as early as 6 h after exposure of radiosensitive leukemic HL-60 cells, radiation-induced changes in cell cycle progression could be measured even with radiation doses as low as 0.25 Gy. As a result, a method to define transition rates for a single cell cycle phase or from one phase to another is described. Even minor changes can be described. Moreover, the BrdUrd assay allows for discrimination of cells irradiated in different phases of the cell cycle. Thus, it is possible to follow the progression in the cell cycle of cells either irradiated in G1, S, or G2 + M phase, respectively. Radiation effects on single cell cycle phases can be analysed separately. A detailed evaluation of the cellular response to irradiation regarding dose, time, and effect is described. The value of cell cycle parameters for assessment of various biological indicators of radiation effect is discussed.

Evaluation of a modified micronucleus assay.

The relationship between ionizing radiation-induced cell killing and DNA damage measured by the micronucleus assay was determined in three established cell lines (L929, HL-60, and Chang). Our data revealed a dose-dependent increase of cells bearing multiple micronuclei. Cells with the same number of micronuclei were counted separately up to 50 h after irradiation. The counts of these subsets showed a parallel increase and decrease throughout the study. In order to transform the peak of the micronucleus frequency, occurring over only a brief time period into a less time dependent value, we calculated ratios between the different subsets of micronucleated cells. These ratios converged to values which were almost constant beyond 30 h after irradiation. The values showed correlations with cell survival (clonogenic assay) and radiation dose which were comparable with the correlations with the peak of the micronucleus frequency (maximum micronucleus yield) when utilizing the conventional evaluation of the micronucleus assay performed without cytochalasin B. This means that large-scale time kinetics and additional drugs like cytochalasin B can be avoided by changing the evaluation procedure of the conventional micronucleus assay. The modified assay described in this manuscript revealed apoptosis-induced limitations as recently detected for the maximum micronucleus yield assay.

Early and late G2 arrest of cells undergoing radiation-induced apoptosis or micronucleation.

Conventional flow cytometric DNA measurements combined with the microscopic detection of cells in the late G2 phase of the cell cycle (characterized by the occurrence of paired kinetochores) enabled us to differentiate and to quantify early and late G2 cells 0-40 h after irradiation using a radioresistant (L929) and a radiosensitive (HL-60) cell line. This approach provided us with (1) a new kind of G2 arrest characteristic revealing changes in the G2 phase which can not be detected by flow cytometric DNA measurements, (2) cell line dependent differences in the radiation-induced transition through G2, accompanied by the occurrence of micronucleation and apoptosis, and (3) the characterization of apoptotic cells occurring probably during early G2 and bearing a rapidly reduced number of kinetochores in contrast to mitotic cells, suggesting processes different from those that operate in mitosis.

Correlation of micronucleus and apoptosis assays with reproductive cell death.

The relationship between ionizing radiation-induced cell killing and DNA damage measured by the micronucleus and apoptosis assays was determined in three established cell lines (L929, HL-60, and Chang). Irradiation experiments revealed a dose-dependent increase of micronucleated cells until a certain dose was reached. Above this dose no further increase of the micronucleus frequency was observed, but in HL-60 and Chang cells additional DNA fragmentation was detected by morphological criteria, characteristic of apoptosis. This change was detected at different doses for the three cell lines examined, suggesting the existence of a cell-type-dependent upper limit for the employment of the micronucleus assay. However, the sum of both kinds of cellular DNA damage (e.g. micronucleation and morphological-like apoptosis) led to a significant cell-type-independent correlation with cell survival, even above the dose where micronuclei levels saturated. Therefore, a total cell damage assay, involving the inclusion of micronuclei and morphological-like apoptotic events, should be considered when evaluating the use of a predictor assay for ionizing radiation-induced cell killing, especially in conditions when apoptosis (-like) processes may occur.

Cell kinetic analysis after irradiation in L929- and LLC-MK2-cells by a BrdU/DNA assay.

Exponentially growing L929- and LLC-MK2-cells were X-irradiated in vitro. Irradiation-induced cell cycle kinetic effects were examined by the calculation of the cell number doubling time (TD), the duration of the cell cycle phases by the BrdU/DNA assay and the number of micronuclei. The number of cells arrested in G2/M and the duration of the delay are dose-related. The effect of irradiation on the duration of cell cycle phases was estimated by following the BrdU-labeled cells through the cell cycle. With increasing radiation doses the duration of the G2/M-phase increases whereas G1- and S-phase show only slight variations. Due to the problems involved in evaluation of radiation effects on the cell cycle a comparison with other methods proves the BrdU/DNA-assay to be a valuable instrument in those experiments. The micronucleus frequency is dependent on irradiation dose. However, after 7.52 Gy the number of micronuclei increases, whereas no cells in G1-phase and no increase of cell number could be detected, indicating a mechanism of micronucleus formation that is not linked with mitosis.