Cell cycle kinetics of irradiated synchronous and asynchronous tumor cells with DNA distribution analysis and BrdUrd-Hoechst 33258-technique.

Radiation-induced progression delay in S-phase and G2-block, both depending on time of irradiation in the cell cycle, were measured in synchronized Ehrlich ascites tumor cells in vitro using flow cytometric analysis of DNA content in single cells. Similar results could be obtained by the BrdUrd-Hoechst 33258 technique after irradiating asynchronous cells. BrdUrd replaces thymidine in newly synthesized DNA which is not stainable by the thymidine-specific dye Hoechst 33258. The temporal development of the fluorescence distributions after addition of BrdUrd to the medium has been measured in the flow cytometer. In asynchronous cells the mean durations of the cell cycle phases and their perturbations after irradiation could be calculated, and the data agreed with the results in synchronized cells. Division delay afer irradiation consists of a small progression delay in S-phase and a block in G2-phase. Both effects depend on time of irradiation during the cell cycle with delay in S-phase increasing in irradiated late S-cells and G2-block increasing in irradiated G2-cells. Data from both types of experiments were used with a simple model describing the fractions of cells in the cell cycle phases after irradiation had been applied

In vitro antitumour activity of cyanidin-3-O-beta-glucopyranoside.

Background: Little is currently known regarding the cancer preventive potential of cyanidin-3-O-b-glucopyranoside (Cy-g) apart from its antioxidant activity. Methods: We tested Cy-g on Jurkat and HL-60 leukemia cell lines and, to help elucidate whether the effects of Cy-g are specific for cancer cells, also on normal T lymphocytes. Results: Cy-g-induced apoptosis on all three cell systems, and this indicated that Cy-g was not selective towards leukemia cells. Moreover, Cy-g caused HL-60 differentiation. The induction of apoptosis and cytodifferentiation involved different proteins, thus suggesting that Cy-g-induced apoptosis and cytodifferentiation are two distinct events. Conclusions: Although in vitro, our findings indicate that Cy-g possess some interesting biological properties that should encourage further investigation as regards its chemotherapeutic potential

Induction of apoptosis in two human leukemia cell lines as well as differentiation in human promyelocytic cells by cyanidin-3-O-betaglucopyranoside.

Little is known about the potentially chemopreventive mechanisms of anthocyanins apart from their antioxidant activity. We investigated the in vitro capacity of the anthocyanin cyanidin-3-O-\u3b2-glucopyranoside (Cy-g) to induce apoptosis in T-lymphoblastoid, as well as apoptosis and differentiation in HL-60 promyelocytic cells. Although Cy-g-induced apoptosis (as well as necrosis) in the two systems, HL-60 cells were much less sensitive than T-lymphoblastoid cells. Moreover, treatment of HL-60 cells with Cy-g caused differentiation into macrophage-like cells and granulocytes. Concerning the mechanism of action, the induction of apoptosis in Jurkat T cells can be explained by a modulation of p53 and bax protein expression. At the molecular level, the induction of apoptosis and cytodifferentiation in HL-60 cells involved different proteins, thus suggesting that the effects of Cy-g on apoptosis and cytodifferentiation induction are two distinct events. These interesting biological properties should encourage further investigation into the chemopreventive and/or chemotherapeutic potential of Cy-g

In vitro anticancer activity of cyanidin-3-O-beta-glucopyranoside: effects on transformed and non-transformed T lymphocytes.

BACKGROUND: Little is known regarding the potentially chemopreventive activity of cyanidin-3-O-beta-glucopyranoside (Cy-g), the main anthocyanin present in the juice of pigmented oranges, apart from its antioxidant activity. After excluding a potential genotoxicity of Cy-g, its ability to induce apoptosis on transformed and normal T cells was analysed. In order to delineate the events leading to apoptosis, the expression of different proteins, known to be involved in apoptosis, was also measured. MATERIALS AND METHODS: The evaluation of genotoxicity was performed by the micronucleus test. Flow cytometry was used for the analysis of apoptotic cells and proteins involved in the modulation of apoptosis. RESULTS: Cy-g was nongenotoxic. Moreover, it induced apoptosis in both cell systems, modulated by an increase of p53 and bax proteins. CONCLUSION: These interesting biological properties should encourage further studies into the chemopreventive potential of Cy-g. Nevertheless, its activity in normal T cells underlines the need for extensive toxicological investigation

Sulforaphane increases the efficacy of doxorubicin in mouse fibroblasts characterized by p53 mutations.

One novel strategy for increasing cancer chemotherapy efficacy and reversing chemoresistance involves co-administration of natural chemopreventive compounds alongside standard chemotherapeutic protocols. Sulforaphane is a particularly promising chemopreventive agent, which has been shown to exert proapoptotic effects on tumor cells containing p53 mutations. The p53(Ser220) mutation has been implicated in reduced efficacy and drug resistance in the context of osteosarcomas and breast tumors treated with doxorubicin-based protocols. We investigated the effects of a combination of doxorubicin and sulforaphane on cell viability and apoptosis induction in fibroblasts characterized by different p53 status (p53 wild-type, p53 knock-out, and p53(Ser220) mutation), and identified some of the molecular pathways triggered by the drug combination. Very high concentrations of doxorubicin were necessary to decrease the viability of p53(Ser220) and p53 knock-out (but not wild-type) cells. Treatment of p53(Ser220) and p53 knock-out cells with doxorubicin did not induce apoptosis, also at very high concentrations (10muM). Sulforaphane restored chemosensitivity and induced apoptosis in doxorubicin-resistant p53(Ser220) and p53 knock-out cells, irrespective of p53 status. The induction of apoptosis was caspase-3 dependent and caspase-8 independent. Bongkrekic acid, a mitochondrial membrane stabilizer, partially prevented the effects of doxorubicin plus sulforaphane on mitochondrial permeability but was unable to prevent the induction of apoptosis. N-acetyl-cysteine, a glutathione precursor, blocked the induction of apoptosis by doxorubicin plus sulforaphane. Considering the negligible safety profile of sulforaphane, our findings could prompt innovative clinical studies designed to investigate whether its coadministration can enhance the efficacy of doxorubicin-based regimens

Automated evaluation of frequencies of aneuploid sperm by laser-scanning cytometry (LSC).

Background: Laser-scanning cytometry (LSC) allows fast automated scoring of fluorescence signals directly on microscopic slides. Frequencies of spontaneous aneuploidies in murine and human sperm were evaluated by using this new LSC technique. Rapid detection may be of great interest in reproductive toxicology, as certain chemicals act as aneugens during meiosis, increasing the production of aneuploid germ cells.Materials and Methods: Selected chromosomes were detected by using fluorescence in situ hybridization (FISH) and fluorochrome-labeled DNA-probes. Sperm chromatin was counterstained with propidium iodide. By scanning across the slide, fluorescence signals within sperm nuclei were detected and counted.Results: In murine sperm, the frequencies of disomies for chromosomes 8 and X were 0.019% and 0.021%, respectively. The automated assessment in human sperm resulted in disomy frequencies of 0.061% and 0.090% for chromosomes 13 and X, respectively. These results were comparable to data obtained from the same samples by manual microscopic scoring and to literature data.Conclusions: Frequencies of genotypically abnormal sperm were not significantly different between automated and manual scoring. In conclusion, sperm aneuploidy was reliably determined and disomic sperm were successfully relocated by LSC. By virtue of rapid and reliable analyses, LSC has the powerful potential to replace manual microscopic FISH analysis in molecular cytogenetics