Effect of caffeine treatment on low-dose γ- irradiation-induced chromatid-type aberrations in human leukemia cells and human normal fibroblast cells in culture

e22113 Background: It is estimated that low dose radiation can increase the risk of cancer as well as mutations. However, the interaction of low dose radiation with caffeine has not been adequately investigated. We investigated the effects of caffeine on low dose- gamma-radiation-induced chromosomal damage in human T leukemia cells (Jurket T-cells) and two normal human fibroblast cell lines (AG1522 and GM 2149). Method: Jurkat cells were maintained in RPMI 1640 medium and fibroblast in alpha-minimal essential medium (MEM) All cells were incubated at 37o C in a humidified atmosphere of 5% CO2 in air. Cells from the exponential phase were treated with 1 mg/ml caffeine ( control cells received same amount of solvent) and irradiated with low doses (3, 5, 10, 20 and 40 cGy,), using a 137 Cs-gamma radiation source. Colcemid at a concentration of 0.1 μg/ml was added to every flask. Cells were fixed in methanol: acetic acid solution and stained with Giemsa. 100 irradiated and un-irradiated metaphase- like cells were scored for chromatid-type aberrations. Results: Low dose gamma-radiation increased the levels of chromatid breaks(dose dependent) in both normal and cancer cells; however, cancer cells appeared to be more sensitive than the normal cells. Caffeine treatment markedly increased chromatid aberrations in Jurkat T-cells at all radiation doses but not in normal cells. Previously, we reported that caffeine eliminates gamma-ray-induced G2 delay in other human tumor cells but not normal cells (Jha, et.al., Radiat. Res. 157, 26–31, 2002). Conclusions: The mechanisms that may underlie this differential effect of caffeine in cancer and normal cells are unknown, but if one result of a G2 delay is to allow more time for chromosome breakage rejoining processes to occur, then elimination of this delay by caffeine in tumor cells but not normal cells might account for the difference. To the extent these observations are generally true for tumor vs normal cells, the differential sensitization could have an impact in improving the efficacy of radiation therapy. No significant financial relationships to disclose.

Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1–Cdk1 at the centrosome

Cdc25 phosphatases are essential for the activation of mitotic cyclin–Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1–Cdk1 and cyclin A–Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1–Cdk1 on centrosomes.

Regulation and Localization of the Bloom Syndrome Protein in Response to DNA Damage

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RecQ-like helicase, presumed to function in DNA replication, recombination, or repair. BLM localizes to promyelocytic leukemia protein (PML) nuclear bodies and is expressed during late S and G2. We show, in normal human cells, that the recombination/repair proteins hRAD51 and replication protein (RP)-A assembled with BLM into a fraction of PML bodies during late S/G2. Biochemical experiments suggested that BLM resides in a nuclear matrix–bound complex in which association with hRAD51 may be direct. DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism. This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed. It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci. After radiation, foci containing BLM and PML formed at sites of single-stranded DNA and presumptive repair in normal cells, but not in cells with defective PML. Our findings suggest that BLM is part of a dynamic nuclear matrix–based complex that requires PML and functions during G2 in undamaged cells and recombinational repair after DNA damage.