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A G1-phase delay after exposure to alpha particles has not been report ed previously, perhaps because immortalized cell lines or cell lines from tumor cells were used in past studies. Therefore, we compared the effects of alpha particles (0.19 or 0.57 Gy) and approximately equitoxic doses of gamma rays (2 or 4 Gy) on progression of cells through the cell cycle in normal human skin fibroblasts. Cell cycle analyses were performed using flow cytometry by measuring incorporation of bromodeoxyuridine (BrdUrd) in each phase of the cell cycle up to 44 h after irradiation. We observed an alpha-particle-induced G1-phase delay in human skin fibroblasts even at the lowest dose, 0.19 Gy. At equitoxic doses, more pronounced and persistent G1-phase delays and arrests were observed in gamma-irradiated cultures in that increased fractions of the G1-phase cells remained BrdUrd- over the course of the study after gamma-ray exposure compared to cells exposed to alpha particles. In addition, G1-phase cells that became BrdUrd+ after gamma irradiation re-arrested in G1 phase, whereas BrdUrd+ G1-phase cells in alpha-particle-irradiated cultures continued cycling. In contrast, comparable percentages of cells were delayed in G2 phase after either alpha-particle or gamma irradiation. Both gamma and alpha-particle irradiation caused increases in cellular p53 and p2lCip1 shortly after the exposures, which suggests that the G1-phase delay that occurs in response to alpha-particle irradiation is dependent on p53 like the initial G1-phase delay induced by gamma rays.

SUMMARY Deuterium oxide (D 2 O) increases both the fluorescence lifetime and the fluorescence intensity of the intercalating dyes propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis coupled with conventional and phase-sensitive flow cytometry to compare the alterations in intensity and lifetime of various DNA-binding fluorochromes bound to DNA and Chinese hamster ovary (CHO) cells in the presence of D 2 O vs phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a differential enhancement of intensity and lifetime based on the mode of fluorochrome-DNA interaction. The fluorescence properties of intercalating probes, such as 7-aminoactinomycin D (7-AAD) and ethidium homodimer II (EthD II) were enhanced to the greatest degree, followed by the probes TOTO and YOYO, and the non-intercalating probes Hoechst 33342 (HO) and 4 Ј ,6-diamidino-2-phenylindole (DAPI). The non-intercalating probe mithramycin (MI) gave unexpected results, showing a great enhancement of fluorescence intensity and lifetime in D 2 O, indicating that when staining is performed in PBS, much of the MI fluorescence is quenched by the solvent environment. Apoptotic subpopulations of HL-60 cells had a shorter lifetime compared to nonapoptotic subpopulations when stained with EthD II. These results indicate that accessibility of the dye molecules to the solvent environment, once bound to DNA, leads to the differential enhancement effects of D 2 O on fluorescence intensity and lifetime of these probes. D na-binding fluorochromes have been used extensively in many applications, including flow cytometry and gel and capillary electrophoresis. Fluorochromes utilized in these applications must satisfy several criteria. They must bind specifically to nucleic acids, there must be a low quantum yield as free dye in solution, and there must be enhancement of quantum yield on binding to nucleic acid structures. Several classes of DNA-specific fluorochromes with different modes of base pair ( bp ) binding are available: (a) the DNA intercalators propidium iodide (PI) and ethidium bromide (EB), which lack appreciable bp specificity; (b) DNA intercalators with A-T bp preference, such as ethidium homodimer II (EthD II) or with G-C preference, such as 7-amino actinomycin D (7-AAD); and (c) non-intercalating probes with either A-T bp specificity, such as Hoechst 33342 (HO) or 4 Ј ,6-diamidino-2-phenylindole (DAPI) or with G-C specificity, such as mithramycin (MI). Two high quantum yield cyanine fluorochromes, TOTO and YOYO, are dimers of the dyes thiazole orange and oxazole yellow, respectively (Lee et al. 1986). These fluorochromes are believed to bind to DNA by intercalation, without base specificity (Haugland 1992) but with some base sequence preference (Netzel et al. 1995).Enhancement of the fluorescence intensity of DNAbinding probes improves the signal-to-noise ratio, allowing the use of lower dye concentrations and thereby reducing potential self-quenching between d...

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Integration-A concept to unify components of a system. Lead Time-Length of time required to replenish the inventory for a material from the time that a need for additional material is felt until the new order for material is received in the inventory and is ready to use.