A method for comparing effects of different synchronizing protocols on mammalian cell cycle traverse. The traverse perturbation index.

After treatment of Chinese hamster cells (line CHO) with various protocols for synchrony induction, the subsequent ability of cells to traverse the cell cycle (i e., to perform, an essential cell cycle process) has been determined by measurement of the DNA distribution pattern among cells in large populations with the Los Alamos flow microfluorometer In the cultures prepared by the various synchronizing techniques the vast majority of cells traversed the cell cycle in a normal fashion; however, in all cultures examined there remained small subpopulations which, though remaining viable for several days, could not carry out normal traverse. After reversible inhibition of DNA synthesis by means of a double-thymidine blockade, approximately 17% of the cells were unable to complete genome replication. After reversal of G(1) arrest resulting from cultivation of cells in isoleucine-deficient medium, 12 4% of the cells commenced synthesis of DNA but were unable to complete the S phase. Cells prepared by mitotic selection yielded a subpopulation (5 5% of the total cells) with a G(1) DNA content which remained viable but noncycling for at least 5 days. We propose a term "traverse perturbation index" which is defined as the fraction of cells converted to a noncycle-traversing state as the result of experimental manipulation. A knowledge of the perturbation index will allow direct comparison of effects on cell cycle traverse of various synchrony-induction protocols

Normal and perturbed Chinese hamster ovary cells: correlation of DNA, RNA, and protein content by flow cytometry.

Quantitative, correlated determinations of DNA, RNA, and protein, as well as RNA to DNA and RNA to protein ratios, were performed on three-color stained cells using a multiwavelength-excitation flow cytometer. DNA-bound Hoechst 33342 (blue), protein-fluorescein isothiocyanate (green), and RNA-bound pyronin Y (red) fluorescence measurements were correlated as each stained cell intersected three spatially separated laser beams. The analytical scheme provided sensitive and accurate fluorescence determinations by minimizing the effects of overlap in the spectral characteristics of the three dyes. Computer analysis was used to generate two-parameter contour density profiles as well as to obtain numerical data for subpopulations delineated on the basis of cellular DNA content. Such determinations allowed for analysis of RNA to DNA and RNA to protein ratios for cells within particular regions of the cell cycle. The technique was used to study the interrelationship of DNA, RNA, and protein contents in exponentially growing Chinese hamster ovary cells as well as in cell populations progressing the cell cycle after release from arrest in G1 phase. The sensitivity of the method for early detection of conditions of unbalanced growth is demonstrated in the comparison of the differential effects of the cycle-perturbing agent, adriamycin, on cells treated either during exponential growth or while reversibly arrested in G1 phase.

Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210.

The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.

Cell-cycle analysis in 20 minutes.

Mithramycin added to mammalian cells fixed in aqueous ethanol is bound to DNA and fluoresces in direct proportion to the cellular DNA content. Quantitative fluorescence measurement by means of a high-speed flow system instrument provides a rapid method for cell-cycle analysis and for the first time permits continuous monitoring of cell-cycle kinetics during ongoing experiments.

Correlated measurements of DNA, RNA, and protein in individual cells by flow cytometry.

A cytochemical method was developed to differentially stain cellular DNA, RNA, and proteins with fluorochromes Hoechst 33342, pyronin Y, and fluorescein isothiocyanate, respectively. The fluorescence intensities, reflecting the DNA, RNA, and protein content of individual cells, were measured in a flow cytometer after sequential excitation by three lasers tuned to different excitation wavelengths. The method offers rapid analysis of changes in the cellular content of RNA and protein as well as in the RNA-protein, RNA-DNA, and protein-DNA ratios in relation to cell cycle position for large cell populations. An analysis of cycling cell populations (exponentially growing CHO cultures) and noncycling CHO cells arrested in the G1 phase by growth in isoleucine-free medium demonstrated the potential of the technique.

Interferon effects upon the adenocarcinoma 38 and HL-60 cell lines: antiproliferative responses and synergistic interactions with halogenated pyrimidine antimetabolites.

In order to gain insight into the mechanisms affecting combination treatment of tumor cells with interferon and halogenated pyrimidine antimetabolites, in vitro studies of murine colon adenocarcinoma 38 and HL-60 cell lines were undertaken. Interferons exhibited modest antiproliferative effects against these lines with DNA synthesis inhibited greater than RNA greater than protein synthesis, as studied by 3H-precursor incorporation. The adenocarcinoma cell line was considerably more sensitive to recombinant gamma-than to purified alpha/beta-interferon, while HL-60 was slightly more sensitive, in short term studies, to antiproliferative effects of recombinant alpha- than to gamma-interferon. Interferon treatment was further associated with suppression of a hyperdiploid component of the adenocarcinoma cell line, as detected by flow cytometry. Combination treatment of the adenocarcinoma cell line with interferon and halogenated pyrimidines, under 4-day continuous exposure conditions, revealed significant synergy for growth inhibition with gamma- much greater than alpha/beta-interferon and with 5-fluorodeoxyuridine greater than 5-fluorouracil much greater than 5-fluorouridine. Thus, synergy was much greater with the more antiproliferative interferon and with the antimetabolite derivative most likely to lead to thymidylate synthetase inhibition rather than RNA incorporation. Sequential 2-day + 2-day treatment revealed greater synergy when interferon preceded 5-fluorouracil or 5-fluorodeoxyuridine rather than the reverse protocol. The synergy of gamma-interferon and 5-fluorodeoxyuridine could be blocked by thymidine, which bypasses inhibition of thymidylate synthetase. HL-60 also exhibited thymidine antagonized synergistic growth-inhibitory effects of interferon and 5-fluorouracil. Analysis of this interaction by [3H]thymidine incorporation, which can reflect thymidylate synthase inhibition, revealed exaggerated responses of interferon-treated cells to either 5-fluorouracil or 5-fluorodeoxyuridine. These results indicate that interferon-halogenated pyrimidine antimetabolite synergistic interactions may be common to several cell types. Evidence is further presented for a mechanism of synergy entailing enhanced thymidylate synthetase inhibition by the antimetabolite of interferon-treated cells.

Comparative effects of three nitrosourea derivatives on mammalian cell cycle progression.

Three nitrosourea analogs, 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, and 1-trans-(2-chloroethyl)-3-cyclohexyl-1-(4-methylcyclohexyl)-1-nitrosourea, were examined for effects on survival and cell cycle traverse capacity in exponentially growing (cycling) populations of line CHO Chinese hamster cells and in cultures arrested in G1 by isoleucine deprivation during treatment with drugs, then returned to the cycling mode by restoration of isoleucine (noncycling cells). Among parameters studied were survival, cell division, DNA initiation capacities, cell cycle distributions, and rates of cell cycle traverse in drug-treated cycling and noncycling cells utilizing a protocol combining autoradiography, cell number enumeration, and flow microfluorometry. The results obtained were in generally good agreement with results obtained in vivo in other studies and included the following. Cells treated with any of these agents accumulated preferentially in late S and G2, primarily the result of a gross increase in duration of these phases of the cell cycle. There was also a prolongation of doubling time during the early stages following drug treatment and return to the proliferating mode of cells which ultimately survived. All three drugs induced mitotic nondisjunction in cells capable of dividing and also induced polyploidy by allowing multiple rounds of progression through the cell cycle in the absence of an intervening cell division. In treated populations, the G2-arrested and polyploid cells were among the first cells to die. Treated, noncycling cells that were returned to cycle exhibited a lower survival capacity than did treated, cycling cells. Finally, 1-(2-chloroethyl-3-cyclohexyl)-1-nitrosourea and 1-trans(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea induced a dramatic alteration in clonal morphology and growth patterns in surviving cells that persisted for at least a week after drug removal. The results obtained suggest that our model system may be useful as a predictive guide for determining response of susceptible tumor cells to treatment with chemotherapeutic agents.

Flow cytometric localization within the cell cycle and isolation of viable cells following exposure to cytotoxic agents.

Viable cell sorting, based on flow cytometric analysis of DNA content and cell volume, was used to evaluate the cycle position and survival potential of Adriamycin (AdR)-treated or 1-beta-D-arabinofuranosylcytosine (ara-C)-treated CHO cells. Drug-treated cells initially stained with the vital, DNA-specific fluorochrome, Hoechst 33342, were analyzed for DNA content and volume, and sorting "windows" were established for subsequent sorting of duplicate unstained cell samples based only on cell volume. Another portion of the cell sample was fixed in ethanol, and stained with three fluorochromes for correlated flow cytometric analysis of DNA, RNA, and protein. Similarities in the viable cell volume distributions and the protein content distributions of the ethanol-fixed samples provided a means for indirectly determining the DNA and RNA contents of the sorted cells. Three regions (S, L, and I) were selected in the cell volume distributions corresponding to the range of near normal cell size (S), larger than normal cell size (I), and the extremely large cells (L). Adriamycin-treated or ara-C-treated cells sorted from the S region had survival values, respectively, 46 times and 7 times greater than the abnormally large cells in region L. Cells from the S region also respectively survived 14-fold (AdR-treated) and 7-fold (ara-C-treated) greater than the cells sorted from the I regions. RNA content levels for cells within the L region were three times and two times greater, respectively, than the AdR-treated and Ara-C-treated subpopulations in the S regions. Survival of subpopulations of G2-arrested, AdR-treated cells (I and L regions) was better correlated with relative abnormality in cell size than with position in the cell cycle. In addition to providing further support for the validity of the "balanced growth hypothesis," the results of this study suggest that two-parameter DNA content and cell volume measurements would be extremely useful for providing general guidelines for judging the effectiveness of therapy, especially in clinical diagnoses where cell sorting is impractical or impossible. From these analyses the frequency and cycle position of cells resistant to therapy can be estimated. Such information would be particularly useful for rapidly detecting drug-resistant cells and design of subsequent therapeutic regimens.

Effects of conditioned medium upon proliferation, deoxynucleotide metabolism, and antimetabolite sensitivity of promyelocytic leukemic cells in vitro.

In order to investigate the interaction of factors from leukocyte-conditioned medium with leukemic cells, effects of an ammonium sulfate-precipitated conditioned medium concentrate were tested upon HL-60 cells. This preparation increased tritiated thymidine incorporation into DNA of HL-60 cells markedly, an effect which was found to be attributable in large part to greater thymidine accumulation in the intracellular nucleotide triphosphate pool. A modestly expanded population of DNA synthetic phase cells was also demonstrated by flow cytometry. Similar effects were noted of the K562 and KG-1 cell lines and were also demonstrable with giant cell tumor-conditioned medium. These effects were not demonstrable with a purified preparation of granulocyte-monocyte colony-stimulated factor. Because of the altered pattern of nucleotide metabolism noted, the effect of the conditioned medium concentrate upon 5-fluorouracil sensitivity was tested. Following continuous 24-h exposure to 5-fluorouracil at 5 X 10(-6) M, tritiated thymidine incorporation of HL-60 cells increased in parallel with depletion of endogenous thymidylate. Conditioned medium concentrate markedly sensitized cells to this effect of 5-fluorouracil and also increased growth retardation, cytotoxicity, and cell cycle arrest as assessed by flow cytometry. These studies thus demonstrated marked effects of a factor in conditioned medium on deoxynucleotide uptake and metabolism of the HL-60 line. These effects occurred in conjunction with, but were relatively more marked than, effects upon cell cycle distribution and were found to influence chemotherapy sensitivity.

Reduction in the radiation-induced late S phase and G2 blocks in HL-60 cell populations by amiloride, an efficient inhibitor of the Na+/H+ transporter.

Recent investigations that showed that amiloride delayed or inhibited apoptosis indicated it might also attenuate cell cycle checkpoints activated by ionizing radiation. In this report, single- and dual-parameter flow cytometry were used to investigate the effects of amiloride on cell cycle progression, and the effectiveness of amiloride to attenuate the S and G2 phase checkpoint responses induced by 2.5, 5.0, and 7.5 Gy of gamma radiation. The late S-phase delay, noted at 8 h following irradiation, and a radiation-induced G2 block, which was maximum at 16 h after irradiation, were both significantly reduced in amiloride-treated samples. Attenuation of the radiation-induced late S phase and G2 blocks resulted in cell division without apparent apoptosis or necrosis over a 24-h period. Results presented indicate that amiloride reduces the radiation-induced G2 block in HL-60 cell populations almost equally well as caffeine and to a greater extent than staurosporine. Immunofluorescent detection and quantitation of cyclin B1 expression demonstrated that amiloride only significantly reduced cyclin B1 expression following 5.0 Gy, when there was a notable induction of a significant G2 delay, followed by a relatively rapid recovery in cycling potential. The results suggest that amiloride affects the radiation-triggered signaling cascades to alter the kinase activity of proteins associated with mitotic progression, particularly the cyclin B1-p34cdc2 complex. Alternatively, alterations in intracellular ion concentrations induced by amiloride may lead to changes in Ca2+-dependent signaling cascades and thereby decrease the radiation-mediated cell cycle perturbations.

Effects of ionizing radiation of RNA metabolism in cultured mammalian cells. III. Specific reduction in mRNA synthesis during resumed division and plateau phases following exposure to 800 rads of x irradiation.

The cell growth response of cultured Chinese hamster ovary cells, line CHO, to 800 rads of X irradiation involves a period of division delay, followed by a period of resumed division which terminates in a plateau phase. Over 95% of the cells die eventually. No direct effects of RNA or protein metabolism are evident during the delay period. During the resumed division and beginning of plateau phases, however, a specific and relatively constant reduction in mRNA synthesis relative to messenger-like RNA and heterogenous nuclear RNA synthesis is evidenced. The ratio of mRNA to messenger-like RNA synthesis ranges from 0.8 to 0.65 during these phases. The effect is not due to altered cell-cycle distribution, and evidence is presented to indicate that it is probably not a compensatory response to the unbalanced growth that occurs during the division delay period.

Characterization of granulosa cell subpopulations from avian preovulatory follicles by multiparameter flow cytometry.

The objective of the present study was to characterize the granulosa cell populations from individual hen (Gallus domesticus) preovulatory follicles at defined stages of follicular maturation using multiparameter flow cytometry. Granulosa cells were fixed and stained with three fluorochromes that selectively bind to DNA (Hoechst 33342, blue), RNA (pyronin Y, red), or protein (fluorescein isothiocyanate, green). A flow cytometer equipped with a three-laser excitation system was used to analyze three colors of fluorescence from stained cells. Forward angle light scatter and axial light loss measurements were made on each cell to determine relative cell size. In addition, the ratios of RNA to protein and DNA to protein were measured. The major findings obtained from correlated measurements of cell cycle (DNA), protein, RNA, cell size, and ratios were: 1) the percentage of proliferating cells decreased while cell size increased during follicular maturation; 2) two subpopulations of granulosa cells were identified within each follicle based on relatively high and low protein contents; the fraction of cells in the high protein subpopulation increased, and the fraction of cells in the low protein subpopulation decreased during follicular maturation; 3) the high and low protein subpopulations also differed in cell cycle distribution, RNA content, and cell size; and 4) the distribution of cells into the two subpopulations and the degree of proliferation were influenced by stage of the ovulatory cycle, primarily in the most mature follicles. The results demonstrate the dynamic heterogeneity of the granulosa cell populations from individual ovarian follicles and show the influences of follicular maturation and stage of the ovulatory cycle on cell growth and metabolism.

Dual-laser flow cytometry of single mammalian cells.

An improved dual-laser flow cytometric system for quantitative analysis and sorting of mammalian cells has been developed using a low-power argon and high-power krypton laser as illumination sources, thus permitting the excitation of fluorescent dyes having absorption regions ranging from the ultraviolet to infrared. Cells stained in liquid suspension with fluorescent dyes enter a flow chamber where they intersect two spatially separated laser beams. Separate pairs of quartz beam-shaping optics focus each beam onto the cell stream. Electro-optical sensors measure fluorescence and light scatter signals from cells that are processed electronically and displayed as frequency distribution histograms. Cells also can be electronically separated and microscopically identified. The ease and versatility of operation designed into this system represent a marked technological improvement for dual-laser excited flow systems. Details of this instrument are described along with illustrative examples of cells stained with mithramycin and rhodamine and analyzed for DNA content, total protein, and nuclear and cytoplasmic diameter.

Fluorescent DNA probes for flow cytometry. Considerations and prospects.

Techniques employing base specific deoxyribonucleic acid (DNA)-binding fluorochromes and flow cytometry (FCM) are potentially useful for obtaining information of the compositional features of chromatin or chromosomes of mammalian cells. Fluorescent compounds which form complexes preferentially at the A-T rich regions (i.e., DNA-reactive Hoechst dyes) or the G-C rich regions (i.e., mithramycin, chromomycin, olivomycin) in DNA are available and compatible with current FCM technology as are other compounds (i.e., ethidium bromide, propidium iodide) which show little or no base specificity and bind by intercalation in the double stranded regions of helical DNA. Energy transfer between appropriate DNA-bound dyes is a reflection of the quantity and proximity of regions containing the respective base pair segments. Since extrinsic fluorescent probes provide only a measure of available binding sites or regions unobstructed by chromatin-associated or chromosomal-associated proteins, interpretations of fluorescence measurements need to be substantiated by adequate control measures.

Flow microfluorometric and light-scatter measurement of nuclear and cytoplasmic size in mammalian cells.

A technique for rapid measurement of nuclear and cytoplasmic size relationships in mammalian cell populations has been developed. Based on fluorescence staining of either the nucleus alone or in combination with the cytoplasm using two-color fluorescence methods, this technique permits the simultaneous determination of nuclear and cytoplasmic diameters from fluorescence and light-scatter measurements. Cells stained in liquid suspension pass through a flow chamber at a constant velocity, intersecting a laser beam which excites cell fluorescence and causes light scatter. Depending upon which analysis procedure is used, optical sensors measure nuclear fluorescence and light scatter (whole cell size) or two-color nuclear and cytoplasmic fluorescence from individual cells crossing the laser beam. The time durations of signals generated by the nucleus and cytoplasm are converted electronically into signals proportional to the respective diameters and are displayed as frequency distribution hitograms. Illustrative examples of measurements on uniform microspheres, cultured mammalian cells and human exfoliated gynecologic cells are presented.

Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells.

Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.

Differential effects of deuterium oxide on the fluorescence lifetimes and intensities of dyes with different modes of binding to DNA.

Deuterium oxide (D2O) 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 D2O 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 4,6-diamidino-2-phenylindole (DAPI). The non-intercalating probe mithramycin (MI) gave unexpected results, showing a great enhancement of fluorescence intensity and lifetime in D2O, 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 non-apoptotic 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 D2O on fluorescence intensity and lifetime of these probes.

Association of G1/S-phase and late S-phase checkpoints with regulation of cyclin-dependent kinases in Chinese hamster ovary cells.

We investigated the time-dependent effects of 8 Gy of gamma radiation on the activities of cyclin-dependent kinases (Cdk's) and the incorporation of the thymidine analog bromodeoxyuridine (BrdU) throughout the S phase in Chinese hamster ovary (CHO) cells. The in vitro Cdk activities of immunoprecipitated cyclin E, cyclin A and Cdk2 were reduced about 30% per cell within 0.5-1 h after irradiation, but they recovered at different rates. The kinase activity of the cyclin E-Cdk2 complex recovered first and exceeded the control values by 1.5-2 h after irradiation. Cyclin A-Cdk activities began to recover at 3-4 h after irradiation, and cyclin E/A-Cdk2 activities recovered at intermediate rates. The super-recovery of cyclin E-Cdk2 coincided with the appearance of a small synchronous population of cells entering into S phase, consistent with transient G1-phase delay/recovery regulated by cyclin E-Cdk2, whereas the activities of cyclin A-Cdk's (75% cyclin A-Cdk2; 25% cyclin A-Cdc2 when inhibition was maximal) were correlated with rates of total DNA synthesis. Multivariate flow cytometry analyses of BrdU incorporation demonstrated that radiation-induced inhibition of DNA synthesis occurred predominantly within the last quarter of S phase and that the majority of the irradiated cells failed to enter G2 phase for 4-5 h. The recovery of cyclin A-Cdk activities coincided with increased levels of total DNA synthesis and BrdU incorporation into cells within the last quarter of S phase. Western blot analysis demonstrated that levels of Waf1/p21 did not increase during inhibition of cyclin A-Cdk's and DNA synthesis in the irradiated p53-mutated CHO cells; however, Cdc2 and Cdk2 exhibited increased levels of phosphotyrosine. The results (1) indicate that the transient G1-phase delay or G1/S-phase checkpoint (Lee et al., Proc. Natl. Acad. Sci. USA 94, 526-531, 1997) is mediated by inhibition of cyclin E-Cdk2 and (2) point to the existence of a radiation-induced S-phase checkpoint located about 75% into S phase involving the inhibition of cyclin A-Cdk's by a p53/Waf1-independent pathway in CHO cells.

Alterations in the progression of cells through the cell cycle after exposure to alpha particles or gamma rays.

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.