A new method to discriminate G1, S, G2, M, and G1 postmitotic cells

A new flow cytometric method combining light scattering measurements, detection of bromodeoxyuridine (BrdU) incorporation via fluorescent antibody, and quantitation of cellular DNA content by propidium iodide (PI) allows identification of additional compartments in the cell cycle. Thus, while cell staining with BrdU-antibodies and PI reveals the G1, S, and G2 + M phases of the cell cycle, differences in light scattering allow separation of G2 phase cells from M phase cells and subdivision of G1 phase into two compartments, i.e., G1A representing postmitotic cells which mature to G1B cells ready to initiate DNA synthesis. The method involves fixation of cells in 70% ethanol, extraction of histones with HC1, and thermal denaturation of DNA. This treatment appears to enhance the differences in chromatin structure of cells in the various phases of the cell cycle to the extent that cells could be separated on the basis of the 90 degrees scatter. Mitotic cells show much lower scatter than G2 phase cells, and G1 postmitotic cells (G1A) show lower scatter than G1 cells about to enter the S phase (G1B). Light scattering is correlated with chromatin condensation, as judged by microscopic evaluation of cells sorted on the basis of light scatter. The method has the advantage over the parental BrdU/DNA bivariate analysis in allowing the G2 and M phases of the cell cycle to be separated and the G1 phase to be analyzed in more detail. The method may also allow separation of unlabeled S phase cells from mitotic cells and distinguish between labeled and unlabeled mitotic cells.     

Analysis of cell kinetics using BrdU monoclonal antibody on cultured tumor cells after irradiation and treatment with anti-cancer drugs

Flow cytometry (FCM) permits instantaneous determination of the percentages of cells in various phases of cell cycle using BrdU-PI double staining method, and allowing rapid evaluation of the effects of irradiation and anti-cancer drugs (ACNU, ADR, BLM) on the cell kinetics. In this study, the growth inhibition and changes in the cell kinetics after irradiation and chemotherapy were examined according to the growth curve analysis and BrdU-PI method to evaluate the usefulness of BrdU-PI method for assessment of the effect of the treatments. By the conventional method based on the DNA histogram, accurate determination of S cell fraction was difficult due to overlapping of the DNA contents of G1 cells and early S cells and those of late S cells and G2 cells. BrdU-PI double staining allowed direct differentiation of G1, S, and G2 + M cells, especially between G1-S and S-G2 + M cells. The analysis of cell kinetics using BrdU is advantageous in comparison to the conventional autoradiographic methods because it allows more rapid assay with very high sensitivity. By the present BrdU method, rapid transition to the G1-S phase was observed within 4 hours after exposure to radiation and anti-cancer drugs. This initial G1 arrest induced by irradiation was confirmed for the first time by the present BrdU-PI double staining. The present method is considered to be indispensable for evaluation of the percentage of S cells in the tumor tissue and analysis of cell kinetics after irradiation and chemotherapy against cancer.     

Cell Cycle Kinetics of Aerated, Hypoxic and Re-Aerated Cells In Vitro Using Flow Cytometric Determination of Cellular Dna and Incorporated Bromodeoxyuridine

Abstract. The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 μM, 20 min, 37°C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G₂ and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. the percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Cell cycle kinetics of aerated, hypoxic and re-aerated cells in vitro using flow cytometric determination of cellular DNA and incorporated bromodeoxyuridine

The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 microM, 20 min, 37 degrees C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G2 and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. The percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Flow cytometric determination of cell cycle parameters of V79 cells by continuous labeling with bromodeoxyuridine

A simple method with which to determine the cell cycle parameters, TG1, TS and TG2M (the durations of the G1, S and G2 + M phases) is described. V79 Chinese hamster lung cells were used to evaluate the method. After continuous labeling with bromodeoxyuridine (BrdU), V79 cells were stained with anti BrdU-monoclonal antibody with FITC (fluorescein isothiocyanate) and with PI (propidium iodide). The individual cells were checked by flow cytometry for green and red fluorescences whose signal intensities corresponded to the BrdU and cellular DNA contents. The durations of G1, S and G2 + M phases of V79 cells were determined by measuring the cell fractions containing the nonlabeled G1, labeled S and nonlabeled G2 + M phases. The reliability of this method is discussed.     

流式细胞术BrdU／DNA双染法测定云芝糖肽诱导的Molt-4细胞周期阻滞

目的：探究云芝糖）Ik（PSP）对人急性淋巴母细胞白血病Molt-4细胞周期的影响。方法：采用流式细胞术BrdU／DNA双染法获得各时相细胞分布状况和细胞周期的动力学参数。结果：0．1mg／mlPSP处理12h后,G2／M期细胞百分比由对照组的11．09％减少至3．69％。DNA合成时间由12．10h延长至108．40h。24h处理组中,S期细胞百分比由对照组的43.29％增加至67．26％,而G0／G1期和G2／M期细胞百分比均减少,G0／G1期细胞百分比由对照组的37．47％减少至27．43％,G2／M期细胞百分比由对照组的19．24％降低至5.31％。DNA合成时间更是由11．95h延长至114．52h。结论：PSP对人急性淋巴母细胞白血病Molt-4细胞周期的阻滞作用在于S期．该作用与DNA合成抑制有关。     

Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase

Competency for self-renewal of human embryonic stem (ES) cells is linked to pluripotency. However, there is a critical paucity of fundamental parameters of human ES cell division. In this study we show that human ES cells (H1 and H9; NIH-designated WA01 and WA09) rapidly proliferate due to a very short overall cell cycle (15-16 h) compared to somatic cells (e.g., normal diploid IMR90 fibroblasts and NT-2 teratocarcinoma cells). The human ES cell cycle maintains the four canonical cell cycle stages G1, S, G2, and M, but the duration of G1 is dramatically shortened. Bromodeoxyuridine (BrdU) incorporation and FACS analysis demonstrated that 65% of asynchronously growing human ES cells are in S phase. Immunofluorescence microscopy studies detecting BrdU labeled mitotic chromosomes, Ki67 domains, and p220(NPAT) containing Cajal bodies revealed that the durations of the S ( approximately 8 h), G2 ( approximately 4 h), and M phases ( approximately 1 h) are similar in ES and somatic cells. We determined that human ES cells remain viable after synchronization with either nocodazole or the anti-tumor drug Paclitaxel (taxol) and have an abbreviated G1 phase of only 2.5-3 h that is significantly shorter than in somatic cells. Molecular analyses using quantitative RT-PCR demonstrate that human ES cells and somatic cells express similar cell cycle markers. However, among cyclins and cyclin-dependent kinases (CDKs), we observed high mRNA levels for the G1-related CDK4 and cyclin D2 genes. We conclude that human ES cells exhibit unique G1 cell cycle kinetics and use CDK4/cyclin D2 related mechanisms to attain competency for DNA replication.     

Study of the Lineage and Cell Cycle of Small Micromeres in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

A study was made of 1st cell cycle of small micromeres, segregated at the 5th cleavage cycle, in the sea urchin embryos of Hemicentrotus pulcherrimus . For identification of small micromeres, the embryos were pulse labeled with 5-bromodeoxyuridine (BrdU) at the 1st cleavage. Using multiparametric microfluorometry equipped with a scanning stage (Tanaka, 1990), DNA content, extent of BrdU incorporation, protein content and the extent of ³H-thymidine labeling were measured on identical individual cells dissociated from an embryo. The findings of the present study are as follows. There is a short period of time between the telophase and onset of DNA replication. The period of DNA replication is 5 hr and after which, asynchronous mitosis takes place to produce 8 cells before hatching. The long S period is 83% the total 6 hr of the cell cycle. The rate of DNA accumulation is quite small during the initial one third of S but increases later in this phase. The degree of chromatin condensation remains high even during the S phase but it is low in large micromeres. The cell cycle may possibly be related causally to the development of small micromeres. The developmental significance of cell cycle duration, particularly that of DNA replication is discussed.     

cAMP-mediated growth inhibition of a B-lymphoid precursor cell line Reh is associated with an early transient delay in G2/M, followed by an accumulation of cells in G1

This study was undertaken to gain more insight into the effects of cyclic adenosine monophosphate (cAMP) on cell-cycle progression in the B-lymphoid precursor cell line Reh. The adenylate cyclase activator forskolin reduced the proliferation of asynchronously growing Reh cells by 50% after 72 hr culture. Growth inhibition was associated with an accumulation of cells in G1. Furthermore, we demonstrated that forskolin provoked a delay of cells for approximately 10 hr in G2/M prior to the G1 arrest. Two different methods were applied to elucidate how cells in different phases of the cell cycle were affected by an elevated cAMP level. One method was based on centrifugal elutriation, whereby synchronous cell populations from the different phases of the cell cycle were isolated. By the other method, S-phase cells were selectively stained by pulsing asynchronously growing cells with bromo-deoxyuridine (BrdU). The data demonstrate that the position of a cell in the cell cycle is critical in determining how the cell will respond to an elevated cAMP level. Thus cells in G1 at the time forskolin is added are not delayed in G2/M, but they will subsequently accumulate in G1 after 48 hr. Cells given forskolin in G2/m, however, are delayed for 10 hr in G2/M, but they do not accumulate in G1. Cells given forskolin in the S phase are delayed in G2/M as well as arrested in G1. The results suggest that cAMP inhibits growth of the Reh cells by preventing the cells from passing important restriction points located in the G1 and G2 phases of the cell cycle.     

Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping

Tape stripping of human skin elicits a proliferative response of a synchronously-dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid-S phase and the other centred around G2 + M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram. The cohort can be described as the recruitment of cells from a pre-existing G0 compartment which consists of 76% of all proliferative cells. The duration of the S phase is calculated to be 10.2 hr and G2 + M phase 5.1 hr. The cell cycle time of 39 hr for normal human keratinocytes derived from these figures is in line with recent values obtained by different techniques.     

用双参数流式细胞光度术(FCM)研究阿克拉霉素对L_(1210)白血病细胞周期的影响

本文用双参数FCM技术,对同一个细胞的DNA和RNA含量进行相关测量,比较了ACM B对小鼠L_(1210)白血病细胞周期和RNA含量的影响.结果发现在一次给药后8小时可导致早、中期S的积累,并抑制S期细胞的DNA合成;到24小时DNA合成恢复正常,并进入G_2期,但由于G_2期细胞进入M期受阻,造成G_2期细胞的积累,这时被阻断在G_2期的细胞RNA含量显著增加,形成正不平衡生长,而给药剂量较大的实验组(1/1.5LD_(50))S期细胞的RNA含量不随着DNA含量的增加而增加,形成负不平衡生长,ACM A和ACM B对体内Li_(210)细胞周期作用相同.     

Regenerative proliferation of mouse epidermal cells following adhesive tape stripping. Micro-flow fluorometry of isolated epidermal basal cells combined with 3H-TdR incorporation and a stathmokinetic method (colcemid).

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

In vitro bromodeoxyuridine labeling of nuclei: application to myotube hybridization

Rat myoblast nuclei were labeled with various concentrations of bromodeoxyuridine (BrdU), an analogue of thymidine, for 24 or 48 hr. Almost every myoblast was labeled with BrdU at concentrations between 10(-7) M and 10(-5) M. When the cells were labeled with 0.5 microM or more, the percentage of labeled cells remained over 90% and 80% at 2 and 5 days, respectively. However, when the cells were labeled with BrdU concentration lower than 10(-7) M the percentage of labeled nuclei decreased more rapidly with time. The BrdU-labeled cells were mixed with an unlabeled population to determine whether their capacity to fuse was reduced. At a BrdU concentration of 0.5 x 10(-6) M, labeled myoblasts fused to a similar extent as unlabeled myoblasts, and a high percentage of marked cells were still perceptively labeled after 5 days. In contrast, the fusion capacity of myoblasts incubated with more than 10(-6) M BrdU was inhibited after only few rounds of DNA synthesis. These myoblasts were eventually able to fuse, however, when the BrdU diminished in the DNA due to cell division. These results indicate that labeling with BrdU at a concentration of 0.5 x 10(-6) M and an incorporation time of 48 hr is optimal to obtain perceptible immunocytochemical staining without affecting myoblast fusion. Such BrdU immunolabeling could be used as a nuclear marker for hybridization studies.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

Early events in murine erythroleukemia cells induced to differentiate: Variation of the cell cycle parameters in relation to p53 accumulation

Among the early events of induced differentiation of murine erythroleukemia cells that we studied was the variations of cell distribution in the cell cycle as a function of the time of induction. Flow-cytofluorimetry measurements of DNA content and BrdU incorporation allowed for a precise determination of the variations of the cell cycle parameters. Cells underwent a transient arrest in both G1 and G2 + M between 6 to 16 h of induction. The progression of the cells through S phase seems not to be affected during this period. After this time cells escaped from G1 and reentered the S phase. We described previously [S. Khochbin et al. (1988) J. Mol. Biol. 200, 55-64], that p53 decreased continuously during the induction of MELC and remained at a steady-state level after 18 to 20 h of induction. In order to look for a possible redistribution of the protein along the cell cycle during the induction process, we measured the accumulation of the protein along the cell cycle. In noninduced cells there were four steps in the accumulation of the protein throughout the cell cycle: the amount of p53 was constant during G1 and it increased as cells progressed through S phase, which is characterized by an increased accumulation at the G1/S transition and a more moderate accumulation during progression through the rest of the S phase. A constant level in G2/M, approximately twice that obtained in G1, was achieved. There was no change in this distribution that correlated with the various modifications of the cell cycle in induced cells. It seems then, that p53 is associated neither with the progression of the cells in the S phase nor with the resumption of the DNA synthesis after the G1 block.     

Effect of cadmium on cell cycle progression in Chinese hamster ovary cells

Chinese hamster ovary K1 (CHO K1) cells are very sensitive to cadmium (Cd) toxicity. They were used to investigate the effect of Cd on cell cycle progression. Cells were cultured with 0.1, 0.4, 1 or 4 microM Cd for various time intervals. There was no difference in growth rate when less than 0.4 microM Cd was given within 24 h. A dose-dependent reduction of cell proliferation was observed when more than 0.4 microM of Cd was given. The cells were pulse-labeled with 5-bromodeoxyuridine (BrdU), and the labeled cells were cultured in the presence of increasing concentrations of Cd. Cell cycle progression was retarded as a function of Cd concentration. G2/M arrest was observed when the BrdU-labeled cells were treated with 1 microM Cd for 8h, whereas cells receiving 4 microM Cd stopped at the S phase within 4 h. Cell cycle analysis of cells treated with Cd for 24 h showed that G2/M arrest occurred only when cells received 0.8 to 2 microM Cd. Despite the occurrence of G2/M arrest in the Cd treatment, only a limited proportion of the cells were blocked in the M phase. However, the increase in M phase cells coincided with an elevation in the cyclin-dependent kinase 1 activity. To examine whether Cd acts on cells at a specific cell stage, they were synchronized at the G1 or G2/M phase then treated with 1 microM Cd for 12 h. The cells were blocked at the G2/M and G1/S phase, respectively. This finding indicates that Cd toxicity is global and not cell phase specific. We also investigated the involvement of Cd-induced reactive oxygen species (ROS) with the occurrence of G2/M block and found a lack of correlation between cell cycle arrest and ROS production. We measured the Cd content that caused G2/M arrest from a series of Cd treatments and determined the ranges of cumulative Cd concentrations that could result in cell cycle arrest.     

Mathematical model analysis of mouse epidermal cell kinetics measured by bivariate DNA/anti-bromodeoxyuridine flow cytometry and continuous [3H]-thymidine labelling

In a previous study the epidermal cell kinetics of hairless mice were investigated with bivariate DNA/anti-bromodeoxyuridine (BrdU) flow cytometry of isolated basal cells after BrdU pulse labelling. The results confirmed our previous observations of two kinetically distinct sub-populations in the G2 phase. However, the results also showed that almost all BrdU-positive cells had left S phase 6-12 h after pulse labelling, contradicting our previous assumption of a distinct, slowly cycling, major sub-population in S phase. The latter study was based on an experiment combining continuous tritiated thymidine [( 3H]TdR) labelling and cell sorting. The purpose of the present study was to use a mathematical model to analyse epidermal cell kinetics by simulating bivariate DNA/BrdU data in order to get more details about the kinetic organization and cell cycle parameter values. We also wanted to re-evaluate our assumption of slowly cycling cells in S phase. The mathematical model shows a good fit to the experimental BrdU data initiated either at 08.00 hours or 20.00 hours. Simultaneously, it was also possible to obtain a good fit to our previous continuous labelling data without including a sub-population of slowly cycling cells in S phase. This was achieved by improving the way in which the continuous [3H]TdR labelling was simulated. The presence of two distinct subpopulations in G2 phase was confirmed and a similar kinetic organization with rapidly and slowly cycling cells in G1 phase is suggested. The sizes of the slowly cycling fractions in G1 and G2 showed the same distinct circadian dependency. The model analysis indicates that a small fraction of BrdU labelled cells (3-5%) was arrested in G2 phase due to BrdU toxicity. This is insignificant compared with the total number of labelled cells and has a negligible effect on the average cell cycle data. However, it comprises 1/3 to 1/2 of the BrdU positive G2 cells after the pulse labelled cells have been distributed among the cell cycle compartments.     

Measurement of c-myc protein content and cell cycle kinetics of normal and spontaneously transformed murine mastocytes by bivariate flow cytometry

Progressive in vitro culturing of interleukin-3 (IL-3) dependent normal murine mastocytes (PB-3) resulted in a variant cell line (PB-1) able to grow without exogenous IL-3 and which was tumorogenic in syngenic mice. Bivariate flow cytometry was used to evaluate the c-myc protein and DNA content of PB-3 and PB-1 cells. The c-myc protein was detected by specific monoclonal antibodies. Kinetic characteristics of PB-3 and PB-1 cell lines, namely, the duration of the G1, S and G2 + M cell cycle phases were also evaluated using the bromodeoxyuridine (BrdU) pulse-chase method and BrdU/DNA flow cytometry. Levels of c-myc protein in PB-1 cells were about two-fold higher than those of PB-3 cells in all cell cycle phases. Mean duration of the cell cycle (Tc) was 15.3 h for PB-3 cells and 12.4 h for PB-1 cells. Shortening in Tc for the transformed cells was due to a decrease of nearly 30% in mean duration of the G1 phase (from 8 h to 5.7 h). No significant differences were found in the duration of the S and G2 + M phases. These results indicate that acquired IL-3 independency in vitro and tumorogenicity of PB-1 cells were accompanied by a doubling of c-myc protein level and by a parallel shortening, or bypass, of the regulatory events within the G1 phase of the cell cycle.     

Flow cytometric analysis of X-ray sensitivity in ataxia telangiectasia

Flow cytometric analysis of 5-bromodeoxyuridine (BrdU) incorporation during DNA synthesis was used to characterize the effects of X-rays on cell-cycle kinetics in the DNA-repair deficiency disease ataxia telangiectasia (AT). Cultured fibroblasts from homozygotes (at/at), heterozygotes (at/+) and normal controls (+/+) were either: (1) irradiated, cultured, then pulsed with BrdU and harvested, or (2) pulsed with BrdU, irradiated, cultured and then harvested. Cells were then fixed and stained with both a fluoresceinated monoclonal antibody against BrdU to identify S-phase cells and with propidium diiodide to measure total DNA content. Irradiation of +/+ and at/+ cells induced a similar, transient G2/M arrest detectable within 8 h, which subsequently delayed by 6-8 h the passage of cells into G1 and depleted early S phase. In contrast, at/at cells failed to arrest in G2/M phase and entered the next cell cycle without pausing to repair radiation-induced damage. X-Rays also blocked entry of +/+ G1 cells into S phase, subsequently reducing the total S-phase population. This effect was not observed in at/at cells. These cell-cycle responses to radiation may be of diagnostic use and ultimately may help explain the basic defect in AT.