The re-incarnation, re-interpretation and re-demise of the transition probability model.

There are two classes of models for the cell cycle that have both a deterministic and a stochastic part; they are the transition probability (TP) models and sloppy size control (SSC) models. The hallmark of the basic TP model are two graphs: the alpha and beta plots. The former is the semi-logarithmic plot of the percentage of cell divisions yet to occur, this results in a horizontal line segment at 100% corresponding to the deterministic phase and a straight line sloping tail corresponding to the stochastic part. The beta plot concerns the differences of the age-at-division of sisters (the beta curve) and gives a straight line parallel to the tail of the alpha curve. For the SC models the deterministic part is the time needed for the cell to accumulate a critical amount of some substance(s). The variable part differs in the various variants of the general model, but they do not give alpha and beta curves with linear tails as postulated by the TP model. This paper argues against TP and for an elaboration of SSC type of model. The main argument against TP is that it assumes that the probability of the transition from the stochastic phase is time invariant even though it is certain that the cells are growing and metabolizing throughout the cell cycle; a fact that should make the transition probability be variable. The SSC models presume that cell division is triggered by the cell's success in growing and not simply the result of elapsed time. The extended model proposed here to accommodate the predictions of the SSC to the straight tailed parts of the alpha and beta plots depends on the existence of a few percent of the cell in a growing culture that are not growing normally, these are growing much slower or are temporarily quiescent. The bulk of the cells, however, grow nearly exponentially. Evidence for a slow growing component comes from experimental analyses of population size distributions for a variety of cell types by the Collins-Richmond technique. These subpopulations existence is consistent with the new concept that there are a large class of rapidly reversible mutations occurring in many organisms and at many loci serving a large range of purposes to enable the cell to survive environmental challenges. These mutations yield special subpopulations of cells within a population. The reversible mutational changes, relevant to the elaboration of SSC models, produce slow-growing cells that are either very large or very small in size; these later revert to normal growth and division. The subpopulations, however, distort the population distribution in such a way as to fit better the exponential tails of the alpha and beta curves of the TP model.     

Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions.

An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.     

Synchronized generation of reactive oxygen species with the cell cycle

A possible appearance of reactive oxygen species (ROS) with the normal cell cycle was studied to find how ROS are generated in cells in relation to the cell cycle. The production of ROS in relation to the cell cycle was examined by determining the changes in intracellular ROS concentrations at different phases of the cell cycle by culturing BALB 3T3 cells in the presence and absence of aphidicolin. The amounts of intracellular ROS and the cell population at specific phases (S and G2/M) were determined as the fluorescence of dichlorodihydrofluorescein and propidium iodide taken up simultaneously by the cells, respectively, by flow cytometry. Although intracellular ROS remained at the control levels when the cell growth was arrested with aphidicolin at the G1 phase, they increased when the arrest was released to result in the increase of the cell population at the S phase. Furthermore, ROS was shown to disturb/stop the cell cycle by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The cell cycle was regulated through oxidative stress by exposure to hydrogen peroxide and glutathione ethyl ester. The cell cycle was prevented more sensitively in metallothionein-null cells than in the wild type cells. Based on the present observations, we proposed for the first time that ROS are generated synchronously with the normal cell cycle, and that they have to be controlled at certain level for normal progress of the cell cycle.     

MHC class I manipulation on cell surfaces by gene transfer of anti-MHC class I intrabodies--a tool for decreased immunogenicity of allogeneic tissue and cell transplants

Intrabodies (IB) are suitable tools to down-regulate the expression of cell surface molecules in general. In this work, the appearance of major histocompatibility (MHC) class I molecules on the cell surface could be prevented by the expression of intracellularly localized anti-MHC class I antibodies. The expression of MHC antigens presenting intracellularly synthetised peptides on the cell surface is the predominant reason for immunologic detection and rejection of allogeneic cell and tissue transplants. Allogeneic keratinocyte sheets might be a suitable tool for skin grafting. Within this study primary rat keratinocytes have been transfected with anti-MHC I-IB. Strong IB-expressing cells showed a MHC I "knockout" phenotype. The cells did not exhibit any significant alterations compared to non-transfected cells: the cell growth and the expression of other surface molecules were unaltered. Merely an enhanced intracellular accumulation of MHC I molecules could be detected. Notably, IB-expressing keratinocytes displayed a reduced susceptibility to allogeneic cytotoxic T cells in vitro compared to unmodified cells with a normal level of MHC I surface expression. These MHC I-deficient keratinocytes might be utilized in tissue-engineered allogeneic non-immunogeneic skin transplants. The principle of MHC class I manipulation in general can be used for other allogeneic cell and tissue-engineered transplants as well.     

Midblastula transition (MBT) of the cell cycles in the yolk and pigment granule-free translucent blastomeres obtained from centrifuged Xenopus embryos

We obtained translucent blastomeres free of yolk and pigment granules from Xenopus embryos which had been centrifuged at the beginning of the 8-cell stage with cellular integrity. They divided synchronously regardless of their cell size until they had decreased to 37.5 microm in radius; those smaller than this critical size, however, divided asynchronously with cell cycle times inversely proportional to the square of the cell radius after midblastula transition (MBT). The length of the S phase was determined as the time during which nuclear DNA fluorescence increased in Hoechst-stained blastomeres. When the cell cycle time exceeded 45 min, S and M phases were lengthened; when the cell cycle times exceeded 70 min, the G2 phase appeared; and after cell cycle times became longer than 150 min, the G1 phase appeared. Lengths of G1, S and M phases increased linearly with increasing cell cycle time. Enhanced green fluorescent protein (EGFP)-tagged proliferating cell nuclear antigen (PCNA) expressed in the blastomeres appeared in the S phase nucleus, but suddenly dispersed into the cytoplasm at the M phase. The system developed in this study is useful for examining the cell cycle behavior of the cell cycle-regulating molecules in living Xenopus blastomeres by fluorescence microscopy in real time.     

Heterogenous expression of a murine B16 melanoma-associated antigen correlates with cell cycle

Summary A murine monoclonal antibody (MM2-3C6) that reacts with a B16 murine melanoma-associated membrane antigen was used to study the relationship of antigen expression to the cell cycle. Dual-parameter flow cytometric measurements of membrane antigen and DNA revealed that antigen-positive cells were present throughout the cell cycle. Peak antigenic expression was noted during the late log phase of the cell growth curve with negligible antigen-negative population. The emergence of a distinct antigen-negative population (30%–40%) was noted in the late stationary phase. Cell cycle analysis indicated that the negative subpopulation was restricted to the G0/G1 phase, thus, demonstrating antigenic heterogeneity within the tumor cell population. Cell sorting was performed to analyze the origin of such heterogeneity. Following two sequential sortings, the antigen-negative cells became antigenic upon reculture. Again, at the late stationary phase, a distinct antigen-negative population (30%–40%) emerged. The sorted antigen-positive cells showed flow cytometric profiles identical to the sorted antigen-negative population upon reculture. Therefore, in this murine model, it appears that antigen expression is cell cycle dependent and such expression seems to be associated with proliferation.     

293 cell cycle synchronisation adenovirus vector production

As the market requirements for adenovirus vectors (AdV) increase, the maximisation of the virus titer per culture volume per unit time is a key requirement. However, despite the fact that 293 cells can grow up to 8 × 10⁶ cell/mL in simple batch mode operations, for optimal AdV infection a maximum cell density of 1 × 10⁶ cell/mL at infection time has usually been utilized due to the so called “cell density effect”. In addition, AdV titer appears to be dependent upon cell cycle phase at the time of infection. To evaluate the dependence of AdV production upon cell cycle phase, 293 cells were chemically synchronised at each phase of the cell cycle; a 2.6‐fold increase on AdV cell specific titer was obtained when the percentage of cells at the S phase of the cell cycle was increased from 36 to 47%; a mathematical equation was used to relate AdV cell specific productivities with cell synchronisation at the S phase using this data. To avoid the use of chemical inhibitors, a temperature shift strategy was also used for synchronisation at the S phase. S phase synchronisation was obtained by decreasing the culture temperature to 31°C during 67 h and restoring it to 37°C during 72 h. By using this strategy we were able to synchronise 57% of the population in the S phase of the cell cycle obtaining an increase of 7.3‐fold on AdV cell specific titer after infection. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cancer chemo-endocrine therapy and its cell biological basis

The aim of our cell kinetic studies is to better understand the effects of chemo-endocrine therapy at the cell biological and molecular level. Cancer cell growth is characterized by uncontrolled proliferation, resulting in DNA distribution pattern in which, at any time, more cells are not G1 phase but in S, G2 and M phase of a shortened cycle. In a recent progress, flow cytometry (FCM) has become a powerful tool for the quantitative analysis of cell cycle parameters by measuring nuclear DNA content in large cell population with high speed. With the aid of FCM in earlier work about 60-80% of ovarian cancers were found to contain aneuploid cells. Now, multi-parameter FCM linked to a computer is available to measure fluorescent intensities not only no base total DNA (Propidium iodide) but also A-T (Hoechst 33342) and G-C (Mithramycin) base pairs in solid cancer nuclei. Since cisplatinum (CDDP) is the most important drug in the treatment of ovarian cancer, we have studied the relationship of CDDP cytotoxicity, pertubations cell cycle kinetis and DNA damage in ovarian adenocarcinoma cells in vitro & in vivo. We employed both CDDP sensitive cell line (KFt) and resistant cell line (KFr) derived from human serous cystoadenocarcinoma of the ovary by Kikuchi et al (JNCI 1986). Comparing cell kinetic pertubations of experimental cells demonstrates a decrease in G1 phase cells concomitant increase in S phase cells. The KFr cells had distinctly a shorter S-phase block up to 24 hrs not A-T but G-C preference in a quick response followed repairing of DNA damage to 48 hrs. However, some fractions of CDDP resistant cell population showed a later onset of G2, M phase accumulation. Comparison with the increase in early S phase cells of KFr in detailed analysis suggests only those damaged cells that are not killed immediately may proceed to G1 phase and start into DNA synthesis in S phase. Measurement of labeling index (L. I.) with Bromodeoxyuridine (BrdU) support our interpretation of differences between sensitivity and resistance to anti-cancer drug. Additionally, we discuss a targeting chemotherapy by coupling cytotoxic drugs with estrogen based on increasing DNA damage into apoptosis and interfares with DNA repair process.     

Cell wall mannoproteins during the population growth phases in Saccharomyces cerevisiae

Mannoproteins from cell walls of Saccharomyces cerevisiae synthesized at successive stages of the population growth cycle have been solubilized with Zymolyase and subsequently analyzed. The major change along the population cycle concerned a large size mannoprotein material; the size of the newly-synthesized molecules varied from 120,000–500,000 (mean of about 200,000) at early exponential phase to 250,000–350,000 (mean of about 300,000) at late exponential phase. These differences are due to modifications in the amount of N-glycosidically linked mannose residues, since the size of the peptide moiety was 90,000–100,000 at all growth stages and the level of O-glycosylation changed only slightly. After, incubation of the purified walls with concanavalin A-ferritin and subsequent analysis by electron microscopy, labelling was localized at the external and internal faces of the walls. The middle space of these was labelled after digestion of the glucan network with Zymolyase, which demonstrate the presence of mannoproteins in close contact with the structural glucan molecules throughout the wall.Abbreviations BSA bovine serum albumin - Con A concanavalin A - SDS sodium dodecyl sulphate     

Effect of conditioned medium factors on productivity and cell physiology in Trichoplusia ni insect cell cultures

The influence of conditioned medium (CM) on cell physiology and recombinant protein production in Trichoplusia ni insect cells (T. ni, BTI-Tn-5B1-4) has been investigated. Cell cycle analysis showed that a high proportion of the cell population (80-90%) was in G1 during the whole culture, indicating that the S and G2/M phases are short relative to the G1 phase. Directly after inoculation, a rapid decrease of the S-phase population occurred, which could be observed as a lag-phase. The following increase in the number of cells in S occurred after 7 h of culture for cells in fresh medium, whereas for cells with the addition of CM it occurred at an earlier time point (5 h) and these cells had therefore a shorter lag-phase. The initial changes in the S-phase population were also affected by the inoculum cell density, as higher seeding cell densities resulted in a more rapid increase in the S-phase population after inoculation. These changes in cell cycle distribution were reflected in the cell size, and the CM-cells were smaller than the cells in fresh medium. Recombinant protein production in T. ni cells was improved by the addition of CM. The specific productivity was increased by 30% compared to cells in fresh medium. This beneficial effect was seen between 20 and 72 h of culture. In contrast, the highest specific productivity was obtained already at 7 h for the cells in fresh medium and then decreased rapidly. The total product concentration was around 30% higher in the culture with CM compared to the culture in fresh medium, and the maximum product concentration was obtained on day 2 compared to day 3 for the cells in fresh medium. Our results indicate that the positive effect on productivity by CM is related to its growth-promoting effect, suggesting that the proliferation potential of the culture determines the productivity.     

Mouse L cell mitochondrial DNA molecules are selected randomly for replication throughout the cell cycle

The number of mitochondrial DNA molecules in a cell population doubles at the same rate as the cell generation time. This could occur by a random selection of molecules for replication or by a process that ensures the replication of each individual molecule in the cell. We have investigated the rate at which mouse L cell mitochondrial DNA molecules labeled with 3H-thymidine during one round of replication are reselected for a second round of replication. Mouse L cells were labeled with 3H-thymidine for 2 hr, chased for various periods of time and then labeled with 5-bromodeoxyuridine for 4 hr immediately before mitochondrial DNA isolation. A constant fraction of 3H-thymidine-labeled mitochondrial DNA incorporated 5-bromodeoxyuridine after chase intervals ranging from 1.5-22 hr. This result demonstrates that mitochondrial DNA molecules replicated in a short time interval are randomly selected for later rounds of replication, and that replication of mitochondrial DNA continues throughout the cell cycle in mouse L cells.     

Prespore cell fate bias in G1 phase of the cell cycle in Dictyostelium discoideum

By generating a population of Dictyostelium cells that are in the G1 phase of the cell cycle we have examined the influence of cell cycle status on cell fate specification, cell type proportioning and its regulation, and terminal differentiation. The lack of observable mitosis during the development of these cells and the quantification of their cellular DNA content suggests that they remain in G1 throughout development. Furthermore, chromosomal DNA synthesis was not detectable these cells, indicating that no synthesis phase had occurred, although substantial mitochondrial DNA synthesis did occur in prespore cells. The G1-phase cells underwent normal morphological development and sporulation but displayed an elevated prespore/prestalk ratio of 5.7 compared to the 3.0 (or 3:1) ratio normally observed in populations dominated by G2-phase cells. When migrating slugs produced by G1-phase cells were bisected, each half could reestablish the 5.7 (or 5.7:1) prespore/prestalk ratio. These results demonstrate that Dictyostelium cells can carry out the entire developmental cycle in the G1 phase of the cell cycle and that passage from G2 into G1 phase is not required for sporulation. Our results also suggest that the population asymmetry provided by the distribution of cells around the cell cycle at the time of starvation is not strictly required for cell type proportioning. Finally, when developed together with G2-phase cells, G1-phase cells preferentially become prespore cells and exclude G2-phase cells from the prespore-spore cell population, suggesting that G1-phase cells have an advantage over G2-phase cells in executing the spore cell differentiation pathway.     

Cell cycle dynamics inferred from the static properties of cells in balanced growth

The duration of a morphological phase of the cell cycle is reflected in the steady state distribution of the sizes of cells in that phase. Relationships presented here provide a method for estimating the timing and variability of any cell cycle phase. It is shown that the mean size of cells initiating and finishing any phase can be estimated from (1) the frequency of cells exhibiting the distinguishing morphological or autoradiographic features of the phase; (2) the mean size of cells in the phase; and (3) their coefficient of variation. The calculations are based on a submodel of the Koch-Schaechter Growth Controlled Model which assumes that (i) the distribution of division sizes is Gaussian; (ii) there is no correlation in division sizes between successive generations; and (iii) every cell division gives rise to two daughter cells of equal size. The calculations should be useful for a wider range of models, however, because the extrapolation factors are not sensitive to the chosen model. Criteria are proposed to allow the user to check the method's applicability for any experimental case. The method also provides a more efficient test of the dependence of growth on cell size than does the Collins-Richmond method. This is because the method uses the mean and coefficient of variation of the size of the total population, in conjunction with those of the cells in a final phase of the cell cycle, to test potential growth laws. For Escherichia coli populations studied by electron microscopy, an exponential growth model provided much better agreement than did a linear growth model. The computer simulations were used to generate rules for three types of cell phases: those that end at cell division, those that start at cell division, and those totally contained within a single cell cycle. For the last type, additional criteria are proposed to establish if the phase is well enough contained for the formulae and graphs to be used. The most useful rule emerging from these computer studies is that the fraction of the cell cycle time occupied by a phase is the product of the frequency of the phase and the ratio of the mean size of cells in that phase to the mean size of all cells in the population. A further advantage of the techniques presented here is that they use the 'extant' distributions that were actually measured, and not hypothesized distributions nor the special distributions needed for Collins-Richmond method that can only be calculated from the observed distributions of dividing or newborn cells on the basis of an assumed growth law.     

cAMP-mediated increase in the critical cell size required for the G1 to S transition in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, cyclic AMP is required for cellular growth. In this study we show that cAMP also specifically inhibits the G1-S transition of the S. cerevisiae cell cycle by increasing the critical cell size required at start, the major yeast cell cycle control step. In fact: (a) addition of cAMP delays the time of entering into the S budded phase of small G1 cells, while it is ineffective on large fast-growing cells. (b) If cell growth is strongly depressed, cAMP permanently inhibits cell cycle commitment of cells arrested at the α-factor-sensitive step. The cell fraction inhibited by cAMP is inversely correlated with the average cell size of treated populations. (c) The critical protein content (P_s) and the critical cell volume (V_B) required for budding in unperturbed exponentially growing yeast populations are largely increased by cAMP. On these bases, we propose a new cAMP role at start.     

Susceptibility of Hep3B cells in different phases of cell cycle to tBid

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC.     

The cell cycle of<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: the role of the commitment point

Chlamydomonas reinhardtii cells can double their size several times during the light period before they enter the division phase. To explain the role of the commitment point (defined as the moment in the cell cycle after which cells can complete the cell cycle independently of light) and the moment of initiation of cell division we investigated whether the timing of commitment to cell division and cell division itself are dependent upon cell size or if they are under control of a timer mechanism that measures a period of constant duration. The time point at which cells attain commitment to cell division was dependent on the growth rate and coincided with the moment at which cells have approximately doubled in size. The timing of cell division was temperature-dependent and took place after a period of constant duration from the onset of the light period, irrespective of the light intensity and timing of the commitment point. We concluded that at the commitment point all the prerequisites are checked, which is required for progression through the cell cycle; the commitment point is not the moment at which cell division is initiated but it functions as a checkpoint, which ensures that cells have passed the minimum cell size required for the cell division.     

The response of malignant B lymphocytes to ionizing radiation: cell cycle arrest, apoptosis and protection against the cytotoxic effects of the mitotic inhibitor nocodazole

Ionizing radiation and mitotic inhibitors are used for the treatment of lymphoma. We have studied cell cycle arrest and apoptosis of three human B-lymphocyte cell lines after X irradiation and/or nocodazole treatment. Radiation (4 and 6 Gy) caused arrest in the G(2) phase of the cell cycle as well as in G(1) in Reh cells with an intact TP53 response. Reh cells, but not U698 and Daudi cells with defects in the TP53 pathway, died by apoptosis after exposure to 4 or 6 Gy radiation (>15% apoptotic Reh cells and <5% apoptotic U698/Daudi cells 24 h postirradiation). Lower doses of radiation (0.5 and 1 Gy) caused a transient delay in the G(2) phase of the cell cycle for the three cell lines but did not induce apoptosis (<5% apoptotic cells at 24 h postirradiation). Cells of all three cell lines died by apoptosis after exposure to 1 microg/ml nocodazole, a mitotic blocker that acts by inhibiting the polymerization of tubulin (>25% apoptotic cells after 24 h). When X irradiation with 4 or 6 Gy was performed at the time of addition of nocodazole to U698 and Daudi cells, X rays protected against the apoptosis-inducing effects of the microtubule inhibitor (<5% and 15% apoptotic cells, respectively, 24 h incubation). U698 and Daudi cells apparently have some error(s) in the signaling pathway inducing apoptosis after irradiation, and our results suggest that the arrest in G(2) prevents the cells from entering mitosis and from apoptosis in the presence of microtubule inhibitors. This arrest was overcome by caffeine, which caused U698 cells to enter mitosis (after irradiation) and become apoptotic in the presence of nocodazole (26% apoptotic cells, 24 h incubation). These results may have implications for the design of clinical multimodality protocols involving ionizing radiation for the treatment of cancer.     

Transition of the blastomere cell cycle from cell size-independent to size-dependent control at the midblastula stage in Xenopus laevis

Dissociated animal cap blastomeres of Xenopus laevis blastulae were cultured at a low Ca level (1 microM) from 9th to 18th cell cycle at 22 +/- 1 degrees C and observed by a time-lapse video recorder. Blastomeres cleaved unequally to increase variability in cell size as cell cycles progressed, but synchronously at a constant cell cycle time of about 30 min up to the 12th cleavage in diploid cells, and up to the 13th cleavage in haploid cells, regardless of their cell sizes. Thereafter, blastomeres cleaved asynchronously at varying cell cycle times in proportion to the inverse square of their radii. The transition from the cell size-independent to -dependent cell cycles occurred at the critical cell radius, 37.5 microm for the diploid and 27.9 microm for the haploid. While the protein synthesis inhibitor, cycloheximide (CHX) lengthened cell cycle times two- to six-fold, epidermal growth factor (EGF) had no significant effect on the cell cycle. CHX-treated blastomeres synchronously cleaved at a constant cell cycle time of 60 min up to the 12th cleavage. Thereafter, cell cycle times became variable in proportion to the inverse square of radii in the presence of CHX at 0.10-0.14 microg/ml, but to the inverse cube of radii at 0.18 microg/ml. The critical cell size of CHX-treated blastomeres for the transition from cell size-independent to -dependent cell cycles remained the same as that of untreated blastomeres. Frequency distributions of cell cycle times of synchronous cell cycles were monomodal with the peak at 30 min, except for CHX-treated blastomeres with the peak at 60 min. In contrast, frequency distributions of asynchronous cell cycles were polymodal with peaks at multiples of a unit time of 30-35 min. To explain these results, we propose that blastomere cytoplasm has 30-min cycles that repeatedly produce mitosis promoting factor (MPF) in a quantity proportional to the cell surface area. MPF is neutralized when it titrates a nuclear inhibitor present in a quantity proportional to the genome size, and sequestered in the nucleus. When the total amount of MPF produced exceeds the threshold required to titrate all of the inhibitor, mitosis is initiated.     

Concentration-dependent effects of potassium dichromate on the cell cycle

Hexavalent chromium is found to be a strong mutagen, and it also is a potential carcinogen in man. DNA flow cytometry, growth measurements, and determinations of mitotic index show that 1-2 microM K2Cr2O7 produces a prolongation of the G2 phase of the cell cycle in NHIK 3025 cells. By increasing the chromate concentrations (greater than 2 microM K2Cr2O7) the cells are also arrested in G2 phase. We have found, using synchronized cells and measuring cell cycle time, that the most chromate-sensitive part of the cell cycle is S phase. This phase is also somewhat prolonged, and the cells became arrested in early S phase at high toxic K2Cr2O7 concentrations (8 microM). Our results thus indicate that K2Cr2O7 has an effect within S phase--maybe on DNA/RNA synthesis--and also interferes with processes necessary for progression through the G2 phase.     

Abrogation of G(2)/M-phase block enhances the cytotoxicity of daunorubicin, melphalan and cisplatin in TP53 mutant human tumor cells

Irradiation of human melanoma (MeWo, Be11) and squamous cell carcinoma (4451, 4197) cells induces cell cycle blocks from which the cells recover to re-enter mitosis after 40-60 h. In the TP53 mutant cell lines, MeWo and 4451, irradiation induces a G(2)-phase block, where the fraction of cells in G(2) phase reaches a maximum after 18-20 h. In the TP53 wild-type cell lines, 4197 and Be11, a G(1)- and G(2)-phase block is reached 12 and 16 h postirradiation, respectively. Addition of pentoxifylline after irradiation at the time when the number of cells in G(2) phase has reached a maximum shortens the normal recovery from G(2)-phase block to approximately 7 h. Addition of daunorubicin, melphalan and cisplatin under these conditions markedly enhanced drug toxicity. In the TP53-mutated cell lines MeWo and 4451, the survival ratio at 7 Gy measured by colony formation was 2.3-2.8, 8.6-85 and 52-74 for daunorubicin, melphalan and cisplatin, respectively. In the TP53 wild-type cell lines, the corresponding survival ratios were found to be 1.3-1.4, 2.3-3.0 and 1.2-2.6, respectively. The survival ratios are for clonogenic survival after 7 Gy and 2 mM pentoxifylline and measure the influence of drug doses that ensure 95% survival in nonirradiated controls. The results indicate that the G(2)-phase block is a crucial event in the damage response that can be manipulated to achieve a significant enhancement of drug toxicity. These effects are particularly pronounced in TP53 mutant cells and are observed at drug doses well below the clinical range.