On the existence of 'arrested G2 cells' in mouse epidermis

It has been postulated that mouse epidermis contains two populations of resting cells, one of which is blocked at the G1-S boundary and the other between G2 and mitosis. The 'arrested G2 cells' were estimated, by the labelled mitosis method, to comprise 5-10% of the epidermal population and presumed to function as a 'reserve pool' which could be activated by wounding. A comprehensive search has now been carried out for arrested G2 cells in mouse epidermis using the direct methods of single cell and flow through cytophotometry. No evidence was obtained which supports the existence of such a cell compartment. Suitable control experiments were carried out to ensure that G2 cells were not lost during the isolation of epidermal nuclei.     

Chalones and the Control of Normal, Regenerative, and Neoplastic Growth of the Skin

SYNOPSIS Chalones,inhibitors of cell dmsion have been isolatedand studied from a number of mammalian tissues, most notably,the epidermis The epidermal rhalone is a glycoprotein It exhibitsconsiderable, but not complete specificity The epidermal chalone decreases mitotic activity by inhibitingcells in the G 2 phase of the cell cycle from entering mitosis,and probably also by inhibiting ceils in the G 1 phase of thecell cycle from entering mitosis To inhibit cells in G 2 fromentering mitosis the chilone requnes adrenalin, and for maximalactivity hydrocortisone It is not known if idrenalin and hydrocortisoneare required for chalone inhibition of cells in G 1 In addition to inhibiting cell division in normal epidermalcells the epidermal chalone can inhibit cell division in regeneratingepidermal cells induced to proliferate by chemical damage Thephase of the cell cycle in which the chalone inhibits legeneratingepidermal cells from entering mitosis is not known Epidermal tumors contain a decreased amount of chalone Mitosisin epidermal tumors is inhibited by treatment with epidermalchalone Tumor cells are inhibitedfrom entering mitosis fromeither the G 1 or G 2 phases of the cell cycle Chalones are said to inhibit mitosis by a negative feedbackmechanism However, experiments which presumably result in adecrease in chalone concentration do not result in an increasein mitotic activity It is suggested that if chalones are physiological controllers of cell division they do not act by a simplenegative feedback mechanism but require the action of a substanceto decrease their concentration     

ON THE EXISTENCE OF 'ARRESTED G2 CELLS' IN MOUSE EPIDERMIS

It has been postulated that mouse epidermis contains two populations of resting cells, one of which is blocked at the G₁-S boundary and the other between G₂ and mitosis. the ‘arrested G₂ cells’ were estimated, by the labelled mitosis method, to comprise 510% of the epidermal population and presumed to function as a ‘reserve pool’ which could be activated by wounding. A comprehensive search has now been carried out for arrested G₂ cells in mouse epidermis using the direct methods of single cell and flow through cytophotometry. No evidence was obtained which supports the existence of such a cell compartment. Suitable control experiments were carried out to ensure that G₂ cells were not lost during the isolation of epidermal nuclei.     

Circadian rhythms in mouse epidermal basal cell proliferation. Variations in compartment size, flux and phase duration.

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G2 phase DNA content were measured by flow cytometry of isolated basal cells, and the [3H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G2 phase and traversed the G2 phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G2 phase and mitosis. Circadian variations in the duration of the S phase, the G2 phase and mitosis were clearly demonstrated.     

The Dictyostelium cell cycle and its relationship to differentiation

Abstract The Dictyostelium vegetative cell cycle is characterized by a short mitotic period followed immediately by a short S-phase (less than 30 min) and a long and variable G2 phase. The cell cycle continues during differentiation despite a decrease in cell mass: DNA replication and mitosis occur early in development and also at the tipped aggregate stage. Cells that are in mitosis, S-phase or early G2, when starved differentiate into prestalk cells and cells that are in the middle of G2 differentiate into prespore cells. We postulate that there is a restriction point late in the G2 phase, about 1–2 h before mitosis, where the cells can be arrested either by starvation and the initiation of development, by growing into stationary phase, or by prolonged incubation at low temperature. During development, this block persists to the tipped aggregate stage, where it is specifically released in prespore cells, and these cells then go through one more round of cell division. Genes encoding components of the cell cycle machinery have recently been isolated and attemps to specifically block the cell cycle by reverse genetics to study the effects on differentiation have been initiated.     

Lethal response of HeLa cells to x-irradiation in the latter part of the generation cycle.

The age-response for the killing of HeLa S3 cells by X-rays during the latter part of the generation cycle has been examined in detail. As synchronous cells move from the G1/S boundary through S phase, the relatively high sensitivity of late G1 cells gradually decreases; minimum sensitivity is reached in mid-S and maintained during the remainder of that phase. The response of cells as they progress from S to the point in G2 at which they are temporarily arrested by radiation (or by inhibitors of protein synthesis) was measured in populations free of both S phase cells and late G2 cells that had passed the arrest point: cells retain their high resistance from early G2 up to the arrest point. The response of G2 cells that have passed the arrest point before being irradiated was examined by exposing randomly growing cultures to X-rays and collecting cells periodically thereafter, as they entered mitosis. Survival values very close to those of sensitive mitotic cells were found in the 2 h period after irradiation during which unarrested cells continued to reach mitosis. Values typical of lateS/early G2 were found only after cells that had been arrested began arriving at mitosis. Thus, HeLa S3 cell undergo an abrupt increase in sensitivity at or near the arrest point. The sensitivity to a second irradiation of cells arrested in G2 by a conditioning X-ray dose increases rapidly in the early part of the arrest period.     

Localization of MPM-2 recognized phosphoproteins and tubulin during cell cycle progression in synchronized Vicia faba root meristem cells.

MPM-2 antibody reacts with a subset of mitotic phosphoproteins. We followed localization of MPM-2 immunoreactive material and localization of microtubules during cell cycle progression in a highly synchronous population of Vicia faba root meristem cells and isolated nuclei. The MPM-2 antibody labelling showed significant cell cycle dependence. MPM-2 nuclear reactivity was weak and homogeneous in G1 and S phase of the cell cycle and became stronger and heterogeneous during G2, resembling staining of the nuclear matrix, with maximum staining at the G2/M interface. Similarly the staining intensity of nucleoli increased from late G1 phase to nucleoli dispersion in early prophase. During mitosis MPM-2 immunoreactivity was associated with spindle configurations and the brightest signal was localized in kinetochores from prophase to metaphase.     

Radioautographic and cytofluorimetric analysis of DNA synthesis during the pre-implantation period of rat and mouse embryonic development]

The patterns of DNA synthesis and kinetics of cell population in the rat and mouse embryos were studied by means of 3H-thymidine autoradiography and cytofluorimetry. The rat and mouse embryos during the period of cleavage consist of a heterogenous population of blastomeres. At all the stages under study, all phases of the cell cycle occur in the blastomeres: G1, S, G2 and mitosis. The embryonic cells were distributed into groups containing 2c, 3c, 4c and more DNA. The ratio of cell number in these groups differed in the mouse and rat embryos. The mouse embryos are characterized by the appearance of a considerable amount of polyploid cells in S phase at the morula stage. The stage and species specific quantitative and qualitative patterns were established for DNA synthesis and kinetics of the cell population of blastomeres.     

The effect of chalone on the cell cycle in the epidermis during wound healing

A cut was made on the ear conch of mouse and an extract containing epidermal chalone was injected subcutaneously 2 days later. The time changes after the chalone administration in the number of cells labeled with ³H-thymidine, in the number of grains on labeled cells and in the number of mitoses within the regenerating epidermis surrounding the wound were investigated by means of autoradiography (ARG). Grain counts decreased temporarily in early phase (0–2 h) after chalone injection. This decrease in grain count resulted in a decrease in the number of labeled cells on the ARG of a short exposure but not in that on the ARG of a long exposure. A decrease in the number of labeled cells on the ARG of a long exposure was evident at 6 h when the grain counts reverted to a level similar to the control without chalone. The number of mitoses reached a minimum at 2 h and then recovered quickly, indicating a rapid disappearance of the inhibition of cells in G 2 from entering M phase. Mitoses decreased again thereafter, presumably as a result caused by inhibition of cells in the preceding S phase from completing DNA synthesis. The extract made similarly from liver or kidney affected neither the mitotic nor the DNA synthetic activities.These results indicate that the epidermal chalone or chalones inhibit the epidermal cell proliferation in, at least, 3 different processes of the cell cycle; the DNA synthesis in S phase, the transition from G 1 to S phase and the transition from G 2 to M phase.     

Coordination of mitosis and morphogenesis: role of a prolonged G2 phase during chordate neurulation

Chordates undergo a characteristic morphogenetic process during neurulation to form a dorsal hollow neural tube. Neurulation begins with the formation of the neural plate and ends when the left epidermis and right epidermis overlying the neural tube fuse to close the neural fold. During these processes, mitosis and the various morphogenetic movements need to be coordinated. In this study, we investigated the epidermal cell cycle in Ciona intestinalis embryos in vivo using a fluorescent ubiquitination-based cell cycle indicator (Fucci). Epidermal cells of Ciona undergo 11 divisions as the embryos progress from fertilization to the tadpole larval stage. We detected a long G2 phase between the tenth and eleventh cell divisions, during which fusion of the left and right epidermis occurred. Characteristic cell shape change and actin filament regulation were observed during the G2 phase. CDC25 is probably a key regulator of the cell cycle progression of epidermal cells. Artificially shortening this G2 phase by overexpressing CDC25 caused precocious cell division before or during neural tube closure, thereby disrupting the characteristic morphogenetic movement. Delaying the precocious cell division by prolonging the S phase with aphidicolin ameliorated the effects of CDC25. These results suggest that the long interphase during the eleventh epidermal cell cycle is required for neurulation.     

Relationship between cell surface protease activity and doubling time in various normal and transformed cells

A sensitive method for measuring cell surface and secreted protease activity utilizing ³H-labelled casein is described. The method is based upon proteolytic degradation of the casein substrate into trichloroacetic acid soluble ³H-labelled peptides. Utilizing the radioassay we found that all cultured cell lines examined contain cell surface proteolytic activity which is not secreted into the media. The protease activity was found to be due to protease(s) other than plasminogen activator or plasmin. A comparison of surface protease activity of normal and transformed mouse epidermal cells indicated that the transformed cells contained approximately 3–4 times more proteolytic activity than the normal cells.Surface protease activity was also correlated with the doubling times of various cultured cells. The results indicated that cultured cells with doubling times of greater than three days possess less surface protease activity than cells with shorter doubling times. In order to determine changes in the levels of surface protease activity during the cell cycle several cell lines were synchronized. In synchronized rabbit aortic fibroblasts, mouse transformed epidermal cells and human melanoma cells, a marked increase in surface protease activity was observed during or before mitosis. The protease levels decreased following mitosis. The results suggest that in culture, cell surface protease(s) may be important factor in regulating the rate of cell growth.     

Growth kinetics in newborn mouse epidermis: response to epidermal chalone.

Epidermal DNA synthesis, the epidermal mitotic rate, and the responsiveness to the epidermal G1 and G2 inhibitors were examined in newborn mice at different times after birth. The rate of epidermal cell renewal was in general low during the first two weeks of life. Later the two growth parameters increased and reached very high values at 32-33 days after birth. The rate of epidermal cell proliferation then decreased to a level comparable with that found in adult hairless mouse epidermis at 40-45 days. A single i.p. injection of skin extract containing the two epidermal growth inhibitors induced varying types of responses. The epidermal G2 inhibitor stimulated the mitotic rate on day 2 and day 10, but inhibited it on all other days. The epidermal G1 inhibitor brought about an increase in epidermal DNA synthesis on day 6 and possibly on the following days. No response at all seen at 2, 4, 17, and 32 days after birth. At the other examined times the inhibition was similar to that found in adult mice. These findings differed from those made in vitro on separated newborn mouse epidermal cells (our own unpublished data), and we suggest that the variability of newborn mouse epidermis could be an expression of the immaturity of the skin as a whole, and that dermis in some way modifies the response of epidermis to exogenous epidermal chalone. Our study did not support the theory that the nonresponsiveness of newborn mouse epidermal at certain times could be due to the presence of nonresponsive stem cells in epidermis.     

Growth regulation in X-irradiated mouse skin; the possible role of chalones.

Extracts of hairless mouse skin were tested for their content of epidermal G1 inhibitor and G2 inhibitor at daily intervals after X-irradiation with 4 500 or 2 250 rad. After either dose the skin extracts lacked G1 inhibitory activity on days 5 and 6 respectively after irradiation. This coincided with the time when the epidermal mitotic rate again became normal and started a period of over-shoot. The time interval of 5-6 days corresponds to the turnover time of the differentiating cells in hairless mouse back epidermis. The findings indicate that the proliferating cells in epidermis can respond to changes in local chalone concentration, even after X-irradiation at the tested doses, and that the irradiated epidermal cell population still retains some important properties inherent in a cybernetically regulated system. The local G2-inhibitory activity also varied after irradiation, but these variations could not be directly related to the corresponding mitotic rates.     

Patterns of mitosis in hamster epidermis.

Alignment of the flattened keratinizing cells of the upper strata of mammalian epidermis leads to the formation of columnar units of structure. In mouse epidermis, mitoses have been found to occur relatively infrequently in the region beneath the center of each cell column where a non-keratinocyte dendritic cell, usually with freatures typical of an epidermal Langerhans cell, is situated. The observed pattern of mitosis could therefore be due either to displacement of central keratinocytes by Langerhans cells or indicate some control of keratinocyte proliferation related either to the Langerhans cells or to the over-lying cell columns. No relationship exists between the position of Langerhans cells and epidermal cell columns in hamster epidermis but measurement of the position of mitosis has shown a reduced frequency of occurrence of mitosis beneath the central region. This pattern of mitosis is therefore unrelated to Langerhans cells and appears to reflect differences in the mitotic potential of basal keratinocytes which could be associated with feedback from the overlying cell columns or with an intrinsic pattern of basal cell activity.     

X-ray Sensitivity of HeLa S3 Cells in the G2 Phase: Comparison of Two Methods of Synchronization

The sensitivity of HeLa S3 cells to 220 kv X-rays was measured in terms of cell survival (colony development) during the G2 phase of the cell generation cycle, employing two procedures designed to free G2 cultures from contaminating cells from other phases of the cycle. Treatment of synchronous cultures (obtained initially by mitotic selection) with high specific activity tritiated thymidine (HSA-³HTdR) selectively eliminated S phase cells, while addition of vinblastine permitted removal of cells as they entered mitosis. It was found that HeLa S3 cells become increasingly sensitive as they progress through G2. The pattern of sensitivity fluctuations observed in synchronous HeLa S3 populations selected by the foregoing method was compared with that found in synchronous cultures prepared by the HSA-³HTdR method of Whitmore. The latter method had been used previously with mouse L cells, which were found to undergo a different pattern of sensitivity fluctuations. The two methods yield similar results for HeLa cells in the S and G2 phases of the cycle. It may be concluded, therefore, that the discrepancies between HeLa and mouse L cells do not arise from methodological factors, but represent fundamental differences between the cell types.     

The molecular basis of drug-induced G2 arrest in mammalian cells

Summary The purpose of this review was to focus mainly on the molecular events related to the progression of cells through the G2 period to examine the cause for G2-arrest in mammalian cells after exposure to various anticancer drugs. With few exceptions, most of the eukaryotic cells exhibit a G2 period in their life cycles. The G2 period, which separates S phase from mitosis, represents the time necessary for the synthesis of the various components related to the condensation of chromosomes, assembly of the mitotic spindle, and cytokinesis. Continued synthesis of RNA and protein is necessary for the successful completion of G2 and the initiation of mitosis. Inhibition of RNA and protein synthesis, replacement of phenylalanine by its analog parafluorophenylalanine, or the elevation of intracellular cAMP concentrations, induce reversible G2 arrest in cultured cells. Exposure of cells to certain antineoplastic drugs also blocks cells preferentially in G2. This irreversible drug-induced G2 arrest is associated with extensive chromosome damage. The G2-arrested cells were found to be deficient in certain proteins that may be specific for the G2-mitotic transition. These mitotic or chromosome condensation factors synthesized during the G2 period, reach their maximum levels at mitosis. A preliminary characterization of the chromosome condensation factor revealed that it is a heat labile, Ca²⁺-sensitive, nondialyzable protein with a sedimentation value of 4–5S.     

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.     

The effect of the epidermal G2 chalone on the mitotic duration in nude mouse epidermis and in a transplanted squamous cell carcinoma.

Balb/c/nu nude mice transplanted with a moderately differentiated squamous cell carcinoma were injected intraperitoneally with different doses of aqueous skin extracts containing the epidermal G2 chalone. The mitotic counts and the mitotic rates were determined in histological sections using a stathmokinetic method with vinblastine sulphate. The mitotic duration was calculated from the mitotic rates and counts. Skin extracts containing epidermal G2 chalone increased the mitotic duration in the epidermis, and a similar trend was seen in the tumour. The higher the dose of chalone, the longer the mitotic duration tended to be. A straight line of best fit used to indicate the dose/response relationship was steeper for the epidermis than for the tumour. The study thus shows that the epidermal G2 chalone not only prevents epidermal cells from entering mitosis, it also prolongs the mitotic duration. Further, the results do not contradict the theory that tumour cells may be less sensitive to chalone than normal cells.     

Relationship between cell surface protease activity and doubling time in various normal and transformed cells.

A sensitive method for measuring cell surface and secreted protease activity utilizing 3H-labelled casein is described. The method is based upon proteolytic degradation of the casein substrate into trichloracetic acid soluble 3H-labelled peptides. Utilizing the radioassay we found that all cultured cell lines examined contain cell surface proteolytic activity which is not secreted into the media. The protease activity was found to be due to protease(s) other than plasminogen activator or plasmin. A comparison of surface protease activity of normal and transformed mouse epidermal cells indicated that the transformed cells contained approximately 3--1 times more proteolytic activity than the normal cells. Surface protease activity was also correlated with the doubling times of various cultured cells. The results indicated that cultured cells with doubling times of greater than three days possess less surface protease activity than cells with shorter doubling times. In order to determine changes in the levels of surface protease activity during the cell cycle several cell lines were synchronized. In synchronized rabbit aortic fibroblasts, mouse transformed epidermal cells and human melanoma cells, a marked increase in surface protease activity was observed during or before mitosis. The protease levels decreased following mitosis. The results suggest that in culture, cell surface protease(s) may be important factor in regulating the rate of cell growth.