The effect of environmental stress stimuli on cell division in rat palatal epithelium

Abstract. The effect on cell proliferation of procedures commonly used in studies in laboratory animals was examined in the palatal epithelium of rats. Groups of rats were exposed to ether anaesthesia, immobilization in the supine position or to cage change. Animals left undisturbed in the cages during the experiment were used as controls. At intervals of 30 min, four animals of each group were killed and histological sections of the palatal mucosa were prepared. The number of prophases, metaphases and ana/telophases in the epithelium was counted and related to 1 mm² of the surface. Thirty minutes after the various procedures the number of metaphases and ana/telophases were extremely low. After a further 30 min a high number of prophases was observed. The number of metaphases rose within the following 30 min, during which time the number of ana/telophases also reached a maximum.
These observations indicate that the environmental stimuli have blocked the entrance of cells into mitosis and have resulted in an accumulation of cells, which, following the raising of the blockade, pass through the various stages of the mitotic phase. In studies of cell population kinetics the blockade of cell division as a result of environmental stress stimuli may lead to misleading results.     

The effect of prolonged stress-inducing electrical stimulation on cell proliferation in rat palatal epithelium

This study examines whether blocking of cell division by stress-inducing electrical stimulation can be maintained for a prolonged period of time and whether this results in the accumulation of a large number of cells ready to divide when the block is released. A total of 96 rats was subjected to electrical stimulation at intervals of 30 min for periods of 2, 4, 6 or 12 hr. Groups of animals were killed every second hour during these stimulation periods. After 2, 4 and 6 hr groups of animals were exempted from further stimulation and killed 2 or 4 hr later. After termination of the 12 hr stimulation period, groups of animals were killed every second hour during the succeeding 24 hr. Forty-eight animals were left undisturbed and served as controls. Two and a half hours prior to death, each of the experimental and control animals was injected intraperitoneally (i.p.) with colchicine. Histological sections of the palatal mucosa were prepared and the numbers of arrested metaphases were counted. The electrical stimulation for 2, 4, 6 and 12 hr consistently resulted in a reduced number of cells entering mitosis, indicating a blockade at the G2/M transition. However, with an increase in the length of electrical stimulation, an increasing number of cells passed the block, while a decreasing number entered mitosis during the first 4 hr following cessation of the stimulation. The sum of metaphases accumulated during the various stress periods and the following 4 hr was always similar and consistently lower than the number of metaphases accumulated in the control animals within the same time periods. This observation indicates that, during stress-inducing electrical stimulation and the succeeding 4 hr, only a defined number of cells can enter the mitotic phase. Thus, in addition to the block at the G2/M transition, there appears to be another block of cell division at an earlier stage in the cell cycle.     

The effect of prolonged stress-inducing electrical stimulation on cell proliferation in rat palatal epithelium

Abstract. This study examines whether blocking of cell division by stress-inducing electrical stimulation can be maintained for a prolonged period of time and whether this results in the accumulation of a large number of cells ready to divide when the block is released.
A total of 96 rats was subjected to electrical stimulation at intervals of 30 min for periods of 2,4,6 or 12 hr. Groups of animals were killed every second hour during these stimulation periods. After 2, 4 and 6 hr groups of animals were exempted from further stimulation and killed 2 or 4 hr later. After termination of the 12 hr stimulation period, groups of animals were killed every second hour during the succeeding 24 hr. Forty-eight animals were left undisturbed and served as controls. Two and a half hours prior to death, each of the experimental and control animals was injected intraperitoneally (i.p.) with colchicine. Histological sections of the palatal mucosa were prepared and the numbers of arrested metaphases were counted.
The electrical stimulation for 2, 4, 6 and 12 hr consistently resulted in a reduced number of cells entering mitosis, indicating a blockade at the G₂/M transition. However, with an increase in the length of electrical stimulation, an increasing number of cells passed the block, while a decreasing number entered mitosis during the first 4 hr following cessation of the stimulation. The sum of metaphases accumulated during the various stress periods and the following 4 hr was always similar and consistently lower than the number of metaphases accumulated in the control animals within the same time periods. This observation indicates that, during stress-inducing electrical stimulation and the succeeding 4 hr, only a defined number of cells can enter the mitotic phase. Thus, in addition to the block at the G₂/M transition, there appears to be another block of cell division at an earlier stage in the cell cycle.     

12-O-tetradecanoylphorbol-13-acetate induces an immediate, but transient increase in mitotic activity uncoupled from DNA synthesis in the monolayer cultures of rat keratinocyte

A single wave of mitotic activity was observed in a monolayer culture of rat keratinocytes immediately after exposure to 12-O-tetradecanoylphorbol-13-acetate. A peak for cells in prophase, observed at 10 min after the exposure, was followed by a peak for metaphase at 20 min, for anaphase at 25 min and telophase at 30 min after the exposure. Thereafter, the mitotic activity began to subside. This transient stimulation of mitotic activity resulted in an increase of population density in the monolayer culture. There was neither a stimulation of DNA synthesis during this period nor a change of the DNA content after the mitotic activity was completed. This single burst of synchronous mitotic activity which did not require a substantial stimulation of DNA synthesis suggests that the effect was on the initiation process of mitosis among a subpopulation of cells, presumably cells delayed in the G2 phase of the cell cycle.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. I. Identification of early and late steps of mitosis

In this series of two articles, the duration of mitosis and that of the cell cycle were examined in a group of proliferating cells located in the mouse pyloric antrum and known as isthmal cells. However, before measuring the duration of mitosis, as described in the second article, it is necessary to identify the early and late steps of the mitotic process. This is attempted in the present article, in which the four phases of mitosis and the interphase are described in semithin (0.5 micron thick) Epon serial sections stained with hemalun. The frequency of these phases is then estimated. The beginning of prophase is indicated by the appearance in the nucleus of numerous 0.2-0.3 micron thick basophilic threads. The threads gradually increase in thickness to become the typical chromosomes (about 0.7-micron thick) observed at the end of prophase. Metaphase and anaphase show no remarkable features. At telophase, chromosomes separate from one another, gradually acquire pale segments along their length eventually to look like rows of alternating dark and light patches, and finally vanish. When prophases and telophases are defined in this manner, the enumeration of isthmal cells yields a high proportion of prophases (28%) and telophases (31%), but a low proportion of metaphases (1%) and anaphases (0.3%). Forty per cent of the cells are in interphase.     

DNA synthesis and mitotic activity in cells of regenerating rat liver following x-irradiation in stages GO and Gl]

Effect of X-rays on DNA synthesis and mitotic activity of regenerating liver cells has been studied. The irradiation was performed at a dose of 630 rad before hepatectomy and 2.5 and 6 hours after the stimulation of liver. With the stimulated liver being irradiated, the number of cells synthetizing DNA and entering into mitosis was seen reduced almost twice, whereas DNA synthesis and entering into mitosis were delayed, resp., by 4 and 6 hours. Irradiation of liver before the stimulation brings about a delay in DNA synthesis and in start of mitosis by 2 and 4 hours, resp., without reducing the numbers of cells capable to synthesize DNA and to enter mitosis.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. I. Identification of early and late steps of mitosis

Abstract. In this series of two articles, the duration of mitosis and that of the cell cycle were examined in a group of proliferating cells located in the mouse pyloric antrum and known as isthmal cells. However, before measuring the duration of mitosis, as described in the second article, it is necessary to identify the early and late steps of the mitotic process. This is attempted in the present article, in which the four phases of mitosis and the interphase are described in semithin (0.5 μ m thick) Epon serial sections stained with hemalun. The frequency of these phases is then estimated.
The beginning of prophase is indicated by the appearance in the nucleus of numerous 0.2-0.3 μ m thick basophilic threads. The threads gradually increase in thickness to become the typical chromosomes (about 0.7- μ m thick) observed at the end of prophase. Metaphase and anaphase show no remarkable features. At telophase, chromosomes separate from one another, gradually acquire pale segments along their length eventually to look like rows of alternating dark and light patches, and finally vanish.
When prophases and telophases are defined in this manner, the enumeration of isthmal cells yields a high proportion of prophases (28%) and telophases (31%), but a low proportion of metaphases (1%) and anaphases (0.3%). Forty per cent of the cells are in interphase.     

Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK1. When the laser power delivered to sample surface was 90-190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360-420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50-70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.     

The effect of bleomycin on rapidly proliferating epidermis. A comparative investigation using micro-flow fluorometry, H3Tdr incorporation and a stathmokinetic method (colcemid).

At different time intervals after injection of Bleomycin (BLM) th effect on several kinetic parameters of the hairless mouse epidermis stimulated to proliferate by previous adhesive tape stripping was measured. Micro-flow fluorometry was used to determine the relative number of cells in the various phases of the cell cycle (G1, S and G2). Tritiated thymidine was used to determine labelling indices and grain counts. Colcemid was used to observe the mitotic rate. An initial decrease followed by a subsequent significant increase compared to the non-BLM-treated controls was observed in all parameters studied except the mitotic rate, which remained lower than in the control animals during all 48 hours. The transit time of the cells through the S-phase was initially slightly prolonged, but thereafter it seemed to be shorter than that of the controls. BLM seems to provoke a partial blocking of cells in the G1 phase. When the block is released, a greater number of cells pass through the S phase in partial synchrony at a higher than normal speed. BLM induced a low mitotic rate which remained below the level of that of the normal animals after stripping, even though there obviously was a considerably higher influx of cells from the S phase to the G2 phase. This resulted in a subsequent accumulation of cells in the G2-phase. Thus, BLM has also a blocking effect on the G2-M boundary of the cell cycle. This inhibitory effect of BLM on the mitotic rate was shown to be independent of the effect of BLM on the DNA synthesis. BLM therefore seems to have complex influence on epidermal cell kinetics in vivo. Cells in G1-phase are partially and transiently blocked, but this block is soon released. These cells thereafter pass through the S-phase and pile up in the G2-phase, because BLM also blocks the passage of cells from the G2-phase to mitosis. The overall reduction in cell proliferation seen after BLM in vivo seems mainly to be due to the effect on the G2-M boundray of the cell cycle.     

Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK₁. When the laser power delivered to sample surface was 90–190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360–420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50–70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.     

Mitosis may be an obligatory route to terminal differentiation in the Friend erythroleukemia cell

In previous studies, it was shown that treatment of Friend erythroleukemia (FEL) cells with dimethylsulfoxide (DMSO) and the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) blocked the differentiation pathway just prior to commitment. These studies show that the exposure of DMSO(+3AB)-induced cells to the mitotic inhibitors colcemid or nocodazole resulted in commitment to terminal differentiation. Expression of differentiated phenotype required further incubation without the mitotic inhibitors. Microscopic examination indicated that the number of cells blocked in mitosis and those that differentiated were approximately equivalent. These observations suggest that commitment had occurred during mitosis and that expression of the differentiated state occurred after completion of mitosis. Since commitment was not inhibited by blocking DNA replication by aphidicolin or cytokinesis by cytochalasin B, mitosis may be the only phase of the cell cycle required for commitment.     

Isolation and partial characterization of a cage of filaments that surrounds the mammalian mitotic spindle

Mitotic cells have been detergent extracted under conditions that support microtubule assembly. When HeLa cells are lysed in the presence of brain tubulin, mitotic-arrested cells nucleate large asters and true metaphase cells yield spindles that remain enclosed within a roughly spherical cage of filamentous material. Detergent-extracted mitotic Chinese hamster ovary (CHO) cells show a similar, insoluble cage but the mitotic apparatus is only occasionally stabilized. In later stages of mitosis, HeLa cages are observed in elongated and furrowed configurations. In the terminal stages of cell division, two daughter filamentous networks are connected by the intercellular bridge. When observed in the electron microscope the cages include fibers 7-11 nm in diameter. The polypeptide composition of cages isolated from mitotic HeLa cells is complex, but the major polypeptides are a group with mol wt ranging from 43,000-60,000 daltons and a high molecular weight polypeptide. CHO cells contain a subset of these proteins which includes a major 58,000-dalton and a high molecular weight polypeptide. Two different antisera directed against the vimentin-containing intermediate filaments bind to polypeptides in the electrophoretic profiles of isolated HeLa and CHO cages and stain the cages, as visualized by indirect immunofluorescence. These results suggest that the HeLa and CHO cages include intermediate filaments of the vimentin type. The polypeptide composition of HeLa cages suggests that they also contain tonofilaments. The cages apparently form as the cells enter mitosis. We propose that these filamentous cages maintain the structural continuity of the cytoplasm while the cell is in mitosis.     

Clonal growth of normal human epidermal keratinocytes in a defined medium

Early studies on the duration of mitotic stages and on metaphase-to-prophase ratios in a number of normal and neoplastic cells indicated that the process of mitosis becomes altered during the course of oncogenesis. However, the nature of these changes and their effects on each of the mitotic stages are still unclear. With the use of time lapse cinemicrography, we have compared the durations of mitotic stages of SV40/Wl-38 and SV40/Wl-26 cells to those of their normal counterparts and to other nontransformed human fibroblasts. We also examined the relative frequencies of the individual mitotic stages in fixed preparations of Wl-38 and SV40/Wl-38 cells. The data show that metaphase durations are increased in the transformed cells and as much as 3-4.7-fold in SV40/Wl-38 cells compared to Wl-38 and other nontransformed cells. Other stages are also prolonged though to lesser degrees. These findings suggest that increased metaphase/prophase ratios observed in many tumors are due to increases in duration of metaphase rather than to shorter prophases, and that increased mitotic indices commonly observed in malignant tumors and sometimes used as an index of growth rate are at least in part due to the prolongation of mitotic stages.     

Stress-induced blocking of cell division in the colchicine arrest technique

Previous studies have shown that procedures commonly used in studies of cell population kinetics in laboratory animals cause a transient block of the entrance of cells into mitosis. The purpose of the present study was, therefore, to examine whether the handling of animals in conjunction with the administration of colchicine affects the results obtained by the metaphase arrest technique. Groups of rats were injected with colchicine or saline at 1300 h and sacrificed at regular intervals during the following 140 min. Histological sections of the palatal mucosa were produced and the number of metaphases was assessed in the epithelium. In both the saline-treated and colchicine-treated groups the numbers of metaphases decreased immediately after injection and reached a minimum within 30 min. After that time a transient increase was observed in the saline group, whereas the number of metaphases in the colchicine-treated group increased continuously during the experimental period. These results indicate that colchicine takes effect after a delay period of 30 min and that the handling procedures in conjunction with the administration of colchicine provoke a transient block of the entry of cells into mitosis. The second part of the study was designed in such a way that the number of metaphases collected during a 2 h period under the influence of stress induced by injection of colchicine could be compared with the number of metaphases collected during the same time period without the influence of stress.(ABSTRACT TRUNCATED AT 250 WORDS)     

The transition point for protein synthesis in late S-G2 may be delayed by genome bromosubstitution without affecting the time of entry into mitosis

Bromosubstitution for most of the S period in synchronous populations of Allium cepa L. meristematic cells resulted in a delay in the late S-G2 transition point where protein synthesis is needed for later mitotic entrance to occur. This retardation in the position of the transition point was not accompanied by the expected delay in the entrance into mitosis, suggesting that such protein synthesis is a requisite, but not a timer for prophase triggering.     

Study of the effect of cAMP on the mitotic regimen in the esophageal epithelium of tumor-bearing mice]

The mitotic activity and number of DNA-synthesizing cells in the epithelium of the esophagus of the tumour-bearing albino mice were studied for 24 hours after the injection of dibutyryl cyclic 3', 5'-AMP. It was shown that injection of the preparation led to the blocking of cells in the G2-phase of the mitotic cycle, and to prolongation of mitosis during the first hours of the experiment without changing the total number of cells undergoing mitosis in the course of 24 hours.     

Arrangement of spindle apparatus in mitoses of different ploidy

In non-hypotonically treated mitoses from tissue cultures of Microtus agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, triploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99% of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45% were bipolar, 29.5% tripolar, 7.5% quadripolar and 18% formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.     

Immunolocalization of phospho-S6 kinases: a new way to detect mitosis in tissue sections and in cell culture

During a study on the mTor pathway in the rat kidney we observed a striking increase of the phosphorylation of the S6 kinase in mitosis. In cryostat sections of perfusion-fixed tissue mitotic cells appeared as bright spots in immunofluorescence using an antibody specific for the phosphorylation site Thr421/Ser424. They were easily spotted in overviews with the objective 4× and 10×. Immunofluorescence was weak during the interphase. During the prophase it increased in both the nucleus and the cytoplasm and it remained bright during the subsequent phases of mitosis. All mitotic cells which were found in tubules and in the interstitium of the kidney using a chromatin stain displayed the bright immunofluorescence for phospho-S6 kinase. The same phenomenon was observed in rat liver and in mouse kidney as well as in a human cell line. Provided a rapid fixation, mitotic cells could be identified with the immunoperoxidase technique in paraffin sections of immersion-fixed tissue. This is the first report of phosphorylation of S6 kinase during mitosis in vivo. Thus, immunohistochemistry with anti-phospho-S6 kinase (Thr421/Ser424) appears to provide a convenient way to detect mitotic cells at low magnification.     

Duration of mitosis in mouse thymus cortex cells under normal conditions and after antigen administration at different times of the day]

The influence of an antigen injected at different periods of the circadian mitotic activity on the cell proliferation in the thymus cortex was studied. The duration of mitosis and the number of cells entering division were determined. A pronounced stimulating effect of immunization with sheep red blood cells entering mitosis increased, while the duration of mitosis diminished (colchamine mitotic index was 29.79 percent in control mice, and 47.99 percent in the immunized ones. The duration of mitosis was 0.4 hours in control animals, and 0.24 hours in the immunized ones. When compared with intact mice, the animals immunized in the evening showed no significant changes in the parameters studied.     

Acceleration of CHO cells into mitosis and reduction of X-ray-induced G2 delay by cordycepin

The mitotic cell selection technique was used to monitor the effect of cordycepin and/or 100 rad of X-rays on the entry of asynchronous or synchronous Chinese hamster ovary cells into mitosis. Continuous exposure of asynchronous cells to 5–50 μg/ml of cordycepin caused a rapid increase in the relative numbers of cells entering mitosis. In irradiated cells, cordycepin also reduced a 120-min mitotic delay by about 80 min and shifted the X-ray transition point about 10 min farther away from mitosis. Further studies showed that synchronous cells, treated continuously with 15 μg/ml of cordycepin starting at mid-to-late S phase, proceeded into mitosis approx. 40 min ahead of controls. This acceleration was associated with a 30-min lengthening of S phase and a reduction in the length of G2 from 80 to about 10 min. Furthermore, cordycepin reduced the 70-min mitotic delay observed for cells irradiated in S phase by 20 min. In contrast to the results for treatment at mid-S phase, continuous treatment during G2 of unirradiated synchronous cells with 15 μg/ml of cordycepin had little effect on accelerating cells into mitosis, yet did reduce by about 60 min the 170-min mitotic delay observed for cells irradiated in G2. Unirradiated synchronous cells treated with cordycepin starting before mid-S did not reach mitosis. Thus, there are the following transition points or intervals for cordycepin: for treatment prior to mid-S phase, cell cycle progression through S is blocked; for treatment between mid-S and late S, progression through S continues but progression through G2 is accelerated; and for treatment during G2, the rate of progression in accelerated only if the cells have been irradiated. These results are discussed in relation to the synthesis during late S and G2 of critical protein molecules essential for mitosis.