G2 Sub-Population In Mouse Liver Induced Into Mitosis By Lead Acetate

A single intracardiac dose of lead acetate (40 μ lead/g body weight) induced a 25-fold increase in mitosis of mouse hepatocytes 5 hr after injection, as determined by autoradiography. the prompt appearance of a mitotic wave and the relatively large number of mitoses suggest that the mitotic cells were derived from a hepatocyte sub-population arrested in the G₂ phase. the injection of lead also stimulated a small increase in labeled hepatocytes within 6 hr. Analysis of grain counts gave no evidence for unscheduled DNA synthesis. the incremental labeled cells may have originated from a small fraction of the G₁ population that was ready to enter the S phase without the usual pre-synthetic delay.     

The dependence of the cytokinetic effects of alkylating antitumor preparations on the phase of the hepatocyte cell cycle in regenerating liver]

Effects of alkylating antitumor drugs on resting (G0 phase of cell cycle) and proliferating (G1, S, G2 and M phases) hepatocytes were studied in regenerating mouse liver. Cell cycle kinetics (fraction of labeled mitoses, labeling and mitotic indices) were determined by 3H-thymidine autoradiography. Dipin and fotrin as a DNA-damaging agents attack mainly resting (G0) and proliferating (G1) cells. Effect of the damage results in the inhibition of DNA synthesis and G2 phase arrest in the following mitotic cycle. An alkylating drug phopurin as well as ara-C both suppress the mitotic progression in proliferating hepatocytes and do not influence the resting cells.     

Differentiation of mitotic melanoma cells from G2 cells and their isolation by use of 5-bromo-2'-deoxyuridine and propidium iodide

This report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5-bromo-2'-deoxyuridine (BrdUrd) and double-stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 micrograms/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti-BrdUrd and PI. PGE1-treated cells monitored at 3-hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti-BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd-negative "S phase" cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move "backwards" in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to over-estimation of S phase cells in kinetic studies because of contaminating mitotic cells.     

Visualization of chromosome assembly during the S and G2 stages of the cycle and chromosome disassembly during the G1 stage in semithin sections of mouse duodenal crypt cells and other cells

Previous examination of dividing cells in the isthmus of the mouse pyloric antrum by using semithin (0.5-micron-thick) Epon sections revealed that the prophasic condensation of chromosomes began early in the DNA-synthesizing (S) stage. In order to examine whether the same observation could be made in other proliferating cell types, the crypt base columnar cells in mouse duodenum and the hepatocytes of the rat 48 hr after partial hepatectomy were investigated by morphologic and radioautographic techniques. When crypt base columnar cells were studied in semithin Epon sections, the four phases of mitosis showed the characteristic features described by classical cytologists. Moreover, the proportion of cells in prophase and telophase was high. To relate the mitotic phases to the stages of the cell cycle, the "frequency of labeled mitoses method" provided the duration of the cell cycle, 12.3 hr, and of the S stage, 7.3 hr. From the frequency of the occurrence of mitotic phases, it was estimated that metaphase lasted 0.3 hr and anaphase 0.11 hr, in line with previous estimates. However, the durations of prophase and telophase were long, 5.9 and 1.9 hr, respectively. The whole mitotic process took over 8 hr. From the duration of prophase and cycle stages, it was calculated that 67% of the S stage was occupied by prophasic cells. In fair agreement with this estimate, 68% of the labeled cells 10 min after a 3H-thymidine injection were found to be in prophase. In regenerating hepatocytes, the morphological features and frequency of prophase and telophase cells were similar to those observed in duodenal crypt cells. While the cycle time was not measured and, therefore, the duration of cycle stages and mitotic phases could not be estimated, it is likely that their duration would be of the same order of magnitude. In conclusion, the mitotic process in duodenal crypt cells takes over 8 hr. Moreover, the crypt cells, like antral isthmal cells, show features of early prophase soon after they enter the S stage of the cycle.     

Lobular and cellular patterns of early hepatic glycogen deposition in the rat as observed by light and electron microscopic radioautography after injection of 3H-galactose

Very low hepatic glycogen levels are achieved by overnight fasting of adrenalectomized (ADX) rats. Subsequent injection of dexamethasone (DEX), a synthetic glucocorticoid, stimulates marked increases in glycogen synthesis. Using this system and injecting 3H-galactose as a glycogen precursor 1 hr prior to sacrifice, the intralobular and intracellular patterns of labeled glycogen deposition were studied by light (LM) and electron (EM) microscopic radioautography. LM radioautography revealed that 1 hr after DEX treatment, labeling patterns for both periportal and centrilobular hepatocytes resembled those in rats with no DEX treatment: 18% of the hepatocytes were unlabeled, and 82% showed light labeling. Two hours after treatment with DEX, 14% of the hepatocytes remained unlabeled, and 78% were lightly labeled; however, 8% of the cells, located randomly throughout the lobule, were intensely labeled. An increased number of heavily labeled cells (26%) appeared 3 hr after DEX treatment; and by 5 hr 91% of the hepatocytes were intensely labeled. Label over the periportal cells at this time was aggregated, whereas centrilobular cells displayed dispersed label. EM radioautographs showed that 2 to 3 hr after DEX injection initial labeling of hepatocytes, regardless of their intralobular location, occurred over foci of smooth endoplasmic reticulum (SER) and small electron-dense particles of presumptive glycogen, and in areas of SER and distinct glycogen particles. After 5 hrs of treatment with DEX, the intracellular distribution of label reflected the glycogen patterns characteristic of periportal or centrilobular regions.     

Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose

The effects of short- and long-term stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of 3H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled 1 hr after labeling. The percentage of heavily labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after administration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.     

Cell Cycle Kinetics and Radiation-Induced Chromosomal Aberrations Studied with C14 and H3 Labels

Chinese hamster cells in vitro were double labeled with C(14)TdR and H(3)TdR. At the time of irradiation with Co(60) gamma rays (600 rad), the cells in the G(2) phase were labeled only with C(14), whereas cells in the late and middle S phases were labeled with both C(14) and H(3). The cells in early S phase were labeled only with H(3) and the G(1) cells were unlabeled. Samples were fixed at various time intervals following irradiation and the metaphases were analyzed for chromosomal damage. The phase in which the cell was located at the time of irradiation was determined by counting grains in the first and second layers of autoradiographic film. In both control and irradiated cells some G(1) cells divided prior to some of the cells which were in the S phase denoting mixing of the populations. The G(2) phase sustained three times more chromosomal damage than the S phase. Little difference in chromosomal damage was found between the G(1) and S phases or among the different parts of the S phase. Cells in G(2) sustained a mitotic delay of 4 hr, while the other phases sustained a delay of 2 to 3 hr. Chromatid and chromosome (dicentrics) exchanges were induced in G(1) cells but only chromatid exchanges were induced in S and G(2) cells; this is consistent with the hypothesis that the chromosome consists of two subunits which separate either slightly before or immediately as the cell enters the S phase.     

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.     

The nature of the lengthening of the G2 phase in the mitotic cycle of parenchymal cells of the mouse liver

The phenomenon of G2 phase prolongation was found in the population of mouse hepatocytes. In normal postnatal liver growth, G2 phase prolongation in not pronounced and occurs in a small fraction of proliferating hepatocytes. In case of liver regeneration after removal of 2/3 of the organ, G2 phase prolongation is observed in a population of hepatocytes, which response to the proliferative stimulus first. Estimation of individual variation in expression of prolonged G2 phase along with the detailed analysis of the structure of the process of proliferation in the main population of hepatocytes (cells with normal G2 phase) allows to define the biological meaning of the "G2-population" observed. The prolongation of G2 phase may result from non-specific cell damage in mitotic cycles, caused by destruction of trophic relations in liver during its growth and regeneration.     

Effects of hydroxyurea on the mitotic activity and the G2 phase in hairless mouse epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid at various times after HU. The animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G2 was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G2 for the first 6 hr after HU injection, then the G2 compartment was emptied. The results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G2 and M. The cell kinetic effects of HU thus seem to be very complex.     

OBSERVATIONS ON HUMAN MONOCYTE KINETICS AFTER PULSE LABELING

Monocyte kinetics were studied in seven hematologically normal individuals using in vivo pulse labeling with tritiated thymidine. Although occasional labeled cells appear in the peripheral blood within 4 or 5 hr of the administration of label, a significant outflow from the marrow begins 13–26 hr later. This interval is occupied by the G₂ and M phases of the mitotic cycle since mitotic cells are not observed in the peripheral blood. The duration of the DNA synthesis phase of monocytes is measured at 34 hr ≈ 1.8 hr. Cells do not enter this phase while circulating since exposure of circulating cells to tritiated thymidine does not result in any uptake. If monocytes are not 'end'cells which have completed their mitotic activity before leaving the marrow they must at least be inhibited from further proliferative activity until they are permanently sequestered in other tissues.
The generation time is probably not less than 40 hr and data derived from the mean grain counts of labeled cells suggest that it is often more than 70 hr. The total daily output of monocytes in man is 9.4 × 10⁸ cells per 24 hr ≈ 3.3 × 10⁸.
Cells leave the bloodstream with a half-time of about 71 hr thereby proving themselves to be considerably more durable than neutrophils which have a half-life in the neighborhood of 6 hr.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration.

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36-40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G2/M phase of the cell cycle was significantly higher than that in G0/G1 phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G2/M phase.     

Inequality of numbers of cells passing daily from S phase into G2 and from G2 into mitosis for a population of young mouse hepatocytes]

The formulas are proposed which allow to verify the equality of the diurnal streams of cells from one to another phase of mitotic cycle for systems with the diurnal rythm of mitotic index. The unequality of the diurnal stream of cells from S into G2 phase and the diurnal streams from G2 into M phase for hepatocytes of 3-weeks old mice is assumed to be caused by the passage of about 75 percent of cells from G2 phase directly to the resting phase R1. Part of these cells may then return from R1 to G1 phase.     

The Circadian Optimal Time for Hepatectomy in the Study of Liver Regeneration

Standardized (light from 0600 to 1800) C3HS mice, hepatectomized at different circadian stages, were killed at 1400 (the peak time of mitotic activity in intact mice). The higher values of mitotic index were those of mice operated at 1400, 48 hr before. The curve of mitotic activity of the regenerating liver of mice operated at 1400 and that of mice operated at 0200 (an opposite time in the circadian stage) are, both, grossly in phase with the curves of mitotic index in young and adult mice liver. The amplitude of the first peak of mitotic activity in mice operated at 0200 was dramatically lower than that of animals operated at 1400. The same applies to hepatocytes as well as to the sinusoid litoral population of cells. It is concluded that 1400 hr, as contrast to 0200 hr, is an optimal time for hepatectomy if one wants to obtain the highest mitotic index first peak during regeneration in a normal phase position (the position of the mitotic index peak in the liver of normal young and adult mice).     

Effects of pulse labelling with tritiated thymidine on the circadian rhythmicity in epidermal cell-cycle distribution in mice

The influence of pulse labelling with 50 microCi tritiated thymidine ( [3H]TdR) (2 microCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G2 phases of the cell cycle were estimated from the obtained DNA frequency distributions. The proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [3H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [3H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G2 and M phase during the first 32 hr after the injection. The number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 microCi [3H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

Studies on gluconeogenesis and ketogenesis in isolated hepatocytes from alloxan diabetic rats.

Gluconeogenesis and ketogenesis were studied in isolated hepatocytes obtained from normal and alloxan diabetic rats. Insulin treatment maintained near-normal blood glucose levels and caused an increase in glycogen deposition. The third day after insulin withdrawal the rats displayed a diabetic syndrome marked by progressive hyperglycemia and glycogen depletion. Net glucose production in liver cells isolated from alloxan diabetic rats progressively increased with time up to 72 hr after the last in vivo insulin injection. Maximal glucose production was observed at 72 hr with 10 mM alanine, lactate, pyruvate, or fructose. Glucose production decreased at 96 hr. The same pattern was observed with the incorporation of labeled bicarbonate into glucose. Ketogenesis in liver cells and hepatic lipid content also peaked at 72 hr.     

The Circadian Optimal Time for Hepatectomy in the Study of Liver Regeneration

Standardized (light from 0600 to 1800) C3HS mice, hepatectomized at different circadian stages, were killed at 1400 (the peak time of mitotic activity in intact mice). The higher values of mitotic index were those of mice operated at 1400, 48 hr before. The curve of mitotic activity of the regenerating liver of mice operated at 1400 and that of mice operated at 0200 (an opposite time in the circadian stage) are, both, grossly in phase with the curves of mitotic index in young and adult mice liver. The amplitude of the first peak of mitotic activity in mice operated at 0200 was dramatically lower than that of animals operated at 1400. The same applies to hepatocytes as well as to the sinusoid litoral population of cells. It is concluded that 1400 hr, as contrast to 0200 hr, is an optimal time for hepatectomy if one wants to obtain the highest mitotic index first peak during regeneration in a normal phase position (the position of the mitotic index peak in the liver of normal young and adult mice).     

G1 and S phase mammalian cells synthesize histones at equivalent rates

To determine the effect of cell cycle position on protein synthesis, synchronized cell populations were metabolically labeled and the synthesis of the basic proteins, including histones, was examined by two-dimensional gel electrophoresis. Exponentially growing S49 mouse lymphoma or Chinese hamster ovary (CHO) cells were separated into G1 and S phase populations by centrifugal elutriation, selective mitotic detachment, fluorescence-activated cell sorting, or a combination of these, and pulse-labeled with radiolabeled amino acids. The histone proteins, both free and chromatin-bound, were completely resolved from some 300 other basic polypeptides in whole-cell lysates by a modification of the NEPHGE technique of O'Farrell, Goodman and O'Farrell (1977). Comparisons of matched autoradiograms from samples of G1 and S phase labeled cells revealed an equivalent rate of histone synthesis through the cell cycle of both S49 and CHO cells. Nuclei isolated from G1 phase S49 cells that were pulse-labeled contained between 13 and 15% of the newly synthesized nucleosomal histones present in S phase nuclei. Nuclei prepared from G1 phase cells that were pulse-labeled and then chased for 5 hr contained more than 90% of the labeled nucleosomal histones present in wholecell lysates. It therefore seems likely that differential alterations in the rate of histone synthesis do not occur to a significant degree as cells proceed through the cycle, but the association of newly synthesized histones with DNA takes place after the onset of DNA replication.     

The cytogenetic and hepatotoxic effects of dioxin on mouse liver cells

The cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo p-dioxin (TCDD) on mouse liver cells were investigated. Male C57BL/6J strain mice, which have TCDD receptors, were given single intraperitoneal injections of 25, 37.5, 75 and 150 g of TCDD/kg body weight or corn oil carrier alone. Two-thirds hepatectomies were carried out at 1 or 7 days after injection and chromosomal aberrations and mitotic indexes of the regenerating hepatocytes were scored 54 hr after hepatectomy. Liver sections from additional intact mice were studied for TCDD-hepatotoxicity at 1, 7 and 30 days after injection. The three high doses of TCDD caused hepatotoxicity with necrosis of liver cells and focal architectural collapse by 30 days after injection. No evidence was obtained of an increase in the frequency of chromosomal structural aberrations at doses that allowed sufficient mitotic activity for cytogenetic evaluation. We conclude that TCDD is not a clastogen for mouse hepatocytes, although high doses cause marked hepatocellular necrosis.Abbreviations CSD chromosome deletion - META metacentric chromosome - TCDD 2,3,7,8-tetrachlorobenzo-p-dioxin     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

The kinetics of isthmal cells in mouse antrum were examined in three ways: the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; the duration of interphase and mitotic phases was determined from how frequently they occurred; and mice were killed at various intervals after an intravenous injection of 3H-thymidine to time the acquisition of label by the various phases of mitosis. The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G1 and G2 stages were 6.8 and 1.0 hr, respectively. From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurrence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr. Ten minutes after an intravenous injection of 3H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase. We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G2. Also, nuclei are in telophase during the early half of G1 but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G1 into early and late periods is practical.