CELL KINETICS OF IRRADIATED EXPERIMENTAL TUMORS: CELL TRANSITION FROM THE NON-PROLIFERATING TO THE PROLIFERATING POOL

Parenchymal tumor cells of murine mammary carcinomas can be divided into two pools, using nucleoli as morphological ‘markers’. Cells with dense nucleoli traverse the cell cycle and divide, thus constituting the proliferating pool. Cells with trabeculate or ring-shaped nucleoli either proceed slowly through G₁ phase or are arrested in it. The role of these non-proliferating, G₁ phase-confined cells in tumor regeneration was studied in vivo after a subcurative dose of X-irradiation in two transplantable tumor lines. Tumor-bearing mice were continuously injected with methyl[³H]thymidine before and after irradiation. Finally, the labeling was discontinued, mice injected with vincristine sulfate and cells arrested in metaphase were accumulated over a 10-hr period. Two clearly delineated groups of vincristinearrested mitoses emerged in autoradiograms prepared from tumor tissue at the time of starting tumor regrowth: one group with the silver-grain counts corresponding to the background level, the other with heavily labeled mitoses. As the only source of unlabeled mitoses was unlabeled G₁ phase-confined cells persisting in the tumor, this observation indicated cell transition from the non-proliferating to the proliferating pool, which took place in the initial phase of the tumor regrowth. Unlabeled progenitors have apparently remained in G₁ phase for at least 5–12 days after irradiation.     

A CRITICAL REVIEW OF THE USE OF VINCRISTINE (VCR) AS A TUMOUR CELL SYNCHRONIZING AGENT IN CANCER THERAPY

Vincristine (VCR) has been used clinically in so-called ‘tumour cell synchronization therapy schedules'. These schedules are based on the assumption that cells, arrested in metaphase by low doses of VCR, subsequently re-enter the proliferative cycle synchronously. However, the evidence that tumour cell synchrony can be achieved under clinical conditions or that ‘cell synchronization therapy schedules’ yield a better therapeutic response than other efficient combination schemes, is scanty. Further, even in experimental systems, the efficacy of VCR as a cell synchronizing agent is disputed. Indeed, in some systems, cells arrested in metaphase by low doses of VCR, do not re-enter a normal proliferative cycle at all following arrest. In addition, the complex nature of the VCR—tumour interaction and the heterogeneous nature of the tumour cell populations against which it is used augurs badly for the successful application of cell synchronization therapy schedules.     

THE EFFECT OF VINCRISTINE ON MOUSE JEJUNAL CRYPT CELLS OF DIFFERING CELL AGE: DOUBLE LABELLING AUTORADIOGRAPHIC STUDIES USING 3H- AND 14C-TdR

The mechanism of action of the alkaloid vincristine (VCR) has been investigated in vitro on HeLa cells in culture and in vivo on jejunal crypt cells of the mouse. The in vitro experiments with HeLa cells show that VCR affects not only mitotic but also interphase cells. The VCR-affected cells first continue their passage through the cell cycle undisturbed but after reaching mitosis they are arrested in metaphase. This agrees well with the results obtained by Madoc-Jones & Mauro (1968) and Madoc-Jones (1973) on synchronized cell cultures. Until now there has been no investigation of the mechanism of action of VCR in vivo. This is due to the absence of a suitable technique for synchronization in vivo. The present study is based on a method which permits the assessment of the VCR sensitivity as a function of the cell age without synchronization in the usual sense. The jejunal crypt epithelium of the normal mouse was double labelled with ³H- and ¹⁴C-thymidine (TdR) in such a way as to produce a narrow subpopulation of crypt cells with a maximum age difference of 1 hr. On autoradiographs these cells can be distinguished by their characteristic labelling from other cells. As this ‘pseudo’-synchronized subpopulation passes through the cycle the effect of VCR can be studied, i.e. one can analyse the effect in well-defined time intervals of the cycle. The results show that the effect of VCR is the same in vivo as in vitro. The crypt cells which are affected by VCR in interphase continue their passage through the cycle, but upon entering mitosis they are arrested in metaphase. VCR has, at the concentration used in the present study, no effect on the duration of the S and G₂ phases. The necrotic cells seen after VCR application are formed from arrested metaphases.     

CHARACTERISTICS OF THE ISOPRENALINE STIMULATED PROLIFERATIVE RESPONSE OF RAT SUBMAXILLARY GLAND

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using ³H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMW,) waves gave a mean G_is transit time of 21-2 hr, mean G_is+ S transit time of 270 hr, and mean G_is+ S + G₂ transit time of 35-8 hr for a single injection of isoprenaline, where G_is is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the G_is phase dropped from 18-1 to 1–3 while the other variances remained unchanged. The estimated proliferative fraction was 0–24 after a single injection of isoprenaline, and 0–31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated ³H-TdR injections, were 0–21 and 0–36 respectively.     

NEW METHODS OF CELL CYCLE ANALYSIS BASED ON THE SELECTIVE TAGGING OF CELLS EITHER AT THE BEGINNING OR END OF S PHASE

New techniques for cell cycle analysis are presented. Using HeLa cells, methods are described for the selection of a narrow window or cohort of lightly [³H]-labeled cells located either at the very beginning or the very end of S phase. The cohort cells are tagged by a labeling procedure which entails alternating pulses of high and low levels of [³H]thymidine and are identified autoradiographically. Additional methods are described for following the progress of cohort cells through the cell cycle. Theoretically, with the methods described, it should be possible to follow the ‘early S cohort’ cells as they exit from S phase, as they enter and exit M and as they enter the subsequent S phase. This would allow a determination of S, S + G₂, S + G₂+ M and T. It should theoretically be possible to follow ‘late S cohort’ cells in a similar manner, allowing a determination of G₂, G₂+ M and G₂+ M + G₁. To test these predictions, several experiments are presented in which the progress of the two cohorts is monitored. The best data were obtained from the mitotic curves of cohort cells. For each of the cohorts, values were obtained for the time required for peak concentration of cells in mitosis, the coefficients of variation and of skew. The curve of cohort cells passing through mitosis is shown to fit a log-normal curve better than a normal curve. In addition, the mitotic curves are used to estimate the length of M and to estimate the loss of cohort synchrony. Other uses of these methods are discussed.     

THE PROLIFERATION KINETICS OF NHIK 1922 CELLS IN VITRO AND IN SOLID TUMOURS IN ATHYMIC MICE

The proliferation kinetics of cells of the line NHIK 1922 grown in vitro and as solid tumours in the athymic mutant nude mouse has been studied. In vitro, growth curves were determined for exponentially growing populations and for populations synchronized by mitotic selection. The phase durations for these populations were determined by flow cytofluorometric measurements of DNA-histograms and pulsed incorporation of [³H]TdR respectively. The generation time and the phase durations for synchronized populations were found to be about equal to those for exponentially growing populations. The duration of the phases G₁, S and G₂+ M was found to be 8·5–9·5, 11·0–12·0 and 6·0–6·5 hr respectively, i.e. the generation time was 26·5–27·0 hr. The proliferation kinetics in vivo were studied by flow cytofluorometry and by the technique of percentage labelled mitoses. The median duration of S-phase and (G₂+ M)-phase in vivo was found to be approximately the same as that observed in vitro, while the median duration of G₁-phase was found to be approximately 5 hr longer in vivo than under the present in vitro growth conditions. The growth fraction in vivo was estimated to be approximately 50%. The non-proliferative compartment of the tumour cells was found to consist mainly of cells with the DNA-content of cells in G₁-phase. It is concluded that the reduced rate of proliferation of NHIK 1922 cells in vivo is correlated with alterations in the duration of G₁-phase and, hence, the proportion of cells in G₁-phase.     

THE EFFECT OF MITOTIC INHIBITORS ON MYOGENESIS IN VITRO

The effect of colchicine and other antimitotic drugs was studied in cultures of 11-day chick embryo breast muscle. Exposure of such cultures to 10^-6 M colchicine results in fragmentation of the elongate myotubes into rounded, cytoplasmic sacs (myosacs) containing various numbers of nuclei. Comparison of the dose-response relation between myotube fragmentation and metaphase arrest suggests that the underlying mechanism may be similar in both cases. Low temperature does not duplicate the effects of colchicine. Glycerinated myotubes are not affected by the mitotic inhibitors. The effect of colchicine on myotubes is reversible. Myosacs elongate within several days after removal from colchicine. However, the regenerated myotubes fail to incorporate additional mononucleated cells. Colchicine does not interfere with the process of fusion itself, but the metaphase block prevents cells from entering that phase of the cell cycle during which fusion can occur. Cells arrested in mitosis by colchicine do not recover when incubated in normal medium. Colcemid-induced arrest is reversible and does not prevent subsequent fusion of the cells.     

TRANSIT TIMES THROUGH THE CYCLE PHASES OF JEJUNAL CRYPT CELLS OF THE MOUSE ANALYSIS IN TERMS OF THE MEAN VALUES AND THE VARIANCES

Mean transit times as well as variances of the transit times through the individual phases of the cell cycle have been determined for the crypt epithelial cells of the jejunum of the mouse. To achieve this the fraction of labelled mitoses (FLM) technique has been modified by double labelling with [³H] and [¹⁴C]thymidine. Mice were given a first injection of [³H]thymidine, and 2 hr later a second injection of [¹⁴C]thymidine. This produces a narrow subpopulation of purely ³H-labelled cells at the beginning of G₂-phase and a corresponding subpopulation of purely ¹⁴C-labelled cells at the beginning of the S-phase. When these two subpopulations progress through the cell cycle, one obtains FLM waves of purely ³H- and purely ¹⁴C-labelled mitoses. These waves have considerably better resolution than the conventional FLM-curves. From the temporal positions of the observed maxima the mean transit times of the cells through the individual phases of the cycle can be determined. Moreover one obtains from the width of the individual waves the variances of the transit times through the individual phases. It has been found, that the variances of the transit times through successive phases are additive. This indicates that the transit times of cells through successive phases are independently distributed. This statistical independence is an implicit assumption in most of the models applied to the analysis of FLM curves, however there had previously been no experimental support of this assumption. A further result is, that the variance of the transit time through any phase of the cycle is proportional to the mean transit time. This implies that the progress of the crypt epithelial cells is subject to an equal degree of randomness in the various phases of the cycle.     

CELL CYCLE-SPECIFIC CHANGES IN HISTONE PHOSPHORYLATION ASSOCIATED WITH CELL PROLIFERATION AND CHROMOSOME CONDENSATION

Preparative polyacrylamide gel electrophoresis was used to examine histone phosphorylation in synchronized Chinese hamster cells (line CHO). Results showed that histone f1 phosphorylation, absent in G₁-arrested and early G₁-traversing cells, commences 2 h before entry of traversing cells into the S phase. It is concluded that f1 phosphorylation is one of the earliest biochemical events associated with conversion of nonproliferating cells to proliferating cells occurring on old f1 before synthesis of new f1 during the S phase. Results also showed that f3 and a subfraction of f1 were rapidly phosphorylated only during the time when cells were crossing the G₂/M boundary and traversing prophase. Since these phosphorylation events do not occur in G₁, S, or G₂ and are reduced greatly in metaphase, it is concluded that these two specific phosphorylation events are involved with condensation of interphase chromatin into mitotic chromosomes. This conclusion is supported by loss of prelabeled ³²PO₄ from those specific histone fractions during transition of metaphase cells into interphase G₁ cells. A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests involvement of f1 phosphorylation in chromatin structural changes associated with a continuous interphase "chromosome cycle" which culminates at mitosis with an f3 and f1 phosphorylation-mediated chromosome condensation.     

EFFECT OF CYCLOHEXIMIDE ON THE CELL CYCLE OF THE CRYPTS OF THE SMALL INTESTINE OF THE RAT

A single injection of 1.5 mg/kg of cycloheximide induces a complete disappearance of mitotic activity in rat intestinal crypts within 1.5–2 hr. No significant necrosis of crypt cells is observed even though this phenomenon is accompanied by a marked decrease in uptake of labeled precursors into protein and DNA. Mitoses reappear 6 hr after injection and recovery then follows a cyclic pattern over a period equivalent to one cell cycle, thereby reflecting at least a partial synchronization of cell division. Concurrent use of colchicine, an agent known to induce metaphase arrest, has demonstrated that cycloheximide, while having no apparent effect on cells already in division, prevents the entrance of new cells into visible mitosis. Analysis of the cell cycle suggests that one block initiated by cycloheximide occurs in G₂, presumably as the result of an interference with the formation of protein(s) required for the normal progression of cells from this phase of the cycle into mitosis.     

STUDIES ON THE MECHANISM OF DIURNAL VARIATION OF PROLIFERATIVE INDICES IN THE SMALL BOWEL MUCOSA OF THE RAT

In the rat small bowel mucosa significant variation was found in both the labelling and the mitotic indices with time of day. The zenith and the nadir of labelling and mitotic activity coincided at 15.00 and 02.00 hours respectively. Small changes were found in the ‘cut-off’ position, but this variation in proliferative compartment size was insufficient to account for the comparatively wider fluctuations in proliferative indices. Measurements of the rate of entry into mitosis, using metaphase arrest with vincristine at three widely separated times during the day, showed no significant change. Changes in the growth fraction or in the birth rate as measured cannot account for diurnal variation in the proliferative activity of the small bowel mucosa. We propose a hypothesis which involves diurnal fluctuations in the transit times through G₁ and through G₂.     

NITROUS OXIDE: EFFECTS ON THE MITOTIC APPARATUS AND CHROMOSOME MOVEMENT IN HELA CELLS

When HeLa cells were grown in the presence of nitrous oxide (N₂O) under pressure (80 lb/in²) mitosis was inhibited and the chromosomes displayed a typical colchicine metaphase (c-metaphase) configuration when examined by light microscopy. When the cells were returned to a 37°C incubator, mitosis was resumed and the cells entered G₁ synchronously. Ultrastructural studies of N₂O-blocked cells revealed a bipolar spindle with centriole pairs at each pole. Both chromosomal and interpolar (pole-to-pole) microtubules were also present. Thus, N₂O, unlike most c-mitotic agents, appeared to have little or no effect upon spindle microtubule assembly. However, the failure of chromo somes to become properly aligned onto the metaphase plate indicated an impairment in normal prometaphase movement. The alignment of spindle microtubules was frequently atypical with some chromosomal microtubules extending from kinetochores to the poles, while others extended out at acute angles from the spindle axis. These ultrastructural studies indicated that N₂O blocked cells at a stage in mitosis more advanced than that produced by Colcemid or other c-mitotic agents. Like Colcemid, however, prolonged arrest in mitosis with N₂O led to an increased incidence of multipolar spindles.     

EFFECTS OF MAPLE SYRUP URINE DISEASE METABOLITES ON MOUSE l-FIBROBLASTS IN VITRO: A FINE STRUCTURAL AND BIOCHEMICAL STUDY

—The branched-chain amino and ketoacids [i.e. l -leucine, l -isoleucine, l -valine, alpha-ketoisocaproic acid (AKICA), alpha-keto-beta-methylvaleric acid (AKBMVA) and alpha-ketoisovaleric acid (AKIVA)] were administered to mouse strain l fibroblasts in tissue culture in an attempt to study the effects of increased levels of the compounds in an in vitro system. All of these compounds are found to be elevated in the blood of patients with Maple Syrup Urine Disease (MSUD). With AKICA, l -leucine, AKIVA and AKBMVA, there was a decreased growth rate at concentrations of 10 to 30 times the levels found in Maple Syrup Urine Disease. Combined administration of the above six compounds at the maximum blood levels noted in MSUD produced a significantly decreased growth rate. Electron microscopic studies revealed numerous annulate lamellae in cells treated with AKICA and in those treated with a combination of all six MSUD compounds. AKICA-treated cells contained elevated concentrations per cell of free fatty acids, triglycerides, sterols and some classes of phospholipids. Isotope labelling experiments were performed using [U-¹⁴C] AKICA and [³H]isoleucine, which were added to l -cell suspension cultures containing various levels of unlabelled AKICA. Labelled AKICA and isoleucine were both taken up by the cells. The net uptake of isoleucine was inhibited by AKICA in concentrations found in MSUD. Folch-Lees extraction of cells treated with labelled AKICA revealed increased ¹⁴C counts only in the lower lipid phase. The growth inhibition and annulate lamellae observed with AKICA treatment may be due to an arrest of the cells in phase G₁ of the cell growth cycle, possibly due to decreased isoleucine uptake. It is proposed that a similarly-mediated arrest in the proliferation of oligodendroglial cells during the critical period of myelination gliosis might account for the myelination abnormalities reported in MSUD.     

cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G₁ phase of the cell cycle instead of G₀. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G₀ phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G₁, 12% were in S and 37% in G₂ phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P <0.01) [³H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P <0.01) [³H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G₁ phase of the cycle to enhance IGF-I effects in cell proliferation.     

Inhibition of proliferation of MCF-7 breast cancer cells by a blocker of Ca2+-permeable channel

In MCF-7 breast cancer cells, insulin-like growth factor-1 (IGF-1) increased the calcium-permeability of the cells by activating a voltage-independent calcium-permeable channel. IGF-1 also induced oscillatory elevation of cytoplasmic free calcium concentration in these cells. An anti-allergic compound, tranilast, reduced the calcium-permeability augmented by IGF-1 in a dose-dependent manner and blocked the oscillatory elevation of cytoplasmic free calcium concentration. Tranilast did not affect early intracellular signals activated by IGF-1, including receptor autophosphorylation, activations of Ras, mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Tranilast inhibited increases in [³H]-thymidine incorporation, DNA content and cell number induced by IGF-1. The ID₅₀ for [³H]-thymidine incorporation and DNA content were about 10 μM. The inhibitory effect of tranilast was reversible, and cell viability was not affected. Treatment with tranilast increased the number of cells in the G₁ phase suggesting that this compound induced G₁ arrest. Tranilast also reduced the phosphorylation of the retinoblastoma protein. These results indicate that tranilast inhibits the IGF-1-induced cell growth in MCF-7 cells by blocking calcium entry.     

SPERMATOGONIAL CHALONE(S): EFFECT ON THE PHASES OF THE CELL CYCLE OF TYPE A SPERMATOGONIA IN THE RAT

Adult rats with X-irradiated testes were used to analyze the effect of the spermatogonial chalone(s) on the phases of the cell cycle of type A spermatogonia. Twelve days after irradiation, the animals were used in two experiments designed to test the existence of hypothetical G₂ and S phase chalones. For the G₂ assay, rats injected twice with testicular extract (Group I), liver extract (Group II) or physiological saline (Group III) were killed 10 hr after the initial injection. Mitoses of type A, Intermediate and type B spermatogonia were counted in whole mounts of dissected seminiferous tubules. To test for an S phase inhibitor, two groups of rats were given multiple injections of either testicular extract (Group IV) or saline solution (Group V). Twenty-two hr after the first injection they were injected with [³H]thymidine and killed 2 hr later. Silver grains over labelled type A nuclei were counted in radioautographed sections of testes from these animals. The average grain counts were identical in Groups IV and V, indicating that the testicular extract did not affect type A spermatogonia during the S phase. Counts of type A mitoses in Groups I, II and III revealed that in the animals injected with the testicular extract (Group I) the number of divisions was 50% lower than in the control groups (Groups II and III). In contrast, mitotic activity of differentiating spermatogonia (In + B) was similar in all three groups of animals. This result is attributed to a testicular chalone which specifically inhibits type A spermatogonia during the G₂ phase of the cell cycle. Indirect evidence for a G₁ spermatogonial chalone is also presented, as a result of an analysis of published data (Clermont & Mauger, 1974).     

The human immunodeficiency virus type 1 Vpr protein and its carboxy-terminally truncated form induce apoptosis in tumor cells

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces apoptosis after cell cycle arrest at the G₂ phase in primate cells. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation and results in apoptosis without G₂ arrest. Here, we investigated whether this property of Vpr and C81 could be exploited for use as a potential anticancer agent. First, we demonstrated that C81 induced G₁ arrest and apoptosis in all tumor cells tested. In contrast, Vpr resulted in G₂ arrest and apoptosis in HeLa and 293 T cells. Vpr also suppressed the damaged-DNA-specific binding protein 1 (DDB1) in HepG2 cells, thereby inducing apoptosis without G₂ arrest. G₂ arrest was restored when DDB1 was overexpressed in cells that also expressed Vpr. Surprisingly, C81 induced G₂ arrest when DDB1 was overexpressed in HepG2 cells, but not in HeLa or 293 T cells. Thus, the induction of Vpr- and C81-mediated cell cycle arrest appears to depend on the cell type, whereas apoptosis was observed in all tumor cells tested. Overall, Vpr and C81 have potential as novel therapeutic agents for treatment of cancer.     

Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G₀/G₁ cell cycle arrest and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G₀/G₁ cell cycle phase arrest and increased levels of p27^kip1 in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G₀ state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.     

EXISTENCE OF TWO CHALONE-LIKE SUBSTANCES IN INTESTINAL EXTRACT FROM THE ADULT NEWT, INHIBITING EMBRYONIC INTESTINAL CELL PROLIFERATION

The inhibiting effect of tissue extract from fully differentiated intestinal mucosa of adult animals on proliferation kinetics of exponentially growing embryonic epithelial gut cell populations was studied in the newt Pleurodeles waltlii. Crude extract was fractionated by G-200 Sephadex chromatography and the effect of fractions on cell proliferation was studied using both mitotic index and ³H-thymidine incorporation methods. The inhibitions we obtained were then displayed by means of cytophotometric study of age distribution of intestinal gut cells around the cell cycle, measuring the Feulgen-DNA content. The results revealed the presence of two chalone-like substances in the intestine of adults. One (factor 1) is characterized by a molecular weight of between 120,000 and 150,000 and inhibits the cell cycle at the end of the G₁ phase, the other (factor 2) is characterized by a molecular weight lower than 2000 and inhibits the cell cycle in the course of the G₂ phase. The cells delayed in the G₂ phase escape from inhibition but the cells delayed in the G₁ phase do not, although availability time of both factor 1 and factor 2 is about 12 hr. It is thus thought that cells prevented from dividing in G₁ phase are indefinitely delayed in this phase and possibly differentiate.     

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

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.