EFFECT OF AN INHIBITING FACTOR ISOLATED FROM RAT LIVER ON DNA POLYMERASES IN REGENERATING RAT LIVER

We partially purified an inhibitory factor (LIF), isolated from 105,000 g supernatant of a saline adult rat liver homogenate. LIF stopped in vitro cell multiplication by blocking the G₁—S transition, and reduced in vivo [³H]thymidine incorporation into liver DNA in two-thirds hepatectomized rats. This reduction in DNA synthesis was observed at 24 hr after hepatectomy, even when the LIF was injected before the beginning of the S phase, 10 hr after hepatectomy, i.e. when DNA polymerase activity had not yet increased. Under these experimental conditions, LIF in vivo treatment prevented α DNA polymerase activity from increasing after partial hepatectomy, so that enzyme activity at 24 hr in LIF-treated rats decreased compared to the controls. No direct inhibitory effect of LIF on α DNA polymerase was detected. LIF did not affect β DNA polymerase. These results suggest that LIF plays a part in controlling liver growth.     

Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage

Background  

P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial.     

Overexpression of WDR79 in non‐small cell lung cancer is linked to tumour progression

WD‐repeat protein 79 (WDR79), a member of the WD‐repeat protein family, acts as a scaffold protein, participating in telomerase assembly, Cajal body formation and DNA double‐strand break repair. Here, we first report that WDR79 is frequently overexpressed in cell lines and tissues derived from non‐small cell lung cancer (NSCLC). Knockdown of WDR79 significantly inhibited the proliferation of NSCLC cells in vitro and in vivo by inducing cell cycle arrest and apoptosis. WD‐repeat protein 79 ‐induced cell cycle arrest at the G0/G1 phase was associated with the expression of G0/G1‐related cyclins and cyclin‐dependent kinase complexes. We also provide evidence that WDR79 knockdown induces apoptosis via a mitochondrial pathway. Collectively, these results suggest that WDR79 is involved in the tumorigenesis of NSCLC and is a potential novel diagnostic marker and therapeutic target for NSCLC.     

Cyclooxygenases and prostaglandin E2 receptors in growth plate chondrocytes in vitro and in situ – prostaglandin E2 dependent proliferation of growth plate chondrocytes

Prostaglandin E₂ (PGE₂) plays an important role in bone development and metabolism. To interfere therapeutically in the PGE₂ pathway, however, knowledge about the involved enzymes (cyclooxygenases) and receptors (PGE₂ receptors) is essential. We therefore examined the production of PGE₂ in cultured growth plate chondrocytes in vitro and the effects of exogenously added PGE₂ on cell proliferation. Furthermore, we analysed the expression and spatial distribution of cyclooxygenase (COX)-1 and COX-2 and PGE₂ receptor types EP1, EP2, EP3 and EP4 in the growth plate in situ and in vitro. PGE₂ synthesis was determined by mass spectrometry, cell proliferation by DNA [³H]-thymidine incorporation, mRNA expression of cyclooxygenases and EP receptors by RT-PCR on cultured cells and in homogenized growth plates. To determine cellular expression, frozen sections of rat tibial growth plate and primary chondrocyte cultures were stained using immunohistochemistry with polyclonal antibodies directed towards COX-1, COX-2, EP1, EP2, EP3, and EP4. Cultured growth plate chondrocytes transiently secreted PGE₂ into the culture medium. Although both enzymes were expressed in chondrocytes in vitro and in vivo, it appears that mainly COX-2 contributed to PGE₂-dependent proliferation. Exogenously added PGE₂ stimulated DNA synthesis in a dose-dependent fashion and gave a bell-shaped curve with a maximum at 10^-8 M. The EP1/EP3 specific agonist sulprostone and the EP1-selective agonist ONO-D1-004 increased DNA synthesis. The effect of PGE₂ was suppressed by ONO-8711. The expression of EP1, EP2, EP3, and EP4 receptors in situ and in vitro was observed; EP2 was homogenously expressed in all zones of the growth plate in situ, whereas EP1 expression was inhomogenous, with spared cells in the reserve zone. In cultured cells these four receptors were expressed in a subset of cells only. The most intense staining for the EP1 receptor was found in polygonal cells surrounded by matrix. Expression of receptor protein for EP3 and EP4 was observed also in rat growth plates. In cultured chrondrocytes, however, only weak expression of EP3 and EP4 receptor was detected. We suggest that in growth plate chondrocytes, COX-2 is responsible for PGE₂ release, which stimulates cell proliferation via the EP1 receptor.     

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro*

Abstract. Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [³H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G₀/G₁, S, and G₂/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [³H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [³H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G₂ tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

Differential Effects of 3,5,3′-Triiodothyronine on Control andmdxMyoblasts and Fibroblasts: Analysis by Flow Cytometry

The possibility of differential effects of triiodothyronine (T3) treatmentin vivoon myoblast and fibroblast cell proliferation was examined in control andmdxmuscle cultures. Cell isolates were purified in a Percoll gradient, sorted by flow cytometry (light scatter), and characterized as myoblasts and fibroblasts using anti-skeletal muscle myosin fluorescence. The two cell types were grown separately or remixed (1:1). Cultures were incubated with or without T3 (10⁻⁹M) for 19 h. Cells were either exposed to [³H]thymidine for 1 h and DNA prepared for scintillation counts or stained with propidium iodide for cell cycle analysis by flow cytometry. Overall [³H]thymidine uptake per cell was greater inmdxthan control cells (mainly fibroblasts and mixed cells) and was decreased by T3 only in myoblast and mixed cultures. Cell cycle data showed that the effects of T3 originated primarily at the G₀/G₁phase. There were moremdxthan control myoblasts at G₀/G₁without T3. After T3 treatment, more control fibroblasts than myoblasts were at G₀/G₁, but moremdxmyoblasts than fibroblasts were at G₀/G₁. In the absence of T3, there were also fewermdxthan control myoblasts in S. After T3, only the proportion ofmdxmyoblasts in S phase was reduced. Results are consistent with distinct T3 effects on muscle regenerationin vivoand support the hypothesis that cycling and proliferation ofmdxand control myoblasts are differentially modulated by T3. As control andmdxfibroblasts also showed distinct responses to T3, muscle regeneration likely occurs by a complex regulation of gene expression endogenous to specific cell types as well as interactions between cells of different lineage.     

Molecular evidence of anti-leukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model

We have shown that gypenosides (Gyp) induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there are no reports showing that show Gyp acts on human leukemia HL-60 cells in vitro and in a murine xenograft model in vivo. In the present study effects of Gyp on cell morphological changes and viability, cell cycle arrest and induction of apoptosis in vitro and effects on Gyp in an in vivo murine xenograft model. Results indicated that Gyp induced morphological changes, decreased cell viability, induced G0/G1 arrest, DNA fragmentation and apoptosis (sub-G1 phase) in HL-60 cells. Gyp increased reactive oxygen species production and Ca²⁺ levels but reduced mitochondrial membrane potential in a dose- and time-dependent manner. Gyp also changed one of the primary indicators of endoplasmic reticulum (ER) stress due to the promotion of ATF6-α and ATF4-α associated with Ca²⁺ release. Gyp reduced the ratio of Bcl-2 to Bax due to an increase in the pro-apoptotic protein Bax and inhibited levels of the anti-apoptotic protein Bcl-2. Oral consumption of Gyp reduced tumor size of HL-60 cell xenograft mode mice in vivo. These results provide new information on understanding mechanisms by which Gyp induces cell cycle arrest and apoptosis in vitro and in vivo.     

Overexpression of Cyclin D1 Blocks Proliferation of Normal Diploid Fibroblasts

Human cyclin D1 forms complexes with several Cdks, with proliferating cell nuclear antigen, and with a recently discovered 21-kDa inhibitor of Cdk activity. Substrates for cyclin D1/Cdks have not been identified in vivo, but it has been proposed that the D class of cyclins might play a role in regulating the activity of the retinoblastoma gene product p105^Rb, Here we report that normal human diploid fibroblasts that endogenously or ectopically express high levels of cyclin D1 are unable to enter S phase in response to normally mitogenic stimuli. Fibroblasts that have reached the end of their in vitro life span (senescent cells) express five-fold higher levels of cyclin D1 protein than low-passage cells and individual cells in mass culture that fail to initiate DNA synthesis in response to serum addition have severalfold higher levels of this cyclin than proliferation-competent cells. These observations provide evidence that cyclin D1 is involved with the regulation of cell proliferation by more than one mechanism.     

The Structural Characterization of a Novel Water-Soluble Polysaccharide from Edible Mushroom Leucopaxillus giganteus and Its Antitumor Activity on H22 Tumor-Bearing Mice

In the present study, a novel cold water-soluble polysaccharide fraction (LGP) with the average molecular weight of 1.78×10⁶ Da was extracted and purified from Leucopaxillus giganteus and its primary structure as well as in vivo antitumor activity was evaluated. The monosaccharide composition of LGP was determined by ion chromatography to be galactose, xylose, glucose and fucose in a molar ratio of 2.568 : 1.209 : 1 : 0.853. Its backbone was composed of α-D-Glu, α-D-Xyl, α-D-Gal and α-L-Fuc. The results of in vivo antitumor experiment demonstrated that LGP could effectively protect immune organs, has excellent antitumor activity, and inhibit the proliferation of H22 solid tumors in a dose-dependent manner. By analyzing Annexin V-FITC/PI staining, cell cycle and mitochondrial membrane potential detection assay, we concluded that LGP induced apoptosis of H22 cells via S phase arrest and mitochondria-mediated apoptotic pathway. Our results could provide valuable information for the potential application of LGP as an anti-hepatoma agent.     

Misexpression of cyclin D1 in embryonic germ cells promotes testicular teratoma initiation

Testicular teratomas result from anomalies in embryonic germ cell development. In the 129 family of inbred mouse strains, teratomas arise during the same developmental period that male germ cells normally enter G1/G0 mitotic arrest and female germ cells initiate meiosis (the mitotic:meiotic switch). Dysregulation of this switch associates with teratoma susceptibility and involves three germ cell developmental abnormalities seemingly critical for tumor initiation: delayed G1/G0 mitotic arrest, retention of pluripotency, and misexpression of genes normally restricted to embryonic female and adult male germ cells. One misexpressed gene, cyclin D1 (Ccnd1), is a known regulator of cell cycle progression and an oncogene in many tissues. Here, we investigated whether Ccnd1 misexpression in embryonic germ cells is a determinant of teratoma susceptibility in mice. We found that CCND1 localizes to teratoma-susceptible germ cells that fail to enter G1/G0 arrest during the mitotic:meiotic switch and is the only D-type cyclin misexpressed during this critical developmental time frame. We discovered that Ccnd1 deficiency in teratoma-susceptible mice significantly reduced teratoma incidence and suppressed the germ cell proliferation and pluripotency abnormalities associated with tumor initiation. Importantly, Ccnd1 expression was dispensable for somatic cell development and male germ cell specification and maturation in tumor-susceptible mice, implying that the mechanisms by which Ccnd1 deficiency reduced teratoma incidence were germ cell autonomous and specific to tumorigenesis. We conclude that misexpression of Ccnd1 in male germ cells is a key component of a larger pro-proliferative program that disrupts the mitotic:meiotic switch and predisposes 129 inbred mice to testicular teratocarcinogenesis.     

Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

Loss of p21 expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21 gene promoter

To answer what is a critical event for higher incidence of tumor development in old than young individuals, primary culture of human diploid fibroblasts were employed and DNA damage was induced by doxorubicin or X-ray irradiation. Response to the damage was different between young and old cells; loss of p21^sdi1 expression in spite of p53^S15 activation in old cells along with [³H]thymidine and BrdU incorporation, but not in young cells. The phenomenon was confirmed by other tissue fibroblasts obtained from different donor ages. Induction of miR-93 expression and reduced p53 binding to p21 gene promoter account for loss of p21^sdi1 expression in senescent cells after DNA damage, suggesting a mechanism of in vivo carcinogenesis in aged tissue without repair arrest.     

In Vivo measurement of unscheduled DNA synthesis and S-phase synthesis as an indicator of hepatocarcinogenesis in rodents

Measurement of chemically induced DNA repair as unscheduled DNA synthesis in rodent liver following in vivo treatment is a useful screen for potential hepatocarcinogens. In addition to measurement of unscheduled DNA synthesis, examination of S-phase synthesis provides an indicator of chemically induced cell proliferation in the liver, which may be a basis for hepatic tumor promotion. Several chemicals and classes of chemicals have been examined using these endpoints. The pyrrolizidine alkaloid riddelline is a potent genotoxic agent in vitro, and in vivo studies confirm this response as riddelline induces significant elevations in unscheduled DNA synthesis and S-phase synthesis in rat liver. Conversely, H. C. Blue dyes #1 and #2 are both potent genotoxic agents in vitro but fail to express this genotoxicity in vivo. H. C Blue #1 induces significant increases in S-phase synthesis in B6C3F1 mouse liver, which correlates with the observed carcinogenicity of this compound. Halogenated hydrocarbons likewise fail to induce unscheduled DNA synthesis in vivo, but many of these compounds do increase hepatic cell proliferation in mice, which may be the principal mechanism of hepatocarcinogenesis in this species.Abbreviations BCMEE bis(2-chloro-l-methylethyl)ether - dThd thymidine - HCB1 H.C. Blue #1 - HCB2 H.C. Blue #2 - UDS unscheduled DNA synthesis     

Exogenous retinoic acid decreases in vivo and in vitro proliferative activity during the early migratory stage of neural crest cells

We have previously demonstrated that directional migration of neural crest cells (NCC) is associated with a high cell density, resulting from an active cell proliferation. It is also known that treatment with retinoic acid (RA) causes a dose-dependent inhibition of proliferation of some cell types, and that administration of RA during the early stages of embryonic development, induces cranio-facial abnormal patterns corresponding to NCC derivatives. In view of these findings, it was of interest to determine if exogenous RA is a potential modulator of the mitotic rate of NCC, and to explore the hypothesis of an inhibitory effect exerted by RA on the proliferative behaviour of NCC in vivo and in vitro. Homogenates of RA-treated chick embryos showed a low [³H]dT incorporation, indicating a generalized diminution of DNA synthesis. The labelling index (LI=number of labelled cells/total number of cells) revealed that NCC from RA-treated and control embryos had higher values of [³H]dT incorporation than neural tube cells (P < 0.0001). Autoradiographs of RA-treated chick embryos showed a significantly lower [³H]dT incorporation in NCC at the prosencephalic and mesencephalic levels, as well as in the neural tube cells at the prosencephalic, mesencephalic and rhombencephalic levels, than in control chick embryos (P < 0.0001). NCC cultures treated with 1 or 10 μm RA had a significantly lower LI than in cultures treated with 0.1 μm RA or control cultures (P < 0.04). In chick embryos, the mitotic index of NCC was 0.026 for RA-treated and 0.033 for controls, while the duration of the cell cycle was significantly longer in the NCC of RA-treated embryos (～ 40 h) than in controls (～ 25 h). The length of the cell cycle phases of NCC was similar in both experimental conditions, except for G₁ phase, which was significantly longer in the RA-treated group than in controls. These results show that RA blocks DNA synthesis and lengthens the proliferative behaviour of NCC both in early chick embryos and in vitro, effects that could modify the morphogenetic patterns of NCC distribution through a decreased cell population.     

TGFβ1 induces growth arrest and apoptosis but not ciliated cell differentiation in rat tracheal epithelial cell cultures

Summary We are studying the regulation of ciliated cell differentiation using an in vitro model of tracheal regeneration. Previously, we reported that removal of growth stimulating compounds such as epidermal growth factor (EGF) and cholera toxin reduced DNA synthesis and cell number while increasing ciliated cell differentiation (Clark et al., 1995). This result suggested that the induction of growth arrest may stimulate terminal differentiation of airway epithelial cells into ciliated cells. Transforming growth factor βs (TGFβs) inhibit epithelial cell proliferation and have also been shown to stimulate epithelial cell differentiation. In this study, the effect of TGFβ₁ on growth and ciliated cell differentiation of rat tracheal epithelial (RTE) cells was examined. TGFβ₁ inhibited [³H]thymidine incorporation by RTE cells in a dose-dependent manner. A 40% inhibition was observed after a 24-h incubation with 10 pM TGFβ₁. Continuous treatment with TGFβ₁ (1–50 pM) also reduced cell number during the time when ciliogenesis occurs. This reduction resulted in part from a loss of cells through exfoliation, in addition to the inhibition of proliferation. The exfoliated cells exhibited several morphological features characteristic of apoptosis, including shrunken cells, condensed and fragmented nuclei, and intact organelles. In addition, electrophoretic analysis of genomic DNA analysis isolated from exfoliated cells demonstrated the presence of a nucleosomal ladder. However, in contrast to the removal of EGF, treatment with TGFβ₁ for 7 d did not increase ciliated cell differentiation. TGFβ1 is, therefore, capable of inhibiting proliferation and increasing apoptosis in RTE cells without stimulating ciliated cell differentiation.     

Temporal relationships between DNA metabolism and the growth-inhibitory response produced by dexamethasone in rat glioma cell cultures

Summary The temporal relationships between aspects of DNA metabolism and the suppression of cell proliferation were investigated in rat glioma (strain C6) monolayer cultures exposed to 10μM dexamethasone. Cell densities (cell number per cm²), rates of DNA synthesis (dpm of [³H]thymidine incorporated per μg DNA per min), and cellular DNA (μg DNA per cm²) were measured daily in control and dexamethasone-treated cultures over a 3-day period. The percentage of cells in metaphase and the proportion of metaphases containing >2n(42) chromosomes also were determined in control and treated cultures. When log-phase C6 cultures were exposed to dexamethasone (day 0), cell densities were not significantly different from controls by day 1. Cell proliferation ceased thereafter in dexamethasone-treated cultures, whereas control cell populations continued to proliferate at log-phaserates. In contrast, cellular DNA increased exponentially in control and treated cultures over the 3-day period. On days 0 and 1, control and treated cells each contained 6 pg DNA. By day 3, the DNA content per treated cell increased to >20 pg; control cells each contained 10 pg DNA. The rates of DNA synthesis in the treated cultures did not differ significantly from controls on days 1 and 2. However, the rate in the treated cultures decreased significantly on day 3, one day after cell proliferation ceased. On day 2, the percentage of cells found in metaphase in the treated cultures was 0.32% compared to 0.64% in control cultures. By day 3, these percentages decreased to 0.20% and 0.22%, respectively. However, the proportion of metaphases containing >42 chromosomes increased 1.5-fold in the treated cultures relative to controls. These results indicate that nonproliferating dexamethasone-treated cells contain elevated amounts of DNA. Thus dexamethasone action appears to arrest the cell cycle at any point between the completion of DNA replication and mitosis. A preliminary report of this work was presented on June 8, 1977, at the 28th Annual Meeting of the Tissue Culture Association in New Orleans, Louisiana. This investigation was supported in part by grants from Merck Sharp & Dohme Research Laboratories, West Point, Pa., the American Cancer Society (IN-113), and NIH (AM 18719).     

Dysfunctional telomeres induce p53‐dependent and independent apoptosis to compromise cellular proliferation and inhibit tumor formation

Aging is associated with progressive telomere shortening, resulting in the formation of dysfunctional telomeres that compromise tissue proliferation. However, dysfunctional telomeres can limit tumorigenesis by activating p53‐dependent cellular senescence and apoptosis. While activation of both senescence and apoptosis is required for repress tumor formation, it is not clear which pathway is the major tumor suppressive pathway in vivo. In this study, we generated Eμ‐myc; Pot1b ^?/? mouse to directly compare tumor formation under conditions in which either p53‐dependent apoptosis or senescence is activated by telomeres devoid of the shelterin component Pot1b. We found that activation of p53‐dependent apoptosis plays a more critical role in suppressing lymphoma formation than p53‐dependent senescence. In addition, we found that telomeres in Pot1b^?/?; p53^?/? mice activate an ATR‐Chk1‐dependent DNA damage response to initiate a robust p53‐independent, p73‐dependent apoptotic pathway that limited stem cell proliferation but suppressed B‐cell lymphomagenesis. Our results demonstrate that in mouse models, both p53‐dependent and p53‐independent apoptosis are important to suppressing tumor formation.     

Cell proliferation in the subependymal layer of the adult mouse in vivo and in vitro

Pulse labelling experiments with [³H] thymidine (dT) and double labelling experiments with [³H]dT and bromodeoxyuridine (BrdUrd) were carried out on cells of the subependymal layer in the brain of adult normal mice in vivo, in vivo/in vitro and in vitro. The results should (i) lead to information about cell cycle parameters of these cells in the brain of adult mice, since these cells have been studied mostly in the rat brain up to now and (ii) answer the question whether results concerning cell proliferation obtained in vivo correspond with those from brain slices incubated in vitro with or without prelabelling in vivo. In vivo an LI of 20.2 ± 2.7% (x?± SEM) and T_s= 7.2 ± 0.7h were found. Furthermore, grain count halving experiments led to a surprisingly short cycle time (T_c) of 11.2–14.2 h. The longer T_c values (18–20 h) reported in the literature for subependymal cells in the rat brain seem to be due to evaluations of different areas around the lateral ventricle without considering the migrating behaviour of these cells which is quite different regionally. The in vitro studies (with or without prelabelling in vivo) showed a significantly reduced LI due to the fact that about 20% of the S phase cells, possibly lying in the middle of S, stopped further DNA synthesis after transfer to culture. This was shown by comparing the cell fluxes at the G₁/S and S/G₂ borders of in vivo vs. in vitro studies.     

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.