Derepression of the cell cycle by starvation is involved in the induction of tobacco pollen embryogenesis

Summary Microspectrophotometry following Feulgen staining and autoradiography following (³H)-thymidine labelling were used to study cell-cycle events during pollen development in tobacco (Nicotiana tabacum L.). During normal gametophytic pollen development in the anther and in vitro the generative nucleus passes through the S phase to the G₂ phase soon after microspore mitosis, while the vegetative nucleus remains arrested in G₁ (=G₀). During embryogenie induction by an in vitro starvation treatment of immature pollen ongoing DNA replication in the generative nucleus is completed and followed by DNA replication in the vegetative cell in a large fraction of the pollen grains. Addition of the DNA replication inhibitor hydroxyurea to the starvation medium postpones S phase entry until the pollen is transferred to a rich medium and does not affect embryo formation. These results demonstrate that one of the crucial events of embryogenic induction is the derepression of the G₁ arrest in the cell cycle of the vegetative cell.     

Reinitiation of DNA synthesis in quiescent mouse keratinocytes; Regulation by polypeptide hormones,cholera toxin,dexamethasone, and retinoic acid

Summary Cloned mouse keratinocytes (MK-1 cells) display density-dependent growth arrest when reaching confluency in a serum-free medium with a calcium concentration <0.1 mM, supplemented only with insulin and transferrin. In this quiescent state, greater than 95% of the cell population is in the G_0/1 phase of the cell cycle. Treatment of quiescent MK-1 cells with 1 to 10 ng/ml epidermal growth factor (EGF) resulted in a sharp burst of DNA synthetic activity. Both insulin and cholera toxin potentiated the mitogenic effect of EGF, but neither agent was necessary or sufficient to induce thymidine incorporation into DNA. Dexamethasone abolished the effect of insulin, but not the mitogenic effect of EGF alone. In contrast, retinoic acid (RA) did not possess any mitogenic effect for quiescent MK-1 cells, nor did it modulate the actions of EGF or dexamethasone. A number of commercially available crude extracts of bovine brain and pituitary were also capable of initiating DNA synthesis in resting MK-1 cells. Finally, transforming growth factor type beta (TGFβ) proved to be a potent inhibitor of the mitogen-induced DNA synthesis in MK-1 cells (IC₅₀∶10pM). This defined culture system is eminently suited to study the regulation of DNA synthesis of epidermal cells. In addition, it can be used as a sensitive bioassay for the detection of epidermal mitogens, as well as inhibitors of DNA synthesis such as TGFβ. Supported by PHS Award CA-41556 from the National Cancer Institute, Bethesda, MD.     

Epidermal Growth Factor Induces Proliferation and DNA Synthesis in Ciliate Cells

We studied the effect of murine epidermal growth factor on cell proliferation and DNA synthesis in macronuclei of ciliate Tetrahymena pyriformis Gl. Mitogenic effect of epidermal growth factor on proliferation-induced tetrahymena cells has been revealed. This effect is due to the induced progression of cells at G ₁ and, consequently, their earlier entering DNA synthesis phase of the first cell cycle. Epidermal growth factor had no mitogenic effect on the resting cells in a stationary culture (G ₀ phase) whose development is independent of the growth factors in the medium.     

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.     

Organ culture of adult rat colonic mucosa on fibrin foam

Summary A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37°C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum,l-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmosphere for culture was 95% O₂ and 5% CO₂. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [³H]thymidine and [³H]leucine into DNA and protein, [¹⁴C]glucosamine and [³H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O₂ retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland. This work was supported by the National Cancer Institute Contract N01-CP-75953 and in part by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, under Contract N01-CO-65341 with the International Union Against Cancer.     

The specificity of epidermal chalone action: the results of in vivo experimentation with two purified skin extracts.

To date two inhibitors of epidermal cell proliferation have been characterized: (1) a factor which depresses DNA synthesis, and (2) a factor which depresses mitotic rate. In the absence of experimental proof it has been assumed that the respective targets for these purified inhibitory factors are in G₁ and G₂ phases of the cell cycle. In the experiments reported here both these fractions were subjected to cell cycle phase specificity tests in order to verify these assumptions. In addition, an epidermally derived “cell line” (the sebaceous gland) and two nonectodermal tissues were examined for a response. The results suggest that the response induced by the inhibitor of DNA synthesis is cell cycle phase-specific, that the target cells are at the G₁-S phase boundary, and that only epidermal cells respond. Similarly the factor which depresses the flow of cells from G₂ into mitosis had no measurable effect on DNA synthesis by any of the tissues tested. The G₂ inhibitor lacks an inhibitory effect on mitosis in the sebaceous gland.The physiological roles which epidermal chalones may play are briefly discussed. It is suggested that a G₁–G₂ chalone system may have been effective in isolating kinetically cell populations with modified function during the evolutionary development in the vertebrates.     

Cell cycle specificity and reversibility of the inhibitory effect of epidermal G1-chalone on DNA synthesis in partially synchronized RTE-2 keratinocytes in vitro

The specific action of a pig skin fraction enriched in epidermal G₁-chalone, a tissuespecific inhibitor of epidermal DNA synthesis, was investigated by means of flow cytofluorometry. The results indicate that G₁-chalone inhibits progression of partially synchronized rat tongue epithelial cells (line RTE-2) through the cell cycle at a point 2 h prior to the beginning of the S-phase. Approximately 8 h after chalone addition, the cells can overcome the inhibition and begin to enter the S-phase. The duration of this delay is concentrationindependent, but the fraction of cells affected is proportional to the chalone concentration. The progression of cells which already have entered S-phase is not affected. In contrast to the G₁-chalone preparation, aphidicolin, a potent inhibitor of DNA polymerase α, clearly shows S-phase-specific inhibition. These results indicate that the epidermal G₁-chalone inhibits epidermal cell proliferation in a fully reversible manner by a highly specific effect on cell cycle traverse.     

Basal-Cell Subpopulations and Cell-Cycle Kinetics In Human Epidermal Explant Cultures

Cultured human epidermal cells were studied by cell sorting and autoradiography after different ³H-thymidine (³H-dThd)-labelling procedures and after labelling with DNA precursors that are incorporated via salvage or de novo pathways. It was shown that ³H-dThd incorporation was the best measure of the rate of DNA replication. Dose-response experiments with pulse and continuous labelling revealed that all S- and G₂-phase cells were cycling, whereas some 20% of the cells stayed in G₁-phase for long periods of time. Most, if not all of these cells were probably non-proliferating differentiated keratinocytes. At least two subpopulations of S-phase cells could be discriminated on the basis of the rate of incorporation of DNA precursors. the difference in precursor incorporation did not seem to be caused by differences in nucleotide metabolism but rather to reflect true differences in the rate of DNA replication. Continuous labelling experiments showed that these subpopulations also were apparent in the G₁- and G₂-phases. Studies of the grain-count distribution revealed that cells that appeared to move rapidly through the S-phase moved slowly through the G₂-phase, and vice versa. Cells stained with acridine orange were subjected to a two-parameter analysis in the cell sorter by simultaneous measurement of the DNA and RNA fluorescence. Autoradiography of sorted cells revealed that, on average, cells with low RNA contents incorporated ³H-dThd at a higher rate than cells with high RNA contents.     

Use of a low-speed,iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations

Summary A simple yet effective method (iso-density percoll centrifugation) has been developed for consistently preparing isolated rat liver parenchymal cells with over 98% initial viability. The method has been applied to cells isolated by a variety of collagenase digestion techniques. This procedure involves the low-speed centrifugation (50 ×g) of the initial cell suspension through a percoll medium having a density of 1.06 g/ml and results in the separation of single and viable parenchymal cells from cell aggregates, debris, and nonparenchymal cells. The enriched parenchymal cells have been shown to be superior to untreated cells by a number of criteria including: preparation homogeneity, cell morphology, maintenance of cytochrome P-450, hormonal responsiveness (measured by the induction of tyrosine aminotransferase after treatment with glucagon or dexamethasone, or both), plasma membrane integrity (determined by both trypan blue exclusion and leakage of glutamic-oxaloacetic transaminase), and the DNA repair capability after treatment with benzo[a]pyrene or 2-acetylaminofluorene. This work was supported in part by the National Institutes of Health Biomedical Research Support Program, and National Institute of Environmental Health Services grant (ES-01737) awarded to M.T.S.H., and by National Cancer Institute grants CA-017175, CA-09135, CA-22484 awarded to H.C.P.N.S. was supported by a Cancer Research Campaign Grant (U. K.) through the International Union Against Cancer. This work was presented in part at the 24th Annual Meeting of the Society of Toxicology, 18–22 March 1985, San Diego, CA.     

Cadmium cytotoxicity and variation in nuclear content of DNA in Euglena gracilis

Cultures of Euglena gracilis (strain Z from French CNRS collection) can be made cadmium resistant if grown in a medium with 5x10^-4M cadmium chloride. This resistance is reflected by the appearance of a second exponential growth phase. The development of this resistance was studied at the cellular level by determining the relative content of DNA at different stages of the cell cycle in an asynchronously grown culture. The culture was followed until the second, cadmium resistant, growth phase had reached its stationary state. During the first exponential growth phase, cells were mostly in the late period of DNA synthesis (stage S of the cell cycle), or in the gap preceding mitosis (stage G₂ of the cell cycle). In addition, some cells contained high multiples of the normal amount of DNA. In the beginning of the second exponential growth phase, a few cells were again in G₁ (the post mitotic stage of the cell cycle preceding DNA synthesis). These G₁ cells were predominant at the end of the second growth period. During the second stationary phase the DNA content of the cadmium treated cells was similar to the stationary phase of the control culture. Cells had stopped growing in G₁ with an unreplicated genome. The implications of these data are discussed.     

The naphthylamidase reaction as a diagnostic tool for the demonstration of cellular injury and autophagy

Summary As a sequence to previous results suggesting that the naphthylamidase (LNase) reaction performed at pH 5.5 mainly demonstrates sites of available lysosomal cathepsin B activity, a study was undertaken both on spontaneously occurring and induced cellular autolysis. This modified LNase reaction forms a useful enzymatic tool for the histochemical visualization of enlarged lysosomes and autolytio vacuoles. Cell injury may thus be demonstrated at an earlier stage long before dye-exclusion tests become positive.Supported by institutional grants from the Jubilee Fund and the Cancer Society of Stockholm.This work was undertaken during the tenure of an Eleanor Roosevelt International Cancer Fellowship of the American Cancer Society awarded to Dr. Niemi by the International Union Against Cancer.     

WEE1 inhibition and genomic instability in cancer

One of the hallmarks of cancer is genomic instability controlled by cell cycle checkpoints. The G₁ and G₂ checkpoints allow DNA damage responses, whereas the mitotic checkpoint enables correct seggregation of the sister chromosomes to prevent aneuploidy. Cancer cells often lack a functional G₁ arrest and rely on G₂ arrest for DNA damage responses. WEE1 kinase is an important regulator of the G₂ checkpoint and is overexpressed in various cancer types. Inhibition of WEE1 is a promising strategy in cancer therapy in combination with DNA-damaging agents, especially when cancer cells harbor p53 mutations, as it causes mitotic catastrophy when DNA is not repaired during G₂ arrest. Cancer cell response to WEE1 inhibition monotherapy has also been demonstrated in various types of cancer, including p53 wild-type cancers. We postulate that chromosomal instability can explain tumor response to WEE1 monotherapy. Therefore, chromosomal instability may need to be taken into account when determining the most effective strategy for the use of WEE1 inhibitors in cancer therapy.     

Effects of insecticide profenophos on germination,early growth,meiotic behavior and chlorophyll mutation of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

The genotoxicity of different concentrations of insecticide, profenophos (O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate) was evaluated at various stages of cell cycle (G₁, S and G₂) by using the seeds of barley (Hordeum vulgare L.). The aim of this work was to investigate the effects of insecticide profenophos at various stages of cell cycle on germination, seedling height and meiotic behavior in M₁ and chlorophyll mutations in M₂ generation. From the present study, it can be concluded that the stages of cell cycle were sensitive for the treatments of chemicals and it also showed that the S-phase of cell cycle is more sensitive than other phases of cell cycle.     

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.     

Non-Circadian Periodicity in the Duration of the Cell Cycle and the G1 Phase in the Hindlimb Epidermis of the Anuran Tadpole,Rana Pipiens

Using the percentage labeled mitoses method, seven cell cycle determinations were initiated at 6-hr intervals over a 36-hr span in order to see if the cell cycle in the tadpole hindlimb epidermis varied with time or showed rhythmicity. There was a pattern of two long cell cycles followed by a shorter one. Total cell cycle length (Tc) and the length of the G₁ phase plus one-half of the mitotic time (TG₁+½M) fluctuated the most, although only TG₁+½M varied significantly with the Chi-square test. The proportion of TC spent in each phase was also calculated. Only TG₁+½M/Tc had statistically significant fluctuations with time.

Rhythmicity was analyzed by a computer program using the method of least squares for cosine curve fitting. Statistically significant ultradian rhythms of 18.4 hr in TC, 18.5 hr in TG₁+½M and 18.6 hr in TG₁+½M/TC and the length of the DNA synthetic phase/total cell cycle length (TS/TC) were found. Circadian rhythmicity was not observed. The acrophases of the ultradian rhythms of TC and TG₁+½M coincided, suggesting that the rhythm of TC was due mainly to variation in TG₁+½M. In the absence of significant variation in TS, the longest phase of the cell cycle, whenever G₁+½M was short, TS/TC increased, so that the 18.6 hr rhythm in TS/TC was also a result of the periodicity in TG₁+½M.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Mouse epidermal cell renewal after topical treatment with different concentrations of the epidermal peptide pyroGlu-Glu-Asp-Ser-GlyOH

Earlier work has shown that epidermal cells contain a peptide, pyroGlu-Glu-Asp-Ser-GlyOH, that induces a moderate but long-lasting inhibition of epidermal cell proliferation when given at low (picomol) doses ip in vivo and in vitro. In the present study, the epidermal pentapeptide was applied topically to the back skin of hairless mice at different concentrations and in a water-miscible cream. A single topical application of either high (0.25% wt/wt) or low (0.004% or 0.02% wt/ wt) doses of the pentapeptide was followed by oscillations in epidermal DNA synthesis and G₂-M cell flux (mitotic rate). In general, epidermal cell proliferation was inhibited during the first 10-day period after treatment with the two lower doses, while the highest concentration of pentapeptide (0.25%) stimulated epidermal cell proliferation. In spite of the effects on epidermal cell proliferation the size of the epidermal cell population in the treated area (number of nucleated cells and epidermal thickness) showed no corresponding alterations. The results could imply that the epidermal pentapeptide modifies epidermal cell proliferation and terminal differentiation in such a way that the two are balance with each other.     

Epidermis Extracts (Chalone) Inhibit Cell Flux At the G1-S, S-G2 and G2-M Transitions In Mouse Epidermis

Epidermal cell flux at the G₁-S, S-G₂ and G₂-M transition was examined during the first 4 hr after injection of epidermis extract. the flux parameters were estimated by a combination of several methods. the G₁-S and S-G₂ transit rates were calculated on the basis of a double labelling technique with [³H]TdR, the G₂-M flux by means of colcemid and the relative proportion of cells in the S or G₂ phase by means of flow cytometry. All experiments were performed both in early morning and late evening, corresponding to maximum and minimum rates of epidermal cell proliferation in the hairless mouse. the epidermis extract inhibited the S-G and G₂-M transit rates to the same degree, while the inhibition of cell flux at the G₁-S transit was consistently stronger. In general, the inhibition of cell flux at the different transitions was most pronounced when the rate of cell proliferation was low and vice versa.     

Improved conditions for murine epidermal cell culture

Summary An improved method for cultivating newborn mouse epidermal cells has been developed that increases the longevity, epithelial nature and efficiency of cell-line establishment. The use of Super Medium, an enriched Waymouth's formulation, increased proliferation for long periods of time, as did incubation at 31°C rather than 37°C. The fetal bovine serum requirement was found to be reduced at the lower temperature. An increase in labeling indices was seen when epidermal growth factor (EGF) or the cyclic nucleotides were added and the presence of EGF receptors was determined. Of the prostaglandins (PG) examined, PGE₁ and PGE₂ produced the greatest increase in DNA synthesis. The PG precursors, arachidonic and 8,11,14-eicosatrienoic acid, were also greatly stimulatory. The use of a lethally irradiated 3T3 feeder layer at 31°C proved superior in maintenance of an epithelial morphology. Subculturable cell lines were established much more readily and reproducibly in carcinogen-treated cultures grown under the improved conditions. Research sponsored jointly by the National Cancer Institute under Interagency Agreement YO1-CP-70227 and the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. Predoctoral Investigator supported by Grant CA 09104 from the National Cancer Institute. Postdoctoral Investigator supported by Carcinogenesis Training Grant CA 05296 from the National Cancer Institute.     

Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: Clonal analyses,growth kinetics,and cell cycle studies

The effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum-free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony-forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24-hour cell generation time with G₁, S, G₂, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (> 30 population doublings) and neonatal prokeratinocytes (> 50 population doublings) is associated with senescence and the G₁ arrest of noncycling cells.