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 tissue-specific 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 concentration-independent, 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 alpha, 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.     

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.     

Immunomorphological analysis of G2-chalone localization during the process of rat epidermis histogenesis

The appearance of G2-chalone in the cytoplasm of the intermediate cell layer and partly in the periderm of 17-day-old rat embryo epidermis has been demonstrated by the indirect method of Coons using a monospecific antiserum. G2-chalone was absent from the basal cell layer of 17--21-day-old embryos and of the newborn rats. It was found in all the epidermal layers in 2--5-day-old postnatal rats, while in 6--9-day-old animals it was primarily detected in the cytoplasm of spinous and basal cells. Thus the localization of epidermal G2-chalone typical for defined tissue becomes stabilized at the end of epidermis histogenesis.     

Purification and characterization of epidermal G2- and G1-chalones]

Highly purified epidermal G1- and G2-chalones from rat skin inhibit the entering of epidermocytes to S and M phases of cell cycle respectively. Their biological activity is characterized by tissue-specificity and not by species-specificity. Both of them are tissue-specific glycoproteins as for their antigenic properties. Molecular weight of G1-chlone is 21 000, G2-chalone--34 000, isoelectric point (pH) 5.55 and 5.85 respectively. G2-chalone is the fastest as compared to G1-chalone in 5% acrylamide gel electrophoresis, pH 8.3. When injected in rabbits, G2-chalone produced monospecific antibodies which have no cross-reactivity with G1-chalone. The amino acid composition of both chalones and immunofluorescent localization of G2-chalone in epidermal tisues are given.     

Integration of tissue-specific inhibitors and thyroid hormones in the regulation of proliferation of gastric gland epithelial cells]

G1- and G2-chalone effects of pig's stomach mucosa extract were registered in mice stomach gland epithelium. The inhibitory activity of chalones on cell's proliferation reduced following increased level of thyroid hormones. Local (chalones) and organism (thyroid hormones) factors cooperate in control of gland epithelial cell proliferation.     

Determination of epidermal G2-chalone in epidermal-type tissues

By means of monospecific immune serum, using the indirect method by Coons, the epidermal G2-chalone was revealed in the corneal epithelium, in the transitory epithelium of the urinary bladder, renal pelvis, as well as in stromal epithelial cells of the cortical substance and in thymic bodies, the facts that suggest epithelial nature of these tissues. In tracheal epithelium the method mentioned failed to reveal G2-chalone. Analysing localization of the epidermal G2-chalone in various tissues of the epidermal origin, it has been stated that in the non-cornified multistratified flat epithelium it is present in cellular cytoplasm of all layers, while in the cornified epithelium - it is predominantly detected in basal and scupular cells. A suggestion is made that distribution of the intratissue epidermal G2-chalone depends on the process of cornification. A possibility to use G2-chalone as a marker for tissues of the epidermal type is discussed.     

A system for the study of epidermal G1-chalone in cell culture. The rat tongue epithelial cell line RTE2

A pig-skin preparation enriched in epidermal G1-chalone when administered to cells of the rat tongue epithelial line RTE2 at concentrations of 3-300 micrograms/ml (dry mass) caused a 60% reduction in cell number. Three other cell lines showed essentially no growth inhibition during chalone treatment. The kinetics of chalone inhibition were similar to those observed in mouse epidermis in vivo. Five hours after the addition of chalone preparation in fresh medium a decrease in the rate of DNA synthesis was observed. Maximum inhibition at 12 h was followed by a subsequent increase in DNA synthesis, reaching control values again after 30 h. The inhibitory effect was dose-dependent up to 3 micrograms/ml. At higher concentrations the degree of inhibition remained constant at about 50% of the control up to 300 micrograms/ml. Removal of added chalone by changing the medium at the time of maximum inhibition gave rise to a complete recovery within 9 h. These results indicate a cell-line specific, non-toxic and reversible inhibitory effect of the chalone preparation which resembles that observed in the living animal. The RTE2 cell line may thus be considered to provide a highly sensitive experimental system suitable for more detailed studies on the mechanism of action of epidermal G1-chalone.     

Immunomorphologic analysis of G2-chalone localization in epidermal-type tissues

Localization of the epidermal G2-chalone in tissues has been established by means of indirect method of Coons using a monospecific immune serum. It has been found in dermal epithelia of the back, external ear, tongue, esophagus, forestomach, vagina, hairy follicles, but it has not been found in the sebaceous glands and derma. These findings are fully in agreement with the results obtained by the method of double diffusion after Ouchterlony. Tissue specificity of G2-chalone is proved by the fact that at places where epithelia differing histogenetically join with each other, it is found only in the epithelia of the epidermal type. Within the epithelial layer G2-chalone is mainly localized in the spinous and partly in the basal cells. Possible mechanisms on regulation of the mitotic activity are discussed in connection with the data obtained.     

Changes in the level of epidermal G2-chalone and mitotic activity in the rat vaginal epithelium]

The content of tissue-specific inhibitor of mitosis in epidermal epithelium (G2-chalone) was estimated by a single radial immunodiffusion test in the rat vagina during various stages of the estrous cycle. The level of chalone was found to correlate with the mitotic index (MI) of vaginal epithelium. The lowest level of G2-chalone is detected in proestrus and the highest one in estrus. The level of G2-chalone in vaginal epithelium was shown to be significantly decreased in aging rats (14--16 month-old) with regular cycles as compared to that in young normal cycle rats (3--4 month-old). The single injection of estradiol benzoate (1 microgram/100 g) into ovariectomized rats led to an increase in the MI following 18 hours. The increased MI is preceeded by a substantial drop of the G2-chalone level 12 hours after estrogen injection.     

Inhibition by epidermal extracts of the 12-O-tetradecanoylphorbol-13-acetate induced peak of ornithine decarboxylase activity in the mouse epidermis

The time course of induction of epidermal ornithine decarboxylase (E.C. 4.1.117) (ODC) activity following a single topical application of 17 nmoles of 12-O-tetradecanoylphorbol-13-acetate (TPA) on hairless mouse skin was established. Prior intraperitoneal (i.p.) administration of a crude epidermal extract prepared from hairless mouse epidermis led to a time-dependent, 50% inhibition of the peak level of TAP-induced ODC activity. Maximum inhibition was observed when the extract was injected 1.5 h before TPA treatment. The crude epidermal extract did not affect ODC activity in vitro. Following the administration of epidermal extracts, the inhibition of the TPA-induced ODC-response correlated positively with the presence of epidermal G2-chalone activity (determined by a stathmokinetic method) whereas myocardial, skeletal muscle, or heat-inactivated epidermal extracts with no epidermal G2-chalone activity, had no effect on TPA-induced ODC activity. These results indicate a possible relationship between ODC-activity and the control of mitotic rate by G2-chalone.     

Purification of epidermal G2-chalone using immunoaffinity chromatography

Epidermal G2-chalone was purified from rat skin extract by immunoaffinity chromatography with the use of mono-specific antibodies coupled to CNBr-activated sepharose 4B. During SDS PAAG electrophoresis, it produced a single band with a molecular weight of 13000 dalton. G2-chalone purified in this way had a 60% antimitotic inhibitory effect on external ear epidermocytes in mice in a dose of 2 micrograms/g bw but had no effect on the mitosis in the sebaceous glands.     

白藜芦醇对人子宫内膜癌细胞系AN3CA增殖和凋亡效应及其机制探讨

目的：探讨白藜芦醇（resveratrol,Res）对人子宫内膜癌细胞AN3CA的增殖抑制和凋亡诱导效应及可能存在的机制。方法：应用噻唑蓝（MTT）法检测Res对AN3CA的增殖影响;流式细胞术检测Res对细胞周期分布和凋亡影响：荧光实时定量PCR检测Res对细胞Bcl-2、Bax和MMP-9mRNA表达水平的影响;WesternBlot方法检测Res对PCNA、Bcl-2、Bax及ERK1／2、P—ERK1／2蛋白表达水平的影响。结果：Res对子宫内膜癌细胞AN3CA具有显著的生长抑制作用（P〈0．01）,呈时间-剂量依赖性;不同浓度Res处理细胞G0／G1期比例显著增加伴随S期细胞数的减少;细胞凋亡率明显增高,200Dmol／lRes处理48h凋亡率可达30．96％±2．041％（P〈0．01）。与对照组相比,Res能抑制PCNA的蛋白表达量,增加Bax和降低Bcl-2转录和蛋白水平的表达量。Res在短时间内（0．5—1h）激活ERK1／2的磷酸化表达但随着作用时间延长（4—48h）其表现为抑制效应。结论：Res具有抑制AN3CA细胞增殖,诱导细胞G0／G1期阻滞和凋亡的效应。Res诱导凋亡可能是通过上调Bax,下调Bcl-2发挥作用,其抗癌作用机制可能与ERK1／2通路失调相关。     

Fractionation of a chalone-containing substance from Ehrlich's ascites tumor using high pressure chromatography]

The effect of various fractions of chalone--containing preparation from ascyte Ehrlich's tumour obtained by high performance liquid chromatography (HPLC) on mitotic activity and DNA synthesis in the tumour has been studied. After filtration the division of active chalone component which inhibits entering cells into M-phase and S-phase took place. The component inhibiting DNA synthesis eluated with G1-chalone.     

The influence of epidermal growth factor and insulin on proliferation and DNA synthesis in ciliates Tetrahymena pyriformis

The influence of the epidermal growth factor (EGE) (10(-8) M), insulin (10(-6) M) and EGF (10(-8) M) in combination with insulin (10(-6) M) on proliferation and DNA synthesis in the nuclei of ciliates Tetrahymena pyriformis GL was studied. Insulin and EGF, known to stimulate growth of many types of mammalian cells revealed a mitogen influence on the unicellular eukaryotes. This effect involves stimulation of DNA synthesis, rising synchronization of cell division (upon the influence of EGF), and increase in cell number during the exponential growth. The mitogen effect may be evoked by cell progression in G1-phase under the action of growth factors and, consequently by earlier entry of cells into S-phase of the first cell cycle. Insulin repressed division of cells that entered into the generative cycle. These cells were delayed in late S-phase and G2-phase of the cycle. Part of these cells perished, while other cells could successively overcome the cell block to start their division by the 4th hours of cultivation. A collateral cytotoxic effect of insulin was found, being most prominent in early periods of Tetrahymena cultivation.     

Tissue-specific depression of DNA synthesis by calcium salt-free liver and lung extracts that are able to increase the stability of cell linkage in the tissue]

The macromolecular fraction of Ca-free salt extracts (Ca-FSE) removed from lungs and liver of adult rats, mice and fishes inhibits DNA synthesis in the embryonic tissue. The effect of Ca-FSE is tissue-specific rather than species-specific. Ca-FSE was earlier reported to increase with the same specificity the tissue stability to mechanical disruption. A concept is proposed that the tissue-specific adhesive factor may play the role of G1-chalone in epithelial tissue.     

Regulation of mitotic activity in rat vaginal epithelium: relationships between the level of its inhibitor (G2-chalone) and estrogens.

The level of a tissue-specific inhibitor of mitotic activity (G2-chalone) and mitotic activity in the vaginal mucosa of cycling rats of varying age and castrated rats were studied. A direct correlation between the level of the inhibitor and mitotic index is found in cycling animals. Both parameters are maximal during estrus and minimal in proestrus, when estrogen level in blood circulation is the highest. The undulating variations in G2 inhibitor level during estrous cycle are less pronounced and the concentrations of the inhibitor in relevant phases are significantly lower in aged females than in adult rats. Administration of estradiol benzoate (1 microgram/100 g) to castrated female rats was followed by a significant decrease in mitotic inhibitor level in vaginal mucosa within 12 hrs. This, in turn, was followed by a rise in mitotic activity 18 hr after estrogen administration. Therefore, the estrogen exerts its effect on mitotic activity in target tissue after it has induced a decrease in the level of the antimitotic factor (G2-chalone).     

Real‐time cell cycle imaging during melanoma growth,invasion, and drug response

Solid cancers are composed of heterogeneous zones containing proliferating and quiescent cells. Despite considerable insight into the molecular mechanisms underlying aberrant cell cycle progression, there is limited understanding of the relationship between the cell cycle on the one side, and melanoma cell motility, invasion, and drug sensitivity on the other side. Utilizing the fluorescent ubiquitination‐based cell cycle indicator (FUCCI) to longitudinally monitor proliferation and migration of melanoma cells in 3D culture and in vivo, we found that invading melanoma cells cycle actively, while G1‐arrested cells showed decreased invasion. Melanoma cells in a hypoxic environment or treated with mitogen‐activated protein kinase pathway inhibitors remained G1‐arrested for extended periods of time, with proliferation and invasion resuming after re‐exposure to a more favorable environment. We challenge the idea that the invasive and proliferative capacity of melanoma cells are mutually exclusive and further demonstrate that a reversibly G1‐arrested subpopulation survives in the presence of targeted therapies.