Epidermal G2 Chalone Research Articles

Epidermal G1‐chalone and transforming growth factor‐β are two different endogenous inhibitors of epidermal cell proliferation

Epidermal G1-chalone and transforming growth factor-beta (TGF beta), two endogenous inhibitors of epidermal cell proliferation, were compared with regard to several effects on epidermis in vivo and in vitro. Both factors inhibited DNA labeling in a rat tongue epithelial cell line, with similar kinetics and half-maximal effects at approximately 1 pg/ml (enriched chalone) and 1 ng/ml (TGF beta). For primary neonatal mouse keratinocytes, TGF beta was found to be a rather strong inhibitor of cell proliferation, whereas chalone showed only a weak effect on cells grown in medium containing 1.2 mM Ca2+ and no effect at all in the presence of 0.06 mM Ca2+. Vice versa, upon i.p. injection, only chalone was able to inhibit mouse epidermal DNA synthesis in vivo, whereas TGF beta had no effect at all. A moderate increase of transglutaminase activity in neonatal primary mouse keratinocytes was induced by both factors at concentrations of about 300 pg TGF beta/ml and 10 pg chalone fraction/ml. Chalone did not compete with [125I]TGF beta for specific binding sites on primary murine keratinocytes. A polyclonal "chalone antiserum" did not interact with TGF beta, and a neutralizing TGF beta antibody that inhibited the effect of TGF beta on cell proliferation could not block the inhibitory effect of chalone on RTE2 cells. In contrast to TGF beta, epidermal G1-chalone did not induce proliferation of NIH-3T3 cells. These results indicate that epidermal G1-chalone and TGF beta are two different inhibitors of epidermal cell proliferation.

The Epidermal G1‐Chalone: An Endogenous Tissue‐Specific Inhibitor of Epidermal Cell Proliferation

An apparently macromolecular factor is isolated from aqueous skin extracts which inhibits DNA synthesis in vivo and in vitro with high efficacy (ID50 in vivo 0.2 pmol/g, in vitro 0.2 pM) and in a highly specific manner showing a point of attack in the late G1-phase of the cell cycle (epidermal G1-chalone). Preliminary characterization indicates an unusual highly amphipathic structure consisting of amino acids and carbohydrate. Despite its apparent molecular weight of approximately 10 kD the chalone is stable against denaturing agents and most enzymes, including proteases. An inverse correlation between chalone responsiveness of mouse epidermis in vivo and the development of hyperplasia due to injury indicates an important role of the factor in the regulation of tissue homeostasis. According to its physicochemical and biological properties the epidermal G1-chalone appears not to be related to other endogenous inhibitors of epidermal cell proliferation such as the pentapeptide pyroGlu-Glu-Asp-Ser-GlyOH and transforming growth factor beta (TGF beta).

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 μg/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 μg/ml. At higher concentrations the degree of inhibition remained constant at about 50% of the control up to 300 μg/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.

Analysis of the circadian rhythm of mitotic activity and epidermal G2-chalone content in the esophageal and lingual epithelium

Analysis of the circadian rhythm of mitotic activity and epidermal G2-chalone content in the esophageal and lingual epithelium

Purification of epidermal G2-chalone by immunoaffinity chromatography

Purification of epidermal G2-chalone by immunoaffinity chromatography

Changes in epidermal G2-chalone content and mitotic activity of vaginal epithelial cells of ovariectomized rats after stimulation of proliferation by estradiol

Changes in epidermal G2-chalone content and mitotic activity of vaginal epithelial cells of ovariectomized rats after stimulation of proliferation by estradiol

Partial purification of the epidermal G2 chalone based on an in vivo assay system

The epidermal G2 chalone was partially purified from hairless mouse skin extracts by means of chromatography and Ultrafiltration and tested in vivo on hairless mouse epidermis. The active component had a molecular weight of less than 10,000 daltons. It significantly inhibited epidermal G2 cells from entering mitosis but had no effect on epidermal cells at the G1/S transition. Extracts from mouse spleen or skeletal muscle treated in the same way as the skin extracts had no inhibitory effect on epidermal G2 cells.

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.

Immunologic determination of epidermal G2-chalone as marker of squamous-cell structures in rat lung tumors

Immunologic determination of epidermal G2-chalone as marker of squamous-cell structures in rat lung tumors

CELL PROLIFERATION KINETICS OF EPIDERMIS AND SEBACEOUS GLANDS IN RELATION TO CHALONE ACTION

Median S-phase lengths of pinna epidermis and sebaceous glands, and of epithelia from the oesophagus and under surface of the tongue of Albino Swiss S mice were estimated by the percentage labelled mitoses method (PLM). The 18.4 and 18,8 hr for the median length of S-phase for pinna epidermis and sebaceous glands respectively made it possible for these two tissues to be used experimentally for testing tissue specificity in chalone assay experiments. The 10.0 and 11.5 hr for oesophagus ang tongue epithelium respectively made experimental design for chalone assay difficult when pinna epidermis was the target tissue. The results of the Labelling Index measured each hour throughout a 24-hr period showed no distinct single peaked diurnal rhythm for pinna epidermis and sebaceous glands. Instead a circadian rhythm with several small peaks occurred which would be expected if an S-phase of approximately 18 hr was imposed on the diurnal rhythm. This indicates that there may be very little change in the rate of DNA synthesis. The results are given for the assay in vivo of purified epidermal G1 and G2 chalones, and the 72--81% ethanol precipitate of pig skin from which they could be isolated. These experiments were performed over a time period which took into account the diurnal rhythm of activity of the mice as well as the S-phase lengths. Extrapolating the results with time of action of the chalone shows that the G1 chalone acts at the point of entry into DNA synthesis and that the S-phase length was approximately 17 hr for both the pinna epidermis and sebaceous glands. This may be a more correct value since the PLM method overestimates the median S-phase length as it is known that in pinna skin the [3H]TdR is available to the tissues for 2 hr and true flash labelling does not take place. The previous reports that epidermal G1 chalone acts some hours prior to entry into S-phase resulted from experiments on back skin where the S-phase is shorter and there is a pronounced diurnal rhythm which could mask the chalone effect. The epidermal G2 chalone had no effect on DNA synthesis even at different times in the circadian rhythm. Thus the circadian rhythms and S-phase lengths of the test tissues need to be considered when experiments are performed with chalones. Ideally, the target tissues selected for cell line specificity tests should have the same cell kinetics for the easier and more accurate assessment and interpretation of results. When the tissues have markedly different cell kinetics, experimental procedures and results need to be evaluated accordingly. The point of action of G1 chalone can only be assessed if the effect is measured over the peak of incorporation of [3H]TdR into DNA. The results of the effects of skin extracts are analysed in relation to changes in the availability of [3H]TdR for the incorporation into DNA and to the possibility of there being two distinct populations of proliferating cells.

Quantitative determination of epidermal G2-chalone-like factor by radial immunodiffusion

A homogeneous high-molecular-weight glycoprotein possessing tissue-specific, but not species-specific antimitotic activity relative to epithelium of dermal type (epidermal G2-chalone) was isolated electrophoretically and immunochemically from rat skin. A method of quantitative immunodiffusion determination of this substance by means of monospecific antiserum in certain tissues is described. Clear correlation between the mitotic index and concentration of G2-chalone in the mucous membrane at various stages of the estrous cycle was demonstrated in the vaginal epithelium.

Changes in the level of epidermal G2 chalone and mitotic activity in the vaginal epithelium of rats

The content of epidermal inhibitor of mitosis (G2 chalone) at different stages of the estrous cycle in rats was determined by the radial immunodiffusion method. The chalone level correlates with the mitotic index of the vaginal mucous membrane and is minimal in proestrus and maximal in estrus. In aging (14–16 months) rats with regular cycles the content of G2 chalone in the vaginal mucous membrane at all phases of the estrous cycle was significantly lower than in young (3–4 months) rats with a regular cycle. In castrated rats the mitotic index begins to rise 18 h after a single injection of estradiol benzoate (1 μg/100 g body weight). This increase is preceded by a significant decrease in the concentration of G2 chalone 12 h after injection of the estrogen.

The effect of the epidermal G2 chalone on the mitotic duration in nude mouse epidermis and in a transplanted squamous cell carcinoma.

Balb/c/nu nude mice transplanted with a moderately differentiated squamous cell carcinoma were injected intraperitoneally with different doses of aqueous skin extracts containing the epidermal G2 chalone. The mitotic counts and the mitotic rates were determined in histological sections using a stathmokinetic method with vinblastine sulphate. The mitotic duration was calculated from the mitotic rates and counts. Skin extracts containing epidermal G2 chalone increased the mitotic duration in the epidermis, and a similar trend was seen in the tumour. The higher the dose of chalone, the longer the mitotic duration tended to be. A straight line of best fit used to indicate the dose/response relationship was steeper for the epidermis than for the tumour. The study thus shows that the epidermal G2 chalone not only prevents epidermal cells from entering mitosis, it also prolongs the mitotic duration. Further, the results do not contradict the theory that tumour cells may be less sensitive to chalone than normal cells.

Partial purification of epidermal G2 chalone.

A partial purification of epidermal G2 chalone from crude epidermal cell extracts from hairless mice is described. The procedure involved the sequential use of ammonium sulphate precipitation, affinity chromatography and gel filtration. Mitosis-inhibiting activity at each stage in the purification was tested in an in vitro assay system employing human epidermoid carcinoma cells in exponential growth phase and Colcemid (Ciba) for arresting mitoses. This assay system is much more rapid and convenient than conventional in vivo systems. By the procedure described, a 10,000-fold material purification of the active component has been obtained. This corresponds to a 3,000-fold purification measured by protein content, and a 300-fold increase in mitosis-inhibiting activity per unit we;ght. The active component is acidic, contains sugar residues and has gel chromatographic properties characteristic of a substance with molecular weight of approximately 20,000. On SDS polyacrylamide gel electrophoresis, however, three weak bands were found. The active component is resistant to trypsin and protease and stable between pH 6.0 and 8.5. It is easily inactivated at pH values below 6.0. At the present stage of purification, several components other than the active one still remain in our material and further purification steps must be eventually employed.

Lack of relationship between Langerhans cells, epidermal cell proliferation and epidermal G1 chalone.

Mouse tail interscale epidermis contains numerous Langerhans cells, whereas the adjacent scale regions are devoid of these cells. No difference in a) proliferative activity and b) inhibitory effect of the epidermal G1 chalone can be demonstrated in both regions. A direct relationship between Langerhans cells and growth control may be excluded.

Specificity of epidermal chalone extracts for human epidermoid tumour cells in vitro: a preliminary report.

In order to test the mitosis-inhibiting effect and the tissue specificity of the epidermal G2 chalone for tumour cells, extracts from hairless mouse epidermis were tested in short-term tissue cultures of cells from human respiratory tract epidermoid carcinomas and adenocarcinomas. The chalone inhibited strongly the mitotic activity in two cases of histologically proven epidermoid carcinoma, and had no effect in two cases of adenocarcinoma. In one case of a supposed epidermoid carcinoma, the chalone had no effect. Revision of the histology, and the result of autopsy 11 months later, showed that in this case the lesion in the lung had been a poorly differentiated metastasis from an adenocarcinoma of the ovary. Liver extracts produced in the same way as the epidermal extracts showed no mitotic inhibition in any of the cultures. These results indicate that epidermal G2 chalone produced from mouse skin is tissue specific for human epidermoid tumour cells, and also indicate that a chalone test might be used as a diagnostic tool for poorly differentiated carcinomas to see whether they are of epidermoid origin or not.

Changes in epidermal chalone activity during response of the skin to application of carcinogen and epilation

A decrease in the biological activity of epidermal G2-chalone, isolated from the dorsal skin of rats, was demonstrated 1–2 days after treatment with the carcinogen methylnitrosourea and one day after epilation. The results of quantitative analysis of mitoses and morphological analysis of the skin which was the source of the chalones showed that this effect is based on different mechanisms.

EFFECT OF MECHANICAL STIMULATION ON CELL PROLIFERATION IN MOUSE EPIDERMIS AND ON GROWTH REGULATION BY ENDOGENOUS FACTORS (CHALONES)

Mechanical stimulation of dorsal mouse skin by skin massage or removal of the horny layer results in a mutually comparable increase in DNA-labelling and mitotic activity. However, only after injury such as removal of the horny layer hyperplasia develops. This phenomenon, called "hyperplastic transformation" is characterized by a transient abolition of the epidermal G1 chalone responsiveness. There is some indication that the susceptibility to a heat labile factor, probably the epidermal G2 chalone, is not affected. Skin massage neither interferes with the responsiveness to epidermal G1 chalone nor induces "hyperplastic transformation". Mouse tail epidermis shows a "functional hyperplasia" and responds to the G1 chalone. To explain these observations, it is assumed that the epidermal stem cell population is heterogeneous consisting of G1 chalone-sensitive and G1 chalone-insensitive cells.

Effects of Methylcholanthrene on Epidermal Growth Regulators II. Variations in the S-Factor

There is increasing evidence to support the existence of tissue-specific growth-inhibitory chemical substances which can be found in extract homogenized cells. In the epidermis, two such tissue-specific factors (which also have cell-cycle specificity) have been found. These factors act specifically on different phases of the cell cycle (epidermal G1 and G2 chalones, respectively). This paper concerns a study of the effect of 20-methylcholanthrene on the epidermal G1 chalone. The variations in epidermal G2 inhibitor after such treatment were described in a previous paper. Hairless mice received a single topical application of 0.2 ml 0.5% solution of the carcinogen. The short time effect of the carcinogen application on epidermal DNA synthesis was first studied. Other groups of carcinogen-treated mice were then killed at similar time intervals, and the treated area of skin was homogenized and extracted with water. The inhibitory effect of these extracts on normal epidermal G1 cells was assayed in normal hairless mice. The obtained inhibition was registered as an expression of G1 inhibitor concentration in the skin extracts. The first experiment confirmed that a single carcinogen application provokes a short block in epidermal DNA synthesis, followed by a high, biomodal peak of increased activity with the first and maximum peak on day 2, and a smaller peak on day 8 after treatment. The second experiment showed that the content of G1 chalone in the skins of treated animals varied inversely to the alterations in epidermal DNA synthesis, revealing almost no chalone activity on day 2, and reduced chalone activity on day 8.

Chalone regulation of the epidermal cell cycle.

Purified epidermal G2 chalone does not inhibit DNA synthesis or influx of S-phase cells and is therefore cell cycle phase-specific, inhibiting only the flow of cells into M-phase.