Immunologic determination of an epidermal G2-chalone-like factor as a marker of squamous cell structures in the urinary bladder and urine in rats

The epidermal G2 chalone-like substance of the rat skin may be determined by immunochemical methods only in squamous epithelia and it is revealed neither in mucosa of the urinary bladder, nor in urine of control rats. The antigenic activity of the chalone was revealed in 37 extracts of 56 tumors of the rat bladder. All antigen-containing tumors were found to be squamous cell carcinomas, transitional cell carcinomas with squamous cell metaplasia or possessed ultramicroscopic signs of squamous cell metaplasia appearing as "pure" transitional cell carcinomas under light microscope. The epidermal G2 chalone-like substance was present in urine of 7 out of 10 rats bearing the antigen-positive tumors of the urinary bladder.     

Chalones and the Control of Normal, Regenerative, and Neoplastic Growth of the Skin

SYNOPSIS Chalones,inhibitors of cell dmsion have been isolatedand studied from a number of mammalian tissues, most notably,the epidermis The epidermal rhalone is a glycoprotein It exhibitsconsiderable, but not complete specificity The epidermal chalone decreases mitotic activity by inhibitingcells in the G 2 phase of the cell cycle from entering mitosis,and probably also by inhibiting ceils in the G 1 phase of thecell cycle from entering mitosis To inhibit cells in G 2 fromentering mitosis the chilone requnes adrenalin, and for maximalactivity hydrocortisone It is not known if idrenalin and hydrocortisoneare required for chalone inhibition of cells in G 1 In addition to inhibiting cell division in normal epidermalcells the epidermal chalone can inhibit cell division in regeneratingepidermal cells induced to proliferate by chemical damage Thephase of the cell cycle in which the chalone inhibits legeneratingepidermal cells from entering mitosis is not known Epidermal tumors contain a decreased amount of chalone Mitosisin epidermal tumors is inhibited by treatment with epidermalchalone Tumor cells are inhibitedfrom entering mitosis fromeither the G 1 or G 2 phases of the cell cycle Chalones are said to inhibit mitosis by a negative feedbackmechanism However, experiments which presumably result in adecrease in chalone concentration do not result in an increasein mitotic activity It is suggested that if chalones are physiological controllers of cell division they do not act by a simplenegative feedback mechanism but require the action of a substanceto decrease their concentration     

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.     

Action of the chalone from Ehrlich's ascites, tumor in mice on the mitotic activity in this tumor after single and double administrations

Chalone from Ehrlich's ascites tumour exerts a short-lived and reversible inhibitory effect on cell proliferation in the tumour both after a single and two-fold administration. 10 hours following single and two-fold injection of chalone (second injection was given at 6 p.m.), the mitotic index in tumour cells rises as compared to controls an evidence of chalone action on G(2) cell population of the mitotic cycle and synchronization of cell division. Repeated injection of chalone at 9 p.m. results in a more prolonged effect on the cells and in a more pronounced synchronization wave of G(2) cell population comparatively to its injection at 6 p.m. Thus the duration of cell inhibition in G(2) phase of the mitotic cycle depends with repeated administration of chalone, on the condition of cell population affected by chalone.     

Interaction of chalones and glucocorticoid hormones in regulation of cell division

The action of hepatic chalone on cell proliferation in inoculated hepatoma 22a of mice was studied in the presence of a changed level of glucocorticoid hormones in experimental animals. Chalone was obtained from the liver of intact rats by ethanol precipitation. The intensity of cell proliferation in hepatoma was evaluated by the colcemide and autoradiography methods. Six hours after chalone injection c-mitosis in the tumor decreased 2.7-fold, and the DNA index 6.8-fold. It may be concluded that the preparation used contains both G1- and G2-chalones. Single or repeated injections of hydrocortisone to mice inhibits cell proliferation to a less degree than administration of chalone alone. Combination of hydrocortisone and chalone produces the same effect as injection of chalone alone. Adrenalectomy diminishes susceptibility of hepatoma cells to exogenous chalone. The degree of tumor proliferative activity in the adrenalectomized animals was half as much after chalone injection, as compared to that in intact animals. Thus, a certain level of glucocorticoid hormones in hepatoma tissue is necessary to reveal the action of chalones.     

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.     

Epidermal chalones and squamous cell carcinomas. The growth inhibitory effects of aqueous epidermal extracts (G1 and G2 chalones) on the epidermis and on a transplantable keratinizing carcinoman in nude mice.

Balb/c/nu nude mice that had been transplanted with a moderately differentiated squamous cell carcinoma were injected i.p. with different doses of epidermal chalone, and control animals were injected with saline. The labelling indices (H3TdR) and the mitotic rate (stathmokinetic method with vinblastine sulphate) were determined. In the untreated animals, both the labelling index and the mitotic rate of the tumor were considerably higher than in the epidermis, and the rate of cell birth was almost twice that of the epidermis. Higher doses of chalone were needed to reduce the labelling index for the tumour than for the epidermis, and there was generally a less pronounced dose/response relationship in the tumours than in the epidermis. The same was true of the mitotic rate but here the results were not as obvious as for the labelling index. A possible explanation of the results may be that the tumour cells are less sensitive than epidermal cells to the injected chalones, or that reduced vascularization of the transplanted tumour may lead to reduced access of chalone, or that tumour necrosis may pay a role. However, it is evident that the tumour cells react less than the epidermis to both the G1 and the G2 chalone, and thus the findings of this study do not provide any evidence against the theory that epidermoid transplanted tumours are less sensitive to epidermal chalones than normal tissue of the same histogenetic origin.     

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.     

Effects of propranolol on mitosis-inhibiting activity of G2-chalone and adrenaline in cell culture of Ehrlich's ascites tumor]

Chalone-containing preparation from ascite Ehrlich's tumour blocks these cell transition from G2-phase to mitosis and its motion in mitosis in vitro (G2-block, M-block). Adrenaline blocks these cell transition from G2-phase to mitosis. Propranolol raises G2-block of chalone or adrenaline. Consequently this way of chalones action on cell division includes beta-adrenergic receptors influence of preparation on cell motion in mitosis doesn't change with addition of propranolol. Consequently this way of chalone system action on mitosis doesn't include beta-adrenergic receptors.     

Quantitative determination of the epidermal G2-chalonelike factor in rat tissues by means of radial immunodiffusion]

A high-molecular glycoprotein (epidermal G2 chalone) shown to be homogeneous by electrophoresis and immunochemistry was isolated from rat skin. It exerts a strong tissue-rather than species-specific antimitotic effect on the keratinizing epithelium. The paper is concerned with its quantitative immunodiffusion determination in some tissues by means of monospecific antiserum. A clear-cut correlation between the mitotic index and the epidermal G2 chalone level in the mucosa is shown with special reference to vaginal epithelium of rats during the estrual cycle.     

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.     

Evaluation of a short term in vitro test for granulocyte chalone activity.

A short term in vitro test for granulocyte chalone activity eas examined for its specificity and reliability. The test used the inhibition, by granulocyte extracts, of 3H-thymidine (3H-Tdr) uptake in to the acid-insoluble material by rat bone marrow cells in vitro to measure possible chalone activity. Among the many possible 3H-Tdr artifacts pool size dilution by Tdr contained in the extracts was excluded using an E. coli mutant requiring thymine. Several amino acids and biogenic amines do not affect the test. However, continuous and pulse labelling of bone marrow cells with 3H-Tdr, viability tests and micro flow fluorometric measurements of the cell cycle distribution following colcemid treatment strongly suggests that the cells do not proliferate in vitro during short term incubation, since practically no cells enter the S-phase, cells in the S-phase die and few if any cells proceed through G2 and mitosis. Moreover, the test cannot exclude cytotoxicity. Thus, the in vitro test may only sceem for an unspecific S-phase inhibitor and must hence be supplemented by another assay to prove the chalone nature of an extract or fraction. The test per se fails to meet most of the requirements of a valid granulocyte chalone assay.     

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.     

The effect of chalone on the cell cycle in the epidermis during wound healing

A cut was made on the ear conch of mouse and an extract containing epidermal chalone was injected subcutaneously 2 days later. The time changes after the chalone administration in the number of cells labeled with ³H-thymidine, in the number of grains on labeled cells and in the number of mitoses within the regenerating epidermis surrounding the wound were investigated by means of autoradiography (ARG). Grain counts decreased temporarily in early phase (0–2 h) after chalone injection. This decrease in grain count resulted in a decrease in the number of labeled cells on the ARG of a short exposure but not in that on the ARG of a long exposure. A decrease in the number of labeled cells on the ARG of a long exposure was evident at 6 h when the grain counts reverted to a level similar to the control without chalone. The number of mitoses reached a minimum at 2 h and then recovered quickly, indicating a rapid disappearance of the inhibition of cells in G 2 from entering M phase. Mitoses decreased again thereafter, presumably as a result caused by inhibition of cells in the preceding S phase from completing DNA synthesis. The extract made similarly from liver or kidney affected neither the mitotic nor the DNA synthetic activities.These results indicate that the epidermal chalone or chalones inhibit the epidermal cell proliferation in, at least, 3 different processes of the cell cycle; the DNA synthesis in S phase, the transition from G 1 to S phase and the transition from G 2 to M phase.     

CONTROL OF GRANULOCYTE PRODUCTION

In normal conditions the granulocytic cell population is prevented from excessive cell proliferation by a humoral mechanism based on a specific feedback inhibitor, granulocytic chalone. In conditions of acute functional demand a tissue-specific stimulator, granulocytic antichalone, replaces chalone in rat serum. Mature granulocytes contain, and presumably produce, the chalone which is also present in fresh normal serum. Thus, the inhibitor is both humoral and present within the same cell system on which it acts: the action of this chalone is target tissue specific as it only inhibits granulocytic precursor cells in normal rat bone marrow in vitro. Granulocytic chalone and antichalone were partly purified by gel filtration on Sephadex; the elution parameters suggested molecular weights of 4000 and 30,000–35,000, respectively. Granulocytic chalone was not separated from the erythrocytic chalone (present in fresh normal serum and in blood erythrocytes) on Sephadex; however, separation at the cellular level was easily achieved.     

Impact of DNA demethylation of the G0S2 gene on the transcription of G0S2 in squamous lung cancer cell lines with or without nuclear receptor agonists

We recently identified that DNA methylation of the G0S2 gene was significantly more frequent in squamous lung cancer than in non-squamous lung cancer. However, the significance of G0S2 methylation levels on cancer cells is not yet known. We investigated the effect of G0S2 methylation levels on cell growth, mRNA expression, and chromatin structure using squamous lung cancer cell lines and normal human bronchial epithelial cells. DNA methylation and mRNA expression of G0S2 were inversely correlated, and in one of the squamous lung cancer cell lines, LC-1 sq, G0S2 was completely methylated and suppressed. Overexpression of G0S2 in LC-1 sq did not show growth arrest or apoptosis. The G0S2 gene has been reported to be a target gene of all-trans retinoic acid and peroxisome proliferator-activated receptor agonists. We treated LC-1 sq with 5-Aza-2′-deoxycytidine, Trichostatin A, all-trans retinoic acid, Wy 14643, or Pioglitazone either alone or in combination. Only 5-Aza-2′-deoxycytidine restored mRNA expression of G0S2. Chromatin immunoprecipitation revealed that histone H3 lysine 9 was methylated regardless of DNA methylation or mRNA expression. In summary, mRNA expression of G0S2 was regulated mainly by DNA methylation in squamous lung cancer cell lines. When the G0S2 gene was methylated, nuclear receptor agonists could not restore mRNA expression of G0S2 and did not show any additive effect on mRNA expression of G0S2 even after the treatment with 5-Aza-2′-deoxycytidine.     

THE CELL SPECIFIC EFFECT OF THE GRANULOCYTE CHALONE DEMONSTRATED WITH THE DIFFUSION CHAMBER TECHNIQUE

The granulocytic chalone is secreted by mature granulocytes and inhibits ³H-thymidine incorporation of proliferating granulocytes in vitro . The effect and the cell line specificity of this chalone was assessed with the in vivo diffusion chamber culture technique. Tests were carried out on cultures from normal mouse bone marrow cells and mouse and rat blood leucocytes. The majority of the DNA synthesizing cells in marrow cultures were proliferating granulocytes. Macrophages and immunoblasts proliferated in rat leucocyte cultures, when the chambers had been carried for 5 days in host mice. Repeated chalone or control injections were given i.p. to the host mice during 6–7 hr prior to ³H-thymidine injection. Isotope uptake of proliferative granulocytes was reduced by the chalone treatment. No such effect was found on the rat immunoblasts and macrophages. The viability of cultured cells was apparently not affected by the chalone treatment.     

Alpha-enolase is restricted to basal cells of stratified squamous epithelium.

We have developed a monoclonal antibody against a 50-kDa protein that binds preferentially to basal cells in the limbus of rat, rabbit, and human corneas (J. D. Zieske, G. Bukusoglu, and M. A. Yankauckas, Invest. Ophthalmol. Visual Sci. 33, 143-152, 1992). Here we report on the purification and identification of the antigen. The 50-kDa antigen was purified from rabbit limbal and corneal epithelium using HPLC methodology including anion exchange (DEAE) followed by reverse-phase (C18) chromatography. The purified 50-kDa protein was then digested with endoproteinase Lys-C, and a reproducible profile comprising approximately 20 peptides was observed by reverse-phase HPLC of the digest. Sequence analysis of five peptides ranging in length from 4 to 20 residues revealed that the 50-kDa protein was alpha-enolase, a glycolytic enzyme. Overall, 57 amino acids were identified with a 95% sequence homology. Localization of alpha-enolase in rat epithelium by immunofluorescence microscopy demonstrated that simple epithelium contained low or undetectable levels of the enzyme. Stratified squamous epithelium, however, showed high levels of alpha-enolase, which was localized specifically to cells of the basal layer. Epidermal, corneal limbal, oral mucosal, vaginal, and laryngeal epithelium all showed cytoplasmic binding specific to the basal cells. These data indicate that the glycolytic enzyme alpha-enolase is preferentially localized in the basal cell layer of stratified squamous epithelium and suggest that glycolytic activity is concentrated in these cells. The localization pattern suggests that a major change in metabolism occurs as cells leave the mitotically active basal cell layer and migrate toward terminal differentiation in the suprabasal cell layers.     

Role of lipocortin I in the glucocorticoid induction of the terminal differentiation of a human squamous carcinoma

The human squamous cell carcinoma SqCC/Y1 undergoes spontaneous terminal differentiation in the confluent state. The degree of maturation was markedly increased by glucocorticoids and by both human recombinant and placental lipocortin I. Western analyses demonstrated cellular secretion of lipocortin into the medium. Glucocorticoid-induced maturation was antagonized by a lipocortin I-specific monoclonal antibody, by phospholipase A2 (PLA2), and by arachidonic acid. Induction of the differentiation of SqCC/Y1 cells by lipocortin I was prevented by arachidonic acid. The PLA2 inhibitor, dibromoacetophenone, caused an increase in envelope-competent cells indicating that inhibition of PLA2 results in induction of differentiation. Epidermal growth factor prevented the induction of differentiation by both lipocortin I and by glucocorticoids. The nonsteroidal lipoxygenase/cyclo-oxygenase inhibitor, phenidone, also increased SqCC/Y1 differentiation, suggesting that leukotrienes, thromboxanes, and/or prostaglandins may be involved in lipocortin-mediated regulation of SqCC/Y1 maturation. The findings support a role for lipocortin I in mediating the effects of glucocorticoids on epidermal cell differentiation.     

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.