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 BLEOMYCIN ON THE EPIDERMAL CONTENT OF GROWTH-REGULATORY SUBSTANCES (CHALONES)

Hairless male mice were given 2 mg Bleomycin i.p. on two successive days. At different time intervals from 1 to 10 days after the last Bleomycin injection, groups of animals were killed and water extracts of homogenized skin were made. These extracts, supposed to contain the epidermal G₁ and G₂ chalones, were injected into female hairless mice, and their growth inhibitory potency determined by two methods. 5 mg of lyophilized crude skin extract were injected i.p. together with Colcemid, and the animals killed 4 hr later. The number of Colcemid-arrested mitoses was determined, and was considered to be a measure of the G₂ inhibitor present in the skin extracts. 10 mg of the same extracts were injected i.p., and these animals also got ³H-TdR i.p. 12 hr later, and were killed after a subsequent 30 min. The epidermal LI was determined, and was considered to be a measure of the epidermal G₁ factor in the skin extracts. The results obtained were compared to the effect of Bleomycin alone and to the effects of skin extracts from non-Bleomycin-treated animals. The results show that Bleomycin provoked slight alterations in the growth-inhibitory potency of the G₁ chalone, whereas significant effects were seen in the G₂ chalone. There was an increased amount of growth-inhibiting factors on days 2 and 3, and on days 8–10. The results are discussed and it is concluded that the most probable hypothesis is that Bleomycin, in addition to its known inhibitory effect on epidermal cell proliferation, exerts growth inhibition by accumulation of cells with high growth inhibitory potency. An initial, additional direct effect of Bleomycin on the chalone system cannot be excluded.     

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.     

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.     

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 and 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 pronounceddiurnal rhythm which could mask the chalone effect. The epidermal G, 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 G, chalone can only be assessed if the effect is measured over the peak of incorporation of 13H]TdR into DNA. The results of the effects of skin extracts are analysed in relation to changes in the availability of i3H]TdR for the incorporation into DNA and to the possibility of there being two distinct populations of proliferating cells.     

ON THE EXISTENCE OF 'ARRESTED G2 CELLS' IN MOUSE EPIDERMIS

It has been postulated that mouse epidermis contains two populations of resting cells, one of which is blocked at the G₁-S boundary and the other between G₂ and mitosis. the ‘arrested G₂ cells’ were estimated, by the labelled mitosis method, to comprise 510% of the epidermal population and presumed to function as a ‘reserve pool’ which could be activated by wounding. A comprehensive search has now been carried out for arrested G₂ cells in mouse epidermis using the direct methods of single cell and flow through cytophotometry. No evidence was obtained which supports the existence of such a cell compartment. Suitable control experiments were carried out to ensure that G₂ cells were not lost during the isolation of epidermal nuclei.     

EHRLICH ASCITES TUMOR (EAT) CHALONE EFFECTS ON NASCENT DNA SYNTHESIS AND DNA POLYMERASE ALPHA AND BETA

EAT chalone effects on nascent DNA synthesis and DNA polymerase were examined. Concentration related inhibition of ³H-thymidine (³H-TdR) incorporation into EAT cell DNA was noted over a chalone range of 50–200 μg/ml. RNA synthesis was not affected, but protein synthesis decreased an average of 82% during 3 hr. Nascent DNA pulse-labeled for 2 min was normally incorporated into bulk DNA in the presence of chalone, but crude α and β-polymerase activities were inhibited. Crude DNA polymerase from C₃H mouse kidney and spleen was also partially inhibited by EAT chalone, suggesting non-specific inhibition of DNA polymerase. Preincubation studies of chalone with crude EAT DNA polymerase or ‘gapped’ DNA primer had no effect on chalone activity. Chalone may control mitotic activity by inhibiting α- and β-polymerase activity, thereby decreasing nascent DNA synthesis. Nascent DNA is incorporated normally into bulk DNA in the presence of chalone, indicating that DNA ligase is not inhibited.     

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.     

Agonist- and mitogen-induced desensitization of isoproterenol-stimulated cyclic AMP formation in mouse epidermis in vivo

Injection of the beta-adrenergic agonist isoproterenol causes a rapid desensitization of cyclic AMP formation to subsequent beta-adrenergic challenge in mouse epidermis. Long-lasting catecholamine refractoriness is also observed after prolonged treatment of mouse skin with certain mitogens such as the phorbol ester TPA (tumor promoter), 12-retinoylphorbol-acetate, the TPA-analogue C14:4phorbol acetate or the divalent cation ionophore A 23187 but not after mitogenic stimulation by phorbol-12,13-dibenzoate and 4-O-methyl-TPA or by means of chemical depilation, removal of the horny layer or skin massage. Thus no clear-cut correlation exists between the desensitizing efficacy of a given treatment and its ability to provoke epidermal hyperplasia and to promote skin tumor formation. Both, agonist- and mitogen-induced desensitization are dependent on protein synthesis in epidermis, the mitogen-induced effect is in addition dependent on RNA synthesis. The putative desensitizing protein is not cyclic AMP phosphodiesterase but could be a feedback inhibitor of receptor-cyclase interaction ("refractoriness protein") which has recently been proposed to be responsible for catecholamine tachyphylaxis in certain in vitro systems. In contrast to epidermal hyperproliferation mitogen-induced tachyphylaxis is not mediated by prostaglandin synthesis and is apparently also independent of initial cyclic AMP formation. It can be prevented, however, by the antimitotic synthetic corticoid fluocinolone acetonide but not by colchicine, vincristine cytochalasin B or adrenergic blockers.     

MITOTIC CONTROL IN ADULT MAMMALIAN TISSUES

Mitotic homeostasis: Mitotic control is maintained by the interaction of a tissue-specific mitosis-inhibiting chalone, which permeates the whole tissue, and a non-tissue-specific mitosis-promoting mesenchymal factor, which originates in the connective tissue and acts only on connective-tissue-adjacent cells. In the basal layer of the epidermis the mitotic rate is determined by the relative concentrations of these two substances; in the distal layers the chalone is dominant so that all cells must become post-mitotic, age, and die. Thus the perfect balance between cell gain and cell loss that is maintained equally in hypoplasia, normality, and hyperplasia is ensured by the fact that all cells forced distally by mitotic pressure enter a chalone concentration that is high enough to direct them into post-mitosis and so to their deaths. The mitotic rate of the basal epidermal cells and the ageing rate of the distal cells are both inversely related to the chalone concentration. A change in the mitotic rate is matched by an equal change in the ageing rate so that, within limits, epidermal thickness (or mass) remains constant. Epidermal thickness is determined by the tissue-specific ratio, mitotic rate: ageing rate; it is influenced by the mitotic rate only when this exceeds a certain critical level. Evidently all epithelial tissues, even when these form solid masses (e.g. liver hepato-cytes), have a similar control mechanism, the ‘basal cells’ being those that are connective-tissue-adjacent and the ‘distal cells' those that are not. Tissues that are not connective-tissue-based (e.g. erythrocytes and granulocytes) have specialized mechanisms involving differentiation from relatively undifferentiated stem cell populations, as also do the connective tissues themselves. Local tissue damage leads via local chalone loss to a temporarily and locally increased mitotic rate; chronic damage leads via chronic chalone loss to hyperplasia, the increase in tissue mass being limited by the reduced life-span of the post-mitotic cells. Compensatory hypertrophy When a tissue mass is so large (e.g. the hepatocytes) in relation to the total body mass that the escaping chalone forms a significant systemic concentration, extensive damage leads to compensatory hypertrophy. The reduced tissue mass (e.g. after partial hepatectomy) produces less chalone, leading to a reduced systemic concentration, and therefore a higher chalone loss from the surviving tissue. This results in a general mitotic response in that tissue, as the relative power of the mesenchymal factor increases, and thus to an increase in tissue mass. Growth ceases when the normal tissue mass is attained. When a large tissue suffers chronic damage (e.g. liver cirrhosis) the chronic chalone lack results in hypertrophy, which is limited by the reduced life-span of the post-mitotic cells. Tumour growth Mitotic control is lost when the chalone concentration falls so low that the ‘distal cells’ remain mitotic; cell gain then exceeds cell loss and a tumour appears. Such chalone loss is related to permanent membrane damage, which may be the central event in carcinogenesis. The evidence is that a tumour continues to produce and to respond to the chalone of its tissue of origin. As a tumour grows the systemic concentration of its chalone rises steadily so that there is an increasing mitotic inhibition, first, in the parent tissue, and second, in the tumour itself. Thus tumour growth may be described as an exponential process limited by an exponential retardation. This means that, if the host survives, the tumour growth will cease and the tumour mass will reach a plateau. This is a negative feedback mechanism which differs from compensatory hypertrophy only in that, at the plateau, the mass attained is greater than normal, and also in that, at any time, further cell damage may cause the tumour to ‘progress’. When this happens the new and higher plateau may be unattainable before the host is killed. Tumour growth is normally slower than would be expected if the mitotic advantage were the only factor involved; clearly tumour growth is usually inhibited by factors other than the chalone, in particular perhaps by the immune response to the altered cell membrane. It is an especial pleasure to acknowledge the constant help and encouragement that has been given by Johanna U. R. Deol.     

SPERMATOGONIAL CHALONE(S): EFFECT ON THE PHASES OF THE CELL CYCLE OF TYPE A SPERMATOGONIA IN THE RAT

Adult rats with X-irradiated testes were used to analyze the effect of the spermatogonial chalone(s) on the phases of the cell cycle of type A spermatogonia. Twelve days after irradiation, the animals were used in two experiments designed to test the existence of hypothetical G₂ and S phase chalones. For the G₂ assay, rats injected twice with testicular extract (Group I), liver extract (Group II) or physiological saline (Group III) were killed 10 hr after the initial injection. Mitoses of type A, Intermediate and type B spermatogonia were counted in whole mounts of dissected seminiferous tubules. To test for an S phase inhibitor, two groups of rats were given multiple injections of either testicular extract (Group IV) or saline solution (Group V). Twenty-two hr after the first injection they were injected with [³H]thymidine and killed 2 hr later. Silver grains over labelled type A nuclei were counted in radioautographed sections of testes from these animals. The average grain counts were identical in Groups IV and V, indicating that the testicular extract did not affect type A spermatogonia during the S phase. Counts of type A mitoses in Groups I, II and III revealed that in the animals injected with the testicular extract (Group I) the number of divisions was 50% lower than in the control groups (Groups II and III). In contrast, mitotic activity of differentiating spermatogonia (In + B) was similar in all three groups of animals. This result is attributed to a testicular chalone which specifically inhibits type A spermatogonia during the G₂ phase of the cell cycle. Indirect evidence for a G₁ spermatogonial chalone is also presented, as a result of an analysis of published data (Clermont & Mauger, 1974).     

CHALONE CONTROL OF MITOTIC ACTIVITY IN SEBACEOUS GLANDS

Extracts of skin with sebaceous glands contain a substance which inhibits mitotic activity in sebaceous glands both in vivo and in vitro. This substance is neither the epidermal chalone nor the melanocyte chalone, both of which are also present in skin extracts. However, it resembles these other chalones in that it is water soluble, is precipitated by ethanol, is activated by the two stress hormones adrenalin and hydrocortisone, and is not species specific. It is present within the sebaceous glands, and it is evidently a sebaceous gland chalone.     

Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes

Mutations in TRPM1, a calcium channel expressed in retinal bipolar cells and epidermal melanocytes, cause complete congenital stationary night blindness with no discernible skin phenotype. In the retina, TRPM1 activity is negatively coupled to metabotropic glutamate receptor 6 (mGluR6) signaling through Gα_o and TRPM1 mutations result in the loss of responsiveness of TRPM1 to mGluR6 signaling. Here, we show that human melanocytes express mGluR6, and treatment of melanocytes with L‐AP4, a type III mGluR‐selective agonist, enhances Ca²⁺ uptake. Knockdown of TRPM1 or mGluR6 by shRNA abolished L‐AP4‐induced Ca²⁺ influx and TRPM1 currents, showing that TRPM1 activity in melanocytes is positively coupled to mGluR6 signaling. Gα_o protein is absent in melanocytes. However, forced expression of Gα_o restored negative coupling of TRPM1 to mGluR6 signaling, but treatment with pertussis toxin, an inhibitor of G_i/G_o proteins, did not affect basal or mGluR6‐induced Ca²⁺ uptake. Additionally, chronic stimulation of mGluR6 altered melanocyte morphology and increased melanin content. These data suggest differences in coupling of TRPM1 function to mGluR6 signaling explain different cellular responses to glutamate in the retina and the skin.     

Relations entre la prolifération et la différenciation cellulaires dans l'intestin embryonnaire et larvaire dePleurodeles waltlii Michah

Résumé Les chalones 1 et 2, extraites de l'intestin de pleurodèle adulte, inhibent en phase G₁ et G₂ respectivement les cycles mitotiques de l'épithélium intestinal embryonnaire. Les effets de ces chalones sur la prolifération et la différenciation cellulaires dans ce tissu ont été étudiés en fonction de la dose injectée, du stade de développement et de la durée du traitement. L'inhibition provoquée par la chalone 2 est proportionnelle à la dose injectée entre deux seuils de concentration. Le quart environ des cellules intestinales en activité mitotique est insensible à la chalone 2 même à la suite d'injections répétées de l'inhibiteur. Seules les cellules intestinales des embryons âgés (stade 34) sont sensibles à cette chalone et répondent par un allongement de la phase G₂ qui, malgré des injections répétées de l'inhibiteur, n'excède pas une vingtaine d'heures. La sensibilité des cellules de l'épithélium intestinal à la chalone 1 se manifeste à la fin du développement (stade 33), comme dans le cas de la chalone 2. A l'égard de la chalone 1, la population cellulaire en activité mitotique dans l'intestin embryonnaire apparaît hétérogène et comprend: 50% de cellules aptes à être inhibées par des doses faibles de chalone 1; 25% de celludes aptes à n'être inhibées que par des doses de chalone 1 cent fois plus élevées et 25% environ de cellules insensibles à cet inhibiteur. Les injections répétées de chalone 1 bloquent définitivement en phase G₁ la moitié environ des cellules en activité mitotique dans l'épithélium intestinal indifférencié au stade 34; en outre, elles accélèrent la consommation du vitellus, favorisent la différenciation des cellules à mucus et diminuent le nombre des cellules constituant les nids sous-épithéliaux qui apparaissent au stade 36 et représentent le compartiment générateur de l'épithélium intestinal. Les réultats obtenus permettent de proposer un modèle de cinétique de la prolifération cellulaire au cours de la différenciation de l'épithélium intestinal du pleurodèle; de plus, ils conduisent à l'hypothèse que le nombre de divisions subies par une cellule embryonnaire et le taux de chalone dans le tissu auquel elle appartient, sont les deux signaux complémentaires qui déclenchent le blocage du cycle mitotique et l'achèvement de la différenciation dans cette cellule.

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Relationships between the cell proliferation and the differentiation in the embryonic and larval intestine ofpleurodeles waltlii michah. II. Effects of intestinal chalones extracted from the intestine of the adult newt

Summary The intestinal chalones 1 and 2, extracted from the intestine of the adult newt, are known to inhibit the G₁ and G₂ phases of the cell cycle in the embryonic intestine. The effects of these intestinal chalones on the proliferation and differentiation of intestinal cells of newt embryos were studied with special attention to the dose-response relationship, the embryonic stage and the duration of treatment. The chalone 2 triggered a linear, dose-dependent inhibition between two concentration thresholds; nevertheless about 25% of the cycling cells were not inhibited either by the highest doses injected or by repeated injections. Sensitivity to chalone 2 appeared in the intestinal epithelium at the end of embryonic development (stage 34) but the cells were not delayed in the G₂ phase for more than about 20 h in spite of repeated injections. It was inferred from the doseresponse curve of the mitotic inhibition by chalone 1, that the intestinal cell population was heterogeneous: about 50% of the cycling cells were inhibited by low concentrations of chalone 1; an additional proportion of about 25% of cycling cells was inhibited by 100 x more concentrated chalone 1 and the remaining 25% was insensitive to the inhibitor. Repeated injections of chalone 1 blocked about 50% of the cycling cells definitively in the G₁ phase, speeded up digestion of yolk platelets, promoted the differentiation of goblet cells and depressed the number of stem cells in the proliferative compartment located beneath the epithelium. A kinetics model of cell proliferation and cell differentiation in the intestinal cell lineages was elaborated and it was suggested that the arrest of mitotic activity and the completion of differentiation in an embryonic cell depends on two incoming signals: one is intracellular and appears when the required number of cell cycles has occured in the cell lineage, leading to a committed stem cell sufficiently differentiated to synthesise chalone and to respond to chalone; the other signal is extracellular and appears when the chalone concentration is high enough: i.e. when the required number of cells is obtained in this tissue.

Ce travail a bénéficié de l'aide du CNRS (ATP N^o A655 1799) et de l'INSERM (AT N^o 74142036)     

Interaction of Liposomes with the Human Skin Lipid Barrier: hSclls as Model System — DSC of Intact Stratum Corneum and in Situ CLSM of Human Skin

Abstract

The interaction of liposomes with the human skin lipid barrier was studied by (i) physico-chemical analyses of lipid vesicles prepared from the complete mixture of human epidermal lipids, (ii) differential scanningcalorimetry of integral horny layers (strata cornea) before and after topical administration of phospholipid vesicles and (iii) confocal laser scanning microscopy of pieces of skin to which Rho-PE labelled DMPC-liposomes were applied. The data imply that the epidermal lipids of the human skin have at body temperature a high propensity of mixing if Ca²⁺ -ions are present, that intact liposomes do not penetrate into the viable epidermis but diffusing/mixing liposomal lipids disorder the complex structure of the upper intercellular lipid sheets thereby causing an increased hydration. The result is an enhancement of penetration of chemicals via the ‘polar and lipid routes'.     

Cell kinetic characterization of cultured human keratinocytes from normal and psoriatic individuals

Psoriasis is a chronic skin disease characterized by epidermal hyperproliferation, disturbed differentiation, and inflammation. It is still a matter of debate whether the pathogenesis of psoriasis is based on immunological mechanisms, on defective growth control mechanisms, or possibly on a combination of both. Several in vivo cell biological differences between psoriatic lesional epidermis and normal epidermis have been reported. However, it is not clear whether these changes are causal or consequential. In case that keratinocytes from psoriatic patients have genetically determined deficiencies or polymorphisms with respect to autocrine growth regulation and the response to inflammatory cytokines, we hypothesize that these differences should be maintained in culture. Here we have started a systematic comparison of first passage keratinocytes cultured from normal skin and uninvolved psoriatic skin to address the question whether there are intrinsic differences in basic cell cycle parameters. In an established, defined culture system using keratinocyte growth medium (KGM) we have determined: (i) cell cycle parameters of exponentially growing keratinocytes, (ii) induction of quiescence by transforming growth factor β₁ (TGF-β₁), and (iii) restimulation from the G₀-phase of the cell cycle. Bivariate analysis of Iodo-deoxyuridine incorporation and relative DNA content was performed by flow cytometry. Within the limitations of this model no gross differences were found between normal and psoriatic keratinocytes with respect to S-phase duration (T_s), total cell cycle duration (T_c), responsiveness to TGF-β₁ and the kinetics for recruitment from G₀. In psoriatic keratinocytes we found a lower amount of cells in S-phase and a shorter duration of G₁, compared to normal keratinocytes. The methodology developed here provides us with a model for further studies on differences between normal and psoriatic keratinocytes in their response to immunological and inflammatory mediators. © 1996 Wiley-Liss, Inc.     

A Flow Cytometric In Vivo Chalone Assay Using Retransplanted Old Murine Jb-1 Ascites Tumour Cells

A flow cytometric in vivo chalone assay is described. Transplantation of old JB-1 ascites tumour cells to new hosts induced an influx of tumour cells, with G₁ DNA content, to the S phase. This induction could be reversibly and specifically blocked by injections of an ultrafiltrate of old JB-1 ascites fluid. The method described is superior to a previously published in vivo chalone assay using regenerating ascites tumours. Owing to a reduced variability in time of onset of DNA synthesis, a smaller scatter of observations is achieved and thus the number of mice per group may be reduced using the new method. In contrast to the older technique, the present one does not necessitate killing of mice during the observation period.     

Arginase activity and other cellular events associated with epidermal hyperplasia

The unknown biochemical role of arginase in epidermal metabolism was probed by examining the association of elevated arginase activty with epidermal hyperplasia and hyperkeratinization. Epidermal hyperplasia as induced experimentally by topical application of 1-decanol to the right side of male hairless mice while the contralateral side served as control. Arginase activity, incorporation of ³ H-thymidine into DNA, DNA and protein content were measured in the separated control and experimental epidermis six hours and on days 1 through 5 and 7 after 1-decanol application. After six hours, the epidermis appears damaged histologically, and DNA synthesis is inhibited. By day 1, incoporation of ³ H-thymidine into DNA is elevated and a new hyperplastic epidermis has formed beneath the original epidermis. Epidermal arginase is elevated two through seven days after 1-decanol application and always is associated with continuing epidermal hyperplasia. The stimulation of DNA synthesis, which parallels the induction of epidermal hyperplasia by 1-decanol, precedes the induction of epidermal arginase activity. An attempt to relate these results with polyamine synthesis and other metabolic events is made.     

NEW METHODS OF CELL CYCLE ANALYSIS BASED ON THE SELECTIVE TAGGING OF CELLS EITHER AT THE BEGINNING OR END OF S PHASE

New techniques for cell cycle analysis are presented. Using HeLa cells, methods are described for the selection of a narrow window or cohort of lightly [³H]-labeled cells located either at the very beginning or the very end of S phase. The cohort cells are tagged by a labeling procedure which entails alternating pulses of high and low levels of [³H]thymidine and are identified autoradiographically. Additional methods are described for following the progress of cohort cells through the cell cycle. Theoretically, with the methods described, it should be possible to follow the ‘early S cohort’ cells as they exit from S phase, as they enter and exit M and as they enter the subsequent S phase. This would allow a determination of S, S + G₂, S + G₂+ M and T. It should theoretically be possible to follow ‘late S cohort’ cells in a similar manner, allowing a determination of G₂, G₂+ M and G₂+ M + G₁. To test these predictions, several experiments are presented in which the progress of the two cohorts is monitored. The best data were obtained from the mitotic curves of cohort cells. For each of the cohorts, values were obtained for the time required for peak concentration of cells in mitosis, the coefficients of variation and of skew. The curve of cohort cells passing through mitosis is shown to fit a log-normal curve better than a normal curve. In addition, the mitotic curves are used to estimate the length of M and to estimate the loss of cohort synchrony. Other uses of these methods are discussed.