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.     

Modified distribution of epidermal glycoproteins in the nude mouse

The nude mouse is an athymic mutant whose immunological deficiency has been exploited for transplantation of normal and diseased xenogeneic tissue. Histologically, its skin has no unusual features apart from the absence of hair. We report here a biochemical study of its epidermis, with comparison to the hairless mouse (which is devoid of hair but otherwise functionally normal). The epidermal glycoproteins were probed with the lectin, concanavalin A (Con A). Fluorescein isothiocyanate (FITC)-Con A overlays of cryostat skin sections gave a similar fluorescent pattern for both mouse strains: all the viable epidermal cell layers were labeled but not the stratum corneum. In contrast, when different populations of keratinocytes that were separated on Percoll gradients were analyzed by gel electrophoresis, and the gels then overlaid with iodinated Con A, all the epidermal layers, including the stratum corneum, were labeled. For all the epidermal cell layers there are substantial differences between the two mouse strains. We observe changes in the glycoprotein distribution with the stage of differentiation. Comparison with our earlier data for human epidermis indicates that the discrepancies between the nude mouse and the hairless mouse are much greater than those between the latter and man. The most striking difference is the absence in the stratum corneum of the nude mouse of a 40 K glycoprotein which is the dominant feature for the hairless mouse and for man. The gel patterns point to functional discrepancies in the epidermis of the nude mouse, particularly in the stratum corneum, not evident histologically or with FITC-Con A.     

Growth kinetics in newborn mouse epidermis: response to epidermal chalone.

Epidermal DNA synthesis, the epidermal mitotic rate, and the responsiveness to the epidermal G1 and G2 inhibitors were examined in newborn mice at different times after birth. The rate of epidermal cell renewal was in general low during the first two weeks of life. Later the two growth parameters increased and reached very high values at 32-33 days after birth. The rate of epidermal cell proliferation then decreased to a level comparable with that found in adult hairless mouse epidermis at 40-45 days. A single i.p. injection of skin extract containing the two epidermal growth inhibitors induced varying types of responses. The epidermal G2 inhibitor stimulated the mitotic rate on day 2 and day 10, but inhibited it on all other days. The epidermal G1 inhibitor brought about an increase in epidermal DNA synthesis on day 6 and possibly on the following days. No response at all seen at 2, 4, 17, and 32 days after birth. At the other examined times the inhibition was similar to that found in adult mice. These findings differed from those made in vitro on separated newborn mouse epidermal cells (our own unpublished data), and we suggest that the variability of newborn mouse epidermis could be an expression of the immaturity of the skin as a whole, and that dermis in some way modifies the response of epidermis to exogenous epidermal chalone. Our study did not support the theory that the nonresponsiveness of newborn mouse epidermal at certain times could be due to the presence of nonresponsive stem cells in epidermis.     

A role for the E-cadherin cell-cell adhesion molecule during tumor progression of mouse epidermal carcinogenesis

The expression of the cell-cell adhesion molecules E- and P-cadherin has been analyzed in seven mouse epidermal keratinocyte cell lines representative of different stages of epidermal carcinogenesis. An inverse correlation between the amount of E-cadherin protein and tumorigenicity of the cell lines has been found, together with a complete absence of E-cadherin protein and mRNA expression in three carcinoma cell lines (the epithelioid HaCa4 and the fibroblastoid CarB and CarC cells). A similar result has been detected in tumors induced in nude mice by the cell lines, where induction of E-cadherin expression takes place in moderately differentiated squamous cell carcinomas induced by HaCa4 cells, although at much lower levels than in well-differentiated tumors induced by the epithelial PDV or PDVC57 cell lines. Complete absence of E-cadherin expression has been observed in spindle cell carcinomas induced by CarB or CarC cells. P-cadherin protein was detected in all cell lines that exhibit an epithelial (MCA3D, AT5, PDV, and PDVC57) or epithelioid (HaCa4) morphology, as well as in nude mouse tumors, independent of their tumorigenic capabilities. However, complete absence of P-cadherin was observed in the fibroblast-like cells (CarB and CarC) and in spindle cell carcinomas. The introduction of an exogenous E-cadherin cDNA into HaCa4 cells, or reactivation of the endogenous E-cadherin gene, leads to a partial suppression of the tumorigenicity of this highly malignant cell line. These results suggest a role for E-cadherin in the progression to malignancy of mouse epidermal carcinogenesis. They also suggest that the loss of both E- and P-cadherin could be associated to the final stage of carcinogenesis, the development of spindle cell carcinomas.     

Growth regulation in X-irradiated mouse skin; the possible role of chalones.

Extracts of hairless mouse skin were tested for their content of epidermal G1 inhibitor and G2 inhibitor at daily intervals after X-irradiation with 4 500 or 2 250 rad. After either dose the skin extracts lacked G1 inhibitory activity on days 5 and 6 respectively after irradiation. This coincided with the time when the epidermal mitotic rate again became normal and started a period of over-shoot. The time interval of 5-6 days corresponds to the turnover time of the differentiating cells in hairless mouse back epidermis. The findings indicate that the proliferating cells in epidermis can respond to changes in local chalone concentration, even after X-irradiation at the tested doses, and that the irradiated epidermal cell population still retains some important properties inherent in a cybernetically regulated system. The local G2-inhibitory activity also varied after irradiation, but these variations could not be directly related to the corresponding mitotic rates.     

Distinctive Uptake of Neutral Red by Mitotic Cancer Cells

Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.     

Luxol Fast Scarlet C as a Stain for Phase-Contrast Microscopy

Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.     

Comparison of the Effects of Epidermal Chalone In Young and Aging Mice

The ability of epidermal chalones to produce inhibition of epidermal mitotic and DNA synthetic activities was investigated in young (2 month old) and old (27 month old) mice. Extracts of epidermal chalone were prepared from the skin of mice of these different ages, and these extracts were then tested for their inhibitory capacities against the same age group from which they were extracted, and also against the mice of the other age group. It was found that the ability of mouse skin to produce tissue-specific agents with mitotic and DNA-synthetic inhibitory capabilities did not change significantly with increasing age. There were however, decreases in both the labeling and mitotic indices with aging in untreated mice. These data suggest that chalone-type inhibitory mechanisms are not primarily responsible for the increased cell cycle times seen to occur with aging in normal tissues.     

Comparison of the effects of epidermal chalone in young and aging mice.

The ability of epidermal chalones to produce inhibition of epidermal mitotic and DNA synthetic activities was investigated in young (2 month old) and old (27 month old) mice. Extracts of epidermal chalone were prepared from the skin of mice of these diferent ages, and these extracts were then tested for their inhibitory capacities against the same age group from which they were extracted, and also against the mice of the other age group. It was found that the ability of mouse skin to produce tissue-specific agents with mitotic and DNA-synthetic inhibitory capabilities did not change significantly with increasing age. There were however, decreases in both the labeling and mitotic indices with aging in untreated mice. These data suggest that chalone-type inhibitory mechanisms are not primarily responsible for the increased cell cycle times seen to occur with aging in normal tissues.     

Effects of hydroxyurea on the mitotic activity and the G2 phase in hairless mouse epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid at various times after HU. The animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G2 was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G2 for the first 6 hr after HU injection, then the G2 compartment was emptied. The results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G2 and M. The cell kinetic effects of HU thus seem to be very complex.     

Adrenaline Increases Cyclic 3′5′-AMP Formation in Hamster Epidermis

CATECHOLAMINES probably influence cell proliferation by delaying cells in the premitotic phase^1,2. Bullough and Laurence found that crude skin extracts contained a tissue-specific protein (chalone) which inhibited epidermal cell proliferation and that the action of this extract was augmented by adrenaline³. They later found that adrenaline alone (0.00025 µg/ml.) reduced epidermal mitotic activity in mouse ears by about 50% in vitro⁴.     

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.     

Acute effects of ultraviolet light B on morphology and epidermal cell kinetics in human skin transplanted to nude mice

Grafts of human skin on nude mice were irradiated with varying doses of ultraviolet light B and at various intervals were subjected to histological examination and determination of the epidermal 3H-thymidine labelling index. The studies showed that the human skin in the foreign milieu of the nude mouse retained its ability to respond to phototoxic damage. The findings suggest that the nude mouse/human skin model could be a valuable tool in human photodermatological research.     

Effects of Hydroxyurea On the Mitotic Activity and the G2 Phase In Hairless Mouse Epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid® at various times after HU. the animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G₂ was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G₂ for the first 6 hr after HU injection, then the G₂ compartment was emptied. the results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G₂ and M. the cell kinetic effects of HU thus seem to be very complex.     

In vivo growth stimulation of collagen gel embedded normal human and mouse primary mammary epithelial cells

A new system for studying growth of normal human mammary epithelial cells in an in vivo environment using athymic nude mice is described. Human mammary epithelial cells dissociated from reduction mammoplasty specimens were embedded within collagen gels and subsequently transplanted subcutaneously into nude mice. Histological sections of recovered collagen gels showed epithelial cells arranged as short tubules with some branching. Proliferation of mammary epithelial cells was quantitated in vivo by 3 days' continuous infusion with 5 bromo-2′-deoxy-uridine followed by immunostaining of sections from recovered gels. Ovarian steroids administered to the host animals, resulting in blood serum levels normally found in the human female, had little or no effect on the proliferation of human mammary epithelial cells. Collagen gel embedded mouse mammary epithelial cells, mouse mammary explants, and host mammary glands all responded similarly to ovarian steroids, suggesting that the unresponsiveness of the human mammary epithelial cells under these conditions was not due to dissociation per se. However, an increased dose of 17β-estradiol or a growth factor combination containing epidermal growth factor, cholera toxin, and cortisol significantly stimulated the proliferation of human outgrowths. The growth factor response was dependent on the location of the cells, with the greatest response seen in the part of the gel proximal to the osmotic pump delivering the growth factors and the effect gradually waning in area more distal to the pump. The effect was especially striking since the mitotic figures could be easily identified and the labeling index was as high as 75%. The host mouse mammary gland also responded to growth factors, resulting in ductal hyperplasia. The proliferative and morphogenetic effects of various agents on normal human mammary epithelial cells embedded in collagen gel can be studied in vivo in nude mice. © 1995 Wiley-Liss, Inc.     

Carcinogen metabolism in human skin grafted onto athymic nude mice: a model system for the study of human skin carcinogenesis

Human skin grafted onto athymic nude mice maintains its major histological features and may provide a useful system with which to assess the carcinogen interaction with human skin. Significant differences were observed in basal levels of cytochrome P-450 and cytochrome P-448-dependent monooxygenase activities between human grafted and nude mouse epidermis. Topical application of crude coal tar (CCT) to human skin transplanted onto nude mice resulted in 3.9 & 3.5; 3.2 & 2.9 and 1.1 & 1.2 fold increases in mouse and human epidermal aryl hydrocarbon hydroxylase (AHH), ethoxyresorufin deethylase (ERD) and ethoxycoumarin deethylase (ECD) activities, respectively. CCT applied topically to mouse skin resulted in 27.8 & 6.4; 12.8 & 3.3 and 1.7 & 2.6 fold increases in mouse and human epidermal AHH, ERD and ECD activities, respectively. Topical application of coal tar either onto human transplanted skin or to mouse skin also resulted in substantial induction of hepatic and pulmonary AHH and ERD activities. These studies indicate that human skin grafted onto nude mice preserves its metabolic capacity and offers a useful model system with which to assess the effects of polycyclic aromatic hydrocarbons and CCT on cutaneous xenobiotic metabolism in the human population.     

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.     

Regional differences in mitotic activity due to injury in mouse skin

Summary Mitotic activity adjacent to a wound inflicted at different sites in the mouse skin was measured 24 h after injury. A regional difference in the epidermal mitotic activity due to injury was noted. Mitotic activity was high in the anterior parts of the body including the head, lower in the middle to posterior regions of the body and lowest in the posterior-most parts of the body. Regional differences in epidermal mitotic activity due to injury were demonstrated in both female and male mice. The existence of a cranio-caudal gradient in epidermal response to injury is suggested.     

The organizational fate of intermediate filament networks in two epithelial cell types during mitosis

Intermediate filaments (IF) appear to be attached to the nuclear envelope in various mammalian cell types. The nucleus of mouse keratinocytes is enveloped by a cagelike network of keratin-containing bundles of IF (IFB). This network appears to be continuous with the cytoplasmic IFB system that extends to the cell surface. Electron microscopy reveals that the IFB appear to terminate at the level of the nuclear envelope, frequently in association with nuclear pore complexes (Jones, J. C .R., A. E. Goldman, P. Steinert, S. Yuspa, and R. D. Goldman, 1982, Cell Motility, 2:197-213). Based on these observations of nuclear-IF associations, it is of interest to determine the fate and organizational states of IF during mitosis, a period in the cell cycle when the nuclear envelope disassembles. Immunofluorescence microscopy using a monoclonal keratin antibody and electron microscopy of thin and thick sections of mitotic mouse keratinocytes revealed that the IFB system remained intact as the cells entered mitosis and surrounded the developing mitotic spindle. IFB were close to chromosomes and often associated with chromosome arms. In contrast, in HeLa, a human epithelial cell, keratin-containing IFB appear to dissemble as cells enter mitosis (Franke, W. W., E. Schmid, C. Grund, and B. Geiger, 1982, Cell, 30:103-113). The keratin IFB in mitotic HeLa cells appeared to form amorphous nonfilamentous bodies as determined by electron microscopy. However, in HeLa, another IF system composed primarily of a 55,000-mol-wt protein (frequently termed vimentin) appears to remain morphologically intact throughout mitosis in close association with the mitotic apparatus (Celis, J.E., P.M. Larsen, S.J. Fey, and A. Celis, 1983, J. Cell Biol., 97:1429-34). We propose that the mitotic apparatus in both mouse epidermal cells and in HeLa cells is supported and centered within the cell by IFB networks.     

The dependence of the cytokinetic effects of alkylating antitumor preparations on the phase of the hepatocyte cell cycle in regenerating liver]

Effects of alkylating antitumor drugs on resting (G0 phase of cell cycle) and proliferating (G1, S, G2 and M phases) hepatocytes were studied in regenerating mouse liver. Cell cycle kinetics (fraction of labeled mitoses, labeling and mitotic indices) were determined by 3H-thymidine autoradiography. Dipin and fotrin as a DNA-damaging agents attack mainly resting (G0) and proliferating (G1) cells. Effect of the damage results in the inhibition of DNA synthesis and G2 phase arrest in the following mitotic cycle. An alkylating drug phopurin as well as ara-C both suppress the mitotic progression in proliferating hepatocytes and do not influence the resting cells.