Heat shock mRNA in mouse epidermis after UV irradiation

Total RNA from murine epidermis was extracted at different times after irradiation with erythemogenic doses of ultraviolet light (UVB or UVB/UVA) and hybridized to a DNA probe from the gene of a heat shock protein (hsp 70). An intense and transitory enrichment in RNA molecules hybridizing to the DNA probe was found between 15 and 120 min after irradiation, followed by a return to control levels over the next 70 h. Dose-response analysis indicates that 30 min after the irradiation, the relative amount of RNA hybridizing to the hsp 70 DNA probe increases with the dose (within the range explored: 0-180 mJ/cm2) up to values greater than 5 times the control.     

Collagen metabolism in mouse lung after X irradiation

Collagen and total protein synthesis rates have been determined in the lungs of CBA mice irradiated with single doses of X rays between 8 and 16 Gy. Mice were injected with [3H]proline accompanied by a large dose of unlabeled proline, and synthesis rates were measured at 2-month intervals from 8 to 31 weeks after irradiation. At 2 months after radiation treatment, collagen and total protein synthesis rates were significantly depressed but they had recovered by 4 months. By 6 months collagen synthesis rates had increased above control in a dose-dependent manner, so that in the 14-Gy dose group the fractional synthesis rate for collagen was 4.6 times higher than in control mice as measured by incorporation of [3H]proline. However, a significant net accumulation of collagen was seen only in the lungs of the highest dose group at 31 weeks, as indicated by total hydroxyproline measurements. There was a slight increase in the ratio of types I and III collagen. Late radiation damage in the CBA mouse lung is characterized by increased collagen metabolism, which may or may not lead to a net accumulation of collagen.     

Changes in the DNA labelling index after beta irradiation of the mouse epidermis

This study looked at the changes in the interfollicular DNA labelling index (LI) with time after strontium-90/yttrium-90 beta irradiation of approximately 100 mm2 of mouse flank skin, after a dose of 100 Gy which produces transitory moist desquamation. Within 24 hr of such a dose the LI of the irradiated area was essentially zero (0.07 +/- 0.03%), whilst those of the side area and of the control area were 15.0 +/- 2.6% and 21.4 +/- 2.7%, respectively. The LI of the side and the control areas then fell within 3-5 days to approximately 4% and approximately 2% respectively, whilst that of the irradiated area rose rapidly to a peak value of 30.2 +/- 1.7% at 10 days post-irradiation. There was a 20% reduction in the diameter of the area with detectable radiation damage within 5 days, and this is primarily due to cell proliferation and migration from the unirradiated margins of the field. In contrast, between days 10 and 20 the major source of repopulation is probably derived from local migration and proliferation of surviving hair follicle basal cells within the irradiated field.     

Evaluation of DNA damage in different stages of mouse spermatogenesis after testicular X irradiation

To evaluate whether DNA alterations in mature spermatozoa could stem from DNA damage induced in immature germ cells, testis cells and spermatozoa were analyzed by the comet assay and by the sperm chromatin structure assay 14, 45 and 100 days after in vivo X irradiation of the testes. These times were selected, according to the mouse seminiferous epithelium cycle, to follow the DNA damage induced in different germ cell compartments. The cytotoxic action was assessed by DNA flow cytometric analysis of testicular cells. A dose-dependent increase of DNA damage in testis cells was observed 14 days after irradiation, whereas mature sperm cells were not affected. On the other hand, an increase in DNA strand breaks was seen in spermatozoa 45 days after treatment. DNA damage returned to the control levels 100 days after irradiation. The methods used to evaluate DNA damage gave comparable results, emphasizing the correlation between DNA fragmentation and susceptibility of sperm chromatin to denaturation. Both techniques showed the high radiosensitivity of differentiating spermatogonia. The overall results showed that DNA damage induced in pre-meiotic germ cells is detectable in primary spermatocytes and is still present in mature spermatozoa.     

Proliferative response of mouse spermatogonial stem cells after irradiation: a quantitative model analysis of experimental data

The testes of CDF₁ mice were irradiated with single doses of X-rays ranging from 2–16 Gy. The number of haploid cells in the testis at different times after irradiation (42–350 days) was determined by one-parameter flow cytometry both for irradiated animals and for age-matched controls. Based on literature data on the kinetics of the spermatogenesis in mice, a mathematical model of the (hierarchical) germ tissue was developed. Using this model, the processes of radiation-induced cell loss and subsequent recovery were simulated and free parameters of the model were estimated by fitting the model prediction to the experimental data. One of the aims of the study was to investigate the kinetic behaviour of spermatogonial stem cells and the corresponding control mechanisms. In order to fit the data, the model has to include the following features: (i) A preferential self-repopulation of spermatogonial stem cells following tissue injury. The model-estimated probability of a self-renewing division rises from 50% (the steady-state value) to 95% if the stem-cell population is reduced to 10% of its normal size. (ii) A relatively low, almost constant turnover rate of the stem-cell compartment. It is suggested by the analysis that less than 10% of the permatogonial stem cells present in the testis divide per day, regardless of the degree of cellular depletion. (iii) A mechanism responsible for incomplete recovery. The observed incomplete recovery of spermatogenesis after single doses exceeding 10 Gy can be described quantitatively assuming that the stem cells are organized into discrete proliferative structures, the number of cells per structure being about 60.     

Development of ATPase-positive,immature Langerhans cells in the fetal mouse epidermis and their maturation during the early postnatal period

Summary The development and maturation of Langerhans cells during the differentiation of skin was studied in mice from fetal day 13 to adult using 3 indices: (1) ATPase activity; (2) ultrastructure; and (3) quantitative evaluation of the cell population.ATPase-positive Langerhans cells appeared in the epidermis at first at fetal day 16, and they increased in number in the differentiating epidermis during the late fetal period. The earliest appearance of Birbeck granules was at postnatal day 4. Cored tubules were also formed in the Langerhans cells in the dermis at around the same age. The cells containing Birbeck granules or cored tubules are considered to be mature Langerhans cells. In the Langerhans-cell lineage, those cells in the epidermis at stages earlier than postnatal day 4 and not yet containing specific organelles are considered to be immature Langerhans cells. These immature Langerhans cells can be identified ultrastructurally in the epidermis at fetal day 16, coinciding with the appearance of ATPase-positive cells. The increase in the number of immature Langerhans cells during the perinatal period was shown by quantitative analysis of nuclear density and relative Langerhans-cell area on the electron micrographs.It is concluded that ATPase is a marker of the Langerhans-cell lineage from the early development stages, while Birbeck granules and cored tubules are markers that identify mature Langerhans cells in electron micrographs.     

Label-retaining keratinocytes and Langerhans cells in mouse epithelia

Summary Slowly cycling cells in murine epithelia can be marked by their retention of a tritiated-thymidine nuclear label. The position and identity of such label-retaining cells in palatal and lingual epithelia and ear epidermis was examined using autoradiography and histochemistry. They were found to be either (a) basally positioned keratinocytes preferentially occupying sites within units of epithelial structure that correspond to those expected for epithelial stem cells, or (b) nonkeratinocytes of the Langerhans cell type which lie suprabasally except in the epidermis where they are present in low numbers and occupy a similar position to label-retaining keratinocytes.This work was supported by NIH-NID-RO1-DEO 5395     

The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation.

The radiosensitivity of spermatogonial stem cells to X rays was determined in the various stages of the cycle of the seminiferous epithelium of the CBA mouse. The numbers of undifferentiated spermatogonia present 10 days after graded doses of X rays (0.5-8.0 Gy) were taken as a measure of stem cell survival. Dose-response relationships were generated for each stage of the epithelial cycle by counting spermatogonial numbers and also by using the repopulation index method. Spermatogonial stem cells were found to be most sensitive to X rays during quiescence (stages IV-VII) and most resistant during active proliferation (stages IX-II). The D0 for X rays varied from 1.0 Gy for quiescent spermatogonial stem cells to 2.4 Gy for actively proliferating stem cells. In most epithelial stages the dose-response curves showed no shoulder in the low-dose region.     

Subchromosomal DNA synthesis in synchronous V-79 chinese hamster cells after X irradiation

A substantial fraction of replicon initiation events in Chinese hamster V-79 cells have been shown to be refractory to the effects of X irradiation immediately after exposure. This study examines the possibility that the initiation radiorefractive portion is the result of changes in replicon radiosensitivity as a function of position in S phase. The data obtained from DNA fiber autoradiograms and kinetic incorporation of radiolabeled thymidine from cells irradiated at various positions in S phase showed only slight changes in the proportion of replicons refractive to X irradiation immediately after exposure. These results indicate that initiation radiorefractive replicons may be an intrinsic property of V-79 cells and that cell-cycle-specific heterogeneity in radiation response cannot fully account for this phenomenon. The results also indicate that delayed inhibition of initiation events may play a larger role in the observed radiorefractive fraction than previously thought.     

The mouse keratin 6 isoforms are differentially expressed in the hair follicle, footpad, tongue and activated epidermis

Keratin 6 (K6) is expressed constitutively in a variety of internal stratified epithelia as well as in palmoplantar epidermis and in specialized cells of the hair follicle. K6 expression can also be induced by hyperproliferative conditions as in wound healing or by conditions that perturb normal keratinocyte function. The functional significance of the expression of K6 on keratinocyte biology under these disparate conditions is not known. Here we report on the characterization of two isoforms of mouse K6 that are encoded by separate genes. The two genes (denoted K6a and K6b) are linked, have the same orientation and are actively transcribed. Sequence analysis revealed, that although they encode almost identical products, they have distinctly different regulatory regions, suggesting that the two K6 genes would be differentially expressed. In an attempt to define the expression characteristics of the K6 isoforms, we produced transgenic mice with each gene after modifying the C-terminal sequences to enable detection of the transgenic proteins with specific antibodies. The constitutive expression of the K6a transgene paralleled that of the endogenous genes in all K6 expressing tissues, except in the tongue. The K6b transgene was also expressed in these tissues but, in contrast to K6a, was only expressed in suprabasal cells. Both K6 transgenes were also induced in the interfollicular epidermis in response to phorbol esters, with K6a induced in all layers of the treated epidermis, while K6b was expressed only in suprabasal cells. These studies suggest that the K6 isoforms have overlapping yet distinct expression profiles.     

A modified adenosine polyphosphatase histochemical method to demonstrate Langerhans cells in fragile epidermis

Adenosine polyphosphatase enzymes provide useful markers for epidermal Langerhans cells. Established adenosine polyphosphatase histochemical methods were refined and applied to demonstrate Langerhans cells in thin sheets of murine dorsal epidermis. The skin was supported during staining by attaching the keratinized surface to polyallyl diglycol carbonate "plastic" slides with cyanoacrylate adhesive and flattening it with pressure from a glass slide on the dermal surface. Optimal specific staining of dendritic Langerhans cells occurred after fixation of ethylenediaminetetraacetic acid-separated epidermal sheets in cacodylate buffered formaldehyde for 20 min and incubation, in the presence of magnesium and lead ions, with 9.36 X 10(-4) M adenosine diphosphate (ADP) for 45 min. Better definition of the cells was obtained with ADP as a substrate than with any concentration of adenosine triphosphate.     

Dose-dependent biphasic accumulation of TP53 protein in normal human embryo cells after X irradiation

The effects of various doses of X radiation on the kinetics of accumulation of TP53 protein (formerly known as p53) were examined in normal human embryo cells. We found that the rate of accumulation of TP53 protein was biphasic at X-ray doses between 1 and 4 Gy, while monophasic accumulation was observed after X irradiation with doses higher than 6 Gy. The first phase of accumulation was detected within 1 h after irradiation, and a second phase of accumulation was detected between 6 and 12 h after irradiation. The induction of CDKN1A (formerly known as p21(WAF1/CIP1)) and MDM2 proteins was also biphasic after doses of 4 Gy or less, while monophasic accumulation was observed after 6 Gy or higher. We found that the proteasome inhibitor ALLN increased the constitutive level of TP53 protein, and no change was observed in the TP53 level after X irradiation when cells were treated with ALLN. These results indicate that the dose-dependent accumulation of TP53 is due to an inhibition of TP53 degradation, and that the induction of MDM2 might be responsible in part for the different kinetics of accumulation of TP53.     

Inhibited differentiation of Langerhans cells in the rat epidermis upon systemic treatment with cyclosporin A

The immunosuppressant drug cyclosporin A (CsA) is known to cause reduction in number, DNA synthesis and function of Langerhans cells (LC). Since also the differentiation of LC is known to be hampered in conditions of acquired immunodeficiency not due to drugs, we investigated whether this occurs with CsA. Rats were injected subcutaneously with CsA (5, 10 and 50 mgxkg(-1) x d(-1)) for three weeks; the skin was analyzed by Ia immunohistochemistry and by electron microscopy. Epidermal immunolabeled cells were 15+/-3.5 (mean +/- SEM) per 100 basal keratinocytes in untreated controls and 8.75+/-1.3, 4.75+/-1.0 and 1.7+/-1.2 upon increasing doses of CsA (p<0.01). By electron microscopy, monocytoid cells with deep invaginations of the plasma membrane and roundish LC poor in Birbeck granules appeared in the epidermis upon treatment. The results suggest that CsA inhibits the differentiation of LC precursors in the epidermis and that this can in part explain the selective increase in the risk of skin viral disease and cancer in chronically treated patients.     

The effects of ultraviolet light and certain drugs on Ia-bearing Langerhans cells in murine epidermis

Langerhans cells and indeterminate cells are immune macrophages of the epidermis and have Ia markers on their surface. Because of their position in the epidermis, they are subject to many environmental toxins like ultraviolet light. Also medications like cortisone applied topically to the skin could have important effects on these cells. We have used an anti-Ia serum and an indirect immunofluorescent technique to study Langerhans cells in epidermal sheets. We found that shortwave ultraviolet light (250–320 nm) and ultraviolet B (280–320nm) increased the density of Ia-bearing cells (Langerhans cells) in the skin. Psoralens and ultraviolet A (PUVA) (320–400 nm) depleted the skin of Ia-bearing cells, an effect which takes 2 weeks to produce but which persists for several weeks after stopping treatment. Triamcinolone acetonide administered topically or intraperitoneally also depletes the skin of Ia-bearing cells. These agents, light and steroids, either destroy the Ia-bearing cells or remove the Ia markers from the cellular surface.