Dexamethasone inhibition of rat hepatoma cell growth and cell cycle traverse is reversed by insulin.

(1) The growth of 7800 C1 Morris hepatoma cells was inhibited by dexamethasone. The inhibition was detectable at 1 nM and half-maximal effect was obtained with approx. 13 nM dexamethasone. About 80% growth inhibition was obtained with 250 nM of the hormone and the growth rate was normalized on cessation of treatment. (2) These hepatoma cells contain dexamethasone receptors with equilibrium dissociation constant of 0.24 nM and a capacity of 24 fmol/mg cell protein. Treatment of the cells with insulin did not change these dexamethasone binding properties. Binding experiments showed that 2, 10 and 100% of the receptors were occupied when the cells were incubated with 1 nM, 7 nM and 250 nM dexamethasone, respectively. (3) Insulin completely counteracted the growth inhibition by dexamethasone and antagonized the induction of peroxisomal acyl-CoA oxidase and tyrosine aminotransferase caused by the glucocorticoid. (4) Micro-flow fluorometry showed that the cultures had a major diploid DNA stem line and a minor tetraploid stem line. Changes in diploid, tetraploid and S phase cells of the diploid stem line were scored. Dexamethasone reduced the proportion of cells in S phase and of tetraploid cells. Insulin partly reversed the action of dexamethasone in S phase, but prevented the reduction in tetraploid cells caused by dexamethasone. (5) The mitotic rate was significantly reduced by dexamethasone and this effect was reversed by insulin. (6) Continuous [3H]methyl-thymidine labelling showed a growth fraction of unity in all treatment groups. (7) It is concluded that dexamethasone induces growth inhibition by reducing the G1-S transition. Insulin is able to counteract this effect and increase the rate of DNA synthesis.

The cyclosporin PSC 833 increases survival and delays engraftment of human multidrug-resistant leukemia cells in xenotransplanted NOD-SCID mice.

Circumvention of chemoresistance in cancer may involve several modulator drugs with high affinity for the multidrug transporter P-glycoprotein (Pgp), which is expressed in a number of multi-resistant malignancies. Pgp acts as a membrane efflux pump with broad substrate specificity including antineoplastic drugs and endogenous substances such as certain cytokines and sphingolipids. Therefore, the consequence of Pgp blockade could be far more complex than intracellular drug retention. In the present study exposure of the Pgp inhibitor, PSC 833 (1200 ng/ml), to Pgp expressing KG1a/200 human leukemia cells provoked cell cycle arrest and apoptosis in vitro. This finding was put to test in vivo using a xenotransplant model of KG1a/200 human cells intravenously inoculated into non-obese diabetic severe combined immunodeficient (NOD-SCID) mice. The animals were randomly allocated to receive treatment with PSC 833 (n = 32) or placebo (n = 24). PSC 833 (30 mg/kg) was subcutaneously injected six or 12 times separated by 48-96 h. The overall mean whole blood concentration of PSC 833 was 1191 +/- 60 ng/ml (s.e.m.) at 20 h after administration. Tumor engraftment was significantly reduced in the treatment group (P = 0.037), which also had prolonged survival compared to control animals (P = 0.0016). This is the first study that demonstrates antileukemic effects of a Pgp inhibitor as single agent therapy in vivo, and the present data raise the possibility of alternative exploitation of modulators in cancer chemotherapy.

Perturbation of cell cycle progression in mouse epidermis prior to the regenerative response.

Cell kinetic perturbations that resulted in a wave of increased cell division during the 6--8 hr lag period prior to regenerative DNA replication in mouse epidermis were examined. The epidermis was stimulated to proliferate by adhesive tape stripping, and flow cytometric DNA measurements of isolated epidermal basal cells, counting of mitoses, of Colcemid arrested metaphases and of labeled mitoses among basal cells in histologic sections were made. The results showed that mitotic peaks that occur in the pre-replicative period subsequent to tape stripping can be explained by a delay in cell progression through the S phase, followed by subsequent release and partial synchrony in further cell cycle progression. Early peaks of mitoses in epidermis stimulated to proliferate should therefore not, without further evidence, be assumed to originate from cells triggered into division from a resting G2 compartment. The results also indicate an initial delayed cell progression through the G2 phase, whereas the mitotic duration seemed to be initially reduced, indicating that the DNA synthesis phase and the G2 phase are parts of the epidermal cell cycle that may be most vulnerable to various types of influences.

Perturbation of cell cycle progression in mouse epidermis prior to the regenerative response.

Cell kinetic perturbations that resulted in a wave of increased cell division during the 6-8 hr lag period prior to regenerative DNA replication in mouse epidermis were examined. The epidermis was stimulated to proliferate by adhesive tape stripping, and flow cytometric DNA measurements of isolated epidermal basal cells counting of mitoses, of Colcemid arrested metaphases and of labeled mitoses among basal cells in histologic sections were made. The results showed that mitotic peaks that occur in the prereplicative period subsequent to tape stripping can be explained by a delay in cell progression through the S phase, followed by subsequent release and partial synchrony in further cell cycle progression. Early peaks of mitoses in epidermis stimulated to proliferate should therefore not, without further evidence, be assumed to originate from cells triggered into division from a resting G2 compartment. The results also indicate an initial delayed cell progression through the G2 phase, whereas the mitotic duration seemed to be initially reduced, indicating that the DNA synthesis phase and the G2 phase are parts of the epidermal cell cycle that may be most vulnerable to various types of influences.

Cell cycle progression kinetics of regenerating mouse epidermal cells: an in vivo study combining DNA flow cytometry, cell sorting, and [3H]dThd autoradiography.

Cantharidin application to mouse skin induces cell injury followed by a regenerative wave of cells entering S phase in partial synchrony about 16 h after application. After pulse labeling with [3H]dThd the synchronized cohort of cells was traced through subsequent cell cycles during regeneration. This was accomplished by DNA flow cytometry of isolated basal cells combined with sorting from G1, S, and G2 phases followed by autoradiography at intervals after pulse labeling. Successive peaks of labeled cells in S phase at about 12-h intervals, followed by subsequent peaks in G2 and G1 phases were seen. This shows that the peaks of S-phase cells seen at 16 and 28 h after cantharidin application represent mother and daughter cells, respectively, the latter still cycling in partial synchrony. These 2 peaks of S-phase cells, therefore, are not keratinocyte subpopulations with different time lags between the stimulus to regeneration and the subsequent response. It is further shown that the mean cell cycle time is reduced from about 55 h in normal epidermis to 12 h during early regeneration. This is mainly due to a considerably reduced G1 phase duration, but the S and G2 phase durations are also reduced, although still within the range of circadian variations seen in normal animals. It is reasonable to assume a causal relationship between the considerably reduced G1 duration and loss of growth restriction. Cells with a slow progression rate through G2 phase (70% of all G2 cells) in normal mouse epidermis seem to maintain a slow progression rate during regeneration. Normal growth homeostasis seems to be gradually reestablished during the second day of regeneration.

Adrenalin has differential effects on epidermal cell cycle progression in mice.

The cell kinetic response after intraperitoneal injection of the 10 micrograms adrenalin was investigated in hairless mouse epidermis. Changes in the proportion of cells in S and G2 phase were studied by means of flow cytometry of isolated basal cells. Changes in the proportion of cells in prophase and metaphase, changes in the mitotic rate (Colcemid method) and in cell cycle progression of 3H-TdR labeled cells were studied in histologic sections. The results showed that adrenalin has a differential effect on cell proliferation in mouse epidermis. The cell progression rate from S phase through G2 phase to metaphase is increased in one cohort of cells shortly after adrenalin injection. Simultaneously another cohort of cells is reversibly delayed or blocked in prophase. In agreement with most previous studies a significantly reduced cell division rate was seen 2-3 hr after adrenalin injection. At this time the proportions of cells in prophase and G2 phase were normalized, whereas a significant increase in the proportion of cells in S phase had cycle progression out of S phase might be responsible for the reduced mitotic rate seen after adrenalin administration.

DNA synthesis in mouse epidermis: S phase cells that remain unlabeled after pulse labeling with DNA precursors progress slowly through S.

Epidermal basal cells from hairless mice were isolated after pulse labeling with tritiated DNA precursors and subjected to DNA flow cytometry combined with cell sorting. Cells were sorted from a window in the middle of the S phase, collected on glass slides, and subjected to autoradiography. Unlabeled cells in the middle of the S phase were found in normal mouse epidermis after optimal pulse labeling with tritiated thymidine [( 3H]dThd), in accordance with previous results. The proportion of unlabeled S phase cells was considerably increased among basal cells from mice treated with growth-inhibitory epidermal extracts. Reanalysis and re-sorting of cells previously sorted from mid S showed that unlabeled cells could not be accounted for by G1 contamination. Furthermore, labeling with precursors incorporated into DNA by "de novo" metabolic pathway [( 3H]Urd) did not reduce the proportion of unlabeled S phase cells, either when given alone or when given in combination with the precursor for DNA incorporated by the "salvage" pathway [( 3H]dThd). This strongly indicates that the unlabeled S phase cells do not synthesize DNA continuously, or are synthesizing DNA at a rate below the level of detection. A reduced proportion of unlabeled S phase cells was found in regenerating epidermis. This may be explained by a dilution effect caused by the 3-fold increase in the total number of cells within S phase at this condition. The observation that essentially all cells in mid S phase were labeled during 4 days of continuous labeling with [3H]dThd, indicates that cells in S phase that remain unlabeled after optimal pulse labeling are cycling, albeit slowly. Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells are larger than the average. These cells may represent a subpopulation of basal cells going through their last division cycle before differentiation.

Evidence of mouse epidermal subpopulations with different cell cycle times.

In order to obtain information on the distribution of total cell cycle times in hairless mouse epidermis, basal cells were isolated and prepared for DNA flow cytometry at intervals after a pulse labeling with 50 microCi of thymidine. The DNA distributions were recorded, and cells were sorted from windows in the S, G2, and G1 phases of the cell cycle, collected on glass slides, and subjected to autoradiography. The proportions of labeled cells were scored in each fraction, and the percentage of labeled mitoses was determined in histologic sections from the same animals. Grain count distributions were recorded at selected time points over labeled cells in sorted fractions and over labeled mitoses. The movement of the labeled S-phase cohort was thus followed through all cell cycle phases. Peaks in labeled cells were observed at about 36 h in S phase, G2 phase, and mitosis, and high levels of labeled G2 cells and mitoses were seen at about 80 h. These results indicate the existence of one rapidly cycling subpopulation of keratinocytes with a cell cycle time slightly less than 30 h, in addition to keratinocytes with considerably longer cell cycle times. The first peak of labeled G2 cells reached only about 30%. This is consistent with earlier findings of about 30% G2 cells with a rapid traverse, and 70% with a considerably delayed traverse through G2 phase. The proportion of labeled G1 cells reached a value corresponding to twice the initial labeling index at 8 h after pulse labeling. This is consistent with previously obtained phase durations, indicating an unperturbed cell cycle traverse of labeled cells from S phase through G2 and mitosis.

Decreased Leydig cell responsiveness in the testicular feminized male rat.

The Stanley-Gumbreck pseudohermaphrodite or testicular feminized male (tfm) rat exhibits a decreased Leydig cell sensitivity to human CG (hCG) measured by androgen and cyclic-3',5',-adenosine monophosphate production in vitro. These changes were associated with an 80% reduction in the number of LH receptors in the tfm testis, when compared on the basis of equivalent amounts of testis particle protein or per 10(6) isolated Leydig cells. Androstenedione and not testosterone is the major androgen secreted by the tfm Leydig cell and androstenedione secretion is, therefore, a more appropriate end point than testosterone secretion for Leydig cell function in tfm animals. A dose of hCG (3 ng/2 ml) which elicited a near maximal response in androgen production from the decapsulated testes and Leydig cell suspensions of normals rats, did not significantly stimulate androgen production from Leydig cells of the tfm animals. A much higher dose of hCG (200 ng/2 ml) gave a response from the tfm Leydig cells which was comparable to that obtained with 3 ng from Leydig cells of normal littermates. This indicates that the small number of LH receptors on the tfm Leydig cell membrane are functional and that the reduction in receptor number results in a decrease in the sensitivity of response to LH rather than a reduction in the maximum steroid response.

Heterogeneity in the mouse epidermal cell cycle analysed by computer simulations.

Different sets of cell kinetic data obtained over many years from hairless mouse epidermis have been simulated by a mathematical model including circadian variations. Simulating several independent sets of data with the same mathematical model strengthens the validity of the results obtained. The data simulated in this investigation were all obtained with the experimental system in a state of natural synchrony. The data include cell cycle phase distributions measured by DNA flow cytometry of isolated epidermal basal cells, fractions of tritiated thymidine ([3H]TdR) labelled cells within the cell cycle phases measured by cell sorting at intervals after [3H]TdR pulse labelling, bivariate bromodeoxyuridine (BrdUrd)/DNA data from epidermal basal cells isolated at intervals after pulse labelling with BrdUrd, mitotic rate and per cent labelled mitosis (PLM) data from histologic sections. The following main new findings were made from the simulations: the second PLM peak observed at about 35 h after pulse labelling is hardly influenced by circadian variations; the peak is mainly determined by persisting synchrony of a rapidly cycling population with a G1-duration (TG1) of 20 h to 30 h; and there is a highly significant population of slowly cycling G1-cells (G1 sigma). However, no significant circadian variations were found in the number of these cells.

Technetium-99m-tetrofosmin for parathyroid scintigraphy: a comparison with sestamibi.

UNLABELLED: Parathyroid scintigraphy with the new myocardial perfusion radiopharmaceutical 99mTc-tetrofosmin was compared with 99mTc-sestamibi scintigraphy using early and delayed imaging. METHODS: The two preparations were administered on different days to the same 16 patients suffering from primary hyperparathyroidism. Anterior view gamma camera planar imaging (10-min acquisition) was performed in the period between 5 min and 3 hr after administration of the radiopharmaceutical. For most of the patients, a pertechnetate image of the thyroid was available for eyeball comparison when reading the tetrofosmin and sestamibi images. Imaging results were compared with those from histopathological examination after surgery. RESULTS: On early images, all the adenomas visualized with sestamibi were equally well seen with tetrofosmin and vice versa. In 6 of 11 scintigraphically detected neck adenomas, delayed imaging improved the adenoma visualization with sestamibi. In contrast, this differential washout was never seen with tetrofosmin. Histopathological examination of excised tissue specimens after neck exploration (15 patients) or thoracotomy (one patient) revealed a parathyroid adenoma in all 16 patients. Our 12 scintigraphic findings were true-positives, while the remaining four scintigraphies were false-negatives, giving a diagnostic sensitivity of 75% with both preparations. The mediastinal adenoma was detected in a patient with a history of two unsuccessful neck explorations and one unsuccessful thoracotomy. CONCLUSION: Tetrofosmin has the same success rate as sestamibi for detection of parathyroid adenomas on scintigrams acquired immediately after injection. In contrast to sestamibi, delayed imaging has no diagnostic impact. Moreover, the thyroid/ parathyroid differential washout of sestamibi failed in 5 of 11 neck adenomas here detected, indicating that delayed sestamibi washout is an unreliable diagnostic criterion. Therefore, whether sestamibi or tetrofosmin is preferred for parathyroid scintigraphy, thyroid scintigraphy seems mandatory.

p53 accumulation confers prognostic information in resectable adenocarcinomas with ductal but not with intestinal differentiation in the pancreatic head.

The aim of the study was to examine the relation between p53 protein accumulation, clinicopathological variables and prognosis in resectable adenocarcinomas of the pancreatic head. The clinical records and tissue specimens of 82 consecutive patients resected for adenocarcinomas located in the head of the pancreas were reviewed retrospectively. Formalin-fixed and paraffin-embedded specimens from each tumour were stained with the monoclonal antibody DO7, and the nuclear p53 positivity within each tumour was assessed. Histopathological reclassification showed that 60 tumours exhibited ductal differentiation and 22 tumours intestinal differentiation. Twenty-five percent (15/60) of the ductal tumours and 50% (11/22) of the intestinal tumours were positive for p53 accumulation. p53 immunoreactivity was significantly correlated to a worse prognosis in the tumours of ductal differentiation, with median survival 0.76 years for p53 positive and 1.44 years for p53 negative patients. The p53 positivity of tumours with intestinal differentiation showed no such correlation. No correlation was found between p53 accumulation and other known prognostic factors in either the ductal or the intestinal type of tumours. Our results indicate that the tumour biology of ductal adenocarcinomas differs significantly from that of adenocarcinomas of the intestinal type located in the pancreatic head, and that p53 accumulation confers a worse prognosis only of ductal tumours. Subclassification of these tumours based on type of differentiation is therefore suggested since periampullary tumours include ductally as well as intestinally differentiated adenocarcinomas.

Characterization of bladder tumours by multiparameter flow cytometry with special reference to grade II tumours.

Sixty-three human transitional cell carcinomas of the urinary bladder were studied by multiparameter flow cytometry (FCM). The cellular DNA content, the cellular protein content, the fraction of cells in S phase, and the nuclear size were registered and correlated to histological grade (WHO) and histologically determined infiltration through the basement membrane. Aneuploidy was found in the great majority of grade III tumours, but in only 24% of grade II tumours. A new, combined variable, viz. the cellular DNA to protein ratio, indicated a possibility for further subdivision of the tumours. Grade II tumours, which constitute a rather heterogeneous group with regard to prognosis, could be classified in two subgroups: One group of diploid tumours with the FCM characteristics of grade I tumours, and another group of diploid and aneuploid tumours with the characteristics of grade III tumours. Infiltration was most frequently seen in the latter subgroup. The putative prognostic relevance of such a subdivision will be the subject of a future study. Compared to FCM measurement of DNA alone, multiparameter FCM, including measurement of the total cellular protein content, has given additional information that may be of prognostic value.

Association of p53 accumulation with TP53 mutations, loss of heterozygosity at 17p13, and DNA ploidy status in 273 colorectal carcinomas.

The aim of this study was to establish an experimentally based cutoff level for assessing p53 immunoreactivity in colorectal tumors. The accumulation of p53 protein in 273 colorectal tumors was correlated with previously obtained data on TP53 mutation and loss of heterozygosity at two 17p13 loci in the same tumors. The monoclonal antibody PAb 1801 was used for p53 staining, and the results obtained by immunohistochemistry and immunoblotting were similar. Mutation analyses of exons 5-8 were performed using constant denaturant gel electrophoresis followed by sequencing. There were no statistically significant differences for any measured TP53 gene alteration between the group of tumors without p53-positive nuclei (n = 83) and the group with <5% positive nuclei (n = 58). The majority of mutations within these groups were deletions/insertions and nonsense mutations without p53 accumulation. Therefore, we assume that 5% p53-positive nuclei is the relevant cutoff level to assess TP53 damage in colorectal tumors. A prerequisite for this recommendation is optimal conditions for p53 protein detection. The parameters for p53 dysfunction were correlated to DNA aneuploidy measured by flow cytometry. TP53 mutations were significantly associated with DNA aneuploidy (P < 0.00001), and a nonrandom distribution of TP53 gene alterations among diploid (DI = 1), hyperdiploid (1.0 < DI < 1.3), and highly aneuploid (DI > 1.3) tumors indicates that DNA hyperdiploid tumors constitute a separate developmental entity different from tumors with gross aneuploidy.

Deoxyribonucleic acid flow cytometry of germ cells in the investigatin of male infertility.

DNA flow cytometry was applied to fine-needle aspiration biopsy specimens from the testes of 20 oligospermic or azoospermic men under investigation for infertility. All aspirations gave sufficient material for DNA flow cytometry, and no complications occurred after aspiration. In both groups many DNA patterns deviated from those observed in control specimens. In general, abnormally small testes were associated with the greatest deviations in DNA patterns. In specimens from certain azoospermic men the DNA distributions did not deviate from those of control samples, suggesting a block in the testicular excurrent ducts. These findings also suggest that DNA flow cytometry of aspirated testicular material can be used in the classification of tubular degeneration in males. The results are obtained within a short time, are quantitative and without bias, and the aspiration causes only little discomfort to the patient. The method can therefore be recommended as a very practical means of obtaining quantitative estimates of spermatogenesis in men being evaluated for infertility.

Application of flow cytometry to studies on the human acrosome.

This study describes the use of flow cytometry combined with specific labelling of the human sperm acrosome using a FITC-labelled plant lectin (Arachis hypogea agglutinin). Localization of the label to the acrosome was encouraged by freezing the sperm for at least 24 hours at -70 degrees C prior to labelling. Studies of sperm from 53 normospermic men revealed that acrosome labelling followed a single normal distribution without the presence of subpopulations. The average fluorescence and degree of variation within the sperm population differed markedly between sperm samples. These differences could not be predicted by any of the normal criteria of sperm quality, including sperm morphology, vitality, and motility. Exposure of washed sperm to the calcium ionophore A 23187 in the presence of calcium at 37 degrees C, caused a time-related leftward shift in the distribution of acrosome fluorescence, indicating that this technique can be also used to monitor the acrosome reaction.

Prognostic significance of proliferative and apoptotic markers in oral tongue squamous cell carcinomas.

The prognostic impact of proliferative and apoptotic markers was studied in 85 T1-4 oral tongue squamous cell carcinomas (SCCs). Ki67 immunoreactivity and AgNOR counts, including mean AgNOR counts (mAgNOR) and the percentage of nuclei with more than one AgNOR (pAgNOR > 1), were used as proliferative parameters. The apoptotic index (AI) was assessed using the TUNEL method. Bax expression was detected immunohistochemically and scored. Bax expression correlated positively with AI (p = 0.0122). Ki67 correlated with both pAgNOR > 1 (p = 0.0042) and mAgNOR (p = 0.0189). Low Bax expression and low AI correlated significantly with the disease-free period (p = 0.0001 and p = 0.0024, respectively). High values for Ki67, pAgNOR > 1 and mAgNOR correlated with poor prognosis (p = 0.0021, p = 0.0001 and p = 0.0244, respectively). Combinations of proliferative and apoptotic parameters were stronger predictors than individual parameters (p < 0.0001). pAgNOR > 1-Bax expression appeared to be the best combination (p < 0.0001). We conclude that proliferative and apoptotic markers, especially their combinations, have prognostic value in tongue SCC.

Establishment of a human meningeal cell line in culture.

A human metastatic meningiosarcoma has been established as a cell line as follows: At autopsy performed 12 hours after death, tumour nodules from the left parietal pleura were obtained under sterile conditions, minced and inoculated into culture vessels. The cells obtained were cloned, and clones with abnormal chromosome stemlines ascertained by G-, C-, and N-banding techniques were then injected subcutaneously into nude mice of the NMRI and BALB/c strains. Tumour nodules from the athymic mice were also minced, established as cell cultures and maintained for further studies. In vitro cytogenetical analysis showed variations in chromosome numbers per cell ranging from 23 to 126; whereas flow cytometric measurements of DNA-specific fluorescence during exponential cell growth in vitro demonstrated the presence of 3 well defined DNA classes around 3C, 6C, and 12C. The xenografts still retained the particular morphological and structural characteristics of the primary tumour, as analyzed by histological sections. The permanent growth integrity of the established meningiosarcoma should render its utilization as a model system for biological studies possible.

Retinoic acid provokes a regeneration-like proliferative response in murine epidermis. A bivariate DNA/bromodeoxyuridine flow cytometric study.

Retinoic acid (RA) is an inducer of epidermal proliferation by a mechanism of action which is not fully known. We examined the proliferative response of hairless mouse epidermis to a single topical application of different doses of RA (0.1-1000 nmol). The mitotic rate was assessed using the stathmokinetic method, and change in epidermal cell numbers were scored per microscopic vision field in tissue sections. Cell cycle parameters were measured by bivariate bromodeoxyuridine/DNA flow cytometry on isolated epidermal basal cells after pulse labelling up to 10 days after RA treatment. The results showed a dose-dependent increase in mitotic activity with a maximum at 3 days after RA application, and a dose-dependent hyperplasia with a maximum at 4 days after RA application. Cell-cycle analysis showed an immediate proliferative response after RA application similar to that following various skin irritants. Although differences in the G2 phase transit were seen, this indicates a similar mechanism of action of RA-induced epidermal proliferation and that associated with epidermal regeneration in general.

Topical application of calcitriol alters expression of filaggrin but not keratin K1 in mouse epidermis.

Calcitriol (1 alpha,25-dihydroxyvitamin D3) and its analogues are antiproliferative agents which promote epidermal differentiation in vitro, possibly reflecting their modes of action in the treatment of psoriasis. We examined the effect of calcitriol on early and late terminal differentiation in mouse epidermis in vivo using an immunofluorescence assay to detect keratin K1 and filaggrin expression. Pulse labelling with the tymidine analogue 5-bromo-2-deoxyuridine (BrdUrd) was performed by intraperitoneal injection of mice immediately or 16 h after a single topical application of 0.72 nmol calcitriol. The BrdUrd labelling index (LI) and keratin K1 or filaggrin expression of postmitotic cell cohorts were scored by paired immunofluorescence staining for up to 72 h after BrdUrd labelling. Calcitriol induced cell proliferation as shown by a 100% increase in the BrdUrd LI 17 h after application. The onset of keratin K1 expression in the postmitotic period was, however, unchanged in both series after calcitriol treatment. Filaggrin expression appeared earlier after calcitriol treatment than in control epidermis, probably reflecting altered cell kinetics with increased epidermal turnover. The results suggest that calcitriol only influences the later stages of the keratinocyte differentiation programme, possibly secondarily to its hyperproliferative effect.