An estrogen-dependent esterase activity in MCF-7 cells

The presence of steroidal esters in hormonally sensitive tissues lends importance to the esterases which convert the biologically inactive adducts to the parent potent forms. Accordingly, esterase-activities were studied in a human breast cancer model--the MCF-7 cell line. Tritiated estradiol esters- estradiol-17-acetate (EA), estradiol-17-valerate (EV) and estradiol-17-stearate (ES) were tested systematically, but 3 beta-ol esters of androgens, and phorbol diesters were also investigated. All compounds tested, except the phorbol diesters were hydrolyzed either when added to growing cultures or to the 28,000 g supernate of homogenized MCF-7 cells. Among the estrogens, the relative rates of hydrolysis were EA greater than EV greater than ES. The esterase for EA was different as it was not inhibited by saturating concentrations of EV or ES, and unlike the others its activity was stimulated by the addition of estradiol to the culture medium. The antiestrogen keoxifene,[(6-Hydroxy-2-(4-hydroxyphenyl)benzo less than b greater than thien-3-yl greater than less than 4- less than 2-(1-piperidinyl)ethoxy greater than phenyl greater than methanone], negated the stimulatory effect. Other major classes of steroids did not influence EA esterase activity. Results of inhibition experiments indicated that the esterases are of the serine active-site types. The significance of the estrogen-dependent esterase activity can be assessed when the natural substrate(s) for the enzyme is elucidated.     

13-cis retinoic acid and all-trans retinoic acid in the regulation of the proliferation and survival of human breast cancer cell line MCF-7.

Retinoids are a group of compounds which inhibit cell proliferation and induce cellular differentiation. The aim of this study was to compare the antiproliferative activity of various concentrations of 13-cis retinoic acid (isotretinoin) and all-trans retinoic acid (tretinoin) in a culture of the estrogen-sensitive human breast cancer cell line MCF-7. Evaluation was based on [3H]thymidine incorporation into the cancer cells and through immunocytochemical analysis of cell cycle-associated PCNA and Ki-67 protein expression. Both retinoids inhibited [3H]thymidine incorporation into the cancer cells most effectively at a concentration of 3x10(-3) M. Two basic substances used for line MCF-7 culture experiments, one stimulating - estradiol - and the other inhibiting - tamoxifen - were applied. Estradiol added to a culture containing decreasing concentrations of isotretinoin (from 3x10(-3) to 3x10(-8) M) caused a statistically significant reduction in the percentage of [3H]thymidine incorporation into the cancer cell line MCF-7, compared to the 17 beta estradiol group (189.25%+/-62.64, control=100%, p<0.05). In the group of decreasing tretinoin concentrations, statistically significant differences were found only at 3x10(-3), 3x10(-4) and 3x10(-8) M. Following culture supplementation with tamoxifen (1 microM), statistically significant differences were observed only at the highest concentrations of both retinoids (3x10(-3) and 3x10(-4) M). The evaluation of breast carcinoma cells with a positive immunocytochemical reaction to PCNA and Ki-67 has revealed that isotretinoin reduces their percentage in the most determined and statistically significant way (38.00%+/-2.58 and 39.25%+/-3.09), compared to the control group (86.50%+/-9.20 and 100%+/-3.87, p<0.001 and p<0.0001) and to the estradiol group (87.00%+/-6.79 and 86.10%+/-7.0, p<0.001). Apart from their blocking effect on the cell cycle, retinoids also induce the apoptotic pathway.     

Induction and inhibition of estradiol hydroxylase activities in MCF-7 human breast cancer cells in culture

The effects of estradiol, progesterone, and tamoxifen on the activity of estradiol 2- and 16 alpha-hydroxylases were studied in human breast cancer cell cultures using a radiometric assay. After 5 days' exposure to these compounds, incubations in the presence of either [2-3H]estradiol or [16 alpha-3H]estradiol as substrate were carried out. In MCF-7 cells, estradiol (10(-8) M), progesterone (10(-6) M) and tamoxifen (10(-6) M) significantly increased 16 alpha-hydroxylase activity (estradiol; 21% progesterone 10% to 32%; tamoxifen 21% to 31%; P less than 0.01). Synergistic effects were observed when the cells were successively exposed to tamoxifen and progesterone. Simultaneous treatment with tamoxifen plus estradiol or estradiol plus progesterone showed no change from estradiol alone. On the other hand, although estradiol had no direct effects on 2-hydroxylase activity, tamoxifen decreased this enzymatic activity significantly at 10(-6) M (23% to 37%). Progesterone acted synergistically to further decrease this reaction. Treatment with only progesterone caused an increase in 2-hydroxylation. In contrast, a subline of MCF-7 cells with low estrogen receptor levels showed only minimal enzyme-hormone responses. Likewise, treatment of the estrogen receptor-negative MDA-MB-231 human breast cancer cell line with these compounds showed no effects on either 2- or 16 alpha-hydroxylase activity. In the progesterone receptor-rich T47D cell line, estradiol decreased both activities while progesterone increased both.(ABSTRACT TRUNCATED AT 250 WORDS)     

An esterase is involved in geraniol production during palmarosa inflorescence development

Total incorporation of exogenously administered [2-14C]acetate into essential oil of palmarosa (Cymbopogon martinii) was found to be relatively higher than that of either [U-14C]sucrose or [U-14C]glucose during inflorescence development. Among the major essential oil constituents, biogenesis of geranyl acetate was much higher than that of geraniol. Alkaline hydrolysis of [14C]labeled geranyl acetate revealed that the majority of the label incorporated into geranyl acetate was present in the geraniol moiety, indicating that only newly synthesized geraniol gets acetylated to form geranyl acetate. Geranyl acetate cleaving esterase (GAE) activity followed a similar pattern during both in vivo and in vitro inflorescence development, with maximum activity at immature inflorescence stages, suggesting the involvement of GAE in geraniol production during inflorescence development. Five esterase isozymes (Est-A to E) were detected in the enzymic fraction of palmarosa inflorescence and all showed GAE activity, with Est-B being significantly increased during inflorescence development. The role of GAE in geraniol production and improving the palmarosa oil quality is discussed.     

Studies on type II progesterone receptor in MCF-7 cells

These experiments demonstrate for the first time the existence of a Type II progesterone receptor (RpII) in MCF-7 human breast cancer cells. RpII was shown to have a lower affinity for tritiated progesterone ([3H]Pg) (Kd greater than or equal to 13 nM) than classical Rp (Kd less than or equal to 3 nM). RpII was detected by cytosolic, nuclear, and whole cell assays of MCF-7 cells. Scatchard analysis of [3H]Pg binding data revealed that classical Rp but not RpII could be recompartmentalized from the cytosolic to the nuclear pool by treating cells 1 h at 37 degrees C with 1 microM Pg. RpII levels were shown to be increased more than two-fold by growing MCF-7 cells for 4 days in 10 nM estradiol (E2) plus 100 nM Pg when compared to either untreated cells or to cells treated with only E2.     

The influence of estradiol on cholesterol processing by the corpus luteum

Recent studies from our laboratory have suggested that estradiol or androgen precursor may stimulate steroidogenesis in the luteal cell by modulating intracellular cholesterol metabolism including mobilization of cholesteryl esters, stimulation of lipoprotein receptor activity and induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity. To test the functionality of cholesteryl ester turnover per se, we measured the activities of acyl CoA:cholesterol acyltransferase (ACAT) and cholesteryl esterase, the enzymes involved in cholesteryl ester synthesis and hydrolysis, respectively; we also measured de novo synthesis of cholesterol, cholesteryl esters, and steroids. Pregnant rats, hypophysectomized and hysterectomized on Day 12, were treated for 72 h with either estradiol or testosterone, and luteal microsomal and cytosolic fractions were utilized to measure ACAT and cholesteryl esterase activity, respectively. Intact corpora luteal were employed for [14C]acetate incorporation experiments. Basal ACAT activity (expressed as pmol.min-1.CL-1 increased from a mean of 78 +/- 16 in vehicle-treated rats to 119 +/- 18 and 197 +/- 16 in the estradiol- and testosterone-treated rats, respectively. Similarly, total ACAT activity (measured in the presence of exogenous cholesterol) was also increased in estradiol- and testosterone-treated groups. On the other hand, cholesterol esterase activity (expressed either pmol.min-1.CL-1 or pmol.min-1.mg protein-1) was similar in all three groups and comparable to corpora lutea from intact pregnant rats. Hypophysectomy and hysterectomy caused a 50-60% reduction in [14C]acetate incorporation into sterols when compared with intact pregnant rat. Treatment with either estradiol or testosterone not only restored the cholesterol biosynthetic capacity but also enhanced the overall rate of [14C]acetate incorporation into steroids as compared to intact pregnant rats. The major (-80%), newly synthesized steroid was identified as progesterone. In conclusion, the present studies suggest that the major function of luteal estradiol is to induce de novo cholesterol biosynthesis, regulate ACAT activity, and channel available free cholesterol (derived from both endogenous and exogenous sources) for steroidogenesis.     

Contribution of aromatase to the deoxyribonucleic acid synthesis of MCF-7 human breast cancer cells and its suppression by aromatase inhibitors.

We have studied the effects of various steroids on DNA synthesis in MCF-7 human breast carcinoma cells, which have aromatase activity and which exert an oestrogen receptor-mediated growth, to assess the significance of intracellular aromatase on growth stimulation as well as inhibition by aromatase inhibitors. The cells were cultured for 96 h in phenol red-free medium containing 10% charcoal-treated fetal bovine serum and test reagents and pulse-labelled with [3H]thymidine. Physiological concentrations of oestradiol, oestrone, testosterone (T) and androstenedione (AD) stimulated thymidine incorporation. However, oestrone-sulphate and dihydrotestosterone (DHT) only stimulated at concentrations greater than the physiological levels. T and DHT stimulation was blocked by tamoxifen, but not by cyproterone acetate, suggesting that the stimulation was mediated via the oestrogen receptor but not by the androgen receptor. Stimulation by T and AD was reduced by aminoglutethimide and 14 alpha-hydroxy-4-androstene-3,6,17-trione, both of which inhibit aromatase activity, however, stimulation by nonaromatizable DHT was not reduced by the inhibitors, suggesting that androgens were converted by the intracellular aromatase to oestrogens which stimulated the thymidine incorporation. It is suggested that intracellular aromatase significantly contributes to the stimulation of DNA synthesis and that aromatase inhibitors suppress the stimulation.     

Metabolism of [11-3H]retinyl acetate in liver tissues of vitamin A-sufficient, -deficient and retinoic acid-supplemented rats

A study was conducted on the incorporation of [11-3H]retinyl acetate into various retinyl esters in liver tissues of rats either vitamin A-sufficient, vitamin A-deficient or vitamin A-deficient and maintained on retinoic acid. Further, the metabolism of [11-3H]retinyl acetate to polar metabolites in liver tissues of these three groups of animals was investigated. Retinol metabolites were analyzed by high-performance liquid chromatography. In vitamin A-sufficient rat liver, the incorporation of radioactivity into retinyl palmitate and stearate was observed at 0.25 h after the injection of the label. The label was further detected in retinyl laurate, myristate, palmitoleate, linoleate, pentadecanoate and heptadecanoate 3 h after the injection. The specific radioactivities (dpm/nmol) of all retinyl esters increased with time. However, the rate of increase in the specific radioactivity of retinyl laurate was found to be significantly higher (66-fold) than that of retinyl palmitate 24 h after the injection of the label. 7 days after the injection of the label, the specific radioactivity between different retinyl esters were found to be similar, indicating that newly dosed labelled vitamin A had now mixed uniformly with the endogenous pool of vitamin A in the liver. The esterification of labelled retinol was not detected in liver tissues of vitamin A-deficient or retinoic acid-supplemented rats at any of the time point studied. Among the polar metabolites analyzed, the formation of [3H]retinoic acid from [3H]retinyl acetate was found only in vitamin A-deficient rat liver 24 h after the injection of the label. A new polar metabolite of retinol (RM) was detected in liver of the three groups of animals. The formation of 3H-labelled metabolite RM from [3H]retinyl acetate was not detected until 7 days after the injection of the label in the vitamin A-sufficient rat liver, suggesting that metabolite RM could be derived from a more stable pool of vitamin A.     

Polyamine requirement for prolactin action on macromolecular synthesis in the MCF-7 human mammary epithelial cell line

The actions of insulin, hydrocortisone, prolactin and growth hormone on the synthesis of macromolecules in MCF-7 cells was determined in a serum-free defined medium. The inclusion of the polyamine spermidine in the medium was shown to enhance the insulin stimulation of the rate of [3H]uridine incorporation into RNA in a manner similar to that demonstrated for hydrocortisone. Spermidine, in addition to insulin and hydrocortisone, was also essential for prolactin to manifest a stimulation of the rate of [3H]uridine incorporation; this effect of spermidine was optimal with spermidine concentrations between 1 and 5 mM. Prolactin also stimulated the rate of [3H]leucine incorporation into total cellular protein and into an isoelectrically precipitable (pH 4.6) phosphoprotein fraction. The actions of prolactin on total protein and phosphoprotein synthesis were only expressed if spermidine, in addition to insulin and hydrocortisone, was contained in the culture medium. All of the prolactin responses were observed employing physiological concentrations of prolactin. Specificity of the prolactin responses was established by demonstrating that porcine growth hormone had no effects on RNA or phosphoprotein synthesis in the MCF-7 cells.     

Aromatase and testosterone fatty acid esters: the search for a cryptic biosynthetic pathway to estradiol esters.

The estradiol fatty acid esters (lipoidal derivatives, LE2) are extremely potent estrogens that accumulate in fat, including fat of menopausal women. These steroidal esters are protected from metabolism and are converted to the free, biologically active steroid through the action of esterases. Previous studies have shown that biosynthetic pathways in the adrenal gland exist in which steroid fatty acid esters are substrates. This led us to determine whether a cryptic aromatase pathway exists in which testosterone esters could be converted directly into LE2. We tested a representative fatty acid ester, testosterone stearate, both as an inhibitor and as a substrate for the aromatase enzyme from human placental microsomes. This ester had neither activity. In addition, we tested [1 beta-3H]testosterone acetate as a substrate for this enzyme complex, measuring the production of 3H2O as evidence of aromatization. Although the rate of reaction was considerably slower than that of testosterone, 3H2O was produced. However, when [2, 4, 6, 7-3H]testosterone acetate was incubated and the steroidal products isolated, we found that hydrolysis of the substrate had occurred. Both [3H]-labeled testosterone and estradiol were found, and very little if any [3H]estradiol acetate was formed. Thus, we conclude that an aromatase pathway involving testosterone esters does not exist and that the sole source of LE2 is through direct esterification of estradiol.     

Enhancement of 2- and 16 alpha-estradiol hydroxylation in MCF-7 human breast cancer cells by 2,3,7,8-tetrachlorodibenzo-P-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin exhibits antiestrogenic activity and induces cytochromes P-450 in estrogen-dependent MCF-7 human breast-cancer cells. To determine whether induction of 2- or 16 alpha-hydroxylation of 17 beta-estradiol has a role in this antiestrogenic activity, MCF-7 cells which were exposed to this xenobiotic for 72 hrs were incubated with either [2-3H] or [16 alpha-3H] 17 beta-estradiol and the extent of tritiated H2O formation, indicative of site-specific hydroxylation, was determined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-treated MCF-7 cultures showed an 8-fold increase in 2-hydroxylation and a 2-fold increase in 16 alpha-hydroxylation. These results support the suggestion that increased hydroxylation of 17 beta-estradiol may have a role in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells.     

Growth factors and hormones which affect survival, growth, and differentiation of the MCF-7 stem cells and their descendants

The human breast tumor cell line was separated by Percoll density gradient centrifugation into six different subpopulations, A to F, one of which (E) appears to contain the stem cells on the basis of several criteria (M. Resnicoff et al. 1987, Proc. Natl. Acad. Sci. USA 84, 7295. We now analyzed the response of the isolated subpopulations to insulin, thrombin, PGF2 alpha, estradiol, and 13-cis-retinal. We demonstrate that the first two growth factors stimulate [3H]thymidine incorporation in the more differentiated subpopulations (D and F), while PGF2 alpha has mitogenic activity in subpopulations C and D. In the absence of any added growth factor, estradiol has the extreme and transient capacity of allowing the stem cell to detach from the tissue culture dish and to grow in suspension as multicellular aggregates (MCF-7/SE cells). 13-cis-Retinal acts as a negative modulator of differentiation and protects the cells from the inhibitory and differentiation activity of Na-butyrate.     

Accumulation of a non-binding form of estrogen receptor in MCF-7 cells under hydroxytamoxifen treatment

It is well known that MCF-7 cells, when incubated with hydroxytamoxifen (OH-Tam) loose their capacity to bind [³H]estradiol. By using Western blotting and [³H]tamoxifen aziridine labeling of KCl extracts from these cells we found that this loss in binding capacity was not associated with a disappearance of the estrogen receptor (ER) protein, an event known to occur after incubation with estradiol. Attempts to label under exchange conditions these ER molecules, which, on the basis of enzyme immunoassays appear to accumulate under OH-Tam treatment, were unsuccessful. Cell fractionation suggested that their origin is nuclear. Assessment of a few triphenylethylenic antiestrogens, as far as their inhibitory potency towards the in vitro MCF-7 cell growth is concerned, indicated a correlation between accumulation of these non-binding ER molecules and the antiestrogen antiproliferative action. However, we were unable to demonstrate absence of such an ER accumulation in two tamoxifen-resistant variants. Impaired folding of the ER protein or impaired phosphorylation of its hormone-binding domain are attractive hypotheses to account for these non-binding ER molecules. Whether these ER molecules have any physiological role, such as competition with the “normal” receptor molecules for the estrogen responsive elements on the DNA is unknown and deserves further study.     

The covalent labeling of proteins by 17 beta-estradiol, retinoic acid, and progesterone in the human breast cancer cell lines MCF-7 and MCF-7/AdrR.

In this study we analyzed the covalent binding to proteins of 17 beta-estradiol (E2), retinoic acid (RA), and progesterone in MCF-7 and MCF-7/AdrR cells. MCF-7 cells have receptors for E2 and progesterone. MCF-7/AdrR cells do not have these receptors. After a 1-day incubation period with either [3H]E2, [3H]progesterone, or [3H]RA the levels of covalently bound radioactivity was between 1.4- to 2-fold greater in MCF-7 cells than in MCF-7/AdrR cells. We analyzed the labeled proteins with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. About 40 proteins were labeled by E2 in MCF-7 cells and about 10 of these proteins were the only proteins labeled by E2 in MCF-7/AdrR cells. We saw that the same 8 proteins were labeled by RA in both cell lines. Progesterone labeled 2 proteins with M(r) values of 37,000 and 20,000 in MCF-7 cells. These 2 proteins had mobilities that were the same as proteins that were labeled by either E2 or RA in both MCF-7 and MCF-7/AdrR cells. Besides these 2 proteins, we saw proteins of M(r) 51,000 (p51) and 55,000 that were covalently labeled by E2 in MCF-7 cells and by RA in both MCF-7 and MCF-7/AdrR cells. The p51 had the same mobility on 2D-PAGE as an 8-azido-[32P]cAMP-labeled protein. This protein is probably RII alpha, the type II cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase. These results suggest that the estrogen receptor, while not obligatory, might still modulate the covalent linkage of E2 to protein. In addition, our results raise the possibility that some effects of some ligands of the thyroid/steroid hormone receptor family may involve the covalent linking of these hormones to proteins, including RII alpha.     

Steroid sulfatase activity and expression in mammary myoepithelial cells

PURPOSE: This investigation examined mRNA expression and enzymatic activity of steroid sulfatase (STS) in human mammary myoepithelial cells (MMECs) and MCF-7 cells and assessed the effects of 17-beta estradiol on the activity of STS. METHODS: The mRNA level of STS in MMECs was determined by RT-PCR analysis using specific primers for STS. STS enzymatic activity prior to and after treatment with 17-beta estradiol was determined by measuring 3H-metabolites formed after exposure to [3H]estrone 3-sulfate (E1S) and [3H]dehydroepiandrosterone-sulfate (DHEA-S). RESULTS: Our data demonstrate the presence of STS in the MMECs. Based on RT-PCR analysis, MMECs had slightly lower levels of STS compared to MCF-7 cells. However, sulfatase activity was about 120 times greater in the MMECs than the MCF-7 cells (E1S V(max)=2640nmol/(mg DNAh) compared to 20.9nmol/(mg DNAh)). Exposure to 17-beta estradiol was associated with 70% reduction in E1S sulfatase activity in the MCF-7 cells and 9% increase in the MMECs after 6 days. DISCUSSION: Our studies indicate for the first time the presence of STS in MMECs. This is suggestive of a previously undetermined role for MMECs in converting precursor hormones into active steroid hormones within mammary tissue. In addition, differential response of the MMECs and the MCF-7 cells to estrogen demonstrates differences in hormone metabolism between these two cell types, perhaps related to the absence of estrogen receptors in the MMECs and their presence in the MCF-7 cells. The MMECs may have an important role in hormonal regulation within mammary tissue.     

Noninvasive monitoring of glutathione turnover in perfused MCF-7 cells

Glutathione metabolism was monitored in proliferating intact, perfused MCF-7 breast cancer cells by (13)C NMR spectroscopy. Label incorporation from [3,3'-(13)C(2)]cystine in the perfusate into intracellular glutathione was monitored in native wild-type MCF-7 (MCF-7wt) cells and sublines resistant to doxorubicin (MCF-7dox) and 4-hydroperoxycyclophosphamide (MCF-7hc). Pulse-chase studies showed non-linear rates of isotope label uptake and washout. Fitting these data to an exponential model of glutathione metabolism allowed calculation of rate constants for glutathione metabolism in these cell lines. Comparison of these rate constants showed glutathione metabolism was increased in both drug-resistant lines. No significant difference was observed between these results for cells growing in three dimensions and results for cells cultured in monolayer.     

Androgens increase osteoblast-stimulating activity of human breast cancer cells in vitro

Bone metastases of breast cancers produce not only osteolytic but also osteosclerotic lesions. The latter are often observed after androgenic treatment of the tumor. Potential production of osteoblast stimulating activity (ObSA) in breast cancer cell lines, and possible androgen control of this activity have been investigated. Conditioned media (CM) collected from 4 breast cancer cell lines (MCF-7, ZR75, MDA-MB 231, BT20) was tested in vitro on ROS 17/2,8 osteoblast-like cells and on osteoblasts derived from human bone biopsies. The parameters monitored in osteoblasts were [3H]thymidine incorporation, alkaline phosphatase activity, and osteocalcin secretion. Serum-free media conditioned during 24 h by MCF-7 cells presented the highest ObSA. CM decreased thymidine incorporation in DNA and increased alkaline phosphatase activity in a dose-dependent manner. Bone GLA protein (osteocalcin) secretion by human osteoblasts was not increased however in the presence of CM. MCF-7 cells were cultured in the presence of dihydrotestosterone (DHT) [1-100 nM] for 5 days. Serum-free, DHT-free CM collected after an additional 24 h, contained alkaline-phosphatase stimulating activity which was DHT dose-dependent. Estradiol and 1,25(OH)2D3 failed to elicit a comparable increase of the ObSA in the CM. In conclusion, MCF-7 cells product factor(s) that interfere with bone remodeling. The DHT modulation of ObSA parallels the estradiol control of MCF-7 cells osteolytic lesions in relation with Prostaglandin E secretion. Sex hormones at physiological and pharmacological levels might thus control both osteosclerotic and osteolytic lesions observed in bone deposits of hormone dependent cancers.     

DNA synthesis in Langerhans' cells of mouse ear epithelium revealed by tritiated thymidine autoradiography and histochemical staining for non-specific esterase and beta-glucuronidase activity

The proportion of Langerhans' cells in DNA synthesis in normal mouse skin was assessed by combining tritiated thymidine [3H]TdR autoradiography with enzyme histochemistry. After injection of [3H]TdR, ear skin was treated in two ways. Epithelial sheet preparations were stained for the presence of non-specific esterase and cytospin preparations of epithelial cell suspensions were stained for beta-glucuronidase activity. The labelling index (+/- SE mean) for cytospins, 40 min after injecting [3H]TdR, was 1.6 +/- 0.15%, doubling to 3-4% from 7-17 days after injection. The sheet preparations showed the proportion of label attributable to paired Langerhans' cells rising from 18% at 40 min after injection, to approximately 45%, on days 1-4 after injection. These results suggest that the proliferation of Langerhans' cells in normal mouse skin might be higher than was previously thought to be the case.