Reduction of Intrafollicular Apoptosis in Chemotherapy-Induced Alopecia by Topical Calcitriol-Analogs
1998; Elsevier BV; Volume: 111; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.1998.00350.x
ISSN1523-1747
AutoresMarkus B. Schilli, Ralf Paus, Andreas Menrad,
Tópico(s)Reproductive System and Pregnancy
ResumoChemotherapy-induced alopecia is thought to result from cytotoxic and apoptosis-related damage to the hair follicle. This study was designed to confirm whether keratinocyte apoptosis is indeed induced in growing (= anagen) hair follicles of C57 BL/6 mice after the injection of cyclophosphamide, using improved methods for histologic detection of apoptotic cells in murine skin. More importantly, we asked whether topical calcitriol-analogs are able to modulate cyclophosphamide-induced apoptosis in vivo, because there are conflicting reports on the effects of calcitriols on apoptosis in vitro. Anagen was induced in telogen mice on day 0 by depilation. Starting on day 5 post-depilation, the back skin of mice was topically treated with either 0.2 μg 1,25-dihydroxyvitamin D3, 2.0 μg calcipotriol, 0.02 μg KH 1060, or vehicle (ethanol) only. On the last day of treatment (i.e., day 9 post-depilation), all mice received 150 mg cyclophosphamide i.p. per kg as a single dose to induce alopecia, or vehicle (aqua dist.). Analysis of the treated skin by in situ-end labeling (using a modified terminal UTP nucleotide end labeling technique suitable for murine skin), by Hoechst 33342 stain, and by DNA electrophoresis on days 10 and 14, revealed the induction of massive apoptosis in cyclophosphamide-treated anagen hair bulbs, which was most prominent on day 10, whereas controls showed no follicular apoptosis. The calcitriol-pretreated groups demonstrated a significant reduction of apoptosis, with a maximal inhibition seen on day 14. This confirms that cyclophosphamide indeed induces massive keratinocyte apoptosis in anagen hair follicles, and provides evidence that topical calcitriol-analogs can suppress epithelial cell apoptosis in vivo. The mouse model employed here offers an excellent tool for dissecting the as yet poorly understood controls of keratinocyte apoptosis in situ and its pharmacologic manipulation. Chemotherapy-induced alopecia is thought to result from cytotoxic and apoptosis-related damage to the hair follicle. This study was designed to confirm whether keratinocyte apoptosis is indeed induced in growing (= anagen) hair follicles of C57 BL/6 mice after the injection of cyclophosphamide, using improved methods for histologic detection of apoptotic cells in murine skin. More importantly, we asked whether topical calcitriol-analogs are able to modulate cyclophosphamide-induced apoptosis in vivo, because there are conflicting reports on the effects of calcitriols on apoptosis in vitro. Anagen was induced in telogen mice on day 0 by depilation. Starting on day 5 post-depilation, the back skin of mice was topically treated with either 0.2 μg 1,25-dihydroxyvitamin D3, 2.0 μg calcipotriol, 0.02 μg KH 1060, or vehicle (ethanol) only. On the last day of treatment (i.e., day 9 post-depilation), all mice received 150 mg cyclophosphamide i.p. per kg as a single dose to induce alopecia, or vehicle (aqua dist.). Analysis of the treated skin by in situ-end labeling (using a modified terminal UTP nucleotide end labeling technique suitable for murine skin), by Hoechst 33342 stain, and by DNA electrophoresis on days 10 and 14, revealed the induction of massive apoptosis in cyclophosphamide-treated anagen hair bulbs, which was most prominent on day 10, whereas controls showed no follicular apoptosis. The calcitriol-pretreated groups demonstrated a significant reduction of apoptosis, with a maximal inhibition seen on day 14. This confirms that cyclophosphamide indeed induces massive keratinocyte apoptosis in anagen hair follicles, and provides evidence that topical calcitriol-analogs can suppress epithelial cell apoptosis in vivo. The mouse model employed here offers an excellent tool for dissecting the as yet poorly understood controls of keratinocyte apoptosis in situ and its pharmacologic manipulation. chemotherapyinduced alopecia cyclophosphamide p.d., postdepilation terminal UTP nucleotide end labeling Alopecia is one of the most distressing side-effects of many chemotherapeutic agents. In addition to the traumatizing stigma of hair loss there often is also a rather defective re-growth of abnormally structured and/or discolored hair. Although several animal studies have reported various drugs to be effective in preventing chemotherapy-induced alopecia (CIA; e.g.,Jimenez and Yunis, 1992Jimenez J.J. Yunis A.A. Protection from chemotherapy-induced alopecia by 1,25-dihydroxyvitamin D3.Cancer Res. 1992; 52: 5123-5125PubMed Google Scholar) or in promoting the re-growth of normal hair shafts (Paus et al., 1994Paus R. Handjiski B. Eichmüller S. Czarnetzki B.M. Chemotherapy-induced alopecia in mice: induction by cyclophosphamide, inhibition by cyclosporine A, and modulation by dexamethasone.Am J Pathol. 1994; 144: 719-734PubMed Google Scholar,Paus et al., 1996Paus R. Schilli M.B. Handjiski B. Menrad A. Henz B.M. Plonka P. Topical calcitriol enhances normal hair re-growth but does not prevent chemotherapy-induced alopecia in mice.Cancer Res. 1996; 56: 4438-4443PubMed Google Scholar), up to now there has been no effective and safe treatment available for this severe, psychologically devastating hair growth disorder in oncologic patients. One prerequisite for therapeutic progress in this rather poorly studied field is a more detailed characterization of the pathomechanisms by which chemotherapeutic agents damage the hair follicle, using carefully chosen animal models (Paus et al., 1994Paus R. Handjiski B. Eichmüller S. Czarnetzki B.M. Chemotherapy-induced alopecia in mice: induction by cyclophosphamide, inhibition by cyclosporine A, and modulation by dexamethasone.Am J Pathol. 1994; 144: 719-734PubMed Google Scholar,Paus et al., 1996Paus R. Schilli M.B. Handjiski B. Menrad A. Henz B.M. Plonka P. Topical calcitriol enhances normal hair re-growth but does not prevent chemotherapy-induced alopecia in mice.Cancer Res. 1996; 56: 4438-4443PubMed Google Scholar). Although the morphologic changes that occur in the hair follicle during chemotherapy were initially described decades ago (Braun-falco, 1961Braun-falco O. Klinik und Pathomechanismus der Endoxan-Alopecie als Beitrag zum Wesen cytostatischer Alopecien.Arch Klin Exp Derm. 1961; 212: 194-216Crossref Scopus (17) Google Scholar;Kostanecki and Kwiatowska, 1966Kostanecki W. Kwiatowska E. Über Wachstums-und Melanogenesestörungen der Haare bei Alopecia areata.Arch Klin Exp Derm. 1966; 226: 21-32Crossref Scopus (2) Google Scholar), little progress has been made in the basic understanding of the pathomechanisms that underlie these morphologic changes. It is now known that chemotherapeutic agents are potent inducers of apoptosis (Barry et al., 1990Barry M.A. Behnke C.A. Eastman A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia.Biochem Pharmacol. 1990; 40: 2353-2362Crossref PubMed Scopus (892) Google Scholar;Hickman, 1992Hickman J.A. Apoptosis induced by anticancer drugs.Cancer Metastasis Rev. 1992; 11: 121-139Crossref PubMed Scopus (896) Google Scholar;Dive et al., 1993Dive C. 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Evidence from both electron microscopy and the terminal UTP nucleotide end labeling (TUNEL) method suggests that the massive induction of apoptosis in the hair follicle is one of the mechanisms by which cytostatic drugs damage growing (=anagen) hair follicles (Lindner et al., 1997Lindner G. Botchkarev V.A. Botchkareva N. Ling G. van der Veen C. Paus R. Analysis of apoptosis during hair follicle regression (catagen).Am J Pathol. 1997; 155: 1601-1617Google Scholar;Searle et al., 1975Searle J. Lawson T.A. Abbott P.J. Harmon B. Kerr J.F.R. An electron-microscope study on the mode of cell death induced by cancer-chemotherapeutic agents in populations of proliferating normal and neoplastic cells.J Path. 1975; 116: 129-138Crossref PubMed Scopus (260) Google Scholar;Goldberg et al., 1990Goldberg M.T. Tackaberry L.E. Hardy M.H. Noseworthy J.H. Nuclear aberrations in hair follicle cells of patients receiving cyclophosphamide.Arch Toxicol. 1990; 64: 116-121Crossref PubMed Scopus (27) Google Scholar;Cece et al., 1996Cece R. Cazzaniga S. Morelli D. et al.Apoptosis of hair follicle cells during doxorubicin-induced alopecia in rats.Lab Invest. 1996; 75: 601-609PubMed Google Scholar). We have recently developed an instructive mouse model for the study of CIA that strikingly mimics key features of human CIA and that allows us to dissect both the response of well-defined, mature, pigmented hair follicle populations to chemotherapy-induced damage as well as their techniques to recover from this trauma. In addition, this model has proven to be very useful as a screening assay for studying candidate drugs that may be employed to manipulate CIA in vivo (Paus et al., 1994Paus R. Handjiski B. Eichmüller S. Czarnetzki B.M. Chemotherapy-induced alopecia in mice: induction by cyclophosphamide, inhibition by cyclosporine A, and modulation by dexamethasone.Am J Pathol. 1994; 144: 719-734PubMed Google Scholar,Paus et al., 1995Paus R. Schilli M.B. Plonka P. Menrad A. Reichrath J. Czarnetzki B.M. Handjiski B. Topical calcitriol, calcipotriol and KH 1060 do not prevent chemotherapy-induced alopecia in mice, but accelerate hair re-growth and re-pigmentation.J Invest Dermatol. 1995; 104: 659aGoogle Scholar,Paus et al., 1996Paus R. Schilli M.B. Handjiski B. Menrad A. Henz B.M. Plonka P. Topical calcitriol enhances normal hair re-growth but does not prevent chemotherapy-induced alopecia in mice.Cancer Res. 1996; 56: 4438-4443PubMed Google Scholar;Slominski et al., 1996Slominski A. Paus R. Plonka P. Handjiski B. Maurer M. Chakraborty A. Mihm M.C. Pharmacological disruption of hair follicle pigmentation by cyclophosphamide as a model for studying the melanocyte response to and recovery from cytostatic drug damage in situ.J Invest Dermatol. 1996; 106: 1203-1211Crossref PubMed Scopus (55) Google Scholar). Using this mouse model, our objective in this study was to confirm whether the chemotherapeutic agent cyclophosphamide (CYP) induces apoptosis in anagen hair follicles in vivo. More importantly, we wished to explore whether topically applied calcitriol-analogs, which do not prevent CIA but promote re-growth of normally pigmented hair in mice (Paus et al., 1995Paus R. Schilli M.B. Plonka P. Menrad A. Reichrath J. Czarnetzki B.M. Handjiski B. Topical calcitriol, calcipotriol and KH 1060 do not prevent chemotherapy-induced alopecia in mice, but accelerate hair re-growth and re-pigmentation.J Invest Dermatol. 1995; 104: 659aGoogle Scholar,Paus et al., 1996Paus R. Schilli M.B. Handjiski B. Menrad A. Henz B.M. Plonka P. Topical calcitriol enhances normal hair re-growth but does not prevent chemotherapy-induced alopecia in mice.Cancer Res. 1996; 56: 4438-4443PubMed Google Scholar), modulate CIA-related apoptosis in the hair follicle. Previous reports had suggested that calcitriols may either induce (Simboli-campbell et al., 1995Simboli-campbell M. Welsh J. 1,25–dihydroxyvitamin D3: a coordinate regulator of active cell death and proliferation in MCF–7 breast cancer cells.in: Tenniswood M. Michna H. Apoptosis in Hormone–Dependent Cancers. Springer, Berlin1995: 181-200Google Scholar) or inhibit apoptosis (Xu et al., 1993Xu H.-M. Tepper C.G. Jones J.B. Fernandez C.E. Studzinski G.P. 1,25 dihydroxyvitamin D3 protects HL-60 cells against apoptosis but downregulates the expression of the bcl-2 gene.Exp Cell Res. 1993; 209: 367-374Crossref PubMed Scopus (89) Google Scholar) in vitro, depending on the cell type studied. In a series of preparatory analyses of murine skin we had observed that most of the commercially available cell death detection kits using the TUNEL method (Gavrieli et al., 1992Gavrieli Y. Sherman Y. Ben-sasson S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (8978) Google Scholar) seem to be customized for cell culture and human tissue applications, because they resulted in a high background staining and unsatisfactory reproducibility when murine skin was studied. Therefore, we have modified and combined two previously described methods (Gavrieli et al., 1992Gavrieli Y. Sherman Y. Ben-sasson S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (8978) Google Scholar;Sträter et al., 1995Sträter J. Günthert A.R. Brüderlein S. Möller P. Microwave irradiation of paraffin-embedded tissue sensitizes the TUNEL method for in situ detection of apoptotic cells.Histochemistry. 1995; 103: 157-160Crossref PubMed Scopus (64) Google Scholar) to improve the sensitivity, specificity, and reproducibility of TUNEL stains in murine tissue. In addition, the usefulness of a very simple staining method for the histologic detection of apoptotic cells for screening purposes was explored, using the DNA-intercalating dye Hoechst 33342. 1,25-dihydroxyvitamin D3, calcipotriol, and KH 1060 (dissolved in absolute ethanol + 5% isopropylmyristate vol/vol) were provided by Leo Pharmaceuticals (Ballerup, Denmark). Cyclophosphamide (Endoxan 200) was obtained from Asta Medica (Frankfurt, Germany). Biotin-16-dUTP was obtained from Boehringer (Mannheim, Germany); terminal deoxynucleotydil transferase was obtained from Promega (Madison, WI); alkaline phosphatase-conjugated streptavidin was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA); and Hoechst 33342-dye, Fast Red Naphthol AS Mix tablets, and BCIP/NBT tablets were obtained from Sigma (Deisenhofen, Germany). The DNA-extraction kit was obtained from Stratagene (Heidelberg, Germany) and the GeneScreenPlus-membrane from DuPont (Cambridge, MA). Female C57 BL/6 mice, 6–8 wk old, were purchased from Charles River (Sulzfeld/Germany). Animals were kept on a 12 h light/dark cycle and were fed with mousechow and water ad libitum. To induce a perfectly synchronized anagen development over the whole back skin area, the hair shafts of the back fur coat of mice with all follicles in telogen were depilated using a warm resin-beeswax mixture as described before (Paus et al., 1990Paus R. Stenn K.S. Link R.E. Telogen skin contains an inhibitor of hair growth.Br J Dermatol. 1990; 122: 777-784Crossref PubMed Scopus (208) Google Scholar,Paus et al., 1994Paus R. Handjiski B. Eichmüller S. Czarnetzki B.M. Chemotherapy-induced alopecia in mice: induction by cyclophosphamide, inhibition by cyclosporine A, and modulation by dexamethasone.Am J Pathol. 1994; 144: 719-734PubMed Google Scholar). On day 5 post-depilation (p.d.), when all hair follicles were in anagen III–IV of the hair cycle (Paus et al., 1990Paus R. Stenn K.S. Link R.E. Telogen skin contains an inhibitor of hair growth.Br J Dermatol. 1990; 122: 777-784Crossref PubMed Scopus (208) Google Scholar), the back skin area was topically treated with either 0.2 μg 1,25-dihydroxyvitamin D3, 2.0 μg calcipotriol, 0.02 μg KH 1060 (dissolved in 100 μl ethanol), or vehicle alone. These doses had previously been established to effectively promote hair re-growth after CIA (Paus et al., 1995Paus R. Schilli M.B. Plonka P. Menrad A. Reichrath J. Czarnetzki B.M. Handjiski B. Topical calcitriol, calcipotriol and KH 1060 do not prevent chemotherapy-induced alopecia in mice, but accelerate hair re-growth and re-pigmentation.J Invest Dermatol. 1995; 104: 659aGoogle Scholar,Paus et al., 1996Paus R. Schilli M.B. Handjiski B. Menrad A. Henz B.M. Plonka P. Topical calcitriol enhances normal hair re-growth but does not prevent chemotherapy-induced alopecia in mice.Cancer Res. 1996; 56: 4438-4443PubMed Google Scholar). Topical treatment was applied once daily for the next four consecutive days. On the last day of treatment (i.e., day 9 p.d.), all test and control mice received 150 mg CYP i.p. per kg as a single dose to induce alopecia (Paus et al., 1994Paus R. Handjiski B. Eichmüller S. Czarnetzki B.M. Chemotherapy-induced alopecia in mice: induction by cyclophosphamide, inhibition by cyclosporine A, and modulation by dexamethasone.Am J Pathol. 1994; 144: 719-734PubMed Google Scholar). An additional control group received only vehicle (topically and intraperitoneally). A total of at least 15 mice per group was studied in 2–3 separate experiments. All experiments yielded the expected macroscopic and microscopic findings previously reported. Animals were sacrificed on day 10 and 14 p.d. (10 animals per group and day). Blood and skin samples were immediately harvested. Punch biopsies of treated skin areas were snap-frozen in liquid nitrogen for DNA extraction. For histology, the skin was fixed in Lillie's formalin for 72 h, dehydrated, and then paraffin-embedded. Paraffin sections (4 μm) of the skin were deparaffinized and rehydrated, then digested with proteinase K (20 μg per ml, Amresco, Solon, OH) for 10 min at room temperature followed by RNAse-digestion (2 μl per ml in Tris-buffered saline; RNAse-it from Stratagene) for 15 min at 37°C. Then the slides were incubated with UTP (40 μM stock diluted to 2 nM; Boehringer, Mannheim, Germany) for 15 min at 37°C. After preincubation with terminal deoxynucleotidyl transferase-buffer for 15 min at room temperature, the sections were incubated with a mixture containing biotin-conjugated dUTP (biotin-16-dUTP, 1.7 μl per 100 μl) and terminal deoxynucleotidyl transferase (10 U per 100 μl) for 2 h at 37°C. The reaction was terminated by rinsing the slides in TB-buffer (300 mM NaCl, 30 mM sodium citrate) for 15 min at room temperature. After washing in Tris-buffered saline the sections were incubated with alkaline-phosphatase streptavidin 1:200 in Tris-buffered saline for 30 min at 37°C. Labeled nuclei were visualized by Fast Red dye. Sections were evaluated anonymously (i.e., in ignorance of their group assignment) and in randomized sequence by light microscopy. The combined treatment of the sections with RNAse, excess-UTP, and TB-Buffer constituted significant changes in this TUNEL protocol substantially improved the staining results on murine skin, compared with the original TUNEL method (Gavrieli et al., 1992Gavrieli Y. Sherman Y. Ben-sasson S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (8978) Google Scholar) and a microwave-based method (Sträter et al., 1995Sträter J. Günthert A.R. Brüderlein S. Möller P. Microwave irradiation of paraffin-embedded tissue sensitizes the TUNEL method for in situ detection of apoptotic cells.Histochemistry. 1995; 103: 157-160Crossref PubMed Scopus (64) Google Scholar). This modified TUNEL protocol provides a sensitive, specific, and well reproducible light microscopic alternative for the histologic detection of apoptotic cells to available fluorescent detection techniques with their more limited visualization of tissue architecture (Lindner et al., 1997Lindner G. Botchkarev V.A. Botchkareva N. Ling G. van der Veen C. Paus R. Analysis of apoptosis during hair follicle regression (catagen).Am J Pathol. 1997; 155: 1601-1617Google Scholar). This method was adapted for histology from previously reported fluorescence-activated cell sorter methods (Chrest et al., 1993Chrest F.J. Buchholz M.A. Kim Y.H. Kwon T.-K. Nordin A.A. Identification and quantification of apoptotic cells following anti-CD3 activation of murine G0 T cells.Cytometry. 1993; 14: 883-890Crossref PubMed Scopus (30) Google Scholar). Paraffin sections (4 μm) of the skin were deparaffinized in xylene and ethanol. After rehydration in phosphate-buffered saline, the tissue sections were incubated with a 10 μg per ml solution of the fluorescent Hoechst 33342 dye in phosphate-buffered saline for 1 h at room temperature in the dark. After washing in tap water, the slides were mounted and evaluated by fluorescence microscopy at 510 nm. Due to the simplicity and cost efficiency of this technique, we wished to explore whether it can serve as a fast method for high-throughput screening of tissue sections for differences in apoptotic cells between test and control groups. For the morphometric analysis of the skin sections, defined, standardized back skin areas of test and control mice were examined and compared with each other. From each animal, at least 20 follicles were evaluated by light- and fluorescence microscopy at 400× magnification using a Zeiss microscope. For the TUNEL analysis only small, condensed, and thus intensively red stained nuclei were considered to represent apoptotic nuclei. This rigid cut-off criterion was selected in order to minimize the risk of false positive counts (e.g., due to necrosis or background staining). Because the Hoechst 33342 dye nonspecifically intercalates with the DNA and thus marks all DNA-containing cells, likewise in the TUNEL analysis, only small and intensively labeled nuclei and apoptotic bodies (in comparison with surrounding cells) were counted as reliable indicators for apoptosis. Statistical evaluation was performed on at least 20 follicles per animal, and the mean for this animal was calculated. Subsequently, the mean (±SEM) of all animals from one group and day was calculated; p values were calculated by using the Mann–Whitney U Wilcoxon test. Total DNA was isolated from snap-frozen skin punches (8 mm) of histology-corresponding back skin areas, using a commercially available DNA-extraction kit. DNA fragments were separated by agarose gel electrophoresis (2% agarose) and visualized by staining with ethidium bromide (Cotter and Martin, 1996Cotter T.G. Martin S.J. Techniques in Apoptosis – a User's Guide. London, Portland Press1996Google Scholar). Due to the relatively small number of apoptotic nuclei compared with the vast majority of nonapoptotic cells in full-thickness mouse skin, as demonstrated by histology (i.e., TUNEL and Hoechst stain), there was no distinct fragmentation (i.e., laddering) detectable on the gel, following this routine protocol. To enhance the detection of fragmented DNA, we therefore combined and modified two highly sensitive, but radioactive methods (Huang and Plunkett, 1992Huang P. Plunkett W. A quantitative assay for fragmented DNA in apoptotic cells.Anal Biochem. 1992; 207: 163-167Crossref PubMed Scopus (62) Google Scholar;Tilly and Hsueh, 1993Tilly J.L. Hsueh A.J.W. Microscale autoradiographic method for the qualitative and quantitative analysis of apoptotic DNA fragmentation.J Cell Pathol. 1993; 154: 519-526Google Scholar) to generate a nonradioactive method, based on 3′-end-labeling. Like in the TUNEL stain, the isolated DNA was incubated with biotin-16-dUTP and terminal deoxynucleotidyl transferase for 2 h at 37°C. The reaction was terminated by heating to 70°C for 10 min. DNA was precipitated by ethanol overnight at –20°C and a gel electrophoresis was performed as described above. The DNA was then blotted by vacuum (25 mmHg, 2 h) on a GeneScreenPlus membrane and air-dried overnight. After blocking for 1 h in phosphate-buffered saline with 0.5% Tween 20 and 2% bovine serum albumin, the membrane was incubated for 1 h with alkaline-phosphatase streptavidin 1:2000 in phosphate-buffered saline with 0.05% Tween 20 and 2% bovine serum albumin. After a washing step, the labeled DNA was visualized using BCIP/NBT as substrate. Densitometry was performed on the negative film of the photographed membrane using the Personal Densitometer SE and ImageQuaNT software (both from Molecular Dynamics, Sunny Vale, CA). The serum calcium level for each individual animal was obtained by flame-photometry (Keller, 1986Keller H. Klinisch-chemische Labordiagnostik für die Praxis. Stuttgart, G. Thieme-Verlag1986Google Scholar), and was found to remain unaltered by the application of calcitriols throughout the course of the experiments (not shown). To confirm that CYP induces apoptosis in murine anagen hair follicles, skin sections of CYP-treated mice were stained by using a modification of the biochemical in situ end labeling technique (TUNEL), specifically developed for light-microscopic evaluation of apoptosis in murine skin sections (see Materials and Methods). Compared with routine TUNEL techniques (Gavrieli et al., 1992Gavrieli Y. Sherman Y. Ben-sasson S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.J Cell Biol. 1992; 119: 493-501Crossref PubMed Scopus (8978) Google Scholar;Sträter et al., 1995Sträter J. Günthert A.R. Brüderlein S. Möller P. Microwave irradiation of paraffin-embedded tissue sensitizes the TUNEL method for in situ detection of apoptotic cells.Histochemistry. 1995; 103: 157-160Crossref PubMed Scopus (64) Google Scholar), the sections were digested with RNAse to reduce background staining. Another step to quench background staining was to saturate unspecific UTP-binding sites in murine skin by providing excess UTP to the sections prior to applying the actual terminal deoxynucleotidyl transferase/biotin-16-dUTP mixture. The TB-buffer was applied to terminate the labeling reaction. These three steps resulted in substantially more specific and highly reproducible staining results for apoptotic cells in murine skin, and can serve as a convenient alternative to fluorescent apoptosis detection techniques with their poor demarcation of tissue architecture (Lindner et al., 1997Lindner G. Botchkarev V.A. Botchkareva N. Ling G. van der Veen C. Paus R. Analysis of apoptosis during hair follicle regression (catagen).Am J Pathol. 1997; 155: 1601-1617Google Scholar). This technique revealed the induction of massive keratinocyte apoptosis by CYP in all anagen hair follicles, with most apoptotic cells located in the hair bulb, as reflected by the amount of stained apoptotic nuclei (Figure 1, 2). Only a few nuclei were stained in the epidermis and interfollicular dermis of treated animals. The maximum of intrafollicular apoptosis was seen on day 10 p.d., i.e., 24 h after CYP administration, with hair follicles being in the anagen VI-phase of the hair cycle. Importantly, only sections from an identical back skin region of test and control mice were compared, and apoptosis was compared only between hair follicles of identical hair cycle stages. Vehicle-treated control mice revealed no TUNEL-positive nuclei in the bulb on both investigated days.Figure 2Statistical analysis of TUNEL morphometry. The morphometry data (see Materials and Methods) were pooled from n = 10 animals per group and day, representing the amount of apoptotic nuclei per follicle as determined by evaluating 200 follicles per group and day. (A) Day 10, (B) day 14. Means ± SEM are given, the level of significance compared with the CYP group (only vehicle as topical treatment) is indicated as ***p < 0.005.View Large Image Figure ViewerDownload (PPT) These data were confirmed histochemically by staining of apoptotic bodies with Hoechst 33342 dye (Figure 3, 4) as well as biochemically by DNA-gel electrophoresis and blotting (Figure 5). A total of 20 μg ethidium bromide-labeled DNA per lane isolated from full-thickness back skin with its predominance of nonapoptotic cells failed to demonstrate "laddering" in a regular agarose gel electrophoresis, yet attested to the absence of loading error (not shown). Therefore, a more sensitive detection method was applied by blotting of DNA, which was end-labeled with biotin-16-dUTP. This clearly displayed a distinct fragmentation of the DNA in the skin of CYP-treated mice, which was absent in vehicle-treated control skin (Figure 5). As positive controls, DNA from HL-60 cells was used, which had been treated with 1 μM of camptothecin for 4 h to induce apoptosis (Li et al., 1994Li X. Traganos F. Darzynkiewicz Z. Simultaneous analysis of DNA replication and apoptosis during treatment of HL-60 cell with camptothecin and hyperthermia and mitogen stimulation of human lymphocytes.Cancer Res. 1994; 54: 4289-4293PubMed Google Scholar).Figure 4Statistical analysis of Hoechst 33342 morphometry. The morphometry data were pooled from n = 10 animals per group and day representing the amount of apoptotic nuclei in 200 follicles per group and day. (A) Day 10, (B) day 14. Means ± SEM are given, the level of significance compared with the vehicle group is indicated as ***p < 0.005.View Large Image Figure ViewerDownload (PPT) As reported before (Paus et al., 1995Paus R. Schilli M.B. Plonka P. Menrad A. Reichrath J. Czarnetzki B.M. Handjiski B. Topical calcitriol, calcipotriol and KH 1060 do not prevent chemotherapy-induced alopecia in mice, but accelerate hair re-growth and re-pigmentation.J Invest Dermatol. 1995; 104: 659aGoogle Scholar,Paus et al., 1996Paus R. Schilli M.B. Handjiski B. Menrad A. Henz B.M. Plonka P. Topical calcitriol enhances normal hair re-growth but does not prevent chemotherapy-induced alopecia in mice.Cancer Res. 1996; 56: 4438-4443PubMed Google Scholar), topical pretreatment with calcitrio
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