[CANCER RESEARCH 60, 5002–5006, September 15, 2000] Advances in Brief p53 Is Essential for Chemotherapy-induced Hair Loss 1 Vladimir A. Botchkarev, 2 Elena A. Komarova, Frank Siebenhaar, Natalia V. Botchkareva, Pavel G. Komarov, Marcus Maurer, Barbara A. Gilchrest, and Andrei V. Gudkov 2 Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts 02118 [V. A. B., F. S., N. V. B., B. A. G.]; Department of Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607 [E. A. K., P. G. K., A. V. G.]; Department of Dermatology, Johannes Gutenberg University, D-55101 Mainz, Germany [F. S., M. M.]; and Quark Biotech, Inc., Pleasanton, California 94566 [P. G. K.] Abstract Anticancer drugs stimulate apoptosis in the hair follicles (HF) and cause hair loss, the most common side effect of chemotherapy. In a mouse model for chemotherapy-induced hair loss, we demonstrate that p53 is essential for this process: in contrast to wild-type mice, p53-deficient mice show neither hair loss nor apoptosis in the HF keratinocytes that main- tained active proliferation after cyclophosphamide treatment. HF in p53 mutants are characterized by down-regulation of Fas and insulin-like growth factor-binding protein 3 and by increased expression of Bcl-2. These observations indicate that local pharmacological inhibition of p53 may be useful to prevent chemotherapy-associated hair loss. Introduction Cancer treatment with chemotherapeutic agents is associated with severe side effects due to the occurrence of apoptosis in several sensitive tissues (such as the hematopoietic system or epithelia of digestive tract) as a result of drug cytotoxicity (1). This apoptosis largely depends on p53, a key mediator of cellular mechanism of stress response (2). p53 accumulation in sensitive cells after a variety of stresses results in growth arrest at one of the cellular checkpoints or induction of programmed cell death (3). p53 acts as a nuclear tran- scription factor altering expression of multiple p53-responsive genes, the activity of which is, at least in part, responsible for cell reaction to stress (2, 3). The major role of p53 in mediating the side effects of cancer treatment was confirmed by the isolation and application of a chemical inhibitor of p53 that reduced the toxicity of cancer treatment in experimental animals by temporary reversible sup- pression of p53 (4). Temporary hair loss (alopecia) is a common side effect of chemo- therapy. HF 3 are “hair shaft-producing miniorgans” showing during postnatal life a unique pattern of cyclic activity with periods of relative resting (telogen), active growth (anagen), and apoptosis- driven involution (catagen) (5). HF are strongly affected by many chemotherapeutic agents because of the rapid proliferative rate of hair matrix keratinocytes during anagen. In the mouse model of chemo- therapy-induced hair loss, the active hair growth phase was first induced by depilation, and cyclophosphamide administration during new anagen phase causes complete alopecia imitating changes seen in human chemotherapy-induced hair loss (6, 7). The drug treatment induces dystrophic changes in growing HF and, in more severely damaged follicles, premature regression as a result of massive apo- ptosis in the entire proximal hair bulb, with subsequent hair shedding (6, 7). Apoptosis of hematopoietic cells and cells of the digestive tract associated with cancer treatment is known to be p53 dependent (2– 4, 8). Radiation- or chemotherapy-induced DNA damage leads to the rapid accumulation of p53 protein in the susceptible cells (3, 9), followed by up-regulation of Fas, IGF-BP3, and Bax, encoded by the corresponding p53-responsive genes (10 –12). Moreover, it was dem- onstrated that Fas and Bax are up-regulated in the HF during cyclo- phosphamide treatment (13) and that p53 is involved in the ionizing radiation-induced apoptosis in the HF (14). To explore the role of p53 in the hair loss induced by chemother- apeutic agents, we used a mouse model for chemotherapy-induced hair loss: cyclophosphamide treatment of C57BL/6 mice after hair cycle synchronization in anagen by depilation (6, 7, 13). We analyzed the expression of p53 in the HF of drug-treated wild-type mice and then compared the dynamics of HF of wild-type versus p53-deficient mice after cyclophosphamide treatment. We demonstrate here that p53 is indeed essential for the development of chemotherapy-induced hair loss in mice. Materials and Methods Animal Models and Tissue Collection. Female 8-week-old C57BL/6 mice (n = 20), 8- to 10-week-old p53 knockout (n = 25), and wild-type mice (n = 25) were purchased from Charles River (Boston, MA) and The Jackson Laboratory (Bar Harbor, ME). p53 knockout mice generated on C57BL/6 background were viable, showed apparently normal fur, and developed spon- taneous tumors not earlier than 12 weeks after birth (15), i.e., after the end of experiment. Mice were housed in community cages at the animal facilities of the Boston University School of Medicine and University of Illinois at Chi- cago. All mice were fed water and murine chow ad libitum and were kept under 12-h light/dark cycles. Active hair growth (anagen) was induced in the back skin by application of a wax-rosin mixture with subsequent depilation, as described before (6, 7, 13). On day 9 after hair cycle induction (at anagen VI stage of the hair cycle), a single i.p. injection of 150 mg/kg cyclophosphamide (Endoxan; Bristol Meyers Squibb, Princeton, NJ) or PBS (vehicle control) was given as described previously (6, 7, 13). Skin samples were harvested at days 1, 3, 5, 7, 9, and 11 after cyclophosphamide administration (at days 10, 12, 14, 16, 18, and 20 postdepilation, respectively). Harvesting of skin and cryosectioning were per- formed by a special technique to obtain longitudinal sections of the HF, as described previously (6, 7, 13). Immunohistochemistry and TUNEL Technique. Expression of p53 pro- tein, p55 TNF receptor, and IGF-BP3 was assessed with the use of rabbit polyclonal antiserum against murine p53 (Novocastra, Newcastle upon Tyne, United Kingdom), rat monoclonal antibody against murine p55 TNF receptor (PharMingen, San Diego, CA), and rabbit polyclonal antiserum against IGF- BP3 (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. Tyramide amplification was used for the visualization of these antigens, as described previously (16, 17). Double immunovisualization of p53 and TUNEL or proliferative marker Ki-67 and TUNEL was performed, as described previ- Received 5/31/00; accepted 8/3/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Grant IRG-72-001-26-IRG from the American Cancer Society to V. A. B., by grants from the National Institutes of Health (CA75179) and Quark Biotech, Inc., to A. V. G., and by a grant from MAIFOR (to M. M.). 2 To whom requests for reprints should be addressed, at Department of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, MA 02118. Phone: 617-638-5548; Fax: 617-638-5515; E-mail: vladbotc@bu.edu [V. A. B.] or Department of Molecular Genetics, University of Illinois at Chicago, 60607-7170. Phone: 312-413-0349; Fax: 312-996-0683; E-mail: gudov@uic.edu [A. V. G.]. 3 The abbreviations used are: HF, hair follicle(s); IGF-BP3, insulin-like growth factor- binding protein 3; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling. 5002 Research. on November 3, 2021. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from