[CANCER RESEARCH 60, 5002–5006, September 15, 2000]

Advances in Brief

p53 Is Essential for Chemotherapy-induced Hair Loss1

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) andcause hair loss, the most common side effect of chemotherapy. In a mousemodel for chemotherapy-induced hair loss, we demonstrate that p53 isessential for this process: in contrast to wild-type mice, p53-deficient miceshow neither hair loss nor apoptosis in the HF keratinocytes that main-tained active proliferation after cyclophosphamide treatment. HF in p53mutants are characterized by down-regulation of Fas and insulin-likegrowth factor-binding protein 3 and by increased expression of Bcl-2.These observations indicate that local pharmacological inhibition of p53may be useful to prevent chemotherapy-associated hair loss.

Introduction

Cancer treatment with chemotherapeutic agents is associated withsevere side effects due to the occurrence of apoptosis in severalsensitive tissues (such as the hematopoietic system or epithelia ofdigestive tract) as a result of drug cytotoxicity (1). This apoptosislargely depends on p53, a key mediator of cellular mechanism ofstress response (2). p53 accumulation in sensitive cells after a varietyof stresses results in growth arrest at one of the cellular checkpoints orinduction 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 tostress (2, 3). The major role of p53 in mediating the side effects ofcancer treatment was confirmed by the isolation and application ofa chemical inhibitor of p53 that reduced the toxicity of cancertreatment in experimental animals by temporary reversible sup-pression of p53 (4).

Temporary hair loss (alopecia) is a common side effect of chemo-therapy. HF3 are “hair shaft-producing miniorgans” showing duringpostnatal life a unique pattern of cyclic activity with periods ofrelative resting (telogen), active growth (anagen), and apoptosis-driven involution (catagen) (5). HF are strongly affected by manychemotherapeutic agents because of the rapid proliferative rate of hairmatrix keratinocytes during anagen. In the mouse model of chemo-therapy-induced hair loss, the active hair growth phase was firstinduced by depilation, and cyclophosphamide administration duringnew anagen phase causes complete alopecia imitating changes seen inhuman chemotherapy-induced hair loss (6, 7). The drug treatment

induces dystrophic changes in growing HF and, in more severelydamaged 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 tractassociated with cancer treatment is known to be p53 dependent (2–4,8). Radiation- or chemotherapy-induced DNA damage leads to therapid accumulation of p53 protein in the susceptible cells (3, 9),followed by up-regulation of Fas, IGF-BP3, and Bax, encoded by thecorresponding 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 ionizingradiation-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-inducedhair loss: cyclophosphamide treatment of C57BL/6 mice after haircycle synchronization in anagen by depilation (6, 7, 13). We analyzedthe expression of p53 in the HF of drug-treated wild-type mice andthen compared the dynamics of HF of wild-typeversusp53-deficientmice after cyclophosphamide treatment. We demonstrate here thatp53 is indeed essential for the development of chemotherapy-inducedhair loss in mice.

Materials and Methods

Animal Models and Tissue Collection.Female 8-week-old C57BL/6mice (n5 20), 8- to 10-week-old p53 knockout (n5 25), and wild-type mice(n 5 25) were purchased from Charles River (Boston, MA) and The JacksonLaboratory (Bar Harbor, ME). p53 knockout mice generated on C57BL/6background were viable, showed apparently normal fur, and developed spon-taneous tumors not earlier than 12 weeks after birth (15),i.e., after the end ofexperiment. Mice were housed in community cages at the animal facilities ofthe Boston University School of Medicine and University of Illinois at Chi-cago. All mice were fed water and murine chowad libitum and were keptunder 12-h light/dark cycles.

Active hair growth (anagen) was induced in the back skin by application ofa 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), asingle i.p. injection of 150 mg/kg cyclophosphamide (Endoxan; Bristol MeyersSquibb, Princeton, NJ) or PBS (vehicle control) was given as describedpreviously (6, 7, 13). Skin samples were harvested at days 1, 3, 5, 7, 9, and 11after cyclophosphamide administration (at days 10, 12, 14, 16, 18, and 20postdepilation, respectively). Harvesting of skin and cryosectioning were per-formed by a special technique to obtain longitudinal sections of the HF, asdescribed 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 rabbitpolyclonal 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. Tyramideamplification was used for the visualization of these antigens, as describedpreviously (16, 17). Double immunovisualization of p53 and TUNEL orproliferative 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 markedadvertisementin accordance with18 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 CancerSociety to V. A. B., by grants from the National Institutes of Health (CA75179) and QuarkBiotech, 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 ofMolecular 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 endlabeling.

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ously (18). For immunovisualization of Ki-67, a rabbit polyclonal antiserumagainst murine Ki-67 was obtained from Dianova (Hamburg, Germany).

Immunodetection of Fas, p75 neurotrophin receptor, Bax, and Bcl-2 wasperformed with corresponding monoclonal antibodies and the avidin-biotin-alkaline phosphatase method, as described previously (13, 17). Histoenzymaticdetection of the endogenous alkaline phosphatase was performed for theprecise identification of dermal papilla morphology in HF of the distinct stagesof apoptosis-driven involution (catagen) (6, 7, 13, 16, 17).

Results and Discussion

p53 Is Up-Regulated in Hair Follicles after CyclophosphamideTreatment. To determine the p53 involvement in the pathogenesis ofchemotherapy-induced hair loss, we first compared the expression ofp53 in actively growing HF in the back skin of vehicle-treated andcyclophosphamide-treated C57BL/6 mice. Active hair growth (ana-gen) was induced by depilation as described before (6, 7, 13), andcyclophosphamide was administered 9 days postdepilation,i.e.,at thetime point when all HF have reached the stage of active hair shaftproduction (anagen VI).

Twenty-four hours after cyclophosphamide administration (10 dayspostdepilation), HF treated by vehicle control showed a relativelyweak expression of p53 in the proximal outer root sheath and hairmatrix, associated with absence of TUNEL-positive cells (Fig. 1A). Incontrast, cyclophosphamide-treated HF displayed a strong up-regula-tion of p53 in the proximal outer and inner root sheaths and hairmatrix (Fig. 1B). In the HF after cyclophosphamide treatment, manyTUNEL-positive apoptotic cells located in the HF matrix were alsop53 positive (Fig. 1B).

In line with previous reports (6, 7, 13), 3 days after cyclophos-phamide administration (12 days post depilation), HF showed a short-ening of their length caused by the massive apoptosis in the proximalhair bulb. This was associated with prominent p53 expression andcolocalization of p53 and TUNEL in the regressing outer and innerroot sheaths (Fig. 1C). At day 7 after cyclophosphamide administra-tion (day 16 postdepilation), all cyclophosphamide-treated animalsshowed massive hair loss in the entire back (not shown), and 2 dayslater the process of cyclophosphamide-driven HF regression was

Fig. 1. Up-regulation of p53 and TUNEL in the cyclophosphamide-treated hair follicles of C57BL/6 mice. The hair cycle was induced in the back skin of 8-week-old C57BL/6 miceby depilation, and cyclophosphamide was administered 9 days after. Skin was harvested at the indicated time points after cyclophosphamide administration, and cryosections (8mmthick) were processed for double immunovisualization of p53 and TUNEL. Cell nuclei were visualized by TO-PRO-3 staining.A, control (day 10 postdepilation). Weak expressionof p53 (red fluorescence) in the HF matrix (arrows) and outer root sheath (ORS; arrowheads);B, 1 day after cyclophosphamide administration (day 10 postdepilation). Prominentexpression of p53 (red fluorescence) and colocalization of p53 and TUNEL (yellow fluorescence) in the HF matrix (small arrows). Prominent expression of p53 alone in the outer rootsheath (large arrowheads) and inner root sheath (small arrowheads). Numerous TUNEL-positive cells are visible in the hair matrix (green fluorescence) and in the hair shaft (largearrow); C, 3 days after cyclophosphamide administration (day 12 postdepilation). Prominent expression of p53 (red fluorescence) and colocalization with TUNEL (yellow-orangefluorescence) in the regressing outer root sheath (large arrowheads) and inner root sheaths (arrow). Numerous TUNEL-positive cells (green fluorescence,small arrowheads) are alsovisible in the inner root sheath;D, 9 days after cyclophosphamide administration (day 18 postdepilation). Shortening of HF length is completed and is associated with absence of TUNELin the HF epithelium, whereas relatively weak expression of p53 is present in the HF connective tissue sheath (arrow).DP, dermal papilla;HM, hair matrix;HS,hair shaft;IRS,innerroot sheath;Mel, melanogenic area.ORS, outer root sheath.Bars,100 mm.

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Fig. 2. p53 knockout mice treated by cyclophosphamide do not display hair loss. Hair cycle was induced in the back skin of 8-week-old wild-type (n5 25) and p53-null mice (n5 25)by depilation, and cyclophosphamide was administered 9 days after. Skin was harvested at day 7 after cyclophosphamide administration (day 16 postdepilation), and cryosections (8mm thick) were processed for the histoenzymatic detection of alkaline phosphatase (B andC) and double immunovisualization of proliferative marker Ki-67 and TUNEL (D andE).Cell nuclei inD andE were visualized by TO-PRO-3 staining.A, severe hair loss over the entire back of the wild-type mice treated by cyclophosphamide, with no hair loss visiblein the p53 knockout mice.B, shortening of the length and reduction in the volume of proximal hair bulb in the wild-type HF (arrows) is associated with dramatic decrease of skinthickness after cyclophosphamide treatment.C, large volume of the proximal hair bulb (arrows) and dermal papilla (red color) in the p53 null HF after cyclophosphamide treatment.D, numerous TUNEL-positive cells (green fluorescence,arrows) and single Ki-67-positive cells (red fluorescence,arrowhead) in the regressing HF compartments of wild-type skin.E, numerous Ki-67-positive cells in the HF matrix (red fluorescence,arrowheads) and single TUNEL-positive granules in the precortical zone (arrow). Some TUNEL-positive cellsare also visible in the subcutis around HF (greenfluorescence).Bars,100 mm.

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almost complete. At this time, no p53 immunoreactivity or TUNELpositivity were seen in the HF (Fig. 1D).

p53 Knockout Mice Do Not Show Cyclophosphamide-inducedHair Loss. To test the role for p53 in the control of cyclophospha-mide-induced apoptosis in the HF and hair loss, we compared theeffects of cyclophosphamide on actively growing HF of p53 knockoutversuswild-type mice. Animals were treated by cyclophosphamide 9days postdepilation as described previously (6, 7, 13). Seven daysafter cyclophosphamide treatment (16 days postdepilation), wild-typemice showed extensive hair loss over the entire back associated withdramatic shortening of HF length and massive apoptosis in the re-gressing HF compartments (Fig. 2,A, B, andD).

However, no hair loss was seen in the cyclophosphamide-treatedp53 knockout mice up to 14 days after cyclophosphamide adminis-tration. In particular, 7 days after cyclophosphamide treatment (16days postdepilation), all HF in p53-null mice were in anagen VI stage.These HF were characterized by a large volume of the proximal hairbulb and dermal papilla, by active keratinocyte proliferation in thehair matrix, and by single TUNEL-positive granules in the melano-genic area above dermal papilla (Fig. 2,A, C, and E). Therefore,anagen HF in p53 knockout mice kept a high rate of keratinocyteproliferation in the hair matrix and active hair shaft production, didnot show intrafollicular apoptosis, and, therefore were resistant to thecyclophosphamide treatment. Most importantly, at days 9–11 aftercyclophosphamide administration (18–20 days postdepilation), all HFin the p53 knockout mice showed apparently normal patterns ofphysiological transformation from the active growth to the regressionand then to the resting phase (not shown).

Hair Follicles in p53-deficient Mice Are Characterized byDown-Regulation of Fas and IGBP3, and by Increased Expressionof Bcl-2. To identify factors that might contribute to p53-dependentcyclophosphamide-induced hair loss, we compared the expression ofnumber of molecules (Fas, p55 kDa TNF receptor, p75 kDa neuro-trophin receptor, Bax, Bcl-2, IGF-BP3) implicated in the controlof cyclophosphamide-induced apoptotic cell death in the HF (5,13). Several of the proteins analyzed (Fas, Bax, Bcl-2, IGF-BP3)are encoded by genes, the transcription of which is regulated byp53 during DNA damage induced by chemotherapeutic agents(10 –12, 19).

We found that, in contrast to the wild-type HF treated by cyclo-phosphamide, p53-null HF showed strongly reduced expression of Fasin the proximal outer and inner root sheaths (Fig. 3,A andB). Thisobservation is consistent with the reported involvement of p53 inintracellular trafficking of Fas receptor (10). Interestingly, immuno-reactivity for p55 kDa TNF receptor was also reduced in the proximalouter root sheath of the HF in p53 knockout mice, compared withwild-type animals (Fig. 3,C andD). However, the expression patternsof another member of “death domain” containing growth factor re-ceptors, namely p75 neurotrophin receptor, showed no differencesbetween wild-type and p53-null HF treated by cyclophosphamide(Fig. 3, E andF).

Furthermore, the immunoreactivity for IGF-BP3 was strongly re-duced in the p53-deficient HF, compared with wild-type HF (Fig. 3,G andH). Bax expression was relatively unchanged in p53-null HF,whereas Bcl-2 was highly expressed in the HF dermal papilla of 53knockout mice, compared with that of wild-type mice (Fig. 3,I–L).

Taken together, our data suggest that p53 is essential for triggeringapoptotic cell death in the HF that is induced by cyclophosphamide inmice. This puts HF in the same category of other organs sensitive togenotoxic stress, such as the hematopoietic system or epithelia of thedigestive tract, which respond to genotoxic stress by p53-dependentapoptosis, thus limiting the tolerable doses of anticancer drugs orradiation (2, 3, 8). Considering the similarity of chemotherapy-induced alterations in HF of humans and mice (6), we presume thatp53 plays a similar role in hair loss occurring in cancer patients duringchemotherapy.

As is true for other sensitive tissues, the exact mechanism ofp53-dependent apoptosis in HF remains unclear. It may involve atleast several p53-responsive genes acting through different mecha-nisms. We speculate that p53 mediates cyclophosphamide-inducedapoptosis by: (a) up-regulation of IGF-BP3 and stimulation of theextracellular neutralization of the insulin-like growth factor-1 (IGF-1),implicated in retarding physiological transition of the HF from activegrowth to apoptosis-driven regression (20); (b) up-regulation of themembrane “death domain” containing receptor Fas/Apo-1 implicatedin mediating apoptosis in the HF (13); and (c) modulation (reduction)of the Bcl-2:Bax ratio in the HF triggering mitochondria-mediated

Fig. 3. Cyclophosphamide-treated HF of p53-null mice show down-regulation of Fas and IGF-BP3 and up-regulation of Bcl-2. Skin sections of wild-type (WT) and p53 knockout(2/2) mice were analyzed at day 5 after cyclophosphamide administration (day 14 postdepilation) for immunoreactivity of Fas, p55 kDa TNF receptor (p55 TNFR), p75 kDaneurotrophin receptor (p75NTR), IGF-BP3, Bax, and Bcl-2.A andB, Fas. Prominent expression of Fas in the HF outer root sheath (A, arrows) and inner root sheath (A, arrowhead)in wild-type skin, and weak expression of Fas in the HF outer root sheath in p53-null skin (B, arrows).C andD, p55 TNFR. Wild-type HF shows p55 TNF receptor immunoreactivityin the outer root sheath (C, arrow). Decrease of immunoreactivity in the p53 null HF (D, arrow).E andF, p75 NTR. Expression in the outer root sheath of wild-type (E, arrows) andp53 null HF (F, arrows).G andH, IGF-BP3. Expression in the proximal outer root sheath (G, arrows) and inner root sheath (G, arrowheads) of the wild-type HF. Down-regulationof IGF-BP3 in the HF of p53 knockout mice (H).I andJ, Bax. Expression in the matrix and dermal papilla (arrows) of the wild-type (I) and p53-null HF (J).K andL, Bcl-2. Expressionin the dermal papilla (K, arrow) and in the hair matrix (K, arrowhead) of the wild-type HF. Up-regulation of Bcl-2 in the dermal papilla (L, arrow) and hair matrix (L, arrowhead)of the p53-null HF.Bar, 100 mm.

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apoptosis (13). The relative impact of the above factors in HF regres-sion after chemotherapy remains to be determined.

Recently, we showed that a small molecule acting as a p53 inhibitorefficiently reduces the side effects of ionizing radiation in mice,presumably by suppressing p53-mediated apoptosis (4). We thuspresume that a local pharmacological blockade of p53 by syntheticantagonists may provide a new therapeutic strategy for the preventionof chemotherapy-induced hair loss, one of the most devastating sideeffects of cancer treatment.

Acknowledgments

The generous help of Dr. T. Golovkina and continued support of Prof. J.Knop are gratefully acknowledged.

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2000;60:5002-5006. Cancer Res   Vladimir A. Botchkarev, Elena A. Komarova, Frank Siebenhaar, et al.   p53 Is Essential for Chemotherapy-induced Hair Loss

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