Testing Chemotherapeutic Agents in the Feather Follicle Identifies a Selective Blockade of Cell Proliferation and a Key Role for Sonic Hedgehog Signaling in Chemotherapy-Induced Tissue Damage Guojiang Xie 1,5 , Hangwei Wang 1,5 , Zhipeng Yan 1 , Linyan Cai 1 , Guixuan Zhou 1 , Wanzhong He 2 , Ralf Paus 3,4 and Zhicao Yue 1 Chemotherapeutic agents induce complex tissue responses in vivo and damage normal organ functions. Here we use the feather follicle to investigate details of this damage response. We show that cyclophosphamide treatment, which causes chemotherapy-induced alopecia in mice and man, induces distinct defects in feather formation: feather branching is transiently and reversibly disrupted, thus leaving a morphological record of the impact of chemotherapeutic agents, whereas the rachis (feather axis) remains unperturbed. Similar defects are observed in feathers treated with 5-fluorouracil or taxol but not with doxorubicin or arabinofuranosyl cytidine (Ara-C). Selective blockade of cell proliferation was seen in the feather branching area, along with a downregulation of sonic hedgehog (Shh) transcription, but not in the equally proliferative rachis. Local delivery of the Shh inhibitor, cyclopamine, or Shh silencing both recapitulated this effect. In mouse hair follicles, those chemotherapeutic agents that disrupted feather formation also downregulated Shh gene expression and induced hair loss, whereas doxorubicin or Ara-C did not. Our results reveal a mechanism through which chemo- therapeutic agents damage rapidly proliferating epithelial tissue, namely via the cell population–specific, Shh-dependent inhibition of proliferation. This mechanism may be targeted by future strategies to manage chemotherapy-induced tissue damage. Journal of Investigative Dermatology (2015) 135, 690–700; doi:10.1038/jid.2014.409; published online 23 October 2014 INTRODUCTION Chemotherapy often damages normal tissue as an adverse effect. Commonly used chemotherapeutic agents such as cyclophosphamide (CYP), 5-fluorouracil (5-FU), taxol, and doxorubicin (Dox) are believed to be toxic to all mitotic cells. Chemotherapy-induced alopecia (CIA) is common in human patients (Hesketh et al., 2004; Yun and Kim, 2007; Trueb, 2009; Chon et al., 2012), and rodent models have been developed to study the molecular mechanism of chemo- therapy-induced tissue damage (Hussein et al., 1990; Botchkarev et al., 2000; Sharov et al., 2004; Jimenez et al., 2008; Paus et al., 1994; 2013). A p53-dependent apoptosis program, which likely involves Fas-dependent signaling events, was suggested to underlie CIA (Botchkarev et al., 2000; Sharov et al., 2004). Other molecules such as ectodysplasin A receptor, epidermal growth factor receptor, and the eukaryotic initiation factor 4E have also been implicated (Brosh et al., 2010; Bichsel et al., 2013; Nasr et al., 2013; Paik et al., 2013). It is likely that cells interact with chemotherapeutic agents in complex ways. For example, in addition to undergoing apoptosis, cells may ‘‘slip’’ out of the cell cycle and thus escape apoptosis (Gascoigne and Taylor, 2008). Also, cell-to- cell variation in response to apoptosis-inducing agents can arise from transcriptional differences (Spencer et al., 2009; Ooi and Ma, 2013). Furthermore, because of the diverse mechanisms of action, it is difficult to rationally predict whether a specific chemotherapeutic agent will cause CIA in a given patient (Hesketh et al., 2004; Yun and Kim, 2007; ORIGINAL ARTICLE 1 Institute of Life Sciences, Fuzhou University, Fuzhou, China; 2 National Institute of Biological Sciences, Beijing, China; 3 Institute of Inflammation and Repair, University of Manchester, Manchester, UK, and 4 Department of Dermatology, University of Muenster, Muenster, Germany Correspondence: Zhicao Yue, Institute of Life Sciences, Fuzhou University, # 2, Xue Yuan Road, University Campus, Fuzhou, Fujian 350108, China. E-mail: raw600@gmail.com 5 These authors contributed equally to this work. Received 25 October 2013; revised 22 August 2014; accepted 29 August 2014; accepted article preview online 18 September 2014; published online 23 October 2014 Abbreviations: 5-FU, 5-fluorouracil; Ara-C, arabinofuranosyl cytidine; BrdU, 5-bromo-2-deoxyuridine; CIA, chemotherapy-induced alopecia; CYP, cyclophosphamide; Dox, doxorubicin; g-H2AX, phosphorylated histone H2AX; LCAM, liver cell adhesion molecule; NCAM, neural cell adhesion molecule; PARP, poly ADP-ribose polymerase; PCNA, proliferating cell nuclear antigen; PI, propidium iodide; qPCR, quantitative PCR; RT-PCR, reverse transcription-PCR; Shh, sonic hedgehog; TEM, transmission electron microscopy 690 Journal of Investigative Dermatology (2015), Volume 135 & 2015 The Society for Investigative Dermatology