**6. Molecular mechanisms of CIA**

Molecular mechanisms of CIA are not well understood, in part due to the lack of appropriate experimental models that mimic human CIA. Much of our understanding on CIA is based on animal and cell culture models, some of which are described below.

#### **6.1 DNA damage**

Most chemotherapeutic agents including cyclophosphamide, doxorubicin and cisplatin induce DNA damage and kill both normal and cancer cells by apoptosis (Muller et al., 1998). p53 is a transcription factor and tumor suppressor protein that plays a critical role in cell cycle progression and apoptosis. Activation of p53 in response to DNA damage is associated with the degradation of Mdm2/p53 complex, leading to increased availability of p53 to bind DNA and consequently transcriptional activation of p53 target genes. Many p53 target genes, including Fas, Bax, Bcl-2, insulin-growth factor receptor type I (IGFR1), and insulinlike growth factor binding protein 3 (IGF-BP3), are expressed in the hair follicles (Lindner et al., 1997). In the adult mouse model for CIA, p53 was shown to be essential in the hair follicle response to DNA damage induced by cyclophosphamide. Specifically, hair loss was not observed and hair follicle cells remained active in p53-deficient mice, as shown by a large volume of hair bulb and dermal papilla, and active keratinocyte proliferation in the hair matrix (Botchkarev et al., 2000).

#### **6.2 Apoptosis**

Chemotherapy-induced apoptosis of hair follicle cells is one of the major findings from CIA animal studies. Although the mechanism of apoptosis is not well understood, p53 and Fas signaling pathways are believed to play a key role.

In adult mice, cyclophosphamide-treated hair follicles show a strong up-regulation of p53 in the hair matrix, particularly in TUNEL-positive apoptotic keratinocytes (Botchkarev et al., 2000). By contrast, in p53-deficient mice, apoptosis in the matrix keratinocytes was not detected after cyclophosphamide treatment, indicating the involvement of p53 in the apoptotic process. The precise mechanism of p53-dependent apoptosis in the hair follicles remains unclear, but likely involves several p53 target genes. Cyclophosphamide-treated

FNK protein constructed from rat Bcl-xL by site-directed mutagenesis (Y22F/Q26N/R165K) localizes to mitochondria and functions to maintain mitochondrial membrane potential (Aosh et al., 2000). Mitochondrial membrane potential regulates the release of cytochrome C, which once binds to caspase-activating proteins such as Apaf-1 initiates the intrinsic caspase cascade and apoptosis (Li et al., 1997). Recently, FNK protein has been fused to protein transduction domain (PTD) to improve its cellular entry. Subcutaneous injection of PTD-FNK protects against CIA induced by etoposide in the neonatal rat model. The fusion protein helps retain hair follicle structures, prevent hair follicle regression and maintain the anagen duration upon etoposide treatment (Nakashima-Kamimura et al., 2008). Indeed, its protective effect on CIA suggests that it could penetrate the epidermis and reach the dermal hair follicles. Localized administration of FNK fusion protein has been suggested as a potential protein therapy

Molecular mechanisms of CIA are not well understood, in part due to the lack of appropriate experimental models that mimic human CIA. Much of our understanding on

Most chemotherapeutic agents including cyclophosphamide, doxorubicin and cisplatin induce DNA damage and kill both normal and cancer cells by apoptosis (Muller et al., 1998). p53 is a transcription factor and tumor suppressor protein that plays a critical role in cell cycle progression and apoptosis. Activation of p53 in response to DNA damage is associated with the degradation of Mdm2/p53 complex, leading to increased availability of p53 to bind DNA and consequently transcriptional activation of p53 target genes. Many p53 target genes, including Fas, Bax, Bcl-2, insulin-growth factor receptor type I (IGFR1), and insulinlike growth factor binding protein 3 (IGF-BP3), are expressed in the hair follicles (Lindner et al., 1997). In the adult mouse model for CIA, p53 was shown to be essential in the hair follicle response to DNA damage induced by cyclophosphamide. Specifically, hair loss was not observed and hair follicle cells remained active in p53-deficient mice, as shown by a large volume of hair bulb and dermal papilla, and active keratinocyte proliferation in the

Chemotherapy-induced apoptosis of hair follicle cells is one of the major findings from CIA animal studies. Although the mechanism of apoptosis is not well understood, p53 and Fas

In adult mice, cyclophosphamide-treated hair follicles show a strong up-regulation of p53 in the hair matrix, particularly in TUNEL-positive apoptotic keratinocytes (Botchkarev et al., 2000). By contrast, in p53-deficient mice, apoptosis in the matrix keratinocytes was not detected after cyclophosphamide treatment, indicating the involvement of p53 in the apoptotic process. The precise mechanism of p53-dependent apoptosis in the hair follicles remains unclear, but likely involves several p53 target genes. Cyclophosphamide-treated

CIA is based on animal and cell culture models, some of which are described below.

**5.2.7.2 Anti-death FNK protein** 

for CIA without affecting the chemotherapy efficacy.

**6. Molecular mechanisms of CIA** 

hair matrix (Botchkarev et al., 2000).

signaling pathways are believed to play a key role.

**6.1 DNA damage** 

**6.2 Apoptosis** 

p53-deficient mice show strongly down-regulated Fas in the hair follicle keratinocytes and highly up-regulated Bcl-2 in the dermal papilla as compared to wild-type mice. The role of Fas in the control of cyclophosphamide-induced apoptosis in keratinocytes was also investigated using Fas-deficient mice (Sharov et al., 2004). These mice show significantly reduced CIA and a parallel decrease in apoptotic keratinocytes and FADD and caspase-8 expression. Similarly, anti-Fas ligand neutralizing antibody inhibits cyclophosphamideinduced keratinocyte apoptosis. These studies indicate that Fas signaling is an important pathway in mediating the apoptosis induced by cyclophosphamide and suggest the crosstalk between p53 and Fas death signaling. However, the eventual hair loss observed in Fasdeficient mice points to the lower resistance of hair follicles to cyclophosphamide as compared to p53-deficient mice. Thus, it is likely that Fas signaling represents only a component of the p53-dependent apoptosis machinery in the hair follicles and that other p53 targets are also involved. Cyclophosphamide treatment also alters the expression of melanogenic proteins and causes apoptosis of hair follicle melanocytes (Sharov et al., 2003). In contrast to matrix keratinocytes, the melanocytes undergo apoptosis primarily through Fas signaling but not p53 signaling.

### **6.3 Reactive oxygen species**

The observation that antioxidants such as NAC protect against CIA in animals suggest the involvement of reactive oxygen species (ROS) in CIA. Various chemotherapeutic agents induce oxidative stress through multiple mechanisms, i.e., activation of NADPH oxidase system and mitochondrial respiration chain. Agents that induce a high level of ROS include anthracyclines (e.g., doxorubicin, epirubicin, and daunorubicin), alkylating agents (e.g., cyclophosphamide), platinum coordination complexes (e.g., cisplatin, carboplatin, and oxaliplatin), and epipodophyllotoxins (e.g., etoposide) (Conklin, 2004). Interestingly, anthracyclines, alkylating agents, platinum complexes, and epipodophyllotoxins also induce CIA more frequently and more severely than most other agents, suggesting a relationship between ROS generation and CIA. The exact mechanism of how ROS induces or promotes CIA is unclear, but likely involves apoptosis regulation since apoptosis of hair follicles is a hallmark of CIA and since ROS generation is generally required for the induction of apoptosis by chemotherapeutic agents (Simon et al., 2000).
