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Departments of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USADepartment of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
Departments of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USADepartment of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
Departments of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USADepartment of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
Centre for Skin Sciences, School of Life Sciences, University of Bradford, Richmond Road, Bradford BD7 1DP, UKDepartment of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, Massachusetts 02118, USA
Affiliations
Departments of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USADepartment of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, USACentre for Skin Sciences, School of Life Sciences, University of Bradford, Bradford, UK
Departments of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USADepartment of Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, USA
Chemotherapy has severe side effects in normal rapidly proliferating organs, such as hair follicles, and causes massive apoptosis in hair matrix keratinocytes followed by hair loss. To define the molecular signature of hair follicle response to chemotherapy, human scalp hair follicles cultured ex vivo were treated with doxorubicin (DXR), and global microarray analysis was performed 3 hours after treatment. Microarray data revealed changes in expression of 504 genes in DXR-treated hair follicles versus controls. Among these genes, upregulations of several tumor necrosis factor family of apoptotic receptors (FAS, TRAIL (tumor necrosis factor–related apoptosis-inducing ligand) receptors 1/2), as well as of a large number of keratin-associated protein genes, were seen after DXR treatment. Hair follicle apoptosis induced by DXR was significantly inhibited by either TRAIL-neutralizing antibody or caspase-8 inhibitor, thus suggesting a previously unreported role for TRAIL receptor signaling in mediating DXR-induced hair loss. These data demonstrate that the early phase of the hair follicle response to DXR includes upregulation of apoptosis-associated markers, as well as substantial reorganization of the terminal differentiation programs in hair follicle keratinocytes. These data provide an important platform for further studies toward the design of effective approaches for the management of chemotherapy-induced hair loss.
Hair loss (alopecia) is a common side effect of many chemotherapeutic treatment protocols and is one of the most distressing aspects of cancer therapy. Because of the rapid proliferation of hair matrix keratinocytes during hair shaft production, the hair follicle represents a ‘bystander’ target for many chemotherapeutic agents (
). Despite significant advances in understanding the mechanisms of the hair follicle response to chemotherapeutic agents achieved within the last decade (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
Modulation of chemotherapy-induced human hair follicle damage by 17-beta estradiol and prednisolone: potential stimulators of normal hair regrowth by ‘dystrophic catagen’ promotion?.
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
), cyclophosphamide administration caused rapid increase in the p53 protein in hair matrix keratinocytes, followed by massive apoptosis, whereas genetic p53 ablation resulted in complete resistance of the hair follicles to chemotherapy (
). Fas (APO-1, CD95) as a p53 target is also involved in mediating apoptosis in the hair matrix keratinocytes and melanocytes during hair follicle exposure to cyclophosphamide (
). In contrast to p53 and its target genes, members of the fibroblast growth factor (FGF) family, including keratinocyte growth factor, as well as pharmacological modulator of the heat-shock proteins geldanomycin show relative protective effects in rodent models of chemotherapy-induced hair loss (
Keratinocyte growth factor is an important endogenous mediator of hair follicle growth, development, and differentiation. Normalization of the nu/nu follicular differentiation defect and amelioration of chemotherapy-induced alopecia.
Modulation of chemotherapy-induced human hair follicle damage by 17-beta estradiol and prednisolone: potential stimulators of normal hair regrowth by ‘dystrophic catagen’ promotion?.
During chemotherapy, the DNA damage response is regulated at several levels, including involvement of the response sensors, transducers, and effectors, which operate in a stage-dependent manner to induce apoptosis, cell cycle arrest, or senescence in target cells (reviewed in
). Analyses of the kinetics of the cellular response to chemotherapy reveal that anti-cancer drugs induce apoptosis in tumor cells within several hours after in vivo administration (
). Cyclophosphamide treatment in mice also induces apoptosis in rapidly proliferating hair matrix keratinocytes within 24 hours after intraperitoneal administration (
In a recently developed an ex vivo human model for chemotherapy-induced hair loss, it was shown that the cyclophosphamide derivative 4-hydroperoxycyclophosphamide induces apoptosis in isolated human hair follicles, followed by their dystrophy, in a manner that resembles the follicular response to cyclophosphamide in the C57BL/6 mouse in vivo model (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
). In this model, microarray analyses of the relatively advanced stage (48 hours) of the hair follicle response to chemotherapy reveal the changes in expression of more than 400 genes including several growth factors (FGF-18, IL-8, and so on), apoptotic regulators (BAX, MDM2, and so on) and adhesion/extracellular matrix–associated molecules (CTNND2, GPC6, and so on), suggesting their involvement in chemotherapy-induced hair loss (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
However, the mechanisms underlying the early phase of the hair follicle response to chemotherapy remain unclear. In this article, we use a global microarray profiling approach to define the molecular signatures of the early phase of response of hair follicles to chemotherapy treatment ex vivo. We show here that the early phase of the hair follicle response to chemotherapy is far more complex than it was previously appreciated and includes upregulation of not only apoptosis-associated genes but also marked reorganization of the terminal differentiation programs in hair follicle keratinocytes. These data provide an important foundation for further research in identification of the mechanisms that trigger hair follicle response to DNA damage and development of effective approaches for management of hair loss induced by anti-cancer drugs.
Results
Doxorubicin treatment induces apoptosis-driven premature catagen development in human hair follicles cultured ex vivo
To further develop an ex vivo human model for chemotherapy-induced hair loss used previously for studying hair follicle response to cyclophosphamide (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
), we focused our studies on doxorubicin (DXR), a broadly used anti-cancer drug, treatment with which induces apoptosis in hair follicles, followed by massive hair loss in patients as well as in rodent models (
), which we considered an advantage that allows direct testing of its effects on hair follicles cultured ex vivo.
To define whether DXR treatment is capable of inducing apoptosis and premature catagen development in human hair follicles cultured ex vivo, hair follicles isolated from the scalp of normal individuals were cultured for 24 hours as described previously (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
) followed by treatment with different concentrations (0.3 or 1.0 μM) of DXR for 1 hour (Figure 1a). DXR concentrations for cultured hair follicles were selected according to recommendations published previously (
). Both DXR concentrations tested induced rapid transition of normal anagen hair follicles into catagen II/III stages within 24 hours after treatment (Figure 1b and c). Treatment of hair follicles with 1.0 μM DXR induced more rapid catagen development compared with the group of follicles treated with 0.3 μM DXR (Figure 1c). After 5–7 days, all hair follicles treated with both concentrations of DXR entered catagen V–VI phase of the hair cycle, whereas about 67% of the follicles in the control group still remained in anagen VI/catagen II phases (Figure 1b and c).
Figure 1Doxorubicin induces premature catagen development in human HFs cultured ex vivo. (a) Scheme illustrating the experimental design of this study. (b) Morphological changes in HFs treated with 1 μM doxorubicin (DXR) for 1 hour at different time points after treatment. Bars=300 μm and 100 μm. (c) Dynamics of catagen development in HFs treated with 0.3 or 1 μM doxorubicin versus the controls (mean±SD, *P<0.05, Student’s t-test). CH, club hair; DP, dermal papilla, DXR, doxorubicin; ES, epithelial strand; HF, hair follicle; HM, hair matrix.
Analysis of the hair follicle apoptosis and cell proliferation performed by double immuno-visualization of apoptotic (TUNEL+) and proliferating (Ki-67+) cells showed appearance of numerous TUNEL+ cells and significant decrease in proliferating cells in the hair follicle matrix within 24 hours after DXR treatment (Figure 2a–c). Further increase in TUNEL+ cells was seen in catagen III/IV hair follicles 2–3 days after DXR treatment (Figure 2a and b). Consistently with morphological data (Figure 1c), hair follicles treated with 1.0 μm of DXR showed more rapid increase in the number of TUNEL+ cells and decrease in cell proliferation in the hair matrix compared with the group of follicles treated with 0.3 μM DXR (Figure 2b and c). However, whereas control hair follicles showed the appearance of single TUNEL+ cells during hair follicle progression to catagen II/III stages only 5 days after beginning of the experiment, lack of TUNEL+ cells and numerous proliferating Ki-67+ cells were seen in the control follicles at the beginning of the study (Figure 2a–c). These data suggest that, similar to 4-hydroperoxycyclophosphamide (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
), DXR treatment induces premature apoptosis-driven catagen development in human hair follicles cultured ex vivo, which is consistent with its effects on the hair follicles observed in the in vivo rodent models or in patients who had received anti-cancer therapy (
Figure 2Increase in apoptosis and decrease in cell proliferation in the hair follicles treated with DXR. (a) Double immuno-detection of apoptotic (TUNEL+, green arrowhead) and proliferating (Ki-67+, red arrow) cells in the HFs treated with DXR versus controls. Scale bar=100 μm. (b) Increase in the number of TUNEL+ cells in the HFs exposed to 0.3 or 1 μM DXR (mean±SD, ***P<0.001, Student’s t-test). (c) Decrease in the number of Ki-67+ cells in the HFs exposed to 0.3 or 1 μM DXR (mean±SD, *P<0.05, **P<0.01, Student’s t-test). DAPI, 4,6-diamidino-2-phenylindole; DXR, doxorubicin; HF, hair follicle.
Early-phase response of hair follicles to DXR includes upregulation of pro-apoptotic FAS and TRAIL receptors and KAP genes
To define the molecular mechanisms underlying the initiation of the hair follicle response to DXR, hair bulbs of the follicles cultured ex vivo were collected 3 hours after DXR treatment and processed for RNA isolation followed by global microarray analyses with Affymetrix GeneChipSystem, as described previously (
). Microarray data were validated by quantitative real-time reverse-transcriptase–PCR (qRT-PCR) and immunohistology and revealed 2-fold and higher changes in the expression of 504 genes in DXR-treated hair follicles compared with controls (Figure 3a). Genes that show changes in expression in the hair follicles after DXR treatment were grouped into several functional categories, which encoded apoptosis/cell cycle regulators, adhesion/extracellular matrix molecules, cytoskeletal proteins, metabolic enzymes, and signaling/transcription regulators (Figure 3a; Supplementary Tables S1 and S2 online).
Figure 3Microarray, quantitative real-time reverse-transcriptase–PCR (qRT-PCR), and immunohistochemical profiling of human hair follicles 3–24 hours after doxorubicin treatment. (a) Microarray analysis of the global gene expression in hair follicles (HFs) collected 3 hours after 1 μm doxorubicin treatment versus control HFs: functional assignments of the genes with altered expression induced by DXR. (b, c) Validation of microarray results by qRT-PCR reveals changes in expression of the genes encoding distinct pro- and anti-apoptotic markers (b) (left and right panels, respectively) and cell differentiation–associated markers (c) after doxorubicin treatment compared with controls. (d) Increase in expression of P53, FAS, TRAIL, TRAIL-R1, caspase-8, cFLIP, P21, and KAP1 in the epithelial (arrows) or mesenchymal (asterisks) portions of the hair bulb 24 hours after doxorubicin treatment. Note, lack of cFLIP expression in hair matrix keratinocytes located at the bottom part of the hair bulb (arrowheads), whereas prominent cFLIP expression is seen in the differentiating hair shaft keratinocytes (arrow) and dermal papilla (asterisk). Scale bar=100 μm.
Genes that are involved in the control of apoptosis were subdivided into the categories of pro- and anti-apoptotic regulators (Figure 3b and c; Supplementary Tables S1 and S2 online). Interestingly, among the group of genes that encoded different pro-apoptotic regulators, several tumor necrosis factor (TNF)-family apoptotic receptors and their ligands including FAS, TNFRSF10A, TNFRSF10B (tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptors 1 and 2, respectively), TNFSF10 (TRAIL), and TNFSF15 showed marked upregulation in DXR-treated hair follicles versus the controls (Figure 3b and d). Immunohistological and quantitative immunohistomorphometric analyses revealed that, after DXR treatment, markedly increased expressions of TRAIL, TRAIL-R1, p53, and caspase-8 were seen in the hair follicle compartments enriched either in proliferating keratinocytes (distal hair matrix) or in post-mitotic differentiating cells (proximal part or precortex of the hair bulb) compared with controls (Figure 3b and d; Supplementary Figure S1a and b online).
However, microarray data validated by qRT-PCR also showed increased expression of anti-apoptotic markers (BCL2, BCL2A1, BCL6B, BCL10, TNFRSF10D, CFLAR) in DXR-treated hair follicles compared with controls (Figure 3b and d). This was not surprising because hair follicles contain the significant number of cells that survive after chemotherapy, including the dermal papilla fibroblasts, post-mitotic differentiating keratinocytes, as well as epithelial progenitor cells protected from apoptosis (
). Quantitative immunohistomorphometric data revealed that one of the anti-apoptotic regulators, cFLIP (CFLAR), that inhibit TNF receptor-mediated apoptosis and caspase-8 activity (
Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex.
) is strongly upregulated in post-mitotic differentiating cells of the inner root sheath and hair shaft (precortex area), as well as in the dermal papilla, whereas its expression is detected at low levels in proliferating hair matrix keratinocytes located at the bottom of the hair bulb (Figure 3d; Supplementary Figure S1a and b online). Because cFLIP expression decreased in differentiating hair bulb keratinocytes 48 and 72 hours after DXR treatment (Supplementary Figure S2 online), these data suggest that the balance of pro- and anti-apoptotic regulators at early time points after chemotherapy (3–24 hours) is critical for the fate (apoptosis versus survival) of distinct hair follicle cell populations (proliferating versus differentiating keratinocytes).
In addition to changes in expression of apoptosis-related genes, two key genes that encode cell cycle inhibitors and promote cell differentiation in the hair follicle (
), such as cyclin-dependent kinase inhibitors CDKN1A (P21) and CDKN1C (P57), also showed marked upregulation in the hair follicle after DXR treatment (Figure 3d; Supplementary Tables S1 and S2 online). Furthermore, DXR treatment resulted in upregulation of expression of 15 genes encoding keratin-associated proteins (KAPs; such as KRTAP9-9, KRTAP4-7, KRTAP3-2, KRTAP1-3 and others) and involved in execution of the terminal differentiation program in hair matrix keratinocytes (Figure 3c and d;
). Consistently with microarray and qRT-PCR data, hair follicles treated with DXR showed earlier onset of expression of KAP1 in differentiating hair matrix keratinocytes compared with controls (Figure 3d). The increased expression of the KAP genes was accompanied by downregulation of the genes encoding selected keratins, including KRT17, KRT16, KRTHA3B, and KRT6A/6B, after DXR treatment, compared with controls (Supplementary Tables S1 and S2 online). These data suggest that DXR induces marked changes in gene expression in hair matrix keratinocytes, which include not only pro- and anti-apoptotic genes but also marked reorganization of the cell differentiation program and premature activation of the KAP genes.
Inhibition of TRAIL receptor signaling and caspase-8 results in decrease in DXR-induced apoptosis in the hair follicles
To assess the impact of TRAIL receptor–mediated signaling in the control of apoptosis induced by DXR in the hair follicles, hair follicles were treated with TRAIL-neutralizing antibody for 24 hours prior to and after DXR treatment. TRAIL-neutralizing antibody has been shown previously to effectively inhibit apoptosis in different models (
Protective effects of neurotrophic factors on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis of murine adrenal chromaffin cell line tsAM5D.
). Treatment of follicles with TRAIL-neutralizing antibody alone did not result in any significant changes in keratinocyte proliferation or apoptosis, whereas expression of TRAIL in the hair matrix was markedly decreased, suggesting the inhibition of TRAIL activity (data not shown). However, hair follicles treated with a combination of DXR and TRAIL-neutralizing antibody showed marked decrease in the number of TUNEL+ cells compared with the follicles treated with DXR alone, suggesting that DXR-induced apoptosis in hair matrix keratinocytes is mediated, at least in part, by TRAIL receptor signaling (Figure 4a–c). Furthermore, pretreatment of hair follicles with TRAIL-R1 and TRAIL-R2 antagonistic antibodies results in significant decrease in the number of TUNEL+ cells in the hair bulb after DXR treatment (Figure 4d), suggesting that both TRAIL-R1 and TRAIL-R2 are involved in mediating DXR-induced apoptosis in hair matrix keratinocytes.
Figure 4TRAIL or TRAIL-R1/R2 antagonistic antibodies and caspase-8 inhibitor decrease apoptosis in the hair follicles treated with doxorubicin (DXR). HFs were treated with TRAIL-neutralizing antibody (5 μg ml-1), TRAIL-R1 or TRAIL-R2 antagonistic antibodies, or with caspase-8 inhibitor (50 μm) for 24 hours prior to doxorubicin treatment. (a) Histomorphology of the HFs exposed for 48 hours to the different treatments. (b) Detection of apoptotic cells by TUNEL (green) reveals numerous apoptotic cells in the HFs treated with doxorubicin (arrow). Scale bars=100 μm. (c) Quantitative analysis of apoptosis shows dramatic elevation in TUNEL+ cells in doxorubicin-treated HFs, whereas treatment with TRAIL-neutralizing antibody and caspase-8 inhibitor significantly reduced doxorubicin-induced apoptosis (mean±SD, ***P<0.001 Student’s t-test). (d) Treatment with TRAIL-R1 or TRAIL-R2 antagonistic antibodies significantly reduced doxorubicin-induced apoptosis and number of TUNEL+ cells in the HFs (mean±SD, *P<0.05, **P<0.01, ***P<0.001; Student’s t-test).
Because activation of the TRAIL receptor pathway is accompanied by the recruitment of the adaptor protein FADD and procaspase-8 to the intracellular death domain of the receptors, followed by procaspase-8 cleavage and activation of the effector caspases (
), the effects of caspase-8 inhibitor on DXR-induced apoptosis in the hair follicle were tested. Similarly to the TRAIL-neutralizing antibody, treatment of hair follicles with caspase-8 inhibitor resulted in significant decrease in the number of TUNEL+ cells in DXR-treated hair follicles compared with the follicles treated with DXR alone (Figure 4a–c). These data suggest that apoptosis induced by DXR in the hair follicles is mediated, at least in part, by the TRAIL receptor 1/2 signaling involving an activation of caspase-8 as its downstream effector.
Discussion
Chemotherapy has severe side effects for normal rapidly proliferating tissues, such as hair follicles, leading to massive apoptosis in hair matrix keratinocytes followed by hair loss (
). Because of the ethical issues in obtaining scalp biopsies from patients treated with chemotherapy, only limited information is available about the molecular events underlying the response of human hair follicles to chemotherapy (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
) and global microarray profiling to study changes in the molecular signature of human hair follicles during the early stages of their response to DXR, a broadly used anti-cancer drug (
). Cytotoxic effects of DXR are based on its binding to and inhibiting activity of the DNA-associated enzymes (topoisomerases I/II), on intercalation with DNA base pairs, as well as on targeting multiple apoptotic regulatory molecules such as Bcl2/Bax, caspases, AMP-activated protein kinase, and so on (
). Consistently with the data obtained from tumor cells, we demonstrate here that within 3 hours after DXR treatment hair follicles show marked changes in the expression levels of the genes encoding several components of the apoptotic/cell cycle machinery, cytoskeleton/cell differentiation markers, signaling/transcription regulators, and so on (Supplementary Tables S1 and S2 online).
In general, these data are consistent with those obtained in the same model that show changes in expression of quite similar groups of genes in the hair follicles 48 hours after 4-hydroperoxycyclophosphamide treatment (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
). Indeed, several key regulators of apoptosis, cell differentiation, and signaling/transcription, such as TNFRSF10B, CDKN1A, FGF-18, or ID4, showed similar changes in their expression in the hair follicles treated with either DXR (Supplementary Tables S1 and S2 online) or 4-hydroperoxycyclophosphamide (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
). However, the differences between the two data sets are also evident and most likely reflect the distinct mechanisms of the DNA damage response activated by DXR versus 4-hydroperoxycyclophosphamide, as well as the distinct time points in response to the treatment (early versus more advanced) selected for analyses (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
Interestingly, among the genes encoding pro-apoptotic regulators, several members of the TNF family of apoptotic receptors (FAS, TRAIL receptors 1 and 2) show upregulation in their expression after DXR treatment. FAS as a direct p53 target gene is involved in the control of apoptosis induced by chemotherapy in many organs, including hair follicles (
). Similarly to FAS receptor, signaling through TRAIL receptor 1/2 activated by the TRAIL ligand induces apoptosis in normal and transformed keratinocytes (
Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex.
Low concentrations of doxorubicin sensitizes human solid cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor (R) 2-mediated apoptosis by inducing TRAIL-R2 expression.
). Apoptotic signaling through the FAS and TRAIL receptors requires their interaction with the intracellular adaptor molecule FADD, followed by activation of procaspase-8, its recruitment into the death-inducing signaling complex, and activation of caspase-3 along the common final pathway of apoptosis (reviewed in
We show here that, similarly to tumor cells, DXR treatment results in upregulation of TRAIL receptor 1 and 2 expressions in the hair matrix keratinocytes, whereas treatment of hair follicles with TRAIL-neutralizing antibodies or TRAIL-R1/R2 antagonistic antibodies markedly reduces a number of TUNEL+ cells in the hair follicles (Figures 3b, c and 4). Because TRAIL and TRAIL-R1 are direct p53 target genes (
), TRAIL receptor–mediated apoptosis is likely to be a part of the p53-regulated apoptotic program triggered by DXR in hair matrix keratinocytes. However, because in some cases the increase in TRAIL-R1/2 expression in tumor cells after chemotherapy occurs also in a p53-independent manner (
Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: evidence for the role of c-Jun N-terminal kinase activation.
), further studies are required to define the contribution of p53 to upregulation of TRAIL receptors in the hair follicles after DXR treatment. In addition, genetically engineered mice with ablation of TRAIL-R1 or TRAIL-R2 might be used to further dissect the role of these receptors in the control of chemotherapy-induced apoptosis in hair matrix keratinocytes.
However, DXR treatment also increases the expression of a number of anti-apoptotic genes in the hair follicle, which is consistent with the data showing that distinct hair follicle cell populations (selected outer root sheath and hair matrix keratinocytes, dermal papilla fibroblasts) characterized by high expression of anti-apoptotic proteins are programed to survive after chemotherapy and contribute to subsequent hair follicle regeneration (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex.
). As cFLIP is strongly upregulated in the post-mitotic differentiating keratinocytes and is not expressed in proliferating hair matrix keratinocytes, these data confirm the previously proposed model suggesting that the balance of pro- and anti-apoptotic factors has a critical role in the control of cell fate decision (apoptosis versus survival) in the distinct hair follicle cell populations after chemotherapy (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
Our data also reveal marked upregulation of 15 genes encoding KAPs in the hair follicles after DXR treatment (Figure 3b and c). In normal human hair follicles, KAPs are expressed in differentiating keratinocytes of the hair matrix, cortex, and cuticle (
), whereas DXR treatment leads to premature activation of KAP genes, as evident by the KAP1 expression in post-mitotic hair matrix keratinocytes (Figure 3c). As recent data reveal that KAPs interact with hair-specific keratins (
), it appears to be interesting to check whether premature activation of KAP genes and early onset of expression of KAPs in the hair follicles after DXR treatment have a role in alterations in the hair shaft structure and breakage during chemotherapy-associated anagen effluvium (
Interestingly, increase in KAP1 expression in the hair matrix keratinocytes after DXR treatment coincides with upregulation of P21 cyclin–dependent kinase inhibitor, a key p53 target gene that promotes mitotic exit and terminal differentiation in hair follicle keratinocytes (
). In addition, microarray analyses reveal marked increase in expression of the related CDKN1C (P57) gene in DXR-treated follicles (Supplementary Table S1 online). Thus, it would be interesting to test whether P21 and P57 contribute to marked upregulation of the KAP genes in the hair follicle after DXR treatment, or whether other mechanisms are involved in mediating these effects of DXR.
Taken together, these data provide insights into the mechanisms underlying the early events in the complex changes induced in human hair follicles by DXR and reveal a new role for TRAIL receptor signaling in the control of chemotherapy-induced hair loss. These data also serve as an important platform for the development of approaches for the prevention or reduction of the toxic effects of chemotherapy on hair follicles and for the search for drug-specific hair-protective paradigms for cancer patients.
Materials and Methods
Hair follicle culture and pharmacological experiments
This study was approved by the Institutional Review Board Committee of Boston University to ensure subject protection and adherence to the Declaration of Helsinki Principles. Patient consent for experiments was not required because human scalp skin samples were obtained from anonymous donors (five different female individuals) undergoing face-lift cosmetic surgery procedures. The hair follicles were microdissected from skin samples and cultured in Williams E medium supplemented with 10 μg ml-1 insulin, 10 ng ml-1 hydrocortisone, 2 mM L-glutamine, and antibiotic/antimycotic mixture as described previously (
). Individual hair follicles were cultured in 0.5 ml of supplemented Williams E medium at 37 °C in a 5% CO2. After 24 hours of culture, DXR HCl (0.3 or 1.0 μM; Thermo Fisher Scientific, Waltham, MA) was added to the microdissected anagen hair follicles for 1 hour, and culture medium was replaced with freshly prepared supplemented Williams E medium after completion of the DXR treatment. Control hair follicles were treated with phosphate-buffered saline instead of DXR. For pharmacological modulation of apoptosis induced by DXR, hair follicles were treated with TRAIL-neutralizing antibody (5 μg ml-1, Abcam, Cambridge, MA) or Caspase-8 inhibitor (50 μM, Santa Cruz Biotech, Dallas, TX), TRAIL-R1 and TRAIL-R2 mAbs (clones HS101 and HS201, respectively, 10 μg ml-1, Enzo Biochem, Farmingdale, NY), and corresponding isotype-matched Ig for 24 hours prior to DXR exposure. For each time point, 20–25 hair follicles from five individuals were studied.
Microarray and qRT-PCR analyses
For microarray analysis, hair bulbs were dissected and processed for RNA isolation 3 hours after completion of the DXR treatment. Total RNA was isolated from the hair follicle bulbs using TriIzol reagent (Invitrogen, San Diego, CA). All experiments were performed using at least three replicates, and total RNA isolated from three experimental and control samples was pooled and processed for microarray analyses using one sample of pooled RNA per experimental and control group. All microarray analyses were performed at the Microarray Core Facility at Boston University School of Medicine using Human Genome U133A 2.0 array (Affimetrix, Santa Clara, CA). After statistical analysis and initial filtering of the microarray data the changes in gene expression after DXR treatment equal to or 2-fold higher with at least one signal equal to or higher than 80 fluorescence units for the gene were considered significant (
). For quantitative RT-PCR analysis, equal amounts of total RNA were used as a template for cDNA synthesis using SupperScript III First-Strand Synthesis System and random primers (Life Technologies, San Diego, CA). PCR primers were designed using Beacon Designer software (Premier Biosoft International, Paolo Alto, CA) and are listed in Supplementary Table S3 online. Real-time PCR was performed using iCycler Thermal Cycler (Bio-Rad, Hercules, CA). Differences between samples and controls were calculated using the Gene Expression Macro program (Bio-Rad) based on the ΔΔCt equitation method, as described previously (
Immunohistochemistry and quantitative immunohistomorphometry
For qRT-PCR, histomorphology, and immunohistochemical analyses, hair follicles were collected at 3, 24, 48, 72 hours and 7 days after DXR treatment. For immunohistochemical analyses, 8 μm longitudinal cryosections of the hair follicles taken through the central part of the dermal papilla were used, as described previously (
). The cryosections were incubated with primary antisera (see Supplementary Table S4 online) overnight at room temperature, followed by application of corresponding TRITC- or Alexa Fluor 555 Conjugate-labeled secondary antibody (Invitrogen, Paisley, UK; diluted 1:200) for 45 min at 37 °C. Incubation steps were interspersed by four washes with phosphate-buffered saline (5 minutes each), as described previously (
). For double immunofluorescence detection of TUNEL-positive cells and Ki-67 immunoreactivity, the ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit (EMD Millipore, Billerica, MA) and anti Ki-67 antibody were used.
Alkaline phosphatase staining was used for identification and quantification of the hair follicles at distinct stages of catagen, as described previously (
). Ki67+ cells were calculated below the distal end of the dermal papilla and were expressed as percentage to the total number of DAPI+ cells in the hair matrix, whereas TUNEL+ cells were calculated per hair bulb, according to recommendations published previously (
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
). In total, 20–25 hair follicles were assessed per time point in each experimental and control group. Image preparation and analysis were performed using a fluorescent microscope (Nikon, Tokyo, Japan) in combination with a DS-C1 digital camera and ACT-2U image analysis software (Nikon).
Immunofluorescence intensity was determined using ImageJ software (NIH, Bethesda, MD), as described previously (
). In brief, red fluorescent signal was collected from experimental tissues in RGB format under the same exposure conditions. To measure the fluorescence intensity at each pixel, the RGB images were converted to 8-bit grayscale format. Regions of interest of distinct size (hair matrix—170 μm2, dermal papilla—180 μm2, and pre-cortex—200 μm2) within the control and DXR-treated hair follicles were selected, and the mean values of intensity (the sum of gray values of all the pixels in the selection divided by the number of pixels) were calculated for each selected area. In total, 20–25 hair follicles were assessed per time point for each marker (TRAIL, TRAIL-R1, cFLIP) in the DXR-treated and control groups, and the differences between mean values obtained from the control and DXR-treated hair follicles were calculated using Student’s t-test.
ACKNOWLEDGMENTS
This study was supported in part by a NIAMS grant to VAB (AR049778) and by a NIH KO1 Award (AR056771) to AAS. We thank J Schweizer, L Langbein, and M Rogers for providing anti-KAP1 antibody.
Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy.
Modulation of chemotherapy-induced human hair follicle damage by 17-beta estradiol and prednisolone: potential stimulators of normal hair regrowth by ‘dystrophic catagen’ promotion?.
Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: evidence for the role of c-Jun N-terminal kinase activation.
Keratinocyte growth factor is an important endogenous mediator of hair follicle growth, development, and differentiation. Normalization of the nu/nu follicular differentiation defect and amelioration of chemotherapy-induced alopecia.
Cellular FLICE-inhibitory protein (cFLIP) isoforms block CD95- and TRAIL death receptor-induced gene induction irrespective of processing of caspase-8 or cFLIP in the death-inducing signaling complex.
Protective effects of neurotrophic factors on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis of murine adrenal chromaffin cell line tsAM5D.
Low concentrations of doxorubicin sensitizes human solid cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor (R) 2-mediated apoptosis by inducing TRAIL-R2 expression.
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