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. 2013 Nov 13;14:58.
doi: 10.1186/2050-6511-14-58.

Suppression of Eukaryotic Initiation Factor 4E Prevents Chemotherapy-Induced Alopecia

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Free PMC article

Suppression of Eukaryotic Initiation Factor 4E Prevents Chemotherapy-Induced Alopecia

Zeina Nasr et al. BMC Pharmacol Toxicol. .
Free PMC article

Abstract

Background: Chemotherapy-induced hair loss (alopecia) (CIA) is one of the most feared side effects of chemotherapy among cancer patients. There is currently no pharmacological approach to minimize CIA, although one strategy that has been proposed involves protecting normal cells from chemotherapy by transiently inducing cell cycle arrest. Proof-of-concept for this approach, known as cyclotherapy, has been demonstrated in cell culture settings.

Methods: The eukaryotic initiation factor (eIF) 4E is a cap binding protein that stimulates ribosome recruitment to mRNA templates during the initiation phase of translation. Suppression of eIF4E is known to induce cell cycle arrest. Using a novel inducible and reversible transgenic mouse model that enables RNAi-mediated suppression of eIF4E in vivo, we assessed the consequences of temporal eIF4E suppression on CIA.

Results: Our results demonstrate that transient inhibition of eIF4E protects against cyclophosphamide-induced alopecia at the organismal level. At the cellular level, this protection is associated with an accumulation of cells in G1, reduced apoptotic indices, and was phenocopied using small molecule inhibitors targeting the process of translation initiation.

Conclusions: Our data provide a rationale for exploring suppression of translation initiation as an approach to prevent or minimize cyclophosphamide-induced alopecia.

Figures

Figure 1
Figure 1
Inducible and reversible suppression of eIF4E in hair follicle cells. (A) Allele configuration at Rosa26 and Col1A1 and loci of shRNA/CAGs-RIK mice designed to exhibit inducible and reversible expression of shRNAs. (B) Representative immunohistochemistry staining showing eIF4E and GFP staining in the hair follicles of vehicle-treated (-Dox) and Dox-treated (+Dox) (5 days) mice. Bar represents 50 μm. (C) Representative immunohistochemistry staining showing eIF4E and GFP staining in the hair follicles of vehicle-treated (-Dox) 4E.389/CAGs-RIK mice, doxycycline-treated (+Dox) (5 days) 4E.389/CAGs-RIK mice, and Dox-treated (5 days) 4E.389/CAGs-RIK mice that were then taken off Dox for two weeks (ON/OFF Dox). Bar represents 50 μm. (D) Representative immunohistochemistry staining showing mKate2 staining in the hair follicles of vehicle-treated (-Dox) and Dox-treated (+Dox) (5 days) 4E.389/CAGs-RIK or FLuc.1309/CAGs-RIK mice. Sections are from the experiment presented in Figure 1B. Bar represents 50 μm.
Figure 2
Figure 2
Suppression of eIF4E protects from CIA. (A) Schematic illustration showing experimental design for inducing shRNA expression and CIA in shRNA/CAGs-RIK mice. Shown are the timelines and the stages of hair growth induced upon depilation. In red is the time of Dox induction and CyP delivery schedule. (B) Mice of the indicated genotypes were depilated, exposed to Dox for 5 days prior to CyP treatment, and allowed to recover in the absence of Dox.
Figure 3
Figure 3
Representative immunostaining of skin sections from Dox-treated shRNA/CAGs-RIK mice 3 days post-CyP. (A) Skin sections from mice of the indicated genotypes were processed for H&E staining, immunostained for the indicated proteins, or processed for visualization of apoptotic bodies as described in the Methods. Bars = 25 μm. (B) Percent of dystrophic hair follicles in mice of the indicated genotype taken 3 days after cyclophosphamide delivery. n = 3 mice. Bars denote S.E.M. (C) Average number of apoptotic cells per hair follicle. Five different fields (5 follicles/field) were analyzed from sections obtained from 2 different mice. Bars denote S.E.M.
Figure 4
Figure 4
Representative immunostaining of skin sections from Dox-treated shRNA/CAGs-RIK mice 12 days post-CyP exposure. Representative immunostaining of skin sections from Dox-treated mice of the indicated genotype taken 12 days after cyclophosphamide delivery. Bars = 50 μm.
Figure 5
Figure 5
Suppression of eIF4F protects against chemotherapy-induced cell death in non-transformed BJ/hTERT cells. (A) Representative Giemsa staining of BJ/hTERT cells pre-treated with nutlin-3a or Cr131-b for 24 hrs, or transfected with siRNA against eIF4E (si4E) (2 days before treatment), followed by removal of compounds and exposure to paclitaxel (PAC) for 48 hrs. Cells were then allowed to recover for 5 days. (B) Western blot analysis of p53 and eIF4E from BJ/hTERT cells treated with nutlin-3a, Cr131-b or transfected with siRNA against eIF4E. (C) Representative cell cycle profiles of BJ/hTERT cells pre-treated as described in Panel A. (D) Quantification of the DNA content of BJ/hTERT cells pre-treated as indicated in Panel A. n = 3. Bars denote S.E.M. (E) Relative viability of BJ/hTERT cells that had been pre-treated with nutlin-3a, Cr131-b or hippuristanol (Hipp) for 24 hours or transfected with si4E or a non-targeting control (siNT) 2 days prior to the indicated drug treatments. Cell counts for VRL, NOCO, and PAC are normalized to controls in which cells were exposed to vehicle. n = 3. Bars denote S.E.M.
Figure 6
Figure 6
Suppression of eIF4F protects against chemotherapy-induced cell death in non-transformed MRC5 cells. (A) Representative Giemsa staining of MRC5 cells that had been pre-treated with nutlin-3a, Cr131-b, Hipp, 4E1RCat or 4E2RCat for 24 hours , followed by exposure to paclitaxel for 48 hrs. (B) Relative viability of MRC5 cells that had been pre-treated with nutlin-3a, Cr131-b, hippuristanol (Hipp), 4E1RCat or 4E2RCat for 24 hours and followed by exposure to 40 nM vinorelbine (VRL), 200 nM nocodazole (NOCO), or 50 nM paclitaxel (PAC) for 48 hrs. Cell counts for VRL, NOCO, and PAC were normalized to controls that had not been pre-treated. n = 3. Bars denote S.E.M. (C) Cell numbers after treatment of MRC5 cells with the indicated compounds for 24 hours and left to recover for 7 days. n = 3. Bars denote S.E.M.

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