Gene Expression in Hair Follicle Dermal Papilla Cells after Treatment with Stanozolol

Abstract

Doping with anabolic agents is a topic in sports where strength is crucial, e.g. sprinting, weight lifting and many more. Testosterone and its functional analogs are the drugs of choice taken as pills, creams, tape or injections to increase muscle mass and body performance, and to reduce body fat. Stanozolol (17β-hydroxy-17α-methyl-5α-androst-2-eno[3,2c]pyrazol) is a testosterone analogue with the same anabolic effect like testosterone but its ring structure makes it possible to take it orally. Therefore, stanozolol is one of the most frequently used anabolic steroids. Common verifi cation methods for anabolic drugs exist, identifying the chemicals in tissues, like hair or blood samples. The idea of this feasibility study was to search for specifi c gene expression regulations induced by stanozolol to identify the possible infl uence of the synthetically hormone on different metabolic pathways. Finding biomarkers for anabolic drugs could be supportive of the existing methods and an additional proof for illegal drug abuse. In two separate cell cultures, human HFDPC (hair follicle dermal papilla cells) from a female and a male donor were treated with stanozolol. In the female cell culture treatment concentrations of 0 nM (control), 1 nM, 10 nM and 100 nM were chosen. Cells were taken 0 h, 6 h, 24 h and 48 h after stimulation and totalRNA was extracted. Learning from the results of the pilot experiment, the male cell culture was treated in 10 nM and 100 nM concentrations and taken after 0 h, 6 h, 24 h and 72 h. Using quantitative real-time RT-PCR expression of characteristics of different target genes were analysed. Totally 13 genes were selected according to their functionality by screening the actual literature and composed to functional groups: factors of apoptosis regulation were Fas Ligand (FasL), its receptor (FasR), Caspase 8 and Bcl-2. Androgen receptor (AR) and both estrogen receptors (ERα, ERβ) were summarized in the steroid receptor group. The growth factor group included the insulin like growth factor receptor (IGF1R) and growth hormone receptor (GHR). Fibroblast growth factor 2 (FGF2) and keratinocyte growth factor (FGF7) were summarized in the hair cycle factor group. 5α-Steroidreductases (SRD5A1, SRD5A2) represented the enzyme group. Three reference genes were taken for relative quantifi cation: ubiquitin (UBQ), glycerinaldehyde-3-phsophate-dehydrogenase (GAPDH), and β-actin (ACTB). In cell culture 1 AR, FasR, FGF2 showed signifi cant regulations within one treatment time, signifi cant gene expressions over time were analysed for Caspase 8. In cell culture 2 AR, FasR and SRD5A2 were signifi cantly regulated within one treatment time. In this feasibility study fi rst biomarker for a screening pattern of anabolic agents could be identifi ed providing the rationality to investigate modifi ed, metabolic pathways in the whole hair follicle.

Novel In Vitro Culture Condition Improves the Stemness of Human Dermal Stem/Progenitor Cells

“…Toma et al demonstrated that small punch biopsies of human scalp proliferate in response to SKP medium supplemented with FGF2 and EGF (Toma et al, 2005). Dermal papilla cells derived from hair follicle show higher proliferative property in AmnioMAX C-100 or HFDPC growth medium compared to 10% FBS-DMEM (Driskell et al, 2012;Reiter et al, 2008). In our study, we demonstrated that SKP medium exhibited higher expression level of dermal progenitor genes compared to other culture media, indicating that SKP medium is appropriate for preserving the stemness of hDSPCs (Fig.…”

“…During in vitro culture, hDSPCs, which exhibited stem celllike characteristics in our previous study, gradually lose the expression of transcription factors associated with multipotency and self-renewal, such as SOX2 and NANOG for maintaining the characteristics of stem cells. Several previous reports suggested that the quality of the stem cells strongly depend on the culture method (Driskell et al, 2012;Grayson et al, 2007;Kuroda et al, 2010;Minchenko et al, 1994;Reiter et al, 2008;Ren et al, 2006;Rendl et al, 2005). Thus, we investigated whether hDSPCs retain their stemness in various culture conditions despite long-term culture.…”

“…The four media evaluated were as follows: (i) DMEM containing 10% FBS; (ii) AmnioMAX C-100, which has been reported to maintain dermal papilla gene expression (Driskell et al, 2012;Rendl et al, 2005). (iii) skinderived precursor (SKP) medium containing fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) (Toma et al, 2001; and (iv) Hair follicle dermal papilla cell (HFDPC) growth medium supplemented with FGF2 and insulin (Reiter et al, 2008). Real-time quantitative RT-PCR was used to measure the mRNA levels of SOX2, NANOG, and S100B.…”

Novel Tissue Types for the Development of Genomic Biomarkers

“…To date most research has been in diseases involving skin conditions (Ohyama et al, 2006). However, hair follicles are also being examined for markers in to quantify exposures to pharmaceuticals (Reiter et al, 2008) or toxicology to certain drug targets (Kim et al, 2006).…”

The analysis of the transcriptome as a new approach for biomarker development to trace the abuse of anabolic steroid hormones

“…Cells were harvested at different time points after treatment, RNA was extracted, and gene expression analysis was done via RT‐qPCR for quantifying 13 different genes. Genes like the androgen receptor (AR), the apoptosis factors Fas receptor (FasR) and caspase 8, the fibroblast growth factor FGF7 and the 5α‐Steroidreductase SRD5A2 were detected as significantly regulated, whereas only AR and FasR were significantly regulated in both female and male cells and the other genes were significantly regulated either in female (FGF7, Caspase 8) or in male cells (SRD5A2) 37. In another study, the expression of different genes in plucked hair follicle samples was quantified in female and male hair follicles and also in a small number of hair samples from weightlifters under doping conditions.…”