Dose and context dependent effects of Myc on epidermal stem cell proliferation and differentiation

Myc is activated in many tumours, yet, paradoxically, stimulates differentiation in mammalian epidermis. To test whether the epidermis responds differently to different levels of Myc, we treated K14MycER transgenic mice with a range of concentrations of the inducing agent, 4-hydroxy-tamoxifen (4OHT). Proliferation was stimulated at all levels of Myc activity; sebocyte differentiation was stimulated at low and intermediate levels; and interfollicular epidermal differentiation at intermediate and high levels. Mutational inactivation of the Myc p21 activated kinase 2 (PAK2) phosphorylation sites increased Myc activity and further enhanced epidermal differentiation. We conclude that Myc induced differentiation acts as a fail-safe device to prevent uncontrolled proliferation and neoplastic conversion of epidermal stem cells expressing high levels of Myc.

Telogen (resting phase of hair growth cycle) skin from male and female mice aged between 6 weeks and 11 months was examined. Mice were treated once with the stated doses of 4OHT dissolved in 100 µl acetone and harvested 4 days later, except when stated. When mice were treated for 8 days 4OHT was applied on days 1, 3, 5 and 7. All experiments were subject to CR-UK ethical review and performed under the terms of a UK Government Home Office licence.

Keratinocyte culture
Primary keratinocytes were isolated from adult mouse skin and cultured with a J2 3T3 feeder layer essentially as described previously (Silva-Vargas et al., 2005).
Spontaneously immortalized keratinocyte lines were generated through repeated passaging of primary keratinocytes as described previously (Silva-Vargas et al., 2005).
To determine colony forming efficiency, 10 3 keratinocytes (passage 7-10), were seeded per well in triplicate wells of 6-well plates. Cells were maintained for 8-12 days in the presence of 4OHT then fixed, stained with 1% Rhodamine B (Acros Organic, Geel, Belgium) and 1% Nile Blue (Acros Organic, Geel, Belgium) (Silva-Vargas et al., 2005) and counted under a Nikon SMZ1000 dissecting microscope. A colony was defined as containing 4 or more cells. Colony forming efficiency was determined in three independent experiments, each performed in triplicate. The size (area) of individual colonies was determined as pixel count using Volocity 4 software. HP siRNA SI01368647 siRNA oligos were used.

Q-PCR
Keratinocytes were either starved for 16hrs in unsupplemented KSFM and then transferred to unsupplemented KSFM ± 4OHT for 5hrs or treated with unsupplemented KSFM ± 4OHT for 16hrs without prior starvation.
Quantitative PCR was performed as described (Frye et al, 2003)  Tissue samples were either fixed overnight in neutral buffered formalin or 4% PFA and embedded in paraffin or else frozen, unfixed, in OCT compound (Miles). Cultured keratinocytes were fixed in 4% PFA and stained with DAPI and anti-ER. Slides were viewed on a Olympus DMI6000 confocal scanning microscope.
For Ki67 quantitation, sections were scanned and analysed using the Ariol SL-50 system (Applied Imaging Corp., San Jose). Six fields (viewed with a 10x objective) of basal layer cells were counted per slide and at least 3 slides (each from a different mouse) were counted per condition. To measure IFE thickness, the distance from the basement membrane to the outermost cornified layer was determined using an Eclipse Nikon standard microscope and NIS Element software. When IFE thickness varied along a section the region of maximum thickness was measured. Data were collected from 20 fields per slide and at least 3 slides per condition. Total SG area was estimated from photographs by measuring the maximum length and diameter of individual sebaceous glands. The FABP5 positive area per SG was determined using Volocity 4 software of scanned sections.

Western blotting
Proteins were extracted from whole skin in RIPA buffer containing a protease inhibitor cocktail (Roche) using a homogenizer (Power Gen 500, Fisher Scientific). Proteins were extracted from cultured keratinocytes by scraping into ice cold HEPES lysis buffer (10mM HEPES pH 8, 10 mM KCl, 0.1 mM EDTA, 0.2% NP-40, Roche protease inhibitor cocktail). Proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to PVDF (NEN) or nitrocellulose (Amersham) membranes. Blots were incubated with primary antibodies at 4°C overnight or for 2 hours at room temperature, washed and incubated with anti-mouse or anti-rabbit horse-radish-peroxidase linked antibodies for 1 hour at room temperature (Amersham Pharmacia). Blots were visualised using the ECL TM detection kit (Amersham Pharmacia). Mouse monoclonal anti-α tubulin (Sigma-Aldrich, clone DM1A T6199) was used to probe blots as a loading control.
For quantitation, blots were analysed using ImageJ software (version 1.34 NIH) (Abramoff et al., 2004). Densitometric values were expressed as fold change relative to control samples, and standardised against the corresponding α-tubulin densitometry values from the same samples.