More specifically, we exposed both wildtype and primary non-immortalized MEFs from exhibit a greater susceptibility to SCC tumorigenesis compared to normal mice in the 7,12-dimethylbenz(a)anthracene/12- em O /em -tetradecanoylphorbol-13-acetate (DMBA/TPA) skin carcinogenesis model (18)

More specifically, we exposed both wildtype and primary non-immortalized MEFs from exhibit a greater susceptibility to SCC tumorigenesis compared to normal mice in the 7,12-dimethylbenz(a)anthracene/12- em O /em -tetradecanoylphorbol-13-acetate (DMBA/TPA) skin carcinogenesis model (18). pLP-IRESneo acceptor using the Creator? kit (Clontech; Mountain View, CA) per manufacturer’s protocol. All plasmids were confirmed by DNA sequencing and immunoblotting after either transfection with FuGENE-6 (Roche Diagnostics; Indianapolis, IN) or Nucleofection (Amaxa; Gaithersburg, MD). Apoptosis assays For both UVB-associated and EphA2-mediated apoptosis, we plated 100,000 cells in 60mm dishes about 21 hours prior to either UVB irradiation (80 mJ/cm2 UVB) or transfection (5 g pLP-IRES-EphA2 or pLP-IRES vectors). Treated cells were analyzed for intracellular casapase-3 activity at 24 hours (UVB irradiation) or 30 hrs (EphA2 transfection) using the Caspase-3-Activity Detection Kit (Upstate; Lake Placid, NY). For propidium iodide (PI) staining, 800,000 cells were harvested and then fixed by 70% ice-cold EtOH overnight. After fixation, cells were washed with PBS and then stained with 100 g/ml PI in PBS containing 100 g/ml RNase A and 0.1% NP40 (all from Sigma; St Louis, MO). PI-stained cells were analysis by FACSCalibur (Becton Dickinson; San Jose, CA). RESULTS UVR-mediated upregulation of EphA2 In order to determine if UVR selectively induces EphA2 among other members of the Eph/ephrin family, we re-analyzed our microarray data(3) to assess the effect of UVR exposure on other ephrins and Eph receptors. As shown in supplemental Figure S1, EphA2 stands out among other Eph/ephrin genes in both level of stimulation and significance of increase. There is no apparent predilection for either Ephrins or Eph receptors, as a group, to be induced. Thus, EphA2, among its relatives, is selectively upregulated by UVR. Our previous data demonstrated an increase in EphA2 mRNA levels in response to UVR(3). Figure 1A shows that EphA2 protein levels are induced by UVR within 5 hours and reach a maximum by approximately 12 hours post-irradiation in normal human melanocytes (NHMs); this level is sustained for at least 48 hours (data not shown). There is also a dose-dependent increase in EphA2 levels starting at about 15mJ/cm2 UVB (Figure 1B) and reaching a maximum around 35-50mJ/cm2. Open in a separate ATF1 window Figure 1 UV-mediated induction of EphA2 is cell autonomous and p53-independentEphA2 induction by UVR in NHMs is time (A; 0, 0.25, 0.5, 2, 9, 12 and 24 hrs) and dose (B; 0, 15, 25, 35, 50 mJ/cm2) dependent. The upregulation of EphA2 is also observed in primary human melanocytes immortalized with p53DD, CDK4R24C and hTERT (data not shown). Since immortalized NHMs have ectopically abrogated p53 function (by p53DD), we also set out to determine if melanoma lines were UVR-responsive. (C) NHMs and immortalized NHMs and NHKs (p53 inactivated by p53DD construct) all show induction of EphA2 by UVR. In the lower bar graph, 8 melanoma cell lines (Lane 1, SK-Mel 28; Lane 2, SK Mel-119; Lane 3, WM35; Lane 4, MM455; Lane 5,A375; Lane 6, UACC903; Lane 7, MM-L; Lane 8, WM164) were also subjected to UVB irradiation and exhibited a range of EphA2 inducible levels. Since the two cell lines with the greatest degree of upregulation (lanes 7 and 8) both harbor 6-O-2-Propyn-1-yl-D-galactose homozygous p53 point mutations, these data suggest that intact p53 function is not needed for EphA2 induction in melanocytic systems. Although not directly mutated, p53 is presumed to 6-O-2-Propyn-1-yl-D-galactose be functionally crippled in cell lines 2-6 where ARF is deleted (ARF). Similar results were obtained with primary human keratinocytes (D). We irradiated NIH-3T3 cells, WT and and to UVR and found that EphA2 protein levels were in fact appropriately induced in all of these MEFs (Figure 1D). Taken together, our results using immortalized melanocytes, p53 and ARF-inactivated melanoma cells and genetically-defined MEFs all strongly support the contention that p53, p63 and p73 are not individually necessary for the observed EphA2 legislation although we can not eliminate the likelihood that there surely is useful compensation between your family 6-O-2-Propyn-1-yl-D-galactose members. It really is worthy of mentioning, however, these results do.