Studies of induced transcription in response to an activated Ha-ras, TPA and UV radiation in keratinocytes

The mutational activation of the Ha-ras gene in keratinocytes has been shown to occur during the initiation step in the mouse skin model for multistage carcinogenesis. Skin tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and ultraviolet radiation (UVR) stimulate a selective outgrowth of initiated keratinocytes to form benign tumours (papillomas). The mechanisms that determine the cooperativity between an activated Ha-ras and a tumor promoter is assumed to depend on altered gene expression caused by these two types of transcriptional activators. This thesis describes the characterization of response elements, transcription factors and signalling pathways activated by an Ha-ras gene and tumour promoters respectively. Paper III and IV work describes the identification and characterisation of a 20 bp sequence (B10 RRE), present in the LTR of an endogenous VL30 retrotransposon, that mediates induced transcription in response to an activated Ha-ras, Raf-1 and EGF. B10 RRE is a composite responsive element composed of a 5' AP-1-like sequence and a 3' sequence unrelated to previously known transcription factor binding sites.

The AP-1-like motif preferentially binds ATF3/c-Jun and ATF3/JunD heterodimers, whereas the 3 sequence binds a novel nuclear factor (TAR) with an apparent size of 178 kDa in solution. DNA-affinity chromatography and southwestern blot analysis revealed that the DNA binding component of TAR is a 50 kDa protein. Interestingly, B10 RRE is unresponsive to both TPA and WUVR. Instead, these two stimuli induce transcription through a previously characterised TPA responsive element (VLTRE), present in the same LTR as B10 RRE. VLTRE is demonstrated tobe a target for TPA-and UVR-induced binding of Rel/NF-KB proteins. When comparing the signalling pathways activated by TPA and UVR respectively, it is shown that TPA and UVB activate different sets of Rel/NF-KB proteins and that UVB induced transcription is independent of PKC activity. Furthermore, UVB transactivation is not dependent on a functional Ras protein, and the Raf-1 kinase has only a permissive role in mediating the UVB response.

In conclusion, this work has led to the identification of different response elements and transcription factors that mediate induced transcription in response to an UVR, (papers I and II) respectively. These results now provide tools to further elucidate differences and similarities in intracellular signals activated during the different phases in mouse skin tumourigenesis.