Increased expression of Dyrk1a in HPV16 immortalized keratinocytes enable evasion of apoptosis

Immortalization is a critical event in virus-related oncogenesis. No enough information, however, is currently available to elucidate the changes that occur in cellular molecules during immortalization. To identify potential cellular markers or regulators involving in immortalization, a paired-cell model of primary foreskin keratinocytes (FK) and HPV16 immortalized foreskin keratinocytes were established. Using mRNA differential display, RT-PCR and Northern blot methods, we have identified and confirmed that Dyrk1a (dual-specificity tyrosine-phosphorylated and regulated kinase 1A) is present and increased in HPV16 immortalized cells, but is absent in primary keratinocytes. Moreover, transfection of E7 siRNA oligo into immortalized cells leads to a diminishing E7 expression and the eventual disappearance of Dyrk1a. Similar results of Dyrk1a expressional differences could also be seen when tissue specimens were compared using LCM/real-time PCR and immunohistochemistry analysis; malignant cervical lesions contain significantly more DYRK1A than normal tissue. It was also demonstrated that raised DYRK1A could rearrange the cellular localization of FKHR (forkhead in rhabdomyosarcoma), an apoptosis activator, and suppress BAD. Importantly, this phenomenon can be reversed when endogenous Dyrk1a was knocked down in immortalized cells by RNA interference. These results suggest that the raised Dyrk1a in HPV16 immortalized keratinocytes and cervical lesions may serve as a candidate antiapoptotic factor in the FKHR regulated pathway and initiate immortalization and tumorigenesis gradually. (c) 2007 Wiley-Liss, Inc.