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

Abstract

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. ' 2007 Wiley-Liss, Inc.Key words: HPV16; keratinocytes; Dyrk1a; immortalization; apoptosis Mammalian cells have a limited life span and proliferating capacity when cultivated in vitro. Replicate senescence or the terminal arrest state, has been found to be accompanied by several molecular changes that lead to an activated suppression of the cell cycle and the shortening of the telomeres. 1 Similar to apoptosis, cellular senescence is thought to be a mechanism of tumor suppression because it prevents the outgrowth of cells that have acquired mutations in genes rendering them cancerous. Consistent with this model, several tumor suppressor genes (such as, overexpressed p16 or ZNF217, activated p53 and deregulated Rassf1a or c-myc, etc) or their products have been reported and shown to be associated in parallel with telomere irregularities at the onset of cellular senescence. 2 Telomere shortening has been shown to generate unstable DNA leading to p53 activation and the subsequent transcriptional induction of the cdk inhibitor p21 CIP13 that moves cells into senescence state. Therefore, preventing telomere erosion by ectopic expression of telomerase is sufficient to immortalized primary cells in vitro and thus extend life span. [4][5][6] Although it works in human fibroblast, telemorase alone, however, is not sufficient to induce immortalization in human keratinocytes and mammary epithelial cells. 7 To identify other factors that are involved ...

MiR-1246: A new link of the p53 family with cancer and Down syndrome

“…The first evidence for the possible involvement of DYRK1A in tumorigenesis was the observation that DYRK1A is highly expressed in HPV16 immortalized keratinocytes and malignant cervical lesions compared with their primary foreskin keratinocytes and normal cervical tissues, respectively. 28 In line with this result is the finding that knockdown of endogenous DYRK1A in HPV16 immortalized keratinocytes leads to apoptosis, 28 suggesting that DYRK1A may act as a cell survival and anti-apoptotic molecule in cells. Consistent with this notion, DYRK1A appears to utilize several different mechanisms to inhibit apoptosis and to promote cell proliferation.…”

“…Since DYRK1A is highly expressed in Down syndrome patients due to trisomy 21 and is shown to promote cell survival and to inhibit apoptosis, 26,28,[31][32][33][34] it would be predicted that these patients might have the high risk of developing cancer. However, the story might not be this simple, as some Down syndrome-associated proteins were also found to induce apoptosis in cells, such as ETS2.…”

Intrinsic Structural Disorder in Adenovirus E1A: a Viral Molecular Hub Linking Multiple Diverse Processes

“…It is unclear why E1A targets Dyrk1A and -1B, but the interaction with E1A stimulates their kinase activity in vitro and their binding overlaps the CtBP binding site (148). In an interesting parallel, the immortalization of keratinocytes by HPV16 E7 results in an increase in Dryk1A expression, and a similar increase in Dyrk1A expression is present in malignant HPV-positive cervical cancers, where it has been proposed to function as an antiapototic factor (15). E1A may utilize Dyrk1A as a survival factor in a manner similar to that of HPV E7, but this remains to be determined.…”

Down-regulation of miR-1246 in cervical cancer tissues and its clinical significance

“…DYRK1A expression level is higher in immortalized HPV16-infected keratinocyte cells. DYRK1A disappears when the HPV E7 oncoprotein is silenced, and this leads to increased cell apoptosis [13]. It has been reported that DYRK1A and DYRK3 may be involved in tumorigenesis through a phosphorylation event that activates SIRT1.…”

“…In addition, DYRK1A is also present and increased in HPV16 immortalized cells, but not in primary keratinocytes. Transfection of E7 siRNA oligo into immortalized cells leads to diminish E7 expression as well as DYRK1A [13]. Given the clues about miR-1246, we are interested with miR-1246 in cervical cancer development.…”