FBXL16 is a novel E2F1-regulated gene commonly upregulated in p16INK4A- and p14ARF-silenced HeLa cells

Two crucial cell cycle regulators, p16INK4A and p14ARF, are produced from the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene locus by alternative reading frames; these regulators act as tumor suppressors during tumorigenesis. However, the molecular events incidental to the acute functional loss of CDKN2A remain a critical issue. Two pivotal regulatory pathways of cell fate determination involving p16INK4A/retinoblastoma protein (pRb)/E2F1 and p14ARF/p53 interact tightly with each other; however, novel factors with an integral or overlapping role in these two pathways remain incompletely defined. To this end, we specifically decreased the expression of p16INK4A or p14ARF proteins using RNA interference (RNAi) in HeLa cells. Using a DNA microarray approach, we showed that several genes are commonly regulated in both p16INK4A and p14ARF knockdown cells, compared with control RNA-treated cells. We focused on the FBXL16 (F-box and leucine-rich repeat protein 16) gene, the expression of which was reproducibly upregulated in p16INK4A and p14ARF knockdown cells, as evaluated using RT-PCR. Interestingly, the promoter region of FBXL16 was shown to be upregulated by activator E2Fs. Finally, RNAi-mediated knockdown of FBXL16 increased the cell proliferation rate of HeLa cells. Together, our results illustrate a unique aspect of the interdependence between the p16INK4A/pRb/E2F1 and p14ARF/p53 pathways at a molecular level.