Expression of p15 and p15.5 products in neuroendocrine lung tumours: relationship with p15^INK4b methylation status

Abstract

The cell cycle inhibitor p15^INK4B is frequently inactivated by homozygous deletions together with p16^INK4a/p14^ARF in many tumour types. Although it is now well established that p16^INK4a and p14^ARF act as tumour suppressor genes, the role of p15^INK4b remains to be well defined. In order to explore the possibility of a selective deregulation of p15^INK4b in human lung carcinogenesis, we studied p15^INK4b status in neuroendocrine (NE) lung tumours where homozygous deletions of the p16^INK4a/p14^ARF locus are rarely observed. Expressions of p15 and p15.5 protein isoforms were analysed in a series of eight control normal lung, 12 tumour-associated normal lung, five low grade and 15 high grade neuroendocrine (NE) lung tumours and relationship with a specific p15^INK4b methylation status was studied. Using Western blot analysis, we showed that p15 and p15.5 isoforms displayed a high heterogeneous pattern of expression in both normal and tumour tissues. P15 and p15.5 expressions were correlated in control normal lung (P<0.04) whereas they were not in tumours and associated normal lung. The level of p15.5 was significantly higher in associated normal lung and in tumours (P<0.02 respectively), specially in low grade tumours (P<0.01), than in control normal lung. Furthermore, p15.5 expression was more variable in tumours than in normal lung (P<0.01) and in low grade than in high grade NE lung tumours (P<0.02). Levels of p15 and p15.5 were distinct (up- or downregulated) from those observed in paired normal lung in 4/12 (33%) and 10/12 (83%) tumours respectively. Aberrant methylation at the 5′ end of p15^INK4b gene was observed in 15% of NE lung tumours using PCR-based assay, in a region proximal to the translation start where methylation did not occur in control and associated normal lung. However, no correlation could be assessed with protein status. MSP analysis of CpG islands proximal to the transcription start revealed methylation in all normal and tumour samples. No correlation was found between p15^INK4b and p16^INK4a or p14^ARF status. These data suggest that complex deregulation of p15.5 is implicated in the carcinogenesis of human NE lung tumours independently of p16^INK4a and p14^ARF status.