The p16INK4a (MTS1) and pl8INK4c gene products are normal, and highly expressed, in human
neuroblastoma cell lines. The
retinoblastoma protein (pRb) was, nonetheless, phosphorylated and functional in these cells. Such high levels of p16INK4a/p18INK4c should normally inhibit
cyclin-dependent kinase (CDK) 4 and 6 activities in cells containing functional pRb, delaying cell cycle progression and growth. These
neuroblastoma cell lines express both CDK4 and CDK6
mRNA and
protein, but only significant
CDK6 protein kinase activity was detected in this study. In addition, CDK6 was not present in p16INK4a
immune complexes in cells with significant
kinase activity, although p16INK4a levels were high. Others have shown that a specific mutation in the NH2-terminal region of the CDK4 gene product can disrupt p16INK4a binding, thereby bypassing its inhibitory activity. To determine whether mutation of the CDK6 gene, or some other mechanism, is responsible for the CDK6
kinase activity in these cell lines, several complementary analyses were performed. The CDK6 gene from each cell line was examined for mutations that might affect p16INK4a binding, whereas p16INKa add-back experiments were performed with CDK6
immune complexes to assess p16INK4a function. A bona fide CDK6 mutation that disrupts p16INK4a binding and prevents inhibition of
CDK6 protein kinase activity was identified in 1 of 17
neuroblastoma cell lines. The mechanism(s) responsible for disruption of p16INK4a inhibitory activity in the remaining cell lines is unknown, but these results suggest that
neuroblastoma cells may bypass the cell cycle block imposed by constitutive expression of wild-type p16INK4a in novel ways.