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Disruption of the Cyclin D/Cyclin-dependent Kinase/INK4/Retinoblastoma Protein Regulatory Pathway in Human Neuroblastoma¹

Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101

The p16^INK4a (MTS1) and p18^INK4c 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 p16^INK4a/p18^INK4c 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 p16^INK4a immune complexes in cells with significant kinase activity, although p16^INK4a levels were high. Others have shown that a specific mutation in the NH₂-terminal region of the CDK4 gene product can disrupt p16^INK4a 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 p16^INK4a binding, whereas p16^INKa add-back experiments were performed with CDK6 immune complexes to assess p16^INK4a function. A bona fide CDK6 mutation that disrupts p16^INK4a 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 p16^INK4a 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 p16^INK4a in novel ways.

¹ This research was supported by NIH Grant CA 67938 (to V. J. K.), Cancer Center Core Grant CA 21765 (to St. Jude Children's Research Hospital), and by the American Lebanese Syrian Associated Charities (to V. J. K. and J. M. L.).

² Present address: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101.

³ To whom requests for reprints should be addressed, at Department of Tumor Cell Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38101. Phone: (901) 495-3597; Fax: (901) 495-2381.

Received 12/ 9/97. Accepted 4/20/98.

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