Correspondence to: Wendell Yarbrough, M.D., University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Room 237, Campus Box 7295, Chapel Hill, NC 27599-7295.
In the September 15, 1999, issue of the Journal, we published our findings of the biologic and biochemical analyses of tumor-associated p16INK4a mutants (1). Dr. Hansson correctly notes that functional analyses of the proline-48 leucine (P48L) mutant differ from report to report. Previously published analyses of p16 mutants are confusing due to conflicting results, different methodologies, and incomplete testing for all known functions of p16namely cyclin-dependent kinase (CDK) binding, inhibition of CDK activity, and ability to cause a cell cycle arrest.
Dr. Hansson suggests that data from three reports contradict our finding that the P48L mutant maintains CDK binding. Castellano et al. (2) show that immunoprecipitated p16, from a cell line known to contain the P48L mutant, is associated with endogenous CDK6 but not CDK4. Reports by Ruas et al. (3) and Hashemi et al. (4) test binding of CDK4 and CDK6 by mixing in vitro-translated (IVT) CDKs and p16 mutants followed by immunoprecipitation with antibodies specific to p16. With the use of the IVT binding assay, Ruas et al. (3) report that the P48L mutant has greatly reduced, but detectable binding to CDK4 and CDK6, whereas Hashemi et al. (4) suggest that CDK binding is lost.
Regarding the ability of the P48L mutant to bind CDKs, these reports are not consistent with one another or with our report. The differences in binding data are likely explained by the use of different methodologies. Our binding assay used affinity precipitation of bacterially produced glutathione S-transferase (GST)-p16 that had been mixed with 35S-labeled in vitro-translated CDKs. Our assay is similar to that described by Ruas et al. (3) and Hashemi et al. (4), but there are some differences that may be critical. First, bacterial production of GST fusion proteins may allow for stabilization of protein structure relative to production of native proteins in a cell-free system. Second, production of proteins by IVT can result in decreased binding, even if the proteins are known to associate in vivo (i.e., CDK4 and cyclin D), an effect that could be enhanced by mutational alteration of protein structure. Third, bacterial production of GST-p16 mutants allows the addition of greater quantities of the p16 mutant in the binding assay relative to the IVT CDKs. Increasing the amount of mutant p16 relative to the CDKs may allow detection of CDK binding by mutants with decreased binding affinity. Our data are internally consistent in that the P48L GST-p16 fusion protein maintained some CDK6 inhibitory activity, a function that relies on CDK binding. Despite CDK binding and inhibitory activity, our data suggest that the P48L mutant has no cell cycle inhibitory properties, even when greatly overexpressed, and, as such, mutations resulting in this amino acid substitution should be considered inactivating (1).
Dr. Hansson's letter supports our contention that multiple assays are necessary to determine the functional activity of p16 mutants and that slight variations in methodology may alter conclusions regarding the activity of individual p16 mutants. Comparison of our assays of p16 activity suggests that the in vivo cell cycle arrest assay is most sensitive and allows detection of p16 mutants with partial loss of function.
REFERENCES
1
Yarbrough WG, Buckmire RA, Bessho M, Liu ET. Biologic and biochemical analyses of p16INK4a mutations from primary tumors. J Natl Cancer Inst 1999;91:156974.
2 Castellano M, Pollock PM, Walters MK, Sparrow LE, Down LM, Gabrielli BG, et al. CDKN2A/p16 is inactivated in most melanoma cell lines. Cancer Res 1997;57:486875.[Abstract]
3 Ruas M, Brookes S, McDonald NQ, Peters G. Functional evaluation of tumour-specific variants of p16INK4a/CDKN2A: correlation with protein structure information. Oncogene 1999;18:542334.[Medline]
4 Hashemi J, Linder S, Platz A, Hansson J. Melanoma development in relation to non-functional p16/INK4A protein and dysplastic naevus syndrome in Swedish melanoma kindreds. Melanoma Res 1999;9:2130.[Medline]
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