CDK4 and CDK6 delay senescence by kinase-dependent and p16INK4a-independent mechanisms.

Replicative senescence of human diploid fibroblasts (HDFs) is largely implemented by the cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(CIP1). Their accumulation results in a loss of CDK2 activity, and cells arrest with the retinoblastoma protein (pRb) in its hypophosphorylated state. It has become standard practice to bypass the effects of p16(INK4a) by overexpressing CDK4 or a variant form that is unable to bind to INK4 proteins. Although CDK4 and CDK6 and their INK4-insensitive variants can extend the life span of HDFs, they also cause a substantial increase in the levels of endogenous p16(INK4a). Here we show that CDK4 and CDK6 can extend the life span of HDFs that have inactivating mutations in both alleles of INK4a or in which INK4a levels are repressed, indicating that overexpression of CDK4/6 is not equivalent to ablation of p16(INK4a). However, catalytically inactive versions of these kinases are unable to extend the replicative life span, suggesting that the impact of ectopic CDK4/6 depends on their ability to phosphorylate as yet unidentified substrates rather than to sequester CDK inhibitors. Since p16(INK4a) deficiency, CDK4 expression, and p53 or p21(CIP1) ablation have additive effects on replicative life span, our results underscore the idea that senescence is an integrated response to diverse signals.

Chemical structure searching: taking it to the next level.

Chemical structure searching can be challenging. The most common question in a searcher's mind is, "is my search comprehensive?" What he least wishes to hear from a client is, "why did you miss this?" This article will discuss ways of expanding the scope of structure searches conducted in the pharmaceutical industry. Patent offices commonly search a broad 'core' structure in the context of a drug indication (e.g., thyroid cancer) or a drug's mechanism of action (e.g., PDE5 inhibitors). Other approaches include patent citation searching of relevant references, the use of pharmaceutical pipeline databases and inventor/assignee-based searches. During analysis of the results, it can be useful for an analyst to include such references in the report (e.g., as 'reference of possible interest'), while also indicating which techniques were used to retrieve them. Regardless of whether these additional search techniques provide further relevant references, the search report is still more comprehensive and, therefore, less likely to omit relevant art.