We have identified a family of resistin-like molecules (RELMs) in rodents and humans. Resistin is a hormone produced by fat cells. RELM␣ is a secreted protein that has a restricted tissue distribution with highest levels in adipose tissue. Another family member, RELM␤, is a secreted protein expressed only in the gastrointestinal tract, particularly the colon, in both mouse and human. RELM␤ gene expression is highest in proliferative epithelial cells and is markedly increased in tumors, suggesting a role in intestinal proliferation. Resistin and the RELMs share a cysteine composition and other signature features. Thus, the RELMs together with resistin comprise a class of tissue-specific signaling molecules.

The tumor suppressor p16INK4a is a potent mediator of cell cycle arrest in transient expression studies, is induced in senescing cells, and can impose morphological features of senescence. Nonetheless, it is unclear whether p16INK4a can block cell proliferation irreversibly. We explored this issue using osteogenic sarcoma cell clones with inducible p16INK4a expression. Induction of p16INK4a for 1 day arrested most cells in G1 phase. If the induction was then interrupted, p16 INK4a levels returned to baseline and robust growth resumed within 3 ± 5 days. When p16INK4a was induced for 6 days DNA synthesis remained strongly inhibited and the cells acquired morphological features of senescence. Moreover, if p16INK4a induction was interrupted at this point and the cells were followed for 12 more days, most cells retained these morphologic features and either failed to divide or died. This occurred despite the prompt return of p16 INK4a expression and retinoblastoma protein phosphorylation toward baseline levels. In fact, some senescing cells appeared to enter S phase. These results demonstrate that a sustained period of p16INK4a expression is sucient in this setting to impose a durable block to cell proliferation and that this state becomes independent of p16 INK4a expression, hypophosphorylation of pRB, or a strict G1 arrest.

The tumor suppressor p16INK4a inhibits cyclin-dependent kinases 4 and 6. This activates the retinoblastoma protein (pRB) and, through incompletely understood events, arrests the cell division cycle. To permit biochemical analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clones in which p16 transcription could be induced. In these clones, binding of p16 to cdk4 and cdk6 abrogated binding of cyclin D1, p27 KIP1 , and p21 WAF1/CIP1 . Concomitantly, the total cellular level of p21 increased severalfold via a posttranscriptional mechanism. Most cyclin E-cdk2 complexes associated with p21 and became inactive, expression of cyclin A was curtailed, and DNA synthesis was strongly inhibited. Induction of p21 alone, in a sibling clone, to the level observed during p16 induction substantially reproduced these effects. Overexpression of either cyclin E or A prevented p16 from mediating arrest. We then extended these studies to HCT 116 colorectal carcinoma cells and a p21-null clone derived by homologous recombination. In the parental cells, p16 expression also augmented total cellular and cdk2-bound p21. Moreover, p16 strongly inhibited DNA synthesis in the parental cells but not in the p21-null derivative. These findings indicate that p21-mediated inhibition of cdk2 contributes to the cell cycle arrest imposed by p16 and is a potential point of cooperation between the p16/pRB and p14 ARF /p53 tumor suppressor pathways.

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