Since its discovery as a CDKI (cyclin-dependent kinase inhibitor) in 1993, the tumor suppressor p16 (INK4A/MTS-1/CDKN2A) has gained widespread importance in cancer. The frequent mutations and deletions of p16 in human cancer cell lines first suggested an important role for p16 in carcinogenesis. This genetic evidence for a causal role was significantly strengthened by the observation that p16 was frequently inactivated in familial melanoma kindreds. Since then, a high frequency of p16 gene alterations were observed in many primary tumors. In human neoplasms, p16 is silenced in at least three ways: homozygous deletion, methylation of the promoter, and point mutation. The first two mechanisms comprise the majority of inactivation events in most primary tumors. Additionally, the loss of p16 may be an early event in cancer progression, because deletion of at least one copy is quite high in some premalignant lesions. p16 is a major target in carcinogenesis, rivaled in frequency only by the p53 tumor-suppressor gene. Its mechanism of action as a CDKI has been elegantly elucidated and involves binding to and inactivating the cyclin D-cyclin-dependent kinase 4 (or 6) complex, and thus renders the retinoblastoma protein inactive. This effect blocks the transcription of important cell-cycle regulatory proteins and results in cell-cycle arrest. Although p16 may be involved in cell senescence, the physiologic role of p16 is still unclear. Future work will focus on studies of the upstream events that lead to p16 expression and its mechanism of regulation, and perhaps lead to better therapeutic strategies that can improve the clinical course of many lethal cancers.

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Murine long-term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM-CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either alpha-MEM with 2% heat-inactivated horse serum alone (alpha) or supplemented with 10% L-cell-conditioned medium (as a source of M-CSF) (L10). Few substrate-attached cells survived in basal alpha medium, but when treated with L10 medium or GM-CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM-CSF had large numbers of multinucleated cells, more than half of which were tartrate-resistant acid phosphatase-positive (TRAP). Osteocalcin further promoted the development of TRAP-positive multinucleated cells; a dose of 0.7 microgram/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate-resistant acid phosphatase activity in the osteocalcin plus GM-CSF group (75.6 +/- 14.2) than in GM-CSF alone (53.3 +/- 7.3). In the absence of M-CSF, GM-CSF stimulated tartrate-resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal-free subpopulation of hematopoietic progenitors in the presence of GM-CSF and L-cell-conditioned medium. These results are consistent with the hypothesis that this bone-matrix constituent plays a role in bone resorption.

Although the hematopoietic origin of the osteoclast is generally accepted, the precise phenotype of the progenitor and the regulation of its differentiation are unclear. This study compares proliferation and differentiation of progenitors in response to macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Nonadherent progenitor cells from murine long-term bone marrow cultures (LTBMC) (as a source of osteoclast progenitors) demonstrated a significant proliferative response to M-CSF. In addition, M-CSF increased the number of multinucleated cells, only a small percent of which (14-16%) were tartrate-resistant, acid phosphatase (TRAP)-positive. In contrast, cells cultured with GM-CSF generated more TRAP-positive multinucleated cells even at concentrations less stimulatory of proliferation than M-CSF. The osteoclast phenotype of these multinucleated cells was also assessed by ultrastructural characterization of ruffled borders in association with bone fragments. The bone-active hormone 1,25-dihydroxyvitamin D3 inhibited the proliferation of this subset of progenitor cells in the presence of M-CSF or GM-CSF. All of these results show effects on progenitors in the absence of the stromal cell microenvironment in this system. These results provide evidence for a divergence in the biological responsiveness of osteoclast progenitor cells to M-CSF compared with GM-CSF; they support the notion that M-CSF has a "priming" effect on osteoclast progenitors whose subsequent differentiation to osteoclastic multinucleated cells is promoted by GM-CSF.