RANKL is a TNF family member that mediates osteoclast formation, activation, and survival by activating RANK. The proresorptive effects of RANKL are prevented by binding to its soluble inhibitor osteoprotegerin (OPG). Recombinant human OPG-Fc recognizes RANKL from multiple species and reduced bone resorption and increased bone volume, density, and strength in a number of rodent models of bone disease. The clinical development of OPG-Fc was discontinued in favor of denosumab, a fully human monoclonal antibody that specifically inhibits primate RANKL. Direct binding assays showed that denosumab bound to human RANKL but not to murine RANKL, human TRAIL, or other human TNF family members. Denosumab did not suppress bone resorption in normal mice or rats but did prevent the resorptive response in mice challenged with a human RANKL fragment encoded primarily by the fifth exon of the RANKL gene. To create mice that were responsive to denosumab, knock-in technology was used to replace exon 5 from murine RANKL with its human ortholog. The resulting ''huRANKL'' mice exclusively express chimeric (human/murine) RANKL that was measurable with a human RANKL assay and that maintained bone resorption at slightly reduced levels versus wildtype controls. In young huRANKL mice, denosumab and OPG-Fc each reduced trabecular osteoclast surfaces by 95% and increased bone density and volume. In adult huRANKL mice, denosumab reduced bone resorption, increased cortical and cancellous bone mass, and improved trabecular microarchitecture. These huRANKL mice have potential utility for characterizing the activity of denosumab in a variety of murine bone disease models.

The increased expression of G 1 cyclins has been associated with the many types of human tumors. In primary solid tumors however, the expression and activity of cyclin E2, the newest member of the G 1 cyclin family, is largely unknown. In this study we have analysed the expression of the E-type cyclins in primary solid tumors from breast, lung, uterus, ovary, colon, and rectal tissues. Relative gene expression was analysed by quantitative real-time reverse transcription polymerase chain reaction (Taqman). The levels of cyclin E1 and cyclin E2 were significantly elevated (23 vs 38%, respectively) in primary breast tumor samples relative to normal breast tissue controls. We also observed an inverse correlation between the expression of cyclin E1/ E2 and estrogen receptor in breast tumors. Our results demonstrate that the expression and associated catalytic activity for both cyclin E1 and cyclin E2 is elevated in primary breast tumors when compared to normal breast tissue. The increased level of cyclin E2 in breast tumors suggests that, similar to cyclin E1, it may contribute to the pathogenesis of breast cancer. Oncogene (2002) 21, 8529 -8534. doi:10.1038/sj.onc. 1206035Keywords: Cyclin E2; Cyclin E1; Cdk2; breast tumor Results and DiscussionA number of studies have demonstrated a strong correlation between elevated levels of cyclin E1 and tumor progression and mortality (Muller-Tidow et al., 2001;Geng et al., 2001;Keyomarsi et al., 1994; GrayBablin et al., 1996;Loden et al., 1999;Nielson et al., 1996;Porter et al., 1997;Bortner and Rosenberg, 1997). In addition, there is a correlation between elevated cyclin E1-Cdk2 kinase activity, increased phosphorylation of Rb, and increased rates of tumor cell proliferation in breast cancers (Loden et al., 1999). However, the expression and activity of cyclin E2 in solid tumors is largely unknown. Our initial characterization of cyclin E2 expression in tumor derived cell lines and primary mouse embryonic fibroblasts lacking Rb demonstrated a strong association between elevated levels of cyclin E2 and the functional inactivation of Rb (Gudas et al., 1999;Geng et al., 2001). We also demonstrated that cyclin E2 expression was elevated in lung tumor-derived cell lines in comparison to cell lines derived from normal lung tissue. Two recent studies evaluated the expression of cyclin E2 in early stage primary Non-Small Cell Lung Carcinoma (NSCLC) and in primary breast carcinoma (Muller-Tidow et al., 2001;Geng et al., 2001). Interestingly, no significant elevation of cyclin E2 transcript levels was found in any of the lung tumors. In contrast, increased expression of cyclin E2 was observed in 25% of primary breast carcinomas.To confirm and extend these recent observations we have analysed the expression of cyclin E2 in a variety of primary tumors and determined the catalytic activity associated with both cyclin E2 and cyclin E1 in primary breast tumors. Quantitative real-time RT -PCR (Taqman) analysis of primary tumor RNAs was used to determine the Relative Gene Expression (RGE) val...