We have analyzed expression conferred by two domains from the cauliflower mosaic virus (CaMV) 35S promoter and found different patterns in seeds, seedlings and seven week old plants. Expression from domain A (‐90 to +8) is strongest in the radicle of the embryo, the radicle pole of the endosperm and in root tissue of seedlings and mature plants. Expression from domain B (‐343 to ‐90) is strongest in the cells adjacent the cotyledon of the endosperm, in the cotyledons of the embryo and seedings and in the leaves and stem of mature plants. When both domain A and domain B are present expression is detectable in most tissues at all stages of development. Thus analysis of a constitutive promoter in transgenic plants can be used to identify cis elements that confer tissue specific and developmentally regulated expression.

The cauliflower mosaic virus (CaMV) 35S enhancer is able to confer strong constitutive expression in plants. We have previously defined two domains within this enhancer that can confer different tissue‐specific expression patterns throughout development. We show here that the upstream domain (B) has a modular organization. It contains at least five subdomains that are able to confer distinct expression patterns when fused to the downstream domain (A). When fused to a minimal promoter only three of the five subdomains give any expression in the early stages of plant development. Comparison of the expression patterns conferred by the subdomains alone, in combination with the downstream domain or in combination with other subdomains provides evidence for synergistic interactions among cis‐elements within the 35S enhancer.

Metastasis continues to be the leading cause of mortality for patients with cancer. High expression of the chemokine receptor CXCR4 correlates with poor prognosis in many cancers, including osteosarcoma and melanoma. CXCL12, the ligand for CXCR4, is expressed at high levels in the lung and lymph node, which are the primary sites to which these tumors metastasize respectively. These findings suggest that therapy aimed at disruption of this specific receptor/ligand complex may lead to a decrease in metastases. CTCE-9908, a small peptide CXCR4 antagonist was utilized in two murine metastasis models to test this hypothesis. Treatment of osteosarcoma cells in vitro with CTCE-9908 led to the following changes: decreased adhesion, decreased migration, decreased invasion, and decreased growth rate. Following tail vein injection of osteosarcoma cells, mice that were treated with CTCE-9908 had a 50% reduction in the number of gross metastatic lung nodules and a marked decrease in micrometastatic disease. Similar findings were observed following injection of melanoma cells and treatment with CTCE-9908. However, these results could only be consistently reproduced when the cells were pre-treated with the inhibitor. A novel ex vivo luciferase assay showed decreased numbers of cells in the lung immediately after injection into mice, when treated with CTCE-9908, suggesting the importance of interactions between the receptor and the ligand. Our findings show that inhibition of the CXCR4/CXCL12 pathway decreases metastatic disease in two murine tumor models and expands on previous reports to describe potential mechanisms of action.

Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. Metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of ezrin, and that ezrin coimmunoprecipitated in cells with PKCa, PKCi and PKCc. Furthermore, phosphorylated forms of ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to ezrin phosphorylation. These data are the first to suggest a dynamic regulation of ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.