BACKGROUND Family history is a significant risk factor for prostate cancer, although the molecular basis for this association is poorly understood. Linkage studies have implicated chromosome 17q21-22 as a possible location of a prostate-cancer susceptibility gene. METHODS We screened more than 200 genes in the 17q21-22 region by sequencing germline DNA from 94 unrelated patients with prostate cancer from families selected for linkage to the candidate region. We tested family members, additional case subjects, and control subjects to characterize the frequency of the identified mutations. RESULTS Probands from four families were discovered to have a rare but recurrent mutation (G84E) in HOXB13 (rs138213197), a homeobox transcription factor gene that is important in prostate development. All 18 men with prostate cancer and available DNA in these four families carried the mutation. The carrier rate of the G84E mutation was increased by a factor of approximately 20 in 5083 unrelated subjects of European descent who had prostate cancer, with the mutation found in 72 subjects (1.4%), as compared with 1 in 1401 control subjects (0.1%) (P = 8.5×10−7). The mutation was significantly more common in men with early-onset, familial prostate cancer (3.1%) than in those with late-onset, nonfamilial prostate cancer (0.6%) (P = 2.0×10−6). CONCLUSIONS The novel HOXB13 G84E variant is associated with a significantly increased risk of hereditary prostate cancer. Although the variant accounts for a small fraction of all prostate cancers, this finding has implications for prostate-cancer risk assessment and may provide new mechanistic insights into this common cancer. (Funded by the National Institutes of Health and others.)

Osteopontin (OP) is a component of extracellular, bone, and urinary stone matrices, but the mechanism by which it is stably incorporated into such matrices remains unknown. By SDS-PAGE analysis of [125I]OP, treated with a catalytic amount of TG, we first demonstrate both intra- and intermolecular covalent cross-linking of OP. Most importantly, the analysis of the products generated from reactions containing OP, Fn, and TG by SDS-PAGE, autoradiography, and Western blotting using either OP or Fn antibody, and quantitation of TG-catalyzed epsilon-(gamma-glutamyl)lysine isopeptide formation between OP and Fn demonstrate, for the first time, covalent cross-linking between these two proteins. Similar reactions in the presence of polyamine substrates of TG show OP-Fn intermolecular cross-linking via N,N-bis-(gamma-glutamyl)polyamine formation. Finally, immunoprecipitation of 125I-labeled NRK cell surface proteins with anti-OP and anti-Fn antibodies, SDS-PAGE analysis, and autoradiography provides critical evidence for nonreducible OP-Fn cross-linking in vivo. These results clearly suggest that TG-mediated cross-linking between OP and Fn represents one of the most likely mechanisms by which OP becomes covalently linked to bone matrix, urinary stone matrix, and to ECM.

No abstract

Osteopontin (OP), purified from rat bone, binds Ca2+ but whether different molecular forms of OPs derived from non-osteogenic sources and non-phosphorylated OP also possess this property remains to be determined. Furthermore, it is not known which specific site or sites of the molecule bind Ca2+. In the present study, following an established procedure, total proteins in the conditioned media from OP-synthesizing cell cultures were separated by SDS-PAGE, transferred to Immobilon-P membranes, and incubated with 45CaCl2, then Ca2+ ions bound to protein bands were analyzed by autoradiography. Purified OPs, and synthetic oligopeptides representing specific domains of the OP molecule were adsorbed on the membrane and processed as described above. Our results show that OPs synthesized by normal rat kidney cells, oncogenically transformed Rat-1 cells, OP purified from human milk, and non-phosphorylated OP secreted by 1 alpha, 25-dihydroxyvitamin D3-treated mouse epidermal JB6 cells all bind detectable levels of Ca2+ with specificity. We also show that a synthetic peptide representing the domain of OP which contains nine consecutive aspartic acid residues binds Ca2+ with specificity. It is probable, therefore, that a Ca(2+)-binding site resides in this region of the OP molecule. We conclude that Ca(2+)-binding is a general property of OP, irrespective of its molecular mass and origin, and the phosphate moieties of OP may not influence the conformation or accessibility of the Ca2+ affinity sites of the molecule.

No abstract

Multimeric ligands are ligands that contain multiple binding domains that simultaneously target multiple cell-surface proteins. Due to cooperative binding, multimeric ligands can have high avidity for cells (tumor) expressing all targeting proteins and only show minimal binding to cells (normal tissues) expressing none or only some of the targets. Identifying combinations of targets that concurrently express in tumor cells but not in normal cells is a challenging task. Here, we describe a novel approach for identifying such combinations using genome-wide gene expression profiling followed by immunohistochemistry. We first generated a database of mRNA gene expression profiles for 28 pancreatic cancer specimens and 103 normal tissue samples representing 28 unique tissue/cell types using DNA microarrays. The expression data for genes that encode proteins with cell-surface epitopes were then extracted from the database and analyzed using a novel multivariate rule-based computational approach to identify gene combinations that are expressed at an efficient binding level in tumors but not in normal tissues. These combinations were further ranked according to the proportion of tumor samples that expressed the sets at efficient levels. Protein expression of the genes contained in the top ranked combinations was confirmed using immunohistochemistry on a pancreatic tumor tissue and normal tissue microarrays. Coexpression of targets was further validated by their combined expression in pancreatic cancer cell lines using immunocytochemistry. These validated gene combinations thus encompass a list of cellsurface targets that can be used to develop multimeric ligands for the imaging and treatment of pancreatic cancer. [Mol Cancer Ther 2008;7(9):3071 -80]

Terameprocol can be safely administered to advanced leukemia patients, sufficient drug exposure was obtained and clinical activity and biomarker modulation were observed.

It has been reported previously that oncogenically transformed cells secrete different molecular forms of osteopontin (OPN), a sialic acid-rich, adhesive, phosphoglycoprotein, than OPNs secreted by their nontransformed counterparts. However, the origin of the OPN isoform secreted by the transformed cells and whether it has different physiological properties which may serve transformation-specific functions remain poorly understood. Here, we report that Rat-1 cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsB77) secrete two discrete molecular forms of OPN, a 69-kDa OPN at the nonpermissive temperature (41 degrees C) and a 62-kDa form at the permissive temperature (34 degrees C). However, tsB77 cells at both temperatures transcribe a single 1.6 kb OPN mRNA and contain only the 69-kDa form of OPN intracellularly, suggesting that the 69-kDa OPN is modified to the 62-kDa form prior to or immediately after secretion by cells at 34 degrees C. We ruled out proteolytic cleavage, differential phosphorylation, or lack of N- or O-linked carbohydrates as the possible mechanism, but found that the 62-kDa OPN contains significantly reduced levels of sialic acid, as compared to its 69-kDa form. The binding assays using 32P-labeled OPN revealed that only the 69-kDa OPN, not its 62-kDa form, undergoes receptor-mediated localization on the cell surface, although tsB77 cells synthesize OPN receptors (alpha(v)beta3 integrins) at both permissive and nonpermissive temperatures. Furthermore, 125I-labeled purified milk OPN, which is highly sialylated and shows cell surface binding, upon digestion with neuraminidase failed to interact with the cell surface. Taken together, these results suggest that the difference between the 69-kDa and 62-kDa isoforms of OPN resides in their sialic acid content, and sialylation of OPN is crucial for its receptor-mediated binding on tsB77 cells. The data presented here demonstrate for the first time a physiological role of sialic acids in this protein, and raise the possibility that oncogenically transformed tsB77 cells may exploit the lack of OPN-receptor interactions for their invasive behavior.