We aimed to analyze the differential gene expression in various murine dental tissues, expecting to find novel factors that are involved in tooth formation. We here describe the identification of a novel ameloblast-specific gene, amelotin (AMTN), by differential display polymerase chain-reaction (DD-PCR) analysis of microdissected ameloblasts, odontoblasts, dental pulp, and alveolar bone cells of 10-day-old mouse incisors. The conceptually translated protein sequence was unique and showed significant homology only with its human orthologue. The amelotin genes from mouse and human displayed a similar exon-intron structure and were expressed from loci on chromosomes 5 and 4, respectively, which have been associated with various forms of amelogenesis imperfecta. Expression of amelotin mRNA was restricted to maturation-stage ameloblasts in developing murine molars and incisors. Amelotin protein was efficiently secreted from transfected cells in culture. Taken together, our findings suggest that amelotin is a novel factor produced by ameloblasts that plays a critical role in the formation of dental enamel.

The RING finger family of proteins possess ubiquitin ligase activity and play pivotal roles in protein degradation and receptor-mediated endocytosis. In this study, we examined whether the breast cancer-associated gene 2 (BCA2), a novel RING domain protein, has E3 ubiquitin ligase activity and investigated its expression status in breast tumors. The fulllength BCA2 gene was cloned from the human breast cancer cell line MDA-MB-468. It encodes an open reading frame of 304 amino acids and contains a RING-H2 domain. BCA2 maps to chromosome 1q21.1, a region known to harbor cytogenetic aberrations in breast cancers. We found that the BCA2 protein has an intrinsic autoubiquitination activity, the hallmark of E3 ligases, whereas mutant RING protein is not autoubiquitinated. This indicates that the BCA2 ubiquitin ligase activity is dependent on the RING-H2 domain. Using tissue microarrays and immunohistochemistry, we found strong to intermediate BCA2 staining in 56% of 945 invasive breast cancers cases, which was significantly correlated with positive estrogen receptor status [odds ratio (OR), 1.51; P = 0.004], negative lymph node status (OR, 0.73; P = 0.02), and an increase in disease-free survival for regional recurrence (OR, 0.45; P = 0.03). Overexpression of BCA2 increased proliferation and small interfering RNA inhibited growth of T47D human breast cancer cells and NIH3T3 mouse cells. The autoubiquitination activity of BCA2 indicates that it is a novel RING-type E3 ligase. Its association with clinical measures and its effects on cell growth indicate that BCA2 may be important for the ubiquitin modification of proteins crucial to breast carcinogenesis and growth. (Cancer Res 2005; 65(22): 10401-12)

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.

Seed size and weight are key traits determining crop yield, which often undergo strongly artificial selection during crop domestication. Although seed sizes differ significantly between oil flax and fiber flax, the genetic basis of morphological differences and artificial selection characteristics in seed size remains largely unclear. Here we resequenced 200 flax cultivated accessions to generate a genome variation map based on chromosome assembly reference genomes. We provide evidence that oil flax group is the ancestor of cultivated flax, and the oil-fiber dual purpose group (OF) is the evolutionary intermediate transition state between oil and fiber flax. Genome-wide association studies (GWAS) were combined with LD Heatmap to identify candidate regions related to seed size and weight, then candidate genes were screened based on detailed functional annotations and estimation of nucleotide polymorphism effects. Using this strategy, we obtained 13 candidate genes related to seed size and weight. Selective sweeps analysis indicates human-involved selection of small seeds during the oil to fiber flax transition. Our study shows the existence of elite alleles for seed size and weight in flax germplasm and provides molecular insights into approaches for further improvement.