Taken together, these findings suggest that Slug plays an important role during wound re-epithelialization in adult skin and indicate that Slug controls some aspects of epithleial cell behavior in adult tissues as well as during embryonic development.

Purpose: Carcinoma progression is linked to a partially dedifferentiated epithelial cell phenotype.As previously suggested, this regulation could involve transcription factors, Snail and Slug, known to promote epithelial-mesenchymal transitions during development. Here, we investigate the role of Snail and Slug in human breast cancer progression. Experimental Design: We analyzed Snail, Slug, and E-cadherin RNA expression levels and protein localization in large numbers of transformed cell lines and breast carcinomas, examined the correlation with tumor histologic features, and described, at the cellular level, Snail and Slug localization in carcinomas using combined in situ hybridization and immunolocalization. Results: In contrast with transformed cell lines, Slug was found to colocalize with E-cadherin at the cellular level in normal mammary epithelial cells and all tested carcinomas. Snail also colocalized at the cellular level with E-cadherin in tumors expressing high levels of Snail RNA. In addition, Snail was significantly expressed in tumor stroma, varying with tumors. Slug and Snail genes were significantly overexpressed in tumors associated with lymph node metastasis. Finally, the presence of semidifferentiated tubules within ductal carcinomas was linked to Slug expression levels similar to or above normal breast samples. Conclusions: These results suggest that Snail or Slug expression in carcinoma cells does not generally preclude significant E-cadherin expression. They emphasize a link between Snail, Slug, and lymph node metastasis in a large sampling of mammary carcinomas, and suggest a role for Slug in the maintenance of semidifferentiated structures. Snail and Slug proteins seem to support distinct tumor invasion modes and could provide new therapeutic targets.

We explored, by cDNA mini-arrays, gene expression measurements of MVLN, a human breast carcinoma cell line derived from MCF-7, after 4 days of exposure to 17 -estradiol (E 2 ) treatment, in order to extend our understanding of the mechanism of the pharmacological action of estrogens. We focused on 22 genes involved in estrogen metabolism, cell proliferation regulation and cell transformation. genes. The temporal response of these gene expression regulations was then investigated after 6 and 18 h of E 2 treatment and this allowed the identification of different time-course patterns. Cycloheximide treatment studies indicated first that estrogen affected the transcript levels of ABCC3 and ABCC5 through dissimilar pathways, and secondly that protein synthesis was needed for modulation of the expression of the CCNA2 and TACC1 genes by estrogens. Western blot analysis performed on TFF1, IRS1, IGFBP4, amphiregulin, PCNA, cyclin A2, TACC1 and ABCC5 proteins confirmed the mini-array and RTQ-PCR data, even for genes harboring low variations of mRNA expression. Our findings should enhance the understanding of changes induced by E 2 on the transcriptional program of human E 2 -responsive cells and permit the identification of new potential diagnostic/prognostic tools for the monitoring of estrogen-related disease conditions such as breast cancer.

Background & Aims: Hints, Histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2.

The molecular basis of capacitative (or store-operated) Ca 2+ entry is still subject to debate. The transient receptor potential proteins have been hypothesized to be structural components of store-operated Ca 2+ channels and recent evidence suggests that Trp3 and its closely related homolog Trp6 are gated by the N-terminal region of the inositol 1,4,5-triphosphate receptors (InsP 3 R). In this study, we report the existence of two isoforms of the human Trp4 protein, referred to as K K-hTrp4 and L L-hTrp4. The shorter variant L L-hTrp4 is generated through alternative splicing and lacks the C-terminal amino acids G 785Ŝ 868 . Using a yeast two-hybrid assay and glutathione-Stransferase-pulldown experiments, we found that the C-terminus of K K-hTrp4, but not of L L-hTrp4, associates in vitro with the Cterminal domain of the InsP 3 receptors type 1, 2 and 3. Thus, we describe a novel interaction between Trp proteins and InsP 3 R and we provide evidence suggesting that the formation of hTrp4^InsP 3 R complexes may be regulated by alternative splicing. ß

Dynein heavy chains (DHCs) are the main components of multisubunit motor ATPase complexes called dyneins. Axonemal dyneins provide the driving force for ciliary and flagellar motility. Recent molecular studies demonstrated that multiple DHC isoforms are produced by separate genes. We describe the isolation of five human axonemal DHC genes. Analysis of the human genomic clones revealed the existence of intronic sequences that were used to demonstrate that human axonemal DHC genes are located on different chromosomes. The cloned human DHC sequences were integrated into an evolutionary approach based on phylogenetic analysis. Tissue expression studies showed that these human axonemal DHCs are expressed in testis and/or trachea, two tissues with axonemal structures that can be altered in primary ciliary dyskinesia, making DHC genes strong candidates in the genesis of these human diseases.

Ca 2؉ signals mediate the hepatic effects of numerous hormones and growth factors. Hepatic Ca 2؉ signals are elicited by the inositol trisphosphate receptor, an intracellular Ca 2؉ channel. Three isoforms of this receptor have been identified; they are expressed and regulated differently. We investigated the effect of liver fibrosis and cirrhosis on the hepatic expression of the inositol trisphosphate receptor isoforms. Two different rat models were used: bile duct ligation (fibrosis) and chronic exposure to CCl 4 /phenobarbital (cirrhosis). Messenger RNA levels were determined by ribonuclease protection assay (RPA), competitive polymerase chain reaction (PCR) followed by Southern blotting, and real-time quantitative PCR. Protein expression was assessed by Western blotting; tissue distribution was assessed by immunohistology. In control animals, isoform 2 was the predominant isoform, isoform 1 represented less than one third, and isoform 3 less than 1%. After bile duct ligation, expression of types 1 and 3 increased 1.9-and 5.7-fold, and expression of type 2 decreased 2.5-fold at the protein level. After exposure to CCl 4 /phenobarbital, expression of types 1, 2, and 3 were 2.4-, 0.9-, and 4.2-fold their expression in control animals. Type 2 was localized to the apical domain of hepatocytes, consistent with a role for Ca 2؉ signals in canalicular function. Type 3 was detectable in intrahepatic bile duct epithelial cells and not in hepatocytes, suggesting that Ca 2؉ signals may be regulated differently in these cells. Signaling through inositol trisphosphate receptor participates in the pathogenesis of cirrhosis, because this process affects the expression of its isoforms. (HEPATOLOGY 1999;30:1018-1026.)Development of cirrhosis implies not only remodeling of the liver architecture, but also dramatic changes in the functions of hepatic cells. These changes are orchestrated by a network of receptors, protein-kinases, and transducers that compose complex signaling pathways. Among them, the inositol 1,4,5-trisphosphate receptor (IP 3 R) plays a central role. 1 Numerous hormones and growth factors bind to receptors on plasma membrane to stimulate the production of inositol 1,4,5-trisphosphate (IP 3 ) and to trigger IP 3 -dependent Ca 2ϩ release through the Ca 2ϩ channel of the IP 3 R. 2 Ca 2ϩ signals regulate many cellular functions from gene expression and cellular division to metabolic pathways and cellular secretion. 1 Both hepatocytes and biliary epithelial cells demonstrate complex Ca 2ϩ signals, which rely essentially on IP 3 R, because the ryanodine receptor, another intracellular Ca 2ϩ channel, could not be detected in liver. 3 In response to hormonal stimulation, intracellular Ca 2ϩ concentration oscillates in hepatocytes and even forms Ca 2ϩ waves through the entire lobule. 4 Hepatocellular Ca 2ϩ signals start in a specific apical location and diffuse in an apical to basal manner. 5 IP 3 -dependent Ca 2ϩ signals stimulate bile canalicular contractions, an effect blocked by nitric oxide. 6 In intrahepatic bil...

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