Genomic cloning, chromosomal mapping, and expression analysis of Msal-2

Mutations of SALL1 related to spalt of Drosophila have been found to cause Townes-Brocks syndrome, suggesting a function of SALL1 for the development of anus, limbs, ears, and kidneys. No function is yet known for SALL2, another human spalt-like gene. The structure of SALL2 is different from SALL1 and all other vertebrate spalt-like genes described in mouse, Xenopus, and Medaka, suggesting that SALL2-like genes might also exist in other vertebrates. Consistent with this hypothesis, we isolated and characterized a SALL2 homologous mouse gene, Msal-2. In contrast to other vertebrate spalt-like genes both SALL2 and Msal-2 encode only three double zinc finger domains, the most carboxyterminal of which only distantly resembles spalt-like zinc fingers. The evolutionary conservation of SALL2/Msal-2 suggests that two lines of sal-like genes with presumably different functions arose from an early evolutionary duplication of a common ancestor gene. Msal-2 is expressed throughout embryonic development but also in adult tissues, predominantly in brain. However, the function of SALL2/Msal-2 still needs to be determined. Received: 1 June 1999 / Accepted: 26 August 1999     

Aldehyde dehydrogenase 1 is a putative marker for cancer stem cells in head and neck squamous cancer

Aldehyde dehydrogenase 1 (ALDH1) has been considered to be a marker for cancer stem cells. However, the role of ALDH1 in head and neck squamous cell carcinoma (HNSCC) has yet to be determined. In this study, we isolated ALDH1-positive cells from HNSCC patients and showed that these HNSCC-ALDH1⁺ cells displayed radioresistance and represented a reservoir for generating tumors. Based on microarray findings, the results of Western blotting and immunofluorescent assays further confirmed that ALDH1⁺-lineage cells showed evidence of having epithelial-mesenchymal transition (EMT) shifting and endogenously co-expressed Snail. Furthermore, the knockdown of Snail expression significantly decreased the expression of ALDH1, inhibited cancer stem-like properties, and blocked the tumorigenic abilities of CD44⁺CD24⁻ALDH1⁺ cells. Finally, in a xenotransplanted tumorigenicity study, we confirmed that the treatment effect of chemoradiotherapy for ALDH1⁺ could be improved by Snail siRNA. In summary, it is likely that ALDH1 is a specific marker for the cancer stem-like cells of HNSCC.     

Genetic marker analysis in head and neck cancer

Prognostication of head and neck cancer (HNCC) involves molecular identification of residual tumor cells, prediction of recurrence, distant metastases or secondary tumors and prediction of the sensitvity to therapy. Biomarkers of HNCC are mutations of p53, p16 and amplification of Cyclin D and E2F4. One hundred and fifty-two HNCC cases have been evaluated for p53, hMLH1, Cyclin D and p16 gene alterations using PCR-SSCP and Western blot analysis. P53 mutations of HNCC have been found in 37.5% of cases. However, 11% of the cases showed p53 mutations in the normal peritumoral mucosa suggesting "field cancerization" process. Mismatch-repair gene mutations (MMR: hMHL1 and hMSH2) occurred with 17 and 8.6% frequency, respectively, while E2F4 mutations were even more frequent (21.4%) in HNCC. Our data suggest that E2F4 overexpression can be caused by the inactivation of the p16 gene in HNCC, while its mutations are most probably associated to the mutations of the MMR genes. These molecular informations can help to predict the biological potential of HNCC as well as the probability of the development of secondary HNCCs.     

Genomic structure and promoter analysis of the mouse neutral ceramidase gene

We report here the molecular cloning of the mouse neutral ceramidase gene and its promoter analysis. The gene, composed of 27 exons ranging in size from 40 to 292 bp, spans more than 70 kb. Analysis of the 5(')-flanking region of the ceramidase genes revealed that the first exon of the gene of mouse liver was exactly the same as that of mouse kidney and Swiss 3T3 fibroblasts but completely different from that of mouse brain. The putative promoter regions of liver and brain ceramidase genes contained several well-characterized promoter elements such as GATA-2, C/EBP, and HNF3beta but lacked TATA and CAAT boxes, a typical feature of a housekeeping gene, although the expression is regulated in a tissue-specific manner. Interestingly, a GC box was exclusively found in the putative promoter of mouse liver whereas potential AP1 and AP4 binding sites were present in that of mouse brain. By a luciferase reporter gene assay, it was shown that the GC-rich region, which exists just upstream of the first exon, conferred the promoter activity in Swiss 3T3 cells.     

Genomic structure,gene expression,and promoter analysis of human multidrug resistance-associated protein 7

The multidrug resistance-associated protein (MRP) subfamily transporters associated with anticancer drug efflux are attributed to the multidrug-resistance of cancer cells. The genomic organization of human multidrug resistance-associated protein 7 (MRP7) was identified. The human MRP7 gene, consisting of 22 exons and 21 introns, greatly differs from other members of the human MRP subfamily. A splicing variant of human MRP7, MRP7A, expressed in most human tissues, was also characterized. The 1.93-kb promoter region of MRP7 was isolated and shown to support luciferase activity at a level 4- to 5-fold greater than that of the SV40 promoter. Basal MRP7 gene expression was regulated by 2 regions in the 5'-flanking region at -1,780-1,287 bp, and at -611 to -208 bp. In Madin-Darby canine kidney (MDCK) cells, MRP7 promoter activity was increased by 226% by genotoxic 2-acetylaminofluorene and 347% by the histone deacetylase inhibitor, trichostatin A. The protein was expressed in the membrane fraction of transfected MDCK cells.     

Genomic structure, chromosome mapping and expression analysis of the human AXIN2 gene

Conductin is a Wnt signalling protein and serves as a negative regulator of beta-catenin stability. We have previously isolated the human homolog (AXIN2) of the murine conductin gene and shown that it is mutated in colorectal cancer (CRC) with defective mismatch repair (MMR). Here we report the detailed genomic structure of this gene by analysis of cDNA and genomic clones. The gene spans > or =25 kb containing ten exons ranging from 96 bp to 904 bp. All splice donor and acceptor sites conform to the GT/AG rule. FISH (Fluorescence in situ Hybridization) analysis localized this gene to human chromosome band 17q24 and showed that it exists as a single copy in the human genome. Northern blot analysis from different human organs demonstrated that the AXIN2 gene is highly expressed in human thymus, prostate, testis, small intestine and ovarian tissues but expressed at a lower level in colon. The data reported here provides a framework for further analysis of this important Wnt signalling protein in vertebrate development and tumorigenesis.     

cDNA cloning, genomic structure, chromosomal mapping, and expression analysis of parotid secretory protein in pig

A novel cDNA has been isolated from pig parotid glands by 3' and 5' rapid amplification of cDNA ends and designated parotid secretory protein (PSP). The open reading frame of this cDNA covers 714 bases, encoding 238 amino acids, which show 56% identity with human PSP at the level of the primary protein structure. The PSP genomic sequence comprises eight exons and seven introns, is approximately 22 kb in size, determined by sequencing, and maps to pig chromosome 17q21-q23. RT-PCR, dot blot, and Northern blot analyses demonstrated that PSP is strongly expressed in parotid glands, but is not present in heart, liver, lung, kidney, muscle, or stomach. A search for functionally significant protein motifs revealed consensus sequences for casein kinase II phosphorylation and N-myristoylation. We observed a unique amino acid sequence pattern consisting of the residues Leu-X(6)-Leu-X(6)-Leu-X(7)-Leu-X(6)-Leu-X(6)-Leu near the amino-terminal portion of the protein, which is similar to the leucine zipper.