Identification of a new tumour-associated antigen TM4SF5 and its expression in human cancer.
Muller-Pillasch F. Wallrapp C. Lacher U. Friess H. Buchler M. Adler G. Gress TM.
Department of Internal Medicine I, University of Ulm, Robert Koch Str. 8, 89081, Ulm, Germany. firstname.lastname@example.org
In a previous large-scale screening for differentially expressed genes in pancreatic cancer, a gene was identified that was highly overexpressed in pancreatic cancer encoding a novel putative tetraspan transmembrane protein highly homologous to the tumour-associated antigen L6. Using a radiation hybrid panel the identified human gene named TM4SF5 (transmembrane 4 superfamily member 5) was localized to chromosome 17 in the region 17p13.3. The cloned TM4SF5 cDNA has a 32bp 5'-untranslated region (UTR), a 591bp open reading frame (ORF) and a 85bp 3'UTR. The predicted TM4SF5 protein with 197 amino acids contains three NH2-terminal hydrophobic transmembrane regions, followed by an extracellular hydrophilic domain containing two potential N-linked glycosylation sites and a COOH-terminal hydrophobic transmembrane region. These structural features are shared by the L6 antigen and a number of related cell surface proteins associated with cell growth. TM4SF5 was overexpressed in pancreatic cancer tissues as compared to both normal pancreas and chronic pancreatitis tissues, and was detected at high levels in other tumour tissues. Although the precise function of TM4SF5 remains to be elucidated it may be useful in a clinical setting for tumour diagnosis and/or therapy. This hypothesis is supported by the strong homology to the L6 antigen, which has proved promising in immunological, therapeutic and diagnostic approaches.
Establishment and characterization of cell lines constitutively expressing hepatitis B virus X-protein.
Lee Y. Bong Y. Poo H. Lee Y. Park J. Oh S. Sohn M. Lee S. Park U. Kim N. Hyun S.
Molecular Cell Biology Research Division, Korea Research Institute of Bioscience and Biotechnology, Korea Institute of Science and Technology, Taejon, South Korea. email@example.com
We prepared human hepatoma cell lines, which expressed the human hepatitis B virus-X gene product. The plasmid pMAMneo-X, containing an HBV-X gene promoter, an enhancer and a structural gene was constructed. Transfected HBV-X gene integration and expression were detected by Southern and Northern blotting, as well as by chloramphenicol acetylase transferase (CAT) assay using various kinds of promoter-CAT reporter systems. HBV-X protein expression in stable transfectants was confirmed by immunofluorescence microscopy. Transfected cell lines showed permanent expression of HBV-X proteins. The HBV-X transfectant activated its target promoters in promoter-CAT constructs as reporters. The HBV-X transfectant enhanced AP-1 transcription factor binding to its target DNA. Therefore, X-transfectants are not only stable, but also have specific biological functions. Cell cycle analysis by flow cytometry showed that the majority of the transfectant cells are arrested in the G1 or G2 phase of the cell cycle. These cell lines may be useful in analyzing the biological functions of HBV-X and its functional role in the formation of hepatocellular carcinomas.
Identification of a novel transcriptional silencer in the protein-coding region of the human CYP2C9 gene.
Xiang S. Parsons HK. Murray M.
Storr Liver Unit, Department of Medicine, University of Sydney, Westmead Hospital, Westmead, NSW 2145, Australia.
A novel regulatory element (27 bp) which confers transcriptional repression was identified within the protein-coding region immediately after the translation start codon in the human cytochrome P450 (CYP) 2C9 gene. Deletion of this element increased transcriptional activity in HepG2 cells by transient transfection assay. Nuclear protein extracts from HepG2 cells and human liver were found in electrophoretic mobility shift assays to bind specifically to the 27 bp element. A putative binding protein was partially purified by DNA-affinity chromatography and was determined by Southwestern blotting to have a molecular weight of approx. 100 kDa. Studies with mutated competitor oligonucleotides established that binding of the nuclear protein to the 27 bp cis-element was dependent upon two 6 bp direct repeats (5'-CTTGTG-3') that were separated by three bases. It is possible that this novel cis-acting element may be involved in the negative regulation of CYP2C9.
Hepatitis C virus core protein represses p21WAF1/Cip1/Sid1 promoter activity.
Ray RB. Steele R. Meyer K. Ray R.
Division of Infectious Diseases and Immunology, Saint Louis University, MO 63110, USA. firstname.lastname@example.org
Hepatitis C virus (HCV) often causes a prolonged and persistent infection, and an association between hepatocellular carcinoma (HCC) and HCV infection has been noted. Recent experimental evidence using a cloned genomic region suggests that the putative core protein of HCV has numerous biological properties and is implicated as a viral factor for HCV mediated pathogenesis. WAF1/Cip1/Sid1 (p21) is the prototype of a family of proteins that inhibit cyclin-dependent kinases (CDK) and regulate cell cycle progression in eukaryotic cells. In this study, we have observed that the HCV core protein represses the transcriptional activity of the p21 promoter when tested separately by an in-vitro transient expression assay using murine fibroblasts (NIH3T3), human hepatocellular carcinoma (HepG2), and human cervical carcinoma (HeLa) cells. A deletion analysis of the p21 promoter suggested that the HCV core responsive region is located downstream of the p53 binding site. A gel mobility shift analysis showed that the HCV core protein does not bind directly to p21 regulatory sequences. Thus, the HCV core protein appears to act as an effector in the promotion of cell growth by repressing p21 transcription through unknown cellular factor(s).
Highly conserved amino-acid sequence between murine STAT3 and a revised human STAT3 sequence.
Pietra LD. Bressan A. Pezzotti AR. Serlupi-Crescenzi O.
Drug Discovery Department, Istituto di Ricerca Cesare Serono, 22 Via di Valle Caia, 00040 Ardea, Rome, Italy.
Signal transducer and activator of transcription 3 (STAT3) is an important mediator of cytokine signaling, whose cDNA and protein sequences have been fully characterized. We sequenced the whole human STAT3 cDNA isolated from HepG2 cells. The new sequence determined contains 43 nucleotide changes overall, corresponding to six modifications at the amino-acid level. The revised amino-acid sequence of human STAT3 is now completely identical to the mouse sequence, except for a single amino-acid change at position 760. Thus STAT3 now results as one of the most evolutionarily conserved among known proteins. By using specific RT-PCR we could discriminate between the original sequence and the new variant. Amplification of regions within the src-homology domain 2 (SH2) of STAT3, from the RNAs of 11 different tissues or cells, revealed only the expression of the new SH2 variant. Besides, only this SH2 variant was amplified from human genomic DNA. We conclude that the new sequence we have determined in this study represents a revised sequence of hSTAT3 or, less likely, a new predominant allele.
Promoter analysis of the human mismatch repair gene hMSH2.
Iwahashi Y. Ito E. Yanagisawa Y. Akiyama Y. Yuasa Y. Onodera T. Maruyama K.
Department of Animal Resource Science, Graduate School of Agriculture, Life Science, The University of Tokyo, Tokyo 113, Japan.
The human DNA mismatch repair gene homologue hMSH2 is involved in hereditary nonpolyposis colorectal cancer. We isolated and characterized the 5' upstream region, about 4.4kbp, of the hMSH2 gene. This region contains CpG islands and a number of elements involved in constitutive expression, but there is no TATA-box nearby the transcription start points. This is the typical structure for many promoters of housekeeping genes. Alu sequences and mononucleotide repeats are clustered in this region and there are two transcription start points. Deletion analysis revealed that less than 300bp was sufficient to initiate transcription. Although no mutation that influences promoter activity of this region was found, a polymorphism was detected by PCR-RFLP analysis. Because informative cases (C/T heterozygous) were relatively high ( approximately 30%), this polymorphism is suitable for a marker to examine allelic losses.
Functional genetic tests of DNA mismatch repair protein activity in Saccharomyces cerevisiae.
Polaczek P. Putzke AP. Leong K. Bitter GA.
Bit, Tech Inc., Westlake Village, CA 91361, USA.
Hereditary non-polyposis colorectal cancer (HNPCC) is associated with mutations in four different genes encoding proteins involved in DNA mismatch repair (DMR). As many as 30% of the observed sequence variations in human DMR genes predict minor alterations in the encoded protein, such as amino acid (aa) replacements or small in-frame deletions/insertions. For such sequence variants, a functional genetic test will be required to discriminate mutations from polymorphisms. We have constructed a series of isogenic yeast strains in which individual genes involved in DMR are disrupted, and have standardized an assay which measures GT tract stability (Strand et al., 1993) to characterize these gene products. Disruptions of the yeast MSH2, MLH1, and PMS1 genes result in, respectively, a 290-, 450- and 390-fold increased tract instability over the wild type (wt) strain under optimized assay conditions. Expression of the wt MSH2 and PMS1 gene from plasmids results in complementation of the corresponding chromosomal gene disruption. Two different aa replacements which correspond to previously observed sequence variants of the human MSH2 gene, and implicated in HNPCC, were created in the conserved aa of the yeast MSH2 gene by site directed mutagenesis. Conversion of the Pro640 in the yeast protein to Leu resulted in a complete loss of protein function. In contrast, a yeast MSH2p protein in which the His658 is changed to Tyr retains full function in this in vivo assay. These results indicate that the Pro-->Leu and His-->Tyr variants observed in humans constitute, respectively, a mutation and a polymorphism. The system described here may be used for further structure/function analysis of yeast DMR proteins. Such studies may provide insight into the effects that specific sequence variations observed in human DMR proteins have on their function.