hSmad5 gene, a human hSmad family member: its full length cDNA, genomic structure, promoter region and mutation analysis in human tumors

Abstract

hSmad (mothers against decapentaplegic)-related proteins are important messengers within the Transforming Growth Factor-b 1 (TGF-b 1 ) superfamily signal transduction pathways. To further characterize a member of this family, we obtained a full length cDNA of the human hSmad5 (hSmad5) gene by rapid ampli®cation of cDNA ends (RACE) and then determined the genomic structure of the gene. There are eight exons and two alternative transcripts; the shorter transcript lacks exon 2. We identi®ed the hSmad5 promoter region from a human genomic YAC clone by obtaining the nucleotide sequence extending 1235 base pairs upstream of the 5' end of the cDNA. We found a CpG island consistent with a promoter region, and we demonstrated promoter activity in a 1232 bp fragment located upstream of the transcription initiation site. To investigate the frequency of somatic hSmad5 mutations in human cancers, we designed intron-based primers to examine coding regions by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Neither homozygous deletions or point mutations were found in 40 primary gastric tumors and 51 cell lines derived from diverse types of human cancer including 20 cell lines resistant to the growth inhibitory eects of TGF-b 1 . These results suggest that the hSmad5 gene is not commonly mutated and that other genetic alterations mediate the loss of TGF-b 1 responsiveness in human cancers.

Aberrant FHIT mRNA transcripts are present in malignant and normal haematopoiesis, but absence of FHIT protein is restricted to leukaemia

“…Some data argue against this notion: (a) at the genomic level point mutations possibly inactivating one allele appear to be extremely rare Gemma et al, 1997;Thiagalingam et al, 1996); (b) aberrant mRNA transcripts are mostly accompanied by an often predominant wt mRNA fragment, and abnormal FHIT mRNA transcripts are also found in various normal tissues (Gayther et al, 1997;Panagopoulos et al, 1997;van den Berg et al, 1997;Wang et al, 1998); (c) aberrant transcripts are not consistently translated into truncated protein; and (d) replacement of FHIT protein in human carcinoma cells does not always suppress tumour cell growth implying that FHIT is perhaps involved in tumorigenesis along pathways distinct from the classical tumour suppressor mechanism (Otterson et al, 1998). On the other hand some data suggest that the wt FHIT gene product might exert tumour-suppressing activity: (a) in contrast to normal cells which invariably express normal FHIT protein, the protein may be absent in solid tumours Greenspan et al, 1997;Sozzi et al, 1997).…”

Reduction of PTEN protein and loss of epidermal growth factor receptor gene mutation in lung cancer with natural resistance to gefitinib (IRESSA)

“…RNA isolation, cDNA array hybridisation and analysis of hybridisation signals Total RNA was isolated from each cell line using standard protocols described previously (Gemma et al, 1996(Gemma et al, , 1998. Messenger RNA was then purified from total RNA by incubation with oligo-dT-magnetic beads (Toyobo Co., Osaka, Japan).…”

“…Polymerase chain reaction -single strand conformation polymorphism (PCR -SSCP) analysis was performed as previously described (Gemma A, et al, 1996(Gemma A, et al, , 1998. Each of the three exons (18, 19 and 21) of the EGFR gene was amplified separately using reported PCR primers (Lynch et al, 2004).…”

“…Following electrophoresis, the gels were analysed using the FluorImager 595 (Amersham Pharmacia Biotech, Uppsala, Sweden). DNA sequence analysis was performed as previously described (Gemma A, et al, 1996(Gemma A, et al, , 1998. Aberrant bands were cut from the gel and further amplified by PCR using sequencing primers with the M13 sequence (TGTAAAACGACGGCCAGT) added to the appropriate PCR primers.…”

Target validation for genomics using peptide‐specific phage antibodies: A study of five gene products overexpressed in colorectal cancer

“…The reason for this apparent anomaly is unclear, however, a recent report has described the existence of an antisense transcript of Smad5, DAMS, which has been suggested to regulate translation of Smad5. 30 Our analysis of 5 different targets has shown that in all cases antibodies specific to gene-derived peptide sequences can be isolated when a maximum of 2 peptides are synthesized per gene. In n ϭ 2/5 (40%) of the gene targets we were able to isolate antibodies that showed specificity for a protein product of the expected molecular weight in Western blotting and gave a specific staining pattern in immunohistochemistry.…”