Molecular cloning, isolation and characterisation of ERK3 gene from chewing-tobacco induced oral squamous cell carcinoma.

The mitogen activated serine/threonine kinases (MAPKs) constitute extracellular signal-regulated protein kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 MAPK, with an important role in cell proliferation and transformation. Earlier studies from our laboratory had indicated a role for MAPK pathway in oral cancer. Our current study was aimed at examining the role of a MAPK-ERK3, in chewing-tobacco associated oral squamous cell carcinoma. We constructed a cDNA library from primary oral cancer tissue, cloned and isolated the ERK3 gene. The gene was sequenced and the sequence submitted to GenBank (Accession number AF420474). The oral cancer ERK3 clone demonstrated 100% homology to human ERK3 isolated from fetal skeletal muscle, with four specific nucleotide alterations in the non-coding region of the gene, comprising deletion of 'TTT' between 2701 and 2705 nt; 'G' to 'T' substitution at 188 nt; insertion of 'A' between 121 and 122 nt, and insertion of 'CTTTA' between 3391 and 3392 nt. Southern analysis of EcoRI genomic digests indicated ERK3 specific fragments of 11, 8.6, 6.5 and 3.2 kb sizes. The mRNA transcript analysis defined a single transcript of 4.5 kb. RT-PCR analysis revealed a three- to eight-fold increase in ERK3 expression in a majority (90%) of oral cancer tissues and peripheral blood cells (61.5%) of the patients, whereas absence or low levels of expression was observed in peripheral blood cells of 74% clinically normal healthy individuals with no tobacco habits, and overexpression in PBC from 26% normal individuals. The alterations in the non-coding region of ERK3 gene cloned from oral cancer tissue, may affect stability or regulation of mRNA, resulting in overexpression in the patient samples. The overexpression of the gene in the normal healthy individuals may be indicative of increased risk of developing oral cancers in this group.