Protein-tyrosine kinase and protein-serine/threonine kinase expression in human gastric cancer cell lines

Abstract

Protein kinases play key roles in cellular functions. They are involved in many cellular functions including; signal transduction, cell cycle regulation, cell division, and cell differentiation. Alterations of protein kinase by gene amplification, mutation or viral factors often induce tumor formation and tumor progression toward malignancy. The identification and cloning of kinase genes can provide a better understanding of the mechanisms of tumorigenesis as well as diagnostic tools for tumor staging. In this study, we have used degenerated polymerase-chain-reaction primers according to the consensus catalytic domain motifs to amplify protein kinase genes (protein-tyrosine kinase, PTK, and protein-serine/threonine kinase, PSK) from human stomach cancer cells. Following amplification, the protein kinase molecules expressed in the gastric cancer cells were cloned into plasmid vectors for cloning and sequencing. Sequence analysis of polymerase-chain-reaction products resulted in the identification of 25 protein kinases, including two novel ones. Expression of several relevant PTK/PSK genes in gastric cancer cells and tissues was further substantiated by RT-PCR using gene-specific primers. The identification of protein kinases expressed or activated in the gastric cancer cells provide the framework to understand the oncogenic process of stomach cancer.

Tyrosine-kinase expression profiles in human gastric cancer cell lines and their modulations with retinoic acids

“…This finding correlated with the reported in situ hybridisation and Northern blot data (Quantin et al, 1995), which validated our PTK profiles. By eliminating cloning and sequencing steps in previous profiling approaches (Lin et al, 1998) and adopting end-labelled PCR primers, this RAGE method is more quantitative as the intensity of each PTK fragments could reflect the PTK molecules expressed in cells. This approach enabled us to establish comprehensive PTK expression profiles from a large number of samples in a single experiment, and thus we were able to generate comprehensive PTK profiles from six different human gastric cancer cell lines in a very limited amount of time.…”

“…Six human gastric cancer cell lines were used in this study, including HR, AGS, KATO III, NUGC (NUGC-3), TSGH (TSGH9201) and SC-M1 (Lin et al, 1998). All cell lines, except HR, were kindly provided by Dr Chin-Wen Chi at Taipei-Veterans General Hospital, Taiwan.…”

“…On the basis of their cellular localisation, these PTKs can be further classified as receptor-type or nonreceptor-type PTK and dual kinases that can phosphorylate both tyrosine and serine/threonine residues. Since PTKs share significant homologies in their kinase catalytic domain, degenerated polymerase chain reaction (PCR) primers can be designed according to the amino-acid sequence submotifs of the kinase catalytic domain (Robinson et al, 1996;Lin et al, 1998). In previous studies, we identified 25 -35 different PTK genes expressed in cells by simple reverse trancriptase -polymerase chain reaction (RT -PCR) reaction (Lin et al, 1998(Lin et al, , 1999Wu et al, 2000).…”

“…Since PTKs share significant homologies in their kinase catalytic domain, degenerated polymerase chain reaction (PCR) primers can be designed according to the amino-acid sequence submotifs of the kinase catalytic domain (Robinson et al, 1996;Lin et al, 1998). In previous studies, we identified 25 -35 different PTK genes expressed in cells by simple reverse trancriptase -polymerase chain reaction (RT -PCR) reaction (Lin et al, 1998(Lin et al, , 1999Wu et al, 2000). Since then, we implemented an improved restriction analysis of gene expression (RAGE) profiling approach, developed at Dr Hsing-Jien Kung's laboratory (Robinson et al, 1996), which utilises restriction enzyme digestion and gel electrophoresis for quick and efficient kinase profiling (manuscript in preparation).…”

Tyrosine kinases and gastric cancer

“…Robinson et al (1996) designed a set of primers based on motifs in subdomain VII and IX of the catalytic domain of tyrosine kinases, and optimized the conditions, such that the reaction is of high ®delity (more than 90% of the amplicons are kinases) and broad speci®city (more than 90% of tyrosine kinases can be primed). Wu et al (2000a) utilized a slight modi®cation of this approach (Lin et al, 1998(Lin et al, , 1999a to compare two pairs of gastric cancers. The data summarized in Figure 1a reveals the expression of 32 dierent tyrosine kinases and a few dual kinases (mkk3, mkk4 etc) in these tissues, including 13 nonreceptor tyrosine kinases, 19 receptor tyrosine kinases.…”