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. 2003 Apr 7;88(7):1058-64.
doi: 10.1038/sj.bjc.6600821.

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

H-W Kao  1 , H-C Chen, C-W Wu, W-C Lin
Affiliations

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

H-W Kao et al. Br J Cancer. .

Abstract

Many protein tyrosine kinases are key regulators involved in cellular growth, differentiation, development, apoptosis and signal transduction pathways. Obtaining a comprehensive tyrosine-kinase expression profile in tumour cells is essential to learning more about their oncogenic potentials and responses to various chemotherapeutic reagents - such as retinoic acid, which has been shown to suppress the growth of gastric cancer cells and modulate gene expression. Expression of tyrosine kinases in retionic acid-treated cancer cells was investigated by reverse trancriptase-polymerase chain reaction (RT-PCR) and a novel restriction analysis of gene expression (RAGE) display technique. We first established comprehensive tyrosine-kinase profiles in different human gastric cancer cell lines. In cells treated with 9-cis-retinoic acid or all-trans-retinoic acid, we found that two PTKs (Eph and Hek5) appeared to be upregulated. In the present study, we demonstrate an efficient and simple RAGE approach for examining tyrosine kinases' expression in tumour cells and their alterations following drug treatments.

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Figures

Figure 1
Figure 1
RAGE PTK profiling of different human tissues. Poly(A) mRNA obtained from Clontech was amplified by RT–PCR as described in Materials and Methods. The 150–170 bp amplicon was purified and used for MwoI digestion. The completed digested products were separated by a denaturing sequencing gel. Specific PTKs were identified by the digested fragment sizes as indicated.
Figure 2
Figure 2
RAGE PTK profiling of different human gastric cancer cell lines. Total RNA obtained from six human gastric cancer cell lines (AGS, HR, Kato III, NUGC, SC-M1 and TSGH) was amplified by RT–PCR as described in Materials and Methods. The 150–170 bp amplicon was purified and used for HaeIII digestion. The completed digestion products were separated by a denaturing sequencing gel. Specific PTKs were identified by their digested fragment sizes as indicated.
Figure 3
Figure 3
RAGE profile and RT–PCR pattern of yes tyrosine kinase in human gastric cancer cell lines. Upper panel: RAGE expression profile of the yes kinase gene. Lower panel: yes kinase expression detected by RT–PCR method with specific primers.
Figure 4
Figure 4
Immunoblot showing the expression of yes tyrosine kinase in human gastric cancer cell lines. An equal amount of cell lysate from human gastric cancer cell lines was resolved by SDS–PAGE. After electrophoresis, proteins were transferred to a PVDF nylon membrane and then probed with anti-yes antibody. The anti-β-actin antibody was used a control.
Figure 5
Figure 5
PTK expression profiles of six human gastric cancer cell lines. Total RNA was isolated from exponentially growing human gastric cancer cell lines (AGS, HR, Kato III, NUGC, SC-M1 and TSGH) and used for RAGE analysis as described in Materials and Methods. A total of 39 different human kinases were identified and the expression level of each kinase was determined using a Fuji BAS phosphoimager and are represented by their radiation dose units (PSL).
Figure 6
Figure 6
Modulation of hek5 kinase in three gastric cancer cell lines treated with 9-cis-RA and all-trans-RA. NUGC, SC-M1 and TSGH cells were treated with 10−6 M 9-cis-RA or 10−6 M all-trans-RA for 36 h. Total RNA was extracted and used for PTK RAGE analysis. The RAGE profile of hek5 expression is shown in the upper panel. In the lower panel, expression of hek5 is demonstrated by RT–PCR using hek5-specific primers.
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