A FISH analysis. Nuclei are stained with DAPI.Preparation of CRKL
A FISH analysis. Nuclei are stained with DAPI.Preparation of CRKL targeting peptideIn this study, we used the peptides, which has been reported to be disrupted complexes between BCR-ABL and CRKL depend on the SH3 domain of CRKL in CML cells [26]. Peptides used in the experiments are followed: CRKL-targeting peptide; KKW KMR RNP FWI KIQ RC ?CGI RVV DNS PPP ALP PKR RRS APS PTR V, control peptide; KKW KMR RNP FWI KIQ RC ?CGI RVV DNS PPG ALG PLL RRS APS PTR V. The KKW KMR RNP FWI KIQ RC was the shuttle tag sequence performing a receptor-independent cell entry. The chimeric peptide was synthesized and purified by using reverse-phase high performance liquid chromatography (HPLC) (Toray Research Center, Otsu, Japan). Peptide stocks were prepared in DMSO and stored in aliquots at -80 .Statistical analysisanalyses. P values less than 0.05 were considered statistically significant.ResultsIdentification of CRKL amplification in gastric cancerThe statistical analysis was performed using an unpaired t-test, chi-square test, or Dunnett’s test. JMP version 7.0.1 software (SAS Institute, Cary, NC) was used for theTo search for highly amplified genes in gastric adenocarcinoma, we adopted a genome-wide high-resolution SNP microarray approach in three cell lines of differentiated gastric adenocarcinoma: MKN7, MKN28, and MKN74. Genotype calls were obtained at more than 95 of the 262,264 SNP sites on the array, meaning that the SNP microarray analysis had been performed properly. The SNP microarray data were then used to determine the chromosomal copy number using the CNAG program (Figures 1A and 1B). Five highly amplified regions with a copy number of more than 6 (9p13, 17q12-q21, 19q12, 19q13, and 22q11) were identified, as shown in Table 1. These regions contained various kinds of genes, a total of 22 genes (Table 1). Among them, we decided to focus on the CRKL gene at chromosome PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28212752 22q11.21, the productNatsume et al. Journal of Translational Medicine 2012, 10:97 http://www.translational-medicine.com/content/10/1/Page 6 ofFigure 4 (See legend on next page)of which is an SH2 and SH3 domain-containing adaptor protein that shares homology with the CRK oncoprotein, because CRKL is a known substrate of BCR-ABL kinase in Philadelphia chromosome-positive leukemia [27,28] and its role in gastric cancer has not been previously analyzed. To confirm that CRKL gene amplification was detectable in the MKN74 cell line, we performed a FISH analysis using a probe specific for CRKL. As expected, an extreme increase in the CRKL copy number was detected in the MKN74 cells using aFISH analysis (Figure 1C). When the level of CRKL mRNA expression was examined in MKN74 cells using a real-time QRT-PCR analysis, the level was much higher than that in non-cancerous gastric tissue (Figure 1D). Moreover, a western blot analysis showed that the level of CRKL protein expression was higher in MKN74 cells than in non-cancerous gastric tissue (Figure 1E). These results suggested that the CRKL gene is highly amplified and that CRKL is overexpressed in a subset of gastric cancer cell lines.Natsume et al. Journal of Translational Medicine 2012, 10:97 http://www.translational-medicine.com/content/10/1/Page 7 of(See figure on previous page) Figure 4 Responses of the MKN74 gastric cancer cell line with CRKL amplification to treatment with BMS354825 (a dual Src/BCR-ABL kinase inhibitor) and CRKL-targeting peptide. (A) Viability of MKN74 cells Avermectin B1a cost treated with BMS354825 but not those treated with AM.

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