Miller Charles T, Chen Guoan, Gharib Tarek G, Wang Hong, Thomas Dafydd G, Misek David E, Giordano Thomas J, Yee John, Orringer Mark B, Hanash Samir M, Beer David G
Department of Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Oncogene. 2003 Sep 11;22(39):7950-7. doi: 10.1038/sj.onc.1206529.
The C-CRK gene, cellular homolog of the avian v-crk oncogene, encodes two alternatively spliced adaptor signaling proteins, CRKI (28 kDa) and CRKII (40 kDa). Both CRKI and CRKII have been shown to activate kinase signaling and anchorage-independent growth in vitro and CRKI transformed cells readily form tumors in nude mice. Affymetrix oligonucleotide arrays were used to analyse 86 lung adenocarcinomas and 10 uninvolved lung tissues. C-CRK mRNA expression was increased in more advanced (stage III versus stage I), larger (T(2-4) versus T(1)), and poorly differentiated tumors and in tumors from patients demonstrating poor survival (P=0.00034). An overlapping series of 93 lung adenocarcinomas (64 stage I and 29 stage III) and 10 uninvolved lung specimens were measured for quantitative differences in CRKI and CRKII protein levels using 2-D PAGE. CRK protein spots were identified using mass spectrometry and 2-D Western blotting. A significant increase in levels of the CRKI oncoprotein and the phosphorylated isoform of CRKII was observed in tumors (P<0.05). No difference in protein level was evident between stages. Concordant with mRNA expression, CRKI and CRKII were increased in poorly differentiated tumors (P<0.05). CRK immunohistochemical analysis of tumor tissue arrays using the same tumor series also demonstrated increased abundance of nuclear and cytoplasmic CRK in more proliferative tumors (P<0.05). This study provides the first quantitative analysis of discrete CRKI and CRKII protein isoforms in human lung tumors and provides evidence that the C-CRK proto-oncogene may foment a more aggressive phenotype in lung cancers.
C-CRK基因是禽v-crk癌基因的细胞同源物，编码两种可变剪接的衔接子信号蛋白，即CRKI（28 kDa）和CRKII（40 kDa）。CRKI和CRKII均已被证明可在体外激活激酶信号传导和不依赖贴壁的生长，并且CRKI转化的细胞在裸鼠中很容易形成肿瘤。使用Affymetrix寡核苷酸阵列分析了86例肺腺癌和10例未受累的肺组织。在更晚期（III期与I期）、更大（T(2-4)与T(1)）、低分化肿瘤以及生存较差的患者的肿瘤中，C-CRK mRNA表达增加（P = 0.00034）。使用二维聚丙烯酰胺凝胶电泳测量了93例肺腺癌（64例I期和29例III期）和10例未受累肺标本的重叠系列，以检测CRKI和CRKII蛋白水平的定量差异。使用质谱和二维蛋白质印迹法鉴定CRK蛋白斑点。在肿瘤中观察到CRKI癌蛋白水平和CRKII磷酸化异构体水平显著增加（P<0.05）。各阶段之间蛋白质水平无明显差异。与mRNA表达一致，CRKI和CRKII在低分化肿瘤中增加（P<0.05）。使用相同肿瘤系列对肿瘤组织阵列进行CRK免疫组织化学分析也表明，在增殖性更强的肿瘤中，核和细胞质CRK的丰度增加（P<0.05）。本研究首次对人肺肿瘤中离散CRKI和CRKII蛋白异构体进行了定量分析，并提供了证据表明C-CRK原癌基因可能在肺癌中促成更具侵袭性的表型。
