Kohashi Kenichi, Oda Yoshinao, Yamamoto Hidetaka, Tamiya Sadafumi, Izumi Teiyu, Ohta Shigeru, Taguchi Tomoaki, Suita Sachiyo, Tsuneyoshi Masazumi
Department of Anatomic Pathology, Pathological Sciences, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan.
J Cancer Res Clin Oncol. 2007 Nov;133(11):817-24. doi: 10.1007/s00432-007-0223-z. Epub 2007 May 8.
SMARCB1/INI1, which negatively regulates cell cycle progression from G0/G1 into the S-phase via the p16INK4a-RB-E2F pathway, has been reported to be inactivated homozygously by deletion and/or mutations in malignant rhabdoid tumor (MRT). In the current study, we investigated the alteration of the SMARCB1/INI1 gene using simple methods, and its gene product at the protein level. Moreover, we investigated the status of hyperphosphorylation in RB protein, known as a key cell cycle molecule.
Three cell lines and 11 formalin-fixed, paraffin-embedded specimens of MRT were investigated. SMARCB1/INI1 gene alteration was analyzed with simple methods as a quantitative real-time PCR and direct sequencing method. Furthermore, SMARCB1/INI1 and RB protein were immunohistochemically evaluated.
In 12 of 14 cases, we detected genetic alterations comprised of nine (including three cell lines) homozygous deletions and three mutations, which can induce abnormal expression of gene products. At the protein level, SMARCB1/INI1 immunohistochemical expressions were not detected in any cases. Twelve out of 14 cases showed high-level (+5) expression of tRB (both hyperphosphorylated and underphosphorylated RB), combined with low-level (+1) expression of uRB (underphosphorylated RB), indicating a high rate of hyperphosphorylation.
We could analyze the SMARCB1/INI1 gene alteration with simple methods, and SMARCB1/INI1 gene alteration was found in 12 of 14 cases. Especially, quantitative real-time PCR was a convenient and accurate method. In addition, a high rate of hyperphosphorylation of RB gene was recognized. These results suggest that the clinically aggressive character of MRT is caused by the inactivation of the SMARCB1/INI1 gene.
SMARCB1/INI1通过p16INK4a-RB-E2F途径负向调控细胞周期从G0/G1期进入S期，据报道在恶性横纹肌样瘤（MRT）中通过缺失和/或突变而纯合失活。在本研究中，我们使用简单方法研究了SMARCB1/INI1基因的改变及其在蛋白质水平的基因产物。此外，我们研究了作为关键细胞周期分子的RB蛋白的过度磷酸化状态。
研究了3种细胞系和11例福尔马林固定、石蜡包埋的MRT标本。采用定量实时PCR和直接测序法等简单方法分析SMARCB1/INI1基因改变。此外，对SMARCB1/INI1和RB蛋白进行免疫组织化学评估。
在14例中的12例中，我们检测到基因改变，包括9例（包括3种细胞系）纯合缺失和3种突变，这些改变可诱导基因产物的异常表达。在蛋白质水平上，所有病例均未检测到SMARCB1/INI1免疫组织化学表达。14例中的12例显示tRB（过度磷酸化和磷酸化不足的RB）高水平（+5）表达，同时uRB（磷酸化不足的RB）低水平（+1）表达，表明过度磷酸化率高。
我们可以用简单方法分析SMARCB1/INI1基因改变，14例中的12例发现有SMARCB1/INI1基因改变。特别是，定量实时PCR是一种方便、准确的方法。此外，还发现RB基因的过度磷酸化率很高。这些结果表明，MRT的临床侵袭性特征是由SMARCB1/INI1基因失活引起的。
