Somatic mutation of hCDC4 gene is rare in lung adenocarcinomas.

Perturbation of cell cycle regulation is important in the development and progression of human cancers. Many human cancers highly express cyclin E [1 3]. Cyclin E coupled with cyclin-dependent kinase 2 (Cdk2) involved in G1 progression of the cell cycle. Degradation of cyclin E depends on ubiquitinmediated proteolysis by the SCF ubiquitin ligase complex [1,2]. hCDC4 (Fbw7) is a component of the SCF complex [1,2]. The hCDC4 gene was reported to be mutated in both cancer cell lines and primary human cancers. The hCDC4 mutations were found in breast, ovary and leukemia cell lines, and functionally the mutations were associated with increased levels of cyclin E protein [1,2]. In primary tumor tissues, hCDC4 mutations were detected in different tumors, including endometrial, colorectal and ovarian tumors (2.9 16% of the endometrial carcinomas, 13% of the colorectal tumors and 2% of the ovarian carcinomas) [4 8]. The hCDC4 gene is considered a tumor suppressor gene, and the inactivation of hCDC4 by the somatic mutations caused increased chromosomal instability of the affected cancer cells [4]. Although the mutational analysis of hCDC4 gene has widely been performed in human tumors, the data on the lung adenocarcinoma is lacking. In this study, we investigated the occurrence of the hCDC4 mutation in lung adenocarcinomas by a polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) assay. Methacarn-fixed tissues of 50 lung adenocarcinomas (including 15 adenocarcinomas with bronchioloalveolar carcinoma features and 1 pure bronchioloalveolar carcinoma) were randomly selected for the study. All of the patients were Asians (Korean). We analyzed the primary tumors, but not the metastatic lesions of the tumors. The male to female ratio was 27:23. Ages of the patients ranged from 36 79 years with an average of 59.6 years. The patients consisted of 15 current smokers, 5 former smokers and 30 non-smokers. Approval was obtained from the Catholic University of Korea, College of Medicine’s institutional review board for this study. Informed consent was provided according to the Declaration of Helsinki. Malignant cells and normal cells from the same patients were selectively procured from hematoxylin and eosin-stained slides using a 30G1/2 hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) affixed to a micromanipulator by microdissection. To date, most of the hCDC4 mutations in the tumor tissues have been reported within the exon 3 11 [4 8]. Thus, we analyzed the hCDC4 mutation in these nine exons. Genomic DNAs from tumor cells and normal cells from the same patients were amplified by PCR with 15 primer pairs covering the exon 3 11 of human hCDC4 gene. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by SSCP autoradiogram. After SSCP, DNAs showing mobility shifts were cut out from the dried gel, and the DNA sequencing of was carried out using the cyclic sequencing kit (Perkin-Elmer, Foster City, CA). On the SSCP autoradiogram, all of the PCR products were clearly seen. However, there was no any aberrantly migrating band compared to the wildtype bands from the normal tissues, indicating no evidence of DNA sequence alterations in the PCR