8%) 29 (55.8%) N.S. G (Arg) 27 (42.2%) 23 (44.2%) In vitro study of Rad18 polymorphism Though there was no Rad18 mutation in human cancer cell line and NSCLC tissue examined except PC3, as Rad18 JNK-IN-8 clinical trial functions as post-replication repair system, we have examined whether there is any difference between wild type Rad18 and Rad18 SNP in vitro. Using Rad18 null cell line PC3, wild type Rad18 or Rad18 SNP was transfected. The expression of introduced Rad18 gene was confirmed by RT-PCR and Western blotting (Fig 4A). The cell morphology of these stable transfectant had no difference (Fig 4B). Additionally, there was no difference in growth, sensitivity or survival
rate against anti-cancer drugs (CDDP or CPT-11) (Fig 4C, 5A, B). Furthermore, the in vitro DNA repair showed that, when PC3 was transfected with Rad18, the DNA repair was induced compared to the control (LacZ transfected PC3). However, there was no difference between the status of the codon 302 (A/A, A/G, G/G) (Fig 5C). Figure 4 In vitro study of Rad18 WT and Rad18 SNP. A: Expression of introduced Rad18 assessed by RT-PCR
(top) and Western blotting (bottom). Lane 1: PC3 + LacZ, 2: PC3-WT Rad18, 3: PC3-SNP Rad18. B: Cell morphology of the three cell lines. C: Growth assay of the three cell lines. D: Sensitivity to CDDP (left) MAPK inhibitor and CPT-11 (right) in the three cell lines. E: Percent survival at day 7 for different dose of CDDP (left) and CPT-11 (right). Figure 5 Drug sensitivity and repair function of Rad18 Org 27569 and the SNP. A: Sensitivity to CDDP (left) and CPT-11 (right) in the three cell lines. B: Percent survival at day 7 for different dose of CDDP (left) and CPT-11 (right). C: DNA repair assay of LacZ, WT(A/A), hetero(A/G), SNP(G/G). The vertical axis is the amount of RPA protein which shows the activity of DNA repair function. Discussion There is no doubt that genetic instability is one of the main causes of cancer development. Genetic instability can be divided in two. One is chromosomal instability and the other is microsatellite instability (MSI). It is reported that chromosomal instability is frequently found
in lung cancer but microsatelite instability is rare . Though 60% of non small cell lung cancer has loss of heterozygosity (LOH) in 3p and it is suggested that several tumor suppressor genes might be mapped in this region, a clear relation between lung cancer development and a single gene mutation has not been reported to date [14, 15]. Concerning microsatellite instability, using microsatellite markers located at 3p or targeting human mismatch repair gene, hMLH1, has been analyzed [16, 17]. They concluded that MSI is not frequently found in lung cancer tissue or pleural effusion of lung cancer patients. We focused on Rad18 which functions as a PRR system and mapped on 3p25. Within the cell lines and lung cancer tissues that we examined, no Rad18 mutation was detected but a homozygous deletion in PC3 (lung cancer cell line).