Within the ten PCR fragment, only PCR fragment No.7 showed abnormal band pattern in 17 cell lines (Figure 2A). Direct sequence of

the abnormal band revealed that all 17 Captisol ic50 cell lines carried a single nucleotide polymorphism (SNP) at the second letter of codon 302 (51.5%) and there were no other mutation (Figure 2B). This SNP was already reported in the SNP database. Though there was no coding region mutation, as lung cancer cell line PC3 had a homozygous deletion in Rad18 genomic lesion and as Rad18 is mapped at chromosome 3p25 which is reported to have frequent LOH in lung cancer [12], we decided to analyze lung cancer tissue for Rad18 mutation. Figure 2 SSCP analysis of human cancer cell line. A: A part of SSCP of primer set 7 is present. The shifted abnormal band is pointed. B: The result of direct sequence of the shifted band. At codon 302, three different patterns were detected. Mutation analysis of Rad18 in NSCLC tissues The clinicopathological characteristics of examined NSCLC patients are shown in Table 2. First we checked the expression of Rad18 by H 89 in vitro RT-PCR The expression of Rad18 was observed in all 32 NSCLC tissues. Doramapimod in vivo RT-PCR SSCP revealed that there was no mutation in Rad18 coding region but the same SNP of codon 302. This SNP was observed in 20 samples of 32 NSCLC tissues (62.5%) and in 15 peripheral blood samples of 26 healthy volunteers (57.7%). Though, the frequency of Rad18 SNP is

tended to be higher in NSCLC tissue than the healthy

volunteers, the difference was not significant. There was no difference in other characters such as sex, histological type, T-stage, lymph node metastasis or p-stage between WT and SNP (Table 2). In addition, there were no difference between the three patterns of codon 302 and lung cancer development (Table 3). Furthermore, Rad18 expression level was also examined using light cycler (Fig 3). No difference was observed between WT and SNP or between the three patterns of codon 302. Figure 3 Rad18 expression level in lung cancer tissues. Left: Expression level according to wild type and SNP. Right: Expression level according to the pattern of codon 302. Table 2 Clinicopathological characteristics of however NSCLC   WT (n = 12) SNP (n = 20)         N.S. Age (years, mean ± SD) 70.6 ± 8.2 70.0 ± 8.8         N.S. Sex          Male 9 12      Female 3 8         N.S. Histological type          Squamous cell carcinoma 7 3      Adenocarcinoma 3 14      Others 2 3         N.S. T stage          T1 4 13      T2 7 7      T3 1 0         N.S. Lymph node metastasis          Positive 2 4      Negative 10 16         N.S. pStage          IA 4 11      IB 3 5      IIA 0 2      IIB 5 2   Table 3 Frequency of Rad18 Gln302Arg polymorphism   Lung cancer tissue Healthy volunteers   No. of samples 32 26   No. of polymorphism 20 (62.5%) 15 (55.7%) N.S. Pattern of codon 302     N.S.    A (Gln) 12 (37.5%) 11 (42.3%)      A/G (Gln/Arg) 13 (40.7%) 7 (26.9%)      G (Arg) 7 (21.9%) 8 (30.