Mutation analysis of Rad18 in human cancer cell lines and non small cell lung cancer tissues

Abstract

Background: Genetic instability is known as a cause of oncogenesis. Though Rad18 is reported to function in a post replication mismatch repair system, the relation between the status of Rad18 and human tumorigenesis has not been described so far.

Methods: Mutation analysis of 34 human cancer cell lines and 32 non small cell lung cancer (NSCLC) tissues were performed by RT-PCR SSCP. Expression level of Rad18 was measured by real time RT-PCR. Stable transfectant was constructed for in vitro study.

Results: No mutation was found in both cancer cell lines and NSCLC tissues. A single nucleotide polymorphism (SNP) at codon 302 was detected in 51.5% of the cell lines and 62.5% of NSCLC tissues. Interestingly, Rad18 was homozygously deleted in a pulmonary adenocarcinoma cell line PC3. Furthermore, there was no difference in the expression level of wild type Rad18 and Rad18 with SNP. The growth, cell morphology, sensitivity to anti-cancer drugs and in vitro DNA repair activity between wild type Rad18 and Rad18 with SNP revealed to have no difference in vitro.

Conclusion: Though the frequency of SNP was tended to be higher in NSCLC patients than healthy volunteers (57.7%), as the difference was not significant, we have concluded that there is no relation between Rad18 SNP and lung cancer development.

Figure 1

The expression of Rad18 in human cancer cell lines. A: RT-PCR analysis of Rad18 in human cancer cell lines. A part of cell lines examined are present. The expression of Rad18 mRNA is observed in all cancer cell lines but PC3 (lane 24). Lane 1: KYSE30, 2: KYSE140, 3: TE1, 4: TE9, 5: TE10, 6: AGS, 7: MKN1, 8: MKN28, 9: NUGC3, 10: NUGC4, 11: Caco2, 12: Colo201, 13: Colo205, 14: DLD-1, 15: HCT116, 16: AsPC-1, 17: Capan1, 18: Capan2, 19: Panc1, 20: SUIT-2, 21: A549, 22: EBC1, 23: LU99, 24: PC3, 25: LCOK. B: Fragment Southern of PC3 (lane 1) and MCF7 (lane 2). Rad18 is homozygously deleted in lung cancer cell line PC3.

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.

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.

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) 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 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.