A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines

Abstract

Background: Tumor cell lines are commonly used as experimental tools in cancer research, but their relevance for the in vivo situation is debated. In a series of 11 microsatellite stable (MSS) and 9 microsatellite unstable (MSI) colon cancer cell lines and primary colon carcinomas (25 MSS and 28 MSI) with known ploidy stem line and APC, KRAS, and TP53 mutation status, we analyzed the promoter methylation of the following genes: hMLH1, MGMT, p16INK4a (CDKN2A alpha-transcript), p14ARF (CDKN2A beta-transcript), APC, and E-cadherin (CDH1). We compared the DNA methylation profiles of the cell lines with those of the primary tumors. Finally, we examined if the epigenetic changes were associated with known genetic markers and/or clinicopathological variables.

Results: The cell lines and primary tumors generally showed similar overall distribution and frequencies of gene methylation. Among the cell lines, 15%, 50%, 75%, 65%, 20% and 15% showed promoter methylation for hMLH1, MGMT, p16INK4a, p14ARF, APC, and E-cadherin, respectively, whereas 21%, 40%, 32%, 38%, 32%, and 40% of the primary tumors were methylated for the same genes. hMLH1 and p14ARF were significantly more often methylated in MSI than in MSS primary tumors, whereas the remaining four genes showed similar methylation frequencies in the two groups. Methylation of p14ARF, which indirectly inactivates TP53, was seen more frequently in tumors with normal TP53 than in mutated samples, but the difference was not statistically significant. Methylation of p14ARF and p16INK4a was often present in the same primary tumors, but association to diploidy, MSI, right-sided location and female gender was only significant for p14ARF. E-cadherin was methylated in 14/34 tumors with altered APC further stimulating WNT signaling.

Conclusions: The present study shows that colon cancer cell lines are in general relevant in vitro models, comparable with the in vivo situation, as the cell lines display many of the same molecular alterations as do the primary carcinomas. The combined pattern of epigenetic and genetic aberrations in the primary carcinomas reveals associations between them as well as to clinicopathological variables, and may aid in the future molecular assisted classification of clinically distinct stages.

Figure 1

Distribution of simultaneously methylated promoters in MSS and MSI colon cancer cell lines and colorectal carcinomas. The two panels illustrate the percentage of MSS and MSI samples displaying methylation of zero to all of the promoters analyzed in the present study in a) cell lines and b) primary colorectal tumors. Abbreviations: MSS, microsattelite stable; MSI, microsattelite instable.

Figure 2

Promoter hypermethylation in colon cancer cell lines and colorectal primary tumors. Methylation was evaluated by methylation-specific PCR (MSP). A visible PCR product in Lanes U indicates the presence of unmethylated alleles whereas a PCR product in Lanes M indicates the presence of methylated alleles. The upper panel (a) illustrates the methylation status of all the loci analyzed in a MSI cell line (RKO) and a MSS cell line (HT29). The lower panel (b) shows the methylation status of representative primary colorectal tumors. Abbreviations: NB, normal blood (positive control for unmethylated samples); MP, methylated placenta (positive control for methylated samples); neg, negative control (containing water as template); U, lane for unmethylated MSP product; M, lane for methylated MSP product.