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. 1998 Jun 9;95(12):6870-5.
doi: 10.1073/pnas.95.12.6870.

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma

J G Herman  1 , A Umar, K Polyak, J R Graff, N Ahuja, J P Issa, S Markowitz, J K Willson, S R Hamilton, K W Kinzler, M F Kane, R D Kolodner, B Vogelstein, T A Kunkel, S B Baylin
Affiliations

Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma

J G Herman et al. Proc Natl Acad Sci U S A. .

Abstract

Inactivation of the genes involved in DNA mismatch repair is associated with microsatellite instability (MSI) in colorectal cancer. We report that hypermethylation of the 5' CpG island of hMLH1 is found in the majority of sporadic primary colorectal cancers with MSI, and that this methylation was often, but not invariably, associated with loss of hMLH1 protein expression. Such methylation also occurred, but was less common, in MSI- tumors, as well as in MSI+ tumors with known mutations of a mismatch repair gene (MMR). No hypermethylation of hMSH2 was found. Hypermethylation of colorectal cancer cell lines with MSI also was frequently observed, and in such cases, reversal of the methylation with 5-aza-2'-deoxycytidine not only resulted in reexpression of hMLH1 protein, but also in restoration of the MMR capacity in MMR-deficient cell lines. Our results suggest that microsatellite instability in sporadic colorectal cancer often results from epigenetic inactivation of hMLH1 in association with DNA methylation.

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Figures

Figure 1
Figure 1
Methylation of hMLH1 promoter region CpG island in cell lines and primary human samples. The presence of a visible PCR product in those lanes marked U indicates the presence of unmethylated genes of hMLH1; the presence of product in those lanes marked M indicates the presence of methylated genes. (A) Normal lymphocytes and colorectal cell lines. Normal lymphocytes and the MSI− colorectal cell line SW480 contain only unmethylated hMLH1. MSI+ cell lines RKO and SW48 contain only methylated hMLH1. MSI+ cell lines Lovo and DLD1, both with mutations in MMR genes, are unmethylated at hMLH1. HT29 contains both unmethylated and methylated hMLH1 genes. (B) Normal colonic mucosa samples, all unmethylated at hMLH1. (C) Primary sporadic colon carcinomas (T), with the MSI phenotype shown above. All primary tumors include amplification with the U primer set, a result of the presence of normal contaminating tissue. Included is one MSI− tumor with adjacent normal mucosa, labeled N. (D) Primary colon carcinomas from patients with either inherited hMSH2 mutations (Left) or hMLH1 mutations (Right).
Figure 2
Figure 2
Methylation of hMSH2 in primary sporadic colorectal cancer. The presence of a visible PCR product in those lanes marked U indicates the presence of unmethylated genes of hMSH2; the presence of product in those lanes marked M indicates the presence of methylated genes, seen only in the in vitro methylated control DNA (∗). All primary colorectal tumors contain only unmethylated hMSH2 genes.
Figure 3
Figure 3
Immunohistochemistry of hMLH1 in primary colon cancer. A is normal colon adjacent to the MSI+ carcinoma shown in C, which is methylated at hMLH1 and does not express any protein within the cancer cells. B is a MSI− tumor that is unmethylated at hMLH1 and expresses the protein. D and E are from a MSI+ tumor with hypermethylation of hMLH1 that expresses hMLH1 in only some of the cancer cells, which are shown near arrows. In E, control vascular structures at the bottom stain for hMLH1, whereas the carcinoma nuclei do not. F is a MSI− tumor that has hypermethylation of hMLH1 and expresses hMLH1 in most cells.
Figure 4
Figure 4
(A) Western blot analysis of hMLH1 in colorectal cell lines. Note detectable protein in SW480 and HT29 before drug treatment (AzaC), but in RKO and SW48 only after drug treatment. (B) Demethylation analysis of cell lines after azacytidine treatment. The presence of U product in RKO and SW48 after 5-aza-2′-deoxycytidine indicates the presence of demethylation of the hMLH1 promoter in these cell lines.
Figure 5
Figure 5
(A) Mismatch repair activity in extracts of tumor cell lines treated with 5′-aza 2′-deoxycytidine. Repair reactions were incubated for 30 min (except G⋅G-3′ for 15 min), and the products were analyzed as described in Materials and Methods. Results are for the mismatched substrates G⋅G-5′/3′ and 2 unpaired bases with a nick 5′ to unpaired bases. DNA substrates contained a nick in the minus strand at either position −264 (for the 3′ nicked substrate) or at position +276 (for the 5′ nicked substrate), where position +1 is the first transcribed base of the lacZα-complementation gene. The G⋅G mismatch is at position 88, and α2 is at 90, 91 of the lacZα gene. HeLa, RKO-pretreatment (RKO-Pre), and SW48-pretreatment (SW48-Pre) are compared with cell extracts of RKO (RKO-5-AzaC) and SW48 (SW48-5-AzaC) made after 5 days of treatment with 5-aza-2′-deoxycytidine. (B) Mismatch repair activity in extracts of the AN3CA tumor cell line either untreated or treated with 5′ azacytidine. Above described substrates are tested in AN3CA cell extracts either pre, 1 day, or 3 days posttreatment with 5′ azacytidine.
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