Epigenetic inactivation of CHFR in human tumors

Abstract

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin-ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected.

Fig. 1.

RT-PCR analysis of CHFR expression in human tumor cell lines. Cell lines are indicated above the data. PCR was performed by using samples prepared with (RT+) or without (RT-) reverse transcriptase. GAPDH was amplified to show the integrity of the RNA.

Fig. 2.

Methylation status of CHFR.(A) CpG island of CHFR: exons 1 and 2 are indicated by solid boxes, the transcription start site is indicated by an arrow, and the region analyzed by bisulfite PCR is indicated by a bar. (B) Re-expression of CHFR after treatment with the methyltransferase inhibitor 5-aza-dC. (C) Aberrant methylation of CHFR in human tumor cell lines. Cell lines are indicated above the data; the percentage of methylation is indicated below. (D) Representative results of analysis of CHFR methylation in human primary colorectal and head and neck cancers. N, normal tissue; T, primary cancers; OSCC, oral squamous cell cancer.

Fig. 3.

Expression and methylation status of CHFR in DNMT knockout cells. (A) Re-expression of CHFR in methyltransferase knockout cells. The genotypes of the cell lines are indicated: MT1KO1 and MT1KO2, DNMT1-/-; 3bKO1 and 3bKO2, DNMT3b-/-; DKO2 and DKO3, DNMT1-/-DNMT3b-/-. (B) Bisulfite PCR analysis of the methylation status of the CHFR CpG island in HCT116 and methyltransferase knockout cells; M, methylated alleles. (C) Bisulfite sequencing of CHFR in WT and DNMT knockout cells. The PCR products were cloned into a vector, and at least nine clones were sequenced for each cell line. Open and closed areas represent unmethylated and methylated CpG dinucleotides, respectively.

Fig. 4.

Expression and histone acetylation status of CHFR in a human tumor cell line. (A) Re-expression of CHFR in the RKO colorectal cancer cell line after treatment with 5-aza-dC and/or TSA. RKO cells were treated as follows: lane 1, mock; lane 2, 0.2 μM 5-aza-dC for 72 h; lane 3, 300 nM TSA for 24 h; and lane 4, 0.2 μM 5-aza-dC for 72 h plus 300 nM TSA for 24 h. The bar graph depicts the ratios of the intensities of the CHFR and GAPDH signals. (B) Chromatin immunoprecipitation analysis of the 5′ region of CHFR. Genomic DNA associated with acetylated histones H3 or H4 was prepared by immunoprecipitation using antibodies specific to acetylated histones H3 or H4. PCR was performed by using oligonucleotides specific for the 5′ region of CHFR. Input DNA was amplified to evaluate the total amount of DNA applied to the immunoprecipitation. As a control, the region around the transcription start site of GAPDH was amplified by using the DNA recovered from the chromatin immunoprecipitation assay.

Fig. 5.

(A) Expression of a truncated form of CHFR in tumor cell lines. The 396-bp products correspond to expected size of WT CHFR; the 241-bp products correspond to the FHA domain-deleted variant (CHFR-ΔFHA). LoVo, SW480, and Colo320 cells express CHFR-ΔFHA. GAPDH was amplified to show the integrity of the cDNA. (B) Schematic diagram showing various CHFR mRNA splicing variants. The names of the transcripts are shown on the left. Closed boxes indicate the coding regions. (C) Schematic diagram of intact CHFR and CHFR-ΔFHA proteins. FHA, fork-head associated domain; RF, RING finger domain; CR, cystein rich domain. Amino acid numbers (A.A) are shown on the top.

Fig. 6.

Suppression of growth by CHFR. (A) Colony formation assay using the T98G human tumor cell line. Cells were transfected with CHFR, CHFR-ΔFHA,or empty vector (pcDNA3.1) and plated. After 2 weeks, the cells were fixed with methanol and stained with Gimsa. (B) Quantitative analyses of colony numbers after transfection of CHFR, CHFR-ΔFHA, or p53 in several human tumor cell lines. Each experiment was repeated three times; the average numbers of colonies are shown.

Fig. 7.

Impairment of checkpoint into mitosis in CHFR-deficient cell lines. (A) Mitotic index of cancer cell lines after treatment with microtubule inhibitor. Cell lines were treated for 16 h with 1 μM docetaxel, after which the mitotic index was determined. (B) Cell cycle analysis. The indicated cell lines were treated for 0, 12, 24, 36, or 48 h and subjected to fluorescence-activated cell sorting analysis. The apoptotic cells were indicated as the sub-G₁ fraction.