Up-regulation of telomere-binding proteins, TRF1, TRF2, and TIN2 is related to telomere shortening during human multistep hepatocarcinogenesis

Abstract

The telomeric repeat-binding factor 1 (TRF1), TRF2, and the TRF1-interacting nuclear protein 2 (TIN2) are involved in telomere maintenance. We describe the regulation of expression of these genes along with their relationship to telomere length in hepatocarcinogenesis. The transcriptional expression of these genes, TRF1 protein, and telomere length was examined in 9 normal livers, 14 chronic hepatitis, 24 liver cirrhosis, 5 large regenerative nodules, 14 low-grade dysplastic nodules (DNs), 7 high-grade DNs, 10 DNs with hepatocellular carcinoma (HCC) foci, and 31 HCCs. The expression of TRF1, TRF2, TIN2 mRNA, and TRF1 protein was gradually increased according to the progression of hepatocarcinogenesis with a marked increase in high-grade DNs and DNs with HCC foci and a further increase in HCCs. There was a gradual shortening of telomere during hepatocarcinogenesis with a significant reduction in length in DNs. Most nodular lesions (52 of 67) had shorter telomeres than their adjacent chronic hepatitis or liver cirrhosis, and the telomere lengths were inversely correlated with the mRNA level of these genes (P </= 0.001). This was more evident in DNs and DNs with HCC foci. In conclusion, TRF1, TRF2, and TIN2 might be involved in multistep hepatocarcinogenesis by playing crucial roles in telomere shortening.

Figure 1

Microscopic feature of human multistep hepatocarcinogenesis showing low-grade DN (A), high-grade DN with small liver cell dysplasia (B), DN with HCC foci in bottom right part (C), and HCC with trabecular pattern (D). H&E; original magnifications, ×200.

Figure 2

Comparison of the TRF1, TRF2, and TIN2 mRNA levels in human multistep hepatocarcinogenesis. The TRF1 (A), TRF2 (B), and TIN2 (C) mRNA levels are shown in normal livers, CH, LC, LRNs, low-grade dysplastic nodules (LGDNs), high-grade dysplastic nodules (HGDNs), DNs with HCC foci, and HCCs. Eleven patients with multiple synchronous nodules are denoted by different symbols according to the patient numbers (patient 1, black square; patient 2, black diamond; patient 3, black triangle; patient 4, black circle; patient 5, gray square; patient 6, gray diamond; patient 7, gray triangle; patient 8, gray circle; patient 9, white square; patient 10, white diamond; patient 11, white triangle; others, white circle).

Figure 3

Immunoreactivity of TRF1. TRF1 protein expression is mild in CH (A), moderate in high-grade dysplastic nodule (B), and further increased in DN with HCC foci (C) with the marked expression in HCC (D). Notice that increased TRF1 protein expression with the progression of human multistep hepatocarcinogenesis. Original magnifications, ×200.

Figure 4

Comparison of the telomere length in human multistep hepatocarcinogenesis. A: The telomere length is shown in normal livers, CH, LC, LRNs, low-grade dysplastic nodules (LGDNs), high-grade dysplastic nodules (HGDNs), DNs with HCC foci, and HCCs. The lesions are denoted by different symbols according to the patient numbers (patient 1, black square; patient 2, black diamond; patient 3, black triangle; patient 4, black circle; patient 5, gray square; patient 6, gray diamond; patient 7, gray triangle; patient 8, gray circle; patient 9, white square; patient 10, white diamond; patient 11, white triangle; others, white circle. B: A representative result of Southern blot analysis in various lesions. The genomic DNA, digested with Hinf I, was hybridized with a digoxigenin-labeled d(TTAGGG₄. The mean telomere restriction fragment length was calculated by a previously described method, using the Image Gauge software (Fujifilm, Tokyo, Japan). The size markers are indicated on the right.

Figure 5

Comparison of TRF1, TRF2, and TIN2 mRNA levels with the telomere length in human multistep hepatocarcinogenesis. The TRF1, TRF2, and TIN2 mRNA levels are compared with the telomere length, shown in A, B, and C, respectively. The analysis was performed with the nodular lesions with shortened telomeres compared to the adjacent CH and LC. This analysis included 4 LRNs, 11 low-grade dysplastic nodules (DNs), 7 high-grade DNs, 6 DNs with HCC foci, 24 HCCs, and their respective adjacent CH (n = 11) and LC (n = 19). CH, LC, and LRNs are indicated with white circles, low- and high-grade DNs and DNs with HCC foci with black circles, and HCCs with gray triangles.