Bong-Kyeong Oh

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

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.

High telomerase activity and long telomeres in advanced hepatocellular carcinomas with poor prognosis

Telomerase reactivation and telomere maintenance are crucial in carcinogenesis and tumor progression. In this study, the relationships between telomere parameters, chromosomal instability and clinicopathological features were evaluated in hepatocellular carcinomas (HCCs). Telomere length (TL), telomerase activity (TA) and human telomerase reverse transcriptase (hTERT) mRNA levels were measured in 49 hepatitis B virus (HBV)-related HCCs and corresponding non-tumorous tissues. The results were compared with clinicopathological data, including differentiation, multipolar mitosis (MM), anaphase bridge, immunohistochemical stain results for cytokeratin 19 (CK19) and patient outcome. TL of HCCs ranged from 4.7 to 13.1 kb, and 44.4% of HCCs showed telomere lengthening. hTERT mRNA levels and TA were closely related (P=0.008), and were significantly higher in HCCs than non-tumorous tissues. TL was significantly higher in HCCs with strong TA (P=0.048), high hTERT mRNA levels (P=0.001) and poor differentiation (P=0.041). Frequent MM was associated with poor differentiation (P=0.007) and advanced stage (P<0.001). TA was positively correlated with MM, anaphase bridges and advanced stage (P=0.019, P=0.017 and P=0.029). Thirteen (28.3%) HCCs were CK19+ and demonstrated longer telomeres than CK19− HCCs (P=0.046). Overall survival was poor in HCCs with MM >0.4 per field (P=0.016), high TA (P=0.009) and high TL ratio (HCC/non-HCC) >0.8 (P=0.044). Our results show that long telomeres, high TA and high mitotic instability are poor prognostic markers for HBV-related HCCs and their close association suggests that telomere maintenance may be important for the progression of HCCs with high chromosomal instability to more aggressive ones.

Quantitative assessment of hTERT mRNA expression in dysplastic nodules of HBV-related hepatocarcinogenesis.

BACKGROUND:Telomerase reverse transcriptase (hTERT) is the rate-limiting determinant of telomerase, which is critical for carcinogenesis. Dysplastic nodules (DNs) appear to be preneoplastic lesions of hepatocellular carcinomas (HCCs). In this study, in order to characterize DNs, hTERT mRNA, hTERT gene dosage, and mRNA for c-myc, a transcriptional activator of hTERT were studied in human multi-step hepatocarcinogenesis.METHODS:Fifty four hepatic nodules including 5 large regenerative nodules, 14 low-grade DNs, 7 high-grade DNs, 11 DNs with HCC foci and 17 HCCs, 23 livers with chronic hepatitis/cirrhosis, and 6 normal livers were examined. Transcript levels were measured by real-time quantitative RT-PCR and gene dosages by real-time PCR and Southern blotting.RESULTS:The hTERT mRNA levels increased with the progression of hepatocarcinogenesis, and a significant induction in the transition between low- and high-grade DNs was seen. Most high-grade DNs strongly expressed hTERT mRNA at levels similar to those of HCCs. Twenty-one percent of low-grade DNs had high levels of hTERT mRNA, up to those of high-grade DNs and there was no difference in the pathological features between low-grade DNs with and without increased hTERT mRNA levels. No correlation was found between hTERT mRNA levels, hTERT gene dosage, and c-myc mRNA levels.CONCLUSIONS:These results suggest that the induction of hTERT mRNA is an important early event and that its measurement by real-time quantitative RT-PCR is a useful tool to detect premalignant/malignant tendencies in hepatic nodules. However, hTERT gene dosage and c-myc expression are not the main mechanisms regulating hTERT expression in hepatocarcinogenesis.

Large liver cell dysplasia in hepatitis B virus x transgenic mouse liver and human chronic hepatitis B virus-infected liver.

Objectives: Large liver cell dysplasia (LCD) is frequently associated with hepatitis B virus (HBV), but it remains uncertain whether it is reactive, senescent or preneoplastic. Methods: The HBX transgenic mice and normal control mice were sacrificed at 1, 3, 5, 7, 9, 11, 13 and 15 months after birth. Twenty-three cases of human B viral chronic hepatitis/cirrhosis with prominent LCD were selected. The immunohistochemical stain of proliferating cell nuclear antigen (PCNA), transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and senescence-associated β-galactosidase (SA-β-Gal) were evaluated. Results: In HBX transgenic mice, LCD was developed since 3 months and formed small nodules of hepatocellular adenoma, which progressed to hepatocellular carcinoma. The hepatocytes with LCD in HBX transgenic mice showed significantly higher PCNA-labeling index (LI) and lower TUNEL-LI than normal hepatocytes of control mice (p < 0.05). In the majority of human B viral chronic hepatitis/cirrhosis, the hepatocytes with LCD revealed higher PCNA-LI and lower TUNEL-LI than those without, when compared in each case using the same tissue block. SA-β-Gal staining showed no difference between hepatocytes with and without LCD. Conclusion: It is suggested that LCD, related to HBV, might not be just an innocent bystander, but closely related to hepatocarcinogenesis.

Human PinX1 Mediates TRF1 Accumulation in Nucleolus and Enhances TRF1 Binding to Telomeres

Human PinX1 (hPinX1) is known to interact with telomere repeat binding factor 1 (TRF1) and telomerase. Here, we report that hPinX1 regulates the nucleolar accumulation and telomeric association of TRF1. In HeLa, HA-hPinX1 was co-localized with fibrillarin, a nucleolar protein, in 51% of the transfected cells and was present in the nucleoplasm of the remaining 48%. Mutant analysis showed that the C-terminal region was important for nucleolar localization, while the N-terminus exhibited an inhibitory effect on nucleolar localization. Unlike HA- and Myc-hPinX1, GFP-hPinX1 resided predominantly in the nucleolus. Nuclear hPinX1 bound to telomeres and other repeat sequences as well but, despite its interaction with TRF1, nucleolar hPinX1 did not bind to telomeres. Nucleolar hPinX1 forced endogenous TRF1 accumulation in the nucleolus. Furthermore, TRF1 binding to telomeres was upregulated in cells over-expressing hPinX1. In an ALT cell line, WI-38 VA-13, TRF1 did not co-localize with hPinX1 in the nucleoli. In summary, hPinX1 likely interacts with TRF1 in both the nucleolus and the nucleoplasm, and excess hPinX1 results in increased telomere binding of TRF1. The PinX1 function of mediating TRF1 nucleolar accumulation is absent from ALT cells, suggesting that it might be telomerase-dependent.

Telomeric 3′ overhangs in chronic HBV-related hepatitis and hepatocellular carcinoma

Telomeric 3′ overhang is a key component of telomere structure, but little is known about its role in hepatocarcinogenesis. We examined the 3′ overhang and telomere length, mRNA levels of hTERT, POT1, TRF1 and cytokeratin 19 (CK19) in 41 hepatitis B virus (HBV)‐related hepatocellular carcinomas (HCCs) and adjacent non‐HCCs of B viral chronic hepatitis/cirrhosis. 3′ overhang length was positively correlated with telomere length (p < 0.001). In non‐HCCs, the 3′ overhang shortened with increasing age (p = 0.043). Twenty‐six HCCs had shorter and 15 HCCs had longer 3′ overhangs than the adjacent non‐HCCs. The mRNA levels of hTERT, POT1 and TRF1 were upregulated in HCCs than in non‐HCCs. HCCs with lengthened 3′ overhangs expressed higher hTERT mRNA levels than those with shortened 3′ overhangs, when compared to 3′ overhangs in non‐HCCs (p = 0.044). POT1 and TRF1 showed no significant difference according to the 3′ overhangs. HCCs with long 3′ overhangs had higher mitosis (p = 0.046) and more frequent multipolar mitosis compared to those with short 3′ overhangs (p = 0.034). HCCs with high cytokeratin 19 mRNA levels, a marker for hepatic progenitor cells, had longer 3′ overhangs than HCCs with low cytokeratin 19 mRNA levels (p= 0.019). In conclusion, the 3′ overhang erosion might be closely related to the number of cell divisions in telomerase‐negative hepatocytes of chronic hepatitis/cirrhosis. In telomerase‐positive HCCs, an altered 3′ overhang are involved in HBV‐related hepatocarcinogenesis and hTERT might be involved in regulation of 3′ overhang.

Induction of telomerase activity during an early burst of proliferation in pancreatic regeneration

Telomerase activity (TA) and telomerase reverse transcriptase (rTERT) were investigated in Sprague-Dawley rats, killed at 6 and 12 h, and 1, 2, 3, 7, and 14 days after 90% pancreatectomy (px), by TRAPeze enzyme-linked immunosorbent assay telomerase detection and reverse transcriptase-polymerase chain reaction. TA increased at 2 days and reached a maximum at 3 days after px, when the small ductules showed the highest proliferation activity. After 3 days, TA decreased to basal levels as the ductules differentiated into new endocrine and exocrine pancreas. The expression of rTERT showed a correlation with TA. These results suggest telomerase is actively regulated during pancreatic regeneration.