Chromosomal instability, telomere shortening, and inactivation of p21(WAF1/CIP1) in dysplastic nodules of hepatitis B virus-associated multistep hepatocarcinogenesis

Systemic analysis for chromosomal instability and inactivation of cell cycle checkpoints are scarce during hepatocarcinogenesis. We studied 24 patients with chronic B viral cirrhosis including 30 cirrhotic regenerative nodules, 35 low-grade dysplastic nodules, 15 high-grade dysplastic nodules, 7 dysplastic nodules with hepatocellular carcinoma foci, and 18 hepatocellular carcinomas. Eight normal livers were studied as the control group. Telomere length and micronuclei were detected by Southern blot and Feulgen-fast green dyeing technique, respectively, and p21(WAF1/CIP1) expression was studied by immunohistochemistry. Micronuclei >1 per 3000 hepatocytes were found in 17% of low-grade dysplastic nodules, 87% of high-grade dysplastic nodules, and 100% of high-grade dysplastic nodules with hepatocellular carcinoma foci and hepatocellular carcinomas in contrast to those of all normal livers, and 90% of cirrhosis showed no micronuclei. The micronuclei index showed a gradual increase during hepatocarcinogenesis and there was a significant increase between cirrhosis and low-grade dysplastic nodules, low-grade dysplastic nodules and high-grade dysplastic nodules, and high-grade dysplastic nodules and hepatocellular carcinomas. Telomere length showed a gradual shortening during hepatocarcinogenesis and a significant reduction was found in high-grade dysplastic nodules (P=0.024) and hepatocellular carcinomas (P=0.031) compared with normal and cirrhotic livers. The micronuclei index was correlated with telomere shortening (P=0.016). The p21(WAF1/CIP1) labeling index was significantly higher in cirrhosis than in normal livers (P=0.024) and markedly decreased in low-grade dysplastic nodules, high-grade dysplastic nodules, and hepatocellular carcinomas compared with cirrhosis (P<0.05). The p21(WAF1/CIP1) labeling index was associated with telomere length (P<0.001) but not micronuclei index. This study shows that telomere shortening, chromosomal instability, and inactivation of p21(WAF1/CIP1) checkpoint function occur in low-grade dysplastic nodules as well as in high-grade dysplastic nodules, and their cooperation is considered to be critical for malignant transformation during hepatitis B virus associated-multistep hepatocarcinogenesisope

Characterization of necleolar protein PinX1 function in Rodent

의과학/석사[한글]이중 나사선과 3’overhang의 단일 가닥구조로 이루어진 사람의 telomere는 염색체 말단 부위에 위치하며, 여러 단백질들과 함께 염색체를 보존하는 역할을 한다. 정상세포의 경우 telomere의 길이가 critical point에 이르면 세포는 더 이상 분열하지 못하고 세포사멸이나 노화에 이르게 된다. 그러나 사람의 불멸의 세포(immortal cell)이나 종양세포에서는 telomerase에 의해 telomere가 일정하게 유지된다. Telomerase는 활성요소인 TERT 단백질과 주형이 되는 RNA TR로 이루어져 있는 ribonucleoprotein 복합체이다.PinX1은 telomere 결합단백질인 TRF1에 결합하는 단백질로서 염색체 8p23에 위치한다. PinX1의 카르복실 만달기에 있는 TID (telomerase inhibitory domain)이 hTERT와 결합하여 telomerase의 활성도를 저해하는 것으로 알려져 있다. 또 PinX1이 과발현된 세포에서는 세포 성장에 영향을 미치면서 telomere 길이감소를 유발하는 것이 관찰되었다. 이와 같이 PinX1이 telomerase 억제자(inhibitor)로서 종양 억제유전자로서의 가능성이 제기되면서 암치료에 대한 이용 가능성이 부각되었다.본 연구실에서는 rodent에서 PinX1기능 규명을 위해 PinX1을 백서의 간줄기세포인 WB-F344에서 클로닝 하였다. 아미노산 331개로 이루어진 rPinX1은 사람과 73%, 쥐와 91%의 유사성을 띄었다. 또 염색체 15p12에 위치하며 핵인 단백질 fibirillarin과 co-localization 하는 것을 확인하였다. 백서 PinX1 전사가 심장, 간과 고환에서 발현하고 있음을 확인하였다. 또 rPinX1의 stable cell line을 제작하여 세포의 성장 및 노화에 미치는 영향과 3’overhang과 telomere 길이의 변화를 살펴보았다. rPinX1 stable cell이 PDL(population doubling) 100이 넘어가면서 세포 모양이 납작하고 커지는 노화 형태를 띄면서 성장이 억제되었고 SA-β-gal assay를 통해 세포의 노화가 확인되었다. Stable cell line은 3’ G-rich overhang 변화는 없었으나 후기 계대에서 telomere 길이 감소를 확인하였다. 대조군 세포주에 비해 stable cell에서 뚜렷한 telomerase 억제효과는 없었으나 PinX1 과발현은 백서에서도 telomere 길이 감소를 이끌었고 짧은 telomere는 궁극적으로 세포 노화를 유도한 것으로 추정된다. 본 연구는 PinX1의 telomere 길이조절 기능이 포유류에서 보존됨을 확인 하였고, 또한 향후 PinX1을 이용한 새로운 항암제 표적 단백질로서의 동물실험의 기초가 될 것이라 기대된다. [영문]Telomere consists of the double strand and a single stranded 3’overhang. Human telomeres and various telomere binding protein complexes protect the ends of linear chromosomes. In somatic cells, telomeres shorten until they reach a critical length at which they are no longer able to be cell division and induce replicative senescence. However immortalized human cell and tumor cell maintains chromosomal stability by telomerase enzyme that is ribonucleoprotein complex. Telomerase identified as the catalytic subunit TERT and RNA template, TR. PinX1 is TRF1 binding nucleolus protein which locates to human chromosome 8p23. TID (telomerase inhibitory domain) of C-terminal directly inhibits cellular telomerase activity and leads to progressive telomere shortening. PinX1 overexpressed cell line has effect on cell growth which is being correlated with telomere shortening. The depletion of endogenous PinX1 increased telomerase activity. These results suggest that PinX1 is a putative tumor suppressor and might be useful for cancer therapy.In our laboratory rat PinX1 gene was cloned from WB-F344 cell line which encodes a protein of 331 amino acids with 73% similarity to human PinX1 and 91% to mouse. rPinX1 locates to chromosome 15p12 and protein located to nucleolus which was assessed by co-localization with fibrillarin. In this study, northern analysis revealed that rPinX1 mRNA is abundant in heart, liver and testis. To examine the rPinX1 effect on cell and telomere maintenance, we established rPinX1 overexpressed NIH-3T3 cell lines. The growth of rPinX1 stable cells in NIH-3T3 slowed down after 100 population doublings. Cells exhibited increased size and a flattened morphology and they were stained positive for the SA-β-gal assay. We found no detectable 3’ G-rich overhang size and the telomerase inhibition in stable cell. However, the telomere length was progressively altered at the late passage of stable cell. The cellular senescence was induced as result of the shorten telomere. Given its ability to induce crisis, telomere shortening, PinX1 might be a new target of cancer therapy as rodent model.ope

Epithelial cell adhesion molecule (EpCAM) marks hepatocytes newly derived from stem/progenitor cells in humans.

Epithelial cell adhesion molecule (EpCAM) is a surface marker on human hepatic stem/progenitor cells that is reported as absent on mature hepatocytes. However, it has also been noted that in cirrhotic livers of diverse causes, many hepatocytes have EpCAM surface expression; this may represent aberrant EpCAM expression in injured hepatocytes or, as we now hypothesize, persistence of EpCAM in hepatocytes that have recently derived from hepatobiliary progenitors. To evaluate this concept, we investigated patterns of EpCAM expression in hepatobiliary cell compartments of liver biopsy specimens from patients with all stages of chronic hepatitis B and C, studying proliferation, senescence and telomere lengths. We found that EpCAM(+) hepatocytes were rare in early stages of disease, became increasingly prominent in later stages in parallel with the emergence of ductular reactions, and were consistently arrayed around the periphery of cords of keratin 19(+) hepatobiliary cells of the ductular reaction, with which they shared EpCAM expression. Proliferating cell nuclear antigen (proliferation marker) and p21 (senescence marker) were both higher in hepatocytes in cirrhosis than in normal livers, but ductular reaction hepatobiliary cells had the highest proliferation rate, in keeping with being stem/progenitor cell-derived transit amplifying cells. Telomere lengths in EpCAM(+) hepatocytes in cirrhosis were higher than EpCAM(-) hepatocytes (P < 0.046), and relatively shorter than those in the corresponding ductular reaction hepatobiliary cells (P = 0.057). CONCLUSION: These morphologic, topographic, immunophenotypic, and molecular data support the concept that EpCAM(+) hepatocytes in chronic viral hepatitis are recent progeny of the hepatobiliary stem/progenitor cell compartment through intermediates of the transit amplifying, ductular reaction hepatobiliary cells.ope

SNPE(Self Natural Posture Exercise) 훈련이 여자 청소년 치어리딩 선수들의 견관절 근력 및 관절가동범위에 미치는 영향

이 연구의 목적은 SNPE(Self Natural Posture Exercise) 적용이 여성 청소년 치어리딩 선수의 어깨 관절 등척성 근력 및 운동 범위에 미치는 영향을 분석하는 것이다. 총 22명의 여성 청소년 참여자가 운동군(EG: Exercise Group, n=11, 15.9± 2.0 yrs, 159.2±7.1 cm, 54.6±16.2 kg), 비운동군(NEG: Non Exercise Group, n=11, 15.9±2.5 yrs, 158.2±8.1 cm, 53.5±9.2 kg)으로 참여하였다. SNPE는 벨트 및 도구 운동으로 구성하여 6주 동안 60분간 주 5회 실시되었으며 운동 강도를 높이기 위해 2주마다 세트와 반복 횟수를 늘렸다. SNPE 훈련 전후에 견관절의 등척성 근력(벌림, 모음, 안쪽돌림, 가쪽돌림) 및 관절가동범위(굽힘, 폄, 안쪽돌림.가쪽돌림)를 측정하였다. 데이터 분석은 대응표본 t 검정과 공분산분석을 실시하였다. 그룹 내의 결과, EG에서 사후 값이 모두 증가하였다. 어깨 관절의 등척성 근력은 벌림, 모음, 안쪽돌림, 가쪽돌림이 NEG보다 EG에서 증가하여 그룹간 유의한 차이가 나타났다(p&lt;.001). 관절가동범위는 왼쪽 굽힘, 양쪽 폄, 오른쪽 안쪽돌림(p&lt;.001), 오른쪽 굽힘, 왼쪽 안쪽돌림(p&lt;.01), 양쪽 가쪽돌림(p&lt;.05)에서 그룹간 유의한 차이가 나타났다. 지속적인 연구를 통해 선수들에게 퍼포먼스 증가와 회복에 도움이 되는 프로그램으로 개발할 수 있을 것이다

Large liver cell change in hepatitis B virus-related liver cirrhosis

Large liver cell change (LLCC) refers to microscopic lesions often found in various chronic liver diseases; however, its nature is still controversial. Thirty-four formalin-fixed and 19 fresh frozen hepatitis B virus (HBV)-related cirrhosis samples were examined for the presence of LLCC, small liver cell change (SLCC), and hepatocellular carcinoma (HCC). The cell cycle checkpoint status (p21, p27, p16, Tp53), cell dynamics (proliferating cell nuclear antigen, Ki-67, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling, M30), DNA damage (gamma-H2AX [H2A histone family, member X]), telomere lengths, chromosomal instability (micronuclei index), and senescence-associated beta-galactosidase (SA-beta-Gal) activity were evaluated using an in situ approach and compared to those in normal liver (n = 5) and liver with chronic cholestasis (34 cases of hepatolithiasis and three cases of primary biliary cirrhosis). In HBV-related cirrhosis, the p21, p27, and p16 cell cycle checkpoint markers were activated in normal-looking cirrhotic hepatocytes (NLCH), but diminished gradually from LLCC, SLCC, to HCC, with an increase in Tp53 expression. There was a general decrease in telomere length from NLCH, LLCC, SLCC, to HCC. Micronuclei, gamma-H2AX foci, and net cellular gain were significantly increased from normal hepatocytes, NLCH, LLCC, SLCC, to HCC. The SA-beta-Gal activity was weaker in LLCC compared to NLCH and absent in SLCC and HCC. In contrast, cholestatic LLCC showed retained expression of cell cycle checkpoint markers and decreased net cellular gain compared to adjacent normal-looking hepatocytes. HBV-related LLCC showed significantly higher Tp53 labeling index, gamma-H2AX labeling index, and micronuclei index; shorter telomere length; decreased SA-beta-Gal activity; and increased net cellular gain compared to cholestatic LLCC. Conclusion: The nature of LLCC is rather heterogeneous depending on the biological setting. The characteristics of HBV-related LLCC are more consistent with dysplastic rather than merely reactive hepatocytes, whereas cholestatic LLCC more likely represents reactive change with more stringent cell cycle checkpoint control.ope