Hepatocellular Telomere Length in Biliary Atresia ... - Springer Link

2 downloads 0 Views 2MB Size Report
Feb 7, 2012 - Naotaka Shimomura • Naoshi Ishikawa • Tomio Arai • Steven S. S. Poon • ... Takeshi Saito • Satoshi Egami • Shuji Hishikawa • Yoshiyuki Ihara •.
World J Surg (2012) 36:908–916 DOI 10.1007/s00268-012-1453-z

Hepatocellular Telomere Length in Biliary Atresia Measured by Q-FISH Yukihiro Sanada • Junko Aida • Youichi Kawano • Ken-ichi Nakamura • Naotaka Shimomura • Naoshi Ishikawa • Tomio Arai • Steven S. S. Poon • Naoya Yamada • Noriki Okada • Taiichi Wakiya • Makoto Hayashida • Takeshi Saito • Satoshi Egami • Shuji Hishikawa • Yoshiyuki Ihara • Taizen Urahashi • Koichi Mizuta • Yoshikazu Yasuda • Hideo Kawarasaki Kaiyo Takubo



Published online: 7 February 2012 Ó Socie´te´ Internationale de Chirurgie 2012

Abstract Background Liver transplantation for biliary atresia is indicated whenever a Kasai portoenterostomy is considered unfeasible. However, the timing of liver transplantation in biliary atresia has not been precisely defined. Excessive shortening of hepatocellular telomeres may occur in patients with biliary atresia, and therefore, telomere length could be a predictor of hepatocellular reserve capacity. Methods Hepatic tissues were obtained from 20 patients with biliary atresia who underwent LT and 10 age-matched autopsied individuals (mean age, 1.7 and 1.2 years, respectively). Telomere lengths were measured by Southern blotting and quantitative fluorescence in situ hybridization using the normalized telomere-centromere ratio. The correlation between the normalized telomere-centromere ratio for the hepatocytes in biliary atresia and the pediatric end-stage liver disease score was analyzed. Results The median terminal restriction fragment length of the hepatic tissues in biliary atresia was not significantly different from that of the control (p = 0.425), whereas the median normalized telomere-centromere ratio of hepatocytes in biliary

atresia was significantly smaller than that of the control (p \ 0.001). Regression analysis demonstrated a negative correlation of the normalized telomere-centromere ratio with the pediatric end-stage liver disease score in biliary atresia (p \ 0.001). Conclusions Telomere length analysis using quantitative fluorescence in situ hybridization could be an objective indicator of hepatocellular reserve capacity in patients with biliary atresia, and excessive telomere shortening supports the early implementation of liver transplantation.

Y. Sanada (&)  Y. Kawano  N. Yamada  N. Okada  T. Wakiya  S. Egami  S. Hishikawa  Y. Ihara  T. Urahashi  K. Mizuta  H. Kawarasaki Department of Transplant Surgery, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke City, Tochigi 329-0498, Japan e-mail: [email protected]

S. S. S. Poon Terry Fox Laboratory, British Columbia Cancer Research Centre, Vancouver, Canada

Introduction Biliary atresia (BA) is a severe cholestatic disease of unknown etiology in neonates, and untreated cases progress to cirrhosis, end-stage liver disease, and death by 2 years of age [1]. Although the initial surgical treatment for BA is an early Kasai portoenterostomy to establish bile flow to the gastrointestinal tract [2, 3], liver transplantation (LT) is indicated in cases where Kasai portoenterostomy fails or is not feasible. However, the timing of LT for BA has not been

M. Hayashida Department of Pediatric Surgery, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

Y. Sanada  J. Aida  Y. Kawano  K. Nakamura  N. Shimomura  N. Ishikawa  K. Takubo Research Team for Geriatric Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan

T. Saito Department of Pediatric Surgery, Graduate School of Medicine, Chiba University, Chuo-ku, Japan

T. Arai Department of Pathology, Tokyo Metropolitan Geriatric Medical Center, Tokyo, Japan

Y. Yasuda Department of Surgery, Jichi Medical University, Tochigi 329-0498, Japan

123

World J Surg (2012) 36:908–916

precisely defined. At present, hepatocellular reserve capacity after Kasai portoenterostomy is usually evaluated in terms of the pediatric end-stage liver disease (PELD) score [4–6]. The PELD score is calculated on the basis of several objective values, the main components being age, growth failure (based on sex, height, and weight), albumin (g/dL), prothrombin time (international normalized ratio), and total bilirubin (mg/dL). The PELD score accurately predicts the 3 month probability of waiting list death for children with chronic liver disease [5]. Although some studies have demonstrated a correlation between the PELD score before LT and outcome after LT [4, 6], the PELD score is not a direct index that reflects the degree of hepatocellular injury, because it does not include histopathological evaluation. If a more objective evaluation of hepatocellular injury could be utilized, it would be possible to judge more precisely the timing of LT for BA. Human telomeric DNA is considered to protect chromosomes against degeneration, recombination, fusion, and loss [7]. Human somatic cells have a limited proliferative life span when serially cultured in vitro. As they approach this limit, they cease to replicate and enter a state of senescence [8]. This replication arrest in vitro is due, at least in part, to telomere shortening, because it can be bypassed by transfection with the gene encoding the telomerase catalytic subunit [9]. The telomere hypothesis of cellular aging suggests that when telomere shortening on a particular chromosome reaches a critical level, a DNA damage checkpoint mechanism may be initiated and the cells stop dividing [10, 11]. Telomere shortening also may promote chromosomal and genetic instability, while increasing the risk of malignancy. Although telomere attrition occurs in liver diseases, such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC) [12, 13], no such data are available for BA. Telomere length has been measured in various human organs and tissues by Southern blotting [14, 15]. Since Lansdorp et al. [16] devised the quantitative fluorescence in situ hybridization (Q-FISH) method, telomere measurements in several tissues have been determined by different groups using various Q-FISH methods [17, 18]. In the present study, we assessed hepatocellular telomere lengths in BA using an improved Q-FISH method [19–22], based on a hypothesis that excessive shortening of hepatocellular telomeres would occur in BA due to continuous biliary retention and chronic inflammation and that telomere length might be able to predict the hepatocellular reserve capacity in BA.

909

females; aged; 0.5–6.1 (mean age, 1.7) years). All patients survived after LT and were managed as outpatients, regardless of the PELD score. Hepatic specimens also were obtained from ten autopsied individuals without liver cirrhosis or HCC as age-matched controls (within 3 h postmortem; five males and five females; aged; 0–5.0 (mean age, 1.2) years). All of the hepatic specimens were stored at -80°C until use. Approval to conduct this study was obtained from the Ethics Committees of Jichi Medical University and Tokyo Metropolitan Institute of Gerontology. Southern blotting Seventeen samples were obtained from the hepatic tissue specimens of the BA patients (four males and 13 females; aged, 0.6–6.1 (mean age, 1.7) years). Southern blotting was not possible for the remaining three cases of BA because of an insufficient volume of hepatic tissue. Samples were obtained from all ten of the controls. Genomic DNA was extracted using the standard method with proteinase K and sodium dodecyl sulfate. The length of the terminal restriction fragment (TRF) derived from Hinf I-digested DNA was measured by the standard Southern blotting method described previously [23]. In the present study, we employed the Telometric software package version 1.2 (Fox Case Cancer Center, USA) to assess the sizes and distribution of the TRF [24]. The median value of the TRF was adopted as representative of telomere length, because the TRF values did not show a normal distribution. The telomere lengths were measured twice for all specimens, and their mean values were determined. Tissue processing and histological assessment Specimens were fixed for 6 h in buffered formalin and then subjected to standard tissue processing and paraffin embedding. The tissue specimens were sliced into sections 2 lm thick for both histological examinations (hematoxylin and eosin staining) and Q-FISH. In histological examination, the slides were checked for autolysis, inflammatory cell accumulation, and fibrosis. Q-FISH and image analysis of telomeres We performed Q-FISH in accordance with the method reported previously [19, 20].

Materials and methods Q-FISH and probes Subjects Specimens of resected hepatic tissue were obtained from 20 patients with BA at the time of LT (four males and 16

The tissue sections on slides were hybridized with 30 lmol/L peptide nucleic acid (PNA) telomere probe (telo C Cy3 probe: 50 -CCCTAACCCTAACCCTAA-30 -, Fasmac, Atsugi

123

910

City, Kanagawa, Japan) conjugated to Cy3 (final concentration 0.32 lmol/L) and the PNA centromere probe (Cemp1 probe: 50 -CTTCGTTGGAAACGGGGT-30 -, Fasmac) conjugated to fluorescein isothiocyanate (FITC; final concentration 0.12 lmol/L) for 3 min at 80°C, and the slides were incubated for 1 h at room temperature. The nuclei were then stained with DAPI (Molecular Probes, Eugene, OR) and mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA). Image analysis of telomeres The Q-FISH digital images were captured using a chargecoupled device camera (ORCAER-1394, Hamamatsu Photonics KK, Hamamatsu, Japan) mounted on an epifluorescence microscope (80i, Nikon, Tokyo, Japan) equipped with a triple band-pass filter set for DAPI/FITC/ Cy3 (Part #61010, Chroma Technology Corp., Rockingham, VT) and a 9 40 objective lens (Plan Fluor 409/0.75, Nikon). Microscope control and image acquisition were performed using the Image-Pro Plus software package (version 5.0, Media Cybernetics Co. Ltd., Silver Spring, MD). The captured images were analyzed with a telomere analysis software package, TissueTelo Ver. 2, which, in summary, defines the telomere length, estimated for each nucleus as the ratio of the detected telomere signal intensity to the centromere signal intensity (telomere centromere ratio; TCR). The TCRs from an average of 302.6 (range, 119–537) cells were analyzed for each case. Normalization by cell block Q-FISH also was performed on a block-section of a cultured cell strain, TIG-1 [25], (population doubling level of 34, 8.6 kbp), placed on the same slides as the hepatic sections, as a control for variations in sample preparation. Every TCR for hepatocytes was divided by the median TCR for the cell block on the same slide to give the normalized TCR (NTCR) [6]. The representative value for each specimen was the median value of the NTCR obtained. Senescence-associated b-galactosidase (SA-b-Gal) staining Sections (4 lm) of frozen hepatic tissues were cut, mounted onto glass slides, fixed in 10% formalin in phosphate-buffered saline for 1 min at room temperature, washed, and incubated overnight at 37°C with fresh SAb-Gal stain solution: 1 mg of 5-bromo-4-chloro-3-indolyl b-D-galactoside/mL stock solution (20 mg/mL dimethylformamide, 40 mmol/L citric acid, sodium phosphate, pH 6, 5 mmol/L potassium ferrocyanide, 5 mmol/L potassium ferricyanide, 150 mmol/L NaCl, 2 mmol/L

123

World J Surg (2012) 36:908–916

MgCl2). After rinsing and counterstaining with eosin, the slides were viewed under a bright field. Evaluation of SA-b-Gal staining Senescent cells were detected as those showing a cytoplasmic blue precipitate. The localization and number of positive cells were assessed independently by two pathologists (KT and JA). In cases of discordance, the same two pathologists performed a consensual evaluation by a simultaneous slide. On the basis of this evaluation, two groups were defined as follows: absent, no or few positive cells (\10%); present, presence of positive cells ([10%). Statistical analysis The NTCRs and TRFs of the hepatic tissues were compared between the BA patients and controls using the Mann–Whitney U test. Correlations between the NTCRs of the hepatocytes in BA and the PELD score were analyzed using Pearson’s correlation coefficient and single regression analysis. In all comparisons, differences at p \ 0.05 were considered to be significant.

Results Histology of BA and control hepatic tissue Histologically, all 20 BA patients were diagnosed by two pathologists (KT and JA) as having biliary liver cirrhosis, showing many inflammatory and interstitial cells (Fig. 1a). The controls showed mild lymphocytic infiltration and extramedullary hematopoiesis (Fig. 1b). Analysis of hepatic tissue telomere length using Southern blotting The median TRF length showed considerable homogeneity among the specimens of BA and the controls (Fig. 2). The TRF length in the hepatic tissue from BA patients diverged from a maximum of 15.9 kbp to a minimum of 10.9 kbp, with a median 13.8 kbp. The TRF length in the controls diverged from a maximum of 15.2 kbp to a minimum of 10.3 kbp, with a median 13.5 kbp. The TRFs for the BA and control specimens are shown in Fig. 3. The box plots show the TRF distributions (TRFs at 10, 25, 50, 75, and 90%). The TRF lengths in BA slightly exceeded those of the controls, but the difference was not significant (p = 0.425). Telomere lengths for BA and control hepatic tissue determined by Southern blotting are shown in Tables 1, 2.

World J Surg (2012) 36:908–916

911

Fig. 1 Representative histological features of biliary atresia (BA) in a 0.6-year-old male (case 4) (a) and the liver of a female control at 41 weeks of gestation (case 6) (b) (hematoxylin-eosin, original magnification 9200). The BA specimen shows many proliferating

fibroblasts and lymphocytes infiltrating into the interstitium. The control shows mild lymphocytic infiltration and extramedullary hematopoiesis

Fig. 2 Southern blotting of hepatic tissue from a patient with biliary atresia (BA) and a control. The terminal restriction fragment (TRF) yields wide smears in all lanes. In BA, median values of the TRF lengths (from the left) were 14.1, 14.3, 13.8, 13.2, 15.3, 10.9, 12.5,

14.6, 14.4, 12.5, 12.4, 11.2, 13.7, 15.9, 15.2, 15.2, and 15.3 kbp. In the control, median values of the TRF lengths (from the left) were 15.2, 10.3, 15.1, 13.0, 13.5, 14.5, 11.4, 13.5, 13.7, and 12.9 kbp

Analysis of hepatocellular telomere length using Q-FISH Figure 4 shows Q-FISH images of the cell block and hepatic tissues from BA patients and the controls. The telomere signals (Cy3) appear red, and the centromere signals (FITC) appear green in the blue DAPI-stained nuclei. Fibroblasts often showed strong signal intensity for Cy3. Figure 5 shows the representative TCR distribution of the hepatocytes in BA and control hepatic tissue (A, B), and the representative NTCR distribution of the TIG-1 cell block (C). The overall TCR distributions were shorter for the hepatocytes of BA patients than for the controls. The NTCRs for BA and control hepatic tissue are shown in Fig. 6. The box plots show the NTCR distributions (NTCRs at 10, 25, 50, 75, and 90%). The NTCRs of

Fig. 3 Box plots showing the median terminal restriction fragment (TRF) distributions in hepatic tissues from patients with biliary atresia (BA) and control (TRFs at 10, 25, 50, 75, and 90%). The TRFs for BA do not differ significantly from those of the control (p = 0.425)

123

912

World J Surg (2012) 36:908–916

Table 1 Telomere length of biliary atresia cases

PELD pediatric end-stage liver disease, NTCR normalized telomere-centromere ratio, TRF terminal restriction fragment, (-) not examined

Case

Age (year)

Sex

1

1.1

Female

2

0.8

Female

3

1.6

Female

23.7

13.8

0.71

4

0.6

Male

0.2

13.2

1.62

5

0.8

Female

20

15.3

0.25

6

0.8

Male

19.8

10.9

0.53

7

0.6

Female

23.1

12.5

0.85

8

0.7

Female

15.5

14.6

0.79

9

0.7

Female

22.8

14.4

0.24

10

1.0

Male

19.2

12.5

0.46

11

4.6

Female

-3.7

(-)

0.61

12

3.8

Male

-7.2

12.4

1.24

13

6.2

Female

-6.2

11.2

1.26

14

3.6

Female

-0.6

13.7

0.76

15 16

1.1 2.7

Female Female

1.2 -9.8

15.9 15.2

0.94 1.03

17

1.8

Female

-6

18

0.9

Female

Median TRF (kbp)

Median NTCR

4.9

14.1

0.24

26.5

14.3

0.47

1.8

15.2

1.8

15.3

1.03

19

0.8

Female

37.3

(-)

0.4

20

0.5

Female

34.6

(-)

0.36

Table 2 Telomere length of age-matched control cases Case

Age

Sex

Median TRF (kbp)

Median NTCR

1

43 wga

Male

15.2

1.77

2

34 wga

Male

10.3

1.95

3

21 wga

Female

15.1

2.12

4

21 wga

Female

13

4.48

5

28 wga

Male

13.5

2.48

6

41 wga

Female

14.5

2.79

7

29 wga

Female

11.4

3.95

8

5.0 year

Female

13.5

1.54

9

5.0 year

Male

13.7

1.2

10

2.0 year

Male

12.9

1.69

wga week of gestational age, NTCR normalized telomere-centromere ratio, TRF terminal restriction fragment

hepatocytes in BA were significantly smaller than those of the hepatocytes in control liver (p \ 0.001). Telomere lengths in BA and control hepatic tissue determined by Q-FISH are shown in Tables 1, 2. Correlation between NTCR in BA and PELD score The regression line estimated from analysis of the scatter plot of NTCR against PELD score for the hepatocytes in BA is shown in Fig. 7. There was a significant negative correlation between NTCR and the PELD score (p \ 0.001).

123

PELD score

Expression of SA-b-Gal in hepatic tissues from BA patients Positive cells were observed in 7 (35%) of the 20 BA cases (cases 3, 4, 5, 7, 9, 19, and 20). Senescent cells were detected as those showing a cytoplasmic blue precipitate, as illustrated in Fig. 8. Correlation between SA-b-Gal in BA and PELD score The mean PELD score for BA cases positive and negative SA-b-Gal was 23.1 and 4.3, respectively. There was a significant correlation between SA-b-Gal in BA and the PELD score (p \ 0.001).

Discussion The present study examined telomere lengths in hepatic tissue from BA patients and controls using Southern blotting and Q-FISH analysis. Because hepatic tissue from BA patients includes a variety of cell types, such as hepatocytes, chronic inflammatory cells and fibroblasts, it is necessary to analyze the length of hepatocellular telomeres in a cell type-specific manner to evaluate hepatocellular injury. Southern blotting does not demonstrate any differences in the telomere length of hepatic tissues among

World J Surg (2012) 36:908–916

913

Fig. 4 Representative Q-FISH images for a 0.7-year-old female patient with biliary atresia (BA) (case 9, 14.4 kbp, TCR = 0.35) (a), a 2.0-year-old male control (case 10, 12.9 kbp, TCR = 1.48) (b), and a cell block (TIG-1 cell, 34 PD, 8.6 kbp, TCR = 1.04) (c) (original

magnification 9400). Red (Cy3) and green (FITC) spots indicate telomere and centromere signals, respectively. Blue (DAPI) reveals the nuclei. Telomere signals are brighter in the control than in the BA sample by visual assessment

Fig. 5 Distributions of the telomere-centromere ratio (TCR) in hepatocytes of a 0.7-year-old female patient with biliary atresia (BA) (case 9) (a) and a female control at 41 weeks of gestation (case 6) (b). The normalized TCR (NTCR) distributions of the two cell

types are shown in (c): hepatocytes in BA (red); hepatocytes in control liver (blue); TIG-1 cells in a cell block (green). The tails of the NTCR distributions are shorter in the hepatocytes of the BA patient than in the control

123

914

Fig. 6 Box plots showing the normalized telomere-centromere ratio (NTCR) distributions in hepatocytes of patients with biliary atresia (BA) and controls (NTCRs at 10, 25, 50, 75, and 90%). The NTCRs of the hepatocytes are significantly smaller in BA than in the controls (p \ 0.001)

Fig. 7 Regression analysis of the normalized telomere-centromere ratio (NTCR) for the hepatocytes in biliary atresia against pediatric endstage liver disease (PELD) score demonstrating a significant negative correlation (NTCR = -0.021*PELD score ? 0.997, p \ 0.001)

Fig. 8 Expression of senescence-associated b-galactosidase in hepatic tissues of a patient with biliary atresia (case 5). Senescent cells were detected as those showing a cytoplasmic blue precipitate

123

World J Surg (2012) 36:908–916

individuals. On the other hand, Q-FISH makes it possible to analyze the length of hepatocellular telomeres in a cell type-specific manner and is therefore considered to be an indispensable method for measurement of telomere length in tissue showing marked inflammation, such as hepatic tissue affected by BA. Some previous studies using Q-FISH have demonstrated the prevalence of shortened telomeres in hepatic tissue affected by chronic diseases or HCC [12, 13]. Rudolph and DePinho [26] proposed a telomere hypothesis for liver cirrhosis in which chronic liver injury induces continual waves of liver destruction and regeneration, resulting in critical telomere shortening, which culminates in hepatocyte replicative senescence or death, and ultimately liver cirrhosis. Using Q-FISH, we demonstrated the presence of excessive hepatocellular telomere shortening in BA, suggesting that hepatocellular telomeres shorten rapidly after birth. Furthermore, senescent cells were detected on the basis of SA-b-Gal expression in hepatic tissues from BA patients, reflecting hepatocellular telomere shortening. Progressive biliary liver cirrhosis as well as chronic liver disease may result in progression of cellular senescence, in parallel with hepatocellular telomere shortening. Therefore, hepatocellular telomere length could be an objective indicator of hepatocellular injury. The telomere hypothesis proposes that telomere shortening initiates carcinoma through induction of chromosomal and genetic instability due to telomere dysfunction [27], and a critical reduction of telomere length has been implicated as one of the causes of hepatocarcinogenesis [28]. Although an association between HCC and BA is not commonly observed, BA complicated by HCC has been reported occasionally in young patients (females aged, 6, 8, 10, and 19 years) [29–31]. Hepatocarcinogenesis in BA has generally been overlooked because of the low long-term survival rate of BA patients with their native liver, and the high likelihood of death before HCC can develop. Therefore, excessive hepatocellular telomere shortening also might have the potential to contribute to the induction of HCC in BA patients. Children with BA who retain their native liver after a Kasai portoenterostomy show 10- and 20 year survival rates of 31–60% and 23–48%, respectively [23, 32–35]. The cause of death is almost always liver failure. These data lend support to the fact that children with BA have a lower hepatocellular reserve capacity than children with a normal liver. The PELD score is an effective and noninvasive indicator of the degree of progression of liver failure, but indications for LT in BA patients with a low PELD score include intractable cholangitis, portal hypertension, and pulmonary vascular disorders (hepatopulmonary syndrome and portopulmonary hypertension) [36]. However, the timing of LT for these patients remains unclear. In the

World J Surg (2012) 36:908–916

present study, hepatocellular telomere shortening was negatively correlated with the PELD score in BA patients, thus underlining its effectiveness as an indicator. However, the hepatocellular telomere length in approximately 90% of patients with BA was excessively shortened compared with the controls, regardless of the PELD score, and all of the patients are currently still alive and being managed as outpatients after LT. Therefore, excessive telomere shortening in BA may be representative of a lower hepatocellular reserve capacity, thus supporting the early implementation of LT, even if the PELD score is low. In conclusion, the present findings support the feasibility of using hepatocellular telomere length determined by Q-FISH as an objective indicator of hepatocellular reserve capacity in BA. Because the native liver of BA patients is cirrhotic and the long-term survival rate of such patients with their native liver is low, coupled with the fact that extremely short telomeres are associated with subsequent carcinogenesis and organ failure, it can be said that prominent telomere shortening in the hepatic tissue of BA patients compared with normal controls supports the early implementation of LT, regardless of the PELD score. Financial support This study was partly supported by a grant for 388research expenses from Jichi Medical University Young Investigator 389Award (to Yukihiro Sanada) and by Grants-in-Aid for Scientific 390Research from the Ministry of Education, Culture, Sports, Science 391and Technology of Japan (Grant Nos. B21390109 to Kaiyo Takubo; 392Grant No. C20590378 to Junko Aida, and Grant No. C20590389 to 393Naoshi Ishikawa). Conflict of interest

None.

References 1. Perlmutter DH, Shepherd RW (2002) Extrahepatic biliary atresia: a disease or a phenotype? Hepatology 35(6):1297–1304 2. Hartley JL, Davenport M, Kelly DA (2009) Biliary atresia. Lancet 374(9702):1704–1713 3. Kasai M, Kimura S, Asakura Y, Szuki H, Taira Y, Ohashi E (1968) Surgical treatment of biliary atresia. J Pediatr Surg 3(6): 665–675 4. Cowles RA, Lobritto SJ, Ventura KA, Harren PA, Gelbard R, Emond JC, Altman RP, Jan DM (2008) Timing of liver transplantation in biliary atresia-results in 71 children managed by a multidisciplinary team. J Pediatr Surg 43(9):1605–1609 5. McDiarmid SV, Anand R, Lindblad AS (2002) Principal investigators and institutions of the studies of pediatric liver transplantation (SPLIT) research group. Development of a pediatric end-stage liver disease score to predict poor outcome in children awaiting liver transplantation. Transplantation 74(2):173–181 6. McDiarmid SV, Merion RM, Dykstra DM, Harper AM (2004) Selection of pediatric candidates under the PELD system. Liver Transpl 10(10 Suppl 2):S23–S30 7. Blackburn EH (1991) Structure and function of telomeres. Nature 350(6319):569–573 8. Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621

915 9. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE (1998) Extension of life-span by introduction of telomerase into normal human cells. Science 279(5349):349–352 10. Harley CB, Futcher AB, Greider CW (1990) Telomeres shorten during ageing of human fibroblasts. Nature 345(6274):458–460 11. Von Zglinicki T (2002) Oxidative stress shortens telomeres. Trends Biochem Sci 27(7):339–344 12. Plentz RR, Caselitz M, Bleck JS, Gebel M, Flemming P, Kubicka S, Manns MP, Rudolph KL (2004) Hepatocellular telomere shortening correlates with chromosomal instability and the development of human hepatoma. Hepatology 40(1):80–86 13. Wiemann SU, Satyanarayana A, Tsahuridu M, Tillmann HL, Zender L, Klempnauer J, Flemming P, Franco S, Blasco MA, Manns MP, Rudolph KL (2002) Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis. FASEB J 16(9):935–942 14. Takubo K, Nakamura K, Izumiyama N, Sawabe M, Arai T, Esaki Y, Tanaka Y, Mafune K, Fujiwara M, Kammori M, Sasajima K (1999) Telomere shortening with aging in human esophageal mucosa. Age 22(3):95–99 15. Takubo K, Nakamura K, Izumiyama N, Furugori E, Sawabe M, Arai T, Esaki Y, Mafune K, Kammori M, Fujiwara M, Kato M, Oshimura M, Sasajima K (2000) Telomere shortening with aging in human liver. J Gerontol A Biol Sci 55(11):B533–B536 16. Lansdorp PM, Verwoerd NP, van de Rijke FM, Lansdorp PM, Dragowska V, Little MT, Dirks RW, Raap AK, Tanke HJ (1996) Heterogeneity in telomere length of human chromosomes. Hum Mol Genet 5(5):685–691 17. Ferlicot S, Youssef N, Feneux D, Delhommeau F, Paradis V, Bedossa P (2003) Measurement of telomere length on tissue sections using quantitative fluorescence in situ hybridization (Q-FISH). J Pathol 200(5):661–666 18. Meeker AK, Gage WR, Hicks JL, Simon I, Coffman JR, Platz EA, March GE, De Marzo AM (2002) Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining. Am J Pathol 160(4):1259–1268 19. Aida J, Izumiyama-Shimomura N, Nakamura K, Ishii A, Ishikawa N, Honma N, Kurabayashi R, Kammori M, Poon SS, Arai T, Takubo K (2007) Telomere length variations in 6 mucosal cell types of gastric tissue observed using a novel quantitative fluorescence in situ hybridization method. Hum Pathol 38(8):1192–1200 20. Aida J, Izumiyama-Shimomura N, Nakamura K, Ishikawa N, Poon SS, Kammori M, Sawabe M, Arai T, Matsuura M, Fujiwara M, Kishimoto H, Takubo K (2008) Basal cells have longest telomeres measured by tissue Q-FISH method in lingual epithelium. Exp Gerontol 43(9):833–839 21. Aida J, Izumo T, Shimomura N, Nakamura K, Ishikawa N, Matsuura M, Poon SS, Fujiwara M, Sawabe M, Arai T, Takubo K (2010) Telomere lengths in the oral epithelia with and without carcinoma. Eur J Cancer 46(2):430–438 22. Takubo K, Fujita M, Izumiyama N, Nakamura K, Ishikawa N, Poon SS, Fujiwara M, Sawabe M, Matsuura M, Grabsch H, Arai T, Aida J (2010) Q-FISH analysis of telomere and chromosome instability in the oesophagus with and without squamous cell carcinoma in situ. J Pathol 221(2):201–209 23. Hung PY, Chen CC, Chen WJ, Lai HS, Hsu WM, Lee PH, Ho MC, Chen TH, Ni YH, Chen HL, Hsu HY, Chang MH (2006) Long-term prognosis of patients with biliary atresia: a 25 year summary. J Pediatr Gastroenterol Nutr 42(2):190–195 24. Grant JD, Broccoli D, Muquit M, Manion FJ, Tisdall J, Ochs MF (2001) Telomeric: a tool providing simplified, reproducible measurements of telomeric DNA from constant field agarose gels. Biotechniques 31(6):1314–1316

123

916 25. Ohashi M, Aizawa S, Ooka H, Ohsawa T, Kaji K, Kondo H, Kobayashi T, Noumura T, Matsuo M, Mitsui Y, Murota S, Yamamoto K, Ito H, Shimada H, Utakoji T (1980) A new human diploid cell strain, TIG-1, for the research on cellular aging. Exp Gerontol 15(2):121–133 26. Rudolph K, DePinho RA (2001) Telomeres and telomerase in experimental liver cirrhosis. In: The liver biology and pathobiology, 4th edn, Lippincott Williams and Wilkins, Philadelphia 27. Yokota T, Suda T, Igarashi M, Kuroiwa T, Waguri N, Kawai H, Mita Y, Aoyagi Y (2003) Telomere length variation and maintenance in hepatocarcinogenesis. Cancer 98:110–118 28. Isokawa O, Suda T, Aoyagi Y, Kawai H, Yokota T, Takahashi T, Tsukada K, Shimizu T, Mori S, Abe Y, Suzuki Y, Nomoto M, Mita Y, Yanagi M, Igarashi H, Asakura H (1999) Reduction of telomeric repeats as a possible predictor for development of hepatocellular carcinoma: convenient evaluation by slot-blot analysis. Hepatology 30(2):408–412 29. Hol L, van den Bos IC, Hussain SM, Zondervan PE, de Man RA (2008) Hepatocellular carcinoma complicating biliary atresia after Kasai portoenterostomy. Eur J Gastroenterol Hepatol 20:227–231 30. Kohno M, Kitatani H, Wada, Kajimoto T, Matuno H, Tanino M, Nakagawa T, Takarada A (1995) Hepatocellular carcinoma complicating biliary cirrhosis caused by biliary atresia: report of a case. J Pediatr Surg 30(12):1713–1716

123

World J Surg (2012) 36:908–916 31. Tatekawa Y, Asonuma K, Uemoto S, Inomata Y, Tanaka K (2001) Liver transplantation for biliary atresia associated with malignant hepatic tumors. J Pediatr Surg 36(3):436–439 32. Howard ER, MacLean G, Nio M, Donaldson N, Singer J, Ohi et al (2001) Survival patterns in biliary atresia and comparison of quality of life of long-term survivors in Japan and England. J Pediatr Surg 36(6):892–897 33. Lykavieris P, Chardot C, Sokhn M, Gauthier F, Valayer J, Bernard O (2005) Outcome in adulthood of biliary atresia: a study of 63 patients who survived for over 20 years with their native liver. Hepatology 41(2):366–371 34. Nio M, Ohi R, Miyano T, Nio M, Ohi R, Miyano T, Saeki M, Shiraki K, Tanaka K, Japanese Biliary Atresia Registry (2003) Five- and 10-year survival rates after surgery for biliary atresia: a report from the Japanese Biliary Atresia Registry. J Pediatr Surg 38(7):997–1000 35. Shinkai M, Ohhama Y, Take H, Shinkai M, Ohhama Y, Take H, Kitagawa N, Kudo H, Mochizuki K, Hatata T (2009) Long-term outcome of children with biliary atresia who were not transplanted after the Kasai operation: [20-year experience at a children’s hospital. J Pediatr Gastroenterol Nutr 48(4):443–450 36. Shinkai M, Ohhama Y, Take H, Fukuzato Y, Fujita S, Nishi T (2003) Evaluation of the PELD risk score as a severity index of biliary atresia. J Pediatr Surg 38(7):1001–1004