Masahiro Tsutsumi

Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets

Hepatic tissue engineering using primary hepatocytes has been considered a valuable new therapeutic modality for several classes of liver diseases. Recent progress in the development of clinically feasible liver tissue engineering approaches, however, has been hampered mainly by insufficient cell-to-cell contact of the engrafted hepatocytes. We developed a method to engineer a uniformly continuous sheet of hepatic tissue using isolated primary hepatocytes cultured on temperature-responsive surfaces. Sheets of hepatic tissue transplanted into the subcutaneous space resulted in efficient engraftment to the surrounding cells, with the formation of two-dimensional hepatic tissues that stably persisted for longer than 200 d. The engineered hepatic tissues also showed several characteristics of liver-specific functionality. Additionally, when the hepatic tissue sheets were layered in vivo, three-dimensional miniature liver systems having persistent survivability could be also engineered. This technology for liver tissue engineering is simple, minimally invasive and free of potentially immunogenic biodegradable scaffolds.

Ki-ras mutations and p53 protein expressions in intrahepatic cholangiocarcinomas : relation to gross tumor morphology

BACKGROUND & AIMS We previously reported that intrahepatic cholangiocarcinomas (ICCs) can be divided into three categories according to their gross appearance with possible links to biological behavior. Ki-ras and p53 gene alterations are thought to be involved in early and late phases of carcinogenesis, respectively. This study was performed to investigate the relationship between the gross appearance and genetic alterations of ICC. METHODS We examined 21 patients with ICC. Ki-ras point mutations were assessed by polymerase chain reaction/single-strand conformation polymorphism methods followed by direct DNA sequencing. Expressions of p53 protein were immunohistochemically assessed. RESULTS Ki-ras point mutations were found in 10 patients (48%), and expressions of p53 protein were detected in 4 (19%). Applying the gross classification that we previously proposed, Ki-ras mutations were prominent in the periductal extension type (4 of 6; 67%) and the spicula-forming type (6 of 10; 60%). On the other hand, none of the five mass-forming-type tumors harbored Ki-ras mutations. Expressions of p53 protein did not show any clear association with gross appearance. CONCLUSIONS Ki-ras gene alterations may be involved in the cholangiocarcinogenesis of periductal extension and spicula-forming but not mass-forming types, suggesting that the underlying processes of development are different.

The response of Parp knockout mice against DNA damaging agents.

Gene-disruption studies involving poly(ADP-ribose) polymerase (Parp) have identified the various roles of Parp in cellular responses to DNA damage. The partial rescue of V[D]J recombination process in SCID/Parp(-/-) double mutant mice indicates the participation of Parp in the repair of DNA strand break. Parp(-/-) mice are more sensitive to the lethal effects of alkylating agents. Parp is also thought to be involved in base-excision repair after DNA damage caused by alkylating agents. On the other hand, resistance of Parp(-/-) mice to DNA damage induced by reactive oxygen species implicates the contribution of Parp to cell death through NAD depletion. Parp(-/-) mice with two different genetic backgrounds also show enhanced sensitivity to the lethal effects of gamma-irradiation. Parp(-/-) mice show more severe villous atrophy of the small intestine compared to the wild-type counterpart in a genetic background of 129Sv/C57BL6. Other forms of enhanced tissue damage have been identified in Parp(-/-) mice with a genetic background of 129Sv/ICR. For example, Parp(-/-) mice exhibit extensive hemorrhage in the glandular stomach and other tissues, such as the testes, after gamma-irradiation. Severe myelosuppression is also observed in both Parp(+/+) and Parp(-/-) mice, but Parp(+/+) mice show extensive extramedullary hematopoiesis in the spleen during the recovery phase of post-irradiation, whereas the spleen of Parp(-/-) mice exhibits severe atrophy with no extramedullary hematopoiesis. The absence of extramedullary hematopoiesis in the spleen is probably the underlying mechanism of hemorrhagic tendency in various tissues of Parp(-/-) mice. These findings suggest that loss of Parp activity could contribute to post-irradiation tissue hemorrhage.

Hypomethylation of CpG Sites and c-myc Gene Overexpression in Hepatocellular Carcinomas, but Not Hyperplastic Nodules, Induced by a Choline-deficient L-Amino Acid-defined Diet in Rats

We have investigated aberrant methylation of CpG nucleotides (CpG sites) and gene expression of c‐myc during hepatocarcinogenesis induced by a choline‐deficient, L‐amino acid‐defined (CDAA) diet in rats. Male Fischer 344 rats, 6 weeks old, were continuously given a CDAA diet for 50 and 75 weeks and then killed. Macroscopically detectable nodules, which were histologically confirmed to be hyperplastic nodules (HNs) or well‐differentiated hepatocellular carcinomas (HCCs), were dissected free from the surrounding tissue. Normal control liver was obtained from 6‐week‐old rats. Methylation of CpG sites of the c‐myc gene was investigated in bisulfite‐treated DNA isolated from normal liver, HNs and HCCs. All 33 cytosines in the 5′‐upstream region of the c‐myc gene were fully methylated in control liver and the 4 HNs. In contrast, these cytosines were completely unmethylated in 5 HCCs. Examination of the c‐myc expression by reverse transcription‐polymerase chain reaction (RT‐PCR) analysis also showed a marked increase as compared to the low levels in normal livers and HNs. These results suggest that hypomethylation of the c‐myc gene might play a critical role in malignant transformation from HN to HCC during CDAA diet‐induced hepatocarcinogenesis in rats.

Inhibitory Effect of Tomato Juice on Rat Urinary Bladder Carcinogenesis after N-Butyl-N-(4-hydroxybutyl)nitrosamine Initiation

The effects of tomato juice on urinary bladder carcinogenesis were studied in male Fischer 344 rats initiated with N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) in rats. The animals (6 weeks old) were given 0.05% BBN in their drinking water for 8 weeks, followed by diluted tomato juice for 12 weeks, and killed at 20 weeks after the beginning of the experiment. Lycopene concentrations in the livers of rats given tomato juice were elevated. Histopathological analysis of urinary bladder lesions revealed the numbers, but not incidences, of urinary bladder transitional cell carcinomas (TCCs) to be decreased in the group given tomato juice. No influence on the incidence of simple and nodullopapillary hyperplasias, invasion or differentiation of TCC was noted. These results indicate that tomato juice, presumably the contained lycopene and other anti‐oxidants in combination, exerts an inhibitory effect on the development of TCCs in the rat urinary bladder.

Parp-1 deficiency implicated in colon and liver tumorigenesis induced by azoxymethane

Poly(ADP‐ribose) polymerase‐1 (Parp‐1) is activated by DNA strand breaks and functions in the maintenance of genomic integrity and cell death control. On the other hand, Parp‐1 is also involved in transcriptional regulation of various genes, and the relationship between Parp‐1 deficiency and susceptibility to tumorigenesis has not been fully elucidated. In the present study, Parp‐1−1− mice, harboring exon 1 disruption in Parp‐1, and Parp‐1+l+ animals were administered azoxymethane (AOM) at a dose of 10 mg/kg body weight once a week for 6 weeks. At 30 weeks after the first carcinogen treatment, mice were sacrificed. The incidence of animals bearing either adenomas or adenocarcinomas in the colon and the average number of colon tumors per mouse were significantly higher in Parp‐1−1− mice than in Parp‐1+l+ animals. β‐Catenin accumulation was observed in 43/44 of Parp‐1−1− tumors and 19/21 of the Parp‐1+l+ tumors and was not statistically different between the genotypes. This suggests that most tumors developed through a pathway involving the alteration of Wnt‐β‐catenin signaling in both Parp‐1−1‐ and Parp‐1+l+ mice. In the liver, where AOM is primarily activated, the incidence of animals bearing nodules and the average number of nodules per section were significantly increased in Parp‐1−1‐ mice compared with Parp‐1+l+ mice. Therefore, the results indicate that susceptibility to AOM‐induced tumorigenesis in the colon and also in the liver is enhanced in Parp‐1−1‐ mice, and Parp‐1 could have a substantial role in colon and liver tumorigenesis.

Development of Hepatocellular Adenomas and Carcinomas Associated with Fibrosis in C57BL/6J Male Mice Given a Choline‐deficient, L‐Amino Acid‐defined Diet

Development of hepatocellular carcinomas in rats caused by a choline‐deficient, L‐amino acid defined (CDAA) diet, usually associated with fatty liver, fibrosis, cirrhosis and oxidative DNA damage, has been recognized as a useful model of hepatocarcinogenesis caused by endogenous factors. In the present study, in order to further explore involved factors and genes, we established an equivalent model in spontaneous liver tumor‐resistant C57BL/6J mice. Six‐week‐old males and females were continuously fed the CDAA diet and histological liver lesions and oxidative DNA damage due to 8‐hydroxydeoxyguanosine (8‐OHdG) were examined after 22, 65 and 84 weeks. In male mice, fatty change and fibrosis were evident at 22 weeks, and preneoplastic foci of altered hepatocytes were seen at an incidence of 8/8 (100%) and a multiplicity of 6.6±4.0 per mouse at 65 weeks. Hepatocellular adenomas and carcinomas developed at incidences of 16/24 (66.7%) and 5/ 24 (20.8%), and multiplicities of 1.421±1.32 and 0.29±0.62, respectively, at 84 weeks. The female mice exhibited resistance to development of these lesions. The CDAA diet also increased 8‐OHdG levels in male but not female mice. These results indicate that a CDAA diet causes hepatocellular preneoplastic foci, adenomas and carcinomas associated with fibrosis and oxidative DNA damage in mice, as in rats, providing a hepatocarcinogenesis model caused by endogenous factors in mice.

K‐ras Gene Mutation in Early Ductal Lesions Induced in a Rapid Production Model for Pancreatic Carcinomas in Syrian Hamsters

The presence of K‐ras gene mutation was examined in experimentally induced preneoplastic pancreatic ductal lesions. Syrian hamsters received 70 mg/kg of N‐nitrosobis(2‐oxopropyl)amine (BOP) followed by repeated exposure to an augmentation pressure regimen consisting of choline‐deficient diet combined with dl‐ethionine and l‐methionine and administration of 20 mg/kg BOP. After two augmentation pressure cycles, pancreatic ductal cell hyperplasias appeared and after three cycles, atypical hyperplasias of pancreatic ductal cells and intraductal carcinomas developed. K‐ras mutations were detected by single‐strand conformation polymorphism analysis of polymerase chain reaction products and nucleotide sequencing. The results showed that K‐ras mutation had occurred in one of 9 simple hyperplasias of pancreatic ductal epithelium, in 5 of 9 atypical hyperplasias, and in 4 of 8 intraductal carcinomas. The findings thus suggested that K‐ras is activated in association with very early stage malignant transformation of pancreatic ductal cells in hamsters.

Age and organ dependent spontaneous generation of nuclear 8-hydroxydeoxyguanosine in male Fischer 344 rats.

8-Hydroxydeoxyguanosine (8-OHdG) is a major oxidative DNA adduct playing roles in senescence, carcinogenesis and various disease processes. High-performance liquid chromatography with an electrochemical detection (HPLC-ECD) method has been widely used to assess organ levels of 8-OHdG, and a recently introduced immunohistochemical approach has made it possible to clarify intra-organ localization. In the present study, these methods were employed to reveal age-dependent changes in nuclear 8-OHdG within various tissues of male Fischer 344 rats between 18 fetal days and 104 weeks of age. 8-OHdG was detected in the nuclei of cerebellar small granule and small cortical cells, cerebral nerve cells, and choroid plexus epithelia of the brain and ependymal cells of the spinal cord; parenchymal cells in the anterior lobe of the pituitary and adrenal glands (mainly cortex); bronchial epithelium of the lung; intra-hepatic bile duct, pancreatic duct, glandular gastric and intestinal epithelial cells; renal tubular epithelial cells (mainly medulla); and spermatogonia and spermatocytes of the testis and seminal vesicle epithelia. The nuclear 8-OHdG levels were high (more than two lesions per 106 deoxyguanosines) from 7 days to 104 weeks of age in the brain, 3 to 6 weeks in the adrenal gland, 6 to 104 weeks in the lung, and 3 to 52 weeks in the testis. In the other organs, the nuclear 8-OHdG levels remained low throughout. These findings provide a basis for research dealing with oxidative stress by indicating organ-specific and age- but not aging-dependent changes in the localization of spontaneously generated nuclear 8-OHdG in intact rats. The immunohistochemical approach has advantages for assessing variation of 8-OHdG formation at the cellular level not accessible to the HPLC-ECD method.

Inhibitory effects of β-carotene, palm carotene, and green tea polyphenols on pancreatic carcinogenesis initiated by N-nitrosobis(2-oxopropyl)amine in Syrian golden hamsters

The effects of alpha-carotene, beta-carotene, palm carotene, and green tea polyphenols (GTP) on the progression stage of pancreatic carcinogenesis after rapid production of ductal lesions were studied in Syrian hamsters. Dose threshold inhibitory effects were noted for beta-carotene, 25 ppm, and palm carotene, 40 ppm, which includes 24 ppm beta-carotene reducing the numbers of putative preneoplastic lesions of duct epithelial hyperplasia and atypical hyperplasia, as well as carcinoma in situ and invasive carcinomas. GTP at doses of 500 and 5000 ppm, but not 100 ppm, also significantly decreased the numbers of hyperplasia and total duct lesions. Combined administration of 40 ppm palm carotene, and 50 ppm GTP similarly inhibited the lesion development. Alpha-carotene, however, did not affect pancreatic carcinogenesis. The results suggest that chemopreventive effects are exerted by beta-carotene and GTP above critical doses and that combined administration of palm carotene and GTP might be a candidate chemoprevention strategy for pancreatic cancer in humans.