ABSTRACT. One-step RT-PCR procedure without initial RNA extraction step is tested for laser microdissected tissue sample. Unfixed cryosections of liver and kidney tissue of male SD rats were cut using laser microdissection system and directly used as templates for RT-PCR study. To check the sensitivity, 5, 25, 125, and 625 hepatocytes were cut and put in PCR-tube. After DNase treatment and cDNA synthesis with pd(N)6 random primer, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were amplified by 60 thermal cycles. GAPDH-specific bands were observed at as few as 25 hepatocytes. Specificity of this procedure was tested for hepatocytes, renal tubular epithelium and glomerular tissue using albumin PCR primers. Approximately 250 cells were cut and albumin cDNA was amplified as described above. Albumin specific band was observed only in hepatocytes sample. To apply this approach to quantitative PCR, various numbers of hepatocytes were cut and put in 0.2 mL PCR tube. After reverse transcription and 10 cycles of GAPDH cDNA amplification by regular thermal-cycler, PCR solution was transferred to 96-well plate designed for real-time PCR system, and further 40 cycles were performed. As a result, GAPDH cDNAs were successfully amplified with a good correlation between the number of template hepatocytes and the intensity of PCR signal. From these results, we concluded this approach would be very useful for the expression analysis of microdissected pathology samples. KEY WORDS: laser microdissection, one-step RT-PCR.

Transforming growth factor a (TGF-a) is a potent stimulator of normal hepatocyte proliferation, considered to have relationship to the liver regeneration or carcinogenesis . In this study, we investigated immunohistochemicall y the association between expression of TGF-a and cell proliferation activity in mouse hepatoblastoma s (HBs) and hepatocellular carcinomas (HCCs) induced in B6C3F1 mice by diethylnitrosamin e and sodium phenobarbital . The TGF-a-positive rate in HBs (29.2%) was signi cantly higher than that in HCCs (12.7%). Likewise, the proliferating cell nuclear antigen-positive rate (22.2%) was higher than the HCC value (14.5%). On the individual data for both TGF-a and PCNA, most of the HBs showed higher positive rates than HCCs. In HBs, TGF-a was localized only in the nuclei, whereas some HCC cells stained positive both in their nuclei and cytoplasm (0.6%). These results suggest expression of TGF-a and its localization might be linked to cell proliferation and play a role in malignant progression of mouse HBs.

Hepatocellular carcinomas (HCCs) were induced in male Fischer 344 rats with dietary 3'-methyl-4-(dimethylamino)-azobenzene treatment and were classified into solid, glandular (well-or

ABSTRACT. Immunohistochemical localization of TGF-alpha and cell proliferation kinetics during liver regeneration after two-thirds partial hepatectomy (PH) were investigated. Twenty-four to 72 hr after PH, appreciable increase in the number of TGF-alpha-positive hepatocytes was observed in zones 1 and 2. At the peak at 36 hr, almost all positive cells were stained in their nuclei. Considerable increase in the BrdU labeling index was observed 24-36 hr after PH with a peak at 24 hr in zones 1 and 2. These results indicated an ass ociation between TGF-alpha expression and hepatocyte regeneration. It is suggested that immunohistochemical localization of TGF-alpha may be a useful marker of cell proliferation activity in rat liver. KEY WORDS: immunohistochemistry, liver, TGF-alpha.J. Vet. Med. Sci. 64(11): 1045-1048, 2002 Transforming growth factor alpha (TGF-alpha), a member of the epidermal growth factor (EGF) family, is a 50 amino acid polypeptide [12]. It shares about 30% structural similarity with EGF and binds to the EGF receptor [3]. Several reports have suggested TGF-alpha to play important roles during hepatic development and regeneration as a potent stimulator of hepatocyte [2,8]. However, there are few reports describing TGF-alpha expression and its intracellular localization in relation to cell proliferation in the liver.In the present immunohistochemical study, we investigated localization of TGF-alpha in different zones of the liver after partial hepatectomy (PH), and analyzed its links with cell kinetics.Male Fischer 344 rats weighing around 200 g were purchased from Charles River Japan (Yokohama, Japan) and acclimatized for 1 week in an air-conditioned animal room at 22°C with a 12-hr light/dark cycle. They were given Oriental MF diet (Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum.All animals underwent two-thirds partial hepatectomy under light ether anesthesia. For the cell proliferation kinetic study, the rats were given an intraperitoneal injection of BrdU (100 mg/kg i.p.) (Sigma Chemical Co., St. Louis, MO) 1 hr before sacrifice at 6, 12, 24, 36, 48 or 72 hr after PH under ether anesthesia. Number of rats in each group was 9.Livers were excised, fixed in 10% neutral-buffered formalin, embedded in paraffin, cut into 3-4 µm sections and examined immunohistochemically using anti-BrdU (Dako Japan Co., Kyoto) and anti-TGF-alpha (Oncogene Research Products, Cambridge, MA, U.S.A.) antibodies by avidinbiotin-peroxidase complex method. For quantitative comparisons, 1,000 nuclei were counted for each zone of the liver, and the percentages of BrdU-positive nuclei at different time points after PH were calculated. Similarly, 1,000 cells were counted for each zone and the percentages of TGF-alpha-positive hepatocytes with staining of their nuclei or cytoplasm were calculated.Representative immunohistochemical localization of TGF-alpha and BrdU labeling in each zone of the liver are illustrated in Fig. 1. Data for the numbers of hepatocytes with TGF-alpha-positive nuclei or cytoplasm are summarized ...

ABSTRACT. Hepatoblastomas (HBs) were induced in B6C3F1 male mice by diethylnitrosamine (DEN) and sodium phenobarbital (PB). Six-week-old mice received a single intraperitoneal dose of DEN followed by a continuous treatment with PB in diet at a concentration of 0 (group 1) or 500 (group 2) ppm for 50 weeks. HBs were observed in 13 of 21 (62%) group 2 mice, with typical histologic features as reported previously, while no such tumors were observed in group 1. Seven of 13 (54%) HBs were found in and/or adjacent to hepatocellular adenomas (HCAs) or hepatocellular carcinomas (HCCs). Immunohistochemically, all HBs were positive for S-100 protein but negative for keratin, α-fetoprotein (AFP), albumin (ALB) and vimentin, while HCC cells occasionally reacted positively for AFP with a mosaic pattern. HCC and HCA cells were occasionally positive for ALB. Non-neoplastic hepatocytes and normal bile ducts were positively stained for ALB and keratin/S-100 protein, respectively. S-100 protein is known to be expressed in many mesenchymal tissues and neoplasms including neuroectodermal elements but negative in cells of the hepatic lineage. Thus, the present immunohistochemical results suggested that mesenchymal differentiation occurs in mouse HB cells as observed in human HBs, one of the most frequent infant liver tumors in humans. Although the susceptibility of mouse HBs to PB-promotion suggests a hepatocytic histogenesis, the present immunohistochemical results support the hypothesis that the mouse HB is derived from pluripotent endodermal stem-like cells. KEY WORDS: hepatoblastoma, immunohistochemistry, mouse, S-100 protein.