OBJECTIVE: To study the effect of retinoid receptor alpha (RXRalpha) transfection plus treatment with the RXRalpha ligand, 9-cis-RA, on the proliferation and phenotype of platelet-derived growth factor (PDGF) activated hepatic stellate cells (HSCs). METHODS: PDGF activated rat hepatic stellate cells were transfected with eukaryotic expression vector pcDNA3.1-human RXRalpha, and confirmed by Western blot. Proliferation of transfected HSC was assayed by bromodeoxyuridine (BrdU) incorporation as well as MTT, and the phenotype (alpha-smooth muscle actin, desmin) was observed by immunocytochemistry with image analysis. RESULTS: Transfection of the RXRalpha gene and treatment with ligand 9-cis-RA of PDGF-activated HSCs extended the increased expression of RXRalpha protein for at least 168 hours. Cell proliferation and expressions of alpha-smooth muscle actin (alpha-SMA) and desmin were blocked, compared with groups of sham-transfected, PDGF-activated, no transfection, no ligand treatment, and irrelevant ligand treated HSCs. CONCLUSION: Transfection with the RXRalpha gene followed by 9-cis-RA ligand treatment will inhibit the proliferation and reverse the phenotype of activated HSC.

In both the primary tumor and associated lymph-node metastases of 40 cases of Dukes'C colorectal adenocarcinomas, exons 5 to 9 of the p53 tumor-suppressor gene were examined by PCR amplification and single-strand-conformation-polymorphism (SSCP) analysis. Mobility shifts indicating p53 mutations, which were confirmed by direct sequencing, were identified in 14 primary cancers (35%) and in 19 of the 40 lymph-node metastases (48%). In 12 cases (30%), the p53-mutation status in the primary cancer and its lymph-node metastases was identical. This result is compatible with the hypothesis that when a p53 mutation occurs before the establishment of lymph-node metastasis, it subsequently persists in the metastatic nodes. In 7 cases (18%), p53 mutations were identified in lymph-node metastases that were not concordant with the p53 status in the primary tumor. This finding can be explained by assuming that (1) p53 heterogeneity existing in the primary tumor is not reflected in all metastases and/or (2) new p53 mutations may occur during the development of metastatic lesions.

Chemically induced DNA fragmentation and unscheduled DNA synthesis were determined in gamma-glutamyltranspeptidase (GGT)-positive and GGT-negative hepatocytes isolated from rat livers subjected to a multistage hepatocarcinogenesis regimen (Solt-Farber), which included 0.05% phenobarbital promotion for 6 weeks (early) or 6 months (late). The results indicated that there was DNA damage in untreated GGT-positive and GGT-negative hepatocytes with either period of promotion compared with normal hepatocytes; however, no statistical difference could be seen between GGT-positive and GGT-negative hepatocytes. DNA damage induced in vitro by the activation-dependent carcinogen dimethylnitrosamine was much less in GGT-positive hepatocytes than in GGT-negative hepatocytes or normal hepatocytes. No significant difference in DNA damage was seen in both GGT-positive and GGT-negative cell populations following treatment with the activation-independent carcinogen ethylnitrosourea (ENU), although DNA damage of GGT-positive hepatocytes was less than that of normal hepatocytes. The background of unscheduled DNA synthesis in both GGT-positive and GGT-negative hepatocytes at either time of promotion was higher than that of normal hepatocytes. The capacity for DNA repair in GGT-positive hepatocytes appeared to be lower than that in GGT-negative hepatocytes. GGT-negative hepatocytes exhibited a lower capacity for DNA repair than that of normal hepatocytes in terms of the rate of unscheduled DNA synthesis elicited by dimethylnitrosamine and ethylnitrosourea in vitro.

Serial observations were performed on the sera α-fetoprotein (A F P) as well as on the immunohistochemical localization of A F P in liver tissues during hepatocarcinogenesis induced with 3'-Me-DAB in rats. Results showed that the dynamics of the sera A F P in rats bearing hepatocellular carcinomas (HCC) revealed saddle-like curve, whereas that of those rats without hearing HCC only increased at a sustained low level. The pathological bases which are responsible for precancerous increase of sera AFP are as follows: the small basophilic hepatocytes, "survival" hepatocytes, hyperplastic nodules, dysplastic hepatocytes, the hepacytic cords within the cholangiofibrosis foci. Solt-Farber model analysis indicated that the increase of sera AFP was related to the proliferation of the initiated cells. Immunoelectronic microscopy demonstrated the AFP was located on the membranes, which may be caused by the absorption of AFP from blood onto the cell membranes.

OBJECTIVE: To study the effects of heparin on the growth, extracellular matrix and matrix metalloproteinase (MMP) gene expression in rat hepatic stellate cells (HSC). METHODS: Activated HSC was treated by heparin or fetal calf serum without heparin. The cell growth was evaluated by actual cell count and BrdU-labelled immunocytochemical stain. The gene expressions of type I and IV procollagen, fibronectin, MMP-2 and membrane type matrix metalloproteinase (MT-MMP) were investigated by immunocytochemical stain and digoxigenin-labeled in situ hybridization technique, respectively. In addition, the gelatinase activity of MMP-2 was examined by zymography. RESULTS: Heparin could obviously reduce HSC growth, inhibit the synthesis of type I procollagen and fibronectin protein, and the gene expressions of type I procollagen, fibronectin and MT-MMP. The expressions of type IV procollagen, MMP-2 and MMP-2 activity were not affected by heparin. CONCLUSION: The results demonstrate that heparin can inhibit HSC proliferation, down-regulate interstitial collagen synthesis and inhibit MT-MMP gene expression.