Recent advances made in the isolation, culture, and transplantation of defined populations of intrahepatic biliary epithelial cells and oval cells have permitted direct analysis of the functions, growth properties, and differentiation potential of these respective cell types in their untransformed or transformed states. This review provides a current and comprehensive examination of the various approaches that have been taken to isolate and culture intrahepatic biliary epithelial cells from normal and cholestatic liver and oval cells from preneoplastic liver. Emphasis is placed on comparing the phenotypic features and growth properties of these various biliary cell types in vitro as well as on describing their transplantation into ectopic tissue sites. In addition, the oval cell is evaluated in terms of its potential role as a ‘facultative stem cell’ during hepatocarcinogenesis.

An extensive bile ductular cell hyperplasia with the formation of well-differentiated bile ductules is the most prominent feature of rat liver at 6 to 15 weeks after bile duct ligation. We have improved our previous cell isolation procedure and are now routinely able to obtain from such livers high yields of viable bile ductular epithelial cells. These cells were characterized with respect to their specific activities of gamma-glutamyl transpeptidase and beta-glucuronidase and of select Phase I and Phase II enzymes of biotransformation. At the time of their isolation, only a very small number of the bile ductular epithelial cells were observed to be in DNA synthesis. In addition, in histological sections prepared from intact hyperplastic bile ductular tissue isolates, only the bile ductular epithelial cells exhibited histochemical staining for gamma-glutamyl transpeptidase activity. Typically, greater than 95% of the cells isolated from this tissue were also found to be histochemically positive for gamma-glutamyl transpeptidase activity, and no hepatocytes were seen contaminating this cell population. Biochemically, the isolated bile ductular cells exhibited a gamma-glutamyl transpeptidase specific activity that was 100 times higher than that of hepatocytes isolated at the same time from the bile duct-ligated rats and more than 300 times higher than the specific activity of the enzyme of freshly isolated normal rat hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

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We analyzed the components of the renin-angiotensin system (RAS) in ocular tissues of normal rabbit eyes and compared the results with those measured in rabbit eyes with proliferative vitreoretinopathy and ocular hypertension. Proliferative vitreoretinopathy was induced by injection of human platelets into the vitreous humor, and ocular hypertension was induced by injection of alpha-chymotrypsin into the posterior chamber. Angiotensinogen, renin, angiotensin converting enzyme (ACE), angiotensin II (Ang II), and Ang II receptors were assessed using conventional biochemical techniques. The vascularized tissues of normal eyes contained high renin and ACE activities concomitant with low concentration of angiotensinogen and Ang II. In general, in the ocular humors, the opposite was found. The Ang II receptor density was highest in the uveal tract [range 35-190 fmol/mg protein]. The AT1 receptor subtype predominated [> 80%]. The RAS was only minimally different in the two pathological models except that, in ocular hypertension, the renin activity in the uveal tract was reduced [-50%]. Also, the ratio of AT1 to AT2 receptors changed as compared to control, although the total receptor density remained unaltered. In conclusion, we present evidence for the presence of a complete local RAS in the rabbit eye, which is only marginally affected by the two pathological models studied.

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The repair of DNA damage in eukaryotic cells is closely coupled with local changes of chromatin structure such that newly synthesized repair patches transiently appear in 'free' DNA domains with increased accessibility to enzymatic and chemical probes. We have isolated these domains from mammalian cells repairing bulky DNA adducts. During the first 3 h of repair, excision of adducts occurred exclusively in free DNA and was closely linked with the appearance of newly synthesized repair patches. Following depletion of chromatin-bound poly(ADP-ribose), the repositioning of repair patches into these domains was completely blocked, although overall repair patch synthesis was unaltered. Concomitantly, DNA adducts were no longer excised and tended to accumulate in free DNA domains. Our results suggest a tight coupling of the excision step with the formation of free DNA domains by a mechanism involving poly ADPrnbosylation of chromatin proteins.In chromatin of mammalian cells, newly synthesized DNA repair patches exhibit a transient micrococcal nuclease hypersensitivity. This hypersensitivity is thought to reflect local disruptions in the tightly packed nucleosomal organization of chromatin, causing exposure of 'free' DNA domains (for review see 1,2). The function of these domains as well as the mechanisms involved in their formation are unknown. We have speculated that the post-translational poly ADP-ribosylation of chromatin proteins might be involved in the formation of free DNA domains in DNA excision repair. Poly ADP-ribosylation is catalyzed by the enzyme poly(ADP-ribose)polymerase (1,3,4; EC 2.4.2.30). Following activation by DNA nicks, this enzyme operates in a strictly processive manner (5). Continuous treatment of living cells with benzamide, a competitive inhibitor of this enzyme (3,4,6), results in the degradation of chromatin-bound ADP-ribose polymers (7) by the enzyme poly(ADP-ribose)glycohydrolase (3,4). Using non-replicating adult rat hepatocytes in primary monolayer culture, we have established conditions for the complete depletion of chromatin-associated poly(ADP-ribose) (7,8). Hepatocytes survive up to 9 days under these conditions and maintain expression of liver-specific functions (8). Thus, poly(ADPribose)-depleted hepatocytes represent a convenient model system to study the role of poly ADP-ribosylation in specific steps of DNA repair.We have previously shown that unfolded free DNA domains can be isolated from the chromatin of intact mammalian cells by taking advantage of their preferential accessibility to 8-methoxypsoralen (9). Upon intercalation into free DNA domains of living cells, 8-methoxypsoralen can be photoactivated to form bifunctional DNA adducts crosslinking the two DNA strands. Crosslinked (free) DNA domains can then be isolated quantitatively following a denaturation/renaturation treatment and subsequent nuclease SI digestion of non-crosslinked DNA strands (for details see 9).Here we have isolated free DNA domains from non-replicating hepatocytes (9

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The chromatin organization of living mammalian cells was probed using 8-methoxypsoralen (MOP). In intact cells, MOP intercalates into DNA domains which are also preferentially accessible to micrococcal nuclease. After UV365 nm irradiation of MOP-treated cells, this chemical forms bifunctional adducts crosslinking the two strands of DNA. Following extraction of cellular DNA, heat denaturation and renaturation at low temperature, the fraction of crosslinked DNA is obtained following enzymatic hydrolysis of unhybridized, non-crosslinked DNA by nuclease S1 treatment. An application of this procedure in the isolation of 8-methoxypsoralen-accessible DNA domains during DNA excision repair is shown.