Search

Type I diabetes is caused by the progressive loss of pancreatic Β cells and is associated with several autoimmune phenomena (1). Although particular alleles closely linked to the major histocompatibility [...], Type I diabetes is an autoimmune disease involving an interaction between an epigenetic event (possibly a viral infection), the pancreatic Β cells, and the immune system in a genetically susceptible host. The possibility that the type I interferons could mediate this interaction was tested with transgenic mice in which the insulin-producing cells expressed an interferon-[Alpha]. These mice developed a hypoinsulinemic diabetes associated with a mixed inflammation centered on the islets. The inflammation and the diabetes were prevented with a neutralizing antibody to the interferon-[Alpha]. Thus, the expression of interferon-a by the cells could be causal in the development of type I diabetes, which suggests a therapeutic approach to this disease.

An Initiation-Promotion Assay in Rat Liver as a Potential Complement to the 2-Year Carcinogenesis Bioassay. DRAGAN, Y. P., RIZVI, T., XU, Y.-H., HULLY, J. R., BAWA, N., CAMPBELL, H. A., MARONPOT, R. R., AND PITOT, H. C. (1991). . 16, 525–547. Several pharmaceutical agents, manufacturing chemicals, and environmental contaminants were found to act primarily as promoting agents in an initiation-promotion paradigm. The phenotypic distribution of four enzyme markers-placental glutathione-S-transferase (PGST), γ-glutamyl trans-peptidase (GGT), canalicular ATPase (ATPase), and glucose-6-phosphatase (G6Pase)-was analyzed in altered hepatic foci (AHF) by quantitative stereology. The number and volume distribution of AHF were determined for each promoter tested. For phenobarbital and 2,3,7,8-tetrachloro--dioxin, PGST and GGT together scored 100% of the AHF; for l-(phenylazo)-2-naphthol (CI solvent yellow 14) and chlorendic acid, PGST alone marked 90% of the AHF; after chronic administration of WY-14,643, ATP and G6Pase were the predominant markers. In rats fed tamoxifen, G6P scored more than half of the AHF. Differences in the number of AHF promoted by each of these agents and in their phenotypic distributions may reflect the differentially responsive nature of individual initiated hepatocytes to the action of specific promoters. Since the chronic bioassay of suspected carcinogens does not allow one to differentiate between weak complete carcinogens and those carcinogenic agents that act in a reversible manner to promote the growth of previously initiated cells, the partial hepatectomy, altered-hepatic-focus model of cancer development is proposed as a supplement to the chronic bioassay for the identification of those carcinogenic agents that are primarily, if not exclusively, promoting agents in rat liver. [ABSTRACT FROM PUBLISHER]

After a 70% partial hepatectomy (PH), the steady-state levels of Connexin (Cx)32, Cx26, and Cx43 messenger RNA (mRNA) transcripts each displayed unique patterns of temporal expression. Within 1 hour after surgical resection, increased expression of all three Cx mRNAs was observed. Subsequently, the level of Cx32 mRNA transcripts transiently decreased to a nadir at 12 hours. Comparisons of the spatial changes with previously reported hepatocyte proliferation kinetics induced by PH demonstrated that hepatocytes before S-phase "remodel" their GJs. Within 1 to 5 hours post-PH, midzonal hepatocytes exhibited diffuse membrane staining different from the normal punctate distribution. Subsequently, midzonal hepatocytes expressed colocalized punctate Cx32 and Cx26 immunostaining. Because the changes occurred in midzonal hepatocytes before 24 hours post-PH, near the peak of hepatocyte DNA synthesis, these findings indicate that Cx26 is enhanced in hepatocytes before the onset of S-phase. In contrast to the restricted expression of Cx43 in Glisson's capsule in adult liver, Cx43 protein and mRNA were enhanced specifically in proliferating bile duct and perisinusoidal cells post-PH. PH performed during continuous administration of 2-acetylaminofluorene (AAF) prevented changes in Cx32 and Cx26 staining observed in the absence of AAF. Proliferating oval cells were found to express diffuse Cx43 immunoreactivity. On day 11 post-PH and AAF, basophilic hepatocytes displayed both punctate Cx32 and Cx26 staining, whereas bile ducts and perisinusoidal cells expressed Cx43. These findings indicate that alterations in Cx32 and Cx26 expression occur rapidly in hepatocytes stimulated to proliferate and that several nonparenchymal liver cell types upregulate Cx43 expression when induced to proliferate. Differentiation of oval cells into basophilic hepatocytes resulted in their expression of Cx32 and Cx26.

Gap-junctional intercellular communication (GJIC) in normal rat liver cells involves at least three different connexins (Cxs)--Cx32, Cx26, Cx43--depending on the cell type, position in the lobule, or both. Whereas rat hepatocyte primary cultures expressed Cx32 and Cx26 as observed in vivo, cell lines derived from normal rat liver (WB-F344, Clone 9, RLEC, and BRL) expressed Cx43 and to a lesser extent Cx26. Hepatoma cells propagated in vitro were either deficient in GJIC and Cx expression (7777, 8994, H4IIE-C3) or communicated via gap junctions composed of Cx43 protein (N1S1-67, 9618A). Analysis of neoplasms that resulted from injection of hepatoma cells into rat femoral muscle showed differences in Cx expression when compared with cells grown in vitro. Whereas hepatoma cells 7777 and H4IIE-C3 failed to express Cx mRNAs in culture, these cells transplanted in vivo expressed levels of Cx32 mRNA comparable to those in normal liver. However, detectable Cx32 immunostaining was observed in less than 5% of the neoplastic cells in vivo. These results indicate that Cx32 protein was posttranscriptionally downregulated in 7777 and H4IIE-C3 tumor cells. Unexpectedly, 9618A cells expressed Cx43 mRNA and protein in cell culture but expressed Cx32 mRNA in vivo. In contrast, N1S1 transplants continued to express Cx43 mRNA and protein in vivo. Unlike the punctate Cx43 staining observed in suspension cultures of N1S1 cells, diffuse intracellular Cx43 staining was observed in N1S1-derived neoplasms in vivo, although the electrophoretic pattern of Cx43 isolated from N1S1 tumors grown in vivo (43 kDa) was different from that observed in suspension cell cultures (43 and 45 kDa). Thus, the findings reported here demonstrate that Cx expression in hepatoma cells depends on the environment, whether in vivo or in vitro, in which the cells are propagated.

Recombinant human activin A, a member of the transforming growth factor-beta superfamily, induced significant cell loss in rodent livers and in primary hepatocyte cultures. Histologically and biochemically the hepatocyte death was mediated by apoptosis, a form of programmed cell death. Male mice were treated with 200 or 500 micrograms recombinant human activin A/kg body wt/day for up to 3 days by means of a subcutaneously implanted minipump. Livers were taken for light and electron microscopy, DNA isolation and in situ nick end-labeling. Primary cultures of rat hepatocytes were treated with 10 ng/ml recombinant human activin A for 24 hr before being harvested for electron microscopy and DNA isolation. Infusion of activin A evoked dose-dependent loss of liver mass due to the atrophy and death of hepatocytes around the central vein. Morphologically, the dying cells demonstrated all the characteristic nuclear and cytoplasmic features of apoptosis. Low molecular weight DNA isolated activin A-treated intact livers and primary cultures exhibited the typical oligosomal ladder. Nick end-labeling of DNA in situ confirmed that virtually all topographical apoptotic hepatocytes had fragmented DNA. The currently accepted criteria for apoptosis (i.e., specific morphological alterations and internucleosomal clipping of DNA) were evident in activin A-treated hepatocytes both in vitro and in vivo, leading to the conclusion that cell loss occurs mainly through apoptosis. These observations suggest that activin A may be important in hepatic homeostasis.

Although transgenic hepatocarcinogenesis has been accomplished in the mouse with a number of genetic constructs targeting the oncogene to expression primarily in the liver, no example of this process has yet been developed in the rat. Because our understanding of the multistage nature of hepatocarcinogenesis is most advanced in the rat, we have developed a strain of transgenic rats carrying the promoter-enhancer sequences of the mouse albumin gene linked 5' to the simian virus-40 T antigen gene. A line of transgenic rats bearing this transgene has been developed from a single founder female. Five to six copies of the transgene, possibly in tandem, occur within the genome of the transgenic animals, which are maintained by heterozygous matings. Livers of transgenic animals are histologically normal after weaning; at 2 months of age, small foci of vacuolated cells appear in this organ. By 4 months of age, all animals exhibit focal lesions and nodules consisting primarily of small basophilic cells, many of which exhibit considerable cytoplasmic vacuolization. Mating of animals each bearing the transgene results in rats with a demyelinating condition that develops acutely in pregnant females and more chronically in males. Ultrastructural studies of these cells indicate that the vacuoles contain substantial amounts of glycogen, with the cells resembling hepatoblasts. Malignant neoplasms with both a glandular and a hepatoblastoma/hepatocellular carcinoma pattern arise from the nodules. Enzyme and immunohistochemical studies of all lesions reveal many similarities in gene expression to comparable lesions in rats subjected to chemically induced hepatocarcinogenesis, with certain exceptions. The placental form of glutathione-S-transferase is absent from all lesions in the transgenic animal, as is the expression of connexin 32. A significant number of lesions express serum albumin, and many, but not all, exhibit the T antigen. Lesions expressing the T antigen also contain stainable amounts of the p53 gene product; by contrast, normal hepatocytes express only very low levels of the T antigen within their nuclei and no demonstrable p53. All of the animals develop hepatic lesions, and approximately one-third also develop adenomas and carcinomas derived from the islet cells of the pancreas. Although there are differences in the morphology, biology, and genetic expression in early and late hepatic lesions in this strain of transgenic rat, many similarities also occur, making this a potential model system with which to study the interactions of environmental factors with a genetic program for hepatocarcinogenesis.

Carcinogenesis is a multistep, multistage process that begins with irreversible, but heritable damage to a single cell. The partial hepatectomy/diethylnitrosamine (DEN) model of rat hepatocarcinogenesis has been well characterized and many aspects of the stage of initiation are known. Recently, it has been suggested that hepatocytes expressing the placental isozyme of glutathione S-transferase (PGST) may be one population of initiated cells. Male Fischer rats were subjected to a 70% partial hepatectomy and at the peak of cell proliferation 24 h later were administered either the solvent trioctanoin, or 10 mg DEN/kg. The rats were administered 100 mg bromodeoxyuridine (BrdU)/kg 1 h prior to death at various times after DEN administration. Since initiation of the carcinogenesis process requires the division of cells containing DNA damage to induce mutations, we examined the concentration of alkylated adducts and the labeling index at various times after DEN administration. In addition, the time course of hepatic PGST expression was determined concurrent with the adduct concentration and labeling index. During the first day after DEN or solvent administration to a rat subjected to a 70% partial hepatectomy, a diurnal variation in labeling index was observed. A recovery to postsurgical labeling index levels was demonstrated for both the solvent- and DEN-treated groups by 7 days. The concentration of three promutagenic lesions was maximal at 6 h after DEN administration. The detectable level of the O6EG adduct was negligible by 24 h after DEN administration, while the two O-alkylpyrimidines, O2ET and O4ET, were retained for much longer periods. Single hepatocytes expressing PGST were observed by 2 days after DEN administration, while small foci of PGST-expressing hepatocytes could be reliably detected by 2 weeks. Two phases of PGST expression in single hepatocytes were observed. The first phase was maximal at day 3 and complete by day 6, while the second reached a plateau by day 8 and was maintained for the 28 days of the study. The presence of the three O-alkylation adducts during a time of enhanced cellular proliferation suggests that all three promutagenic adducts may contribute to the initiation that results in the partial hepatectomy/DEN model of rat hepatocarcinogenesis.

Considerable experimental evidence has developed to argue that carcinogenesis occurs in a series of stages whose characteristics allow division into the distinct stages of initiation, promotion, and progression. The stage of initiation results from subtle genetic alterations in individual target cells caused both by experimental perturbations as well as by ambient environmental factors. Initiation of a cell may result from the mutation of one or a few key genes whose expression is modified by many other genes. Promoting agents induce the clonal development of initiated cells susceptible to the proliferative effects of the specific promoting agent. The stage of progression is characterized by evolving karyotypic instability and may be induced by specific clastogenic effects and/or agents, the latter termed progressor agents. Carcinogens may induce cancer by an action at all three stages or selectively by an action at any one or two of the individual stages of carcinogenesis. Cancer then develops as a result of fortuitous, endogenous, or ambient environmental factors, thus accounting for the action of both genotoxic and nongenotoxic agents as carcinogens.

Although numerous biochemical markers can identify putative preneoplastic altered hepatic foci (AHF) in rat liver, no consistent pattern of expression during hepatocarcinogenesis has emerged. Using quantitative stereologic analyses we demonstrated that decreased expression of the major hepatocyte gap junction protein, connexin 32 (Cx32), in rat AHF is a consistent observation in several protocols of multistage hepatocarcinogenesis. This change was observed after initiation by either ethylnitrosourea (ENU) or diethylnitrosamine (DEN), followed by promotion with phenobarbital (PB), dioxin, chlorendic acid, C.I. Solvent Yellow, or tamoxifen. AHF generated by Wy-14,643, ciprofibrate, and a choline/methionine-deficient dietary regimen also showed decreased Cx32 expression. The decrease of Cx32 in AHF was rapidly reversible after withdrawal of PB, and this change preceded a reduction in placental isozyme of glutathione-S-transferase (GST) expression in the same AHF. Within 20 days of withdrawal, fewer than 4% of GST-positive AHF were Cx32 deficient, while the volume of total AHF decreased 30%. Chronic PB treatment also resulted in a reversible decrease in Cx32 specifically in mid- and centro-lobular hepatocytes. Continuous thymidine labeling demonstrated that Cx32 could be uncoupled from the cell cycle, suggesting that some liver promoters may act directly to alter the expression of Cx32. These observations suggest that a decrease in Cx32 content was a relatively common epigenetic change in AHF induced during hepatocarcinogenesis by a number of initiating and promoting agents but that this change was not sufficient for carcinogenesis. This change, however, may be necessary for the mechanism(s) of tumor promotion, since Cx32-positive AHF did not proliferate as readily as Cx32-deficient AHF.

The proliferative response to isoprenaline in the submaxillary and parotid glands of the Balb/c mouse has been studied in the intact male and female, and also in the male castrated one month prior to stimulation. The hyperplastic response of the acinar cells has been monitored by serial measurements of the flash tritiated thymidine labelling index and the mitotic index. Castration caused the atrophy of the granular ducts in the submaxillary gland, and therefore an increased predominance of the acini. At one month after castration the acini occupied an area almost 1.5-fold greater than that of the granular ducts, but this was not as great as in the intact female gland where acini occupied twice the area of the granular ducts. Hyperplasia was induced by a single injection of isoprenaline (0.3 mM/kg body weight). The response of the submaxillary gland in the intact male and intact female was very similar, DNA synthesis commencing 21-24 h after stimulation and mitotic activity first noted after 33-36 h. On the other hand, in the submaxillary gland of the castrated male, DNA synthesis began after only 18-21 h and mitotic activity after only 27-30 h. A metaphase arrest experiment with vincristine confirmed the more prompt response in the castrated animals; between 33-36 h after isoprenaline injection, the rate of entry of cells into mitosis was 4 cells/100 cells/h in the castrated group but only 0.4 cells/100 cells/h in the intact males. Thus castration appears to bestow a unique state of responsiveness upon the submaxillary gland to isoprenaline stimulation. The mechanisms underlying this change are not yet understood, for it is paradoxical that atrophy of a structural component rich in specific protein growth factors can alter the format of isoprenaline-induced hyperplasia in acinar cells that produce secretory glycoproteins.