Differentiation of rat oval cells after activation of peroxisome proliferator-activated receptor alpha43.

Peroxisome proliferators (PPs) act as nongenotoxic tumor promoters in rodents. Their hepatocarcinogenicity requires the presence of the PP-activated receptor alpha (PPARalpha); however, the exact role played by this transcription factor in the liver, more precisely in liver cell growth and differentiation, is not known. The aim of this study was to investigate the role of PPARalpha in oval cells, which are considered to be closely related to liver stem cells, act as bipotential progenitors for the two main hepatic lineages, and have been implicated as playing a role in several models of liver carcinogenesis. We studied the PPARalpha-mediated response of primary oval cells isolated from rats fed a choline-deficient ethionine-supplemented diet (CDE diet, a regimen commonly used for the induction of oval cell proliferation in rodents) with or without cotreatment with WY14,643, a prototype PPARalpha-activator. PPARalpha was expressed at relatively low levels in primary oval cells from rats fed the CDE diet alone. In vivo treatment with WY14,643 for 2-6 weeks induced, in the oval cells, the expression of PPARalpha as well as that of the PPARalpha-responsive genes encoding fatty acyl-CoA oxidase and cytochrome P450 4A1. Moreover, the oval cell response to WY14,643 was accompanied by an overall phenotypic modulation toward the hepatocyte lineage. In addition, the PPARalpha activator induced, among the oval cells, a subpopulation of transitional cells showing features of maturing hepatocytes expressing the oncofetal marker, alpha-fetoprotein. These results show that oval cells are responsive to PPs and strongly argue for a role of PPARalpha in the differentiation/maturation of rat oval cells. In the absence of the CDE diet regimen, 9-week treatment with WY14,643 lead to the appearance of a population of large-sized cells somewhat similar to the transitional cells. However, these cells showed little expression of markers of mature hepatocytes, consistent with a block during their maturation process, i.e., they are resistant to PPARalpha-mediated differentiation. Interestingly, the phenotype of these cells resembled that of the cells usually found in neoplastic foci induced by PPs. Our results, together with previous reports, suggest the involvement of oval cells in the hepatocarcinogenicity of PPs.

Immunophenotype of the canine transmissible venereal tumour.

The canine transmissible venereal tumour is a naturally occurring contagious round-cell neoplasia which is primarily located in the mucous membrane of the external genitalia in dogs of either sex. In order to specify the controversial cytogenetic origin of this round-cell tumour, 14 cases of canine transmissible venereal tumour, formalin- or Bouin-fixed and paraffin-embedded, were subjected to extensive immunophenotypic analysis using reagents specific to a variety of cytoplasmic or surface antigens: lysozyme, ACM1 antigen, vimentin, neuron-specific enolase, glial fibrillary acidic protein, desmin, alpha smooth muscle actin, CD3, IgG, kappa and lambda light chains, and keratin. Lysozyme immunoreactivity was detected in all cases, ACM1 antigen in 11 of 14, neuron-specific enolase in 11 of 14, vimentin in 10 of 14, glial fibrillary acidic protein in 4 of 14 and desmin in 1 of 14. All the sections were negative to keratins, alpha smooth muscle actin and CD3, whereas in five cases, perivascular tumour cells contained Ig G, kappa and lambda light chains. The immunoreactivity to lysozyme and ACM1 antigen supports the hypothesis of a histiocytic immunophenotype for the canine transmissible venereal tumour.

Frequent rearrangements at minisatellite loci D1S7 (1p33-35), D7S22 (7q36-ter) and D12S11 (12q24.3-ter) in hepatitis B virus-positive hepatocellular carcinomas from Thai patients.

Primary hepatocellular carcinoma (HCC) is one of the most common cancers in Thailand; chronic infection with hepatitis B virus (HBV) is endemic and represents a major risk factor for the development of this cancer. Several mechanisms for HBV-related hepatocarcinogenesis have been proposed, among them a direct role of HBV in the promotion of genetic recombination leading to chromosomal alterations. Minisatellite DNA sequences are hypervariable regions dispersed throughout the genome which are susceptible to genetic recombination events. In the present study, somatic rearrangements affecting minisatellite sequences were examined in a total of 26 HCC from Thai patients. Multilocus DNA fingerprinting using probes 33.15 and 33.6 detected rearrangements in 11 and 12 HCC, respectively, all of them carrying integrated HBV DNA. The frequency of rearranged bands was calculated for each probe based on the total number of rearrangements observed in the 26 tumours and the total number of bands revealed by DNA fingerprinting in the non-tumour DNA. With each probe a total of 23 rearrangements was observed, yielding rearrangement frequencies of 3.7% and 4.2% for the 33.15 and 33.6 minisatellite families, respectively. To test for possible clustering of these rearrangements at specific loci, we used minisatellite locus-specific probes previously cloned from 33.15 and 33.6. Minisatellites located at 1p33-35, 7q36-ter and 12q24.3-ter were shown to be frequently affected by rearrangement events in this series of HBV-positive HCC. Frequent rearrangements at minisatellite locus D7S22 (7q36-ter) in HBV-positive human HCC have not been reported so far.

Minisatellite rearrangements are increased in liver tumours induced by transplacental aflatoxin B1 treatment of hepatitis B virus transgenic mice, but not in spontaneously arising tumours.

Transgenic mice carrying an integrated subgenomic human hepatitis B virus (HBV) DNA fragment coding for the viral envelope polypeptides, represent a model for the study of the mechanisms involved in hepatocarcinogenesis. The mice develop a progressive liver injury characterized by inflammation, regenerative hyperplasia and dysplasia terminating in hepatocellular carcinoma (HCC) at around 18-21 months of age. No alterations in specific oncogenes and tumour suppressor genes in the HCC arising in this transgenic model have been observed. However, onset of liver tumours is significantly earlier in mice treated with aflatoxin B1 (AFB1). In order to examine more generally for genetic rearrangements during the natural history of the disease, DNA multilocus fingerprinting was performed using probes recognizing mouse minisatellites. Liver tumour samples from HBV transgenic mice either untreated or treated with AFB1 transplacentally were included in the study. In a total of 28 tumour samples from HBV transgenic mice receiving no carcinogen treatment, using three minisatellite probes, no alterations were detected. The frequency of rearrangements using any one of the three probes is calculated to be below 0.2%. This result demonstrates that genetic instability in minisatellite sequences is not a common event associated with HBV gene expression and liver injury in this model. In 11 liver tumours from mice exposed to AFB1 transplacentally six had minisatellite alterations (band gains and losses) revealed by at least one of the three probes used. The frequency of rearrangements was between 1.1% and 2% depending on the minisatellite probe. These data show that genetic alterations can be induced by transplacental exposure to AFB1 and suggest that genetic instability could be important in hepatocarcinogenesis with combined exposures to AFB1 and HBV.

Rearrangements in minisatellite sequences induced by aflatoxin B1 in a metabolically competent strain of Saccharomyces cerevisiae.

The role of aflatoxin B1 (AFB1) in the induction of rearrangements affecting minisatellite sequences was studied in an in vitro yeast model. The Saccharomyces cerevisiae strain used expresses human cytochrome P450 1A2 and NADPH-cytochrome P450 oxidoreductase and has previously been used to study genetic recombination events induced by AFB1. DNA multilocus fingerprinting was performed using probe M13 core hybridizing to a set of hypervariable minisatellite sequences in S. cerevisiae. Frequent spontaneous genomic alterations that affect the minisatellite fingerprint pattern were observed. Control cultures showed 15.8% rearrangements in minisatellites, and this frequency increased to 40.0% in cultures exposed to AFB1 (80 microg/ml). A total of approximately 29 minisatellite loci were visualized for each culture. Given the number of cultures examined (40 AFB1-treated and 38 controls) the rearrangement frequency per detectable minisatellite was 2.59% in the AFB1-treated group and 0.73% in the control group, which represents a statistically significant (P = 0.001) difference. Thus, our data strongly suggest that AFB1 can promote the genetic events responsible for minisatellite rearrangements in the yeast genome. Such genetic rearrangements may be important events during the etiology of liver carcinogenesis in people chronically exposed to dietary aflatoxins.