Malignantly transformed non-parenchymal liver epithelial cells and transformed oval cells suppress the homotypical gap junctional intercellular communication of co-cultured rat liver parenchymal cells.

Isolated rat liver parenchymal cells (PC) were co-cultured with a non-parenchymal rat liver epithelial cell line (NEC) or with an oval cell line. The homotypical gap junctional intercellular communication (GJIC) between the liver PC was measured after microinjection of Lucifer Yellow by dye transfer. The rat liver PC were dye coupled between 87% and 100% for at least 1 week in both co-cultures, in contrast to PC In monoculture between which no dye coupling was left after 1 week. When liver PC were co-cultured with a transformed and tumorigenic NEC or with a transformed and tumorigenic oval cell line the homotypical GJIC between the liver PC was drastically decreased with culture time, and t…

C-myc mRNA Expression in Epithelial Ovarian Carcinomas in Relation to Estrogen Receptor Status, Metastatic Spread, Survival Time, FIGO Stage, and Histologie Grade and Type

Recently, it has been suggested that c-myc expression might correlate with estrogen receptor (ER) status and metastatic spread in ovarian cancer. In this study, expression of c-myc mRNA in 90 epithelial ovarian carcinomas was determined using the S1 nuclease protection assay. Expression of c-myc mRNA was detectable in 27 of 90 tumors. There was no significant association between c-myc mRNA expression and metastatic spread, survival time, FIGO stage, or histologic grade and type. C-myc mRNA was expressed in 45% of ER-positive tumors but only 24% of ER-negative tumors (p = 0.094; Fisher's exact test). Similarly, 44% of progesterone receptor (PR)-positive and 23% of PR-negative tumors expresse…

Studies on the importance of microsomal epoxide hydrolase in the detoxification of arene oxides using the heterologous expression of the enzyme in mammalian cells.

In order to investigate the role of the microsomal epoxide hydrolase (mEH) in the detoxification of arene oxides in the presence of a high endogenous glutathione S-transferase (GST) activity-a situation found in several organs--we expressed the rat mEH cDNA in BHK21 Syrian hamster cells. These cells have high GST activities but contain an extremely low endogenous mEH enzyme activity. We obtained several cell clones which expressed the mEH heterologously, as determined by immunoblotting. The cell clone BHK21-mEH/Mz1 had the highest level of mEH protein. Immunofluorescence showed that the level of expression was almost homogeneous throughout the cell population. Total protein isolated from th…

Evaluation of c-erbB-2-oncogene expression as a prognostic factor in ovarian cancer

mdm2 mRNA expression is associated with survival in ovarian cancer

Expression of mdm-2 mRNA was measured in 90 ovarian-cancer tissue specimens using the S1 nuclease assay, to investigate a possible association between MDM2 expression and prognosis. mdm-2 mRNA expression was an independent prognostic factor for patients with primary ovarian cancer, FIGO (International Federation of Gynecology and Obstetrics) stages III and IV (n = 57), who all received chemotherapy with carboplatin or cisplatin and cyclophosphamide. Median survival time for patients (FIGO stages III and IV) with no detectable expression of mdm-2 mRNA (n = 14) was 171 days, as compared with 839 days for patients (n = 43) with detectable mdm-2 mRNA (p = 0.0194; log-rank test). However, no ass…

Prognostic significance of c-erB-2 mRNA in ovarian carcinoma.

The oncogene specific mRNA of c-erbB-2 was detected by the S1 nuclease protection assay in 95 ovarian cancer specimens. In 79 primary carcinomas, we found 16 (20%) with strong expression, 13 (17%) with weak expression, 4 (5%) with very weak expression, and 46 (58%) with no expression. In 3 of 16 recurrencies (19%) a strong expression of c-erbB-2 mRNA was detected, in 2 (12%) weak expression was detected, and in 11 (69%) no expression of c-erbB-2 mRNA was detected. Kaplan-Meier analysis revealed no significant association between strong expression of c-erbB-2 mRNA and survival of the 79 patients with primary cancer. However, in the subgroup of patients with FIGO (International Federation of …

The human p53 gene mutated at position 249per se is not sufficient to immortalize human liver cells

A particular point mutation of the tumor suppressor gene p53, namely a G→T transversion at the third base of codon 249, is frequently detected in primary hepatocellular carcinomas from patients living in areas where the levels of dietary exposure to aflatoxin B 1 and the rates of infection with the hepatitis B virus are very high. Very recently, a nontumorigenic liver epithelial cell line (HACL-1) with a finite life-span and expressing a number of hepatocyte-specific markers was established from a human hepatocellular adenoma in our laboratory. To analyze the role of mutated p53 in the immortalization of human liver cells, we transfected HACL-1 cells with an expression vector containing a h…

Ha-rasVa112 but not p53Ser247 leads to a significant neoplastic transformation rate of the putative rat liver stem cells (oval cell)

In order to test the controversially discussed hypothesis that oval cells are part of a liver stem cell compartment and can give rise to cholangiocellular as well as hepatocellular carcinomas in the course of liver carcinogenesis, we transfected an oval cell line established in our laboratory with an oncogenically activated genomic Ha-ras clone (pUC EJ 6.6), carrying a valine at position 12 instead of the wild-type glycine, or a rat p53 cDNA mutated by site-directed mutagenesis at codon 247, which corresponds to codon 249 in the human p53. This codon is of particular interest since it represents a mutation hotspot observed in hepatocellular carcinoma especially in regions with high aflatoxi…

The role of insulin-like growth factor II in the malignant transformation of rat liver oval cells

Oval cells are small nonparenchymal epithelial cells that first appear in the periportal areas of the liver and thereafter invade the whole parenchyma when mice or rats are exposed to a variety of chemical carcinogens. In the present study we have analyzed the expression of insulin-like growth factor II (IGF II) in the recently established oval cell line OC/CDE 22 and its malignantly transformed counterpart (the M22 cells) and the biological consequences of the constitutive expression of IGF II in oval cells. OC/CDE 22 cells do not express the above-mentioned growth factor, whereas the M22 cells do and addition of a neutralizing anti-IGF II antibody to M22 cells resulted in an almost comple…

The microsomal epoxide hydrolase has a single membrane signal anchor sequence which is dispensable for the catalytic activity of this protein

The microsomal epoxide hydrolase (mEH) catalyses the hydrolysis of reactive epoxides which are formed by the action of cytochromes P-450 from xenobiotics. In addition it has been suggested that mEH might mediate the transport of bile acids. For the mEH it has been shown that it is co-translationally inserted into the endoplasmic reticulum. Here we demonstrate that the N-terminal 20 amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can also be supplied by a cytochrome P-450 (CYP2B1) anchor signal sequence. The evidence supporting this conclusion is as follows: (i) the rat mEH and a CYP2B1-mEH fusion protein, in whic…

Stable Expression of Heterologous Microsomal Epoxide Hydrolase in BHK21 Cells: Influence on the Mutagenicity of Benzo[a]pyrene 4,5-Oxide

Most environmental mutagens and carcinogens require metabolic activation to electro- philic intermediates capable of reacting with cellular target structures, such as DNA. These electrophilic intermediates are in addition subject to metabolic detoxification. This metabolism is mainly controlled by enzymes whose expression is very variable. Among other things, various enzymes are inducible by environmental chemicals. Understanding the toxicology of chemicals (for example, species differences, idiosyncrasias, organotropisms) therefore requires knowledge of critical host factors. One approach towards this goal involves the use of purified enzymes in metabolism and toxicological studies (Glatt …

cAMP-dependent phosphorylation of CYP2B1 as a functional switch for cyclophosphamide activation and its hormonal controlin vitro andin vivo

An important feature of cytochrome P450 (CYP) 2B1 is its high ability to convert the prodrug cyclophosphamide (CPA) to therapeutically cytotoxic metabolites, resulting in interstrand DNA-cross-linking and cell death. We have examined whether and how the phosphorylation of CYP2B1 influences CPA metabolic activation in vitro and in vivo. We found first that only part of the total CYP2B1 pool undergoes phosphorylation. This part is fully inactivated. Second, phosphorylation of CYP2B1 in intact hepatocytes reduced by up to 75% toxification of CPA to mutagenic metabolites (totally dependent on the same preferentially CYP2B-catalyzed 4-hydroxylation of CPA as is the generation of highly cytotoxic…

Differential modulation of CYP2E1 activity by cAMP-dependent protein kinase upon Ser129 replacement.

Many toxic compounds are activated by cytochrome P450 (CYP) 2E1 to reactive metabolites, which represents a potential hazard for cellular homeostasis. Therefore knowledge about CYP2E1 regulation could be of great biological importance. It has been shown that CYP2E1 is controlled transcriptionally and post-translationally by phosphorylation. In the present study we investigated the role of serine-129 (Ser129) in the protein kinase A (PKA) recognition sequence motif Arg-Arg-Phe-Ser129. To gain further insights into the possible relevance of Ser129 for CYP2E1 function, Ser129 was replaced by alanine (Ala) or glycine (Gly) by site-directed mutations of the cDNA coding for CYP2E1. The mutant cDN…

Investigating the Role of the Microsomal Epoxide Hydrolase Membrane Topology and Its Implication for Drug Metabolism Pathways

The microsomal epoxide hydrolase (mEH) catalyzes the hydrolysis of reactive epoxides which are formed by the action of cytochromes P450 from xenobiotics. In addition the mEH has been found to mediate the transport of bile acids. For the mEH it has been shown that it is cotranslationally inserted into the endoplasmic reticulum. Here we demonstrate that the amino-terminal twenty amino acid residues of this protein serve as its single membrane anchor signal sequence and that the function of this sequence can be also supplied by a cytochrome P450 (CYP2B1) anchor signal sequence.