Biomedical subjects

A Buchmann

Publications and source records attributed to A Buchmann.

At least 19 recordsLinked to original sources

PubMed2001

Selective pressure during tumor promotion by phenobarbital leads to clonal outgrowth of beta-catenin-mutated mouse liver tumors.

Tumor promoters are non-mutagenic chemicals which increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. Phenobarbital (PB) is an antiepileptic drug which promotes hepatocarcinogenesis in rodents when administered subsequent to an initiating carcinogen like diethylnitrosamine (DEN). Here we have investigated the prevalence and patterns of mutations in two genes, Ha-ras and beta-catenin, both known mutational targets in mouse hepatocarcinogenesis. Liver tumors were generated by a single administration of DEN to 6 week old mice followed by feeding of PB (0.05%) containing or control diet for 39 weeks. Mutations at Ha-ras codon 61 were screened by allele-specific oligonucleotide hybridization; beta-catenin mutations were detected by direct sequencing of PCR products spanning exon 2. In tumors from mice treated with DEN alone, the prevalence of Ha-ras mutations was approximately 30% (6/20), while no beta-catenin mutations (0/13) were detectable in tumors of this treatment group. By contrast, Ha-ras mutations were undetectable in tumors from mice treated with DEN/PB (0/32), while approximately 80% (37/46) of tumors from this group showed beta-catenin mutations. These results demonstrate that PB strongly affects the prevalence of mutations in the two cancer-related genes, presumably by positive and negative selection for cells harboring the respective mutation.

Animals↗

PubMed2001

Transforming growth factor-beta1-induced Smad signaling, cell-cycle arrest and apoptosis in hepatoma cells.

Transforming growth factor-beta1 (TGFbeta) is involved in the regulation of liver cell proliferation and apoptosis, and escape of hepatoma cells from the growth restraining signals of TGFbeta has been suggested to contribute to tumor development. TGFbeta modulates gene transcription by receptor-mediated activation of Smad proteins which act as transcription factors. TGFbeta-mediated primary signaling responses as well as effects on the cell cycle and apoptosis were investigated in the human hepatoblastoma line HepG2, the rat hepatoma line FTO-2B and the mouse hepatoma line 55.1c. Activation of a Smad (Sma and Mad homolog) response-element-driven luciferase reporter by TGFbeta was very similar in all three cell lines, indicating functionality of the primary TGFbeta signaling pathway. Moreover, TGFbeta-inducible early gene was transiently activated by TGFbeta in all cell lines as shown by RT-PCR. HepG2 cells, however, were completely resistant to TGFbeta-induced growth arrest and apoptosis and 55.1c cells were only slightly susceptible to TGFbeta-induced apoptosis. By contrast, treatment of FTO-2B cells with TGFbeta led to a partial G0/G1 arrest and a strong induction of apoptosis. TGFbeta-induced apoptosis of FTO-2B cells was inhibited by dexamethasone, insulin, phenobarbital and dieldrin. Of these agents, only insulin led to a significant reduction of TGFbeta-stimulated Smad-reporter activity, suggesting that the other compounds interfere with TGFbeta-induced apoptosis downstream of Smad-mediated primary transcriptional responses at a level that may be constitutively altered in apoptosis-resistant hepatoma cell lines.

Apoptosis↗

PubMed2000

Lack of phenobarbital-mediated promotion of hepatocarcinogenesis in connexin32-null mice.

Connexin32 (Cx32) is the major gap junction forming protein in liver. We have recently shown that hepatocarcinogenesis is strongly enhanced in mice deficient in Cx32, demonstrating that lack of functional Cx32 accelerates liver tumorigenesis. Many tumor-promoting agents, including phenobarbital, block gap junctional intercellular communication in vitro, and it has been suggested that this effect is relevant for clonal expansion of neoplastic cells in vivo. We have now tested this hypothesis by analyzing the potency of phenobarbital as a liver tumor promoter in male Cx32-wild-type (Cx32(Y/+)) and Cx32-null (Cx32(Y/-)) mice. Preneoplastic and neoplastic liver lesions were induced in 6-week-old male mice by a single injection of 90 microg/g body weight of N-nitrosodiethylamine, and groups of mice were subsequently kept on phenobarbital-containing (0.05%) or control diet for 39 weeks. Frozen liver sections were prepared, and (pre)neoplastic lesions were identified by their deficiency in glucose-6-phosphatase staining. In addition, the number and size of macroscopically visible tumors were monitored. Phenobarbital led to a approximately 5-fold increase in the volume fraction occupied by glucose-6-phosphatase-deficient liver lesions in Cx32(Y/+) mice, whereas there was no such increase in Cx32(Y/-) mice. Even more pronounced differences were observed with respect to tumor response. Whereas phenobarbital clearly promoted the occurrence of numerous large hepatomas in Cx32(Y/+) mice, no such effect was seen in Cx32(Y/-) mice. These results demonstrate, for the first time, that functional Cx32 protein is required for tumor promotion by phenobarbital.

Animals↗

PubMed2000

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on initiation and promotion of GST-P-positive foci in rat liver: A quantitative analysis of experimental data using a stochastic model.

We use a stochastic model describing initiation and clonal growth of altered cells to analyze data from an initiation-promotion hepatocarcinogenesis experiment in female Wistar rats. Starting at 7 weeks of age, the animals were treated for 10 days with the initiating agent diethylnitrosamine (DEN, 10 mg/kg body wt per day). After a 10-week resting period, the animals were treated either with corn oil or with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via biweekly sc injections of 1.4 microg/kg body wt of TCDD dissolved in corn oil. Groups of four or five animals were euthanized 3, 17, 31, 73, and 115 days after start of TCDD/corn oil treatment. The data analyzed consist of the number and sizes of GST-P-positive focal transections at various time points. By fitting the model to the data, we estimate the rates of initiation, cell division, and cell death during different time periods of the experiment. The model estimates of cell kinetic parameters are consistent with directly made experimental observations of cell division and cell death. The model predicts that DEN-induced initiation of GST-P-positive cells is highly protracted in controls and TCDD-treated animals alike. We also find that TCDD interferes with the normal rate at which cells with (DEN-inflicted) DNA damage are converted into cells expressing the GST-P-positive phenotype, suggesting a TCDD-mediated "acceleration" of the appearance of de novo GST-P-positive initiated cells from damaged precursor cells. Furthermore, the model predicts a significant reduction in the rate of apoptosis within the first 4 to 5 weeks of TCDD treatment, and after 10 weeks of TCDD treatment, but not in between.

Animals↗

PubMed2000

Ah receptor ligands and tumor promotion: survival of neoplastic cells.

A number of agonists of the aryl hydrocarbon or dioxin receptor (AhR) are potent tumor promoters in rodent liver. The prototype compound is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Tumor promotion by TCDD is likely to be AhR-mediated. Tumor promoters may affect the rate of division, terminal differentiation or death (apoptosis) of tumor precursor cells. The present paper reviews some of the effects of TCDD on liver cell homeostasis that have been observed under diverse experimental settings and discusses some of the possible underlying mechanisms.

Animals↗

PubMed2000

Hepatocarcinogenesis in female mice with mosaic expression of connexin32.

Mice deficient for connexin32 (Cx32), the major gap junction forming protein in liver, are highly susceptible to hepatocarcinogenesis. Because the Cx32 gene is located on the X-chromosome, heterozygous females show mosaicism with respect to Cx32 expression; this enables their use in studying the effect of Cx32-deficiency in a mixed Cx32-plus/Cx32-minus environment in vivo. Female C3H/He mice (Cx32(+/+)) were crossed with Cx32-deficient C57BL/129Sv males (Cx32(Y/-)) to yield F1 females heterozygous with respect to Cx32 (Cx32(+/-)). Patches of hepatocytes were observed in normal liver that either expressed Cx32 or failed to express the protein. The mean fraction of Cx32-negative tissue in liver was about 60% and did not change significantly with age of mice. Neoplastic liver lesions, induced in weanling mice, were identified in serial liver sections by their deficiency in glucose-6-phosphatase staining. Parallel sections were used for immunohistochemical demonstration of Cx32 protein. Smaller lesions were either homogenously Cx32-negative or showed unchanged to slightly elevated levels of Cx32 protein. There were no major differences in number and size distribution between lesions of these 2 phenotypes. In addition, larger lesions were mostly Cx32-negative but often contained embedded patches of Cx32-positive cells. Staining for the proliferation-associated nuclear antigen Ki-67 did not reveal significant differences between Cx32-negative and Cx32-positive hepatocytes in Cx32-mosaic tumors. This suggests that expression of Cx32 within a subpopulation of tumor cells does not negatively regulate their growth nor does it seem to affect the proliferation of their directly neighboring Cx32-negative counterparts.

Animals↗

PubMed2000

Suppression of apoptosis in C3H mouse liver tumors by activated Ha-ras oncogene.

Liver tumors were induced in male C3H mice by a single injection of N-nitrosodiethylamine and characterized with respect to the presence of base substitutions in the hot-spot position at codon 61 of the Ha-ras proto-oncogene. An increase in Ha-ras mutation prevalence was found with time after induction of tumors, suggesting that the activated ras gene provides a selective growth advantage. However, no significant differences in 5-bromodeoxyuridine labeling indices were evident between ras mutated and ras wild-type tumors, demonstrating that cell division rates in the two tumor populations were very similar. Apoptotic indices were determined by counting eosinophilic apoptotic bodies. The frequency of occurrence of apoptotic bodies was found to be approximately five times lower in tumors with Ha-ras mutations when compared with tumors not showing the mutation. This demonstrates that the activated p21(Ras) protein has anti-apoptotic activity in transformed mouse hepatocytes in vivo and suggests that the preferential outgrowth of Ha-ras-mutated hepatoma cells is mediated by suppression of apoptosis rather than by stimulation of cell division.

Animals↗

PubMed1999

Inhibition of transforming growth factor beta1-induced hepatoma cell apoptosis by liver tumor promoters: characterization of primary signaling events and effects on CPP32-like caspase activity.

The effects of the liver tumor promoters phenobarbital, clofibrate, dieldrin, and DDT on transforming growth factor-beta1 (TGFbeta)-induced apoptosis were studied in FTO-2B hepatoma cells. Inhibition of apoptosis by these compounds was strongly correlated with a decrease in CPP32-like caspase activity. Similar effects were obtained with insulin and dexamethasone. CPP32-like activity may thus provide a useful tool for quantiation of apoptosis under various treatment conditions. Diverse effects on apoptosis-associated cellular signaling proteins were observed: insulin led to an activation of the MAP kinases ERK1/2, of PKB/Akt and of NF-kappaB, phenobarbital and clofibrate enhanced NF-kappaB activity solely, while dexamethasone slightly enhanced NF-kappaB activity and increased the expression of Bcl-xL. Since inhibition of apoptosis was still detectable if the anti-apoptotic compounds were administered more than 10 h after TGFbeta, the diverse primary signals appear to converge at a presumably late stage of apoptosis, but upstream of activation of CPP32 or related caspases.

Animals↗

PubMed1999

The effect of connexin32 null mutation on hepatocarcinogenesis in different mouse strains.

Connexin32 (Cx32) is the major gap junctional protein in mouse liver. We have shown recently that the formation of liver tumours in Cx32-deficient mice is strongly increased in comparison with control wild-type mice, demonstrating that the deficiency in gap junctional communication has an enhancing effect on hepatocarcinogenesis. We have now compared the effect of Cx32 deficiency on liver carcinogenesis in two strains of mice with differing susceptibility to hepatocarcinogenesis. Heterozygous Cx32(+/-) females were crossed with male Cx32 wild-type C57BL/6J (low susceptibility) or C3H/He (high susceptibility) mice. Since the Cx32 gene is located on the X-chromosome, the resulting F1 males segregated to the genotypes Cx32(Y/+) and Cx32(Y/-). Genotyping was performed by PCR-analysis using tail-tip DNA. Weanling male mice were i.p. injected with a single dose of N-nitrosodiethylamine and were killed 16, 21 or 26 weeks later. The number, volume fraction and size distribution of precancerous liver lesions characterized by a deficiency in the marker enzyme glucose-6-phosphatase were quantitated. The results demonstrate that Cx32 deficiency only slightly affects the number of enzyme-altered lesions, but strongly enhances their growth, both in the resistant and the susceptible mouse strain, suggesting that decreased intercellular communication results in tumour promoting activity irrespective of the genetic background of the mouse strain used. Since Cx32-deficient C3H/He hybrids were approximately 5-10 times more sensitive than C3H/He hybrids with an intact Cx32 gene, this mouse strain may prove very useful for toxicological screening purposes.

Animals↗

PubMed1998

p21Ras downstream effectors are increased in activity or expression in mouse liver tumors but do not differ between ras-mutated and ras-wild-type lesions.

Mouse liver tumors frequently harbor activating ras gene mutations. Downstream effector molecules of p21Ras include Raf-1 kinase which mediates external signals via kinase signaling pathways to nuclear transcription factors including c-Fos and c-Jun. Mouse liver tumors with differing ras-mutational status were analyzed for alterations in Ras/Raf-1 signal transduction. Tumors were characterized with respect to the presence of base substitutions in the 3 known hot-spot positions at codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras. Ha-ras codon 61 or Ki-ras codon 13 mutations, but no N-ras mutations, were detected in 23 out of 33 tumors analyzed, while no ras-mutations were found in 10 of the tumors. There was no significant difference in the expression of p21RaS proteins between ras-mutated tumors and tumors without detectable ras mutations. To allow for determination of Raf-1 kinase activity in tumors, a sensitive and specific assay was developed for measurements with tissue homogenates. Raf-1 kinase activity was increased about four-fold in liver tumors as compared with normal liver tissue. No significant differences in kinase activity, however, were evident between ras-mutated and ras-wild-type tumors. The same was true with respect to the levels of c-fos and c-jun mRNAs. Moreover, there were no significant differences in cell division (5-bromo-2'-deoxyuridine-labeling indices) of hepatocytes from ras-mutated and ras-wild-type tumors. The similar degree of constitutive activation of the Ras/Raf-1 signaling pathway in liver tumors, with and without detectable ras mutations, suggests that other molecules within the signaling pathway may substitute for ras-mutations during oncogenic conversion of ras-wild-type hepatocytes.

Animals↗

PubMed1998

Expression of hypoxia-inducible genes in tumor cells.

Tumor tissue oxygenation impacts on proliferation of cancer cells and their sensitivity towards radio- and chemotherapy. Under low oxygen, mammalian cells show an adaptive response that leads to the induction of a number of genes with well-defined roles in oxygen supply and energy maintenance, e.g. genes encoding enzymes of the glycolytic pathway. The hypoxia-inducible factor 1 (HIF-1), a transcription factor consisting of the two proteins HIF-1alpha and HIF-1beta, plays a major role in the pleiotropic response observed under low oxygen. We have determined, by Northern analysis, the mRNA levels of HIF-1alpha and of two glycolytic enzymes known to be transcriptionally activated by HIF-1, namely phosphoglycerate kinase 1 (PGK 1) and pyruvate kinase M2 (PKM2), in different hepatoma cell lines and in mouse and human tissues. Hypoxic treatment of various mouse and human hepatoma cell lines led to the expected increase in the amount of PGK1 and PKM2 mRNA, while HIF-1alpha mRNA levels were not significantly elevated. Analysis of mouse liver tumors demonstrated no tumor-specific increases in HIF-1alpha or PGK1 mRNA levels. In five of eight human colorectal cancers investigated, PGK1 and PKM2 mRNA levels were increased in comparison to the corresponding normal tissues, while HIF-1alpha mRNA levels were not significantly changed. The majority of the colorectal cancers demonstrated p53 immunoreactivity, presumably due to mutation of the gene; there was, however, no correlation between the p53 staining pattern and mRNA expression levels of glycolytic enzymes.

Animals↗

PubMed1998

The immunoreactivity of glial fibrillary acidic protein in mesangial cells and podocytes of the glomeruli of rat kidney in vivo and in culture.

In this study the presence of glial fibrillary acidic protein (GFAP) in kidney is for the first time demonstrated in cryostat sections and cultures of isolated glomerular explants derived from rat kidneys. In double immunolabelling analysis of adult rat kidney sections using antiserum against GFAP and monoclonal antibody (mAb) against vimentin or desmin, the presence of immunoreactivity for GFAP could be observed in the glomerulus of the kidney and vascular cells situated in the peritubular space which expressed vimentin and desmin. Labelling of the sections with absorbed antiserum against GFAP completely abolished the staining in all these cells. The mAb against GFAP, clone GF12.24 which is known to label GFAP both in neural and non-neural cells, recognised its antigen only in the cells located in glomeruli. The investigations performed on early 2- or 3-day-old cultures from glomerular explants revealed different patterns of staining for GFAP in mesangial cells and podocytes: weak filamentous in mesangial cells and a strong non-filamentous perinuclear pattern in podocytes. Due to prominent perinuclear expression in podocytes GFAP may be considered as a marker of these cells. A different pattern of distribution of immunoreactivity for GFAP in podocytes and mesangial cells might be due to function-related posttranslational modifications of GFAP resulting in assembly or disassembly of GFAP filaments. The different pattern of staining for GFAP in the podocytes and mesangial cells, cells which exert a different influence on the capillaries of the glomeruli, suggests a role for GFAP in regulation of the tension and permeability of vascular walls. Previous investigations and present studies hint at GFAP as being a general marker of perivascular cells.

Animals↗

PubMed1998

Wild-type function of the p53 tumor suppressor protein is not required for apoptosis of mouse hepatoma cells.

The role of the tumor suppressor protein p53 in apoptosis of mouse hepatoma cells was studied. Different lines were used which were either p53 wild-type or carried various types of heterozygous or homozygous p53 mutations. The presence of mutations was demonstrated to correlate with a lack in transactivating activity of p53. While UV-light effectively produced apoptosis in cells of all lines, irrespective of their p53 mutational status, gamma-irradiation induced the formation of micronuclei but failed to induce apoptosis. Both UV- and gamma-irradiation led to nuclear accumulation and increases in p53 protein in p53 wild-type cells. Similarly, no significant differences in apoptotic response between p53 wild-type and p53 mutated cells were seen with other apoptotic stimuli like CD95/APO-1/Fas or TNFalpha. These data suggest that wild-type p53 is not required for induction of apoptosis in mouse hepatoma cells which may explain the apparent lack of p53 mutations in mouse liver tumors.

Animals↗

PubMed1997

High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32.

Connexins are subunits of gap junction channels, which mediate the direct transfer of ions, second messenger molecules and other metabolites between contacting cells. Gap junctions are thought to be involved in tissue homeostasis, embryonic development and the control of cell proliferation [1,2]. It has also been suggested that the loss of intercellular communication via gap junctions may contribute to multistage carcinogenesis [3-5]. We have previously shown that transgenic mice that lack connexin32 (Cx32), the major gap junction protein expressed in hepatocytes, express lower levels of a second hepatic gap junction protein, Cx26, suggesting that Cx32 has a stabilizing effect on Cx26 [6]. Here, we report that male and female one-year-old mice deficient for Cx32 had 25-fold more and 8-fold more spontaneous liver tumors than wild-type mice, respectively. Incorporation of bromodeoxyuridine (BrdU) into the liver was higher for Cx32-deficient mice than for wild-type mice, suggesting that their hepatocyte proliferation rate was higher. Furthermore, intraperitoneal injection, two weeks after birth, of the carcinogen diethylnitrosamine (DEN) led, after one year, both to more liver tumors in Cx32-deficient mice than in controls, and to accelerated tumor growth. Loss of Cx32 protein from hepatic gap junctions is therefore likely to cause enhanced clonal survival and expansion of mutated ('initiated') cells, which results in a higher susceptibility to hepatic tumors. Our results demonstrate that functional gap junctions inhibit the development of spontaneous and chemically induced tumors in mouse liver.

Animals↗

PubMed1996

Quantitative analysis of enzyme-altered liver foci in rats initiated with diethylnitrosamine and promoted with 2,3,7,8-tetrachlorodibenzo-p-dioxin or 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin.

A quantitative method based upon a stochastic model was used to estimate rates of initiation (alteration to express the ATPase-deficient phenotype) and of clonal growth of altered cells in an initiation promotion experiment in the livers of female Wistar rats. Diethylnitrosamine (DEN) was used as the initiating agent followed by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HCDD) as promoters. Two distinct versions of the stochastic model, called Model I and Model II, were fitted to the data. Model I made the assumption that, after the initial phase of acute initiation with DEN, background rates of initiation were equal in animals treated with DEN and controls that were not so treated. Model II, which fit the data substantially better than Model I, assumed that background rates of initiation were different in DEN-treated animals and animals not so treated, even after the acute phase of initiation was over. Both models indicate that the rates of cell division and apoptosis of altered cells are increased during TCDD treatment. In contrast, the rate of division remains more or less constant during treatment with HCDD, but the rate of apoptosis is decreased. The background rate of initiation during treatment with HCDD is equal to that in controls not administered promoters. With TCDD treatment, however, the rate of initiation estimated from the model is substantially increased over controls. The analysis also suggests that there is heterogeneity within foci of the rates of cell division, with cells on the surface of foci dividing faster than cells in the interior.

Adenosine Triphosphatases↗

PubMed1996

Expression of hepatocyte-enriched nuclear transcription factors in mouse liver tumours.

Preneoplastic and neoplastic lesions in rodent liver show alterations in the expression of various enzymes which can be used for their identification. To address the question whether these enzymatic alterations result from specific changes in the levels of hepatocyte-enriched nuclear factors (HNF), we analysed the mRNA levels of six different HNFs (HNF-1alpha, beta, HNF-3alpha, beta, gamma, and HNF-4) by RNase protection assay in chemically induced liver tumours and corresponding normal liver tissue from mice of three different strains. When compared with the normal liver tissue, HNF-1beta, HNF-3alpha and HNF-3beta showed unchanged expression levels in the various liver tumours, which HNF-1alpha and HNF-4 mRNAs were lowered by 20-30%, and HNF-3gamma mRNA was increased by 50%. There were no significant differences in HNF-expression between tumours harbouring point mutations at codon 61 of the Ha-ras protooncogene and tumours without detectable Ha-ras mutations.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1995

Induction of cytochrome P4501A by smoking or omeprazole in comparison with UDP-glucuronosyltransferase in biopsies of human duodenal mucosa.

Drug-metabolizing enzymes were investigated in duodenal biopsy specimens. Cytochrome P4501A (CYP1A) activity was determined by measuring 7-ethoxyresorufin O-deethylase (EROD) activity in biopsies from 20 smokers (3-30 cigarettes per day), 21 nonsmokers, and 10 nonsmokers receiving omeprazole treatment (20-60 mg/day for at least 1 week). Omeprazole is known to act as a polycyclic aromatic hydrocarbon (PAH)-type inducer in humans. EROD activity was found to be significantly induced in smokers and omeprazole-treated patients, with medians of 2.1 and 1.1 pmol.min-1.mg protein-1, respectively, compared with 0.5 pmol.min-1.mg protein-1 in nonsmokers. Immunoblot analysis substantiated that EROD activity was correlated with CYP1A protein. In contrast, UDP-glucuronosyltransferase (UGT) activity towards 4-methylumbelliferone (an overlapping substrate of several constitutive and inducible UGTs) was not significantly affected. The results demonstrate CYP1A induction by omeprazole and by constituents of cigarette smoke in the human duodenum and support the utility of duodenal biopsies to monitor CYP1A induction by PAH-type inducers.

Cytochrome P-450 CYP1A1↗

PubMed1995

Role of receptors in human and rodent hepatocarcinogenesis.

The liver is a major target organ in rodent carcinogenicity assays. Amongst the agents that are effective in producing rodent liver tumours are many chemicals which are not mutagenic, but are believed to mediate their effects by promoting the clonal outgrowth of initiated cells. Some of these chemicals, such as dibenzo-p-dioxins and certain PCBs, have been demonstrated to interact with specific cellular receptors and receptor binding appears crucial for their tumourigenic activity. Enzyme-altered foci in rat liver may serve as a sensitive means to estimate the promoting activity of these agents in rodents. Mechanistic considerations are of relevance when extrapolating these data from rodents to humans.

Animals↗