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Biomedical subjects

P Amstad

Publications and source records attributed to P Amstad.

At least 19 recordsLinked to original sources
PubMed1992

Oxidant carcinogenesis and antioxidant defense.

Growth promotion by oxidants is observed with cultured human and mouse fibroblasts as well as epidermal cells. It is expected to play a role in inflammation, fibrosis, and tumorigenesis. Indeed, oxidants trigger (patho)physiological reactions that resemble those induced by growth and differentiation factors. For example, active oxygen activates protein kinases, causes DNA breakage, and induces the growth competence-related protooncogenes c-fos and c-myc. The cellular antioxidant defenses affect the consequences of oxidant exposure. Transfectants of mouse epidermal cells that overproduce Cu,Zn-superoxide dismutase (SOD) were sensitized to the toxic effects of an extracellular burst of O2-. plus H2O2, whereas overproducers of catalase (CAT) were protected. Transfection of SOD overproducers with CAT corrected their hypersensitivity. Inducibility of the protooncogene c-fos by oxidants was diminished in SOD and CAT overproducers, albeit probably for different reasons. It is concluded that a fine balance of the multiple components of the antioxidant defense determines the growth response of cells to oxidative stress. In studies of the mechanism of the transcriptional induction of c-fos by oxidants, we identified the joint DSE-AP1 elements (dyad symmetry element, DSE) as major enhancer motifs in the 5'-upstream regulatory sequences of c-fos. Oxidants also increased the de novo synthesis of protein factors that bind to the fos-AP1 enhancer motif. Protein kinase and ADPR transferase inhibitors suppressed the transcriptional induction of c-fos as well as the increase in factor binding to fos-AP1. We conclude that protein phosphorylation and protein polyADP-ribosylation are required for the transcriptional induction of c-fos and the synthesis of protein factors that bind to fos-AP1. It is likely that the FOS and JUN proteins are among these factors and that they participate in the regulation of c-fos expression by oxidants.

Animals↗
PubMed1992

Expression of the proto-oncogene c-fos in three-dimensional fetal brain cell cultures and the lack of correlation with maturation-inducing stimuli.

Previous work has shown that aggregating fetal brain cell cultures are able to attain a highly differentiated state, and that their development is greatly enhanced by growth and/or differentiation factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and the protein kinase C-activating tumor promoter mezerein. The present study shows that in these 3-dimensional cultures the peptide growth factors EGF and bFGF as well as mezerein are able to induce the expression of the proto-oncogene c-fos. This induction was rapid and transient, in good agreement with observations reported from a wide variety of cell types in vitro. The maximal levels of c-fos mRNA found after stimulation were low in immature cultures and increased greatly as maturation progressed. Of the three factors tested, mezerein was the most potent inducer of c-fos. In contrast to the peptide growth factors EGF and bFGF which were found to induce c-fos only in glial cells, mezerein was stimulatory in glial cells as well as in neurons. A similar cell type specificity has been observed previously for the maturation-enhancing response in immature aggregate cultures. However, in the present study no correlation was found between the degree of c-fos induction and the extent of the maturation-enhancing stimulation. Immature cultures known to be most sensitive and responsive to these maturation-enhancing agents required relatively high doses of peptide growth factors for the induction of c-fos, and the maximal levels of c-fos mRNA elicited were much lower than those in differentiated cultures which did not show any long-term response to these stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

The balance between Cu,Zn-superoxide dismutase and catalase affects the sensitivity of mouse epidermal cells to oxidative stress.

Oxidants are toxic, but at low doses they can stimulate rather than inhibit the growth of mammalian cells and play a role in the etiology of cancer and fibrosis. The effect of oxidants on cells is modulated by multiple interacting antioxidant defense systems. We have studied the individual roles and the interaction of Cu,Zn-superoxide dismutase (SOD) and catalase (CAT) in transfectants with human cDNAs of mouse epidermal cells JB6 clone 41. Since only moderate increases in these enzymes are physiologically meaningful, we chose the following five clones for in-depth characterization: CAT 4 and CAT 12 with 2.6-fold and 4.2-fold increased catalase activities, respectively, SOD 15 and SOD 3 with 2.3-fold and 3.6-fold increased Cu,Zn-SOD activities, respectively, and SOCAT 3 with a 3-fold higher catalase activity and 1.7-fold higher Cu,Zn-SOD activity than the parent JB6 clone 41. While the increases in enzyme activities were moderate, the human cDNAs were highly expressed in the transfectants. As demonstrated for the clone SOD 15, this discordance between message concentrations and enzyme activities may be due to the low stability of the human Cu,Zn-SOD mRNA in the mouse recipient cells. According to immunoblots the content of Mn-SOD was unaltered in the transfectants. While the activities of glutathione peroxidase were comparable in all strains, the concentrations of reduced glutathione (GSH) were significantly lower in SOD 3 and SOD 15. This decrease in GSH may reflect a chronic prooxidant state in these Cu,Zn-SOD overproducers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

A genotypic mutation system measuring mutations in restriction recognition sequences.

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the PvuII recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'PvuII mutant standard'. Following amplification with Taq-polymerase and cloning into lambda gt10, plaques containing wild-type sequence, PvuII mutant standard or Taq-polymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 PvuII mutant standard copies can be rescued from 10(8) to 10(9) wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the MspI-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 10(8) copies of wild-type DNA.

Base Sequence↗
PubMed1990

Neoplastic transformation of a human kidney epithelial cell line transfected with v-Ha-ras oncogene.

We have recently shown that normal human kidney epithelial (NHKE) cells were immortalized by treatment with Ni(II) alone. In the present study the immortalized human kidney cell line (IHKE) was transfected with a plasmid construct containing the v-Ha-ras oncogene (pZipras). After transfection, the cell lines formed tumors in athymic nude mice, whereas the ZipNeoSV(X)-transfected IHKE control cultures formed no tumors. Tumor cell lines (THKE) were established from the tumors in nude mice. These cells appear to be of human epithelial origin and express high levels of Ha-ras transcript. Karyotypic analysis was performed. The cell lines were tri-, tetra- or pentaploid. A consistent finding in the IHKE, IHKZE and THKE cells was increased numbers of chromosomes 17 and 7p+. Some marker chromosomes were identical in the IHKE and THKE cell lines, underlining their common origin and their possible importance in the carcinogenic process. This shows that the combined action of a chemical carcinogen [i.e., Ni(II)] and v-Ha-ras oncogene resulted in fully transformed human kidney epithelial cells, consistent with a step-wise progression of human epithelial cell transformation.

Animals↗
PubMed1990

Genetic modulation of the cellular antioxidant defense capacity.

Oxidants are ubiquitous in our aerobic environment. While they are always toxic, they can also exert pathophysiological effects at low concentrations and play an etiological role in human disease. For example, oxidants can stimulate cell growth and act as tumor promoters. The cellular antioxidant defense system attenuates the effect of oxidants and consists of low molecular weight components and several enzymes. Most important are catalase (CAT), superoxide dismutases (SOD), and glutathione peroxidase. We are attempting to elucidate the role of CAT and Cu,Zn-SOD in oxidant tumor promotion of mouse epidermal cells JB6. We have found that the promotable clone 41 possesses 2- to 3-fold higher levels of activity, protein, and stationary mRNA of CAT and Cu,Zn-SOD than does the nonpromotable clone 30. We propose that the growth-stimulatory effect of oxidants is more pronounced in promotable clone 41 because it is better protected from oxidant toxicity. In order to corroborate this model, we have constructed JB6 cells with higher levels of Cu,Zn-SOD and CAT by transfection with expression vectors containing cDNA for these genes. On the other hand, cells with decreased amounts of Cu,Zn-SOD have been obtained by their stable transfection with a vector containing SOD-cDNA in the antisense orientation. These cell clones with modified antioxidant enzyme complements are being characterized. In particular, their promotability by oxidants and their sensitivity to killing and oxidative macromolecular damage are being measured. Certain tumor promoters that lack oxidizing properties may generate a cellular prooxidant state by a variety of mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Pathophysiological mechanisms of active oxygen.

Besides being toxic, oxidants can induce pathophysiological effects in mammalian cells. For example they can stimulate rather than inhibit cell growth. Since oxidants are ubiquitous they may represent 'natural' tumour promoters. Our work with xanthine/xanthine-oxidase as an extracellular source of active oxygen (AO) and promotable (clone 41) and non-promotable (clone 30) mouse epidermal cells JB6 allows insights into the mechanism of action of oxidant promoters. We found that AO stimulated the growth only of promotable clone 41 after an initial period of moderate inhibition while it was strongly cytostatic for non-promotable clone 30. Active oxygen induced larger amounts of DNA-strand breaks and poly ADP-ribosylation of chromosomal proteins in non-promotable cells. In addition, AO was capable of inducing the growth- and differentiation-related proto-oncogenes c-fos and c-myc in promotable and non-promotable JB6 cells. We speculate that these genes can exert their functions only in the promotable clone 41 because the general cytostatic effects of AO are moderate. A possible explanation for the differences between these 2 clones was discovered when we compared the constitutive activities, protein concentrations and mRNA levels for the antioxidant enzymes catalase (CAT), Cu,Zn-superoxide dismutase (SOD) and glutathione-peroxidase (GPx). We found that CAT and SOD (but not GPx) levels were 2-3-fold higher in the promotable clone 41. We propose that promotable cells possess a superior antioxidant defence which protects them from excessive cytostatic effects of AO.

Animals↗
PubMed1989

Cooperation of c-raf-1 and c-myc protooncogenes in the neoplastic transformation of simian virus 40 large tumor antigen-immortalized human bronchial epithelial cells.

Overexpression of c-raf-1 and the myc family of protooncogenes is primarily associated with small cell carcinoma, which accounts for approximately 25% of human lung cancer. To determine the functional significance of the c-raf-1 and/or c-myc gene expression in lung carcinogenesis and to delineate the relationship between protooncogene expression and tumor phenotype, we introduced both protooncogenes, alone or in combination, into human bronchial epithelial cells. Two retroviral recombinants, pZip-raf and pZip-myc, containing the complete coding sequences of the human c-raf-1 and murine c-myc genes, respectively, were constructed and transfected into simian virus 40 large tumor antigen-immortalized bronchial epithelial cells (BEAS-2B); this was followed by selection for G418 resistance. BEAS-2B cells expressing both the transfected c-raf-1 and c-myc sequences formed large cell carcinomas in athymic nude mice with a latency of 4-21 weeks, whereas either pZip-raf- or pZip-myc-transfected cells were nontumorigenic after 12 months. Cell lines established from tumors (designated RMT) revealed the presence of the cotransfected c-raf-1 and c-myc sequences and expressed morphological, chromosomal, and isoenzyme markers, which identified BEAS-2B cells as the progenitor line of the tumors. A significant increase in the mRNA levels of neuron-specific enolase was detected in BEAS-2B cells containing both the c-raf-1 and c-myc genes and derived tumor cell lines. The data demonstrate that the concomitant expression of the c-raf and c-myc protooncogenes causes neoplastic transformation of human bronchial epithelial cells resulting in large cell carcinomas with certain neuroendocrine markers. The presented model system should be useful in studies of molecular events involved in multistage lung carcinogenesis.

Animals↗
PubMed1988

Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral Harvey ras.

Activated ras oncogenes have previously been implicated in the pathogenesis of human lung carcinomas. A v-Ha-ras-containing retrovirus, Zip-ras, was generated by inserting the coding region of the v-Ha-ras oncogene into the Zip-NeoSV(X) [Cepko et al., Cell 37:1053-1062, 1984] retroviral vector. Amphotrophic Zip-ras retrovirus was used to infect an SV40 large T antigen-positive immortalized cell line, BEAS-2B, derived from normal bronchial epithelial cells, the predominant progenitor cells of human lung carcinomas. Zip-ras-infected BEAS-2B cells selected for G418 resistance formed anaplastic carcinomas in 12 of 15 athymic nude mice (latency 3 wk), whereas Zip-NeoSV(X)-infected BEAS-2B control cultures inoculated into 12 nude mice formed no tumors after a minimum of 7 mo. Tumor cell lines were established and demonstrated to be of human epithelial origin and to express v-Ha-ras p21 protein. A common feature of the tumor cell lines was an increase in ploidy. The increased efficiency of neoplastic transformation by v-Ha-ras of cell lines as compared with our previous results with normal bronchial epithelial cells [Yoakum et al., Science 227:1174-1179, 1985] is consistent with the hypothesis that the "immortalization" step is rate-limiting in in vitro human epithelial cell carcinogenesis.

Animals↗
PubMed1987

Induction of granulocyte-macrophage colony-stimulating factor by lipopolysaccharide and anti-immunoglobulin M-stimulated murine B cell lines.

The present study was undertaken to elucidate whether B cell lymphoma and hybridoma cell lines can be stimulated by lipopolysaccharides (LPS) or by antibodies against immunoglobulin M (IgM) to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF activity was assayed on the basophil/mast cell line PT-18 which is GM-CSF- and interleukin 3-dependent. Antibodies against murine recombinant GM-CSF were used to identify the colony-stimulating factor activity present in the supernatants of the stimulated B cell lines. When these cell lines were stimulated with LPS, two of five lymphoma and five of six hybridoma lines produced GM-CSF. Two cell lines, the B cell lymphoma M12.4.1 and the hybridoma TH2.2, were analyzed more extensively under serum-free conditions. In these two cell lines, the production of GM-CSF was dependent on the dose of LPS used and time of exposure. Antibodies against IgM stimulated the TH2.2 (IgM+) but not the M12.4.1 (IgM-) cells to produce GM-CSF. Northern blot analysis of the M12.4.1 and TH2.2 cells showed that mRNA of GM-CSF can be detected in LPS-stimulated but not in unstimulated cells. Our data show that transformed B cells can be stimulated to produce GM-CSF. The present data and previous studies on GM-CSF production by normal bone marrow-derived B cells suggest a possible participation of B cells in granulopoiesis.

Animals↗
PubMed1987

Differential regulation of colony-stimulating factors and interleukin 2 production by cyclosporin A.

Stimulation of T lymphocytes with mitogens or antigens is followed by proliferation and lymphokine production. Although cyclosporin A (CsA), an immunosuppressive drug, has been shown to inhibit the production of certain lymphokines, including interleukin 2 (IL-2), interleukin 3 (IL-3), and gamma-interferon, its effect on the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) has not been evaluated. In the current study, concanavalin A (Con A)-stimulated murine spleen cells secreted GM-CSF, IL-3, and IL-2, and in the presence of CsA (0.1-1.0 micrograms/ml), IL-2 and IL-3 activities were inhibited. In contrast, significant activity was detected when the CsA-treated culture supernatants were assayed on a cell line that is dependent on GM-CSF and/or IL-3. Similar CsA-resistant activity was observed when the EL-4 thymoma cells were stimulated with a phorbol ester [phorbol 12-myristate 13-acetate (PMA)] in the presence of CsA. The activity resistant to CsA was identified as GM-CSF by the ability of specific antibodies against murine recombinant GM-CSF to neutralize its activity. These findings indicate that GM-CSF, in contrast to IL-2 and IL-3, was not inhibited by CsA. In additional experiments, transfer blot of poly(A)+ RNA isolated from PMA-induced EL-4 cells in the presence or the absence of CsA was hybridized with GM-CSF and IL-2 cDNA probes. Expression of the GM-CSF gene in EL-4 cells was detected independent of CsA, whereas CsA inhibited the expression of the IL-2 gene. The present data show that production of IL-2 and IL-3, but not that of GM-CSF, is inhibited by CsA and suggest a differential control mechanism for lymphokine synthesis in T lymphocytes.

Animals↗
PubMed1985

In vitro studies of human lung carcinogenesis.

Advances in the methodology to culture normal human lung cells have provided opportunities to investigate fundamental problems in biomedical research, including the mechanism(s) of carcinogenesis. Using the strategy schematically shown in Figure 1, we have initiated studies of the effects of carcinogens on the normal progenitor cells of the human cancers caused by these carcinogens. Extended lifespans and aneuploidy were found after exposure of mesothelial cells to asbestos and bronchial epithelial cells to nickel sulfate. These abnormal cells may be considered to be preneoplastic and at an intermediate position in the multistage process of carcinogenesis. Human bronchial epithelial cells can also be employed to investigate the role of specific oncogenes in carcinogenesis and tumor progression. Using the protoplast fusion method for high frequency gene transfection, vHa-ras oncogene initiates a cascade of events in the normal human bronchial cells leading to their apparent immortality, aneuploidy, and tumorigenicity in athymic nude mice. These results suggest that oncogenes may play an important role in human carcinogenesis.

Animals↗
PubMed1984

Evidence for membrane-mediated chromosomal damage by aflatoxin B1 in human lymphocytes.

The hepatocarcinogen aflatoxin B1 (AFB1) was found to be a potent clastogen for phytohemagglutinin stimulated human lymphocytes. It also induced sister chromatid exchanges. These types of chromosomal damage were induced at very low levels of covalent AFB1 - DNA adducts suggesting that AFB1 operates in part by indirect action because of its membrane-active character. The membrane-active character of AFB1 is documented by the following results: (i) AFB1 stimulated the excretion of hydroxy- and/or hydroperoxy-arachidonic acid (AA) and free AA into the culture medium; (ii) the phospholipase A2 inhibitor p-bromophenacylbromide was anticlastogenic ; (iii) the inhibitors of the oxidative metabolism of AA indomethacin, flufenamic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid and BN 1015 were anticlastogenic . These results are compatible with the induction of DNA damage by indirect action or the formation of covalent adducts via metabolic activation by cooxygenation . The observation that CuZn superoxide dismutase was anticlastogenic indicates the intermediacy of superoxide in DNA damage formation and supports the former mechanism.

Aflatoxin B1↗