Biomedical subjects

R J Isfort

Publications and source records attributed to R J Isfort.

At least 37 records · Page 2Linked to original sources

PubMed1996

The pH 6.7 Syrian hamster embryo cell transformation assay for assessing the carcinogenic potential of chemicals.

Cell transformation models have been established for studying the cellular and molecular basis of the neoplastic process. Transformation models have also been utilized extensively for studying mechanisms of chemical carcinogenesis and, to a lesser degree, screening chemicals for their carcinogenic potential. Complexities associated with the conduct of cell transformation assays have been a significant factor in discouraging broad use of this approach despite their reported good predictivity for carcinogenicity. We previously reported that many of the experimental difficulties with the Syrian hamster embryo (SHE) cell transformation assay could be reduced or eliminated by culturing these cells at pH 6.7 culture conditions compared to the historically used pH 7.1-7.3. We and others have shown that morphological transformation (MT), the earliest recognizable phenotype in the multi-step transformation process and the endpoint used in the standard assay to indicate a chemical's transforming activity, represents a pre-neoplastic stage in this model system. In the collaborative study reported here, in which approx. 50% of the chemicals were tested under code in one laboratory (Hazelton) and the other 50% evaluated by several investigators in the second laboratory (P & G), we have evaluated 56 chemicals (30 carcinogens, 18 non-carcinogens, 8 of inconclusive carcinogenic activity) in the SHE cell transformation assay conducted at pH 6.7 culture conditions with a standardized, Good Laboratory Practices-quality protocol. An overall concordance of 85% (41/48) between SHE cell transformation and rodent bioassay results was observed with assay sensitivity of 87% (26/30) and specificity of 83% (15/18), respectively. The assay exhibited a sensitivity of 78% (14/18) for Salmonella assay negative carcinogens, supporting its value for detecting non-mutagenic carcinogens. For maximum assay sensitivity, two exposure durations were required, namely a 24-h exposure and a 7-day exposure assay. Depending on the duration of chemical treatment required to induce transformation, insight into the mechanism of transformation induction may also be gained. Based on the data reported here, as well as the larger historical dataset reviewed by Isfort et al. (1996), we conclude that the SHE cell transformation assay provides an improved method for screening chemicals for carcinogenicity relative to current standard genotoxicity assays.

3T3 Cells↗

PubMed1996

Calcium functions as a transcriptional and mitogenic repressor in Syrian hamster embryo cells: roles of intracellular pH and calcium in controlling embryonic cell differentiation and proliferation.

We have investigated the role played by the growth and differentiation factor (GDF)-induced calcium ion second messenger signal in the control of cellular differentiation and proliferation in Syrian hamster embryo (SHE) cells. Blocking the platelet-derived growth factor (PDGF)-induced calcium ion signal with either extracellular/intracellular acidification or pharmacological agents resulted in increased immediate early gene expression and mitogenesis. The increase in immediate early gene expression resulted from a change in the level of immediate early gene transcription and not immediate early gene mRNA stability. Analysis of the promoter elements that control immediate early gene expression indicated that the calcium ion effect is mediated through the CaRE/CRE and AP1 promoter elements. The calcium signal-mediated reduction in PDGF A/B-stimulated SHE cell immediate early gene expression resulted in a reduction in PDGF A/B-induced cellular proliferation. These results demonstrate that in SHE cells, calcium functions to suppress mitogen-induced proliferation at the level of immediate early gene expression, an effect related to the control of cellular proliferation and differentiation by GDFs through the calcium ion second messenger.

Animals↗

PubMed1996

Use of the Syrian hamster embryo cell transformation assay for determining the carcinogenic potential of heavy metal compounds.

Cobalt sulfate hydrate, gallium arsenide, molybdenum trioxide, vanadium pentoxide, and nickel sulfate heptahydrate were tested in the Syrian hamster embryo (SHE) assay in order to increase the SHE assay database for heavy metals. All five compounds produced significant morphological transformation at one or more doses in a dose-responsive manner. Cobalt sulfate hydrate, gallium arsenide, molybdenum trioxide, and nickel (II) sulfate heptahydrate were all positive with a 24-hr exposure, suggesting direct DNA perturbation. Vanadium pentoxide was negative with a 24-hr exposure, but positive with a 7-day exposure. This pattern of response (24-hr SHE negative/7-day SHE positive) has been seen with other chemicals which have tumor promotion-like characteristics. Since the inception of the use of the SHE cell transformation assay for detecting the neoplastic transformation potential of chemicals, over 42 heavy metal compounds have been tested in this assay. Based on the 24 metal compounds which have been tested in the SHE, Salmonella, and some type of rodent bioassay, the SHE assay is 92% concordant with rodent bioassay carcinogenicity results, including a sensitivity of 95% (21/22) and a specificity of 50% (1/2). At this time, the measure of SHE assay specificity for rodent carcinogenicity of metals is limited by the paucity of metal compounds which are rodent noncarcinogens. The Salmonella assay results are only 33% concordant with the rodent bioassay for these same chemicals. This relatively high concordance between the SHE assay and the rodent bioassay carcinogenicity results demonstrates the utility of the SHE assay for determining the carcinogenic potential of heavy metal compounds in rodent cancer bioassays.

Animals↗

PubMed1996

Application of in vitro cell transformation assays to predict the carcinogenic potential of chemicals.

Genotoxicity test batteries have become a standard fool for identifying chemicals that may have potential carcinogenic risk to humans. It is now apparent, however, that the use of genotoxicity batteries for assessing carcinogenic potential has limitations including an overall low specificity and a limited ability to detect carcinogens acting via 'nongenotoxic' mechanisms. In vitro cell transformation models, because they measure a chemical's ability to induce preneoplastic or neoplastic endpoints regardless of mechanism, may fulfil the current need for an in vitro biologically relevant model with increased predictiveness for determining carcinogenic potential. This review will focus on data demonstrating the similarities of chemically induced cell transformation in vitro to carcinogenesis in vivo. Furthermore, a growing database demonstrating a high overall correlation between cell transformation results with those of the rodent bioassay will also be discussed. Finally, the inclusion of cell transformation approaches for assessing the carcinogenic potential of chemicals relative to currently used genotoxicity batteries will be presented.

Animals↗

PubMed1996

Isolation and biological characterization of morphological transformation-sensitive Syrian hamster embryo cells.

Investigations were carried out designed to isolate and biologically characterize the subpopulation of cells within the Syrian hamster embryo (SHE) cell population which are sensitive to morphological transformation (MT). Biological cloning of MT-sensitive cells demonstrated that within the complex SHE cell population, MT-sensitive cells comprise approximately 27% of the clonally plateable cellular population. Importantly, MT-sensitive cells display MT rates of approximately 7% following benzo[a]pyrene exposure, a rate which falls to <1% with additional passage of the cells, indicating that the ability to undergo MT is a transient phenomenon. Biological characterization of the clonal MT-sensitive cells indicates that these cells are relatively undifferentiated, since they express both cytokeratins and vimentin and respond to a variety of stem cell growth and differentiation factors, although the majority appear to be committed progenitor cells, since they express either mesenchymal or epithelial cell characteristics. Together these data demonstrate that MT-sensitive cells comprise a subpopulation of the cells within the complex SHE cell population, that the ability to undergo MT is a transient phenomenon and that MT-sensitive cells are relatively undifferentiated committed progenitor stem-like cells, all of which gives rise to the hypothesis that MT is an alteration in the cellular differentiation state.

Animals↗

PubMed1996

pH effects on the lifespan and transformation frequency of Syrian hamster embryo (SHE) cells.

We have investigated the effects of modification of the growth media pH on Syrian hamster embryo (SHE) cellular lifespan and transformation. Culturing SHE cells in Dulbecco's modified Eagle's medium (DMEM) at pH 6.7 relative to pH 7.3 results in a 4-fold increase in the lifespan of SHE cells before senescence occurs. This effect was observed both with the wild type SHE cell population and with the morphological transformation sensitive subpopulations derived from the wild type SHE cell mixture (cl9 and cl17). The increase in SHE cell lifespan at pH 6.7 was observed both as an increase in days in culture and an increase in the number of total population doublings before cellular senescence occurred. The tumor promoting agent TPA increased the lifespan of the SHE cell population and one of the MT sensitive subpopulations when these cells were cultured in pH 7.3 medium. Interestingly, treatment of the SHE cell population, and the two MT sensitive SHE cell clones with TPA at culture medium pH of 6.7 reduced the lifespan of each cell type. Finally, culturing SHE cells in medium of pH 6.7 increased the potential of the cells to undergo morphological transformation in response to treatment with carcinogens, demonstrating a potential connection between SHE cell lifespan and carcinogen induced morphological transformation, probably through a mechanism involving a pH induced block in cellular differentiation. We believe this is the first report demonstrating the role of pH in controlling cellular lifespan.

Animals↗

PubMed1996

Use of the Syrian hamster embryo cell transformation assay for carcinogenicity prediction of chemicals currently being tested by the National Toxicology Program in rodent bioassays.

The Syrian hamster embryo (SHE) cell transformation assay was used to predict the carcinogenicity of 26 chemicals currently being tested in the rodent bioassay by the National Toxicology Program as part of its program titled "Strategies for Predicting Chemical Carcinogenesis in Rodents." Of these 26 chemicals, 17 were found to be positive in the SHE cell transformation assay while 9 were negative. Carcinogenicity predictions were made for these chemicals, based upon the SHE cell transformation assay results. Our predictions will be compared with the rodent bioassay results as they become available.

Animals↗

PubMed1996

Alpha-quartz-induced chemokine expression by rat lung epithelial cells: effects of in vivo and in vitro particle exposure.

Chemokines are chemotactic cytokines that can play a key role in leukocyte recruitment to sites of tissue injury or infection. Previous studies have demonstrated that exposure to alpha-quartz as well as other noxious particles increases chemokine gene expression in rat lung, although the cells responsible for chemokine expression and the mechanisms underlying this response have remained unclear. The present studies demonstrate that exposure of rats to alpha-quartz induced expression of mRNA for the chemokine macrophage-inflammatory protein (MIP)-2 in epithelial cells lining the terminal bronchioles and alveolar ducts as well as macrophages and alveolar type II cells in the more distal lung. Treatment of rats with an anti-MIP-2 antiserum before alpha-quartz exposure markedly attenuated neutrophilic infiltration of the lungs demonstrating an important role for MIP-2 in alpha-quartz-induced pulmonary inflammation. In vitro exposure of primary cultures of rat alveolar type II cells or the rat alveolar type II cell line RLE-6TN to tumor necrosis factor-alpha, endotoxin, or alpha-quartz increased mRNA for MIP-2 as well as the structurally and functionally similar chemokine cytokine-induced neutrophil chemoattractant but not the chemokine MIP-1 alpha. The alpha-quartz-induced increase in epithelial MIP-2 mRNA resulted, at least in part, from increased gene transcription and was associated with the release of active MIP-2 protein. Induction of RLE-6TN MIP-2 and cytokine-induced neutrophil chemoattractant mRNA expression was not unique to alpha-quartz, being also increased by crocidolite asbestus fibers but not by titanium dioxide or MMVF-10 glass fibers. These findings indicate that epithelial cells contribute to chemokine expression in rat lung after exposure to alpha-quartz and potentially other noxious particles and suggest that alpha-quartz-activated MIP-2 expression in vivo results, at least in part, from a direct action of the particles on the lung epithelium.

Animals↗

PubMed1996

Oval cell proliferation associated with the murine insertional mutation TgN737Rpw.

The Tg737 gene was identified by its direct association with a transgene-induced insertion mutation in the mouse. This mutation causes pleiotropic phenotypes including a syndrome similar to autosomal recessive polycystic kidney disease in humans. This syndrome, in addition to renal cyst formation, includes the presence of an invariably associated liver abnormality. The liver pathology in TgN737Rpw mice is characterized by a biliary hyperplasia that includes the proliferation of cells that morphologically and immunologically resemble oval cells, a liver progenitor cell. This abnormality is first observed at approximately 5 days of age in the portal region and then progresses into the periportal regions. Additionally, the formation and proliferation of dysplastic ductular structures are observed from the onset of the phenotype. Serum chemistry indicated that the primary defect is likely to be of biliary origin, and hepatic function appears normal in the mutant mice. Therefore, this mutation is unlike other causes of oval cell proliferation in that the hepatic parenchyma is relatively unaffected. The identification of the Tg737 gene associated with this mutation suggests that it functions in regulating the proliferation/differentiation of oval cells within the liver, which further indicates that this gene may function in pathological conditions that include oval cell proliferation, such as hepatocellular carcinogenesis.

Animals↗

PubMed1995

Modulation of the platelet-derived-growth-factor-induced calcium signal by extracellular/intracellular pH in Syrian hamster embryo cells. Implications for the role of calcium in mitogenic signalling.

Studies have been performed to understand the interactions and the role which intracellular calcium and intracellular pH have in mediating mitogen-stimulated cellular proliferation. Stimulation of Syrian hamster embryo (SHE) cells with the mitogen platelet-derived growth factor A/B (PDGF) results in intracellular acidification and capacitative calcium entry involving the intracellular release of calcium via the inositol trisphosphate gamma receptor calcium channel, followed by an extracellular influx of calcium through a dihydropyridine-sensitive plasma membrane calcium channel. Chronic extracellular/intracellular acidification results in the inactivation of both these calcium channels due to slowly reversible protein alterations. Paradoxically, transient intracellular acidification, like that following PDGF stimulation, could not stimulate the activation of either calcium channel. In addition, even though intracellular calcium fluxes by themselves could intiate intracellular acidification, loss of the PDGF-induced calcium signal did not result in the loss of the PDGF-induced transient intracellular acidification. Importantly with regard to the role intracellular calcium and pH have in mediating the mitogenic signal leading to cellular proliferation, chronic extracellular/intracellular acidification, which leads to a complete loss of the PDGF-induced calcium signal, did not result in the loss of PDGF-induced mitogenesis. These results indicate that the PDGF-induced calcium signal is not essential for PDGF-stimulated mitogenesis in Syrian hamster embryo cells. In contrast, blocking the PDGF-induced transient intracellular acidification completely blocks PDGF-induced mitogenesis, indicating that the mitogen-induced transient intracellular acidification, unlike the intracellular calcium ion signal, is indispensible for cellular proliferation in Syrian hamster embryo cells.

Animals↗

PubMed1995

Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen.

Macrophage inflammatory protein-2 (MIP-2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses. To clone the cDNA for rat MIP-2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide. Reverse transcription-polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP-2 cDNA sequence. A cDNA containing the coding region of rat MIP-2 was cloned and sequenced. Comparison to the mouse MIP-2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level. The rat MIP-2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity. The recombinant rat MIP-2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes. In addition, the recombinant rat MIP-2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro.

Amino Acid Sequence↗

PubMed1995

Analysis of oncogenes, tumor suppressor genes, autocrine growth-factor production, and differentiation state of human osteosarcoma cell lines.

Human osteosarcoma and fibrosarcoma cell lines were investigated for alterations in oncogenes, tumor suppressor genes, and growth factors, all of which have been implicated in tumor formation. Characterization of oncogenes that are involved in osteosarcoma formation, including the c-fos and c-myc oncogenes, indicated that all six osteosarcoma cell lines examined had 5- to 20-fold amplification of the c-myc oncogene, whereas neither of two fibrosarcoma cell lines c-myc amplification. Interestingly, only three of six osteosarcoma cell lines displayed altered c-myc immediate-early gene function. c-fos was found to be normal, both at the gene and functional levels, in all six osteosarcoma and both fibrosarcoma cell lines tested. Characterization of two tumor suppressor genes, p53 and RB1, that have been implicated in osteosarcoma formation indicated that p53 was altered in five of six osteosarcoma cell lines, whereas RB1 was altered in only two or six of these cell lines. Neither RB1 nor p53 was found to be altered in the fibrosarcoma cell lines tested. An additional transformation marker, autocrine growth-factor production, was observed in all six osteosarcoma cell lines and both fibrosarcoma cell lines examined. Finally, the differentiation state of the osteosarcoma cell lines was investigated via the bone differentiation markers alkaline phosphates and osteocalcin. Alkaline phosphatase activity was observed in four of six osteosarcoma cell lines but not in the two fibrosarcoma cell lines examined. The alkaline phosphatase activity was a result of the expression of the bone/liver/kidney alkaline phosphatase isoform. High-level osteocalcin expression was observed in one of the osteosarcoma cell lines but not in the two fibrosarcoma cell lines examined, although all cell lines demonstrated low-level osteocalcin expression. Together, these data demonstrate that relatively undifferentiated osteosarcomas commonly display c-myc amplification, p53 and RB1 mutation, and autocrine growth-factor production, all of which may play a role in osteosarcomagenesis.

Base Sequence↗

PubMed1995

The Syrian hamster embryo (SHE) cell transformation system: a biologically relevant in vitro model--with carcinogen predicting capabilities--of in vivo multistage neoplastic transformation.

Neoplastic transformation is a multistep process that can be modeled in vitro using Syrian hamster embryo (SHE) cells. SHE cells multistage transformation involves several intermediate stages, including morphological transformation, immortality, acquisition of tumorigenicity, and malignant progression. Analysis of the molecular alterations that occur at each stage indicated that morphological transformation results from both carcinogen-induced irreversible chromosomal/genetic mutations and reversible genetic events, including altered DNA methylation. Morphological transformation results from a block in the cellular differentiation of progenitor and determined stem-like cells in the SHE cell population via alternation in the expression of the H19 tumor suppressor gene and other genes. Immortality results from genetic mutations in growth factor responsiveness, including loss of growth suppression by TGF beta and autocrine growth factor production, and genomic stability, resulting in genomic instability and an increased mutation rate. Acquisition of tumorigenicity involves loss of tumor suppressor gene function, altered mitogenic signal transduction, mutation of oncogenes, acquisition of anchorage independent growth, and chromosomal aberrations. Malignant progression is associated with alterations in extracellular matrix growth characteristics, alterations in cytoskeleton structure, elevated fibrinolytic activity, secretion of proteases, and changes in extracellular matrix protein secretion. Together, these changes model the alterations observed during in vivo neoplastic transformation and possibly explain why the SHE assay, as a carcinogen screening tool, is able to identify carcinogens with a 80 to 85% accuracy.

Animals↗

PubMed1994

Alterations in cellular differentiation, mitogenesis, cytoskeleton and growth characteristics during Syrian hamster embryo cell multistep in vitro transformation.

In vitro Syrian hamster embryo (SHE) cell transformation is a neoplastic process that proceeds through several identifiable consecutive stages including in vitro morphological transformation (mt), acquisition of immortality (I+), acquisition of tumorigenicity (T+) and tumor-derived cells (I'TD). Eight transformed lineages consisting of cells at the mt, I+, T+ and I'TD stages were assayed for alterations in general markers of cell differentiation, mitogenic signaling pathways, cytoskeleton and cellular growth in 3D matrix. Alterations in cellular differentiation markers included a decrease in H19 gene expression and placental alkaline phosphatase enzymatic activity at the mt stage in all lineages examined with a complete absence of H19 gene expression and placental alkaline phosphatase enzymatic activity by the I'TD stage in a majority of transformed lineages. Changes in mitogenic signaling pathways included the production of autocrine growth factors and alterations in growth factor-induced immediate early gene expression by the I'TD stage of transformation in the majority of transformed lineages investigated. By the I'TD stage of transformation in most lineages, changes in both the cytoskeleton (including a decrease in tropomyosin-I gene expression) and the Matrigel growth characteristics of SHE cells were observed.

Actins↗

PubMed1994

Integration of multiple chicken retroviruses into multiple chicken herpesviruses: herpesviral gD as a common target of integration.

Integration of two different avian retroviruses, reticuloendotheliosis virus (REV) and avian leukosis virus (ALV), into the genome of two different avian herpesviruses, the herpesvirus of turkeys (HVT) and Marek's disease virus (MDV), was investigated. Integration events occurred by the fourth and sixth in vitro passage of cells coinfected with REV/HVT and ALV/MDV, respectively. In order to further characterize the integration events, integrated proviruses and surrounding herpesviral genetic material were cloned and analyzed. In the REV/HVT coinfection experiment, one of the three unique integrated proviruses was found to have integrated into the HVT gD gene, resulting in disruption of the coding region of this gene. The two additional unique integrations were localized to the UL and IRL border regions of HVT, two previously described common sites of REV integration into MDV. Interestingly, one of the integrated proviruses in the HVT genome appeared to be full length, was infectious when transfected into CEF cells, and therefore could potentially function to produce infectious REV from an HVT infectious platform. In the ALV/MDV coinfection experiment, one of two unique integrated proviruses was found to have integrated into the gD gene, resulting in disruption of the coding region of this gene. The second unique integration site was in the polyadenylation site of the SORF2 gene at the boundary of the IRS and US, once again a common site of REV integration into MDV. These results demonstrate that the U/IR-TR border regions of herpesviruses are common sites of retroviral integration. In addition gD, in the US region of the herpesvirus, is a common site of retroviral integration in multiple herpesvirus, indicating a possible selective advantage for disruption of this gene in the in vitro growth of a herpesvirus. Finally, this is the first instance of a full-length provirus found integrated into a herpesvirus genome, indicating that a retrovirus could alter its route of infection by being carried in a herpesvirus genome.

Amino Acid Sequence↗

PubMed1994

Retrovirus insertion into herpesviruses.

Recent studies have indicated that retroviruses can integrate into and mutate the genomes of herpesviruses during co-infection. This interaction has the potential to change the host range and pathogenicity of both viruses and result in novel infectious agents and diseases. This phenomenon also allows genetic material to be exchanged between these viruses and their hosts.

Animals↗

PubMed1994

Growth factor responsiveness and alterations in growth factor homeostasis in Syrian hamster embryo cells during in vitro transformation.

Syrian hamster embryo (SHE) cells were investigated for their growth factor responsiveness as well as changes in growth factor homeostasis, including alterations in autocrine growth factor production and growth factor responsiveness, during in vitro transformation. For wild-type SHE cells, fetal bovine serum (FBS), epidermal growth factor (EGF) family members, platelet derived growth factor (PDGF) family members, fibroblast growth factor family members, interleukin-4, interleukin-9, oncostatin M, hepatocyte growth factor, erythropoietin and pituitary extract were found to be mitogenic. SHE cell mitogenesis was inhibited in response to transforming growth factor beta (TGF-beta) family members, interleukin-1 alpha, interleukin-1 beta and nerve growth factor. Additional experiments were conducted to study alterations in growth factor responsiveness to three SHE cell mitogens (FBS, EGF and PDGF) and one inhibitor of mitogenesis (TGF-beta) during SHE cell in vitro transformation. Alterations in either EGF, PDGF or TGF-beta responsiveness were observed in 7/8 SHE transformed lineages during the stepwise transformation process. Finally, 6/8 lineages underwent alterations which resulted in the production of autocrine growth factors during the transformation process. These results indicate that multiple alterations in growth factor homeostasis occur during the in vitro transformation process.

Animals↗

PubMed1993

Induction of protein phosphorylation, protein synthesis, immediate-early-gene expression and cellular proliferation by intracellular pH modulation. Implications for the role of hydrogen ions in signal transduction.

In Syrian hamster embryo cells, intracellular acidification (but not alkalization) results in proliferation, immediate-early-gene expression and tyrosine phosphorylation. In addition, both intracellular acidification and alkalization result in serine/threonine phosphorylation and de novo protein synthesis of specific proteins. Calcium is not mobilized in response to either intracellular alkalization or acidification. Neither intracellular acidification nor alkalization altered the serum proliferative signal while intracellular alkalization (but not acidification) reduced the epidermal-growth-factor-induced proliferative signal, tyrosine phosphorylation and immediate-early-gene expression. Finally, intracellular acidification (but not alkalization) could induce immediate-early-gene expression in cells growing in the presence of serum, indicating that the pH signalling pathway is not down modulated by the serum signalling pathway. These results, while indirect, indicate that hydrogen ions may play an important role in mitogen-signal transduction in Syrian hamster embryo cells.

Animals↗