Maximal force is unaffected by emphysema-induced atrophy in extensor digitorium longus.

Patients with chronic obstructive pulmonary disease (COPD) demonstrate a limited exercise capacity. It is unknown whether muscle fiber atrophy and subsequent decrease in force production contributes to this functional limitation. Therefore, the purpose of this investigation was to determine whether emphysema-induced muscle fiber atrophy leads to a reduction in locomotory muscle force production. Maximal muscle force production and fiber cross-sectional area were measured in the almost exclusively fast-twitch extensor digitorium longus muscles at 4 and 8 months following saline (control, n=8/time period) or elastase (emphysema, n=15/time period) instillation in the lungs of hamsters. Excised lung volume increased 145 and 161% with emphysema at 4 and 8 months, respectively (both P<0.01). Muscle mass, maximal force, and fiber cross-section were unaltered at 4 months. However, absolute mass (-15%) and fiber cross-sectional area (-18%) were reduced at 8 months (both P<0.01). Surprisingly, maximal force was preserved in emphysema animals. These data demonstrate that maximal muscle force may be preserved in the face of emphysema-induced fiber atrophy.

The tetratricopeptide repeat containing Tg737 gene is a liver neoplasia tumor suppressor gene.

The Tg737 gene was investigated for gross alterations in a series of rodent/human liver tumors and human tumorigenic cell lines. The Tg737 gene was found to be altered in approximately 40% of the rodent chemically-induced liver tumors, 40% of the human liver tumors, and in liver, kidney and pancreatic human tumor cell lines. Ectopic re-expression of the Tg737 gene in a Tg737 deleted mouse liver tumor cell line resulted in suppression of tumorigenic growth, without altering in vitro cell culture growth. Treatment of mice which are either homozygous normal or heterozygous deleted at the Tg737 locus with the carcinogen diethylnitrosamine resulted in an increase in preneoplastic foci formation in the Tg737 heterozygous deleted mice. Ectopic expression of the Tg737 gene results in multinucleated cells, loss of Tg737 gene expression results in the proliferation of liver stem cells (oval cells) without concomitant differentiation, and reexpression of the Tg737 gene reestablished responsiveness to external differentiation factors. We believe this is the first report demonstrating tumor suppression activity for a tetratricopeptide repeat gene family member and provides insights into the function of this family of genes in mammalian cells.

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.

Serum and growth factors stimulate ribosomal RNA processing in Syrian hamster embryo cells: divergence of this signalling pathway from immediate-early gene expression.

Growth factor stimulation of cells results in multiple intracellular biochemical changes including increases in calcium levels, phosphatidylinositol turnover, protein phosphorylation, immediate-early gene transcription and intracellular pH alterations. We report here that serum and growth factor stimulation of Syrian hamster embryo (SHE)-derived cell lines increases the ribosomal RNA (rRNA) processing rates. In addition, in several transformed SHE cell lines, the immediate-early response of fos/actin transcription could be separated from the immediate-early rRNA processing response. These results indicate that in these cell lines a mutation(s) which uncouples these responses has occurred. This is the first report describing (i) rRNA processing as an immediate-early effect following growth factor stimulation and (ii) the identification of a mutation(s) which can uncouple two immediate-early signalling responses.

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.

Enzymatic purification of plasmid DNA.

A novel method for plasmid DNA purification using enzymes that degrade all major types of contaminating nucleic acids present in crude plasmid DNA mixtures (but not plasmid DNA) has been devised. This method is quick (can be accomplished in two hours), requires no expensive laboratory equipment (an ultracentrifuge is not necessary) and is inexpensive. Plasmid DNA purified by this methodology can be used in a variety of molecular biological techniques including restriction enzyme digestions, subcloning, sequencing, nick translation and end labeling. This plasmid purification technique will be very useful for the molecular biologist performing cloning experiments.

The 4-6-8 method of sequence analysis.

A new method to obtain more sequence data from a single gel run is described. This method allows the reading of over 500 bases of sequence data from a single gel by taking advantage of the differential migration of specific sized dideoxy terminated chain lengths in sequencing gels containing variable percentages of acrylamide. The method is easy to use, requires no special equipment and requires no special technical abilities. We feel the methodology described will be useful for the average laboratory doing sequencing work.

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.

Mechanisms of cell transformation in the Syrian hamster embryo (SHE) cell transformation system.

The Syrian hamster embryo (SHE) cell transformation system has been used for investigational studies of basic mechanisms of neoplastic transformation, as well as determining the carcinogenic potential of chemical, physical, and biological agents. Many of these investigations utilize an intermediate step in the SHE cell neoplastic transformation process, known as morphological transformation, as an indicator that the cells have acquired an increased potential to progress to malignancy. While the nature of the morphologically transformed phenotype is not completely understood, it is believed to result from a block in the cellular differentiation of stem cells present within the SHE cell population. In terms of determination of the transforming potential of biological/chemical/physical agents, more than 500 agents have been tested in the SHE cell transformation assay with an 80-90% correlation between MT and carcinogenic potential. As such, the SHE cell transformation assay has utility as a test to provide short-term information on the carcinogenic potential of chemicals. One class of agents of current interest with regard to SHE cell transformation assay utilization consists of growth and differentiation factors (GDFs). Analysis of the SHE cell transformation potential of the GDFs, epidermal growth factor (EGF), fibroblast growth factor 4 (FGF-4), platelet-derived growth factor AA (PDGF AA), PDGF AB, PDGF BB, and the antimitogenic GDF, transforming growth factor beta one (TGF-beta1), was performed. All GDFs, with the exception of TGF-beta1, induced SHE cell transformation. However, an interesting difference between the GDFs was observed--PDGF A/B and PDGF B/B, but not PDGF A/A, EGF, or FGF-4, induced transformation after both a transient 1-day exposure and a continuous 7-day exposure, while continuous 7-day exposure was required for transformation by PDGF A/A, EGF, and FGF-4. Interestingly, both transient 1-day and continuous 7-day TGF-beta1 exposure resulted in suppression of transformation induced by a variety of transforming agents including growth factors, Ames assay-positive carcinogens, Ames assay-negative carcinogens, and spontaneous transformation. Interestingly TGF-beta1 was not able to suppress transformation by the tumor promoter, TPA. Together, these data demonstrate the utility of the Syrian hamster embryo cell transformation system for analyzing the transforming potential of GDFs and for characterizing differences in transforming mechanisms between different GDFs.

Assessing the predictiveness of the Syrian hamster embryo cell transformation assay for determining the rodent carcinogenic potential of single ring aromatic/nitroaromatic amine compounds.

The pH 6.7 Syrian hamster embryo (SHE) cell transformation assay was used to test the morphological transformation potential of 5 rodent carcinogenic single ring aromatic/nitroaromatic amine compounds: 2-amino-4-nitrotoluene, 2,4-diaminotoluene, 2,4-dinitrotoluene, o-anisidine hydrochloride, and o-toluidine; and 5 noncarcinogenic single ring aromatic/nitroaromatic amine compounds: 2,6-diaminotoluene, 2,4-dimethoxyaniline hydrochloride, 4-nitro-o-phenylenediamine, p-phenylenediamine dihydrochloride, and HC Blue No. 2. All 5 rodent carcinogens produced significant morphological transformation in a dose-responsive manner. None of the 5 noncarcinogens yielded significant transformation at any of the doses tested. Therefore, the concordance between the pH 6.7 SHE cell transformation assay and rodent carcinogenicity for these 10 single ring aromatic/nitroaromatic amine compounds is 100%. In contrast, the concordance between the standard SHE cell transformation assay and rodent carcinogenicity for 13 single ring aromatic/nitroaromatic amine compounds was 62%. For 5 aromatic/nitroaromatic amine compounds which were tested in both standard and pH 6.7 SHE cell transformation assays (i.e., a subset of the above two databases), the concordance between the standard SHE cell transformation assay and the rodent bioassay was 40%, while the concordance between the pH 6.7 SHE cell transformation assay and the rodent bioassay was 100%. This relatively high concordance between the pH 6.7 SHE cell transformation assay and rodent bioassay results demonstrates the utility of the pH 6.7 SHE cell transformation assay for predicting the rodent carcinogenic potential of single ring aromatic/nitroaromatic amine compounds.

Purification of genomic sized herpesvirus DNA using pulse-field electrophoresis.

A new method for the separation of genomic sized herpesvirus DNA was developed. This method utilizes the improved separation of extremely high molecular weight DNA molecules by pulse-field electrophoresis. This method has allowed us to separate and purify herpesvirus DNA (greater than 100 kbp) free of cellular DNA, directly from infected cells. In the case of cell associated herpesviruses (such as Marek's disease herpesvirus), this method is more efficient than present isolation procedures. The DNA obtained is able to be restriction enzyme digested and used in cloning experiments.

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.

Comparison of the standard and reduced pH Syrian hamster embryo (SHE) cell in vitro transformation assays in predicting the carcinogenic potential of chemicals.

A comprehensive review of the Syrian Hamster Embryo (SHE) cell transformation literature was performed in order to catalogue the chemical/physical entities which have been evaluated for in vitro cell transformation potential. Both reduced pH (pH 6.7) and standard pH (pH 7.1-7.3) SHE cell testing protocols were considered. Based upon this analysis, over 472 individual chemical/physical agents and 182 combinations of chemical/physical agents have been tested under the standard pH conditions, while over 56 chemical/physical agents have been tested under reduced pH conditions. Of the 472 chemical/physical agents tested at the standard pH, 213 had in vivo carcinogenicity data available. Of these 213 chemical/physical agents, 177 were carcinogens while 36 were non-carcinogens. The results of testing the SHE transformability of these 213 chemical/physical agents indicates that the standard pH SHE cell transformation assay had a concordance of 80% (171/213), a sensitivity of 82% (146/177), and a specificity of 69% (25/36). Of these 213 chemical/physical agents, 53% (112/213) were tested more than once often in more than one laboratory, with a 82% (92/112) interlaboratory agreement rate, thus providing confirmatory results. Carcinogenicity data were available for 48 of the 56 chemical/physical agents tested for SHE cell transformation under the reduced pH conditions. The SHE cell transformation assay under reduced pH conditions had a concordance of 85% (41/48), a sensitivity of 87% (26/30), and a specificity of 83% (15/18). For Salmonella-negative carcinogens, the standard pH SHE assay correctly predicted carcinogenicity 75% (48/64) of the time while the reduced pH SHE assay correctly predicted carcinogenicity for Salmonella-negative carcinogens 78% (14/18) of the time. For chemical/physical agents tested under both the reduced pH and standard pH conditions, the standard pH and reduced pH SHE cell assays had a 69% (22/32) agreement rate. Under the reduced pH conditions, the SHE assay correctly predicted rodent carcinogenicity in 86% (25/29) of the chemicals tested under both reduced and standard pH conditions. Under standard pH conditions, the SHE assay correctly predicted rodent carcinogenicity in 69% (20/29) of the chemicals tested under both reduced and standard pH conditions. Collectively, these data indicate that the SHE cell transformation assay is predictive for rodent carcinogenicity under either reduced or standard pH conditions. Importantly, the assay displays better performance and appears to have improved carcinogen prediction capability under reduced pH conditions.

The low pH Syrian hamster embryo (SHE) cell transformation assay: a revitalized role in carcinogen prediction.

A series of publications of the results of National Toxicology Program (NTP) studies (Tennant et al. (1987) Science, 236, 933-941; Haseman et al. (1990) J. Am. Stat. Assoc., 85, 964-971; Shelby et al. (1993) Environ. Mol. Mutagen., 21, 160-179) show that the commonly used short-term genotoxicity tests are less predictive of rodent carcinogenicity than once thought. These results have fueled a great deal of debate in the field of genetic toxicology regarding appropriate strategies for assessing the potential carcinogenicity of chemicals. The debate has continued in the recent discussion of harmonized genotoxicity test strategies (Ashby (1993) Mutation Res., 298, 291-295 and Ashby (1994) 308, 113-114; Madle (1993) Mutation Res., 300, 73-76 and Madle (1994) 308, 111-112; Zeiger (1994) Mutation Res., 304, 309-314) since the underlying problem still has not been resolved. The underlying problem is the fact that the current short-term genotoxicity tests in any combination do not provide both the necessary high sensitivity and high specificity needed for accurate rodent carcinogen detection. In this discussion, we describe the utility of the newly revised Syrian hamster embryo (SHE) cell transformation assay alone and in combination with the Salmonella mutation assay for improved accuracy of screening of rodent carcinogens relative to standard short-term genotoxicity tests. The accompanying papers provide details of improved methodologies for the conduct of the SHE cell transformation assay and an extensive review of the databases which support our conclusion that the SHE cell transformation assay provides an improved prediction of rodent bioassay results relative to other in vitro genotoxicity test batteries.

A comprehensive protocol for conducting the Syrian hamster embryo cell transformation assay at pH 6.70.

Studies from our laboratory have demonstrated several advantages of conducting the Syrian hamster embryo (SHE) cell transformation assay at pH 6.70 compared to that done historically at higher pH values (7.10-7.35). These include reduction of the influence of SHE cell isolates and fetal bovine serum lot variability on the assay, an increase in the frequency of chemically induced morphological transformation (MT) compared to controls, and an increased ease in scoring the MT phenotype. The purpose of this paper is to report a comprehensive protocol for conduct of the pH 6.70 SHE transformation assay including experimental procedures, a description of criteria for an acceptable assay and statistical procedures for establishing treatment-related effects. We have also identified several assay parameters in addition to pH which can affect transformation frequencies, particularly the critical role colony number per plate can have on transformation frequency. Control of this parameter, for which details are provided, can greatly increase the reproducibility and predictive value of the assay.

Induction of micronuclei in Syrian hamster embryo cells: comparison to results in the SHE cell transformation assay for National Toxicology Program test chemicals.

Sixteen chemicals currently being tested in National Toxicology Program (NTP) carcinogenicity studies were evaluated in the Syrian hamster embryo (SHE) cell in vitro micronucleus assay. Results from these studies were compared to the results from the SHE cell transformation assay for the same chemicals The overall concordance between induction of micronuclei and transformation of SHE cells was 56%, which is far lower that the 93% concordance between these two tests reported previously by Fritzenschaf et al. (1993; Mutation Res. 319, 47-53). The difference between our results appears to be due to differences in the types of chemicals in the two studies. Overall, there is good agreement between the SHE cell micronucleus and transformation assays for mutagenic chemicals, but, as our study highlights, the SHE cell transformation assay has the added utility of detecting nonmutagenic carcinogens. The utility of a multi-endpoint assessment in SHE cells for carcinogen screening is discussed.

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.

Establishment of immortalized alveolar type II epithelial cell lines from adult rats.

We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37 degrees C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham's F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial-derived oxidants and quartz activation of chemokine gene expression.

Macrophage inflammatory protein 2 (MIP-2) is a chemotactic cytokine which mediates neutrophil recruitment in the lung and other tissues. Pneumotoxic particles such as quartz increase MIP-2 expression in rat lung and rat alveolar type II epithelial cells. Deletion mutant analysis of the rat MIP-2 promoter demonstrated quartz-induction depended on a single NFkappaB consensus binding site. Quartz activation of NFkappaB and MIP-2 gene expression in RLE-6TN cells was inhibited by anti-oxidants suggesting the responses were dependent on oxidative stress. Consistent with anti-oxidant effects, quartz was demonstrated to increase RLE-6TN cell production of hydrogen peroxide. Rotenone treatment of RLE-6TN cells attenuated hydrogen peroxide production, NFkappaB activation and MIP-2 gene expression induced by quartz indicating that mitochondria-derived oxidants were contributing to these responses. Collectively, these findings indicate that quartz and crocidolite induction of MIP-2 gene expression in rat alveolar type II cells results from stimulation of an intracellular signaling pathway involving increased generation of hydrogen peroxide by mitochondria and subsequent activation of NFkappaB.

Skin stem cell hypotheses and long term clone survival--explored using agent-based modelling.

Epithelial renewal in skin is achieved by the constant turnover and differentiation of keratinocytes. Three popular hypotheses have been proposed to explain basal keratinocyte regeneration and epidermal homeostasis: 1) asymmetric division (stem-transit amplifying cell); 2) populational asymmetry (progenitor cell with stochastic fate); and 3) populational asymmetry with stem cells. In this study, we investigated lineage dynamics using these hypotheses with a 3D agent-based model of the epidermis. The model simulated the growth and maintenance of the epidermis over three years. The offspring of each proliferative cell was traced. While all lineages were preserved in asymmetric division, the vast majority were lost when assuming populational asymmetry. The third hypothesis provided the most reliable mechanism for self-renewal by preserving genetic heterogeneity in quiescent stem cells, and also inherent mechanisms for skin ageing and the accumulation of genetic mutation.