Robert J. Isfort

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.

Skeletal muscle hypertrophy and anti-atrophy effects of clenbuterol are mediated by the β2-adrenergic receptor

Analyses were performed to evaluate the roles of the β1‐ and β2‐adrenergic receptors in the skeletal muscle hypertrophy and anti‐atrophy response to the β‐adrenergic agonist, clenbuterol. Treatment of wild‐type mice with clenbuterol resulted in statistically significant hypertrophy of the innervated tibialis anterior and medial gastrocnemius muscles and inhibition of denervation‐induced atrophy of these muscles. Treatment of β1‐adenergic receptor knockout mice with clenbuterol also resulted in statistically significant hypertrophy of the innervated tibialis anterior and medial gastrocnemius muscles and inhibition of denervation‐induced atrophy of these muscles. In contrast, in β2‐adrenergic receptor knockout mice and in mice lacking both the β1‐ and β2‐adrenergic receptors, clenbuterol treatment did not result in hypertrophy of the innervated tibialis anterior and medial gastrocnemius muscles, nor did it inhibit denervation‐induced atrophy in these muscles. Together these data demonstrate that the β2‐adrenergic receptor is responsible for both the skeletal muscle hypertrophy and anti‐atrophy effects of the β‐adrenergic agonist clenbuterol.

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 chemicals 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

SummaryWe 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° 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. However, at later passages the 6T cells became polyploid, while the 6TN genotype remained stable. The RLE-6T and 6TN cells were not tumorigenic in nude mice. The cell isolation methods reported and the novel cell lines produced represent potentially useful tools to study the role of pulmonary epithelial cells in neoplastic and nonneoplastic lung disease.

Limb Immobilization Induces a Coordinate Down-Regulation of Mitochondrial and Other Metabolic Pathways in Men and Women

Advancements in animal models and cell culture techniques have been invaluable in the elucidation of the molecular mechanisms that regulate muscle atrophy. However, few studies have examined muscle atrophy in humans using modern experimental techniques. The purpose of this study was to examine changes in global gene transcription during immobilization-induced muscle atrophy in humans and then explore the effects of the most prominent transcriptional alterations on protein expression and function. Healthy men and women (N = 24) were subjected to two weeks of unilateral limb immobilization, with muscle biopsies obtained before, after 48 hours (48 H) and 14 days (14 D) of immobilization. Muscle cross sectional area (∼5%) and strength (10–20%) were significantly reduced in men and women (∼5% and 10–20%, respectively) after 14 D of immobilization. Micro-array analyses of total RNA extracted from biopsy samples at 48 H and 14 D uncovered 575 and 3,128 probes, respectively, which were significantly altered during immobilization. As a group, genes involved in mitochondrial bioenergetics and carbohydrate metabolism were predominant features at both 48 H and 14 D, with genes involved in protein synthesis and degradation significantly down-regulated and up-regulated, respectively, at 14 D of muscle atrophy. There was also a significant decrease in the protein content of mitochondrial cytochrome c oxidase, and the enzyme activity of cytochrome c oxidase and citrate synthase after 14 D of immobilization. Furthermore, protein ubiquitination was significantly increased at 48 H but not 14 D of immobilization. These results suggest that transcriptional and post-transcriptional suppression of mitochondrial processes is sustained throughout 14 D of immobilization, while protein ubiquitination plays an early but transient role in muscle atrophy following short-term immobilization in humans.

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.

Proteomic analysis of rat soleus muscle undergoing hindlimb suspension‐induced atrophy and reweighting hypertrophy

A proteomic analysis was performed comparing normal rat soleus muscle to soleus muscle that had undergone either 0.5, 1, 2, 4, 7, 10 and 14 days of hindlimb suspension‐induced atrophy or hindlimb suspension‐induced atrophied soleus muscle that had undergone 1 hour, 8 hour, 1 day, 2 day, 4 day and 7 days of reweighting‐induced hypertrophy. Muscle mass measurements demonstrated continual loss of soleus mass occurred throughout the 21 days of hindlimb suspension; following reweighting, atrophied soleus muscle mass increased dramatically between 8 hours and 1 day post reweighting. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 29 soleus proteins. Reweighting following atrophy demonstrated statistically significant changes in the relative levels of 15 soleus proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both atrophied and hypertrophied soleus muscle. Five differentially regulated proteins from the hindlimb suspended atrophied soleus muscle were identified while five proteins were identified in the reweighting‐induced hypertrophied soleus muscles. The identified proteins could be generally grouped together as metabolic proteins, chaperone proteins and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the skeletal muscle proteome occur during disuse‐induced soleus muscle atrophy and reweighting hypertrophy.

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.

Proteomic analysis of the atrophying rat soleus muscle following denervation

A proteomic analysis was performed comparing normal rat soleus muscle to denervated soleus muscle at 0.5, 1, 2, 4, 6, 8 and 10 days post denervation. Muscle mass measurements demonstrated that the times of major mass changes occurred between 2 and 4 days post denervation. Proteomic analysis of the denervated soleus muscle during the atrophy process demonstrated statistically significant (at the p < 0.01 level) changes in 73 soleus proteins, including coordinated changes in select groups of proteins. Sequence analysis of ten differentially regulated proteins identified metabolic proteins, chaperone and contractile apparatus proteins. Together these data indicate that coordinated temporally regulated changes in the proteome occur during denervation‐induced soleus muslce atrophy, including changes in muscle metabolism and contractile apparatus proteins.

Sex Differences in Global mRNA Content of Human Skeletal Muscle

Women oxidize more fat as compared to men during endurance exercise and several groups have shown that the mRNA content of selected genes related to fat oxidation are higher in women (e.g. hormone sensitive lipase, β-hydroxyacyl-CoA dehydrogenase, CD36). One of the possible mechanisms is that women tend to have a higher area percentage of type I skeletal muscle fibers as compared with men. Consequently, we hypothesized that sex would influence the basal mRNA and protein content for genes involved in metabolism and the determination of muscle fiber type. Muscle biopsies from the vastus lateralis were collected from healthy men and women. We examined mRNA content globally using Affymetrix GeneChips, and selected genes were examined and/or confirmed by RT-PCR. Furthermore, we examined protein content by Western blot analysis. Stringent gene array analysis revealed 66 differentially expressed genes representing metabolism, mitochondrial function, transport, protein biosynthesis, cell proliferation, signal transduction pathways, transcription and translation. Stringent gene array analysis and RT-PCR confirmed that mRNA for; acyl-coenzyme A acyltransferase 2 (ACAA2), trifunctional protein β (HADHB), catalase, lipoprotein lipase (LPL), and uncoupling protein-2 (UCP-2) were higher in women. Targeted gene analysis revealed that myosin heavy chain I (MHCI), peroxisome proliferator-activated receptor (PPAR)δ were higher in women compared with men. Surprisingly, there were no significant sex based differences in protein content for HADHB, ACAA2, catalase, PPARδ, and MHC1. In conclusion, the differences in the basal mRNA content in resting skeletal muscle suggest that men and women are transcriptionally “primed” for known physiological differences in metabolism however the mechanism behind sex differences in fiber type remains to be determined.