Hyperthermia induces expression of transforming growth factor-beta s in rat cardiac cells in vitro and in vivo.

Hyperthermia causes changes in expression of TGF-beta mRNA and protein in cultured cardiac cells, as well as in the heart in vivo. 12 h after hyperthermia, primary cultures of neonatal rat cardiomyocytes show a two- to threefold decreased expression of TGF-beta mRNAs which returns to control levels by 48 h after heat shock. In cultures of cardiac fibroblasts, expression of TGF-beta mRNAs increases 5-25-fold, 12-48 h after heat shock, while fetal bovine heart endothelial cells show little change in TGF-beta expression after hyperthermia. In each case, mRNAs for TGF-beta s 1, 2, and 3 are regulated similarly. Hearts isolated from rats exposed to hyperthermia show an initial 20-fold decrease in TGF-beta 1 and 3 mRNA levels which return to control levels by 24 h and subsequently are elevated two- to threefold above normal 48-72 h after heat shock; there is little change in TGF-beta 2 mRNA. Expression of immunoreactive TGF-beta 1 and 3 protein, localized intracellularly in myocytes, follows the same pattern as the mRNA expression. By 72 h, some myocytes show hyperstaining for TGF-beta 1. Staining for extracellular TGF-beta 1/3 exhibits the opposite time course, being most intense 3-6 h after heat shock and returning to control levels by 48 h. The increase in TGF-beta s after hyperthermia could play a role in mediating the reported cardioprotective effects of heat shock.

Recombinant latent transforming growth factor beta 1 has a longer plasma half-life in rats than active transforming growth factor beta 1, and a different tissue distribution.

Transforming growth factor beta 1 (TGF-beta 1) is a key regulator of cell growth and differentiation. Under normal physiological conditions, it is made as a biologically latent complex whose significance is unknown. Previous work has indicated that active TGF-beta 1 has a very short plasma half-life in rats (Coffey, R. J., L. J. Kost, R. M. Lyons, H. L. Moses, and N. F. La-Russo. 1987. J. Clin. Invest. 80:750-757). We have investigated the possibility that latent complex formation may extend the plasma half-life of TGF-beta 1 and alter its organ distribution. Radiolabeled latent TGF-beta 1 was formed by noncovalent association of 125I-TGF-beta 1 with the TGF-beta 1 precursor "pro" region from recombinant sources. TGF-beta 1 in this latent complex had a greatly extended plasma half-life (greater than 100 min) in rats compared with active TGF-beta 1 (2-3 min). Whereas active TGF-beta 1 was rapidly taken up by the liver, kidneys, lungs, and spleen and degraded, TGF-beta 1 in the latent complex was largely confined to the circulation, and was less than 5% degraded after 90 min. The pharmacokinetics of TGF-beta 1 in the latent complex were shown to be critically dependent on the degree of sialylation of the complex. The results suggest that formation of latent complexes may switch endogenous TGF-beta 1 from an autocrine/paracrine mode of action to a more endocrine mode involving target organs distant from the site of synthesis.

Role of transforming growth factor-beta in maintenance of function of cultured neonatal cardiac myocytes. Autocrine action and reversal of damaging effects of interleukin-1.

The three isoforms of transforming growth factor-beta (TGF-beta) have previously been implicated in embryonic development of the heart as well as in repair of myocardial damage after ischemia/reperfusion injury. TGF-beta 1 has also been localized intracellularly to both mitochondria and contractile filaments of cardiac myocytes, although its role in these structures has not been defined. We now report that exogenous TGF-beta stabilizes the beating rate of neonatal rat cardiac myocytes cultured on fibroblast matrix, and sustains their spontaneous rhythmic beating in serum-free medium. Moreover, using blocking antibodies to TGF-beta, we show that endogenous TGF-beta secreted by these myocytes acts in an autocrine fashion to maintain their beating rate. In contrast, IL-1 beta, an inflammatory mediator secreted by immune cells during myocardial injury, inhibits the beating of cardiac myocytes, and TGF-beta can overcome this inhibition. The antagonistic effects of TGF-beta and IL-1 were not observed when the myocytes were cultured on gelatin, as compared to native fibroblast matrix. The data indicate that TGF-beta is an important regulator of contractile function of the heart and have significant implications for understanding cardiac physiology in health and disease.

Overexpression of transforming growth factor-beta in transgenic mice carrying the human T-cell lymphotropic virus type I tax gene.

Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia as well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-cell leukemia patients express high levels of the type 1 isoform of transforming growth factor-beta (TGF-beta 1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-beta 1 promoter. To understand further the regulation of TGF-beta 1 expression by Tax, we examined its expression in transgenic mice carrying the HTLV-I tax gene. We show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-beta 1 mRNA and protein. Moreover, TGF-beta 1 significantly stimulated the incorporation of tritiated thymidine into one of three cell lines derived from neurofibromas of tax-transgenic mice, which suggests that the excessive production of TGF-beta 1 may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-beta in vivo.

Expression of sulfated glycoprotein 2 is associated with carcinogenesis induced by N-nitroso-N-methylurea in rat prostate and seminal vesicle.

To understand the molecular mechanism of carcinogenesis in androgen-dependent tumors, we have searched for new markers which are associated with this process. In normal rat prostate and seminal vesicle, sulfated glycoprotein 2 (SGP-2) messenger RNA is barely detectable. However, we have found high levels of SGP-2 expression in the epithelial component of carcinomas of the prostate and seminal vesicle after initiation with N-nitroso-N-methylurea and promotion with testosterone propionate. We have also observed induction of SGP-2 expression in epithelial cells at early stages in carcinogenesis when cytologically malignant cells first begin to appear. SGP-2 has been reported previously to be associated with a variety of models of programmed cell death (apoptosis), including the prostate following castration. Our present findings provide a novel marker for carcinogenesis in the rat prostate and seminal vesicle.

A prospective trial of telepathology for intraoperative consultation (frozen sections).

Telepathology is a maturing technology that, for a variety of reasons, has not been widely deployed. In addition, clinical validation is relatively modest compared with accepted telemedicine applications such as teleradiology. A prototype telepathology system (Tele-Path(sm)) featuring high-resolution images selected from a remote microscope site has been developed at the University of Alabama at Birmingham (UAB). To validate the diagnostic efficacy of the system, a prospective study was undertaken of parallel diagnoses by conventional microscopy and telepathology with a remotely operated microscope. Slides from 99 intraoperative consultations from 29 tissue/ organ sites in the University of Alabama Hospitals by 9 academic pathologists were used in the study. Each microscopic and telepathology diagnosis was compared with the final diagnosis rendered by a referee pathologist. Diagnoses were classified as correct, false positive, or false negative or classification error. Of the 99 frozen sections evaluated, 3 cases were deferred. Of the remaining 96 cases, 2 received incorrect diagnoses in both the microscopic and telepathology arms of the study. Three errors occurred only in the telepathology arm. There was 1 false-positive diagnosis, 1 false-negative diagnosis, and 1 classification error. Statistical analysis indicated no significant difference between telepathology and conventional microscopy. Qualitative data indicated that the pathologists were generally satisfied with the performance of the system. Telepathology using this system paradigm is sufficiently accurate for real time utilization in a complex surgical environment. Telepathology therefore may be an effective model to support the surgical services of hospitals lacking full-time pathology coverage, resulting in full-time access to anatomic pathology services.

Cardiomyopathy in a carrier of Duchenne's muscular dystrophy.

During the third trimester of her pregnancy, a 25-year-old carrier of Duchenne's muscular dystrophy developed severe cardiac failure and required mechanical circulatory support and transplantation. Her cardiac function improved during 311 days of circulatory support. However this improvement was not sufficient to allow removal of her left ventricular assist device before transplantation.

An initial trial of a prototype telepathology system featuring static imaging with discrete control of the remote microscope.

Routine diagnosis of pathology images transmitted over telecommunications lines remains an elusive goal. Part of the resistance stems from the difficulty of enabling image selection by the remote pathologist. To address this problem, a telepathology microscope system (TelePath, TeleMedicine Solutions, Birmingham, Ala) that has features associated with static and dynamic imaging systems was constructed. Features of the system include near real time image transmission, provision of a tiled overview image, free choice of any fields at any desired optical magnification, and automated tracking of the pathologist's image selection. All commands and images are discrete, avoiding many inherent problems of full motion video and continuous remote control. A set of 64 slides was reviewed by 3 pathologists in a simulated frozen section environment. Each pathologist provided diagnoses for all 64 slides, as well as qualitative information about the system. Thirty-one of 192 diagnoses disagreed with the reference diagnosis that had been reached before the trial began. Qf the 31, 13 were deferrals and 12 were diagnoses of cases that had a deferral as the reference diagnosis. In 6 cases, the diagnosis disagreed with the reference diagnosis yielding an overall accuracy of 96.9%. Confidence levels in the diagnoses were high. This trial suggests that this system provides high-quality anatomic pathology services, including intraoperative diagnoses, over telecommunications lines.

Immunofluorescent analysis of gamma-glutamyl transpeptidase and glutathione-S-transferase-P during the initial phase of experimental hepatocarcinogenesis.

We used double-label immunofluorescence to evaluate the expression of glutathione-S-transferase-P (GST-P) and gamma-glutamyl transferase (gamma GT) in individual liver cells in rats placed on a modified Solt-Farber hepatocarcinogenesis protocol. Four days after treatment with diethylnitrosamine (DEN), single GST-P+ cells were found in the centrilobular and midzonal regions. Most of these were gamma GT-, but the percentage of gamma GT+ cells increased with DEN dose to a high of 32%. At later times (7-8 days) doublets and small clusters of GST-P+ cells predominated; all cells in each pair or cluster displayed the same phenotype (GST-P+ or GST-P+/gamma GT+). Following administration of acetylaminofluorene and partial hepatectomy, lesions were larger and a greater percentage were GST-P+/gamma GT+. However, within these lesions individual cells displayed different phenotypes. These results indicate that the expression of GST-P and gamma GT is discordant in individual cells and small clusters soon after treatment, and suggest that different molecular events may be involved in their expression.

Regional synthesis of monohydroxy eicosanoids by the kidney.

Arachidonic acid was incubated with 10,000 X g and 100,000 X g supernatants prepared from the medullae of hydronephrotic, normal, and contralateral rabbit kidneys. Metabolites whose production was calcium dependent but not inhibited by indomethacin were identified by thin layer chromatography. Preparative separation was carried out by silicic acid high pressure liquid chromatography. The methyl ester trimethylsilyl derivatives were prepared and examined by gas chromatography-mass spectrometry. The mass spectra matched previously published mass spectra for 12-hydroxy-5,8,10,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. The renal cortex does not metabolize arachidonic acid through the lipoxygenase pathway. Thus, there is regional metabolism of arachidonate to monohydroxy eicosanoids in the kidney.

Case report: pineocytoma in a male Fischer 344 rat.

An enlarged pineal gland was observed in a 112-wk-old male Fischer 344 rat from the low-dose treatment group in a 2-yr bioassay. Formalin-fixed, paraffin-embedded sections of the gland were stained with hematoxylin and eosin along with the immunohistochemical biomarkers synaptophysin, placental alkaline phosphatase, glial fibrillary acidic protein, and vimentin. Based on its histomorphological features and on positive staining with synaptophysin, the lesion was diagnosed as a malignant pineal gland parenchymal cell tumor or pineocytoma of incidental origin.

Transforming growth factor-betas block cytokine induction of catalase and xanthine oxidase mRNA levels in cultured rat cardiac cells.

We examined the effects of transforming growth factor-beta (TGF-beta) on the mRNA expression of the antioxidative enzymes, catalase, manganese superoxide dismutase (MnSOD), and copper-zinc superoxide dismutase (CuZnSOD), as well as the oxidative enzyme, xanthine oxidase (XO), in cultures of cardiomyocytes, cardiac non-myocytes, and fetal bovine heart endothelial cells. TGF-betas alone had little effect on expression of these enzymes, but treatment with a combination of interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha increased expression of MnSOD, catalase, and XO in some cell types with little effect on CuZnSOD expression. When TGF-betas were added along with these inflammatory cytokines there was a return to control levels of catalase expression, as well as a dramatic reduction in XO expression. In fetal bovine heart endothelial cells, treatment with inflammatory cytokines increased XO mRNA expression 11.5-fold and inclusion of TGF-betas reduced this 4-5-fold: effects on XO enzyme activity paralleled those seen on mRNA expression. Similar changes in XO expression were seen in cardiomyocytes. In contrast, TGF-betas did not change cytokine-induced MnSOD expression. All three mammalian isoforms of TGF-beta showed similar effects. In summary, TGF-betas may be able to decrease superoxide anion production and subsequent tissue damage by decreasing levels of XO.

Autoinduction of mRNA and protein expression for transforming growth factor-beta S in cultured cardiac cells.

Although transforming growth factor-beta s (TGF-beta s) are expressed widely in both adult and embryonic rat heart, both mRNA and protein expression increase following ischemic injury. Furthermore, exogenous administration of TGF-beta decreases cardiac damage following ischemia-reperfusion in rats. We have found that treatment of primary cultures of neonatal rat cardiomyocytes or cardiac fibroblasts with TGF-beta 1, 2, or 3 results in increased expression of TGF-beta 1, 2, and 3 mRNA. TGF-beta 2 was generally the least effective isoform in inducing TGF-beta expression. In cardiac fibroblasts mRNA expression of all TGF-beta s increased 2-3-fold following 1 h of treatment and decreased to control levels by 8 h which was accompanied by a 2.5- and 2.3-fold increase in TGF-beta 1 and 2 protein secretion, respectively. By 48 h of treatment mRNA levels for TGF-beta s 2 and 3 were less than 10% of control levels. In cardiomyocytes two-five-fold increases in mRNA levels were observed following 1-24 h of TGF-beta 1 treatment, but TGF-beta 1 and 3 mRNA levels returned to control values by 48 h while TGF-beta 2 mRNA expression remained elevated. TGF-beta 1 and 2 protein secreted by the cardiac myocytes was increased 2.9- and 1.7-fold, respectively. Autoinduction of TGF-beta s may play a beneficial role in cardiac wound healing by sustaining transient increases in TGF-beta levels from either endogenous synthesis or exogenous application.

Differential regulation of the expression of transforming growth factor-beta s 1 and 2 by retinoic acid, epidermal growth factor, and dexamethasone in NRK-49F and A549 cells.

Although most biological activities of transforming growth factor-beta s 1 and 2 (TGF-beta 1 and TGF-beta 2) examined in vitro are similar or identical, recent studies suggest that each of these factors may be independently regulated in vivo. In this study we have used highly sensitive and specific sandwich enzyme-linked immunosorbent assays for TGF-beta 1 and TGF-beta 2 to examine the effects of a variety of treatments on expression of these two TGF-beta isoforms. We show that epidermal growth factor (EGF) induces secretion of TGF-beta 1 and not TGF-beta 2, whereas retinoic acid (RA) induces secretion of TGF-beta 2 and not TGF-beta 1 in NRK-49F normal rat kidney fibroblasts and A549 human lung carcinoma cells. Moreover, treatment with EGF diminishes the levels of TGF-beta 2, while RA decreases the levels of TGF-beta 1 in both cell lines. Dexamethasone (Dex), on the other hand, inhibits the secretion of both TGF-beta 1 and TGF-beta 2 in A549 cells, while selectively inhibiting TGF-beta 1 secretion in NRK-49F cells. The interactive effects of EGF, RA, and Dex on the production of TGF-beta 1 and TGF-beta 2, which were studied on NRK-49F cells, demonstrate that EGF blocks the induction of TGF-beta 2 mRNA and peptide by RA, while Dex inhibits the induction of TGF-beta 1 mRNA and peptide by EGF. These results demonstrate that RA, EGF and Dex are each unique, differential, and interactive regulators of the expression of TGF-beta s 1 and 2.

Addition of a C-terminal extension sequence to transforming growth factor-beta 1 interferes with biosynthetic processing and abolishes biological activity.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized and secreted as a biologically latent complex. It has been proposed that one role of the latent complex is to prevent premature interaction of ligand and receptor intracellularly during biosynthesis (Wakefield et al., J. Cell Biol. (1987) 105, 965-975). To test this hypothesis, the endoplasmic reticulum retention sequence Lys-Asp-Glu-Leu (KDEL) was added to the C-terminus of the wildtype TGF-beta 1 coding sequence, and to a construct in which mutagenesis of two cysteine residues in the precursor pro region results in the synthesis and secretion of active, as opposed to latent, TGF-beta. Addition of either SEKDEL, or the control sequence SEKDVS to the TGF-beta 1 protein abolished biological activity. Western blot analysis indicated that the extended gene products are synthesized, but that the extension sequence partially interferes with the normal dimerization of the protein product, and totally inhibits the normal proteolytic processing and glycosylation of the precursor protein. The data suggest that correct folding of the highly conserved C terminus of TGF-beta 1 is critical for subsequent proteolytic cleavage and glycosylation at sites that are quite distant in the primary sequence. Thus molecular strategies for the generation of TGF-beta antagonists or superagonists should avoid extensive modification of this region of the molecule. Since synthesis of the endogenous TGF-beta 1 is unaffected by the presence of the mutated analog, the data further indicate that transfection with the KDEL-extended TGF-beta 1 sequence cannot be used as a dominant negative mutation to prevent secretion of the endogenous TGF-beta protein.

Evaluation of DNA ploidy in dysplastic and Spitz nevi by flow cytometry.

As many as 40% of all primary cutaneous melanomas can have histologic remnants of nevomelanocytic nevi adjacent to the tumor. There is increasing evidence that dysplastic nevi are at least a clinical marker for melanoma risk. Spitz nevi are not known for such an association, but are noteworthy because of their histologic appearances. Spitz and dysplastic nevi were studied by flow cytometry to search for DNA abnormality. The study material consisted of formalin-fixed, paraffin embedded material of 41 dysplastic nevi and 14 Spitz nevi. Four cases of dysplastic nevi were excluded for technical reasons. Of the 37 interpretable histograms from dysplastic nevi, 28 (76%) were diploid and nine (24%) were aneuploid. All the Spitz nevi were diploid. Thus, dysplastic nevi, but not Spitz nevi, share aneuploidy features in some cases with melanoma. Previous authors have demonstrated aneuploidy in melanoma with aggressive behavior and in those in deep vertical growth phase. Aneuploidy may be a feature of early as well as late stages of tumor progression regarding the nevomelanocyte system.

In vitro and in vivo regulation of liver epithelial cells carrying a metallothionein-rasT24 fusion gene.

We inserted a zinc-responsive metallothionein-rasT24 fusion gene in both orientations into a retroviral vector (SVX) and infected Fisher rat liver epithelial cells. Only the construction in which the viral long terminal repeat and the metallothionein promoters were in opposite orientations resulted in cell lines with biologically altered behavior. Two lines (MTR-1 and MTR-6) showed altered morphology in vitro when ZnSO4 was added to the medium. These cell lines grew in soft agarose in a dose-dependent manner. Immunoprecipitation experiments revealed dose-dependent increases in the rate of synthesis of the mutant p21Ha-ras protein in these lines in response to ZnSO4. Both lines produced poorly differentiated metastatic adenocarcinomas when injected subcutaneously in Fisher rats, and tumors derived from MTR-6 cells grew more rapidly in animals on a zinc-supplemented diet than on a zinc-deficient diet. Uninfected liver epithelial cells showed no change in morphology in vitro after ZnSO4 addition and did not grow in soft agarose or after subcutaneous transplantation during the 14-wk experimental period. These results indicate that altered levels of expression of a single gene (rasT24) can have profound effect on the biologic behavior of tumor cells both in vitro and in vivo.

MTrasT24, a metallothionein-ras fusion gene, modulates expression in cultured rat liver cells of two genes associated with in vivo liver cancer.

We studied the effects of a zinc-inducible metallothionein-ras fusion gene (MTrasT24) in cultured rat liver epithelial (RLE) cells on expression of two genes induced during liver carcinogenesis in vivo: gamma-glutamyltransferase [(5-glutamyl)-peptide:amino acid 5-glutamyltransferase, EC 2.3.2.2] and glutathione S-transferase-P (RX:glutathione R-transferase, EC 2.5.1.18). Expression of MTrasT24 increased steady-state RNA levels of gamma-glutamyltransferase and glutathione transferase-P 6- to 100-fold and 1.6- to 6-fold, respectively; in contrast, levels of alpha-tubulin RNA fell slightly or were unchanged. RNA gel blots verified that gamma-glutamyltransferase and glutathione transferase-P RNAs were of the appropriate size, and results from immunocytochemistry on transfected cells demonstrated that RLE cells carrying MTrasT24 synthesized immunoreactive, appropriately localized gamma-glutamyltransferase and glutathione transferase-P. Zinc induction studies indicated that gamma-glutamyltransferase and glutathione transferase-P RNA levels were directly dependent on MTrasT24 RNA levels. These data suggest that expression of gamma-glutamyltransferase and glutathione transferase-P expression are part of a reorientation of cellular gene expression during carcinogenesis and that activated ras expression, like chemical carcinogens, can bring about this change.

Expression of transforming growth factor-beta s 1-4 in chicken embryo chondrocytes and myocytes.

cDNA probes and antibodies for TGF-beta s 1, 2, 3, and 4 were used to study the expression of these different TGF-beta isoforms in cultured chicken embryo chondrocytes and cardiac myocytes, as well as in developing cartilage and heart tissues. TGF-beta s 2, 3, and 4 mRNAs, but not TGF-beta 1 mRNA, were detected in cultured chondrocytes and myocytes. Expression of TGF-beta s 2 and 4 mRNAs increased with age, while expression of TGF-beta 3 mRNA was independent of age in chondrocytes cultured from 12- to 17-day-old embryos. In contrast, expression of TGF-beta s 2, 3, and 4 mRNAs was constitutive in myocytes cultured from 7- to 9-day-old embryonic hearts; expression of TGF-beta s 3 and 4 mRNAs increased, while expression of TGF-beta 2 mRNA remained unchanged in myocytes from 10-day-old embryos. Immunoprecipitation studies demonstrated expression of TGF-beta in both the conditioned media and the cell lysates of metabolically labeled chondrocyte and myocyte cell cultures. Immunohistochemical staining of cultured chondrocytes and myocytes and of cartilage and heart tissues of developing chicken embryos with antibodies specific for each TGF-beta isoform showed immunoreactive TGF-beta s 1, 2, 3, and 4. Our results demonstrate coordinate expression of these four TGF-beta isoforms in chicken embryo chondrocytes and myocytes, both in vitro and in vivo, with expression of TGF-beta s 2, 3, and 4 mRNA and protein more prominent than that of TGF-beta 1.

Inhibition of protein phosphatases blocks myogenesis by first altering MyoD binding activity.

To examine the role of protein phosphatases in skeletal muscle differentiation, C2C12 myoblasts were treated with okadaic acid, a potent in vitro inhibitor of protein phosphatases 1 and 2A which regulate various cellular events in intact cells. We now show that okadaic acid treatment of the mouse myoblast C2C12 cell line reversibly altered the morphology of the cells and blocked differentiation. At a molecular level, it extinguished expression of the myogenic determination genes, MyoD1 and myogenin, but induced the expression of an inhibitor of differentiation, Id. Analysis of the MyoD1 promoter showed that inhibition of MyoD1 expression by okadaic acid occurs at the transcriptional level. These changes occur 10-20 h after okadaic acid treatment. However, within 1 h of treatment the ability of muscle extracts to support a specific MyoD-dependent gel mobility shift using a MyoD DNA binding site is lost. These data suggest that protein phosphatases play an important role during myogenic differentiation.