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The most common sarcomas in the thorax are metastasis from an extrathoracic primary malignancy. Primary intrathoracic sarcomas are rare albeit aggressive malignancies that are diagnosed on histopathology. Although a few imaging characteristics have been described that are common to sarcomas, it is still a diagnosis of exclusion as other tumors are much more common. Like elsewhere, primary thoracic sarcomas are also classified according to their histologic features. They are a rare group of tumors that can arise from the mediastinal structures, lung, pleura, or chest wall. On imaging, differentiating these from more common malignancies like lung cancer is difficult and often requires multimodality workup and tissue sampling. A few sarcomas are very specific to their locations, such as angiosarcoma in the right atrium, leiomyosarcoma in the pulmonary artery, where imaging has high accuracy for the diagnosis. Despite being nonspecific in a majority of cases, imaging plays a pivotal role in determining the organ of origin, tumor extent, invasion of adjacent structures, and thus help to assess the surgical resectability. Although sarcomas arising from chest wall are the most common primary sarcomas in the chest, they are excluded from this review to focus only on primary intrathoracic sarcomas. The article provides a comprehensive imaging and pathology review of the rare primary intrathoracic sarcomas, including but not limited to angiosarcoma, Kaposi sarcoma, fibrosarcoma, malignant transformation of fibrous tumor of pleura, sarcomatoid mesothelioma, leiomyosarcoma, and malignant small round blue cell tumors.Key points:Primary intrathoracic sarcomas are rare but clinically important.Imaging helps to determine local extent, invasion, metastases and appropriate site/mode of biopsy.Role of pathology is paramount in diagnosis and guiding treatment based on immunogenetic/molecular typing.

Background: With the increased use of molecular testing of thyroid fine-needle biopsies, the frequency and extent of thyroid resection for thyroid nodules has changed. Although the role of frozen-section analysis of the thyroid has been reduced markedly in recent years, many surgeons still routinely use it intraoperatively. We sought to determine the utility of frozen section during thyroidectomy in the era of molecular testing., Study Design: We reviewed 236 consecutive patients who had thyroidectomy with intraoperative frozen-section analysis at our institution between November 2015 and October 2017. We re-reviewed the preoperative diagnosis, frozen-section diagnosis, final pathology, and whether operative management changed from the initial plan based on frozen section., Results: Mean age of the patients was 55.6 ± 14.1 years, and 83% were female. Of the 236 patients, frozen section did not change the intraoperative management in 225 (95%). Of the 11 patients whose thyroid operation was modified, the operation was either too much or not enough in 6 patients. In only 5 (2.1%) patients, frozen-section analysis correctly changed the extent of thyroidectomy., Conclusions: Thyroid frozen-section analysis adds cost and time to thyroid operations without notable benefit. In our cohort, only 2.1% of frozen sections accurately changed intraoperative management. We recommend against its routine use., (Copyright © 2018 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: With rapid advances in genomic medicine, the complexity of delivering precision medicine to oncology patients across a university health system demanded the creation of a Molecular Tumor Board (MTB) for patient selection and assessment of treatment options. The objective of this report is to analyze our progress to date and discuss the importance of the MTB in the implementation of personalized medicine., Materials and Methods: Patients were reviewed in the MTB for appropriateness for comprehensive next generation sequencing (NGS) cancer gene set testing based on set criteria that were in place. Because profiling of stage IV lung cancer, colon cancer, and melanoma cancers were standard of care, these cancer types were excluded from this process. We subsequently analyzed the types of cases referred for testing and approved with regards to their results., Results: 191 cases were discussed at the MTB and 132 cases were approved for testing. Forty-six cases (34.8%) had driver mutations that were associated with an active targeted therapeutic agent, including BRAF, PIK3CA, IDH1, KRAS , and BRCA1 . An additional 56 cases (42.4%) had driver mutations previously reported in some type of cancer. Twenty-two cases (16.7%) did not have any clinically significant mutations. Eight cases did not yield adequate DNA. 15 cases were considered for targeted therapy, 13 of which received targeted therapy. One patient experienced a near complete response. Seven of 13 had stable disease or a partial response., Conclusions: MTB at University of Alabama-Birmingham is unique because it reviews the appropriateness of NGS testing for patients with recurrent cancer and serves as a forum to educate our physicians about the pathways of precision medicine. Our results suggest that our detection of actionable mutations may be higher due to our careful selection. The application of precision medicine and molecular genetic testing for cancer patients remains a continuous educational process for physicians., Competing Interests: CONFLICTS OF INTEREST None.

Background: The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports., Methods: Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges., Results: Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45-60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations., Conclusions: Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician-supervised resident/fellow programming projects as learning opportunities and workflow improvements in the molecular laboratory., Competing Interests: There are no conflicts of interest.

Pleomorphic hyalinizing angiectatic tumor of soft parts is a recently recognized, low-grade mesenchymal neoplasm of uncertain lineage. It is characterized by clusters of ectatic, fibrin-lined, thin-walled vessels surrounded by pleomorphic but mitotically inert spindled cells with frequent intranuclear inclusions and a variable inflammatory component. Here, we report a case of recurrent pleomorphic hyalinizing angiectatic tumor in a 37-year-old white woman. Touch imprint cytologic analysis revealed oval and spindled cells with fine chromatin, occasional prominent intranuclear inclusions, and intracytoplasmic pigment. The histologic features of the cells constituting the bulk of tumor were those characteristic of a putative precursor lesion ("early pleomorphic hyalinizing angiectatic tumor") as well as another recently described entity known as hemosiderotic fibrohistiocytic lipomatous lesion. Cytogenetic analysis revealed 2 unbalanced translocations involving chromosomes 1 and 3 and chromosomes 1 and 10, with a karyotype of 45,XX,der(1)t(1;3)(p31;q12),-3,der(10)t(1;10)(p31;q25)[11]/46,XX[4], thus suggesting that this disease is neoplastic. The controversies in classification of these lesions are also discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: The objective was to identify whether repeat positron emission tomography scan after neoadjuvant chemoradiotherapy in patients with esophageal cancer predicted a complete response., Methods: A retrospective study using a prospective database was performed. Patients had esophageal cancer and underwent neoadjuvant chemoradiotherapy, an initial and repeat positron emission tomography, endoscopic ultrasound with fine-needle aspiration (at the same institution), and Ivor Lewis esophagogastrectomy with lymph node resection., Results: There were 221 patients who underwent Ivor Lewis, 86 of whom had their initial and repeat positron emission tomography scans performed at the same center. Of these, 37 patients (43%) were complete responders. The median maximum standardized uptake value of esophageal cancer decreased by 72% in the 37 patients who were complete responders, by 58% in the 31 patients who were partial responders, and by 37% in the 18 patients who had a minimal pathologic response. When the maximum standardized uptake value decreased by more than 64%, the patient was likely to be a complete responder (P = .003, area under the curve = 0.75)., Conclusion: When initial and repeat positron emission tomography scans are performed at the same center at least 30 days after the completion of preoperative chemoradiotherapy, the percent change in the maximum standardized uptake value is a predictor of the response to chemoradiotherapy by a patient with esophageal cancer. When the maximum standardized uptake value decreases by 64% or more, it is likely that the patient is a complete responder. These data may help guide neoadjuvant therapy and identify patients for a future randomized study that compares observation with surgical resection in patients with esophageal cancer who appear to be complete responders.

Angiomyolipoma is a benign tumor composed of varying proportions of smooth muscle cells, blood vessels, and adipose tissue that most commonly occurs in the kidney. Sporadic lesions and lesions arising in the setting of the tuberous sclerosis complex have been reported in extrarenal sites. We present the case of an incidentally discovered angiomyolipoma in the anterior mediastinum. Thymoma was suspected clinically, and the lesion was composed mainly of spindled-to-epithelioid cells arranged in a histologic pattern reminiscent of hemangiopericytoma, a pattern that has been described in thymoma. Immunohistochemical stains revealed positivity for smooth muscle actin and HMB-45, revealing the expression of smooth muscle and melanocytic markers characteristic of angiomyolipoma and other lesions in the PEComa family.

This report describes the surgical management of a tumor that filled the left chest of a 58-year-old man. Histopathologic examination showed that this was an angiomyolipoma, a tumor that most commonly occurs in the kidney. The preoperative evaluation and intraoperative management are presented, along with a brief review of this unusual neoplasm.

This preliminary study demonstrates the superiority of live three-dimensional transthoracic echocardiography (3D TTE) over two-dimensional (2D) TTE in the assessment of left atrial (LA) tumors in four patients studied by us (three myxomas, one hemangioma, all subsequently pathologically proven). Because of the unique ability of live 3D TTE to systematically section and view the contents of an intracardiac mass, LA myxomas in the three patients studied could be more confidently diagnosed by noting isolated echolucent areas consistent with hemorrhage/necrosis in the tumor mass. On the other hand, a definite echolucent area was found by 2D TTE in only two of the three patients with myxoma. In the fourth patient with a hemangioma, live 3D TTE showed much more extensive and closely packed echolucencies with little solid tissue as compared to a myxoma consistent with a highly vascularized tumor. In contrast, 2D TTE demonstrated only two isolated echolucencies in the tumor suggesting an erroneous diagnosis of myxoma.

Background: Repeat positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) and chest computed tomography (CT) are used to assess the effectiveness of chemoradiotherapy in patients with non-small cell lung cancer (NSCLC); however, the change in the standardized uptake values (SUV) has not been correlated with the pathologic change of the primary tumor., Methods: This is a retrospective cohort study of a prospective database of 56 patients who had NSCLC, FDG-PET, and chest CT scans both before and after neoadjuvant therapy, followed by complete resection of their cancer. Maximum SUVs (maxSUV) and tumor size were measured, and the percentage of change was compared with the percentage of nonviable tumor cells. The primary objective was to measure the degree of correlation between these values., Results: The change in the maxSUV has a near linear relationship to the percent of nonviable tumor cells in the resected tumors. FDG-PET's maxSUV is better correlated to pathology than the change in size on CT scan (r2 = 0.75, r2 = 0.03, p < 0.001). When the maxSUV decreased by 80% or more, a complete pathologic response could be predicted with a sensitivity of 90%, specificity of 100%, and accuracy of 96%., Conclusions: The change in maxSUV on FDG-PET scan after neoadjuvant therapy holds a near linear relationship with pathologic response. It is a more accurate predictor than the change of size on CT scan. When the maxSUV decreases by 80% or more it is likely that the patient is a complete responder irrespective of cell type, neoadjuvant treatment, or the final absolute maxSUV. These findings may help guide treatment strategies.

Assays that conveniently quantify invasion of carcinoma cells in vitro have generally measured the passage of dissociated cells into a matrix. Although these assays have been helpful in identifying relative differences between different carcinoma cell lines or types, the requirement for dissociation overlooks the possible modulation of invasion by cell-cell interactions among carcinoma cells. Described here is a novel assay that quantifies invasion of a matrix placed above intact, multilayered raft cultures of lung carcinoma cell lines A549 and H520. The assay was performed by placing a porous membrane coated with matrix at the air interface of the raft cultures for varying lengths of time, after which the cells invading the matrix were enumerated. The numbers of cells invading increased in a relatively linear fashion from 24 to 72 h, and the absolute numbers within each cell line were reproducible with multiple sets of raft cultures prepared at different times. It was also found that this assay could quantify differences in invasion caused by changes in matrix composition. It is concluded that this assay can reproducibly quantify carcinoma cell invasion from three dimensional raft cultures.

A 12-year-old girl with presumed myocarditis was supported with right and left ventricular assist devices for 68 days before device removal. During this time, the patient underwent echocardiography and right heart catheterization for evaluation of cardiac recovery. This case report serves as the basis for a discussion of criteria for deciding when to terminate mechanical circulatory support in a patient with recovery after acute myocarditis.

Adenocarcinoma (AC), squamous cell carcinoma (SCC) and adenosquamous carcinoma (ASC) of the lung are morphologically distinguished in part by cyto-architectural features. However, little is known about the relative expression and distribution of cyto-architectural proteins among AC, SCC and ASC. Initial microarray analysis revealed significant differences in expression of two cyto-architectural genes in AC, SCC and ASC. Desmoplakin (DP) 1 and 2, which link desmosomes to intermediate filaments, was strongly expressed in SCC relative to AC and ASC. Cytokeratin 18 (CK18), an intermediate filament that is commonly linked to desmoplakin, was strongly expressed in AC and ASC relative to SCC. Western blot analysis demonstrated that AC and ASC had abundant CK18 protein, whereas CK18 was weakly detected in SCC. DP 1 and 2 are strongly expressed in SCC and minimally expressed in AC and ASC. However, the ratio of one to the other is the same in SCC and AC, but DP2 is lost in ASC. Microscopic analysis with fluorescence-labeled antibodies for CK18 and DP 1 and 2 revealed abundant membrane localization of DP and minimal perinuclear localization of CK18 in SCC. In contrast, in both AC and ASC, the CK18 protein was diffusely distributed within the cytoplasm, and DP showed both membranous and cytoplasmic localization. In conclusion, the data here shows that AC, SCC and ASC each have specific patterns of DP 1 and 2 and CK18 gene expression, protein content and biodistribution.

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.

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.

We review the literature on Lambl's excrescences (valvular strands). With the widespread use of cardiac imaging modalities, most importantly, with transesophageal echocardiography, abnormal valvular structures are frequently identified on both native and prosthetic heart valves. However, there is no consensus in the literature on the correct terminology of these structures. The relationship between valvular strands (the so-called Lambl's excrescences) and papillary fibroelastomas has not been well established. In this review, we attempt to summarize the available echocardiographic descriptors and the gross macroscopic and histological features of Lambl's excrescences (valvular strands). In addition, we review the etiology, pathogenesis, and clinical implications of these cardiac excrescences. Also, we describe features that help to distinguish between Lambl's excrescences and papillary fibroelastomas. Because valvular strands (Lambl's excrescences) have been implicated in systemic thromboembolism, we also review the available management principles for patients with valvular strands; unfortunately, no specific therapeutic guidelines can be proposed at the present time.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.