Chromosomal aberrations and gene expression profiles in non-small cell lung cancer.

Alterations in genomic content and changes in gene expression levels are central characteristics of tumors and pivotal to the tumorigenic process. We analyzed 23 non-small cell lung cancer (NSCLC) tumors by array comparative genomic hybridization (array CGH). Aberrant regions identified included well-characterized chromosomal aberrations such as amplifications of 3q and 8q and deletions of 3p21.31. Less frequently identified aberrations such as amplifications of 7q22.3-31.31 and 12p11.23-13.2, and previously unidentified aberrations such as deletion of 11q12.3-13.3 were also detected. To enhance our ability to identify key acting genes residing in these regions, we combined array CGH results with gene expression profiling performed on the same tumor samples. We identified a set of genes with concordant changes in DNA copy number and expression levels, i.e. overexpressed genes located in amplified regions and underexpressed genes located in deleted regions. This set included members of the Wnt/beta-catenin pathway, genes involved in DNA replication, and matrix metalloproteases (MMPs). Functional enrichment analysis of the genes both overexpressed and amplified revealed a significant enrichment for DNA replication and repair, and extracellular matrix component gene ontology annotations. We verified the changes in expressions of MCM2, MCM6, RUVBL1, MMP1, MMP12 by real-time quantitative PCR. Our results provide a high resolution map of copy number changes in non-small cell lung cancer. The joint analysis of array CGH and gene expression analysis highlights genes with concordant changes in expression and copy number that may be critical to lung cancer development and progression.

Stress activated protein kinase p38 is involved in IL-6 induced transcriptional activation of STAT3.

The pleiotropic cytokine interleukin-6 (IL-6) induces acute phase protein expression in HepG2 human hepatoma cells and promotes the growth of mouse B9 hybridoma. The signaling cascades leading to these biological functions are only partially known. We analysed the involvement of MAPK homologues in IL-6 transduction pathways and found that interleukin-6 triggered activation of p38 stress-activated protein kinase (p38) but not of jun kinase. p38 activity was required for biological functions including acute phase protein secretion from HepG2 hepatoma and proliferation of B9 hybridoma cells. Using a reporter gene construct containing a 190 bp promoter fragment of the acute phase protein haptoglobin we found that p38 is involved in transcriptional activation of the haptoglobin promoter by STAT3 but not by NF-IL6. Thus, we present evidence for a role of p38 in IL-6 induced functions and a possible cross-talk between this MAPK homologue and the STAT pathway.

The effect of prostaglandin E2 on cystine uptake and glutathione synthesis by human lung fibroblasts.

Prostaglandin E2 (PGE2) is an inflammatory mediator capable of regulating fibroblast cell proliferation, matrix protein production, and system A amino acid transport. System x-c amino acid transport is regulated by electrophilic agents and oxygen. The effect of PGE2 on the x-c system transport of cystine and the synthesis of glutathione by human lung fibroblasts was examined. Preincubation of fibroblast cultures with PGE2 decreased cystine uptake by 42%. Kinetic studies revealed a 42% decrease in the Vmax of the x-c system transporter in PGE2-treated fibroblasts; however, the apparent Km was not affected. The glutathione content of PGE2-treated fibroblasts was decreased by up to 25% of control. These results demonstrate that system x-c transport of cystine is regulated by PGE2 and suggest that the limited availability of intracellular cysteine inhibited glutathione synthesis.

Retinoic acid-induced inhibition of type I collagen gene expression by human lung fibroblasts.

We examined alpha 1(I) collagen gene expression in all-trans retinoic acid (RA)-treated human lung fibroblast cultures. RA (10(-5)M) decreased steady-state levels for alpha 1(I) collagen mRNA by at least 75% after 24 h. The inhibition was evident within 8 h after addition of RA, was dose dependent and reversible. Treatment with 9-cis-retinoic acid did not affect alpha 1(I) collagen mRNA levels. RA also inhibited the increases in alpha 1(I) mRNA stimulated by transforming growth factor-beta (TGF-beta). The RA-mediated decrease in alpha 1(I) collagen mRNA was blocked by cycloheximide treatment, suggesting that synthesis of a protein intermediate is required for the inhibition. The RA-induced decrease in alpha 1(I) collagen mRNA levels was not mediated by increases in prostaglandin E2 production. RA decreased alpha 1(I) gene transcription as determined by nuclear run-off assays but did not significantly alter the rate of degradation of the alpha 1(I) transcript as determined by actinomycin D treatment. Studies employing cells stably transfected with constructs containing portions of the alpha 1(I) collagen promoter indicate that the DNA sequences which mediate the inhibitory effect are located within 900 bases from the transcription start site.

The promotion of plasmacytoma tumor growth by mesenchymal stroma is antagonized by basic fibroblast growth factor induced activin A.

The mesenchymal stroma has been shown to play a crucial role in the development of multiple myeloma, partly by secretion of interleukin (IL)-6, that serves as a growth factor for myeloma cells. However, it is still unclear which other stromal molecules are involved in the pathogenesis of this disease. We chose, as a model system, a mouse plasmacytoma cell line, which does not respond to IL-6. We found that the formation of mouse plasmacytoma tumors, in an in vivo skin transplantation model, is facilitated by co-injection of these tumor cells along with a mesenchymal stromal cell. The tumor promoting effect of the stroma was reproduced in an in vitro model; stromal cells induced the proliferation of plasmacytoma cells under serum-free conditions. This growth promotion could not be mimicked by a series of cytokines including IL-6 and insulin-like growth factor (IGF)-I implying a role for yet unidentified stromal factors. The in vivo formation of plasmacytoma tumors was reduced following administration of activin A, a cytokine member of the transforming growth factor (TGF)beta superfamily. Furthermore, the in vitro growth promoting effect of the stroma was abrogated by basic fibroblast growth factor (bFGF) which induced a higher stromal expression of activin A. Our results thus show that mesenchymal stroma expresses plasmacytoma growth stimulating activities that overcome the low constitutive level of the plasmacytoma inhibitor, activin A. The expression of activin A is upregulated by bFGF rendering the stroma suppressive for plasmacytoma growth. The balance between the expression of these regulators may contribute to mesenchymal stroma activity and influence the progression of multiple myeloma.

Lipoid pneumonia: a preventable form of drug-induced lung injury.

We report a case where recurrent "pneumonia" was eventually diagnosed as lipoid pneumonia in an elderly patient with cerebrovascular disease. The discontinuation of paraffin oil laxative led to clinical improvement. Lipoid pneumonia, a foreign body-type reaction to the presence of lipid within lung parenchyma, is probably underdiagnosed and underreported, and paraffin oil laxative is the main causative agent. Paraffin oil is marketed as a food additive, and no information about its hazards is provided to clinicians or patients. We suggest that a change in paraffin oil licensing may decrease the incidence of lipoid pneumonia.

Left pleural hemorrhagic effusion. A presenting sign of thoracic aortic dissecting aneurysm.

Left hemorrhagic pleural effusion was the presenting sign of painless aortic dissecting aneurysm in two elderly hypertensive patients. Computed tomography (CT) of the chest revealed the aneurysmal dilatation of the thoracic aorta and an intimal flap connecting its descending part with the left pleural space. The patients were treated conservatively with blood transfusions and drugs directed to control blood pressure. The first reported 71-year-old patient remains in stable condition for 16 months without evidence of recurrent active aortic dissection. The second 85-year-old patient remained in stable condition for 28 days, but finally had a second fatal episode of dissection into the left pleural space. The differential diagnosis of nontraumatic left hemorrhagic pleural effusion in an elderly hypertensive patient should include dissecting aneurysm of the descending thoracic aorta and CT of the chest should be performed as the next preferable diagnostic procedure.

Wegener's granulomatosis with peripheral eosinophilia. Atypical variant of a classic disease.

A patient with Wegener's granulomatosis (WG) diagnosed by ultrasound-guided transthoracic biopsy of a pulmonary nodule is reported. The case is atypical because of marked eosinophilia in the peripheral blood and the pleural effusion. The granulomatous infiltrate of the lung showed the classic picture of WG without eosinophils. The patient responded dramatically to treatment with steroids and cyclophosphamide. This variant form of WG poses problems in its distinction from Churg-Strauss syndrome, and the differential diagnosis between these two entities is discussed.

Nuclear antigen expressed by proliferating cells.

We describe a novel mouse monoclonal antibody (PRA-72) that recognizes a nuclear antigen associated with cell proliferation. The monoclonal antibody stained the nuclei of logarithmically growing cultured stromal cells. The nuclear staining disappeared when these cells entered Gzero phase of the cell cycle. Western blot analysis revealed a nuclear protein which appeared as a doublet at 35-40 KD, which was undetectable in extracts from confluent cells. Immunocytological study of purified cell populations from various cell cycle phases revealed peripheral nuclear staining in all stages except mitosis, when the chromosomes were observed enveloped with the antigen. In co-cultures of quiescent stromal cells and proliferating hemopoietic precursors, only the latter showed nuclear staining by PRA-72 monoclonal antibody. Further indications for selective expression of the antigen by proliferating cells were found by an immunohistochemical study of various tissues including newborn mouse bone marrow and its surrounding connective tissue, mouse tongue epithelium, and human carcinoma of the colon. This antibody may, therefore, prove useful in the evaluation of human tumors.

[Ceftriaxone or combined cefazolin-gentamicin for complicated urinary tract infections].

40 seriously ill patients with complicated urinary tract infections were randomly assigned to receive either a single daily dose of ceftriaxone or combined therapy with cefazolin and gentamicin administered every 8 hours. The groups were of equal size, similar in age (both averaged 75 years) and the sex ratio was about 1:1 in each group. 32.5% had proven bacteremia and the overall mortality was 17.5%. Both regimens were similarly effective in terms of mortality, duration of fever (3.0-3.1 days), and sterilization of the urine prior to discharge from hospital. However, the single daily dose of ceftriaxone was much more convenient to administer.

[Pulmonary alveolar microlithiasis presenting with prolonged cough].

A 40-year-old man had been followed in the pulmonary clinic for prolonged cough. Chest X-ray showed bilateral diffuse interstitial infiltrates with accentuation toward the bases. CT-scan demonstrated a fine diffuse reticulonodular pattern. Transbronchial lung biopsy showed pulmonary alveolar microlithiasis, a rare disease characterized by the presence of concentric calcifications within the pulmonary alveoli. This is the second case of the disease reported in Israel.

Stromal cell effects on clonal growth of tumors.

Clonal growth of tumor cell lines originating from a variety of solid tumors was studied. The seeding efficiency of these tumors in methylcellulose medium was in the range of 0.036 to 0.177. Stromal cell lines from mouse bone marrow as well as primary stromal cells from human bone marrow stimulated the growth of HCT and oat human carcinoma cells 32-fold and 25-fold, respectively. In contrast, these stromal cells inhibited the in vitro cloning of human and mouse sarcoma cell lines. Both activities of the stromal cells diffused through agar layers and operated across species barriers. Despite the diffusable nature of the factors involved, no biologic activity was observed in concentrated conditioned media prepared in the presence or absence of serum. Human foreskin fibroblasts tested under identical conditions, could neither stimulate nor inhibit the clonal growth of tumors. This preferential growth of tumor cells in the presence of tissue specific stroma may be used as an in vitro model for the study of the role of stromal cells in tumor cell spread.

Musculoskeletal symptoms as a presenting sign of long-standing hypothyroidism.

Muscle and joint pains and/or weakness are not usually stressed as central symptoms in hypothyroidism. Two cases of long-standing hypothyroidism presenting with prominent myopathic symptoms are described. The first patient presented with a 12-year history of proximal myopathy, arthropathy and skin abnormalities, and florid primary myxedema was diagnosed. No evidence for a systemic autoimmune process was found. The second patient had been treated with irradiation to the neck 23 years before admission and presented with clinical and laboratory signs of both proximal myopathy and hypothyroidism. Thyroid hormone replacement resulted in a complete recovery of all the musculoskeletal symptoms, with reversion to normal of the very high muscle enzyme levels in both patients. The cases presented illustrate that hypothyroidism can lead to the development of a variety of muscular, rheumatic and dermatologic syndromes easily confused with dermatomyositis or other collagen diseases.

Late appearance of thrombotic thrombocytopenic purpura after autoimmune hemolytic anemia and in the course of chronic autoimmune thrombocytopenic purpura: two case reports.

The association between thrombotic thrombocytopenic purpura (TTP) and autoimmune hematological conditions is reported in 2 patients. In a 35-year-old man, acute autoimmune hemolytic anemia (AIHA) was diagnosed in 1960; until 1965 he was free of disease, when he abruptly developed TTP and failed to respond to blood transfusions and corticosteroids. In a 14-year-old girl, autoimmune thrombocytopenic purpura (AITP) was diagnosed in 1981 and treated with corticosteroids and splenectomy. Four years later the patient was admitted with acute catastrophic signs and symptoms of TTP and failed to respond to plasmapheresis and plasma transfusions. The present case reports of associations between AIHA and AITP with TTP support the connection of the latter with abnormalities of the immune system.

Regulation of type I collagen mRNA by amino acid deprivation in human lung fibroblasts.

The steady state levels of alpha1(I) collagen mRNA are decreased by retinoic acid and prostaglandin E2. These effector substances decrease the uptake of A system amino acids. We examined the effect of amino acid deprivation on the steady state levels of alpha1(I) collagen in human lung fibroblasts. Maintenance of fibroblasts in amino acid-free medium decreased alpha1(I) collagen mRNA levels by 29% at 24 h and 78% at 72 h. Frequent refeeding of cultures with amino acid-free medium resulted in more rapid decreases in intracellular amino acids and in alpha1(I) collagen mRNA levels. The decrease in alpha1(I) collagen mRNA levels was mediated by decreases in mRNA stability as assessed by a half-life determination using actinomycin D and by decreases in the rate of transcription as assessed by nuclear run-on assay. Treatment of fibroblasts with medium containing amino acids resulted in rapid restoration of alpha1(I) collagen mRNA levels. This increase in alpha1(I) collagen mRNA expression required protein synthesis as determined by cycloheximide sensitivity and was inhibited by prostaglandin E2. These data indicate that alpha1(I) collagen mRNA levels are sensitive to alterations in the amount of intracellular amino acids and suggest a potential mechanism whereby alpha1(I) collagen accumulation may be regulated independent of inflammatory mediators following lung injury.

The effect of retinoic acid on amino acid uptake and protein synthesis by lung fibroblasts.

The effect of retinoic acid (RA) on the uptake and utilization of extracellular amino acids by fetal lung fibroblasts was examined. RA decreased the incorporation of [3H]proline into collagen and other proteins. The effect was maximal at a RA concentration of 10(-5) M; smaller decreases were observed at a RA concentration of 10(-6) M. This decrease in collagen formation was associated with a large decrease in intracellular [3H] proline. The decrease in intracellular [3H]proline was first observed at 2 h following the addition of RA to cell cultures. Transport studies employing radiolabeled amino acids revealed that RA decreased the uptake of proline, 2-aminoisobutyric acid, and 2-(methylamino)isobutyric acid but not leucine or methionine. Kinetic analysis of 2-aminoisobutyric acid uptake indicated that this effect was mediated primarily by an increase in apparent Km, with a lesser decrease in Vmax, RA-induced inhibition of proline uptake was not abolished by the presence of cycloheximide nor by pretreatment with indomethacin. Na+,K(+)-ATPase activity was not affected by RA treatment. These results suggest that RA modulates protein production in fibroblasts by altering the function of the Na(+)-dependent A transport system for amino acid uptake.

Effect of okadaic acid on elastin gene expression in interstitial lung fibroblasts.

Okadaic acid (OA), a specific serine/threonine protein phosphatase inhibitor, downregulated tropoelastin formation and elastin mRNA levels in a dose-related and cycloheximide-sensitive fashion in cultured lung fibroblasts. Treatment with a tyrosine phosphatase inhibitor at high concentrations did not alter elastin mRNA levels, however. Nuclear run-on analysis indicated that OA primarily suppressed elastin gene expression through a transcriptional mechanism. In contrast to its effects on elastin expression, OA downregulated alpha 1(I) mRNA to significantly lesser degrees. The mechanism by which OA decreased elastin mRNA levels did not appear to involve protein kinase C or share the signaling pathway of IL-1 beta. Prolonged treatment with phorbol ester promoted the inhibitory effects of OA on elastin, as did shorter treatment with IL-1 beta. Moreover, transient transfection studies indicated that OA and IL-1 beta do not act through the same cis-acting element in the elastin promoter. Finally, unlike the transient effects of IL-1 beta, OA induced persistent inhibition of elastin expression by a transcriptional mechanism. Taken together, these data indicate that serine/threonine protein phosphorylation can regulate the amount and composition of extracellular matrix secreted by fibroblasts into the interstitium of the lung.

Regulation of type I collagen production by insulin and transforming growth factor-beta in human lung fibroblasts.

The effects and interaction of transforming growth factor-beta (TGF-beta) and insulin on collagen production in human fetal lung fibroblasts was examined. Fibroblasts were labeled with [3H]proline and collagen production was analyzed by polyacrylamide gel electrophoresis. The addition of insulin (2 micrograms/ml) increased collagen production 5 fold and TGF-beta (5 ng/ml) increased collagen production 6-fold. The combination of TGF-beta and insulin further increased type I collagen production (12 fold). We found that TGF-beta increased pro-alpha 1 (I) collagen mRNA levels 2-3 fold, insulin increased mRNA levels by less than 2 fold, and the combination stimulated a 3-4 fold increase. In a nuclear run-on assay, we found a 1.7 fold increase in the rate of transcription for the pro-alpha 1 (I) collagen gene in insulin-treated cultures and a 2-fold increase in TGF-treated cultures. In fibroblasts transfected with a plasmid containing 2.4 kb of the 5' flanking sequences of the human pro-alpha 1 (I) collagen gene, TGF-beta stimulated a 2.8 fold increase in promoter activity. In contrast, the addition of insulin stimulated a small increase (less than 2 fold) in the pro-alpha 1 (I) collagen promoter activity when administered alone or in combination with TGF-beta. Insulin prolonged the half-life of pro-alpha 1 (I) collagen mRNA from 9.1 h to 14.3 h as assessed by treatment with actinomycin D. The insulin-induced increase in pro-alpha 1 (I) collagen mRNA was blocked by the presence of cycloheximide indicating a requirement for new protein synthesis. These results show that the combination of TGF-beta and insulin stimulate large increases in type I collagen formation by acting at different sites in the collagen biosynthetic pathway.