Phosphatidylinositol-3-OH Kinase (PI3K)/AKT2, activated in breast cancer, regulates and is induced by estrogen receptor alpha (ERalpha) via interaction between ERalpha and PI3K.

We have shown previously that the AKT2 pathway is essential for cell survival and important in malignant transformation. In this study, we demonstrate elevated kinase levels of AKT2 and phosphatidylinositol-3-OH kinase (PI3K) in 32 of 80 primary breast carcinomas. The majority of the cases with the activation are estrogen receptor alpha (ERalpha) positive, which prompted us to examine whether AKT2 regulates ERalpha activity. We found that constitutively activated AKT2 or AKT2 activated by epidermal growth factor or insulin-like growth factor-1 promotes the transcriptional activity of ERalpha. This effect occurred in the absence or presence of estrogen. Activated AKT2 phosphorylates ERalpha in vitro and in vivo, but it does not phosphorylate a mutant ERalpha in which ser-167 was replaced by Ala. The PI3K inhibitor, wortmannin, abolishes both the phosphorylation and transcriptional activity of ERalpha induced by AKT2. However, AKT2-induced ERalpha activity was not inhibited by tamoxifen but was completely abolished by ICI 164,384, implicating that AKT2-activated ERalpha contributes to tamoxifen resistance. Moreover, we found that ERalpha binds to the p85alpha regulatory subunit of PI3K in the absence or presence of estradiol in epithelial cells and subsequently activates PI3K/AKT2, suggesting ERalpha regulation of PI3K/AKT2 through a nontranscriptional and ligand-independent mechanism. These data indicate that regulation between the ERalpha and PI3K/AKT2 pathway (ERalpha-PI3K/AKT2-ERalpha) may play an important role in pathogenesis of human breast cancer and could contribute to ligand-independent breast cancer cell growth.

AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells.

Extensive studies have demonstrated that the Akt/AKT1 pathway is essential for cell survival and inhibition of apoptosis; however, alterations of Akt/AKT1 in human primary tumors have not been well documented. In this report, significantly increased AKT1 kinase activity was detected in primary carcinomas of prostate (16 of 30), breast (19 of 50), and ovary (11 of 28). The results were confirmed by Western blot and immunohistochemical staining analyses with phospho-Ser473 Akt antibody. The majority of AKT1-activated tumors are high grade and stage III/lV (13 of 16 prostate, 15 of 19 breast, and 8 of 11 ovarian carcinomas). Previous studies showed that wild-type AKT1 was unable to transform NIH3T3 cells. To demonstrate the biological significance of AKT1 activation in human cancer, constitutively activated AKT1 (Myr-Akt) was introduced into NIH3T3 cells. Overexpression of Myr-Akt in the stably transfected cells resulted in malignant phenotype, as determined by growth in soft agar and tumor formation in nude mice. These data indicate that AKT1 kinase, which is frequently activated in human cancer, is a determinant in oncogenesis and a potential target for cancer intervention.

NR8383 alveolar macrophage toxic growth arrest by hydrogen peroxide is associated with induction of growth-arrest and DNA damage-inducible genes GADD45 and GADD153.

Breathing air exposes humans and other mammals to various toxic agents including oxidative contaminants associated with fine particles of less than 2.5 micron which may be deposited in the deep lung and have been implicated in the increased morbidity and mortality correlated with air pollution. Oxidative damage from inhaled particles may include damage to DNA, thereby adversely affecting the immunosurveillance provided by alveolar macrophages. Using the rat alveolar macrophage cell line NR8383, we demonstrated that cell proliferation was inhibited by exogenous hydrogen peroxide, an oxidant naturally produced in cellular respiration and phagocytosis. Mercaptosuccinate, a specific inhibitor of the antioxidant enzyme glutathione peroxidase, also inhibited cell growth. Genes known to be coordinatively regulated in response to growth arrest and DNA damage, GADD45 and GADD153, were induced compared to the housekeeping gene beta-ACTIN by equitoxic doses of hydrogen peroxide and mercaptosuccinate. Hydrogen peroxide treatment of cells in which glutathione peroxidase was inhibited by mercaptosuccinate resulted in even greater induction of both GADD genes. This approach using the NR8383 alveolar macrophage cell line provides a model for studying genotoxicity at the mechanistic level at which stress-responsive genes involved in growth arrest and DNA-damage response are modulated.

Lysozyme is an ozone-sensitive component of alveolar type II cell lamellar bodies.

Exposure of rats to 3 ppm ozone for up to 8 h results in significant changes in lamellar bodies, the surfactant storing organelles of type II cells. We have previously shown that a 14 kDa lamellar body protein is decreased as early as 4 h after the onset of ozone exposure. We have isolated this ozone-sensitive protein from rat lung lamellar bodies and identified it as lysozyme by immunochemical methods, as well as by its amino acid composition, N-terminal amino acid sequence and bacteriolytic activity. Reduced lysozyme activity in isolated lamellar bodies is detected as early as 4 h after the start of ozone exposure. Following an 8 h ozone exposure, the activity does not return to control levels for at least 48 h. Lamellar body lysozyme is expected to be secreted with surfactant phospholipids, thereby contributing to the antimicrobial defense of the alveolar lining layer. The acute lysozyme deficiency seen in ozone-induced oxidant injury may reduce the resistance of the lung to infection.

Surfactant-associated glycoproteins accumulate in alveolar cells and secretions during reparative stage of hyaline membrane disease.

Surfactant-associated (SA) glycoproteins are lung-specific proteins produced in the human lung by alveolar type II cells and Clara cells. The distribution of these proteins was studied immunohistochemically in lung tissue obtained postmortem from 12 stillborn fetuses and 49 infants with hyaline membrane disease (HMD). By 21 weeks of gestation, SA glycoproteins were detected in the fetal alveolar epithelium and within Clara cells. The staining increased in intensity and extent with advancing gestational age. Infants with HMD who survived less than 48 hours did not generally exhibit stainable material either within type II cells or secretions, but staining was often noted in Clara cells as well as focally beneath hyaline membranes. In infants surviving more than 48 hours, intense staining of hyaline membranes, alveolar secretions, proliferating alveolar type II cells, and Clara cells was evident. Immunoreactivity was intense in hypertrophic type II cells that formed a continuous alveolar epithelial lining in lungs with bronchopulmonary dysplasia. Included in the population of infants with HMD were 15 infants with pulmonary hypoplasia. The lungs of these infants showed minimal staining for SA glycoproteins regardless of postnatal survival time. The results provide an immunomorphologic basis for defining normal and abnormal lung maturation. They also indicate that enhanced SA glycoprotein production is a sustained response of regenerating and hypertrophic type II cells in premature infants.

Ozone-induced alterations of lamellar body lipid and protein during alveolar injury and repair.

Alveolar Type II cells in the rat respond to severe, acute ozone injury (3 ppm ozone for eight hours) by increasing their intracellular pool of surfactant; however, the newly stored surfactant is abnormal in composition. Lamellar bodies isolated between 24 and 96 hours after ozone exposure contained significantly more cholesterol in relation to phosphatidylcholine than did controls. By contrast, the cholesterol content of surfactant isolated from alveolar lavage remained unchanged throughout an 8-day post-ozone period. The total protein content of lamellar bodies in relation to phosphatidylcholine was significantly decreased at 24 and 48 hours post-ozone. Analysis of lamellar body proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the amount of a 14 kDa proteolipid was greatly reduced at the end of the eight-hour ozone exposure and remained low for at least 48 hours. This proteolipid appeared to be a specific lamellar body component since it was not detected in extracellular surfactant. The findings indicate that oxidative alveolar stress initiates characteristic alterations in both lipid and protein constituents of stored surfactant, without perturbation in the composition of extracellular surfactant.

Prenatal relationship of surfactant lipid and protein constituents in infants with respiratory distress syndrome: a preliminary communication.

The prenatal relationships between surfactant disaturated phosphatidylcholine (DSPC) and surfactant-associated proteins of preterm infants with respiratory distress syndrome (RDS) have not been well documented. In the present study we measured the concentration of DSPC, surfactant glycoproteins (GP), and surfactant proteolipids (PLP) in amniotic fluids obtained within 6 hours prior to delivery of 16 newborn infants with gestational ages between 27 and 32 weeks. In control infants of 27-32 weeks gestation without RDS, the values of DSPC, GP, and PLP per milliliter of amniotic fluid were 20 +/- 2.9 micrograms, 684 +/- 115.3 ng and 289 +/- 62.5 ng, respectively. These values were significantly higher, threefold for PLP, fourfold for DSPC, and fivefold for GP, than amniotic fluid levels in infants with RDS. The findings support the concept that immaturity of surfactant in RDS involves both phospholipids and surfactant-associated proteins. Measurements of surfactant lipid-protein complex appear to enhance the reliability for identifying prenatally, infants at risk of developing hyaline membrane disease. More extensive studies are warranted to assess the usefulness of these assays for clinical application.

Lung surfactant-associated glycoproteins and proteolipids in human amniotic fluids evaluated by dot immunobinding assays.

Dot immunobinding assays for the quantitation of two classes of proteins associated with lung surfactant phospholipids in human amniotic fluid are described. With the use of these assays it was determined that the two classes of surfactant proteins accumulate in the amniotic fluid at the same rate. The concentrations of disaturated phosphatidylcholine and the surfactant-associated proteins are less closely correlated. Centrifugation of amniotic fluids either before or after freezing resulted in a loss ranging from 10 to 35 percent of both surfactant disaturated phosphatidylcholine and proteins depending on the relative centrifugal force used. Preterm amniotic fluids contained significantly less of both surfactant-associated proteins, as well as disaturated phosphatidylcholine, than did term amniotic fluids which suggests that the use of these specific protein markers may enhance the assessment of fetal lung maturity.

Distribution and subcellular localization of surfactant-associated glycoproteins in human lung.

Human surfactant contains lung-specific, high molecular weight glycoproteins which are composed of disulfide-linked 34-kilodalton peptide subunits. We prepared antibodies to both isolated HMW glycoproteins and 34-kilodalton peptides and tested the antisera for specificity with immunochemical procedures. In the present study we have investigated the cellular distribution and subcellular localization of these glycoproteins in surgically excised human lung tissue with or without type II cell hyperplasia. An immunoperoxidase technique was used, and cytoplasmic staining reflecting labeling with antibodies to either high molecular weight glycoproteins or the 34-kilodalton peptide subunits was consistently observed in normal type II cells, Clara cells, and some alveolar macrophages and was more intense and diffuse in hyperplastic type II cells. The ultrastructural localization of surfactant-associated glycoproteins was investigated using the periodate-lysine-paraformaldehyde fixative and a peroxidase-labeled antibody technique. In both normal and proliferating type II cells the staining was localized in the rough endoplasmic reticulum, perinuclear cisternas, vesicles of the Golgi complex, vesicles and lamellar membranes of the multivesicular bodies, and some multivesicular body-lamellar body forms. In addition, staining was frequently found in peripheral portions of partially preserved lamellar bodies including those at the stage of secretion, as well as in association with of alveolar tubular myelin. Labeling was also observed in the rough endoplasmic reticulum of Clara cells. We conclude that antibodies against human surfactant-associated glycoproteins are markers for normal and regenerating type II cells, as well as for Clara cells which apparently retain limited ability to produce surfactant-associated glycoproteins independently of surfactant phospholipids. The results indicate that, in type II cells, synthesis and secretion of these glycoproteins involve the same cytoplasmic organelles that are responsible for synthesis, packaging, storage, and exocytosis of surfactant phospholipids. However, maturation of the lamellar bodies, known to be characterized by progressive accumulation of phospholipids, may not require parallel storage of surfactant-associated glycoproteins within the entire lamellar body compartment.

Immunologically related multimeric forms of 30-40 kDa peptides associated with lung surfactant in various mammalian species.

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000-40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high-molecular-weight proteins (greater than 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30-40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.

Ultrastructural and immunohistochemical studies of bronchiolo-alveolar carcinoma.

A detailed ultrastructural study was made of seven cases of bronchiolo-alveolar carcinoma, and the findings were correlated with histochemical and immunohistochemical data. By electron microscopic examination all seven tumors displayed glandular differentiation, manifested by the presence of microvilli and intercellular junctions, with or without mucin production. Variable proportions of tumor cells retained ultrastructural characteristics of alveolar type II cells and Clara cells. In addition, some tumor cells revealed desmosomes and tonofilaments consistent with squamous differentiation. Immunohistochemical evaluation was carried out using a peroxidase-antiperoxidase technique and specific antibodies against surfactant high molecular weight glycoproteins, keratin proteins, IgA + secretory piece, carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), and alpha-fetoprotein (AFP). Four tumors with type II cell-like differentiation stained with anti-surfactant glycoprotein sera. All seven tumors stained focally with anti-keratin and IgA + anti-secretory piece antibodies, and diffusely with CEA. These tumors failed to stain with antisera against HCG and AFP. It is concluded that bronciolo-alveolar carcinomas are primarily composed of cells with alveolar and bronchiolar cell differentiation. Adequate criteria were established for ultrastructural identification of tumor cells with differentiation to type II alveolar cell or Clara cell. Moreover, the findings of this study indicate that the surfactant glycoprotein marker, when present in a given tumor either diffusely or focally, is diagnostic of bronchiolo-alveolar carcinoma.

Lung surfactant phospholipids in different animal species.

A comparative study of adult mammalian lung surfactants was undertaken to determine which animal species might serve as appropriate models for surfactant alterations in human lung diseases. Phosphatidylcholine (PC) comprised 80-87% of the phospholipid and contained more than 65% palmitic acid in all species studied. Phosphatidylglycerol (PG) was found to vary significantly in fatty acid composition among the species. Rabbit, dog and rat surfactant PG contained 50-60% palmitic acid, while human and cat surfactant contained much lower levels of saturated fatty acids. Both the PC and PG of all species contained 2 positional isomers of fatty acids with 16 carbons and one double bond, but the relative amounts of the unusual isomer, 16:1 delta 7, and palmitoleic acid, 16:1 delta 9, varied among the different animal species. Only cat and dog surfactant phospholipids contained 18:1 delta 5. Cat surfactant phospholipids also differed by the absence of 20:4 and the presence of small amounts of several 20- and 22-carbon fatty acids. These results explain some discrepancies found in the literature concerning surfactant composition and delineate limiting factors in extrapolating results from animal studies for the evaluation of maturation and pathological alterations in human surfactant.

Synthesis of lung surfactant-associated glycoproteins by A549 cells: description of an in vitro model for human type II cell dysfunction.

Immunocytochemical, ultrastructural, and biochemical approaches were used in a series of in vitro and in vivo experiments designed to identify characteristic changes reflecting differentiated type II cell function of A549 cells. Monolayers of A549 cells and A549 clones were maintained in culture for up to 3 weeks. Using an immunoperoxidase (PAP) technique, we demonstrated that variable proportions of A549 cells and of cells in several A549 clones reacted specifically with antibodies to high molecular weight (greater than 400,000) human surfactant-associated glycoproteins (HSAG). The cells of one clone, A549-C12, were consistently negative for HSAG, but their lamellar bodies were similar in appearance and distribution to those found in a PAP-positive clone, A549-C11, as well as in A549 cells. In addition, both C11 and C12 clones displayed time-dependent, divergent differentiation predominantly toward type II epithelium and nonciliated bronchiolar and bronchial cells. Surfactant isolated from either C11 or C12 cells revealed reduced content of disaturated phosphatidylcholine and phosphatidylglycerol when compared to human surfactant; however, a 95,000-dalton peptide immunologically related to HSAG was identified in surfactant from C11 cells but not from the PAP-negative C12 clone. Tumor xenografts produced in athymic (nude) mice following inoculation with cells from C11 and C12 clones exhibited prominent immunoperoxidase staining involving most tumor cells. Cell lines derived from these xenografts (T-11 and T-12) were also enriched in PAP-positive cells. Immunoelectron microscopy indicated that HSAG was localized in the rough endoplasmic reticulum, multivesicular bodies (MVB), intermediate MVB-lamellar forms, and abnormal pleomorphic inclusions. Moreover, two HSAG peptides, both larger than the 34,000-dalton peptide subunit found in normal human surfactant, were present in cells and media from monolayers of the T-11 cell line. We conclude that A549 cells synthesize "defective" HSAG and that the synthesis may be modulated by host factors. The results indicate that appropriate A549 clones can be used effectively as model systems for selected type II cell dysfunctions.

Surfactant "apoproteins" in human amniotic fluid: an enzyme-linked immunosorbent assay for the prenatal assessment of lung maturity.

We have developed an enzyme-linked immunosorbent assay for the quantitation of human lung surfactant high--molecular weight proteins and used this assay to evaluate changes in surfactant "apoprotein" content of amniotic fluid during the last trimester of pregnancy. The amount of surfactant apoprotein in human amniotic fluid increased with advancing gestational age and correlated well with other parameters of fetal lung maturity, the lecithin:sphingomyelin ratio, and the presence of phosphatidylglycerol. The findings suggest that this test, which is relatively simple to perform, may prove useful as an additional clinical parameter for assessing fetal lung maturity and predicting more accurately fetuses at risk of developing hyaline membrane disease.

Secretory IgA is a component of rabbit lung surfactant.

Secretory IgA is a major protein component of rabbit lung surfactant purified by NaBr density gradient centrifugation from endobronchial lavage and minced lung tissue. Secretory IgA was found in both surfactant and non-surfactant fractions obtained from endobronchial lung washings. By contrast in minced-lung washings, which are not contaminated with proteins from the upper respiratory tree, secretory IgA is prominent only in the surfactant fraction. These findings indicate that in rabbit lung secretory IgA is present in the alveoli and is intimately associated with the surfactant system.

Clinical evaluation of UNB 3-state myoelectric control for arm prostheses.

This report describes an attempt to conduct (in 1978 during a period of one week) an intensive, thorough, and objective evaluation of a prosthetic control system in such a manner that the evaluation avoids what are seen as shortcomings common among evaluation procedures described in the literature. The evaluation, in terms of benefits to patients, involved consideration on an interdisciplinary basis among an engineering team, prosthetics team, and therapy team. Nine below-elbow and two forequarter amputees participated. The device evaluated was the University of New Brunswick 3-state myoelectric control system, in the 12-volt version designed in 1975. This system is intended for use where there are not enough control sites to permit use of an Otto Bock or similar control system, and permits on/off control of a powered hand or other device in two directions from a single muscle. Observations on each patient by the 14-person evaluation team are summarized, and an Appendix presents questionnaires with summarized responses of the subjects and their families.

Sequential changes of surfactant phosphatidylcholine in hyaline-membrane disease of the newborn.

Although reduced levels of lung surfactant are known to predispose to hyaline-membrane disease, the role of biochemical changes in surfactant composition has not been defined. We found that surfactant isolated from pharyngeal and tracheal aspirates of newborns with hyaline-membrane disease had a distinctly different phosphatidylcholine fatty acid composition from surfactant of control infants. Surfactant phosphatidylcholine from newborns with hyaline-membrane disease had a lower percentage of palmitic acid and higher percentages of the 18-carbon and 20-carbon fatty acids, irrespective of gestational age. Evaluation of serial aspirates for 18 days revealed that in hyaline-membrane disease the surfactant phosphatidylcholine fatty acids followed a predictable pattern of change, gradually becoming similar to those of the control group. Evaluation of surfactant from tracheal and pharyngeal aspirates effectively monitors the biochemical maturation of the surfactant system in hyaline-membrane disease.