Sue A. Shelley

AKT1/PKBα 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.

Biochemical composition of adult human lung surfactant.

Surfactant was isolated from minced adult human lung tissues by repetitive centrifugation on NaBr density gradients. The surface active fraction contained 12 mg phospholipid per mg of protein. Eighty percent of the phospholipid was phosphatidylcholine and 9% was phosphatidylglycerol. The phosphatidylcholines, 55% of which were disaturated, contained more than 70% palmitic acid. In contrast, phosphatidylglycerol contained 22% palmitic acid and 52% oleic acid, suggesting that the importance of phosphatidylglycerol in surfactant function relates to its acidic head group. The most abundant proteins in human surfactant were high molecular weight (>400,000) glycoproteins which on reduction with dithiothreitol yielded peptides of 34,000 daltons. Antibodies to the high molecular weight proteins were prepared in rabbits and using immunoperoxidase methods were shown to stain alveolar type II cells and the alveolar and bronchial surfaces, but no other human tissues. A low molecular weight protein was also present in lung surfactant and was most apparent in surfactant subfractions which had higher phospholipid to protein ratios. The results of this study provide a basis to further investigate the role of normal and altered surfactant constituents in disease states of the human lung.

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.

Positive Feedback Regulation between Akt2 and MyoD during Muscle Differentiation CLONING OF Akt2 PROMOTER

Akt2 is a member of the Akt/PKB family, which is involved in a variety of cellular events including cell survival, proliferation, and differentiation. During skeletal muscle differentiation, the Akt2 but not Akt1 expression was significantly increased. Microinjection of anti-Akt2 but not anti-Akt1 antibody efficiently abrogated myogenesis, indicating that Akt2 plays a specific role in muscle differentiation. It has been well documented that ectopic expression of MyoD is sufficient to induce muscle differentiation in myoblasts. However, the mechanism of induction of Akt2 during muscle differentiation and the significance of Akt2 protein in MyoD-induced myogenesis are largely unknown. In this study, we provide direct evidence that Akt2 is transcriptionally regulated by MyoD and activates MyoD-myocyte enhancer binding factor-2 (MEF2) transactivation activity. The Akt2 promoter was isolated and found to contain nine putative E-boxes (CANNTG), which are putative MyoD binding sites. Electrophoretic mobility shift analyses revealed that MyoD bound to eight of the sites. The expression of MyoD significantly enhanced Akt2 promoter activity and up-regulated Akt2 mRNA and protein levels. Moreover, Akt2 but not Akt1 was activated during differentiation. The expression of Akt2 activated MyoD-MEF2 transcriptional activity and induced myogenin expression. These data indicate that there is a positive feedback regulation loop between Akt2 and MyoD-MEF2 during muscle differentiation, which is essential for MyoD-induced myogenesis.

Inhibition of JNK by cellular stress- and tumor necrosis factor alpha-induced AKT2 through activation of the NF kappa B pathway in human epithelial Cells

Previous studies have demonstrated that AKT1 and AKT3 are activated by heat shock and oxidative stress via both phosphatidylinositol 3-kinase-dependent and -independent pathways. However, the activation and role of AKT2 in the stress response have not been fully elucidated. In this study, we show that AKT2 in epithelial cells is activated by UV-C irradiation, heat shock, and hyperosmolarity as well as by tumor necrosis factor α (TNFα) through a phosphatidylinositol 3-kinase-dependent pathway. The activation of AKT2 inhibits UV- and TNFα-induced c-Jun N-terminal kinase (JNK) and p38 activities that have been shown to be required for stress- and TNFα-induced programmed cell death. Moreover, AKT2 interacts with and phosphorylates IκB kinase α. The phosphorylation of IκB kinase α and activation of NFκB mediates AKT2 inhibition of JNK but not p38. Furthermore, phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 significantly enhances UV- and TNFα-induced apoptosis, whereas expression of constitutively active AKT2 inhibits programmed cell death in response to UV and TNFα stimulation with an accompanying decreased JNK and p38 activity. These results indicate that activated AKT2 protects epithelial cells from stress- and TNFα-induced apoptosis by inhibition of stress kinases and provide the first evidence that AKT inhibits stress kinase JNK through activation of the NFκB pathway.

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∶1Δ7, and palmitoleic, acid, 16∶1Δ9, varied among the different animal species. Only cat and dog surfactant phospholipids contained 18∶1Δ5. 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.

Immunocytochemical localization of lysozyme and surfactant protein A in rat type II cells and extracellular surfactant forms.

Using immunogold labeling of fixed, cryosubstituted tissue sections, we compared the distribution of lysozyme, an oxidant-sensitive lamellar body protein, with that of surfactant protein A (SP-A) in rat Type II cells, extracellular surfactant forms, and alveolar macrophages. Morphometric analysis of gold particle distribution revealed that lysozyme and SP-A were present throughout the secretory and endosomal pathways of Type II cells, with prominent localization of lysozyme in the peripheral compartment of lamellar bodies. All extracellular surfactant forms were labeled for both proteins with preferential labeling of tubular myelin and unilamellar vesicles. Labeling of tubular myelin for SP-A was striking when compared with that of lamellar bodies and other extracellular surfactant forms. Lamellar body-like forms and multilamellar structures were uniformly labeled for lysozyme, suggesting that this protein is rapidly redistributed within these forms after secretion of lysozyme-laden lamellar bodies. By contrast, increased labeling for SP-A was observed over peripheral membranes of lamellar body-like forms and multilamellar structures, apparently reflecting progressive SP-A enrichment of these membranes during tubular myelin formation. The results indicate that lysozyme is an integral component of the lamellar body peripheral compartment and secreted surfactant membranes, and support the concept that lysozyme may participate in the structural organization of lung surfactant.

Inhibition of JNK by Cellular Stress- and Tumor Necrosis Factor α-induced AKT2 through Activation of the NFκB Pathway in Human Epithelial Cells

Abstract Previous studies have demonstrated that AKT1 and AKT3 are activated by heat shock and oxidative stress via both phosphatidylinositol 3-kinase-dependent and -independent pathways. However, the activation and role of AKT2 in the stress response have not been fully elucidated. In this study, we show that AKT2 in epithelial cells is activated by UV-C irradiation, heat shock, and hyperosmolarity as well as by tumor necrosis factor α (TNFα) through a phosphatidylinositol 3-kinase-dependent pathway. The activation of AKT2 inhibits UV- and TNFα-induced c-Jun N-terminal kinase (JNK) and p38 activities that have been shown to be required for stress- and TNFα-induced programmed cell death. Moreover, AKT2 interacts with and phosphorylates IκB kinase α. The phosphorylation of IκB kinase α and activation of NFκB mediates AKT2 inhibition of JNK but not p38. Furthermore, phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 significantly enhances UV- and TNFα-induced apoptosis, whereas expression of constitutively active AKT2 inhibits programmed cell death in response to UV and TNFα stimulation with an accompanying decreased JNK and p38 activity. These results indicate that activated AKT2 protects epithelial cells from stress- and TNFα-induced apoptosis by inhibition of stress kinases and provide the first evidence that AKT inhibits stress kinase JNK through activation of the NFκB pathway.

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‐surfactant 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.

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.