A431 Cell Lysates Research Articles

Comparisons of (±)-Benzo[a]pyrene-trans-7,8-Dihydrodiol Activation by Human Cytochrome P450 and Aldo−Keto Reductase Enzymes: Effect of Redox State and Expression Levels

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are metabolically activated to proximate carcinogenic trans-dihydrodiols. PAH trans-dihydrodiols are further activated in humans by cytochrome P450 (P450) 1A1 and 1B1 to yield diol-epoxides or by aldo-keto reductases (AKR) 1A1 and 1C1-1C4 to yield reactive and redox-active o-quinones. Reconstituted in vitro systems were used to compare the steady-state kinetic constants for human P450 (P450 1A1 and 1B1) and AKR (AKR1A1, AKR1C1-1C4) mediated metabolism of (+/-)- trans-7,8-dihydroxy-7,8-dihydrobenzo[ a]pyrene ((+/-)-B[ a]P-7,8-diol) at physiological pH. It was found that P450 isoforms yielded much greater k cat/ K m values than AKR enzymes. Initial rates of (+/-)-B[ a]P-7,8-diol oxidation were measured for AKR1A1, AKR1C2, P450 1A1, and P450 1B1 as the ratio of NADPH/NAD (+) cofactors was varied to determine the redox state necessary for AKRs to successfully compete for trans-dihydrodiols. P450 and AKR enzymes equally competed for (+/-)-B[ a]P-7,8-diol substrate at an NADPH/NAD (+) ratio equal to 0.001. The resting NADPH/NAD (+) ratio was determined in A549 human lung adenocarcinoma cells to be 0.28. These data suggest that the P450 pathway would be favored over the AKR pathway if the enzymes were equally expressed. Basal mRNA transcript levels of AKR1C1-1C3 exceed those of both basal and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)-induced P450 1A1 and 1B1 by up to 90-fold in A549 cells as measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods. AKR expression levels were comparable to TCDD-induced P450 1A1 and 1B1 in HBEC-KT immortalized normal human bronchial epithelial cells. Functional assays of both A549 and HBEC-KT cell lysates demonstrated a lack of TCDD-inducible P450 1A1/1B1 activity but robust basal expression of AKR1A1 and AKR1C activities, where the functional assay for P450 detection is 300-fold more sensitive than the functional assay for AKR isoforms. These data suggest that AKR enzymes may effectively compete with P450 1A1/1B1 for PAH trans-dihydrodiol activation in human lung cells.

DJ-1 Decreases Bax Expression through Repressing p53 Transcriptional Activity

DJ-1, originally identified as an oncogene product, is a protein with various functions in cellular transformation, oxidative stress response, and transcriptional regulation. Although previous studies suggest that DJ-1 is cytoprotective, the mechanism by which DJ-1 exerts its survival functions remains largely unknown. Here we show that DJ-1 exerts its cytoprotection through inhibiting p53-Bax-caspase pathway. DJ-1 interacts with p53 in vitro and in vivo. Overexpression of DJ-1 decreases the expression of Bax and inhibits caspase activation, whereas knockdown of DJ-1 increases Bax protein levels and accelerates caspase-3 activation and cell death induced by UV exposure. Our data provide evidence that the protective effects of DJ-1 on apoptosis are associated with its ability of decreasing Bax level through inhibiting p53 transcriptional activity.

A new subtype-specific monoclonal antibody for IAP-survivin identifies high-risk patients with diffuse large B-cell lymphoma and improves the prognostic value of bcl-2

Anti-apoptotic factors including IAP-survivin and bcl-2 are involved in carcinogenesis and predict for disease outcome for patients with cancer. We used RT-PCR and specific primers to generate two recombinant IAP-survivin proteins; one encoding for the full-length protein and the second comprising the survivin sequence incorporating amino acids 98 to 142. Both proteins were used to immunize mice and as capture antigens to screen NS1/immune splenocyte hybridoma supernatants for anti-survivin antibody in ELISA assays. The antibody designated F2-9C3 was most effective and reacted with both recombinant proteins and with the native protein present in lysates of A549 (lung carcinoma) and Jurkat cells in Western blots, immunoprecipitation and formalin-fixed tissue sections. Immunohistochemical staining of normal and neoplastic tissues showed association of the F2-9C3 antibody with the mitotic spindles. Expression of survivin was not detected elsewhere in sections of normal tissue while all neoplastic tissues examined, including those from patients with diffuse large B-cell lymphoma (DLBCL), showed significant expression of survivin. The intensity and localization of staining in these tumours varied and was observed in cytoplasm and/or nuclei. High nuclear expression of survivin predicted the disease outcome in patients with DLBCL. This association was evident when relating intensity to patient survival (p=0.0321) and strengthened when a score was calculated based on both staining intensity and the proportion of the reactive tumour cells (p=0.0128; reduction in the mean survival times: 35% and 46%, respectively). Elevated expression of bcl-2 protein also identified the high-risk patients (p=0.0095; reduction in mean survival time: 37%). Over-expression of both factors was a more powerful indicator of poor prognosis than either marker alone (p=0.0054, 70% reduction in mean survival time). In conclusion, our novel F2-9C3 monoclonal antibody is effective in determination of expression of IAP-survivin in neoplastic tissue. Nuclear overexpression of IAP-survivin using this antibody predicts the disease outcome in patients with DLBCL and significantly improves the predictive power of bcl-2 in these patients.

RelA/NF-κB transcription factor associates with α-actinin-4

The NF-κB/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-κB in the cytoplasm and the transport mechanism to the nucleus. We found that NF-κB is associated with the actin-binding protein α-actinin-4. NF-κB and α-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-α, α-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-α led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-κB co-immunoprecipitated α-actinin-4 from A431 cell lysates and nuclear extracts, but α-actinin-1 and β-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-κB can bind to matrix-bound chicken gizzard α-actinin. We suggest that the α-actinin-4 is important for the NF-κB nuclear translocation and its functions inside the nucleus.

Autoantibody to alanyl‐tRNA synthetase in patients with idiopathic pulmonary fibrosis

The pathogenesis of IPF is unknown and it is hypothesized that immunological responses are involved. The purpose of this study was to detect autoantibodies in IPF patients and to identify the relevant antigens. Sera from 37 healthy subjects and 22 IPF patients who had no clinical symptoms of collagen vascular disease were examined for immunostaining of A549 human type II cells and human lung tissue. Immunoprecipitation and proteome analysis were performed to identify the antigen. Fifty per cent of the patient sera and none of the control sera exhibited positive staining. Sera from 10 of the 22 IPF patients showed positive immunohistochemistry and immunoprecipitated a 110-kDa protein from the A549 cell lysate. Sera from only two of 41 patients with collagen vascular disease showed positive immunoreactivity. Proteome analysis using tandem mass spectrometry revealed that the protein was alanyl-tRNA synthetase. Transfection of cDNA of this enzyme into CHO-K1 cells conferred positive staining on these cells with the patients' IgG. The 135-kDa fusion protein consisting of 108-kDa enzyme protein and 27-kDa YFP from the cell lysate of the transfected cells was immunoprecipitated by the patient IgG. In addition, sera from IPF patients significantly inhibited the enzyme activity of alanyl-tRNA synthetase. A significant number of IPF patients possess circulating autoantibodies against alanyl-tRNA synthetase, suggesting the involvement of an autoimmune background in the pathogenesis of IPF.

Protein Kinase Cζ Abrogates the Proapoptotic Function of Bax through Phosphorylation

Protein kinase Czeta (PKCzeta) is an atypical PKC isoform that plays an important role in supporting cell survival but the mechanism(s) involved is not fully understood. Bax is a major member of the Bcl-2 family that is required for apoptotic cell death. Because Bax is extensively co-expressed with PKCzeta in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells, it is possible that Bax may act as the downstream target of PKCzeta in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKCzeta activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKCzeta specific inhibitor (a myristoylated PKCzeta pseudosubstrate peptide). Purified, active PKCzeta directly phosphorylates Bax in vitro. Overexpression of wild type or the constitutively active A119D but not the dominant negative K281W PKCzeta mutant results in Bax phosphorylation at serine 184. PKCzeta co-localizes and interacts with Bax at the BH3 domain. Specific depletion of PKCzeta by RNA interference blocks nicotine-stimulated Bax phosphorylation and enhances apoptotic cell death. Intriguingly, forced expression of wild type or A119D but not K281W PKCzeta mutant results in accumulation of Bax in cytoplasm and prevents Bax from undergoing a conformational change with prolonged cell survival. Purified PKCzeta can directly dissociate Bax from isolated mitochondria of C2-ceramide-treated cells. Thus, PKCzeta may function as a physiological Bax kinase to directly phosphorylate and interact with Bax, which leads to sequestration of Bax in cytoplasm and abrogation of the proapoptotic function of Bax.

Phosphorylation of Pirh2 by Calmodulin-dependent kinase II impairs its ability to ubiquitinate p53

Although the recently identified Pirh2 protein is known as a p53-induced ubiquitin-protein E3 ligase, which negatively regulates p53, the detailed mechanism underlying the regulation of Pirh2 remains largely unknown. Here, we demonstrate that while Pirh2 is mostly detected in the phosphorylated form in normal tissues, it is predominantly present in the unphosphorylated form in majority of tumor cell lines and tissues examined. Phosphorylated Pirh2 is far more unstable than its unphosphorylated form. We further identified that Calmodulin-dependent kinase II (CaMK II) phosphorylates Pirh2 on residues Thr-154 and Ser-155. Phosphorylation of Pirh2 appears to be regulated through cell cycle-dependent mechanism. CaMK II-mediated Pirh2 phosphorylation abrogates its E3 ligase activity toward p53. Together, our data suggest that phosphorylation of Pirh2 may act as a fine-tuning to maintain the balance of p53-Pirh2 autoregulatory feedback loop, which facilitates the tight regulation of p53 stability and tumor suppression.

Enrichment of phosphorylated proteins from cell lysate using a novel phosphate‐affinity chromatography at physiological pH

While phosphoproteins have attracted great interest toward the post-genome research (e.g. clinical diagnosis and drug design), there have been few procedures for the specific enrichment of native phosphoproteins from cells or tissues. Here, we describe a simple and efficient protocol to enrich phosphoproteins comprehensively from a complex mixture containing solubilized cellular proteins. This method is based on immobilized metal affinity chromatography using a phosphate-binding tag molecule (i.e. a dinuclear zinc(II) complex) attached on a highly cross-linked agarose. The binding, washing, and elution processes were all conducted without a detergent or a reducing agent at pH 7.5 and room temperature. An additive, 1.0 M CH3COONa, was necessary in the binding and washing buffers (0.10 M Tris-CH3COOH, pH 7.5) to prevent the nonphosphorylated protein from binding. The absorbed phosphoproteins were eluted using a mixed buffer solution (pH 7.5) consisting of 0.10 M Tris-CH3COOH, 10 mM NaH2PO4-NaOH, and 1.0 M NaCl. In this study, we demonstrate a typical example of phosphate-affinity chromatography using an epidermal growth factor-stimulated A431 cell lysate. The total time for the column chromatography (1 mL gel scale) was less than 1 h. The strong enrichment of the phosphoproteins into the elution fraction was evaluated using SDS-PAGE followed by Western blotting analysis.

Antitumor Activity of ZSTK474, a New Phosphatidylinositol 3-Kinase Inhibitor

We previously synthesized a novel s-triazine derivative, ZSTK474 [2-(2-difluoromethylbenzimidazol-1-yl)-4,6-dimorpholino-1,3,5-triazine], that strongly inhibited the growth of tumor cells. We identified its molecular target, investigated its effects on cellular signaling pathways, and examined its antitumor efficacy and toxicity in vivo. We used COMPARE analysis of chemosensitivity measurements from 39 human cancer cell lines and identified phosphatidylinositol 3-kinase (PI3K) as a molecular target for ZSTK474. PI3K was immunoprecipitated from A549 cell lysates, and its activity was measured by assessing the incorporation of 32P into phosphatidylinositol. We used the crystal structure of the PI3K-LY294002 complex to model the binding of ZSTK474 to PI3K (where LY294002 is a known PI3K inhibitor). PI3K downstream activity was analyzed by immunoblotting. Antitumor activity of ZSTK474 was examined against A549, PC-3, and WiDr xenografts in nude mice. Phosphorylation of Akt, a serine/threonine protein kinase and a major signaling component downstream of PI3K, was assessed in vivo by immunohistochemistry. PI3K was identified as a molecular target for ZSTK474 by COMPARE analysis. We confirmed that ZSTK474 directly inhibited PI3K activity more efficiently than the PI3K inhibitor LY294002. At concentrations of 1 microM, ZSTK474 and LY2194002 reduced PI3K activity to 4.7% (95% confidence interval [CI] = 3.2% to 6.1%) and 44.6% (95% CI = 38.9% to 50.3%), respectively, of the untreated control level. Molecular modeling of the PI3K-ZSTK474 complex indicated that ZSTK474 could bind to the ATP-binding pocket of PI3K. ZSTK474 inhibited phosphorylation of signaling components downstream from PI3K, such as Akt and glycogen synthase kinase 3beta, and mediated a decrease in cyclin D1 levels. ZSTK474 administered orally to mice had strong antitumor activity against human cancer xenografts without toxic effects in critical organs. Akt phosphorylation was reduced in xenograft tumors after oral administration of ZSTK474. ZSTK474 is a new PI3K inhibitor with strong antitumor activity against human cancer xenografts without toxic effects in critical organs. ZSTK474 merits further investigation as an anticancer drug.

Hemoglobin Is Expressed by Alveolar Epithelial Cells

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the alpha- and beta-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both alpha- and beta-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

The N‐terminus of phosphoinositide 3‐kinase‐C2β regulates lipid kinase activity and binding to clathrin

The class II phosphoinositide 3-kinase (PI3K)-C2beta is recruited to polypeptide growth factor receptors following ligand stimulation. In contrast to the class I A p85/p110 heterodimers, this interaction is dependent upon proline residues present within the N-terminal sequence of the 3-phosphoinositide kinase. However, the mechanism by which PI3K-C2beta catalytic activity is regulated currently remains unknown. In many tumours, increased expression of ErbB receptors confers a poor prognosis. We demonstrate that increased expression of EGFR enhanced its association with PI3K-C2beta following stimulation with EGF. Deletion of the first proline rich region within the N-terminus precluded recruitment of PI3K-C2beta to activated EGFR. Although deletion of the first proline rich motif also rendered the enzyme catalytically inactive, further deletions (residues 1-148 and 1-261) that removed the second and third proline rich motifs increased kinase activity. These data confirm a regulatory role for the N-terminus of class II PI3K enzymes suggesting that catalytic activity is regulated by factors that associate with this region during recruitment to activated growth factor receptors. Using an N-terminal PI3K-C2beta-GST fusion protein, clathrin heavy chain was affinity purified from A431 cell lysates. Association of PI3K-C2beta with clathrin was confirmed by co-immunoprecipitation from cell lysates while intracellular co-localisation of PI3K-C2beta and clathrin was confirmed by confocal microscopy. Our findings demonstrate for the first time that the PI3K-C2beta isoform associates with clathrin and thus provides a link between receptor mediated intracellular signalling and clathrin coated vesicle transport.

Comparison of Elisa-Based Tyrosine Kinase Assays for Screening EGFR Inhibitors

Receptor tyrosine kinases (PTKs) play key roles in the pathogenesis of numerous human diseases, including cancer, and therefore PTK inhibitors are currently under intense investigation as potential drug candidates. PTK inhibitor screening data are, however, poorly comparable because of the different assay technologies used. Here we report a comparison of ELISA-based assays for screening epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitory compound libraries to study interassay variations. All assays were based on the same protocol, except for the source of EGFR-TK enzymes. In the first protocol, the enzyme was isolated from A431 cells without affinity purification. In the second protocol, commercial EGFR-TK (Sigma) isolated from A431 cells by affinity-purification was employed. In the third protocol, an enzyme preparation obtained from a recombinant (Baculovirus transfected Sf9 cells) expression system was used. All assays employed the synthetic peptide substrate poly-(Glu, Tyr)1:4 and an ELISA-based system to detect phosphorylated tyrosine residues by a monoclonal antibody. We observed significant differences in both the activity of the enzymes and in the EGFR-TK inhibitory effect of our reference compound PD153035. The differences were significant in case of A431 cell lysate compared to affinity purified EGFR-TKs derived from either A431cells or Baculovirus transfected Sf9 cells, whereas the latter two showed comparable results. Our data suggest that differences in terms of interassay variation are not related to the source of the enzyme but to its purity; changes in the mode of detection can markedly influence the reproducibility of results. In conclusion, normalization of the EGFR activity used for inhibitor screening and standardization of detection methods enable safe comparison of data.

Photopolymerized microtips for sample preparation in proteomic analysis

We demonstrate a novel method for the fabrication of disposable plastic microtips, which we name "EasyTip", by a photopolymerization technique. C18 reversed-phase (C18) and ion metal affinity chromatography (IMAC) beads were immobilized on a plastic pipette tip, made of polypropylene materials, by photo-initiated polymerization. The fabricated EasyTips can be manipulated using commercial pipettes for wash/elution of minute amount of biological samples (< 10 microL) and can be applied for mass spectrometry (MS)-based proteomic analysis, in which the detection sensitivity depends critically on the optimal sample preparation. The recovery of a sample of 25 fmol of tryptic hemoglobin digest loaded in a C18 EasyTip was near 100% and we estimated the loading capacity to be around 0.4-2.0 microg of total proteins or peptides, which is well above a sufficient quantity for MS analysis. The effectiveness of the C18 EasyTips in enhancing the detection sensitivity of matrix-assisted laser desorption/ionization (MALDI)-MS signal, and thus providing a greater sequence coverage, was also demonstrated by the analysis of hemoglobin digest and the in-gel digested epidermal growth factor receptor (EGFR) protein from A431 cell lysate. We also demonstrated the usefulness of the immobilized IMAC EasyTips in extracting the signal of tryptic phosphopeptides of beta-casein (10 pmol) having one and four phosphorylation sites by using an IMAC EasyTip prior to off-line analysis by MS. The combination of IMAC EasyTips and MALDI-MS allowed the unambiguous identification of phosphopeptides based on the phosphatase assay as well as the post-source decay. Compared to other miniaturized devices, this fabrication method is simple, cheap, and requires less human intervention. Moreover, the method of manipulating the EasyTips is straightforward and can be automated readily by a robotic system for high-throughput analysis.

Cytoplasmic dynein mediates adenovirus binding to microtubules.

During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% +/- 3.5% to 80.7% +/- 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding.

755. Cytoplasmic Dynein-Dependent Adeno-Associated Virus Interaction with Microtubules

The importance of adeno-associated virus (AAV)-based gene therapy vectors as therapeutic vehicles is derived in part from efficient cell membrane penetration and translocation of the adeno-associated virus capsid to the nucleus where its genetic cargo is delivered. Other viruses have enhanced intracellular trafficking to the nucleus through interaction with the microtubule cytoskeleton with subsequent energy-dependent translocation in a retrograde manner towards the nucleus. The retrograde molecular motor, cytoplasmic dynein, participates in the microtubule-based nuclear-directed translocation of numerous viruses. Based on the hypothesis that the cytoplasmic dynein motor complex mediates a biochemical linkage of the AAV capsid with microtubules, a microtubule binding assay was developed in which binding of fluorophore-conjugated AAV capsids to microtubules was evaluated by mixing fluorophore-conjugated AAV capsid with polymerized microtubules, centrifuging to pellet the microtubules, and measuring the presence of the AAV-associated fluorescence in either the microtubule-free supernatant or microtubule-containing pellet. SDS-polyacrylamide gel electrophoresis of supernatant and pellet fractions showed that fluorophore-labeled AAV capsid was located predominantly in the supernatant when AAV was incubated with taxol-stabilized microtubules. However, when purified bovine microtubule-associated proteins were combined with microtubules, the majority of AAV was found in the pellet after centrifugation (69 ± 4 % vs 23 ± 4% in the absence of microtubule-associated proteins). The interaction of microtubule-associated proteins and AAV with microtubules did not occur in the presence of 500 mM NaCl. Using freshly isolated A549 lung epithelial cell lysate, the direct involvement of cytoplasmic dynein was evaluated. A549 cell lysate was depleted of endogenous cytoplasmic dynein by immunoprecipitation with a monoclonal antibody against the 74.1 kDa intermediate chain of cytoplasmic dynein. Microtubule-associated proteins isolated from whole A549 cell lysate versus cytoplasmic dynein-depleted A549 cell lysate were compared for the ability to support AAV-microtubule interaction. Whereas AAV capsid pelleted with microtubules when incubated with microtubule-associated proteins from whole A549 cell lysate, AAV capsid was found predominantly in the supernatant when cytoplasmic dynein-depleted cell lysate was used. No effect was observed when an irrelevant primary antibody was used for immunprecipitation. To confirm the involvement of cytoplasmic dynein in AAV-microtubule interaction, the nucleotide-sensitive binding property of cytoplasmic dynein was utilized. Under conditions that are known to disrupt cytoplasmic dynein interaction with microtubules (10 mM ATP), the efficiency of the AAV-microtubule interaction was reduced. However, the presence of an identical concentration of AMP-PNP, a non-hydrolyzable analog of ATP, had no effect on AAV-microtubule interactions. The results of this study suggest that the AAV capsid interacts with microtubules in a cytoplasmic dynein-dependent manner, providing a potential mechanism for the efficient translocation of the AAV capsid to the nucleus during infection.

Interleukin-1β responses to Mycoplasma pneumoniae infection are cell-type specific

Interleukin-1β (IL-1β) is a major proinflammatory cytokine that is involved in many important cellular functions such as proliferation, differentiation, and activation of different cell types. Its mature form is released from the cells in response to various bacterial and viral infections, and it plays a significant role in host defense. Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans following attachment to respiratory epithelium, as well as extrapulmonary infections. Very little is known about the role of cytokines in pathogenesis or the response of target cells to M.pneumoniae attachment. The purpose of this study was to investigate the ability of M. pneumoniae to induce IL-1β in human lung epithelial carcinoma A549 and in human monocytic U937 cell lines. Following M. pneumoniae infection, both IL-1β mRNA and protein were induced in A549 cells vs. no induction in uninfected cells; however, the protein remained inside the A549 cells. Similarly, M. pneumoniae infection strongly increased mRNA and extracellular protein levels in U937 cells, which unlike A549 cells did exhibit baseline constitutive levels. De novo IL-1β protein expression was verified by cycloheximide studies. M. pneumoniae infection did not affect constitutive caspase-1 mRNA or protein levels in either cell line. Reduced caspase-1 activity in A549 cell lysates suggests the presence of an endogenous caspase-1 inhibitory component in the A549 cells. These collective data confirm previous studies that show that M. pneumoniae is a potent inducer of cytokines following adherence to host target cells, and establish that IL-1β release in response to M. pneumoniae infection is cell-type specific, thus emphasizing the importance of carefully considering multiple cell types in M. pneumoniae pathogenesis studies involving both immune cells and cytokine release patterns.

Activation of Polycyclic Aromatic Hydrocarbontrans-Dihydrodiol Proximate Carcinogens by Human Aldo-keto Reductase (AKR1C) Enzymes and Their Functional Overexpression in Human Lung Carcinoma (A549) Cells

Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants and suspected human lung carcinogens. In patients with non-small cell lung carcinoma, differential display shows that aldo-keto reductase (AKR1C) transcripts are dramatically overexpressed. However, whether AKR1C isoforms contribute to the carcinogenic process and oxidize potent PAH trans-dihydrodiols (proximate carcinogens) to reactive and redox active o-quinones is unknown; nor is it known whether these reactions occur in human lungs. We now show that four homogeneous human recombinant aldo-keto reductases (AKR1C1-AKR1C4) are regioselective and oxidize only the relevant non-K region trans-dihydrodiols. However, these enzymes are not stereo-selective, since they oxidized 100% of these racemic substrates. The highest utilization ratios (V(max)/K(m)) were observed for some of the most potent proximate carcinogens known (e.g. 7,12-dimethylbenz[a]anthracene-3,4-diol (DMBA-3,4-diol) and benzo[g]chrysene-11,12-diol). In vitro, DMBA-3,4-diol was oxidized by AKR1C4 to the highly reactive 7,12-dimethylbenz[a]anthracene-3,4-dione (DMBA-3,4-dione), which was trapped in situ as its mono- and bis-thioether conjugates, which arise from the sequential 1,6- and 1,4-Michael addition of thiol nucleophiles. Human multiple tissue expression array analysis showed that AKR1C isoform transcripts were highly expressed in the human lung carcinoma cell line A549. Isoform-specific reverse transcriptase-PCR showed that AKR1C1, AKR1C2, and AKR1C3 transcripts were all expressed. Western blot analysis and functional assays confirmed high expression of AKR1C protein and enzyme activity in these lung cells. A549 cell lysates were found to convert DMBA-3,4-diol to the corresponding o-quinone. In trapping experiments, LC/MS analysis identified peaks in the cell lysates that corresponded to the synthetically prepared mono- and bis-thioether conjugates of DMBA-3,4-dione. This quinone is one of the most electrophilic and redox-active o-quinones produced by AKRs. Its unique ability to form bis-thioether conjugates parallels the formation of bis- and tris-glutathionyl conjugates of hydroquinone, which display end organ toxicity. The ability to measure DMBA-3,4-dione formation in A549 cells implicates the AKR pathway in the metabolic activation of PAH in human lung.

Recruitment of the class II phosphoinositide 3-kinase C2beta to the epidermal growth factor receptor: role of Grb2.

Previously we demonstrated that the class II phosphoinositide 3-kinase C2beta (PI3K-C2beta) is rapidly recruited to a phosphotyrosine signaling complex containing the activated receptor for epidermal growth factor (EGF). Although this association was shown to be dependent upon specific phosphotyrosine residues present on the EGF receptor, the underlying mechanism remained unclear. In this study the interaction between PI3K-C2beta and the EGF receptor is competitively attenuated by synthetic peptides derived from each of three proline-rich motifs present within the N-terminal region of the PI3K. Further, a series of N-terminal PI3K-C2beta fragments, truncated prior to each proline-rich region, bound the receptor with decreased efficiency. A single proline-rich region was unable to mediate receptor association. Finally, an equivalent N-terminal fragment of PI3K-C2alpha that lacks similar proline-rich motifs was unable to affinity purify the activated EGF receptor from cell lysates. Since these findings revealed that the interaction between the EGF receptor and PI3K-C2beta is indirect, we sought to identify an adaptor molecule that could mediate their association. In addition to the EGF receptor, PI3K-C2beta(2-298) also isolated both Shc and Grb2 from A431 cell lysates. Recombinant Grb2 directly bound PI3K-C2beta in vitro, and this effect was reproduced using either SH3 domain expressed as a glutathione S-transferase (GST) fusion. Interaction with Grb2 dramatically increased the catalytic activity of this PI3K. The relevance of this association was confirmed when PI3K-C2beta was isolated by coimmunoprecipitation with anti-Grb2 antibody from numerous cell lines. Using immobilized, phosphorylated EGF receptor, recombinant PI3K-C2beta was only purified in the presence of Grb2. We conclude that proline-rich motifs within the N terminus of PI3K-C2beta mediate the association of this enzyme with activated EGF receptor and that this interaction involves the Grb2 adaptor.

Plk3 functionally links DNA damage to cell cycle arrest and apoptosis at least in part via the p53 pathway.

Polo-like kinase 3 (Plk3, previously termed Prk) contributes to regulation of M phase of the cell cycle (Ouyang, B., Pan, H., Lu, L., Li, J., Stambrook, P., Li, B., and Dai, W. (1997) J. Biol. Chem. 272, 28646-28651). Plk3 physically interacts with Cdc25C and phosphorylates this protein phosphatase predominantly on serine 216 (Ouyang, B., Li, W., Pan, H., Meadows, J., Hoffmann, I., and Dai, W. (1999) Oncogene 18, 6029-6036), suggesting that the role of Plk3 in mitosis is mediated, at least in part, through direct regulation of Cdc25C. Here we show that ectopic expression of a kinase-active Plk3 (Plk3-A) induced apoptosis. In response to DNA damage, the kinase activity of Plk3 was rapidly increased in an ATM-dependent manner, whereas that of Plk1 was markedly inhibited. Recombinant Plk3 phosphorylated in vitro a glutathione S-transferase fusion protein containing p53, but not glutathione S-transferase alone. Recombinant Plk1 also phosphorylated p53 but on residues that differed from those targeted by Plk3. Co-immunoprecipitation and pull-down assays demonstrated that Plk3 physically interacted with p53 and that this interaction was enhanced upon DNA damage. In vitro kinase assays followed by immunoblotting showed that serine 20 of p53 was a target of Plk3. Furthermore, expression of a kinase-defective Plk3 mutant (Plk3(K52R)) resulted in significant reduction of p53 phosphorylation on serine 20, which was correlated with a decrease in the expression of p21 and with a concomitant increase in cell proliferation. These results strongly suggest that Plk3 functionally links DNA damage to cell cycle arrest and apoptosis via the p53 pathway.

αIIb’s cytoplasmic domain is not required for ligand-induced clustering of integrin αIIbβ3

The platelet integrin αIIbβ3 exhibits bidirectional signaling, in that intracellular messengers enable adhesive macromolecules to bind to its ectodomain, while ligation promotes the association of cytoskeletal proteins with its cytoplasmic domains. In order to understand the linkage between these distant regions, we investigated the effects of receptor occupancy on the solution structure of both full-length recombinant αIIbβ3 and αIIbΔ991β3, an integrin truncation mutant which lacks one cytoplasmic domain. Lysates of 35S-labeled human A549 cells expressing either full-length αIIbβ3 or αIIbΔ991β3 were examined by sucrose density gradient sedimentation followed by immunoprecipitation to determine the distributions of integrin protomers and oligomers. Recombinant αIIbβ3 exhibited a weight-average sedimentation coefficient, S w=11.3±1.4 S with 73% sedimenting as protomers/dimers (9.1±1.0 S) and 27% as oligomers (15.4±0.4 S). Truncation mutant αIIbΔ991β3 exhibited a similar pattern with 65% sedimenting as protomers/dimers. Upon ligation with eptifibatide, both full-length αIIbβ3 and αIIbΔ991β3 sedimented mainly at >14 S, indicating 2–3-fold increased oligomerization. Thus we have demonstrated that αIIb’s cytoplasmic region is not required for integrin clustering, a key event in outside-in signaling.