Hannah Alexander

Immunogenic structure of the influenza virus hemagglutinin

We chemically synthesized 20 peptides corresponding to 75% of the HA1 molecule of the influenza virus. Antibodies to the majority (18) of these peptides were capable of reacting with the hemagglutinin molecule. These 18 peptides are not confined to the known antigenic determinants of the hemagglutinin molecule, but rather are scattered throughout its three-dimensional structure. In contrast, antibody raised to intact hemagglutinin did not react with any of the 20 peptides. Taken together these results suggest that the immunogenicity of an intact protein molecule is not the sum of the immunogenicity of its pieces.

Proteomic Analysis to Identify Breast Cancer Biomarkers in Nipple Aspirate Fluid

Purpose: Proteomic analysis of breast nipple aspirate fluid (NAF) holds promise as a noninvasive method to identify markers of breast cancer. The objectives of the study were to: (a) describe the NAF proteome, (b) identify candidate markers of breast cancer in NAF by using proteomic analysis, and (c) validate the markers identified by using a quantitative, high-throughput ELISA analysis. Experimental Design: For proteome analysis, NAF proteins from a single subject without breast cancer were separated by two-dimensional PAGE and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectometry identification. A total of 41 different proteins were identified, 25 of which were known to be secreted. To identify breast cancer markers, we separated 20 NAF samples (10 normal, 10 cancer) by two-dimensional PAGE. Three protein spots were detected that were up-regulated in three or more cancer samples. These spots were identified to be gross cystic disease fluid protein (GCDFP)-15, apolipoprotein D (apoD), and α1-acid glycoprotein (AAG). To validate these three potential biomarkers, 105 samples (53 from benign breasts and 52 from breasts with cancer) were analyzed using ELISA. Results: Among all of the subjects, GCDFP-15 levels were lower (P < 0.001) and AAG levels were higher (P = 0.001) in breasts with cancer. This was also true in premenopausal (GCDFP-15, P = 0.011; AAG, P = 0.002) but not in postmenopausal women. GCDFP-15 levels were lowest (P = 0.003) and AAG levels highest (P < 0.001) in women with ductal carcinoma in situ (DCIS). Menopausal status influenced GCDFP-15 and AAG more in women without breast cancer than in women with breast cancer. apoD levels did not correlate significantly with breast cancer. Conclusions: Our study revealed that the NAF proteome, as defined by two-dimensional PAGE, consists of a limited number of proteins, and that the expression of AAG and GCDFP-15 correlates with disease presence and stage.

(Dihydro)ceramide synthase 1-regulated sensitivity to cisplatin is associated with the activation of p38 mitogen-activated protein kinase and is abrogated by sphingosine kinase 1

Resistance to chemotherapeutic drugs often limits their clinical efficacy. Previous studies have implicated the bioactive sphingolipid sphingosine-1-phosphate (S-1-P) in regulating sensitivity to cisplatin [cis-diamminedichloroplatinum(II)] and showed that modulating the S-1-P lyase can alter cisplatin sensitivity. Here, we show that the members of the sphingosine kinase (SphK1 and SphK2) and dihydroceramide synthase (LASS1/CerS1, LASS4/CerS4, and LASS5/CerS5) enzyme families each have a unique role in regulating sensitivity to cisplatin and other drugs. Thus, expression of SphK1 decreases sensitivity to cisplatin, carboplatin, doxorubicin, and vincristine, whereas expression of SphK2 increases sensitivity. Expression of LASS1/CerS1 increases the sensitivity to all the drugs tested, whereas LASS5/CerS5 only increases sensitivity to doxorubicin and vincristine. LASS4/CerS4 expression has no effect on the sensitivity to any drug tested. Reflecting this, we show that the activation of the p38 mitogen-activated protein (MAP) kinase is increased only by LASS1/CerS1, and not by LASS4/CerS4 or LASS5/CerS5. Cisplatin was shown to cause a specific translocation of LASS1/CerS1, but not LASS4/CerS4 or LASS5/CerS5, from the endoplasmic reticulum (ER) to the Golgi apparatus. Supporting the hypothesis that this translocation is mechanistically involved in the response to cisplatin, we showed that expression of SphK1, but not SphK2, abrogates both the increased cisplatin sensitivity in cells stably expressing LASS1/CerS and the translocation of the LASS1/CerS1. The data suggest that the enzymes of the sphingolipid metabolic pathway can be manipulated to improve sensitivity to the widely used drug cisplatin. (Mol Cancer Res 2007;5(8):801–12)

Molecular basis for resistance to the anticancer drug cisplatin in Dictyostelium

The efficacy of the widely used chemotherapeutic drug cisplatin is limited by the occurrence of drug-resistant tumour cells. To fully exploit the potential of this drug in cancer therapy, it is imperative to understand the molecular basis of cisplatin resistance. Using an insertional mutagenesis technique in cells of Dictyostelium discoideum, we have identified six genes which are involved in cisplatin resistance. None of these genes has been previously linked to resistance to this drug. Several of these genes encode proteins that are involved in signal transduction pathways which regulate cell death, cell proliferation or gene regulation. The resistance of these mutant strains is specific for cisplatin, since deletion of these genes does not confer resistance to other DNA-damaging agents. Significantly, the disruption of three of these genes, encoding the sphingosine-1-phosphate lyase, the RegA cAMP phosphodiesterase and a phosphatidylinositol-4-phosphate 5-kinase, also results in abnormalities in the multicellular development of this organism, although there is no change in the rate of mitotic cell growth. This study has identified previously unsuspected molecular pathways which function in the cellular response to cisplatin and are required for normal morphogenesis, and underscores the complexity of the cellular response to cisplatin. These pathways provide potential targets for modulating the response to this important drug.

Sphingosine-1-Phosphate Lyase Regulates Sensitivity of Human Cells to Select Chemotherapy Drugs in a p38-Dependent Manner

Resistance to cisplatin is a common problem that limits its usefulness in cancer therapy. Molecular genetic studies in the model organism Dictyostelium discoideum have established that modulation of sphingosine kinase or sphingosine-1-phosphate (S-1-P) lyase, by disruption or overexpression, results in altered cellular sensitivity to this widely used drug. Parallel changes in sensitivity were observed for the related compound carboplatin but not for other chemotherapy drugs tested. Sensitivity to cisplatin could also be potentiated pharmacologically with dimethylsphingosine, a sphingosine kinase inhibitor. We now have validated these studies in cultured human cell lines. HEK293 or A549 lung cancer cells expressing human S-1-P lyase (hSPL) show an increase in sensitivity to cisplatin and carboplatin as predicted from the earlier model studies. The hSPL-overexpressing cells were also more sensitive to doxorubicin but not to vincristine or chlorambucil. Studies using inhibitors to specific mitogen-activated protein kinases (MAPK) show that the increased cisplatin sensitivity in the hSPL-overexpressing cells is mediated by p38 and to a lesser extent by c-Jun NH2-terminal kinase MAPKs. p38 is not involved in vincristine or chlorambucil cytotoxicity. Measurements of MAPK phosphorylation and enzyme activity as well as small interfering RNA inhibition studies show that the response to the drug is accompanied by up-regulation of p38 and c-Jun NH2-terminal kinase and the lack of extracellular signal-regulated kinase up-regulation. These studies confirm an earlier model proposing a mechanism for the drug specificity observed in the studies with D. discoideum and support the idea that the sphingosine kinases and S-1-P lyase are potential targets for improving the efficacy of cisplatin therapy for human tumors.

Differential developmental expression and cell type specificity of Dictyostelium catalases and their response to oxidative stress and UV-light.

Cells of Dictyostelium discoideum are highly resistant to DNA damaging agents such as UV-light, gamma-radiation and chemicals. The genes encoding nucleotide excision repair (NER) and base excision repair (BER) enzymes are rapidly upregulated in response to UV-irradiation and DNA-damaging chemicals, suggesting that this is at least partially responsible for the resistance of this organism to these agents. Although Dictyostelium is also unusually resistant to high concentrations of H(2)O(2), little is known about the response of this organism to oxidative stress. To determine if transcriptional upregulation is a common mechanism for responding to DNA-damaging agents, we have studied the Dictyostelium catalase and Cu/Zn superoxide dismutase antioxidant enzymes. We show that there are two catalase genes and that each is differentially regulated both temporally and spatially during multicellular development. The catA gene is expressed throughout growth and development and its corresponding enzyme is maintained at a steady level. In contrast, the catB gene encodes a larger protein and is only expressed during the final stages of morphogenesis. Cell type fractionation showed that the CatB enzyme is exclusively localized to the prespore cells and the CatA enzyme is found exclusively in the prestalk cells. Each enzyme has a different subcellular localization. The unique developmental timing and cell type distribution suggest that the role for catB in cell differentiation is to protect the dormant spores from oxidative damage. We found that exposure to H(2)O(2) does not result in the induction of the catalase, superoxide dismutase, NER or BER mRNAs. A mutant with greatly reduced levels of catA mRNA and enzyme has greatly increased sensitivity to H(2)O(2) but normal sensitivity to UV. These results indicate that the natural resistance to oxidative stress is not due to an ability to rapidly raise the level of antioxidant or DNA repair enzymes and that the response to UV-light is independent from the response to reactive oxygen compounds.

Overexpression of Sphingosine-1-Phosphate Lyase or Inhibition of Sphingosine Kinase in Dictyostelium discoideum Results in a Selective Increase in Sensitivity to Platinum-Based Chemotherapy Drugs

ABSTRACT The efficacy of the chemotherapy drug cisplatin is often limited due to resistance of the tumors to the drug, and increasing the potency of cisplatin without increasing its concentration could prove beneficial. A previously characterized Dictyostelium discoideum mutant with increased resistance to cisplatin was defective in the gene encoding sphingosine-1-phosphate (S-1-P) lyase, which catalyzes the breakdown of S-1-P, an important regulatory molecule in cell function and development and in the regulation of cell fate. We hypothesized that the increased resistance to cisplatin was due to an elevation of S-1-P and predicted that lowering levels of S-1-P should increase sensitivity to the drug. We generated three strains that stably overexpress different levels of the S-1-P lyase. The overexpressor strains have reduced growth rate and, confirming the hypothesis, showed an expression-dependent increase in sensitivity to cisplatin. Consistently, treating the cells with d-erythro-N,N,-dimethylsphingosine, a known inhibitor of sphingosine kinase, increased the sensitivity of mutant and parent cells to cisplatin, while addition of exogenous S-1-P or 8-Br-cyclic AMP made the cells more resistant to cisplatin. The increased sensitivity of the overexpressors to cisplatin was also observed with the cisplatin analog carboplatin. In contrast, the response to doxorubicin, 5-flurouracil, or etoposide was unaffected, indicating that the involvement of the sphingolipid metabolic pathway in modulating the response to cisplatin is not part of a global genotoxic stress response. The augmented sensitivity to cisplatin appears to be the result of an intracellular signaling function of S-1-P, because D. discoideum does not appear to have endothelial differentiation growth (EDG/S1P) receptors. Overall, the results show that modulation of the sphingolipid pathway at multiple points can result in increased sensitivity to cisplatin and has the potential for increasing the clinical usefulness of this important drug.

CERAMIDE SYNTHASE 1 IS REGULATED BY PROTEASOMAL MEDIATED TURNOVER

Ceramide is an important bioactive lipid, intimately involved in many cellular functions, including the regulation of cell death, and in cancer and chemotherapy. Ceramide is synthesized de novo from sphinganine and acyl CoA via a family of 6 ceramide synthase enzymes, each having a unique preference for different fatty acyl CoA substrates and a unique tissue distribution. However, little is known regarding the regulation of these important enzymes. In this study we focus on ceramide synthase 1 (CerS1) which is the most structurally and functionally distinct of the enzymes, and describe a regulatory mechanism that specifically controls the level of CerS1 via ubiquitination and proteasome dependent protein turnover. We show that both endogenous and ectopically expressed CerS1 have rapid basal turnover and that diverse stresses including chemotherapeutic drugs, UV light and DTT can induce CerS1 turnover. The turnover requires CerS1 activity and is regulated by the opposing actions of p38 MAP kinase and protein kinase C (PKC). p38 MAP kinase is a positive regulator of turnover, while PKC is a negative regulator of turnover. CerS1 is phosphorylated in vivo and activation of PKC increases the phosphorylation of the protein. This study reveals a novel and highly specific mechanism by which CerS1 protein levels are regulated and which directly impacts ceramide homeostasis.

Antibodies against synthetic peptides react with the second Epstein-Barr virus-associated nuclear antigen.

Five peptides were synthesized on the basis of amino acid sequences predicted from the transformation‐associated BamHI WYH region of the genome of the Epstein‐Barr virus (EBV). Antisera to two peptides deduced from a 1.6‐kb open reading frame in the BamHI H fragment identified an 87 000‐dalton nuclear polypeptide that was present in EBV‐carrying cell lines that expressed the second EBV‐determined nuclear antigen (EBNA‐2). This polypeptide was not detected in cell lines that carried EBV variants with a deleted BamHI WYH region or in EBV‐negative cell lines. Three peptides deduced from the 1.6‐kb open reading frame reacted with human EBNA‐positive sera, but not with EBNA‐negative sera. Following affinity purification with the peptides, two of the corresponding human antibodies also reacted with the 87 000‐dalton polypeptide.

Proteomics of Canine Lymphoma Identifies Potential Cancer-Specific Protein Markers

Purpose: Early diagnosis of cancer is crucial for the success of treatment of the disease, and there is a need for markers whose differential expression between disease and normal tissue could be used as a diagnostic tool. Spontaneously occurring malignancies in pets provide a logical tool for translational research for human oncology. Lymphoma, one of the most common neoplasms in dogs, is similar to human non–Hodgkins lymphoma and could serve as an experimental model system. Experimental Design: Thirteen lymph nodes from normal dogs and 11 lymph nodes from dogs with B-cell lymphoma were subjected to proteomic analysis using two-dimensional PAGE separation and matrix-assisted laser desorption/ionization time-of-flight analysis. Results: A total of 93 differentially expressed spots was subjected to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis, and several proteins that showed differential expression were identified. Of these, prolidase (proline dipeptidase), triosephosphate isomerase, and glutathione S-transferase were down-regulated in lymphoma samples, whereas macrophage capping protein was up-regulated in the lymphoma samples. Conclusions: These proteins represent potential markers for the diagnosis of lymphoma and should be further investigated in human samples for validation of their utility as diagnostic markers.