A p53 homologue and a novel serine proteinase inhibitor are over-expressed in lung squamous cell carcinoma.

LSCC is a common type of lung cancer and accounts for approximately 30% of all lung cancers. We have used a combination of subtraction and cDNA microarray technology to identify genes preferentially over-expressed in LSCC. Here we report extensive molecular characterization of two novel full-length cDNA sequences, L530S and L531S. Although L530S and L531S were found to be differentially over-expressed in LSCC, the expression profiles for these two genes were not identical. L530S expression was specifically elevated in LSCC whereas L531S transcript was up regulated in both LSCC and head and neck squamous cell carcinoma samples. L530S is a homologue of p53, and L531S belongs to a new member of serine proteinase inhibitors with significant homology to SCCA1 and SCCA2. Furthermore, L531S protein was found to be expressed in lung cancers by IHC analysis. The distinct as well as similar expression profiles exhibited by L530S and L531S suggest that each gene may play a unique role for tumorgenesis of LSCC. Identification of these genes not only allows us to further explore their diagnostic and therapeutic potentials for LSCC, but also provides us with additional tools and reagents for understanding the biology behind LSCC, and differentiating LSCC from other types of lung cancer at the molecular level.

Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy.

BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.

WT1-specific serum antibodies in patients with leukemia.

WT1 is an oncogenic protein expressed by the Wilms' tumor gene and overexpressed in the majority of acute myelogenous leukemias (AMLs) and chronic myelogenous leukemias (CMLs). The current study analyzed the sera of patients with AML and CML for the presence of antibodies to full-length and truncated WT1 proteins. Sixteen of 63 patients (25%) with AML had serum antibodies reactive with WT1/full-length protein. Serum antibodies from all 16 were also reactive with WT1/NH2-terminal protein. By marked contrast, only 2 had reactivity to WT1/COOH-terminal protein. Thus, the level of immunological tolerance to the COOH terminus may be higher than to the NH2 terminus. The WT1/COOH-terminal protein contains four zinc finger domains with homology to other self-proteins. By implication, these homologies may be related to the increased immunological tolerance. Results in patients with CML were similar with antibodies reactive to WT1/full-length protein detectable in serum of 15 of 81 patients (19%). Antibodies reactive with WT1/NH2-terminal protein were present in the serum of all 15, whereas antibodies reactive with WT1/COOH-terminal protein were present in only 3. By contrast to results in leukemia patients, antibodies reactive with WT1/full-length protein were detected in only 2 of 96 normal individuals. The greater incidence of antibody in leukemia patients provides strong evidence that immunization to the WT1 protein occurred as a result of patients bearing malignancy that expresses WT1. These data provide further stimulus to test therapeutic vaccines directed against WT1 with increased expectation that the vaccines will be able to elicit and/or boost an immune response to WT1.

Chemical deglycosylation can induce methylation, succinimide formation, and isomerization.

Interpretation of deglycosylation studies relies heavily on the absence of modifications to the polypeptide chain. We have found that by using a common chemical deglycosylation technique, one can effect at least three changes in a peptide's structure: methylation, isomerization, and ring formation. It was determined that the conditions of chemical deglycosylation introduce a +14 Da shift in the masses of our model peptides, RKDVY, RKEVY, and horseradish peroxidase. This shift is localized to acidic functional groups and is interpreted as methylation of the free carboxylates in our models. An additional shift in mass of -18 Da is found in the model peptide RKDVY consistent with the loss of water associated with succinimide ring formation in this peptide. Chemical treatment induced isomerization of aspartyl residues to isoaspartyl residues in another model peptide, tetragastrin. These results indicate that one should use caution when interpreting the results of chemical deglycosylation experiments.

Cloning of a Mycobacterium tuberculosis gene encoding a purifed protein derivative protein that elicits strong tuberculosis-specific delayed-type hypersensitivity.

The purified protein derivative (PPD) skin test has been used for the diagnosis of tuberculosis for more than 75 years. However, the test lacks specificity because all mycobacteria share antigens present in PPD. Therefore, sensitization with nontuberculous pathogenic or with environmental nonpathogenic mycobacteria can lead to positive skin tests. This communication describes a novel PPD protein present only in tuberculous complex mycobacteria. A recombinant protein was obtained and named DPPD on the basis of the first 4 amino acids of its N-terminus sequence. DPPD elicited delayed-type hypersensitivity (DTH) in 100% of Mycobacterium tuberculosis-infected guinea pigs but in no animals sensitized with several organisms representative of all members of the Mycobacterium genus. Preliminary results indicate that DPPD induces strong and specific DTH in humans. This work points to the definition of a single recombinant M. tuberculosis protein that may be an alternative to the PPD test.

Identification of differentially expressed genes in human prostate cancer using subtraction and microarray.

We have identified human prostate cancer- and tissue-specific genes using cDNA library subtraction in conjunction with high throughput microarray screening. Subtracted cDNA libraries of prostate tumors and normal prostate tissue were generated. Characterization of subtracted libraries showed enrichment of both cancer- and tissue-specific genes. Highly redundant clones were eliminated by colony hybridization. The remaining clones were selected for microarray to determine gene expression levels in a variety of tumor and normal tissues. Clones showing overexpression in prostate tumors and/or normal prostate tissues were selected and sequenced. Here we report the identification of two genes, P503S and P504S, from subtracted libraries and a third gene, P510S, by subtraction followed by microarray screening. Their expression profiles were further confirmed by Northern blot, real-time PCR (TaqMan), and immunohistochemistry to be overexpressed in prostate tissues and/or prostate tumors. Full-length cDNA sequences were cloned, and their subcellular locations were predicted by a bioinformatic algorithm, PSORT, to be plasma membrane proteins. The genes identified through these approaches are potential candidates for cancer diagnosis and therapy.

Molecular cloning, expression, and immunogenicity of MTB12, a novel low-molecular-weight antigen secreted by Mycobacterium tuberculosis.

Proteins secreted into the culture medium by Mycobacterium tuberculosis are thought to play an important role in the development of protective immune responses. In this report, we describe the molecular cloning of a novel, low-molecular-weight antigen (MTB12) secreted by M. tuberculosis. Sequence analysis of the MTB12 gene indicates that the protein is initially synthesized as a 16.6-kDa precursor protein containing a 48-amino-acid hydrophobic leader sequence. The mature, fully processed form of MTB12 protein found in culture filtrates has a molecular mass of 12. 5 kDa. MTB12 protein constitutes a major component of the M. tuberculosis culture supernatant and appears to be at least as abundant as several other well-characterized culture filtrate proteins, including members of the 85B complex. MTB12 is encoded by a single-copy gene which is present in both virulent and avirulent strains of the M. tuberculosis complex, the BCG strain of M. bovis, and M. leprae. Recombinant MTB12 containing an N-terminal six-histidine tag was expressed in Escherichia coli and purified by affinity chromatography. Recombinant MTB12 protein elicited in vitro proliferative responses from the peripheral blood mononuclear cells of a number of purified protein derivative-positive (PPD+) human donors but not from PPD- donors.

Identification of rat prostatic steroid-binding protein as a target antigen of experimental autoimmune prostatitis: implications for prostate cancer therapy.

The long term goal of this study is to develop autoimmune prostatitis as a therapy for prostate cancer. An immune attack capable of destroying normal prostate epithelial cells should also destroy malignant prostate tissue and provide therapeutic benefit in cancer patients. The current study was initiated to identify antigenic targets for experimental autoimmune prostatitis on the assumption that such proteins might also be suitable targets for immunotherapy of prostate cancer. Male Lewis rats were immunized with syngeneic prostate homogenates, and the immune sera were used to screen prostate proteins for immunoreactivity by Western blot analysis. The dominant protein recognized by the immune sera was purified by ion exchange chromatography and reverse phase HPLC. Microsequence analysis of two polypeptide components of this immunodominant protein demonstrated N-terminal sequences identical with two of the three component chains of rat prostatic steroid-binding protein (PSBP). T cell responses to PSBP were also detected in rats immunized with prostate homogenate. Immunizing male rats with purified PSBP induced vigorous Ab and T cell responses. Significant prostate inflammation was observed in some rats immunized with PSBP. Adoptive transfer of T cells immune to PSBP induced rapid and severe destructive autoimmune prostatitis. These results demonstrate that PSBP is a major target Ag of experimental autoimmune prostatitis in a rat model and may serve as a target Ag for vaccine and T cell therapy against prostate cancer.

Molecular cloning of a major cockroach (Blattella germanica) allergen, Bla g 2. Sequence homology to the aspartic proteases.

Inhalation of allergens produced by the German cockroach (Blattella germanica) elicits IgE antibody formation and the development of asthma in genetically predisposed individuals. We compared the allergenic importance of two cockroach (CR) allergens, Bla g 1 and Bla g 2, and determined the complete amino acid sequence of the major 36-kDa allergen, Bla g2. A survey of 106 sera from CR allergic patients showed the prevalence of IgE antibodies to Bla g 1 and Bla g 2 to be 30.2% and 57.6%, respectively. Immediate skin tests on 7 selected patients gave positive reactions using 10(-3) micrograms/ml either allergen, whereas controls showed no response to 10 micrograms/ml. Natural Bla g 2 was purified and the sequence of the NH2 terminus and tryptic peptides, comprising 36% of the molecule, was determined. The cDNA for Bla g 2 was cloned from a B. germanica expression library and encoded a 24-amino acid signal peptide and a 328-amino acid mature protein, which showed the highest degree of identity to mosquito (Aedes aegypti) lysosomal aspartic protease (30.8%), with similar identity to pepsin, cathepsins D and E, renin, and chymosin. Bla g 2 mRNA and protein were detected in B. germanica, but not in Periplaneta americana, the other principal domiciliary CR species in the U.S. High concentrations of Bla g 2 were found in CR digestive organs (esophagus, gut, and proventriculus). The results show that Bla g 2 is a major species-specific allergen of B. germanica and suggest that the allergen functions as a digestive enzyme in the cockroach.

Structural analysis of the light subunit of the Entamoeba histolytica galactose-specific adherence lectin.

Adherence of Entamoeba histolytica trophozoites to colonic mucins and resistance to lysis by the membrane attack complex of complement are mediated by a galactose- and N-acetyl-D-galactosamine-specific cell-surface lectin. This lectin is a heterodimeric glycoprotein of heavy (170 kDa) and light (35/31 kDa) subunits. In this work, the amino acid sequence and membrane anchor of the light subunit were analyzed. The light subunit cDNA encoded a protein with a calculated molecular mass of 32 kDa containing two potential sites for N-linked glycosylation and putative amino- and carboxyl-terminal signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored proteins. No classical carbohydrate-binding domains common to C- or S-type eukaryotic lectins were detected by sequence analysis of either the heavy or light subunits, leaving the location of the ligand-binding site of the lectin unknown. Analysis of restriction enzyme-digested E. histolytica DNA by Southern blotting was consistent with the presence of more than one light subunit gene. Two light subunit isoforms of 31 and 35 kDa were identified by SDS-polyacrylamide gel electrophoresis analysis of affinity-purified lectin, and the isoforms were shown on two-dimensional gel analysis to form distinct 170/35- and 170/31-kDa heterodimers. The amino acid compositions and cyanogen bromide peptide patterns of the two light subunit isoforms were nearly identical. The 35-kDa isoform labeled more efficiently than the 31-kDa isoform with [3H]glucosamine, while only the 31-kDa isoform labeled with [3H]myristate and [3H]palmitate. Nitrous acid deamination released lipid from the 31-kDa isoform, which co-migrated on thin layer chromatography with acylphosphatidylinositol, a component of some GPI anchors. Gas chromatography and mass spectrometry of the deamination product from the 31-kDa subunit identified both myo- and chiro-inositols, supporting the presence of a GPI membrane anchor. The covalent association of a transmembrane protein with a GPI-anchored protein, as suggested by the cDNA sequences of the lectin heavy and light subunits, is novel and suggests unique roles for the two subunits in the pathogenesis of amebiasis.

Recent advances in the expression of foreign genes in Pichia pastoris.

The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. Recent advances in our understanding and application of the system have improved its utility even further. These advances include: (1) methods for the construction of P. pastoris strains with multiple copies of AOX1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P. pastoris secreted proteins. In this review, these advances along with basic features of the P. pastoris system are described and discussed.

Rapid comparison of the cytochrome c3 gene from nine strains of Desulfovibrio vulgaris using polymerase chain reaction amplification.

Polymerase chain reaction amplification was used to compare different regions of the cytochrome c3 gene from nine strains of Desulfovibrio vulgaris, to examine homology within the species. Six 30-base polymerase chain reaction primers and three probes were synthesized on the basis of the published nucleic acid sequence of the cytochrome c3 gene from D. vulgaris, NCIMB 8303. Amplifications were performed on genomic DNA isolated from NCIMB 8303 as well as eight other strains. Six strains, NCIMB 8302, 8305, 8306, 8311, 11779, and DSM 2119, showed amplification products of equal size and quantity to those of strain 8303. Two other strains, NCIMB 8456 and DSM 1744, either showed reduced levels or no detectable amplification products. These results were confirmed by hybridization of amplified DNA to radiolabeled probes specific for each product. DNA sequencing of a 145-bp polymerase chain reaction fragment from strains NCIMB 8302, 8303, 11779, and DSM 2119 revealed complete sequence homology between these strains, whereas slight differences were seen with strain NCIMB 8456. Amino acid sequencing of the first 20 residues of cytochrome c3 purified from strains NCIMB 8456 and 8303 also showed differences in the two proteins. In contrast to the results obtained with strain NCIMB 8456, limited homology was observed between the first 20 amino acid residues of cytochrome c3 from strain DSM 1744 and strain NCIMB 8303.

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening.

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.

High level expression, purification, and characterization of the Kunitz-type protease inhibitor domain of protease nexin-2/amyloid beta-protein precursor.

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.

Gene expression in yeast: Pichia pastoris.

Recent studies have shown the versatility and utility of the Pichia pastoris expression system. Improvements in strains have boosted the yield of proteins and peptides to the commercially feasible range. The Pichia pastoris expression system will soon be used to manufacture proteins for human clinical trials.

Sequence of a cysteine-rich galactose-specific lectin of Entamoeba histolytica.

Entamoeba histolytica trophozoites adhere to human colonic mucins and epithelial cells by a cell surface galactose-specific lectin. This lectin, which is composed of two subunits linked by disulfide bonds, has been shown to be a protective antigen in an animal model of amebiasis. We have determined the sequence of the mature form of the 170-kDa heavy subunit from cDNA clones and PCR-amplified fragments. The heavy subunit sequence consisted of a putative extracellular domain containing 1209 amino acids with 16 potential sites for N-linked glycosylation, a 26-amino acid hydrophobic region, and a 41-amino acid cytoplasmic tail. The presence of N-linked oligosaccharides was confirmed by culturing amebae with tunicamycin, which resulted in a decrease in the heavy subunit molecular mass to 160 kDa and a loss of lectin activity. The extracellular domain was remarkable for an extensive cysteine-rich domain that shared identify with similar regions of several other cell surface proteins and appeared to confer protease resistance to the subunit.

Structural characterization of the dihydropyridine-sensitive calcium channel alpha 2-subunit and the associated delta peptides.

Upon disulfide bond reduction, the alpha 2-subunit of the dihydropyridine-sensitive Ca2+ channel undergoes a characteristic mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with the concurrent appearance of the three delta peptides delta 1 (25,000 Da), delta 2 (22,000 Da), and delta 3 (17,000 Da). Densitometric scanning of Coomassie Blue-stained gels shows a stoichiometric ration of 1.0:0.31.47:0.08 for the alpha 2-subunit and the delta peptides 1, 2, and 3, respectively. Characterization of the delta peptides using antibodies, photoincorporation of a hydrophobic probe, and lectin staining shows tham to be antigenically similar hydrophobic glycoproteins. Amino-terminal sequence analysis of the delta peptides reveals three identical sequences that match the predicted amino acid sequence of the alpha 2-subunit starting at Ala935. Enzymatic deglycosylation of the reduced alpha 2.delta complex produces individual core peptides of 105,000 and 17,000 Da, respectively. Treatment of skeletal muscle membranes with high pH in the presence of reducing agents is able to extract the larger amino-terminal peptide but not the smaller carboxyl (delta) peptide, consistent with a single transmembrane domain in the carboxyl (delta) region. The data support a model of the alpha 2-subunit in which the propeptide is processed into two chains that remain attached through disulfide linkages.

Primary structure of the gamma subunit of the DHP-sensitive calcium channel from skeletal muscle.

Affinity-purified, polyclonal antibodies to the gamma subunit of the dihydropyridine (DHP)-sensitive, voltage-dependent calcium channel have been used to isolate complementary DNAs to the rabbit skeletal muscle protein from an expression library. The deduced primary structure indicates that the gamma subunit is a 25,058-dalton protein that contains four transmembrane domains and two N-linked glycosylation sites, consistent with biochemical analyses showing that the gamma subunit is a glycosylated hydrophobic protein. Nucleic acid hybridization studies indicate that there is a 1200-nucleotide transcript in skeletal muscle but not in brain or heart. The gamma subunit may play a role in assembly, modulation, or the structure of the skeletal muscle calcium channel.

Peptide mapping of conformational epitopes in a human malarial parasite heat shock protein.

A protein of 75 kDa is found in large quantities throughout the blood stages of the human malarial parasite, Plasmodium falciparum. Based on a partial amino acid sequence for p75, previously deduced from a cDNA clone encoding approximately 40% of the molecule, secondary structural predictions were made. The potential role of long range effects on the tertiary structure of the protein stabilized by disulfide bridges was determined by reduction and alkylation of the fusion protein. Five regions were then chosen for peptide modeling. Peptides of 16, 28, 49, 64, and 76 residues were synthesized and used to immunize rabbits. All but the 16-residue peptides were capable of stimulating boostable IgG antibody responses in rabbits, but the antibody produced against the 49 mer did not react with the native parasite protein. Thus, the 28, 64, and 76 residue peptides represent good immunologic models for portions of the P. falciparum 75-kDa protein capable of stimulating both T and B cells in rabbits. The peptides were also used to probe whether any of the selected regions contain epitopes which react with antibodies from owl monkeys immune to P. falciparum. Of these peptides, two were found to be consistently recognized in ELISA by four owl monkey antisera raised in response to malarial infection. Because these two peptides model a cysteine-containing region of the protein, owl monkey sera were also used as probes of the importance of disulfide bonding in maintaining the native structure. The results obtained were consistent with a folding pattern for p75 that incorporates a disulfide bond between cysteines 161 and 194. These results also suggest that most of the epitopes recognized in this part of p75 by the immune system of the monkey are created by folding of the molecule.