Extracellular Gene Products Research Articles

Abstract 5058: Expansion and validation of the prostate-specific antigen substrate degradome.

Abstract Kallikrein-related peptidase 3 (KLK3), better known as prostate-specific antigen (PSA), remains the most important serum biomarker in all of cancer. Its continued expression by tumors throughout the progression of prostate cancer in most patients has led to some speculation as to the possibility of PSA playing an active role in the disease. Although the use of PSA as a biomarker continues to be the subject of great discussion, our understanding of its functions in physiological and pathophysiological contexts is in its infancy. PSA is a secreted serine protease with substrate cleavage preferences similar to chymotrypsin. The principal physiological substrates of PSA in reproduction are semenogelins I and II. Currently, the number of PSA substrates, the so-called substrate degradome, has been identified by a candidate gene approach, and is limited to less than 20 gene products. Our working hypothesis is that the PSA substrate degradome is greatly understated, and that candidate identification by a bioinformatics approach, followed by biochemical validation would serve to dramatically expand the known substrates and help further our understanding of the roles played by this protease in reproduction and prostate cancer biology. Using the Merops database, a series of 32 unique consensus PSA substrate recognition sites was generated, based on reported P4-P1 residues on the N-terminal of known substrate cleavage sites. These peptide sequences were each used to search the PIR (Protein information resource) and IPA (Ingenuity Pathway Analysis) databases for the corresponding genes containing matching sequences. The resulting gene list was consolidated and filtered based on cellular localization. Cell-surface and extracellular gene products were retained for further analysis. Novel, highly active recombinant PSA was expressed, isolated, and used to biochemically validate a subset of the candidate substrates identified in silico. Finally, immunoblotting was used to determine PSA-induced cleavage of candidate substrates. The series of 32 consensus PSA substrate recognition sequences matched almost 10,000 gene products. Of these, approximately 90% were proteins not secreted or found on cell surfaces, and therefore not considered accessible to PSA. The remaining 900 gene products included cytokines, cell surface receptors, proteases, extracellular matrix proteins and immume modulators, many with reported roles in prostate cancer. They were organized by function prior to being subjected to cell-free biochemical validation for time-dependant cleavage. This study has used a combination of bioinformatics-based strategies and biochemistry to dramatically increase the list of potential PSA substrates. The information will be of great use in increasing our understanding of PSA and its functions in reproduction and cancer biology. Citation Format: Sangmi Nam, Niquiche Sangster-Guity, Theodore E. Ewachiw, Samuel R. Denmeade, Simon A. Williams. Expansion and validation of the prostate-specific antigen substrate degradome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5058. doi:10.1158/1538-7445.AM2013-5058

Gene transfer to the pleural mesothelium as a strategy to deliver proteins to the lung parenchyma.

The pleura covers the lung parenchyma, chest wall, and diaphragm with a single layer of flat cells that are easy to genetically modify with adenovirus (Ad) vectors. Although intrapleural gene therapy has been used to treat intrapleural disorders, we hypothesized that it may also be used to deliver extracellular gene products to the lung parenchyma. In this context, this study is based on the concept that administration of adenovirus gene transfer vectors into the pleural cavity will mediate expression of gene products in mesothelial cells, and that the extracellular products produced by these genetically modified cells will reach the lung parenchyma. To assess this concept, Ad(beta)gal (expressing beta-galactosidase [beta-Gal]) or AdLuc (expressing luciferase) was administered into the right pleural cavity of BALB/c mice, as compared with intravenous injection via the jugular vein or the intratracheal route. Histologic assessment of lungs and pleural surface after intrapleural administration of Ad(beta)gal demonstrated beta-Gal expression limited to the pleural mesothelium and cells adjacent to the pleural surface. Right intrapleural administration of AdLuc showed higher level of luciferase in both the right and left lung (right vs. left, p > 0.8), compared with the intratracheal (p < 0.05) or intravenous routes (p < 0.02), that is, unilateral intrapleural administration is sufficient to transfer genes bilaterally to the pleura. To assess the ability of intrapleural gene transfer to modify lung parenchymal processes, CT26.CL25 tumor cells (3 x 10(5)) were injected via the jugular vein to generate tumor metastases in the lung parenchyma followed 24 hr later by administration to the right pleura of 5 x 10(8) PFU of Adsflt (an Ad "antiangiogenesis" vector expressing a soluble, secreted, extracellular portion of the Flt-1 receptor for vascular endothelial growth factor). Compared with phosphate-buffered saline, or the control vector AdNull (no transgene), mice receiving Adsflt by the intrapleural route had a marked suppression of tumor growth in the parenchyma of both lungs as assessed 12 days after tumor administration (p < 0.005). Treatment of preestablished lung metastases with Adsflt administered by the intrapleural route significantly improved long-term survival as compared with control animals (p < 0.0001). Thus, although intrapleural administration of an Ad vector encoding an intracellular protein mediates gene expression only in mesothelial cells and the local tissues, intrapleural delivery of a vector expressing a secreted protein can be used to modify processes throughout the lung parenchyma. In the context that intravascular gene transfer is an ineffective strategy to deliver gene products to the lung parenchyma, and that intratracheal administration is associated with alveolar inflammation secondary to host defenses against Ad vectors, these findings demonstrate that intrapleural administration represents a strategy that can be used to effectively deliver extracellular gene products to the lung parenchyma via a site that is readily accessible, and where inflammation against the vector will not have significant pathophysiologic consequences.

Prodrug-activating systems in suicide gene therapy.

Cancer chemotherapy encompasses a large number of well-documented and clinically established methods for the treatment of malignant diseases. However, the efficacy of these approaches is often hampered by an insufficient therapeutic index, lack of specificity, and the emergence of drug-resistant cell subpopulations. One approach aimed at enhancing the selectivity of cancer chemotherapy for solid tumors relies on the application of gene therapy technologies. Gene therapies are techniques for modifying the cellular genome for therapeutic benefit. In cancer gene therapy, both malignant and nonmalignant cells may be suitable targets. The possibility of rendering cancer cells more sensitive to chemotherapeutics or toxins by introducing “suicide genes” was suggested in the late 1980s. This approach has two alternatives: toxin gene therapy, in which the genes for toxic products are transfected directly into tumor cells; and enzyme-activating prodrug therapy, in which the transgenes encode enzymes that activate specific prodrugs to create toxic products. The latter approach, known as gene-directed enzyme prodrug therapy (GDEPT) (1, 2) or virus-directed enzyme prodrug therapy (VDEPT) (3), may be used in isolation or combined with other strategies, such as the biotherapies described elsewhere in this Perspective series. VDEPT using selectively replicating viruses as vectors represents a promising means to target suicide genes specifically to tumor cells, an approach that is only beginning to be explored (for examples, see Hermiston, this Perspective series, ref. 4). GDEPT and VDEPT are two-step treatments for solid tumors (Figure ​(Figure1).1). In the first step, the gene for a foreign enzyme is delivered and targeted in a variety of ways to the tumor where it is to be expressed. In the second step, a prodrug is administered that is selectively activated to the drug by the foreign enzyme expressed in the tumor. Ideally, the gene for the enzyme should be expressed exclusively in the tumor cells and should reach a concentration sufficient to activate the prodrug for clinical benefit. The catalytic activity of the expressed protein must be adequate to activate the prodrug under physiological conditions. Because expression of the foreign enzymes will not occur in all cells of a targeted tumor in vivo, a bystander effect (BE) is required, whereby the prodrug is cleaved to an active drug that kills not only the tumor cells in which it is formed, but also neighboring tumor cells that do not express the foreign enzyme (5). Figure 1 GDEPT, a form of suicide gene therapy, aims to maximize the effect of a toxic drug and minimize its systemic effects by generating the drug in situ within the tumor. In the first step in this procedure, the gene for an exogenous enzyme is delivered and ... The genes can be engineered to express their products either intracellularly or extracellularly in the recipient cells (6). There are potential advantages to each approach. When the enzyme is intracellularly expressed, the prodrug must enter the cells for activation, and subsequently the active drug must diffuse through the interstitium across the cell membrane to elicit a BE. Cells in which the enzyme is expressed (tethered to the outer surface) are able to activate the prodrug extracellularly. A more substantial BE could therefore be generated with extracellular gene product expression, but spread of the active drug into the general circulation is a possible disadvantage (1, 6).

Characterization of a hemoglobin protease secreted by the pathogenic Escherichia coli strain EB1.

Many pathogenic bacteria can use heme compounds as a source of iron. Pathogenic Escherichia coli strains are capable of using hemoglobin as an iron source. However, the mechanism of heme acquisition from hemoglobin is not understood for this microorganism. We present the first molecular characterization of a hemoglobin protease (Hbp) from a human pathogenic E. coli strain. The enzyme also appeared to be a heme-binding protein. Affinity purification of this bifunctional protein enabled us to identify the extracellular gene product, and to clone and analyze its gene. A purification procedure developed for Hbp allowed us to perform functional studies. The protein interacted with hemoglobin, degraded it and subsequently bound the released heme. These results suggest that the protein is involved in heme acquisition by this human pathogen. Hbp belongs to the so-called IgA1 protease-like proteins, as indicated by the kinetics of its membrane transfer and DNA sequence similarity. The gene of this protein appears to be located on the large pColV-K30 episome, that only has been isolated from human and animal pathogens. All these characteristics indicate that Hbp may be an important virulence factor that may play a significant role in the pathogenesis of E. coli infections.

Altered expression of plasminogen activator and plasminogen activator inhibitor during cellular senescence.

Fibroblast senescence is associated with a loss of proliferative potential and an alteration in extracellular gene expression. Because the expression of extracellular gene products are frequently growth state dependent, we undertook a comparative study of the regulation of the components of the plasminogen activation system in young and senescent cells under controlled conditions of growth. Young and senescent cells were compared in quiescent and activated growth conditions for the secretion of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2). Whereas young cells showed decreased levels of PAI-1 in the secreted and extracellular matrix pools upon serum deprivation, senescent cells showed a more constitutive pattern of gene expression, with no noticeable decrease of the levels in a low concentration of serum. RNA analysis revealed that senescent lung and skin cells, independent of the growth state, constitutively express levels of u-PA and PAI-1 comparable to the expression levels in young mitotically growing cells. These expression levels are down-regulated in quiescent young cells. In contrast, both t-PA and PAI-2 were markedly overexpressed in senescent skin lung cells under all growth conditions. Total plasminogen activator activity in conditioned medium was 50-fold higher in senescent-cell medium compared to young when cultured in 0.5% fetal calf serum (FCS) for five days, with the majority of the activity co-migrating on zymograms with u-PA. Increases in PAI-1 was also observed in senescent human umbilical vein endothelial cells. In summary, cells of various types display alterations in plasminogen activator activity during replicative senescence. The inappropriate over-expression of plasminogen activator activity in vivo may be expected to lead to a progressive disruption of extracellular matrix maintenance. Thus, our observations suggest that cellular replicative senescence is associated with an altered expression of several genes regulating tissue maintenance which, in turn, could lead to degenerative changes in tissue in age-related disease(s).

Recent advances in the study of the taxonomy, pathogenicity, and infectious syndromes associated with the genus Aeromonas

Over the past decade, the emergence of Aeromonas species as bona fide human pathogens and their probable role as etiologic agents of bacterial gastroenteritis have resulted in an explosion of scientific interest in the genus. Major accomplishments occurring in this field during that interval include a more refined taxonomy, identification of new cell-associated factors (surface layers, pili), and the molecular analysis of selected extracellular gene products that may play a critical role in pathogenesis (hemolysins, enterotoxins). This review provides an updated overview of recent systematic, clinical, and pathophysiologic advances and defines key areas of medical and scientific interest in which major questions remain unanswered.

Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase)

We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, α-galactosidase (melibiase) (α-Gal). The predicted translation product of MEL1 is a pre-α-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL8O. There are three TATA boxes between UASm and the initiation codon of pre-α-Gal, as well as a typical yeast cleavage/poly- adenylation sequence in the 3'-flank of the gene.

Focal Dermal Hypoplasia: Abnormal Growth Characteristics of Skin Fibroblasts in Culture

Focal dermal hypoplasia is an inherited disease affecting the skin, bones, ocular and dental structures. Histologically, the skin lesions are characterized by a marked decrease in dermal connective tissue. Fibroblast cultures initiated from both the skin lesions and the unaffected skin from a patient with focal dermal hypoplasia were used to assess the growth characteristics of these cells as well as potential abnormalities in the metabolism of collagen, the major extracellular gene product of skin fibroblasts. Cells from affected skin were characterized by a markedly compromised growth potential with a mean population doubling time twice that of the controls and a final saturation density of one-fifth that of control cultures. Phase contrast microscopy revealed that fibroblasts derived from the skin lesions were strikingly abnormal and characterized by a large granular cytoplasm with cytoplasmic vacuoles. Despite these findings, the rate of collagen synthesis, measured as the formation of [ 3 H]hydroxyproline in relation to DNA and cell protein was undisturbed in focal dermal hypoplasia. Furthermore, the relative synthesis of genetically distinct pro-collagens of type I and III, isolated by DEAE-cellulose chromatography, was unaltered in the affected cell strains. These findings indicate that, although the synthesis of collagen by individual fibroblasts is normal, an abnormality in cell kinetics may have relevance to the absence of collagen and other connective tissue components of the dermis in focal dermal hypoplasia.

Environmental pH modulation of collagenase in normal human fibroblast cultures

Normal human skin fibroblast cultures have been used to assess the effects of relatively minor changes in environmental pH on collagenase, a major extracellular gene product. Collagenase accumulation in the culture medium, measured both as enzyme activity and immunoreactive material, was 2- to 10-fold greater at pH 7.6–8.2 than at pH 6.8–7.2. The pH-associated increase in collagenase was parallel by an increase in general protein synthesis. Nevertheless, prototypic lysosomal and cytoplasmic enzymes changed very little under identical culturing conditions. Although substantial intracellular protein degradation occurred at all pH values, the small differences either in general protein degradation or in specific collagenase degradation in the medium were of insufficient magnitude to account for the increased accumulation of collagenase.