Combined MET inhibition and topoisomerase I inhibition block cell growth of small cell lung cancer.

Small cell lung cancer (SCLC) is a devastating disease, and current therapies have not greatly improved the 5-year survival rates. Topoisomerase (Top) inhibition is a treatment modality for SCLC; however, the response is short lived. Consequently, our research has focused on improving SCLC therapeutics through the identification of novel targets. Previously, we identified MNNG HOS transforming gene (MET) to be overexpressed and functional in SCLC. Herein, we investigated the therapeutic potential of combinatorial targeting of MET using SU11274 and Top1 using 7-ethyl-10-hydroxycamptothecin (SN-38). MET and TOP1 gene copy numbers and protein expression were determined in 29 patients with limited (n = 11) and extensive (n = 18) disease. MET gene copy number was significantly increased (>6 copies) in extensive disease compared with limited disease (P = 0.015). Similar TOP1 gene copy numbers were detected in limited and extensive disease. Immunohistochemical staining revealed a significantly higher Top1 nuclear expression in extensive (0.93) versus limited (0.15) disease (P = 0.04). Interestingly, a significant positive correlation was detected between MET gene copy number and Top1 nuclear expression (r = 0.5). In vitro stimulation of H82 cells revealed hepatocyte growth factor (HGF)-induced nuclear colocalization of p-MET and Top1. Furthermore, activation of the HGF/MET axis enhanced Top1 activity, which was abrogated by SU11274. Combination of SN-38 with SU11274 dramatically decreased SCLC growth as compared with either drug alone. Collectively, these findings suggest that the combinatorial inhibition of MET and Top1 is a potentially efficacious treatment strategy for SCLC.

The database of chromosome imbalance regions and genes resided in lung cancer from Asian and Caucasian identified by array-comparative genomic hybridization.

BACKGROUND: Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis. METHODS: Array-comparative genomic hybridization (array-CGH) was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR), chromogenic in situ hybridization (CISH), reverse transcriptase-qPCR (RT-qPCR), and immunohistochemistry (IHC) in more patients. RESULTS: We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1) functioning in Rho activity control, FRAT2 (10q24.1) involved in Wnt signaling, PAFAH1B1 (17p13.3) functioning in motility control, and ZNF322A (6p22.1) involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (P<0.001~P=0.06). In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of PAFAH1B1 protein overexpression was 68% in Asian and 70% in Caucasian. CONCLUSIONS: Our study provides an invaluable database revealing common and differential imbalance regions at specific chromosomes among Asian and Caucasian lung cancer patients. Four validation methods confirmed our database, which would help in further studies on the mechanism of lung tumorigenesis.

Catecholamine biosynthesis and secretion: physiological and pharmacological effects of secretin.

Pituitary adenylyl cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) augment the biosynthesis of tyrosine hydroxylase (TH). We tested whether secretin belonging to the glucagon/PACAP/VIP superfamily would increase transcription of the tyrosine hydroxylase (Th) gene and modulate catecholamine secretion. Secretin activated transcription of the endogenous Th gene and its transfected promoter (EC(50) ∼4.6 nM) in pheochromocytoma (PC12) cells. This was abolished by pre-treatment with a secretin receptor (SCTR) antagonist and by inhibition of protein kinase A (PKA), mitogen-activated protein kinase, or CREB (cAMP response element-binding protein). In agreement, secretin increased PKA activity and induced phosphorylation of CREB and binding to Th CRE, suggesting secretin signaling to transcription via a PKA-CREB pathway. Secretin stimulated catecholamine secretion (EC(50) ∼3.5 µM) from PC12 cells, but this was inhibited by pre-treatment with VIP-preferring receptor (VPAC1)/PACAP-preferring receptor (PAC1) antagonists. Secretin-evoked secretion occurred without extracellular Ca(2+) and was abolished by intracellular Ca(2+) chelation. Secretin augmented phospholipase C (PLC) activity and increased inositol-1,4,5-triphosphate (IP(3)) levels in PC12 cells; PLC-ß inhibition blocked secretin-induced catecholamine secretion, indicating the participation of intracellular Ca(2+) from a phospholipase pathway in secretion. Like PACAP, secretin evoked long-lasting catecholamine secretion, even after only a transient exposure. Thus, transcription is triggered by nanomolar concentrations of the peptide through SCTR, with signaling along the cAMP-PKA and extracellular-signal-regulated kinase 1/2 pathways and through CREB. By contrast, secretion is triggered only by micromolar concentrations of peptide through PAC1/VPAC receptors and by utilizing a PLC/intracellular Ca(2+) pathway.

Proteomic characterization of non-small cell lung cancer in a comprehensive translational thoracic oncology database.

BACKGROUND: In recent years, there has been tremendous growth and interest in translational research, particularly in cancer biology. This area of study clearly establishes the connection between laboratory experimentation and practical human application. Though it is common for laboratory and clinical data regarding patient specimens to be maintained separately, the storage of such heterogeneous data in one database offers many benefits as it may facilitate more rapid accession of data and provide researchers access to greater numbers of tissue samples. DESCRIPTION: The Thoracic Oncology Program Database Project was developed to serve as a repository for well-annotated cancer specimen, clinical, genomic, and proteomic data obtained from tumor tissue studies. The TOPDP is not merely a library-it is a dynamic tool that may be used for data mining and exploratory analysis. Using the example of non-small cell lung cancer cases within the database, this study will demonstrate how clinical data may be combined with proteomic analyses of patient tissue samples in determining the functional relevance of protein over and under expression in this disease. Clinical data for 1323 patients with non-small cell lung cancer has been captured to date. Proteomic studies have been performed on tissue samples from 105 of these patients. These tissues have been analyzed for the expression of 33 different protein biomarkers using tissue microarrays. The expression of 15 potential biomarkers was found to be significantly higher in tumor versus matched normal tissue. Proteins belonging to the receptor tyrosine kinase family were particularly likely to be over expressed in tumor tissues. There was no difference in protein expression across various histologies or stages of non-small cell lung cancer. Though not differentially expressed between tumor and non-tumor tissues, the over expression of the glucocorticoid receptor (GR) was associated improved overall survival. However, this finding is preliminary and warrants further investigation. CONCLUSION: Though the database project is still under development, the application of such a database has the potential to enhance our understanding of cancer biology and will help researchers to identify targets to modify the course of thoracic malignancies.

Generation of comprehensive thoracic oncology database--tool for translational research.

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

Inhibition of Sonic hedgehog and Notch pathways enhances sensitivity of CD133(+) glioma stem cells to temozolomide therapy.

Malignant gliomas are currently treated with temozolomide (TMZ), but often exhibit resistance to this agent. CD133(+) cancer stem cells, a population believed to contribute to the tumor's chemoresistance, bear the activation of Notch and Sonic hedgehog (SHH) pathways. In this study, we examined whether inhibition of both pathways enhances the efficacy of TMZ monotherapy in the context of glioma stem cells. Transcriptional analysis of Notch and SHH pathways in CD133(+)-enriched glioma cell populations showed the activity of these pathways. CD133(+) cells were less susceptible to TMZ treatment than the unsorted glioma counterparts. Interestingly, Notch and SHH pathway transcriptional activity in CD133(+) glioma cells was further enhanced by TMZ exposure, which led to NOTCH 1, NCOR2, and GLI1 upregulation (6.64-, 3.73-, and 2.79-fold, respectively) and CFLAR downregulation (4.22-fold). The therapeutic effect of TMZ was enhanced by Notch and SHH pathway pharmacological antagonism with GSI-1 and cyclopamine. More importantly, simultaneous treatment involving TMZ with both of these compounds led to a significant increase in CD133(+) glioma cytotoxicity than treatment with any of these agents alone (P < 0.05). In conclusion, CD133(+) glioma cells overexpress genes involved in Notch and SHH pathways. These pathways contribute to the chemoresistant phenotype of CD133(+) glioma cells, as their antagonism leads to an additive effect when used in combination with TMZ.

Short hairpin RNA-mediated fibronectin knockdown delays tumor growth in a mouse glioma model.

Glioblastoma multiforme is the most common and lethal primary brain tumor. Glioma progression depends on the rapid proliferation of tumor cells accompanied by an acute immunosuppressive environment, facilitated mainly by tumor infiltration of regulatory T cells (Tregs). In this study, we characterize the role of fibronectin, a high-molecular weight extracellular matrix glycoprotein secreted by tumor cells, in controlling glioma progression and in mediating immunosuppression. Fibronectin binds to membrane-spanning integrin receptors and plays an important role in cell signaling, in defining cellular shape, in mobility, and in regulating the cell cycle. We found that inhibition of fibronectin expression in glioma cells, using short hairpin RNA-mediated silencing of gene expression, delayed cell proliferation in vitro. This delayed growth is explained, in part, by the observed reduced expression of integrin ß(1) fibronectin receptor, which was restored by the inhibition of proteosomal activity. In our analysis of the downstream signaling targets of integrin ß(1), we demonstrated reduced phosphorylation of Src kinase and STAT-3. We also observed reduced survivin expression that induced a three-fold increased accumulation of fibronectin-knockdown cells in the G(2)/M phase. In an experimental animal model, the fibronectin knockdown tumors had a mean survival advantage of 23 days over wild-type tumors. Moreover, brain samples of animals bearing fibronectin-knockdown tumors showed delayed Treg recruitment. Collectively, we propose that fibronectin is a key mediator of glioma progression because its inhibition delays both tumor progression and immunosuppression.

CBL is frequently altered in lung cancers: its relationship to mutations in MET and EGFR tyrosine kinases.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a heterogeneous group of disorders with a number of genetic and proteomic alterations. c-CBL is an E3 ubiquitin ligase and adaptor molecule important in normal homeostasis and cancer. We determined the genetic variations of c-CBL, relationship to receptor tyrosine kinases (EGFR and MET), and functionality in NSCLC. METHODS AND FINDINGS: Using archival formalin-fixed paraffin embedded (FFPE) extracted genomic DNA, we show that c-CBL mutations occur in somatic fashion for lung cancers. c-CBL mutations were not mutually exclusive of MET or EGFR mutations; however they were independent of p53 and KRAS mutations. In normal/tumor pairwise analysis, there was significant loss of heterozygosity (LOH) for the c-CBL locus (22%, n = 8/37) and none of these samples revealed any mutation in the remaining copy of c-CBL. The c-CBL LOH also positively correlated with EGFR and MET mutations observed in the same samples. Using select c-CBL somatic mutations such as S80N/H94Y, Q249E and W802* (obtained from Caucasian, Taiwanese and African-American samples, respectively) transfected in NSCLC cell lines, there was increased cell viability and cell motility. CONCLUSIONS: Taking the overall mutation rate of c-CBL to be a combination as somatic missense mutation and LOH, it is clear that c-CBL is highly mutated in lung cancers and may play an essential role in lung tumorigenesis and metastasis.

The role of chemokine receptor CXCR4 in lung cancer.

Lung cancer is the leading cause of cancer deaths worldwide. Small cell lung cancer (SCLC), which comprises 15% of all lung cancers, is almost exclusively due to smoking and is highly aggressive due to early widespread metastasis. While combination chemotherapy has lead to modest improvements in outcome, the five-year overall survival for SCLC remains at 5%. Identifying distinct biochemical pathways of metastasis and chemotherapy resistance in SCLC may lead to novel therapeutic approaches and improve survival in SCLC patients. The chemokine receptor CXCR4 is emerging as an important target in cancer growth, metastasis, relapse and resistance to therapy. In this article, we review the structure and function of CXCR4 and its ligand, CXCL12, as well as mechanisms of CXCR4/CXCL12 signal transduction in lung cancer. We review the current preclinical and translational research involving this pathway in lung cancer and the clinical development of several novel agents targeting the CXCR4/CXCL12 pathway. Further understanding of the CXCR4/CXCL12 pathway in SCLC and NSCLC may provide a rationale for innovative research on the CXCR4 receptor as a potential novel therapeutic target in lung cancer.

A chimeric adenovirus with an Ad 3 fiber knob modification augments glioma virotherapy.

BACKGROUND: Malignant gliomas remain refractory to treatment despite advances in chemotherapy and surgical techniques. Viral vectors developed to treat gliomas have had low transduction capabilities, limiting their use. Gliomas over-express CD46, CD80, and CD86, all of which bind adenovirus serotype 3. METHODS: To increase the infectivity and replication of oncolytic vectors in malignant brain tumors, we created a conditionally replicating adenovirus, CRAd-Survivin-5/3, which contains a survivin promoter-driving E1A and a chimeric fiber consisting of adenovirus serotype 3 knob. RESULTS: In vitro, this modified CRAd showed ten- to 100-fold increased cytotoxicity against glioma cells. Ex vivo analysis of primary glioblastoma multiforme samples infected with CRAd-Survivin-5/3 showed an increase in cytotoxicity of 20-30% compared to adenovirus wild-type (AdWT). In normal human astrocytes and normal brain tissues, CRAd-Survivin-5/3 exhibited 30-40% and 10-15% lower cytotoxicity than AdWT, respectively. In an intracranial xenograft model of glioma, this oncolytic virus increased tumor-free survival and overall lifespan by 50% compared to controls (p < 0.05). CONCLUSIONS: CRAd-Survivin-5/3 represents an attractive alternative to existing vectors and should be tested further in the pre-clinical setting.

Adenoviral virotherapy for malignant brain tumors.

Glioblastoma multiforme is the most common form of primary brain cancer. In the past decade, virotherapy of tumors has gained credence, particularly in glioma management, as these tumors are not completely resectable and tend to micro-metastasize. Adenoviral vectors have an advantage over other viral vectors in that they are relatively non-toxic and do not integrate in the genome. However, the lack of coxsackie and adenovirus receptors on surface of gliomas provides for inefficient transduction of wild-type adenoviral vectors in these tumors. By targeting receptors that are overexpressed in gliomas, modified adenoviral constructs have been shown to efficiently infect glioma cells. In addition, by taking advantage of tumor-specific promoter elements, oncolytic adenoviral vectors offer the promise of selective tumor-specific replication. This dual targeting strategy has enabled specificity in both laboratory and pre-clinical settings. This review examines current trends in adenoviral virotherapy of gliomas, with an emphasis on targeting modalities and future clinical applications.

Low-dose radiation enhances survivin-mediated virotherapy against malignant glioma stem cells.

To improve the efficacy and selectivity of virotherapy for malignant glioma, we designed a strategy to amplify adenoviral replication in conjunction with radiotherapy using a radioinducible promoter. First, we compared the radiation-inducible activity of FLT-1, vascular endothelial growth factor, DR5, Cox2, and survivin. We then examined the capacity of the optimal promoter to modulate transgene expression followed by E1A activity in vitro and in vivo in a glioma stem cell model. In the presence of radiation, survivin mRNA activity increased 10-fold. Luciferase transgene expression was dose dependent and optimal at 2 Gy. A novel oncolytic adenovirus, CRAd-Survivin-pk7, showed significant toxicity and replication against a panel of passaged and primary CD133(+) glioma stem cells. On delivery of radiation, the toxicity associated with CRAd-Survivin-pk7 increased by 20% to 50% (P < 0.05). At the same time, the level of E1A activity increased 3- to 10-fold. In vivo, treatment of U373MG CD133(+) stem cells with CRAd-Survivin-pk7 and radiation significantly inhibited tumor growth (P < 0.05). At the same time, the level of E1A activity was 100-fold increased versus CRAd-Survivin-pk7 alone. Selected genes linked to radioinducible promoters whose expression can be regulated by ionizing radiation may improve the therapeutic ratio of virotherapy. In this study, we have identified a new radioinducible promoter, survivin, which greatly enhances the activity of an oncolytic adenovirus in the presence of low-dose radiotherapy.

The oncogene mcts1.

The oncogene MCTS1, discovered as an amplified product in a subset of T-cell lymphoma lines, has been implicated in cell cycle progression and conferring a growth advantage in lymphomas and breast cancer. Recent research shows that it modulates the MAPK pathway and acts as a translational activator both in vivo and in vitro. In breast cancer cells, expression of MCTS1 confers aggressive properties and inhibits apoptosis. This article will review these data and its implications on our understanding of cancer.

Expression of DNA mismatch repair proteins in transformed non-Hodgkin's lymphoma: relationship to smoking.

It has been hypothesized that defects in DNA-mismatch repair are associated with smoking in certain types of transformed non-Hodgkin lymphoma (NHL). We have analyzed biopsy samples from two indolent B-cell lymphomas, follicular lymphoma (FL) and chronic lymphocytic leukemia/small lymphocytic leukemia (CLL/SLL), that have transformed to diffuse-large B-cell lymphoma (DLBCL). We correlated the presence or absence of DNA-mismatch repair enzymes by immunostaining as well as the p53 status to smoking history. Of all patients (n = 30), 37% showed negative immunostaining of MLH1, 16% showed negative immunostaining of MSH2 and 63% had p53 mutations and/or protein expression. Eighteen out of 20 transformed follicular lymphomas and seven out of 10 CLL/SLL that have transformed to DLBCL (Richter's syndrome) were informative for smoking histories. We found that the relative risk of negative immunostaining for either MLH1 or MSH2 was 2.2 times higher in smokers than non-smokers (relative risk = 2.2041, 95% confidence interval: 0.89714, 5.41491). No direct correlation was found between smoking and the mutations in the p53 gene. These results suggest that cigarette smoking may play a role in the development of transformed lymphomas through defective mismatch repair.

MCT-1 protein interacts with the cap complex and modulates messenger RNA translational profiles.

MCT-1 is an oncogene that was initially identified in a human T cell lymphoma and has been shown to induce cell proliferation as well as activate survival-related pathways. MCT-1 contains the PUA domain, a recently described RNA-binding domain that is found in several tRNA and rRNA modification enzymes. Here, we established that MCT-1 protein interacts with the cap complex through its PUA domain and recruits the density-regulated protein (DENR/DRP), containing the SUI1 translation initiation domain. Through the use of microarray analysis on polysome-associated mRNAs, we showed that up-regulation of MCT-1 was able to modulate the translation profiles of BCL2L2, TFDP1, MRE11A, cyclin D1, and E2F1 mRNAs, despite equivalent levels of mRNAs in the cytoplasm. Our data establish a role for MCT-1 in translational regulation, and support a linkage between translational control and oncogenesis.

Characterization of the MCT-1 pseudogene: identification and implication of its location in a highly amplified region of chromosome 20.

The MCT-1 oncogene was initially identified as an amplified gene on chromosome Xq22-24 in a T-cell lymphoma. MCT-1 is over-expressed in a subset of diffuse large B-cell lymphoma (DLBCL), a common form of Non-Hodgkin's Lymphoma (NHL). We have identified a pseudogene for MCT-1 (PsiMCT-1) that is located on chromosome 20q11.2, a region within an amplicon containing several important genes frequently amplified in certain breast and ovarian cancers. Genomic analysis revealed that PsiMCT-1 is a processed pseudogene. Interestingly, both MCT-1 and its pseudogene are located on regions of the genome that are frequently amplified in several different human malignancies. MCT-1 is the oldest known oncogene and its insertion as a pseudogene occurred at a later time point in evolution. Existence of PsiMCT-1 should be considered when analyzing genomic amplification and or expression of MCT-1. Analysis of MCT-1 and PsiMCT-1 might provide clues to cancer genes and their evolution across species.

Integration of metal-resistant determinants from the plasmid of an Acidocella strain into the chromosome of Escherichia coli DH5alpha.

Acidophilic bacteria of mine origin are ideal systems for studying microbial metal resistance because of their ability to grow in the presence of high concentrations of metal salts. We have previously shown that the metal-resistant transformants obtained after transformation of Escherichia coli DH5alpha with plasmid DNA preparation from Acidocella sp. strain GS19h did not contain any plasmid suggesting chromosomal integration of the plasmid(s) (Appl Environ Microbiol 1997; 63: 4523-4527). The present study provides evidence in support of this suggestion. The pulsed field gel electrophoresis (PFGE) pattern of genomic DNA of the plasmidless metal-resistant transformants differed markedly from that of the untransformed DH5alpha strain. Moreover, when the recombinant plasmids constructed by cloning plasmid DNA fragments of the Acidocella strain GS19h in the vector pBluescript II KS+ were used to transform E. coli DH5alpha strain, no plasmid DNA was detected in some of the zinc- and ampicillin-resistant (ZnrAmpr) clones. The PFGE pattern of genomic DNA of such a transformed clone also differed markedly from that of the parent strain, suggesting chromosomal integration of the recombinant plasmid(s) containing both ampicillin- and zinc-resistance determinants. This observation was further supported by hybridization of chromosomal DNA of the plasmidless ZnrAmpr E. coli DH5alpha clone with the probes made from the plasmid DNA of strain GS19h and the vector DNA. Thus, this study corroborates our previous finding and documents the phenomenon of integration of metal-resistant determinants from the Acidocella GS19h plasmid(s) into the chromosome of E. coli DH5alpha.

Mutation in the relA gene of Vibrio cholerae affects in vitro and in vivo expression of virulence factors.

The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relA(VCH)) was cloned and sequenced. The relA(VCH) gene encodes a 738-amino-acid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A deltarelA::kan allele was generated by replacing approximately 31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.

Genesis of variants of Vibrio cholerae O1 biotype El Tor: role of the CTXphi array and its position in the genome.

The gene encoding cholera toxin, the principal virulence factor of Vibrio cholerae, is encoded by a filamentous, lysogenic bacteriophage known as CTXphi. The genome of V. cholerae, the host for CTXphi, consists of two chromosomes, one large and one small. Here, it is shown that localization and array of CTX prophage DNA in either the large or small chromosome of V. cholerae is likely to be one of the reasons for the emergence of O1 biotype El Tor variants isolated just before and after the V. cholerae O139 cholera outbreak in 1992. Analyses of the organization of the CTX region of the genome of pre-O139 El Tor strains revealed that these strains carry two distinct CTX prophages integrated in the small chromosome in tandem: CTX(ET), the prophage having a conserved NotI site in its repeat sequence segment which seems to be specific for the El Tor strains so far examined, followed by CTX(calc)-like genome, the prophage found in recent O139 clinical isolates from Calcutta. In sharp contrast, in post-O139 El Tor strains only one copy of the CTX(ET) prophage was found to be integrated in the large chromosome. To the authors' knowledge, the presence of CTX prophage in the small chromosome of O1 El Tor strains has not been reported previously. It is also shown that the difference in the CTX copy number and the position of the bacteriophage on the genomes of pre- and post-O139 El Tor strains have an effect on cholera toxin production. While a pre-O139 strain produced maximum cholera toxin in yeast extract/peptone medium at 30 degrees C, a post-O139 El Tor strain showed maximal yield at 37 degrees C, indicating differential regulation of cholera toxin between the strains. It appears from this study that the variation in the integration site of the CTX prophage, its copy number and the presence of diverse phage genomes in V. cholerae O1 biotype El Tor may be strategically important for generating variants with subtle phenotypic modulations of virulence factor production in this longest-ruling seventh pandemic strain.