Anti-tumorigenic Activity Of Adenovirus E1A
John M Routes, Associate Professor Medicine And Immunol
Pediatricsmedical College Of Wisconsin
Grant 5R01CA125666-02 from National Cancer Institute, IRG: ZRG1
Abstract: Adenovirus (Ad) E1A is presently in phase l/ll clinical trials for the treatment of human malignancy. In order to optimize this form of therapy, we must understand the molecular basis for the anti-tumorigenic effect of E1A. We established that the capacity of E1A to elicit a vigorous NK cell and T cell anti-tumor immune response is an important component of the anti-tumorigenic activity of E1A. The expression of E1A, but not mutant forms of E1A unable to interact with the cellular transcriptional co-adaptor molecules p300 or CBP (abbreviated E1A-Ap300), increases the expression of NKG2D ligands on the surface of tumor cells. Consequently, tumor cells that express E1A are eliminated by NK cells in vivo in a NKG2D-dependent manner. The upregulation of NKG2D ligands contributes to the immune-mediated decrease in tumorigenicity mediated by E1 A. The present use of E1A in the treatment of human malignancy does not exploit the immune-mediated, anti-tumorigenic activity of E1A. Studies in this proposal will define the molecular basis for the immune-mediated, anti-tumorigenic activity of E1A. In the first aim of the proposal we will explore the molecular mechanism whereby the interaction of E1A with p300 or the highly related transcriptional coadaptor protein, CBP, increases the expression of NKG2D ligands on the surface of tumor cells. In the second aim we will determine if the upregulation of NKG2D ligands on tumor cells by E1A is sufficient to induce a CD8+, E1 A-specific T cell response or if other pro-immunogenic activities of E1A are involved. In third aim, we will ascertain if E1A can be used as a molecular adjuvant to elicit antigen-specific anti-tumor immune responses Aim 1 Determine the molecular mechanisms for the ability of E1A, but not E1A-Ap300, to increase the expression of NKG2D ligands. Aim 2 Determine the molecular basis for the robust, E1 A-specific, CD+8 T cell response elicited by tumor cells that express E1A. Aim 3 Determine if the pro-immunogenic activities of E1A can be harnessed to elicit vigorous tumor antigen-specific immune responses
Project start date: 2007-08-14
Project end date: 2012-07-31
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Grants awarded to John M Routes
Lymphoproliferative Disorders In Primary Immunodeficiencies
John M Routes, Associate Professor Medicine And Immunol
Pediatricsmedical College Of Wisconsin
8701 Watertown Plank Rd
milwaukee, Wi 532260509
Grant 1R01CA122539-01A2 from National Cancer Institute, IRG: HAI
Abstract: We demonstrated that patients with common variable immunodeficiency (CVID) and granulomatous and lymphocytic interstitial lung disease (GLILD) are at high risk for the development of B cell lymphomas and early mortality. We also found that a majority of patients with CVID and GLILD (CVID-GLILD) were infected with human herpes virus type 8 (HHV8). In Aim 1, we will determine the prevalence of HHV8 infection in patients with a larger cohort of patients with CVID and broad spectrum of primary immunodeficiencies. To identify the source of HHV8, we will determine the prevalence of HHV8 infection in family members and household contacts of infected and uninfected patients with CVID and determine the molecular phylogeny of HHV8 in infected cohorts by amplifying the highly polymorphic HHV8 K1 ORF by PCR and performing DNA sequencing of the K1 amplicon. In Aim 2, we will expand our observations on the role of HHV8 infection in lymphoproliferative disorders, lung and liver disease in patients with CVID by examining lung, liver or lymph node tissue biopsies for evidence of HHV8 infection. In Aim 3, we will determine if abnormalities in cellular immunity identify patients with CVID at risk for infection with HHV8 and determine if specific promoter polymorphisms in the IL- 6, TNF-1 or IL-10 genes or mutations in the TACI gene predispose patients with CVID to infection with HHV8. Aim 1 Ascertain the molecular phylogeny of HHV8 in CVID patients infected with HHV8 and determine if HHV8 is an opportunistic pathogen in a larger cohort of patients with CVID and other primary immunodeficiencies. Aim 2 Ascertain the role of HHV8 infection in the etiology of lung disease, liver disease, lymphoproliferative disorders in patients with CVID. Aim 3 Determine if promoter polymorphisms of cytokine genes, dysregulated inflammatory cytokine production, and defects in cellular immunity or mutations in the TACI gene predispose patients with CVID to infection with HHV8.Project Narrative People with impaired immune systems (primary immunodeficiency) have an increased risk of developing and dying from cancer. In this application, we will try to determine the cause of the cancer that occurs in patients with primary immunodeficiency
Project start date: 2008-01-01
Project end date: 2012-12-31
EXAMINING HELICOBACTER PYLORI GASTRIC INFECTION IN PATIENTS WITH IMMUNODEFICENNCY
John M Routes, Associate Professor Medicine And Immunol
University Of Colorado Denver Grants And Contracts, Mail Stop F428 Aurora, Co 800450508
Grant 2M01RR000051-360998 from National Center For Research Resources, IRG:
Abstract: The purpose of this study is to determine if the prevalence of H. Pylori infection (of the stomach) is increased iin patients with common variable immunodeficiency compared to normal controls. This is to be determined by the 14 C breath test.
Keywords: Helicobacter, epidemiology, gastritis, immunodeficiency, prognosis, breath test, clinical research, human subject
Project start date: 1997-02-14
Project end date: 1997-11-30
DISSIMILAR IMMUNOGENICITIES OF ELA AND E7 ONCOPROTEINS
John M Routes, Associate Professor Medicine And Immunol
National Jewish Medical & Res Ctr
denver, Co 80206
Grant 5R01CA076491-05 from National Cancer Institute, IRG: EI
Abstract: The purpose of the project is to use the MCA-102 model system to elucidate the immunologic mechanisms involved in the rejection of E1A- but not E7-expressing tumor cells, genetically define the components of ElA that are required to induce robust cellular immunity, and further characterize the molecular basis by which ElA but not E7 induces a vigorous cellular immune response in two specific aims. Specific Aim 1 Elucidate the key immunologic mechanisms involved in the rejection of tumor cells expressing ElA but not E7. Specifically they will determine the interrelationship and function of NK cells, T cells, perforin- and Fas-mediated lysis and the cytokines, IL-12 and IFNg, in the rejection of E1A- but not E7-expressing tumor cells. Specific Aim 2 Define the molecular mechanisms by which E1A-p300 binding and ElA- exon 2 expression activate the NK cell response, thereby triggering a robust cellular immune response against E1A-expressing tumor cells
Keywords: cellular immunity, neoplasm /cancer immunology, oncoprotein, tumor antigen CD95 molecule, T lymphocyte, cytolysin, cytotoxic T lymphocyte, interferon gamma, interleukin 12, natural killer cell, pore forming protein Adenoviridae, gene targeting, human papillomavirus, laboratory mouse, neoplastic cell, transfection, transgenic animal
Project start date: 1998-09-01
Project end date: 2004-06-30
5R01CA076491-05 (2002): $253204
5R01CA076491-04 (2001): $245831
5R01CA076491-03 (2000): $238667
1R01CA076491-01A1 (1998): $224968
VIRAL ONCOGENES, INTERFERON, AND IMMUNITY
John M Routes, Associate Professor Medicine And Immunol
National Jewish Medical & Res Ctr
denver, Co 80206
Grant 5R29AI032136-03 from National Institute Of Allergy And Infectious Diseases, IRG: EI
Abstract: Natural killer (NK) cells are an important component of cellular antiviral immunity. Recent studies show that interferon (IFN) promotes the selective lysis of Ad2/5 infected cells by activating NK cells and protecting uninfected cells (IFN mediated cytoprotection or IFN MCP) but not Ad infected cells from NK cell mediated lysis. Expression of a single Ad gene, early region 1A (EIA), blocks IFN MCP. This offers the first opportunity to study the interference of IFN MCP by a single viral gene. Preliminary genetic analysis reveals that expression of sequences in the first exon of EIA is necessary for inhibition of IFN MCP. Expression of SV40 LT, which shares homologous sequences with the first exon of EIA, also inhibits IFN MCP. The homologous regions of EIA and SV40 LT have been recently shown to bind specific cellular proteins (e.g., p105 retinoblastoma (Rb) gene product; p107; p60, cyclin A) which has con-elated with some of the biological activities of these viral oncoproteins. Based on these studies, it appears that the inhibition of IFN MCP by SV40 LT and Ad EIA is mediated through the same cellular pathway, likely by interacting with specific, common cellular protein(s). The first specific aim of this proposal To map, by mutational analysis, the genetic subregion(s) responsible for ElAs inhibition of IFN MCP and to determine if the ability of mutant EIA proteins to bind specific cellular proteins (p60 (cyclin A), p105 (Rb), pl07, p300) correlates with ElA mediated inhibition of IFN MCP. Human papillomavirus (HPV) early region 7 (E7), like SV40 LT, share homologous sequences with the first exon of ElA . However, preliminary studies show that HPV E7 expression in HeLa cells does not block IFN MCP. The second specific aim of this proposal To determine if expression of HPV16 or 18 E7 gene products in other types of human cells inhibits IFN MCP. The following approaches will be used a) Comparison of IFN MCP in SV40 LT or HPV16 E7 expressing keratinocyte and fibroblast cell lines. b) Determination of the significance of IFN-induced, downregulation of E7 expression in HPV16/18 transformed human cell lines in modulating the inhibition of IFN MCP. c) Determination of the relationship between level of E7 expression and IFN MCP
Keywords: Adenoviridae, cellular immunity, human papillomavirus, interferon, oncogene, simian virus 40, virus genetics cell cycle protein, chemical binding, gene mutation, genetic regulation, natural killer cell, oncoprotein genetic mapping, human tissue, tissue /cell culture, transfection
Project start date: 1992-07-01
Project end date: 1997-04-30
5R29AI032136-03 (1994): $97940
AMID In Apoptosis And P53-Mediated Downstream Effects
John M Routes, Associate Professor Medicine And Immunol
National Jewish Medical And Res Ctr Denver, Co 80206
Grant 5R01CA108771-02 from National Cancer Institute, IRG: CDF
Abstract: Apoptosis is a cell suicide process involved in various physiological and pathological activities, such as embryonic development, cancer and autoimmune diseases. Induction of apoptosis can be mediated through both caspase dependent and independent processes. The mechanisms of caspase-independent apoptosis have not been well understood. AIF is a mitochondrial flavoprotein that triggers caspase-independent apoptosis. We have cloned a novel AIF homologous molecule designated as AMID. AMID is localized to the outer membrane of mitochondria. Overexpression of AMID induces caspase-independent apoptosis. Moreover, AMID is induced by the tumor suppressor p53 and expression of AMID is down-regulated in tumors in comparison to their individually matched normal tissues. We hypothesize that AMID induces caspase-independent apoptosis through novel mechanisms and AMID is a tumor suppressor involved in p53-mediated downstream effects. To test our hypotheses, we have proposed three specific aims 1). To investigate the mechanisms of AMID-induced caspase-independent apoptosis; 2). To investigate the roles of AMID in p53-mediated apoptosis and cell growth arrest; 3). To determine whether AMID is a tumor suppressor gene using in vitro cell culture systems and in vivo gene knock-out studies in mice. Successful completion of the proposed studies will help to understand the molecular mechanisms of caspase-independent apoptosis and roles of AMID in p53-mediated biological effects and tumorigenesis.
Keywords: apoptosis, flavoprotein, genetic regulation, mitochondria, neoplasm /cancer genetics, p53 gene /protein, protein structure function, cell cycle, gene expression, genetic promoter element, guanine nucleotide binding protein, neoplastic transformation, protein protein interaction, athymic mouse, genetically modified animal, immunoprecipitation, western blotting, yeast two hybrid system
Project start date: 2004-07-14
Project end date: 2009-04-30
5R01CA108771-02 (2005): $279702
Anti-tumorigenic Activity Of Adenovirus E1A
John M Routes, Associate Professor Medicine And Immunol
Pediatricsmedical College Of Wisconsin
Grant 5R01CA125666-02 from National Cancer Institute, IRG: ZRG1
Abstract: Adenovirus (Ad) E1A is presently in phase l/ll clinical trials for the treatment of human malignancy. In order to optimize this form of therapy, we must understand the molecular basis for the anti-tumorigenic effect of E1A. We established that the capacity of E1A to elicit a vigorous NK cell and T cell anti-tumor immune response is an important component of the anti-tumorigenic activity of E1A. The expression of E1A, but not mutant forms of E1A unable to interact with the cellular transcriptional co-adaptor molecules p300 or CBP (abbreviated E1A-Ap300), increases the expression of NKG2D ligands on the surface of tumor cells. Consequently, tumor cells that express E1A are eliminated by NK cells in vivo in a NKG2D-dependent manner. The upregulation of NKG2D ligands contributes to the immune-mediated decrease in tumorigenicity mediated by E1 A. The present use of E1A in the treatment of human malignancy does not exploit the immune-mediated, anti-tumorigenic activity of E1A. Studies in this proposal will define the molecular basis for the immune-mediated, anti-tumorigenic activity of E1A. In the first aim of the proposal we will explore the molecular mechanism whereby the interaction of E1A with p300 or the highly related transcriptional coadaptor protein, CBP, increases the expression of NKG2D ligands on the surface of tumor cells. In the second aim we will determine if the upregulation of NKG2D ligands on tumor cells by E1A is sufficient to induce a CD8+, E1 A-specific T cell response or if other pro-immunogenic activities of E1A are involved. In third aim, we will ascertain if E1A can be used as a molecular adjuvant to elicit antigen-specific anti-tumor immune responses Aim 1 Determine the molecular mechanisms for the ability of E1A, but not E1A-Ap300, to increase the expression of NKG2D ligands. Aim 2 Determine the molecular basis for the robust, E1 A-specific, CD+8 T cell response elicited by tumor cells that express E1A. Aim 3 Determine if the pro-immunogenic activities of E1A can be harnessed to elicit vigorous tumor antigen-specific immune responses
Project start date: 2007-08-14
Project end date: 2012-07-31
1R01CA125666-01A1 (2007): $287850
VIRAL ONCOGENES, INTERFERON, AND IMMUNITY
John M Routes, Associate Professor Medicine And Immunol
National Jewish Medical And Res Ctr Denver, Co 80206
Grant 5R29AI032136-05 from National Institute Of Allergy And Infectious Diseases, IRG: EI
Project start date: 1992-07-01
Project end date: 1997-04-30
5R29AI032136-05 (1996): $106575
Related Publications
E1A oncogene enhancement of caspase-2-mediated mitochondrial injury sensitizes cells to macrophage nitric oxide-induced apoptosis. J Immunol. 2008 Jun 15; 180( 12): 8272-9. PMID: 18523293 Immunodeficiency overview. Prim Care. 2008 Mar; 35( 1): 159-73, viii. Review. PMID: 18206723 Measurement of natural-killer cell lytic activity of adenovirus-infected or adenovirus-transformed cells. Methods Mol Med. 2007; 131: 213-9. PMID: 17656785 Pulmonary infection with Mycobacterium neoaurum identified by 16S ribosomal DNA sequence. J Infect. 2007 Apr; 54( 4): e227-31. Epub 2007 Feb 15. Review. PMID: 17306883 The interaction of adenovirus E1A with p300 family members modulates cellular gene expression to reduce tumorigenicity. J Cell Biochem. 2007 Mar 1; 100( 4): 929-40. PMID: 17063489 
Adenovirus serotype 5 E1A sensitizes tumor cells to NKG2D-dependent NK cell lysis and tumor rejection. J Exp Med. 2005 Dec 5; 202( 11): 1477-82. Epub 2005 Nov 28. PMID: 16314433 Possible role of human herpesvirus 8 in the lymphoproliferative disorders in common variable immunodeficiency. J Exp Med. 2005 Aug 15; 202( 4): 479-84. PMID: 16103407 Granulomatous disease in common variable immunodeficiency. Curr Allergy Asthma Rep. 2005 Sep; 5( 5): 370-5. Review. PMID: 16091208 
Practice parameter for the diagnosis and management of primary immunodeficiency. Ann Allergy Asthma Immunol. 2005 May; 94( 5 Suppl 1): S1-63. No abstract available. Erratum in: Ann Allergy Asthma Immunol. 2006 Mar;96(3):504. PMID: 15945566 Uncommon conundrum in common variable immunodeficiency. Clin Immunol. 2005 Sep; 116( 3): 208-10. No abstract available. PMID: 15905130 Adenovirus E1A gene-induced tumor cell rejection through cellular sensitization to immune and nonimmune apoptotic injuries. Front Biosci. 2005 May 1; 10: 1396-414. Review. PMID: 15769634 Pre-event smallpox vaccination and postevent exposure and disease: a report of the Joint Task Force on Smallpox Vaccination for Allergists. Ann Allergy Asthma Immunol. 2005 Jan; 94( 1): 4-7. Review. No abstract available. PMID: 15702806 Macrophages kill human papillomavirus type 16 E6-expressing tumor cells by tumor necrosis factor alpha- and nitric oxide-dependent mechanisms. J Virol. 2005 Jan; 79( 1): 116-23. PMID: 15596807 Dose dependence and time course of the immunologic response to administration of standardized cat allergen extract. J Allergy Clin Immunol. 2004 Dec; 114( 6): 1339-44. PMID: 15577832 Granulomatous-lymphocytic lung disease shortens survival in common variable immunodeficiency. J Allergy Clin Immunol. 2004 Aug; 114( 2): 415-21. PMID: 15316526 Expression of an E1A/E7 chimeric protein sensitizes tumor cells to killing by activated macrophages but not NK cells. J Virol. 2004 May; 78( 9): 4646-54. PMID: 15078947 
Expression of human herpesvirus 8 in primary pulmonary hypertension. N Engl J Med. 2003 Sep 18; 349( 12): 1113-22. PMID: 13679525 E1A oncogene-induced sensitization of human tumor cells to innate immune defenses and chemotherapy-induced apoptosis in vitro and in vivo. Cancer Res. 2003 Jun 15; 63( 12): 3435-43. PMID: 12810682 Adenovirus E1A, not human papillomavirus E7, sensitizes tumor cells to lysis by macrophages through nitric oxide- and TNF-alpha-dependent mechanisms despite up-regulation of 70-kDa heat shock protein. J Immunol. 2003 Apr 15; 170( 8): 4119-26. PMID: 12682242