E-cadherin inhibits cell surface localization of the pro-migratory 5T4 oncofetal antigen in mouse embryonic stem cells.

Epithelial-mesenchymal transition (EMT) events occur during embryonic development and are important for the metastatic spread of epithelial tumors. We show here that spontaneous differentiation of mouse embryonic stem (ES) cells is associated with an E- to N-cadherin switch, up-regulation of E-cadherin repressor molecules (Snail and Slug proteins), gelatinase activity (matrix metalloproteinase [MMP]-2 and -9), and increased cellular motility, all characteristic EMT events. The 5T4 oncofetal antigen, previously shown to be associated with very early ES cell differentiation and altered motility, is also a part of this coordinated process. E- and N-cadherin and 5T4 proteins are independently regulated during ES cell differentiation and are not required for induction of EMT-associated transcripts and proteins, as judged from the study of the respective knockout ES cells. Further, abrogation of E-cadherin-mediated cell-cell contact in undifferentiated ES cells using neutralizing antibody results in a reversible mesenchymal phenotype and actin cytoskeleton rearrangement that is concomitant with translocation of the 5T4 antigen from the cytoplasm to the cell surface in an energy-dependent manner. E-cadherin null ES cells are constitutively cell surface 5T4 positive, and although forced expression of E-cadherin cDNA in these cells is sufficient to restore cell-cell contact, cell surface expression of 5T4 antigen is unchanged. 5T4 and N-cadherin knockout ES cells exhibit significantly decreased motility during EMT, demonstrating a functional role for these proteins in this process. We conclude that E-cadherin protein stabilizes cortical actin cytoskeletal arrangement in ES cells, and this can prevent cell surface localization of the promigratory 5T4 antigen.

Hypoxia response element-driven cytosine deaminase/5-fluorocytosine gene therapy system: a highly effective approach to overcome the dynamics of tumour hypoxia and enhance the radiosensitivity of prostate cancer cells in vitro.

BACKGROUND: We proposed to exploit hypoxia-inducible factor (HIF)-1alpha overexpression in prostate tumours and use this transcriptional machinery to control the expression of the suicide gene cytosine deaminase (CD) through binding of HIF-1alpha to arrangements of hypoxia response elements. CD is a prodrug activation enzyme, which converts inactive 5-fluorocytosine to active 5-fluorouracil (5-FU), allowing selective killing of vector containing cells. METHODS: We developed a pair of vectors, containing either five or eight copies of the hypoxia response element (HRE) isolated from the vascular endothelial growth factor (pH5VCD) or glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (pH8GCD) gene, respectively. The kinetics of the hypoxic induction of the vectors and sensitization effects were evaluated in 22Rv1 and DU145 cells in vitro. RESULTS: The CD protein as selectively detected in lysates of transiently transfected 22Rv1 and DU145 cells following hypoxic exposure. This is the first evidence of GAPDH HREs being used to control a suicide gene therapy strategy. Detectable CD levels were sustained upon reoxygenation and prolonged hypoxic exposures. Hypoxia-induced chemoresistance to 5-FU was overcome in both cell lines treated with this suicide gene therapy approach. Hypoxic transfectants were sensitized to prodrug concentrations that were ten-fold lower than those that are clinically relevant. Moreover, the surviving fraction of reoxygenated transfectants could be further reduced with the concomitant delivery of clinically relevant single radiation doses. CONCLUSIONS: This strategy thus has the potential to sensitize the hypoxic compartment of prostate tumours and improve the outcome of current therapies.

Immune evasion mechanisms in colorectal cancer liver metastasis patients vaccinated with TroVax (MVA-5T4).

We have recently reported the results of a phase II trial in which two TroVax [modified vaccinia ankara (MVA) encoding the tumour antigen 5T4] vaccinations were given to patients both pre- and post-surgical resection of liver metastases secondary to colorectal cancer (CRC). 5T4-specific cellular responses were assessed at the entry and 2 weeks after each vaccination by proliferation of fresh lymphocytes and ELISA for antibody responses; 18 from the 19 CRC patients mounted a 5T4-specific cellular and/or humoral response. Here, we present a comparison of individual and between patient responses over the course of the treatments using cryopreserved peripheral blood mononuclear cells (PBMC) samples from the baseline until after the fourth vaccination at 14 weeks. Assays used were proliferation assay with 5T4-Fc fusion protein, overlapping 32mer 5T4 peptides, MVA-LacZ and MVA-5T4 infected autologous monocytes. Responses to 5T4 protein or one or more peptide pools were pre-existing in 12/20 patients and subsequently 10 and 12 patients showed boosted and/or de novo responses, respectively. Cumulatively, 13/20 patients showed proliferative responses by week 14. We also assessed the levels of systemic T regulatory cells, plasma cytokine levels, phenotype of tumour-infiltrating lymphocytes including T regulatory cells and tumour HLA class I loss of expression. More than half of the patients showed phenotypes consistent with relative immune suppression and/or escape highlighting the complexity of positive and negative factors challenging any simple correlation with clinical outcome.

O6-Methylguanine-DNA methyltransferase inactivation and chemotherapy.

INTRODUCTION: Alkylating agents are frequently used in the chemotherapy of many types of cancer. This group of drugs mediates cell death by damaging DNA and therefore, understandably, cellular DNA repair mechanisms can influence both their antitumour efficacy and their dose-limiting toxicities. SOURCES OF DATA: This review focuses on the mechanism of action of the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT) and its exploitation in cancer therapy and reviews the current literature. AREAS OF AGREEMENT: MGMT can provide resistance to alkylating agents by DNA damage reversal. Inhibition of tumour MGMT by pseudosubstrates to overcome tumour resistance is under clinical evaluation. In addition, MGMT overexpression in haematopoietic stem cells has been shown in animal models to protect normal cells against the myelosuppressive effects of chemotherapy: this strategy has also entered clinical trials. AREAS OF CONTROVERSY: MGMT inhibitors enhance the myelotoxic effect of O(6)-alkylating drugs and therefore reduce the maximum-tolerated dose of these agents. Retroviral vectors used for chemoprotective gene therapy are associated with insertional mutagenesis and leukaemia development. GROWING POINTS: The results of ongoing preclinical and clinical research involving various aspects of MGMT modulation should provide new prospects for the treatment of glioma, melanoma and other cancer types. AREAS TIMELY FOR DEVELOPING RESEARCH: Tissue- and tumour-specific approaches to the modulation of MGMT together with other DNA repair functions and in combination with immuno- or radiotherapy are promising strategies to improve alkylating agent therapy.

A cancer cell-specific inducer of apoptosis.

Human papillomavirus (HPV) DNA is found in virtually all cervical cancers, strongly suggesting that these viruses are necessary to initiate this disease. The HPV E2 protein is required for viral replication, but E2 expression is usually lost in HPV-transformed cells because of the integration of viral DNA into the host chromosome. Several studies have shown that the reintroduction of E2 into HPV-transformed cells can induce growth arrest and apoptotic cell death. This raises the possibility that E2 could be useful in the treatment of HPV-induced disease. However, the effects of E2 on cell proliferation are not limited to HPV-transformed cells. The E2 protein from HPV type 16 can induce apoptosis via at least two pathways. One pathway involves the binding of E2 to p53 and operates in HPV-transformed cells, many non-HPV-transformed cell lines, and untransformed normal cells. The second pathway requires the binding of E2 to the viral genome and operates only in HPV-transformed cells. A mutation in E2 that significantly reduces the binding of this protein to p53 abrogates the induction of apoptosis in non-HPV-transformed cells and normal cells, but has no effect on the ability of the mutated protein to induce apoptosis in HPV-transformed cells. Here we show that a chimeric protein consisting of this mutant of E2, fused to the herpes simplex virus type 1 VP22 protein, can traffic between cells in a three-dimensional tumor model and induce apoptosis in HPV-transformed cells with high specificity. This cancer cell-specific inducer of apoptosis may be useful in the treatment of cervical cancer and other HPV-induced diseases.

CXCR4 mediated chemotaxis is regulated by 5T4 oncofetal glycoprotein in mouse embryonic cells.

5T4 oncofetal molecules are highly expressed during development and upregulated in cancer while showing only low levels in some adult tissues. Upregulation of 5T4 expression is a marker of loss of pluripotency in the early differentiation of embryonic stem (ES) cells and forms an integrated component of an epithelial-mesenchymal transition, a process important during embryonic development and metastatic spread of epithelial tumors. Investigation of the transcriptional changes in early ES differentiation showed upregulation of CXCL12 and down-regulation of a cell surface protease, CD26, which cleaves this chemokine. CXCL12 binds to the widely expressed CXCR4 and regulates key aspects of development, stem cell motility and tumour metastasis to tissues with high levels of CXCL12. We show that the 5T4 glycoprotein is required for optimal functional cell surface expression of the chemokine receptor CXCR4 and CXCL12 mediated chemotaxis in differentiating murine embryonic stem cells and embryo fibroblasts (MEF). Cell surface expression of 5T4 and CXCR4 molecules is co-localized in differentiating ES cells and MEF. By contrast, differentiating ES and MEF derived from 5T4 knockout (KO) mice show only intracellular CXCR4 expression but infection with adenovirus encoding mouse 5T4 restores CXCL12 chemotaxis and surface co-localization with 5T4 molecules. A series of chimeric constructs with interchanged domains of 5T4 and the glycoprotein CD44 were used to map the 5T4 sequences relevant for CXCR4 membrane expression and function in 5T4KO MEF. These data identified the 5T4 transmembrane domain as sufficient and necessary to enable CXCR4 cell surface expression and chemotaxis. Furthermore, some monoclonal antibodies against m5T4 can inhibit CXCL12 chemotaxis of differentiating ES cells and MEF which is not mediated by simple antigenic modulation. Collectively, these data support a molecular interaction of 5T4 and CXCR4 occurring at the cell surface which directly facilitates the biological response to CXCL12. The regulation of CXCR4 surface expression by 5T4 molecules is a novel means to control responses to the chemokine CXCL12 for example during embryogenesis but can also be selected to advantage the spread of a 5T4 positive tumor from its primary site.

Hypoxia-targeted over-expression of carboxylesterase as a means of increasing tumour sensitivity to irinotecan (CPT-11).

The induced expression of carboxylesterase (CE) enzymes, which convert the prodrug irinotecan (CPT-11) into its active cytotoxic metabolite SN-38, constitutes a promising strategy for cancer gene therapy. By incorporating hypoxia-responsive elements (HREs) in conjunction with the transgene, expression can be targeted specifically to hypoxic tissues (such as solid tumours), expressing the hypoxia-inducible factor 1 (HIF-1). We have constructed a recombinant adenoviral vector, AdHRE-rCE, encoding the cDNA for the highly efficient rabbit liver CE (rCE), under the control of a HRE derived from the human phosphoglycerate kinase 1 (PGK-1) gene in conjunction with a minimal SV40 promoter. In vitro, HT1080 fibrosarcoma and SW480 colon carcinoma cells demonstrated an approximately 10-fold hypoxia-dependent induction in CE expression following pre-infection with AdHRE-rCE, which led to a15-30-fold increased sensitivity to CPT-11. Furthermore, in vivo, SW480 tumour xenografts infected with AdHRE-rCE demonstrated a 2-fold decrease in tumour doubling time, when combined with 7 days of CPT-11 treatment, in comparison to mock-infected controls, with rCE expression shown to be limited to hypoxic regions only. As the cytotoxicity of CPT-11 is reduced under hypoxic conditions, over-expression of a highly efficient CE such as rCE under hypoxia control within these hypoxic cells could reverse this effect and, therefore, form the basis for future clinical treatment strategies.

The P140K mutant of human O(6)-methylguanine-DNA-methyltransferase (MGMT) confers resistance in vitro and in vivo to temozolomide in combination with the novel MGMT inactivator O(6)-(4-bromothenyl)guanine.

The O(6)-methylguanine-DNA-methyltransferase (MGMT) inactivator O(6)-benzylguanine (O(6)-beG) is currently under clinical investigation as a potential tumour-sensitising agent. In clinical trials its use has been associated with increased myelotoxicity and a reduced maximum tolerated dose (MTD) for BCNU. Thus the concept of myeloprotection by gene therapy with an O(6)-beG-insensitive mutant of MGMT is soon to be tested. Recently, an alternative inactivator has been described (O(6)-(4-bromothenyl)guanine, PaTrin-2), which shows potential advantages over O(6)-beG in terms of higher activity against wild-type MGMT and oral formulation. The use of PaTrin-2 has also been associated with increased myelotoxicity in clinical trials and thus PaTrin-2 may also be a candidate for use in conjunction with mutant MGMT gene transfer in genetic chemoprotective strategies. However, its activity against mutant MGMTs has not been reported. We show here that the P(140)K mutant of MGMT is highly resistant to inactivation by PaTrin-2. Furthermore, we show that a human haemopoietic cell line (K562) transduced with a retroviral vector encoding MGMT(P140K) is highly resistant to the cytotoxic effects of PaTrin-2 in combination with the methylating agent temozolomide, and that cells expressing MGMT(P140K) can be effectively enriched in vitro following challenge with this drug combination. Finally, we show that animals reconstituted with bone marrow expressing MGMT(P140K) exhibit haemopoietic resistance to PaTrin-2/temozolomide, which results in in vivo selection of gene-modified cells. All of these effects were comparable to those also achieved using O(6)-beG/temozolomide. Thus our data show that MGMT(P140K) is a suitable candidate for chemoprotective gene therapy where PaTrin-2 is being used in conjunction with temozolomide.

Dual agent chemoprotection by retroviral co-expression of either MDR1 or MRP1 with the P140K mutant of O6-methylguanine-DNA-methyl transferase.

Tumour resistance to chemotherapeutic agents results in most chemotherapy being administered in a multi-agent fashion that is often associated with a high level of toxicity in highly proliferative tissues such as the haematopoietic compartment. Thus, whilst many genetic manipulation strategies aim to protect normal tissue against a single component of a multi-agent regime, it is clearly preferable to protect normal cells against all toxicities. In this study we have used retroviral gene transfer to achieve co-expression of either p-glycoprotein (MDR1) or multi-drug resistance-related protein 1 (MRP1) with the P140K mutant form of O6-methylguanine-DNA-methyl transferase (MGMT) which, unlike the wild-type protein, is insensitive to inactivation by tumour sensitisers such as O6-benzylguanine (O6-BeG) or PaTrin2. The combination of certain MDR1/MRP1 substrate drugs with O6-alkylating agents (against which MGMT confers resistance) is particularly myelotoxic. We show here that haematopoietic progenitors co-expressing mutant MGMT with an ABC-transporter exhibit resistance to combination chemotherapy in vitro. This combination of drug transporter and DNA repair function may provide an effective in vivo protection of the haematopoietic compartment during tumour ablation using combination chemotherapy.

Radioprotective gene therapy through retroviral expression of manganese superoxide dismutase.

BACKGROUND: Radiotherapy for the control of cancer, either alone or in conjunction with chemotherapy, is often limited by normal tissue toxicity including haematopoietic toxicity. Exposure of cells to ionizing radiation leads to the formation of reactive oxygen species that are associated with radiation-induced cytotoxicity. The antioxidant enzyme manganese superoxide dismutase (SOD2) catalyzes the dismutation of the superoxide anions into hydrogen peroxide. METHODS: We have investigated the potential of SOD2 overexpression, through retroviral gene transfer using a retrovirus optimized for transcription in early haematopoietic cells, to enhance the radioresistance of a human erythroleukaemic cell line and primary murine bone marrow. Using these as in vitro models we have investigated whether SOD2 gene therapy may be suitable for the protection of the haematopoietic compartment from the effects of ionizing radiation. RESULTS: Here we demonstrate using both biological and physical assays that overexpression of SOD2 protects haematopoietic cells from ionizing radiation injury. Our results show that an increase in the levels of SOD2 enzymatic activity within K562 cells (from 160.7 +/- 23.6 to 321.8 +/- 45.2 U/mg protein) or primary murine haematopoietic progenitor cells leads to both a significant decrease in DNA fragmentation and a significant increase in clonogenic survival, as evident by a significant increase in Dbar (from 2.66 to 3.42Gy), SF2 (from 0.52 to 0.73) values, and a significant decrease in the alpha value (from 0.3040 +/- 0.037 to 0.0630 +/- 0.037 Gy(-1)) when compared either to cells transduced with a retroviral vector encoding eGFP alone or to the parental line. CONCLUSIONS: The results presented suggest that retroviral radioprotective gene therapy may be applicable to the haematopoietic compartment, enabling radiation dose escalation in cancer therapy.