Nitroreductase-based therapy of prostate cancer, enhanced by raising expression of heat shock protein 70, acts through increased anti-tumour immunity.

Gene-directed enzyme-prodrug therapy (GDEPT) using nitroreductase (NTR), with efficient adenoviral delivery, and CB1954 (CB), is an effective means of directly killing tumours. However, an immune-mediated bystander effect remains an important product of GDEPT since it is often critical to the elimination of untransduced tumour cells both locally and at distal metastatic sites through generation of tumour-specific immunity without the need for tumour antigen identification or the generation of a personalised vaccine. The mode of induced tumour cell death is thought to contribute to the immunisation process, together with the induction and release of stress proteins. Here, RM-9 murine prostate tumour cells were efficiently killed by adenovirally delivered NTR/CB treatment both in vitro and in vivo, and bystander effects were observed. Cells appeared to die by pathways that suggest necrosis more than that of classical apoptosis. NTR/CB-induced expression of a range of stress proteins was determined by proteomic analysis, revealing chiefly heat shock protein (HSP)25 and HSP70 upregulation, whilst immune responses in vivo were weak. In an attempt to enhance the anti-tumour effect, an adenoviral vector was constructed that co-expressed NTR and HSP70, the latter being a known immune stimulator and chaperone of antigen. This combination elicited significantly enhanced protection over NTR alone for both the treated tumour and a subsequent re-challenge. Protection was CD4+ and CD8+ T cell-dependent and was associated with tumour-specific CTL, IFNgamma and IL-5 responses. The use of such a cytotoxic and immunomodulatory gene combination in cancer therapy warrants further pursuit.

Antitumor immune responses mediated by adenoviral GDEPT using nitroreductase/CB1954 is enhanced by high-level coexpression of heat shock protein 70.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising approach to local management of cancer through targeted chemotherapy. Killing localized tumors by GDEPT in a manner that induces strong antitumor cellular immune responses might improve local management and allow benefit in disseminated cancer. Here we evaluated the combination of nitroreductase (NTR)/CB1954 GDEPT with high-level expression of heat shock protein 70 (HSP70, a stress protein that can shuttle cytosolic peptides into antigen-presenting cells) for induction of antitumor immunity using adenovirus gene delivery in an aggressive and nonimmunogenic BALB/c syngeneic 4T1 breast cancer model. The mechanism of cell death and spectrum of stress proteins induced are likely to be important determinants of the resulting immune responses. We showed that NTR/CB1954 treatment of 4T1 cells gave both apoptotic and nonapoptotic killing. In vivo killing of 4T1 cells expressing NTR gave weak antitumor immunity and very limited induction of stress proteins including HSP70. High-level coexpression of HSP70 during NTR/CB1954-mediated killing of 4T1 cells in vivo gave much greater protection from tumor challenge (67% long-term survivors compared to 17%) and induced 4T1-specific cytotoxic T-cell responses. The enhancement of antitumor responses resulting from HSP70 coexpression was similar to that conferred by coexpression of GM-CSF.

Optimization of a synthetic beta-catenin-dependent promoter for tumor-specific cancer gene therapy.

We recently published the construction and evaluation of a beta-catenin-dependent, highly active promoter, CTP1, and its possible application for the treatment of colorectal cancer using gene-directed enzyme prodrug therapy with adenoviral (Ad) vectors. Alternative Ad-based approaches such as tumor-specific, replication-competent vectors and/or exploiting therapeutic gene products with intrinsic toxic activity, such as gibbon ape leukemia virus fusogenic membrane glycoprotein, diphtheria toxin A (DTA), and ricin, would demand a very tightly regulated promoter to avoid breakthrough replication and toxicity in nontumor tissue and Ad producer cell lines. In this study we optimized the activity/specificity profile of the synthetic beta-catenin-dependent promoter by varying its basal promoter, the number of Tcf binding sites, and the distance between these and the basal promoter. The optimal promoter, CTP4, showed virtually undetectable expression in cells with normal beta-catenin regulation but high level expression in cells deregulated for beta-catenin. Using CTP4 we were able to generate, for the first time to our knowledge, an Ad vector expressing fully active wild-type DTA without the need for time-consuming and cumbersome production systems. CTP4 should be the promoter of choice for Ad-based gene therapies of tumors deregulated for beta-catenin. We provide preliminary evidence that these may include prostate and ovarian as well as colorectal cancer.

A novel regulatory switch mediated by the FNR-like protein of Lactobacillus casei.

FNR (regulator for fumarate and nitrate reduction) and CRP (cAMP receptor protein) are global regulators which regulate the transcription of overlapping modulons of target genes in response to anaerobiosis and carbon source in Escherichia coli. An ORF, designated flp because it encodes an FNR-like protein of the FNR-CRP family, has been found in Lactobacillus casei. The product of the flp coding region (FLP) was overproduced in E. coli, purified and crystallized. FLP is a homodimeric protein in which each subunit can form an intramolecular disulphide bond. The isolated protein also contains non-stoichiometric amounts of Cu and Zn. Although the DNA recognition helix of FLP resembles that of FNR, the flp gene failed to complement the anaerobic respiratory deficiency of an fnr mutant when expressed in E. coli and it neither activated nor interfered with transcription from FNR- or CRP-dependent promoters in E. coli. Site-specific DNA binding by oxidized FLP (the form containing intrasubunit disulphide bonds) was abolished by reduction. The interconversion between disulphide and dithiol forms thus provides the basis for a novel redox-mediated transcriptional switch. Two non-identical FLP-binding sites, distinct from FNR- and CRP-binding sites, were identified in the meIR region of E. coli by gel-retardation analysis. A further eight FLP-binding sites were selected from a random library. A synthetic oligonucleotide conforming to a putative FLP site consensus, CA/CTGA-N4-TCAG/TG (the most significant bases are underlined), was retarded by FLP. Functional tests showed that FLP represses the aerobic transcription of a semi-synthetic promoter in E. coli. A C5S variant of FLP lacking the ability to form intramolecular disulphide bonds was unable to bind to FLP sites and failed to repress transcription in vivo.