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.

Synergistic effects of imatinib (STI 571) in combination with chemotherapeutic drugs in head and neck cancer.

The tyrosine kinase inhibitor imatinib (STI 571; glivec) is a potent inhibitor of bcr-abl, c-kit and platelet-derived growth factor receptors. Imatinib was evaluated both alone and in combination with established chemotherapeutic agents in adenoid cystic carcinoma (ACC) primary cultures and established cell lines representing squamous cell carcinoma of the head and neck (HNSCC). Over 90% of ACC tumors are c-kit-positive, and these primary cultures proved to be of short-term usefulness in assessing chemosensitivity. Interaction was determined over a wide range of drug combinations using a statistical three-dimensional analysis model. Both ACC short-term cultures and HNSCC cell lines were demonstrated to have a response ranging from additive to synergistic when imatinib and cisplatin were combined. The interaction of imatinib on cisplatin-induced DNA cross-linking was further investigated using the comet-X assay. In contrast, significant antagonism was observed when imatinib and gemcitabine were combined. Since gemcitabine is activated by deoxycytidine kinase (dCK), the effect of imatinib on this enzyme was investigated. A dose-dependent inhibition of dCK was observed, highlighting this kinase as a possible additional secondary molecular target for imatinib. This work demonstrates a synergistic interaction between cisplatin and imatinib, which may prove to be clinically relevant in the future management of both ACC and HNSCC.

Preclinical evaluation of the pharmacodynamic properties of 2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone.

PURPOSE: The purpose of our study was to investigate the cellular accumulation, DNA cross-linking ability, and cellular toxicity of RH1 (2,5-diaziridinyl-3-[hydroxymethyl[-6-methyl-1,4-benzoquinone), a novel DNA alkylating agent currently in clinical trials. In addition, the in vivo efficacy of RH1 formulated in different vehicles was also compared. EXPERIMENTAL DESIGN: RH1 is activated by the two-electron reducing enzyme NQO1 [NADPH:quinone oxidoreductase] forming a potent cytotoxic agent that cross-links DNA. We have used whole blood, cell lines, and primary explanted tumor cultures to measure both the cellular accumulation, DNA cross-linking, and cytotoxicity of RH1. Furthermore, the pharmacokinetic and pharmacodynamic characteristics of RH1 formulated in different vehicles were measured in vivo using the validated comet-X assay in mice bearing human tumor xenografts. RESULTS: Accumulation of RH1 was shown to be both time and concentration dependent, reaching a maximum after 2 hours and correlated well with DNA cross-linking measurements. DNA cross-linking in vitro could be detected at low (1-10 nmol/L) concentrations after as little as 2 hours exposure. In primary tumor cultures, RH1 induces much higher levels of DNA cross-links at lower doses than either mitomycin C or cisplatin. In vivo efficacy testing using polyvinyl pyrrolidone, saline, or cyclodextrin as vehicles showed DNA cross-links readily detectable in all tissues examined and was enhanced when given in cyclodextrin compared with polyvinyl pyrrolidone or saline. CONCLUSIONS: RH1 represents a potent bioreductive anticancer drug, which may prove effective in the treatment of cancers, particularly those that overexpress NQO1. DNA cross-linking can be reliably measured in tissue using the validated comet-X assay.

Activity profile of the novel aziridinylbenzoquinones MeDZQ and RH1 in human tumour xenografts.

BACKGROUND: RH1 and MeDZQ represent novel aziridinylbenzoquinones that can be activated by DT-diaphorase to form unique DNA lesions. RH1 is due to enter a phase 1 clinical trial in the United Kingdom in the summer of 2003, where pharmacodynamic monitoring of DT-diaphorase will be performed. MATERIALS AND METHODS: The antitumour efficacy of RH1 and MeDZQ has been studied in 4 human xenografts (3 non-small cell lung cancer and 1 colon cancer), and compared to the level of constitutive DT-Diaphorase activity measured by the DCPIP assay. RESULTS: The 4 xenografts exhibited a wide range of DT-diaphorase activity (4.8-303 nmol/min/mg). Greater antitumour activity was recorded in the xenografts expressing high levels of DT-diaphorase (e.g. NX002, DT-diaphorase activity, 303 +/- 52 nmol/min/mg, T/C to MeDZQ, 33.3% and to RH1, 43.4%). CONCLUSION: These data add in vivo support to a role for DT-Diaphorase in the antitumour activity of RH1.

3-substituted-5-aziridinyl-1-methylindole-4,7-diones as NQO1-directed antitumour agents: mechanism of activation and cytotoxicity in vitro.

Indolequinone agents are a unique class of bioreductive cytotoxins that can function as dual substrates for both one- and two-electron reductases. This endows them with the potential to be either hypoxia-selective cytotoxins or NAD(P)H:quinone oxidoreductase 1 (NQO1)-directed prodrugs, respectively. We have studied the structure-activity relationships of four novel indolequinone analogues with regard to one- and/or two-electron activation. Single-electron metabolism was achieved by exposing the human carcinoma cell line T47D to each agent under hypoxic conditions, whilst concerted two-electron metabolism was assessed by stably expressing the cDNA for human NQO1 in a cloned cell line of T47D. The C-3 and C-5 positions of the indolequinone nucleus were modified to manipulate reactivity of the reduction products and the four prodrugs were identified as NQO1 substrates of varying specificity. Two of the four prodrugs, in which both C-3 and C-5 groups remained functional, proved to be NQO1-directed cytotoxins with selectivity ratios of 60- to 80-fold in the T47D (WT) versus the NQO1 overexpressing T47D cells. They also retained selectivity as hypoxic cytotoxins with oxic/hypoxic ratios of 20- to 22-fold. Replacement of the C-3 hydroxymethyl leaving group with an aldehyde group ablated all selectivity in air and hypoxia in both cell lines. Addition of a 2-methyl group on the C-5 aziridinyl group to introduce steric hinderance reduced but did not abolish NQO1-dependent metabolism. However, it enhanced single-electron metabolism-dependent DNA cross-linking in a manner that was independent of cytotoxicity. These data demonstrate that subtle structure-activity relationship exists for different cellular reductases and under certain circumstances distinct forms of DNA damage can arise, the cytotoxic consequences of which can vary. This study identifies a candidate indolequinone analogue for further development as a dual hypoxia and NQO1-directed prodrug.