Viral delivery of P450 reductase recapitulates the ability of constitutive overexpression of reductase enzymes to potentiate the activity of mitomycin C in human breast cancer xenografts

Indolequinones such as mitomycin C (MMC) require enzymatic bioreduction to yield cytotoxic moieties. An attractive approach to overcome the potential variability in reductive bioactivation between tumors is to exploit specific enzyme-bioreductive drug combinations in an enzyme-directed gene therapy (GDEPT) approach. To this end, human breast cancer cell lines (T47D, MDA468, and MDA231) that overexpress either DT-diaphorase (DTD) or NADPH:cytochrome P450 reductase (P450R) have been developed. Cytotoxicity of MMC was evaluated in the panel of cell lines following aerobic or anoxic exposure in vitro. DTD and/or P450R overexpression sensitized cells to MMC in air with no further increase in the cytotoxicity of MMC under anoxia. The most profound effect was seen in the MDA468 cells, where a 27-fold increase in potency was observed for MMC in the DTD-overexpressing cell line. The MMC sensitization achieved through DTD and P450R overexpression in MDA468 cells was maintained in vivo. Xenografts established from the clonal lines exhibited significant tumor control following MMC treatment (treated/control [T/C] 17% and 51% for DTD and P450R xenografts, respectively) that was not seen in wild-type tumors (T/C 102%). Delivery of a clinically relevant adenoviral vector encoding P450R to MDA468 wild-type tumors yielded comparable P450R activity to that seen in the P450R clonal xenografts and resulted in greater MMC sensitization (T/C 46%). The model systems developed will facilitate the identification of novel indolequinone agents that are targeted toward a specific enzyme for bioactivation and are consequently of potential use in a GDEPT approach.     

Longitudinal genotype analysis and quantification of hepatitis C virus in haemophilia patients receiving interferon-α therapy

SUMMARY. Haemophilic patients have a high prevalence of hepatitis C virus (HCV) infection because of the use of unsterilized clotting factor concentrates. Six major genotypes of HCV have been distinguished so far, with epidemiological evidence suggesting that genotypes 1–3 are common in the indigenous UK and US populations. The aim of this study was to analyse the changes in viral load and composition of the HCV quasispecies in haemophilic patients receiving therapy with interferon-α (IFN-α) using the four major methods currently available for HCV genotyping. The most consistent genotype results were obtained using restriction fragment-length polymorphism (RFLP) analysis when compared with the DNA sequence analysis, and showed that the dominant genotype can change in patients with mixed genotype infections treated with IFN-α. This study indicates the difficulties in studying this group of patients with mixed HCV genotype infections, and that frequent sampling is necessary, together with viral load measurement to monitor response to IFN-α therapy.     

Ovarian autografts in sheep as a model for studying folliculogenesis

The studies outlined in this review indicate that the cortical autograft, in addition to its clinical application as a means to restore fertility, represents a valuable experimental model that can be exploited to examine aspects of both early and terminal follicle development. The autograft procedure is a means to experimentally deplete the follicle population in an individual and this procedure results in similar endocrine changes and reproductive cycle perturbances as those observed in aged sheep and women with incipient ovarian failure. This methodology therefore represents a non-primate large animal model to study the consequences of, and possible interventions to overcome, reproductive problems associated with depleted ovarian follicular reserves. Without the necessity of keeping animals for large periods of time so that this depletion can occur naturally. In terms of early follicle development, the fact that the ischaemia that occurs during revascularisation of the autograft effectively synchronises follicle development at the primordial stages of development means that the autograft can be used as a model to study the control of early follicle development. This model has been used to examine the role of FSH (follicle stimulating hormone) in the control of early follicle development and the preliminary data presented provides strong evidence that FSH does indeed modulate early folliculogenesis, confirming the value of this model as a means of performing experimental investigations in this area. Further work using this model will concentrate on the role of other endocrine and local factors in the control of early folliculogenesis and the identification of the key developmental checkpoints during this process, with a view to designing physiological culture systems to support early follicle and oocyte development.     

Determining Demographic Effects of Cypermethrin in the Marine Copepod Acartia tonsa: Stage-Specific Short Tests Versus Life-Table Tests

Short-term lethal and sublethal responses of the calanoid copepod Acartia tonsa to cypermethrin were compared with life-table responses to assess whether or not it is necessary to use exposure periods longer than 5 days to estimate demographic responses to stress. More specifically, by limiting exposure periods to sensitive age classes (eggs, nauplii, copepodids, and adults) and including measurements on survival, egg production, and feeding rates, it was possible to derive a short test design of similar sensitivity and ecological relevance as full life-table tests. Short-term exposures to cypermethrin reduced copepodid's feeding rates at concentrations well below those affecting egg production rates and survival of eggs and adult stages. Lethal effects on naupliar stages occurred at lower concentrations than any other effect observed on eggs and adults. Life-table sensitivities of the intrinsic rate of increase (r _m) to cypermethrin were similar to those observed in short-term exposures. More specifically, exposure to cypermethrin impaired r _m responses at concentrations (7.4 ng L⁻¹) that also affected feeding and naupliar responses. Our results show that by quantifying and separating combined toxic effects on ecologically relevant individual life-history traits, it is possible to develop toxicity test designs of similar ecological relevance yet that are less labor-intensive and costly than existing demographic tests. Received: 18 December 2001/Accepted: 5 April 2002