A dual function fusion protein of Herpes simplex virus type 1 thymidine kinase and firefly luciferase for noninvasive in vivo imaging of gene therapy in malignant glioma

BACKGROUND: Suicide gene therapy employing the prodrug activating system Herpes simplex virus type 1 thymidine kinase (HSV-TK)/ ganciclovir (GCV) has proven to be effective in killing experimental brain tumors. In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive in vivo monitoring of the activity of a therapeutic gene in brain tumor cells. METHODS: The HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper. RESULTS: Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo. CONCLUSION: This approach may represent a valuable tool for the in vivo evaluation of gene therapy strategies for treatment of malignant disease

Expression of soluble guanylyl cyclase Catalytic activity requires two enzyme subunits

AbstractPurified soluble guanylyl cyclase consists of two subunits (70 and 73 kDa) whose primary structures were recently determined. The availability of cDNA clones coding for either subunit allowed to study the question of the functional roles of the two subunits in expression experiments. Enzyme subunits were expressed in COS-7 cells by transfection with expression vectors containing the coding region for the 70 of 73 kDa subunit of the enzyme. No significant elevation in the activity of soluble guanylyl cyclase was observed in cells transfected with cDNA coding for one of the subunits. In contrast, transfection of cells with cDNAs coding for both subunits resulted in a marked increase in activity of soluble guanylyl cyclase. Enzyme activity was stimulated about 50-fold by sodium nitroprusside. The results indicate that formation of cyclic GMP by soluble guanylyl cyclase requires'both 70 and 73 kDa subunits

Welfare Assessment following Heterotopic or Orthotopic Inoculation of Bladder Cancer in C57BL/6 Mice

<div><p>Few studies have assessed whether mice used as cancer models experience pain. Despite this possibility, the usual practice is to withhold analgesics as these are generally viewed as confounding. However, pain also alters cancer progression, so preventing it might not only be beneficial to welfare but also to study validity. Establishing the extent to which different cancer models result in pain is an important first step towards their refinement. We used conditioned place preference (CPP) testing and body-weight and behaviour analyses to evaluate the assumption that heterotopically implanted tumours result in less pain and fewer welfare concerns than those implanted orthotopically. C57Bl/6 mice received MB49^Luc luciferase expressing bladder cancer cells or saline implanted subcutaneously or into the bladder. These tumour-bearing or control groups underwent 2 daily 45 minute conditioning trials to saline or morphine (2mg/kg) and then a 15 minute drug-free preference test on day 3 of a 3 day cycle, continuing until the study ended. Tumours were imaged and behaviour data obtained following preference tests. Development of preference for the morphine-paired chamber (morphine-seeking) was determined over time. Heterotopic tumour development had no effect on morphine-seeking, and although the restraint used for heterotopic inoculation caused greater initial weight losses than anaesthesia, these mice steadily gained weight and behaved comparatively normally throughout the study. Orthotopic tumour inoculation caused no initial weight losses, but over the final 7 days these mice became less active and lost more body weight than cancer-free controls. This indicated orthotopic implantation probably caused a more negative impact on welfare or conceivably pain; but only according to the current test methods. Pain could not be confirmed because morphine-seeking in the tumour-bearing groups was similar to that seen in controls. Imaging was not found to be an effective method of monitoring tumour development surpassing manual tumour inspection.</p></div

Behaviour results.

<p>The mean frequency of rearing (±SEM) in the orthotopic (left axis; circles) and heterotopic study (right axis; triangles) in tumour bearing and control groups (filled versus open symbols); showing the significantly greater decline in activity in the tumour-bearing mice in the orthotopic study from CPP test 3 (P_3) to just prior to euthanasia (P_1).</p

Body weight results.

<p>Mean percentage changes from baseline (pre-inoculation) body weight (±SEM) in mice inoculated with MB49^luc bladder cancer (Tum) or DPBS (Ctrl) and conditioned to morphine (Mor) or saline (Sal). Panels respectively illustrate data spanning the first 18 days (6 CPP Tests) and the final 6 CPP tests prior to euthanasia (16 days) in mice inoculated orthotopically (a, b) or heterotopically (c, d).</p

Imaging results.

<p>(a) the mean total flux (TF ±SEM) of bioluminescent signals emanating from tumours implanted orthotopically (Orth; left axis, circles) or heterotopically (Het; right axis, triangles) in mice conditioned to morphine (Mor; closed symbols) or saline (Sal; open symbols) and imaged every 3 days (beginning on day 3) for 18 days (Day_CPP Test); (b) Mean TF (±SEM) during the final 6 CPP tests (P_6 to P_1; 18 days) before euthanasia; (c) Caliper measurements showing the mean tumour surface area (mm² ±SEM) of heterotopically implanted tumours over the first 18 post-inoculation days (6 CPP tests) and (d) last 16 days (6 CPP tests) in mice conditioned to morphine or saline.</p

CPP results.

<p>Proportionate S+ chamber residence time (mean ±SEM) over the final 6 CPP cycles (P_6 to P_1; 16 days) before euthanasia in mice inoculated orthotopically (a) or heterotopically (b) with MB49^luc bladder cancer (Tum) or DPBS (Ctrl) and conditioned over a repeated 3 day cycle to morphine (Mor) or saline (Sal).</p