Assessment of the hepatic CYP reductase null mouse model and its potential application in drug discovery.

CYP Oxidoreductase (Por) is the essential electron donor for all CYP enzymes and is responsible for the activation of CYP. The Taconic Hepatic CYP Reductase Null (HRN) mouse model possesses a targeted mutation that results in liver-specific deletion of the Por gene thereby resulting in a disruption of CYP metabolism in the liver. The objectives of these studies were to further characterize the HRN mouse using probe drugs metabolized by CYP. In addition, tumor exposure in xenograft tumor bearing HRN immune-compromised (nude) mice was also determined. In HRN mice following intravenous (iv) administration of midazolam, clearance (CL) was reduced by ∼ 80% compared to wild-type mice (WT). After oral administration, the AUC of midazolam was increased by ∼ 20-fold in HRN mice compared to WT mice; this greater effect suggests that hepatic first pass plays a role in the oral CL of midazolam. A 50% and an 80% decrease in CL were also observed in HRN mice following iv administration of docetaxel and theophylline, respectively, compared to WT mice. In addition, a 2- to 3-fold increase in tumor concentrations of G4222, a tool compound, were observed in tumor bearing HRN nude mice compared to tumor bearing nude WT mice. The observations from these experiments demonstrate that, for compounds that are extensively metabolized by hepatic CYP, the HRN mouse model could potentially be valuable for evaluating in vivo efficacy of tool compounds in drug discovery where high hepatic CL and low exposure may prevent in vivo evaluation of a new chemical entity.

Malignant fibrous histiocytoma and fibrosarcoma of bone: a re-assessment in the light of currently employed morphological, immunohistochemical and molecular approaches.

Malignant fibrous histiocytoma (MFH) and fibrosarcoma (FS) of bone are rare malignant tumours and contentious entities. Sixty seven cases labelled as bone MFH (57) and bone FS (10) were retrieved from five bone tumour referral centres and reviewed to determine whether recent advances allowed for reclassification and identification of histological subgroups with distinct clinical behaviour. A panel of immunostains was applied: smooth muscle actin, desmin, h-caldesmon, cytokeratin AE1-AE3, CD31, CD34, CD68, CD163, CD45, S100 and epithelial membrane antigen. Additional fluorescence in situ hybridisation and immunohistochemistry were performed whenever appropriate. All cases were reviewed by six bone and soft tissue pathologists and a consensus was reached. Follow-up for 43 patients (median 42 months, range 6-223 months) was available. Initial histological diagnosis was reformulated in 18 cases (26.8 %). Seven cases were reclassified as leiomyosarcoma, six as osteosarcoma, three as myxofibrosarcoma and one each as embryonal rhabdomyosarcoma and interdigitating dendritic cell sarcoma. One case showed a peculiar biphasic phenotype with epithelioid nests and myofibroblastic spindle cells. Among the remaining 48 cases, which met the WHO criteria for bone FS and bone MFH, we identified five subgroups. Seven cases were reclassified as undifferentiated pleomorphic sarcoma (UPS) and 11 as UPS with incomplete myogenic differentiation due to positivity for at least one myogenic marker. Six were reclassified as spindle cell sarcoma not otherwise specified. Among the remaining 24 cases, we identified a further two recurrent morphologic patterns: eight cases demonstrated a myoepithelioma-like phenotype and 16 cases a myofibroblastic phenotype. One of the myoepithelioma-like cases harboured a EWSR1-NFATC2 fusion. It appears that bone MFH and bone FS represent at best exclusion diagnoses.

Therapeutic efficacy as predicted by quantitative assessment of murine RIF-1 tumour pH and phosphorous metabolite response during hyperthermia: an in vivo 31P NMR study.

Described herein are the initial findings from an 'in-magnet' 31P NMR compatible hyperthermia system capable of concurrently heating and monitoring the metabolic response of murine tumours; the murine radiation induced fibrosarcoma (RIF-1) was employed for these studies. At thermal doses sufficient to raise tumour temperature to 41.5 and 43 degrees C for a period of 30 min, a marked and rapid decrease in nucleoside triphosphate concentration and in pH was observed during the heating period, while inorganic phosphate concentration increased significantly but more gradually. These 31P NMR determined metabolic indices remained depressed/elevated throughout a 1.5 h post-hyperthermia monitoring period. Importantly, these metabolic indices correlated significantly with specific growth delay. This suggests a possible role for NMR spectroscopy in early assessment, and perhaps control, of therapeutic response to hyperthermia.

Fluorescence-based assessment of LRP activity: a comparative study.

BACKGROUND: Broad resistance to anticancer drugs is a major cause of failure in cancer treatment. The Lung Resistance-related Protein (LRP) is a protein associated with drug resistance, which is involved in nucleo-cytoplasmic transport and is known to predict a poor response to chemotherapy in acute myeloid leukaemia. The only method allowing the detection of LRP activity is based on radio-labelled daunorubicin incorporation. Our goal was to develop a fluorescence-based assay to analyse LRP function. MATERIALS AND METHODS: We used human colon carcinoma cell lines treated with sodium butyrate (NaB) in order to induce LRP expression. Daunorubicin efflux in isolated nuclei was measured by flow cytometry, the localization and quantification of Daunorubicin analysed by confocal laser scanning microscopy (CLSM) and the diffusion coefficient of this drug estimated by Fluorescence Correlation Spectrometry (FCS). RESULTS: According to the method using [14C] Doxorubicin cells incubated with NaB displayed an efflux of Daunorubicin out of isolated nuclei demonstrated by flow cytometry or CLSM. The FCS method was able to evaluate kinetics of Daunorubicin molecules in nucleus and cytoplasm and showed a higher dispersion of Daunorubicin kinetics with cells previously NaB-treated. This argument is in favour of an increase of nucleo-cytoplasmic exchange. CONCLUSION: Using CLSM we showed that LRP was able to modify anticancer drug repartition in the cells. LRP activity assessment needs either isolated nuclei if flow cytometry is employed, or FCS, and only a few cells may be analysed.

Assessment of DNA damage produced by 125I-triplex-forming oligonucleotides in cells.

PURPOSE: Triplex-forming oligodeoxyribonucleotides (TFOs) bind specifically to their target sequences by forming hydrogen bonds within the major groove of the target duplex. When labeled with Auger-electron-emitting radioisotopes, TFOs are able to damage the target gene in a process named antigene radiotherapy. We compared radiotoxicity and the amount of DNA damage produced within cultured cells by two 125I-labeled TFOs, one with a single target in the genome and another with multiple targets. MATERIALS AND METHODS: Radiotoxicity was measured by clonogenic assay while DNA damage was assessed by the number of histone gamma-H2AX foci formed at the sites of DNA double strand breaks (DSBs). RESULTS: The TFO with multiple nuclear targets was 1.7 fold more radiotoxic and produced on average 1.9 fold more gamma-H2AX foci per cell than the TFO with a single target. CONCLUSION: Since the two methods gave comparable results, measuring the number of gamma-H2AX foci per decay may be a useful procedure for the assessment of cytotoxic effects and the intranuclear localization of radionuclides when they produce DSBs.

The use of needle biopsies for radiobiological assessment of oxygen levels in KHT-C tumors.

Hypoxia affects the sensitivity of cells to radiation. Hence there is considerable interest in the development and assessment of techniques for measuring oxygen levels. In the work described here, we explore the use of tumor needle biopsies (fine needle aspirates) in an assay that is standard in the field of radiation biology: the paired survival assay. We found that needle biopsies are a feasible option for estimating cell survival when conducting this assay, and that the variability in cell survival between tumors was greater than that between different biopsies from the same tumor. Using this technique, we then compared measurements of tumor hypoxia using the paired survival assay and the growth delay assay in the same individual tumors. We found a significant correlation between these two techniques.

Assessment of changes in murine tumor oxygenation in response to nicotinamide using 19F NMR relaxometry of a perfluorocarbon emulsion.

The oxygen dependencies of the 19F NMR spin-lattice relaxation rates (R1 = 1/T1) of a perfluorocarbon emulsion sequestered in a murine tumor model has been used to evaluate nicotinamide, a radiosensitizer believed to act through enhanced tissue oxygenation. Fluorine-19 NMR spectroscopic measurements from solid Radiation-Induced Fibrosarcoma (RIF-1) tumors in C3H mice showed a statistically significant improvement in tumor pO2 for a Nicotinamide-treated group, with a delta pO2 = 4.7 +/- 3 torr ( = mm Hg) (Mean +/- SEM) at t = 60 min (P < .01), and 4.5 +/- 3 at t = 70 min post intraperitoneal injection (P < 0.02) as compared with saline-treated Controls, while several other time points for which t > 30 min were significant at the P < 0.05 level. Both groups had n = 10, and the statistics were based on Student's one-tailed group t test. By comparison, in another study group where breathing gas was switched from air to 100% O2, a statistically insignificant increase of 2 torr was realized in tumor pO2 (n = 9). The maximal treatment effect occurs at a delay of 60 to 70 min, consistent with results obtained by other investigators using radiobiology techniques. Fluorine-19 spectroscopic relaxometry can measure therapeutically meaningful changes in in vivo tumor pO2 and represents an improvement in expenditures of time, animal resources, and statistical power over conventional radiobiological methods.

A rapid method for assessment of a macrophage chemotactant produced by SaD2 fibrosarcoma cells in vitro.

A new rapid method for assessing murine macrophage chemotaxis was developed using peritoneal exudate cells labeled with [5,6-3H]uridine in modified Boyden chambers with double polycarbonate filters. The method gave positive results with endotoxin-treated mouse serum and with serum-free culture supernatant of the DBA/2 SaD2 fibrosarcoma. These results correlated with results obtained with conventional microscopic assessment of chemotaxis. Using this method, the SaD2 supernatant was shown to be chemotactic rather than chemokinetic.