Human Xenografts In Nude Mice Research Articles

Flavones and flavonols may have clinical potential as CK2 inhibitors in cancer therapy

The serine-threonine kinase CK2, which targets over 300 cellular proteins, is overexpressed in all cancers, presumably reflecting its ability to promote proliferation, spread, and survival through a wide range of complementary mechanisms. Via an activating phosphorylation of Cdc373, a co-chaperone which partners with Hsp90, CK2 prolongs the half-life of protein kinases that promote proliferation and survival in many cancers, including Akt, Src, EGFR, Raf-1, and several cyclin-dependent kinases. CK2 works in other ways to boost the activity of signaling pathways that promote cancer aggressiveness and chemoresistance, including those driven by Akt, NF-kappaB, hypoxia-inducible factor-1, beta-catenin, TGF-beta, STAT3, hedgehog, Notch1, and the androgen receptor; it promotes the epidermal-mesenchymal transition and aids efficiency of DNA repair. Several potent and relatively specific inhibitors of CK2 are now being evaluated as potential cancer drugs; CX-4945 has shown impressive activity in cell culture studies and xenograft models, and is now entering clinical trials. Moreover, it has long been recognized that the natural flavone apigenin can inhibit CK2, with a Ki near 1 µM; more recent work indicates that a range of flavones and flavonols, characterized by a planar structure and hydroxylations at the 7 and 4′ positions – including apigenin, luteolin kaempferol, fisetin, quercetin, and myricetin - can inhibit CK2 with Ki s in the sub-micromolar range. This finding is particularly intriguing in light of the numerous studies demonstrating that each of these agents can inhibit the growth of cancer cells lines in vitro and of human xenografts in nude mice. These studies attribute the cancer-retardant efficacy of flavones/flavonols to impacts on a bewildering array of cellular targets, including those whose activities are boosted by CK2; it is reasonable to suspect that, at least in physiologically achievable concentrations, these agents may be achieving these effects primarily via CK2 inhibition. Inefficient absorption and rapid conjugation limit the bioefficacy of orally administered flavonoids; however, the increased extracellular beta-glucuronidase of many tumors may give tumors privileged access to glucuronidated flavonoids, and nanopartical technology can improve the bioavailability of these agents. Enzymatically modified isoquercitrin has particular promise as a delivery vehicle for quercetin. Hence, it may be worthwhile to explore the clinical potential of flavones/flavonols as CK2 inhibitors for cancer therapy.

Abstract A54: Pantoprazole enhances the activity of docetaxel chemotherapy for solid tumors by inhibition of autophagy

Abstract Purpose: Resistance to anti-tumour agents is a major cause of treatment failure in patients with cancer. Luciani and colleagues (JNCI 2004; 96:1702-13) have shown that PPIs can augment the effects of chemotherapy in vitro and in vivo, most likely by modifying the acid-base balance across the cell membrane and/or of intracellular organelles such as lysosomes and endososmes. Endosomes are important intermediaries of the process of autophagy, a survival mechanism for stressed cells and a possible target for anticancer therapy. Here we evaluated the effect of pantoprazole (PTP) (i) to modify cytotoxic effects of the commonly-used anticancer drug docetaxel (DOC) for cultured cells and for human tumour xenografts. (ii) to modify the distribution of biomarkers of activity of docetaxel in relation to tumour blood vessels. (iii) to inhibit autophagy. Methods: Endosomal pH of cells was measured by fluorescence spectroscopy following exposure to the endosomal probe Lysosensor. Levels of autophagy were evaluated by quantifying the level of the LC3B protein in Western blots. Human prostate cancer PC3 cells were transfected with a construct where LC3B was linked to red (RFP) and green (GFP) fluorescent protein (mRFP-GFP-LC3) such that cells with high levels of autophagy stained red while those with low levels stained green. Cytotoxicity of DOC +/-PTP was evaluated in vitro by a colony-forming assay for PC3 parental cells, and for cells transfected with shRNAs to inhibit autophagy. Growth delay of three human xenografts in nude mice was evaluated following treatment with single or multiple doses of DOC+/- PTP 3. The distribution of drug effects in tumour sections in relation to functional blood vessels (recognized by Dioc7) was quantified (at early intervals of ~10 minutes) by γH2AX, a marker of DNA damage, (at 24 hours) by cleaved caspase 3, a marker of apoptosis, Ki67, a marker of cell proliferation and LC3, a marker of autophagy. Results: PTP increased endosomal pH and inhibited autophagy in a dose and time dependent manner. PTP was not cytotoxic when used alone but increased the cytotoxicity of DOC for cultured PC3 cells; the effects of PTP were reduced against genetically-modified cells that have reduced ability to undergo autophagy. Pre-treatment with PTP increased markedly the growth delay due to DOC of three different xengografts, especially after three treatments at weekly intervals, with minimal effects on DOC toxicity. Cellular effects of DOC evaluated by γH2AX and by cleaved caspase 3 decreased with increasing distance from tumour blood vessels, but were enhanced substantially by pre-treatment with PTP. There is less cell proliferation and marked reduction in hypoxia in xenografts generated from autophagy-deficient PC3 cells compared to PC3 wild type xenografts. Conclusions: Autophagy is a mechanism of resistance of chemotherapy that may be modified by pantoprazole to improve therapeutic. Docetaxel is the primary chemotherapy for men with metastatic castration resistant prostate cancer, and we plan a phase II study of PTP + DOC in such patients. Citation Format: QIAN TAN, Anthony Joshua, Jasdeep K. Saggar, Yu Man, Carol CM Lee, Bradly R. Wouters, Ian F. Tannock. Pantoprazole enhances the activity of docetaxel chemotherapy for solid tumors by inhibition of autophagy. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A54. doi:10.1158/1538-7445.CHTME14-A54

Abstract 3829A: Pantoprazole enhances the activity of chemotherapy for solid tumors by inhibition of autophagy.

Abstract Purpose: Resistance to anti-tumour agents is a major cause of treatment failure in patients with cancer. Luciani have shown that PPIs can augment the effects of chemotherapy in vitro and in vivo, most likely by modifying the acid-base balance across the cell membrane and/or of intracellular organelles such as lysosomes and endososmes. Endosomes are important intermediaries of the process of autophagy, a survival mechanism for stressed cells and a possible target for anticancer therapy. Here we evaluated the effect of pantoprazole (PTP) to modify cytotoxic effects of the commonly-used anticancer drug docetaxel (DOC) for cultured cells and for human tumour xenografts, to modify the distribution of biomarkers of activity of docetaxel in relation to tumour blood vessels and inhibit autophagy. Methods: Endosomal pH of cells was measured by fluorescence spectroscopy following exposure to the endosomal probe Lysosensor. Levels of autophagy were evaluated by quantifying the level of the LC3B protein in Western blots. Human prostate cancer PC3 cells were transfected with a construct where LC3B was linked to red (RFP) and green (GFP) fluorescent protein (mRFP-GFP-LC3) such that cells with high levels of autophagy stained red while those with low levels stained green. Cytotoxicity of DOC +/-PTP was evaluated in vitro by a colony-forming assay for PC3 parental cells, and for cells transfected with shRNAs to inhibit autophagy. Growth delay of three human xenografts in nude mice was evaluated following treatment with single or multiple doses of DOC+/- PTP. The distribution of drug effects in tumour sections in relation to blood vessels was quantified (at early intervals of ∼30 minutes) by γH2AX, a marker of DNA damage, and (at 24 hours) by cleaved caspase 3, a marker of apoptosis. Results: PTP increased endosomal pH and inhibited autophagy in a dose and time dependent manner. PTP was not cytotoxic when used alone but increased the cytotoxicity of DOC for cultured PC3 cells; the effects of PTP were reduced against genetically-modified cells that have reduced ability to undergo autophagy. Pre-treatment with PTP increased markedly the growth delay due to DOC of three different xengografts, especially after three treatments at weekly intervals, with minimal effects on DOC toxicity. Cellular effects of DOC evaluated by γH2AX and by cleaved caspase 3 decreased with increasing distance from tumour blood vessels, but were enhanced substantially by pre-treatment with PTP. There is less cell proliferation and marked reduction in hypoxia in xenografts generated from autophagy-deficient PC3 cells compared to PC3 wild type xenografts. Conclusions: Pantoprazole increases the therapeutic effectiveness of docetaxel in vitro and in vivo, and a potential mechanism is inhibition of autophagy. Docetaxel is the primary chemotherapy for men with metastatic castration resistant prostate cancer, and we plan a phase II study of PTP + DOC in such patients. Citation Format: Qian Tan, Anthony Joshua, Jasdeep Saggar, Man Yu, Carol M Lee, Marianne Koritzinsky, Bradly R Wouters, Ian F Tannock. Pantoprazole enhances the activity of chemotherapy for solid tumors by inhibition of autophagy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3829A. doi:10.1158/1538-7445.AM2013-3829A

Anticancer Activity of New Haloalkyl Camptothecin Esters against Human Cancer Cell Lines and Human Tumor Xenografts Grown in Nude Mice

All chemotherapeutic agents currently in use have a narrow window of therapeutic index of 1 to 1.2. Camptothecin ester compounds are reported to have a wider therapeutic index when being used to treat human xenografts in nude mice. As a continuous effort in searching for better chemotherapeutic agents for treating cancers, new haloalkyl camptothecin and 9-nitrocamptothecin ester derivatives 2a-b and 3a-d were prepared by respective acylation of camptothecin 1a and 9-nitrocamptothecin 1b with the corresponding acylating agents. These new derivatives were tested in vitro against 8 human cancer cell lines using 7 different concentrations ranging from 5 to 300 nM and also in vivo against various types of human tumor xenografts grown in nude mice. Most of these new compounds started showing inhibitory effects on the growth of 8 cancer cell lines at concentration of 80 nM and achieved greater than 70% inhibitions against these cell lines when the concentration increased to 300 nM. Compound 2a and 3a showed good activity against human tumor xenografts in nude mice. Compared to mother compound camptothecin, 3a was much less toxic in mice with a better therapeutic index, having the potential to be further developed as a safer treatment for cancers.

Abstract B33: Intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a novel prodrug activator gene transfer technology in an immune-competent model of glioblastoma

Abstract Background: Patients with the most common and aggressive form of brain cancer, glioblastoma multiforme (GBM), have poor prognosis and current treatments remain only palliative. Previous gene therapy approaches using replication-deficient viral vectors have demonstrated limited efficacy likely due to poor gene delivery to the tumor. Methods: We used a novel replication-competent retroviral vector (Toca 511) designed and engineered to efficiently deliver a modified cytosine deaminase (CD) prodrug activating gene to glioma cells. Previous studies showed that such a replicating vector can preferentially infect intracranial human xenografts in nude mice (Tai et al. Mol Ther. 2005;12:842) and lead to increased survival in treatment groups compared to controls. In preparation for clinical trials we have improved the CD gene activity and further stabilized the vector structure to create Toca 511. We investigated the functionality of Toca 511 using stereotactic, intratumoral delivery in a syngeneic, orthotopic glioma model in immune-competent B6C3F1 mice. Freshly excised tumors were analyzed by quantitative PCR, Western, and HPLC. Results: Brain tumors isolated from mice treated with Toca 511 and fluorocytosine (5FC) had observable expression of CD protein and vector copy numbers per genome that demonstrate efficient tumor infection. Extensive tumor specific viral spread with almost no spread of vector from the site of injection was observed in the first 24 days. Highly efficient intratumoral conversion of the 5FC prodrug into the anticancer drug 5-fluorouracil (5FU) was demonstrated after intraperitoneal or oral dosing with 5FC. Studies with Toca 511 at high and mid dose levels in combination with 5FC resulted in prolonged survival compared to controls (> 5 months, p<0.0036 and <0.0354 respectively) in this syngeneic glioma model. The highest Toca 511 dose level tested in combination with 5-FC yielded no safety related findings in the mice. Conclusions: Toca 511 efficiently delivers a functional CD gene for expression in glioma cells in a syngeneic, orthotopic model in immune-competent mice. Toca 511 increases survival through the delivery of CD gene and subsequent efficient conversion of 5FC into 5FU within the brain tumor with no safety related findings. A phase 1 ascending dose trial investigating the safety and tolerability of Toca 511 in combination with 5FC in patients with recurrent GBM is about to initiate. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B33.

Tissue inhibitor of metalloproteinase-2 inhibits ameloblastoma growth in a new mouse xenograft disease model

Ameloblastomas are odontogenic neoplasms characterized by local invasiveness. This study was conducted to develop a new animal model of ameloblastoma and to address the role of tissue inhibitor of metalloproteinase-2 (TIMP-2) and matrix metalloproteinase-2 (MMP-2) in the growth and invasiveness of ameloblastomas. Donated fresh human ameloblastoma tissue was finely minced, screened, and subcutaneously implanted in three locations on each of 10 BALB/c-nu/nu nude mice. Newly established tumors on each mouse were injected with: (i) transfection reagent; (ii) liposome and transfection reagent; or (iii) liposome, transfection reagent, and the expression plasmid pcDNA3.1(+)/green fluorescent protein (GFP)-TIMP-2. Tumors were monitored for 5 weeks and excised for histopathology, RNA, and protein analyses. The ameloblastoma xenografts were established with high frequency and contained a variety of typical features, validating this new model system. Xenografts injected with the TIMP-2 expression plasmid showed reduced growth, increased TIMP-2 mRNA and protein, and decreased MMP-2 protein compared with the control groups. We successfully established a new experimental model of ameloblastoma consisting of subcutaneous human xenografts in nude mice. In addition, we demonstrated the successful introduction of the TIMP-2 gene in tumor xenograft cells in vivo, resulting in xenograft growth inhibition. This growth inhibition may have resulted from TIMP-2 overexpression specifically inhibiting MMP-2 protein expression and activity.

Tumor Interstitial Fluid Pressure as an Early-Response Marker for Anticancer Therapeutics

Solid tumors have a raised interstitial fluid pressure (IFP) due to high vessel permeability, low lymphatic drainage, poor perfusion, and high cell density around the blood vessels. To investigate tumor IFP as an early-response biomarker, we have tested the effect of seven anticancer chemotherapeutics including cytotoxics and targeted cytostatics in 13 experimental tumor models. IFP was recorded with the wick-in-needle method. Models were either ectopic or orthotopic and included mouse and rat syngeneic as well as human xenografts in nude mice. The mean basal IFP was between 4.4 and 15.2mm Hg; IFP was lowest in human tumor xenografts and highest in rat syngeneic models. Where measured, basal IFP correlated positively with relative tumor blood volume (rTBV) determined by dynamic contrast-enhanced magnetic resonance imaging. Most chemotherapeutics sooner (2 or 3 days) or later (6 or 7 days) lowered tumor IFP significantly, and the cytotoxic patupilone caused the greatest decrease in IFP. In rat mammary orthotopic BN472 tumors, significant drug-induced decreases in IFP and rTBV correlated positively with each other for both patupilone and the cytostatic vatalanib. In the two orthotopic models studied, early decreases in IFP were significantly (P ≤ .005) correlated with late changes in tumor volume. Thus, drug-induced decreases in tumor IFP are an early marker of response to therapy, which could aid clinical development.

The Development of a Selective Cyclin-Dependent Kinase Inhibitor That Shows Antitumor Activity

Normal progression through the cell cycle requires the sequential action of cyclin-dependent kinases CDK1, CDK2, CDK4, and CDK6. Direct or indirect deregulation of CDK activity is a feature of almost all cancers and has led to the development of CDK inhibitors as anticancer agents. The CDK-activating kinase (CAK) plays a critical role in regulating cell cycle by mediating the activating phosphorylation of CDK1, CDK2, CDK4, and CDK6. As such, CDK7, which also regulates transcription as part of the TFIIH basal transcription factor, is an attractive target for the development of anticancer drugs. Computer modeling of the CDK7 structure was used to design potential potent CDK7 inhibitors. Here, we show that a pyrazolo[1,5-a]pyrimidine-derived compound, BS-181, inhibited CAK activity with an IC(50) of 21 nmol/L. Testing of other CDKs as well as another 69 kinases showed that BS-181 only inhibited CDK2 at concentrations lower than 1 micromol/L, with CDK2 being inhibited 35-fold less potently (IC(50) 880 nmol/L) than CDK7. In MCF-7 cells, BS-181 inhibited the phosphorylation of CDK7 substrates, promoted cell cycle arrest and apoptosis to inhibit the growth of cancer cell lines, and showed antitumor effects in vivo. The drug was stable in vivo with a plasma elimination half-life in mice of 405 minutes after i.p. administration of 10 mg/kg. The same dose of drug inhibited the growth of MCF-7 human xenografts in nude mice. BS-181 therefore provides the first example of a potent and selective CDK7 inhibitor with potential as an anticancer agent.

The Essential Role of the Mitochondria and Reactive Oxygen Species in Cisplatin-Mediated Enhancement of Fas Ligand-Induced Apoptosis in Malignant Pleural Mesothelioma

Cytotoxic chemotherapeutic drugs such as cisplatin (CDDP) synergistically interact with soluble Fas ligand (sFasL) to mediate profound induction of apoptosis in cancer cells, particularly those refractory to this death-inducing ligand. The goal of this study was to evaluate the roles of the mitochondria-dependent apoptotic cascade and the CDDP-generated reactive oxygen species (ROS) in mediating the supra-additive enhancement of cytotoxicity and apoptosis in combination-treated malignant pleural mesothelioma (MPM) cells. MPM cells were treated with sequential CDDP/sFasL in vitro. Cell viability and apoptosis were determined by MTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays. Stable transfectants expressing high levels of Bcl2 were created by retroviral gene transfer. Specific proteolytic activity of caspases 3, 8, and 9 were measured using fluorescent substrates. Pretreating MPM cells with CDDP increased their susceptibility to sFasL by 2- to more than 20-fold. Overexpression of either Bcl-2, the selective caspase 9 inhibitor z-LEHD-fmk, or the antioxidant N-acetylcysteine significantly abrogated combination-induced cytotoxicity and apoptosis. Moreover, the robust activation of caspase 8 in combination-treated cells was completely suppressed by Bcl-2 overexpression, thus implicating a mitochondria-mediated amplification feedback loop. As an in vivo correlate, sequential intraperitoneal administration of CDDP and sFasL significantly inhibited the growth of intraperitoneal MPM human xenografts in nude mice. Our data indicate that the mitochondria-dependent feedback loop of the caspase activation cascade and the generation of ROS are both essential in mediating profound cytotoxicity and apoptosis of MPM cells treated with CDDP and sFasL. This mechanistic study establishes a the translational framework for the clinical application of sequential CDDP/sFasL in the treatment of MPM.

A class of iron chelators with a wide spectrum of potent antitumor activity that overcomes resistance to chemotherapeutics.

Novel chemotherapeutics with marked and selective antitumor activity are essential to develop, particularly those that can overcome resistance to established therapies. Iron (Fe) is critical for cell-cycle progression and DNA synthesis and potentially represents a novel molecular target for the design of new anticancer agents. The aim of this study was to evaluate the antitumor activity and Fe chelation efficacy of a new class of Fe chelators using human tumors. In this investigation, the ligands showed broad antitumor activity and could overcome resistance to established antitumor agents. The in vivo efficacy of the most effective chelator identified, di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT), was assessed by using a panel of human xenografts in nude mice. After 7 weeks, net growth of a melanoma xenograft in Dp44mT-treated mice was only 8% of that in mice treated with vehicle. In addition, no differences in these latter animals were found in hematological indices between Dp44mT-treated mice and controls. No marked systemic Fe depletion was observed comparing Dp44mT- and vehicle-treated mice, probably because of the very low doses required to induce anticancer activity. Dp44mT caused up-regulation of the Fe-responsive tumor growth and metastasis suppressor Ndrg1 in the tumor but not in the liver, indicating a potential mechanism of selective anticancer activity. These results indicate that the novel Fe chelators have potent and broad antitumor activity and can overcome resistance to established chemotherapeutics because of their unique mechanism of action.

Therapeutic potential of an adenovirus expressing p73beta, a p53 homologue, against human papilloma virus positive cervical cancer in vitro and in vivo

Human papilloma virus (HPV) infection is the most important risk factor for cervical cancer development. p53 based gene therapy is not suitable for cervical cancer because HPV oncoprotein E6 inactivates p53 protein by targeting it for ubiquitin mediated degradation. Here we evaluated the efficiency of Ad-p73, a replication deficient adenovirus expressing p73?, a p53 homologue, to inhibit the growth of HPV positive cervical cancer cells in vitro using tissue culture system and in vivo using human xenografts in nude mice. Ad-p73, but not Ad-p53 (p53 adenovirus), inhibited the growth in vitro of three different HPV positive cervical cancer cell lines, HeLa, ME180, and SiHa, efficiently, which correlated with stable expression of functional p73 protein. However, the growth of a HPV negative cervical cancer cell line, C33A, was inhibited equally by both Ad-p73 and Ad-p53. In addition, we show that Ad-p73 pre-infected HeLa cells and HCT116 E6 cells, an E6 stable cell line, failed to form tumors in nude mice unlike Ad-p53 or Ad-LacZ pre-infected cells. Moreover, Ad-p73, but not Ad-p53, inhibited completely the growth of already established tumors of HeLa or HCT116 E6 cells. Furthermore, the ability of p73 to inhibit the growth of these tumors correlated with the stable expression of p73 protein with the concomitant induction of its target gene p21WAF1/CIP1 and induction of apoptosis in tumor cells. These results suggest that Ad-p73 inhibits efficiently the growth in vitro and tumorigenicity and tumor growth in vivo of HPV positive cervical cancer cells and that p73-based approach should be explored as a potential therapeutic model for the treatment of cervical cancer.

Gastric penetration of amoxicillin in a human Helicobacter pylori-infected xenograft model.

The delivery of antibiotics into Helicobacter pylori-infected human stomachs is still poorly understood. Human embryonic gastric xenografts in nude mice have recently been proposed as a new model for the study of H. pylori infection. Using this model, we compared the penetration of amoxicillin, after intraperitoneal administration of a dose of 20 mg/kg of body weight, into the gastric mucosae of infected and uninfected xenografts. The concentrations of this drug in serum and superficial gastric mucosae were determined at 20 min and 1 and 3 h after injection. Ten mice with H. pylori-infected grafts (n = 5) or uninfected grafts (n = 5) were studied. Mucosal samples were obtained by cryomicrotomy. The concentrations in serum were similar to those obtained in the serum of humans after oral administration of 1 g of amoxicillin. The mean area under the tissue concentration-versus-time curve from 0 to 3 h obtained for mice with infected grafts was significantly higher than that obtained for the animals with uninfected grafts (P = 0.01). These results suggest that the penetration of amoxicillin into the superficial gastric mucosa may be substantially increased in the case of H. pylori infection. Thus, human xenografts in nude mice represent a new, well-standardized model for investigation of systemic delivery of drugs into H. pylori-infected gastric mucosa.

The clinical application of an ATP assay to predict the chemosensitivity of gastric cancer

The chemosensitivity of human xenografts in nude mice and fresh surgical specimens of gastric cancer was evaluated in vitro using the ATP assay and the MTT assay. The in vitro sensitivity of 6 human xenografts was detected by the ATP assay and compared with the in vivo sensitivity of the xenografts in nude mouse. The assay showed a 56.3% true-positive rate and a 85.7% true-negative rate, with 90.0% sensitivity and 46.2% specificity. When 10 surgical specimens obtained from gastric cancer patients were divided into two groups and sensitivities assessed by the ATP and the MTT assays, the overall correlation of both assays was 81.3%. The ATP assay might be useful in evaluating the chemosensitivity of human gastric carcinomas.

MDM2 gene amplification and transcript levels in human sarcomas: relationship to TP53 gene status.

Alterations of the TP53 tumor suppressor gene appear to be implicated in the tumorigenesis and progression of several types of human cancer, including different histologic subtypes of sarcomas. The MDM2 (murine double minute-2) gene encodes a nuclear phosphoprotein that may interact with both mutant and wild-type p53 proteins, thereby inhibiting p53-mediated transactivation in a dose-dependent manner. Recently it has been suggested that mdm2 and p53 proteins are components of an autoregulatory loop in which the MDM2 gene is transactivated by p53. Our purpose was to examine the frequency of MDM2 amplifications in larger panels of sarcomas, determine if the mRNA level could be elevated in tumors without concomitant gene amplification, and relate MDM2 findings to the TP53 status of the tumors. Sarcoma tissue of different histologic subtypes was obtained from 68 patients at the time of surgery and from 26 human xenografts in nude mice. In addition, two human sarcoma cell lines (OSA and U2OS) were studied. Genomic DNA from tumor tissue, in vitro cell lines, and peripheral blood cells were isolated by Southern-blot analysis methods to determine MDM2 gene amplification. Tumor DNA was analyzed for possible TP53 gene mutations in exons 5, 7, and 8 by constant denaturing gel electrophoresis. To determine the MDM2 and TP53 mRNA levels, Northern-blot analysis was performed. Amplification of the MDM2 gene was detected in 10 tumors (10.3%). Whereas MDM2 amplification and/or over-expression were found only in two (U2OS and OSA cell lines) of 18 osteosarcomas, one of 20 malignant fibrous histiocytomas (MFHs), and in none of 14 leiomyosarcomas, such alterations were observed in two of two fibrosarcomas, three of six malignant schwannomas, three of 19 liposarcomas, and in the one hemangiopericytoma examined. MDM2 overexpression was found in all nine examined cases with and in three tumors without amplification. TP53 mutations were detected in 12 cases (five osteosarcomas, four MFHs, and three leiomyosarcomas), of which none showed amplification, but one had increased levels of MDM2 mRNA. None of the fibrosarcomas, malignant schwannomas, and liposarcomas examined had mutated TP53. The six sarcomas that showed high TP53 mRNA expression in the absence of gene mutation also had elevated levels of MDM2 mRNA. The present data provide further indications that increased MDM2 expression level, caused by gene amplification or altered regulation of transcription, is involved in tumor progression of some, but not all, sarcoma subtypes.

Dosimetry of a new holding device for fast d (14) + Be—neutron irradiation of human xenografts in nude mice

Fast neutron irradiation of human xenografts in the flanks of nude mice is mainly a technical problem. The feasibility is dependant on the distribution of the tumor absorbed dose D T and on an effective shielding of the bodies of the animals. Therefore a special holding device for simultaneous irradiation of two tumour-bearing animals was developed. Meticulous dosimetric investigations using TLD-300 detectors in a mouse phantom showed that a relatively homogeneous dose distribution could be achieved in the xenografts and that effective shielding of the mice from direct irradiation would be achieved; the dose for the bodies of the animals being less than 10% of the dose to the tumour. Thus survival and a sufficient follow-up period of the irradiated animals could be guaranteed for all preclinical dose ranges used.