Anticancer Drug Response Research Articles

Abstract A117: Chk1-dependent up-regulation of ribonucleotide reductase R2 in response to camptothecin-induced DNA damage

Abstract To investigate drug mechanisms of action and identify molecular targets for the development of rational drug combinations, we are conducting synthetic siRNA-based RNAi screens to identify genes whose silencing affects anti-cancer drug responses. One such screen indicated that the silencing of RRM1 and RRM2, which encode the large and small subunits of the human ribonucleotide reductase (RNR) complex respectively, markedly enhances the cytotoxicity of the Topoisomerase 1 (Top1) inhibitor camptothecin (CPT). Silencing of RRM2 was also found to enhance DNA damage as measured by -H2AX. Further studies showed that CPT up-regulates both RRM1 and RRM2 mRNA and protein levels, and induces the nuclear translocation of RRM2. The Checkpoint kinase 1 (Chk1) was also up-regulated and activated in response to CPT. CHEK1 down-regulation by siRNA and small molecule inhibition of Chk1 activity blocked induction of RRM2 by CPT. CHEK1 siRNA also suppressed E2F1 up-regulation by CPT, and silencing of E2F1 suppressed the up-regulation of RRM2. Silencing of ATR or ATM, and inhibition of ATM activity by KU-55933 blocked Chk1 activation and RRM2 up-regulation. This study links the function of known components of CPT-induced DNA damage response with proteins required for the synthesis of dNTPs, presumably in preparation for DNA repair. Specifically, we propose that, upon DNA damage, Chk1 activation, mediated by ATM and ATR, regulates RRM2 expression through the E2F1 transcription factor. Up-regulation in RRM2 levels, coupled with its nuclear recruitment suggests an active role for RNR in the cellular response to CPT-mediated DNA damage that could potentially be exploited as strategy for enhancing the efficacy of Top 1inhibitors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A117.

Escape from hsa-miR-519c enables drug-resistant cells to maintain high expression of ABCG2.

Overexpression of ABCG2 has been reported in cell lines selected for drug resistance and it is widely believed to be important in the clinical pharmacology of anticancer drugs. We and others have previously identified and validated two microRNAs (miRNA; hsa-miR-519c and hsa-miR-520h) targeting ABCG2. In this study, the shortening of the ABCG2 3' untranslated region (3'UTR) was found to be a common phenomenon in several ABCG2-overexpressing resistant cell lines, which as a result removes the hsa-miR-519c binding site and its repressive effects on mRNA stability and translation blockade, thereby contributing to drug resistance. On the other hand, reduced expression of hsa-miR-520h, previously thought to have allowed ABCG2 overexpression, was found to be caused by the sequestering of the miRNA by the highly expressed ABCG2. In drug-sensitive cells, inhibitors against hsa-miR-519c and hsa-miR-520h could augment the cytotoxic effect of mitoxantrone, suggesting a substantial role for both miRNAs in controlling ABCG2 level and thereby anticancer drug response. However, in drug-resistant cells, altering the levels of the two miRNAs did not have any effect on sensitivity to mitoxantrone. Taken together, these studies suggest that in ABCG2-overexpressing drug-resistant cells, hsa-miR-519c is unable to affect ABCG2 expression because the mRNA lacks its binding site, whereas hsa-miR-520h is sequestered and unable to limit ABCG2 expression. Given the recent observation that a truncated 3'UTR is also observed in ABCG2-overexpressing human embryonic stem cells, our results in drug-resistant cell lines suggest that 3'UTR truncation is a relatively common mechanism of ABCG2 regulation.

Cancer Pharmacoethnicity: Ethnic Differences in Susceptibility to the Effects of Chemotherapy

A long-term goal of pharmacogenomics research is the design of individualized therapy based on the genomic sequence of the patient, in order to maximize response and minimize adverse drug reactions. Pharmacoethnicity, or ethnic diversity in drug response or toxicity, is becoming increasingly recognized as an important factor accounting for interindividual variation in anticancer drug responsiveness. Although pharmacoethnicity is determined by genetic and nongenetic factors, there is rapidly accumulating clinical evidence about ethnic differences in the frequencies of polymorphisms within many of the important cancer drug-related genes. This article reviews the current clinical evidence for ethnic differences in anticancer drug disposition and sensitivity while highlighting the challenges, and potential solutions, to acquiring such knowledge. The discovery of "ethnic-specific genetic signatures," representing unique sets of drug susceptibility-governing polymorphisms, may be the outcome of such work. Ultimately, such understanding will further the lofty goal of individualization of chemotherapy based on a person's unique genetic make-up to improve the tolerability and effectiveness of chemotherapy for all patients.

Implication of Checkpoint Kinase-dependent Up-regulation of Ribonucleotide Reductase R2 in DNA Damage Response

To investigate drug mechanisms of action and identify molecular targets for the development of rational drug combinations, we conducted synthetic small interfering RNA (siRNA)-based RNAi screens to identify genes whose silencing affects anti-cancer drug responses. Silencing of RRM1 and RRM2, which encode the large and small subunits of the human ribonucleotide reductase complex, respectively, markedly enhanced the cytotoxicity of the topoisomerase I inhibitor camptothecin (CPT). Silencing of RRM2 was also found to enhance DNA damage as measured by histone gamma-H2AX. Further studies showed that CPT up-regulates both RRM1 and RRM2 mRNA and protein levels and induces the nuclear translocation of RRM2. The checkpoint kinase 1 (Chk1) was up-regulated and activated in response to CPT, and CHEK1 down-regulation by siRNA and small molecule inhibitors of Chk1 blocked RRM2 induction by CPT. CHEK1 siRNA also suppressed E2F1 up-regulation by CPT, and silencing of E2F1 suppressed the up-regulation of RRM2. Silencing of ATR or ATM and inhibition of ATM activity by KU-55933 blocked Chk1 activation and RRM2 up-regulation. This study links the known components of CPT-induced DNA damage response with proteins required for the synthesis of dNTPs and DNA repair. Specifically, we propose that upon DNA damage, Chk1 activation, mediated by ATM and ATR, up-regulates RRM2 expression through the E2F1 transcription factor. Up-regulation in RRM2 expression levels coupled with its nuclear recruitment suggests an active role for ribonucleotide reductase in the cellular response to CPT-mediated DNA damage that could potentially be exploited as a strategy for enhancing the efficacy of topoisomerase I inhibitors.

Involvement of gene polymorphisms of the folate pathway enzymes in gene expression and anticancer drug sensitivity using the NCI-60 panel as a model

Folate, a vitamin of the B group involved in one-carbon group metabolism, plays an important role in DNA synthesis and methylation. Several polymorphisms in the genes involved in folate uptake and biotransformations have been shown to be associated to the risk of cancer and to anticancer drug response. We studied common polymorphisms in MTHFR (N 5,10-methylene-tetrahydrofolate reductase), MTHFD1 (N 5,10-methylene-tetrahydrofolate dehydrogenase), MTR (methionine synthetase) and SLC19A1 (reduced folate carrier) in the panel of 60 human tumour cell lines established by the NCI for anticancer drug screening and we tentatively associated these polymorphisms with gene expression and drug cytotoxicity as extracted from the public database of the Developmental Therapeutic Programme. We observed a consistent and highly significant association between the presence of the variant C allele of the A>C1298 polymorphism of MTHFR and the sensitivity to many anticancer drugs belonging to the classes of antifolates, antimetabolites, alkylating agents and, to a lesser extent, topoisomerase inhibitors. In contrast, the T variant allele of the C>T677 variation of MTHFR was rather associated to lower sensitivity of the cell lines towards anticancer drugs (alkylating agents, antifolates and antimetabolites) but with much lower effects than the A>C1298 variation. The polymorphisms of the other genes studied were not associated with differences in anticancer drug sensitivity, but the expression of the SLC19A1 gene was significantly correlated with the sensitivity to several drugs (antifolates, thiopurines, nitrosoureas, and DACH-platinum drugs). We concluded that the NCI-60 panel may constitute a good starting point for implementing clinical studies aimed at discovering and validating predictive genetic markers of drug efficacy and/or toxicity.

Genomics and Pharmacogenomics in Anticancer Drug Development and Clinical Response

pharmacoGENOMICS, ANTICANCER DRUG DISCOVERY, AND RESPONSE Book editor: Federico Innocenti, M.D., Ph.D. Genomic experimental approaches in oncology Toward the realisation of the promise of microarrays in oncology Neil Winegarden Cell based models to identify genetic variants contributing to anticancer drug response M. Eileen Dolan Proteomic analysis in cancer patients Kazuyuki Nakamura MicroRNAs and discovery of new targets Gozoh Tsujimoto Pharmacogenomics of the National Cancer Institute's 60 tumor cell panel David G. Covell Use of single nucleotide polymorphism array for tumor aberrations in gene copy numbers Kwong-Kwok Wong Pharmacogenomics of toxicity and response of chemotherapy Concordance between tumor and germline DNA Sharon Marsh Epidermal growth factor receptor mutations and sensitivity to selective kinase inhibitors in human lung cancer Jeffrey Settleman 3. BCR-ABL mutations and imatinib resistance in chronic myeloid leukemia patients Mark Litzow Role of thymidylate synthase gene variations in colorectal cancer patients Heinz-Josef Lenz Thiopurines in the treatment of childhood acute lymphoblastic leukemia and genetic variants in the thiopurine S-methyltransferase gene Martin Stanulla The impact of polymorphisms on the clinical outcomes of monoclonal antibody therapy against hematologic malignancies Dong Hwan Kim DNA repair and mitotic check point genes as potential predictors of chemotherapy response in non-small-cell lung cancer Rafael Rosell Dihydropyrimidine dehydrogenase (DPYD) gene polymorphism: portrait of a serial killer. Gerard Milano Impact of UDP-glucuronosyltransferase 1A haplotypes on irinotecan treatment Kimie Sai Microarray profiling in breast cancer patients Lan Guo 11. Role of the folate pathway and the thymidylate synthase genes in pediatric acute lymphoblastic leukemia treatment response Richard Aplenc Pharmacogenomics in clinical drug development in oncology Pharmacogenomics in drug development: an pharmaceutical industry perspective Tal Zaks Identification of a pharmacogenomic biomarker classifiers in cancer drug development Richard Simon Toxicogenomics application to oncology drug development Teresa Lettieri Strategies to identify pharmacogenomic biomarkers: candidate gene, pathway-based, and genome-wide approaches Xifeng Wu

Cell density-dependent alterations in tumorigenic potential of a murine T-cell lymphoma: implication in the evolution of multidrug resistance in tumor cells

We have previously demonstrated that cells of murine T-cell lymphoma, when grown in vivo or in vitro in an environment of high cell density, undergo phenotypic alterations, providing them with survival benefits. However, it is unclear whether the acquisition of such growth-related phenotypic alterations is inheritable in successive cell generations and if these alterations are associated with an irreversible alteration in their tumorigenic ability and evolution of multidrug resistance. To investigate this, tumor cells of a murine model of a T-cell lymphoma, designated as Dalton's lymphoma, and obtained from high and low cell density environment in vitro and in vivo, were transplanted in mice with or without the administration of anticancer drugs followed by analysis of their phenotypic properties and tumorigenic potential as measured by kinetics of tumor growth and survival of the tumor-bearing host. Kinetics of tumor progression was comparatively rapid in tumor-bearing mice transplanted with tumor cells from a high cell density environment, causing an early death of the host. Moreover, under these conditions the antitumor response of anticancer drugs, cisplatin, doxorubicin, and methotrexate, was found to be less effective compared with mice transplanted with tumor cells from a low cell density environment. The tumor cells from a high cell density source showed a long-term alteration in their survival properties both in vitro and in vivo, indicating that such alterations were sustainable over successive cell cycles. The study also discusses the possible mechanisms indicating the role of MDR1, Hsp70 and 90, Bcl-2, IL-1, IL-6, IL-10, IFNgamma, and TGFbeta in the evolution of multidrug resistance in tumor cells obtained from a high cell density environment.

Correlation between genetic variations and anticancer drug response

Correlation between genetic variations and anticancer drug response

MicroRNAs modulate the chemosensitivity of tumor cells

MicroRNAs are strongly implicated in such processes as development, carcinogenesis, cell survival, and apoptosis. It is likely, therefore, that they can also modulate sensitivity and resistance to anticancer drugs in substantial ways. To test this hypothesis, we studied the pharmacologic roles of three microRNAs previously implicated in cancer biology (let-7i, mir-16, and mir-21) and also used in silico methods to test pharmacologic microRNA effects more broadly. In the experimental system, we increased the expression of individual microRNAs by transfecting their precursors (which are active) or suppressed the expression by transfection of antisense oligomers. In three NCI-60 human cancer cell lines, a panel of 60 lines used for anticancer drug discovery, we assessed the growth-inhibitory potencies of 14 structurally diverse compounds with known anticancer activities. Changing the cellular levels of let-7i, mir-16, and mir-21 affected the potencies of a number of the anticancer agents by up to 4-fold. The effect was most prominent with mir-21, with 10 of 28 cell-compound pairs showing significant shifts in growth-inhibitory activity. Varying mir-21 levels changed potencies in opposite directions depending on compound class; indicating that different mechanisms determine toxic and protective effects. In silico comparison of drug potencies with microRNA expression profiles across the entire NCI-60 panel revealed that approximately 30 microRNAs, including mir-21, show highly significant correlations with numerous anticancer agents. Ten of those microRNAs have already been implicated in cancer biology. Our results support a substantial role for microRNAs in anticancer drug response, suggesting novel potential approaches to the improvement of chemotherapy.

Pharmacogenetics/genomics of membrane transporters in cancer chemotherapy

Inter-individual variability in drug response and the emergence of adverse drug reactions are main causes of treatment failure in cancer therapy. Recently, membrane transporters have been recognized as an important determinant of drug disposition, thereby affecting chemosensitivity and -resistance. Genetic factors contribute to inter-individual variability in drug transport and targeting. Therefore, pharmacogenetic studies of membrane transporters can lead to new approaches for optimizing cancer therapy. This review discusses genetic variations in efflux transporters of the ATP-binding cassette (ABC) family such as ABCB1 (MDR1, P-glycoprotein), ABCC1 (MRP1), ABCC2 (MRP2) and ABCG2 (BCRP), and uptake transporters of the solute carrier (SLC) family such as SLC19A1 (RFC1) and SLCO1B1 (SLC21A6), and their relevance to cancer chemotherapy. Furthermore, a pharmacogenomic approach is outlined, which using correlations between the growth inhibitory potency of anticancer drugs and transporter gene expression in multiple human cancer cell lines, has shown promise for determining the relevant transporters for any given drugs and predicting anticancer drug response.

Estimating the False Discovery Rate Using Mixed Normal Distribution for Identifying Differentially Expressed Genes in Microarray Data Analysis

The recent development of DNA microarray technology allows us to measure simultaneously the expression levels of thousands of genes and to identify truly correlated genes with anticancer drug response (differentially expressed genes) from many candidate genes. Significance Analysis of Microarray (SAM) is often used to estimate the false discovery rate (FDR), which is an index for optimizing the identifiability of differentially expressed genes, while the accuracy of the estimated FDR by SAM is not necessarily confirmed. We propose a new method for estimating the FDR assuming a mixed normal distribution on the test statistic and examine the performance of the proposed method and SAM using simulated data. The simulation results indicate that the accuracy of the estimated FDR by the proposed method and SAM, varied depending on the experimental conditions. We applied both methods to actual data comprised of expression levels of 12,625 genes of 10 responders and 14 non-responders to docetaxel for breast cancer. The proposed method identified 280 differentially expressed genes correlated with docetaxel response using a cut-off value for achieving FDR <0.01 to prevent false-positive genes, although 92 genes were previously thought to be correlated with docetaxel response ones.

352 POSTER Development of web-based bioinformatics tools to reveal relationships between gene expression, gene function and anticancer drug response

352 POSTER Development of web-based bioinformatics tools to reveal relationships between gene expression, gene function and anticancer drug response

Synthesis and structure–activity relationship studies of 4,11-diaminonaphtho[2,3- f]indole-5,10-diones

We describe the synthesis of derivatives of 4,11-diaminonaphtho[2,3- f]indole-5,10-dione and their cytotoxicity for human tumor cells that express major determinants of altered anticancer drug response, the efflux pump P-glycoprotein, and non-functional p53. Nucleophilic substitution of methoxy groups in 4,11-dimethoxynaphtho[2,3- f]indole-5,10-dione with various ethylenediamines yielded the derivatives of 4,11-diaminonaphtho[2,3- f]indole-5,10-dione, the indole containing analogues of the antitumor agent ametantrone. The cytotoxicity of novel compounds for multidrug resistant, P-glycoprotein-expressing tumor cells is highly dependent on the N-substituent at the terminal amino group of the ethylenediamine moiety. Whereas p53 null colon carcinoma cells were less sensitive to the reference drug doxorubicin than their counterparts with wild type p53, the majority of novel naphthoindole derivatives were equally potent for both cell lines, regardless of the p53 status.

Membrane transporters and channels in chemoresistance and -sensitivity of tumor cells

Membrane transporters play important roles in mediating chemosensitivity and -resistance of tumor cells. ABC transporters, such as ABCB1/MDR1, ABCC1/MRP1 and ABCG2/BCRP, are frequently associated with decreased cellular accumulation of anticancer drugs and multidrug resistance of tumors. SLC transporters, such as folate, nucleoside, and amino acid transporters, commonly increase chemosensitivity by mediating the cellular uptake of hydrophilic drugs. Ion channels and pumps variably affect sensitivity to anticancer therapy by modulating viability of tumor cells. A pharmacogenomic approach, using correlations between drug potency and transporter gene expression in multiple cancer cell lines, has shown promise for identifying potential drug–transporter relationships and predicting anticancer drug response, in an effort to optimize chemotherapy for individual patients.

Associations Between Drug Metabolism Genotype, Chemotherapy Pharmacokinetics, and Overall Survival in Patients With Breast Cancer

To evaluate associations between patient survival, pharmacokinetics, and drug metabolism-related genetic polymorphisms in patients receiving a combination chemotherapy regimen for breast cancer. A genotype association study was conducted on 85 chemotherapy-naïve patients with metastatic or inflammatory breast cancer that were evaluated for an extended period after receiving standard-dose chemotherapy followed by high-dose cyclophosphamide, cisplatin, and carmustine. Blood pharmacokinetics were evaluated, and DNA was genotyped for 29 polymorphisms in 17 drug metabolism genes. Patients with cyclophosphamide plasma exposures above the median (implying slower metabolic activation) had a shorter survival than those below the median (1.8 v 3.8 years, respectively; P = .042). Patients having a variant genotype of cytochrome P450 3A4 displayed higher blood concentrations of parent (inactive) cyclophosphamide with the second and third doses (P = .024 and .028, respectively) in addition to slower cyclophosphamide activation over the three doses (P = .031). Median survival for these patients was 1.3 years compared with 2.7 years for those without the variant (P = .043). Similar results were observed for patients carrying a genetic variant of P450 3A5. Median survival for patients with deletions of glutathione-S-transferase M1 gene was 3.5 v 1.5 years for patients with one or both copies (P = .041). Patients with a polymorphism in a gene regulating metallothionein had lower platinum concentrations and shorter survival (P = .033). These data suggest that pretreatment evaluation of drug metabolism genes may explain some interindividual differences in both anticancer drug pharmacokinetics and response. The correlations found here may have implications for other commonly used anticancer drugs.

Novel proteomics approach for functional identification of regulators of cancer drug response and apoptosis in ex vivo tissue cultures of human colorectal cancer

9658 Background: It is notoriously difficult to study the molecular effects of anticancer drug treatment in vivo. So far, this has been only possible by performing serial biopsies in tumor patients undergoing treatment. An approach to address this problem is to perform ex vivo tissue cultures, where patient tumor tissue is obtained at the occasion of a routine operation and then processed in vitro. The molecular program of apoptosis and the activation status of various signal transduction pathways has been identified as a major determinant of chemotherapy responsiveness in cancer. Methods: To analyze genetic pathways potentially involved in anticancer drug response, we have treated tumor samples from colorectal cancer patients ex vivo with antitumor agents with various mechanisms of action. Cytotoxicity was quantified using a colorimetric dye assay. Proteome profiles of the untreated/treated and normal/cancerous patient samples have been generated by reverse-phase protein microarrays, which are a novel approach to analyze signaling pathways using small numbers of cells microdissected from biopsy specimens. For the feasibility phase of the project, we looked at well established growth factor activation pathways such as p-ERK, p-SAPK/JNK, p-AKT, p-p38, p-JNK, p-IkBa, p-STAT3, proliferation regulatory pathways such as p-histone H3, p-Rb, p-p53, cyclin D1, and pathways involved in apoptosis regulation such as cleaved caspase 3, cleaved PARP, and Bcl-2. Results: We found higher cytotoxic activity of most anticancer agents in cancer tissue as compared to normal tissue of the same organ (colon or liver). There was a prominent down-regulation of p-SAPK/JNK and a less pronounced down-regulation of p-ERK and p-p38 with chemotherapy in tumor samples but not in normal tissue samples. Conclusions: This culture technique allows to treat human tissue with various agents ex vivo to simulate the in vivo treatment situation and to assess cytotoxicity by a simple colorimetric dye assay. With the reverse-phase protein microarray technology activated signalling transduction pathways can be identified on low quantity tissue samples. No significant financial relationships to disclose.

Prediction of anticancer drug response by transcriptome analysis. The new pathways of pharmacogenomics

Predicting drug response is a challenging problem in oncology. Between 1975-1985, important efforts were devoted to the generation of cellular assays able to predict, on an individual basis, the in vitro response of tumour cells to chemotherapeutic agents, but such methods could not be adopted in routine. New molecular biology techniques have been developed in order to identify the genes involved in drug sensitivity and resistance. The availability of techniques of gene expression profiling has allowed to establish correlations between gene expression and drug sensitivity of tumour cells or human cancers. This type of approach has been initiated on in vitro systems by the National Cancer Institute (NCI) in the USA and is pursued by a growing number of public and private laboratories around the world. In the clinical setting, gene expression profiles are currently developed for the diagnosis and overall prognosis of human cancers, but should rapidly allow the prediction of drug response, so that clinicians would be able to prescribe individual treatments on a rational basis.

Quantification of Viability in Organotypic Multicellular Spheroids of Human Malignant Glioma using Lactate Dehydrogenase Activity: A Rapid and Reliable Automated Assay

Organotypic spheroids from malignant glioma resemble the biological complexity of the original tumor and are therefore appealing to study anticancer drug responses. Accurate and reproducible quantification of response effect has been lacking to determine drug responses in this three-dimensional tumor model. Lactate dehydrogenase (LDH) activity was demonstrated in cryostat sections of spheroids using the tetrazolium salt method. Calibrated digital image acquisition of the stained cryostat sections enables quantification of LDH activity. Fully automated image cytometry reliably demarcates LDH-active and LDH-inactive tissue areas by thresholding at specific absorbance values. The viability index (VI) was calculated as ratio of LDH-active areas and total spheroid tissue areas. Duplicate staining and processing on the same tissue showed good correlation and therefore reproducibility. Sodium azide incubation of spheroids induced reduction in VI to almost zero. We conclude that quantification of viability in cryostat sections of organotypic multicellular spheroids from malignant glioma can be performed reliably and reproducibly with this approach.

Membrane transporters and channels: role of the transportome in cancer chemosensitivity and chemoresistance.

Membrane transporters and channels (collectively the transportome) govern cellular influx and efflux of ions, nutrients, and drugs. We used oligonucleotide arrays to analyze gene expression of the transportome in 60 human cancer cell lines used by the National Cancer Institute for drug screening. Correlating gene expression with the potencies of 119 standard anticancer drugs identified known drug-transporter interactions and suggested novel ones. Folate, nucleoside, and amino acid transporters positively correlated with chemosensitivity to their respective drug substrates. We validated the positive correlation between SLC29A1 (nucleoside transporter ENT1) expression and potency of nucleoside analogues, azacytidine and inosine-glycodialdehyde. Application of an inhibitor of SLC29A1, nitrobenzylmercaptopurine ribonucleoside, significantly reduced the potency of these two drugs, indicating that SLC29A1 plays a role in cellular uptake. Three ABC efflux transporters (ABCB1, ABCC3, and ABCB5) showed significant negative correlations with multiple drugs, suggesting a mechanism of drug resistance. ABCB1 expression correlated negatively with potencies of 19 known ABCB1 substrates and with Baker's antifol and geldanamycin. Use of RNA interference reduced ABCB1 mRNA levels and concomitantly increased sensitivity to these two drugs, as expected for ABCB1 substrates. Similarly, specific silencing of ABCB5 by small interfering RNA increased sensitivity to several drugs in melanoma cells, implicating ABCB5 as a novel chemoresistance factor. Ion exchangers, ion channels, and subunits of proton and sodium pumps variably correlated with drug potency. This study identifies numerous potential drug-transporter relationships and supports a prominent role for membrane transport in determining chemosensitivity. Measurement of transporter gene expression may prove useful in predicting anticancer drug response.

Anticancer drug response and expression of molecular markers in early-passage xenotransplanted colon carcinomas

Despite some success in the treatment of colorectal carcinomas, novel rational therapies targeting specific cancer-related molecules are under development and urgently needed. These approaches need careful preclinical evaluation in models that closely mirror the clinical situation. Therefore, we established a panel of 15 xenotransplantable tumours directly from fresh surgical material. We showed that both the histology and expression of tumour-associated markers (Epithelial Cell Adhesion molecule (EpCAM), E-cadherin, carcinoembryonic antigen (CEA)) could be maintained during passaging in nude mice. Xenotransplanted tumours were characterised for chemosensitivity and revealed a response rate of 5/15 (33%) for 5-fluorouracil (5-FU), 15/15 (100%) for irinotecan and 8/14 (57%) for oxaliplatin. 5 patients out of 15 were treated with cytostatics because of synchronous metastases. The response to chemotherapy in these patients coincided very closely with the response of the individual xenografts. All of the xenografts expressed the proliferation marker Ki67 and the nuclear enzyme, Topoisomerase IIα (Topo IIα) at the protein level. Most of the xenografts also expressed the tumour suppressor, p53 (9/14) and the nuclear enzyme Topoisomerase Iα (Topo Iα) (13/14) at the protein level. Interestingly, the presence of a K-ras mutation in codon 12 (5/15 xenografts) coincided with a low response rate towards oxaliplatin. This observation needs further confirmation using a larger number of tumours. In conclusion, we were able to establish transplantable xenografts suitable to mimic the clinical situation. These well characterised models are useful tools for the preclinical development of novel therapeutic approaches and for investigating translational research aspects.