(Yondelis, Trabectedin) in Human Sarcoma Cells Explanted from Chemo-Naı¨Ve Patients

814

Transcriptional signature of Ecteinascidin 743 (Yondelis, Trabectedin) in human sarcoma cells explanted from chemo-naı¨ve patients

Nerea Martı´nez,1 Margarita Sa´ nchez-Beato,1 of ET-743 downstream mediators and transcription Amancio Carnero,2 Victoria Moneo,2 regulators and the proposal of strategies by which ET- Juan C. Tercero,3 Isabel Ferna´ ndez,1 743–sensitive tumors may be identified. [Mol Cancer Mercedes Navarrete,1 Jose´ Jimeno,3 Ther 2005;4(5):814–23] and Miguel A. Piris1

1Molecular Pathology Programme and 2 Experimental Therapeutics Programme, Centro Nacional de Investigaciones Oncolo´gicas and Introduction 3 Pharmamar R&D, Madrid, Spain Ecteinascidin 743 (ET-743; Yondelis, Trabectedin) is a tetrahydroisoquinoline alkaloid with antiproliferative effects, isolated from the tunicate Ecteinascidia turbinata Abstract (1). This compound binds to the minor groove of DNA Ecteinascidin 743 (ET-743; Yondelis, Trabectedin) is a and binds the N2 position of guanine (2–4), producing a marine anticancer agent that induces long-lasting objec- bend towards the major groove of the DNA, in a way tive remissions and tumor control in a subset of patients that differs from that of other known alkylating agents. with pretreated/resistant soft-tissue sarcoma. Drug- This alteration in the DNA structure could be the cause induced tumor control is achievable in 22% of such of the broad inhibition of inducible transcription, previ- patients, but there is no clear indication of the molecular ously described at pharmacologically achievable concen- features correlated with clinical sensitivity/resistance to trations, probably interfering with transcription factors ET-743. Nine low-passage, soft-tissue sarcoma cell lines, (5–7). In addition, an efficient DNA repair function is explanted from chemo-naı¨ve patients with different known to be required for ET-743 cytotoxicity. In fact, the patterns of sensitivity, have been profiled with a cDNA mode of action of ET-743 is different from that of all microarray containing 6,700 cancer-related genes. The other DNA-interacting antitumorals because nucleotide- molecular signature of these cell lines was analyzed at excision-repair deficiencies that increase the susceptibility baseline and at four different times after ET-743 of cell lines to cisplatin, UV, and alkylating reagents are exposure. The association of levels of TP53 mutation more resistant to ET-743. and TP73 expression with ET-743 sensitivity and cell At the cellular level, ET-743 produces cell cycle cycle kinetics after treatment was also analyzed. Gene perturbations with a decreased rate of S-phase progres- expression profile analysis revealed up-regulation of 86 sion and a G2-M blockade (3, 8–11). Experiments with genes and down-regulation of 244 genes in response to synchronized cells showed that cells in G1 phase are more ET-743. The ET-743 gene expression signature identified sensitive to the cytotoxic effects of ET-743 than are cells in a group of genes related with cell cycle control, stress, G2 or S phase. and DNA-damage response (JUNB, ATF3, CS-1, SAT, Phase I clinical trials started back in 1996 and phase II GADD45B, and ID2) that were up-regulated in all the and comparative trials are currently in progress (12). The cell lines studied. The transcriptional signature 72 hours data available thus far confirm a therapeutic potential in after ET-743 administration, associated with ET-743 patients with advanced breast cancer pretreated with sensitivity, showed a more efficient induction of genes anthracyclines and taxanes and in women with ovarian involved in DNA-damage response and apoptosis, such cancer that is resistant, or has experienced relapse, to as RAD17, BRCA1, PAR4, CDKN1A, and P53DINP1, platin-taxane–based therapy (13–15). Additionally, a large in the sensitive cell line group. The transcriptional phase II program, conducted in patients with advanced signature described here may lead to the identification soft-tissue sarcoma that is resistant, or has undergone relapse, to conventional agents, has shown long-lasting objective responses and tumor control in a clinically significant proportion of cases (16–18); such a rate of ET- Received 11/29/04; revised 2/22/05; accepted 3/9/05. 743–induced tumor control seems to have an effect on The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked median survival and particularly on the proportion of advertisement in accordance with 18 U.S.C. Section 1734 solely to patients (29%) who were alive after 2 or more years. indicate this fact. This clinical data set suggests major constitutive/ Requests for reprints: Miguel Angel Piris, Molecular Pathology Programme, molecular differences between a group of patients in whom Centro Nacional de Investigaciones Oncolo´gicas, C/ Melchor Ferna´ndez Almagro 3, E-28029 Madrid, Spain. Phone: 34-91-224-69-00; the compound is able to modulate progression of the Fax: 34-91-224-69-23. E-mail: [email protected] disease for very long periods and a cohort that is fully Copyright C 2005 American Association for Cancer Research. resistant to ET-743.

Mol Cancer Ther 2005;4(5). May 2005

Given the shown clinical benefit of ET-743 treatment Table 1. Details of the cell lines treated with ET-743 and for soft-tissue sarcoma, we decided to investigate the analyzed by cDNA microarrays mechanism of action of this drug in a panel of chemo- Cell line Tumor origin IC (nmol/L) Doubling naı¨ve, low-passage soft-tissue sarcoma cell lines estab- 50 96 h time (h) lished from biopsies of newly diagnosed patients with different sarcoma histology types. We characterized the SR2103/1A Liposarcoma 0.7 48 expression profiles of those cell lines and estimated their SR2103/1B Liposarcoma 0.7 48 correlation with in vitro sensitivity to ET-743 both in the SW872 Liposarcoma 0.5 24 absence of the drug and in time-course experiments. This CA1010 Leiomyosarmoa 0.4 36 should make it possible to develop a predictive ectei- SR2205 Solitary Fibrous Tumor 1 96 nascidin sensitivity model for subsequent validation in SR2410 MPNST >100 96 tumor samples from patients treated with ET-743. SR2910 Ewing Sarcoma 1 24 SR0312 Osteosarcoma 0.4 24 SR0306 Nephroblastoma* >100 96 Materials and Methods CS-1R Chondrosarcoma 34 ND Cell Lines CS-1S Chondrosarcoma 0.9 ND Eleven cell lines were studied, eight of which were directly established from surgical tumor-tissue specimens Abbreviations: MPNST, malignant peripheral nerve sheath tumor; ND, not from sarcoma patients (Table 1). Sensitive and isogenic determined. *Rhabdomyoblastic differentiation. resistant chondrosarcoma cell lines (CS-1S and CS-R) were kindly provided by Dr. Lawrence Weissbach (Massachu- Superscript System for cDNA synthesis (Life Technologies). setts General Hospital, Boston, MA). The sarcoma cell line In vitro transcription was carried out using the T7 Mega- SW872 was obtained from the American Type Culture script in vitro transcription kit (Ambion, Austin, TX). The Collection (Rockville, MD). The in vitro sensitivity of each quality of the amplified RNA produced was checked by cell line to ET-743 was previously established.4 Cells were electrophoresis and its concentration was measured. maintained in RPMI medium supplemented with 10% fetal cDNA Microarray Hybridizations and Analysis bovine serum, fungizone, and penicillin/streptomycin. CS- 1R and CS-1S were grown without antibiotics or fungizone. Amplified RNA was hybridized on the CNIO-Oncochip A For time-course analysis, cells were treated with as previously described (20). Briefly, 5 g of amplified RNA 10 nmol/L ET-743 for 6, 24, 48, and 72 hours. were directly labeled with cyanine 3–conjugated dUTP A TP53 gene mutations were analyzed in accordance with (Cy3), whereas 5 g of aRNA from the Universal Human described protocols (19). Reference RNA (Stratagene) were labeled with cyanine 5– Real-time ReverseTranscription-PCR conjugated dUTP (Cy5) as reference. Hybridizations were done as described (21). Hybridized slides were scanned Quantitative reverse transcription-PCR (RT-PCR) was and analyzed using the Scanarray 5000 XL (GSI Lumonics, done for ATF3, BRCA1, GADD45B, GAPDH, JUNB, Kanata, Ontario, Canada) and GenePix Pro 4.0 software RAD17, SAT, TAp73,andDNp73 using the Applied (Axon Instruments, Inc., Union City, CA), respectively. Biosystems 7900HT (Applied Biosystems, Foster City, Data Analysis CA). GADPH expression was used as a reference for calculating expression levels of the other genes. Raw microarray data were processed as previously Flow CytometryAnalysis described. Data obtained from each hybridization were stored in a database for analysis. Cy3/Cy5 ratios were Cells were cultured for 24 to 72 hours in the absence of normalized with respect to the median ratio value of all of the drug. ET-743, 10 nmol/L, was then added to the the spots in the array. After normalization, spots with medium and incubation was continued for 6, 24, 48, and intensities for both channels (sum of medians) less than 72 hours. For flow cytometry analysis, cells corresponding that of the local background were discarded. The ratios of to each treatment time were fixed in 70% ethanol, and the remaining spots were log transformed (base 2), and RNase (0.6 Ag/AL final concentration) and propidium duplicated spots on the OncoChip were averaged to the iodide (50 Ag/mL) were added to the fixed cells. The median (20). After this first processing, results at all DNA content was quantified with a flow cytometer treatment times were compared with those in untreated (FACSCalibur, Becton Dickinson, Franklin Lakes, NJ). RNA Extraction and Amplification cells, and genes were deemed to be up-regulated or down- regulated if the ratio of the difference with untreated cells Total RNA from cells was extracted in two steps after 6, was more than twice that at any of the four treatment times 24, 48, and 72 hours of treatment using Trizol (Life studied in at least 72% (8 of 11) of the cell lines Technologies, Inc., Grand Island, NY) and then purified analyzed. Biological functions of selected genes were with RNeasy (Qiagen, Inc., Valencia, CA). Double-strand assigned using the GeneCards5 and Gene Ontology6 cDNA was synthesized from 4 Ag of total RNA using the

5 http://bioinfo.cnio.es/genecards/index.html 4 V. Moneo and A. Carnero et al., unpublished data. 6 http://fatigo.bioinfo.cnio.es/

Mol Cancer Ther 2005;4(5). May 2005

databases. Sensitivity/resistance signatures were obtained SAT. SAT [spermidine/spermine N1-acetyltransferase by dividing the median expression level for each gene in all (SSAT)] was up-regulated shortly after the start of sensitive cell lines by that of all resistant cell lines under treatment (6 hours) and the amount of expression grew basal conditions (i.e., in the absence of the drug) and over time (from 1.17 at 6 hours to 2.63 at 48 and 72 hours, throughout the time course of treatment with ET-743. log scale). This gene codes for a protein involved in the Selected genes were defined as those with at least 2-fold regulation of the intracellular concentration of polyamines up-regulation or down-regulation in the sensitive group of (24), a key factor required for tumor cell growth, regulated cell lines relative to the resistant group. by a complex system that involves homeostatic balancing of Clustering Analysis biosynthesis catabolism, uptake, and export of polyamines. To classify the temporal profiles of gene expression, Experiments producing selective down-regulation of SSAT cluster analysis was done using the Self-Organizing Tree by small interfering RNA revealed the requirement for Algorithm7 (22), assuming Euclidian distances between genes.

Results Gene Signature of ET-743 Among the 11 human sarcoma cell lines studied, the SR2103/1A, SR2103/1B, CA1010, SR2205, SR2410, SR2910, SR0312, and SR0306 lines had been established at the Centro Nacional de Investigaciones Oncolo´gicas from tissue samples of sarcoma patients and are considered low-passage cell lines. Cellular phenotype and ET-743 sensitivity data of all the cell lines are detailed in Table 1. The 11 human sarcoma cell lines were exposed to 10 nmol/L ET-743; this concentration may be achieved and maintained for more than 72 hours in patients plasma below the recommended clinical dose incorporat- ed in the phase II trials in sarcoma and is therefore considered to be therapeutically appropriate for this experimental study (23). ET-743 in vitro treatment produced deregulation of 330 genes in the majority of these cell lines (8 of 11; 72% of samples). Most of the deregulated genes were down- regulated (244; 74%) and only 86 genes (26%) were up- regulated (Fig. 1; raw data and selected gene data can be found in Table 1 at the website http://redlinfomas.cnio.es/ publications/ET743).8 The number of genes of which expression level changed increased with the time elapsed after treatment. Thus, early on (6 hours of treatment), only four genes were noted as being consistently changed in most of the cell lines in response to treatment with ET-743: two genes (SAT and EST) were up-regulated and two genes (BCAR3 and DDX20) were down-regulated. The upward or downward tendency of these genes was constant throughout the treatment. The genes up-regulated after treatment with ET-743 are mainly involved in signal transduction, molecule and ion transport, and energy pathways, whereas the down-regulated genes are predominantly associated with nucleic acid binding, gene transcription, signal transduction, and cell cycle control. Notably, the most significantly overexpressed downstream mediators after treatment with ET-743 were CS-1, ATF3, SAT, JUNB, GADD45, and ID2, all of which are involved in cell cycle arrest and apoptosis (Fig. 2A).

Figure 1. Expression profile of deregulated genes after treatment with 7 http://gepas.bioinfo.cnio.es/cgi-bin/sotarray ET-743. Eighty-six genes were up-regulated and 244 genes were down- 8 http://redlinfomas.cnio.es/publications/ET743 regulated as a consequence of the treatment.

Mol Cancer Ther 2005;4(5). May 2005

SSAT for apoptosis induction after treatment with poly- induced in response to a variety of stress conditions in amines analogues (25). The induction of SSAT after many different tissues, such as toxic chemicals, anticancer treatment with polyamine analogues has been proposed drugs, proteasome inhibitors, and genotoxic agents. This as a prognostic indicator of drug response (26) because it is p53 target is involved in drug-induced apoptosis, specifi- known to be induced by 5-fluorouracil, TDX, cisplatin, and cally after treatment with DNA topoisomerase inhibitors, oxaliplatin (27, 28). It has also been shown that over- such as camptothecin or etoposide, inducing activation of expression of SSAT, which is dependent on p53 status (27), caspase proteases (30, 31). produces cell cycle arrest in G2-M (29). ID2. ID2 is a member of a family of helix-loop-helix ATF3. ATF3 was up-regulated after 24 hours of treatment transcription factors that regulate cell differentiation and with ET-743 in 8 of 11 cell lines. ATF3 is a stress-inducible proliferation (32–34). Id2 binds to Rb family members, transcriptional repressor of which levels are dramatically abolishing their growth-suppressing function. It has been

Figure 2. ET-743 downstream genes. A, average of gene expression log-ratios (base 2) of the most significantly up-regulated genes in response to treatment with ET-743 in 11 sarcoma cell lines. The ratio for each time point is calculated by dividing the average expression at the corresponding time by the average of expression before treatment. B, expression pattern of four genes identified as ET-743 downstream genes were validated in two cell lines by RT-PCR.

Mol Cancer Ther 2005;4(5). May 2005

shown that trichostatin A, a histone deacetylase inhibitor, Table 2. Genes constitutively overexpressed or down-regulated induces the expression of ID2 (35–37). Overexpression of in sensitive compared with resistant cell lines ID2 augments apoptosis in myeloid and osteogenic Gene symbol Gene description S/R sarcoma-derived cell lines (38). . CS-1 CS-1 is a calcineurin-binding protein. Calcineurin IL13RA2 Interleukin-13 receptor a2 5.25 is a calcium- and calmodulin-dependent serine/threonine MMP3 Matrix metalloproteinase 3 3.43 phosphatase that plays an important role in transducing SAMSN1 SAM-domain protein SAMSN-1 2.94 calcium-dependent signals in a variety of cell types. This EDG1 Endothelial differentiation, sphingolipid 1.95 enzyme is required for G1 progression and it was recently G-protein–coupled receptor 1 identified as an important link between Ca2+ signaling and IGFBP3 Insulin-like growth 1.74 NF-nB activation in response to mitochondrial stress (39). factor-binding protein 3 Overexpression of Cs-1 inhibits calcineurin activity, pro- PRKAG2 Protein kinase, AMP-activated, 1.71 ducing cell cycle arrest (40). g2 noncatalytic subunit PDGFRA Platelet-derived growth factor À3.13 JUNB. Activator protein 1 is composed of homodimers receptor, a polypeptide or heterodimers formed by related Jun (c-Jun, JunB, and TIMP3 Tissue inhibitor of metalloproteinase 3 À2.83 JunD), Fos (c-Fos, FosB, Fra-1, and Fra-2), and ATF AHR Aryl hydrocarbon receptor À2.65 family proteins (41). Jun is known to be up-regulated NBL1 Neuroblastoma, suppression À1.93 in response to the exposure to genotoxic stress, such of tumorigenicity 1 as alkylating agents or short-wavelength UV radiation CCND1 Cyclin D1 À1.90 (42, 43). CDH6 Cadherin 6, type 2, K-cadherin À1.84 GADD45B. Gadd45 proteins cooperate in the activation NID2 Nidogen 2 (osteonidogen) À1.81 PCDH7 BH-protocadherin À1.77 of S and G2-M checkpoints following exposure of cells to UV irradiation and other genotoxic stresses, producing PTPRM Protein tyrosine phosphatase À1.69 receptor-type M growth arrest and apoptosis (8, 44). The expression changes for four selected genes were NOTE: S/R is the log-ratio (base 2) of median expression in the sensitive verified by real-time RT-PCR (Fig. 2B) in two cell lines. The versus resistant cell lines. pattern of gene expression modulation was comparable for real-time RT-PCR and microarray results, although the former technique was more sensitive in detecting gene molecular alterations that lead to ecteinascidin sensitivity expression changes (Fig. 2B). model resistance. Thus, ET-743–sensitive sarcoma cells Differences between ET-743^ Sensitive and ET-743^ constitutively express the following markers: IL-13, Resistant Sarcoma Primary Cell Lines MMP3, SAMSN1, EDG1, IGFBP3, and PRKAG2, whereas As shown in Table 1, six cell lines were considered as ET- ecteinascidin-resistant cells show increased expression of 743 sensitive (those with LC50 < 1 nmol/L), and three were PTPRM, PCDH7, NID2, CDH6, CCND1, NBL1, AHR, resistant to ET-743. TIMP3, and PDGFRA (Table 2). Effect of ET-743 on the Cell Cycle of Resistant and Interestingly, matrix metalloproteinase 3 and its Sensitive Cell Lines inhibitor, tissue inhibitor of metalloproteinase 3, are All cell lines were analyzed by flow cytometry at differentially expressed in the sensitive and resistant cell different treatment times with ET-743. The cell cycle of lines, respectively. Metalloproteinase activity influences resistant cell lines was undisturbed throughout treatment cellular sensitivity to extrinsic death in different cell (data not shown). A cytotoxic effect of ET-743 was types through proteolytic shedding of cell-surface revealed in sensitive cell lines as a G2-M blockade, or signaling molecules. Metalloproteinase defective expres- apoptosis. These effects varied among the cell lines sion has already been described in the resistant studied, thereby confirming previous observations (8). chondrosarcoma cell line although its role in ET-743 Transcriptional Signature Associated with ET-743 Sen- resistance has not been elucidated (45). Matrix metal- sitivity loproteinase 3, a regulator of matrix remodeling, plays a The gene expression profiles of the sensitive and role in tumor metastasis through the degradation of resistant cell lines were obtained under basal conditions fibronectin, laminin, gelatins, collagens, and cartilage (i.e., in the absence of the drug). The median expression in proteoglycans. Matrix metalloproteinase 3 has been shown sensitive cell lines was compared with the corresponding to selectively modulate Fas-mediated neuronal apoptosis median expression of the resistant cell lines by way of the induced by doxorubicin (46) and induces apoptosis when S/R expression ratio. Differences between resistant and overexpressed in mammary epithelial cells (47). On the sensitive cell lines were also reflected in changes of the other hand, tissue inhibitor of metalloproteinase 3, which expression level of several genes (Table 2). The results at acts as an inhibitor of matrix metalloproteinase 3, was each time for each cell line and the median ratios and SDs expressed at higher levels in the resistant cell lines. This are presented in Table 3 at the website (http://redlinfo- inhibitor of metalloproteinase can either enhance or mas.cnio.es/publications/ET743).8 These data represent a suppress apoptosis, depending on the cell type or context mixture of signals concerning cell differentiation and analyzed (48–50).

Mol Cancer Ther 2005;4(5). May 2005

Table 3. Genes with a higher level of expression in sensitive than 72 hours of treatment with ET-743. By contrast, Table 4 resistant cell lines after 72 hours of treatment illustrates a group of genes of which expression increased in the resistant cell lines after 72 hours of treatment with Gene symbol Gene description S/R Trend ET-743, and decreased in the sensitive cell lines. . SR RAD17 RAD17 is a component of the DNA damage G2- M checkpoint, interacting with RAD1 (protein-protein RAD17 RAD17 (S. pombe) homologue 1.81 interaction in the cell cycle checkpoint machinery), and is PTPN3 Protein tyrosine phosphatase, 1.78 required for S-phase and G2-M arrest in response to DNA nonreceptor type 3 damage or incomplete DNA replication (57, 58). RANBP2L1 RAN-binding protein 2–like 1 1.76 BRCA1. BRCA1 has been purified as part of a large BRCA1 Breast cancer 1, early onset 1.65 protein complex known as BRCA1-associated genome PAWR PRKC, apoptosis, WT1, regulator 1.28 surveillance complex, which contains a wide range of p53DINP1 p53-inducible p53DINP1 2.06 DNA repair and replication proteins (44, 59). An interaction CASP8AP2 Caspase 8–associated protein 2 1.77 with p53-BRCA1 (60) has been noted in the repair after HSA250839 Gene for serine/threonine 1.65 DNA damage. protein kinase CDKN1A Cyclin-dependent kinase 1.61 PAR4. PAR4 (PAWR) is a cell death modulator that inhibitor 1A (p21, Cip1) inhibits the atypical protein kinase C isoforms, leading to reduced cellular NF-nB activation and increased apop- NOTE: S/R column shows the log-ratio (base 2) of the median expression tosis (61). level of sensitive and resistant cell lines at 72 hours; trend column shows the CASP8AP2. CASP8AP2 is involved in Fas-mediated change in the level of expression of these genes during the treatment. activation of caspase 8 during apoptosis. The transcription of TP53DINP1 is increased during IGFBP-3, which is also overexpressed in sensitive cell cellular stress by p53-mediated activation of transcription. lines, enhances the p53-dependent apoptotic response of TP53DINP1, in association with homeodomain-interacting colorectal cells to DNA damage (51). protein kinase 2, regulates p53 transcriptional activity on The aryl hydrocarbon receptor is a ubiquitously P21, MDM2, PIG3, and BAX promoters, inducing G1 arrest expressed transcriptional regulatory protein, which indu- and increasing p53-mediated apoptosis (62). Homeodo- ces the transcription of CYP1A1, CYP1A2, and CYP1B1, main-interacting protein kinase has been shown to play a which are genes that encode different enzyme isoforms of critical role in triggering p53-dependent apoptosis in cytochrome P-450. These enzymes catalyze the oxidation response to the antineoplastic drug cisplatin (63). of certain classes of xenobiotics, resulting in the genera- tion of highly reactive electrophilic metabolites that bind specific residues of DNA, causing DNA-adduct formation Table 4. Genes with a higher level of expression in resistant than in sensitive cell lines after 72 hours of treatment that may lead to mutation and subsequent cellular transformation (52). The findings described here are Gene symbol Gene description S/R Trend consistent with those previously published showing ET- 743 to be metabolized by P450, most predominantly by the SR cytochrome P-450 3A subfamily (53). Additionally, the higher AHR expression values observed here in the ET- H4FG H4 histone family, À1.51 743–resistant cell lines are consistent with the previous member G observation of an increased aryl hydrocarbon receptor PTPRK Protein tyrosine phosphatase, À1.53 expression associated with an adverse outcome in soft- receptor type, K tissue sarcoma patients (16). MAL Mal, T-cell À1.55 differentiation protein Platelet-derived growth factor receptor A, which is more T1A-2 Lung type-I cell À1.88 strongly expressed in the resistant cell lines, is a receptor membrane–associated tyrosine kinase overexpressed in a subset of solid tumors, glycoprotein and is therefore the target of drugs inhibiting this function, H4FI H4 histone family, À1.52 such as imatinib mesylate (Gleevec). Platelet-derived member I growth factor A selectively binds to platelet-derived APOC1 Apolipoprotein C-I À1.53 growth factor receptor A, regulating cell proliferation, CAV1 Caveolin 1, caveolae À1.56 survival, and chemotaxis (54–56). protein, 22 kDa Ecteinascidin-Inducible Genes in the Sensitive Cell Lines ISG15 IFN-stimulated À1.68 In addition to the constitutive differences that exist protein, 15 kDa between ET-743–sensitive and ET-743–resistant cells, the MMP10 Matrix metalloproteinase À1.69 10 (stromelysin 2) soft-tissue sarcoma cell lines studied showed variable expression of a series of genes in response to treatment NOTE: The S/R column shows the log-ratio (base 2) of sensitive to resistant with ET-743. Thus, Table 3 shows a group of genes of which cell lines at 72 hours; the trend column shows the change in the level of expression level increased in the sensitive cell lines after expression of these genes during the treatment.

Mol Cancer Ther 2005;4(5). May 2005

CDKN1A. CDKN1A (p21) wild-type p53-activated frag- Discussion ment 1, p21, regulator of cell cycle, cyclin-dependent ET-743 is a potent cytotoxic agent that acts as a transcrip- kinases 2 and 4 (CDK2/CDK4) inhibitor protein, inhibiting tion-interfering agent (5). Different clinical and experimen- cell cycle progression by interacting with G1 cyclin/CDK tal studies have begun to provide an explanation of the complexes and proliferating cell nuclear antigen. mechanism underlying the cytotoxic effect of this drug, The expression levels of Rad17 and BRCA1 were verified although further effort is still necessary to fully understand by real-time RT-PCR (Fig. 3) in one sensitive and one its mechanism of action. resistant cell line, confirming the results obtained by cDNA One of the most striking findings here is the massive microarray analysis. down-regulation of gene transcription, confirming previ- Ecteinascidin-Inducible Genes in the Resistant Cell Lines ous studies done with a smaller selection of genes (5). We MAL and CAV1 are components of membrane rafts. These have used a cDNA Oncochip, enriched in cancer-relevant rafts are membrane microdomains that play a central role in genes, containing a total of 6,800 genes. Analysis of the signal transduction by acting as a scaffold in which mole- changes induced by ET-743 revealed a common signature cules involved in signal transduction pathways can interact. of 244 down-regulated and 86 up-regulated genes in the Analysis of TP53 Status majority (8 of 11) of these cell lines. TP53 is a critical cellular gatekeeper that mediates DNA High-throughput screening has allowed the identifica- damage–induced cell cycle arrest and apoptosis. As time- tion of genes that are induced after treatment with ET-743. course experiments reveal the expression in ET-743– These genes (CS-1, ATF3, SAT, JUNB, GADD45, and ID2) sensitive cell lines of p53 targets, we tested TP53 status in have a common role as transcription regulators, leading this panel of cell lines by sequencing exons 5 to 8. As shown to cell cycle arrest in G -M and apoptosis (25, 29–31, 38, in Table 5, only three cell lines (SR2205, SR2910, and 2 40, 42–44), which can be correlated with the cell cycle SR2410) had wild-type TP53, whereas all other cell lines arrest and apoptosis induced after treatment of different featured mutated TP53. In this panel of cell lines, TP53 cell types with ET-743 in our experiments and those of status did not correlate with ET-743 sensitivity, thereby others (3, 8–11). confirming previous observations (64). The approach taken here, using low-passage sarcoma cell TP73 Expression lines, makes this study of even greater interest because To determine whether TP73 expression was induced in response to treatment with ET-743, and to study a possible such cell lines have characteristics that are closest to those relation of TP73 expression with sensitivity to ET-743, of tumors. quantitative PCR was done at all the time points studied This study has also attempted to identify specific drug- (0, 6, 24, 48, and 72 hours). induced genes of which expression is associated with ET- Basal levels of TAp73 and DNp73 varied among cell lines, 743 sensitivity. To this end, tumor cells were treated with and did not correlate with sensitivity or resistance to ET-743 ET-743, and basal and ET-743–induced genes were (see Fig. 4). ET-743 seems to induce a decrease in the level of compared in sensitive and resistant cells. Although the expression of full-length TAp73, unlike other chemothera- cell lines display a constellation of ET-743–induced peutic drugs inducing DNA damage (65). By contrast, three changes at several time intervals, at 72 hours, all the cells of the studied cell lines showed a striking increase in the lines showed a relatively uniform signature induced by expression of DNp73, which is derived from an alternative the drug, which also revealed a set of genes that were promoter in intron 3 and lacks the transactivation domain differentially expressed in sensitive and resistant cells. of full-length TAp73. DNp73 is known to counteract Thus, after 72 hours of treatment, ET-743–sensitive soft- apoptosis and growth suppression mediated by wild-type tissue sarcoma cells expressed a set of DNA damage p53 and TAp73, inducing drug resistance to wild-type p53- sensors, including RAD17 and BRCA1, which underlies harboring tumor cells (66). No relation was observed the unique mechanism of ET-743 action. ET-743 is known between the induction of DNp73 and ET-743 sensitivity. to subvert normal nucleotide excision repair by generating

Figure 3. Quantitative RT-PCR of RAD17 and BRCA1 genes in one resistant cell line (SR0306) and one sensitive cell line (SR2103).

Mol Cancer Ther 2005;4(5). May 2005

Table 5. TP53 status in the cell lines studied

Cell lines

SW872 CS-R* CS SR0306* SR2103A SR2103B SR0312 CA1010 SR2410* SR2205 SR2910

Exon 5 WT WT WT WT WT WT WT WT WT WT WT Exon 6 WT WT WT WT WT WT WT WT WT WT WT Exon 7 251:T-A 245:G-A 245:G-A WT WT WT WT WT WT WT WT Exon 8 WT WT WT 273:G-A 273:G-A 273:G-A 273:G-A 273:G-A WT WT WT Exon 9 WT WT WT WT WT WT WT WT WT WT WT

NOTE: Mutations are indicated by the corresponding nucleotide change. Abbreviations: WT, wild-type TP53. *ET-743 – resistant cell lines. lethal DNA breaks during transcription-coupled nucleo- TheconfirmationinthisseriesthatET-743isnot tide excision repair (67). ET-743–induced adducts are associated with TP53 status led us to investigate whether recognized by the nucleotide excision repair system (67), the p73 pathway is involved in the response to ET-743. We which leads to apoptosis of the tumor cells, instead of hypothesized that p73 might substitute the apoptosis- repairing the damage caused by the adducts. The increased inducer role of p53, as has been shown in cells lacking expression of both RAD17 and BRCA1 indicates the functional p53 (65). Thus, we measured the expression activation of a sensor for DNA damage including both levels of p73 throughout the treatment with ET-743 in proteins. Our results are consistent with an explanation sensitive and resistant cell lines. Treatment of the panel of whereby BRCA1 functions as a molecular determinant of human soft-tissue sarcoma cell lines with ET-743 resulted response to a range of different chemotherapeutic agents in the inhibition of TAp73 and the induction of the (68), now including ET-743. oncogenic form Np73, unlike what has been shown for Genes found to be overexpressed in sensitive soft-tissue cisplatinum and other DNA-damaging drugs. The expres- sarcoma cells after ET-743 treatment include apoptosis sion levels of TAp73 and Np73 genes were not related to inducers, such as PAR4 and CASP8AP2, and some known TP53 status or sensitivity to ET-743, which differs from the p53 targets, such as TP53DINP1 and CDKN1A. This observations for other DNA-damaging drugs (65, 69–71). prompted us to investigate further the potential role of These results strongly suggest that the response of the p53 and p73 in regulating apoptosis in response to ET-743 sarcoma cells to ET-743 does not occur through p53/p73 treatment. Cell lines harboring TP53 mutations showed no pathways. The significance of p73 down-regulation after detectable changes in ET-743 sensitivity, confirming previ- treatment with ET-743 is not yet clear, and functional ous observations (3, 9, 11), although p21 induction was experiments with p73 are needed to clarify the effect of greater in the TP53 wild-type cell lines. different p73 transcripts on ET-743 response.

Figure 4. Quantitative RT-PCR showing the expression levels of Tap73 and DNp73 in different sensitive and resistant cell lines during ET-743 treatment. The level of expression is heterogeneous but there is a general inhibition of full-length p73, whereas DNp73 seems to be induced after treatment with ET-743.

Mol Cancer Ther 2005;4(5). May 2005

The comparison of sensitive and resistant cells has also sarcomas of soft tissues refractory to chemotherapy. J Clin Oncol revealed a set of genes, including MMP3, AHR, and 2004;22:1480 – 90. 18. Yovine A, Riofrio M, Blay JY, et al. Phase II study of ecteinascidin-743 TIMP3, which are differentially expressed in the absence in advanced pretreated soft tissue sarcoma patients. J Clin Oncol of the drug. Results obtained here for this panel of low- 2004;22:890 – 9. passage soft-tissue sarcoma cell lines need to be corrobo- 19. Saez AI, Artiga MJ, Romero C, et al. Development of a real-time rated in tumoral specimens before they are translated to reverse transcription polymerase chain reaction assay for c-myc expression that allows the identification of a subset of c-myc+ diffuse large B- clinical trials. This method may help to identify the subset cell lymphoma. Lab Invest 2003;83:143 – 52. of soft-tissue sarcoma tumors that potentially show an 20. Martinez N, Camacho FI, Algara P, et al. The molecular signature of increased sensitivity to ET-743 (17). mantle cell lymphoma reveals multiple signals favoring cell survival. Cancer Res 2003;63:8226 – 32. 21. Tracey L, Villuendas R, Ortiz P, et al. Identification of genes involved in resistance to interferon-a in cutaneous T-cell lymphoma. Am J Pathol References 2002;161:1825 – 37. 1. Guan Y, Sakai R, Rinehart KL, Wang AH. Molecular and crystal 22. Herrero J, Valencia A, Dopazo J. A hierarchical unsupervised growing structures of ecteinascidins: potent antitumor compounds from the neural network for clustering gene expression patterns. Bioinformatics Caribbean tunicate Ecteinascidia turbinata. J Biomol Struct Dyn 1993; 2001;17:126 – 36. 10:793 – 818. 23. van Kesteren C, Mathjt RA, Lopez-Lazaro L, et al. A comparison of 2. Pommier Y, Kohlhagen G, Bailly C, Waring M, Mazumder A, Kohn KW. limited sampling strategies for prediction of ecteinascidin 743 clearance DNA sequence- and structure-selective alkylation of guanine N2 in the when administered as a 24-h infusion. Cancer Chemother Pharmacol DNA minor groove by ecteinascidin 743, a potent antitumor compound 2001;48:459 – 66. from the Caribbean tunicate Ecteinascidia turbinata. Biochemistry 24. Vujcic S, Halmekyto M, Diegelman P, et al. Effects of conditional 1996;35:13303 – 9. overexpression of spermidine/spermine N1-acetyltransferase on poly- 3. Takebayashi Y, Goldwasser F, Urasaki Y, Kohlhagen G, Pommier Y. amine pool dynamics, cell growth, and sensitivity to polyamine analogs. Ecteinascidin 743 induces protein-linked DNA breaks in human colon J Biol Chem 2000;275:38319 – 28. carcinoma HCT116 cells and is cytotoxic independently of topoisomerase I 25. Chen Y, Kramer DL, Jell J, Vujcic S, Porter CW. Small interfering RNA expression. Clin Cancer Res 2001;7:185 – 91. suppression of polyamine analog-induced spermidine/spermine N1-acetyl- 4. Aune GJ, Furuta T, Pommier Y. Ecteinascidin 743: a novel anticancer transferase. Mol Pharmacol 2003;64:1153 – 9. drug with a unique mechanism of action. Anticancer Drugs 2002;13: 26. Gabrielson EW, Pegg AE, Casero RA Jr. The induction of spermidine/ 545 – 55. spermine N1-acetyltransferase (SSAT) is a common event in the response 5. Minuzzo M, Marchini S, Broggini M, Faircloth G, D’Incalci M, of human primary non-small cell lung carcinomas to exposure to the new Mantovani R. Interference of transcriptional activation by the antineo- antitumor polyamine analogue N1,N11-bis(ethyl)norspermine. Clin Cancer plastic drug ecteinascidin-743. Proc Natl Acad Sci U S A 2000;97: Res 1999;5:1638 – 41. 6780 – 4. 27. Maxwell PJ, Longley DB, Latif T, et al. Identification of 5-fluorouracil- 6. Jin S, Gorfajn B, Faircloth G, Scotto KW. Ecteinascidin 743, a inducible target genes using cDNA microarray profiling. Cancer Res transcription-targeted chemotherapeutic that inhibits MDR1 activation. 2003;63:4602 – 6. Proc Natl Acad Sci U S A 2000;97:6775 – 9. 28. Marverti G, Bettuzzi S, Astancolle S, Pinna C, Monti MG, Moruzzi MS. 7. Friedman D, Hu Z, Kolb EA, Gorfajn B, Scotto KW. Ecteinascidin-743 Differential induction of spermidine/spermine N1-acetyltransferase activity inhibits activated but not constitutive transcription. Cancer Res 2002;62: in cisplatin-sensitive and -resistant ovarian cancer cells in response to 3377 – 81. N1,N12-bis(ethyl)spermine involves transcriptional and post-transcriptional 8. Gajate C, An F, Mollinedo F. Differential cytostatic and apoptotic regulation. Eur J Cancer 2001;37:281 – 9. effects of ecteinascidin-743 in cancer cells. Transcription-dependent cell 29. Scorcioni F, Corti A, Davalli P, Astancolle S, Bettuzzi S. Manipulation cycle arrest and transcription-independent JNK and mitochondrial mediated of the expression of regulatory genes of polyamine metabolism results apoptosis. J Biol Chem 2002;277:41580 – 9. in specific alterations of the cell-cycle progression. Biochem J 2001;354: 9. Erba E, Bergamaschi D, Bassano L, et al. Ecteinascidin-743 (ET-743), 217 – 23. a natural marine compound, with a unique mechanism of action. Eur 30. Amundson SA, Bittner M, Chen Y, Trent J, Meltzer P, Fornace AJ Jr. J Cancer 2001;37:97 – 105. Fluorescent cDNA microarray hybridization reveals complexity and 10. Li WW, Takahashi N, Jhanwar S, et al. Sensitivity of soft tissue heterogeneity of cellular genotoxic stress responses. Oncogene sarcoma cell lines to chemotherapeutic agents: identification of ecteinascidin- 1999;18:3666 – 72. 743 as a potent cytotoxic agent. Clin Cancer Res 2001;7:2908 – 11. 31. Mashima T, Udagawa S, Tsuruo T. Involvement of transcriptional 11. Martinez EJ, Corey EJ, Owa T. Antitumor activity- and gene repressor ATF3 in acceleration of caspase protease activation during expression-based profiling of ecteinascidin Et 743 and phthalascidin Pt DNA damaging agent-induced apoptosis. J Cell Physiol 2001;188: 650. Chem Biol 2001;8:1151 – 60. 352 – 8. 12. Jimeno J, Lopez-Martin JA, Ruiz-Casado A, Izquierdo MA, Scheuer 32. Sun XH. Constitutive expression of the Id1 gene impairs mouse B cell PJ, Rinehart K. Progress in the clinical development of new marine-derived development. Cell 1994;79:893 – 900. anticancer compounds. Anticancer Drugs 2004;15:321 – 9. 33. Benezra R, Davis RL, Lassar A, et al. Id: a negative regulator of helix- 13. Hendriks HR, Fiebig HH, Giavazzi R, Langdon SP, Jimeno JM, loop-helix DNA binding proteins. Control of terminal myogenic differenti- Faircloth GT. High antitumour activity of ET743 against human tumour ation. Ann N Y Acad Sci 1990;599:1 – 11. xenografts from melanoma, non-small-cell lung and ovarian cancer. Ann 34. Christy BA, Sanders LK, Lau LF, Copeland NG, Jenkins NA, Nathans D. Oncol 1999;10:1233 – 40. An Id-related helix-loop-helix protein encoded by a growth factor-inducible 14. Izbicka E, Lawrence R, Raymond E, et al. In vitro antitumor activity of gene. Proc Natl Acad Sci U S A 1991;88:1815 – 9. the novel marine agent, ecteinascidin-743 (ET-743, NSC-648766) against 35. Strait KA, Dabbas B, Hammond EH, Warnick CT, Iistrup SJ, Ford CD. human tumors explanted from patients. Ann Oncol 1998;9:981 – 7. Cell cycle blockade and differentiation of ovarian cancer cells by the 15. Valoti G, Nicoletti MI, Pellegrino A, et al. Ecteinascidin-743, a new histone deacetylase inhibitor trichostatin A are associated with changes in marine natural product with potent antitumor activity on human ovarian p21, Rb, and Id proteins. Mol Cancer Ther 2002;1:1181 – 90. carcinoma xenografts. Clin Cancer Res 1998;4:1977 – 83. 36. Eickhoff B, Germeroth L, Stahl C, et al. Trichostatin A-mediated 16. Delaloge S, Yovine A, Taamma A, et al. Ecteinascidin-743: a marine- regulation of gene expression and protein kinase activities: reprogramming derived compound in advanced, pretreated sarcoma patients—preliminary tumor cells for ribotoxic stress-induced apoptosis. Biol Chem 2000;381: evidence of activity. J Clin Oncol 2001;19:1248 – 55. 1127 – 32. 17. Garcia-Carbonero R, Supko JG, Manola J, et al. Phase II and 37. Eickhoff B, Ruller S, Laue T, et al. Trichostatin A modulates pharmacokinetic study of ecteinascidin 743 in patients with progressive expression of p21waf1/cip1, Bcl-xL, ID1, ID2, ID3, CRAB2, GATA-2,

Mol Cancer Ther 2005;4(5). May 2005

hsp86 and TFIID/TAFII31 mRNA in human lung adenocarcinoma cells. Biol 55. Lindahl P, Bostrom H, Karlsson L, Hellstrom M, Kalen M, Betsholtz C. Chem 2000;381:107 – 12. Role of platelet-derived growth factors in angiogenesis and alveogenesis. Curr Top Pathol 1999;93:27 – 33. 38. Florio M, Hernandez MC, Yang H, Shu HK, Cleveland JL, Israel MA. Id2 promotes apoptosis by a novel mechanism independent of 56. Lindahl P, Betsholtz C. Not all myofibroblasts are alike: revisiting the dimerization to basic helix-loop-helix factors. Mol Cell Biol 1998;18: role of PDGF-A and PDGF-B using PDGF-targeted mice. Curr Opin Nephrol 5435 – 44. Hypertens 1998;7:21 – 6. 39. Biswas G, Anandatheerthavarada HK, Zaidi M, Avadhani NG. 57. Post SM, Tomkinson AE, Lee EY. The human checkpoint Rad protein Mitochondria to nucleus stress signaling: a distinctive mechanism of Rad17 is chromatin-associated throughout the cell cycle, localizes to DNA NFnB/Rel activation through calcineurin-mediated inactivation of InBh.J replication sites, and interacts with DNA polymerase epsilon. Nucleic Cell Biol 2003;161:507 – 19. Acids Res 2003;31:5568 – 75. 40. Frey N, Richardson JA, Olson EN. Calsarcins, a novel family of 58. Post S, Weng YC, Cimprich K, Chen LB, Xu Y, Lee EY. Phosphory- sarcomeric calcineurin-binding proteins. Proc Natl Acad Sci U S A 2000; lation of serines 635 and 645 of human Rad17 is cell cycle regulated and is 97:14632 – 7. required for G(1)/S checkpoint activation in response to DNA damage. Proc Natl Acad Sci U S A 2001;98:13102 – 7. 41. Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nat Cell Biol 2002;4:E131 – 6. 59. Wang Y, Cortez D, Yazdi P, Neff N, Elledge SJ, Qin J. BASC, a super complex of BRCA1-associated proteins involved in the recognition and 42. Shaulian E, Schreiber M, Piu F, Beeche M, Wagner EF, Karin M. The repair of aberrant DNA structures. Genes Dev 2000;14:927 – 39. mammalian UV response: c-Jun induction is required for exit from p53- imposed growth arrest. Cell 2000;103:897 – 907. 60. MacLachlan TK, Takimoto R, El-Deiry WS. BRCA1 directs a selective 43. Karin M. Mitogen-activated protein kinase cascades as regulators of p53-dependent transcriptional response towards growth arrest and DNA stress responses. Ann N Y Acad Sci 1998;851:139 – 46. repair targets. Mol Cell Biol 2002;22:4280 – 92. ,44. Vairapandi M, Balliet AG, Hoffman B, Liebermann DA. GADD45b and 61. Garcia-Cao I, Lafuente MJ, Criado LM, Diaz-Meco MT, Serrano GADD45g are cdc2/cyclinB1 kinase inhibitors with a role in S and G /M M, Moscat J. Genetic inactivation of Par4 results in hyperactivation 2 n cell cycle checkpoints induced by genotoxic stress. J Cell Physiol 2002; of NF- B and impairment of JNK and p38. EMBO Rep 2003;4: 192:327 – 38. 307 – 12. 45. Shao L, Kasanov J, Hornicek FJ, Morii T, Fondren G, Weissbach L. 62. Tomasini R, Samir AA, Carrier A, et al. TP53INP1s and homeodomain- Ecteinascidin-743 drug resistance in sarcoma cells: transcriptional and interacting protein kinase-2 (HIPK2) are partners in regulating p53 activity. cellular alterations. Biochem Pharmacol 2003;66:2381 – 95. J Biol Chem 2003;278:37722 – 9. 46. Wetzel M, Rosenberg GA, Cunningham LA. Tissue inhibitor of 63. Di Stefano V, Rinaldo C, Sacchi A, Soddu S, D’Orazi G. Homeodomain- metalloproteinases-3 and matrix metalloproteinase-3 regulate neuronal interacting protein kinase-2 activity and p53 phosphorylation are sensitivity to doxorubicin-induced apoptosis. Eur J Neurosci 2003;18: critical events for cisplatin-mediated apoptosis. Exp Cell Res 2004; 1050 – 60. 293:311 – 20. 47. Alexander CM, Howard EW, Bissell MJ, Werb Z. Rescue of mammary 64. Poindessous V, Koeppel F, Raymond E, Comisso M, Waters SJ, epithelial cell apoptosis and entactin degradation by a tissue inhibitor of Larsen AK. Marked activity of irofulven toward human carcinoma cells: metalloproteinases-1 transgene. J Cell Biol 1996;135:1669 – 77. comparison with cisplatin and ecteinascidin. Clin Cancer Res 2003;9: 2817 – 25. 48. Fata JE, Leco KJ, Voura EB, et al. Accelerated apoptosis in the Timp- 3-deficient mammary gland. J Clin Invest 2001;108:831 – 41. 65. Irwin MS, Kondo K, Marin MC, Cheng LS, Hahn WC, Kaelin WG Jr. Chemosensitivity linked to p73 function. Cancer Cell 2003;3:403 – 10. 49. Smith MR, Kung H, Durum SK, Colburn NH, Sun Y. TIMP-3 induces cell death by stabilizing TNF-a receptors on the surface of human colon 66. Zaika AI, Slade N, Erster SH, et al. DNp73, a dominant-negative carcinoma cells. Cytokine 1997;9:770 – 80. inhibitor of wild-type p53 and TAp73, is up-regulated in human tumors. J Exp Med 2002;196:765 – 80. 50. Bond M, Murphy G, Bennett MR, Newby AC, Baker AH. Tissue inhibitor of metalloproteinase-3 induces a Fas-associated death domain-dependent 67. Takebayashi Y, Pourquier P, Zimonjic DB, et al. Antiproliferative type II apoptotic pathway. J Biol Chem 2002;277:13787 – 95. activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair. Nat Med 2001;7:961 – 6. 51. Williams AC, Collard TJ, Perks CM, et al. Increased p53-dependent apoptosis by the insulin-like growth factor binding protein IGFBP-3 in 68. Quinn JE, Kennedy RD, Mullan PB, et al. BRCA1 functions as a human colonic adenoma-derived cells. Cancer Res 2000;60:22 – 7. differential modulator of chemotherapy-induced apoptosis. Cancer Res 2003;63:6221 – 8. 52. MacDonald CJ, Ciolino HP, Yeh GC. The drug salicylamide is an antagonist of the aryl hydrocarbon receptor that inhibits signal transduc- 69. Costanzo A, Merlo P, Pediconi N, et al. DNA damage-dependent tion induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cancer Res 2004; acetylation of p73 dictates the selective activation of apoptotic target 64:429 – 34. genes. Mol Cell 2002;9:175 – 86. 53. Reid JM, Kuffel MJ, Ruben SL, et al. Rat and human liver cytochrome 70. Gong JG, Costanzo A, Yang HQ, et al. The tyrosine kinase c-Abl P-450 isoform metabolism of ecteinascidin 743 does not predict gender- regulates p73 in apoptotic response to cisplatin-induced DNA damage. dependent toxicity in humans. Clin Cancer Res 2002;8:2952 – 62. Nature 1999;399:806 – 9. 54. Heldin CH, Ostman A, Ronnstrand L. Signal transduction via 71. Yuan ZM, Shioya H, Ishiko T, et al. p73 is regulated by tyrosine platelet-derived growth factor receptors. Biochim Biophys Acta 1998; kinase c-Abl in the apoptotic response to DNA damage. Nature 1999; 1378:F79 – 113. 399:814 – 7.

Mol Cancer Ther 2005;4(5). May 2005

Downloaded from mct.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Transcriptional signature of Ecteinascidin 743 (Yondelis, Trabectedin) in human sarcoma cells explanted from chemo-naïve patients

Nerea Martínez, Margarita Sánchez-Beato, Amancio Carnero, et al.

Mol Cancer Ther 2005;4:814-823.

Updated version Access the most recent version of this article at: http://mct.aacrjournals.org/content/4/5/814

Cited articles This article cites 69 articles, 32 of which you can access for free at: http://mct.aacrjournals.org/content/4/5/814.full#ref-list-1

Citing articles This article has been cited by 5 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/4/5/814.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/4/5/814. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.