Eur Arch Otorhinolaryngol (2006) 263: 127–134 DOI 10.1007/s00405-005-0936-z
HEAD AND NECK ONCO L OGY
Marianne Schmidt Æ Gabriele Schler Petra Gruensfelder Æ Florian Hoppe
Differential gene expression in a paclitaxel-resistant clone of a head and neck cancer cell line
Received: 10 September 2004 / Accepted: 15 December 2004 / Published online: 28 December 2005 Springer-Verlag 2005
Abstract The anti-neoplastic drug paclitaxel (taxol), which is known to block cells in the G2/M phase of the cell cycle through stabilization of microtubules, is meanwhile commonly used for chemotherapy of advanced head and neck cancer. Chemotherapy is primarily used in order to preserve laryngeal and/or pharyngeal structures. Although paclitaxel generally seems to be a powerful agent, it failed to reach a loco-regional tumor control in a sufficient percentage of patients. In order to investigate molecular resistance mechanisms, we have established a paclitaxel-resistant subline originating from the larynx carcinoma cell line HLaC79, which seemed to be partially dependent on taxol. The original and the descendant cell line were characterized by growth inhibition assays. We used western blotting and the cDNA subtraction (SSH) technique to identify genes differentially expressed in the taxol-resistant cell clone. cDNA subtraction revealed increased expression of six genes, including clathrin heavy chain, a3-tubulin, a neuroblastoma-specific Thymosin b, the ribosomal protein L7a, HLA-B associated transcript 3 and collagen IIIa1 in the taxol-resistant cell line. Furthermore, western blots showed an overexpression of MDR-1 in the taxol-resistant clone, while a- and b-tubulins and p48/IRF9 were expressed in equal amounts in both cell lines. Keywords Head and neck cancer Æ Paclitaxel Æ Resistance Æ Gene expression
M. Schmidt (&) Æ G. Schler Æ P. Gruensfelder Æ F. Hoppe Department of Otorhinolaryngology, University of Wu¨rzburg, Josef-Schneider-Straße 11, 97080 Wu¨rzburg, Germany E-mail:
[email protected] Tel.: +49-931-20121365 Fax: +49-931-20121369
Introduction The antineoplastic drug paclitaxel is a naturally occurring diterpenoid, isolated from the Pacific yew (taxus brevifolia). Paclitaxel accelerates the polymerization of tubulin monomers, thus stabilizing microtubules and inhibiting the normal dynamic microtubule network reorganization. Cells treated with taxanes are arrested in the G2/M-phase of the cell cycle. In practice, paclitaxel is used as a chemotherapeutic agent for the treatment of head and neck cancer patients predominantly applied in combination with other cytotoxic agents, such as cisplatin and/or radiotherapy. Recently a prospective study of paclitaxel/cisplatin chemotherapy in combination with computerized tomography-based radiation therapy was published [21], in which the effects of the combined therapy were investigated with special regard to local tumor control, survival and larynx preservation. According to this investigation, a complete tumor volume reduction was reached in 10% of the patients, and a partial volume reduction in 78% of the patients after paclitaxel/cisplatin chemotherapy. Twelve percent of patients did not respond to chemotherapy. Single-agent paclitaxel was tested in several studies and proved to be active in patients with squamous cell carcinoma of the head and neck. Response rates varied from 20 to 40% (for a review, see [22]). There are several mechanisms that have been proposed to play a role in the development of paclitaxel resistance. Taxanes are substrates for the P-glycoprotein efflux pump (P-gp/MDR). An overexpression of P-gp therefore may inhibit drug retention in the cell [6]. Other mechanisms concern the paclitaxel target protein btubulin, which may be affected by point mutations at the paclitaxel binding site [4] or by expression of tubulin isotypes such as class III b-tubulin, which inhibit the assembly of b-tubulin subunits promoted by paclitaxel [20]. Recently, Luker et al. [15] showed an overexpression of the interferon (IFN) regulatory factor IRF9/p48/
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ISGF3c in taxol-resistant breast cancer cells. We established a paclitaxel-resistant clone of the larynx carcinoma cell line HLaC79 and characterized the descendant by proliferation assays, cell cycle analysis, western blot and cDNA subtraction analysis.
according to the manufacturer’s protocol. mRNA was prepared with the Oligotex mRNA Midi Kit of Qiagen (Hilden, Germany) according to the kit manual. Subtraction hybridization (SSH)
Material and methods Cell lines and cell culture The head and neck squamous carcinoma cell line HLaC79 was established from a lymph node metastasis of a laryngeal squamous cell carcinoma [32]. The cell line was grown with RPMI 1640 medium (Seromed, Munich), supplemented with 10% fetal calf serum (FCS). HLaC79 cells were treated with 10 nM paclitaxel. A resistant clone was isolated using the ‘‘ring technique;’’ in brief, a metal ring was placed over the clonal population and isolated by trypsination inside the ring [18]. The permanent HLaC79 clonal cell line HLaC79-clone1 was kept with RPMI 1640 medium, supplemented with 10% FCS and 10 nM paclitaxel. Cell viability and proliferation assay Cells were seeded at 5,000 cells/well in 96-well plates. They were treated with increasing concentrations of paclitaxel (10–200 nM) in RPMI medium for 24 h. Controls were kept in the medium without paclitaxel. Cell viability was measured by replacing the culture medium with medium containing 1 mg/ml MTT. After 4 h incubation, MTTstaining solution was replaced by isopropanol, and cells were incubated at 37C overnight. The color conversion of MTT to a blue formazon dye was measured with an ELISA reader at a wavelength of 570 nm. The amount of formazon dye is in direct proportion to the number of metabolically active cells in the culture.
Cell cycle distribution Cells were treated with 10 nM paclitaxel for 24 h. Controls received medium. After this incubation, medium was changed and cells were grown for a further 24 h. DNA content was measured by staining with 40 lg/ml Propidium iodide (Sigma Aldrich, Taufkirchen, Germany) and 100 lg/ml RNAse (Qiagen, Hilden, Germany) in PBS for 30 min at 37C. FACS analysis was performed with a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson, Heidelberg). mRNA isolation Total RNA was isolated from tissue samples using TRIZOL-reagent (GIBCO BRL, Eggenstein, Germany)
Subtraction hybridization was performed using the PCR-Select cDNA Subtraction Kit of Clontech (Becton Dickinson, Heidelberg) according to the manufacturer’s protocol. Briefly, mRNA of HLaC79 and HLaC79clone1 was reverse transcribed into cDNA. Both cDNA populations were hybridized, and the hybrid sequences were then removed. Consequently, the remaining unhybridized cDNAs represent genes that are differentially expressed in one of the two cDNA populations. These cDNAs were cloned into the pCR 2.1-TOPO cloning vector (Invitrogen, Paisley, U.K.) and subsequently verified by dot blot hybridization and northern blot hybridizations. Differentially expressed cDNAs were sequenced and sequence data evaluated using the BLAST tool. Sequences were compared with the EMBL database. Dot blot hybridization Bacterial clones arising from the cDNA subtraction library were picked and grown overnight in 100 ll LB medium containing 100 lg/l ampicillin in 96-well plates. Of each bacterial culture, 1 ll was used to amplify the cDNA insert by PCR, using the nested primers of the PCR-Select cDNA Subtraction Kit. The rest of bacterial culture was archived as glycerol cultures at 80C. Amplification was tested on a 1% agarose gel. From the clones with positive cDNA insert 4 ll of the amplification reaction was mixed with 4 ll of freshly prepared 0.6 N NaOH; 1 ll of these mixtures was spotted on two identical nylon membranes. The dot blots were neutralized in 0.5 M Tris-HCl (pH7.5), UV-crosslinked and used for hybridization with 32P-labeled cDNA mixtures of HLaC79 and HLaC79-clone1. Equal counts of each labeled cDNA population were used for hybridization. Radioactive labeling was performed with the Hexa-Labeling Kit of MBI (MBI Fermentas, St. Leon-Rot, Germany) according to the manufacturer’s handbook. Dot-blots were exposed to a bio-imaging analyzer (Fuji, Tokyo) for at least 1 h. Evaluation of imaging data was effected with the AIDA imaging software (Raytest GmbH, Straubenhardt, Germany). Northern hybridizations Bacterial cDNA clones that proved to be differentially expressed in dot blots were grown in liquid cultures. Plasmid DNA was isolated with the Qiagen Plasmid Mini Kit (Qiagen, Hilden, Germany). Inserts were excised with EcoRI, gel purified and subsequently
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Fig. 1 Absorbance curves ( y-axis) of the MTT assay performed on HLaC79 and HLaC79-clone1 with increasing concentrations of paclitaxel ( x-axis) at 570 nm. Increasing absorbance correlates
directly with the number of living cells. One hundred percent cell growth corresponds to 0 nm paclitaxel. a HlaC79 and b HLaC79clone1; each diagram displays three independent experiments
labeled with dig-dUTP according to the random primer labeling protocol for non-radioactive labeling of the Hexa Label DNA Labeling Kit (MBI Fermentas, St.Leon-Rot, Germany). Of each mRNA (HLaC79 and HLaC79-clone1, see above) sample, 2 lg was size-fractionated on a 1.2% formaldehyde agarose gel. RNA was transferred to a positively charged nylon membrane (Tropix, Bedford, Mass.) by capillary transfer with 20· SSC as transfer buffer. Blots were UV-fixed at 1,200 mJ (Stratalinker, Stratagene, La Jolla, Calif.) and hybridized with 5 ll denaturated digoxygenin-labeled probe at 50C in Dig Easy Hyb hybridization buffer (Clontech, Becton Dickinson, Heidelberg). Blots were washed twice for 15 min with 2· SSC, 1% sodium dodecyl sulfate at room temperature and twice for 15 min in 0.5· SSC, 1% sodium dodecyl sulfate at 50C. Detection of digoxygenin-labeled RNA-RNA-hybrids was carried out according to Boehringer-Dig-Manual (Boehringer Mannheim, Germany) with an anti-digoxygenin antibody conjugated with alkaline phosphatase (Boehringer, Mannheim, Germany) and CSPD (Tropix, Bedford, Mass.) as chemoluminescent detection substrate. Developed northern-blots were exposed to Kodak X-AR X-ray films for 30 min.
used a HRP-conjugated goat anti-rabbit IgG serum (Santa Cruz Biotechnology, Heidelberg). Detection of antibody conjugates was performed with the enhanced chemiluminescence system (ECL, Amersham Biosciences, Freiburg, Germany), according to the manufacture’s protocol.
Results Establishment of a taxol-resistant cell line The larynx carcinoma cell line HLaC79 was treated with 10 nM paclitaxel diluted in complete culture medium. In nine out of ten culture flasks treated with taxol, all cells died and detached from the flask surface. In one flask a few cells survived, and one of these surviving cells originated in a cell clone, optically not distinguishable from the parental cell line. The cell clone was isolated and kept with 10 nM paclitaxel as a permanent clonal cell line.
Western blot analysis For western blot analysis, cells were harvested by trypsination and dissolved in RIPA buffer (PBS, containing 1% NP40, 0.5% sodium deoxycholate and 0.1% SDS), supplemented with 10 lg/ml phenylmethanesulfonyl fluoride (PMSF). The protein content was determined according to the method of Lowry [14]. Equal amounts of total protein lysates were loaded on 10% SDS-polyacrylamide gels and run at a constant current of 20 mA. Gels were blotted onto nitrocellulose membranes according to the semidry method of Kyhse-Andersen [12]. Blots were blocked over night with TBST (10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 8.0), containing 5% nonfat dry milk. For detection of a/b-tubulin, MDR-1/P-gp and IRF9 polyclonal rabbit sera were used (Santa Cruz Biotechnology, Heidelberg). As secondary antibody we
Fig. 2 Growth of HLaC79-clone1 in the presence of increasing concentrations of paclitaxel (nm) within the graphic. Results indicate that cells grow better in the presence of taxol
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Fig. 3 Cell cycle analysis of HLaC79 and HLaC79-clone1, treated with 10 nM paclitaxel for 24 h. M1 G0/G1; M2 G2/M; M3 apoptotic fraction; tax controls treated with cell culture medium only; + tax cells treated with 10 nm paclitaxel
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Cell proliferation and viability assay
Western blot analysis
Both the parental cell line and the resistant clone were treated with increasing concentrations of paclitaxel. After 48 h incubation, the cell viability and cytotoxicity of paclitaxel were measured with the MTT assay. Paclitaxel suppressed the growth of HLaC79 cells significantly at the low dose of 10 nM (Fig. 1a). HLaC79-clone1 in contrast showed nearly no growth inhibition up to concentrations of 100 nM paclitaxel (Fig. 1b). Results in Fig. 2 show that cells grow somewhat better in the presence of paclitaxel, which may indicate a partial dependency on the drug.
There are several factors/genes that have previously been related to taxol resistance, among others variations in the expression of tubulin subtypes a and b, overexpression of the P-glycoprotein efflux pump (MDR-1) and overexpression of IRF9, an IFN (type I IFNs) regulatory factor in the IFN-mediated signaling cascade [15]. In order to analyze expression levels of these factors, we performed western blots, using antibodies against atubulin, b-tubulin, MDR/P-gp and IRF9. Results are displayed in Fig. 4. Western blot analysis revealed overexpression of MDR-1 in the paclitaxel-resistant cell clone of HLaC79. All other factors tested were nearly equally expressed (Fig. 4). IRF9 was not expressed in any of the cell lines (data not shown).
FACS analysis In order to determine if there was any alteration in the cell cycle distribution, cells were cultured with or without paclitaxel for 24 h, incubated for a further 24 h without toxic agents and subsequently permeabilized and stained with propidium iodide. As shown in Fig. 3, both the parental cell line HLaC79 and the taxolresistant clone display similar cell cycle profiles in the absence of paclitaxel (- tax). G0/G1 (diploid) is indicated by M1, S and G2/M by M2, apoptotic cell fraction by M3 (Fig. 3) in percent of gated cells. It is obvious that paclitaxel acts toxically in the parental cell line HLaC79, indicated by the high M3 score of 23.24% apoptotic cells after 24-h treatment. The cell cycle was shifted towards G2/M (M2 fraction) as expected. The HLaC79 subline, however, proved to be not significantly affected by paclitaxel and was marked by an unaltered cell cycle distribution as well as a low apoptotic fraction.
Fig. 4 Western blot of HLaC79 and HLaC79-clone1. PA gels were loaded equally. Western blots were incubated with antibodies against MDR-1, b-tubulins, a-tubulins or IRF9, respectively
cDNA subtraction hybridization In order to investigate further differences between the parental cell line and the descending taxol-resistant clone we decided to search for differentially expressed genes using cDNA subtraction hybridization. The subtraction hybridization was performed with the PCR Select system of Clontech (Becton Dickinson, Heidelberg), which is based on one round of substractive hybridization and a selective PCR amplification of differentially expressed genes. We yielded a subtraction library, from which we isolated 600 single bacterial clones. The cDNA of each clone was amplified by PCR, and amplified cDNA was spotted on nylon membranes. With these blots we performed dot blot hybridization, using 32P-labelled total cDNA of HLaC79 and HLaC79clone1 as radioactive hybridization probes. Clones that seemed to be overexpressed in the taxolresistant clone of HLaC79 were picked, cultivated and plasmid DNA was isolated. From each clone putatively differentially expressed, we produced a digoxygenin-labeled cDNA probe for northern hybridizations on mRNA of both cell lines. cDNA clones that proved to be differentially expressed in the paclitaxel-resistant clone of HLaC79 were subsequently sequenced. Sequences were compared with the EMBL databases using the BLAST tool. Northern blotting confirmed in the end six genes, differentially expressed in the taxol-resistant cell clone (Fig. 5). cDNA clones identified as differentially expressed were sequenced; sequences were subsequently compared with the EMBL databases, using the NCBI Blast engine. Sequencing identified the following six genes as differently expressed in the two cell lines: collagen type IIIa1 (ColIIIA1), ribosomal protein L7a (RPL7A), tubulin a3 (TUBAIII), HLA-B associated transcript 3 (BAT3), a subtype of thymosine b, identified in neuroblastoma cells (TMSNB), and clathrin heavy chain (CLTC) (Fig. 5).
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Fig. 5 Northern blot analysis of differentially expressed cDNA clones. mRNA of HLaC79 ( A) and HLaC79-clone1 ( B) were blotted and probed with dig-dUTP labeled cDNA. Formaldehyde containing agarose gels was equally loaded with mRNA of HLaC79 and the paclitaxel resistant subline, respectively
Discussion In advanced laryngeal and hypopharyngeal cancer the functional and cosmetic deformations produced by surgery can be very impairing for the patients. The chemotherapeutic agent paclitaxel is meanwhile commonly used for chemotherapy of advanced head and neck cancer in order to preserve laryngeal and/or pharyngeal structures [21]. Although paclitaxel generally seems to be a powerful agent, it failed to reach a locoregional tumor control in 12% of the patients according to a previously published study [21]. Chemotherapeutic failure may be related either to inherited resistance against the drug or/and the acquirement of resistance during the therapy. Drug resistance is mostly a multifactorial procedure; in the case of paclitaxel several mechanisms have been described. One mechanism is the overexpression of P-glycoprotein (coded by the multi drug resistance gene 1, MDR-1, [25]). We were able to verify MDR-1 overexpression in HLaC79-clone1 too. P-glycoprotein is a transmembrane protein, consisting of two subunits, each containing six transmembrane domains and two intracellular ATP binding sites. It acts as a drug efflux pump for a wide range of lipophilic drugs, such as anthracyclines, vinca alkaloids and taxanes [5], which are thought to enter cells by passive diffusion. In head and neck cancer a correlation between clinical stage and MDR-1 expression [25] and a correlation with the response to MDR-1 dependent drugs [9] was shown previously, albeit the number of patients in that study was very low (seven out of eight patients). In a further study [30] concerning non-small-cell lung cancer, only 68% of the tumor specimen with poor response to paclitaxel showed positive MDR-1 expression, which suggests, that other factors also play a role in paclitaxel resistance. MDR-1 overexpression has not been detected by SSH in our study. This might be explained by the particular feature of the SSH technique enriching lowly expressed genes, which was clearly not the case for MDR-1. Furthermore, it has to be considered, that more than 600 cDNA clones have to be analyzed by dot blot hybridization, and artifacts cannot be excluded. In 2001 Luker and colleagues [15] demonstrated that transcriptional activation of IRF-9, which is an IFN regulatory factor, corresponded with resistance to
paclitaxel and other antimicrotubule agents in breast cancer cells. The authors suggested that overexpression of IRF-9 is involved in downstream regulation of IFNresponsive genes in breast cancer cells and may be associated with drug resistance. However, overexpression of IRF-9 could not be detected in the taxol-resistant cell clone of HLaC79. Main players involved in paclitaxel resistance are the microtubules. Alterations in tubulin isotype expression or introduction of point mutations in one or both alleles of specific tubulin genes have been shown to be associated with paclitaxel resistance [27]. Microtubules are built of tubulina/b heterodimers. There are six a- and seven b-tubulin isotypes known to occur in human tissue. We examined the overall expression level of a- and b-tubulin isotypes, respectively. We were not able to detect striking differences in the expression of total a- and b-tubulin between the parental taxol-sensitive cell line and the resistant clone. This is in contradiction to the results of Han et al. [13], who showed an overexpression of total a- and b-tubulin in paclitaxel-resistant lung cancer cell lines. These resistant cell lines showed, similar to the HLaC79 subline, a MDR-1 overexpression. The authors tested tubulin expression using northern and western blotting and were able to show that tubulin-overexpression derived from a posttranslational mechanism. Probably several cellular changes can enhance paclitaxel resistance. In order to screen for further expression differences between paclitaxel-sensitive and -resistant cell lines, we performed cDNA subtraction hybridization. Using this technique we were able to isolate six cDNA clones overexpressed in HLaC79-clone1. The paclitaxel-resistant clone was marked by a distinct overexpression of ribosomal protein L7A (RPL7A). RPL7A up-regulation has not yet been described in context with drug resistance, albeit this protein seems to be a general marker for malignant transformed tissue, as has been shown in colorectal cancer [29], brain tumors [11] and prostate carcinomas [26], each compared to non-malignant tissue. A further gene with enhanced expression in HLaC79clone1 was identified as tubulin-a3 (TUBA3), predominantly found in morphologically differentiated neuron cells. Recent studies have implicated b-tubulin as the site of paclitaxel binding [4]. However, not very much
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information is available about the function and importance of a-tubulins in drug resistance. Transfection of antisense a1-tubulin-cDNA resulted in an increased sensitivity of the cells towards paclitaxel, which indicates at least a certain function of a-tubulins in drug resistance. Furthermore, several mutations in a-tubulins have been described so far, which lead to increased microtubule instability and may therefore compensate the microtubule-stabilizing properties of paclitaxel [17]. The special neuronal isoform tubulin-a3 has not been associated with cancer so far. Since the total amount of tubulins was not changed in the western blots, we checked the specificity of the broad-reacting a-tubulin antibody used in our western blots. Sequence alignment revealed that the antibody reacts with TUBA3 as well. So the question remains open if there is either a shift in expression of a-tubulins towards the TUBA3 isoform or if the up-regulation of TUBA3 is only of transcriptional nature. In this respect, it has been observed that overexpression of a- and b-tubulins in taxol-resistant lung cancer cell lines has been attributed to a posttranscriptional mechanism [13]. To differentiate between the two possibilities, a TUBA3 specific antibody has to be used, but is not commercially available so far. The third gene identified to be up-regulated was the HLA-B associated transcript (BAT3). It encodes a large proline-rich protein with yet unknown function and is located in the human major histocompatibility complex (MHC) on human chromosome 6. Manchen and Hubberstay [16] demonstrated that BAT3 has a functional nuclear localization signal and remains in the nucleus even during apoptosis. Under conditions of cellular stress the chaperones Hsp70/Hsc70 accumulate in the nuclei. The authors propose that BAT3 functions in the nucleus as a coupling factor with Hsp70 and plays a role in proteolytic control during stress. BAT3 has not been related to cancer or drug resistance either, but treatment of cells with drugs is accompanied by cellular stress and therefore up-regulation of stress/apoptosis factors cannot be excluded. A further cDNA isolated encoded collagen type III a-1 (COL3A1), which is a component of soft connective tissue. It has been related to carcinogenic progression such as ovarian cancer [24], breast cancer [8] and v-fos transformed NIH3T3 cells. Increased expression of COL3A1 has been suggested to play a role in tumor invasion [8]. In 2001 Tannock et al. [23] published an interesting article about the limited penetration of anticancer drugs through the tumor microenvironment. This article points to a strategy for building up drug resistance by producing extracellular matrix, but direct evidence for this hypothesis is not yet available. Clathrin heavy polypeptide (CLTC) proved to be a further gene that is overexpressed in HLaC79-clone1. Clathrin is a major protein component of the cytosolic surface of intracellular organelles, called coated vesicles and coated pits. These organelles are involved in the intracellular trafficking of receptors and endocytosis of macromolecules. It has been shown that the MDR-1
product P-glycoprotein occurs under steady state conditions for 70% on the cell surface, and for 30% in the cytoplasm. Surface P-glycoprotein undergoes constitutive endocytosis and recycling, which involves clathrin and adaptin complex [10]. For that reason, it seems plausible that clathrin polypeptides are overexpressed in the taxol-resistant clone expressing MDR-1 at a very high level. The sixth gene that showed increased expression in the taxol-resistant clone was a subtype of thymosin b, previously identified in neuroblastoma cells [31] by expression profiling. Thymosins are actin-binding proteins, widely distributed and highly conserved in nature. They have been shown to increase motility of cells, which is a critical factor in metastatic spreading (for review see [3]). Several thymosin b isotypes, especially thymosin b4 and 10, have been shown to be up-regulated in diverse cancer types such as gastric cancer [19], non small cell lung cancer [7] or thyroid cancer [2]. In colon cancer cells, thymosin b4 expression correlated with malignant progression [28] and in prostatic cancer expression of thymosin b15 was associated with metastatic potential [1]. The cDNA sequence isolated from HLaC79-clone1 was identified in a neuroblastoma cell line by compiled expression profiling. An association between the expression of thymosins and drug resistance has not been described yet, thus not delivering satisfactory explanations why HLaC79-clone1 is marked by increased thymosin b expression. In summary, it is obvious that one of the main players in taxol resistance in our cell line is the expression of MDR-1 in the descendant taxol restistant cell clone. Additional cDNAs showed a changed expression profile and therefore may contribute to the paclitaxel-resistant phenotype of the cell line. Several pathways seem to exist for cells to achieve drug resistance using different metabolic strategies, which makes it difficult to develop general therapeutic approaches in order to recover taxol sensitivity.
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