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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Children's Cancer Institute Australia for Medical Research, Randwick, New South Wales, Australia and 2 University of New South Wales, Sydney, New South Wales, Australia
Requests for reprints: Maria Kavallaris, Children's Cancer Institute Australia for Medical Research, Randwick, 2031 New South Wales, Australia. Phone: 61-29382-1823; Fax: 61-29382-1850; E-mail: m.kavallaris{at}ccia.unsw.edu.au.
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Abstract |
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/ß-tubulin, inducing mitotic arrest and apoptosis. Up-regulation of class III ß-tubulin (ßIII-tubulin) has been implicated in clinical resistance in NSCLC, ovarian and breast tumors treated in combination with a TBA and DNA-damaging agent. To investigate the functional significance of ßIII-tubulin in resistance to both these classes of agents, small interfering RNA (siRNA) was used to silence the expression of this isotype in two NSCLC cell lines, NCI-H460 and Calu-6. Reverse transcription-PCR and immunoblotting showed that ßIII-siRNA potently inhibited the expression of ßIII-tubulin, without affecting the expression of other major ß-tubulin isotypes. Clonogenic assays showed that ßIII-siRNA cells were significantly more sensitive to TBAs, paclitaxel, vincristine, and vinorelbine, and for the first time, DNA-damaging agents, cisplatin, doxorubicin, and etoposide compared with controls. Cell cycle analysis of H460 ßIII-siRNA cells showed reduced accumulation at the G2-M boundary and an increase in the sub-G1 population in response to TBA treatment compared with control cells. Importantly, ßIII-siRNA cells displayed a significant dose-dependent increase in Annexin V staining when treated with either paclitaxel or cisplatin, compared with controls. These findings have revealed a novel role for ßIII-tubulin in mediating response to both TBA and DNA-damaging agent therapy and may have important implications for improving the targeting and treatment of drug-refractory NSCLC. [Cancer Res 2007;67(19):9356–63] |
Introduction |
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TBAs such as taxanes and Vinca alkaloids constitute the most important group of chemotherapeutic agents, with activity against a broad range of solid tumors and hematologic malignancies. These agents target the ß-tubulin subunit of the /ß-tubulin heterodimers that assemble to form microtubules. Microtubules are dynamic polymers that play a key role in many fundamentally important cellular processes such as vesicular transport, maintenance of cell shape, and cell division. In mammals, there are at least six - and seven ß-tubulin isotypes, encoded by multiple genes that display differential tissue expression. Selection of cancer cells for resistance to TBAs could lead to alterations in ß-tubulin isotype composition. In particular, increased expression of the neuronal specific class III ß-tubulin (ßIII-tubulin) has been frequently implicated in drug-resistant cancer cell lines (5, 6). Importantly, there is mounting clinical evidence that ßIII-tubulin expression is involved in the resistance to taxanes and Vinca alkaloids in lung (7–9), breast (10, 11), and ovarian cancers (12–14). In addition, in nonneuronal cancers such as NSCLC, the expression of ßIII-tubulin is associated with poorly differentiated tumor tissue and high-grade malignancy, as well as increased metastatic potential (15). Despite strong correlational evidence in preclinical and clinical studies, its role in drug response is far from clear. Stable overexpression of ßIII-tubulin in human prostate cells failed to confer resistance to TBAs (16–18). In contrast, transfection of ßIII-tubulin into Chinese hamster ovary (CHO) cells confers low-level resistance to paclitaxel (19). A recent study showed that CHO cells exogenously overexpressing ßIII-tubulin had reduced suppression of microtubule dynamics in the presence of paclitaxel compared with control cells (20). We have previously shown that antisense oligonucleotides targeted against the ßIII-tubulin isotype partially reversed paclitaxel resistance in a NSCLC cell line, A549-T12 (21). To date, the available functional data largely focuses on the role of ßIII-tubulin in paclitaxel resistance, and the relevance of this isotype in resistance to other TBAs is not known. In addition, despite the common use of cisplatin and related agents in combination therapy with TBAs in NSCLC, the role of ßIII-tubulin in response to these agents has not been addressed.
To identify the functional role of ßIII-tubulin in response to chemotherapy in NSCLC, we employed the RNA interference technique to knock down ßIII-tubulin expression in two independent lung cancer cell lines that express this isotype. Knockdown of ßIII-tubulin corresponded with increased sensitivity to TBAs. Interestingly, the knockdown cells also exhibited increased sensitivity to DNA-damaging agents that are structurally and functionally unrelated to TBAs. This report shows that ßIII-tubulin expression could mediate the response to microtubule-stabilizing and -destabilizing agents, and for the first time, DNA-damaging agents in NSCLC.
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Materials and Methods |
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The ßIII small interfering RNA (siRNA) SMARTpool reagent was obtained from Dharmacon Research, Inc. The nonsilencing control siRNA, which has no sequence homology to any known human gene sequence, was used as a negative control in all experiments (Qiagen). Cells were transfected with ßIII siRNA at a final concentration of 25 nmol/L (Calu-6) and 100 nmol/L (H460) using LipofectAMINE 2000 (Invitrogen) following the manufacturer's instructions. The optimal amount of siRNA used for transfection was determined empirically for each cell line and the lowest siRNA concentration that gave effective silencing of the targeted protein and caused minimal stress to the cells was used in all subsequent experiments. Control experiments were done in parallel by transfecting the cells with a nonsilencing control siRNA (Qiagen) at equivalent concentrations as the target siRNA. All experiments involved the use of a LipofectAMINE-only control (mock-transfected) and a nonsilencing control siRNA (negative control).
Cytotoxic drugs. Cytotoxic drugs were obtained as previously described (22). Paclitaxel (Calbiochem, Merck Biosciences) was prepared at a stock concentration of 2 mmol/L in DMSO. Vincristine (Sigma-Aldrich) was prepared at a stock concentration of 2 mmol/L in saline (0.9% wt/vol NaCl); vinorelbine (kindly provided by Dr. B. Hill, Division of Experimental Cancer Research, Pierre Fabre Research Centre, Castres, France) was solubilized in water at a stock concentration of 2 mmol/L. Doxorubucin (doxorubicin hydrochloride; Pfizer) was prepared at a stock concentration of 3.45 mmol/L in saline. Etoposide (Sigma-Aldrich) was prepared at a stock concentration of 68 mmol/L in DMSO and cisplatin (Pharmacia) was prepared at a stock concentration of 3.3 mmol/L in saline.
Reverse transcription-PCR. Total RNA was extracted from the transfected cells using the Trizol reagent (Invitrogen), according to the manufacturer's instructions. RNA samples were DNase-treated, reverse-transcribed for reverse transcription-PCR (RT-PCR) analysis using methodology and specific primers as described in detail previously (12). ß2-Microglobulin was used as an internal control.
Western blot analysis. Preparation of protein lysates as well as Western blot analyses were done as described previously (23), using antibodies directed against ßI-tubulin (clone SAP 4G5; Abcam, Ltd.), ßII-tubulin (clone 7B9; Chemicon), ßIII-tubulin (clone TUJ1, Chemicon), ßIV-tubulin (clone ONS 1A6, Sigma-Aldrich), and glyceraldehyde-3-phosphate dehydrogenase (Abcam Ltd.). Enhanced chemiluminescence (GE Healthcare) was used for detection. The blots were scanned using the Typhoon scanner and quantified using ImageQuant software version 5.2 (Molecular Dynamics, Inc.).
Immunofluorescence staining. Briefly, siRNA-transfected cells were plated in glass chamber slides and allowed to reach 70% confluence. Cells were then treated with 10 nmol/L of paclitaxel and vincristine for 1 h. Drug was then removed by washing cells with PBS. Immunofluorescence staining was then done as described previously (23). For dual staining, cells were first stained with ßIII-tubulin followed by Cy3 anti-mouse fluorescent-tagged antibody (GE Healthcare). This was then followed by staining with -tubulin and Cy2 anti-mouse fluorescent-tagged antibody (GE Healthcare). Slides were mounted on a coverslip using DAPI II Counterstain (Vysis, Inc.). Immunofluorescence microscopy was done using a Zeiss Axioplan 2 Microscope (Zeiss), and images were captured using a Sensicam Charged Coupled Device camera (PCO Imaging) and the Image-Pro Plus 4.1 software (Media Cybernetics, L.P.).
Clonogenic assays. Twenty-four hours after siRNA transfection, cells were harvested and plated into six-well plates for 6 h, prior to the addition of various drugs as indicated in the figure legends. After 72 h of incubation, the drug-containing medium was removed and replaced with complete growth medium. Medium was changed every 3 days for 7 to 10 days until visible colonies formed. Colonies were simultaneously fixed and stained with 0.5% crystal violet in methanol, and manually counted. Individual stained colonies in each well were counted and the surviving fraction was calculated as follows: colony number / (number of cells seeded x plating efficiency), where plating efficiency is equivalent to the colony number divided by the number of cells seeded in the drug-free medium.
Drug accumulation assays. Cellular uptake and retention of tritiated substrate [3H]paclitaxel and [3H]vincristine were measured as previously described (22). Briefly, cells were transfected in 12-well plates for 48 h. Drug uptake was monitored by adding [3H]paclitaxel (14.7 Ci/mmol; final concentration, 50 nmol/L; Moravek Biochemicals, Inc.) or [3H]vincristine (7.1 Ci/mmol; final concentration of 12.5 nmol/L; GE Healthcare) to the transfected cells for 2 h at 37°C. Cells were washed, hydrolyzed, and counted as previously described. The amount of tritiated drug accumulated in the cells was determined for duplicate samples and expressed as picomoles of the drug per milligram of protein. At least three independent experiments were done. Relevant positive controls were included, together with vincristine-resistant neuroblastoma (BE/VCR10) and etoposide-resistant breast cancer cells (MCF7-VP16), which have previously been shown to overexpress multidrug resistance 1 (MDR1) and multidrug resistance–associated protein 1 (MRP1), respectively (23, 24).
Cell cycle analysis by flow cytometry. Cell cycle analysis was determined by transfecting H460 cells with siRNA for 72 h and harvesting (adherent and suspension) cells 24 h after drug treatment. DNA content was stained for 15 min at 37°C with a solution containing 0.4% Triton X-100 (Sigma-Aldrich), 50 µg/mL of propidium iodide (Sigma-Aldrich), and 2 µg/mL of DNase-free RNase (Roche). The cells were then analyzed for cell cycle perturbation using a FACSCalibur (Becton Dickinson). The CellQuest program was used to quantitate the distribution of cells in each cell cycle phase: sub-G1 (apoptotic cells), G1, S, and G2-M.
Detection of apoptosis by Annexin V-FITC staining. Apoptosis induction was determined by transfecting H460 cells with siRNA for 72 h and harvesting (adherent and suspension) cells 48 h after drug treatment, as previously described with slight modifications (25). Briefly, 1 x 105 cells were incubated with Annexin V-FITC and propidium iodide for 15 min in the dark (Becton Dickinson), immediately followed by flow cytometry using a FACSCalibur (Becton Dickinson). Cytogram analysis was done using Cell Quest software.
Statistical analysis. A two-tailed Student's t test was used for statistical analysis of comparative data using the GraphPad Prism program. Data were expressed as means of at least three independent experiments ± SE, with P < 0.05 considered statistically significant.
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Results |
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To examine the effect of TBAs on ßIII-tubulin siRNA-treated cells, cells were exposed for 1 h to either 10 nmol/L of paclitaxel or vincristine, and cellular morphology was examined. As shown in Fig. 2 , ßIII siRNA-transfected Calu-6 cells showed extensive disruption of the microtubule cytoskeleton as compared with the control-treated cells. The majority of the ßIII siRNA-treated cells were rounded and many of these cells displayed abnormal cellular or nuclear morphology (Fig. 2, arrows). The control-treated cells occasionally showed minimal bundling of microtubules normally associated with paclitaxel treatment but the frequency of rounded cells and the extent of microtubule disruption was minimal compared with ßIII transfectants.
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Knockdown of ßIII-tubulin abrogates paclitaxel- and vincristine-induced G2-M arrest and induces an increase in sub-G1 population. To determine whether ßIII-tubulin silencing affects the cell cycle profiles, cell cycle analysis using flow cytometry was done. The cell cycle profiles of H460 cells were not affected by ßIII-tubulin silencing (Fig. 4 ). To determine whether the TBAs affected the cell cycle profiles of ßIII-tubulin siRNA transfectants, cells were treated with either paclitaxel or vincristine. Following 24 h of incubation with 5 nmol/L of paclitaxel, the ßIII-tubulin–silenced cells had a higher sub-G1 content (apoptotic cells) compared with the control siRNA-treated cells (P < 0.05; Fig. 4A), although both the ßIII-tubulin siRNA and control siRNA-treated cells had a similar increase in G2-M content compared with untreated samples. A major difference was observed with 40 nmol/L of paclitaxel, with the control siRNA-treated cells showing a marked G2-M block whereas the ßIII-tubulin siRNA-treated cells had a marked increase in the sub-G1 population reflective of apoptotic cells (Fig. 4A). Similar results were observed when siRNA-treated cells were exposed to vincristine (Fig. 4B), suggesting that there is a common mechanism that enhances the effects of both the taxanes and Vinca alkaloids following ßIII-tubulin silencing.
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Discussion |
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One approach to identifying the role of specific ß-tubulin isotypes in drug resistance is to overexpress these proteins in cell lines. However, altering the expression of specific tubulin isotypes is far from straightforward due to the complexity of tubulin autoregulation (28). As previously noted, overexpression of ßIII-tubulin in human prostate carcinoma cells failed to confer resistance to a range of TBAs including colchicine, estramustine, paclitaxel, and vinblastine (18). Of particular note, class II and IVb tubulins were also increased collaterally in these transfected cells (18). The reasons for this nonspecific regulation were unclear, but as suggested by the investigators, might possibly be due to a compensatory mechanism that overcomes the effects of the elevated ßIII levels. Expression of ß-tubulin isotypes is tightly regulated by an autoregulatory mechanism through cotranslational degradation of ß-tubulin mRNAs in response to an increase in the level of soluble tubulin (28, 29). Hence, it is of particular importance to analyze the total isotype profile to confirm the contribution of the target isotype in drug response studies. Importantly, the siRNA used in this study resulted in specific down-regulation of the ßIII-tubulin expression with no compensatory changes observed with other ß-tubulin isotypes examined, further strengthening the role of ßIII-tubulin in contributing to chemosensitivity. In addition, the cell lines used in this study have not been subjected to prior drug selection, and are more likely to represent intrinsic drug resistance observed in lung carcinoma.
We have previously shown that blocking ßIII-tubulin expression in drug-selected NSCLC cells by antisense oligonucleotides partially restored paclitaxel sensitivity in these paclitaxel-resistant cells (21). The results presented here using ßIII-tubulin–specific siRNA supports our previous study with regard to the role of ßIII-tubulin in paclitaxel response. The finding that ßIII-tubulin silencing sensitizes NSCLC cells to Vinca alkaloids is novel. At their lowest effective concentrations, TBAs suppress microtubule dynamics in cancer cells, leading to mitotic arrest and subsequent cell death, without changing the polymer mass (30). Given the crucial role of microtubule dynamics in the pharmacologic action of these drugs, one mechanism by which ßIII-tubulin has been thought to mediate resistance to TBAs is to constitutively increase microtubule dynamics (31, 32). However, a recent study showed that inducible stable overexpression of ßIII-tubulin in CHO cells did not significantly alter any variables of microtubule dynamic stability, but reduced the ability of paclitaxel to suppress the microtubule dynamics in these cells (20). It remains to be determined whether down-regulation of ßIII-tubulin in NSCLC cells plays a dominant role in modulating microtubule dynamics in a way that could enhance or reduce the effectiveness of TBAs.
Of particular interest is the novel finding that ßIII-tubulin silencing significantly increases sensitivity to DNA-damaging agents. Although these agents are not known to directly target the tubulin/microtubule system, the results are perhaps not surprising due to frequent overexpression of ßIII-tubulin in drug-refractory tumors which are commonly treated with a TBA in combination with a DNA-damaging agent (8, 13). Ferrandina et al. found that ßIII-tubulin serves as a potent prognostic marker for patients with ovarian cancer, regardless of whether the chemotherapeutic regimens include a taxane (14). The ßIII-tubulin–silenced NSCLC cells examined in the current study exhibited a significant increase in apoptosis induction when exposed to paclitaxel or cisplatin as revealed by Annexin-V staining. The mechanistic basis for the increased sensitivity is not clear, however, one possibility is that ßIII-tubulin may serve as a survival factor to rescue tumor cells from death signals triggered by chemotherapeutic agents. In support of this hypothesis, a pool of tubulin has been shown to localize in the mitochondria membranes where it is associated with voltage-dependent anion channels (33). Interestingly, this pool of mitochondrial tubulin is enriched with ßIII-tubulin, suggesting that this tubulin isotype may be involved in mitochondrial-mediated cell death. Therefore, we propose that altered expression of ßIII-tubulin isotypes may provide a cellular defense not only against TBAs but also against diverse classes of DNA-targeting drugs.
TBAs are known to promote cell death by inducing a potent mitotic block causing cells to accumulate at the G2-M phase of the cell cycle (34). In the present study, the extent of the mitotic block induced by incubation with either paclitaxel or vincristine seemed to be markedly reduced in ßIII-tubulin siRNA-treated cells as compared with control siRNA-transfected cells. In contrast, the level of apoptosis induction was significantly higher in ßIII-tubulin–depleted cells. These results suggest that suppression of ßIII-tubulin may enhance TBA-induced apoptotic cell death via a separate pathway that is independent of classical mitotic arrest. Although mitotic arrest is a hallmark cellular response to TBAs, Milross et al. showed that the antitumor efficacy of paclitaxel is dependent on its ability to induce apoptosis, but not mitotic arrest (35). Previous studies with NSCLC A549 cells also found that low concentrations of paclitaxel are sufficient to induce cell death without an apparent G2-M block (36, 37). The exact mechanism is not fully understood, although aberrant mitosis and p53 induction have been suggested as possible mechanisms (36, 38).
Thus, the sensitization effects of ßIII down-regulation on apoptosis induced by TBAs may have broad significance and may also explain, in part, its hypersensitivity to DNA-damaging agents. In fact, similar results were obtained when treating the cells with cisplatin in which ßIII knockdown abrogated cisplatin-induced G2-M arrest but showed an increase in the sub-G1 populations (data not shown). So why does repression of mitotic block result in apoptotic cell death in ßIII-tubulin–depleted cells following drug treatment? One possibility is that decreasing ßIII-tubulin levels may change the expression of microtubule-associated or interacting proteins. We addressed this possibility by examining for altered expression of two important regulators of microtubule function after ßIII-tubulin knockdown, microtubule-associated protein-4 and stathmin, and found no significant change (data not shown). We cannot exclude the possibility that changes in other components of the cytoskeleton are affected. Potentially, ßIII-tubulin may serve as a survival factor to rescue tumor cells from death signals triggered by chemotherapeutic agents. Therefore, by knocking down this survival factor, cells are hypersensitive to drug-induced cell death. The mechanisms that enhance drug-induced cell death after suppressing ßIII-tubulin expression are currently under active investigation.
Because TBAs and DNA-damaging agents have distinct cellular targets, it is also possible that ßIII-tubulin may enhance the drug action of these agents via activation of signaling pathways shared by both drugs or those specific to each drug. One potential explanation for the novel enhancement of drug sensitivity by down-regulation of ßIII-tubulin in NSCLC cells is through regulation of gene expression, e.g., for genes that are involved in apoptosis, DNA repair pathway, or mitosis.
The finding that ßIII-tubulin is involved in the sensitivity of both taxanes and Vinca alkaloids highlights a common effect of this isotype on microtubules and the action of TBAs. Importantly, ßIII-tubulin seems to play a broader role in chemosensitivity as shown by its effects on DNA-damaging agent response. These results have mapped out new challenges and opportunities for circumventing drug resistance to improve the efficacy of current chemotherapeutic agents in the treatment of cancer.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Footnotes |
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Received 2/ 7/07. Revised 5/17/07. Accepted 7/18/07.
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References |
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-actin and tubulin-targeted drug resistance in childhood leukemia. J Natl Cancer Inst 2006;98:1363–74.This article has been cited by other articles:
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  C. Dumontet, M. A. Jordan, and F. F.Y. Lee Ixabepilone: targeting {beta}III-tubulin expression in taxane-resistant malignancies Mol. Cancer Ther., January 1, 2009; 8(1): 17 - 25. [Abstract] [Full Text] [PDF]
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 S. Mozzetti, R. Iantomasi, I. De Maria, S. Prislei, M. Mariani, A. Camperchioli, S. Bartollino, D. Gallo, G. Scambia, and C. Ferlini Molecular Mechanisms of Patupilone Resistance Cancer Res., December 15, 2008; 68(24): 10197 - 10204. [Abstract] [Full Text] [PDF]
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P. P. Gan and M. Kavallaris Tubulin-Targeted Drug Action: Functional Significance of Class II and Class IVb {beta}-Tubulin in Vinca Alkaloid Sensitivity Cancer Res., December 1, 2008; 68(23): 9817 - 9824. [Abstract] [Full Text] [PDF]
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A. L. Risinger, E. M. Jackson, L. A. Polin, G. L. Helms, D. A. LeBoeuf, P. A. Joe, E. Hopper-Borge, R. F. Luduena, G. D. Kruh, and S. L. Mooberry The Taccalonolides: Microtubule Stabilizers That Circumvent Clinically Relevant Taxane Resistance Mechanisms Cancer Res., November 1, 2008; 68(21): 8881 - 8888. [Abstract] [Full Text] [PDF]
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 C. M. Galmarini, I. Treilleux, F. Cardoso, C. Bernard-Marty, V. Durbecq, D. Gancberg, M.-C. Bissery, M. Paesmans, D. Larsimont, M. J. Piccart, et al. Class III {beta}-Tubulin Isotype Predicts Response in Advanced Breast Cancer Patients Randomly Treated Either with Single-Agent Doxorubicin or Docetaxel Clin. Cancer Res., July 15, 2008; 14(14): 4511 - 4516. [Abstract] [Full Text] [PDF]
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L. Cicchillitti, R. Penci, M. Di Michele, F. Filippetti, D. Rotilio, M. B. Donati, G. Scambia, and C. Ferlini Proteomic characterization of cytoskeletal and mitochondrial class III {beta}-tubulin Mol. Cancer Ther., July 1, 2008; 7(7): 2070 - 2079. [Abstract] [Full Text] [PDF]
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S. Prislei, S. Mozzetti, F. Filippetti, M. De Donato, G. Raspaglio, L. Cicchillitti, G. Scambia, and C. Ferlini From plasma membrane to cytoskeleton: a novel function for semaphorin 6A Mol. Cancer Ther., January 1, 2008; 7(1): 233 - 241. [Abstract] [Full Text] [PDF]
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), control siRNA- (
), and ßIII-transfected cells (- - -) as described in Materials and Methods. Clonogenic survival, expressed as surviving fractions, for treatment with TBAs, such as paclitaxel (A) and vincristine (B), and for treatment with DNA-damaging agents (C), such as cisplatin (top), doxorubicin (middle), and etoposide (VP-16; bottom). Points, means of at least four individual assays; bars, SE. Statistics were calculated by comparing the surviving fraction of the siRNA-treated cells with the mock-transfected cells at each drug concentration. *, P < 0.05; **, P < 0.005; ***, P < 0.005.
