p53及neu在膀胱癌細胞抗癌藥性作用中扮演之角色

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(8) >??@ABC RS, T UV&W Abstract. . Background: Chemotherapy for the treatment of most recurrent and metastatic bladder cancer remains ineffective. One of the major causes is the development of drug resistance in tumor cells. Neu gene has been linked to chemoresistance in non-small-cell lung cancer and breast cancer. Overexpression of oncogene neu was frequently detected in bladder tumors. Purpose: The purpose of this study is to investigate the relationship between drug resistance and the expression of neu in bladder cancer cell lines and stable neu-transfectants. Methods: The exogenous neu gene was introduced into a very low neu p185 -expressing TCC-SUP bladder cancer neu cell line and several high p185 -expressing stable transfectants were established. We perfomed drug sensitivity tests on parental TCC-SUP, the control trsnafectant, and the stable transfectants. Drug sensitivity tests for methotrexate was carried out on six bladder cancer cell lines: TCC-SUP, Sca-BER, TSGH8301, BFTC-905, HT1197, and T24. Results: Increased chemoresistance to cisplatin was observed in the stable neu transfectants comparing with that of parental TCC-SUP cells. In contrast, the stable transfectants revealed high susceptibility to methotrexate. In addition, low-expression of. neu. p185 was correlated with chemoresistance to methotrexate in six bladder cancer cell neu lines. Conclusion: The expression of p185 in bladder cancer cells enhanced chemoresistance to cisplatin, but decreased chemoresistance to methotrextate. Keywords: Neu, methotrexate, bladder cancer.

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(10) E-mail: a1211207@mail.ncku.edu.tw Bladder cancer is the most common cancer of the urinary tract. The treatment of recurrent and metastatic cases remains mostly ineffective, nearly all of the patients with metastasis die from the diseases with or without chemotherapy. One of the major causes is the development of drug resistance in the tumor cells (1). The neu gene encodes a neu transmembrane protein of 185 kD (p185 ), which shares extensive sequence homology with epidermal growth factor receptor (2, 3). neu P185 contains intrinsic tyrosine kinase activity, and belongs to growth factor receptor tyrosine kinase gene family (2, 3, 4). Amplification or overexpression of neu gene has been detected in many cancers including bladder cancer (5-9). Progression of bladder cancer was associated with the overexpression of neu (7-9). The relationship between overexpression of neu and drug resistance has been investigated in non-small-cell lung cancer and breast cancer. Correlation of intrinsic chemoresistance with the level of neu p185 was first identified in non-small-cell. 1.

(11) lung cancer (10). Elevated p185neu level in neu-transfected human lung cancer cells confers chemoresistance to cisplatin, doxorubicin, mitomycin and etoposide (11). A selective neu tyrosine kinase inhibitor tryphostinAG825 can enhance chemosensitivity to cisplatin, doxorubicin, neu and etoposide in high p185 -expressing non-small-cell lung cacer cells (12). Overexpression of neu in breast cancer cells also confers increased resistance to Taxol (13).. Establishment of TCC-SUP-neu transfectants. Either pSV-neu or pSV-neu(activated) and pMam-neo (Clonetech Inc.) were cotransfected into TCC-SUP cells at the ratio of 10:1 by the calcium phosphate method as described before (16). G418-resistant transfectants were selected and isolated in the medium containing 400 µg/ml of G418.. Drug sensitivity testing. Cells were seeded on 24 well plates at the density of To determine whether overexpression 5000 cells/per well. Various concentrations of neu affects the chemosensitivity of bladder of anticancer drugs were added to the cancer cells to anticancer drugs, we created medium 12 hours after seeding. Cells were neu stable clones expressing high level of p185 . incubated with drugs further for 96 hours and Plasmids expressing normal neu or active viable cell number was determined by MTT neu (14) was delivered into a low p185neu assay as described (17). In brief, 200 µl of expressing bladder cancer cell line MTT (3-4,5 dimethylthiazol-2,5-diphenyl (TCC-SUP). The chemosensitivity to tetrazolium bromide, Sigma; 2 mg/ml) was anticancer drugs of the stable transfectants added to each well. After 4 hours of and the parental cells were compared. incubation, the MTT medium was removed and 600 µl of dimethyl sulfoxide per well was added to dissolve the formazan precipitate. The optical density was determined by absorbance spectrometry at 540 nm. Three separate experiments were Cell lines, plasmids, cytotoxic drugs, performed with triplicate data for each drug antibodies. Sca-BER, TCC-SUP, HT1376, concentration. HT1197, T24, 5637, and J82 cell lines were Western blotting. Total cell lysate was obtained from the American Type Culture prepared by direct adding 150 µl of 2x SDS Collection (Rockville, MD). BFTC-905 and loading buffer to confluent cells on 10cm TSGH8301 cell lines were obtained from Dr. dish, and detaching the cells with Ching-Cherng Tzeng as described before rubberpoliceman. Protein concentration was (15). All cell lines were cultured in determinhed by the Bio-Rad protein assay. Dulbecco’s modified Eagles medium with 10% fetal calf serum in a humidified Twenty five µg of total cell lysates was separated by electrophoresis on a 8% SDS atmosphere containing 5% CO2 and 95% air o polyacrylamide minigel. The proteins were C. Plasmids for the expression of at 37 electro-transfered onto a nitrocellulose filter normal neu and activated neu were generous at 100 mA for 2 hr by using a TE70 semidry gifts from Dr. Mien-Chie Hung, M. D. Anderson Hospital (14). The activated neu transfer unit (Hoefer, Pharmacia Biotech.). The nitrocellulose filter was preblocked with gene contains a point mutation in the 5% skim milk (Difco Laboratories, Inc., transmembrane domain, leading to Detroit, MI) for 1 hr and probed with primary constitutive activation of kinase activity. Methotrexate, adriamycin, and cisplatin were antibody for 2 hr. The protein bands were visualized with an enhanced purchased from Sigma. The antibody used chemilumnescence detection kit (Amersham for this study was HER2/neu (Ab-3) from Corp. Arlington Heights, IL) using Oncogene Science. 2.

(12) horseradish peroxidase-labeled secondary antibody as suggested by the manufacturer. Equal loading of cell lysates was confirmed by staining the nitrocellulose membrane with amido black after western blotting analysis.. 2A and 2B). In contrast to the responses to adriamycin and cisplatin, the neu transfectants were more sensitive to methotrextae than the control transfectants and parental TCC-SUP cells (Figure 2C).. Establishment of TCC-SUP-neu transfectants. The level of p185neu in nine bladder cancer cell lines was determined by western blotting (Fig. 1A). The TCC-SUP bladder carcinoma cell line expressed the neu lowest level of p185 in these bladder cell lines, and was used as parental cells to express exogenous neu gene. We obtained three stable transfectants (TCC-SUP-N5, TCC-SUP-N10, and TCC-SUP-N14) neu showing high expression of p185 . As neu point-mutated active p185 has more neu transforming ability than normal p185 protein (18), plasmid expressing the active neu gene was also delivered into TCC-SUP cells. Two stable transfectants for activated neu (TCC-SUP-NA1, TCC-SUP-NA2) were neu established. Overexpression of p185 was observed in these five neu-transfectants but not in the control transfectant with pMam-neo (Figure 1B). The anchorage-independent growth of these transfectants was determined in vitro by soft agar assay. The ability of these cells to grow in soft agar showed an order of: TCC-SUP active neu-transfectants > TCC-SUP normal neu-transfectants > control transfectants and TCC-SUP cells (data not shown). These results indicated that overexpression of either normal neu or activated neu in bladder cancer cells further enhanced the transforming ability.. Drug sensitivity of methotrexate in six bladder carcinoma cell lines with wild-type p53 status. We investigated neu whether the expression of p185 is associated with the chemosensitivity to methotrextate in cells with different genetic backgrounds. P53 can affect the chemosensitivity to many chemotherapeutic drugs in many cell types (19-23). To exclude the effects of p53, we chose six bladder cancer cell lines with wild-type p53 status: TCC-SUP, Sca-BER, TSGH8301, BFTC-905, HT1197, and T24 cells (15, 24, 25). TCC-SUP and HT-1197 cell lines with lower neu expression of p185 were much more resistant to methotrexate-induced growth inhibition than the other four cell lines. The results suggested that the expression of neu p185 was correlated with the chemosensitivity to methotrexate in bladder cancer cells even under various genetic backgrounds. Some genetic alterations may affect the responses of tumor cells to chemotherapeutic drugs. Among these alterations, p53 and neu were shown to confer some tumor cells the resistance to many chemotherapeutic drugs. Previous reports indicated that neu overexpression of p185 can confer chemoresistance to several chemotherapeutic drugs in non-small-cell lung cancer and breast cancer (10-13). The expression of neu p185 can only wealkly enhance chemoresistance to cisplatin in bladder cancer cells (Fig. 2). Enhanced chemoresistance to adriamycin by the neu expression of p185 was not observed in our study because the control transfectant showed similar chemoresistance to adriamycin as the neu-transfectants (Fig. 2). In this report, however, we demonstrated that neu the expression of exogenous p185 could confer chemosensitivity to methotrexate in. Drug sensitivity of TCC-SUP and TCC-SUP-neu transfectants. The chemosensitivity of the neu-transfectants and the parental cells to cisplatin, adriamycin, and methotrexate was determined by MTT assay. The neu-transfectants were more resistant to adriamycin and cisplatin than the parental TCC-SUP cells. The control transfectant showed similar chemoresistance to adriamycin as the neu-transfectants (Fig. 3.

(13). . TCC-SUP bladder cancer cells. Correlation neu of the expression of p185 with the chemosensitivity to methotrexate was also observed in six bladder cancer cell lines. These results indicated that expression of neu p185 may have variable responses to different chemotherapeutic drugs in bladder cancer cells. If the clinical relevance of our finding is confirmed, methotrexate may be the drug of choice for treating high neu-expressing bladder cancer. The effects of expression of neu on the chemosensitivity to methotrexate in breast cancer cell lines or other types of cancer cell lines have not been determined yet. It will be of therapeutic interests to study the effects of expression of neu on the response to methotrexate in these types of cells.. 1.. Yagoda A: Chemotherapy of urothelial tract tumors. Cancer 60:574-585, 1987. 2. Bargmann CI, Hung MC, Weinberg RA: The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature 319:226-228, 1986. 3. Hung MC, Schechter AL, Chevray PYM, Stern DF, Weinberg RA: Molecular cloning of the neu gene. Proc Natl Acad Sci 83:261-265, 1986. 4. Akiyama T, Sudo C, Ogawara H, Toyoshima K, Yamamoto T: The product of the human c-erbB2-2 gene: a 185-kilodalton glycoprotein with tyrosine kinase activity. Science 232:1644-1646, 1986. 5. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, et al: Studies of the HER2/neu proto-oncogene in human breast cancer and ovarian cancer. Science 244:707-712. 1989. 6. Kameda T, Yasui W, Yoshida K, Tsujino T, Nakayama H, Ito M, et al: Expression of ERBB2 in human gastric carcinomas: relationship between p185ERBB2 expression and the gene amplification. Cancer Res 50:8002-8009, 1990. 7. Lunec J, Challen C, Wright C, Mellon K, Neal DE: C-erbB2 amplification and identical p53 mutations in concomitant transitional carcinomas of renal pelvis and urinary bladder. Lancet 339:439-440, 1992. 8. Coombs LM, Pigott DA, Sweeny E, Proctor AJ, Eydmann ME, Parkinson C, et al: Amplification and over-expression of c-erbB2 in transitional cell carcinoma of the urinary bladder. Br J Cancer 63:601-608, 1991. 9. Wright C, Mellon K, Chang KT, Wu LH, Neal DE, et al: Expression of c-erbB2 protein product in bladder cancer. Br J Cancer 62:764-765, 1990. 10. Tsai CM, Chang KT, Wu LH, Chen JY., Gazdar AF, Mitsudomi T, et al: Correlation of intrinsic chemoresistance of non-small cell lung cancer cell lines. The mechanism of chemosensitivity to neu methotrexate induced by p185 is currently unknown. As one of major cellular targets for methotrexate is dihydrofolate reductase, altering expression of dihydrofolate reductase may affect the biological response of methotrexate. However, we did not detect any significant difference in the expression of dihydrofolate reductase between the neu-transfectants, the control transfectant, and parental TCC-SUP cells by reverse-transcription polymerase chain reaction (data not shown). Though the active-neu transfectants (TCC-SUP-NA1 and -NA2) had higher anchorage-independent growth in soft agar, the active-neu transfectants were not more sensitive to methotrexate than the normal neu-transfectants (Fig. 2C). This suggests neu that the ability of p185 to confer chemosensitivity for methotrexate is not directly related to its transforming ability in bladder cancer cells. neu P185 is the prefered heterodimerization partner of all ErbB receptors and has a role in the lateral transmission of signals between Erb receptors (26). The interactions between neu ErbB-1, p185 , ErbB-3, and ErbB-4 is probably important for regulation of neu-induced drug resistance or drug sensitivity in bladder cancer cells. 4.

(14) 11.. 12.. 13.. 14.. 15.. 16.. 17.. 18.. 19.. with HER2/neu gene expression but not with ras mutations. J Natl Cancer Inst 85:897-901, 1993. Tsai CM, Yu D, Chang KT, Wu LH, Perng RP, Ibrahim NK, et al: Enhanced chemoresistance by elevation of the neu levels of p185 in the HER2/neu transfected human lung cancer cells. J Natl Cancer Inst 87:682-684, 1995. Tsai CM, Levitzki A, Wu L-H, Chang K-T, Chang C-C., Cheng C-C, et al: Enhancement of chemosensitivity by neu tryphostin AG825 in highp185 expressing non-small lung cancer cells. Cancer Res 56:1068-1074, 1996. Yu D, Liu B, Tan M, Li J, Wang S-S, Hung MC: Overexpression of c-erbB2/neu in breast cancer cells confers increased resistance to taxol via mdr-1-independent mechanisms. Oncogene 13:1359-1365, 1996. Yu D, Hung M-C: Expression of activated rat neu oncogene is sufficient to induce experimental metastasis in 3T3 cells. Oncogene 6: 1991-1996, 1991. Cheng Y-T, Li Y-L, Wu JD, Long S-B, Tzai T-S, Tzeng C-C, et al: Overexpression of MDM-2 mRNA and mutation of the p53 tumor suppressor gene in bladder carcinoma cell lines. Mol. Carcinogen 13:173-181, 1995. Shaw Y-T, Chang S-H, Chiou S-T, Chang W-C, Lai M-D: Partial reversion of transformed phenotype of B104 cancer cells by antisense nucleic acids. Cancer Letters 69:27-32, 1993. Carmichael J, Degraff WG, Gazdar AF, Minna JD, Mitchell JB: Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 47:936-942, 1987. Bargmann CI, Hung MC, Weinberg RA: Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell 45:649-657, 1986. Shelling AN: Role of p53 in drug resistance in ovarian cancer. Lancet 349:744-745, 1997.. 20. Benhatter J, Cerottini JP, Saraga E, Metthez G, Givel JC: p53 mutations as a possible predictor of response to chemotherapy in metastatic colorectal carcinomas. Int J Cancer 69:190-192, 1996. 21. Lowe SW, Ruley HE, Jacks T, Housman DE: p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74:957-967, 1993. 22. El Rouby S, Thomas A, Costin D, Rosenberg CR, Potmesil M, Silber R, et al: P53 mutations in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression. Blood 82:3452-3459, 1993. 23. Lowe SW, Bodis S, Mcclatchey A, Remington L, Ruley HE, Fisher DE, et al: p53 status and the efficacy of cancer therapy in vivo. Science 266:807-810, 1994. 24. Rieger KM, Little AF, Swart JM, Kastrinakis WV, Fitzgerald JM, Hess DT, et al: Human bladder carcinoma cell lines as indicators of oncogenic change relevant to urothelial neoplastic progression. Br J Cancer 72:683-690, 1995. 25. Haapajarvi T, Kivinen L, Pitkanen K, Laiho M: Cell cycle dependent effects of u.v.-radiation on p53 expression and retinoblastoma protein phosphorylation. Oncogene 11:151-159, 1995. 26. Graus-Porta D, Beerli RR, Daly JM, Hynes NE: ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J 16:1647-1655, 1997.. . 5.

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