MDRl ribozyme gene mediated by retrovirus *

VOI. 40 NO. 2 SCIENCE IN CHINA (Series C) ~pri~ 1997 Selective reversal of drug resistance in drug-resistant lung adenocarcinoma cells by tumor-specific expression of MDRl ribozyme gene mediated by retrovirus * GAO Zhenqiang (I@%$&), GAO Zhiping (g&%), LIU ~ifu(2qgg) and ZHANG Tao (3# %) (Department of Pathology, Beijing Medical University, Beijing 100083, China) Received September 3,1996 Abstract According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (SEAMR) was constructed which carried the CEA promoter coupled to MDRl ribozyme gene. pceamr was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pceamr and drug resistance in the infected cells were analyzed in vitro and in vim ; pceamr expressed only in CEA-producing GAOK cells and not in non-cea-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5 % in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDRl ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effective treatment method for patients with CEA-producing lung cancers which was usually refractory to conventional chemotherapy. Keywords: noma, gene therapy. tumor-specific expression vector, MDRl ribozyme, drug resistance, human lung adenocarci- Lung adenocarcinorna, accounting for 50 % of all lung cancers, is one of the common malignant tumors, and often has resistance to chemotherapy, which is a main obstacle in clinical treatment[']. Overexpression of P-glycoprotein ( Pgp ), the product of multidrug resistance gene (MDRl), is the main mechanism for multidrug resistance"]. In clinical studies, overexpression of MDRl gene was found in about 50 % of untreated NSCLCs and in more than 50% of treated NSCLCs. This finding suggests that overexpression of MDRl gene and Pgp is closely correlative with the multidrug resistance in lung cancersl2]. It is the key to inhibiting MDRl/Pgp expression and overcoming drug resistance for treatment of lung cancers. Ribozyme is a kind of RNA molecules which possess the activity of restriction enzyme and recognizing the GUX sequence (X was one of A, C, U) and cleaving it, furthermore blocking protein synthesis[31. We previously reported that MDRl ribozyme retrovirus (pmr) could reverse the multidrug resistance in multidrug-resistant lung cancer cells GAOK in ~itro'~]. Because of the expressing without tumorspecificity, pmr vector also inhibited the MDRl expression, which could protect the normal cells from harmful substances such as carcinogen, in normal tracheal epithelial cells'']. Thus, in clinical treatment it is very important to reverse selectively the drug resistance of lung cancers without affecting the MDRl expression in normal epithelial cells. CEA has been used as a tumor marker of various adenocarcinomas. About 50 % of lung cancers, and about 90 % of lung adenocarcin~mas[~] are serologically positive to CEA. CEA overexpressed in lung adenocarcinoma cells but did not

No. 2 SELECTIVE REVERSAL OF DRUG RESISTANCE BY MDRl RIBOZYME 123 express in normal tracheal or lung cells. If the expression of MDRl ribozyme was regulated by CEA promoter, MDRl ribozyme could express only in CEA-expressing lung adenocarcinoma cells and specifically reversed the drug resistance, and had no effect on MDRl expression in normal lung cells. In the present study, MDRl ribozyme gene was linked with CEA promoter and the retroviral vector (pceamr) was constructed; then pceamr was introduced into CEA-expressing human lung adenocarcinoma cells as well as non-cea-expressing cancer cells. The drug resistance of the infected cells was measured in vitro and in vivo. 1 Materials and methods 1.1 Cell lines GAOK multidrug-resistant human lung adenocarcinoma cell line was induced by doxorubicin (DOX) and had crossresistance to vinblastine and colchicine; the parental cell line was GAO, whose CEA expression was strongly positive by immunohistochemistry[21. HeLa cell line was kept in our lab and had no CEA expression; HeLaK was resistant-hela cell subline induced by DOX. The cells were maintained in RPMI 1640 medium containing 10 % ( v/ v) fetal bovine serum. 1.2 Construction of retroviral vector Based on the sequence of CEA pr~moter'~], the primers were synthesized as follows: CEAPl 5'-AATTGGATCCAGCCCCCAGAGCCACCTCTC-3' ( containing BamH I site ), CEAP2 5'- ATTAAGCTTATGGTCTCTGCTGTCTGCTCT-3' ( containing Hind III cleavage site ). CMV promoter was removed from retroviral vector LNCX by digestion with BamH I and Hind III enzymes; then CEA promoter was prepared by PCR and directionally inserted into the BamH I / Hind III site in LNCX. MDRl ribozyme gene was synthesized by DNA synthesizer (Gibco) as follows : RP15 ' -CCCTCAAGCTTGTTGCCATTCTGATGAGTCCGTGAGG AGA-3'(containing Hind III site), RP2 5'-ATCGATCTTTCAGTTTCGTCCTCACGGACTCA- TCAGAATGGCAACG-3' (containing Cla I site). The fragment of MDRl ribozyrne gene was digested and directionally inserted into Hind B/Cla I site of LNCX, and the retroviral vector of MDRl ribozyme promoted by CEA promoter was constructed, named pceamr ( figure 1 ). 5'-LTR BamH I Hind I11 Cla 1 - r - neo CEA promoter MDR 1 nibozyme Fig. 1. Construct of retroviral vector pceamr. LTR. MoMLV long terminal repeat; neo, neomycin resistance gene. 1.3 pceamr transfer"] The recombinant vector pceamr was introduced into the virus package cell PA317 (NIH) by Lipofectin method and selected by G418 (500 pg/ml, Sigma) for 2 weeks. The viral titers of supernatants from the resistant colonies were tested by infecting NIH 3T3 cells. The highest vitus-t~tel~ supernatant was used to infect GAOK and HeLaK cells. The transfected cells were selected by G418 (0.5 mg/ml) for 2 weeks and the resistant colonies were used to test drug resistance. The infected cells of GAOK and HeLaK were named GAOKMR and HeLaKMR, respec-

124 SCIENCE IN CHINA (Series C) Vol. 40 tively. 1.4 Expression of CEA gene and MDRl ribozyme gene To detect the expression of CEA and MDRl ribozyme genes, total RNA was extracted and subjected to Northern blot according to previous method[". The conserved sequence of ri- bo~~me['~ and Pst I fragment of human CEA cdna served as probes. 1.5 Drug resistance test in vitro To detect the drug resistance, the transfected cells (1 x lo4) were added to the wells of 96- well microplates in 100 pl medium. The medium containing different concentrations of DOX or VBL or COL was added until the cells in control were confluent; three days later the viability was assessed by the reduction of MTT'". The relative drug resistance was determined by comparing the ICso of transfected cells and untransfected cells. ICso was defined as the concentration of cytotoxic drug that caused 50% inhibition of cell growth as compared with untreated control. 1.6 Drug resistance test in vivo Male BALB/c nu/nu 6-week-old nude mice were purchased from Department of Experimental Animal, Beijing Medical University. The cells were inoculated into nude mice as previously describedl9]. After 3-d inoculation, DOX (6 mg/kg per day) in 0.2 ml or 5 % glucose solution (GS) was i. p. injected into mice once a day for 28d. Each group consisted of 10 mice. Tumor volume (TV) was measured every 2 d, and calculated by the formula, TV= a2 x b/2, where a is the tumor width and b is its length in mm. The effect of the drug was expressed as relative tumor volume (RTV), RTV= TV,/TVo, where TV, is the TV at any given time and TVo is the TV at the onset of treatment. 2 Results 2.1 Expression of CEA gene Northern blot analysis was performed with specific CEA cdna probe. Two bands, one at 4.2 kb and the other at 2.9 kb, were detected in GAOK cells, but there were no bands found in HeLaK cells, showing that CEA gene expressed only in GAOK cells, not in HeLaK cells (fig. 2). CEA gene family contained CEA and NCA. The 4.2-kb band was mrna of CEA and the 2.9-kb band was mrna of NCA'"]. There were two bands in GAOK cells, showing that GAOK cells expressed not only CEA but also NCA. 2.2 Specific-expression of MDRl ribozyme gene Total RNA was isolated from GAOKMR and HeLaKMR colonal cells infected with pceamr retrovirus. By Northern blot, expression of the ribozyme was demonstrated in the GAOKMR cells and not in HeLaKMR cells ( fig. 2). By Southern blot hybridization, the ribozyme gene was detected in the DNA isolated from HeLaKMR cells ( fig. 2 ), showing that MDRl ribozyme was present in the DNA of HeLaKMR cells but did not express. These results indicated that pceamr vector expressed specifically only in CEA-expressing GAOK cells.

No. 2 SELECTIVE REVERSAL OF DRUG RESISTANCE BY MDRl RIBOZYME 125 Fig. 2. Analysis of CEA expression, Northern blot of MDRl ribozyme mrna, Southern blot of MDRl ribozyme DNA. (a) Northern blot of CEA mrna; (b) Northern blot of MDRl ribozyme mrna; (c) Southern blot of MDRl ribozyme DNA. 1, HeLa cells; 2, HeLaKMR cells; 3, GAOK cells; 4, GAOKMR cells. 2.3 In vitro drug resistance of the infected cells GAOK and HeLaK cells were both infected with pceamr, and the resistant colonies had no changes in morphology and growth speed after selection by (3418 (data not shown). The resistances to DOX, VBL and COL are shown in table 1. In GAOKMR cells, the resistance decreased 91.5 % to DOX, 85.0 % to VBL and 76.4 % to COL respectively. In HeLaKMR cells, the resistance decreased only 10.2 % to DOX, 12.8 % to VBL and 11.1 % to COL respectively. These resistance changes were slight. These results showed that pceamr vector expressed and reversed selectively the drug resistance only in CEA-expressing GAOK cells and had no effect on the resis- tance in non-cea-expressing HeLaK cells. Table 1 Relative drug resistance of pceamr infected cells Cell Relative drug resistance') line doxorubicin ( ICso) vinblastine ( ICs0) colchicine ( ICso) GAO 1.0 (30) 1.0 (0.026) l.o(l.00) GAOK 13.0 (390) 8.0 (0.208) 5.5(5.50) GAOKMR 1.1 (33) 1.2 (0.031) 1.3 (1.30) HeLa 1.0 (24) 1.0 (0.024) 1.0 (1.00) HeLaK 11.8 (283) 9.4 (0.226) 4.5 (4.50) HeLaKMR 10.6 (254) 8.2 (0.197) 4.0 (4.00) a) Relative drug resistance: ICSo of GAOK, GAOKMR relative to GAO or HeLaK, HeLaKMR to HeLa; the values of GAO and HeLa were set arbitrarily at 1 ; ( ICSo) were ICso absolute values (nmol). 2.4 In vivo effect of MDRl ribozyme on the infected cells To investigate the resistance to DOX in vitro, the infected cells were inoculated s. c. at the flank of nude mice and treated with DOX or glucose solution. In the groups treated with glucose solution, GAOKMR cells grew as well as GAOK and GAO cells. In the groups treated with DOX, GAOKMR cells were significantly suppressed as GAO cells did, and GAOK cells grew well. Similar experiments were performed using HeLaKMR cells. HeLa cells grew very slowly in

126 SCIENCE IN CHINA (Series C) Vol. 40 mice given W X treatment. Neither HeLaK nor HeLaKMR cells were suppressed in mice given DOX injection (fig. 3). These results were the same in vivo as in vitro, showing that pceamr expressed only in GAOK cells and not in HeLaK cells. 3 Discussion Chemotherapy is one of the main.treatment of lung adenocarcinomas, which are often resistant to anticancer drugs because of overexpression of. MDRl gene[111. In previous study, Timeld MDRl antisense RNA mediated by Fig. 3. The effects of DOX on the growth of GAO. GAOKMR, HeLa and retrovirus was introduced into mul- HeLaKMR tumor xenografts in nude mice. 0, GAO ;, GAO + DOX ; 0, GAOK; x, GAOK + DOX; *, GAOKMR; a, GAOKMR + tidrug-resistant GAoK and inhib- DOX; +, H ~L~; a, H ~ L DOX; ~ + A, H~L~K; +, H ~L~K + DOX; ited the drug resistance efficiently but, HeLaKMR ; A, HeLaMR + DOX. not completely[121. Ribozyme could inhibit gene expression and block protein synthesis. HIV ribozyme was successfully used to inhibit the synthesis of p24 antigen protein; ras ribozyme reversed the malignant phenotype of human bladder car~inorna"~~. ~oba~ashi['~] used MDRl ribozyme to reverse the drug resistance of human acute lymphoblastic leukemia cells; the drug sensitivity increased 700-fold. So MDRl ribozyme is a good molecule against tumor drug resistance. However, if expression of MDRl ribozyme was not tumor-specific, MDRl ribozyme also inhibited the MDRl expression in normal tracheal epithelial cells or other cells, which protected the normal cells from harmful agents. If MDRl ribozyme expressed tumor-specifically and did not affect MDRl expression of normal cells, it would have wide applications in future. CEA is a common tumor marker of human adenocarcinomas. Although CEA expressed in normal colonic mucosa, the level of CEA expression is far higher in adenocarcinoma than in colonic mucosa[61. It was shown that CEA was a good monitoring ruler for the recurrence and metastasis of adenocarcinomas. CEA was a good marker of lung adenocarcinomas because it expressed only in adenocarcinomas but not in normal lung cellst6]. The element of CEA gene in the upstream was correlated with high expression of CEA; the cis-acting sequences conveying cell type-specific expression was within the putative promoter region of CEA'~]. The CEA promoter appeared to selectively up-regulate gene expressing in CEA-producing cells. It was shown that in CEA-producing tumor cells, the HSV-TK expression driven by CEA promoter was much higher than that by other promoteri151. In the present study, we constructed the tumor-specific retrovi- ral vector pceamr of MDRl ribozyme driven by CEA promoter. pceamr specifically expressed only in CEA-producing GAOK cells and not in non-cea-producing HeLaK cells. The drug resistance of GAOK cells was reversed and the drug sensitivity was restored selectively by pceamr vector which had not any effects on drug resistance of HeLaK cells in vitro and in

No. 2 SELECTIVE REVERSAL OF DRUG RESISTANCE BY MDRl RIBOZYME 127 vivo. An retrovirus system has potential advantages for gene delivery, such as easiness of producing high titer virus, high infection efficiency, infectivity for many types of cells, innocuousness for host cells. So retrovirus was often used as gene expression vector. Retroviral vector of MDRl ribozyme regulated by CEA promoter expressed only in CEA-producing cells, and selectively reversed drug resistance, and had no effect on non-cea-producing cells. This was very important for normal epithelial cells against harmful agents and might lead to a new possible treatment for patients with CEA-producing lung cancers which are usually refracto- ry to conventional chemotherapy. References 1 Gao, Z. Q., Gao Z. P., Advance in gene therapy of lung cancer, Foreign Med. Sci-Respir. (in Chinese), 1996, 3: 148. 2 Gao, Z. Q., Zhang, Z. H., Gao, Z. P., Clinical correlates of MDRl gene expression in human lung cancer, Chin. J. Internal Med. (in Chinese), 1996.7: 482. 3 Symons, R., Self-cleavage of RNA in the replicative of small pathogens of plants and animals, TIBS, 1989, 14:445. 4 Gao, Z. Q., Gao, Z. P., Liu, X. F. et a1., Reversal of drug sensitivity in multidrug-resistant lung cancer cells by an MDRl ribozyme retrovirus, Chin. Sci. Bull., 1996, 22 : 220. 5 Shinkai, T., Saijo, N., Tominaga, K. et a1., Serial plasma CEA measurement for momitoring patients with advanced lung cancer during chemotherapy, Cancer, 1986, 57: 1318. 6 Schrewe, H., Thompson, J., Bona, M. et a1., Cloning of the complete gene for carcinoembryonic antigen: analysis of its promoter indicates a region conveying cell type-specific expression, Mol. Cell Biol., 1990, 10:2738. 7 Gao, Z. Q., Gao, Z. P., Zhang, X. B., Inhibitow effect of drug resistance in multidrug-resistant human lung cancer cells by GST-x antisense RNA mediated by retrovirus, Chin. I. Prev. & Control Chronic Non-communi. Dis. ( in Chinese), 1996, 3:llO. 8 Gao, Z. Q., Gao. Z. P., Zhang, X. B., Suppression of human lung cancer by wild-type p53, Chin. J. Prev. & Control ~hrbnic Non-communi. Dis. (in Chinese), 1996, 2 : 63. 9 Gao, Z. Q., Zheng, J., Wu, B. Q. et al., Study on the biology of xenograft of human lung cancer induced by benzo(a) pyrene in nude mouse, Chin. J. Pathol. (in Chinese), 1996, 2: 106. 10 Sato, C., Miyaki, M., Oikawa, S. et a1., Differential expression of CEA and nonspecific crossreacting antigen genes in human colon adenocarcinomas and normal colon mucosa, Jpn. 1. Cancer Res., 1987, 79:433. 11 Gao, Z.Q., Gao, Z.P., Advance in multidrug resistance, Chin. J. Pathol. (in Chinese), 1996, 3:185. 12 Gao, Z. Q., Gao, Z. P., Liu, X. F., Reversal of drug resistance in multidrug-resistant human lung cancer cells by retrovirusmediated MDRl antisense RNA, Chin. Sci. Bull., 1996, 41 (20): 2166. 13 Kashami-sabet, M., Funato, T., Tone, T. et al., Reversal of the malignant phenotype by an anti-ras ribozyme, Aptise Res. Dew., 1992, 2:3 14 Kobayashi, H., Dorai, T., Holland, J. et a1., Reversal of drug sensitivity in multidrug-resistant cells by an MDRl ribozyme, Cancer Res., 1994, 54 : 1271. 15 Dimaio, J. M., Clary, B. M., Via, D. F. et a1., Directed enzyme pro-drug gene therapy for pancreatic cancer in vim, Surgery. 1994. 116:205.