Cong-Mei Wu, Tian-Hua Huang, Research Center of Reproductive Medicine, Shantou University Medical College (SUMC), Shantou 515041, Guangdong Province, China
Xiu-Yi Li, The Ministry of Public Health Radiobiology Research Unit of Jilin University, Changchun 130021, Jilin Province, China
Supported by the National Natural Science Fundation of China, No. 39970229
Correspondence to: Dr. Cong-Mei Wu, Research Center of Reproductive Medicine, Shantou University Medical College, Shantou 515041, Guangdong Province, China.  cmwu@stu.edu.cn
Telephone: +86-754-8900442    Fax: +86-754-8557562
Received: 2003-12-10    Accepted: 2004-03-02

Abstract
AIM: To construct a pEgr-IFNg plasmid and to investigate its expression properties of interferon-g (INF-g) induced by irradiation and the effect of gene-radiotherapy on the growth of melanoma.

METHODS: A recombined plasmid, pEgr-IFNg, was constructed and transfected into B16 cell line with lipofectamine. The expression properties of pEgr-IFNg were investigated by ELISA. Then, a B16 melanoma-bearing model was established in mice, and the plasmid was injected into the tumor tissue. The tumor received 20 Gy X-ray irradiation 36 h after injection, and IFN-g expression was detected from the tumor tissue. A tumor growth curve at different time points was determined.

RESULTS: The eukaryotic expression vector, pEgr-IFNg, was successfully constructed and transfected into B16 cells. IFN-g expression was significantly increased in transfected cells after X-ray irradiation in comparison with 0 Gy group (77.73-94.60 pg/mL, P<0.05-0.001), and was significantly higher at 4 h and 6 h than that of control group after 2 Gy X-ray irradiation (78.90-90.00 pg/mL, P<0.01-0.001). When the transfected cells were given 2 Gy irradiation 5 times at an interval of 24 h, IFN-g expression decreased in a time-dependent manner. From d 3 to d 15 after IFNg gene-radiotherapy, the tumor growth was significantly slower than that after irradiation or gene therapy alone.

CONCLUSION: The anti-tumor effect of pEgr-IFNg gene-radiotherapy is better than that of genetherapy or radiotherapy alone for melanoma. These results may establish an important experimental basis for gene-radiotherapy of cancer.

Wu CM, Li XY, Huang TH. Anti-tumor effect of pEgr-IFNg gene-radiotherapy in B16 melanoma-bearing mice. World J Gastroenterol  2004; 10(20): 3011-3015
http://www.wjgnet.com/1007-9327/10/3011.asp

INTRODUCTION
Radiotherapy is one of the treatments for cancer. However, its therapeutic effect is still unsatisfactory, and thus new therapeutic strategy must be adopted. Gene therapy in combination with radiotherapy is one of the most important advances^[1-4]. The introduction of Egr-1 promoter induced by irradiation has provided a possible approach to this combination therapy^[5-6].
      IFNg is the first cytokine produced by gene engineering and used for treatment of carcinoma, and has anti-tumor effects. Its antitumor mechanism includes direct inhibition of tumor cell proliferation, and indirect action by activating cytotoxic activities^[7-18]. In the present study we constructed the pEgr-IFNg plasmid by connecting IFNg cDNA to Egr-1 promoter to investigate its expression properties in B16 cells and its antitumor effect in mice.

MATERIALS AND METHODS
Construction of pEgr-IFNg Plasmid
The expression vector for pEgr-IFNg is shown in Figure 1.

Cell line and transfection
B16 cell line was cultured in MH Radiobiology Research Unit of Jilin University and maintained in RPMI 1640 (Life Technologies) with 100 mL/L fetal bovine serum (Hyclone Laboratories), L-glutamine, 100 mg/mL of streptomycin, and 100 U/mL of penicillin. The cell line was incubated at 37 °C in 50 mL/L CO₂.
      B16 cells were transfected in a 6-well plate when the cells reached 70% confluence. Solution A was prepared by addition of 10 mg of pEgr-IFNg or pcDNA3.1+ to 100 mL serum-free medium (SFM), and solution B by addition of 10 mL liposome to 100 mL SFM. Solutions A and B were mixed at room temperature for 30 min, then mixed with 0.8 mL SFM, the mixture was added to the rinsed cells. The medium was replaced with fresh and complete medium 6 h after transfection.

Protein determination
Supernatants from different groups were collected for detection of the IFNg expression with ELISA kit (Genzyme).

Establishment of B16 melanoma-bearing model
Adult female Kunming mice were provided by the Experimental Animal Center of Jilin University, with an average weight of 18±2 g.
      A melanoma-bearing model was established by subcutaneous injection at right hind limb with 0.1 mL B16 cells ( 5×10⁶ /mL), 10 d later, tumor tissue received multi-focus injection of plasmids packaged with liposome (20 mg plasmid and 0.1 mL liposome per mouse) for the experimental groups.
     Tumor size was measured. Then, tumor volume (V) was calculated according to the formula: V (mm³) = L×W²/2, where, L: the longest diameter of tumor; W: the diameter at right angles on the largest horizontal section. Tumor growth rate (f) was the ratio of the volume at different time points over the initial volume (V₀).

Ionizing irradiation
X-rays of 200 kV and 10 mA with 0.5 mm copper and 1.0 mm aluminum filter were given at a dose-rate of 0.8639 Gy/min for a total dose of 2--20 Gy.

Figure 1(PDF) Construction of plasmid pEgr-IFNg.

      B16 cells were seeded in a 6-well plate and randomly divided into different groups. The experiment groups received X-ray irradiation of various doses or at different time points, control groups received sham irradiation simultaneously.
      Mice bearing B16 xenografts (n = 40) were randomly divided into five groups: control group, 20 Gy group, pcDNA3.1+20 Gy group, pEgr-IFNg group and pEgr-IFNg+20 Gy group. Thirty-six hours before irradiation, melanoma tissue was injected with plasmids or buffer at 5 separate sites. Tumor beds were given 20 Gy X-ray irradiation. Animals of 20 Gy group, pcDNA3.1+20 Gy group and pEgr-IFNg+20 Gy group were shielded with lead except for the tumor-bearing hind limb, animals in the other 2 groups were given sham-irradiation at the same time. Tumors were measured and recorded as previously described.

RT-PCR
Total RNA were extracted from EC9706 cells and tumor tissue for RT-PCR. GAPDH was used as an internal reference. Primers were as follows: GAPDH, forward primer 5’-TGCACCACCAACTGCTTAGC –3’ and reverse one 5’GGCATGGACTGTGG TCATGAG-3’ mouse IFNg cDNA, forward primer 5’GATCCTTTGGACCCTCTG ACTT-3’and reverse one 5’AGACAGTGATAAACTATAAATGAGCG-3’
      RT-PCR was performed as following: denaturation at 95 °C for 3 min, 30 cycles at 95 °C for 45 s, at 56 °C for 45 s, at 72 °C for 40 s and extension at 72 °C for 10 min.

Statistical analysis
Student’s t test was used to determine comparability between groups. P values less than 0.05 were considered statistically significant.

RESULTS
B16 cell line transfected with pEgr-IFNg plasmid
Pre- and post-transfection of B16 cells are shown in Figure 2.

IFNg expressions in B16 cells transfected with pEgr-IFNg after different doses of X-irradiation
After transfection B16 cells received different doses of X-ray irradiation. The cells of control group were transfected with pcDNA3.1+ plasmid. Six hours after irradiation IFNg expression and mRNA level were detected.
     The results showed that IFNg expression in 2-20 Gy groups was significantly higher than that in 0 Gy group (P<0.05-0.01) (Figure 3).

Figure 2  Pre- and post-transfection of B16 cells. A:   B:
Figure 3(PDF) Expression of IFNg in B16 cells after different doses X-ray irradiation. ( mean±SD, n = 3)  ^aP<0.05 and ^bP<0.01  vs 0 Gy group.

      After irradiation IFNg mRNA could be detected in B16 cells(Figure 4). The level of IFNg mRNA was compared with that of GAPDH, and their ratios are shown in Table 1. The IFNg mRNA levels in 2-20 Gy groups were higher than that of 0 Gy group.

Figure 4(PDF) IFNg RNA level in B16 cells after different doses of X-ray irradiation. Lane 1: DL2000 Marker; Lane 2: 0 Gy group; Lane 3: 2 Gy group; Lane 4: 5 Gy group; Lane 5: 10 Gy group; Lane 6: 20 Gy group.

IFNg expressions in B16 cells transfected with pEgr-IFNg at different time points after 2Gy irradiation
After transfection B16 cells received 2 Gy of X-ray irradiation while the control group received sham irradiation. IFNg protein was detected at different time points after irradiation. ELISA results showed that the IFNg expression increased with time from 2 h to 6 h in a time-dependent manner, and peaked at 6 h, about 1.8 times of that in control group (P<0.001). However, from 8 h to 48 h post-radiation IFNg expressions were not significantly different from that in control group (Figure 5).

Table 1  IFNg mRNA level in B16 cells after irradiation with different doses 

Figure 5(PDF) Expression time course of IFNg in B16 cells after 2 Gy X-ray irradiation ( mean±SD, n = 3) ^bP<0.01 and ^dP<0.001 vs control group.

Expression of IFNg in B16 cells at different time points after X-ray irradiation
After transfection B16 cells received 2 Gy irradiation while the control group received sham irradiation. IFNg expression was detected 6 h later. Irradiation and detection were repeated 5 times at an interval of 24 h.
      The result showed that the IFNg expression after the first irradiation was the highest, then decreased in a time-dependent manner. The expressions after the first 2 times of irradiation were higher than that in control group (P<0.01-0.001) (Figure 6).

Figure 6(PDF) Expression of IFNg in B16 cells at different time points after X-ray irradiation ( mean±SD, n = 3)  ^bP<0.01 and ^dP<0.001  vs 0 Gy groups.

Effect of gene-radiotherapy on tumor growth
Melanoma-bearing mice of different groups were shown in Figure 7.
      Tumor growth rate of pEgr-IFNg group was significantly slower than that of control group (P<0.001) between 6 d and 15 d after irradiation (Table 2), so was pEgr-IFNg plus 20 Gy group compared with control and 20 Gy groups between 3 d and 15 d after irradiation (P<0.001).

Table 2  Tumor growth rate after gene-radiotherapy ( mean±SD, n = 8) 

^aP<0.05, ^bP<0.001 vs control group; ^dP<0.01, ^fP<0.001 vs 20 Gy group.

Figure 7 Melanoma-bearing mice 15 d after treatment.  A:  B:  C:  D:

RT-PCR analysis of IFNg in tumor tissue
Melanoma-bearing mice were injected with plasmids, and the tumor received 20 Gy X-ray irradiation, 3 d later total RNA from tumor tissue was extracted for RT-PCR.
      GAPDH bands were shown in all groups, but FNg cDNA bands were shown only in pEgr-IFNg and pEgr-IFNg+20 Gy groups (Figure 8).

Figure 8(PDF) RT-PCR analysis of intratumor IFNg. Lane 1: DL2000 Marker; Lane 2: control; Lane 3: 20 Gy; Lane 4: pcDNA3.1+20 Gy; Lane 5: pEgr-IFNg; Lane 6: pEgr-IFNg+20 Gy.

DISCUSSION
In 1992 Weichselbaum put forward the new therapeutic strategy that took advantage of the dual tumor-killing effects of genetherapy and radiotherapy, namely, to choose certain exogenous genes that could be activated by irradiation and then transcript some cytotoxic proteins to kill the tumor cells. They also established the techniques that might be used for target gene therapy of carcinomas^[19-25].
      It had been reported that Egr-1 was transcriptionally induced by exposure to irradiation, and its induction by irradiation was conferred by serum response or CC (A/T) rGG elements in its promoter region^[26-30]. Based on this finding, we firstly connected IFNg cDNA with Egr-1 promoter to construct pEgr-IFNg plasmid to investigate the expression properties in B16 melanoma cells. Furthermore, a melanoma model was established by subcutaneous injection of B16 cells, and then plasmids were injected to observe its antitumor effect in vivo.
      Firstly, B16 cells transfected with pEgr-IFNg received different doses of X-ray irradiation and the IFNg expression was detected. The results showed that the IFNg expression level in B16 cells post-transfection induced by irradiation was higher than that of sham-irradiation group (P<0.05-0.01). Time-course studies revealed that IFNg expression reached its peak at 6 h after 2Gy irradiation, and the maximal level was 1.8 times of that in control group (P<0.01). Furthermore, after repeated irradiation the IFNg expression in B16 cells post-transfection reached the peak level just after the first irradiation, and then decreased in time-dependent manner. All of these demonstrated that pEgr-IFNg plasmid could enhance IFNg expression.  
      Secondly, the results of in vivo experiments showed that the proliferation of melanoma was significantly inhibited in pEgr-IFNg group in comparison with control group between 6 and 15 d after irradiation (P<0.001). So was pEgr-IFNg gene-radiotherapy group compared with control and 20 Gy groups between 3 and 15 d after irradiation (P<0.001). IFNg expression was detected in melanoma tissue having receivied injection of pEgr-IFNg plasmid. These results demonstrated that injection of pEgr-IFNg improved antitumor effect, and combined pEgr-IFNg and irradiation showed the most optimal effect.
      This study combined pEgr-IFNg plasmid and irradiation, and demonstrated much more enhanced antitumor efficacy than either one in the melanoma model. It was very easy to administer directly the plasmid into melanoma tissue, and therapeutic dose could also be administered as required. The tumor could be effectively exposed to radiation with external beam or intratumoral sources, or both, to enhance local IFNg expression and boost local tumor control. Increased IFNg levels might elicit systemic mediators, such as cytokines and matrix proteinases, which target occult distant metastases and thereby further enhance the therapeutic ratio. The absence of systemic toxicities with intratumoral administration of IFNg supports the safe addition of pEgr-IFNg gene radiotherapy to current antitumor protocols.

REFERENCES
1    Weichselbaum RR, Hallahan DE, Beckett MA, Mauceri HJ, Lee H, Sukhatme VP, Kufe DW. Gene therapy targeted by 
      radiation preferentially radiosensitizes tumor cells. Cancer Res  1994; 54: 4266-4269
2    Liu XF, Zou SQ, Qiu FZ. Construction of HCV-core gene vector and its expression in cholangiocarcinoma. World J 
      Gastroenterol  2002; 8: 135-138
3    Gou WJ, Yu EX, Liu LM, Li J, Chen Z, Lin JH, Meng ZQ, Feng Y. Comparison between chemoembolization combined with 
      radiotherapy and chemoembolization alone for large hepatocellular carcinoma. World J Gastroenterol 
      2003; 9: 1697-1701
4    Ido A, Uto H, Moriuchi A, Nagata K, Onaga Y, Onaga M, Hori T, Hirono S, Hayashi K, Tamaoki T, Tsubouchi H. Gene 
      therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a 
      hypoxia-inducible enhancer linked to a human alpha-fetoprotein promoter. Cancer Res 2001; 61: 3016-3021
5    Datta R, Rubin E, Sukhatme V, Qureshi S, Hallahan D, Weichselbaum RR, Kufe DW. Ionizing radiation activates 
      transcription of the EGR1 gene via CArG elements. Proc Natl Acad Sci U S A 1992; 89: 10149-10153
6    Tsai Morris CH, Cao XM, Sukhatme VP. 5' flanking sequence and genomic structure of Egr-1, a murine mitogen 
      inducible zinc finger encoding gene. Nucleic Acids Res 1988; 16: 8835-8846
7    Lokshin A, Mayotte JE, Levitt ML. Mechanism of interferon beta-induced squamous differentiation and programmed 
      cell death in human non-small-cell lung cancer cell lines. J Natl Cancer Inst 1995; 87: 206-212
8    Shiau AL, Lin CY, Tzai TS, Wu CL. Postoperative immuno-gene therapy of murine bladder tumor by in vivo 
      administration of retroviruses expressing mouse interferon-gamma. Cancer Gene Ther 2001; 8: 73-81
9    Siesjo P, Visse E, Sjogren HO. Cure of established, intracerebral rat gliomas induced by therapeutic immunizations 
      with tumor cells and purified APC or adjuvant IFN-gamma treatment. J Immunother Emphasis Tumor Immunol 
      1996; 19: 334-345
10  Saleh M, Jonas NK, Wiegmans A, Stylli SS. The treatment of established intracranial tumors by in situ retroviral 
      IFN-gamma transfer. Gene Ther 2000; 7: 1715-1724
11  Li XM, Chopra RK, Chou TY, Schofield BH, Wills Karp M, Huang SK. Mucosal IFN-gamma gene transfer inhibits 
      pulmonary allergic responses in mice. J Immunol 1996; 157: 3216-3219
12  Fujinami K, Ikeda I, Miura T, Kondo I. Combination therapy with 5-fluorouracil (5-FU), cisplatin (CDDP) and interferon 
      alpha-2B (IFN alpha-2B) for advanced renal cell carcinoma. Gan To Kagaku Ryoho 1996; 23: 1689-1691
13  Yeow WS, Lawson CM, Beilharz MW. Antiviral activities of individual murine IFN-alpha subtypes in vivo: intramuscular 
      injection of IFN expression constructs reduces cytomegalovirus replication. J Immunol 1998; 160: 2932-2939
14  Ahn EY, Pan G, Vickers SM, McDonald JM. IFN-gammaupregulates apoptosis-related molecules and enhances 
      Fas-mediated apoptosis in human cholangiocarcinoma. Int J Cancer 2002; 1: 445-451
15  Blanck G. Components of the IFN-gamma signaling pathway in tumorigenesis. Arch Immunol Ther Exp 
      2002; 50: 151-158
16  Nayak SK, McCallister T, Han LJ, Gangavalli R, Barber J, Dillman RO. Transduction of human renal carcinoma cells with 
      human gamma-interferon gene via retroviral vector. Cancer Gene Ther 1996; 3: 143-150
17  Tada H, Maron DJ, Choi EA, Barsoum J, Lei H, Xie Q, Liu W, Ellis L, Moscioni AD, Tazelaar J, Fawell S, Qin X, Propert KJ, 
      Davis A, Fraker DL, Wilson JM, Spitz FR. Systemic IFN-beta gene therapy results in long-term survival in mice with 
      established colorectal liver metastases. J Clin Invest 2001; 108: 83-95
18  Paradis TJ, Floyd E, Burkwit J, Cole SH, Brunson B, Elliott E, Gilman S, Gladue RP. The anti-tumor activity of anti-CTLA-4 
      is mediated through its induction of IFN gamma. Cancer Immunol Immunother 2001; 50: 125-133
19  Weichselbaum RR, Hallahan DE, Sukhatme VP, Kufe DW. Gene therapy targeted by ionizing radiation. Int J Radiat Oncol 
      Biol Phys 1992; 24: 565-567
20  Weichselbaum RR, Kufe DW, Advani SJ, Roizman B. Molecular targeting of gene therapy and radiotherapy. Acta Oncol 
      2001; 40: 735-738
21  Khodarev NN, Park JO, Yu J, Gupta N, Nodzenski E, Roizman B, Weichselbaum RR. Dose-dependent and independent 
      temporal patterns of gene responses to ionizing radiation in normal and tumor cells and tumor xenografts. Proc Natl 
      Acad Sci U S A 2001; 98: 12665-12670
22  Gupta VK, Park JO, Jaskowiak NT, Mauceri HJ, Seetharam S, Weichselbaum RR, Posner MC. Combined gene therapy 
      and ionizing radiation is a novel approach to treat human esophageal adenocarcinoma. Ann Surg Oncol 
      2002; 9: 500-504
23  Hanna NN, Seetharam S, Mauceri HJ, Beckett MA, Jaskowiak NT, Salloum RM, Hari D, Dhanabal M, Ramchandran R, 
      Kalluri R, Sukhatme VP, Kufe DW, Weichselbaum RR. Antitumor interaction of short- course endostatin and ionizing 
      radiation. Cancer J 2000; 6: 287-293
24  Takahashi T, Namiki Y, Ohno T. Induction of the suicide HSV-TK gene by activation of the Egr-1 promoter with 
      radioisotopes. Hum Gene Ther 1997; 8: 827-833
25  Griscelli F, Li H, Cheong C, Opolon P, Bennaceur- Griscelli A,Vassal G, Soria J, Soria C, Lu H, Perricaudet M, Yeh P. 
      Combined effects of radiotherapy and angiostatin gene therapy in glioma tumor model. Proc Natl Acad Sci U S A 
      2000; 97: 6698-6703
26  Christy B, Nathans D. DNA binding site of the growth factor- inducible protein Zif268. Proc Natl Acad Sci U S A 
      1989; 86: 8737-8741
27  Seyfert VL, Sukhatme VP, Monroe JG. Differential expression of a zinc finger-encoding gene in response to positive 
      versus negative signaling through receptor immunoglobulin in murine B lymphocytes. Mol Cell Biol 1989; 9: 2083-2088
28  Joseph LJ, Le-Beau MM, Jamieson GA Jr, Acharya S, Shows TB, Rowley JD, Sukhatme VP. Molecular cloning, 
      sequencing, and mapping of EGR2, a human early growth response gene encoding a protein with “zinc-binding finger” 
      structure. Proc Natl Acad Sci U S A 1988; 85: 7164-7168
29  Sukhatme VP. Early transcriptional events in cell growth: the Egr family. J Am Soc Nephrol 1990; 1: 859-866
30  Cao XM, Koski RA, Gashler A, McKiernan M, Morris, CF, Gaffney R, Hay RV, Sukhatme VP. Identification and 
      characterization of the Egr-1gene product, a DNA-binding zinc finger protein induced by differentiation and growth 
      signals. Mol Cell Biol 1990; 10: 1931-1939

   Edited by Ren SY and Wang XL  Proofread by Xu FM  

Reviews	Add
more>>