481
책임저자：장석균, 서울시 영등포구 여의도동 62
150-713, 여의도성모병원 외과 Tel: 02-3779-1175, Fax: 02-786-0802 E-mail: [email protected]
접수일：2007년 1월 17일, 게재승인일：2007년 8월 7일
The Synergistic Cell Killing Effects by the Transduction of the w-p53 Gene and 5-FU Administration in Colon Cancer Cell Lines
Department of Surgery, College of Medicine, The Catholic University of Korea, Seoul, Korea
Hyung Jin Kim, M.D., Sang Chul Lee, M.D., In Kyu Lee, M.D., Won Kyung Kang, M.D., Seong Taek Oh, M.D. and Suk Kyun Chang, M.D.
대장암 세포주에서 w-p53 유전자 이입 및 5 FU 투여에 의한 세포사 효과
김형진ㆍ이상철ㆍ이인규ㆍ강원겸ㆍ오승택ㆍ장석균
Purpose: It is known that the wild-type p53 (w-p53) gene has several functions such as suppression of tumor cell growth, control of the cell cycle, stabilization of the genes and cellular differentiation. Recombinant w-p53 adenovirus was transfected and 5-FU was administered into the LoVo (w-p53 gene positive) and SW-837 (mutant-p53 gene pos- itive) colon cancer cell lines to determine the cell death ef- fects according to the presence or absence of the w-p53 gene.
Methods: The transduction of the p53 gene was done using recombinant adenovirus and liposomes, and the cell death effect was determined by performing MTT assay.
Results: The cell death effect by 5-FU was higher in the LoVo cell line than that in the SW-837 cell line. The rate of w-p53 gene transduction was about 90%. The cell death effect by w-p53 gene transduction was shown by the admin- istration of 10μM of 5-FU. The cell death effect according to the administration of 5-FU after w-p53 gene transduction was 10 fold of that with 5-FU administration in the both cell lines, and there was a more significant effect in the LoVo cell line.
Conclusion: The cell killing effect by 5-FU administration af- ter w-p53 gene transduction showed a synergistically higher effect than those of w-p53 transduction only or 5-FU admin- istration only in the colon cancer cell lines. Gene therapy using w-p53 gene transduction requires more extensive clin- ical trials. (J Korean Surg Soc 2007;73:481-489)
Key Words: P53 Gene, Transduction, Gene therapy, Color- ectal neoplasm
중심 단어: P53 유전자, 형질도입, 유전자요법, 대장암
가톨릭대학교 의과대학 외과학교실
INTRODUCTION
Colorectal cancer represents the fourth most com- mon cancer in Korea and is one of the most rapidly increasing cancer. Although the diagnostic and treat- ment methods have been developed in recent years, the mortality and survival rates have not changed sig- nificantly and 40∼50% of patients eventually develop metastatic disease.(1,2) The majority of these patients develop metastatic disease to liver, lung and peritoneum.
Many series have demonstrated that only surgical resection of metastasis confined to the liver offers the real chance of long term survival. The overall 5-year survival rate of patients undergoing resection for iso- lated hepatic metastasis ranges from 25% to 40%.
Because only the surgical resection can offer a real chance of long-term survival, it should be considered the standard therapy for all patients with hepatic col- orectal metastasis isolated to the liver. Unfortunately, only 5∼10% of these patients are potentially curable by liver resection due to extrahepatic involvement or numerous hepatic lesions.(3) Several non-surgical mo- dalities such as cryoablation, radiofrequency ablation, chemotherapy, percutaneous ethanol injection (PEI), hepatic arterial infusion therapy, chemoemobolization, immunologic therapy and gene therapy are used for
the treatment of metastatic colorectal cancers not ame- nable to surgical resection.(3,4) Newly developed non-surgical modalities for metastatic colorectal can- cers, especially hepatic metastases, such as im- munotherapy using HLA-B7, IL-2 cytokine gene, CEA gene, or gene therapy with p53 gene, K-ras gene,(5-7) and suicide gene therapy with HSV-TK gene and cyto- sine deaminase were introduced experimentally in clinical settings in recent years.(8-10)
The wild type p53 (w-p53) gene is known to have several functions such as suppression of tumor cell growth, control of the cell cycle, DNA production and duplication, stabilization of genes and cellular differentiation. Applying the w-p53 gene to cancer treatments through the p53-mediated apoptosis has been tried in the experimental and clinical settings.(7,11,12) Also several investigators reported that the w-p53 gene has a synergistic cell killing effect with the chemotherapy or radiation therapy to the can- cer cells.(13,14)
We conducted the study to elucidate the cell killing effect by the transduction of w-p53 gene and the syner- gistic effect with 5-FU administration in colon cancer cell lines.
METHODS 1) Cell lines and cell culture
The cell lines used in this study were obtained from the Cancer Center at Thomas Jefferson University Hospital and American Type Culture Collection.
Several cell lines such as LoVo and RKO (w-p53 pos- itive), SW-1222 (w-p53 negative), SW-1463 and SW-837 (m-p53 positive) were tested with MTT assay. LoVo and SW-837 cell lines were selected for this study be- cause results of MTT assay and growth patterns were stable with small deviation.
The LoVo cells were maintained in HamF-12 liquid medium supplemented with 10% FBS containing pen- icillin and streptomycin at 37oC under a humidified 5% CO2 atmosphere. The SW837 cells were maintained in Leibovitz-15 liquid medium supplemented with 10% FBS containing penicillin and streptomycin at 37oC under a humidified atmosphere only.
2) Adenovirus encoding recombinant p53 gene (AdCMVp53)
The AdCMVp53 viruses were provided by generous gift from Professor Uoong-Shick Ahn at the Depart- ment of Obstetrics and Gynecology, The Catholic Uni- versity of Korea.
3) Transduction and β-galactosidase staining in the LoVo and SW837 cells
The w-p53 gene transduction was done using a lip- osome method. Transduction, its rates and expressions of AdCMVp53 were detected by β-galactosidase stain- ing and western blotting.
β-galactosidase staining was done to confirm the AdCMVLacZ virus transduction and its rates. Briefly, the smeared cancer cells on the slide were fixed in fix- ative solution for 5 min at room temperature and washed out with PBS. The slide fixed with cancer cells was put in the X-Gal reaction solution prepared by mixing of the dissolved X-Gal staining solute (Gibco BRL, Gaithersburg, MD, USA) in dimethylformamide as much as 20 mg/ml in concentration and the stain- ing solution and was incubated at 30oC for 24 h. The slide was dehydrated in 70∼100% alcohol after fixing in 10% formalin for more than a day and was washed out under the running water. Then, the slide was re-dehydrated in 100∼70% alcohol after treating in xy- lene solution for 2 min for 3 times. Finally, the slide was stained with hematoxylin and eosin, mounted, and was observed by optical microscope under ×100 and ×200 magnifications.
4) Expressions of the w-p53 gene in LoVo and SW837 cells
Western blotting was done to confirm the protein expression of the w-p53 gene. Briefly, the total protein was harvested from lysed whole cancer cells with lysis buffer, and then 50μg of whole protein was applied on 7.5% acrylamide gel and was electrophoresed at room temperature for 1 h at 100V. The electroeluted proteins separated by different molecular weights were transferred onto PVDF membrane for 2 h at 4oC, 350 mA. The first antibody (DO-1, Santa Cruz Biotechnology, Santa Cruz, CA, USA) against to the w-p53 protein was diluted with 5% skim milk until its concentration to 0.4μg/ml and blocking was done at
Fig. 1. Normal growth pattern of the LoVo and SW837 cells on day 2, 4, 6, 8 and 10 of the culture. It shows gradual growth initially and drastically increasing growth on day 8 of the culture.
4oC overnight. After washing the PVDF membrane 3 times for 15 min with TBS-T (Tris buffered saline with 0.1% Tween-20), the first Ab was applied to PVDF membrane and was incubated at room temperature for 2 h followed by washing with TBS-T 4 times each for 15 min. The second antibody against to human protein (anti-mouse monoclonal Ab, Santa Cruz Biotechnolo- gy, Santa Cruz, CA, USA) was diluted with 5% skim milk until its concentration to 0.15 mg/ml and was ap- plied to the PVDF membrane following by incubation at room temperature for 1 h and 30 min and washed out with TBS-T 4 times each for 15 min. The reacted protein was detected using an ECL detection kit (Amersham, Uppsala, Sweden).
5) Experimental groups and cell viability (cell killing effect)
Cell viabilities were estimated on day 2, 4, 6, 8 and 10 of the culture for each of following 4 groups of the experimental subjects. Group 1, normal control group;
Group 2, 5-FU administration at concentration of 0.1μ M, 1μM, 10μM, 100μM and 1,000μM; Group 3, w-p53 gene transduction only; Group 4, 5-FU admin- istration at concentration of 0.1μM, 1μM and 10μM after w-p53 gene transduction.
The cell viability presented in percentages was esti- mated by MTT assay which reflects cell proliferation and viability.(9) The cell viabilities by AdCMVLacZ vi- ruses of 20 moi, 50 moi and 100 moi concentrations were measured to estimate the cell killing effects of ad- enovirus itself, and the adequate concentration of AdCMVp53 with higher transduction rate and least cell killing effect was determined (data not shown).
A pilot study to determine the number of cells to be used in the MTT assay was done with 1.0×103, 3.0×103, 5.0×103, 1.0×104 and 2.5×104 cells. 3.0×103 cells were chosen because of the stability with low range of deviation of measured absorbance.
The MTT assay was done as follows：the LoVo and the SW837 cells harvested on the day 2, 4, 6, 8 and 10 of the culture were plated in the 90-well and 10μl of MTT reagent (R & D Systems, Inc., Minneapolis, MN, USA) was added and incubated for 2∼4 h at 37oC until the purple-colored precipitation was seen.
When precipitation was seen, 100μl of detergent was added to the cells and incubated again for 2 h at 37oC in the dark. The absorbance of the plates was meas-
ured at 570 nm using Bio-Kinetics Reader (Bio-Tek Instruments, Winooski, VT, USA). B0 was absorbance of the culture medium only without cells at each day of the culture.(9)
Absorbance of the LoVo and the SW837 cells was measured on day 2, 4, 6, 8 and 10 of the culture. The growth curves of the LoVo and the SW837 cells in the group 1 were illustrated by measured absorbance. In the group 2, 3 and 4, cell viability was estimated as follows：the calculated mean absorbance of each day of the culture in the group 1 was placed in the denom- inator and the measured absorbance of corresponding day of the culture in the group 2, 3 and 4 (experimental groups) was placed in the numerator, divided and multiplied by 100 to calculate percentages.
Each test was triplicated and the mean±standard devi- ation (SD) was calculated.
Cell viability (%) =
(Each absorbance of day of the culture in group 2, 3 and 4) - B0
×100 Mean of (each absorbance of day of the culture
in group 1 - B0) (B0=Absorbance of culture medium only without cells)
6) Statistical analysis
The student t-test was used for each period. The re- sult among different groups was compared with multi- ple comparison Scheffe's and Dunnet method.
Table 1. The cell viabilities of the LoVo and the SW837 cells by the AdCMVLacZ virus itself. The cell killing effect was 6∼9%
in 20 moi, 15∼22% in 50 moi and 27∼30% in 100 moi with over 80% of transduction rate.
Days of the culture
20 moi 50 moi 100 moi
LoVo SW837 LoVo SW837 LoVo SW837
2 4 6 8 10
112.3±3.5 107.3±3.1 96.7±5.0 90.7±3.1 90.7±1.5
111.0±3.6 105.0±4.5 98.0±4.0 97.3±4.7 94.0±4.0
112.7±1.2 98.7±2.1 87.7±4.0 87.0±4.4 77.3±7.0
109.0±2.6 105.3±3.0 95.3±3.0 90.0±1.0 83.7±3.8
105.0±2.6 92.7±3.0 83.3±3.1 72.0±4.0 70.0±2.0
110.3±1.5 101.0±1.0 89.7±2.5 72.7±3.1 72.7±3.1
Fig. 2. Microphotography of the transducted w-p53 genes into the LoVo (A) and SW837 (B) cells confirmed by ß-galactocidase staining on day 8 of the culture (×100).
Statistical significance was determined at P values less than 0.05.
RESULTS
1) Growth pattern of LoVo and SW837 cells The LoVo cells (w-p53 positive) showed a gradual growth curve maintaining each cell shape but drasti- cally increasing growth curve on day 8 of the culture.
Similarly, SW837 cells (m-p53 positive) also showed a gradual growth curve initially but drastically increas- ing growth curve on day 8 of the culture with more dense and not distinguishable in cell shapes compared to the LoVo cells (Fig. 1).
2) Cell viability by AdCMVLacZ virus itself
The AdCMVLacZ viruses at concentration of 20 moi, 50 moi and 100 moi were transduced into LoVo and SW837 cells. And cell killing effects of the virus itself and transduction rates were evaluated to select ad-
equate concentration of AdCMVp53 virus. The cell killing effect was 6∼9% in 20 moi, 15∼22% in 50 moi and 27∼30% in 100 moi with over 80% of transduction rate. We determined to use the 20 moi concentration of AdCMVp53 virus in the groups 3 and 4 (Table 1).
3) Transduction of AdCMVLacZ viruses and its rates.
The β-glactosidase staining was done to confirm the transduction of AdCMVLacZ viruses and its rates. The blue to green colored transducted viruses were seen from day 2 of the culture followed by progressively increasing transduction rates of over 90% in day 8 and 10 of the culture (Fig. 2).
4) P53 protein expressions after w-p53 gene transduction The p53 protein expressions were confirmed success- fully by western blotting analysis on day 2, 4, 6 and 8 of the culture.
In LoVo cells, pre-existed (endogenous) p53 proteins were expressed in moderate amounts from day 4 of
Fig. 3. Western blotting analysis with DO-1 anti-p53 antibody in the LoVo (upper) and SW837 (lower) cells (a,b,c,d
; before w-p53 transduction and A,B,C,D ; after w-p53 transduction on day 2, 4, 6 and 8 of the culture). The transduction of the w-p53 gene into LoVo and SW837 cells express p53 proteins.
Fig. 4. The cell viabilities of the LoVo and the SW837 cells by 5-FU administration of several different concentrations on day 2, 4, 6, 8 and 10 of the culture. The cell killing effects by 5-FU against LoVo and SW837 cells were gradually increased by concentration of 5-FU and duration of exposure, and LoVo cells were more sensitive than SW837 cells.
the culture followed by the expression of increased amounts on day 6 and 8 before AdCMVp53 transduction. But considerably increased amounts of p53 proteins were expressed from day 2 to 8 of the culture after AdCMVp53 transduction (Fig. 3. upper).
In SW837 cells, p53 proteins were expressed in a few or small amounts on day 2 and 4 of the culture and not expressed on day 6 and 8 before AdCMVp53 transduction. But p53 proteins were expressed in mod-
erately increased amounts after AdCMVp53 trans- duction from day 2 to 8 of the culture (Fig. 3. lower).
These results mean that the transduction of the w-p53 gene into LoVo and SW837 cells expressed p53 proteins.
5) Cell viabilities after 5-FU administration of various concentrations into LoVo and SW837 cells
In LoVo cells of group 2, cell viabilities after 5-FU administration were significantly decreased from day 4 of the culture at concentrations of 100μM and 1000 μM (78.9±13.1% and 78.9±8.6%), and from day 6 at concentrations of 0.1μM, 1μM and 10μM (79.6±5.0%, 64.9±4.7%, 51.3±16.1%) and then after followed by pro- gressively decreased cell viabilities at all concen- trations (41.3±3.0%∼19.6±0.6%) until day 10 of the cul- ture, except 0.1μM (78.1±4.4%). In SW837 cells of group 2, cell viabilities were also decreased from day 4 of the culture at all concentrations of 5-FU (93.9±4.3%
∼65.2±4.3%) and then were progressively decreased until day 10 of the culture (77.8±5.8%∼31.4±3.8%), but the degree of decrement was less than that of LoVo cells (Fig. 4). These results implied that the cell killing effects by 5-FU against LoVo cells or SW837 cells were gradually increased by concentration of the agent and duration of exposure and LoVo cells (w-p53 positive) were more sensitive than SW837 cells (m-p53 positive).
We decided to apply the 5-FU concentrations of 0.1μM, 1μM and 10μM to this study for the synergistic effect by 5-FU and w-p53 gene in group 4.
Fig. 6. The cell viability by the 5-FU administration after w-p53 gene transduction on day 2, 4, 6, 8 and 10 of the culture in the LoVo and the SW837 cell. The synergistic cell killing effects of w-p53 gene with 5-FU was shown in both the LoVo and SW837 cells.
Fig. 5. The cell viability after AdCMVp53 transduction in the LoVo and the SW837 cells. The cell killing effects of transduced w-p53 gene were similar to those of 5-FU administration of 10 μM in LoVo and SW837 cells.
6) Cell viabilities after w-p53 gene transduction Cell viabilities after AdCMVp53 transfection were similar to normal cell of group 1 on day 2 and 4 of the culture (108.6±2.3% and 116.1±1.5%), and after then were slightly decreased until day 8 (65.8±1.4%) fol- lowed by a considerable decrease in cell viabilities on day 10 (32.4±2.2%) in LoVo cells (Fig. 5) (P＜0.01).
On the other hand, cell viabilities after AdCMVp53 transfection were decreased slightly from day 2 of cul- ture (82.9± 2.1%) followed by gradual decrease until day 10 (52.1±1.0%) in SW837 cells (Fig. 5) (P＜0.01).
These cell killing effects of transduced w-p53 gene
were similar to those of 5-FU administration of 10μM in LoVo and SW837 cells (Fig. 4).
7) Cell viabilities by administration of 5-FU after w-p53 gene transduction
In LoVo cells, cell viabilities by 5-FU administration after w-p53 gene transduction were similar to that of 5-FU administration only (group 2) or AdCMVp53 transduction only (group 3) through days of the cul- ture at the concentration of 0.1μM and 1μM. But cell viabilities at the concentration of 10μM were sig- nificantly decreased from day 6 to 10 of the culture (54.4±0.9%, 37.6±0.4%, 17.4±1.1%) when compared to those of other concentrations (P＜0.05∼0.001) (Fig. 6).
On the other hand, in SW837 cells, cell viabilities were also similar to those of 5-FU administration only (group 2) or AdCMVp53 transfection only (group 3) from day 2 to 6 of culture at all concentrations of 5-FU.
But from day 8 of the culture, cell viabilities were sig- nificantly lower than those of group 3, especially at concentration of 10μM (52.1±1.0%∼28.3± 0.2%), 1μM (80.2±1.7%∼47.5±2.1%), 0.1μM (81.3±3.3%∼44.0±1.8%) on day 10 of the culture (P＜0.001), although cell via- bilities of day 10 were higher than those of LoVo cells (P＜0.01) (Fig. 6).
DISCUSSION
In colorectal cancer, gene therapies as CD suicide gene therapy, p53 gene therapy and cytokine generat-
ing gene therapy have been studied in laboratories and were applied in clinical trials as well.(6-9,12,15) Especially, several investigators reported that the w-p53 gene per se is effective in the induction of can- cer cell apoptosis and has synergistic effects with che- motherapeutic agents or radiations by induction of DNA- damage.(13,14,16)
In order to evaluate the cell killing effects of 5-FU in the presence of the w-p53 gene, we transduced w-p53 gene using recombinant adenovirus and lip- osome to w-p53 positive LoVo cells and the m-p53 positive SW837 cells and also examined the synergistic effect of w-p53 transduction and 5-FU administration.
The transduction of the w-p53 gene into the cancer cells have been studied from the early 1990's, but re- markable advances was made in recent years by use of adenovirus and liposome method.(6,17,18) Roth et al.(16) and Fujiwara et al.(17) reported that the w-p53 gene has suppressing effects on cancer cell growth and w-p53 gene transduction into the cancer cells provided an anti-cancer effect in colon, lung, and pancreatic cancers.
Levine(19) described that many factors such as m-p53 protein, mdm2 gene, HPV-16 could inactivate the function of the w-p53 gene, and additionally, the half-life of the w-p53 protein was only 20 minutes and unstable. On the other hand, the m-p53 proteins have greater stability and hours of half-life in the cells.
Thus, the ratio of the m-p53 protein to w-p53 protein in cells could be an important variable in regulating cell division, probably because the phenotype of m-p53 positive has less w-p53 proteins than m-p53 proteins and vice versa. Also the author mentioned pre-existed endogenous w-p53 gene and the transduction of exog- enous w-p53 gene using some vectors. Many inves- tigators reported that the transducted exogenous w-p53 genes into the cancer cells were well expressed and functioned to induce the cell growth arrest or apoptosis.(11,13,15,17)
We confirmed successful transduction of w-p53 genes (AdCMVLacZ) by the β-galactosidase staining in the LoVo and the SW837 cancer cells and the trans- duction rates were gradually increased over 90% until day 8 of the culture. A few investigators reported the toxicities of the AdCMVp53 viruses described as 'not significant' or 'minimum toxicities' and it account for 20% by 100 moi of adenoviruses.(12-14) We used 20
moi of AdCMVp53 viruses chosen from 20 moi, 50 moi and 100 moi because the minimum cell killing effects (6∼9%) was not significant and the maximum trans- duction rates of adnoviruses itself.
The expression of the transduced w-p53 genes was confirmed by the western blotting analysis. The p53 proteins were shown in moderate amounts before w-p53 gene transduction followed by the great amounts of increased p53 proteins after the w-p53 gene transduction in the LoVo cells. And in the SW837 cells, small amounts of p53 proteins were shown be- fore w-p53 gene transduction followed by the great amounts of increased p53 proteins through days of the culture after w-p53 gene transduction. These mean that the pre-existed and transduced w-p53 genes expressed the p53 proteins and w-p53 proteins or m-p53 proteins reacted together also because the DO-1 antibody used in this study can react with both of wild and mutant p53 proteins. Anyway, it was confirmed that w-p53 proteins were more expressed after w-p53 gene trans- duction in the LoVo and the SW837 cells.
On the other hand, Ogawa et al.(13) reported that the w-p53 gene is an essential factor during apoptotic process as the cell death mechanism starting from the DNA damage in the chemotherapy or radiotherapy, and also reported synergistically increased cell killing effects by the cisplatin administration after w-p53 gene transduction in the WiDr cells. Several investigators re- ported the cell growth inhibition after transduction of w-p53 gene into the m-p53 gene positive human tumor cells but a little or little inhibition into the w-p53 gene positive cells.(15,17,18,20)
In the present study, the cell killing effects by the transduction of w-p53 gene itself were as effective as the 10 uM of 5-FU administration from day 2 to 10 of the culture (82.9%∼52.1%) in the m-p53 positive SW837 cells. But in the w-p53 positive LoVo cells, there were no effects on day 2 and 4 followed by mod- erate effects as in the SW837 cells on day 6 and 8 (72.8% & 65.8%) and significant effects on day 10 of culture (32.4%). These results means transduced w-p53 proteins seemed to have an action or may be influ- enced by adenovirus itself in the later days of culture in the LoVo cells (Fig. 6).
On the other hand, the cell killing effect by the 5-FU administration after w-p53 gene transduction showed synergistically higher effects than those of the w-p53
transduction only or 5-FU administration only in the cell lines. The synergistic effect was as high as 10 times of the same 5-FU concentration and was enhanced by the increase of the 5-FU concentration and the duration of the culture in both cell lines. But the pattern of ef- fect was different like as the considerable effects from day 2 to day 10 of the culture (81.3%∼28.3%) in the SW837 cells, but no effects on the day 2 and 4 followed by the almost similar effects to the 5-FU administration only (76.9%∼37.6%) on the day 6 and 8 and significant synergistic effects on the day 10 of the culture (25.6%
∼17.4%) in the LoVo cells.
We observed that the cell killing effects by the trans- duced w-p53 gene and the synergistic cell killing ef- fects with 5-FU were considerable and significant from day 2 to 10 of the culture in the SW837 cells, but in the LoVo cells, although there were no synergistic ef- fects in the early days but the considerable and sig- nificant effects greater than that of the SW837 cells were observed in the later days of the culture espe- cially at 10μM of 5-FU concentration. On the effects of the 5-FU administration only, the LoVo cells (w-p53 positive) were more sensitive than the SW837 cells (m-p53 positive) only in later days of the culture.
In conclusion, we confirmed the successful trans- duction and expression of w-p53 gene using the ad- enoviral-mediated liposome method, β-galactocidase staining, western blotting. We ascertained the consid- erable cell killing effects of w-p53 gene and the syner- gistic effects with 5-FU in both the SW837 cells and the LoVo cells. When added to regional chemotherapy, in- cluding intra-arterial 5-FU, w-p53 gene transduction may provide a synergistic effect. Gene therapy using w-p53 gene transduction awaits more extensive clinical trials.
REFERENCES
1) Doci R, Gennari L, Bignami P, Montalto F, Morabito A, Bozzetti F. One hundred patients with hepatic metastases from colorectal cancer treated by resection: analysis of prognostic determinants. Br J Surg 1991;78:797-801.
2) Pawlik TM, Izzo F, Cohen DS, Morris JS, Curley SA.
Combined resection and radiofrequency ablation for advanced hepatic malignancies: results in 172 patients. Ann Surg Oncol 2003;10:1059-69.
3) Wren SM. Non-operative treatment of hepatic metastasis from colorectal carcinoma. In: Silberman H, Silberman AW,
editors. Surgical Oncology: Multidisciplinary Approach to Difficult Problems. 1st ed. London: Arnold publication; 2002.
p.668-81.
4) Weber SM, Jarnagin WR, DeMatteo RP, Blumgart LH, Fong Y. Survival after resection of multiple hepatic colorectal metastases. Ann Surg Oncol 2000;7:643-50.
5) Aoki K, Yoshida T, Sugimura T, Terada M. Liposome- mediated in vivo gene transfer of antisense K-ras construct inhibits pancreatic tumor dissemination in the murine peritoneal cavity. Cancer Res 1995;55:3810-6.
6) Bookstein R, Demers W, Gregory R, Maneval D, Park J, Wills K. p53 gene therapy in vivo of herpatocellular and liver metastatic colorectal cancer. Semin Oncol 1996;23:
66-77.
7) el-Deiry WS, Harper JW, O'Connor PM, Velculescu VE, Canman CE, Jackman J, et al. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 1994;
54:1169-74.
8) Huber BE, Austin EA, Good SS, Knick VC, Tibbels S, Richards CA. In vivo antitumor activity of 5-fluorocytosine on human colorectal carcinoma cells genetically modified to express cytosine deaminase. Cancer Res 1993;53:4619-26.
9) Rowley S, Lindauer M, Gebert JF, Haberkorn U, Oberdorfer F, Moebius U, et al. Cytosine deaminase gene as a potential tool for the genetic therapy of colorectal cancer. J Surg Oncol 1996;61:42-8.
10) Garcia-Sanchez F, Pizzorno G, Fu SQ, Nanakorn T, Krause DS, Liang J, et al. Cytosine deaminase adenoviral vector and 5-fluorocytosine selectively reduce breast cancer cells 1 million-fold when they contaminate hematopoietic cells: a potential purging method for autologous transplantation.
Blood 1998;92:672-82.
11) Zhang WW, Fang X, Mazur W, French BA, Georges RN, Roth JA. High-efficiency gene transfer and high-level expression of wild-type p53 in human lung cancer cells mediated by recombinant adenovirus. Cancer Gene Ther 1994;1:5-13.
12) Chung-Faye GA, Kerr DJ, Young LS, Searle PF. Gene therapy strategies for colon cancer. Mol Med Today 2000;6:82-7.
13) Ogawa N, Fujiwara T, Kagawa S, Nishizaki M, Morimoto Y, Tanida T, et al. Novel combination therapy for human colon cancer with adenovirus-mediated wild-type p53 gene transfer and DNA-damaging chemotherapeutic agent. Int J Cancer 1997;73:367-70.
14) Xu L, Pirollo KF, Chang EH. Tumor-targeted p53-gene the- rapy enhances the efficacy of conventional chemo/ radio- therapy. J Control Release 2001;74:115-28.
15) Harris MP, Sutjipto S, Wills KN, Hancock W, Cornell D, Johnson DE, et al. Adenovirus-mediated p53 gene transfer inhibits growth of human tumor cells expressing mutant p53 protein. Cancer Gene Ther 1996;3:121-30.
16) Roth JA, Grammer SF, Swisher SG, Komaki R, Nemunaitis J, Merritt J, et al. P53 gene replacement for cancer--interactions with DNA damaging agents. Acta Oncol 2001;40:739-44.
17) Fujiwara T, Grimm EA, Mukhopadhyay T, Zhang WW, Owen-Schaub LB, Roth JA. Induction of chemosensitivity in human lung cancer cells in vivo by adenovirus-mediated transfer of the wild-type p53 gene. Cancer Res 1994;54:
2287-91.
18) Nielsen LL, Maneval DC. P53 tumor suppressor gene therapy for cancer. Cancer Gene Ther 1998;5:52-63.
19) Levine AJ. Tumor suppressor gene. In: Mendelsohn J,
Howley PM, Israel MA, Liotta LA, editors. The Molecular Basis of Cancer. Philadelphia: W.B. Saunders Co; 1995.
p.86-99.
20) He M, Rennie PS, Dragowska V, Nelson CC, Jia W. A mutant P53 can activate apoptosis through a mechanism distinct from those induced by wild type P53. FEBS Lett 2002;517:151-4.
