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. 1999 Mar;44(3):366–371. doi: 10.1136/gut.44.3.366

Adenovirus mediated p53 tumour suppressor gene therapy for human gastric cancer cells in vitro and in vivo

M Ohashi 1, F Kanai 1, H Ueno 1, T Tanaka 1, K Tateishi 1, T Kawakami 1, Y Koike 1, T Ikenoue 1, Y Shiratori 1, H Hamada 1, M Omata 1
PMCID: PMC1727416  PMID: 10026322

Abstract

BACKGROUND/AIMS—Gastric cancer is one of the most prevalent forms of cancer in East Asia. Point mutation of the p53 gene has been reported in more than 60% of cases of gastric cancer and can lead to genetic instability and uncontrolled cell proliferation. The purpose of this investigation was to evaluate the potential of p53 gene therapy for gastric cancer. 
METHODS—The responses of human gastric cancer cell lines, MKN1, MKN7, MKN28, MKN45, and TMK-1, to recombinant adenoviruses encoding wild type p53 (AdCAp53) were analysed in vitro. The efficacy of the AdCAp53 treatment for MKN1 and MKN45 subcutaneous tumours in nude mice was assessed in vivo. 
RESULTS—p53-specific growth inhibition was observed in vitro in two of four gastric cancer cell lines with mutated p53, but not in the wild type p53 cell line. The mechanism of the killing of gastric cancer cells by AdCAp53 was found, by flow cytometric analysis and detection of DNA fragmentation, to be apoptosis. In vivo studies showed that the growth of subcutaneous tumours of p53 mutant MKN1 cells was significantly inhibited by direct injection of AdCAp53, but no significant growth inhibition was detected in the growth of p53 wild type MKN45 tumours. 
CONCLUSIONS—Adenovirus mediated reintroduction of wild type p53 is a potential clinical utility in gene therapy for gastric cancers. 



Keywords: gastric cancer; p53; adenovirus; gene therapy

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Figure 1 .

Figure 1

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Transduction efficiency of adenovirus in gastric cancer cells. MKN1 (A) and MKN45 (B) cells were plated on six-well plates and infected with AdCAlacZ at a multiplicity of infection of 30. After 24 hours, cells were fixed and stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside to demonstrate lacZ gene expression. Original magnification × 100. 


Figure 2 .

Figure 2

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Effect of AdCAp53 on gastric cancer cell viability. Gastric cancer cells, MKN1(A), MKN7 (B), and MKN45 (C), were infected with either AdCAlacZ or AdCAp53 at a multiplicity of infection (moi) ranging from 1 to 300. Five days after infection, effects on cell viability were measured by the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay. Data are expressed as a percentage (mean (SD)) of the absorbance (A570 − A620) of untreated cells. 


Figure 3 .

Figure 3

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Adenovirus mediated p53 expression in human gastric cancer cells. MKN45, MKN7, and MKN1 cells were left untreated (lanes 1, 4, and 7) or were infected with AdCAp53 (lanes 2, 5, and 8) or AdCAlacZ (lanes 3, 6, and 9) at a multiplicity of infection of 30. Western blot analysis of cell lysates was performed with the p53-specific antibody. 


Figure 4 .

Figure 4

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Detection of apoptotic cells by flow cytometry. About 1 × 106 MKN1 and MKN45 cells were left untreated (A, D) or were treated with AdCAlacZ (B, E) or AdCAp53 (C, F) at a multiplicity of infection of 30. After 24 hours, cells were centrifuged and fixed in 70% ethanol. Samples were then treated with RNase, stained with propium iodide, and analysed by flow cytometry. Untreated control (A) or AdCAlacZ treated (B) MKN1 cells showed little sub-G1 peak. AdCAp53 treated MKN1 cells showed a significant sub-G1 peak indicative of apoptosis (C). The flow cytometry histograms for untreated control, AdCAlacZ treated, and AdCAp53 treated MKN45 cells show no significant hypodiploid fraction (D, E, F). 


Figure 5 .

Figure 5

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DNA fragmentation induced by AdCAp53 infection. MKN1 and MKN45 cells were untreated (lanes 1 and 4) or treated with AdCAlacZ (lanes 2 and 5) or AdCAp53 (lanes 3 and 6) at a multiplicity of infection of 30. The positions of molecular mass markers (lane M) are indicated on the right (base). DNA was prepared from 5 × 105 cells as described in the Methods section; 10 µl aliquots from 50 µl DNA solution were separated on a 2.5% agarose gel in the presence of 0.5 µg/ml ethidium bromide. 


Figure 6 .

Figure 6

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Effect of AdCAp53 on established tumours. After reaching a size of 4 mm in diameter, tumours were treated with three injections (arrows) on days 8, 10, and 12 of control vehicle or adenovirus (total dose 3 × 107 plaque forming units). Tumour volume was plotted for each treatment animal group (n = 6) against days after inoculation (mean (SE)). (A) Over the 30 day experimental period, MKN1 tumour growth was significantly suppressed in AdCAp53 treated mice in comparison with those treated with control vehicle or AdCAlacZ (p<0.001). (B) In MKN45 tumour growth, there was no significant difference between untreated and AdCAp53 treated animals. 


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