GTMB 7 - Gene Therapy & Molecular Biology

Jekunen et al: Strategy of sensitizing tumor cells with adenovirus-p53 transfectionRecently, a report using isobologram modelling haveshowed that the combination of adeno-p53 + radiationproduced significantly synergistic effects in NSCL celllines, whereas the combination of docetaxel + adeno-p53and docetaxel + radiation produced mixed effects rangingbetween additive and synergistic (Nguyen al., 1996). Thethree-agent combination also produced significantlysynergistic effects.Brown and Wouters have criticized the sensitizingresults obtained in cell cultures. They have pointed out theneed for further evidence in relating p53 to the sensitivityof anticancer agents (Brown and Wouters, 1999). Becauseapoptosis, particularly p53–dependent apoptosis, can occurrapidly after drug exposure, short-term growth rate assaystend to underestimate overall death of cells with mutantp53 or of cells not undergoing apoptosis. This may resultin a situation where short-term assays may incorrectlyassess overall cell death in tumor cells with differentprobabilities of undergoing early apoptosis. Thus, resultsmay have a bias toward increased cell death in wild-typep53 cells and decreased cell kill in mutant p53 cells.Results of experiments with normal cells transformed withdominant oncogenes have often been extrapolated totumor cells, instead of initially using cancer cell models.Transformed normal cells are usually apoptotically moresensitive than cancer cells. Therefore, in sensitizingexperiments, both long term clonogenic assays and tumorcell models with solid tumors should be used rather thangrowth rate assays and transformed normal cells.However, the more widely accepted conclusion drawnfrom studies conducted in cancer cell lines and tumors ofdifferent origin is still that restoration of normal p53function in tumors restores the apoptotic pathway andleads to an increased response to chemotherapy (Peller,1998; Ferreira, 1999; Chang, 2000).C. Transfection of cell cultures with theadenovirus p53 gene constructAdenovirus vectors have many advantages over otherviral and non-viral vectors. Their transfection efficacy ishigh, in both dividing and resting cells, and they showhigh expression levels (Hwu, 2001). As adenoviral DNAis not incorporated into the cell genome, expression of thetransgene is transient, but adenoviral vectors can beproduced at high titers. Introduction of wild-type p53 intotumors with non functional p53 offers a novel strategy fortreating cancer, by inducing apoptotic death in neoplasticcells.Genomic instability accompanied by loss of p53-mediated apoptosis can also lead to therapy resistance. Thesupport for this rationale is that loss of p53 coulddesensitize cells to the damaging effects of drugs. Normaltransgenic hematopoetic cells (Lotem and Sachs, 1993),E1A-expressing transgenic fibroblasts (Lowe et al, 1993),and transformed transgenic fibroblasts (Lowe et al, 1994)were all more resistant to apoptosis following treatmentwith any of a wide variety of anticancer agents, than werecomparable cells from the parental strain of mice, whichexpressed wild-type p53. Apoptosis seemed to beenhanced in cells that expressed wild-type p53 and wereable to trigger their own cell death program.In cell culture models, adenovirus-mediated p53 genetransfer alone inhibits cell growth and promotes apoptosis,regardless of the endogenous p53 status of the ovariancancer cells (Santoso et al, 1995). In tumor cells, mutatedp53 and also loss of p53 function were associated withresistance to chemotherapeutic agents. There are severalreports of at least an additive interaction between adenop53and cisplatin in bladder cancer (Miyake et al, 2000),between adeno-p53 and cisplatin, SN-38 (a metabolite ofirinotecan), 5-fluorouracil, taxanes, bleomycin, andcyclophosphamide in NSCLC (Fujiwara et al, 1994)(Horio et al, 2000), and between adeno-p53 and paclitaxelin ovarian cancer (Nielsen et al, 1998). In the ovariancancer model, enhanced efficacy has been reported in athree-drug combination of adeno-p53, cisplatin, andpaclitaxel (Gurnani et al, 1999).There is some evidence that chemosensitivity can beincreased by replacement of the p53 gene. Roth (Roth,1996) reported that recombinant-adenovirus-mediatedtransfer of the wild-type p53 gene into several human cellswith homozygous deletions of p53 markedly increasedcellular chemosensitivity to the major chemotherapeuticdrugs. An additive antiproliferative effect was reported inp53null H358 lung cancer cells when cultured withcisplatin for 24 h before transduction with adeno-p53(Fujiwara et al, 1994). Enhanced apoptosis, detected byDNA fragmentation, was reported for the combinationcompared with each agent alone.A viability assay demonstrated that a replicationdefectiveadenovirus encoding the wild-type p53 gene(INGN 201, Introgen Therapeutics, Inc.) suppressesgrowth and enhances sensitivity to DNA-damagingchemotherapeutic drugs (5-fluorouracil, doxorubicin,cisplatin) in p53-mutant-expressing cell lines (Gjerset andMercola, 2000). These cells lines represent DLD-1 coloncancer, T47D breast cancer, PC-3 prostate cancer, andT98G glioblastoma. Transfection efficiencies were 60-70%. It seems that restoration of the wild-type p53 tomutant p53-expressing or p53null cells results in markedenhancement of sensitivity to several DNA damagingagents. This enhancement of sensitivity was not observedin two wild-type p53-expressing cell lines, MCF7 andLS174T, suggesting that, in this model, wild-type p53gene transfer is effective as therapy sensitization only intumors that have lost wild-type p53 function.1. Glioma and pancreatic cancerSomatic gene therapy based on the reintroduction ofp53 limits the proliferation of human malignant gliomacells, but is unlikely to induce clinically relevantsensitization to chemotherapy in these tumors. Wild-typep53 failed to sensitize glioma cells to cytotoxic drugsincluding BCNU, cytarabine, doxorubicin, teniposide, andvincristine. The combined effects of the wild-type p53gene transfer and drug treatment were less than additiverather than synergistic, suggesting that the intracellularcascades activated by p53 and chemotherapy wereredundant. Unexpectedly, forced expression of mutant-28