Gene Therapy and Molecular Biology Vol 7, page 27 vivo and may strongly suggest the presence of synergy in vivo. Nielsen et al, used three-dimensional statistical modeling to evaluate the presence of synergistic, additive, or antagonistic efficacy between adenovirus-mediated p53 gene transfer and paclitaxel in a panel of human tumor cell lines, including those for ovarian, head and neck, prostate, and breast cancer (Nielsen et al, 1998). Cells were either pretreated with paclitaxel 24 h or not, before proliferation was measured 3 days later. Paclitaxel had synergistic or additive efficacy with p53 transfer, independently of whether the cells expressed mutant p53 protein or no p53 protein at all. Cell cycle analysis demonstrated that, prior to apoptotic cell death, p52 transfection arrested cells in the G0/G1 stage, whereas paclitaxel arrested cells in the G2-M stage. When combined, the relative concentrations of the two agents determined the dominant cellular response. The observed synergy remained unexplained; however, some speculations were offered. P53 has been shown to down regulate the expression of the antiapoptotic bcl-2 gene and up regulate the expression of the proapoptotic bax gene in other tumor cells (Selter and Montenarh 1994). Thus, p53 and paclitaxel may potentiate each other in stimulating the apoptotic pathway in neoplastic cells (Nielsen et al, 1998). It may also be that paclitaxel increased the number of cells transfected by the adenovirus. Particularly, the concentrations of paclitaxel responsible for increased adenovirus transduction are lower than the concentrations required for microtubule condensation. Moreover, the rate of change in the number of cells transduced by adenovirus appears to be independent of paclitaxel-induced cell death. The authors also determined the efficacy of the combination therapy in vivo. In some instances, it seems that loss of p53 may increase resistance to one agent, while simultaneously increasing sensitivity to another. Bunz et al, (1999) have reported that deletion of p53 in colorectal cancer cell lines maintained the cells that were resistant to 5-fluorouracil, but increased the sensitivity to doxorubicin and radiation in vitro. If the compound exerts it effects by apoptosis, as does 5-fluorouracil, loss of the apoptotic pathway may lead to resistance.
These genes include ribonucleotide reductase, dihydrofolate reductase, DNA-dependent RNA polymerase, thymidylate synthase, c-myc, c-fos, and cmyb. Activation of these gene products facilitates the entry of the cell into the S phase. There is much evidence in support of the idea that a mutation in p53 may lead to resistance to cytotoxic agents. In premenopausal women with node negative breast cancer, it has been shown by immunohistochemistry that p53(+) tumors are less sensitive to treatment with a regimen including 5-fluorouracil, doxorubicin, and cyclophosphamide than p53 (-) tumors. (Clahsen et al, 1998). Under in vitro conditions Koechli et al, have shown that mutant p53 can increase chemoresistance to 5fluorouracil, cyclophosphamide, and methotrexate (Koechli, 1994). Cisplatin resistance seems to be connected with p53 mutations, and in advanced ovarian cancer, the p53 mutational status is a predictor of the responsiveness to platinum-based chemotherapy (Calvert, 1999). However, there are also reports that apparently disagree with the chemoresistance effect of p53 (Fan et al, 1995; Stal 1995; Hawkins et al, 1996). Human fibroblasts lacking functional p53 were more sensitive to cisplatin, carboplatin, paclitaxel, nitrogen mustard or melphalan than cells with functional p53 (Hawkins et al, 1996). Similar results, loss of p53 function and the sensitizing effect of cisplatin, have been demonstrated in MCF-7 breast cancer cells and RKO methotrexate, and 5-fluorouracil have been reported in colon cancer cell lines with or without disruption of p53 function by a dominant negative p53 transgene (Fan et al, 1995). Increased rates of response to cyclophosphamide, patients with breast cancer who were determined to be immunohistochemically p53(+) (Stal,1995).
B. In vitro interactions Synergy between two chemical agents in vitro is an empirical phenomenon, in which the observed effect of the combination is greater than would be predicted from the effect of each agent working alone. While synergy is not directly measurable in clinical practice, it may predict a favorable outcome when two treatments are combined in
Figure 2. Two examples of cell cycle arrest via p53 activation. P53 mediated cell-cycle arrest is demonstrated with two examples: A) inhibition of cdk4 and cdk2 resulting G1-S and G2-M arrest, respectively. B) p53 activation increases the transcription of the cyclindependent kinase (cdk) inhibitor p21. Increase levels of p21 protein prevent cdk’s from phosphorylating their substrates, such as the retinoblastoma protein (RB) and thus block cell-cycle progression from G1 into S phase. (Kirsch 1998; Brown and Wouters 1999)
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