Methioninase gene therapy of human cancer cells is synergistic with recombinant methioninase treatment

Results obtained over the past 40 years have demonstrated that tumor cells of all types tested have an elevated growth requirement for methioninase co mpared with normal cells. Recombinant methioninase (rMETase) cloned from Ps eudomonas putida has been found previously to he an effective antitumor age nt attributable to deprivation of the extracellular methionine source of th e tumor. To degrade intracellular methioninase, we have nom developed an ad enoviral vector inserted with the P. putida methioninase (MET) gene (rAd-ME T). The in vitro efficacy of rAd-MET was tested on the OVCAR-8 human ovaria n cancer cell line, the HT1080 human fibrosarcoma tell line, and human norm al fibroblasts. rAd-MET transduction of OVACAR-8 and HT1080 resulted in hig h levels of methioninase expression up to 10% or more of the total protein of the cells, depending on the multiplicity of infection. The IC50 of rAd-M ET for OVCAR-8 cells in 96-well plates was approximately 2 x 10(6) plaque-f arming units (pfu)/well. The IC50 of control adenovirus (control-rAd) was 4 x 10(7) pfu/well, 20 times higher than rAd-MET. In the presence of the IC5 0 of 2 x 10(6) pfu/well of rAd-MET, the addition of 0.025 units/ml of rMETa se, which is 25% of the IC50, resulted in a 90% inhibition of tumor cell nu mber. This indicated that rAd-MET enhanced the efficacy of rMETase, In cont rast, 2 x 10(6) pfu/well of control-rAd in combination with 0.025 units/ml of rMETase had an efficacy of only 10% inhibition of cell number. The syner gistic effect of the combination of rMETase and rAd-MET was quantitated by calculating the combination index (CI), The CIs for all combinations of rAd -MET and rMETase tested on OVCAR-8 were <0.7 with a mean of 0.5, indicating synergy. Similar synergy of rAd-MET and rMETase was seen on HT1080 human f ibrosarcoma cells with a mean of 0.74. In contrast, the CIs of all combinat ions of rMETase and control adenovirus concentrations tested on both cell l ines had a mean CI of similar to 1, which indicated that this combination h ad only an additive effect. The normal fibroblasts, on the other hand, appe ared relatively resistant to the MET gene because in the presence of rMETas e, 2.5 x 10(7) pfu/well of rAd-MET or control rAd had almost an identical e ffect on cell survival. The selectively strong synergy of rAd-MET and rMETa se on cancer cells allows reduced levels of each agent to be used, thus dec reasing potential side effects.