DIFFERENT PH DEPENDENCY OF MITOMYCIN-C ACTIVITY IN MONOLAYER AND 3-DIMENSIONAL CULTURES

Purpose. Previous studies by other investigators have shown an enhance ment of mitomycin C (MMC) activity at acidic extracellular pH (pH(e)) in monolayer cultures of human cells. The goal of the present study wa s to determine if the efficacy of intravesical MMC therapy in patients treated for superficial bladder cancer can be enhanced by using acidi fied dosing solutions. We evaluated (a) the effect of pH(e) on MMC act ivity in patient bladder tumors in vitro, and (b) the pH dependency of MMC activity in 2-dimensional monolayer and 3-dimensional multilayer cultures of human bladder RT4 tumor cells. Methods. Patient bladder tu mors were maintained as 3-dimensional histocultures. RT4 cells were ha rvested and maintained as monolayer cultures or as 3-dimensional cell pellets an a collagen gel matrix. The cell pellets were 300-450 cell l ayers and 4,000-5,000 mu m in diameter. Tumors or cells were incubated for 2 hr with MMC-containing media at pH(e) of 5, 6, and 7.4. The dru g effect was measured by the inhibition of DNA precursor (thymidine) i ncorporation. The stability of MMC as a function of pH(e) was determin ed. About 24% of MMC was degraded following 2 hr exposure at pH(e) 5 a nd less than or equal to 2% at pH(e) 6 and 7.4. Results. The drug conc entrations required to inhibit thymidine incorporation by 50% (IC50) w ere corrected for the degraded MMC at acidic pH(e). The results showed no pH-dependent MMC activity in human patient bladder tumors nor in R T4 multilayer cultures; the ICS, values were about 10 mu g/ml at all t hree pH(e). In contrast, the monolayer RT4 cultures showed a pa-depend ent MMC cytotoxicity; the IC50 were 0.1, 0.8 and 1.2 mu g/ml at pH(e) 5, 6 and 7.4, respectively (p < 0.05). Pre-incubation of multilayered RT4 cultures in acidic pH medium for 8 hr enhanced the MMC activity; t he IC50, was reduced by about 5 fold at pH(e) 5 and about 3 fold at pH (e) 6. Similar pH-dependent MMC activity was found when multilayers we re pre-treated for 1 hr with 0.5 mu g/ml nigericin, a proton ionophore known to cause the intracellular pH (pH(i)) to equilibrate with pH(e) . Conclusions. These data suggest that the difference in the pH depend ency of MMC activity in the monolayer and multilayer systems was due t o the different experimental conditions. The time lag for pH(i) to equ ilibrate with pH(e) in the multilayer systems and the instability of M MC at low pH(e) imply that the efficacy of intravesical MMC therapy is unlikely to be enhanced by using acidic dosing solution.