Effect of D,L-alpha-difluoromethylornithine (DFMO) enhanced [3H]putrescine uptake on 9L tumor cell growth and colony forming efficiency.

OBJECTIVE

This study explored the possible use of D,L- alpha-difluoromethylornithine (DFMO) to enhance the uptake of [3H] putrescine in order to selectively kill brain tumor cells.

METHODS

Gliosarcoma cells (9L) were grown for 4 or 20 day periods in monolayer cultures with or without [3H] putrescine and/or DFMO. Cells in culture incubated for 20 days were replated at 4-day intervals. Cells were counted on a Coulter Electronic Particle Counter and percent viability was determined by eosin dye exclusion. Survival of cells with proliferative capacity was assayed by their colony. Forming ability and surviving fraction was calculated. The radioactive counts due to [3H] putrescine were measured in 9L cells and in medium and expressed as cpm/100 cells or cpm/ml, respectively.

RESULTS

As previously reported (15), DFMO treatment resulted in termination of cell proliferation that was reversible by the addition of exogenous putrescine. Specifically, after 4 days in culture, cell counts in groups exposed to 10 mM DFMO were 55% of those in control groups and addition of 3 mM putrescine reversed the DFMO effects. Uptake of [3H] putrescine into untreated cells increased in proportion to the amount of exogenous putrescine present during 4 days of culture (range 0.01 nmol to 100 nmol) and the presence of DFMO in the medium enhanced the uptake 9 fold throughout these ranges. At activities greater than 100 cpm/100 cells the cell count was reduced to 23 to 48% of control after 4 days in culture. Extending the treatment to 20 days of incubation increased the killing of 9L cells. During the 20-day incubation, control cells increased from 5 x 10(5) to 13 x 10(12) of which 90% were colony forming cells. Treatment with either 25 microCi [3H] putrescine or 1 mM DFMO for 4 days followed by removal of these agents and incubation for an additional 16 days for a total of 20 days resulted in 31 x 10(8) or 18 x 10(7) colony forming cells, respectively. Combining [3H] putrescine and DFMO treatments during the first 4 days of the 20 day incubation reduced the colony forming cells to 21 x 10(5) (surviving fraction to 67%). When the DFMO treatment was present during the entire 20 days, it became cytotoxic since the colony forming cells were reduced to 35 x 10(3) (surviving fraction was 17%). The combination of the 4-day [3H putrescine and the 20 day DFMO treatments resulted in only 1200 surviving colony forming cells (surviving fraction was only 2%).

CONCLUSIONS

DFMO treatment of 9L cells for 20 days resulted in increased uptake of [3H] putrescine, a 10(10) fold inhibition of colony forming cells and extensive 9L cell killing relative to untreated controls.