Enhanced uptake of [3H] spermidine by 9L rat brain tumors after direct intratumoral infusion of inhibitors of enzymes of the polyamine biosynthetic pathway.

We have been exploring the feasibility of delivering ionizing radiation to brain tumor cells by using tritium labeled polyamines. Polyamines are taken up preferentially by dividing cells and form noncovalent bonds with DNA. Their uptake can be enhanced by drugs which deplete endogenous polyamines. To test this in vivo, 9L cells were implanted in the striatal region of the brain in male Fisher 344 rats. Osmotic pumps containing trace amounts of [3H] spermidine or [3H] putrescine with either difluoromethylornithine or combinations of 3 inhibitors of enzymes of the polyamine biosynthetic pathway were implanted subcutaneously and were connected to intratumoral cannulas. After 14-16 days the brains were removed and sliced in the coronal plane. The diameters of the tumors were measured and tumor tissue was dissected from each slice, weighed and lysed for scintillation counting. It was found that difluoromethylornithine enhanced the uptake of [3H] putrescine while a combination of inhibitors of enzymes of the polyamine biosynthetic pathway enhanced the uptake of [3H] putrescine and [3H] spermidine producing a localized region of radioactivity in the 9L tumor. It is estimated that if the [3H] polyamines were at higher specific activity (commercially available), instead of the trace dose given here, the [3H] polyamine uptake would be sufficient to kill 9L tumor cells within a 2 to 3 week period.

Difluoromethylornithine enhanced uptake of tritiated putrescine in 9L rat brain tumors.

Difluoromethylornithine (DFMO) depletes endogenous putrescine and enhances the uptake of and retention of [3H] putrescine in vitro. To determine if DFMO also enhances uptake of [3H] putrescine in vivo, DFMO and trace doses of [3H] putrescine, dissolved in artificial CSF, were infused into growing (6-9 day) 9L brain tumors by means of osmotic pumps. When 7-day osmotic pumps were loaded with 1 microCi [3H] putrescine, with or without 10 or 100 mM DFMO, pumped at 1 microl/h, the mean uptake after 3 days was 168 +/- 62 cpm/mg tumor (17 rats) without DFMO, 300 +/- 197 cpm/mg tumor (11 rats) with 10 mM DFMO and 1088 +/- 421 cpm/mg tumor (11 rats) with 100 mM DFMO (p < or = 0.05 vs. control). Significantly less radioactivity was detected in the contralateral brain and in nonbrain tissues (0.5 +/- 0.1 to 14 +/- 5 cpm/mg). To measure the extent of [3H] putrescine distribution in the tumor, the same dose of drugs was delivered for a longer period of time, using 14-day pumps to allow tumors to become large enough to be divided into 1.4 mm thick transections. The mean radioactivity in the sections from eight control rats receiving [3H] putrescine without DFMO were not significantly different between the sections (174 +/- 61 cpm/mg tumor for sections containing the cannulas, 273 +/- 61 and 259 +/- 91 cpm/mg for adjacent sections). In the six rats given 100 mM DFMO there was a significant increase in mean radioactivity in the cannula containing section (2251 +/- 919 cpm/mg tumor). Mean counts from adjacent sections in these rats were 97 +/- 44 and 33 +/- 13 cpm/mg. Values for contralateral corpus striatum and nonbrain tissues ranged from 0.7 +/- 0.3 to 4.3 +/- 1.5 cpm/mg tissue. When DFMO was delivered directly to the tumors while [3H] putrescine was infused intraperitoneally, the uptake in the tumor slices was low (5-10 cpm/mg in different slices). These results demonstrate that infusion of DFMO directly into growing 9L brain tumors can selectively enhance the uptake of exogenous [3H] putrescine by rapidly dividing cells which are within a 1.4 mm diameter area at the cannula tip. Although these studies used [3H] putrescine at trace doses, it is estimated that infusion of higher doses of [3H] putrescine plus DFMO will selectively kill tumor cells.