CYTOTOXICITY AND METABOLISM OF ETA-D-RIBOFURANOSYLAMINO)PYRIMIDO[5,4-D]PYRIMIDINE IN HCT-116 COLON-CANCER CELLS

We examined the cytotoxicity, biochemical effects and metabolism of 4- methoxy-8-(beta-D-ribofuranosylamino)pyrimido[5, 4-d]pyrimidine (MRPP) , a synthetic nucleoside inhibitor of phosphoribosylpyrophosphate synt hetase, in HCT 116 human colorectal cancer cells. A 4-hr exposure to 1 and 10 mu M MRPP inhibited cell growth over a 72-hr period by 76 and 89%, and inhibited clonogenic capacity by 36 and 65%, respectively. MR PP was avidly metabolized to the 5'-monophosphate derivative (MRPP-MP) , and MRPP-MP formation increased with increasing MRPP exposure (mu M. hr). MRPP-MP was stable, and the intracellular half-life was in excess of 48 hr. A 4-hr exposure to 10 mu M MRPP resulted in significant dec reases in ATP, UTP, GTP, CTP, dATP, dTTP, and PRPP pools. Near maximal ribonucleotide triphosphate depletion was achieved with greater than or equal to 24 mu M.hr MRPP, and growth inhibition as a function of MR PP mu M hr closely reflected the biochemical effects. Ribonucleotide t riphosphate pools remained depleted for up to 48 hr after drug removal , apparently as a consequence of the prolonged retention of MRPP-MP. M RPP (10 mu M) inhibited the salvage of [H-3]guanine, [H-3]adenine and [H-3]guanosine, and concurrent exposure to MRPP and either 100 mu M ad enine, hypoxanthine, or guanine did not reverse ATP or GTP depletion. Concurrent exposure to 10 mu M MRPP and either 10 mu M adenosine, urid ine or thymidine was accompanied by repletion of ATP, UTP, and dTTP po ols, respectively, but depletion of other nucleotide pools was not cor rected. In contrast, 10 mu M guanosine did not correct GTP depletion i n the presence of MRPP. The combination of 10 mu M each of thymidine, uridine, adenosine and guanosine during and following a 24-hr exposure to MRPP provided partial protection against 0.1 or 1 mu M MRPP, but d id not affect the cytotoxicity associated with 10 mu M MRPP. MRPP is a novel antimetabolite that inhibits both de novo and salvage pathways for purine synthesis and de novo pyrimidine synthesis.