Abstract
Cisplatin is a mainstay of cancer chemotherapy. It forms DNA adducts, thereby activating poly(ADP-ribose) polymerases (PARPs) to initiate DNA repair. The PARP substrate NAD+ is synthesized from 5-phosphoribose-1-pyrophosphate (PRPP), and we found that treating cells for 6 h with cisplatin reduced intracellular PRPP availability. The decrease in PRPP was likely from (1) increased PRPP consumption, because cisplatin increased protein PARylation and PARP1 shRNA knock-down returned PRPP towards normal, and (2) decreased intracellular phosphate, which down-regulated PRPP synthetase activity. Depriving cells of a single essential amino acid decreased PRPP synthetase activity with a half-life of ~ 8 h, and combining cisplatin and amino acid deprivation synergistically reduced intracellular PRPP. PRPP is a rate-limiting substrate for purine nucleotide synthesis, and cisplatin inhibited de novo purine synthesis and DNA synthesis, with amino acid deprivation augmenting cisplatin’s effects. Amino acid deprivation enhanced cisplatin’s cytotoxicity, increasing cellular apoptosis and DNA strand breaks in vitro, and intermittent deprivation of lysine combined with a sub-therapeutic dose of cisplatin inhibited growth of ectopic hepatomas in mice. Augmentation of cisplatin’s biochemical and cytotoxic effects by amino acid deprivation suggest that intermittent deprivation of an essential amino acid could allow dose reduction of cisplatin; this could reduce the drug’s side effects, and allow its use in cisplatin-resistant tumors.
Highlights
Cisplatin is a mainstay of cancer chemotherapy
The assay is dependent on the enzyme hypoxanthine–guanine phosphoribosyl-transferase (HPRT), and we showed that HPRT activity measured in cell extracts did not change after 6 h of 100 μM cisplatin treatment (Table 1)
We found that cisplatin and deprivation for a single essential amino acid individually reduced cellular availability of PRPP
Summary
Cisplatin is a mainstay of cancer chemotherapy. It forms DNA adducts, thereby activating poly(ADPribose) polymerases (PARPs) to initiate DNA repair. PRPP is a rate-limiting substrate for purine nucleotide synthesis, and cisplatin inhibited de novo purine synthesis and DNA synthesis, with amino acid deprivation augmenting cisplatin’s effects. In part, by covalently binding to the N7 position of adenine and guanine in DNA, generating intra- and inter-strand adducts[1, 3] In response to such DNA damage, poly(ADP-ribose) polymerases (PARPs) are activated, catalyzing both auto-poly(ADP-ribosyl)ation (PARylation) of a central glutamate-rich domain, as well as PARylation of chromatin-associated proteins. This generates branching polymers of ADPribose, which serve as scaffolds for DNA repair factors, including those of the mismatch repair and nucleotide exchange repair p athways[3]. Of 5-phosphoribose-1-pyrophosphate (PRPP), we hypothesized that cisplatin could deplete intracellular PRPP, a rate-limiting substrate for purine, pyrimidine, and pyridine nucleotide synthesis
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