Abstract 3583: 5-Amino-4-imidazolecarboxamide riboside-potentiated methotrexate uptake in CCRF-CEM cells
Authors: Lim Vun-Sin , Lo Crystal Pui-Kwan , Black Jennifer D. , McGuire John J. 
Published: February 2011

Abstract
We previously showed that 5-amino-4-imidazolecarboxamide riboside (Z) potentiates methotrexate (MTX) uptake in human leukemia CCRF-CEM cells via the reduced folate carrier (RFC) and also enhances MTX growth inhibitory potency. We have now studied the role of protein kinases (PK) in Z-mediated potentiation of MTX uptake. AMP-activated PK (AMPK), the prototypical target of Z, was not involved because an AMPK activator (D942) alone did not potentiate MTX uptake and an AMPK inhibitor (compound C) did not inhibit Z-mediated potentiation. We then assessed the ability of a PK inhibitor library to inhibit Z-mediated potentiation of [3H]MTX uptake. None of the 80 PK inhibitors at 10 µM affected MTX uptake in the absence of Z. At 10 µM, triciribine (Akt), GF 109203X and Ro 31-8220 (both pan-PKC) demonstrated the greatest inhibition (&gt;78%) of Z potentiation, with IC50 values of 4 µM, 1.8 µM and 1.7 µM, respectively. These data suggest that Akt and/or PKC (classical, c; novel, n; or atypical, a) could be involved in Z potentiation. Further studies with phorbol 12-myristate 13-acetate, which can selectively modulate activity of cPKC and nPKC (but not aPKC), and with PKC isotype-specific inhibitors suggested that cPKC and nPKC are not involved, but aPKC (PKC-ι and/or PKC-ζ) are. Expression of PKC-ι but not PKC-ζ in CCRF-CEM cells indicated PKC-ι to be the most likely candidate.
Our PK inhibitor screen would identify inhibitors of Z transport and metabolism, as well as of Z-potentiated RFC function. Our previous findings revealed that Z must enter CCRF-CEM cells via equilibrative nucleoside transporter-1 (ENT-1) and that Z potentiates MTX uptake in a concentration-dependent manner. It was reported by others that PKC-δ and/or -ε enhance nucleoside uptake through ENT-1 in other cell types. To investigate if PKC can regulate transport/metabolism of Z, which could ultimately affect Z potentiation, a modified transport assay and HPLC analysis were used to quantitate intracellular [14C]Z ± pan-PKC inhibitor GF 109203X. GF 109203X decreased cellular Z accumulation by 54 ± 0.7% at 20 min, with an 86 ± 6.5% lower Z monophosphate (ZMP) level. Together with the results above, these data suggest that PKC-ι enhances Z accumulation and ZMP levels, which could potentiate MTX uptake. Further studies are underway investigating the effect of PKC-ι on Z transport alone by measuring the effect of GF 109203X on [14C]Z uptake at 10 sec. In addition, Akt inhibitor, triciribine, also decreased cellular [14C]Z accumulation by 48 ± 7.8%.
Current evidence showing a role for PKC-ι and/or Akt in Z-potentiated MTX uptake may be partly or largely due to their effects on Z transport/metabolism, rather than on RFC. Because potentiation of MTX uptake requires Z transport and metabolism and at least 1 potentiating process, direct studies on posttranslational modification of RFC may provide better insight into possible mechanisms of Z-potentiated MTX uptake.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3583.