formycins and Osteosarcoma

Two subtypes of equilibrative transporters, es (equilibrative inhibitor-sensitive) and ei (equilibrative inhibitor-insensitive), are responsible for the majority of nucleoside flux across mammalian cell membranes. Sequence analyses of the representative genes, ENT1 {equilibrative nucleoside transporter 1; also known as SLC29A1 [solute carrier family 29 (nucleoside transporters), member 1]} and ENT2 (SLC29A2), suggest that protein kinase CK2-mediated phosphorylation may be involved in the regulation of es- and ei-mediated nucleoside transport. We used human osteosarcoma cells transfected with catalytically active or inactive alpha' and alpha subunits of CK2 to assess the effects of CK2 manipulation on nucleoside transport activity. Expression of inactive CK2alpha' (decreased CK2alpha' activity) increased the number of binding sites (approximately 1.5-fold) for the es-specific probe [3H]NBMPR ([3H]nitrobenzylthioinosine), and increased (approximately 1.8-fold) the V(max) for 2-chloro[3H]adenosine of the NBMPR-sensitive (es) nucleoside transporter. There was a concomitant decrease in the V(max) of the NBMPR-resistant (ei-mediated) uptake of 2-chloro[3H]adenosine. This inhibition of CK2alpha' activity had no effect, however, on either the K(D) of [3H]NBMPR binding or the K(m) of 2-chloro[3H]adenosine uptake. Quantitative PCR showed a transient decrease in the expression of both hENT1 (human ENT1) and hENT2 mRNAs within 4-12 h of induction of the inactive CK2alpha' subunit, but both transcripts had returned to control levels by 24 h. These data suggest that inhibition of CK2alpha' reduced ei activity by attenuation of hENT2 transcription, while the increase in es/hENT1 activity was mediated by post-translational action of CK2. The observed modification in es activity was probably due to a CK2alpha'-mediated change in the phosphorylation state of the ENT1 protein, or an interacting protein, effecting an increase in the plasma membrane lifetime of the transport proteins.

Topics: 2-Chloroadenosine; Bone Neoplasms; Casein Kinase II; Catalytic Domain; Cell Line, Tumor; Computer Systems; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Formycins; Gene Expression Regulation, Neoplastic; Humans; Nucleosides; Osteosarcoma; Polymerase Chain Reaction; Substrate Specificity; Thioinosine; Transfection; Tritium

Formycin 5'-triphosphate (FoTP), a fluorescent analog of ATP, is shown to be a substrate for the membrane-bound adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from rat osteosarcoma cells. The formation of the adenylate cyclase reaction product, 3',5'-cyclic formycin monophosphate (cFoMP), was followed by the conventional radioimmunoassay (RIA) procedure used to detect cAMP and by an assay procedure in which the reaction product was separated from the substrate by reverse-phase high-pressure liquid chromatography (HPLC) and the reaction product was detected by fluorometry. Because the HPLC--fluorometric procedure can determine the amount of cFoMP present in the reaction mixture within 6 min, the enzymatic conversion of FoTP to cFoMP can be followed directly during the course of a typical 15-min incubation. The amount of cFoMP detected by this procedure was found to be within 2% of the values obtained by the RIA. The rate of product formation with FoTP was similar to that observed with ATP and the activity of the enzyme was enhanced about 5-fold with guanyl-5'-yl imidodiphosphate when either ATP or FoTP was used as the substrate. Kinetic studies revealed values for the Vmax of 120 pmol/min per mg of protein and apparent Km values of 220 microM with both substrates. In addition to suggesting that the recognition of the substrate by the adenylate cyclase may not require a specific chemical structure of the 5-membered ring of the base or a unique configuration about either the glycosyl or the C(5')-C(4') bond, the results of this study are consistent with the idea that the cytotoxicity observed with the adenosine analog formycin may be the result of its metabolism to cFoMP. Furthermore, these studies indicate that the fluorescent analog FoTP can be used, in combination with HPLC, to provide an alternative, nonradioactive direct method for the assay of adenylate cyclase catalytic activity.

Topics: Adenylyl Cyclases; Antibiotics, Antineoplastic; Cell Membrane; Formycins; Neoplasms, Experimental; Osteosarcoma; Radioimmunoassay; Ribonucleotides; Substrate Specificity