Role of the Phosphorolysis of Deoxyadenosine in the Cytotoxic Effect of the Combination of Deoxyadenosine and Deoxycoformycin on a Human Colon Carcinoma Cell Line (LoVo) Francesco Giorgelli, 1 Michela Giannecchini, 1 Valentina Bemi, 1 Gino Turchi, 2 Francesco Sgarrella, 3 Maria Grazia Tozzi, 3 and Marcella Camici 1 * 1 Dipartiment o di Fisiologi a e Biochimica , Pisa , Italy 2 Istitut o di Mutagenes i e Differenziament o del CNR , Pisa , Italy 3 Dipartiment o di Scienz e del Farmaco , Sassari , Italy Abstract I n LoV o cells, phosphorolyti c activit y actin g on deoxyadenosin e play s a majo r rol e in th e resistanc e to th e cytotoxic effect of th e combinatio n of deoxynucleoside wit h deoxycoformycin. I n fact , th e observed dependence of toxicit y on cell densit y appear s to be relate d to th e metaboli c conversion of deoxyadenosin e int o adenine . The phosphorylatio n of th e deoxynucleoside, which represent s th e firs t step toward s th e formatio n of th e cytotoxic agent dATP , proceeds at a significantl y lower rat e as compared to th e phosphorolysi s of deoxyadenosine . Th e analysi s of the levels of deoxyadenosin e an d it s derivative s in th e incubatio n media reveal s tha t th e rate s of disappearanc e of deoxyadenosin e and of formatio n of adenin e increas e in concert with the reductio n of the effect on cell survival . J . Cell. Biochem . 80:241–247, 2000. © 2000 Wiley-Liss , Inc. Key words: deoxyadenosine ; deoxycoformycin; LoVo; purin e enzymes; adenosin e phosphorylase The combination of deoxyadenosine (dAdo) and deoxycoformycin (dCF), a powerful inhibi- tor of adenosine deaminase (ADA) [Agarwal et al., 1977], has proven to affect the growth of a human colon carcinoma cell line (LoVo) [Camici et al., 1995]. The cytotoxicity seems to be dependent on phosphorylation of dAdo, be- cause inhibitors of adenosine kinase activity significantly reduced the effect of the combina- tion [Camici et al., 1995]. In general, when dealing with compounds that exert their effect after metabolic conversion into the effective cytotoxic agent (pro-drugs), the complete met- abolic pattern of the pro-drug must be taken into account. Indeed, not only the “activation”, but also the “inactivation” pathways play a cru- cial role in the determination of the efficacy of the cytotoxic agent. In particular, the efficacy of the combination of dAdo and dCF is likely due to the balance between the “activating” enzymes (adenosine kinase, deoxycytidine ki- nase) and “inactivating” enzymes (Fig. 1). In the latter class, besides the nucleotidase and phosphatase activities, which subtract the nucleoside monophosphate from the subse- quent phosphorylation steps that lead to the formation of the cytotoxic agent dATP, in LoVo cells we have recently isolated a phosphorolytic enzyme activity acting on dAdo [Bemi et al., 1998] distinct from methylthioadenosine phos- phorylase, purine nucleoside phosphorylase, and S-adenosyl homocysteine hydrolase. In mammalian tissues, the existence of this activ- ity appears to be a novelty because its presence has been reported in several biologic sources [Jensen, 1978; Trembacz and Jezewska, 1994], however, with the only exception of an adeno- sine phosphorylase activity described in Sar- coma 180 cells and rat liver [Divekar, 1976], not in mammals. Although the phosphorolysis of dAdo appears to be a secondary metabolic pathway in LoVo cells when the deamination Grant sponsors: CNR (Target Project Biotecnologie), MURST. *Correspondence to: Marcella Camici, Dipartimento di Fi- siologia e Biochimica, Via S. Maria 55, 56100 Pisa, Italy. E-mail: camici@dfb.unipi.it Received 27 July 1999; Accepted 5 May 2000 Journal of Cellular Biochemistry 80:241–247 (2000) © 2000 Wiley-Liss, Inc.