Deoxyadenosine metabolism in a human colon-carcinoma cell line (LoVo) in relation to its cytotoxic effect in combination with deoxycoformycin

Abstract

We have assessed the intracellular metabolism of 2′‐deoxyadenosine in a human colon‐carcinoma cell line (LoVo), both in the absence and in the presence of deoxycoformycin, the powerful inhibitor of adenosine deaminase. The combination of 2′‐deoxyadenosine and deoxycoformycin has been reported to inhibit the growth of LoVo cells in culture. In this paper we demonstrate that the observed toxic effect is strictly dependent on cell density. In the absence of deoxycoformycin, 2′‐deoxyadenosine is primarily deaminated to 2′‐deoxyinosine and then converted into hypoxanthine. In the presence of the inhibitor, the deoxynucleoside, in addition to a phosphorylation process, undergoes phosphorolytic cleavage giving rise to adenine. The conversion of 2′‐deoxyadenosine to adenine might represent a protective device, emerging when the activity of adenosine deaminase is reduced or inhibited. There is much evidence to indicate that the enzyme catalyzing this process may be distinct from methylthioadenosine phosphorylase and S‐adenosyl homocysteine hydrolase, which are the enzymes reported to be responsible for the formation of adenine from 2′‐deoxyadenosine in mammals. Int. J. Cancer 75:713–720, 1998.© 1998 Wiley‐Liss, Inc.

Adenosine deaminase, a key enzyme in DNA precursors control, is a new p73 target

“…To induce an ADA deficit, we used 2 0 deoxycoformycin (dCF), a potent ADA inhibitor plus ADA substrate 2 0 deoxyadenosine (dAdo). We found that when used alone, neither dCF nor dAdo causes cellular stress, while their combination determines the inhibition of cell growth (data not shown), as reported in the literature for different cell lines (Benveniste and Cohen, 1995;Camici et al, 1995;Bemi et al, 1998). The extent of inhibition increased with the increasing time of incubation and concentration of the ADA inhibitor and substrate.…”

“…We observed only a slight increase in p21 level after dCF treatment, but we found a more evident increase in the cyclin-dependent kinase inhibitor p57Kip2, which was recently found to be modulated by p73b and not by p53 (Balint et al, 2002). The increases of p73 and ADA mRNA levels were found to vary somewhat with cell culture conditions, which are likely to reflect the complex regulation of the ADA gene (Bemi et al, 1998). To demonstrate that the increasing level of p73 is the cause of the increase in ADA mRNA expression, we transfected H1299 cells with the recombinant reporter plasmid pBLCAT2 containing ADA-RE.…”

“…The abnormal accumulation of dATP leads to the inhibition of ribonucleotide reductase (RD) and an imbalance in dNTP levels because of the lower levels of the other three deoxynucleoside triphosphates. As a result of this unbalance, both DNA repair and replicative DNA synthesis are inhibited (Camici et al, 1995;Bemi et al, 1998). We found for the first time that the ADA deficit and dAdo accumulation, caused by dCF plus dAdo, induces the arrest of the cells in the G1 and S phase with the activation of p73 and an increase in ADA mRNA expression in the p53-null H1299 cell line (Figure 7).…”

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)

“…In the latter class, besides the nucleotidase and phosphatase activities, which subtract the nucleoside monophosphate from the subsequent 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 phosphorylase, purine nucleoside phosphorylase, and S-adenosyl homocysteine hydrolase. In mammalian tissues, the existence of this activity 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 adenosine phosphorylase activity described in Sarcoma 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 process is operating, it becomes relevant in the presence of dCF. We have shown [Bemi et al, 1998] that the effect of the combination of dAdo and dCF on LoVo cell growth is strictly dependent on the initial cell density. We show here that the adenosine phosphorylase activity, which subtracts dAdo from the "activation" pathway, plays a major role in this dependence of cytotoxicity on cell density.…”