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Restoration of Adenosine Deaminase-Deficient Human Thymocyte Development In Vitro by Inhibition of Deoxynucleoside Kinases^1,2

Michelle L. Joachims^*, Patrick A. Marble^*, Aletha B. Laurent^*, Peter Pastuszko^3,, Marco Paliotta, Michael R. Blackburn and Linda F. Thompson^4,*

^* Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; Department of Thoracic Surgery; University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, Houston, TX 77030

Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an "in vivo-like" environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health grants F32 HD008709 (to M.L.J.), HD36044 (to L.F.T.), and AI43572 (to M.R.B.) and was conducted in a facility constructed with support from Research Facilities Improvement Program Grant C06 RR14570-01 from the National Center for Research Resources, National Institutes of Health. L.F.T. holds the Putnam City Schools Distinguished Chair in Cancer Research.

² Parts of this work were presented in the Purine and Pyrimidine Society Meeting, July 2007, in Chicago, IL.

³ Current address: Rady Children’s Hospital, San Diego, CA 92123.

⁴ Address correspondence and reprint requests to Dr. Linda F. Thompson, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104. E-mail address: Linda-Thompson{at}omrf.ouhsc.edu

⁵ Abbreviations used in this paper: ADA, adenosine deaminase; Ado, adenosine; AK, adenosine kinase; dAdo, 2'-deoxyadenosine; 5'A5'dAdo, 5'-amino-5'-deoxyadenosine; dCF, 2'-deoxycoformycin; dCK, deoxycytidine kinase; dCyd, 2'-deoxycytidine; DN, double negative; DP, double positive; EDP, early double positive; FTOC, fetal thymic organ culture; hu/moFTOC, human/mouse chimeric fetal thymic organ culture; ISP, immature single positive; SAH, S-adenosylhomocysteine; TCRβ^ic, intracellular TCRβ.