Proliferation of Acute Lymphoblastic Leukemic (ALL) Cells Is Dependent on Exogenous Purine Administration.
Authors: Goselink Henriette M. , Nijmeijer Bart A. , Hokke Cornelis H. , van der Heijden Rob , Willemze Roel , Falkenburg J.H. Frederik 
Published: October 2019

Abstract
To study the biology of ALL, primary leukemic cells were cultured in a longterm serum-free culture system. In 10 out of 25 primary precursor B-ALL cells we succeeded to establish ALL cell lines, phenotypically identical to their original clone. The cultured leukemic cells proliferated for more than two years with a constant doubling time of 2.0 ± 0.3 days, but only in cultures of moderate to high densities, suggesting the production of an autocrine proliferation inducing factor. To study the possible autocrine production of a proliferation inducing factor low cell concentrations (1,000 cells/ml/well) were cultured in the absence or presence of autologous conditioned medium(CM, 20%v/v). In the absence of CM, no proliferation was observed as determined by 3H-thymidine uptake as well as in a limiting dilution assay in 9 out of 10 ALL samples. In the presence of CM, proliferation was induced in all ALL cell lines. CM generated from a variety of other unrelated cell sources, as well as CM derived from lysed cells also induced identical proliferation in all ALL samples demonstrating that the samples proliferated and responded to a factor present in all cell populations.To characterize this autocrine factor CM was prepared from ALL cell lysates. Chemical analysis of this CM revealed that the factor was a heat stable molecule with a molecular size less than 500 Dalton. The factor was further purified on a carbograph column and finally on a Superdex Peptide size-exclusion column. The active fractions were analysed by NMR spectroscopy and the pattern of the spectrum strongly suggested the presence of a heterocycle purine. CE-MS analysis confirmed this observation by detecting the presence of a molecule with a mass of 136.05, identical to the theoretical mass of Hypoxanthine. By MS/MS fragmentation analysis of both the isolated compound and synthetic Hypoxanthine the compounds were found to be identical. Addition of the chemically synthetised Hypoxanthine (0.45 μM) to low cell concentrations of ALL cell cultures as well as to cultures of primary ALL cells induced proliferation comparable to cell cultures in the presence of CM. Proliferation of ALL cells could also be induced by Deoxyinosine or Inosine comparable to Hypoxanthine but only moderate to low proliferation was observed after addition of Adenine or Adenosine and no proliferation in the presence of Guanine, Guanosine or Inosinemonophosphate. In conclusion, we developed a culture system allowing identification of the requirements of ALL for proliferation in vitro to study the biology of these leukemic cells. Using this culture system, we illustrated that ALL cells appeared to be highly dependent on an exogenous source of Hypoxanthine or its metabolic component. This dependency may open new perspectives in the development of treatment for ALL, and may result in a more specific control of the different metabolic pathways of purines in malignant cells.