Leukemia Research Vol. 16, No. 8, pp. 797-806, 1992. 0145-2126192 $5.00 + .00 Printed in Great Britain. Pergamon Press Ltd OLIGOCLONALITY OF MOLONEY LEUKEMIAS RIVKA OFIR, JACOB GOPAS,* ESTHER AFLALO and YACOB WEINSTEIN Department of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev and * Department of Oncology, Soroka Medical Center, Beer Sheva, Israel (Received 7 April 1992) Abstract--Induction of leukemia by non-transforming retroviruses results in the appearance of various hematopoietic tumors. It is believed that these tumors are monoclonal, In this work, the clonal nature of Moloney leukemia virus (MoLV)-induced tumors was studied. Two genetic parameters were used in order to identify leukemic clones: the pattern of the proviral integration sites and the rearrangement of the T-cell receptor complex (TCR). In more than 60% of the mice, different leukemic clones populated tumors developed in different organs of the same animal. Genotypic analysis of cell lines derived from a leukemic organ revealed that the tumor is composed of more than one clone. Phenotypic analysis of subclones which were derived from a monoclonal cell line showed variability in the expression of the Thy 1.2 and MHC antigens. The results indicate that MoLV-induced tumors are of oligoclonal nature. Each leukemic organ contains a mixture of leukemic clones, of which one is dominant. Key words: MoLV, leukemic cells, rearrangement, lymphokines. INTRODUCTION THE mechanisms of induction of leukemia by non- acute retroviruses are largely unknown. Active viremia is one of the factors which are important in this process; viremic mice develop tumors 3-12 months after infection [1, 2]. Most tumors were reported to be of monoclonal origin [3, 4]; however, there were reports which raise the possibility of the existence of oligiclonal tumors [5]. It is still unknown whether the monoclonal nature of the leukemia results from a single 'hit' or from a selective process of multiple 'hits'. Statistically, in a viremic mouse, there is a chance that more than one cell will be transformed during the preleukemic state. If the leu- kemic process begins with multiple 'hits', it is reason- able to assume that the transformed cell with the best selective advantages will be dominant in the tumor. However, the presence of other leukemic cells at a lower frequency in the tumor cannot be excluded. In order to answer such a question, a detailed study of the clonal nature of the tumor is needed. Several methods were used to define the clonality Abbreviations: MoLV, Moloney leukemia virus; TCR, T-cell receptor complex; pfu, plaque forming unit; PBS, phosphate-buffered saline; FCS, fetal calf serum; IL, interleukin; Con A, Concanavlin A. Correspondence to: Dr Y. Weinstein, Department of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva, Israel. 797 of tumors. One method is based on the identification of the proviral integration sites, since each infected cell and its progeny will have a unique pattern of integrated proviruses [6]. The second method is based on the identification of the structure of the T- cell receptor gene complex, since the rearrangement of the T-cell receptor genes is a random process and unique to the progeny of each clone [7]. Therefore, DNA probes which hybridize with the proviral genes or with the T-cell receptor genes are used in Southern-blot analysis to define the clonal origin of the leukemia. The results in this study show a high percentage of heterogeneity among MoLV-induced leukemias. This heterogeneity was detected either between two leukemic organs in the same mouse or even in a leukemic tissue from a specific site (spleen, thymus, etc.). MATERIALS AND METHODS Mice Balb/C mice were obtained from the animal breeding facilities of the Weizmann Institute of Science, Rehovot and the Hebrew University, Jerusalem. Virus MoLV-NIH/3T3 mouse fibroblasts chronically pro- ducing MoLV were plated at a density of 2 × 106 per 9 cm diameter tissue culture dish (Nunc, Denmark) and 24 h later, the culture medium was changed. The fresh medium was collected after 16 h, clarified of cell debris by cen- trifugation and used as a virus stock. It contained 1-