Expression of the c-myc Protein Is Down-Regulated at the Terminal Stages During In Vitro Differentiation of B-Type Chronic Lymphocytic Leukemia Cells z By Lars-Gunnar Larsson, Marina Schena, Mats Carlsson, Jan Sallstrom, and Kenneth Nilsson The translocated c-myc oncogene in Burkitt's lymphoma (BL) and murine plasmacytoma (MPC) has been proposed t o be expressed at a stage of differentiation at which the gene is normally silent, resulting in a continuous proliferation and an inhibited terminal differentiation. To determine whether c-myc is differently expressed at the various stages of the differentiation pathway, we used B-type chronic lymphocytic leukemia (8-CLL) cells, representing resting B lymphocytes, inducible to proliferation and/or differentiation in vitro. The c-myc protein, and lg blight chain and PCA-1 antigen as markers of B-cell maturation, were analyzed in single, mor- phologically defined cells by immunocytochemical double- staining. The proliferation of individual cells was determined by 3H-thymidine incorporation and zyxwvuts by analysis of Ki-67 anti- zyxwvuts OST HEMATOPOIETIC tumors represent clonal M expansions of cells arrested at a preterminal stage of Chromosomal aberrations probably play a role in deregulation of the genetic programs control- ling growth and differentiation. In B-cell tumors, transloca- tions involving the c-myc and Ig loci are common features in Burkitt's lymphoma (BL), murine plasmacytoma (MPC), and rat imm~nocytoma~" but also occur in acute B-lympho- blastic leukemia (BALL).' Similar translocations of c-myc, involving the T-cell receptor loci, have been reported in T-ALL.' The crucial role of deregulated c-myc expression in malignant transformation of B cells is also suggested by the retroviral integration into the c-myc locus demonstrated in avian' and murine" lymphomas and by the high incidence of B lymphomas in transgenic mice expressing a Ek/c-myc transgene." Although the precise function of the nuclear myc oncopro- tein is unknown, observations made during recent years suggest that it is involved in the control of growth and/or differentiation.'* In vitro differentiation of hematopoietic cell lines, eg, the murine erythroleukemia (MEL)I3and the human HL-6014 and U-937,15316 is associated with a downreg- dation of c-myc expression. The significance of this reduc- tion was further suggested by the observations that terminal differentiation is inhibited in MEL" and U-937 cellsI6 constitutively expressing a transferred c- or v-myc gene. In BL and MPC, the normal c-myc allele is transcriptionally silent,18 indicating that the translocated myc oncogene may be expressed at a stage of B-cell differentiation at which c-myc expression normally is nonpermissive. Little is known, however, about c-myc regulation during B-cell differentia- tion. Selected B-type chronic lymphocytic leukemia (B-CLL) tumors may be used as model systems for studies of differentiation-associated c-myc expression because B-CLL clones represent resting (Go) B lymphocytes inducible to activation, proliferation, and differentiation closely resem- bling that of normal B cells.'' We previously showed that 12-0-tetradecanoyl-phorbol-13-acetate (TPA) induction of B-CLL cells results in a transition of the cells from Go to GI, an increase in RNA and protein synthesis, an orderly expression of B-cell activation and differentiation surface gen expression. The results show that the level of c-myc expression correlates t o the stage of differentiation and t o the proliferative activity. Uninduced resting cells did not express c-myc. The c-myc protein was observed in the highest amount at the proliferative B-lymphoblast stage of maturation and was reduced in plasmablasts and undetect- able in plasma cells. The results suggest that maturation of B cells into nonproliferative, terminally differentiated plasma cells is associated with a downregulated c-myc expression and thus support the view that the deregulated c-myc gene in BL and MPC is expressed at an inappropriate stage of maturation and thereby inhibits terminal differentiation. zy 0 1991 by The American Society of Hematology. antigens, an increase in the ratio secret0ry:membrane Ig pheavy chain mRNA, a morphologic transition of the cells into lymphoblasts and plasmablasts and secretion of IgM.20~21 The process is accompanied by a dramatic increase in expression of c-myc mRNA and protehw This increase contrasts with the observed downregulation of c-myc during differentiation of MEL, HL-60, and U-937 cells, and suggests that B-cell differentiation is compatible with c-myc expression. Induction of differentiation in B-CLL cells by TPA gives rise to a heterogeneous population of cells representing different stages of maturation, however, and only a few percent of the cells will differentiate terminally into cells with a plasma cell morphology. To determine whether c-myc expression is preferentially confined to certain stages of B-cell differentiation, we studied the expression of c-myc protein in individual B-CLL cells by immunocytochemical techniques. We used four different protocols for induction of a selected B-CLL clone, I83**: (a) StuphyZococcus zyxw aureus Cowan strain I (SAC) + B-cell stimulatory factor (BSF-MP6) + tumor necrosis factor-a (TNF-a), resulting in poor differentiation and moderate DNA synthesis; (b) SAC + BSF-MP6 + interleu- kin-2 (IL-2), leading to moderate differentiation and very high DNA synthesis; (c) TPA, inducing moderate differen- tiation and no DNA synthesis; and (d) TPA + BSF-MP6 + IL-4, resulting in an efficient differentiation and moderate DNA synthesis. The results suggest that c-myc expression in ~ From the Department of Patholo@, University of Uppsala, Univer- Submitted April 18, zyxwv 1990; accepted October 24, 1990. Supported by grants from the Swedish Cancer Society, the Children's Cancer Foundation of Sweden, and the National Swedish Board for Technical Development. Address reprint requests to Lars-Gunnar Lamon, PhD. Department of Pathology, University zyxwv of Uppsala, Universiiy Hospital, S- 751 85 Uppsala, Sweden. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with zyxwv 18 U.S.C. section 1734 solely to indicate this fact. sity Hospital, Uppsala, Sweden. 0 1991 by The American Society of Hematology. 0006-4971 /91l7705-0024$3.00/0 zyxw Blood, zyxwvutsrqponm Vol77, No 5 (March 1). 1991: pp 1025-1032 1025 Downloaded from http://ashpublications.org/blood/article-pdf/77/5/1025/604983/1025.pdf by guest on 29 October 2022