Lars-Gunnar Larsson

Analysis of the DNA-binding activities of Myc/Max/Mad network complexes during induced differentiation of U-937 monoblasts and F9 teratocarcinoma cells

The bHLHZip protein Max interacts with both the Myc and Mad family proteins forming heterodimers which specifically bind certain E-box DNA recognition sequences, thereby regulating transcription. Whereas Myc proteins actively promote cell proliferation, Mad complexes have the opposite function. Although the main regulation of this network seems to be the control of myc- and mad family gene expression, regulation at the level of DNA-binding and transactivation may also be in operation. Few studies on the DNA-binding activity of native Mycu2009:u2009Max or Maxu2009:u2009Mad complexes have been reported mainly due to technical difficulties. To overcome these problems we have developed a specific and sensitive solid phase DNA-binding assay based on partial purification of native Myc, Max and Mad1 complexes by immunological methods. Using this technique we report that the DNA-binding activity of c-Myc-containing complexes is reduced during induced differentiation of U-937 monoblasts and F9 embryonic teratocarcinoma cells. In contrast, the DNA-binding of Mad1-containing complexes increases during monocytic differentiation. In general, the DNA-binding activity of c-Myc and Mad1 correlate with their expression. However, our studies of early kinetics of TPA-induced differentiation of U-937 cells as well as of late events during F9 differentiation suggest that post-translational regulation of Myc and Max DNA-binding may also occur. The solid phase DNA-binding assay may thus provide a tool to study the regulation of DNA-binding in more detail.

On the Role of Endogenously Produced TNF-α and IL-6 as Regulators of Growth and Differentiation of B-Type Chronic Lymphocytic Leukemia Cells In Vitro

Chronic lymphocytic leukemia of CD5+ B cells is clinically a heterogeneous disease [1]. This heterogeneity is also easily demonstrable in vitro [2]. In the vast majority of the cases B-CLL is a malignancy of CD5+ B cells with a very low capacity for proliferation in peripheral blood and with a phenotype resembling that of virgin [2] and mantle zone memory [3] B cells. In some cases the stage of differentiation is more advanced, as reflected by the capacity of the leukemic cells to secrete immunoglobulin [Ig]. In other rare cases Ig production may be very low and undetectable by immunofluorescence. The possibility that B-CLL might be a disease of differentiating leukemic B cells has not been extensively studied, but our own unpublished observations suggest that the stage of differentiation of the B-CLL cells may be different in peripheral lymphoid organs and in the bone marrow of the patient than in the peripheral blood, and the capacity for proliferation seems often to be about a 10-fold higher in these tissues.

Expression of c- myc and c- fos During Phorbol Ester Induced Differentiation of B-type Chronic Lymphocytic Leukemia Cells

The development of the various functionally specialized cell types within the hematopoietic system involves not only stepwise, cell lineage specific phenotypic alterations towards the fully mature end cells, but also proliferation to allow for the necessary expansion of the stem cell progeny (Mak and McCulloch 1982). The two processes appear to be regulated by two independent but coupled genetic programs. In malignant hematopoietic cell clones, in contrast, cells often are arrested at a particular stage of differentiation (Greaves 1979). This arrest was until recently assumed to be irreversible. However, it is now clear from many studies in vitro of mouse and human leukemia/1ymphoma cells that further, and in the case of some myelo-monocytic human tumor cell lines, even terminal differentiation may be induced by several inducers including phorbol esters (i.e. TPA), dimethylsulphoxide (DMSO), vitamin D3 and retinoic acid (Sachs 1980, Huberman and Callaham 1979, Nilsson et al. 1980).

Cytokine-induced Inhibition of Myc Activity in Monocytic Cells

The c-myc proto-oncogene plays an important role in the regulation of cell proliferation, differentiation and apoptosis. It encodes a transcription factor of the basic region/helix-loop-helix/leucine zipper (bHLHZip) family and binds specifically to certain E-box DNA elements and activates transcription from promoters containing such elements by forming heterodimers with the bHLHZip protein Max (Blackwood et al. 1991, Prendergast et al. 1991, for review see Henriksson and Luscher 1996). Max on the other hand was recently shown to form heterodimers with a number of other bHLHZip proteins; Mad1, Mxi1, Mad3 and Mad4, referred to as the Mad family (Ayer et al. 1993, Zervos et al. 1993, Hurlin et al. 1995a). These are, like c-Myc, dependent on Max for their activity and bind the same DNA elements as Myc:Max heterodimers. The Mad proteins seem to act as negative regulators of growth and inhibit the transactivating and transforming capacity of Myc (for review see Henriksson & Luscher 1996). This repressive activity depends on the N-terminus of Mad which is required for interaction with mammalian homologues of the yeast repressor protein Sin3 (Ayer et al. 1995, Schreiber-Agus et al. 1995).

Differentiation Associated c-myc Expression in Phorbol Ester and Lymphokine Stimulated B-Type Chronic Lymphocytic Leukemia Cells

Detailed phenotypic studies of human leukemia/lymphoma cells over the last decade have demonstrated that most hematopoietic tumors represent clonal expansions of malignant cells arrested at a pre-terminal stage of differentiation (Greaves 1979, Nilsson et al 1985a). The importance of this blocked differentiation for the aberrant growth behaviour of the tumors is further suggested by in vitro experiments with established leukemia/lymphoma cell lines (e.g. HL-60 and U-937). These demonstrate that induction of differentiation will lead to maturation and irreversible growth arrest, similar to what occurs in normal hematopoietic cells undergoing terminal differentiation (Nilsson et al 1985b).