Determination of the dissociation constants for recombinant c-Myc, Max, and DNA complexes: the inhibitory effect of linoleic acid on the DNA-binding step

c-Myc, the protein product of protooncogene c-myc, functions in cell proliferation, differentiation, and neoplastic disease. In this study, recombinant c-Myc and Max proteins, encompassing DNA binding (basic region) and dimerization (helix-loop-helix/leucine zipper) domain of human origin, were expressed in bacteria as Myc87 and Max85. Myc87 was purified under denatured conditions and was renatured again. The dissociation constant for the protein dimers and for dimer/DNA complexes were not detectable by isothermal titration calorimetry because of the low degree of solubility of Myc87 and Max85. Therefore, we set up equations which were used to determine the dissociation constants from the proportion of protein-DNA complexes. The dimer dissociation constants in TBS were 5.90(+/-0.54)x10(-7)M for Max85/Max85 homodimer, 6.85(+/-0.25)x10(-3)M for Myc87/Myc87 homodimer, and 2.55(+/-0.29)x10(-8)M for Myc87/Max85 heterodimer, and the DNA-binding dissociation constants in TBS were 1.33(+/-0.21)x10(-9)M for Max85/Max85/DNA, 2.27(+/-0.08)x10(-12)M for Myc87/Myc87/DNA, and 4.43(+/-0.37)x10(-10)M for Myc87/Max85/DNA. In addition, we revealed that linoleic acid which is known as an inhibitor for the formation of Max/Max/DNA complex reduced the affinity of Max homodimer for DNA. This result indicates that linoleic acid may bind to the DNA-binding region of Max homodimer.     

c-Myc inactivation by mutant max alters growth and morphology of NCI-H-630 colon cancer cells

The myc gene family has been implicated in multiple cell processes including proliferation, differentiation, tumorigenesis, and apoptosis. For its cellular growth promoting function, Myc must heterodimerize with Max. To study the effect of Myc inactivation on the growth and differentiation properties of epithelial tumor cells, we transfected the H-630 human colon cancer cell line with bm-max, a mutant Max protein in which DNA-binding activity has been abolished. Cells expressing high levels of bm-Max grow poorly, and the morphology of both colonies and single cells is altered. Moreover, increased bm-Max expression results in a prolonged G₀/G₁ phase accompanied by induced expression of p21 (WAF1/CIP1), elevated levels of alkaline phosphatase (ALP) activity, and accumulation of large fat granuli within the cells. These distinctive cell characteristics are associated with differentiation processes in numerous malignant cell lines. The results of this study support a model in which sequestering of endogenous Myc and Max proteins into “basic mutant” dimers lacking DNA-binding activity is sufficient both to inhibit proliferation and to induce changes in cell behavior consistent with differentiation. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Protein tracking-induced supercoiling of DNA: A tool to regulate DNA transactions in vivo?

An interplay between DNA-dependent biological processes appears to be crucial for cell viability. At the molecular level, this interplay relies heavily on the communication between DNA-bound proteins, which can be facilitated and controlled by the dynamic structure of double-stranded DNA. Hence, DNA structural alterations are recognized as potential tools to transfer biological information over some distance within a genome. Until recently, however, direct evidence for DNA structural information as a mediator between cellular processes was lacking. This changed when the concept of transient waves of DNA supercoiling, induced by proteins tracking along the right-handed DNA double helix, came into the limelight. Indeed, a number of observations now suggest that helix tracking-induced DNA structural information might be exploited to participate in the regulation of a variety of DNA transactions in vivo.