Expression and Mutation Analysis of the Myc Antagonist Gene Mad1 in Acute Leukemia.

Objectives: Overexpression of Myc genes has been observed in many solid tumors and hematological malignancies, and Myc proteins are believed to act as transcription factors. In the past decade, several Myc-related genes have been identified, and the interaction mechanisms between these genes have been clarified. Members of Myc superfamily proteins belong to four different families: the Max family of common dimerization partners (including Max and MLx), the Myc family of proto-oncoproteins (including c-, N-, and L-Myc), the Myc antagonists including Mad family (including Mad1 and Mxi1) and Rox (Mnt). Mad1 gene maps to human chromosome 2p13 and encodes a 221-amino acid protein. Mad1 protein was shown to counteract the effects of Myc proteins and to inhibit the proliferation and tumorigenicity of glioblastoma and astrocytoma cells, and therefore was considered to be a kind of tumor suppressor. All Myc super-family members share a highly conserved structural domain, basic helix-loop-helix leucine zipper domain (bHLHZip). This bHLHzip domain is essential for these proteins to function during molecular switching from proliferation to differentiation. To function in vivo, Myc and Mad1 must heterodimerize with Max through the HLHZip motif. Then through the b (basic) motif, the heterodimers bind with CACGTG (E-box) containing DNA to activate or inactivate transcription. The present study is thus designed to examine the expression of Mad1 gene and to search for mutations in bHLHzip encoding domain.

Methods: we screened bone marrow mononuclear cells (BMMNC) from 26 patients with AL and peripheral blood mononuclear cells (PBMNC) from 30 healthy volunteers, using reverse transcription- polymerase chain reaction, single strand conformation polymorphism analysis and sequencing.

Results: Mad1 gene was expressed in all samples. Two polymorphisms of Mad1 were found in BMMNC from AL patients and PBMNC from health volunteers. A point mutation was detected at a very important position, a substitution of A397G inducing Lys78Glu in two patients. This might have significant implication for the function of Mad1, as Mad1 will heterodimerize with Max at this very point. Mutation at this point could thus disrupt proper intermolecular alignment between Mad1 and Max. The two patients with Mad1 gene mutation had poor clinical outcomes, patient No.9 never achieved CR and died 2 months after diagnosis, while patient No.11 got CR, after two courses chemotherapy, but relapsed quickly and died 3 months after diagnosis.

Conclusion: Mad1 gene expressed and displayed mutations in acute leukemia patients. It would be interesting to know whether these mutations influence either the heterodimerizing or DNA binding potentials of Mad1 protein, by performing functional analysis of the mutant protein.