Abstract
The c-myc oncogene product (c-Myc) is a transcription factor that dimerizes with Max and recognizes the E-box sequence, and it plays key functions in cell proliferation, differentiation, and apoptosis. We previously showed that MM-1 bound to myc box II within the transactivation domain of c-Myc and repressed the E-box-dependent transcriptional activity of c-Myc. Here we report that MM-1 showed features of a tumor suppressor. In an EST data base search for cDNAs homologous to MM-1, we found a frequent substitution of amino acid 157 of MM-1, from alanine to arginine (A157R), and the substitution was observed more in tumor cells than in normal cells. A survey of the A157R mutation of MM-1 in 57 cultured cancer cells and 90 tissues from cancer patients showed that the A157R was present in about 50-60% of leukemia/lymphoma cells and in more than 75% of squamous cell carcinoma of tongue cancer. Although both the A157R and the wild-type MM-1 bound to c-Myc, only A157R lost the activities to repress both the E-box-dependent transcriptional activity of c-Myc and the myc/ras cooperative transforming activity in rat 3Y1 cells. Furthermore, the wild-type MM-1, but not A157R, arrested the growth of 3Y1 cells. The human MM-1 gene was mapped at chromosome 12q12-12q13, where many chromosome abnormalities in cancer cells have been reported. The results suggest that MM-1 is a novel candidate for a tumor suppressor that controls the transcriptional activity of c-Myc.
Highlights
C-Myc is a transcription factor, and it plays key functions in cell proliferation, differentiation, and apoptosis. c-Myc complexed with Max at the Cproximal region recognizes the E-box sequence in the target genes to be transactivated
We found that a point mutation from alanine to arginine at amino acid 157 in MM-1 frequently occurred in cells from lymphoma, leukemia, and tongue cancer and that this mutation abrogated the inhibitory functions of MM-1 to c-Myc
MM-1 genomic DNA isolated in this study revealed that a “new MM-1” comprises 154 amino acids that lack the first 13 amino acids of an “old MM-1.”
Summary
Cell Culture—Human HeLa, rat 3Y1, and mouse Balb/3T3 cells were cultured in Dulbecco’s-modified Eagle’s medium supplemented with 10% calf serum. The resultant PCR fragments were digested with EcoRI and XhoI and inserted into the respective site, of pBluescript SK-, and their nucleotide sequences were determined. Focus Forming and Growth Arrest Assays—Rat 3Y1 cells cultured in Dulbecco’s modified Eagle’s medium in a 10-cm dish were transfected with 1 g each of pEF-c-myc(ATG), pEJ6.6, pCMV-F-MM-1, pCMV-FMM1-A157T, or pCMV-F-MM1-A157R by LipofectAMINE PLUS (Life Technologies), and the medium was changed every 2 days. Osoegawa and P. de Jong at the Roswell Park Cancer Institute with human MM-1 cDNA as a probe, and 6 positive clones were obtained from the Roswell Park Cancer Institute Since these clones contain an insert of more than 100 kilobases at the BamHI site of pPAC4, one clone, 57-L5, was digested with BamHI and hybridized with a labeled MM-1 cDNA probe, and the hybridized fragments were inserted into the BamHI site of pBluescript SK(Ϫ). FISH signals and DAPI banding pattern were recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes [40]
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