c-Myc is a proto-oncogene that acts as a transcription factor, regulating cell proliferation and apoptosis through dimerization with Max. The Mad family of transcriptional repressors acts as c-Myc endogenous inhibitors by sequestrating Max. Mad/Max dimers bind to the same DNA consensus sequence (called the Myc E box) as cMyc/Max dimers, and cause transcriptional repression by bringing histone deacetylase to genes normally regulated by c-Myc/Max dimers. While screening a senescent cell cDNA library, a cDNA 90% homologous to the mouse Mad4 protein was isolated, and therefore was named human Mad4 (hMad4). It shows 48% and 52% amino acid homology to the previously described hMad1 and hMxi1 proteins, respectively. hMad4 is highly expressed in brain, lung, kidney and muscle as well as in confluent and senescent fibroblasts while nearly absent in replicating fibroblasts. Reminiscent of other Mad family members, hMad4 co-immunoprecipitates with Max and represses transcription of a reporter gene driven by E box sequences. Moreover, hMad4 represses cell growth and transformation of tumor cell lines. UV- and staurosporine-induced apoptosis of hMad4-infected cells is inhibited upstream of cytochrome c released from the mitochondria and downstream of mitochondrial cytochrome c release but upstream of caspase-3 activation, respectively. This effect is not caused by an increase in any of the lAPs tested. Overexpression of hMad4 in early passage WI38 fibroblasts completely abrogates cell growth, an effect dependent on its transcriptional repression activity. In hMad4 infected fibroblasts, growth-arrest is accompanied by the appearance of the acidic pgalactosidase marker, PAI-1 up-regulation, and an increase in Pml nuclear bodies. Northern blot and RT-PCR analysis demonstrate that, while following serum stimulation, hMad4 expression is decreased in young fibroblasts, levels are unchanged in senescent fibroblasts. However, c-Myc response is similar in both young and senescent fibroblasts, suggesting that hMad4 might interfere with c-Myc transcriptional activity in senescent fibroblasts. Chromatin immunoprecipitation experiments suggest that, while in young cells there is a switch from hMad4 to c-Myc when these cells are serum-stimulated, this switch might be selectively lost for some genes in senescent cells. To study the mechanism behind the difference of hMad4 induction in senescent cells, the full length hMad4 promoter was isolated and serial deletion mapped the minimal region to a fragment of 289 nucleotides upstream of the transcriptional start site, previously located by primer extension assay. Several transcription factors bind to the promoter region, including Spl and c-Myc. Addition of c-Myc inhibits promoter activity while Sp1 slightly increases expression. Mutation of the unique E-box sequence located 369 nucleotides from the transcriptional start site did not affect expression from the promoter, while mutation of a Sp1 site located 6 nt downstream of the transcription start site decreased basal activation by Sp1. These results demonstrate for the first time a cross-regulation between c-Myc and one of its inhibitors.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.19387 |
| Date | January 2003 |
| Creators | Marcotte, Richard |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001989258, Theses scanned by McGill Library. |
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