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Dwarfism and dysregulated proliferation in mice overexpressing the MYC antagonist MAD1
C Quéva1, G A McArthur, L S Ramos
1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024, USA. christophe.queva@astra-zeneca.com
Abstract:
The four members of the MAD family are bHLHZip proteins that heterodimerize with MAX and act as transcriptional repressors. The switch from MYC-MAX complexes to MAD-MAX complexes has been postulated to couple cell-cycle arrest with differentiation. The ectopic expression of Mad1 in transgenic mice led to early postnatal lethality and dwarfism and had a profound inhibitory effect on the proliferation of the hematopoietic cells and embryonic fibroblasts derived from these animals. Compared to wild-type cells, Mad1 transgenic fibroblasts arrested with altered morphology and reduced density at confluence, cycled more slowly, and were delayed in their progression from G0 to the S phase. These changes were accompanied by accumulation of hypophosphorylated retinoblastoma protein and p130. Cyclin D1-associated kinase activity was dramatically reduced in MAD1-overexpressing fibroblasts. However, wild-type cell-cycle distribution and morphology could be rescued in the Mad1 transgenic cells by the introduction of HPV-E7, but not an E7 mutant incapable of binding to pocket proteins. This indicates that the activities of the retinoblastoma family members, via the cyclin D pathway, are likely to be the major targets for MAD1-mediated inhibition of proliferation in primary mouse fibroblasts.
Insights
The MAD1 protein, a transcriptional repressor, inhibits cell proliferation by targeting retinoblastoma family members and the cyclin D pathway. Ectopic Mad1 expression in mice caused dwarfism and impaired cell growth, highlighting its role in cell-cycle regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- MAD proteins are bHLHZip transcriptional repressors that heterodimerize with MAX.
- The transition from MYC-MAX to MAD-MAX complexes is linked to cell-cycle arrest and differentiation.
Purpose of the Study:
- To investigate the role of Mad1 in cell proliferation and its underlying molecular mechanisms.
- To determine the effect of ectopic Mad1 expression in transgenic mice.
Main Methods:
- Generation of Mad1 transgenic mice.
- Analysis of hematopoietic cells and embryonic fibroblasts from transgenic and wild-type mice.
- Cell cycle analysis, including progression from G0 to S phase.
- Western blot analysis for retinoblastoma protein, p130, and cyclin D1-associated kinase activity.
- Rescue experiments using HPV-E7 in Mad1 transgenic fibroblasts.
Main Results:
- Ectopic Mad1 expression in mice resulted in postnatal lethality and dwarfism.
- Mad1 transgenic fibroblasts exhibited inhibited proliferation, altered morphology, and slower cell cycling.
- Accumulation of hypophosphorylated retinoblastoma protein and p130, and reduced cyclin D1-kinase activity were observed.
- HPV-E7 expression rescued the cell-cycle defects in Mad1 transgenic fibroblasts.
Conclusions:
- Mad1 acts as a potent inhibitor of cell proliferation in primary mouse fibroblasts.
- Mad1-mediated inhibition involves targeting retinoblastoma family proteins via the cyclin D pathway.
- The MAD1-MAX complex plays a crucial role in regulating cell growth and differentiation.