DNA replication licensing factor minichromosome maintenance deficient 5 rescues p53-mediated growth arrest.
Inactivation of p53 signaling by mutation of p53 itself or abrogation of its normal function by other transfactors, such as MDM2, is a key event in the development of most human cancers. To identify novel regulators of p53, we have used a phenotype-based selection in which a total cDNA library in a retroviral vector has been introduced into TR9-7ER cells, which arrest when p53 is expressed from a tetracycline-regulated promoter. We have isolated several clones derived from cells that are not growth-arrested when p53 is overexpressed. In one clone, the levels of p53, p21, and MDM2 are comparable with those in TR9-7ER cells and, therefore, the abrogation of growth arrest by an exogenous cDNA is likely to be distal to p21. Using reverse transcription-PCR, we were able to isolate a cDNA of approximately 2.2 kb, which was found to have 99% identity to the nucleotides between about 80 and 2,288 of the open reading frame of a gene encoding DNA replication licensing factor. It encodes complete peptide of 734 residues of this protein also called minichromosome maintenance deficient 5 (MCM5) or cell division cycle 46 (Saccharomyces cerevisiae). Northern and Western blot analyses revealed that the expression of MCM5 and its transcriptional regulator, E2F1, is negatively regulated by p53. When MCM5 cDNA was reintroduced into fresh TR9-7ER cells, numerous colonies that grow in the absence of tetracycline were formed. This novel observation establishes a role for MCM5 in negating the growth arrest function of p53.
Agarwal
Mukesh K
MK
Department of Genetics, Case Western Reserve University, Cleveland, OH 44106, USA.
Ruhul Amin
A R M
AR
Agarwal
Munna L
ML
D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics*; D048708:Cell Growth Processes / Q000235:genetics; D004261:DNA Replication / Q000502:physiology*; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D015536:Down-Regulation; D050687:E2F1 Transcription Factor / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C489423:MCM5 protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53