Inducible expression of a degradation-resistant form of p27Kip1 causes growth arrest and apoptosis in breast cancer cells.
The cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (p27) is an important regulator of cell cycle progression controlling the transition from G to S-phase. Low p27 levels or accelerated p27 degradation correlate with excessive cell proliferation and poor prognosis in several forms of cancer. Phosphorylation of p27 at Thr187 by cyclin E-CDK2 is required to initiate the ubiquitination-proteasomal degradation of p27. Protecting p27 from ubiquitin-mediated proteasomal degradation may increase its potential in cancer gene therapy. Here we constructed a non-phosphorylatable, proteolysis-resistant p27 mutant containing a Thr187-to-Ala substitution (T187A) which is not degraded by ubiquitin-mediated proteasome pathway, and compared its effects on cell growth, cell-cycle control, and apoptosis with those of wild-type p27. In muristerone A-inducible cell lines overexpressing wild-type or mutant p27, the p27 mutant was more resistant to proteolysis in vivo and more potent in inducing cell-cycle arrest and other growth-inhibitory effects such as apoptosis. Transduction of p27(T187A) in breast cancer cells with a doxycycline-regulated adenovirus led to greater inhibition of proliferation, more extensive apoptosis, with a markedly reduced protein levels of cyclin E and increased accumulation of cyclin D1, compared with wild-type p27. These findings support the potential effectiveness of a degradation-resistant form of p27 in breast cancer gene therapy.
Zhang
Qingxiu
Q
Department of Molecular Therapeutics, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Tian
Ling
L
Mansouri
Abdel
A
Korapati
Anita L
AL
Johnson
Terry J
TJ
Claret
Francois X
FX
D000256:Adenoviridae / Q000235:genetics; D000409:Alanine / Q000737:chemistry; D017304:Annexin A5 / Q000737:chemistry; D017209:Apoptosis; D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D042846:CDC2-CDC28 Kinases / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000378:metabolism; D019927:Cyclin E / Q000737:chemistry / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004318:Doxycycline / Q000494:pharmacology; D004441:Ecdysterone / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D011379:Prognosis; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011506:Proteins / Q000737:chemistry; D016196:S Phase; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism
D017304:Annexin A5; D014408:Biomarkers, Tumor; D018797:Cell Cycle Proteins; D019927:Cyclin E; D011506:Proteins; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013912:Threonine; C055348:muristerone A; D004441:Ecdysterone; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D046988:Proteasome Endopeptidase Complex; D004318:Doxycycline; D000409:Alanine