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A novel gene expression system using transcription amplification to examine cdk2‐associated cell cycle regulator role in cancer cell apoptosis
Author(s) -
Figueiredo Marxa L,
Sato Makoto,
Wu Lily
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.438.10
Subject(s) - cyclin dependent kinase 2 , cancer research , downregulation and upregulation , cell cycle , biology , cell growth , regulator , microbiology and biotechnology , apoptosis , cell cycle checkpoint , chemistry , gene , biochemistry
In order to improve understanding of the contribution of p27kip1, a cyclin‐dependent kinase 2 (cdk2) regulator to growth inhibition in prostate cancer cells, we have modulated p27 expression using a new two‐step transcription amplification system. p27 expression usually is associated with G1/S arrest through cdk2 inhibition, and downregulated during prostate cancer progression by its increased targeting for ubiquitin proteasome degradation. We selected a degradation‐resistant mutant (T187M/P188I;p27mt), hypothesized to be more effective in inhibiting tumor growth. We adapted an adenoviral‐based transcriptional amplification system containing a prostate‐specific promoter (PSE‐BC) driving a GAL4‐VP16 activator, which in turn activates expression of a bidirectional Luc/p27 (or p27mt) cassette. p27mt showed higher growth inhibition, with ~80% tumor growth reduction in vivo, and the main mechanism of growth inhibition was through apoptosis induction. Using a qPCR array, we observed several proapoptotic gene expression changes, including death receptors/ligands, caspases, and TP73 upregulation. Prosurvival changes also were observed, for example, Birc8 upregulation. Further optimization of this system is in progress and includes combination vectors for enhanced antitumor effectiveness, including cell cycle regulators (p12/cdk2ap1) and antisurvival strategies (shHer2).