Adenoviral p16/CDKN2 gene transfer to malignant glioma: role of p16 in growth, invasion, and senescence.
In gliomas, a high frequency of homozygous p16 gene deletions have been demonstrated, which are believed to be linked with malignant progression. The aim of this study was to assess the role of p16 in growth, invasion, and senescence. The human glioma cell lines U87 MG and U373 MG were transduced with Ad-p16, and cell viability was assessed by trypan blue staining. To examine the mechanism of cell growth inhibition, cell cycle analyses and annexin assays were performed. The invasive potential of Ad-p16 transduced cells was evaluated using a Matrigel invasion assay, and trimolecular complex (MMP-2/MT1-MMP/TIMP-2) synthesis was proven by zymography and Western blotting. To establish the link between p16 and cell senescence, we stained for Senescence-Associated beta-galactosidase activity. A cell proliferation assay demonstrated that Ad-p16 treatment significantly inhibits cell growth. Moreover, this cell growth inhibition was induced by cell cycle arrest, not by apoptosis. In vitro treatment of malignant glioma cells with Ad-p16 significantly decreased their invasive potential by Matrigel invasion assay. However, we were unable to demonstrate any differences in the constitutive productions and secretions of MMP-2, MT1-MMP, and TIMP-2, among the mock-treated, Ad-lacZ-transduced, and Ad-p16-transduced cells. p16 expression caused an enlargement of all cells, and these were morphologically similar to senescent cells. Staining for Senescence-Associated beta-galactosidase activity showed that the enlarged cells stained positively. Taken together these data strongly suggest that the anti-cancer effect of p16 is modulated by p16-mediated cell cycle arrest and by the induction of senescence.
Kim
Seung-Ki
SK
Department of Neurosurgery, Seoul National University, College of Medicine, Seoul, Korea.
Wang
Kyu-Chang
KC
Cho
Byung-Kyu
BK
Lim
Su-Young
SY
Kim
Young-Yim
YY
Oh
Chang-Wan
CW
Chung
You-Nam
YN
Kim
Chae-Yong
CY
Lee
Choon-Taek
CT
Kim
Hyun Jib
HJ
D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D002453:Cell Cycle; D002455:Cell Division; D045744:Cell Line, Tumor; D016922:Cellular Senescence; D003094:Collagen / Q000737:chemistry / Q000494:pharmacology; D004396:Coloring Agents / Q000494:pharmacology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D018450:Disease Progression; D004338:Drug Combinations; D018014:Gene Transfer Techniques; D005910:Glioma / Q000628:therapy*; D006801:Humans; D007797:Laminin / Q000737:chemistry / Q000494:pharmacology; D009361:Neoplasm Invasiveness; D011509:Proteoglycans / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D017304:Annexin A5; D004396:Coloring Agents; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; D001616:beta-Galactosidase