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Phenotypic changes associated with exogenous expression of p16INK4a in human glioma cells
1Department of Neurosurgery, Kyorin University School of Medicine, Mitaka, Tokyo, Japan. nogu-kyr@umin.ac.jp
Abstract:
The tumor suppressor p16/CDKN2A/INK4a gene is frequently mutated, mostly by homozygous deletions in high-grade gliomas. Although the p16 protein suppresses cell proliferation primarily through inhibition of cell-cycle progression at the G1 phase, other phenotypic changes in glioma cells associated with p16INK4a alterations have not been fully described. To determine the roles of p16 alterations in glioma formation, we have established ecdysone-driven inducible p16 expression in the human glioblastoma cell line CL-4, which were derived from p16-null U87MG cells. Here we show that exogenous p16 expression in CL-4 cells results in morphological changes, with large and flattened cytoplasm, which are associated with increased formation of cytoplasmic actin-stress fibers and vinculin accumulation in the focal adhesion contacts. Adhesion of CL-4 cells to extracellular matrix proteins, such as laminin, fibronectin, and type IV collagen, significantly increased upon exogenous p16 expression, which correlated with increased expression of integrin alpha5 and alphav. Expression of a small GTP-binding protein, Rac, also decreased. Following epidermal growth factor stimulation, phosphorylation of MAP kinases ERK1 and 2 and induction of an early immediate gene product, c-Fos, were significantly reduced in CL-4 cells with p16 expression. These results suggest that the tumor suppressor p16 may exert its antitumor effects through modulation of multiple aspects of glioblastoma phenotypes, including proliferation, invasiveness, and responsiveness to extracellular growth stimuli.
Insights
The tumor suppressor p16INK4a gene impacts glioblastoma by altering cell shape, adhesion, and growth factor response. Its expression influences multiple tumor characteristics, suggesting broader antitumor effects beyond cell cycle inhibition.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- The p16/CDKN2A/INK4a gene, a tumor suppressor, is frequently deleted in high-grade gliomas.
- p16 protein primarily inhibits cell cycle progression at G1 phase, but its broader effects on glioma phenotypes are not fully understood.
Purpose of the Study:
- To investigate the multifaceted roles of p16 alterations in glioma formation and progression.
- To characterize phenotypic changes in glioblastoma cells upon inducible p16 expression.
Main Methods:
- Established ecdysone-inducible p16 expression in p16-null human glioblastoma CL-4 cells.
- Analyzed morphological changes, actin-stress fiber formation, focal adhesion dynamics, and cell adhesion to extracellular matrix proteins.
- Assessed expression of integrins (alpha5, alphav), Rac GTPase, and downstream signaling pathways (ERK1/2, c-Fos) following epidermal growth factor stimulation.
Main Results:
- Exogenous p16 expression induced morphological changes (larger, flattened cytoplasm) and increased actin-stress fibers and vinculin accumulation.
- Cell adhesion to laminin, fibronectin, and type IV collagen increased, correlating with elevated integrin alpha5 and alphav expression.
- p16 expression decreased Rac GTPase levels and significantly reduced ERK1/2 phosphorylation and c-Fos induction upon EGF stimulation.
Conclusions:
- The tumor suppressor p16 influences glioblastoma phenotypes beyond cell cycle arrest, affecting cell morphology, adhesion, and invasiveness.
- p16 modulation impacts cellular responsiveness to extracellular growth stimuli, suggesting a broader role in antitumor activity.
- These findings highlight p16 as a key regulator of multiple glioblastoma characteristics, offering potential therapeutic insights.