PTEN negatively regulates neural stem cell self-renewal by modulating G0-G1 cell cycle entry

Abstract

Previous studies have demonstrated that a small subpopulation of brain tumor cells share key characteristics with neural stem/progenitor cells in terms of phenotype and behavior. These findings suggest that brain tumors might contain "cancer stem cells" that are critical for tumor growth. However, the molecular pathways governing such stem cell-like behavior remain largely elusive. Our previous study suggests that the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) tumor suppressor gene, one of the most frequently mutated genes in glioblastomas, restricts neural stem/progenitor cell proliferation in vivo. In the present study, we sought to determine the role of PTEN in long-term maintenance of stem cell-like properties, cell cycle entry and progression, and growth factor dependence and gene expression. Our results demonstrate an enhanced self-renewal capacity and G(0)-G(1) cell cycle entry and decreased growth factor dependency of Pten null neural/stem progenitor cells. Therefore, loss of PTEN leads to cell physiological changes, which collectively are sufficient to increase the pool of self-renewing neural stem cells and promote their escape from the homeostatic mechanisms of proliferation control.

Fig. 1.

PTEN loss leads to greater and persistent self-renewal capacity. (A) A schematic illustration of the serial neurosphere culture system. (B) Percentages of WT and MUT sphere cultures that can be serially passaged. (C) Sphere formation relative to WT sphere counts at each passage. (D) Histograms show the neurogenic potential of WT (in blue) and MUT (in red) neurosphere cultures. WT spheres demonstrated a loss of neurogenic potential over time with serial, low-density passages. Filled bars, spheres with neuron; hatched bars, spheres without neuron. (E) Representative images of the neurogenic potential of control and mutant neurospheres, with propridium iodide staining in Left and TuJ1 immunostaining in Right. (Scale bar: 17 μm.) A, GFAP⁺ astrocytes; O, O4⁺ oligodendrocytes; N, Tuj1⁺ neurons.

Fig. 2.

Pten null neurospheres are hypersensitive to growth factor stimulation. Identical number of cells from WT and MUT E14.5 front brains were seeded in neurosphere cultures with the indicated concentration of FGF2. Neurosphere numbers (A) and size (B) were measured 7 days after initial culture and presented as mean ± SD.

Fig. 3.

Loss of PTEN leads to increased G₀/G₁ A to G₁B cell cycle transition as well as increased cell growth. (A) Flowcytometric analysis of cell cycle status in primary cells from E14.5 cortices (Upper) and cells that have gone through one generation of neurosphere culture (Lower). Left, WT; Right, MUT. N-butyrate treated neurosphere cultures, which are arrested at the G₁ A to G₁B transition, were used to demarcate the line (vertical axes) between cells falling into the G₀/G₁A stage (quiescence, Lower Left) and cells which have entered the G₁B stage of the cell cycle (Lower Right). (B Left) A comparison of cell cycle profiles. Statistic analysis for brain: G₀/G₁A, P = 0.05; G₁B, P = 0.08; S/G₂/M, P = 0.43; neurosphere: G₀/G₁A, P = 0.05; G₁B, P = 0.72; S/G₂/M, P = 0.00003. *, statistically significant. (Right) Cell size profiles at different cell cycle stages. Data include all experiments conducted. For brain, WT, n = 10; Mut, n = 5; for neurosphere, WT, n = 11; Mut, n = 7.

Fig. 4.

Gene expression analysis. (A) Microarray data can be classified into two major groups, genes that are up-regulated in Pten null neurosphere cultures (upper box) and genes that are down-regulated in Pten null neurosphere cultures (lower box). (B) Gene Ontology analysis.