Cell surface area and membrane folding in glioblastoma cell lines differing in PTEN and p53 status

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Abstract

Glioblastoma multiforme (GBM) is characterized by rapid growth, invasion and resistance to chemo-/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM), the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT) technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN) exhibited the lowest degree of membrane folding, probed by the area-specific capacitance Cm = 1.9 μF/cm 2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest Cm values of 3.7-4.0 μF/cm2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the migration and invasion of GBM and other tumor types. © 2014 Memmel et al.

Figures

  • Table 1. Mutational status,* cell radius and dielectric properties of 5 GBM lines studied here.
  • Figure 1. Representative scanning electron micrographs of 5 GBM lines used in the present study. For each cell line 2 views are shown, with the scale bars given in the bottom edges of each image. Scale bars in A–C and E–I correspond to 10 mm, in D and K the scale bars are 1 and 20 mm, respectively. doi:10.1371/journal.pone.0087052.g001
  • Figure 2. Rotation spectra of 5 GBM lines measured in isotonic 300-mOsm inositol solutions of the same conductivity of 50 mS/cm. Each spectrum (symbols) is the mean (6SE) of 8–10 single cell measurements. Curves are best fits with a double or triple Lorentzian function (Eq. 1). The fitted parameters are given in Table S1. The fci symbols denote three ROT peaks dominated by the plasma membrane charging (fc1), and by the polarization of the cytosol and cell nucleus (fc2 and fc3). doi:10.1371/journal.pone.0087052.g002
  • Figure 3. Cumulative plots of the radius-normalized fc1 values (fc1?a) of the indicated GBM lines versus the external conductivity se. The measurements were performed in isotonic 300-mOsm inositol medium. The fc1 data were obtained by the CRF-technique. Each symbol is the mean (6SE) from 20 cells measured at closely similar conductivities. The lines are best fits of Eq. 2 to the CRF data sets, each containing 300–420 cells. The line slopes are inversely proportional to Cm values. The steeper slope obtained for DK-MG cells implies a much smaller Cm = 1.9 mF/cm 2 (A, empty circles), as compared to those of U373-MG (4.0 mF/cm2) and SNB19 cell lines (3.7 mF/cm2) (B). U87-MG and GaMG showed intermediate Cm values of 3.2 and 2.8 mF/cm 2, respectively (A). The fitted Cm values are summarized in Table 1. doi:10.1371/journal.pone.0087052.g003
  • Figure 4. Volume changes in GaMG cells induced by strongly hypotonic sucrose- (A–C) and inositol-substituted solutions (D–F) of osmolalities 100 and 50 mOsm, respectively. The microphotographs were taken before (A and D, isotonic CGM), about 3 min (B, E) and 20 min (C, F) after an acute hypotonic shock. In hypotonic sucrose (A–C), the volume of the indicated cell increased 2.1-fold within 3 min after hypotonic shock (B). Thereafter, the cell gradually shrank via the RVD mechanism and reduced its volume to ,120% of the original isotonic value (C). In sharp contrast to the disaccharide sucrose, the small organic osmolyte inositol fully abolished RVD in GaMG cells (D–F). Thus, upon exposure to 50-mOsm inositol, the cell volume increased first rapidly 1.6-fold at 3 min (E), and then slower, more than 3-fold within the following 20 min (F). The scale bars correspond to 10 mm. doi:10.1371/journal.pone.0087052.g004
  • Figure 5. Changes of the normalized volume V/V0 in glioblastoma cell lines in response to sucrose- and inositol-substituted solutions of different osmolalities (LHS and RHS columns, respectively). All cells were bathed initially (time ,0) in isotonic culture medium (300 mOsm) and then exposed at zero time to solutions having osmolalities of 300, 100 or 50 mOsm. As expected, the initial swelling (2–3 min) of all cell lines increased in magnitude with decreasing osmolality of hypotonic media. But the secondary volume changes (time .5 min) were found to be strongly dependent on both the cell type and the extracellular osmolyte. In general, all tested cell lines were able to undergo regulatory volume decrease (RVD) in sucrose-substituted media over the entire tonicity range (LHS column). In sharp contrast to sucrose, inositol not only abolished RVD but also caused continuous secondary cell swelling, resulting in an up to 3-fold volume increase of GaMG and SNB19 cells 20 min after hypotonic shock (D). Each data point represents the mean 6 SE of 50–120 individual cells. Continuous curves in A show best least-square fits of the Lúcio-model [50] to the data. The fitted parameters (Pw and a) are given in Table 2. The slower initial swelling of DK-MG cells (black symbols) indicates a lower osmotic water permeability of the plasma membrane in this cell line (Pw in Table 2). doi:10.1371/journal.pone.0087052.g005
  • Table 2. Osmotic parameters of glioblastoma cell lines.
  • Figure 6. Representative Western blot analysis of the expression of p53, MDM2, PTEN, PI3K, phospho-AKT, phospho-mTOR and FAS proteins. For each cell line, cell lysates were prepared from exponentially growing cells, 20–24 h after splitting the culture. Each protein band was normalized to the intensity of b-actin used as loading control, and the ratios protein/actin are depicted by numbers. The experiments were repeated at least three times. doi:10.1371/journal.pone.0087052.g006

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Memme, S., Sukhorukov, V. L., Höring, M., Westerling, K., Fiedler, V., Katzer, A., … Djuzenova, C. S. (2014). Cell surface area and membrane folding in glioblastoma cell lines differing in PTEN and p53 status. PLoS ONE, 9(1). https://doi.org/10.1371/journal.pone.0087052

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