Growth inhibition of human malignant glioma cells induced by the PI3-K—specific inhibitor

2003 ◽  
Vol 98 (1) ◽  
pp. 154-161 ◽  
Author(s):  
Takashi Shingu ◽  
Kazuo Yamada ◽  
Nobumasa Hara ◽  
Kouzo Moritake ◽  
Harumi Osago ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Malignant Glioma ◽  
Cell Lines ◽  
Akt Pathway ◽  
Content Type ◽  
Phosphorylated Akt ◽  
Human Malignant Glioma ◽  
The Status ◽  
Malignant Glioma Cells ◽  
Fine Print

Object. The phosphatase and tensin homolog deleted from chromosome 10 (PTEN) functions as a tumor suppressor by negatively regulating the growth/survival signals of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The PI3-K/Akt pathway in PTEN-deficient tumors may be one of the key targets for anticancer therapy. The authors examined the effects of the PI3-K inhibitor 2-(4-morpholinyl)-8-phenylchromone (LY294002) on human malignant glioma cells, and compared these effects on PTEN-deficient cells with those on PTEN—wild-type (PTEN-wt) cells. Methods. Using human malignant glioma cell lines, including the PTEN-deficient cells A172 and U87MG and the PTEN-wt cells LN18 and LN229, the effects of LY294002 on cell growth, apoptosis, and chemotherapeutic agent—induced cytotoxicity were evaluated. The LY294002 inhibited the growth of U87MG cells associated with reduced phosphatidylinositol 3,4,5,-trisphosphate and phosphorylated Akt, and also induced growth inhibition in three other cell lines. Although LY294002 caused apoptosis in all four cell lines, apoptosis seemed to contribute to only a small portion of growth inhibition induced by LY294002. There was no link between the status of PTEN and the median inhibitory concentration values for LY294002 or between the gene status and the extent of LY294002-induced apoptosis. The LY294002 significantly augmented the cytotoxicity induced by etoposide in PTEN-deficient cells, but not in PTEN-wt cells. Enhancement of 1,3-bis(2-chloroethyl)-1-nitrosourea— and cisplatin-induced cytotoxicity by LY294002 was not linked to the status of PTEN. No marked difference in the amounts of phosphorylated Akt was found between PTEN-deficient and PTEN-wt cells. Conclusions. The findings show that PI3-K is a possible target for therapy in patients with gliomas, and PI3-K inhibitors in combination with chemotherapeutic agents could be potent therapeutic modalities for patients with malignant gliomas.

Resistance to growth inhibition by transforming growth factor—β in malignant glioma cells with functional receptors

1998 ◽  
Vol 88 (3) ◽  
pp. 529-534 ◽  
Author(s):  
Shiro Isoe ◽  
Hirofumi Naganuma ◽  
Shin Nakano ◽  
Atsushi Sasaki ◽  
Eiji Satoh ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Malignant Glioma ◽  
Cell Lines ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Content Type ◽  
Glioma Cell Lines ◽  
Malignant Glioma Cells ◽  
Tgf Β1 ◽  
Fine Print

Object. The aim of this study was to investigate the mechanism by which malignant glioma cells escape from growth inhibition mediated by transforming growth factor-β (TGF-β), a ubiquitous cytokine that inhibits cell proliferation by causing growth arrest in the G1 phase of the cell cycle. Methods. The authors measured the response of eight malignant glioma cell lines to the growth-inhibiting activity of TGF-β in vitro and the expression of TGF-β Types I and II receptors in malignant glioma cells. The effect of TGF-β on the expression of a p27Kip1 cyclin-dependent kinase inhibitor was also investigated to assess the downstream signal transmission from TGF-β receptors. All malignant glioma cell lines were insensitive to growth inhibition by TGF-β1 and TGF-β2. Analyses of TGF-β receptors by means of affinity labeling in which 125I-TGF-β1 was used showed that six glioma lines had both TGF-β Types I and II receptors on their cell surfaces, whereas two lines had very small amounts of TGF-β Type I and/or Type II receptors. Northern blot analysis showed that all tumor lines expressed variable levels of messenger RNAs for both TGF-β Types I and II receptors. Flow cytometric analyses revealed that treatment of malignant glioma cells with TGF-β1 significantly downregulated the expression of p27Kip1 protein in all malignant glioma cell lines except one. Conclusions. The authors suggest that most malignant glioma cells express TGF-β Types I and II receptors, which can transmit some signals downstream and that the loss of response to TGF-β growth inhibition may not be caused by an abnormality of the TGF-β receptors.

Isolation and characterization of human malignant glioma cells from histologically normal brain

1997 ◽  
Vol 86 (3) ◽  
pp. 525-531 ◽  
Author(s):  
Daniel L. Silbergeld ◽  
Michael R. Chicoine
Keyword(s):  
Malignant Glioma ◽  
Cell Lines ◽  
Growth Rates ◽  
Glioma Cells ◽  
Normal Brain ◽  
Content Type ◽  
Human Malignant Glioma ◽  
Malignant Glioma Cells ◽  
Fine Print ◽  
Neuropathological Evaluation

✓ Brain invasion prevents complete surgical extirpation of malignant gliomas; however, invasive cells from distant, histologically normal brain previously have not been isolated, cultured, and characterized. To evaluate invasive human malignant glioma cells, the authors established cultures from gross tumor and histologically normal brain. Three men and one woman, with a mean age of 67 years, underwent two frontal and two temporal lobectomies for tumors, which yielded specimens of both gross tumor and histologically normal brain. Each specimen was acquired a minimum of 4 cm from the gross tumor. The specimens were split: a portion was sent for neuropathological evaluation (three glioblastomas multiforme and one oligodendroglioma) and a portion was used to establish cell lines. Morphologically, the specimens of gross tumor and histologically normal brain were identical in three of the four cell culture pairs. Histochemical staining characteristics were consistent both within each pair and when compared with the specimens sent for neuropathological evaluation. Cultures demonstrated anchorage-independent growth in soft agarose and neoplastic karyotypes. Growth rates in culture were greater for histologically normal brain than for gross tumor in three of the four culture pairs. Although the observed increases in growth rates of histologically normal brain cultures do not correlate with in vivo behavior, these findings corroborate the previously reported stem cell potential of invasive glioma cells. Using the radial dish assay, no significant differences in motility between cultures of gross tumor and histologically normal brain were found. In summary, tumor cells were cultured from histologically normal brain acquired from a distance greater than 4 cm from the gross tumor, indicating the relative insensitivity of standard histopathological identification of invasive glioma cells (and hence the inadequacy of frozen-section evaluation of resection margins). Cell lines derived from gross tumor and histologically normal brain were usually histologically identical and demonstrated equivalent motility, but had different growth rates.

Communication between malignant glioma cells and vascular endothelial cells through gap junctions

2003 ◽  
Vol 98 (4) ◽  
pp. 846-853 ◽  
Author(s):  
Wei Zhang ◽  
Joseph A. DeMattia ◽  
Hua Song ◽  
William T. Couldwell
Keyword(s):  
Malignant Glioma ◽  
Gap Junction ◽  
Vascular Endothelial Cells ◽  
Glioma Cells ◽  
Tube Formation ◽  
Vascular Endothelial ◽  
Content Type ◽  
Human Malignant Glioma ◽  
Malignant Glioma Cells ◽  
Fine Print

Object. Extensive invasion and angiogenesis are hallmark features of malignant gliomas. Communication between malignant glioma cells and surrounding astrocytes occurs, resulting in transformation of the astrocytic phenotype. In the present study, the authors examined whether malignant glioma cells and vascular endothelial cells (VECs) communicate through the formation of gap junctions and whether this communication influences angiogenesis. Methods. Connexin43 (Cx43), a gap junction protein expressed in glioma cells, was identified in human umbilical VECs (HUVECs). Immunocytochemical staining for Cx43 demonstrated immunoreactive plaques at areas of cell—cell contact among HUVECs as well as between HUVECs and Cx43-expressing malignant glioma cells. Dye transfer, performed using the gap junction—permeable dye dicarboxy-dichlorofluorescein diacetate (CDCF), among these cocultures indicated that these were functional communications. Calcium signaling also occurred from malignant glioma cells to HUVECs. Tube formation by HUVECs cocultured with Cx43-transfected T98G malignant glioma cells (T98G-Cx43 cells) or with U87MG malignant glioma cells, which naturally express Cx43, was significantly increased compared with tube formation by HUVECs alone. The difference in tube formation by HUVECs cocultured with empty vector—transfected T98G glioma cells (T98G-mock cells) or with Cx43-deficient U373MG malignant glioma cells and tube formation by HUVECs alone was not statistically significant. Furthermore, the concentration of vascular endothelial growth factor (VEGF), an angiogenic factor important for the induction of angiogenesis and blood vessel formation, was significantly higher in medium harvested from cultures of T98G-Cx43 cells than in that harvested from cultures of control T98G-mock cells. Human malignant glioma U87MG cells also secreted increased concentrations of VEGF as compared with HUVECs alone. Nevertheless, there was no statistically significant difference in tube formation by HUVECs cultured in medium conditioned by either Cx43-expressing or Cx43-deficient glioma cells, suggesting that the direct gap junction communication between glioma cells and HUVECs may play a much more significant role than the increased VEGF secretion in vascular tube formation in this assay. Conclusions. These results indicate that functional gap junction formation between human malignant glioma cells and VECs occurs. This communication appears to influence tumor angiogenesis. Targeting gap junction signaling may offer a potential mechanism for therapy in patients with these tumors.

Enhanced radiosensitivity of malignant glioma cells after adenoviral p53 transduction

1999 ◽  
Vol 91 (6) ◽  
pp. 997-1004 ◽  
Author(s):  
William C. Broaddus ◽  
Yue Liu ◽  
Laura L. Steele ◽  
George T. Gillies ◽  
Peck-Sun Lin ◽  
...  
Keyword(s):  
Malignant Glioma ◽  
Glioma Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Wild Type ◽  
Content Type ◽  
Cranial Radiation ◽  
Adenoviral Transduction ◽  
Wild Type P53 ◽  
Malignant Glioma Cells ◽  
Fine Print

Object. The goal of this study was to determine whether adenoviral vector—mediated expression of human wildtype p53 can enhance the radiosensitivity of malignant glioma cells that express native wild-type p53.The p53 gene is thought to function abnormally in the majority of malignant gliomas, although it has been demonstrated to be mutated in only approximately 30%. This has led to studies in which adenoviral transduction with wild-type human p53 has been investigated in an attempt to slow tumor cell growth. Recent studies suggest that reconstitution of wild-type p53 can render cells more susceptible to radiation-mediated death, primarily by p53-mediated apoptosis.Methods. Rat RT2 glioma cells were analyzed for native p53 status by reverse transcriptase—polymerase chain reaction and sequence analysis and for p53 expression by Western blot analysis. Clonogenic survival and the terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate nick-end labeling assay were used to characterize RT2 cell radiosensitivity and apoptosis, respectively, with and without prior transduction with p53-containing and control adenoviral vectors. Animal survival length was monitored after intracerebral implantation with transduced and nontransduced RT2 cells, with and without cranial radiation.The RT2 cells were demonstrated to express native rat wild-type p53 and to markedly overexpress human p53 following adenoviral p53 transduction. The combination of p53 transduction followed by radiation resulted in marked decreases in RT2 cell survival and increases in apoptosis at radiation doses from 2 to 6 Gy. Animals receiving cranial radiation after intracerebral implantation with RT2 cells previously transduced with p53 survived significantly longer than control animals (p < 0.01).Conclusions. The ability to enhance the radiosensitivity of malignant glioma cells that express wild-type p53 by using adenoviral transduction to induce overexpression of p53 offers hope for this approach as a therapeutic strategy, not only in human gliomas that express mutant p53, but also in those that express wild-type p53.

Motility factor produced by malignant glioma cells: role in tumor invasion

1990 ◽  
Vol 73 (6) ◽  
pp. 881-888 ◽  
Author(s):  
Takanori Ohnishi ◽  
Norio Arita ◽  
Toru Hayakawa ◽  
Shuichi Izumoto ◽  
Takuyu Taki ◽  
...  
Keyword(s):  
Cell Migration ◽  
Malignant Glioma ◽  
Tumor Invasion ◽  
Cytochalasin B ◽  
Glioma Cells ◽  
Actin Microfilaments ◽  
Content Type ◽  
Motility Factor ◽  
Malignant Glioma Cells ◽  
Fine Print

✓ To better understand the cellular mechanism of tumor invasion, the production of a cell motility-stimulating factor by malignant glioma cells was studied in vitro. Serum-free conditioned media from cultures of rat C6 and human T98G cell lines contained a factor that stimulated the locomotion of the producer cells. This factor was termed the “glioma-derived motility factor.” The glioma-derived motility factor is a heat-labile protein with a molecular weight greater than 10 kD and has relative stability to acid. The factor showed not only chemotactic activity but also chemokinetic (stimulated random locomotion) activity in the two types of glioma cells studied. Although glioma-derived motility factors in conditioned media obtained from two different cell origins are likely to be the same, chemokinetic migration of T98G cells to their conditioned medium was much stronger than that of C6 cells to theirs. Coincubation of cells with cytochalasin B, which disrupts the assembly of cellular actin microfilaments, almost completely inhibited the cell migration stimulated by glioma-derived motility factor. Cytochalasin B also induced marked alterations in cell morphology, including cell retraction and arborization, while the drug did not affect cell attachment to culture dishes. These results indicate that glioma cells produce a motility factor which may play a role particularly when tumor cells are detached and migrate away from the original tumor mass, thus promoting tumor invasion. Also, glioma cell migration stimulated by the motility factor requires the normal organization of cytoskeletons such as actin microfilaments.

Increased invasive capacity of connexin43-overexpressing malignant glioma cells

2003 ◽  
Vol 99 (6) ◽  
pp. 1039-1046 ◽  
Author(s):  
Wei Zhang ◽  
Chiedozie Nwagwu ◽  
Duc Minh Le ◽  
V. Wee Yong ◽  
Hua Song ◽  
...  
Keyword(s):  
Malignant Glioma ◽  
Gap Junction ◽  
Cell Invasion ◽  
Malignant Gliomas ◽  
Glioma Cells ◽  
C6 Glioma Cells ◽  
Glioma Invasion ◽  
Content Type ◽  
Malignant Glioma Cells ◽  
Fine Print

Object. Malignant glioma cells, similar to astrocytes, express connexin43 (Cx43) universally but at widely varied levels. Data from previous studies have demonstrated that malignant glioma cells form functional gap junction channels among themselves as well as with astrocytes and that such a communication has the potential to modulate the phenotypic characteristics of astrocytes. Recently, gap junctions have been demonstrated to play a role in the invasive phenotype of malignant gliomas. In this study, the authors have further investigated the motility and invasion ability of Cx43-overexpressing and Cx43-deficient malignant glioma cells. Methods. Using a standard invasion system of a Matrigel transwell invasion chamber, the authors found that the number of Cx43-transfected C6 glioma cells (C6-Cx43 cells) migrating through the Matrigel-coated membrane was similar to that of mock-transfected control cells (C6-mock cells) during the first 24 hours, but increased significantly thereafter. When these cells were cocultured with astrocytes, the number of invading C6-Cx43 cells was more than threefold greater than the number of invading C6-mock cells. Results of an in vitro cell motility assay also demonstrated that C6-Cx43 cells were more motile and scatter-active than C6-mock cells. Furthermore, zymographic analysis of MMPs, an important determinant in glioma invasion, demonstrated that the amounts of MMP-2 and MMP-9 in culture medium collected from C6-Cx43 cells were orders of magnitude higher than those from C6-mock cells. In addition, BB-94, a synthetic MMP inhibitor, significantly inhibited C6-Cx43 cell invasion. Conclusions. The overexpression of gap junction proteins in glioma cells and the intercellular communication between tumor and nontumor glia cells may play important roles in the facilitation of glioma cell invasion.

Participation of an abnormality in the transforming growth factor–β signaling pathway in resistance of malignant glioma cells to growth inhibition induced by that factor

2006 ◽  
Vol 105 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Lei Zhang ◽  
Eiji Sato ◽  
Kenichi Amagasaki ◽  
Atsuhito Nakao ◽  
Hirofumi Naganuma
Keyword(s):  
Growth Inhibition ◽  
Malignant Glioma ◽  
Cell Line ◽  
Cell Lines ◽  
A549 Cell ◽  
Glioma Cell ◽  
Nuclear Translocation ◽  
A549 Cell Line ◽  
Glioma Cell Lines ◽  
Malignant Glioma Cells

Object Malignant glioma cells secrete and activate transforming growth factor–β (TGFβ) and are resistant to growth inhibition by that factor. Nevertheless, the mechanism underlying this effect remains poorly understood. In this study, the mechanism of the resistance to growth inhibition induced by TGFβ was investigated. Methods The authors examined the expression of downstream components of the TGFβ receptor, including Smad2, Smad3, Smad4, and Smad7, and the effect of TGFβ1 treatment on the phosphorylation of Smad2 and the nuclear translocation of Smad2 and Smad3 by using 10 glioma cell lines and the A549 cell line, which is sensitive to TGFβ-mediated growth inhibition. The expression of two transcriptional corepressor proteins, SnoN and Ski, and the effect of TGFβ1 treatment on the expression of the SnoN protein and the cell cycle regulators p21, p15, cyclin-dependent kinase–4 (CDK4), and cyclin D1 were also examined. Expression of the Smad2 and Smad3 proteins was lower in the glioma cell lines than in the A549 cell line and in normal astrocytes. In particular, Smad3 expression was low or very low in nine of the 10 malignant glioma cell lines. Expression of Smad4 was low in four glioma cell lines, and expression of the Smad7 protein was similar when compared with protein expression in the A549 cell line and in normal astrocytes. The levels of Smad2 phosphorylation after TGFβ1 treatment were lower in glioma cell lines than in the A549 cell line, except for one glioma cell line. Seven of the 10 glioma cell lines exhibited lower levels of nuclear translocation of Smad2 and Smad3, and two cell lines that expressed very low levels of Smad3 protein showed no nuclear translocation. All glioma cell lines expressed the SnoN protein and its expression was unaltered by treatment with TGFβ1. Three glioma cell lines expressed high levels of the Ski protein. The expression of the p21cip1, p15INK4B, CDK4, and cyclin D1 proteins was not altered by TGFβ1 treatment, except in one cell line that displayed a slight increase in p21 protein. Overall, the expression of the Smad2 and Smad3 proteins was low in the glioma cell lines, the phosphorylation and nuclear translocation of Smad2 and Smad3 were impaired, and the TGFβ receptor signal did not affect the expression of the SnoN, p21, p15, cyclin D1, and CDK4 proteins. Conclusions These results suggest that the ability to resist TGFβ-mediated growth inhibition in malignant glioma cells is due to abnormalities in the TGFβ signaling pathway.

Application of p27 gene therapy for human malignant glioma potentiated by using mutant p27

2004 ◽  
Vol 101 (3) ◽  
pp. 505-510 ◽  
Author(s):  
Kyung-Ho Park ◽  
Jaeho Lee ◽  
Chul-Gyu Yoo ◽  
Young Whan Kim ◽  
Sung Koo Han ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Gene Therapy ◽  
Malignant Glioma ◽  
Cell Lines ◽  
Wild Type ◽  
Antitumor Effects ◽  
Poor Prognostic Factor ◽  
Content Type ◽  
Fine Print

Object. Malignant glioma could be an ideal candidate for local gene therapy because its invasion is local and it has little metastatic potential. A low expression level and high degradation activity of p27 are known to constitute an independent poor prognostic factor in patients with malignant glioma. In this study, the authors investigated the roles of wild-type p27 and mutant p27 on the treatment of malignant glioma. Methods. The authors used two adenoviruses: one expressed wild-type p27 (ad-p27wt) and the other, containing a mutation at the major metabolic site, expressed mutant p27 (ad-p27mt). The antitumor effects of the two adenoviruses were compared with respect to cell growth arrest, cell cycle alteration, apoptosis induction, and in vitro tumorigenicity in three glioblastoma mutiforme (GBM) cell lines and in a primary GBM cell line. Transduction with ad-p27wt or ad-p27mt induced the production of p27 and the dephosphorylation of pRB. The protein level of mutant p27 was significantly higher than that of wild-type p27. The ad-p27wt induced cell cycle arrest at the G1—S transition point, whereas the ad-p27mt induced arrest at the G2—M point. Both ad-p27wt and ad-p27mt induced a growth-inhibiting effect, apoptosis, and suppression of in vitro tumorigenicity; however, ad-p27mt displayed a stronger antitumor effect than ad-p27wt in brain tumor cell lines. Conclusions. Gene therapy involving p27, especially mutant p27, has the potential to become a novel and powerful therapy for malignant glioma.

Lovastatin Induces Growth Inhibition and Apoptosis in Human Malignant Glioma Cells

1994 ◽  
Vol 205 (3) ◽  
pp. 1681-1687 ◽  
Author(s):  
K.D. Jones ◽  
W.T. Couldwell ◽  
D.R. Hinton ◽  
Y.H. Su ◽  
S.K. He ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Malignant Glioma ◽  
Glioma Cells ◽  
Human Malignant Glioma ◽  
Malignant Glioma Cells
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