The progression of mammalian cells through G1 phase of the cell cycle is governed by the D-type cyclins (D1, D2, D3). These proteins are induced at the beginning of the G1 phase and associate with serine/threonine cyclin-dependent kinases to form holoenzymes. Overexpression ofcyclin D1 in human cancers as well as in several cancer cell lines has been reported. Here, we employed mitotic selection to synchronize the C6 glioma cell cycle at the start of the G1 phase and assessed the effects of neomycin on cyclin D1 protein detection by immunocytochemical analysis. Cyclin D1 activation as well as cell proliferation were already significantly reduced after 3 h of incubation of the cells with neomycin. These findings suggested that the antiproliferative effects of neomycin in gliomas could be mediated by inhibition of the expression of cyclin D1 gene and support further consideration of therapeutic use of neomycin in a Phase I clinical study for patients with recurrent glioblastoma. [Neurol Res 2003; 25: 691-693]
Keywords: Glioma cells; cyclin D1 activation; neomycin; immunocytochemistry
Targeted therapy directed to intracellular signaling pathways presents new opportunities for the treatment of glioblastoma, the most common brain cancer in adults and also the most lethal of all cancers1. Cyclins are a family of proteins playing an essential role in cell cycle regulation because of their specific and periodic expression during cell cycle progression2. D-type cyclins expression is regulated by extracellular mitogenic signals via mitogen activated protein kinases (MAPKs) pathways3. Experimental evidence also shows that nuclear factor-kappa B (NF-kB) binding to the cyclin D1 gene promoter is critical for the cyclin D1 expression . Cyclin D1 overexpression has been reported to play a key role in the growth and progression of several human cancers such as B-cell lymphoma5, breast cancer6, esophageal carcinoma7 and hepatocellular carcinoma8. Furthermore, overexpression of cyclin D1 protein is associated with poor prognosis in malignancies9. Recently a direct correlation between cell cycle progression and cyclin D1 expression has been reported in glioblastoma cells10. These data suggested that cyclin D1 inhibition could be a novel therapy for the treatment of solid cancers. Previously we have reported that the aminoglicoside antibiotic, neomycin, inhibits glioma growth in vitro as well as in vivo11,12 at least in part by negative regulation of MAPKs, cyclic AMP response element binding protein (CREB) and NF-kB signaling pathways13,14. In the present study, the effect of neomycin on cyclin D1 protein expression in glioma cells was investigated via cyclin D1 immunocytochemistry.
MATERIALS AND METHODS
Fetal calf serum and medium for cell culture were obtained from Gibco (Paisley, Scotland, UK); neomycin from Sigma Chemicals (St. Louis, MO, USA); penicillin and streptomycin from Gibco, and other general laboratory chemicals from Sigma Chemicals. Antibody to cyclin D1 was obtained from BioMol (Plymouth Meeting, PA, USA); biotinconjugated goal anti-mouse IgG secondary antibody from Zymed (San Francisco, CA, USA); ABC staining kit from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and diaminobenzidine (DAB) from Sigma Chemicals.
Cell culture and drug exposure
C6 glioma cells were cultured in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 7.5% fetal calf serum and penicillin (10 units ml^sup -1^) and streptomycin (10 [mu]g ml^sup -1^). Cultures were maintained in a humidified atmosphere of 5% CO2 and 37[degrees]C. Glioma cell cultures were synchronized using a mitotic selection procedure as reported15, seeded at a density of 5 x 10^sup -4^ on 12 mm-diameter cover glasses and exposed to 10 mM neomycin sulfate for 3 h or not (control cultures). This time point was selected to correspond to early CDKs activation induced after proteosomal inhibition in cortical neuronal cultures16. This concentration of neomycin was chosen on the basis of our previous experiments11. Cell viability was determined by trypsan blue exclusion. The viability of untreated cells was found to exceed 95% consistently. The viability of cells treated with neomycin sulfate was found to exceed 93%.
For immunocytochemical detection of cyclin D1 protein, cultures were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100 and incubated in Tris-buffered saline (TBS) containing 5% normal goat serum. Cells were labeled by the sequential application of the primary mouse monoclonal anti-cyclin D1 antibody (1:50 overnight at 4[degrees]C) and with biotin-conjugated goat antimouse IgG secondary antibody followed by the ABC staining method. The glasses were reacted with DAB, rinsed, dehydrated in alcohol, cleared in xylene and mounted for light microscopy. Staining was performed at least three times to ensure accurate interpretation. Negative controls were exposed to blocking solution instead of primary antibody. To quantify cyclin D1 activation, we assessed the percentage of C6 cells in the cultures that show intense nuclear accumulation of cyclin D1. Triplicate coverslips were assessed counting at least 300 C6 cells each by someone blinded to the experimental conditions.
Data are expressed as means + or - SEM. Statistical comparisons were made using the Student's t-test. p
This study corroborates the antiproliferative effect of neomycin in glioma C6 cells as previously reported11,14. To determine if this antiproliferative effect of neomycin was mediated at the level of cyclin D1, glioma cells were exposed to 10 mM neomycin for 3 h. To this end, immunocytochemical staining for cyclin D1 was performed in mitotic selection cultures. As expected glioma C6 cells showed cyclin D1-specific nuclear immunoreactivity. Cyclin D1-specific nuclear staining was distributed in many cells of control cultures (Figure 1A), that may be indicative of functional activation. In contrast, in C6 cell cultures exposed to neomycin for 3 h cyclin D1 had a characteristic perinuclear localization (Figure 1B), indicating that cyclin D1 is inactivated. Neomycin produced significant inhibition of cyclin D1 translocation from the perinuclear region to the nucleus of the C6 cells as compared to control cultures. Furthermore, in neomycin-treated cultures, nuclear cyclin D1 immunoreactivity is less intense than in control cultures.
We quantified numbers of cyclin D1 positive cells in all cultures and evaluated as the percentage of total counted cells. This analysis demonstrated that 3 h after neomycin administration, percentage of cyclin D1-positive cells decreased significantly when compared with control cultures (53.21% + or - 3.30% in control cultures versus 15.14 %+ or - 1.32% in neomycin-treated cultures; p
The major findings to emerge from this study are that cyclin D1 gene activation influences the growth of glioma C6 cells and that inhibition of cyclin D1 expression results in a diminished capacity to glioma proliferation. Passage of cells through the cell cycle is a highly ordered process involving the sequential activation of different cyclin/cyclin-dependent kinase (CDK) complexes17,18. At a molecular level, this process mainly depends on the synthesis and degradation of cyclin proteins, modification of the kinase subunits by phosphorylation-dephosphorylation, and translocation of cyclin CDK complexes19. The D- and E-type cyclin proteins are believed to control progression through the G1 phase, while A- and B-type cyclins regulate cyclin from S phase (DNA synthesis) through G2 and into M phase. Experimental evidence indicates that several antiproliferative agents mediate cell cycle arrest through effects on G1 cyclins. CDKs and/or CDK inhibitory proteins17. Previously we have reported that neomycin acts as an antiproliferative agent through inhibition of MAPKs, CREB and NF-kB activation13,14. CREB is a transcription factor that modulates transcription of genes which possess the cAMP response element in their promoter region as cyclin D1 which plays an important role in cancer development and progression20-22. Among a variety of signaling mechanisms, it appears that MARK cascade, specifically ERK1/2 pathways might play an essential role in the activation of NF-kB, cyclins and CREB19-22. Strong experimental evidence indicates that CREB-binding protein is a transcriptional coactivator that is required by NF-kB for maximal transcriptional activity23. On the other hand, cyclin D1 expression was induced by NF-kB4,24. Thus, cellular signaling pathways which activate ERK1/2, NF-kB, CREB and cyclins participate in the activation of genes playing essential roles in proliferation, angiogenesis and apoptosis, key events for tumor growth and progression.
The results presented here clearly demonstrate that glioma C6 cells overexpress nuclear cyclin D1, another NF-kB-regulated gene, and that this expression is down-regulated by neomycin. Given that cyclin D1 is needed for cells to advance from the G1 to the S phase of the cell cycle, it is not surprising we found that neomycin induced G1/S arrest and this caused suppression of cell proliferation. Since neomycin can suppress ERK1/2, CREB, NF-kB, cyclin D1 intracellular signals resulting in inhibition of cell proliferation of glioma cells, our studies provide enough rationale for considering neomycin worthy of clinical trial in patients with glioma.
We thank Chantai Bourdier for editorial assistance and Concha Muela and Argentina Fernandez-Ayerdi for technical work. This study was partially supported by Fundacion Futuro (Madrid).
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Pedro Cuevas*[dagger], Diana Diaz-Gonzalez* and Manuel Dujovny
* Departamento de Investigation, Hospital Universitario Ramon y Cajal, Universidad de Alcala de Henares, Madrid, Spain [dagger] Department of Neurosurgery, Wayne State University, Detroit, MI, USA
Correspondence and reprint requests to: Dr Pedro Cuevas, Departamento de Investigation, Hospital Universitario Ramon y Cajal, Universidad de Alcala de Henares, Ctra. de Colmenar, km. 9.100, E-28034-Madrid, Spain, [firstname.lastname@example.org] Accepted for publication June 2003.
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