Differential expression and role of p21cip/waf1 and p27kip1 in TNF-α-induced inhibition of proliferation in human glioma cells

<p>Abstract</p> <p>Background</p> <p>The role of TNF-α in affecting the fate of tumors is controversial, while some studies have reported apoptotic or necrotic effects of TNF-α, others provide evidence that endogenous TNF-α promotes growth and development of tumors. Understanding the mechanism(s) of TNF-α mediated growth arrest will be important in unraveling the contribution of tissue associated macrophages in tumor resistance. The aim of this study was to investigate the role of Cyclin Dependent Kinase Inhibitors (CDKI) – p21^cip/waf1and p27^kip1in TNF-α mediated responses in context with p53 and activation of NF-κB and Akt pathways. The study was done with human glioma cell lines -LN-18 and LN-229 cells, using monolayer cultures and Multicellular Spheroids (MCS) as <it>in vitro </it>models.</p> <p>Results</p> <p>TNF-α induced inhibition of proliferation and enhanced the expression of p21^cip/waf1and p27^kip1in LN-18 cells. p21 was induced on exposure to TNF-α, localized exclusively in the nucleus and functioned as an inhibitor of cell cycle but not as an antiapoptotic protein. In contrast, p27 was constitutively expressed, localized predominantly in the cytoplasm and was not involved in arrest of proliferation. Our data using IκBα mutant LN-18 cells and PI3K/Akt inhibitor-LY294002 revealed that the expression of p21 is regulated by NF-κB. Loss of IκBα function in LN-229 cells (p53 positive) did not influence TNF-α induced accumulation of pp53 (Ser-20 p53) suggesting that p53 was not down stream of NF-κB. Spheroidogenesis enhanced p27 expression and p21 induced by TNF-α was significantly increased in the MCS compared to monolayers.</p> <p>Conclusion</p> <p>This study demarcates the functional roles for CDKIs-p21^cip/waf1and p27^kip1during TNF-α stimulated responses in LN-18 glioma cells. Our findings provide evidence that TNF-α-induced p21 might be regulated by NF-κB or p53 independently. p21 functions as an inhibitor of cell proliferation and does not have a direct role in rendering the cells resistant to TNF-α mediated cytotoxicity.</p

Upregulation of Survivin in G2/M Cells and Inhibition of Caspase 9 Activity Enhances Resistance in Staurosporine-Induced Apoptosis

Survivin, a member of the inhibitor of apoptosis (IAP) gene family, plays an important role in both the regulation of cell cycle and the inhibition of apoptosis, and is frequently overexpressed in many tumor types. In neuroblastomas, the expression of survivin correlates with a more aggressive and histologically unfavorable disease. Survivin is predominantly a cytoplasmic protein that is expressed in a cell cycle-dependent manner, increasing in the G2/M phase of the cell cycle followed by a rapid decline in the G1 phase. Recently, the role of survivin in resistance to chemotherapy has become an area of intensive investigation. In this study, we demonstrate a phase-specific resistance due to survivin in staurosporine (STS)-induced apoptosis in the human neuroblastoma cell line SK-N-MC. G2/M-arrested cultures show an upregulation of survivin expression and are more resistant, whereas G1-phase cells that show decreased levels of survivin are more sensitive to apoptosis. Localization studies revealed differences in the distribution of survivin in two synchronized populations, with G1 cells having weakly positive staining confined to the nucleus, in contrast to G2/M cells that depicted a more uniform and intense expression of survivin throughout the cell. In our experimental system, STS induced apoptosis through the mitochondrial-caspase 9-mediated pathway. Retention of survivin in G1 cells by inhibition of the ubiquitin-proteosome pathway or inhibition of caspase 9 protected the cells against apoptosis. Our data suggest that survivin exerts its antiapoptotic effect by inhibiting caspase 9 activity, an important event in STS-mediated apoptosis. In context with cell cycle-dependent responses to chemotherapy, the data from this study suggest the possibility of exploiting the survivin pathway for inducing apoptosis in tumor cells

TNF-α and IFN-γ Together Up-Regulates Par-4 Expression and Induce Apoptosis in Human Neuroblastomas

The objective of this study was to examine the combined effect of Interferon-gamma (IFN-γ) and Tumor Necrosis factor-alpha (TNF-α) on cytotoxicity and expression of prostate apoptosis response-4 (Par-4) and Par-4 interacting proteins B-cell lymphoma (Bcl-2), nuclear factor kappa-light-chain-enhancer of activated B cells/p65 subunit (NF-κB/p65), Ak mouse strain thymoma (Akt) in human neuroblastoma (NB) cells. Materials and methods included human neuroblastoma cell lines-SK-N-MC, SK-N-SH, and SH-SY5Y, which were treated with IFN-γ and TNF-α individually, or in combination, and were assessed for viability by tetrazolium (MTT) assay. Apoptosis was monitored by hypodiploid population (by flow cytometry), DNA fragmentation, Poly (ADP-ribose) polymerase (PARP) cleavage, and caspase-8 activity. Transcript level of Par-4 was measured by RT-PCR. Protein levels of Par-4 and suppressor of cytokine signaling 3 (SOCS-3) were assessed by immunoblotting. Cellular localization of Par-4 and p65 was examined by immunofluorescence. Unbiased transcript analysis for IFN-γ, TNF-α, and Par-4 were analyzed from three independent clinical datasets from neuroblastoma patients. In terms of results, SK-N-MC cells treated with a combination of, but not individually with, IFN-γ and TNF-α induced apoptosis characterized by hypodiploidy, DNA fragmentation, PARP cleavage, and increased caspase-8 activity. Apoptosis was associated with up-regulation of Par-4 mRNA and protein expression. Immunofluorescence studies revealed that Par-4 was localized exclusively in cytoplasm in SK-N-MC cells cultured for 24 h. but showed nuclear localization at 48 h. Treatment with IFN-γ and TNF-α together enhanced the intensity of nuclear Par-4. In gene expression, data from human neuroblastoma patients, levels of IFN-γ, and TNF-α have strong synergy with Par-4 expression and provide good survival advantage. The findings also demonstrated that apoptosis was associated with reduced level of pro-survival proteins–Bcl-2 and Akt and NF-κB/p65. Furthermore, the apoptotic effect induced by IFN-γ-induced Signal Transducer and Activator of Transcription-1(STAT-1), and could be due to down-regulation of suppressor of cytokine signaling-3 (SOCS3). The study concludes that a combinatorial approach using IFN-γ and TNF-α can be explored to maximize the effect in chemotherapy in neuroblastoma, and implies a role for Par-4 in the process

Expression and regulation of prostate apoptosis response-4 (Par-4) in human glioma stem cells in drug-induced apoptosis.

Gliomas are the most common and aggressive of brain tumors in adults. Cancer stem cells (CSC) contribute to chemoresistance in many solid tumors including gliomas. The function of prostate apoptosis response-4 (Par-4) as a pro-apoptotic protein is well documented in many cancers; however, its role in CSC remains obscure. In this study, we aimed to explore the role of Par-4 in drug-induced cytotoxicity using human glioma stem cell line--HNGC-2 and primary culture (G1) derived from high grade glioma. We show that among the panel of drugs- lomustine, carmustine, UCN-01, oxaliplatin, temozolomide and tamoxifen (TAM) screened, only TAM induced cell death and up-regulated Par-4 levels significantly. TAM-induced apoptosis was confirmed by PARP cleavage, Annexin V and propidium iodide staining and caspase-3 activity. Knock down of Par-4 by siRNA inhibited cell death by TAM, suggesting the role of Par-4 in induction of apoptosis. We also demonstrate that the mechanism involves break down of mitochondrial membrane potential, down regulation of Bcl-2 and reduced activation of Akt and ERK 42/44. Secretory Par-4 and GRP-78 were significantly expressed in HNGC-2 cells on exposure to TAM and specific antibodies to these molecules inhibited cell death suggesting that extrinsic Par-4 is important in TAM-induced apoptosis. Interestingly, TAM decreased the expression of neural stem cell markers--Nestin, Bmi1, Vimentin, Sox2, and Musashi in HNGC-2 cell line and G1 cells implicating its potential as a stemness inhibiting drug. Based on these data and our findings that enhanced levels of Par-4 sensitize the resistant glioma stem cells to drug-induced apoptosis, we propose that Par-4 may be explored for evaluating anti-tumor agents in CSC

Differential expression and role of p21and p27in TNF-α-induced inhibition of proliferation in human glioma cells-6

<p><b>Copyright information:</b></p><p>Taken from "Differential expression and role of p21and p27in TNF-α-induced inhibition of proliferation in human glioma cells"</p><p>http://www.molecular-cancer.com/content/6/1/42</p><p>Molecular Cancer 2007;6():42-42.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1904457.</p><p></p>es (200 nM) for 6 hr and cultured as monolayers or spheroids for 18 hr. Cells were treated with TNF-α (10 ng/ml) for 24 hr, and tritiated thymidine incorporation assay was done as described in Fig. 1. * p < 0.05 inhibition of proliferation with TNF-α in transfected versus untransfected cells. The data is representative of three experiments done in triplicates

Differential expression and role of p21and p27in TNF-α-induced inhibition of proliferation in human glioma cells-3

<p><b>Copyright information:</b></p><p>Taken from "Differential expression and role of p21and p27in TNF-α-induced inhibition of proliferation in human glioma cells"</p><p>http://www.molecular-cancer.com/content/6/1/42</p><p>Molecular Cancer 2007;6():42-42.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1904457.</p><p></p>B) LN-229 cells were transfected with IκBα dominant negative construct and the expression of nuclear p65 was determined after exposure to TNF-α for 6 hr

Differential expression and role of p21and p27in TNF-α-induced inhibition of proliferation in human glioma cells-0

<p><b>Copyright information:</b></p><p>Taken from "Differential expression and role of p21and p27in TNF-α-induced inhibition of proliferation in human glioma cells"</p><p>http://www.molecular-cancer.com/content/6/1/42</p><p>Molecular Cancer 2007;6():42-42.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1904457.</p><p></p>or 24 hr, during the last 18 hr of treatment, 1 μCi of triatiated thymidine was added to the wells. The cells were harvested and the radio activity-counts per minute (CPM) was measured and percent inhibition of proliferation was calculated considering the CPM in untreated cells as 100%. B) Expression of TNFR1 and TNFR2 was analyzed by RT-PCR in LN-18 cells and compared with human glioma cell line-U87MG, used as positive control. β-actin was used as control. C) Expression of TNFR1 and TNFR2 in LN-18 cells and U87MG cells analyzed by flowcytometry, bold lines represent TNFR1/TNFR2 expression and dotted lines depict profiles with isocontrol antibody. D) Monolayers and spheroids were preincubated with neutralizing antibodies to TNFR1 and TNFR2 (50 μg/ml) or isocontrol antibody for 2 hr and treated with TNF-α (10 ng/ml) for 24 hr and proliferation assay was done as described above. E) Monolayers and spheroids were treated with serial concentrations of TNFR1 agonistic antibody or isocontrol antibody for 24 hr and proliferation was determined by triatiated thymidine incorporation assay. The data for A, D and E is the mean ± SE of at least three independent experiments done in triplicates. * p < 0.05-comparison of inhibition of proliferation in TNF-α treated cells in the presence and absence of TNFR neutralizing antibodies for D and between treated and controls cells for A and E. F) Levels of TNFR1 expression by Western blotting in monolayers (M) and spheroids (S) cultured for 24 hr. The blots were stripped and reprobed with β-actin, which served as a loading control of proteins

Effect of TNF-α on pp53 and p21 levels was studied in LN-229 cells transfected with IκBα dominant negative construct by confocal laser scanning microscopy

<p><b>Copyright information:</b></p><p>Taken from "Differential expression and role of p21and p27in TNF-α-induced inhibition of proliferation in human glioma cells"</p><p>http://www.molecular-cancer.com/content/6/1/42</p><p>Molecular Cancer 2007;6():42-42.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1904457.</p><p></p> Treatment with primary antibodies was followed by incubation with appropriate secondary antibodies tagged to Cy3. DAPI was used for staining nucleus (Magnification 63×)

Differential expression and role of p21and p27in TNF-α-induced inhibition of proliferation in human glioma cells-2

<p><b>Copyright information:</b></p><p>Taken from "Differential expression and role of p21and p27in TNF-α-induced inhibition of proliferation in human glioma cells"</p><p>http://www.molecular-cancer.com/content/6/1/42</p><p>Molecular Cancer 2007;6():42-42.</p><p>Published online 12 Jun 2007</p><p>PMCID:PMC1904457.</p><p></p>ed by MTT assay. The percent cell death was determined considering viability of control cells as 100%. (B) IκBα mutant LN-18 cells were treated with TNF-α (10 ng/ml) for different time points and cleavage of PARP (89 kD fragment) was determined by Western blotting. C) IκBα mutant LN-18 cells grown on coverslips were treated with TNF-α (10 ng/ml) for 3 hr and stained with p21 followed by secondary antibody labeled with Cy3 and DAPI was used for staining nucleus (Magnification 63×). D) Expression of p21 and p27 determined by Western blotting in monolayers (M) and spheroids (S) derived from IκBα mutant LN-18 cells treated with TNF-α for 3 hr. The blots were stripped and reprobed with β-actin, which served as control for equal loading of proteins