The inhibitor of growth 1 (ING1) is involved in trichostatin A-induced apoptosis and caspase 3 signaling in p53-deficient glioblastoma cells.

Prognosis for patients with glioblastoma multiforme (GBM) is poor. Inhibitors of histone deacetylases (HDACi) like trichostatin A (TSA) are promising alternatives to conventional treatment. Deficient tumor suppressor functions, such as TP53 mutations and p14(ARF)/p16(INK4a) deletions, are characteristic for GBM and can cause resistance to DNA damaging agents such as cisplatin and to HDACi like TSA. The type II tumor suppressor Inhibitor of growth 1 (ING1) is involved in DNA damage response and histone modification. We have previously shown that ING1 is downregulated in GBM and involved in glioma-induced angiogenesis and in cisplatin-induced apoptosis in malignant glioma cells. Hence, the goal of our present study was to investigate whether TSA affects ING1 protein expression and also whether modulating ING1 levels affects TSA-induced apoptosis in malignant glioma cells that contain deficient p53 function and inactive pl4(ARF)/p16(INK4a) signaling. If so, we asked, which apoptotic pathway might be the major mediator beyond this interaction. To test whether ING1 proteins function in TSA-induced apoptosis in GBM, we analyzed TSA effects in LN229 GBM cells, which harbor TP53 mutations and INK4a deletion, following ING1 knockdown by siRNA. Expression of ING1, acetylated core histones H3 and H4, and the proapoptotic proteins caspase 3 and Fas-associated death domain (FADD) was determined by Western blotting. Percentages of apoptotic cells were obtained by flow cytometry. TSA induced the major ING1 isoform p33(ING1b) and increased levels of both histone acetylation and apoptosis in LN229 cells. ING1 knockdown cells revealed marked resistance to TSA-induced apoptosis, impairment of caspase 3 activation, and suppression of FADD. The data suggest that ING1 contributes to TSA-induced apoptosis in GBM cells with deficient p53 and p14(ARF)/p16(INK4a) functions, possibly by regulating FADD/caspase 3 signaling.

Selective inactivation of DNA-dependent protein kinase with antisense oligodeoxynucleotides: consequences for the rejoining of radiation-induced DNA double-strand breaks and radiosensitivity of human cancer cell lines.

The inhibition of DNA-dependent protein kinase activity with antisense-oligodeoxynucleotide (As-ODN) and its consequences for the rejoining of DNA-double-strand breaks (Dsbs) and radiation sensitivity was studied in human non-small cell lung cancer (NSCLC) cell lines. Cells were transfected with As-ODNs specific for the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). In comparison, cells were treated with Wortmannin, a potent but nonspecific inhibitor of DNA-PK activity. As-ODN efficiently reduced the kinase activity with an IC50 of about 100-200 IC50 of Wortmannin was at approximately 5-10 micro M. Treatment of cells with 300 nM As-ODN increased the fraction of residual Dsb at 4 h after irradiation by a factor of 4.4, 2.6, and 1.7 in A549, H460, and H661 cells, respectively. The respective values after treatment with 20 micro M Wortmannin were 5.3, 4.3, and 2.2. Inhibition of DNA-PK activity by As-ODN and Wortmannin also decreased the surviving fraction of the NSCLC cell lines. These data show that kinase activity of DNA-PKcs can be specifically inhibited with As-ODN as effective as Wortmannin and results in marked inhibition of DNA-Dsb rejoining and radiosensitization of NSCLC cell lines.

Increased radiation-induced apoptosis and altered cell cycle progression of human lung cancer cell lines by antisense oligodeoxynucleotides targeting p53 and p21(WAF1/CIP1).

Lung cancer is difficult to control locally by radiotherapy and is known to have frequently p53 mutations. Previous results have shown that non-small-cell lung cancer (NSCLC) cell lines with nonfunctional p53 have a higher fraction of radiation-induced apoptosis and that apoptosis follows after the release from the G2/M arrest. The aim of the present work was to study whether inhibition of the p53 response in NSCLC cell lines can modulate the G2/M arrest and the induction of apoptosis after ionizing radiation. Antisense oligodeoxynucleotides (As-ODNs) were used to inhibit the p53 response in the cell lines H460 and A549 with functional p53. In addition, H661 with nonfunctional p53 was used. The results have shown that As-ODNs targeting mRNA of p53 and p21 downregulate radiation-induced expression of p53 and p21(WAF1/CIP1). Delayed apoptosis (35.7+/-4.2% in H460, 1.2+/-0.4% in A549 and 72.2+/-6.5% in H661) was observed after cell cycle progression beyond the G2 block, either in the late G2 phase of the same cell cycle being irradiated (H661) or in the G1 phase of the subsequent cell cycle (H460, A549). As-p53 significantly decreased the fraction of G2/M-arrested cells in H460 cells and increased radiation-induced apoptosis at 96 hours by 17.9+/-8.5 and 9.1+/-3.3% to 53.6+/-7.4 and 10.8+/-2.9% in H460 and A549 cells (P<.01), respectively, but had no effect in H661 cells with nonfunctional p53. In addition, As-p21 decreased the fraction of G2-arrested A549 and H460 cells and increased apoptosis by 23.8+/-5.2 and 31.6+/-7.3% to 59.4+/-3.1 and 32.8+/-7.3%, respectively (P<.01). In conclusion, these data show that radiation-induced G2 arrest is decreased in NSCLC cells and radiation-induced apoptosis is increased when p53-responsive pathways are blocked via As-ODN targeting p53 or p21(WAF1/CIP1) mRNA. In view of the fact that p53 and p21 As-ODN had similar effects on radiation-induced apoptosis normalized by their ability to inhibit radiation-induced p21 expression, we concluded that p21 is an important trigger of late ionizing radiation-induced apoptosis.

Caffeine confers radiosensitization of PTEN-deficient malignant glioma cells by enhancing ionizing radiation-induced G1 arrest and negatively regulating Akt phosphorylation.

PTEN mutations are frequently found in malignant glioma and can result in activated phosphatidylinositol-3-kinase/Akt survival signaling associated with resistance to radiotherapy. Strategies to interfere with aberrant PI3K/Akt activity are therefore being developed to improve the therapeutic efficacy of radiotherapy in patients with malignant glioma. The methylxanthine caffeine has been described as a PI3K inhibitor and is also known to sensitize cells to ionizing radiation. However, a direct association between these two caffeine-mediated effects has not been reported yet. Therefore, we asked whether caffeine or its derivative pentoxifylline differentially affect the radiosensitivity of malignant gliomas with different PTEN status. As models, we used the radiosensitive EA14 malignant glioma cell line containing wild-type PTEN and the radioresistant U87MG malignant glioma cell line harboring mutant PTEN. Our study revealed that caffeine and pentoxifylline radiosensitized PTEN-deficient but not PTEN-proficient glioma cells. Radiosensitization of PTEN-deficient U87MG cells by caffeine was significantly correlated with the activation of the G(1) DNA damage checkpoint that occurred independently of de novo synthesis of p53 and p21. The p53 independency was also confirmed by a significant caffeine-mediated radiosensitization of the glioma cell lines T98G and U373MG that are deficient for both PTEN and p53. Furthermore, caffeine-mediated radiosensitization was associated with the inhibition of Akt hyperphosphorylation in PTEN-deficient cells to a level comparable with PTEN-proficient cells. Our data suggest that the methylxanthine caffeine or its derivative pentoxifylline are promising candidate drugs for the radiosensitization of glioma cells particularly with PTEN mutations.

Pifithrin-α as a potential cytoprotective agent in radiotherapy: protection of normal tissue without decreasing therapeutic efficacy in glioma cells.

Activation of p53 has been causally linked to normal tissue damage after irradiation. Pifithrin-α (PFT-α), a specific inhibitor of p53, has been suggested as a combinatory agent in the treatment of p53-deficient tumors in which inhibition of p53 would not compromise therapeutic efficacy but would decrease p53-mediated side effects in normal tissue. We tested this concept for radiotherapy of p53-deficient and -proficient glioma. We observed significant interaction of PFT-α with radiation-induced G(1) checkpoint activation and plating efficiency only in glioma cells expressing at least one wild-type allele of p53. This interaction was correlated with PFT-α-mediated inhibition of radiation-induced expression of the p53 target gene p21(Waf1). Despite inhibition of p53 function we did not observe significant changes in radiosensitivity after treatment with PFT-α in either p53-deficient or p53-proficient tumor cells. We confirmed these results in p53-proficient lung cancer cells. In contrast, PFT-α significantly increased the fraction of normal astrocytes and fibroblasts surviving irradiation; this was accompanied by improved DNA damage repair, speaking against an accumulation of cells with genetic lesions after PFT-α treatment. In conclusion, PFT-α might prove useful in protecting normal tissue from the side effects of radiotherapy without reducing the efficacy of treatment for both p53-proficient and -deficient tumors.

Association between intestinal tight junction permeability and whole-body electrical resistance in healthy individuals: a hypothesis.

OBJECTIVE: Intestinal permeability describes non-carrier-mediated modes of transport through the intestinal epithelium. Wrist-ankle bioimpedance analysis (BIA) is a standard method to determine body composition based on the measurements of whole-body electrical resistance and reactance values. The present report deals with the coincidentally observed associations between permeability results and electrical raw values of BIA and their subsequent reproduction in a larger group of individuals. METHODS: Tetrapolar wrist-ankle BIA was performed on day 1 in the initial sample (12 women, 36 +/- 11 y of age) and the validation sample (36 healthy subjects, 26 women and 10 men, 35 +/- 14 y of age). Intestinal permeability tests (lactulose and mannitol) were implemented within 1 wk thereafter. Wrist-ankle electrical resistance plus electrical resistance between current-conducting electrodes and voltage-sensing electrodes (Rtotal) was measured at 5 kHz and 100 kHz. RESULTS: Permeability and bioimpedance raw values were normal, indicating normal tight junction permeability and normal hydration. Lactulose correlated to R(50total) in the initial sample (rho = 0.639, P = 0.025) and in the validation sample (rho = 0.673, P < 0.001). Weaker associations to R(50total) were observed with mannitol (rho = 0.381, P = 0.008) and lactulose/mannitol (rho = 0.369, P = 0.010) in the total group of individuals. Regression analyses demonstrated that R(50total) alone accounted for 41.3% of the variance in lactulose permeability. CONCLUSION: The nature of the observed positive association between intestinal tight junction permeability and whole-body electrical resistance is unclear. We hypothesize that regulation involving submolecular mechanisms based on the principles of quantum physics might have caused the observed association. Such coherent mechanisms might possibly play a role in basal physiologic regulation in humans.

Down-regulation of the inhibitor of growth 1 (ING1) tumor suppressor sensitizes p53-deficient glioblastoma cells to cisplatin-induced cell death.

Impaired tumor suppressor functions, such as deficient p53, are characteristic for glioblastoma multiforme (GBM) and can cause resistance to DNA-damaging agents like cisplatin. We have recently shown that the INhibitor of Growth 1 (ING1) tumor suppressor is down-regulated in malignant gliomas and that the decrease of ING1 expression correlates with histological grade of malignancy, suggesting a role for ING1 in the pathogenesis and progression of malignant gliomas. Based on this background, the purpose of our current study was to examine the potential impact of ING1 protein levels on DNA-damage response in GBM. Using LN229 GBM cells, which express ING1 proteins and harbor mutant TP53, we are the first to show that DNA damage by cisplatin or ionizing radiation differentially induced the two major ING1 splicing isoforms. The p47 ING1a isoform, that promotes deacetylation of histones, thus formation of heterochromatic regions of DNA, which are less susceptible to DNA damage, was preferentially induced by >50-fold. This might represent a response to protect DNA from damage. Also, ING1 knockdown by siRNA accelerated transit of cells through G1 phase, consistent with ING1 serving a tumor suppressor function, and caused cells to enter apoptosis more rapidly in response to cisplatin. Our results indicate that malignant gliomas may down-regulate ING1 to allow more efficient tumor growth and progression. Also, ING1 down-regulation may sensitize GBM cells with deficient p53 to treatment with cisplatin.