Opposite effects of the triple target (DNA-PK/PI3K/mTOR) inhibitor PI-103 on the radiation sensitivity of glioblastoma cell lines proficient and deficient in DNA-PKcs.

BACKGROUND: Radiotherapy is routinely used to combat glioblastoma (GBM). However, the treatment efficacy is often limited by the radioresistance of GBM cells. METHODS: Two GBM lines MO59K and MO59J, differing in intrinsic radiosensitivity and mutational status of DNA-PK and ATM, were analyzed regarding their response to DNA-PK/PI3K/mTOR inhibition by PI-103 in combination with radiation. To this end we assessed colony-forming ability, induction and repair of DNA damage by γH2AX and 53BP1, expression of marker proteins, including those belonging to NHEJ and HR repair pathways, degree of apoptosis, autophagy, and cell cycle alterations. RESULTS: We found that PI-103 radiosensitized MO59K cells but, surprisingly, it induced radiation resistance in MO59J cells. Treatment of MO59K cells with PI-103 lead to protraction of the DNA damage repair as compared to drug-free irradiated cells. In PI-103-treated and irradiated MO59J cells the foci numbers of both proteins was higher than in the drug-free samples, but a large portion of DNA damage was quickly repaired. Another cell line-specific difference includes diminished expression of p53 in MO59J cells, which was further reduced by PI-103. Additionally, PI-103-treated MO59K cells exhibited an increased expression of the apoptosis marker cleaved PARP and increased subG1 fraction. Moreover, irradiation induced a strong G2 arrest in MO59J cells (~ 80% vs. ~ 50% in MO59K), which was, however, partially reduced in the presence of PI-103. In contrast, treatment with PI-103 increased the G2 fraction in irradiated MO59K cells. CONCLUSIONS: The triple-target inhibitor PI-103 exerted radiosensitization on MO59K cells, but, unexpectedly, caused radioresistance in the MO59J line, lacking DNA-PK. The difference is most likely due to low expression of the DNA-PK substrate p53 in MO59J cells, which was further reduced by PI-103. This led to less apoptosis as compared to drug-free MO59J cells and enhanced survival via partially abolished cell-cycle arrest. The findings suggest that the lack of DNA-PK-dependent NHEJ in MO59J line might be compensated by DNA-PK independent DSB repair via a yet unknown mechanism.

Differential effects of the Akt inhibitor MK-2206 on migration and radiation sensitivity of glioblastoma cells.

BACKGROUND: Most tumor cells show aberrantly activated Akt which leads to increased cell survival and resistance to cancer radiotherapy. Therefore, targeting Akt can be a promising strategy for radiosensitization. Here, we explore the impact of the Akt inhibitor MK-2206 alone and in combination with the dual PI3K and mTOR inhibitor PI-103 on the radiation sensitivity of glioblastoma cells. In addition, we examine migration of drug-treated cells. METHODS: Using single-cell tracking and wound healing migration tests, colony-forming assay, Western blotting, flow cytometry and electrorotation we examined the effects of MK-2206 and PI-103 and/or irradiation on the migration, radiation sensitivity, expression of several marker proteins, DNA damage, cell cycle progression and the plasma membrane properties in two glioblastoma (DK-MG and SNB19) cell lines, previously shown to differ markedly in their migratory behavior and response to PI3K/mTOR inhibition. RESULTS: We found that MK-2206 strongly reduces the migration of DK-MG but only moderately reduces the migration of SNB19 cells. Surprisingly, MK-2206 did not cause radiosensitization, but even increased colony-forming ability after irradiation. Moreover, MK-2206 did not enhance the radiosensitizing effect of PI-103. The results appear to contradict the strong depletion of p-Akt in MK-2206-treated cells. Possible reasons for the radioresistance of MK-2206-treated cells could be unaltered or in case of SNB19 cells even increased levels of p-mTOR and p-S6, as compared to the reduced expression of these proteins in PI-103-treated samples. We also found that MK-2206 did not enhance IR-induced DNA damage, neither did it cause cell cycle distortion, nor apoptosis nor excessive autophagy. CONCLUSIONS: Our study provides proof that MK-2206 can effectively inhibit the expression of Akt in two glioblastoma cell lines. However, due to an aberrant activation of mTOR in response to Akt inhibition in PTEN mutated cells, the therapeutic window needs to be carefully defined, or a combination of Akt and mTOR inhibitors should be considered.

Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status.

Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion.

A prospective study on histone γ-H2AX and 53BP1 foci expression in rectal carcinoma patients: correlation with radiation therapy-induced outcome.

BACKGROUND: The prognostic value of histone γ-H2AX and 53BP1 proteins to predict the radiotherapy (RT) outcome of patients with rectal carcinoma (RC) was evaluated in a prospective study. High expression of the constitutive histone γ-H2AX is indicative of defective DNA repair pathway and/or genomic instability, whereas 53BP1 (p53-binding protein 1) is a conserved checkpoint protein with properties of a DNA double-strand breaks sensor. METHODS: Using fluorescence microscopy, we assessed spontaneous and radiation-induced foci of γ-H2AX and 53BP1 in peripheral blood mononuclear cells derived from unselected RC patients (n = 53) undergoing neoadjuvant chemo- and RT. Cells from apparently healthy donors (n = 12) served as references. RESULTS: The γ-H2AX assay of in vitro irradiated lymphocytes revealed significantly higher degree of DNA damage in the group of unselected RC patients with respect to the background, initial (0.5 Gy, 30 min) and residual (0.5 Gy and 2 Gy, 24 h post-radiation) damage compared to the control group. Likewise, the numbers of 53BP1 foci analyzed in the samples from 46 RC patients were significantly higher than in controls except for the background DNA damage. However, both markers were not able to predict tumor stage, gastrointestinal toxicity or tumor regression after curative RT. Interestingly, the mean baseline and induced DNA damage was found to be lower in the group of RC patients with tumor stage IV (n = 7) as compared with the stage III (n = 35). The difference, however, did not reach statistical significance, apparently, because of the limited number of patients. CONCLUSIONS: The study shows higher expression of γ-H2AX and 53BP1 foci in rectal cancer patients compared with healthy individuals. Yet the data in vitro were not predictive in regard to the radiotherapy outcome.

Hypoxia induced CA9 inhibitory targeting by two different sulfonamide derivatives including acetazolamide in human glioblastoma.

HIF-1α regulated genes are mainly responsible for tumour resistance to radiation- and chemo-therapy. Among these genes, carbonic anhydrase isoform IX (CA9) is highly over expressed in many types of cancer especially in high grade brain cancer like Glioblastoma (GBM). Inhibition of the enzymatic activity by application of specific chemical CA9 inhibitor sulphonamides (CAI) like Acetazolamide (Aza.), the new sulfonamide derivative carbonic anhydrase inhibitor (SU.D2) or indirect inhibitors like the HIF-1α inhibitor Chetomin or molecular inhibitors like CA9-siRNA are leading to an inhibition of the functional role of CA9 during tumorigenesis. Human GBM cells were treated with in vitro hypoxia (1, 6, or 24 h at 0.1%, O2). Aza. application was at a range between 250 and 8000 nM and the HIF-1α inhibitor Chetomin at a concentration range of 150-500 nM. Cell culture plates were incubated for 24 h under hypoxia (0.1% O2). Further, CA9-siRNA constructs were transiently transfected into GBM cells exposed to extreme hypoxic aeration conditions. CA9 protein expression level was detectable in a cell-type specific manner under normoxic conditions. Whereas U87-MG exhibited a strong aerobic expression, U251 and U373 displayed moderate and GaMG very weak normoxic CA9 protein bands. Aza. as well as SU.D2 displayed inhibitory characteristics to hypoxia induced CA9 expression in the four GBM cell lines for 24 h of hypoxia (0.1% O2) at concentrations between 3500 and 8000 nM, on both the protein and mRNA level. Parallel experiments using CA9-siRNA confirmed these results. Application of 150-500 nM of the glycolysis inhibitor Chetomin under similar oxygenation conditions led to a sharply reduced expression of both CA IX protein and CA9 mRNA levels, indicating a clear glucose availability involvement for the hypoxic HIF-1α and CA9 expression in GBM cells. Hypoxia significantly influences the behaviour of human tumour cells by activation of genes involved in the adaptation to hypoxic stress. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Aza., SU.D2, Chetomin or CA9-siRNA possesses functional CA9 inhibitory characteristics when applied against human cancers with hypoxic regions like GBM. They may be used as alternative or in conjunction with other direct inhibitors possessing similar functionality, thereby rendering them as potential optimal tools for the development of an optimized therapy in human brain cancer treatment.

Small interfering RNA targeting HIF-1α reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro.

BACKGROUND: Hypoxia inducible factor-1 has been identified as a potential target to overcome hypoxia-induced radioresistance The aim of the present study was to investigate whether selective HIF-1 inhibition via small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α) affects hypoxia-induced radioresistance in HT 1080 human fibrosarcoma cells. MATERIAL AND METHODS: HIF-1α expression in HT 1080 human fibrosarcoma cells in vitro was silenced using HIF-1α siRNA sequence primers. Quantitative real-time polymerase chain reaction assay was performed to quantify the mRNA expression of HIF-1α. HIF-1α protein levels were studied by Western blotting at 20% (air) or after 12 hours at 0.1% O2 (hypoxia). Cells were assayed for clonogenic survival after irradiation with 2, 5, or 10 Gy, under normoxic or hypoxic conditions in the presence of HIF-1α-targeted or control siRNA sequences. A modified oxygen enhancement ratio (OER´) was calculated as the ratio of the doses to achieve the same survival at 0.1% O(2) as at ambient oxygen tensions. OER´ was obtained at cell survival levels of 50%, 37%, and 10%. RESULTS: HIF-1α-targeted siRNA enhanced radiation treatment efficacy under severely hypoxic conditions compared to tumor cells treated with scrambled control siRNA. OER was reduced on all survival levels after treatment with HIF-1α-targeted siRNA, suggesting that inhibition of HIF-1 activation by using HIF-1α-targeted siRNA increases radiosensitivity of hypoxic tumor cells in vitro. CONCLUSION: Inhibition of HIF-1 activation by using HIF-1α-targeted siRNA clearly acts synergistically with radiotherapy and increase radiosensitivity of hypoxic cells in vitro.

Rapid detection of the hypoxia-regulated CA-IX and NDRG1 gene expression in different glioblastoma cells in vitro.

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of tumor cell hypoxia. It regulates the expression of several genes related to oxygen homeostasis in response to hypoxic stress. Carbonic anhydrase IX (Ca-IX) has been found to be a stable marker of acute or chronic hypoxia. N-Myc down-regulated gene 1 (NDRG1) has been shown to possess more specific characteristics for clinical analysis and identification purposes. HIF-1 activates gene expression of the two genes and promotes tumor cell survival under hypoxic conditions. Herein, we modified a flow cytometry protocol to separate NDRG1- and CA-IX-negative and -positive cells in vitro to sort chronically hypoxic cells from glioblastoma tumors. The FITC-anti-CA-IX fluorescence differed between positive and negative cells by a factor of 60-160 in U373, U87-MG, U251 and GaMG, respectively. A clear effect of the O2 concentration on CA-IX expression was visible in GaMG and U251 cell lines whereas U373 showed a less differentiated pattern. NDRG1 expression was present in U373, U251 and GaMG with the lowest expression rate in GaMG. It was stable over 48 h of reoxygenation after 24 h of extreme hypoxia (0.1% O2). During reoxygenation NDRG1 was relatively stable in the four tumor cell lines with the lowest expression in GaMG. An oxygen- and time-dependent elevation of nuclear HIF-1alpha binding on HRE was displayed. FACS analysis of CA-IX and NDRG1 expression may be a new approach to determining the hypoxic state of tumor cells. However, an extensive analysis of other hypoxia-regulated genes in different tumors is required to identify additional markers for the detection of the oxygenation state in human tumors in order to tailor effective tumor-specific therapeutic strategies.

MEK-inhibitor PD184352 enhances the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922: the role of cell type and drug-irradiation schedule.

Targeting MEK protein in cancer cells usually leads to acquired resistance to MEK inhibitors and activation of the prosurvival protein Akt. Since both MEK and Akt are clients of the Hsp90 chaperone system, the present study explores the responses of irradiated lung carcinoma A549 and glioblastoma SNB19 cell lines to combined MEK and Hsp90 inhibition. Unexpectedly, the MEK inhibitor PD184352 administered 24 h prior to irradiation, enhanced cell survival through upregulation of not only MEK and Erk1/2 but also of Akt. In contrast, PD184352 added 1 h before irradiation strongly reduced the expression of Erk and did not upregulate Akt in both cell lines. As a result, the MEK inhibitor increased the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in glioblastoma SNB19 cells. Possible reasons for the enhanced cell killing under this short-term pretreatment schedule may be a down-regulation of Erk during or directly after irradiation, increased DNA damage and/or a strong G2/M arrest 24 h after irradiation. In addition, an 1-h pretreatment with PD184352 and/or NVP-AUY922 under schedule II induced neither G1 arrest nor up-regulation of p-Akt in both cell lines as it did under schedule I. Yet, a long-term treatment with the MEK inhibitor alone caused a strong cytostatical effect. We conclude that the duration of drug pretreatment before irradiation plays a key role in the targeting of MEK in tumor cells. However, due to an aberrant activation of prosurvival proteins, the therapeutic window needs to be carefully defined, or a combination of inhibitors should be considered.

Modulation of glucose metabolism inhibits hypoxic accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha).

BACKGROUND AND PURPOSE: The hypoxic accumulation of the transcription factor subunit hypoxia-inducible factor-1alpha (HIF-1alpha), a potential endogenous hypoxia marker and therapeutic target, has recently been shown to strongly depend on glucose availability. The aim of this study was to investigate the underlying mechanism of this effect. MATERIAL AND METHODS: HIF-1alpha protein levels were studied by Western blotting in HT 1080 human fibrosarcoma cells and in a hypoxia-responsive element green fluorescent protein (HRE-GFP) reporter assay in stably transfected HT 1080 cells treated with hypoxia (0.1% O(2), 12 h) and glycolysis inhibitors 2-deoxyglucose (2-DG) or iodoacetate (IAA). HIF-1alpha mRNA expression was quantified via real-time polymerase chain reaction (RT-PCR). RESULTS: Both inhibitors drastically reduced hypoxic HIF-1alpha accumulation (2-DG + hypoxia 2% mean HIF-1alpha protein level vs. 59% hypoxia alone; IAA + hypoxia 13% mean HIF-1alpha protein level vs. 96% hypoxia alone), an effect not rescued by the addition of pyruvate and confirmed in an HRE-GFP reporter assay in stably transfected HT 1080 cells. RT-PCR under identical conditions showed no effect of glycolysis inhibition on HIF-1alpha mRNA levels, suggesting a translational or posttranslational mechanism. CONCLUSION: The effect of glycolysis modulation on the HIF-1alpha levels in tumor cells may provide a novel approach to therapeutically target HIF-1alpha.

Effects of HIF-1 inhibition by chetomin on hypoxia-related transcription and radiosensitivity in HT 1080 human fibrosarcoma cells.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) overexpression has been linked to tumor progression and poor prognosis. We investigated whether targeting of HIF-1 using chetomin, a disrupter of the interaction of HIF-1 with the transcriptional coactivator p300, influences the radiosensitivity of hypoxic HT 1080 human fibrosarcoma cells. METHODS: Optimal dose of chetomin was determined by EGFP-HRE gene reporter assay in stably transfected HT 1080 cells. Cells were assayed for expression of the hypoxia-inducible genes carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) by RT-PCR and for clonogenic survival after irradiation with 2, 5 or 10 Gy, under normoxic or hypoxic (0.1% O2, 12 h) conditions in the presence or absence of chetomin (150 nM, 12 h, pre-treatment of 4 h). RESULTS: Chetomin treatment significantly reduced CA9 and VEGF mRNA expression in hypoxic cells to 44.4 +/- 7.2% and 39.6 +/- 16.0%, respectively, of untreated hypoxic controls. Chetomin clearly reduced the modified oxygen enhancement ratio (OER') compared to untreated cells, from 2.02 to 1.27, from 1.86 to 1.22 and from 1.49 to 1.06 at the 50%, 37% and 10% clonogenic survival levels, respectively. CONCLUSION: HIF-1 inhibition by chetomin effectively reduces hypoxia-dependent transcription and radiosensitizes hypoxic HT 1080 human fibrosarcoma cells in vitro.

Time- and oxygen-dependent expression and regulation of NDRG1 in human brain cancer cells.

N-myc downstream-regulated gene 1 (NDRG1) is a tumor suppressor with the potential to suppress metastasis, invasion and migration of cancer cells. It is regulated under stress conditions such as starvation or hypoxia. NDRG1 regulation is both induced and controlled by HIF-1α-dependent and -independent pathways under hypoxic conditions. However, there are profound differences in the way NDRG1 expression is regulated by HIF-1α and other transcription factors. Therefore, we aimed to define the time-dependent pattern of NDRG1 mRNA and protein expression in human glioblastoma cell lines in extreme hypoxia and after re-oxygenation as well as under normoxic conditions. Furthermore, we ascribe the regulation of NDRG1 to the transcription factors HIF-1α, SP1, CEBPα, YB-1 and Smad7 in a time-dependent manner. The human malignant glioma cell lines U87-MG, U373 and GaMG were cultured for 1, 6 and 24 h under hypoxic (0.1% O2) conditions and then they were re-oxygenated. The mRNA expression of NDRG1, HIF-1α SP1, CEBPα, YB-1 and Smad7 was measured using semi-quantitative RT-PCR analysis. Their protein expression was analyzed using western blotting. Our experiments revealed that long-term (24 h), but not short-term hypoxia led to the induction of NDRG1 expression in human glioma cell lines. NDRG1 expression was found to correlate with the protein expression of HIF-1α, SP1, CEBPα, YB-1 and Smad7. The present study suggests for the first time that SP1 regulates NDRG1 expression in glioma cells under hypoxia in a time-dependent manner along with HIF-1α, CEBPα, YB-1 and Smad7. These molecules, each separately or in combination, may possess the potential to become target molecules for antitumor therapeutic approaches particularly in human brain tumors.

Migration pattern, actin cytoskeleton organization and response to PI3K-, mTOR-, and Hsp90-inhibition of glioblastoma cells with different invasive capacities.

High invasiveness and resistance to chemo- and radiotherapy of glioblastoma multiforme (GBM) make it the most lethal brain tumor. Therefore, new treatment strategies for preventing migration and invasion of GBM cells are needed. Using two different migration assays, Western blotting, conventional and super-resolution (dSTORM) fluorescence microscopy we examine the effects of the dual PI3K/mTOR-inhibitor PI-103 alone and in combination with the Hsp90 inhibitor NVP-AUY922 and/or irradiation on the migration, expression of marker proteins, focal adhesions and F-actin cytoskeleton in two GBM cell lines (DK-MG and SNB19) markedly differing in their invasive capacity. Both lines were found to be strikingly different in morphology and migration behavior. The less invasive DK-MG cells maintained a polarized morphology and migrated in a directionally persistent manner, whereas the highly invasive SNB19 cells showed a multipolar morphology and migrated randomly. Interestingly, a single dose of 2 Gy accelerated wound closure in both cell lines without affecting their migration measured by single-cell tracking. PI-103 inhibited migration of DK-MG (p53 wt, PTEN wt) but not of SNB19 (p53 mut, PTEN mut) cells probably due to aberrant reactivation of the PI3K pathway in SNB19 cells treated with PI-103. In contrast, NVP-AUY922 exerted strong anti-migratory effects in both cell lines. Inhibition of cell migration was associated with massive morphological changes and reorganization of the actin cytoskeleton. Our results showed a cell line-specific response to PI3K/mTOR inhibition in terms of GBM cell motility. We conclude that anti-migratory agents warrant further preclinical investigation as potential therapeutics for treatment of GBM.

Plasma osteopontin levels in patients with head and neck cancer and cervix cancer are critically dependent on the choice of ELISA system.

BACKGROUND: The tumor-associated glycoprotein osteopontin (OPN) is discussed as a plasma surrogate marker of tumor hypoxia and as an indicator of the presence of pleural mesothelioma in asbestos-exposed individuals. The clinical introduction of plasma OPN measurements requires the availability of a reliable enzyme-linked immunosorbence assay (ELISA). METHODS: We compared previously described and currently available ELISA systems on 88 archival plasma samples obtained from patients with head and neck or cervix cancer between 20 days before and 171 after the start of radiotherapy. RESULTS: Median (range) plasma OPN levels were 667 (148.8-2095) ng/ml and 9.8 (3.5-189.5) ng/ml for a previously described and a newly marketed assay, respectively. Although results for different assays were significantly correlated (r = 0.38, p < 0.05, Spearman rank test), between-assay factors ranged from 2.0 to 217.9 (median 74.6) in individual patients. OPN levels in cervix cancer patients were comparable to those of head and neck cancer patients. CONCLUSION: Commercially available OPN ELISA systems produce different absolute plasma OPN levels, compromising a comparison of individual patient data with published results. However, different assays appear to have a similar capacity to rank patients according to plasma OPN level. A review of literature data suggests that plasma OPN levels measured even with identical ELISA systems can only be compared with caution.

Dual PI3K- and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule.

Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells.

Characterization of carbonic anhydrase IX (CA IX) as an endogenous marker of chronic hypoxia in live human tumor cells.

PURPOSE: Published clinical studies provide conflicting data regarding the prognostic significance of carbonic anhydrase IX (CA IX) overexpression as an endogenous marker of tumor hypoxia and its comparability with other methods of hypoxia detection. We performed a systematic analysis of CA IX protein levels under various in vitro conditions of tumor hypoxia in HT 1080 human fibrosarcoma and FaDu human pharyngeal carcinoma cells. Because sorting of live CA IX positive cells from tumors provides a tool to study the radiosensitivity of chronically hypoxic cells, we modified and tested a CA IX flow cytometry protocol on mixed hypoxic/aerobic suspensions of HT 1080 and FaDu cells. METHODS AND MATERIALS: HT 1080 and FaDu cells were treated with up to 24 h of in vitro hypoxia and up to 96 h of reoxygenation. To test the effect of nonhypoxic stimuli, glucose and serum availability, pH and cell density were modified. CA IX protein was quantified in Western blots of whole-cell lysates. Mixed suspensions with known percentages of hypoxic cells were prepared for CA IX flow cytometry. The same mixtures were assayed for clonogenic survival after 10 Gy. RESULTS: Hypoxia-induced CA IX protein expression was seen after >6 h at < or =5% O(2), and protein was stable over 96 h of reoxygenation in both cell lines. Glucose deprivation abolished the hypoxic CA IX response, and high cell density caused CA IX induction under aerobic conditions. Measured percentages of CA IX-positive cells in mixtures closely reflected known percentages of hypoxic cells in HT 1080 and were associated with radioresistance of mixtures after 10 Gy. CONCLUSION: CA IX is a stable marker of current or previous chronic hypoxia but influenced by nonhypoxic stimuli. Except the time course of accumulation, all properties of this marker resembled our previous findings for hypoxia-inducible factor-1alpha. A modified flow cytometry protocol provided good separability of CA IX-negative and -positive cells in vitro and can be applied to sort chronically hypoxic cells from tumors.

Glucose requirement for hypoxic accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha).

Hypoxia-inducible factor-1alpha (HIF-1alpha) is both a potential endogenous marker of tumor hypoxia and therapeutic target, and elevated HIF-1alpha protein levels have been shown to be associated with increased hypoxic radiation resistance in FaDu human pharyngeal carcinoma cells in vitro. Here, we found that in FaDu xenografts, no significant HIF-1alpha protein accumulation was detectable by either flow cytometry or Western blot, despite the presence of hypoxic (pimonidazole-positive, radiation resistant) cells. To investigate the effect of different tumor microenvironment conditions on hypoxic HIF-1alpha accumulation, we performed in vitro hypoxia experiments (0.1% O2, 24 h) with manipulation of pH (7.4 vs. 6.7), glucose (0-5.5 mM) and serum (0 or 10%) availability in FaDu and HT 1080 human fibrosarcoma cells. Hypoxic induction of HIF-1alpha protein was strongly dependent on glucose availability and largely abolished at 0.55 mM glucose or less in both cell lines. This glucose effect was confirmed in a hypoxia-responsive-element (HRE)/enhanced-green-fluorescent-protein (EGFP) reporter assay in transfected HT 1080 cells and possibly explains a lack of HIF-1alpha protein in hypoxic tumor cells.

Response of the plasma hypoxia marker osteopontin to in vitro hypoxia in human tumor cells.

BACKGROUND: Osteopontin (OPN) is recognized as a tumor-associated protein and has recently been shown to be a potential plasma marker of tumor hypoxia in head and neck cancer patients. We sought to detect patterns of OPN accumulation and secretion in human tumor cells during in vitro hypoxia. MATERIALS AND METHODS: The human tumor cell lines A 549 lung carcinoma, U 87 malignant glioma, HT 1080 fibrosarcoma, FaDu pharyngeal carcinoma and HT 29 and HCT 116 colorectal carcinoma were treated with 1, 6 or 24 h of hypoxia (0.1% O2) or 24 h followed by 4 h or 20 of reoxygenation. OPN concentration in the supernatant was measured by ELISA, OPN protein and mRNA levels by Western and Northern blotting. RESULTS: In FaDu, HT 29 and HCT 116, OPN levels in the supernatant remained below 10 ng/ml under all conditions. In A 549, HT 1080 and U 87, mean aerobic OPN concentrations were 2296, 164 and 115 ng/ml, respectively. No increase of OPN in the medium during 24h of hypoxia, but moderate increases during subsequent 24-hour-reoxygenation were observed in these three cell lines. Intracellular OPN protein was present to a similar extent in all six-cell lines under aerobic conditions and also did not accumulate during hypoxia treatment. OPN mRNA response to hypoxia and reoxygenation was very heterogeneous between cell lines. CONCLUSION: Reoxygenation rather than hypoxia appears to induce OPN secretion from human tumor cells in a cell-type specific manner.

Cell type-specific association of hypoxia-inducible factor-1 alpha (HIF-1 alpha) protein accumulation and radiobiologic tumor hypoxia.

PURPOSE: The transcription factor subunit hypoxia-inducible factor-1 alpha (HIF-1 alpha) is currently discussed as a potential endogenous marker of tumor hypoxia to select patients for modified treatment. Despite an association of immunohistochemical HIF-1 alpha overexpression and poor prognosis after radiotherapy in many tumor entities, the reported pattern of HIF-1 alpha staining was often not consistent with tumor hypoxia. To explain this discrepancy, we studied the in vitro conditions under which HIF-1 alpha protein accumulates. METHODS AND MATERIALS: FaDu human pharyngeal carcinoma and HT 1080 human fibrosarcoma cells were treated with different schedules of in vitro hypoxia at 5%, 1%, and 0.1% O(2) and reoxygenation. HIF-1 alpha protein levels were determined in nuclear extracts. Cellular radiation sensitivity was assessed by clonogenic survival assay after single-dose irradiation at the above oxygen concentrations. RESULTS: In both cell lines, weak HIF-1 alpha expression was observed at 20% O(2) and after 10 min of hypoxia. Increased HIF-1 alpha protein levels were observed at 1 h of hypoxia, remained stable over 24 h, and decreased to baseline within 15 min of reoxygenation. HIF-1 alpha protein at 5% O(2) was half-maximal in FaDu but indistinguishable from 0.1% O(2) in HT 1080. A good correlation of HIF-1 alpha protein level and hypoxic radiation resistance, with equal ranking of data points by both assays, was observed in FaDu cells but not in HT 1080 cells. CONCLUSIONS: The ability of HIF-1 alpha to indicate radiobiologically relevant levels of tumor hypoxia seems to be cell type specific. This finding may explain the inconsistent results regarding the pattern of HIF-1 alpha expression in tumor sections.

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

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 C m = 1.9 µF/cm(2). In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19) showed the highest C m values of 3.7-4.0 µF/cm(2), 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.

Radiosensitivity in breast cancer assessed by the histone γ-H2AX and 53BP1 foci.

BACKGROUND: High expression of constitutive histone γ-H2AX, a sensitive marker of DNA damage, might be indicative of defective DNA repair pathway or genomic instability. 53BP1 (p53-binding protein 1) is a conserved checkpoint protein with properties of a DNA double-strand breaks sensor. This study explores the relationship between the clinical radiosensitivity of tumor patients and the expression/induction of γ-H2AX and 53BP1 in vitro. METHODS: Using immunostaining, we assessed spontaneous and radiation-induced foci of γ-H2AX and 53 BP1 in peripheral blood mononuclear cells derived from unselected breast cancer (BC) patients (n=57) undergoing radiotherapy (RT). Cells from apparently healthy donors (n=12) served as references. RESULTS: Non-irradiated cells from controls and unselected BC patients exhibited similar baseline levels of DNA damage assessed by γ-H2AX and 53BP1 foci. At the same time, the γ-H2AX assay of in vitro irradiated cells revealed significant differences between the control group and the group of unselected BC patients with respect to the initial (0.5 Gy, 30 min) and residual (2 Gy, 24 h post-radiation) DNA damage. The numbers of 53BP1 foci analyzed in 35 BC patients were significantly higher than in controls only in case of residual DNA damage. A weak correlation was found between residual foci of both proteins tested. In addition, cells from cancer patients with an adverse acute skin reaction (grade 3) to RT showed significantly increased radiation-induced γ-H2AX foci and their protracted disappearance compared to the group of BC patients with normal skin reaction (grade 0-1). The mean number of γ-H2AX foci after 5 clinical fractions was significantly higher than that before RT, especially in clinically radiosensitive patients. CONCLUSIONS: The γ-H2AX assay may have potential for screening individual radiosensitivity of breast cancer patients. TRIAL REGISTRATION: http://www.krebshilfe.de/wir-foerdern.html.