Impact of dimethyl sulfoxide on irradiation-related DNA double-strand-break induction, -repair and cell survival.

Dimethyl sulfoxide (DMSO) is an effective radical scavenger and, when added to cells, reduces the initial number of radiation-induced DNA double-strand breaks (DSB). The aim of this study was to investigate modification by DMSO of both DSB induction and DSB repair by means of pulsed-field gel electrophoresis (PFGE) as well as gamma-H2AX immunofluorescence staining. WiDr cells (human colon carcinoma provided by DKFZ) were incubated with 2% DMSO for 2 h (or mock-treated) prior to irradiation with varying X-ray doses and subsequent incubation for repair. Sample processing for PFGE analysis or counting of γ-H2AX foci was performed according to standard protocols. Effects on apoptosis induction and cell survival were investigated additionally by standard protocols. DMSO reduced DSB yield after 20-80 Gy measured by PFGE. A qualitatively similar result was found after low-dose irradiation (1 Gy) using γ-H2AX immunofluorescence staining. During incubation for repair, both DNA fragment rejoining (PFGE) as well as γ-H2AX foci removal occurred at a reduced rate when cells had been pre-treated with DMSO. But this effect was clearly more pronounced for the PFGE-analyzed double-strand breakage, particularly at early repair times. WiDr cells treated with DMSO (2%) showed a significantly increased clonogenic survival after irradiation doses above 8 Gy. Apoptosis rates were not changed by DMSO. The radio-protective effect of DMSO, well known from other PFGE studies, could be confirmed for the formation of γ-H2AX foci. DSB generated in the presence of DMSO were less rapidly repaired. DMSO showed radio-protective effects on clonogenic survival but not on apoptosis.

Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.

Carbon ion radiation is a promising new form of radiotherapy for cancer, but the central question about the biologic effects of charged particle radiation is yet incompletely understood. Key to this question is the understanding of the interaction of ions with DNA in the cell's nucleus. Induction and repair of DNA lesions including double-strand breaks (DSBs) are decisive for the cell. Several DSB repair markers have been used to investigate these processes microscopically, but the limited resolution of conventional microscopy is insufficient to provide structural insights. We have applied superresolution microscopy to overcome these limitations and analyze the fine structure of DSB repair foci. We found that the conventionally detected foci of the widely used DSB marker γH2AX (Ø 700-1000 nm) were composed of elongated subfoci with a size of ∼100 nm consisting of even smaller subfocus elements (Ø 40-60 nm). The structural organization of the subfoci suggests that they could represent the local chromatin structure of elementary DSB repair units at the DSB damage sites. Subfocus clusters may indicate induction of densely spaced DSBs, which are thought to be associated with the high biologic effectiveness of carbon ions. Superresolution microscopy might emerge as a powerful tool to improve our knowledge of interactions of ionizing radiation with cells.-Lopez Perez, R., Best, G., Nicolay, N. H., Greubel, C., Rossberger, S., Reindl, J., Dollinger, G., Weber, K.-J., Cremer, C., Huber, P. E. Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.

Phytotherapeutics Oridonin and Ponicidin show Additive Effects Combined with Irradiation in Pancreatic Cancer in Vitro.

Background: Chemoradiation of locally advanced non-metastatic pancreatic cancer can lead to secondary operability by tumor mass reduction. Here, we analyzed radiomodulating effects of oridonin and ponicidin in pancreatic cancer in vitro. Both agents are ent-kaurane diterpenoids, extracted from Isodon rubescens, a plant that is well known in Traditional Chinese Medicine. Cytotoxic effects have recently been shown in different tumor entities for both agents. Materials and methods: Pancreatic cancer cell lines AsPC-1, BxPC-3, Panc-1 and MIA PaCa-2 were pretreated with oridonin or ponicidin and irradiated with 2 Gy to 6 Gy. Long-term survival was determined by clonogenic assay. Cell cycle effects and intensity of γH2AX as indicator for DNA double-strand breaks were investigated by flow cytometry. Western blotting was used to study the DNA double-strand break repair proteins Ku70, Ku80 and XRCC4. Results: Oridonin and ponicidin lead to a dose-dependent reduction of clonogenic survival and an increase in γH2AX. Combined with irradiation we observed additive effects and a prolonged G2/M-arrest. No relevant changes in the levels of the DNA double-strand break repair proteins were detected. Conclusions: Pretreatment with oridonin or ponicidin followed by irradiation lead to an additional reduction in survival of pancreatic cancer cells in vitro, presumably explained by an induced prolonged G2/M-arrest. Both agents seem to induce DNA double-strand breaks but do not interact with the non-homologous end joining (NHEJ) pathway.

Metformin Enhanced in Vitro Radiosensitivity Associates with G2/M Cell Cycle Arrest and Elevated Adenosine-5'-monophosphate-activated Protein Kinase Levels in Glioblastoma.

BACKGROUND: It is hypothesized that metabolism plays a strong role in cancer cell regulation. We have recently demonstrated improved progression-free survival in patients with glioblastoma who received metformin as an antidiabetic substance during chemoradiation. Although metformin is well-established in clinical use the influence of metformin in glioblastoma is far from being understood especially in combination with other treatment modalities such as radiation and temozolomide. MATERIALS AND METHODS: In this study, we examined the influence of metformin in combinations with radiation and temozolomide on cell survival (clonogenic survival), cell cycle (routine flow cytometric analysis, FACScan), and phosphorylated Adenosine-5'-monophosphate-activated protein kinase (AMPK) (Phopho-AMPKalpha1 - ELISA) levels in glioblastoma cell lines LN18 and LN229. RESULTS: Metformin and temozolomide enhanced the effectiveness of photon irradiation in glioblastoma cells. Cell toxicity was more pronounced in O6-methylguanine DNA methyltransferase (MGMT) promoter non-methylated LN18 cells. Induction of a G2/M phase cell cycle block through metformin and combined treatments was observed up to 72 h. These findings were associated with elevated levels of activated AMPK levels in LN229 cells but not in LN18 cells after irradiation, metformin, and temozolomide treatment. CONCLUSIONS: Radiosensitizing effects of metformin on glioblastoma cells treated with irradiation and temozolomide in vitro coincided with G2/M arrest and changes in pAMPK levels.

Kallikrein-related peptidase 6 regulates epithelial-to-mesenchymal transition and serves as prognostic biomarker for head and neck squamous cell carcinoma patients.

BACKGROUND: Dysregulated expression of Kallikrein-related peptidase 6 (KLK6) is a common feature for many human malignancies and numerous studies evaluated KLK6 as a promising biomarker for early diagnosis or unfavorable prognosis. However, the expression of KLK6 in carcinomas derived from mucosal epithelia, including head and neck squamous cell carcinoma (HNSCC), and its mode of action has not been addressed so far. METHODS: Stable clones of human mucosal tumor cell lines were generated with shRNA-mediated silencing or ectopic overexpression to characterize the impact of KLK6 on tumor relevant processes in vitro. Tissue microarrays with primary HNSCC samples from a retrospective patient cohort (n = 162) were stained by immunohistochemistry and the correlation between KLK6 staining and survival was addressed by univariate Kaplan-Meier and multivariate Cox proportional hazard model analysis. RESULTS: KLK6 expression was detected in head and neck tumor cell lines (FaDu, Cal27 and SCC25), but not in HeLa cervix carcinoma cells. Silencing in FaDu cells and ectopic expression in HeLa cells unraveled an inhibitory function of KLK6 on tumor cell proliferation and mobility. FaDu clones with silenced KLK6 expression displayed molecular features resembling epithelial-to-mesenchymal transition, nuclear ß-catenin accumulation and higher resistance against irradiation. Low KLK6 protein expression in primary tumors from oropharyngeal and laryngeal SCC patients was significantly correlated with poor progression-free (p = 0.001) and overall survival (p < 0.0005), and served as an independent risk factor for unfavorable clinical outcome. CONCLUSIONS: In summary, detection of low KLK6 expression in primary tumors represents a promising tool to stratify HNSCC patients with high risk for treatment failure. These patients might benefit from restoration of KLK6 expression or pharmacological targeting of signaling pathways implicated in EMT.

Impaired aldehyde dehydrogenase 1 subfamily member 2A-dependent retinoic acid signaling is related with a mesenchymal-like phenotype and an unfavorable prognosis of head and neck squamous cell carcinoma.

BACKGROUND: An inverse correlation between expression of the aldehyde dehydrogenase 1 subfamily A2 (ALDH1A2) and gene promoter methylation has been identified as a common feature of oropharyngeal squamous cell carcinoma (OPSCC). Moreover, low ALDH1A2 expression was associated with an unfavorable prognosis of OPSCC patients, however the causal link between reduced ALDH1A2 function and treatment failure has not been addressed so far. METHODS: Serial sections from tissue microarrays of patients with primary OPSCC (n = 101) were stained by immunohistochemistry for key regulators of retinoic acid (RA) signaling, including ALDH1A2. Survival with respect to these regulators was investigated by univariate Kaplan-Meier analysis and multivariate Cox regression proportional hazard models. The impact of ALDH1A2-RAR signaling on tumor-relevant processes was addressed in established tumor cell lines and in an orthotopic mouse xenograft model. RESULTS: Immunohistochemical analysis showed an improved prognosis of ALDH1A2(high) OPSCC only in the presence of CRABP2, an intracellular RA transporter. Moreover, an ALDH1A2(high)CRABP2(high) staining pattern served as an independent predictor for progression-free (HR: 0.395, p = 0.007) and overall survival (HR: 0.303, p = 0.002), suggesting a critical impact of RA metabolism and signaling on clinical outcome. Functionally, ALDH1A2 expression and activity in tumor cell lines were related to RA levels. While administration of retinoids inhibited clonogenic growth and proliferation, the pharmacological inhibition of ALDH1A2-RAR signaling resulted in loss of cell-cell adhesion and a mesenchymal-like phenotype. Xenograft tumors derived from FaDu cells with stable silencing of ALDH1A2 and primary tumors from OPSCC patients with low ALDH1A2 expression exhibited a mesenchymal-like phenotype characterized by vimentin expression. CONCLUSIONS: This study has unraveled a critical role of ALDH1A2-RAR signaling in the pathogenesis of head and neck cancer and our data implicate that patients with ALDH1A2(low) tumors might benefit from adjuvant treatment with retinoids.

Phosphorylation of AKT(Ser473) serves as an independent prognostic marker for radiosensitivity in advanced head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by high resistance to radiotherapy, which critically depends on both altered signaling pathways within tumor cells and their dynamic interaction with the tumor microenvironment. This study evaluated the prognostic value of the phosphorylation status of AKT on Ser473 and Thr308 for the clinical outcome of patients with advanced HNSCC on radiotherapy. Furthermore, we investigated the impact of AKT(Ser473) phosphorylation [p-AKT(Ser473)] in the context of radioresistance using ex vivo tissue cultures that resemble the complex tissue architecture and paracrine interaction with the tumor microenvironment. In a cohort of 120 patients with advanced HNSCC, who were treated with primary or adjuvant radiotherapy, a significant association was found between relative p-AKT(Ser473) levels and overall survival (p = 0.006) as well as progression-free survival (p = 0.021), while no significant correlation was revealed for relative p-AKT(Thr308) levels. In ex vivo tissue cultures p-AKT(Ser473) levels were increased upon irradiation and treatment with the PI3K inhibitor LY294002 inhibited both basal and irradiation induced AKT(Ser473) phosphorylation. Strikingly, pretreatment with LY294002 sensitized tissue cultures derived from primary and recurrent tumors to radiotherapy as determined by impaired tumor cell proliferation and enhanced DNA damage. In conclusion, phosphorylation status of AKT(Ser473) in tumor specimens serves as a novel biomarker to identify patients with advanced HNSCC at high risk for treatment failure following radiotherapy, and our data from ex vivo tissue cultures support the assumption that pharmacological inhibition of AKT(Ser473) phosphorylation might circumvent radioresistance to improve efficiency and reduce toxicity of current treatment modalities.

Integrin-based meningioma cell migration is promoted by photon but not by carbon-ion irradiation.

PURPOSE: Sublethal doses of photon irradiation (IR) are suspected to increase tumor cell migration and support locoregional recurrence of disease, which has already been shown in other cell lines. This manuscript describes the effect of photon and carbon-ion IR on WHO class I meningioma cell migration and provides an approach to the underlying cellular mechanisms. MATERIALS AND METHODS: Meningioma cells were gained operatively at the university hospital in Homburg/Saar, Germany. For migration, membranes (8-µm pore sizes) were coated with collagen I, with collagen IV, and with fibronectin. Cells were analyzed in migration experiments with or without serum stimulation, with or without photon and carbon IR 24 h prior to experiments, and with or without integrin antibodies. Fluorescence-activated cell sorting (FACS) analyses of the integrins ανß1, ανß3, and ανß5 were performed without IR and 6, 12 and 24 h after IR. Enzyme-linked immunosorbent assay (ELISA) analyses of matrix metalloproteinases (MMP)-2 and MMP-9 were realized with and without IR after cells were cultured on collagen I, collagen IV, or fibronectin for 24 h. Cells and supernatants for FACS and ELISA were stored at - 18 °C. The significance level was set at 5 % using both Student's t test and two-way ANOVA. RESULTS: Migration of meningioma cells was serum-inducible (p < 0.001). It could be increased by photon IR (p < 0.02). The integrins ανß1 and ανß5 showed a 21 and 11 % higher expression after serum stimulation (not significant), respectively, and ανß1 expression was raised by 14 % (p = 0.0057) after photon IR. Antibody blockage of the integrins ανß1 and ανß5 inhibited serum- and photon-induced migration. Expression of MMP-2 and MMP-9 remained unchanged after both IR and fetal bovine serum (FBS). Carbon-ion IR left both integrin expression and meningioma cell migration unaffected. CONCLUSION: Photon but not carbon-ion IR promotes serum-based meningioma cell migration. Fibronectin receptor integrin ανß1 signaling can be identified as an important mechanism for serum- and photon-induced migration of WHO class I meningioma cells.

Pathogen-triggered activation of plasmacytoid dendritic cells induces IL-10-producing B cells in response to Staphylococcus aureus.

Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell-independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the V(H)3(+) BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A-bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scid(Prkdc)/γc(-/-) mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell-derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell-mediated immune tolerance.

Hippocampal serotonin-1A receptor function in a mouse model of anxiety induced by long-term voluntary wheel running.

We have recently demonstrated that, in C57/Bl6 mice, long-term voluntary wheel running is anxiogenic, and focal hippocampal irradiation prevents the increase in anxiety-like behaviors and neurobiological changes in the hippocampus induced by wheel running. Evidence supports a role of hippocampal 5-HT1A receptors in anxiety. Therefore, we investigated hippocampal binding and function of 5-HT1A receptors in this mouse model of anxiety. Four weeks of voluntary wheel running resulted in hippocampal subregion-specific changes in 5-HT1A receptor binding sites and function, as measured by autoradiography of [(3) H] 8-hydroxy-2-(di-n-propylamino)tetralin binding and agonist-stimulated binding of [(35) S]GTPγS to G proteins, respectively. In the dorsal CA1 region, 5-HT1A receptor binding and function were not altered by wheel running or irradiation. In the dorsal dentate gyrus and CA2/3 region, 5-HT1A receptor function was decreased by not only running but also irradiation. In the ventral pyramidal layer, wheel running resulted in a decrease of 5-HT1A receptor function, which was prevented by irradiation. Neither irradiation nor wheel running affected 5-HT1A receptors in medial prefrontal cortex or in the dorsal or median raphe nuclei. Our data indicate that downregulation of 5-HT1A receptor function in ventral pyramidal layer may play a role in anxiety-like behavior induced by wheel running.

In Vivo Evaluation of Combined CK2 Inhibition and Irradiation in Human WiDr Tumours.

BACKGROUND/AIM: Casein kinase 2 (CK2) which sustains multiple pro-survival functions in cellular DNA-damage response, is strictly regulated in normal cells but elevated in cancer. CK2 is considered as a potential therapeutic target, and its inhibition has been associated with radiosensitization in mammalian cells in vitro. Here, we investigated potential radiosensitization by CK2 inhibition in vivo. MATERIALS AND METHODS: The effect of CK2 inhibition in vivo was investigated in human WiDr-xenograft tumours grown subcutaneously on BALB/c nu/nu mice with and without fractionated irradiation. CK2 inhibition was performed using the specific inhibitor tetra-bromobenzotriazole (TBB). Histological examinations included staining for apoptosis and double-strand breaks. RESULTS: Both TBB treatment alone and radiation alone significantly reduced tumour growth, which was reflected by increased apoptosis rates. However, TBB treatment did not boost radiation-induced tumour growth suppression in combined treatment, although the apoptosis rate increased and repair of double-strand breaks was reduced. This was in stark contrast to previous data on in vitro radiosensitization. CONCLUSION: The absence of radiosensitization by CK2 inhibition should be investigated in different tumour models.

Device for the local radiation of tumor-bearing laboratory rodents.

Since no standard method for reproducibly irradiating local tumors of laboratory rodents exists, the authors designed a device to accurately irradiate rodent tumors. Laboratory personnel can easily assemble this inexpensive device. The new device delivers highly focused treatment and avoids the need for anesthesia and sedation. It represents an improvement over former improvised procedures done at the authors' institutions, because it allows for more accurate irradiation of rodent tumors. The authors used the device to irradiate single tumors on the left hind legs of 60 mice and 10 rats with a dosage of 7 gray (Gy) ionizing radiation. At time of irradiation, the clinical condition of the animals was very poor, but all animals survived the treatment.

Radiobiological evaluation and correlation with the local effect model (LEM) of carbon ion radiation therapy and temozolomide in glioblastoma cell lines.

PURPOSE: To investigate the cytotoxic effect of high linear-energy transfer (LET) carbon irradiation on glioblastoma cells lines in combination with temozolomide (TMZ). METHODS AND MATERIALS: The cell lines U87-MG expressing wild-type p53 and LN229 expressing both mutant and wild-type p53 were irradiated with monoenergetic carbon ion beams (LET 172 keV/microm) or an extended Bragg peak (LET 103 keV/microm) after treatment with 10 microM or 20 microM TMZ. Cytotoxicity was measured by a clonogenic survival assay, and cell growth as well as cell cycle progression, were examined. RESULTS: The p53 mutant was more sensitive to X-ray irradiation than the p53 wild type cell line, which was also expressed in a shorter G2 block. High LET carbon ions show an increased biological effectiveness in both cell lines, which is consistent with the predictive calculations by the Local Effect Model (LEM) introduced by Scholz et al. The cell line LN229 was more sensitive to TMZ treatment than the U87MG cell line expressing wild-type p53 only. The combination of TMZ and irradiation showed an additive effect in both cell lines. CONCLUSION: High LET carbon ion irradiation is significantly more effective for glioblastoma cell lines compared to photon irradiation. An additional treatment with TMZ may offer a great chance especially for several tumor types.

DNA damage response of clinical carbon ion versus photon radiation in human glioblastoma cells.

BACKGROUND AND PURPOSE: Carbon ion radiotherapy is a promising therapeutic option for glioblastoma patients due to its high physical dose conformity and greater biological effectiveness than photons. However, the biological effects of carbon ion radiation are still incompletely understood. Here, we systematically compared the biological effects of clinically used carbon ion radiation to photon radiation with emphasis on DNA repair. MATERIALS AND METHODS: Two human glioblastoma cell lines (U87 and LN229) were irradiated with carbon ions or photons and DNA damage response was systematically analyzed, including clonogenic survival, induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and apoptosis or autophagy. γH2AX foci were analyzed by flow cytometry, conventional light microscopy and 3D superresolution microscopy. RESULTS: DSBs were repaired delayed and with slower kinetics after carbon ions versus photons. Carbon ions caused stronger and longer-lasting cell cycle delays, predominantly in G2 phase, and a higher rate of apoptosis. Compared to photons, the effectiveness of carbon ions was less cell cycle-dependent. Homologous recombination (HR) appeared to be more important for DSB repair after carbon ions versus photons in phosphatase and tensin homolog (PTEN)-deficient U87 cells, as opposed to PTEN-proficient LN229 cells. CONCLUSION: Carbon ions induced more severe DSB damage than photons, which was repaired less efficiently in both cell lines. Thus, carbon ion radiotherapy may help to overcome resistance mechanisms of glioblastoma associated with DNA repair for example in combination with repair pathway-specific drugs in the context of personalized radiotherapy.

S-phase cell-specific modification by gemcitabine of PFGE-analyzed radiation-induced DNA fragmentation and rejoining.

PURPOSE: To assess the cell cycle-dependent influence of gemcitabine on ionizing radiation-induced DNA double-strand breakage (DSB) and rejoining measured by pulsed-field gel electrophoresis (PFGE). MATERIALS AND METHODS: WIDR cells (human colon carcinoma) were synchronized by serum starvation/stimulation providing populations with 7% (G1) or 50% S-phase cells, respectively. Following drug treatment (0.5 microg/ml for 2 hours) cells were irradiated (up to 90 Gy) or incubated for repair (up to 6 h after 40 Gy). Cell cycle changes were monitored by flow cytometry, DNA fragmentation was assessed by PFGE as fraction of electrophoretically mobile DNA. RESULTS: Without drug treatment, irradiated S-phase cells exhibited lower PFGE signals than the G1 cells due to the well known electrophoretic immobility of replicative DNA fragments, but DSB rejoining was not different. Gemcitabine pretreatment increased the apparent initial radiation-induced DNA fragmentation specifically for S-phase cells. This effect was rapidly reversed (1 h) during incubation for repair. CONCLUSIONS: The data indicate that gemcitabine causes the formation of additional radiation-induced DSB in S-phase cells or destabilizes the replicative structures that otherwise prevent DNA fragment migration during PFGE. The latter would be rapidly restituted superimposing DSB rejoining. This is discussed in relation to the recently proposed role of mismatch repair in gemcitabine radiosensitization.

Human mesenchymal stem cells lose their functional properties after paclitaxel treatment.

Mesenchymal stem cells (MSCs) are an integral part of the bone marrow niche and aid in the protection, regeneration and proliferation of hematopoietic stem cells after exposure to myelotoxic taxane anti-cancer agents, but the influence of taxane compounds on MSCs themselves remains incompletely understood. Here, we show that bone marrow-derived MSCs are highly sensitive even to low concentrations of the prototypical taxane compound paclitaxel. While MSCs remained metabolically viable, they were strongly impaired regarding both their proliferation and their functional capabilities after exposure to paclitaxel. Paclitaxel treatment resulted in reduced cell migration, delays in cellular adhesion and significant dose-dependent inhibition of the stem cells' characteristic multi-lineage differentiation potential. Cellular morphology and expression of the defining surface markers remained largely unaltered. Paclitaxel only marginally increased apoptosis in MSCs, but strongly induced premature senescence in these stem cells, thereby explaining the preservation of the metabolic activity of functionally inactivated MSCs. The reported sensitivity of MSC function to paclitaxel treatment may help to explain the severe bone marrow toxicities commonly caused by taxane-based anti-cancer treatments.

Activation of telomerase by ionizing radiation: differential response to the inhibition of DNA double-strand break repair by abrogation of poly (ADP-ribosyl)ation, by LY294002, or by Wortmannin.

PURPOSE: Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. METHODS AND MATERIALS: COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10 Gy. Phosphorylation status of PKB/Akt and of PKCalpha/beta(II) was assessed by western blotting. RESULTS: No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. CONCLUSIONS: Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than alpha/beta(II) cannot be ruled out.

In vitro responsiveness of glioma cell lines to multimodality treatment with radiotherapy, temozolomide, and epidermal growth factor receptor inhibition with cetuximab.

BACKGROUND: The majority of glioblastoma multiforme (GBM) cells express the epidermal growth factor receptor (EGFR). The present study evaluates the combination of temozolomide (TMZ), EGFR inhibition, and radiotherapy (RT) in GBM cell lines. METHODS AND MATERIALS: Human GBM cell lines U87, LN229, LN18, NCH 82, and NCH 89 were treated with various combinations of TMZ, RT, and the monoclonal EGFR antibody cetuximab. Responsiveness of glioma cells to the combination treatment was measured by clonogenic survival. RESULTS: Overall, double and triple combinations of RT, TMZ, and cetuximab lead to additive cytotoxic effects (independent toxicity). A notable exception was observed for U87 and LN 18 cell lines, where the combination of TMZ and cetuximab showed substantial antagonism. Interestingly, in these two cell lines, the combination of RT with cetuximab resulted in a substantial increase in cell killing over that expected for independent toxicity. The triple combination with RT, cetuximab, and TMZ was nearly able to overcome the antagonism for the TMZ/cetuximab combination in U87, however only marginally in LN18, GBM cell lines. CONCLUSION: It appears that EGFR expression is not correlated with cytotoxic effects exerted by cetuximab. Combination treatment with TMZ, cetuximab and radiation resulted in independent toxicity in three out of five cell lines evaluated, the antagonistic effect of the TMZ/cetuximab combination in two cell lines could indicate that TMZ preferentially kills cetuximab-resistant cells, suggesting for some cross-talk between toxicity mechanisms. Expression of EGFR was no surrogate marker for responsiveness to cetuximab, alone or in combination with RT and TMZ.

Telomerase activity in peripheral blood mononuclear cells after whole body irradiation.

Telomerase activity in vitro represents a radiation-inducible function. To test this effect after irradiation in vivo, we measured telomerase activity in peripheral blood mononuclear cells (PBMC) of 25 patients with leukemia or lymphoma before and 1 h after whole body irradiation. Telomerase activity of the patients was compared to telomerase activity in PBMC of 15 healthy volunteers. Peripheral blood of the patients was taken before and 1 h after the first fraction of whole body irradiation (2 Gy). Blood samples of the volunteers were irradiated ex vivo with 2 Gy. After Ficoll-Paque density gradient centrifugation telomerase activity of 10(4) PBMC per sample was measured using the telomerase PCR ELISA method. No age-dependence of telomerase activity was detected for both the volunteer and the patient group. Telomerase activity in patients was not statistically significantly increased compared to healthy individuals, and this parameter was also no prognostic factor for patient survival. After whole body irradiation an induction of telomerase activity was observed for only 7 patients (28%), or in PBMC of 2 volunteers (13%), respectively. In patients with radiation-inducible telomerase activity, a slightly better survival was indicated, but this difference did not reach statistical significance. The feasibility to assess in vivo radiation-induction of telomerase activity in PBMC of leukemia or lymphoma patients was demonstrated. An unexpectedly low number of whole body irradiated patients displayed this phenotype, and the treatment impact of telomerase upregulation in PBMC upon radiation exposure needs to be further analyzed.

Dedicated high dose rate 192Ir brachytherapy radiation fields for in vitro cell exposures at variable source-target cell distances: killing of mammalian cells depends on temporal dose rate fluctuation.

Afterloading brachytherapy is conducted by the stepwise movement of a radioactive source through surgically implanted applicator tubes where at predefined dwell positions calculated dwell times optimize spatial dose delivery with respect to a planned dose level. The temporal exposure pattern exhibits drastic fluctuations in dose rate at a given coordinate and within a single treatment session because of the discontinuous and repeated source movement into the target volume. This could potentially affect biological response. Therefore, mammalian cells were exposed as monolayers to a high dose rate 192Ir source by utilizing a dedicated irradiation device where the distance between a planar array of radioactive source positions and the plane of the cell monolayer could be varied from 2.5 mm to 40 mm, thus varying dose rate pattern for any chosen total dose. The Gammamed IIi afterloading system equipped with a nominal 370 GBq (10 Ci) 192-Ir source was used to irradiate V79 Chinese hamster lung fibroblasts from both confluent and from exponential growth phase with dose up to 12 Gy (at room temperature, total exposure not exceeding 1 h). For comparison, V79 cells were also exposed to 6 MV x-rays from a clinical linear accelerator (dose rate of 2.5 Gy min-1). As biological endpoint, cell survival was determined by standard colony forming assay. Dose measurements were conducted with a diamond detector (sensitive area 7.3 mm2), calibrated by means of 60Co radiation. Additionally, dose delivery was simulated by Monte Carlo calculations using the EGSnrc code system. The calculated secondary electron fluence spectra at the cell location did not indicate a significant change of radiation quality (i.e. higher linear energy transfer) at the lower distances. Clonogenic cell survival curves obtained after brachytherapy exhibited an altered biological response compared to x-rays which was characterized by a significant reduction of the survival curve shoulder when dose rate fluctuations were high. Therefore, also for the time scale of the present investigation, cellular effects of radiation are not invariant to the temporal pattern in dose rate. We propose that with high dose rate variation the cells activate less efficiently their DNA damage response than after continuous irradiation.