Klaus J. Weber

Combination of Vascular Endothelial Growth Factor Receptor/Platelet-Derived Growth Factor Receptor Inhibition Markedly Improves Radiation Tumor Therapy

Purpose: Investigations on the combination of radiotherapy with vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) antiangiogenic agents, which has the potential to improve the clinical outcome in cancer patients. Experimental Design: Here, we analyze the combined VEGF (SU5416) and PDGF (SU6668) receptor tyrosine kinase inhibition with irradiation in human endothelium (HUVEC), prostate cancer (PC3), and glioblastoma (U87) in vitro and in vivo. Results: Combined inhibition of VEGF and PDGF signaling resulted in enhanced apoptosis, reduced cell proliferation, and clonogenic survival as well as reduced endothelial cell migration and tube formation compared with single pathway inhibition. These effects were further enhanced by additional irradiation. Likewise, in PC3 and U87 tumors growing s.c. on BALB/c nu/nu mice, dual inhibition of VEGF and PDGF signaling significantly increased tumor growth delay versus each monotherapy. Interestingly, radiation at ∼20% of the dose necessary to induce local tumor control exerts similar tumor growth-inhibitory effects as the antiangiogenic drugs given at their maximum effective dose. Addition of radiotherapy to both mono- as well as dual-antiangiogenic treatment markedly increased tumor growth delay. With respect to tumor angiogenesis, radiation further decreased microvessel density (CD31 count) and tumor cell proliferation (Ki-67 index) in all drug-treated groups. Of note, the slowly growing PC3 tumor responded better to the antiangiogenic drug treatments than the faster-growing U87 tumor. In addition to the beneficial effect of abrogating VEGF survival signaling when combined with radiation, we identified here a novel mechanism for the tumor escape from radiation damage. We found that radiation induced up-regulation of all four isoforms of PDGF (A-D) in endothelial cells supporting adjacent smooth muscle cells resulting in a prosurvival effect of radiation. The addition of SU6668 attenuated this undesirable paracrine radiation effect, which may rationalize the combined application of radiation with PDGF signaling inhibition to increase antitumor effects. Conclusion: A relative low radiation dose markedly enhances local antitumor effects of combined VEGF and PDGF signaling inhibition, suggesting a promising combination regimen for local tumor treatment with radiotherapy remaining an essential element.

Enhancement of Radiation Response in Osteosarcoma and Rhabomyosarcoma Cell Lines by Histone Deacetylase Inhibition

PURPOSE Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation treatment (XRT) by altering numerous molecular pathways. We investigated the effect of pan-HDACIs such as suberoylanilide hydroxamic acid (SAHA) on radiation response in two osteosarcoma (OS) and two rhabdomyosarcoma (RMS) cell lines. METHODS AND MATERIALS Clonogenic survival, cell cycle analysis, and apoptosis were examined in OS (KHOS-24OS, SAOS2) and RMS (A-204, RD) cell lines treated with HDACI and HDACI plus XRT, respectively. Protein expression was investigated via immunoblot analysis, and cell cycle analysis and measurement of apoptosis were performed using flow cytometry. RESULTS SAHA induced an inhibition of cell proliferation and clonogenic survival in OS and RMS cell lines and led to a significant radiosensitization of all tumor cell lines. Other HDACI such as M344 and valproate showed similar effects as investigated in one OS cell line. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In all investigated sarcoma cell lines, SAHA attenuated radiation-induced DNA repair protein expression (Rad51, Ku80). CONCLUSION Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair, as well as increased apoptosis induction after exposure to HDACIs, can be mechanisms of radiosensitization by HDACIs.

Differential effects of dose rate and superfractionation on survival and cell cycle of V79 cells from spheroid and monolayer culture

Recent developments concerning brachytherapy suggest conditions for an equivalence between the common continuous low dose rate (CLDR) exposure and pulsed irradiation regimens (PDR), provided that total dose is administered in the same overall time. The respective theoretical considerations have been based solely on the phenomenon of sublethal damage recovery. The present study, therefore, aimed to assess a possible influence of growth state/cell cycle progression when CLDR and different super fractionation protocols are compared. The respective experiments were performed with V79 cells that can be grown as a rapidly proliferating monolayer culture or as small spheroids (without hypoxia) where most of the cells are out of cycle. Differential changes in cell cycle distribution occurring during the compared exposure schemes and their impact on cell survival were expected to be expressed most clearly with this model system because of the short G1 phase. Cell irradiations were performed with brachytherapy sources either continuously (137Cs) or with high dose rate pulses (192Ir) at different (1 h and 4 h) pulse repetitions whereby the overall dose rate was kept constant to approximately 1 Gy/h. Cell survival curves were generated by sampling cells at different exposure times or number of pulses, respectively. For spheroid cells an unequivocal decrease of effectivity was demonstrated with decreasing dose per pulse, and the dose effect relation obtained with hourly pulses of 1 Gy was indistinguishable from the CLDR response. For monolayer cells, on the contrary, the scheme of hourly pulses was significantly more effective than the CLDR irradiation. As measured by flow cytometry, this different behaviour could be attributed to the accumulation of cycling cells in the radiosensitive G2/M phase (G2 block) during protracted exposure which was drastically more pronounced for the pulsed scheme compared to the CLDR condition. The observed principle phenomenon of a block to cell cycle progression from high dose rate pulses (at low overall dose rate) may be less expressed in (human) cells having a long G1 period, but if applicable to a clinical situation, an increase of acute effectiveness of a superfractionated brachytherapy protocol has to be considered.

Radiosensitization by pemetrexed of human colon carcinoma cells in different cell cycle phases

PURPOSE The novel folate antimetabolite Alimta (pemetrexed disodium, LY231514) exhibits antitumor activity in a broad array of human malignancies and was recently found to enhance radiation-induced cell killing in vitro. In the present study, a possible cell cycle phase-specific radiosensitization by pemetrexed was assessed. METHODS AND MATERIALS Widr human colon carcinoma cells were synchronized by serum withdrawal/stimulation that yielded about 80% cells with G1 DNA content 6 h after replating and more than 60% S-phase cells after 22 h, as assessed by flow cytometry. The respective cultures were irradiated with doses up to 12 Gy in combination with a subtoxic pemetrexed exposure (1.06 microM for 2 h: about 80% survival), or after mock treatment. Survival curves were generated by the clonogenic assay; apoptosis was measured by sub-G1 DNA flow cytometry. RESULTS The combination treatment of the G1 cells and of the more radioresistant S-phase cell preparations yielded survival rates that were lower than expected for independent cell killing. Radiosensitization, calculated as the ratio of the mean inactivation doses without or with drug exposure (enhancement ratio), was not significantly different for the two cell preparations (enhancement ratio of 2.1 and 2.3, respectively) and was similar to the previously reported value for log-phase cells. Pemetrexed exposure was unable to stimulate an apoptotic response of these cells to radiation. CONCLUSIONS Radiosensitization by pemetrexed is not cell cycle phase-specific, and the relative radioresistance of S-phase cells is retained. Apoptosis seems to have no influence on radiosensitization in this cell line.

Carbon ion irradiation inhibits glioma cell migration through downregulation of integrin expression.

PURPOSE To investigate the effect of carbon ion irradiation on glioma cell migration. METHODS AND MATERIALS U87 and Ln229 glioma cells were irradiated with photons and carbon ions. Migration was analyzed 24 h after irradiation. Fluorescence-activated cell sorting analysis was performed in order to quantify surface expression of integrins. RESULTS Single photon doses of 2 Gy and 10 Gy enhanced α(ν)β(3) and α(ν)β(5) integrin expression and caused tumor cell hypermigration on both vitronectin (Vn) and fibronectin (Fn). Compared to integrin expression in unirradiated cells, carbon ion irradiation caused decreased integrin expression and inhibited cell migration on both Vn and Fn. CONCLUSION Photon radiotherapy (RT) enhances the risk of tumor cell migration and subsequently promotes locoregional spread via photon induction of integrin expression. In contrast to photon RT, carbon ion RT causes decreased integrin expression and suppresses glioma cell migration on both Vn and Fn, thus promising improved local control.

Targeting ανβ3 and ανβ5 inhibits photon-induced hypermigration of malignant glioma cells

BackgroundSublethal photon irradiation was recently suspected to increase tumor cell motility and promote locoregional recurrence of disease. This study was set up to describe mechanisms underlying increased glioma cell migration through photon irradiation and to analyse the modifiability of photon-altered glioma cell motility by integrin inhibition.MethodsEight μm pore size membranes were coated with vitronectin (VN), collagen I and collagen IV. U87 and Ln229 glioma cells were analysed in migration experiments with and without radiotherapy (RT), serum stimulation and addition of monoclonal antibodies directed to human integrins ανβ3 and ανβ5. Quantitative FACS analysis of integrins was performed in U87 and Ln229 glioma cells following RT. Statistical analysis was performed using Students t- test.ResultsGlioma cell migration is serum-dependent and can be increased by photon RT which leads to enhanced expression of Vn receptor integrins. Blocking of either ανβ3 or ανβ5 integrins by antibodies inhibits Vn-based migration of both untreated and photon-irradiated glioma cells.ConclusionsPeripheral glioma cells are at risk of attraction into the adjacent healthy brain by serum components leaking through the blood brain barrier (BBB). Radiation therapy is associated with upregulation of Vn receptor integrins and enhanced glioma cell migration at sublethal doses. This effect can be inhibited by specific integrin blockade. Future therapeutical benefit may be derived from pharmacological integrin inhibition in combination with photon irradiation.

A specific inhibitor of protein kinase CK2 delays gamma-H2Ax foci removal and reduces clonogenic survival of irradiated mammalian cells.

BackgroundThe protein kinase CK2 sustains multiple pro-survival functions in cellular DNA damage response and its level is tightly regulated in normal cells but elevated in cancers. Because CK2 is thus considered as potential therapeutic target, DNA double-strand break (DSB) formation and rejoining, apoptosis induction and clonogenic survival was assessed in irradiated mammalian cells upon chemical inhibition of CK2.MethodsMRC5 human fibroblasts and WIDR human colon carcinoma cells were incubated with highly specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB), or mock-treated, 2 hours prior to irradiation. DSB was measured by pulsed-field electrophoresis (PFGE) as well as gamma-H2AX foci formation and removal. Apoptosis induction was tested by DAPI staining and sub-G1 flow cytometry, survival was quantified by clonogenic assay.ResultsTBB treatment did not affect initial DNA fragmention (PFGE; up to 80 Gy) or foci formation (1 Gy). While DNA fragment rejoining (PFGE) was not inhibited by the drug, TBB clearly delayed gamma-H2AX foci disappearence during postirradiation incubation. No apoptosis induction could be detected for up to 38 hours for both cell lines and exposure conditions (monotherapies or combination), but TBB treatment at this moderately toxic concentration of 20 μM (about 40% survival) enhanced radiation-induced cell killing in the clonogenic assay.ConclusionsThe data imply a role of CK2 in gamma-H2AX dephosporylation, most likely through its known ability to stimulate PP2A phosphatase, rather than DSB rejoining. The slight but definite clonogenic radiosensitization by TBB does apparently not result from interference with an apoptosis suppression function of CK2 in these cells but could reflect inhibitor-induced uncoupling of DNA damage response decay from break ligation.

Radiation induced chromosome aberrations and clonogenic survival in human lymphoblastoid cell lines with different p53 status

PurposeTo better understand the relation of radiation induced chromosome aberrations and clonogenic survival in cells with different p53 status.Materials and MethodsThe human lymphoblasts TK6 and WTK1 were derived from the same donor, but differ in radiosensitivity, p53 status and kinetics of apoptosis. TK6 cells have wild type p53 (p53wt), whereas WTK1 cells have a mutated, non-functional p53 (p53mut). Additionally, a HPV16 E6 transfected TK6 cell line (TK6E6), which is also negative for p53 function (p53neg), was studied. The cells were irradiated, incubated with colcemid, hypotonically lysed and fixed. After staining with Giemsa, asymmetric chromosomal exchange type aberrations were counted in 50 mitoses each per dose point (0 to 4 Gy). Clonogenic survival was determined using the microtiter plate assay. All experiments were performed in triplicate.ResultsWTK1 (p53mut) show a higher spontaneous frequency of chromosome aberrations than TK6 (p53wt). No significant differences were noted in radiation induced aberration frequency. TK6E6 (p53neg) show comparable aberration frequencies like TK6. However, the dose required to reduce survival to 10% (D10) was about 2 Gy for TK6 and TK6E6, whereas the D10 for WTK1 was approximately 3 Gy.ConclusionThe p53 status influences the radiosensitivity in this lymphoblast cell system showing a high rate of radiation induced apoptosis. Cells with p53mut (WTK1), survive with a damaged genome, because they do not undergo apoptosis to loose their clonogenicity. There was no difference between the p53wt (TK6) and p53neg cells (TK6E6) suggesting a suppression of radiation induced apoptosis by p53mut.ZusammenfassungZielZielsetzung dieser Untersuchungen war, den Zusammenhang zwischen bestrahlungsinduzierten Chromosomenaberrationen und dem klonogenen Überleben in Zellen mit unterschiedlichem p53-Status näher zu klären.Material und MethodeDie humanen Lymphoblastenlinien TK6 und WTK1 stammen ursprünglich vom selben Spender, unterscheiden sich jedoch in Strahlenresistenz, p53-Stätus und Kinetik bestrahlungsinduzierter Apoptose. TK6Zellen sind Wildtyp für p53 (p53wt), WTK1-Zellen haben ein mutiertes, nichtfunktionales p53 (p53mut). Zusätzlich wurde noch eine mit HPV16 E6 transfizierte TK6-Zellinie (TK6E6) untersucht, welche ebenso negativ für die p53Funktion ist. Die Zellen wurden bestrahlt und in Colcemid inkubiert, hypoton lysiert und fixiert. Nach Giemsa-Färbung wurden asymmetrische chromosomale Austauschaberrationen in jeweils 50 Mitosen pro Dosispunkt (0 bis 4 Gy) ausgezählt. Mittels des Mikrotiterplattenassays wurde das klonogene Überleben nach Bestrahlung bestimmt. Alle Experimente wurden dreimal wiederholt.ErgebnisseWTK1 (p53mut) zeigen eine höhere Frequenz bestrahlungsinduzierter Chromosomenaberrationen als TK6 (p53wt) sowohl vor als auch nach Bestrahlung (Abbildung 1). Nach Abzug der erhohten Spontanaberrationsfrequenz ergeben sich nahezu deckungsgleiche Werte. Ebenso zeigen TK6E6 (p53rieg) vergleichbare Aberrationsraten wie TK6. Im klonogenen Überleben ergaben sich jedoch deutliche Unterschiede zwischen den Zellinien. Für TK6 und TK6E6 errechnete sich eine D10 von ca. 2 Gy und für die WTK1 von ca. 3 Gy (Abbildung 2). 1Laboratory for Molecular and Cellular Radiobiology (Chairman: Prof. Dr. Dr. M. Wannenmacher), Department of Clinical Radiology, University of Heidelberg, Germany.SchlußfolgerungDer p53-Status beeinflußt die zelluläre Strahlensensibilität in diesem Lymphoblastenzellsystem, welches eine hohe Rate bestrahlungsinduzierter Apoptose zeigt. Zellen mit p53mut, welche eine verzögerte und verminderte Apoptoserate haben, überleben mit einem geschadigten Genom, da ihnen mit der Apoptose der Mechanismus zum Verlust der Klonogenität fehlt. Allerdings unterscheiden sich die p53-negativen Zellen nicht von den p53wt-Zellen. Dies würde auf eine Unterdrilckung der bestrahlungsinduzierten Apoptose durch p53mut in diesem Zellsystem hinweisen.

Combined effects of ionizing radiation and 4-hydroperoxyifosfamide in vitro

BACKGROUND AND PURPOSE Combined radiochemotherapy has gained increasing interest in clinical applications. The effects of combined exposure of ionizing radiation and 4-hydroperoxyifosfamide (4HOOIF) on cell survival were investigated in vitro. MATERIALS AND METHODS Clonogenic survival of log phase V79, Caski (squamous carcinoma), Widr (colon carcinoma) and MRI-221 cells (human melanoma) was determined after combined exposure to 4HOOIF and radiation. Measurement of cell survival for different cell cycle phases was performed after mitotic shake-off (V79) or appropriate intervals after serum stimulation of plateau phase cells (Widr). Control of cell cycle distribution was performed using flow cytometry. RESULTS In all cell lines tested, a combined exposure resulted in cell killing that was greater than for independent action. While this type of radiosensitization was of minor magnitude for log-phase cells or cells in G1 substantial radiosensitization was detected for S-phase cells with enhancement ratios (calculated from the respective mean inactivation doses) of up to 1.5. CONCLUSIONS The results demonstrate the interaction of 4HOOIF and radiation-induced cell damage with marked cell cycle specificity. Since the largest combination effect was observed for the most radioresistant S-phase cells, damage interaction could be mediated by an interference of 4HOOIF with the repair/fixation pathway of radiation-induced potentially lethal damage.

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 [3H] 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin binding and agonist‐stimulated binding of [35S]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. Synapse 67:648–655, 2013.