Split-dose Irradiation Research Articles

Tumor radioresistance caused by radiation-induced changes of stem-like cell content and sub-lethal damage repair capability

Cancer stem-like cells (CSCs) within solid tumors exhibit radioresistance, leading to recurrence and distant metastasis after radiotherapy. To experimentally study the characteristics of CSCs, radioresistant cell lines were successfully established using fractionated X-ray irradiation. The fundamental characteristics of CSCs in vitro have been previously reported; however, the relationship between CSC and acquired radioresistance remains uncertain. To efficiently study this relationship, we performed both in vitro experiments and theoretical analysis using a cell-killing model. Four types of human oral squamous carcinoma cell lines, non-radioresistant cell lines (SAS and HSC2), and radioresistant cell lines (SAS-R and HSC2-R), were used to measure the surviving fraction after single-dose irradiation, split-dose irradiation, and multi-fractionated irradiation. The SAS-R and HSC2-R cell lines were more positive for one of the CSC marker aldehyde dehydrogenase activity than the corresponding non-radioresistant cell lines. The theoretical model analysis showed that changes in both the experimental-based ALDH (+) fractions and DNA repair efficiency of ALDH (−) fractions (i.e., sub-lethal damage repair) are required to reproduce the measured cell survival data of non-radioresistant and radioresistant cell lines. These results suggest that the enhanced cell recovery in SAS-R and HSC2-R is important when predicting tumor control probability in radiotherapy to require a long dose-delivery time; in other words, intensity-modulated radiation therapy is ideal. This work provides a precise understanding of the mechanism of radioresistance, which is induced after irradiation of cancer cells.

TP53 modulates radiotherapy fraction size sensitivity in normal and malignant cells

Recent clinical trials in breast and prostate cancer have established that fewer, larger daily doses (fractions) of radiotherapy are safe and effective, but these do not represent personalised dosing on a patient-by-patient basis. Understanding cell and molecular mechanisms determining fraction size sensitivity is essential to fully exploit this therapeutic variable for patient benefit. The hypothesis under test in this study is that fraction size sensitivity is dependent on the presence of wild-type (WT) p53 and intact non-homologous end-joining (NHEJ). Using single or split-doses of radiation in a range of normal and malignant cells, split-dose recovery was determined using colony-survival assays. Both normal and tumour cells with WT p53 demonstrated significant split-dose recovery, whereas Li-Fraumeni fibroblasts and tumour cells with defective G1/S checkpoint had a large S/G2 component and lost the sparing effect of smaller fractions. There was lack of split-dose recovery in NHEJ-deficient cells and DNA-PKcs inhibitor increased sensitivity to split-doses in glioma cells. Furthermore, siRNA knockdown of p53 in fibroblasts reduced split-dose recovery. In summary, cells defective in p53 are less sensitive to radiotherapy fraction size and lack of split-dose recovery in DNA ligase IV and DNA-PKcs mutant cells suggests the dependence of fraction size sensitivity on intact NHEJ.

Comparison of Cellular Sensitivity to a Split Radiation Dose and a Combination of a Single Radiation Dose and Electromagnetic Field Exposure

Comparison of Cellular Sensitivity to a Split Radiation Dose and a Combination of a Single Radiation Dose and Electromagnetic Field Exposure

DIFFERENCE IN DEGREE OF SUB-LETHAL DAMAGE RECOVERY BETWEEN CLINICAL PROTON BEAMS AND X-RAYS.

Fractionated proton beam radiotherapy is spreading worldwide these days. However, biological data of sub-lethal damage recovery (SLDR) after proton irradiation is not known yet. We here conducted split-dose experiments (20-360 min intervals) to clarify SLDR kinetics, and also compared the kinetics between cells with different repairability of DNA double-strand breaks. CHO and 51D1 cell lines but not V3 cell line showed significant SLDR, which reached plateau in 4-6 h. The recovery rates and recovery halftime of SLDR after X-rays were significantly higher and shorter than proton beams for CHO and 51D1 cells, respectively. Additionally, the frequency of remaining gamma-H2AX foci after two fractions was remarkably higher for X-rays than proton beams. These data suggest that there is a difference between proton beam and X-rays in SLDR and the retained DNA double-strand breaks after split-dose irradiation.

Kinetics of cytokine mRNA and protein expression by plastic adherent cells in the thymus after split-dose irradiation

Whole body irradiation causes significant apoptosis in various tissues such as the thymus. If apoptotic cells outnumber the phagocytic capacity of macrophages, apoptosis becomes secondary necrosis, inducing inflammatory cytokine expression in macrophages. Radiation also induces thymic lymphomas in C57BL/6 mice after four consecutive irradiations with 1.6 Gy X-rays with nearly 100% incidence. Since cancer development is modulated by a microenvironment involving macrophages, we examined the kinetics of thymocyte number and plastic adherent cell number in the thymus as well as cytokine mRNA expression by plastic adherent cells in the thymus after split-dose irradiation. Upon split-dose irradiation, thymocyte number changed dramatically, whereas plastic adherent cell number did not. Among cytokine mRNAs tested, IL-1β, IL-11 and IL-12p40 mRNAs were up regulated 2 days after the 1st and 2nd, 3rd and 4th, and 2nd and 3rd irradiations, respectively. On the other hand, TNF-α mRNA was up regulated 2 days after the 3rd irradiation and 2 weeks after the 4th irradiation. The level of IL-11 protein was also increased 2 days after 3rd and 4th irradiations. These results suggest that, upon split-dose irradiation, macrophages in the thymus produce various cytokines in a time-dependent manner, thereby contributing to induction of thymic lymphomas.

029. - Evaluation of cellular sensitivity to split radiation dose and a combination of a single radiation dose and electromagnetic field exposure

029. - Evaluation of cellular sensitivity to split radiation dose and a combination of a single radiation dose and electromagnetic field exposure

Mutations in the FHA-domain of ectopically expressed NBS1 lead to radiosensitization and to no increase in somatic mutation rates via a partial suppression of homologous recombination

Ionizing radiation induces DNA double-strand breaks (DSBs). Mammalian cells repair DSBs through multiple pathways, and the repair pathway that is utilized may affect cellular radiation sensitivity. In this study, we examined effects on cellular radiosensitivity resulting from functional alterations in homologous recombination (HR). HR was inhibited by overexpression of the forkhead-associated (FHA) domain-mutated NBS1 (G27D/R28D: FHA-2D) protein in HeLa cells or in hamster cells carrying a human X-chromosome. Cells expressing FHA-2D presented partially (but significantly) HR-deficient phenotypes, which were assayed by the reduction of gene conversion frequencies measured with a reporter assay, a decrease in radiation-induced Mre11 foci formation, and hypersensitivity to camptothecin treatments. Interestingly, ectopic expression of FHA-2D did not increase the frequency of radiation-induced somatic mutations at the HPRT locus, suggesting that a partial reduction of HR efficiency has only a slight effect on genomic stability. The expression of FHA-2D rendered the exponentially growing cell population slightly (but significantly) more sensitive to ionizing radiation. This radiosensitization effect due to the expression of FHA-2D was enhanced when the cells were irradiated with split doses delivered at 24-h intervals. Furthermore, enhancement of radiation sensitivity by split dose irradiation was not seen in contact-inhibited G0/G1 populations, even though the cells expressed FHA-2D. These results suggest that the FHA domain of NBS1 might be an effective molecular target that can be used to induce radiosensitization using low molecular weight chemicals, and that partial inhibition of HR might improve the effectiveness of cancer radiotherapy.

A Model of Photon Cell Killing Based on the Spatio-Temporal Clustering of DNA Damage in Higher Order Chromatin Structures

We present a new approach to model dose rate effects on cell killing after photon radiation based on the spatio-temporal clustering of DNA double strand breaks (DSBs) within higher order chromatin structures of approximately 1–2 Mbp size, so called giant loops. The main concept of this approach consists of a distinction of two classes of lesions, isolated and clustered DSBs, characterized by the number of double strand breaks induced in a giant loop. We assume a low lethality and fast component of repair for isolated DSBs and a high lethality and slow component of repair for clustered DSBs. With appropriate rates, the temporal transition between the different lesion classes is expressed in terms of five differential equations. These allow formulating the dynamics involved in the competition of damage induction and repair for arbitrary dose rates and fractionation schemes. Final cell survival probabilities are computable with a cell line specific set of three parameters: The lethality for isolated DSBs, the lethality for clustered DSBs and the half-life time of isolated DSBs.By comparison with larger sets of published experimental data it is demonstrated that the model describes the cell line dependent response to treatments using either continuous irradiation at a constant dose rate or to split dose irradiation well. Furthermore, an analytic investigation of the formulation concerning single fraction treatments with constant dose rates in the limiting cases of extremely high or low dose rates is presented. The approach is consistent with the Linear-Quadratic model extended by the Lea-Catcheside factor up to the second moment in dose. Finally, it is shown that the model correctly predicts empirical findings about the dose rate dependence of incidence probabilities for deterministic radiation effects like pneumonitis and the bone marrow syndrome. These findings further support the general concepts on which the approach is based.

Use of the γ-H2AX assay to investigate DNA repair dynamics following multiple radiation exposures.

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.

SU-E-T-292: In-Vivo Blood Oxygen Measurements Via Interstitial Fiber Optic Probe and Photoacoustic Imaging

Purpose: Better understanding of hemodynamics and oxygen transport in response to radiation will enable clinicians to optimize radiotherapy treatment regimens. It is well‐known that oxygenated tissue is significantly more responsive to damage caused by irradiation. We sought to explore the real‐time hemodynamics and blood oxygenation dynamics in response to radiation using a novel interstitial fiber‐optic probe and a photoacoustic imaging system. Methods: Mice bearing xenografted tumors composed of a head and neck cancer cell line (UM‐SCC47) and a patient‐derived xenograft (UW‐SCC36) were irradiated (10 Gy). Blood oxygena saturation (S) and blood volume fraction (B) were monitored in real time using an interstitial fiber optic probe as well as a photoacoustic imaging system (using a 21 MHz transducer, and infra red wavelengths of 750 and 850nm on the Vevo LAZR (R)). Results: A significant drop in blood oxygen saturation was found within 15 minutes following irradiation using the interstitial fiber optic probe. Meanwhile, Blood volume fraction remained constant. Preliminary photoacoustic imaging provided evaluation of the spatial characteristics of blood‐oxygen dynamics and showed qualitative agreement with probe measurements. The measurements were compared to gain a better understanding of the fiber optic probe's spatial averaging, also. Conclusion: While development of the interstitial fiber optic probe continues, it appears to provide real‐time assessment of blood‐oxygen dynamics. Correlation of optimal reoxygenation with split dose radiation response is ongoing and may provide insight that could lead to improvements in the delivery of radiation therapy. This is particularly valuable for hypofractionated treatments as this information can guide the timing, size, and number of fractions for radiotherapy treatments. Minalini Lakshman is an employee of the VisualSonics company; however, the company did not provide any financial support for this work.

SU-E-T-01: Applications of 6MV FFF Photon Beams in Optimizing Radiobiological Response for Respiratory-Gated Liver SBRT.

To establish a radiobiological basis for gated stereotactic body radiotherapy of primary and metastatic liver cancers using volumetric arc radiotherapy in a flattening filter free (FFF) mode. Human cervical carcinoma, SiHa, non-small cell lung carcinoma, H460, and Chinese hamster V79 cells were irradiated in a water bath with 6MV photons from a Varian TrueBeam linear accelerator. To establish dose-response and its sensitivity to dose rate following acute irradiation, doses of 2, 4, 6, 8 and 10 Gy were delivered in FFF mode at 400 and 1200 MU/min. To investigate whether removal of the flattening filter affects cell response, doses of 5 and 10 Gy were delivered to SiHa and H460 cells in FFF and filtered modes at 400 MU/min. Finally, to assess the effect of protracting dose delivery by gating, a dose of 10 Gy was delivered to SiHa and H460 cells acutely and also over 15, 30 and 60 min. Dose-response over doses examined was independent of dose rate in FFF mode. Differences in cell survival following irradiation in FFF and filtered modes were not significant. However a significant increase in survival for both H460 and SiHa cells was observed for 15 min split-dose irradiation compared to acute irradiation but further increase in irradiation time to 60 min did not affect cell survival. Dose rate and presence of a flattening filter showed no effect on cell survival, however, survival was significantly affected when dose delivery time was protracted to that typical of conformal field therapy. Volumetric arc based gated SBRT may be beneficial for tumor cell kill, though the gating window and duty cycle have to be balanced against the effect of dose delivery protraction. Research Support (Varian Medical Systems).

A nucleoside anticancer drug, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (TAS106), sensitizes cells to radiation by suppressing BRCA2 expression

BackgroundA novel anticancer drug 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (ECyd, TAS106) has been shown to radiosensitize tumor cells and to improve the therapeutic efficiency of X-irradiation. However, the effect of TAS106 on cellular DNA repair capacity has not been elucidated. Our aim in this study was to examine whether TAS106 modified the repair capacity of DNA double-strand breaks (DSBs) in tumor cells.MethodsVarious cultured cell lines treated with TAS106 were irradiated and then survival fraction was examined by the clonogenic survival assays. Repair of sublethal damage (SLD), which indicates DSBs repair capacity, was measured as an increase of surviving cells after split dose irradiation with an interval of incubation. To assess the effect of TAS106 on the DSBs repair activity, the time courses of γ-H2AX and 53BP1 foci formation were examined by using immunocytochemistry. The expression of DNA-repair-related proteins was also examined by Western blot analysis and semi-quantitative RT-PCR analysis.ResultsIn clonogenic survival assays, pretreatment of TAS106 showed radiosensitizing effects in various cell lines. TAS106 inhibited SLD repair and delayed the disappearance of γ-H2AX and 53BP1 foci, suggesting that DSB repair occurred in A549 cells. Western blot analysis demonstrated that TAS106 down-regulated the expression of BRCA2 and Rad51, which are known as keys among DNA repair proteins in the homologous recombination (HR) pathway. Although a significant radiosensitizing effect of TAS106 was observed in the parental V79 cells, pretreatment with TAS106 did not induce any radiosensitizing effects in BRCA2-deficient V-C8 cells.ConclusionsOur results indicate that TAS106 induces the down-regulation of BRCA2 and the subsequent abrogation of the HR pathway, leading to a radiosensitizing effect. Therefore, this study suggests that inhibition of the HR pathway may be useful to improve the therapeutic efficiency of radiotherapy for solid tumors.

The effect on the small bowel of 5-FU and oxaliplatin in combination with radiation using a microcolony survival assay

BackgroundIn locally advanced rectal cancer, 5-Fluorouracil (5-FU)-based chemoradiation is the standard treatment. The main acute toxicity of this treatment is enteritis. Due to its potential radiosensitizing properties, oxaliplatin has recently been incorporated in many clinical chemoradiation protocols. The aim of this study was to investigate to what extent 5-FU and oxaliplatin influence the radiation (RT) induced small bowel mucosal damage when given in conjunction with single or split dose RT.MethodsImmune competent balb-c mice were treated with varying doses of 5-FU, oxaliplatin (given intraperitoneally) and total body RT, alone or in different combinations in a series of experiments. The small bowel damage was studied by a microcolony survival assay. The treatment effect was evaluated using the inverse of the slope (D0) of the exponential part of the dose-response curve.ResultsIn two separate experiments the dose-response relations were determined for single doses of RT alone, yielding D0 values of 2.79 Gy (95% CI: 2.65 - 2.95) and 2.98 Gy (2.66 - 3.39), for doses in the intervals of 5-17 Gy and 5-10 Gy, respectively. Equitoxic low doses (IC5) of the two drugs in combination with RT caused a decrease in jejunal crypt count with significantly lower D0: 2.30 Gy (2.10 - 2.56) for RT+5-FU and 2.27 Gy (2.08 - 2.49) for RT+oxaliplatin. Adding both drugs to RT did not further decrease D0: 2.28 Gy (1.97 - 2.71) for RT+5-FU+oxaliplatin. A clearly higher crypt survival was noted for split course radiation (3 × 2.5 Gy) compared to a single fraction of 7.5 Gy. The same difference was seen when 5-FU and/or oxaliplatin were added.ConclusionCombining 5-FU or oxaliplatin with RT lead to an increase in mucosal damage as compared to RT alone in our experimental setting. No additional reduction of jejunal crypt counts was noted when both drugs were combined with single dose RT. The higher crypt survival with split dose radiation indicates a substantial recovery between radiation fractions. This mucosal-sparing effect achieved by fractionation was maintained also when chemotherapy was added.

Damage to Aging Thymic Stroma and De Novo T Cell Development.

Successful T lymphopoiesis is essential for maintaining resistance to infection, mediating graft versus tumor activity, and as a platform for some immune based therapies. Among the most profound age-related change affecting the hemopoietic system is involution of the thymus and a decline in T cell production. Coordinated interaction of marrow-derived lymphoid progenitors with thymic stromal cells is required for successful de novo T lymphopoiesis. Current models of thymic involution indicate that aging alters the capacity of thymic stromal cells to support this de novo T cell lymphopoiesis. Loss of specific sets of thymic stromal cells, or loss of specific lympho-stromal signaling interactions may partially explain age-based impaired T cell reconstitution. Prior models examining the potential to reconstitute the aged thymus have been conducted either in vitro or in cyto-reduced hosts, and thus largely risk confounding cytotoxic damage with age-related damage. We have established an in vivo functional assessment of the ability of thymic stroma to support de novo T lymphocyte development. In the absence of radiation or chemotherapy this system supports robust thymic reconstitution after transplantation of limited numbers of T cell depleted wild type precursors. We have previously demonstrated that the capacity for reconstitution in this model system depends on the presence of normal, but empty, stromal niches. Using this model we have demonstrated that a variety of chemotherapeutic agents damage the thymic stroma and impair robust reconstitution. We now present data demonstrating that with increasing age reconstitution becomes less robust. Thus in mice older than 16 months unlike in those younger than twelve months, donor derived T lymphocytes can be identified, but the absolute cellularity of the thymus does not increase after transplant. In addition, we demonstrate that there are agent specific defects in the stromal compartments of treated aged mice. We have proceeded to identify a subset of agents (including split dose irradiation) that impair reconstitution in aged recipients but do not do so in the young. Thus while reconstitution of the thymus of young mice subjected to hyperfractionated total body irradiation declines 28 +/− 3% that of old mice treated in the same manner declines 91 +/− 6%. This last finding has enabled us to identify specific “at risk” stromal populations that normally appear to support the DN2 to DN3 transition in T lymphopoiesis. In a series of experiments designed to ask whether these at risk stromal populations can be protected from injury we identify agent specific differences in mechanisms of injury. It is anticipated that results from these investigations will augment our understanding of lympho-stromal interactions crucial to normal intra-thymic T cell development.

Split Dose Recovery Studies using Homologous Recombination Deficient Gene Knockout Chicken B Lymphocyte Cells

To understand the role of proteins involved in DSB repair modulating SLD recovery, chicken B lymphoma (DT 40) cell lines either proficient or deficient in RAD52, XRCC2, XRCC3, RAD51C and RAD51D were subjected to fractionated irradiation and their survival curves charted. Survival curves of both WT DT40 and RAD52 (-/-) cells had a big shoulder while all the other cells exhibited small shoulders. However, at the higher doses of radiation, RAD51C(-/-) cells displayed hypersensitivity comparable to the data obtained for the homologous recombination deficient RAD54(-/-) cells. Repair of SLD was measured as an increase in survival after a split dose irradiation with an interval of incubation between the radiation doses. All the cell lines (parental DT40 and genetic knockout cell lines viz., RAD52(-/-), XRCC2(-/-), XRCC3(-/-) RAD51C(-/-) and RAD51D(-/-)) used in this study demonstrated a typical split-dose recovery capacity with a specific peak, which varied depending on the cell type. The maximum survival of WT DT40 and RAD52(-/-) was reached at about 1-2 hours after the first dose of radiation and then decreased to a minimum thereafter (5h). The increase in the survival peaked once again by about 8 hours. The survival trends observed in XRCC2 (-/-), XRCC3(-/-), RAD51C (-/-) and RAD51D(-/-) knockout cells were also similar, except for the difference in the initial delay of a peak survival for RAD51D(-/-) and lower survival ratios. The second phase of increase in the survival in these cell lines was much slower in XRCC2(-/-) , XRCC3(-/-), RAD51C(-/-) and RAD51D(-/-) and further delayed when compared with that of RAD52(-/-) and parental DT40 cells suggesting a dependence on their cell cycle kinetics. This study demonstrates that the participation of RAD52, XRCC2, XRCC3, RAD51C and RAD51D in the DSB repair via homologous recombination is of less importance in comparison to RAD54, as RAD54 deficient cells demonstrated complete absence of SLD recovery.

Nuclear Accumulation and Activation of Nuclear Factor κB after Split-dose Irradiation in LS174T Cells

Although radiation-induced gene expression has been extensively studied, most of the studies to date have focused on that after single-dose irradiation. As split-dose irradiation, rather than single-dose irradiation, is usual in clinical situations, we investigated the effects of split-dose irradiation on nuclear factor kappaB (NF-kappaB) in the human rectum carcinoma cell line, LS174T. After either single- or split-dose irradiation with a different interval, nuclear localization of NF-kappaB was examined by Western blot and immunofluorescence and its DNA-binding activity was measured by ELISA-based assay. Irradiation-induced NF-kappaB nuclear accumulation and DNA binding activity increased in a dose-dependent manner. The peak of NF-kappaB nuclear accumulation and DNA binding activity was seen 2 to 6 hours after a single dose of 4 Gy irradiation and returned to control levels after 12 hours. In split-dose irradiation, NF-kappaB activity was similar after the first and second doses of 4 Gy irradiation separated by 12 hours. In addition, NF-kappaB activity was decreased by lengthening the interval between irradiation. The cell survival, which was assessed by colony formation assay, showed inverse correlation to this: the surviving fraction was higher after split-dose irradiation than after single-dose irradiation of the same total dose and it increased as the interval between irradiation was lengthened. Thus the present results showed a correlation between NF-kappaB activation and the repair of sublethal damage in split-dose irradiation.

Synergistic Effects of Radiation and β-Lapachone in DU-145 Human Prostate Cancer CellsIn Vitro

It has been reported that beta-lapachone (beta-lap), a bioreductive anti-cancer drug, synergistically interacts with ionizing radiation and that the sensitivity of cells to beta-lap is closely related to the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1). Here we report the results of our studies of mechanisms underlying the synergistic interaction of beta-lap and radiation in killing cancer cells using the DU-145 human prostate cancer cell line. The clonogenic cell death caused by the combination of radiation and beta-lap was synergistic when beta-lap was administered 0-10 h after irradiation but not when it was given before irradiation. The expression and activity of NQO1 increased significantly and remained elevated for longer than 12 h after 4 Gy irradiation, suggesting that the long-lasting elevation of NQO1 sensitized the cells to beta-lap. Studies with split-dose irradiation demonstrated that beta-lap given immediately after irradiation effectively inhibited sublethal radiation damage (SLD) repair. Taken together, these results lead us to conclude that the synergistic interaction between beta-lap and radiation in killing cells is the result of two distinct mechanisms: First, radiation sensitizes cells to beta-lap by up-regulating NQO1, and second, beta-lap sensitizes cells to radiation by inhibiting SLD repair. The combination of beta-lap and radiotherapy is potentially promising modality for the treatment of cancer in humans.

Radiosensitization of Squamous Cell Carcinoma by the Alkylphospholipid Perifosine in Cell Culture and Xenografts

Combined modality treatment has improved outcome in various solid tumors. Besides classic anticancer drugs, a new generation of biological response modifiers has emerged that increases the efficacy of radiation. Here, we have investigated whether perifosine, an orally applicable, membrane-targeted alkylphospholipid, enhances the antitumor effect of radiation in vitro and in vivo. Several long-term and short-term in vitro assays (clonogenic survival, sulforhodamine B cytotoxicity, apoptosis, and cell cycle analysis) were used to assess the cytotoxic effect of perifosine in combination with radiation. In vivo, the response of human KB squamous cell carcinoma xenografts was measured after treatment with perifosine, irradiation, and the combination. Radiolabeled perifosine was used to determine drug disposition in tumor and normal tissues. At various intervals after treatment, tumor specimens were collected to document histopathologic changes. In vitro, perifosine reduced clonogenic survival, enhanced apoptosis, and increased cell cycle arrest after radiation. In vivo, radiation and perifosine alone induced a dose-dependent tumor growth delay. When combining multiple perifosine administrations with single or split doses of radiation, complete and sustained tumor regression was observed. Histopathologic analysis of tumor specimens revealed a prominent apoptotic response after combined treatment with radiation and perifosine. Radiation-enhanced tumor response was observed at clinically relevant plasma perifosine concentrations and accumulating drug disposition of >100 microg/g in tumor tissue. Perifosine enhances radiation-induced cytotoxicity, as evidenced by reduced clonogenic survival and increased apoptosis induction in vitro and by complete tumor regression in vivo. These data provide strong support for further development of this combination in clinical studies.

Alteration of cellular DNA-repair by interferon-beta

Type I interferons have been investigated as radiosensitizer over a long time. Recent clinical data in pancreatic cancer are suggesting an enhanced efficacy of radiation (XRT) and chemotherapy by the simultaneous application with IFN-alpha. On the other hand, a randomized clinical study on the addition of IFN-beta to XRT for the treatment of lung cancer showed a negative result. These observations are suggesting, that the interaction of XRT with type I interferons might be determined by the genetic background of the treated tumors. Since the enhancement of radiation by IFN-beta is associated with an increase of the alpha-component of the survival curves; we assumed that the interaction of IFN-beta with ionizing irradiation is characterized by interference with DNA repair. A number of cell lines representing normal tissue and different tumor types and with known genetic alterations in DNA repair (DNA-PK and Brca2) or cell cycle control (TP53, RB) were used. Cellular repair capacity was modulated by wortmannin (WMN) treatment (in M059K cells) or PRCDK gene transfection (in M059J cells). For experiments, cells were incubated with natural IFN-beta (IFN) (0, 300 I.U./ml or 3000 I.U./ml) 24 h before irradiation. Cycling or confluent cell cultures were used. We measured cellular survival after graded single dose irradiation, split dose (SD), and low dose rate irradiation (LDR), with colony formation as an endpoint. Survival curves were fitted according to the linear-quadratic equation. Sensitizer enhancement ratios were calculated at the 37% survival level, and isobologram analysis was applied to test the IFN-radiation interactions. Apoptosis and DNA-rejoining were measured by standard assays. On the survival level, additive as well as supraadditive interactions of IFN and XRT were observed, depending on the cell lines used. The IFN effectiveness was more pronounced in cycling compared to resting cells. The expression of mutated TP53 or RB did not influence the IFN-induced radiation cytotoxicity. Incubation with 3000 I.U./ml IFN could induce apoptosis, but it did sparsely account for increased radiation toxicity. Supraadditivity of IFN and XRT was always accompanied by increased alpha-values of the respective survival curves. The involvement of repair processes in the IFN effectiveness was supported by the results of the SD and LDR experiments, indicating an inhibition of sublethal damage repair. Initial DSBs or DNA rejoining were not altered by IFN-treatment. DNA-PK deficient M059J cells were radiosensitive and showed a supraadditive IFN-XRT interaction that could be abrogated by the re-transfection of a functional PRKDC gene (M059J-Fus1 cells). DNA-PK proficient M059K cells were radioresistant and showed a solely additive IFN-XRT interaction, which could be converted into supraadditivity by repair-inhibition with WMN. Brca2-deficient CAPAN-1 pancreatic tumor cells were extremely IFN sensitive but showed additive IFN-XRT interaction, while Brca2 proficient pancreatic tumor cells were less IFN sensitive and showed a supraadditive IFN-XRT interaction. Our results are pointing towards an interaction of IFN with the regulation of the repair of radiation-induced damage as the predominant mechanism of IFN-related radiosensitization. Inhibition of the Non-Homologous-End-Joining repair (NHEJ) pathway, as shown in MO59J cells and WMN inhibited MO59K-cells, led to a supraadditive interaction of IFN and XRT, whereas cells with intact NHEJ (MO59K-cells) showed an additive interaction of IFN and XRT. Likely IFN is interfering with the second major pathway of double strand break repair, homologous recombination (HR). This was supported by the observation that mutation in the Brca2-gene in CAPAN-1 cells, which is already associated with impaired HR, did not alter the interaction of IFN with radiation repair

Changes in the effect of dose fractionation during daily fractionated irradiation: studies in mouse oral mucosa

Purpose The aim of the present study was to quantify the fractionation effect in mouse oral mucosa during a daily fractionated protocol. Methods and materials Irradiation of the snout of C3H mice was performed with 5 × 3 Gy/week. In the first experiment, graded test doses were applied to the lower tongue on Days 4, 7, 11, 14, or 18. Subsequently, a split-dose experiment was performed on the same days with 1, 2, 3, or 5 fractions (graded doses). Results The ED50 (dose expected to induce ulcer in 50% of the mice) for untreated mucosa was 10.7 ± 1.0 Gy. Fractionated irradiation with 5 × 3 Gy/week yielded an ED50 for top-up irradiation of 6.5 ± 1.8 Gy, 6.8 ± 0.9 Gy, 5.3 ± 2.1 Gy, 7.3 ± 1.9 Gy, and 7.5 ± 1.3 Gy on Days 4, 7, 11, 14, and 18. The ED50 values for split-dose irradiation on Day 4 increased from 6.24 ± 1.48 Gy (1 fraction) to 9.96 ± 1.47 Gy (5 fractions). Similarly, an increase from 4.04 ± 1.56 Gy to 10.07 ± 1.9 Gy was found on Day 11, and from 5.84 ± 2.37 Gy to 9.81 ± 2.29 Gy on Day 18. After the weekend breaks, values between 6.22 ± 1.60 Gy (1 fraction) and 4.7 ± 1.95 Gy (5 fractions) were observed on Day 7, and between 5.62 ± 1.36 Gy and 5.98 ± 2.01 Gy on Day 14. Conclusions These results indicate that the fractionation effect in oral mucosa is consistently lost over the weekends and restored during the treatment weeks.