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Results for “Radiosensitivity”

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PubMed2005

ErbB3 expression predicts tumor cell radiosensitization induced by Hsp90 inhibition.

The ability to identify tumors that are susceptible to a given molecularly targeted radiosensitizer would be of clinical benefit. Towards this end, we have investigated the effects of a representative Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG), on the radiosensitivity of a panel of human tumor cell lines. 17DMAG was previously shown to enhance the radiosensitivity of a number of human cell lines, which correlated with the loss of ErbB2. We now report on cell lines in which 17DMAG induced the degradation of ErbB2, yet had no effect on radiosensitivity. In a comparison of ErbB family members, ErbB3 protein was only detectable in cells resistant to 17DMAG-induced radiosensitization. To determine whether ErbB3 plays a casual role in this resistance, short interfering RNA (siRNA) was used to knockdown ErbB3 in the resistant cell line AsPC1. Whereas individual treatments with siRNA to ErbB3 or 17DMAG had no effect on radiosensitivity, the combination, which reduced both ErbB2 and ErbB3, resulted in a significant enhancement in AsPC1 radiosensitivity. In contrast to siRNA to ErbB3 or 17DMAG treatments only, AsPC1 cell exposure to the combination also resulted in a decrease in ErbB1 kinase activity. These results indicate that ErbB3 expression predicts for tumor cell susceptibility to and suggests that the loss of ErbB1 signaling activity is necessary for 17DMAG-induced radiosensitization. However, for cell lines sensitized by 17DMAG, treatment with siRNA to ErbB2, which reduced ErbB1 activity, had no effect on radiosensitivity. These results suggest that, whereas the loss of ErbB1 signaling may be necessary for 17DMAG-induced radiosensitization, it is not sufficient.

Benzoquinones↗

PubMed2004

Is there a link between telomere maintenance and radiosensitivity?

Several recent studies point to the possibility that telomere maintenance may constitute a potential genetic marker of radiosensitivity. For example, the human diseases ataxia telangiectasia and Nijmegen breakage syndrome, which are characterized by clinical radiosensitivity, show alterations in telomere maintenance. In addition, Fanconi's anemia patients, who are characterized by mild cellular radiosensitivity and in some cases marked clinical radiosensitivity, have altered telomere maintenance. Similarly, a correlation between telomere maintenance and cellular radiosensitivity was reported in a group of breast cancer patients. Another study demonstrated that radiosensitivity may be more pronounced in human fibroblasts with short telomeres than in their counterparts with long telomeres. Several mouse models including mice deficient in Ku, DNA-PKcs (Prkdc), Parp and Atm, all of which are radiosensitive in vivo, show clear telomere alterations. The link between telomere maintenance and radiosensitivity is also apparent in mice genetically engineered to have dysfunctional telomeres. Finally, studies using non-mammalian model systems such as C. elegans and yeast point to the link between radiosensitivity and telomere maintenance. These results warrant further investigation to identify the extent to which these two phenotypes, namely radiosensitivity and telomere maintenance, are linked.

Animals↗

PubMed2000

The role of cell cycle progression in radiosensitization by 2',2'-difluoro-2'-deoxycytidine.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) has been shown to be a potent radiosensitizer in tumor cells both in vitro and in vivo. We evaluated the ability of dFdCyd to enhance the radiosensitivity of two human glioblastoma cell lines. The results demonstrated that U251 cells were more sensitive to the cytotoxicity of dFdCyd, and that dFdCyd was able to radiosensitize these cells. In contrast, D54 cells were more resistant to the cytotoxic effect of dFdCyd, and no radiosensitization occurred at any concentration of dFdCyd tested. Because radiosensitization by dFdCyd has been correlated with its ability to deplete dATP pools through inhibition of ribonucleotide reductase by dFdCyd diphosphate, we evaluated the metabolism of dFdCyd in both cell lines. At equitoxic concentrations of dFdCyd, both cell lines accumulated similar levels of the cytotoxic metabolite, dFdCyd triphosphate, as well as similar levels of dFdCyd monophosphate in DNA. In U251 cells, radiosensitizing concentrations of dFdCyd (10 or 25 nM; IC10 or IC50) depleted dATP by approximately 80% within 4 h. In contrast, 80 nM (IC50) was unable to deplete dATP by >30% within 4 h in D54 cells. Higher concentrations of dFdCyd or hydroxyurea, an inhibitor of ribonucleotide reductase that depleted dATP >90%, also did not produce radiosensitization in D54 cells. D54 cells were not resistant to radiosensitization because bromodeoxyuridine was able to induce radiosensitization. Because D54 cells express wild-type p53, whereas U251 cells express a mutant p53, the effect of dFdCyd and ionizing radiation on cell cycle progression was evaluated. Radiation alone produced a G1 block in D54 cells and a transient G2-M block in U251 cells. After a 24 h incubation with dFdCyd alone or in combination with ionizing radiation, U251 cells readily accumulated in S-phase, which remained elevated for at least 72 h, consistent with previous results in other mutant p53 cell lines. In addition, radiation enhanced the ability of dFdCyd to induce S-phase-specific cell death in U251 cells. In contrast, D54 cells showed a G1 block after dFdCyd and radiation exposure, with fewer cells in S-phase for at least 48 h after drug washout/irradiation. Furthermore, treatment with dFdCyd and/or radiation did not increase the amount of S-phase-specific cell death in D54 cells compared with control cells. These results suggest that the G1 block in D54 cells resulting from wild-type p53 induction prevented radiosensitization by dFdCyd.

Adenosine Triphosphate↗

PubMed1990

Iododeoxyuridine radiosensitization by low- and high-energy photons for brachytherapy dose rates.

The dependence of iododeoxyuridine (IUdR) radiosensitization on photon energy and dose rate in the range of interest to brachytherapy was investigated by irradiating Chinese hamster cells in vitro under aerobic conditions. The radiosensitization produced by 10(-5) and 10(-4) M IUdR for 28-keV (average) photons from 125I, 60-keV photons from 241Am, and 830-keV (average) photons from 226Ra was measured at nominal dose rates of 0.17, 0.30, 0.57, and 0.73 Gy/h. Radiosensitization factors for IUdR were essentially independent of dose rate from 0.30 to 0.73 Gy/h for all cases except for 10(-4) M IUdR plus 241Am, in which case the radiosensitization factor increased from 2.5 +/- 0.2 to 3.0 +/- 0.1. In all cases, the radiosensitization factor decreased significantly as the dose rate was lowered from 0.30 to 0.17 Gy/h e.g., the radiosensitization factor for 241Am dropped to 1.9 +/- 0.2 at a dose rate of 0.17 Gy/h. Moreover, at 0.17 Gy/h the radiosensitization factors were essentially the same for all three photon energies. As the dose rate increased from 0.17 to 0.73 Gy/h, the difference between the radiosensitization factors for the three photon energies became larger; radiosensitization factors for 241Am were higher than those for 226Ra and 125I. In temporary brachytherapy the tumor is irradiated at the higher dose rate of about 0.50-0.70 Gy/h, while the normal tissues are irradiated at lower dose rates; the dose rate dependence of the radiosensitization factor may therefore lead to an improvement in the therapeutic ratio for brachytherapy in combination with IUdR.

Americium↗

PubMed2003

The non-homologous end-joining pathway is not involved in the radiosensitization of mammalian cells by heat shock.

A synergistic increase in cell killing is observed when a heat-shock is administered prior to, during, or immediately after exposure to ionizing radiation (IR). This phenomenon, known as heat-radiosensitization, is believed to be mediated by inhibition of repair of radiation-induced double strand breaks (DSB) when cells are exposed to temperatures above 42 degrees C. However, the mechanism by which heat inhibits DSB repair is unclear. The bulk of radiation-induced DSBs are repaired via the non-homologous end-joining pathway (NHEJ). Several reports indicate that the Ku70 and Ku80 subunits of the mammalian DNA-dependent protein kinase (DNA-PK), a complex involved in NHEJ, appear to be susceptible to a heat-induced loss of DNA-binding activity, with Ku80 representing the heat-sensitive component. Since the heat-induced loss and subsequent recovery of Ku-DNA binding activity correlates well with heat-radiosensitization, a role for Ku80 and NHEJ in heat-radiosensitization has been proposed. However, direct evidence implicating Ku80 (and NHEJ) in heat-radiosensitization has been indeterminate. In this study, we demonstrate that equitoxic heat treatments at 42.5-45.5 degrees C induce a similar amount of aggregation of Ku80 in human U-1 melanoma cells. These data suggest that the time-temperature-dependent relationship between heat lethality and Ku80 aggregation are similar. However, the aggregation/disaggregation of Ku80 and its transient or permanent inactivation is unrelated to heat-radiosensitization. When survival curves were obtained for irradiated or irradiated and heated Ku80(-/-) mouse embryo fibroblasts (MEFs) and compared with survival curves obtained for wild-type (WT) cells, we found that heat-radiosensitization was not reduced in the Ku80(-/-) cells, but actually increased. Thus, our findings indicate that Ku80 is not essential for heat-radiosensitization. Non-involvement of Ku-dependent or Ku-independent NHEJ pathways in heat-radiosensitization was confirmed by comparing clonogenic survival between DNA ligase IV-defective and WT human cells. Our data therefore implicate homologous recombination in inhibition of repair of radiation-induced DSBs and as a target for heat-radiosensitization.

Animals↗

PubMed1998

Radiosensitivity of dermal and tumor fibroblasts derived from the same patient.

The in vitro radiosensitivity of dermal fibroblasts has been found to vary between individuals, and a number of studies have also shown that this parameter correlates with radiation-induced late injuries in clinical radiotherapy. In addition, certain genetic disorders are known to effect radiosensitivity, e.g. normal tissues of patients homozygous or heterozygous for the ataxia teleangiectasia gene show unusual sensitivity to radiation both in vivo and in vitro. Thus, it has been assumed that there is a genetically determined component resulting in a certain intrinsic cellular radiation response in an individual. To study this possible relationship between different cells of a specific patient, we established eight pairs of dermal and tumor fibroblast cultures. The donor patients had either adenocarcinoma of the uterus or squamous cell carcinoma (SCC) of the head and neck. The radiosensitivity of these strains was determined by a 96-well plate clonogenic assay, previously used by us for radiosensitivity testing of cancer cells. From a paired comparison, the values for the cell fraction surviving 2.0 Gy (SF2), of both fibroblast strains, were found to be on the same level in five out of eight cases. In patient 6, the SF2 of tumor fibroblasts was significantly higher than that of dermal fibroblasts (P=0.0014). In two additional cases the tendency was the same, but not statistically significant. As groups, the two types of fibroblasts did not differ from each other, mean SF2 values of 0.24+/-0.07 and 0.21+/-0.05, respectively. The SF2 of tumor fibroblasts from SCC patients proved to be significantly higher than that of the adenocarcinoma patients (P=0.030). These preliminary results indicate that the in vitro radiosensitivity of tumor fibroblasts correlates with normal cell sensitivity in many cases, but not in all. The radiosensitivity of tumor fibroblasts also seems to follow the level of in vitro radiosensitivity determined for the corresponding histological type of tumor cells. Further studies are needed to determine more closely the relationship between the radiosensitivities of tumor cells and tumor fibroblasts, thus evaluating the possibility of testing radiosensitivity from tumor fibroblasts in order to estimate tumor response.

Adenocarcinoma↗

PubMed2003

Individual radiosensitivity measured with lymphocytes may be used to predict the risk of fibrosis after radiotherapy for breast cancer.

BACKGROUND AND PURPOSE: To analyse the relationship of individual cellular radiosensitivity and fibrosis after breast conserving therapy. A new model was used describing the percentage of patients developing fibrosis per year and per patient at risk. PATIENTS AND METHODS: In a retrospective study, 86 patients were included, who had undergone breast conserving surgery and irradiation of the breast with a median dose of 55 Gy (54-55 Gy) given at 2.5 Gy/fraction (n=57) or 2 Gy/fraction (n=29). Median age was 62 years (range 44-86) and median follow-up was 7.5 years (range 5-17). Patients were examined for fibrosis according to the LENT/SOMA score. For analysis, fibrosis was classified as grade 0 and grade 1 (G0-1) or present grade 2 and grade 3 (G2-3). The time to complete development of fibrosis was determined by analysis of yearly mammograms. Individual cellular radiosensitivity was determined by scoring lethal chromosomal aberrations in in vitro irradiated (6 Gy) lymphocytes using metaphase technique. Patients with low/intermediate cellular radiosensitivity were compared with patients with high cellular radiosensitivity using actuarial methods. RESULTS: Ten patients developed fibrosis at 1-8 years after radiotherapy. Individual cellular radiosensitivity was described by normal distribution of lethal chromosomal aberrations, the average was 5.47 lethal aberrations per cell (standard deviation (SD) 0.71). Cellular radiosensitivity was defined as low/intermediate (< or =6.18 lethal aberrations) in 73 patients and high (>6.18 lethal aberrations; mean+SD) in 13 patients. In both groups, the actuarial rate of fibrosis-free patients decreased exponentially with time after radiotherapy. Patients with high cellular radiosensitivity showed a 2.3-fold higher annual rate for fibrosis than patients with intermediate and low radiosensitivity (3.6 versus 1.6% per year). CONCLUSIONS: In breast cancer patients, high individual cellular radiosensitivity as determined by the number of lethal chromosome aberrations in in vitro irradiated lymphocytes might be associated with an enhanced annual rate of fibrosis.

Adult↗

PubMed2003

Cell cycle effect of gemcitabine and its role in the radiosensitizing mechanism in vitro.

PURPOSE: The mechanism of radiosensitization by gemcitabine is still unclear. It has been hypothesized that the accumulation of cells in early S phase may play a role in enhancing radiosensitivity. METHODS AND MATERIALS: The schedule dependency of the radiosensitizing effect was studied in ECV304, human bladder cancer cells, and H292, human lung cancer cells, by varying the incubation time and time interval between gemcitabine and radiation treatment. To determine the role of cell cycle perturbations in the radiosensitization, the influence of gemcitabine on the cell cycle at the moment of radiation was investigated by flow cytometry. RESULTS: The radiosensitizing effect increased with a longer incubation period: Dose enhancement factors varied from 1.30 to 2.82 in ECV304 and from 1.04 to 1.78 in H292 after treatment during 8-32 h, respectively. Radiosensitization decreased with an increasing interval: Dose enhancement factors varied from 2.26 to 1.49 in ECV304 and from 1.45 to 1.11 in H292 after an interval 0-24 h, respectively. Cells were blocked in the early S phase of the cell cycle by gemcitabine. The highest percentage S-phase cells was observed after treatment with the schedules that resulted in the highest radiosensitizing effect. CONCLUSIONS: We observed a clear schedule-dependent radiosensitization by gemcitabine. Our findings demonstrated a correlation between gemcitabine-induced early S-phase block and the radiosensitizing effect.

Cell Cycle↗

PubMed2004

Radiosensitization of tumour cells by cantharidin and some analogues.

PURPOSE: Mammalian cells at mitosis contain chromatin in compacted form and are hypersensitive to ionizing radiation. Previous research had shown some chemicals that induce chromatin compaction within interphase cells act as radiosensitizers. Of these agents, cantharidin (LS-1), which is an inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), showed good radiosensitizing activity at non-toxic doses. Cantharidin and 13 additional structural analogues (LS-2-14) were tested for their radiosensitizing activity on tumour cells in vitro. MATERIALS AND METHODS: Twelve of the 14 cantharidin analogues were synthesized in the authors' laboratory. Various concentrations of the drugs were screened for toxicity and radiosensitizing effectiveness with asynchronous DU-145 (human prostate carcinoma) cells. More detailed radiobiological studies of the more potent agents were performed with HT-29 (human colon carcinoma) cells since they could be readily synchronized. The radiosensitization of G1 phase HT-29 cells was measured after a 2-h exposure to the more potent drugs and reductions of the surviving fraction after an acute dose of 2 Gy (SF2Gy) served to estimate their relative effectiveness. The increase in phosphorylation of histone 1 (H1) and histone 3 (H3) induced by these drug exposures was measured by Western blotting of protein extracts. Drug-induced change in chromatin morphology was visualized by electron microscopy, and the alkaline comet assay (which measures DNA single-strand breaks) was employed to measure the radiation sensitivity of cellular chromatin in the drug-treated cells. RESULTS: Of the 14 cantharidin analogues tested, LS-1, LS-2 and LS-5 at concentrations of 3-20 microM showed little or no toxicity, produced elevated levels of H1 and H3 phosphorylation, and effected significant radiosensitization at low radiation dose. The chromatin in tumour cells treated with LS-5 became visibly compacted and its DNA was about 1.6 times more sensitive to radiation-induced strand breakage relative to that of control cells. CONCLUSIONS: The results confirm the authors' earlier studies that showed an increase in tumour cell intrinsic radiosensitivity by exposure to agents that promote chromatin compaction. LS-5 was identified as the optimal radiosensitizing agent of this class of compounds. Radiosensitization was correlated with chromatin compaction and elevated phosphorylation of H1 and H3. The DNA in drug-treated cells exhibited an enhanced sensitivity to radiation-induced single-strand breakage.

Cantharidin↗

PubMed1997

Oral platinum analogue JM216, a radiosensitizer in oxic murine cells.

This study was designed to compare radiosensitization by the oral platinum compound JM216 with cisplatin. RIF1 mouse tumour cells were treated at various doses and at various exposure times with JM216 and irradiated 15 min before the end of drug exposure. The fraction of cells surviving treatment was assessed by colony formation. Results were compared with those for equivalent treatments with cisplatin. JM216 alone showed exponential killing of RIF1 cells, being approximately three times less efficient than cisplatin on a molar basis. For radiosensitization studies, drug doses used gave approximately 50 or 90% cell killing alone. No radiosensitization was seen after 2-h drug exposures, but significant radiosensitization occurred after 1- and 0.5-h exposures (shorter times required proportionally higher drug doses, giving equivalent drug kill). The enhancement ratio and time dependence were similar for the two platinum compounds, reaching 1.5 at the highest concentrations tested. Drug DNA adduct formation was assessed using immunocytochemistry with the NKI-A59 antiserum raised to cisplatin-DNA adducts. The antiserum was shown to recognize JM216-DNA adducts in a dose-dependent manner and maximum nuclear staining was found to be correlated with cell kill for both drugs. However, neither the level of staining at the time of irradiation nor at the time of maximum adducts correlated with radiosensitization, indicating that the number of DNA adducts did not determine radiosensitization. Intracellular glutathione levels were shown to be decreased by the drug, but only by approximately 50%, implying that this was not the cause of the increased radiosensitivity. In summary, JM216 was shown capable of radiosensitizing a platinum-sensitive tumour line to an extent similar to cisplatin. Radiosensitization was exposure-time and drug-concentration dependent, but was not dependent on DNA adduct levels nor glutathione depletion. In contrast, cell kill after drug alone was well correlated with adduct levels. These data suggest that JM216 could replace cisplatin in combined radiotherapy-chemotherapy studies, and also indicate that the NKI-A59 antibody could be used to monitor exposure levels in vivo.

Animals↗

PubMed2006

The interaction between two radiosensitizers: 5-iododeoxyuridine and caffeine.

5-Iododeoxyuridine (IUdR) and caffeine are recognized as potential radiosensitizers with different mechanisms of interaction with ionizing radiation (IR). To assess the interaction of these two types of radiosensitizers, we compared treatment responses to these drugs alone and in combination with IR in two p53-proficient and p53-deficient pairs of human colon cancer cell lines (HCT116 versus HCT116 p53-/- and RKO versus RKO E6). Based on clonogenic survival, the three single agents (IR, IUdR, and caffeine) as well as IUdR or caffeine combined with IR are less or equally effective in p53-deficient human tumor cells compared with p53-proficient tumor cells. However, using both radiosensitizers, a significantly greater radiosensitization was found in p53-deficient human tumor cells. To better understand the interaction of these two radiosensitizers, additional studies on DNA repair and cell cycle regulation were done. We found that caffeine enhanced IUdR-DNA incorporation and IUdR-mediated radiosensitization by partially inhibiting repair (removal) of IUdR in DNA. The repair of IR-induced DNA double-strand breaks was also inhibited by caffeine. However, these effects of caffeine on IUdR-mediated radiosensitization were not found in p53-proficient cells. Cell cycle analyses also showed a greater abrogation of IR-induced S- and G2-phase arrests by caffeine in p53-deficient cells, particularly when combined with IUdR. Collectively, these data provide the mechanistic bases for combining these two radiosensitizers to enhance tumor cytotoxicity. This differential dual mode of radiosensitization by combining IUdR and caffeine-like drugs (e.g., UCN-01) in p53-deficient human tumors may lead to a greater therapeutic gain.

Base Pair Mismatch↗

PubMed1992

5-Nitro-4-(N,N-dimethylaminopropylamino)quinoline (5-nitraquine), a new DNA-affinic hypoxic cell radiosensitizer and bioreductive agent: comparison with nitracrine.

Targeting of electron-affinic radiosensitizers to DNA via noncovalent binding (e.g., intercalation) may offer the potential for increasing sensitizing efficiency. However, it has been suggested that high-affinity DNA binding may compromise sensitization by restricting the mobility of sensitizers along the DNA, and by decreasing rates of extravascular diffusion in tumors. The weak DNA intercalator nitracrine (1-NC) is a more efficient radiosensitizer than related nitroacridines with higher DNA-binding affinities (Roberts et al., Radiat. Res. 123, 153-164, 1990). The present study investigates whether electron-affinic agents of even lower DNA-binding affinity may be superior to nitroacridines. The quinoline analog of 1-NC, 5-nitraquine (5-NO), was shown to have an intrinsic association constant for calf thymus DNA in 20 mM phosphate buffer which was 12-fold lower than that of 1-NC. 5-Nitraquine was not accumulated as efficiently as 1-NC by AA8 cells, but, despite a similar one-electron reduction potential, was 2- to 3-fold more potent than 1-NC as a hypoxia-selective radiosensitizer in vitro when compared on the basis of average intracellular concentration. Thus the radiosensitizing potency of 5-NQ appears not to be compromised by its low DNA-binding affinity. The cytotoxic mechanisms of 5-NQ and 1-NC appear to be similar (hypoxia-selective formation of DNA monoadducts), but 5-NQ is 1200-fold less potent than 1-NC as a cytotoxin. Despite this advantage, 5-NQ was not active in vivo as a radiosensitizer in SCCVII tumors. This lack of activity appears to be due to its relatively high toxicity in vivo (intraperitoneal LD50 of 105 mumol kg-1 in C3H/HeN mice), high one-electron reduction potential (-286 mV), and rapid metabolism to the corresponding amine in mice. The in vitro therapeutic index (hypoxic radiosensitizing potency/aerobic cytotoxic potency) of this weak DNA binder was lower than that of the non-DNA targeted radiosensitizer misonidazole, suggesting that DNA targeting enhances cytotoxicity more than radiosensitization. Development of useful DNA-targeted radiosensitizers may require the exploitation of DNA binding modes different from those of the nitroacridines and nitroquinolines.

Aminoquinolines↗

PubMed2003

Enhanced cell killing induced by the combination of radiation and the heat shock protein 90 inhibitor 17-allylamino-17- demethoxygeldanamycin: a multitarget approach to radiosensitization.

PURPOSE: Current strategies for tumor cell radiosensitization focus on a target-based approach. However, the radioresponse of a tumor cell is influenced by a wide variety of signaling molecules existing in a number of different survival pathways. Therefore, in an attempt to increase the probability and/or degree of radiosensitization, we have begun to investigate a multitarget approach using the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG). EXPERIMENTAL DESIGN: The effect of 17AAG on the levels of three proteins (Raf-1, ErbB2, and Akt) previously implicated in the regulation of radiosensitivity was determined in four human tumor cell lines. Tumor cell survival after exposure to corresponding concentrations of 17AAG combined with clinically relevant doses of X-rays was then evaluated using a clonogenic assay. The radiosensitivity of a nonimmortalized, normal fibroblast cell line was also determined after exposure to 17AAG. RESULTS: Exposure to nanomolar concentrations of 17AAG reduced the levels of the three radiosensitivity-associated proteins in a cell type manner. Using corresponding concentrations, 17AAG enhanced the radiosensitivity of each of the tumor cell lines with enhancement factors ranging from 1.3 to 1.7. The enhancement appeared to be related to the number of radioresponse-regulatory proteins affected. In contrast to the tumor cell lines, 17AAG had no effect on the radiosensitivity of a normal, nonimmortalized human fibroblast cell line. CONCLUSIONS: These data suggest that heat shock protein 90 may be an appropriate target for selectively enhancing the radiosensitivity of tumor cells over normal cells. Furthermore, they illustrate the potential of a multitarget approach to radiosensitization.

Benzoquinones↗

PubMed2003

Enhanced radiosensitization with gemcitabine in mismatch repair-deficient HCT116 cells.

Gemcitabine [2',2'-difluoro-2'-deoxycytidine (dFdCyd)] is a potent ionizing radiation sensitizer in solid tumor cells in vitro and in vivo. Previously, we have demonstrated (Shewach et al., Cancer Res., 54: 3218-3223, 1994) a strong correlation between depletion of dATP (caused by dFdCyd diphosphate-mediated inhibition of ribonucleotide reductase) and radiosensitization. In addition, we and others (Latz et al., Int. J. Radiat. Oncol. Biol. Phys., 41: 875-882, 1998; Ostruszka and Shewach, Cancer Res., 60: 6080-6088, 2000) have shown that the accumulation of cells in S phase prior to irradiation is also important for radiosensitization with dFdCyd. This led us to hypothesize that the incorporation of incorrect nucleotides because of the dATP pool imbalance was important for radiosensitization with dFdCyd, and, therefore, cells deficient in mismatch repair (MMR) would exhibit greater radiosensitization. We tested this hypothesis by evaluating the ability of HCT116 colon carcinoma cell lines, which differ in MMR proficiency, to be radiosensitized by dFdCyd. The MMR-proficient cell line (HCT116 + ch3) was more sensitive to dFdCyd alone than were the MMR-deficient cell lines (HCT116, HCT116 + ch2, and HCT116 p53(-/-)). Interestingly, the MMR-proficient cells could not be radiosensitized at concentrations of dFdCyd IC(96)) enhanced cell killing with radiation. In contrast, the MMR-deficient cells were radiosensitized at concentrations of dFdCyd or=80% decrease in dATP within 4 h after drug addition, and this low dATP level was maintained for another 12-20 h. Although the IC(50) of dFdCyd was unable to sustain a >80% decrease in the dATP level in the MMR-proficient cells, the IC(90) did achieve this level of dATP depletion; however, it was unable to radiosensitize the MMR-proficient cells. Similar results were obtained with HCT116 cells, in which the MMR deficiency was corrected by transfection with a vector containing the hMLH1 cDNA. In addition, the deletion of p53 did not increase radiation enhancement ratios. These results demonstrate that MMR deficiency promotes radiosensitization with dFdCyd. We suggest that dATP depletion produces errors of replication in MMR-deficient cells, which, if left unrepaired, enhances cell death by ionizing radiation.

Base Pair Mismatch↗

PubMed1998

Comparison between pulsed-field gel electrophoresis and the comet assay as predictive assays for radiosensitivity in fibroblasts.

The radiosensitivity of skin fibroblasts derived from patients as measured in vitro by a clonogenic survival assay appears to correlate with the risk of developing severe late reactions to radiation. Unfortunately, these assays are clinically impractical as a predictive test for radiosensitivity. The purpose of this study was to assess the utility of two possible surrogate assays for radiosensitivity, pulsed-field gel electrophoresis (PFGE) and single-cell gel electrophoresis (comet assay), both of which can be used to measure DNA double-strand breaks. Twenty-three nontransformed human fibroblast cell lines exhibiting a range of radiosensitivities were studied with both of these assays. The results were correlated with measurements of radiosensitivity obtained as part of a larger study examining the correlation between cellular radiosensitivity and clinical response. [2-(14)C]Thymidine-labeled confluent cultures were irradiated at 1.0 Gy/min with doses of 0 to 150 Gy. After allowing 4 h for repair at 37 degrees C, cells were trypsinized and aliquots were used for preparing slides for the comet assay. After neutral lysis and electrophoresis, the slides were stained with ethidium bromide and 50 comet moments were measured for each dose. The remainder of the cells were formed into agarose plugs and, after neutral lysis, were subjected to PFGE. The fraction of activity released (FAR) from the well was measured by scintillation counting of appropriate segments of each gel lane. Cellular radiosensitivity was measured with a standard clonogenic assay at a low dose rate of 1.2 cGy/min, and the dose that resulted in a surviving fraction of 0.01 (D0.01) was calculated. The slope of the plot of comet moment as a function of dose for each cell line did not correlate with D0.01 (R = 0.36, P > 0.1). In contrast, the slope of the FAR as a function of dose had a weak inverse correlation with D0.01 (R = 0.43 and P = 0.05) such that the more radiosensitive cell lines exhibited a steeper dose response for FAR. Although the correlation between the slope of the dose response for FAR and D0.01 was weak, refinement of the PFGE technique may provide a potentially useful predictive assay for radiosensitivity.

Breast↗

PubMed2002

The influence of chromatin structure on initial DNA damage and radiosensitivity in CHO-K1 and xrs1 cells at low doses of irradiation 1-10 Gy.

Mitotic compaction of chromatin was generated by treatment of cells with nocodazole. Alternatively, chromatin structure was altered by incubating cells in 500 mM NaCl. The irradiation response in the dose range of 1-10 Gy was measured by colony assay and by a modified fluorometric analysis of DNA unwinding (FADU) assay which measures the amount of undamaged DNA by EtBr fluorescence. Cell survival curves of irradiated CHO-K1 cells showed that treatment with nocodazole increases radiosensitivity as indicated by a decrease of the mean inactivation dose (D) from 4.446 to 4.376. Nocodazole treatment increased the initial radiation-induced DNA damage detected by the FADU assay from 7% to 13%. In repair-defective xrs1 cells, the same conditions increased the radiosensitivity from 1.209 to 0.7836 and the initial DNA damage from 43% to 57%. Alterations to chromatin structure by hypertonic medium increased radiosensitivity in CHO-K1 cells from of 4.446 to 3.092 and the initial DNA damage from 7% to 15%. In xrs1 cells these conditions caused radiosensitivity to decrease from 1.209 to 1.609 and the initial DNA damage to decrease from 43% to 36%. Disruption of chromatin structure by hypertonic treatment was found to be time-dependent. A threefold increase of exposure time to hypertonic medium from 40 to 120 min increased the initial DNA damage in CHO-K1 cells from 7% to 18% but decreased initial DNA damage in xrs1 cells from 43% to 21%. Perturbation of chromatin structure with hypertonic treatment has been shown to increase the radiosensitivity and the initial DNA damage in repair-competent CHO-K1 cells and decrease the radiosensitivity and DNA damage in repair-defective xrs1 cells. Hypertonic treatment thus abolishes differences in chromatin structure between cell lines and differences in initial DNA damage. Radiosensitivity and initial DNA damage are correlated ( r(2)=0.92; p=0.0026) and this correlation also holds when chromatin compaction is altered. The experiments demonstrate that initial DNA damage and chromatin structure are major determinants of radiosensitivity.

Animals↗

PubMed1992

Heterogeneity of 5-fluorouracil radiosensitivity modulation in cultured mammalian cell lines.

There is ample evidence that 5-fluorouracil (5-FU) improves both local control and survival of a variety of gastrointestinal tumors when added to radiotherapy. However, the modulation of radiosensitivity by 5-FU is incompletely understood and some reports are apparently contradictory. Therefore, we have reevaluated the modulation of radiosensitivity by 5-FU in a variety of mammalian cells. HT-29 and WiDr (human colon adenocarcinoma), DU-145 (human prostate adenocarcinoma), V-79 (Chinese hamster lung fibroblast), and HeLa cell lines were maintained in exponential growth as monolayer cultures. Cell survival following treatment with drug and/or radiation was determined by colony formation assay. Radiation was delivered either alone; midway through a 1 hr exposure to 7-25 micrograms/ml 5-FU (pulse); or following initiation of 0.1-1.5 micrograms/ml 5-FU present throughout the entire incubation for assay of colony forming ability (continuous exposure). These 5-FU levels were selected to approximate those achieved in vivo in humans. The results indicate that mammalian cell lines may vary substantially insofar as modulation of their radiosensitivity by 5-FU is concerned. Radiosensitization, defined by reduction in D0, was observed for continuous exposure only in V-79, WiDr, and HT-29 cell lines, was observed for both pulse exposure and continuous exposure in DU-145, and was not present in HeLa cells. Radioenhancement, defined by reduction in n, was observed in V-79, WiDr, and HT-29 but not in the other cell lines. This effect, characterized by reduction in the shoulder portion of the curve, is naturally accompanied by a decrease of Dq. This indicates that mammalian cell lines may have different responses to radiosensitivity modulation by 5-FU. Though the cell lines may exhibit radiosensitivity by either alterations in the slope or shoulder of the cell survival curve, the mechanisms responsible for both the heterogeneity as well as the radiosensitization itself are completely unknown at this time. Insight into the mechanisms for both the heterogeneity and the radiosensitization will be important areas for further investigation.

Animals↗

PubMed1993

Prospective comparison of in vitro normal cell radiosensitivity and normal tissue reactions in radiotherapy patients.

PURPOSE: This pilot study was undertaken to assess the relationship between in vitro radiosensitivity of different normal cell types and the type and severity of normal tissue reactions in individual patients after radiotherapy. METHODS AND MATERIALS: Twenty-one patients with head and neck cancer were studied prospectively; four with head and neck and two with breast cancer were studied retrospectively. The retrospective cases were chosen because they exhibited unusual (severe or minimal) normal tissue reactions after radiotherapy. Small skin biopsies and blood samples were obtained and used to generate in vitro fibroblast and lymphocyte cultures, respectively. Clonogenic assays were used to measure in vitro fibroblast and lymphocyte radiosensitivity after high- and low-dose rate irradiation. Head and neck patients were treated by conventional, hyperfractionated, or concomitant boost regimens, which have been found to yield an equal probability of late normal tissue reactions. The highest dose received by each normal tissue in the target volume was estimated using computed tomography treatment plans. The median patient follow-up time was 19 months (range: 13-25). RESULTS: The distributions of in vitro radiosensitivity parameters and the grade of tissue reaction scores in the patients showed a broad range between individuals. When in vitro parameters were compared to the acute and late tissue reactions, the radiosensitivity of fibroblasts, measured as surviving fraction at 2 Gy after high-dose rate irradiation, showed a highly significant correlation with the maximum grade of late effects (p < 0.0001 for the whole group and p = 0.0013 for the group of patients studied prospectively). No significant correlation was found between fibroblast radiosensitivity and maximum grade of acute effects or between lymphocyte radiosensitivity and either acute or late effects. CONCLUSION: We conclude that individuals vary in normal cell radiosensitivity, and that in vitro measurements of fibroblast radiosensitivity may predict the magnitude of late normal tissue reactions after radiotherapy. These preliminary results, however, need to be validated in a larger group of patients.

Biopsy↗