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PubMed2006

[Chloroquine increased radiosensitivity of radioresistant MDA-MB 231 cells and its molecular mechanism].

OBJECTIVE: To study the genes associated with CQ (Chloroquine) increased radiosensitivity in radioresistant MDA-MB 231 cells. METHODS: The survival curve of MDA-MB 231 breast cancer cells was establish in the presence or absence of CQ after irradiation by colongenic assay. The relationship between certain protein expressions (MLH1, ERCC1, PARP, RAD51, XRCC4) and CQ increased radiosensitivity in radioresistant MDA-MB 231 cells was examined by Western blot method. The genes which are associated with CQ increased radiosensitivity were studied by microarray analysis. RESULTS: CQ increased deaths of parental MDA-MB 231 cells, radiosensitive MDA-MB 231 cells and radioresistant MDA-MB 231 cells were compared with those of the CQ-untreated control cells after irradiation, and the Radiation Potentiation Factor were 1.21-1.23. The expression of ERCC1 in the presence and absence of CQ in the radioresistant cells was significantly higher than that of sham-irradiated control cells by Western blot method. After irradiation, RAD51 level was increased up to 3-hour radiation and then gradually reduced. The expression of MLH1 was lower in 24 hours than that of other time points after irradiation. There were no differences on the expressions of MLH1, ERCC1, RAD51 in cells with and without CQ, which suggested that these genes weren't correlated with CQ increased radiosensitivity. XRCC4 and PARP levels were not changed by irradiation. Microarray analysis showed that TNP3, FRIH, CA21, ADTA genes might be related to CQ increased radiosensitivity in radioresistant cells. CONCLUSION: CQ can increase radiosensitivity in radioresistant MDA-MB 231 breast cancer cells. It is not related to MLH1, ERCC1, PARP, RAD51, XRCC4 genes. TNP3 may play an important role in CQ increase radiosensitivity in radioresistant MDA-MB 231 breast cancer cells.

Adaptor Proteins, Signal Transducing↗

PubMed1988

Oxygen- and temperature-dependent cytotoxic and radiosensitizing effects of cis-dichlorodiammineplatinum(II) on human NHIK 3025 cells in vitro.

The radiosensitizing effect of the chemotherapeutic drug cis-dichlorodiammineplatinum(II) (cis-DDP) was tested on human NHIK 3025 cells cultivated in vitro. cis-DDP was found to exert a radiomodifying effect under hypoxic but not under aerobic conditions. These results confirm that cis-DDP may act as a radiosensitizer of hypoxic cells; however, the radiosensitizing effect was seen only at concentrations of cis-DDP having a considerable cytotoxic activity, and for practical reasons concerning survival level the highest drug concentration that was investigated was 15 microM at 37 degrees C. The radiosensitizing effect was of a dose-modifying type and with a dose-modifying factor (DMF) of 1.2 at 15 microM in hypoxic cells. The radiosensitizing as well as the cytotoxic effect of cis-DDP was found to be strongly temperature dependent. Isoeffect doses of cis-DDP was reduced with a factor of 3 at 22 as compared to 37 degrees C. We also found that hypoxic cells were less sensitive to cis-DDP than cells treated in the presence of oxygen. To test the correlation between cytotoxicity and radiosensitization on the one hand and cellular uptake of cis-DDP on the other, cell-associated Pt was measured by atomic absorption spectroscopy. From these studies the cytotoxicity of cis-DDP at 22 and 37 degrees C under aerobic conditions was found to be the same as long as the amount of cell-associated Pt (i.e., the cellular uptake) was the same. However, whether the cells were treated under hypoxic or aerobic conditions, the cellular uptake of Pt was the same. While the radiosensitizing effect was present at 37 and at 40 degrees C, no such effect could be found at 22 degrees C. Since the cytotoxicity of cis-DDP as well as the drug uptake was reduced about three times at 22 as compared to 37 degrees C, we increased the concentration threefold, to 50 microM at 22 degrees C. Still no radiosensitizing effect was found at this temperature.

Antineoplastic Agents↗

PubMed1987

Some complexes of cobalt(III) and iron(III) are radiosensitizers of hypoxic EMT6 cells.

The radiosensitizing potential in hypoxic EMT6 cells of several complexes of Co(III) and Fe(III) has been examined. The cytotoxicity of each of the agents toward oxygenated and hypoxic EMT6 cells was tested over the concentration range of 1 to 500 micron for 1-h drug exposure. There was no statistically significant difference between the cytotoxicity of these complexes toward oxygenated and hypoxic cells. Based on these findings, 100 micron was selected as the drug concentration for the initial assessment of radiosensitizing potential. The radiation survival of EMT6 cells in the presence of 100 microM drug for a series of Co(III) complexes in which the number of nitro ligands was varied showed that the hexanitro and the triamine-trinitro complexes are very effective radiosensitizers. The trans-tetrammine dinitro complex was a more effective radiosensitizer than the corresponding cis-dinitro complex. The diethylenetriamine and 1,10-phenanthroline complexes were very effective radiosensitizers, producing dose-modifying factors of 2.4. The trans-tetrammine dichloro complex was moderately effective, giving a dose-modifying factor of 1.9. On the other hand, the hexammine and triammine tricyano complexes and the trans-dinitro complex with negatively charged acetylacetonate ligands were ineffective as radiosensitizers in this system. Finally, three complexes with cyclopentadienyl ligands were examined. The ferricenium salt itself was a moderately effective radiosensitizer, giving a dose-modifying factor of 2.0. However, both the dimethylferricenium salt and the analogous cobalt complex were ineffective. The FSaIIC fibrosarcoma was used to study radiosensitizing potential in vivo. The trans-tetramminedinitro complex was administered at doses of 100, 200, or 300 mg/kg as a single ip injection 1 h prior to irradiation or as three daily ip injections. There was increasing dose modification with increasing drug dosage. With a fractionated radiation protocol in which five daily fractions of 2, 3, or 4 Gy were administered to the tumor-bearing limb with ip drug injections of 100 or 200 mg/kg given 1 h prior to irradiation, a dose-modifying effect of 1.6 was observed with 5 X 200 mg/kg of the drug.

Animals↗

PubMed2000

Chromosomal radiosensitivity, cancer predisposition and response to radiotherapy.

AIM: This paper briefly summarizes the research on this topic, undertaken in the Department of Cancer Genetics, Paterson Institute for Cancer Research, Manchester, England, over the previous 6 years. PATIENTS AND METHOD: Patients with the recessively-inherited disease, ataxia-telangiectasia (A-T), who are cancer-prone and suffer severe reactions after radiotherapy, also have highly radiosensitive cells, particularly when chromosome damage is used as the measure of radiosensitivity. Enhanced chromosomal radiosensitivity is also a feature of many other cancer-prone disorders. We have investigated the possible role of such radiosensitivity as a marker of cancer predisposition and response to radiotherapy in the general population. RESULTS: We found that 42% (57/135) of breast cancer patients exhibit chromosomal radiosensitivity when lymphocytes are irradiated in the G2 phase of the cell cycle, compared with 6% (6/105) of healthy controls (Figure 1). These figures are much higher than the estimated frequencies of carriers of the ataxia-telangiectasia gene (heterozygotes) amongst breast cancer patients (< 5%) and controls (0.5%). We have also obtained evidence of heritability of G2 sensitivity by studying relatives of breast cancer cases (Figures 2 and 3). The pattern of inheritance is relatively simple and attributable to 1 or 2 genes segregating in each family (Figure 4). In a prospective study of 123 breast cancer patients, 9 (7%) had severe acute reactions to radiotherapy and their mean G2 sensitivity was significantly greater (p = 0.001) than that of the remaining patients (Figure 5). In 16 patients with adverse acute reactions we found no mutations of the ataxia-telangiectasia gene (ATM). Using another chromosomal assay (micronucleus induction in G0 lymphocytes) we found that the mean radiosensitivity of patients with severe late reactions was higher than that of normal reactors. For example, 8 patients with severe fibrosis were more sensitive (p = 0.055) than 39 patients with a normal response (Figure 6). However, the discriminatory power of these chromosomal assays is too low for them to be used alone in a clinical setting. CONCLUSION: Our results provide good evidence that genes other than ATM, that confer chromosomal radiosensitivity, are involved in low penetrance predisposition to breast cancer in a high proportion of cases and contribute to adverse reactions after radiotherapy.

Ataxia Telangiectasia↗

PubMed1991

SR 4233: a tumor specific radiosensitizer active in fractionated radiation regimes.

The benzotriazine SR 4233, in addition to preferential killing of hypoxic cells both in vitro and in vivo, also radiosensitizes aerobic cells in vitro if the cells are exposed to the drug under hypoxic conditions, either before or after irradiation. We have attempted to exploit this aerobic radiosensitization in vivo, by giving SR 4233 with the hypoxia inducing agent, hydralazine, after each radiation dose in a 8 x 2.5 Gy fractionated regime. The results show greater than additive cytotoxicity using both cell survival and regrowth delay as the endpoints of radiation response, but no radiosensitization in parallel groups treated with the hypoxic cell radiosensitizer SR 2508. The data are, therefore, consistent with radiosensitization of the tumor aerobic cells by the SR 4233 treatments. Significantly, the effect occurred with equal magnitude with or without hydralazine. Further, there was no radiosensitization to radiation induced leg contraction in the thighs of mice, a late responding normal tissue endpoint. The results, therefore, demonstrate a selective radiosensitization of tumors to a multifraction regime and suggest that SR 4233, or a close analog, may be useful in radiation therapy.

Animals↗

PubMed1996

Relationship between the in vitro radiosensitivity of skin fibroblasts and the expression of subcutaneous fibrosis, telangiectasia, and skin erythema after radiotherapy.

OBJECTIVE: To investigate if the occurrence of subcutaneous fibrosis after radiotherapy in an unselected group of breast cancer patients is related to cellular radiosensitivity of skin fibroblasts as measured in a clonogenic assay. MATERIALS AND METHODS: An in vitro colony-forming assay of normal fibroblast radiosensitivity was applied to primary skin biopsies from 31 breast cancer patients who received post-mastectomy radiotherapy with large doses per fraction (2.7-3.9 Gy) more than 10 years earlier. Three clinical normal-tissue endpoints were assessed. Two late endpoints, subcutaneous fibrosis and telangiectasia, were evaluated in three treatment fields by a single experienced clinician. In addition, skin erythema had been assessed at the end of treatment by members of the staff and junior staff. From previous analyses of normal tissue response, individual clinical radiosensitivity could be assessed as "excess risk' of each of the three reactions. This was defined as the difference between the actual observed response in the patient and the expected response estimated from individual treatment characteristics in a linear quadratic (LQ) mixture model and, for the two late endpoints, with correction for the follow-up time. This clinical radioresponsiveness was compared with the in vitro radiosensitivity of the skin fibroblasts. To this end, the fractions of colony-forming cells after graded single doses were fitted by an LQ survival curve using non-linear regression from which the surviving fraction at 3.5 Gy (SF3.5) was estimated. Assessment at 3.5 Gy was chosen to reflect the fraction size during clinical radiotherapy. RESULTS: A statistically significant variability of in vitro radiosensitivity between patients could be detected for both SF2 (P = 0.0095) and SF3.5 (P = 0.0008). A significant correlation was observed between SF3.5 and excess risk of fibrosis (rs = -0.46, P = 0.009) while no association was found between fibroblast radiosensitivity and either the occurrence of severe skin telangiectasia or the acute endpoint skin erythema. CONCLUSION: These results suggest that variability in the occurrence of subcutaneous fibrosis, but not telangiectasia or erythema, after radiotherapy is partly accounted for by differences in cellular radiosensitivity of normal skin fibroblasts.

Adult↗

PubMed1989

Keynote address: hypoxic cell radiosensitizers: where next?

The second generation hypoxic cell radiosensitizers SR 2508 (etanidazole) and Ro 03-8799 (pimonidazole) are now undergoing Phase III clinical trials and are predicted from in vitro, animal, and clinical studies to be equivalent to an increase in the tolerable dose of Miso of 5-6 fold. Nonetheless, this will only produce an SER of the hypoxic cells of approximately 1.5 when given with each dose of a 24 fraction course of radiotherapy. There is ample opportunity, therefore, for a sufficiently improved third generation radiosensitizer to warrant the considerable time, effort, and money that would be involved for its clinical testing. Many groups are now involved in the synthesis and testing of drugs with the goal of producing this third generation sensitizer. The purpose of the present review is to summarize what we have learned from the many studies of new radiosensitizers and attempt to draw conclusions about the most fruitful directions for further work. One of the conclusions is that in vitro testing of radiosensitizers, essential though it is as a first screen, can lead to many false predictions. Fortunately, these in vitro systems tend to "overpredict" (i.e., produce false positives rather than false negatives) for in vivo activity. There is only one class of drugs, so far, (those that increase tumor oxygenation) which radiosensitize tumors in vivo, but not hypoxic cells in vitro. Another major conclusion is that the physico-chemical characteristics of drugs that have been shown to be important in enhancing radiosensitization or reducing toxicity (electron affinity, pKa, partition coefficient, DNA binding), have not been systematically optimized in any one compound. Systematic rational drug design is needed to achieve this. The search for new hypoxic cell radiosensitizers must not detract from the fact that a sensitizer of aerobic cells to low radiation doses is needed. A major challenge in achieving a useful drug for radiotherapy is the desirability for such a drug to be tumor specific. We present data which show that tumor hypoxia might be exploited to achieve this goal.

Cells, Cultured↗

PubMed1991

Radiosensitization by the combination of etanidazole (SR-2508) and pimonidazole (Ro 03-8799) in human tumor xenografts.

The two current second generation radiosensitizers SR-2508 (etanidazole) and Ro 03-8799 (pimonidazole) have different dose-limiting toxicities in humans. SR-2508 produces peripheral neuropathy, whereas Ro 03-8799 causes acute CNS toxicity. Overall toxicity can be minimized while increasing maximal radiosensitization by combining the two sensitizers. The additivity of their radiosensitizing properties was tested using clinically relevant SR-2508 and Ro 03-8799 concentrations in two human tumor xenografts: a rectal adenocarcinoma (HRT18) and a pigmented melanoma (Na11+). Drug concentrations were determined by a High Performance Liquid Chromatography method. Tumor response was assayed by clonogenic cell survival. The maximal radiosensitizing effect was determined using a single dose of radiation and high doses of drugs. For HRT18, the maximal radiosensitization was achieved when SR-2508 and Ro 03-8799 were given 45 and 30 min, respectively, before irradiation. For Na11+, the maximal radiosensitizing effect was lower than for HRT18 and not significant, but a trend was observed at about 30 min before irradiation for both drugs. Using clinically relevant doses, mean Sensitizing Enhancement Ratio (SER) values for HRT18 were: 1.3 (Ro 03-8799: 0.1 mg/gram body weight (gbw) or SR-2508: 0.2 mg/gbw), and 1.5 (Ro 03-8799: 0.1 mg/gbw + SR-2508: 0.2 mg/gbw). Mean SER values for Ro 03-8799 0.2 mg/gbw, SR-2508 0.4 mg/gbw, and Ro 03-8799 0.2 mg/gbw + SR-2508 0.4 mg/gbw were 1.5, 1.5, and 1.8, respectively. No significant radiosensitizing effect was observed on Na11+ with either drug administered singly or in combination and at the same concentrations. Our results suggest that the effectiveness of the two sensitizers administered alone or in combination may be tumor-dependent and that melanomas may not be good candidates.

Adenocarcinoma↗

PubMed1992

Correlation of the radiosensitization potency afforded by nitroacridine intercalators with their electron scavenging efficiency in DNA.

Nitracrine (1-NC) and its nitro-positional analogues are electron-affinic DNA intercalating compounds that act as hypoxic cell radiosensitizers in cell culture, but are too rapidly metabolized to provide radiosensitization in vivo. We have explored the electron-trapping efficiencies of nitroacridines to see if such compounds can scavenge radicals formed in irradiated DNA and if the efficiency of such trapping is related to their radiosensitization properties. We have shown that a correlation does indeed exist as 1-NC, the most potent radiosensitizer, scavenges approximately 45% of the migrating electrons (formed by attachment of eaq- to the DNA) compared with approximately 4% for 4-NC, the least efficient radiosensitizer. The quenching of the delayed fluorescence from acridine orange bound to DNA was also used to probe intracellular uptake and association with DNA. The results are in accord with earlier measurements of average uptake and a suggestion that intercalators which form DNA complexes with short residence times will be preferable to highly bound agents as radiosensitizers. Since 1-NC possesses the smallest association constant with DNA of the four compounds, it is suggested that its high radiosensitization potency may well be related to it being able to interact with more radical sites on the DNA than the other analogues, in addition to its capture of migrating electrons in DNA.

Animals↗

PubMed1995

Normal tissue radiosensitivity in breast cancer patients.

PURPOSE: To investigate whether in vitro radiosensitivity of lymphocytes derived from a blood sample will predict late effects from radiotherapy in breast cancer patients. METHODS AND MATERIALS: Blood samples were collected from consenting patients who had received radiotherapy for breast cancer. Lymphocytes were extracted and transformed by the Epstein-Barr virus. The resulting lymphoblastoid cell lines (LCLs) were assessed for in vitro radiosensitivity using a tetrazolium-based colorimetric assay (MTT). Survival curves were generated using doses of 0.5 to 2 Gy. For each analysis an LCL derived from an individual with the radiosensitive condition ataxia-telangiectasia (AT) was run as control. Some patients also consented to give skin biopsies from which fibroblast cultures were derived. Clonogenic assays were performed to generate survival curves using doses in the range of 0.5 to 4 Gy. Comparison was made with the data obtained from LCLs. Late effects of radiotherapy were assessed using the Radiation Therapy Oncology Group (RTOG) scoring scheme and compared with the in vitro radiosensitivity data. RESULTS: LCLs from 56 breast cancer patients were assessed for in vitro radiosensitivity. Surviving fraction to 2 Gy (SF2) generated from survival curves ranged from 0.04-0.35 with coefficient of variation for the whole group of 41%. None of the LCLs equalled the sensitivity of the AT line, but 16% showed equal or greater sensitivity to a line derived from an AT heterozygote. Comparison of LCL and fibroblast radiosensitivity showed reasonable correlation for 12 paired samples (r = 0.64). The majority of patients showed no or minimal effects after radiotherapy (Grade 0, 1 effects) but seven developed a Grade 2 reaction and four a Grade 3 or 4 reaction. Patients with a Grade 2-4 reaction were found to be more sensitive in vitro than those with a Grade 0-1 reaction (p < 0.02). CONCLUSIONS: The use of the MTT assay to assess LCL radiosensitivity has demonstrated considerable heterogeneity amongst the breast cancer population. The presence of a proportion of patients showing in vitro sensitivity but normal clinical response to radiotherapy would limit the usefulness of the assay for predictive purposes.

Breast Neoplasms↗

PubMed1994

The interaction of epidermal growth factor and radiation in human head and neck squamous cell carcinoma cell lines with vastly different radiosensitivities.

PURPOSE: This study was performed to characterize the interaction of epidermal growth factor and radiation in two human head and neck squamous cell cancer cell lines of vastly different radiosensitivities (UM-SCC-6 Radiosensitive; UM-SCC-1 radioresistant). METHODS AND MATERIALS: The two human head and neck squamous cell cancers (UM-SCC-1 and UM-SCC-6) were grown in medium and following the appropriate treatments, cell survival was assessed by a standard colony formation assay. Growth inhibition was assessed by monitoring cell counts following treatment and flow cytometry was used to assess cell cycle distributions. RESULTS AND CONCLUSION: It was determined that exposure to epidermal growth factor (10 ng/ml) for 24 h prior to radiation resulted in radiosensitization in both cell lines, however, the magnitude of radiosensitization was greater in the radiosensitive UM-SCC-6 cells compared to the radioresistant UM-SCC-1 cells. Treatment of the UM-SCC-6 cells with epidermal growth factor (EGF) (10 ng/ml) for 24 h resulted in a growth delay, however, cell growth returned to normal approximately 24 h following removal of EGF. Similar treatment of the UM-SCC-1 cells resulted in no growth inhibition. The 24 h pre-radiation exposures to EGF (10 ng/ml) did not affect the radiation-induced growth delay in either cell line. Additionally, the 24 h exposures to EGF (10 ng/ml) did not affect the radiation-induced growth delay in either cell line. Additionally, the 24 h exposures to EGF (10 ng/ml) did not cause the cells to enter a more radiosensitive cell cycle phase. Further work will be necessary to determine whether events associated with the EGF-induced growth delay in the UM-SCC-6 cells are associated with the enhanced EGF-induced radiosensitization in these cells compared to UM-SCC-1 cells.

Carcinoma, Squamous Cell↗

PubMed1996

Effect of cisplatin on the clinically relevant radiosensitivity of human cervical carcinoma cell lines.

PURPOSE: To evaluate the effect of clinically relevant levels of cisplatin on the radiosensitivity of human cervical tumor cells, and to estimate what changes in local control rates might be expected to accrue from the concomitant use of cisplatin during fractionated radiotherapy. METHODS AND MATERIALS: The effects of concomitant cisplatin (1 microgram/ml, a typical intratumor concentration) on the clinically relevant radiosensitivity, i.e., surviving fraction after 2 G (SF2) values, was determined in 19 cloned human cervical tumor cell lines. These early passage cell lines had SF2 values ranging from 0.26 to 0.87. RESULTS: The concomitant administration of cisplatin reduced the clinically relevant radiosensitivity in the majority (11 out of 19) of the human tumor cell lines investigated. In only 4 out of 19 was any radiosensitization observed, and in 4 out of 19 cell lines there was no significant change in radiosensitivity. However, the sum of the independent cell killing by radiation and cisplatin, was approximately twofold higher than after radiation alone. There was no apparent dependence of the cisplatin-induced changes in SF2 values upon the level of cell killing by cisplatin. However, there is a suggestion that concomitant cisplatin administration may have a differential effect in inherently radiosensitive and resistant human tumor cell lines. CONCLUSIONS: Our data suggest that concomitant cisplatin/radiotherapy regimens may result in a higher level of local tumor control, but primarily through additive toxicity and not through radiosensitization. Future improvements in local tumor control may, thus, be derived by increasing the total dose of cisplatin.

Antineoplastic Agents↗

PubMed1990

Glyoxylic compounds as radiosensitizers of hypoxic cells.

The radiosensitizing effect of five glyoxal derivatives on the survival of TC-SV40 cells has been measured, under aerobic and hypoxic conditions. A toxicity study was previously performed in order to use nontoxic concentrations. The OER for the TC-SV40 cells was 2.74. None of the glyoxylic compounds showed radiosensitizing activity under aerobic conditions while in hypoxia their radiosensitizing factors decreased in the order phenylglyoxylic acid (1.68 at 8 x 10(-3) mole dm-3) greater than phenylglyoxal (1.55 at 5 x 10(-6) mole dm-3) greater than 2-2' furil (1.48 at 5 x 10(-5) mole dm-3) greater than glyoxylic acid (1.39 at 1 x 10(-3) mole dm-3) greater than glyoxal (1.30 at 5 x 10(-5) mole dm-3). The dose-modifying factors were also determined at two equimolar concentrations 5 x 10(-5) and 5 x 10(-6) mole dm-3. A concentration effect was noticed for all the compounds although their relative radiosensitizing activity kept, independently of the concentration, the same order noted above. Glyoxals with aromatic or heterocyclic rings exert a greater radiosensitization than the others. The acidic compounds have less radiosensitizing activity than their aldehydic counterparts. Interaction of these glyoxals with NPSH cellular groups was tested and the low degree of inhibition shows that this mechanism would contribute very little, if any, to the radiosensitization effect.

Animals↗

PubMed2005

Radiation-induced damage to normal tissues after radiotherapy in patients treated for gynecologic tumors: association with single nucleotide polymorphisms in XRCC1, XRCC3, and OGG1 genes and in vitro chromosomal radiosensitivity in lymphocytes.

PURPOSE: To examine the association of polymorphisms in XRCC1 (194Arg/Trp, 280Arg/His, 399Arg/Gln, 632Gln/Gln), XRCC3 (5' UTR 4.541A>G, IVS5-14 17.893A>G, 241Thr/Met), and OGG1 (326Ser/Cys) with the development of late radiotherapy (RT) reactions and to assess the correlation between in vitro chromosomal radiosensitivity and clinical radiosensitivity. METHODS AND MATERIALS: Sixty-two women with cervical or endometrial cancer treated with RT were included in the study. According to the Common Terminology Criteria for Adverse Events, version 3.0, scale, 22 patients showed late adverse RT reactions. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays were performed to examine polymorphic sites, the G2 assay was used to measure chromosomal radiosensitivity, and patient groups were compared using actuarial methods. RESULTS: The XRCC3 IVS5-14 polymorphic allele was significantly associated with the risk of developing late RT reactions (odds ratio 3.98, p = 0.025), and the XRCC1 codon 194 variant showed a significant protective effect (p = 0.028). Patients with three or more risk alleles in XRCC1 and XRCC3 had a significantly increased risk of developing normal tissue reactions (odds ratio 10.10, p = 0.001). The mean number of chromatid breaks per cell was significantly greater in patients with normal tissue reactions than in patients with no reactions (1.16 and 1.34, respectively; p = 0.002). Patients with high chromosomal radiosensitivity showed a 9.2-fold greater annual risk of complications than patients with intermediate chromosomal radiosensitivity. Combining the G2 analysis with the risk allele model allowed us to identify 23% of the patients with late normal tissue reactions, without false-positive results. CONCLUSION: The results of the present study showed that clinical radiosensitivity is associated with an enhanced G2 chromosomal radiosensitivity and is significantly associated with a combination of different polymorphisms in DNA repair genes.

Adult↗

PubMed2006

ErbB receptor tyrosine kinase network inhibition radiosensitizes carcinoma cells.

PURPOSE: The expression of epidermal growth factor receptor (EGFR)-CD533, a truncation mutant of the wild-type EGFR, radiosensitizes carcinoma and malignant glioma cell lines. This deletion mutant disrupts EGFR activation and downstream signaling through the formation of inhibitory dimerizations. In this study, the effects of EGFR-CD533 on other ErbB receptor tyrosine kinase (RTK) family members were quantified to better understand the mechanism of EGFR-CD533-mediated radiosensitization. METHODS AND MATERIALS: Breast carcinoma cell lines with different ErbB RTK expression profiles were transduced with EGFR or ErbB2 deletion mutants (EGFR-CD533 and ErbB2-CD572) using an adenoviral vector. ErbB RTK activation, mitogen activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/p70S6K signaling, and clonogenic survival were determined for expression of each deletion mutant. RESULTS: EGFR-CD533 radiosensitizes carcinoma cells with either high EGFR expression (MDA-MB231) or low EGFR expression (T47D) through significant blockade of the ErbB RTK network. Analysis of clonogenic survival demonstrate significant enhancement of the alpha/beta ratios, as determined by the linear-quadratic model. Split-dose survival experiments confirm that EGFR-CD533 reduces the repair of cellular damage after ionizing radiation. CONCLUSION: Expression of EGFR-CD533 inhibits the ErbB RTK network and radiosensitizes carcinoma cells irrespective of the ErbB RTK expression patterns, and ErbB2-CD572 does not radiosensitize cells with low EGFR expression. These studies demonstrate that the mechanism of action for EGFR-CD533-mediated radiosensitization is inhibition of the ErbB RTK network, and is an advantage for radiosensitizing multiple malignant cell types.

Breast Neoplasms↗

PubMed2005

Radiosensitization by a dominant negative to DNA polymerase beta is DNA polymerase beta-independent and XRCC1-dependent.

BACKGROUND AND PURPOSE: DNA base damages and single strand breaks after ionizing radiation are repaired by base excision repair (BER) and single strand break repair (SSBR), with both DNA polymerase beta (polbeta) and XRCC1 playing key roles. We previously showed that a dominant negative to polbeta (polbetaDN) sensitized human tumor cells to ionizing radiation. However, polbeta-deficient cells, in contrast to XRCC1-deficient cells, are not more radiosensitive. The purpose of the present study was to further elucidate the mechanism of action of the polbetaDN to better understand the roles of BER and SSBR in determining radiosensitivity. MATERIALS AND METHODS: Mouse embryonic fibroblasts, both polbeta wildtype and knockout, and hamster XRCC1-deficient EM9 cells together with its parental line, were transfected with the polbetaDN. Clones with equal polbetaDN expression levels were selected and used in clonogenic assays to determine radiosensitivity. RESULTS: Radiosensitization of polbeta deficient cells by the polbetaDN is shown here, demonstrating inhibition of a polbeta-independent pathway. In addition, we observed radiosensitization of wildtype hamster cells but no radiosensitization of the XRCC1-deficient EM9 cells. CONCLUSIONS: The polbetaDN acts independently of polbeta status and inhibits a pathway, which is dependent on XRCC1, consistent with inhibition of BER and/or SSBR. The data further indicate involvement of other polymerases, which are inhibited by polbetaDN. Finally, they demonstrate that inhibition of BER and SSBR can increase radiosensitivity, with potential clinical relevance.

Animals↗

PubMed1997

Potential clinical impact of normal-tissue intrinsic radiosensitivity testing.

A critical appraisal is given of the possible benefit from a reliable pre-treatment knowledge of individual normal-tissue sensitivity to radiotherapy. The considerations are in part, but not exclusively, based on the recent experience with in vitro colony-forming assays of the surviving fraction at 2 Gy, the SF2. Three strategies are reviewed: (1) to screen for rare cases with extreme radiosensitivity, so-called over-reactors, and treat these with reduced total dose, (2) to identify the sensitive tail of the distribution of 'normal' radiosensitivities, refer these patients to other treatment, and to escalate the dose to the remaining patients, or (3) to individualize dose prescriptions based on individual radiosensitivity, i.e. treating to isoeffect rather than to a specific dose-fractionation schedule. It is shown that these strategies will have a small, if any, impact on routine radiotherapy. Screening for over-reactors is hampered by the low prevalence of these among otherwise un-selected patients that leads to a low positive predictive value of in vitro radiosensitivity assays. It is argued, that this problem may persist even if the noise on current assays could be reduced to (the unrealistic value of) zero, simply because of the large biological variation in SF2. Removing the sensitive tail of the patient population, will only have a minor effect on the dose that could be delivered to the remaining patients, because of the sigmoid shape of empirical dose-response relationships. Finally, individualizing dose prescriptions based exclusively on information from a normal-tissue radiosensitivity assay, leads to a nearly symmetrical distribution of dose-changes that would produce a very small gain, or even a loss, of tumor control probability if implemented in the clinic. From a theoretical point of view, other strategies could be devised and some of these are considered in this review. Right now the most promising clinical use of in vitro radiosensitivity assays may be as a guide for the prescription of treatment schedules that are costly or involves a high risk of complications. Examples of this are certain strategies attempting to widen the therapeutic window, the use of very high doses or re-irradiation of a previously irradiated region, or the selection of patients for experimental strategies like the use of biological response modifiers to reduce normal-tissue toxicity. Finally, published data are summarized on the possible correlation between the radiosensitivities of tumor and normal tissues or between the sensitivities of various normal tissues.

Colony-Forming Units Assay↗

PubMed2002

In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme: mechanistic considerations.

PURPOSE: Selective cyclooxygenase-2 inhibitors have been reported to enhance the tumor response to radiation in vivo, but the cellular mechanisms underlying the radiosensitizing effect are not understood. In the present study, we investigated several possible mechanisms using a murine sarcoma cell culture system. METHODS AND MATERIALS: Cells derived from a murine sarcoma, designated NFSA, were cultured in vitro and exposed to different (either single or split) doses of radiation with and without a pretreatment of SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-l-yl] benzene sulfonamide), a selective cyclooxygenase-2 (COX-2) inhibitor. The cells were assayed for clonogenic survival to determine the radiosensitizing effect of SC-236. In addition, MTT assay and TUNEL assay were performed to determine the effects of SC-236 and radiation on the cell survival and cell cycle distribution. RNase protection assay was performed on the total RNA extract using probes that encoded for selected cell cycle regulatory proteins, such as cyclins and cyclin-dependent kinases. To monitor the extent of COX-2 activity and its role in radiosensitization, the cellular content of prostaglandin E2, a major metabolite of COX-2 activity on arachidonic acid, was also determined. RESULTS: The cell clonogenic survival assay showed that SC-236 significantly enhanced tumor cell radiosensitivity: 50 microM SC-236 increased it by a factor of 1.51 at the 0.1 cell survival level. Treatment with SC-236 (50 microM, 3 days) removed the "shoulder" region on the radiation survival curve, suggesting that the drug inhibited repair of sublethal radiation damage. The inhibition was confirmed by split-dose experiments where two doses (3 Gy each) of radiation were given 4 h apart. The cells exposed to radiation only repaired the damage by a factor of 1.44, whereas those treated with SC-236 plus radiation repaired it by a factor of 1.1 only. Whereas SC-236 induced apoptosis in these NFSA cells, radiation did not. No further increase in apoptosis was observed when the cells were exposed to both SC-236 and radiation, suggesting that SC-236 did not render tumor cells more susceptible to radiation-induced apoptosis. The RNase protection assay showed that SC-236 (50 microM, 3 days) inhibited the expression of cyclins A and B, as well as cyclin-dependent kinase-1. Inhibition of these cell cycle regulatory elements by SC-236 was associated with the arrest of cells in the radiosensitive G2-M phase (67%), determined by flow cytometry. CONCLUSIONS: SC-236 significantly enhanced radiosensitivity of tumor cells; the magnitude of sensitivity was dependent on the drug's concentration. The likely mechanisms involve accumulation of cells in the radiosensitive G2-M phase of the cell cycle and inhibition of repair from sublethal radiation damage.

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