Capacity For Repair Of Sublethal Damage Research Articles

Re-evaluation of cellular radiosensitization by 5-fluorouracil: High-dose, pulsed administration is effective and preferable to conventional low-dose, chronic administration

Purpose: It is widely believed that the anticancer drug 5- fluorouracil (5-FU) must be administered chronically and in low doses to maximize radiosensitization during chemoradiotherapy. The rationale is based upon cell experiments that assumed identical mechanisms of 5-FU action between low-dose chronic (LDC) and high-dose pulsed (HDP) exposures. Here we challenge the paradigm and demonstrate the effectiveness of HDP 5-FU as a radiosensitizer and the wide range of dose/time schedules that can be used to synergize with radiation as compared to the relatively restrictive protocols prescribed for current LDC administrations.Materials and methods: Clonogenic survival of human glioblastoma and colon cancer cell lines, U87MG-VIII and HCT-116, respectively, was used to assess temporal and dose effects of 5-FU on radiosensitivity and in split-dose experiments to characterize changes in sublethal damage repair.Results: We show that HDP 5-FU administration does indeed radiosensitize both the highly radioresistant U87MG-VIII and HCT-116. Additionally, we show that this radiosensitization lasts for at least 24 h if cells are pre-irradiated with 2 Gy immediately after HDP 5-FU exposure as a result of a decrease in sublethal damage repair capacity for subsequent irradiations, suggesting the ideal combination of 5-FU bolus injection with fractionation radiotherapy schemes.Conclusions: 5-FU bolus administration protocols combined with radiation would not only help improve treatment outcomes and reduce development of 5-FU resistance, but it would greatly benefit patients by shortening clinical stays and lowering overall therapeutic costs.

Radiosensitivity and capacity for radiation-induced sublethal damage repair of canine transitional cell carcinoma (TCC) cell lines

Understanding the inherent radiosensitivity and repair capacity of canine transitional cell carcinoma (TCC) can aid in optimizing radiation protocols to treat this disease. The objective of this study was to evaluate the parameters surviving fraction at 2 Gy (SF(2) ), α/β ratio and capacity for sublethal damage repair (SLDR) in response to radiation. Dose-response and split-dose studies were performed using the clonogenic assay. The mean SF(2) for three established TCC cell lines was high at 0.61. All the three cell lines exhibited a low to moderate α/β ratio, with the mean being 3.27. Two cell lines exhibited statistically increased survival at 4 and 24 h in the dose-response assay. Overall, our results indicate that the cell lines are moderately radioresistant, have a high repair capacity and behave similarly to a late-responding normal tissue. These findings indicate that the radiation protocols utilizing higher doses with less fractionation may be more effective for treating TCC.

Radiosensitization and Anti-tumour Effects of Cytosine Deaminase and Thymidine Kinase Fusion Suicide Gene in Human Adenoid Cystic Carcinoma Cells

Herpes simplex virus thymidine kinase (HSV-TK) and Escherichia coli cytosine deaminase (CD) can convert innocuous prodrugs into cytotoxic metabolites and are being investigated for use in gene therapy for cancer. Human adenoid cystic carcinoma (ACC-2) cells transduced with a CD/HSV-TK fusion gene (ACC-2/CD-TK cells) were found to be more sensitive to radiation than ACC-2 cells when exposed to 5-fluorocytosine (5-FC; 40 microg/ml) plus ganciclovir (0.1 microg/ml) for 48 h before irradiation. Analysis of radiation survival curves for cells exposed to 5-FC plus ganciclovir before irradiation showed that ACC-2 cells had a higher capacity for sublethal damage repair (D(q) value) and greater cellular radiosensitivity (D(0) value) than ACC-2/CD-TK cells. Colony formation rate after 2 Gy of irradiation was significantly greater for ACC-2 than for ACC-2/CD-TK cells when cells were treated with 5-FC plus ganciclovir before irradiation. This study, therefore, indicates that addition of radiation might substantially improve the therapeutic potential of CD-TK fusion gene therapy of human adenoid cystic carcinomas.

Intrinsic radiosensitivity and repair of sublethal radiation-induced damage in canine osteosarcoma cell lines

To characterize the radiosensitivity and capacity for sublethal damage repair (SLDR) of radiation-induced injury in 4 canine osteosarcoma cell lines. 4 canine osteosarcoma cell lines (HMPOS, POS, COS 31, and D17). A clonogenic colony-forming assay was used to evaluate the cell lines' intrinsic radiosensitivities and SLDR capacities. Dose-response curves for the cell lines were generated by fitting the surviving fractions after radiation doses of 0 (control cells), 1, 2, 3, 6, and 9 Gy to a linear quadratic model. To evaluate SLDR, cell lines were exposed to 2 doses of 3 Gy (split-dose experiments) at an interval of 0 (single 6-Gy dose), 2, 4, 6, or 24 hours, after which the surviving fractions were assessed. Mean surviving fraction did not differ significantly among the 4 cell lines at the radiation doses tested. Mean surviving fraction at 2 Gy was high (0.62), and the alpha/beta ratios (predictor of tissue sensitivity to radiation therapy) for the cell lines were low (mean ratio, 3.47). The split-dose experiments revealed a 2.8- to 3.9-fold increase in cell survival when the radiation doses were applied at an interval of 24 hours, compared with cell survival after radiation doses were applied consecutively (0-hour interval). Results indicated that these canine osteosarcoma cell lines are fairly radioresistant; alpha/beta ratios were similar to those of nonneoplastic, late-responding tissues. Future clinical investigations should involve increasing the fraction size in a manner that maximizes tumor killing without adverse effects on the nonneoplastic surrounding tissues.

Radiation-induced effects on telomerase in gynecological cancer cell lines with different radiosensitivity and repair capacity

Purpose: Telomerase activation in response to irradiation might enhance the radioresistance of cells. Thus, we have investigated radiation-induced effects on telomerase in six gynecological cancer cell lines, with different intrinsic radiosensitivity and capacity for sublethal damage repair (SLDR).Materials and methods: Three endometrial adenocarcinoma (UM-EC-1, UT-EC-2B and UT-EC-3) and three vulvar squamous cell carcinoma (A431, UM-SCV-2 and UM-SCV-7) cell lines were irradiated with doses of 5, 10 and 25 Gy and the effects on telomerase were evaluated at 0.5, 6, 24 and 48 h post-irradiation. Telomerase activity was quantitatively measured by SYBR Green real-time telomeric repeat amplification protocol.Results: The most radioresistant cell line A431 had the strongest stimulatory effects (∼2.0 – 2.5-fold) on telomerase activity 24 and 48 h post-irradiation with the highest radiation doses. In contrast to that, telomerase activities in the highly radiosensitive cell line UT-EC-2B remained below the basal level throughout the 48-h period of post-irradiation with the highest doses, and even a decline to ∼50% of the basal level was found 24 h after exposure. In other cell lines being either moderately or highly radiation resistant, telomerase activity levels in response to irradiation remained mainly at the basal level or gradually increased.Conclusions: The present findings indicate that there might be a connection between the radiation-induced telomerase response and radiosensitivity. However, no correlation was found between the radiation-induced effects on telomerase and the sublethal damage repair capacity of the cells.

Sublethal damage repair capacity in carcinoma cell lines with p53 mutations.

The previous findings that sublethal damage repair (SLDR) capacity varies between carcinoma cell lines and that the inherent radiosensitivity of these lines tends to be higher in connection with p53 mutations lead us to study the possible role of p53 gene in the regulation of SLDR. The activation of p53 gene by irradiation is known to cause changes in cell cycle progression. Thus, p53 status probably has effects on cellular radiosensitivity, theoretically through modulating repair processes. The SDLR capacity of 17 head and neck carcinoma cell lines was determined in split-dose experiments using a 96-well plate clonogenic assay. The SLDR capacity as well as the inherent radiosensitivity were compared with the p53 status of the cells. The SLDR capacity varied markedly also between cell lines of similar radiosensitivity, but there was a tendency of the more sensitive cells to be more SLDR proficient .(r = -.69; p = .0016). The (beta-values obtained from linear quadratic equation correlated well with the observed amount of SLDR (r = .73; p = .0006). With one exception, those cell lines having p53 mutations showed higher SLDR than those with no mutations (p = .0017). In many of these cell lines, the mutations caused either total loss of the p53 protein or major, probably functional changes in it. The cell line UT-SCC-16A, showing no SLDR in the experiments, had two mutation points in different alleles, perhaps having less effect on the protein function. This extended material confirmed the previous result that the SLDR capacity tends to increase with increasing radiosensitivity in carcinoma cell lines. A clear correlation between p53 mutations and SLDR capacity was found. The SLDR depended, however, on loss of normal p53 function, which implies that the p53-mediated G1 arrest is not as important in this repair process, as would have been expected.

Hypothermia-Enhanced Human Tumor Cell Radiosensitivity

Ablation of neoplastic disease by freezing has found increasing utility as a potential therapeutic modality. To assess the effect of cooling temperatures on cellular radiation response, an established human cervical carcinoma cell line (HTB35) was subjected to holding temperatures of 0, 5, or 15°C for up to 24 h before irradiation. Survival was measured byin vitroclonogenic assay of colonies containing at least 50 cells. Cooling for up to 12 h did not significantly decrease survival, but after 24 h survival fell to 75% of control cultures grown at 37°C. X-irradiation immediately after cooling for 24 h resulted in 1.6-fold enhanced radiosensitivity. However, the radiosensitizing effect decayed rapidly if the cooled cells were returned to normal growth temperature for 6 h or longer before irradiation and subculture. Both temperature and cooling duration influenced the radiation response. With 0, 5, or 15°C, radiosensitivity increased after 3, 6, or 12 h, respectively, and progressively rose with up to 24 h of cooling. By flow cytometric analysis, no statistically significant difference was observed in the S-phase fraction between control cells and those cooled to 0°C for 24 h. These data demonstrate cooling-enhancedin vitroradiation sensitivity which is dependent upon cooling temperature, duration, and rewarming interval before irradiation. While cell cycle redistribution does not appear to be a factor in the increased radiosensitivity, differences in the radiation survival curves between cooled versus normothermic cells suggest that diminished capacity for sublethal damage repair may be a significant influence on the changes which were observed.

Cellular radiosensitivity of small-cell lung cancer cell lines

The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based on the construction of continuous exponential growth curves. Fifteen SCLC cell lines were studied, applying a slightly modified clonogenic assay and a growth extrapolation method. A dose-survival curve was obtained for each experiment and used for calculating several survival parameters. The multitarget single hit model was applied to calculate the cellular radiosensitivity (D0), the capacity for sublethal damage repair (Dq), and the extrapolation number (n). Values for alpha and beta were determined from best-fit curves according to the linear-quadratic model and these values were applied to calculate the surviving fraction after 2-Gy irradiation (SF2). In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines studied were radiobiologically heterogeneous with no discrete features of the examined parameters including the repair capacity. The results indicate that SCLC tumors per se are not generally candidates for hyperfractionated radiotherapy.

Sublethal damage repair after fractionated irradiation in endometrial cancer cell lines tested with the 96-well plate clonogenic assay.

Two long-established and seven newly established endometrial adenocarcinoma cell lines were tested for their capacity to repair sublethal damage after fractionated irradiation. Cell survival was determined with the 96-well plate clonogenic assay based on limiting dilutions. Total radiation doses of 0.75 Gy, 1.25 Gy, 2.50 Gy, 5.00 Gy and 7.50 Gy were used either as a single dose or divided into two or three equal fractions with a 24 h interval. Survival data were fitted to the linear quadratic model, and the area under the survival curve (AUC), equivalent to the mean inactivation dose, was obtained with numerical integration. The amount of sublethal damage repair (SLDR) was expressed as an area-under-the-curve (AUC) ratio comparing survivals from fractionated-dose with those from single-dose experiments. SLDR capacity of the cell lines expressed as an AUC ratio varied between 1.00 and 1.59, and the mean was 1.17. Two highly radiosensitive cell lines were found to be SLDR-deficient, but most of the cell lines studied had some SLDR capacity. We have earlier shown that endometrial cancer cell lines as a group are more radiosensitive than squamous-cell carcinoma (SCC) lines. Data obtained in this study suggest that the capacity for SLDR in these cell lines is rather limited compared with the majority of SCC lines tested. This finding underlines further the high radioresponsiveness of endometrial cancer.

Comparison of Thermoradiosensitization in Two Human Melanoma Cell Lines and One Fibroblast Cell Line by Concurrent Mild Hyperthermia and Low-Dose-Rate Irradiation

Two human melanoma cell lines, one radioresistant (SK-MEL-3) and one radiosensitive (HT-144), and a normal human fibroblast line (AG1522) were evaluated for thermoradiosensitization of low-dose-rate irradiation by concurrent mild hyperthermia (39-41 degrees C). None of the cell lines expressed chronic thermotolerance during heating at 39-41 degrees C. The SK-MEL-3 cells were the most heat sensitive, while AG1522 and HT-144 cells had the same sensitivity at 39 and 40 degrees C but HT-144 cells were more sensitive at 41 degrees C. All cell lines expressed thermal enhancement of radiosensitivity with heating during irradiation which increased with heating temperature. The SK-MEL-3 cells, which were the most resistant to radiation and demonstrated the greatest repair of sublethal damage (SLD) during low-dose-rate irradiation, had the greatest thermal enhancement of radiosensitivity, while the HT144 cells, which were the most sensitive and expressed little repair of SLD during low-dose-rate irradiation, had the smallest thermal enhancement of radiosensitivity. These data show that concurrent mild hyperthermia during low-dose-rate irradiation may be most efficacious in radiation-resistant tumor cells which express resistance through an enhanced capacity for repair of SLD.

Effects of v-src Oncogene Activation on Radiation Sensitivity in Drug-Sensitive and in Multidrug-Resistant Rat Fibroblasts

Recent work has implicated the activated ras oncogene, whose gene product is a G-protein located in the plasma membrane, as well as the activated raf oncogene, whose gene product is a membrane-associated protein kinase, in contributing to radioresistance. Another transforming oncogene whose gene product is localized to the plasma membrane is v-src. We have examined a rat fibroblast line (RAT-1) infected with an avian sarcoma virus carrying a temperature-sensitive mutation in the v-src tyrosine kinase domain (LA-24). At 40 degrees C, LA-24 cells have a flat morphology and grow as a contact-inhibited monolayer, while at 35 degrees C, LA-24 cells have a transformed morphology, lose contact inhibition, grow in soft agar, and exhibit 3.5-fold higher tyrosine kinase activity. The parental RAT-1 line, not infected by the virus, grows at both temperatures as a contact-inhibited monolayer. This well-characterized system represents a good model for examining the effect of v-src transformation on radiosensitivity. RAT-1 and LA-24 cells grown at 35 and 40 degrees C were irradiated with graded doses of radiation, and clonogenic survival was assayed. For LA-24 cells grown at 35 and 40 degrees C, and for RAT-1 cells grown at 35 and 40 degrees C, calculated D0, n, alpha, and beta values did not differ significantly. To determine whether there might be differences in radiation damage repair capacity too subtle to detect by comparing radiation survival curves, sublethal damage repair capacity was assessed. There was no difference in sublethal damage repair capacity for LA-24 cells grown at 35 or 40 degrees C. Other studies have associated multidrug resistance with radioresistance. We have examined the radiation sensitivity of two colchicine-resistant LA-24 clones with four- to fivefold amplification of the P-glycoprotein gene, which are four-to fivefold more resistant to colchicine than the parental LA-24 line. In these multidrug-resistant clones, v-src activation does appear to increase radiation resistance. This did not appear to be due to alteration in cell cycle kinetics. We conclude that oncogene activation, or even protein kinase activity per se, does not necessarily lead to radiation resistance. Rather, radiation resistance following oncogene activation depends upon the oncogene and cell line studied, and perhaps upon specific protein phosphorylation.