Radiosensitization Dependent on p53 Function in Bronchial Carcinoma Cells by the Isoflavone Genistein and Estradiol in Vitro*

BACKGROUND AND PURPOSE: Simultaneous radiotherapy with chemotherapy is a standard treatment for inoperable non-small cell lung cancer (NSCLC), but the clinical outcome still remains poor. To further intensify treatment, substances need to be identified, which increase the effect of radiation on tumor cells without further enhancing toxicity to normal tissue. Hormones have a different toxicity profile than radiation or cytostatic drugs. As NSCLC often express estrogen receptors (ERs), the combination of genistein or estradiol and radiation in vitro was investigated. MATERIAL AND METHODS: A549 NSCLC cells with an inducible expression of a mutated TP53 and fibroblasts of a male donor (DF-18) were examined. ER expression was immunocytologically confirmed in all studied cell lines. Clonogenic survival was measured after incubation of the cells with genistein or estradiol (0.01 microM and 10 microM as maximum clinically applicable dose) and irradiation with different doses (0-4 Gy). The differentiation state of fibroblasts after combined therapy was analyzed. RESULTS: A549 cells expressing mutated TP53 were more radioresistant than TP53 wild-type cells. Incubation of nonfunctional TP53 cells with genistein or estradiol increased radiosensitivity in both tested concentrations. By contrast, radiosensitivity of A549 with wild-type TP53 and DF-18 was not altered by hormonal incubation. In DF-18 radiation induced growth arrest that was not increased by additional hormonal incubation. CONCLUSION: NSCLC cells with nonfunctional TP53 might be sensitized against radiation by genistein or estradiol. As genistein is better tolerable than estradiol in patients, additional studies are warranted to assess potential gains of this combination therapy.     

Radiosensitivity and TP53, EGFR Amplification and LOH10 Analysis of Primary Glioma Cell Cultures

AIM: Determination of in-vitro radiosensitivity and genetic alterations of cell cultures derived from human glioma biopsy tissue and established glioma cell lines. MATERIAL AND METHODS: Fresh brain tumor specimens of six patients were processed to early passage cell cultures. In addition the cell lines D 384 and Gli 6 were used. Cell cultures were irradiated with doses from 2 to 10 Gy. Following irradiation, cell survival was determined by clonogenic assay and survival curves were generated. The surviving fractions after 2 Gy (SF2) and 4 Gy (SF4) were used as radiosensitivity parameters. Genetic analysis included determination of the mutational and loss of heterozygosity (LOH) status of TP 53 (exons 5-8), the LOH 10- and epidermal growth factor receptor gene (EGFR) amplification status. RESULTS: The SF2 and SF4 values ranged from 0.54 to 0.88 (mean: 0.70) and from 0.13 to 0.52 (mean: 0.32), respectively. Genetic alterations were found in the Gli 6 cell line and in two primary cell cultures. The genetic profile of Gli 6 showed LOH but no TP 53 mutation, complete LOH 10 and no EGFR amplification. The VU 15 cell culture showed TP 53 mutation but no LOH 10 or EGFR amplification, while VU 24 showed incomplete LOH 10, EGFR amplification and no TP 53 mutation. In the other four cell cultures and D 384 cell line no genetic alterations were diagnosed. Histopathological classification of glioblastoma multiforme and/or genetic alterations resulted in lower radiosensitivity. CONCLUSION: In this small series of early passage glioma cell cultures low radiosensitivity and alterations in cell regulatory genes were seen. Further testing of biological behavior in larger series of patient-derived material is ongoing.     

Sequentially-induced responses define tumour cell radiosensitivity

Purpose:?Our aim was to define dose-dependent and genotype-dependent components of radiosensitivity by resolving patterns of radiation-induced clonal inactivation into specific responses.

Methods:?In a set of 10 tumour cells with varying expression of radiosensitivity and genotype, we identified doses at which all tumour cells change in their rate of clonogenic inactivation. We tested intervening dose-segments as to whether inactivation was constant, expressing inactivation as a log-linear function of dose. We compared these segments to components proposed in the Hit-target (HT) model and the Linear-quadratic (LQ) model. Temporal changes in redistribution in cell-cycle prevalence and apoptosis were examined as essential components of cellular radiosensitivity.

Results:?We identified four distinct responses induced sequentially in all cells independent of genotype. Rates of inactivation within each response varied with expression of genotype and identified: (i) A hypersensitive component H (0.0–0.10 Gy); (ii) a resistant component R (0.1–0.2 Gy); (iii) an induced repair response alpha* (0.2 Gy and higher); and (iv) a more sensitive component omega* (3.0 Gy and higher). The H, alpha* and omega* components were fitted well by log-linear patterns, the R response did not.

Conclusions:?Four distinct, sequentially-induced responses comprise cellular radiosensitivity. H and R responses are associated with low dose hyper-radiosensitivity and early apoptosis, while the alpha* and omega* responses share characteristics of the HT and LQ models and are associated with post-repair apoptosis. Radiation induces these four responses at the same doses in all cells, but the rate of inactivation over each response depends on genotype.     

Preferential radiosensitization in p53-mutated human tumour cell lines by pentoxifylline-mediated disruption of the G2/M checkpoint control

Purpose : There is evidence that the duration of the G2/M delay following irradiation is correlated with cell survival. We studied the radiosensitizing potential of pentoxifylline (PTX) and the PTX-mediated modulation of cell-cycle progression dependent on the p53 status of various human tumour cell lines. Materials and methods : The cellular radiosensitivity of human MCF-7 (wild-type p53) and HT-29 (p53-defective) tumour cells, which were exposed to PTX (2 mM) immediately after γ-irradiation was determined by colony forming assay. The influence on cell cycle progression after irradiation (6 Gy) was assessed by flow cytometric analysis using p53 wild-type MCF-7 and HPR600 cells, and p53-defective HT-29 and WiDr cells. Results : Clonogenic survival assays up to 8 Gy demonstrated that p53-defective HT-29 cells (sensitizer enhancement ratio [SER]=1.54) were sensitized by PTX (2 mM) to a significantly higher degree than p53 wild-type MCF-7 (SER=1.14) cells. Exposure of irradiated (6 Gy) cells to PTX (2 mM) resulted in abrogation of the radiation-induced G2/M arrest in the p53-defective HT-29 and WiDr cells, whereas the p53 wild-type-expressing MCF-7 and HPR600 cells showed less significant impairment of the G2/M checkpoint. In HT-29 cells, the rate of transition into mitosis was even higher than in the sham-treated control cells. G2/M abrogation was accompanied by an increase of apoptosis only in HPR600 cells. Conclusions : Since PTX was less effective in cells expressing intact p53, the application of PTX suggests a promising strategy of pharmacological disruption of the G2/M checkpoint control by which preferentially radiation-resistant tumours with defective p53 function might be rendered more sensitive to ionizing radiation.     

Preferential radiosensitization in p53-mutated human tumour cell lines by pentoxifylline-mediated disruption of the G2/M checkpoint control

PURPOSE: There is evidence that the duration of the G2/M delay following irradiation is correlated with cell survival. We studied the radiosensitizing potential of pentoxifylline (PTX) and the PTX-mediated modulation of cell-cycle progression dependent on the p53 status of various human tumour cell lines. MATERIALS AND METHODS: The cellular radiosensitivity of human MCF-7 (wild-type p53) and HT-29 (p53-defective) tumour cells, which were exposed to PTX (2 mM) immediately after gamma-irradiation was determined by colony forming assay. The influence on cell cycle progression after irradiation (6 Gy) was assessed by flow cytometric analysis using p53 wild-type MCF-7 and HPR600 cells, and p53-defective HT-29 and WiDr cells. RESULTS: Clonogenic survival assays up to 8 Gy demonstrated that p53-defective HT-29 cells (sensitizer enhancement ratio [SER]=1.54) were sensitized by PTX (2 mM) to a significantly higher degree than p53 wild-type MCF-7 (SER=1.14) cells. Exposure of irradiated (6 Gy) cells to PTX (2 mM) resulted in abrogation of the radiation-induced G2/M arrest in the p53-defective HT-29 and WiDr cells, whereas the p53 wild-type-expressing MCF-7 and HPR600 cells showed less significant impairment of the G2/M checkpoint. In HT-29 cells, the rate of transition into mitosis was even higher than in the sham-treated control cells. G2/M abrogation was accompanied by an increase of apoptosis only in HPR600 cells. CONCLUSIONS: Since PTX was less effective in cells expressing intact p53, the application of PTX suggests a promising strategy of pharmacological disruption of the G2/M checkpoint control by which preferentially radiation-resistant tumours with defective p53 function might be rendered more sensitive to ionizing radiation.     

Ukrain, an alkaloid thiophosphoric acid derivative of Chelidonium majus L. protects human fibroblasts but not human tumour cells in vitro against ionizing radiation.

PURPOSE: Ukrain, an alkaloid thiophosphoric acid derivative of Chelidonium majus L., has demonstrated a promising impact on chemotherapy in a variety of malignancies. The effects of the drug on cell survival, alteration of the cell cycle and induction of apoptosis were examined without and in combination with ionizing radiation (IR). The TP53 status of the cell lines used was also investigated. MATERIALS AND METHODS: Exponentially growing human tumour cell lines MDA-MB-231 (breast), PA-TU-8902 (pancreas), CCL-221 (colorectal), U-138MG (glioblastoma), and human skin and lung fibroblastic cells, HSF1, HSF2 and CCD32-LU were studied by colony assay, flow cytometry (cell-cycle, annexin-V staining for apoptosis) and Western blotting. Ukrain was used in concentrations from 0.1 to 50 microg ml(-1) for 1, 3 and 24 h and radiation as single doses of 1-10Gy. Combined drug-radiation exposure employed 1 microg ml(-1) Ukrain for 24h plus 2-8 Gy. RESULTS: Ukrain cytotoxicity was time- and dose-dependent. The combination of Ukrain plus IR gave enhanced toxicity in CCL-221 and U-138MG cells, but not in MDA-MB-231 and PA-TU-8902 cells. Most strikingly, a radioprotective effect was found in normal human skin and lung fibroblasts. Flow-cytometry analyses supported the differential and cell line-specific cytotoxicity of Ukrain. CCL-221 and U-138MG cells accumulated in G2 after 24-h Ukrain treatment, whereas no alterations were detected in the other tumour cells and normal fibroblasts tested. Western blotting of TP53 demonstrated non-functional overexpression in all tumour cell lines without affecting p21. HSF1 presented wild-type TP53 and a p21 response after IR. Flowcytometric analyses of annexin-V staining showed no induction of apoptosis after Ukrain treatment in comparison with untreated controls. CONCLUSIONS: Differential effects of Ukrain in modulating radiation toxicity of human cancer cell lines and its protective effect in normal human fibroblasts suggest that this alkaloid may have potential properties for clinical radiochemotherapy.     

Radiation-induced apoptosis in human non-small-cell lung cancer cell lines is secondary to cell-cycle progression beyond the G2-phase checkpoint

PURPOSE: To characterize the relationship between cell-cycle progression and radiation-induced apoptosis in NSCLC cell lines with different p53 status. MATERIALS AND METHODS: Cell lines with functional (H460, A549) and non-functional p53 (H661 and H520) were irradiated with 20 Gy. Multiparameter flow-cytometry was used to follow the progression of synchronized cells through the cell cycle after irradiation. RESULTS: Delayed apoptosis was observed after cell-cycle progression beyond the G2 block, either in the late G2/M-phase of the same cell cycle being irradiated (H661, H520) or in the G1-phase of the subsequent cell cycle (H460, A549). The apoptotic fraction in H661 and H520 was 60-80% at 144h after irradiation, higher than in A549 and H460 (5 and 35%, respectively). As an alternative to apoptosis in cells cycling beyond the G2 restriction point, hyperploid cells were generated by all cell lines. Inhibition of cell-cycle progression through the G2/M-phase efficiently reduced the induction of late apoptosis. After irradiation in S-phase, 50-60% of cells with functional p53 remained arrested at the G2 restriction point until 144 h post-irradiation, while only 20% of the H661 or H520 did so. CONCLUSIONS: These data characterize radiation-induced apoptosis in NSCLC cell lines as a removal pathway of clonogenically inactivated cells secondary to cell-cycle progression beyond G2/M, and is unlikely to be a critical factor for cellular radiation sensitivity.     

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

PURPOSE: To better understand the relation of radiation induced chromosome aberrations and clonogenic survival in cells with different p53 status. MATERIALS AND METHODS: The human lymphoblasts TK6 and WTK1 were derived from the same donor, but differ in radiosensitivity, p53 status and kinetics of apoptosis. TK6 cells have wild type p53 (p53wt), whereas WTK1 cells have a mutated, non-functional p53 (p53mut). Additionally, a HPV16 E6 transfected TK6 cell line (TK6E6), which is also negative for p53 function (p53neg), was studied. The cells were irradiated, incubated with colcemid, hypotonically lysed and fixed. After staining with Giemsa, asymmetric chromosomal exchange type aberrations were counted in 50 mitoses each per dose point (0 to 4 Gy). Clonogenic survival was determined using the microtiter plate assay. All experiments were performed in triplicate. RESULTS: WTK1 (p53mut) show a higher spontaneous frequency of chromosome aberrations than TK6 (p53wt). No significant differences were noted in radiation induced aberration frequency. TK6E6 (p53neg) show comparable aberration frequencies like TK6. However, the dose required to reduce survival to 10% (D10) was about 2 Gy for TK6 and TK6E6, whereas the D10 for WTK1 was approximately 3 Gy. CONCLUSION: The p53 status influences the radiosensitivity in this lymphoblast cell system showing a high rate of radiation induced apoptosis. Cells with p53mut (WTK1), survive with a damaged genome, because they do not undergo apoptosis to loose their clonogenicity. There was no difference between the p53wt (TK6) and p53neg cells (TK6E6) suggesting a suppression of radiation induced apoptosis by p53mut.     

Radiation-induced apoptosis in human non-small-cell lung cancer cell lines is secondary to cell-cycle progression beyond the G2-phase checkpoint

Purpose : To characterize the relationship between cell-cycle progression and radiation-induced apoptosis in NSCLC cell lines with different p53 status. Materials and methods : Cell lines with functional (H460, A549) and non-functional p53 (H661 and H520) were irradiated with 20 Gy. Multiparameter flow-cytometry was used to follow the progression of synchronized cells through the cell cycle after irradiation. Results : Delayed apoptosis was observed after cell-cycle progression beyond the G2 block, either in the late G2/M-phase of the same cell cycle being irradiated (H661, H520) or in the G1-phase of the subsequent cell cycle (H460, A549). The apoptotic fraction in H661 and H520 was 60-80% at 144 h after irradiation, higher than in A549 and H460 (5 and 35%, respectively). As an alternative to apoptosis in cells cycling beyond the G2 restriction point, hyperploid cells were generated by all cell lines. Inhibition of cell-cycle progression through the G2/M-phase efficiently reduced the induction of late apoptosis. After irradiation in S-phase, 50-60% of cells with functional p53 remained arrested at the G2 restriction point until 144 h post-irradiation, while only 20% of the H661 or H520 did so. Conclusions : These data characterize radiation-induced apoptosis in NSCLC cell lines as a removal pathway of clonogenically inactivated cells secondary to cell-cycle progression beyond G2/M, and is unlikely to be a critical factor for cellular radiation sensitivity.     

p53 -Dependent thermal enhancement of cellular sensitivity in human squamous cell carcinomas in relation to LET

Purpose : To investigate the dependence on p53 gene status of the thermal enhancement of cellular sensitivity against different levels of linear energy transfer (LET) from X-rays or carbon-ion (C-) beams. Materials and methods : Two kinds of human squamous cell carcinoma cell lines were used with an identical genotype except for the p53 gene. SAS/m p53 cells were established by transfection with mutated p53 (m p53) gene to SAS cells having functional wild-type p53 (wtp53). As the control, a neo vector was transfected to the SAS cells (SAS/ neo cells). Both cells were exposed to X-rays or accelerated C-beams (30-150 KeV w m -1) followed by heating at 44°;C. Cellular sensitivity was determined by colony-forming activity. Induction of apoptosis was analysed by Hoechst 33342 staining of apoptotic bodies and agarose-gel electrophoresis for the formation of DNA ladders. Results : It was found that (1) there was no significant difference in cellular sensitivity between SAS/ neo and SAS/m p53 cells to LET radiation of >30 KeV w m -1, although the radiosensitivity of SAS/ neo cells to X-rays was higher (1.2-fold) than that of SAS/m p53 cells; (2) there was an interactive thermal enhancement of radiosensitivity below an LET of 70 KeV w m -1 in SAS/ neo cells, although only additive thermal enhancement was observed in SAS/m p53 cells through all LET levels examined; (3) low-LET radiation induced apoptosis only in SAS/ neo cells; (4) high-LET radiation at an isosurvival dose-induced apoptosis of SAS/ neo cells at a higher frequency compared with that with low-LET radiation; (5) high-LET radiation-induced p53-independent apoptosis in SAS/m p53 cells; and (6) thermal enhancement of cellular sensitivity to X-rays was due to induction of p53-dependent apoptosis. Conclusions : The findings suggest that thermal enhancement of radiosensitivity may result from p53-dependent apoptosis induced by inhibition of p53-dependent cell survival system(s) through either regulation of the cell cycle or induction of DNA repair. It is also suggested that the analysis of p53 gene status of cancer cells may predict response to combined therapies with low-LET radiation and hyperthermia.     

p53-dependent thermal enhancement of cellular sensitivity in human squamous cell carcinomas in relation to LET.

PURPOSE: To investigate the dependence on p53 gene status of the thermal enhancement of cellular sensitivity against different levels of linear energy transfer (LET) from X-rays or carbon-ion (C-) beams. MATERIALS AND METHODS: Two kinds of human squamous cell carcinoma cell lines were used with an identical genotype except for the p53 gene. SAS/mp53 cells were established by transfection with mutated p53 (mp53) gene to SAS cells having functional wild-type p53 (wtp53). As the control, a neo vector was transfected to the SAS cells (SAS/neo cells). Both cells were exposed to X-rays or accelerated C-beams (30-150 KeV microm(-1)) followed by heating at 44 degrees C. Cellular sensitivity was determined by colony-forming activity. Induction of apoptosis was analysed by Hoechst 33342 staining of apoptotic bodies and agarose-gel electrophoresis for the formation of DNA ladders. RESULTS: It was found that (1) there was no significant difference in cellular sensitivity between SAS/neo and SAS/mp53 cells to LET radiation of >30 KeV microm(-1), although the radiosensitivity of SAS/neo cells to X-rays was higher (1.2-fold) than that of SAS/mp53 cells; (2) there was an interactive thermal enhancement of radiosensitivity below an LET of 70 KeV microm(-1) in SAS/neo cells, although only additive thermal enhancement was observed in SAS/mp53 cells through all LET levels examined; (3) low-LET radiation induced apoptosis only in SAS/neo cells; (4) high-LET radiation at an isosurvival dose-induced apoptosis of SAS/neo cells at a higher frequency compared with that with low-LET radiation; (5) high-LET radiation-induced p53-independent apoptosis in SAS/mp53 cells; and (6) thermal enhancement of cellular sensitivity to X-rays was due to induction of p53-dependent apoptosis. CONCLUSIONS: The findings suggest that thermal enhancement of radiosensitivity may result from p53-dependent apoptosis induced by inhibition of p53-dependent cell survival system(s) through either regulation of the cell cycle or induction of DNA repair. It is also suggested that the analysis of p53 gene status of cancer cells may predict response to combined therapies with low-LET radiation and hyperthermia.     

Influence of dose-rate, post-irradiation incubation time and growth factors on interphase cell death by apoptosis and clonogenic survival of human peripheral lymphocytes.

PURPOSE: To investigate the effects of dose-rate, post-irradiation incubation time and growth factors on radiation-induced interphase cell death by apoptosis and reproductive cell death in human peripheral lymphocytes. MATERIALS AND METHODS: Lymphocytes in G0-phase were exposed in vitro to 1-3Gy 137Cs gamma-radiation at a high- (HDR, 45Gy/h) or a low dose-rate (LDR, 0.024Gy/h). HDR exposures were performed either on day 1 (HDR1) simultaneously with the start of the 3 Gy LDR exposure, or on day 6 (HDR6) when the LDR exposures ended. Apoptosis was studied at different times after irradiation by measuring (1) cellular membrane integrity, (2) morphological changes and (3) cell size reduction. Clonogenic survival was analysed for cells plated directly after irradiation (LDR, HDR6, HDR1 day 1) or after 5 days post-irradiation incubation (HDR1 day 6). RESULTS: A significant decrease in reproductive cell death was observed after 3 Gy LDR exposure as compared with the HDR1 exposure for cells plated day 6. For the lower doses applied, the dose-rate effect could not be statistically verified. A decrease in apoptosis for all three doses applied (i.e. 1, 2 and 3Gy) was observed when the LDR exposures were compared with the HDRI analysed day 6, although not of statistical significance. Radiation-induced apoptosis was efficiently counteracted by growth factors up to 24-48 h after 3 Gy HDR exposure. The prevention of radiation-induced cell death by growth factors was dependent on dose and post-irradiation time in G0. When the growth factors were added after a prolonged post-irradiation incubation in G0 (HDR 1 cells plated day 6), a significant increase in reproductive cell death was found (3 Gy) as compared with HDR protocols where the growth factors were added directly after irradiation (HDR 1 plated day 1 and HDR6). CONCLUSIONS: A dose-rate effect on radiation-induced apoptosis was indicated but not statistically verified. A significant dose-rate effect on reproductive cell death was observed. This dose-rate effect was, however, inverted when growth factors were added directly after the HDR irradiations.     

No association between radiosensitivity and TP53 status, G1 arrest or protein levels of p53, myc, ras or raf in human melanoma lines.

PURPOSE: First, to investigate whether TP53 status and/or radiation-induced G1 arrest are associated with radiosensitivity, and, second, to detect possible associations between protein levels of p53, myc, ras or raf and radiosensitivity and to investigate whether hypoxia-induced changes in the levels of these proteins are related to hypoxia-induced changes in radiosensitivity in human melanoma lines. MATERIALS AND METHODS: Radiosensitivity was assessed by clonogenic assays. TP53 status was investigated at the genomic level by constant denaturant gel electrophoresis and at the cDNA level by sequencing. G1 arrest was investigated by flow cytometric analysis of DNA. Protein expression of hypoxia-treated and untreated cells was assessed by flow cytometric measurements and Western blotting. RESULTS: Considerable differences in radiosensitivity were detected among melanoma lines with wild-type TP53. Only a fraction of the melanoma cells, differing between the lines, was arrested in G1. No association between the fraction of arrested cells and radiosensitivity was detected. Protein levels of p53, myc, ras or raf were not associated with radiosensitivity. Hypoxia-induced changes in p53, ras and raf levels were detected in all cell lines. Changes in the level of myc protein were detected for two of the four cell lines, while hypoxia-induced changes in radiosensitivity were observed only for one. CONCLUSIONS: Differences in radiosensitivity among melanoma lines cannot be elucidated by TP53 status, differences in G1 arrest or different levels of p53, myc, ras or raf proteins. Hypoxia-induced changes in p53, myc, ras or raf levels do not seem to be related to hypoxia-induced changes in radiosensitivity.     

No association between radiosensitivity and TP53 status,G1 arrest or protein levels of p53, myc,ras or raf in human melanoma lines

Purpose: First, to investigate whether TP53 status and/or radiation-induced G1 arrest are associated with radiosensitivity, and, second, to detect possible associations between protein levels of p53, myc, ras or raf and radiosensitivity and to investigate whether hypoxia-induced changes in the levels of these proteins are related to hypoxia-induced changes in radiosensitivity in human melanoma lines. Materials and methods: Radiosensitivity was assessed by clonogenic assays. TP53 status was investigated at the genomic level by constant denaturant gel electrophoresis and at the cDNA level by sequencing. G1 arrest was investigated by flow cytometric analysis of DNA. Protein expression of hypoxia-treated and untreated cells was assessed by flow cytometric measurements and Western blotting. Results: Considerable differences in radiosensitivity were detected among melanoma lines with wild-type TP53. Only a fraction of the melanoma cells, differing between the lines, was arrested in G1. No association between the fraction of arrested cells and radiosensitivity was detected. Protein levels of p53, myc, ras or raf were not associated with radiosensitivity. Hypoxia-induced changes in p53, ras and raf levels were detected in all cell lines. Changes in the level of myc protein were detected for two of the four cell lines, while hypoxia-induced changes in radiosensitivity were observed only for one. Conclusions: Differences in radiosensitivity among melanoma lines cannot be elucidated by TP53 status, differences in G1 arrest or different levels of p53, myc, ras or raf proteins. Hypoxia-induced changes in p53, myc, ras or raf levels do not seem to be related to hypoxia-induced changes in radiosensitivity.     

Huge differences in cellular radiosensitivity due to only very small variations in double-strand break repair capacity

The DNA double-strand break (DSB) repair capacity of normal human fibroblasts was compared with that of cell lines with different genetic alterations. These cell lines are affected either in non-homologous end-joining (180BR), homology directed repair (C2352, C2395), base excision repair (CS1TAN, 46BR) or signalling (AT3, AT2BE, LFS2675, LFS2800, 95P558). Cellular radiosensitivity was determined by colony formation assay, DSB by constant-field gel electrophoresis and apoptosis was detected by caspase3 activity. For the mutated cell lines, the survival fraction at 2 Gy (SF2) varied between 0.013 and 0.49 in contrast to a variation of only 0.15-0.53 for normal fibroblasts. There was no variation in the number of initial DSB and only a small variation in the number of DSB remaining 24 h after irradiation. At 100 Gy, the latter number varied between 2 and 5 Gy-equivalents for normal fibroblasts and only between 3 and 7 Gy-equivalents for the mutated cell lines, corresponding to repair capacities of 95-98 and 93-97%, respectively. There were, however, two outliers (LFS2800, 180BR) where the number of remaining DSB was much higher with 22 and 30 Gy-equivalents, respectively. This elevated number resulted from a delayed repair and apoptotic cells. For all but these two cell lines, the relationship between the number of DSB remaining 24 h after irradiation and SF2 could be described by an identical correlation (r2 = 0.86, p < 0.0001). This result indicates that the relationship between DSB repair capacity and cellular radiosensitivity appears to be the same for normal and mutated cell lines, and that in both cases huge differences in cellular radiosensitivity result from only a very small variation in DSB repair capacity.     

Influence of dose-rate,post-irradiation incubation time and growth factors on interphase cell death by apoptosis and clonogenic survival of human peripheral lymphocytes

Purpose: To investigate the effects of dose-rate, post-irradiation incubation time and growth factors on radiation-induced interphase cell death by apoptosis and reproductive cell death in human peripheral lymphocytes. Materials and methods : Lymphocytes in G0-phase were exposed in vitro to 1-3Gy 137Cs gamma-radiation at a high- (HDR, 45Gy/h) or a low dose-rate (LDR, 0.024Gy/h). HDR exposures were performed either on day 1 (HDR1) simultaneously with the start of the 3Gy LDR exposure, or on day 6 (HDR6) when the LDR exposures ended. Apoptosis was studied at different times after irradiation by measuring (1) cellular membrane integrity, (2) morphological changes and (3) cell size reduction. Clonogenic survival was analysed for cells plated directly after irradiation (LDR, HDR6, HDR1 day 1) or after 5 days post-irradiation incubation (HDR1 day 6). Results: A significant decrease in reproductive cell death was observed after 3Gy LDR exposure as compared with the HDR1 exposure for cells plated day 6. For the lower doses applied, the dose-rate effect could not be statistically verified. A decrease in apoptosis for all three doses applied (i.e. 1, 2 and 3Gy) was observed when the LDR exposures were compared with the HDR1 analysed day 6, although not of statistical significance. Radiation-induced apoptosis was e fficiently counteracted by growth factors up to 24-48h after 3Gy HDR exposure. The prevention of radiation-induced cell death by growth factors was dependent on dose and post-irradiation time in G0. When the growth factors were added after a prolonged post-irradiation incubation in G0 (HDR1 cells plated day 6), a significant increase in reproductive cell death was found (3Gy) as compared with HDR protocols where the growth factors were added directly after irradiation (HDR1 plated day 1 and HDR6). Conclusions: A dose-rate effect on radiation-induced apoptosis was indicated but not statistically verified. A significant dose-rate effect on reproductive cell death was observed. This dose-rate effect was, however, inverted when growth factors were added directly after the HDR irradiations.     

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 (approximately 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 approximately 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.