Adenovirus-mediated wild-type p53 gene expression radiosensitizes non-small cell lung cancer cells but not normal lung fibroblasts

Purpose : We compared the ability of adenoviral-mediated wildtype p53 RPR/INGN201(Ad5/CMV/p53) to radiosensitize nonsmall cell lung carcinoma (NSCLC) and normal lung fibroblast cells. Materials and methods : NSCLC cell lines (A549 and H322) and human lung fibroblast cells (MRC-9 and CCD-16) were used in this study. Radiosensitivity was determined by clonogenic assay and tumor growth delay. Expression of p53, Bax, and p21 WAF1 protein were evaluated by immunoblot. A FITC conjugate of annexin V was used for flow cytometric detection of apoptosis. Results : Clonogenic and apoptotic assays indicated that Ad5/CMV/p53 enhanced the radiosensitivity of both NSCLC cell lines. On the other hand, the two normal human fibroblast cell lines appeared to be resistant to the cytotoxic effects of Ad5/CMV/p53 and were not radiosensitized compared to the NSCLC cells. According to immunoblot analysis, Bax expression was increased in the NSCLC cells treated with the combination therapy; Bax expression, however, was unchanged in normal cells. In in vivo studies, tumor growth suppression was enhanced by this combination strategy in xenograft tumors growing in nude mice compared to Ad5/CMV/p53 or radiation therapy when used alone. Conclusions : Our data indicate that therapy using Ad5/CMV/p53 and irradiation in combination is more effective than either treatment when used alone on NSCLC cells, is not limited to cells with defective endogenous p53, and does not enhance the radiosensitivity of normal cells.     

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.     

外源性野生型p53基因对不同p53功能状态的人肺腺癌细胞株的放射增敏作用

目的 观察外源性野生型p53基因(wtp53)对人肺腺癌细胞株的放射增敏作用,比较不同p53基因状态对照射的影响。 方法 免疫组织化学法、聚合酶链反应-单链构象多态性分析(PCR-SSCP法)筛选p53基因状态不同的两种人肺腺癌细胞系A549及GLC-82,用腺病毒介导wtp53 (Ad-p53)转染后分别给予0、2和4 Gy照射,测定集落形成率,流式细胞仪检测细胞周期分布和凋亡。结果 A549细胞的p53基因正常,而GLC-82细胞的p53基因第7外显子突变,转染后wtp53在两种细胞内均成功表达。Ad-p53对A549及GLC-82细胞抑制率分别为55%和88%,对GLC-82抑制作用较强(P<0.01)。转染Ad-p53后照射,两种细胞集落形成率较对照组明显下降(P<0.001)。流式细胞仪分析Ad-p53使G₁期细胞比例增加和凋亡指数增高,Ad-p53＋照射组最为明显(P<0.001)。结论 Ad-wtp53可以抑制人肺腺癌细胞株的生长,增加其放射敏感性,其放射增敏作用并不依赖于细胞内源性p53基因的状态。     

LINC01296/miR-1255b-5p促进非小细胞肺癌进展的研究

目的 探究下调LINC01296的表达对非小细胞肺癌（non-small cell lung cancer,NSCLC）进展的影响,以及LINC01296调控miR-1255b-5p在NSCLC发生发展中的分子机制。方法 培养6种NSCLC细胞系;实时荧光定量聚合酶链反应法检测LINC01296在NSCLC细胞系以及在48例患者样本中表达水平的高低;细胞计数试剂盒-8法检测稳转细胞株的细胞增殖情况;克隆形成实验检测细胞的生长以及增殖状况;划痕实验检测细胞的迁移情况;FITC-Annexin-V法检测细胞凋亡;动物实验,包括裸鼠尾静脉和皮下瘤注射,观察癌细胞的转移以及成瘤情况;双荧光素酶报告基因实验验证LINC01296与miR-1255b-5p之间存在靶向结合关系。 结果 LINC01296在NSCLC中高表达;LINC01296基因敲低抑制NSCLC细胞生长、迁移;LINC01296敲低抑制体内瘤体的生长和转移;LINC01296与miR-1255b-5p的之间存在海绵吸附作用;LINC01296与miR-1255b-5p的表达呈负相关。结论 LINC01296作为NSCLC的促癌分子,通过抑制miR-1255b-5p的表达水平,促进了NSCLC的发展。     

Hypoxia-regulated p53 and its effect on radiosensitivity in cancer cells

PURPOSE: To determine the response of tumor suppressor p53 to hypoxia in different tumor cell lines and the involvement of p53 activity regulation in the effect of hypoxia on tumor cell sensitivity to radiation and hyperthermia. MATERIALS AND METHODS: Three tumor cell lines with functional p53 were treated with chronic or cyclic hypoxia followed by radiation or hyperthermia to investigate p53 activity and cell survival. Flow cytometry was used to investigate the effect of hypoxia-induced cell cycle arrest on radiosensitivity in KHT-C (mouse fibrosarcoma) cells. Transient transfection was performed to determine the role of altered p53 activity in KHT-C and SCC VII (mouse squamous-cell carcinoma) radiosensitivity. RESULTS: Aerobic radiosensitivity was decreased in KHT-C and SCC VII cells after in vitro chronic or cyclic hypoxia pretreatment, but in HT1080 cells, it was slightly increased after chronic hypoxia, and was unchanged after acute hypoxia pretreatment. Decreased radiosensitivity in hypoxia-pretreated KHT-C and SCC VII cells was unlikely due to hypoxia-induced cell cycle arrest, but rather seemed to be associated with increased expression of Mdm2 (mouse double minute-2) and decreased p53. Furthermore, hypoxia pretreatment inhibited the activation of p53 by radiation. Similar results were observed in hyperthermia treated KHT-C cells. Finally, decreased radiosensitivity was observed in both KHT-C and SCC VII cells transiently transfected with Mdm2 or anti-sense p53 cDNA. CONCLUSION: We demonstrated that hypoxia may decrease tumor cell radiosensitivity through the suppression of p53 activity in some tumor cell lines. These results suggested the response of p53 to hypoxia can be cell type specific and contribute to radiosensitivity of hypoxic cells.     

Hypoxia-regulated p53 and its effect on radiosensitivity in cancer cells

Purpose: To determine the response of tumor suppressor p53 to hypoxia in different tumor cell lines and the involvement of p53 activity regulation in the effect of hypoxia on tumor cell sensitivity to radiation and hyperthermia.

Materials and methods: Three tumor cell lines with functional p53 were treated with chronic or cyclic hypoxia followed by radiation or hyperthermia to investigate p53 activity and cell survival. Flow cytometry was used to investigate the effect of hypoxia-induced cell cycle arrest on radiosensitivity in KHT-C (mouse fibrosarcoma) cells. Transient transfection was performed to determine the role of altered p53 activity in KHT-C and SCC VII (mouse squamous-cell carcinoma) radiosensitivity.

Results: Aerobic radiosensitivity was decreased in KHT-C and SCC VII cells after in vitro chronic or cyclic hypoxia pretreatment, but in HT1080 cells, it was slightly increased after chronic hypoxia, and was unchanged after acute hypoxia pretreatment. Decreased radiosensitivity in hypoxia-pretreated KHT-C and SCC VII cells was unlikely due to hypoxia-induced cell cycle arrest, but rather seemed to be associated with increased expression of Mdm2 (mouse double minute-2) and decreased p53. Furthermore, hypoxia pretreatment inhibited the activation of p53 by radiation. Similar results were observed in hyperthermia treated KHT-C cells. Finally, decreased radiosensitivity was observed in both KHT-C and SCC VII cells transiently transfected with Mdm2 or anti-sense p53 cDNA.

Conclusion: We demonstrated that hypoxia may decrease tumor cell radiosensitivity through the suppression of p53 activity in some tumor cell lines. These results suggested the response of p53 to hypoxia can be cell type specific and contribute to radiosensitivity of hypoxic cells.     

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.     

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 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.     

miR-30a-5p和NUAK1在非小细胞肺癌中的表达及对A549细胞增殖、迁移和侵袭的影响

 目的 研究miR-30a-5p和靶基因NUAK1在非小细胞肺癌中的表达及对A549细胞增殖、迁移侵袭的影响。方法 收集2016-09至2019-03在保定市第一医院行手术切除治疗的非小细胞肺癌患者癌组织和癌旁组织标本64例,QPCR和Western blot分别检测非小细胞肺癌组织样本和细胞中miR-30a-5p和NUAK1的表达;TargetScan网站预测miR-30a-5p与NUAK1间的靶向关系,双荧光素酶报告基因实验和Western blot进行验证;MTT和Transwell实验检测miR-30a-5p及联合过表达NUAK1对A549细胞增殖、迁移和侵袭的影响。结果 非小细胞肺癌组织中miR-30a-5p的相对表达量(0.33±0.14)低于癌旁组织(1.00±0.21),差异有统计学意义(t=21.237,P＜0.05);非小细胞肺癌组织中NUAK1 mRNA表达(4.13±1.87)和蛋白表达(3.41±1.62)均高于癌旁组织(1.00±0.17、1.00±0.16),差异有统计学意义(t=13.335、11.844,P＜0.05)。非小细胞肺癌细胞系H460、H1299、A549中miR-30a-5p表达量(0.35±0.03、0.51±0.05、0.28±0.03)低于人正常肺上皮细胞BEAS-2B(1.00±0.11),差异有统计学意义(F=77.100,P＜0.05);H460、H1299、A549细胞中NUAK1 mRNA相对表达量(2.98±0.30、2.39±0.24、3.42±0.36)和蛋白相对表达量(3.06±0.31、2.27±0.23、4.12±0.41)均显著高于BEAS-2B细胞(1.00±0.09、1.00±0.11),差异有统计学意义(F=46.660、63.070,P＜0.05)。结论 miR-30a-5p可通过靶向NUAK1抑制非小细胞肺癌细胞A549增殖、迁移和侵袭,提示miR-30a-5p和NUAK1可能成为一种新的临床诊断和治疗非小细胞肺癌的靶点。     

Determination of the initial DNA damage and residual DNA damage remaining after 12 hours of repair in eleven cell lines at low doses of irradiation

Purpose : To determine the relationship between DNA damage and radiosensitivity at low doses (1-10Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. Materials and methods : Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60 Co γ-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). Results : The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r 2 =0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. Conclusion : It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.     

Determination of the initial DNA damage and residual DNA damage remaining after 12 hours of repair in eleven cell lines at low doses of irradiation

PURPOSE: To determine the relationship between DNA damage and radiosensitivity at low doses (1-10 Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. MATERIALS AND METHODS: Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60Co gamma-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). RESULTS: The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r2 = 0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. CONCLUSION: It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.     

Relationship between cellular radiosensitivity and DNA damage measured by comet assay in human normal, NBS and AT fibroblasts

PURPOSE: To study the relationship between cellular radiosensitivity and DNA damage measured by the comet assay. MATERIALS AND METHODS: Experiments were performed with nine human fibroblast lines (six normal, one NBS, and two AT). Cellular radiosensitivity was determined by colony assay and DNA damage was assessed by the comet assay. RESULTS: The cellular radiosensitivity of the fibroblast lines used covered a broad range with SF2 values varying between 1.3% and 53%. The comets analysed immediately after irradiation with doses up to 5 Gy showed marked differences among the cell lines; the relative initial tail moment at a dose of 5 Gy, ITM5, varied from 2.7+/-0.2 to 5.0+/-0.3. This variation was considered not to result from different numbers of DNA strand breaks induced but from differences in chromatin structure. There was an inverse correlation between SF2 and ITM5, i.e. radiosensitive cell lines exhibited a higher initial tail moment than radioresistant cell lines. In contrast, the repair kinetics measured with the comet assay for a dose of 2Gy followed by an incubation of up to 2h showed little variation and were found not to correlate with SF2. Repair kinetics as well as the amount of residual damage measured by this version of the comet assay were fairly similar to those measured by the alkaline unwinding technique and unlike that measured by neutral gel electrophoresis, indicating that this comet assay detects primarily single-strand breaks and alkali-labile sites, not double-strand breaks. CONCLUSIONS: The correlation between SF2 and the initial tail moment at 5 Gy found here suggests that the cellular radiosensitivity of human fibroblasts also depends on the chromatin structure.     

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.     

重组人p53腺病毒注射液对人淋巴瘤细胞的作用及辐射敏感性的影响

目的 探讨重组人p53腺病毒注射液对人淋巴瘤细胞的抑制作用及其辐射增敏性。方法 利用四氮唑盐(MTT)比色法分析重组人p53腺病毒注射液、放射治疗以及两者联合应用对人淋巴瘤细胞Raji 和Daudi的抑制作用;通过蛋白质的Western印迹分析法(Western blotting)分析淋巴瘤细胞内P53蛋白的表达;应用反转录-聚合酶链反应(RT-PCR)检测淋巴瘤细胞内p53基因的mRNA表达。结果 MTT结果显示重组人p53腺病毒注射液对人淋巴瘤细胞具有一定的抑制作用,但抑制作用不显著,Raji 及Daudi 细胞最大剂量的抑制率分别为27.5%±4.1%和28.1%±1.6%,也未见明显的辐射增敏效应。Western blotting和RT-PCR结果表明,外源性P53蛋白和p53基因的mRNA均有所表达,但未见高效表达和明显的辐射增敏性。结论 重组人p53腺病毒注射液对人淋巴瘤细胞的抑制作用及辐射增敏性不显著。     

Relationship between cellular radiosensitivity and DNA damage measured by comet assay in human normal,NBS and AT fibroblasts

Purpose : To study the relationship between cellular radiosensitivity and DNA damage measured by the comet assay. Materials and methods : Experiments were performed with nine human fibroblast lines (six normal, one NBS, and two AT). Cellular radiosensitivity was determined by colony assay and DNA damage was assessed by the comet assay. Results : The cellular radiosensitivity of the fibroblast lines used covered a broad range with SF2 values varying between 1.3% and 53%. The comets analysed immediately after irradiation with doses up to 5 Gy showed marked differences among the cell lines; the relative initial tail moment at a dose of 5 Gy, ITM5, varied from 2.7 ±0.2 to 5.0 ±0.3. This variation was considered not to result from different numbers of DNA strand breaks induced but from differences in chromatin structure. There was an inverse correlation between SF2 and ITM5, i.e. radiosensitive cell lines exhibited a higher initial tail moment than radioresistant cell lines. In contrast, the repair kinetics measured with the comet assay for a dose of 2Gy followed by an incubation of up to 2h showed little variation and were found not to correlate with SF2. Repair kinetics as well as the amount of residual damage measured by this version of the comet assay were fairly similar to those measured by the alkaline unwinding technique and unlike that measured by neutral gel electrophoresis, indicating that this comet assay detects primarily single-strand breaks and alkali-labile sites, not double-strand breaks. Conclusions : The correlation between SF2 and the initial tail moment at 5 Gy found here suggests that the cellular radiosensitivity of human fibroblasts also depends on the chromatin structure.     

p53 status,cellular recovery and cell cycle arrest as prognosticators of in vitro radiosensitivity in human pancreatic adenocarcinoma cell lines

Purpose: To investigate the factors contributing to the in vitro radiosensitivity of four human pancreatic adenocarcinoma cell lines differing in p53 status, and the basis for the lack of post-irradiation G1 arrest in the two cell lines that have retained a wild-type p53 allele. Materials and methods: Cells were X-irradiated and the parameters related to radiosensitivity, as well as the modulation of gene products linked to regulation of cell cycle transit (p53, p21/WAF1/CIP1, pRb) or DNA replication and repair (DNA topoisomerase I and II), were determined. Results: Both cell lines expressing either mutant (mt) R248W or R273H p53 proteins were more radioresistant. All the cell lines arrested in G2. None of the cell lines arrested in G1 and this was linked to the inability to upregulate p21/WAF1/CIP1. There were no correlations between p53 status and the magnitude or time of maximum G2 arrest. However, there was a negative correlation between a protracted arrest in G2 and the ability to recover from potentially lethal damage (PLD). Conclusions: Variation in radiosensitivity is related to p53 status, but the survival advantage conferred by having mutant p53 status is not readily explained neither by recovery from PLD nor by cell cycle arrest kinetics. There is no p53-independent pathway for the recruitment of p21 in these cell lines following irradiation.     

p53 status, cellular recovery and cell cycle arrest as prognosticators of in vitro radiosensitivity in human pancreatic adenocarcinoma cell lines

PURPOSE: To investigate the factors contributing to the in vitro radiosensitivity of four human pancreatic adenocarcinoma cell lines differing in p53 status, and the basis for the lack of post-irradiation G1 arrest in the two cell lines that have retained a wild-type p53 allele. MATERIALS AND METHODS: Cells were X-irradiated and the parameters related to radiosensitivity, as well as the modulation of gene products linked to regulation of cell cycle transit (p53, p21/WAF1/CIP1, pRb) or DNA replication and repair (DNA topoisomerase I and II), were determined. RESULTS: Both cell lines expressing either mutant (mt) R248W or R273H p53 proteins were more radioresistant. All the cell lines arrested in G2. None of the cell lines arrested in G1 and this was linked to the inability to upregulate p21/WAF1/CIP1. There were no correlations between p53 status and the magnitude or time of maximum G2 arrest. However, there was a negative correlation between a protracted arrest in G2 and the ability to recover from potentially lethal damage (PLD). CONCLUSIONS: Variation in radiosensitivity is related to p53 status, but the survival advantage conferred by having mutant p53 status is not readily explained neither by recovery from PLD nor by cell cycle arrest kinetics. There is no p53-independent pathway for the recruitment of p21 in these cell lines following irradiation.