辐射增强启动子调控的p53基因联合照射对肿瘤细胞的作用

目的 研究辐射增强启动子调控的野生型-p53抑癌基因系统联合照射对人肿瘤细胞系HeLa和A549细胞的特异性杀伤作用。方法 构建辐射增强启动子pE6（TATA）-p53,Western blot检测不同射线剂量诱导下人肺腺癌A549细胞系和人宫颈癌HeLa细胞系中P53蛋白的表达水平,筛选出最适的照射剂量;AnnexinV-FITC试剂盒检测肿瘤细胞系早期凋亡率;利用克隆形成实验检测此系统对肿瘤细胞放射敏感性的影响。结果 在HeLa和A549细胞中,P53蛋白表达均受放射线诱导增高,且在6 Gy时辐射诱导活性最高;实验组质粒的细胞早期凋亡率与转染对照组质粒的细胞早期凋亡率相比有明显提高（F=11.018、10.736,P<0.05）。HeLa细胞和A549细胞的放射增敏比（SER）分别为2.56和2.36。结论 辐射增强启动子调控的p53基因系统具有显著的诱导肿瘤细胞凋亡的作用,可以提高肿瘤细胞的辐射敏感性,对肿瘤的治疗提供了新思路。     

Kinetics of depopulation, repopulation and host cell infiltration in the rhabdomyosarcoma R1H after 14 MeV neutron irradiation.

The kinetics of depopulation and repopulation of the solid transplantable rhabdomyosarcoma R1H in the rat was studied following irradiation with 5 Gy of 14 MeV neutrons. Several parameters were sequentially measured over a time period of 4 weeks after irradiation: the tumour volume was assessed by in situ caliper measurements; the numerical density of tumour cells was obtained by morphometry; the clonogenic fraction of tumour cells was derived from in vitro colony assay; and the numerical ratio of host to tumour cells was determined by flow cytometry. From these primary parameters the number of clonogenic tumour cells, non-clonogenic tumour cells, and nucleated host cells per tumour, as well as their variation with time, were derived. The results were compared with two sets of data obtained previously for the same tumour exposed to 15 Gy of 200 kVp X-rays. Survival of tumour cells was reduced to 5.5 +/- 0.5% by 5 Gy neutrons and to 4.5 +/- 0.5% by 15 Gy X-rays, i.e. an RBE of close to 3. There was a lag period before the onset of repopulation (4.9 +/- 0.4 days and 4.9 +/- 0.5 days, respectively), followed by a high initial rate of repopulation corresponding to a doubling time of 2.0 +/- 0.2 days for neutrons and 2.1 +/- 0.2 days for X-rays. The rate of depopulation was significantly different for the two treatment modalities; the halving time for the number of non-clonogenic tumour cells was 11 +/- 4 days for neutrons and 2.8 +/- 0.5 days for X-rays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inhibition of the epidermal growth factor receptor tyrosine kinase by BIBX1382BS and the improvement of growth delay, but not local control, after fractionated irradiation in human FaDu squamous cell carcinoma in the nude mouse

PURPOSE: To investigate the effect of BIBX1382BS, an inhibitor of the epidermal growth factor receptor tyrosine kinase, on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumour growth and local tumour control after fractionated irradiation over 6 weeks in nude mice. FaDu human squamous cell carcinoma is epidermal growth factor receptor positive and significant repopulation during fractionated irradiation was demonstrated in previous experiments. MATERIALS AND METHODS: Receptor status, receptor phosphorylation, cell cycle distribution, cell proliferation and clonogenic cell survival after irradiation were assayed with and without BIBX1382BS (5 microM) in vitro. Tumour volume doubling time, BrdUrd and Ki67 labelling indices and apoptosis were investigated in unirradiated tumours growing in NMRI nude mice treated daily with BIBX1382BS (50 mg kg(-1) body weight orally) or carrier. Tumour growth delay and dose-response curves for local tumour control were determined after irradiation with 30 fractions within 6 weeks. RESULTS: BIBX1382BS blocked radiation-induced phosphorylation of the epidermal growth factor receptor and reduced the doubling time of FaDu cells growing in vitro by a factor of 4.9 (p=0.008). Radiosensitivity in vitro remained unchanged after incubation with BIBX1382BS for 3 days and decreased moderately after 6 days (p=0.001). BIBX1382BS significantly reduced the volume doubling time of established FaDu tumours in nude mice by factors of 2.6 when given over 15 days (p<0.001) and 3.7 when applied over 6 weeks (p<0.001). When given simultaneously to fractionated irradiation, growth delay was significantly prolonged by an average of 33 days (p=0.003). Local tumour control was not improved by BIBX1382BS. The radiation doses necessary to control 50% of the tumours locally were 63.6 Gy (95% confidence interval 55; 73) for irradiation alone and 67.8 Gy (60; 77) for the combined treatment (p=0.5). CONCLUSIONS: Despite clear antiproliferative activity in rapidly repopulating FaDu human squamous cell carcinoma and significantly increased tumour growth delay when combined with fractionated irradiation, local tumour control was not improved by BIBX1382BS. The results do not disprove that epidermal growth factor receptor inhibition might enhance the results of radiotherapy. However, the results imply that further preclinical investigations using relevant treatment schedules and appropriate endpoints are necessary to explore the mechanisms of action and efficacy of such combinations.     

DNA-dependent protein kinase: effect on DSB repair,G2/M checkpoint and mode of cell death in NSCLC cell lines

Abstract

Purpose: To evaluate the effect of NU7026, a specific inhibitor of DNA-PKcs, on DNA-double strand break (DSB) repair in a cell cycle specific manner, on the G2/M checkpoint, mitotic progression, apoptosis and clonogenic survival in non-small-cell lung carcinoma (NSCLC) cell lines with different p53 status.

Material and methods: Cell cycle progression, and hyperploidy were evaluated using flow cytometry. Polynucleation as a measure for mitotic catastrophe (MC) was evaluated by fluorescence microscopy. DSB induction and repair were measured by constant-gel electrophoresis and γH2AX assay. The efficiency of DSB rejoining during the cell cycle was assessed by distinguishing G1 and G2/M phase cells on the basis of the DNA content in flow cytometry. The overall effect on cell death was determined by apoptosis and the surviving fraction after irradiation with 2?Gy (SF2) assessed by clonogenic survival.

Results: DSB signaling upon treatment with NU7026, as measured by γH2AX signaling, was differently affected in G1 and G2/M cells. The background level of γH2AX was significantly higher in G2/M compared to G1 cells, whereas NU7026 had no effect on the background level. The steepness of the initial dose effect relation at 1?h after irradiation was less pronounced in G2/M compared to G1 cells. NU7026 had no significant effect on the initial dose-effect relation of γH2AX signaling. In comparison, NU7026 significantly slowed down the repair kinetics and increased the residual γH2AX signal at 24?h after irradiation in the G1 phase of all cell lines, but was less effective in G2/M cells. NU7026 significantly increased the fraction of G2/M phase cells upon irradiation. Moreover, NU7026 significantly increased mitotic catastrophe and hyperploidy, as a measure for mitotic failure after low irradiation doses of about 4?Gy, but decreased both at higher doses of 20?Gy. In addition, radiation induced apoptosis increased in A549, H520 and H460 but decreased in H661 upon NU7026 treatment, with a significant reduction of SF2 in all NSCLC cell lines.

Conclusion: Overall, NU7026 significantly influences the cell cycle progression through the G2- and M-phases and thereby determines the fate of cells. The impairment of DNA-PK upon treatment with NU7026 affects the efficiency of the NHEJ system in a cell cycle dependent manner, which may be of relevance for a clinical application of DNA-PK inhibitors in tumor therapy.     

DEGRO-ÖGRO 2008

BACKGROUND AND PURPOSE: Tumor-induced anemia often occurs in cancer patients, and is corrected by recombinant human erythropoietins (rHuEPOs). Recent studies indicated that, besides erythroid progenitor cells, tumor and endothelial cells express erythropoietin receptor (EPOR) as well; therefore, rHuEPO may affect their functions. Here, the effect of rHuEPOalpha on irradiation in EPOR-positive human squamous cell carcinoma xenograft was tested. MATERIAL AND METHODS: A431 tumor-bearing SCID mice were treated from the tumor implantation with rHuEPOalpha at human-equivalent dose. Xenografts were irradiated (5 Gy) on day 14, and the final tumor mass was measured on day 22. The systemic effects of rHuEPOalpha on the hemoglobin level, on tumor-associated blood vessels and on hypoxia-inducible factor-(HIF-)1alpha expression of the tumor xenografts were monitored. The proliferation, apoptosis and clonogenic capacity of A431 cancer cells treated with rHuEPOalpha and irradiation were also tested in vitro. RESULTS: In vitro, rHuEPOalpha treatment alone did not modify the proliferation of EPOR-positive A431 tumor cells but enhanced the effect of irradiation on proliferation, apoptosis and clonogenic capacity. In vivo, rHuEPOalpha administration compensated the tumor-induced anemia in SCID mice and decreased tumoral HIF-1alpha expression but had no effect on tumor growth. At the same time rHuEPOalpha treatment significantly increased the efficacy of radiotherapy in vivo (tumor weight of 23.9 +/- 4.7 mg and 34.9 +/- 4.6 mg, respectively), mediated by increased tumoral blood vessel destruction. CONCLUSION: rHuEPOalpha treatment may modulate the efficacy of cancer radiotherapy not only by reducing systemic hypoxia and tumoral HIF-1alpha expression, but also by destroying tumoral vessels.     

Radiobiological analysis of human melanoma cells on the 62 MeV CATANA proton beam

PURPOSE: To measure the ability of protons and gamma-rays to effect cell viability and cell survival of human HTB140 melanoma cells. MATERIALS AND METHODS: Exponentially growing HTB140 cells were irradiated close to the Bragg peak maximum of the 62 MeV protons or with 60Co gamma-rays with single doses, ranging from 8 - 24 Gy. Cell viability using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay was evaluated at 6 h, 24 h, 48 h or 7 days after irradiation and clonogenic survival was assessed at 7 days after irradiation. Cell cycle phase redistribution and the level of apoptosis were evaluated at 6 h and 48 h after irradiation. RESULTS: The study of cell viability as a function of time (cell survival progression) and cell survival, using a clonal assay, demonstrated the considerably stronger inactivation effect of protons compared to gamma-rays with a relative biological effectiveness (RBE) of approximately 1.64. Cell cycle phase distribution and apoptosis levels with time enabled us to investigate the development and the character of the damage induced by irradiation. Due to the high radio-resistance of HTB140 cells, cell cycle phase redistribution exhibited only a modest cell accumulation in G2/M phase. Protons but not gamma-rays induced apoptosis. CONCLUSIONS: It appears that protons reduce the number of HTB140 cells by apoptosis as well as by severe DNA damage, while gamma-rays eliminate viable cells primarily by the production of irreparable DNA damage. Protons have an increased RBE relative to gamma-rays.     

13-cis Retinoic Acid in Combination with Interferon-α Enhances Radiation Sensitivity of Human Squamous Cell Carcinoma Cells of the Oral Cavity

BACKGROUND: Preclinical and clinical trials demonstrated the antiproliferative and chemopreventive potential of 13-cis retinoic acid in combination with interferon-alpha. The present study was designed to determine the radiosensitizing potential of both drugs after single and combined treatment of human squamous-cell carcinoma cells of the oral cavity in vitro. MATERIAL AND METHODS: The study was performed using the human squamous-cell carcinoma cell line SCC4, which was originally established from a tumor of the oral cavity. Based on clonogenic assays, the inhibition of clonogenic activity and radiosensitizing potential of 13-cis retinoic acid and interferon-alpha after single or combined treatment without and with subsequent irradiation was determined. RESULTS: 13-cis retinoic acid (10 microM) and interferon-alpha (50 IU/ml) showed significant inhibition of clonogenic activity after single treatment. A combined treatment protocol resulted at least in a highly significant additive inhibition of clonogenicity. Treatment with both drugs (5 microM 13-cis retinoic acid, 25 IU/ml IFN-alpha) prior and post irradiation of the cells resulted in a pronounced enhancement of radiation toxicity resulting in significantly decreased SF2- and alpha-values. Combined treatment with both drugs was significantly more effective than single drug treatment. CONCLUSIONS: The data presented indicate that pre- and post-irradiation treatment with 13-cis retinoic acid and interferon-alpha significantly enhance the radiosensitivity of human squamous-cell carcinoma cells, SCC4, in vitro. Therefore, they support the initiation of clinical trials to test the radio-oncological value of such a treatment regime for squamous-cell carcinomas.     

Low-Dose Irradiation and Short-Exposure Suboptimal-Dose Paclitaxel Adversely Modulate Metastatic Potential of Squamous Carcinoma Cells

BACKGROUND AND PURPOSE: Low-dose irradiation and suboptimal drug concentrations may induce unexpected biological responses. Paclitaxel (PTX) is a widely used drug, which has a range of antitumoral effects and which is also regarded as a radiation sensitizer. In this study, it was tested how "suboptimal" short exposure to PTX modifies the biological effects of low-dose irradiation on human epithelial carcinoma cell lines. MATERIAL AND METHODS: 2 Gy irradiation, 7 and 100 nM PTX treatment, and their combinations were tested on squamous and transitional cell carcinoma lines A431, KB and ECV304. Cytoskeleton of interphase cells was investigated with immunocytochemistry and confocal laser scanning microscopy; viability and clonogenicity were assessed with MTT test and standard clonogenic assay. Effects on tumor growth and metastatic potential of A431 cells were tested in vivo using a liver metastasis model in SCID mice. RESULTS: Exposure of human tumor cells to irradiation induced bundling of microtubules, similar to PTX. Combined treatments suspended each other's effect independently of treatment combinations. Single or combination treatments with low-dose PTX did affect cell proliferation to no relevant extent in vitro or in vivo (primary tumor xenografts of A431 cells). However, exposure to irradiation of A431 cells inhibited while 100 nM PTX stimulated their liver-metastatic potential. Combination of 100 nM PTX with irradiation suspended the effect of 100 nM PTX and exhibited the highest antimetastatic activity. CONCLUSION: Short exposure to PTX and irradiation can interfere with each other's effects on tumor cells without significant modulation of proliferation and tumorigenicity. However, PTX and irradiation may significantly modulate the metastatic property of tumor cells.     

Radio- and chemo-sensitization of human erythroleukemic K562 cells by the histone deacetylase inhibitor Trichostatin A

Histone deacetylase inhibitors are emerging therapeutic agents for cancer. In addition to effecting hyperacetylation of core histones, they have been shown to induce biologic effects such as cell cycle redistribution, cytostasis and in certain cases apoptosis in a variety of cell lines. In this study, the purpose was to investigate the effects of Trichostatin A (TSA) - the most potent histone deacetylase inhibitor identified to date - in human erythroleukemic K562 cells. Further aims were to examine the effect of TSA pre-treatment on the chemosensitivity of the cells to the anthracycline, doxorubicin, and on radiosensitivity. In all experiments the cells were treated with 0.2, 0.5 and 2 mM TSA for 24 h prior to analysis for histone acetylation status, cell growth and survival. In parallel assays, TSA treated cells were exposed to doxorubicin or g-radiation and subsequently analyzed for clonogenic survival. The findings indicated that TSA exhibits potent histone deacetylase inhibitor activity in K562 cells, resulting in hyperacetylation of histones 3 and 4 at the concentrations tested. Furthermore, treatment of cells with TSA resulted in dose-dependent inhibition of proliferation, reduction in clonogenic survival and induction of apoptosis. Moreover, the findings of clonogenic survival assays indicated that pre-treatment of K562 cells with TSA augmented the cytotoxic potency of doxorubicin. The magnitude of sensitization to 10 mM doxorubicin-mediated cell death was approximately 2-fold in cells that were treated with 0.2 mM TSA and 5-fold in cells exposed to 0.5 and 2 mM TSA, compared to cells that had not been pre-treated with the histone deacetylase inhibitor. Similarly, exposure of K562 cells to TSA prior to irradiation resulted in dose-dependent radiosensitization. The dose modification factors at D(37) were calculated to be 1.3, 1.6 and 2.5 for cells treated with 0.2, 0.5 and 2 mM TSA, respectively. These findings provide additional evidence that histone deacetylase inhibitors can increase the cytotoxic efficiency of chemotherapeutic drugs, particularly those which target DNA, and can enhance the sensitivity of cells to g-radiation. More generally, the results support the development of histone deacetylase inhibitors as potential clinical chemo- and radio-sensitizers.     

Effect of Doxorubicin on Cell Survival and Micronuclei Formation in HeLa Cells Exposed to Different Doses of Gamma-Radiation

PURPOSE: The present study was undertaken to obtain an insight into the combined effects of doxorubicin with radiation on the cell survival and micronuclei induction in HeLa cells. MATERIAL AND METHODS: HeLa S3 cells were allowed to grow till they reached plateau phase, inoculated with 10 micrograms/ml doxorubicin hydrochloride and then exposed to 0, 0.5, 1, 2 and 3 Gy gamma-radiation. Clonogenicity of cells was measured using the colony forming assay, micronuclei formation using the micronucleus assay. RESULTS: The treatment of HeLa cells with doxorubicin (adriamycin) for 2 hours before exposure to different doses of gamma-radiation resulted in a significant and dose-dependent decline in the cell survival and cell proliferation when compared to the PBS + irradiation group. Conversely, the frequency of micronuclei increased in a dose-related manner in both the PBS + irradiation and doxorubicin + irradiation groups. The pretreatment of HeLa cells with doxorubicin before irradiation to various doses of gamma-rays resulted in a significant elevation in the frequency of micronuclei when compared with the concurrent PBS + irradiation group. The dose-response relationship for both PBS + irradiation and doxorubicin + irradiation groups was linear. The correlation between cell survival and micronuclei induction was also determined for PBS or doxorubicin + irradiation group, where the clonogenicity of cells declined with the increase in micronuclei formation. The correlation between cell survival and micronuclei induction was linear quadratic for both PBS + irradiation and doxorubicin + irradiation groups. CONCLUSION: From our study it can be concluded that combination treatment with doxorubicin and radiation increased the genotoxic effect of the either treatment given alone.     

Cell death induced by 131 I in a differentiated thyroid carcinoma cell line in vitro: necrosis or apoptosis?

AIM: The apoptotic and necrotic dose-response of thyroid carcinoma cells following irradiation with I was evaluated. METHODS: In our in-vitro model, cells of well-differentiated papillary thyroid carcinoma (B-CPAP) were incubated with increasing activity concentrations of I for 2 days. Changes in cell viability and the extents of necrosis and apoptosis were evaluated both immediately and 2 days after irradiation. RESULTS: Viability of B-CPAP cells diminished with increasing I activity concentration. No apoptosis was detectable immediately after irradiation. Two days after irradiation significant apoptosis was found. The lowest I activity concentration at which apoptosis was detectable corresponds to about 1 MBq . ml. At higher activity concentrations a larger percentage of cells became apoptotic but the proportion decreased again at activity concentrations >10 MBq . ml. Likewise, necrosis was minimal at low activity concentrations and showed an exponential increase with rising I activity concentrations (>5-10 MBq . ml). Necrosis was already detectable immediately after irradiation and was the predominant form of cell death at high activity concentrations. CONCLUSION: The data suggest that the nature of the cytotoxic effect of I and whether it leads to apoptotic or necrotic cell death is dose-dependent. High I doses seem to produce mainly necrotic phenomena, whereas at low I activity concentrations apoptotic phenomena prevail. The predominance of delayed apoptosis could explain why radioiodine therapy at lower doses is often linked to delayed onset and possible continuation of thyroid volume reduction over some months and even up to a year.     

Changes in epidermal radiosensitivity with time associated with increased colony numbers

Epidermal clonogenic cell survival and colony formation following irradiation were investigated and related to radiosensitivity. A rapid in vivo/in vitro assay was developed for the quantification of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. Bromodeoxyuridine (BrdU)-labelled cells in full thickness epidermal sheets were visualized using standard immunohistochemistry. In unirradiated skin, approximately 900 BrdU-positive cells mm(-2) were counted. In a time sequence experiment, BrdU-positive cell numbers increased from an average of 900 cells mm(-2) to approximately 1400 cells mm(-2) after BrdU-labelling for 2-24 h. In irradiated skin, colonies containing >/=16 BrdU-positive cells were seen for the first time at days 14/15 after irradiation. The number of these colonies per cm(2) as a function of skin surface dose yielded a cell survival curve with a D(0)-value (+/-SE) of 3.9+/-0.6 Gy. This relatively high D(0)-value is possibly due to a rapid fall off in depth dose distribution for the iridium-192 source and consequently a substantial contribution of hair follicular epithelium to colony formation. At 14/15 days after irradiation, the ED(50) level of 33.6 Gy for the in vivo response of moist desquamation corresponded with 2.7 colonies cm(-2). Surprisingly, the number of colonies increased with time after irradiation with an estimated doubling time of approximately 4 days, while the D(0)-value remained virtually unchanged. This increase in colony numbers could be due to migration of clonogenic cells, to the recruitment of dormant clonogenic cell survivors by elevated levels of cytokines, or to both. Although frequent biopsying caused increased cytokine levels, which had a systemic effect on unirradiated skin, it had no influence on colony formation in irradiated skin. Smaller colonies, containing 4-8 cells or 9-15 cells, were abundant, particularly after higher doses, which resulted in higher D(0)-values. The majority of these small colonies were abortive and did not progress to larger colonies. There was no statistical evidence for significant variations in the interanimal responses.     

华蟾素诱导人膀胱癌细胞株T24凋亡研究

目的研究华蟾素注射液（cinobufacin,Cino）对膀胱癌细胞株T24的体外增殖抑制及诱导凋亡作用,探讨其对膀胱癌的l临床应用价值。方法不同浓度Cino单独作用和配伍丝裂霉素C（MMC）作用于膀膀癌T24细胞株后,通过流式细胞术（FCM）以及DNA凝胶电泳检测细胞的凋亡情况,应用光学显微镜观察细胞形态学的变化。结果除Cino（25mg／m1）组外,其它处理组均可看到较为典型的细胞凋亡的形态学变化。DNA琼脂糖电泳可见到凋亡的特征性“梯子”条带。流式细胞仪结果示,除高浓度的Cino（25mg／m1）组外．其余各组均见到在G0／G1期前出现亚二倍体峰（APO峰）,且阻滞细胞生长在G0／G1期。各药物浓度组与对照组的S期比率（SPF）和增殖指数（PI）分别进行比较（P〈O．05）有统计学差异。结论Cino对膀胱肿瘤1、24细胞有诱导凋亡和直接杀死的双重作用。具体表现为高浓度（25mg／m1）作用于细胞时,对细胞表现为直接杀死作用。而在较低浓度范围内（O．001～2．5mg/ml）,表现为对细胞诱导凋亡的作用,且具有一定的时效和量效关系：配伍丝裂霉素应用时．对T24细胞的诱导凋亡作用明显增强．两药有协同作用。具有治疗膀胱癌的临床价值     

Selective inhibition of the epidermal growth factor receptor tyrosine kinase by BIBX1382BS and the improvement of growth delay,but not local control,after fractionated irradiation in human FaDu squamous cell carcinoma in the nude mouse

Purpose: To investigate the effect of BIBX1382BS, an inhibitor of the epidermal growth factor receptor tyrosine kinase, on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumour growth and local tumour control after fractionated irradiation over 6 weeks in nude mice. FaDu human squamous cell carcinoma is epidermal growth factor receptor positive and significant repopulation during fractionated irradiation was demonstrated in previous experiments.

Materials and methods: Receptor status, receptor phosphorylation, cell cycle distribution, cell proliferation and clonogenic cell survival after irradiation were assayed with and without BIBX1382BS (5?µM) in vitro. Tumour volume doubling time, BrdUrd and Ki67 labelling indices and apoptosis were investigated in unirradiated tumours growing in NMRI nude mice treated daily with BIBX1382BS (50?mg?kg^?1 body weight orally) or carrier. Tumour growth delay and dose–response curves for local tumour control were determined after irradiation with 30 fractions within 6 weeks.

Results: BIBX1382BS blocked radiation‐induced phosphorylation of the epidermal growth factor receptor and reduced the doubling time of FaDu cells growing in vitro by a factor of 4.9 (p=0.008). Radiosensitivity in vitro remained unchanged after incubation with BIBX1382BS for 3 days and decreased moderately after 6 days (p=0.001). BIBX1382BS significantly reduced the volume doubling time of established FaDu tumours in nude mice by factors of 2.6 when given over 15 days (p<0.001) and 3.7 when applied over 6 weeks (p<0.001). When given simultaneously to fractionated irradiation, growth delay was significantly prolonged by an average of 33 days (p=0.003). Local tumour control was not improved by BIBX1382BS. The radiation doses necessary to control 50% of the tumours locally were 63.6?Gy (95% confidence interval 55; 73) for irradiation alone and 67.8?Gy (60; 77) for the combined treatment (p=0.5).

Conclusions: Despite clear antiproliferative activity in rapidly repopulating FaDu human squamous cell carcinoma and significantly increased tumour growth delay when combined with fractionated irradiation, local tumour control was not improved by BIBX1382BS. The results do not disprove that epidermal growth factor receptor inhibition might enhance the results of radiotherapy. However, the results imply that further preclinical investigations using relevant treatment schedules and appropriate endpoints are necessary to explore the mechanisms of action and efficacy of such combinations.     

Oxic and anoxic enhancement of radiation-mediated toxicity by horseradish peroxidase/indole-3-acetic acid gene therapy

PURPOSE: To evaluate the interaction of horseradish peroxidase (HRP)/indole-3-acetic acid (IAA) gene therapy with therapeutically relevant doses of radiation. MATERIALS AND METHODS: Human T24 bladder and FaDu nasopharyngeal carcinoma cells were transiently transfected with the HRP cDNA using a non-viral delivery method. HRP expression was confirmed by immunostain and enzyme activity assay. The cells were exposed to IAA or the analogue 1-Me-IAA in conjunction with X-rays in air or in anoxic conditions, and cytotoxicity was determined by clonogenic assay. RESULTS: A significant and selective enhancement of radiation-mediated cytotoxicity was observed. Pre-incubation with the prodrugs induced sensitizer enhancement ratios (SER) ranging from 2.6 (0.1mM IAA) to 5.4 (0.5 mM IAA). Radiosensitization was also observed when prodrug exposure was performed immediately after irradiation (SER=2.1-5.6), or in anoxic conditions (SER=3.6). CONCLUSIONS: The use of gene therapy protocols to enhance the effect of ionizing radiation holds promise for anticancer therapy. The data suggest that the combination of HRP/IAA gene therapy with ionizing radiation could present therapeutic advantages in the treatment of solid malignancies, in particular to target the hypoxic population, which has been shown to correlate with poor outcome after radiotherapy.     

Determination of colony numbers in pig epidermis as an estimate for radiosensitivity. A rapid assay based on in vitro BrdU-labelling

A rapid assay has been developed for the quantitation of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. The number of surviving clonogenic cells per unit area was related to the epidermal in vivo response of moist desquamation. After irradiation with single doses, ranging from 20 to 36 Gy, skin biopsies were taken and incubated in dispase for enzymatic separation of the epidermis and dermis. Full thickness epidermal sheets were labelled with bromodeoxyuridine (BrdU) in vitro. Proliferating cells were visualized using standard immunohistochemical procedures. Cell groups containing > or = 16 cells were counted as colonies. These colonies were first seen on day 14/15 after irradiation. The number of colonies per cm2, as a function of skin surface dose, yielded a cell survival curve with a D0 (+/- SE) of 3.87 +/- 0.57 Gy. The ED50 for the epidermal in vivo reaction of moist desquamation corresponded with a colony density of 2.7 colonies per cm2. After higher doses, abundant smaller colonies of 4-8 BrdU-positive cells were seen and these were more radioresistant, as represented by higher D0 values.     

富氢水对电离辐射引起胸腺细胞损伤的影响

目的 探讨富氢水对6 Gy照射小鼠胸腺细胞内活性氧水平、细胞凋亡及DNA损伤程度的影响。 方法 实验分为对照组、6 Gy照射组、6 Gy照射+富氢水组;对照组和6 Gy照射组小鼠照射前10 min灌胃正常饮用水0.5 ml,6 Gy照射+富氢水组小鼠灌富氢水0.5 ml,照射后连续灌胃,给予小鼠正常饮用水和富氢水7 d,于照射后4、7、15 d处死小鼠获取胸腺细胞。采用流式细胞术检测胸腺细胞内活性氧水平、凋亡细胞比例及磷酸化组蛋白H2AX（γ-H2AX）平均荧光强度。 结果 与对照组的小鼠比较,6 Gy照射组小鼠在照射后4、7、15 d胸腺细胞中的活性氧水平升高,早期凋亡细胞[AnnexinⅤ阳性、碘化丙啶（PI）阴性]和晚期凋亡细胞（AnnexinⅤ阳性、PI阳性）的细胞比例明显增加,γ-H2AX平均荧光强度增加。与6 Gy照射组小鼠相比,6 Gy照射+富氢水组小鼠的胸腺细胞中活性氧水平下降,早期凋亡和晚期凋亡的细胞比例降低,细胞内γ-H2AX平均荧光强度则是下降的,差异均具有统计学意义。 结论 富氢水可以有效减轻电离辐射引起的胸腺细胞损伤,为富氢水作为辐射损伤防护剂提供了实验依据。     

Cell–cell contact increases radioresistance in head and neck carcinoma cell lines

Purpose : To investigate the in vitro radiosensitivity of microcolonies from head and neck cancers of varying degrees of diÚerentiation. Materials and methods : The location of individual squamous-cell carcinoma (SCC) cells in tissue culture flasks was recorded using computerized microscopy. This allowed the positions of the cells to be continually revisited, which enabled manual sizing of individual microcolonies, both at the time of irradiation (0-5 Gy) and following a post-irradiation incubation period during which the microcolonies were assessed for clonogenic survival. Results : When irradiated as microcolonies with 0-3 Gy, malignant cells had plating eÝciencies (PE; applying multiplicity corrections) that were lower than expected when compared with the PE of cells irradiated individually. However, at between 2 Gy and 3Gy the PE values became similar and at higher doses the 'corrected PE' of microcolonies became higher than that of individually irradiated cells. Conclusion : These data show that cell-cell interactions can occur in malignant cells in a manner similar to that demonstrated previously in non-malignant cells. Cells within microcolonies demonstrate increased radioresistance compared with cells irradiated singly.     

肝细胞生长因子在大鼠心肌细胞放射损伤中的抗凋亡作用

目的 研究肝细胞生长因子(HGF)对60Co γ射线照射诱导的体外培养心肌细胞凋亡的保护作用.方法 体外培养新生Wistar大鼠心肌细胞,分为未照射组(A组)、单纯照射组(B组)、HGF(20ng/ml)处理照射组(C组)、HGF(40ng/ml)处理照射组(D组).B、C、D组分别用60Co γ射线20Gy单剂量照射心肌细胞,C、D组细胞在照射前3h分别用终浓度为20、40ng/ml的HGF孵育.照射后48h检测培养上清中的LDH浓度,用流式细胞仪检测心肌细胞生长周期及细胞凋亡率变化.结果 照射后48h,B、C、D组细胞培养上清中的LDH浓度较A组升高,C、D组的LDH浓度较B组下降;B、C、D组细胞生长周期较A组无明显变化,但凋亡细胞比例较A组升高,经HGF孵育的心肌细胞凋亡率则呈下降趋势.结论 60Co γ射线单剂量照射可造成体外培养的心肌细胞损伤,促进心肌细胞凋亡,HGF可抑制60Co γ射线对体外培养的大鼠心肌细胞的促凋亡作用,其机制仍需进一步研究.     

Cell-cell contact increases radioresistance in head and neck carcinoma cell lines

PURPOSE: To investigate the in vitro radiosensitivity of microcolonies from head and neck cancers of varying degrees of differentiation. MATERIALS AND METHODS: The location of individual squamous-cell carcinoma (SCC) cells in tissue culture flasks was recorded using computerized microscopy. This allowed the positions of the cells to be continually revisited, which enabled manual sizing of individual microcolonies, both at the time of irradiation (0-5 Gy) and following a post-irradiation incubation period during which the microcolonies were assessed for clonogenic survival. RESULTS: When irradiated as microcolonies with 0-3 Gy, malignant cells had plating efficiencies (PE; applying multiplicity corrections) that were lower than expected when compared with the PE of cells irradiated individually. However, at between 2 Gy and 3 Gy the PE values became similar and at higher doses the 'corrected PE' of microcolonies became higher than that of individually irradiated cells. CONCLUSION: These data show that cell cell interactions can occur in malignant cells in a manner similar to that demonstrated previously in non-malignant cells. Cells within microcolonies demonstrate increased radioresistance compared with cells irradiated singly.