Analysis of cell kinetics after gamma ray irradiation using anti-BrdU monoclonal antibody

The cell cycle was analyzed using anti-BrdU monoclonal antibody, and changes in cell kinetics after gamma ray irradiation as evaluated by this BrdU-PI double staining were compared with those evaluated by the DNA histogram method based on PI staining. The effect of irradiation on the cell kinetics has been studied according primarily to the number of G2 blocked cells. By the present BrdU method, rapid transition of the G1-S phase was observed within 2 hours of irradiation, and then G1 block was observed. Cells in the S phase progressed to the G2 + M phase, in which they were arrested, resulting in a decrease in the percentage of S cells to 5% or less. After 8 hours, release of G1 block was observed, and G2 + M cells returned to the G1 phase after 18 or more hours. These initial G1 blocked cells induced by irradiation were confirmed for the first time by the present BrdU-PI double staining. By the conventional method based on the DNA histogram, accurate determination of S cell fraction was difficult due to overlapping of the DNA contents of G1 cells and early S cells and those of late S cells and G2 cells. On the other hand, BrdU-PI double staining allowed direct differentiation of G1, S, and G2 + M cells, especially between G1-S and S-G2 + M cells. The analysis of cell kinetics using BrdU is advantageous over the conventional autoradiographic methods in that it allowed more rapid assay with very high sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

G2-block after irradiation of cells with different p53 status

Background

Although it is clear that functional p53 is not required for radiation-induced G₂ block, certain experimental findings suggest a role for p53 in this context. For instance, as we also confirm here, the maximum accumulation in the G₂ compartment after X-ray exposure occurs much later in p53 mutants than in wild types. It remains to be seen, however, whether this difference is due to a longer block in the G₂ phase itself.

Material and methods

We observed the movement of BrdU-labeled cells through G₂ and M into G₁. From an analysis of the fraction of labeled cells that entered the second posttreatment cell cycle, we were able to determine the absolute duration of the G₂ and M phases in unirradiated and irradiated cells.

Results

Our experiments with four cell lines, two melanomas and two squamous carcinomas, showed that the radiation-induced delay of transition through the G₂ and M phases did not correlate with p53 status.

Conclusion

We conclude that looking at the accumulation of cells in the G₂ compartment alone is misleading when differences in the G₂ block are investigated and that the G₂ block itself is indeed independent of functional p53.     

快中子诱导人鼻咽癌细胞系凋亡的研究

目的　研究中子不同剂量照射人鼻咽癌（ＣＮＥ２） 细胞凋亡发生特点及与Ｘ射线所致凋亡的差异。探讨凋亡在中子治疗肿瘤中的作用及临床意义。方法　采用琼脂糖凝胶电泳及ＤＮＡ特异性荧光染色方法（Ｈｏｅｃｈｓｔ３３３４２） 检测照射后不同时间人鼻咽癌（ＣＮＥ２）细胞。结果　发现中子可诱导人鼻咽癌细胞发生凋亡,这种凋亡发生存在着一定的时间剂量相关性。在相同剂量照射下,同一时间点上,中子照射所致的凋亡反应强于Ｘ 射线所致的凋亡。结论　快中子照射离体细胞可引起较强的凋亡反应。中子杀伤肿瘤的机理可能也是主要通过凋亡途径来实现的。     

Results after split-course irradiation of bronchial carcinoma (2 series) (author's transl)

This report concerns 1092 patients with bronchial carcinoma, exclusively irradiated by means of the split-course method. Only squamous cell carcinomas have a chance to survive the 5-year limit: all stages in 9/328 = 2.7%, T1 and T2 in 5/48 = 10%. The dependence of the effect on the radiation dose can be confirmed for squamous cell carcinoma but not for anaplastic tumors. Doses higher than 4500 rad do not bring about a longer survival for the latter. Advantages of the split-course method are: The treatment is better tolerated, operability of questionably operable cases can be checked once again after the first irradiation, series, the dose may be determined from the therapy effect in every individual case.     

Hp/DNA双参数分析放射损伤小鼠骨髓细胞周期、凋亡和Hp

目的：建立结合珠蛋白(Hp)／DNA双参数流式细胞技术，以探讨放射损伤小鼠骨髓细胞中Hp含量与细胞周期和凋亡的关系。方法：小鼠骨髓细胞经70％乙醇固定后，Triton-X100破膜，间接免疫荧光技术标记Hp，PI标记DNA，上流式细胞仪测试。用ModFit软件设双门法分析Hp^ 和Hp^-细胞的细胞周期和凋亡，CELLQuest软件分析Hp^ 细胞比例。结果：γ射线照射后6h小鼠骨髓有核细胞中Hp^ 细胞的比例明显高于未照射对照组，骨髓细胞的凋亡来自于Hp^-细胞，Hp^ 细胞未见凋亡，Hp^-细胞发生的G2/M阻滞比Hp6 细胞严重。这些信息是单用Westem印迹法或单纯DNA分析所不能提示的。结论：Hp/DNA双参数流式细胞技术可用于分析Hp与细胞周期和凋亡的关系，是一种快速、客观的分析方法。     

辐射诱导人细胞系CEM、外周血单个核细胞DNA修复基因hHR21~(sp)的表达研究

目的　研究辐射对人T淋巴细胞白血病细胞系 (CEM)、外周血单个核细胞的hHR2 1sp基因转录表达水平的影响及意义。方法　分别对人T淋巴细胞白血病细胞系CEM和正常人外周血单核细胞在UV或γ辐射后不同时间提取细胞总RNA ,通过RT PCR与hHR2 1sp 基因特异引物杂交 ,以 β actin为内参照放射影像密度扫描检测人T淋巴细胞白血病细胞系CEM、单核细胞DNA修复基因表达。结果　在UV 辐射、γ 辐射后早期 (3～ 6h) ,人T淋巴细胞白血病细胞系CEM和淋巴细胞hHR2 1sp基因的表达水平明显增加 ,照射后 6hhHR2 1sp基因的表达水平增加最多且UV辐射更明显 ,在晚期 (9h)降低。比较人细胞系CEM和淋巴细胞两者hHR2 1sp基因的表达水平 ,γ辐射 (3Gy)后淋巴细胞对hHR2 1sp基因表达高于人细胞系CEM ,且表达增加时间较长 ,达 9h ;而人细胞系CEM受到γ辐射后早期表达增加 ,在 6～ 9h后表达降低。结论　人T淋巴细胞白血病细胞系 (CEM)和人淋巴细胞的DNA修复基因hHR2 1sp基因在一定剂量辐射 (UV、γ辐射 )范围内其表达水平随辐射剂量增加而诱导表达水平增高 ,且对UV辐射更敏感 ;提示hHR2 1sp基因在人细胞系CEM细胞和人单核细胞在照射损伤后表达增加 ,可能是促进单核细胞损伤修复的原因之一。     

Copper(II) bis(thiosemicarbazone) complexes as potential tracers for evaluation of cerebral and myocardial blood flow with PET

Wider application of positron emission tomography would be facilitated by the availability of positron-emitting radiopharmaceuticals labeled with nuclides, like 62Cu, that are available from parent/daughter generator systems. Using a longer-lived copper isotope (67Cu) we have examined three derivatives of copper(II) pyruvaldehyde bis(thiosemicarbazone) as potential tracers for evaluation of cerebral and myocardial blood flow: Cu(PTS), Cu(PTSM), and Cu(PTSM2) (where PTS = pyruvaldehyde bis(thiosemicarbazone), PTSM = pyruvaldehyde bis(N4-methylthiosemicarbazone), and PTSM2 = pyruvaldehyde bis(N4-dimethylthiosemicarbazone). All three lipophilic radiocopper complexes were obtained in high yield via a procedure that could be adapted to a "kit" formulation. In animal model systems Cu(PTSM) and Cu(PTSM2) show excellent uptake in the brain and heart following i.v. injection. These tracers differ in that Cu(PTSM) exhibits microsphere-like retention in the brain and heart, whereas Cu(PTSM2) substantially clears from these organs. The relative cerebral pharmacokinetics of [67Cu]Cu(PTSM) and [67Cu]Cu(PTSM2) are consistent with their known reactivity towards intracellular sulfhydryl groups.     

The induction of DNA double-strand breaks in CHO cells by Pvu II: kinetics using neutral filter elution (pH 9.6)

Chinese hamster CHO K1 cells were treated with the restriction endonuclease Pvu II during electroporation and assayed for DNA double-strand breaks (dsb). Dsb were measured by the non-denaturing filter elution technique (pH 9.6) at various times up to 24 h after restriction endonuclease (RE) treatment. The frequency of dsb following electroporation in the presence of 200 units/ml Pvu II increased over the post-treatment incubation period. This was found not to be due to cell or DNA degradation, indicating that Pvu II remains active for at least 24 h inside the cell. We suggest that these kinetics of dsb result from a competition between incision (by Pvu II) and dsb repair.     

荧光原位杂交技术分析大剂量照射后Calyculin A诱导的早熟凝集染色体

目的 探索用荧光原位杂交(FISH)技术分析大剂量照射后Calyculin A(CA)诱导的早熟凝集染色体的可行性.方法 采用X射线照射离体外周血,吸收剂量为0、1、5、10、15和20 Gy.RPMI 1640培养基培养,CA诱导染色体早熟凝集,1、4号全染色体探针荧光原位杂交,荧光显微镜下观察,计数畸变阳性细胞数及两条染色体断片数,拟合剂量效应曲线.结果 以阳性细胞作为观察对象,剂量范围在0~15 Gy时,阳性细胞数和照射剂量呈现良好的剂量-效应关系,Y=0.008+0.065D+1.858×10^-5D²(R²=0.994).以断片数作为观察对象,剂量范围在0~20 Gy 时,同样呈现良好的剂量-效应关系:Y=-0.032+0.216D-0.01D²(R²=1.0).结论 用FISH分析CA诱导的早熟凝集染色体,可用于估算大剂量照射后的生物剂量.     

Stunning of iodide transport by (131)I irradiation in cultured thyroid epithelial cells.

The existence of thyroid stunning (i.e., inhibited thyroidal iodide uptake after administration of diagnostic amounts of (131)I) is controversial and is currently a subject of debate. To our knowledge, the stunning phenomenon has not been investigated previously in vitro. METHODS: Growth-arrested porcine thyroid cells that formed a tight and polarized monolayer in a bicameral chamber were irradiated with 3-80 Gy (131)I present in the surrounding culture medium for 48 h. The iodide transport capacity after irradiation was evaluated 3 d later by measuring the transepithelial (basal to apical) flux of trace amounts of (125)I. RESULTS: The basal-to-apical (125)I transport decreased with increasing absorbed dose acquired from (131)I; a nearly 50% reduction was observed already at 3 Gy. Stable iodide at the same molarity as (131)I (10(-8) mol/L) had no effect on the (125)I transport. Cell number and epithelial integrity were not affected by irradiation. CONCLUSION: Stunning of iodide transport is detected after (131)I irradiation of cultured thyroid cells. The degree of inhibition of transport is dependent on the absorbed dose.     

辐射诱导人细胞系CEM、外周血单个核细胞DNA修复基因hHR21sp的表达研究

目的：研究辐射对人T淋巴细胞白血病细胞系（CEM）、外周血单个核细胞的hHR21^sp基因转录表达水平的影响及意义。方法：分别对人T淋巴细胞白血病细胞系CEM和正常人外周血单核细胞在UV或γ辐射后不同时间提取细胞总RNA,通过RT－PCR与hHR21^sp基因特异引物杂交,以β-actin为内参照射像密度扫描检测人T淋巴细胞白血病细胞系CEM、单核细胞DNA修复基因表达,结果：UV－辐射、γ－辐射后早期（3－6h）, 人T淋巴细胞白血病细胞系CEM和淋巴细胞hHR21^sp基因的表达水平明显增加,照射后6hhHR21^sp基因的表达水平增加最多且UV辐射更明显,在晚期（9h)降低。比较人细胞系CEM和淋巴细胞两者hHR21^sp基因的表达水平,γ辐射（3Gy)后淋巴细胞对hHR21^sp基因表达高于人细胞系CEM,且表达增加时间较长,达9h;而人细胞系CEM受到γ辐射后早期表达增加,在6－9h后表达降低,结论：人T淋巴细胞白血病细胞系（CEM）和人淋巴细胞的DNA修复基因hHR21^sp基因在一定剂量辐射（UV、γ辐射）范围内其表达水平随辐射剂量增加而诱导表达水平增高,且对UV辐射更敏感,提示hHR21^sp基因在人细胞系CEM细胞和人单核细胞在照射损后表达增加,可能是促进单核细胞损伤修复的原因之一。     

Catalytic inhibition of topoisomerase II in Werner's syndrome cell lines enhances chromosomal damage induced by X-rays in the G2 phase of the cell cycle

PURPOSE: To investigate whether catalytic topoisomerase II activity by ICRF187, a compound that interferes with the catalytic cycle of topoisomerase II without causing DNA damage, could result in a modulation of X-ray-induced chromosomal damage in Werner's syndrome (WS) cell lines. MATERIALS AND METHODS: Two WS (KO375, DJG) and one normal lymphoblastoid cell line (SNW646) were exposed to X-rays, post-treated with ICRF187 and harvested after various recovery times. Cell progression to mitosis was monitored by 5-bromo-2'-deoxyuridine (BrdUrd) and fluorescent immmunodetection to analyse chromosomal damage in homogeneous treated cell populations in the G1, S or G2 phase of the cell cycle. RESULTS: In WS cell lines, catalytic inhibition of topoisomerase II activity by ICRF187 resulted in potentiation of X-ray- induced chromosomal damage in the G2 phase of the cell cycle. This potentiation was not observed in the G1 or S phases of the cell cycle, neither in WS nor normal cells. CONCLUSION: These results point out the possibility that Werner's syndrome protein (WRNp) might play a role in a G2 recombinational pathway of double-strand break repair, cooperating with topoisomerase II and thus contributing to maintain genomic integrity.