大鼠急性肾功能衰竭时肾小管上皮细胞凋亡及其意义

目的探讨肾小管上皮细胞凋亡在急性肾功能衰竭（ａｃｕｔｅ ｒｅｎａｌ ｆａｉｌｕｒｅ,ＡＲＦ）中的意义。方法以体积分数为５０％的甘油分别按５ｍｌ／ｋｇ和１０ｍｌ／ｋｇ的剂量注射于Ｗｉｓｔａｒ大鼠后肢肌肉,造成不同程度的ＡＲＦ,在注射后１,３,６,１２,２４,４８,９６ｈ测定肾功能,并取肾脏分别以原位末端标记法（ＴＵＮＥＬ）、流式细胞仪（ＦＣＭ）及瑞氏染色等检测肾小管上皮细胞的凋亡。结果甘油注射后有肌     

Relationships between clonogenic cell survival,DNA damage and chromosomal radiosensitivity in nine human cervix carcinoma cell lines

Purpose : To compare clonogenic cell survival, DNA damage and chromosomal radiosensitivity in nine cervix carcinoma cell lines. Materials and methods : Initial and residual (after 24h repair) radiation-induced DNA damage was evaluated using pulsed field gel electrophoresis. Chromosome damage was measured by micronucleus (MN) induction in cytochalasin-B-induced binucleate cells. Results : Significant differences between the cell lines were obtained in the induced levels of initial damage, residual damage and MN. Values for SF2 for the nine cell lines ranged from 0.36 to 0.92. No correlation was found between clonogenic measurements of radiosensitivity and initial DNA damage dose-response slopes. However, borderline significant correlations were seen between clonogenic radiosensitivity data and the levels of residual DNA damage. There was no correlation between clonogenic radiosensitivity and the levels of radiation-induced MN. Cell lines with high levels of initial damage had high yields of MN induced by radiation and the correlation seen was significant. Conclusions : The poor correlation between the different endpoints precludes their use in a clinical setting on primary tumour samples in vitro. It may be that tumour cell lines in vitro are a poor model for tumours in vivo. Studies aimed at assessing assays for measuring tumour radiosensitivity therefore should employ clinical samples. In vitro cell line work should concentrate on unravelling the complex mechanisms involved in determining a radiosensitive or radioresistant phenotype.     

Relationships between clonogenic cell survival, DNA damage and chromosomal radiosensitivity in nine human cervix carcinoma cell lines

PURPOSE: To compare clonogenic cell survival, DNA damage and chromosomal radiosensitivity in nine cervix carcinoma cell lines. MATERIALS AND METHODS: Initial and residual (after 24h repair) radiation-induced DNA damage was evaluated using pulsed field gel electrophoresis. Chromosome damage was measured by micronucleus (MN) induction in cytochalasin-B-induced binucleate cells. RESULTS: Significant differences between the cell lines were obtained in the induced levels of initial damage, residual damage and MN. Values for SF2 for the nine cell lines ranged from 0.36 to 0.92. No correlation was found between clonogenic measurements of radiosensitivity and initial DNA damage dose response slopes. However, borderline significant correlations were seen between clonogenic radiosensitivity data and the levels of residual DNA damage. There was no correlation between clonogenic radiosensitivity and the levels of radiation-induced MN. Cell lines with high levels of initial damage had high yields of MN induced by radiation and the correlation seen was significant. CONCLUSIONS: The poor correlation between the different endpoints precludes their use in a clinical setting on primary tumour samples in vitro. It may be that tumour cell lines in vitro are a poor model for tumours in vivo. Studies aimed at assessing assays for measuring tumour radiosensitivity therefore should employ clinical samples. In vitro cell line work should concentrate on unravelling the complex mechanisms involved in determining a radiosensitive or radioresistant phenotype.     

Differences in repair of radiation induced damage in two human tumor cell lines as measured by cell survival and alkaline DNA unwinding

We studied the relationship between the repair of radiation induced DNA strand breaks and cellular repair kinetics in two human tumor cell lines, NB-100 (neuroblastoma) and HN-1 (squamous cell carcinoma). Damage was quantified using the fluorometric analysis of DNA unwiding (FADU) for DNA damage, and cell survival was assessed using a clonogenic assay. In plateau phase cells repair of sublethal damage was virtually absent in NB-100 after 4 Gy (recovery ratio 1.0), whereas HN-1 cells did show sublethal damage repair (recovery ratio 1.4). Repair of potentially lethal damage was more pronounced in NB-100 cells (recovery ratio 2.3) than in HN-1 cells (recovery ratio 1.7) after 4 Gy. Graded doses of X-rays induced comparable levels of DNA damage in both tumor cell lines. However, in HN-1 cells more DNA strand breaks were repaired after 4 Gy, leaving about 25% of the initial damage unrepaired, whereas in NB-100 about 50% was unrepaired. This higher fraction of unrepaired DNA damage correlated well with the degree of sublethal damage repair which was lower in NB-100 than in HN-1 cell, but it did not correlate with the repair of potentially lethal damage, which was higher in NB-100 than in HN-1. Since the level of damage remaining post-irradiation may be the critical variable for survival, the FADU technique can contribute in elucidating the relationship between radiosensitivity and DNA damage repair capacity.     

Radiation damage to glucose concentrating capacity and cell survival in kidney tubule cells: effects of fractionation

The glucose concentrating capacity of cultured LLC-PK1 kidney epithelial cells has been measured after single and fractionated doses of X-rays. Steady-state glucose concentrating capacity (ratio of glucose concentration inside to outside cell) can be measured using radiolabelled analogues of glucose which are actively transported but not metabolized. These cells can be stimulated to increase their glucose concentrating capacity (up-regulation) by a reduction in the glucose concentration of the growth medium. However, after X-ray irradiation the cells have a reduced capacity to respond to up-regulation. This effect can be measured 7 days after irradiation and before radiation-induced cell killing affects the cell population. The previously reported radiosensitivity of this function to single doses of X-rays (in the range 1-16 Gy) was confirmed. Surprisingly, no significant sparing of this effect could be measured by fractionation of the X-ray dose into two or four fractions. However, the cells showed a significant fractionation effect if clonogenic survival was measured using the standard cell survival assay. These early effects have different fractionation response from the later phases of tissue damage, measured months to years after irradiation, which do show sparing due to fractionation and are thought to be mainly due to changes in cell survival. The lack of sparing by fractionation to the functional damage may suggest a different target from that which determines cell survival. These results support the hypothesis that radiation damages cellular functions, separately from cell replication.     

The relationship of DNA double-strand break induction to radiosensitivity in human tumour cell lines

Recent data suggest that the differences in radiosensitivity between cell lines can be related to differences in dsb induction (Radford 1986a). In the light of this we have set out to assess the extent to which differences in radiation survival between human tumour cell lines can be attributed to differences in dsb induction. For nine human tumour lines survival was assayed by clonogenic assay and compared with dsb induction by irradiation at ice-bath temperature as measured by neutral filter elution. The lines varied widely in their sensitivity, ranging from a sensitive neuroblastoma (surviving fraction at 2 Gy, SF2 = 0.13) to a resistant bladder carcinoma (SF2 = 0.62). Dsb induction was found to vary between the cell lines, such that resistant cells generally suffered less damage than sensitive ones. However, the relationship between damage induction and cellular sensitivity was not a simple one, and other factors which may influence sensitivity need to be invoked. These data suggest that, in human tumour cell lines, differences in radiosensitivity may at least in part be due to different levels of damage induction, but that some lines may vary in their tolerance of damage due to differences in biological characteristics such as repair capacity.     

Acute response of mouse kidney clonogens to fractionated irradiation in situ and then assayed in primary culture

Although the difference in sensitivity to the changes in dose fraction size between early-responding and late-responding tissues is well established, the underlying mechanisms in terms of target-cell responses are not yet clearly identified for any tissue. The radiosensitivity of mouse kidney cells after in situ single-dose, 2, 8, and 16 fraction X-irradiations was measured in primary culture using a clonogenic assay. The assay was made 12 h after single doses or 12 h after the last dose of the multifraction regimens. When analysed using the linear-quadratic model, as predicted the individual alpha components for all the different fractionation schedules were not significantly different, and the changes in the beta values were consistent with those expected on the basis of the reciprocal fraction numbers. When all four data sets were integrated to derive a common alpha/beta ratio, the result was 4.4 +/- 1.3 (1SE) Gy, or 2.8 +/- 0.9 Gy (a better fit) if the single-dose data set was excluded. These values fall into the range reported for kidney using assays of tissue function at long times after irradiation. Hence, it has been shown for the first time that the fractionation sensitivity of a late-responding organ is mimicked by that of a clonogenic cell population in that organ. The evidence also suggests that the time available prior to fixation of potentially lethal damage does not influence the low alpha/beta ratio observed for the kidney.     

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.     

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.     

Linear quadratic model of radiocurability on multicellular spheroids of human lung adenocarcinoma LCT1 and mouse fibrosarcoma FSA.

The LCT1 cells derived from a human lung adenocarcinoma and the FSA cells from a mouse fibrosarcoma were found to form spheroids. The cure-dose relationship of spheroids and the survival curves of their component cells were analysed by using a linear-quadratic model for cell survival and a Poisson distribution for cure. The analysis resulted in three conclusions: (1) the double minus logarithm of cure probability was linearly related to radiation dose, (2) the critical cell number was constant at any given cure probability, and (3) cellular radiosensitivity was also constant. The experiments seem to meet these conditions for each of two kinds of spheroids. Control doses (50%) were 20 Gy for LCT1 spheroids and 21 Gy for FSA spheroids, both 400 microns in diameter. The analysis showed that the lower cellular radiosensitivity and the higher number of clonogenic cells made LCT1 spheroids more radioresistant than FSA spheroids and that the higher critical number of 130 cells made the LCT1 spheroids more sensitive than the FSA spheroids with 18 such cells. The overall radiocurability of spheroids was a result of these three opposing effects, indicating that the critical cell number can be one important factor in determining the radiocurability of multicellular systems.     

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.     

3-氨基苯甲酰胺对人食管癌细胞放射增敏作用的实验研究

目的 探讨多聚(ADP-核糖)聚合酶(PARP)抑制剂3-氨基苯甲酰胺(3-AB)对人食管癌细胞株CaEs-17的放射增敏作用及其机制。方法 利用人食管癌细胞CaEs-17为靶细胞, 给予0、2.5、7.5 mmol/L 3-AB及0、3、6、9、12Gy照射剂量, 采用集落形成法及噻唑蓝(MTT)法观察3-AB的放射增敏效应;彗星电泳法、中期染色体分析法分别观察3-AB对放射所致细胞DNA断裂及细胞染色体畸变的影响。结果 根据多靶单击数学模型拟合细胞存活曲线, 得出2.5、7.5mmol/L 3-AB作用于CaEs-17细胞24h的放射增敏比分别为1.21和1.52;MTT实验结果显示3-AB可降低放射细胞的存活分数;3-AB可增加放射所致细胞DNA断裂程度以及放射所致细胞染色单体断裂数。结论3-AB对体外培养人食管癌细胞株CaEs-17有一定放射增敏效应;放射增敏的机制可能与3-AB抑制 PARP的功能从而降低细胞对放射所致DNA断裂的修复水平有关。     

The Relationship of DNA Double-strand Break Induction to Radiosensitivity in Human Tumour Cell Lines

Summary

Recent data suggest that the differences in radiosensitivity between cell lines can be related to differences in dsb induction (Radford 1986a). In the light of this we have set out to assess the extent to which differences in radiation survival between human tumour cell lines can be attributed to differences in dsb induction. For nine human tumour lines survival was assayed by clonogenic assay and compared with dsb induction by irradiation at ice-bath temperature as measured by neutral filter elution. The lines varied widely in their sensitivity, ranging from a sensitive neuroblastoma (surviving fraction at 2 Gy, SF₂ = 0·13) to a resistant bladder carcinoma (SF₂ = 0·62). Dsb induction was found to vary between the cell lines, such that resistant cells generally suffered less damage than sensitive ones. However, the relationship between damage induction and celluar sensitivity was not a simple one, and other factors which may influence sensitivity need to be invoked. These data suggest that, in human tumour cell lines, differences in radiosensitivity may at least in part be due to different levels of damage induction, but that some lines may vary in their tolerance of damage due to differences in biological characteristics such as repair capacity.     

Genotype-dependent radiosensitivity: clonogenic survival, apoptosis and cell-cycle redistribution

PURPOSE: We describe variations of three radiation-induced endpoints on the basis of cell genotype: Clonogenic survival, expression of apoptosis and cell-cycle redistribution. METHODS: Clonogenic survival, apoptosis and cell-cycle redistribution are measured in multiple cell lines after exposure to radiation between 2 and 16 Gy. Cell lines varied in clonogenic radiosensitivity and expression of specific genes. RESULTS: Clonal radiosensitivity is genotype-dependent, associating with four specific genes: A mutated form of Ataxia telangiectasia mutated (mutATM); with two forms of TP53, the gene that is template for tumor protein p53, wildtype TP53 (wtTP53) and mutated TP53 (mutTP53); and an unidentified gene in radioresistant glioblastoma cells. Apoptosis is also genotype-dependent showing elevated levels in cells that express mutATM and abrogated 14-3-3sigma (an isoform of the 14-3-3 gene) but less variation for different forms of TP53. Cell-cycle redistribution varied in mutATM cells. Kinetics of apoptosis are biphasic for both time and dose; cell lines did not express apoptosis at doses below 5 Gy or times before 24 hours. Kinetics of cell-cycle redistribution changed dynamically in the first 24 hours but showed little change after that time. CONCLUSIONS: Clonogenic survival, radiation-induced apoptosis and radiation-induced redistribution in the cell-cycle vary with cell genotype, but not the same genotypes. There is temporal, not quantitative, correlation between apoptosis and clonal radiosensitivity with apoptosis suppressed by lower, less toxic doses of radiation (<5 Gy) but enabled after larger, more toxic doses. Kinetic patterns for apoptosis and redistribution show a common change at approximately 24 hours.     

慧星电泳用于肿瘤细胞辐射敏感性检测的研究

目的：探讨彗星电泳方法检测肿瘤细胞对γ射线的辐射敏感性的可行性。方法：以自行设计的图像分析系统，用彗星电泳方法检测4种人肿瘤细胞受γ射线照射后细胞初始DNA损伤，以及细胞径30min修复时DNA损伤残余率；用细胞集落存活法检测2Gy γ射线照射后细胞存活率。结果：4种肿瘤细胞初始DNA损伤与辐射敏感性无关，2Gyγ射线照射后4种细胞存活率（SF2）与细胞经30min修复后的DNA损伤残余率相关明显（r=-0.87)，结论：本实验方法有可能成为一种快速、准确检测肿瘤细胞内在辐射敏感性的方法。     

Changes in renal function and oxidative damage in methamphetamine-treated rat

In this study, we observed renal damage and peroxidative injury as the acute or sub-acute effect of methamphetamine (MA) to determine whether MA intoxication can be diagnosed from immunohistochemical changes in the kidney. In addition, renal function was investigated in relation to the immunohistochemical changes. A single administration of MA (group I) (50mg/kg/ (i.p.)) and repeated administration (group II) (10mg/kg/day (i.p.) for 5 days) were designed as an acute model and a sub-acute or chronic model. Immunohistochemically, cell damage markers were observed. Then, renal function markers and minerals in blood were measured. Myoglobin and creatinine phosphokinase (CPK) in blood were also analyzed. In group I, ubiquitin immunoreactivity was enhanced only in the renal tubules. Creatinine increased, while K, Ca, and P decreased (P<0.01). CPK increased significantly (P<0.01). Therefore, it was suspected that MA might induce renal dysfunction with renal tubule damage. This damage might be related to leakage of CPK from muscle. In group II, 8-hydroxy-2'-deoxyguanosine (8-OH-dG) increased immunohistochemically and quantitatively (P<0.01). It was considered that oxidative DNA damage might be induced by repeated administration. It was considered that this study offers basic information for the evaluation of pathological changes in the kidney in MA-related autopsy cases.     

Chronic exposure to bryostatin-1 increases the radiosensitivity of U937 leukaemia cells ectopically expressing Bcl-2 through a non-apoptotic mechanism

PURPOSE: Ionizing radiation (IR) produced a dose-dependent increase in apoptosis in U937/pCEP4 cells which was attenuated by the stable over expression of Bcl-2 (U937/Bcl-2). A dose of 2 Gy IR was selected for further analyses to determine if subsequent exposure to 10nM bryostatin- would overcome the resistance to IR-induced apoptosis conferred by Bcl-2 over expression. METHODS AND RESULTS: Although bryostatin- did not increase IR-induced apoptosis in U937/pCEP4 or U937/Bcl-2 cells, it impaired mitochondrial function and increased the antiproliferative effects of IR in both cell lines. The effects were more pronounced in U937/Bcl-2 cells. Bryostatin-1 also exerted differential effects on cell-cycle distributions of U937 transfectant cells, producing a significant G0/G1 arrest in U937/Bcl-2 cells, while decreasing IR-induced G2/M arrest in U937/pCEP4 cells. Although Bcl-2 over expression attenuated IR-induced apoptosis, clonogenic survival was similar in U937/pCEP4 and U937/Bcl-2 cells following 2 Gy IR treatment. Treatment with 10nM bryostatin-1 after 2 Gy IR further reduced clonogenic survival in both cell lines. Moreover, U937/Bcl-2 cells were more susceptible to the growth-inhibitory effects of IR/bryostatin-1 than U937/pCEP4 cells. CONCLUSIONS: Bryostatin-1 increased the radiosensitivity of U937 transfectant cell lines without enhancing apoptosis; furthermore, U937/Bcl-2 cells were more susceptible to IR/bryostatin-1-mediated antiproliferative effects than their empty-vector counterparts.     

Chronic exposure to bryostatin-1 increases the radiosensitivity of U937 leukaemia cells ectopically expressing Bcl-2 through a non-apoptotic mechanism

Purpose : Ionizing radiation (IR) produced a dose-dependent increase in apoptosis in U937/pCEP4 cells which was attenuated by the stable over expression of Bcl-2 (U937/Bcl-2). A dose of 2 Gy IR was selected for further analyses to determine if subsequent exposure to 10 n m bryostatin-1 would overcome the resistance to IR-induced apoptosis conferred by Bcl-2 over expression. Methods and results : Although bryostatin-1 did not increase IR-induced apoptosis in U937/pCEP4 or U937/Bcl-2 cells, it impaired mitochondrial function and increased the antiproliferative effects of IR in both cell lines. The effects were more pronounced in U937/Bcl-2 cells. Bryostatin-1 also exerted differential effects on cell-cycle distributions of U937 transfectant cells, producing a significant G 0 /G1 arrest in U937/Bcl-2 cells, while decreasing IR-induced G 2 /M arrest in U937/pCEP4 cells. Although Bcl-2 over expression attenuated IR-induced apoptosis, clonogenic survival was similar in U937/pCEP4 and U937/Bcl-2 cells following 2Gy IR treatment. Treatment with 10nm bryostatin-1 after 2 Gy IR further reduced clonogenic survival in both cell lines. Moreover, U937/Bcl-2 cells were more susceptible to the growth-inhibitory effects of IR/bryostatin-1 than U937/pCEP4 cells. Conclusions : Bryostatin-1 increased the radiosensitivity of U937 transfectant cell lines without enhancing apoptosis; furthermore, U937/Bcl-2 cells were more susceptible to IR/bryostatin1-mediated antiproliferative effects than their empty-vector counterparts.