Inhibition of radiation-induced apoptosis in vitro by tumor promoters.
Inhibition of radiation-induced apoptosis in vitro by tumor promoters.
复制标题
肿瘤促进剂在体外抑制辐射诱导的细胞凋亡。
DOI：
10.1016/s0006-291x(88)81088-x
复制
发表时间：
1988
影响因子：
3.1
通讯作者：
Wenner,CE
中科院分区：
文献类型：
--
作者：
Tomei,LD;Kanter,P;Wenner,CE
材料和方法C3 H-10 T1/2（CLS）小鼠成纤维细胞的培养物如（14）所述使用补充有10%热灭活胎牛血清（Sterile Systems，Logan，UT）的基础Eagle培养基（GIBCO）维持。每3-4天更新生长培养基。在8-10天达到汇合，汇合后的细胞用于实验（汇合后48-72小时）。在60 mm直径培养板（Falcon Plastics）中对一式两份培养物进行实验。药物：DHTB是T. Sugimura和H. Fujita（国立癌症研究所，东京）。TPA和类似物获自Chemical Carcinogens，伊甸园草原，MN。3 H-胸苷（3 H-dThd）购自ICN。环己酰亚胺（试剂级）获自Sigma Chemicals。如先前由Kanter和Schwartz（15，16）所述测量DNA片段化，并且片段化指数计算为从单链DNA提取的放射性与从单链和双链DNA提取的总量的商。分别于T= 0 ~ 24 h、24 ~ 48 h和48 ~ 72 h测定~ 3 H-dThd的24 h累积掺入量。如表1所示，在血清抽取后的前24小时内观察到的放射性标记细胞核的密度（1 μ Ci/5 ml，5 Ci/mmole 3 H-dThd）为2.23 x 103标记细胞核/cmL，而在用10-sM TPA处理的细胞中，该值增加至6.13 x 103，反映了预期的促有丝分裂活性。随着细胞达到静止，在第二和第三个24小时期间观察到的标记核的数量减少至0.52和0.88标记核/cm z。总细胞密度显示出类似的降低，从8.38 × 104个细胞核/cm 2降低到第三个和最后一个24小时周期结束时的5.9 × 104个细胞核/cm 2的稳定平台。当TPA在血清后的第二或第三阶段加入时，
MATERIALS AND METHODS Cells: Cultures of C3H-10T1/2 (CLS) mouse fibroblasts were maintained as described (14) using Basal Eagle's Medium (GIBCO) supplemented with 10% heat-inactivated fetal bovine serum (Sterile Systems, Logan, UT). Growth medium was renewed every 3-4 days. Confluence was reached at 8-10 days, and postconfluent cells were used for the experiments (48-72 hours after the time of confluence). Experiments were performed on duplicate cultures in 60 mm diameter culture plates (Falcon Plastics). Drugs: DHTB was a gift of T. Sugimura and H. Fujita (National Cancer Research Institute, Tokyo). TPA and analogs were obtained from Chemical Carcinogens, Eden Prairie, MN. 3H-Thymidine (3H-dThd) was purchased from ICN. Cycloheximide {reagent grade) was obtained from Sigma Chemicals. DNA fragmentation is measured as previously described by Kanter and Schwartz (15, 16) and a fragmentation index is calculated as the quotient of the radioactivity extracted from the single stranded DNA and the total amount extracted from both single and double stranded DNA.RESULTS Serum deprivation: Replicate cultures of exponentially proliferating 10T1/2 cells were transferred to serum-free medium at T= O. The 24h cumulative incorporation of 3H-dThd was then determined for three sequential periods from T= O to 24h, 24h to 48h, and 48h to 72h respectively. As seen in Table 1, the density of radiolabelled nuclei observed (1 uCi/5ml, 5 Ci/mmole 3H-dThd) during the first 24h period following serum withdrawal was found to be 2.23 x 103 labeled nuclei/cmL whereas, in those treated with 10-sM TPA this value increased to 6.13 x 103 reflecting the expected mitogenic activity. The number of labeled nuclei observed in the second and third 24h period was reduced to 0.52 and 0.88 labeled nuclei/cm z as the cells reached quiescence. The total cell density showed a similar reduction from 8.38 x 104 nuclei/cm 2, to a stable plateau of 5.9 x 104 nuclei/cm z at the end of the third and final 24h period. When TPA was added during the second or third periods after serum