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Limitations of the fluorescent probe viability assay. 荧光探针活力测定的局限性。
Pub Date : 1989-10-01
E J Massaro, K H Elstein, R M Zucker, K W Bair

In vitro assessment of the efficacy/capacity of toxicants (e.g., cancer chemotherapeutic agents, environmental pollutants, etc.) to damage/kill cells and/or inhibit growth (cell duplication) requires accurate measurement of target cell viability as a function of exposure. Rapid measurement of viability, such as can be achieved employing fluorescent probes of metabolic function in combination with instrumental analysis, is highly desirable. However, we observe that exposure to chemicals (of unrelated type) complicates the interpretation of viability data and, in the case of perturbed cells, questions the validity of viability growth assays based on intrinsic enzyme activity. Viability commonly is determined flow cytometrically (FCM) by the carboxyfluorescein diacetate (CFDA)/propidium iodide (PI) assay. Nonfluorescent CFDA is taken up by diffusion and converted via cytoplasmic esterase-catalyzed hydrolysis to carboxyfluorescein (CF), a negatively charged fluorescent molecule that is retained (incompletely) by the cell. As such, if CF fluorescence intensity is a relative measure of enzyme activity, it also can be considered an index of cellular vigor (metabolic rate). It is generally accepted that the viable cell excludes both basic dyes, such as PI, and acidic dyes, such as trypan blue, and uptake is indicative of irreversible cellular injury presaging cell death. We observe that, following incubation for 4 h with 0.5-1.0 microM tributyltin (TBT), a potent environmental toxicant, murine erythroleukemic cells (MELC) exhibit enhanced (supranormal) CF fluorescence compared to control cells. Apparent cell volume (ACV) is unaltered, and because such cells exclude PI, they are considered viable in terms of the CFDA/PI assay. However, rate of growth (increase in cell number over 48 h) is depressed, suggesting that supranormal CF fluorescence, even in the absence of PI uptake, is indicative of cellular perturbation. In effect, although CF fluorescence is the product of an enzyme-catalyzed reaction and, therefore, an indicator of vital function (enzyme activity), it apparently is not a reliable index of cellular vigor. At higher TBT concentrations (greater than 1.0, but less than 50.0 microM), the cells exhibit both increased CF fluorescence and PI fluorescence and are growth inhibited. MELC exposed to the cancer chemotherapeutic agents adriamycin, m-AMSA, or crisnatol (Burroughs Wellcome 770U82) also exhibit increased cellular CF fluorescence. However, rate of growth is decreased and ACV increased. The latter, measured either as a function of electrical resistance (Coulter volume) or by the FCM parameter axial light loss could account for the increase in mean CF fluorescence.(ABSTRACT TRUNCATED AT 400 WORDS)

在体外评估有毒物质(如癌症化疗药物、环境污染物等)损害/杀死细胞和/或抑制生长(细胞复制)的功效/能力,需要准确测量靶细胞活力作为暴露的函数。非常需要快速测量活力,例如可以使用代谢功能的荧光探针与仪器分析相结合来实现。然而,我们观察到,暴露于化学物质(不相关类型)使活力数据的解释复杂化,并且在受干扰细胞的情况下,质疑基于内在酶活性的活力生长测定的有效性。生存能力通常是通过二乙酸羧基荧光素(CFDA)/碘化丙啶(PI)测定的流式细胞术(FCM)来确定的。非荧光CFDA被扩散吸收,并通过胞质酯酶催化水解转化为羧基荧光素(CF),这是一种带负电的荧光分子,被细胞保留(不完全保留)。因此,如果CF荧光强度是酶活性的相对度量,它也可以被认为是细胞活力(代谢率)的指标。人们普遍认为,活细胞不包括碱性染料,如PI和酸性染料,如台盼蓝,摄取是不可逆的细胞损伤预示着细胞死亡。我们观察到,与0.5-1.0微米的三丁基锡(TBT)(一种强效环境毒物)孵育4小时后,小鼠红细胞白血病细胞(MELC)与对照细胞相比表现出增强的(异常的)CF荧光。表观细胞体积(ACV)不变,由于这些细胞排除了PI,因此根据CFDA/PI检测,它们被认为是活的。然而,生长速度(超过48小时的细胞数量增加)下降,表明即使在没有PI摄取的情况下,异常的CF荧光也表明细胞受到扰动。实际上,虽然CF荧光是酶催化反应的产物,因此是一种重要功能(酶活性)的指标,但它显然不是细胞活力的可靠指标。在较高的TBT浓度(大于1.0,但小于50.0微米)下,细胞的CF荧光和PI荧光均增加,生长受到抑制。暴露于癌症化疗药物阿霉素、m-AMSA或crisnatol (Burroughs Wellcome 770U82)的MELC也表现出细胞CF荧光增加。但是,生长速率降低,ACV增加。后者,作为电阻(库尔特体积)的函数或通过FCM参数测量,轴向光损失可以解释平均CF荧光的增加。(摘要删节为400字)
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引用次数: 0
Xenobiotic regulation of glutathione S-transferase Ya gene expression. 谷胱甘肽s -转移酶Ya基因表达的外源调控。
Pub Date : 1989-10-01
K E Paulson

The gene for the rat GST Ya subunit has been examined in detail as a model for understanding the molecular mechanisms of inducibility by xenobiotics and their tissue-specific regulation. The focus of this article is to describe our current understanding of these mechanisms. The discussion will begin with the classification of the types of inducing agents. These pioneering studies suggested that there were multiple mechanisms for the inducibility of GSTs. In fact, the analysis of GST Ya gene expression has identified two different upstream activating elements and putative protein factors through which different classes of inducers act. Finally, the position-specific and tissue-specific regulation of the GST Ya gene will be discussed.

大鼠GST Ya亚基基因已被详细研究,作为理解外源性药物诱导及其组织特异性调控的分子机制的模型。本文的重点是描述我们目前对这些机制的理解。讨论将从诱导剂类型的分类开始。这些开创性的研究表明,gst的诱导有多种机制。事实上,对GST Ya基因表达的分析已经确定了两种不同的上游激活元件和假定的蛋白质因子,不同类型的诱导剂通过它们起作用。最后,将讨论GST Ya基因的位置特异性和组织特异性调控。
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引用次数: 0
Induction of a novel damage-specific DNA binding protein correlates with enhanced DNA repair in primate cells. 一种新的损伤特异性DNA结合蛋白的诱导与灵长类细胞中DNA修复的增强相关。
Pub Date : 1989-10-01 DOI: 10.1201/9781003075387-2
M. Protic, S. Hirschfeld, A. P. Tsang, M. Wagner, K. Dixon, A. Levine
Pretreatment of mammalian cell with DNA-damaging agents, such as UV light or mitomycin C, but not the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), results in the enhanced repair of subsequently transfected UV-damaged expression vectors. To determine the cellular factors that are responsible for this enhancement, we have used a modified gel retardation assay to detect the proteins that interact with damaged DNA. We have identified a constitutive DNA binding protein in extracts from primate cells that has a high affinity for UV-irradiated double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin, but not TPA or serum starvation, have higher levels of this damage-specific DNA binding (DDB) protein. These results suggest that the signal for induction of DDB protein can either be damage to the DNA or interference with cellular DNA replication. The induction of DDB protein varies among primate cells with different phenotypes: (1) virus-transformed repair-proficient cells have partially or fully lost the ability to induce DDB protein above constitutive levels; (2) primary cells from repair-deficient xeroderma pigmentosum (XP) group C, and transformed XP groups A and D, show constitutive DDB protein, but do not show induced levels of this protein 48 h after UV; and (3) primary and transformed repair-deficient cells from one XP E patient are lacking both the constitutive and the induced DDB activity. The correlation between the induction of the DDB protein and the enhanced repair of UV-damaged expression vectors implies the involvement of the DDB protein in this inducible cellular response.
用dna损伤剂预处理哺乳动物细胞,如紫外线或丝裂霉素C,而不是肿瘤启动子12- o -tetradecanoyl- phorboll -13-acetate (TPA),结果增强了随后转染的紫外线损伤表达载体的修复。为了确定导致这种增强的细胞因素,我们使用了一种改进的凝胶延迟试验来检测与受损DNA相互作用的蛋白质。我们已经从灵长类细胞的提取物中鉴定出一种对紫外线照射的双链DNA具有高亲和力的DNA结合蛋白。用紫外光、丝裂霉素C或阿霉素预处理的细胞,而不是TPA或血清饥饿处理的细胞,具有更高水平的这种损伤特异性DNA结合(DDB)蛋白。这些结果表明,诱导DDB蛋白的信号可能是DNA损伤或干扰细胞DNA复制。在不同表型的灵长类动物细胞中,DDB蛋白的诱导存在差异:(1)病毒转化的修复熟练细胞部分或完全丧失了诱导DDB蛋白高于组成水平的能力;(2)修复缺陷性着色性干皮病(XP) C组和转化后的XP A、D组原代细胞均可见组成性DDB蛋白,但在紫外线照射48 h后未显示该蛋白的诱导水平;(3)来自一名XP E患者的原代和转化修复缺陷细胞既缺乏构成型活性,也缺乏诱导型活性。DDB蛋白的诱导与uv损伤表达载体的增强修复之间的相关性表明DDB蛋白参与了这种可诱导的细胞反应。
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引用次数: 18
Review: gene amplification--a cellular response to genotoxic stress. 综述:基因扩增——对基因毒性应激的细胞反应。
Pub Date : 1989-10-01
C Lüke-Huhle

Recent years of cancer research have defined the role of key regulatory genes in oncogenesis. Oncogenes and suppressor genes are affected in the process of carcinogenesis either by mutations within the coding region, promoter mutations, or gene amplification. This review describes our studies on gene amplification in mammalian cells, with emphasis on the initiating events induced by carcinogenic chemicals and various types of radiation. The influence of genomic instability, cell dedifferentiation, and the malignant potential of a cell on their capacity to amplify genes is demonstrated by molecular biologic and cytogenetic studies on human and rodent cells. Cells that contain amplified DNA are at risk for chromosomal aberrations, sister chromatid exchanges, and rearrangements. Surviving cells show such cancer-prone genetic consequences.

近年来的癌症研究明确了关键调控基因在肿瘤发生中的作用。致癌基因和抑制基因在癌变过程中受到编码区突变、启动子突变或基因扩增的影响。本文综述了我们在哺乳动物细胞中基因扩增的研究,重点介绍了致癌化学物质和各种类型辐射诱导的启动事件。对人类和啮齿动物细胞进行的分子生物学和细胞遗传学研究证明了基因组不稳定性、细胞去分化和细胞的恶性潜能对其基因扩增能力的影响。含有扩增DNA的细胞存在染色体畸变、姐妹染色单体交换和重排的风险。存活的细胞显示出这种易患癌症的遗传后果。
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引用次数: 0
Induction of a novel damage-specific DNA binding protein correlates with enhanced DNA repair in primate cells. 一种新的损伤特异性DNA结合蛋白的诱导与灵长类细胞中DNA修复的增强相关。
Pub Date : 1989-10-01
M Protić, S Hirschfeld, A P Tsang, M Wagner, K Dixon, A S Levine

Pretreatment of mammalian cell with DNA-damaging agents, such as UV light or mitomycin C, but not the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA), results in the enhanced repair of subsequently transfected UV-damaged expression vectors. To determine the cellular factors that are responsible for this enhancement, we have used a modified gel retardation assay to detect the proteins that interact with damaged DNA. We have identified a constitutive DNA binding protein in extracts from primate cells that has a high affinity for UV-irradiated double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin, but not TPA or serum starvation, have higher levels of this damage-specific DNA binding (DDB) protein. These results suggest that the signal for induction of DDB protein can either be damage to the DNA or interference with cellular DNA replication. The induction of DDB protein varies among primate cells with different phenotypes: (1) virus-transformed repair-proficient cells have partially or fully lost the ability to induce DDB protein above constitutive levels; (2) primary cells from repair-deficient xeroderma pigmentosum (XP) group C, and transformed XP groups A and D, show constitutive DDB protein, but do not show induced levels of this protein 48 h after UV; and (3) primary and transformed repair-deficient cells from one XP E patient are lacking both the constitutive and the induced DDB activity. The correlation between the induction of the DDB protein and the enhanced repair of UV-damaged expression vectors implies the involvement of the DDB protein in this inducible cellular response.

用dna损伤剂预处理哺乳动物细胞,如紫外线或丝裂霉素C,而不是肿瘤启动子12- o -tetradecanoyl- phorboll -13-acetate (TPA),结果增强了随后转染的紫外线损伤表达载体的修复。为了确定导致这种增强的细胞因素,我们使用了一种改进的凝胶延迟试验来检测与受损DNA相互作用的蛋白质。我们已经从灵长类细胞的提取物中鉴定出一种对紫外线照射的双链DNA具有高亲和力的DNA结合蛋白。用紫外光、丝裂霉素C或阿霉素预处理的细胞,而不是TPA或血清饥饿处理的细胞,具有更高水平的这种损伤特异性DNA结合(DDB)蛋白。这些结果表明,诱导DDB蛋白的信号可能是DNA损伤或干扰细胞DNA复制。在不同表型的灵长类动物细胞中,DDB蛋白的诱导存在差异:(1)病毒转化的修复熟练细胞部分或完全丧失了诱导DDB蛋白高于组成水平的能力;(2)修复缺陷性着色性干皮病(XP) C组和转化后的XP A、D组原代细胞均可见组成性DDB蛋白,但在紫外线照射48 h后未显示该蛋白的诱导水平;(3)来自一名XP E患者的原代和转化修复缺陷细胞既缺乏构成型活性,也缺乏诱导型活性。DDB蛋白的诱导与uv损伤表达载体的增强修复之间的相关性表明DDB蛋白参与了这种可诱导的细胞反应。
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引用次数: 0
Characteristics of lead adaptation in a rat kidney cell line. II. Effect on DNA synthesis, protein synthesis, and gene expression. 大鼠肾细胞系对铅的适应特性。2对DNA合成、蛋白质合成和基因表达的影响。
Pub Date : 1989-07-01
B Hitzfeld, D M Taylor

The effects of adaptation of normal rat kidney cells (NRK 52-E) to growth in 5 or 10 microM lead nitrate on the rates of DNA synthesis and on the rate and pattern of protein synthesis was studied. The rate of [3H]thymidine incorporation into DNA was increased in normal cells, but remained unchanged in one lead-adapted cell line (only 5 microM NRK studied). Increased rates of [3H]leucine incorporation into nonadapted NRK cells were found only at times up to 3 h; in contrast, the lead-adapted cells showed such increases only at longer times. The most pronounced differences between normal and lead-adapted cells were found with lead concentrations of 10 or 50 microM lead nitrate. Lead-adapted control cells incorporated 170% of the [3H]leucine taken up by nonadapted cells. In both adapted and nonadapted cells the pattern of synthesis of specific proteins showed varied and dose-dependent differences between the three cell sublines examined. The changed sensitivity of both DNA and protein synthesis following lead exposure appears to be a potent parameter in the development of resistance, perhaps through the development of specific lead-binding proteins.

研究了正常大鼠肾细胞(NRK 52-E)在5和10 μ m硝酸铅中生长的适应性对其DNA合成速率、蛋白质合成速率和模式的影响。正常细胞中[3H]胸苷并入DNA的速率增加,但在一种铅适应细胞系中保持不变(仅研究了5微米NRK)。[3H]亮氨酸掺入非适应性NRK细胞的比率仅在3小时内增加;相比之下,适应铅的细胞仅在较长时间内表现出这种增长。正常细胞和铅适应细胞之间最明显的差异是在铅浓度为10或50微米硝酸铅时发现的。铅适应对照细胞吸收了未适应细胞吸收的170%的[3H]亮氨酸。在适应和非适应细胞中,特定蛋白质的合成模式在所检查的三种细胞亚系之间表现出不同的剂量依赖性差异。铅暴露后DNA和蛋白质合成敏感性的变化似乎是耐药性发展的一个有力参数,可能是通过特定铅结合蛋白的发展。
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引用次数: 0
Plasmid-aided insertion of MMTV-LTR and ras DNAs to NIH 3T3 fibroblast cells makes them responsive to 2,3,7,8-TCDD causing overexpression of p21ras and down-regulation of EGF receptor. 质粒辅助将MMTV-LTR和ras dna插入NIH 3T3成纤维细胞,使其对2,3,7,8- tcdd产生应答,导致p21ras过表达和EGF受体下调。
Pub Date : 1989-07-01
J Jankun, F Matsumura, H Kaneko, J E Trosko, A Pellicer, A H Greenberg

TCDD administered to NIH 3T3 fibroblast cells transfected with a plasmid containing MMTV-LTR and mouse ras DNAs caused an increased level of p21ras protein and down-regulation of EGF receptor. This effect occurred only in the cells with introduced N-ras or Ha-ras under transcriptional control of glucocorticoid-sensitive MMTV-LTR but not ones without these DNAs. The MMTV-LTR ras-incorporated cells treated with either dexamethasone or TCDD grew in soft agar to form colonies (anchorage independent growth), while nontreated cells did not, indicating profound cellular changes due to activation of N-ras by these two agents.

用含有MMTV-LTR和小鼠ras dna的质粒转染NIH 3T3成纤维细胞,TCDD可引起p21ras蛋白水平升高和EGF受体下调。这种效应只发生在糖皮质激素敏感的MMTV-LTR转录控制下引入N-ras或Ha-ras的细胞中,而不存在这些dna的细胞中。用地塞米松或TCDD处理的MMTV-LTR ras结合细胞在软琼脂中生长形成集落(锚定独立生长),而未处理的细胞则没有,表明这两种药物激活N-ras导致细胞发生了深刻的变化。
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引用次数: 0
Characteristics of lead adaptation in a rat kidney cell line. I. Uptake and subcellular and subnuclear distribution of lead. 大鼠肾细胞系对铅的适应特性。铅的摄取和亚细胞和亚核分布。
Pub Date : 1989-07-01
B Hitzfeld, D M Taylor

Two sublines of normal rat kidney cell (NRK 52-E) that are resistant to 5 and 10 microM lead nitrate have been developed and characterized. The cellular and nuclear uptake of 210Pb, as well as the subnuclear distribution, was unchanged by lead adaptation: it remained at values of 2.3-6.6 pmole 210Pb/microgram protein/h. Also no difference in the slope of the curve of the uptake rate of the three cell types could be detected. Studies of the nuclear uptake of 210Pb showed that in both normal and lead-adapted cells the major lead fraction was associated with the chromatin and mostly (63-67%) with the nuclear proteins. Electron microscopic studies demonstrated that particulate lead was taken up in all lead-exposed cells. It was concluded that development of resistance to lead does not arise from a reduced rate of cellular uptake, from increased excretion, or from changes in nuclear uptake or in the subnuclear distribution within the cells.

研究了正常大鼠肾细胞NRK 52-E对5和10 μ m硝酸铅的耐药亚系。铅适应后,细胞和细胞核对210Pb的摄取以及亚核分布没有变化,保持在2.3-6.6摩尔210Pb/微克蛋白/小时。三种细胞摄取速率曲线的斜率也无差异。对210Pb核摄取的研究表明,在正常细胞和铅适应细胞中,铅的主要部分与染色质有关,大部分(63-67%)与核蛋白有关。电子显微镜研究表明,微粒铅在所有铅暴露的细胞中被吸收。结论是,对铅的抗性的发展不是由于细胞摄取率的降低、排泄量的增加或细胞内核摄取或亚核分布的变化引起的。
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引用次数: 0
Differential DNase I hypersensitivity of ras oncogenes in B6C3F1, C3H/He, and C57BL/6 mouse liver. B6C3F1、C3H/He和C57BL/6小鼠肝脏中ras癌基因的差异DNase I超敏性
Pub Date : 1989-07-01
R L Vorce, J I Goodman

The male hybrid B6C3F1 mouse exhibits a 30% spontaneous hepatoma incidence, whereas the paternal C3H/He strain and the maternal C57BL/6 strain exhibit a 60% and a negligible incidence, respectively. In addition, both male and female B6C3F1 mice are extremely sensitive to chemical induction of hepatocarcinogenesis. The Ha-ras, Ki-ras, and myc oncogenes have been implicated in a variety of solid tumors. Specifically, Ha- and, less frequently, Ki-ras have been reported to be activated in B6C3F1 mouse liver tumors. The objective of this study was to examine a possible point of transcriptional control of Ha-ras, Ki-ras, and myc in all three mouse strains, our hypothesis being that these oncogenes may be primed for expression in the nascent liver of those strains exhibiting a high spontaneous hepatoma incidence. A positive correlation has been established between gene expression and the presence of DNAase I hypersensitive sites. DNase I hypersensitive sites were observed in the Ha-ras and myc oncogenes in the three mouse strains. However, Ha-ras appears to possess an additional site in B6C3F1 and C3H/He as compared to C57BL/6. Similarly, the Ki-ras oncogene exhibited a DNase I hypersensitive site only in B6C3F1 and C3H/He mouse liver. These results indicate that the hepatoma-prone strains (B6C3F1 and C3H/He) may have a greater potential for Ha- and Ki-ras expression than does the non-hepatoma-prone strain (C57BL/6).

雄性杂交B6C3F1小鼠的自发性肝癌发生率为30%,而父系C3H/He株和母系C57BL/6株的自发性肝癌发生率分别为60%和可忽略不计。此外,雄性和雌性B6C3F1小鼠对化学诱导肝癌发生都极为敏感。Ha-ras、Ki-ras和myc癌基因与多种实体瘤有关。具体来说,Ha-和较少出现的Ki-ras在B6C3F1小鼠肝肿瘤中被激活。本研究的目的是研究在所有三种小鼠品系中Ha-ras、Ki-ras和myc可能的转录控制点,我们的假设是这些癌基因可能在那些表现出高自发性肝癌发病率的品系的新生肝脏中被启动表达。基因表达与DNAase I超敏位点存在正相关。在3株小鼠的Ha-ras和myc癌基因中均观察到dna酶I的超敏位点。然而,与C57BL/6相比,Ha-ras似乎在B6C3F1和C3H/He中拥有一个额外的位点。同样,Ki-ras癌基因仅在B6C3F1和C3H/He小鼠肝脏中表现出DNase I超敏位点。这些结果表明,肝癌易发菌株(B6C3F1和C3H/He)可能比非肝癌易发菌株(C57BL/6)具有更大的Ha-和Ki-ras表达潜力。
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引用次数: 0
Meeting report: thirteenth annual interdisciplinary cancer research workshop. 会议报告:第十三届跨学科癌症研究研讨会。
Pub Date : 1989-07-01
C Parkanyi, P Politzer
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引用次数: 0
期刊
Molecular toxicology
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