Leung Euphemia Y, Askarian-Amiri Marjan E, Singleton Dean C, Ferraro-Peyret Carole, Joseph Wayne R, Finlay Graeme J, Broom Reuben J, Kakadia Purvi M, Bohlander Stefan K, Marshall Elaine, Baguley Bruce C
Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand.
Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand.
Front Oncol. 2018 Oct 12;8:425. doi: 10.3389/fonc.2018.00425. eCollection 2018.
Most human breast cancer cell lines currently in use were developed and are cultured under ambient (21%) oxygen conditions. While this is convenient in practical terms, higher ambient oxygen could increase oxygen radical production, potentially modulating signaling pathways. We have derived and grown a series of four human breast cancer cell lines under 5% oxygen, and have compared their properties to those of established breast cancer lines growing under ambient oxygen. Cell lines were characterized in terms of appearance, cellular DNA content, mutation spectrum, hormone receptor status, pathway utilization and drug sensitivity. Three of the four lines (NZBR1, NZBR2, and NZBR4) were triple negative (ER-, PR-, HER2-), with NZBR1 also over-expressing EGFR. NZBR3 was HER2+ and ER+ and also over-expressed EGFR. Cell lines grown in 5% oxygen showed increased expression of the hypoxia-inducible factor 1 (HIF-1) target gene carbonic anhydrase 9 () and decreased levels of ROS. As determined by protein phosphorylation, NZBR1 showed low AKT pathway utilization while NZBR2 and NZBR4 showed low p70S6K and rpS6 pathway utilization. The lines were characterized for sensitivity to 7-hydroxytamoxifen, doxorubicin, paclitaxel, the PI3K inhibitor BEZ235 and the HER inhibitors lapatinib, afatinib, dacomitinib, and ARRY-380. In some cases they were compared to established breast cancer lines. Of particular note was the high sensitivity of NZBR3 to HER inhibitors. The spectrum of mutations in the NZBR lines was generally similar to that found in commonly used breast cancer cell lines but mutations were absent and mutations in , and , which have not been reported in other breast cancer lines, were detected. The results suggest that the properties of cell lines developed under low oxygen conditions (5% O) are similar to those of commonly used breast cancer cell lines. Although reduced ROS production and increased HIF-1 activity under 5% oxygen can potentially influence experimental outcomes, no difference in sensitivity to estrogen or doxorubicin was observed between cell lines cultured in 5 vs. 21% oxygen.
目前使用的大多数人乳腺癌细胞系都是在环境（21%）氧气条件下建立并培养的。虽然这在实际操作上很方便，但较高的环境氧气水平可能会增加氧自由基的产生，从而潜在地调节信号通路。我们在5%氧气条件下衍生并培养了一系列四种人乳腺癌细胞系，并将它们的特性与在环境氧气条件下生长的已建立乳腺癌细胞系的特性进行了比较。细胞系在外观、细胞DNA含量、突变谱、激素受体状态、信号通路利用情况和药物敏感性方面进行了表征。四个细胞系中的三个（NZBR1、NZBR2和NZBR4）是三阴性（ER-、PR-、HER2-），其中NZBR1还过表达EGFR。NZBR3是HER2+和ER+，也过表达EGFR。在5%氧气条件下生长的细胞系显示缺氧诱导因子1（HIF-1）靶基因碳酸酐酶9（）的表达增加，活性氧（ROS）水平降低。通过蛋白质磷酸化测定，NZBR1显示AKT信号通路利用率低，而NZBR2和NZBR4显示p70S6K和rpS6信号通路利用率低。这些细胞系对7-羟基他莫昔芬、阿霉素、紫杉醇、PI3K抑制剂BEZ235以及HER抑制剂拉帕替尼、阿法替尼、达可替尼和ARRY-380的敏感性进行了表征。在某些情况下，将它们与已建立的乳腺癌细胞系进行了比较。特别值得注意的是NZBR3对HER抑制剂的高敏感性。NZBR细胞系中的突变谱与常用乳腺癌细胞系中发现的突变谱总体相似，但未检测到突变，并且检测到了、和中的突变，这些突变在其他乳腺癌细胞系中尚未报道。结果表明，在低氧条件（5% O）下建立的细胞系的特性与常用乳腺癌细胞系的特性相似。虽然在5%氧气条件下ROS产生减少和HIF-1活性增加可能会潜在地影响实验结果，但在培养于5%与21%氧气条件下的细胞系之间，未观察到对雌激素或阿霉素敏感性的差异。
