Wolf Steven J, Huynh Tony, Bryce Nicole S, Hambley Trevor W, Wakelin Laurence P G, Stewart Bernard W, Catchpoole Daniel R
Biospecimens Research and Tumour Bank, Children's Cancer Research Unit, The Children's Hospital at Westmead, Westmead, NSW 2774, Australia.
BMC Cell Biol. 2011 Aug 24;12:36. doi: 10.1186/1471-2121-12-36.
Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma derived from skeletal muscle precursor cells, which accounts for 5-8% of all childhood malignancies. Disseminated RMS represents a major clinical obstacle, and the need for better treatment strategies for the clinically aggressive alveolar RMS subtype is particularly apparent. Previously, we have shown that the acridine-4-carboxamide derivative AS-DACA, a known topoisomerase II poison, is potently cytotoxic in the alveolar RMS cell line RH30, but is 190-fold less active in the embryonal RMS cell line RD. Here, we investigate the basis for this selectivity, and demonstrate in these RMS lines, and in an AS-DACA- resistant subclone of RH30, that AS-DACA-induced cytotoxicity correlates with the induction of DNA double strand breaks.
We show that inhibition of the multidrug-resistance associated protein (MRP1) has no effect on AS-DACA sensitivity. By exploiting the pH-dependent fluorescence properties of AS-DACA, we have characterized its intracellular distribution, and show that it concentrates in the cell nucleus, as well as in acidic vesicles of the membrane trafficking system. We show that fluorescence microscopy can be used to determine the localization of AS-DACA to the nuclear and cytoplasmic compartments of RMS cells grown as spheroids, penetrance being much greater in RH30 than RD spheroids, and that the vesicular signal leads the way into the spheroid mass. EEA1 and Rab5 proteins, molecular markers expressed on early-endosomal vesicles, are reduced by >50% in the sensitive cell lines.
Taking the evidence as a whole, suggests that endosomal vesicle trafficking influences the toxicity of AS-DACA in RMS cells.
横纹肌肉瘤（RMS）是一种源自骨骼肌前体细胞的恶性软组织肉瘤，占所有儿童恶性肿瘤的5 - 8%。播散性RMS是一个主要的临床障碍，对于临床上侵袭性较强的肺泡型RMS亚型，更优治疗策略的需求尤为明显。此前，我们已经表明，吖啶-4-甲酰胺衍生物AS-DACA是一种已知的拓扑异构酶II毒物，在肺泡型RMS细胞系RH30中具有强大的细胞毒性，但在胚胎型RMS细胞系RD中的活性低190倍。在此，我们研究这种选择性的基础，并在这些RMS细胞系以及RH30的AS-DACA抗性亚克隆中证明，AS-DACA诱导的细胞毒性与DNA双链断裂的诱导相关。
我们表明，抑制多药耐药相关蛋白（MRP1）对AS-DACA敏感性没有影响。通过利用AS-DACA的pH依赖性荧光特性，我们表征了其细胞内分布，并表明它集中在细胞核以及膜运输系统的酸性囊泡中。我们表明，荧光显微镜可用于确定AS-DACA在作为球体生长的RMS细胞的核和细胞质区室中的定位，在RH30球体中的穿透率远高于RD球体，并且囊泡信号引导进入球体团块。早期内体囊泡上表达的分子标记物EEA1和Rab5蛋白在敏感细胞系中减少>50%。
综合现有证据表明，内体囊泡运输影响AS-DACA在RMS细胞中的毒性。
