title: Intracellular trafficking as a determinant of AS-DACA cytotoxicity in rhabdomyosarcoma cells.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Taking the evidence as a whole, suggests that endosomal vesicle trafficking influences the toxicity of AS-DACA in RMS cells.
authors.affiliation: Biospecimens Research and Tumour Bank, Children's Cancer Research Unit, The Children's Hospital at Westmead, Westmead, NSW 2774, Australia.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Wolf
authors.forename: Tony
authors.initials: T
authors.lastname: Huynh
authors.forename: Nicole S
authors.initials: NS
authors.lastname: Bryce
authors.forename: Trevor W
authors.initials: TW
authors.lastname: Hambley
authors.forename: Laurence P G
authors.initials: LP
authors.lastname: Wakelin
authors.forename: Bernard W
authors.initials: BW
authors.lastname: Stewart
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Catchpoole
mesh_terms: D000620:Aminoimidazole Carboxamide / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000473:pathology; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000473:pathology; D012208:Rhabdomyosarcoma / Q000188:drug therapy* / Q000473:pathology; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
chemical_list: D000620:Aminoimidazole Carboxamide; D000970:Antineoplastic Agents; D015415:Biomarkers; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins; C093846:early endosome antigen 1; C421109:multidrug resistance-associated protein 1