ASNA1, an ATPase targeting tail-anchored proteins, regulates melanoma cell growth and sensitivity to cisplatin and arsenite.
OBJECTIVE
ASNA1 is homologous to E. coli ArsA, a well characterized ATPase involved in efflux of arsenite and antimonite. Cells resistant to arsenite and antimonite are cross-resistant to the chemotherapeutic drug cisplatin. ASNA1 is also an essential ATPase for the insertion of tail-anchored proteins into ER membranes and a novel regulator of insulin secretion. The aim of this study was to determine if altered ASNA1 levels influenced growth and sensitivity to arsenite and cisplatin in human melanoma cells.


METHODS
Cultured melanoma T289 cells were transfected with plasmids containing sense or antisense ASNA1. Cells were exposed to cisplatin, arsenite and zinc. Cell growth and chemosensitivity were evaluated by the MTT assay and apoptosis by a TUNEL assay.


RESULTS
ASNA1 expression was necessary for growth. T289 clones with decreased ASNA1 expression exhibited 51 +/- 5% longer doubling times than wildtype T289 (P = 0.0091). After exposure to cisplatin, ASNA1 downregulated cells displayed a significant increase in apoptosis. The cisplatin IC(50) in ASNA1 underexpressing cells was 41.7 +/- 1.8% compared to wildtype (P = 0.00097) and the arsenite IC(50) was 59.9 +/- 3.2% of wildtype IC(50) (P = 0.0067).


CONCLUSIONS
Reduced ASNA1 expression is associated with significant inhibition of cell growth, increased apoptosis and increased sensitivity to cisplatin and arsenite.
Hemmingsson
O
O
Department of Surgical and Perioperative Sciences, Umeå University Hospital, Umeå, Sweden. oskar.hemmingsson@surgery.umu.se
Zhang
Y
Y
Still
M
M
Naredi
P
P
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053501:Arsenite Transporting ATPases / Q000502:physiology*; D018053:Arsenites / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division / Q000502:physiology*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D006224:Cricetinae; D017931:DNA Primers; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008545:Melanoma / Q000473:pathology*; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010957:Plasmids
D018053:Arsenites; D017931:DNA Primers; C489667:GET3 protein, human; D016376:Oligonucleotides, Antisense; D053501:Arsenite Transporting ATPases; C015001:arsenite; D002945:Cisplatin