Redefining the subcellular location and transport of APC: new insights using a panel of antibodies.
Adenomatous polyposis coli (APC) is a tumour suppressor involved in colon cancer progression. We and others previously described nuclear-cytoplasmic shuttling of APC. However, there are conflicting reports concerning the localization of endogenous wild-type and tumour-associated, truncated APC. To resolve this issue, we compared APC localization using immunofluorescence (IF) microscopy and cell fractionation with nine different APC antibodies. We found that three commonly used APC antibodies showed nonspecific nuclear staining by IF and validated this conclusion in cells where APC was inactivated using small interfering RNA or Cre/Flox. Fractionation showed that wild-type and truncated APC from colon cancer cells were primarily cytoplasmic, but increased in the nucleus after leptomycin B treatment, consistent with CRM1-dependent nuclear export. In contrast to recent reports, our biochemical data indicate that APC nuclear localization is not regulated by changes in cell density, and that APC nuclear export is not prevented by truncating mutations in cancer. These results verify that the bulk of APC resides in the cytoplasm and indicate the need for caution when evaluating the nuclear accumulation of APC.
Brocardo
Mariana
M
Westmead Institute for Cancer Research, Westmead Millennium Institute at Westmead Hospital, University of Sydney, Darcy Road (PO Box 412), Westmead, New South Wales 2145, Australia.
Näthke
Inke S
IS
Henderson
Beric R
BR
D011125:Adenomatous Polyposis Coli / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000906:Antibodies / Q000276:immunology*; D002467:Cell Nucleus / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D017491:Genes, APC; D006801:Humans
D000906:Antibodies