Redefining the subcellular location and transport of APC: new insights using a panel of antibodies

Abstract

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.

Figure 1

Detection of ectopic and endogenous APC with different antibodies by IF microscopy. (A) Diagram of APC protein showing the different epitopes recognized by antibodies tested in this study. (B) Full-length pAPC–YFP was transfected into SW480 colon cancer cells. After 48 h, ectopic YFP localization of APC–YFP was compared using different antibodies against APC protein by immunostaining. Positive colocalization of each antibody with APC–YFP expression is indicated (+). (C) Cellular staining patterns observed with different APC antibodies. SW480, HCT116 or MDCK cells were fixed on glass coverslips in formalin and analysed by IF microscopy. The staining shows variability between different antibodies, although all except for Ab4 detected APC at membrane protrusions in MDCK cells.

Figure 2

M-APC nuclear staining is not specific for APC. (A) SW480 cells, untransfected or transfected with an anti-APC siRNA for 48 h, were fixed and stained with affinity-purified M-APC antibody. APC siRNA did not affect the cell staining profile of M-APC antibody. (B) In parallel, equivalent amounts of cytoplasmic or nuclear extracts from cells treated ±siRNA (APC, or control BRCA1 siRNA) were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and analysed by immunoblot. Truncated APC (150 kDa) was detected with antibody M-APC. The same membrane was reprobed for topoisomerase IIα (170 kDa) as a fractionation control. APC siRNA effectively reduced cytoplasmic and nuclear APC by 80% relative to untransfected cells. Similar results were observed in three independent experiments. (C) Comparison of APC nuclear expression as measured by immunoblots quantified by densitometry, and for cell staining with quantification of nuclear fluorescence using ScionImage software. The degree of nuclear APC inhibition was expressed in arbitrary units and shows that the nuclear staining signal obtained with M-APC antibody was not specific for APC. (D) We confirmed the lack of specificity of M-APC antibody in floxed mouse fibroblasts in which APC expression was inducibly silenced by Cre recombinase (Sansom et al, 2004). In such cells, the loss of full-length APC (as shown by immunoblot with N-APC antibody, see Midgley et al (1997); the lower band is nonspecific and confirms equal loading of proteins in the samples) was accompanied by the predicted increase in cellular β-catenin levels, but there was no loss of nuclear fluorescence with M-APC antibody.

Figure 3

Nuclear export of endogenous APC in different cell lines. (A) SW480 cells were treated ±5 h LMB, fixed with formalin and stained with antibodies Ali 12–28 (1:50) and Ab7 (1:50) followed by Texas red secondary conjugate. LMB induced a visible shift to the nucleus as shown by IF. The nucleus is stained with Hoechst chromatin dye. (B) Nuclear and cytoplasmic cell extracts were prepared from SW480 (APC^mut/mut), HT29 (APC^mut/mut) and HCT116 (APC^wt/wt) colon cancer cell lines. The endogenous truncated or full-length forms of APC were separated by SDS–PAGE (or agarose gels for HCT116 extracts, see Methods) and detected by western blot using M-APC antibody. The APC bands were quantified by densitometry and the cytoplasmic/nuclear ratios and LMB-dependent nuclear shift are shown. Similar results were observed in two independent experiments.

Figure 4

The localization of endogenous APC is not influenced by cell density in SW480, HCT116 or MDCK cells. (A) SW480 and MDCK cells were seeded at different densities and harvested at 40% confluence (subconfluent) or confluence. Cells were fixed with formalin and stained with M-APC antibody (1:1,000 dilution). The M-APC staining pattern shifted from nuclear–cytoplasmic to cytoplasmic with increased cell density, as described by others (Fagman et al, 2003; Davies et al, 2004). (B) SW480, HCT116 and MDCK cells were grown under the same conditions as above, and processed for immunoblotting. Equivalent amounts of cytoplasmic (60 μg) or nuclear (20 μg) extracts were separated by SDS–PAGE or agarose gel (for HCT116 and MDCK) and analysed by western blot. Truncated and full-length APC were detected with M-APC antibody (1:4,000). Neither form of APC redistributed to the nucleus after changes in density. The same membrane was reprobed for topoisomerase II (TOPOII) as a fractionation control. The same results were obtained in two independent experiments.