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

Mariana Brocardo, Inke S. Näthke, Beric R. Henderson

Research output: Contribution to journalArticleResearchpeer-review

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.

LanguageEnglish
Pages184-190
Number of pages7
JournalEMBO Reports
Volume6
Issue number2
DOIs
Publication statusPublished - 2005
Externally publishedYes

Fingerprint

Adenomatous Polyposis Coli
Fractionation
Tumors
Antibodies
Small Interfering RNA
Microscopic examination
Cells
Cell Nucleus Active Transport
Colonic Neoplasms
Cell Fractionation
Neoplasms
Fluorescence Microscopy
leptomycin B
Fluorescent Antibody Technique
Cytoplasm

Keywords

  • Antibodies
  • APC
  • Colon cancer
  • Mutations
  • Subcellular localization

Cite this

Brocardo, Mariana ; Näthke, Inke S. ; Henderson, Beric R. / Redefining the subcellular location and transport of APC : New insights using a panel of antibodies. In: EMBO Reports. 2005 ; Vol. 6, No. 2. pp. 184-190.
@article{b573a5a8efff4c59a311b5211dd0e4b6,
title = "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.",
keywords = "Antibodies, APC, Colon cancer, Mutations, Subcellular localization",
author = "Mariana Brocardo and N{\"a}thke, {Inke S.} and Henderson, {Beric R.}",
year = "2005",
doi = "10.1038/sj.embor.7400329",
language = "English",
volume = "6",
pages = "184--190",
journal = "EMBO Reports",
issn = "1469-221X",
publisher = "Springer, Springer Nature",
number = "2",

}

Redefining the subcellular location and transport of APC : New insights using a panel of antibodies. / Brocardo, Mariana; Näthke, Inke S.; Henderson, Beric R.

In: EMBO Reports, Vol. 6, No. 2, 2005, p. 184-190.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Redefining the subcellular location and transport of APC

T2 - EMBO Reports

AU - Brocardo, Mariana

AU - Näthke, Inke S.

AU - Henderson, Beric R.

PY - 2005

Y1 - 2005

N2 - 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.

AB - 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.

KW - Antibodies

KW - APC

KW - Colon cancer

KW - Mutations

KW - Subcellular localization

UR - http://www.scopus.com/inward/record.url?scp=14644390357&partnerID=8YFLogxK

U2 - 10.1038/sj.embor.7400329

DO - 10.1038/sj.embor.7400329

M3 - Article

VL - 6

SP - 184

EP - 190

JO - EMBO Reports

JF - EMBO Reports

SN - 1469-221X

IS - 2

ER -