In the current study, we have generated purified polyclonal antiserum that is specific for APC. APC-M2 pAb is versatile and can be used to detect APC in immunostaining, immunoblotting and immunoprecipitation. Using this antibody, we identified a novel interaction between APC and IFs. The APC and IF protein interactions were confirmed by co-precipitation and colocalization using both endogenous and GFP-tagged exogenous APC in cultured epithelial cells. Association of APC with IFs is not dependent on actin-containing microfilaments and microtubules.
APC mutations are found in over 80% of all colorectal cancers and have been assessed for screening and prognosis purposes in the clinical setting. For such research, access to an APC antibody that accurately depicts levels and subcellular localizations of APC in cells and especially in tissues is extremely important. According to Wakeman et. al. , many commercially available antibodies also recognize an unidentified protein that localizes to the apical region of polarized epithelial cells. Most currently available commercial APC antibodies were raised against several hundred amino acids at either the N- or C-terminus of the APC protein. However, most of these antibodies have not been proven to be specific for APC . Furthermore, many of these antibodies are only useful for a single application. For example, anti-APC Ab-7 is only effective when used for immunostaining. Therefore, we generated a new antibody against the 15 amino acid repeat region in the middle of APC (Additional file 1). The affinity-purified APC-M2 pAb recognizes junctional, cytoplasmic and nuclear APC by immunostaining (Figure 2A) without producing the non-specific apical staining pattern previously seen . Although there was a great deal of overlap, immunofluorescent signals from APC-M2 pAb and anti-APC ali12-28 were not 100% coincident. The ali12-28 antibody was raised against a different portion of APC protein than M2-APC. The most likely explanation for the slight signal disparity is that the epitopes recognized by APC-M2 pAb are masked by APC folding, or the binding of another protein to a subset of the APC protein. In a similar manner, it would be expected that the epitope recognized by ali12-28 might be inaccessible in some fraction of the APC protein.
One of the most clinically relevant applications for APC antibodies is for immunohistochemical analysis of tissues. We have validated the use of APC-M2 pAb for immunostaining sections of frozen human colonic tissue and mouse small intestinal tissues. Previously, APC has been detected in both the nucleus and cytoplasm of human colonic tissue, with more prevalent cytoplasmic staining in colonic tumors compared to normal tissue . In our study of human colon tissue, APC-M2 pAb revealed APC in both nuclei and at cell-cell junctions, partially colocalized with β-catenin, similar to previous reports [14, 30] (Figure 5B). APC protein also appeared concentrated near the basal surface of epithelial cells (Figure 5A and 5B) as previously reported . APC staining became most intense in cells at the top of villi in the mouse small intestine (Figure 7A-B) and in cells at the luminal surface of the human colon (Figure 5A), consistent with a role for APC in differentiated cells. Most importantly, APC-M2 pAb did not recognize protein at the apical surface of intestinal tissues. Thus, APC-M2 pAb does not appear to exhibit the cross reactivity previously reported for some commercially available APC antibodies . Taken together, the APC-M2 pAb is a useful antibody for future examination of APC in mouse and human tissue sections. At present, APC-M2 pAb does not appear to recognize APC in formalin-fixed paraffin-embedded tissues (data not shown).
Intermediate filaments are essential components of cytoskeletal structure. Recent reports have implicated APC in the regulation of cellular structural integrity through affects on microtubule growth directionality and polarity [32, 33]. APC also associates with microtubule-interacting protein EB1, and actin-associated protein ASEF [11, 18]. Our own previous screen for APC-interacting proteins identified keratin 18 (Stecklein and Neufeld, personal communication). Although classified as a hair cuticle-specific keratin, keratin 81 is expressed in a wide variety of tissues including mammary gland and lung and in human embryonic kidney 293T cells [34–37]. Keratin 82 is also thought to be expressed predominantly in hair and nail. However, when flag-tag specific antibodies were used to immunoprecipitate flag-tagged human homolog 3 of Drosophila Disc-large (hDlg) from human embryonic kidney 293 cells, keratin 82 was identified as a protein partner . This observation is significant because it indicates that keratin 82 has a broader expression pattern than initially suggested and because hDlg protein itself is a binding partner of APC .
Downregulation of β-catenin activity by APC is not limited to the cytoplasmic destruction complex. Association of APC with β-catenin in the nucleus appears to result in sequestration of β-catenin from the TCF/LEF transcription factor leading to inactivation of Wnt-responsive genes . Furthermore, chromatin immunoprecipitation analysis revealed that APC associated with the enhancer region of a Wnt-responsive gene in a similar temporal manner as several co-repressors, suggesting that APC can oppose a Wnt signal by associating with co-repressors at the transcription complex . Given the emerging role of the nuclear lamina in gene regulation , it is tempting to speculate that APC association with lamin B1 is yet another mechanism by which expression of Wnt-responsive genes is regulated. Moreover, finding APC associated with the nucleus after extraction suggests APC might be bound to the nuclear matrix. This observation is consistent with our previous finding that the majority of nuclear APC purified with the nuclear matrix .
As a vital part of the cytoskeleton, intermediate filaments are dynamic and can stabilize cellular organelles such as the nucleus . Mutations in genes encoding IF proteins are associated with a number of diseases such as Monilethrix (K81) , Autosomal dominant leukodystrophy (LMNB1) , acquired partial lipodystrophy (LMNB2) , and various other lipodystrophies and cardiomyopathies . APC potentially cross-links IFs to improve their stability or binds IFs to other structures such as actin filaments or microtubules. Therefore, truncated APC found in the majority of colorectal cancers and also in other cancers, might have a compromised ability to link APC binding partners with different IF proteins. It is worth noting that full-length APC has been found near IFs in epithelial cells from human colon tissue using immunogold electron microscopy . Given the evidence for an association of APC with IFs, it is predicted that disruption of IFs would result in APC redistribution. Our attempts to disrupt IFs by treating cells with acrylamide led to cell shrinking, but not elimination of IFs (data not shown). Further study of this novel APC/IF interaction using a related approach might illuminate the underlying mechanisms of intermediate filament protein-associated diseases.