Fig 1.
In vitro translation of MUC1 mRNA yields both MUC1-TM protein and MUC1-ARF protein.
(A) RNAs transcribed from MUC1 cDNA [comprising a single 60 nucleotide (20 amino acid) repeat unit] cut with HindIII, PvuII, BalI, PstI or AccI (H, Pv, B, Ps and A, lanes 1–5 respectively, see Fig 1D and E) were translated in vitro with [35S]-methionine and [35S] cysteine and the products resolved by SDS-PAGE. Molecular masses of protein markers run in a parallel gel are indicated to the right of the autoradiogram, and are also shown in (B) and (C). (B) MUC1 mRNAs circled in red lettering a and b, that differ from each other by 27 nucleotides downstream from the MUC1-TM initiation codon (Fig 1D, a and b) were translated in vitro with [35S]-methionine and [35S]-cysteine and the products resolved by SDS-PAGE. (C) MUC1 mRNA containing a single 20 amino acid repeat was translated for 30 minutes in an in vitro reticulocyte protein translation system with [35S]-methionine and [35S]-cysteine and labelled proteins chased with an excess of unlabeled methionine and cysteine. Samples were removed (times indicated) and resolved by SDS-PAGE. (D) Locations of the initiating (AUG) and terminating (STOP) codons in the MUC1 mRNA driving translation of MUC1-TM are as indicated. The TransMembrane (TM) and cytoplasmic (CYT) domains, Variable Number of Tandem Repeats (VNTR) of MUC1-TM are as indicated. (E) MUC1-ARF, lacking these domains, is shown by VNTR (yellow color) flanked by 5' and 3' domains (red color). The initiation and stop codons (AUGARF and STOPARF) are shown (boxed). Regions bound by anti-MUC1-TM tandem repeats antibodies, anti-MUC1-ARF tandem repeats antibodies and anti-MUC1-SEA module antibodies (in MUC1-TM) are indicated by downward facing dark green, orange and light green arrowheads, respectively. (F) Amino acid sequence of MUC1-ARF. The MUC1-ARF twenty amino-acid-long repeat sequence is shown as three repeats (light and dark brown fonts) whereas in the actual MUC1-ARF protein the number of repeats may vary between fifteen and one hundred twenty five. The peptide sequence used for generating anti-MUC1-ARF monoclonal antibodies is underlined (dashed).
Fig 2.
Nucleotide and amino acid sequence of MUC1-TM and MUC1-ARF.
MUC1-TM initiation codon is shown in black font and green highlight. Downstream to this initiation codon, three potential MUC1-ARF initiation codons in a +1 frame are shown in red font and green highlight. Amino acid sequences are in black and red beneath the nucleotide sequence, represent MUC1-TM protein and MUC1-ARF protein, respectively. The red arrow indicates the signal peptide cleavage site of the MUC1-TM protein. Kozak sequences upstream to the initiation codons of both proteins are in bold fonts and underlined. The sequences shown for both MUC1-TM and MUC1-ARF extend from their respective initiation codons to their tandem repeat domains; three such repeats are shown indicated by square brackets. The EcoR1 site and grey highlighted regions are not part of the actual MUC1 cDNA sequence.
Fig 3.
Detection of nuclear MUC1-ARF protein with polyclonal anti-MUC1-ARF antibodies and with three distinct anti-MUC1-ARF monoclonal antibodies, MPR2G10, MPR4B3 and MPR5C9.
(A) Polyclonal anti-MUC1-ARF antibodies and (B) three independently isolated anti-MUC1-ARF monoclonal antibodies, MPR2G10, MPR4B3 and MPR5C9, were reacted in the presence of competing ARF peptide (B, panels 4), in its absence (B, panels 2), or with a non-relevant peptide (B, panels 3) with mouse DA3 cells stably transfected with and expressing human MUC1 DNA (DA3-MUC1) and with T47D human breast cancer cells that endogenously express MUC1. Parental DA3 cells (DA3-PAR) which do not express human MUC1 are shown in B, panels 1. Immunofluorescence of secondary antibody (red), DAPI staining of nuclei (blue) are shown in the merged images. (C) Mouse DA3 cells expressing human MUC1 reacted with anti-MUC1-ARF repeat monoclonal antibody MPR2G10 (left panel), compared with anti-MUC1-TM tandem repeat monoclonal antibody H23 (right panel), both followed by red-labelled secondary antibody.
Fig 4.
Mouse cell lines transfected with human MUC1 DNA express MUC1-ARF.
(A, and C) DA3 cells transfected with MUC1 cDNA; (B) DA3 cells transfected with human MUC1 genomic DNA, and (D) non-transfected mouse parental DA3 cells were immunostained with anti MUC1-ARF monoclonal antibody MPR2G10 followed by red-labeled secondary antibody. DAPI (blue) and green-labeled phalloidin are shown as merged images. In (C), anti-MUC1-ARF monoclonal antibody was added together with competing ARF peptide. (E) Immunoblots with anti-MUC1-ARF antibodies of cell lysates prepared from either untransfected DA3 mouse cells (DA3-P) or from cells transfected with MUC1 genomic DNA (DA3-G). MUC1-ARF is indicated by the arrowhead. (F) Mouse 3T3 cells transfected with human MUC1 DNA containing differently sized VNTRs (diagrams designated MUC1-I, MUC1-II) express differently sized MUC1-ARF proteins: Cell lysates from either parental 3T3 cells or 3T3 transfectants stably expressing human MUC1 DNA (lanes 1, 2 and 3, designated PAR, MUC1-I and MUC1-II, respectively) were resolved by SDS-PAGE and immunoblots probed with anti-MUC1-ARF alone (F1) or together with competing ARF peptide (F2). Following probing, the immunoblot was stripped, reprobed with an antibody recognizing an epitope within the MUC1-TM tandem repeat sequence, and redeveloped with ECL (F3). Filled red arrows indicate MUC1-ARF proteins and filled green arrows indicate MUC1-TM proteins (F1 and F3, respectively). Stippled red arrows designate the positions of the MUC1-ARF proteins that have been specifically competed out. Protein loading with anti-actin antibodies confirmed that equal amounts of protein were present in each lane. MUC1-I and MUC1-II cDNAs used for transfections contains approximately 17 and 28 repeats respectively. The two cartoons at the bottom of the Figure designating the MUC1-I and MUC1-II cDNAs are intended for illustrative purposes only. They demonstrate the fact that the two cDNAs differ one from the other in the number of repeats each contains.
Fig 5.
Human cancer cells express MUC1-ARF in both the nucleus and cytoplasm.
T47D breast cancer cells (Panels A and C) and COLO357 pancreatic cancer cells (Panels B) were immunostained with anti-MUC1-ARF mAb MPR2G10 followed by red-labeled secondary antibody. DAPI (blue, demonstrating nuclei: white stippled ovals) and green-labeled phalloidin (labeling actin filaments) are shown in the merged images (Panels A' and B'). Immunostaining of T47D cells with anti-MUC1-ARF mAb MPR2G10 is abrogated when done in the presence of competing MUC1-ARF peptide (compare Panels C with Panels D). Simultaneous immunostaining of ZR75 breast cancer cells with anti-MUC1-TM antibodies (DMB5F3 mAbs directly green labeled) and anti-MUC1-ARF antibodies (MPR2G10, red labeled) in the absence of MUC1-TM-junction peptide is shown in Panels E, while the effect of adding MUC1-TM-junction peptide to an identical immunostaining of ZR75 breast cancer cells is shown in Panels F. (Panel G) Lysates of human COLO357 cancer cells were resolved on SDS-PAGE, western blotted, and probed with anti-MUC1-ARF MPR2G10. MUC1-ARF protein is indicated by the filled red arrow head (left panel). Immunoreactivity is abrogated by addition of competing free MUC1-ARF peptide (right panel). (Panel H, left side, labeled MUC1-ARF) Equivalent amounts of protein from either cytoplasmic or nuclear (cyt or nuc) T47D cell extracts were analyzed by a sandwich ELISA that detects MUC1-ARF. Competing MUC1-ARF peptide (ARF pep) added to the detecting biotinylated anti-MUC1-ARF MPR4B3, abolished signal in both cytoplasmic and nuclear samples, whereas non-relevant peptide (non-rel. pep) had no effect. (Panel H, right side, labeled MUC1-TM)- analysis of nuclear and cytoplasmic T47D cell extracts with a sandwich ELISA detecting MUC1-TM protein. (Panel I) Mouse DA3 mammary tumor cells expressing human MUC1 cDNA were immunostained with anti MUC1-ARF antibody MPR2G10 and red-labeled secondary antibody followed by DAPI staining. High magnification images of orthogonal projections of confocal laser microscopy are shown for anti-MUC1-ARF (Panel I-i), DAPI (Panel I-ii) and merged images (Panel I-iii). (Panel J) Untransfected mouse DA3 mammary tumor cells (DA3-PAR, left panels) or transfected with human MUC1 cDNA (DA3-MUC1, J, right panels) were stained with DAPI and immunostained with anti-MUC1-ARF monoclonal followed by red-labeled secondary antibody. DAPI staining alone (blue) and the merged images of DAPI plus red anti-ARF immunostaining (DAPI + anti-ARF) are shown. A parallel set of cells (Panel J, lower panels, plus etop.) were treated with Etoposide, a DNA topoisomerase II inhibitor.
Fig 6.
MUC1-ARF protein is observed only in those human cell lines that express the MUC1 gene.
Lysates prepared from human cell lines A431, MCF7, KB, SY5Y, HK293 and U87 (Panels 'A' to 'E') were resolved on SDS-PAGE, western blotted, and probed with anti-MUC1-ARF MPR2G10. MUC1-ARF protein is indicated by the filled red arrowheads. Addition of competing free MUC1-ARF peptide (indicated beneath the lanes by a 'plus' sign) competed out reactivity of the MUC1-ARF protein (white arrowheads with dotted red outline designate the positions of MUC1-ARF protein that has been competed out by added ARF peptide). (Panel F): Expression levels of the MUC1 gene in the various cell lines were assessed in triplicate by quantitative PCR (qPCR). Expression in MCF7 cells was set to be 100. (Immunoblot analysis of Colo357 for MUC1-ARF expression appears in Fig 5G).
Fig 7.
MUC1-TM and MUC1-ARF expression in normal human kidney and pancreas.
Serial sections of paraffin-embedded human pancreatic and renal tissues were immunohistochemically stained with anti-MUC1-TM SEA module antibodies (anti-MUC1-TM [DMB5F3]) and anti-MUC1-ARF antibodies (anti-MUC1-ARF [MPR2G10]) as indicated. Normal kidney is shown in Panels A-i (MUC1-TM) and A-ii (MUC1-ARF); larger fields and higher magnifications are shown in Panels C-i, C-i’ (MUC1-TM), and D-i, D-i’ (MUC1-ARF)]. Glomerulus, proximal tubule, and distal tubule are designated by G, PT and DT respectively. Filled green arrows designate sites of MUC1-TM-SEA protein at the cell surface whereas filled red arrows designate MUC1-ARF protein; absence of anti-MUC1-ARF immunoreactivity is shown by filled red and white arrows. Normal pancreatic tissue reacted with anti-MUC1-TM in the presence of ARF peptide (plus ARF pep) or in the presence of MUC1-TM SEA peptide (plus SEA pep), are shown in Panels B-i and B-i", respectively. MUC1-TM protein on the cell surface of ductal epithelial cells is competed out by MUC1-TM SEA peptide (B-i") but not by MUC1-ARF peptide (B-i). Conversely MUC1-ARF protein in the nuclei of pancreatic epithelial cells is competed out by MUC1-ARF peptide (B-ii") but not by MUC1-SEA peptide (B-ii). Larger fields and higher magnifications of pancreatic tissue is shown in E-i, E-i’ (MUC1-TM), and E-ii, E-ii’ (MUC1-ARF).
Fig 8.
MUC1-TM and MUC1-ARF proteins are expressed solely in the exocrine pancreas and not in the endocrine pancreatic islets.
Serial sections of paraffin-embedded human pancreatic tissues were immunohistochemically stained with anti-MUC1-TM SEA module antibodies (anti-MUC1-TM [DMB5F3]), anti-MUC1-ARF antibodies (anti-MUC1-ARF [MPR2G10]) and normal mouse immunoglobulin as indicated. Filled green arrows designate sites of MUC1-TM-SEA protein at the cell surface whereas filled red arrows designate MUC1-ARF protein; absence of anti-MUC1-ARF immunoreactivity is shown by filled red and white arrows. The pancreatic islets (Islets of Langerhans) are demarcated by the dotted blue lines.
Fig 9.
Immunohistochemical analyses of MUC1-TM and MUC1-ARF expression in breast and pancreatic cancer.
(A) Serial sections of breast cancer tissues from three distinct individuals were immunohistochemically stained with anti-MUC1-TM antibodies (anti-MUC1-TM [DMB5F3]), anti-MUC1-ARF antibodies (anti-MUC1-ARF [MPR2G10]) and with hematoxylin/eosin (H and E). Green arrows indicate sites of MUC1-TM reactivity at the cell surface, and red arrows designate MUC1-ARF reactivity in the nuclei. Absence of anti-MUC1-ARF immunoreactivity is shown by filled red and white arrows. (B) Binding specificity of anti-MUC1-ARF [MPR2G10] antibody is demonstrated by addition of either MUC1-ARF peptide or a non-relevant peptide, as indicated. Only MUC1-ARF peptide abrogates immunoreactivity. Serial sections of normal pancreas (C) or pancreatic cancer tissue (D), were immunohistochemically stained with anti-MUC1-TM [DMB5F3] or anti-MUC1-ARF [MPR2G10]. Both MUC1-TM and MUC1-ARF are expressed in the normal pancreatic tissue (C). In contrast, cancer tissue expresses only MUC1-TM. MUC1-ARF was not detected (red and white arrows).
Fig 10.
Cytokines upregulate MUC1-ARF expression and result in its relocalization.
(A) COLO357 pancreatic cancer cells were either untreated (Control, Panel a), or incubated for 8 hours with interferon-gamma and interleukin1-beta (Panel b); interleukin 6 and TNFalpha (Panel c); interferon-gamma and TNFalpha (Panel d). This was followed by immunostaining with anti MUC1-ARF antibody MPR2G10 and red-labeled secondary detection. (B a.) COLO357 cells were either untreated or incubated for 8 hours with interferon-gamma and interleukin1-beta, (blue and green tracings, respectively), permeabilized, and analyzed by flow cytometry with anti-MUC1-ARF MPR-2G10, and detection with fluorescently labeled secondary anti-mouse antibody. Addition of secondary antibodies alone to untreated and cytokine-treated cells is shown by the orange and red tracings, respectively. (B b. c.) MUC1-ARF protein in untreated and cytokine-treated COLO357 cells was quantitatively assessed by sandwich ELISA (b), and shown in block graphs in (c). Specificity of antibody binding to MUC1-ARF protein was confirmed by addition of competing MUC1-ARF peptide (b. plus ARF peptide). (C) COLO357 cells were either untreated (Panels a, a', designated 'no cytokine') or incubated for 24 hours with interferon-gamma plus interleukin1-beta (Panels b, b', designated 'plus IFNgamma plus IL1beta'). Immunostaining with anti-MUC1-ARF MPR2G10 and detection by red-labeled secondary antibody (a' and b') was followed by green-labeled anti-MUC1-SEA module DMB5F3 (a and b). DAPI (blue) and merged images are presented in the lower left and right panels, respectively. (D) COLO357 cells were treated for forty-eight hours with IFNgamma and IL1beta, and actin filaments visualized by staining with green-labeled phalloidin (a), and MUC1-ARF protein immunostained with anti MUC1-ARF MPR2G10 followed by detection with red-labeled secondary antibody (b). DAPI-stained nuclei and merged images are shown in (c) and (d), respectively. Following prolonged cytokine treatment, MUC1-ARF expression shifted in part to membrane protrusions, indicated by yellow arrows. (E) Cell lysates were prepared from HK293 cells transiently transfected with an expression vector driving expression of MUC1 cDNA (lanes 2) or with a control empty expression vector (lanes 1). Anti-MUC1-ARF immunprecipitates were resolved on SDS-PAGE, western blotted, and probed with antibodies as indicated.