Aberrant Expression of Fetal RNA-Binding Protein p62 in Liver Cancer and Liver Cirrhosis
2001; Elsevier BV; Volume: 159; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)61770-1
ISSN1525-2191
AutoresMaolong Lu, Robert M. Nakamura, E. DuBose Dent, Jian-Ying Zhang, Finn Cilius Nielsen, Jan Christiansen, Edward K. L. Chan, Eng M. Tan,
Tópico(s)Cancer-related molecular mechanisms research
Resumop62 is a RNA-binding protein that was isolated by immunoscreening a cDNA expression library with autoantibodies from patients with hepatocellular carcinoma (HCC). This autoantigen binds to mRNA encoding insulin-like growth factor II, which has been found to be overexpressed in HCC and is tumorigenic in transgenic animals. Immunohistochemical analysis of HCC liver showed that 33% (9 of 27) exhibited readily detectable staining of p62 protein in the cytoplasm of all malignant cells in cancer nodules, whereas it was undetectable in adjacent nonmalignant liver cells. In addition one of two patients with cholangiocarcinoma expressed p62 in malignant bile duct epithelial cells. p62 expression was also detected in scattered cells in cirrhotic nodules in contrast to uniform expression in all cells in HCC nodules. In HCC nodules, p62 mRNA was also detected by reverse transcriptase-polymerase chain reaction analysis. Nine normal adult livers did not contain detectable p62 mRNA or p62 protein whereas five fetal livers were all positive for mRNA and protein. The observations show that p62 is developmentally regulated, expressed in fetal, but not in adult liver, and aberrantly expressed in HCC and could be playing a role in abnormal cell proliferation in HCC and cirrhosis by modulating expression of growth factors such as insulin-like growth factor II. p62 is a RNA-binding protein that was isolated by immunoscreening a cDNA expression library with autoantibodies from patients with hepatocellular carcinoma (HCC). This autoantigen binds to mRNA encoding insulin-like growth factor II, which has been found to be overexpressed in HCC and is tumorigenic in transgenic animals. Immunohistochemical analysis of HCC liver showed that 33% (9 of 27) exhibited readily detectable staining of p62 protein in the cytoplasm of all malignant cells in cancer nodules, whereas it was undetectable in adjacent nonmalignant liver cells. In addition one of two patients with cholangiocarcinoma expressed p62 in malignant bile duct epithelial cells. p62 expression was also detected in scattered cells in cirrhotic nodules in contrast to uniform expression in all cells in HCC nodules. In HCC nodules, p62 mRNA was also detected by reverse transcriptase-polymerase chain reaction analysis. Nine normal adult livers did not contain detectable p62 mRNA or p62 protein whereas five fetal livers were all positive for mRNA and protein. The observations show that p62 is developmentally regulated, expressed in fetal, but not in adult liver, and aberrantly expressed in HCC and could be playing a role in abnormal cell proliferation in HCC and cirrhosis by modulating expression of growth factors such as insulin-like growth factor II. Liver cancers are one of the most common cancers in the world, especially in developing countries, with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCC) being the most frequent types of liver malignancy. Liver cirrhosis related to chronic viral hepatitis (hepatitis B and C) has been recognized as one of the major risk factors leading to the development of HCC, but the genetic defects and proteins involved in the development of liver cancer are not completely understood.1Berk PD Okuda K Wands JR Hepatocellular carcinoma.Semin Liver Dis. 1999; 19: 233-311Crossref Scopus (4) Google Scholar Using serum antibody from a patient with HCC to screen a cDNA expression library we recently identified a 62-kd RNA-binding protein that elicited a humoral immune response in ∼20% of HCC patients.2Zhang JY Chan EKL Peng XX Tan EM A novel cytoplasmic protein with RNA-binding motifs is an autoantigen in human hepatocellular carcinoma.J Exp Med. 1999; 189: 1101-1110Crossref PubMed Scopus (184) Google Scholar p62 contains one set of the RNA recognition motif3Query CC Bentley RC Keene D A common RNA recognition motif identified within a defined U1 RNA-binding domain of the 70K U1 snRNP protein.Cell. 1989; 57: 89-101Abstract Full Text PDF PubMed Scopus (524) Google Scholar and four hnRNP K homology (KH) domains4Siomi H Matunis MJ Michael WM Dreyfuss G The pre-mRNA binding K protein contains a novel evolutionarily conserved motif.Nucleic Acids Res. 1993; 21: 1193-1198Crossref PubMed Scopus (469) Google Scholar, 5Siomi H Choi M Siomi MC Nussbaum RL Dreyfuss G Essential role for KH domains in RNA binding: impaired RNA binding by a mutation in the KH domain of FMR1 that causes fragile X syndrome.Cell. 1994; 77: 33-39Abstract Full Text PDF PubMed Scopus (389) Google Scholar, 6Adinolfi S Bagni C Castiglione Morelli MA Fraternali F Musco G Pastore A Novel RNA-binding motif: the KH module.Biopolymers. 1999; 51: 153-164Crossref PubMed Scopus (60) Google Scholar and belongs to the family of IMPs (insulin-like growth factor II mRNA-binding proteins).7Nielsen J Christiansen J Lykke-Andersen J Johnsen AH Wewer UM Nielsen FC A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development.Mol Cell Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (575) Google Scholar The human IMPs have high sequence identity with and similar RNA-binding domain distribution as other RNA-binding proteins such as the Xenopus Vg1RBP/Vera protein,8Deshler JO Highett MI Abramson T Schnapp BJ A highly conserved RNA-binding protein for cytoplasmic mRNA localization in vertebrates.Curr Biol. 1998; 8: 489-496Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 9Havin L Git A Elisha Z Oberman F Yaniv K Schwartz SP Standart N Yisraeli JK RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization.Genes Dev. 1998; 12: 1593-1598Crossref PubMed Scopus (183) Google Scholar the chicken zipcode-binding protein-1,10Ross AF Oleynikov Y Kislauskis EH Taneja KL Singer RH Characterization of a beta-actin mRNA zipcode-binding protein.Mol Cell Biol. 1997; 17: 2158-2165Crossref PubMed Google Scholar and the mouse c-myc- coding region instability determinant-binding protein.11Doyle GAR Betz NA Leeds PF Fleisig AJ Prokipcak RD Ross J The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins.Nucleic Acids Res. 1998; 26: 5036-5044Crossref PubMed Scopus (145) Google Scholar p62 represents a splice variant of IMP-2 and lacks 43 amino acids between the KH 2 and KH 3 domains, but the functional significance of the alternative splicing is currently unknown. These RNA-binding proteins have been implicated in posttranscriptional events such as RNA localization, stability, and translatability. Intracellular localization of Xenopus Vg1 and chicken β-actin mRNAs takes place via cis-elements in their 3′UTR that associate with Vg1RBP/Vera and zipcode-binding protein-1, respectively. The instability of mouse c-myc mRNA is partly mediated through endonucleolysis of an element in the coding region that is shielded from degradation by coding region instability determinant-binding protein. Finally, the human Koc (K homology protein overexpressed in cancer) protein, that is identical to IMP-3, was originally isolated by screening for genes differentially expressed in pancreatic cancer.12Muller-Pillasch F Lacher U Wallrapp C Micha A Zimmerhack F Hameister S Varga G Friess H Bucher M Geger HG Vila MR Adler G Gress M Cloning of a gene highly overexpressed in cancer coding for a novel KH-domain containing protein.Oncogene. 1997; 14: 2729-2733Crossref PubMed Scopus (242) Google Scholar In this study we have examined the role of p62 in hepatic tumorigenesis. We show that p62 is expressed at high levels in fetal liver but is not detectable in adult liver. However, p62 is aberrantly and uniformly expressed in malignant cells of HCC nodules and in some cells in cirrhotic nodules. The results indicate that p62 has features associated with oncofetal proteins and its role in tumorigenesis could be by way of regulation of mRNA stability. Liver tissue from 27 HCC patients were obtained from Henan Medical University, Henan Province, People's Republic of China and from the Department of Pathology, the Scripps Clinic, La Jolla, CA. Biopsies or surgically removed specimens were fixed in 10% formalin and embedded in paraffin for routine histological examination. The clinical data (20 males and 7 females; mean age, 49.3 years; range, 27 to 82 years) and pathological diagnoses are summarized in Table 1. HCC grading criteria were according to those described.13Edmondson HA Steiner PE Primary carcinoma of the liver: a study of 100 cases among 48,900 necropsies.Cancer. 1954; 7: 462-503Crossref PubMed Scopus (2751) Google Scholar Additional paraffin blocks from the Department of Pathology, Scripps Clinic, La Jolla, consisted of nine liver specimens from normal donors, two from patients with poorly differentiated CCC (see Table 1 for clinical data), and 23 from patients with liver cirrhosis. Patients with liver cirrhosis included 18 males and 5 females ranging from 30 to 66 years (mean age, 53.9 years). Five fetal livers ranging from 50 to 125 days were obtained from the Central Laboratory for Human Embryology (University of Washington, Seattle, WA), which is a National Institutes of Health funded center for collecting fetal embryos for research purposes. Hematoxylin and eosin (H&E)-stained sections of all specimens including cancer and noncancer cases were examined by two senior pathologists followed by separately conducted immunohistochemical analysis by other authors in this report.Table 1Clinical Pathological Data on Patients with HCC and CCC and Immunohistochemistry of Liver TissueCaseAgeSexDiagnosisAFP (μg/ml)GradingAnti-p62C IHC*IHC: immunohistochemical analysis.150MHCC>400III+239MHCC>400II−334MHCC40II−442FHCC 400III+667FHCC 400II−858FHCC104II+960MHCC>400III+1029MHCC>400II−1158MHCC>400III−1227FHCC>400II−1354MHCC>400I+1447MHCC>400II−1546FHCC>400III−1646MHCC>400I−1738MHCC200IV−1848MHCC 400II−2051MHCC400III−2151MHCCN/AII−2262MHCCN/AII−2346MHCCN/AI+2437FHCCN/AIV−2582MHCCN/AIII+2666FHCCN/AIII−2760MHCCN/AII+2869MCCCN/A+2950MCCCN/A−HCC, hepatocellular carcinoma; CCC, cholangiocarcinoma; AFP, alpha fetoprotein; N/A, data not available.Cases 1 to 20 were tissue specimens from patients in China and 21 to 29 from patients in the United States.* IHC: immunohistochemical analysis. Open table in a new tab HCC, hepatocellular carcinoma; CCC, cholangiocarcinoma; AFP, alpha fetoprotein; N/A, data not available. Cases 1 to 20 were tissue specimens from patients in China and 21 to 29 from patients in the United States. Paraffin-embedded liver tissue specimens were sectioned at 5 μm and picked up on Superfrost plus slides (Fisher Scientific, Pittsburgh, PA). Antigen retrieval was performed by microwave-heating methods in a citrate-based antigen retrieval solution (BioGenex, San Ramon, CA) according to the manufacturer's recommendation. Briefly, the sectioned slides were deparaffinized using xylene (Fisher Scientific), hydrated by dipping 20 times in each concentration of ethyl alcohol (100, 95, and 75%), and rinsed in distilled water several times. The slides were put into boiling citrate-based antigen retrieval solution and heated in a microwave oven for 15 minutes with 5- to 10-second sudden boiling at 20- to 30-second intervals. The heated slides were cooled down to room temperature in a water bath, rinsed with distilled water, and washed three times with phosphate-buffered saline (PBS) (10 mmol/L KH2PO4, 2 mmol/L NaH2PO4, 140 mmol/L NaCl, 40 mmol/L KCl, pH7.4). The sections were reacted with anti-p62C antibodies (diluted at 1:100 in PBS) and stained with fluorescein isothiocyanate-conjugated secondary antibody (Caltag, Burlingame, CA). Immunofluorescence was observed in an Olympus epifluorescence microscope BHT (Olympus Optical Co., Tokyo, Japan) using a ×20 or ×50 water immersion lens. Confocal microscopy was performed with a Zeiss LSM510 confocal laser-scanning microscope. IMP-1 and Koc/IMP-3 cDNAs were inserted into pET28 vectors as described.2Zhang JY Chan EKL Peng XX Tan EM A novel cytoplasmic protein with RNA-binding motifs is an autoantigen in human hepatocellular carcinoma.J Exp Med. 1999; 189: 1101-1110Crossref PubMed Scopus (184) Google Scholar p62 and golgin-97 expression vectors, the latter encodes a Golgi autoantigen used as an unrelated control,14Griffith KJ Chan EKL Lung CC Hamel JC Gao X Miyachi K Fritzler MJ Molecular cloning of a novel 97-Kd Golgi complex autoantigen associated with Sjogren's syndrome.Arthritis Rheum. 1997; 40: 1693-1702Crossref PubMed Scopus (145) Google Scholar were constructed as previously described.2Zhang JY Chan EKL Peng XX Tan EM A novel cytoplasmic protein with RNA-binding motifs is an autoantigen in human hepatocellular carcinoma.J Exp Med. 1999; 189: 1101-1110Crossref PubMed Scopus (184) Google Scholar The expression vectors were transformed into Escherichia coli BL21 (DE3) cells and recombinant proteins were expressed by induction with IPTG for 4 hours. The recombinant proteins were affinity-purified on a Ni-NTA column and eluted with a 7 mol/L-urea-containing solution according to the manufacturer's instructions (Qiagen, Santa Clarita, CA). Rabbit anti-p62C was made against a peptide containing the C-terminal 10 amino acids of p62 (PQGVASQRSK), which shares no amino acid homology with IMP-1 and Koc/IMP-3, and anti-golgin-97 was made against full-length golgin-97. Female New Zealand White rabbits were immunized with 100 to 250 μg of synthesized peptide or purified recombinant protein in Freund's complete adjuvant, and boosted 1 month later with the same amount of peptide or protein in Freund's incomplete adjuvant. Antisera were collected 10 days after the booster injection. One hundred ng of recombinant protein was run on each lane of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk for 30 minutes, incubated with diluted rabbit anti-p62C (1:1500) for 1 hour, followed by horseradish peroxidase-conjugated secondary antibody (Caltag, Burlingame, CA). Reactivities were detected by the chemiluminescence method using enhanced chemiluminescence reagents (Amersham Life Science, Buckinghamshire, UK). Five hundred μg of recombinant proteins (p62 or golgin-97) were run on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes. The membranes containing only the recombinant protein were cut into small slices. The slices were blocked in 5% milk overnight, and divided into three parts. Five hundred μl of diluted anti-p62C antibody was incubated with the first part of these membrane slices for 8 hours, the absorbed supernatant recovered, and repeated absorption performed with the other two membrane sections in an identical manner. The absorbed antibody was kept in a −20°C freezer until use for immunofluorescent staining. The method for RNA extraction was a modification of a method previously described.15Tsuji S Hisaoka M Morimitsu Y Hashimoto H Shimajiri S Komiya S Ushijima M Nakamura T Detection of SYT-SSX fusion transcripts in synovial sarcoma by reverse transcription-polymerase chain reaction using archival paraffin-embedded tissues.Am J Pathol. 1998; 153: 1807-1812Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar Twenty sections of 5-μm thick paraffin-embedded liver specimens were used and areas were selected that contained at least 80% tumor hepatocytes as determined by examining adjacent H&E-stained sections. To avoid contamination, a separate razor blade was used to dissect out the tumor nodules of each liver specimen. The sections were transferred to 1.5-ml Eppendorf tubes, deparaffinated in three changes of 1.0 ml xylene (Fisher Scientific) and three washes of 1.0 ml ethanol. After brief drying, tissues were incubated with 300 ml of lysis buffer (10 mmol/L Tris-HCl, pH 8.0, 20 mmol/L ethylenediaminetetraacetic acid, 2% sodium dodecyl sulfate), and homogenized. The homogenized tissues were mixed with 15 μl of proteinase K solution (100 mg/ml) and incubated in 55°C water bath overnight. The solution was cooled to room temperature and 1 ml of Trizol reagent (Life Technologies, Inc., Gaithersburg, MD) was added. Total RNA was extracted according to the manufacturer's suggestions. The RNA pellet was dissolved in 20 μl of distilled water and one-tenth aliquots were used for quantitation. Poly(A)+ mRNAs from human fetal and adult livers were purchased from Clontech (Palo Alto, CA). Fetal liver RNA was reported by the manufacturer to have been extracted from a pool of 32 tissue specimens at age 18 to 24 weeks, whereas adult liver poly(A)+ RNA was extracted from a pool of two tissue specimens at age 15 and 35 years. Actin and p62 primers were synthesized as follows: actin forward primer AGGCCAACCGCGAGAAGATGACC, actin reverse primer GAAGTCCAGGGCGACGTAGCAC, p62FL forward primer 5′-TTGAATTCGCCATGGTGAACAAGCTTTACATCGGGAACC-3′, p62FL reverse primer 5′-TTTATGTCGACGGTGTTGGAAGGGCTACATT-3′, p62SL forward primer 5′-ATTGAACATGAAACAGGGACC-3, and p62SL reverse primer 5′-ACGTCTGGGCCTTCCGCAGG-3′. The p62FL primers were previously used for the amplification of the full-length coding region of p62 and generated a 1.7-kb PCR product.2Zhang JY Chan EKL Peng XX Tan EM A novel cytoplasmic protein with RNA-binding motifs is an autoantigen in human hepatocellular carcinoma.J Exp Med. 1999; 189: 1101-1110Crossref PubMed Scopus (184) Google Scholar The p62SL primers were designed to readily distinguish between p62 and IMP2 splice variants. The p62 cDNA does not contain the extra 129-bp sequence present in the longer splice variant IMP2 cDNA.2Zhang JY Chan EKL Peng XX Tan EM A novel cytoplasmic protein with RNA-binding motifs is an autoantigen in human hepatocellular carcinoma.J Exp Med. 1999; 189: 1101-1110Crossref PubMed Scopus (184) Google Scholar, 7Nielsen J Christiansen J Lykke-Andersen J Johnsen AH Wewer UM Nielsen FC A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development.Mol Cell Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (575) Google Scholar For p62-type and IMP2-type splice variants using the p62SL primers, the expected PCR products are 386 bp and 515 bp, respectively. Reverse transcription and PCR were performed as described with slight modifications.16Lu M Johnson RR Iatrou K Trans-activation of a cell housekeeping gene promoter by the IE1 gene product of baculovirus.Virology. 1996; 218: 103-113Crossref PubMed Scopus (58) Google Scholar Briefly, half μg of poly(A)+ RNA or 1 μg of total RNA was reverse-transcribed in 20-μl volume containing 20 pmol oligo(dT)12–18 primer. Two μl of cDNA aliquot was pretreated with 5 U of RNase-free DNase (Boehringer Mannheim, Mannheim, Germany) at room temperature and heated at 95°C for 5 minutes. The reaction was then used for PCR in a 50-μl volume containing 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 0.01% (w/v) gelatin, and 0.2 μg of each pair of PCR primers. The PCR conditions used were 25 cycles with denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 2 minutes. The PCR products were run in 1 or 1.5% agarose gel and stained with ethidium bromide. Specificity of anti-p62C raised against its C-terminal peptide was first tested using Western blots. Anti-p62C specifically recognized recombinant p62 protein (Figure 1, lane 1), but had no cross-reactivity with recombinant IMP-1 and Koc/IMP-3 (lanes 2 to 3). Anti-p62C also reacted with native 62-kd protein in HepG2 cells with a weak reaction with a lower molecular size band that was unidentified (lane 4). It was not reactive with many other cellular antigens in the HepG2 cell extract as shown by the multiple reactivity of a lupus autoimmune serum (lane 5). Human HCC liver specimens were subsequently examined by immunohistochemical analysis. The cancer nodule from a liver specimen with a poorly differentiated carcinoma (Figure 2A), uniformly showed strong reactivity to anti-p62C antibody (Figure 2, B and C), but not to preimmune rabbit sera (data not shown). Nonmalignant liver epithelial cells adjacent to the HCC nodule and fibrous and connective tissue cells showed absent or barely detectable fluorescence so that the borders between tumor nodule and nontumor tissue could be clearly demarcated. The nuclei of tumor hepatocytes were stained with 4,6-diamidino-2-phenylindole (DAPI) (Figure 2D) to distinguish between nuclear and cytoplasmic domains. Comparison of signals in Figure 2, C and D, indicated that p62 was expressed mostly in the cytoplasm of tumor hepatocytes and visualized most clearly with confocal microscopy comparing negative contrast and immunohistochemistry (Figure 3). The cytoplasmic localization of p62 protein in tumor hepatocytes was consistent with expression in cultured HEp-2 cells and with that of two homologous proteins (IMP-1 and IMP-3/Koc) in cultured NIH 3T3 cells as previously reported.2Zhang JY Chan EKL Peng XX Tan EM A novel cytoplasmic protein with RNA-binding motifs is an autoantigen in human hepatocellular carcinoma.J Exp Med. 1999; 189: 1101-1110Crossref PubMed Scopus (184) Google Scholar, 7Nielsen J Christiansen J Lykke-Andersen J Johnsen AH Wewer UM Nielsen FC A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development.Mol Cell Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (575) Google Scholar Anti-p62C antibody also recognized the cytoplasmic p62 protein in tumor hepatocytes of a well-differentiated carcinoma (Figure 4, A and B). Antibody absorption experiments were performed to confirm the specificity of anti-p62C antibody. The anti-p62C antiserum preabsorbed with recombinant p62 protein lost reactivity to p62 protein (Figure 4C), whereas the antiserum preabsorbed with control, irrelevant recombinant golgin-97 (a Golgi protein) retained reactivity (Figure 4D). A total of 27 HCC liver specimens (20 from China and 7 from the United States) were examined using anti-p62C antibodies (Table 1). Six of 20 Chinese specimens and 3 of 7 United States specimens (total of 33% in the combined groups) were shown to have p62 expressed in HCC nodules. These results were confirmed using anti-p62F antibody that was raised against the full-length p62 protein (data not shown). Statistically, there was no correlation of p62 expression with age, sex, and α-fetoprotein expression level (Table 1).Figure 2p62 detection in the cytoplasm of tumor hepatocytes of a poorly differentiated HCC specimen (case 1). A: H&E staining showing HCC nodule of case 1 (Table 1). B: Immunohistochemical analysis for p62. The section adjacent to that used for H&E staining was antigen-retrieved in a citrate-based solution, and subjected to immunofluorescent staining with the anti-p62C antibody (1:100 dilution), followed by fluorescein isothiocyanate-conjugated secondary antibody. Original magnification, ×200. C: Enlargement (original magnification, ×500) of the fluorescent area boxed in B to show that cytoplasm of cancer cells were positive and not nuclei. D: Counterstaining with DAPI showing the staining of nuclei in the same section as in C. The demarcation between tumor nodule and nontumor tissue is outlined by dashed lines. C and D represent enlargement of area boxed in B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Subcellular localization of p62 in hepatic carcinoma. Paraffin-embedded sections of hepatic tissue were stained with anti-p62 antibody and subcellular distribution was examined by confocal microscopy. A and C show the Nomarski contrast pictures and cytoplasmic p62 is shown as an overlay in B and D. The immunofluorescent B and D show different areas of cancer nodules illustrating both the intensity of fluorescent signals for p62 and its distribution in cell cytoplasm. Scale bars: 14 μm (A and B), 20 μm (C and D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Anti-p62C absorbed with recombinant p62 or with golgin-97, a Golgi autoantigen used as a control, in a well-differentiated HCC specimen (case 13). A: p62 was detected in the cytoplasm of cells in the cancer nodule by immunohistochemistry. B: Enlargement (original magnification, ×500) of the area boxed in A. C: Anti-p62C antiserum was absorbed with recombinant p62 protein. The fluorescent signal was primarily removed whereas staining was not removed by absorption with recombinant golgin-97 (D). Arrows in A and B point to cells in the same area.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Nine liver specimens from normal adult donors were also examined for p62 expression. No staining with either anti-p62F or anti-p62C antibodies was observed in all cases (data not shown). Taken together, these data demonstrated that p62 gene is down-regulated or not expressed in differentiated hepatocytes of adult normal livers but aberrantly expressed in the tumor hepatocytes of one-third of HCC patients investigated. To examine whether p62 is expressed in other liver cancers, we performed immunohistochemical analysis on two available liver specimens with CCC using anti-p62C antibodies. Proliferating bile ducts were abundant in the specimen depicted in Figure 5A, and as shown in Figure 5, B to D, p62 was expressed in the cytoplasm of epithelial cells of bile ducts (case 28 in Table 1). However, p62 was not detectable in the epithelial cells of bile ducts in case 29. Liver cirrhosis is considered a high-risk precursor for development of HCC. Twenty-three specimens from patients with liver cirrhosis were examined for the expression of p62. As shown in the upper panels of Figure 6, cytoplasmic staining was observed in ∼5% of hepatocytes in the periphery of cirrhotic nodules. This pattern of expression was observed in eight cases and designated as low focal positivity (Table 2). The focal expression pattern of these cirrhotic livers was in clear contrast to the uniform expression of p62 in HCC (Figure 2, Figure 4). Three other cirrhotic liver specimens displayed p62 expression not only in hepatocytes in the periphery of nodules but also in hepatocytes located in the centers of cirrhotic nodules that were not observed in the low focal positive cases (Figure 6, bottom panels). The level of p62 expression varied from high to low intensity in different cells and the total number of p62-expressing cells varied from 20 to 50%. This expression pattern in the three cirrhotic livers was designated as high focal positivity (Table 2).Table 2Immunohistochemistry of p62 on Liver TissueTotal number of casesImmunohistochemistryUniform fluorescence of all nodular cellsHigh focal positive fluorescenceLow focal positive fluorescenceHCC27900CCC2100Liver cirrhosis23038Normal liver9000 Open table in a new tab To determine whether the p62 gene was developmentally regulated, five human liver specimens at different fetal stages (days 50 to 125) were examined for the expression of p62 gene at the protein level. As shown in Figure 7, A to D, p62 protein was detectable in hepatocytes in a day 50 fetal liver. Similar to the day 50 fetus, p62 was expressed in the other four fetal livers ranging from day 50 to day 125. In contrast, p62 protein was undetectable in nine normal adult livers (Table 2). These data indicated that p62 gene is expressed in human fetal livers and seems to be down-regulated in adult hepatocytes during development. To investigate whether the p62 gene is regulated at the transcriptional level, RT-PCR was performed using an equal amount of poly(A)+ mRNA from either human fetal or adult livers. A 1.7-kb PCR product containing the open reading frame of p62/IMP-2 cDNA from fetal livers could be abundantly amplified with p62FL primers (Figure 8, lane 5), whereas the product from adult livers was barely detected (lane 6). As a control, an actin fragment amplified from the same batch of cDNA showed no difference in amount of cDNA (Figure 8A, lanes 3 and 4). The 1.7-kb PCR DNA product from fetal livers was subcloned and five recombinant clones were identified. The nucleotide sequences of all five recombinant clones were identical to those of original p62 cDNA,2Zhang JY Chan EKL Peng XX Tan EM A novel cytoplasmic protein with RNA-binding motifs is an autoantigen in human hepatocellular carcinoma.J Exp Med. 1999; 189: 1101-1110Crossref PubMed Scopus (184) Google Scholar a splice variant that does not contain the extra 129-bp nucleotide present in IMP-2 cDNA,7Nielsen J Christiansen J Lykke-Andersen J Johnsen AH Wewer UM Nielsen FC A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development.Mol Cell Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (575) Google Scholar suggesting that the shorter p62 rather than the longer IMP-2 form was selectively expressed in the fetus. We further performed RT-PCR using total RNA extracted from paraffin-embedded tissues. Using the p62SL primers, a 386-bp p62 PCR product rather than the 515-bp IMP-2 product was amplified from each of two immunohistochemically p62-positive HCC tissues (Figure 8B, lanes 3 and 4). No PCR fragment was detected from a normal adult liver and a p62-negative HCC tissue (lanes 1 and 2, respectively). Thus, the data from RT-PCR were consistent with those from immunohistochemical analysis an
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