Position of the 62-kd protein is marked by the arrow

Position of the 62-kd protein is marked by the arrow. livers were all positive for mRNA and protein. The observations show that p62 is Cenicriviroc Mesylate 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 Cenicriviroc Mesylate factors leading to the development of HCC, but the genetic defects and proteins involved in the development of liver cancer are not completely understood. 1 Using serum antibody from a patient with HCC to screen a cDNA expression library Cenicriviroc Mesylate we recently identified a 62-kd RNA-binding protein that elicited a humoral immune response in 20% of HCC patients. 2 p62 contains one set of the RNA recognition motif 3 and four hnRNP K homology (KH) domains 4-6 and belongs to the family of IMPs (insulin-like growth factor II mRNA-binding proteins). 7 The human IMPs have high sequence identity with and similar RNA-binding domain distribution as other RNA-binding proteins such as the Vg1RBP/Vera protein, 8,9 the chicken zipcode-binding protein-1, 10 and the mouse c-Vg1 and chicken -actin mRNAs takes place via 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. 12 In this Cenicriviroc Mesylate 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. Materials and Methods Patients and Tissue Specimens Liver tissue from 27 HCC patients were obtained from Henan Medical University, Henan Province, Peoples Republic of China and from the Department of Pathology, the Scripps Clinic, La Jolla, CA. IFNW1 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. 13 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 Cenicriviroc Mesylate noncancer cases were examined by two senior pathologists followed by separately conducted immunohistochemical analysis by other authors in this report. Table 1. Clinical Pathological Data on Patients with HCC and CCC and Immunohistochemistry of Liver Tissue 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 manufacturers 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 g of synthesized peptide or purified recombinant protein in Freunds complete adjuvant, and boosted 1 month later with the same amount of peptide or protein in Freunds incomplete.