Analysis of Intracytoplasmic Hyaline Bodies in a Hepatocellular Carcinoma

Abstract

Intracytoplasmic hyaline bodies (IHBs) resemble inclusions in hepatocellular carcinoma cells, which so far have escaped further characterization. A relationship to Mallory bodies was suggested on the basis of light microscopy and filamentous ultrastructure. A hepatocellular carcinoma containing numerous IHBs was studied. Our studies revealed immunoreactivity of IHBs with the monoclonal antibodies SMI 31 and MPM-2, which recognize hyperphosphorylated epitopes present on paired helical filaments in Alzheimer's disease brains (SMI 31) or on diverse proteins hyperphosphorylated by mitotic kinases in the M-phase of the cell cycle (MPM-2). One- and two-dimensional gel electrophoresis of tumor extracts followed by immunoblotting with SMI 31 and MPM-2 antibodies revealed a major immunoreactive protein with an apparent molecular weight between 62 and 65 kd, which was resolved into several highly acidic (pH 4.5) protein components in two-dimensional gels. This protein was undetectable in non-neoplastic liver tissue. Sequence analysis identified the SMI 31 and MPM-2 immunoreactive material as p62, indicating that p62 is a major constituent of IHBs. p62 is an only recently discovered protein that is a phosphotyrosine-independent ligand of the SH2 domain of p56(lck), a member of the c-src family of cytoplasmic kinases. Moreover, p62 binds ubiquitin and may act as an adapter linking ubiquitinated species to other proteins. These features suggest a role of p62 in signal transduction and possibly also carcinogenesis. IHBs observed in the hepatocellular carcinoma cells presented are the first indications of a role of p62 in disease.

In vitroproduction of Mallory bodies and intracellular hyaline bodies: The central role of sequestosome 1 / p62

“…In Vitro Reproduction of MBs and IHBs by Cotransfections. Cotransfection of p62 and ubiquitin resulted in globular cytoplasmic aggregates resembling IHBs, 2,4 with an indistinct fibrillar to granular ultrastructure and colocalization of p62 and ubiquitin (Fig. 3A,B).…”

“…MBs and IHBs associated with diseased liver differ with respect to chemical composition, light microscopy, and ultrastructure. 1,2,4,34,35 In IHBs, the indistinct fibrillar and granular ultrastructure prevails. 2 The ultrastructure of MBs is variable.…”

“…Western blotting was performed as described. 2 Equal amounts of total lysates of transfected CHO-K1 cells were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by protein transfer onto polyvinylidine fluoride membranes. The following antibodies were used: (1) primary antibodies to keratin 8 (Troma I) to detect transfected keratin 8, p62 (Transduction Laboratories) to detect transfected p62, p62CT 2 to detect endogenous and transfected p62, myctag (Abcam) to detect transfected ubiquitin, and ␤-actin (Sigma-Aldrich, Vienna, Austria) to detect endogenous ␤-actin; (2) as secondary antibodies, horseradish peroxidase-conjugated rabbit anti-mouse Ig, swine anti-rabbit Ig, and rabbit anti-guinea pig Ig (Dako) were used.…”

“…1,2 They differ in their light and electron microscopic appearance and chemical composition but share sequestosome 1/p62 (p62) as a major component. [1][2][3][4] Whereas MBs contain in addition abnormal keratins, ubiquitin, and heat shock proteins, keratins are lacking in classical IHBs.…”

“…1,2 They differ in their light and electron microscopic appearance and chemical composition but share sequestosome 1/p62 (p62) as a major component. [1][2][3][4] Whereas MBs contain in addition abnormal keratins, ubiquitin, and heat shock proteins, keratins are lacking in classical IHBs. 1,4 P62 is a multifunctional protein 5,6 with several structural motifs that determine its various functions: the SH2 domain binds the tyrosine kinase p56 lck in a phosphotyrosine-independent manner, an acidic interaction domain associates with the atypical protein kinase C and mediates the role of p62 as adapter in tumor necrosis factor alpha-initiated, interleukin-1-initiated, and nerve growth factor-initiated signal transduction, and a ZZ-type zinc finger binds the receptor interactive protein involved in tumor necrosis factor-induced apoptosis.…”