Mutation analysis of human cytokeratin 8 gene in malignant rhabdoid tumor: A possible association with intracytoplasmic inclusion body formation

The rhabdoid cell, which is typically observed in malignant rhabdoid tumor (MRT) and other malignant neoplasms, has an eosinophilic cytoplasm containing a spheroid perinuclear inclusion body. This distinct cell is known to act as a highly aggressive indicator in many types of malignant tumors and is characterized by aggregates of intermediate filaments, comprising both vimentin and cytokeratin (CK) 8, which is mainly expressed in simple-type epithelium such as liver and intestine. To clarify the cause of the inclusion body formation, we analyzed the alteration of the complete human CK8 gene (KRT 8: 1724 base pairs) in seven samples of MRT (three from frozen materials and four from cultured cell lines) by reverse-transcriptase polymerase chain reaction, followed by direct sequencing. In addition, the two cell lines, Huh7 and HeLa, which lacked rhabdoid feature, six pediatric malignant tumors, including three cases of primitive neuroectodermal tumor (PNET) and three of Wilms' tumor; and 15 normal liver tissue (as a control) were also analyzed. All MRT samples had missense mutations in the human KRT 8 gene, i.e., Arg89 → Cys (5/7); Arg → Cys251 (3/7); Glu267 → Lys (6/7); Ser290 → Ile, Met; (7/7) and Arg301 → His(4/7), none of which was detected in any control samples. Among these mutations, the most noteworthy findings were that Arg89 belongs to the H1 subdomain of the head domain and that Arg251 belongs to the short non-helical linker segment, or L1-2. Both these mutations are noted for their relationships to lateral protofilament-protofilament interactions. In addition, Ser290 has been previously reported to be a phosphorylation site, which has been recognized to play an important role in filament organization, leading to conformational change of the CK8 filaments. In conclusion, mutated codons of CK8 gene in MRT were located in the important region involved in the conformational change of intermediate filament.