Characterization of the human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) oncogene, Kaposin (ORF K12)

Background: Human herpesvirus 8 (HHV-8) has been implicated in the etiology of Kaposi's sarcoma (KS), a highly angiogenic tumor of complex histology, and two lymphoproliferative diseases, primary effusion lymphoma (PEL) and m ulticentric Castleman's disease (MCD). A number of HHV-8 encoded genes have been proposed to be involved in the pathogenesis of KS and PEL and a few h ave been shown to be oncogenic in heterologous systems (Reyes GR, LaFemina R, Hayward SD, Hayward GS. Morphological transformation by DNA fragments of human herpesviruses: evidence for two distinct transforming regions in her pes simplex virus types 1 and 2 and lack of correlation with biochemical tr ansfer of the thymidine kinase gene. Cold Spring Harbor Symp Quant Biol 198 0;44:629-641; Moore PS, Boshoff C, Weiss RA, Chang Y. Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV. Science 1996;27 4:1739-1744; Cheng EH, Nicholas J, Bellows DS, Hayward GS, Guo HG, Reitz MS , Hardwick JM. A Bcl-2 homolog encoded by Kaposi sarcoma-associated virus, human herpesvirus 8, inhibits apoptosis but does not heterodimerize with Ba r or Bak. Proc Natl Acad Sci USA 1997;94:690-694; Li M, Lee H, Yoon DW, Alb recht JC, Fleckenstein B, Neipel F, Jung JU. Kaposi's sarcoma-associated he rpesvirus encodes a functional cyclin. J Virol 1997;71:1984-1991; Neipel F, Albrecht J-C, Fleckenstein B. Cell-homologous genes In the Kaposi's sarcom a-associated rhadinovirus human herpesvirus 8. determinants of its pathogen icity? J Virol 1997;71:4187-4192; Nicholas J, Ruvolo VR, Burns WH, Sandford G, Wan X, Ciufo D, Hendrickson SE, Guo HG, Hayward GS, Reitz MS. Kaposi's sarcoma-associated human herpesvirus-8 encodes homologues of macrophage inf lammatory protein-1 and interleukin-6. Nat Med 1997;3:287-292; Nicholas J, Zong J, Alcendor DJ, Ciufu DM, Poole LJ, Sarisky RT, Chiuo C, Zhang X, Wan X, Guo H, Reitz MS, Hayward GS. Novel organizational features, captured cel lular genes, and strain variability within the genome of KSHV/HHV-8. JNCI M onographs 1998;23:79-88; Muralidhar S, Pumfery AM, Hassani M, Sadaie MR, Az umi N, Kishishita M, Brady JN, Doniger J, Medveczky P. Rosenthal LJ. Identi fication of kaposin (ORF K12) as a human herpesvirus 8 (Kaposi's sarcoma as sociated herpesvirus) transforming gene. J Virol 1998;72:4980-4988). The ka posin gene (ORF K12) encoded by the abundant latency-associated HHV-8 trans cript. T0.7. has been previously shown to induce tumorigenic transformation of Rat-3 cells (Muralidhar S, Pumfery AM, Hassani M, Sadaie MRI Azumi N, K ishishita M, Brady JN, Doniger J, Medveczky P, Rosenthal LJ. Identification of kaposin (ORF K12) as a human herpesvirus 8 (Kaposi's sarcoma associated herpesvirus) transforming gene. J Virol 1998;72:4980-4988). The current st udy is a further characterization of kaposin protein. Objectives: Character ization of kaposin expression in transformed and tumor-derived Rat-3 cells as well as PEL cell lines, BCBL-1, BC-3 and KS-1 and analysis of mechanism( s) of transformation. Dc sign: The presence of kaposin DNA in transformed c ells was determined by fluorescent in situ hybridization (FISH). Expression of kaposin protein was analyzed by Western blot analysis and indirect immu nofluorescence assay (IFA). Activation of cellular kinases in kaposin-trans formed cells was analyzed using Phosphospot peptide strips (Jerini Biotools ). Results: Kaposin DNA was integrated at a single locus in the genome of t ransformed Rat-3 cells as determined by FISH. Kaposin protein was expressed predominantly in the cytoplasm and colocalized with the 58 kDa Golgi membr ane protein in transformed Rat-3 cells. Western blot analysis of transformed Rat-3 cells revealed predominant prote in bands of approximately 16-18 kDa. Predominant 16-18 kDa bands were also detected in PEL cell lines BCBL-1, BC-3 and KS-I. In addition, bands of hig her molecular weight were detected in both transformed Rat-3 cells and PEL cells. Kaposin-transformed Rat-3 cells showed a 3-fold increase in the acti vities of serine-threonine kinases such as protein kinase C (PKC), calcium: calmodulin-dependent kinase II (CAM kinase II) and myosin light chain kinas e (MLCK). In addition, a 2-fold increase in the activities of Cdc2-kinase a nd cyclic-GMP (c-GMP)-dependent protein kinase was also observed. Conclusio ns: Results indicated that kaposin DNA was retained in transformed Rat-3 ce lls and expressed as predominantly cytoplasmic proteins of 16-18 kDa. Impor tantly, kaposin protein expression was detected by Western blot analysis in PEL cell lines, BCBL-1. BC-3 and KS-1. Preliminary studies indicated that kaposin may be involved in the activation of cellular serine-threonine kina ses which play an important role in cell proliferation such as PKC, CAM kin ase 11 and cdc3-kinase. (C) 2000 Elsevier Science B.V. All rights reserved.