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. 2001 Oct 9;98(21):12032-7.
doi: 10.1073/pnas.211026798. Epub 2001 Sep 25.

SV40 replication in human mesothelial cells induces HGF/Met receptor activation: a model for viral-related carcinogenesis of human malignant mesothelioma

P Cacciotti  1 , R Libener, P Betta, F Martini, C Porta, A Procopio, L Strizzi, L Penengo, M Tognon, L Mutti, G Gaudino
Affiliations

SV40 replication in human mesothelial cells induces HGF/Met receptor activation: a model for viral-related carcinogenesis of human malignant mesothelioma

P Cacciotti et al. Proc Natl Acad Sci U S A. .

Abstract

Recent studies suggested that simian virus 40 (SV40) may cause malignant mesothelioma, although the pathogenic mechanism is unclear. We found that in SV40-positive malignant mesothelioma cells, the hepatocyte growth factor (HGF) receptor (Met) was activated. In human mesothelial cells (HMC) transfected with full-length SV40 DNA (SV40-HMC), Met receptor activation was associated with S-phase entry, acquisition of a fibroblastoid morphology, and the assembly of viral particles. Coculture experiments revealed the ability of SV40-HMC to infect permissive monkey cells (CV-1), HMC, and murine BNL CL cells. Cocultured human and murine SV40-positive cells expressed HGF, showed Met tyrosine phosphorylation and S-phase entry, and acquired a spindle-shaped morphology (spBNL), whereas CV-1 cells were lysed. Cocultured HMC inherited from SV40-HMC the infectivity, as they induced lysis in cocultured CV-1 cells. Treatment with suramin or HGF-blocking antibodies inhibited Met tyrosine phosphorylation in all large T antigen (Tag)-positive cells and reverted the spindle-shaped morphology of spBNL. This finding indicated that Met activation and subsequent biological effects were mediated by an autocrine HGF circuit. This, in turn, was causally related to Tag expression, being induced by transfection with the SV40 early region alone. Our findings suggest that when SV40 infects HMC it causes Met activation via an autocrine loop. Furthermore, SV40 replicates in HMC and infects the adjacent HMC, inducing an HGF-dependent Met activation and cell-cycle progression into S phase. This may explain how a limited number of SV40-positive cells may be sufficient to direct noninfected HMC toward malignant transformation.

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Figures

Figure 1
Figure 1
Expression of Met and Tag by MM cell lines. (A) Equal amounts of solubilized proteins were immunoprecipitated with Met antibodies, separated by SDS/PAGE, transferred to nitrocellulose filter, and probed with the same antibodies. Asterisks on the right indicate the pr170MET precursor (**) and the mature β-chain (p145MET, *). (B) The same filter was reprobed with antiphosphotyrosine antibodies. (C) Tag sequence (*, nucleotides 4402–4574) was amplified by PCR from genomic DNA. (D) RT-PCR was performed on MM cells and COS-7 cells to amplify a fragment of Tag cDNA (172 bp), indicated by the asterisk on the right. (E) Electron microscopy of MMP cells. (Original magnification: ×40,000.)
Figure 2
Figure 2
Transfection of HMC cells with Tag and SV40 DNA. (A) Tag sequence (*) was amplified by PCR from genomic DNA of COS-7 (control), HMC, Tag-HMC, and SV40-HMC cells. (B) Tag protein (*) was detected in cell lysates immunoprecipitated and probed with Tag antibodies. (C) Immunoblotting was performed on Met immunoprecipitates with Met antibodies. GTL-16 cell line was used as a control of Met expression and phosphorylation. Asterisks on the right of the blot indicate the positions of the pr170MET precursor (**) and p145MET (*). (D) The same filter was reprobed with antiphosphotyrosine antibodies. (E) Citofluorimeter analysis of HMC cells before (Left) and after (Right) transfection with SV40 DNA.
Figure 3
Figure 3
Morphology after cocultures with SV40-HMC. The morphology of target cells CV-1 (A), BNL CL (D), and HMC (G) changed after coculture with SV40-HMC cells (B, E, and H, respectively). SV40 particles were observed by electron microscopy (C and I). (F) No virions were detected in nonpermissive BNL CL cells. CV-1 cells underwent vacuolization and massive lysis, accompanied by Tag expression. Tag protein (*) was detected in cell lysates immunoprecipitated and probed with Tag antibodies. (Original magnifications: A, B, D, E, G, and H, ×320; C, ×50,000; F and I, ×40,000.)
Figure 4
Figure 4
Coculture experiments. (A) Tag sequence (*) was amplified by PCR from genomic DNA of CV-1, BNL CL, and HMC. PCR without DNA (lane 1) and with COS-7 DNA (lane 2) were also performed as controls. Shown is the product of PCR amplification performed on lysates from cells cocultured with Tag-HMC (a) and SV40-HMC (b). The same PCR amplification was also performed on the SV40-HMC coculture medium (c). (B) Solubilized proteins from cell lysates were immunoprecipitated with Met antibodies and probed with the same Met antibodies and (C) with the antiphosphotyrosine antibodies. Asterisks on the right indicate the positions of the pr170MET precursor (**) and p145MET (*). (D) Tag protein (*) was detected in cell lysates immunoprecipitated and probed with Tag antibodies. (E) Electron microscopy of SV40-HMC cells. (Original magnification: ×60,000.)
Figure 5
Figure 5
Cell cycle of HMC and BNL CL cocultured cells. Cytofluorimeter analysis before (A and C) and after (B and D) coculture with SV40-HMC cells.
Figure 6
Figure 6
Met/HGF autocrine loop. Scatter assay was performed on the MDCK cell line. Shown are untreated MDCK cells (A) and MDCK stimulated with recombinant HGF (B), with MMP-conditioned medium (C), with MMM-conditioned medium (D), and with spBNL-conditioned medium (E). (Original magnification: ×320.) (F) RT-PCR and Southern hybridization specific for HGF (nucleotides 646-1533). Cell lines expressing activated Met (MMP, lane 4; MMCa, lane 5; Tag-HMC, lane 6; SV40-HMC, lane 7; HMC/SV40, lane 9; and spBNL, lane 11) display HGF expression (*), whereas HGF cDNA was not amplified in MMM (lane 3), HMC/Tag (lane 8), and BNL CL (lane 10) cells as well as in RT-PCR control without RNA (lane 1). MRC5 cell line (lane 2) was used as a control of HGF expression.
Figure 7
Figure 7
Met/HGF autocrine loop. (A) spBNL cells were photographed before (Left) and after (Right) treatment with anti-HGF blocking antibodies. (Original magnification: ×320.) (B) Met phosphorylation in MMP (lane 1), SV40-HMC (lane 2), HMC/SV40 (lane 3), and spBNL cells (lane 4) with (+) and without (−) treatment with anti-HGF blocking antibodies. Equal amounts of proteins from cell lysates were immunoblotted with antiphosphotyrosine antibodies. GTL-16 cell line was used as a control of Met expression and phosphorylation. Asterisks on the right indicate the positions of the pr170MET precursor (**) and p145MET (*).
Figure 8
Figure 8
Rb dependence of HGF autocrine circuit. (A) Solubilized proteins were immunoprecipitated with Met antibodies and probed with the same Met antibodies. Asterisks on the right of the blot indicate the positions of the pr170MET precursor (**) and p145MET (*). The same filter was reprobed with antiphosphotyrosine antibodies (B) and anti-Tag antibodies, using COS-7 as a control of Tag expression (C). (D) RT-PCR and Southern hybridization specific for HGF (nucleotides 646-1533). The MRC5 cell line was used as a control of HGF expression.
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