Kaposi's sarcoma-like tumors in a human herpesvirus 8 ORF74 transgenic mouse

Abstract

The product of human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is related structurally and functionally to cellular chemokine receptors. ORF74 activates several cellular signaling pathways in the absence of added ligands, and NIH 3T3 cells expressing ORF74 are tumorigenic in nude mice. We have generated a line of transgenic (Tg) mice with ORF74 driven by the simian virus 40 early promoter. A minority (approximately 30%) of the Tg mice, including the founder, developed tumors resembling Kaposi's sarcoma (KS) lesions, which occurred most typically on the tail or legs. The tumors were highly vascularized, had a spindle cell component, expressed VEGF-C mRNA, and contained a majority of CD31(+) cells. CD31 and VEGF-C are typically expressed in KS. Tumors generally (but not always) occurred at single sites and most were relatively indolent, although several mice developed large visceral tumors. ORF74 was expressed in a minority of cells in the Tg tumors and in a few other tissues of mice with tumors; mice without tumors did not express detectable ORF74 in any tissues tested. Cell lines established from tumors expressed ORF74 in a majority of cells, expressed VEGF-C mRNA, and were tumorigenic in nude mice. The resultant tumors grew rapidly, metastasized, and continued to express ORF74. Cell lines established from these secondary tumors also expressed ORF74 and were tumorigenic. These data strongly suggest that ORF74 plays a role in the pathology of KS and confirm and extend previous findings on the tumorigenic potential of ORF74.

FIG. 1.

ORF74 transgene in Tg mice. (A) DNA was prepared from the following: a tail snip from founder mouse 383 (lane a); a tail snip from a non-transgenic littermate (lane b); cell lines GR3 (lane c) and GR4 (lane d), derived from serially transplanted tumors of nude mice (Table 2); cell line GR1 (lane e), derived from a Tg mouse primary tumor; or cell line GR5 (lane f), derived from a non-Tg mouse. DNA was digested with EcoRI and analyzed by Southern blotting, as described in Materials and Methods, with a probe consisting of the ORF74 coding region. The sizes of the observed fragments (in kilobase pairs) are indicated. (B) Schematic structure of the ORF74 transgene. The large arrow represents the ORF74 coding sequences, and the SV40 promoter, poly(A) signal, and β-globin intron are represented as boxes. The small arrowheads indicate the positions of the primers used for RT-PCR assays of the transgene.

FIG. 2.

ORF74 tumors in mice. (A and B) Foot and tail tumors, respectively, of ORF74 Tg mice. (C) A tumor in a nude mouse 3 weeks after injection of 5 × 10⁶ cells from the GR1 cell line, which was derived from a tumor from an ORF74 Tg mouse. (D) A hematoxylin-and-eosin-stained section from an external tumor near the ear. Characteristic spindle-shaped cells are present. The large elongated shapes are muscle fibers. (E) Staining of a 4-μm slice of a snap-frozen section from a tumor behind the ear. The section was stained with a murine monoclonal antibody to CD31 followed with Texas red-conjugated goat anti-mouse IgG and was examined in an Axiophot microscope (40×). (F) An adjacent section treated in the same way but using an irrelevant isotype control.

FIG. 3.

Expression of ORF74 RNA in Tg mouse tumor tissue. (A) RNA was purified from tissues of a Tg mouse (mouse 470) with tumors (lanes 1 to 5) and a Tg mouse (mouse 2830) with no tumors (lanes 6 to 9). RNA was from tumor tissue (lane 1), leg muscle (lane 2), kidney (lane 3), brain (lane 4), and intestine (lane 5) of mouse 470 and from leg muscle (lane 6), kidney (lane 7), brain (lane 8), and liver (lane 9) of mouse 2830. Lane 10 contains RNA from a normal cell line (GR5) and lane 11 contains RNA from GR1, a tumor-derived cell line from Tg mouse 116. RT-PCR was performed separately for ORF74 and 18S RNA, and both PCR mixtures were run on the same gel. First, 18S amplicons were loaded and electrophoresed until they had visibly migrated into the gel. The ORF74 amplicons were then loaded, and electrophoresis was resumed. The lower bands represent 18S cDNA and the upper bands represent ORF74 cDNA, as indicated. (B) RNA was purified from a nude mouse tumor (lane 1), a tumor from Tg mouse 470 (lane 2), leg muscle from Tg mouse 470 (lane 3), leg muscle from a Tg mouse (mouse 460) with no tumors (lane 4), liver tissue from Tg mouse 470 (lane 5), liver tissue from a non-Tg mouse (lane 6), a cell line (GR1) from the tumor of a Tg mouse (lane 7), a cell line (GR3) from a nude mouse tumor (lane 8), and a cell line (GR5) from a normal mouse tail (lane 9). RT-PCR was performed with VEGF-C primers as described in Materials and Methods. Amplicons were electrophoresed on 1% agarose gels and stained with ethidium bromide.

FIG. 4.

Expression of ORF74 protein in Tg tumors. Sections from a KS-like tumor behind the ear were analyzed by immunostaining with a rabbit polyclonal antipeptide antiserum to ORF74 as described in Materials and Methods. (A) ORF74 expression both inside and outside areas containing spindle cells (magnification, ×25). (B) Expression in cytoplasm of spindle cell (magnification, ×40). (C) Expression in cytoplasmic area of a nonspindle cell (magnification, ×40).

FIG. 5.

Expression of ORF74 protein in tumor-derived cell line. Uninduced BCBL-1 cells (A), cells from tumor-derived cell line 2362 (GR2) (B), and NIH 3T3 cells (C) were analyzed by immunostaining with a rabbit polyclonal antipeptide antiserum to ORF74 as described in Materials and Methods. The intensely labeled cell shown in panel A is a BCBL-1 cell presumably undergoing spontaneous lytic phase viral expression. (D) RNA from the GR5 (mouse 2380) and GR3 (mouse 2363) cell lines was analyzed by real time RT-PCR for expression of VEGF-A and VEGF-C by using 18S rRNA as an internal reference standard as described in Materials and Methods. Values were normalized so that expression in GR5 equaled 1. Error bars show the standard deviations with triplicate samples.