Kaposi's sarcoma-associated herpesvirus latency-associated nuclear antigen prolongs the life span of primary human umbilical vein endothelial cells

Abstract

Tumor spindle cells in all clinical types of Kaposi's sarcoma (KS) are infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Although KSHV contains more than 80 genes, only a few are expressed in tumor spindle cells, including latency-associated nuclear antigen (LANA) and k-cyclin (kCYC). To assess the oncogenic potential of LANA and kCYC, primary human umbilical vein endothelial cells (HUVEC) and murine NIH 3T3 cells were stably transduced by using recombinant retroviruses expressing these genes or the known viral oncogene simian virus 40 large T antigen (LTAg). Interestingly, LANA-transduced HUVEC proliferated faster and demonstrated a greatly prolonged life span (mean +/- standard deviation, 38.3 +/- 11.0 passages) than untransduced cells and vector-transduced cells (<20 passages). By contrast, kCYC-transduced HUVEC did not proliferate faster or live longer than control cells. LANA- and kCYC-transduced HUVEC, but not LTAg-transduced HUVEC, retained the ability to form normal vessel-like structures in an in vitro model of angiogenesis. In cellular assays of transformation, LANA- and kCYC-transduced NIH 3T3 cells demonstrated minimal or no anchorage-independent growth in soft agar and no tumorigenicity when injected into nude mice, unlike LTAg-transduced NIH 3T3 cells. Lastly, gene expression profiling revealed down-regulation, or silencing, of a number of genes within LANA-transduced HUVEC. Taken together, these results suggest that KSHV LANA is capable of inducing prolonged life span, but not transformation, in primary human cells. These findings may explain why LANA-expressing spindle cells proliferate within KS tumors, yet most often do not demonstrate biologic characteristics of transformation or true malignant conversion.

FIG. 1.

LANA, kCYC, and LTAg protein expression in cells following infection with recombinant retroviruses and selection in the presence of G418. (A) Proteins were extracted from either transduced or untransduced cells and immunoblotted with specific antibodies as indicated. (B) LANA protein expression within all nuclei of LANA-transduced and G418-selected HUVEC (upper left), but not in vector (pLXRN)-transduced HUVEC (upper right). LANA-transduced HUVEC stained with rabbit IgG (lower left) and the KSHV-infected PEL cell line BCBL-1 stained with anti-LANA antibodies (lower right) were used as additional negative and positive controls, respectively.

FIG. 2.

LANA-transduced HUVEC proliferate faster than kCYC- or vector (pLXRN)-transduced HUVEC. A total of 30 × 10³ stably transduced HUVEC were subcultured on day zero, and absolute cell counts were determined daily for the next 5 days by using a hemocytometer. Triangles = LTAg-transduced cells; squares = LANA-transduced cells; circles = kCYC-transduced cells; diamonds = vector (pLXRN)-transduced cells. Values shown represent the means plus SD of four separate experiments. P = 0.029 when comparing LANA-transduced HUVEC to vector-transduced cells at day 5.

FIG. 3.

LANA-transduced primary HUVEC have a prolonged life span. Primary HUVEC were transduced as indicated and cultured under similar standard conditions. Data represent means ± SD for pLXRN- and LANA-transduced cells. Transduction of HUVEC with kCYC and LTAg was performed only once. LANA protein expression was periodically confirmed in LANA-transduced cells by immunofluorescence antibody staining (data not shown).

FIG. 4.

LANA- and kCYC-transduced HUVEC retain the ability to form normal vessel-like structures in an in vitro angiogenesis assay, unlike LTAg-transduced HUVEC. Representative fields are shown.

FIG. 5.

No anchorage-independent growth of kCYC-transduced cells and minimal anchorage-independent growth of LANA-transduced cells in soft agar. (A) Representative photographs of soft agar plates 4 weeks following culture of transduced HUVEC, demonstrating no colony formation by pLXRN- and kCYC-transduced cells and rare colony formation by LANA-transduced HUVEC. (B) Mean colony number in five separate soft agar plates ± SD 4 weeks following culture of either transduced HUVEC (open bars) or transduced NIH 3T3 cells (closed bars). Only colonies consisting of more than five cells were counted.

FIG. 6.

LANA- and kCYC-transduced NIH 3T3 cells do not form tumors when injected into nude mice. A total of 5 × 10⁵ transduced NIH 3T3 cells were subcutaneously injected into nude mice on day zero, and tumor sizes were assessed at regular intervals. There were five mice in each group. Each mouse is represented by a different symbol. Transduced HUVEC (including LTAg-transduced HUVEC) injected subcutaneously on day zero did not form tumors (data not shown).