Lymphokines modulate the growth and survival of thymic tumor cells containing a novel feline leukemia virus/Notch2 variant

Abstract

Tumorigenesis occurs through a multistep process initiated by genetic lesions and facilitated by endogenous and external growth/survival signals. In many malignancies, specific oncogenic mutations correlate with phenotypic characteristics, inferring lineage-specific pathogenic mechanisms. To characterize these relationships in a unique feline tumor, we studied primary cells and two-cell lines independently-derived from a thymic lymphoma that contained and actively expressed a novel feline leukemia virus (FeLV) recombinant with transduced host Notch2 sequences. All three tumor cell populations contained similar FeLV/Notch2 proviral variants and phenotypically resembled mature thymocytes. Multiple Notch2 transcripts were expressed in the cell lines, including species that correspond to viral genomes and spliced subgenomic viral mRNA. Tumor cell line FeLV/Notch2 virus was packaged into virions; however, the variant was not efficiently transmitted to feline cells in vitro. Primary tumor cells constitutively expressed mRNA for interleukin-4 (IL-4), IL-6 and the p40 subunit of IL-12. Lymphokine mRNA was not detected in established tumor cell lines nor was T-cell growth-promoting activity found in culture supernatants. Exogenous IL-4 enhanced primary tumor cell survival, but inhibited proliferation of the cell lines. Interleukin-4 abrogated hydrocortisone-induced apoptosis in all three populations and had divergent effects on cell line clonogenic colony formation. Exogenous IL-7 and, to a lesser degree, IL-6 also had variable positive effects on the growth and viability of the tumor cell populations. Collectively, these data suggest that thymocytes are susceptible to the transforming potential of dysregulated Notch2 and that thymopoietic factors could, through overlapping and distinct mechanisms, promote the survival and outgrowth of FeLV/Notch2-containing neoplastic cells.

Introduction

Animal tumors induced by slow-transforming retroviruses provide valuable models of the oncogenic and biological mechanisms contributing to clonal neoplasia. In cats, recurring proviral and host genetic alterations have been identified in both spontaneous and experimental feline leukemia virus (FeLV)-associated thymic tumors (Athas et al., 1994). These genetic determinants include enhancer duplications in the retroviral long terminal repeat (LTR) (Rohn and Overbaugh, 1995, Pantginis et al., 1997), insertional disruption of cellular genes (Tsatsanis et al., 1994), env mutations (Rohn et al., 1994) and recombination with endogenous env or cellular gene sequences (Tsatsanis et al., 1994).

In contrast to our understanding of molecular mechanisms in feline tumors, little is known about the potential facilitating effects of autocrine and microenvironmental growth and survival factors. Neoplastic cells from human leukemias and murine lymphomas frequently retain responsiveness to microenvironmental signals (Hassuneh et al., 1997, Raziuddin et al., 1998). Furthermore, the action of specific factors on malignant cells in vitro often correlates with the phenotype, differentiation status and, in some cases, the cytogenetic profile and clinical behavior of the tumor (O’Connor et al., 1991). By extension, thymopoietic lymphokines could play a role in promoting the outgrowth of neoplastic FeLV-infected cells. This effect may collaborate with, or be independent of, growth alterations related to specific oncogenic mutations.

To begin to address these issues in a feline model, we studied tumor cells derived from a novel FeLV-induced thymic lymphoma. The original tumor contained recombinant proviruses with transduced sequences encoding the intracellular portion of the feline homologue of Notch2 replacing most of env (Rohn et al., 1996). Northern blot analyses revealed that Notch2 sequences were expressed in the tumor cells, but not in uninvolved tissues from the cat, indicating that this recombinant was transcriptionally active (Rohn et al., 1996). Additional studies with molecular clones of the FeLV/Notch2 variant indicated that the host sequences were encoded in a context that permits expression of a truncated, nuclear form of the Notch2 protein (Rohn et al., 1996). While the normal function of Notch2 is unknown, structurally similar Notch1 mutations, which generate activated intracellular forms of the Notch1 protein, have been implicated in the pathogenesis of spontaneous T-cell leukemias in humans (Ellisen et al., 1991) and experimental T-cell lymphomas in mice (Pear et al., 1996, Girard et al., 1996). Analysis of DNA from the primary feline tumor identified no additional putative oncogenic determinants in either intact proviral env or LTR sequences (Rohn et al., 1994, 1995). Therefore, we hypothesized that the FeLV/Notch2 recombinant played an important pathogenic role in tumorigenesis.

In order to correlate the molecular and phenotypic features of the FeLV/Notch2-containing tumor, we analyzed primary cells and two derived continuous cell lines. The recombinant proviral integration patterns and expressed Notch2 mRNA species were characterized. We determined if FeLV/Notch2 virus could be transmitted to naive feline cells and whether phenotypic alterations occurred in cells exposed to tumor-derived virus. Furthermore, we defined the phenotypic profiles of the tumor cells, their cytokine expression patterns and their growth and viability responses to exogenous lymphokines.