Lymphokines modulate the growth and survival of thymic tumor cells containing a novel feline leukemia virus/Notch2 variant
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.
Section snippets
Primary tumor cells, cell lines and cell line conditioned media
Pleural fluid tumor cells from domestic cat 40836 were cultured in a 1 : 1 mixture of RPMI : L15 media (both from GIBCO, Grand Island, NY) with 20% heat-inactivated fetal calf serum (FCS, Summit Laboratories, Fort Collins, CO); 1% penicillin/streptomycin/fungizone (GIBCO); 2 mM glutamine (GIBCO); and 5 × 10−5 M β-mercaptoethanol. Additional tumor cells were frozen in 5% dimethyl sulfoxide in liquid nitrogen for later use. Two continuous cell lines, designated 836-L1 and 836-L2, were independently
Primary tumor cells and established tumor cell lines phenotypically resemble mature thymocytes
The morphologic, histochemical and immunophenotypic profiles of the primary tumor cells and established cell lines were similar. These lymphoblastoid cells (Fig. 1B) expressed AP (100%), but were negative for Alk P, MPO, NSE, PAS and TdT. Flow cytometry revealed a high level of membrane CD5 expression (95–100%) with low/variable expression of CD4 (5% in primary cells and 5–25% in established cell lines) and no expression of CD8 (Fig. 1A). All three populations were uniformly positive for MHC
Discussion
Neoplastic transformation occurs through a multistep process (Magrath, 1992). This process is often initiated by genetic events that alter normal cell growth controls. Exogenous stimuli, such as microenvironmental cytokines and hormones, may facilitate tumor development. Together, endogenous and external signals promote the expansion of preneoplastic populations. Eventually, a fully malignant clone emerges from this preneoplastic pool.
In this study, we sought to characterize the relationships
Acknowledgements
This research was supported by IRG-185 from the American Cancer Society and NIH Grants DK49888 (M.L.L.) and CA51080 (J.O.). Jennifer L. Rohn was supported, in part, by a Helen Riaboff Whitely Graduate Fellowship Award. Julie Overbaugh is a scholar of the Leukemia Society of America. We thank Zeny Sisk for assistance in preparation of the figures.
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