Mechanism of autocrine stimulation in hematopoietic cells producing interleukin-3 after retrovirus-mediated gene transfer.

Abstract

Endogenous expression of the interleukin-3 (IL3) gene introduced with a retrovirus vector renders hematopoietic cells autonomous of exogenous growth factor. To investigate the mechanism of autocrine stimulation, 25 clones were isolated after retrovirus transduction of IL3 into 32D-c123 or FDC-P1 cells. Medium conditioned by these autonomous IL3-producing clones supported the growth of factor-dependent 32D cells. Although there was a severalfold variation in the amount of IL3 secreted (some clones secreted barely detectable levels), the doubling time of each clone in the absence of added IL3 was identical to that of the parental cell line maximally stimulated by exogenous IL3. Concentrated monoclonal and polyclonal antibodies, both highly effective in neutralizing exogenous IL3, were assayed for ability to inhibit autocrine growth. Minimal inhibitory effects were observed only on washed autocrine clones secreting low levels of IL3. To test the activity of cytoplasmically synthesized IL3, the nucleotides encoding the signal sequence of IL3 were deleted and replaced with an in-frame ATG in the context of a consensus translation initiation sequence. Ten 32D clones expressing this restructured IL3 genome were obtained. Despite the presence of biologically active IL3 in cell lysates, all clones remained dependent on exogenous IL3, with the same dose-response as that found for 32D cells. Our data are most compatible with a mechanism whereby endogenously produced IL3 interacts with its receptor prior to surface display.Normal as well as neoplastic cell growth has been shown to be modulated by growth factors, the interplay of which coordinates myriad genes leading to proliferation and differentiation (15, 46). Such substances either are elaborated by tissues at variable distances from their targets (endocrine or paracrine) or derive from the target cells themselves (autocrine). In neoplasia, the novel expression of a growth factor by a cell expressing a functional receptor is one mechanism for the interruption of normal restraints on growth (45). Amplified or dysregulated mitogenic signals resulting from abnormal synthesis of growth factors have been demonstrated in certain human neoplasms (3,6,9,25,35,50,51).Early in the study of factor-dependent cell lines, infrequent mutants that had acquired the ability to secrete growth factor and grow autonomously were discovered (20,41). With the cloning of the genes for growth factors and their receptors, the steps that link growth factor expression and tumorigenesis have become accessible to investigation. Many reports on the mechanism of autocrine stimulation have shown that autonomous cells can be further stimulated by the addition of exogenous growth factor, that cell growth can be inhibited by neutralizing antibody, and that at low cell density, growth and plating efficiency are diminished (3,9,11,12,18,25,28,36,37,40,42,50). These three criteria are compatible with the secretion of growth factor which only then binds and transduces the mitogenic signal. However, other inves...

Retrovirus-mediated transfer and expression of the interleukin-3 gene in mouse hematopoietic cells result in a myeloproliferative disorder.

“…Analogous results have been obtained with the murine IL-3 gene (14,15,38). In some cases, autonomous growth appeared to depend on secretion of the growth factor (14,15,18), whereas cell proliferation of other growth factor-producing lines was insensitive to cell density and neutralizing antibody (6,20).…”

Aberrant Expression of the Multi-CSF Gene in Hematopoietic Precursor and Stem Cell Lines Initiates Leukemogenic Progression

“…Work by Browder et al [12) is in accordance with this hypothesis. Infection of multi-CSF-dependent 32D cells with a construct encoding a modified multi-CSF protein that does not direct the CSF molecule into the ER for processing did not lead to factor independence, although the gene product localized in the cytosol proved to be biologically active.…”

“…Analysis of leukemic cells from both AML and CML patients have provided evidence in support of the hypothesis that aberrant expression of growth factor genes (e.g., GM-CSF and G-CSF) may contribute to the uncontrolled growth of the leukemic cells [13,14]. Indeed, two experimental studies have demonstrated the causal correlation of aberrant factor production with leukemogenic growth: unregulated expression of an IL-6 gene conjugated to the human Ig heavy chain gene enhancer (E~) in transgenic mice triggers generation of plasmacytoma/myeloma (Suematsu and Kishimoto, cited in [15]) and expression of IL-3 in a retroviral construct introduced in vivo via murine hematopoietic stem cells leads to a hematopoietic disorder similar to CML [12].…”

Interleukin-6 functions as an intracellular growth factor in hairy cell leukemia in vitro.

“…In addition, the blocking ofthis response by IL-6 antisense oligonucleotide, but not by anti-IL-6 neutralizing mAb, suggests that it is mediated by IL-6 in an intracytoplasmic mechanism. This is analogous to previous reports of autocrine systems wherein cell growth is insensitive to exogenous growth factors and neutralizing antibodies are unable to inhibit proliferation (45)(46)(47)(48)(49). Such systems are compatible with a mechanism whereby receptor association and signal transduction both occur internally.…”