Regulation of a heterologous glucose transporter promoter in chicken embryo fibroblasts.

GLUT1 is a glucose transporter responsible for increased cellular glucose uptake upon oncogenic transformation or mitogenic stimulation. This is associated with transcriptional activation of the GLUT1 gene. Three regions in the mouse GLUT1 gene mediate this regulation (the promoter and two enhancers). In contrast, chicken GLUT1 transcription is completely uninducible, suggesting either avian signal transduction events are different or the chicken GLUT1 gene lacks these transcriptional control elements. To distinguish between these possibilities, reporter plasmids containing mouse GLUT1 control elements in rodent and avian fibroblasts were compared. These elements function within chicken cells, suggesting that signaling pathways are similar to mouse cells but that the avian GLUT1 gene lacks the control elements necessary to respond to them.

Characterization of the avian GLUT1 glucose transporter: differential regulation of GLUT1 and GLUT3 in chicken embryo fibroblasts.

Vertebrate cells that are transformed by oncogenes such as v-src or are stimulated by mitogens have increased rates of glucose uptake. In rodent cells, the mechanisms whereby glucose transport is up-regulated are well understood. Stimulation of glucose transport involves an elevation in mRNA encoding the GLUT1 glucose transporter that is controlled at the levels of both transcription and mRNA stability. Cloning and sequencing of chicken GLUT1 cDNA showed that it shares 95% amino acid sequence similarity to mammalian GLUT1s. Nevertheless, unlike mammalian GLUT1 mRNA, it was not induced by v-src, serum addition, or treatment with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in chicken embryo fibroblasts. Rather, the induction of glucose transport in chicken embryo fibroblasts by v-src, serum, and 12-O-tetradecanoylphorbol 13-acetate was associated with induction of GLUT3 mRNA level and GLUT3 transcription. Rat fibroblasts were also found to express both GLUT1 and GLUT3 isoforms, but v-src induced GLUT1 and not GLUT3. This suggests that animal cells require both a basal and an upregulatable glucose transporter and that these functions have been subsumed by different GLUT isoforms in avian and mammalian cells.

Differential-effects of viral and cellular oncogenes on the growth factor-dependency of hematopoietic-cells.

The effects of different viral and cellular oncogenes on the cytokine-dependency of murine hematopoietic cell lines were compared. The myeloid FDC-P1 cell line was sensitive to abrogation of growth factor-dependency by the constitutive expression of viral oncogenes (v-abl, v-src, v-Ha-ras, and v-fms) and the activated cellular oncogene BCR-ABL and Delta Nraf. The Delta Nraf encoded serine-threonine kinase was approximately 100-fold less efficient in relieving the factor-dependency of FDC-P1 cells than the other oncogenes examined. The synthesis of autocrine cytokines was not detected in the factor-independent FDC-P1 lines, indicating that the oncogene-mediated transformation occurred by a non-autocrine mechanism. A low frequency of cells were isolated after infection with the chronic retrovirus, murine leukemia virus and approximately 40% of these clones synthesized the autocrine lymphokine, granulocyte-macrophage colony stimulating factor. In contrast, only the v-abl and BCR-ABL oncogenes relieved the cytokine-dependency of the lymphoid FL5.12 cell line. In all the transformed cell lines, the rate of glucose transport was elevated above the basal level seen in uninfected cells indicating that this pivotal growth-regulated protein was associated with malignant transformation. In summary, these cell lines varied with respect to abrogation of growth factor-dependency as the myeloid FDC-P1 line was sensitive to transformation by all oncogenes examined whereas only the abl-family members would relieve the cytokine-requirement of lymphoid FL5.12 cells.

Autocrine growth-factor secretion after transformation of human cytokine-dependent cells by viral and cellular oncogenes.

The effects of viral and cellular oncogenes on a human erythroleukemic cell line (TF-1) were investigated. The TF-1 cell line required granulocyte/macrophage-colony stimulating factor (GM-CSF) for growth but this factor-dependency was abrogated by the constitutive expression of either viral (v-fms, v-Ha-ras and v-src) or cellular oncogenes (BCR-ABL and Delta N-raf). Furthermore the overexpression of the human insulin-like growth factor-1 (IGF-1) receptor could substitute the dependency on GM-CSF with a requirement for either IGF-1 or insulin as a proliferative signal. An autocrine cytokine, (GM-CSF) was found in the supernatant of cells transformed by Delta N-raf (and to a lesser extent in cells infected with other oncogenes. The level of GM-CSF secreted by the Delta N-raf transformants was sufficient to support the proliferation of the parental cell line. GM-CSF mRNA transcripts were detected in the Delta N-raf-infected but not in the parental cells. No structural alterations of the GMCSF locus were seen in these cells. Together these observations indicated that overexpression of a raf oncogene resulted in the expression of GM-CSF transcripts. The rates of glucose transport were elevated above basal levels by GMCSF and by oncogene expression indicating that this pivotal control point of metabolism correlated with mitogenesis and malignant transformation. These studies indicate the importance of raf in growth regulation as its deregulation can lead to autocrine synthesis of cytokines in certain hematopoietic cells. Furthermore these results suggest a synergy between oncogene and cytokine gene regulation leading to autocrine growth factor expression and tumor progression.