A v-H-ras-dependent hemopoietic tumor model involving progression from a clonal stage of transformation competence to autocrine interleukin 3 production.

Abstract

Autocrine interleukin 3 (IL-3)-secreting tumors were generated from an IL-3-dependent mouse mast cell line (PB-3c) after introduction of the v-H-ras oncogene. Tumor progression was characterized by four distinct phenotypes. The first corresponded to immortalized mast cells unresponsive to the oncogenic effect of v-H-ras. The second was expressed in a clonable subpopulation of PB-3c cells and was marked by the competence to form v-H-ras-dependent tumors (immortalized transformation competence). The third was a direct effect of v-H-ras expression on all PB-3c cells and was characterized in vitro by a reduced IL-3 requirement. Upon injection of v-H-ras-expressing, transformation-competent cells into mice, the final, fully malignant phenotype developed with a long latency period and was marked in vitro by independence of exogenous IL-3 and by autocrine IL-3 stimulation. Northern (RNA) blot analysis and an RNase A-T1 protection assay showed that IL-3 production was strictly associated with the tumor phenotype. Two of six tumors showed an alteration at the 5' region of the IL-3 gene. We conclude that v-H-ras required complementation by IL-3 gene rearrangement or an alternate event to generate autocrine mastocytomas.

Regulation of interleukin 3 mRNA expression in mast cells occurs at the posttranscriptional level and is mediated by calcium ions.

“…The selective stabilization of GM-CSF mRNA has been described in monocytic tumors and involvement of the 3' untranslated region in GM-CSF mRNA in this process has been suggested (28). Work in our laboratory has shown that PB-3c cells can progress to IL-3-producing autocrine mastocytomas after introduction of the v-Ha-ras oncogene (15). In addition, production of IL-3 and GM-CSF can be induced in PB-3c cells transfected with the vector expressing the EJ c-Ha-ras oncogene at high levels (36).…”

“…The following plasmid probes were used: pBR322; pBR/actin4, containing a 567-bp Pst I fragment of a ,8-actin cDNA (22) (20); and pUC/c-myc, containing a 4.8-kb Xba I-BamHI fragment of c-myc (23). Linearized and denatured plasmids (3 ,ug) were spotted on 9-mm nitrocellulose filters and hybridized with labeled RNA (=2 x 107 cpm/ml) at 45°C for (15). IL-3 mRNA was also not detected in the cells treated with PMA (lanes 2 and 6), irrespective of concentration (5-100 ng/ml) and time of treatment (1-16 hr) (data not shown).…”

“…PB-3c cells, an IL-3-dependent murine mastocyte line (14), were cultured as described (15) in Iscove's modified Dulbecco's medium supplemented with a concentrated conditioned medium from WEHI-3B cells, which served as a source of IL-3 (16).…”

Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism

“…The levels of the ⌬Raf:ER protein often increases after ␤-estradiol treatment, this has been shown to be due to protein stabilization induced by ␤-estradiol. [39][40][41] Thus there was a dose-dependent activation of Raf and MEK activities in these cells which correlated with …”

“…35 (7) pBP3⌬B-Raf [DD] :ER which encodes the catalytic domain of murine B-Raf fused to the hbER and inserted into pBP3. 41 (8 and 9) pBP3⌬B-Raf [FF] :ER and pBP3⌬B-Raf [YY] :ER in which the wild-type [DD] amino acids of B-Raf at positions 492 and 493 were replaced by [FF] or [YY], respectively; and (10) pBP3⌬Raf-1301:ER kinase-inactive, which contains the lysine → typtophan substitution at the ATP binding site of the catalytic domain of Raf-1. 35,50 Puro r FDC-P1 cells were isolated by selection in 500 ng/ml puromycin (Sigma) in the presence of IL-3.…”