Suppression of apoptosis induced by growth factor withdrawal by an oncogenic form of c-Cbl.
The v-Cbl oncogene induces myeloid and B-cell leukemia; however, the mechanism by which transformation occurs is not understood. An oncogenic form of c-Cbl (Cbl-DeltaY371) was expressed in the interleukin-3 (IL-3)-dependent cell line 32Dcl3 to determine whether it was able to induce growth factor-independent proliferation. We were unable to isolate clones of transfected 32Dcl3 cells expressing Cbl-DeltaY371 that proliferated in the absence of IL-3. In contrast, 32Dcl3/Cbl-DeltaY371 cells did not undergo apoptosis like parental 32Dcl3 cells when cultured in the absence of IL-3. Both 32Dcl3 and 32D/CblDeltaY371 cells arrested in G(1) when cultured in the absence of IL-3. Approximately 18% of the 32Dcl3 cells cultured in the absence of IL-3 for 24 h were present in a sub-G(1) fraction, while only 4% of the 32D/Cbl-DeltaY371 and 2% of the 32D/Bcl-2 cells were found in a sub-G(1) fraction. There was no difference in the pattern of tyrosine-phosphorylated proteins observed following stimulation of either cell type with IL-3. The phosphorylation of JAK2, STAT5, and endogenous c-Cbl was identical in both cell types. No differences were detected in the activation of Akt, ERK1, or ERK2 in unstimulated or IL-3-stimulated 32D/Cbl-DeltaY371 cells compared with parental 32Dcl3 cells. Likewise, there was no difference in the pattern of phosphorylation of JAK2, STAT5, ERK1, ERK2, or Akt when 32Dcl3 and 32D/CblDY371 cells were withdrawn from medium containing IL-3. The protein levels of various Bcl-2 family members were examined in cells grown in the absence or presence of IL-3. We observed a consistent increased amount of Bcl-2 protein in five different clones of 32D/Cbl-DeltaY317 cells. These data suggest that the Cbl-DeltaY371 mutant may suppress apoptosis by a mechanism that involves the overexpression of Bcl-2. Consistent with this result, activation of caspase-3 was suppressed in 32D/Cbl-DeltaY371 cells cultured in the absence of IL-3 compared with 32Dcl3 cells cultured under the same conditions.
Hamilton
E
E
Department of Pathology, University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Miller
K M
KM
Helm
K M
KM
Langdon
W Y
WY
Anderson
S M
SM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D003470:Culture Media; D007377:Interleukin-3 / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases
D003470:Culture Media; D007377:Interleukin-3; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D019000:Phosphotyrosine; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; C493849:Cbl protein, mouse