Burgess Gs

Identification of a ligand for the c-kit proto-oncogene

We report the purification and N-terminal amino acid sequence of a novel mast cell growth factor, termed MGF, from the supernatants of a murine stromal cell line. A panel of interleukin 3-dependent cell lines were screened for responsiveness to partially purified MGF in [3H]thymidine incorporation assays; proliferative stimulation of these cells in response to MGF correlated with expression of mRNA for the c-kit protooncogene. MGF was shown to be a ligand for c-kit by cross-linking 125I-labeled MGF to c-kit-expressing cells with subsequent immunoprecipitation of the complex with antiserum specific for the C-terminus of c-kit. This establishes MGF as a ligand for the c-kit protein.

Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation

A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) π, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.

Cyclic AMP negatively controls c-myc transcription and G1 cell cycle progression in p210 BCR-ABL transformed cells: inhibitory activity exerted through cyclin D1 and cdk4

Raised intracellular cyclic AMP (cAMP) has been demonstrated to exert an antiproliferative effect in myeloid cells. How the antiproliferative activity of cAMP is exerted in p210 BCR-ABL transformed myeloid cells was the subject of this investigation. It was hypothesized that cyclin dependent kinase 4, cdk4, might be a critical target enzyme to affect the related events of c-myc transcription and progression through G1 phase of the cell cycle within cells transformed by p210 BCR-ABL, and further, that cdk4 might be downregulated by cAMP to inhibit proliferation. In order to investigate the regulatory role of cdk4, synchronized cells were studied. In p210 BCR-ABL transformed cells transiting early G1 phase, treatment with a cAMP analogue led to inhibition of cyclin D1 synthesis, and marked reduction of cdk4 kinase activity. Within cells in which cdk4 was inhibited by cAMP, there was augmented interaction of E2F1 with the retinoblastoma protein, pRb in a nuclear matrix-associated cell fraction. As a result of E2F1 sequestration, raised intracellular cAMP was found to inhibit c-myc transcription in p210 BCR-ABL transformed myeloid cells synchronously transiting the early G1 phase of the cell cycle. A target of this transcriptional suppression exerted by cAMP was the E2F site of the c-myc P2 promoter. On the other hand, cyclin D1 content was not reduced by cAMP in these cells when it was applied at a later cell cycle stage at the interface between G1 and S. Corresponding to lack of cyclin D1 inhibition in these later G1-to-S phase cells, cdk4 activity was only modestly suppressed, and c-myc mRNA expression was also inhibited to a lesser degree. These studies show that Rb interaction with E2F1 is regulated by cdk4 and cyclin D1 within p210 BCR-ABL transformed leukemia cells in early G1 phase of the cell cycle. In this context, both cyclin D1 and cdk4 are subject to the level of intracellular cAMP. This interaction between Rb and E2F1, which is subject to the level of cAMP, is critical to transcriptional control of c-myc. Further, pRb regulation of E2F activity affects cellular potential for G1-S phase transition in p210 BCR-ABL transformed myeloid cells, in part, via its effect on c-myc transcription.

Mutant H-ras over-expression inhibits a random apoptotic nuclease in myeloid leukemia cells.

Cell suicide, or apoptosis, is now recognized as an essential regulatory step in such diverse developmental processes as embryogenesis, thymocyte restriction, and hematopoiesis. One of the major features of apoptosis is the activation of an endogenous nuclease that cleaves DNA into nucleosomal fragments. Little is known about the activation or specificity of the apoptotic endonuclease. In this study, we investigated signalling pathways and the specificity of the apoptotic nuclease. We found that forced over-expression of activated H-ras inhibited activation of the apoptotic endonuclease. Since a high percentage of myelodysplasias and leukemias have mutations that activate ras, this finding lends insight into how ras might be leukemogenic. In addition, the phorbol ester TPA and a cyclic AMP analogue also slowed activation of this endonuclease. Interestingly, protein synthesis inhibition stimulated the endonuclease activity. In addition, by cloning and sequencing apoptotic fragments we found that the apoptotic nuclease has no sequence specificity. Thus, the apoptotic nuclease inhibited by H-ras over-expression was random in nature.

In leukemogenesis by bcr-abl cell cycle progression via cyclin D1 involves cooperation between akt/gsk3β and jnk and is c-jun-mediated

Abstract Leukemic transformation by tyrosine kinase oncogenes (eg. Bcr/Abl) involves c-jun upregulation by c-jun-promoted transcriptional feedback. This has been closely linked to activity of c-jun N-terminal kinase (JNK), which phosphorylates c-jun serines 63, 73 to augment interaction with transcriptional coactivator p300/CBP. However, p21rac-/JNK-independent activation of PI-3-kinase and akt, both downstream of p210 Bcr-Abl, potentially bear upon c-jun and its transcription. Akt phosphorylates and inactivates glycogen synthase kinase 3β GSK3β), which then can no longer mediate inhibitory phosphorylation of the C-terminal jun DNA-binding domain. We transformed IL-3-dependent H7 cells by dual expression of activated gag-akt and RafCAAX (H7gag-akt/RafCAAX 1 & 2). We compared these cells to H7 cells transformed by p210 Bcr-Abl as well as with parental H7 cells, for enzyme activity of JNK and GSK3β. Extracts of these cells were also immunoblotted for quantity of total GSK3β vs. akt-inactivated phosphoserine 9 GSK3β, as well as for total c-jun and JNK-activated phosphoserine 73 c-jun. JNK activity for GST c-jun was 2-fold greater in Bcr-Abl.A54 cells compared with H7gag-akt/RafCAAX 1&2, and yet total c-jun expression was greater in the latter cells, which had more abundant akt-inactivated phosphoserine 9 GSK3β. However, the relative level of phosphoserine 73 c-jun was higher in Bcr-Abl.A54 vs. H7gag-akt/RafCAAX, and both cells overexpressed c-jun relative to H7 parent. Corresponding to the importance of JNK toward c-jun transactivation potential, Bcr-Abl.A54 cells overexpressed the putative c-jun transcriptional target cyclin D1, and these cells underwent more rapid cell cycle transit. Confirmation was sought for the importance of JNK in c-jun-dependent cyclin D1 expression by transfecting and overexpressing a dominant-negative mutant (serines 63,73 to alanine) c-jun not phosphorylatable by JNK (JNP). In these cells with JNP and p210 Bcr-Abl, cyclin D1 expression was blunted and proliferation was slowed. Taken together, these data point to collaboration of JNK-independent/akt-mediated and JNK-dependent pathways in leukemic transformation, acting on quantitative and qualitative levels, respectively, for c-jun regulation of cell cycle progression.