Timothy J. Ernst

BCR/ABL induces multiple abnormalities of cytoskeletal function.

The BCR/ABL oncogene causes human chronic myelogenous leukemia (CML), a myeloproliferative disease characterized by massive expansion of hematopoietic progenitor cells and cells of the granulocyte lineage. When transfected into murine hematopoietic cell lines, BCR/ABL causes cytokine-independence and enhances viability. There is also growing evidence that p210(BCR/ABL) affects cytoskeletal structure. p210(BCR/ABL) binds to actin, and several cytoskeletal proteins are tyrosine phosphorylated by this oncoprotein. Also, at least one aspect of cytoskeletal function is abnormal, in that the affinity of beta1 integrins for fibronectin is altered in CML cells. However, isolated changes in beta1 integrin function would be unlikely to explain the clinical phenotype of CML. We used time-lapse video microscopy to study cell motility and cell morphology on extracellular cell matrix protein-coated surfaces of a series of cell lines before and after transformation by BCR/ABL. BCR/ABL was associated with a striking increase in spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia) and accelerated the rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. Also, while untransformed cells were sessile for long periods, BCR/ABL-transformed cells exhibited persistent motility, except for brief periods during cell division. Using cell lines transformed by a temperature-sensitive mutant of BCR/ABL, these kinetic abnormalities of cytoskeletal function were shown to require BCR/ABL tyrosine kinase activity. Similar abnormalities of cytoskeletal function on fibronectin-coated surfaces were observed when hematopoietic progenitor cells purified by CD34 selection from patients with CML were compared with CD34 positive cells from normal individuals. Interestingly, alpha-interferon treatment was found to slowly revert the abnormal motility phenotype of BCR/ABL-transformed cells towards normal. The increase in spontaneous motility and other defects of cytoskeletal function described here will be useful biological markers of the functional effects of BCR/ABL in hematopoietic cells.

Expression of ras oncogenes in cultured human cells alters the transcriptional and posttranscriptional regulation of cytokine genes.

Autonomous production of cytokines such as the hematopoietic colony-stimulating factors (CSFs), IL-1, or IL-6 has been demonstrated in numerous human and murine neoplasms, and may be involved in the pathogenesis of several paraneoplastic syndromes such as leukocytosis, fever, and hypercalcemia. Because of the high frequency with which mutations in ras protooncogenes have been detected in human tumors, as well as evidence linking ras gene products to activation of certain cellular functions, we investigated whether ras mutations might influence the regulation of cytokine genes. Normal human fibroblasts transfected with a mutant val12 H-ras oncogene expressed increased levels of mRNA transcripts encoding granulocyte-CSF (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and IL-1 beta compared with controls. Human mesothelioma cells transfected with a mutant asp12 N-ras oncogene exhibited similar alterations in cytokine gene expression. Estimates of transcriptional activity by nuclear run-on analysis revealed a selective increase in transcription only for the IL-1 gene. Analysis of mRNA half-life demonstrated a marked increase in the stability of numerous cytokine transcripts, including G-CSF, GM-CSF, IL-1, and IL-6. The addition of anti-IL-1 neutralizing antibody to cultures of cells expressing ras mutants did not block the expression of any of the cytokines examined, suggesting that the baseline expression of GM-CSF, G-CSF, and IL-6 was not a secondary event due to the increased transcription of IL-1. These results indicate that mutations in ras genes may alter expression of several cytokine genes through both transcriptional and posttranscriptional mechanisms.

The human granulocyte-macrophage colony-stimulating factor receptor is capable of initiating signal transduction in NIH3T3 cells.

The ability of the receptor for the hematopoietic cytokine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) to function in non‐hematopoietic cells is unknown. NIH3T3 fibroblasts were transfected with cDNAs encoding the alpha and beta subunit of the human GM‐CSF receptor and a series of stable transformants were isolated that bound GM‐CSF with either low (KD = 860 − > 1000 pM) or high affinity (KD = 20–80 pM). Low affinity receptors were not functional. However, the reconstituted high affinity receptors were found to be capable of activating a number of signal transduction pathways, including tyrosine kinase activity, phosphorylation of Raf‐1, and the transient induction of c‐fos and c‐myc mRNAs. The activation of protein tyrosine phosphorylation by GM‐CSF in NIH3T3 cells was rapid (< 1 min) and transient (peaking at 5–20 min) and resulted in the phosphorylation of proteins of estimated molecular weights of 42, 44, 52/53 and 58–60 kDa. Some of these proteins co‐migrated with proteins from myeloid cells that were phosphorylated on tyrosine residues in response to GM‐CSF. In particular, p42 and p44 were identified as mitogen‐activated protein kinases (MAP kinases), and the phosphorylation on tyrosine residues of p42 and p44 MAP kinases occurred at the same time as the phosphorylation of Raf‐1. However, despite evidence for activation of many mitogenic signal transduction molecules, GM‐CSF did not induce significant proliferation of transfected NIH3T3 cells. These results suggest that murine fibroblasts contain signal transducing molecules that can effectively interact with the human GM‐CSF receptor, and that are sufficient to activate at least some of the same signal transduction pathways this receptor activates in myeloid cells, including activation of one or more tyrosine kinase(s). However, the level of activation of signal transduction is either below a threshold of necessary activity or at least one mitogenic signal necessary for proliferation is missing.

Treatment of the blastic phase of chronic myelogenous leukemia with high-dose cytosine arabinoside.

Sixteen patients with accelerated or blastic phase chronic myelogenous leukemia were treated with high-dose cytosine arabinoside given at 3 g/m2 every 12 h for two to eight doses per course. All patients had an immediate clearing of peripheral blood blast cells. Five patients achieved a complete response for 1–14 months, median 4.5 months. Toxicity was significant and predictable on the basis of dose given. We have identified both an effective and a toxic dosing schedule. The rapid response is dramatic but relatively short-lived, and suggests that additional agents in conjunction with reinfusion of autologous stem cells may offer these patients a greater chance of significant response.