Abl Tyrosine Kinase Activity Research Articles

The tyrosine kinase inhibitor STI571, like interferon-α, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors from patients with chronic myeloid leukemia

Objective To determine whether the compound STI571 (formerly known as CGP571418B), a selective inhibitor of the protein tyrosine kinase (PTK) activity of ABL and BCR-ABL proteins, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors (CFU-GM) from patients with chronic myeloid leukemia while sparing normal CFU-GM and to compare responses of CML and normal cells with STI571 and IFN-α. Materials and Methods Chronic phase CML and normal CFU-GM were grown with and without STI571, IFN-α, or the two agents in combination. Colonies were plucked and replated in 96-well microtiter plates. Secondary colonies were scored, and the results were expressed as the area-under-the-curve (AUC) of the distribution of secondary colony numbers per primary CFU-GM. This value gives an overall measure of the replating ability or amplification of the original CFU-GM population. Results STI571 selectively inhibits the formation of granulocyte-macrophage colony-forming cells (CFU-GM) from CML patients. It also significantly inhibits the amplification of CML CFU-GM (p = 0.002) as measured by secondary colony formation after replating primary CFU-GM colonies. In contrast, amplification of normal CFU-GM was enhanced (p = 0.001) at low concentrations (0.1 μM) of STI571 with a return to baseline at 10 μM STI571. Addition of interferon (IFN)-α to STI571 abolished the increase in normal CFU-GM amplification seen with either agent alone. There was a highly significant correlation between the in vitro response to STI571 and the in vitro response to IFN-α (r = 0.74 for CML cells, and 0.77 for normal cells). Conclusion We conclude that STI571, like IFN-α, preferentially suppresses amplification of CML CFU-GM while sparing normal CFU-GM.

Inactivation of wild-type BCR/ABL tyrosine kinase in hematopoietic cells by mild hyperthermia.

Temperature-sensitive mutants of BCR/ABL tyrosine kinase have been extensively used to study the mechanisms of cell transformation and signal transduction. However, little is known about the effect of temperature on the activity of wild-type BCR/ABL gene product. In this study, we demonstrate that in vivo tyrosine kinase activity of p210, p190 BCR/ABL and v-abl are temperature-sensitive when expressed in hematopoietic cells and decline when temperature is raised 2 degrees C above normal range. In vitro tyrosine kinase activities of purified recombinant Abl and immunoprecipitated p210 BCR/ABL were also sensitive to increased temperature. Tyrosine phosphorylation of cellular proteins was markedly reduced in BCR/ABL transformed cells after 16 h at 39 degrees C, whereas the expression of BCR/ABL was unchanged. Temperature-induced downregulation of BCR/ABL kinase activity was reversible when cells were shifted back to 37 degrees C. The downregulation of Abl tyrosine kinase activity was not influenced by mutation or deletion of SH2 or SH3 domains or mutation of the GRB2 binding site. No increase in functional activity or expression of protein-tyrosine phosphatases, PTP-1B, SH-PTP1 or SH-PTP2 was detected in cells grown at 39 degrees C. Temperature-induced downregulation in tyrosine kinase activity correlated with decline in phosphotyrosine-associated PI 3-kinase whereas there was no change in growth factor independence of transformed hematopoietic cells. In conclusion, Abl tyrosine kinase has intrinsic sensitivity to temperature and BCR/ABL expressed in hematopoietic cells is downregulated by increasing temperature 2 degrees C. These observations provide a unique opportunity to identify cellular factor(s) which regulate BCR/ABL kinase in vivo and suggests possible novel treatment of CML by a mild hyperthermia.

Use of glutathione S-Transferase and break point cluster region protein as artificial dimerization domains to activate tyrosine kinases

Publisher Summary This chapter describes the artificial activation of tyrosine kinase enzymes by tagging sequences such as glutathione S -transferase (GST) or the break point cluster region (BCR) sequence that can confer oligomerizing abilities on them. Glutathione S -transferases appear to play an essential role in the biotransformation of xenobiotics. They catalyze reactions in which the sulfur atom of glutathione provides electrons for reactive electrophiles, resulting in the formation of glutathione conjugates. BCR has been identified as a fusion partner of ABL in the formation of the BCR-ABL oncogene found in human chronic myelogenous leukemias. The ABL gene encodes a tyrosine kinase whose activity is highly regulated in vivo . The fusion to BCR constitutively activates the ABL tyrosine kinase activity, and the amino-terminal BCR sequence l-60 with its oligomerizing property makes an essential contribution to activation. Artificial oligomerization of tyrosine kinases by fusion to GST or BCR result in their activation. Molecular mechanisms may involve disruption of negative regulation or mimic physiological activation as in the case of receptor tyrosine kinases.

A chimeric receptor/oncogene that can be regulated by a ligand in vitro and in vivo.

The BCR/ABL oncogene encodes an activated tyrosine kinase that causes human chronic myelogenous leukemia. The mechanism of transformation, however, is complex and not well understood. One of the important contributions of BCR to transformation is believed to be dimerization or oligomerization of ABL, thereby activating ABL tyrosine kinase activity. We reasoned that if ABL was dimerized through other mechanisms, activation of the tyrosine kinase activity should also result, and the activated kinase may also be transforming. Erythropoietin is known to activate its receptor by causing dimerization, and therefore a synthetic oncogene was created by linking the extracytoplasmic and transmembrane domains of the EPO receptor with c-ABL. This chimeric receptor was stably expressed in Ba/F3 cells and, in the absence of EPO, had no detectable biological effect on the cells. EPO, however, induced a rapid, dose-dependent activation of ABL tyrosine kinase activity and phosphorylation of several cellular proteins. The major target proteins have been identified, and are very similar to the known substrates of BCR/ABL, including Shc, CBL, CRKL, and several proteins in the cytoskeleton. EPO treatment also resulted in biological effects that were remarkably similar to those of BCR/ABL, including improved viability, altered integrin function, and a weak mitogenic signal. The biological effects were in part dose-dependent, in that low EPO concentrations enhanced viability but did not cause proliferation. At high EPO doses, kinase activation was maximal, and a mitogenic effect was also revealed. In nude mice, Ba/F3 cells expressing this chimeric receptor did not cause detectable disease without administration of pharmacologic doses of EPO. If EPO was given intraperitoneally 5 days a week, however, a dose-dependent lethal leukemia resulted. This ligand-regulatable oncogene mimics some of the biological effects of BCR/ABL, and analysis of ABL mutants in this system will be useful to dissect the signaling pathways that cause CML.

An Activating Mutation in the ATP Binding Site of the ABL Kinase Domain

A number of structural alterations have been shown to activate the leukemogenic potential of the ABL oncogene, but there is little understanding of the regulatory mechanisms that are subverted by such changes. We have used directed mutagenesis to examine a potential regulatory motif in cABL, which could directly influence ABL tyrosine kinase activity. A tyrosine to phenylalanine substitution within the ATP binding fold of the ABL kinase domain is sufficient to activate cABL enzymatic activity, and the mutant protein will alleviate growth factor dependence when expressed in the BA/F3 cell line. This growth promotion is dependent upon the structure of the amino terminus of the protein, and the ABL mutation will cooperate with certain BCR sequences in BCR/ABL fusion proteins to deregulate ABL kinase activity.

Crkl is the major tyrosine-phosphorylated protein in neutrophils from patients with chronic myelogenous leukemia.

The Philadelphia chromosome (Ph1), detected in virtually all cases of chronic myelogenous leukemia (CML), is formed by a reciprocal translocation between chromosome 9 and 22 that fuses Bcr-encoded sequences upstream of exon 2 of c-Abl. This oncogene produces a fusion protein, p210bcr-abl, in which the Abl tyrosine kinase activity is elevated. Using anti-phosphotyrosine immunoblotting, we have compared the pattern of phosphotyrosine-containing proteins from freshly prepared neutrophils of patients in the stable phase of CML to normal controls. The only consistent difference was the presence of a 39-kDa tyrosine-phosphorylated protein in 18 out of 18 neutrophil samples from CML patients that was not seen in normal controls. This same protein, as assessed by two-dimensional anti-phosphotyrosine immunoblotting, was also present in cell lines expressing p210bcr-abl, including K562 cells. Using K562 cells as a source of protein, the 39-kDa protein was purified and identified by microsequencing as Crkl, an SH2/SH3 adaptor protein related to the crk oncogene of the avian sarcoma virus, CT10. A direct interaction between Crkl and Abl has also been shown using a yeast two-hybrid screen.

Integration of molecular and biological abnormalities in quest for selective treatment of chronic myelogenous leukemia (CML).

CML is an excellent target for development of selective treatment because of its highly consistent genetic abnormality t(9;22) and unique fusion gene product, p210bcr/abl, although it is not yet clear what form of specific therapy might be effective. Several components of p210bcr/abl are thought to be essential for its transforming activity: These include the constitutive tyrosine kinase activity of abl and the ability of the first exon for bcr both to specifically bind to abl's SH2 binding domain and possibly also to function as a novel type of serine kinase. Relatively little is yet known about what specific abnormalities in the regulatory pathways are caused by the altered tyrosine kinase activity of p210bcr/abl and other bcr/abl oncoproteins, but whatever its precise mode of action proves to be, p210bcr/abl presumably somehow changes the normal pattern of phosphorylation of key regulatory proteins in the signaling pathways so that the genes which normally direct the orderly sequence of proliferation and maturation of the myeloid progenitors are not properly regulated. The end results of this 'disregulation' are that there is asynchronous or discordant maturation; relative to comparable normal progenitors, a higher proportion of CML progenitors exhibit earlier cytoplasmic and delayed nuclear maturation. The leukemic progenitors do not proliferate more rapidly than comparable normal progenitors or have increased ultimate proliferative potential, but they go through one or more additional divisions during passage through the later maturation compartments and also live longer, resulting in overexpansion of the leukemic population. It is important to recognize the close linkage between maturation and proliferation in designing experiments to correlate the molecular and biological abnormalities and in seeking novel therapies to selectively affect the leukemic progenitors.

Tyrosine phosphorylation of rasGAP and associated proteins in chronic myelogenous leukemia cell lines

The Philadelphia chromosome (Ph1), detected in virtually all cases of chronic myelogenous leukemia, is formed by a reciprocal translocation between chromosomes 9 and 22 that fuses BCR encoded sequences upstream of exon 2 of c-ABL. This oncogene produces a fusion protein (p210BCR/ABL) in which the ABL tyrosine kinase activity is elevated. This elevated kinase activity is essential for transformation, but the mechanisms involved are unknown. We report here that p21ras GTPase activating protein (rasGAP) or rasGAP-associated proteins p190 and p62 are phosphorylated on tyrosine in Ph1 (+) cell lines. Further, rasGAP coimmunoprecipitates with p210BCR/ABL in these cell lines. These results suggest that rasGAP or associated proteins are potential substrates for p210BCR/ABL kinase and thus directly link p210BCR/ABL with a signal transduction pathway known to be activated by hematopoietic growth factors (p21ras).

RAPID COMMUNICATION Tyrosine Phosphorylation of rasGAP and Associated Proteins in Chronic Myelogenous Leukemia Cell Lines

AbstractThe Philadelphia chromosome (Ph1), detected in virtually all cases of chronic myelogenous leukemia, is formed by a reciprocal translocation between chromosomes 9 and 22 that fuses BCR encoded sequences upstream of exon 2 of c-ABL. This oncogene produces a fusion protein (p210BCR/ABL) in which the ABL tyrosine kinase activity is elevated. This elevated kinase activity is essential for transformation, but the mechanisms involved are unknown. We report here that p21ras GTPase activating protein (rasGAP) or rasGAP- associated proteins p190 and p62 are phosphorylated on tyrosine in Ph1 (+) cell lines. Further, rasGAP coimmunopre-cipitates with p210BCR/ABL in these cell lines. These results suggest that rasGAP or associated proteins are potential substrates for p210BCR/ABL kinase and thus directly link p210BCR/ABL with a signal transduction pathway known to be activated by hematopoietic growth factors (p21ras)

Inhibition of abl Oncogene Tyrosine Kinase Induces Erythroid Differentiation of Human Myelogenous Leukemia K562 Cells

The human chronic myelogenous leukemia cell line K562 expresses a structurally altered c‐abl protein with tyrosine kinase activity. Erythroid differentiation of K562 cells was induced by tyrosine kinase inhibitors, but not by other kinase inhibitors. Treatment of K562 cells with 5'd(TACTGGCCGCTG‐AAGGGC)3′, complementary to the second exon (codons 2 to 7) of c‐abl mRNA, inhibited cell growth and induced benzidine‐positive cells in a dose‐dependent manner. However, exposure to the sense oligomer did not induce erythroid differentiation of the cells. These results suggest that inhibition of abl tyrosine kinase activity is closely related to induction of erythroid differentiation of K562 cells. A multidrug‐resistant subline (K562R) was induced to undergo erythroid differentiation by tyrosine kinase inhibitors such as genistein or herbimycin A as effectively as the parent K562 cells were. Therefore, tyrosine kinase inhibitors might be useful as cancer chemotherapeutic agents against some multidrug‐resistant leukemias having abnormally high activity of oncogene tyrosine kinase(s).

The bcr-c- abl tyrosine kinase activity is extinguished by TPA in K562 leukemia cells

Tyrosine kinase activity is associated with the transforming potential of several oncogenes. Human chronic myeloid leukemia (CML) cells and cell lines have been shown to contain an active bcr-c- abl p210 tyrosine kinase as a consequence of the Philadelphia chromosomal translocation. In the present work the activity of the c- abl and c- src oncogene-encoded tyrosine kinases was investigated during phorbol diester (TPA) induced differentiation of the K562 CML cells. The high tyrosine kinase activity of p210 bcr -c- abl is strongly reduced during the initial 24 h of TPA treatment. In contrast, the activity of the c- src tyrosine kinase is not changed. No change occurs in the expression of the c- abl-specific RNAs during this period. Following the reduction of bcr-c- abl kinase activity, cell proliferation is arrested and megakaryoblastic antigens appear on the cells. Sodium butyrate caused a slight decrease in growth rate and of bcr-c- abl kinase activity during erythroid differentiation whereas no changes in c- src or c- abl tyrosine kinase activities were seen in DMSO-treated control cells.