Inhibitors of tyrosine phosphorylation induce apoptosis in human leukemic cell lines

Experimental evidence suggests that hematopoietic growth factors promote cell survival by suppressing apoptosis or programmed cell death. Since interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) induce tyrosine phosphorylation of a common set of proteins in the factor-dependent cell line M07e, we have investigated whether growth-factor-induced tyrosine phosphorylation is involved in the promotion of cell survival and suppression of apoptosis. Experiments were carried out with the leukemic cell lines HL-60 and M07e and the tyrosine kinase inhibitors genistein and tyrphostin AG82. Both the tyrosine kinase inhibitors induced apoptosis of HL-60 and M07e cells. This was indicated by the appearance of DNA degradation and morphologic evidence of nuclear condensation and fragmentation. It was also confirmed by flow cytometry of DNA, which showed apoptotic cells as a fraction of cells characterized by a diminished DNA stainability, represented on the DNA frequency histograms as a distinct peak below the G0/G1 population. Kinase inhibitors also reduced the fraction of cells in the S phase of the cell cycle. That tyrphostin specifically inhibited tyrosine kinases was further suggested by the prevention of its effects by the tyrosine phosphatase inhibitor sodium orthovanadate (vanadate), at least during the first 18-24 h of treatment. The incomplete prevention of genistein effects by vanadate suggests that genistein is a less specific inhibitor of tyrosine kinases than tyrphostin, and may also act as an inhibitor of topoisomerase II. Vanadate also prevented apoptosis and reduction of the S phase in M07e cells cultured for 24 h in the absence of growth factors. These results suggest that tyrosine phosphorylation is an essential step in IL-3 and GM-CSF signal transduction. Since in our experimental model the effects of tyrosine kinase inhibition and growth factor deprivation could be reversed by concomitant inhibition of tyrosine phosphatases, it is suggested that a balance between tyrosine kinases and tyrosine phosphatases establishes whether a cell will survive or undergo apoptosis.     

Agonist-specific tyrosine phosphorylation of Cbl in human neutrophils

The effects of soluble and particulate agonists on the tyrosine phosphorylation levels of the proto-oncogene Cbl in human neutrophils were examined. Experimental conditions allowing the maintenance of Cbl as well as of its tyrosine phosphorylation status were first established. Their use allowed us to observe that Cbl was tyrosine phosphorylated in response to some (FcgammaRII ligation, opsonized bacteria and zymosan, granulocyte-macrophage colony-stimulating factor, monosodium urate, and calcium pyrophosphate microcrystals), but not all (fMet-Leu-Phe, interleukin-8) neutrophil agonists. Cbl was also shown to account for a varying proportion of the 120-kDa phosphoprotein(s) observed in response to the above stimuli. These data establish that Cbl is present in human neutrophils and that its level of tyrosine phosphorylation is modulated by some of these cells' agonists, and in particular by phagocytic particles. Furthermore, the signaling pathways activated by chemotactic factors and the other neutrophil stimuli tested in this investigation diverge at or downstream from the tyrosine phosphorylation of Cbl.     

Depolarization-induced tyrosine phosphorylation in PC12h cells

Depolarization induced by KCl was found to induce tyrosine phosphorylation of cellular proteins in PC12h cells. By Western blotting with anti-phosphotyrosine antibody, we detected tyrosine phosphorylation of proteins with molecular weights of 120, 110, 105, 95, 75, 70, 66, 44, and 42 kDa in response to KCl. The immunoprecipitates from KCl-treated cells with the antibody contained large amounts of tyrosine-phosphorylated proteins and increased activity of tyrosine kinase. Incubation of the immunoprecipitates with [gamma-32P]ATP resulted in tyrosine phosphorylation of two proteins with the molecular weights of 120 and 140 kDa. These effects were completely abolished by the addition of EGTA before KCl treatment, suggesting that the depolarization-induced tyrosine phosphorylation may require calcium entry into the cells from the medium. Increased activity of tyrosine kinase phosphorylating the 120 and 140 kDa proteins was also recovered from cells stimulated with nerve growth factor, basic fibroblast growth factor, epidermal growth factor, and vasoactive intestinal peptide. Among them, depolarization by KCl elicited the strongest effect. These results indicate that a protein tyrosine kinase that phosphorylate the 120 and 140 kDa proteins is phosphorylated or activated in response to calcium ion, cAMP, and growth factors acting through tyrosine kinase receptors.     

Shrinkage-induced protein tyrosine phosphorylation in Chinese hamster ovary cells

To investigate the signal transduction of osmotic stress, we examined hypertonicity-induced tyrosine phosphorylations in Chinese hamster ovary cells. Hyperosmosis elicited characteristic phosphotyrosine accumulation in at least 3 proteins (approximately 42, approximately 85, and approximately 120 kDa). The most prominent response occurred in the 85-kDa band (p85) whose phosphorylation was rapid, sustained, apparent already at mild hypertonicity (350 mosM), proportional to the extracellular osmotic concentration, and reversible. Hyperosmotic environment could not induce tyrosine phosphorylation if cell shrinkage was prevented by nystatin and appropriately composed media. Conversely, isotonic shrinkage caused strong tyrosine phosphorylation. Thus, the initial signal is a decrease in cell volume and not an increase in the intra- or extracellular osmotic concentration, or a rise in cytosolic K+ and Cl- levels. Tyrosine phosphorylation of p85 was not due to the hypertonicity-induced protein kinase C-dependent stimulation of the extracellular signal-regulated protein kinase, nor to the activation of stress-activated protein kinases. Tonicity-responsive proteins interacted with Grb2-glutathione S-transferase fusion proteins: the 120-kDa protein complexed with the SH2 and both SH3 domains, whereas p85 associated with the SH2 and the N-terminal SH3 domains of the adapter. Tyrosine phosphorylation of p85 is a sensitive indicator of reduced intracellular hydration and might signify a hitherto unrecognized, early volume-dependent signaling event.     

Protein tyrosine phosphorylation and activation of primary response genes by interleukin 11 in B9-TY1 cells

Interleukin (IL) 11 is a multifunctional cytokine derived from bone marrow stromal cells. To understand the mechanisms by which IL-11 exerts its pleiotropic actions, we have analyzed IL-11-mediated signal transduction pathways in IL-11-dependent B9-TY1, which is a subclone of an IL-6-dependent B-cell hybridoma, B9. IL-11 stimulation of B9-TY1 cells resulted in tyrosine phosphorylation of a 97/95 kilodalton cellular protein in a dose-dependent manner, and this effect was inhibited by tyrosine kinase inhibitors genistein and herbimycin A, but not by a serine/threonine kinase inhibitor, H7. We next examined the early nuclear events in the IL-11-triggered intracellular signaling cascade. The data showed that tis11, tis21, and junB early response genes were rapidly activated following IL-11 treatment. The kinetic studies indicated that activation of tis11 and junB genes peaked at 30-60 min and then declined slowly afterward. The tis21 gene was constitutively expressed, and the level of tis21 mRNA was significantly increased and maintained at the elevated level following IL-11 stimulation. Inhibitor studies with genistein, herbimycin A, and H7 revealed that tyrosine kinases and H7-sensitive serine/threonine kinases are required for the IL-11-mediated activation of tis11, tis21, and junB genes. Using a variety of known protein kinase inhibitors or activators, we have demonstrated that H7-sensitive protein kinases activated by IL-11 are distinct from those of well-characterized protein kinase-second messenger systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human type 1 inositol 1,4,5-trisphosphate receptor from T lymphocytes. Structure, localization, and tyrosine phosphorylation

Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular calcium release channels involved in diverse signaling pathways. An IP3R is thought to play a role in mobilizing calcium required for activation of T lymphocytes. The IP3R is a tetrameric structure comprised of four approximately 300-kDa subunits encoded by a approximately 10-kilobase mRNA. In the present study we determined the structure of the human type 1 IP3R expressed in T lymphocytes (Jurkats). The IP3R in human T cells had a predicted molecular mass of 308 kDa and was most similar to the non-neuronal form of the rodent type 1 IP3R. Two putative tyrosine phosphorylation sites were identified, one near the amino terminus and one near the putative channel pore at the carboxyl terminus. During T cell activation the IP3R was tyrosine phosphorylated. A site-specific anti-IP3R antibody was used to localize the carboxyl terminus of the IP3R to the cytoplasm in T cells.     

Angiotensin II-induced tyrosine phosphorylation in mesangial and vascular smooth muscle cells

It has previously been suggested that paradigms investigating schema theory should include tests of the effects of variability of practice on tasks experienced during the acquisition of learning. The present aim was to test this effect using a sports-related task. Two groups of children (n = 13 each) aged between 3 and 5 yr. old were pretested on an aiming task. They then received ten practice trials per day for ten days. One group (designated the specific group) practised only at the specified target distance, while another group (designated the specific plus variable group) practised at the target distance and at distances around the target. At the end of the practice period both groups were again tested. The data were analysed for distance and lateral errors, using two-way (group x time) analyses of covariance with one repeated measure on the latter factor. The interaction of groups x time was significant for the distance errors, indicating that at the end of the practice period the specific plus variable group had improved more than the specific group. This finding extends previous work on a similar variability paradigm to a sports-related setting.     

Distribution and subcellular localization of a lysosome-associated protein in human genital organs

The management of breast cancer is becoming increasingly complex, almost year by year. Previous paradigms have held that issues were dichotomous (e.g., good versus bad). These are being challenged constantly by more complex models that demand more than a duality of choice in therapeutic decision making. Most importantly, the determinants of local failure after conservative treatment are quite different from the determinants of survival. While we develop enhanced chemotherapy for immediate life-threatening disease, we also accepting subtypes of breast cancer that are of little threat to life and need little treatment. With the establishment of the efficacy of systemic chemotherapy, the question of whether some patients may be helped with chemotherapy becomes extremely important. The list of possible clinically useful subcategories is growing and is under active development. However, the prognostic factors in use have been validated repeatedly. The two major elements of anatomic staging--size and lymph node status--interact powerfully with histologic categories of grade and special type. In the area of small tumors, these associations indicate lesions that have almost no likelihood of association with distant failure, at least within 5 years.     

Depolarization-induced tyrosine phosphorylation of paxillin in PC12h cells

Treatment of PC12h cells with a high concentration of KC1 induces depolarization of the plasma membrane and Ca2+ influx into the cells. We have previously shown that KC1 induced tyrosine phosphorylation of cellular proteins of 120, 110, 68, 44 and 42 kDa. In the present study, we found that the 68-kDa protein is paxillin, a tyrosine kinase substrate associated with the actin cytoskeleton. A calcium ionophore, A23187, also induced tyrosine phosphorylation of the 68-kDa protein, while KC1 did not in the presence of EGTA or nifedipine, indicating that the effect of KC1 was due to the Ca2+ influx into the cells. Tyrosine phosphorylation of paxillin was also induced by nerve growth factor and epidermal growth factor, but its migration patterns on an SDS/polyacrylamide gel were different, that is, nerve growth factor and epidermal growth factor caused upward shifts of the bands, while KC1 did not. However, both forms could associate with Csk and Crk. The effect of KC1 was blocked by cytochalasin D, indicating that tyrosine phosphorylation required the integrity of actin filaments. These results suggest that tyrosine phosphorylation of paxillin may be involved in Ca2+ -dependent events in neuronal and neuroendocrine cells.     

Ultrastructural localization of DNA in leukemic cells using osmium ammine B

In order to determine whether there were any differences in distribution of nuclear DNA between acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML), the localization of DNA in blasts from the bone marrow or buffy coat of 30 patients with ALL and 30 patients with AML was examined ultrastructurally by staining with osmium ammine B. By the ultrastructural cytochemistry, DNA in ALL cells was clumped in the nuclei, while in AML cells, it was dispersed. DNA had accumulated around the nucleoli of some blasts, and flecks of DNA were observed in nucleoli of a majority of blasts. The perinucleolar and intranucleolar DNA distribution could be classified into four types. The types with abundant perinucleolar DNA were frequently observed in ALL blasts, while the majority of AML blasts showed scant perinucleolar DNA. The types with intranucleolar flecks of DNA were more prominent in leukemic cells than in normal immature leukocytes. In conclusion, the pattern of distribution of DNA in the nuclei of leukemic cells differs between ALL and AML.     

Cloning, subcellular localization and functional expression of human RNase HII

Recently we showed that the major mammalian RNase H, RNase HI, is evolutionarily related to prokaryotic RNase HII (Frank et al., FEBS-Lett. 421, 23-26, 1998), an enzyme described to be a minor activity in E. coli. As a consequence we addressed the question of whether a human RNase H exists, sharing homology with the main E. coli enzyme, RNase HI. Employing sequence analysis of expressed sequence tags, followed by specific PCR amplification of human cDNA, we cloned, sequenced and expressed a human open reading frame, coding for a 32 kDa protein. Purification of the recombinant His(6)-tagged protein from E. coli extracts using Ni(2+)-chelating chromatography and subsequent renaturation gel assay proved that it is an active RNase H. The properties of this enzyme suggest that it is identical with the human RNase HII, previously purified by one of us (Frank et al., Nucleic Acids Res. 22, 5247-5254, 1994). Studies using a green fluorescent protein-fusion construct reveal that this protein is located in the nucleus.     

Apoptosis and resistance to daunorubicin in human leukemic cells

We compared test methods based on specific mechanisms of daunorubicin (DNR) resistance to more global procedures. Assessment of P-glycoprotein (P-gp) expression and function by means of immunocytochemistry, DNR accumulation, and modulation of resistance and accumulation by the P-gp inhibitor cyclosporin A (CsA) were selected as parameters for multidrug resistance (MDR). On the other hand, we used the MTT assay and measured apoptosis and proliferative activity (S- and G2M-phases of the cell cycle) by flow cytometry. Validation of test methods was achieved for four leukemic cell lines (HL-60, KG-1a, K562/WT, K562/ADM). This battery of tests was then applied to mononuclear cells (MNC) from 18 leukemic patients. Low proficiency of MNC to undergo apoptosis and low proliferative activity rather than P-gp-mediated MDR correlated with DNR resistance as measured by the MTT assay. Bell-shaped dose-response curves for apoptosis, however, which reflect a switch from the apoptotic to the necrotic death mode with increasing cellular damage tend to limit practicability in clinical testing, because appropriate dose range and time points need to be explored. Thus, measurement of apoptosis by flow cytometry may be less convenient than the MTT assay for determination of chemosensitivity, if clinical samples with unknown patterns of responsiveness are to be tested. Spontaneous apoptosis in untreated MNC following 24 h incubation in vitro correlated significantly with DNR sensitivity in the MTT assay. A lack of essential viability factors (eg growth factors or cytokines) in vitro which are known to prevent apoptosis may contribute to DNR sensitivity.     

Metabolism of 6-mercaptopurine in human leukemic cells

The PRPP concentrations, PRPP formation, and phosphorylation of 6-mercaptopurine in leukocyte suspensions and homogenates prepared from leukemic patients were studied...     

Inhibition of neuropeptide-stimulated tyrosine phosphorylation and tyrosine kinase activity stimulates apoptosis in small cell lung cancer cells

Small cell lung cancer (SCLC) cell growth is sustained by multiple autocrine and paracrine growth loops involving neuropeptides. The bombesin family of peptides are autocrine growth factors in H345 SCLC cells and provide a paradigm for the study of growth factors and mitogenic signaling in SCLC cells. We show that bombesin (and other neuropeptides) stimulates protein tyrosine phosphorylation (particularly focal adhesion kinase) and protein tyrosine kinase (PTK) activity in intact SCLC cells. Furthermore, the broad spectrum neuropeptide receptor antagonist [D-Arg, D = Phe, D-Trp, Leu11]substance P inhibits all neuropeptide-mediated signals (including PTK activation), SCLC cell growth in vivo and in vitro, and also increases the natural rate of apoptosis seen in growing SCLC cell lines. Hence the effect of selective PTK inhibition on SCLC cell growth and apoptosis was examined. We show that selective inhibition of PTK activity, with genistein and (3,4,5-tri-hydroxyphenyl)-methylene(-propanedinitrile) tyrphostin-25 inhibits basal and neuropeptide-stimulated SCLC cell growth. Genistein and tyrphostin-25 also stimulate apoptosis in SCLC cells. Inhibition of proliferation in these cells is intimately linke to apoptosis, because these changes occurred without any effect on SCLC cell cycle kinetics, suggesting that apoptosis occurs independently of the cell cycle and that failure to progress through the cell cycle results in apoptosis. Because tyrphostin-25 fails to influence p53 or Bcl-2 expression in these cells, this mode of programmed cell death appears to be via a p53- and Bcl-2-independent mechanism. These results provide evidence that tyrosine phosphorylation is a mitogenic signal in SCLC cells and suggest that regulation of the level of protein tyrosine phosphorylation represents a critical determinant of whether SCLC cells survive and proliferate or die by apoptosis. Thus PTK inhibition may provide a novel therapeutic option in SCLC that has become resistant to conventional chemotherapeutic agents.     

Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates

Diabetic cardiovascular autonomic neuropathy increases the risk of deterioration in renal function and is associated with increased mortality in patients with renal failure. Type 1 diabetic patients with long diabetes duration, matched for age (38 +/- 9 years) and diabetes duration (28 +/- 8 years) were studied regarding the association between cardiovascular autonomic nerve function and different degrees of diabetic nephropathy. Eighteen patients were normo- (< 30 mg/l), six micro- (30-300 mg/l), and 13 macroalbuminuric (> 300 mg/l) based on urinary albumin concentrations in three separate morning samples. They were compared with 33 control subjects with similar age. Autonomic nerve function was evaluated by measuring the response of heart rate to deep breathing and active standing. Beat-to-beat finger artery blood pressure (Finapres) was tested during active standing. During deep breathing both change in heart rate (17 +/- 11, 9 +/- 7 and 4 +/- 3 beats/min) and ratio between expiratory and inspiratory R-R intervals (1.32 +/- 0.24, 1.14 +/- 0.15 and 1.05 +/- 0.04) decreased from normo- over micro- to macroalbuminuria (p < 0.05 vs normoalbuminuric and control subjects [17 +/- 5 beats/min and 1.28 +/- 0.10, respectively]). Similar results were obtained during active standing with respect to change in systolic arterial blood pressure (3 +/- 8, 2 +/- 13 and -6 +/- 11 mmHg; p < 0.05 vs control subjects [8 +/- 11 mmHg]). However, the response of diastolic arterial blood pressure or mean heart rate to standing up did not differ between any of the groups. The ratio of maximum to minimum R-R interval during the dynamic response of heart rate to active standing decreased with the degree of nephropathy (1.27 +/- 0.17, 1.11 +/- 0.11 and 1.05 +/- 0.06) with significantly higher values in patients with normo- compared with patients with macroalbuminuria (p < 0.05). All patients groups had significantly lower values than control subjects (1.46 +/- 0.22, p < 0.05). The overshoot of the blood pressure after an initial fall during active standing decreased with the degree of diabetic nephropathy. In conclusion, type 1 diabetic patients with long duration of diabetes have signs of cardiovascular autonomic neuropathy, the severity of which is related to the degree of nephropathy.     

Stimulation of tyrosine phosphorylation after ligation of beta7 and beta1 integrins on human B cells

B lymphocytes express several members of the integrin family of adhesion molecules that mediate cell-cell and cell-extracellular matrix interactions. In addition to beta1 integrins, predominantly alpha4 beta1, mature B cells also express alpha4 beta7, which is a receptor for vascular cell adhesion molecule-1 and fibronectin, and is also involved in the homing of B cells to mucosal sites through binding to a third ligand, mucosal address in cell adhesion molecule-1. Here we describe that crosslinking of alpha4 beta7 integrins on B cell lines and normal tonsillar B cells, induces tyrosine phosphorylation of multiple substrates of 105-130 kD, indicating that beta7 integrin plays a role as signaling molecule in B cells. This pattern of phosphorylated proteins was very similar to that induced following ligation of alpha4 beta1. Interestingly, ligation of alpha5 beta1 or alpha6 beta1 also stimulated the 105-125 kD group of phosphorylated proteins, whereas ligation of beta2 integrins did not. The focal adhesion tyrosine kinase p125FAK was identified as one of these substrates. Beta1 or beta7 mediated tyrosine phosphorylations were markedly decreased when the microfilament assembly was inhibited by cytochalasin B. These results suggest that intracellular signals initiated by different integrins in B cells may converge, to similar cytoskeleton-dependent tyrosine phosphorylated proteins.