RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38.

Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondrial apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug.

Discrepant NOXA (PMAIP1) transcript and NOXA protein levels: a potential Achilles' heel in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm with transient response to conventional chemotherapy. We here investigated the role of the Bcl-2 homology domain 3-only protein NOXA for life-death decision in MCL. Surprisingly, NOXA (PMAIP1) mRNA and NOXA protein levels were extremely discrepant in MCL cells: NOXA mRNA was found to be highly expressed whereas NOXA protein levels were low. Chronic active B-cell receptor signaling and to a minor degree cyclin D1 overexpression contributed to high NOXA mRNA expression levels in MCL cells. The phoshatidyl-inositol-3 kinase/AKT/mammalian target of rapamycin pathway was identified as the major downstream signaling pathway involved in the maintenance of NOXA gene expression. Interestingly, MCL cells adapt to this constitutive pro-apoptotic signal by extensive ubiquitination and rapid proteasomal degradation of NOXA protein (T½âˆ¼15-30 min). In addition to the proteasome inhibitor Bortezomib, we identified the neddylation inhibitor MLN4924 and the fatty acid synthase inhibitor Orlistat as potent inducers of NOXA protein expression leading to apoptosis in MCL. All inhibitors targeted NOXA protein turnover. In contrast to Bortezomib, MLN4924 and Orlistat interfered with the ubiquitination process of NOXA protein thereby offering new strategies to kill Bortezomib-resistant MCL cells. Our data, therefore, highlight a critical role of NOXA in the balance between life and death in MCL. The discrepancy between NOXA transcript and protein levels is essential for sensitivity of MCL to ubiquitin-proteasome system inhibitors and could therefore provide a druggable Achilles' heel of MCL cells.

Pharmacological inhibition of c-Abl compromises genetic stability and DNA repair in Bcr-Abl-negative cells.

Imatinib inhibits the kinase activity of Bcr-Abl and is currently the most effective drug for treatment of chronic myeloid leukemia (CML). Imatinib also blocks c-Abl, a physiological tyrosine kinase activated by a variety of stress signals including damaged DNA. We investigated the effect of pharmacological inhibition of c-Abl on the processing of irradiation-induced DNA damage in Bcr-Abl-negative cells. Cell lines and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were treated with imatinib or dasatinib before gamma-irradiation. Inhibition of c-Abl caused an enhanced irradiation-induced mutation frequency and slowdown of DNA repair, whereas imatinib was ineffective in cells expressing a T315I variant of c-Abl. Mutation frequency and repair kinetics were also studied in c-Abl-/- murine embryonic fibroblasts (MEFs) retransfected with wild-type c-Abl (wt-Abl) or a kinase-defect variant of Abl (KD-Abl). Enhanced mutation frequency as well as delayed DNA repair was observed in cells expressing KD-Abl. These data indicate that pharmacological inhibition of c-Abl compromises DNA-damage response.

Role of poly(ADP-ribose) polymerase activity in imatinib mesylate-induced cell death.

Imatinib targets Bcr-Abl, the causative event of chronic myelogenous leukemia (CML), and addresses leukemic cells to growth arrest and cell death. The exact mechanisms responsible for imatinib-induced cell death are still unclear. We investigated the role of poly(ADP-ribose) polymerase (PARP) activity in imatinib-induced cell death in Bcr-Abl-positive cells. Imatinib leads to a rapid increase of poly(ADP-ribosyl)ation (PAR) preceding loss of integrity of mitochondrial membrane and DNA fragmentation. The effect of imatinib on PAR can be mimicked by inhibition of phosphatidylinositol 3-kinase (PI3-K) implicating a central role of the PI3-K pathway in Bcr-Abl-mediated inhibition of PAR. Importantly, inhibition of PAR in imatinib-treated cells partially prevented cell death to an extent comparable to that observed after caspase inhibition. Simultaneous blockade of both caspases and PAR revealed additive cytoprotective effects indicating that both pathways function in parallel. In conclusion, our results suggest that in addition to the well-documented caspase-dependent pathway, imatinib also induces a PARP-mediated death process.

Requirement for Mdm2 in the survival effects of Bcr-Abl and interleukin 3 in hematopoietic cells.

The p53/Mdm2 pathway plays an important role in the induction of cell cycle arrest or apoptosis in response to genotoxic stress. Both the oncogene Bcr-Abl and physiological growth factors such as interleukin (IL)-3 can modulate the outcome of cellular exposure to DNA damage. To determine whether Bcr-Abl and growth factors can affect the p53/Mdm2 pathway, we studied the expression of Mdm2 in the IL-3-dependent pre-B cell line BaF3 and its bcr-abl-transfected derivative BaF3p185 after IL-3 deprivation or treatment with the c-Abl tyrosine kinase inhibitor STI571. We found that both growth factor withdrawal and inhibition of Bcr-Abl kinase lead to a down-regulation of Mdm2 preceding the induction of apoptosis. Apoptotic cell death induced by STI571 is partially dependent on p53. The early decrease of Mdm2 protein was not attributable to transcriptional regulation or to caspase-mediated cleavage. On the other hand, it could be completely blocked by the proteasomal inhibitor lactacystin. Targeted down-regulation of Mdm2 protein by antisense oligodeoxynucleotides overcame the survival effects of IL-3 and Bcr-Abl and resulted in accelerated apoptosis. Taken together, survival signals provided either by physiological growth factors or by oncogenic Bcr-Abl can positively regulate Mdm2, whereas Mdm2 ablation can reduce cell survival. These findings imply that, similarly to physiological growth factors such as IL-3, Bcr-Abl can promote cell survival through modulating the p53-Mdm2 pathway.

Adhesion to fibronectin selectively protects Bcr-Abl+ cells from DNA damage-induced apoptosis.

The phenotype of Bcr-Abl-transformed cells is characterized by a growth factor-independent survival and a reduced susceptibility to apoptosis. Furthermore, Bcr-Abl kinase alters adhesion features by phosphorylating cytoskeletal and/or signaling proteins important for integrin function. Integrin-mediated adhesion to extracellular matrix molecules is critical for the regulation of growth and apoptosis. However, effects of integrin signaling on regulation of apoptosis in cells expressing Bcr-Abl are largely unknown. The influence of adhesion on survival and apoptosis in Bcr-Abl+ and Bcr-Abl- BaF3 cells was investigated. p185bcr-abl-transfected BaF3 cells preadhered to immobilized fibronectin had a significant survival advantage and reduced susceptibility to apoptosis following gamma-irradiation when compared with the same cells grown on laminin, on polylysin, or in suspension. Both inhibition of Bcr-Abl kinase by STI571 and inhibition of specific adhesion reversed the fibronectin-mediated antiapoptotic effect in BaF3p185. The DNA damage response of Bcr-Abl- BaF3 cells was not affected by adhesion to fibronectin. In contrast to parental BaF3 cells, BaF3p185 adherent to fibronectin did not release cytochrome c to the cytosol following irradiation. The fibronectin-mediated antiapoptotic mechanism in Bcr-Abl-active cells was not mediated by overexpression of Bcl-XL or Bcl-2 but required an active phosphatidylinositol 3-kinase (PI-3K). Kinase-active Bcr-Abl in combination with fibronectin-induced integrin signaling led to a hyperphosphorylation of AKT. Thus, cooperative activation of PI-3K/AKT by Bcr-Abl and integrins causes synergistic protection of Bcr-Abl+ cells from DNA damage-induced apoptosis.

The C3435T mutation in the human MDR1 gene is associated with altered efflux of the P-glycoprotein substrate rhodamine 123 from CD56+ natural killer cells.

P-glycoprotein (PGP) is a membrane protein which determines drug disposition in humans (e.g. digoxin). It is also expressed in various leukocyte lineages with highest expression in CD56+ natural killer cells. Recently, a polymorphism in exon 26 (C3435T) of this gene was shown to correlate with intestinal PGP expression and function in humans. Carriers homozygous for this polymorphism (TT) showed more than two-fold lower PGP expression and higher digoxin plasma concentrations compared to the CC group. However, it is not known whether this mutation in the MDR1 gene is also associated with altered PGP function in peripheral blood cells. We therefore assessed efflux of the PGP-substrate rhodamine 123 from CD56+ natural killer cells. Leukocytes were isolated from whole blood of 10 CC, 10 CT and 11 TT healthy Caucasian individuals. Using flow cytometry, rhodamine fluorescence was determined in CD56+ cells. Moreover, MDRI mRNA was quantified in leukocytes by real-time polymerase chain reaction. Subjects with CC genotype revealed a significantly lower rhodamine fluorescence (i.e. higher PGP function) compared to individuals with TT genotype (51.1 +/- 11.4% versus 67.5 +/- 9.5%, p < 0.01). Heterozygous individuals had an intermediate rhodamine fluorescence (61.4 +/- 6.3%). MDR1 mRNA normalized for cyclophilin was lowest in the TT population (1.29 +/- 1.01), intermediate in heterozygous subjects (1.60 +/- 0.76) and highest in the CC group (1.91 +/- 0.94; not significant). In summary, subjects being homozygous for C in position 3435 of the MDR1 gene have a more pronounced efflux of rhodamine from CD56+ natural killer cells and a higher MDR1 mRNA expression in leukocytes than subjects with the TT genotype. Measurement of rhodamine efflux using flow-cytometry from peripheral blood cells allows assessment of genetically determined differences in P-glycoprotein function.

Bcr-Abl kinase down-regulates cyclin-dependent kinase inhibitor p27 in human and murine cell lines.

Chronic myeloid leukemia (CML) is a malignant stem cell disease characterized by an expansion of myeloid progenitor cells expressing the constitutively activated Bcr-Abl kinase. This oncogenic event causes a deregulation of apoptosis and cell cycle progression. Although the molecular mechanisms protecting from apoptosis in CML cells are well characterized, the cell cycle regulatory event is poorly understood. An inhibitor of the cyclin-dependent kinases, p27, plays a central role in the regulation of growth factor dependent proliferation of hematopoietic cells. Therefore, we have analyzed the influence of Bcr-Abl in the regulation of p27 expression in various hematopoietic cell systems. An active Bcr-Abl kinase causes down-regulation of p27 expression in murine Ba/F3 cells and human M07 cells. Bcr-Abl blocks up-regulation of p27 after growth factor withdrawal and serum reduction. In addition, p27 induction by transforming growth factor-beta (TGF-beta) is completely blocked in Bcr-Abl positive M07/p210 cells. This deregulation is directly mediated by the activity of the Bcr-Abl kinase. A Bcr-Abl kinase inhibitor completely abolishes p27 down-regulation by Bcr-Abl in both Ba/F3 cells transfected either with a constitutively active Bcr-Abl or with a temperature sensitive mutant. The down-regulation of p27 by Bcr-Abl depends on proteasomal degradation and can be blocked by lactacystin. Overexpression of wild-type p27 partially antagonizes Bcr-Abl-induced proliferation in Ba/F3 cells. We conclude that Bcr-Abl promotes cell cycle progression and activation of cyclin-dependent kinases by interfering with the regulation of the cell cycle inhibitory protein p27. (Blood. 2000;96:1933-1939)

The tyrosine kinase inhibitor CGP 57148 (ST1 571) induces apoptosis in BCR-ABL-positive cells by down-regulating BCL-X.

CGP 57148 is a potent inhibitor of the ABL protein tyrosine kinase and a promising new compound for the treatment of a variety of BCR-ABL-positive leukemias. We used this enzyme inhibitor to characterize the biological effects of BCR-ABL in primary cells and two growth factor-dependent BCR-ABL-transfected cell lines. The effect of CGP 57148 on primary cells is dependent on the stage of differentiation. The growth of maturing chronic myeloid leukemia cells is independent of BCR-ABL in the presence of growth factors. However, the proliferation of leukemic immature cobblestone-forming area cells is almost completely blocked after the inhibition of the BCR-ABL kinase. In the BCR-ABL-transfected cell lines, M07/ p210 and Ba/F3/p185, CGP 57148 induces apoptosis by releasing cytochrome c, activating caspase 3, and cleavage of PARP. No alteration of the expression level of the apoptosis regulator BCL-2 was observed. In contrast, BCL-X was down-regulated after exposure to CGP 57148. Inhibitors of signal transduction proteins such as PI-3 kinase, mitogen-activated protein/extracellular signal-regulated kinase kinase, and Janus-activated kinase 2 pathways were not capable of a comparable down-regulation of BCL-X. The Fas/Fas ligand system was not involved either in the induction of apoptosis by CGP 57148. We conclude that the inhibition of the BCR-ABL kinase by CGP 57148 (a) preferentially inhibits the growth of immature leukemic precursor cells, (b) efficiently reverts the antiapoptotic effects of BCR-ABL by down-regulation of BCL-X, and (c) is more effective than the inhibition of the downstream signal transduction pathways of PI-3 kinase, mitogen-activated protein/extracellular signal-regulated kinase kinase, and Janus-activated kinase 2.

The DNA-binding subunit p140 of replication factor C is upregulated in cycling cells and associates with G1 phase cell cycle regulatory proteins.

The DNA-binding subunit of replication factor C (RFCp140) plays an important role in both DNA replication and DNA repair. The mechanisms regulating activation of RFCp140 thereby controlling replication and cellular proliferation are largely unknown. We analyzed protein expression of RFCp140 during cell cycle progression and investigated the association of RFCp140 with cell cycle regulatory proteins in cell lines of various tissue origin and in primary hematopoietic cells. Western and Northern blot analyses of RFCp140 from synchronized cells showed downregulation of RFCp140 when cells enter a G0-like quiescent state and upregulation of RFCp140 in cycling cells. Translocation from the cytoplasmic compartment to the nucleus did not account for the significant increase in RFCp140 protein levels observed in cycling cells. To investigate a potential association of RFCp140 with cell cycle regulatory proteins coimmunoprecipitation assays were performed. These studies demonstrated specific binding of RFCp140 to cdk4-kinase in hematopoietic and fibroblast cell lines. Additional coimmunoprecipitation studies revealed specific association of RFCp140 with cyclin D1, p21, proliferating cell nuclear antigen, and retinoblastoma protein. These findings link DNA replication and repair factor RFCp140 to G1 phase cell cycle regulatory elements critically involved in cell cycle control.

Bcr-Abl kinase promotes cell cycle entry of primary myeloid CML cells in the absence of growth factors.

Cell cycle control of both immature and differentiated primary myeloid normal and chronic-phase chronic myeloid leukaemia (CML) cells to growth factor deprivation was studied. CD34+ cells were cultured in liquid culture. After removal of growth factors for 48 h normal cells were very efficiently arrested with the fraction of cells in S phase reduced by 70.8 +/- 6.5% in CD34+ and 50.5 +/- 4.2% in CD34- cells. In contrast, a significantly higher proportion of leukaemic cells remained in S phase. The fraction of S-phase cells was reduced by only 29.3 +/- 5.7% in CD34+ CML cells and 21.2 +/- 3.8% in CD34- cells. This abnormal negative cell cycle control in leukaemic cells was specific for growth factor deprivation. Reaction to IFN-alpha and TNF-alpha treatment was identical both in normal and CML cells. Equal quantities of the cytokines TNF-alpha, IL-1alpha, IL-1RA and IL-6 were produced by CML and normal cells. However, production of GM-CSF, with a median of 11 +/- 5 pg/ml, was found only in the supernatants of CML cells. But antibodies to GM-CSF did not restore growth factor dependence of the leukaemic cells. The defect was completely corrected by the abl-specific tyrosine kinase inhibitor CGP 57148 without effecting cell cycle control of normal cells. Our results demonstrate a directly Bcr-Abl-dependent defective response of both immature and differentiated primary myeloid CML cells to growth factor deprivation.

Induction of the pro-myelocytic leukaemia gene by type I and type II interferons.

The physiological role of the pro-myelocytic leukaemia (PML) gene product is poorly defined. Among other functions, PML is involved in haematopoietic differentiation and in control of cell growth and tumorigenesis. We investigated the regulation of human PML expression by interferons (IFNs) and IL-1 in various human haematopoietic lines (U937, THP1, HL60, NB4), in human diploid fibroblasts and in human peripheral blood leukocytes. Cytokine-induced modulation of PML expression was assessed by Northern blot analyses, flow cytometry studies and in situ immunolabelling. Our data show that IFNs and IL-1 upregulate PML transcript and protein expression in a time and dose-dependent manner. In situ immunolabelling revealed that upregulation of protein expression by IFN-alpha is a consequence of a marked increase in both the number and the intensity of the staining of so-called PML nuclear bodies. Our data suggest that stimulation of PML expression by interferons and IL-1 may account for upregulation of PML proteins observed in inflammatory tissues and in proliferative states.

Induction of interferon regulatory factors 2'-5' oligoadenylate synthetase, P68 kinase and RNase L in chronic myelogenous leukaemia cells and its relationship to clinical responsiveness.

The genes crucially determining the therapeutic response of chronic myelogenous leukaemia (CML) to interferon-alpha (IFN-alpha) are unknown. Recently, two independent IFN-alpha signalling pathways were identified: the classic pathway mediates induction of 2'-5' oligoadenylate synthetase (2-5 OAS), p68 kinase and IFN regulatory factor-2 (IRF-2), whereas the alternate pathway leads to activation of IFN regulatory factor-1 (IRF-1). We investigated whether deficient or imbalanced expression of components of these two pathways is associated with resistance of CML cells to antiproliferative action of IFN alpha/beta. Constitutive and IFN-induced transcript levels of IFN-dependent genes in mononuclear cells, granulocytes, monocytes, lymphocytes and CD34+ cells of chronic-phase CML and blast crisis patients were assessed by Northern blot techniques and were correlated with subsequent clinical responses to IFN therapy. Our results demonstrated that IFN-alpha or -beta treatment in vitro and in vivo leads to an enhanced expression of IRF-1, IRF-2. RNase L, p68 and 2-5 OAS which was independent of the degree of cellular differentiation and clonal evolution of CML. Neither the magnitude of induction of these genes nor the IRF-1/IRF-2 mRNA balance differed between chronic-phase CML patients responding or failing IFN-alpha therapy. These results indicate that failure of IFN-alpha treatment is not due to defects in mRNA induction of the above-mentioned candidate genes for the direct antiproliferative response to IFN type I.

Overexpression of mouse p140 subunit of replication factor C accelerates cellular proliferation.

Screening of a murine cDNA expression library with an IFN-stimulated response element (ISRE), as a recognition site DNA probe, resulted in the isolation of a cDNA encoding a polypeptide of 1145 amino acids designated ISRE-binding factor-1. This was subsequently shown to be identical to the M(r) 140,000 subunit of replication factor C (RFC). RFC is required, along with the proliferating cell nuclear antigen and DNA polymerase delta, for the synthesis of the leading strand during DNA replication. RFC exhibits a structure-specific DNA-binding activity that has been localized to its M(r) 140,000 subunit (p140). Sequence-specific binding of this polypeptide to the ISRE occurs only with low affinity. Based on DNA-binding activity of the truncated RFC-p140 encoded by the partial cDNA isolated, the DNA-binding domain of this polypeptide was mapped to a region encoded by amino acids 366 to 540. Transfection of NIH 3T3 cells with an expression plasmid containing murine RFC-p140 driven by cytomegalovirus early promoter led to the establishment of stable cell lines that expressed a 2.5- to 3.0-fold higher RFC-p140 protein level in comparison with control cells. The stable clones exhibited significantly accelerated cell proliferation, indicating that RFC-p140 is the limiting subunit of an active RFC complex in normal cells.

Non-radioactive organization and transcript analysis of the ATPase subunit 6 gene region in the mitochondrial genome from fertile and sterile (CMS) forms of wheat and triticale.

In sterile triticale forms [with cytoplasmic male-sterile (CMS)-inducing timopheevi cytoplasm], fertile orms (with normal cytoplasm) and the corresponding wheat cross parents mitochondrial DNA (mtDNA) and RNA (messenger mtRNA) were characterized using total DNA and RNA material for Southern and Northern blots. A novel non-radioactive technique was applied by marking the probes with digoxenin. The fertile and sterile Triticum and triticale forms were analysed in three genes, atp 6, coxIII and rps13. These forms can be distinguished in the apt6 gene at the mtDNA and mtRNA levels.