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P Salomoni

Publications and source records attributed to P Salomoni.

At least 19 recordsLinked to original sources
PubMed2001

Caspase cleavage enhances the apoptosis-inducing effects of BAD.

The function of BAD, a proapoptotic member of the Bcl-2 family, is regulated primarily by rapid changes in phosphorylation that modulate its protein-protein interactions and subcellular localization. We show here that, during interleukin-3 (IL-3) deprivation-induced apoptosis of 32Dcl3 murine myeloid precursor cells, BAD is cleaved by a caspase(s) at its N terminus to generate a 15-kDa truncated protein. The 15-kDa truncated BAD is a more potent inducer of apoptosis than the wild-type protein, whereas a mutant BAD resistant to caspase 3 cleavage is a weak apoptosis inducer. Truncated BAD is detectable only in the mitochondrial fraction, interacts with BCL-X(L) at least as effectively as the wild-type protein, and is more potent than wild-type BAD in inducing cytochrome c release. Human BAD, which is 43 amino acids shorter than its mouse counterpart, is also cleaved by a caspase(s) upon exposure of Jurkat T cells to anti-FAS antibody, tumor necrosis factor alpha (TNF-alpha), or TRAIL. Moreover, a truncated form of human BAD lacking the N-terminal 28 amino acids is more potent than wild-type BAD in inducing apoptosis. The generation of truncated BAD was blocked by Bcl-2 in IL-3-deprived 32Dcl3 cells but not in Jurkat T cells exposed to anti-FAS antibody, TNF-alpha, or TRAIL. Together, these findings point to a novel and important role for BAD in maintaining the apoptotic phenotype in response to various apoptosis inducers.

Animals↗
PubMed2000

Bcl-2 expression restores the leukemogenic potential of a BCR/ABL mutant defective in transformation.

Growth factor-dependent hematopoietic cell lines expressing the BCR/ABL oncoprotein of the Ph chromosome show growth factor-independent proliferation and resistance to apoptosis. Apoptosis resistance of BCR/ABL-expressing cells may depend on enhanced expression of anti-apoptotic proteins as well as reduced expression and/or inactivation of pro-apoptotic proteins. Compared to myeloid precursor 32Dcl3 cells expressing wild type BCR/ABL, cells expressing a BCR/ABL mutant lacking amino acids 176-426 in the BCR domain (p185 delta BCR) are susceptible to apoptosis induced by interleukin-3 (IL-3) deprivation. These cells exhibited the hypophosphorylated apoptotic BAD and markedly reduced levels of Bcl-2. Upon ectopic expression of Bcl-2, these cells showed no changes in BAD phosphorylation, but they became apoptosis-resistant and proliferated in the absence of IL-3, albeit more slowly than cells expressing wild type BCR/ABL. Moreover, the p185 delta BCR/Bcl-2 double transfectants were leukemogenic when injected into immunodeficient mice, but Bcl-2 expression did not restore the leukemia-inducing effects of p185 delta BCR to the levels of wild type BCR/ABL. Leukemic cells recovered from the spleen of mice injected with p185 delta BCR/Bcl-2 cells did not show rearrangements in the Bcl-2 genomic locus, but they exhibited enhanced proliferation in culture and induced a rapidly fatal disease process when inoculated in secondary recipient mice. Together, these data support the importance of anti-apoptotic pathways for BCR/ABL-dependent leukemogenesis and suggest that Bcl-2 expression promotes secondary changes leading to a more aggressive tumor phenotype. (Blood. 2000;96:3915-3921)

Animals↗
PubMed2000

Versatility of BCR/ABL-expressing leukemic cells in circumventing proapoptotic BAD effects.

BAD, the proapoptotic member of the "BH3-only" subfamily of BCL-2 proteins, is inactivated by phosphorylation at serines 112 and 136 and by sequestration in the cytoplasm where it interacts with members of the 14-3-3 family. In BCR/ABL-expressing cells, BAD is constitutively phosphorylated and mainly cytoplasmic, whereas in cells expressing BCR/ABL mutants unable to protect from apoptosis, BAD is nonphosphorylated. We show here that both the wild-type (WT) and the S112A/ S136A double mutant (DM) BAD are more potent inducers of apoptosis in parental than in BCR/ABL-expressing 32D myeloid precursor cells. Stable lines of parental cells expressing DM BAD could not be established and most clones from WT BAD retrovirus-infected parental cells lost BAD expression. On IL-3 withdrawal from parental 32D cells, BAD was rapidly dephosphorylated by the serine-threonine phosphatase 1 alpha, and localized in the mitochondria, whereas it remained phosphorylated and did not localize to the mitochondria in the cohort of BCR/ABL-expressing cells escaping apoptosis induced by WT BAD. Moreover, these cells showed high levels of BCL-2 and BCL-X(L) expression. The cohort of BCR/ABL-expressing cells resistant to apoptosis induced by DM BAD showed only high levels of BCL-2 and BCL-X(L). These findings suggest that BCR/ABL-expressing cells are more versatile than normal hematopoietic progenitors in counteracting the apoptotic potential of BAD, and raise the possibility that tumor cells activate multiple antiapoptotic pathways for survival in the face of death-inducing stimuli. (Blood. 2000;96:676-684)

Animals↗
PubMed2000

Promyelocytic leukemia protein (PML) and Daxx participate in a novel nuclear pathway for apoptosis.

The promyelocytic leukemia protein (PML) gene of acute promyelocytic leukemia (APL) encodes a cell growth and tumor suppressor essential for multiple apoptotic signals. Daxx was identified as a molecule important for the cytoplasmic transduction of the Fas proapoptotic stimulus. Here, we show that upon mitogenic activation of mature splenic lymphocytes, Daxx is dramatically upregulated and accumulates in the PML nuclear body (NB) where PML and Daxx physically interact. In the absence of PML, Daxx acquires a dispersed nuclear pattern, and activation-induced cell death of splenocytes is profoundly impaired. PML inactivation results in the complete abrogation of the Daxx proapoptotic ability. In APL cells, Daxx is delocalized from the NB. Upon retinoic acid treatment, which induces disease remission in APL, Daxx relocalizes to the PML NBs. These results indicate that PML and Daxx cooperate in a novel NB-dependent pathway for apoptosis and shed new light in the role of PML in tumor suppression.

Animals↗
PubMed2000

The transcriptional role of PML and the nuclear body.

The PML gene encodes a tumour suppressor protein associated with a distinct subnuclear domain, the nuclear body. Various functions have been attributed to the PML nuclear body, but its main biochemical role is still unclear. Recent findings indicate that PML is essential for the proper formation of the nuclear body and can act as a transcriptional co-factor. Here we summarize the current understanding of the biological functions of PML and the nuclear body, and discuss a role for these intra-nuclear structures in the regulation of transcription.

Animals↗
PubMed2000

The function of PML in p53-dependent apoptosis.

The PML gene of acute promyelocytic leukaemia (APL) encodes a growth- and tumour-suppresor protein that is essential for several apoptotic signals. The mechanisms by which PML exerts its pro-apoptotic function are still unknown. Here we show that PML acts as a transcriptional co-activator with p53. PML physically interacts with p53 both in vitro and in vivo and co-localizes with p53 in the PML nuclear body (PML-NB). The co-activatory role of PML depends on its ability to localize in the PML-NB. p53-dependent, DNA-damage-induced apoptosis, transcriptional activation by p53, the DNA-binding ability of p53, and the induction of p53 target genes such as Bax and p21 upon gamma-irradiation are all impaired in PML-/- primary cells. These results define a new PML-dependent, p53-regulatory pathway for apoptosis and shed new light on the function of PML in tumour suppression.

Animals↗
PubMed1999

Activation of mitochondrial Raf-1 is involved in the antiapoptotic effects of Akt.

The Akt serine/threonine kinase is required for the survival of many cell types and for transformation of hematopoietic cells by the BCR/ABL oncogenic tyrosine kinase. Analysis of the potential mechanisms whereby Akt promotes survival of hematopoietic cells revealed that it induced the activity of plasma membrane and mitochondrial Raf-1 in a Ras-independent, but PKC-dependent manner. Inhibition of plasma membrane Raf-1-dependent mitogen-activated protein kinase activity had no effect on the enhanced survival of cells expressing Akt. By contrast, suppression of mitochondrial Raf-1 enzymatic activity by expression of a mitochondria-targeted Raf-1 dominant-negative mutant rendered Akt-expressing cells susceptible to apoptosis induced by growth factor deprivation and was accompanied by inhibition of BAD, but not mitogen-activated protein kinase, phosphorylation. Together, these data indicate that PKC-dependent activation of Raf-1 plays an important role in Akt-dependent antiapoptotic effects.

Animals↗
PubMed1999

Resistance to apoptosis in CTLL-2 cells overexpressing B-Myb is associated with B-Myb-dependent bcl-2 induction.

Transcriptional regulators of the Myb family play important roles in cell proliferation, differentiation, and survival. To investigate the role of Myb proteins in the regulation of apoptosis, we studied the apoptotic response of interleukin 2-dependent CTLL-2 cells stably transfected with B-Myb. B-Myb-overexpressing cells showed a diminished cytokine dependence and were resistant to apoptosis induced by doxorubicin, ceramide, and dexamethasone. Overexpression of B-Myb was associated with enhanced expression of bcl-2, which was dependent, at least in part, on increased transcription. In transient transfection assays in T-lymphoblastic cells, B-Myb was able to stimulate the promoter activity of the bcl-2 5' flanking region linked to the chloramphenicol acetyltransferase reporter gene. A segment of the bcl-2 promoter (nucleotides +34 to +58 relative to the transcription initiation site) contained a putative Myb-binding site and was shown to specifically interact with B-Myb and to confer B-Myb responsiveness to a bcl-2/chloramphenicol acetyltransferase reporter construct. These results indicate that B-Myb promotes T cells survival by enhancing the expression of bcl-2 and identify bcl-2 as a B-Myb target gene regulated in a DNA binding-dependent manner.

Animals↗
PubMed1999

Signal transducer and activator of transcription (STAT)5 activation by BCR/ABL is dependent on intact Src homology (SH)3 and SH2 domains of BCR/ABL and is required for leukemogenesis.

Signal transducer and activator of transcription (STAT)5 is constitutively activated in BCR/ ABL-expressing cells, but the mechanisms and functional consequences of such activation are unknown. We show here that BCR/ABL induces phosphorylation and activation of STAT5 by a mechanism that requires the BCR/ABL Src homology (SH)2 domain and the proline-rich binding site of the SH3 domain. Upon expression in 32Dcl3 growth factor-dependent myeloid precursor cells, STAT5 activation-deficient BCR/ABL SH3+SH2 domain mutants functioned as tyrosine kinase and activated Ras, but failed to protect from apoptosis induced by withdrawal of interleukin 3 and/or serum and did not induce leukemia in severe combined immunodeficiency mice. In complementation assays, expression of a dominant-active STAT5B mutant (STAT5B-DAM), but not wild-type STAT5B (STAT5B-WT), in 32Dcl3 cells transfected with STAT5 activation-deficient BCR/ABL SH3+SH2 mutants restored protection from apoptosis, stimulated growth factor-independent cell cycle progression, and rescued the leukemogenic potential in mice. Moreover, expression of a dominant-negative STAT5B mutant (STAT5B-DNM) in 32Dcl3 cells transfected with wild-type BCR/ABL inhibited apoptosis resistance, growth factor-independent proliferation, and the leukemogenic potential of these cells. In retrovirally infected mouse bone marrow cells, expression of STAT5B-DNM inhibited BCR/ABL-dependent transformation. Moreover, STAT5B-DAM, but not STAT5B-WT, markedly enhanced the ability of STAT5 activation-defective BCR/ABL SH3+SH2 mutants to induce growth factor-independent colony formation of primary mouse bone marrow progenitor cells. However, STAT5B-DAM did not rescue the growth factor-independent colony formation of kinase-deficient K1172R BCR/ABL or the triple mutant Y177F+R522L+ Y793F BCR/ABL, both of which also fail to activate STAT5. Together, these data demonstrate that STAT5 activation by BCR/ABL is dependent on signaling from more than one domain and document the important role of STAT5-regulated pathways in BCR/ABL leukemogenesis.

Animals↗
PubMed1999

Multiple signaling pathways of the insulin-like growth factor 1 receptor in protection from apoptosis.

The type 1 insulin-like growth factor receptor (IGF-1R), activated by its ligands, protects several cell types from a variety of apoptotic injuries. The main signaling pathway for IGF-1R-mediated protection from apoptosis has been previously elucidated and rests on the activation of phosphatidylinositol 3-kinase, Akt/protein kinase B, and the phosphorylation and inactivation of BAD, a member of the Bcl-2 family of proteins. In 32D cells (a murine hemopoietic cell line devoid of insulin receptor substrate 1 [IRS-1]), the IGF-1R activates alternative pathways for protection from apoptosis induced by withdrawal of interleukin-3. One of these pathways leads to the activation of mitogen-activated protein kinase, while a third pathway results in the mitochondrial translocation of Raf and depends on the integrity of a group of serines in the C terminus of the receptor that are known to interact with 14.3.3 proteins. All three pathways, however, result in BAD phosphorylation. The presence of multiple antiapoptotic pathways may explain the remarkable efficacy of the IGF-1R in protecting cells from apoptosis.

Apoptosis↗
PubMed1998

BCR/ABL-mediated leukemogenesis requires the activity of the small GTP-binding protein Rac.

The phenotype of hematopoietic cells transformed by the BCR/ABL oncoprotein of the Philadelphia chromosome is characterized by growth factor-independent proliferation, reduced susceptibility to apoptosis, and altered adhesion and motility. The mechanisms underlying this phenotype are not fully understood, but there is evidence that some of the properties of BCR/ABL-expressing cells are dependent on the activation of downstream effector molecules such as RAS, PI-3k, and bcl-2. We show here that the small GTP-binding protein Rac is activated by BCR/ABL in a tyrosine kinase-dependent manner. Upon transfection with a vector carrying the dominant-negative N17Rac, BCR/ABL-expressing myeloid precursor 32Dcl3 cells retained the resistance to growth factor deprivation-induced apoptosis but showed a decrease in proliferative potential in the absence of interleukin-3 (IL-3) and markedly reduced invasive properties. Moreover, compared with BCR/ABL-expressing cells, fewer BCR/ABL plus N17Rac double transfectants were capable of homing to bone marrow and spleen. Consistent with these findings, survival of SCID mice injected with the BCR/ABL plus N17Rac double transfectants was markedly prolonged as compared with that of mice injected with BCR/ABL-expressing cells. Together, these data support the important role of a Rac-dependent pathway(s) controlling motility in BCR/ABL-mediated leukemogenesis.

Animals↗
PubMed1998

TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis.

The leukemogenic potential of BCR/ABL oncoproteins depends on their tyrosine kinase activity and involves the activation of several downstream effectors, some of which are essential for cell transformation. Using electrophoretic mobility shift assays and Southwestern blot analyses with a double-stranded oligonucleotide containing a zinc finger consensus sequence, we identified a 68 kDa DNA-binding protein specifically induced by BCR/ABL. The peptide sequence of the affinity-purified protein was identical to that of the RNA-binding protein FUS (also called TLS). Binding activity of FUS required a functional BCR/ABL tyrosine kinase necessary to induce PKCbetaII-dependent FUS phosphorylation. Moreover, suppression of PKCbetaII activity in BCR/ABL-expressing cells by treatment with the PKCbetaII inhibitor CGP53353, or by expression of a dominant-negative PKCbetaII, markedly impaired the ability of FUS to bind DNA. Suppression of FUS expression in myeloid precursor 32Dcl3 cells transfected with a FUS antisense construct was associated with upregulation of the granulocyte-colony stimulating factor receptor (G-CSFR) and downregulation of interleukin-3 receptor (IL-3R) beta-chain expression, and accelerated G-CSF-stimulated differentiation. Downregulation of FUS expression in BCR/ABL-expressing 32Dcl3 cells was associated with suppression of growth factor-independent colony formation, restoration of G-CSF-induced granulocytic differentiation and reduced tumorigenic potential in vivo. Together, these results suggest that FUS might function as a regulator of BCR/ABL leukemogenesis, promoting growth factor independence and preventing differentiation via modulation of cytokine receptor expression.

Amino Acid Sequence↗
PubMed1998

Expression of constitutively active Raf-1 in the mitochondria restores antiapoptotic and leukemogenic potential of a transformation-deficient BCR/ABL mutant.

The oncogenic BCR/ABL protein protects hematopoietic cells from apoptosis induced by growth factor deprivation, but the mechanisms are only partially understood. A BCR/ABL mutant lacking amino acids 176-426 in the BCR domain (p185DeltaBCR) failed to protect interleukin 3-deprived 32Dcl3 myeloid precursor cells from apoptosis, although it possessed tyrosine kinase activity and was capable of activating the Ras-Raf-MAP kinase pathway. Compared to p185 wild-type transfectants, p185DeltaBCR-transfected cells showed markedly reduced levels of Bcl-2 and expressed the hypophosphorylated, proapoptotic form of BAD. Bcl-2 expression in the mitochondrial fraction of p185DeltaBCR cells was also markedly diminished and mitochondrial RAF was undetectable. In p185DeltaBCR cells transfected with a mitochondria-targeted, constitutively active RAF (M-Raf) BAD was expressed in the hyperphosphorylated form and released from the mitochondria into the cytosol. p185DeltaBCR/M-Raf-transfected cells were completely resistant to apoptosis induced by growth factor deprivation in vitro. Moreover, constitutive expression of dominant-negative M-Raf (K375W) enhanced the susceptibility of 32Dcl3 cells expressing wild-type BCR/ABL to apoptosis. In severe combined immunodeficiency (SCID) mice, p185DeltaBCR/M-Raf double transfectants were leukemogenic, whereas cells expressing only p185DeltaBCR showed no leukemogenic potential. Together, these data support the existence of a BCR/ABL-dependent pathway that leads to expression of an active RAF in the mitochondria and promotes antiapoptotic and leukemia-inducing effects of BCR/ABL.

Animals↗
PubMed1998

Growth properties and growth factor responsiveness in skin fibroblasts from centenarians.

Human fibroblast cultures, which have a finite replicative lifespan in vitro, are the most widely used model for the study of senescence at the cellular level. An inverse relationship between replicative capability and donor age has been reported in human fibroblast strains. We studied the growth capacity of fibroblast primary cultures derived from people whose lifespan was as closer as possible to the expected maximum human lifespan, i.e. people over one hundred. Our data suggest that outgrowth of fibroblasts from biopsies, growth kinetics at different population doubling levels, capability to respond to a classical mitogenic stimulus (such as 20% serum) and a variety of growth factors, were remarkably similar in fibroblasts from centenarians and young controls. On the whole, our data challenge the tenet of a simple and strict relationship between in vivo aging and in vitro proliferative capability of human fibroblasts, at least at the individual level.

Adolescent↗
PubMed1998

The SH3 domain contributes to BCR/ABL-dependent leukemogenesis in vivo: role in adhesion, invasion, and homing.

To determine the possible role of the BCR/ABL oncoprotein SH3 domain in BCR/ABL-dependent leukemogenesis, we studied the biologic properties of a BCR/ABL SH3 deletion mutant (delta SH3 BCR/ABL) constitutively expressed in murine hematopoietic cells. delta SH3 BCR/ABL was able to activate known BCR/ABL-dependent downstream effector molecules such as RAS, PI-3kinase, MAPK, JNK, MYC, JUN, STATs, and BCL-2. Moreover, expression of delta SH3 BCR/ABL protected 32Dcl3 murine myeloid precursor cells from apoptosis, induced their growth factor-independent proliferation, and resulted in transformation of primary bone marrow cells in vitro. Unexpectedly, leukemic growth from cells expressing delta SH3 BCR/ABL was significantly retarded in SCID mice compared with that of cells expressing the wild-type protein. In vitro and in vivo studies to determine the adhesive and invasive properties of delta SH3 BCR/ABL-expressing cells showed their decreased interaction to collagen IV- and laminin-coated plates and their reduced capacity to invade the stroma and to seed the bone marrow and spleen. The decreased interaction with collagen type IV and laminin was consistent with a reduced expression of alpha 2 integrin by delta SH3 BCR/ABL-transfected 32Dcl3 cells. Moreover, as compared with wild-type BCR/ABL, which localizes primarily in the cytoskeletal/membrane fraction, delta SH3 BCR/ABL was more evenly distributed between the cytoskeleton/membrane and the cytosol compartments. Together, the data indicate that the SH3 domain of BCR/ABL is dispensable for in vitro transformation of hematopoietic cells but is essential for full leukemogenic potential in vivo.

Animals↗
PubMed1997

Resistance to apoptosis in CTLL-2 cells constitutively expressing c-Myb is associated with induction of BCL-2 expression and Myb-dependent regulation of bcl-2 promoter activity.

c-Myb, the cellular homologue of the transforming gene of the avian myeloblastosis virus, is preferentially expressed in all hematopoietic lineages, including T and B lymphocyte lineages. In T lymphocytes, c-Myb expression appears to be required for cell cycle progression and proliferation. To further investigate the role of c-Myb in T cell proliferation and survival, interleukin (IL) 2-dependent CTLL-2 cells were transfected with a constitutively active c-myb or with a c-myb antisense construct able to down-regulate endogenous Myb levels, and the transfectants were assessed for proliferation and survival in low concentrations of IL-2 and for susceptibility to dexamethasone-induced apoptosis. Compared with control cells, CTLL-2 cells constitutively expressing c-Myb proliferate in low concentrations of IL-2 and are less susceptible to apoptosis induced by IL-2 deprivation or treatment with dexamethasone. In contrast, cells transfected with an antisense c-myb construct do not proliferate in low concentrations of IL-2 and undergo apoptosis upon IL-2 deprivation or dexamethasone treatment more rapidly than parental cells. Overexpression of c-Myb was accompanied by up-regulation of BCL-2 expression. In transient transfection assays, the murine bcl-2 promoter was efficiently transactivated by c-Myb, but such effect was observed also in cells transfected with a DNA binding-deficient c-myb construct. Moreover, in gel retardation assays, a 38-bp oligomer in the shortest bcl-2 promoter segment regulated by c-Myb formed a specific complex with nuclear extracts from c-Myb-transfected CTLL-2 cells. Thus, these results strongly suggest that c-Myb, in addition to regulating T cell proliferation, protects T lymphocytes from apoptosis by induction of BCL-2 expression, which involves a c-Myb-dependent mechanism of promoter regulation.

Animals↗
PubMed1996

c-fos/c-jun expression and AP-1 activation in skin fibroblasts from centenarians.

In vitro replicative senescence is characterized by an irreversible growth arrest due to the inability of the cell to induce some key regulators of cell cycle progression, such as c-fos and AP-1, in response to mitogenic stimuli. In vitro replicative senescence and in vivo aging have been assumed to be two related phenomena, likely controlled by overlapping or interacting genes. As a corollary, fibroblasts from centenarians, which have undergone a long process of senescence in vivo should have very limited proliferative capability. On the contrary, in a previous work we found that fibroblasts from centenarians exhibited the same capacity to respond to different mitogenic stimuli as fibroblasts from young donors. Here we provide evidences that the well preserved proliferative response is likely due to the fact that some pivotal regulators- c-fos, c-jun and AP-1-are still fully inducible, despite a long process of in vivo senescence. Our data therefore suggest that in vivo and in vitro aging are separate phenomena whose possible relationships, if any, have to be ascertained very carefully.

Adolescent↗