A switching mechanism in doxorubicin bioactivation can be exploited to control doxorubicin toxicity.

Although doxorubicin toxicity in cancer cells is multifactorial, the enzymatic bioactivation of the drug can significantly contribute to its cytotoxicity. Previous research has identified most of the components that comprise the doxorubicin bioactivation network; however, adaptation of the network to changes in doxorubicin treatment or to patient-specific changes in network components is much less understood. To investigate the properties of the coupled reduction/oxidation reactions of the doxorubicin bioactivation network, we analyzed metabolic differences between two patient-derived acute lymphoblastic leukemia (ALL) cell lines exhibiting varied doxorubicin sensitivities. We developed computational models that accurately predicted doxorubicin bioactivation in both ALL cell lines at high and low doxorubicin concentrations. Oxygen-dependent redox cycling promoted superoxide accumulation while NADPH-dependent reductive conversion promoted semiquinone doxorubicin. This fundamental switch in control is observed between doxorubicin sensitive and insensitive ALL cells and between high and low doxorubicin concentrations. We demonstrate that pharmacological intervention strategies can be employed to either enhance or impede doxorubicin cytotoxicity in ALL cells due to the switching that occurs between oxygen-dependent superoxide generation and NADPH-dependent doxorubicin semiquinone formation.

Activation of cAMP signaling inhibits DNA damage-induced apoptosis in BCP-ALL cells through abrogation of p53 accumulation.

In lymphocytes, the second messenger cyclic adenosine monophosphate (cAMP) plays a well-established antiproliferative role through inhibition of G(1)/S transition and S-phase progression. We have previously demonstrated that, during S-phase arrest, cAMP inhibits the action of S phase-specific cytotoxic compounds, leading to reduction in their apoptotic response. In this report, we provide evidence that cAMP can also inhibit the action of DNA-damaging agents independently of its effect on S phase. Elevation of cAMP in B-cell precursor acute lymphoblastic leukemia cells is shown to profoundly inhibit the apoptotic response to ionizing radiation, anthracyclins, alkylating agents, and platinum compounds. We further demonstrate that this effect depends on the ability of elevated cAMP levels to quench DNA damage-induced p53 accumulation by increasing the p53 turnover, resulting in attenuated Puma and Bax induction, mitochondrial outer membrane depolarization, caspase activation, and poly(ADP-ribose) polymerase cleavage. On the basis of our findings, we suggest that cAMP levels may influence p53 function in malignant cells that retain wild-type p53, potentially affecting p53 both as a tumor suppressor during cancer initiation and maintenance, and as an effector of the apoptotic response to DNA-damaging agents during anticancer treatment.

Expression and functional role of inhibitor-of-apoptosis protein livin (BIRC7) in neuroblastoma.

We evaluated the expression of the inhibitor-of-apoptosis protein (IAP)livin (BIRC7)in 59 cases ofneuroblastoma (NBL) by quantitative RT-PCR. We also examined the role of livin in protecting tumor cells from chemotherapy drugs. Livin expression varied significantly amongtumors. High levels of expression were observed in 17 of 39 patients with advanced stages (stages 3 and 4) and 6 of 20 patients with localized stages (stages 1 and 2). Livin-transfected, MYCN-amplified NBL cells showed increased resistance to doxorubicin and etoposide. Conversely, livin knockdown with siRNA enhanced spontaneous and drug-induced apoptosis in NBL cells. Multivariate analysis of prognostic factors showed that high livin expression worsened prognosis for patients with MYCN-amplified tumors. Our data suggest that (i) livin is frequently expressed in NBL and protects tumor cells with amplified MYCN oncogene from genotoxic agents; (ii) the antiapoptotic effect of livin in NBL is blocked by siRNA; (iii) in the sample studied, high livin expression enhanced the adverse prognostic impact of MYCN amplification. These findings suggest that livin may contribute to drug resistance in NBL.

The MDM2 antagonist nutlin-3 sensitizes p53-null neuroblastoma cells to doxorubicin via E2F1 and TAp73.

Neuroblastoma (NB) is a primitive neuroectodermal tumor and the second most common solid tumor in children. NB exhibits heterogeneous behavior and spontaneous regression can occur in patients under 12 months of age. Response to treatment is both age- and stage-specific; however, patients over 1 year of age are generally considered high risk. NB tumors from these patients are often characterized by alterations in p53 expression and murine double minute (MDM2) activity with concomitant resistance to chemotherapy. We evaluated the ability of nutlin-3 to sensitize a p53-null and doxorubicin-resistant NB cell line, LA155N, to doxorubicin. Nutlin-3 treatment upregulated TAp73 and E2F1 protein levels. It potentiated the ability of doxorubicin to block cell proliferation and activate apoptosis and TAp73 knockdown resulted in a reduction of this sensitization. Additionally, PUMA expression was induced by the combination treatment, but reduced by knockdown of either TAp73 or E2F1. We conclude that, following nutlin-3 treatment, TAp73 and E2F1 are released from MDM2 and activated by doxorubicin to induce PUMA and apoptosis. This study addresses p53-independent mechanisms of nutlin-3 action in chemoresistant NB, especially in combination with chemotherapeutics. We believe that this model has strong clinical relevance for chemoresistant and p53 dysfunctional NB.

PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells.

BACKGROUND: c-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated. METHODS: Expression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-gamma agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migration RESULTS: High c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation. CONCLUSION: c-Met is highly expressed in most tumors from patients with advanced-stage, metastatic NBL. Furthermore, using the NBL cell line SH-EP as a model, PHA665752 was shown to inhibit cMet/HGF/SF signaling in vitro, suggesting c-Met inhibitors may have efficacy for blocking local progression and/or metastatic spread of c-Met-positive NBL in vivo. These are novel findings for this disease and suggest that further studies of agents targeting the c-Met/HGF axis in NBL are warranted.

MycN is a transcriptional regulator of livin in neuroblastoma.

Our previous studies have suggested that MycN may have a role in regulating livin expression in neuroblastoma. Here, we show that siRNA-mediated repression of MycN in neuroblastoma cells with both elevated MycN and livin expression resulted in significant downregulation of livin. Conversely, induction of MycN in neuroblastoma cells with low endogenous levels of MycN and livin protein upregulated livin expression. MycN directly associated with its regulatory motif (E-box) within the putative livin promoter. Based on these results, we hypothesize that MycN is required for livin expression and that livin may counteract the proapoptotic effects of MycN.

Contribution of STAT3 to the activation of survivin by GM-CSF in CD34+ cell lines.

OBJECTIVE: Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to specifically stimulate proliferation of CD34(+) hematopoietic progenitor cells. Although signal transducers and activators of transcription 3 (STAT3) is believed essential for transduction of GM-CSF-induced cell proliferation, the signaling mediated by STAT3 is not completely understood. Because survivin regulates cell proliferation and survival via its antiapoptotic function, we studied the link between STAT3 signaling and survivin expression in CD34(+) cells. METHODS: GM-CSF-induced STAT3 and survivin expression in CD34(+) cells was examined by Western blot assay. GM-CSF-activated survivin promoter activity was analyzed by gene transfection and reporter assays. The binding of STAT3 to the survivin promoter was evaluated by chromatin immunoprecipitation and electrophoretic mobility shift assay. Western blotting and flow cytometry were utilized to test the effect of Janus family of tyrosine kinases (JAK) inhibitor and STAT3 small interfering RNA (siRNA) on cell apoptosis. RESULTS: We found that GM-CSF stimulates survivin promoter activity in CD34(+) KG-1 cells, and STAT3 binds to the core survivin promoter containing a STAT response element TT(N)(5)AA at sites -264 to -256. Mutation or deletion of this STAT response element completely abolished the effects of GM-CSF on survivin promoter activity. Furthermore, addition of either JAK inhibitor or STAT3 siRNA was able to inhibit GM-CSF-induced survivin promoter activity and survivin expression. Inhibition of survivin by STAT3 siRNA or by withdrawal of GM-CSF in a GM-CSF-dependent, CD34(+) line TF-1 decreased cell growth and increased apoptosis. CONCLUSION: Altogether, our results suggest that survivin is a transcriptional target of STAT3, and that GM-CSF-stimulated CD34(+) cell proliferation is regulated by the JAK/STAT3/survivin signaling pathway.

PTEN reverses MDM2-mediated chemotherapy resistance by interacting with p53 in acute lymphoblastic leukemia cells.

The tumor suppressor PTEN has been associated with the cellular localization of MDM2 in regulation of apoptosis through inhibiting PI3k/Akt signaling. To investigate whether expression of PTEN is involved in MDM2-mediated chemoresistance, we examined a set of acute lymphoblastic leukemia (ALL) cell lines for the expression of PTEN and sensitivity to doxorubicin. Testing 9 ALL cell lines selected for wild-type p53 phenotype and uniformly high levels of MDM2 expression, we initially demonstrated that cell lines with high levels of PTEN expression were sensitive to doxorubicin, whereas lines lacking PTEN expression were generally resistant. Forced expression of PTEN in a PTEN-negative and doxorubicin-resistant ALL line (EU-1) resulted in decreased cell growth and enhanced sensitivity to doxorubicin. Examining the cellular localization of MDM2, we confirmed that the majority of MDM2 is localized in the nucleus in PTEN-negative doxorubicin-sensitive ALL cells, whereas MDM2 is expressed predominantly in the cytoplasm in either PTEN-positive or PTEN-transfected cells. Furthermore, by coimmunoprecipitaton and cotransfection assays, we found that PTEN physically binds p53 in vitro as well as in vivo. Binding of PTEN to p53 attenuated MDM2-mediated p53 inhibition. These results suggest that PTEN inhibits MDM2 and protects p53 through both p13k/Akt-dependent and -independent pathways. Furthermore, loss of PTEN can result in resistance to apoptosis by activating MDM2-mediated antiapoptotic mechanism.

Transfection of a dominant-negative mutant NF-kB inhibitor (IkBm) represses p53-dependent apoptosis in acute lymphoblastic leukemia cells: interaction of IkBm and p53.

To investigate the possible role of inhibiting NF-kB activation in sensitizing tumor cells to chemotherapy-induced apoptosis, we transfected the dominant-negative mutant inhibitor of NF-kB (IkBm) into the EU-1 cell line, an acute lymphoblastic leukemia (ALL) line with constitutive NF-kB activation. Overexpression of IkBm significantly reduced constitutive NF-kB activity in EU-1 cells, resulting in decreased cell growth. In response to apoptosis induced by chemotherapeutic drugs, IkBm-transfected cells (EU-1/IkBm) exhibited increased sensitivity to vincristine (VCR), whereas sensitivity to doxorubicin (Dox) was not changed as compared to neo-transfected control (EU-1/neo) cells. To further evaluate the link between IkBm and sensitivity to Dox and VCR, we demonstrated that both endogenous IkBalpha and ectopic IkBm bind to p53. In response to Dox, the cytosolic p53.IkBalpha complex rapidly dissociated due to downregulation of IkBalpha. However, the p53.IkBm complex did not dissociate under these conditions. Although treatment of EU-1/IkBm cells with Dox increased the expression of p53, the nondissociating p53.IkBm complex resulted in decreased p53 function, as demonstrated by absence of cell-cycle arrest and induction of p53 target genes. Contrastingly, VCR-induced cell death neither downregulated IkBalpha nor induced p53, as shown by the lack of NF-kB activation and p53-mediated gene expression in VCR-treated cells. Our data suggest that IkBm simultaneously downregulates NF-kB activation and sequesters p53 in the cytoplasm, thus enhancing NF-kB-regulated apoptosis but blocking p53-dependent apoptosis.

An alternatively spliced survivin variant is positively regulated by p53 and sensitizes leukemia cells to chemotherapy.

Survivin is a unique member of the inhibitor of apoptosis protein family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DeltaEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild-type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53-143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53-143. When EU-4/p53-143 cell culture was shifted from 37.5 degrees C to the wt-p53-permissive temperature (32.5 degrees C), the expression of survivin and survivin-DeltaEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a proapoptotic factor and its expression is upregulated by p53.

Comparison of DR5 and Fas expression levels relative to the chemosensitivity of acute lymphoblastic leukemia cell lines.

The relationship between p53 gene status and the expression of DR5 and Fas was evaluated as a function of sensitivity of 11 acute lymphoblastic leukemia cell lines to adriamycin, etoposide, vincristine, methotrexate and dexamethasone. There was up to a 37-fold increase in expression of DR5 following treatment with ADR or VP-16 only in cells with wt p53. A direct correlation was observed between enhanced DR5 expression and sensitivity to ADR and VP-16. There was no induction of DR5 following treatment with VCR, MTX or DEX. There was up to a 51-fold increase in the median level of expression of Fas following treatment with ADR and VP-16, and unlike DR5 this occurred in cells with either wild-type or mutant p53. Nevertheless, a direct correlation was observed between Fas expression and drug-sensitivity. Conversely, there was only a two-fold increase in expression of Fas after exposure to VCR, MTX and DEX. These findings suggest that DR5 mediates sensitivity to ADR and VP-16 in a p53-dependent manner, whereas, Fas appears to mediate sensitivity to these two drugs independent of p53 status. DR5 and Fas do not appear to play a major role as determinants of chemosensitivity to VCR, MTX and DEX.

Molecular mechanism of serial VH gene replacement.

The molecular mechanism of serial VH replacement was analyzed using a human B cell line, EU12, that undergoes continuous spontaneous differentiation from pro-B to pre-B and then to B cell stage. In earlier studies, we found that this cell line undergoes intraclonal V(D)J diversification. Analysis of the IgH gene sequences in EU12 cells predicted the occurrence of serial VH replacement involving the cryptic recombination signal sequences (cRSS) embedded within framework 3 regions and concurrent extension of the CDR3 region. Detection of double-stranded DNA breaks at the cRSS site and different VH replacement excision circles confirm the ongoing nature of this diversification process in the EU12 cells. In vitro binding and cleavage assays using recombinant RAG-1 and RAG-2 proteins further validated the cRSS participation in this RAG-mediated recombination process. Serial VH replacements may represent an additional mechanism for diversification of the primary B cell repertoire.

Variable Expression and Hypermethylation of p16 Gene in Patients with T-ALL and Cell Lines.

The multi tumor suppressor genes MTS1 (CDKN2, p16INK4A) and MTS2 (CDKN1, p15INK4B) located at 9p21-22 are inactivated in some human cancers via several mechanisms including deletion and hypermethylation. In hematological malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid malignancies, and more particularly to T-cell acute lymphoblastic leukemia (TALL). We have investigated the deletion, methylation and p16 protein expression status of MTS1 in Tcell childhood acute lymphoblastic leukemia (19 cases) and cell lines[11]. On Southern blot homozygous deletions or hemizygous deletion with rearangement were detected in 4/19 T-ALL. The expression of p16 protein was not observed on Western blot in 4/15 T-ALL with intact p16 gene. The p16 gene was methylated 3/15 in T-ALL. Only one of three expressed p16 protein. The other 11/15 T-ALL had p16 protein expression but different level. Loss of MTS1 was observed in 3/11 cell lines. Cell line with MTS1 gene had p16 protein expression in 6/8. After treatment with the demethylating agent (5-AzoCyt) RD cell line showed p16 expression. This has not been observed with the other cell lines. Thus hypermethylation of MTS1 is rare in childhood T-ALL. Although inactivation of MTS1 by deletion is common in T-ALL and cell lines. Furthermore our data show that the p16 gene inactivation by hypermethylation and deletion may play a role in the leukemogenesis.

The PI-3 kinase-Akt-MDM2-survivin signaling axis in high-risk neuroblastoma: a target for PI-3 kinase inhibitor intervention.

PURPOSE: Studies of SF1126, an RGDS targeted, water-soluble prodrug of LY294002, are currently nearing completion in two adult Phase I trials. Herein, we performed a preclinical evaluation of SF1126 as a PI-3K inhibitor for Phase I trials in the treatment of recurrent neuroblastoma (NB). METHODS: The effects of SF1126 on pAkt-MDM2 cell signaling, proliferation, apoptosis, and migration were determined using a panel of NB cell lines, and anti-tumor activity was determined using a xenograft model of NB. RESULTS: SF1126 blocks MDM2 activation, IGF-1 induced activation of Akt, and the upregulation of survivin induced by IGF-1. It also increases sensitivity to doxorubicin in vitro and was found to exhibit marked synergistic activity in combination with doxorubicin. Treatment disrupts the integrin αvß3/αvß5-mediated organization of the actin cytoskeleton as well as the α4ß1/α5ß1-mediated processes essential to metastasis. In vivo, SF1126 markedly inhibits tumor growth in NB xenografted mice (P < 0.05). CONCLUSIONS: A pan PI-3 kinase inhibitor has potent antitumor activity and induces apoptosis in multiple neuroblastoma cell lines. The observed effects of SF1126 on the p-Akt-MDM2-survivin axis suggest a patient selection paradigm in which NB tumors with increased pAkt-MDM2-survivin signaling may predict response to SF1126 alone or in combination with standard chemotherapy regimens that contain anthracyclines.

Anti-CD22 immunotoxin RFB4(dsFv)-PE38 (BL22) for CD22-positive hematologic malignancies of childhood: preclinical studies and phase I clinical trial.

PURPOSE: Although most children with B-lineage acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma are cured, new agents are needed to overcome drug resistance and reduce toxicities of chemotherapy. We hypothesized that the novel anti-CD22 immunotoxin, RFB4(dsFv)-PE38 (BL22, CAT-3888), would be active and have limited nonspecific side effects in children with CD22-expressing hematologic malignancies. We conducted the first preclinical and phase I clinical studies of BL22 in that setting. EXPERIMENTAL DESIGN: Lymphoblasts from children with B-lineage ALL were assessed for CD22 expression by flow cytometry and for BL22 sensitivity by in vitro cytotoxicity assay. BL22 was evaluated in a human ALL murine xenograft model. A phase I clinical trial was conducted for pediatric subjects with CD22+ ALL and non-Hodgkin lymphoma. RESULTS: All samples screened were CD22+. BL22 was cytotoxic to blasts in vitro (median IC(50), 9.8 ng/mL) and prolonged the leukemia-free survival of murine xenografts. Phase I trial cohorts were treated at escalating doses and schedules ranging from 10 to 40 microg/kg every other day for three or six doses repeated every 21 or 28 days. Treatment was associated with an acceptable safety profile, adverse events were rapidly reversible, and no maximum tolerated dose was defined. Pharmacokinetics were influenced by disease burden consistent with rapid drug binding by CD22+ blasts. Although no responses were observed, transient clinical activity was seen in most subjects. CONCLUSIONS: CD22 represents an excellent target and anti-CD22 immunotoxins offer therapeutic promise in B-lineage hematologic malignancies of childhood.

High level MycN expression in non-MYCN amplified neuroblastoma is induced by the combination treatment nutlin-3 and doxorubicin and enhances chemosensitivity.

MYCN gene amplification is a negative prognostic indicator in neuroblastoma and high level MycN expression in Stage IV neuroblastoma is generally a hallmark of poor patient outcome. However, high level expression of the MycN protein in neuroblastoma cells lacking MYCN amplification suppresses growth and drives apoptosis; this, in part, explains the absence of clinical observations of high level MycN in neuroblastoma lacking MYCN amplification. In the current study, we found that combination treatment with nutlin-3 and doxorubicin upregulated MycN expression in non-MYCN-amplified neuroblastoma cells at both the protein and mRNA levels. The induced expression of MycN in non-MYCN-amplified cells inhibited cell proliferation and increased apoptosis. MycN induction also upregulated p53, p21 and Bax protein levels, as well as mRNA levels for the positive neuroblastoma prognostic factors CD44 and EFNB3. Blocking MycN reversed these effects. These results were corroborated by findings using a MycN-inducible system in SHEP cells, another MYCN non-amplified neuroblastoma cell line. Our results indicate that doxorubicin/nutlin-3 combination treatment both induces expression of MycN in a non-MYCN-amplified background and sensitizes neuroblastoma cells to chemotherapy. These findings support the idea that induction of MycN in non-MYCN-amplified cells drives neuroblastoma cells toward apoptosis and suggest that combination nutlin-3/doxorubicin treatment may be clinically important.

KLF5 Interacts with p53 in regulating survivin expression in acute lymphoblastic leukemia.

The Kruppel-like factor 5 (KLF5) is a transcription factor that regulates cellular signaling involved in cell proliferation and oncogenesis. Here, we report that KLF5 interacts with tumor suppressor p53 in regulating the expression of the inhibitor-of-apoptosis protein survivin, which may play a role in pathological process of cancer. The core promoter region of survivin contains multiple GT-boxes that have been characterized as KLF5 response elements. Deletion and mutation analyses as well as chromatin immunoprecipitation and electronic mobility shift assay indicated that KLF5 binds to the core survivin promoter and strongly induces its activity. Furthermore, we demonstrated that KLF5 protein is able to bind to p53 and abrogate the p53-regulated repression of survivin. Transfection of KLF5 into a KLF5-negative acute lymphoblastic leukemia cell line EU-8 enhanced survivin expression, and conversely, silencing of KLF5 by small interfering RNA in a KLF5-overexpressing acute lymphoblastic leukemia cell line EU-4 down-regulated survivin expression. The KLF5 small interfering RNA-mediated down-regulation of survivin sensitized EU-4 cells to apoptosis induced by chemotherapeutic drug doxorubicin. These findings identify a novel regulatory pathway for the expression of survivin under the control of KLF5 and p53. Deregulation of this pathway may result in overexpression of survivin in cancer, thus contributing to drug resistance.

Transcriptional repression of the eukaryotic initiation factor 4E gene by wild type p53.

The eukaryotic initiation factor 4E (eIF4E) plays important roles in transformation and cancer progression. It is frequently overexpressed in malignant cells, one mechanism of which is through transcriptional activation by c-myc. Here, we report that high level of eIF4E expression and its tumorigenicity could be alternatively associated with defects of p53, since we found that induction of wt-p53 repressed eIF4E expression. Gene transfection of p53 inhibited eIF4E promoter activity, while inactivation of p53 either by mutation or by over-expression of MDM2 resulted in stimulation of eIF4E promoter activity. We demonstrated that p53-repression of eIF4E was regulated by c-myc. The wt-p53 can physically bind to c-myc, which inhibited binding of c-myc to eIF4E promoter and c-myc-stimulated promoter activity. These results suggest that the expression of eIF4E is reciprocally regulated by p53 and c-myc, and loss of p53-mediated control over c-myc-dependent transactivation of eIF4E may represent a novel mechanism for eIF4E-mediated neoplastic transformation and cancer progression.

Expression of inhibitor-of-apoptosis protein (IAP) livin by neuroblastoma cells: correlation with prognostic factors and outcome.

Livin is a recently identified member of the Inhibitor-of-Apoptosis protein (IAP) family of antiapoptosis proteins, and expression has been reported in melanoma and some types of carcinoma. We evaluated livin expression in paraffin-embedded tumor tissue from 68 patients with neuroblastoma (NB) and 7 NB cell lines by immunoperoxidase using an anti-livin monoclonal antibody. Eighteen (26.5%) of the 68 NB tumor tissues showed high livin expression, 36 (53%) showed low-intermediate expression, and 14 (20.5%) were negative. Similarly, 4 NB cell lines showed high livin expression, and 3 showed intermediate expression. In primary NB tissue, livin was observed mainly in tumor neuropil, an extension of tumor cell cytoplasm, and the cytoplasm itself. By reverse transcriptase-polymerase chain reaction, livin expression was confirmed in all 7 NB lines and in frozen tissue from 1 of 3 primary tumors examined to date, in agreement with immunohistochemical data; both livin alpha and beta isoforms were detected. In the NB cases, we further analyzed the correlation between livin expression and clinical and biological features with established prognostic significance (i.e., age at diagnosis, stage, histology, and MYCN oncogene status), and patients' outcome. Livin expression alone did not appear to have an effect on survival; however, patients with high livin expression and amplified MYCN had significantly decreased survival compared with patients lacking both markers or with either of these markers alone. These results suggest that (a) livin is expressed in primary and cultured neuroblastoma cells and (b) high livin expression may identify a subset of neuroblastoma patients with a particularly poor prognosis among those with MYCN amplified tumors.

MDM2 induces NF-kappaB/p65 expression transcriptionally through Sp1-binding sites: a novel, p53-independent role of MDM2 in doxorubicin resistance in acute lymphoblastic leukemia.

MDM2 protein is thought to exhibit tumorigenic activity by binding to the p53 tumor-suppressor protein and inhibiting its function. Alternatively, MDM2 may have oncogenic roles other than those resulting from p53 interactions. Here we report that MDM2 can induce expression of the p65 subunit of NF-kappaB, which is an anti-apoptotic factor expressed in certain neoplastic cells in response to chemotherapy. Initially, we noted that the overexpression of MDM2 protein in leukemic bone marrow cells of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and an ALL cell line (EU-4) transfected with the MDM2 gene was associated with elevated expression of p65 and in vitro resistance to doxorubicin (Adriamycin). By cotransfection of the MDM2 gene and p65-promoter-reporter constructs into EU-4 cells, we found that transient and high-level MDM2 expression induced p65 promoter activity. In the presence of wild-type (wt) p53, MDM2 increased p65 promoter activity by reversing p53-mediated suppression of p65. In the absence of p53, MDM2 directly increased p65 promoter activity. Deletion and mutation analysis of the p65 promoter indicated that the region between nt -575 and -178, which contains the first and second Sp1-binding sites, was required for activation by MDM2. Further studies using chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assay (EMSA) showed that MDM2 was able to directly bind to the Sp1 site of the p65 promoter. Our findings suggest that by inducing p65 expression, MDM2 has a p53-independent role in tumorigenesis, which may further elucidate the association between MDM2 overexpression and resistant disease in childhood ALL.