Allelism and linkage studies of murine leukemia virus activation genes in low leukemic strains of mice.

Previously, we identified two genes, termed Inc-1 and Inb-1, that interact to enhance ecotropic murine leukemia virus induction in low virus strains of mice. Mice related to BALB/c in origin carry a locus termed Inc-1, whereas mice related to B6 carry an Inb-1 locus. Mice that carry both Inc-1 and Inb-1 yield 10- to 50-fold more virus-producing cells than parental strains on induction with halogenated pyrimidines in vitro and demonstrate enhanced murine leukemia virus production in vivo. Here, we show that mice related to BALB/c in origin, i.e., A, C3H/He, and SEC, have an Inc-1 locus that is allelic with that of BALB/c. The C57BR mouse strain has an Inb-1 locus that is allelic with that of B6, located on chromosome 8, 30 cM from Es-1. We also show that the Inc-1 locus of BALB/c mice is located on chromosome 5, 24 cM from Pgm-1 and 43 cM from Gus. Kozak and Rowe (6,8) and Ihle and co-workers (3) have shown that the ecotropic virus-inducing genes in BALB/c and B10 mice are located on chromosomes 5 and 8, respectively, with similar distances from the previously mentioned biochemical markers. Our data are consistent with two possibilities: Inc-1 and Inb-1 are part of the virus-inducing genes Cv-1 and Bv-1, respectively , or Inc-1 and Inb2-1 are tightly linked regulatory genes.

Genetic interactions in the spontaneous production of endogenous murine leukemia virus in low leukemic mouse strains.

The spontaneous expression of ecotropic murine leukemia virus (MuLV) in spleen cells of BALB/c, C57BL/6 (B6), and derivative mice was examined as a function of age. The patterns of spontaneous virus induction in vivo correlate with the patterns of virus induction in vitro, which result from the action of two loci, Inc-l and Inb-l (7). Whereas mice carrying Inc-l or Inb-l have similar phenotypes in vitro, they have significantly different phenotypes in vivo. Mice of the Inb-l+/+ genotype, e.g., B6, rarely expressed MuLV, and the titer of MuLV recovered from rare MuLV-positive mice of this genotype was usually low. Mice of the Inc-l+/+ genotype, e.g., BALB/c, expressed low amounts of MuLV early in life, however, from 6-12 mo of age approximately one-half of the Inc-l+/+ mice expressed virus, frequently of high titer. Equal numbers of N-tropic and B-tropic MuLV were recovered from Inb-l+ mice, but predominantly N-tropic MuLV was recovered from Inc-l+ mice. Strains that carry dominant (+) alleles at both Inc-l and Inb-l show higher titers of MuLV earlier in life than strains that carry only Inc-l or Inb-l. The presence of dominant alleles at both loci results in the appearance of predominantly N-tropic virus early in life. These results demonstrate that the principal determinants of spontaneous virus expression in these low leukemic strains of mice are the In loci or genes linked to them. A further inference that can be drawn from these studies is that the appearance of B-tropic virus is by no means a random process but rather results from predictable patterns of MuLV expression and alteration.

Structure and expression of endogenous ecotropic murine leukemia viruses in RF/J mice.

High leukemic mouse strains possess proviral genomes that are more inducible for virus expression by halogenated pyrimidines than the proviral genomes harbored by low leukemic mice. We investigated the induction and arrangement of ecotropic proviruses in RF mice, a strain of mouse that develops a moderate incidence of leukemia late in life. We found that RF mice, unlike either high or low leukemic inbred strains, carried both a gene for high efficiency virus induction (Rjv-1) and a gene for low efficiency virus induction (Rjv-2). Virus induction from mice that contained Rjf-2 alone was observed only in crosses with two other strains that carried ecotropic proviruses, i.e., DBA/2 and C57BL/6, and not in crosses performed with mice that lacked ecotropic proviruses, i.e., 129, SWR, and NFS. Inheritance of the Rjv-1 gene frequently resulted in viremia when a virus-suppressive gene(s) of RF (most likely Fv-1) was not present in the same individual. Rjv-1 and Rjv-2 virus induction genes co-segregated with ecotropic proviruses integrated in different cellular DNA sequences. Rjv-2, the less inducible ecotropic provirus in RF mice, is located in cellular DNA sequences very similar to those found adjacent to the ecotropic provirus of BALB/c. These results document a second system of virus interaction or complementation and demonstrate that ecotropic proviruses of different phenotypes can be found within an individual mouse strain.

The insulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 induces apoptosis in acute myeloid leukemia cells exhibiting autocrine insulin-like growth factor-I secretion.

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) have been implicated in the pathophysiology of many human cancers, including those of hematopoietic lineage. We investigated the therapeutic potential of the novel IGF-IR tyrosine kinase activity inhibitor, NVP-AEW541, on human acute myeloid leukemia (AML) cells. NVP-AEW541 was tested on a HL60 cell subclone, which is dependent on autocrine secretion of IGF-I for survival and drug resistance, as well as primary drug resistant leukemia cells. NVP-AEW541 treatment (24 h) induced dephosphorylation of IGF-IR. NVP-AEW541 also caused Akt dephosphorylation and changes in the expression of key regulatory proteins of the cell cycle. At longer incubation times (48 h), NVP-AEW541-induced apoptotic cell death, as demonstrated by caspase-3 cleavage. Apoptosis was accompanied by decreased expression of anti-apoptotic proteins. NVP-AEW541 enhanced sensitivity of HL60 cells to either cytarabine or etoposide. Moreover, NVP-AEW541 reduced the clonogenic capacity of AML CD34(+) cells cultured in the presence of IGF-I. Chemoresistant AML blasts displayed enhanced IGF-I secretion, and were sensitized to etoposide-induced apoptosis by NVP-AEW541. Our findings indicate that NVP-AEW541 might be a promising therapeutic agent for the treatment of those AML cases characterized by IGF-I autocrine secretion.

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts.

A high incidence of relapses following induction chemotherapy is a major hindrance to patient survival in acute myelogenous leukemia (AML). There is strong evidence that activation of the phosphoinositide 3 kinase (PI3K)/Akt signaling network plays a significant role in rendering AML blasts drug resistant. An important mechanism underlying drug resistance is represented by overexpression of membrane drug transporters such as multidrug resistance-associated protein 1 (MRP1) or 170-kDa P-glycoprotein (P-gp). Here, we present evidence that MRP1, but not P-gp, expression is under the control of the PI3K/Akt axis in AML blasts. We observed a highly significant correlation between levels of phosphorylated Akt and MRP1 expression in AML cells. Furthermore, incubation of AML blasts with wortmannin, a PI3K pharmacological inhibitor, resulted in lower levels of phosphorylated Akt, downregulated MRP1 expression, and decreased Rhodamine 123 extrusion in an in vitro functional dye efflux assay. We also demonstrate that wortmannin-dependent PI3K/Akt inhibition upregulated p53 protein levels in most AML cases, and this correlated with diminished MRP1 expression and enhanced phosphorylation of murine double minute 2 (MDM2). Taken together, these data suggest that PI3K/Akt activation may lead to the development of chemoresistance in AML blasts through a mechanism involving a p53-dependent suppression of MRP1 expression.

Systematic analysis of GSK-3 signaling pathways in aging of cerebral tissue.

Glycogen synthase kinase-3 (GSK-3) is a constitutively active kinase, involved in regulation of multiple physiological processes. In brain, changes in GSK-3 signaling are related to neurodegenerative issues, including Alzheimer's disease. Due to the wide range of GSK-3 cellular targets, a therapeutic use of the enzyme inhibitors entails significant risk of side effects. Thus, altering the ratio of specific pool of GSK-3 or specific substrates instead of changing the global activity of GSK-3 in brains might be a more appropriate strategy. This paper provides a comprehensive data on abundances of proteins involved in GSK-3 signaling in three regions of young and old mouse brains. It might help to identify novel protein targets with the highest therapeutic potential for treatment of age-related neurodegenerative diseases.

Therapeutic targeting of CK2 in acute and chronic leukemias.

CK2 is a ubiquitously expressed, constitutively active Ser/Thr protein kinase, which is considered the most pleiotropic protein kinase in the human kinome. Such a pleiotropy explains the involvement of CK2 in many cellular events. However, its predominant roles are stimulation of cell growth and prevention of apoptosis. High levels of CK2 messenger RNA and protein are associated with CK2 pathological functions in human cancers. Over the last decade, basic and translational studies have provided evidence of CK2 as a pivotal molecule driving the growth of different blood malignancies. CK2 overexpression has been demonstrated in nearly all the types of hematological cancers, including acute and chronic leukemias, where CK2 is a key regulator of signaling networks critical for cell proliferation, survival and drug resistance. The findings that emerged from these studies suggest that CK2 could be a valuable therapeutic target in leukemias and supported the initiation of clinical trials using CK2 antagonists. In this review, we summarize the recent advances on the understanding of the signaling pathways involved in CK2 inhibition-mediated effects with a particular emphasis on the combinatorial use of CK2 inhibitors as novel therapeutic strategies for treating both acute and chronic leukemia patients.

Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin module for acute myelogenous leukemia therapy: from bench to bedside.

The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB)/mammalian Target Of Rapamycin (mTOR) signaling pathway plays a critical role in many cellular functions which are elicited by extracellular stimuli. However, constitutively active PI3K/Akt/mTOR signaling has also been firmly established as a major determinant for cell growth, proliferation, and survival in an wide array of human cancers. Thus, blocking the PI3K/AKT/mTOR signal transduction network could be an effective new strategy for targeted anticancer therapy. Pharmacological inhibitors of this signaling cascade are powerful antineoplastic agents in vitro and in xenografted models of tumors, and some of them are now being tested in clinical trials. Recent studies showed that PI3K/Akt/mTOR axis is frequently activated in acute myelogenous leukemia (AML) patient blasts and strongly contributes to proliferation, survival, and drug-resistance of these cells. Both the disease-free survival and overall survival are significantly shorter in AML cases with PI3K/Akt/mTOR upregulation. Therefore, this signal transduction cascade may represent a target for innovative therapeutic treatments of AML patients. In this review, we discuss the possible mechanisms of activation of this pathway in AML cells and the downstream molecular targets of the PI3K/Akt/mTOR signaling network which are important for blocking apoptosis, enhancing proliferation, and promoting drug-resistance of leukemic cells. We also highlight several pharmacological inhibitors which have been used to block this pathway for targeted therapy of AML. These small molecules induce apoptosis or sensitize AML cells to existing drugs, and might be used in the future for improving the outcome of this hematological disorder.

Synergy between an IGF-1R antibody and Raf/MEK/ERK and PI3K/Akt/mTOR pathway inhibitors in suppressing IGF-1R-mediated growth in hematopoietic cells.

The Insulin-like growth factor-1 receptor (IGF-1R) is overexpressed in a variety of tumors including breast, prostate and myeloma. Thus, IGF-1R and its downstream signaling effectors are good candidates for molecular-based targeted antitumor therapies. Indeed, protein inhibitors of IGF-1R signaling and IGF-1R blocking antibodies are undergoing clinical trials. Herein, the molecular basis for antibody-mediated IGF-1R signal inhibition has been investigated in a hematopoietic cell line model, FDC-P1, that has been rendered interleukin-3 independent in a ligand-dependent manner through retroviral-mediated expression of IGF-1R (FD/IGF-1R). Furthermore, the ability of an anti-IGF-1R antibody to synergize with signal-transduction pathway inhibitors and induce apoptosis was determined. The alphaIGF-1R antibody, A12, was capable of arresting IGF-1 or insulin-induced FD/IGF-1R cell proliferation in the G1 phase of the cell cycle and resulted in apoptotic induction. A12 effectiveness could be potentiated through combination treatment with small molecule inhibitors of the Ras/Raf/MEK/ERK or PI3K/Akt/mTOR pathways. These results validate the use of the FD/IGF-1R cells to evaluate the effectiveness and mechanisms of targeted IGF-1R therapeutic strategies.

Inhibition of PI3K, mTOR and MEK signaling pathways promotes rapid apoptosis in B-lineage ALL in the presence of stromal cell support.

Bone marrow stromal cells are essential for the differentiation, survival and proliferation of normal and leukemic human B-lineage cells. Leukemic cells require stromal cell support for optimal proliferation and apoptotic resistance. Stromal cell contact can promote resistance to chemotherapeutic agents. In this study, we have made use of small molecular weight inhibitors and an established stromal cell-dependent pre-B-ALL cell line, BLIN-2, to investigate the role of the MAP kinase, PI3K/Akt, JAK/STAT and mTOR pathways in the promotion of leukemic cell growth in the presence of stromal cell support. Treatment with PI3K+JAK, PI3K+MEK, or MEK+JAK inhibitor combinations resulted in an inhibition of proliferation as measured by DNA synthesis. However, only inhibition of both PI3K and MEK or both mTOR and MEK resulted in a dramatic increase in the number of annexinV(+)/PI(+) apoptotic events within a 24 h period. Our data suggest that stromal cell-mediated apoptotic protection in B-lineage ALL is mediated by PI3K/mTOR and MEK via a synergistic mechanism(s).

Raf and VEGF: emerging therapeutic targets in Kaposi's sarcoma-associated herpesvirus infection and angiogenesis in hematopoietic and nonhematopoietic tumors.

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with several cancers including Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease. KSHV-mediated pathogenesis is dependent mainly on KSHV infection as well as on the microenvironment provided by the growth factors (GFs)/inflammatory cytokines (ICs). Recently, we determined that oncoprotein Raf enhances KSHV infection of target cells. Interestingly, Raf regulates the expression of a variety of GFs/ICs including those involved in angiogenesis such as vascular endothelial growth factor (VEGF). In this review, we discuss the effect of the Raf-GF/IC autocrine/paracrine loop on KSHV infection of both hematopoietic and nonhematopietic cells, and associated disease conditions.

NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance.

Sorafenib, an oral multikinase inhibitor, is the only approved agent for the treatment of advanced hepatocellular carcinoma (HCC). However, its benefits are modest, and as its mechanisms of action remain elusive, a better understanding of its anticancer effects is needed. Based on our previous study results, we investigated here the implication of the nuclear protein 1 (NUPR1) in HCC and its role in sorafenib treatment. NUPR1 is a stress-inducible protein that is overexpressed in various malignancies, but its role in HCC is not yet fully understood. We found that NUPR1 expression was significantly higher in primary human HCC samples than in the normal liver. Knockdown of NUPR1 significantly increased cell sensitivity to sorafenib and inhibited the cell growth, migration and invasion of HCC cells, both in vitro and in vivo. Moreover, NUPR1 silencing influenced the expression of RELB and IER3 genes. Unsurprisingly, RELB and IER3 knockdown also inhibited HCC cell viability, growth and migration. Using gene expression profiling of HCC cells following stable NUPR1 knockdown, we found that genes functionally involved in cell death and survival, cellular response to therapies, lipid metabolism, cell growth and proliferation, molecular transport and cellular movement were mostly suppressed. Network analysis of dynamic gene expression identified NF-κB and ERK as downregulated gene nodes, and several HCC-related oncogenes were also suppressed. We identified Runt-related transcription factor 2 (RUNX2) gene as a NUPR1-regulated gene and demonstrated that RUNX2 gene silencing inhibits HCC cell viability, growth, migration and increased cell sensitivity to sorafenib. We propose that the NUPR1/RELB/IER3/RUNX2 pathway has a pivotal role in hepatocarcinogenesis. The identification of the NUPR1/RELB/IER3/RUNX2 pathway as a potential therapeutic target may contribute to the development of new treatment strategies for HCC management.

Autocrine transformation of hemopoietic cells resulting from cytokine message stabilization after intracisternal A particle transposition.

Cell lines that no longer require exogenous interleukin 3 (IL-3) for growth were isolated from an IL-3-dependent cell line that possesses characteristics of early lymphoid cells. Unlike the parental cells (FL5.12), these autocrine transformed lines (FL-IL3-R) constitutively secreted IL-3, were rearranged at the IL-3 locus and formed tumors upon injection into syngeneic mice. The rearrangement and IL-3 expression resulted from the transposition of an intracisternal A particle (IAP) provirus into the 3' untranslated region of the IL-3 gene. This region contained ATTTA sequence motifs that have been associated with cytokine and oncogene mRNA instability. IL-3 transcripts from the autocrine transformed cell lines had a longer half-life than similar transcripts isolated from either phorbol ester-stimulated T cells or the WEHI-3B myelomonocytic cell line. IAP proviral transposition did not alter the transcription rate of the IL-3 gene in FL-IL3-R cells. Therefore, IAP proviral transposition can activate IL-3 gene expression by prolonging mRNA stability, and this mechanism can contribute to the autocrine transformation of the hemopoietic cells.

P21(Cip1) induced by Raf is associated with increased Cdk4 activity in hematopoietic cells.

To investigate the functions of the different Raf genes in hematopoietic cell proliferation, the capacities of beta-estradiol-regulated Delta Raf:ER genes to induce cell cycle regulatory gene expression and cell cycle progression in FDC-P1 cells were examined. Raf activation increased the expression of Cdk2, Cdk4, cyclin A, cyclin D, cyclin E, p21(Cip1) and c-Myc and decreased the expression of p27(Kip1) which are associated with G(1) progression. However only the cell clones with moderate Raf activation, i.e. FD/Delta Raf-1:ER and FD/Delta A-Raf:ER, successfully underwent cell proliferation. The cell clones with the highest Delta Raf activity, FD/Delta B-Raf:ER, underwent apoptosis before cell proliferation. p21(Cip1) induced by Raf activation specifically bound with Cdk4/cyclin D complexes but not Cdk2/cyclin E complexes and this binding was associated with the increased Cdk4 activity. However, no binding of p27(Kip1) with either Cdk2/cyclin E or Cdk4/cyclin D was observed. Thus Raf mediated growth was associated with elevated p21(Cip1) expression, which may specifically bind with and activate Cdk4/cyclin D complexes and with decreased p27(Kip1) expression.

Phorbol esters support the proliferation of a hematopoietic cell line by upregulating c-jun expression.

FD/PMA, a derivative of the interleukin-3 (IL-3) dependent FDC-P1 cell line, proliferates in response to either phorbol esters (phorbol 12-myristate 13-acetate, PMA) or IL-3. Analysis of immediate-early gene expression revealed that FD/PMA cells contained elevated levels of c-jun transcripts when grown in the presence of phorbol esters. Upregulation of c-jun mRNA was specific since other jun family members (namely jun-B and jun-D) displayed similar patterns of gene expression as observed in the parental cell line. The accumulation of c-jun RNA was due to increased c-jun transcription and not the result of altered message stability. Furthermore, elevated c-jun expression resulted in increased AP-1 binding activity in FD/PMA cells. Antisense c-jun oligonucleotides suppressed proliferation of FD/PMA cells by 80% and resulted in a significant reduction in both AP-1 binding activity and c-jun message levels. Collectively, these results demonstrate that FD/PMA cells require elevated levels of c-jun expression for PMA-responsive proliferation and indicate that tumor promoters have the ability to abrogate IL-3 dependency by elevating AP-1 activity.

Retroviral infection can abrogate the factor-dependency of hematopoietic cells by autocrine and non-autocrine mechanisms depending on the presence of a functional viral oncogene.

The mechanisms responsible for abrogation of the growth factor-dependency of a hematopoietic cell line were investigated. FDC-P1 cells were infected with retroviral constructs containing the neo gene and either a wild-type or a temperature-sensitive v-src oncogene. v-srcwt abrogated the factor-dependency of these cells since each G418r colony gave rise to factor-independent cells and no autocrine growth factor activity was detected. Moreover, the vast majority (< 99%) of cells infected with the v-srcts mutant gave rise to conditional factor-independent cells. Therefore a functional v-src gene product was required for growth factor-independence which occurred by a non-autocrine mechanism. A minority of factor-independent cells which arose after v-srcts infection, grew at the non-permissive temperature and one-half secreted granulocyte/macrophage-colony stimulating factor (GM-CSF) which supports the growth of the parental cells. Since the v-srcts viral stock contained a helper virus, Murine Leukemia Virus (MuLV), the ability of this virus to relieve factor-dependency was examined. A low frequency of factor-independent transformants was recovered after MuLV infection and one-half secreted GM-CSF. Therefore, retroviruses such as MuLV which lack an oncogene, can transform cells by stimulating autocrine growth factor secretion. Subsequent experiments performed with helper-free v-src preparations indicated that they could abrogate factor-dependency directly by a non-autocrine mechanism. These results demonstrate that a hematopoietic cell line can be transformed by two different mechanisms after retroviral infection and may be relevant for understanding hematopoietic cell transformation after persistent viral infection.

A conditionally-active form of MEK1 results in autocrine tranformation of human and mouse hematopoietic cells.

The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using deltaMEK1:ER, a conditionally-active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of the human and murine hematopoietic cells lines TF-1, FDC-P1 and FL5.12. Cytokine-independent cells were obtained from TF-1, FDC-P1 and FL5.12 cells at frequencies of 2.5 x 10(-3), 5 x 10(-5) and 10(-7) respectively, indicating that not all cells expressing deltaMEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to deltaMEK1:ER activation than those that remained cytokine-dependent. deltaME-K1:ER-responsive cells could be maintained long-term in the presence of beta-estradiol as well as the estrogen-receptor antagonist 4-Hydroxy-Tamoxifen and the anti-estrogen ICI 164383. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. Treatment of deltaMEKI:ER-responsive cells with a specific and selective inhibitor, PD98059, prevented growth in response to beta-estradiol. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that the activated deltaMEK1:ER may induce a pathway leading to autocrine proliferation. Treatment of MEK1-responsive cells with an anti-GM-CSF antibody, but not a control antibody, suppressed cell growth. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells.

The Raf signal transduction cascade as a target for chemotherapeutic intervention in growth factor-responsive tumors.

This review focuses on the Ras-Raf-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signal transduction pathway and the consequences of its unregulation in the development of cancer. The roles of some of the cell membrane receptors involved in the activation of this pathway, the G-protein Ras, the Raf, MEK and ERK kinases, the phosphatases that regulate these kinases, as well as the downstream transcription factors that become activated, are discussed. The roles of the Ras-Raf-MEK-ERK pathway in the regulation of apoptosis and cell cycle progression are also analyzed. In addition, potential targets for pharmacological intervention in growth factor-responsive cells are evaluated.

The Raf/MEK/ERK signal transduction cascade as a target for chemotherapeutic intervention in leukemia.

The Raf/MEK/ERK (MAPK) signal transduction cascade is a vital mediator of a number of cellular fates including growth, proliferation and survival, among others. The focus of this review centers on the MAPK signal transduction pathway, its mechanisms of activation, downstream mediators of signaling, and the transcription factors that ultimately alter gene expression. Furthermore, negative regulators of this cascade, including phosphatases, are discussed with an emphasis placed upon chemotherapeutic intervention at various points along the pathway. In addition, mounting evidence suggests that the PI3K/Akt pathway may play a role in the effects elicited via MAPK signaling; as such, potential interactions and their possible cellular ramifications are discussed.