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PubMed2001

Secretion in alveolar type II cells at the interface of constitutive and regulated exocytosis.

Long-term, simultaneous, measurements of cytoplasmic free Ca(2+) concentrations and single exocytotic fusion events in surfactant-secreting type II cells were performed. All fusion (constitutive, phorbol ester-induced, and agonist-induced) was Ca(2+)-dependent. Kinetic analysis revealed that agonist (adenosine triphosphate [ATP])-induced fusion exhibited a kinetic pattern that correlated well with the Ca(2+) signal. The effects of Ca(2+) release from intracellular stores (early) and Ca(2+) entry (late) could be demonstrated for the first time by dissecting the slow (10-to-15-min) fusion response to ATP into these two components. Bath Ba(2+) or Sr(2+) could replace Ca(2+) to elicit a fusion response in thapsigargin-pretreated cells lacking ATP-induced Ca(2+) release from stores. Although the late response was partially inhibited by interrupting the phospholipase D-protein kinase C axis, a high Ca(2+) dependence of the entire secretory course was demonstrated by a significant correlation between the integrated Ca(2+) signal and the fusion response. There was also a highly significant correlation between constitutive and ATP-stimulated fusion activity in individual cells. We propose a common mechanistic model for all types of fusion in this slow secretory cell, in which constitutive and regulated forms of exocytosis are subject to the same principles of regulation.

Adenosine Triphosphate↗
PubMed1997

Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells.

The pathogenesis and etiology of Hodgkin's disease, a common human malignant lymphoma, is still unresolved. As a unique characteristic, we have identified constitutive activation of the transcription factor nuclear factor (NF)-kappaB p50-RelA in Hodgkin/Reed-Sternberg (H/RS) cells, which discriminates these neoplastic cells from most cell types studied to date. In contrast to other lymphoid and nonlymphoid cell lines tested, proliferation of H/RS cells depended on activated NF-kappaB. Furthermore, constitutive NF-kappaB p50-RelA prevented Hodgkin's lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkin's lymphoma cells depleted of constitutive nuclear NF-kappaB revealed strongly impaired tumor growth in severe combined immunodeficient mice. Our findings identify NF-kappaB as an important component for understanding the pathogenesis of Hodgkin's disease and for developing new therapeutic strategies against it.

Animals↗
PubMed1997

B lymphocytes from patients with chronic lymphocytic leukemia contain signal transducer and activator of transcription (STAT) 1 and STAT3 constitutively phosphorylated on serine residues.

To explore the pathogenesis of chronic lymphocytic leukemia (CLL), we examined whether phosphorylation of one or more signal transducer and activator of transcription (STAT) factors was abnormal in cells from CLL patients. No constitutive tyrosine phosphorylation was detected on any STAT in CLL cells. To assess the phosphorylation of serine residues of STAT1 and STAT3 in CLL cells, we raised antibodies that specifically recognize the form of STAT1 phosphorylated on ser-727 and the form of STAT3 phosphorylated on ser-727. We found that in 100% of patients with CLL (n = 32), STAT1 and STAT3 were constitutively phosphorylated on serine. This was in contrast to normal peripheral blood B lymphocytes or CD5+) B cells isolated from tonsils, in which this phosphorylation was absent. Serine phosphorylation of STAT1 and STAT3 was seen occasionally in other leukemias, but it was a universal finding only in CLL. The serine phosphorylation of these STATs was a continuous process, as incubation of CLL cells with the kinase inhibitor H7 led to the dephosphorylation of these serine residues. The STAT serine kinase in CLL cells has not been identified, and appears to be neither mitogen-activated protein kinase nor pp70(s6k). In summary, the constitutive serine phosphorylation of STAT1 and STAT3 is present in all CLL samples tested to date, although the physiologic significance of this modification remains to be determined.

Antibody Formation↗
PubMed2006

Loss of constitutive activity of the growth hormone secretagogue receptor in familial short stature.

The growth hormone (GH) secretagogue receptor (GHSR) was cloned as the target of a family of synthetic molecules endowed with GH release properties. As shown recently through in vitro means, this receptor displays a constitutive activity whose clinical relevance is unknown. Although pharmacological studies have demonstrated that its endogenous ligand--ghrelin--stimulates, through the GHSR, GH secretion and appetite, the physiological importance of the GHSR-dependent pathways remains an open question that gives rise to much controversy. We report the identification of a GHSR missense mutation that segregates with short stature within 2 unrelated families. This mutation, which results in decreased cell-surface expression of the receptor, selectively impairs the constitutive activity of the GHSR, while preserving its ability to respond to ghrelin. This first description, to our knowledge, of a functionally significant GHSR mutation, which unveils the critical importance of the GHSR-associated constitutive activity, discloses an unusual pathogenic mechanism of growth failure in humans.

Adolescent↗
PubMed2002

HSP70 constitutive expression in rat central nervous system from postnatal development to maturity.

We studied the level of the basal (constitutive) HSP70 expression (inducible and constitutive forms) in the central nervous system (CNS) of male and female rats from the postnatal period to maturity. HSP70 levels were analyzed by immunoblotting in five different areas (cortex, hippocampus, hypothalamus, cerebellum, and spinal cord). The highest levels of HSP70 were found in juvenile rats and decreased progressively until reaching baseline levels between 2 and 4 months. A slight and nonsignificant increase in aged (2-year-old) rats compared with adult subjects was observed in some cerebral areas (cerebral cortex, hippocampus, and cerebellum). In the first weeks of postnatal development, HSP70 immunoreactivity was distributed throughout CNS sections and no specific immunopositive cells could be clearly determined. In adult animals, strong immunostaining was observed in some large neurons (Purkinje neurons and mesencephalic and spinal cord motor neurons), some perivascular and subpial astrocytes, and ependymocytes. Immunoelectron microscopy revealed that HSP70 in these cells is located in the perinuclear area and in mitochondria, rough endoplasmic reticulum, and microtubules. In neurons, strong immunolabeling was also observed in synaptic membranes. The postnatal time course of HSP70 levels and the location and size of HSP70-immunopositive cells suggest that HSP70 constitutively expressed in the rat CNS may be mainly determined by the degree of development and metabolic activity of the neural cells.

Animals↗
PubMed1987

Constitutive and induced synthesis of heat shock proteins in transplantable hepatomas.

The synthesis of heat shock proteins (HSP) was studied in rat liver and in a series of transplantable Morris hepatomas with different growth rates, subjected to heat shock in vivo and in vitro. Different from the liver, hepatomas synthesized HSP constitutively, i.e., also before exposure to heat. This constitutive synthesis was low and limited to one HSP in the slowest-growing tumor, more marked and involving other HSP in the intermediate- and fast-growing hepatomas. In tumor that synthesized HSP constitutively, the induction of HSP in response to heat was proportionately reduced. These patterns of reaction were essentially similar in vivo ad in vitro. The amount of HSP 68 was well correlated to the levels of its mRNA in liver and in all hepatomas, whereas the increase in HSP 89 was accompanied by a corresponding increase in the related mRNA in liver and in slow-growing hepatoma, not in the other tumors, thus suggesting a different mechanism of control of HSP 89 synthesis in the more malignant hepatomas.

Animals↗
PubMed2004

Constitutive STAT6 activation in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMBL), currently recognized as a diffuse large B-cell lymphoma (DLBCL) subtype, shows increased expression of interleukin 4 (IL-4)/IL-13 signaling effectors and targets, suggesting constitutive activation of these pathways. We therefore investigated the functional state of the signal transducer and activator of transcription 6 (STAT6), mediating IL-4/IL-13 transcriptional effects. Constitutive STAT6 phosphorylation and DNA-binding activity were detected in PMBL cell lines but not DLBCL cell lines. Moreover, immunohistochemical analysis revealed nuclear phosphorylated STAT6 (P-STAT6) in 8 of 11 PMBL, compared with 1 of 10 DLBCL primary tumors (P =.01). IL-4 and IL-13 transcripts were absent in PMBL cell lines and expressed at low levels in tumors, indicating that, contrary to classical Hodgkin lymphoma (cHL), STAT6 activation is not due to an autocrine IL-4/IL-13 secretion. We demonstrated an amplification of the JAK2 gene in 2 of 6 PMBL cases, and showed higher JAK2 mRNA levels in PMBL compared with DLBCL (P =.005). The Janus kinase 2 (JAK2) was constitutively phosphorylated in the PMBL MedB1 cell line. MedB1 treatment with JAK2 inhibitor AG490 partially decreased STAT6 phosphorylation, suggesting that JAK2 is partially involved in STAT6 activation in these cells. Our findings highlight phosphorylated STAT6 as a characteristic distinguishing PMBL from DLBCL, but a common feature to PMBL and cHL, supporting the hypothesis of common pathogenic events in these 2 lymphomas.

Cell Line, Tumor↗
PubMed2004

Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival.

Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukemogenesis, and their presence is associated with poor prognosis in acute myeloid leukemia (AML). To better understand FLT3 signaling in leukemogenesis, we have examined the changes in gene expression induced by FLT3/ITD or constitutively activated wild-type FLT3 expression. Microarrays were used with RNA harvested before and after inhibition of FLT3 signaling. Pim-1 was found to be one of the most significantly down-regulated genes upon FLT3 inhibition. Pim-1 is a proto-oncogene and is known to be up-regulated by signal transducer and activator of transcription 5 (STAT5), which itself is a downstream target of FLT3 signaling. Quantitative polymerase chain reaction (QPCR) confirmed the microarray results and demonstrated approximately 10-fold decreases in Pim-1 expression in response to FLT3 inhibition. Pim-1 protein also decreased rapidly in parallel with decreasing autophosphorylation activity of FLT3. Enforced expression of either the 44-kDa or 33-kDa Pim-1 isotypes resulted in increased resistance to FLT3 inhibition-mediated cytotoxicity and apoptosis. In contrast, expression of a dominant-negative Pim-1 construct accelerated cytotoxicity in response to FLT3 inhibition and inhibited colony growth of FLT3/ITD-transformed BaF3 cells. These findings demonstrate that constitutively activated FLT3 signaling up-regulates Pim-1 expression in leukemia cells. This up-regulation contributes to the proliferative and antiapoptotic pathways induced by FLT3 signaling.

Animals↗
PubMed2005

NF-kappaB constitutes a potential therapeutic target in high-risk myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a preneoplastic condition that frequently develops into overt acute myeloid leukemia (AML). The P39 MDS/AML cell line manifested constitutive NF-kappaB activation. In this cell line, NF-kappaB inhibition by small interfering RNAs specific for p65 or chemical inhibitors including bortezomib resulted in the down-regulation of apoptosis-inhibitory NF-kappaB target genes and subsequent cell death accompanied by loss of mitochondrial transmembrane potential as well as by the mitochondrial release of the caspase activator cytochrome c and the caspase-independent death effectors endonuclease G and apoptosis-inducing factor (AIF). Bone marrow cells from high-risk MDS patients also exhibited constitutive NF-kappaB activation similar to bone marrow samples from MDS/AML patients. Purified hematopoietic stem cells (CD34+) and immature myeloid cells (CD33+) from high-risk MDS patients demonstrated the nuclear translocation of the p65 NF-kappaB subunit. The frequency of cells with nuclear p65 correlated with blast counts, apoptosis suppression, and disease progression. NF-kappaB activation was confined to those cells that carried MDS-associated cytogenetic alterations. Since NF-kappaB inhibition induced rapid apoptosis of bone marrow cells from high-risk MDS patients, we postulate that NF-kappaB activation is responsible for the progressive suppression of apoptosis affecting differentiating MDS cells and thus contributes to malignant transformation. NF-kappaB inhibition may constitute a novel therapeutic strategy if apoptosis induction of MDS stem cells is the goal.

Active Transport, Cell Nucleus↗
PubMed2005

Down-regulating constitutive activation of the NF-kappaB canonical pathway overcomes the resistance of cutaneous T-cell lymphoma to apoptosis.

Constitutive activation of the nuclear factor-kappaB (NF-kappaB) pathway has been shown to be involved in the resistance of tumor cells to apoptosis in several human malignancies of the hematopoietic lineage. By using electrophoretic mobility shift assay (EMSA) and confocal microscopic analysis, we demonstrate that NF-kappaB is constitutively activated in cutaneous T-cell lymphoma (CTCL) cell lines HuT-78, MyLa, and SeAx and in peripheral blood lymphocytes (PBLs) from patients with Sézary syndrome (SS) presenting a high ratio of tumor cells, with evidence of p50 and RelA/p65 in DNA-linked complexes. Transfection of SeAx line with a kappaB/luciferase reporter plasmid showed that translocated NF-kappaB complexes were functional. Selective inhibition of NF-kappaB, by transfecting CTCL cell lines with a super-repressor form of IkappaB alpha, led to apoptosis. We evidenced down-regulation of NF-kappaB activation and induction of CTCL cell apoptosis in the presence of proteasome 26S inhibitors ALLN, MG132, and bortezomib. Bortezomib at nanomolar concentrations inhibited constitutive activation of NF-kappaB and induced apoptosis of CTCL cells, with evidence of an upregulation of Bax expression. These results demonstrate the key role played by NF-kappaB in the resistance of CTCL to apoptosis and suggest that bortezomib might be useful for the treatment of patients with advanced stages of CTCL refractory to standard antineoplastic chemotherapy.

Adult↗
PubMed2006

Constitutive NF-kappaB and NFAT activation leads to stimulation of the BLyS survival pathway in aggressive B-cell lymphomas.

B-lymphocyte stimulator (BLyS), a relatively recently recognized member of the tumor necrosis factor ligand family (TNF), is a potent cell-survival factor expressed in many hematopoietic cells. BLyS binds to 3 TNF-R receptors, TACI, BCMA, BAFF-R, to regulate B-cell survival, differentiation, and proliferation. The mechanisms involved in BLYS gene expression and regulation are still incompletely understood. In this study, we examined BLYS gene expression, function, and regulation in B-cell non-Hodgkin lymphoma (NHL-B) cells. Our studies indicate that BLyS is constitutively expressed in aggressive NHL-B cells, including large B-cell lymphoma (LBCL) and mantle cell lymphoma (MCL), playing an important role in the survival and proliferation of malignant B cells. We found that 2 important transcription factors, NF-kappaB and NFAT, are involved in regulating BLyS expression through at least one NF-kappaB and 2 NFAT binding sites in the BLYS promoter. We also provide evidence suggesting that the constitutive activation of NF-kappaB and BLyS in NHL-B cells forms a positive feedback loop associated with lymphoma cell survival and proliferation. Our findings indicate that constitutive NF-kappaB and NFAT activations are crucial transcriptional regulators of the BLyS survival pathway in malignant B cells that could be therapeutic targets in aggressive NHL-B.

Apoptosis Regulatory Proteins↗
PubMed2006

SALL4, a novel oncogene, is constitutively expressed in human acute myeloid leukemia (AML) and induces AML in transgenic mice.

SALL4, a human homolog to Drosophila spalt, is a novel zinc finger transcriptional factor essential for development. We cloned SALL4 and its isoforms (SALL4A and SALL4B). Through immunohistochemistry and real-time reverse-transcription-polymerase chain reaction (RT-PCR), we demonstrated that SALL4 was constitutively expressed in human primary acute myeloid leukemia (AML, n = 81), and directly tested the leukemogenic potential of constitutive expression of SALL4 in a murine model. SALL4B transgenic mice developed myelodysplastic syndrome (MDS)-like features and subsequently AML that was transplantable. Increased apoptosis associated with dysmyelopoiesis was evident in transgenic mouse marrow and colony-formation (CFU) assays. Both isoforms could bind to beta-catenin and synergistically enhanced the Wnt/beta-catenin signaling pathway. Our data suggest that the constitutive expression of SALL4 causes MDS/AML, most likely through the Wnt/beta-catenin pathway. Our murine model provides a useful platform to study human MDS/AML transformation, as well as the Wnt/beta-catenin pathway's role in the pathogenesis of leukemia stem cells.

Alternative Splicing↗
PubMed2001

Constitutive NF-kappaB maintains high expression of a characteristic gene network, including CD40, CD86, and a set of antiapoptotic genes in Hodgkin/Reed-Sternberg cells.

Constitutively activated nuclear factor (NF)-kappaB is observed in a variety of neoplastic diseases and is a hallmark of the malignant Hodgkin and Reed-Sternberg cells (H/RS) in Hodgkin lymphoma. Given the distinctive role of constitutive NF-kappaB for H/RS cell viability, NF-kappaB-dependent target genes were searched for by using adenoviral expression of the super-repressor IkappaBDeltaN. A surprisingly small but characteristic set of genes, including the cell-cycle regulatory protein cyclin D2, the antiapoptotic proteins Bfl-1/A1, c-IAP2, TRAF1, and Bcl-x(L), and the cell surface receptors CD86 and CD40 were identified. Thus, constitutive NF-kappaB activity maintains expression of a network of genes, which are known for frequent, marker-like expression in primary or cultured H/RS cells. Intriguingly, CD40, which is able to activate CD86 or Bcl-x(L) via NF-kappaB, is itself transcriptionally regulated by NF-kappaB through a promoter proximal binding site. NF-kappaB inhibition resulted in massive spontaneous and p53-independent apoptosis, which could be rescued by ectopic expression of Bcl-x(L), underscoring its dominant role in survival of H/RS cells. Hence, NF-kappaB controls a signaling network in H/RS cells, which promotes tumor cell growth and confers resistance to apoptosis.

Antigens, CD↗
PubMed2001

STAT3 is constitutively activated in Hodgkin cell lines.

Hodgkin disease (HD) represents a malignant lymphoma in which the putative malignant Hodgkin and Reed-Sternberg cells are rare and surrounded by abundant reactive nonmalignant cells. It has been suggested that cytokines such as interleukin-6 (IL-6) are involved in the pathogenesis of the disease. The expression of the IL-6 receptor (IL-6R) complex and its link to the activation of signal transducers and activators of transcription (STAT) molecules in HD cell lines was investigated. Gel retardation and Western blot analyses revealed a high level of constitutively activated STAT3 in 5 of 7 HD cell lines, which could not be detected in Burkitt lymphoma cell lines. Different levels of IL-6R protein were measured in various HD cell lines: L428 and Dev cells were characterized by very low levels of gp80 and gp130, on KMH2 cells only gp130 but no gp80 was detected, whereas L540, L591, HDLM2, and L1236 were positive for both gp80 and gp130, suggesting a possible autocrine stimulation of STAT3. However, a further increase in STAT3 activation on IL-6 or IL-6/soluble IL-6R stimulation was not observed. Neutralizing monoclonal antibodies against IL-6, gp80, gp130, or both receptor subunits did not affect the proliferation or the constitutive activation of STAT molecules in HD cell lines. However, the tyrosine kinase inhibitor AG490 blocked the constitutive activation of STAT3 and inhibited spontaneous growth of HD tumor cells. The evidence suggests abnormal STAT signaling and growth regulation in Hodgkin cell lines. (Blood. 2001;98:762-770)

Antibodies, Monoclonal↗
PubMed2002

Spontaneous interleukin-5 production in cutaneous T-cell lymphoma lines is mediated by constitutively activated Stat3.

Mycosis fungoides is a low-grade cutaneous T-cell lymphoma (CTCL) of unknown etiology. In advanced stages of CTCL, a shift in cytokine profile from T(H)1 to T(H)2 is observed, which coincides with eosinophilia, high levels of immunoglobulin E, and increased susceptibility to bacterial infections. It is, however, unknown why T(H)2 cytokines predominate in advanced CTCL, and the cellular source of these cytokines also remains to be identified. In several leukemias and lymphomas, constitutively activated signal transducer and activator of transcription (Stat) signaling pathways have been detected. In a previous study, constitutive activation of Stat3 was found in tumor cells isolated from affected skin and blood from CTCL patients. Here, it is shown that CTCL tumor cell lines, but not nonmalignant cell lines, spontaneously produce interleukin-5 (IL-5), IL-6, and IL-13. Transfection of tumor cells with dominant-negative Stat3 almost completely blocks IL-5 production and strongly inhibits IL-13 production, whereas IL-6 production is unaffected. Thus, the data show that malignant CTCL cells themselves might contribute to the change in cytokine pattern accompanying progression of CTCL. In conclusion, constitutively activated Stat3 is found to mediate a spontaneous IL-5 production and regulate IL-13 production in CTCL cell lines, pointing toward a new role of Stat3 in malignant transformation.

Cytokines↗
PubMed2002

Etanercept, a soluble tumor necrosis factor receptor, palliates constitutional symptoms in patients with myelofibrosis with myeloid metaplasia: results of a pilot study.

Patients with myelofibrosis with myeloid metaplasia (MMM) often experience debilitating constitutional symptoms such as drenching night sweats, profound fatigue, unexplained fevers, and unintentional weight loss. Tumor necrosis factor (TNF) contributes to organ fibrosis and hypercatabolic symptoms in a variety of disease states. We conducted an open-label pilot study of etanercept, a soluble TNF receptor, administered at a dose of 25 mg subcutaneously twice weekly for up to 24 weeks in 22 patients with MMM. Of 20 evaluable patients, 12 (60%) experienced an improvement in constitutional symptoms, and 4 (20%) had an objective response (improvement in peripheral cytopenias or spleen size). The degree of marrow fibrosis was unchanged, and only minor changes in overall marrow cellularity were observed. Toxicity was mild, with injection site reactions (20%) and minor infections (10%) as the most common side effects. One patient developed reversible pancytopenia. Etanercept may be useful for palliation of constitutional symptoms in MMM.

Aged↗
PubMed2007

A strong constitutive ethylene-response phenotype conferred on Arabidopsis plants containing null mutations in the ethylene receptors ETR1 and ERS1.

BACKGROUND: The ethylene receptor family of Arabidopsis consists of five members, falling into two subfamilies. Subfamily 1 is composed of ETR1 and ERS1, and subfamily 2 is composed of ETR2, ERS2, and EIN4. Although mutations have been isolated in the genes encoding all five family members, the only previous insertion allele of ERS1 (ers1-2) is a partial loss-of-function mutation based on our analysis. The purpose of this study was to determine the extent of signaling mediated by subfamily-1 ethylene receptors through isolation and characterization of null mutations. RESULTS: We isolated new T-DNA insertion alleles of subfamily 1 members ERS1 and ETR1 (ers1-3 and etr1-9, respectively), both of which are null mutations based on molecular, biochemical, and genetic analyses. Single mutants show an ethylene response similar to wild type, although both mutants are slightly hypersensitive to ethylene. Double mutants of ers1-3 with etr1-9, as well as with the previously isolated etr1-7, display a constitutive ethylene-response phenotype more pronounced than that observed with any previously characterized combination of ethylene receptor mutations. Dark-grown etr1-9;ers1-3 and etr1-7;ers1-3 seedlings display a constitutive triple-response phenotype. Light-grown etr1-9;ers1-3 and etr1-7;ers1-3 plants are dwarfed, largely sterile, exhibit premature leaf senescence, and develop novel filamentous structures at the base of the flower. A reduced level of ethylene response was still uncovered in the double mutants, indicating that subfamily 2 receptors can independently contribute to signaling, with evidence suggesting that this is due to their interaction with the Raf-like kinase CTR1. CONCLUSION: Our results are consistent with the ethylene receptors acting as redundant negative regulators of ethylene signaling, but with subfamily 1 receptors playing the predominant role. Loss of a single member of subfamily 1 is largely compensated for by the activity of the other member, but loss of both subfamily members results in a strong constitutive ethylene-response phenotype. The role of subfamily 1 members is greater than previously suspected and analysis of the double mutant null for both ETR1 and ERS1 uncovers novel roles for the receptors not previously characterized.

Arabidopsis↗
PubMed2003

Expression of constitutively active 4EBP-1 enhances p27Kip1 expression and inhibits proliferation of MCF7 breast cancer cells.

BACKGROUND: Eukaryotic initiation factor 4E (eIF4E) is essential for cap-dependent initiation of translation. Cell proliferation is associated with increased activity of eIF4E and elevated expression of eIF4E leads to tumorigenic transformation. Many tumors express very high levels of eIF4E and this may be a critical factor in progression of the disease. In contrast, overexpression of 4EBP, an inhibitor of eIF4E, leads to cell cycle arrest and phenotypic reversion of some transformed cells. RESULTS: A constitutively active form of 4EBP-1 was inducibly expressed in the human breast cancer cell line MCF7. Induction of constitutively active 4EBP-1 led to cell cycle arrest. This was not associated with a general inhibition of protein synthesis but rather with changes in specific cell cycle regulatory proteins. Cyclin D1 was downregulated while levels of the CDK inhibitor p27Kip1 were increased. The levels of cyclin E and CDK2 were unaffected but the activity of CDK2 was significantly reduced due to increased association with p27Kip1. The increase in p27Kip1 did not reflect changes in p27Kip1 mRNA or degradation rates. Rather, it was associated with enhanced synthesis of the protein, even though 4EBP-1 is expected to inhibit translation. This could be explained, at least in part, by the ability of the p27Kip1 5'-UTR to mediate cap-independent translation, which was also enhanced by expression of constitutively active 4EBP-1. CONCLUSIONS: Expression of active 4EBP-1 in MCF7 leads to cell cycle arrest which is associated with downregulation of cyclin D1 and upregulation of p27Kip1. Upregulation of p27Kip1reflects increased synthesis which corresponds to enhanced cap-independent translation through the 5'-UTR of the p27Kip1 mRNA.

Journal Article↗