9-(2-Phosphonylmethoxyethyl)adenine induces tumor cell differentiation or cell death by blocking cell cycle progression through the S phase.
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In addition to its inhibitory activity against viral DNA polymerases and reverse transcriptase, the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA) also markedly inhibits the replicative cellular DNA polymerases alpha, delta, and epsilon. We have previously shown that PMEA is a strong inducer of differentiation in several in vitro tumor cell models and has marked antitumor potential in vivo. To elucidate the molecular mechanism of the differentiation-inducing activity of PMEA, we have now investigated the effects of the drug on cell proliferation and differentiation, cell cycle regulation, and oncogene expression in the human erythroleukemia K562 cell line. Terminal, irreversible erythroid differentiation of PMEA-treated K562 cells was evidenced by hemoglobin production, increased expression of glycophorin A on the K562 cell membrane, and induction of acetylcholinesterase activity. After exposure to PMEA, K562 cell cultures displayed a marked retardation of S-phase progression, leading to a severe perturbation of the normal cell cycle distribution pattern. Whereas no substantial changes in c-myc mRNA levels and p21, PCNA, cdc2, and CDK2 protein levels were noted in PMEA-treated K562 cells, there was a marked accumulation of cyclin A and, most strikingly, cyclins E and B1. A similar picture of cell cycle deregulation was also observed in PMEA-exposed human myeloid THP-1 cells. However, in contrast to the strong differentiation-inducing activity of PMEA in K562 cells, the drug completely failed to induce monocytic maturation of human myeloid THP-1 cells. On the contrary, THP-1 cells underwent apoptotic cell death in the presence of PMEA, as demonstrated by prelytic, intracellular DNA fragmentation and the binding of annexin V to the cell surface. We hypothesize that, depending on the nature of the tumor cell line, PMEA triggers a process of either differentiation or apoptosis by the uncoupling of normally integrated cell cycle processes through inhibition of DNA replication during the S phase. (+info)
SIAH-1 promotes apoptosis and tumor suppression through a network involving the regulation of protein folding, unfolding, and trafficking: identification of common effectors with p53 and p21(Waf1).
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We have previously described biological model systems for studying tumor suppression in which, by using H-1 parvovirus as a selective agent, cells with a strongly suppressed malignant phenotype (KS or US) were derived from malignant cell lines (K562 or U937). By using cDNA display on the K562/KS cells, 15 cDNAs were now isolated, corresponding to genes differentially regulated in tumor suppression. Of these, TSAP9 corresponds to a TCP-1 chaperonin, TSAP13 to a regulatory proteasome subunit, and TSAP21 to syntaxin 11, a vesicular trafficking molecule. The 15 cDNAs were used as a molecular fingerprint in different tumor-suppression models. We found that a similar pattern of differential regulation is shared by activation of p53, p21(Waf1), and the human homologue of Drosophila seven in absentia, SIAH-1. Because SIAH-1 is differentially expressed in the various models, we characterized it at the protein and functional levels. The 32-kDa, mainly nuclear protein encoded by SIAH-1, can induce apoptosis and promote tumor suppression. These results suggest the existence of a common mechanism of tumor suppression and apoptosis shared by p53, p21(Waf1), and SIAH-1 and involving regulation of the cellular machinery responsible for protein folding, unfolding, and trafficking. (+info)
3'-Azido-3'-deoxythymidine reduces the rate of transferrin receptor endocytosis in K562 cells.
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K562 cells, exposed for at least 24 h to 5 microM 3'-azido-3'-deoxythymidine (AZT), gave rise to an overall increase in the number of cell surface transferrin binding receptors (18-20%). This effect was ascertained either with binding experiments by using 125I-transferrin and with immunoprecipitation by using a specific monoclonal antibody against the transferrin receptor. At higher AZT concentrations (20 and 40 microM), a further increase was found, that is, up to 23% by binding experiments and up to 110% by immunoprecipitation. However, Scatchard analysis of the binding data indicated that although the number of cell surface transferrin receptors increased, the affinity of transferrin for its receptor did not change (Ka=4.0x108 M). Surprisingly, immunoprecipitation of total receptor molecules showed that the synthesis of receptor was not enhanced by the drug treatment. The effect of AZT on transferrin internalization and receptor recycling was also investigated. In this case, data indicated that the increase in the number of receptors at the cell surface was probably due to a slowing down of endocytosis rate rather than to an increased recycling rate of the receptor to cell surface. In fact, the time during which half the saturated amount of transferrin had been endocytosed (t1/2) was 2.15 min for control cells and 3.41, 3.04, and 3.74 min for 5, 20, and 40 microM AZT-treated cells, respectively. Conversely, recycling experiments did not show any significant differences between control and treated cells. A likely mechanism through which AZT could interfere with the transferrin receptor trafficking, together with the relevance of our findings, is extensively discussed. (+info)
LightCycler technology for the quantitation of bcr/abl fusion transcripts.
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Quantifying bcr/abl fusion transcripts in chronic myelogenous leukemia is thought to serve as a powerful parameter for monitoring the kinetic nature of this clonal disease in vivo and in vitro. Recently, we demonstrated the technical advantages as well as the clinical relevance of quantitating bcr/abl fusion mRNA using the 5-nuclease assay and a real-time fluorescence reverse transcriptase-PCR (RT-PCR) detection system (ABI PRISM 7700 SDS). Meanwhile, another technique was introduced (LightCycler technology) that may be used for the same purpose. To investigate whether this method may be an appropriate alternative to the described procedure, we have established bcr/abl LightCycler RT-PCR for major and minor bcr/abl fusion transcripts. We found that, with only minor modifications, TaqMan RT-PCR and fluorescent probe design can be used to obtain comparable results in the LightCycler system. The developed method could quantitate as little as 10 bcr/abl copies per 100 ng cDNA and was as safe and reproducible as the previously described technique. Because reaction efficiency was identical within different bcr/abl major fusions, one single RT-PCR could be established that simultaneously detects b2a3, b2a2, b3a2, and b3a3 fusion RNA with equal specificity and sensitivity. Compared to results generated by the ABI PRISM 7700 SDS, absolute amounts of bcr/abl did not differ significantly, and there was a linear correlation between the respective values. We conclude that TaqMan chemistry can be used in the LightCycler and that both real-time fluorescence PCR detection systems equally fulfill the criteria for the safe and reliable quantitation of bcr/abl fusion RNA in clinical samples. This may be of help for further standardization of quantitative bcr/abl RT-PCR, which, again, is necessary for the comparison of results generated by different investigators. (+info)
Molecular cloning of transferrin receptor 2. A new member of the transferrin receptor-like family.
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Transferrin receptor (TfR) plays a major role in cellular iron uptake through binding and internalizing a carrier protein transferrin (Tf). We have cloned, sequenced, and mapped a human gene homologous to TfR, termed TfR2. Two transcripts were expressed from this gene: alpha (approximately 2.9 kilobase pairs), and beta (approximately 2.5 kilobase pairs). The predicted amino acid sequence revealed that the TfR2-alpha protein was a type II membrane protein and shared a 45% identity and 66% similarity in its extracellular domain with TfR. The TfR2-beta protein lacked the amino-terminal portion of the TfR2-alpha protein including the putative transmembrane domain. Northern blot analysis showed that the alpha transcript was predominantly expressed in the liver. In addition, high expression occurred in K562, an erythromegakaryocytic cell line. To analyze the function of TfR2, Chinese hamster ovary TfR-deficient cells (CHO-TRVb cells) were stably transfected with FLAG-tagged TfR2-alpha. These cells showed an increase in biotinylated Tf binding to the cell surface, which was competed by nonlabeled Tf, but not by lactoferrin. Also, these cells had a marked increase in Tf-bound (55)Fe uptake. Taken together, TfR2-alpha may be a second transferrin receptor that can mediate cellular iron transport. (+info)
Differentiation-induced internal translation of c-sis mRNA: analysis of the cis elements and their differentiation-linked binding to the hnRNP C protein.
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In previous reports we showed that the long 5' untranslated region (5' UTR) of c-sis, the gene encoding the B chain of platelet-derived growth factor, has translational modulating activity due to its differentiation-activated internal ribosomal entry site (D-IRES). Here we show that the 5' UTR contains three regions with a computer-predicted Y-shaped structure upstream of an AUG codon, each of which can confer some degree of internal translation by itself. In nondifferentiated cells, the entire 5' UTR is required for maximal basal IRES activity. The elements required for the differentiation-sensing ability (i.e., D-IRES) were mapped to a 630-nucleotide fragment within the central portion of the 5' UTR. Even though the region responsible for IRES activation is smaller, the full-length 5' UTR is capable of mediating the maximal translation efficiency in differentiated cells, since only the entire 5' UTR is able to confer the maximal basal IRES activity. Interestingly, a 43-kDa protein, identified as hnRNP C, binds in a differentiation-induced manner to the differentiation-sensing region. Using UV cross-linking experiments, we show that while hnRNP C is mainly a nuclear protein, its binding activity to the D-IRES is mostly nuclear in nondifferentiated cells, whereas in differentiated cells such binding activity is associated with the ribosomal fraction. Since the c-sis 5' UTR is a translational modulator in response to cellular changes, it seems that the large number of cross-talking structural entities and the interactions with regulated trans-acting factors are important for the strength of modulation in response to cellular changes. These characteristics may constitute the major difference between strong IRESs, such as those seen in some viruses, and IRESs that serve as translational modulators in response to developmental signals, such as that of c-sis. (+info)
Enhancement of beta-globin locus control region-mediated transactivation by mitogen-activated protein kinases through stochastic and graded mechanisms.
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Activation of the mitogen-activated protein kinase (MAPK) pathway enhances long-range transactivation by the beta-globin locus control region (LCR) (W. K. Versaw, V. Blank, N. M. Andrews, and E. H. Bresnick, Proc. Natl. Acad. Sci. USA 95:8756-8760, 1998). The enhancement requires tandem recognition sites for the hematopoietic transcription factor NF-E2 within the hypersensitive site 2 (HS2) subregion of the LCR. To distinguish between mechanisms of induction involving the activation of silent promoters or the increased efficacy of active promoters, we analyzed basal and MAPK-stimulated HS2 enhancer activity in single, living cells. K562 erythroleukemia cells stably transfected with constructs containing the human Agamma-globin promoter linked to an enhanced green fluorescent protein (EGFP) reporter, with or without HS2, were analyzed for EGFP expression by flow cytometry. When most cells in a population expressed EGFP, MAPK augmented the activity of active promoters. However, under conditions of silencing, in which cells reverted to a state with no measurable EGFP expression, MAPK activated silent promoters. Furthermore, studies of populations of EGFP-expressing and non-EGFP-expressing cells isolated by flow cytometry showed that MAPK activation converted nonexpressing cells into expressing cells and increased expression in expressing cells. These results support a model in which MAPK elicits both graded and stochastic responses to increase HS2-mediated transactivation from single chromatin templates. (+info)
Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells.
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The chronic myelogenous leukemic K562 cell line carrying Bcr-Abl tyrosine kinase is considered as pluripotent hematopoietic progenitor cells expressing markers for erythroid, granulocytic, monocytic, and megakaryocytic lineages. Here we investigated the signaling modulations required for induction of erythroid differentiation of K562 cells. When the K562 cells were treated with herbimycin A (an inhibitor of protein tyrosine kinase), ras antisense oligonucleotide, and PD98059 (a specific inhibitor of MEK), inhibition of ERK/MAPK activity and cell growth, and induction of erythroid differentiation were observed. The ras mutant, pZIPRas61leu-transfected cells, K562-Ras61leu, have shown a markedly decreased cell proliferation rate with approximately 2-fold doubling time, compared with the parental K562 cells, and about 60% of these cells have shown the phenotype of erythroid differentiation. In addition, herbimycin A inhibited the growth rate and increased the erythroid differentiation, but did not affect the elevated activity of ERK/MAPK in the K562-Ras61leu cells. On the other hand, effects of PD98059 on the growth and differentiation of K562-Ras61leu cells were biphasic. At low concentration of PD98059, which inhibited the elevated activity of ERK/MAPK to the level of parental cells, the growth rate increased and the erythroid differentiation decreased slightly, and at high concentration of PD98059, which inhibited the elevated activity of ERK/MAPK below that of the parental cells, the growth rate turned down and the erythroid differentiation was restored to the untreated control level. Taken together, these results suggest that an appropriate activity of ERK/MAPK is required to maintain the rapid growth and transformed phenotype of K562 cells. (+info)