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ABSTRACT: The polycomb group protein BMI1 has been shown to support normal stem cell proliferation via its putative stem cell factor function, but it is not known if BMI1 may also act as a cancer stem cell factor to promote cancer development. To determine the role of human BMI1 in cancer growth and survival, we performed a loss-of-function analysis of BMI1 by RNA interference (RNAi) in both normal and malignant human cells. Our results indicate that BMI1 is crucial for the short-term survival of cancer cells but not of normal cells. We also demonstrated that loss of BMI1 was more effective in suppressing cancer cell growth than retinoid-treatment, and surviving cancer cells showed significantly reduced tumorigenicity. The cancer-specific growth retardation was mediated by an increased level of apoptosis and a delayed cell cycle progression due to the loss of BMI1. By comparison, BMI1 deficiency caused only a moderate inhibition of the cell cycle progression in normal lung cells. In both normal and cancer cells, the loss of BMI1 led to an upregulation of INK4A-ARF, but with no significant effect on the level of telomerase gene expression, suggesting that other BMI1-cooperative factors in addition to INK4A-ARF activation may be involved in the BMI1-dependent cancer-specific growth retardation. Thus, human BMI1 is critical for the short-term survival of cancer cells, and inhibition of BMI1 has minimal effect on the survival of normal cells. These findings provide a foundation for developing a cancer-specific therapy targeting BMI1.
Oncogene 08/2006; 25(31):4370-5. · 6.37 Impact Factor
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ABSTRACT: Current standard cancer therapies (chemotherapy and radiation) often cause serious adverse off-target effects. Drug design strategies are therefore being developed that will more precisely target cancer cells for destruction while leaving surrounding normal cells relatively unaffected. Telomerase, widely expressed in most human cancers but almost undetectable in normal somatic cells, provides an exciting drug target. This review focuses on recent pharmacogenomic approaches to telomerase inhibition. Antisense oligonucleotides, RNA interference, ribozymes, mutant expression, and the exploitation of differential telomerase expression as a strategy for targeted oncolysis are discussed here in the context of cancer therapeutics. Reports of synergism between telomerase inhibitors and traditional cancer therapeutic agents are also analyzed.
Current Medicinal Chemistry 02/2006; 13(24):2875-88. · 4.86 Impact Factor
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ABSTRACT: Although telomerase, which maintains the ends of chromosomes, is down-regulated as cells differentiate leading to attrition of chromosomal termini and ultimate replicative senescence, it is up-regulated in most cancer cells which show no net loss of average telomere length. The mRNA level of the catalytic component of telomerase, hTERT, is the major determinant of telomerase activity but little is known about control of hTERT transcription. We propose mechanisms whereby cytosine methylation may alter the binding of activators such as c-Myc or repressors such as WT1 which interact with the hTERT gene regulatory region to modulate telomerase activity in aging cells and tumorigenesis. Mechanisms are also proposed for control of hTERT expression through changes in the collective binding of its transcription factors in aging and tumorigenic cells. Elucidation of telomerase regulation should facilitate advances in understanding age-related diseases such as cancer and in potential therapeutic modalities.
Medical Hypotheses 07/2001; 56(6):630-7. · 1.39 Impact Factor
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ABSTRACT: Recent interest in the regulation of telomerase, the enzyme that maintains chromosomal termini, has lead to the discovery and characterization of the catalytic subunit of telomerase, hTERT. Many studies have suggested that the transcription of hTERT represents the rate-limiting step in telomerase expression and key roles for hTERT have been implied in cellular aging, immortalization, and transformation. Before the characterization of the promoter of hTERT in 1999, regulatory mechanisms suggested for this gene were limited to speculation. The successful cloning and characterization of the hTERT 5' gene regulatory region has enabled its formal investigation and analysis of potential mechanisms controlling hTERT expression. Although these studies have provided important information about hTERT gene regulation, there has been some confusion regarding the nucleotide boundaries of this region, the location, number, and importance of various transcription factor binding motifs, and the results of promoter activity assays. We feel that this uncertainty, combined with the sheer volume of recent publications on hTERT regulation, calls for consolidation and review. In this analysis we examine recent advances in the regulation of the hTERT gene and attempt to resolve discrepancies resulting from the nearly simultaneous nature of publications in this fast-moving area. Additionally, we aim to summarize the extant knowledge of hTERT gene regulation and its role in important biological processes such as cancer and aging.
Gene 06/2001; 269(1-2):1-12. · 2.34 Impact Factor
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Methods in molecular biology (Clifton, N.J.) 02/1999; 118:233-44.
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ABSTRACT: 3T3-L1 preadipocytes ectopically expressing the mammalian RNA-binding protein Hel-N1 expressed up to 10-fold more glucose transporter (GLUT1) protein and exhibited elevated rates of basal glucose uptake. Hel-N1 is a member of the ELAV-like family of proteins associated with the induction and maintenance of differentiation in various species. ELAV proteins are known to bind in vitro to short stretches of uridylates in the 3' untranslated regions (3'UTRs) of unstable mRNAs encoding growth-regulatory proteins involved in transcription and signal transduction. GLUT1 mRNA also contains a large 3'UTR with a U-rich region that binds specifically to Hel-N1 in vitro. Analysis of the altered GLUT1 expression at the translational and posttranscriptional levels suggested a mechanism involving both mRNA stabilization and accelerated formation of translation initiation complexes. These findings are consistent with the hypothesis that the Hel-N1 family of proteins modulate gene expression at the level of mRNA in the cytoplasm.
Molecular and Cellular Biology 03/1997; 17(2):954-62. · 5.53 Impact Factor
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ABSTRACT: Purine nucleoside phosphorylase deficiency is an inherited disease of purine metabolism characterized clinically as combined immunodeficiency. The molecular defects have been published for 4 different alleles in 3 patients. We report four new mutations including two amino acid substitutions, A174P and G190V, a single codon deletion, delta I129, and a point mutation in intron 3 which leads to aberrant splicing and creation of a premature stop codon in exon 4 (286-18G-->A). Of the previously reported mutations, E89K was found in one additional patient, and R234P was found in 3 unrelated patients, making R234P the most common mutation reported to date in this disease.
Human Mutation 02/1997; 9(2):118-21. · 5.69 Impact Factor
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ABSTRACT: Hel-N1, is an RRM protein which is a mammalian homologue of the Drosophila melanogaster RNA binding protein, ELAV (embryonic lethal abnormal vision). Hel-N1 binds to RNA containing short stretches of uridylates similar to those found in the 3' untranslated regions (3'-UTRs) of oncoprotein and cytokine mRNAs. The GLUT1 glucose transporter has an extensive 3' UTR that is AU-rich reminiscent of the 3'UTR of an oncogene mRNA. An in vitro RNA binding assay using Hel-N1 demonstrated binding to a specific portion of the GLUT1 3'UTR. Analysis of the folding pattern of this region depicted the retention of a stem loop structure, wherein the loop is composed of a stretch of uridylates. To further analyze the potential function of Hel-N1, stable transfectants were made in the 3T3-L1 cell line. The transfectants have been characterized, and the presence of the Hel-N1 DNA and protein verified. Data indicate Hel-N1 is a ligand for GLUT1 and its binding affects the stability and translatability of the GLUT1 message.
Nucleic Acids Symposium Series 02/1995;
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ABSTRACT: Gene silencing is often mediated by CpG methylation of key protein binding sites within gene regulatory sequences (GRSs). An aging mechanism is proposed based on this gene-silencing phenomenon whereby accumulation over time of methylation within GRSs contributes to cellular senescence. The proposed molecular mechanism for age-related gene silencing is the spreading of methylation through the regulatory sequences of genes resulting in progressive reduction of gene transcription. There is considerable experimental evidence for methylation spreading and its role in gene silencing, but the mechanism responsible for this process has not been elucidated. A four-step mechanism is proposed whereby an original methylation occurs, methyltransferase (MTase) molecules progressively move 5' to 3' from this site, neighboring CpG dinucleotides become methylated, and diminished gene expression ensues. Over time, this process may lead to widespread gene silencing in diverse dividing and nondividing cell types contributing to aging of the organism.
Medical Hypotheses 08/1993; 41(1):83-92. · 1.39 Impact Factor
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ABSTRACT: We have investigated the RNA binding specificity of Hel-N1, a human neuron-specific RNA-binding protein, which contains three RNA recognition motifs. Hel-N1 is a human homolog of Drosophila melanogaster elav, which plays a vital role in the development of neurons. A random RNA selection procedure revealed that Hel-N1 prefers to bind RNAs containing short stretches of uridylates similar to those found in the 3' untranslated regions (3' UTRs) of oncoprotein and cytokine mRNAs such as c-myc, c-fos, and granulocyte macrophage colony-stimulating factor. Direct binding studies demonstrated that Hel-N1 bound and formed multimers with c-myc 3' UTR mRNA and required, as a minimum, a specific 29-nucleotide stretch containing AUUUG, AUUUA, and GUUUUU. Deletion analysis demonstrated that a fragment of Hel-N1 containing 87 amino acids, encompassing the third RNA recognition motif, forms an RNA binding domain for the c-myc 3' UTR. In addition, Hel-N1 was shown to be reactive with autoantibodies from patients with paraneoplastic encephalomyelitis both before and after binding to c-myc mRNA.
Molecular and Cellular Biology 07/1993; 13(6):3494-504. · 5.53 Impact Factor
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ABSTRACT: Purine nucleoside phosphorylase (PNP) deficiency is an inherited autosomal recessive disorder resulting in severe combined immunodeficiency. The purpose of this study was to determine the molecular defects responsible for PNP deficiency in one such patient. The patient's PNP cDNA was amplified by PCR and sequenced. Point mutations leading to amino acid substitutions were found in both alleles. One point mutation led to a Ser-to-Gly substitution at amino acid 51 and was common to both alleles. In addition, an Asp-to-Gly substitution at amino acid 128 and an Arg-to-Pro substitution at amino acid 234 were found in the maternal and paternal alleles, respectively. In order to prove that these mutations were responsible for the disease state, each of the three mutations was constructed separately by site-directed mutagenesis of the normal PNP cDNA, and each was transiently expressed in COS cells. Lysates from cells transfected with the allele carrying the substitution at amino acid 51 retained both function and immunoreactivity. Lysates from cells transfected with PNP alleles carrying a substitution at either amino acid 128 or amino acid 234 contained immunoreactive material but had no detectable human PNP activity. In summary, molecular analysis of this patient identified point mutations within the PNP gene which are responsible for the enzyme deficiency.
The American Journal of Human Genetics 11/1992; 51(4):763-72. · 10.60 Impact Factor
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ABSTRACT: We report a defect in splicing of precursor messenger RNA (pre-mRNA) resulting from a naturally occurring mutation of the gene encoding purine nucleoside phosphorylase (PNP) in a patient with PNP-deficient severe combined immunodeficiency. This defects results from a G to T transversion at the terminal nucleotide of exon 2 within the 5' splice site of intron 2 and causes skipping of exon 2 during processing of PNP pre-mRNA. Translation of the misspliced mRNA results in a reading frameshift at the exon 1-exon 3 junction. The predicted polypeptide encoded by the aberrant mRNA is severely truncated, terminating at 31 amino acids. Only 4 residues at the NH2 terminus of the polypeptide correspond to PNP amino acids. Otherwise the translation product of the misspliced mRNA differs completely from PNP in amino acid sequence and has no PNP activity. The finding of exon skipping in PNP is the first report of a splicing defect resulting in PNP-deficient severe combined immunodeficiency. Analysis of the genomic context of the G-1 to T mutation of the 5' splice site lends support for the exon definition model of pre-mRNA splicing and contributes to the understanding of splice site selection.
Journal of Biological Chemistry 05/1992; 267(11):7834-8. · 4.77 Impact Factor
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ABSTRACT: Gene silencing is often mediated by CpG methylation of key protein binding sites within gene regulatory sequences (GRSs). An aging mechanism is proposed based on this gene-silencing phenomenon whereby accumulation over time of methylation within GRSs contributes to cellular senescence. The proposed molecular mechanism for age-related gene silencing is the spreading of methylation through the regulatory sequences of genes resulting in progressive reduction of gene transcription. There is considerable experimental evidence for methylation spreading and its role in gene silencing, but the mechanism responsible for this process has not been elucidated. A four-step mechanism is proposed whereby an original methylation occurs, methyltransferase (MTase) molecules progressively move 5′ to 3′ from this site, neighboring CpG dinucleotides become methylated, and diminished gene expression ensues. Over time, this process may lead to widespread gene silencing in diverse dividing and nondividing cell types contributing to aging of the organism.
Medical Hypotheses.
