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Background: Multiple mechanisms regulate cancer-associated telomerase activity at the level of human telomerase reverse transcriptase (hTERT) transcription which may serve as novel targets for anticancer approaches., Materials and Methods: The effects of prolonged all-trans retinoic acid (ATRA) exposure on hTERT regulation in estrogen receptor-negative SK-BR-3 breast cancer cells were examined., Results: ATRA had a profound effect on the morphology and proliferation rate of the SK-BR-3 cells. ATRA also hindered the ability of these cancer cells to grow independently, rendering them more like normal somatic cells. The effect of ATRA on the decrease of telomerase activity was found to be associated with a rapid decrease in histone H3-lysine 9 acetylation (H3-K9-Ac) of the hTERT promoter. Extended-exposure to ATRA in these cells also caused the initiation of a putative compensatory mechanism, counteracting the induced surge in apoptosis., Conclusion: A rapid decrease of H3-K9 acetylation at the hTERT promoter could be an important mechanism by which ATRA shuts down telomerase activity and mediates its antitumor effects in estrogen receptor-negative breast cancer cells.

Telomerase, a ribonucleoprotein important to neoplastic immortality, is up-regulated in approximately 85% of cancers, including leukemias. In this study, 9cUAB30, a novel retinoic acid, resulted in differentiation of HL60 leukemia cells as indicated by morphologic changes characteristic of granulocytes. It also caused a down-regulation of hTERT gene expression and a decrease in telomerase activity. Telomerase inhibition was followed by loss of proliferative capacity, induction of apoptosis, and partial differentiation. These findings demonstrate the effectiveness of 9cUAB30 at inhibiting telomerase activity by down-regulating hTERT gene expression in human leukemic cells.

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.

Telomerase activity is one of the most important factors that have been linked to multiple developmental processes, including cell proliferation, differentiation, aging and senescence. Dysregulation of telomerase has often been found in developmental abnormalities, such as cancer, loss of function in the hematopoietic system, and low success rate of somatic cloning. A comprehensive network of transcription factors has been shown to be involved in the genetic control of telomerase expression and activity. Epigenetic mechanisms have recently been shown to provide an additional level of regulation, and may be responsible for the diverse expression status of telomerase that is manifested in a tissue and cell-type-dependent manner. This article summarizes the recent developments in the field of telomerase research with a focus on the coregulation of the telomerase gene by both genetic and epigenetic pathways. Developmental consequences of aberrant telomerase activity will also be summarized and discussed.

Human promyelocytic leukemia HL60 cells display high telomerase activity, a phenotype related to their immortal status. All-trans retinoic acid (ATRA) is a clinically effective cytodifferentiating agent. To understand the mechanism underlying ATRA-induced cytodifferentiation, we did a kinetic analysis of the role of ATRA in inhibiting telomerase in HL60 cells. Our studies indicate that telomerase inhibition by ATRA occurred relatively early after treatment of HL60 cells due to a rapid decrease in hTERT gene expression. More importantly, however, we found through monitoring the expression of CD11b, a marker for granulocytic differentiation of HL60 cells, that down-regulation of telomerase preceded the differentiation of HL60 cells. These observations suggest that the hTERT gene may be a primary target of ATRA regulation of cellular differentiation and the antileukemia activity of ATRA may be mediated by its ability to induce the differentiation of the promyelocytic leukemia cells through down-regulation of the hTERT gene.

Elucidation of the mechanisms governing expression of the human telomerase reverse transcriptase (hTERT) is important for understanding cancer pathogenesis. Approximately 90% of tumors express hTERT, the major catalytic component of telomerase. Activation of telomerase is an early event, and high levels of this activity correlate with poor prognosis. Recent studies have shown that the transcription factors c-Myc and Mad1 activate and repress hTERT, respectively. It is not clear how these transcription factors compete for the same recognition sequence in the hTERT core promoter region. Studies have shown that the combined expression of SV40 large T antigen (T-Ag), hTERT, and H-Ras is able to transform human cells. In this study, we used a distinct human cell type, WI-38 fetal lung fibroblasts used extensively for senescence studies. We transduced cells with amphotropic retroviral constructs containing SV40 T antigen, hTERT, and activated H-ras. Transduced cells exhibited anchorage independence in soft agar and expressed increased levels of c-Myc and endogenous hTERT. These effects were observed by 25 population doublings (PDs) following the establishment of the neoplastic cell line. During the process of transformation, we observed a switch from Mad1/Max to c-Myc/Max binding to oligonucleotide sequences containing the hTERT promoter distal and proximal E-boxes. c-Myc can bind specifically to the hTERT promoter in vitro, indicating that c-Myc expression in tumors may account for the increased expression of hTERT observed in vivo. These findings indicate that the widely used model system of WI-38 fibroblasts can be employed for transformation studies using defined genetic elements and that the endogenous hTERT and c-Myc are induced in these cells during early tumorigenesis. Such studies should have important implications in the mechanisms of hTERT and c-Myc induction in the beginning stages of tumorigenesis and facilitate extension of these studies to novel models of tumorigenesis in cellular senescence.

Short strands of tandem hexameric repeats known as telomeres cap the ends of linear chromosomes. These repeats protect chromosomes from degradation and prevent chromosomal end-joining, a phenomenon that could occur due to the end-replication problem. Telomeres are maintained by the activity of the enzyme telomerase. The total number of telomeric repeats at the terminal end of a chromosome determines the telomere length, which in addition to its importance in chromosomal stabilization is a useful indicator of telomerase activity in normal and malignant tissues. Telomere length stability is one of the important factors that contribute to the proliferative capacity of many cancer cell types; therefore, the detection and estimation of telomere length is extremely important. Until relatively recently, telomere lengths were analyzed primarily using the standard Southern blot technique. However, the complexities of this technique have led to the search for more simple and rapid detection methods. Improvements such as the use of fluorescent probes and the ability to sort cells have greatly enhanced the ease and sensitivity of telomere length measurements. Recent advances, and the limitations of these techniques are evaluated. Drugs that assist in telomere shortening may contribute to tumor regression. Therefore, factors that contribute to telomere stability may influence the efficiency of the drugs that have potential in cancer therapy. These factors in relation to telomere length are also examined in this analysis.

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.

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.

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.

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.

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.

A case of Down's syndrome in association with rheumatoid arthritis is described. The patient has spastic diplegia and atlanto-axial dislocation, probably the result of ligamentous laxity which is common in both Down's syndrome and rheumatoid arthritis. This laxity may have been enhanced by tonsillar or deep cervical infection. The myelopathy was thought to be due to the atlanto-axial dislocation. Spinal fusion may reverse the neurological abnormality in some cases.