Loss of the human polycomb group protein BMI1 promotes cancer-specific cell death.

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.

Hel-N1: an autoimmune RNA-binding protein with specificity for 3' uridylate-rich untranslated regions of growth factor mRNAs.

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.

Ectopic expression of Hel-N1, an RNA-binding protein, increases glucose transporter (GLUT1) expression in 3T3-L1 adipocytes.

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.

Telomerase inhibition in cancer therapeutics: molecular-based approaches.

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.

Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT).

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.

A patient with partial duplication 2q and partial deficiency 11q.

A patient with partial duplication 2q and partial deficiency 11q is reported. The propositus was delivered at 30 weeks gestation, with a birth weight of 1,390 g. He had severe hyaline membrane disease, intraventricular hemorrhage, bronchopulmonary dysplasia, hypotonia, psychomotor retardation, hearing loss, and other anomalies including a short bitemporal diameter, prominent occiput, low-set ears, exophthalmos, short nose with depressed nasal root, downturned mouth corners, narrow high-arched palate, micrognathia, a deep longitudinal groove over the sacrococcygeal region, clinodactyly, and abnormal dermatoglyphics. Chromosome analysis showed the following karyotype: 46,XY,der11,t(2:11)(q32.2;q25)pat.

Evaluation of families wherein a single male manifests a phenotype of X-linked lymphoproliferative disease (XLP).

The Epstein-Barr virus (EBV)-induced diseases of males with X-linked lymphoproliferative disease (XLP) include fatal infectious mononucleosis (IM), non-Hodgkin lymphoma (ML), agammaglobulinemia, and aplastic anemia. These phenotypes also occur as sporadic cases in families, and EBV seronegative males in these families must be considered at risk for XLP until they seroconvert normally to EBV. Given that 50% of males inheriting the defective XLP gene die following primary EBV infection, it is vital that they be identified pre-EBV infection. Here we report results using molecular genetic techniques to provide information as to the relative risks of EBV negative males and potential carrier females in ten families wherein a single male had died of IM.

Sex-linked hereditary ataxic deplegia, the borderland between cerebral palsy and Pelizaeus-Merzbacher disease.

After a review of the literature concerning hereditary cases of cerebral palsy, a family is reported in which ataxic diplegia appears to be inherited as a sex-linked and probably recessive condition occurring in 3 males in successive generations. This ataxic diplegia, occurring after an unremarkable perinatal course, is associated with mild to moderate mental retardation, congenitial nystagmus and significantly small stature and prevents the acquisition of free walking. Associated extrapyramidal features may gradually become more marked, while the nystagmus may subside. The condition is similar to that described in three previous reports in the literature. No evidence of linkage with other sex-linked disorders has been found, Xga typing showed that recombination between the Xg locus and the locus for hereditary ataxic deplegia has occurred once out of three possible opportunities. In the absence of neuropathological findings or specific biochemical tests, the differential diagnosis from Pelizaeus-Merzbacher disease cannot be made with certainty. The differentiation from other progressive sex-linked neurological disorders is discussed.

Mechanisms for methylation-mediated gene silencing and aging.

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.

Mechanisms for telomerase gene control in aging cells and tumorigenesis.

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.

A system for checking radiation therapy calculations for treatment time or monitor setting.

Errors in the calculation of radiation therapy treatment time or monitor setting are likely to occur with regularity unless adequate quality control procedures are utilized. These errors can be greatly reduced if appropriate computer systems are employed with programs which utilize analytical representations of dosimetric quantities. A system wherein dosimetry technologists routinely check treatment calculations using a computer terminal has been in operation in our department for several years and has proved helpful in uncovering calculation errors.

Myelopathy due to atlanto-axial dislocation in a patient with Down's syndrome and rheumatoid arthritis.

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.

Spinal cord injuries in children in British Columbia.

Fifteen children who sustained traumatic spinal cord injury in British Columbia over a 13-year period have been reviewed. The aetiology, incidence of spinal fracture, length of hospitalisation and subsequent spinal surgery, and their self-care, transfer and ambulatory abilities, bowel and bladder management, schooling, employment and place of abode have been determined.

Exon skipping in purine nucleoside phosphorylase mRNA processing leading to severe immunodeficiency.

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.

Molecular analysis of mutations in a patient with purine nucleoside phosphorylase deficiency.

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.

Hel-N1, an RNA-binding protein, is a ligand for an A + U rich region of the GLUT1 3' UTR.

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.

COMPARISON OF THE EFFICIENCY OF TWO DOSES OF BOVINE PPD TUBERCULIN IN SINGLE CAUDAL FOLD TESTS ON AUSTRALIAN CATTLE.

The sensitivity and specificity of 0.2 mg and 0.4 mg doses of bovine PPD tuberculin were compared in Northern Territory beef cattle from tuberculous herds and herds with a prevalence of tuberculosis of less than 0.1%. Reactions were interpreted subjectively by observation and palpation, and were also measured to the nearest mm with calipers at 24 h, 48 h, 72 h and 96 h after injection of tuberculin. All cattle were examined post mortem for the presence of macroscopic and microscopic tuberculous lesions. The apparent specificity of caudal fold tests with 0.2 mg and 0.4 mg doses was determined in cattle in Victoria from tuberculosis-free dairy and beef herds. Victorian cattle reacting to the caudal fold tests were subjected to a comparative intradermal test with 0.1 mg bovine PPD and 2,500 IU avian PPD not less than 42 days later. Tests with the 0.2 mg dose achieved the highest level of sensitivity of 95.6% at 48 h, 72 h and 96 h, while in tests with 0.4 mg the maximum reached was 94.7% at 72 h. The specificity of tests in Northern Territory cattle ranged from 85.0% to 88.3% with the 0.2 mg dose and from 80.6% to 82.3% with the 0.4 mg dose. The highest specificity was achieved with both doses at 96 h. The apparent specificity of 0.2 and 0.4 mg doses of bovine PPD in tuberculosis-free herds in Victoria was high, a false-positive reactor rate of only 0.6% occurring with caudal fold tests. All false-positive reactions were shown to be non-specific or due to previous experimental sensitisation.