Amyloid precursor protein mRNA stability is controlled by a 29-base element in the 3'-untranslated region

In the accompanying paper (Zaidi, S. H. E., Denman, R., and Malter, J. S. (1994) J. Biol. Chem. 269, 24000-24006) we demonstrate that in tumor and normal cells, multiple cytosolic proteins interact with a 29-base sequence in the 3'-untranslated region of amyloid precursor protein (APP) mRNA. These data suggested that APP gene expression may be modulated by regulated APP mRNA decay. We have investigated this prediction by measuring the decay rates of APP mRNA in resting and mitogen-treated peripheral blood mononuclear cells and H4 and K562 tumor cell lines. In resting peripheral blood mononuclear cells, APP mRNA decayed with a half-life of 4 h. Under these conditions, the activity of APP mRNA-binding proteins was not detectable. After activation, binding protein activities were induced, and APP mRNA decay was blocked with a half-life of > 12 h. In log phase neuronal or lymphoid tumor cell lines, binding activity was constitutively present and APP mRNA displayed a half-life of > 12 h. Protein synthesis inhibition by cycloheximide had no effect on APP mRNA decay in normal or tumor cells. Transfected wild type or mutant APP mRNAs that lacked the 29-base region were stable (t1/2 > 10 h) in K562 tumor cells. Therefore, we conclude that the 29-base region functions in cis to destabilize APP mRNA in resting, normal cells. Upon activation APP mRNA-binding proteins are induced, interact with the 29-base region, and likely participate in stabilization of the mRNA.     

The 3'-untranslated region of human type 2 iodothyronine deiodinase mRNA contains a functional selenocysteine insertion sequence element

Type 2 deiodinase (D2) catalyzes the 5'-deiodination of thyroxine to form 3,5,3'-triiodothyronine. Two mammalian D2 cDNAs have been identified containing 2 kilobases (kb) of the 7-kb mRNA including the complete coding sequence. Both contain in-frame TGA codons, which should serve as selenocysteine codons. However, the selenocysteine insertion sequence (SECIS) elements required for the decoding of UGA as a selenocysteine in the 3'-untranslated region (UTR) of the mRNA are not present. We have identified two overlapping expressed sequence tag clones, which contain the missing 4.4-kb 3'-UTR of the human D2 (hD2) cDNA. Computer analysis predicts a stem loop structure 280 base pairs 5' to the polyadenylation site, which has potent SECIS activity. A fragment containing these sequences hybridizes to D2 mRNA in human thyroid. A G to A mutation in the essential AUGA motif of this element abolished its function. Transfection of the hD2 coding region plus the 3'-UTR results in the expression of D2, and its in vitro transcribed mRNA programs D2 activity in Xenopus oocytes. This is the first identification of a SECIS element in a mammalian D2 cDNA and establishes that hD2 is a bona fide selenoprotein.     

Negative control of the poly(A)-binding protein mRNA translation is mediated by the adenine-rich region of its 5'-untranslated region

Translation of the mRNA for the poly(A)-binding protein (PABP) may be autoregulated by the binding of PABP to the A-rich segment of its 5'-untranslated region (UTR). To test this hypothesis, we examined the effect of different fragments of the 5'-UTR from human PABP cDNA on the translation of the beta-galactosidase (beta-Gal) reporter gene. Presence of the A-rich sequence from the 5'-UTR of PABP mRNA inhibited expression of the chimeric beta-Gal gene in transfected HeLa cells. The differences in expression of beta-Gal polypeptide was due to the translational repression of beta-Gal mRNA containing the A-rich 5'-UTR of PABP mRNA. The A-rich region of the 5'-UTR located within nucleotides 58-146 of PABP mRNA was sufficient to mediate translational control of this mRNA expression. We also examined the effect of overexpression of PABP mRNA in HeLa cells. The ectopic PABP mRNA without the A-rich 5'-UTR region was translated efficiently, whereas the translation of the endogenous PABP mRNA was substantially inhibited in the transfected cells. In contrast, the ectopic PABP mRNA containing the A-rich 5'-UTR region did not show similar effect on the translation of the endogenous PABP mRNA in these cells. These results suggest that feedback control of mRNA translation is involved in regulating PABP expression in HeLa cells.     

Protein kinase A stimulates binding of multiple proteins to a U-rich domain in the 3'-untranslated region of lactate dehydrogenase A mRNA that is required for the regulation of mRNA stability

We have explored the molecular basis of the cAMP-induced stabilization of lactate dehydrogenase A (LDH-A) mRNA and identified four cytoplasmic proteins of 96, 67, 52, and 50 kDa that specifically bind to a 30-nucleotide uridine-rich sequence in the LDH 3'-untranslated region with a predicted stem-loop structure. Mutational analysis revealed that specific protein binding is dependent upon an intact primary nucleotide sequence in the loop as well as integrity of the adjoining double-stranded stem structure, thus indicating a high degree of primary and secondary structure specificity. The critical stem-loop region is located between nucleotides 1473 and 1502 relative to the mRNA cap site and contains a previously identified cAMP-stabilizing region (CSR) required for LDH-A mRNA stability regulation by the protein kinase A pathway. The 3'-untranslated region binding activity of the proteins is up-regulated after protein kinase A activation, whereas protein dephosphorylation is associated with a loss of binding activity. These results imply a cause and effect relationship between LDH-A mRNA stabilization and CSR-phosphoprotein binding activity. We propose that the U-rich CSR is a recognition signal for CSR-binding proteins and for an mRNA processing pathway that specifically stabilizes LDH mRNA in response to activation of the protein kinase A signal transduction pathway.     

Identification of cellular proteins that bind the central conserved region of p53

The bacterial fusion protein between glutathione S-transferase and the central conserved region of human p53(GST-p53) was purified and fixed on the beads and then used in the binding assay with radiolabeled cell extract from human hepatocarcinoma cell line, Hep3B. The binding assay disclosed the presence of cellular proteins that interact with GST-p53 but not with GST. SV40 large T antigen abrogated the bindings of two cellular proteins with molecular weights of 50 kda and 40 kda. The binding of the proteins to p53 was observed in a cell cycle-dependent manner. These two proteins are candidate cellular proteins which regulate the function of p53.     

Expression of p21(waf1/cip1) protein in transitional cell bladder tumours and its prognostic value

Selective antagonists to the Type 3 serotonin receptor (5HT3) in combination with corticosteroids are now considered the standard of care for the prevention of emesis from moderately to highly emetogenic chemotherapy. Here we address issues of optimal dose, schedule and route of administration of four currently available selectable 5HT3 antagonists. This paper utilizes an evidence based medicine approach to the literature regarding this class of drugs, emphasizing the results large, randomized, controlled trials to make formal recommendations concerning optimal use of this important new class of anti-emetic agents. We conclude that for each drug there is a plateau in therapeutic efficacy at a definable dose level above which further dose escalation does not improve outcome. Furthermore, a single dose is as effective as multiple doses or continuous infusion, and finally, emerging data demonstrate that the oral route is equally efficacious as the intravenous route of administration, even with highly emetogenic chemotherapy.     

Variation in the AU(AT)-rich element within the 3'-untranslated region of PPP1R3 is associated with variation in plasma glucose in aboriginal Canadians

We are investigating associations between variations in candidate genes on chromosome 7q and diabetes-related phenotypes in Canadian Oji-Cree. One of these genes encodes the skeletal muscle regulatory G subunit of the glycogen-associated form of protein phosphatase 1 (PPPIR3), which may play a key role in muscle glycogen metabolism. There is a common 5-bp insertion-deletion polymorphism in a messenger ribonucleic acid-stabilizing AU(AT)-rich element within the 3'-untranslated region (UTR) of PPPIR3. The D allele had a frequency of 0.30 in the Oji-Cree. We found that this 3'-UTR variation of PPPIR3 was significantly associated with variation in 2-h postprandial glucose in adult Oji-Cree with type 2 diabetes or impaired glucose tolerance (IGT). Specifically, Oji-Cree with diabetes or IGT who were D/D homozygotes had significantly lower 2-h postprandial plasma glucose than subjects with the other genotypes. There was no association of the PPPIR3 genotype either with the presence of type 2 diabetes or IGT or with other quantitative traits in this sample. These findings suggest that common PPPIR3 3'-UTR variation that potentially affects messenger ribonucleic acid stability is associated with variation in glycemia in Oji-Cree subjects with type 2 diabetes.     

Selection of a cDNA clone for chicken high-mobility-group 1 (HMG1) protein through its unusually conserved 3'-untranslated region, and improved expression of recombinant HMG1 in Escherichia coli

Screening of cDNA libraries for the homologous vertebrate proteins high mobility group (HMG) 1 and 2 using DNA probes based on the coding sequences is likely to result in isolation of both HMG1 and HMG2 clones, as well as pseudogenes, which may be transcribed at low levels. However, the 3'-untranslated regions (UTRs) of HMG1 and 2 are quite distinct, and unusually conserved across species. We have used this property to select the true chicken HMG1 cDNA clone from a chicken lymphocyte cDNA library in lambdagt11, using a probe based on the 3'-UTR of rat HMG1 cDNA. The chicken HMG1 cDNA clone is very similar to all the complete HMG1 cDNA clones isolated so far. We suggest that the sequence designated chicken HMG1 in the GenBank Data Library (Accession number D14314) is, in fact, that of HMG2a [and moreover that the recently reported mouse clone (Accession number AF022465), proposed to encode a new HMG protein, HMG4, is also likely to encode an HMG2a, based on the translated amino-acid sequence and 3'-UTR]. We also report much improved expression of intact recombinant HMG1 in Escherichia coli by the use of chloramphenicol rather than ampicillin selection and conditions that limit cell growth. This should be general for all members of the HMG1 (and 2) family which may be toxic to cells (possibly because of the long acidic tail), and may also prove useful in the production of other such proteins.     

Regulation of tyrosine hydroxylase mRNA stability by protein-binding, pyrimidine-rich sequence in the 3'-untranslated region

The stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is regulated by oxygen tension in the pheochromocytoma-derived PC12 cell line. We previously identified a pyrimidine-rich 27-base-long protein-binding sequence in the 3'-untranslated region of TH mRNA that is associated with hypoxia-inducible formation of a ribonucleoprotein complex (hypoxia-inducible protein-binding site (HIPBS)). In this study, we show that HIPBS is an mRNA stabilizing element necessary for both constitutive and hypoxia-regulated stability of TH mRNA. The mutations within this sequence that abolish protein binding markedly decrease constitutive TH mRNA stability and ablate its hypoxic regulation. A short fragment of TH mRNA that contains the wild-type HIPBS confers the increased mRNA stability to the reporter chloramphenicol acetyltransferase mRNA. However, it is not sufficient to confer hypoxic regulation. The HIPBS element binds two isoforms of a 40-kDa poly(C)-binding protein (PCBP). Hypoxia induces expression of the isoform 1, PCBP1, but not the isoform 2, PCBP2, in PC12 cells.     

Developmental regulation of the Drosophila Tropomyosin I (TmI) gene is controlled by a muscle activator enhancer region that contains multiple cis-elements and binding sites for multiple proteins

We have analyzed in Paramecium cells the occurrence and intracellular distribution of the high capacity/low affinity calcium-binding proteins, calsequestrin (CS) and calreticulin (CR) using antibodies against CS from rat skeletal muscle and against CR from rat liver, respectively. As revealed by Western blots, a CS-like protein isolated by affinity chromatography from Paramecium cells comigrated with CS isolated from rat skeletal muscle. The immunoreactivity of this 53 kDa protein band was blocked when the antibodies had been preadsorbed with purified rat CS. A band of identical molecular size was shown to bind 45Ca in overlays. By immunofluorescence and immunogold labeling this CS-like protein was localized selectively to the extended subplasmalemmal calcium stores, the "alveolar sacs", which cover almost the entire cell surface. Concomitantly the 53 kDa 45Ca-binding band became increasingly intense in overlays as we increasingly enriched alveolar sacs. Antibodies against rat CR react with a 61 kDa band but do not cross-react with CS-like protein in Paramecium. These antibodies selectively stained intracellular reticular structures, identified bona fide as endoplasmic reticulum.     

Expression and alteration of p53 and p21(waf1/cip1) influence the sensitivity of chemoradiation therapy for esophageal cancer

BACKGROUND/AIMS: In vitro analyses have been demonstrated that wild type p53 and p21(waf1/cip1) proteins regulate cellular proliferation and sensitivity of anticancer agents, however, the roles of p53 and p21(waf1/cip1) expression on the response to the chemoradiation therapy for human esophageal squamous cell cancer have not been investigated. METHODOLOGY: With an immunohistochemical method using specimens before and after the chemoradiation therapy, we investigate in this report the influence of the p53 and p21(waf1/cip1) expression on the chemoradiation therapy response or alteration of these protein expressions by chemoradiation therapy in thirteen esophageal squamous cell cancer patients who received our recently developed chemoradiation therapy. RESULTS: In the biopsy specimens before the chemoradiation therapy, 82% of the responders and 71% of patients with down T-classification had either a p53-/p21+ or p53+/p21+ phenotype. Eighty two percent and 46% of the cases with these two phenotypes showed complete or partial response and down T-classification, respectively. After the chemoradiation therapy, 73% of the responders and 71% of the patients with down T-classification had either a p53-/p21+ or p53+/p21+ phenotype. Eighty eight percent and 56% of the cases with these two phenotypes showed complete or partial response and down T-classification, respectively. Comparative analysis before and after the chemoradiation therapy revealed that four patients had an alteration of p53 and p21(waf1/cip1) expression in association with the therapeutic response. CONCLUSIONS: These results suggest that wild type p53 or p21(waf1/cip1) expression relates with and can be altered by the chemoradiation therapy, which could influence the therapeutic efficacy.     

Abnormal accumulation of copper in LEC rat liver induces expression of p53 and nuclear matrix-bound p21(waf 1/cip 1)

The LEC rat is an inbred mutant strain which spontaneously develops liver injury and subsequent liver cancer. Liver injury in LEC rats has recently been shown to be closely related to abnormal copper accumulation in the liver. Previously, we reported that LEC rat hepatocytes lose their growth potential, probably allowing selective growth of preneoplastic cells. In this study, to elucidate the effects of copper accumulation on the growth activity of LEC rat hepatocytes, we examined the growth activity and the expression of p53 and p21(waf 1/cip 1) in the livers of LEC rats fed on either a control or a low-copper diet. Potential for cell proliferation of hepatocytes obtained from normal diet fed LEC rats was almost comparable to that of the cells from age-matched Sprague-Dawley (SD) rats. Northern blot analysis showed that the expression of p53 and p21(waf 1/cip 1) was significantly high in the livers of LEC rats fed a control diet, while the expression of p53 and p21(waf 1/cip 1) in the LEC rats fed a low-copper diet was as low as that of SD rat livers. Western blot analysis consistently showed that the amount of p21(waf 1/cip 1) bound to the nuclear matrix scaffold of the LEC rat liver was reduced by feeding a low-copper diet. These findings suggest that abnormal accumulation of copper induced the expression of p53 and p21(waf 1/cip 1), resulting in the inhibition of cell proliferation of LEC rat hepatocytes.     

Interaction between a cellular protein that binds to the C-terminal region of adenovirus E1A (CtBP) and a novel cellular protein is disrupted by E1A through a conserved PLDLS motif

Adenovirus E1A proteins immortalize primary animal cells and cooperate with several other oncogenes in oncogenic transformation. These activities are primarily determined by the N-terminal half (exon 1) of E1A. Although the C-terminal half (exon 2) is also essential for some of these activities, it is dispensable for cooperative transformation with the activated T24 ras oncogene. Exon 2 negatively modulates in vitro cooperative transformation with T24 ras as well as the tumorigenic and metastatic potentials of transformed cells. A short C-terminal sequence of E1A governs the oncogenesis-restraining activity of exon 2. This region of E1A binds with a cellular phosphoprotein, CtBP, through a 5-amino acid motif, PLDLS, conserved among the E1A proteins of human adenoviruses. To understand the mechanism by which interaction between E1A and CtBP results in tumorigenesis-restraining activity, we searched for cellular proteins that complex with CtBP. Here, we report the cloning and characterization of a 125-kDa protein, CtIP, that binds with CtBP through the PLDLS motif. E1A exon 2 peptides that contain the PLDLS motif disrupt the CtBP-CtIP complex. Our results suggest that the tumorigenesis-restraining activity of E1A exon 2 may be related to the disruption of the CtBP-CtIP complex through the PLDLS motif.