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Tumor Biology |
Departments of Neurology [L. B. N., P. H. K.], Neurosurgery [G. Y. G.], and Physiology and Biophysics [P. H. K.], University of Alabama at Birmingham, Birmingham, Alabama 35233-7340, and Birmingham Veterans Affairs Medical Center [L. B. N., L. H., P. H. K.], Birmingham, Alabama
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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as potential RNA ligands. Our results indicated overall a very high binding affinity to these RNA targets. A comparison of these ligands revealed a hierarchy of binding affinities with the angiogenic factors, and TGF-ß showing the highest (Kd of 1.8–3.4 nM), and TNF- the lowest (Kd of 18.3 nM). The expression pattern of HuR, coupled with the RNA binding data, strongly suggests a role for this protein in the posttranscriptional regulation of these genes in CNS tumors. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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mRNA in epithelial cancer cells may contribute to the acquired resistance of these cells to the cytolytic effects of exogenous TNF- (6)
. Recently, HuR, a member of the Elav family of RNA-binding proteins, has been identified as a potential trans-acting factor in RNA stabilization. This observation stems from two lines of evidence. First, members of the Elav family have a strong binding affinity to AREs (7
, 8) . Second, the overexpression of HuR, in certain cell systems, has led to the stabilization of c-fos and VEGF transcripts that contain AREs in their 3'-UTR (9, 10, 11)
. A recent study of cyclin A and B1 regulation linked HuR with the RNA stabilization of these genes and enhanced tumor proliferation (12)
. The potential role of HuR as a stabilizer of growth-related mRNAs is consistent with its strong expression pattern in proliferating cells of the developing CNS (13)
. As demonstrated in mutational studies in Drosophila, moreover, the Elav family is essential for the normal growth and development of the CNS (14
, 15)
. Aberrant RNA stabilization of growth-related genes, however, may promote the uncontrolled growth of a CNS tumor. We were, therefore, interested in determining whether the expression pattern of HuR in primary brain tumors correlated with histological type or grade. To further delineate the potential role of HuR in posttranscriptional gene regulation in brain tumors, we then selected angiogenic and cytokine genes that are up-regulated in CNS tumors and determined whether HuR could bind to their 3'-UTRs. |
MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Preparation of Recombinant Proteins.
The cDNA for HuR was subcloned into the pGEX-5X-1 vector (Amersham Pharmacia Biotech, Piscataway, NJ) in frame with the GST tag and verified by sequence and restriction analysis. The plasmid was transformed into the BL21 strain of Escherichia coli and induced as described previously (19)
. The protein was purified using a glutathione-Sepharose column based on the manufacturer’s specifications (Amersham Pharmacia Biotech). The protein was dialyzed extensively in PBS and quantified by the DC protein assay (Bio-Rad, Hercules, CA) using BSA as a standard. The biosynthesis of recombinant proteins HuC, HuD, Hel-N1, and HuR using the histidine tag have been reported previously (19)
.
RPA.
The HuR riboprobe template was obtained by subcloning a PstI/SacI digestion fragment of the HuR cDNA (U38175; nucleotide 159–416; Ref. 8
) into Bluescript SK (Stratagene, La Jolla, CA). Hel-N1, HuC, and HuD templates were used as described previously (20)
. The GAPDH gene was used as an internal standard for comparison purposes. Riboprobes were synthesized with [-32P]UTP as described previously (20)
. RNA hybridizations and RNase digestions were carried out as described previously (20
, 21)
. To quantify RNA levels, test and control bands were evaluated by phosphorimaging (Molecular Dynamics, Sunneyvale, CA).
Preparation of Polyclonal Antibodies to HuR.
Rabbit polyclonal antibodies to HuR were prepared commercially (Genosys, The Woodlands, TX). Animals were immunized with a synthetic peptide to the HuR NH2 terminus containing the initial 15 amino acid residues (MSNGYEDHMAEDCRG). This region of HuR diverges significantly from the other human homologues of Elav (HuC, HuD, and Hel-N1; Ref. 22
). Serum was affinity-purified on a HuR NH2 terminus peptide column, concentrated, and then stored in 50% glycerol.
Immunocytochemistry.
Tissue sections from tumor samples (obtained at the time of tumor biopsy or resection) were fixed in 10% buffered formalin, processed, and embedded in paraffin. Sections were cut at 8 µm and dried overnight in a 50°C oven. Sections were deparaffinized in xylene and dehydrated through a series of alcohols and hydrated in water. Sections were pretreated with pepsin (2.5 mg/ml; Biogenex Laboratories, San Ramon, CA) at 37°C for 4 min and were rinsed with distilled water. Sections underwent epitope recovery using a modified protocol for Antigen Retrieval Citra Buffer (Biogenex Laboratories, San Ramon, CA). Briefly, citra buffer was prewarmed to 80°C in microwave oven. Sections were incubated in prewarmed buffer immersed in a 48°C water bath for 150 min. Sections were rinsed and blocked with 3% hydrogen peroxide (Sigma, St. Louis, MO). Sections were rinsed with Tris buffer (pH 7.6) and blocked with FC receptor block (Innovex Bioscience, Richmond, CA) for 15 min at room temperature. Purified rabbit anti-HuR antibody was applied at 1:80 dilution and incubated in the refrigerator (at 2–8°C) overnight. Sections were incubated with horseradish peroxidase-enhancing wash buffer (Innovex Bioscience). Detection and amplification of signal was achieved using Non-biotin Amplification Kit (Zymed Laboratories, South San Francisco, CA). Signal was developed using liquid DAB (Innovex Bioscience), counterstained with hematoxylin I (Richard-Allan Scientific, Kalamazoo, MI), and rinsed with tap water. Sections were dehydrated through a series of alcohols into xylene and mounted with Secure Mount (Biosciences, Inc, Swedesboro, NJ).
ELISA-based RNA-binding Assay.
The 3'-UTR riboprobe templates of the following growth factors and cytokines (accession number; nucleotide sequence) were obtained by reverse transcription-PCR from the U251 malignant glioma cell line: VEGF (Y08736; 1282–1913), COX-2 (U04636; 6904–7483), IL-6 (M54894; 691-1102), IL-8 (M28130; 3389–4198), TGF-ß (M60315; 1697–2859), and TNF- (M10988; 795-1565). PCR products were cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA). All of the clones were verified by restriction mapping and sequence analysis. Biotinylated riboprobes were synthesized as described previously (23)
. All of the probes were verified by visualization on an ethidium-stained polyacrylamide gel. Riboprobe concentrations were determined using Ribogreen RNA Quantitation reagent (Molecular Probes, Eugene, OR) in a fluorometer. The ELISA-based RNA binding assay has been described previously (23)
. Briefly, 500 ng of recombinant GST-HuR fusion protein was plated in a volume of 50 µl into individual ELISA wells and allowed to adsorb overnight. On the following day, the plates were washed, and biotinylated riboprobes were added at various amounts (range, 0.1–3.0 pmol) to 50 µl of RNA-binding buffer (25 mM HEPES, 0.5 mM EGTA, 100 mM NaCl, 0.5 mM DTT, 4 mM MgCl2, 20 mM KCl, 0.05% NP40, 0.5 mg/ml yeast tRNA, 0.05 mg/ml poly(A) RNA, 0.4 mM VRC, and 5% glycerol) and placed in the ELISA well for 30 min at room temperature. After extensive plate washing, a streptavidin-alkaline phosphatase conjugate was added and allowed to incubate for 30 min at room temperature. The plates were then developed with a p-nitrophenyl phosphate (Sigma, St. Louis, MO) solution, and the absorbance was determined at 405 nm. These values (in arbitrary units) were plotted against RNA concentrations, and binding curves were estimated with DeltaGraph software (SSPS, Chicago, IL). At first approximation, the probes were in excess of the protein, and affinity constants were estimated from the curves.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, we detected nearly universal expression of HuR in primary brain tumors and cultured tumor cells, regardless of cell origin or degree of invasiveness. The only sample in which HuR was absent also had a weak GAPDH signal (Fig. 1
, Gb, Lane 9). Densitometric analysis of the bands, compared with the internal control GAPDH, indicated that the highest HuR expression was observed in meningiomas, suggesting that elevated RNA levels did not correlate with higher-grade malignancies (Fig. 2)
. This observation, however, is based on the assumption that GAPDH is equally expressed among tumors. The other samples, including GBMs and medulloblastomas, had lower but similar HuR RNA levels. The ubiquitous expression pattern is consistent with previous observations for HuR in lung cancer (24)
. Because the neuronal-associated homologues of the Elav family, including Hel-N1, HuC, and HuD, are restricted to neurons and neuroendocrine tumors (20
, 21
, 25
, 26)
, we were interested in comparing the relative expression patterns of these homologues to that of HuR in brain tumors of different cell origins. We quantified the RNA level of each family member within a tumor sample and expressed the value as a percentage of total Elav RNA expression for that sample (Fig. 3)
. Because tissue samples were limited, we analyzed only those with sufficient RNA to test all four of the Elav members. Thus, the percentage values shown in Fig. 3B
indicate relative rather than absolute expression levels. Representative RPAs for each family member with different tumor samples are shown (Fig. 3A)
. Three protected bands were consistently observed with HuC and are most likely attributable to alternative splicing (22)
. As shown in Fig. 3B
, cells of glial or arachnoidal lineage, including primary astrocytes, malignant glioma cell lines, and meningiomas, had nearly exclusive expression of HuR. One of the cell lines (D54 MG) and two meningioma samples expressed low levels of Hel-N1 but none of the other neuronal-associated homologues. The data, therefore, indicate that these glial and arachnoidal tumor types have the capability of expressing neuroendocrine markers. The GBM samples, on the other hand, had expression of all of the three neuronal-associated members, with HuR predominating. Because GBMs are highly invasive, some of the biopsy specimens may have contained normal cortex, thus accounting for detection of the neuronal-associated homologues. To help discern this possibility, we compared the relative Elav expression patterns between normal cortex (Fc and Oc) and the tumor samples. The expression patterns of the Elav members in Fc and Oc, with HuC showing the highest and HuR the lowest relative RNA levels, corresponded well with previous in situ hybridization studies in mouse brain (27)
. The predominant expression of HuR in all of the GBMs is, therefore, reflective of tumor-specific expression and not normal brain tissue [compare HuR levels in GBM (Fig. 1
, Gb, Lanes 1–7) to Fc and Oc controls]. In four of the GBM samples (Lanes 3, 5, 6, and 7), all of the three neuronal-associated homologues were detected, with HuC and HuD levels being comparatively higher than that for Hel-N1. This suggested the possibility that these specimens contained variable numbers of neuroglial cells. In samples 2 and 4, however, neither Hel-N1 nor HuD was detected, indicating selective expression of some neuronal-associated markers with these GBM tumor specimens. The two pilocytic astrocytomas, considered lower-grade glial tumors, contained reduced HuR levels relative to the GBMs but higher levels than did cortex (Fig. 1
, PA, samples 1–2). The presence of all of the three neuronal-associated homologues in pilocytic astrocytoma, with a pattern similar to Fc and Oc, suggests that these specimens may also have included normal neuroglial elements. The predominance of HuD expression, coupled with low HuC expression, was also observed in two of the three ependymomas (Fig. 1
, EP, Lanes 2 and 3), indicating that this tumor type also has the capacity to express neuronal-associated markers.
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, MD, samples 1–5). The expression pattern of these homologues, however, substantially differed from normal cortical tissues. In sample 1, for example, Hel-N1 was the only neuronal homologue detected, similar to the glioma cell lines and meningiomas. On the basis of developmental studies in Xenopus and mouse, sole expression of this neuronal homologue reflects a very early stage of neuronal differentiation (27
, 28) . This expression pattern was also observed in the poorly neuroendocrine-differentiated variant of small-cell lung cancer (20)
. In the other four medulloblastoma samples, HuD expression predominated (in contrast to HuC in normal cortex), suggesting more advanced neuronal maturation (13
, 20
, 27
, 28)
. Higher levels of HuD have been correlated with favorable prognostic features in a study of neuroblastoma tumors (21)
. Although the number of specimens investigated here were too few to establish definitive conclusions, the variable expression pattern of neuronal-associated Elav family members within medulloblastomas may be an important genetic footprint of tumor grade and prognosis. In summary, all of the primary brain tumors expressed HuR RNA, with the highest levels detected in tumors of glial and arachnoid origin. HeL-N1, HuC, and HuD, on the other hand, were detected at the highest levels in medulloblastoma. The variable detection of these homologues in GBMs and ependymomas underscores the capacity of these tumors for expressing markers of neuronal differentiation.
HuR Is Expressed at the Protein Level in Highly Proliferative Brain Tumors.
The conserved RNA-binding domains within the Elav family have made it difficult to develop specific antibodies to individual members. The monoclonal antibody Mab16A11, e.g., immunoreacts with all of the three neuronal-associated homologues (29)
. Polyclonal anti-Hu (or ANNA-1) serum from patients with paraneoplastic neurological disease, on the other hand, reacts with all of the four family members (19
, 24
, 27)
. Thus, to analyze HuR protein expression in primary brain tumors, we developed an anti-HuR polyclonal rabbit antibody using a unique peptide sequence in the NH2 terminus of the protein. This portion of the Elav protein is the least conserved among the different family members (22)
. We tested the monospecificity of this antibody by Western blot analysis using all of the four recombinant Elav proteins (Fig. 4)
. A reactive band was observed for HuR but not for the neuronal-associated members Hel-N1, HuC, or HuD (Fig. 4A
, upper panel). Preabsorption of the anti-HuR IgG with recombinant HuR abrogated this immunoreactivity (not shown). The blot was stripped and probed with biotinylated anti-Hu paraneoplastic IgG. Fig. 4A
, lower panel, demonstrates the typical immunoreactive pattern with all of the four family members (19)
. We next tested the antibody by immunocytochemistry using U251 glioma cells in culture because this cell line robustly expressed HuR RNA (see Fig. 1B
: Gc, Lane 6). As shown in Fig. 4B
, left panel, there was intense staining, predominantly in the nucleus and to a lesser extent in the cytoplasm. This localization pattern has been observed with the neuronal-associated homologues, using either anti-Hu paraneoplastic serum or Mab16A11 (29, 30, 31)
. Preabsorption of the antibody with recombinant HuR protein abolished the reactivity, underscoring the specificity of this immunostaining (Fig. 4B
, right panel). With this novel antibody, we then analyzed different primary brain tumors for HuR expression (summarized in Table 1
). We chose immunocytochemistry rather than Western blot analysis to allow accurate localization of immunoreactivity within the tissue sample. The most striking finding was the intense staining of the high-grade tumors (GBMs and medulloblastomas) and the minimal reactivity in the low-grade tumors (pilocytic astrocytomas and meningiomas; Fig. 5
). The predominant nuclear pattern of immunoreactivity in the GBMs and medulloblastomas was similar to that of U251 cells (Fig. 4B)
, and preabsorption of the antibody with recombinant HuR abrogated this reactivity as shown in Fig. 5
, the (-) panels. This pattern was consistently observed in all of the eight GBM tumors analyzed (see Table 1
). Histologically, the GBM and medulloblastoma tumors demonstrated high cellular density with many mitotic figures. The association between HuR protein expression and cell proliferation was recently demonstrated in colorectal carcinoma cells in which reduced HuR expression levels (by transfection of antisense HuR) correlated with reduced tumor growth (12)
. Reduced stabilization of cyclin A and B1 mRNAs was implicated in the altered growth phenotype. Another recent report of HuR expression in malignant lung tumors also indicated a correlation of HuR protein expression with degree of malignancy (32)
.
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. Interestingly, tumor cells adjacent to regions of necrosis showed intense staining. These regions, which are relatively hypoxic, have increased expression of angiogenic genes including VEGF, COX-2, and IL-8 (33, 34, 35)
. The colocalization and up-regulation of HuR in these regions meshes with the previous observation in other tumor cells in which HuR participates in the hypoxia-induced stabilization and up-regulation of VEGF (9)
. In contrast to the high-grade tumors, very little immunostaining was detected in low-grade astrocytoma and meningioma tumors. Those cells that were positive, moreover, were not the primary tumor and did not have a predominant nuclear pattern. In pilocytic astrocytoma, for example, the subset of cells that stained positive was morphologically different from the tumor cells. They demonstrated a plump cytoplasm where most of the immunostaining was localized. They resembled reactive astrocytes rather than neoplastic cells. In the meningioma tumor, the small subset of positively staining cells morphologically resembled endothelial cells rather than tumor cells. This pattern of low HuR immunoreactivity was observed in three of three low-grade astrocytomas and eight of nine meningiomas. The dissociation between HuR RNA and protein expression in low-grade brain tumors and normal brain has also been observed in normal, nonproliferating mouse tissues, suggesting that HuR is regulated at the translational level (27)
. In summary, the correlation of HuR expression with highly proliferative CNS tumors meshes with other findings in other tumors and supports a role for HuR in the posttranscriptional regulation of gene expression in malignant brain tumors.
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and show the number and distribution of AREs (as defined by an AUUUA or AUUUUA motif). We used the VEGF regulatory sequence (VRS) within the VEGF 3'-UTR, which has previously been shown to bind HuR by gel-shift assay (9)
. HuR had a high binding affinity to all of the three angiogenic factor 3'-UTRs (Fig. 7B)
. No binding was detected to the control transcript, pBSK, underscoring the specificity of this binding. The affinity to IL-8 (Kd of 1.8 nM) was slightly higher than for VEGF and COX-2 (Kd of 3.4 and 3.1 nM, respectively). As with VEGF, however, we analyzed only a segment of the entire 3'-UTR for COX-2, which is 
2.5 kb. The IL-8 3'-UTR was full length based on published sequence (37)
. Both COX-2 and IL-8 have not previously been shown to bind HuR, although the 3'-UTR probes contained a similar number of AREs as that of VEGF (Fig. 7A)
. The colocalization of HuR with the angiogenic factor genes in GBM tumors, coupled with these high binding affinities, supports, but does not prove, a role for HuR in their posttranscriptional regulation. The relative hypoxia present in the perinecrotic tumor zones of the GBM raises the intriguing possibility that HuR may be involved in the hypoxia-induced stabilization of VEGF in GBM (as it is in other tumor cells; Refs. 4
, 5
, 9
, 35
, 38
). Although posttranscriptional regulation of COX-2 is an important mechanism for its up-regulation in other tumors, the role for this pathway in COX-2 (and IL-8) expression in GBMs has not been elucidated (39, 40, 41)
.
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, and IL-6, were more variable. These autocrine factors play an important role in the interplay between the immune system and the brain tumor, and may confer a growth advantage to the tumor (42
, 43)
. TGF-ß, e.g., had one of the highest affinities for HuR (Kd of 2.3 nM) whereas TNF- and IL-6 had the lowest (18.3 and 10.6, respectively). TGF-ß had an 8-fold higher-binding affinity for HuR than did TNF-. The TGF-ß 3'-UTR also had the fewest AREs (six in total) which were dispersed over the entire probe, whereas the TNF- had eight clustered AREs (Fig. 7A)
. This discrepancy in binding affinities suggests that optimal RNA binding may be related to other cis elements within the UTR or its secondary structure, rather than to the absolute number of AREs. The original RNA-binding studies with Hel-N1, using an immunoprecipitation technique, indicated that this family may have binding preferences for other U-rich motifs (7)
. The TGF-ß 3'-UTR had many of these additional motifs (not shown). Additionally, the Elav proteins have three RNA recognition motifs, each of which may bind to a different RNA ligand (44)
. The maximal saturation levels for each of the curves (including the angiogenic factor mRNAs), however, were similar, indicating that the RNA ligands were likely binding to the same site, but with different affinities. Qualitatively, HuR and other members of the Elav family have been shown to bind a number of 3' UTRs using different assays (7
, 8
, 25
, 26
, 45, 46, 47, 48)
. The ELISA-based RNA binding assay, although still in vitro, permits a rapid comparison of binding affinities with different RNA ligands (23)
. The hierarchy of affinities observed here suggests that ARE-bearing 3'-UTRs are not alike with respect to HuR binding. Specificity of HuR binding, therefore, may partly be dictated by these affinity differences. Our results indicate that: (a) HuR, unlike the neuronal-associated Elav homologues, is ubiquitously expressed in tumors of the CNS at the RNA level; (b) HuR protein is strongly expressed in highly proliferative tumors including GBMs and medulloblastomas in contrast to weak expression in low-grade brain tumors; (c) intense HuR immunoreactivity was observed in perinecrotic regions within the GBMs in which angiogenic growth factors are up-regulated; and (d) HuR can bind with high affinity to the 3'-UTRs of critical cytokine and growth factor mRNAs involved in CNS tumor proliferation and angiogenesis, suggesting a posttranscriptional regulatory role for this protein in malignant brain tumors.
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FOOTNOTES |
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1 Supported by a Department of Veterans Affairs VISN 7 Career Development Award (to L. B. N.), by American Cancer Society RPG-97-111-01-CCE (to P. H. K.) and IR6-60-001-41 (to L. B. N.), by NIH CA71933 (to G. Y. G.), and grants from the Pediatric Brain Tumor Foundation of the United States and from JCR Biopharmaceutical, Inc. Generous support provided by the Judge Richard Holmes Brain Tumor Research Fund. 
2 To whom requests for reprints should be addressed, at Department of Neurology, University of Alabama at Birmingham, 1235 Jefferson Tower, Birmingham, AL 35233-7340. Phone: (205) 975-8116; Fax: (205) 934-0928.
3 The abbreviations used are: AU, adenine and uridine; ARE, AU-rich element; CNS, central nervous system; UTR, untranslated region; GBM, glioblastoma multiforme; VEGF, vascular endothelial growth factor; TNF, tumor necrosis factor; GAPDH, glyceraldehyde-3 phosphate dehydrogenase; TGF, transforming growth factor; IL, interleukin; GST, glutathione S-transferase; RPA, RNase protection assay; Fc, frontal cortex; Oc, occipital cortex; Kd, dissociation constant.
Received 6/14/00. Accepted 1/ 4/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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 C. Segrelles, S. Ruiz, M. Santos, J. Martinez-Palacio, M. F. Lara, and J. M. Paramio Akt mediates an angiogenic switch in transformed keratinocytes Carcinogenesis, July 1, 2004; 25(7): 1137 - 1147. [Abstract] [Full Text] [PDF]
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J.-Y. Lu and R. J. Schneider Tissue Distribution of AU-rich mRNA-binding Proteins Involved in Regulation of mRNA Decay J. Biol. Chem., March 26, 2004; 279(13): 12974 - 12979. [Abstract] [Full Text] [PDF]
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I. L. de Silanes, M. Zhan, A. Lal, X. Yang, and M. Gorospe Identification of a target RNA motif for RNA-binding protein HuR PNAS, March 2, 2004; 101(9): 2987 - 2992. [Abstract] [Full Text] [PDF]
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D. Campa, S. Zienolddiny, V. Maggini, V. Skaug, A. Haugen, and F. Canzian Association of a common polymorphism in the cyclooxygenase 2 gene with risk of non-small cell lung cancer Carcinogenesis, February 1, 2004; 25(2): 229 - 235. [Abstract] [Full Text] [PDF]
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C. Denkert, W. Weichert, S. Pest, I. Koch, D. Licht, M. Kobel, A. Reles, J. Sehouli, M. Dietel, and S. Hauptmann Overexpression of the Embryonic-Lethal Abnormal Vision-like Protein HuR in Ovarian Carcinoma Is a Prognostic Factor and Is Associated with Increased Cyclooxygenase 2 Expression Cancer Res., January 1, 2004; 64(1): 189 - 195. [Abstract] [Full Text] [PDF]
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 J. R. Brody, S. S. Kadkol, M. C. Hauer, F. Rajaii, J. Lee, and G. R. Pasternack pp32 Reduction Induces Differentiation of TSU-Pr1 Cells Am. J. Pathol., January 1, 2004; 164(1): 273 - 283. [Abstract] [Full Text] [PDF]
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T.-L. Erkinheimo, H. Lassus, A. Sivula, S. Sengupta, H. Furneaux, T. Hla, C. Haglund, R. Butzow, and A. Ristimaki Cytoplasmic HuR Expression Correlates with Poor Outcome and with Cyclooxygenase 2 Expression in Serous Ovarian Carcinoma Cancer Res., November 15, 2003; 63(22): 7591 - 7594. [Abstract] [Full Text] [PDF]
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K. Subbaramaiah, T. P. Marmo, D. A. Dixon, and A. J. Dannenberg Regulation of Cyclooxgenase-2 mRNA Stability by Taxanes: EVIDENCE FOR INVOLVEMENT OF p38, MAPKAPK-2, and HuR J. Biol. Chem., September 26, 2003; 278(39): 37637 - 37647. [Abstract] [Full Text] [PDF]
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 E.-S. Akool, H. Kleinert, F. M. A. Hamada, M. H. Abdelwahab, U. Forstermann, J. Pfeilschifter, and W. Eberhardt Nitric Oxide Increases the Decay of Matrix Metalloproteinase 9 mRNA by Inhibiting the Expression of mRNA-Stabilizing Factor HuR Mol. Cell. Biol., July 15, 2003; 23(14): 4901 - 4916. [Abstract] [Full Text] [PDF]
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L. B. Nabors, E. Suswam, Y. Huang, X. Yang, M. J. Johnson, and P. H. King Tumor Necrosis Factor {alpha} Induces Angiogenic Factor Up-Regulation in Malignant Glioma Cells: A Role for RNA Stabilization and HuR Cancer Res., July 15, 2003; 63(14): 4181 - 4187. [Abstract] [Full Text] [PDF]
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 D. C. Mitchell and N. H. Ing Estradiol Stabilizes Estrogen Receptor Messenger Ribonucleic Acid in Sheep Endometrium via Discrete Sequence Elements in Its 3'-Untranslated Region Mol. Endocrinol., April 1, 2003; 17(4): 562 - 574. [Abstract] [Full Text] [PDF]
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M. Kullmann, U. Gopfert, B. Siewe, and L. Hengst ELAV/Hu proteins inhibit p27 translation via an IRES element in the p27 5'UTR Genes & Dev., December 1, 2002; 16(23): 3087 - 3099. [Abstract] [Full Text] [PDF]
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H. Li, S. Park, B. Kilburn, M. A. Jelinek, A. Henschen-Edman, D. W. Aswad, M. R. Stallcup, and I. A. Laird-Offringa Lipopolysaccharide-induced Methylation of HuR, an mRNA-stabilizing Protein, by CARM1 J. Biol. Chem., November 15, 2002; 277(47): 44623 - 44630. [Abstract] [Full Text] [PDF]
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 Y. Moon and J. J. Pestka Vomitoxin-Induced Cyclooxygenase-2 Gene Expression in Macrophages Mediated by Activation of ERK and p38 but Not JNK Mitogen-Activated Protein Kinases Toxicol. Sci., October 1, 2002; 69(2): 373 - 382. [Abstract] [Full Text] [PDF]
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 J. Chung, R. E. Bachelder, E. A. Lipscomb, L. M. Shaw, and A. M. Mercurio Integrin ({alpha}6{beta}4) regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells J. Cell Biol., July 8, 2002; 158(1): 165 - 174. [Abstract] [Full Text] [PDF]
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W. Liu, J. Seto, G. Donovan, and M. Toth Jerky, a Protein Deficient in a Mouse Epilepsy Model, Is Associated with Translationally Inactive mRNA in Neurons J. Neurosci., January 1, 2002; 22(1): 176 - 182. [Abstract] [Full Text] [PDF]
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