Heterogeneous Nuclear Ribonucleoprotein B1 Research Articles

Immunohistochemical study of the hnRNP A2 and B1 in the hippocampal formations of brains with Alzheimer's disease

To elucidate the post-transcriptional regulation in the subjects with Alzheimer's disease (AD), we employed immunohistochemical techniques and examined the expression of the heterogeneous nuclear ribonucleoprotein (hnRNP) A2 and B1 in the hippocampus with neurofibrillary tangle (NFT) neuropathology. In the mildly affected subjects (Braak stages I and II), the most intense A2 immunoreactivity was observed in the CA3 to CA1 neurons. In the moderately (Braak stages III and IV) and severely affected subjects (Braak stages V and VI), the CA1 region demonstrated a decrease in the number of A2 immunoreactive neurons and in immunoreactivity in the remaining neurons, while within the CA4 to CA2 in the severely affected subjects, the majority of neurons showed increased A2 immunoreactivity. An intense B1 immunoreactivity was observed throughout the CA subfields. In the CA1 subfield of the moderately affected subjects and in the extensive hippocampal regions of the severely affected subjects, a decrease in B1 immunoreactivity was observed. Double-immunolabeling studies demonstrated that tangle-bearing neurons reduced A2 and B1 immunoreactivity. Our study suggests that hnRNP A2 and B1 display different responses in the AD hippocampus, and further suggests that the post-transcriptional regulation is disturbed in neurons of the AD hippocampus.

Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.

Detection of plasma hnRNP B1 mRNA, a new cancer biomarker, in lung cancer patients by quantitative real-time polymerase chain reaction

Circulating cell-free nucleic acids are noninvasive diagnostic tools for cancer detection. Heterogeneous nuclear ribonucleoprotein (hnRNP) B1, an RNA binding protein, has been found overexpressed in the early stage of lung cancer, including bronchial dysplasia, a premalignant lesion of lung squamous cell carcinoma. To determine the utility of plasma hnRNP B1 RNA and as cancer detection markers for lung cancer, we analyzed plasma hnRNP B1 mRNA of lung cancer patients by real-time RT-PCR. Plasma RNA was extracted from plasma of 44 lung cancer patients, 7 lung neoplasm patients, 24 benign lung diseases and 25 healthy volunteers. Mean concentration of plasma hnRNP B1 mRNA in lung cancer patients was 0.99 pg/microg RNA, whereas that in healthy volunteers and in benign lung diseases was 0.23 pg/microg RNA and 0.30 pg/microg RNA, respectively (p<0.05). Twenty of 44 (45.5%) lung cancer patients showed more than 0.70 pg/microg RNA of plasma hnRNP B1 mRNA, compared with only 3 of 25 (12.0%) healthy volunteers. Looking at histological subtype, squamous cell carcinoma patients showed higher hnRNP B1 mRNA in the plasma than did adenocarcinoma patients, which is consistent with our previous immunohistochemistry results. These results indicate that plasma hnRNP B1 mRNA is a useful non-invasive markers for detection of lung cancer.

Autoantibodies against heterogeneous nuclear ribonucleoprotein B1 in CSF of MS patients

Heterogeneous nuclear ribonucleoproteins (hnRNPs) play an important role as the autoantigens in certain autoimmune disorders including neurological diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis and paraneoplastic neurological syndromes. To clarify their implication in multiple sclerosis (MS), we assayed antibodies (Abs) against hnRNP A and B proteins in sera and cerebrospinal fluid (CSF) of MS patients and compared the results with 25 patients with other neurological diseases (ONDs). Using recombinant hnRNP A1, A2, and B1 proteins and Western blotting for the assay, we found Abs against hnRNP B1 in CSF from 32 of 35 MS patients (91.4%) but not in any sera or CSF of the 25 OND patients. Most notably, no Abs against hnRNP B1 were found in sera of all 22 MS patients examined. Although Abs against hnRNP A1 and A2 were concomitantly found in CSF reacting with B1, their incidence and immunoreactivity were lower or weaker than those of anti-hnRNP B1 Abs. There was no correlation between the reactivity of CSF with hnRNP B1 and CSF parameters-such as the number of the cells and the IgG level-or clinical parameters-such as duration of illness and disease activity. The selective generation of Abs against hnRNP B1 in CSF was shown to be highly specific for MS, which makes them a disease marker.

Immunohistochemical study of hnRNP B1 in the postmortem temporal cortices of patients with Alzheimer's disease

In order to examine the post-transcriptional regulations in Alzheimer's disease, we employed immunohistochemical techniques and examined the expression of heterogeneous nuclear ribonucleoprotein (hnRNP) B1 in the inferior temporal cortex of subjects with Alzheimer's disease. In the mild cases, intense B1 immunoreactivity was observed in neurons of layer V, and less intense immunoreactivity was observed in layers II and III. The overall distributions and intensities of B1 immunoreactivity were undistinguishable among mild, moderate, and severe cases. Double-immunolabeling with MC1 and B1 demonstrated that B1 immunoreactivity was preserved in the majority of neurofibrillary tangle (NFT)-bearing neurons. Our study suggests that hnRNP B1-associated post-transcriptional regulations are preserved in the inferior temporal cortex of Alzheimer's disease.

Alterations of heterogeneous nuclear RNP A2 and B1 in the hippocampus of the rat after perforant pathway lesion

We examined alterations in post-transcriptional regulation following deafferentation of the perforant pathway by focusing on heterogeneous nuclear ribonucleoprotein (hnRNP) A2 and B1 in rat hippocampi subjected to perforant pathway lesions. In control brains, immunoreactivity to both was observed in the nuclei of neurons throughout the hippocampus using immunohistochemical techniques. From 1 to 14 days post-lesion, a slight increase in A2 immunoreactivity was observed in neurons within the dentate granular layer as well in the pyramidal cells of the cornus Ammon fields ipsilateral to the lesion. In contrast, we observed a marked decrease in B1 immunoreactivity in the same regions at 1, 3 and 7 days post-lesion. All these alterations, however, were transient. A2 immunoreactivity returned to normal levels by 30 days post-lesion, and B1 immunoreactivity had completely recovered by 14 days post-lesion. The results of immunoblot analysis for A2 and B1 were wholly consistent with immunohistochemical observations. Our study suggests that post-transcriptional regulation in the hippocampal neurons changes after a perforant pathway lesion. Our study further suggests that the functions of hnRNPA2 and B1 are different, as each was differentially involved in the plastic response to deafferentation of the perforant pathway.

Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing

The genetic elements that are responsible for establishing a transcriptionally competent, open chromatin structure at a region of the genome that consists only of ubiquitously expressed, housekeeping genes are currently unknown. We demonstrate for the first time through functional analysis in stably transfected tissue culture cells that transgenes containing methylation-free CpG islands spanning the dual divergently transcribed promoters from the human TATA binding protein ( TBP)–proteasome component-B1 ( PSMB1) and heterogeneous nuclear ribonucleoprotein A2/B1 ( HNRPA2B1)–heterochromatin protein 1Hs-γ (chromobox homolog 3, CBX3) gene loci are sufficient to prevent transcriptional silencing and a variegated expression pattern when integrated within centromeric heterochromatin. In addition, only transgene constructs extending over both the HNRPA2B1 and the CBX3 promoters, and not the HNRPA2B1 promoter alone, were able to confer high and stable long-term EGFP reporter gene expression. These observations suggest that methylation-free CpG islands associated with dual, divergently transcribed promoters possess an independent dominant chromatin opening function and may therefore be major determinants in establishing and maintaining a region of open chromatin at housekeeping gene loci.

HnRNP B1 protein may be a possible prognostic factor in squamous cell carcinoma of the lung

Heterogeneous nuclear ribonucleoprotein (hnRNP) B1 is an RNA-binding protein that is required for the maturation of mRNA precursor. It was previously reported that hnRNP A2/B1 was overexpressed at the early clinical stage of lung cancer, and that hnRNP B1 protein, a splicing variant of hnRNP A2 mRNA, was elevated in lung cancer tissues. In this study, we applied the immunohistochemical method, using anti-hnRNP B1 antibody to analyze the usefulness of the hnRNP B1 antibody as a prognostic marker and also as a marker useful for early detection. A total of 206 specimens were examined. Histological examination revealed this protein to be positive in 79 (71.2%) of 111 squamous cell carcinomas and in 45 (64.3%) of 70 adenocarcinomas, respectively. This protein was also expressed in 24 (63.2%) of 38 roentgenographically occult carcinomas and in seven (63.6%) of 11 dysplastic lesions. These findings suggest the possible participation of this protein in early carcinogenesis. Furthermore, the survival curve of the squamous cell carcinoma patients with hnRNP B1 overexpresseion showed a better prognosis compared with that of the patients without hnRNP B1 expression ( P=0.014), whereas in adenocarcinoma patients, there was no such a difference between them ( P=0.889). These findings indicate that hnRNP B1 could be a useful marker for the early detection of bronchogenic squamous cell carcinoma and that it may be a prognostic factor in this cell type.

Detection and discrimination of preneoplastic and early stages of lung adenocarcinoma using hnRNP B1 combined with the cell cycle-related markers p16, cyclin D1, and Ki-67

Heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), an RNA binding protein, is a useful marker for early detection of lung squamous cell carcinoma because it is overexpressed in the early stages of lung cancer, including bronchial dysplasia, a premalignant lesion of lung squamous cell carcinoma. In the case of adenocarcinoma, we investigated the utility of hnRNP B1 for both detection of early adenocarcinoma and discrimination of non-invasive lesion, atypical adenomatous hyperplasia (AAH) from adenocarcinoma. hnRNP B1, cyclin D1, p16, and Ki-67 were analyzed in lung adenocarcinoma tissues and divided into early and overt adenocarcinoma and AAH, using immunohistochemistry. The intensity of these molecular markers was compared among three groups and also analyzed for 4 patients who showed both adenocarcinoma and AAH. Thirty-six of 54 (67%) adenocarcinoma patients showed positive staining of hnRNP B1: 14/20 (70%) early adenocarcinoma and 22/34 (65%) overt adenocarcinoma. In contrast, overexpression of hnRNP B1 in non-invasive lesion, AAH was observed in only 9% (1/11). Overexpression of cyclin D1 and decrease of p16 were frequently observed in both adenocarcinoma and AAH. These results suggest that hnRNP B1 would be a candidate of molecular marker for detection of early lung adenocarcinoma. In addition, combined analysis of hnRNP B1 and cell cycle-related genes, such as cyclin D1 and p16, might aid in discrimination of AAH from early adenocarcinoma.

Imbalance Between Apoptosis and Telomerase Activity in Myelodysplastic Syndromes: Possible Role in Ineffective Hemopoiesis

The myelodysplastic syndromes (MDS) are a group of disorders characterized by peripheral pancytopenia despite normo- or hyper-cellular bone marrow. This is thought to be due to apoptosis of hematopoietic bone marrow cells, resulting in ineffective hematopoiesis. The heterogeneous nuclear ribonucleoprotein (hnRNP) B1 is involved in pre-mRNA processing and binds to telomeric cDNA repeats. The hnRNP B1 is a marker for early cancer. The aim of our study was to clarify the relationships between prognosis and apoptosis, telomerase activity (TA) and hnRNP expression in the bone marrow. The subjects were 51 patients with MDS, including patients with refractory anemia (RA) <formula>(<italic>n</italic>=32),</formula> refractory anemia with ringed sideroblasts (RARS) <formula>(<italic>n</italic>=1),</formula> refractory anemia with excess blasts (RAEB) <formula>(<italic>n</italic>=7),</formula> refractory anemia with excess blasts in transformation (RAEB-t) <formula>(<italic>n</italic>=8)</formula> and chronic myelomonocytic leukemia (CMMoL) <formula>(<italic>n</italic>=3).</formula> We also studied 6 cases with acute myelogenous leukemia (AML) arising from MDS (AML-MDS) and 10 control subjects. Bone marrow biopsies were stained immunohistochemically for caspase-3 (marker of apoptotic activity) and human telomerase reverse transcriptase (hTERT), and hnRNP B1. Fatal pancytopenia was the cause of death in 19 of the 51 patients. The caspase-3 positive cell rate was higher in MDS (16.3%) than in controls (4.4%) and AML-MDS (0.5%). The percentage of hnRNP B1-positive cells was higher in MDS (15.3%) and AML-MDS (56.3%) than in controls (5.6%). In MDS, hnRNP B1 levels were higher in RAEB and RAEB-t subtypes than in RA and RARS. The percentage of hTERT-positive cells was higher in AML-MDS (50.0%) than in controls (20.2%) and MDS (23.6%). Our findings suggest that activation of apoptosis occurs in MDS in the absence of hTERT expression, implicating high apoptosis in the absence of high TA with ineffective hematopoiesis. Poor prognosis correlated with higher caspase-3 and lower hTERT rates. In MDS, hnRNP B1 activity may be associated with leukemic transformation.

Overexpression of heterogeneous nuclear ribonucleoprotein B1 in lymphoproliferative disorders: High expression in cells of follicular center origin

It is reported that overexpression of hnRNP A2 and B1 proteins is useful for detecting early cancers, and that B1, a splicing minor isoform of A2, is more specific than A2. The B1 expression is still undetermined in human lymphoid tissues. We quantitatively studied the B1 expression in 85 lymph node specimens, comprising reactive lymphoid hyperplasia (RLH; n=8), B-cell lymphoma (n=23), T-cell lymphoma (n=22), and metastatic carcinoma (n=32). Immunostaining and immunoblotting analyses with an anti-B1 monoclonal antibody, 2B2 were performed, and the two sets of results correlated with each other (p<0.05). In RLH specimens, B1 expression rate was significantly higher in follicular centers (FC; 44%) than in mantle zone (MZ; 15%) and paracortex (16%) (p<0.01). B1 expression was statistically higher in B-cell lymphoma than in T-cell lymphoma (p<0.01). In B-cell lymphomas, B1 expression rates were 51% in diffuse large B-cell lymphoma (DLBL; n=5) and 45% in follicular lymphoma (FL; n=16), and they were almost the same as that of the FC. Especially in DLBLs, CD10+ FC-origin lymphomas expressed greater amount of B1 than CD10- non-FC-origin lymphomas. B1 expression rate was low in mantle cell lymphoma (MCL; n=2) and similar to that of MZ in RLH. These results suggest that B1 expression is associated with differentiation in lymphoid tissue rather than transformation. B1 expression increases during the process of B-cell differentiation in the FC, and that high B1 expression is maintained in B-cell lymphomagenesis, especially in cells of FC-origin DLBL.

Lung cancer prevention with (-)-epigallocatechin gallate using monitoring by heterogeneous nuclear ribonucleoprotein B1

Considering the problems involved in prevention of human lung cancer, growth inhibition of human lung cancer cell line A549 was studied with emphasis on two parameters: green tea polyphenols, such as (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG); and heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), a new biomarker of human lung cancer which is highly expressed in the very early stages of human lung cancer. The inhibitory potencies of green tea polyphenols were compared with those of genistein as a control. EGCG or ECG and genistein as a control dose-dependently inhibited the growth of A549 cells, which strongly elevated hnRNP B1 protein, and increased G2/M phase cells associated with induction of apoptotic cells. The results were confirmed by previous evidence with human lung cancer cell line PC-9. Some larger differences in mechanisms of action between green tea polyphenols and genistein were presented. Treatment of A549 cells with EGCG, ECG or genistein significantly inhibited the expression levels of hnRNP B1 mRNA and the elevated levels of hnRNP B1 protein, both of which are constitutively elevated in cancer cells. Furthermore, both EGCG and genistein inhibited the promoter activity of hnRNP A2/B1 gene expression, with IC50 values 29 microM for EGCG and 66 microM for genistein, suggesting the interaction of EGCG or genistein with the transcriptional complex. Looking at our results here, and those of previously reported epidemiological studies with green tea, we discuss the steadily accumulating evidence that clinical trials with green tea extract would be an efficient means of lung cancer prevention.

Heterogeneous nuclear ribonucleoprotein B1 expressed in esophageal squamous cell carcinomas as a new biomarker for diagnosis.

We recently reported that heterogeneous nuclear ribonucleoprotein (hnRNP) B1 was overexpressed in most human lung cancers, especially squamous cell carcinoma (SCC), as well as human oral SCC. To find the significance of hnRNP B1 in cancer diagnosis, we studied hnRNP B1 expression in 16 paraffinized sections of esophageal SCC, using immunohistochemical staining with antihnRNP B1 polyclonal antibody, raised in a rabbit. We compared the expression of hnRNP B1 in cancerous and noncancerous regions of the same specimen: enhanced expression was observed in 63% of cancerous regions (10/16), whereas none of the noncancerous regions showed enhanced expression. The enhanced expression of hnRNP B1 in cancerous regions was compared with that in noncancerous tissue in relation to histopathological grade: 83% for well differentiated (5/6), 83% for moderately differentiated (5/6) and 0% for poorly differentiated (0/4). Histologically, enhanced expression of hnRNP B1 was observed around cancer pearls, as well as in the cells of nests lacking keratinization in well and moderately differentiated SCC. Western blotting analysis revealed enhanced expression in three frozen specimens of moderately differentiated SCC. Using esophageal cancer cell lines, we further confirmed the decreased expression in poorly differentiated SCC cells, compared with other differentiation types. All our results support the significance of hnRNP B1 expression in esophageal SCC as a unique diagnostic marker with regard to association between expression level and histopathological grading.

Significance of heterogeneous nuclear ribonucleoprotein B1 as a new early detection marker for oral squamous cell carcinoma.

The development of an early tumor detection marker for oral cancer is an obvious need due to the high recurrence rate and poor survival rate. Based on our previous report that overexpression of heterogeneous nuclear ribonucleoprotein (hnRNP) B1 protein was found in 100% of squamous cell carcinomas of human lung, we applied the same immunohistochemical method, using anti‐hnRNP B1 antibody, to human oral squamous cell carcinoma (OSCC). Seven human tissue sections of OSCC showed strong staining with anti‐hnRNP B1 antibody, and hnRNP B1 protein of 37 kDa was identified in protein fractions isolated from six of the cancerous tissue sections, while it was not found in adjacent noncancerous tissue. Moreover, three non‐homogeneous (nodular) leukoplakia sections showed significant anti‐hnRNP B1 staining. The results suggest that this antibody detects precancerous lesions as well as advanced lesions (stages I to IV) of OSCC. We also present positive results of cytodiagnosis for two smear specimens. All of the above results indicate that hnRNP B1 is a new and useful marker for early detection of OSCC.

Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts.

We have isolated cDNAs for the major heterogeneous nuclear ribonucleoprotein (hnRNP) A2, B1, and C2 proteins and determined their nucleotide and deduced amino acid sequences. The A2 and B1 cDNAs are identical except for a 36-nucleotide in-frame insert in B1. Similarly, the sequence of the C2 protein cDNA is related to that of C1 in that C2 contains an extra 39 in-frame nucleotides. Therefore, the B1 amino acid sequence is identical to A2 except for the insertion of 12 amino acids near its amino terminus, and C1 and C2 are also identical to each other except for an extra 13 amino acids near the middle of C2. All three proteins are members of a large family of RNA binding proteins that contain the consensus sequence-type RNA binding domain (CS-RBD). The A2 and B1 proteins have a modular structure similar to that of the hnRNP protein A1: they contain two CS-RBDs and a glycine-rich auxiliary domain at the carboxyl terminus. The CS-RBDs of A2 and B1 have approximately 80% amino acid identity with those of A1, whereas the glycine-rich auxiliary domain is considerably more divergent with less than 30% of the amino acids being identical. These findings indicate that the addition of small peptides, probably by alternative pre-mRNA splicing, generates some of the diversity apparent among hnRNP proteins.