hnRNP B1 Research Articles

Expression and value of hnRNP B1 in sputum of patients with lung cancer by immunohistochemistry method

The expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 increases significantly in lung cancer tissues. The aim of this study is to investigate the expression of hnRNP B1 in exfoliative cells in the sputum of patients with lung cancer and its sensitivity and specificity in diagnosis of lung cancer. The expression of hnRNP B1 in sputum was detected by immunohistochemistry (IHC) using anti-hnRNP B1 monoclonal antibody in 70 patients with lung cancer and 30 normal subjects (control group). The results of sputum exfoliative cytology were 27 positive in 70 samples diagnosed with lung cancer, whose sensitivity and specificity were 38.6% and 100.0% respectively. The results of sputum immunohistochemistry were 50 positive in 70 samples, with 23 positive in 29 squamous cell carcinoma and 17 positive in 26 adenocarcinoma and 10 positive in 15 small cell lung carcinoma. Sputum IHC of hnRNP B1 gave a sensitivity of 71.4% and specificity of 93.3%. Sputum IHC of hnRNP B1 was more sensitive than sputum exfoliative cytology (P < 0.05). hnRNP B1 overexpresses in sputum exfoliative cells and its sensitivity is better than sputum exfoliative cytology in diagnosis of lung cancer.

Alternative lengthening of telomeres in the human adrenocortical carcinoma cell line H295R

Telomere maintenance can occur in the absence of telomerase by a mechanism referred to as alternative lengthening of telomeres (ALT). ALT is considered to be rare in tumors of epithelial origin. The biological significance and molecular mechanism of ALT have not been well studied in human cancers. It has been reported that clinical samples of adrenocortical carcinoma show a low incidence of telomerase positivity. We characterized an adrenocortical carcinoma cell line, H295R, focusing on the telomere maintenance mechanism, and compared it with telomerase-positive 293 cells and HeLa cells. Telomerase activity could not be detected in H295R cells by the TRAP assay. Among the essential components of the telomerase holoenzyme, only hTERT expression was undetectable by RT-PCR, indicating that the lack of telomerase activity is due to suppression of hTERT. H295R cells had long and heterogeneous telomere DNA, and FISH revealed large nuclear bodies in a few interphase nuclei, which presumably represented ALT-associated PML bodies. These results suggest that H295R cells have the ALT phenotype. Several proteins that are possibly associated with the telomere maintenance mechanism were examined. TRF1 and TRF2 were expressed in H295R cells as well as in 293 cells and HeLa cells. The ratio of hnRNP B1 to A2 was higher in H295R cells than in 293 cells and HeLa cells. In conclusion, the H295R adrenocortical carcinoma cell line is negative for telomerase and maintains its telomeres by the ALT mechanism. We anticipate that H295R cells will be a good model for understanding the significance and mechanism of ALT in human cancers.

Combined Detection of CEA, CK-19 and c-met mRNAs in Peripheral Blood: A Highly Sensitive Panel for Potential Molecular Diagnosis of Non-Small Cell Lung Cancer

Objective: Detection of tumor-related mRNA in blood has become a potential cancer diagnostic approach. However, the sensitivity of single-marker assays is not high enough for clinical applications. The present study was aimed to evaluate the efficacy of a multimarker panel for molecular diagnosis of non-small cell lung cancer (NSCLC). Methods: Carcinoembryonic antigen (CEA), cytokeratin 19 (CK-19), c-met and heterogeneous nuclear ribonucleoprotein (hnRNP) B1 mRNAs were quantified by quantitative real-time reverse transcriptase polymerase chain reaction in 34 tumor tissues and 69 peripheral blood samples of NSCLC patients. Results: All four markers displayed high overexpression rates (range 82.3–97.1%) in NSCLC tumors. When used as single markers in blood for NSCLC diagnosis, CEA, CK-19, c-met and hnRNP B1 could only reach sensitivities of 52.2, 50.7, 42 and 17.4%, respectively. However, the sensitivity was enhanced up to 85.5% when CEA, CK-19 and c-met were combined in a 3-marker panel. Moreover, the expression of c-met and hnRNP B1 in blood was significantly correlated with patients’ pathological stages. Conclusions:The combined detection of CEA, CK-19 and c-met mRNAs in blood provided a valuable tool for molecular diagnosis of NSCLC. In addition, our results also suggested that hnRNP B1 was not a valuable diagnostic marker but a potential prognostic marker for NSCLC.

Prognostic and diagnostic relevance of hnRNP A2/B1, hnRNP B1 and S100 A2 in non-small cell lung cancer

Background: S100 A2 and hnRNP A2/B1 (heterogeneous ribonucleoprotein A2/B1) with its splicing variant hnRNP B1 are proteins which are involved in cellular proliferation, differentiation and protein synthesis and are up-regulated in non-small cell lung cancer (NSCLC). Since previous studies using paraffin-embedded tissues indicated a high potential of these markers for diagnosis and screening the present analysis intended to validate these data applying cryostat sections. Methods: 78 tumor-infiltrated lung cancer specimens and 66 adjacent histologically tumor-free tissues were analyzed; 11 autopsy specimens from patients who did not suffer from a malignant disease served as a control group. Cryostat sections were stained with monoclonal antibodies against hnRNP A2/B1, hnRNP B1 and S100 A2 and were compared with the previously established immunohistochemical profile of the same patients including EGFR, EGFRvIII, pEGFR, c-erbB-2, c-erbB-3, p53, Ki-67, bcl-2, p120 and microvessel density. Furthermore, these results were correlated with clinical parameters. Results: Expression of hnRNP A2/B1, hnRNP B1 and S100 A2 was increased in the tumor group when compared with the microscopic tumor-free specimens in 10% versus 5% (n.s.), 91% versus 5% and 65% versus 6%, respectively. Increased S100 and A2 hnRNP A2/B1 expressions were negative prognostic factors. With the exception of an increased EGFR expression in hnRNP A2/B1 negative cases the three analyzed markers did not correlate with the immunohistochemical parameters tested previously. Conclusion: Comparison between tumor probes and tumor-free specimens of NSCLC patients failed to approve the diagnostic relevance of hnRNP A2/B1 shown in previous studies, whereas hnRNP B1 revealed a high tumor specificity that could be helpful for tumor cell screening. Moreover, S100 A2 and hnRNP A2/B1 confirmed to be valuable prognostic parameters.

Roles of heterogeneous nuclear ribonucleoproteins A and B in cell proliferation

Overexpression of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and B1 has been observed in a variety of tumour types, however, it is unknown whether this dysregulation is a consequence of, or a driving force for, unregulated cell proliferation. We have shown that the levels of hnRNPs A1, A2 and B1, but not A3, are modulated during the cell cycle of Colo16 squamous carcinoma cells and HaCaT immortalized keratinocytes, suggesting that A1, A2 and B1 are needed at particular cell cycle stages. However, the levels of hnRNP A1, A2 and B1 mRNAs were constant, indicating that regulation of protein levels was controlled at the level of translation. RNAi suppression of hnRNP A1 or A3 alone did not affect the proliferation of Colo16 cells but the proliferation rate was significantly reduced when both were suppressed simultaneously, or when either was suppressed together with hnRNP A2. Reducing hnRNP A2 expression in Colo16 and HaCaT cells by RNAi led to a non-apoptotic-related decrease in cell proliferation, reinforcing the view that this protein is required for cell proliferation. Suppression of hnRNP A2 in Colo16 cells was associated with increased p21 levels but p53 levels remained unchanged. In addition, expression of BRCA1 was downregulated, at both mRNA and protein levels. The observed effects of hnRNP A2 and its isoforms on cell proliferation and their correlation with BRCA1 and p21 expression suggest that these hnRNP proteins play a role in cell proliferation.

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.

Expression of Heterogeneous Nuclear Ribonucleoproteins A2 and B1 in the Thyroid Follicular Cells

Several proteins implicated in hormonogenesis of the thyroid have alternatively spliced isoforms. Alternative splicing of pre-mRNA is considered to be important to regulate the hormonal activity. Heterogeneous nuclear ribonucleoproteins (hnRNP) A2 and B1 are two of the abundant nuclear RNA-binding proteins involved in alternative splicing. We examined the expression of hnRNP A2 and B1 in the thyroid, paying particular attention to the relationship between their function and the cellular morphology. B1 was expressed more frequently in cuboidal follicular cells that are hormonally active than in flat follicular cells in normal thyroid, although A2 expression showed no significant difference in two cell types. In Graves' disease, the patients who had high serum levels of triiodothyronine and thyroxine showed significantly increased expression of B1. B1 expression did not differ significantly between normal thyroids and thyroid neoplasms, except undifferentiated (anaplastic) carcinoma. Conclusively, B1 expression varied in relation to hormonal activity in thyroid follicular cells. B1 protein is a good immunohistological marker to detect hormonal activity of follicular cells, and may provide a key to elucidate the splicing mechanisms involved in thyroid hormonogenesis.

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.

Expression of hnRNP A2/B1 in human lung cancer cell lines

To explore the mRNA expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 in human lung cancer cell lines. Expression of hnRNP A2/B1 and hnRNP B1 mRNA was detected by RT-PCR using specific primers. Expression of hnRNP B1 protein was examined by immunocytochemistry using specific anti-hnRNP B1 antibody. RT-PCR indicated that the hnRNP A2/B2 and hnRNP B1 mRNA existed in the lung adenocarcinoma cell lines SPC-A1 and Y-90, squamous cell carcinoma cell line A431 and small cell lung cancer cell line NCI-H446. According to immunocytochemistry results, the expression of hnRNP B1 was observed in lung cancer cell lines, but the expression in squamous cell carcinoma cell line A431 and small cell lung cancer cell line NCI-H446 was markedly higher than that in adenocarcinoma cell lines SPC-A1 and Y-90. The results suggest that the human lung cancer cell lines detected have overexpression of hnRNP A2/B1 and hnRNP B1.

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.

HnRNP B1 expression in benign and malignant lung disease.

Evidence is accumulating to suggest that hnRNP B1 expression may be a useful tool in the early diagnosis of lung cancer. This study examined the immunohistochemical expression of hnRNP B1 in archived sections of resected lung cancers and compared the patterns of expression with those seen in similar archived sections of non-neoplastic lung. Particular attention was paid to the expression of hnRNP B1 in the benign bronchial cells in both cases, to establish if overexpression of this protein in respiratory epithelial cells is specific for malignancy. Nineteen cases of different types of non-small cell carcinoma were examined (eight squamous cell, six adenocarcinomas, two carcinosarcomas, two undifferentiated large cell carcinomas, and one mucoepidermoid carcinoma) and compared with sections from 16 open lung biopsies (three cases of cryptogenic fibrosing alveolitis, two cases of sarcoidosis, two cases of organizing pneumonia, and one case each of tuberculosis, extrinsic allergic alveolitis, non-specific interstitial pneumonitis, pneumocystis pneumonia, aspergilloma, respiratory bronchiolitis-interstitial lung disease, mineral dust disease, Sjögren's syndrome and systemic sclerosis vascular variant). All the tumours showed positive staining, with the vast majority, 16/19 (84%), showing strong diffuse nuclear staining. The background cells of these cases showed positive staining in alveolar macrophages, lymph node germinal centres, bronchial mucous glands, and bronchial epithelial cells. No significant difference was seen in the percentage of positive bronchial epithelial cells in bronchi adjacent to the tumour compared with the resection margins. In the benign lung cases, positive bronchial epithelial cells were seen in a small percentage, 3/16 (18%), of cases, but the majority of cases showed no or very focal staining. The levels of expression between benign epithelial cells of malignant cases, compared with benign, showed a significant difference when the staining was assessed in percentage of positive nuclei (p = 0.001, Fisher's exact test). The results confirm that hnRNP B1 is widely expressed in a range of lung carcinomas; that expression is seen in benign bronchial epithelial cells and inflammatory cells; and that expression in background bronchial epithelial cells appears to be higher in malignant than in benign lung disease. It is feasible that this biomarker may be of use in the detection of early lung cancer, provided that levels of expression can be accurately quantified.

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.

Heterogeneous nuclear ribonucleoprotein particle A/B proteins and the control of alternative splicing of the mammalian heterogeneous nuclear ribonucleoprotein particle A1 pre-mRNA.

Mammalian pre-messenger RNAs (pre-mRNAs) interact with a distinct set of proteins to form heterogeneous nuclear ribonucleoprotein particles (hnRNPs). More than 25 different hnRNP proteins have been found associated with these complexes (Pinol-Roma et al. 1988; Dreyfuss et al. 1993). The core hnRNP proteins A1, A2, B1, B2, C1, and C2 are the most abundant species and are expressed in actively growing cells to levels that are comparable to core histones (Beyer et al. 1977; Kiledjian et al. 1994). Although several observations support the notion that core hnRNP proteins participate in pre-mRNA processing, the question of whether these proteins act in a sequence-independent or a sequence-specific manner is still a matter of debate (Abdul-Manan and Williams 1996; Abdul-Manan et al. 1996; McAfee et al. 1997). Early investigations have suggested that hnRNP proteins associate randomly with many different types of pre-mRNAs and ribopolymers to form particles sharing common structures and sedimentation properties (McKnight and Miller 1979; Lamb and Daneholt 1979; Pullman and Martin 1983; Thomas et al. 1983; Wilk et al. 1983). While the great abundance of core hnRNP proteins in the nucleus of growing cells (approximately 0.2 mM in a HeLa cell nucleus) can be taken as an argument in favor of generic RNA binding activity, it is unclear what fraction of this population of hnRNP proteins is free to interact with a nascent RNA sequence that exits the RNA polymerase II transcription complex. More recently, the use of antibodies recognizing individual hnRNP proteins has revealed that some hnRNP proteins can be deposited nonrandomly on nascent pre-mRNAs (Pinol-Roma et al. 1989; Matunis et al. 1993; Wurtz et al. 1996), suggesting that binding can be sequence-specific. Given the high abundance of hnRNP proteins and their ability to bind to many types of sequences with a range of affinities (Cobianchi et al. 1988; Conway et al. 1988; Pinol-Roma et al. 1988; LeStourgeon et al. 1990; Casas-Finet et al. 1993; Abdul-Manan and Williams 1996; Abdul-Manan et al. 1996), it has remained unclear to what extent the functions of core hnRNP proteins in nuclear RNA processing always depend on their ability to bind RNA in a sequence-specific manner.

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 A3, a Novel RNA Trafficking Response Element-binding Protein

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41-, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.