BackgroundHeterogeneous nuclear ribonucleoproteins (hnRNPs) of the A/B type (hnRNP A1, A2/B1, A3) are highly related multifunctional proteins participating in alternative splicing by antagonising other splicing factors, notably ASF/SF2. The altered expression pattern of hnRNP A2/B1 and/or splicing variant B1 alone in human lung cancer and their potential to serve as molecular markers for early diagnosis remain issues of intense investigation. The main objective of the present study was to use paired tumour/non-tumour biopsies from patients with non-small cell lung cancer (NSCLC) to investigate the expression profiles of hnRNP A1, A2/B1 and A3 in conjunction with ASF/SF2.MethodsWe combined western blotting of tissue homogenates with immunohistochemical examination of fixed tissue sections and quantification of mRNA expression levels in tumour versus adjacent normal-looking areas of the lung in the same patient.ResultsOur study, in addition to clear evidence of mostly uncoupled deregulation of hnRNPs A/B, has revealed hnRNP A1 to be the most deregulated protein with a high frequency of over-expression (76%), followed by A3 (52%) and A2/B1 (43%). Moreover, direct comparison of protein/mRNA levels showed a lack of correlation in the case of hnRNP A1 (as well as of ASF/SF2), but not of A2/B1, suggesting that different mechanisms underlie their deregulation.ConclusionOur results provide strong evidence for the up-regulation of hnRNP A/B in NSCLC, and they support the existence of distinct mechanisms responsible for their deregulated expression.

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Spliceosomal small nuclear ribonucleoproteins (U-snRNPs) are frequent and specific targets of autoantibodies in systemic rheumatic diseases. The abundant, functionally related heterogeneous nuclear ribonucleoprotein complexes (hnRNPs) have later defined as a new target of autoantibodies, of which their immunochemical/immunogenic and pathogenic properties are still under investigation. Among hnRNP proteins, those belonging to the A/B type are considered as the major autoantigens targeted by antibodies in sera of patients suffering with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). By performing an extensive screening using rat liver 40S hnRNP antigenic material, we document here the existence of multiple specificities of anti-hnRNP A/B autoantibodies in sera of Greek patients suffering with a spectrum of systemic rheumatic diseases. This included patients with SLE, Sjogren's syndrome (SS), Scleroderma (SSc) and a specific group of patients mostly with undifferentiated disease (UD patients). In total, four distinct types of anti-hnRNP A/B autoantibodies have been recognized. The first two referred to the known anti-hnRNPA2(RA33) and anti-hnRNP A1; the latter appearing very rarely. The third was of the new type selectively reacting with hnRNP B2 and an hnRNP A3 variant, while the fourth was a rare case of anti-hnRNP B2 alone. In addition, a novel specificity of autoantibodies against hnRNP L protein was identified in association with anti-hnRNP A/B antibodies. The co-existence within a serum of autoantibodies having variable specificity for hnRNP A/B and L autoantigens was shown. Specific immunochemical features of the identified autoantibodies are presented and a possible mechanism of autoepitope spreading within protein components of hnRNP complexes is discussed.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) constitute an important group of RNA-binding proteins (RBPs) that play an active role in post-transcriptional gene regulation. Here, we focus on representative members of the hnRNP group of RBPs, namely hnRNP A1 and hnRNP C1/C2, which participate mainly in RNA splicing, as well as on HuR, a prototype of the AU-rich element-binding proteins (ARE-BP), which has an established role in regulating the stability and translation of target mRNAs. HuR and most hnRNPs are primarily localized in the nucleoplasm, and they can shuttle between the nucleus and the cytoplasm. Herein, we have extended our recently reported findings on the ability of HuR to associate with the immunopurified from mammalian cell extracts hnRNP and mRNP complexes by the application of an anti-HuR antibody that selects HuR-RNP complexes. We find that the protein components precipitated by the anti-HuR antibody are very similar to the hnRNP-HuR complexes reported previously. The in vivo association of HuR and hnRNP proteins is examined in the presence and the absence of thermal stress by confocal microscopy of intact cells and by in situ nuclear matrix preparation. We find notable heat-induced changes of HuR and of hnRNP A1, which exit the nucleus and co-localize to large cytoplasmic foci that represent heat-induced stress granules. The functional implications of HuR-hnRNP interactions in stressed and unstressed cells are discussed.

New therapeutic approaches are needed for lung cancer, the leading cause of cancer death. Methylating agents constitute a widely used class of anticancer drugs, the effect of which on human non small cell lung cancer (NSCLC) has not been adequately studied. N-methyl-N-nitrosourea (MNU), a model S(N)1 methylating agent, induced cell death through a distinct mechanism in two human NSCLC cell lines studied, A549(p53(wt)) and H157(p53(null)). In A549(p53(wt)), MNU induced G2/M arrest, accompanied by cdc25A degradation, hnRNP B1 induction, hnRNP C1/C2 downregulation. Non-apoptotic cell death was confirmed by the lack of increase in the sub-G1 DNA content, Poly (ADP-ribose) polymerase cleavage and caspase-3, -7 activation. In H157(p53(null)), MNU induced apoptotic cell death, confirmed by cytofluorometry of DNA content and immunodetection of apoptotic markers, accompanied by overexpression of hnRNP B1 and C1/C2. Thus, the mechanism of the cell death induced by S(N)1 methylating agents is cell type-dependent and must be assessed prior treatment.