ABSTRACT
Immunocytochemical studies have revealed that overexpression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/ B1 in exfoliated epithelial cells is a potentially useful marker of early lung cancer. This study analyzed the correlation of hnRNP A2/B1 expression with molecular alterations in phenotypically different epithelial cells of paraffin-embedded pulmonary tissues. Sections from 20 human subjects were analyzed immunohistochemically for expression of hnRNP A2/B1. Normal-appearing, hyperplastic, and malignant epithelial cells with and without hnRNP A2/B1 expression (n = 78) were microdissected and assessed for microsatellite alterations (MA) and loss of heterozygosity (LOH) (n = 14 markers) as well as for clonality. Results showed that (1) hnRNP A2/B1 immunoreactive cells contained a significantly higher frequency of MA and LOH than did comparable cells that lacked detectable hnRNP A2/B1; (2) over 80% of MA and LOH seen in hnRNP A2/B1 immunoreactive normal-appearing and hyperplastic cells persisted in malignant cells; (3) preliminary analysis of methylation status of the androgen receptor gene in non-neoplastic cells was suggestive of hnRNP A2/B1-expressing cells being of clonal origin; and (4) cells with cytoplasmic hnRNP A2/B1 immunoreactivity had a 3-fold higher frequency of MA and LOH than did cells with nuclear hnRNP A2/B1 immunoreactivity. These findings suggest that phenotypically different respiratory epithelial cells with hnRNP A2/B1 overexpression might be clonally derived, and that the subcellular localization of hnRNP A2/B1 might be an important factor associated with tumor progression.
Subject(s)
Gene Expression Regulation, Neoplastic , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung/metabolism , Ribonucleoproteins, Small Nuclear/biosynthesis , Ribonucleoproteins, Small Nuclear/genetics , Cell Division , Humans , Hyperplasia , Immunohistochemistry , Lung/pathology , Lung Neoplasms/pathologyABSTRACT
High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC cell lines tested expressed both isoforms of cyclooxygenases (COX). The pan-COX inhibitor ketorolac continuously and significantly decreased PGE(2) production and IL-6 and IL-8 levels in all OPC cell lines tested, but did not affect IL-1alpha, GM-CSF levels, or in vitro tumor cell growth. In contrast, ketorolac reduced OPC growth in vivo. The OPC cell lines used express the IL-6 receptor, and IL-6 stimulation of these cells causes transduction to occur via STAT3 pathway activation. Coincubation with OPC cell lines with conditioned medium from a TPA-exposed HL-60 cells stimulated growth proportional to the IL-6 levels measured in the conditioned medium. This growth effect was specifically inhibited by anti-IL-6 antibody. These results are consistent with cytokine products of inflammatory cells having paracrine growth effects on OPC. If chronic inflammation plays a role in promoting the development of OPC, this mechanism may also apply to other epithelial tumor systems modulated by COX activity.
Subject(s)
Inflammation/metabolism , Interleukin-6/metabolism , Oropharyngeal Neoplasms/enzymology , Oropharyngeal Neoplasms/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Animals , Antibodies/immunology , Antibodies/pharmacology , Arachidonic Acid/metabolism , Cell Division/drug effects , Clinical Trials, Phase II as Topic , Culture Media, Conditioned/metabolism , Culture Media, Conditioned/pharmacology , Cyclooxygenase Inhibitors/pharmacology , DNA-Binding Proteins/metabolism , Dinoprostone/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , HL-60 Cells , Humans , Interleukin-1/metabolism , Interleukin-1/pharmacology , Interleukin-6/antagonists & inhibitors , Interleukin-6/immunology , Interleukin-6/pharmacology , Interleukin-8/metabolism , Interleukin-8/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Ketorolac/pharmacology , Mice , Mice, Inbred BALB C , Models, Biological , Oropharyngeal Neoplasms/pathology , Paracrine Communication/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Interleukin-6/metabolism , STAT3 Transcription Factor , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Trans-Activators/metabolism , Tumor Cells, CulturedABSTRACT
We have reported that a mouse monoclonal antibody 703D4, detects lung cancer 2 years earlier than routine chest x-ray or cytomorphology. We purified the 703D4 antigen to elucidate its role in early lung cancer biology, using Western blot detection after SDS-polyacrylamide gel electrophoresis. Purification steps included anion exchange chromatography, preparative isoelectric focusing, polymer-based C18-like, and analytical C4 reverse phase high performance liquid chromatography. After 25-50,000-fold purification, the principal immunostaining protein was > 95% pure by Coomassie staining. The NH2 terminus was blocked, so CNBr digestion was used to generate internal peptides. Three sequences, including one across a site of alternate exon splicing, all identified a single protein, heterogeneous nuclear ribonucleoprotein-A2 (hnRNP-A2). A minor co-purifying immunoreactive protein resolved at the final C4 high performance liquid chromatography step is the splice variant hnRNP-B1. Northern analysis of RNA from primary normal bronchial epithelial cells demonstrated a low level of hnRNP-A2/B1 expression, consistent with immunohistochemical staining of clinical samples, and increased hnRNP-A2/B1 expression was found in lung cancer cells. hnRNP-A2/B1 expression is under proliferation-dependent control in normal bronchial epithelial cell primary cultures, but not in SV40-transformed bronchial epithelial cells or tumor cell lines. With our clinical data, this information suggests that hnRNP-A2/B1 is an early marker of lung epithelial transformation and carcinogenesis.
Subject(s)
Antibodies, Monoclonal/immunology , Carcinoma, Non-Small-Cell Lung/diagnosis , DNA-Binding Proteins/isolation & purification , Lung Neoplasms/diagnosis , Neoplasm Proteins/isolation & purification , Amino Acid Sequence , Base Sequence , Blotting, Northern , Blotting, Western , Carcinoma, Non-Small-Cell Lung/metabolism , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Cyanogen Bromide , DNA Primers , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , DNA-Binding Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Heterogeneous-Nuclear Ribonucleoprotein Group A-B , Humans , Lung Neoplasms/metabolism , Molecular Sequence Data , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , Peptide Mapping , RNA, Messenger/genetics , Tumor Cells, CulturedABSTRACT
Signal transduction pathways shared by different autocrine growth factors may provide an efficient approach to accomplish clinically significant control of lung cancer growth. In this study, we demonstrate that two autocrine growth factors activate 5-lipoxygenase action of the arachidonic acid metabolic pathway in lung cancer cell lines. Both growth factors increased the production of 5(S)-hydrooxyeicosa-6E,8Z,11Z,14Z-tetraeno ic acid (5-HETE), a major early 5-lipoxygenase metabolic product. Exogenously added 5-HETE stimulated lung cancer cell growth in vitro. Inhibition of 5-lipoxygenase metabolism by selective antagonists resulted in significant growth reduction for a number of lung cancer cell lines. Primary clinical specimens and lung cancer cell lines express the message for the 5-lipoxygenase enzymes responsible for the generation of active metabolites. In vivo evaluation demonstrated that interruption of 5-lipoxygenase signaling resulted in enhanced levels of programmed cell death. These findings demonstrate that 5-lipoxygenase activation is involved with growth factor-mediated growth stimulation for lung cancer cell lines. Pharmacological intervention with lipoxygenase inhibitors may be an important new clinical strategy to regulate growth factor-dependent stages of lung carcinogenesis.
