Purification and characterization of a protein that permits early detection of lung cancer. Identification of heterogeneous nuclear ribonucleoprotein-A2/B1 as the antigen for monoclonal antibody 703D4.
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.
Zhou
J
J
Biomarkers and Prevention Research Branch, National Cancer Institute, Rockville, Maryland 20850-3300, USA.
Mulshine
J L
JL
Unsworth
E J
EJ
Scott
F M
FM
Avis
I M
IM
Vos
M D
MD
Treston
A M
AM
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003488:Cyanogen Bromide; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D010449:Peptide Mapping; D012333:RNA, Messenger / Q000235:genetics; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004268:DNA-Binding Proteins; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D003488:Cyanogen Bromide