Identification of genes differentially expressed in breast cancer cell line SKBR3: potential identification of new prognostic biomarkers.
The identification of differentially expressed genes in tumour cells should have important implications in understanding carcinogenesis and developing new therapeutic and prognostic biomarkers. We have combined PCR-based cDNA subtraction and Northern blotting to identify truly differentially expressed genes in breast cancer cell line SKBR3 as compared to normal human mammary epithelial cells (HMEC). Hybridizing probe molecules were rescued from the Hybond N+ membranes and then PCR reamplified. The PCR reamplification is possible due to the fact that all probe molecules contain the same pair of adapter sequences on both ends. After cloning and sequencing three known genes, ribosomal protein L19 (RPL19), ADP/ATP carrier protein and ErbB-2 with high-elevated mRNA levels in SKBR3 were identified. In addition, two overexpressed genes with unknown functions, CXYorf1-related protein and hypothetical protein PRO2605, were found. High-titer andibodies against the recombinant RPL19 were detected in 5 patients out of 50 patients investigated. Thus, the present novel strategy based on the combination of PCR-based cDNA subtraction and Northern blotting should facilitate the identification of truly differentially expressed biomarkers, which may offer the potential to determine the proper drug for an individual patient at a given stage of disease or treatment.
Leirdal
Marianne
M
Institute for cancer Research, Department of Immunology, Molecular Medicine Group, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
Shadidy
Mohsen
M
Røsok
Øystein
Ø
Sioud
Mouldy
M
D014408:Biomarkers, Tumor / Q000378:metabolism*; D015152:Blotting, Northern; D001940:Breast / Q000378:metabolism; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D000226:Mitochondrial ADP, ATP Translocases / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011379:Prognosis; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D013997:Time Factors
D014408:Biomarkers, Tumor; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D000226:Mitochondrial ADP, ATP Translocases; D011506:Proteins; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C489092:RPL19 protein, human; C407225:SPRY3 protein, human