Elevated expression of S100P, CAPL and MAGE 3 in doxorubicin-resistant cell lines: comparison of mRNA differential display reverse transcription-polymerase chain reaction and subtractive suppressive hybridization for the analysis of differential gene expression.
Subtractive suppressive hybridization (SSH) and mRNA differential display reverse transcription-polymerase chain reaction (DDRT-PCR) were compared for their ability to detect the expression of drug-resistance associated genes in a doxorubicin-resistant and -sensitive colon carcinoma cell line (LoVo H67P). The expression pattern of more than 9000 bands obtained by DDRT-PCR were identical in both cell lines by more than 95%. Of the remaining differentially expressed DDRT-PCR products, 21 cDNA fragments were further analyzed after cloning. A total of 210 clones were sequenced resulting in 40 different sequences of which only five were differentially expressed as revealed by Northern blot analysis. SSH, on the other hand, resulted in 30 different sequences of 37 clones analyzed. Thirteen of 30 sequences (43%) could be identified by databank analysis (excluding expressed sequence tags) in contrast to nine of 40 clones (23%) obtained by DDRT-PCR. Of the clones identified by SSH, 60% exhibited a differential expression comparing the doxorubicin-resistant and -sensitive cell line, respectively, as compared to only 13% of the DDRT-PCR derived clones. The application of SSH resulted in the identification of differentially expressed genes in three doxorubin-resistant cell lines (LoVo DxR, ARH D60 and KB-V1) as compared to the sensitive parental cell lines. A significant higher expression of S100P, a protein involved in calcium metabolism, as well as MAGE 3 (melanoma antigen gene) was found in the resistant cell lines using this methodology. The expression of CAPL, a second protein involved in calcium metabolism, was only moderately elevated in the doxorubicin-resistant cells. We found that subtractive suppressive hybridization proved to be a more rapid and reliable method for the detection of differentially expressed mRNAs in our system.
Bertram
J
J
Department of Hematology/Oncology, University of Göttingen, Germany.
Palfner
K
K
Hiddemann
W
W
Kneba
M
M
D000951:Antigens, Neoplasm / Q000096:biosynthesis; D002135:Calcium-Binding Proteins / Q000096:biosynthesis*; D002460:Cell Line; D003001:Cloning, Molecular; D003110:Colonic Neoplasms; D018076:DNA, Complementary / Q000737:chemistry; D004317:Doxorubicin / Q000633:toxicity*; D019008:Drug Resistance, Neoplasm; D006801:Humans; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; C072013:MAGEA3 protein, human; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C103003:S100P protein, human; C077591:S100A4 protein, human; D004317:Doxorubicin