Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray.
To identify new diagnostic markers and drug targets for pancreatic cancer, we compared the gene expression patterns of pancreatic cancer cell lines growing in tissue culture with those of normal pancreas using cDNA microarray analysis. Fluorescently (cyanine 5) labeled cDNA probes, made individually from mRNA samples of nine pancreatic cell lines, were each combined with fluorescently (cyanine 3) labeled universal reference mRNA. The mixed probes of each sample were then hybridized with 5760 cDNA arrays (5289 unique cDNA sequences) printed on individual microscope slides. Fluorescently (cyanine 5) labeled normal pancreas mRNA was also compared with the same universal reference mRNA reference pool. The expression ratios of neoplastic versus normal pancreas cells were then calculated by multiplying the ratio of cancer versus the universal reference mRNA and the ratio of the universal reference mRNA cell versus normal pancreas. For 5289 different genes interrogated by the arrays, 30 of them showed an expression ratio 2 SD from the mean in at least three of the nine pancreatic cell lines studied. To confirm the expression profiles of these genes, quantitative reverse transcription-PCR and Northern blot were carried out for 25 of the overexpressed genes. To verify the overexpression in patient samples, two of the overexpressed genes, c-Myc and Rad51, were selected to undergo analysis by reverse transcription-PCR in frozen tumor tissues and by immunostaining in paraffin-embedded tissue section microarrays. The results of these experiments are in agreement with the microarray data. Potential up-regulated targets of note from this study include urokinase-type plasminogen activator receptor, serine/threonine kinase 15, thioredoxin reductase, and CDC28 protein kinase 2, as well as several others.
Han
Haiyong
H
Department of Pathology, Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA.
Bearss
David J
DJ
Browne
L Walden
LW
Calaluce
Robert
R
Nagle
Raymond B
RB
Von Hoff
Daniel D
DD
D014408:Biomarkers, Tumor / Q000235:genetics; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D020869:Gene Expression Profiling; D006367:HeLa Cells; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; C495153:RAD51 protein, human; D051135:Rad51 Recombinase