Expression profiling in transformed human B cells: influence of Btk mutations and comparison to B cell lymphomas using filter and oligonucleotide arrays.
We have used both Clontech Atlas Human Hematology/Immunology cDNA microarrays, containing 588 genes, and Affymetrix oligonucleotide U95Av2 human array complementary to more than 12,500 genes to get a global view of genes expressed in Epstein-Barr virus (EBV)-transformed B cells and genes regulated by Bruton's tyrosine kinase (Btk). We compared EBV-transformed wild-type (WT) B cells from a healthy individual, WT1 and an X-linked agammaglobulinemia (XLA) patient cell line, XLA1, using the Clontech filters arrays. Eleven genes were > or =1.9-fold induced in absence of functional Btk. Furthermore, we analyzed a second patient cell line, XLA2, and compared this to two WT cell lines using oligonucleotide arrays. A total of 391 genes were found to be differentially expressed, including kinases and transcriptions factors. Furthermore, one expressed sequence tag and eight complementary DNA clones with unknown function were down-regulated in XLA2, indicating their biological role. Higher-fold inductions, Fyn (39.5), Hck (15.5) and Cyp1B1 (5.8), were observed using oligonucleotide array and were confirmed using real-time PCR for Fyn (20.8), Hck (6.7) and Cyp1B1 (10). Two genes, B cell translocation gene1 (BTG1) and B cell-specific OCT binding factor-1 (OBF-1) were induced > or =1.9-fold in both XLA1 and XLA2 analyzed by Atlas filter arrays andAffymetrix chips, respectively. Data from both filter and oligonucleotide arrays were compared to the gene clusters of a previously published lymphoma expression profile by linking to the UniGene transcript database. Our findings demonstrate for the first time the use of microarray to study the influence of Btk mutations and the use of functional annotation and validation of expression data by comparison of microarray analyses.
Islam
Tahmina C
TC
Center for BioTechnology, Department of Biosciences, Karolinska Institutet, NOVUM, Huddinge, Sweden. tahmina.islam@csb.ki.se
Lindvall
Jessica
J
Wennborg
Anders
A
Brandén
Lars J
LJ
Rabbani
Hodjatallah
H
Smith
C I Edvard
CI
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000361:Agammaglobulinemia / Q000201:enzymology / Q000235:genetics*; D002461:Cell Line, Transformed / Q000378:metabolism; D002472:Cell Transformation, Viral; D003199:Computer Systems; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000502:physiology; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D013382:Subtraction Technique; D014960:X Chromosome / Q000235:genetics
D018076:DNA, Complementary; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human