Microarray and pathway analysis reveals decreased CDC25A and increased CDC42 associated with slow growth of BCL2 overexpressing immortalized breast cell line.
Bcl-2 is an anti-apoptotic protein that is frequently overexpressed in cancer cells but its role in carcinogenesis is not clear. We are interested in how Bcl-2 expression affects non-cancerous breast cells and its role in the cell cycle. We prepared an MCF10A breast epithelial cell line that stably overexpressed Bcl-2. We analyzed the cells by flow cytometry after synchronization, and used cDNA microarrays with quantitative reverse-transcription PCR (qRT-PCR) to determine differences in gene expression. The microarray data was subjected to two pathway analysis tools, parametric analysis of gene set enrichment (PAGE) and ingenuity pathway analysis (IPA), and western analysis was carried out to determine the correlation between mRNA and protein levels. The MCF10A/Bcl-2 cells exhibited a slow-growth phenotype compared to control MCF10A/Neo cells that we attributed to a slowing of the G(1)-S cell cycle transition. A total of 363 genes were differentially expressed by at least two-fold, 307 upregulated and 56 downregulated. PAGE identified 22 significantly changed gene sets. The highest ranked network of genes identified by IPA contained 24 genes. Genes that were chosen for further analysis were confirmed by qRT-PCR, however, the western analysis did not always confirm differential expression of the proteins. Downregulation of the phosphatase CDC25A could solely be responsible for the slow growth phenotype in MCF10A/Bcl-2 cells. Increased levels of GTPase Cdc42 could be adding to this effect. PAGE and IPA are valuable tools for microarray analysis, but protein expression results do not always follow mRNA expression results.
Department of Biology, East Carolina University, Greenville, North Carolina 27858, USA.
Jacquelyn M
JM
Long
Charles W
CW
Bell
W Samuel
WS
Fagg
Mary E
ME
Kushman
Kevin G
KG
Becker
James A
JA
McCubrey
Mary A
MA
Farwell
D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D053263:Gene Regulatory Networks; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D020687:cdc25 Phosphatases / Q000235:genetics* / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000235:genetics* / Q000378:metabolism
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D020687:cdc25 Phosphatases; D020764:cdc42 GTP-Binding Protein