Transcriptional changes associated with breast cancer occur as normal human mammary epithelial cells overcome senescence barriers and become immortalized.
BACKGROUND
Human mammary epithelial cells (HMEC) overcome two well-characterized genetic and epigenetic barriers as they progress from primary cells to fully immortalized cell lines in vitro. Finite lifespan HMEC overcome an Rb-mediated stress-associated senescence barrier (stasis), and a stringent, telomere-length dependent, barrier (agonescence or crisis, depending on p53 status). HMEC that have overcome the second senescence barrier are immortalized.


METHODS
We have characterized pre-stasis, post-selection (post-stasis, with p16 silenced), and fully immortalized HMEC by transcription profiling and RT-PCR. Four pre-stasis and seven post-selection HMEC samples, along with 10 representatives of fully immortalized breast epithelial cell lines, were profiled using Affymetrix U133A/B chips and compared using both supervised and unsupervised clustering. Datasets were validated by RT-PCR for a select set of genes. Quantitative immunofluorescence was used to assess changes in transcriptional regulators associated with the gene expression changes.


RESULTS
The most dramatic and uniform changes we observed were in a set of about 30 genes that are characterized as a "cancer proliferation cluster," which includes genes expressed during mitosis (CDC2, CDC25, MCM2, PLK1) and following DNA damage. The increased expression of these genes was particularly concordant in the fully immortalized lines. Additional changes were observed in IFN-regulated genes in some post-selection and fully immortalized cultures. Nuclear localization was observed for several transcriptional regulators associated with expression of these genes in post-selection and immortalized HMEC, including Rb, Myc, BRCA1, HDAC3 and SP1.


CONCLUSIONS
Gene expression profiles and cytological changes in related transcriptional regulators indicate that immortalized HMEC resemble non-invasive breast cancers, such as ductal and lobular carcinomas in situ, and are strikingly distinct from finite-lifespan HMEC, particularly with regard to genes involved in proliferation, cell cycle regulation, chromosome structure and the DNA damage response. The comparison of HMEC profiles with lines harboring oncogenic changes (e.g. overexpression of Her-2neu, loss of p53 expression) identifies genes involved in tissue remodeling as well as proinflamatory cytokines and S100 proteins. Studies on carcinogenesis using immortalized cell lines as starting points or "normal" controls need to account for the significant pre-existing genetic and epigenetic changes inherent in such lines before results can be broadly interpreted.
Li
Yizheng
Y
Section of Bioinformatics, Department of Biological Technologies, Wyeth Research, 87 Cambridge Park Drive, Cambridge, MA 02140, USA. yli@wyeth.com
Pan
Jing
J
Li
Jian-Liang
JL
Lee
Jee Hyung
JH
Tunkey
Chris
C
Saraf
Katie
K
Garbe
James C
JC
Whitley
Maryann Z
MZ
Jelinsky
Scott A
SA
Stampfer
Martha R
MR
Haney
Steven A
SA
D001943:Breast Neoplasms / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D016000:Cluster Analysis; D004847:Epithelial Cells / Q000378:metabolism* / Q000502:physiology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D018719:Erb-b2 Receptor Tyrosine Kinases; D016159:Tumor Suppressor Protein p53