Cyclooxygenase-2 expression induces genomic instability in MCF10A breast epithelial cells.
BACKGROUND
Cyclooxygenase-2 (COX-2) is induced in many breast cancers and COX-2 expression correlates with a worse outcome in the clinic. We hypothesized that the induction of genomic instability is a major mechanism through which COX-2 contributes to breast cancer progression.


METHODS
We transfected a normal immortalized breast epithelial cell line of Basal B subtype, MCF10A, with the pSG5-COX-2 vector and established the stably transfected cell line MCF10A/COX-2. We analyzed the genomic instability phenotype by chromosomal analysis of metaphase-arrested MCF10A and MCF10A/COX-2 cells after Giemsa staining. Groups were compared using chi(2) tests. To investigate the DNA damage checkpoint signaling, we analyzed the phosphorylation status of CHK1 protein with a phospho-specific antibody.


RESULTS
Cytogenetic analysis of early passage transfected cells showed that COX-2 expression increased genomic instability compared with the MCF10A cells transfected with a luciferase vector alone. COX-2 overexpression was associated with a significant increase in chromosomal aberrations (fusions, breaks, and tetraploidy). There was a statistically significant increase in the number of polyploid cells in the COX-2 transfected cells versus the control (P=0.004). We also found that an inhibitory CHK1 phosphorylation at Ser-280 was dramatically increased upon COX-2 overexpression in MCF10A cells, thus explaining the mechanism of inactivation of an important cell cycle checkpoint. Further analysis of the MCF10A/COX-2 cells showed that these cells have acquired a premalignant phenotype characterized by a morphological transformation, a resistance to anoikis, a reduced requirement of epidermal growth factor for growth in culture, but their inability to establish tumors in a nude mouse model of malignancy.


CONCLUSIONS
We found that COX-2 expression in MCF10A breast epithelial cells confers a premalignant phenotype that includes enhanced genomic instability and altered cell-cycle regulation.
Singh
Balraj
B
Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Vincent
Laura
L
Berry
Jacob A
JA
Multani
Asha S
AS
Lucci
Anthony
A
D000818:Animals; D023102:Anoikis / Q000235:genetics; D001940:Breast / Q000166:cytology / Q000201:enzymology*; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D002453:Cell Cycle; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D018450:Disease Progression; D004815:Epidermal Growth Factor / Q000502:physiology; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D042822:Genomic Instability / Q000235:genetics*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D010641:Phenotype; D011230:Precancerous Conditions / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous
D004815:Epidermal Growth Factor; D051546:Cyclooxygenase 2; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse