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Detection of eIF4E gene amplification in breast cancer by competitive PCR
D L Sorrells1, D R Black, C Meschonat
1Department of Surgery, Louisiana State University Medical Center, Shreveport 71130, USA.
Background:
Initiation factor eIF4E binds to mRNA as the initial step for protein translation. Overexpression of the eIF4E oncoprotein has been found in breast cancer but not in benign breast tissue. The objective of this study is to determine if eIF4E oncoprotein overexpression is associated with eIF4E gene amplification in breast cancer using Western blots and competitive polymerase chain reaction (PCR).
Methods:
Unknown concentrations of DNA extracted from breast specimens were amplified by PCR using a set of primers spanning intron 2/exon 3 of the eIF4E gene. In the same PCR tube, an internal control consisting of a known concentration of an eIF4E DNA template with 20-base pair (bp) deletion was used as the competitive reference standard (CRS) for competitive PCR. Gel electrophoresis of the PCR products was performed and the bands quantified by densitometry. eIF4E gene amplification was then determined relative to a nonamplified gene (gastrin). Using an anti-eIF4E rabbit antibody, Western blots were performed on benign and malignant breast specimens. Quantification was accomplished by developing blots with a color assay using nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), scanned and analyzed by densitometry.
Results:
Twenty-two breast specimens (14 cancer, 8 control) from patients were examined for eIF4E gene amplification and oncoprotein expression. In all fourteen specimens from stage I-III breast cancer patients, eIF4E overexpression was detected at 3- to 30-fold ( 16.71 +/- 7.83) elevations. Similarly, all 14 specimens demonstrated eIF4E gene amplification by competitive PCR (3.69 +/- 1.27). In the eight benign breast specimens examined, all were negative for eIF4E overexpression and gene amplification.
Conclusions:
Overexpression of eIF4E was associated with eIF4E gene amplification in breast cancer specimens. No overexpression or gene amplification was detected in benign breast tissues. eIF4E gene amplification may be one mechanism for eIF4E oncoprotein overexpression.
