DETECTION OF EIF4E GENE AMPLIFICATION IN BREAST-CANCER BY COMPETITIVEPCR

Background: Initiation factor eIF3E binds to mRNA as the initial step for protein translation. Overexpression of the eIF3E oncoprotein has b een found in breast cancer but not in benign breast tissue. The object ive of this study is to determine if eIF3E oncoprotein overexpression is associated with elF4E gene amplification in breast cancer using Wes tern blots and competitive polymerase chain reaction (PCR). Methods: U nknown concentrations of DNA extracted from breast specimens were ampl ified by PCR using a set of primers spanning intron 2/exon 3 of the eI F4E gene. In the same PCR tube, an internal control consisting of a kn own concentration of an eIF4E DNA template with 20-base pair (bp) dele tion was used as the competitive reference standard (CRS) for competit ive PCR. Gel electrophoresis of the PCR products was performed and the bands quantified by densitometry. eIF4E gene amplification was then d etermined relative to a nonamplified gene (gastrin). Using an anti-eIF 4E rabbit antibody, Western blots were performed on benign and maligna nt breast specimens. Quantification was accomplished by developing blo ts with a color assay using nitro blue tetrazolium (NBT) and 5-bromo-3 -chloro-3-indolyl phosphate (BCIP), scanned and analyzed by densitomet ry. Results: Twenty-two breast specimens (14 cancer, 8 control) from p atients were examined for eIF4E gene amplification and oncoprotein exp ression. In all fourteen specimens from stage I-III breast cancer pati ents, eIF4E overexpression was detected at 3- to 30-fold (16.71 +/- 7. 83) elevations. Similarly, all 14 specimens demonstrated eIF4E gene am plification by competitive PCR (3.69 +/- 1.27). In the eight benign br east specimens examined, all were negative for eIF4E overexpression an d gene amplification. Conclusions: Overexpression of eINE was associat ed with elF4E gene amplification in breast cancer specimens. No overex pression or gene amplification was detected in benign breast tissues. elF4E gene amplification may be one mechanism for eIF4E oncoprotein ov erexpression.