Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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Human breast tissues and reverse transcription
Eighteen cases were selected in the Manitoba Breast Tumor Bank, which had well separated and histopathologically characterized normal and adjacent neoplastic components. The presence of normal ducts and lobules as well as the absence of any proliferative lesion were confirmed in all 18 normal specimens. Within the eighteen adjacent primary invasive ductal breast carcinomas, seven were ER negative (< 3 fmol/mg protein) and eleven were ER positive (> 3 fmol/mg protein), as determined by the ligand binding assay. Within the ER negative tumor subgroup, 6 tumors were progesterone receptor (PR) negative (<10 fmol/mg protein) and 1 was PR positive (>10 fmol/mg protein), as measured by the ligand binding assay. In the ER positive subgroup, 2 tumors were PR negative and 9 were PR positive. Total RNA was extracted from frozen tissue sections and reverse transcribed in a final volume of 25 ml.

Polymerase chain reaction (PCR)
ERC4 primer set was used to co-amplify wild-type ER-a (WT-ER) and ERC4 cDNAs. This set consisted of ERU primer (5'-TGTGCAATGACTATGCTTCA-3'; sense; located in WT-ER), ERL primer (5'-GCTCTTCCTCCTGTTTTTAT-3'; antisense; located in WT-ER exon 3), and C4L primer (5'-TTTCAGTCTTCAGATACCCCAG-3'; antisense; located in C4ER sequence). ERD3 primer set, used to co-amplify WT-ER and ERD3 cDNAs, consisted of D3U primer (5'-TGTGCAATGACTATGCTTCA-3'; sense; located in WT-ER exon 2) and D3L primer (5'-TGTTCTTCTTAGAGCGTTTGA-3'; antisense; located in WT-ER exon 4). ERD5 primer set, used to co-amplify WT-ER and ERD5 cDNAs, consisted of D5U primer (5'-CAGGGGTGAAGTGGGGTCTGCTG-3'; sense; located in WT-ER exon 4) and D5L primer (5'-ATGCGGAACCGAGATGATGTAGC-3'; antisense; located in WT-ER exon 6).

0.2 µl of reverse transcription mixture was amplified in a final volume of 15 µl, in the presence of 1.5 mCi of [a-32P] dCTP (3000Ci/mmol), 4 ng/µl of each primer of the primer set considered (ERC4, ERD3 or ERD5 primer set) and 0.3 unit of Taq DNA polymerase. Each PCR consisted of 30 cycles: 1 min at 94°C, 30 sec at 60°C and 1 min at 72°C for ERC4 primer set; and 30 sec 94°C, 30 sec at 60°C and 30 sec at 72°C for ERD3 and ERD5 primer sets. PCR products were then separated on 6% polyacrylamide gels containing 7M urea (PAGE). Following electrophoresis, the gels were dried and autoradiographed. For each PCR, two PCR products were obtained, which were identified by subcloning and sequencing. PCR products migrating with the apparent size of 149 bp, 354 bp and 483 bp, using ERC4, ERD3 and ERD5 primer set, respectively, were shown to correspond to WT-ER cDNA. PCR products migrating with the apparent size of 536 bp, 237 bp and 344 bp, using ERC4, ERD3 and ERD5 primer set, were shown to correspond to ERC4, ERD3 and ERD5 cDNAs, respectively. For each experiment, bands corresponding to the variant mRNA (i.e ERC4, ERD3 or ERD5) and to WT-ER, were excised from the gel and counted in a scintillation counter.

Quantitation and statistical analysis
For each set of primers (i.e ERC4, ERD3 and ERD5 primer set) and for each sample, 4 independent PCR assays were performed. The ratios between ERC4, ERD3 or ERD5 signals and corresponding WT-ER signal were calculated. For each experiment, in order to correct for overall inter-assay variations, the ratio observed in the same particular tumor (case number 12) was arbitrarily given the value of one and all other ratios expressed relatively, in arbitrary units (see Fig. 2). Under our experimental conditions, some samples did not have measurable levels (i.e signal lower than twice the background value) of D3ER or D5ER variant mRNAs (see figure 1 and 2) in any of the 4 repetitions performed. Only cases having detectable levels in at least 3 of the replicates in both their normal and tumor compartments were included in the statistical analysis. The significance of the differences in the relative levels of expression of ERC4, ERD3 and ERD5 mRNAs between matched normal and tumor components was determined using the Wilcoxon signed rank test.

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