Identification of two estrogen receptor transcripts with novel 5' exons isolated from a MCF7 cDNA library.

Two novel transcripts of human estrogen receptor (ER) have been identified that differ in the 5' untranslated sequence. It has previously been determined that an alternate ER transcript is generated from transcription initiated upstream of the main ER cap site (P1), and utilizes a splice acceptor site at +163. Here we report the isolation of 21 ER clones from a MCF7 cDNA library. Eleven of these clones correspond to transcripts that initiate at the P1 cap site, whereas the remaining 10 clones are derived from two previously unidentified ER transcripts (designated E and H) that both utilize the +163 splice acceptor site. A panel of breast and endometrial carcinoma cell lines were screened by reverse transcriptase-polymerase chain reaction (RT-PCR) for expression of the E and H transcripts. It was found that all ER-positive cell lines expressed both of the novel transcripts. In addition, 10 primary human breast cancers were analyzed, of which six expressed the E transcript and five abundantly expressed the H transcript. These data indicate that expression of ER in human breast cancers can be dependent upon an alternate promoter at least 20 kb upstream of the primary cap site for ER.

Analysis of estrogen receptor messenger RNA in breast carcinomas from archival specimens is predictive of tumor biology.

As the size of breast tumors continues to decrease, it has become more difficult to obtain adequate tumor tissue for molecular studies. We have used the estrogen receptor (ER) gene as a model to study the ability to perform a quantitative analysis of ER mRNA extracted from archival breast carcinoma specimens using reverse transcriptase polymerase chain reaction. Based upon ER mRNA abundance, tumors were characterized as having low, medium, or high ER mRNA expression. These data were compared with ER and progesterone receptor (PR) status determined by enzyme immunoassay, tumor histology, and Bloom-Richardson grade. Comparing the low and high ER mRNA groups, there were statistically significant differences in ER-positive status (10% versus 95%; P = 0.0001), PR-positive status (10% versus 90%; P = 0.0001), and tumor grade (2.67 +/- 0.12 versus 2.09 +/- 0.14; P = 0.0025). Of the 28 tumors in the high ER mRNA group, 5 (18%) were invasive lobular carcinomas whereas all 24 tumors with low ER mRNA were invasive ductal carcinomas. These data demonstrate that archival breast tumor specimens can be characterized for ER mRNA abundance. In addition, we conclude that the mechanisms regulating ER gene transcription influence the phenotype of breast carcinomas. These results also suggest that this technique can be designed to provide a quantitative analysis of gene expression for any gene of interest utilizing archival tumor specimens.

Quantitative analysis of the transcriptional start sites of estrogen receptor in breast carcinoma.

Recent studies have identified an estrogen receptor (ER) promoter upstream of the transcriptional start site originally mapped for the ER gene. We have examined promoter use in a number of breast carcinoma cell lines. MCF7, T47D, BT474, and MDA-MB-361 cell lines all use the P0 promoter, whereas BT20 and ZR75-1 do not demonstrate transcription from this upstream start site. S1 nuclease analysis was used to quantitate the amount of ER mRNA originating from the two different promoters. In MCF7, one-third of the ER mRNA results from transcription originating upstream of the major ER promoter and in T47D, 12% of the message originates from upstream transcription. Promoter use was analyzed in human mammary epithelial cells and human late proliferation endometrium. Upstream promoter use was found to be characteristic of human late proliferation endometrium but not human mammary epithelial cells. These results indicated that certain breast carcinomas demonstrate ER transcription from a promoter not normally active in normal breast epithelium. This activation may involve a factor active in normal human endometrium.

Transcriptional regulation of estrogen receptor in breast carcinomas.

An important transcriptional regulatory element of the estrogen receptor (ER) gene that binds a protein expressed in ER-positive breast carcinomas has been identified. Using a transient expression assay, we identified a 75-bp region of the 5' untranslated leader of the ER gene which augments expression from the ER promoter. This region contains two binding sites for a protein, estrogen receptor factor 1 (ERF-1), which is expressed in ER-positive breast carcinomas. A concatenated ERF-1 binding site probe identified a 30,000-Da protein. Low-level ERF-1 expression was detected in normal human mammary epithelial cells. Abundant ERF-1 expression was also found in endometrial carcinoma cell lines that express the ER-positive phenotype. These results indicate that ERF-1 expression represents a common mechanism of ER regulation in hormonally responsive carcinomas.

Transcriptional control of estrogen receptor in estrogen receptor-negative breast carcinoma.

The mechanisms which control estrogen receptor (ER) expression in breast carcinoma have not been elucidated. The MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) breast carcinoma cell lines have been used to examine ER gene expression. Northern blot analysis of mRNA isolated from these two cell lines demonstrates that MCF-7 cells express an expected 6.5-kilobase ER mRNA whereas MDA-MB-231 do not make detectable ER message. Reverse polymerase chain reaction, which offers greater sensitivity, confirms these Northern blot results. The nuclear run-on assay was used to examine directly transcription in these two cell lines. MCF-7 cells actively transcribe the ER gene whereas no ER transcription is detected in MDA-MB-231. Southern analysis of genomic DNA confirms that the ER gene is present in MDA-MB-231. We conclude that the ER gene is controlled transcriptionally in this ER-negative breast carcinoma line.