Conditional expression of Ki-RasG12V in the mammary epithelium of transgenic mice induces estrogen receptor alpha (ERα)-positive adenocarcinoma.

Appropriate 'in vivo' models are crucial for studying breast cancer biology and evaluating the efficacy of therapeutic agents. Thus we engineered a novel transgenic mouse line expressing the human Ki-Ras bearing an activating mutation (Ki-Ras(G12V)) selectively in the mammary epithelium after lactation. These mice develop invasive ductal adenocarcinomas with 100% incidence within 3-9 months after Ki-Ras(G12V) induction. Immunophenotyping revealed that the mammary tumors express luminal markers, are positive for estrogen and progesterone receptors, negative for HER2 and have a low proliferation index. Moreover, cell lines derived from such tumors are estrogen-responsive and, when transplanted into nude mice, form tumors that respond to the antiestrogen ICI 182780. In conclusion, the mammary tumors of these transgenic mice and the derived cell lines exhibit key features of the major form of human breast cancer, that is, luminal A subtype and thus have a high potential for breast cancer research and treatment.

Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases.

Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ER(T) or Cre-ER(T2) under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).

Characterization of the amino-terminal transcriptional activation function of the human estrogen receptor in animal and yeast cells.

We have previously reported that the transcriptional activation function AF-1, located in the A/B region of the human estrogen receptor, exhibits cell-type and promoter context specificity in both animal cells and yeast. To further characterize AF-1, we have constructed a number of deletion mutants spanning the A/B region in the context of either the whole human estrogen receptor or the A/B region linked to the GAL4 DNA binding domain, and tested their transcriptional activity in chicken embryo fibroblasts and in yeast cells, two cell types in which AF-1 efficiently activates transcription on its own. Additionally, we utilized HeLa cells in which AF-1 is poorly active but can synergize with the transcriptional activation function AF-2 located in the hormone binding domain. We show that in animal cells the "independent" activity of AF-1 is embodied in a rather hydrophobic proline-rich 99-amino acid activating domain (amino acids 51-149), whereas amino acids 51-93 and 102-149 can independently synergize with AF-2. Interestingly, in yeast, three discrete activating domains (amino acids 1-62, 80-113, and 118-149) are almost as active on their own as the whole A/B region, indicating that multiple activating domains can operate independently in yeast. Our study also demonstrates that, within the context of the whole human estrogen receptor, the same AF-1 activating domains are "induced" by either estradiol or 4-hydroxytamoxifen.

Modulation of transcriptional activation by ligand-dependent phosphorylation of the human oestrogen receptor A/B region.

Using a transient co-transfection system, we show that the human oestrogen receptor (hER) becomes phosphorylated in the presence of oestradiol (E2) as well as in the presence of the anti-oestrogens 4-hydroxy-tamoxifen (OHT) and ICI 164, 384 (ICI), although at lower efficiencies than with E2. There are multiple sites of phosphorylation in hER; using deletion and point mutants one of these sites has been mapped in the N-terminal A/B region at serine 118. Mutation of this serine to alanine caused, in a number of cell types, a significant reduction in transcriptional activation by hER from reporter genes containing an oestrogen response element (ERE), but did not affect the DNA binding properties or nuclear localization of hER. Thus phosphorylation of serine 118 is important for the action of the transcription activation function 1 (AF-1) located in the A/B region of the oestrogen receptor.

Promoter specificity of the two transcriptional activation functions of the human oestrogen receptor in yeast.

We have previously demonstrated that the human oestrogen receptor (hER) contains two transcriptional activation functions located in the N-terminal region (TAF-1) and in the hormone binding domain (TAF-2), which can act both independently and synergistically in a promoter- and cell-specific manner in animal cells. We have also demonstrated that hER can activate transcription from chimaeric oestrogen-responsive GAL1 promoters in yeast, and shown that transcriptional activation was due to TAF-1, whereas TAF-2 showed little, if any, transcriptional activity on these promoters. By using a more complex promoter derived from the URA3 gene, we now show that TAF-2 is also active in yeast, and that the activities of TAF-1 and TAF-2 are promoter-context-specific in yeast. We also confirm that the agonistic activity of 4-hydroxytamoxifen (OHT) can be ascribed to the activity of TAF-1.

Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A.

We have cloned and sequenced the complete complementary DNA of the oestrogen receptor (ER) present in the breast cancer cell line MCF-7. The expression of the ER cDNA in HeLa cells produces a protein that has the same relative molecular mass and binds oestradiol with the same affinity as the MCF-7 ER. There is extensive homology between the ER and the erb-A protein of the oncogenic avian erythroblastosis virus.

The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors.

A chicken oviduct cDNA clone containing the complete open reading frame of the oestrogen receptor (ER) has been isolated and sequenced. The mol. wt of the predicted 589-amino acid protein is approximately 66 kd which is very close to that of the human ER. Comparison of the human and chicken amino acid sequences shows that 80% of their amino acids are identical. There are three highly conserved regions; the second and third of which probably represent the DNA- and hormone-binding domains of the receptor. The putative DNA-binding domain is characterised by its high cysteine and basic amino acid content, and the hormone-binding domain by its overall hydrophobicity. These two domains of homology are also present in the human glucocorticoid receptor (GR) and the product of the avian erythroblastosis virus (AEV) gene, v-erbA, indicating that c-erbA, the cellular counterpart of v-erbA, belongs to a multigene family of transcriptional regulatory proteins which bind steroid-related ligands. The first highly conserved ER region is not present in the truncated v-erbA gene, but shares some homology with the N-terminal end of the GR. The function of the v-erbA gene product is discussed in relation to its homology with the ER and GR sequences.

The SV40 TC-II(kappa B) enhanson binds ubiquitous and cell type specifically inducible nuclear proteins from lymphoid and non-lymphoid cell lines.

We have characterized the complexes resulting from the specific binding in vitro of proteins present in nuclear extracts of several lymphoid and non-lymphoid cell lines to the TC-I and TC-II sequences of the simian virus 40 (SV40) enhancer. No proteins could be detected, binding selectively to the TC-I sequence, but two proteins TC-IIA and TC-IIB were identified interacting specifically with both the TC-II/kappa B enhanson, 5'-GGAAAGTCCCC-3' (important for the activity of the SV40 enhancer in vivo), and with the related H-2Kb enhanson, 5'-TGGGGATTCCCCA-3'. The binding of these two proteins to mutated TC-II enhansons correlates with the effect of these mutations in vivo, suggesting that both proteins may be important for SV40 enhancer activity. The TC-IIA binding activity was present in nuclear extracts of mature lymphoid B cells and was increased in pre-B cell nuclear extracts by lipopolysaccharide (LPS) and cycloheximide treatment. Furthermore, complex formation between the TC-IIA protein and the TC-II enhanson was efficiently competed by the kappa B motif from the kappa chain enhancer, indicating that TC-IIA is the NF-kappa B factor or a closely related protein. However, in contrast to previous reports, a TC-IIA/NF-kappa B-like protein whose properties could not be distinguished from those of the TC-IIA protein present in lymphoid B cells, was found in nuclear extracts of several untreated non-lymphoid cell lines, notably of HeLa cells, but not of undifferentiated F9 embryonal carcinoma (EC) cells [F9(ND)]. The TC-IIA binding activity which was moderately increased in HeLa cell nuclear extracts by 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or cycloheximide treatment could be induced in nuclear extracts of F9(ND) cells by cycloheximide, but not by TPA. Moreover, the TC-IIA binding activity could be induced in cytosolic fractions from F9(ND) cells by treatment with deoxycholate, indicating that these cells contain an inhibitor protein similar to the previously described NF-kappa B inhibitor, I kappa B. The second TC-II enhanson binding protein, TC-IIB, which could be clearly distinguished from the TC-IIA/NF-kappa B-like protein, by a number of differential properties, resembles the previously described KBF1/H2TF1 protein as it binds with a higher affinity to the H-2Kb enhanson than to the TC-II/kappa B enhanson, and its pattern of methylation interference on the H-2Kb and TC-II/kappa B enhansons is identical to that reported for the KBF1/H2TF1 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning of the human estrogen receptor cDNA.

Poly(A)+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and fractions enriched in estrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt10 and lambda gt11 vectors. Clones corresponding to ER sequence were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five were isolated after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kilobase pairs) and lambda OR8 (2.1 kilobase pairs), isolated by using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains the DNA sequence expected from the two ER peptides and crosshybridizes with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Use of lambda OR8 as a hybridization probe revealed a single poly(A)+ RNA band of approximately equal to 6.2 kilobase pairs in the ER-containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-negative cell line HeLa. The same probe hybridizes to a chicken gene that is expressed in oviduct tissue as a 7.5-kilobase-pair poly(A)+ RNA.

Targeted somatic mutagenesis in mouse epidermis.

Gene targeting in the mouse is a powerful tool to study mammalian gene function. The possibility to efficiently introduce somatic mutations in a given gene, at a chosen time and/or in a given cell type will further improve such studies, and will facilitate the generation of animal models for human diseases. To create targeted somatic mutations in the epidermis, we established transgenic mice expressing the bacteriophage P1 Cre recombinase or the tamoxifen-dependent Cre-ER(T2) recombinase under the control of the human keratin 14 (K14) promoter. We show that LoxP flanked (floxed) DNA segments were efficiently excised in epidermal keratinocytes of K14-Cre transgenic mice. Furthermore, Tamoxifen administration to adult K14-Cre-ER(T2) mice efficiently induced recombination in the basal keratinocytes, whereas no background recombination was detected in the absence of ligand treatment. These two transgenic lines should be very useful to analyse the functional role of a number of genes expressed in keratinocytes.