) were crossed with Brca1lox/lox
) or p53lox/lox
) mice. Triple transgenic Amhr2Cre/+
mice were generated by crossing Amhr2Cre/+
mice. The resulting transgenic mice were maintained on a mixed background. All mice were genotyped by PCR using tail or ear DNA. Kaplan-Meier survival curves were drawn using GraphPad PRISM software. Mean survival time was calculated using the Log-rank test.
Confirmation of gene recombination
Genomic DNA extracted from tumors or normal tissues of the female reproductive tract was used to detect Cre-mediated recombination of the p53 and Brca1 genes. Cre-mediated deletion of p53 displayed a 612 bp PCR product amplified with primers p53-a (5′-CAC AAA AAC AGG TTA AAC CCA-3′) and p53-c (5′-GAA GAC AGA AAA GGG GAG GG-3′). PCR amplification of the recombined Brca1 gene resulted in a 621 product using the primers Brca1-d (5′-CTG GGT AGT TTG TAA GCA TCC-3′) and Brca1-g (5′-CTG CGA GCA GTC TTC AGA AAG-3′), which flanked Brca1 exon 11. The presence of wild-type Brca1 was determined by PCR using primers within exon 11 (Brca1-e: 5′-ATC AGT AGT AGA AAT CCA AGC CCA CC-3′; Brca1-f: 5′-TGC CAC TCC CAG CAT TGT TAG-3′).
Formalin-fixed paraffin-embedded archival human specimens were obtained from the following institutions: Massachusetts General Hospital, Boston, MA; Baylor College of Medicine, Houston, TX; Memorial Sloan-Kettering Cancer Center, New York, NY; Cedars-Sinai Medical Center, Los Angeles, CA; Olive View Medical Center, Los Angeles, CA; Inova Fairfax Hospital, Falls Church, VA; Universita Cattolica, Rome, Italy; and Istituto di Anatomia e Istologia Pathologica, Ancona, Italy.
H&E staining and immunohistochemistry
Tissues were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections were deparaffinized in a graded xylene/ethanol series and used for H&E staining or immunohistochemistry with an ABC antibody staining kit (Vector Laboratories) according to the manufacturer’s instructions. After color development, the slides were counterstained with hematoxylin and mounted with mounting medium (Permount, Fisher Sciences). To determine the proliferative index of the tumors, mice were intraperitoneally injected with 100 mg/kg 5′-bromo-3′-deoxyuridine (BrdU) (Zymed Laboratories). Tissues and tumors were collected after two hours and fixed in 10% formalin overnight. Paraffin-embedded sections were deparaffinized, followed by hydrogen chloride (2N HCl) digestion, trypsinization (0.1% Trypsin), and immunohistochemistry with an ABC antibody staining kit. The following primary antibodies were used: α-smooth muscle actin (1:200 dilution, Sigma); β-catenin (H-102) (1:100 dilution, Santa Cruz); BRCA1 (Ab-1) (1:100 dilution, Calbiochem); BrdU (1:100 dilution, Vector Laboratories); p16 (M-156) (1:100 dilution, Santa Cruz); p53 (Ab-1) (1:100 dilution, Calbiochem); phospho-estrogen receptor α (Ser167) (1:100 dilution, Cell Signaling); and TROMA-1 (keratin 8) (1:25 dilution, Developmental Studies Hybridoma Bank at the University of Iowa). Hematoxylin and Eosin (H&E) and immuno-stained uterine tumor sections were reviewed by two independent observers (D.X. and E.O.).
BRCA1 promoter methylation analysis
The methylation status of ULMS specimens was determined using the EZ DNA Methylation Gold kit (Zymo Research) following manufacturer’s instructions. Ovarian cancer tissues with known BRCA1
methylation status (12
) were used as a control. Bisulfite-modified DNA PCR amplification and primers have been previously described (12