The cDNA clone (Pg8) corresponding to the 3'-end of fatty acid synthetase (FAS) mRNA is one of the few probes to study in cell lines the mechanism by which an endogenous gene is regulated by progestins. The steady-state level of FAS mRNA is known to be increased by the synthetic progestin R5020 in the human breast cancer cell line MCF7. We show here that it is also increased in another progesterone-receptor-positive cell line T47D but not in progesterone-receptor-negative cell lines BT20, MDA-MB231, and HBL100. In R5020-treated MCF7 cells, the FAS mRNA concentration increased with cell density. Protein synthesis inhibitors did not abolish progestin induction of FAS suggesting a primary effect of the hormone. In vitro nuclear run-on transcription assays showed that the FAS gene transcription rate was increased about 4-fold by progestin. This stimulation of transcription was detectable 30 min after addition of R5020 and was maximal after 4 h, but could not totally account for the total increase in the mRNA steady state level. Chase experiments in the presence of Actinomycin D or Cordycepin showed that progestin also increased the half-life of FAS mRNA from 6-9 h to 24-33 h. We conclude that progestin stimulates the FAS concentration both by increasing the transcription rate of the gene and by stabilizing the mRNA.
We have shown previously that fatty acid synthetase (FAS) is specifically induced by progestins in human breast cancer cell lines. To test the potential value of FAS as a clinical marker in breast diseases, we measured FAS expression in frozen sections of 22 benign and 27 malignant mammary tumors using in situ hybridization with the [35S]UTP alpha S-labeled FAS anti-sense mRNA. The hybridized RNA was quantified with an IMSTAR computerized image analyzer. We found FAS RNA in epithelial cells, but no labeling was detected in the connective tissue. In breast cancer, we found no correlation between FAS expression and estrogen receptor and progesterone receptor concentrations or status. However, the level of FAS was significantly (P less than .02) higher in premenopausal than in post-menopausal patients and increased with the grade of tumor differentiation (P less than .005 between the poorly and well-differentiated tumors). In benign mastopathies, high levels of FAS RNA were found in some cysts (mostly with apocrine metaplasia). In lobules, the FAS RNA level increased proportionally to the degree of proliferation determined by histological examination (P less than .015) and correlated with the H4 histone level measured in an adjacent section using in situ hybridization (r = 0.85, P less than .001). In ductal structures, a lower correlation (r = 0.64, P less than .01) was found between FAS and H4 RNA levels. We conclude that FAS RNA is overexpressed in some mammary tumors and may be useful in predicting high-risk mastopathies and less aggressive breast cancers.
Fatty acid synthetase (FAS) is induced by progestins in human breast cancer cell lines. To study its regulation in normal mammary glands, the FAS level was estimated by immunohistochemistry, using the biotin-streptavidin method, in ducts and lobules of normal tissues adjacent to nonproliferative benign breast lesions collected by biopsy. Rabbit polyclonal antibodies to human FAS specifically recognized the 250-kDa FAS from MCF7 cells, as shown by Western immunoblotting. An excess of purified FAS totally switched off FAS immunostaining of R5020-treated MCF7 cells, demonstrating the validity of FAS immunocytochemical detection. FAS labeling was quantified using a computer-aided image analyzer (SAMBA 2005) in 18 patients receiving progestin therapy from the 15th to the 25th day of the menstrual cycle and 26 untreated patients. In the 2 groups, FAS staining, absent of fibroblasts, was observed in the cytoplasm of epithelial cells. It was higher in lobules than in ducts and increased significantly from the follicular to the luteal phase in both structures. Progestin treatment increased FAS expression in both structures. Using monoclonal antibodies, progesterone receptor expression was measured in frozen serial sections. In patients receiving progestin treatment, the progesterone receptor level increased from the beginning of the cycle to day 14 and then decreased during the second part of the menstrual cycle, probably down-regulated by progestin, indicating a regulation similar to that in the endometrium. We conclude that FAS is induced by progestins in the ducts and lobules of human normal mammary glands as it is in human breast cancer cells. FAS may, therefore, be useful for studying the effect of progesterone in normal human mammary glands.
Fatty acid synthetase (FAS) is induced by progesterone in MCF7 and T47D breast cancer cell lines. We studied a possible in vivo regulation of expression of this gene by looking for FAS RNA in human endometrial biopsies at various periods of the menstrual cycle, using a cloned cDNA FAS probe. By Northern blot analysis, we detected the 8-kilobase FAS RNA throughout the cycle in 7 uterine samples. RNA in situ hybridization analysis of frozen sections from 22 endometrial biopsies showed that FAS RNA was present during follicular and luteal phases of the menstrual cycle in stromal and epithelial cells. RNA levels were quantified by counting autoradiographic silver grains using a computer-aided image analyzer. FAS RNA levels were significantly higher in epithelial cells than in fibroblasts (P less than 2 x 10(-5]. Furthermore, in both cell types, mean FAS RNA concentrations were higher in biopsies removed during the luteal phase than the follicular phase of the menstrual cycle (P = 2 x 10(-3) and 9 x 10(-5), respectively). A 2- to 3-fold increase in FAS RNA levels between days 8-14 and days 22-24 was detected in 2 normal patients who had previously undergone 2 successive biopsies. This increase was not observed in 2 patients with low plasma estradiol and progesterone concentrations, indicating a probable dysovulation. We conclude that FAS normally increases in both stromal and epithelial endometrial cells during the luteal phase. This increase is probably due to progesterone, which implies that FAS is induced in normal endometrium, as demonstrated in breast cancer.
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