Progress 03/02/00 to 02/28/06
Outputs
Estrogens regulate normal reproductive biology and development and environmental estrogens can interfere with these mechanisms. The objectives of the project were to characterize estrogen response mechanisms by using estrogen action in human breast cancer cell lines, to study estrogen and xenoestrogen action mechanisms in animals and in human breast cancer, and to study the developmental effects of endogenous estrogens and xenoestrogens in fetal exposure via maternal exposure in animals including mice. Both natural estrogens and environmental estrogens act through estrogen receptors that include, but are not limited to, nuclear-acting forms of estrogen receptor alpha and estrogen receptor beta. In the project we have examined the role of feeds on the determination of fetal estrogenic activities of xenoestrogens such as bisphenol A rodents, and detailed how major, uncontrolled variation in estrogenic activities present in nominally estrogen-free feeds interferes with
endocrine experiments, and creates substantial variation in reported results. In collaboration involving exercise physiology in the pig model, we have determined that the boar circulates total estrogenic activity that is in within the range of the normal human male, indicating that the boar is an appropriate model for the study of endocrine effects on exercise physiology. These findings indicate that in the boar, high reported levels of estradiol are not reflected in circulating estrogenic activity. We have also collaborated with several labs to use the unique estrogen-independent cell lines developed at the University of Missouri in the prior project, for the study of mechanisms of progression from hormone dependence to hormone independence in human breast cancer cells. Overall, our work on mechanisms of natural estrogen and environmental estrogen action has aided in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal
reproduction, and normal estrogen action in both.
Impacts
This work on mechanisms of natural estrogen and environmental estrogen action in tissue culture, in mice during fetal development, and applied to understanding circulating estrogens and estrogen action in swine, has aided in understanding natural estrogen and environmental estrogen actions in human and domestic animal reproduction. In addition, the work on circulating estrogenic activity in the boar has provided basic endocrine information on this domestic animal.
Publications
- Perry, G.A., Welshons, W.V., Bott, R.C. and Smith, M.F., 2005. Basis of melengestrol acetate action as a progestin. Domestic Animal Endocrinology 28: 147-161.
- vom Saal, F.S., Richter, C.A., Ruhlen, R.L., Nagel, S.C., Timms, B.G. and Welshons, W.V., 2005. The importance of appropriate controls, animal feed, and animal models in interpreting results from low-dose studies of bisphenol A. Birth Defects Res. A Clin. Mol. Teratol. 73: 140-145.
- Dhulipala, V.C., Maddali, K.K., Welshons, W.V. and Reddy, C.S., 2005. Secalonic acid D blocks embryonic palatal mesenchymal cell cycle by altering the activity of CDK2 and the expression of p21 and cyclin E. Birth Defects Res. B Dev. Reprod. Toxicol. 74: 233-242.
- vom Saal, F.S., Nagel, S.C., Timms, B.G. and Welshons, W.V., 2005. Implications for human health of the extensive bisphenol A literature showing adverse effects at low doses: A response to attempts to mislead the public. Toxicology: 212: 244-252.
- vom Saal, F.S., Richter, C.A., Mao, J. and Welshons, W.V., 2005. Commercial animal feed: Variability in estrogenic activity and Effects on body weight in mice. Birth Defects Res. A Clin. Mol. Teratol. 73: 474-475.
- Reizner, N., Maor, S., Sarfstein, R., Abramovitch, S., Welshons, W.V., Curran, E.M., Lee, A.V. and Werner, H., 2005. The WT1 Wilms' tumor suppressor gene product interacts with estrogen receptor-alpha and regulates IGF-I receptor gene transcription in breast cancer cells. J. Mol. Endocrinol. 35: 135-144.
- Welshons, W.V., 2005. Evaluation and prediction of risks of environmental estrogens. Program of the Third International Conference of the Collegium Ramazzini, Framing the Future in Light of the Past: Living in a Chemical World, Bologna, Italy, Page 52, Abstract 38.
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Progress 01/01/04 to 12/31/04
Outputs
The objectives of the project are to characterize estrogen response mechanisms by using estrogen action in human breast cancer cell lines, to study estrogen and xenoestrogen action mechanisms in animals and human breast cancer, and in fetal estrogen exposure via maternal exposure in animals including mice. Both natural estrogens and environmental estrogens act through estrogen receptors that include, but are not limited to, nuclear-acting forms of estrogen receptor alpha and estrogen receptor beta. In the last year we have examined the role of feeds on the determination of fetal estrogenic activities of xenoestrogens such as bisphenol A rodents, and detailed how major, uncontrolled variation in estrogenic activities present in nominally estrogen-free feeds interferes with endocrine experiments, and creates substantial variation in reported results. In an additional collaboration involving exercise physiology in the pig model, we have determined that the boar
circulates total estrogenic activity that is in within the range of the normal human male, indicating that the boar is an appropriate model for the study of endocrine effects on exercise physiology. These findings indicate that in the boar, high reported levels of estradiol are not reflected in circulating estrogenic activity. We have also collaborated with several labs to supply unique estrogen-independent cell lines developed at the University of Missouri, for the study of mechanisms of progression from hormone dependence to hormone independence in human breast cancer cells. Overall, our work on mechanisms of natural estrogen and environmental estrogen action will aid in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal reproduction, and normal estrogen action in both.
Impacts
This work on mechanisms of natural estrogen and environmental estrogen action in tissue culture, in mice during fetal development, and applied to understanding circulating estrogens and estrogen action in swine, will aid in understanding natural estrogen and environmental estrogen actions in human and domestic animal reproduction. In addition, the work on circulating estrogenic activity in the boar will provide basic endocrine information on this domestic animal.
Publications
- Liu, Y., Rector, R.S., Thomas, T.R., Taylor, J.A., Holiman, D.A., Henderson, K.K., Welshons, W.V. and Sturek, M.S. 2004. Lipoproteins during the estrous cycle in swine. Metabolism 53:140-141.
- Xu, X., Murdoch, F.E., Curran, E.M., Welshons, W.V. and Fritsch, M.K. 2004. Transcription factor accessibility and histone acetylation of the progesterone receptor gene differs between parental MCF-7 cells and a subline that has lost progesterone receptor expression. Gene 328:143-151.
- Dougherty, M.K., Schumaker, L.M., Jordan, V.C., Welshons, W.V., Curran, E.M., Ellis, M.J. and El-Ashry, D. 2004. Estrogen receptor expression and sensitivity to Paclitaxel in breast cancer. Cancer Biol. Ther. 3(5):460-467.
- Philips, B.J., Ansell, P.J., Newton, L.G., Harada, N., Honda, S.I., Ganjam, V.K., Rottinghaus, G.E., Welshons, W.V. and Lubahn, D.B. 2004. Estrogen receptor-independent catechol estrogen binding activity: Protein binding studies in wild-type, estrogen receptor-alpha KO, and aromatase KO mice tissues. Biochemistry 43:6698-6708.
- Dhulipala, V.C., Welshons, W.V. and Reddy, C.S. 2004. Inhibition of human embryonic palatal mesenchymal cell cycle by secalonic acid D: a probable mechanism of its cleft palate induction. Orthod. Craniofac. Res. 7:227-236.
- Leu, Y.-W., Yan, P.S., Fan, M., Jin, V.X., Liu, J.C., Curran, E.M., Welshons, W.V., Wei, S.H., Davuluri, R.V., Plass, C., Nephew, K.P. and Huang, T.H.-M. 2004. Loss of estrogen receptor signaling triggers epigenetic silencing of downstream targets in breast cancer. Cancer Res. 64:8184-8192.
- Cui, Y., Zhang, M., Pestell, R., Curran, E.M., Welshons, W.V. and Fuqua, S.A. 2004. Phosphorylation of estrogen receptor alpha blocks its acetylation and regulates estrogen sensitivity. Cancer Res. 64:9199-9208.
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Progress 01/01/03 to 12/31/03
Outputs
The objectives of the project are to characterize estrogen response mechanisms by using estrogen action in human breast cancer cell lines, to study fetal estrogen exposure via maternal exposure in animals including mice. Both natural estrogens and environmental estrogens act through estrogen receptors that include nuclear-acting forms of estrogen receptor alpha and estrogen receptor beta. In the last year we have reviewed the estrogenic mechanism of action of the widely studied environmental estrogen, bisphenol A, in animals including rodents, and described in a major review how the lack of standard endocrine experimental controls, both positive and negative, has led to substantial quantitative errors in the toxicological assessment of estrogenic endocrine disruptors including bisphenol A, to substantially underestimate the current exposure levels and risks. In collaboration we have shown that some fetal effects of estrogens we described in the review may be modeled
in organ and cell culture. In an additional collaboration involving exercise physiology in the pig model, we have published that, while the boar has been reported to circulate high levels of estradiol, our specific analytical tools and approaches indicate that the circulating total estrogenic activity is in fact much lower, in the range of the normal human male, indicating that the boar is an appropriate model for the study of endocrine effects on exercise physiology. The sow on the other hand circulates estradiol and estrogenic activity that is significantly lower than the human female level. These findings indicate that in the boar, high reported levels of estradiol are not reflected in circulating estrogenic activity. Overall, our work on mechanisms of natural estrogen and environmental estrogen action will aid in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal reproduction and normal estrogen action in both.
Impacts
This work on mechanisms of natural estrogen and environmental estrogen action in tissue culture, in mice during fetal development, and applied to understanding circulating estrogens and estrogen action in swine, will aid in understanding natural estrogen and environmental estrogen actions in human and domestic animal reproduction. In addition, the work on circulating estrogenic activity in the boar will provide basic endocrine information on this domestic animal.
Publications
- Welshons WV, Thayer KA, Judy BM, Taylor JA, Curran EM and vom Saal FS. 2003. Large effects from small exposures. I. Mechanisms for endocrine-disrupting chemicals with estrogenic activity. Environ. Health Perspect. 111: 994-1006.
- Laughlin MH, Welshons WV, Sturek M, Rush JWE, Turk JR, Judy BM, Taylor JA, Henderson KK and Ganjam VK. 2003. Gender, exercise training, and eNOS expression in porcine skeletal muscle arteries. J. Applied Physiol. 95: 250-264.
- Howdeshell KL, Peterman PH, Judy BM, Taylor JA, Orazio CE, Ruhlen RL., vom Saal FS and Welshons WV. 2003. Bisphenol A is released from used polycarbonate animal cages into water at room temperature. Environ. Health Perspect. 111: 1180-1187.
- Kostoryz EL, Eick JD, Glaros AG, Judy BM, Welshons WV, Burmaster S and Yourtee DM. 2003. Biocompatibility of hydroxylated metabolites of BISGMA and BFDGE. J. Dental Research (Biological) 82: 367-371.
- Pamidimukkala J, Taylor JA, Welshons WV, Lubahn DB and Hay M 2003. Estrogen modulation of baroreflex function in conscious mice. American Journal of Physiology-Regulatory Integrative and Comparative Physiology 284: R983-R989.
- Welshons WV, Thayer KM, Judy BM, Taylor JA, Curran EM and vom Saal FS. 2003. Endocrine mechanisms mediating errors in estimating low-dose responses from high-dose studies. Program of the Low Dose Workshop on Endocrine Active Compounds, Harnack-Haus Berlin, p. 34.
- vom Saal FS, Richter CA, Welshons WV and Timms BG. 2003. Bisphenol A, ethinylestradiol and DES produce identical malformations in the urogenital sinus of male mouse fetuses. Program of the Low Dose Workshop on Endocrine Active Compounds, Harnack-Haus Berlin, p. 19.
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Progress 01/01/02 to 12/31/02
Outputs
The objectives of the project are to characterize estrogen response mechanisms by using estrogen action in human breast cancer cell lines, to study fetal estrogen exposure via maternal exposure in animals including mice. Both natural estrogens and environmental estrogens act through estrogen receptors that include nuclear-acting forms of estrogen receptor alpha and estrogen receptor beta. In the last year we have reviewed the estrogenic mechanism of action of the widely studied environmental estrogen, bisphenol A, in animals including rodents, and described in a major review how the lack of standard endocrine experimental controls, both positive and negative, has led to substantial quantitative errors in the toxicological assessment of estrogenic endocrine disruptors including bisphenol A, to substantially underestimate the current exposure levels and risks. In collaboration we have shown that some fetal effects of estrogens we described in the review may be modeled
in organ and cell culture. In an additional collaboration involving exercise physiology in the pig model, we have described that, while the boar has been reported to circulate high levels of estradiol, our specific analytical tools and approaches indicate that the circulating total estrogenic activity is in fact much lower, in the range of the normal human male, suggesting that the boar is an appropriate model for endocrine effects on exercise physiology. The sow on the other hand appears to circulate both estradiol equal total estrogenic activity that is significantly lower than the human female level. These findings are in press at the Journal of Applied Physiology. Overall, our work on mechanisms of natural estrogen and environmental estrogen action will aid in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal reproduction and normal estrogen action in both.
Impacts
This work on mechanisms of natural estrogen and environmental estrogen action in tissue culture, in mice during fetal development, and applied to understanding circulating estrogens and estrogen action in swine, will aid in understanding natural estrogen and environmental estrogen actions in human and domestic animal reproduction. In addition, the work on circulating estrogenic activity in the boar will provide new basic endocrine information on this domestic animal.
Publications
- Welshons, W.V., Thayer, K.A., Judy, B.M., Taylor, J.A., Curran, E.M. and vom Saal, F.S. Large effects from small exposures. I. Mechanisms for endocrine-disrupting chemicals with estrogenic activity. Environ. Health Perspect., in press 2002.
- Laughlin, M.H., Welshons, W.V., Sturek, M., Rush, J.W.E., Turk, J.R., Judy, B.M., Taylor, J.A., Henderson, K.K. and Ganjam, V.K. Gender, exercise training, and eNOS expression in porcine skeletal muscle arteries. J. Applied Physiol, in press 2002.
- Welshons, W.V., 2002. Large effects from small exposures: Implications for adult behaviors following fetal exposure to low doses of the environmental estrogen bisphenol A (BPA). Program of the Erice Meeting on Environmental Endocrine Disruptors and Behavior, Erice, Silicy, Abstract.
- Richter, C.A., Ruhlen, R.L., Welshons, W.V. and vom Saal, F.S., 2002. Androgen receptor mRNA is upregulated by estrogen in mouse prostate primary cell culture. Program of the 2002 Ann. Meeting of the Society of Toxicology, Abstract.
- Hanumegowda, U.M., Judy, B.M., Welshons, W.V. and Reddy, C.S., 2002. Selective inhibition of murine palatal mesenchymal cell proliferation in vitro by secalonic acid D. Toxicological Sciences 66: 159-165.
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Progress 01/01/01 to 12/31/01
Outputs
The objectives of the project are to characterize estrogen receptor proteins and estrogen response mechanisms, by using estrogen action in breast cancer cell lines, and fetal estrogen exposure via maternal exposure in mice. Both natural estrogens and environmental estrogens act through estrogen receptors that include nuclear-acting forms of estrogen receptor alpha and estrogen receptor beta. In the last year we have examined the metabolism and kinetics of the widely studied environmental estrogen, bisphenol A, in pregnant mice and reported in abstract form how the molecule bioaccumulates in circulation of the pregnant but not the nonpregnant female mouse, leading to high circulating levels of the molecule at a time of increased sensitivity to its effects in the fetus. We have also shown that the synthetic estrogen ethinyl estradiol, used in birth control pills, effects reproductive tract development in fetal mice at doses below those that women are exposed to if they
continue to take birth control pills after they become pregnant. In collaboration we have published that estrogen action in clonal cell lines that we developed from human breast cancer cell lines can be studied with respect to the involvement of growth factor signaling pathways, and that estrogen action through estrogen receptor alpha and beta in these cells allow differences in these pathways to be examined. Overall, our work on mechanisms of natural estrogen and environmental estrogen action in human breast cancer cells in tissue culture and in mice during fetal development will aid in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal reproduction.
Impacts
This work on mechanisms of natural estrogen and environmental estrogen action in human breast cancer cells in tissue culture, and in mice during fetal development, will aid in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal reproduction.
Publications
- Gobert, G.N., Hueser, C.N., Curran, E.M., Sun, Q.-Y., Glinskii, S., Welshons, W.V., Eisenstark, A. and Schatten, H., 2001. Immunolocalization of NuMA and phosphorylated proteins during the cell cycle in human breast and prostate cancer cells as analyzed by immunofluorescence and postembedding immunoelectron microscopy. Histochemistry and Cell Biology 115: 381-395.
- Oesterreich, S., Zhang, P., Guler, R.L., Sun, X., Curran, E.M., Welshons, W.V., Osborne, C.K. and Lee, A.V., 2001. Re-expression of estrogen receptor alpha in estrogen receptor alpha-negative MCF-7 cells restores both estrogen and insulin-like growth factor-mediated signaling and growth. Cancer Res. 61: 5771-5777.
- Thayer, K.A., Ruhlen, R.L., Howdeshell, K.L., Buchanan, D.L., Cooke, P.S., Preziosi, D.E., Welshons, W.V., Haseman, J.K. and vom Saal, F.S., 2001. Altered prostate growth and daily sperm production in male mice exposed prenatally to sub-clinical doses of 17alpha-ethinyl estradiol. Human Reproduction 16: 988-996.
- Thayer, K.A., Ruhlen, R.L., Howdeshell, K.L., Buchanan, D.L., Cooke, P.S., Preziosi, D., Welshons, W.V., Haseman, J. and vom Saal, F.S. , 2001. Altered prostate growth and daily sperm production in male mice exposed prenatally to subclinical doses of 17 alpha-ethinyl oestradiol. APMIS 109 (Suppl 103): S278-S286. [Acta Pathologica Microbiologica Immunologica Scandanavica]
- Phillips, B.J., Ansell, P.J., Newton, L.G., Taylor, J.A., Ganjam, V.K., Rottinghaus, G.E., Welshons, W.V. and Lubahn, D.B., 2001. 4-Hydroxylated catechol estrogen binding in ERalphaWT, ERalphaKO and ArKO mice: Evidence for "ERgamma"?. Program of the 83rd Annual Meeting of the Endocrine Society.
- Yuan, X., Curran, E.M., Ansell, P.J., Welshons, W.V. and Lubahn, D.B., 2001. Both in vivo ligand-binding and transcriptional activities of human ERalpha and ERbeta are cell-type specific. Program of the 83rd Annual Meeting of the Endocrine Society.
- Dougherty, M.K., Schumaker, L., Jordan, V.C., Curran, E., Welshons, W.V., El-Ashry, D. and Ellis, M.J., 2001. Estrogen-regulated proliferation influences paclitaxel sensitivity in breast cancer. Proc. 92nd Ann. Meeting of the Am. Assoc. for Cancer Res. Abstract 1947.
- Ganjam, V.K., Taylor, J.A., Judy, B.M., Laughlin, M.H., and Welshons, W.V., 2001. Biologically active estrogen in the boar. Program of the 2001 APS Conference on Genome and Hormones: An Integrative Approach to Gender Differences in Physiology.
- Ganjam, V.K., Alworth, L.C., Howdeshell, K.L., Ruhlen, R.L., Slight, S.H., vom Saal, F.S. and Welshons, W.V., 2001. Fetal programming of 11beta-hydroxysteroid dehydrogenase by maternal exposure to estrogen. Program of the 83rd Annual Meeting of the Endocrine Society.
- vom Saal, F.S., Howdeshell, K.L., Ruhlen, R.L., Welshons, W.V., 2001. Fetal levels of endogenous estradiol and exposure to environmental estrogens influence age-related changes in the male and female reproductive system. Program of the India Meeting on Hormones, Development and Behavior.
- Welshons, W.V., Nagel, S.C., Judy, B.M., Taylor, J.A., Howdeshell, K.L., Ruhlen, R.L. and vom Saal, F.S., 2001. In vitro assessment of endocrine disruptors: Activity of the environmental estrogen bisphenol A at levels of current human exposure. Program of the 2001 Congress on In Vitro Biology.
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Progress 01/02/00 to 12/31/00
Outputs
The objectives of the project are to characterize estrogen receptor proteins and estrogen response mechanisms, by using estrogen action in breast cancer cell lines, and fetal estrogen exposure via maternal exposure in mice. Both natural estrogens and environmental estrogens act through estrogen receptors that include nuclear-acting forms of estrogen receptor alpha and estrogen receptor beta. In the last year we have described how the physiology of delivery of the environmental estrogen genistein to target cells substantially changes its effective estrogenic activity, leading to both increases and decreases in the biological impact of genistein in rodents and potentially in humans. In addition we have examined the metabolism and kinetics of another widely studied environmental estrogen, bisphenol A, in pregnant mice and reported in abstract form how the molecule bioaccumulates in circulation of the pregnant but not the nonpregnant female mouse, leading to high
circulating levels of the molecule at a time of increased sensitivity to its effects in the fetus. In collaboration we have also published that estrogen action in clonal cell lines that we developed from human breast cancer cell lines can be studied with respect to the involvement of growth factor signaling pathways, and that estrogen action through estrogen receptor alpha and beta in these cells allows differences in these pathways to be examined. Overall, our work on mechanisms of natural estrogen and environmental estrogen action in human breast cancer cells in tissue culture and in mice during fetal development will aid in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal reproduction.
Impacts
This work on mechanisms of natural estrogen and environmental estrogen action in human breast cancer cells in tissue culture, and in mice during fetal development, will aid in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal reproduction.
Publications
- Lee, A.V., Guler, B.L., Sun, X., Oesterreich, S., Zhang, Q.P., Curran, E.M. and Welshons, W.V. 2000. Oestrogen receptor is a critical component required for insulin-like growth factor (IGF)-mediated signalling and growth in MCF-7 cells. Eur. J. Cancer 36 (Suppl 4):109-110.
- Welshons, W.V., Nagel, S.C. and vom Saal, F.S. 2000. Protein binding interactions - Approaches to quantifying "free" and "bound" fractions of genistein and other xenobiotic estrogens in serum. J. Med. Food 2 (3 & 4):119-121.
- vom Saal, F.S. and Welshons, W.V. 2000. NIH Panel Confirms That Endocrine Disrupting Chemicals Cause Effects at Very Low Doses. Risk Policy Report 7:47-50.
- Yuan, X., Curran, E.M., Judy, B.M., Welshons, W.V. and Lubahn, D.B. 2000. Comparison of in vitro ligand-binding and transactivation properties of human ER-alpha and ER-beta. Program of the 82nd Annual Meeting of the Endocrine Society, p. 351, abstr. 1456.
- Bolden-Tiller, O.U., Welshons, W.V., Lucy, M.C. and Smith, M.F. 2000. Progesterone receptor mRNA and protein expression in bovine corpora lutea. Program of the 33rd Annual Meeting of the Society for the Study of Reproduction, Biol. Reprod., abstr.
- Welshons, W.V., Taylor, J.A., Judy, B.M. and vom Saal, F.S. 2000. Why bisphenol A is weak in the adult yet strong in the fetus. Program of the Meeting on Bisphenol A: Low Dose Effects-High Dose Effects, p. 21.
- vom Saal, F.S., Howdeshell, K.L., Ruhlen, R.L., Taylor, J.A., Timms, B.G. and Welshons, W.V. 2000. High sensitivity of the fetal prostate to endogenous and environmental estrogens. Program of the Meeting on Bisphenol A: Low Dose Effects-High Dose Effects, p. 20.
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Progress 01/01/99 to 12/31/99
Outputs
The objectives of the project are to characterize estrogen receptor proteins and estrogen response mechanisms, by using estrogen action in breast cancer cell lines, and fetal estrogen exposure via maternal exposure in mice. Both natural estrogens and environmental estrogens act through estrogen receptors that include nuclear-acting forms of estrogen receptor alpha and estrogen receptor beta. In the last year we have described how the physiology of delivery of environmental estrogens to target cells substantially changes their effective estrogenic activity, leading to increases in the biological impact of many environmental estrogens including phytoestrogens and the current use pesticide methoxychlor. These results explain the surprisingly high biological activity of some xenoestrogens, including the plastic monomer bisphenol A, the synthetic estrogen DES and methoxychlor. We have also published this year that fetal exposure of mice to low doses of methoxychlor can
disrupt normal development of the reproductive tract, which can lead to enlargement of the prostate and reduced sperm production in subsequent adult mice. In collaboration we have also published that hypermethylation of a tumor suppressor gene accompanies development of human breast cancer when compared to normal mammary tissue from the same patient. Overall, our work on mechanisms of natural estrogen and environmental estrogen action in breast cancer cells and in human breast cancer cells in tissue culture will aid in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal reproduction.
Impacts
Our work on mechanisms of natural estrogen and environmental estrogen action in human breast cancer cells in tissue culture, and in mice during fetal development, will aid in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal reproduction.
Publications
- Welshons, W.V., Nagel, S.C., Thayer, K.A., Judy, B.M. and vom Saal, F.S., 1999. Low-dose bioactivity of xenoestrogens in animals: Fetal exposure to low doses of methoxychlor and other xenoestrogens increases adult prostate size in mice. Toxicol. Industrial Health 15: 12-25.
- Nagel, S.C., vom Saal, F.S. and Welshons, W.V., 1999. Developmental effects of estrogenic chemicals are predicted by an in vitro assay incorporating modification of cell uptake by serum. J. Steroid Biochem. Molec. Biol. 69: 343-357.
- Cunha, G.R., Forsberg, J.G., Golden, R., Haney, A., Iguchi, T., Newbold, R., Swan, S. and Welshons, W., 1999. New approaches for estimating risk from exposure to diethylstilbestrol. Environ. Health Perspect. 107 (Suppl 4): 625-630.
- Laux, D.E., Curran, E.M., Welshons, W.V., Lubahn, D.B. and Huang, T.H.M., 1999. Hypermethylation of the Wilms' tumor suppressor gene CpG island in human breast carcinomas. Breast Cancer Res. Treat. 56: 35-43.
- deGraffenried, L.A., Welshons, W.V., Curran, E.M. and Fuqua, S.A., 1999. Transcriptional regulation of the estrogen receptor gene minimal promoter in human cancer cells. Program of the 81st Annual Meeting of the Endocrine Society, p. 194, abstr. P1-280.
- Ganjam, V.K., Slight, S.H., Hong, G. and Welshons, W.V., 1999. Inhibition of 11-beta-hydroxysteroid dehydrogenase (11-beta-HSD) in MCF-7 human breast cancer cells as a sensitive marker for estrogenic endocrine disrupters. Program of the 81st Annual Meeting of the Endocrine Society, p. 336, abstr. P2-266.
- Lee, A.V., Guler, R.L., Oesterreich, S., deGraffenried, L.A., Fuqua, S.A., Curran, E.M. and Welshons, W.V., 1999. Loss of estrogen receptor in MCF-7 breast cancer cells is associated with reduced IGFR1 and IRS-1 expression, diminished IGF signaling, and a failure to respond mitogenically to IGFs. Program of the 81st Annual Meeting of the Endocrine Society, p. 478, abstr. P3-190.
- Ruhlen, R.L., Thayer, K.A., Preziosi, D.E., Howdeshell, K.L., vom Saal, F.S. and Welshons, W.V., 1999. Prenatal ethinyl estradiol exposure increases adult prostate size and prostatic androgen receptors in adult male mice. Program of the 81st Annual Meeting of the Endocrine Society, p. 493, abstr. P3-258.
- Lee, A.V., Guler, B.L., Curran, E. and Welshons, W., 1999. Loss of estrogen receptor (ER) in MCF-7 breast cancer cells results in reduced insulin-like growth factor receptor (IGFR1) and insulin receptor substrate (IRS)-1 expression, diminished IGF signaling, and a failure to respond mitogenically to IGFs. Program of International IGF Meeting, p. nnn, abstr. nnn.
- Taylor, J.A., Judy, B.M., Rottinghaus, B.A., Blackwell, K.J., Rottinghaus, G.E., Alworth, L.C., vom Saal, F.S. and Welshons, W.V., 1999. Bisphenol A bioaccumulates in the serum of pregnant mice. Program of the Symposium on Environmental Hormones: Past, Present, Future, p. 18-19.
- Howdeshell, K.L., Peterman, P.H., Ruhlen, R.L., Judy, B.M., Welshons W.V. and vom Saal, F.S., 1999. Bisphenol A is released from used polycarbonate rodent cages. Program of the Symposium on Environmental Hormones: Past, Present, Future, p. 9.
- Ruhlen, R.L., Thayer K.A., Preziosi, D.E., Howdeshell, K.L., Welshons, W.V., vom Saal, F.S., 1999. Fetal exposure to very low doses of ethinyl estradiol increases prostate size and androgen receptors in CF-1 and CD-1 mice. Program of the Symposium on Environmental Hormones: Past, Present, Future, p. 14.
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Progress 01/01/98 to 12/31/98
Outputs
The objectives of the project are to characterize estrogen receptor proteins and estrogen response mechanisms. Both natural estrogens and environmental estrogens act through estrogen receptors that include nuclear-acting forms of estrogen receptor alpha and estrogen receptor beta, but also may include other estrogen receptors and postulated non-nuclear, nongenomic forms of ER alpha and beta (for example membrane-associated forms of the receptors). In the last year we have published a review on the potential nongenomic estrogen receptor mechanisms. With respect to the less controversial genomic mechanisms, we have described how the physiology of delivery of environmental estrogens to target cells substantially changes their effective estrogenic activity, leading to increases in the biological impact of many environmental estrogens including phytoestrogens. These results explain the surprisingly high biological activity of some xenoestrogens, including the plastic
monomer bisphenol A, the synthetic estrogen DES and the current-use insecticide methoxychlor. We have also published this year that fetal exposure of mice to low doses of bisphenol A and methoxychlor can disrupt normal development of the reproductive tract, which can lead to reduced sperm production in subsequent adult mice. This may represent an estrogenic mechanism for reductions in human sperm counts that have been reported and debated recently. Overall, our work on mechanisms of natural estrogen and environmental estrogen action will aid in understanding the impact of developmental exposures to environmental estrogens on human and domestic animal reproduction.
Impacts
(N/A)
Publications
- Judy, B.M. and Welshons, W.V. 1998. Cellular localization of receptors mediating actions of steroid hormones. In: Handbook of Physiology, Section 7: The Endocrine System, Volume I: Cellular Endocrinology, ed. P.M. Conn and H.M. Goodman, American Physiology Society, pp. 437-460.
- Nagel, S.C., vom Saal, F.S. and Welshons, W.V. 1998. The effective free fraction of estradiol and xenoestrogens in human serum measured by whole cell uptake assays: physiology of delivery modifies estrogenic activity. Proc. Soc. Exp. Biol. Med. 217:300-309.
- Welshons, W.V., vom Saal, F.S. and Nagel, S.C. 1998. The importance of protocol design and data reporting to research on endocrine disruption. Response. Environ. Health Perspect. 106:A315-A317.
- vom Saal, F.S., Cooke, P.S., Buchanin, D.L., Palanza, P., Thayer, K.A., Nagel, S.C., Parmigiani, S. and Welshons, W.V. 1998. A physiologically based approach to the study of bisphenol A and other estrogenic chemicals on the size of reproductive organs, daily sperm production and behavior. Toxicol. Industrial Health 14:239-260.
- vom Saal, F.S., Welshons, W.V. and Hansen, L.G. 1998. Organochlorine levels and breast cancer. New England J. Med. 338:988.
- Thayer, K.A, Howdeshell, K.L., vom Saal, F.S., Lubahn, D.B., Buchanan, D., Cooke, P. and Welshons, W.V. 1998. Low dose effects of prenatal exposure to estrogens. Program of the first Gordon Research Conference on Endocrine Disruptors, abstr. 21.
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Progress 01/01/97 to 12/31/97
Outputs
The objectives of the project are to characterize estrogen receptor proteins and estrogen response mechanisms. Both natural estrogens and xenobiotic, environmental estrogens act through the estrogen receptor mechanism. In the last year we have published how characterizing natural estrogen and environmental estrogen action in MCF-7 human breast cancer cells can be used to understand and predict environmental estrogen action in the intact animal. We found that how estrogenic chemicals are carried in serum to estrogen responsive cells, combined with the low estrogen receptor occupancy required for biological response, made it possible for us to predict accurately the in vivo estrogenic activity of chemical estrogens that included bisphenol A, 4-octylphenol and diethylstilbestrol (DES), in acting to disrupt normal fetal development of the reproductive tract in mice. The endocrine disruption we predicted and observed with bisphenol A occurred at doses that were thousands
of fold lower than have been reported for this chemical in the past, and occurred at oral doses which include current human exposures to this chemical which is widely-used in plastics. This work on the fundamental mechanisms of natural estrogen and environmental estrogen action will substantially aid in understanding the effects and impact of environmental estrogen exposures on human and domestic animal reproduction.
Impacts
(N/A)
Publications
- Nagel SC, et al, 1997. Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vitr . Envir Health Perspect 105:70-76.
- vom Saal, FS, et al. 1997. Prostate enlargement in mice due to fetal exposure to low doses of estradiol or . Proc Natl Acad Sci (USA)
- Welshons, WV, vom Saal, FS and Nagel, SC, 1997. Bisphenol A in food cans:an update. Envir Health Perspect 105:571-572.
- Ruh, MF, Taylor, JA, Howlett, AC, Welshons, WV, 1997. Failure of cannabinoid compounds to stimulate estrogen receptors. Biochem
- Gray, LE, et al. 1997. Endocrine screening methods workshop report - Detection of estrogenic and androgenic hormonal and antihormonal activity for . Repro Tox 11:719-750.
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Progress 01/01/96 to 12/30/96
Outputs
The objectives of the project are to characterize estrogen receptor proteins andestrogen responsiveness in hormone-dependent MCF-7 human breast cancer cells. isolated novel sublines derived from estrogen-dependent, estrogen receptor-positive MCF-7 cells that yielded a series of cell lines that progress through two intermediate steps to estrogen-independence and complete estrogen receptor used these cell lines for the study of breast cancer progression and regulation of the expression of estrogen receptors. Loss of expression of estrogen receptors in the estrogen receptor-negative cell lines was accompanied by inc the receptors for epidermal growth factor (EGF receptors), and by progressively altered response to EGF. The role of estrogen receptor gene methylation was examined in the control of estrogen receptor expression in these cells. We found that loss of estrogen receptor expression preceded changes in methylation of the estrogen receptor gene in these cell
lines.
Impacts
(N/A)
Publications
- Judy B, Greene G, Welshons W. 1996. Characterization of non-nuclear estrogen receptors (ERNN) in MCF Pgm 10th Internat Congr of Endocrinol, p575, abstr P2-683. PB Nagel S, vom Saal F, Sharpe-Timms K, Welshons W. 1996. Free estradiol in human cord serum measured at less than .05% using... Pgm 10th Internat Congr of Endocrinol p855, abstr P3-401.
- Curran E, Judy B, Golomb M, Welshons W. 1996. Regulation of estrogen receptor mRNA by EGF, estradiol and TPA in human breast ... Pgm. 10th Internat. Congr of.
- Ruh, M.F., Taylor, J.A., Howlett, A.C., Welshons, W.V. 1997. Failure of cannabinoid compounds to stimulate estrogen receptors. Biochem. Pharmacol (In Press ).
- Nagel S, vom Saal F, Thayer K, Dahr M, Boechler M, Welshons, W. 1997. Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vitro... Envir Hlth Perspec (in press).
- vom Saal F, Timms B, Montano M, Palanza P, Thayer K, Nagel S, Dahr M, Ganjam V, Parmigiani S, Welshons W. 1997. Prostate enlargement in mice due to fetal... Proc Natl Acad Sci(USA)(in press).
- Ruh, M., Taylor, J., Howlett, A., Welshons, W., 1996. Estrogen receptors fail torespond to cannabinoid agonists. Pgm. 10th Internat. Congr. of Endocrinol., p. 573, abstr. P2-676.
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Progress 01/01/95 to 12/30/95
Outputs
The objectives of the project are to characterize estrogen receptor proteins andestrogen responsiveness in hormone-dependent MCF-7 human breast cancer cells. Approximately 15% of estrogen receptors (ER) in MCF-7 cells are non-nuclear estrogen receptors (ERnn). We used sucrose density sedimentation to compare ERnn to ER, and to determine how both receptor forms interact with anti-ER antibodies which bind to epitopes in different ER domains. The monomer form of both receptors in high salt (0.4 M KCl) migrated at 3.6S, however, in low salt buffer, oligomeric ERnn migrated 1.2S-1.4S faster than oligomeric ER, 8.2S vs. 6.8S, respectively. All antibodies tested recognized both ER and ERnn. We conclude that ERnn are associated in the cytoplasm of the cell with one or more ER-associated proteins (ERAP) with which nuclear ER are not associated, and that ERnn are positioned to mediate nongenomic estrogen actions. Analysis of novel hormone-independent sublines of MCF-7 cells
isolated in prior work indicated that hormone-responsiveness was lost in two steps that preceded loss of expression of estrogen receptors in the final ER-negative cell lines. While ER gene deletion had not occurred, mRNA for the ER was not expressed in the hormone-nonresponsive cells. The role of methylation will be examined in the control of ER expression in these cells. Studies of the relationship of mitogen-stimulated MCF-7 cell proliferation to changes in the phosphoinositide pathway were also published.
Impacts
(N/A)
Publications
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Progress 01/01/94 to 12/30/94
Outputs
We have described that approximately 15% of estrogen receptors (ER) in MCF-7 human breast cancer cells are non-nuclear estrogen receptors (ER(subscript NN)), and these may represent a nongenomic form of steroid receptor. The activities of the past year have focused on characterizing these receptors, and examining whether MCF-7 cells show rapid or other nongenomic estrogenic responses that may be mediated by nongenomic receptors. Compared to the major population of ER, ER(subscript NN) could not bind to the nucleus in intact cells even when ligand-occupied, showed lack of receptor dimerization, and showed differences in several physical properties. As determined by aqueous two-phase partitioning, ER(subscript NN) do not appear to bind to DNA as well as did the major form of ER. Size and immunoreactivity on Western blots are the same as for the major form of ER. Despite careful evaluation, it did not appear that in MCF-7 cells, estrogen could provoke a rapid mobilization
of the phosphoinositide pathway, a nongenomic steroid response that has been reported in other cells. However, estrogens did strongly mobilize the phosphoinositide pathway as a long term, presumably genomic response. Additional work continues to examine a recently-reported nongenomic mobilization of cAMP in MCF-7 cells by estrogens.
Impacts
(N/A)
Publications
- WELSHONS, W.V., JUDY, B.M., STRNAD, R.L., and GRADY, L.H. 1994. Non-nuclear estrogen receptors in cytoplasts from MCF-7 human breast cancer cells. Estrogens in the Environment III: Global Health Implications, ed. JA McLachlan.
- TIMMS, B.G., WELSHONS, W.V., NAGEL, S.C., PETERSEN, S.L., PETERSON, R.E., and VOM SAAL, F.S. 1994. Prostrate morphogenesis correlates with blood estradiol levels in fetal male and female rats. Estrogens in the Environment III:.
- CURRAN, E.M., JUDY, B.M., GOLOMB, M., and WELSHONS, W.V. 1994. A hormone-independent, estrogen- receptor-negative cell line isolated from MCF-7 human breast cancer cells: Relation to tumor progression. Endocrinology 134 (Suppl):390.
- WELSHONS, W.V., JUDY, B.M., STRNAD, R.L., GRADY, L.H., and CURRAN, E.M. 1994. Non-nuclear estrogen receptors (ER(subscript NN)) in MCF-7 human breast cancer cells. Endocrinology 134 (Suppl):628 (abstract 1711).
- TAYLOR, J.A., GRADY, L.H., ENGLER, K.S., and WELSHONS, W.V. 1995. Relationship of growth stimulated by lithium, estradiol and EGF to phospholipase C activity in MCF-7 human breast cancer cells. Breast Cancer Res. Treat (in press).
- WELSHONS, W.V. and JUDY, B.M. 1995. Nuclear vs translocating steroid receptor models and the excluded middle. Endocrine (in-press).
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Progress 01/01/93 to 12/30/93
Outputs
Research is summarized by the specific project objectives. 1) Enucleation of estrogen-responsive MCF-7 cells was established to separate the synthesis of estrogen receptors (ER) in cytoplasts from the degradation of ER, which occurred in nucleoplasts. 2) Determination of kinetics indicated that down-regulation of the ER by ligand was partially mediated by increases in the rate of receptor degradation. ER degradation was independent of the lysosomal, ATP-ubiquitin, and other ATP-dependent protein degradative pathways of the cell. The kinetics of progesterone and glucocorticoid receptor turnover were much slower than those of ER. The first change in ER kinetics observed after initiation of receptor up-regulation was a decrease in the rate of receptor protein degradation within 18 h, followed by slower changes in actual receptor protein level over 4 d. 3) By immunoassay compared to ligand binding, there was no evidence for intermediates in ER synthesis. However, we
unexpectedly observed that 15% of MCF-7 cell ER were non-nuclear (ER(subscript NN)). Compared to the major population of ER, ER(subscript NN) could not bind to the nucleus in intact cells even when ligand-occupied, showed lack of receptor dimerization, and showed differences in several physical properties. Size and immunoreactivity on Western blots were the same as for the major form of ER. ER(subscript NN) may mediate recently-described nongenomic estrogen actions in cells.
Impacts
(N/A)
Publications
- WELSHONS, W.V., GRADY, L.H., JUDY, B.M., JORDAN, V.C. and PREZIOSI, D.E., 1993. Subcellular compartmentalization of MCF-7 estrogen receptor synthesis and degradation. Molec. Cell. Endocrinol. 94: 183-194.
- MOFFATT, R.J., ZOLLERS, W.G., JR., WELSHONS, W.V., KEIBORZ, K.R., GARVERICK, H.A. and SMITH, M.F., 1993. Basis of norgestomet action as a progestin in cattle. Domestic Animal Endocrinol. 10: 21-30.
- WELSHONS, W.V., JUDY, B.M., STRNAD, R.L. and GRADY, L.H., 1993. Cytoplasmic, non-nucleophilic estrogen receptors in cytoplasts from MCF-7 human breast cancer cells. Endocrinology 132 (Suppl): 517, abstr. 1865.
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Progress 01/01/92 to 12/30/92
Outputs
We have separated estrogen receptor (ER) synthesis from ER degradation by using enucleation of MCF-7 human breast cancer cells to separate the cytoplasmic and nuclear compartments as cytoplasts and nucleoplasts. Synthesis of the ER protein occurs in the cytoplasm, while degradation of the protein appears to be restricted to the nuclear compartment. The cytoplasmic compartment (cytoplasts) appears to contain a novel form of the ER protein, approximately 15% of the total ER's in the MCF-7 cell. These cytoplastic ER's are not subject to ligand-dependent down-regulation (processing) in the cytoplast, and are non-nucleophilic in that incubation with hormone does not transfer any of the cytoplastic ER to the nuclear compartment. We used aqueous two-phase partitioning (ATPP) to compare solution properties of the cytoplastic receptors to the total receptors in whole cells. The ATPP partitioning coefficients (Kobs) for total MCF-7 ER were 1.97/+-/ 0.03, 1.30/+-/ 0.01 and
0.45/+-/0.02 (n=2) for unoccupied ER, estradiol-occupied, nontransformed ER, and estradiol-occupied, heat-transformed ER, respectively. For cytoplastic ER, the Kobs for unoccupied ER was lower than that of the whole cell ER, and the decrease in Kobs upon transformation of the cytoplastic ER was less than half of that seen for the whole cell ER. These data indicate that transformation of the cytoplast ER may be incomplete or absent.
Impacts
(N/A)
Publications
- WELSHONS, W.V., GRADY, L.H. and JUDY, B.M. 1992. Control of proliferation of MCF-7 breast cancer cells in a commercial preparation of charcoal-stripped adult bovine serum. Breast Cancer Res Treat 23:97-104.
- NONNEMAN, D.J., GANJAM, V.K., WELSHONS, W.V. and VOM SAAL, F.S. 1992. Intrauterine position effects on steroid metabolism and steroid receptors of reproductive organs in male mice. Biol Reprod 47:723-729.
- TAYLOR, J.A. and WELSHONS, W.V. 1992. MCF-7 breast cancer cell proliferation and membrane-associated phospholipase C activity: Implications for mitogenic signalling mechanisms. Endocrinol 130 (Suppl):324, Abstr 1092.
- ZOLLERS, W.J., JR., SMITH, M.F., MOFFATT, R.J., WELSHONS, W.V., KIEBORZ, K.R. and GARVERICK, H.A. 1992. Norgestomet binds to the progesterone but not to the estrogen receptor and has no significant estrogenic activity. J Animal Sci 70.
- MONTANO, M., WELSHONS, W., KEEL, B. and VOM SAAL, F.S. 1992. Estradiol passes from blood into brain cells and binds to estrogen receptors in newborn female rats. 25th Ann Mtg of Society for Study of Reproduction, Biol Reprod 46 (Suppl 1).
- GANJAM, V.K., GOFF, A.L., NONNEMAN, D.J., WEST, D.A., SWEENY, C.L., WILSON, D.A., DAWSON, L. and WELSHONS, W.V. 1992. Regional distribution of androgen receptors and 5 (alpha)-reductase in the equine epididymis. Proc. 5th Int'l Symposium.
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Progress 01/01/91 to 12/30/91
Outputs
We used cell enucleation to separate the degradation of estrogen receptors in the nucleus of MCF-7 cells, from new receptor synthesis in the cytoplasm, to study these usually concurrent processes in live, intact cytoplasts and nucleoplasts. Kinetic and Scatchard binding analyses of estradiol to nucleoplast estrogen receptors indicated that the ligand affinity of estrogen receptors in whole cells and in nucleoplasts formed from those cells were essentially the same, with apparent Kd's of 0.1 to 0.2nM for the receptors in both. In the course of these studies, we observed a pH-dependent cytotoxicity present as a contaminant in the pH indicator phenol red. The cytotoxicity was 100-fold greater for the hormone-dependent breast cancer MCF-7 cells, than for other cell lines. The pH-dependent cytotoxicity of phenol red may need to be considered as a factor in many physiological studies in tissue culture, and this specific toxicity for a breast cancer cell line may have
application in anticancer therapy. During the current year we initiated work to study estrogen receptor mRNA stability. We obtained clones for the estrogen receptor message (pOR3) and for two necessary controls (36B4 and pS2). Restriction fragments of the full-length clones were subcloned for use as probes.
Impacts
(N/A)
Publications
- GRADY, L.H., NONNEMAN, D.J., ROTTINGHAUS, G.E. and WELSHONS, W.V. 1991. pH-dependent cytotoxicity of contaminants of phenol red for MCF-7 breast cancer cells. Endocrinology 129:3321-3330.
- WELSHONS, W.V. , GRADY, L.H., NONNEMAN, D.J. and ROTTINGHAUS, G.E. 1991. Specific pH-dependent toxicity, as well as estrogenic activity, of contaminants of phenol red for hormone-dependent human breast cancer cells. Endocrinology 128.
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Progress 01/01/90 to 12/30/90
Outputs
We have used cell enucleation to separate the degradation of estrogen receptors in the nucleus of the MCF-7 cells, from new receptor synthesis in the cytoplasm, to study the isolated degradation of the receptors in live, intact nucleoplasts. MCF-7 nucleoplasts incorporated ?(superscript 3 )H?leucine at 1/8 the rate seen in whole cells, indicating that most of the cytoplasm had been removed. The nucleoplasts degraded estrogen receptors rapidly (t 1/2 of 4-8 hrs), but the longer-lived progesterone receptors were not degraded, indicating specificity to the receptor turnover. The degradation of estrogen receptors in nucleoplasts was compared to the degradation of estrogen receptors in whole cells during accelerated turnover, brought on by incubation in serum-free media, and the rates of degradation measured by these 2 techniques were the same. A substantial fraction of receptor degradation in whole cells during accelerated turnover appeared to be blocked by the
proteinase-inhibitor iodoacetic acid (0.1 mM), in estrogen-treated but not in control cells. These data may link changes in the rate of degradation of estrogen receptors in vivo to 1 or more stimulated down-regulation of estrogen receptors. In the course of these studies, we have observed a pH-dependent toxicity present as a contaminant in the pH indicator phenol red. This activity is distinct from the several estrogenic contaminants that have already been described, and the toxicity is specific for the MCF-7 hormone-dependent breast cancer cells.
Impacts
(N/A)
Publications
- WELSHONS, W.V. and GRADY, L.H. 1990. Regulation of the steady-state level and degradation rates of estrogen receptors in MCF-7 breast cancer cells. Endocrinology 126(Suppl), Abstract 935.
- WELSHONS, W.V., GRADY, L.H., NONNEMAN, D.J. and ROTTINGHAUS, G.E. 1991. Specific pH-dependent toxicity, as well as estrogenic activity, of contaminants of phenol red for hormone-dependent human breast cancer cells, Endocrinology 128:abstra.
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Progress 01/01/89 to 12/30/89
Outputs
Cell enucleation was used to separate the cytoplasmic (cytoplast) and nuclear (nucleoplast) compartments of estrogen-responsive MCF-7 cells, in order to study the degradation of steroid receptor proteins in the enriched nuclear compartment represented in nucleoplasts. Estrogens down-regulated the steady state level of estrogen receptors maintained in the cells. Down-regulated, occupied estrogen receptors were degraded or inactivated in nucleoplasts with a half-life of 4 hrs (rate constant -0.17/hr). In up-regulated cells (unoccupied receptors), which maintained approximately 3 times as many estrogen receptors per cell, the receptors were degraded in nucleoplasts with a half-life of 7-8 hrs (rate constant -0.09 to -0.10/hr). Therefore, changes in receptor degradation/inactivation rate account for approximately half of the increase in receptor content in up-regulated cells. Receptor degradation in nucleoplasts could not be prevented by inhibitors of the lysosomal,
ubiquitin-ATP, or other ATP-dependent protein degradative pathways. However, in serum free media, approximately 25% of receptor degradation came to be blocked by these inhibitors (chloroquine, NH(4)Cl, dinitrophenol). Interestingly, the protein synthesis inhibitor cycloheximide did not reduce the already-maximal rate of receptor degradation in nucleoplasts.
Impacts
(N/A)
Publications
- NO PUBLICATIONS REPORTED THIS PERIOD.
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Progress 09/01/88 to 12/30/88
Outputs
Work was initiated to use the enucleated MCF-7 cell system to study steroid receptor synthesis and degradation. Nucleoplasts and cytoplasts of MCF-7 cells showed changes in estrogen receptor content that were consistent with receptor inactivation or degradation in nucleoplasts, and consistent with receptor synthesis in cytoplasts (shown to be cycloheximide-inhibitable in other studies). In the present studies, the nucleoplast receptor content decreased with a T1/2 of 3-4 hrs, and the decreases (presumably receptor inactivation/degradation) were not affected by cycloheximide. In cytoplasts, approximately linear estrogen receptor synthesis was observed for 3-5 hrs. 343HiLeucine incorporation was used to measure general protein synthesis in cytoplasts and nucleoplasts compared to that in control whole cells. 343HiLeucine incorporation in cytoplasts indicated approximately linear protein synthesis for 3 hrs, and strong incorporation continued beyond 5 hours. Nucleoplasts
incorporated leucine at a lower rate, consistent with reduced cytoplasm. Work continues on steroid receptor kinetics in up- and down-regulated receptor states of the MCF-7 cell, and on which cellular pathway(s) are involved in the degradation of the receptors.
Impacts
(N/A)
Publications
- WELSHONS, W.V. and JORDAN, V.C. (1988). Estrogen receptor dynamics in enucleated MCF-7 cells. Endocrinology 122(Suppl), Abstr. 1287.
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