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Functional analysis of a mutant estrogen receptor isolated from T47Deo breast cancer cells
Citations from the Literature Fun&onaI anaIysia of a mutant estrogen receptor isolated from T47Dco breast cancer cells Leslie K.K.; Tasset D.M.; Horwitz K.B. USA AM J OBSTET GYNECOL 1992 16614(1053-1061) Objective: Estrogen receptor-positive cancers that initially respond to hormone therapy often progress to a resistant state. The breast cancer cell line T47Dco is a model for such resistance. It is a polymorphic line, composed of multiple cell populations that demonstrate the presence of mutant estrogen receptors by cloning and sequencing techniques. Our objective was to isolate and analyze the structural and functional characteristics of the T47Dco mutant estrogen receptor complementary deoxyribonucleic acid clones. Study design: We constructed two independent T47Dco complementary deoxyribonucleic acid libraries. We isolated and sequenced T47Dco estrogen receptors and have identified a mutant receptor that is truncated near the end of the deoxyribonucleic acid binding domain. This mutant has now been recreated with site-directed mutagenesis and tested for its ability to bind to deoxyribonucleic acid, dimerixe with other estrogen receptors, and activate gene transcription by the chloramphenicol acetyltransferase assay and by gel shift assays. Results: The chloramphenicol acetyltransferase assays reveal that in the absence of estradiol low levels of conversion of chloramphenicol to acetylated products occur when the mutant estrogen receptor is used to activate chloramphenicol acetyltransferase gene transcription, supporting that it has some constitutive function. Also, gel mobility shift assays demonstrate low levels of deoxyribonucleic acid binding with the mutant protein. Conclusion: This mutant estrogen receptor may contribute to the estrogen receptor-positive, hormone-resistant phenotype of T47Dc0 cells by, constitutively binding to and activating genes that were previously estradiol dependent.
GYNECOLOGICAL ENDOCRINOLOGY wtddl bormonc tsstsfor tbc dlagmis of golyeyatic ovary syn4rolne? Robinson !I.; Rodin D.A.; Deacon A.; Wheeler M.J.; Clayton R.N. GBR BR J OBSTET GYNAECOL 1992 9913 (232-238) Objective: To assess the frequency of abnormal values for hormone measurements commonly used in the biochemical diagnosis of polycystic ovary syndrome (PCOS). Design: Hormone measurements in 63 unselected women with clinical and ultrasound diagnosis of PCOS attending gynaecological and general endocrine clinics in a District General Hospital were compared with those from a group of 20 normal ovulatory controls in the early follicular phase of their cycles. Measurements: Serum levels of luteinixing hormone (LH), follicle stimulating hormone (FSH), LWFSH ratio, total testosterone, derived free testosterone, sex hormone binding globulin androstenedione and dehydroepiandrosterone (DHEA) were, measured by radioimmunoassays. LH and FSH measured by two different assays. Results: The mean serum LH and LWFSH ratio were
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significantly (P c 0.01) higher in the women with PCOS compared with the normal group, but these two measurements were in the abnormal range for only 35% and 41-44%, respectively. Absolute gonadotrophin values were significantly different using the two assay methods, which employed the same reference preparation as standard. Mean serum total testosterone concentration was significantly higher in the PCOS group and was the most frequently (70%) abnormal biochemical marker for PCOS. Sex hormone binding globulin (SHBG) did not differ significantly between the two groups but showed a negative correlation with body mass’index in women with PCOS. The combination of SHBG and testosterone to derive a free testosterone value did not further aid the biochemical diagnosis of PCOS. Androstenedione was significantly higher in the PCOS group than in the control group, with a frequency of 53%. There was no significant difference in DHEA-S between the two groups. Conclusion: When typical ovarian ultrasound appearances plus the clinical features of oligomenorrhoea and/or hirsuitism were used to define PCOS total testosterone was the best single honnonal marker of the condition. Testosterone androstenedione or LH, either alone or in combination, were raised in 86% of women with PCOS and these should be. the definitive hormonal tests. Using LI-UFSH ratio as a biochemical criterion for diagnosis of PCOS should be abandoned because of its low sensitivity. To be of value the normal range for all hormones should be precisely defined in a group of regularly ovulating women in the early follicular phase of the cycle for the assay used in each laboratory. Lipid amI apolipoproteinabnmmaIitIea In birsnte women. I. The assedatIon &II IrmuIInreshance Wild R.A.; Alaupovic P.; Parker I.J. USA AM J OBSTET GYNECOL 1992 166/4 (1191-1197) Objective: We attempted to evaluate apolipoprotein lipid levels in hirsute hyperandrogenic women and to clarity the influence of insulin resistance versus endogenous sex steroids on these profiles. Study design: Forty-seven women seen for evaluation of hirsutism in the Reproductive Endocrine Clinic were contrasted with 15 normal women. Thirty-one patients were treated with the gonadotropin-releasing hormone analog, leuprolide acetate, 7.5 mg intramuscularly monthly for 3 months. Insulin resistance was assessed before and after GnRH analog administration. Mean concentrations were compared with the Student’s t-test and analysis of variance. Results: Hirsute women were heavier and had higher and diastolic blood pressures, more menstrual irregularities (all P < 0.01) and higher waistihip ratios (P < 0.05). They had higher triglyceride, very-low-density lipoprotein cholesterol and apolipoprotein C-III levels and lower highdensity lipoprotein cholesterol concentrations (all P c 0.01). They had lower, apolipoprotein A-I/A-II ratios (P < 0.01). After administration of GnRH analog androgen and estrogens were suppressed and insulin resistance was unaltered. Insulin correlated with apolipoprotein lipid abnormalities. Conclusion: Lipid and apolipoprotein abnormalities in these hirsute women seem to be associated more with insulin resistance than with endogenous androgens or estrogens. Int J Gynecol Obstet 39
GYNECOLOGICAL ENDOCRINOLOGY wtddl bormonc tsstsfor tbc dlagmis of golyeyatic ovary syn4rolne? Robinson !I.; Rodin D.A.; Deacon A.; Wheeler M.J.; Clayton R.N. GBR BR J OBSTET GYNAECOL 1992 9913 (232-238) Objective: To assess the frequency of abnormal values for hormone measurements commonly used in the biochemical diagnosis of polycystic ovary syndrome (PCOS). Design: Hormone measurements in 63 unselected women with clinical and ultrasound diagnosis of PCOS attending gynaecological and general endocrine clinics in a District General Hospital were compared with those from a group of 20 normal ovulatory controls in the early follicular phase of their cycles. Measurements: Serum levels of luteinixing hormone (LH), follicle stimulating hormone (FSH), LWFSH ratio, total testosterone, derived free testosterone, sex hormone binding globulin androstenedione and dehydroepiandrosterone (DHEA) were, measured by radioimmunoassays. LH and FSH measured by two different assays. Results: The mean serum LH and LWFSH ratio were
361
significantly (P c 0.01) higher in the women with PCOS compared with the normal group, but these two measurements were in the abnormal range for only 35% and 41-44%, respectively. Absolute gonadotrophin values were significantly different using the two assay methods, which employed the same reference preparation as standard. Mean serum total testosterone concentration was significantly higher in the PCOS group and was the most frequently (70%) abnormal biochemical marker for PCOS. Sex hormone binding globulin (SHBG) did not differ significantly between the two groups but showed a negative correlation with body mass’index in women with PCOS. The combination of SHBG and testosterone to derive a free testosterone value did not further aid the biochemical diagnosis of PCOS. Androstenedione was significantly higher in the PCOS group than in the control group, with a frequency of 53%. There was no significant difference in DHEA-S between the two groups. Conclusion: When typical ovarian ultrasound appearances plus the clinical features of oligomenorrhoea and/or hirsuitism were used to define PCOS total testosterone was the best single honnonal marker of the condition. Testosterone androstenedione or LH, either alone or in combination, were raised in 86% of women with PCOS and these should be. the definitive hormonal tests. Using LI-UFSH ratio as a biochemical criterion for diagnosis of PCOS should be abandoned because of its low sensitivity. To be of value the normal range for all hormones should be precisely defined in a group of regularly ovulating women in the early follicular phase of the cycle for the assay used in each laboratory. Lipid amI apolipoproteinabnmmaIitIea In birsnte women. I. The assedatIon &II IrmuIInreshance Wild R.A.; Alaupovic P.; Parker I.J. USA AM J OBSTET GYNECOL 1992 166/4 (1191-1197) Objective: We attempted to evaluate apolipoprotein lipid levels in hirsute hyperandrogenic women and to clarity the influence of insulin resistance versus endogenous sex steroids on these profiles. Study design: Forty-seven women seen for evaluation of hirsutism in the Reproductive Endocrine Clinic were contrasted with 15 normal women. Thirty-one patients were treated with the gonadotropin-releasing hormone analog, leuprolide acetate, 7.5 mg intramuscularly monthly for 3 months. Insulin resistance was assessed before and after GnRH analog administration. Mean concentrations were compared with the Student’s t-test and analysis of variance. Results: Hirsute women were heavier and had higher and diastolic blood pressures, more menstrual irregularities (all P < 0.01) and higher waistihip ratios (P < 0.05). They had higher triglyceride, very-low-density lipoprotein cholesterol and apolipoprotein C-III levels and lower highdensity lipoprotein cholesterol concentrations (all P c 0.01). They had lower, apolipoprotein A-I/A-II ratios (P < 0.01). After administration of GnRH analog androgen and estrogens were suppressed and insulin resistance was unaltered. Insulin correlated with apolipoprotein lipid abnormalities. Conclusion: Lipid and apolipoprotein abnormalities in these hirsute women seem to be associated more with insulin resistance than with endogenous androgens or estrogens. Int J Gynecol Obstet 39