Benjamin S. Leung

Estrogen receptor α and β expression in the vaginal walls and uterosacral ligaments of premenopausal and postmenopausal women

Abstract Objective: To assess the expression of messenger RNA (mRNA) for estrogen receptors α and β in the vaginal walls and uterosacral ligaments of premenopausal and postmenopausal women. Design: In vitro experiment. Setting: Academic research environment. Patient(s): Sixteen consecutively seen women who underwent hysterectomy. Intervention(s): Samples of anterior vaginal walls and uterosacral ligaments were obtained during hysterectomy. Main Outcome Measure(s): Reverse transcriptase-polymerase chain reaction analysis for estrogen receptor mRNA. Result(s): Messenger RNA transcripts for estrogen receptor α were present in all samples of vaginal walls (16/16) and uterosacral ligaments (16/16). Estrogen receptor β mRNA was detected in all samples of vaginal walls from premenopausal women (12/12) but in none of those from postmenopausal women (0/4). Estrogen receptor β mRNA was found in most samples of uterosacral ligaments from premenopausal women (10/12) and in some of those from postmenopausal women (2/4). Conclusion(s): Estrogen receptors α and β were expressed in the vaginal walls and uterosacral ligaments of premenopausal and postmenopausal women. Estrogen receptor β was absent from the vaginal walls of postmenopausal women.

Survey of oncogene and growth factor/receptor gene expression in cancer cells by intron-differential RNAPCR

To elucidate the possible roles of proto-oncogenes and growth factors in estrogen-regulated cell proliferation of human breast and gynecologic cancers, we have determined the gene expressions of c-myc, transforming growth factor-alpha and beta 1 (TGF-alpha, beta 1) and epidermal growth factor receptor (EGFR) in a number of these cancer cell lines by using an intron-Differential (ID) RNA/PCR method, which differentially identifies the amplified cDNA from PCR products of genomic DNA contaminants. With this method, we demonstrated the expression of these genes, except EGFR, in an estrogen-dependent breast cancer cell line (CAMA-1). Our results show that TGF-alpha/EGF does not function as an autocrine factor in this cell line. Accordingly, it is unlikely that the TGF-alpha/EGFR system participates as a mediator in the estrogen-induced cell proliferation of CAMA-1 cells. The ID RNA/PCR method is a rapid, sensitive and specific technique for mRNA phenotyping and will have great clinical utility.

Characterization of uterine estrogen receptors by size-exclusion and ion-exchange high-performance liquid chromatography.

This report presents the first application of ion-exchange high-performance liquid chromatography in the study of ER from the rabbit uterus. In the presence of sodium molybdate (20 mM), native ER was eluted as a sharp peak at 0.29 M NaCl by a linear salt gradient, but without molybdate, it resolved into 4 major peaks. Molybdate-stabilized ER from the DEAE column, similar to ER from crude cytosol, sedimented at the 6-8S region in low salt and 4S region in high salt linear sucrose gradients, and was excluded from size-exclusion HPLC. In contrast, dissociated ER subunits from DEAE eluates ranged from 3.5 to 4.5S, and showed differences in molecular weights in a size-exclusion column. These results show that the native ER is a large molecule which dissociates into smaller subunits in the absence of molybdate; each of the steroid-bound moieties differs in molecular weight and surface charge from the native molecule.

Characterization of nuclear estradiol receptors released by micrococcal nuclease and deoxyribonuclease I

Interaction of estradiol receptor with chromatin was probed by nuclease digestion of receptor-chromatin complex. The complex was formed by incubating partially purified receptor with chromatin. Micrococcal nuclease digestion of the complex released a 7S form of receptor which could be converted to 2.8S form by DNAase I. Digestion of the complex with DNAase I yielded different forms of receptors ranging from 7S to 2.8S depending on the digestion time. Receptor distribution was also examined by isolating nuclei form tissue pre-incubated with radioactive estradiol. Micrococcal nuclease digestion of receptor-filled nuclei released 7S, 5.5S and 3.5S forms of receptors. Collectively, these results indicate that the 7S nuclear receptor may have an associated chromatin fragment which is sensitive to DNAase I activity. The desirable features of receptor-chromatin method over conventional methods for studies relating to receptor interaction at the gene site are discussed.

Characterization of Molybdate-Stabilized Estrogen Receptors by Hydrophobic Interaction HPLC: Resolution of Two 8S Subunits

This report describes the separation and characterization of estrogen receptors (ER) according to their degree of hydrophobicity and surface charges. Molybdate-stabilized [3H]ER from rabbit uterine cytosol was sequentially purified by passage through a size-exclusion pre-column, an anion-exchange column, and a hydrophobic interaction column. With fresh cytosol, a major radioactive peak was eluted from the DEAE columns; a major peak and a minor, less hydrophobic, peak were eluted from the hydrophobic column. In contrast, ER from frozen cytosol showed one peak in the DEAE-column and exhibited four radioactive peaks in the hydrophobic column. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, [3H] tamoxifen aziridine(TA)-labelled ER showed radioactive bands at 62 and 48 kd. The subunits which were characterized by these radioactive bands were successfully separated by the hydrophobic column; the more hydrophobic subunit corresponded to the 62 kd band. The HPLC-purified [3H]TA-labelled ER subunits sediment at a 7.4-8.5S region in a low-salt sucrose gradient. These results show that differential negative surface charge and hydrophobic areas exist in the holo-receptor and its subunits, and the hydrophobic interaction HPLC column separates the two major 8S steroid binding subunits of ER.

Purification of rabbit uterine cytosolic estrogen receptors by affinity chromatography

Abstract Progress to elucidate the mechanism of estrogen action in uterine tissues has been hampered because of the formidable task of receptor purification. This report describes our recent success in the purification of ERc from estrogen-primed rabbit uterus. Crude cytosolic ERc are purified sequentially by affinity chromatography and gel filtration, using heparin-Sepharose 4B, 17β-estradiol-17-hemisuccinyl-oval-bumin-Sepharose 4B, and Sephadex G-50. This purified ERc sediments as a 4.5 S molecule on sucrose gradients in low-salt buffer, and exhibits a major radioactive peak in both polyacrylamide gel electrophoresis under nondenaturing conditions and in HPLC eluates. The radioactivity pattern in HPLC coincides with that of the u.v. scan, showing the elimination of other u.v. absorbing materials of the crude cytosol. These results show that the bulk of cytosolic proteins have been removed by this procedure and indicate the feasibility of using HPLC for the large scale purification and analysis of ERc. Attempts are initiated in this laboratory to produce antibodies against this purified receptor by hybridoma technique, and to further analyze the biochemical nature of the purified receptor.

Characterization of Progesterone Receptor Subunits in Breast Cancer Cell Line, CAMA-1N

In order to study whether cytosolic progesterone receptor (PRc) is involved in progesterone-induced proliferative arrest of breast cancer cells (CAMA-1N), subunit composition of PRc in these cells was characterized. In the TSK G3000 high-performance liquid chromatography (HPLC) column, molybdate-stabilized PRc eluted as a single sharp peak immediately following the void volume. PRc from this peak had a sedimentation coefficient of 7.6 S and was eluted at 0.22 M salt from a linear gradient (0.1 to 0.4 M NaCl) in a DEAE column. When covalently labeled with [3H]R5020, analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by fluorography, PRc from crude cytosol exhibited two major protein bands with molecular weights of 105 +/- 2.1 and 83 +/- 3.2 kD, respectively. However, HPLC-purified PRc showed only one major band of 105 kD. These results show that PRc in CAMA-1N cells has steroid-binding components similar to other target tissues and provides a basis for further quantitative and qualitative analyses of PRc during cell proliferation and progesterone-induced growth arrest of CAMA cells.

Direct positive effect of epidermal growth factor on the cytoplasmic maturation of mouse and human oocytes**Supported in part by the research grant RO1 AA07733 (awarded to B.S.L.) from the National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland.

OBJECTIVE Immature mammalian oocytes cultured in vitro undergo inadequate cytoplasmic maturation and hence have a limited potential for fertilization. Our primary objective was to determine if the addition of epidermal growth factor (EGF) to the in vitro culture system would have a positive effect on oocyte cytoplasmic maturation. DESIGN We studied the effect of different EGF concentrations on both denuded and cumulus-enclosed mouse oocytes cultured in vitro. MAIN OUTCOME MEASURES The percentage of oocytes undergoing germinal vesicle breakdown (GVBD) and polar body one formation over time as a function of EGF concentration was determined. RESULTS A dose-related positive effect of EGF on both GVBD and polar body one formation over time was observed for mouse oocytes. As well, a similar effect of EGF was seen on immature human oocytes that had not been stimulated with exogenous gonadotropins. CONCLUSIONS The use of EGF may allow for the performance of successful in vitro fertilization procedures using immature human oocytes retrieved during unstimulated cycles.

Association of Monoclonal Antibody-Recognized Antigen with Steroid Receptors in Gynecologic Tumors

In an attempt to differentiate between the biochemical characteristics of hormonal-dependent and independent gynecologic tumors, monoclonal antibodies (mABs) MBr1 and MBr3 were used to detect tumor antigens. Steroid receptors and MBr1 and MBr3 determinants were assayed independently in a double-blinded manner. Of the 45 gynecologic tumors analyzed, 16 of 20 (80%) with both progesterone receptors (PR) (greater than 10 fm/mg protein) and estrogen receptors (ER) (greater than 5 fm/mg protein) also contained high levels of MBr1 determinant. However, these receptors were not quantitatively related to MBr1 activity. The 15 of 17 (88%) tumors that contained receptors below the discriminant levels also had low or undetectable levels of MBr1 activity. In tumors where only either PR or ER was present, 2 of 2 PR+/ER- tumors and 2 of 6 PR-/ER+ tumors expressed MBr1 antigen. No relationship of MBr3 antigenic activity to the receptors was established. These results show that MBr1-recognized antigen is expressed largely in steroid receptor-positive tumors and these data suggest that either the antigen itself or its carrier protein may be regulated by estrogen. Like steroid receptors, MBr1 antigens may have important prognostic value in gynecologic tumors.

Analysis of Rat Uterine Estrogen Receptors by High-Pressure Liquid Chromatography Methods

Cytosolic (ERc) and nuclear (ERn) estrogen receptors prepared from rat uteri were characterized by size-exclusion and ion-exchange HPLC. The oligomeric ERc eluted as a single, sharp peak near the exclusion volume of the gel column; ERn eluted as a broad peak. When salt-extracted ERn was partially purified sequentially by Sephadex G-200, DEAE-cellulose chromatography and polyacrylamide gel electrophoresis, the partially purified receptor moieties were not distinguishable by the sucrose gradient method, but showed characteristic retention times in the size-exclusion HPLC column. Further distinction in net surface charges was observed between ERc and ERn moieties by ion-exchange high-pressure liquid chromatography (HPLC). Molybdate-stabilized ERc was eluted as sharp peak at 0.27 M salt gradient. In contrast, fresh extracts of ERn emerged as a broad peak in the region of 0.1-0.2 M salt gradient. In the absence of molybdate, ERc dissociated into several 4-5 S molecules, which were well resolved in the DEAE column. This report, therefore, demonstrates the usefulness of size-exclusion and ion-exchange HPLC for steroid receptor analysis.