Feasibility, pitfalls and results of a structured concept-development phase for a randomized controlled phase III trial on radiotherapy in primary prostate cancer patients.

OBJECTIVE: Failure rate in randomized controlled trials (RCTs) is > 50%, includes safety-problems, underpowered statistics, lack of efficacy, lack of funding or insufficient patient recruitment and is even more pronounced in oncology trials. We present results of a structured concept-development phase (CDP) for a phase III RCT on personalized radiotherapy (RT) in primary prostate cancer (PCa) patients implementing prostate specific membrane antigen targeting positron emission tomography (PSMA-PET). MATERIALS AND METHODS: The 1 yr process of the CDP contained five main working packages: (i) literature search and scoping review, (ii) involvement of individual patients, patients' representatives and patients' self-help groups addressing the patients' willingness to participate in the preparation process and the conduct of RCTs as well as the patient informed consent (PIC), (iii) involvement of national and international experts and expert panels (iv) a phase II pilot study investigating the safety of implementation of PSMA-PET for focal dose escalation RT and (v) in-silico RT planning studies assessing feasibility of envisaged dose regimens and effects of urethral sparing in focal dose escalation. RESULTS: (i) Systematic literature searches confirmed the high clinical relevance for more evidence on advanced RT approaches, in particular stereotactic body RT, in high-risk PCa patients. (ii) Involvement of patients, patient representatives and randomly selected males relevantly changed the PIC and initiated a patient empowerment project for training of bladder preparation. (iii) Discussion with national and international experts led to adaptions of inclusion and exclusion criteria. (iv) Fifty patients were treated in the pilot trial and in- and exclusion criteria as well as enrollment calculations were adapted accordingly. Parallel conduction of the pilot trial revealed pitfalls on practicability and broadened the horizon for translational projects. (v) In-silico planning studies confirmed feasibility of envisaged dose prescription. Despite large prostate- and boost-volumes of up to 66% of the prostate, adherence to stringent anorectal dose constraints was feasible. Urethral sparing increased the therapeutic ratio. CONCLUSION: The dynamic framework of interdisciplinary working programs in CDPs enhances robustness of RCT protocols and may be associated with decreased failure rates. Structured recommendations are warranted to further define the process of such CDPs in radiation oncology trials.

Improving interinstitutional and intertechnology consistency of pulmonary SBRT by dose prescription to the mean internal target volume dose.

PURPOSE: Dose, fractionation, normalization and the dose profile inside the target volume vary substantially in pulmonary stereotactic body radiotherapy (SBRT) between different institutions and SBRT technologies. Published planning studies have shown large variations of the mean dose in planning target volume (PTV) and gross tumor volume (GTV) or internal target volume (ITV) when dose prescription is performed to the PTV covering isodose. This planning study investigated whether dose prescription to the mean dose of the ITV improves consistency in pulmonary SBRT dose distributions. MATERIALS AND METHODS: This was a multi-institutional planning study by the German Society of Radiation Oncology (DEGRO) working group Radiosurgery and Stereotactic Radiotherapy. CT images and structures of ITV, PTV and all relevant organs at risk (OAR) for two patients with early stage non-small cell lung cancer (NSCLC) were distributed to all participating institutions. Each institute created a treatment plan with the technique commonly used in the institute for lung SBRT. The specified dose fractionation was 3â€¯× 21.5 Gy normalized to the mean ITV dose. Additional dose objectives for target volumes and OAR were provided. RESULTS: In all, 52 plans from 25 institutions were included in this analysis: 8 robotic radiosurgery (RRS), 34 intensity-modulated (MOD), and 10 3D-conformal (3D) radiation therapy plans. The distribution of the mean dose in the PTV did not differ significantly between the two patients (median 56.9 Gy vs 56.6 Gy). There was only a small difference between the techniques, with RRS having the lowest mean PTV dose with a median of 55.9 Gy followed by MOD plans with 56.7 Gy and 3D plans with 57.4 Gy having the highest. For the different organs at risk no significant difference between the techniques could be found. CONCLUSIONS: This planning study pointed out that multiparameter dose prescription including normalization on the mean ITV dose in combination with detailed objectives for the PTV and ITV achieve consistent dose distributions for peripheral lung tumors in combination with an ITV concept between different delivery techniques and across institutions.

Stereotactic body radiotherapy (SBRT) in recurrent or oligometastatic pancreatic cancer : A toxicity review of simultaneous integrated protection (SIP) versus conventional SBRT.

BACKGROUND: Stereotactic body radiotherapy (SBRT) in pancreatic cancer can be limited by its proximity to organs at risk (OAR). In this analysis, we evaluated the toxicity and efficacy of two different treatment approaches in patients with locally recurrent or oligometastatic pancreatic cancer. MATERIALS AND METHODS: According to the prescription method, patients were divided in two cohorts (C1 and C2). The planning target volume (PTV) was created through a 4 mm expansion of the internal target volume. In C2, a subvolume was additionally created, a simultaneous integrated protection (SIP), which is the overlap of the PTV with the planning risk volume of an OAR to which we prescribed a reduced dose. RESULTS: In all, 18 patients were treated (7 with local recurrences, 9 for oligometastases, 2 for both). Twelve of 23 lesions were treated without SIP (C1) and 11 with SIP (C2). The median follow-up was 12.8 months. Median overall survival (OS) was 13.2 (95% confidence interval [CI] 9.8-14.6) months. The OS rates at 6 and 12 months were 87 and 58%, respectively. Freedom from local progression for combined cohorts at 6 and 12 months was 93 and 67% (95% CI 15-36), respectively. Local control was not statistically different between the two groups. One patient in C2 experienced grade ≥3 acute toxicities and 1 patient in C1 experienced a grade ≥3 late toxicity. CONCLUSION: The SIP approach is a useful prescription method for abdominal SBRT with a favorable toxicity profile which does not compromise local control and overall survival despite dose sacrifices in small subvolumes.

Correlation between double and nonresonant single ionization.

We performed simultaneous measurements of electrons and ions produced in Xe photoionization driven by an 800-nm, 100-fs laser pulse. The obtained energy and angular resolved electron spectra allow the identification of the electronic states populated during the ionization. Xe2+ ions appear at the same laser intensity as electrons emerging from a nonresonant 9-photon ionization process of Xe. Similar to optical tunneling ionization, the nonresonant ionization delivers low-energy electrons needed for the formation of Xe2+ by a backscattering process.

Differential expression of uterine progesterone receptor forms A and B during the menstrual cycle.

Recent studies suggest that the progesterone receptor isoforms (PR-A and PR-B) activate genes differentially and that PR-A may act as a repressor of PR-B function. Hence, the absolute and relative expression of the two isoforms will determine the response to progesterone. We have measured their relative expression in the uterus of cycling women who underwent endometrial biopsy. PR isoforms were identified on blots of SDS-PAGE gels by reaction with the AB-52 antibody after immunoprecipitation from endometrial extract. Both isoforms were highest in the peri-ovulatory phase, but levels of PR-A were always higher than those of PR-B. The ratio of PR-A to PR-B changed during the menstrual cycle. Between days 2 and 8, PR-B is almost undetectable and the A:B ratio is >10:1. From days 9 to 13, the ratio is about 5:1, and it is about 2:1 between days 14 and 16. Thereafter, PR-B dwindles rapidly and is virtually undetectable at the end of the cycle. In various hypoestrogenic environments, PR-B expression was reduced. However, exogenous estrogens in the follicular phase in the form of oral contraceptives, enhanced PR-B expression. These data support the possibility that progesterone acts through cycle-specific PR isoforms.

Insulin-like growth factor binding proteins in peritoneal fluid of women with minimal and mild endometriosis.

This prospective cohort study was carried out in a university-based infertility clinic to determine the profile of insulin-like growth factor binding proteins (IGFBPs) in patients with mild endometriosis and no obvious mechanical factor contributing to infertility. A total of 26 patients with minimal and mild endometriosis and 10 controls contributed peritoneal fluid at surgery. The variety, expression and levels of IGFBPs were determined by radio-immunoassay and Western ligand blots (WLBs) with quantitation by laser densitometer. A 27 kDa species was significantly lower and 31 kDa species tended to be lower in patients with endometriosis as determined by quantitative laser densitometer. The levels of IGFBP-3 detected by radioimmunoassay and by WLB were correlated in the control group and in the patients with endometriosis in the follicular phase but not in patients with endometriosis in the luteal phase. The level of 27 kDa species seen on WLBs did not appear to correspond to IGFBP-1 determined by radioimmunoassay and IGFBP-3 levels in luteal phase endometriosis patients also departed from values determined by radioimmunoassay. These discrepancies suggest a complex system to control levels of IGF in the peritoneum involving multiple binding proteins and proteases. The IGFBPs of patients with endometriosis may contribute to reproductive dysfunction and be able to serve as markers.

Relation between cathepsin D expression and other prognostic factors in breast carcinomas.

We evaluated cathepsin D concentrations in 318 breast carcinoma specimens with a standardized IRMA and established distribution values of 5.9-217.8 nmol/g (median 51.8). Concentrations of cathepsin D did not correlate with age or with concentrations of HER-2/neu oncoprotein, estrogen receptor, or epidermal growth factor receptors. A significant correlation was observed between cathepsin D and progestin receptor (P = 0.009), but only in postmenopausal patients. In our role as a National Reference Laboratory for conducting interlaboratory comparisons of tumor markers, we evaluated cathepsin D assay proficiency by using control samples with intra- and interassay CVs of 2-8% and 10-13%, respectively. Human reference specimens containing known quantities of cathepsin D were developed to facilitate standardized testing. The IRMA procedure and the use of quality-assurance samples permits evaluation of the clinical significance of cathepsin D in human breast cancer trials.

Marker proteins in the particulate fraction of third-trimester amniotic fluid.

The present clinical evaluation of fetal lung maturity relies largely on the determination of the amniotic surfactant phospholipids phosphotidylglycerol, lecithin, and sphingomyelin, but there are many false negatives as well as false positives among diabetics. The use of other components of lung surfactant, namely, the hydrophobic surfactant proteins (SPs) has long been suggested as an alternative to the classical assay, but tests based on the detection of immunoreactive SP-A have not proved superior or supplanted phospholipid ratios as an index. This report investigates the proteins in a fraction of third-trimester human amniotic fluid (the particulate fraction) enriched in the SP complexes that form the surfactant monolayer. The proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining and immunoblotting. Eight proteins are of particular interest. Three novel proteins (termed AFPP-1, AFPP-4, and AFPP-8) and the alpha-fetoprotein/human serum albumin complex (AFPP-7) can be detected throughout the 28- to 38-week gestational window. The protein that is referred to as AFPP-2 could be identified as SP-A on the basis of immunologic cross-reactivity as well as size and charge characteristics. The time course of appearance of AFPP-2 was also followed in patients with Rh isoimmunization syndrome and was found to be the same as that seen for SP-A. The SP-A was detected as at least five major charged isoforms with multiple subisoforms of different molecular weight and can be distinguished from a related set of proteins (AFPP-5) that appear with a different time course but are possible precursors. Two other proteins (AFPP-3, AFPP-6), which are detectable inconsistently bear some similarity to others reported previously but not extensively characterized. These results define both constant and variable proteins of the particulate fraction of the amniotic fluid and indicate that certain protein isoforms are changing throughout the third trimester. These data enhance the possibility of the utilization of these proteins as markers of lung maturity in conditions such as maternal diabetes.

Emergency contraception alters progesterone-associated endometrial protein in serum and uterine luminal fluid.

OBJECTIVE: To evaluate the effect of high-dose oral contraceptives on serum and uterine luminal fluid progesterone-associated endometrial protein in the luteal phase. METHODS: Five ovulatory women participated in the study. In a control cycle, serum and uterine lavage samples were collected on luteal day 11. In the next cycle, on luteal day 9, the participants were given two 50-micrograms ethinyl estradiol-norgestrel tablets, repeated 12 hours later. Serum and uterine lavage samples were collected 48 hours (luteal day 11) after the last dose and analyzed by two-dimensional polyacrylamide gel electrophoresis and radioimmunoassays of the serum. RESULTS: Progesterone-associated endometrial protein levels were lower in sera from treated compared with control cycles. Analysis of serum levels of this protein by two-dimensional polyacrylamide gel electrophoresis did not reveal bands corresponding to the known size and charge characteristics (27 kd and pI of 4.9) in either control or treatment samples. On the other hand, in uterine lavage samples, a complete suppression of the 27-kd, pI-4.9 species was evident after treatment. CONCLUSION: High-dose ethinyl estradiol-norgestrel emergency contraception effectively suppresses progesterone-associated endometrial protein in the midluteal uterus, potentially altering the endometrial environment unfavorably and affecting the survival of the early embryo.

High doses of oral contraceptives do not alter endometrial alpha 1 and alpha v beta 3 integrins in the late implantation window.

OBJECTIVE: To assess the effects of an emergency contraceptive agent on the distribution of integrin heterodimers during that part of the implantation window. DESIGN: Prospective, case-controlled study in a university-based Population Program. In the first ovulatory control cycle after the detection of LH surge, patients had endometrial sampling 11 days after the surge. In the next cycle the procedure was repeated 2 days after the administration of a postcoital contraceptive agent on day 9 after LH surge (100 g ethinyl E2 and 2 mg norgestrel). MAIN OUTCOME MEASURES: The effects of postcoital contraceptives on the expression of integrin heterodimers (alpha 1 and alpha v beta 3 subunits) reported to be unique to secretory phase was determined. RESULTS: All six specimens were consistent histologically with days 24 and 25 of the menstrual cycle by light microscopy. Using immunohistochemistry, strong membrane staining of endometrial glandular cells and superficial epithelium for both alpha 1 subunit and vitronectin (alpha v beta 3) receptor was observed in treatment and controls. No diminution of intensity or distribution was observed relative to pretreatment controls. CONCLUSIONS: There is no apparent change in the level of these two integrins in the human endometrium when high-dose oral contraceptives are given in the later stages of the implantation window. This suggests that the high doses of steroids used in emergency contraceptives may exert their effect through more complex mechanisms than endometrial cell surface changes.

PIP: In the human female, high doses of estrogen have been used to prevent implantation of the fertilized egg. Integrins function as receptors and play an important role in blastocyst attachment to the uterine wall. This article reports the findings of a study which tested what effects an emergency contraceptive agent has on integrin spread during the effective implantation window. Three women with regular menstrual cycles volunteered for this study. Endometrial biopsies were performed before any hormonal treatment; these samples were considered controls. These same patients were then orally given 100 g ethinyl E2 and 2 mg norgestrel. 11 days later a second endometrial biopsy was taken. All samples were divided into two groups. One group was fixed in glutaraldehyde and prepared for light microscopy examination. The second group was frozen in liquid nitrogen and subsequently used for cryosection examination. Immunohistochemistry techniques were performed on the tissues. No morphological differences were observed between the two groups. Immunohistochemistry results were unremarkable. Expected luteal phase endometrial changes were not observed. These results show that there is no apparent change in the level of different integrins in the human endometrium when high doses of oral contraceptives are administered. This further suggests that high doses of steroids used for preventing blastocyst implantation may not work through changes in the endometrial cell surface receptivity as previously believed.

Establishment of a temperature-dependent cell line from rat endometrium by retroviral infection.

We have established a cell line, RENTts3.1, by infection of primary rat endometrial cells with a retrovirus carrying a temperature-sensitive mutant of SV40 large T-antigen. These cells show a temperature-dependent phenotype with respect to morphology, growth and expression of macrophage colony-stimulating factor 1 (CSF-1) mRNA. At the permissive temperature, the cells grow with a doubling time of 24 h and exhibit an elongated, fibroblast-like morphology. They express vimentin and type III collagen mRNA. At the non-permissive temperature, the cells stop growing and exhibit an epitheloid morphology with flat enlarged cytoplasm. At the higher temperature, the cells continue to express type III collagen, but also express CSF-1 mRNA, and the cellular content of this transcript is influenced by glucocorticoid treatment. No expression of the epithelial markers cytokeratin, uteroglobin, beta-endorphin or preproenkephalin was detected, suggesting a stromal origin of the cell line. Electron microscopic data of cells cultivated on different substrates also support this conclusion. This cell line may be useful for the study of the molecular processes involved in decidual transformation of the endometrial mucosa.

Expression of the uteroglobin promoter in epithelial cell lines from endometrium.

To understand the molecular mechanism of endometrial differentiation we have initiated an analysis of the uteroglobin promoter. Uteroglobin is normally expressed in endometrial tissues under the control of ovarian hormones. In gene transfer experiments with the Ishikawa cell line, derived from a human endometrial adenocarcinoma, we have identified several regions in the promoter of the uteroglobin gene that are responsible for its endometrium-specific expression. To evaluate the generality of these findings, we have begun cloning the promoter regions of potential endometrial markers, including the rat, mouse, and human uteroglobin gene. In the rat, expression of the uteroglobin-like gene, CC10, is dominant in the lung but is also observed in the endometrium of progesterone treated animals. A comparison of the 5'-flanking sequence of the rat and rabbit uteroglobin gene resulted in the detection of similarities and differences that could explain their differential expression in vivo. To substantiate these findings we have established several cell lines from rat endometrium using murine retroviral vectors containing a positive selection marker and various viral oncogenes, such as SV40 large T antigen, adenovirus E1A, and Ha-ras. Cell lines immortalized by SV40 T-antigen were subsequently transformed with the Ha-ras oncogene. Several cell lines exhibit properties of epithelial endometrial cells. Two cell lines generated with a temperature sensitive mutant of the SV40 large T-antigen grow as transformed cells at the permissive temperature, but differentiate upon shifting to the non-permissive temperature. These rat endometrial cell lines should be useful for the analysis of endometrium-specific gene expression and as model systems for endometrial carcinoma.

Establishment of rat endometrial cell lines by retroviral mediated transfer of immortalizing and transforming oncogenes.

To study hormone responsive genes in differentiated epithelial cells and as a model for endometrial carcinoma, lines were established from primary rat endometrial cells by infection with replication-defective retroviruses carrying oncogenes and the selectable gene neo. The initial step involved immortalization through the large T antigen of SV40 to generate a line we designate RENT4, or with the E1a gene of adenovirus to generate lines referred to as RENE1 and RENE2. Additionally, lines generated by large T antigen of SV40 were superinfected with a replication-defective retrovirus harboring the v-Ha-ras oncogene and selected by the ability to form colonies in soft agar. The latter cell lines appeared fully transformed and were designated RENTR01 and RENTR03. Five established lines were characterized for steroid hormone receptors, alkaline phosphatase activity and their complement of the intermediate filaments vimentin and cytokeratin. With the exception of the RENE1 cells all other lines have normal levels of glucocorticoid receptor, whereas only RENE1, RENE2 and RENT4 were positive for the progesterone receptor. RENTR01, RENTR03 and, to a lesser extent, RENE1 exhibited differential expression of cytokeratins dependent upon whether the cells were grown on a substrata of NIH3T3 cells. When grown on formalin-fixed NIH3T3 cells, RENTR01 and RENTR03 cells appeared to differentiate or rearrange themselves in culture. Individual islands of cells showed a heterogeneous pattern of intermediate filaments with vimentin-positive cells localized to the outer portion of the islands whereas cytokeratin-positive cells are seen on the insides of these structures.

Alterations in estrogen receptor isoforms in the mammary gland and uterus of the rat during differentiation.

1. Estrogen receptors in lactating mammary glands and uteri of rats which were 10 and 19 days postpartum exhibited molecular heterogeneity based on their surface charge properties. 2. The polymorphism of estrogen receptors detected by high-performance ion exchange chromatography may be monitored in-line with a radioisotope detector. 3. Estrogen receptors from the mammary gland and uterus of rats at 10 days of lactation exhibited primarily two receptor isoforms eluting at 200-250 mM and 250-300 mM phosphate, whereas three ionic isoforms (eluting at 50-150, 200-250 and 325-375 mM phosphate) were found in the mammary glands of rats at 19 days of lactation. Similar changes in the profiles of estrogen receptor isoforms were observed in uterine cytosol preparations at each stage of postpartum differentiation. 4. The elution pattern of receptor-associated radioactivity was not altered by the addition of diisopropylphosphate, a potent inhibitor of trypsin-like proteases, either before, during or immediately after homogenization. This indicates that the differences observed in the receptor elution profile of 10 and 19 day postpartum lactating mammary glands were not due to artifactual proteolysis. 5. In summary, our data indicate that the differentiation stage of lactating mammary glands may dictate the final profile of receptor isoforms detected.

HPLC analysis of estrogen receptor by a multidimensional approach.

Previously we demonstrated the polymorphism of estrogen receptors (ER) in cytosol of various tissues based upon properties of size, shape and surface charge. This study describes the application of a multidimensional approach utilizing HPLC for characterization of ER. Cytosols from human uterus and endometrial carcinomas were characterized sequentially by high performance size exclusion chromatography (HPSEC) on Spherogel TSK-3000 SW, and high performance ion-exchange chromatography (HPIEC) using SynChropak AX-1000 anion exchange columns. Using HPSEC, specific estrogen binding was exhibited by a 30 A isoform and by one appearing after the V0 (approximately 60 A) in human uterus. However, in endometrial carcinoma other smaller binding components with Stoke's radii of less than 20 A were observed also. In buffers containing 400 mM KCl, predominantly a 28-30 A species was observed by HPSEC. Further characterization of the 28-30 A isoform from low and high salt elution from HPSEC was accomplished with an AX-1000 column. With either condition, 2 forms were eluted on HPIEC, 1 in the column wash (retention time 8-9 min), and the other at 50-70 mM phosphate. The elution profile of the larger species (approximately 60 A by HPSEC) on the ion-exchange column was time dependent. Immediate analysis (within 15 min) showed a profile similar to that of the original cytosol which contained minor components eluting in wash buffer and at 50-70 mM phosphate and a major isoform at 180 mM phosphate. However delayed analysis (after 2 h) of the 60 A isoform showed a similar profile (components in buffer wash and at 50-70 mM phosphate) obtained with the 30 A species. This time dependent change was not observed for the 30 A species or for the original cytosol. Estrogen receptors in cytosol sedimented at 10S and 4S in low ionic strength gradients and at 4S in sucrose gradients containing 400 mM KCl. The 28-30 A and 60 A species recovered from HPSEC sedimented at 3.5S. This multidimensional approach indicates that native estrogen receptors dissociated into a number of smaller molecular isoforms, which were distinguishable by different surface charge properties.

High-performance hydrophobic-interaction chromatography of steroid hormone receptors.

The use of high-performance hydrophobic-interaction chromatography (HPHIC) on SynChropak 500 propyl columns has been evaluated for the first time in the analysis of estrogen receptors labeled with [125I]iodoestradiol-17 beta. These receptors were extracted from reproductive tissues with 500 mM phosphate buffer and applied to the stationary phase. Utilizing an inverse phosphate gradient (500 to 10 mM), elution resulted in rapidly excluded components in the void volume followed by a second radioactive peak at 400 mM phosphate. Both peaks appeared to contain specific estrogen-binding components in that steroid association was inhibited by diethylstilbestrol and free ligand was eluted with a different retention time. A great deal of [125I]iodoestradiol-17 beta was retained by the column. Inclusion of acetonitrile (20%) in the mobile phase resulted in the elution of [125I]iodoestradiol-receptor complexes at a different position from free ligand. Distribution of specific estrogen-binding components appeared to be tumor-dependent. These preliminary results indicate that HPHIC may be useful for isolating various isoforms of steroid hormone receptors so that detailed information regarding their intrinsic properties may be ascertained.

Rapid, high-resolution procedure for assessment of estrogen receptor heterogeneity in clinical samples.

Approximately two-third of the women with breast cancer may benefit from hormone therapies if the lesion contains estrogen receptor in reasonable levels (greater than 10 fmol/mg cytosol protein). Our lab and others have suggested that not only the concentration, but the various molecular form(s) of the receptor may be an important factor in predicting patient responsiveness. Until now, this heterogeneity has been determined by analysis on sucrose density gradients requiring 16 h of centrifugation. Other methodologies which can disclose the profile of receptor isoforms are compromised by irreproducibility, poor recovery, or the length of time required to perform the analysis. We believe that high-performance liquid chromatography (HPLC) in the anion-exchange and chromatofocusing modes may be able to supply more insight into receptor structure than currently available by any other method. Utilizing the high activity ligand [16 alpha-125I]iodoestradiol-17 beta (2200 Ci/mmol) and flow-through equipment to monitor conductivity, pH, and radioactivity, we are able to describe the distribution of ionic isoforms in crude cytosolic preparations of breast cancer and uterus. This "on-line" technology is rapid (chromatogram is complete within 120 min), sensitive (receptor isoforms equivalent to 1 fmol are apparent), efficient (columns typically return greater than 90% of applied radio-labeled receptor) and reproducible (due to the sophistication of modern HPLC equipment). It is clear these techniques may be used in the clinical setting to better describe the profile of estrogen receptor isoforms and should be explored as a method of correlating receptor structure with hormone function in responsive tissues.

Isoforms of estrogen receptors by high-performance ion-exchange chromatography.

SynChropak AX-300, AX-500 and AX-1000 columns were used to separate ionic forms of estrogen receptors by high-performance ion-exchange chromatography. Cytosols from hormone-responsive tissues were incubated for 4-10 h with 3-4 nM [16 alpha-125I]iodoestradiol-17 beta, cleared of unbound ligand and applied to an anion-exchange column. Components were eluted at pH 7.4 using a gradient of phosphate buffer at 4-6 degrees C. Non-specific binding components were identified by chromatographing, the identical cytosol, incubated with [125I]iodoestradiol and a 500-fold excess of diethylstilbestrol, which blocks specific binding sites. [125I]Iodoestradiol was applied to the column in the absence of cytosol and eluted normally to determine the behavior of free ligand. Each column exhibited a different elution pattern for the estrogen receptor. The various isoforms of estrogen receptor were eluted differently from each column usually in the 15-120 mM and 180-250 mM region of the gradient. Often one non-specific binding component was not retained whereas other non-specific species were retained and eluted from the column in a salt-dependent manner; their position in the gradient varied from column to column. Similarly, free [125I]iodoestradiol was eluted at different positions in the gradients, dependent upon which column was employed. In general, the high flow-rates, reproducibility, good recovery and the apparent differential selectivity of each of the columns appear valuable in the investigation of the nature and subunit composition of the estrogen receptor molecule.

High-performance size exclusion chromatography as a rapid method for the separation of steroid hormone receptors.

High-performance size exclusion chromatography ( HPSEC ) on TSK 3000SW molecular sieve columns was used to separate estrogen, progestin, and androgen receptors from several target tissues within 50 min on the basis of size and shape (Stokes radius). Moreover, this system provided for the detection of heterogeneity of receptor species (isoforms) in a manner superior to that observed with sucrose density gradient centrifugation. HPSEC separated various estrogen receptor isoforms having Stokes radii of greater than 61 A, approximately 48 A and 29-32 A. Agents such as potassium chloride and sodium molybdate which alter the distribution of estrogen receptor species on sucrose density gradient centrifugation, promote similar alterations in receptor profiles when HPSEC was employed. Our investigations suggest the use of [125I]iodoestradiol-17 beta and HPSEC allows the sequential analysis of estrogen receptor species providing new insights into receptor composition and structure. It is concluded that HPSEC has a broad application in the field of steroid hormone receptors. This method should be useful in studies ranging from measurements of molecular and kinetic properties to their mode of cellular interaction and regulation.

Rapid analysis of estrogen receptor heterogeneity by chromatofocusing with high-performance liquid chromatography.

Chromatofocusing principles have been utilized to develop a high-performance liquid chromatographic technique for the rapid and routine analysis of steroid receptor heterogeneity. Two anion-exchange columns (SynChropak AX-300 and AX-500) were compared for analytical and preparative chromatofocusing of estrogen receptor components. As many as ten different [125I]iodoestradiol-labeled binding proteins were identified in cytosols prepared from mammary gland and uterus. Estrogen receptors were well separated from other cytosolic proteins and recovery of activity routinely exceeded 90%. Parallel analyses of these cytosols to determine receptor size and shape indicated that HPLC chromatofocusing can be used effectively to study receptor isoforms with Stokes radii ranging from 30 A to greater than 70 A. In contrast to isoelectric focusing, this technique is compatible with the inclusion of a commonly used receptor-stabilizing agent, sodium molybdate. Inclusion of molybdate during chromatofocusing of molybdate-stabilized receptor allowed the identification of two acidic receptor species not previously reported.