NH2-terminally truncated HER-2/neu protein: relationship with shedding of the extracellular domain and with prognostic factors in breast cancer.

We identified an NH2-terminally truncated HER-2/neu product of M(r) 95,000 with in vitro kinase activity by Western blotting and immunoprecipitations using domain-specific antibodies. p95 levels correlated with the extracellular domain (ECD) shed from different cells under varied conditions. Both ECD and p95 were at approximately 20-fold lower levels in SKOV3 ovarian carcinoma cells, as compared to BT474 breast carcinoma cells. Both were stimulated by treatment of cells with the phorbol ester tumor promoter phorbol 12-myristate 13-acetate and the lysosomotrophic agent chloroquine. The hydroxamate inhibitor of metalloproteases, TAPI, suppressed both p95 and ECD in a dose-dependent fashion, with maximal inhibition at < or = 10 microM in BT474 cells. Cancer tissues were analyzed by Western blotting and scored for p95HER-2/neu and for p185HER-2/neu expression. Breast and ovarian cancer tissues were both found to express p95HER-2/neu in addition to p185HER-2/neu. Of 161 breast cancer tissues, 22.4% expressed p95, 21.7% overexpressed p185, and 14.3% were p95 positive and overexpressed p185. A higher proportion of node-positive patients (23 of 78) than node-negative patients (9 of 63) expressed p95 in all tumors combined (P = 0.032). In the group that overexpressed p185, those that contained p95 were associated with node-positive patients (15 of 21), whereas those that were p95 negative were associated with node-negative patients (8 of 11; P = 0.017). Neither p95- nor p185-rich patients significantly correlated with tumor size or with hormone receptor status in this study. Our findings show that breast cancers, which express the HER-2/neu oncogene, are heterogeneous with respect to HER-2/neu protein products. p95HER-2/neu appears to distinguish tumors that have metastasized to the lymph nodes from those in node-negative patients.

Estrogen receptor gene expression in human uterine leiomyomata.

Estrogen and progestin are believed to be important physiological regulators of uterine leiomyoma growth. We recently showed that progesterone receptor messenger ribonucleic acid (mRNA) and protein levels are increased in human uterine leiomyomas compared with those in myometrial biopsy tissue obtained from the same patient. To further characterize the molecular mechanisms underlying abnormal growth of uterine leiomyomas, we analyzed biopsy samples of tumor and adjacent normal myometrium for estrogen receptor (ER) gene expression. Northern analysis indicated that ER mRNA levels were increased 1.4-to 12.6-fold in leiomyoma compared with myometrium in all patients examined (n = 11), whereas beta-actin mRNA was not different between the two groups. The size of the primary ER mRNA transcript was 6.2 kilobases in both leiomyoma and myometrium, indicating no gross mutation of the ER gene. An ER protein of 66 kilodaltons was detected by Western blot analysis, and quantitative immunoassay of ER revealed 9448 +/- 1955 fmol/mg DNA in leiomyoma compared to 2827 +/- 979 fmol/mg DNA in myometrial tissue. Scatchard analysis of 17 beta-estradiol binding to cell-free extracts revealed enhanced binding capacity (per mg DNA) in leiomyoma tissue (n = 6) of about 6-fold, whereas ER binding affinity was not substantially different between the leiomyoma and adjacent myometrial tissues. We propose that increased expression of progesterone receptor in leiomyoma is most likely a consequence of overexpression of functional ER that results in increased end-organ sensitivity to estradiol.

Progesterone receptor messenger ribonucleic acid and protein are overexpressed in human uterine leiomyomas.

OBJECTIVE: Our purpose was to identify molecular mechanisms underlying abnormal growth of uterine leiomyomas. STUDY DESIGN: Biopsy samples of tumor and adjacent "normal" myometrium from nine patients were analyzed for progesterone receptor gene expression and for proliferation-associated antigen Ki-67. RESULTS: Northern analysis indicated that progesterone receptor messenger ribonucleic acid levels were increased twofold to 15-fold in leiomyoma compared with adjacent myometrial biopsy tissue from all patients (n = 9), whereas beta-actin messenger ribonucleic acid was at similar levels in these samples. Quantitative immunoassay, immunohistochemistry studies, and Western blot analyses revealed increased amounts of progesterone receptor protein in the tumor tissue. Both the progesterone receptor A and B forms were expressed in the leiomyoma and adjacent myometrium. Corresponding to increased progesterone receptor gene expression, the proliferation-associated antigen Ki-67 was also significantly elevated in the leiomyoma tissue. CONCLUSION: These data provide the first evidence that progesterone receptor messenger ribonucleic acid is overexpressed in uterine leiomyomas, suggesting that amplified progesterone-mediated signaling is instrumental in the abnormal growth of these tumors.

Androgen receptors in osteoblast-like cell lines.

Although androgens exert major effects on bone remodeling, the mechanisms by which they exert their effects remain unclear. Recently, it has become apparent that receptors for sex steroids may be present in osteoblastic cells. We have examined several cell lines with osteoblastic phenotypes to determine if specific, high affinity androgen receptors are present. Two cell lines of human origin (Saos-2 and U2-OS) and one of rat origin (UMR-106.01) were studied. Androgen binding sites were present in all cell lines. Binding affinities were high (KD = 1.6 - 2.5 x 10(-10) M), and similar to those in classical androgen target tissues (prostate, kidney). Concentrations were greater in the human cell lines (1277 and 1605 sites/cell) than in the rodent line (74 sites/cell). In the human cell lines androgen binding was also specific and typical of androgen receptors in other tissues. Specific estrogen binding was not present in the UMR-106.01 cells, and no estrogen receptors were detectable in the human cell lines using an enzyme-linked receptor immunoassay. Specific binding for progesterone was also absent in the UMR-106.01 cells, but progesterone receptors were detected immunologically in the Saos-2 (119 sites/cell) and U2-OS (118 sites/cell) lines. These findings indicate the presence of androgen receptors that are of similar character to those in classical androgen target tissues, and suggest that the study of these cell lines may be useful in the study of the regulation of androgen effects in osteoblasts.

Effect of clomiphene citrate treatment on endometrial estrogen and progesterone receptor induction in women.

A direct adverse effect of clomiphene citrate on the endometrium has been presumed, and interference with estrogen receptor-mediated endometrial estrogen receptor and progesterone receptor induction has been implicated as the mechanism responsible for an increased incidence of luteal phase deficiency in association with clomiphene citrate treatment. To clarify the net influence of clomiphene administration on endometrial steroid receptor induction, we studied five normal ovulatory women, in both a spontaneous and clomiphene-induced (150 mg/day, cycle days 5 to 9) ovulatory cycle. From cycle day 11 blood samples were obtained daily and urinary luteinizing hormone determinations were performed twice daily. Endometrial biopsy was performed on the day of the urinary luteinizing hormone surge and again 13 days after the surge. Serum levels of follicle-stimulating hormone and luteinizing hormone were determined by immunoradiometric assay, estradiol and progesterone by radioimmunoassay, and clomiphene citrate isomer concentrations in treatment cycles by reversed-phase high-performance liquid chromatography and fluorescence detection. Total, cytosolic, and salt-extracted nuclear endometrial estrogen receptor and progesterone receptor concentrations were determined by enzyme-linked immunoassay. Serum estradiol was threefold to fivefold higher (p less than 0.05) in clomiphene-induced than in spontaneous cycles 8 and 10 days before the luteinizing hormone surge, and progesterone was increased (p less than 0.05) from the day of the surge to end of the cycle. Serum enclomiphene rose to plateau between 12 and 6 days before the luteinizing hormone surge (4.1 +/- 0.8 ng/ml, mean +/- SE, n = 19) and fell thereafter to less than 1.0 ng/ml. Zuclomiphene levels increased rapidly between 14 and 8 days before the surge (53.9 +/- 2.8 ng/ml, mean +/- SE, n = 5) and then decreased gradually but remained elevated throughout the luteal phase (29.0 +/- 1.2 ng/ml, mean +/- SE, n = 33). Late luteal endometrial histology was abnormal in one of four available treatment cycle specimens, but the endocrine characteristics and number and subcellular distribution of estrogen receptor and progesterone receptor in the abnormal cycle were not different from those of normal, in-phase cycles.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione transferase activity and isoenzyme composition in primary human breast cancers.

The human glutathione transferases (GSTs) are a multigene family of detoxication enzymes with patterns of expression that are both tissue specific and genetically determined. Changes in the levels of one or more GST isoenzymes have been associated with the development of anticancer drug resistance in cultured cell lines. In this study, total GST activity and GST isoenzyme composition have been determined for 45 primary human breast carcinomas using a 1-chloro-2,4-dinitrobenzene substrate assay and Western blotting, respectively. The GST activity ranged from 5-208 mU/mg protein with a mean of 67 mU/mg protein (+/- 44 SD). GST-pi) isoenzyme protein was detectable on Western blots in 44 of 45 samples. Mu Class GST protein was detected in 18 of 38 samples and undetectable in 20 of the 38 samples tested. By polymerase chain reaction analysis of genomic DNA, the absence of mu class GST in breast tumors was determined to be due to the deletion of the gene for GST-mu in the DNA of those tumors. None of the 43 primary human breast cancer samples tested contained detectable alpha class GST protein. Neither the total GST activity of tumor samples, the quantity of GST-pi protein, nor the presence or absence of mu class GST correlated with other factors known to be of prognostic significance including tumor size, nodal status, estrogen receptor protein positivity, or progesterone receptor protein positivity. Substantial differences exist among primary breast carcinomas in both the amount of GST activity and GST isoenzyme composition. However, these are not tightly linked either to tumor stage or to hormone receptor status. Whether the levels of these enzymes are independent predictors of either risk of recurrence or response to anticancer therapy has yet to be tested directly.

The mechanism of hormone-sensitive breast cancer progression on antiestrogen therapy. Implications for treatment and protocol planning.

Fifteen patients whose tumors progressed while they received tamoxifen citrate therapy were studied by serial determinations of serum levels of estrone (E1), estradiol (E2), and dehydroepiandrosterone (DHEA) obtained during progression after withdrawal from tamoxifen therapy and total endocrine ablation or suppression. Discontinuation of tamoxifen therapy resulted in reductions of DHEA, E1, and E2 levels by 44%, 49%, and 42%, respectively. Ablation or suppression reduced sex steroids to minimal levels and produced responses in all patients. Elevations of DHEA, E1, and E2 could be provoked by readministering tamoxifen to hypophysectomized and oophorectomized, but not adrenalectomized, patients, indicating that the adrenal gland is the source of these sex steroids. We conclude that tamoxifen stimulates adrenal production of DHEA, which is aromatized to E1 and E2. Buildup of E1 and E2 overwhelms the competitive binding of tamoxifen to the estrogen receptor, resulting in tumor progression.

Marked decline in trabecular bone mineral content in healthy men with age: lack of association with sex steroid levels.

To define the association of age-related changes in bone mineral content to gonadal function in normal men, we measured radial (largely cortical) and vertebral (largely trabecular) bone mineral content (BMC), testosterone (total and free), estrone and estradiol-17B levels in 62 healthy subjects, ages 30 to 92. Radial BMC fell 2 to 3.4% per decade but vertebral trabecular BMC declined more rapidly at 12% per decade. Of the sex steroids measured the only statistically significant change occurred in free testosterone levels which decreased with age (r = -.57, P less than .0001). Free testosterone levels correlated significantly with trabecular vertebral BMC (r = .458, P less than .0002) but not with bone mineral measures at the predominantly cortical radial sites. However, by multiple regression analysis free testosterone did not add to the effect of age on vertebral BMC. There were no associations of total testosterone, estrone, or estradiol levels to bone mineral content at any of the three sites measured in these healthy men. Age-related declines in male gonadal function do not appear to be of primary importance in male age-related bone loss.

Alpha 2-adrenergic receptor levels in obstructive and spastic Raynaud's syndrome.

The present study examines the hypothesis that alterations in the activity of alpha 2-adrenergic receptors (A2R) may underlie the clinical vasospasm seen in patients with Raynaud's syndrome. Platelets were isolated from 13 normal subjects, from 50 patients with vasospastic Raynaud's syndrome, and from 20 patients with obstructive Raynaud's syndrome and A2R levels measured. Binding capacity as determined in femtomoles per milligram of protein (fmol/mg of protein) and affinity were measured by Scatchard plot analysis. In a separate experiment normal human platelets were incubated with either buffer, normal serum, or serum from patients with spastic Raynaud's syndrome and A2R levels were measured. A2R levels in normal subjects averaged 112 +/- 18 fmol/mg; in the patients with spastic Raynaud's syndrome, 191 +/- 14 fmol/mg, p less than 0.01; and in the patients with obstructive Raynaud's syndrome, 164 +/- 31 fmol/mg, p greater than 0.05 (ns). Of the patients with spastic Raynaud's syndrome, 26% had values that were less than the mean value of the normal subjects (69 +/- 7 fmol/mg, p less than 0.05). The A2R levels decreased after incubation with serum from patients who had spastic Raynaud's syndrome by 17.4 +/- 3.1 fmol/mg (p less than 0.05). These results indicate that most patients with vasospastic Raynaud's syndrome have increased platelet A2R levels, which may constitute a primary pathophysiologic abnormality underlying this condition. The presence of subnormal A2R levels in a portion of the patients and the finding of a decrease in measurable A2R levels after incubation in serum from patients with spastic Raynaud's syndrome suggests the possibility of receptor modulation as a mechanism for increased cellular receptor synthesis.

Determination of estrogen receptor in breast cancer using monoclonal antibody technology: results of a multicenter study in the United States.

Three laboratories compared their routine steroid binding assays with the Abbott estrogen receptor-enzyme immunoassay (ER-EIA) to determine ER in breast cancer cytosols. Each laboratory was first trained to use the ER-EIA kit and then performed routine proficiency panels to determine assay reproducibility. The frozen panels prepared from MCF-7 cytosol produced good intraassay results but the between assay coefficients of variation were frequently above 10%. Lyophilized MCF-7 cytosols produced better reproducibility upon repeated assay. One laboratory demonstrated that New England Nuclear steroid binding kits and the ER-EIA produced comparable results when MCF-7 lyophilized cytosols were used. The analysis of breast tumor samples demonstrated excellent linear correlation coefficients for each laboratory (greater than 0.92 N approx. 60 samples each) but different slopes. The comparison of the ER-EIA with the New England Nuclear steroid binding assay produced a slope of 1.13. The ER-EIA appears to produce comparable results to the conventional steroid binding assays for the determination of ER in breast tumor cytosols.

Alpha-adrenergic receptors in platelets from patients with Raynaud's syndrome.

The pathophysiologic basis for spastic Raynaud's syndrome remains undefined but may be related to altered adrenergic activity. The relationship between Raynaud's syndrome and alterations in adrenergic receptor populations was examined in the present study by evaluation of alpha 2-adrenergic receptors in platelets. Direct binding assays revealed 78 +/- 8 fm/mg protein of alpha 2-adrenergic receptors in the platelets from control subjects. Similar levels of alpha 2-adrenergic receptors were observed in patients with obstructive Raynaud's syndrome. In contrast, platelet alpha 2-adrenergic receptor levels were significantly elevated (182 +/- 15 fm/mg protein) in the platelets from patients with spastic Raynaud's syndrome. These results indicate the presence of an altered alpha 2-adrenergic receptor population in the platelets from patients with spastic Raynaud's syndrome, which may be related to the vasospasm experienced by these patients.

Hormone changes during the menstrual cycle in abetalipoproteinemia: reduced luteal phase progesterone in a patient with homozygous hypobetalipoproteinemia.

Progesterone synthesis by the human corpus luteum requires a source of cholesterol, which can be derived from both local synthesis and uptake of low density lipoproteins (LDL). When the corpus luteum is maintained in organ culture, progesterone synthesis is primarily dependent on LDL and the rate of progesterone production during growth in a LDL-free media is suboptimal. An in vivo situation analogous to that of corpus luteum grown in LDL-depleted media exists naturally in patients with abetalipoproteinemia. To determine whether a complete deficiency of plasma LDL affects serum concentrations of progesterone (particularly during the luteal phase) or those of other hormones, we have measured the serum concentrations of luteinizing hormone, follicle-stimulating hormone, prolactin, estradiol, estrone, and progesterone during the menstrual cycle in a patient with phenotypic abetalipoproteinemia (on the basis of homozygous hypobetalipoproteinemia). Our results show a normal cyclical pattern with midcycle increases in the concentrations of luteinizing and follicle-stimulating hormones, prolactin, and estrogens but a distinctly subnormal increase in the luteal phase concentrations of progesterone. These results suggest that, in patients with phenotypic abetalipoproteinemia, the absence of LDL leads to an impairment in the maximal rates of production of progesterone by the corpus luteum.

Multiple simultaneous and sequential estrogen receptor values in patients with breast cancer.

Fifty-nine women had multiple estrogen receptor assays done, either simultaneously or sequentially. Eighty-six percent of the patients who had multiple synchronous estrogen receptor assays from various metastatic sites showed no significant discrepancy in estrogen receptor values. When estrogen receptor assays were done sequentially without intervening therapy, 83.5 percent of the patients maintained their initial positivity or negativity. However, when the second estrogen receptor determination was preceded by either chemotherapy or hormonal therapy, 33 percent of the patients had a significant discrepancy in estrogen receptor values. The most common discrepancy was estrogen receptor-positive tumors becoming estrogen receptor-negative, although a small number of patients were found whose receptor values became more positive after hormonal ablation.

Results of oophorectomy by menstrual and estrogen receptor states in patients with metastatic breast cancer.

The records of 204 women with metastatic breast carcinoma treated by oophorectomy were analyzed. Premenopausal women had a response rate of 50 percent. Forty-one percent of postmenopausal women responded. Those who responded had an average duration of response of 22 months and a length of survival twice that of the nonresponders. There was a better than 60 percent correlation between response to oophorectomy and response to further endocrine ablation. Response to endocrine manipulation is more a function of the hormonal sensitivity of the carcinoma than of menopausal status.

Hormone receptors in male breast carcinoma.

It is well established that approximately 60% of female breast cancers contain estrogen receptors (ER+). The hormonal receptor status of male breast cancers remains relatively unknown. Tumor tissue from 14 patients has been reviewed at the University of Oregon Health Sciences Center. Specimens of all 14 patients were ER+ with a range of 5.5 to 374 fmol/mg protein. Eight of 12 patients had tumors which also contained progesterone receptors (PR+) with a range of 42 to 1852 fmol/mg protein. Nine patients were treated by modified radical mastectomy, two by radical mastectomy, one by simple mastectomy, and two by biopsies only. Twelve patients remain free of disease with a mean follow-up of less than 2 years. The two patients who developed metastases both responded to endocrine ablation by orchiectomy and subsequent adrenalectomy. These data suggest that male breast cancer is highly endocrine sensitive and that endocrine ablation can play an integral part in those men unfortunate enough to develop disseminated disease.

Endocrine ablation for metastatic breast cancer: a reappraisal of hormone receptors.

PIP: Other indicators of hormone sensitivity, besides estrogen receptor (ER) content, such as response to oophorectomy, antiestrogens, prolactin suppression, and correlation with progesterone receptors (PR), were evaluated in the hope of further improving selectivity and response of patients undergoing endocrine ablation for metastatic breast cancers. 225 patients have undergone full endocrine ablation in the last 30 years at this hospital, 208 by adrenalectomy and 17 by hypophysectomy. 206 of these patients could be retrospectively reviewed, and of these there were objective responses to therapy in 50% of patients. ER analyses were performed in 1 or more breast cancer specimens in 113 of these patients. ER study showed that a patient who was ER+ and responded to a functional test of endocrine sensitivity had a 70-80% chance of also benefiting from adrenalectomy or hypophysectomy. Conversely, patients with absent or unknown hormone receptors who failed therapeutic trials of endocrine sensitivity had little or no chance of responding to major ablation; these cases are best treated with multiagent chemotherapy. The value of sequentially treating selected patients with endocrine manipulation in addition to chemotherapy was also studied. Patients who failed to respond to endocrine manupulation survived slightly over 2 years on chemotherapy, whereas patients who responded to major ablation lived with metastases an average of 4 years, whereas complete responders lived with metastatic disease an average of 6 years. By life table analysis, total survival of ER+ vs. ER- patients as well as responders vs. nonresponders was highly significant^ieng

Specific binding of prolactin by the prostate gland of the rat and man.

Specific binding sites for 125iodine-prolactin are present in membrane particles obtained from the rat ventral prostate, human benign prostatic hyperplasia and prostatic adenocarcinoma. In the ventral prostate glands of young rats (1 to 4 months old) specific binding of 125iodine-prolactin is higher than in older animals (greater than 8 months old). Subcellular distribution studies revealed that specific 125iodine-prolactin binding activity is associated primarily with the 15,000 and 100,000 g particulate membrane fractions of the rat ventral prostate and human prostate glands. In rats between 2 and 4 months old significant increases in the prolactin binding activity in the 100,000 g membrane fraction of the ventral prostate are observed to occur without concomitant increases in prolactin binding activity in the 15,000 g fraction. The level of specific 125iodine-prolactin binding activity present in the human prostate gland is lower than that observed in the rat ventral prostate gland. Localization of specific prolactin binding sites in the rat ventral psotate and the human prostate gland suggests that prolactin could influence the function of these tissues directly.