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.

The effects of familial relationships, age, body weight, and diet on blood pressure and the 24 hour urinary excretion of sodium, potassium, and creatinine in men, women, and children of randomly selected families.

Two hundred thirty-three randomly selected families provided a population for studying the effects of familial relationships, age, diet, body weight, and urinary electrolyte excretion on blood pressure. There was a strong familial component for urinary sodium, potassium, and creatinine excretion and for systolic blood pressure. In individuals, age, heart rate, and body weight were independently related to blood pressure. In women, urinary sodium and potassium levels were related to diastolic blood pressure. These individual relationships persisted when age was accounted for but were no longer significant after adjusting for both age and body weight, suggesting that heavier people eat more food, which in our culture means greater sodium intake. In fact, our randomly selected families were eating as much sodium (130 to 170 meq/day) and as little potassium (50 to 70 meq/day) as consumed by Americans several decades ago. Furthermore, this study again documented the rise in blood pressure with age, which may represent the effect of environmental influences on blood pressure over time. The familial aggregation of urinary sodium, potassium, and creatinine excretion, along with the well-established familial aggregation of body weight, emphasizes the importance of the entire family in the treatment and prevention of hypertension.

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.

Interaction between prolactin and androgens in the accessory sex organs of male mice.

Treatment of adult male mice with varying doses of prolactin increased the weights of the seminal vesicles and the anterior prostate gland. Only in the seminal vesicles were these increases in organ weights associated with increased levels of DNA. In castrated mice, prolactin alone failed to alter the weights of the accessory sex organs or DNA content. However, the simultaneous administration of prolactin and testosterone resulted in enhanced androgenic stimulation of seminal vesicle weights and their DNA content. Prostatic weights, but not DNA content, were augmented by treatment with prolactin and testosterone. Although the kidneys exhibited androgen sensitivity, prolactin failed to enhance the effect of testosterone upon the kidney. Augmentation of androgen action in the accessory sex organs was observed only after treatment with prolactin or growth hormone. Prolactin also enhanced the effects of dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol, but not of 5 alpha-androstane-3 beta, 17 beta-diol. These studies revealed that prolactin enhances the proliferative actions of androgens in mouse seminal vesicles, but not in the anterior prostate glands or the kidneys.

Effects of prolactin on DNA synthesis and growth of the accessory sex organs in male mice.

The role of PRL in the development of male accessory sex organs remains unresolved. In the present studies, the influence of PRL and testosterone upon DNA synthesis and cell proliferation was examined in the anterior prostate gland (AP) and the seminal vesicles (SV) of mice. Hormonal effects on DNA synthesis were evaluated by examining the in vitro incorporation of [3H]thymidine into DNA in relation to temporal alterations in tissue DNA content. In intact mice, PRL (300 IU/kg daily) produced peak stimulation of [3H]thymidine incorporation into DNA by SV after 3 days of treatment. In contrast, PRL failed to alter [3H]thymidine incorporation by the AP. Only in SV did PRL injections lead to elevated levels of tissue DNA. Injections of testosterone (0.75 or 7.5 mg/kg daily for 3 days) to castrated mice also produced significant stimulation of labeled thymidine incorporation into DNA by AP and SV. Concomitant injections of PRL (150 IU/kg) and testosterone (0.75 or 7.5 mg/kg) enhanced the stimulatory effects of this androgen on DNA synthesis and DNA content in the SV, but not in the AP. When PRL alone was administered to castrated mice, it failed to affect either [3H]thymidine incorporation into DNA by AP and SV or accessory sex organ weights and DNA contents. The results of these studies suggest that PRL enhances the effects of androgens upon DNA synthesis and growth of the seminal vesicles, but not of the anterior prostate glands of mice.

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.