Decreased nocturnal plasma melatonin peak in patients with estrogen receptor positive breast cancer.

Plasma melatonin concentrations were determined over a period of 24 hours in 20 women with clinical stage I or II breast cancer. In ten of the patients, whose tumors were estrogen receptor positive, the nocturnal increase in plasma melatonin was much lower than that observed in eight control subjects. Women with the lowest peak concentration of melatonin had tumors with the highest concentrations of estrogen receptors. A significant correlation was found between the peak plasma melatonin concentration and the tumor estrogen receptor concentration in 19 of the patients. These data suggest that low nocturnal melatonin concentrations may indicate the presence of estrogen receptor positive breast cancer and could conceivably have etiologic significance.

Effect of raloxifene on bone mineral density in premenopausal women at increased risk of breast cancer.

CONTEXT: Raloxifene is a promising breast cancer prevention agent in postmenopausal women at increased risk for breast cancer. The effects of raloxifene in premenopausal women are unknown. OBJECTIVE: We evaluated the effect of raloxifene in premenopausal women at increased risk for breast cancer on bone mineral density (BMD). DESIGN: This was a phase II clinical trial. SETTING: This study was conducted at an academic medical center. PARTICIPANTS: Thirty-seven premenopausal women at increased risk for breast cancer enrolled in the trial. Thirty subjects began treatment and 27 were evaluable. INTERVENTION: Raloxifene (60 mg daily) and elemental calcium (500 mg daily) were given for 2 yr. Subjects were followed up off medications for 1 yr. MAIN OUTCOME MEASURE: The primary end point was the intrasubject percent change in BMD at 1 yr measured by dual-energy x-ray absorptiometry. RESULTS: The mean baseline lumbar spine density was 1.027 g/cm(2). Lumbar spine density decreased 2.3% at 1 yr (P < 0.00001) and 3.5% at 2 yr (P < .00001). Percent change from yr 2 to 3 was +1.4%. The mean baseline total hip bone density was 0.905 g/cm(2). Total hip density decreased 0.3% at 1 yr and 1.0% at 2 yr (P = 0.033). Percent change from yr 2 to 3 was +1.7%. CONCLUSIONS: Raloxifene use is associated with a decrease in BMD in premenopausal women at increased risk for breast cancer. The clinical significance of this decrease is unknown and is attenuated with stopping raloxifene.

Interleukin-1 alpha and interleukin-6 act additively to inhibit growth of MCF-7 breast cancer cells in vitro.

We studied the effects of interleukin-1 alpha (IL-1) and interleukin-6 (IL-6) on MCF-7 breast cancer cells to determine whether these cytokines act additively/synergistically to alter cell growth and metabolism. We found that IL-1 alone (1000 units/ml) inhibited cell growth to a greater degree (83.8%) than IL-6 alone (29.2%, P < 0.001). The combination of IL-1 + IL-6 caused greater inhibition of growth (92.9%, P < 0.02) than either cytokine alone. The additive effect was dose dependent for both IL-1 and IL-6. IL-1 and IL-6 also antagonized estradiol (10(-9) M) stimulated growth. Antagonism by the combination was greater than for either cytokine alone (P < 0.001). IL-1 or IL-6 alone each down-regulated the estrogen receptor (36.7%, P < 0.01, and 23.2%, P < 0.05, respectively), but the combination IL-1 + IL-6 did not cause a significantly greater effect than IL-1 alone. Neither IL-1 or IL-6 blocked estradiol stimulation of progesterone receptor (PR) synthesis; however, the combination IL-1 + IL-6 increased PR content by 28.4% (P < 0.01). IL-1, but not IL-6, increased secretion of transforming growth factor-beta (TGF-beta) by 2.45-fold over 72 h (P < 0.01). The increase was time dependent (detectable at 24 h) and dose dependent (maximum increase of 5.3-fold, 10,000 units/ml, P < 0.02). IL-1-induced TGF-beta secretion was blocked by estradiol (10(-9) M). Neither cytokine altered secretion of insulin-like growth factor-1. These findings indicate that IL-1 and IL-6 act additively to inhibit growth in the absence or presence of estradiol and modulate the estrogen receptor and progesterone receptor content of these cells. TGF-beta may mediate the effects of IL-1; however, other pathways appear to be required for the additive effects of these cytokines.

Interleukin 1 alpha blocks estradiol-stimulated growth and down-regulates the estrogen receptor in MCF-7 breast cancer cells in vitro.

We studied the effect of interleukin 1 alpha (IL-1 alpha) on estradiol stimulation of cell growth and estrogen receptor (ER) content in MCF-7 human breast cancer cells in vitro to determine if IL-1 alpha altered cellular estradiol responsiveness. We found that IL-1 alpha blocked estradiol-stimulated growth of these cells in a dose-dependent manner (complete antagonism at 1000 units/ml: day 7 mean growth = vehicle, 47.7 micrograms DNA; estradiol 10(-10) M, 95.1; IL-1 alpha/estradiol, 44.6) and at all concentrations of estradiol from 10(-8) to 10(-11) M. IL-1 alpha in combination with trans-hydroxytamoxifen further inhibited estradiol-stimulated growth (vehicle = 44.8 micrograms DNA, estradiol = 108.3, estradiol/trans-hydroxytamoxifen = 47.8, IL-1 alpha/estradiol/trans-hydroxytamoxifen = 3.0, P less than 0.01). Inhibition with trans-hydroxytamoxifen was IL-1 alpha dose dependent (maximum = 97% at 1000 units/ml, P less than 0.01) and estradiol dose dependent (reversible with 10(-8) M estradiol, maximum inhibition at 10(-10) M estradiol). Concomitantly, IL-1 alpha down-regulated ER concentration by 38.0-43.7% (P less than 0.01) as measured by immunoreactivity or Scatchard analysis, respectively. This occurred as early as 3 h without a change in the Kd (vehicle = 0.23 nM, IL-1 alpha = 0.24 nM), persisted for at least 48 h, was dose dependent (maximum, 43.7% at 1000 units/ml, P less than 0.01), and was blocked by cycloheximide. IL-1 alpha, however, did not block estradiol stimulation of progesterone receptor content (vehicle = 221.9, IL-1 alpha = 238.9 fmol/mg protein) and did not block estradiol down-regulation of ER content. Furthermore, IL-1 alpha alone did not alter levels of ER mRNA and did not alter estradiol down-regulation of ER mRNA. These findings indicate that while IL-1 alpha antagonizes estradiol stimulation of growth and reduces ER content, its mechanism may involve other non-estrogen-regulated pathways.

Neoadjuvant chemotherapy in the combined modality approach of locally advanced nonmetastatic breast cancer.

We have treated 76 patients with locally advanced breast cancer, 31 with stage IIIA, 41 with stage IIIB, and 4 with stage IV disease, with primary induction chemotherapy including an attempted hormonal synchronization in 70 patients. All were treated to maximum objective clinical response before proceeding to any local therapy. Patients achieving a complete response with a negative repeat biopsy generally received radiation therapy while patients with residual disease, partial response (PR) or no change (NC) status received debulking surgery prior to radiation therapy. Regardless of response to induction chemotherapy, patients received at least 6 additional months of chemotherapy following local therapy. Initial doses of combination chemotherapy were escalated to targeted myelosuppression. The objective response rate to induction chemotherapy was 93% with 49% complete response (CR), 44% PR, and 7% NC. The median numbers of cycles of chemotherapy to achieve a CR, PR, or NC were 5, 3, and 5, respectively. Three patients who currently have PRs are still on chemotherapy with continued tumor regression. Of 37 patients achieving a CR to chemotherapy, 35 were assessed by biopsies to determine pathological evidence of response. Twenty-three of the 37 patients (62%) were proven to be complete responders with negative biopsies. Twenty-four patients have relapsed, 6 with stage IIIA, 16 with stage IIIB, and 2 with stage IV. Five patients have had locoregional relapses alone, 4 locoregional and distant, and 15 distant alone. Median time to progression is 35.9 months for stage IIIA and 34.2 months for stage IIIB. Median survival is 35.3 months for stage IIIB and is indeterminate for stage IIIA. This aggressive primary chemotherapy regimen with hormonal synchronization followed by local therapy appears to provide excellent local control and encouraging early results on systemic disease control.

Plasma melatonin and the hormone-dependency of human breast cancer.

We studied the 24-hour plasma melatonin profile in three groups of women: normal individuals, women with breast cancer, and women at high risk for breast cancer, to determine the relationship of plasma melatonin to this malignancy. The mean daytime (nadir) and mean nighttime (peak) plasma levels for the normal subjects were 9.1 pg/mL and 70.9 pg/mL, respectively. The mean daytime and nighttime plasma levels, and the range of melatonin day to night differences for women with breast cancer and women at high risk for breast cancer were comparable to each other and to the normal subjects, with no statistically significant differences noted. The patients with breast cancer demonstrated a striking correlation between the melatonin diurnal rhythm and the steroid receptor content of the primary tumor. Women with estrogen (ER) or progesterone (PR) receptor-positive tumors had a significantly lower mean plasma melatonin day to night difference than did patients with ER- or PR-negative tumors. Further, a strong inverse correlation was observed between the plasma melatonin concentration and the quantities of ER and PR in the primary tumor: the lower the plasma melatonin concentration the greater the amount of either receptor in the primary tumor. Plasma melatonin did not correlate with tumor glucocorticoid receptor content or stage of breast cancer among these patients, or with menopausal status, age, parity, or the plasma levels of estrone, estradiol, progesterone, follicle-stimulating hormone (FSH), or luteinizing hormone (LH) among all individuals studied. Plasma melatonin was also independent of the degree of risk for breast cancer among the high-risk patients. These findings suggest an important relationship between the plasma melatonin diurnal rhythm and the hormone dependency of human breast cancer, and may have implications for both the prognosis and treatment of this malignancy.

Complete axillary lymph node dissection for stage I-II carcinoma of the breast.

We reviewed the complete axillary dissection specimens of 136 patients with stage I-II breast cancer to clarify the distribution of axillary lymph node metastases in this disease. Our series included 71 patients undergoing axillary dissection as part of a modified radical mastectomy (MRM) and 65 patients undergoing axillary dissection in conjunction with conservative surgery of the breast and definitive postoperative breast radiotherapy (CAD). These two groups of patients were comparable according to age, menopausal status, tumor size, and clinical stage. In all patients the pectoralis minor muscle was excised and all axillary tissue removed. Each specimen contained a median of 23 lymph nodes. The axillary levels (I, II, III) were determined according to the relationship of axillary tissue to the pectoralis minor muscle (lateral, inferior, medial). Thirty-nine percent of the lymph nodes were contained in level I, 41% in level II, and 20% in level III. There were no significant differences noted in the number of lymph nodes or in the distribution of lymph nodes according to axillary level between dissections performed as part of the MRM or those done as a single procedure (CAD). Sixty-five patients (47.8%) had one or more positive lymph nodes in their axillary specimen. The clinical and pathologic stage was determined and compared for all patients. Among patients judged to have a clinically negative axilla, 37.6% had histologically positive lymph nodes (clinical false-negative rate). For patients with a clinically positive axilla, 11.1% had, histologically, no evidence of metastatic disease (clinical false-positive rate). When the distribution of lymph node metastases according to axillary level was studied, it was found that 29.2% of lymph node-positive patients (or 14.0% of all patients) had metastases only to level II and/or III of the axilla, with level I being negative (skip metastases). This incidence of skip metastases was greater among clinically node-negative than among clinically node-positive patients, but was not related to the size or location of the primary tumor in the breast. In addition, it was found that 20.0% of lymph node-positive patients (or 9.6% of all patients) were converted from three or fewer to four or more positive nodes by analysis of lymph nodes contained in levels II and III. This conversion from three or fewer to four or more positive nodes was due primarily to information contained in level II, with level III contributing to a smaller degree.(ABSTRACT TRUNCATED AT 400 WORDS)

Mastectomy versus breast-conserving therapy in the treatment of stage I and II carcinoma of the breast: a randomized trial at the National Cancer Institute.

PURPOSE: Mastectomy versus excisional biopsy (lumpectomy) plus radiation for the treatment of stage I and II breast cancer was compared in a prospective randomized study. PATIENTS AND METHODS: From 1979 to 1987, 247 women were randomized and 237 were treated on this study. All patients received a full axillary dissection and all node-positive patients received adjuvant chemotherapy with cyclophosphamide and doxorubicin. Radiation consisted of external-beam therapy to the whole breast with or without supraclavicular nodal irradiation followed by a boost to the tumor bed. RESULTS: The minimum time on the study was 18 months and the median time on the study was 68 months. No differences in overall survival or disease-free survival were observed. Actuarial estimates at 5 years showed that 85% of mastectomy-treated patients were alive compared with 89% of the lumpectomy/radiation patients (P2 = .49; 95% two-sided confidence interval [CI] about this difference, 0% to 9% favoring lumpectomy plus radiation). The probability of failure in the irradiated breast was 12% by 5 years and 20% by 8 years according to actuarial estimates. Of 15 local breast failures, 14 were treated with and 12 were controlled by mastectomy; the ultimate local-regional control was similar in both arms of the trial. CONCLUSION: These data add further weight to the conclusion that breast conservation using lumpectomy and breast irradiation is equivalent to mastectomy in terms of survival and ultimate local control for stage I and II breast cancer patients.

Adjuvant chemotherapy for patients with high-grade soft-tissue sarcomas of the extremity.

We have previously reported the results of a randomized trial that demonstrated the survival benefit of adjuvant chemotherapy in the treatment of patients with high-grade extremity sarcomas compared with no chemotherapy. This regimen included doxorubicin, cyclophosphamide, and methotrexate. This report updates and extends our experience. The median follow-up of this trial is now 7.1 years and reveals a 5-year disease-free survival of 75% and 54% for chemotherapy and no chemotherapy groups, respectively (two-sided P [P2] = .037). The 5-year overall survival for patients in this trial was 83% and 60% for the chemotherapy and no chemotherapy groups, respectively, with a trend towards improved survival in the chemotherapy arm (P2 = .124). Because of doxorubicin-induced cardiomyopathy we performed a subsequent randomized trial comparing this high-dose regimen to reduced cumulative doses of doxorubicin and cyclophosphamide without methotrexate. Eighty-eight patients were entered into this trial which has a median follow-up of 4.4 years. The 5-year disease-free and overall survival for patients treated with the reduced doses of chemotherapy was 72% and 75%, respectively, and was not significantly different from the high-dose regimen. No patients developed congestive heart failure on this study. We conclude that adjuvant chemotherapy improves disease-free survival in patients with extremity soft-tissue sarcomas. The overall survival advantage in patients receiving adjuvant chemotherapy in our initial randomized high-dose chemotherapy trial has diminished though it continues to favor the chemotherapy group. A reduced-dose chemotherapy regimen was found to be comparable to the high-dose regimen.

Randomized prospective study of the benefit of adjuvant radiation therapy in the treatment of soft tissue sarcomas of the extremity.

PURPOSE: This randomized, prospective study assesses the impact of postoperative external-beam radiation therapy on local recurrence (LR), overall survival (OS), and quality of life after limb-sparing resection of extremity sarcomas. PATIENTS AND METHODS: Patients with extremity tumors and a limb-sparing surgical option were randomized to receive or not receive postoperative adjuvant external-beam radiotherapy. Patients with high-grade sarcomas received postoperative adjuvant chemotherapy whereas patients with low-grade sarcomas or locally aggressive nonmalignant tumors were randomized after surgery alone. RESULTS: Ninety-one patients with high-grade lesions were randomized; 47 to receive radiotherapy (XRT) and 44 to not receive XRT. With a median follow-up of 9.6 years, a highly significant decrease (P2 = .0028) in the probability of LR was seen with radiation, but no difference in OS was shown. Of 50 patients with low-grade lesions (24 randomized to resection alone and 26 to resection and postoperative XRT), there was also a lower probability of LR (P2 = .016) in patients receiving XRT, again, without a difference in OS. A concurrent quality-of-life study showed that extremity radiotherapy resulted in significantly worse limb strength, edema, and range of motion, but these deficits were often transient and had few measurable effects on activities of daily life or global quality of life. CONCLUSION: This study indicates that although postoperative external-beam radiotherapy is highly effective in preventing LRs, selected patients with extremity soft tissue sarcoma who have a low risk of LR may not require adjuvant XRT after limb-sparing surgery (LSS).

Pilot trial of the safety, tolerability, and retinoid levels of N-(4-hydroxyphenyl) retinamide in combination with tamoxifen in patients at high risk for developing invasive breast cancer.

PURPOSE: N-(4-hydroxyphenyl) retinamide (¿4-HPR, Fenretinide; R.W. Johnson Pharmaceutical Research Institute, Springhouse, PA) and tamoxifen (TAM) have synergistic antitumor and chemopreventive activity against mammary cancer in preclinical studies. We performed a pilot study of this combination in women at high risk for developing breast cancer. PATIENTS AND METHODS: Thirty-two women were treated with four cycles of 4-HPR, 200 mg orally (PO) for 25 days of each 28-day cycle, and TAM, 20 mg PO once daily for 23 months beginning after 1 month of 4-HPR alone. Tolerability, dark adaptometry, tissue biopsies, and retinoid plasma concentrations (Cp) were evaluated. RESULTS: Symptomatic reversible nyctalopia developed in two patients (6%) on 4-HPR, but 16 (73%) of 22 patients had reversible changes in dark adaptation, which correlated with relative decrease in Cp retinol (P

Tumor necrosis factor alpha enhances secretion of transforming growth factor beta2 in MCF-7 breast cancer cells.

We studied the effect of tumor necrosis factor alpha (TNF-alpha) on transforming growth factor beta (TGF-beta) secretion by human breast cell lines to further characterize the antitumor effects of TNF-alpha. We found that TNF-alpha increased the secretion of TGF-beta in two established breast cancer cell lines (MCF-7 and ZR-75-1) but not in two immortalized human mammary epithelial cell lines (184B5 and MCF-10A). In MCF-7 cells, TNF-alpha increased the secretion of total TGF-beta 6.1-fold within 72 h in a dose-dependent manner. The secretion of both latent and active forms of TGF-beta was increased, and their ratio altered from 25:1 to 12:1 in the medium. TNF-alpha converted the secretory pattern of TGF-beta by MCF-7 cells from the heterodimeric form TGF-beta1.2 to the homodimeric form TGF-beta2. Immunoblot analysis under nonreducing conditions identified four molecular mass species of TGF-beta secreted in the culture media of untreated MCF-7 cells (238, 210, 40-55, and 25 kDa). Under reducing conditions, three molecular mass species of TGF-beta were identified: 88, 44, and 12 kDa. Gel filtration analysis demonstrated that the secreted TGF-beta within the range of 12-88 kDa was biologically active. TNF-alpha treatment did not alter the size of molecular mass species secreted by MCF-7 cells and did not change steady-state levels of mRNA for TGF-beta1 or TGF-beta2. These findings indicate that TNF-alpha may regulate quantitatively and qualitatively TGF-beta secretion by human breast cancer cells in vitro. The diverse biological activities of TGF-beta may also allow TNF-alpha to regulate the growth and metabolism of human mammary epithelial cells and/or stromal cells in a paracrine manner.

Interleukin-1 alpha and tumor necrosis factor-alpha (TNF alpha) inhibit growth and induce TNF messenger RNA in MCF-7 human breast cancer cells.

We studied the effects of interleukin-1 alpha (IL-1) and tumor necrosis factor-alpha (TNF), alone and in combination, on MCF-7 breast cancer cells to determine whether these cytokines alter cell growth, TNF gene expression, and TNF secretion. We found that IL-1 alone and TNF alone inhibited cell growth in a dose-dependent manner. Each cytokine arrested growth in the G0/G1 phase of the cell cycle, with maximum growth inhibition at 1000 U/ml (P less than 0.05) and 100 U/ml (P less than 0.01), respectively. However, the combination of these two cytokines did not result in greater growth inhibition or a greater percentage of cells arrested in the G0/G1 phase of the cell cycle compared with each cytokine alone. We examined the effect of exogenous IL-1 and TNF on TNF gene expression by Northern blot analysis. In the absence of any cytokine, these cells do not express TNF mRNA. Exposure to IL-1 (1000 U/ml) induced TNF mRNA at 3 h; however, mRNA levels diminished thereafter to barely detectable levels by 24 h. Exposure to TNF (1000 U/ml) also induced TNF mRNA at 3 h, but in contrast to IL-1, the level of enhanced expression persisted at these levels through 72 h of exposure. Secretion of TNF by these cells is induced by exogenous TNF, but not by IL-1. IL-1 and TNF in combination do not produce greater inhibition of growth, greater amounts of TNF mRNA at 3 h, or greater secretion of TNF than that produced by TNF alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of a candidate gonadotropin surge inhibiting factor from porcine follicular fluid.

Several lines of evidence suggest that the ovaries of many species produce a nonsteroidal substance, termed gonadotropin surge inhibiting factor (GnSIF), which inhibits the midcycle gonadotropin surge and attenuates the pituitary response to endogenous or exogenous GnRH. We have previously reported the partial purification of GnSIF from porcine follicular fluid (pFF) and its differentiation from inhibin. We present now the purification of GnSIF to homogeneity and determination of the partial NH2-terminal amino acid sequence. The bioassay for GnSIF used rat pituitary cells in short-term culture that were incubated with test fractions for 48 h, washed, and then incubated with 10 nM GnRH plus test fractions for 4 h. GnSIF activity is defined as the suppression of GnRH-stimulated LH secretion. GnSIF was purified from 500 ml of pFF using sequential heparin-Sepharose, anion-exchange, and cation-exchange liquid chromatography followed by gel permeation, hydrophobic interaction, and mono-Q HPLC steps. Using these six purification steps, we have obtained an apparently homogenous preparation that stains as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GnSIF has an apparent mol wt of 69K. Limited NH2-terminal sequence analysis reveals that GnSIF has no sequence homology with other reproductive hormones including the inhibins, activins, and follistatins. Over the dose range tested, GnSIF had no effect on basal LH or FSH secretion by pituitary cells in culture and only slightly inhibited GnRH-stimulated FSH secretion at the highest dose tested. In addition, there was no inhibin or follistatin immunoactivity in the GnSIF preparation. As such, GnSIF appears to be a novel protein in pFF that inhibits GnRH-stimulated LH secretion, and which may participate along with other ovarian proteins and steroids in the regulation of pituitary gonadotropin secretion.

Evidence for two populations of masked gonadotropin-binding sites in the corpus luteum of the rhesus monkey (Macaca mulatta).

To evaluate the possible existence of masked gonadotropin receptors in the corpus luteum, we characterized the effects of alcohols and neuraminidase on [125I]iodohuman LH binding to in vitro preparations of luteal tissue from the rhesus monkey and pseudopregnant rat. The presence of 1-8% (vol/vol) ethanol enhanced specific LH binding to macaque luteal particulates under steady state conditions (25 C, 20-h incubation), with a maximal effect at 8% ethanol (166% of control uptake; P less than 0.05). However, 1-8% ethanol had no effect on LH binding to rat luteal tissue. Higher concentrations of ethanol (20%) decreased LH binding relative to control values in both species. Ethanol modulation of LH binding to macaque luteal particulates and dispersed cells was a time- and temperature-dependent process. At 4 and 25 C, ethanol increased LH uptake at all times during a 32-h incubation. However, at 37 C, ethanol increased LH uptake at 30 min; binding peaked at 2 h and then returned to control levels within 20 h. The optimal concentration of ethanol for enhancing LH uptake was inversely related to the incubation temperature. The increase in LH binding to macaque luteal particulates in the presence of ethanol was reversible; binding returned to control levels if ethanol was removed before the addition of labeled LH. Longer straightchain alcohols (butanol, pentanol, and octanol) were progressively more potent than ethanol in enhancing LH binding to macaque luteal particulates and dispersed luteal cells. Pretreatment of luteal particulates from either the rat or monkey with neuraminidase increased LH uptake, with a maximal effect (160% of control) at 1 mg/ml enzyme. Scatchard analyses revealed that both ethanol and neuraminidase increased (P less than 0.05) the number of LH-binding sites without altering the affinity for gonadotropin. Moreover, the effects of ethanol and neuraminidase were additive, i.e. increased LH binding during combination of the two treatments approximated the sum of the individual effects. The data suggest that two distinct populations of LH-binding sites are masked within the membranes of the monkey corpus luteum. The ability of two markedly different agents, alcohol and neuraminidase, to increase LH binding indicates that diverse mechanisms may modulate the masking/unmasking of gonadotropin receptors in target cell membranes. Finally, the inability of ethanol to enhance LH binding in the rat suggests species differences in the receptor population or milieu of luteal membranes.

Inhibition of pituitary gonadotropin secretion by the gonadotropin-releasing hormone antagonist antide. I. In vitro studies on mechanism of action.

The GnRH antagonist antide is among the most promising "third generation" compounds available for clinical evaluation. In primates, antide manifests prolonged (several weeks) and reversible inhibition of pituitary gonadotropin secretion after a single high dose injection. In the present study, we have examined the effects of antide on pituitary gonadotropin secretion in vitro. Dispersed anterior pituitary cells from adult female rats were plated (48 h; 5 x 10(5) cells/well), washed, and exposed to increasing concentrations of antide for up to 48 h. Media were removed, and cells were washed twice and then incubated with GnRH (1 x 10(-8) M) plus antide for 4 h. Media and cell lysates were assayed for LH/FSH by RIA. Antide had no effect on basal LH/FSH secretion at any dose tested (10(-6)-10(-12) M). In contrast, GnRH-stimulated LH/FSH secretion was inhibited by this GnRH antagonist in a dose- and time-dependent manner. When incubated simultaneously, antide blocked GnRH-stimulated gonadotropin secretion, with a maximal effect at 10(-6) M (ED50, 10(-7) M). Preincubation of pituitary cells with antide for 6-48 h before GnRH exposure shifted the dose-response curve to the left; the maximally effective dose was 10(-8) M; the ED50 was 10(-10) M antide after 48-h preincubation. Intracellular LH/FSH levels increased concomitant with the decrease in secreted gonadotropins. Total LH/FSH levels (secreted plus cell content) remained unchanged. The inhibition of LH secretion by antide was specific for GnRH-stimulated gonadotropin secretion; antide had no effect on K(+)-stimulated LH secretion. Moreover, antide had little or no residual effect on LH secretion; full recovery of GnRH responsiveness in vitro occurred within 4 h after removal of antide. Lineweaver-Burke analysis of antide inhibition of GnRH-stimulated LH secretion indicated that antide is a direct competitor of GnRH at the level of the pituitary GnRH receptor. In summary, antide is a pure antagonist of GnRH stimulation of gonadotropin secretion; no agonistic actions of antide were manifest in vitro. Moreover, antide has no apparent noxious or toxic effect on pituitary cells in culture; the actions of antide are immediately reversible upon removal of antide from pituitary gonadotropes. We conclude that the long term inhibition of gonadotropin secretion by antide in vivo is not due to deleterious effects of this compound at the level of the pituitary gonadotrope.

Melatonin-induced increase in cytoplasmic estrogen receptor activity in hamster uteri.

The pineal gland hormone, melatonin, has been shown to influence the growth and metabolism of uterine tissue in the Syrian hamster. Because the uterus is an estrogen-responsive target tissue, we studied the effect of melatonin on the unoccupied estrogen receptor (ER) activity of immature hamster uteri in vivo and in vitro. A single sc injection of 2.5 micrograms melatonin increased the ER activity of uterine cytosol (Rc) 30% within 60 min [from 147.4 +/- 14.1 (SE), to 191.8 +/- 18.5 fmol/mg protein]. Scatchard analysis showed the induced population of receptor to be homogeneous and of high affinity, with an equilibrium dissociation constant (Kd = 0.08 nM) similar to that for uteri for vehicle-treated animals. The induction of Rc by melatonin persisted for 90 min, but Rc returned to control levels by 2 h after injection. Scatchard analysis of unoccupied nuclear receptor showed no significant differences either in the quantity of receptor (vehicle = 379.7 +/- 46.7, melatonin = 396.9 +/- 76.7 fmol/mg protein) or in the Kd (vehicle = 0.75 nM, melatonin = 0.60 nM) between these two groups. In vitro incubation of fresh whole uteri for 60 min in media containing 10(-5) M melatonin increased Rc by 83% (from 41.3 +/- 10.7 to 75.4 +/- 11.0 fmol/mg protein), supporting a direct effect of melatonin on uterine tissue. The Rc induced by melatonin both in vivo and in vitro was sensitive to the degree of homogenization used in preparation of the cytosol. Whereas conservative homogenization (two 10-sec bursts with an intermittent 40-sec cooling period) of uteri from melatonin-treated animals was associated with a significant increase in Rc, homogenization for 60 sec without intermittent cooling showed a significant decrease (from 251.1 +/- 17.4 to 182.7 +/- 5.8 fmol/mg protein) as compared with uteri from vehicle-treated animals. Whole organ analysis of uteri from animals injected with melatonin followed by [3H]estradiol, in which no homogenization was used, demonstrated a 26% increase in Rc specific binding, confirming induction as the primary response to melatonin. These findings demonstrate the ability of melatonin to modulate ER activity in hamster uteri, and may help explain the effects of melatonin on uterine growth and metabolism.

In vivo and in vitro modulation of gonadotropin-releasing hormone metabolism by estradiol and progesterone.

The metabolism of GnRH by membrane-bound and serum-degrading enzymes may play an important role in the regulation of pituitary gonadotropin secretion. We examined GnRH metabolism by pituitary cells in vitro and the metabolism of exogenously administered GnRH in vivo in the presence and absence of estradiol (E2) and progesterone (P4). Ovariectomized cynomolgus monkeys were implanted with E2- and/or P4-filled Silastic capsules to simulate the estrogen and progesterone patterns of the normal menstrual cycle. Peripheral levels of GnRH after a 1-microgram iv injection were highest in the E2-replaced monkeys. Peripheral GnRH levels reached a higher peak and remained in circulation longer in monkeys treated with E2-filled Silastic implants than in those treated with E2 plus P4 or nonsteroid-replaced ovariectomized monkeys. In agreement with the in vivo data, GnRH was rapidly metabolized by acutely dispersed cells isolated from pituitaries removed from nonsteroid-replaced ovariectomized monkeys. Priming with E2 followed by P4 in vivo attenuated the clearance of GnRH in vitro, and E2 treatment alone almost completely blocked the ability of pituitary cells to bind and/or degrade GnRH in vitro. In a parallel study, cells prepared from rat pituitaries removed on the morning of proestrus (when serum E2 is highest) metabolized GnRH in vitro more slowly than pituitary cells removed at estrus, diestrus, or metestrus. In summary, our data suggest that E2 inhibits GnRH metabolism by monkey and rat pituitary cells in vitro and exogenously administered GnRH in vivo. Although the precise mechanism of action of E2 is unknown, inhibition of membrane-bound and serum proteases seems likely. The action of E2 may be to increase GnRH presentation to the pituitary and enhance LH and FSH secretion under conditions where circulating levels of the hormone are elevated, such as at midcycle.

Modulation of membrane fluidity in the primate (Macaca mulatta) corpus luteum: correlation with changes in gonadotropin binding.

Addition of alcohols to particulate or cellular preparations of the monkey corpus luteum unmasks gonadotropin-binding sites via a temperature-sensitive process. Since alcohols and temperature are known modulators of membrane fluidity, we measured the fluidity of luteal membranes and determined whether the effects of ethanol and temperature on gonadotropin binding correlated with changes in the fluid state of the membrane. The fluidity of membranes from the macaque and rat corpus luteum was estimated from the fluorescence polarization of the lipophilic membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The absorption and emission spectra of DPH incorporated into luteal membranes were typical of those in other systems. Fluorescence intensity increased rapidly during the first 60 min of incubation and reached steady state conditions within 3 h. In contrast, polarization was constant within minutes and was insensitive to pH, ionic strength, tissue concentration, or DPH levels over the ranges tested. Fluorescence polarization was acutely sensitive to the temperature of the assay medium; polarization decreased as temperature increased from 4-50 C, and no phase transitions were observed. Addition of 4-20% and 8-20% ethanol to monkey and rat membranes, respectively, decreased (P less than 0.05) polarization relative to control values. However, ethanol was less effective on rat membranes, such that 20% ethanol was required to elicit a similar change in polarization as 8% ethanol in macaque membranes. The decrease in polarization was reversed to control levels when ethanol was removed from the incubation medium. Changes in fluorescence polarization of DPH-labeled macaque membranes elicited by ethanol and temperature correlated significantly (r = -0.97) with changes in specific [125I]iodohuman LH binding. In contrast, pretreatment of luteal membranes from the monkey and rat with neuraminidase, which unmasks another population of LH-binding sites in both species, did not alter polarization. We conclude that the fluorescence polarization of DPH is a useful tool for estimating membrane fluidity in the corpus luteum. Furthermore, changes in membrane fluidity may play an important role in the masking/unmasking of alcohol-sensitive (but not neuraminidase-sensitive) gonadotropin-binding sites in the macaque corpus luteum. Finally, the lesser effects of ethanol in the rat suggest important species differences in the receptor milieu and composition of luteal membranes.

Inhibition of pituitary gonadotropin secretion by the gonadotropin-releasing hormone antagonist antide. II. Development of an in vitro bioassay for characterization of pharmacokinetics and pharmacodynamics of antide in circulation.

Previous data from this laboratory revealed a rapid and unexpectedly long inhibition of pituitary gonadotropin secretion in ovariectomized monkeys after a single high dose injection of the GnRH antagonist antide. This extended action of antide may correlate with an extended presence of antide in the peripheral circulation. We have reported on use of a RRA for antide in serum; however, during such a prolonged presence in the body, the possibility of catabolic loss of biological activity remained to be analyzed. In the present study, we have developed an in vitro pituitary cell bioassay for antide to investigate the pharmacokinetics and possible mechanism(s) contributory to its long action. Dispersed anterior pituitary cells from adult female rats were plated (48 h; 5 x 10(5) cells/well), washed, and incubated with 0.024-6 ng antide for 24 h. Media were removed, and cells were washed twice and then incubated with GnRH (1 x 10(-8) M) plus antide standards or serum samples for 4 h. Before antide injection into long term ovariectomized monkeys, peripheral GnRH antagonist levels were undetectable. One day after a single injection (3.0 mg/kg, sc, in 50% propylene glycol-water), the level of antide was 31 +/- 13 ng/ml (n = 3). Thereafter, antide levels declined slowly and were still detectable (greater than 1.4 ng/ml) in two of three monkeys 31 days after injection. After iv administration (3.0 mg/kg; n = 2), peripheral antide levels followed a similar pharmacokinetic profile and declined slowly. Detectable antide concentrations were still present 36 days after single iv injection in both monkeys. The circulating half-lives of antide were 1.7 and 14.5 days for the first and second phases, respectively. Peripheral LH levels were suppressed to the limits of detectability within 1 day and slowly recovered to pretreatment levels within 30 +/- 5 days after sc or iv antide treatment. The ratio of bioactive antide to antide levels measured by RRA was similar throughout the study (chi = 1.24 +/- 0.09; range, 0.40-2.22), although there was a trend toward an increased B/R ratio at the end of the study. In summary, we have developed an in vitro bioassay using cultured rat pituitary cells to measure biologically active antide concentrations in peripheral circulation after sc and iv treatments. The prolonged action of antide on pituitary gonadotropin secretion in vivo is apparently due to the continued presence of biologically active antide in circulation after a single injection.(ABSTRACT TRUNCATED AT 400 WORDS)