Alpha-fetoprotein derived from a human hepatoma prevents growth of estrogen-dependent human breast cancer xenografts.

Alpha-fetoprotein (AFP) is a transport protein that has growth-regulatory properties in many different tissues. It is known to interfere with responses stimulated by estrogen. The purpose of this study was to determine whether human AFP would inhibit the growth of human breast cancer. AFP was isolated from the culture supernatant of human hepatoma cells (HepG2) grown in serum-free medium and was purified by immunoaffinity chromatography. Human breast cancers were grown as xenografts under the kidney capsule of severe combined immunodeficient mice. The minimum inhibitory dose of AFP against estradiol (E2)-stimulated growth of human MCF-7 breast cancer xenografts was 10 microg/mouse/day, and maximum inhibition (no growth) was achieved with 100 microg/mouse/day. Daily treatment was required to sustain inhibition. This 100-microg dose of AFP also inhibited xenograft growth of E2-dependent T47 human breast carcinoma. Estrogen receptor-negative MDA MB 231 and BT20 human breast carcinoma xenografts were not inhibited by AFP (100 microg/mouse/day). Elevation in serum E2 occurred during AFP treatment. AFP did not compete with agonists for the estrogen receptor. These laboratory results are consistent with the findings of a literature search, which consistently showed an association between elevated pregnancy levels of AFP and subsequent reduced risk for breast cancer later in life. We conclude that AFP can inhibit growth of estrogen-dependent breast cancer and warrants further development as an agent for the treatment and perhaps even the prevention of human breast cancer.

Estradiol and progesterone effects on relative luteinizing hormone and follicle stimulating hormone release induced from superfused anterior pituitary cell cultures by defined LHRH pulse regimens.

These studies examined the capacity of estradiol and progesterone to modulate relative luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretion from superfused anterior pituitary cells when stimulated with luteinizing hormone releasing hormone (LHRH) pulse regimens of specific amplitude, duration and frequency. There was particular interest in whether such steroid and LHRH treatments induced evidence of divergent LH or FSH secretion. Pituitaries were recovered from adult, 2 week ovariectomized rats and cultured for 48 h with collagen coated Cytodex microcarrier beads. Cultures were preincubated either with or without estradiol (1 or 10 nM) for 48 h and were subsequently incubated for 3,6 or 12 h with 100 nM progesterone. All groups were then pulsed with 1 of 3 LHRH regimens; regimen 1 delivered 8 ng in a single 100 microliters bolus once/h; regimen 2 divided the 8 ng dose of regimen 1 into 3 equal doses administered at 4 min intervals thereby maintaining the 8 ng mass of regimen 1 while extending the duration of exposure; regimen 3 was the same as regimen 2 except that the 3 equal doses were administered at a pulse frequency of 1 per 2 h rather than 1 per h thereby not only maintaining the duration of exposure as in regimen 2 but also reducing the pulse frequency. 1 nM estrogen alone for 48 h had no effect on LHRH stimulated LH release regardless of regimen; however, FSH was increased when hourly pulses of increased duration were applied (regimen 2). When estrogen was increased to 10 nM, regardless of regimen, LH was predominantly inhibited while FSH was unaffected. When 1 nM estrogen was followed by progesterone, both LH and FSH were elevated at 6 h progesterone in response to regimen 2; with 10 nM estrogen however, a divergent response was observed in that LH release was elevated at 6 h while FSH was elevated at 3 h in response to regimens 2 and 3. These results first of all confirm that progesterone in combination with estrogen is capable of exerting both inhibitory and stimulatory effects on gonadotropin secretion; secondly, these studies show that, as a direct pituitary effect, the LHRH regimen and the gonadal steroid milieu are capable of interacting to significantly influence the relative secretion of LH and FSH. The data therefore suggest that the divergent gonadotropin secretion seen in various physiological states in vivo is due likely in part to a combination of estrogen and progesterone priming in combination with the hypothalamic LHRH secretory pattern.

The induction of divergent gonadotropin secretion in vitro by variations in luteinizing hormone releasing hormone pulse regimen.

The effects of luteinizing hormone releasing hormone (LHRH) pulse amplitude, duration, and frequency on divergent gonadotropin secretion were examined using superfused anterior pituitary cells from selected stages of the rat estrous cycle. Cells were stimulated with one of five LHRH regimens. With low-amplitude LHRH pulses (regimen 1) in the presence of potentially estrogenic phenol red, LH response in pituitary cells from proestrus 1900, estrus 0800, and diestrus 1,0800 were all significantly larger (P less than 0.05) than the other stages tested. In the absence of phenol red, responsiveness at proestrus 1900 was significantly larger than proestrus 0800, proestrus 1500, and estrus 0800 (P less than 0.01, 0.05, and 0.05, respectively); other cycle stages tested were smaller. No significant differences were observed between cycle stages for follicle-stimulating hormone (FSH) secretion in the presence or absence of phenol red. Because pituitary cells at proestrus 1900 were the most responsive to low-amplitude 4 ng LHRH pulses, they were also used to study the effects of LHRH pulses of increased amplitude or duration and decreased frequency. Increasing the amplitude (regimen 2) or the duration (regimens 3 to 5) increased FSH secretion; this effect was greatest with regimens 3 and 5. When regimens 3 and 5 were studied in pituitary cells obtained at proestrus 1500, FSH was significantly increased by both regimes, but most by regimen 5; furthermore, LH release was significantly reduced. When regimens 3 and 5 were studied in pituitary cells obtained at estrus 0800, FSH release was elevated most significantly by regimen 5. Thus, variations in LHRH pulse regimen were found to be capable of inducing significant divergence in FSH release from superfused anterior pituitary cells derived from specific stages of the estrous cycle.

Effect of luteinizing hormone releasing hormone pulse characteristics on comparative luteinizing hormone and follicle stimulating hormone secretion from superfused rat anterior pituitary cell cultures.

We have shown that 4 ng luteinizing hormone releasing hormone (LHRH) pulses induced significantly greater luteinizing hormone (LH) release from proestrous rat superfused anterior pituitary cells with no cycle related differences in follicle stimulating hormone (FSH). Current studies gave 8 ng LHRH in various pulse regimens to study amplitude, duration and frequency effects on LH and FSH secretion from estrous 0800, proestrous 1500 and proestrous 1900 cells. Regimen 1 gave 8 ng LHRH as a single bolus once/h; regimen 2 divided the 8 ng into 3 equal 'minipulses' given at 4 min intervals to extend duration; regimen 3 gave the 3 'minipulses' at 10 min intervals, thereby further extending duration: regimen 4 was the same as regimen 2, except that the 3 'minipulses' were given at a pulse frequency of 2 h rather than 1 h. In experiment 1, all four regimens were employed at proestrus 1900. FSH was significantly elevated by all 8 ng regimens as compared to 4 ng pulses; further, 8 ng divided into 3 equal 'minipulses' separated by 4 min at 1 and 3 h frequencies (regimens 2 and 4) resulted in FSH secretion that was significantly greater than with either a single 8 ng bolus (regimen 1) or when the 'minipulses' were separated by 10 min (regimen 3). In experiment 2, at proestrus 1500, FSH response to the second pulse of regimen 4 was significantly greater than in regimen 2; LH release was significantly suppressed at pulse 2 compared to regimen 2 accentuating divergent FSH secretion. At estrus 0800, FSH response to the second pulse of regimen 4 was significantly stimulated FSH at proestrus 1900, 1500 and estrus 0800, FSH divergence was most marked at proestrus 1500. These data indicate a potential role for hypothalamic LHRH secretory pattern in inducing divergent gonadotropin secretion in the rat.

Cycle-related LHRH responsiveness of superfused pituitary cells in a Phenol red free medium.

Anterior pituitary cell cultures are frequently used in studying the control of gonadotropin secretion. Historically, many (if not most) of these studies have been performed in the presence of Phenol red as a pH indicator. Phenol red preparations, because of their potential estrogenic activity, may have influenced the results of previous studies defining the relative luteinizing hormone releasing hormone responsiveness of rat anterior pituitary-cells derived from various stages of the estrous cycle. We therefore felt it of interest to investigate this possibility by repeating our previous cycle-related superfusion studies [(1988) Life Sci. 42, 61-72] in the absence of these Phenol red preparations. Comparisons of data obtained in the presence or absence of Phenol red revealed cells derived from late proestrous (19.00) and cultured in the absence of Phenol red continued to evidence the highest LH responsiveness. However, diestrous 1 08.00 cells cultured in the absence of Phenol red were lower in responsiveness than previously observed in the presence of the substance and the responsiveness of proestrous 08.00 and 15.00 in the presence was lower in comparison to the same stages in the absence of Phenol red. The results suggest that Phenol red preparations are capable of modulating LHRH responsiveness in superfusion and that the effect is more pronounced at certain cycle stages than at others.

Ether as an anesthetic for decapitation in the rat: gonadotropin secretion by subsequently established anterior pituitary cell cultures.

Historically, for the establishment of dispersed anterior pituitary cell cultures, rodents have been killed by decapitation without anesthesia. Because decapitation fails to induce immediate unconsciousness, the American Veterinary Medical Association Panel on Euthanasia has recently recommended that rodents should not be decapitated without previous anesthesia or sedation. Investigators are therefore confronted with the dilemma of wishing to euthanize rodents humanely yet not wishing to potentially compromise experimental data through the use of anesthetics. We present our observations on the effects of diethyl ether anesthesia administered prior to decapitation on the gonadotropin secretory characteristics exhibited in vitro by cultured rat anterior pituitary cells. Neither light nor complete (surgical level) ether anesthesia had any statistically significant effect on either luteinizing hormone or follicle-stimulating hormone responsiveness or cell content of luteinizing hormone and follicle-stimulating hormone or DNA content. The data indicate that ether anesthesia would appear to be acceptable for those studies involving subsequent in vitro luteinizing hormone and follicle-stimulating hormone secretion.

Superfused pituitary cell cultures: comparative responsiveness of cells derived from various stages of the estrous cycle to LHRH stimulation administered as short duration pulses.

We have reinvestigated the question of maintenance of differential LHRH sensitivity in culture and further investigated the role of pulsatile LHRH in the in vitro release of pulsatile LH and FSH at different stages of the estrous cycle. Pituitaries were collected on each day of the 4 day cycle at 0800. In addition, pituitaries were also collected at 1500 and 1900 on proestrous. The cells were dispersed and exposed 48 hrs later to short duration 4 ng LHRH pulses; this dose was optimized for LH release and was applied at a frequency of 1 pulse/60 min. In terms of absolute magnitude of LH response, observed responsiveness was ranked in the following order: proestrous 1900 greater than estrous 0800 greater than diestrous 1 0800 greater than proestrous 1500 greater than diestrous 2 0800. Responsiveness was significantly greater at proestrous 1900 (p greater than 0.01), estrous 0800 (p greater than 0.05) and diestrous 1 0800 (p greater than 0.05) when compared to either of the other stages tested. The heightened LHRH sensitivity of proestrous was therefore maintained in cell culture indicating that the system should be valid for conducting studies on the control of gonadotropin secretion during this period. FSH did not respond in pulsatile manner to the LHRH levels employed further substantiating recent evidence that LHRH seems to function somehow less directly in FSH as compared to LH secretion.

Superfused pituitary cell cultures: effects of culture conditions on apparent responsiveness to LHRH stimulation administered as short duration pulses.

We wished to study estrous cycle related differences in LH and FSH responsiveness to pulsatile LHRH. Such studies are very difficult to perform in vivo under controlled conditions; therefore, an in vitro superfused anterior pituitary cell culture system was evaluated for its capacity to support differences in estrous stage associated LHRH responsiveness. Three vital culture system parameters were evaluated; these parameters were (1) culture medium composition, (2) duration allowed for cell attachment to microcarrier beads and (3) superfusion flow rate utilized during pulsatile LHRH stimulation. It was found that a culture system which utilized 10% Nu Serum in DMEM (final protein concentration of 1.8 mg/ml; final serum concentration of 2.5%), an attachment time of 48 hrs and a flow rate of 0.125 ml/min most successfully maximized LH responsiveness at the lowest serum concentration. These studies indicated that although one may be able to observe LHRH responsiveness under a wide range of culture conditions, responsiveness may nonetheless be maximized by judicious adjustment of culture conditions.