Expression of oncodevelopmental gene products by human tumor cells in culture.

Sixty-seven human tumor cell lines and 15 lines derived from normal tissue were examined for the production of the oncodevelopmental markers carcinoembryonic antigen, alpha and beta subunits of chorionic gonadotropin, placental and nonplacental forms of alkaline phosphatase, gamma-glutamyltransferase, cystyl aminopeptidase, and calcitonin. Both intracellular and extracellular levels of these markers were determined at three phases during the growth of each culture. Sixty-eight percent of the cell lines produced elevated levels (greater than or equal to 90th percentile) of at least one marker. Of those, 46% produced elevated levels of one marked only, 29% produced two, 22% produced three, and 4% produced four markers. No cell line produced more than four markers at elevated levels. In most instances, however, the expression of any two particular markers was discordant. For approximately 50% of the possible marker pairs, Spearman rank-ordered correlation analyses showed significant negative correlations, indicating that when one marker was produced at elevated levels by a given cell line, other markers were usually absent ot produced at relatively low levels. In no instance was a significant positive correlation found between two markers. These data indicated that, although most human malignant cells examined produced one or more oncodevelopmental gene markers at elevated levels, no predictable coexpression of any two of the markers was seen.

Effect of hypophysectomy on immunocytochemically demonstrated gonadotropin-releasing hormone in the rat brain.

The objective was to determine the effect of hypophysectomy on the store of gonadotropin-releasing hormone (GnRH) in certain parts of the brain as revealed by immunocytochemistry. The antiserum used was prepared against synthetic GnRH conjugated with limpet hemocyanin. No change was observed in the store of GnRH in the organum vasculosum of the lamina terminalis or in the cephalic segment of the median eminence GnRH was depleted severely from the central and caudal (junction with the infundibular stem) segments of the median eminence. GnRH was not found in the axons of magnocellular neurons that regenerate during repair of the median eminence-pituitary stalk after hypophysectomy.

Distribution of gonadotropin-releasing hormone in the rat brain as observed with immunocytochemistry.

The distribution of gonadotropin-releasing hormone (GnRH) was studied in the brain of adult female rats with three immunocytochemical techniques using antisera to unconjugated synthetic GnRH and to GnRH conjugated with limpet hemocyanin. GnRH was found in nervous tissue surrounding blood vessels of the organum vasculosum of the lamina terminalis. In the median eminence it occurred in nervous tissue associated primarily with the tuberoinfundibular sulci throughout their extent. Cephalic to the pars tuberalis GnRH often spread across the median eminence from sulcus to sulcus. Caudally, with widening of the median eminence, GnRH occurred dorsal to the tuberoinfundibular sulci, and especially in the external lamina medial to the sulci. A broad median zone of the median eminence was rather free of GnRH. GnRH was most concentrated in the region of continuity between the dorsolateral walls of the infundibulum and floor of the third ventricle where the tuberoinfundibular sulci are deep. Caudal to the infundibulum GnRH was disposed in a flat zone through the cephalic portion of the floor of the mammillary recess. In the median eminence GnRH appeared to be located in axons that terminated there. The amount of demonstrable GnRH varied significantly from rat to rat. The distributions of GnRH as revealed by use of antisera to unconjugated and conjugated GnRH were essentially the same. The apparent order of sensitivity of the immunocytochemical methods was: the peroxidase-antiperoxidase (PAP) (Sternberger et al.) procedure greater than the immunoglobulin-enzyme bridge (Mason et al.) procedure smaller than the conjugated antibody (Nakane and Pierce) procedure.

Analysis of the radioimmunoassay for gonadotropin-releasing hormone (GnRH): studies on the effect of radioiodinated GnRH.

When GnRH is radioiodinated by the chloramine-T method, two immunoreactive labeled species are formed at pH 6.5 with a chloramine-T: GnRH molar ratio of 11:1, whereas four bands (I, IIa, IIb, and III) are separated by polyacrylamide gel electrophoresis when the hormone is iodinated at pH 7.5 in a system containing a 97:1 molar ratio of chloramine-T:GnRH. Because they were more stable and were more immunoreactive than the other products, band I and band IIa from the latter system were used separately as tracers with Niswender antiserum R-42 in radioimmunoassays for GnRH. The standard curves of each tracer are distinct: when analyzed after log-logit transformation, the band I curve had a mean slope of -3.31 +/- 0.2 (SE) and a 50% B/Bt level of 9 +/- 0.8 pg (n=8) of synthetic GnRH, whereas the band IIa standard curve had a slope of -2.30 +/- 0.6 and a 50% B/Bt value of 20 +/- 0.9 pg (n=11). The sensitivity of both assays is approximately 2.0 pg. Gn RH concentrations in plasma and serum samples assayed with band I were consistently greater than those assayed with band IIa. Normal adult male plasmas assayed with band I measured 21 +/- 0.9 pg/ml, whereas band IIa values were 8 +/- 0.4 pg/ml. No difference between plasma and serum was detected, nor was there any difference among adult men, adult women, prepubertal children, hypogonadal patients, or hypopituitary patients with either assay. Plasma GnRH concentrations were also similar in jugular and vena cava samples from intact and castrated male rats. Because many of the samples were at or below the sensitivity of the band IIa assay, they were concentrated after extraction with either methanol or acid-ethanol. However, endogenous immunoreactive GnRH could not be concentrated by these extraction procedures. As measured in the band IIa assay, hypothalamic extracts from control adult male rats contained 3.1 +/- 0.4 ng while hypothalami from castrated rats contained 1.4 +/- 0.1 ng. Similar but slightly lower values were obtained with band I. In contrast, the GnRH content of pineal glands from intact and castrated male rats was similar (approximately 150 pg) when determined in either assay. These studies emphasize that: 1) the characteristics of the radioiodinated hormone can influence the quantitation of GnRH; and 2) endogenous plasma concentrations of GnRH are much lower than previously reported.

Characterization of calcitonin- and adrenocorticotropin-producing human cloned cell lines.

We have developed three human cloned cell lines that produce immunoreactive human calcitonin (ihCT) and ACTH (iACTH) as well as exhibit characteristics of cultured neural cells. Clones HMS-41/I, -78/2, and -98/2 were developed from cell lines HeLa AV3, MBA 9812 (bronchogenic carcinoma), and SW 267 (pheochromocytoma), respectively. Karyological analysis of both the parent and the cloned cell lines confirmed the identity of HeLa AV3 and MBA 9812. When grown in serum-free media designed for culturing neural cells, the patterns of production for both ihCT and iACTH varied among the clones. The multiple patterns of hormone production suggest that the mechanisms involved in the biosynthesis, processing, and secretion of these hormones differ among the clones. The clones contain neuron-specific enolase and the putative neurotransmitters beta-alanine and gamma-amino butyric acid, and they respond to cAMP analogs by differentiating, as noted by the extension of neurites (except the HeLa-derived HMS-41/I). The iACTH extracted from cells and synthetic ACTH exhibited equivalent profiles upon isoelectric focusing. The forms of ihCT noted in cell extracts were similar to those observed in extracts of human tumor tissue. Our rabbit antiserum to hCT failed to detect ihCT in those cell extracts prepared for ACTH determination or in extracts of rat pituitaries, but it did detect CT in rat thyroids by both RIA and immunofluorescent procedures. We concluded that our antisera to hCT do not detect the precursor form of ACTH. The availability of these cloned cell lines provides model systems for examining the production of these peptide hormones and the concomitant expression of neural and endocrine characteristics.

Synthesis and biological activity of peptide antagonists of luliberin (luteinizing hormone-releasing hormone).

A series of des-His2 octa- and nonapeptide analogues of luliberin (luteinizing hormone-releasing hormone) with modifications in the 1 and 6 positions, and in some instances the 10 position, has been prepared. Some of these analogues are potent inhibitors of luliberin in vitro and in vivo. The use of ultraviolet absorption measurements for evaluating peptides containing tyrosine and tryptophan is described. An efficient synthesis of O-methyl-d-tyrosine is reported.

Luteinizing hormone-releasing hormone versus human chorionic gonadotropin: differential effect on the development of ovulatory refractoriness and antibodies.

Cyclic intramuscular injections of 25 IU of human chorionic gonadotropin (HCG) at 3-week intervals induced ovulatory refractoriness and HCG antibodies after five to eight treatment cycles. Two of fifty rabbits failed to ovulate following two successive injections of luteinizing hormone-releasing hormone (LH-RH); however, no LH-RH antibodies were detected in the sera of these two animals, suggesting that these observations were due to chance alone. Thus, 0.5 mug of LH-RH injected intramuscularly at 3-week intervals did not induce ovulatory refractoriness or antibody formation after as many as 18 successive treatment cycles.

Potency of synthetic luteinizing hormone releasing hormone preparations in rat anterior pituitary cell cultures.

Selected synthetic luteinizing hormone releasing hormone preparations were assayed, and their potencies were determined relative to one sample utilizing primary cultures of enzymatically dispersed rat anterior pituitary cells. Preliminary cell culture experiments indicated that luteinizing hormone releasing hormone had to be in constant contact with cells for continued luteinizing hormone secretion. Luteinizing hormone levels in media reached a maximum concentration after 4 hr of continuous luteinizing hormone releasing hormone exposure. Cell culture bioassay was selected over the bioassay employing chronically ovariectomized steroid-blocked rats due to greater sensitivity and economy. The assay of each luteinizing hormone releasing hormone preparation was replicated four to seven times. Preparations from several companies were less potent (p less than 0.05) than the reference product. Contaminants were disclosed by TLC in preparations with potencies lower than the reference product.

Stimulation of LH release and ovulation in the rat by cutaneous application of luteinizing hormone-releasing hormone (LH-RH) in dimethyl sulfoxide (DMSO).

The efficacy of cutaneously applied luteinizing hormone releasing hormone (LH-RH) in stimulating LH release in the chronically ovariectomized, estrogen/progesterone-blocked rat and ovulation in the chlorpromazine-blocked, proestrous rat was investigated. Following the cutaneous application of 25 or 100 mug of LH-RH in 100% dimethyl sulfoxide (DMSO), serum LH rose to a peak at 1 hr, then declined toward basal levels in the ensuing 3 to 4 hr. LH-RH applied cutaneously in either 100% DMSO or 0.9% NaCl was also capable of inducing ovulation in the chlorpromazine-blocked, proestrous rat; however, LH-RH in 0.9% NaCl did not yield a linear log-dose-response relationship, therefore a valid ED50 potency ratio for DMSO/0.9% NaCl could not be estimated. Nonetheless, 0.9% NaCl was a considerably less effective cutaneous vehicle than DMSO. Cutaneous application of LH-RH in DMSO in doses of 100, 50, 25, 10, 5, 2.5 and 1 mug induced ovulation in 100%, 100%, 90%, 73%, 30%, 10% and 0% of the rats, respectively. When the percent ovulation response was transformed to probits and plotted against the logarithm of the LH-RH dose an approximately linear log dose-response was obtained; the same relationship also held true for ovulation induction following sc LH-RH administration. In turn, the ED50's of these two parallel dose-response curves yielded a potency ratio estimate (sc/cutaneous) of 0.025, suggesting that 2.5% of the cutaneously applied LH-RH dose was absorbed through the skin in a biologically active form. These data indicate that DMSO privdes a convenient cutaneous vehicle for the administration of LH-RH.

Heterogeneity of chloramine T- and lactoperoxidase-radioiodinated human calcitonin.

Radioiodination reportedly damages peptides, but the nature of the damage has not been adequately examined. Utilizing isoelectric focusing, we examined the products of Chloramine T- and lactoperoxidase-directed radioiodinations of human calcitonin. Initially, the reaction products were purified by adsorption onto and elution from microfine silica (QUSO-G32). Radioiodination of the calcitonin by Chloramine T and lactoperoxidase produced a heterogeneous population of 125I-labeled peptides exhibiting apparent isoelectric points that were more acidic than that of unlabeled synthetic calcitonin. Variation in the products among radioiodinations and the inability of QUSO-G32 to resolve the components of the reaction mixture prompted our examination of alternative purification procedures. Anion-exchange chromatography on QAE-Sephadex effectively separated [125I]diiodotyrosine containing calcitonin from free iodine and [125I]iodolactoperoxidase. Our data indicate that: (a) radioiodination of human calcitonin by Chloramine T and lactoperoxidase induced alteration in the peptide as evidenced by isoelectric point, (b) specific [125I]iodopeptides vary in incidence and relative abundance among radioiodinations, (c) identification of the labeled amino acid in [125I]iodopeptides cannot ensure intergrity of the molecule, and (d) isoelectric focusing provides a method of comparing the products of peptide radioiodinations among laboratories.

Multiple sequential biomarkers in monitoring patients with carcinoma of the lung.

To correlate serial biomarkers and disease activity in carcinoma of the lung, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), adrenocorticotropic hormone (ACTH), C3-derived protein (C3DP-C), and LDH were assayed in 43 patients with small cell lung carcinoma (SCLC) and in 20 patients with non-small cell lung cancer (NSCLC) (15 with adenocarcinoma, three with squamous cell carcinoma, and two with mixed histology). Disease status after treatment was rated as one of the following: complete response, partial response, minor regression, stable disease, and progressive disease. Significant correlations between disease status and markers in SCLC were found for CEA, NSE, LDH, and ACTH. In NSCLC, only CEA and LDH showed significant correlation. Marker-marker correlations were significant in SCLC for CEA and NSE (P less than 0.05), CEA and LDH (P = 0.01), and NSE and LDH (P less than 0.01); in NSCLC none were significant. None of the markers exhibited significant correlations with specific metastatic sites. Certain biomarkers (CEA, NSE, and LDH in SCLC; CEA and LDH in NSCLC) can be used alone or in combination to monitor disease activity but appear to be no more sensitive than standard clinical investigational methods.