Mean Maximal Binding Capacity Research Articles

Effective treatment of H838 human non-small cell lung carcinoma with a targeted cytotoxic somatostatin analog, AN-238

The accumulation of radioactive somatostatin analog [111In]pentetreotide in non-small cell lung cancer (non-SCLC) during scintigraphy of patients provides a rationale for investigating the efficacy of somatostatin receptor-based chemotherapy in non-SCLC. Consequently, in this study, we evaluated the antitumor effects of cytotoxic somatostatin analog AN-238 on H838 human non-SCLC xenografted into nude mice in comparison with its cytotoxic radical, 2-pyrrolinodoxorubicin (AN-201). The expression of messenger RNA (mRNA) for human somatostatin receptor subtypes 2 (hsst2) and 5 (hsst5) in H838 cells, and tumors was also investigated using reverse-transcription polymerase chain reaction (RT-PCR). Somatostatin receptors on H838 tumors were characterized by ligand competition assay using radiolabeled somatostatin analog, RC-160. Three i.v. injections of AN-238 at 150 nmol/kg, given on days 1, 7 and 21, resulted in a significant (p<0.05) tumor growth inhibition, the final tumor volume being 60% smaller than in the controls. The tumor doubling time was also extended significantly (p<0.05) from 9.65+/-0.56 days in the controls to 17.52+/-3.3 days. Only one of 8 mice died due to toxicity. In contrast, cytotoxic radical AN-201 was ineffective and more toxic, killing 2 of 7 animals. mRNA for hsst2 was found in H838 xenografts, but not in H838 cells from which the xenografts originated. Interestingly, H838 cells grown in a special, serum-free medium did express mRNA for hsst2. mRNA for hsst5 was not found in any samples tested. Binding studies demonstrated the presence of high affinity (K(d) = 7.3+/-1.2 nM) binding sites for RC-160 with a mean maximal binding capacity (B(max)) of 953.3+/-45.3 fmol/mg protein. AN-238 at 3.14+/-0.93 nM concentration displaced 50% of radiolabeled RC-160 binding to somatostatin receptors in H838 tumors. Our results indicate that patients with inoperable non-SCLC may benefit from chemotherapy targeted to somatostatin receptors based on AN-238.

Expression of growth hormone-releasing hormone and its receptor splice variants in human prostate cancer.

Antagonists of GHRH inhibit the growth of various human tumors, including prostate cancer, but the tumoral receptors mediating the antiproliferative effect of GHRH antagonists have not been clearly identified. Recently, we demonstrated that human cancer cell lines express splice variants (SVs) of receptors for GHRH, of which SV1 exhibits the greatest similarity to the pituitary GHRH receptors. In this study we investigated the expression of GHRH and SVs of GHRH receptor and the binding characteristics of the GHRH receptor isoform in 20 surgical specimens of organ-confined and locally advanced human prostatic adenocarcinomas. The mRNA expression of GHRH and SVs of GHRH receptor was investigated by RT-PCR. The affinity and density of receptors for GHRH were determined by ligand competition assays based on binding of (125)I-labeled GHRH antagonist JV-1-42 to tumor membranes. Twelve of 20 tumors (60%) exhibited specific, high affinity binding for JV-1-42, with a mean dissociation constant (K(d)) of 0.81 nmol/liter and a mean maximal binding capacity of 185.2 fmol/mg membrane protein. The mRNA of SV1 was detected in 13 of 20 (65%) prostate cancer specimens and was consistent with the presence of GHRH binding. RT-PCR analyses also revealed the expression of mRNA for GHRH in 13 of 15 (86%) prostatic carcinoma specimens examined. The presence of GHRH and its tumoral receptor SVs in prostate cancers suggests the possible existence of an autocrine mitogenic loop. The antitumor effects of GHRH antagonists in prostate cancer could be exerted in part by interference with this local GHRH system.

High Expression of Somatostatin Receptors and Messenger Ribonucleic Acid for Its Receptor Subtypes in Organ-Confined and Locally Advanced Human Prostate Cancers1

To evaluate the potential application of somatostatin (SST) analogs as an adjuvant treatment for prostate cancer, we characterized the binding sites for SST octapeptide analogs on prostate cancers in patients treated with radical prostatectomy. The affinity and density of binding sites for SST analog RC-160 on 80 surgical specimens of prostate cancers were determined by ligand competition assays. The expression of messenger ribonucleic acid (mRNA) for SST receptor subtype 1 (SSTR1), subtype 2 (SSTR2), and subtype 5 (SSTR5) was also investigated in 22 samples by RT-PCR. Fifty-two of 80 specimens (65%), showed a single class of specific binding sites for RC-160 with a mean dissociation constant (K(d)) of 9.44 nmol/L and a mean maximal binding capacity of 754.8 fmol/mg membrane protein. The mRNA for SSTR1 was detected in 86% of samples, whereas the incidences of mRNA for SSTR2 and SSTR5 were 14% and 64%, respectively. The expression of SSTR2 and/or SSTR5 was 100%, consistent with the presence of RC-160 binding. In patients at high risk of cancer recurrence (stage pT3 and/or Gleason score of 8-10), the incidence of RC-160 binding (65.7%) was similar to that observed in the low risk group (64.3%). The demonstration of the high incidence of octapeptide-preferring SSTRs in organ-confined and locally advanced prostate cancers supports the merit of further investigations of the application of SST analogs and their radionuclide and cytotoxic derivatives for adjuvant treatment of patients at high risk of cancer recurrence after radical prostatectomy. Such approaches could be also considered for patients with advanced prostate cancer at the time of relapse.

Characterization of bombesin/gastrin-releasing peptide receptors in membranes of MKN45 human gastric cancer

Binding of the radiolabeled bombesin analog [ 125I-Tyr 4]bombesin to crude cell membranes of MKN45 human gastric cancer grown in nude mice was investigated in vitro. Scatchard analyses of multipoint binding data, performed by complete displacement method demonstrated the presence of two classes of [Tyr 4]bombesin binding sites. The high-affinity binding sites had a mean dissociation constant ( K d1) of 2.75 nM with a mean maximal binding capacity ( B max1) of 492 fmol/mg membrane protein, while the low-affinity binding sites showed a mean dissociation constant ( K d2) of 0.41 μM with a mean maximal binding capacity ( B max2) of 41.4 pmol/mg membrane protein. Binding of [ 125I-Tyr 4]bombesin was specific, reversible and linearly related to the protein concentration of tumor membrane. In displacement studies, the binding of radiolabeled [Tyr 4]bombesin was inhibited in a dose-dependent manner by gastrin releasing peptide (GRP)(14–27) and two synthetic antagonists of bombesin/GRP, RC-3095 and RC-3950-II. Both antagonists exhibited high affinity in nearly the same concentration range as GRP(14–27). The presence of receptors for bombesin/GRP on human gastric cancer membranes suggests that bombesin-like peptides may play a role in growth of gastric cancer.

Alterations of human placental epidermal growth factor receptor in intrauterine growth retardation.

We studied human placental microvillous EGF receptor (EGFR) and its relationship with maternal and placental features in 14 cases of intrauterine growth retardation. Placental EGFR phosphorylation was significantly decreased or absent in 12 cases of small for gestational age neonates, as shown by SDS-PAGE, autoradiography, and scanning analysis. Specific [125I]EGF binding and Scatchard plots of the binding data showed a decreased number of EGFR in 6 of the 12 cases, with a mean maximal binding capacity of 1.09 +/- 0.32 pmol/mg for high affinity sites (mean control value = 2.30 +/- 0.23 pmol/mg). Most of the hypertensive women and smokers belonged to this subgroup. In three of the remaining six cases of small gestational age placentas with low EGFR phosphorylation, there was no maternal pathology or significant parenchymatous placental lesions. Five showed a 175-kD EGFR species when probed by [125I]EGF cross-linking and Western blotting with RK2 and C-Term, two polyclonal anti-EGFR antibodies, suggesting abnormal transduction of the EGF-induced signal. The sixth placenta yielded a single 145-kD EGFR band consistent with an abnormal EGFR structure; Western blot analysis showed no immunoreactive band. In conclusion, maternal and placental pathologies in intrauterine growth retardation are associated with various alterations of placental EGFR, pointing out the importance of EGFR ligands in the regulatory pathway of placental and fetal growth.

Abnormal Induction of Aromatase Activity by Dexamethasone in Fibroblasts from a Patient with Cortisol Resistance*

Cortisol resistance is a rare condition due to abnormal glucocorticoid receptor function. Stimulation of aromatase activity by dexamethasone (DEX) in cultured human skin fibroblasts provides a model for studying the biological activity of glucocorticoid receptors in cells. Skin fibroblasts derived from an affected father and his less severely affected son with cortisol resistance were used for this study. Saturation analysis of DEX receptor binding was performed after incubation of cells with various DEX concentrations (1-50 nmol/L). In normal cells, the mean maximal binding capacity (Bmax) and dissociation constant (Kd) were 24 +/- 3 pmol/mg DNA and 14 +/- 3 nmol/L, respectively. Although the Bmax in cells of the father (33 +/- 2 pmol/mg DNA) was normal, the Kd (31 +/- 7 nmol/L) was abnormally elevated. By contrast, both the Bmax and Kd in cells of the son were normal. The dose response of aromatase activity to DEX stimulation was determined by assay of aromatase activity after incubation of cells in the absence or presence of DEX (0.25-500 nmol/L) for 14 h. In three strains of normal fibroblasts, the mean concentration of DEX that produced a half-maximal response was 6 +/- 1 nmol/L). In cells from the father, the mean concentration of DEX that produced half-maximal stimulation of aromatase (27 +/- 4 nmol/L) was abnormally elevated. By contrast, the concentration of DEX that half-maximally stimulated aromatase activity (6.0 and 5.9 nmol/L) was normal in cells from the son. These data provide additional evidence of abnormal glucocorticoid action in the father, but not in his son, and demonstrate the potential usefulness of determining aromatase induction by DEX as a means of assessing the biological activity of the glucocorticoid receptor.

α-Human atrial natriuretic polypeptide binding sites in human adrenal membrane fractions

In a previous study evidence was presented that synthetic α-human atrial natriuretic polypeptide (α-hANP) significantly inhibits the secretion of aldosterone, cortisol, and dehydroepiandrosterone (DHEA) from cultured human adrenal cells. In the present work using crude membrane fractions prepared from human adrenal tissues obtained at autopsy, we noted the existence and molecular weight of specific binding sites for [ 125I]α-hANP. The mean maximal binding capacity ( B max) and dissociation constant ( K d) of 4 human adrenal membrane fractions were 8.0 ± 1.6 fmol/mg protein and 25.7 ± 7.4 pM, respectively, as calculated by Scatchard plot analysis. The interaction of [ 125I]α-hANP with the high-affinity binding sites in human adrenal membrane fractions was unaffected by the addition of lysine vasopressin (LVP), somatostatin-14 and angiotensin-II (A-II). When the membrane fractions were incubated with [ 125I]α-hANP and then cross-linked with disuccinimidyl suberate (5 mM), the 67,000-Da protein was specifically radiolabeled. The very high affinity of [ 125I]α-hANP binding sites suggests that human adrenal steroidogenesis may be influenced by plasma levels of hANP, under physiological conditions.

Characterization of growth hormone-releasing hormone receptors in pituitary adenomas from patients with acromegaly.

GHRH receptors in pituitary adenoma cell membranes from five patients with acromegaly were characterized using [125I] [His1,Nle27]GHRH-(1-32)NH2 ([125I]GHRHa) as a ligand. Specific binding of [125I]GHRHa to adenoma cell membranes was maximal within 20 min at 24 C, remained stable for 60 min, and was reversible in the presence of 500 nmol/L human GHRH-(1-44)NH2 (hGHRH). The specific binding increased linearly with 10-160 micrograms cell membrane protein. This binding was inhibited by 10(-11)-10(-6) mol/L hGHRH in a dose-dependent manner, with an ID50 of 0.20 nmol/L, but not by 10(-7) mol/L vasoactive intestinal peptide, glucagon, somatostatin-14, somatostatin-28, TRH, LHRH, and CRH. The specific binding of [125I]GHRHa to the membranes was saturable, and Scatchard analysis of the data revealed an apparent single class of high affinity GHRH receptors in five adenomas from acromegalic patients; the mean dissociation constant was 0.30 +/- 0.07 (+/- SE) nmol/L, and the mean maximal binding capacity was 26.7 +/- 7.0 (+/- SE) fmol/mg protein. In three nonfunctioning pituitary adenomas, GHRH receptors were not detected. The plasma GH response to hGHRH (100 micrograms) injection was studied in four acromegalic patients before surgery. Plasma GH levels increased variably in response to hGHRH injection in all four patients. However, there was no correlation between the characteristics of the tumor GHRH receptors and plasma GH responsiveness in these patients. We conclude that pituitary GH-secreting adenomas have specific GHRH receptors. Exogenously administered GHRH presumably acts via these receptors, but the variations in plasma GH responsiveness to hGHRH in these patients cannot be directly related to the variations in binding characteristics of the GHRH receptors on the GH-secreting adenoma cells.

Plasma growth hormone responses to somatostatin (SRIH) and SRIH receptors in pituitary adenomas in acromegalic patients.

The plasma GH response to somatostatin (SRIH) infusion and SRIH receptors in pituitary adenoma cell membranes were investigated in six acromegalic patients. Infusion of 0.3 and 1.0 microgram/kg . h SRIH increased plasma SRIH concentrations in these patients in a dose-related manner. In five of the six patients, mean plasma GH levels decreased to 65.5 +/- 5.0% (+/- SEM) and 43.7 +/- 3.1% of the basal level when 0.3 or 1.0 microgram/kg . h SRIH was infused, respectively. In the remaining patient, plasma GH levels did not change, even when a larger dose of SRIH was infused. High density and specific SRIH receptors, with a mean dissociation constant of 0.92 +/- 0.17 nM and a mean maximal binding capacity of 523.8 +/- 174.6 fmol/mg protein, were identified in GH-secreting adenomas from the five SRIH-responsive patients. On the other hand, in the adenoma from the SRIH-nonresponsive patient, the maximal binding capacity (40.5 fmol/mg protein) was as low as those of nonfunctioning adenomas, as reported previously (undetectable to 48.0 fmol/mg protein). We conclude that the differential responses of plasma GH to SRIH in acromegalic patients may be related to variations in the binding capacity for SRIH in adenoma cell membranes.

Modulation of myocardial L-triiodothyronine receptors in normal, hypothyroid, and hyperthyroid rats

To characterize the nuclear receptor believed to mediate the thyroid hormones' actions on the heart, binding of L-[ 125I]T3 to extracts of myocardial cell nuclei from normal, propylthiouracil, and T4-treated rats has been investigated. Analysis of in vitro iodothyronine binding to this solubilized nuclear preparation revealed multiple saturable, specific binding sites for T3. High affinity binding for T3 (Kd = 4.2 ± 1.0 × 10 −10 mol/L), and lower affinity (Kd ∼ 10 −8 mol/L) binding activity appeared to be independent (Hill plot slope = 1). The mean maximal binding capacity of the high affinity binding site for T3 in euthyroid rats (84 ± 8 fmol/mg DNA) was increased by approximately 50% in hypothyroidism (120 ± 6 fmol/mg DNA) and unchanged in hyperthyroidism (88 ± 25 fmol/mg DNA). The molecular weight of this T3 binding site is estimated to be 50,000–55,000 daltons. The properties of this solubilized binding activity from rat myocardial nuclei are consistent with its putative role as the biologic thyroid hormone receptor. The increase in binding capacity with hypothyroidism suggest regulation by thyroid hormone of its nuclear receptor in myocardium.

3H]‐tetracaine binding on rat synaptosomes and sodium channels

[3H]-tetracaine binding was studied in a rat synaptosomal preparation. [3H]-tetracaine bound to a single class of binding sites with a mean KD of 188 +/- 28 nM and a mean maximal binding capacity of 13 +/- 0.7 pmol mg-1 protein. [3H]-tetracaine binding was inhibited by tetracaine, procaine and by beta-adrenoceptor blocking agents which possess local anaesthetic properties. [3H]-tetracaine binding was not modified by neurotoxins interacting specifically with the sodium channels.

In vitro 3,5,3'-triiodothyronine binding to rat cerebrocortical neuronal and glial nuclei suggests the presence of binding sites unavailable in vivo.

The maximal binding capacity (MBC) of the rat cerebrocortical nuclear T3 receptor, as determined by in vivo saturation techniques, is approximately half that measured in vitro on isolated nuclei or solubilized receptors. To investigate this disparity, the MBC values determined in vivo and in vitro for both rat cerebral cortex and liver were compared, taking into account nuclear receptor loss or inactivation and the presence of endogenous T3. By Scatchard analysis of T3 binding to isolated nuclei in vitro at 37 C, the uncorrected MBC values (mean +/- SEM; n = 3) for the cerebrocortical nuclear T3 receptor in euthyroid and hypothyroid rats were 0.80 +/- 0.14 and 0.66 +/- 0.07 ng T3/mg DNA, respectively, and were not significantly different. The Kd values were also not significantly different (5.6 +/- 0.3 and 5.2 +/- 0.9 X 10(-10) M, respectively). After corrections for incomplete dissociation and receptor inactivation under the in vitro conditions, the overall mean MBC increased by approximately 33% to 0.97 ng T3/mg DNA, or about 3.6 times the in vivo MBC. In addition, cerebrocortical nuclei prelabeled in vivo with +/- 131I]T3 at near-saturating levels and subsequently incubated with [125I]T3 in vitro at concentrations up to 10 times the Kd were shown to bind as much as 4 times more T3 in vitro relative to the amount of endogenous hormone which dissociated, thus exceeding the in vivo MBC by a factor of two. Parallel experiments with isolated liver nuclei did not show the existence of nuclear T3 receptors which were available only in vitro, even when the corrected MBC (0.77 ng T3/mg DNA) was compared with the MBC obtained by the in vivo saturation technique (0.76 ng T3/mg DNA). The experiments with liver nuclei were done at 25 C to reduce the rate of inactivation or loss of nuclear T3 receptors in this tissue. By fractionating isolated cerebrocortical nuclei into neuronal and glial subpopulations on discontinuous sucrose gradients, the high affinity, limited capacity nuclear T3 receptor could only be identified in the neuronal fraction. No consistent specific binding of T3 was observed in glial nuclei that were 80% pure, suggesting that either glial cells in the adult rat are not likely to be direct targets of thyroid hormone or that thyroid hormone may act via nonnuclear receptor-mediated pathways. We conclude that only neurons have specific high affinity, limited capacity nuclear T3 receptors and that as many as half of these receptors may not be accessible to plasma T3.(ABSTRACT TRUNCATED AT 400 WORDS)

3H]Vasopressin binding to rat hippocampal synaptic plasma membrane : Kinetic and pharmacological characterization

Characterization of specific vasopressin binding sites to rat hippocampal membranes has been assayed using tritiated lysine-vasopressin labelled on the tyrosyl residue. At 30°C specific [ 3H]vasopressin binding was saturable. The estimated equilibrium dissociation constant was 7.1 nM, the mean maximal binding capacity was 78 fmol/mg protein. Arginine-vasopressin has a high affinity ( K d = 2.8 nM) and dDAVP has a low affinity ( K d = 249 nM) for hippocampal synaptic membranes. (OH)AVP and Phe 2Orn 8VT are at least as active as AVP in inhibiting [ 3H]vasopressin binding. Adenylate cyclase was activated by VIP and inhibited by PIA, but not affected by lysine-vasopressin.

Triiodothyronine binding to lymphocyte nuclei and plasma cyclic AMP response to intravenous glucagon in patients with peripheral resistance to thyroid hormones

In vitro studies of nuclear binding of triiodothyronine (T3) in lymphocytes were performed in three members of a family with hereditary peripheral resistance to thyroid hormone action. Ficoll-Hypaque purified lymphocytes were used; the binding characteristics were analyzed by Scatchard's methods. In 5 euthyroid subjects the apparent mean equilibrium association constant (Ka) was 6.2 x 10(9) l/mol and the mean maximal binding capacity (Cap) 14.4 x 10(-15) mol/100 microgram DNA. In the 3 members of the family one single set os saturable T3 nuclear binding sites with affinity constants similar to those in the controls (mean Ka = 3.2 x 10(9) l/mol; mean Cap = 17.4 x 10(-15) mol/100 microgram DNA) were found. The glucagon stimulated increase in plasma cyclic AMP was studied in 6 healthy subjects and the four members of the family. The plasma cyclic AMP levels of the patients with hormone resistance were generally within the normal range. These observations demonstrate that in these patients with peripheral resistance to thyroid hormone binding of T3 to the receptor in the nucleus of lymphocytes is normal; in relation to the high circulating thyroid hormone levels, the thyroid hormone mediated cyclic AMP response is disturbed, suggesting that the defect is at the post-receptor effector level.

Triiodothyronine binding to lymphocytes from euthyroid subjects and a patient with peripheral resistance to thyroid hormone.

Triiodothyronine (T3) binding to Ficoll-Isopaque purified human lymphocytes was studied. During incubation of lymphocytes with [125I]T3 in a calcium-free medium at 37 degrees C, maximal uptake of T3 in nuclei occurrred after 2 h and declined after prolonged incubationd incubation . Incubation of lymphocytes with T3 concentrations ranging from 1 X 10(-11) TO 1 X 10(-9) mol/l and subsequent treatment with Triton X-100 to strip off [125I]T3 bound with low affinity was used for the estimation of affinity and capacity of nuclear T3 binding sites. The mean equilibrium affinity constant (Ka) estimated with the Scatchard method in 11 euthyroid healthy subjects was 4.5 X 10(9) l/mol, and the mean maximal binding capacity 25 X 10(-5) mol/100 mug DNA. In a female patient with peripheral resistance to thyroid hormone action, the estimated Ka was 3.5 X 10(9) l/mol and the number of T3 binding sites 37 X 10(-15) mol/100 mug DNA. Although not statistically different from the mean value in euthyroid subjects, this Ka value was outside the range of control values observed and was considered presumptive evidence that the nuclear T3 receptors in this patient have abnormally low affinity for its ligand. The nuclear T3 binding capacity in this patient was significantly increased.