Sankar P. Mitra

Neurotensin receptor expression in prostate cancer cell line and growth effect of NT at physiological concentrations

Neurotensin (NT), a neuroendocrine peptide, exerts trophic effects in vivo and stimulates growth of some tumor cells in vitro. Androgen‐sensitive prostate cells derived from lymph node carcinoma of the prostate (LNCaP) secrete NT and exhibit growth responses to NT. This study examines NT secretion, NT receptor and NT‐growth responses in androgen‐independent prostatic carcinoma (PC3) cells derived from prostate adenocarcinoma metastatic to bone.

Posttranslational processing of the neurotensin/neuromedin-N precursor.

Posttranslational processing can be a key regulatory step in determining the quantity and types of products derived from precursors to biologically active peptides. 1-4 Although the “limited proteolysis” involved in this maturation process usually occurs at double basic residues within the parent molecule, there are exceptions to this rule.5 Adding additional complexity is the fact that these reactions are often tissue-specific and that they can occur in an intraor extra-cellular manner.6.’ Furthermore, they can be coupled with a number of other modifications such as glycosylation, amidation, acetylation, pyrrolation, phosphorylation, and sulfation.* In regard to precursor processing for neurotensin (NT) and neuromedin N (NMN), we would ultimately like to address the following questions. What are the products of processing in various tissues and are any precursor-derived peptides, other than NT and NMN, biologically active? What enzymes mediate the maturation process, and where do the reactions take place? Are there instances where precursor processing is rate-limiting for operation of the NT/NMN system and is it, like gene expression, subject to hormonal regulation? This short summary reviews some of our recent efforts to gather information pertaining to a few of these issues.

Antiproliferative effects of c-myc antisense oligonucleotide in prostate cancer cells: A novel therapy in prostate cancer

OBJECTIVESnTo explore the possibility of using antisense oligonucleotide therapy for prostate cancer, we investigated the effect of c-myc-antisense-oligonucleotide (c-myc-As-ODN) in human prostate cancer cell lines such as LNCaP, PC3, and DU145.nnnMETHODSnLNCaP, PC3, and DU145 cells were incubated in the presence of c-myc-As-ODN. Dose (0 to 10 microM) and time dependent (1 to 6 days) effects on proliferation and viability were examined by [3H]thymidine incorporation and MTT assay, respectively. Flow cytometry analysis was carried out to analyze cell cycle status by determining the DNA content in LNCaP cells. Control cultures received either c-myc-sense-ODN or scrambled (nonsense) nucleotides.nnnRESULTSnTime- and dose-dependent decreases in DNA synthesis and cell viability were noted for all three prostate cancer cell lines after c-myc-As-ODN treatment. Further studies using LNCaP cells indicated that these changes were accompanied by an increase in the percentage of cells with less than 2N DNA content after c-myc-As-ODN treatment. The results suggest that c-myc-As-ODN induces cell death. Comparison of a c-myc-As-ODN-treated group with a group subjected to isoleucine deprivation revealed that thymidine incorporation was almost the same in c-myc-As-ODN-treated LNCaP cells and in LNCaP cells at early S phase.nnnCONCLUSIONSnThese results suggest that c-myc-As-ODN inhibits prostate cancer cell growth and proliferation mainly by decreasing cell viability.

Precursor forms of neurotensin (NT) in cat: processing with pepsin yields NT-(3-13) and NT-(4-13).

Basic proteins present in 0.1 N HCl extracts of feline CNS and intestine were found to liberate immunoreactive neurotensin (iNT) when treated with hog pepsin. These protein substrates were separated using Sephadex G-25, Sephadex G-75 and reverse-phase HPLC. In a calibrated SDS-polyacrylamide gel electrophoresis system, the major substrate from cat ileum exhibited a molecular weight of ca 16 kDa and minor substrates were observed at 30, 40 and 65 kDa. As shown previously for synthetic NT, pepsin-treatment of feline ileal NT converted it into the fully immunoreactive NT-(4-13) fragment (yield, 95%). When treated with pepsin, the partially purified ileal substrates gave rise to 4 immunoreactive peptides, one of which (ca 15% of total) eluted with the same retention time as NT-(4-13) while the major peptide formed (ca 40% of total) eluted near to the position of NT-(3-13). Both these products reacted equally well with two different antisera towards the C-terminal 5- and 8-residues of NT and were not recognized by an N-terminal antiserum. Experiments using various proteases demonstrated that the NT-related sequence(s) were located internally in each substrate and suggested that they were bounded by double basic residues. Substrate activity in isotonic homogenates of feline spinal cord, brain, adrenal and ileum cosedimented with iNT during equilibrium centrifugation, apparently in association with vesicle and/or synaptosomal particles. These findings indicate that basic proteins, colocalized with NT in vesicle-like particles of CNS, adrenals and ileum, could serve as precursors to this peptide, being liberated by pepsin-related enzyme(s).

Characterization of large neuromedin-N using antisera towards regions of the neurotensin/neuromedin-N precursor

Processing of the precursor to neurotensin/neuromedin-N was studied in brain and intestine from four mammalian species (dog, cat, guinea pig and rat) using previously characterized immunoassays for neurotensin and neuromedin-N, as well as newly developed assays towards the 35-44 sequence (P1) and the 70-85 sequence (P2) of the canine precursor. While neurotensin was the major product (approximately 98%) with neurotensin immunoreactivity in brain and ileum, a large molecular form of neuromedin-N was found to comprise 55-91% of the neuromedin-N activity in the ileum of these species and only 2-8% that in brain. Large neuromedin-N, which behaved as a single substance during multiple chromatographic steps, was found to cross-react in the assays for P1 and P2, indicating that this molecule extended at least from residues 40-148, neuromedin-N being located at its C-terminus. Western blots confirmed the results obtained by immunoassay. Partially purified preparations of large neuromedin-N from dog, cat and rat were also found to contract the isolated guinea pig ileum, exhibiting potencies near to that of neuromedin-N. These results indicate that tissue-specific storage of large neuromedin-N, a biologically active molecule with greater than 100 amino acids, occurs in these four mammals.

Purification of large neuromedin N (NMN) from canine intestine and its identification as NMN-125

A large molecular form of neuromedin N (NMN), a neurotensin (NT)-related peptide, was purified to homogeneity from canine ileal mucosa. The amino acid sequence of its N-terminal 20 residues was found to be SDSEEEMKALEADLLTNMHT, which is identical to residues 24-43 of the cDNA-predicted NT/NMN precursor. Prior work had indicated that the NMN moiety was located at the C-terminus of large NMN. Having now defined both the N-terminus and C-terminus of large NMN, we note that this molecule must be comprised of 125 amino acids (including residues 24-148 of prepro NT/NMN) and we suggest that it be named NMN-125. This information also defines the signal peptide cleavage site as the cysteine-serine bond within the precursor. The amino acid composition of the isolated peptide and its molar extinction coefficient for absorbance at 280 nm were similar to those for the 24-148 segment of prepro NT/NMN. However, the empirically determined molecular weight (17 kDa) and the isoelectric point (pI = 5.4) were slightly higher than those predicted solely from the peptides amino acid content (14.43 kDa and pI = 4.65, respectively). In total, these results indicate that the major NMN-related peptide found in canine ileum is 125 residues in length, extending from the putative signal peptide to the C-terminus of NMN, and that it might contain non-amino acid substituents.

Effects of GTP analogs and metal ions on the binding of neurotensin to porcine brain membranes

Using 125I-labeled neurotensin (NT), porcine brain membranes were found to contain two types of high-affinity receptors, one class (approximately 1/3 of total) with an apparent Kd of 0.12 nM and another with an apparent Kd of 1.4 nM. Nonhydrolyzable analogs of GTP inhibited NT binding in a dose-dependent manner. In the presence of 60 microM guanosine 5-(3-thio) 5-(beta, gamma-imino) triphosphate. NT binding was decreased by 35% with an associated decrease in the number of binding sites and little change in the Kd. Cross-linking of 125I-labeled NT to brain membranes using disuccinimidyl suberate was found to specifically label two substances of approximately 120 kDa and approximately 160 kDa, which could represent different binding proteins or complexes. For a series of NT analogs, there was close agreement between the IC50 in the binding assay and the ED50 in a bioassay based on ability to contract the guinea pig ileum. In addition, metal ions inhibited NT binding and the contractile action of NT with the same order of potency (Hg++ > Zn++ > Cu++ > Mn++ > Mg++ > Li++). There was a linear relationship between the standard reduction potential for these ions and the logarithm of the IC50 in the binding assay. The results suggest that porcine brain contains high-affinity, G-protein-linked receptors for NT, the functioning of which depends upon group(s), perhaps sulfhydryl(s), which can interact strongly with certain heavy metal ions.

Neurotensin enhances agonist-induced cAMP accumulation in PC3 cells via Ca2+-dependent adenylyl cyclase(s)

A human prostate cancer cell line (PC3) with abundant neurotensin (NT) receptors was used to demonstrate that NT potentiated 3,5-cyclic adenosine monophate (cAMP) accumulation in response to a variety of stimuli, including both direct forskolin (F) and indirect (prostaglandin, (PGE2), isoproterenol (ISO) and cholera toxin (CTx)) activators of adenylyl cyclase. Several mechanisms were investigated and our results indicated an effect on the rate of cAMP formation and not on degradation or extrusion. For each stimulus, NT enhanced efficacy without altering EC50. The effect of NT did not involve stimulatory G-protein (Gs)-activation or interference with a tonic inhibitory G-protein (Gi)-mediated inhibition. A similar response was obtained when NT was added with the stimulus or given as a two minute pulse which was removed prior to addition of stimulus. The potentiating activity disappeared with a t1,2 of approximately 15 min. NT transiently elevated cellular [Ca2+]i and its effects on cAMP could be mimicked by [Ca2+]i-elevating agents (uridine triphosphate (UTP), thapsigargin and ionomycin). Buffering cellular [Ca2+]i with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA-AM) inhibited cAMP responses to ISO and F in presence and absence of NT. These data support the idea that NT potentiated cAMP formation in response to a variety of stimuli by facilitating the activation of Ca2+ -dependent adenylyl cyclases.

Xenopsin-related peptide generated in avian gastric extracts

Two avian counterparts to amphibian xenopsin have been identified as H-Phe-His-Pro-Lys-Arg-Pro-Trp-Ile-Leu-OH (XP-2) and its partial sequence H-His-Pro-Lys-Arg-Pro-Trp-Ile-Leu-OH (XP-1) isolated from extracts of turkey proventriculus and skin. Both peptides were shown to be present within these and other tissues primarily (99%) in precursor form(s), from which they were liberated by the action of endogenous enzyme(s) during extraction. Synthetic and native preparations of XP-2 increased vascular permeability in rats and released histamine from isolated rat mast cells at submicromolar concentrations. The ubiquitous distribution of this XP-related sequence and its pharmacologic capabilities suggest potential roles in the general regulation of tissue blood flow and fluid exchange.

Binding and biologic activity of neurotensin in guinea pig ileum

Experiments were performed to relate receptor binding to biologic activity for the contractile effect of neurotensin (NT) in guinea pig ileum. The contractile response was examined on pieces of ileum under 1 g tension in a 5 ml bath of oxygenated Tyrodes at 38 degrees C. NT contracted the longitudinal muscle (ED50, approximately 0.3 nM), the 2-3 g response peaking at 1 min and fading rapidly. In the presence of atropine (1 microM), > or = 50% of the response was blocked and the residual effect gave an ED50 of approximately 1.4 nM. In the presence of atropine and CP-96,345, a substance P receptor antagonist (0.2 microM), no contraction was observed at 20 nM NT. Thus, there were two components to the response, one involving acetylcholine (ED50, 0.3 nM) and one substance P (ED50, 1.4 nM). Using membrane preparations and 125I-labeled NT, specific, high affinity receptors for NT were demonstrated in the muscle and myenteric plexus. Scatchard analyses indicated the presence of two binding sites (Kds: approximately 0.1 nM and approximately 2 nM). Sodium ion and GTP analogs inhibited binding. Binding and biologic activity were similar in regard to dependence on specific groups within NT and sensitivity to metal ions. The high potency of Hg++ was consistent with an involvement of free sulfhydryl group(s) in the binding reaction; this was supported by work with SH-directed agents. The results suggest that two receptor types or configurations may mediate the two components of the contractile effect of NT on guinea pig ileum.