Selective anticancer strategies via intervention of the death pathways relevant to cell transformation.

Apoptosis is an important physiological process that promotes tissue homeostasis by eliminating unnecessary or malfunctioning cells. Abnormality in this process contributes to tumorigenesis, as well as the resistance to cancer treatment by radiation and chemotherapy. Restoration of normal apoptosis would not only promote cancer cell death and halt tumor progression, but also increase the response to many current cancer therapies. Although apoptosis induction is an important principle of currently used radiation and chemotherapy treatment, uncovering the mechanisms that govern this process, and which are lost during transformation, represents an important direction for realizing improved therapies for the future. This article first briefly reviews aspects of current discovery strategies for new anticancer therapeutics based on intervening in cell death pathways, and then discusses in more detail several cancer-relevant death pathways, which are disabled during transformation and which can be targeted therapeutically. These include anoikis/cell adhesion; energy metabolism and the unfolded protein response. Finally, we introduce a new concept, which utilizes cancer-specific apoptosis induced by oncolytic viruses. The discussion of these topics involves novel targets, compounds and virotherapy.

Involvement of a metalloprotease in low-affinity nerve growth factor receptor truncation: inhibition of truncation in vitro and in vivo.

The mechanism of low-affinity NGF receptor (LNGFR) truncation was investigated in cultured Schwann cells. Affinity labeling of Schwann cells with 125I-NGF or metabolic labeling with 35S-cysteine showed that truncated NGF receptor (NGF-Rt) was derived from the cell surface form of the receptor. Addition of full-length, exogenous NGF receptor (M(r) = 80 kDa) to Schwann cell membranes resulted in cleavage of the exogenous substrate to NGF-Rt. Investigations into the mechanism of truncation revealed that metalloprotease inhibitors such as phenanthroline, bathophenanthroline, and 8-hydroxyquinoline (8-OHQ) blocked LNGFR truncation in a concentration-dependent fashion. Inhibitors of other protease classes had no effect on truncation. In addition, truncation did not occur at 4 degrees C. It was found that truncation could also occur in Schwann cell membrane preparations, indicating that the putative protease was membrane bound and closely associated with the LNGFR. Metal reconstitution experiments revealed a strong preference toward zinc for the truncating activity, with iron and manganese having slight reconstitution activity in phenanthroline-quenched membranes. To determine if apparent truncation could be inhibited in vivo, the metalloprotease inhibitor 8-OHQ was administered to neonatal rats. 8-OHQ resulted in decreased urine and blood NGF-Rt levels and increased the sciatic nerve LNGFR content; this effect was dose dependent. In adult rats with sciatic nerve crush lesions, 8-OHQ (30-300 mg/kg, t.i.d.) significantly enhanced the rate of sensory neuron regeneration as assessed by the nerve pinch assay. This was accompanied by increased levels of LNGFR in distal nerve segments. These results suggest that Schwann cells possess a metalloprotease-like activity that serves to cleave LNGFR from the surface of these cells. We propose that the putative metalloprotease represents a novel mechanism by which the Schwann cell regulates this particular cell surface protein. Furthermore, increasing the amount of Schwann cell surface LNGFR appears to be of functional significance in that sensory nerve regeneration can be enhanced by inhibition of truncation.

Actions of A-75200, a novel catecholamine uptake inhibitor, on norepinephrine uptake and release from bovine adrenal chromaffin cells.

The balance between catecholamine (CA) release and reuptake is closely regulated and determines the effective level of transmitter at the synaptic cleft. Drugs that block CA uptake have potential utility as antidepressant medications. One such drug is racemic (+/-)-(1' R*,3R*)-3-phenyl-1-[1',2',3',4'-tetrahydro-5',6'- methylenedioxy-1'-naphthalenyl-methyl]-pyrrolidine methanesulfonate (A-7500), a novel polycyclic compound developed at Abbott Laboratories. This compound is known to bind to CA transporters in the central nervous system, however, its effects on an intact neurosecretory system have not been studied. In this regard, norepinephrine (NE) release from bovine adrenal chromaffin cells (BACC) is a classic model system for CA release and is an excellent system in which to examine the effects of drugs which modulate neurotransmitter release. We compared the effects of A-75200 and its two constituent enantiomers, A-74111 and A-74112, to the effects of three well-characterized uptake inhibitors, desipramine (DMI), nomifensine and cocaine. We found that the Abbott compounds inhibit [3H]norepinephrine ([3H]NE) uptake with an EC50 comparable to cocaine. In addition, unlike nomifensine and cocaine, these compounds inhibited nicotine- and K(+)-stimulated NE release, whereas histamine-stimulated release was preserved. Thus, the Abbott compounds block the effects on secretion of two agonists (nicotine and K+) which depend on a depolarization-dependent influx of extracellular calcium. We conclude that in addition to blocking NE uptake by inhibiting the NE transporter, the Abbott compounds may modulate peripheral NE release by inhibiting calcium flux through voltage-gated channels. This study demonstrates the utility of bovine adrenal chromaffin cells for preclinical trials of drugs that affect catecholaminergic neurotransmission.

In vivo biosynthesis and transport of oxytocin, vasopressin and neurophysin from the hypothalamus to the spinal cord.

The biosynthesis of oxytocin, vasopressin and their associated neurophysins were studied in the projection from the paraventricular nucleus of the hypothalamus to the spinal cord in individual freely-moving adult male rats. Neuropeptide biosynthesis was studied in vivo by the delivery of [35S]cysteine through stereotaxically implanted indwelling cannulae using an osmotic minipump delivery system. Following the appropriate chase times, the neural lobe and spinal cord segments T1-T4 and T12-L2 were removed from fresh tissue; in addition, the nucleus of the solitary tract was punched from frozen coronal sections. The radiolabeled peptides were purified from the tissue homogenates by sequential linear and exponential gradient elution from reverse-phase high performance liquid chromatography columns. This approach has allowed us to purify radiolabeled oxytocin and vasopressin from both the upper and lower spinal cord. However, the kinetics of oxytocin and vasopressin biosynthesis appeared to be remarkably different, as judged by their differential labeling with different pulse and chase times. Additionally, the use of different chase periods following the pulse of radiolabel has allowed us to determine that oxytocin reaches the spinal cord via the fast component of axonal transport (greater than 8 mm h-1). Using immunoprecipitation and purification by high performance liquid chromatography, we were also able to purify radiolabeled neurophysins from spinal cord tissue homogenates. These results lend further support to a role for oxytocin and vasopressin in the modulation of autonomic nervous system function and to the role of the paraventricular nucleus as an integration center for endocrine and autonomic function.

Enkephalin biosynthesis and processing during lactation.

The biosynthesis of three Met-enkephalin-containing peptides was studied in the hypothalamo-hypophysial system of normal and lactating female rats by the techniques of in vivo radiolabeling and sequential high-performance liquid chromatographic purification of radiolabeled peptides. Met-enkephalin biosynthesis was stimulated in the lactating animals; however, the products derived from the post-translational processing of the enkephalin precursor appeared to remain constant between the two states, as judged by the ratio of radioactivity present in Met5-enkephalin, Met5Arg6Gly7Leu8-enkephalin and Met5Arg6Phe7-enkephalin. Additionally, 8-fold more radioactive enkephalin was harvested from the median eminence than the posterior pituitary. Measurement of preproenkephalin mRNA in the two states also revealed an increase during lactation. All of these data suggest a physiologically important role for the enkephalins in the regulation of anterior pituitary function.

In vivo biosynthesis and transport of oxytocin, vasopressin, and neurophysins to posterior pituitary and nucleus of the solitary tract.

A growing body of literature suggests that oxytocin (OXY) and arginine-vasopressin (AVP), in addition to their neuroendocrine roles, may serve as neuromodulators within the central nervous system of mammals. The present study investigated the biosynthesis of OXY, AVP, and their associated neurophysins in the paraventricular nucleus of the hypothalamus (PVN) and the transport of these peptides to the neural lobe and the nucleus of the solitary tract (NTS) in the brainstem of the rat. Rats were cannulated bilaterally in the PVN, and 24 hr later a 2-hr pulse of [35S]cysteine was administered using an Alzet minipump delivery system. After a 10-hr chase period, the neural lobe was removed and the PVN and NTS were punched. Tissue homogenates were adsorbed to and eluted from octadecyl-silica cartridges and analyzed by linear high performance liquid chromatography (HPLC) gradient elution, chemical and enzymatic modification, and exponential gradient HPLC elution followed by linear HPLC gradient analysis using an ion-pairing buffer system. This rigorous approach has allowed us to identify 35S-labeled material which co-purifies with OXY and AVP from the PVN, neural lobe, and NTS samples. Specific transport of the nonapeptides to the NTS was demonstrated when a unilateral transection of the hypothalamic fibers resulted in a unilateral depletion of the radiolabeled peptides from the NTS samples. Additionally, each of the neurophysins was purified from the neural lobe and NTS samples after linear HPLC gradient analysis, ion-pairing buffer linear gradient analysis, then tryptic digestion followed by exponential gradient HPLC analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of in vivo synthesis of pro-opiomelanocortin-, beta-endorphin-, and alpha-melanotropin-like species in the adult rat brain.

An in vivo labeling technique was utilized to demonstrate the in situ biosynthesis of pro-opiomelanocortin (POMC)-, beta-endorphin- and alpha-melanotropin (MSH)-like molecular species in rat brain. Unrestrained adult female rats were bilaterally cannulated in the hypothalamic arcuate nuclear region; [35S]methionine was infused either over a 15-min period with sacrifice 2 hr subsequently, or at a constant rate for 6 hr prior to sacrifice. Sequential immune-affinity chromatography and several chromatographic and electrophoretic techniques were employed to detect and characterize POMC-related material in the hypothalamic arcuate nuclear region, preoptic area, and median eminence. Four molecular species containing both corticotropin (ACTH) and beta-endorphin antigenic determinants within the same molecules were detected in the arcuate nuclear region and preoptic area. Two forms were similar to rat pituitary POMC with respect to apparent molecular weight (35,000 and 31,500) and [35S]methionine-containing tryptic fragments (one methionine each in N-terminal glycopeptide, ACTH, and beta-endorphin sequences of rat POMC). The other two forms (apparent Mr of 21,000 and 19,000) contained only the labeled tryptic fragments characteristic of ACTH and beta-endorphin. The detection of the latter forms suggests that POMC in brain, unlike its post-translational processing in rat pituitary, undergoes primary cleavage between the N-terminal peptide and the ACTH sequence. Peptides physicochemically indistinguishable from authentic beta-endorphin and des-acetyl alpha-MSH were detected in approximately equimolar amounts in all three brain regions. The ratio of POMC-like material to the alpha-MSH- and beta-endorphin-sized peptides was highest in the arcuate nuclear region, suggesting that POMC-like proteins are synthesized in the arcuate nuclear region and are processed into the smaller molecular species during axonal transport.

Evidence that endopeptidase-catalyzed luteinizing hormone releasing hormone cleavage contributes to the regulation of median eminence LHRH levels during positive steroid feedback.

In previous studies we provided evidence that the degradation of LHRH is regulated so as to contribute to the establishment of appropriate levels of the decapeptide during the events leading to gonadotropin secretion in the first estrous cycle at puberty in the rat. In the present report, we present the first evidence that this apparent regulation of LHRH degradation can be studied in an experimental positive feedback model. We show that LHRH degradation in the median eminence was decreased 3 h after progesterone administration, at a time when LHRH content in this region is increasing, and when serum levels of LH remained at basal levels. Six h after progesterone administration, at the time of the LH surge, median eminence LHRH degradation was still low and LHRH content had fallen to basal levels. Additionally, we exploited this model to examine the mechanism of peptidase activity change by showing that blockade of noradrenergic neurotransmission by diethyldithiocarbamate abolishes the inhibition of LHRH degradation observed prior to the secretion of LH. We conclude that the degradation of LHRH by an endopeptidase may contribute to the regulation of LHRH levels appropriate for gonadotropin release, and that this can be studied in the ovariectomized, estrogen-progesterone-treated rat.

In vivo biosynthesis of hypothalamic luteinizing hormone releasing hormone in individual free-running female rats.

The present studies examined the feasibility of performing in vivo biosynthetic studies of hypothalamic LHRH in preoptic area cannulated individual ovariectomized, estradiol benzoate (EB)-progesterone (P) treated rats. One day after EB treatment, rats were bilaterally cannulated in the anterior ventromedial preoptic area (POA). The following day, rats were treated with P, and a mix of 3H-proline and 3H-leucine (500 muCi) was continuously infused (for 8 hr) into the POA using an Alzet osmotic minipump delivery system at a rate of 1 microliter/h/cannula. 3H-LHRH was acid-extracted from discrete hypothalamic regions (POA, medial basal hypothalamus [MBH]and median eminence [ME]) and was purified to constant specific activity by sequential High Performance Liquid Chromatography. 3H-LHRH accounted for 0.010%, 0.018% and 1.6% of the total tissue 3H recovered in the POA, MBH and ME, respectively. These results demonstrate the feasibility of performing physiological studies on the biosynthesis and transport of hypothalamic LHRH using POA cannulated free-running female rats.

An aminopeptidase from bovine brain which catalyzes the hydrolysis of enkephalin.

An aminopeptidase from bovine brain which catalyzes the hydrolysis of the tyrosyl1-glycine2 bond of methionine5-enkephalin has been purified to electrophoretic homogeneity. The enzyme also catalyzes the hydrolysis of dipeptides, tripeptides, and amino acid beta-naphthylamides. The enzyme can be inactivated by dialysis against EDTA, and reconstituted with divalent metal ions. Inhibition of the enzyme is observed in the presence of p-chloromercuribenzoate and puromycin, the latter compound not being hydrolyzed by the enzyme. The enzyme is composed of a single polypeptide chain of molecular weight approx. 100,000. The properties of this enzyme are similar to those reported for other brain aminopeptidases active on enkephalin, although distinct differences are observed.

Enzymes involved in the degradation of thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH) in bovine brain.

As part of an investigation of neuropeptide inactivation mechanisms, we have resolved an enzymatic activity in bovine brain which catalyzes the deamidation of thyrotropin releasing hormone (TRH) and the hydrolysis of the Pro9--Gly10--NH2 bond of luteinizing hormone releasing hormone (LH-RH) from a second LH-RH degrading activity which does not degrade TRH. The former activity is similar, if not identical to, the post-proline cleaving enzyme in kidney as it is active toward the post-proline cleaving enzyme substrate CbzGly--Pro--Leu--Gly and inhibited by CbzPro--Phe and diisopropylfluorophosphate. In addition, products derived from the degradation of TRH and LH-RH by this activity show a specific cleavage on the carboxyl side of a proline residue. The latter activity has not yet been characterized with respect to its site of cleavage of the LH-RH molecule due to the presence of other contaminating peptidases.