Terry W. Moody

HIV envelope protein-induced neuronal damage and retardation of behavioral development in rat neonates

Cognitive and motor impairment are common symptoms among patients infected with the human immunodeficiency virus (HIV), including children who suffer neurological deficits and are frequently developmentally impaired. The HIV envelope protein, gp120, which has been shown to be toxic to neurons in culture, is shed in abundance by infected cells, and thus may play a significant role in the neuropathology of AIDS. To test this possible mechanism, neonatal rats were injected systemically with purified gp120 and the following consequences were observed: (1) radiolabeled gp120 and toxic fragments thereof were recovered in brain homogenates; (2) dystrophic changes were produced in pyramidal neurons of cerebral cortex; (3) retardation was evident in developmental milestones associated with complex motor behaviors. In parallel studies, co-treatment with peptide T, a gp120-derived peptide having a pentapeptide sequence homologous with vasoactive intestinal peptide, prevented or attenuated the morphological damage and behavioral delays associated with gp120 treatment. These studies suggest that gp120 and gp120-derived toxic fragments may contribute to the neurological and neuropsychiatric impairment related to HIV infection, and that peptide T appears to be effective in preventing gp120-associated neurotoxicity in developing rodents.

Factors influencing murine embryo bioassay: effects of proteins, aging of medium, and surgical glove coatings

The 2-cell murine embryo bioassay as quality control for human in vitro fertilization (IVF) was used to evaluate the effects of protein supplements, medium aging, and surgical glove coatings. Hams F-10 medium (GIBCO, Grand Island, NY) without protein supplementation supported growth of the 2-cell embryos to blastocysts. Addition of bovine serum albumin (BSA), fetal cord serum (FCS), or maternal serum (MS) did not enhance or reduce the blastulation rates (medium alone, 89.4%; BSA, 86.4%; FCS, 90%; MS, 74.7%). Unsupplemented Hams F-10 medium was found to contain three major peaks of approximately 50,000 daltons and several minor peaks, analyzed on high-performance liquid chromatography (HPLC) and sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), suggesting the presence of protein(s) in the medium itself. The storing of medium up to 425 days at 4 degrees C affected neither the HPLC profile nor its ability to support embryo growth (blastulation rates: fresh, 84%; stored 150 to 425 days, 77.7%). The coating of surgical gloves affected embryo growth. Both talc-coated (TC) and talc-free, starch-coated (SC) surgical gloves were found to be progressively embryotoxic when they touched the medium for increasing lengths of time, compared with uncoated latex (UL) gloves and untouched control medium. Quality control of medium preparation and handling in murine embryo bioassay is reemphasized, with requirements for protein supplementation, use of fresh medium, and possible contamination with even talc-free, SC surgical gloves reevaluated.

Protease inhibitors suppress in vitro growth of human small cell lung cancer

The effect of the protease inhibitors Bowman Birk inhibitor (BBI) and aprotinin on the in vitro clonal growth of two human small cell lung cancer (SCLC) cell lines was investigated. In addition, the effect of BBI on the growth factor processing of proGRP by SCLC cells and on mRNA levels for prohormone convertase 1 and 2 (PC1 and PC2) in SCLC cells was examined. The protease inhibitors BBI and aprotinin significantly decreased growth in both SCLC cell lines studied. In NCI-H345 cells, BBI appears to inhibit the processing of proGRP to GRP, as indicated by Western blot analysis. NCI-H345 cells, when treated with BBI (100 micrograms/ml), also showed highly significant decreases of mRNA for PC1 and PC2 of about 50%. These data suggest that proteases serve an important role in the growth regulation of SCLC and that inhibitors of these proteases may be potent suppressors of SCLC growth at the level of the gene.

Bombesin-Related Peptides and Neurotensin: Effects on Cancer Growth/Proliferation and Cellular Signaling in Cancer

ABSTRACT Mammalian bombesin-related peptides and neurotensin are regulatory peptides that have important roles in a number of physiological and pathological processes. In this chapter their effects on human cancer are briefly reviewed, including evidence that these peptides and/or their receptors occur in cancer, stimulate growth, are involved in cellular signaling cascades, and have potential roles in cancer treatment.

Solubilization and purification of bombesin/gastrin releasing peptide receptors from human cell lines.

Bombesin/gastrin releasing peptide (BN/GRP) receptors were solubilized and purified from human glioblastoma (U-118) and lung carcinoid cell lines (NCI-H720). The U-118 cells, when extracted with CHAPS/cholesterol hemisuccinate (CHS), bound (125I-Tyr4)BN with high affinity (Kd=2 nM) to a single class of sites (Bmax)=150 fmol/mg protein). Specific (125I-Tyr4)BN binding was inhibited with high affinity by BN, GRP, GRP14–27, and receptor antagonists such as (D-Phe6)BN6–13methyl ester(ME) and (D-Phe6)BN6–13 propylamide(PA) (IC50=2, 22, 3, 1 and 2 nM, respectively) but not GRP1–16 or BN1–12. The solubilized and cellular receptor bound peptides with similar affinity. The solublized receptor was purified using (Lys0, Gly1–4, D-Ala5)BN and (Lys3, Gly4,5, D-Tyr6)BN3–13 PA affinity resins. When eluted from the affinity resins by NaCl, the receptor bound (125I-D-Tyr6) BN6–13 ME with high affinity. The NCI-H720 BN/GRP receptor was purified 86,000-fold after extraction with CHAPS/CHS and purification using both affinity resins. SDS-PAGE analysis indicated that major 65 and 115 kDa proteins were purified. These data indicate that BN/GRP receptors can be solubilized from human cells and purified using affinity chromatography techniques with retention of ligand binding activity.

The in vitro dissociation kinetics of (R,R)-[125I]4IQNB is reflected in the in vivo washout of the radioligand from rat brain

We have determined the kinetics of dissociation of (R)-3-Quinuclidinyl (R)-4-[125I]Iodobenzilate ((R,R)-[125I]4IQNB) from muscarinic acetylcholine receptor preparations from the cortex, hippocampus, caudate/putamen, thalamus, pons and colliculate bodies. The dissociation curves are well described by a biexponential function and are consistent with subtype selectivity favoring slow dissociation from the M1, M3, and M4 receptors with a 20-fold faster dissociation rate for the M2 receptor. Following intravenous injection, (R,R)-[125I]4IQNB binds to receptor in the rat brain in concentrations which reflect the receptor concentration present in a structure. We determined the extent of radioligand present at two times, 2 and 24 hrs, as an indication of the relative proportions of m-AChR which exhibits rapid vs. slow dissociation of (R,R)-[125I]4IQNB. A good correlation between in vitro and in vivo results suggests that the relative populations of receptor subtypes can be imaged using in vivo pharmacokinetics of (R,R)-[125I]4IQNB.

Bombesin-induced hypothermia: A dose-response and receptor antagonist study

Bombesin infusion into the preoptic area (POA) has previously been shown to induce hypothermia in rats that are food deprived or made hypoglycemic with insulin. The present study evaluated the potency and receptor specificity of this response. Bombesin was microinfused into the POA of food-deprived rats (n = 7) and insulin-pretreated rats (n = 7) at doses of 0, 5, 12, 25, and 50 ng/0.5 microliters. Changes in core body temperature (rectal) were assessed at 1 h. Hypothermia was observed under both conditions with doses as low as 5 ng (3.1 pM) as compared to vehicle (0 ng). In a separate study, infusion of the reduced peptide bond analog (Psi13,14 Leu14)bombesin (2.5 micrograms) prior to bombesin injection (25 ng) was found to prevent the hypothermic response observed in the bombesin control condition. These data suggest that bombesin is a potent hypothermic agent that interacts with gastrin-releasing peptide receptors localized within the POA region to impact thermoregulation.

An atypical endocrine tumor of the lung responsive to radiation therapy and 5‐fluorouracil‐streptozotocin

A case is reported of a patient who presented with a peripheral left upper‐lobe lung mass, a thyroid nodule, and multiple enlarged cervical and supraclavicular lymph nodes. Fine‐needle aspiration cytology of the lung lesion, the thyroid nodule, and several of the lymph nodes was interpreted as small cell cancer of the lung (SCCL). The patient was treated with Cytoxan (cyclophosphamide), Adriamycin (doxorubicin), and vincristine (CAV), alternating with VP‐16 + cisplatin. When progressive disease was documented after three cycles of chemotherapy, an involved cervical lymph node was biopsied. By light microscopy (LM) the tumor appeared to be a poorly differentiated adenocarcinoma, but by transmission electron microscopy (TEM) it was found to have both neuroendocrine and glandular features. Biochemical analysis of the biopsy specimen revealed immunoreactive bombesin, and on immunoperoxidase staining many tumor cells contained neuron‐specific enolase. The tumor was therefore classified as an atypical endocrine tumor of the lung (AETL), a recently described morphologic variant for which no therapy has yet been established. The patient was treated with radiation therapy (RT) followed by chemotherapy including 5‐fluorouracil (5‐FU) (500 mg/m2 IV, d 1–5) and streptozotocin (STZ) (500 mg/m2 IV, d 1–5) every 5–6 weeks, with objective evidence of tumor regression following each modality. This report illustrates the importance of ultrastructural study in the characterization of lung cancer, and indicates the need for the further evaluation of RT and 5FU + STZ in the treatment of neuroendocrine tumors of the lung.

Effects of neonatal blockade of bombesin (BN) receptors with [d-Phe6,ΨLeu13-Cpa14]BN(6–14) on adult behavior and sensitivity to BN

Long-term consequences of neonatal blockade of bombesin (BN) receptors were examined in the present study. Rat pups were injected twice daily with [D-Phe6, phi Leu13-Cpa14]BN(6-14), a BN receptor antagonist, at either high (10 mg/kg; HD group) or low (5 mg/kg; LD group) doses from postnatal day 1 through 8. Their behavioral responses to a variety of conditions were compared to those of rats neonatally injected with saline (SAL group) or animals handled but not injected during infancy (UNT group). Adult HD rats entered and spent more time on the open arms of the elevated plus maze than LD, SAL, or UNT animals. Under the conditions of a water deprivation schedule, neither central nor peripheral injections of BN differentiated the neonatally pretreated groups as determined by measures of grooming, feeding, and drinking behaviors. These results indicate that at the dosage regimen employed, neonatal injections of [D-Phe6, phi Leu13-Cpa14]BN(6-14) had little effect on adult sensitivity to BN, but that such treatments could alter activity on the elevated plus maze through as yet unknown mechanisms.

VIP and PACAP as Autocrine Growth Factors in Breast and Lung Cancer

ABSTRACT VIP and PACAP are synthesized by several human breast and lung cancer cell lines. These peptides bind to cell surface receptors and stimulate adenylylcyclase activity. This leads to increased expression of nuclear oncogenes such as c-fos and c-jun, ultimately leading to cellular proliferation. The growth of breast and lung cancer cell lines is inhibited by VIP receptor antagonists and VIP-chemotherapeutic conjugates.