Michael J. Fay

Vasopressin gene related products are markers of human breast cancer

SummaryImmunohistochemical analysis for products of vasopressin and oxytocin gene expression was performed on acetone-fixed tissues from 19 breast cancers representing a variety of tumor sub-types. Studies employed the avidin-biotin complex (ABC) immunohistochemical procedure and utilized rabbit polyclonal antibodies to arginine vasopressin (VP), provasopressin (ProVP), vasopressin-associated human glycopeptide (VAG), oxytocin (OT), oxytocin-associated human neurophysin (OT-HNP), and a mouse monoclonal antibody to vasopressin-associated human neurophysin (VP-HNP). Western Blot analysis was performed on protein extracts of fresh-frozen tissues from 12 additional breast tumors. While VP gene related proteins were not detected in normal breast tissue, immunohistochemistry revealed the presence of VP, Pro VP, and VAG in all neoplastic cells for all of the tumor tissues examined. Vasopressin-associated human neurophysin was evident in only one of 19 acetone-fixed tumor preparations. However, Western blot analysis for all 12 fresh-frozen tumor samples showed the presence of two proteins, 42,000 and 20,000 daltons, that were immunoreactive with antibodies to VP, VP-HNP, and VAG. Oxytocin and OT-HNP, by immunohistochemistry, were found to be common to cells of normal breast tissues. For tumors, positive staining for OT was observed in 8 of 18 tumors, while OT-HNP was not detected in any of the tumors examined. These findings indicate that VP gene expression is a selective feature of all breast cancers, and that products of this expression might therefore be useful as markers for early detection of this disease and as possible targets for immunotherapy.

Evidence for expression of vasopressin V2 receptor mRNA in human lung

Studies using fetal sheep, goats, and guinea pigs indicate that vasopressin may play a role in preparing the fetal lung for the transition from a uterine to an air-breathing environment by slowing lung liquid secretion. The mechanism of vasopressin action is believed to occur through V2 receptors with subsequent activation of amiloride-sensitive sodium channels. However, the presence of the V2 receptor in human lung has not yet been documented. In the present study, expression of the vasopressin V2 receptor in fetal and adult human lung was examined using reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis, and DNA sequencing. Using RT-PCR and primer pairs specific for the human V2 receptor, PCR products of the predicted sizes of 512 and 862 bp were obtained from adult human lung. DNA sequencing of the cloned PCR products revealed exact identity with the published sequence for the V2 receptor. Northern blot analysis revealed the expression of a approximately 1.9 kb mRNA in adult human lung as well as in kidney, but not in fetal human lung at 22-24 weeks of gestation. However, using the more sensitive RT-PCR assay the 862-bp product was successfully amplified from human fetal lung, although the data indicate the mRNA for this receptor is expressed in lower levels than in adult human lung or kidney. Using RT-PCR and primers specific for the rat V2 receptor, a PCR product of the predicted size of 461 bp was amplified from adult rat lung and kidney, despite an earlier report that this receptor mRNA is absent from the lung of this species. The role for the V2 receptor in adult human lung is unknown at this time, but, as in the human kidney and lungs of fetal sheep, goats, and guinea pigs, this receptor may play a role in fluid balance.

PRESENCE OF FUNCTIONAL NMDA RECEPTORS IN A HUMAN NEUROBLASTOMA CELL LINE

Data are presented that provide convincing evidence for the expression of structurally normal and functional NMDA receptors by acetylcholine-producing human LA-N-2 neuroblastoma cells in culture. Reverse transcription and polymerase chain reaction (RT-PCR), followed by cloning and DNA sequencing, revealed the presence in these cells of mRNA representing the key subunit, NMDAR1, of the receptor. This mRNA was further demonstrated by Northern analysis to be the same size as that described for human neurons. The neutral red cytotoxicity assay was utilized to examine the influence on these neuroblastoma cells of a 48-h incubation with either L-glutamic acid or the specific NMDA agonist N-phthalamoyl-L-glutamic acid (NPG). Cell cytotoxicity was shown by this assay to be increased through incubation with glutamate at 1 and 10 mM by 27 and 37%, and through incubation with NPG at 0.1 and 1 mM by 28 and 46%. A possible mechanism of these toxic effects was further evaluated using the whole-cell configuration of the patch-clamp technique and the specific NMDA agonists (+/-)1-aminocyclobutane-cis-1,3-dicarboxylic acid (ACDA) and NPG. Using this procedure, a voltage-dependent tetrodotoxin-sensitive inward sodium current was found to be increased (x 1.5) by L-glutamic acid and by both NMDA agonists in the presence of glycine. Another voltage-gated inward current, probably carried by calcium ions, was increased three- to fourfold. Hence, these glutamate activities observed in human LA-N-2 neuroblastoma cells appear to occur through the activation of functional NMDA receptors in much the same way as reported for neurons, and both glutamate and NMDA agonists can be toxic to these neuroblastoma cells. Our findings, therefore, suggest this cell line will provide a model suitable for investigating the mechanisms of NMDA-related long-term potentiation (LTP) in neurons and of the NMDA-related neurotoxic effects of glutamate in disease states that involve a reduction in cholinergic function.

Functional Vasopressin V1 Type Receptors are Present in Variant as Well as Classical Forms of Small-Cell Carcinoma

Vasopressin and other neuropeptides are believed to serve as autocrine growth factors for small-cell carcinoma of the lung (SCCL), and these mitogenic influences are reported to involve increases in intracellular Ca2+. Of the classical and variant forms of SCCL, the latter is not only more drug-resistant but also refractory to vasopressin, and other peptides, with respect to changes in intracellular Ca2+. It is currently unclear if this refractiveness of variant SCCL is due to the absence of involved peptide receptors, to the production of abnormal receptors, or to abnormalities in components of induced transduction cascades. In this study, the presence of structurally-normal and functional vasopressin V1a receptors, was examined in a classical SCCL cell line (NCI H345) that is Ca(2+)-responsive to vasopressin, and a variant SCCL cell line (NCI H82) that is unresponsive in this regard to the peptide. Both cell lines were shown to express an mRNA of 1.9 Kb for the vasopressin V1a receptor. RT-PCR, cloning, and DNA sequencing revealed the structure of the mRNA was identical for both cell lines, and, in turn, identical to the mRNA expressed for this receptor by human liver cells. In both cell lines and liver, this mRNA was shown by Western analysis and RIA to generate major protein products of approximately 70,000 and 43,000 daltons. Vasopressin action on NCI H82 cells resulted in a substantial rise in the levels of total inositol phosphates. However, it was reaffirmed that these changes in inositol phosphates were not accompanied by a rise in Ca2+ levels. All of these data indicate that variant SCCL, as well as classical SCCL, expresses structurally-normal and functional vasopressin V1a receptors, but their activation in variant SCCL raises IP3 levels without a corresponding rise in intracellular Ca2+. This difference between the two SCCL sub-types therefore involves either steps in the inositol triphosphate cascade beyond the activation of phospholipase C, or alternatively, components of other transduction events that might be involved with changes in intracellular Ca2+.

Factors regulating the production of vasopressin-associated human neurophysin by small-cell carcinoma of the lung: Evaluation by computer-enhanced quantitative immunocytochemistry

Expression of the vasopressin gene appears to be a property common to all small-cell lung tumours. For some cultures of small-cell lung carcinoma (SCCL), Northern and Western Blot analyses have revealed that expression of this gene and its protein products are regulated by cAMP and glucocorticoids. In this study, these evaluations have been extended by examining the production of vasopressin-associated human neurophysin (VP-HNP) by computer-enhanced quantitative immunocytochemistry in a classical cell-line (H69) of SCCL, and defining the amount of protein in cells by area of positive staining above an arbitrarily set threshold. Intracellular cAMP was raised by incubating cells with either 8,Br-cAMP (0.5 mM) and IBMX (0.5 mM), or with forskolin (25 microM) and IBMX (0.5 mM). Both of these treatments caused a significant increase in the amount of positive VP-HNP immunoreactivity in the cells, an increase that was further enhanced by simultaneous administration of dexamethasone (0.1 microM). Addition of dexamethasone alone, however, caused a significant decrease in VP-HNP levels. Results confirm earlier findings from Western Blot analysis revealing the influence these agents have on production of vasopressin gene-related proteins by H69 cells, and indicate that computer-enhanced quantitative immunocytochemistry can be effectively used to provide a suitable index of this production.

Vasopressin and vasopressin-receptor immunoreactivity in small-cell lung carcinoma (SCCL) cell lines: disruption in the activation cascade of V1a-receptors in variant SCCL

Four classical and three variant small-cell carcinoma of the lung (SCCL) cell lines were examined for vasopressin and vasopressin V1a-receptor immunoreactivity. One of these classical cell lines, NCI-H345, and one variant cell line, NCI-H82, were further investigated for binding of V1 and V2 vasopressin-receptor antagonists, vasopressin-induced calcium mobilization, and vasopressin-induced thymidine uptake. All classical and variant SCCL cell lines examined contained vasopressin and vasopressin-receptors as determined by immunocytochemistry. Both NCI-H82 and NCI-H345 demonstrated similar binding patterns with the V1 and V2 vasopressin-receptor antagonists, indicating the presence of both receptor subtypes. For the classical cell line (NCI-H345), vasopressin (1 microM) induced an increase in cytosolic free calcium, while the peptide was ineffective at increasing cytosolic calcium in the variant cell line (NCI-H82). However, vasopressin (0.1 or 1 microM) was unable to stimulate thymidine uptake in the classical (NCI-H345) or variant (NCI-H82) cell lines for the conditions used. These results indicate that both classical and variant SCCL produce vasopressin, and vasopressin V1a and V2 receptors. In the variant cell line, there appears to be a disruption in the activation cascade for V1a receptors as indicated by the lack of vasopressin-induced calcium mobilization.

Effect of aldonic acids on the uptake of ascorbic acid by 3T3 mouse fibroblasts and human T lymphoma cells

1. Previously, we reported that calcium L-threonate caused a dose-related increase in uptake of ascorbic acid (AA) by human T-lymphoma cells. Preincubation of mouse fibroblasts with calcium L-threonate also resulted in a dose-related augmentation in uptake of AA as compared to non-treated controls. 2. Potassium L-lyxonate increased AA uptake by lymphoma cells, but did not significantly affect uptake by fibroblasts. Tartaric acid decreased uptake of AA by both cell lines. 3. Ouabain and dinitrophenol had no effect on AA uptake nor on the ability of threonate to augment AA uptake by fibroblasts. However, in T-lymphoma cells ouabain and dinitrophenol reduced AA uptake and prevented augmentation of AA uptake by calcium L-threonate.