Neuroblastoma cells clones

Studies in murine neuroblastoma cells (clone NIE-115) have shown that histamine can produce a rapid and marked (up to 50-fold above basal levels) increase in the formation of cyclic [3H] GMP following preloading of cells with [3H]guanine [251], The peak response to histamine is normally observed 30 s after application of histamine and then rapidly declines towards basal levels over the next 2 or 3 min [251,252]. When neuroblastoma cells are pre-incubated with the phosphodiesterase inhibitor 3-isobutyl- 1-methylxan-thine, the rate of decay of cyclic [3H]GMP is significantly reduced [252]. 

Of the effector systems that have been implicated in the transduction mechanisms for opioid receptors, the best studied is opioid inhibition of adenylyl cyclase (see Refs. 69, 97-99 for reviews). Thus binding of an agonist to opioid receptors inhibits the activity of adenylyl cyclase and decreases intracellular cAMP in a number of different tissues. Pertussis toxin sensitivity of opioid inhibition of adenylyl cyclase has been demonstrated in many systems, indicating the involvement of either Gi or Go in the transduction mechanism. Agonist activation of all three types of cloned opioid receptors to inhibit adenyl cyclase has been demonstrated (see Ref 100 and references cited therein). There is also some evidence that (M and 8 opioid receptors can stimulate adenylyl cyclase in certain tissues (see Refs. 69, 97 for reviews). There are conflicting reports on whether k opioid receptors stimulate or inhibit phosphatidylinositol turnover in some tissues (see Ref 100) 8 and fx receptors, however, do not appear to be coupled to phosphatidylinositol turnover in neuroblastoma cell lines NG108-15 and SK-N-SH (101). 

The human Y1 mRNA is about 3.5-4 kb and was detected in the neuroblastoma cell line SK-N-MC by Northern hybridization (Larhammar et al., 1992). An in situ hybridization study with a riboprobe reported widespread distribution in human fetal and adult organs, e.g. colon, kidney, heart, placenta and adrenal (Wharton etal., 1993). However, a Northern hybridization in the same study detected a single mRNA of only 2.2 kb suggesting that the specificity of the probe for Y1 mRNA may be questioned. Curiously, in the same study the size for NPY mRNA was reported as 3.3 kb, which disagrees with the size seen in a human pheochromocytoma (0.8 kb) as well as the size of the human cDNA clone which ends with a poly(A) tract (Minth et al., 1984). 

FIGURE 17.2 Effect of pectenotoxins on F-actin cytoskeleton. Confocal imaging of (a) BE(2)-M17 human neuroblastoma cells and (b) clone 9 rat hepatocarcinoma cells treated for 4 h, and 3 h, respectively with 200 nM PTX-2 or 200 nM PTX-2 SA. F-actin was stained with Oregon Green 514 Phalloidin. 

J. Brennand/ A.C. Chinault/ D.S. Konecki/ D.W. Melton and C.T. Caskey/ Cloned cDNA sequences of the hypoxanthine/guanine phos-phoribosyltransferase gdfne from a mouse neuroblastoma cell line found to have amplified genomic sequences/ Proc. Natl. Acad. 

The existence of cannabinoid receptors was confirmed when it was observed that cannabinoids decreased cAMP levels in neuroblastoma cell cultures (Howlett, 1984). This finding was followed by the determination and characterization of a cannabinoid receptor in rat brain (Devane et al., 1988). Shortly after, the stmcture of this CBj receptor and functional expression of the cloned cDNA was reported (Matsuda et al, 1990). The CBj receptor is found in the central nervous system as well as in several peripheral tissues, including testis, small intestine, vascular endothelium, utems and vas deferens (Herkenham et al., 1990). The distribution and localization of CBj receptor in the brain correlates well with the known effects of cannabinoids on memory, perception and the control of movement. CBj receptors are highly expressed in the hippocampus, association cortex, cerebellum and basal ganglia. 

Cell Culture. Human neuroblastoma IMR-32 (passaged through nude mice the cells were donated by Dr. Steven E. Brooks, Kingsbrook Jewish Medical Center, Brooklyn) and mouse neuroblastoma clones NIE-115, NS-20, and N-18) (donated by Dr. Shraga Makover, Hoffmann LaRoche, Inc., Nutley, New Jersey) were maintained in our laboratory as described previously (30,31). Confluent monolayers (6 to 8 x 10 cells per 250-ml Falcon plastic flask) were harvested for enzymatic studies with phosphate-buffered saline [7.0 mM potassium phosphate/0.14 M NaCl - buffer, pH 7.2 (Pi/NaCl)] containing 0.1% EDTA. 

In order to obtain some idea about the nature of gly-coconjugates and their gross topographical orientation on the cell surfaces, we measured the binding of 125j labeled lectins and toxin to human neuroblastoma IMR-32 and mouse neuroblastoma N1E-115, NS-20, and N-18 clones (Tables VII and VIII). The "5% TCA Wash" column (Table VII) represents 1251-iabeled lectin or toxin bound to both glycoprotein and glycolipid. The "5%... 

Both groups used expression cloning to screen cDNA libraries obtained from NG 108-15 mouse neuroblastoma-rat glioma hybridoma cells. These cells were a logical choice for delta opioid receptor cloning efforts, since they express high density of delta receptors and can be produced in great quantities in cell culture. 

Cloned cDNA for HPRT (3) from a mouse neuroblastoma line was used as a probe against DNA from human fibroblasts and leukocytes digested with a variety of restriction enzymes and transferred to nitrocellulose by the method of Southern. The probe cross-hybridizes well with human genomic DNA (4). Fibroblasts were obtained from the Mutant Cell Repository, Camden, New Jersey, and from patients and their families. 

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