Dual-specificity phosphatase 26 is a novel p53 phosphatase and inhibits p53 tumor suppressor functions in human neuroblastoma.

Chemoresistance is a major cause of treatment failure and poor outcome in neuroblastoma. In this study, we investigated the expression and function of dual-specificity phosphatase 26 (DUSP26), also known as mitogen-activated protein kinase phophatase-8, in human neuroblastoma. We found that DUSP26 was expressed in a majority of neuroblastoma cell lines and tissue specimens. Importantly, we found that DUSP26 promotes the resistance of human neuroblastoma to doxorubicin-induced apoptosis by acting as a p53 phosphatase to downregulate p53 tumor suppressor function in neuroblastoma cells. Inhibiting DUSP26 expression in the IMR-32 neuroblastoma cell line enhanced doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression as well as apoptosis. In contrast, DUSP26 overexpression in the SK-N-SH cell line inhibited doxorubicin-induced p53 phosphorylation at Ser20 and Ser37, p21, Puma, Bax expression and apoptosis. Using in vitro and in vivo assays, we found that DUSP26 binds to p53 and dephosphorylates p53 at Ser20 and Ser37. In this report, we show that DUSP26 functions as a p53 phosphatase, which suppresses downstream p53 activity in response to genotoxic stress. This suggests that inhibition of this phosphatase may increase neuroblastoma chemosensitivity and DUSP26 is a novel therapeutic target for this aggressive pediatric malignancy.

Major histocompatibility complex-restricted lysis of neuroblastoma cells by autologous cytotoxic T lymphocytes.

Vigorous host immune reactivity to neuroblastoma may correlate with better prognosis, but identification of human cytotoxic T-lymphocyte (CTL) responses has been relatively unsuccessful. We generated neuroblastoma-reactive CTL lines from two human leukocyte antigen (HLA) A2+ neuroblastoma patients by stimulation of peripheral blood lymphocytes (PBLs) with irradiated autologous tumor cells pretreated with interferon-gamma in the presence of low concentrations of interleukin-2 (5 U/mL). These lines lyse autologous tumor cells but do not kill HLA mismatched allogeneic tumor cells, Epstein-Barr virus-transformed autologous B cells, or standard natural killer cell targets. Cytotoxic T lymphocytes generated from one patient recognize tumor cells from several HLA-A2 matched children, although the other patient's CTLs do not kill tumor cells from other HLA-A2+ individuals. Pretreatment of CTLs or target cells with appropriate standard monoclonal antibodies demonstrates that these CTLs are major histocompatibility complex class I (HLA-A2) restricted and that the effector cell population is CD8+. Our findings suggest that these tumor cells express at least one common HLA-A2 restricted antigen and at least one unique private epitope. Autologous tumor-specific CTLs can be readily generated from patients' PBLs and maintained in long-term culture using standard techniques.

Bile acid stimulation of cyclic AMP and ion transport in developing rabbit colon.

Bile acids elicit Cl secretion and increases in short circuit current (Isc) in rabbit distal colon in vitro in adult but not newborn animals. In this investigation we found that concentrations of taurodeoxycholic acid (TDC) as low as 50 microM significantly increase cyclic AMP (cAMP) in adult but not newborn colon. Further, blocking the increases of cAMP in adult colon with 3,4,5-trimethoxybenzoate 8-(N,N-dimethylamino) octyl ester (TMB-8), partially inhibited the effect of TDC on Cl secretion. TMB-8 did not block the effect of increases in cAMP seen with vasoactive intestinal peptide (VIP), theophylline, or forskolin. When newborn colon was exposed to 1 mM TDC, limited Cl secretion was elicited. Increased cAMP is not seen in newborn colon where TDC-induced secretion is absent. Thus, increases in cAMP may represent one part of the coupling of TDC stimulation to Cl secretion.

Potassium secretion in response to taurodeoxycholic acid in the newborn rabbit colon.

The newborn colon fails to secrete Cl in response to concentrations of dihydroxy bile acid that cause Cl secretion in adult colonic tissue in vitro. Bile acids also cause secretion of potassium in adult tissues, but there is no information concerning bile acid effects on potassium transport in newborn colon. We mounted newborn rabbit distal colon in Ussing chambers specially designed for newborn colon and measured potassium transport. Basal potassium transport was secretory. Taurodeoxycholic acid, 100 microM, (TDC) decreased JKnet from -0.76 +/- 0.07 to -0.94 +/- 0.11 microEq cm-2 h-1, p less than 0.05, without increasing Isc. Serosal ouabain, 0.1 mM, abolished the secretory response to TDC. Mucosal Ba2+, a potassium channel blocker in many epithelia, did not inhibit K secretion. Similar serosal exposure to TDC in adult colon tissues decreased JKnet from -0.09 +/- 0.29 to -1.63 microEq cm-2 h-1, p less than 0.01, and increased Isc. We conclude that, although the chloride secretory response to dihydroxy bile acids is absent in the newborn, K secretion is elicited in the newborn, similar to the adult colon.

Electrogenic sodium transport in the developing rabbit cecum.

The adult rabbit cecum absorbs Na by an electrogenic, Cl-independent process that is inhibited by the amiloride analogue phenamil. In the colon, this transport system is unique to the cecum. Because the developing colon exhibits many specialized functions, we have now examined the development of electrogenic Na transport in the newborn rabbit. Cecal tissue from animals between 7 and 38 days old was mounted in modified Ussing chambers for measurement of Na and Cl flux (J) and the short-circuit current (Isc). At 7-10 days, the (Isc) was only 0.8 +/- 0.18 microEq cm-2 h-1, but by 35-38 days it had increased to 4.6 +/- 0.79 microEq cm-2 h-1. The Na transport increased in parallel with Isc; JNanet = -0.49 +/- 0.33 microEq cm-2 h-1 at 7-10 and 4.7 +/- 1.6 microEq cm-2 h-1 at 35-38 days. The Na transport was not inhibited by phenamil (10-4 M) at 7-10 days, but by the 35-38-day period, phenamil reduced the JNanet to 1.6 +/- 0.37 microEq cm-2 h-1. The Cl secretion was not stimulated by theophylline in the 14-16-day-old cecum, nor was Na absorption stimulated by epinephrine. The rabbit cecum does not demonstrate electrogenic Na absorption until after the 4th week of life.

Taurodeoxycholate and the developing rabbit distal colon: absence of secretory effect.

Failure to absorb bile acids by the ileum leads to fluid secretion by the colon and diarrhea in adults. The infant ileum, however, does not actively transport bile acids. Therefore, we investigated the effect of taurodeoxycholic acid (TDCA) on ion transport in the colon of rabbits 7-10 days old. We mounted distal colon from infant and adult rabbits in modified Ussing chambers and exposed the mucosal or serosal surfaces to TDCA. In the adult, 50 microM TDCA produced an increase in short-circuit current (delta Isc = 1.0 +/- 0.3 mu eq . h-1 . cm-2, P less than 0.05) and Cl secretion. In the infant, the effect was different, Isc was reduced (delta Isc = -1.1 +/- 0.2 mu eq . h-1 . cm-2, P less than 0.01) and ion flux was not altered. Microscopy demonstrated that the infant epithelium was not significantly damaged by exposure to TDCA at these concentrations. The infant colon was, however, capable of a secretory response to a variety of agonists including theophylline, carbachol, bradykinin, serotonin, and 12,13-dibutyryl phorbol ester. The infant rabbit distal colon lacks a secretory response to TDCA during that period when the ileum cannot transport bile acids.

Ion transport by neonatal rabbit distal colon.

The neonatal small intestine is characterized by electrical conductance and permeability to ions higher than in the corresponding adult intestine. To investigate whether this property of the neonate is limited to the small intestine, or extends to the colon, a modified Ussing chamber for determination of transmucosal potential difference (PD), short-circuit current (Isc), transepithelial conductance (Gt), and ion fluxes in the neonatal rabbit distal colon was constructed. After care to reduce edge damage, Gt for the neonatal colon was found to be 8.4 +/- 0.3 mS . cm2 and for adult colon in the same chamber, 7.4 +/- 0.5 (P greater than 0.05). Net Na and Cl fluxes under short-circuit conditions were similar to those obtained in adult colon. Unidirectional ion fluxes were also similar to those of the adult. Net Na flux (JNanet) was incompletely inhibited by 10(-4) M of amiloride. Response to replacement of Na, Cl, and HCO3-, respectively, in the bathing solutions was not different from that expected in adult rabbit colon. Thus differences between adult and neonatal rabbit colon were small, and the increased conductance and unidirectional ion fluxes characteristic of the neonatal small intestine were not evident in the neonatal rabbit distal colon.

Glucose-coupled sodium absorption in the developing rat colon.

The developing mammalian colon is lined by villi and is capable of glucose and amino acid absorption at birth in the rat. Neither the point at which this capacity is lost nor the effect of the capacity for glucose transport on Na absorption has been studied. We have now applied a system for perfusion of the lumen of in vitro segments of colon from 20-day-old fetal rats, and pups between 6 and 8 days old, to measure Na transport and transepithelial potential difference (PD). The lumens of colons from animals at both ages were perfused with solutions containing glucose or mannitol and 22Na. Net Na transport was 164 +/- 37 mu eq X h-1 X g dry weight tissue perfused-1, as determined by the difference between lumen-to-bath and bath-to-lumen flux in fetal rat colons at day 20. Glucose increased the lumen-to-bath flux by 90 +/- 35 mu eq X h-1 X g-1. PD was immediately increased from -1.7 +/- 0.16 to -8.0 +/- 0.96 mV (lumen with respect to bath) by the addition of glucose, and the change in PD was inhibited by 10(-4) M phlorizin. The PD response to glucose was lost at day 2 of life, but the villus epithelium persisted. Amiloride, 10(-4) M, did not alter PD or Na transport at either age. We conclude that the fetal rat colon exhibits glucose-dependent Na flux at birth but that this property is lost by 6-8 days.(ABSTRACT TRUNCATED AT 250 WORDS)