ATP dependence of the ICl,swell channel varies with rate of cell swelling. Evidence for two modes of channel activation.
(9/5127)
Swelling-induced activation of the outwardly rectifying anion current, ICl, swell, is modulated by intracellular ATP. The mechanisms by which ATP controls channel activation, however, are unknown. Whole cell patch clamp was employed to begin addressing this issue. Endogenous ATP production was inhibited by dialyzing N1E115 neuroblastoma cells for 4-5 min with solutions containing (microM): 40 oligomycin, 5 iodoacetate, and 20 rotenone. The effect of ATP on current activation was observed in the absence of intracellular Mg2+, in cells exposed to extracellular metabolic inhibitors for 25-35 min followed by intracellular dialysis with oligomycin, iodoacetate, and rotenone, after substitution of ATP with the nonhydrolyzable analogue AMP-PNP, and in the presence of AMP-PNP and alkaline phosphatase to dephosphorylate intracellular proteins. These results demonstrate that the ATP dependence of the channel requires ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15-55%/min in the absence of intracellular ATP, current activation was slow (0.3-0.8 pA/pF per min). ATP concentration increased the rate of current activation up to maximal values of 4-6 pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a saturable, hyperbolic function of ATP concentration. The EC50 for ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4-6 pA/pF per min) in the absence of ATP when cells were swollen at rates >/=65%/min. Intracellular ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous ATP was dialyzed out of the cytoplasm of cells swollen at 15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory proteins are capable of sensing the rate of cell volume increase. We suggest that channel activation occurs via ATP-dependent and -independent mechanisms. Increasing the rate of cell swelling appears to increase the proportion of channels activating via the ATP-independent pathway. These findings have important physiological implications for understanding ICl, swell regulation, the mechanisms by which cells sense volume changes, and volume homeostasis under conditions where cell metabolism is compromised. (+info)
Busulphan is active against neuroblastoma and medulloblastoma xenografts in athymic mice at clinically achievable plasma drug concentrations.
(10/5127)
High-dose busulphan-containing chemotherapy regimens have shown high response rates in children with relapsed or refractory neuroblastoma, Ewing's sarcoma and medulloblastoma. However, the anti-tumour activity of busulfan as a single agent remains to be defined, and this was evaluated in athymic mice bearing advanced stage subcutaneous paediatric solid tumour xenografts. Because busulphan is highly insoluble in water, the use of several vehicles for enteral and parenteral administration was first investigated in terms of pharmacokinetics and toxicity. The highest bioavailability was obtained with busulphan in DMSO administered i.p. When busulphan was suspended in carboxymethylcellulose and given orally or i.p., the bioavailability was poor. Then, in the therapeutic experiments, busulphan in DMSO was administered i.p. on days 0 and 4. At the maximum tolerated total dose (50 mg kg(-1)), busulphan induced a significant tumour growth delay, ranging from 12 to 34 days in the three neuroblastomas evaluated and in one out of three medulloblastomas. At a dose level above the maximum tolerated dose, busulphan induced complete and partial tumour regressions. Busulphan was inactive in a peripheral primitive neuroectodermal tumour (PNET) xenograft. When busulphan pharmacokinetics in mice and humans were considered, the estimated systemic exposure at the therapeutically active dose in mice (113 microg h ml(-1)) was close to the mean total systemic exposure in children receiving high-dose busulphan (102.4 microg h ml(-1)). In conclusion, busulphan displayed a significant anti-tumour activity in neuroblastoma and medulloblastoma xenografts at plasma drug concentrations which can be achieved clinically in children receiving high-dose busulphan-containing regimens. (+info)
Improved methods using the reverse transcriptase polymerase chain reaction to detect tumour cells.
(11/5127)
Reverse transcriptase polymerase chain reaction (RT-PCR) is increasingly used to detect small numbers of circulating tumour cells, though the clinical benefit remains controversial. The largest single contributing factor to the controversy of its value is the different approaches to sample processing. The aim of this study was to compare the sensitivity and reproducibility of RT-PCR for the detection of tumour cells after four commonly used different methods of sample processing. Using RT-PCR, one tumour cell spiked in 2 ml of whole blood was detected after analysis of separated mononuclear cell RNA, whole blood total or poly-A+ RNA. No false positives were identified with any method. However, the reproducibility of tumour cell detection was reduced after isolation of the mononuclear cell fraction. Only analysis of poly-A+ RNA had a sensitivity of 100% in all the cell spiking experiments. In patient blood samples, analysis of poly-A+ RNA increased the number of blood samples positive for tyrosine hydroxylase (TH) mRNA compared with those positive after analysis of total RNA. This may reflect high levels of cDNA reducing the efficiency of the PCR. Isolation of poly-A+ RNA increases the sensitivity and reproducibility of tumour cell detection in peripheral blood. (+info)
A possible contributory role of BK virus infection in neuroblastoma development.
(12/5127)
The tumor suppressor protein p53 is aberrantly localized to the cytoplasm of neuroblastoma cells, compromising the suppressor function of this protein. Such tumors are experimentally induced in transgenic mice expressing the large tumor (T) antigen of polyomaviruses. The oncogenic mechanisms of T antigen include complex formation with, and inactivation of, the tumor suppressor protein p53. Samples from 18 human neuroblastomas and five normal human adrenal glands were examined. BK virus DNA was detected in all neuroblastomas and none of five normal adrenal glands by PCR. Using DNA in situ hybridization, polyomaviral DNA was found in the tumor cells of 17 of 18 neuroblastomas, but in none of five adrenal medullas. Expression of the large T antigen was detected in the tumor cells of 16 of 18 neuroblastomas, but in none of the five adrenal medullas. By double immunostaining BK virus T antigen and p53 was colocalized to the cytoplasm of the tumor cells. Immunoprecipitation revealed binding between the two proteins. The presence and expression of BK virus in neuroblastomas, but not in normal adrenal medulla, and colocalization and binding to p53, suggest that this virus may play a contributory role in the development of this neoplasm. (+info)
Persistent infection of human oligodendrocytic and neuroglial cell lines by human coronavirus 229E.
(13/5127)
Human coronaviruses (HuCV) cause common colds. Previous reports suggest that these infectious agents may be neurotropic in humans, as they are for some mammals. With the long-term aim of providing experimental evidence for the neurotropism of HuCV and the establishment of persistent infections in the nervous system, we have evaluated the susceptibility of various human neural cell lines to acute and persistent infection by HuCV-229E. Viral antigen, infectious virus progeny and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, and oligodendrocytic MO3.13 cell lines, were all susceptible to an acute infection by HuCV-229E. The CHME-5 immortalized fetal microglial cell line was not susceptible to infection by this virus. The MO3.13 and H4 cell lines also sustained a persistent viral infection, as monitored by detection of viral antigen and infectious virus progeny. Sequencing of the S1 gene from viral RNA after approximately 130 days of infection showed two point mutations, suggesting amino acid changes during persistent infection of MO3.13 cells but none for H4 cells. Thus, persistent in vitro infection did not generate important changes in the S1 portion of the viral spike protein, which was shown for murine coronaviruses to bear hypervariable domains and to interact with cellular receptor. These results are consistent with the potential persistence of HuCV-229E in cells of the human nervous system, such as oligodendrocytes and possibly neurons, and the virus's apparent genomic stability. (+info)
A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking.
(14/5127)
Appropriate subcellular localization is crucial for regulating p53 function. We show that p53 export is mediated by a highly conserved leucine-rich nuclear export signal (NES) located in its tetramerization domain. Mutation of NES residues prevented p53 export and hampered tetramer formation. Although the p53-binding protein MDM2 has an NES and has been proposed to mediate p53 export, we show that the intrinsic p53 NES is both necessary and sufficient for export. This report also demonstrates that the cytoplasmic localization of p53 in neuroblastoma cells is due to its hyperactive nuclear export: p53 in these cells can be trapped in the nucleus by the export-inhibiting drug leptomycin B or by binding a p53-tetramerization domain peptide that masks the NES. We propose a model in which regulated p53 tetramerization occludes its NES, thereby ensuring nuclear retention of the DNA-binding form. We suggest that attenuation of p53 function involves the conversion of tetramers into monomers or dimers, in which the NES is exposed to the proteins which mediate their export to the cytoplasm. (+info)
Establishment and characterization of human neuroblastoma cell lines.
(15/5127)
Three new tissue culture cell lines, CHP-100, CHP-126, and CHP-134, have been established from explant cultures of human neuroblastoma. The cell lines have been characterized with respect to morphology, chromosomes constitution, growth, neural enzyme content, and their ability to grow in nude mice. The cells grow as dense masses comprised of fibroblast-or neuroblast-like cells with small processes. The cell lines differ in their neural enzyme acitivity. The chromosomal content of the 3 cell lines is near diploid, and all are capable of forming tumors in nude mice. The morphological findings indicate that the cells in culture resemble those found in the tumor, and the enzyme activities are consistent with those of nervous tissue. This the morphological, biochemical, and tumorigenic properties confirm that the 3 cell lines are neoplastic cells of neural origin. (+info)
Neuroblastoma metastatic to the liver in infants.
(16/5127)
Four infants are described who presented with rapid enlargement of the liver. This was found to be due to neuroblastoma which had metastasized to the liver; the condition was associated with high levels of urinary vanillylmandelic acid (VMA). In 3 infants the primary tumour was in the adrenal gland and in one it was not identified. One infant died after laparotomy and 2 infants survive disease free with normal VMA levels, one after adrenalectomy and hepatic irradiation and one after a short course of chemotherapy. The fourth patient responded initially to hepatic irradiation and chemotherapy, but relapsed 2 years later with recurrent disease; at the same time the VMA level which had been normal, again rose. The importance of screening for an abnormal VMA level in any infant with a rapidly enlarging liver in order to obtain an early diagnosis is stressed. Careful follow-up, with serial VMA estimations, is essential to detect recurrent disease. The prognosis for some infants with this distribution of neuroblastoma which has metastasized to the liver, but not to the bones or oribt, is good. (+info)