Vasoactive intestinal peptide stimulates proliferation in HT29 human colonic adenocarcinoma cells: concomitant activation of Ras/Rap1-B-Raf-ERK signalling pathway

The vasoactive intestinal peptide (VIP) has been shown to regulate cell proliferation and differentiation in many cell types. We previously reported that this neuropeptide inhibited proliferation in HT29 adenocarcinoma cells cultured in serum-containing medium. In addition, it has been demonstrated that VIP induced a potent stimulation of intracellular cAMP production in these cells cultured either in the absence or in the presence of serum. We also demonstrated that VIP induced phosphorylation of the small GTPase Rap1 in these cancerogenous cells. In the present study, the effects of VIP on the proliferation of HT29 cells cultured in the absence of growth factors and various concomitant signalling events were investigated. Under serum-free conditions VIP stimulates HT29 cell proliferation and induced a time- and concentration-dependent ERK activation. Furthermore, VIP induced the activation of the small GTPase Rap1 and of a 95 kDa isoform of the serine/threonine kinase B-Raf. Ras GTPase is also activated in VIP-stimulated cells. We hypothesize that VIP-induced proliferation in HT29 adenocarcinoma cells may involve a cAMP-Rap1/Ras-B-Raf-ERK signalling pathway.     

Intravesical morphine analgesia is not effective after bladder surgery in children: results of a randomied double-blind study

PURPOSE: Intravesical morphine was recently recommended to reduce postoperative pain after reimplantation surgery for vesicoureteral reflux in children. The efficacy of such treatment, so far solely evaluated by open study, needed to be confirmed. MATERIALS AND METHODS: After parental informed consent was obtained, 80 children requiring Cohen cross-trigonal reimplantation were considered for inclusion in a double-blind study. On the day of surgery patients were randomly assigned to receive either 0.04 mg./kg. morphine per hour or placebo (normal saline) at a constant intravesical infusion rate of 0.08 ml./kg. per hour. Postoperative pain was assessed every 3 hours using a pain score adapted to patient age. If the score was above a predefined limit, patients received intravenous acetaminophen and nalbuphine alternately every 3 hours. Bladder infusion was discontinued after 48 hours. RESULTS: Mean and maximum pain scores as well as the number of scores above the limit were not statistically different when comparing the morphine and placebo groups. There was no difference in the number of doses of analgesics administered. Urine output, voiding frequency and the number of painful voiding episodes were not significantly different between the 2 groups. Plasma morphine concentrations were 3.0 +/- 2.7 and 1.9 +/- 1.9 ng./ml. at 24 and 48 hours in the morphine group and undetectable in the placebo group. CONCLUSIONS: Intravesical administration of morphine is not effective for relieving postoperative pain during the first 48 hours after intravesical ureteral reimplantation. This study emphasizes the importance of controlled studies in evaluating the effectiveness of a new drug or procedure before recommending its use for all patients.     

Hyaluronan increases glomerular cyclooxygenase-2 protein expression in a p38 MAP-kinase-dependent process

BACKGROUND: Accumulation of the matrix glycosaminoglycan hyaluronan occurs in many types of renal injury but could follow any provision of hyaluronan substrate to the kidney, for example, through widespread use of supplementary glucosamine in osteoarthritic conditions. Hyaluronan can increase cyclooxygenase-2 (COX-2) protein and prostaglandin production. This effect was characterized in rat renal glomeruli to determine the cellular mechanism of activation. METHODS: Isolated glomeruli were treated with purified hyaluronan (molecular mass 2 x 105 D) for up to 24 hours. RESULTS: An increase in cyclooxygenase capacity and COX-2 protein was shown to follow the activation of p38-mitogen-activated protein (MAP) kinase and to be inhibited by a specific pyridinyl imadazole inhibitor (SB 202190). Hyaluronan-induced activation of cytosolic phospholipase A2 also was shown to be a p38 MAP kinase effect in these preparations. Prostaglandin production was inhibited by COX-2-specific non-steroidal anti-inflammatory compounds (NS-398 and celecoxib) but, as shown for many non-steroidal anti-inflammatory drugs (NSAIDs), an increase in COX-2 protein accompanied this inhibition. CONCLUSIONS: We propose that these findings have clinical relevance. Prostaglandins have a number of important intrarenal regulatory effects leading to some debate over renal function with the use of NSAIDs. Where hyaluronan is increased, p38 MAP-kinase-dependent provision of prostaglandin substrate, via activation of cytosolic phospholipase A2, and a concomitant increase in cyclooxygenase-2 protein would raise renal prostaglandin levels. While NSAID treatment can prevent a rise in prostaglandin levels, it needs to be maintained to avoid possible exacerbation of pro-inflammatory conditions due to increased COX-2 protein levels.     

New targets of beta-catenin signaling in the liver are involved in the glutamine metabolism

Inappropriate activation of the Wnt/beta-catenin signaling has been implicated in the development of hepatocellular carcinoma (HCC), but exactly how beta-catenin works remains to be elucidated. To identify, in vivo, the target genes of beta-catenin in the liver, we have used the suppression subtractive hybridization technique and transgenic mice expressing an activated beta-catenin in the liver that developed hepatomegaly. We identified three genes involved in glutamine metabolism, encoding glutamine synthetase (GS), ornithine aminotransferase (OAT) and the glutamate transporter GLT-1. By Northern blot and immunohistochemical analysis we demonstrated that these three genes were specifically induced by activation of the beta-catenin pathway in the liver. In different mouse models bearing an activated beta-catenin signaling in the liver known to be associated with hepatocellular proliferation we observed a marked up-regulation of these three genes. The cellular distribution of GS and GLT-1 parallels beta-catenin activity. By contrast no up-regulation of these three genes was observed in the liver in which hepatocyte proliferation was induced by a signal-independent of beta-catenin. In addition, the GS promoter was activated in the liver of GS(+/LacZ) mice by adenovirus vector-mediated beta-catenin overexpression. Strikingly, the overexpression of the GS gene in human HCC samples was strongly correlated with beta-catenin activation. Together, our results indicate that GS is a target of the Wnt/beta-catenin pathway in the liver. Because a linkage of the glutamine pathway to hepatocarcinogenesis has already been demonstrated, we propose that regulation of these three genes of glutamine metabolism by beta-catenin is a contributing factor to liver carcinogenesis.     

Prevention of diabetes-induced albuminuria in transgenic rats overexpressing human aldose reductase

Studies using pharmacologic inhibitors have implicated the enzyme aldose reductase in the pathogenesis of albuminuria and diabetic renal disease. However, a clear conclusion is not easily drawn from such studies since these pharmacologic inhibitors have nonspecific properties. To examine further the role of aldose reductase, we have overexpressed the human enzyme in a transgenic rat model. Transgene expression in the kidney was predominantly localized to the outer stripe of the outer medulla, compatible with the histotopography of the straight (S3) proximal tubule. The effect of enzyme overexpression on diabetes-induced renal function and structure was then investigated. Contrary to what may have been anticipated from the previous enzyme inhibition studies, diabetes-induced albuminuria was completely prevented by the overexpression of aldose reductase. No effect of overexpression of aldose reductase on renal structure nor on urinary excretion of β₂-microglobulin and N-acetyl-β-d-glucosaminidase was observed in this transgenic rat model. In conclusion, our study strongly suggests that multiple roles for aldose reductase may give it a more complex place in diabetic nephropathy than is currently recognized.     

Comparative Preclinical Study of Three Bone Marrow Purging Methods Using PCR Evaluation of Residual t(14;18) Lymphoma Cells

The t(14;18) chromosomal translocation occuring in most follicular lymphomas can be exploited by a Bcl2/JH polymerase chain reaction (PCR) to detect residual disease and to monitor the effectiveness of ex-vivo tumor cell immunological purging. We first demonstrated the 10^-5 Bcl2/JH PCR sensitivity with serial dilutions of OCY-LY8 lymphoma cell lines in normal mononuclear cells; and then the specificity and reproductibility of this technique by analysing follicular and non follicular lymphoma samples. With the Bcl2/JH PCR, we tested the efficiency of three marrow purging protocols with an experimentally contaminated bone marrow either treated by three anti-B cell monoclonal antibodies (mAb) followed by three rounds of rabbit complement or two rounds of immunomagnetics beads. Samples obtained after each purging were amplified by Bcl2/JH PCR and hybridized with PFL3 probe. We were able to produce a 2 to 3 log tumor cell reduction after three rounds of complement and a 4 to 5 log reduction after two rounds of beads.

This study showed that it is feasible to use the Bcl2/JH PCR technique for residual cell lymphoma detection in patients undergoing intensive chemotherapy or BM transplantation. These results indicate that ex-vivo immunomagnetic BM purging is probably superior to complement mediated lysis for the eradication of B lymphoma cells from the marrow of patients undergoing autologous transplantation.     

Genetics: Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities

In the present study we investigated the results of cyto-geneticanalysis in male and female patients included in an intracytoplasmicsperm injection (ICSI) programme for severe male infertilityas well as in conceptuses resulting from these ICSI treatments.In the 261 couples treated, 11 male (4.2%) and three female(1.2%) abnormal karyotypes were found, all consisting of structuralchromosome anomalies. Chromosomal translocation exhibited thehighest frequency (eight males and two females), and there werealso three cases of chromosomal inversion (two males and onefemale) and one male with one additional marker chromosome.There was no difference in fertilization rates among coupleswith abnormal (n = 14) and normal (n = 147) cytogenetic results,and the rates of clinical pregnancy per ICSI attempt were 25.0%(5/20) and 20.6% (78/378) respectively. In pregnancies obtainedin couples with normal karyotypes, all of the 108 fetuses werefree of chromosomal abnormalities. Among the eight fetuses fromcouples with chromosome structural anomalies, three out of fiveand two out of three inherited the cytogenetic defects foundin their father or mother respectively. In this series of 83ICSI pregnancies there were no chromosomal abnormalities otherthan those inherited from the parents. These findings suggestthat normal pregnancy rates can be obtained by ICSI in casesof chromosomal translocation in couples with severe male infertility.However, until further evaluations of available data can beperformed, cytogenetic analysis must be conducted prior to ICSIin men with low sperm counts, and genetic counselling must includeprenatal diagnosis for all growing conceptuses.