Regulation of chondrogenesis in the developing inner ear: a role for sonic hedgehog.
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Animals↗
Biomedical subjects
W Liu
Publications and source records attributed to W Liu.
Chemosignal transduction in the vomeronasal organ of garter snakes: cloning of a gene encoding adenylate cyclase from the vomeronasal organ of garter snakes.
We previously reported that ES20-receptor binding activates phosphoinositide (PI) turnover, resulting in an increase in inositol-1,4,5-trisphosphate, which in turn mobilizes intracellularly stored calcium in the vomeronasal (VN) sensory epithelium of garter snakes. We also found that the activity of adenylate cyclase (AC) in the VN organ is very sensitive to Ca2+ but insensitive to calmodulin regulation. A 250-bp fragment of adenylate cyclase type VI (AC-VI) was obtained from brain cDNA of garter snake by RT-PCR with degenerate primers. The 250-bp fragments were amplified, cloned, and sequenced. Both Northern blot and RNase protection assays revealed that the vomeronasal organ (VNO) and brain contained more abundance of AC type VI than the main olfactory epithelium. A 3.8-kb cDNA was then cloned from the vomeronasal cDNA library of garter snakes and sequenced. The 5' cDNA was obtained by means of 5' RACE PCR and sequenced. We have successfully cloned a 5200-nucleotide cDNA from VNO of garter snakes containing an open reading frame++ encoding 1150 amino acids of AC-VI protein. The vomeronasal AC is termed AC(VN) . AC(VN) shows a high degree of homology with type VI AC of rat, mouse, or human. In situ hybridization with digoxigenin-labeled cRNA demonstrated that AC(VN) mRNA was abundant in the sensory epithelium but not in the nonsensory epithelium of the mushroom body of the vomeronasal organ of garter snakes.
Adenylyl Cyclases↗
Transgenic mouse model for skin malignant melanoma.
We report here on a novel metallothionein-I (MT)/ret transgenic mouse line in which skin melanosis, benign melanocytic tumor and malignant melanoma metastasizing to distant organs develop stepwise. The process of tumor development and its malignant transformation in this line may resemble that of the human giant congenital melanocytic nevus that is present at birth and that frequently gives rise to malignant melanoma during aging. We observed an increase in the expression level and activity of the ret transgene during the disease progression. That increase in transgene expression accompanied an activation of mitogen-activated protein kinases (MAPKs) and c-Jun as well as matrix metalloproteinases. These results suggest that progressive dysregulation of the expression level of the ret transgene might play a crucial role in the malignant transformation of melanocytic tumors developed in the MT/ret transgenic mouse line.
Animals↗
Loss of imprinting and allele switching of p73 in renal cell carcinoma.
p73, a protein that has substantial structural and functional similarity to p53, has recently been identified. It was found to be monoallelically expressed in all cell lines and normal individuals tested. To elucidate its role in cancer development and as a potential imprinted tumor suppressor, we investigated the allele-specific expression of the human p73 gene in 28 cases of renal cell carcinoma and its imprinting status in fetal pancreatic and thymic tissues. Of 12 informative pairs of renal cell carcinoma and matched normal tissues identified by StyI restriction fragment length polymorphism (RFLP) in exon 2, p73 showed monoallelic expression in 11 out of 12 normal tissues but biallelic expression in 8/12 and switched allele expression in 2/12 of the matched corresponding cancers. An imprinting study of the p73 gene in two families using a newly identified exonic BanI RFLP indicated that expression of p73 was limited to the maternal allele in RNA from fetal pancreas and thymus, demonstrating that p73 is imprinted in at least these two tissues. These findings strongly suggest that loss of imprinting or switching of allelic expression of the p73 gene is associated with the development of renal cell carcinoma.
Alleles↗
Histological characterization and pharmacological control of chronic rejection in xenogeneic and allogeneic heart transplantation.
BACKGROUND: Chronic allograft rejection remains a major barrier to successful long-term allograft transplantation in humans. Chronic allograft rejection is characterized by the appearance of arterial lesions with concentric intimal thickening. This study investigates the development and control of chronic rejection in hamster cardiac xenografts transplanted into Lewis rats. METHODS: Chronic rejection in the xenograft model involves transplantation of hamster hearts into Lewis rats treated with leflunomide (Lef) continuously at 15 mg/kg/day. The allograft model involves transplantation of Lewis hearts into Fisher-334 rats treated with cyclosporine (CsA) at 2.5 mg/kg for 5 days. RESULTS: The average scores of arterial intimal thickening on day 45 after transplantation were 1.89+/-0.43 in the xenograft and 2.50+/-0.72 in the allograft. The basic pathology of both xenografts and allografts undergoing chronic rejection was arterial intimal thickening comprising smooth muscle cell proliferation, mononuclear cell infiltration, and fibrosis. The majority of cells infiltrating the arterial intima and myocardium were T cells and macrophages. Compared with the allograft, intimal edema, matrix deposition and fibrinoid necrosis were specifically presented in the xenografts and generally involved the larger arteries. The predominant isotype of antibody deposited was IgM in xenografts and IgG in allografts. When combined Lef and CsA therapy was initiated on day 45 after transplantation, the changes of chronic rejection were reversed in both xenografts and allografts by day 90. The scores of intimal thickening were significantly reduced to 0.97+/-0.45 and 1.48+/-0.56, respectively. CONCLUSIONS: We conclude that chronic rejection can be induced in xenografts under conditions of inadequate immunosuppression. Chronic rejection in xenografts involves arterial lesions that bear some histological similarities to, as well as differences from, those observed in chronically rejected allografts. Finally, combination therapy with CsA and Lef reduced the incidence and severity of the intimal lesions in both chronically rejecting xenografts and allografts.
Animals↗
Norepinephrine inhibits neurons of the intermediate subnucleus of the lateral septum via alpha2-adrenoceptors.
The physiological and pharmacological actions of norepinephrine (NE) on neurons of the intermediate subnucleus of the lateral septum (LSI) were examined using intracellular recordings in rat brain-slices. Bath-applied NE inhibited 72.5%, excited 5.5% and had no effect on 22% of LSI neurons tested; this study focused on the inhibitory effects of NE. In current clamp recordings, 100 microM NE produced a hyperpolarization of 10.82+/-0.72 mV (n=84) with a decrease in input resistance. In voltage-clamp, NE produced a direct, post-synaptic outward current of 206.8+/-22 pA (n=37) with a 64. 3+/-4.9% increase in input conductance (IC50-17.7+/-4 microM). The NE-induced inhibition was mimicked by the alpha2-agonist, UK14,304, but not by the alpha1- or beta-adrenoceptor agonists. The alpha2-agonist, clonidine, had a weak effect in LSI neurons. Interestingly, the magnitude of the UK14,304-induced response varied between cells (ranging from 29.5 to 320% of the maximal NE inhibition), possibly suggesting the involvement of alpha2A-(high affinity for UK14,304) and non-alpha2A (low affinity for UK14,304) adrenoceptor subtypes. While the alpha2-antagonists, yohimbine, rauwolscine and idazoxan blocked NE-induced inhibition in all neurons tested, the prototypical alpha1-antagonist, prazosin produced a variable degree of block (9-58%), further indicating the possible involvement of alpha2A (prazosin-insensitive) and non-alpha2A (prazosin-sensitive) receptors. However a lack of more selective pharmacological tools precludes definitive classification of the alpha2-receptor-mediated responses into different subtypes. The alpha2-receptor-mediated current in LSI neurons displayed Ba2+-sensitive inward rectification, reversed polarity near EK and was sensitive to external K+. In conclusion, NE inhibits LSI neurons via alpha2-adrenoceptor subtypes.
Adrenergic alpha-Agonists↗
Excitatory effects of muscarine on septohippocampal neurons: involvement of M3 receptors.
Cholinergic mechanisms in the septohippocampal pathway contribute to several cognitive functions and impaired cholinergic transmission in this pathway may be related to the memory loss and dementia that accompanies normal aging and Alzheimer's disease and behavioral studies suggest that muscarinic mechanisms in the medial septum/diagonal band of Broca (MSDB) may contribute to these functions. The goal of the present study was to begin a characterization of the physiological and pharmacological effects of muscarine on antidromically identified septohippocampal neurons (SHNs). Muscarinic agonists produced a concentration-dependent excitation in >90% of SHNs tested using extracellular recordings in an in vitro rat brain slice preparation. The SHNs excited by muscarine had a broad range of conduction velocities (0.2 to 3.7 m/s; mean: 1.6+/-0.06 m/s; n=110), suggesting involvement of neurons with both slow (possibly cholinergic) and fast (possibly GABAergic) conducting fibers. The muscarine-induced excitations in SHNs were found not to be mediated via M1, M2 or M4 receptors, as they were not blocked by the M1-selective antagonists, pirenzepine or telenzepine or by the M2/M4-selective antagonist, methoctramine. In contrast, the M3-selective antagonist, 4-DAMP-mustard, blocked muscarinic excitations in a majority of SHNs, indicating the presence of M3 as well as non-M3-type responses. McN-A-343, an M1 and M5-selective agonist, excited 33% of neurons tested, confirming involvement of non-M3 receptors (possibly M5) and M3 receptors. Since the cholinergic and GABAergic MSDB neurons together innervate almost every type of hippocampal neuron, the effects of muscarine on SHNs would also have a profound effect on hippocampal circuitry.
Animals↗
Regulation of vascular endothelial growth factor expression in human colon cancer by insulin-like growth factor-I.
We investigated the role of insulin-like growth factor (IGF)-I and IGF-binding proteins (IGFBPs) in the regulation of vascular endothelial growth factor (VEGF) expression in colon cancer cells and the mechanism by which this regulation occurs. HT29 human colon cancer cells were treated with IGF-I for various time periods. VEGF mRNA expression increased within 2 h and peaked at 24 h. SW620 colon cancer cells exhibited a peak induction of VEGF mRNA 8 h after IGF-I treatment. IGF-I induction of VEGF was confirmed at the protein level. In experiments using transient transfection of VEGF promoter-reporter constructs into HT29 cells, IGF-I increased the activity of the VEGF promoter, and pretreatment of HT29 cells with dactinomycin abrogated the induction of VEGF mRNA by IGF-I. The half-life of VEGF mRNA was not prolonged by treatment with IGF-I. Blocking the activity of IGFBP-4 did not significantly modulate the effect of IGF-I induction of VEGF mRNA in HT29 cells. Treating cells with des-(1-3)-IGF-I (an active derivative of IGF-I that does not bind to binding proteins) had effects on VEGF mRNA expression that were similar to those of IGF-I. These findings suggest that IGF-I regulates VEGF expression in human colon cancer cells by induction of transcription of the VEGF gene. IGFBPs do not significantly affect IGF-I induction of VEGF.
Colonic Neoplasms↗
Heparan sulfate proteoglycans as adhesive and anti-invasive molecules. Syndecans and glypican have distinct functions.
ARH-77 cells do not adhere to type I collagen and readily invade into collagen gels, but following expression of the transmembrane heparan sulfate proteoglycan syndecan-1, they bind collagen and fail to invade. We now show that cells transfected with syndecan-2 or syndecan-4 also bind collagen and are non-invasive. In contrast, cells transfected with the glycosylphosphatidylinositol-anchored proteoglycan glypican-1 do not bind to collagen and remain invasive, even though glypican- and syndecan-expressing cells have similar surface levels of heparan sulfate, and their proteoglycans have similar affinities for collagen. Analysis of cells expressing syndecan-1-glypican-1 chimeric proteoglycans reveals that inhibition of invasion requires the extracellular domain of syndecan but not its transmembrane or cytoplasmic domain. Surprisingly, cells bearing a chimera composed of the glypican extracellular domain fused to the syndecan transmembrane and cytoplasmic domains bind to collagen but remain invasive, implying that adhesion to collagen is not by itself sufficient to inhibit invasion. Apparently, the extracellular domain of syndecan-1, presumably by interacting with cell-surface signal transducing molecules, directly regulates complex cell behaviors such as motility and invasiveness. These results also show for the first time that syndecans and glypicans can have distinct functions, even when expressed by the same cell type.
Animals↗
Caspase inhibitors prevent cisplatin-induced apoptosis of auditory sensory cells.
In vitro studies tested the efficacy of three caspase inhibitors, Ac-VAD-cmk (caspase-1 inhibitor), z-DEVD-fmk (caspase-3 inhibitor) and B-D-fmk (BOCDFK, a general inhibitor), for protecting auditory sensory cells from cisplatin-damage induced loss. Treatment of 3-day-old rat organ of Corti explants with these caspase inhibitors protected > 80% of the auditory hair cells from cisplatin-damage initiated apoptosis. Dissociated cell cultures of 3-day-old rat spinal ganglia treated with any of these three caspase inhibitors in addition to exogenous neurotrophin have highly significant increases in neuronal survival following cisplatin exposure. These results indicate that loss of auditory sensory cells as a result of cisplatin-induced damage involves apoptosis and that blocking of this cell death pathway at the caspase level effectively rescues both hair cells and neurons.
Animals↗
Genomic organization and mutation analysis of p73 in oligodendrogliomas with chromosome 1 p-arm deletions.
p73, a protein having substantial structural and functional similarity to p53, has recently been identified and demonstrated to be a potential tumor suppressor. Its location on human chromosome 1p36.33 implicates p73 as a candidate for neuroblastoma. Like neuroblastoma, oligodendrogliomas also show a high frequency of deletions in chromosome 1p36.3. To determine whether p73 is a potential tumor suppressor gene involved in the development of oligodendrogliomas, we performed mutation analysis of p73 in oligodendrogliomas with chromosome 1 p-arm deletions. We first determined the genomic organization and the intron-exon boundary sequences of the p73 gene by long PCR, vectorette PCR, and Southern hybridization. This gene spans about 65 kb with a large first intron. Primer pairs for the amplification of each of the 13 p73 encoding exons were designed in corresponding introns. The amplicons were then analyzed using the denaturing high-performance liquid chromatography system for mutations in the p73 gene. Twenty oligodendroglioma samples with 1p36.3 deletions were screened, but no mutations were detected except for several polymorphisms. It is thus clear that p73 is not a candidate gene for oligodendroglioma despite its location in the frequently deleted 1p36.3 region.
Chromosomes, Human, Pair 1↗
Discovery of estrogen-responsive genes using an improved method which combines subtractive hybridization and PCR.
Here we describe a reliable method for isolating genes that are differentially expressed in two cell populations. The method is a combination of subtractive hybridization and PCR. Among many improvements to previously described methods is the incorporation of a new technology into the procedure which sterilizes(inactivates) PCR amplicons, and thereby overcomes the limitation of similar procedures. To test this improved method, we conducted a search for estrogen-responsive genes. Estrogen-regulated genes dominated the subtracted libraries after four rounds of subtractive hybridizations. Four estrogen-regulated genes were identified from the initial screening.
Estrogens↗
PTEN/MMAC1 mutations identified in small cell, but not in non-small cell lung cancers.
A putative tumor suppressor, PTEN/MMAC1 gene at 10q23 was recently identified and found to be mutated in many different human tumors. To determine the role of the PTEN/MMAC1 gene in lung cancer, we screened 34 small cell lung cancer (SCLC) cell lines, 10 SCLC tumors, 13 non-small cell lung cancer (NSCLC) cell lines and 10 NSCLC tumors using Denaturing HPLC (DHPLC) and direct sequencing methods. In SCLC, six (18%) of the cell lines and one of the primary tumor samples (10%) showed alterations of the PTEN/MMAC1 gene including point mutations, small fragment deletions, and homozygous deletions. All of the point mutations and small fragment deletions were observed in hemizygously deleted cell lines. In contrast to SCLC, none of the NSCLC tumors or cell lines had mutations in the PTEN/MMAC1 gene. These data indicate that PTEN/MMAC1 mutations contribute to the pathogenesis and neoplastic evolution in SCLC but not in NSCLC.
Carcinoma, Non-Small-Cell Lung↗
5-HT1A receptors mediate inhibition of ethanol-induced ascorbic acid release in rat striatum studied by microdialysis.
Our previous study showed that the serotonergic system was involved in the ethanol-induced striatal ascorbic acid release in rat. In the present study, the 5-HT1A agonists and antagonists were used to analyze the possible mechanism of ethanol-induced ascorbic acid release in rat striatum. The results showed that ethanol (3.0 g/kg, i.p.) significantly increased striatal ascorbic acid release. Buspirone (5.0 mg/kg, s.c.), a partial agonist of 5-HT1A receptors, and 8-OH-DPAT (0.5 mg/kg, s.c.), a selective agonist of 5-HT1A receptors, showed no effect on basal ascorbic acid release in striatum, but both drugs significantly antagonized the ascorbic acid release induced by ethanol in striatum. WAY 100635 (0.5 mg/kg, s.c.), a selective antagonist of 5-HT1A receptors, affecting neither the basal nor the ethanol-induced ascorbic acid release per se, antagonized the suppressing effect of 8-OH-DPAT on ethanol-induced ascorbic acid release in striatum. This study gives the first evidence that activation of 5-HT1A receptors is involved in ethanol-induced ascorbic acid release in rat striatum.
Animals↗
[Studies of microsatellite instability in Chinese gastric cancer tissues].
OBJECTIVE: To identify the microsatellite instability(MSI) rates in Chinese gastric cancer samples. METHODS: 29 microsatellite markers were selected to examine 42 paired gastric cancer tissues for MSI on all chromosomes except Y. RESULTS: The total frequency of MSI in all 42 gastric cancers was 33.9% with higher rates at loci of D3S1577, D3S1067,D8S279,D9S257, D1S248, D7S520 and D2S147,and the highest rate at D3S1577 and D3S1067(51.35%). MSI varied with different pathological types. The frequencies of MSI were signi- ficantly higher in poorly differentiated tumors and signet cell types, compared with well differentiated tumors(P=0.0026 and 0.0013 by chi-square test),and no difference was noted between poorly differentiated and signet cell types. CONCLUSION: MSI may play an important role in Chinese gastric cancer, particularly the poorly differentiated adenocarcinomas. The data presented here further support the previous hypothesis that pathologically distinct subtypes of gastric cancer undergo different genetic pathways during tumorigenesis.
Adult↗
Autocrine and paracrine apoptosis are mediated by differential regulation of Fas ligand activity in two distinct Jurkat T cell populations.
Fas ligand (FasL) produced by activated T cells mediates autocrine-induced apoptosis to limit T cell expansion. To investigate the regulation of FasL activity, Jurkat cells were stably transfected with a 2.3-kb fragment of human FasL promoter that controlled the expression of a GFP reporter gene. Two populations of Jurkat cells with different levels of GFP expression were obtained. One population constitutively expressed high levels of GFP (GFP+), while the other population expressed low levels of GFP (GFP-). The level of GFP expression in the two populations correlated with their levels of FasL transcription and its functional activity. Autocrine regulation of apoptosis was demonstrated by increased FasL activity after stimulation of GFP- cells with anti-CD3, phorbyl myristyl acetate plus ionomycin, or Con A. Paracrine regulation of apoptosis was suggested by the induction of apoptosis of GFP- cells after coculture with unstimulated GFP+ cells. GFP+ cells exhibited a decreased sensitivity to FasL-mediated apoptosis compared with GFP- cells. Furthermore, the cell surface expression of Fas and CD4 was lower on GFP+ cells than GFP- cells, whereas the expression of CD45RO was higher. A decreased level of IL-2 was produced by GFP+ cells after phorbyl myristyl acetate and ionomycin stimulation. Our results indicate that a subpopulation of T cells that express low levels of FasL and IL-2, which are responsive to up-regulation of these molecules after activation, can undergo apoptosis either by suicide after activation or by a paracrine pathway mediated by T cells that constitutively express higher levels of FasL.
Apoptosis↗
Adenovirus-mediated wild-type p53 gene transfer down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human colon cancer.
Recent studies have indicated that angiogenesis may be regulated, in part, by p53 tumor suppressor gene function. We hypothesized that wild-type p53 replacement would down-regulate vascular endothelial growth factor (VEGF) expression and inhibit angiogenesis. KM12L4 and SW620, human colon cancer cell lines with p53 mutations, were transduced with a replication-defective adenoviral vector containing the wild-type p53 gene (Ad5/CMV/p53). Reverse transcription-PCR confirmed the presence of p53 in Ad5/CMV/p53-transduced cells. Transduction of colon cancer cells with wild-type p53 decreased VEGF RNA expression compared with that of controls. The decrease in VEGF expression in SW620 cells was dose dependent, with a 49% decrease observed at a multiplicity of infection of 50, and a 71% decrease observed at a multiplicity of infection of 100. Similar effects were seen in KM12L4 cells. VEGF supernatant protein levels were significantly reduced compared with those in nontransduced controls 48 h after the introduction of wild-type p53. Ad5/CMV/p53 inhibited tumor cell-induced angiogenesis in vivo. Restoration of wild-type p53 expression may decrease tumor growth by inhibiting the angiogenic response. These findings may explain, in part, the bystander effect seen with p53 tumor suppressor gene therapy.
Adenoviridae↗
Activation of p73 silent allele in lung cancer.
p73, a first p53 relative, has recently been identified and demonstrated to be monoallelically expressed. This protein shows significant amino acid sequence and functional similarities to p53. However, it is unclear whether this protein functions as a tumor suppressor. To elucidate the role of p73 in tumor development, we investigated the expression of the p73 gene in lung cancer. In a comparison between normal lung and tumor tissues, p73 was more highly expressed in tumors. Moreover, using a C/T polymorphism in exon 2 for allele-specific expression analysis in 21 pairs of lung tumors and matched normal tissues, we found that five heterozygous samples exclusively expressed both alleles in tumors while showing monoallelic expression in matched normal tissues. This result was confirmed by single-nucleotide primer extension analysis. Mutation analysis of all 13 coding exons of the gene in 21 lung tumor DNAs revealed several polymorphisms, but no tumor-specific mutations were detected. These findings strongly suggest that p73 may play an important role in lung tumorigenesis through activation of a silent allele and overexpression of wild-type p73 rather than as a tumor suppressor.
Alleles↗