The organization, distribution, and function of neural progenitor cells (NPCs) in the adult spina... more The organization, distribution, and function of neural progenitor cells (NPCs) in the adult spinal cord during motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remain largely unknown. Using nestin promoter controlled LacZ reporter transgenic mice and mutant G93A-SOD1 transgenic mice mimicking ALS, we showed that there was an increase of NPC proliferation, migration, and neurogenesis in the lumbar region of adult spinal cord in response to motor neuron degeneration. The proliferation of NPCs detected by BrdU incorporation and LacZ staining was restricted to the ependymal zone surrounding central canal (EZ). Once the NPCs moved out from the EZ, they lost the proliferative capability, but maintained migratory function vigorously. During ALS-like disease onset and progression, NPCs in the EZ migrated initially toward the dorsal horn direction, and then to the ventral horn regions, where motor neurons have degenerated. More significantly, there was an increased de novo neurogenesis from NPCs during ALS-like disease onset and progression. The enhanced proliferation, migration, and neurogenesis of (from) NPCs in the adult spinal cord of ALSlike mice may play an important role in attempting to repair the degenerated motor neurons and restore the dysfunctional circuitry which resulted from the pathogenesis of mutant SOD1 in ALS.
Background The Na1.7 subtype of voltage-gated sodium channels is specifically expressed in sensor... more Background The Na1.7 subtype of voltage-gated sodium channels is specifically expressed in sensory and sympathetic ganglia neurons where it plays an important role in the generation and transmission of information related to pain sensation. Human loss or gain-of-function mutations in the gene encoding Na1.7 channels (SCN9A) are associated with either absence of pain, as reported for congenital insensitivity to pain, or with exacerbation of pain, as reported for primary erythromelalgia and paroxysmal extreme pain disorder. Based on this important human genetic evidence, numerous drug discovery efforts are ongoing in search for Nav1.7 blockers as a novel therapeutic strategy to treat pain conditions. Results We are reporting here a novel approach to study Na1.7 function in cultured rat sensory neurons. We used live cell imaging combined with electrical field stimulation to evoke and record action potential-driven calcium transients in the neurons. We have shown that the tarantula veno...
CGRP is a potent vasodilator and nociceptive neuropeptide linked to migraine. CGRP receptors are ... more CGRP is a potent vasodilator and nociceptive neuropeptide linked to migraine. CGRP receptors are heterodimers of receptor activity-modifying protein-1 (RAMP1) and either calcitonin receptor-like receptor (CLR), which forms the canonical CGRP receptor, or calcitonin receptor (CTR), which forms the amylin-1 (AMY1 ) receptor. The goal of this study was to test whether transgenic mice globally expressing human RAMP1 (hRAMP1) have increased CGRP receptor activity and whether the receptors are sensitive to the human selective antagonist telcagepant. cAMP production was measured in primary cultures of aortic smooth muscle and trigeminal ganglia neurons from global hRAMP1 mice and nontransgenic littermates. Functional activity and inhibition was compared with clonal cell lines expressing combinations of CLR or CTR with RAMP1. Cultured smooth muscle from global hRAMP1 mice had a 10-fold greater CGRP-induced cAMP maximal response (Rmax) than non-transgenic littermates, with similar EC50 '...
Thirty percent of human breast cancers have amplification of ERBB2, often in conjunction with mut... more Thirty percent of human breast cancers have amplification of ERBB2, often in conjunction with mutations in p53. The most common p53 mutation in human breast cancers is an Arg-to-His mutation at codon 175, an allele that functions in a dominant oncogenic manner in tumorigenesis assays and is thus distinct from loss of p53. Transgenic mice expressing mouse mammary tumor virus-driven neu transgene (MMTV-neu) develop clonal mammary tumors with a latency of 234 days, suggesting that other events are necessary for tumor development. We have examined the role of mutations in p53 in tumor development in these mice. We have found that 37% of tumors arising in these mice have a missense mutations in p53. We have directly tested for cooperativity between neu and mutant p53 in mammary tumorigenesis by creating bitransgenic mice carrying MMTV-neu and 172Arg-to-His p53 mutant (p53-172H). In these bitransgenic mice, tumor latency is shortened to 154 days, indicating strong cooperativity. None of t...
Loss of heterozygosity or mutation at the p53 tumor suppressor gene locus is frequently associate... more Loss of heterozygosity or mutation at the p53 tumor suppressor gene locus is frequently associated with advanced human prostate cancer. Hence, replacement p53 gene therapy may prove to be efficacious for this disease. While many mutations result in p53 molecules with oncogenic properties, other variants may possess wild-type properties with increased tumor suppressor activity. We have chosen to investigate the activity of a naturally occurring variant p53 molecule, p53(R172L), carrying an arginine-to-leucine mutation at codon 172. We demonstrate that p53(R172L) can differentially activate expression of genes involved in cell cycle control and apoptosis in vitro. Transgenic mice expressing a subphysiological level of a p53(R172L) minigene (PB-p53(R172L)) in the prostate epithelium were generated and bred to the transgenic adenocarcinoma mouse prostate (TRAMP) model of prostate cancer. While PB-p53(R172L) transgenic mice developed normally with no detectable prostate gland phenotype, ...
Bone metastases from hepatocellular carcinoma (HCC) seem to be increasing. Previous studies showe... more Bone metastases from hepatocellular carcinoma (HCC) seem to be increasing. Previous studies showed that soluble factors secreted by host cells and direct cell-to-cell interactions contributed to the preferential metastasis and growth of cancer cells in bone, while the underlying mechanism(s) of the metastasis of HCC in the bone are poorly understood. Here, we determined the effect of HS-5 cells on Huh7 cell proliferation, and investigated the role of CCL5 from HS-5 cells on the development of Huh7 cells. In addition, the underlying mechanisms on the influence in Huh7 cells were investigated. Our results showed that HS-5 cells could promote the proliferation, migration and invasion of Huh7 cells, and inhibited apoptosis. CCL5 downregulation was able to inhibit the effects of HS-5 cells on Huh7 cell migration and invasion via the PI3K-Akt signaling pathway and reduce MMP-2 expression. Therefore, these findings suggest that CCL5 secreted from MSCs can promote the migration and invasion...
ER81 is a transcription factor that may contribute to breast cancer; however, little known about ... more ER81 is a transcription factor that may contribute to breast cancer; however, little known about the role of ER81 in breast carcinogenesis. To investigate the role of ER81 in breast carcinogenesis, we examined ER81 expression in IDC, DCIS, ADH, HUT, and normal breast tissues by immunohistochemical staining. We found that ER81 overexpression was detected in 25.7% (9/35) of HUT, 41.2% (7/17) of ADH, 54.5% (12/22) of DCIS, and 63.0% (51/81) of IDC. In 20 of breast cancer tissues combined with DCIS, ADH, and HUT, ER81 expression was found in 14/20 (70%) IDC. In these 14 cases all cases were ER81 positive expression in DCIS, 13 of 14 cases were positively expressed of ER81 in ADH and 8 of 14 were positive for ER81 in HUT components. A statistical significance was found between NBT and HUT () and HUT and ADH (). Clinical-pathological features analysis of breast cancer revealed that ER81 expression was significantly associated with Her2 amplification and was negatively associated with ER a...
Objective: Investigate a role for calcitonin gene-related peptide (CGRP) in osteoarthritis (OA)-r... more Objective: Investigate a role for calcitonin gene-related peptide (CGRP) in osteoarthritis (OA)-related pain. Design: Neutralizing antibodies to CGRP were generated de novo. One of these antibodies, LY2951742, was characterized in vitro and tested in pre-clinical in vivo models of OA pain. Results: LY2951742 exhibited high affinity to both human and rat CGRP (K D of 31 and 246 pM, respectively). The antibody neutralized CGRP-mediated induction of cAMP in SK-N-MC cells in vitro and capsaicin-induced dermal blood flow in the rat. Neutralization of CGRP significantly reduced pain behavior as measured by weight bearing differential in the rat monoiodoacetate model of OA pain in a dose-dependent manner. Moreover, pain reduction with neutralization of CGRP occurred independently of prostaglandins, since LY2951742 and NSAIDs worked additively in the NSAID-responsive version of the model and CGRP neutralization remained effective in the NSAID non-responsive version of the model. Neutralization of CGRP also provided dose-dependent and prolonged (>60 days) pain reduction in the rat meniscal tear model of OA after only a single injection of LY2951742. Conclusions: LY2951742 is a high affinity, neutralizing antibody to CGRP. Neutralization of CGRP is efficacious in several OA pain models and works independently of NSAID mechanisms of action. LY2951742 holds promise for the treatment of pain in OA patients.
Accumulating evidence has suggested that cellular production of superoxide acts as an intracellul... more Accumulating evidence has suggested that cellular production of superoxide acts as an intracellular messenger to regulate gene expression and modulate cellular activities. In this report, we set out to investigate the role of active H-ras-mediated superoxide production on tumor cell malignancy in a SV-40 transformed human lung WI-38 VA-13 cell line. Stable transfection and expression of constitutively active mutant V12-H-ras (V12-H-ras) dramatically increased intracellular production of superoxide. The expression of V12-H-ras signi®cantly enhanced cell proliferation, migration and resistance to TNF-a treatment compared to that of parental and vector control cells, while expression of wild type H-ras (WT-H-ras) only had modest eects. Upon scavenging by superoxide dismutase and other molecules that decrease the intracellular level of active H-ras mediated superoxide production, cell proliferation, migration and resistance to TNF-a were signi®cantly reduced. Furthermore, we demonstrated that the activation of membrane NADPH oxidase activity by expression of active H-ras contributed to the intracellular superoxide production. The causal relationship between membrane superoxide production and increased cell proliferation, migration, and resistance to TNF-a by the expression of active H-ras, has provided direct evidence to demonstrate that superoxide acts as an intracellular messenger to cascade ras oncogenic signal relay and to modulate tumor malignant activity.
Missense mutations in the p53 tumor suppressor occur frequently in human breast cancer and in¯uen... more Missense mutations in the p53 tumor suppressor occur frequently in human breast cancer and in¯uence both the prognosis and response to chemotherapy. Amino acid 175 (equivalent to murine 172) is the second most common site of missense mutations in p53 in human breast cancer. Over 95% of these mutations are arginine-to-histidine (R-H) substitutions resulting in a gain-of-function, and not merely a dominant-negative phenotype. Transgenic mice expressing a p53 172 R ± H construct targeted to the mammary gland by means of a whey acidic protein (WAP) promoter were characterized as a model system in order to determine the speci®c eects of this mutation on mammary tumorigenesis. Although transgene expression alone had no apparent eect on normal mammary development, transgenic mice treated with the chemical carcinogen dimethylbenz(a)anthracene developed tumors with much shorter latency than did control littermates and had a greater tumor burden. Tumors arising in transgenic mice did not exhibit either decreased apoptosis or increased cell proliferation relative to tumors arising in nontransgenic littermates, but did display increased genomic instability. Large pleiomorphic nuclei were visible in many tumors from transgenic mice, and DNĀ ow analysis con®rmed the presence of signi®cant aneuploid cell populations. Since these transgenic mice develop very few spontaneous tumors, while accelerating carcinogen-and oncogene-mediated tumorigenesis, this mouse model will, therefore, be useful in the investigation of early events in mammary tumorigenesis. It may also be used as a preclinical model to test newly developed chemotherapeutic strategies.
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1994
Regulatory sequences derived from the rat whey acidic protein gene have been used to preferential... more Regulatory sequences derived from the rat whey acidic protein gene have been used to preferentially overexpress a murine 172Arg-->Leu mutant p53 in the mammary gland of transgenic mice. Several different lines of mice expressing the 172Arg-->Leu mutant p53 displayed an impaired ability to lactate, and the mice expressing the highest levels of mutant p53 were unable to nurse their young. This failure was related to the inhibition of normal lobuloalveolar development that occurred during late pregnancy and a marked decrease in milk protein gene expression at early lactation. Interestingly, immunohistochemical analysis revealed that the mutant p53 was localized predominantly in the cytoplasm of alveolar cells. Ductal development was not overtly impaired in these mice. Expression of the 172Arg-->Leu mutant p53 resulted in radiation-induced apoptosis, and transactivation or repression of the expression of a number of genes, including mdm-2 and proliferating cell nuclear antigen,...
It has been demonstrated that GSK3b is involved in Alzheimer Disease (AD) pathogenesis. In order ... more It has been demonstrated that GSK3b is involved in Alzheimer Disease (AD) pathogenesis. In order to understand the underlying mechanism, we have generated and characterized transgenic mice in which the constitutively active human GSK3b (with S9A mutation) was overexpressed in the brain under the control of the platelet-derived growth factor (PDGF) B-chain promoter. Varying levels of human GSK3b S9A transgene protein expression was observed in six of the seven founders generated. Line 3083, 3107, 3112 and 3125 displayed higher GSK3b S9A protein expression levels. Immunostaining analysis demonstrated that transgene expression was observed mainly in cortex and hippocampus of transgenic brain. Expression of human GSK3b transgene did not significantly change the brain total GSK3b protein levels in any of the generated mouse lines, as comparing to age matched wild type mice. Although significant kinase activity was detected in human GSK3b S9A transgene protein extracted from brains of all six expressing lines, significant increase in total GSK3b S9A kinase activity was observed only in the offspring of line 3083 and 3107. By analyzing the offspring from several transgenic mouse lines, including lines other than 3083 and 3107, it was found that overexpressed constitutively active human GSK3b S9A resulted in hyperphosphorylation of tau and morphology reminiscent of pretangle-like neurons in cortex and hippocampus.
Cleavage of the amyloid precursor protein (APP) by βand γ-secretases results in the generation of... more Cleavage of the amyloid precursor protein (APP) by βand γ-secretases results in the generation of the amyloid-β protein (Aβ), which is characteristically deposited in the brain of Alzheimer's disease (AD) patients. Inhibitors of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase (the statins) reduce the levels of cholesterol and isoprenoids such as geranylgeranyl pyrophosphate (GGPP). Previous studies have demonstrated that cholesterol increases and statins reduce Aβ levels, mostly by regulating β-secretase activity. In this study, we focused on the role of geranylgeranyl isoprenoids GGPP and geranylgeraniol (GGOH), in the regulation of Aβ production. Our data show that the inhibition of GGPP synthesis by statins plays an important role in statin-mediated reduction of Aβ secretion. Consistent with this finding, the geranylgeranyl isoprenoids preferentially increase the yield of Aβ of 42 residues (Aβ42), in a dose-dependent manner. Our studies further demonstrated that geranylgeranyl isoprenoids increase the yield of APP-CTFγ (a.k.a. AICD) as well as Aβ by stimulating γ-secretase mediated cleavage of APP-CTFα and APP-CTFβ in vitro. Furthermore, GGOH increases the levels of the active γ-secretase complex in the detergent insoluble membrane fraction along with its substrates: APP-CTFα and APP-CTFβ. Our results indicate that geranylgeranyl isoprenoids may be an important physiological facilitator of γ-secretase activity that can foster the production of the pathologically important Aβ42.
show 6-to 60-fold Nova-dependent effects on alternative splicing. The magnitude of Nova's effect ... more show 6-to 60-fold Nova-dependent effects on alternative splicing. The magnitude of Nova's effect on alternative splicing of these transcripts is unprecedented in mammals (26). Moreover, each of these genes are expressed both within and outside the brain, and previous work has identified exons preferentially used (or excluded) in the brain. Our results indicate that Nova is the primary, if not sole, determinant of brainspecific alternative splicing of these and perhaps other transcripts.
Animal and clinical studies have revealed that focal peripheral nerve axon demyelination is accom... more Animal and clinical studies have revealed that focal peripheral nerve axon demyelination is accompanied by nociceptive pain behavior. C-C and C-X-C chemokines and their receptors have been strongly implicated in demyelinating polyneuropathies and persistent pain syndromes. Herein, we studied the degree to which chronic nociceptive pain behavior is correlated with the neuronal expression of chemokines and their receptors following unilateral lysophosphatidylcholine (LPC)-induced focal demyelination of the sciatic nerve in rats.
The molecular mechanisms of selective motor neuron degeneration in human amyotrophic lateral scle... more The molecular mechanisms of selective motor neuron degeneration in human amyotrophic lateral sclerosis (ALS) disease remain largely unknown and effective therapies are not currently available. Mitochondrial dysfunction is an early event of motor neuron degeneration in transgenic mice overexpressing mutant superoxide dismutase (SOD)1 gene and mitochondrial abnormality is observed in human ALS patients.
Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, ... more Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger-leucine-rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase-3a/b (GSK-3a/b) resulting in the inhibition of GSK-3 kinase activity and increase of b-catenin levels. RIFLE expression also is associated with elevated E-cadherin protein levels but not N-cadherin. The regulation of these cell adhesion-associated molecules by RIFLE is accompanied by a significant increase in cell-cell and cellmatrix adhesion. Moreover, increase in cell-cell adhesion but not cell-matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK-3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells.
Recently, LiCl has been shown to inhibit amyloid L L peptide secretion in association with dimini... more Recently, LiCl has been shown to inhibit amyloid L L peptide secretion in association with diminished glycogen synthase kinase L L (GSK3L L) activity. However, it remains unclear if direct inhibition of GSK3L L activity will result in decreased AL L production. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) protein is a negative regulator of GSK3K K/L L kinase activity. To examine whether direct inhibition of GSK3K K/L L kinase activity can lower AL L production, a FRAT1 peptide was expressed in swAPP 751 cells that produce high levels of AL L. Our data demonstrate that cellular expression of FRAT1 peptide in swAPP 751 cells increases both GSK3K K and L L phosphorylation on Ser21 and Ser9, respectively, while inhibiting kinase activity of both isoforms. Moreover, as a result of FRAT1 expression, the production of both total AL L and AL L 1À42 was signi¢cantly decreased. Thus, we provide evidence that direct regulation of GSK3K K/L L by FRAT1 peptide signi¢cantly decreases AL L production in swAPP 751 cells. ß
The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a cr... more The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a critical role in the proteolytic cleavage of APP, which generates either soluble APP L (sAPP L ) and L L-amyloid peptide (AL L), the major component of senile plaques in patient brains in£icted with Alzheimer's disease (AD), or soluble APP K (sAPP K ) and a peptide smaller than AL L. One of the protein kinases known to phosphorylate APP Thr668 is cyclin-dependent kinase 5 (Cdk5). Cdk5 is activated by the association with its regulatory partner p35 or its truncated form, p25, which is elevated in AD brains. The comparative e¡ects of p35 and p25 on APP Thr668 phosphorylation and APP processing, however, have not been reported. In this study, we investigated APP Thr668 phosphorylation and APP processing mediated by p35/Cdk5 and p25/Cdk5 in the human neuroblastoma cell line SH-SY5Y. Transient overexpression of p35 and p25 elicited distinct patterns of APP Thr668 phosphorylation, speci¢cally, p35 increasing the phosphorylation of both mature and immature APP, whereas p25 primarily elevated the phosphorylation of immature APP. Despite these di¡erential e¡ects on APP phosphorylation, both p35 and p25 overexpression enhanced the secretion of AL L, sAPP L , as well as sAPP K . These results con¢rm the involvement of Cdk5 in APP processing, and suggest that p35-and p25-mediated Cdk5 activities lead to discrete APP phosphorylation. ß
The organization, distribution, and function of neural progenitor cells (NPCs) in the adult spina... more The organization, distribution, and function of neural progenitor cells (NPCs) in the adult spinal cord during motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remain largely unknown. Using nestin promoter controlled LacZ reporter transgenic mice and mutant G93A-SOD1 transgenic mice mimicking ALS, we showed that there was an increase of NPC proliferation, migration, and neurogenesis in the lumbar region of adult spinal cord in response to motor neuron degeneration. The proliferation of NPCs detected by BrdU incorporation and LacZ staining was restricted to the ependymal zone surrounding central canal (EZ). Once the NPCs moved out from the EZ, they lost the proliferative capability, but maintained migratory function vigorously. During ALS-like disease onset and progression, NPCs in the EZ migrated initially toward the dorsal horn direction, and then to the ventral horn regions, where motor neurons have degenerated. More significantly, there was an increased de novo neurogenesis from NPCs during ALS-like disease onset and progression. The enhanced proliferation, migration, and neurogenesis of (from) NPCs in the adult spinal cord of ALSlike mice may play an important role in attempting to repair the degenerated motor neurons and restore the dysfunctional circuitry which resulted from the pathogenesis of mutant SOD1 in ALS.
Background The Na1.7 subtype of voltage-gated sodium channels is specifically expressed in sensor... more Background The Na1.7 subtype of voltage-gated sodium channels is specifically expressed in sensory and sympathetic ganglia neurons where it plays an important role in the generation and transmission of information related to pain sensation. Human loss or gain-of-function mutations in the gene encoding Na1.7 channels (SCN9A) are associated with either absence of pain, as reported for congenital insensitivity to pain, or with exacerbation of pain, as reported for primary erythromelalgia and paroxysmal extreme pain disorder. Based on this important human genetic evidence, numerous drug discovery efforts are ongoing in search for Nav1.7 blockers as a novel therapeutic strategy to treat pain conditions. Results We are reporting here a novel approach to study Na1.7 function in cultured rat sensory neurons. We used live cell imaging combined with electrical field stimulation to evoke and record action potential-driven calcium transients in the neurons. We have shown that the tarantula veno...
CGRP is a potent vasodilator and nociceptive neuropeptide linked to migraine. CGRP receptors are ... more CGRP is a potent vasodilator and nociceptive neuropeptide linked to migraine. CGRP receptors are heterodimers of receptor activity-modifying protein-1 (RAMP1) and either calcitonin receptor-like receptor (CLR), which forms the canonical CGRP receptor, or calcitonin receptor (CTR), which forms the amylin-1 (AMY1 ) receptor. The goal of this study was to test whether transgenic mice globally expressing human RAMP1 (hRAMP1) have increased CGRP receptor activity and whether the receptors are sensitive to the human selective antagonist telcagepant. cAMP production was measured in primary cultures of aortic smooth muscle and trigeminal ganglia neurons from global hRAMP1 mice and nontransgenic littermates. Functional activity and inhibition was compared with clonal cell lines expressing combinations of CLR or CTR with RAMP1. Cultured smooth muscle from global hRAMP1 mice had a 10-fold greater CGRP-induced cAMP maximal response (Rmax) than non-transgenic littermates, with similar EC50 '...
Thirty percent of human breast cancers have amplification of ERBB2, often in conjunction with mut... more Thirty percent of human breast cancers have amplification of ERBB2, often in conjunction with mutations in p53. The most common p53 mutation in human breast cancers is an Arg-to-His mutation at codon 175, an allele that functions in a dominant oncogenic manner in tumorigenesis assays and is thus distinct from loss of p53. Transgenic mice expressing mouse mammary tumor virus-driven neu transgene (MMTV-neu) develop clonal mammary tumors with a latency of 234 days, suggesting that other events are necessary for tumor development. We have examined the role of mutations in p53 in tumor development in these mice. We have found that 37% of tumors arising in these mice have a missense mutations in p53. We have directly tested for cooperativity between neu and mutant p53 in mammary tumorigenesis by creating bitransgenic mice carrying MMTV-neu and 172Arg-to-His p53 mutant (p53-172H). In these bitransgenic mice, tumor latency is shortened to 154 days, indicating strong cooperativity. None of t...
Loss of heterozygosity or mutation at the p53 tumor suppressor gene locus is frequently associate... more Loss of heterozygosity or mutation at the p53 tumor suppressor gene locus is frequently associated with advanced human prostate cancer. Hence, replacement p53 gene therapy may prove to be efficacious for this disease. While many mutations result in p53 molecules with oncogenic properties, other variants may possess wild-type properties with increased tumor suppressor activity. We have chosen to investigate the activity of a naturally occurring variant p53 molecule, p53(R172L), carrying an arginine-to-leucine mutation at codon 172. We demonstrate that p53(R172L) can differentially activate expression of genes involved in cell cycle control and apoptosis in vitro. Transgenic mice expressing a subphysiological level of a p53(R172L) minigene (PB-p53(R172L)) in the prostate epithelium were generated and bred to the transgenic adenocarcinoma mouse prostate (TRAMP) model of prostate cancer. While PB-p53(R172L) transgenic mice developed normally with no detectable prostate gland phenotype, ...
Bone metastases from hepatocellular carcinoma (HCC) seem to be increasing. Previous studies showe... more Bone metastases from hepatocellular carcinoma (HCC) seem to be increasing. Previous studies showed that soluble factors secreted by host cells and direct cell-to-cell interactions contributed to the preferential metastasis and growth of cancer cells in bone, while the underlying mechanism(s) of the metastasis of HCC in the bone are poorly understood. Here, we determined the effect of HS-5 cells on Huh7 cell proliferation, and investigated the role of CCL5 from HS-5 cells on the development of Huh7 cells. In addition, the underlying mechanisms on the influence in Huh7 cells were investigated. Our results showed that HS-5 cells could promote the proliferation, migration and invasion of Huh7 cells, and inhibited apoptosis. CCL5 downregulation was able to inhibit the effects of HS-5 cells on Huh7 cell migration and invasion via the PI3K-Akt signaling pathway and reduce MMP-2 expression. Therefore, these findings suggest that CCL5 secreted from MSCs can promote the migration and invasion...
ER81 is a transcription factor that may contribute to breast cancer; however, little known about ... more ER81 is a transcription factor that may contribute to breast cancer; however, little known about the role of ER81 in breast carcinogenesis. To investigate the role of ER81 in breast carcinogenesis, we examined ER81 expression in IDC, DCIS, ADH, HUT, and normal breast tissues by immunohistochemical staining. We found that ER81 overexpression was detected in 25.7% (9/35) of HUT, 41.2% (7/17) of ADH, 54.5% (12/22) of DCIS, and 63.0% (51/81) of IDC. In 20 of breast cancer tissues combined with DCIS, ADH, and HUT, ER81 expression was found in 14/20 (70%) IDC. In these 14 cases all cases were ER81 positive expression in DCIS, 13 of 14 cases were positively expressed of ER81 in ADH and 8 of 14 were positive for ER81 in HUT components. A statistical significance was found between NBT and HUT () and HUT and ADH (). Clinical-pathological features analysis of breast cancer revealed that ER81 expression was significantly associated with Her2 amplification and was negatively associated with ER a...
Objective: Investigate a role for calcitonin gene-related peptide (CGRP) in osteoarthritis (OA)-r... more Objective: Investigate a role for calcitonin gene-related peptide (CGRP) in osteoarthritis (OA)-related pain. Design: Neutralizing antibodies to CGRP were generated de novo. One of these antibodies, LY2951742, was characterized in vitro and tested in pre-clinical in vivo models of OA pain. Results: LY2951742 exhibited high affinity to both human and rat CGRP (K D of 31 and 246 pM, respectively). The antibody neutralized CGRP-mediated induction of cAMP in SK-N-MC cells in vitro and capsaicin-induced dermal blood flow in the rat. Neutralization of CGRP significantly reduced pain behavior as measured by weight bearing differential in the rat monoiodoacetate model of OA pain in a dose-dependent manner. Moreover, pain reduction with neutralization of CGRP occurred independently of prostaglandins, since LY2951742 and NSAIDs worked additively in the NSAID-responsive version of the model and CGRP neutralization remained effective in the NSAID non-responsive version of the model. Neutralization of CGRP also provided dose-dependent and prolonged (>60 days) pain reduction in the rat meniscal tear model of OA after only a single injection of LY2951742. Conclusions: LY2951742 is a high affinity, neutralizing antibody to CGRP. Neutralization of CGRP is efficacious in several OA pain models and works independently of NSAID mechanisms of action. LY2951742 holds promise for the treatment of pain in OA patients.
Accumulating evidence has suggested that cellular production of superoxide acts as an intracellul... more Accumulating evidence has suggested that cellular production of superoxide acts as an intracellular messenger to regulate gene expression and modulate cellular activities. In this report, we set out to investigate the role of active H-ras-mediated superoxide production on tumor cell malignancy in a SV-40 transformed human lung WI-38 VA-13 cell line. Stable transfection and expression of constitutively active mutant V12-H-ras (V12-H-ras) dramatically increased intracellular production of superoxide. The expression of V12-H-ras signi®cantly enhanced cell proliferation, migration and resistance to TNF-a treatment compared to that of parental and vector control cells, while expression of wild type H-ras (WT-H-ras) only had modest eects. Upon scavenging by superoxide dismutase and other molecules that decrease the intracellular level of active H-ras mediated superoxide production, cell proliferation, migration and resistance to TNF-a were signi®cantly reduced. Furthermore, we demonstrated that the activation of membrane NADPH oxidase activity by expression of active H-ras contributed to the intracellular superoxide production. The causal relationship between membrane superoxide production and increased cell proliferation, migration, and resistance to TNF-a by the expression of active H-ras, has provided direct evidence to demonstrate that superoxide acts as an intracellular messenger to cascade ras oncogenic signal relay and to modulate tumor malignant activity.
Missense mutations in the p53 tumor suppressor occur frequently in human breast cancer and in¯uen... more Missense mutations in the p53 tumor suppressor occur frequently in human breast cancer and in¯uence both the prognosis and response to chemotherapy. Amino acid 175 (equivalent to murine 172) is the second most common site of missense mutations in p53 in human breast cancer. Over 95% of these mutations are arginine-to-histidine (R-H) substitutions resulting in a gain-of-function, and not merely a dominant-negative phenotype. Transgenic mice expressing a p53 172 R ± H construct targeted to the mammary gland by means of a whey acidic protein (WAP) promoter were characterized as a model system in order to determine the speci®c eects of this mutation on mammary tumorigenesis. Although transgene expression alone had no apparent eect on normal mammary development, transgenic mice treated with the chemical carcinogen dimethylbenz(a)anthracene developed tumors with much shorter latency than did control littermates and had a greater tumor burden. Tumors arising in transgenic mice did not exhibit either decreased apoptosis or increased cell proliferation relative to tumors arising in nontransgenic littermates, but did display increased genomic instability. Large pleiomorphic nuclei were visible in many tumors from transgenic mice, and DNĀ ow analysis con®rmed the presence of signi®cant aneuploid cell populations. Since these transgenic mice develop very few spontaneous tumors, while accelerating carcinogen-and oncogene-mediated tumorigenesis, this mouse model will, therefore, be useful in the investigation of early events in mammary tumorigenesis. It may also be used as a preclinical model to test newly developed chemotherapeutic strategies.
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1994
Regulatory sequences derived from the rat whey acidic protein gene have been used to preferential... more Regulatory sequences derived from the rat whey acidic protein gene have been used to preferentially overexpress a murine 172Arg-->Leu mutant p53 in the mammary gland of transgenic mice. Several different lines of mice expressing the 172Arg-->Leu mutant p53 displayed an impaired ability to lactate, and the mice expressing the highest levels of mutant p53 were unable to nurse their young. This failure was related to the inhibition of normal lobuloalveolar development that occurred during late pregnancy and a marked decrease in milk protein gene expression at early lactation. Interestingly, immunohistochemical analysis revealed that the mutant p53 was localized predominantly in the cytoplasm of alveolar cells. Ductal development was not overtly impaired in these mice. Expression of the 172Arg-->Leu mutant p53 resulted in radiation-induced apoptosis, and transactivation or repression of the expression of a number of genes, including mdm-2 and proliferating cell nuclear antigen,...
It has been demonstrated that GSK3b is involved in Alzheimer Disease (AD) pathogenesis. In order ... more It has been demonstrated that GSK3b is involved in Alzheimer Disease (AD) pathogenesis. In order to understand the underlying mechanism, we have generated and characterized transgenic mice in which the constitutively active human GSK3b (with S9A mutation) was overexpressed in the brain under the control of the platelet-derived growth factor (PDGF) B-chain promoter. Varying levels of human GSK3b S9A transgene protein expression was observed in six of the seven founders generated. Line 3083, 3107, 3112 and 3125 displayed higher GSK3b S9A protein expression levels. Immunostaining analysis demonstrated that transgene expression was observed mainly in cortex and hippocampus of transgenic brain. Expression of human GSK3b transgene did not significantly change the brain total GSK3b protein levels in any of the generated mouse lines, as comparing to age matched wild type mice. Although significant kinase activity was detected in human GSK3b S9A transgene protein extracted from brains of all six expressing lines, significant increase in total GSK3b S9A kinase activity was observed only in the offspring of line 3083 and 3107. By analyzing the offspring from several transgenic mouse lines, including lines other than 3083 and 3107, it was found that overexpressed constitutively active human GSK3b S9A resulted in hyperphosphorylation of tau and morphology reminiscent of pretangle-like neurons in cortex and hippocampus.
Cleavage of the amyloid precursor protein (APP) by βand γ-secretases results in the generation of... more Cleavage of the amyloid precursor protein (APP) by βand γ-secretases results in the generation of the amyloid-β protein (Aβ), which is characteristically deposited in the brain of Alzheimer's disease (AD) patients. Inhibitors of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase (the statins) reduce the levels of cholesterol and isoprenoids such as geranylgeranyl pyrophosphate (GGPP). Previous studies have demonstrated that cholesterol increases and statins reduce Aβ levels, mostly by regulating β-secretase activity. In this study, we focused on the role of geranylgeranyl isoprenoids GGPP and geranylgeraniol (GGOH), in the regulation of Aβ production. Our data show that the inhibition of GGPP synthesis by statins plays an important role in statin-mediated reduction of Aβ secretion. Consistent with this finding, the geranylgeranyl isoprenoids preferentially increase the yield of Aβ of 42 residues (Aβ42), in a dose-dependent manner. Our studies further demonstrated that geranylgeranyl isoprenoids increase the yield of APP-CTFγ (a.k.a. AICD) as well as Aβ by stimulating γ-secretase mediated cleavage of APP-CTFα and APP-CTFβ in vitro. Furthermore, GGOH increases the levels of the active γ-secretase complex in the detergent insoluble membrane fraction along with its substrates: APP-CTFα and APP-CTFβ. Our results indicate that geranylgeranyl isoprenoids may be an important physiological facilitator of γ-secretase activity that can foster the production of the pathologically important Aβ42.
show 6-to 60-fold Nova-dependent effects on alternative splicing. The magnitude of Nova's effect ... more show 6-to 60-fold Nova-dependent effects on alternative splicing. The magnitude of Nova's effect on alternative splicing of these transcripts is unprecedented in mammals (26). Moreover, each of these genes are expressed both within and outside the brain, and previous work has identified exons preferentially used (or excluded) in the brain. Our results indicate that Nova is the primary, if not sole, determinant of brainspecific alternative splicing of these and perhaps other transcripts.
Animal and clinical studies have revealed that focal peripheral nerve axon demyelination is accom... more Animal and clinical studies have revealed that focal peripheral nerve axon demyelination is accompanied by nociceptive pain behavior. C-C and C-X-C chemokines and their receptors have been strongly implicated in demyelinating polyneuropathies and persistent pain syndromes. Herein, we studied the degree to which chronic nociceptive pain behavior is correlated with the neuronal expression of chemokines and their receptors following unilateral lysophosphatidylcholine (LPC)-induced focal demyelination of the sciatic nerve in rats.
The molecular mechanisms of selective motor neuron degeneration in human amyotrophic lateral scle... more The molecular mechanisms of selective motor neuron degeneration in human amyotrophic lateral sclerosis (ALS) disease remain largely unknown and effective therapies are not currently available. Mitochondrial dysfunction is an early event of motor neuron degeneration in transgenic mice overexpressing mutant superoxide dismutase (SOD)1 gene and mitochondrial abnormality is observed in human ALS patients.
Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, ... more Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger-leucine-rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase-3a/b (GSK-3a/b) resulting in the inhibition of GSK-3 kinase activity and increase of b-catenin levels. RIFLE expression also is associated with elevated E-cadherin protein levels but not N-cadherin. The regulation of these cell adhesion-associated molecules by RIFLE is accompanied by a significant increase in cell-cell and cellmatrix adhesion. Moreover, increase in cell-cell adhesion but not cell-matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK-3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells.
Recently, LiCl has been shown to inhibit amyloid L L peptide secretion in association with dimini... more Recently, LiCl has been shown to inhibit amyloid L L peptide secretion in association with diminished glycogen synthase kinase L L (GSK3L L) activity. However, it remains unclear if direct inhibition of GSK3L L activity will result in decreased AL L production. Frequently rearranged in advanced T-cell lymphomas 1 (FRAT1) protein is a negative regulator of GSK3K K/L L kinase activity. To examine whether direct inhibition of GSK3K K/L L kinase activity can lower AL L production, a FRAT1 peptide was expressed in swAPP 751 cells that produce high levels of AL L. Our data demonstrate that cellular expression of FRAT1 peptide in swAPP 751 cells increases both GSK3K K and L L phosphorylation on Ser21 and Ser9, respectively, while inhibiting kinase activity of both isoforms. Moreover, as a result of FRAT1 expression, the production of both total AL L and AL L 1À42 was signi¢cantly decreased. Thus, we provide evidence that direct regulation of GSK3K K/L L by FRAT1 peptide signi¢cantly decreases AL L production in swAPP 751 cells. ß
The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a cr... more The phosphorylation status of amyloid precursor protein (APP) at Thr668 is suggested to play a critical role in the proteolytic cleavage of APP, which generates either soluble APP L (sAPP L ) and L L-amyloid peptide (AL L), the major component of senile plaques in patient brains in£icted with Alzheimer's disease (AD), or soluble APP K (sAPP K ) and a peptide smaller than AL L. One of the protein kinases known to phosphorylate APP Thr668 is cyclin-dependent kinase 5 (Cdk5). Cdk5 is activated by the association with its regulatory partner p35 or its truncated form, p25, which is elevated in AD brains. The comparative e¡ects of p35 and p25 on APP Thr668 phosphorylation and APP processing, however, have not been reported. In this study, we investigated APP Thr668 phosphorylation and APP processing mediated by p35/Cdk5 and p25/Cdk5 in the human neuroblastoma cell line SH-SY5Y. Transient overexpression of p35 and p25 elicited distinct patterns of APP Thr668 phosphorylation, speci¢cally, p35 increasing the phosphorylation of both mature and immature APP, whereas p25 primarily elevated the phosphorylation of immature APP. Despite these di¡erential e¡ects on APP phosphorylation, both p35 and p25 overexpression enhanced the secretion of AL L, sAPP L , as well as sAPP K . These results con¢rm the involvement of Cdk5 in APP processing, and suggest that p35-and p25-mediated Cdk5 activities lead to discrete APP phosphorylation. ß
Uploads
Papers by Baolin Li