Biomedical subjects

Z P Chen

Publications and source records attributed to Z P Chen.

At least 19 recordsLinked to original sources

PubMed2001

In vitro evidence for homologous recombinational repair in resistance to melphalan.

BACKGROUND: The generation of DNA interstrand cross-links is thought to be important in the cytotoxicity of nitrogen mustard alkylating agents, such as melphalan, which have antitumor activity. Cell lines with mutations in recombinational repair pathways are hypersensitive to nitrogen mustards. Thus, resistance to melphalan may require accelerated DNA repair by either recombinational repair mechanisms involving Rad51-related proteins (including x-ray repair cross-complementing proteins Xrcc2, Xrcc3, and Rad52) or by nonhomologous endjoining involving DNA-dependent protein kinase (DNA-PK) and Ku proteins. We investigated the role of DNA repair in melphalan resistance in epithelial tumor cell lines. METHODS: Melphalan cytotoxicity was determined in 14 epithelial tumor cell lines by use of the sulforhodamine assay. Homologous recombinational repair involving Rad51-related proteins was investigated by determining the levels of Rad51, Rad52, and Xrcc3 proteins and the density of nuclear melphalan-induced Rad51 foci, which represent sites of homologous recombinational repair. Nonhomologous endjoining was investigated by determining the levels of Ku70 and Ku86 proteins and DNA-PK activity. Linear regression analysis was used to analyze correlations between the various protein levels, DNA-PK activity, or Rad51 foci formation and melphalan cytotoxicity. All statistical tests were two-sided. RESULTS: Melphalan resistance was correlated with Xrcc3 levels (r =.587; P =.027) and the density of melphalan-induced Rad51 foci (r =.848; P =.008). We found no correlation between melphalan resistance and Rad51, Rad52, or Ku protein levels or DNA-PK activity. CONCLUSION: Correlations of melphalan resistance in epithelial tumor cell lines with Xrcc3 protein levels and melphalan-induced Rad51 foci density suggest that homologous recombinational repair is involved in resistance to this nitrogen mustard.

Antigens, Nuclear↗

PubMed2001

Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation.

Endothelial nitric-oxide synthase (eNOS) is phosphorylated at Ser-1179 (bovine sequence) by Akt after growth factor or shear stress stimulation of endothelial cells, resulting in increased eNOS activity. Purified eNOS is also phosphorylated at Thr-497 by purified AMP-activated protein kinase, resulting in decreased eNOS activity. We investigated whether bradykinin (BK) stimulation of bovine aortic endothelial cells (BAECs) regulates eNOS through Akt activation and Ser-1179 or Thr-497 phosphorylation. Akt is transiently activated in BK-stimulated BAECs. Activation is blocked completely by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase, suggesting that Akt activation occurs downstream from phosphatidylinositol 3-kinase. BK stimulates a transient phosphorylation of eNOS at Ser-1179 that is correlated temporally with a transient dephosphorylation of eNOS at Thr-497. Phosphorylation at Ser-1179, but not dephosphorylation at Thr-497, is blocked by wortmannin and LY294002. BK also stimulates a transient nitric oxide (NO) release from BAECs with a time-course similar to Ser-1179 phosphorylation and Thr-497 dephosphorylation. NO release is not altered by wortmannin. BK-stimulated dephosphorylation of Thr-497 and NO release are blocked by the calcineurin inhibitor, cyclosporin A. These data suggest that BK activation of eNOS in BAECs primarily involves deinhibition of the enzyme through calcineurin-mediated dephosphorylation at Thr-497.

Androstadienes↗

PubMed2001

Morphine tolerance and dependence in nociceptin/orphanin FQ transgenic knock-out mice.

It has been hypothesized that morphine tolerance and dependence in mice following chronic exposure may reflect increased compensatory activity of antiopioid systems. The endogenous peptide nociceptin/orphanin FQ has been shown to have anti-opioid effects, for example antagonizing morphine analgesia. Moreover, chronic morphine administration increases synthesis of the peptide, and morphine tolerance and dependence can be attenuated or reversed by antagonists and agonists of the nociceptin/orphanin FQ receptor, respectively. The present study seeks to confirm a role for nociceptin/orphanin FQ in opioid tolerance and dependence by comparing morphine ED(50) values and naloxone-precipitated withdrawal jumping in mice homozygous (knock-out) and heterozygous for a null mutation of the Npnc1 gene encoding the nociceptin/orphanin FQ propeptide, and their wild type littermates, following chronic morphine exposure. Relative to morphine-naive control mice, significant rightward shifts in the morphine dose-response curve, resulting in increased morphine ED(50) values (approximately two to three-fold), was observed for all genotypes following three days of repeated systemic morphine injections. However, no differences between genotypes in the magnitude of tolerance were observed. In contrast, knock-out mice displayed significantly increased naloxone-precipitated withdrawal jumping relative to heterozygous and wild-type mice following implantation with a morphine pellet (25mg) for 72h. Use of nociception/orphaninFQ transgenic knock-out mice thus demonstrate the differential involvement of nociceptin/orphanin FQ in morphine tolerance and dependence.

Analgesics, Opioid↗

PubMed2001

Antitumor efficacy of SarCNU in a human glioma xenograft model expressing both MGMT and extraneuronal monoamine transporter.

Treatment of malignant brain tumors with chloroethylnitrosoureas (CENUs) in addition to surgical resection and radiotherapy remains the foundation of glioma therapy. However, the clinical response to CENUs is at best modest. A novel analogue of nitrosoureas, 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU), as compared to the standard CENU, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), has been demonstrated to have increased anticancer effects both in vitro and in vivo. Unfortunately, many human tumors have been known to be resistant to CENUs since they express DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). In order to assess whether SarCNU has an effect on MGMT positive tumors, we evaluated its antitumor efficacy using an MGMT positive human glioma (SF-767) nude mouse xenograft model. Since SF-767 has high MGMT levels, BCNU treatment (20 mg/kg, Q4D x 3 i.p.) alone did not result in a satisfactory anticancer effect (p > 0.05). As expected, O6-benzylguanine (O6-BG) (100 mg/kg), which was given prior to BCNU treatment, by depleting MGMT activity, significantly enhanced BCNU antitumor efficacy (p < 0.001). Moreover, SarCNU treatment (167 mg/kg, Q4D x 3 i.p.) alone had a better antitumor effect than O6-BG plus BCNU treatment (F = 51.7, p = 0.0004). However, in this xenograft model, O6-BG did not significantly enhance the anticancer efficacy of SarCNU (F = 0.8, p = 0.411). The SF-767 human glioma xenograft is positive for extraneuronal monoamine transporter EMT (EMT) as determined by reverse-transcription polymerase chain reaction (RT-PCR). Our present results suggest that SarCNU is also effective for MGMT positive tumor if they exhibit EMT.

Animals↗

PubMed2001

Both extraneuronal monoamine transporter and O(6)-methylguanine-DNA methyltransferase expression influence the antitumor efficacy of 2-chloroethyl-3-sarcosinamide- 1-nitrosourea in human tumor xenografts.

We previously have found that 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) is a selective cytotoxin that enters cells via the extraneuronal transporter for monoamine transmitters (EMT). Both in vitro and in vivo studies demonstrated that SarCNU was more effective than BCNU against human gliomas. To clarify whether EMT expression correlates with antitumor efficacy of SarCNU, we determined human EMT (EMTh) and O(6)-methylguanine-DNA methyltransferase (MGMT) expression in nine human xenograft models using semiquantitative reverse-transcription polymerase chain reaction. These results were compared with the antitumor effects of SarCNU and the standard chloroethylnitrosourea antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). There was no significant correlation between EMTh expression and antitumor efficacy of SarCNU or BCNU. Also, there was no significant correlation between MGMT expression and SarCNU efficacy. However, a significant correlation was found between MGMT expression and BCNU antitumor efficacy. Interestingly, multiple regression analysis demonstrated a significant correlation between SarCNU efficacy and EMTh plus MGMT expression, whereas there was no correlation between BCNU efficacy and MGMT plus EMTh expression. Thus, the absence of a linear correlation between SarCNU efficacy and EMTh expression appears to be due, at least in part, to the presence of DNA repair, specifically, MGMT, in these xenograft models. These studies suggest that MGMT expression alone correlates with BCNU activity, whereas both EMTh and MGMT expression are important determinants of SarCNU activity against human tumor xenograft models. SarCNU is in clinical trials and these results may have important clinical implications.

Animals↗

PubMed2000

Aminohexanoic hydroxamate is a potent inducer of the differentiation of mouse neuroblastoma cells.

Deoxyhypusine synthase is the key enzyme for modifying a lysine residue to hypusine in the cellular protein eukaryotic initiation factor 5A (eIF-5A). Deletion of the deoxyhypusine synthase or the eIF-5A gene in yeast produces lethal phenotype. Inhibition of deoxyhypusine synthase by 1-guanidino-7-aminoheptane (GC7) suppresses tumor cell growth. Hypusine formation represents one of the most specific polyamine-dependent biochemical reactions. In view of the importance of polyamines in growth regulation and cancer biology, deoxyhypusine synthase has been considered to be a good target for chemotherapeutic drug design. Using GC7 as a prototype we have synthesized and tested three classes of diamine analogs, namely, guanidino-, pyrimidino-, and hydroxamate derivatives, as potential inhibitors for deoxyhypusine synthase. Our study shows that (i) among all the compounds tested, GC7 remained to be the most potent inhibitor for deoxyhypusine synthase; (ii) N,N'-bispyrimidino-1, 9-diaminononane, although a poor inhibitor of deoxyhypusine synthase, was a potent growth inhibitor; and (iii) one of the hydroxamate derivatives, 6-aminohexanoic hydroxamate (HC6), prominently induced the differentiation of mouse neuroblastoma cells at sub-millimolar concentrations. Interestingly, other hydroxamates with different chain length were not nearly as effective as HC6 in inducing neuroblastoma cell differentiation. The effect of HC6 was also unique in that it could induce neurite outgrowth and the expression of neuron-specific genes such as synapsin I and MAP-2 in neuroblastoma cells in the absence of other promoting agents such as cAMP. The effect of HC6 on neuroblastoma cell differentiation was comparable with, or better than that of N(6),O(2)'-dibutyryl cAMP (Bt(2)cAMP), a standard reagent commonly used for inducing the differentiation of mouse and human neuroblastoma cells in culture.

3T3 Cells↗

PubMed2000

The preclinical pharmacologic study of dopamine transporter imaging agent [99mTc]TRODAT-1.

The purpose of this study was to investigate the pharmacologic characteristics of TRODAT-1 (2beta-((N,N'-bis(2-mercaptoethyl)ethylene diamino)methyl), 3beta-(4-chlorophenyl)tropane) labeled with [99mTc] as an imaging agent for dopamine transporter (DAT). Radiochemical purity of [99mTc]TRODAT-1 was over 90%. The partition coefficients in octanol and buffer were 2.12 and 2.19 at pH 7.0 and 7.4, respectively. Animal studies have been performed in rats, rabbits, and normal and hemi-Parkinsonian model monkeys. Biodistribution displayed moderate uptake in rat brain (0.28 %ID/organ at 2 min) and the striatal uptake was 0.193, 0.142, and 0.136 %ID/g at 2, 60, and 120 min, respectively. The ratios of striatal/cerebellar (ST/CB) uptake were 2.4, 4.45, and 2.45 %ID/g at 60, 120, and 240 min, respectively. The major radioactivity was excreted by the hepatobiliary system. Blood clearance kinetics was performed in rabbits, and the initial half-life of 1.18 min and late half-life of 367.8 min were obtained. Brain single photon emission computed tomography imaging studies in normal monkeys showed the ratios of ST/CB uptake were 1.56-2.0 %ID/g and indicated that both uptake and retention in the striatal area were associated with the DAT. The imaging of hemi-Parkinsonian model monkeys also displayed the expected selectivity, the highest uptake being observed in the basal ganglia area of the normal side. Thereby, it is suggested that [99mTc]TRODAT-1 is a safe and useful imaging agent for localization of the presynaptic DAT in the brain.

Animals↗

PubMed2000

A PARAFAC algorithm using penalty diagonalization error (PDE) for three-way data array resolution.

A modified parallel factors analysis (PARAFAC) algorithm with the penalty diagnolization error (PDE) was developed. This algorithm can overcome the slow convergence problem of the traditional PARAFAC method and is insensitive to the number of components, i.e., it is much faster than PARAFAC and insensitive to overestimation of the dimensionality of the model. The characteristic performance was demonstrated by treating simulated and real excitation-emission fluorescence data for samples of naphthalene, 1-naphthol and 2-naphthol with satisfactory results.

Journal Article↗

PubMed2000

AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation.

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing protein kinase responsible for coordinating metabolism and energy demand. In rodents, exercise accelerates fatty acid metabolism, enhances glucose uptake, and stimulates nitric oxide (NO) production in skeletal muscle. AMPK phosphorylates and inhibits acetyl-coenzyme A (CoA) carboxylase (ACC) and enhances GLUT-4 translocation. It has been reported that human skeletal muscle malonyl-CoA levels do not change in response to exercise, suggesting that other mechanisms besides inhibition of ACC may be operating to accelerate fatty acid oxidation. Here, we show that a 30-s bicycle sprint exercise increases the activity of the human skeletal muscle AMPK-alpha1 and -alpha2 isoforms approximately two- to threefold and the phosphorylation of ACC at Ser(79) (AMPK phosphorylation site) approximately 8.5-fold. Under these conditions, there is also an approximately 5.5-fold increase in phosphorylation of neuronal NO synthase-mu (nNOSmu;) at Ser(1451). These observations support the concept that inhibition of ACC is an important component in stimulating fatty acid oxidation in response to exercise and that there is coordinated regulation of nNOSmu to protect the muscle from ischemia/metabolic stress.

AMP-Activated Protein Kinases↗

PubMed1999

AMP-activated protein kinase phosphorylation of endothelial NO synthase.

The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.

AMP-Activated Protein Kinases↗

PubMed1999

Dealing with energy demand: the AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that is found in all eukaryotes. AMPK activity is regulated by vigorous exercise, nutrient starvation and ischemia/hypoxia, and modulates many aspects of mammalian cell metabolism. The AMPK yeast homolog, Snf1p, plays a major role in adaption to glucose deprivation. In mammals, AMPK also has diverse roles that extend from energy metabolism through to transcriptional control.

AMP-Activated Protein Kinases↗

PubMed1999

Enhanced antitumor activity of sarCNU in comparison to BCNU in an extraneuronal monoamine transporter positive human glioma xenograft model.

A novel analogue of nitrosoureas, 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU), has demonstrated increased anticancer effects in vitro and in vivo. Our previous work suggested that SarCNU enters cells via the extraneuronal monoamine transporter (EMT), that contributes to its enhanced cytotoxicity. In the present study, comparative activities of SarCNU and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) were evaluated in an EMT positive human glioma xenograft model. Athymic nude mice implanted subcutaneously or intracranially with human glioma SHG-44, a cell line that has been confirmed EMT positive by using reverse-transcription polymerase chain reaction (RT-PCR) assay, were treated with SarCNU at an optimal dose of 167 mg/kg, or BCNU at 20 mg/kg or 30 mg/kg, q4d x 3 intraperitoneally (i.p.). In 17 animals with subcutaneous tumor grafts treated with SarCNU, 9 animals became tumor free and 8 demonstrated tumor regression. While in the BCNU treated group, there were only 2 out of 10 mice in the 20 mg/kg group and 2 out of 7 in the 30 mg/kg group, which demonstrated some tumor regression. There were 4 drug related deaths in the BCNU (30 mg/kg) group, while there were no drug related deaths in the SarCNU group. In the intracranially implanted mice, the median survival time in the SarCNU group was more than 130 days, while in the BCNU treated group it was only 22 days which was similar to the control group (18 days). This is the first demonstration that SarCNU, in comparison to BCNU, has enhanced anticancer activity in an EMT positive human glioma xenograft model.

Animals↗

PubMed1999

Pharmacology study of the neutral myocardial imaging agent technetium-99m-N(NOEt)2.

OBJECTIVE: The biological properties of a new neutral myocardial imaging agent 99mTcN(NOEt)2 were evaluated. METHODS: Blood clearance in rabbits, biodistribution in rats, and initial myocardial imaging in dogs were performed. RESULTS: Radiochemical purity of 99mTcN(NOEt)2 was more than 90% and stable for 6 h at room temperature. Blood disappearance was analyzed with a biexponential model and T1/2(alpha) = 2.53 min, T1/2(beta) = 330 min and CI = 378 ml/h were obtained. Biodistribution studies demonstrated that 99mTcN(NOEt)2 localized selectively in the rat myocardium. Cardiac uptakes were 4.69, 4.20, 3.95 and 3.43% ID/g at 5, 30, 60 and 90 min postinjection, respectively. The mean heart-to-lung activity ratios were 1.69, 2.40 and 2.55 at 10 min, 30 min and 60 min postinjection, respectively. CONCLUSION: Technetium-99m-N(NOEt)2 exhibited favorable stability and biological properties. Further study in humans is required.

Animals↗

PubMed1999

Relationship between O6-methylguanine-DNA methyltransferase levels and clinical response induced by chloroethylnitrosourea therapy in glioma patients.

BACKGROUND: Adjuvant nitrosourea chemotherapy fails to prolong survival significantly as many tumors demonstrate resistance to these drugs. It has been documented in cell lines that O6-methylguanine DNA methyltransferase (MGMT) plays an important role in chloroethylnitrosourea (CENU) drug resistance. METHODS: We evaluated MGMT expression in 22 glioma specimens by using an immunofluorescence assay and compared the results with clinical responses of the patients to CENU-based chemotherapy. RESULTS: Eight tumor samples had no detectable MGMT, whereas other samples had from 9,989 to 982,401 molecules/nucleus. In one group (12 patients), the tumor decreased in size or was stable (effective group), whereas in the other group (10 patients), the tumor demonstrated continuous growth during chemotherapy (progressive group). The Mer- patients (MGMT < 60,000 molecules/nucleus) appeared to have more chance of stable disease or response to CENU therapy than the Mer+ patients (MGMT > 60,000 molecules/nucleus) (X2 = 4.791, p = 0.0286). In patients with glioblastomas multiforme (GBMs), the median time to progression (TTP) of Mer+ patient was shorter than that of Mer- patient (t = 2.04, p = 0.049). As a corollary, the MGMT levels were significantly higher in GBM tumors from the progressive group than those from the effective group (t = 2.26, p = 0.029). However, there was no significant correlation between MGMT levels and either the survival time (r = 0.04, p = 0.8595) or TTP (r = 0.107, p = 0.6444). CONCLUSION: This study suggests that being MGMT positive is indicative of a more aggressive disease that progresses more rapidly with CENU therapy. However, MGMT negative tumors are not always sensitive to CENU agents, suggesting that other factors are also important.

Adult↗

PubMed1999

Extraneuronal monoamine transporter expression and DNA repair vis-à-vis 2-chloroethyl-3-sarcosinamide-1-nitrosourea cytotoxicity in human tumor cell lines.

We previously found that 2-chloroethyl-3-sarcosin-amide-1-nitrosourea (SarCNU), a new chloroethylnitrosourea analogue presently in phase I clinical trials, is a selective cytotoxin that enters cells via the extraneuronal transporter for monoamine transmitters (EMT). In this study, we assessed whether EMT expression correlates with SarCNU cytotoxicity by determining EMT expression in 23 human tumor cell lines with reverse-transcription PCR. Western blot analysis was used to measure protein levels of the DNA repair genes, O6-methylguanine-DNA methyltransferase (MGMT), and excision repair cross-complementing rodent repair deficiency gene 2 (ERCC2). SarCNU cytotoxicity was determined by the sulforhodamine B colorimetric anti-cancer-drug screening assay and correlated with gene expression. Almost all of the cell lines screened were positive for EMT expression. However, seven cell lines (MGR-1, MGR-2, T98-G, SKI-1, SKNSH, 297, and GBM) expressed low levels of EMT. Although there was no linear correlation between SarCNU cytotoxicity and EMT expression, SarCNU cytotoxicity significantly correlated with ERCC2 protein levels, and MGMT-rich (Mer+) cell lines (MGMT protein level >0.1) were more resistant to SarCNU than MGMT-poor (Mer-) cell lines (MGMT protein level <0.1). Moreover, multiple regression analysis indicated that the best correlation with SarCNU cytotoxicity was attainable with EMT plus MGMT and ERCC2 expression. This study suggests that in human tumor cell lines both EMT and DNA repair factors, specifically, MGMT and ERCC2, are important determinants of SarCNU activity. Because EMT is expressed in a wide variety of human tumors, SarCNU should be a more widely effective alternative chemotherapeutic agent.

Antineoplastic Agents↗

PubMed1998

Green tea epigallocatechin gallate shows a pronounced growth inhibitory effect on cancerous cells but not on their normal counterparts.

(-)-Epigallocatechin gallate (EGCG), a catechin polyphenol compound, represents the main ingredient of green tea extract. Although EGCG has been shown to be growth inhibitory in a number of tumor cell lines, it is not clear whether the effect is cancer-specific. In this study we compared the effect of EGCG on the growth of SV40 virally transformed WI38 human fibroblasts (WI38VA) with that of normal WI38 cells. The IC50 value of EGCG was estimated to be 120 and 10 microM for WI38 and WI38VA cells, respectively. Thus, EGCG at 40 microM completely inhibited the growth of WI38VA cells, but had little or no inhibitory effect on the growth of WI38 cells. Similar differential growth inhibition was also observed between a human colorectal cancer cell line (Caco-2), a breast cancer cell line (Hs578T) and their respective normal counterparts. EGCG at a concentration range of 40-200 microM induced a significant amount of apoptosis in WI38VA cultures, but not in WI38 cultures, as determined by terminal deoxynucleotidyl transferase assay. After exposure to EGCG at 200 microM for 8 h, more than 50% of WI38VA cells in a confluent culture became apoptotic. In contrast, less than 1% of WI38 cells displayed apoptotic labeling under the same condition. EGCG did not affect the serum-induced expression of c-fos and c-myc genes in normal WI38 cells. However, it significantly enhanced their expression in transformed W138VA cells. It is possible that differential modulation of certain genes, such as c-fos and c-myc, may cause differential effects of EGCG on the growth and death of cancer cells.

Anticarcinogenic Agents↗

PubMed1998

Ligand- and DNA-induced dissociation of RXR tetramers.

Unliganded bacterially expressed RXR alpha lacking the N-terminal region AB (apo-RXR alpha delta AB) was found in solution as an apparent mixture of 165 kDa tetramers and 42 kDa monomers which could be quantitatively separated by gel filtration and non-denaturing gel electrophoresis. Under identical conditions both liganded (holo-) and apo-RAR alpha delta AB were present as single monomeric species. apo-RXR alpha delta AB tetramers, as well as dimers of the apo-RXR ligand binding domain (apo-LBD), dissociated readily into monomers when exposed to their cognate ligand 9-cis retinoic acid (9c-RA). The apo-RXR alpha delta AB tetramer bound only transiently to a cognate DR1 response element, and was converted into DR1-apo-RXR alpha delta AB homodimer complexes indistinguishable from those generated by cooperative DNA binding of apo-RXR alpha delta AB monomers. In the absence of DNA, the addition of 9c-RA greatly accelerated the formation of heterodimers with the apo-RAR alpha delta AB heterodimerization partner. No RXR alpha delta AB or RAR alpha delta AB homodimers could be observed in solution, but upon mixing of the two receptor monomers stable heterodimers could be isolated which bound to DR5 response elements in a highly cooperative manner. In these heterodimers, RXR alpha delta AB interacted with its cognate ligand as efficiently as in RXR alpha delta AB homodimers. The presence of ligand did not alter the stability of RXR alpha delta AB homodimer or RXR alpha delta AB-RAR alpha delta AB heterodimer complexes on DR1 and DR5 response elements, respectively. These in vitro data support a model in which RXR tetramers could serve as an inactive pool with the dual function of: (i) rapidly supplying large amounts of RXR heterodimerization partners upon 9c-RA generation; and (ii) allowing RXR homodimer formation on "accessible" cognate response elements in the absence of 9c-RA. These events may represent a ligand-dependent regulatory mechanism controlling the availability of the promiscuous RXR dimerization partner that is engaged in multiple nuclear receptor signalling pathways.

Amino Acid Substitution↗

PubMed1998

Tubular epithelial-myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats.

BACKGROUND: Tubulointerstitial fibrosis is the final common pathway to end-stage renal failure. The present study investigated the potential role of tubular epithelial cells (TEC) in progressive fibrosis in the rat remnant kidney model. METHODS: Rats underwent 5/6 nephrectomy or a sham operation (control), and groups of six animals were killed at weeks 1, 3, 5, 9, 13, 17 and 21. RESULTS: Immunohistochemistry staining and in situ hybridization at week 3 after nephrectomy demonstrated de novo expression of alpha-smooth muscle actin (alpha-SMA)--a marker of smooth muscle cells and myofibroblasts--by TEC that was invariably associated with disruption of the tubular basement membrane (TBM). This phenotypic evidence of tubular epithelial-myofibroblast transdifferentiation was supported by ultrastructural studies identifying the presence of characteristic actin microfilaments and dense bodies within TEC with a transformed morphology. In the late stage of this apparent tubular epithelial-myofibroblast transdifferentiation, TEC lost apical-basal polarity and tight junctions, became elongated, detached from the TBM, separated from neighboring cells and appeared to migrate into the peritubular interstitium through the damaged basement membrane. Indeed, focal peritubular accumulation of alpha-SMA+ myofibroblasts and local tubulointerstitial fibrosis was closely associated with alpha-SMA+ tubules, suggesting a tubular epithelial origin for some of these cells. Quantitative analysis found a significant correlation between the number of alpha-SMA+ TEC and the accumulation of interstitial alpha-SMA+ myofibroblasts and the severity of tubulointerstitial fibrosis (both P < 0.001). CONCLUSIONS: This study provides phenotypic and morphological evidence to support the hypothesis that TEC are pro-fibrogenitor cells capable of tubular epithelial-myofibroblast transdifferentiation in progressive renal fibrosis. In addition, we postulate that disruption of the TBM, which facilitates epithelial cell contact with the interstitial matrix, promotes this process of transdifferentiation.

Actins↗