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PubMed2004

Multiple modifications allow high-titer production of retroviral vectors carrying heterologous regulatory elements.

Tumor-specific expression of therapeutic genes is a prerequisite in many approaches to retrovirus-mediated cancer gene therapy. However, tissue specificity is often associated with a reduction in viral titer. To overcome this problem, we constructed a series of murine leukemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors carrying either the simian virus 40 poly(A) signal trimer (3pA) inserted in the 3' long terminal repeat (LTR) of these vectors or the human cytomegalovirus enhancer region (CMVe) inserted 5' and 3' of the retroviral LTRs. Furthermore, an extended AT stretch/attachment site (AT/att) of wild-type MLV was introduced into the vector. In the vector-producing cells, insertion of the CMVe and/or the 3pA resulted in a three- to fourfold-enhanced marker gene expression compared to the parental vector, whereas insertion of the AT/att gave a slight decrease in expression. The combination of all three modifications had no additional effects. In contrast, however, neomycin selection of infected cells revealed only a slight increase in virus titer with vectors carrying the 3pA modification; the titer was increased by 1 with vectors containing the extended AT/att, although the viral DNA copy numbers in infected cells were similar with both types of vectors. Thus, insufficient integration rather than insufficient reverse transcription and/or production of virus RNA is the major cause for the low titer obtained with the ProCon vectors. The combination of all three modifications resulted in a 2- to 3-log increase in the virus titer. These modifications result in expression targeted ProCon vectors with titers similar to those of nonmodified MLV-based vectors.

Animals↗
PubMed1993

Use of transgenic mice reveals cell-specific transformation by a simian virus 40 T-antigen amino-terminal mutant.

We have used the multifunctional transforming protein, simian virus 40 T antigen, as a probe to study the mechanisms of cell growth regulation in the intact organism. T antigen appears to perturb cell growth, at least in part, by stably interacting with specific cellular proteins that function to maintain normal cell growth properties. Experiments in cultured cells indicate that at least three distinct regions of simian virus 40 T antigen have roles in transformation. Two regions correlate with the binding of known cellular proteins, p53, pRB, and p107. A third activity, located near the amino terminus, has been defined genetically but not biochemically. By targeting expression of wild-type and mutant forms of T antigen to distinct cell types in transgenic mice, we have begun to systematically determine which activities play a role in tumorigenesis of each cell type. In this study, we sought to determine the role of the amino-terminal transformation function with such an analysis of the T-antigen mutant dl1135. This protein, which lacks amino acids 17 to 27, retains the p53-, pRB-, and p107-binding activities yet fails to transform cells in culture. To direct expression in transgenic mice, we used the lymphotropic papovavirus transcriptional signals that are specific for B and T lymphocytes and the choroid plexus epithelium of the brain. We show here that although defective in cell culture, dl1135 specifically induced the development of thymic lymphomas in the mouse. Expression of the protein was routinely observed in B- and T-lymphoid cells, although B-cell abnormalities were not observed. Choroid plexus tumors were observed only infrequently; however, dl1135 was not consistently expressed in this tissue. Within a given transgenic line, the penetrance of T-cell tumorigenesis was 100% but appeared to require secondary events, as judged from the clonal nature of the tumors. These experiments suggest that the amino-terminal region of T antigen has a role in the transformation of certain cell types (such as fibroblasts in culture and B lymphocytes) but is dispensable for the transformation of T lymphocytes.

Animals↗
PubMed1994

Induction versus progression of brain tumor development: differential functions for the pRB- and p53-targeting domains of simian virus 40 T antigen.

The ability of simian virus 40-encoded large T antigen to disrupt the growth control of a variety of cell types is related to its ability to interfere with certain cellular proteins, such as p53 and the retinoblastoma susceptibility gene product (pRB). We have used wild-type and mutant forms of T antigen in transgenic mice to dissect the roles of pRB, p53, and other cellular proteins in tumorigenesis of different cell types. In this study, using a cell-specific promoter to target expression specifically to brain epithelium (the choroid plexus) and to B and T lymphoid cells, we characterize the tumorigenic capacity of a T-antigen fragment that comprises only the amino-terminal 121 residues. This fragment (dl1137) retains the ability to interact with pRB and p107 but lacks the p53-binding domain. While loss of the p53-binding region results in loss of the capacity to induce lymphoid abnormalities, dl1137 retains the ability to induce choroid plexus tumors that are histologically indistinguishable from those induced by wild-type T antigen. Tumors induced by dl1137 develop much more slowly, however, reaching an end point at around 8 months of age rather than at 1 to 2 months. Analysis of tumor progression indicates that tumor induction by dl1137 does not require secondary genetic or epigenetic events. Rather, the tumor growth rate is significantly slowed, indicating that the T-antigen C-terminal region contributes to tumor progression in this cell type. In contrast, the pRB-binding region appears essential for tumorigenesis as mutation of residue 107, known to disrupt pRB and p107 binding to wild-type T antigen, abolishes the ability of the dl1137 protein to induce growth abnormalities in the brain.

Animals↗
PubMed1996

GATA factors are essential for activity of the neuron-specific enhancer of the gonadotropin-releasing hormone gene.

The multicomponent neuron-specific enhancer of the gonadotropin-releasing hormone (GnRH) gene specifically targets expression to the GnRH-secreting neurons of the hypothalamus, a small population of specialized cells which play a central role in regulating reproductive function. Utilizing the GnRH-secreting hypothalamic neuronal cell line, GT1, as a model system, we show that members of the GATA family of transcription factors regulate GnRH transcription through two GATA factor-binding motifs that occur in a tandem repeat within the GnRH neuron-specific enhancer. Although GT1 cells contain GATA-2 and GATA-4 mRNAs, only GATA-4 was detected in a GnRH enhancer GATA site-specific complex. Cotransfection experiments with wild-type and mutant GnRH enhancer reporter plasmids with wild-type and dominant negative GATA factor expression vectors demonstrated that both GATA-binding elements are functional in the context of the enhancer. We conclude that GATA-binding proteins are important factors in regulating the neuron-specific expression of the GnRH gene in hypothalamic cells. Although the presence of GATA-2 in a neuronal cell type is not unusual, the presence of GATA-4 in GT1 cells is novel for a neuronal cell type. However, the presence of GATA-4 is consistent with the unique developmental origin of GnRH neurons and may provide insight into the transcriptional mechanisms mediating the differentiation of this limited population of GnRH-secreting neurons.

3T3 Cells↗
PubMed2000

DNA excision repair and DNA damage-induced apoptosis are linked to Poly(ADP-ribosyl)ation but have different requirements for p53.

Poly(ADP-ribose) polymerase (PARP) is a DNA binding zinc finger protein that catalyzes the transfer of ADP-ribose residues from NAD(+) to itself and different chromatin constituents, forming branched ADP-ribose polymers. The enzymatic activity of PARP is induced upon DNA damage and the PARP protein is cleaved during apoptosis, which suggested a role of PARP in DNA repair and DNA damage-induced cell death. We have generated transgenic mice that lack PARP activity in thymocytes owing to the targeted expression of a dominant negative form of PARP. In the presence of single-strand DNA breaks, the absence of PARP activity correlated with a strongly increased rate of apoptosis compared to cells with intact PARP activity. We found that blockage of PARP activity leads to a drastic increase of p53 expression and activity after DNA damage and correlates with an accelerated onset of Bax expression. DNA repair is almost completely blocked in PARP-deficient thymocytes regardless of p53 status. We found the same increased susceptibility to apoptosis in PARP null mice, a similar inhibition of DNA repair kinetics, and the same upregulation of p53 in response to DNA damage. Thus, based on two different experimental in vivo models, we identify a direct, p53-independent, functional connection between poly(ADP-ribosyl)ation and the DNA excision repair machinery. Furthermore, we propose a p53-dependent link between PARP activity and DNA damage-induced cell death.

Animals↗
PubMed1998

Pigmented uveal tumours in a transgenic mouse model.

AIMS/BACKGROUND: The authors have developed transgenic mouse strains at the Arizona Cancer Center using a tyrosinase promoter to target expression of the mutated T24 Ha-ras gene in melanin producing cells. Histopathology and electron microscopy (EM) were performed to characterise the intraocular tumours observed phenotypically. METHODS: Transgenic TPras mice (n = 8) and normal, age matched control mice (n = 6) were sacrificed at 3 weeks, 6 weeks, 7 weeks, 4 months, 5 months, 9 months, and 11 months. Six were processed in formalin for light microscopic examination and eight in a glutaraldehyde/formalin solution for electron microscopic examination. RESULTS: Six of the TPras mice were found to have bilateral pigmented melanocytic/RPE proliferations of the uveal tract. The cytological characteristics of the tumours included low nuclear to cytoplasmic ratios (N:C ratios), bland nuclei, and abundant intracytoplasmic melanin. By EM two populations of cells were identified, including spindle-shaped cells with round to oval melanin granules and cuboidal cells with apically located, cigar-shaped, melanin granules, and basement membrane formation. A 3 week and an 11 month old TPras mouse had a higher grade, bilateral, melanocytic proliferation of the uveal tract which, although not metastatic, was morphologically melanoma. Cytological features included increased N:C ratios, nuclear pleomorphism, and prominent nucleoli. The uveal tract was normal in both eyes in all of the control animals. CONCLUSION: Pigmented intraocular tumours developed in transgenic strains of mice that express a mutated Ha-ras gene in melanin producing cells. The morphology was most consistent with a melanoma in two of the mice and a benign melanocytic/RPE proliferation in the remaining mice.

Animals↗
PubMed2004

Targeted overexpression of G protein-coupled receptor kinase-2 in osteoblasts promotes bone loss.

To investigate the role of G protein-coupled receptor kinases (GRKs) in regulating bone formation in vivo, we overexpressed the potent G protein-coupled receptor (GPCR) regulator GRK2 in osteoblasts, using the osteocalcin gene-2 promoter to target expression to osteoblastic cells. Using the parathyroid hormone (PTH) receptor as a model system, we found that overexpression of GRK2 in osteoblasts attenuated PTH-induced cAMP generation by mouse calvaria ex vivo. This decrease in GPCR responsiveness was associated with a reduction in bone mineral density (BMD) in transgenic (TG) mice compared with non-TG littermate controls. The decrease in BMD was most prominent in trabecular-rich lumbar spine and was not observed in cortical bone of the femoral shaft. Quantitative computed tomography indicated that the loss of trabecular bone was due to a decrease in trabecular thickness, with little change in trabecular number. Histomorphometric analyses confirmed the decrease in trabecular bone volume and demonstrated reduced bone remodeling, as evidenced by a decrease in osteoblast numbers and osteoblast-mediated bone formation. Osteoclastic activity also appeared to be reduced because urinary excretion of the osteoclastic activity marker deoxypyridinoline was decreased in TG mice compared with control animals. Consistent with reduced coupling of osteoblast-mediated bone formation to osteoclastic bone resorption, mRNA levels of both osteoprotegrin and receptor activator of NF-kappaB ligand were altered in calvaria of TG mice in a pattern that would promote a low rate of bone remodeling. Taken together, these data suggest that enhancing GRK2 activity and consequently reducing GPCR activity in osteoblasts produces a low bone-turnover state that reduces bone mass.

Animals↗
PubMed2003

Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart.

The small G protein Ras-mediated signaling pathway has been implicated in the development of hypertrophy and diastolic dysfunction in the heart. Earlier cellular studies have suggested that the Ras pathway is responsible for reduced L-type calcium channel current and sarcoplasmic reticulum (SR) calcium uptake associated with sarcomere disorganization in neonatal cardiomyocytes. In the present study, we investigated the in vivo effects of Ras activation on cellular calcium handling and sarcomere organization in adult ventricular myocytes using a newly established transgenic mouse model with targeted expression of the H-Ras-v12 mutant. The transgenic hearts expressing activated Ras developed significant hypertrophy and postnatal lethal heart failure. In adult ventricular myocytes isolated from the transgenic hearts, the calcium transient was significantly depressed but membrane L-type calcium current was unchanged compared with control littermates. The expressions of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a and phospholamban (PLB) were significantly reduced at mRNA levels. The amount of SERCA2a protein was also modestly reduced. However, the expression of PLB protein and gross sarcomere organization remained unchanged in the hypertrophic Ras hearts, whereas Ser(16) phosphorylation of PLB was dramatically inhibited in the Ras transgenic hearts compared with controls. Hypophosphorylation of PLB was also associated with a significant induction of protein phosphatase 1 expression. Therefore, our results from this in vivo model system suggest that Ras-induced contractile defects do not involve decreased L-type calcium channel activities or disruption of sarcomere structure. Rather, suppressed SR calcium uptake due to reduced SERCA2a expression and hypophosphorylation of PLB due to changes in protein phosphatase expression may play important roles in the diastolic dysfunction of Ras-mediated hypertrophic cardiomyopathy.

Animals↗
PubMed1998

Hormonally controlled chloride movement across Drosophila tubules is via ion channels in stellate cells.

Anion conductance across the Drosophila melanogaster Malpighian (renal) tubule was investigated by a combination of physiological and transgenic techniques. Patch-clamp recordings identified clusters of 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive "maxi-chloride" channels in a small domain of the apical membrane. Fluid secretion assays demonstrated sensitivity to the chloride channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, diphenylamine-2-carboxylate, anthracene-9-carboxylic acid, and niflumic acid. Electrophysiological analysis showed that the calcium-mediated increase in anion conductance was blocked by the same agents. Vibrating probe analysis revealed a small number of current density hot spots, coincident with "stellate" cells, that were abolished by low-chloride saline or the same chloride channel blockers. GAL-4-targeted expression of an aequorin transgene revealed that the neurohormone leucokinin elicits a rapid increase in intracellular calcium levels in stellate cells that precedes the fastest demonstrable physiological effect. Taken together, these data show that leucokinins act on stellate cells through intracellular calcium to increase transcellular chloride conductance through channels. As electrogenic cation conductance is confined to principal cells, the two pathways are spatially segregated in this tissue.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2004

Differential modulation of baroreflex control of heart rate by neuron- vs. glia-derived angiotensin II.

We developed transgenic mice with targeted expression of human renin (hREN) and human angiotensinogen (hAGT) to either neurons (N-AII mice) or glia (G-AII mice) to test the hypothesis that neuronal and glial ANG II may have differential function. Since baseline blood pressure (BP) did not differ between the models (109 +/- 3 vs. 114 +/- 4 mmHg), we stressed the BP regulatory pathway by measuring the heart rate (HR) (baroreflex) response to phenylephrine- and nitroprusside-induced changes in arterial BP. The midpoint of the baroreflex curve (BP50) was reset to a significantly higher BP in N-AII mice (131 +/- 5 mmHg) compared with littermate controls (115 +/- 3 mmHg). Baroreflex gain (slope of BP-HR relation) was similar in N-AII and control mice (12 +/- 1 vs. 14 +/- 2 beats x min(-1) x mmHg(-1)). In contrast, G-AII mice exhibited less of an increase in BP50 (125 +/- 5 mmHg) but a larger decrease in baroreflex gain (8 +/- 1 beats x min(-1) x mmHg(-1)) compared with both control and N-AII mice. Differences in BP50 and gain between N-AII, G-AII, and control mice persisted after parasympathetic blockade with atropine but were eliminated after sympathetic blockade with propranolol, indicating the effects of ANG II were selective for cardiosympathetic arm of the reflex. ANG II-like immunoreactivity was observed more prominently around the paraventricular nucleus and nucleus tractus solitarii in G-AII mice but more prominently in the ventrolateral medulla in N-AII mice. We conclude that ANG II differentially modulates baroreflex control of HR in mice producing ANG II in neurons vs. glia, and its differential function may reflect regional differences in the production of ANG II in cardiovascular control nuclei of the brain.

Angiotensin II↗
PubMed2004

High frequency of functionally active Melan-a-specific T cells in a patient with progressive immunoproteasome-deficient melanoma.

Tumor-reactive T cells play an important role in cancer immunosurveillance. Applying the multimer technology, we report here an unexpected high frequency of Melan-A-specific CTLs in a melanoma patient with progressive lymph node metastases, consisting of 18 and 12.8% of total peripheral blood and tumor-infiltrating CD8+ T cells, respectively. Melan-A-specific CTLs revealed a high cytolytic activity against allogeneic Melan-A-expressing target cells but failed to kill the autologous tumor cells. Loading of the tumor cells with Melan-A peptide reversed the resistance to killing, suggesting impaired function of the MHC class I antigen processing and presentation pathway. Mutations of the coding region of the HLA-A2 binding Melan-A26-35 peptide or down-regulation of the MHC class I heavy chain, the antigenic peptide TAP, and tapasin could be excluded. However, PCR and immunohistochemical analysis revealed a deficiency of the immunoproteasomes low molecular weight protein 2 and low molecular weight protein 7 in the primary tumor cells, which affects the quantity and quality of generated T-cell epitopes and might explain the resistance to killing. This is supported by our data, demonstrating that the resistance to killing can be partially reversed by pre-exposure of the tumor cells to IFN-gamma, which is known to induce the immunoproteasomes. Overall, this is the first report of an extremely high frequency of tumor-specific CTLs that exhibit competent T-cell-effector functions but fail to lyse the autologous tumor cells. Immunotherapeutic approaches should not only focus on the induction of a robust antitumor immune response, but should also have to target tumor immune escape mechanisms.

Antigens, Neoplasm↗
PubMed2006

Estrogen receptor positive breast cancer metastasis: altered hormonal sensitivity and tumor aggressiveness in lymphatic vessels and lymph nodes.

Breast cancers commonly spread to lymph nodes (LNs). If the primary tumors are estrogen receptor (ER) and/or progesterone receptor (PR) positive, then the likelihood that LN metastases express receptors exceeds 80%. However, due to lack of ER+ models, little is known about the role of hormones in breast cancer spread or the effects of the LN microenvironment on hormone responsiveness. We have developed metastasis models using ZsGreen labeled MCF-7 and T47D human breast cancer cells. Tumors are tracked in living mice by whole-body imaging, and macrometastases or micrometastases are detected by intravital imaging or fluorescence microscopy. Tumor growth is estrogen dependent and required for intratumoral lymphangiogenesis. Seventy-five percent of all tumors and >95% of larger tumors generate LN metastases. Occasionally more distant metastases are also observed. "Triads" of primary tumors, tumor-filled draining lymphatic vessels, and tumor-filled LNs from the same mouse show that (a) proliferation, as measured by 5-bromo-2'-deoxyuridine uptake, is higher in the LN than in the primary tumor. (b) High ER levels are extensively down-regulated by estradiol in primary tumors. However, there is partial failure of ER down-regulation in LNs associated with (c) reduced PR expression. This suggests that ER are dysfunctional in the LN microenvironment and perhaps hormone resistant. (d) CD44 is sparsely expressed in primary tumor cells but homogeneously overexpressed in cells transiting the lymphatics and populating LNs. We hypothesize that CD44 expression targets tumor cells for transport to, and uptake in, LNs. If so, the CD44 pathway could be targeted therapeutically to slow or prevent LN metastases.

Animals↗
PubMed2006

Detection of Wilms' tumor antigen--specific CTL in tumor-draining lymph nodes of patients with early breast cancer.

PURPOSE: The Wilms' tumor antigen (WT1) is overexpressed in approximately 90% of breast tumors and, thus, is a potential target antigen for the immunotherapy of breast cancer. We have tested the working hypotheses that WT1 can be immunogenic in patients with breast cancer and can stimulate CTL of sufficient avidity to kill tumor cells. EXPERIMENTAL DESIGN: Paired tumor-draining lymph node and peripheral blood samples were analyzed from five HLA-A2-positive patients with stage I/II breast cancer. Fluorescent HLA-A*0201/WT1 tetramers were used to quantify WT1-specific CTL and the functional capacity of the CTL was assessed using cytotoxicity assays and intracellular cytokine staining. RESULTS: WT1 tetramer-binding T cells expanded from all lymph node samples but none of the corresponding peripheral blood samples. Functional assays were carried out on T cells from the patient who had yielded the highest frequency of HLA-A*0201/WT1 tetramer-positive cells. The cytotoxicity assays showed WT1 peptide--specific killing activity of the CTL, whereas intracellular cytokine staining confirmed that the tetramer--positive T cells produced IFN-gamma after stimulation with WT1 peptide. These WT1-specific T cells killed HLA-A2-positive breast cancer cell lines treated with IFN-gamma but no killing was observed with untreated tumor cells. CONCLUSIONS: These results show that WT1-specific CTL can be expanded from the tumor-draining lymph nodes of breast cancer patients and that they can display peptide-specific effector function. However, the CTL only killed IFN-gamma-treated tumor targets expressing high levels of HLA-A2 and not tumor cells with low HLA expression. This suggests that induction of autologous WT1-specific CTL may offer only limited tumor protection and that strategies that allow a high level of peptide/MHC complex presentation and/or improve CTL avidity may be required.

Aged↗
PubMed2006

Restriction of adenoviral replication to the transcriptional intersection of two different promoters for colorectal and pancreatic cancer treatment.

In our current study, we developed oncolytic adenoviruses which preferentially lyse pancreatic and colon cancer cells by replacing viral E1 and/or E4 promoter with the tumor/tissue-specific promoters, cyclooxygenase-2 (COX-2), midkine (MK), or the cell cycle-dependent promoter, E2F1. We generated three sets of recombinant adenoviral vectors. In the first set, only the native E1A promoter was replaced by the COX-2, MK, or E2F1 promoter, respectively. In the second set, the viral E4 promoter was substituted by these heterologous promoters and the viral E1A promoter was substituted by the ubiquitously active cytomegalovirus-IE promoter. In the third set, we substituted the viral E1A and E4 promoters with the COX-2, MK, or E2F1 promoter, respectively. In our system, transcriptional targeting of solitary viral E1A resulted in 50% enhanced restricted vector replication when compared with an unrestricted replication-competent adenovirus. Furthermore, a targeted expression of the viral E1A gene products had a greater effect on restricted adenoviral replication than that of the E4 region. With our vectors, Ad.COX.MK and Ad.MK.COX, using two different heterologous promoters to control E1A and E4 expression, we showed enhanced viral replication specificity when compared with Ad.COX.COX or Ad.MK.MK, respectively. In a s.c. xenograft tumor model, there was no significant difference in the antineoplastic efficacy of the double heterologous promoter-controlled vectors when compared with our unrestricted replication-competent control adenovirus or vectors with only E1A transcriptionally driven by a heterologous promoter.

Adenoviridae↗
PubMed2004

Phenotypic characterization and distribution of dendritic cells in parotid gland tumors.

Evidence on the relationship between the infiltration of dendritic cells (DCs) and prognosis in head and neck tumors exists. Interestingly, only limited information is available regarding the maturation state and distribution of DCs in parotid gland tumors. The purpose of our study was therefore to extend these observations and to investigate in more detail the density and distribution of mature DCs and Langerhans cells (LCs) in parotid gland tumors. We present immunohistochemical evidence of characterization and distribution of DCs and LCs in parotid gland tumors, enclosed pleomorphic adenomas and malignant parotid tumors. Two populations of mature DCs could be identified, P55(+)-DCs and DC-LAMP(+)-DCs, whereas LCs could be identified as Langerin(+)-LCs. The overall impression was that parotid gland tumors contained only few mature DCs and LCs. Considering the sparsity of mature DCs in the malignant tissues, anti-tumor response can be only limited. On the basis of our data, we imply that the application of DC vaccination in combination with other modalities for treatment of parotid gland carcinoma should be taken into account. In this regard, the utilization of DC immunotherapy for management of minimal residual disease after resection of primary tumor can be promising. Putative targets expressed in this type of tumors have to be defined.

Adenocarcinoma↗
PubMed2005

Dyserythropoietic anemia and thrombocytopenia due to a novel mutation in GATA-1.

Hematopoiesis is a complex process regulated by nuclear proteins that coordinate lineage-specific patterns of gene expression. Targeted mutagenesis has revealed critical roles for the X-linked transcription factor GATA-1 in erythrocyte and megakaryocyte differentiation. GATA-1 has two zinc fingers essential for normal function. The C-terminal finger is necessary for DNA binding. The N-terminal finger mediates interaction with FOG-1, a cofactor for GATA-1. Mutations in the N-terminal zinc finger of GATA-1 result in abnormal hematopoiesis. Here we report a family with a novel single base mutation that results in an amino acid substitution (Gly208Arg) within the highly conserved portion of the GATA-1 N-terminal finger domain, leading to dyserythropoietic anemia and macrothrombocytopenia. Another mutation described at the same codon (208) has been found to be associated with thrombocytopenia only. Our data support and extend the effect of the amino acid substitution at codon 208 on GATA-1 function not only regarding megakaryocyte but also regarding erythroid development.

Amino Acid Substitution↗
PubMed2002

Inhibition of calcineurin and sarcolemmal Ca2+ influx protects cardiac morphology and ventricular function in K(v)4.2N transgenic mice.

BACKGROUND: Cardiac-targeted expression of truncated K(v)4.2 subunit (K(v)4.2N) reduces transient outward current (I(to)) density, prolongs action potentials (APs), and enhances contractility in 3- to 4-week-old transgenic mice. By 13 to 15 weeks of age, these mice develop severely impaired cardiac function and signs of heart failure. In this study, we examined whether augmented contractility in K(v)4.2N mice results from elevations in intracellular calcium ([Ca2+]i) secondary to AP prolongation and investigated the putative roles of calcineurin activation in heart disease development of K(v)4.2N mice. METHODS AND RESULTS: At 3 to 4 weeks of age, L-type Ca2+ influx and peak [Ca2+]i were significantly elevated in K(v)4.2N myocytes compared with control because of AP prolongation. Cardiac calcineurin activity was also significantly elevated in K(v)4.2N mice by 5 weeks of age relative to controls and increased progressively as heart disease developed. This was associated with activation of protein kinase C (PKC)-alpha and PKC-theta but not PKC-epsilon, as well as increases in beta-myosin heavy chain (beta-MHC) and reductions in sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-2a expression. Treatment with either cyclosporin A or verapamil prevented increases in heart weight to body weight ratios, interstitial fibrosis, impaired contractility, PKC activation, and changes in the expression patterns of beta-MHC and SERCA2a. CONCLUSIONS: Our results demonstrate that AP prolongation caused by I(to) reduction results in enhanced Ca2+ cycling and hypercontractility in mice and suggests that elevations in [Ca2+]i via I(Ca,L) and activation of calcineurin play a central role in disease development after I(to) reduction using the K(v)4.2N construct.

Action Potentials↗
PubMed2004

Endothelium-restricted overexpression of human endothelin-1 causes vascular remodeling and endothelial dysfunction.

BACKGROUND: Endothelin (ET)-1 is a potent vasoconstrictor that contributes to vascular remodeling in hypertension and other cardiovascular diseases. Endogenous ET-1 is produced predominantly by vascular endothelial cells. To directly test the role of endothelium-derived ET-1 in cardiovascular pathophysiology, we specifically targeted expression of the human preproET-1 gene to the endothelium by using the Tie-2 promoter in C57BL/6 mice. METHODS AND RESULTS: Ten-week-old male C57BL/6 transgenic (TG) and nontransgenic (wild type; WT) littermates were studied. TG mice exhibited 3-fold higher vascular tissue ET-1 mRNA and 7-fold higher ET-1 plasma levels than did WT mice but no significant elevation in blood pressure. Despite the absence of significant blood pressure elevation, TG mice exhibited marked hypertrophic remodeling and oxidant excess-dependent endothelial dysfunction of resistance vessels, altered ET-1 and ET-3 vascular responses, and significant increases in ET(B) expression compared with WT littermates. Moreover, TG mice generated significantly higher oxidative stress, possibly through increased activity and expression of vascular NAD(P)H oxidase than did their WT counterparts. CONCLUSIONS: In this new murine model of endothelium-restricted human preproET-1 overexpression, ET-1 caused structural remodeling and endothelial dysfunction of resistance vessels, consistent with a direct nonhemodynamic effect of ET-1 on the vasculature, at least in part through the activation of vascular NAD(P)H oxidase.

Animals↗