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At least 1,189 records · Page 66Linked to original sources
PubMed2003

Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region.

Donor-derived T lymphocytes directed against minor histocompatibility antigens (mHags) exclusively expressed on cells of the hematopoietic lineages can eliminate hematologic malignancies. Transfer of T-cell receptors (TCRs) directed against these mHags into T lymphocytes may provide a strategy to generate antileukemic T cells. To investigate the feasibility of this strategy the TCR usage of mHag HA-2-specific T-cell clones was characterized. Thirteen different types of HA-2-specific T-cell clones were detected, expressing TCRs with diversity in TCR alpha- and beta-chain usage, however, containing in the TCR alpha chain a single conserved gene segment J alpha 42, indicating that J alpha 42 is involved in HA-2-specific recognition. We transferred various HA-2 TCRs into T lymphocytes from HLA-A2-positive HA-2-negative individuals resulting in T cells with redirected cytolytic activity against HA-2-expressing target cells. Transfer of chimeric TCRs demonstrated that the HA-2 specificity is not only determined by the J alpha 42 region but also by the N-region of the alpha chain and the CDR3 region of the beta chain. Finally, when HA-2 TCRs were transferred into T cells from HLA-A2-negative donors, the HA-2 TCR-modified T cells exerted potent antileukemic reactivity without signs of anti-HLA-A2 alloreactivity. These results indicate that HA-2 TCR transfer may be used as an alternative strategy to generate HA-2-specific T cells to treat hematologic malignancies of HLA-A2-positive, HA-2-expressing patients that received transplants from HLA-A2-matched or -mismatched donors.

Amino Acid Sequence↗
PubMed1999

Stereotactic radiosurgery, X: clinical isodosimetry of gamma knife versus linear accelerator X-knife for pituitary and acoustic tumours.

Several review articles have compared gamma unit versus linear accelerator (linac)-based radiosurgery systems, concluding that the dose gradient 'fall-off' at the margin of the target (expressed as the distance between isodoses) is very similar for both techniques as far as single isocentre treatment volumes up to 1.5 cm diameter are concerned, and that the two radiosurgical systems are, in general, comparable. 'Fine tuning' of the gamma unit can be carried out by using multiple isocentre plans, the differential use of small collimator sizes (down to 4 mm) and field weightings, and adroit use of the gamma angle, and selective beam blocking. Multiple isocentre plans, beam modification, restriction of gantry angles and arc lengths, and microcollimation can similarly improve the isodose gradients from linac units. In both instances, the dosimetric advantages occur along selected aspects of the target perimeter border. However, the more frequent use of multiple isocentred 'shots' on the gamma unit achieves greater conformity indices for more complex target volumes, but at the expense of steeper internal dose gradients. We studied two patients with tumours close to or arising from radiosensitive special sensory nerves (optic and cochlear) to compare and contrast fine tuning of the two technologies. In a previously irradiated patient with a pituitary adenoma, the dose gradient achieved at the rostral margin, adjacent to the optic chiasma, was steeper on the gamma unit (due to the concentration of small collimator shots rostrally and beam blocking), which was therefore the dosimetrically preferred technique. In contrast, the vastly smaller internal dose gradient (11% for linac/X-knife versus 100% for Gamma Knife) and the ability to fractionate on the X-knife system, gave a large dosimetric advantage to the X-knife plan in the treatment of an acoustic neuroma, where the intracanalicular component of the cochlear nerve traversed the target volume. This advantage also pertains to the cochlear ramus of the internal auditory (labyrinthine) artery and the facial nerve. Our published work on X-knife radiosurgery of acoustic neuroma has documented improvement of hearing after therapy and may be relevant in this regard. That there are advantages in physical dose distribution and fractionation, producing a reduction in the biological dose in normal tissue, argues for the use of linac technology in acoustic neuromas. Craniopharyngiomas enveloping the optic nerve/chiasma will similarly be better treated by the linac X-knife system. It is apparent that different radiosurgery systems may be indicated in particular neuro-oncological situations.

Adenoma↗
PubMed2002

Assessment of antiangiogenic effect using 99mTc-EC-endostatin.

PURPOSE: Tumor vascular density may provide a prognostic indicator of metastatic potential or survival. The purpose of this study was to develop 99mTc-ethylenedicysteine-endostatin (99mTc-EC-endostatin) for the evaluation of anti-angiogenesis therapy. METHOD: 99mTc-EC-endostatin was prepared by conjugating ethylenedicysteine (EC) to endostatin, followed by adding pertechnetate and tin chloride. Radiochemical purity was > 95%. In vitro cell viability, affinity and TUNEL assays were performed. Tissue distribution and planar imaging of radiolabeled endostatin were determined in tumor-bearing rats. To assess anti-angiogenic treatment response, rats were treated with endostatin, paclitaxel and saline, followed by imaging with 99mTc-EC-endostatin. Tumor response to endostatin therapy in tumor-bearing animal models was assessed by correlating tumor uptake dose with microvessel density, VEGF, bFGF and IL-8 expression during endostatin therapy. RESULTS: In vitro cell viability and TUNEL assays indicated no marked difference between EC-endostatin and endostatin. Cellular uptake assay suggests that endostatin binds to endostatin receptor. Biodistribution of 99mTc-EC-endostatin in tumor-bearing rats showed increased tumor-to-tissue count density ratios as a function of time. Tumor uptake (%ID/g) of 99mTc-EC-endostatin was 0.2-0.5. Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-endostatin. The optimal time for imaging using radiolabeled endostatin was 2 hrs. 99mTc-EC-endostatin could assess treatment response. There was a correlation between tumor uptake and cellular targets expression. CONCLUSION: The results indicate that it is feasible to use 99mTc-EC-endostatin to assess efficiency of anti-angiogenesis therapy.

Angiogenesis Inhibitors↗
PubMed2004

Multiple and overlapping combinatorial codes orchestrate hormonal responsiveness and dictate cell-specific expression of the genes encoding luteinizing hormone.

Normal reproductive function in mammals requires precise control of LH synthesis and secretion by gonadotropes of the anterior pituitary. Synthesis of LH requires expression of two genes [alpha-glycoprotein subunit (alphaGSU) and LHbeta] located on different chromosomes. Hormones from the hypothalamus and gonads modulate transcription of both genes as well as secretion of the biologically active LH heterodimer. In males and females, the transcriptional tone of the genes encoding alphaGSU and LHbeta reflects dynamic integration of a positive signal provided by GnRH from hypothalamic neurons and negative signals emanating from gonadal steroids. Although alphaGSU and LHbeta genes respond transcriptionally in the same manner to changes in hormonal input, different combinations of regulatory elements orchestrate their response. These hormone-responsive regulatory elements are also integral members of much larger combinatorial codes responsible for targeting expression of alphaGSU and LHbeta genes to gonadotropes. In this review, we will profile the genomic landscape of the promoter-regulatory region of both genes, depicting elements and factors that contribute to gonadotrope-specific expression and hormonal regulation. Within this context, we will highlight the different combinatorial codes that control transcriptional responses, particularly those that mediate the opposing effects of GnRH and one of the sex steroids, androgens. We will use this framework to suggest that GnRH and androgens attain the same transcriptional endpoint through combinatorial codes unique to alphaGSU and LHbeta. This parallelism permits the dynamic and coordinate regulation of two genes that encode a single hormone.

Animals↗
PubMed1991

Angular homeostasis. VIII. Pursuit of a slowly moving target in a plane: relevance to lateralization in cardiovascular ontogeny.

We explore the pursuit in a plane of a target moving at constant slow speed in a straight line. Two models of the pursuit are given. In the continuous case, the pursuer is moving at constant speed and is subject to proportionate angular homeostasis with correction constant b. In the discrete version movement occurs at a constant speed in a sequence of straight line segments of constant length (called the step size, s) the end of the segments being called the vertices. The pattern considered is not the absolute position of the pursuer, but its distance and orientation relative to the target. Both the transients and the asymptotic orbit are addressed. A key quantity is r, the speed of the target expressed as a fraction of that of the pursuer. If the speed of the pursuer is defined as unity, r is also the ratio of the speeds. There exists a critical speed fraction, R(b,s), a function of b and s, that defines what the term slow designates. R(b,s), which has to be found numerically, has the following property. For r less than R(b,s), the asymptotic path is a simple closed curve. In the discrete case the vertices converge to a simple closed curve. The larger r, the more the path (or in the discrete analogue its set of vertices) departs from a circle, and the more eccentric the target is with respect to it. Interest centers on two issues. First we address the transient patterns of the path, notably whether or not the sense of any particular path (clockwise or counterclockwise) is the same throughout, or changes at some stage.(ABSTRACT TRUNCATED AT 250 WORDS)

Dextrocardia↗
PubMed2005

Ectonucleotidases of CD39 family modulate vascular inflammation and thrombosis in transplantation.

Transplantation results in exposure of the graft vasculature to warm and cold ischemia, followed by perfusion by circulating blood constituents and obligatory oxidant stress. Further graft injury occurs as consequences of acute humoral cellular rejection or chronic transplant vasculopathy, or both. Extracellular nucleotide stimulation of purinergic type 2 (P2) receptors are key components of platelet, endothelial cell (EC), and leukocyte activation resulting in vascular thrombosis and inflammation in vivo. CD39, the prototype nucleoside triphosphate diphosphohydrolase (NTPDase-1) is highly expressed on endothelium; in contrast, CD39L1/NTPDase-2 (a preferential adenosine triphosphatase [ATPase]) is found on vascular adventitial cells. Both ectoenzymes influence thrombogenesis by the regulated hydrolysis of extracellular nucleotides that differentially regulate P2-receptor activity and function in platelets and vascular cells. The intracytoplasmic domains of NTPDase-1 may also independently influence cellular activation and proliferation. NTPDase activity is substantively lost in the vasculature of injured or rejected grafts. A role for NTPDase-1 in thromboregulation has been validated by generation of mutant mice either null for cd39 or overexpressing human CD39. Administration of soluble NTPDase or induction of CD39 by adenoviral vectors, or both, are also of benefit in several models of transplantation. Administration of soluble CD39 or targeted expression may have future therapeutic application in transplantation-associated and other vascular diseases.

Adenosine Triphosphatases↗
PubMed1993

cis-acting DNA elements involved in oocyte-specific expression of mouse sperm receptor gene mZP3 are located close to the gene's transcription start site.

We report that cis-acting DNA elements involved in oocyte-specific expression of the mouse sperm receptor gene (mZP3) are located close to the gene's transcription start site. Mice bearing a transgene that consists of only 153 nt of mZP3 5'-flanking region fused to the firefly luciferase gene (153-ZP3/LUC) expressed the reporter gene in ovary not in a wide variety of tissues; although two of three lines carrying 153-ZP3/LUC also expressed the transgene in forebrain and hypothalamus. Within the ovaries of transgenic mice, luciferase activity was restricted to growing oocytes. However, levels of luciferase activity in these oocytes were lower than those in oocytes from mice bearing transgenes that contain a larger segment of mZP3 5'-flanking region (470-6,500 nt) fused to the firefly luciferase gene. Mice bearing a transgene that consists of 470 nt of mZP3 5'-flanking region and mZP3 intragenic sequences (ZDT) were also analyzed. The presence of mZP3 intragenic sequences did not result in significantly increased levels of firefly luciferase activity in oocytes of mice carrying the ZDT transgene. Overall, these results suggest that as little as 153 nt of mZP3 5'-flanking region is sufficient to target expression of the firefly luciferase gene to mouse oocytes and that the mZP3 intragenic sequences probably do not contain enhancer elements. Rather, enhancer elements are probably present between-153 and -470 nt of the mZP3 5'-flanking region.

Animals↗
PubMed2006

Deregulated minichromosomal maintenance protein MCM7 contributes to oncogene driven tumorigenesis.

Minichromosomal maintenance protein 7 (MCM7) is an essential component of the replication helicase complex (MCM2-7) required for DNA replication. Although this function is highly conserved among eukaryotes, additional functions for the MCM molecules continue to be described. Minichromosomal maintenance protein 7 is a marker for proliferation and is upregulated in a variety of tumors including neuroblastoma, prostate, cervical and hypopharyngeal carcinomas. To further investigate the general role of MCM7 in tumorigenesis, we generated a mouse model with deregulated MCM7 expression targeted to the basal layer of the epidermis using the keratin 14 (K14) promoter (K14.MCM7). When subjected to a two-stage chemical carcinogenesis protocol (dimethylbenz[alpha]anthracene (DMBA) initiation with 12-ortho-tetradecanoylphorbol-13-acetate promotion), K14.MCM7 mice showed significantly increased incidence and prevalence of tumor development relative to controls. Furthermore, within 40 weeks of treatment over 45% K14.MCM7 mice exhibited tumors that had converted to squamous cell carcinomas versus none in the control group. As predicted from previous skin carcinogenesis studies using DMBA as the initiating agent, Ras mutations where found in more than 90% of tumors isolated from K14.MCM7 mice. Whereas previous studies have shown that MCM7 is useful as a proliferation marker, our data suggest that deregulated MCM7 expression actively contributes to tumor formation, progression and malignant conversion.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2006

Gene therapy for treatment of inherited haematological disorders.

Gene therapy, a molecular medicine based on vector-mediated transfer of therapeutic genes, holds promise for a cure of monogenetic inherited diseases. In recent years, tremendous progress has been reported in the treatment of haematological disorders: clinical trials in severe combined immune deficiencies have been successful by using retroviral vectors to express target genes in haematopoietic stem cells, which after transplantation efficiently reconstituted the immune system concomitant with substantial improvement in the clinical status of patients. Conversely, unexpected adverse events were also encountered. In other work, progress towards clinical studies on ex vivo gene transfer for Fanconi anaemia and haemoglobinopathies has been made. Each approach features a unique treatment strategy and also faces various impediments to success. In the case of the X-linked bleeding disorder haemophilia, several Phase I/II clinical trials were conducted, including in vivo administration of viral vectors to skeletal muscle and liver. Adeno-associated viral gene transfer of coagulation Factor IX has been documented in human subjects, reaching therapeutic levels after infusion into a hepatic blood vessel. However, sustained expression of therapeutic levels (as shown in large animal models of haemophilia) has not yet been achieved in humans. In general, long-term follow-up will be important for assessment of the safety of all existing gene therapy strategies.

Animals↗
PubMed2002

Multiple internalization motifs differentially used by prolactin receptor isoforms mediate similar endocytic pathways.

Prolactin (PRL) regulates a variety of physiological processes, including mammary gland growth and differentiation, modulation of behavior, and immune function. A long PRL receptor (lPRLR) and short (sPRLR) isoform were identified in ruminants and rodents, which differ in their distal cytoplasmic domains and possess markedly distinct signaling capacities. Here we compared endocytosis of the bovine isoforms and found that the lPRLR internalized faster than the sPRLR, which would contribute to short-term down-regulation of lPRLR signaling at targets expressing both isoforms. Multiple motifs were required to mediate internalization of the lPRLR, including a phenylalanine (F290) plus a nearby dileucine, and three dileucines proximal to amino acid 272. This is different from the closely related GH receptor that requires only the phenyl-alanine-containing motif for endocytosis. Truncated lPRLR (cT272), which is the same length as the sPRLR and contained the proximal three dileucines, internalized at the same rate as the full-length lPRLR. Finally, the two dileucines shared by the sPRLR were able to mediate similar endocytic pathways as the lPRLR, as revealed by overexpression of mutant dynamin and clathrin hub, despite the slower rate. These studies define the basis of cellular trafficking of PRLR isoforms and increase our understanding of control of target cell responsiveness by PRL.

Amino Acid Sequence↗
PubMed2026

VHL synthetic lethality screens uncover CBF-β as a negative regulator of STING.

Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor β (CBF-β) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-β. Mechanistically, CBF-β loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-β at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-β in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling.

Humans↗
PubMedDate not supplied

Hemochromatosis due to mutations in transferrin receptor 2.

A rare recessive disorder which leads to iron overload and severe clinical complications similar to those reported in HFE-related hemochromatosis has been delineated and sometimes called hemochromatosis type 3. The gene responsible is Transferrin Receptor 2 (TFR2), which maps to chromosome 7q22. The TFR2 gene presents a significative homology to transferrin receptor (TFRC) gene, encodes for a transmembrane protein with a large extracellular domain, is able to bind transferrin, even if with lower affinity than TFRC. The TFR2 function is still unclear. The transcript does not contain IRE elements and is not modified by the cellular iron status. At variance with TFRC, interactions between TFR2 and HFE do not occur, at least in their soluble forms. TFR2 is spliced in two alternative forms, alfa and beta. The alfa form is strongly expressed in the liver. The beta form, codified from a start site in exon 4 of the alpha, has a low and ubiquitous expression. Using anti-TFR2 monoclonal antibodies we have confirmed expression of the protein in the liver but also in duodenal epithelial cells, and studied the protein functional behaviour in cell lines, in response to iron addition, iron deprivation and olo-transferrin exposure. Our results suggest a regulatory role of TFR2 in iron metabolism. Five TFR2 homozygous mutations have been documented in HFE3 patients: a nonsense mutation (Y250X); a C insertion that causes a frameshift and a premature stop codon (E60X); a missense mutation (M172K); a 12 basepair deletion in exon 16, that causes 4 aminoacid loss (AVAQ 594-597del) in the extracellular domain of TFR2; a missense mutation in exon 17 (Q690P). The mutation analysis supports the hypothesis that all are private mutations. The pathogenetic role of TFR2 in hemochromatosis has been recently further demonstrated through the targeted expression of the Y250X human mutation in mice, which develop sings of iron overload identical to the human disease. Although the rarity of TFR2 mutations limits their usefulness in diagnostic/screening programs, their study can contribute to a better understanding of the protein function.

Hemochromatosis↗
PubMed2001

Expression of Q227L-Galpha(s) inhibits intimal vessel wall hyperplasia after balloon injury.

Interaction between signaling pathways regulates many cellular functions, including proliferation. The Galpha(s)/cAMP pathway is known to inhibit signal flow from receptor tyrosine kinases to mitogen-activated protein kinase (MAPK)-1,2 and, thus, inhibit proliferation. Elevation of cAMP or adenovirus-directed expression of mutant (Q227L)-Galpha(s) (alpha(s)*) inhibits the proliferation of rat vascular smooth muscle cells (VSMCs) in culture. Platelet-derived growth factor (PDGF) stimulated MAPK activation and DNA synthesis was also blocked by expression of alpha(s)*. However, it is not known whether such mechanisms are operative in vivo. Proliferation of vascular smooth muscle cells in vivo was induced by balloon injury of carotid arteries in the rat. Recombinant adenovirus encoding beta-galactosidase (beta-gal) or alpha(s)* was applied to arterial segments injured by the balloon catheters. The alpha(s)*-treated vessels showed decreased phospho-MAPK staining in the intima as compared with beta-gal-treated vessels. Application of alpha(s)*, but not beta-gal containing adenovirus, inhibited formation of neointima by 50%. No change was observed in total vessel diameter or in the media or adventitia. These results suggest that the interaction between the Galpha(s) and MAPK pathways can regulate proliferation in vivo and that targeted expression of activated Galpha(s) may have therapeutic potential for the treatment of vascular pathophysiologies that arise from intimal hyperplasia.

Adenoviridae↗
PubMed1997

Class I- and class II-reactive TCRs coexpressed on CD4+ T cells both trigger CD4/CD8-shared and CD4-unique functions.

CD4+ and CD8+ T cells emerge from thymic selection expressing a TCR restricted by MHC class II (TCRII) and MHC class I (TCRI), and upon Ag stimulation develop respectively into Th and CTL effector cells. The influence of thymic differentiation and antigenic stimulation on the determination of T cell functions was studied, with CD4+ T cells expressing a transgenic TCRI that reacts with the class I alloantigen H-2K(b) in a CD8-independent fashion. Such T cells additionally express a TCR, probably TCRII, in which the transgenic TCR beta-chain is associated with endogenously rearranged TCR alpha-chains. Upon in vitro stimulation with H-2K(b)-expressing cells, both CD8+ and CD4+ transgenic TCR+ T cells developed into CTL capable of killing Ag-expressing target cells through a perforin-dependent mechanism, and secreted IL-2 and IFN-gamma. Fas ligand-dependent killing could also be induced in both CD8+ and CD4+ in vitro stimulated T cells. The capacity to secrete IL-4 was restricted to the CD4+ T cells, however, suggesting that both CD8/CD4-shared and CD4-unique programs can be elicited by stimulation of CD4 T cells through a TCRI. Acquisition of CTL function was also induced upon class II alloantigen stimulation through the endogenously rearranged TCRII, which represents a polyclonal set of TCRs. IL-2, IFN-gamma, and after restimulation, IL-4, were also produced. Thus: 1) events associated with intrathymic selection influence the gene program activated in response to the same TCRI/APC interaction; and 2) CD4+ T cells expressing a TCRI and a TCRII can activate the same gene program after engagement of either one of these TCRs.

Animals↗
PubMed1997

Antigen-presenting cells and the selection of immunodominant epitopes.

Cellular immune responses are directed against a narrow set of immunodominant peptides derived from complex antigens. By contrast, epitopes that are hidden or infrequent targets of immune responses have been termed cryptic or subdominant. Although the identification of immunodominant epitopes is important for vaccine development, understanding immunological reactivity to cryptic or subdominant epitopes may hold clues to autoimmunity. We have examined the role of antigen-presenting (APC) cells in the selection of class Ii-restricted epitopes for display to T lymphocytes. The formation and MHC-restricted presentation of distinct antigenic epitopes is directly dependent upon processing and ligand binding reactions within APC. A novel MHC heterodimer, HLA-DM, facilitates the binding and presentation of peptides by class II DR, DP, and DQ molecules. We have demonstrated that some epitopes derived from endogenous antigens bind class II proteins independent of DM, whereas the presentation of other endogenous peptides is greatly influenced by DM expression. Targeting of exogenous antigens into specialized processing compartments within APC appears to overcome the requirement for DM in antigen presentation. These studies suggest HLA-DM may play a role in epitope selection and immunodominance.

Animals↗
PubMed1992

Ectopic expression of ultraviolet-rhodopsins in the blue photoreceptor cells of Drosophila: visual physiology and photochemistry of transgenic animals.

We have generated transgenic flies expressing R7 cell-specific opsins in the major class of photoreceptor cells of the Drosophila retina and characterized their spectral properties using high-resolution microspectrophotometry and sensitivity recordings. We show that the Rh3 and Rh4 opsin genes encode UV-sensitive opsins with similar spectral properties (lambda max = 345 nm and 375 nm), and that Rh3 corresponds to the R7p and R7marg class of visual pigments. We have also generated Rh3 and Rh4 isoform-specific antibodies and present an R7 cell map of the Drosophila retina. In a related set of experiments, we show that it is possible to coexpress two different visual pigments functionally in the same cell and produce photoreceptors that display the summed spectral response of the individual pigments. These findings open up the possibility of tuning an animal's visual behavior by targeted expression of combinations of opsin genes to selective types of photoreceptors.

Animals↗
PubMedDate not supplied

Efficient translocation and apoptosis induction by adenovirus encoded VP22-p53 fusion protein in human tumor cells in vitro.

BACKGROUND: p53 mutations are one of the most frequent genetic alterations in cancer. Various mechanisms of delivering p53 protein into tumor cells, such as plasmids, retroviruses and adenoviruses, have been widely used in experimental studies. Although these methods are relatively successful, the transduction rate into surrounding cells is still a limiting factor. Recent studies have shown that fusing VP22 (important for intercellular transport) to p53 and delivering this protein to cells in the form of an adenovirus is a very efficient method of getting p53 into cells. RESULTS: In the present study, the effect of adenovirus encoded VP22-p53 fusion protein on p53 negative human cancer cells (LNCaP, SK-OV-3, OVCAR-3, DU 145) was investigated. A functional VP22-p53 fusion protein was produced as verified by immunofluorescence and analysis of p21 expression. Induction of p21 expression (target gene for p53) confirmed p53 was functional and immunofluorescence staining using p53 antiserum demonstrated the intercellular trafficking ability of the VP22 portion of the fusion protein. CONCLUSION: In conclusion, VP22-p53 showed efficient translocation into tumor cells, inhibition of tumor cell proliferation and induction of apoptosis. These characteristics make the fusion protein an attractive method for introducing p53 into human cells as well as a potential candidate for gene therapy.

Adenoviridae↗
PubMed1997

Large fragment of the probasin promoter targets high levels of transgene expression to the prostate of transgenic mice.

BACKGROUND: Androgen regulation and prostate-specific expression of targeted genes in transgenic mice can be controlled by a small DNA fragment of the probasin (PB) promoter (-426 to +28 base pairs, bp). Although the small PB fragment was sufficient to direct prostate-specific expression, the low levels of transgene expression suggested that important upstream regulatory sequences were missing. METHODS: To enhance transgene expression, a large fragment of the PB promoter (LPB, -11,500 to +28 bp) was isolated, linked to the bacterial chloramphenicol acetyl transferase (CAT) gene, and microinjected into CD1 mouse oocytes to generate transgenic mouse lines. RESULTS: As shown by the immunohistochemical studies, CAT gene expression was restricted to the prostatic epithelial cells in a tissue-specific manner. High levels of CAT gene expression were observed in two of the six LPB-CAT transgenic lines. In Line 1, developmental regulation of LPB-CAT was detected early, from 1 to 4 weeks of age, with the activity of CAT increasing from 3 to 40,936 dpm/min/mg protein. Upon sexual maturation and elevated serum androgen levels (7 weeks of age), a further 18-fold rise in CAT activity occurred. Hormone ablation by castration in mature mice dramatically reduced transgene expression, whereas treatment with androgens returned LPB-CAT expression to precastration levels. In contrast, treatment with glucocorticoids had no significant effect on CAT gene expression. Zinc treatment of the castrated animals also increased LPB-CAT expression three- to four-fold in two prostatic lobes. CONCLUSIONS: This study demonstrates that important regulatory DNA sequences located in the LPB fragment contribute to tissue-specific expression and greatly increase levels of transgene expression induced by androgens and zinc.

Aging↗