scholarly journals Tissue-specific promoters of the α human folate receptor gene yield transcripts with divergent 5′ leader sequences and different translational efficiencies

1997 ◽  
Vol 326 (2) ◽  
pp. 439-447 ◽  
Author(s):  
Susan J. ROBERTS ◽  
Koong-Nah CHUNG ◽  
Kirsten NACHMANOFF ◽  
Patrick C. ELWOOD
Keyword(s):  
Wheat Germ ◽  
Folate Receptor ◽  
Receptor Gene ◽  
Leader Sequence ◽  
Tissue Specific ◽  
Promoter Usage ◽  
Human Folate Receptor ◽  
Wheat Germ Extracts

The α human folate receptor (αhFR), or KB cell folate receptor, gene contains two major promoters that produce transcripts, KB1 and KB4, varying only in the length and sequence of their 5′ untranslated regions (UTRs). Using RNase protection assays specific for each isoform, we show that the level of expression of these two transcripts is tissue-specific, indicating that promoter usage is regulated, not constitutive. RNA stabilities and translational efficiencies of the KB1 and KB4 transcripts were compared to determine the functional significance of the different 5′ UTRs. Analyses of RNA turnover in vivo with actinomycin D to block new transcription and in vitro with a cytoplasmic extract indicate no discernible differences in the stabilities of the two transcripts. However, the KB4 transcript is 2–3-fold more efficiently translated in wheat germ extracts in vitro and transfected CHO cells in vivo. Also, high ionic strength, which favours the formation of RNA secondary structure, differentially affects the translational efficiencies of the two transcripts. Translation of the longer KB1 mRNA is 2–5-fold more inhibited by hypertonic conditions than translation of the KB4 mRNA. Because the 5′ UTR of KB1 is approximately four times longer than the 5′ UTR of KB4, 149 bp (75%) of the KB1 5′ UTR were deleted to determine whether the long leader sequence inhibited translation. The resulting derivative, dKB1, has a 5′ UTR similar in length, but not sequence, to the 5′ UTR of KB4. dKB1 is translated at a level approaching that of KB4 in wheat germ extracts, indicating that the upstream portion of the 5′ leader sequence contributes to the relative translational inefficiency of KB1. Hence, one consequence of tissue-specific promoter usage is the production of αhFR transcripts with different 5′ non-coding regions that affect translational efficiency.

scholarly journals The Radiation-Induced Regenerative Response of Adult Tissue-Specific Stem Cells: Models and Signaling Pathways

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 855
Author(s):  
Paola Serrano Martinez ◽  
Lorena Giuranno ◽  
Marc Vooijs ◽  
Robert P. Coppes
Keyword(s):  
Signaling Pathways ◽  
Progenitor Cells ◽  
Tissue Regeneration ◽  
Normal Tissue ◽  
Cellular Response ◽  
Adult Tissue ◽  
Tissue Specific ◽  
Radiation Induced

Radiotherapy is involved in the treatment of many cancers, but damage induced to the surrounding normal tissue is often inevitable. Evidence suggests that the maintenance of homeostasis and regeneration of the normal tissue is driven by specific adult tissue stem/progenitor cells. These tasks involve the input from several signaling pathways. Irradiation also targets these stem/progenitor cells, triggering a cellular response aimed at achieving tissue regeneration. Here we discuss the currently used in vitro and in vivo models and the involved specific tissue stem/progenitor cell signaling pathways to study the response to irradiation. The combination of the use of complex in vitro models that offer high in vivo resemblance and lineage tracing models, which address organ complexity constitute potential tools for the study of the stem/progenitor cellular response post-irradiation. The Notch, Wnt, Hippo, Hedgehog, and autophagy signaling pathways have been found as crucial for driving stem/progenitor radiation-induced tissue regeneration. We review how these signaling pathways drive the response of solid tissue-specific stem/progenitor cells to radiotherapy and the used models to address this.

Repeated CT elements bound by zinc finger proteins control the absolute and relative activities of the two principal human c-myc promoters

1993 ◽  
Vol 13 (9) ◽  
pp. 5710-5724
Author(s):  
E DesJardins ◽  
N Hay
Keyword(s):  
Zinc Finger ◽  
Tandem Repeats ◽  
Zinc Finger Protein ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Maximal Activity ◽  
Single Copy ◽  
Promoter Usage

Transcription of the human proto-oncogene c-myc is governed by two tandem principal promoters, termed P1 and P2. In general, the downstream promoter, P2, is predominant, which is in contrast to the promoter occlusion phenomenon usually observed in genes containing tandem promoters. A shift in human c-myc promoter usage has been observed in some tumor cells and in certain physiological conditions. However, the mechanisms that regulate promoter usage are not well understood. The present studies identify regulators which are required to promote transcription from both human c-myc promoters, P1 and P2, and have a role in determining their relative activities in vivo. A novel regulatory region located 101 bp upstream of P1 was characterized and contains five tandem repeats of the consensus sequence CCCTCCCC (CT element). The integrity of the region containing all five elements is required to promote transcription from P1 and for maximal activity from P2 in vivo. A single copy of this same element, designated CT-I2, also appears in an inverted orientation 53 bp upstream of the P2 transcription start site. This element has an inhibitory effect on P1 transcription and is required for P2 transcription. The transcription factor Sp1 was identified as the factor that binds specifically to the tandem CT elements upstream of P1 and to the CT-I2 element upstream of P2. In addition, the recently cloned zinc finger protein ZF87, or MAZ, was also able to bind these same elements in vitro. The five tandem CT elements can be functionally replaced by a heterologous enhancer that only in the absence of CT-I2 reverses the promoter usage, similar to what is observed in the translocated c-myc allele of Burkitt's lymphoma cells.

scholarly journals c-Jun N-Terminal Kinase Activation of Activator Protein-1 Underlies Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene in αT3-1 Cells

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 839-849 ◽  
Author(s):  
Buffy S. Ellsworth ◽  
Brett R. White ◽  
Ann T. Burns ◽  
Brian D. Cherrington ◽  
Annette M. Otis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Cell Model ◽  
Receptor Gene ◽  
Critical Role ◽  
Dominant Negative ◽  
Activator Protein 1 ◽  
Activator Protein

Reproductive function is dependent on the interaction between GnRH and its cognate receptor found on gonadotrope cells of the anterior pituitary gland. GnRH activation of the GnRH receptor (GnRHR) is a potent stimulus for increased expression of multiple genes including the gene encoding the GnRHR itself. Thus, homologous regulation of the GnRHR is an important mechanism underlying gonadotrope sensitivity to GnRH. Previously, we have found that GnRH induction of GnRHR gene expression in αT3-1 cells is partially mediated by protein kinase C activation of a canonical activator protein-1 (AP-1) element. In contrast, protein kinase A and a cAMP response element-like element have been implicated in mediating the GnRH response of the GnRHR gene using a heterologous cell model (GGH3). Herein we find that selective removal of the canonical AP-1 site leads to a loss of GnRH regulation of the GnRHR promoter in transgenic mice. Thus, an intact AP-1 element is necessary for GnRH responsiveness of the GnRHR gene both in vitro and in vivo. Based on in vitro analyses, GnRH appeared to enhance the interaction of JunD, FosB, and c-Fos at the GnRHR AP-1 element. Although enhanced binding of cFos reflected an increase in gene expression, GnRH appeared to regulate both FosB and JunD at a posttranslational level. Neither overexpression of a constitutively active Raf-kinase nor pharmacological blockade of GnRH-induced ERK activation eliminated the GnRH response of the GnRHR promoter. GnRH responsiveness was, however, lost in αT3-1 cells that stably express a dominant-negative c-Jun N-terminal kinase (JNK) kinase, suggesting a critical role for JNK in mediating GnRH regulation of the GnRHR gene. Consistent with this possibility, we find that the ability of forskolin and membrane-permeable forms of cAMP to inhibit the GnRH response of the GnRHR promoter is associated with a loss of both JNK activation and GnRH-mediated recruitment of the primary AP-1-binding components.

scholarly journals A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish

2020 ◽  
Author(s):  
Yueyang Wang ◽  
Alan Y. Hsu ◽  
Eric M. Walton ◽  
Ramizah Syahirah ◽  
Tianqi Wang ◽  
...  
Keyword(s):  
Cell Motility ◽  
Neutrophil Migration ◽  
Transgenic Fish ◽  
Subcellular Location ◽  
Vector System ◽  
Specific Gene ◽  
Tissue Specific ◽  
Biological Studies

AbstractTissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.

scholarly journals Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects upon proliferation, differentiation and hormone sensitivity

2006 ◽  
Vol 191 (1) ◽  
pp. 101-111 ◽  
Author(s):  
David J Flint ◽  
Nadine Binart ◽  
Stephanie Boumard ◽  
John J Kopchick ◽  
Paul Kelly
Keyword(s):  
Adipose Tissue ◽  
Receptor Gene ◽  
Metabolic Effects ◽  
Wild Type ◽  
Extrinsic Factors ◽  
Differentiation Potential ◽  
Site Specific ◽  
Proliferation And Differentiation

Direct metabolic effects of GH on adipose tissue are well established, but effects of prolactin (PRL) have been more controversial. Recent studies have demonstrated PRL receptors on adipocytes and effects of PRL on adipose tissue in vitro. The role of GH in adipocyte proliferation and differentiation is also controversial, since GH stimulates adipocyte differentiation in cell lines, whereas it stimulates proliferation but inhibits differentiation of adipocytes in primary cell culture. Using female gene disrupted (ko) mice, we showed that absence of PRL receptors (PRLRko) impaired development of both internal and s.c. adipose tissue, due to reduced numbers of adipocytes, an effect differing from that of reduced food intake, where cell volume is decreased. In contrast, GHRko mice exhibited major decreases in the number of internal adipocytes, whereas s.c. adipocyte numbers were increased, even though body weight was decreased by 40–50%. The changes in adipose tissue in PRLRko mice appeared to be entirely due to extrinsic factors since preadipocytes proliferated and differentiated in similar fashion to wild-type animals in vitro and their response to insulin and isoproterenol was similar to wild-type animals. This contrasted with GHRko mice, where s.c. adipocytes proliferated, differentiated, and responded to hormones in identical fashion to controls, whereas parametrial adipocytes exhibited markedly depressed proliferation and differentiation potential and failed to respond to insulin or noradrenaline. Our results provide in vivo evidence that both GH and PRL stimulate differentiation of adipocytes but that the effects of GH are site specific and induce intrinsic changes in the precursor population, which are retained in vitro.

scholarly journals Structural characterization, antioxidant and cytotoxic effects of iron nanoparticles synthesized using Asphodelus aestivus Brot. aqueous extract

2020 ◽  
Vol 9 (1) ◽  
pp. 153-163 ◽  
Author(s):  
Burcu Sumer Tuzun ◽  
Tugce Fafal ◽  
Pelin Tastan ◽  
Bijen Kivcak ◽  
Besra Ozmen Yelken ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
Receptor Gene ◽  
Control Cell ◽  
Antioxidant Properties ◽  
Gene Expressions ◽  
Vis Spectroscopy ◽  
Mcf 7

AbstractASP was used to synthesize FeNPA. They were characterized by UV-vis spectroscopy, FT-IR, TEM, SEM, XRD and ZP. The aim of this study was to evaluate in vitro cytotoxic activity and antioxidant acitivities of FeNPA and ASP. The antioxidant properties were evaluated using DPPH, ABTS+ and H2O2 assays. FeNPA had higher antioxidant activity comparing to ASP according to DPPH (IC50: 3.48 μg/mL) and ABTS+ (60.52%) assays. Anti-cancer activities of FeNPA and ASP were investigated in breast cancer, melanoma and control cell lines. FeNPA was more cytotoxic than ASP in MCF-7, MeWo, CHL-1, and HEL 299 cells. FeNPA had shown that mitochondria induce apoptosis through stress in MDA-MB-231, and cells MeWo. ASP also induced apoptosis 2.23-fold in MCF-7 cells. Progesterone receptor gene expression showed a 10-fold increase in a hormone-dependent MCF-7 cell line in ASP, and FeNPA treatment. Expressions of BCL6, CXCL12, DNAJC15, RB1 and TPM1 in melanoma cancer cell lines were significantly increased in ASP and FeNPA administration. It had been shown that FeNPA regulates gene expressions that may be considered important in terms of prognosis in breast cancer and melanoma cell lines and it is suggested that gene expressions regulated by FeNPA are also evaluated in animal models in vivo.

scholarly journals New 55Co-labeled Albumin-Binding Folate Derivatives as Potential PET Agents for Folate Receptor Imaging

2019 ◽  
Vol 12 (4) ◽  
pp. 166 ◽  
Author(s):  
Lauren L. Radford ◽  
Solana Fernandez ◽  
Rebecca Beacham ◽  
Retta El Sayed ◽  
Renata Farkas ◽  
...  
Keyword(s):  
Folate Receptor ◽  
Small Animal ◽  
Small Animal Pet ◽  
Receptor Imaging ◽  
Albumin Binding ◽  
Liver Uptake ◽  
Biodistribution Studies ◽  
Positron Emission

Overexpression of folate receptors (FRs) on different tumor types (e.g., ovarian, lung) make FRs attractive in vivo targets for directed diagnostic/therapeutic agents. Currently, no diagnostic agent suitable for positron emission tomography (PET) has been adopted for clinical FR imaging. In this work, two 55Co-labeled albumin-binding folate derivatives-[55Co]Co-cm10 and [55Co]Co-rf42-with characteristics suitable for PET imaging have been developed and evaluated. High radiochemical yields (≥95%) and in vitro stabilities (≥93%) were achieved for both compounds, and cell assays demonstrated FR-mediated uptake. Both 55Co-labeled folate conjugates demonstrated high tumor uptake of 17% injected activity per gram of tissue (IA/g) at 4 h in biodistribution studies performed in KB tumor-bearing mice. Renal uptake was similar to other albumin-binding folate derivatives, and liver uptake was lower than that of previously reported [64Cu]Cu-rf42. Small animal PET/CT images confirmed the biodistribution results and showed the clear delineation of FR-expressing tumors.

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