Terry Elton - Academia.edu (original) (raw)
Papers by Terry Elton
ASPET 2023 Annual Meeting Abstract - Cancer Pharmacology
PLOS ONE
DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly mul... more DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 an important target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2α/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2α/90, due to intronic polyadenylation (IPA; within intron 19) in an acquired etoposide-resistant K562 clonal cell line, K/VP.5. We previously reported that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2α exon 19/intron 19 5′ splice site in drug-resistant K/VP.5 cells by gene-editing restored TOP2α/170 levels, diminished TOP2α/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19/intro...
Cancers
Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, pr... more Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated with a reduction of nuclear TOP2α/170 levels. In leukemia cell lines with acquired resistance to TOP2α-targeted drugs and reduced TOP2α/170 expression, variant TOP2α mRNA transcripts have been reported due to IPA that resulted in the translation of C-terminal truncated isoforms with altered nuclear-cytoplasmic distribution or heterodimerization with wild-type TOP2α/170. This review provides an overview of the various mechanisms regulating pre-mRNA processing and alternative polyadenylation, as well as the utilization of CRISPR/Cas9 specific gene editing through homology directed repair (HDR) to decrease IPA when splice sites are intri...
Cancer Drug Resistance, 2020
DNA topoisomerase IIα (170 kDa, TOP2α/170) induces transient DNA double-strand breaks in prolifer... more DNA topoisomerase IIα (170 kDa, TOP2α/170) induces transient DNA double-strand breaks in proliferating cells to resolve DNA topological entanglements during chromosome condensation, replication, and segregation. Therefore, TOP2α/170 is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to chemoresistance. Although many resistance mechanisms have been defined, acquired resistance of human cancer cell lines to TOP2α interfacial inhibitors/poisons is frequently associated with a reduction of Top2α/170 expression levels. Recent studies by our laboratory, in conjunction with earlier findings by other investigators, support the hypothesis that a major mechanism of acquired resistance to TOP2α-targeted drugs is due to alternative RNA processing/splicing. Specifically, several TOP2α mRNA splice variants have been reported which retain introns and are translated into truncated TOP2α isoforms lacking nuclear localization sequences and subsequent dysregulated nuclear-cytoplasmic disposition. In addition, intron retention can lead to truncated isoforms that lack both nuclear localization sequences and the active site tyrosine (Tyr805) necessary for forming enzyme-DNA covalent complexes and inducing DNA damage in the presence of TOP2α-targeted drugs. Ultimately, these truncated TOP2α isoforms result in decreased drug activity against TOP2α in the nucleus and manifest drug resistance. Therefore, the complete characterization of the mechanism(s) regulating the alternative RNA processing of TOP2α pre-mRNA may result in new strategies to circumvent acquired drug resistance. Additionally, novel TOP2α splice variants and truncated TOP2α isoforms may be useful as biomarkers for drug resistance, prognosis, and/or direct future TOP2α-targeted therapies.
Information about reprints can be found online at: Reprints: document. Permissions and Rights Que... more Information about reprints can be found online at: Reprints: document. Permissions and Rights Question and Answer this process is available in the click Request Permissions in the middle column of the Web page under Services. Further information about Office. Once the online version of the published article for which permission is being requested is located, can be obtained via RightsLink, a service of the Copyright Clearance Center, not the EditorialHypertensionin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions: by guest on March 5,
Research article Physician reported perception in the treatment of high blood pressure does not c... more Research article Physician reported perception in the treatment of high blood pressure does not correspond to practice
EXCLI Journal, 2015
Functionally matured microRNAs (miRNAs) are small single-stranded non-coding RNA molecules which ... more Functionally matured microRNAs (miRNAs) are small single-stranded non-coding RNA molecules which are emerging as important post-transcriptional regulators of gene expression and consequently are central players in many physiological and pathological processes. Since the biological roles of individual miRNAs will be dictated by the mRNAs that they regulate, the identification and validation of miRNA/mRNA target interactions is critical for our understanding of the regulatory networks governing biological processes. We promulgate the combined use of prediction algorithms, the examination of curated databases of experimentally supported miRNA/mRNA interactions, manual sequence inspection of cataloged miRNA binding sites in specific target mRNAs, and review of the published literature as a reliable practice for identifying and prioritizing biologically important miRNA/mRNA target pairs. Once a preferred miRNA/mRNA target pair has been selected, we propose that the authenticity of a func...
Molecular Pharmacology
DNA topoisomerase IIa protein (TOP2a) 170 kDa (TOP2a/170) is an important target for anticancer a... more DNA topoisomerase IIa protein (TOP2a) 170 kDa (TOP2a/170) is an important target for anticancer agents whose efficacy is often attenuated by chemoresistance. Our laboratory has characterized acquired resistance to etoposide in human leukemia K562 cells. The clonal resistant subline K/VP.5 contains reduced TOP2a/170 mRNA and protein levels compared with parental K562 cells. The aim of this study was to determine whether microRNA (miRNA)-mediated mechanisms play a role in drug resistance via decreased expression of TOP2a/170. miRNAsequencing revealed that human miR-9-3p and miR-9-5p were among the top six of those overexpressed in K/VP.5 compared with K562 cells; validation by quantitative polymerase chain reaction demonstrated overexpression of both miRNAs. miRNA recognition elements (MREs) for both miRNAs are present in the 3ʹ-untranslated region (UTR) of TOP2a/170. Transfecting K562 cells with a reporter plasmid harboring the TOP2a/170 3ʹ-UTR together with either miR-9-3p or miR-9-5p mimics resulted in a statistically significant decrease in luciferase expression. Mutating the miR-9-3p or miR-9-5p MREs prevented this decrease, demonstrating direct interaction between these miRNAs and TOP2a/170 mRNA. Transfection of K562 cells with miR-9-3p or miR-9-5p mimics led to decreased TOP2a/170 protein levels without a change in TOP2a/170 mRNA and resulted in attenuated etoposide-induced DNA damage (gain-of-miRNAinhibitory function). Conversely, transfection of miR-9-3p or miR-9-5p inhibitors in K/VP.5 cells (overexpressed miR-9 and low TOP2a/170) led to increased TOP2a/170 protein expression without a change in TOP2a/170 mRNA levels and resulted in enhancement of etoposide-induced DNA damage (loss-of-miRNA-inhibitory function). Taken together, these results strongly suggest that these miRNAs play a role in and are potential targets for circumvention of acquired resistance to etoposide. SIGNIFICANCE STATEMENT Results presented here indicate that miR-9-3p and miR-9-5p decrease DNA topoisomerase IIa protein 170 kDa expression levels in acquired resistance to etoposide. These findings contribute new information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. Furthermore, increased expression of miR-9-3p and miR-9-5p in chemoresistant cancer cells may support their validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy.
Molecular Pharmacology, 2021
An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topo... more An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR...
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](Molecular pharmacology, 2018
DNA topoisomerase II (170 kDa, TOP2/170) is essential in proliferating cells by resolving DNA top... more DNA topoisomerase II (170 kDa, TOP2/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2/90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2/90 (786 aa) is the translation product of a TOP2 mRNA that retains a processed intron 19. TOP2/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2/170 isoform (1531 aa). Here, we found that TOP2/90, like TOP2/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2/90 and TOP2/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2...
Molecular and Cellular Endocrinology, 2016
The Faseb Journal, Mar 1, 2008
American Journal of Physiology Gastrointestinal and Liver Physiology, 2010
Studies have demonstrated that angiotensin II (Ang II) can regulate intestinal fluid and electrol... more Studies have demonstrated that angiotensin II (Ang II) can regulate intestinal fluid and electrolyte transport and control intestinal wall muscular activity. Ang II is also a proinflammatory mediator that participates in inflammatory responses such as apoptosis, angiogenesis, and vascular remodeling; accumulating evidence suggests that this hormone may be involved in gastrointestinal (GI) inflammation and carcinogenesis. Ang II binds to two distinct G protein-coupled receptor subtypes, the AT 1R and AT 2R, which are widely expressed in the GI system. Together these studies suggest that Ang II-AT 1R/-AT2R actions may play an important role in GI tract physiology and pathophysiology. Currently it is not known whether miRNAs can regulate the expression of the human AT 1R (hAT1R) in the GI system. PCR and in situ hybridization experiments demonstrated that miR-802 was abundantly expressed in human colon and intestine. Luciferase reporter assays demonstrated that miR-802 could directly interact with the bioinformatics-predicted target site harbored within the 3=-untranslated region of the hAT 1R mRNA. To validate that the levels of miR-802 were physiologically relevant in the GI system, we demonstrated that miR-802 "loss-offunction" experiments resulted in augmented hAT 1R levels and enhanced Ang II-induced signaling in a human intestinal epithelial cell line. These results suggest that miR-802 can modulate the expression of the hAT 1R in the GI tract and ultimately play a role in regulating the biological efficacy of Ang II in this system. gastrointestinal system; miRNAs ANGIOTENSIN II (Ang II), an octapeptide hormone, is the biologically active component of the renin-angiotensin system (RAS) (13, 47). Ang II has emerged as a critical hormone that affects the function of virtually all organs, including heart, kidney, vasculature, brain, adrenal, liver, reproductive organs, and the gastrointestinal (GI) system (13, 47). Pharmacological and morphological studies have demonstrated that Ang II plays an important role in GI system epithelial transport processes (11, 33, 34). For example, in the GI tract, Ang II has been shown to mediate epithelial sodium and water absorption in the jejunum, ileum, and distal colon (33). More recent studies have demonstrated that in the jejunum, the effect of Ang II on sodium and water transport is dose dependent (5, 25, 35, 36).
Circulation, Oct 28, 2008
The Faseb Journal, Apr 1, 2010
Molecular and Cellular Endocrinology, 2006
At least four alternatively spliced mRNAs can be synthesized from the human AT 1 R (hAT 1 R) gene... more At least four alternatively spliced mRNAs can be synthesized from the human AT 1 R (hAT 1 R) gene that differ only in the inclusion or exclusion of exon 2 and/or 3. RT-PCR experiments demonstrate that splice variants harboring exon 2 accounts for at least 30% of all the hAT 1 R mRNA transcripts expressed in the human tissues investigated. Since exon 2 contains two upstream AUGs or open reading frames (uORFs), we hypothesized that these AUGs would inhibit the translation of the downstream hAT 1 R protein ORF harbored in exon 4. This study demonstrates that the inclusion of exon 2 in hAT 1 R mRNA transcripts dramatically reduces hAT 1 R protein levels (nine-fold) and significantly attenuates Ang II responsiveness (∼four-fold). Interestingly, only when both AUGs were mutated in combination were the hAT 1 R density and Ang II signaling levels comparable with those values obtained using mRNA splice variants that did not include exon 2. This observation is consistent with a model where the majority of the ribosomes likely translate uORF#1 and are then unable to reinitiate at the downstream hAT 1 R ORF, in part due to the presence of AUG#2 and to the short intercistronic spacing. Importantly, TGF- 1 treatment (4 ng/ml for 4 h) of fibroblasts up-regulated hAT 1 R mRNA splice variants, which harbored exon 2, six-fold. Since AT 1 R activation is closely associated with cardiovascular disease, the inclusion of exon 2 by alternative splicing represents a novel mechanism to reduce the overall production of the hAT 1 R protein and possibly limit the potential pathological effects of AT 1 R activation.
Biochimica Et Biophysica Acta N Gene Structure and Expression, Jan 18, 1999
Transcriptional mechanisms regulating the expression of the rat angiotensin II type 1A receptor (... more Transcriptional mechanisms regulating the expression of the rat angiotensin II type 1A receptor (rAT 1A R) gene were investigated in cultured rat vascular smooth muscle cells (VSMC). Transcriptional analyses of various 5P-deletion mutants of the rAT 1A R promoter region, fused upstream from the firefly luciferase gene, demonstrated that a 71 base pair (bp) region (3557 to 3486 bp, with respect to transcription initiation) was necessary for expression of this gene in VSMC. Electrophoretic mobility shift assays demonstrated that specific protein-DNA complexes were formed with the 3516 to 3486 bp region of the rAT 1A R promoter when incubated with VSMC extract. Computer analysis of this region indicated the presence of an A/T-rich sequence (i.e., TTTAAAAATAAA) which is similar to a myocyte enhancer binding factor 2 (MEF2) cis-regulatory element (i.e., CTTAAAAATAAC). Site-directed mutagenesis of this A/T-rich sequence inhibited rAT 1A R promoter activity in VSMC, suggesting that this region was necessary for expression of this gene in these cells. Immuno-gel shift experiments suggest that MEF2 heterodimers may interact with the A/T-rich sequence in the rAT 1A R promoter. Additionally, it was demonstrated that a transcription factor non-homologous to MEF2 can also interact with this A/T-rich site in the rAT 1A R promoter. Taken together, our results suggest that MEF2 heterodimers, and/or transcription factors nonhomologous to MEF2, are required to regulate the expression of the rAT 1A R gene in VSMC.
Circulation, Oct 28, 2008
Trends in Endocrinology and Metabolism, Jan 3, 2003
Activation of the angiotensin II type 1 (AT 1) receptor is closely involved in the pathogenesis o... more Activation of the angiotensin II type 1 (AT 1) receptor is closely involved in the pathogenesis of cardiovascular diseases; therefore, aberrant regulation of the production of this receptor might play a role in these disorders. Currently, there is strong evidence to suggest that the predominant mechanism regulating the number of AT 1 receptors is the modulation of mRNA stability. Here, we discuss the importance of alternative splicing as an additional post-transcriptional mechanism regulating human AT 1 receptor number and function.
ASPET 2023 Annual Meeting Abstract - Cancer Pharmacology
PLOS ONE
DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly mul... more DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 an important target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2α/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2α/90, due to intronic polyadenylation (IPA; within intron 19) in an acquired etoposide-resistant K562 clonal cell line, K/VP.5. We previously reported that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2α exon 19/intron 19 5′ splice site in drug-resistant K/VP.5 cells by gene-editing restored TOP2α/170 levels, diminished TOP2α/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19/intro...
Cancers
Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, pr... more Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated with a reduction of nuclear TOP2α/170 levels. In leukemia cell lines with acquired resistance to TOP2α-targeted drugs and reduced TOP2α/170 expression, variant TOP2α mRNA transcripts have been reported due to IPA that resulted in the translation of C-terminal truncated isoforms with altered nuclear-cytoplasmic distribution or heterodimerization with wild-type TOP2α/170. This review provides an overview of the various mechanisms regulating pre-mRNA processing and alternative polyadenylation, as well as the utilization of CRISPR/Cas9 specific gene editing through homology directed repair (HDR) to decrease IPA when splice sites are intri...
Cancer Drug Resistance, 2020
DNA topoisomerase IIα (170 kDa, TOP2α/170) induces transient DNA double-strand breaks in prolifer... more DNA topoisomerase IIα (170 kDa, TOP2α/170) induces transient DNA double-strand breaks in proliferating cells to resolve DNA topological entanglements during chromosome condensation, replication, and segregation. Therefore, TOP2α/170 is a prominent target for anticancer drugs whose clinical efficacy is often compromised due to chemoresistance. Although many resistance mechanisms have been defined, acquired resistance of human cancer cell lines to TOP2α interfacial inhibitors/poisons is frequently associated with a reduction of Top2α/170 expression levels. Recent studies by our laboratory, in conjunction with earlier findings by other investigators, support the hypothesis that a major mechanism of acquired resistance to TOP2α-targeted drugs is due to alternative RNA processing/splicing. Specifically, several TOP2α mRNA splice variants have been reported which retain introns and are translated into truncated TOP2α isoforms lacking nuclear localization sequences and subsequent dysregulated nuclear-cytoplasmic disposition. In addition, intron retention can lead to truncated isoforms that lack both nuclear localization sequences and the active site tyrosine (Tyr805) necessary for forming enzyme-DNA covalent complexes and inducing DNA damage in the presence of TOP2α-targeted drugs. Ultimately, these truncated TOP2α isoforms result in decreased drug activity against TOP2α in the nucleus and manifest drug resistance. Therefore, the complete characterization of the mechanism(s) regulating the alternative RNA processing of TOP2α pre-mRNA may result in new strategies to circumvent acquired drug resistance. Additionally, novel TOP2α splice variants and truncated TOP2α isoforms may be useful as biomarkers for drug resistance, prognosis, and/or direct future TOP2α-targeted therapies.
Information about reprints can be found online at: Reprints: document. Permissions and Rights Que... more Information about reprints can be found online at: Reprints: document. Permissions and Rights Question and Answer this process is available in the click Request Permissions in the middle column of the Web page under Services. Further information about Office. Once the online version of the published article for which permission is being requested is located, can be obtained via RightsLink, a service of the Copyright Clearance Center, not the EditorialHypertensionin Requests for permissions to reproduce figures, tables, or portions of articles originally publishedPermissions: by guest on March 5,
Research article Physician reported perception in the treatment of high blood pressure does not c... more Research article Physician reported perception in the treatment of high blood pressure does not correspond to practice
EXCLI Journal, 2015
Functionally matured microRNAs (miRNAs) are small single-stranded non-coding RNA molecules which ... more Functionally matured microRNAs (miRNAs) are small single-stranded non-coding RNA molecules which are emerging as important post-transcriptional regulators of gene expression and consequently are central players in many physiological and pathological processes. Since the biological roles of individual miRNAs will be dictated by the mRNAs that they regulate, the identification and validation of miRNA/mRNA target interactions is critical for our understanding of the regulatory networks governing biological processes. We promulgate the combined use of prediction algorithms, the examination of curated databases of experimentally supported miRNA/mRNA interactions, manual sequence inspection of cataloged miRNA binding sites in specific target mRNAs, and review of the published literature as a reliable practice for identifying and prioritizing biologically important miRNA/mRNA target pairs. Once a preferred miRNA/mRNA target pair has been selected, we propose that the authenticity of a func...
Molecular Pharmacology
DNA topoisomerase IIa protein (TOP2a) 170 kDa (TOP2a/170) is an important target for anticancer a... more DNA topoisomerase IIa protein (TOP2a) 170 kDa (TOP2a/170) is an important target for anticancer agents whose efficacy is often attenuated by chemoresistance. Our laboratory has characterized acquired resistance to etoposide in human leukemia K562 cells. The clonal resistant subline K/VP.5 contains reduced TOP2a/170 mRNA and protein levels compared with parental K562 cells. The aim of this study was to determine whether microRNA (miRNA)-mediated mechanisms play a role in drug resistance via decreased expression of TOP2a/170. miRNAsequencing revealed that human miR-9-3p and miR-9-5p were among the top six of those overexpressed in K/VP.5 compared with K562 cells; validation by quantitative polymerase chain reaction demonstrated overexpression of both miRNAs. miRNA recognition elements (MREs) for both miRNAs are present in the 3ʹ-untranslated region (UTR) of TOP2a/170. Transfecting K562 cells with a reporter plasmid harboring the TOP2a/170 3ʹ-UTR together with either miR-9-3p or miR-9-5p mimics resulted in a statistically significant decrease in luciferase expression. Mutating the miR-9-3p or miR-9-5p MREs prevented this decrease, demonstrating direct interaction between these miRNAs and TOP2a/170 mRNA. Transfection of K562 cells with miR-9-3p or miR-9-5p mimics led to decreased TOP2a/170 protein levels without a change in TOP2a/170 mRNA and resulted in attenuated etoposide-induced DNA damage (gain-of-miRNAinhibitory function). Conversely, transfection of miR-9-3p or miR-9-5p inhibitors in K/VP.5 cells (overexpressed miR-9 and low TOP2a/170) led to increased TOP2a/170 protein expression without a change in TOP2a/170 mRNA levels and resulted in enhancement of etoposide-induced DNA damage (loss-of-miRNA-inhibitory function). Taken together, these results strongly suggest that these miRNAs play a role in and are potential targets for circumvention of acquired resistance to etoposide. SIGNIFICANCE STATEMENT Results presented here indicate that miR-9-3p and miR-9-5p decrease DNA topoisomerase IIa protein 170 kDa expression levels in acquired resistance to etoposide. These findings contribute new information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. Furthermore, increased expression of miR-9-3p and miR-9-5p in chemoresistant cancer cells may support their validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy.
Molecular Pharmacology, 2021
An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topo... more An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR...
Molecular pharmacology, 2018
DNA topoisomerase II (170 kDa, TOP2/170) is essential in proliferating cells by resolving DNA top... more DNA topoisomerase II (170 kDa, TOP2/170) is essential in proliferating cells by resolving DNA topological entanglements during chromosome condensation, replication, and segregation. We previously characterized a C-terminally truncated isoform (TOP2/90), detectable in human leukemia K562 cells but more abundantly expressed in a clonal subline, K/VP.5, with acquired resistance to the anticancer agent etoposide. TOP2/90 (786 aa) is the translation product of a TOP2 mRNA that retains a processed intron 19. TOP2/90 lacks the active-site tyrosine-805 required to generate double-strand DNA breaks as well as nuclear localization signals present in the TOP2/170 isoform (1531 aa). Here, we found that TOP2/90, like TOP2/170, was detectable in the nucleus and cytoplasm of K562 and K/VP.5 cells. Coimmunoprecipitation of endogenous TOP2/90 and TOP2/170 demonstrated heterodimerization of these isoforms. Forced expression of TOP2/90 in K562 cells suppressed, whereas siRNA-mediated knockdown of TOP2...
Molecular and Cellular Endocrinology, 2016
The Faseb Journal, Mar 1, 2008
American Journal of Physiology Gastrointestinal and Liver Physiology, 2010
Studies have demonstrated that angiotensin II (Ang II) can regulate intestinal fluid and electrol... more Studies have demonstrated that angiotensin II (Ang II) can regulate intestinal fluid and electrolyte transport and control intestinal wall muscular activity. Ang II is also a proinflammatory mediator that participates in inflammatory responses such as apoptosis, angiogenesis, and vascular remodeling; accumulating evidence suggests that this hormone may be involved in gastrointestinal (GI) inflammation and carcinogenesis. Ang II binds to two distinct G protein-coupled receptor subtypes, the AT 1R and AT 2R, which are widely expressed in the GI system. Together these studies suggest that Ang II-AT 1R/-AT2R actions may play an important role in GI tract physiology and pathophysiology. Currently it is not known whether miRNAs can regulate the expression of the human AT 1R (hAT1R) in the GI system. PCR and in situ hybridization experiments demonstrated that miR-802 was abundantly expressed in human colon and intestine. Luciferase reporter assays demonstrated that miR-802 could directly interact with the bioinformatics-predicted target site harbored within the 3=-untranslated region of the hAT 1R mRNA. To validate that the levels of miR-802 were physiologically relevant in the GI system, we demonstrated that miR-802 "loss-offunction" experiments resulted in augmented hAT 1R levels and enhanced Ang II-induced signaling in a human intestinal epithelial cell line. These results suggest that miR-802 can modulate the expression of the hAT 1R in the GI tract and ultimately play a role in regulating the biological efficacy of Ang II in this system. gastrointestinal system; miRNAs ANGIOTENSIN II (Ang II), an octapeptide hormone, is the biologically active component of the renin-angiotensin system (RAS) (13, 47). Ang II has emerged as a critical hormone that affects the function of virtually all organs, including heart, kidney, vasculature, brain, adrenal, liver, reproductive organs, and the gastrointestinal (GI) system (13, 47). Pharmacological and morphological studies have demonstrated that Ang II plays an important role in GI system epithelial transport processes (11, 33, 34). For example, in the GI tract, Ang II has been shown to mediate epithelial sodium and water absorption in the jejunum, ileum, and distal colon (33). More recent studies have demonstrated that in the jejunum, the effect of Ang II on sodium and water transport is dose dependent (5, 25, 35, 36).
Circulation, Oct 28, 2008
The Faseb Journal, Apr 1, 2010
Molecular and Cellular Endocrinology, 2006
At least four alternatively spliced mRNAs can be synthesized from the human AT 1 R (hAT 1 R) gene... more At least four alternatively spliced mRNAs can be synthesized from the human AT 1 R (hAT 1 R) gene that differ only in the inclusion or exclusion of exon 2 and/or 3. RT-PCR experiments demonstrate that splice variants harboring exon 2 accounts for at least 30% of all the hAT 1 R mRNA transcripts expressed in the human tissues investigated. Since exon 2 contains two upstream AUGs or open reading frames (uORFs), we hypothesized that these AUGs would inhibit the translation of the downstream hAT 1 R protein ORF harbored in exon 4. This study demonstrates that the inclusion of exon 2 in hAT 1 R mRNA transcripts dramatically reduces hAT 1 R protein levels (nine-fold) and significantly attenuates Ang II responsiveness (∼four-fold). Interestingly, only when both AUGs were mutated in combination were the hAT 1 R density and Ang II signaling levels comparable with those values obtained using mRNA splice variants that did not include exon 2. This observation is consistent with a model where the majority of the ribosomes likely translate uORF#1 and are then unable to reinitiate at the downstream hAT 1 R ORF, in part due to the presence of AUG#2 and to the short intercistronic spacing. Importantly, TGF- 1 treatment (4 ng/ml for 4 h) of fibroblasts up-regulated hAT 1 R mRNA splice variants, which harbored exon 2, six-fold. Since AT 1 R activation is closely associated with cardiovascular disease, the inclusion of exon 2 by alternative splicing represents a novel mechanism to reduce the overall production of the hAT 1 R protein and possibly limit the potential pathological effects of AT 1 R activation.
Biochimica Et Biophysica Acta N Gene Structure and Expression, Jan 18, 1999
Transcriptional mechanisms regulating the expression of the rat angiotensin II type 1A receptor (... more Transcriptional mechanisms regulating the expression of the rat angiotensin II type 1A receptor (rAT 1A R) gene were investigated in cultured rat vascular smooth muscle cells (VSMC). Transcriptional analyses of various 5P-deletion mutants of the rAT 1A R promoter region, fused upstream from the firefly luciferase gene, demonstrated that a 71 base pair (bp) region (3557 to 3486 bp, with respect to transcription initiation) was necessary for expression of this gene in VSMC. Electrophoretic mobility shift assays demonstrated that specific protein-DNA complexes were formed with the 3516 to 3486 bp region of the rAT 1A R promoter when incubated with VSMC extract. Computer analysis of this region indicated the presence of an A/T-rich sequence (i.e., TTTAAAAATAAA) which is similar to a myocyte enhancer binding factor 2 (MEF2) cis-regulatory element (i.e., CTTAAAAATAAC). Site-directed mutagenesis of this A/T-rich sequence inhibited rAT 1A R promoter activity in VSMC, suggesting that this region was necessary for expression of this gene in these cells. Immuno-gel shift experiments suggest that MEF2 heterodimers may interact with the A/T-rich sequence in the rAT 1A R promoter. Additionally, it was demonstrated that a transcription factor non-homologous to MEF2 can also interact with this A/T-rich site in the rAT 1A R promoter. Taken together, our results suggest that MEF2 heterodimers, and/or transcription factors nonhomologous to MEF2, are required to regulate the expression of the rAT 1A R gene in VSMC.
Circulation, Oct 28, 2008
Trends in Endocrinology and Metabolism, Jan 3, 2003
Activation of the angiotensin II type 1 (AT 1) receptor is closely involved in the pathogenesis o... more Activation of the angiotensin II type 1 (AT 1) receptor is closely involved in the pathogenesis of cardiovascular diseases; therefore, aberrant regulation of the production of this receptor might play a role in these disorders. Currently, there is strong evidence to suggest that the predominant mechanism regulating the number of AT 1 receptors is the modulation of mRNA stability. Here, we discuss the importance of alternative splicing as an additional post-transcriptional mechanism regulating human AT 1 receptor number and function.