scholarly journals Mutagenesis Analysis of ABCG2 Gene Promoter of Zebrafish (Danio Rerio)

2020 ◽  
Vol 3 (2) ◽  
pp. a53-59
Author(s):  
NABILA ZURAIN BINTI MD YUSNI ◽  
LEONARD WHYE KIT LIM ◽  
HUNG HUI CHUNG
Keyword(s):  
Breast Cancer ◽  
Transcription Factor ◽  
Danio Rerio ◽  
Chemotherapeutic Agent ◽  
Chemotherapeutic Agents ◽  
Site Directed Mutagenesis ◽  
Luciferase Assay ◽  
Directed Mutagenesis ◽  
Cancer Resistance ◽  
Abcg2 Gene

Breast cancer is the commonest cancer among women worldwide and the probability of a woman dying from breast cancer is high (about 1 in 38 of total human population (2.6%)).The main factor for mortality is due to the resistance of this particular disease to chemotherapeutic agents. One of the most well-known proteins to be found to correlate significantly with breast cancer resistance to chemotherapeutic agent is the ATP-binding cassette super-family G member 2 (ABCG2). Knowledge on ABCG2 gene regulation is still lacking in terms of how the increased cytotoxic levels are closely related to induce a hype in gene transcript levels and ultimately cause of the reduction in chemotherapeutic agents. The approach taken in this study is through mutational analysis of selected transcription factor governing the expression of ABCG2. In order to achieve this, a previously cloned ABCG2 promoter which has been isolated (around 1500 bp in size) from Danio rerio and inserted into pGL3.0 plasmid, was subjected to site-directed mutagenesis. Selected transcription factor which is AP-1 was successfully mutated by deletion of 5'- TGACGCG -3' sequence at position 1113 bp from TSS+1 where it would bind in order to define their role in ABCG2 physiological function. Sequencing result after site-directed mutagenesis shows high similarities about 98% with ABCG2 gene of Danio rerio. Upon validation, it was found that the intended AP-1 binding site has been mutated. In future work, the mutated clone here will be subjected to transfection analysis where dual-luciferase assay will be conducted to verify the loss of activity from the ABCG2 promoter upon mutation of the targeted AP-1 site. Hence, the mutagenesis analysis of ABCG2 promoter are able to provide information on the involvement of AP-1 transcription factor in multidrug resistance mechanism of breast cancer and thus will be a potential target for chemotherapeutic agent.

Synergy between the NF-E1 erythroid-specific transcription factor and the CACCC factor in the erythroid-specific promoter of the human porphobilinogen deaminase gene

1990 ◽  
Vol 10 (7) ◽  
pp. 3838-3842
Author(s):  
J Frampton ◽  
M Walker ◽  
M Plumb ◽  
P R Harrison
Keyword(s):  
Transcription Factor ◽  
Base Pair ◽  
Promoter Activity ◽  
Transient Transfection ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Porphobilinogen Deaminase ◽  
Specific Transcription Factor ◽  
Specific Manner ◽  
Two Factors

A 114-base-pair promoter fragment of the human porphobilinogen deaminase gene functioned in an erythroid-specific manner in transient transfection experiments. Site-directed mutagenesis of the binding site for the erythroid-specific transcription factor (NF-E1) or an adjacent CACCC motif abolished the promoter activity. Increasing the spacing between these sites progressively reduced promoter activity, but there was no evidence that a critical alignment of the two factors on the DNA helix was required.

scholarly journals BTG4 is A Novel p53 Target Gene That Inhibits Cell Growth and Induces Apoptosis

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 217 ◽  
Author(s):  
Na Zhang ◽  
Tinghui Jiang ◽  
Yitao Wang ◽  
Lanyue Hu ◽  
Youquan Bu
Keyword(s):  
Transcription Factor ◽  
Cell Growth ◽  
Binding Sites ◽  
Transcription Initiation ◽  
Core Promoter ◽  
Site Directed Mutagenesis ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Colorectal Cancers ◽  
P53 Overexpression

BTG4 is the last cloned and poorly studied member of BTG/Tob family. Studies have suggested that BTG4 is critical for the degradation of maternal mRNAs in mice during the process of maternal-to-zygotic transition, and downregulated in cancers, such as gastric cancer. However, the regulatory mechanism of BTG4 and its function in cancers remain elusive. In this study, we have for the first time identified the promoter region of the human BTG4 gene. Serial luciferase reporter assay demonstrated that the core promoter of BTG4 is mainly located within the 388 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the BTG4 promoter contains binding sites for canonical transcription factors, such as Sp1, whereas its first intron contains two overlapped consensus p53 binding sites. However, overexpression of Sp1 has negligible effects on BTG4 promoter activity, and site-directed mutagenesis assay further suggested that Sp1 is not a critical transcription factor for the transcriptional regulation of BTG4. Of note, luciferase assay revealed that one of the intronic p53 binding sites is highly responsive to p53. Both exogenous p53 overexpression and adriamycin-mediated endogenous p53 activation result in the transcriptional upregulation of BTG4. In addition, BTG4 is downregulated in lung and colorectal cancers, and overexpression of BTG4 inhibits cell growth and induces apoptosis in cancer cells. Taken together, our results strongly suggest that BTG4 is a novel p53-regulated gene and probably functions as a tumor suppressor in lung and colorectal cancers.

scholarly journals Identification of a Novel Estrogen Response Element in the Breast Cancer Resistance Protein (ABCG2) Gene

2004 ◽  
Vol 64 (4) ◽  
pp. 1247-1251 ◽  
Author(s):  
Pui Lai Rachel Ee ◽  
Sitharthan Kamalakaran ◽  
Debra Tonetti ◽  
Xiaolong He ◽  
Douglas D. Ross ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Resistance Protein ◽  
Estrogen Response Element ◽  
Cancer Resistance ◽  
Response Element ◽  
Estrogen Response ◽  
Resistance Protein ◽  
Abcg2 Gene

scholarly journals ACOX1, regulated by C/EBPα and miR-25-3p, promotes bovine preadipocyte adipogenesis

2021 ◽  
Author(s):  
Feng Zhang ◽  
Qi Xiong ◽  
Hu Tao ◽  
Yang Liu ◽  
Nian Zhang ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Luciferase Assay ◽  
Directed Mutagenesis ◽  
Loss Of Function ◽  
Mobility Shift ◽  
Promoter Deletion Analysis ◽  
Factor C ◽  
Peroxisomal Fatty Acid

Acyl-Coenzyme A oxidase 1 (ACOX1) is the first and rate-limiting enzyme in peroxisomal fatty acid β-oxidation of fatty acids. Previous studies have reported that ACOX1 was correlated with the meat quality of livestock, while the role of ACOX1 in intramuscular adipogenesis of beef cattle and its transcriptional and post-transcriptional regulatory mechanisms remain unclear. In the present study, gain-of-function and loss-of-function assays demonstrated that ACOX1 positively regulated the adipogenesis of bovine intramuscular preadipocytes. The C/EBPα-binding sites in the bovine ACOX1 promoter region at -1142 to -1129 bp, -831 to -826 bp, and -303 to -298 bp were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) further showed that these three regions are C/EBPα-binding sites, both in vitro and in vivo, indicating that C/EBPα directly interacts with the bovine ACOX1 promoter and inhibits its transcription. Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the ACOX1 3’untranslated region (3’UTR). Taken together, our findings suggest that ACOX1, regulated by transcription factor C/EBPα and miR-25-3p, promotes adipogenesis of bovine intramuscular preadipocytes via regulating peroxisomal fatty acid β-oxidation.

MCF-7 Resistant Doxorubicin are Characterized by Lamelapodia, Strong Adhesion on Substrate and P-gp Overexpression

2011 ◽  
Vol 2 (3) ◽  
pp. 304
Author(s):  
Dyaningtyas Dewi Pamungkas Putri ◽  
Sarmoko Sarmoko ◽  
Rifki Febriansah ◽  
Endah Puspitasari ◽  
Nur Ismiyati ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Chemotherapeutic Agents ◽  
Cancer Resistance ◽  
Breast Cancer Patients ◽  
Western Blot Assay ◽  
Chemotherapy Agent ◽  
Strong Adhesion ◽  
Low Sensitivity ◽  
Mcf 7

The prognosis of breast cancer patients is closely associated with the response of tumor cells to chemotherapy agent. Doxorubicin is one of the primary chemotherapeutic agents used for the treatment of breast cancer. Resistance to chemotherapy is believed to cause treatment failure in cancer patients. Furthermore, long time exposure to chemotherapeutic agent induces cancer cells resistance. MCF-7 sensitive cells used as chemoresistance model have overexpression P-gp (P-glycoprotein). Chemoresistance was established by treating MCF-7 cells with 0.5 µg/ml doxorubicin-contained medium for a week. 50% inhibiting concentration (IC50) doxorubicin on MCF-7 cells/DOX were determined using MTT assay. Western blot assay and immunocytochemistry assay was performed to determine the expression of P-gp. Morphological of MCF-7 cell/DOX was changing to become larger and have lamellapodia. IC50 value of doxorubicin was 700 nM on MCF-7/DOX and 400 nM on sensitive MCF-7 cells. The MCF-7/DOX sensitivity to doxorubicin was decreased, shown by 1.5 fold higher IC50 of doxorubicin on MCF-7/DOX compared to MCF-7 sensitive cells. Treatment doxorubicin to sensitive MCF-7 cells leads to the increasing P-gp expression. The P-gp level expression has strong correlation with the low sensitivity of MCF-7/DOX to doxorubicin.Keywords: doxorubicin, resistance cells, sensitive MCF-7 cell

scholarly journals Effects of ABCB1 and ABCG2 polymorphisms on the pharmacokinetics of abemaciclib

2021 ◽  
Author(s):  
Akimitsu Maeda ◽  
Hitoshi Ando ◽  
kei irie ◽  
Naoya Hashimoto ◽  
Jun-ichi Morishige ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Adverse Events ◽  
Gene Polymorphisms ◽  
Cancer Resistance ◽  
Wild Type ◽  
Glycoprotein P ◽  
In The Wild ◽  
Trough Concentrations ◽  
Dose Dependent ◽  
Abcg2 Gene

Aim: The adverse events of the CKD4/6 inhibitor abemaciclib are known to be dose dependent. However, its pharmacokinetics vary among individuals. Abemaciclib is reported to be transported by P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP). Therefore, we evaluated whether ABCB1 and ABCG2 gene polymorphisms could be pharmacokinetic predictive factors of abemaciclib. Methods: A total of 45 patients with breast cancer able to take abemaciclib (150 mg twice daily) for 2 weeks were evaluated to determine the association among abemaciclib concentrations, adverse events, and ABCB1 1236T>C, 2677G>T/A, 3435C>T, and ABCG2 421C>A gene polymorphisms. Results: The trough concentrations of abemaciclib were higher in the group with grade 2 or greater neutropenia, anemia, and thrombocytopenia as compared with the group with grades 0 or 1. No significant association was observed between ABCB1 1236T>C, 3435C>T, and ABCG2 421C>A gene polymorphisms and abemaciclib concentrations. However, in ABCB1 2677G>T/A polymorphisms, the concentrations of abemaciclib tended to be higher in the homozygous group (AA + AT) as compared with that in the wild-type and heterozygous group (GG + GA + GT) [222.8 (80.5–295.8) ng/mL vs. 115.8 (23.6–355.2) ng/mL, P = 0.11]. Hence, the ABCB1 2677G>T/A homozygous group had a significantly higher incidence of abemaciclib withdrawal and dose reduction within 4 weeks as compared than the wild-type and heterozygous group (67% vs. 33%, P = 0.03). Conclusions: The gene polymorphism of ABCB1 2677G> T/A might be a predictor of the pharmacokinetics and tolerability of abemaciclib.

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