Bidirectional reporter assay using <i>HAL</i> promoter and TOPFLASH improves specificity in high‐throughput screening of Wnt inhibitors

Yamaguchi, Kiyoshi; Zhu, Ce; Ohsugi, Tomoyuki; Yamaguchi, Yuko; Ikenoue, Tsuneo; Furukawa, Yoichi

doi:10.1002/bit.26394

Cited by 11 publications

(24 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Regarding the luciferase assay using fly cell‐optimized TCF reporter and Drosophila imaginal disc‐derived clone 8 cells, the Z′‐factor was 0.77 . In the reciprocal assay, the Z′‐factors for TOPFlash and the HAL promoter luciferase assays were 0.69 and 0.79, respectively …”

Section: Application Of Reporter Assay For Htsmentioning

confidence: 99%

“…Rac1 activates JNK2 that in turn phosphorylates β‐catenin at Ser191 and regulates its nuclear translocation, suggesting that the reduced nuclear translocation of β‐catenin by GGTI‐286 might be associated with its interference with Rac1 protein. A reciprocal reporter assay using TOPFlash coupled with HAL reporter led to a decrease in the number of false positives, and identified brefeldin A (BFA), a fungal metabolite, that suppresses the expression of β‐catenin . Although BFA was reported to inhibit protein secretion by blocking transport from the endoplasmic reticulum to the Golgi, the precise mechanism(s) underlying the reduction in β‐catenin by BFA needs further investigation.…”

Section: Small Molecules That Affect β‐Catenin Function or Expressionmentioning

confidence: 99%

“…Intriguingly, identification of genes negatively regulated by the β-catenin/TCF complex led to the development of a new reporter plasmid ( Figure 1B). 23 This plasmid contains 8 copies of the promoter region of histidine ammonia-lyase (HAL) upstream of the luciferase gene. Unlike TOPFlash, the HAL reporter activity is inversely correlated with Wnt/β-catenin signaling activity ( Figure 1C).…”

Section: Asmentioning

confidence: 99%

“…26 In the reciprocal assay, the Z′-factors for TOPFlash and the HAL promoter luciferase assays were 0.69 and 0.79, respectively. 23 Bioluminescent assays have been used in HTS as a major strategy, due to their high sensitivity, broad linearity, and robustness to chemicals. 27 Firefly luciferase (Photinus pyralis) and Renilla luciferase (Renilla reniformis) are commonly used as reporter genes.…”

Section: Appli C Ati On Of Rep Orter a Ssay For Htsmentioning

confidence: 99%

“…A reciprocal reporter assay using TOPFlash coupled with HAL reporter led to a decrease in the number of false positives, and identified brefeldin A (BFA), a fungal metabolite, that suppresses the expression of β-catenin. 23 Although BFA was reported to inhibit protein secretion by blocking transport from the endoplasmic reticulum to the Golgi, 66 the precise mechanism(s) underlying the reduction in β-catenin by BFA needs further investigation.…”

Section: Inhib Itor S Of Tr Anscrip Tional Coac Tivator S Of β -C Amentioning

confidence: 99%

See 4 more Smart Citations

Discovery of chemical probes that suppress Wnt/β‐catenin signaling through high‐throughput screening

et al. 2020

Self Cite

View full text Add to dashboard Cite

Aberrant activation of the Wnt/β‐catenin signaling pathway has been observed in a wide range of human tumors. Deregulation of the pathway is closely linked to various aspects of human carcinogenesis such as cell viability, regulation of cell cycle, epithelial‐mesenchymal transition, and maintenance of stemness. In addition, recent studies have disclosed the involvement of Wnt signaling in immune evasion of tumor cells. The accumulation of β‐catenin in the nucleus is a common feature of cancer cells carrying defects in the pathway, which leads to the continuous activation of T‐cell factor (TCF)/LEF transcription factors. Consequently, a genetic program is switched on, leading to the uncontrolled growth, prolonged survival, and acquisition of mesenchymal phenotype. As β‐catenin/TCF serves as a signaling hub for the pathway, β‐catenin/TCF‐dependent transcriptional activity is a relevant readout of the pathway. To date, a wide variety of synthetic TCF/LEF reporters has been developed, and high‐throughput screening (HTS) using these reporters has made significant contributions to the discovery of Wnt inhibitors. Indeed, HTS led to the identification of chemical probes targeting porcupine, a membrane bound O‐acyltransferase, and CREB‐binding protein, a transcriptional coactivator. This review focuses on various screening strategies for the discovery of Wnt inhibitors and their mode of action to help the creation of new concepts for assay/screening methods.

show abstract

Section: Application Of Reporter Assay For Htsmentioning

confidence: 99%

Section: Small Molecules That Affect β‐Catenin Function or Expressionmentioning

confidence: 99%

Section: Asmentioning

confidence: 99%

Section: Appli C Ati On Of Rep Orter a Ssay For Htsmentioning

confidence: 99%

Section: Inhib Itor S Of Tr Anscrip Tional Coac Tivator S Of β -C Amentioning

confidence: 99%

See 3 more Smart Citations

Discovery of chemical probes that suppress Wnt/β‐catenin signaling through high‐throughput screening

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

Visinin‐like 1, a novel target gene of the Wnt/β‐catenin signaling pathway, is involved in apoptosis resistance in colorectal cancer

et al. 2023

Self Cite

View full text Add to dashboard Cite

Background Abnormal activation of Wnt/β‐catenin signaling is associated with various aspects of cancer development. This study explored the roles of novel target genes of the Wnt/β‐catenin signaling pathway in cancer cells. Methods Using the haploid chronic myelogenous leukemia cell line HAP1, RNA sequencing (RNA‐seq) was performed to identify genes whose expression was increased by APC disruption and reversed by β‐catenin knockdown (KD). The regulatory mechanism and function of one of the candidate genes was investigated in colorectal cancer (CRC) cells. Results In total, 64 candidate genes whose expression was regulated by Wnt/β‐catenin signaling were identified. Of these candidate genes, the expression levels of six were reduced by β‐catenin KD in HCT116 CRC cells in our previous microarray. One of these genes was Visinin‐like 1 (VSNL1), which belongs to the neuronal calcium‐sensor gene family. The expression of VSNL1 was regulated by the β‐catenin/TCF7L2 complex via two TCF7L2‐binding elements in intron 1. VSNL1 KD‐induced apoptosis in VSNL1‐positive CRC cells. Additionally, forced expression of wild‐type VSNL1, but not a myristoylation, Ca2+‐binding, or dimerization‐defective mutant, suppressed the apoptosis induced by camptothecin and doxorubicin in VSNL1‐negative CRC cells. Conclusion Our findings suggest that VSNL1, a novel target gene of the Wnt/β‐catenin signaling pathway, is associated with apoptosis resistance in CRC cells.

show abstract

SFRP5 inhibits melanin synthesis of melanocytes in vitiligo by suppressing the Wnt/β-catenin signaling

et al. 2021

View full text Add to dashboard Cite

Secreted frizzled-related protein 5 (SFRP5) plays a pivotal role in regulating the development of many tissues and organs, however, as an inhibitor of Wnt signaling, the role of SFRP5 in vitiligo remains unknown. Hence, we speculated that SFRP5 might be associated with melanogenesis in melanocytes by regulating Wnt signaling in vitiligo. In this study, we found that SFRP5 was overexpressed in the skin lesions of patients with vitiligo. Compared with that in normal epidermal melanocytes (PIG1), the expression of SFRP5 was increased in vitiligo melanocytes (PIG3V). To investigate the effect of SFRP5 on melanin synthesis, PIG1 cells were infected with recombinant SFRP5 adenovirus (AdSFRP5), and PIG3V cells were infected with recombinant siSFRP5 adenovirus (AdsiSFRP5). The results showed that SFRP5 overexpression inhibited melanin synthesis in PIG1 cells through downregulation of microphthalmia-associated transcription factor (MITF) and its target proteins via suppression of the Wnt/β-catenin signaling pathway. Accordingly, SFRP5 silencing increased melanin synthesis and activated the Wnt signaling pathway in PIG3V cells. Moreover, SFRP5 overexpression also downregulated the transcriptional activity of T cell factor/lymphoid enhancer factor (TCF/LEF) in PIG1 cells. Furthermore, this inhibitory effect of SFRP5 on melanin synthesis was reversed by treatment with the β-catenin agonist, SKL2001. The inhibitory action of SFRP5 in pigmentation was further confirmed in vivo using a nude mouse model. Hence, our results indicate that SFRP5 can inhibit melanogenesis in melanocytes. Additionally, our findings showed that SFRP5 plays a vital role in the development of vitiligo, and thus may serve as a potential therapeutic target for vitiligo.

show abstract

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high‐throughput screening of Wnt inhibitors

Cited by 11 publications

References 41 publications

Discovery of chemical probes that suppress Wnt/β‐catenin signaling through high‐throughput screening

Discovery of chemical probes that suppress Wnt/β‐catenin signaling through high‐throughput screening

Visinin‐like 1, a novel target gene of the Wnt/β‐catenin signaling pathway, is involved in apoptosis resistance in colorectal cancer

SFRP5 inhibits melanin synthesis of melanocytes in vitiligo by suppressing the Wnt/β-catenin signaling

Contact Info

Product

Resources

About