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Confirmatory assay for modulation of the human telomerase reverse transcriptase promoter in HCT116 cells

PubChem AID
1259346
Source
Keith Lab, Institute of Cancer Science, University of Glasgow
External ID
TELO_04
BioAssay Type
Confirmatory
Tested Substances
Version
Status
Live
Dates
  • Deposit:
    2017-10-23
  • Hold-Until:
    2019-06-13
  • Modify:
    2019-06-13
Description
Please note that the bioassay record (AID 1259346) is presented as provided to PubChem by the source(depositor). When possible, links to additional information have been provided by PubChem.

1 Description

Immortalisation is a hallmark of cancer commonly achieved by transcriptional reactivation of the telomerase reverse transcriptase gene TERT, leading to inappropriate telomere maintenance. Multiple transcription factors modulate the TERT promoter, orchestrated by the activities of a range of upstream signalling pathways. Previous studies have identified many of the pathways which individually contribute to activate or repress telomerase levels in cancer cells, resulting in a highly complex picture of TERT regulation. The pathways of TERT activation or suppression could be appropriate therapeutic targets in cancer or in telomeropathies such as dyskeratosis which are associated with loss of telomere stability.

To identify small molecule regulators of TERT transcriptional regulation, activity of the TERT core gene promoter region was monitored using firefly luciferase. Reporter pGL3-TERT contains the TERT promoter region -585/-9, relative to the translational start site, cloned in the Promega vector PGL3-basic. Telomerase positive A2780 ovarian carcinoma cells are transiently transfected with the reporter construct and subsequently treated with small molecules to detect up- or down-regulation of luciferase activity. Derivatives of compound C430-0073, identified in screen TELO-01 were tested in dose responses in the HCT116 cell line.

2 Protocol

50000 HCT116 cells were seeded in wells white 96 well plates (Greiner) in complete RPMI medium (Life Technologies) for 24h prior to transfection. Each well was transiently transfected with 250ng of reporter plasmid using Superfect reagent (Qiagen) at 2.5:1 ratio of reagent (ul): plasmid (ug), according to the manufacturer's protocol. Transfection complexes were allowed to form for 15 minutes prior to addition to cells. Cells were incubated with the transfection complex for 2.5h then rinsed in PBS and returned to fresh growth medium. 32h post transfection, compounds dissolved in DMSO and diluted in growth medium were added in triplicate in a dose response from 10uM. In each plate, 12 wells were treated with DMSO only. Cells were incubated with compounds for a further 16h prior to harvest. Cells were then rinsed once in PBS, lysed for 15 minutes in 30ul Passive Lysis Buffer per well (Promega) with shaking on a vortex with plate attachment. Luciferase activity was detected using 50ul per well of freshly reconstituted Luciferase Assay Reagent (Promega). Plates were read on a GloMax-96 microplate luminometer. Luciferase activity of compound-treated wells was compared with the average of the DMSO controls to generate fold-change readout.

3 Comment

Compounds with IC50 < 0.001 micromolar were taken to be hits.

4 Result Definitions

5 Data Table

6 Identity

6.1 BioAssay Name

Confirmatory assay for modulation of the human telomerase reverse transcriptase promoter in HCT116 cells

6.2 Source

Keith Lab, Institute of Cancer Science, University of Glasgow

6.3 External ID

TELO_04

6.4 Project Category

Other

6.5 BioAssay Type

Confirmatory

6.6 Deposit Date

2017-10-23

6.7 Hold-Until Date

2019-06-13

6.8 Modify Date

Version 1.1
Version 1.2
Version 1.3
Version 1.4
Version 1.5
Version 1.6
Version 1.7
2019-06-13 (currently shown)

6.9 Status

Live

7 Same-Project BioAssays

8 Information Sources

CONTENTS