Detection of Aggregation-Prone Behavior in Mutant P53 V157F Breast Cancer Cells Using Multipoint Thioflavin T Fluorescence

Summary

Hs578T breast cancer cells harboring the p53 V157F mutation exhibit significantly higher Thioflavin T fluorescence compared to MCF7 cells, indicating enhanced protein aggregation. Multipoint fluorescence measurements improve detection accuracy and reliability in identifying β-sheet-rich aggregates, underscoring the importance of aggregation-prone p53 mutations in cancer research and the development of therapeutic strategies.

Abstract

The tumor suppressor p53, encoded by the TP53 gene, plays a central role in maintaining genomic stability. TP53 mutations, particularly hotspot variants, are found in approximately 50% of human cancers and may lead to the loss of tumor-suppressive functions or the acquisition of gain-of-function properties, including aggregation into prion-like structures.

In this study, we assessed the aggregation tendency of the p53 V157F mutation in Hs578T breast cancer cells compared to MCF7 cells expressing wild-type p53. Protein aggregation was evaluated using Thioflavin T (ThT) staining followed by fluorescence quantification in 96-well plate assays. Cells were stained with a ThT/Hoechst solution, washed, and analyzed using a microplate reader under defined excitation/emission parameters. Quantitative analysis using both single-point and four-point fluorescence readings revealed that Hs578T cells exhibited a 3.20- to 4.26-fold increase in ThT fluorescence intensity relative to MCF7, indicating significantly elevated levels of β-sheet-rich or amyloid-like aggregates. Multipoint measurements confirmed the widespread and consistent presence of protein aggregates across the well surface. These findings support the use of multipoint plate-reading to accurately detect protein aggregation in cell-based assays.

Introduction

The TP53 gene, which encodes the tumor suppressor protein p53, is mutated in approximately 50% of human cancers^1,2. Such mutations not only abolish the tumor-suppressive functions of p53 but, in certain hotspot variants, also promote the formation of prion-like aggregates^3,4. These aggregated forms of mutant p53 frequently acquire gain-of-function properties, including increased resistance to anticancer therapies^5,6. Therefore, identifying which p53 hotspot mutations are prone to aggre....

Protocol

1. Cell seeding

1. To assess cell viability, mix 10 µL of Trypan Blue with 10 µL of resuspended cells in 1x Dulbecco's Phosphate-Buffered Saline (DPBS) in a microcentrifuge tube and mix thoroughly.
2. Load 10 µL of the mixture onto a counter slide, then count the live cells using an automated cell counter.
3. Based on the viability count, seed 30,000 viable cells per well into a 96-well culture plate, with one well designated as a non-stained control and the remaining three wells used for staining.

2. Incubation

1. Place the culture plate in a CO₂ incubator ....

Results

To investigate the presence of β-sheet-rich protein aggregates in breast cancer cells, MCF7 and Hs578T cell lines were subjected to ThT staining followed by fluorescence quantification. As shown in Figure 1, cells were seeded into four wells of a 96-well plate, with one well designated as an unstained control. The remaining three wells were stained with ThT according to the standard protocol. Fluorescence was measured using two approaches: (1) a single-point reading taken from the center of .......

Discussion

When seeding cells into 96-well plates, it is often difficult to achieve uniform distribution across the well surface, as gently shaking or tapping the plate to spread the cells is not always feasible. This uneven distribution can introduce variability in signal intensity across the well. Multipoint fluorescence reading revealed significant variation between different regions within the same well, highlighting this bias and the potential for misleading results when only a single point is measured. Relying on a single cen.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well culture plate	Simply	PC396-0100
counter slide	Invitrogen	C10228
Countess II FL Automated Cell Counter	Thermo Fisher Scientific	AMQAF1000
dPBS	Simply	CC704-0500
Eppendorf tube	Simply	PC101-5000
Hoechst 33342	AAT Bioquest	17530
SpectraMax iD3	Molecular Devices	735-0391
Thioflavin T	Sigma-Aldrich	T3516
Trypan Blue	Invitrogen	T10282