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 aggregation is critical for guiding the development of effective treatment strategies in p53-mutant cancers^7,8.

In our previous studies, we observed that specific p53 mutations in head and neck squamous cell carcinoma (HNSCC) and breast cancer cell lines exhibit strong Thioflavin T (ThT) staining signals, indicating the presence of aggregated proteins. ThT is a benzothiazole dye that specifically binds to β-sheet-rich amyloid structures, producing enhanced fluorescence upon binding⁹. For example, the Detroit 562 HNSCC cell line, which carries the p53 R175H mutation, exhibited strong ThT fluorescence signals¹⁰. Similarly, two other HNSCC cell lines, TW01 and HONE-1, both harboring the p53 R280T mutation, also showed pronounced ThT staining¹¹. In these cases, ThT signals colocalized with p53, supporting the presence of aggregated p53^10,11.

In breast cancer cells, MDA-MB-231 (p53 R280K mutation) and T47D (p53 L194F mutation) likewise displayed strong ThT staining with clear p53/ThT co-localization¹². By contrast, MCF7 cells, which express wild-type p53, and MDA-MB-468 cells (p53 R273H mutation) both showed only weak ThT signals¹². Similarly, OECM-1 HNSCC cells with the p53 V173L mutation demonstrated low levels of ThT staining¹⁰.

Compared with other protein aggregation detection methods-such as filter trap assays, transmission electron microscopy (TEM), or immunostaining with aggregate-specific antibodies-ThT fluorescence analysis offers several advantages. It is non-destructive, rapid, and applicable to both live and fixed cells without extensive processing. While filter trap assays effectively capture insoluble aggregates, they require harsh denaturation and are unsuitable for live-cell use¹³. TEM provides direct visualization of fibril morphology but is labor-intensive and low-throughput¹⁴. Aggregate-specific immunostaining enables in situ detection using conformation-dependent antibodies, though antibody availability and specificity can be limiting¹⁵. In contrast, ThT fluorescence-especially when combined with multipoint plate reading-offers a simple, quantitative, and spatially reliable approach for detecting β-sheet-rich or amyloid-like structures.

Practical considerations are also key for reproducibility. ThT fluorescence correlates linearly with amyloid concentration across a wide concentration range (0.2-500 µM), and peak signal is typically achieved at 20-50 µM, depending on the protein-though ThT self-fluorescence can emerge at ≥5 µM, and higher concentrations may impact aggregation kinetics¹⁶. Therefore, a working ThT concentration around 10-20 µM is generally recommended to balance sensitivity while minimizing potential effects on aggregation processes¹⁶. In our protocol, the 1,000x ThT stock (12. mM) is diluted 1:1,000 to yield a working concentration of approximately 12. µM, which falls within this recommended range.

ThT preferentially binds to β-sheet-rich amyloid structures, which are characteristic of amyloid fibrils. This specificity arises from the dye's interaction with the cross-β architecture of amyloid fibrils, where the surfaces formed by β-strands provide binding sites for ThT. Consequently, ThT may not effectively detect other misfolded or non-amyloid aggregates that do not exhibit this β-sheet-rich structure¹⁷. Therefore, while ThT is a valuable tool for identifying amyloid fibrils, it is advisable to employ complementary validation methods-such as co-localization with p53 immunofluorescence or biophysical approaches-to verify the identity and specificity of the aggregates detected^10,18.

Structural prediction studies suggest that the p53 V157F mutation adopts a conformation more prone to aggregation than the wild-type protein¹⁹. In this study, we compared ThT fluorescence signals between Hs578T breast cancer cells, which harbor the aggregation-prone p53 V157F mutation, and MCF7 breast cancer cells expressing wild-type p53. Using a multipoint plate reader-based ThT staining assay, we sought to assess the aggregation propensity of p53 V157F relative to wild-type p53.

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 and incubate 12 h at 37 °C.

3. Preparation of fresh ThT staining stock (1,000x)

1. Weigh 0.02 g (20 mg) of ThT powder and dissolve it in 5.0 mL of deionized water. Mix thoroughly until the powder is completely dissolved.
   NOTE: This preparation yields a 1,000x ThT stock solution with a final concentration of 4 mg/mL, equivalent to 12.5 mM (ThT has a molecular weight of ~318.85 g/mol. A solution of 4 mg/mL equals 4 g/L. Dividing 4 g/L by 318.85 g/mol gives 0.0125 mol/L, or ~12.5 mM).

4. Preparation of ThT staining buffer with nuclear counterstain

1. Prepare the staining solution by adding the following to 1 mL of 1x DPBS and mix gently to ensure homogeneity.
   1. Add 1 µL of freshly prepared 1,000x ThT stock, giving a final ThT concentration of 12.5 µM (1x).
   2. Add 1 µL of 1 mg/mL Hoechst 33342 stock solution, giving a final concentration of ~1 µg/mL (=1.6 µM).

5. Staining

1. Remove the culture medium from each well. Add 100 µL of the prepared ThT/Hoechst staining buffer to each staining well. Add 100 µL of 1x DPBS to non-stained control.

6. Incubation

1. Place the plate in a dark place at room temperature (25 °C) for 30 min.

7. Washing

1. Carefully aspirate or discard the staining solution from each well, taking care not to disturb the cell monolayer.
2. Add 100 µL of 1× DPBS to each well and allow an optional gentle 30-second soak.
3. Carefully aspirate or discard the DPBS wash solution.
4. Repeat the wash by adding 100 µL of fresh 1x DPBS to each well, again allowing an optional 30-second gentle soak, then discard the DPBS wash solution.
5. After the second wash, add 100 µL of fresh 1x DPBS to each well.

8. Fluorescence measurement

1. Before reading the fluorescence signal, remove the cap from the 96-well plate. Measure fluorescence using one of two methods: single-point reading or four-point reading.
2. Place the 96-well plate into a microplate reader, and measure fluorescence using the following settings: ThT fluorescence: Excitation at 450 nm, emission at 490 nm; Hoechst 33342 fluorescence: Excitation at 360 nm, emission at 460 nm; reader settings: Shaking: 5 s prior to reading; Read direction: Top read; Integration time: 140 ms; Read height: 1.00 mm; Points per well: 1 (endpoint) or 4 (well scan); Well scan setting: (Density setting: 2; Point spacing: 2.5).

9. Data analysis

1. Calculate protein aggregation as follows:
   
2. Normalize ThT fluorescence intensity by setting the ratio in MCF7 cells as 1 (baseline control).

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