Single cell imaging reveals cisplatin regulating interactions between transcription (co)factors and DNA

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• Cisplatin is a vital anticancer drug, primarily targeting DNA.
• The precise mechanisms by which cisplatin-induced DNA lesions affect transcription factor-DNA interactions are not fully understood.

Purpose of the Study:

• To investigate the in situ formation of ternary complexes involving HMGB1, Smad3, and cisplatin-crosslinked DNA within single cells.
• To elucidate how cisplatin lesions impact the binding of transcription factors like Smad3 to specific DNA sequences.

Main Methods:

• Developed a dual-modal microscopy strategy combining confocal fluorescence imaging and time-of-flight secondary ion mass spectrometry (ToF-SIMS).
• Mapped EYFP-tagged HMGB1 and DNA using confocal microscopy, followed by high-resolution visualization of cisplatin distribution using ToF-SIMS in the same cells.

Main Results:

• Confirmed the formation of HMGB1-Pt-DNA ternary complexes in cells through superimposed fluorescence and mass spectrometry signals.
• Demonstrated that cisplatin lesions at Smad-binding elements (e.g., GGC(GC)/(CG), AGAC) significantly disrupt Smad3-DNA interactions.
• Observed a marked decrease in Smad-specific luciferase reporter expression following cisplatin treatment, indicating pathway disruption.

Conclusions:

• Cisplatin-induced DNA damage interferes with the binding of transcription factor Smad3 to its target DNA sequences.
• The Smad3 signaling pathway is likely involved in the intracellular response to DNA damage induced by cisplatin.
• Dual-modal imaging provides a powerful approach to study molecular complex formation and drug-DNA interactions in situ.