Introduction

Genomic instability, a hallmark of cancer cells, used for clinical classification can be exploited for targeted therapies. Knowledge of which genome stability process is active or altered in tumour cells during their progression helps in the evaluation of therapeutic regimens. There are several assays to evaluate the general activation of DNA damage response pathways or the detection of DNA breaks, based on antibody detection of the phosphorylated form of the histone protein H2Ax or on the nuclear staining pattern of proteins that are effectors of the pathways in response to DNA damage. However, there are no methods allowing for the molecular evaluation of the transduction pathways linked to the replicative stress induced by oncogene, a process driven by the alteration of DNA replication due to the aberrant ignition and activity of the machinery during replication, which gives rise to collisions of replicating DNA with transcriptional complexes.

Technical features

It was found that cyclin-dependent kinase 12, CDK12, is selectively recruited in the sites of DNA breaks within transcribed loci, indicating that it can be used as a marker to detected DNA damage. Methods have been developed to identifying and / or quantifying said marker in association with DNA damage events. The method is based on the detection of the CDK12 protein at the damaged sites either by immunofluorescence assays or by using the ectopic expression of chimeric proteins derived from the fusion of CDK12 with fluorescent proteins. These assays allow for the detection of CDK12 near DNA Damage Response foci, in both live and processed single cells. The assay is selective because it does not detect DNA damage in loci of genomic regions that are not transcribed.

Possible Applications

• Can be used to diagnose or classify tumors, evaluate their progression or highlight tumor sensitization a certain therapy;
• Targeted tumour therapy.

Advantages

• Simple, single biomarker test;
• Easily adaptable to specific test conditions;
• Scalable for high-throughput / high-information microscopic analysis;
• Easily integrated into the automated processing of samples for clinical / industrial use.
• Superior selectivity compared to single marker detection methods.
• Allows the quantitative of endogenous CDK12 foci with low background.