Apoptosis, the natural death of cells, and cancer drugs can induce double strand breaks (DSBs) that lead to DNA damage. Current methods of identifying apoptosis involve microscopy and have restricted quantitative capabilities because of inadequate ratios between signals and noise. However, scientists at the National Cancer Institute's branches in Bethesda and Frederaick, Maryland, have developed a process that uses immunofluorescence microscopy to analyze fixed tumor biopsy tissues. It can distinguish between DSBs that are induced by apoptosis and therapeutic treatments.

The Problem With Current Apoptosis Detection

Scientists had previously established yH2AX as a biomarker for DSBs. It's the serine 139-phosphorylated structure of histone H2AX at DSB sites and arises as a result of DNA damage or laddering from apoptosis. Positive yH2AX signals in clinical and preclinical fixed tumor tissues are associated with tumor progression and shrinkage.

However, yH2AX-positive cells alone can't tell the difference between DSBs induced by apoptosis and cytotoxic anticancer agents. Activated cleaved caspase-3 is another marker, which promotes DNA fragmentation, plasma membrane blebbing and nuclear condensation. A sandwich enzyme-linked immunosorbent assay uses activated CC3 as an apoptosis marker, but researchers have struggled to use the process as a definitive, quantitative marker for natural cell death.

About the Quantitative Pharmacodynamic Assay

The new immunofluorescence assay works by measuring the colocalization of both yH2AX and CC3. The National Cancer Institute researchers have been able to exploit the morphological changes of apoptosis to develop an assay based around CC3 aggregates to detect apoptotic cells. Using an image analysis spot algorithm, they can accurately and precisely quantitate the cells.

Research Study and Results

In a study, the researchers used xenograft tumor specimens and canine biopsy tissues that received various treatment agents. They found that the yH2AX and CC3 blebbing cellular colocalization assay is very sensitive and can detect relatively low amounts of apoptosis. During the assessment, the assay accurately captured the quantitative differences in apoptosis between the samples before and after they received cancer therapies.

These results provide evidence that the immunofluorescence assay produces dependable readouts of tumor reduction-related cell death. It can define the biochemical interaction of cancer agents in clinical tumor tissues that have the yH2AX marker, and it can report quantitative measurements of apoptosis. This will give scientists important information into how various classes of anticancer therapeutics perform. The National Cancer Institute researchers believe that the assay is ready to be used in clinical trials for that purpose.