DNA damage from replication stress, telomere shortening, UV radiation or chemical toxins is a part of everyday life, but in some cases, it can have the catastrophic effect of causing cancer. Studying proliferation, DNA damage and repair, apoptotic death and their interplay is a cornerstone of oncology research.
Certain acquired cellular behaviors can lead to the development of cancer. These acquired behaviors include a shift towards increased cell proliferation and decreased cell elimination, which upsets homeostatic balance. Accumulation of DNA damage/mutations can cause these shifts.
Researchers who investigate cancer susceptibility or treatment efficacy rely on accurate means of imaging and following proliferation, DNA damage/repair, and apoptosis.
Cell Proliferation
Many types of cancer cells proliferate more quickly than normal cells, and advanced research tools can help measure their proliferation. For example, the Ki-67 proliferation index (PI) measures the number of cells that are dividing by assessing the levels of Ki-67.
Only cells that are dividing produce the protein Ki-67. A high Ki-67 (basically anything over 30%) means that many cells are dividing quickly, and this suggests that the cancer is likely to grow and spread. The Ki-67 proliferation index may also help predict how well some cancers (such as breast cancer) will respond to treatment – tumors with a high Ki-67 may be more responsive to chemotherapy.
Encoded by the MKI67 gene, antigen Ki-67 is one of the main tumor biomarkers in breast cancer. Strongly associated with cancer proliferation, the expression of this protein is most abundant in S, G2 and M of the cell cycle. Ki-67 is also an indicator of prognosis and outcome. Pathologists, oncology professionals, and researchers use Ki-67 as a marker of proliferation and to establish prognosis in many types of neoplasms.
Proliferating cell nuclear antigen (PCNA) is essential for DNA replication, as it acts like a clamp to hold important required proteins to the DNA during division or repair. Expression of PCNA by cells during the S and G2 phases of the cell cycle make it a good cell-proliferation marker and can be essential in the diagnosis and prognosis of breast cancer.
4T1 mouse breast cancer lung metastases staining with Ki-67
Staining Method/Antigen:
Immunohistochemical staining with Ki-67 (Cell signaling #9027T,1:1000)
PCNA staining in human breast tissue explant
Staining Method/Antigen:
Immunohistochemical staining PCNA (# ab29, Abcam,1:10,000)
DNA Damage & Repair
DNA Damage Cell Cycle Arrest
The fidelity of the genetic code relies on the ability of DNA to repair itself after environmental assault.
Two complex signaling processes – DNA damage sensing and the cell cycle regulation – occurs through a number of biochemical reactions. The cyclin-dependent kinase inhibitor 1 (p21Cip1) protein plays an important role in DNA damage-related cell cycle arrest. P21 halts the replication of DNA and cell division before the cells proliferate replicating the damaged or mutant DNA in an uncontrolled manner.
DNA Damage Tumor Suppressor
Maintaining the fidelity of the genetic code is a big job, considering the almost-constant environmental assaults that can damage the DNA. When the cell cycle arrest after DNA damage is working correctly, DNA damage is recognized early; tumor suppressor p53 instructs the cell to express p21. If the cell has sustained too much damage or is not repaired quickly enough, p53 should instruct the cell to undergo apoptosis (cellular suicide) to avoid replicating the mutations
Normal human breast duct, p21 staining
Staining Method/Antigen:
Immunohistochemical staining with p21 antibody (Cell Signaling #2947, 1:50)
Human breast explant tissue treated with 5Gy radiation, p53 staining
Staining Method/Antigen:
Immunohistochemical staining with p53 (Cell Signaling #488185,1:100)
DNA Repair
DNA repair is a collection of processes in which a cell identifies and corrects DNA damage. Specific markers can identify where and when DNA damage and repair are occurring. The markers gH2AX and 53BP-1 will accumulate where DNA damage, particularly double-strand breaks (DSBs), occur. gH2AX forms as an early cellular response and contributes to genome stability. Detection of gH2AX is a highly specific and sensitive molecular marker for monitoring the initiation and resolution of DNA damage.
Tumor suppressor p53-binding protein 1 (53BP1) also accumulates in response to DNA damage. It helps regulate the balance among the DSB repair pathway choices. Together gH2AX and 53BP1 provide complementary measures of DNA damage and repair. Both gH2AX and 53BP1 markers decrease as the DNA is repaired.
Immunofluorescent staining of mouse mammary tissue to determine whether estrogen drives more DNA damage in the mammary tissue of BALB/c mouse strain compared to the tumor resistant C57BL/6 mouse strain.
Staining Method/Antigen: Immunohistochemical staining with gH2AX antibody (Cell Signaling #9718S, 1:100) and ERα antibody (Millipore, 06-935) followed by anti-mouse AlexaFluoro-488-conjugated (Cell Signaling #4408S) or anti-rabbit AlexaFluoro-488-conjugated secondary antibody (Cell Signaling #8889S).
Apoptosis
About 300 billion cells (1% or so) undergo apoptosis each day to maintain homeostasis and balance proliferation. Apoptosis can also provide valuable clues about the development of cancer. Scientists and doctors can observe the process of apoptosis through immunohistochemical analysis. Immunohistochemistry using active-caspase 3 antibody or a TUNEL assay kit provides the best views of apoptosis.
If the cell is too damaged for repair, the caspase system takes over to execute apoptosis, thereby killing the cell and preventing replication of the damaged DNA. Caspases are a family of protease enzymes that that provide critical links in cell regulatory networks controlling inflammation and cell death.
Caspase-3 is an executioner caspase. Activated by initiator caspases, caspases-3 can break up (cleave) DNA to cause DNA fragmentation and cell condensation, which are hallmarks of apoptosis. This makes caspase-3 a key marker of apoptosis, detectable via active-caspase 3 antibody.
TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, also called the TUNEL assay, detects breaks in DNA. More specifically, the terminal deoxynucleotidyl transferase (TdT) enzymes used in TUNEL assays recognize and bind to the ends of the DNA strands cleaved by caspases. Once bound to the DNA, the TdT will add a nucleotide to the end of the strand in a process known as nick translation; the fluorescent dye used in TUNEL staining allows for detection of this cleaved strand.
These tests and others are shedding light on the potential synergy between the DNA damage response and immune responses; leveraging research results using these tests could boost clinical efficacy of immunotherapies.
TUNEL staining of orthotopic breast cancer growth
Staining Method/Antigen:
Staining with TUNEL (Millipore #S7101)
Active caspase 3 staining in mouse lung metastases
Staining Method/Antigen:
Immunohistochemical staining with active caspase 3 (Cell Signaling # ASP175, 1:50)
Optimizing Antibodies for IHC Research
HistoSpring is leading the way in the advancement of oncology research and precision medicine by providing comprehensive biomedical services that fuel discovery, preclinical, and clinical research. Our full-service histology core specializes in immunohistochemical testing and analysis for proliferation, DNA damage and apoptosis.
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