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111In-Labeled DTPA-conjugated Tat-linked anti-phosphorylated histone protein H2AX antibody.

Authors

Chopra A.

Source

Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.
2011 Jul 28 [updated 2011 Aug 25].

Excerpt

Chemotherapy and radiotherapy, or a combination of the two, are often used to treat cancer and are known to either damage cellular DNA itself or to interfere with the DNA metabolic pathways and generate DNA double-strand breaks (dsb) that are fatal for the neoplastic cells (1). Because the generation of dsb by these therapies is the main cause of cell death, the variable capacity of a cell to repair the dsb influences its survival response to these treatments. In other words, the cell will survive only if the dsb is quickly repaired; otherwise, the cell will probably die (2). Therefore, the visualization of DNA dsb in tumor cells can provide useful information regarding the efficacy of a cancer treatment regimen and can also predict the prognosis for a patient (3). The repair of DNA dsb in the cell is initiated by the phosphorylation of H2AX, a histone protein, to γH2AX, which initiates a signaling pathway to attract dsb repair proteins (such as DNA-dependent protein kinase, breast cancer type 1 susceptibility protein, etc.) at the site of DNA damage. As a consequence, compared to normal cells, the expression and phosphorylation of H2AX is elevated in the neoplastic cells that are subject to genotoxic insult, which results in the accumulation of this protein and the formation of foci at the location of DNA injury (2). Current immunohistochemical and flow cytometric methods using an anti-γH2AX antibody (Ab) are utilized to detect dsb in tumor cells before and after an anti-cancer treatment of a patient, but these procedures are invasive and time-consuming (1). Visualization of γH2AX in the cell with non-invasive imaging techniques before and after anti-cancer treatment can be an excellent alternative to invasive procedures and can help in the rapid assessment of an intervention (4). However, the major limitations of imaging DNA dsb are that the strand breaks are located in the nucleus and are protected by the cell and the nuclear membranes. In addition to this limitation, imaging agents (including antibodies) that target γH2AX may not be able to penetrate the protective membranes and generate a signal that can be captured to visualize the dsb. Cornelissen et al. hypothesized that if the anti-γH2AX antibody was to be used as a probe to detect DNA dsb within the cellular nucleus, it will not only require assistance to penetrate the membranes but will have to be labeled with an appropriate tracer, such as a radionuclide, to visualize the dsb with imaging techniques (4). To test this hypothesis, the anti-γH2AX Ab was tagged with the Tat peptide, which contains a short HIV-1 transactivator of the transcription amino acid sequence that facilitates the transport of proteins or other molecules across the cell and nuclear membranes. The anti-γH2AX Ab-Tat complex was then labeled with 111In and Cy3, AF555, or AF888 (a group of fluorophores), respectively (4). In a set of proof-of-principal studies, the fluorophore-labeled Ab-Tat conjugate was then evaluated for the optical imaging of dsb under in vitro conditions (using human mouse embryonic cell lines). In addition, the fluorophore- or radionuclide-labeled Ab probes were investigated for the visualization of MDA-MB-468 cell xenograft tumors (a human breast cancer cell line) in mice, using fluorescence imaging (for animals injected with the Cy3-Ab-Tat conjugate) and single-photon emission computed tomography (SPECT; for animals injected with the [111In]-Ab-Tat conjugate). The biodistribution of [111In]-DTPA-anti-γH2AX-Tat in the tumor-bearing mice was also investigated.

PMID:
21882403
[PubMed]
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