Environmental Factor, July 2010, National Institute of Environmental Health Sciences
Little Things That Do a Lot
By Emily Zhou
The July 7 presentation in the Laboratory of Signal Transduction (LST) Seminar Series featured Les Hanakahi, Ph.D., from the Department of Medicinal Chemistry and Pharmacognosy at the University of Illinois College of Pharmacy at Rockford. Her talk, "Little Things that Do a Lot: Inositol Polyphosphates in DNA Repair," was one of the highlights of the Laboratory's 2010 seminar program.
According to Hanakahi, inositol polyphosphates are important molecules that are crucial in a vast array of cellular physiologies including DNA repair. In a healthy organism, deficits in DNA repair promote development of cancers triggered by accumulated environmental exposures. Conversely, a targeted reduction of DNA repair efficiency in cancer cells by using analogs of inositol hexakisphosphate (IP6) may offer clinicians a novel way to augment traditional therapies.
Hanakahi's research on the role of inositol polyphosphates in DNA repair has advanced the understanding of how a molecule such as IP6, while small, can nevertheless have a considerable role in regulating some of the multi-protein interaction that are so critical to the complex process of DNA repair.
Double-stranded DNA repair
Over 95 percent of cells in human body reside in the G1 (gap 1)/G0 (resting) phase of the cell cycle. Upon ionizing radiation (IR) exposure or other environmental intrusion, double-stranded DNA break (DSB) occurs. Non-homologous end joining (NHEJ) is the process that is responsible for repairing DSB in G0 and G1 phases of the cell cycle.
"Consequences of DSB repair failure are gross chromosomal aberrations that may lead to cancer," said Hanakahi. NHEJ involves multi-protein complexes such as Ku70/80, DNA-protein kinase (DNA-PK), X-ray repair complementing defective repair in Chinese hamster cells 4 (XRCC4), Ligase IV, and XRCC4-like factor (XLF). The presence of IP6 significantly stimulates NHEJ in vitro in a time dependent manner. Furthermore, it is the specific binding of IP6 to Ku70/80 complex, not other components of NHEJ, that results in the stimulation of NHEJ.
Mutants of Ku70/80 that cannot bind IP6 fail to stimulate NHEJ. Future studies that involve knock-down of Ku70/80 in rodent cell lines and knock-in mutants of Ku70/80 in human cells are being carried out to more clearly define the role of IP6 in NHEJ. The potential role in NHEJ of diphosphorylated derivatives of IP6, such as IP7 or IP8, is also under investigation in Hanakahi's laboratory.
Human adenovirus infection and NHEJ
Scientists frequently explore the details of cellular physiology through the interaction between a pathogen and its host. "By using human adenovirus as a tool to study mammalian NHEJ," Hanakahi explains, "we have uncovered a novel aspect of NHEJ - in human cells, DNA recognition by XRCC4 and XLF requires the ligase IV polypeptide."
Experiments from Hanakahi's laboratory have also shown that infection of human cells with human adenovirus type 5 causes inhibition of NHEJ both in vitro and in vivo, loss of DNA ligase IV, and loss of DNA binding by XRCC4 and XLF. Hanakahi hypothesizes that intrinsic DNA binding activities by XRCC4 and XLF are regulated through phosphorylation. Further experiments are underway to identify these putative kinases. Whether this phosphorylation involves the Ku70/80-IP6 complex is being explored.
"The discovery that IP6 stimulates mammalian NHEJ in vitro suggests the exciting possibility that synthetic or natural products resembling IP6 may be used to control the efficiency of NHEJ in human cells," said Hanakahi. "An example of the utility of such IP6-analogs is reduction of NHEJ efficiency in tumor cells, which could augment traditional radiation therapy by preventing DSB repair in cancer cells. This would decrease tumor cell viability and promote reductions in tumor mass."
As seminar host LST Principal Investigator Stephen Shears, Ph.D., observed afterwards, Hanakahi engaged her audience's interest because of her high quality science, infectious enthusiasm for scientific research, and engaging presentation skills.
(Yixing [Emily] Zhou, Ph.D., is a postdoctoral research fellow in the NIEHS Laboratory of Signal Transduction.)