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Title: Genetic Control of the Trigger for the G2/M Checkpoint

The work undertaken in this project addressed two seminal areas of low dose radiation biology that are poorly understood and controversial. These areas are the challenge to the linear-no-threshold (LNT) paradigm at low doses of radiation and, the fundamental elements of radiation bystander effect biology Genetic contributions to low dose checkpoint engagement: The LNT paradigm is an extrapolation of known, measured cancer induction endpoints. Importantly, data for lower doses is often not available. Debatably, radiation protection standards have been introduced which are prudently contingent on the adherence of cancer risk to the established trend seen at higher doses. Intriguing findings from other labs have hinted at separate DNA damage response programs that engage at low or high levels of radiation. Individual radiation sensitivity commensurate with hemizygosity for a radiation sensitivity gene has been estimated at 1-2% in the U.S.. Careful interrogation of the DNA damage response at low doses of radiation became important and served as the basis for this grant. Several genes were tested in combinations to determine if combined haploinsufficiency for multiple radiosensitizing genes could render a cell more sensitive to lower levels of acute radiation exposure. We measured a classical radiation response endpoint, cell cycle arrest priormore » to mitosis. Mouse embryo fibroblasts were used and provided a uniform, rapidly dividing and genetically manipulable population of study. Our system did not report checkpoint engagement at acute doses of gamma rays below 100 mGy. The system did report checkpoint engagement reproducibly at 500 mGy establishing a threshold for activation between 100 and 500 mGy. Engagement of the checkpoint was ablated in cells nullizygous for ATM but was otherwise unperturbed in cells combinatorially haploinsufficient for ATM and Rad9, ATM and PTEN or PTEN and Rad9. Taken together, these experiments tell us that, in a sensitive fibroblast culture system, the engagement of the G2/M checkpoint only occurs at doses where most of the cells are bound for mitotic catastrophe. Further, compound haploinsufficiency of various radiosensitizing genes does not impact the threshold of activation. The experiments confirm a threshold of activation for the G2/M checkpoint, hinting at two separate radiation response programs acting below and above this threshold. Small RNA transfer in bystander effect biology: Small regulatory RNA molecules have now risen in prominence and utility. Specific examples are small interfering RNAs (siRNA) which are employed in cell level expression ablation projects and micro-RNAs (miRNA) which are a pool of short transcription products which serve to modulate the expression of other transcripts emerging from the genome in a meta-regulatory fine tuning of gene expression. The existing tenets of bystander effect radiation biology involve the communication of inflammatory mediators or direct intercellular communication of reactive oxygen/nitrogen species in cell-to-cell communicative organelles called gap junctions. By ablating gap junctions, reducing the ROS/inflammatory cytokine expression one can attenuate bystander effect signaling in cell culture systems. We hypothesized that miRNAs are a competent intercellular communication molecule and therefore a possible component of the bystander response. This view is supported by the observation that miRNA are secreted from cells in exosomes found in the circulation. This circulating pool reports disease type and severity in humans. We proposed use of microbeam irradiation technology at our facilities and enhancement of this capability with a new sorting technology which would allow us to sort irradiated and non-irradiated cells with absolute fidelity. Pursuing direct quantitative transfer assessment, we succeeded in designing and constructing a new add-on sorting appliance which harmonized with our existing instruments. The sorter allowed us to gently sort single fluorescently labeled cells. The plans for this appliance were published and are now available for use in other laboratories for single-cell analyses. Our microfluidic cell sorting modality is being integrated into subsequent microbeam irradiation experiments that are planned and ongoing. We generated and irradiated pools of specially engineered Donor-Recipient cell lines in co-culture that would report a small RNA transfer event by modulation of fluorescent protein expression. Both induction and reciprocal silencing designs were tested. We observed elevation of miRNA/siRNA transfer in response to radiation at doses of 5Gy in experiments to date. The reproducibility of these findings has not been good. Future studies will involve refinement of the reporting systems and a decrease in acute dose of radiation used to determine the lowest dose at which miRNA transfer between cells contributes to radiation bystander effect biology.« less
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  1. Columbia University
Publication Date:
OSTI Identifier:
Report Number(s):
DOE Contract Number:
Resource Type:
Technical Report
Research Org:
Trustees of Columbia University in the City of New York
Sponsoring Org:
USDOE USDOE Office of Science (SC)
Country of Publication:
United States
61 RADIATION PROTECTION AND DOSIMETRY; cell cycle, cell cycle checkpoint control, G2/M, haploinsufficiency, ATM, Rad9, BRCA1, miRNA radiation, miRNA transfer, cell sorting chip, cell sorting device