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Why this Project:

Multicellular crosstalk following exposure to low-dose or low-dose rates of low-LET ionizing radiations may trigger signal pathways that will disrupt normal cell function in the irradiated, as well as neighboring unirradiated cells, leading to bystander effects. These studies in a relevant human cell system help develop low-dose radiation risk models that include positive and negative effects on multicellular targets.

Project Goals:

To use the unique synchrotron-based source of a 12.5 keV X-ray Microbeam line 10.3.1 at the Advanced Light Source ( ALS) at Lawrence Berkeley National Laboratory (LBNL) will be used to expose defined cells and cell populations to low-doses of low-LET radiation.
These studies will be conducted in complex cell systems including:

1. a novel tissue-like model of human mammary epithelial cells (HMEC)
2. normal human fibroblast cells (HFC)
3. both cells types grown together in a co-culture system.

Experimental Approach:

Radiation-induced changes in gene expression will be characterized using:

1. Gene array coupled with the quantitative RT- PCR.
2. Cultures grown in microwell slide chambers will be irradiated with precise 100 mm wide exposure stripes of dose to define the responses in exposed and bystander cells.
3. The time course of the expression of radiation-induced protein markers will be measured with fluorescent immunohistochemistry.
4. Fluorescence microscopy on a high-precision-controlled microscope stage combined with fiducial marked references will be used to map the location of both the radiation exposure and the biological response.

Outcomes:

1. Dose-dependent induction of bystander effects in several classes of radiation-induced signals will be quantified so that cell signaling changed by radiation exposure at both early and late times after exposure could be examined.
2. Evidence demonstrating cell-type specificity in the constitutive expression of genes, as well as dose-dependent Xray-induced genes, known to be involved in ATM/ ATR damage responsive pathways will be determined.
3. Differences in the constitutive, as well as low radiation-induced (10 cGy) expression of gap-junction connexin genes, suggest a cell-type specificity in mechanisms of cell communication.
4. Computer-based fluorescent analysis of radiation-induced signals in thousands of cells has revealed a time dependent statistically significant difference in the broadening of the effects of the dose stripes to neighboring unirradiated cells. The broadening of the dose stripe to involve cells not in the irradiated field will be quantitatively evaluated as a radiation-induced bystander effect.