Significant biological effects can occur in animals, cells, immortalized human cell lines, and primary human cells after exposure to low doses
(< 1 –10 cGy) of ionizing radiation. To apply these cellular observations to radiation risk, cell experiments must be applied and related to humans. This project uses cells near the fields of human patients undergoing radiation therapy as a model to evaluate the effects of low dose radiation on gene regulation in vivo.

1. To develop a human in vivo model using a skin cell system to measure changes in gene expression.
   
2. To determine if human genes are modulated in vivo, by radiation the same way as they are in vitro.
   
3. If so, to determine if the same genes are responsive both in vitro and in vivo.

Experimental Approach:

Our project uses human skin, irradiated in vivo during therapeutic radiation as a model system. Preliminary studies have focused on verifying the accuracy of the dosimetry in the low dose, out of field areas, optimizing RNA and protein extraction from the samples, assessing RNA amplification strategies and performing microarray analyses to ensure the robustness of the physics and biology components of the project prior to obtaining patient samples.

Preliminary biologic studies have focused on obtaining global gene expression data from small volume human skin samples. Samples have been obtained from resected tissue in known radiation fields from elective surgical procedures. Tissue samples from in vivo radiation are harvested and incubated up to 24 hours to assess stability of the message. Other unexposed samples are subjected to immediate ex vivo IR at 1, 10 or 100 cGy and then incubated for equivalent times. RNA is extracted, processed, and hybridized to cDNA microarrays containing over 12,500 unique sequence validated human cDNA clones to assess gene expression changes in the samples. Expression profiles generated from amplified and unamplified RNA are being compared to confirm the fidelity of amplification schemes that are required for samples containing limited RNA.

Expected outcomes:

1. Develop a human in vivo model system to compare radiation effects to those from studies of human cells in vitro.
   
2. Determine whether skin cells are radioresponsive in vivo.
   
3. Determine which, if any, genes are modulated following exposure to low dose radiation both in vivo and in vitro.

Project findings:

Preliminary findings indicate that following 1 cGy exposure ex vivo skin cells have as many as 116 genes with at least 2 times higher gene expression than observed in unexposed normal cells.