Sensitivity to bleomycin induced chromatic breaks is associated with susceptibility to head and neck cancer. This so-called mutagen sensitivity was found to be an inheritable genetic factor as assessed in a combined family and twins study. A mutagen hypersensitive phenotype is associated with an increased risk for head and neck cancer in particular for the development of second primary tumors (as assessed in retrospective and prospective studies). The current focus of this project is to find the genotype behind this functional phenotype of mutagen sensitivity. As a model system EBV-immortalized lymphoblastoid cell lines are used from patients with a hypersensitive and patients with an insensitive phenotype. Indications that genes involved in cell cycle control are involved were obtained from functional studies in which we compared 10 hypersensitive and 10 insensitive cell lines in their cell cycle progression after bleomycin treatment. A less strong G2/M block was shown in hypersensitive cells as measured by flowcytometry which was also substantiated by an increased thymidine incorporation after bleomycin exposure (Cloos et al. submitted for publication). Moreover, cell growth inhibition experiments showed a damage resistant growth in hypersensitive cells. To elucidate specific pathways which may be involved in this impaired cell cycle control we are comparing gene expression profiles between three sensitive and three insensitive cell lines with and without bleomycin exposure using Human Cancer AtlasTM cDNA expression arrays. We have exposed the cells to 10 µM bleomycin for 2, 4, 6, 8 and 24 hr. Already after 2 hr differences in gene expression were detected. After 4 hr bleomycin incubation we find important cell cycle control genes to be differentially expressed in the hypersensitive versus the insensitive cell lines. Currently we are focusing on sophisticated data analysis such as time series to allow for the identification of putative impaired cell cycle pathways. In the near future we will perform micro-array analysis with more cell lines to allow a more detailed assessment.