transendothelial migration (containing Myl7, Cldn5, Jam5 and Vcam1), and tight junction (also containing Myl7 and Cldn5, as well as Jam2, Pten and Myh7) (see Additional file 2 and Table 2). However, it should be noted that there is some overlap between the groups of genes assigned by KEGG to each pathways: melanoma shares 5 genes with either p53 signaling pathway or cytokine-cytokine receptor interaction, it also shares 3 genes with tight junction, and the latter shares 20 genes with leukocyte transendothelial migration. This is likely to result from the fact that the KEGG classification is somewhat arbitrary, and some- times assigns genes on the basis of particular biologic manifestations rather than regulatory pathways or func- tions. Nonetheless, overlap between KEGG pathways sug- gests that a common biological process underlies the selection of these particular genes. Of note, one additional limitation of the Set Enrichment Test is that the coverage of KEGG pathways is still incomplete at the current time. For instance, although Dbp, Tef, Nr1d2 and Rora are all well-known downstream effectors of clock genes [15,16], none of them have been included yet within the KEGG cir- cadian rhythm pathway. Likewise, Myl4 has not been assigned to either leukocyte transendothelial migration or tight junction despite being highly homologous to Myl7. Interestingly, 14 out of the 17 interaction genes enriched within KEGG pathways display strain-dependent differ- ences or surgery-dependent changes that were greater than 1.5 fold (see Additional file 2), which may constitute one other indication that these particular genes play biologi- cally important roles. Circadian rhythm, in addition to being the KEGG pathway showing the most significant enrichment for interactions genes, also comprised genes showing some of the largest differences in expression among interaction genes. If we consider all 14 genes that could be classified as "circa- dian" on the basis of both KEGG pathway database and additional annotation from the literature, we found that expression of 14 of them were significantly affected by CX in C57.YA mice in contrast to only one gene in C57BL/6J (Table 3 and Figure 5). Although only 10 genes were included in the interaction genes set, the post-hoc tests revealed evidence for interaction in two extra circadian genes, i.e. Csnk1e and Csnk1d, that showed 20% increase in castrated animals but only in the C57.YAstrain. As a consequence of this differential response to post-pubertal testosterone, most circadian genes showed strain-depend- ent differences in cardiac expression between intact C57BL/6J and C57.YA mice, but not in their CX counter- parts (Figure 6). One caveat concerning these experimen- tal results is that expression of circadian genes is, by definition, very sensitive to time. Although we took care of killing all mice at the same time of day (between 9:00 and 10:00 AM), further validation requires additional experi- ments using mice from both strains killed at different times of the 24-hour nyctemer. Nonetheless, there are cur- rently other lines of evidence suggesting that the current results are biologically pertinent, since: 1) recent reports showed that androgens are major regulators of clock genes in C57BL/6J mice and that they are responsible for sex-dependent differences in circadian activity [17,18], and 2) male C57BL/6J mice do not show (in contrast to male A/J counterparts) circadian oscillations of heart rate (corrected for locomotor activity) [19]. Moreover, it has also been reported recently that Arntl/Bmal1 is a major reg- ulator of Cdkn1a (whose expression is also greatly affected by CX in a C57.YA-specific fashion, as discussed below) and that Arntl-dependent changes in the concentration of the corresponding p21Waf1/Kip1 protein correlate with changes in the rate of liver cell proliferation, in keeping with the known functions of this cyclin inhibitor [16]. Within the p53 signaling pathway (second in importance in terms of enrichment for interacting genes), Ccnd1, Pten and Cdkn1a may be of particular interest. Indeed, Ccnd1 encodes for cyclin D1 which is considered as one of the few bona fide AR-specific co-repressors, both in androgen- dependent and-independent environments [20-22]. Cyc- lin D1 (down-regulated in CX C57BL/6J vs. CX C57.YA) can be silenced by Akt [23], and the latter may be more active in CX C57BL/6J because its inhibitor Pten is down- regulated vs. CX C57.YA. Cyclin D1 can also be silenced by the p21Waf1/Kip1 cyclin inhibitor protein (encoded by Cdkn1a) [24], the latter being upregulated in CX Expected Count Count P-value 0.33 1.02 1.05 2.30 1.71 2.40 6 5 4 6 5 6 1.8E-07 0.0028 0.0190 0.0249 0.0257 0.0299 BMC Genomics 2009, 10:150 http://www.biomedcentral.com/1471-2164/10/150 Listed KEGG pathways correspond to those showing significant enrichment (p < 0.05) for "interaction genes". Size corresponds to the number of genes assigned by KEGG within each particular pathway. Expected count corresponds to the number of interaction genes could be expected to be found if there was no significant enrichment, count corresponds to the effective number of genes showing significant enrichment (p < 0.05) (see also Additional file 2). Page 5 of 11 (page number not for citation purposes) Table 2: Selected KEGG pathways showing enrichment for interaction genes KEGG ID Term 4710 4115 5218 4060 4670 4530 Circadian rhythm p53 signaling pathway Melanoma Cytokine-cytokine receptor interaction Leukocyte transendothelial migration Tight junction Size 10 31 32 70 52 73