Hierarchical clustering method identifies adaptation of mitochondrial signaling to the treatment of mitochondrial blockers that regulates the expression of cell cycle genes.

The cross-talk between the cell nucleus and mitochondria appears to control hormone-induced signaling involved in the apoptosis, proliferation, and differentiation of both normal and malignant cells. Some pharmacological agents such as tamoxifen; antibiotics such as tetracycline or chloramphenicol (inhibitors of the mitochondrial protein synthesis); and other pharmacological agents exert their effects on cell growth by their influence on mitochondria. We propose to investigate how mitochondrial signaling pathways in breast cancer cells may contribute to molecular mechanisms of mitochondrial inhibitor resistance. We have examined the innovative hypothesis that mitochondria through its signaling to the nucleus control the expression of cell cycle genes. Mitochondrial transcription/replication was impaired in MCF7 cells by culturing cells in routine growth medium supplemented with a low dose of ethidium bromide (50 ng/ml), pyruvate (1 mM), and uridine (50 µg/ml) for 20 days. An alternative approach to blocking mitochondrial transcription/replication was to use rhodamine 6G (R6G) that inhibits oxidative phosphorylation. Acute (1 to 2 h) exposure of cells to inhibitors of oxidative phosphorylation was also given prior to 17ß-estradiol (E2) treatment. Gene expression was measured using the Non-radioactive GEArray Q Series Kit for human cell cycle, genes. MCF7 cells and ethidium bromide (EtBr) or rhodamine 6G (R6G) treated MCF7 cells were exposed to E2 (100pg/ml) for 6 and 24 h. Data were analyzed using the hierarchical clustering method. This method performs a hierarchical cluster analysis using a set of dissimilarities for the gene profiles being clustered. Initially, each object is assigned to its own cluster and then the algorithm proceeds iteratively, at each stage joining the two most similar clusters, continuing until there is just a single cluster. At each stage distances between clusters are recomputed by the Lance-Williams dissimilarity update formula according to the particular clustering method being used. Both of the mitochondrial inhibitors attenuated the expression of early cell cycle genes. Acute treatment of MCF7 cells with R6G produced a drastic inhibition of cell cycle genes; whereas a sub-chronic treatment with EtBr showed a recovery of cell cycle expression compared to R6G. Similar effects were observed in E2 co-treated MCF7 cells. These findings suggest that mitochondria may play an important role in the progression of the breast cancer cell cycle through altering the expression of cell cycle genes and breast cancer cells may adapt to the mitochondrial stress produced by sub-chronic or chronic treatment with inhibitors. In conclusion, the use of mitochondrial inhibitors to study signal transduction networks requires attention to the altered expression of nuclear genes as well as adaptive responses that are attributed to acute versus chronic exposure.