BMC Cancer 2005, 5:102 http://www.biomedcentral.com/1471-2407/5/102 hypervariable regions of the displacement loop (D-loop) [19-21]. Alterations in these hypervariable regions are unlikely to alter mitochondrial function or produce a dis- eased phenotype [22]. These hypervariable regions corre- spond to the OL and Arg tRNA in mouse mtDNA [23]. Otto Warburg originally emphasized that the high glyco- lytic rate of tumors resulted from diminished or disturbed respiration [24,25]. Recent studies suggest that mtDNA mutations may contribute to the respiratory defects in cancer [18]. Furthermore, studies of human tumors have demonstrated an association between mtDNA somatic mutations and tumor progression [26-28]. Few studies have evaluated mtDNA mutations in brain cancer. Mitochondrial genome instability was found in the hypervariable regions in the D-loop of human glio- mas, meningiomas, schwannomas, gliomatosis cerebri, glioblastomas, astrocytomas, and neurofibromas [29-33]. Other studies of the mitochondrial genome focused on mtDNA copy number in brain tumors [34]. In the only study examining the entire mitochondrial genome, Wong et al. found that the majority of mtDNA somatic muta- tions in medulloblastomas existed in regions of mononu- cleotide repeats, rather than in genes encoding proteins for the respiratory chain complexes [35]. A recent study in mouse tumorigenic fibroblasts, suggest that nuclear muta- tions rather than mtDNA mutations contribute to the tumor phenotype [36]. It is not yet clear if mtDNA muta- tions contribute to brain tumor progression. In contrast to most tissues where mitochondrial morphol- ogy and function is relatively homogeneous, mitochon- dria in brain are heterogeneous and exist as non-synaptic free mitochondria (FM), synaptic light mitochondria (LM), and synaptic heavy mitochondria (HM) [37,38]. This morphological heterogeneity is thought to reflect the metabolic heterogeneity of mitochondria within and between the various neuronal and non-neuronal (glial) cell populations [39]. Besides morphological heterogene- ity, genetic heterogeneity in mtDNA has been reported in normal human brain tissue [40]. In this study, we sequenced the entire mitochondrial genome in a series of chemically induced and spontaneous mouse brain tumors that differ in metastasis, malignancy, and vascular- ity. To our knowledge, no prior studies have compared the sequence of the entire mitochondrial genome in brain tumor cell lines with that of the syngeneic brain tissue. Although we found several mutations, none of these were considered pathogenic. Methods Mice and brain tumors The inbred VM and C57BL/6J (B6) mouse strains were obtained originally from Professor H Fraser, University of Edinburgh, and from the Jackson Laboratory, Bar Harbor, ME, respectively. All mice used in the study were propa- gated in the animal care facility at Boston College using animal husbandry conditions described previously [41]. All animal-use procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee. The EPEN and CT-2A brain tumors were produced origi- nally from implantation of 20-methylcholanthrene into the brains of B6 mice as previously described [42,43]. The EPEN tumor arose in the cerebral ventricle and was char- acterized as an ependymoblastoma, whereas the CT-2A tumor arose in the cerebral cortex and was characterized as a poorly differentiated astrocytoma [42,44]. The CT-2A tumor contains complex gangliosides, is highly ang- iogenic in vivo, and grows in vitro as a fusiform monolayer [42,45,46]. In contrast, the EPEN tumor expresses only the simple ganglioside GM3, is less angiogenic in vivo, and grows in vitro as cuboidal cell islands [41,42,46-49]. The nonmetastatic (VM-NM) and the metastatic (VM-M) tumors arose spontaneously in the brains of VM mice as previously described for other VM brain tumors [50,51]. Tumor cell culture Cultured cell lines were prepared from each tumor as we previously described [52,53]. The number of passages for CT-2A and EPEN cell lines were greater than 40, where as VM-NM and VM-M were passaged 9 and 11 times, respec- tively. The VM-M cell line maintained metastatic capacity when implanted into VM mice. The tumor cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a humidified atmosphere containing 95% air and 5% CO2 at 37°C. Upon reaching confluency, the cells were trypsinized and collected. Mitochondrial isolation from brain tissue and cultured tumor cells Mitochondria were isolated from VM and B6 cerebral cor- tex according to previously described procedures [38,54]. Cerebral cortex mitochondria were separated into three populations following ficoll gradient centrifugation. These populations included non-synaptic free mitochon- dria (FM), synaptic light mitochondria (LM), and synaptic heavy mitochondria (HM). Mitochondria were isolated from the tumor cell lines as a single enriched mitochon- drial fraction as previously described [54,55]. Mitochon- dria were isolated in duplicate independent samples from each of the brain tumor cell lines for sequence comparison. Isolation and amplification of mouse mtDNA mtDNA from cerebral cortex and cultured tumor cells was isolated using the DNeasy Tissue Kit (QIAGEN, Valencia, Page 2 of 8 (page number not for citation purposes)