BMC Cancer 2005, 5:102 http://www.biomedcentral.com/1471-2407/5/102 Table 4: mtDNA alterations in human brain tumors compared to matched tissue controls Type of tumor Number of tumors Region of mutation Reference Number of tumors with mtDNA somatic mutationsa Number of somatic mutations causing amino acid substitutionsc Neurofibroma Glioma Meningioma Schwannoma Medulloblastoma Gliomatosis cerebri Glioma Astrocytoma 37 53 11 5 15 6 55 12 23/37 20/53 5/11 1/5 6/15 2/6 12/55 11/12b NAd 2/18 D-loop HVRII D-loop, Asp tRNA, COXI, CoxII, Thr tRNA, ND4 HVRII HVRII, D-loop HVRII Kurtz et al. 2004 Vega et al. 2004 Wong et al. 2003 Kirches et al. 2003 Kirches et al. 2001 Kirches et al. 1999 a Number of tumors with mutations/number of tumors b 2 of the 12 astrocytomas were compared to adjacent brain, the remaining 10 were aligned to Anderson et al. 1981 for mutation analysis c Number of mutations causing amino acid substitutions/total number of mutations found in all tumors d NA, not applicable because in hypervariable region of the D-loop (HVR) region [23]. The second mutation in this cell line involved a T->C transition in the D-loop at position 15584. Neither of these mutations have functional consequence. whole tumor tissue. Hence, our experimental design will detect any mtDNA mutation that could alter mitochon- drial function. A single mutation was found in the metastatic VM-M cell line involving an A->G transition in the 16srRNA gene at position 2159. In contrast to the other mtDNA mutations found in either the B6 or the VM tumor cell lines, the A- >G transition in the VM-M cell line was heteroplasmic (Figure 1). This heteroplasmic mutation, however, was not found in another independently derived cell line from this metastatic tumor. Viewed together, these data indi- cate that only a few mtDNA somatic mutations exist in the chemically induced tumors in the B6 mice or in the spon- taneous brain tumors in VM mice. Also, most of these mutations were found in hypervariable regions and none are considered pathogenic. Discussion The purpose of this study was to determine if mtDNA mutations existed in chemically induced and spontane- ous mouse brain tumors and whether such mutations might provide insight on the development or progression of these experimental brain tumors. To reduce the proba- bility of detecting spurious pathogenic mutations, we compared the mtDNA sequence of each brain tumor cell line with that of normal mtDNA from the syngeneic mouse host strains. Furthermore, to avoid the coamplifi- cation of mitochondrial pseudogenes that exist in nuclear DNA, we amplified only large mtDNA fragments (1–1.8 kb in length) in isolated mitochondria [18,57,58]. Because the solid tumors become infiltrated with host stromal cells containing wildtype mitochondria [59], we used cultured tumor cell lines for these studies instead of Several mtDNA mutations were found in the mouse brain tumor cell lines, but most of these were located in hyper- variable mononucleotide hotspot regions, such as in the OL and in the Arg tRNA for mouse mtDNA. Moreover, none of these mutations were considered pathogenic since they did not change amino acid sequence and there- fore could not alter gene function. Tumor cells in each line were selected from original tumors that may have con- tained multiple transformed cell types. The lines therefore represent only those cells with the most aggressive malig- nant growth in vitro and in vivo. The absence of pathogenic mutations in these tumor cells, further suggests that mtDNA mutations do not likely contribute to the initia- tion or the progression of these diverse mouse brain tumors. Most tumors including brain tumors express abnormali- ties in the number and function of their mitochondria [60,61]. Warburg originally emphasized that the high gly- colytic rate of tumors results from diminished or dis- turbed respiration [24,25]. Later studies in a variety of neural and non-neural tumor systems showed that these respiratory disturbances could involve abnormalities in TCA cycle components, alterations in electron transport, and deficiencies in oxidative phosphorylation [62-66]. Recent studies also suggest that mtDNA mutations may contribute to the respiratory defects in cancer [18]. Our findings indicate that diminished respiration and the glycolytic dependence of the chemically induced CT-2A and EPEN brain tumors or the two spontaneous VM brain Page 5 of 8 (page number not for citation purposes)