Keizo Takenaga

ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis.

Mutations in mitochondrial DNA (mtDNA) occur at high frequency in human tumors, but whether these mutations alter tumor cell behavior has been unclear. We used cytoplasmic hybrid (cybrid) technology to replace the endogenous mtDNA in a mouse tumor cell line that was poorly metastatic with mtDNA from a cell line that was highly metastatic, and vice versa. Using assays of metastasis in mice, we found that the recipient tumor cells acquired the metastatic potential of the transferred mtDNA. The mtDNA conferring high metastatic potential contained G13997A and 13885insC mutations in the gene encoding NADH (reduced form of nicotinamide adenine dinucleotide) dehydrogenase subunit 6 (ND6). These mutations produced a deficiency in respiratory complex I activity and were associated with overproduction of reactive oxygen species (ROS). Pretreatment of the highly metastatic tumor cells with ROS scavengers suppressed their metastatic potential in mice. These results indicate that mtDNA mutations can contribute to tumor progression by enhancing the metastatic potential of tumor cells.

Expression of antisense RNA to S100A4 gene encoding an S100-related calcium-binding protein suppresses metastatic potential of high-metastatic Lewis lung carcinoma cells

S100A4 (also known as pEL98/mts1/p9Ka/18A2/42A/calvasculin/FSP1/CAPL), a member of S100-related calcium-binding proteins, has been implicated to play a role in metastasis. In the present study, we examined the effect of antisense S100A4 RNA on metastatic potential of Lewis lung carcinoma (LLC) cells. High-metastatic A11 cells were transfected with the expression vector containing S100A4 cDNA in an inverted (antisense) orientation under the transcriptional control of the mouse metallothionein promoter. Treatment of a stably transfected clone (AS10 cells) with Zn2+ resulted in the suppression of the experimental metastatic ability, which was accompanied with the expression of antisense S100A4 RNA and the suppression of the S100A4 expression at both the mRNA and the protein levels. To further confirm the effect of antisense S100A4 RNA, we established several clones after retroviral transduction with an antisense S100A4 construct. Notably, reduced metastatic potential was also evident in these clones. In the antisense S100A4 RNA-expressing cells, cell motility and in vitro invasiveness were found to be suppressed.

Induction of differentiation of cultured human promyelocytic leukemia cells by retinoids.

Abstract Human promyelocytic leukemia cells (HL-60) were induced to phagocytize, reduce NBT dye(nitroblue tetrazolium), and change into forms that were morphologically similar to mature granulocytes by retinoic acid and related retinoids, but not by the pyridyl analog of retinoic acid. Induction of differentiation could be detected after 4 days of treatment of the cells with retinoic acid at as low a dose as 4 × 10 −8 M. Thus, retinoids may be used in studies on the control of cell differentiation and malignancy of human myeloid leukemia cells.

Reactive Oxygen Species-generating Mitochondrial DNA Mutation Up-regulates Hypoxia-inducible Factor-1α Gene Transcription via Phosphatidylinositol 3-Kinase-Akt/Protein Kinase C/Histone Deacetylase Pathway

Lewis lung carcinoma-derived high metastatic A11 cells constitutively overexpress hypoxia-inducible factor (HIF)-1α mRNA compared with low metastatic P29 cells. Because A11 cells exclusively possess a G13997A mutation in the mitochondrial NADH dehydrogenase subunit 6 (ND6) gene, we addressed here a causal relationship between the ND6 mutation and the activation of HIF-1α transcription, and we investigated the potential mechanism. Using trans-mitochondrial cybrids between A11 and P29 cells, we found that the ND6 mutation was directly involved in HIF-1α mRNA overexpression. Stimulation of HIF-1α transcription by the ND6 mutation was mediated by overproduction of reactive oxygen species (ROS) and subsequent activation of phosphatidylinositol 3-kinase (PI3K)-Akt and protein kinase C (PKC) signaling pathways. The up-regulation of HIF-1α transcription was abolished by mithramycin A, an Sp1 inhibitor, but luciferase reporter and chromatin immunoprecipitation assays indicated that Sp1 was necessary but not sufficient for HIF-1α mRNA overexpression in A11 cells. On the other hand, trichostatin A, a histone deacetylase (HDAC) inhibitor, markedly suppressed HIF-1α transcription in A11 cells. In accordance with this, HDAC activity was high in A11 cells but low in P29 cells and in A11 cells treated with the ROS scavenger ebselene, the PI3K inhibitor LY294002, and the PKC inhibitor Ro31-8220. These results suggest that the ROS-generating ND6 mutation increases HIF-1α transcription via the PI3K-Akt/PKC/HDAC pathway, leading to HIF-1α protein accumulation in hypoxic tumor cells.

Involvement of Sl00–related Calcium–binding Protein pEL98 (or mts1) in Cell Motility and Tumor Cell Invasion

We examined the relationship between cell motility and the expressions of pEL9S (mtsl) mRNA and protein in various imirine normal and transformed cells. The expression of pEL98 (mtsl) in v–Ha–ras–transformed NIH3T3 cells and in normal rat kidney cells transformed by either v–Ha–ras or v–src was increased over that in the corresponding parental cells at both mRNA and protein levels. The expression in normal rat fibroblasts (3Y1) transformed by v–Ha–ras was also increased compared with that in 3Y1 cells. However, the expression of pEL98 (mtsl) in 3Y1 cells transformed by v–src was increased in one clone (src 3Y1–K), but decreased in another clone (src 3Y1–H). The expression level of pEL98 (mtsl) correlated well with cell motility, which was examined by measuring cell tracks by phagokinesis. In order to test direct involvement of the pEL98 (mtsl) protein in cell motility, src 3Y1–H cells that showed low cell motility were transfected with pEL98 cDNA. The transfectants expressing large amounts of the pEL98 protein showed significantly higher cell motility than src 3Y1–H cells. The expression of pEL98 (mtsl) was also found to be correlated with motile and invasive abilities in various clones derived from Lewis lung carcinoma. These results suggest that the pEL98 (mtsl) protein plays a role in regulating cell motility and tumor cell invasiveness.

Mitochondrial DNA Mutations Regulate Metastasis of Human Breast Cancer Cells

Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously expressed a highly metastatic potential, mitochondrial respiration defects, and ROS overproduction. Since mitochondrial respiratory function is controlled by both mtDNA and nuclear DNA, it is possible that nuclear DNA mutations contribute to the mitochondrial respiration defects and the highly metastatic potential found in MDA-MB-231 cells. To examine this possibility, we carried out mtDNA replacement of MDA-MB-231 cells by normal human mtDNA. For the complete mtDNA replacement, first we isolated mtDNA-less (ρ0) MDA-MB-231 cells, and then introduced normal human mtDNA into the ρ0 MDA-MB-231 cells, and isolated trans-mitochondrial cells (cybrids) carrying nuclear DNA from MDA-MB-231 cells and mtDNA from a normal subject. The normal mtDNA transfer simultaneously induced restoration of mitochondrial respiratory function and suppression of the highly metastatic potential expressed in MDA-MB-231 cells, but did not suppress ROS overproduction. These observations suggest that mitochondrial respiration defects observed in MDA-MB-231 cells are caused by mutations in mtDNA but not in nuclear DNA, and are responsible for expression of the high metastatic potential without using ROS-mediated pathways. Thus, human tumor cells possess an mtDNA-mediated metastatic pathway that is required for expression of the highly metastatic potential in the absence of ROS production.

S100A4: A novel negative regulator of mineralization and osteoblast differentiation

S100A4 is an intracellular calcium‐binding protein expressed by osteoblastic cells. However, its roles in bone physiology are unknown. Because before matrix mineralization, its expression is markedly diminished, we hypothesized that S100A4 negatively regulates the mineralization process. In this study, we investigated the effects of the inhibition of S100A4 synthesis on osteoblast differentiation and in vitro mineralized nodule formation. Inhibition of S100A4 synthesis was achieved by an antisense approach in the mouse osteoblastic cell line MC3T3‐E1. Cell clones that synthesized low levels of S100A4 (AS clones) produced markedly increased number of mineralized nodules at much earlier stages in comparison with controls as demonstrated by Alizarin red S and von Kossa staining. The expression of type I collagen (COLI) and osteopontin (OPN) increased in AS clones compared with controls. Bone sialoprotein (BSP) and osteocalcin (OCN), molecules associated with mineralization and markers for mature osteoblastic phenotype, were expressed in AS clones before their detection in controls. Because S100A4 was not localized in the nucleus of MC3T3‐E1 cells and AS clones, it is unlikely that S100A4 directly regulates the expression of these genes. Moreover, the expression of Cbfa1/Osf‐2 and Osx, transcription factors necessary for the expression of osteoblast‐associated genes, remained unchanged in AS clones, indicating that S100A4 may be downstream to these transcription factors. These findings indicate that S100A4 is a novel negative regulator of matrix mineralization likely by modulating the process of osteoblast differentiation.

Hypoxic induction of hypoxia-inducible factor-1α and oxygen-regulated gene expression in mitochondrial DNA-depleted HeLa cells

Hypoxia increases the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) and induces transcription of a variety of genes including vascular endothelial growth factor (VEGF) gene through oxygen sensing mechanisms. In one model, mitochondria-derived reactive oxygen species (ROS) are suggested to be involved in oxygen sensing. Here, we found that hypoxia increased ROS generation and VEGF gene expression in HeLa cells. To further investigate the role of ROS in oxygen sensing, we compared the hypoxic response between rho(0) HeLa cells lacking mitochondrial DNA (EB8) and rho(0) HeLa cells containing mitochondrial DNA from wild-type HeLa cells (HeEB1). The results showed that, although hypoxia markedly increased ROS generation in HeEB1 cells but not in EB8 cells, EB8 cells showed essentially a normal response to hypoxia, as assessed by VEGF gene promoter activity, HIF-1alpha accumulation, and HIF-1 target gene expressions. These results indicate that mitochondria-derived ROS generated in response to hypoxia are not necessary for oxygen sensing in HeLa cells.

Constitutive upregulation of hypoxia-inducible factor-1α mRNA occurring in highly metastatic lung carcinoma cells leads to vascular endothelial growth factor overexpression upon hypoxic exposure

Neoangiogenesis is crucial for tumor growth and metastasis and is regulated by various angiogenic factors including vascular endothelial growth factor (VEGF). However, little is known whether highly metastatic cells express higher level of VEGF in response to various stimuli, thereby increasing neoangiogenesis compared to low-metastatic cells. Here we report that hypoxia markedly induced the expression of VEGF mRNA in the cell lines with high-metastatic potential (A11 and D6 cells) compared to the cell lines with low-metastatic potential (P29 and P34 cells) established from Lewis lung carcinoma. A11 cells exhibited higher VEGF gene-promoter activity, produced a larger amount of VEGF and showed higher activity to induce neoangiogenesis than P29 cells. Although the degradation rate of VEGF mRNA under hypoxic conditions was similar in both cell lines, hypoxia-inducible factor-1α (HIF-1α) mRNA, but not HIF-1β mRNA, was found to be constitutively upregulated in A11 cells compared to P29 cells. Accordingly, the level of HIF-1α protein in response to hypoxia was higher in A11 cells than in P29 cells. Upregulation of HIF-1α mRNA was also observed in D6 cells but not in P34 cells. Thus, the high-metastatic cells produced a larger amount of VEGF under hypoxic conditions through constitutive HIF-1α mRNA upregulation compared to the low-metastatic cells, thereby leading to extensive neoangiogenesis.

Specific mitochondrial DNA mutation in mice regulates diabetes and lymphoma development

It has been hypothesized that respiration defects caused by accumulation of pathogenic mitochondrial DNA (mtDNA) mutations and the resultant overproduction of reactive oxygen species (ROS) or lactates are responsible for aging and age-associated disorders, including diabetes and tumor development. However, there is no direct evidence to prove the involvement of mtDNA mutations in these processes, because it is difficult to exclude the possible involvement of nuclear DNA mutations. Our previous studies resolved this issue by using an mtDNA exchange technology and showed that a G13997A mtDNA mutation found in mouse tumor cells induces metastasis via ROS overproduction. Here, using transmitochondrial mice (mito-mice), which we had generated previously by introducing G13997A mtDNA from mouse tumor cells into mouse embryonic stem cells, we provide convincing evidence supporting part of the abovementioned hypothesis by showing that G13997A mtDNA regulates diabetes development, lymphoma formation, and metastasis—but not aging—in this model.