Transmitochondrial Cybrid Generation Using Cancer Cell Lines

Soler-Agesta, Ruth; Marco-Brualla, Joaquín; Fernández‐Silva, Patricio; Mozas, Pilar; Anel, Alberto; Loshuertos, Raquel Moreno

doi:10.3791/65186

Cited by 3 publications

(1 citation statement)

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“…Lymphoblastoid cell lines were immortalized by transformation with Epstein–Barr virus (EBV), as described elsewhere [ 20 , 21 ]. Cell lines were derived from six affected matrilineal relatives (Pedigree 1: II-1, II-4 and III-6; Pedigree 2: II-4, III-6 and IV-1) and four controls (Pedigree 1: III-1 and III-2; Pedigree 2: III-1 and III-2).…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Diabetes Is Associated with the ND4 G11696A Mutation

et al. 2023

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Type 2 diabetes mellitus (T2DM) is a common endocrine disorder which remains a large challenge for clinicians. Previous studies have suggested that mitochondrial dysfunction plays an active role in T2DM progression, but a detailed mechanism is still elusive. In the current study, two Han Chinese families with maternally inherited T2DM were evaluated using clinical, genetic, molecular, and biochemical analyses. The mitochondrial genomes were PCR amplified and sequenced. Phylogenetic and bioinformatic analyses were used to assess the potential pathogenicity of mitochondrial DNA (mtDNA) mutations. Interestingly, the matrilineal relatives of these pedigrees exhibited variable severity of T2DM, in particular, the age at onset of T2DM varied from 26 to 65 years, with an average of 49 years. Sequence analysis revealed the presence of ND4 G11696A mutation, which resulted in the substitution of an isoleucine for valine at amino acid (AA) position 312. Indeed, this mutation was present in homoplasmy only in the maternal lineage, not in other members of these families, as well as 200 controls. Furthermore, the m.C5601T in the tRNAAla and novel m.T5813C in the tRNACys, showing high evolutional conservation, may contribute to the phenotypic expression of ND4 G11696A mutation. In addition, biochemical analysis revealed that cells with ND4 G11696A mutation exhibited higher levels of reactive oxygen species (ROS) productions than the controls. In contrast, the levels of mitochondrial membrane potential (MMP), ATP, mtDNA copy number (mtDNA-CN), Complex I activity, and NAD+/NADH ratio significantly decreased in cell lines carrying the m.G11696A and tRNA mutations, suggesting that these mutations affected the respiratory chain function and led to mitochondrial dysfunction that was involved in T2DM. Thus, our study broadened the clinical phenotypes of m.G11696A mutation.

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Section: Methodsmentioning

confidence: 99%

Mitochondrial Diabetes Is Associated with the ND4 G11696A Mutation

et al. 2023

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Generation of transmitochondrial cybrids in cancer cells

Soler-Agesta,

Ripollés-Yuba,

Marco-Brualla

et al. 2024

Methods in Cell Biology

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Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis

Shelton,

House,

Ramesh

et al. 2023

Preprint

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Mitochondrial DNA (mtDNA) mutations are frequently observed in cancer, but their contribution to tumor progression is controversial. To evaluate the impact of mtDNA variants on tumor growth and metastasis, we created human melanoma cytoplasmic hybrid (cybrid) cell lines transplanted with wildtype mtDNA or pathogenic mtDNA encoding variants that partially or completely inhibit oxidative phosphorylation. Homoplasmic pathogenic mtDNA cybrids reliably established tumors despite dysfunctional oxidative phosphorylation. However, pathogenic mtDNA variants disrupted spontaneous metastasis of subcutaneous tumors and decreased the abundance of circulating melanoma cells in the blood. Pathogenic mtDNA did not induce anoikis or inhibit organ colonization of melanoma cells following intravenous injections. Instead, migration and invasion were reduced, indicating that limited circulation entry functions as a metastatic bottleneck amidst mtDNA dysfunction. Furthermore, analysis of selective pressure exerted on the mitochondrial genomes of heteroplasmic cybrid lines revealed a suppression of pathogenic mtDNA allelic frequency during melanoma growth. Collectively, these findings demonstrate that functional mtDNA is favored during melanoma growth and enables metastatic entry into the blood.

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Transmitochondrial Cybrid Generation Using Cancer Cell Lines

Cited by 3 publications

References 0 publications

Mitochondrial Diabetes Is Associated with the ND4 G11696A Mutation

Mitochondrial Diabetes Is Associated with the ND4 G11696A Mutation

Generation of transmitochondrial cybrids in cancer cells

Pathogenic mitochondrial DNA mutations inhibit melanoma metastasis

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