Ewa Bartnik

Mitochondrial DNA mutations in human neoplasia

Many models of tumour formation have been put forth so far. In general they involve mutations in at least three elements within the cell: oncogenes, tumour suppressors and regulators of telomere replication. Recently numerous mutations in mitochondria have been found in many tumours, whereas they were absent in normal tissues from the same individual. The presence of mutations, of course, does not prove that they play a causative role in development of neoplastic lesions and progression; however, the key role played by mitochondria in both apoptosis and generation of DNA-damaging reactive oxygen species might indicate that the observed mutations contribute to tumour development. Recent experiments with nude mice have proven that mtDNA mutations are indeed responsible for tumour growth and exacerbated ROS production. This review describes mtDNA mutations in main types of human neoplasia.

Human mitochondrial RNA turnover caught in flagranti: involvement of hSuv3p helicase in RNA surveillance

The mechanism of human mitochondrial RNA turnover and surveillance is still a matter of debate. We have obtained a cellular model for studying the role of hSuv3p helicase in human mitochondria. Expression of a dominant-negative mutant of the hSUV3 gene which encodes a protein with no ATPase or helicase activity results in perturbations of mtRNA metabolism and enables to study the processing and degradation intermediates which otherwise are difficult to detect because of their short half-lives. The hSuv3p activity was found to be necessary in the regulation of stability of mature, properly formed mRNAs and for removal of the noncoding processing intermediates transcribed from both H and L-strands, including mirror RNAs which represent antisense RNAs transcribed from the opposite DNA strand. Lack of hSuv3p function also resulted in accumulation of aberrant RNA species, molecules with extended poly(A) tails and degradation intermediates truncated predominantly at their 3′-ends. Moreover, we present data indicating that hSuv3p co-purifies with PNPase; this may suggest participation of both proteins in mtRNA metabolism.

Resistance to tyrosine kinase inhibitors in clear cell renal cell carcinoma: From the patient's bed to molecular mechanisms ☆

The introduction of anti-angiogenic drugs especially tyrosine kinase inhibitors (TKIs) was a breakthrough in the treatment of renal cell carcinoma (RCC). Although TKIs have significantly improved outcome in patients with metastatic disease, the majority still develop resistance over time. Because different combinations and sequences of TKIs are tested in clinical trials, resistance patterns and mechanisms underlying this phenomenon should be thoroughly investigated. From a clinical point of view, resistance occurs either as a primary phenomenon (intrinsic) or as a secondary phenomenon related to various escape/evasive mechanisms that the tumor develops in response to vascular endothelial growth factor (VEGF) inhibition. Intrinsic resistance is less common, and related to the primary redundancy of available angiogenic signals from the tumor, causing unresponsiveness to VEGF-targeted therapies. Acquired resistance in tumors is associated with activation of an angiogenic switch which leads to either upregulation of the existing VEGF pathway or recruitment of alternative factors responsible for tumor revascularization. Multiple mechanisms can be involved in different tumor settings that contribute both to evasive and intrinsic resistance, and current endeavor aims to identify these processes and assess their importance in clinical settings and design of pharmacological strategies that lead to enduring anti-angiogenic therapies.

Organization of the ribosomal RNA gene cluster in Aspergillus nidulans

DNA coding for ribosomal RNA in Aspergillus nidulans was found to consist of a unit 7.8 kb in size which is tandemly repeated in the genome and codes for 5.8S, 18S and 26S rRNA. The repeat unit has been cloned, and its restriction map and the location of the individual rRNA coding sequences within the unit have been established.

The S. cerevisiae nuclear gene SUV3 encoding a putative RNA helicase is necessary for the stability of mitochondrial transcripts containing multiple introns.

The product of the nuclear gene SUV3 is implicated in a variety of post-transcriptional processes in yeast mitochondria. We have analysed the effect of SUV3 gene-disruption on the expression of intron-containing alleles of the mitochondrial cytb and coxl genes. We have constructed several strains with mitochondrial genomes containing different combinations of cytb and cox1 introns, and associated these genomes with the disruption of SUV3. The resulting strains were tested for their respiratory competence and spectral cytochrome content. All the strains containing only two or three introns showed normal expression of cytb and cox1, whereas the strains containing more introns were unable to express the appropriate gene. The analysis of mitochondrial RNAs by Northern hybridisation showed that the loss of respiratory competence in the strains containing more introns is due to the decrease of mRNA level with no over-accumulation of high-molecular-weight precursors. However, the transcription of the genes was not affected. These results led us to the notion that SUV3 is required for the stability of intron-containing cytb and cox1 transcripts in a cumulative way, not dependent on any particular intron.

Homoplasmic MELAS A3243G mtDNA mutation in a colon cancer sample

We have analyzed mtDNA variation in various cancer samples, comparing them with normal tissue controls, and identified mutations and polymorphisms, both known and novel, in mitochondrial tRNA, rRNA and protein genes. Most remarkably, in a colon cancer sample we have found the A3243G mutation in the homoplasmic state. This mutation is known to cause severe mitochondrial dysfunction and, until now, has not been found in cancer cells, nor in the homoplasmic state in living subjects. The mutation was absent from normal tissue, suggesting that mtDNA mutation and resulting respiratory deficiency played a role in carcinogenesis.

The suv3 nuclear gene product is required for the in vivo processing of the yeast mitochondrial 21s rRNA transcripts containing the r1 intron

We have constructed a yeast mitochondrial genome containing only one group-I intron, r1, from the 21s rRNA gene and introduced this genome into a strain bearring a disruption of the suv3 gene. The presence of the r1 intron alone causes a block in respiration, while the isogenic strain containing the intronless genome is respiratory competent. Northern analysis indicates that the functional suv3 protein is necessary for the yeast cell in order to process the r1-containing transcripts: in the absence of the suv3 protein the hybridization pattern of the excised r1 intron is altered and the amount of mature 21s rRNA is 50-fold lower. We suggest that the multifunctional suv3 protein, which displays motifs of ATP-dependent RNA helicases, is necessary for the in vivo pathway leading to formation of mature 21s rRNA from the transcripts containing the r1 intron in mitochondria of Saccharomyces cerevisiae.

The Role of the Mitochondrial Genome in Ageing and Carcinogenesis

Mitochondrial DNA mutations and polymorphisms have been the focus of intensive investigations for well over a decade in an attempt to understand how they affect fundamental processes such as cancer and aging. Initial interest in mutations occurring in mitochondrial DNA of cancer cells diminished when most were found to be the same mutations which occurred during the evolution of human mitochondrial haplogroups. However, increasingly correlations are being found between various mitochondrial haplogroups and susceptibility to cancer or diseases in some cases and successful aging in others.

Current approaches in identification and isolation of human renal cell carcinoma cancer stem cells

In recent years, cancer stem cells (CSCs)/tumor initiating cells (TICs) have been identified inside different tumors. However, currently used anti-cancer therapies are mostly directed against somatic tumor cells without targeting CSCs/TICs. CSCs/TICs also gain resistance to chemotherapies/radiotherapies. For the development of efficient treatment strategies, choosing the best method for isolation and characterization of CSCs/TICs is still debated among the scientific community. In this review, we summarize recent data concerning isolation techniques for CSCs using magnetic cell sorting and flow cytometry. The review focuses on the strategies for sample preparation during flow cytometric analysis, elaborating biomarkers such as CXCR4, CD105, and CD133. In addition, functional properties characteristic of CSCs/TICs using side population selection through Hoechst 33342 dye, aldehyde dehydrogenase 1, dye-cycle violet, and rhodamine 123 are also discussed. We also include a special focus on enriching CSCs/TICs using three-dimensional cell culture models such as agarose–agarose microbeads and sphere formation.

Genotype-phenotype correlations in Leber hereditary optic neuropathy.

Leber hereditary optic neuropathy (LHON), acute or subacute vision loss due to retinal ganglion cell death which in the long run leads to optic nerve atrophy is one of the most widely studied maternally inherited diseases caused by mutations in mitochondrial DNA. Although three common mutations, 11778G>A, 14484T>C or 3460G>A are responsible for over 90% of cases and affect genes encoding complex I subunits of the respiratory chain, their influence on bioenergetic properties of the cell is marginal and cannot fully explain the pathology of the disease. The following chain of events was proposed, based on biochemical and anatomical properties of retinal ganglion cells whose axons form the optic nerve: mitochondrial DNA mutations increase reactive oxygen species production in these sensitive cells, leading to caspase-independent apoptosis. As LHON is characterized by low penetrance and sex bias (men are affected about 5 times more frequently than women) the participation of the other factors-genetic and environmental-beside mtDNA mutations was studied. Mitochondrial haplogroups and smoking are some of the factors involved in the complex etiology of this disease.