Mitochondria and cancer: past, present, and future.

The area of mitochondrial genomics has undergone unprecedented growth over the past several years. With the advent of the age of omics, investigations have reached beyond the nucleus to encompass the close biological communication and finely coordinated interactions between mitochondria and their nuclear cell mate. Application of this holistic approach, to all metabolic interactions within the cell, is providing a more complete understanding of the molecular transformation of the cell from normal to malignant behavior, before histopathological indications are evident. In this review the surging momentum in mitochondrial science, as it relates to cancer, is described in three progressive perspectives: (1) Past: the historical contributions to current directions of research; (2) Present: Contemporary findings, results and approaches to mitochondria and cancer, including the role of next generation sequencing and proteomics; (3) FUTURE: Based on the present body of knowledge, the potential assets and benefits of mitochondrial research are projected into the near future.

Improved methods and standards for telomerase detection: quantitative histopathology using antibody staining.

Evaluation of telomerase as an early detection biomarker for cancer has been hindered by a lack of reliable methods and standards for in situ histochemical measurement. Improved histochemical methods for measuring telomerase could expedite the acceptance of telomerase as a biomarker for use in diagnostic and clinical applications. The lack of a crystal structure for telomerase coupled with high variability in the antibodies available for immunohistochemical analysis has led to confusion in the literature regarding the binding specificity of these antibodies. We have developed an automated fluorescence microscopy protocol to assess the specificity of three fluorescently labeled telomerase antibodies and to quantify telomerase in cultured human tumor cells and in human fibroblast cells as a control. Significant differences in staining intensity and distribution were observed. Fluorescence measurements in these cell lines were compared to telomerase measured by the telomerase repeat amplification protocol, reverse transcription-polymerase chain reaction, and flow cytometry. This combination of measurements ensured a more complete quantitation of telomerase levels in each of the cell lines and could also be used as a model for validation of other biomarkers for clinical use.

Forensic application of the Affymetrix human mitochondrial resequencing array.

In the field of forensic DNA testing, sequencing regions of the mitochondrial genome is performed when insufficient genomic DNA is present for traditional autosomal short tandem repeat (STR) testing. Sequencing coding region polymorphisms in the mitochondrial genome can be useful for resolving individuals who have the identical HV1 and HV2 control region sequence. Various methods and strategies have been established to interrogate coding region polymorphisms. These range from SNP assays probing sites most likely to differentiate individuals based on their HV1/HV2 sequence to the use of mass spectrometry to pyrosequencing. Here we evaluate the potential of the Affymetrix GeneChip Mitochondrial Resequencing Array (version 2.0) for forensic applications.

Standardization of PCR amplification for fragile X trinucleotide repeat measurements.

To provide the clinical diagnostics community with accurate protocols and measurements for the detection of genetic disorders, we have established a quantitative measurement program for trinucleotide repeats associated with human disease. In this study, we have focused on the triplet repeat associated with fragile X syndrome. Five cell lines obtained from the Coriell Cell Repository were analyzed after polymerase chain reaction (PCR) amplification and size separation. These cell lines were reported to contain CGG repeat elements (ranging from 29 to 110 repeats). Our initial measurements focused on measurement variability: (a) between slab-PAGE and capillary (CE) separation systems (b) interlane variability (slab-PAGE) (c) intergel variability, and (d) variability associated with amplification. Samples were run in triplicate for all measurements, and the analysis performed using Gene Scan analysis software. The repeat sizes were verified by DNA sequence analyzes. The standard deviations for interlane measurements in slab-gels ranged from 0.05 to 0.35. There was also little variation in size measurements performed on different gels and among PCR amplifications. The CGG repeat measurements performed by capillary electrophoresis were more precise, with standard deviations ranging from 0.02 to 0.29. The slab-PAGE and CE size measurements were in agreement except for the pre-mutation alleles, which yielded significantly smaller sizes by CE.

Gene conversion (recombination) mediates expansions of CTG[middle dot]CAG repeats.

Genetic recombination is a robust mechanism for expanding CTG.CAG triplet repeats involved in the etiology of hereditary neurological diseases (Jakupciak, J. P., and Wells, R. D. (1999) J. Biol. Chem. 274, 23468-23479). This two-plasmid recombination system in Escherichia coli with derivatives of pUC19 and pACYC184 was used to investigate the effect of triplet repeat orientation on recombination and extent of expansions; tracts of 36, 50, 80, and 36, 100, and 175 repeats in length, respectively, in all possible permutations of length and in both orientations (relative to the unidirectional replication origins) revealed little or no effect of orientation of expansions. The extent of expansions was generally severalfold the length of the progenitor tract and frequently exceeded the combined length of the two tracts in the cotransformed plasmids. Expansions were much more frequent than deletions. Repeat tracts bearing two G-to-A interruptions (polymorphisms) within either 171- or 219-base pair tracts substantially reduced the expansions compared with uninterrupted repeat tracts of similar lengths. Gene conversion, rather than crossing over, was the recombination mechanism. Prior studies showed that DNA replication, repair, and tandem duplication also mediated genetic instabilities of the triplet repeat sequence. However, gene conversion (recombinational repair) is by far the most powerful expansion mechanism. Thus, we propose that gene conversion is the likely expansion mechanism for myotonic dystrophy, spinocerebellar ataxia type 8, and fragile X syndrome.

Genetic instabilities of triplet repeat sequences by recombination.

The expansion of triplet repeat sequences is an initial step in the disease etiology of a number of hereditary neurological disorders in humans. Diseases such as myotonic dystrophy, Huntington's, several spinocerebellar ataxias, fragile X syndrome, and Friedreich's ataxia are caused by the expansions of CTG.CAG, CGG.CCG, or GAA.TTC repeats. The mechanisms of the expansion process have been investigated intensely in E. coli, yeast, transgenic mice, mammalian cell culture, and in human clinical cases. Whereas studies from 1994-1999 have implicated DNA replication and repair at the paused synthesis sites due to the unusual conformations of the triplet repeat sequences, recent work has shown that homologous recombination (gene conversion) is a powerful mechanism for generating massive expansions, in addition to, or in concert with, replication and repair.

Genetic instabilities in (CTG.CAG) repeats occur by recombination.

The expansion of triplet repeat sequences (TRS) associated with hereditary neurological diseases is believed from prior studies to be due to DNA replication. This report demonstrates that the expansion of (CTG.CAG)(n) in vivo also occurs by homologous recombination as shown by biochemical and genetic studies. A two-plasmid recombination system was established in Escherichia coli with derivatives of pUC19 (harboring the ampicillin resistance gene) and pACYC184 (harboring the tetracycline resistance gene). The derivatives contained various triplet repeat inserts ((CTG.CAG), (CGG.CCG), (GAA.TTC), (GTC.GAC), and (GTG.CAC)) of different lengths, orientations, and extents of interruptions and a control non-repetitive sequence. The availability of the two drug resistance genes and of several unique restriction sites on the plasmids enabled rigorous genetic and biochemical analyses. The requirements for recombination at the TRS include repeat lengths >30, the presence of CTG.CAG on both plasmids, and recA and recBC. Sequence analyses on a number of DNA products isolated from individual colonies directly demonstrated the crossing-over and expansion of the homologous CTG.CAG regions. Furthermore, inversion products of the type [(CTG)(13)(CAG)(67)].[(CTG)(67)(CAG)(13)] were isolated as the apparent result of "illegitimate" recombination events on intrahelical pseudoknots. This work establishes the relationships between CTG.CAG sequences, multiple fold expansions, genetic recombination, formation of new recombinant DNA products, and the presence of both drug resistance genes. Thus, if these reactions occur in humans, unequal crossing-over or gene conversion may also contribute to the expansions responsible for anticipation associated with several hereditary neurological syndromes.