Clonal architecture of human prostatic epithelium in benign and malignant conditions

The location of stem cells in the epithelium of the prostatic acinus remains uncertain, as does the cellular origin of prostatic neoplasia. Here, we apply lineage tracing to visualize the clonal progeny of stem cells in benign and malignant human prostates and understand the clonal architecture of this epithelium. Cells deficient for the mitochondrially-encoded enzyme cytochrome c oxidase (CCO) were identified in 27 frozen prostatectomy specimens using dual colour enzyme histochemistry and individual CCO-normal and -deficient cell areas were laser-capture microdissected. PCR-sequencing of the entire mitochondrial genome (mtDNA) of cells from CCO-deficient areas found to share mtDNA mutations not present in adjacent CCO-normal cells, thus proving a clonal origin. Immunohistochemistry was performed to visualize the three cell lineages normally present in the prostatic epithelium. Entire CCO-deficient acini, and part-deficient acini were found. Deficient patches spanned either basal or luminal cells, but sometimes also both epithelial cell types in normal, hyperplastic or atrophic epithelium, and prostatic intraepithelial neoplasia (PIN). Patches comprising both PIN and invasive cancer were observed. Each cell area within a CCO-deficient patch contained an identical mtDNA mutation, defining the patch as a clonal unit. CCO-deficient patches in benign epithelium contained basal, luminal and endocrine cells, demonstrating multilineage differentiation and therefore the presence of a stem cell. Our results demonstrate that the normal, atrophic, hypertrophic and atypical (PIN) epithelium of human prostate contains stem cell-derived clonal units that actively replenish the epithelium during ageing. These deficient areas usually included the basal compartment indicating the basal layer as the location of the stem cell. Importantly, single clonal units comprised both PIN and invasive cancer, supporting PIN as the pre-invasive lesion for prostate cancer.     

Respiratory chain supercomplexes set the threshold for respiration defects in human mtDNA mutant cybrids

Mitochondrial DNA (mtDNA) mutations cause heterogeneous disorders in humans. MtDNA exists in multiple copies per cell, and mutations need to accumulate beyond a critical threshold to cause disease, because coexisting wild-type mtDNA can complement the genetic defect. A better understanding of the molecular determinants of functional complementation among mtDNA molecules could help us shedding some light on the mechanisms modulating the phenotypic expression of mtDNA mutations in mitochondrial diseases. We studied mtDNA complementation in human cells by fusing two cell lines, one containing a homoplasmic mutation in a subunit of respiratory chain complex IV, COX I, and the other a distinct homoplasmic mutation in a subunit of complex III, cytochrome b. Upon cell fusion, respiration is recovered in hybrids cells, indicating that mitochondria fuse and exchange genetic and protein materials. Mitochondrial functional complementation occurs frequently, but with variable efficiency. We have investigated by native gel electrophoresis the molecular organization of the mitochondrial respiratory chain in complementing hybrid cells. We show that the recovery of mitochondrial respiration correlates with the presence of supramolecular structures (supercomplexes) containing complexes I, III and IV. We suggest that critical amounts of complexes III or IV are required in order for supercomplexes to form and provide mitochondrial functional complementation. From these findings, supercomplex assembly emerges as a necessary step for respiration, and its defect sets the threshold for respiratory impairment in mtDNA mutant cells.     

The human urothelium consists of multiple clonal units, each maintained by a stem cell

Little is known about the clonal architecture of human urothelium. It is likely that urothelial stem cells reside within the basal epithelial layer, yet lineage tracing from a single stem cell as a means to show the presence of a urothelial stem cell has never been performed. Here, we identify clonally related cell areas within human bladder mucosa in order to visualize epithelial fields maintained by a single founder/stem cell. Sixteen frozen cystectomy specimens were serially sectioned. Patches of cells deficient for the mitochondrially encoded enzyme cytochrome c oxidase (CCO) were identified using dual-colour enzyme histochemistry. To show that these patches represent clonal proliferations, small CCO-proficient and -deficient areas were individually laser-capture microdissected and the entire mitochondrial genome (mtDNA) in each area was PCR amplified and sequenced to identify mtDNA mutations. Immunohistochemistry was performed for the different cell layers of the urothelium and adjacent mesenchyme. CCO-deficient patches could be observed in normal urothelium of all cystectomy specimens. The two-dimensional length of these negative patches varied from 2-3 cells (about 30 μm) to 4.7 mm. Each cell area within a CCO-deficient patch contained an identical somatic mtDNA mutation, indicating that the patch was a clonal unit. Patches contained all the mature cell differentiation stages present in the urothelium, suggesting the presence of a stem cell. Our results demonstrate that the normal mucosa of human bladder contains stem cell-derived clonal units that actively replenish the urothelium during ageing. The size of the clonal unit attributable to each stem cell was broadly distributed, suggesting replacement of one stem cell clone by another.     

An intragenic suppressor in the cytochrome c oxidase I gene of mouse mitochondrial DNA

We report here the identification of a cell line containing single and double missense mutations in cytochrome c oxidase (COX) subunit I gene of mouse mitochondrial DNA. When present in homoplasmy, the single mutant displays a normal complex IV assembly but a significantly reduced COX activity, while the double mutant almost completely compensates the functional defect of the first mutation. We discuss the potential structural consequences of those mutations based on the modeled structure of mouse complex IV. Based on genetic, biochemical and molecular analyses of cultured mouse cells we infer that: (i) deleterious mutations can arise and become predominant; (ii) cultured cells can maintain several mtDNA haplotypes at stable frequencies; (iii) the respiratory chain has little spare COX capacity; and (iv) the size of a cavity in the vicinity of Val421 in CO I of animal COX may affect the function of the enzyme.     

Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells

It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser‐capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi‐lineage differentiation within CCO‐deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO‐deficient areas appeared large within areas of ductal carcinoma in situ, suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Analysis of the clonal architecture of the human small intestinal epithelium establishes a common stem cell for all lineages and reveals a mechanism for the fixation and spread of mutations

Little is known about the clonal structure or stem cell architecture of the human small intestinal crypt/villus unit, or how mutations spread and become fixed. Using mitochondrial DNA (mtDNA) mutations as a marker of clonal expansion of stem cell progeny, we aimed to provide answers to these questions. Enzyme histochemistry (for cytochrome c oxidase and succinate dehydrogenase) was performed on frozen sections of normal human duodenum. Laser‐capture microdissected cells were taken from crypts/villi. The entire mitochondrial genome was amplified using a nested PCR protocol; sequencing identified mutations and immunohistochemistry demonstrated specific cell lineages. Cytochrome c oxidase‐deficient small bowel crypts were observed within all sections: negative crypts contained the same clonal mutation and all differentiated epithelial lineages were present, indicating a common stem cell origin. Mixed crypts were also detected, confirming the existence of multiple stem cells. We observed crypts where Paneth cells were positive but the rest of the crypt was deficient. We have demonstrated patches of deficient crypts that shared a common mutation, suggesting that they have divided by fission. We have shown that all cells within a small intestinal crypt are derived from one common stem cell. Partially‐mutated crypts revealed some novel features of Paneth cell biology, suggesting that either they are long‐lived or a committed Paneth cell‐specific long‐lived progenitor was present. We have demonstrated that mutations are fixed in the small bowel by fission and this has important implications for adenoma development. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Mitochondrial DNA background modulates the assembly kinetics of OXPHOS complexes in a cellular model of mitochondrial disease

Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disorder, is mostly due to three mitochondrial DNA (mtDNA) mutations in respiratory chain complex I subunit genes: 3460/ND1, 11778/ND4 and 14484/ND6. Despite considerable clinical evidences, a genetic modifying role of the mtDNA haplogroup background in the clinical expression of LHON remains experimentally unproven. We investigated the effect of mtDNA haplogroups on the assembly of oxidative phosphorylation (OXPHOS) complexes in transmitochondrial hybrids (cybrids) harboring the three common LHON mutations. The steady-state levels of respiratory chain complexes appeared normal in mutant cybrids. However, an accumulation of low molecular weight subcomplexes suggested a complex I assembly/stability defect, which was further demonstrated by reversibly inhibiting mitochondrial protein translation with doxycycline. Our results showed differentially delayed assembly rates of respiratory chain complexes I, III and IV amongst mutants belonging to different mtDNA haplogroups, revealing that specific mtDNA polymorphisms may modify the pathogenic potential of LHON mutations by affecting the overall assembly kinetics of OXPHOS complexes.     

Respiratory chain complex I deficiency caused by mitochondrial DNA mutations

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.     

Measuring stem cell dynamics in the human colon – where there's a wiggle,there's a way

The last decade has seen huge improvements in our understanding of intestinal stem cell biology, with major advances arising from the ability to transgenically label, and thus identify, murine stem cells and their progeny. In the human, transgenic labelling is not an available option and stem cell dynamic observations have been based on rare hereditary mutations and polymorphisms. Somatic mitochondrial DNA mutations cause a histochemically detectable, but neutrally selected, change in cytochrome c oxidase (CCO) enzyme activity and when this occurs in an intestinal stem cell, it can be used as an effective clonal marker in both health and disease. The intestinal crypt is the functional unit of the gut. Daughter cells are ‘born’ in the stem cell niche at the crypt base and proliferate, differentiate, and then apoptose as they migrate along the vertical crypt axis over 5–7 days. This stereotypical architecture provides a historical record of cell dynamics, as the distance travelled along the crypt axis is proportional to the time since the daughter cell was born. By staining, identifying, and carefully reconstructing crypt maps from serial en face sections of partially mutated mtDNA crypts, clonal ribbon images can be generated. ‘Wiggles’ in the width of the clonal ribbon reflect mtDNA mutated stem cell expansion or contraction events and these biological observations are applied in mathematical models. This clever approach is able to infer temporal evolutionary dynamics from a static, single time point measurement, in both normal and familial adenomatous polyposis tissue. As we have seen in the mouse, the simple ability to identify stem cell progeny can lead to a vast expansion in our understanding of stem cell evolution. The use of these techniques to trace recent stem cell dynamics in the human colon makes some headway into the knowledge gap in our understanding of murine and human intestinal stem cell biology. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Dimensional study of prostate cancer using stereological tools

This study analyzes the dimensional changes of the glands from prostate cancer by applying stereology to estimate the variations in volume, length, surface, and cellular densities of tumor acini. Normal and tumor acini were visualized using immunohistochemistry for cytokeratin18. On immunostained sections, parameters related to the dimensions and cell population of prostate acini were measured. The immunohistochemical expression of proliferative cell nuclear antigen was also measured to correlate the quantitative changes estimated with the proliferative activity of the epithelium. The average cell volume in normal and tumor epithelium was estimated using the method of the nucleator. The relative size of the acini was similar in the carcinoma compared with the normal prostate. Within the acini, the fraction of acinar volume occupied by the epithelium was significantly higher in cancer than in the nontumor prostate. Conversely, the glandular lumen of the cancer acini is lower than in the normal acini. The significant increase of acinar length density in the carcinoma indicates that the glandular tree's growth in the carcinoma is higher and with more branches than in the case of nonneoplastic glands. The basal surface density is higher in the carcinoma than in the controls. The number of epithelial cells per unit length of acini was significantly decreased in the neoplastic glands. This "dilution" of the cell population along the cancer acinus can be explained by the significant increase in the tumor cell's mean cell volume.     

Light-microscopic stereology for the study of the ventral and dorsal prostates of the syrian hamster: Strategy,techniques, and applications

A stereological approach to studying the architecture of the ventral and dorsal prostates of the Syrian hamster is described. In this approach, the prostate is considered to consist of acinar and interacinar compartments. The acinar compartment is divided into luminal, epithelial, lamina proprial, and muscular stromal compartments. Volume fractions of acini and interacinar tissue are calculated with reference to the volume of the gland; those of the acinar components are calculated with reference to the acinar volume. Volume fractions are determined from point counts. The surface fraction of the secretory epithelium is determined from intercept counts, with reference to the volume of the epithelium. By assuming that the acini are cylindrical, the acinar length fraction is calculated with reference to the volume of the gland. Absolute values for the volumes of different components, epithelial surface area, and acinar length can be determined from the weight of the gland, its specific gravity, and the volume fraction of that component, or epithelial surface fraction, or acinar length fraction, respectively. Finally, from these values and assuming a cylindrical shape for acini, characteristics of the typical acinus, including its radius, the thicknesses of its epithelium, lamina propria, and muscular stroma, and the amount of mucosal folding can be calculated from simple geometrical formulae. This approach is illustrated from a consideration of the ventral and dorsal prostates of 20-week-old Syrian hamsters.     

mtDNA lineage analysis of mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and intermolecular recombination

The role of mitochondrial DNA (mtDNA) mutations and mtDNA recombination in cancer cell proliferation and developmental biology remains controversial. While analyzing the mtDNAs of several mouse L cell lines, we discovered that every cell line harbored multiple mtDNA mutants. These included four missense mutations, two frameshift mutations, and one tRNA homopolymer expansion. The LA9 cell lines lacked wild-type mtDNAs but harbored a heteroplasmic mixture of mtDNAs, each with a different combination of these variants. We isolated each of the mtDNAs in a separate cybrid cell line. This permitted determination of the linkage phase of each mtDNA and its physiological characteristics. All of the polypeptide mutations inhibited their oxidative phosphorylation (OXPHOS) complexes. However, they also increased mitochondrial reactive oxygen species (ROS) production, and the level of ROS production was proportional to the cellular proliferation rate. By comparing the mtDNA haplotypes of the different cell lines, we were able to reconstruct the mtDNA mutational history of the L-L929 cell line. This revealed that every heteroplasmic L-cell line harbored a mtDNA that had been generated by intracellular mtDNA homologous recombination. Therefore, deleterious mtDNA mutations that increase ROS production can provide a proliferative advantage to cancer or stem cells, and optimal combinations of mutant loci can be generated through recombination.     

Novel mitochondrial DNA mutations responsible for maternally inherited nonsyndromic hearing loss

Some cases of maternally inherited isolated deafness are caused by mtDNA mutations, frequently following an exposure to aminoglycosides. Two mitochondrial genes have been clearly described as being affected by mutations responsible for this pathology: the ribosomal RNA 12S gene and the transfer RNA serine (UCN) gene. A previous study identified several candidate novel mtDNA mutations, localized in a variety of mitochondrial genes, found in patients with no previous treatment with aminoglycosides. Five of these candidate mutations are characterized in the present study. These mutations are localized in subunit ND1 of complex I of the respiratory chain (m.3388C>A [p.MT-ND1:Leu28Met]), the tRNA for Isoleucine (m.4295A>G), subunit COII of complex IV (m.8078G>A [p.MT-CO2:Val165Ile]), the tRNA of Serine 2 (AGU/C) (m.12236G>A), and Cytochrome B, subunit of complex III (m.15077G>A [p.MT-CYB:Glu111Lys]). Cybrid cell lines have been constructed for each of the studied mtDNA mutations and functional studies have been performed to assess the possible consequences of these mutations on mitochondrial bioenergetics. This study shows that a variety of mitochondrial genes, including protein-coding genes, can be responsible for nonsyndromic deafness, and that exposure to aminoglycosides is not required to develop the disease, giving new insights on the molecular bases of this pathology.     

Nuclear genetic control of mitochondrial translation in skeletal muscle revealed in patients with mitochondrial myopathy

Oxidative phosphorylation deficiencies can be caused by mutations in either the nuclear genome or the mitochondrial genome (mtDNA); however, most pathogenic mutations reported in adults occur in mtDNA. Such mutations often impair mitochondrial translation, and are associated with a characteristic muscle pathology consisting of a mosaic pattern of normal fibres interspersed with fibres that show mitochondrial proliferation (ragged-red fibres) and little or no complex IV (COX) activity. We investigated two adult patients with a severe mitochondrial myopathy in whom all muscle fibres showed mitochondrial proliferation with barely detectable COX activity - a pattern never before reported. Biochemical studies of the respiratory chain in muscle showed decreased activities of complexes I and IV (5% of control) and complex II+III (41% of control). Immunoblot analysis of nuclear and mitochondrial subunits of complexes I, III and IV showed a greater than 90% decrease in the steady-state level of these subunits in mature muscle, but no change in nuclear-encoded subunits of complexes II and V. A generalized mitochondrial translation defect was identified in pulse-label experiments in myotubes, but not in myoblasts cultured from both patients. This defect moved with the nucleus in patient cybrid cells. Myoblasts from one patient transplanted into the muscle bed of SCID mice differentiated into mature human muscle fibres that displayed a defect similar to that seen in the patient muscle. These results suggest a defect in a developmentally regulated nuclear factor important for mitochondrial translation in skeletal muscle.     

Maternally inherited mitochondrial DNA disease in consanguineous families

Mitochondrial respiratory chain disease represents one of the most common inborn errors of metabolism and is genetically heterogeneous, with biochemical defects arising from mutations in the mitochondrial genome (mtDNA) or the nuclear genome. As such, inheritance of mitochondrial respiratory chain disease can either follow dominant or recessive autosomal (Mendelian) inheritance patterns, the strictly matrilineal inheritance observed with mtDNA point mutations or X-linked inheritance. Parental consanguinity in respiratory chain disease is often assumed to infer an autosomal recessive inheritance pattern, and the analysis of mtDNA may be overlooked in the pursuit of a presumed nuclear genetic defect. We report the histochemical, biochemical and molecular genetic investigations of two patients with suspected mitochondrial disease who, despite being born to consanguineous first-cousin parents, were found to harbour well-characterised pathogenic mtDNA mutations, both of which were maternally transmitted. Our findings highlight that any diagnostic algorithm for the investigation of mitochondrial respiratory chain disease must include a full and complete analysis of the entire coding sequence of the mitochondrial genome in a clinically relevant tissue. An autosomal basis for respiratory chain disease should not be assumed in consanguineous families and that 'maternally inherited consanguineous' mitochondrial disease may thus be going undiagnosed.     

Respiratory chain deficiency precedes the disrupted calcium homeostasis in chronic doxorubicin cardiomyopathy

BackgroundDoxorubicin causes a chronic cardiomyopathy in which genetic and functional lesions of mitochondria accumulate in the long-term. A disrupted Ca²⁺ homeostasis is also implicated in doxorubicin cardiotoxicity. We investigated if the alterations in myocellular Ca²⁺ are primary or secondary to the respiratory chain dysfunction in chronic doxorubicin cardiomyopathy.Methods and ResultsA “long-observation group” of rats was treated with intravenous doxorubicin (1 mg/kg per week) for 7 weeks, starting at 11 weeks of age. Controls received equivalents of saline. A “short-observation group” received seven injections of doxorubicin, starting at 41 weeks of age. All rats were euthanized at 48 weeks of age. Only the long-observation rats developed a significant clinical, macroscopic, histological, and ultrastructural cardiomyopathy. Their intramyocardial cytochrome c oxidase (COX) activity was lowest; they had the highest loss of mitochondrial DNA (mtDNA) and its encoded respiratory chain subunit COX I, and the highest amount of ultrastructural and intracellular calcium accumulation resembling hydroxyapatite. The short-term-group hearts had fewer alterations of the cardiomyopathy score, COX-activity, and mtDNA-content than the long-observation group. Despite a measurable loss of mtDNA and its encoded respiratory chain activity, however, there was virtually no detectable calcium accumulation in the hearts of the short-term group.ConclusionsmtDNA depletion and secondary respiratory chain dysfunction precedes the precipitation of mitochondrial calcium deposits in chronic doxorubicin cardiotoxicity.