Defective intramitochondrial NADH oxidation in skin fibroblasts from an infant with fatal neonatal lacticacidemia.

A small-for-gestational-age female infant born at term developed severe lactic acidosis and died on day 13 of life. Two previous sibs had also died of overwhelming lactic acidosis in the neonatal period. The lactate-to-pyruvate and 3-hydroxybutyrate-to-acetoacetate ratios were elevated at 136 and 42 to one, respectively. The activities of the pyruvate dehydrogenase complex and pyruvate carboxylase in cultured skin fibroblasts were normal but a defect in respiration was indicated by the low rates of conversion of 1-[14C]pyruvate, glutamate, and lactate to 14CO2 in these cells. Skin fibroblast cultures also displayed an elevated lactate-to-pyruvate ratio (72:1) when incubated with glucose as substrate compared to control cell cultures (20:1). When mitochondrial preparations of skin fibroblasts (prepared by digitonin extraction) were tested for their ability to synthesize ATP from a variety of substrates, it was found that those of the patient made adequate amounts of ATP with either succinate or ascorbate/tetramethyl-phenylenediamine as substrate but not with the NAD-linked substrates pyruvate, isocitrate, and palmitoyl carnitine. We propose that this is indicative of a defect in the respiratory chain between NADH and coenzyme Q, for the first time demonstrable in cultured skin fibroblasts.     

Respiratory chain analysis of skin fibroblasts in mitochondrial disease

NADH:ubiquinone dehydrogenase (complex I) deficiency can be diagnosed from cultured skin fibroblasts using a number of methods, the most commonly used is a linked assay of rotenone-sensitive complex I + III activity (NADH:cytochrome c reductase). Because of interference from diaphorases, this method requires either the isolation of mitochondria (or at least partial purification). For a suitable mitochondrial preparation from skin fibroblasts, this requires the culturing of 4-20 individual 100mm tissue culture plates, depending on the purity of preparation required. These assays are therefore time-consuming, and do not assist in a rapid diagnosis. There is also no clear demarkation between the normal range of activity and the deficient range since mild mutations can produce only partial decreases in complex I activity. Equally, assaying patient cells that do not have a specific deficiency may prove to be time-wasting in the process of providing a quick, definitive clinical diagnosis. The lactate/pyruvate ratio of fibroblasts has been used to indicate the extent of respiratory chain involvement, as cells with a metabolic defect usually produce more lactate with an increased ratio from 25:1 to much higher values [Methods Enzymol. 264 (1996) 454]. This measurement may not always be conclusive, as the values can fluctuate as a result of culture medium, cell passage number, cell number and viability. In this report, we evaluate the use of pyruvate oxidation measurements from whole cells prepared from a single plate of cultured fibroblasts as an alternative to lactate/pyruvate ratios, or other methods both direct and indirect as indicators of the extent of respiratory chain involvement and the possibility of a defect within complex I. Whole cell 2-14C pyruvate oxidation appears to indicate the presence of a complex I defect in patients compared to normal controls more reliably than L/P ratios, but this has some puzzling exceptions.     

Menadione partially restores NADH-oxidation and ATP-synthesis in complex I deficient fibroblasts

In this paper we report our studies on the effects of menadione in cultured fibroblasts treated with rotenone to block complex I. A normalization of the lactate to pyruvate ratio after incubation with glucose, an increased production of 14CO2 from [6-14C]glucose and an increased intra-cellular concentration of ATP was observed in the presence of micromolar concentrations of menadione. These results not only demonstrate the potential value of menadione in complex I deficient patients but also suggest that this system can be used advantageously for the in vitro assessment of therapeutic agents for disorders of the mitochondrial respiratory chain.     

Cell culture studies on patients with mitochondrial diseases: Molecular defects in pyruvate dehydrogenase

There is a group of inborn errors of metabolism that result in the condition of chronic lacticacidemia of childhood. Nearly all of the defects that can be identified occur in mitochondrial proteins, and can be demonstrated in cultured skin fibroblasts from the patients concerned. One approach to the diagnosis of these defects involves a simple incubation of the fibroblast culture with glucose-containing medium followed by the measurement of accumulated lactate and pyruvate. The total amounts of lactate and pyruvate and the ratio between them is different in cells from patients with defects in the pyruvate dehydrogenase complex or the respiratory chain. Measurement of 1-¹⁴C-pyruvate oxidation to¹⁴CO₂ can also reveal defective oxidative metabolism. Localization of the defects can be achieved using individual assays for the enzymes concerned. The clinical sequelae of the different defects is discussed.     

Citrinin affects the oxidative metabolism of BHK-21 cells

The effects of citrinin on energy production along the respiratory chain and on glycolytic lactate production were examined in BHK-21 cultured cells. Citrinin inhibited the oxygen consumption rate by about 45 per cent. The respiratory rate of digitonin-treated cells energized with succinate, in the presence of ADP, was reduced by about 39 per cent. The mycotoxin inhibited the glucose utilization of BHK-21 cells by about 86 per cent. Cells treated with citrinin produced a small quantity of pyruvate, but were unable to produce lactate. It is concluded that BHK-21 cells cannot generate lactate when oxidative metabolism is inhibited by citrinin. The perturbations in BHK-21 cells caused by citrinin are due to alterations in mitochondrial function and in the glycolytic anaerobic pathway.     

Glucose transport and metabolism in cultured human skin fibroblasts

Human skin fibroblast cultures, seeded at $\text{10}{}^5\text{cells}\text{5}\text{cm}$ plate and allowed to grow to confluence at approx. $\text{10}{}^6\text{cells}\text{5}\text{cm}$ plate, utilized a glycolytic mode of metabolism where the ratio of glucose utilized to lactate produced wa 0.62±0.05 (Zielke, R.H., Ozand, P.T., Tyldon, J.I., Sevdalian, D.A. and Cornblath, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4110–4114) (mean±S.E.). When the glucose in the medium was exhausted, the lactate produced during the highly glycolytic phase was then reutilized. In monolayer cultures that had been washed with phosphate-buffered saline, rates of glucose utilization were measured at 0.25 and 2 mM glucose by monitoring the appearance of ³H₂O from [5-³H]glucose. Rate of utilization for each concentration of glucose decreased markedly as the cultures became more confluent. This decrease also correlated with a reduced ability to transport glucose as measured by 2-deoxy-[³H]glucose uptake in washed monolayer cultures. In washed confluent culture of fibroblasts, glucose utilization was markedly decreased by the presence of pyruvate and lactate but not by glutamine. The respiratory inhibitors, rotenone and antimycin, did not increase the rate of glucose utilization except when added in combination with pyruvate. We conclude that cultured skin fibroblasts posses a highly glycolytic mode of metabolism but that this mode can become more oxidative in the presence of sufficient quantities of pyruvate and lactate.     

Glucose,Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.     

The dynamic provision of different energy substrates improves development of one-cell random-bred mouse embryos in vitro.

Preliminary observations showed that one-cell embryos from random-bred MF1 mice avoid cleavage arrest at the two-cell stage ('in vitro two-cell block') when cultured in modified M16 culture medium containing lactate and pyruvate but lacking glucose. The roles of lactate, pyruvate and glucose during preimplantation development of embryos from random-bred mice in vitro were therefore examined. When all three substrates were present continuously during culture, one-cell embryos arrested at the two- to four-cell stages. Improved development to the morula stage after 96 h in culture was obtained in media containing pyruvate alone, lactate and pyruvate, pyruvate and glucose, lactate pyruvate and glucose for the first 24 h, and medium containing lactate and pyruvate for the remaining 72 h. In a second experiment, embryos were cultured in medium containing pyruvate alone, lactate and pyruvate or pyruvate and glucose for the first 24 h, and lactate plus pyruvate medium for the second 24 h. Subsequent transfer to medium containing lactate, pyruvate and glucose supported the morula to blastocyst transition. These results show that developmental arrest in vitro can be overcome by changing the combination of energy substrates at different stages of preimplantation development.     

A novel syndrome affecting multiple mitochondrial functions, located by microcell-mediated transfer to chromosome 2p14-2p13

We have studied cultured skin fibroblasts from three siblings and one unrelated individual, all of whom had fatal mitochondrial disease manifesting soon after birth. After incubation with 1 mM glucose, these four cell strains exhibited lactate/pyruvate ratios that were six times greater than those of controls. On further analysis, enzymatic activities of the pyruvate dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, NADH cytochrome c reductase, succinate dehydrogenase, and succinate cytochrome c reductase were severely deficient. In two of the siblings the enzymatic activity of cytochrome oxidase was mildly decreased (by approximately 50%). Metabolite analysis performed on urine samples taken from these patients revealed high levels of glycine, leucine, valine, and isoleucine, indicating abnormalities of both the glycine-cleavage system and branched-chain alpha-ketoacid dehydrogenase. In contrast, the activities of fibroblast pyruvate carboxylase, mitochondrial aconitase, and citrate synthase were normal. Immunoblot analysis of selected complex III subunits (core 1, cyt c(1), and iron-sulfur protein) and of the pyruvate dehydrogenase complex subunits revealed no visible changes in the levels of all examined proteins, decreasing the possibility that an import and/or assembly factor is involved. To elucidate the underlying molecular defect, analysis of microcell-mediated chromosome-fusion was performed between the present study's fibroblasts (recipients) and a panel of A9 mouse:human hybrids (donors) developed by Cuthbert et al. (1995). Complementation was observed between the recipient cells from both families and the mouse:human hybrid clone carrying human chromosome 2. These results indicate that the underlying defect in our patients is under the control of a nuclear gene, the locus of which is on chromosome 2. A 5-cM interval has been identified as potentially containing the critical region for the unknown gene. This interval maps to region 2p14-2p13.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

Nuclear complementation restores mtDNA levels in cultured cells from a patient with mtDNA depletion.

We have studied cultured skin fibroblasts from a patient with a fatal mitochondrial disease manifesting soon after birth. These fibroblasts were found to grow only in the presence of pyruvate and uridine, a characteristic of cells lacking mtDNA (rho0 cells). Southern blot and PCR analyses confirmed that the patient's fibroblasts contained less than 2% of control levels of mtDNA. Biochemical analyses indicated that the activities of all the respiratory-chain enzymes were severely decreased in mitochondria isolated from these fibroblasts. In order to elucidate the underlying molecular defect, cell fusions were performed between enucleated fibroblasts from this patient and a human-derived rho0 cell line (rho0 A549.B2). The resulting cybrids were plated in medium lacking pyruvate and uridine, to select for the restoration of respiratory-chain function. Complementation was observed between the nuclear genome of the rho0 A549.B2 cells and the mtDNA of the patient's cells, restoring mtDNA levels and respiratory-chain function in the cybrid cells. These results indicate that mtDNA depletion in our patient is under the control of the nuclear genome.     

Effect of exogenous carbohydrates in a serum-free culture medium on the development of in vitro matured and fertilized porcine embryos

This study was conducted to examine the effect of energy substrates in a serum-free culture medium on in vitro development of porcine embryos. Presumptive zygotes derived from in vitro fertilization were cultured in glucose-free North Carolina State University (NCSU)-23 medium with glucose, pyruvate, fructose and lactate added to the culture medium singly or in various combinations. In experiment 1, a higher percentage of embryos cleaved (53-63% vs 10-13%) and developed to the blastocyst stage (18-27% vs 0) after the single addition of glucose (5.6 mM), pyruvate (0.5 mM) or lactate (10 mM) than with no energy substrate addition or the addition only of fructose (5.6 mM). In experiment 2, the addition of pyruvate and lactate resulted in higher blastocyst formation (25%) than other combinations (6-22%), while the addition of glucose and pyruvate significantly inhibited blastocyst formation. Increasing lactate concentration, as a single energy supplement, from 5 to 20 mM significantly improved blastocyst formation (7% vs 14-18%), while no benefit was achieved from increasing pyruvate concentration up to 2 mM (experiment 3). Glucose-free NCSU-23 medium supplemented with 0.5 mM pyruvate and 5 mM lactate significantly improved blastocyst formation (28% vs 17%) compared with NCSU-23 medium supplemented with 5.6 mM glucose (experiment 4). In conclusion, pyruvate and lactate are preferable energy substrates to support in vitro development of porcine embryos cultured in a serum-free NCSU-23 medium.     

Gluconeogenic and lipogenic properties of isolated kidney tubules from the domestic fowl (Gallus domesticus)

Isolated kidney tubules synthesize glucose actively from fructose, lactate, glycerol and pyruvate and, to a lesser extent, from a variety of amino acids. Ethanol stimulated gluconeogenesis from pyruvate and inhibited it from lactate. The aminotransferase inhibitor, aminooxyacetate, greatly reduced synthesis from lactate but not from pyruvate. Quinolinate inhibited gluconeogenesis from both precursors, indicating an active role for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenic pathway. Incorporation of lactate or glucose into triglycerides was relatively low, and since no fatty acid synthase (FAS) activity could be detected, probably represented chain elongation or reesterification.     

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.     

Carbohydrate metabolism of malarial parasites--I. Metabolism of lactate in Plasmodium knowlesi infected monkey erythrocytes

Lactate metabolism by Plasmodium knowlesi infected erythrocytes was examined after careful removal of leucocytes from cell preparations. Infected cells were able to metabolize glucose, pyruvate and lactate. Respiration of infected erythrocytes was maximally stimulated by lactate and to a lesser degree by pyruvate and glucose. Respiration of infected cells was insensitive to stimulation by succinate or glutamate or inhibition by malonate. Mepacrine was found to be a potent respiratory inhibitor. Chromatographic analysis of end products of lactate metabolism showed incorporation of carbon from [2-C14]lactate into phosphoenol pyruvate, 3-phosphoglycerate and malate. Experimental data failed to provide evidence for the presence of a functional citric acid cycle activity in infected cells.     

Early Deficits in Glycolysis Are Specific to Striatal Neurons from a Rat Model of Huntington Disease

In Huntington disease (HD), there is increasing evidence for a link between mutant huntingtin expression, mitochondrial dysfunction, energetic deficits and neurodegeneration but the precise nature, causes and order of these events remain to be determined. In this work, our objective was to evaluate mitochondrial respiratory function in intact, non-permeabilized, neurons derived from a transgenic rat model for HD compared to their wild type littermates by measuring oxygen consumption rates and extracellular acidification rates. Although HD striatal neurons had similar respiratory capacity as those from their wild-type littermates when they were incubated in rich medium containing a supra-physiological glucose concentration (25 mM), pyruvate and amino acids, respiratory defects emerged when cells were incubated in media containing only a physiological cerebral level of glucose (2.5 mM). According to the concept that glucose is not the sole substrate used by the brain for neuronal energy production, we provide evidence that primary neurons can use lactate as well as pyruvate to fuel the mitochondrial respiratory chain. In contrast to glucose, we found no major deficits in HD striatal neurons’ capacity to use pyruvate as a respiratory substrate compared to wild type littermates. Additionally, we used extracellular acidification rates to confirm a reduction in anaerobic glycolysis in the same cells. Interestingly, the metabolic disturbances observed in striatal neurons were not seen in primary cortical neurons, a brain region affected in later stages of HD. In conclusion, our results argue for a dysfunction in glycolysis, which might precede any defects in the respiratory chain itself, and these are early events in the onset of disease.     

Human cultured skin fibroblasts survive profound inherited ubiquinone depletion

Beside its role in electron transfer in the mitochondrial respiratory chain, ubiquinone is known to prevent lipid peroxidation and DNA damage by trapping cellular free radicals. Thanks to its antioxidant properties, ubiquinone may represent an important factor controlling both necrotic and apoptotic processes. We have investigated the consequences of a profound inherited ubiquinone depletion on cultured skin fibroblasts of a patient presenting with encephalomyopathy. Interestingly, cell respiration, mitochondrial oxidation of various substrates, and cell growth of ubiquinone-deficient fibroblasts were only partially decreased. Moreover, these cells did not apparently overproduce superoxide anions or lipoperoxides. Finally, apoptosis did not increase as compared to control, even after serum deprivation. These observations suggest that ubiquinone may not play a major role in the antioxidant defenses of cultured fibroblasts and that its role in controlling oxidative stress and apoptosis may greatly vary across cell types, especially as not all tissues were equally affected in the patient despite the widespread ubiquinone depletion in vivo.     

Evidence that the flux control coefficient of the respiratory chain is high during gluconeogenesis from lactate in hepatocytes from starved rats. Implications for the hormonal control of gluconeogenesis and action of hypoglycaemic agents.

1. Increasing concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a mild respiratory-chain inhibitor [Halestrap (1987) Biochim. Biophys. Acta 927, 280-290], caused progressive inhibition of glucose production from lactate + pyruvate by hepatocytes from starved rats incubated in the presence or absence of oleate and gluconeogenic hormones. 2. No significant changes in tissue ATP content were observed, but there were concomitant decreases in ketone-body output and cytochrome c reduction and increases in NADH fluorescence and the ratios of [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate]. 3. The inhibition by DCMU of palmitoylcarnitine oxidation by isolated liver mitochondria was used to calculate a flux control coefficient of the respiratory chain towards gluconeogenesis. In the presence of 1 mM-oleate, the calculated values were 0.61, 0.39 and 0.25 in the absence of hormone and in the presence of glucagon or phenylephrine respectively, consistent with activation of the respiratory chain in situ as previously suggested [Quinlan & Halestrap (1986) Biochem. J. 236, 789-800]. 4. Cytoplasmic oxaloacetate concentrations were shown to decrease under these conditions, implying inhibition of pyruvate carboxylase. 5. Inhibition of gluconeogenesis from fructose and dihydroxyacetone was also observed with DCMU and was accompanied by an increased output of lactate + pyruvate, suggesting that activation of pyruvate kinase was occurring. With the latter substrate, measurements of tissue ADP and ATP contents showed that DCMU caused a small fall in [ATP]/[ADP] ratio. 6. Two inhibitors of fatty acid oxidation, pent-4-enoate and 2-tetradecylglycidate, were shown to abolish and to decrease respectively the effects of hormones, but not valinomycin, on gluconeogenesis from lactate + pyruvate, without changing tissue ATP content. 7. It is concluded that the hormonal increase in mitochondrial matrix volume stimulates fatty acid oxidation and respiratory-chain activity, allowing stimulation of pyruvate carboxylation and thus gluconeogenesis to occur without major changes in [ATP]/[ADP] or [NADH]/[NAD+] ratios. 8. The high flux control coefficient of the respiratory chain towards gluconeogenesis may account for the hypoglycaemic effect of mild respiratory-chain inhibitors.     

Abnormal kinetic behavior of cytochrome oxidase in a case of Leigh disease.

Cultured skin fibroblasts from a child with fatal lacticacidemia displayed an abnormally high lactate:pyruvate ratio of 77:1, compared with control values of 22:1-27:1. When protease-treated isolated mitochondria were used, activity of the respiratory-chain enzymes was found to be approximately 60% of normal, and adenosine triphosphate synthesis was found to be normal with all substrates tested. In mitochondria prepared by means of digitonin treatment, adenosine triphosphate synthesis was depressed with all substrates tested, suggesting a defect in the operation of the cytochrome oxidase complex. In disrupted whole cells from the patient, cytochrome oxidase activity was 56% of the activity in the control cell line with the lowest activity. In the presence of a twofold excess of oxidized cytochrome c, patient cells showed 31% of the activity in controls. Cytochrome oxidase activity in both sonicated whole-cell preparations and in sonicated mitochondria displayed abnormal kinetics with regard to the substrate-reduced cytochrome c, which was particularly evident in the presence of excess oxidized cytochrome c. We believe that kinetically abnormal cytochrome oxidase complex is responsible for the biochemical and clinical abnormalities present in this patient.     

Chromosomal instability and cellular hypersensitivity to X-radiation of cultured fibroblasts derived from porokeratosis patients'' skin

Porokeratosis is a rare genetic skin disorder known to be associated with a propensity to develop skin cancer. To further elucidate the previously reported cytogenetic and cellular abnormalities, we studied karyotypic changes and the sensitivity to X-ray irradiation of cultured fibroblasts derived from skin lesions and normal-appearing skin of 3 patients with porokeratosis. Cultured fibroblasts from normal-appearing skin of 9 controls were similarly examined. Porokeratosis subjects had a greater number of cells with chromosomal abnormalities than controls. Two porokeratosis strains which were derived from the normal-appearing skin of a patient had a noticeable clone of abnormal cells. Porokeratosis fibroblasts were hypersensitive to the lethal effects of X-radiation. This hypersensitivity was common to both the lesion-derived strains and the ones derived from normal-appearing skin. The 2 strains with clonal abnormal cells were also similarly hypersensitive to X-radiation. These results suggest that chromosomal instability is strongly related to porokeratosis and that X-ray hypersensitivity is an inherent abnormality in cultured fibroblasts of porokeratosis patients.