Diagnosis and molecular basis of mitochondrial respiratory chain disorders: exome sequencing for disease gene identification.

Mitochondrial disorders have the highest incidence among congenital metabolic diseases, and are thought to occur at a rate of 1 in 5000 births. About 25% of the diseases diagnosed as mitochondrial disorders in the field of pediatrics have mitochondrial DNA abnormalities, while the rest occur due to defects in genes encoded in the nucleus. The most important function of the mitochondria is biosynthesis of ATP. Mitochondrial disorders are nearly synonymous with mitochondrial respiratory chain disorder, as respiratory chain complexes serve a central role in ATP biosynthesis. By next-generation sequencing of the exome, we analyzed 104 patients with mitochondrial respiratory chain disorders. The results of analysis to date were 18 patients with novel variants in genes previously reported to be disease-causing, and 27 patients with mutations in genes suggested to be associated in some way with mitochondria, and it is likely that they are new disease-causing genes in mitochondrial disorders. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells.

ATP is released from cells in response to various stimuli. Our previous studies on pancreas indicated that pancreatic acini could be major stores of secreted ATP. In the present study, our aim was to establish the role of the vesicular nucleotide transporter (VNUT), SLC17A9, in storage and release of ATP. Freshly prepared acini from mice and AR42J rat acinar cells were used in this study. We illustrate that in AR42J cells, quinacrine (an ATP store marker) and Bodipy ATP (a fluorescent ATP analog) co-localized with VNUT-mCherry to vesicles/granules. Furthermore, in acini and AR42J cells, a marker of the zymogen granule membranes, Rab3D, and VNUT co-localized. Dexamethasone treatment of AR42J cells promoted formation of acinar structures, paralleled by increased amylase and VNUT expression, and increased ATP release in response to cholinergic stimulation. Mechanical stimulus (pressure) and cell swelling also induced ATP release, but this was not influenced by dexamethasone, most likely indicating different non-zymogen-related release mechanism. In conclusion, we propose that VNUT-dependent ATP release pathway is associated with agonist-induced secretion process and downstream purinergic signalling in pancreatic ducts.

Tonsillectomy and steroid pulse therapy for recurrent IgA nephropathy in renal allograft.

We experienced two cases of steroid pulse therapy combined with tonsillectomy for recurrent IgA nephropathy (IgAN) in a renal allograft. We defined recurrent IgAN in renal allograft as IgA deposits in glomeruli with persistent proteinuria (> 0.5 g/ day) and microscopic hematuria in renal transplant recipients with biopsy-proven IgAN of their native kidneys. We performed steroid pulse therapy following tonsillectomy as therapeutic protocol for recurrent IgAN. The first patient was diagnosed with recurrent IgAN by allograft biopsy 3 years after renal transplantation, and a second patient was diagnosed after one year. The former patient's proteinuria disappeared 4 months after treatment and the latter patient's proteinuria disappeared after one month. Tonsillectomy combined with steroid pulse therapy can induce clinical remission in patients with recurrent IgAN after renal transplantation.

Simvastatin enhances bone formation around titanium implants in rat tibiae.

Statins are cholesterol-lowering drugs that have been reported to promote bone formation. The purpose of this study was to investigate the effect of simvastatin on the enhancement of bone formation around titanium implants. Thirty-week-old female rats received pure titanium implants in both tibiae. The animals were intra-peritoneally administered 0, 0.125, 1, 5 or 10 mg kg(-1) of simvastatin daily. After 30 days, the animals were sacrificed, and specimens were prepared. The bone contact ratio of the implant, bone density in the medullary canal and percentage of cortical bone were obtained. Markers for bone turnover were also measured using sera collected at the time of euthanasia. In the medullary canal, a scanty amount of bone was observed in the 0, 0.125 and 1 mg kg(-1) groups. In contrast, in both the 5 and 10 mg kg(-1) groups, thicker bone trabeculae were abundant. Histometric observations showed that the bone contact ratio and the bone density of both groups were significantly greater than those of the other groups (anova, P < 0.01). However, no significant difference in the percentage of cortical bone was found between groups. Serum chemistry showed that statin increased bone formation markers and decreased bone resorption markers. In conclusion, although the dose equivalent to that used in human patients with hypercholesterolemia was not effective, a simvastatin dose of 5 mg kg(-1) or higher increased medullary bone formation around the titanium. In contrast, no effect of simvastatin on pre-existing cortical bone was indicated.

Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the human digestive tract, but their molecular etiology and cellular origin are unknown. Sequencing of c-kit complementary DNA, which encodes a proto-oncogenic receptor tyrosine kinase (KIT), from five GISTs revealed mutations in the region between the transmembrane and tyrosine kinase domains. All of the corresponding mutant KIT proteins were constitutively activated without the KIT ligand, stem cell factor (SCF). Stable transfection of the mutant c-kit complementary DNAs induced malignant transformation of Ba/F3 murine lymphoid cells, suggesting that the mutations contribute to tumor development. GISTs may originate from the interstitial cells of Cajal (ICCs) because the development of ICCs is dependent on the SCF-KIT interaction and because, like GISTs, these cells express both KIT and CD34.

Multidrug and toxic compound extrusion (MATE)-type proteins as anchor transporters for the excretion of metabolic waste products and xenobiotics.

1. Multidrug and toxic compound extrusion (MATE)-type transporters, which were first identified as a bacterial drug transporter family, are present in almost all prokaryotes and eukaryotes, and are thus one of the mostly conserved transporter families in nature. 2. Recently, a mammalian MATE transporter was shown to be a long hypothesized electroneutral H(+)/organic cation exporter that is responsible for the excretion of metabolic waste products and xenobiotics at renal brush border membranes and bile canaliculi. Plant MATE-type transporters are involved in the detoxification of metals and secondary metabolites such as phenols through their vesicular storage or extrusion at the plasma membrane. 3. Thus, MATE transporters are involved in one of the basic mechanisms that maintain homeostasis through the excretion of metabolic waste products and xenobiotics in nature.

Genetic cholesteryl ester transfer protein deficiency caused by two prevalent mutations as a major determinant of increased levels of high density lipoprotein cholesterol.

Genetic determinants of HDL cholesterol (HDL-C) levels in the general population are poorly understood. We previously described plasma cholesteryl ester transfer protein (CETP) deficiency due to an intron 14 G(+1)-to-A mutation(Int14 A) in several families with very high HDL-C levels in Japan. Subjects with HDL-C > or = 100 mg/dl (n = 130) were screened by PCR single strand conformational polymorphism analysis of the CETP gene. Two other mutations were identified by DNA sequencing or primer-mediated restriction map modification of PCR products: a novel intron 14 splice donor site mutation caused by a T insertion at position +3 from the exon14/intron14 boundary (Int14 T) and a missense mutation (Asp442 to Gly) within exon 15 (D442G). The Int14 T mutation was only found in one family. However, the D442G and Int14 A mutations were highly prevalent in subjects with HDL-C > or = 60 mg/dl, with combined allele frequencies of 9%, 12%, 21% and 43% for HDL-C 60-79, 80-99, 100-119, and > or = 120 mg/dl, respectively. Furthermore, prevalences of the D442G and Int14 A mutations were extremely high in a general sample of Japanese men (n = 236), with heterozygote frequencies of 7% and 2%, respectively. These two mutations accounted for about 10% of the total variance of HDL-C in this population. The phenotype in a genetic compound heterozygote (Int14 T and Int14 A) was similar to that of Int14 A homozygotes (no detectable CETP and markedly increased HDL-C), indicating that the Int14 T produces a null allele. In four D442G homozygotes, mean HDL-C levels (86 +/- 26 mg/dl) were lower than in Int14 A homozygotes (158 +/- 35 mg/dl), reflecting residual CETP activity in plasma. In 47 D442G heterozygotes, mean HDL-C levels were 91 +/- 23 mg/dl, similar to the level in D442G homozygotes, and significantly greater than mean HDL-C levels in Int14 A heterozygotes (69 +/- 15 mg/dl). Thus, the D442G mutation acts differently to the null mutations with weaker effects on HDL in the homozygous state and stronger effects in the heterozygotes, suggesting dominant expression of a partially defective allele. CETP deficiency, reflecting two prevalent mutations (D442G and Int14 A), is the first example of a genetic deficiency state which is sufficiently common to explain a significant fraction of the variation in HDL-C in the general population.

Correlation of serum pepsinogens and gross appearances combined with histology in early gastric cancer.

The correlation between serum pepsinogen (PG) levels and the gross types was investigated in 128 consecutive patients with early gastric cancer. Although there was no significant difference in age, gender, cancer location, or cancer depth among gross appearances, the distribution of histological type was significantly different between polypoid and depressed cancers: all polypoid cancers except one were intestinal type, whereas nearly a third of depressed cancers were diffuse type. All the patients in whom Helicobacter pylori status was investigated had Helicobacterpylori infection. Combination of gross appearances and histology (polypoid cancer with intestinal type, depressed cancer with intestinal type and depressed cancer with diffuse type) showed a clear difference in distribution of serum PG levels and a ratio between levels of PG I and PG II (I/II ratio). In polypoid cancer with intestinal type, a PG I level and a I/II ratio were significantly lower than those of the others. In depressed cancer with diffuse type, PG I and PG II levels were significantly higher. These findings revealed that backgrounds such as intragastric acidity and extent of gastric atrophy might differ among early gastric cancers with different morphology and histology.

Activity of the cytosolic isozyme of thymidine kinase in human primary lung tumors with reference to malignancy.

Activity increase of the cytosolic isozyme of thymidine kinase (TK) in resected specimens of lung tumor patients would be a useful marker for tumor malignancy and prognosis. In 24 resected cases of malignant lung tumors, the whole enzyme extracts of the tumorous part of the specimens showed that the activities of TK, thymidylate synthetase, and ribonucleotide reductase increased at an average of 469 (P less than 0.001), 208 (not significant), and 193% (P less than 0.02) of the corresponding enzymes in the tumor-uninvolved lung parts, respectively. Two TK isozymes, cytosolic and mitochondrial TKs, were separated better by means of p-aminophenyl 3'-TMP:CH-Sepharose gel affinity column chromatography for precise quantitation of the activity than by polyacrylamide disc gel electrophoresis. These separated isozymes from the tumorous part of the specimens were characteristically very similar to the isozymes of cytosolic and mitochondrial fractions of the xenograft (CPX-101) of human lung tumor transplanted in athymic nude mice, respectively. The cytosolic isozyme activity isolated by this method from the tumorous part was remarkably higher and more varied than that of the tumor-uninvolved part, while that of the mitochondrial isozyme was lower and less agitated. The tumor doubling time showed a good inverse correlation to the activity of the cytosolic isozyme of TK when compared logarithmically (r = -0.798, P less than 0.01). Poorly differentiated tumors exhibited significantly higher activities of the TK cytosolic isozyme than did well-to-moderately differentiated tumors (766.0 +/- 379.1 and 308.1 +/- 119.5 pmol/mg of protein/h, mean +/- SE, respectively), a phenomenon also seen in the activities of the tumors with versus without recurrences within 12 mo after resection (803.6 +/- 278.7 and 124.1 +/- 42.1 pmol/mg of protein/h, respectively). The levels of these relationships using the cytosolic TK activity provided a clearer indication of prognosis and the state of the malignancy than those using the whole extract TK activity.

Lysosomal H+-translocating ATPase has a similar subunit structure to chromaffin granule H+-ATPase complex.

Subunit structure of the lysosomal H+-ATPase was investigated using cold inactivation, immunological cross-reactivity with antibodies against individual subunits of the H+-ATPase from chromaffin granules and chemical modification with N,N'-dicyclohexyl[14C]carbodiimide. The lysosomal H+-ATPase was irreversibly inhibited when incubated at 0 degrees C in the presence of chloride or nitrate and MgATP. Inactivation in the cold resulted in the release of several polypeptides (72, 57, 41, 34 and 33 kDa) from the membrane, which had the same electrophoretic mobility as the corresponding subunits of chromaffin granule H+-ATPase. Cross-reactivity of antibodies revealed that the 72, 57 and 34 kDa polypeptides were immunologically identical to the corresponding subunits of chromaffin granule H+-ATPase. Dicyclohexylcarbodiimide, which inhibits proton translocation in the vacuolar ATPase, predominantly labeled two polypeptides of 18 and 15 kDa, which compose the membrane sector of the enzyme. These results suggest that the lysosomal H+-ATPase is a multimeric enzyme, whose subunit structure is similar to the chromaffin granule H+-ATPase. The subunit structure of other vacuolar H+-ATPases, revealed by cold inactivation and immunological cross-reactivity, is also presented.

Similarity of lysosomal H+-ATPase to mitochondrial F0F1-ATPase in sensitivity to anions and drugs as revealed by solubilization and reconstitution.

Lysosomal H+-translocating ATPase (H+-ATPase) was solubilized with lysophosphatidylcholine and reconstituted into liposomes (Moriyama, Y., Takano, T. and Ohkuma, S. (1984) J. Biochem. (Tokyo) 96, 927-930). In this study, the sensitivities of membrane-bound, solubilized and liposome-incorporated ATPase to various anions and drugs were measured in comparison with those of similar forms of mitochondrial H+-ATPase (mitochondrial F0F1-ATPase) with the following results. (1) Bicarbonate and sulfite activated solubilized lysosomal H+-ATPase, but not the membrane-bound ATPase or ATPase incorporated into liposomes. All three forms of mitochondrial F0F1-ATPase were activated by these anions. (2) All three forms of both lysosomal H+-ATPase and mitochondrial F0F1-ATPase were strongly inhibited by SCN-, NO3- and F-, but scarcely affected by Cl-, Br- and SO2-4. (3) The solubilized lysosomal H+-ATPase was strongly inhibited by azide, quercetin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and oligomycin. Its sensitivity was almost the same as that of mitochondrial F0F1-ATPase. Neither membrane-bound ATPase nor ATPase incorporated into liposomes was affected appreciably by these drugs. These results indicate that the sensitivity to anions and drugs of lysosomal H+-ATPase depends on the form of the enzyme and that the sensitivity of the solubilized lysosomal H+-ATPase is very similar to that of mitochondrial F0F1-ATPase. On the other hand, the two ATPases differ in their sensitivity to N-ethylmaleimide and pyridoxal phosphate: only the mitochondrial ATPase is inhibited by pyridoxal phosphate whereas only the lysosomal ATPase is inhibited by N-ethylmaleimide.

Co-purification of thymidylate kinase and cytosolic thymidine kinase from human term placenta by affinity chromatography.

It was revealed that thymidylate kinase was purified together with cytosolic thymidine kinase from human term placenta by p-aminophenyl thymidine-3'-phosphate-CH-Sepharose affinity column chromatography, which has been commonly used for purification of thymidine kinase. In addition, it was noted that mitochondrial thymidine kinase and nucleoside diphosphate kinase were concurrently eliminated. In the presence of ATP, cytosolic thymidine kinase and thymidylate kinase could be separated from each other by Ultrogel AcA 34 filtration, and their molecular weights were estimated to be 70,000 and 50,000, respectively. On SDS-polyacrylamide gel electrophoresis, thymidine kinase protein exhibited a band of 26,000, which was compatible with the molecular weight of the enzyme subunit calculated from its cDNA, while thymidylate kinase protein showed 24,000. Thymidylate kinase could utilize either ATP or dATP as an efficient phosphate donor, and showed substrate specificity for dTMP.

Circulating levels of secretory type II phospholipase A(2) predict coronary events in patients with coronary artery disease.

Background-The circulating levels of secretory nonpancreatic type II phospholipase A(2) (sPLA(2)) are increased in various chronic inflammatory diseases and the increase in the levels correlates with the disease severity. sPLA(2) may possibly play a role in atherogenesis and is highly expressed in atherosclerotic arterial walls that are known to have inflammatory features. Thus, this study prospectively examined whether circulating levels of sPLA(2) may have a significant risk and prognostic values in patients with coronary artery disease (CAD). Methods and Results-Plasma levels of sPLA(2) were measured in 142 patients with CAD and in 93 control subjects by a radioimmunoassay. The sPLA(2) levels had a significant and positive relations with serum levels of C-reactive protein, a marker of systemic inflammation, and with the number of the traditional coronary risk factors associated with individuals. Multivariate logistic regression analysis showed that higher levels of sPLA(2) (>246 ng/dL; 75th percentile of sPLA(2) distribution in controls) were a significant and independent risk factor for the presence of CAD. In multivariate Cox hazard analysis, the higher levels of sPLA(2) were a significant predictor of developing coronary events (ie, coronary revascularization, myocardial infarction, coronary death) during a 2-year follow-up period in patients with CAD independent of other risk factors, including CRP levels, an established inflammatory predictor. Conclusions-The increase in circulating levels of sPLA(2) is a significant risk factor for the presence of CAD and predicts clinical coronary events independent of other risk factors in patients with CAD; these results may reflect possible relation of sPLA(2) levels with inflammatory activity in atherosclerotic arteries.

Ca2+-dependent exocytosis of L-glutamate by alphaTC6, clonal mouse pancreatic alpha-cells.

Pancreatic islet cells express receptors and transporters for L-glutamate and are thus believed to use L-glutamate as an intercellular signaling molecule. However, the mechanism by which L-glutamate appears in the islets is unknown. In the present study, we investigated whether L-glutamate is secreted through exocytosis by alphaTC6 cells (clonal mouse pancreatic alpha-cells). An appreciable amount of L-glutamate was released from cultured cells after the addition of KCl or A23187 in the presence of Ca2+ and 10 mmol/l glucose in the medium. The KCl-induced glutamate release was significantly reduced when assayed in the absence of Ca2+ or when the cells were pretreated with EGTA-AM. The KCl-induced Ca2+-dependent glutamate release was inhibited approximately 40% by voltage-gated Ca2+ channel blockers, such as nifedipine at 20 micromol/l. The degree of KCl-induced Ca2+-dependent glutamate release was correlated with an increase in intracellular [Ca2+], as monitored by fura-2 fluorescence. Botulinum neurotoxin type E inhibited 55% of the KCl-induced Ca2+-dependent glutamate release, followed by specific cleavage of 25 kDa synaptosomal-associated protein. Furthermore, bafilomycin A1, a specific inhibitor of vacuolar H+-ATPase, inhibited 40% of the KCl-induced Ca2+-dependent glutamate release. Immunoelectronmicroscopy with antibodies against synaptophysin, a marker for neuronal synaptic vesicles and endocrine synaptic-like microvesicles, revealed a large number of synaptophysin-positive clear vesicles in cells. Digitonin-permeabilized cells took up L-glutamate only in the presence of MgATP, which is sensitive to bafilomycin A1 or 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (a proton conductor) but insensitive to either oligomycin or vanadate. From these results, it was concluded that alphaTC6 cells accumulate L-glutamate in the synaptophysin-containing vesicles in an ATP-dependent manner and secrete it through a Ca2+-dependent exocytic mechanism. The Ca2+-dependent glutamate release was also triggered when cells were transferred in the medium containing 1 mmol/l glucose, suggesting that low glucose treatment stimulates the release of glutamate. Our results are consistent with the idea that L-glutamate is secreted by alpha-cells through Ca2+-dependent regulated exocytosis.

Vitamin E administration improves impairment of endothelium-dependent vasodilation in patients with coronary spastic angina.

OBJECTIVES: We examined the effects of oral administration of vitamin E, an antioxidant, on endothelium-dependent vasodilation in patients with coronary spastic angina. BACKGROUND: We have recently reported that endothelium-dependent vasodilation is impaired in patients with coronary spastic angina (CSA). Furthermore, it is known that oxidative stress may play an important role in the impairment of endothelium-dependent vasodilation in cardiovascular diseases. METHODS: With the ultrasound technique, flow-dependent vasodilation of the brachial arteries during reactive hyperemia was examined before and after treatment for a month with either oral administration of vitamin E (alpha-tocopherol acetate, 300 mg/day) or placebo, which is randomly assigned, in patients with CSA (n=60). RESULTS: Before treatment, patients with CSA had impaired flow-dependent vasodilation, lower plasma levels of alpha-tocopherol and higher plasma levels of thiobarbituric acid reactive substances (TBARS), as compared with age- and sex-matched control subjects (n=60) (flow-dependent vasodilation: 3.1+/-1.8 vs. 7.1+/-2.5%, p < 0.001; alpha-tocopherol levels: 8.9+/-1.8 vs. 10.8+/-1.8 microg/ml, p < 0.001). In patients with CSA, treatment with vitamin E restored flow-dependent vasodilation (3.1+/-1.7 vs. 8.3+/-2.0%, p < 0.001), and this improvement was associated with the decreases in plasma TBARS levels and anginal attacks. CONCLUSIONS: The results indicate that vitamin E treatment improved endothelium-dependent vasodilation and decreased plasma TBARS levels in patients with CSA. Thus, increased oxidative stress may contribute to endothelial dysfunction and anginal attacks in patients with CSA.

Improvement of endothelial vasomotor dysfunction by treatment with alpha-tocopherol in patients with high remnant lipoproteins levels.

OBJECTIVES: This study sought to examine whether oral intake of alpha-tocopherol, an antioxidant, could improve endothelium-dependent vasorelaxation in patients with high remnant lipoproteins levels. BACKGROUND: Remnant lipoproteins are known to be atherogenic and impair endothelium-dependent arterial relaxation, but the underlying mechanisms remain unclear. Oxidative stress is a common feature of various risk factors for atherosclerosis. METHODS: Flow-mediated vasodilation of the brachial artery during reactive hyperemia was examined by high resolution ultrasound technique before and at the end of 4 weeks treatment with oral administration of alpha-tocopherol acetate (300 IU/day) or placebo, which was randomly assigned, in 40 patients with high serum levels of remnants and in 30 patients with low remnants levels in the fasting state (>75th percentile and <25th percentile, respectively, of the distribution of remnants levels in 150 consecutive hospitalized patients). RESULTS: Before treatment, flow-mediated vasodilation was lower in patients with high remnants levels than in those with low levels (4.1 +/- 0.3% vs. 6.0 +/- 0.5%, p < 0.01). Treatment with alpha-tocopherol but not with placebo significantly increased flow-mediated dilation in patients with high remnants levels (7.5 +/- 0.4% after alpha-tocopherol vs. 4.2 +/- 0.4% after placebo, p < 0.01). In patients with low remnants levels, alpha-tocopherol was not effective. The beneficial effect with alpha-tocopherol in high remnants patients was associated with decrease in plasma levels of thiobarbituric acid reactive substances, an indicator of lipid peroxidation (6.6 +/- 0.3 nmol/ml before alpha-tocopherol vs. 4.6 +/- 0.3 after alpha-tocopherol, p < 0.05). CONCLUSIONS: Alpha-tocopherol improved impairment of endothelium-dependent vasodilation in patients with high remnants levels. The increase in oxidative stress may at least partly contribute to endothelial vasomotor dysfunction, in patients with high remnants levels.

Rapid, selective digestion of mitochondrial DNA in accordance with the matA hierarchy of multiallelic mating types in the mitochondrial inheritance of Physarum polycephalum.

Although mitochondria are inherited uniparentally in nearly all eukaryotes, the mechanism for this is unclear. When zygotes of the isogamous protist Physarum polycephalum were stained with DAPI, the fluorescence of mtDNA in half of the mitochondria decreased simultaneously to give small spots and then disappeared completely approximately 1.5 hr after nuclear fusion, while the other mitochondrial nucleoids and all of the mitochondrial sheaths remained unchanged. PCR analysis of single zygote cells confirmed that the loss was limited to mtDNA from one parent. The vacant mitochondrial sheaths were gradually eliminated by 60 hr after mating. Using six mating types, the transmission patterns of mtDNA were examined in all possible crosses. In 39 of 60 crosses, strict uniparental inheritance was confirmed in accordance with a hierarchy of relative sexuality. In the other crosses, however, mtDNA from both parents was transmitted to plasmodia. The ratio of parental mtDNA was estimated to be from 1:1 to 1:10(-4). Nevertheless, the matA hierarchy was followed. In these crosses, the mtDNA was incompletely digested, and mtDNA replicated during subsequent plasmodial development. We conclude that the rapid, selective digestion of mtDNA promotes the uniparental inheritance of mitochondria; when this fails, biparental inheritance occurs.

Effect of hyperthermia on both primary proliferation and self-renewal of human leukemic progenitor cells in vitro: its application to in vitro purging.

Self-renewal, as defined by the capacity to yield new colonies following replating, is an important function of leukemic progenitors (L-CFU) to originate self-maintaining clones. In this work, we studied the effect of hyperthermia (41-44 degrees C) on the growth of human L-CFU derived primary colonies and of secondary colonies formed by replating to evaluate the purging effect of human L-CFU by heat. The survival curves clearly demonstrated much greater hyperthermic sensitivity of L-CFU compared to normal granulocyte-macrophage progenitors (CFU-GM) at all temperatures (41-44 degrees C) studied. At 42 degrees C or higher, L-CFU decreased (by more than 2 log reduction) dramatically and therefore were unable to form colonies in vitro. At 42 degrees C and 43 degrees C, 65 and 30%, respectively, of CFU-GM obtained from remission and normal marrows were left after 1 h exposure. At 44 degrees C, however, CFU-GM derived colonies disappeared after a 2 h exposure. In the four available patients with acute myelogenous leukemia, secondary colonies formed by replating were decreased in proportion to the decreasing primary colonies during heating (42 and 43 degrees C). However, their self-renewal capacity was retained in vitro until the primary colonies disappeared. These observations suggest that heat exposure at 43 degrees C for 1 h could be the most effective conditions for in vitro purging of human L-CFU because of the wide difference between surviving fractions of CFU-GM and L-CFU.

Tumor-suppressor effect of interferon regulatory factor-1 in human hepatocellular carcinoma.

IFN regulatory factor-1 (IRF-1) regulates the IFN system, inhibits cell growth, and has tumor-suppressor activities. p21 is a universal cyclin-dependent kinase inhibitor, the induction of which depends on both p53 and IRF-1 in mouse embryonic fibroblasts. The expression of p21 in hepatocellular carcinomas (HCCs) is regulated by wild-type p53. We examined the expressions of IRF-1 and p21 in 32 HCCs by quantitative reverse transcription-PCR and the mutation p53 gene in 32 HCCs by single-strand conformation polymorphism and direct sequencing. The expression of IRF-1 mRNA in 15 of 32 HCCs was lower than that in adjacent noncancerous tissue. IRF-1 mRNA expression was reduced in 0 of 3 specimens of well-differentiated HCC, 9 of 21 (42%) specimens of moderately differentiated HCC, and 6 of 8 (75%) specimens of poorly differentiated HCC. IRF-1 mRNA expression was significantly lower in tumors with portal thrombus than in those without portal thrombus (P = 0.003). p53 mutations were detected in 7 of 32 HCCS: p21 expression was reduced in 6 of the 7 (86%) HCCs with p53 mutations. In contrast, p21 expression was reduced in 13 of 25 (52%) HCCs with wild-type p53. IRF-1 expression was reduced in 7 of 13 (53%) HCCs with both wild-type p53 and reduced expression of p21. These results suggest that IRF-1 may be a tumor-suppressor gene for HCC and that IRF-1 is related to p21 expression in HCC with wild-type p53.