Depletion of mitochondrial DNA alters glucose metabolism in SK-Hep1 cells

Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho(0) SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho(0) cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho(0) cells compared with parental rho(+) cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization.     

Expression of microtubule networks in normal cells, transformed cells, and their hybrids

Microtubules play an important role in several cellular functions including cellular architecture and chromosome movement in cell division. Tubulin which polymerizes to form mictobules can be purified to homogeneity and used to raised antisera. Antisera prepared against porcine or chicken tubulin reacts well with mammalian tubulin. We have examined normal and transformed cells of mouse and human origin for microtubules by indirect immunofluorescence methods. Extensive networks of microtubules (MN) are easily detectable in normal and some transformed cells. The fixation procedure employed and the morphology and the cellular attachment properties seem to determine the ease of detection of MN in these cells. Cells derived from tumors and exhibiting several transformed phenotypes contained MN comparable to those of normal cells. Hybrids between transformed mouse cells and normal human cells were examined. They showed a variability in morphology, but all contained MN. These hybrids exhibited several transformed phenotypes. We conclude that in the cell lines we have examined there is no correlation between the transformed phenotypes and the organization of tubulin.     

Intracellular adenosine triphosphate as a measure of human tumor cell viability and drug modulated growth

Summary Adenosine triphosphate is the primary energy unit for cells, and levels of this compound offer a potential marker for cell viability and growth. The availability of a bioluminescence assay allows for a rapid, sensitive, and reproducible measurement of ATP. A method is described for the quantification of intracellular ATP levels in human cancer cells. ATP levels were linearly related to the number of viable cells and increased with time in human cancer cell line cultures correlating with growth kinetics. The effect of 5-fluorouracil, doxorubicin, methotrexate, cytosine arabinoside, nitrogen mustard, melphalan, vinblastine, and cisplatin on the growth of human cancer cell lines was studied utilizing ATP levels. ATP levels and colony formation in agar of drug-exposed cells were compared. Overall there was a significant correlation between drug effects on colony formation and ATP levels. The ATP assay is rapid, simple, reproducible, and a relatively inexpensive method of quantifying drug effects on malignant cells. This makes it a potentially useful method for screening new anticancer drugs in human cancer cell lines.     

The use of the firefly bioluminescent reaction for the rapid detection and counting of mycobacterium BCG

A rapid test for the counting of Mycobacterium BCG, based on firefly luciferase assay of bacterial ATP has been evaluated. Three different methods for the extraction of ATP from mycobacterial cells were examined. Extraction with n-butanol proved to be the best method. The amount of ATP extracted correlated well with the number of colony forming units. It was found that the ATP content per colony forming particle of BCG varied only slightly after various periods of cultivation.     

p100蛋白表达抑制的HepG2肝癌细胞稳定株的建立

目的建立p100表达抑制的HepG2肝癌细胞稳定株,并初步探讨p100在HepG2肝癌细胞中的功能。方法用脂质体将含有真核细胞筛选标记Neo和GFP的p100 shRNA表达质粒转染入HepG2细胞。经G418耐药筛选稳定整合抗药基因的细胞单克隆;荧光镜检GFP阳性细胞单克隆,挑取单克隆;Western检测HepG2细胞稳定株HepG2（p100I）中p100表达的抑制效果;平板细胞克隆形成实验检测细胞克隆形成能力;MTS法检测细胞存活;划痕实验检测细胞迁移能力。结果成功获得了p100表达抑制的HepG2肝癌细胞稳定株HepG2（p100I）,其中p100的表达明显降低。并且实验表明,该p100表达抑制稳定株的克隆形成能力,抵抗化疗药物Cisplatin诱导的细胞死亡的能力和迁移能力明显低于对照组细胞。结论p100表达抑制的HepG2肝癌细胞稳定株的建立为研究p100蛋白在肝癌中的作用提供了体外细胞系模型,基于此稳定株的研究,发现p100能够影响HepG2肝癌细胞的多种细胞功能。     

Neoplastic transformation and characterization of human fibroblasts by treatment with60Co gamma rays and the human c-Ha-ras oncogene

Summary Human fibroblasts (KMST-6) immortalized by treatment with⁶⁰Co gamma rays were further neoplastically transformed by transfection of the c-Ha-ras oncogene from human lung cancer. The ras-transfected cells formed undifferentiated fibrosarcoma in nude mice. One of the tumors was recultured and a neoplastic human fibroblast line, KMST-6/RAS, was established. To analyze multistep carcinogenesis of human cells, the cellular characteristics of these genetically matched immortalized (KMST-6) and neoplastic (KMST-6/RAS) cell lines were studied in detail. KMST-6/RAS cells showed an increased saturation density, colony formation on confluent monolayers of normal human fibroblasts, proliferation in neomycin-containing medium, anchorage-independent growth, and enhanced expression of the transfected c-Ha-ras oncogene, whereas the immortalized cells did not demonstate these characteristics. Unexpectedly, growth of KMST-6/RAS cells was serum-dependent, although they were neoplastic. Interestingly, the neoplastic cells did not show the criss-crossing or piling up growth pattern characteristic of transformed rodent fibroblasts.     

A small stress protein acts synergistically with trehalose to confer desiccation tolerance on mammalian cells

The ability to desiccate mammalian cells while maintaining a high degree of viability would be very important in many areas of biological science, including tissue engineering, cell transplantation, and biosensor technologies. Certain proteins and sugars found in animals capable of surviving desiccation might aid this process. We report here that human embryonic kidney (293H) cells transfected with the gene for the stress protein p26 from Artemia and loaded with trehalose showed a sharp increase in survival during air-drying. Further, we find vacuum-drying greatly improved the ability of the cells to survive, and that the physical shape and structure of the cellular sample had a large influence on recovery following rehydration. Cells suspended in a rounded droplet survived desiccation markedly better than those spread as a thin film. Finally, we used alamarBlue to monitor cellular metabolism and Hema 3 to assess colony formation after vacuum-drying. AlamarBlue fluorescence indicated that the transfected 293H cells expressing p26 (E11'L) grew much better than the control 293H cells. In fact, immediate survival and colony formation in E11'L cells increased as much as 34-fold compared with control cells when the samples were dried to a water content of 0.2 g H2O/g dry weight, as measured by gravimetric analysis. These results indicate that p26 improves cell survival following drying and rehydration, and suggest that dry storage of mammalian cells is a likely possibility in the future.     

Recombinant human tumor necrosis factors alpha and beta stimulate fibroblasts to produce hemopoietic growth factors in vitro

The influences of TNF alpha and TNF beta were evaluated for their stimulatory and inhibitory effects on in vitro colony formation by human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells. Both TNF alpha and TNF beta induced fibroblasts to produce stimulators of CFU-GM, BFU-E, and CFU-GEMM in a dose-dependent fashion. Similar results were seen when equivalent concentrations of TNF alpha and TNF beta were used. Prior incubation of the TNF alpha and TNF beta with their respective antibodies inactivated the ability of the TNF preparations to induce the release of granulocyte-macrophage, erythroid, and multipotential colony-stimulating activity from fibroblasts. In addition, incubation of the TNF-induced fibroblast supernatant with antibody before colony assay resulted in enhanced colony formation, suggesting that the TNF carried over into the colony assay suppressed colony formation. Additional proof of this suppression by TNF was evident when TNF was added directly to the CFU-GM, BFU-E, and CFU-GEMM colony assays. IL-1 does not appear to function as an intermediary in growth factor production by fibroblasts stimulated with TNF because antibody to IL-1 displayed no effect. Furthermore, assay of TNF-induced fibroblast supernatant was negative for IL-1. These results suggest that TNF alpha and TNF beta exert both a positive and negative influence on in vitro hemopoietic colony formation.     

Effect of selenite on basic mitochondrial function in human osteosarcoma cells with chronic mitochondrial stress

Mitochondrial chronic stress that originates from defective mitochondria is implicated in a growing list of human diseases. To enhance understanding of pathophysiology of chronic mitochondrial dysfunction we investigated human osteosarcoma cells with 2 types of chronic stress: corresponding to the mutation in ATP synthase subunit 6 encoded by mtDNA (NARP syndrome-mild stress) and to a total lack of mtDNA (Rho0 cells-heavy stress). We previously found that selenium influenced mitochondrial stress response and lowered ROS production. Therefore, in this study effect of selenite on other mitochondrial parameters was investigated. We showed that presence of selenium improved survival of starved cells, modified organization of mitochondrial network in NARP cybrids and decreased cytosolic calcium level in NARP and Rho0 cells. Selenium did not affect mitochondrial membrane potential, ATP level, activity of ATP synthase and activity of complex II of the respiratory chain.     

Segregation of mitochondrial DNA in human somatic cell hybrids

Summary The maintenance of mtDNA has been examined in human intraspecific hybrid cells constructed from the fusion of HEB7A, a HeLa tumor cell line carrying the mitochondrially coded chloramphenical (CAP) resistance mutation, and GM 2291, a limited lifespan human diploid fibroblast which is CAP sensitive. These two cells can be distinguished by a polymorphism in a site for the restriction endonuclease, HaeIII. Independently isolated clones of hybrid cells were characterized for their growth properties (either normal limited lifespan or transformed and immortal). Whole cell DNA preparations were made from each hybrid, digested with HaeIII, and the resultant fragments were detected by hybridization to ³²P labelled mouse mtDNA as probe. Experiments with mixtures of HEB7A and GM2291 DNA reveal that HEB7A mtDNA can be detected when it constitutes as little as 5% of the total cell mtDNA.The results indicate that the HEB7A mtDNA is lost from most hybrids, and when it does persist it is usually a minor component of total mtDNA. The addition of CAP at the time of fusion slightly increases the quantity of HEB7A mtDNA, but not enough to confer CAP resistance. Furthermore, five limited lifespan hybrids contained no detectable HEB7A mtDNA, while three transformed hybrids contained varying quantities of HEB7A mtDNA, suggesting that retention of this tumor form of mtDNA is associated with tumor growth behavior. These results suggest that cytoplasmic genetic incompatibility occurs in intraspecific hybrids.     

Intracellular heteroplasmy for disease-associated point mutations in mtDNA: implications for disease expression and evidence for mitotic segregation of heteroplasmic units of mtDNA

Studies in vitro have shown that a respiratorydeficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (GA) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (⁰) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy.     

NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract

Background

We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-κB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-κB in mediating cell survival in response to cigarette smoke exposure in HBECs.

Methods

Both the pharmacologic inhibitor of NF-κB, curcumin, and RNA interference targeting p65 were used to block NF-κB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.

Results

Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-κB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-κB by the pharmacologic inhibitor curcumin (20 μM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.

Conclusion

The current study demonstrates that CSE activates NF-κB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-κB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.     

Fibroblast colonies in monolayer cultures of human bone marrow

In a liquid culture of human bone marrow, the development of fibroblast colonies takes place on days 6 to 9. Twenty percent fetal calf serum is used as the stimulus for fibroblast colony growth. Human bone marrow cells are plated as 2 × 10₅ cells in the culture. Normal human bone marrow yields 47 ± 4 fibroblasts colonies per 2 × 10₅ cells plated. Bone marrow fibroblast cultures using agar or methylcellulose restrict colony formation. Marked colony suppression was observed in acute leukemia, and a discrete colony number was observed in hypoplastic anemia. This fibroblast culture method should be applied to a larger number of patients to determine whether it has a pathognomonic value and clinical significance.     

多瘤病毒癌基因产物对PDGF刺激应答的影响

多瘤病毒癌基因产物对ＰＤＧＦ刺激应答的影响于爱鸣冈野幸雄＊于秉治（中国医科大学基础医学院生化教研室,沈阳１１０００１）＊（日本岐阜大学医学部生化学教室,日本岐阜５００）多瘤病毒癌基因主要表达三种转化蛋白．其中,中等分子肿瘤抗原（ｍｉｄｄｌｅｓｉｚｅｄ...     

Mitochondrial DNA in mortal and immortal human cells. Genome number, integrity, and methylation

Mitochondrial DNA was quantitated in total DNA of various normal and mutant strains of human diploid fibroblasts (finite replicative lifespan) and permanent cell lines, using Southern-transfer hybridization to 32P-labeled pure mtDNA probe and saturation hybridization to 3H-labeled cRNA copied from mtDNA. In six normal fibroblast strains, mtDNA copy number increased during serial passage roughly in proportion to cell volume or protein content, whereas normalized mtDNA content per pg of protein depended upon in vivo donor age but not passage level ("in vitro" age). Copy numbers for mtDNA varied much more widely in individual fibroblast clones than in mass cultures, but were not well correlated with longevity or growth rate. Five mutant fibroblast strains associated with reduced replicative lifespan, and four permanent cell lines, were also examined; in each group, mtDNA values were observed both lower and higher than any obtained for normal fibroblasts. No evidence was found of petite-type deletions from human mtDNA, either at late passage or in individual clones of fibroblasts. Methylation of mtDNA genomes was strikingly non-random and apparently decreased with culture age.     

低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制研究

目的初步探讨低浓度丰加霉素对人白血病K562细胞集落形成抑制作用的机制。方法甲基纤维素集落形成实验检测低浓度丰加霉素对人白血病K562细胞集落形成能力的影响;CCK-8法检测低浓度丰加霉素对K562细胞的生长抑制率;AnnexinV／PI双染流式细胞仪检测低浓度丰加霉素作用下的K562细胞凋亡率;PI单染流式细胞仪检测药物作用后细胞的周期分布改变;Western免疫印迹和实时定量PCR检测周期相关分子表达水平变化。结果低浓度丰加霉素对人白血病K562细胞具有较强的集落形成抑制作用;可明显抑制K562细胞的生长,呈时间一剂量依赖性;尽管短时间（48h）的药物处理仅出现轻度的细胞凋亡和周期阻滞,但10nmol/L和30nmol/L的丰加霉素长时间（7d）作用后,K562细胞G0／G1期比例分别是（62．3±1．7）％和（76．9±0．7）％,与对照组（38．9±1．1）％相比差异具有高度统计学意义（P〈0．01）;低浓度丰加霉素长时间作用后诱导K562细胞周期相关分子P16蛋白水平和转录水平的高表达。结论丰加霉素在低浓度,长时间作用于人白血病K562细胞后,具有较强的集落形成抑制和生长抑制作用,此作用可能与诱导细胞周期相关分子p16高表达,导致细胞G0／G1期阻滞有关。     

Analysis of mammalian viable cell biomass based on cellular ATP

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.     

RecA-like activity in mammalian cell extracts of different origin

The occurrence of a RecA-like activity similar to the one detected in the fibroblast cell line GM1492 derived from a patient suffering from the autosomal recessive disease Bloom's syndrome has been investigated in cell extracts of different origin. The formation of D-loop containing joint molecules from phi X174 RFI DNA and fragments of phi X174 single-stranded DNA by partially purified extracts was measured by a filter binding assay. The RecA-like activity, dependent on ATP and Mg2+, was detected at an elevated level only in the human and rodent cell lines, GM1492 and CHO respectively. The level of activity in DNA-cellulose-purified cell extracts from these cell lines was 4-7-fold higher compared to normal human fibroblasts. Low levels of activity were also detected in extracts from two additional Bloom's syndrome fibroblast cell lines, Fanconi's anemia fibroblasts, virus- (Epstein-Barr virus, Simian virus 40) transformed human cells and human placenta. Cell extracts from rat testis, spleen and calf thymus were also of low activity.     

Depletion of mitochondrial DNA causes impaired glucose utilization and insulin resistance in L6 GLUT4myc myocytes

Mitochondrial dysfunction contributes to a number of human diseases, such as hyperlipidemia, obesity, and diabetes. The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of diabetes. To elucidate the association of cellular mtDNA content and insulin resistance, we produced L6 GLUT4myc myocytes depleted of mtDNA by long term treatment with ethidium bromide. L6 GLUT4myc cells cultured with 0.2 mug/ml ethidium bromide (termed depleted cells) revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. Interestingly, the mtDNA-depleted cells showed a drastic decrease in basal and insulin-stimulated glucose uptake, indicating that L6 GLUT4myc cells develop impaired glucose utilization and insulin resistance. The repletion of mtDNA normalized basal and insulin-stimulated glucose uptake. The mRNA level and expression of insulin receptor substrate (IRS)-1 associated with insulin signaling were decreased by 76 and 90% in the depleted cells, respectively. The plasma membrane (PM) GLUT4 in the basal state was decreased, and the insulin-stimulated GLUT4 translocation to the PM was drastically reduced by mtDNA depletion. Moreover, insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B were drastically reduced in the depleted cells. Those changes returned to control levels after mtDNA repletion. Taken together, our data suggest that PM GLUT4 content and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content, and the reductions in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes.