Expression Pattern of Mitochondrial ND2 Gene in Human Leukemia and in HL60 Cells During Growth and Differentiation

We differentially screened 5,000 clones from a cDNA library of acute myelogenous leukemia (AML) cell line HL60 using cDNA probes derived from normal granulocytes or from acute myelomonocytic leukemia cells, the objective being to identify genes preferentially expressed in myeloid lineage leukemic cells. One clone, corresponding to a mitochondrial DNA fragment, including NADH dehydrogenase subunit 2 (ND2) gene, was selected for use as a probe. We examined expression of the ND2 gene in various leukemic cell populations and in normal peripheral blood cells. DNA-RNA hybridization studies revealed that ND2 messenger RNA (mRNA) was more markedly expressed in AML cells than in other leukemic cells and normal peripheral blood granulocytes. The expression of ND2 mRNA decreased in HL60 cells several hours after treatment with phorbol myristate acetate (PMA), or dimethyl sulfoxide (DMSO). However, the ND2 gene expression did not depend on the growth-state of HL60 cells because the steady-state level of its expression was observed during transitions of growth. These results suggest that ND2 mRNA is involved in the maturation of myeloid cells and in cellular differentiation, in a lineage-preferential manner. A comparison of the nucleotide sequence of this clone with the documented human mitochondrial DNA sequence revealed several single-base substitutions, insertions and a 39-bases insertion.     

Differentiation of HT-29 human colonic adenocarcinoma cells correlates with increased expression of mitochondrial RNA: effects of trehalose on cell growth and maturation.

The HT-29 human adenocarcinoma cell line has been used extensively in the study of colonic cell differentiation and colon cancer. We report here that substitution of glucose with trehalose (alpha-D-glucopyranosyl-alpha-D-glucopyranoside) depresses growth and promotes mucin-producing, goblet-like maturation of HT-29. An initial characterization of this process was made by analyzing several cDNA clones whose RNA templates were differentially expressed at elevated levels in cells grown in trehalose-containing medium. Seven of the 9 clones examined corresponded to 6 mitochondrial genes whose expression levels, relative to those from glucose-grown cells, ranged from approximately 3-fold for 16S rRNA to 8-23-fold for NADH dehydrogenase subunit 4. On the other hand, levels of mitochondrial DNA copy, measured by using NADH dehydrogenase subunit 4 cDNA as probe, were shown to be unaffected by trehalose treatment. Elevation of cellular NADH dehydrogenase subunit 4 RNA in HT-29 cultures grown in medium containing different components (sodium butyrate, galactose, no-sugar, glucose, cellobiose) generally correlated with depressed growth levels and specifically with increased numbers of mucin-producing cells present. Like butyrate, the sugar, trehalose, is an effective inducer of HT-29 differentiation, and may prove useful as a dietary therapeutic, and as a probe for elucidating mitochondrial involvement in colonic cell differentiation and transformation.     

Rotenone, a mitochondrial NADH dehydrogenase inhibitor, induces cell surface expression of CD 13 and CD38 and apoptosis in HL-60 cells

We previously demonstrated that the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene was overexpressed in human acute myelogenous leukemia (AML) cells. Since this finding suggested that ND2 gene expression was related to myeloid differentiation, we here investigated the effects of rotenone, a specific NADH dehydrogenase inhibitor, on HL-60 cell growth, differentiation and death. Fifty nM rotenone inhibited the growth of HL-60 cells and caused an increase in the cell population in the Gz +M phase. In the quantitative comparison of myeloid antigen, the expression of CD13 and CD38 were relatively increased in the rotenone-treated cells. These findings suggest that the inhibition of NADH dehydrogenase changes the cell cycle and induces some specific surface antigens of HL-60 cells. On the other hand, the expression of ND2 gene remained unchanged after the rotenone treatment, suggesting the rotenone-mediated mitochondrial inhibition did not affect the mitochondrial gene expression. Five pM rotenone strongly inhibited the cellular proliferation. Electron microscopy and an electrophoretic analysis of DNA showed that the majority of the HL-60 cells were induced into typical apoptosis within 24-48 hours. On the basis of this and other studies, we believe that mitochondrial function is directly involved in both cellular differentiation and apoptotic cell death.     

Crypt-restricted loss and decreased protein expression of cytochrome C oxidase subunit I as potential hypothesis-driven biomarkers of colon cancer risk.

There is an increasing demand for the development of intermediate biomarkers to assess colon cancer risk. We previously determined that a live cell bioassay, which assesses apoptosis resistance in the nonneoplastic colonic mucosa, detects approximately 50% of patients with colon cancer. A hypothesis-driven biomarker that reflects apoptosis resistance in routine formalin-fixed, paraffin-embedded tissue would be easier to use. Cytochrome c oxidase is a critical enzyme that controls mitochondrial respiration and is central to apoptosis. We did an immunohistochemical study of cytochrome c oxidase subunit I expression in 46 colonic mucosal samples from 16 patients who had undergone a colonic resection. These included five patients without evidence of colonic neoplasia (three normal and two diverticulitis), three patients with tubulovillous adenomas, and eight patients with colonic adenocarcinomas. Analysis of aberrancies in expression of cytochrome c oxidase subunit I showed that, compared with nonneoplasia, the patients with neoplasia had a higher mean incidence of crypts having decreased expression (1.7 versus 22.8, P = 0.03) and a higher mean incidence having crypt-restricted loss (0.6 versus 3.2, P = 0.06). The percentage with segmented loss was low and was similar in the two groups. Combining these results, the mean % normal (i.e., with none of the three types of abnormality) was 96.7 in nonneoplasia versus only 73.2 in patients with neoplasia (P = 0.02). It should be noted that a defect in cytochrome c oxidase subunit I immunostaining was not detected in all biopsy samples from each patient for whom some abnormality was found, indicating a "patchiness" in the cytochrome c oxidase subunit I field defect. As a result of this "patchiness," the increased variability in the incidence of crypt-restricted loss of cytochrome c oxidase subunit I expression was a statistically significant feature of the neoplasia group. Crypt-restricted loss of cytochrome c oxidase subunit I has not been previously reported in colonic mucosa and is presumably the result of a crypt-restricted stem cell mutation. Decreased cytochrome c oxidase subunit I expression also significantly correlated with apoptosis resistance, a factor known to contribute to carcinogenesis. The results suggest, however, that aberrant cytochrome c oxidase subunit I expression may be a better biomarker than loss of apoptosis competence for increased colon cancer risk.     

Expression of mitochondrial cytochrome c oxidase in human colonic cell differentiation, transformation, and risk for colonic cancer

In a panel of eight cloned complementary DNA sequences whose level of expression characterize colon cells as transformed in vivo and in vitro, one which may also serve as a marker of risk in familial polyposis and familial colon cancer flat mucosa has been identified as mitochondrial cytochrome c oxidase subunit 3. Mean level of expression of cytochrome c oxidase subunit 3 decreases progressively in colon adenomas and carcinomas relative to normal mucosa in vivo, and returns to higher levels present in biopsies of normal mucosa when the HT29 human colonic adenocarcinoma cell line is induced to differentiate with sodium butyrate. Quantitation of cytochrome c oxidase subunit 3 DNA by dot blots indicated that these changes in expression were not associated with alterations in the number of mitochondrial genomes.     

Identification and characterization of genes involved in the carcinogenesis of human squamous cell cervical carcinoma

We utilized RT-PCR differential display and cDNA microarrays to identify cellular genes involved in the multi-step carcinogenesis of squamous cell cervical carcinoma. Thirty-eight cervical cancer patients in various stages of the disease and 5 non-cervical cancer patients were studied. Twenty-five cDNA clones were identified and these were subsequently demonstrated to be consistently over-expressed in squamous cell cervical carcinoma biopsies of various FIGO stages. To further evaluate the possible role that these genes may play in the progression of disease, we performed Northern blot analysis and RNA-RNA in situ hybridization studies using cervical cancer biopsies of various FIGO stages. Of particular interest are the 2 clones G32C4B and G30CC that have been identified to be the NADH dehydrogenase 4 gene and the gene that encodes ribosomal protein S12 respectively when compared to sequences available in the GenBank database. Increased expression of these 2 genes were detected in the matched normal tissues collected together with the late FIGO stages of cervical cancer biopsies. In comparison, upregulation of these 2 genes was not detected in cervical squamous epithelium collected from patients admitted for surgery for non-malignant conditions, suggesting that expression of these 2 genes may have altered in the adjacent histopathologically "normal" cervical squamous epithelial tissue from cervical cancer patients. The ribosomal protein S12 and the NADH dehydrogenase 4 genes may therefore be potentially useful as early pre-transformation diagnostic markers for human cervical cancer.     

Biochemical studies on mitochondria isolated from Normal and Neoplastic Tissues of the Mouse Mammary Gland.

Mitochondria isolated from spontaneous and transplanted mammary adenocarcinomas of two strains of mice were compared, by various biochemical criteria, to mitochondria from mammary glands of midpregnant or hormonally stimulated, cancer-free mice. The specific activities of several mitochondrial enzymes including cytochrome oxidase, alpha-glycerophosphate oxidase, and succinate dehydrogenase were twofold to threefold lower, whereas the activity of monoamine oxidase was two fold higher in tumor mitochondria. Malate dehydrogenase, adenylate kinase, and NADH oxidase showed similar levels of activity in tumor and midpregnant mammary gland mitochondria. In addition, mitochondrial polypeptide composition was analyzed by electrophoresis on sodium dodecyl sulfate-urea polyacrylamide gels. Midpregnant mammary gland and mammary tumor mitochondria were similar in polypeptide composition; however, several differences were observed. A high-molecular-weight polypeptide, present in mid-pregnant mammary gland mitochondria was absent from tumor mitochondria. Also, tumor mitochondria contained an additional high-molecular-weight polypeptide not found in the midpregnant mammary gland. There were numerous differences in the relative proportions of many polypeptides common to both tumor and midpregnant mammary gland mitochondria.     

Role of SCOX in determination of Drosophila melanogaster lifespan

In man, COX (cytochrome c oxidase) deficiency is reported to be related to mutation of the SCO2 (synthesis of cytochrome c oxidase 2) gene, which encodes one of the copper-donor chaperones involved in the assembly of mitochondrial cytochrome c oxidase. Such COX deficiency due to the genetic condition leads to heart disease and the Leigh syndrome and is frequently fatal in childhood. Synthesis of cytochrome c oxidase X (SCOX) is a Drosophila orthologue of human SCO2. Here, we generated SCOX-knockdown flies and the full length SCOX transgenic flies to investigate the in vivo roles of SCOX. Our results demonstrated knockdown of SCOX gene in all cells and tissues to be associated with lethality at larval or pupal stages and this correlated with a decrease in ATP level. In contrast, the full length SCOX transgenic flies showed a longer lifespan than wild type flies and control flies carrying Act5C-GAL4 alone and this correlated with an increase in ATP level. Finally, when cultured on paraquat-added medium, full length SCOX transgenic flies also exhibited an elongated lifespan. Therefore, we hypothesized that SCOX plays an important role in ATP production and consumption, which helps to prevent production of mitochondrial reactive oxygen species and/or impairment of mitochondrial activity under oxidative stress.     

In vitro photosensitization of tumour cell enzymes by photofrin II administered in vivo

The ability of injected Photofrin II, a preparation enriched in hydrophobic dihaematoporphyrin ethers and esters, to photosensitize selected mitochondrial and cytosolic enzymes during illumination in vitro was examined. Preparations of R3230AC mammary tumours, obtained at designated times after a single dose of Photofrin II, displayed a time-dependent photosensitivity. Maximum inhibition of mitochondrial enzymes occurred at 24 hours post-treatment, whereas no inhibition of the cytosolic enzyme, pyruvate kinase, was observed over the 168 hour time course. At the selected 24 hour time point, mitochondrial enzyme photosensitisation was found to be drug dose (5.25 mg kg-1 Photofrin II) and light dose dependent, the rank order of inhibition being cytochrome c oxidase greater than F0F1 ATPase greater than succinate dehydrogenase greater than NADH dehydrogenase. We conclude that porphyrin species contained in Photofrin II accumulate in mitochondria of tumour cells in vivo and produce maximum photosensitisation at 24-72 hours after administration to tumour-bearing animals. The time course observed here with Photofrin II is similar to that seen previously with the more heterogenous haematoporphyrin derivative preparation in this in vivo-in vitro model.     

Loss of complex I due to mitochondrial DNA mutations in renal oncocytoma.

PURPOSE: Many solid tumors exhibit abnormal aerobic metabolism characterized by increased glycolytic capacity and decreased cellular respiration. Recently, mutations in the nuclear encoded mitochondrial enzymes fumarate hydratase and succinate dehydrogenase have been identified in certain tumor types, thus demonstrating a direct link between mitochondrial energy metabolism and tumorigenesis. Although mutations in the mitochondrial genome (mitochondrial DNA, mtDNA) also can affect aerobic metabolism and mtDNA alterations are frequently observed in tumor cells, evidence linking respiratory chain deficiency in a specific tumor type to a specific mtDNA mutation has been lacking. EXPERIMENTAL DESIGN: To identify mitochondrial alterations in oncocytomas, we investigated the activities of respiratory chain enzymes and sequenced mtDNA in 15 renal oncocytoma tissues. RESULTS: Here, we show that loss of respiratory chain complex I (NADH/ubiquinone oxidoreductase) is associated with renal oncocytoma. Enzymatic activity of complex I was undetectable or greatly reduced in the tumor samples (n = 15). Blue Native gel electrophoresis of the multisubunit enzyme complex revealed a lack of assembled complex I. Mutation analysis of the mtDNA showed frame-shift mutations in the genes of either subunit ND1, ND4, or ND5 of complex I in 9 of the 15 tumors. CONCLUSION: Our data indicate that isolated loss of complex I is a specific feature of renal oncocytoma and that this deficiency is frequently caused by somatic mtDNA mutations.     

Superoxide anion production by adriamycinol from cardiac sarcosomes and by mitochondrial NADH dehydrogenase

This study investigates the effects of both adriamycin and its 13-hydroxylated metabolite adriamycinol on superoxide anion production from cardiac sarcosomes and by mitochondrial NADH dehydrogenase. Superoxide anion production was determined by using the succinoylated cytochrome c reduction assay. Both adriamycin and adriamycinol stimulated superoxide formation in cardiac sarcosomes and by mitochondrial NADH dehydrogenase. In the first case only NADPH was required as a co-factor and in the second case only NADH. From sarcosomes as well as by NADH dehydrogenase, the superoxide production followed Michaelis-Menten kinetics. With both activating enzymatic systems, the Vmax of adriamycinol was found to be similar to that of adriamycin, but the Km for the former anthracycline was higher than for the latter. Adriamycinol also increased the rate of NADPH and NADH consumption, by sarcosomal fractions and by NADH dehydrogenase respectively. At equimolar consentrations, adriamycinol consumed less NADPH and NADH than adriamycin. These results suggest that adriamycinol could contribute to the chronic cardiac toxicity of adriamycin by forming superoxide anions in cardiac cells constituents.     

Mitochondrial DNA quantity increases with histopathologic grade in premalignant and malignant head and neck lesions.

PURPOSE: Mitochondria are highly susceptible to oxidative damage. Although mitochondrial function decreases with oxidative damage, overall mitochondrial DNA (mtDNA) content increases to compensate for general mitochondrial dysfunction. We performed quantitative polymerase chain reaction for genes specific to mitochondrial and nuclear genomes to investigate relative mitochondrial abundance in a spectrum of dysplastic head and neck lesions. EXPERIMENTAL DESIGN: DNA from mild, moderate, and severe dysplasias, as well as invasive tumors and normal mucosal cells, was extracted. Using quantitative polymerase chain reaction, mitochondrial to nuclear DNA ratios were determined by quantification of cytochrome c oxidase subunit 1 (CoxI) and beta-actin genes. RESULTS: Mean CoxI/beta-actin DNA ratios for mild, moderate, and severe premalignant lesions were 0.0529, 0.0607, and 0.1021, respectively. The mean ratio for the normal mucosal cells contained in saliva was 0.0537, whereas the mean ratio for tumors was 0.1667. As a whole, our experimental model demonstrated significance (P = 0.0358). Comparisons between individual categories showed borderline significance when compared with the normal group, with P values of 0.0673, 0.0747, and 0.0824 for moderate and severe dysplasia and invasive tumor, respectively. CONCLUSIONS: Head and neck squamous cell carcinomas arise through premalignant intermediates and may be merely morphologic manifestations of accumulated genetic alterations. In keeping with this molecular tumor progression model, our study shows that mtDNA increases according to histopathologic grade, a phenomenon that may be a feedback mechanism that compensates for a generalized decline in respiratory chain function. Therefore, high mtDNA content may be another marker of genetic alteration, a measure of relative DNA injury, and a surrogate measure of histopathologic grade.     

Biochemical properties of simian virus 40-transformed 3T3 cell mitochondria.

Mitrochondria isolated from simian virus 40-transformed 3T3 and nontransformed 3T3 cells were compared by various biochemical criteria. Transformed and nontransformed cell mitochondria had identical densities in linear sucrose and discontinuous bovine serum albumin gradients. The activities of several mitochondria-specific enzymes including cytochrome oxidase, adenylate kinase, nicotinamide adenine dinucleotide (NADH)-cytochrome c reductase, and NADH oxidase were similar in both cell types. However, the activity of the mitochondrial outer membrane enzyme, monoamine oxidase. In the virus-transformed cell mitochondria was reduced to 50% of that in nontransformed cell mitochondria.     

Binding of mitochondrial and nuclear proteins from mouse and human-cells to gre-like elements of mouse mitochondrial-DNA

In the context of the hypothesis that the mitochondria represent primary sites of steroid hormone action we have evaluated the possible role of two nucleotide sequences partially homologous to Glucocorticoid Responsive Elements (GREs) present in the cytochrome oxidase subunit I gene of mouse and human mitochondrial DNA, as binding sites for mitochondrial and nuclear regulatory protein(s). Using a gel retardation assay, we examined whether proteins contained in mitochondrial and nuclear extracts from untreated and dexamethasone treated MCF7 and LATK- cell lines bind to these oligonucleotides. Both the mitochondrial and nuclear extracts from these cell lines contain protein(s) that bind to the oligonucleotides, as well as to the GRE of the human metallothionein II(A) promoter, binding which was considerably enhanced when the extracts were derived from cells previously treated with dexamethasone.