Ribonucleotides in closed circular mitochondrial DNA from HeLa cells

Closed circular mitochondrial DNA from HeLa cells is sensitive to both alkali and ribonucleases. The kinetics of ring opening in alkali suggest at least two classes of molecules. One class undergoes rapid breakdown, ultimately to fragments smaller than unit length, in contrast to the second class, which is more resistant to alkaline cleavage and is converted in large part to unit length single strands. Ribonucleases A, T₁ and H relax the supercoiled molecules, indicating that the alkali susceptibility is due to the presence of ribonucleotides in the DNA. By comparison with the rate of hydrolysis of RNA, the alkali-resistant class of mitochondrial DNA molecules is estimated to contain approximately 3 ribonucleotides and the alkalisensitive class 10–18.     

Expression of the mitochondrial genome in HeLa cells. V. Transcription of mitochondrial DNA in relationship to the cell cycle

An investigation of the rate of incorporation of [5-³H]ur dine into mitochondrial RNA in synchronized HeLa cells in different phases of the cell cycle has revealed a considerable acceleration of this incorporation in cells in S and especially in G₂ phase. An analysis of the labeling of the intramitochondrial UTP pool has shown that this acceleration reflects a true increase in the rate of synthesis of mitochondrial RNA: this increase is considerably greater than can be accounted for by the expected doubling of mit-DNA^‡ templates during the S and G₂ phases.     

Hydrogen peroxide induces apoptosis in HeLa cells through mitochondrial pathway

Cervical cancer is the most common cancer amongst females in India and is associated with high risk HPVs, reactive oxygen species (ROS), and excessive inflammation in most cases. ROS in turn affects the expression of pro- and anti-apoptotic proteins. The objective of the present study was to elucidate the effect of hydrogen peroxide (H(2)O(2)) on apoptotic signaling molecules in vitro. HeLa cell line expresses the Human papilloma virus - 18, E6 oncoprotein which causes the ubiquitin mediated degradation of p53 protein and is thus p53 deficient. p53 is known to act as a cellular stress sensor and triggers apoptosis. p73, a member of the p53 family also induces apoptosis in response to DNA damaging agents but unlike p53, it is infrequently mutated in human tumors. We demonstrate here, that in HeLa cells, apoptosis is triggered by H(2)O(2) via the mitochondrial pathway involving upregulation of p73, and its downstream target Bax. This was accompanied by upregulation of ERK, JNK, c-Myc, Hsp-70 and down regulation of anti-apoptotic Bcl-XL, release of cytochrome c from mitochondria and activation of caspases-9 and -3.     

Effect of specific inhibitors on mitochondrial DNA replication in HeLa cells

The crystal structure of an orthorhombic form of 2′-0-methyl cytidine was determined from three dimensional X-ray diffraction data. The two molecules in each asymmetric unit have C2-$\text{endo}$ C3-$\text{exo}$ puckered furanose rings. This differs from the C3-$\text{endo}$ puckering observed for cytidine (1) and it may have some relevance to the kinks that appear at the two 2′-0-methylated nucleotides in the anticodon phosphate ester backbone of the phe tRNA structure (2). This work and other studies (3,4) show that the presence of a 2′-0-methyl group does not prevent the furanose moiety from adopting its most commonly observed configurations. 2′-0-methyl nucleotides make up a small percentage of the residues in HnRNA, rRNA, tRNA and mRNA and therefore their conformational nuances are of interest.     

Sodium arsenite induces ATP depletion and mitochondrial damage in HeLa cells

Our present results show that treatment with sodium arsenite apparently decreases cellular ATP levels in a dose- and time-dependent manner in HeLa S-3 cells. The reduction in ATP induced by sodium arsenite was possibly through mitochondrial damage, since treatment with sodium arsenite resulted in reduction of rhodamine 123 accumulation and disruption of the structure of the cristae in mitochondria. However, all of these changes could be reversed by removing sodium arsenite from the culture medium. The levels of ATP depletion were correlated with the killing effects of sodium arsenite in HeLa S-3 cells.     

Identification of discrete electrophoretic components among the products of mitochondrial protein synthesis in HeLa cells.

Proteins synthesized in vivo by HeLa cell mitochondria in the presence of emetine, an inhibitor of cytoplasmic protein synthesis, have been characterized as to their electrophoretic mobility, solubility properties in organic solvents, and kinetics of labeling with [³H]isoleucine.Ten distinct electrophoretic components in the molecular weight range from about 11,000 to 42,000 have been identified with high reproducibility among the products of mitochondrial protein synthesis. Control experiments involving direct sodium dodecyl sulfate lysis of whole cells, or the use of the protease inhibitor phenylmethylsulfonylfluoride during cell homogenization and fractionation, have excluded a significant role of enzymic degradation during extraction in producing the observed electrophoretic pattern of mitochondrial protein products.A selective solubilization of mitochondrial protein products, representing a 20 to 30-fold purification with respect to the cytoplasmically synthesized proteins, has been obtained by using a neutral chloroform/methanol mixture. At least six of the electrophoretic components with a molecular weight greater than 20,000 were found to be extracted to an appreciable extent with neutral chloroform/methanol.A fairly co-ordinate labeling of the discrete electrophoretic components has been observed during a one-hour exposure period of the cells to [³H]isoleucine in the presence of emetine, without any clear evidence of intorconversion.     

Properties of mitochondrial thymidine kinases of parental and enzyme-deficient HeLa cells

HeLa(BU25), a mutant subline of HeLa S3 cells, contains mitochondrial thymidine (dT) kinase, despite a marked deficiency in the dT kinase activity of the “cytosol” (high-speed supernatant) cell fraction. The HeLa(BU25) mitochondrial dT kinase differs from the “cytosol” enzyme of parental HeLa S3 cells in sedimentation coefficient, ability to utilize ribonucleoside 5′-triphosphates other than ATP as phosphate donors, sensitivity to inhibition by dCTP, and in disc polyacrylamide gel electrophoretic (disc PAGE) patterns. Two dT kinase activities [relative mobilities (Rm) of 0.4 and 0.6–0.7] were detected after disc PAGE of HeLa(BU25) mitochondrial extracts and both activities migrated more rapidly than the typical cytosol enzyme (Rm = 0.2) of dT kinase-positive human cells. The 0.6 to 0.7-Rm dT kinase of HeLa(BU25) mitochondria, but not the 0.4-Rm activity, utilized GTP and UTP, as well as ATP, as phosphate donors. HeLa S3 mitochondrial fractions contained the 0.6–0.7 Rm and the 0.4-Rm activities, and in addition, a “cytosol-like” 0.2-Rm activity. The 0.6 to 0.7-Rm dT kinase of HeLa S3 mitochondria utilized either UTP or ATP as phosphate donors, but the 0.4- and 0.2-Rm dT kinases utilized only ATP. Similarly, the HeLa S3 cytosol dT kinase efficiently utilized ATP, but not UTP, as a phosphate donor.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.