Repair of radiation-induced DNA damage in thermotolerant and nonthermotolerant HeLa cells

The effect of heat exposure on the repair of radiation-induced DNA damage which inhibits the ability of nuclear DNA to undergo supercoiling changes was studied using the fluorescent halo assay in thermotolerant and nonthermotolerant (normal) cells. The assay utilizes an intercalating, fluorescent dye to unwind and rewind endogenous DNA supercoils. When HeLa cells are exposed to 17.3 Gy radiation the ability of DNA to be rewound into supercoils is completely inhibited. However, the ability of DNA to rewind is 70% restored by 30 min after irradiation. Both thermotolerant and normal cells exposed to 45 degrees C for 30 min prior to irradiation had a rewinding ability intermediate between control and unheated cells, but there was no restoration of rewinding ability up to 3 h postirradiation. Thus, when irradiation immediately followed heating, there was no difference between thermotolerant and normal cells. However, when various time intervals were imposed between heating and irradiation, a difference in the ability of the cells to recover from heat-induced alterations became apparent. In normal cells after 6 h of postheat incubation the cells' ability to restore DNA supercoiling was approximately the same as that of control cells, while in thermotolerant cells only 2 h was required to repair the ability to restore supercoiling at the same rate. The rate of repair of DNA remained correlated with relative nuclear protein content as measured by fluorescein isothiocyanate staining in both thermotolerant and normal cells, indicating a possible relationship between the two.     

S-phase dependent forms of DNA - nuclear membrane complexes in HeLa cells.

DNA - nuclear membrane complexes were isolated from HeLa cells and examined by either zone sedimentation analysis or isopycnic centrifugation in sucrose/CsCl gradients. The data suggest that the complexes formed during the first 10 min of the S-phase remain as stable structures throughout the cell cycle. Other DNA - nuclear membrane complexes are formed at later times during replication. These later complexes appear as multiple species and the association of DNA and the nuclear membrane seems to be of a transient nature. Together, these results suggest that both the replicative origins and the replication points of the DNA are associated with the nuclear membrane. Although the complexes formed at the start of the S-phase and at later times during the S-phase appear to differ, these differences may provide them with the needed properties to serve as spatial organizers for the temporal regulation of DNA replication.     

Ceramide synthases 2, 5, and 6 confer distinct roles in radiation-induced apoptosis in HeLa cells

The role of ceramide neo-genesis in cellular stress response signaling is gaining increasing attention with recent progress in elucidating the novel roles and biochemical properties of the ceramide synthase (CerS) enzymes. Selective tissue and subcellular distribution of the six mammalian CerS isoforms, combined with distinct fatty acyl chain length substrate preferences, implicate differential functions of specific ceramide species in cellular signaling. We report here that ionizing radiation (IR) induces de novo synthesis of ceramide to influence HeLa cell apoptosis by specifically activating CerS isoforms 2, 5, and 6 that generate opposing anti- and pro-apoptotic ceramides in mitochondrial membranes. Overexpression of CerS2 resulted in partial protection from IR-induced apoptosis whereas overexpression of CerS5 increased apoptosis in HeLa cells. Knockdown studies determined that CerS2 is responsible for all observable IR-induced C_24:0 CerS activity, and while CerS5 and CerS6 each confer ～ 50% of the C_16:0 CerS baseline synthetic activity, both are required for IR-induced activity. Additionally, co-immunoprecipitation studies suggest that CerS2, 5, and 6 might exist as heterocomplexes in HeLa cells, providing further insight into the regulation of CerS proteins. These data add to the growing body of evidence demonstrating interplay among the CerS proteins in a stress stimulus-, cell type- and subcellular compartment-specific manner.     

Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids.     

The greater lethality of UVB radiation to cultured human cells is associated with the specific activation of a DNA damage-independent signaling pathway

UV radiation causes cell death through the activation of various intracellular signaling molecules in both DNA damage-dependent and -independent manners. The ability of middle-wavelength UV (UVB) radiation to form DNA photoproducts is less than that of short-wavelength UV (UVC) radiation; however, the differences between UVB and UVC radiation in the extent of DNA damage-independent signaling and its contribution to cell death have not been well characterized. When cells were irradiated with UVB or UVC radiation at doses that generated equivalent amounts of DNA photoproducts, UVB radiation induced more clonogenic cell death, apoptotic cells, mitochondrial cytochrome C release, and intracellular oxidative stress. Among the signaling molecules examined, levels of p53 phosphorylated at Ser-392 and p38 were higher in UVB-irradiated cells than in UVC-irradiated cells. Both phosphorylations were reduced by treating cells with an antioxidant. Furthermore, an inhibitor of p38 also blocked the phosphorylation of p53 at Ser-392. These results suggest that UVB radiation activates the p38 pathway through the generation of oxidative stress, which merges with the DNA p53 pathway by phosphorylation of p53 at ser392. This greater contribution of the DNA damage-independent pathway in UVB-irradiated cells may explain the greater lethality of UVB radiation.     

Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells

In Xenopus laevis egg extracts, TPX2 is required for the Ran-GTP-dependent assembly of microtubules around chromosomes. Here we show that interfering with the function of the human homologue of TPX2 in HeLa cells causes defects in microtubule organization during mitosis. Suppressing the expression of human TPX2 by RNA interference leads to the formation of two microtubule asters that do not interact and do not form a spindle. Our results suggest that in vivo, even in the presence of duplicated centrosomes, spindle formation requires the function of TPX2 to generate a stable bipolar spindle with overlapping antiparallel microtubule arrays. This indicates that chromosome-induced microtubule production is a general requirement for the formation of functional spindles in animal cells.     

Increased alkaline phophatase activity in HeLa cells mediated by aliphatic monocarboxylates and inhibitors of DNA synthesis

Alkaline phosphatese activity of HeLa cells is increased from 3- to 8-fold during growth in medium with certain aliphatic monocarboxylates. The four-carbon fatty acid salt, sodium butyrate, is the most effective “inducer” with propionate (C₃), pentanoate (C₅) and hexanoate (C₆) having lesser effects. Other straight-chain aliphatic monocarboxylates, branched-chain analogues of inducers, hydroxylated derivatives, and metabolytes structurally related to butyrate are ineffective in mediating an increase in enzyme activity, indicating stringent structural requirements for inducers. The kinetics of increase in alkaline phosphatase activity in HeLa cells shows a 20–30 h lag period after adding the aliphatic acid followed by a rapid linear increase of enzyme activity. Protein synthesis is required for “induction”. The isozyme of HeLa alkaline phosphatase induced by monocarboxylates is the carcinoplacental form of the enzyme as determined by stereospecific inhibition by the l-enantiomorphs of phenylalanine and tryptophan, heat stability, and immunoreactivity with antibody against the human placental enzyme.Monocarboxylates that mediate increased alkaline phosphatase activity inhibit HeLa cell multiplication. Inhibition of HeLa cell growth may be necessary for induction and this hypothesis is supported by the findings that three different inhibitors of DNA synthesis, i.e. hydroxyurea, 1-β-d-arabinfuranosyl cytosine and methotrexate, also increase alkaline phosphatase activity. These inhibitors are synergistic with butyrate in causing HeLa cells to assume a more spindle-like shape and in producing an up-to 25-fold increase of enzyme activity. Studies on the modulation of carcinoplacental alkaline phosphatase by monocarboxylates commonly used as antimicrobial food additives and by anti-neoplastic agents may provide methods to evoke “tumor markers” of human occult malignancies. These drug-induced elevations of fetal isozyme activity may further our understanding of gene expression in human cells.     

Cytocidal effect of interleukin 1 (IL-1) on HeLa cells is mediated by both soluble and transmembrane tumor necrosis factor (TNF)

Interleukin 1 (IL-1) is a pleiotropic cytokine able to induce cytocidal effect. The aim of the presented work was to analyze the mechanism of IL-1-induced cytocidal effect in HeLa cells in the presence of cycloheximide (CHX). We found that the pattern of IL-1-induced cell death shares significant similarities with the effect of tumor necrosis factor (TNF) in these cells. Subsequently, we identified IL-1 cytotoxicity as an indirect effect. The supernatant collected from the cells treated with IL-1 and CHX showed toxic activity towards IL-1-resistant while TNF-sensitive A9 cells. Furthermore, antibodies neutralizing TNF blocked HeLa cell death induced by IL-1/CHX. TNF was then detected in HeLa cells by means of flow cytometry, fluorescence microscopy and ELISA of detergent-soluble cell extracts. In the presence of an inhibitor of TNF sheddase (TACE), the cytotoxic effect of IL-1/CHX and the amount of TNF protein in detergent-soluble cell extracts were enhanced. These results suggest that in response to interleukin 1/CHX, the amount of transmembrane TNF is increased. Taken together, we demonstrated that the mechanism of IL-1 cytotoxic activity in HeLa cells in the presence of CHX depends on the function of soluble and transmembrane TNF.     

Properties of a transplasma membrane electron transport system in HeLa cells

A transmembrane electron transport system has been studied in HeLa cells using an external impermeable oxidant, ferricyanide. Reduction of ferricyanide by HeLa cells shows biphasic kinetics with a rate up to 500 nmoles/min/g w.w. (wet weight) for the fast phase and half of this rate for the slow phase. The apparentK _m is 0.125 mM for the fast rate and 0.24 mM for the slow rate. The rate of reduction is proportional to cell concentration. Inhibition of the rate by glycolysis inhibitors indicates the reduction is dependent on glycolysis, which contributes the cytoplasmic electron donor NADH. Ferricyanide reduction is shown to take place on the outside of cells for it is affected by external pH and agents which react with the external surface. Ferricyanide reduction is accompanied by proton release from the cells. For each mole of ferricyanide reduced, 2.3 moles of protons are released. It is, therefore, concluded that a transmembrane redox system in HeLa cells is coupled to proton gradient generation across the membrane. We propose that this redox system may be an energy source for control of membrane function in HeLa cells. The promotion of cell growth by ferricyanide (0.33–0.1 mM), which can partially replace serum as a growth factor, strongly supports this hypothesis.     

Dopamine release in PC12 cells is mediated by Ca(2+)-dependent production of ceramide via sphingomyelin pathway

A presynaptic membrane disturbance is an essential process for the release of various neurotransmitters. Ceramide, which is a tumor suppressive lipid, has been shown to act as a channel-forming molecule and serve as a precursor of ceramide-1-phosphate, which can disturb the cellular membrane. This study found that while permeable ceramide increases the rate of dopamine release in the presence of a Ca(2+)-ionophore, A23187, permeable ceramide-1-phosphate provoked its release even without the ionophore. The treatment of PC12 cells with the ionophore at concentrations < 2 microM produced ceramide via the sphingomyelin (SM) pathway with a concomitant release of dopamine, and no cell damage was observed. The addition of a Ca(2+) chelator, EGTA, to the medium inhibited the increase in the release of both the ceramide and dopamine. This suggests that ceramide might be produced by Ca(2+) and is implicated in the membrane disturbance associated with the release of dopamine as a result of its conversion to ceramide-1-phosphate. Consistent with these results, this study detected a membrane-associated and neutral pH optimum sphingomyelinase (SMase) whose activity was increased by Ca(2+). Together, these results demonstrate that ceramide can be produced via the activation of a neutral form of SMase through Ca(2+), and is involved in the dopamine release in concert with Ca(2+).     

Translocation of beta-galactosidase mediated by the cell-penetrating peptide pep-1 into lipid vesicles and human HeLa cells is driven by membrane electrostatic potential

The cell-penetrating peptide (CPP) pep-1 is capable of introducing large proteins into different cell lines, maintaining their biological activity. Two possible mechanisms have been proposed to explain the entrance of other CPPs in cells, endosomal-dependent and independent types. In this work, we evaluated the molecular mechanisms of pep-1-mediated cellular uptake of beta-galactosidase (beta-Gal) from Escherichia coli in large unilamellar vesicles (LUV) and HeLa cells. Fluorescence spectroscopy was used to evaluate the translocation process in model systems (LUV). Immunofluorescence microscopy was used to study the translocation in HeLa cells. Enzymatic activity detection enabled us to monitor the internalization of beta-Gal into LUV and the functionality of the protein in the interior of HeLa cells. Beta-Gal translocated into LUV in a transmembrane potential-dependent manner. Likewise, the extent of beta-Gal incorporation was extensively decreased in depolarized cells. Furthermore, beta-Gal uptake efficiency and kinetics were temperature-independent, and beta-Gal did not colocalize with endosomes, lysosomes, or caveosomes. Therefore, beta-Gal translocation was not associated with the endosomal pathway. Although an excess of pep-1 was mandatory for beta-Gal translocation in vivo, transmembrane pores were not formed as concluded from the trypan blue exclusion method. These results altogether indicated that protein uptake both in vitro with LUV and in vivo with HeLa cells was mainly, if not solely, dependent on negative transmembrane potential across the bilayer, which suggests a physical mechanism governed by electrostatic interactions between pep-1 (positively charged) and membranes (negatively charged).     

Synthesis and in vitro behavior of multivalent cationic lipopeptide for DNA delivery and release in HeLa cells

We report here that a synthesized multivalent cationic lipopeptide can be used for the delivery and release of gene assembled into lipopeptide vesicles. It is found experimentally that the synthesized lipopeptide is safe for gene therapy because of its biocompatibility and the innocuity of the hydrolysis products, such as lipids and amino acids. The experimental results also show that the assembled DNA/lipopeptide complex has high transfection efficiency in HeLa cells compared to that of the selected commercial reagent, which represents a novel vector for the delivery of therapeutic DNA.     

A Ca2+ signal is found upstream of cytochrome c release during apoptosis in HeLa cells

We showed previously that a cytosolic Ca(2+) signal is involved in regulating UV-induced apoptosis in HeLa cells. In this study, we found evidence that this Ca(2+) signal occurs upstream of the release of cytochrome c from mitochondria. First, when we abolished [Ca(2+)](i) increases by injecting BAPTA or heparin into UV-treated HeLa cells, cytochrome c release was either blocked or severely delayed. Second, using a living cell imaging technique, we observed a series of transient [Ca(2+)](i) increases (typically lasting about 40-60s) in many apoptotic cells induced by either UV- or TNFalpha-treatment. Third, using GFP-tagged cytochrome c, we found that the Ca(2+) spikes appear in a time window before cytochrome c was released. Finally, by fixing the TNFalpha-treated cell at the time when it started to display Ca(2+) spikes, we examined the distribution of its endogenous cytochrome c using immunostaining. We found that cytochrome c was not yet released from mitochondria. These findings suggest the existence of certain apoptotic pathways, in which an early Ca(2+) signal is activated upstream of cytochrome c release.     

The mediating role of cPLA2 in IL-1 beta and IL-6 release in LPS-induced HeLa cells

Studies were conducted to characterize a HeLa cell model by which the roles of the 85-kDa phospholipase A2 (cPLA2) in interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) release could be evaluated. At first, untreated HeLa cells were compared with lipopolysaccharide (LPS)-treated HeLa cells. The latter resulted in cPLA2 overexpression and an increased trend of IL-1 beta and IL-6 release. The indicated doses of 85-kDa cPLA2 antisense oligonucleotide directed against the initiation site were then used to block cPLA2 in LPS-induced HeLa cells. The process led to a dose-dependent decrease in cPLA2 protein with no noticeable change of cPLA2 mRNA. Compared with that of LPS added only, a reduction of IL-1 beta and IL-6 levels in the supernatants of transfected cells following the repression of cPLA2 was observed. These results suggested that 85-kDa cPLA2 may mediate the signalling cascades by which IL-1 beta and IL-6 were released in LPS-induced HeLa cells.     

Evidence for the existence of a stable association between nascent DNA and the nuclear membrane of HeLa cells.

Nascent DNA-nuclear membrane complexes isolated from HeLa cells and solubilized in a sodium dodecyl sulfate-urea solution were examined by gel electrophoresis, column chromatography, isopycnic centrifugation, and by extraction with chloroform/methanol. Radioactivity attributable to [3H]DNA co-migrated with three protein peaks during electrophoresis. This radioactivity was eliminated by prior treatment with DNAase. In addition, all of the radioactivity attributable to nascent DNA eluted with a specific protein on Sepharose 4B columns. This DNA - protein complex banded at a density of 1.58 gm/cm3 in sucrose-CsCl gradients. Treatment with DNAase, phospholipase A and C, and dilute alkali disrupted the complex. Moreover, 93% of the radioactivity attributable to protein and 70% of that attributable to DNA could be extracted from the complex with a chloroform/methanol solution. The results suggest that nascent DNA may be in a stable association with a proteolipid moiety of the nuclear membrane.     

DNA ligase I is associated with the 21 S complex of enzymes for DNA synthesis in HeLa cells.

Approximately 80% of the DNA ligase activity in HeLa cell extracts is associated with the 21 S enzyme complex that functions in simian virus 40 DNA replication in vitro (Malkas et al., Biochemistry 29, 6362-6374., 1990). The DNA ligase associated with the 21 S complex was purified extensively and its physical, enzymic and immunological properties characterized as DNA ligase I. The association of DNA ligase I with the 21 S complex of enzymes for DNA synthesis provides evidence for the physiological function of this DNA ligase in DNA replication in human cells.