ATM-mediated mitochondrial damage response triggered by nuclear DNA damage in normal human lung fibroblasts

Ionizing radiation (IR) elevates mitochondrial oxidative phosphorylation (OXPHOS) in response to the energy requirement for DNA damage responses. Reactive oxygen species (ROS) released during mitochondrial OXPHOS may cause oxidative damage to mitochondria in irradiated cells. In this paper, we investigated the association between nuclear DNA damage and mitochondrial damage following IR in normal human lung fibroblasts. In contrast to low-doses of acute single radiation, continuous exposure of chronic radiation or long-term exposure of fractionated radiation (FR) induced persistent Rad51 and γ-H2AX foci at least 24 hours after IR in irradiated cells. Additionally, long-term FR increased mitochondrial ROS accompanied with enhanced mitochondrial membrane potential (ΔΨm) and mitochondrial complex IV (cytochrome c oxidase) activity. Mitochondrial ROS released from the respiratory chain complex I caused oxidative damage to mitochondria. Inhibition of ATM kinase or ATM loss eliminated nuclear DNA damage recognition and mitochondrial radiation responses. Consequently, nuclear DNA damage activates ATM which in turn increases ROS level and subsequently induces mitochondrial damage in irradiated cells.

In conclusion, we demonstrated that ATM is essential in the mitochondrial radiation responses in irradiated cells. We further demonstrated that ATM is involved in signal transduction from nucleus to the mitochondria in response to IR.     

Oxidative insults disrupt OPA1-mediated mitochondrial dynamics in cultured mammalian cells

Objective: To explore the impact of oxidative insults on mitochondrial dynamics. In mammalian cells, oxidative insults activate stress response pathways including inflammation, cytokine secretion, and apoptosis. Intriguingly, mitochondria are emerging as a sensitive network that may function as an early indicator of subsequent cellular stress responses. Mitochondria form a dynamic network, balancing fusion, mediated by optic atrophy-1 (OPA1), and fission events, mediated by dynamin-related protein-1 (DRP1), to maintain homeostasis.

Methods: Here, we examine the impact of oxidative insults on mitochondrial dynamics in 143B osteosarcoma and H9c2 cardiomyoblast cell lines via confocal microscopy, flow cytometry, and protein-based analyses.

Results: When challenged with hydrogen peroxide (H₂O₂), a ROS donor, both cell lines display fragmentation of the mitochondrial network and loss of fusion-active OPA1 isoforms, indicating that OPA1-mediated mitochondrial fusion is disrupted by oxidative damage in mammalian cells. Consistent with this, cells lacking OMA1, a key protease responsible for cleavage of OPA1, are protected against OPA1 cleavage and mitochondrial fragmentation in response to H₂O₂ challenge.

Discussion: Taken together, these findings indicate that oxidative insults damage OPA1-mediated mitochondrial dynamics in mammalian cells via activation of OMA1, consistent with an emerging role for mitochondrial dynamics as an early indicator of cellular stress signaling.     

Inhibitory effect of Tanshinone IIA on inverted formin-2 protects HaCaT cells against oxidative injury via regulating mitochondrial stress

Context: Epidermal cells play an important role in regulating the regeneration of skin after burns and wounds.

Objective: The aim of our study is to explore the role of Tanshinone IIA (Tan IIA) in the apoptosis of epidermal HaCaT cells induced by H₂O₂, with a focus on mitochondrial homeostasis and inverted formin-2 (INF2).

Materials and methods: Cellular viability was determined using the MTT assay, TUNEL staining, western blot analysis and LDH release assay. Adenovirus-loaded INF2 was transfected into HaCaT cells to overexpress INF2 in the presence of Tan IIA treatment. Mitochondrial function was determined using JC-1 staining, mitochondrial ROS staining, immunofluorescence and western blotting.

Results: Oxidative stress promoted the death of HaCaT cells and this effect could be reversed by Tan IIA. At the molecular levels, Tan IIA treatment sustained mitochondrial energy metabolism, repressed mitochondrial ROS generation, stabilized mitochondrial potential, and blocked the mitochondrial apoptotic pathway. Furthermore, we demonstrated that Tan IIA modulated mitochondrial homeostasis via affecting INF2-related mitochondrial stress. Overexpression of INF2 could abolish the protective effects of Tan IIA on HaCaT cells viability and mitochondrial function. Besides, we also reported that Tan IIA regulated INF2 expression via the ERK pathway; inhibition of this pathway abrogated the beneficial effects of Tan IIA on HaCaT cells survival and mitochondrial homeostasis.

Conclusions: Overall, our results indicated that oxidative stress-mediated HaCaT cells apoptosis could be reversed by Tan IIA treatment via reducing INF2-related mitochondrial stress in a manner dependent on the ERK signaling pathway.     

Renal mitochondrial oxidative stress is enhanced by the reduction of Sirt3 activity,in Zucker diabetic fatty rats

Objectives: Mitochondrial oxidative stress is involved in the pathogenesis of diabetic kidney disease. The objective of our study is to identify the mechanisms of renal mitochondrial oxidative stress, focusing on Sirt3, which is nicotinamide adenine dinucleotide (NAD⁺; oxidized NAD)-dependent deacetylase in mitochondria.

Methods: Renal mitochondrial oxidative stress and Sirt3 activity, using Zucker diabetic fatty rats (ZDFRs) and cultured proximal tubular cells under high-glucose condition were evaluated.

Results: At 28 weeks of age, ZDFRs exhibited the increased urinary albumin/liver-type fatty acid-binding protein (L-FABP)/8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion, histological tubular cell damage, compared to non-diabetic Zucker Lean rats. In renal mitochondria, acetylated isocitrate dehydrogenase2 (IDH2) and superoxide dismutase2 (SOD2), accompanied with mitochondrial oxidative stress and mitochondrial morphologic alterations, were increased in ZDFRs, indicating inactivation of Sirt3. Additionally, expression of the NAD-degrading enzyme, CD38, was increased, and the NAD⁺/NADH (reduced NAD) ratio was reduced in the renal cortex of ZDFRs. High-glucose stimulation in cultured proximal tubular cells also resulted in an increase in acetylated IDH2/SOD2, CD38 overexpression and a reduction in the NAD⁺/NADH ratio.

Conclusions: Enhancement of mitochondrial oxidative stress in the diabetic kidney was mediated by the reduction of Sirt3 activity. CD38 overexpression may be related to a reduction in the NAD⁺/NADH ratio in the diabetic kidney.     

Yap overexpression attenuates septic cardiomyopathy by inhibiting DRP1-related mitochondrial fission and activating the ERK signaling pathway

Context: Yes-associated protein (Yap) has been linked to several cardiovascular disorders, but the role of this protein in septic cardiomyocytes is not fully understood.

Objective: The aim of our study was to explore the influence of Yap in septic cardiomyopathy in vivo and in vitro.

Materials and methods: In the current study, Yap transgenic mice and Yap adenovirus-mediated gain-of-function assays were used in an LPS-established septic cardiomyopathy model. Mitochondrial function and mitochondrial fission were determined through western blotting, immunofluorescence analysis and ELISA.

Results: Our results demonstrated that Yap expression was downregulated by LPS, whereas Yap overexpression sustained cardiac function and attenuated cardiomyocyte death. The functional exploration revealed that LPS treatment induced cardiomyocyte mitochondrial stress, as manifested by mitochondrial superoxide overproduction, cardiomyocyte ATP deprivation, and caspase-9 apoptosis activation. Furthermore, we demonstrated that LPS-mediated mitochondrial damage was controlled by mitochondrial fission. However, Yap overexpression reduced mitochondrial fission and therefore improved mitochondrial function. A molecular investigation revealed that Yap overexpression inhibited mitochondrial fission by reversing ERK activity, and the inhibition of the ERK pathway promoted DRP1 upregulation and thereby mediated mitochondrial fission activation in the presence of Yap overexpression.

Conclusions: Overall, our results suggest that the cause of septic cardiomyopathy appears to be connected with Yap downregulation. The overexpression of Yap can attenuate myocardial inflammation injury through the reduction of DRP1-related mitochondrial fission in an ERK pathway activation-dependent manner.     

Downregulation of IDH2 exacerbates H2O2-mediated cell death and hypertrophy

Objectives: Reactive oxygen species-mediated cell death contributes to the pathophysiology of cardiovascular disease and myocardial dysfunction. We recently showed that mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) functions as an antioxidant and anti-apoptotic protein by supplying NADPH to antioxidant systems.

Methods: In the present study, we demonstrated that H₂O₂-induced apoptosis and hypertrophy of H9c2 cardiomyoblasts was markedly exacerbated by small interfering RNA (siRNA) specific for IDH2.

Results: Attenuated IDH2 expression resulted in the modulation of cellular and mitochondrial redox status, mitochondrial function, and cellular oxidative damage. MitoTEMPO, a mitochondria-targeted antioxidant, efficiently suppressed increased caspase-3 activity, increased cell size, and depletion of cellular GSH levels in IDH2 siRNA-transfected cells that were treated with H₂O₂.

Discussion: These results indicated that the disruption of cellular redox balance might be responsible for the enhanced H₂O₂-induced apoptosis and hypertrophy of cultured cardiomyocytes by the attenuated IDH2 expression.     

Mesenchymal stromal cells having inactivated RB1 survive following low irradiation and accumulate damaged DNA: Hints for side effects following radiotherapy

Following radiotherapy, bone sarcomas account for a significant percentage of recurring tumors. This risk is further increased in patients with hereditary retinoblastoma that undergo radiotherapy. We analyzed the effect of low and medium dose radiation on mesenchymal stromal cells (MSCs) with inactivated RB1 gene to gain insights on the molecular mechanisms that can induce second malignant neoplasm in cancer survivors.

MSC cultures contain subpopulations of mesenchymal stem cells and committed progenitors that can differentiate into mesodermal derivatives: adipocytes, chondrocytes, and osteocytes. These stem cells and committed osteoblast precursors are the cell of origin in osteosarcoma, and RB1 gene mutations have a strong role in its pathogenesis. Following 40 and 2000 mGy X-ray exposure, MSCs with inactivated RB1 do not proliferate and accumulate high levels of unrepaired DNA as detected by persistence of gamma-H2AX foci. In samples with inactivated RB1 the radiation treatment did not increase apoptosis, necrosis or senescence versus untreated cells. Following radiation, CFU analysis showed a discrete number of cells with clonogenic capacity in cultures with silenced RB1.

We extended our analysis to the other members of retinoblastoma gene family: RB2/P130 and P107. Also in the MSCs with silenced RB2/P130 and P107 we detected the presence of cells with unrepaired DNA following X-ray irradiation. Cells with unrepaired DNA may represent a reservoir of cells that may undergo neoplastic transformation. Our study suggests that, following radiotherapy, cancer patients with mutations of retinoblastoma genes may be under strict controls to evaluate onset of secondary neoplasms following radiotherapy.     

Global DNA methylation profiling in peripheral blood cells of South African women with gestational diabetes mellitus

Background/Objective: Recently, several studies have reported that DNA methylation changes in tissue are reflected in blood, sparking interest in the potential use of global DNA methylation as a biomarker for gestational diabetes mellitus (GDM). This study investigated whether global DNA methylation is associated with GDM in South African women.

Methods: Global DNA methylation was quantified in peripheral blood cells of women with (n?=?63) or without (n?=?138) GDM using the MDQ1 Imprint® DNA Quantification Kit.

Results: Global DNA methylation levels were not different between women with or without GDM and were not associated with fasting glucose or insulin concentrations. However, levels were 18% (p?=?0.012) higher in obese compared to non-obese pregnant women and inversely correlated with serum adiponectin concentrations (p?=?0.005).

Discussion: Contrary to our hypothesis, global DNA methylation was not associated with GDM in our population. These preliminary findings suggest that despite being a robust marker of overall genomic methylation that offers opportunities as a biomarker, global DNA methylation profiling may not offer the resolution required to detect methylation differences in the peripheral blood cells of women with GDM. Moreover, global DNA methylation in peripheral blood cells may not reflect changes in placental tissue. Further studies in a larger sample are required to explore the candidacy of a more targeted approach using gene-specific methylation as a biomarker for GDM in our population.     

MGMT hypomethylation is associated with DNA damage in workers exposed to low-dose benzene

Objective: This study aims to assess the effects of low-dose benzene on DNA damage and O⁶-methylguanine-DNA methyltransferase (MGMT) methylation in occupational workers.

Materials and methods: We recruited 96 nonsmoking male petrochemical industry workers exposed to low-dose benzene and 100 matched control workers. Urinary S-phenylmercapturic acid (SPMA) and S-benzylmercapturic acid (SBMA) were measured for indicating internal exposure of benzene and toluene. The degree of DNA damage was determined by the Comet assay. The levels of MGMT methylation were detected quantitatively by bisulphite-PCR pyrosequencing assay.

Results: The benzene-exposed workers had significantly higher levels of urinary SPMA, degree of DNA damage but decreased MGMT methylation than the controls (all p?<?0.05). In contrast, the level of urinary SBMA does not differ between benzene-exposed workers and the controls. In all participants, MGMT methylation was negatively associated with the urinary SPMA and the degree of DNA damage, indicating that epigenetic regulation might be involved in response to low-dose benzene exposure-induced genetic damage.

Discussion and conclusion: MGMT methylation could be a potent biomarker associated with low-dose benzene exposure and benzene-induced DNA damage.     

Comet assay for assessment of DNA damage in greenhouse workers exposed to pesticides

Purpose: The main goal of the present study was to determine DNA damage in pesticide-exposed greenhouse workers and pesticides non-exposed controls.

Materials and methods: The DNA damage was measured by alkaline comet assay method (pH?>?13) in 41 greenhouse workers and 45 non-exposed individuals as the control. Pesticide exposure was assessed by duration of working in the greenhouse and pesticide application in the greenhouse time. DNA damage was estimated by arbitrary unit and damage frequency.

Results: Arbitrary unit and damage frequency were consistently significantly higher in greenhouse workers than those of the controls (p?=?0.001). In terms of gender in greenhouse, DNA damage of female workers was significantly higher than those in male workers (p?<?0.05). We found significant correlation between DNA damage and working hours spent. Multiple linear regression analysis showed that working hours in the greenhouse as an indication of pesticide exposure were significantly associated with the DNA damage, which can be attributed to the genotoxic potential of the pesticide mixture.

Conclusions: The comet assay is sensitive to detect the damage exposed to chronic effect of pesticides in greenhouse workers. Significant DNA damage was obtained for the exposed group, which was associated with the pesticide exposure.     

Micronucleus frequency and content in healthy relatives of cancer patients

Objective: To compare chromosomal damage in lymphocytes of individuals who did or did not report first-degree relatives with cancer.

Materials and methods: Cases and controls (68 each) were matched for sex, age and radon exposure. Chromosomal damage was quantified as frequency of micronucleus-containing cells and proportion of centromere-free micronuclei.

Results: Individuals not reporting cancer in their families showed lower values of both the frequency of micronucleus-containing cells (n.s.) and the proportion of centromere-free micronuclei (p?<?0.05) in some subgroups, but not in all.

Conclusion: The chromosomal damage observed may be due to inheritable genomic instability, but environmental influences cannot be excluded.     

Micronuclei as a marker for medical screening of subjects continuously occupationally exposed to low doses of ionizing radiation

Context: Genotoxicity assays are widely employed in human biomonitoring studies to assess genetic damage inflicted by genotoxic agents.

Objective: Evaluation of micronuclei (MN) as a screening marker of occupational ionizing radiation (IR) exposure.

Materials and methods: Using micronucleus test, peripheral blood lymphocytes (PBL) of 402 control and exposed subjects were screened for genetic damage.

Results: The mean frequencies of micronucleus test parameters were significantly higher in exposed persons. Increase of micronucleus yield with duration of exposure (DOE) by 0.303MN/year was revealed.

Discussion and conclusion: The obtained data encourage us to consider MN as valuable markers for preventive medical screening of occupationally exposed groups.     

CDK1 inhibition sensitizes normal cells to DNA damage in a cell cycle dependent manner

Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner.

Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.     

Evaluation of genetic diversity and population structure of Macqueen’s Bustard Chlamydotis macqueenii in Iran

Capsule: There is low genetic diversity in the Macqueen’s Bustard Chlamydotis macqueenii in Iran.

Aims: To investigate the genetic diversity and population structure of Macqueen’s Bustard in Iran, using two mitochondrial DNA loci.

Methods: Molecular diversity of the mitochondrial cytochrome oxidase c subunit I (COI) gene and part of the mitochondrial control region D-loop (in total 1183 base pairs) were analysed from 26 individual Macqueen’s Bustards from three regions of Iran.

Results: There was little variation in nucleotides and haplotypes in the populations for genes of both CR and COI. The population had free breeding and gene flow between the three study regions in Iran: Petregan, Ferdows and Yazd.

Conclusion: The use of molecular and genetic studies is essential to strengthen the protection of genetic diversity of the Macqueen’s Bustard.     

Proteomic analysis of mitochondria: biological and clinical progresses in cancer

Introduction: Mitochondria play important roles in regulating multiple biological processes and signalling pathways in eukaryotic cells, and mitochondrial dysfunction may result in a wide range of serious diseases, including cancer. With improvements in the identification of mitochondrial proteins, mitochondrial proteomics has made great achievements. In particular, this approach has been widely used to compare tumour cells at different stages of malignancy. Therefore, there is an urgent need to identify and characterize the function of mitochondrial proteins in cancer progression and to determine the involved mechanisms.

Areas covered: We provide an overview of recent progress related to mitochondrial proteomics in cancer and the application of comparative mitochondrial proteomics in various biological processes, including apoptosis, necroptosis, autophagy and metastasis, as well as clinical progress in cancer. Proteomics-related reports were found using PubMed and Google Scholar databases.

Expert commentary: Understanding both post-translational modification and post-translational processing is important in the comprehensive characterization of protein function. The application of comparative mitochondrial proteomics to investigate clinical samples and cancer cells will contribute to our understanding of the molecular interplay of mitochondrial proteins in the development of cancer. This approach will mine more biomarkers for diagnosis and prognosis and improve therapeutic outcomes among cancer patients.     

Decellularized spleen matrix for reengineering functional hepatic-like tissue based on bone marrow mesenchymal stem cells

Background and Aims: Decellularized liver matrix (DLM) hold great potential for reconstructing functional hepatic-like tissue (HLT) based on reseeding of hepatocytes or stem cells, but the shortage of liver donors is still an obstacle for potential application. Therefore, an appropriate alternative scaffold is needed to expand the donor pool. In this study, we explored the effectiveness of decellularized spleen matrix (DSM) for culturing of bone marrow mesenchymal stem cells (BMSCs), and promoting differentiation into hepatic-like cells.

Methods: Rats' spleen were harvested for DSM preparation by freezing/thawing and perfusion procedure. Then the mesenchymal stem cells derived from rat bone marrow were reseeded into DSM for dynamic culture and hepatic differentiation by a defined induction protocol.

Results: The research found that DSM preserved a 3-dimensional porous architecture, with native extracellular matrix and vascular network which was similar to DLM. The reseeded BMSCs in DSM differentiated into functional hepatocyte-like cells, evidenced by cytomorphology change, expression of hepatic-associated genes and protein markers, glycogen storage, and indocyanine green uptake. The albumin production (2.74±0.42 vs. 2.07±0.28 pg/cell/day) and urea concentration (75.92±15.64 vs. 52.07±11.46 pg/cell/day) in DSM group were remarkably higher than tissue culture flasks (TCF) group over the same differentiation period, P< 0.05.

Conclusion: This present study demonstrated that DSM might have considerable potential in fabricating hepatic-like tissue, particularly because it can facilitate hepatic differentiation of BMSCs which exhibited higher level and more stable functions.     

Hesperetin mitigates acrolein-induced apoptosis in lung cells in vitro and in vivo

Objectives: A number of studies have suggested that acrolein-induced lung injury and pulmonary diseases are associated with the depletion of antioxidants and the production of reactive oxygen species. Therefore, compounds that scavenge reactive oxygen species may exert protective effects against acrolein-induced apoptosis. Because hesperetin, a natural flavonoid, has been reported to have an antioxidant activity, we investigated the effect of hesperitin against acrolein-induced apoptosis of lung cells.

Methods: We evaluated the protective role of hesperetin in acrolein-induced lung injury using Lewis lung carcinoma (LLC) cells and mice.

Results: Upon exposure of LLC cells and mice to acrolein, hesperetin ameliorated the lung inbjury through attenuation of oxidative stress.

Conclusion: In the present report, we demonstrate that hesperetin exhibits a protective effect against acrolein-induced apoptosis of lung cells in both in vitro and in vivo models. Our study provides a useful model to investigate the potential application of hesperetin for the prevention of lung diseases associated with acrolein toxicity.     

Mitochondrial reactive oxygen species-mediated genomic instability in low-dose irradiated human cells through nuclear retention of cyclin D1

Mitochondria are associated with various radiation responses, including adaptive responses, mitophagy, the bystander effect, genomic instability, and apoptosis. We recently identified a unique radiation response in the mitochondria of human cells exposed to low-dose long-term fractionated radiation (FR). Such repeated radiation exposure inflicts chronic oxidative stresses on irradiated cells via the continuous release of mitochondrial reactive oxygen species (ROS) and decrease in cellular levels of the antioxidant glutathione. ROS-induced oxidative mitochondrial DNA (mtDNA) damage generates mutations upon DNA replication. Therefore, mtDNA mutation and dysfunction can be used as markers to assess the effects of low-dose radiation. In this study, we present an overview of the link between mitochondrial ROS and cell cycle perturbation associated with the genomic instability of low-dose irradiated cells. Excess mitochondrial ROS perturb AKT/cyclin D1 cell cycle signaling via oxidative inactivation of protein phosphatase 2A after low-dose long-term FR. The resulting abnormal nuclear accumulation of cyclin D1 induces genomic instability in low-dose irradiated cells.     

Novel ortho ester-based,pH-sensitive cationic lipid for gene delivery in vitro and in vivo

Context: Cationic lipoplexes are less toxic than viral gene vectors and more convenient to prepare but their efficiencies of gene delivery are generally lower.

Objective: To develop ortho ester-based, pH-sensitive lipoplexes for efficient gene delivery both in cultured cells and in vivo.

Materials and methods: A novel cationic and acid-labile lipid (DOC) containing a cationic headgroup and a cholesterol-derived lipid tail joined together by an acid-labile ortho ester linker was designed and synthesized. DOC was formulated into liposomes with the conical helper lipid DOPE, and then into lipoplexes with plasmid DNA encoding a luciferase reporter gene. The physicochemical properties of the lipoplexes (size, surface charge and pH-sensitivity) were characterized. Gene delivery by DOC/DOPE/DNA lipoplexes was also evaluated in CV-1 cells and in CD-1 mice following intratracheal injection. Lipoplexes consisting of the acid-stable cationic lipid DC-Chol were characterized as a control.

Results: DOC formed cationic lipoplexes with DOPE and DNA. After incubation at acidic pH 4.6, DOC/DOPE/DNA lipoplexes lost their positive charges and aggregated with one another as a result of DOC hydrolysis. Both in CV-1 cell culture and in CD-1 mice, DOC/DOPE/DNA lipoplexes increased the luciferase gene expression by 5- to 10-fold compared with the analogous but acid-stable DC-Chol/DOPE/DNA lipoplexes.

Discussion and conclusion: Incorporation of an acid-labile ortho ester linker into a cationic lipid is a viable approach to enhance gene delivery by the corresponding lipoplexes both in cultured cells and in vivo.