WI-38 VA13 Cells Research Articles

A Knockout of the IFITM3 Gene Increases the Sensitivity of WI-38 VA13 Cells to the Influenza A Virus.

One of the ways to regulate the sensitivity of human cells to the influenza virus is to knock out genes of the innate immune response. Promising targets for the knockout are genes of the interferon-inducible transmembrane protein (IFITM) family, in particular the IFITM3 gene, whose product limits the entry of a virus into the cell by blocking the fusion of the viral and endosomal membranes. In this study, by means of genome-editing system CRISPR/Cas9, monoclonal cell lines with an IFITM3 knockout were obtained based on WI-38 VA13 cells (human origin). It was found that such cell lines are more sensitive to infection by influenza A viruses of various subtypes. Nevertheless, this feature is not accompanied by an increased titer of newly formed viral particles in a culture medium.

Tempol Inhibits the Growth of Lung Cancer and Normal Cells through Apoptosis Accompanied by Increased O2•- Levels and Glutathione Depletion.

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a stable, cell-permeable redox-cycling nitroxide water-soluble superoxide dismutase (SOD) mimetic agent. However, little is known about its cytotoxic effects on lung-related cells. Thus, the present study investigated the effects of Tempol on cell growth and death as well as changes in reactive oxygen species (ROS) and glutathione (GSH) levels in Calu-6 and A549 lung cancer cells, normal lung WI-38 VA-13 cells, and primary pulmonary fibroblast cells. Results showed that Tempol (0.5~4 mM) dose-dependently inhibited the growth of lung cancer and normal cells with an IC50 of approximately 1~2 mM at 48 h. Tempol induced apoptosis in lung cells with loss of mitochondrial membrane potential (MMP; ∆Ψm) and activation of caspase-3. There was no significant difference in susceptibility to Tempol between lung cancer and normal cells. Z-VAD, a pan-caspase inhibitor, significantly decreased the number of annexin V-positive cells in Tempol-treated Calu-6, A549, and WI-38 VA-13 cells. A 2 mM concentration of Tempol increased ROS levels, including O2•- in A549 and WI-38 VA-13 cells after 48 h, and specifically increased O2•- levels in Calu-6 cells. In addition, Tempol increased the number of GSH-depleted cells in Calu-6, A549, and WI-38 VA-13 cells at 48 h. Z-VAD partially downregulated O2•- levels and GSH depletion in Tempol-treated these cells. In conclusion, treatment with Tempol inhibited the growth of both lung cancer and normal cells via apoptosis and/or necrosis, which was correlated with increased O2•- levels and GSH depletion.

Amphotericin b effect on the sensitivity to influenza infection of WI-38 VA-13 cells with IFITM3 gene knockout

BACKGROUND: The application of CRISPR/Cas9 is one of the most rapidly developing areas in biotechnology. This method was used to obtain clones of а human origin cell line with knockout of one or more genes of the IFITM family, representing host restriction factors for influenza infection. Amphotericin B has previously been shown to promote influenza infection by blocking IFITM3 function.
 AIM: The aim of this study was to evaluate the effect of amphotericin B on the sensitivity of IFITM knockout cells to influenza A virus infection.
 MATERIALS AND METHODS: WI-38 VA-13 cells and mutant clones with IFITM3 knockout (F3 clone) or IFITM1, IFITM3 knockout (clone E12) were infected with influenza virus A/PR/8/34 (H1N1) in the presence or absence of amphotericin B. Forty-four hours after infection, the culture medium was taken to determine the infectious activity of the virus by titration in the MDCK cell culture, as well as the hemagglutinating activity of the virus. The infected cells were stained with fluorescently labeled antibodies against the viral NP protein, and the number of NP-positive cells was determined by flow cytometry.
 RESULTS: The addition of amphotericin B increased the hemagglutinating and infectious activity of the virus in WI-38 VA-13cells, while the difference was insignificant for clones with IFITM gene knockout. A similar dependency was obtained for the percent of infected cells.
 CONCLUSIONS: Mutant cells with a knockout of one or several genes of the IFITM family were equally susceptible to influenza infection regardless of the addition of amphotericin B, which confirms the crucial importance of a defect in the IFITM3 protein in increasing the permissiveness of cells to influenza A virus.

Tempol differently affects cellular redox changes and antioxidant enzymes in various lung-related cells

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a potential redox agent in cells. The present study investigated changes in cellular reactive oxygen species (ROS) and glutathione (GSH) levels and in antioxidant enzymes, in Tempol-treated Calu-6 and A549 lung cancer cells, normal lung WI-38 VA-13 cells, and primary pulmonary fibroblasts. Results demonstrated that Tempol (0.5–4 mM) either increased or decreased general ROS levels in lung cancer and normal cells at 48 h and specifically increased O2•− levels in these cells. In addition, Tempol differentially altered the expression and activity of antioxidant enzymes such as superoxide dismutase, catalase, and thioredoxin reductase1 (TrxR1) in A549, Calu-6, and WI-38 VA-13 cells. In particular, Tempol treatment increased TrxR1 protein levels in these cells. Tempol at 1 mM inhibited the growth of lung cancer and normal cells by about 50% at 48 h but also significantly induced cell death, as evidenced by annexin V-positive cells. Furthermore, down-regulation of TrxR1 by siRNA had some effect on ROS levels as well as cell growth inhibition and death in Tempol-treated or -untreated lung cells. In addition, some doses of Tempol significantly increased the numbers of GSH-depleted cells in both cancer cells and normal cells at 48 h. In conclusion, Tempol differentially increased or decreased levels of ROS and various antioxidant enzymes in lung cancer and normal cells, and induced growth inhibition and death in all lung cells along with an increase in O2•− levels and GSH depletion.

Nagilactone D ameliorates experimental pulmonary fibrosis in vitro and in vivo via modulating TGF-β/Smad signaling pathway

Pulmonary fibrosis is a prototypic chronic progressive lung disease with high morbidity and mortality worldwide. Novel effective therapeutic agents are urgently needed owing to the limited treatment options in clinic. Herein, nagilactone D (NLD), a natural dinorditerpenoid obtained from Podocarpus nagi, was found to suppress transforming growth factor-β1 (TGF-β1)-mediated fibrotic process in vitro and bleomycin (BLM)-induced pulmonary fibrosis in vivo. NLD attenuated TGF-β1-induced expression of fibrotic markers including type I and III collagen, fibronectin, α-SMA, and CTGF in human pulmonary fibroblasts (WI-38 VA-13 and HLF-1 cells). Mechanism study indicated that NLD suppressed TGF-β1-induced up-regulation of TβR I, and Smad2 phosphorylation, nuclear translocation, and transcriptional activation. Moreover, NLD ameliorated BLM-induced histopathological abnormalities in the lungs of experimental fibrotic mice, suppressed synthesis of relative fibrotic markers and fibroblast-to-myofibroblast transition, as well as BLM-induced up-regulation of TβR I expression and Smad signaling in mouse lungs. These data collectively support NLD to be a potential therapeutic agent for pulmonary fibrosis.

Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts

Nonmuscle myosin II (NMII) plays an important role in cytokinesis by constricting a contractile ring. However, it is poorly understood how NMII isoforms contribute to cytokinesis in mammalian cells. Here, we investigated the roles of the two major NMII isoforms, NMIIA and NMIIB, in cytokinesis using a WI-38 VA13 cell line (human immortalized fibroblast). In this cell line, NMIIB tended to localize to the contractile ring more than NMIIA. The expression level of NMIIA affected the localization of NMIIB. Most NMIIB accumulated at the cleavage furrow in NMIIA-knockout (KO) cells, and most NMIIA was displaced from this location in exogenous NMIIB-expressing cells, indicating that NMIIB preferentially localizes to the contractile ring. Specific KO of each isoform elicited opposite effects. The rate of furrow ingression was decreased and increased in NMIIA-KO and NMIIB-KO cells, respectively. Meanwhile, the length of NMII-filament stacks in the contractile ring was increased and decreased in NMIIA-KO and NMIIB-KO cells, respectively. Moreover, NMIIA helped to maintain cortical stiffness during cytokinesis. These findings suggest that appropriate ratio of NMIIA and NMIIB in the contractile ring is important for proper cytokinesis in specific cell types. In addition, two-photon excitation spinning-disk confocal microscopy enabled us to image constriction of the contractile ring in live cells in a three-dimensional manner.

A Study on VOCs Released by Lung Cancer Cell Line Using GCMS-SPME

The gas chromatography mass spectrometry (GCMS) with combination of Solid Phase Micro-extraction (SPME) was used to study the volatile organic compounds (VOCs) which emitted by the in-vitro cultured human cells and compared with documented volatile biomarker of lung cancer. For this purpose, the lung cancer cell (A549) and non-cancerous lung cell (WI38VA13) were cultured in identical growth medium, concurrently. The VOCs in the headspace of the cell cultures and the blank growth media (reference sample) were collected directly from the culture flask using SPME for 15minutes. The results show that two different volatile metabolites were screened out between A549 cells and Wi38VA13 cells. A549 cell found to emit 2 noticeable VOC which are decane and heneicosane. While for WI38VA13, the VOCs released were 1-Heptanol and heptadecane. The acquired VOCs were compared with the previous studies. The findings in this work conclude that the specific VOC of cells can be act as their odour signature and can be used to provide non-invasively screening of lung cancer using gas array sensor devices.

Relevance of simultaneous mono-ubiquitinations of multiple units of PCNA homo-trimers in DNA damage tolerance.

DNA damage tolerance (DDT) pathways, including translesion synthesis (TLS) and additional unknown mechanisms, enable recovery from replication arrest at DNA lesions. DDT pathways are regulated by post-translational modifications of proliferating cell nuclear antigen (PCNA) at its K164 residue. In particular, mono-ubiquitination by the ubiquitin ligase RAD18 is crucial for Polη-mediated TLS. Although the importance of modifications of PCNA to DDT pathways is well known, the relevance of its homo-trimer form, in which three K164 residues are present in a single ring, remains to be elucidated. Here, we show that multiple units of a PCNA homo-trimer are simultaneously mono-ubiquitinated in vitro and in vivo. RAD18 catalyzed sequential mono-ubiquitinations of multiple units of a PCNA homo-trimer in a reconstituted system. Exogenous PCNA formed hetero-trimers with endogenous PCNA in WI38VA13 cell transformants. When K164R-mutated PCNA was expressed in these cells at levels that depleted endogenous PCNA homo-trimers, multiple modifications of PCNA complexes were reduced and the cells showed defects in DDT after UV irradiation. Notably, ectopic expression of mutant PCNA increased the UV sensitivities of Polη-proficient, Polη-deficient, and REV1-depleted cells, suggesting the disruption of a DDT pathway distinct from the Polη- and REV1-mediated pathways. These results suggest that simultaneous modifications of multiple units of a PCNA homo-trimer are required for a certain DDT pathway in human cells.

Possible involvement of loss of imprinting in immortalization of human fibroblasts

Disruption of the normal pattern of parental origin-specific gene expression is referred to as loss of imprinting (LOI), which is common in various cancers. To investigate a possible role of LOI in the early stage of human cell transformation, we studied LOI in 18 human fibroblast cell lines immortalized spontaneously, by viral oncogenes, by chemical or physical carcinogens, or by infection with a retrovirus vector encoding the human telomerase catalytic subunit, hTERT cDNA. LOI was observed in all the 18 immortal cell lines. The gene most commonly exhibiting LOI was NDN which displayed LOI in 15 of the 18 cell lines (83%). The other genes exhibiting LOI at high frequencies were PEG3 (50%), MAGE-L2 (61%) and ZNF 127 (50%). Expression of NDN that was lost in the immortal cell lines was restored by treatment with 5-aza-2'-deoxycytidine. The ratio of histone H3 lysine 9 methylation to histone H3 lysine 4 methylation of the chromatin containing the NDN promoter in the immortal WI-38VA13 cells was greater than that in the parental cells, suggesting chromatin structure-mediated regulation of NDN expression. We previously demonstrated that inactivation of the p16INK4a/pRb pathway is necessary for immortalization of human cells. Human fibroblasts in the pre-crisis phase and cells with an extended lifespan that eventually senesce, both of which have the normal p16INK4a/pRb pathway, did not show LOI at any imprinted gene examined. Although it is not clear if LOI plays a causal role in immortalization of human cells or is merely coincidental, these findings indicate a possible involvement of LOI in immortalization of human cells or a common mechanism involved in both processes.

A method for detecting genetic toxicity using the RNA synthesis response to DNA damage

To date, biological risk assessment studies of chemicals that induce DNA lesions have been primarily based on the action of DNA polymerases during replication. However, DNA lesions interfere not only with replication but also with transcription. Therefore, detecting the damaging effects of DNA lesions during transcription might be important for estimating the safety of chemical mutagens and carcinogens. However, methods to address these effects have not been developed. Here, we report a simple, non-isotopic method for determining the toxicity of chemical agents by visualizing transcription in a mammalian cell system. The method is based on the measurement of the incorporation of bromouridine (as the uridine analogue) into the nascent RNA during RNA synthesis inhibition (RSI) induced by the stalling of RNA polymerases at DNA lesions on the transcribed DNA strand, which triggers transcription-coupled nucleotide excision repair (TC-NER). When we tested chemical agents (camptothecin, etoposide, 4-nitroquinoline-1-oxide, mitomycin C, methyl methanesulfonate, and cisplatin) in HeLa cells by the method, RSI indicative of genomic toxicity was observed in the nucleoli of the tested cells. This procedure provides the following advantages: 1) it uses common, affordable mammalian cells (HeLa cells, WI38VA13 cells, human dermal fibroblasts, or Chinese hamster ovary cells) rather than genetically modified microorganisms; 2) it can be completed within approximately 8 hr after the cells are prepared because RNA polymerase responses during TC-NER are faster than other DNA damage responses (replication, recombination, and apoptosis); and 3) it is safe because it uses non-radioactive bromouridine and antibodies to detect RNA synthesis on undamaged transcribed DNA strands.

Identification and characterization of WSG, a fusion gene associated with the proliferation of the WI-38 VA13 cells

ABSTRACTIt is well known that human fibroblasts can be immortalized using simian virus 40 (SV40) T antigen. However, the mechanisms of the SV40-immortalization processes remain unclear. In the present study, the authors identified and characterized a fusion gene, WSG (WI-38 VA13 Specific Gene), which has an integrated sequence of SV40 and chromosome 16p13. WSG is only detectable in WI-38 VA13 cells and not in other human cell lines or tissues. Transient transfection of the constructed pEGFP-WSG certified the WSG localization at the nuclear of HeLa cells. The growth assays and the knockdown experiment indicate that WSG is involved in the WI-38 VA13 cell proliferation. These results support potential capacities of WSG to be a candidate gene involved in proliferation of the WI-38 VA13 cells.

Fabrication of a microarray using a combination of the large circular sense and antisense DNA

In the present study, single-stranded large circular (LC)-sense molecules were utilized as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes. A microarray experiment using 284 LC-sense DNA probes found 6 upregulated and 7 downregulated genes in A549 cells as compared to WI38VA13 cells. Repeated experiments showed largely consistent results, and microarray data strongly correlated with data acquired from quantitative real-time RT-PCR. A large array comprising 5,079 LC-sense DNA was prepared, and analysis of the mean differential expression from dye-swap experiments revealed 332 upregulated and 509 downregulated genes in A549 cells compared to WI38VA13 cells. Subsequent functional analysis using an LC-antisense library of overexpressed genes identified 28 genes involved in A549 cell growth. These experiments demonstrated the proper features of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense and -antisense libraries for an effective functional validation of genes.

Epidermal growth factor competes with EGF receptor inhibitors to induce cell death in EGFR-overexpressing tumor cells

Epidermal growth factor receptor (EGFR) signaling plays an important role in cell growth and differentiation. Mutations in the EGFR gene and EGFR gene amplifications have been associated with increased responsiveness to selective EGFR tyrosine kinase inhibitors (EGFR-TKIs). By contrast, EGF may also stimulate apoptosis in tumor cells, depending on EGFR and Her2 ( erbB-2) expression levels. In the present study, we investigated cellular responses after EGFR activation by EGF, or inhibition by cetuximab and gefitinib. EGF treatment induced a near-immediate increase in p38 MAPK phosphorylation together with inactivation of ERK1/2. In contrast, gefitinib- and cetuximab-induced phosphorylation of p38 MAPK was much delayed, and gefitinib also induced a delayed activation of ERK1/2. EGF induced progressive cell death of A431 cells with prolonged treatment, whereas cetuximab- or gefitinib-treated cells showed temporary growth arrest and subsequent re-growth. Moreover, in combination treatment experiments, cetuximab or gefitinib competitively inhibited EGF-induced cell death. Normal WI38-VA13 cells did not display any noticeable changes in cell proliferation in response to EGF, gefitinib or cetuximab. EGF-induced death signaling is apparently irreversible: EGF induced significant EGFR phosphorylation/internalization and activated caspase-3, -8 and -9, effects that were not observed in cetuximab- or gefitinib-treated cells. Collectively, these results indicate that EGF may be a more potent cytotoxic agent than EGFR blockers in EGFR-overexpressing cancer cells.

Gene expression profiling in human lung fibroblast following cadmium exposure

Cadmium is a naturally occurring metallic element with food and smoking being the main sources of exposure in the non-occupationally exposed population. Chronic exposure to cadmium leads to tumors in a number of tissues including lung. In the present study we investigated genes whose expression is modified by Cd exposure in human lung fibroblast WI38-VA13 cells. We employed a cDNA microarray hybridization method to identify changes in the gene expression profile. Thirty five genes were identified as cadmium-responsive. Their level of expression differed significantly from controls (significance analysis of microarray; SAM, q < 5%). The largest groups of gene products affected by cadmium exposure were those involved in cell cycle, immunity and defense, nucleoside metabolism and signal transduction. Repressed expression of E2f1, Tubb and Actg2 following cadmium exposure may contribute to the cell cycle arrest. Down-regulation of Eno1 indicates a potential for causing protooncogene expression and possibly for cadmium-induced carcinogenicity. These results may contribute to better understand the toxic mechanism of cadmium toxicity. Moreover, the gene expression profile of cadmium could provide potential biomarkers for cadmium exposure.

A Peptide Derived from Tenascin-C Induces β1 Integrin Activation through Syndecan-4

Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated beta1 integrin-mediated cell adhesion of nonadherent and adherent cell types, by inducing activation of beta1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate (HS) proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced beta1 integrin activation: 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photoaffinity labeling; 2) heparitinase I treatment of cells abrogated beta1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) small interfering RNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced beta1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of beta1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with protein kinase Calpha inhibitor Gö6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of the TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains and then induced conformational change in beta1 integrin necessary for its functional activation. A lateral interaction of beta1 integrin with the extracellular region of the syndecan-4 molecule may be involved in this conformation change.

DNA uptake and repair enzyme access to transfected DNA is under reported by gene expression

Gene expression is the typical biological end point of interest following transfection. However, transcription may not accurately assess DNA uptake, or the ability of transfected DNA to be acted on by other enzymatic pathways. We have compared DNA uptake to gene expression and the unrelated enzymatic process of DNA double strand break (DSB) repair. Transfection efficiency (at limiting DNA concentration) was assessed as a function of DNA uptake and gene expression in the DSB repair proficient WI38VA13 and MO59K cell lines and the DSB repair defective cell line MO59J, by comparing eGFP expression from the pHygEGFP expression vector with uptake of rhodamine labeled linear pSP189 plasmid (3:1). Repair proficient cells expressed eGFP most efficiently, but never approached DNA uptake levels (⩾90%). Although transfected DNAs were stable in repair proficient cells and degraded in MO59J cells, most cells did not express eGFP, but in the repair proficient cells linear DNA did undergo DSB repair.

Overexpression of the FK506-Binding Immunophilin FKBP51 Is the Common Cause of Glucocorticoid Resistance in Three New World Primates

Many New World primates have high circulating levels of cortisol to compensate for the expression of glucocorticoid receptors (GRs) with low activity. Recent work in squirrel monkeys has suggested that this may be due to either the expression of GRs that are transcriptionally incompetent or the expression of an FK506-binding immunophilin that inhibits GR binding. The goal of this study was to resolve this controversy by determining the molecular basis of glucocorticoid resistance not only in species of squirrel monkeys but also in other glucocorticoid-resistant New World primates. First, the transcriptional activity of the GR from the Bolivian squirrel monkey was compared to that of the human GR. Incubation of COS-7 cells transfected with the squirrel monkey GR with 10 nM dexamethasone resulted in a robust stimulation of MMTV-luciferase activity (up to 260-fold), which was similar in magnitude to that achieved with the human GR. Second, the effect of FK506 on GR binding was determined in cytosol from cells from two species of squirrel monkeys as well as glucocorticoid-resistant cotton-top tamarins and owl monkeys. Incubation with 10 μM FK506 increased GR binding by at least 4-fold in cytosol from cells of each of the New World primates but had no effect on GR binding in cytosol from human WI-38 VA13 cells. Third, Western blots showed elevated expression of FKBP51 in New World primate cells and liver samples from two squirrel monkey species. On the other hand, the levels of FKBP52 were significantly lower in cells and liver from New World primates. The sequences of FKBP51 from the cotton-top tamarin, owl monkey and squirrel monkey are closely related and share differences from the human, rhesus monkey, mouse, and lemur FKBP51 sequences in the same 18 positions. Fourth, the relative activities of FKBP51 from the cotton-top tamarin, owl monkey and squirrel monkey were determined in cytosol mixing and GR transactivation studies and showed that FKBP51 from each of these primates was a potent inhibitor of GR activity. These results indicate that the elevated expression of FKBP51 contributes to glucocorticoid resistance in three New World primate genera.

Elevated superoxide production by active H-ras enhances human lung WI-38VA-13 cell proliferation, migration and resistance to TNF-alpha.

Accumulating evidence has suggested that cellular production of superoxide acts as an intracellular messenger to regulate gene expression and modulate cellular activities. In this report, we set out to investigate the role of active H-ras-mediated superoxide production on tumor cell malignancy in a SV-40 transformed human lung WI-38 VA-13 cell line. Stable transfection and expression of constitutively active mutant V12-H-ras (V12-H-ras) dramatically increased intracellular production of superoxide. The expression of V12-H-ras significantly enhanced cell proliferation, migration and resistance to TNF-alpha treatment compared to that of parental and vector control cells, while expression of wild type H-ras (WT-H-ras) only had modest effects. Upon scavenging by superoxide dismutase and other molecules that decrease the intracellular level of active H-ras mediated superoxide production, cell proliferation, migration and resistance to TNF-alpha were significantly reduced. Furthermore, we demonstrated that the activation of membrane NADPH oxidase activity by expression of active H-ras contributed to the intracellular superoxide production. The causal relationship between membrane superoxide production and increased cell proliferation, migration, and resistance to TNF-alpha by the expression of active H-ras, has provided direct evidence to demonstrate that superoxide acts as an intracellular messenger to cascade ras oncogenic signal relay and to modulate tumor malignant activity.

Gene targeted DNA double-strand break induction by (125)I-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells.

A parallel binding motif 16mer triplex-forming oligonucleotide (TFO) complementary to a polypurine-polypyrimidine target region near the 3'-end of the SupF gene of plasmid pSP189 was labeled with [5-(125)I]dCMP at position 15. Following triplex formation and decay accumulation, radiation-induced site-specific double-strand breaks (DSBs) were produced in the pSP189 SupF gene. Bulk damaged DNA and the isolated site-specific DSB-containing DNA were separately transfected into human WI38VA13 cells and allowed to repair prior to recovery and analysis of mutants. Bulk damaged DNA had a relatively low mutation frequency of 2.7 x 10(-3). In contrast, the isolated linear DNA containing site-specific DSBs had an unusually high mutation frequency of 7.9 x 10(-1). This was nearly 300-fold greater than that observed for the bulk damaged DNA mixture, and >1.5 x 10(4)-fold greater than background. The mutation spectra displayed a high proportion of deletion mutants targeted to the(125)I binding position within the SupF gene for both bulk damaged DNA and isolated linear DNA. Both spectra were characterized by complex mutations with mixtures of changes. However, mutations recovered from the linear site-specific DSB-containing DNA presented a much higher proportion of complex deletion mutations.

Cell–Substrate Contact: Another Factor May Influence Transepithelial Electrical Resistance of Cell Layers Cultured on Permeable Filters

Transepithelial resistance (TER) measurement has often been used to study the paracellular transport properties of epithelia grown on permeable filters, especially the barrier function of tight junctions. However, the TER value includes another source, the resistance caused by cell–substrate contact, that may give rise to a high TER value if cell–substrate separation is small. In this study we use electric cell–substrate impedance sensing (ECIS) to measure both paracellular resistance and the average cell–substrate distance of MDCK (II), HEp-2, and WI-38 VA13 cells. Comparing ECIS data with those from TER measurements of cell layers cultured on polycarbonate filters, we can obtain the approximate extra resistance resulting from cell–substrate contact for each cell type. The value of cell–substrate resistance was also estimated by two theoretical calculations that bracket the true values. Our results demonstrate that cell–substrate contact substantially influences the TER data measured using polycarbonate filters and that the extra resistance due to cell–substrate spaces depends on both cell type and filter property.