HeLa S3 Research Articles

Veluflavanones A-P, Cytotoxic Geranylated Flavanones from Dalbergia velutina Stems.

Sixteen new geranylated flavanones, named veluflavanones A-P (1-16), and a known analogue (17), were isolated from Dalbergia velutina. The chemical structures of 1-17, as well as their absolute configurations, were determined by spectroscopic analysis and experimental ECD data. All isolated compounds were tested for their cytotoxicity against five human cancer cell lines. Compound 9 showed cytotoxicity toward KB, HeLa S3, and MCF-7 cells with IC50 values of 9.9, 8.1, and 10.0 μM, respectively. In addition, compounds 10, 11, 14, and 16 exhibited selective cytotoxicity against HeLa S3 cells with IC50 values of 6.6-9.9 μM.

Inhibitors of the VEGF Receptor Suppress HeLa S3 Cell Proliferation via Misalignment of Chromosomes and Rotation of the Mitotic Spindle, Causing a Delay in M-Phase Progression.

Cell division is the process by which replicated chromosomes are separated into two daughter cells. Although regulation of M phase has been extensively investigated, not all regulating factors have been identified. Over the course of our research, small molecules were screened to identify those that regulate M phase. In the present study, the vascular endothelial growth factor receptor (VEGFR) inhibitors A83-01, SU4312, and Ki8751 were examined to determine their effects on M phase. Treatment of HeLa S3 cells with these inhibitors suppressed cell proliferation in a concentration-dependent manner, and also suppressed Akt phosphorylation at Ser473, a marker of Akt activation. Interestingly, cleaved caspase-3 was detected in Adriamycin-treated cells but not in inhibitor-treated cells, suggesting that these inhibitors do not suppress cell proliferation by causing apoptosis. A cell cycle synchronization experiment showed that these inhibitors delayed M phase progression, whereas immunofluorescence staining and time-lapse imaging revealed that the M phase delay was accompanied by misalignment of chromosomes and rotation of the mitotic spindle. Treatment with the Mps1 inhibitor AZ3146 prevented the SU4312-induced M phase delay. In conclusion, the VEGFR inhibitors investigated here suppress cell proliferation by spindle assembly checkpoint-induced M phase delay, via misalignment of chromosomes and rotation of the mitotic spindle.

Collaborative study of thresholds for mutagens: proposal of a typical protocol for detection of hormetic responses in cytotoxicity tests

BackgroundAccording to the linear no-threshold model (LNT), even the smallest amount of radiation is hazardous. Although the LNT is not based on solid data, this hypothesis has been applied to mutagens and carcinogens. As a result, it has been postulated that there are no thresholds for these chemicals. To demonstrate negativity by experiments is practically impossible, because negative data may leave behind the possibility that additional data might make the resolution power high enough to change negativity to positivity. Furthermore, additional data collection may be endless and we may be trapped in agnosticism. When hormesis is established, in which biological responses are higher at low-doses and lower at high-doses than the control, thresholds could be established between the low- and high-doses. Before examination of thresholds in chemical mutagenesis, hormetic responses in cytotoxicity were tested using cultured mammalian cells.MethodHuman cells (HeLa S3 and TK6) or Chinese hamster cells (CHL/IU) were cultured in 96-well plates and treated with mitomycin C (MMC) or ethyl methanesulfonate (EMS) at various dose levels and optical density was measured after addition of a reagent to detect cellular activity. In hormetic responses, data might fluctuate to and fro; therefore, experimental conditions were examined from various aspects to eliminate confounding factors including cell numbers, detection time, the edge effect of 96-well plates, and measurement time after addition of the reagent for detection.ResultsThe dose response relationship was never linear. Cellular activities after treatment with MMC or EMS were generally higher at lower doses levels and lower at higher doses than the control, showing hormesis and allowing the establishment of thresholds. Dose response curves sometimes showed two or three peaks, probably reflecting different cellular responses.ConclusionHormetic responses in cytotoxicity tests were observed and thresholds could be established. Based on the results of this investigation, we put forward a tentative protocol to detect chemical hormesis in cytotoxicity tests, i.e., inoculate 2000 cells per well, add various doses of a test chemical 48 h after inoculation, add a detection dye 10 h after treatment, and measure optical density 2 h after addition of the reagent for detection.

A Flow Cytometry-Based Method to Determine the Titer of Adenoviruses Expressing an Extraneous Gene.

In recent times, oncolytic viruses expressing an extraneous gene have attracted great interest; in fact, they have been engaged in multiple applications, such as medicine for cancer. Our group made an oncolytic adenovirus, namely, OBP-301, for use in treating solid cancers and press clinical trial to get approval for a pharmaceutical product. In this study, we applied a flow cytometry-based method to determine the titer of adenoviruses expressing an extraneous gene as well as assess their quality. We considered using the green fluorescent protein (GFP)50 titer as a measure of viral quality. The GFP50 titer (GFP50/mL) is the viral load required to render the HeLa S3 cell line 50% GFP-positive by analysing flow cytometry data. We measured the GFP50 titers for three types of recombinant adenoviruses (OBP-401, OBP-1101, and OBP-1106). We compared GFP50/mL and tissue culture infectious dose (TCID50/mL), a conventional titration index, and found that these titers showed a linear correlation, with a correlation coefficient of >0.9. Moreover, GFP50/mL showed high repetitive accuracy. We expect this flow cytometry-based method to be useful in case of clinically relevant viruses expressing an extraneous gene, in particular, to control viral quality.

Specific binding of PCBP1 to heavily oxidized RNA to induce cell death

In aerobically growing cells, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine (8-oxoG), which induces alteration in their gene expression. We previously demonstrated that the human AUF1 protein binds to 8-oxoG in RNA to induce the selective degradation of oxidized messenger RNA. We herein report that the poly(C)-binding protein PCBP1 binds to more severely oxidized RNA to activate apoptosis-related reactions. While AUF1 binds to oligoribonucleotides carrying a single 8-oxoG, PCBP1 does not bind to such oligoribonucleotides but instead binds firmly to oligoribonucleotides in which two 8-oxoG residues are located nearby. PCBP1-deficient cells, constructed from the human HeLa S3 line using the CRISPR-Cas9 system, exhibited higher survival rates than HeLa S3 cells when small doses of hydrogen peroxide were applied. The levels of caspase-3 activation and PARP-1 cleavage in the PCBP1-deficient cells were significantly lower than those in wild-type cells. The structure-function relationship of PCBP1 was established with the use of PCBP1 mutant proteins in which the conserved KH domains were defective. Human cells appear to possess two distinct mechanisms, one controlled by AUF1 and the other by PCBP1, with the former functioning when messenger RNA is moderately oxidized and the latter operating when the RNA is more severely damaged.

Spatio-temporal dynamics of calcium electrotransfer during cell membrane permeabilization.

Pulsed electric fields (PEFs) are applied as physical stimuli for DNA/drug delivery, cancer therapy, gene transformation, and microorganism eradication. Meanwhile, calcium electrotransfer offers an interesting approach to treat cancer, as it induces cell death easier in malignant cells than in normal cells. Here, we study the spatial and temporal cellular responses to 10μs duration PEFs; by observing real-time, the uptake of extracellular calcium through the cell membrane. The experimental setup consisted of an inverted fluorescence microscope equipped with a color high-speed framing camera and a specifically designed miniaturized pulsed power system. The setup allowed us to accurately observe the permeabilization of HeLa S3 cells during application of various levels of PEFs ranging from 0.27 to 1.80kV/cm. The low electric field experiments confirmed the threshold value of transmembrane potential (TMP). The high electric field observations enabled us to retrieve the entire spatial variation of the permeabilization angle (θ). The temporal observations proved that after a minimal permeabilization of the cell membrane, the ionic diffusion was the prevailing mechanism of the delivery to the cell cytoplasm. The observations suggest 0.45kV/cm and 100 pulses at 1kHz as an optimal condition to achieve full calcium concentration in the cell cytoplasm. The results offer precise levels of electric fields to control release of the extracellular calcium to the cell cytoplasm for inducing minimally invasive cancer calcium electroporation, an interesting affordable method to treat cancer patients with minimum side effects.

Augmentation of Myc-Dependent Mitotic Gene Expression by the Pygopus2 Chromatin Effector

Mitotic segregation of chromosomes requires precise coordination of many factors, yet evidence is lacking as to how genes encoding these elements are transcriptionally controlled. Here, we found that the Pygopus (Pygo)2 chromatin effector is indispensable for expression of the MYC-dependent genes that regulate cancer cell division. Depletion of Pygo2 arrested SKOV-3 cells at metaphase, which resulted from the failure of chromosomes to capture spindle microtubules, a critical step for chromosomal biorientation and segregation. This observation was consistent with global chromatin association findings in HeLa S3 cells, revealing the enrichment of Pygo2 and MYC at promoters of biorientation and segmentation genes, at which Pygo2 maintained histone H3K27 acetylation. Immunoprecipitation and proximity ligation assays demonstrated MYC and Pygo2 interacting in nuclei, corroborated in a heterologous MYC-driven prostate cancer model that was distinct from Wnt/β-catenin signaling. Our evidence supports a role for Pygo2 as an essential component of MYC oncogenic activity required for mitosis.

Icotinib hydrochloride enhances chemo- and radiosensitivity by inhibiting EGFR signaling and attenuating RAD51 expression and function in Hela S3 cells.

BackgroundRadiotherapy and cisplatin-based chemotherapy are currently considered as standard treatments employed for advanced cervical cancer (CC). However, patients with local recurrence or distant metastasis continue to have poor outcomes. EGFR overexpression correlated with chemo/radioresistance, and disease failure has been well proved in the previous studies. Hence, the aim of this study was to explore the therapeutic efficacy and underlying mechanism of the sensitization to radiation or cisplatin of icotinib hydrochloride (IH), a high-selective EGFR tyrosine kinase inhibitor (TKI), in the Hela S3 human CC cell line.MethodsCell proliferation was measured with cell counting kit-8 (CCK-8) assay. Flow cytometry analysis was performed to examine cell cycle distribution and apoptosis. The phosphorylation of EGFR and its downstream signaling molecules were measured by Western blot analysis. γ-H2AX foci and RAD51 foci in the cellular nucleus were visualized using immunofluoresence staining. Expression levels of RAD51 in the whole cells and subceullar fractions were detected to demonstrate the impact of IH on DNA repair.ResultsIH can significantly inhibit cell proliferation, redistribute cell cycle, enhance apoptosis and impair DNA damage response of Hela S3 cells following radiation or cisplatin treatment through suppressing the activation of the EGFR signaling pathway and attenuating the expression and function of homologous recombination (HR) protein RAD51.ConclusionThis study suggests that IH is a potential sensitizer in radiotherapy and cisplatin-based chemotherapy for CC and RAD51 may serve as a prognosis biomarker for this combination treatment.

Full-length myocilin protein is purified from mammalian cells as a dimer

Myocilin (MYOC) is a secreted protein found in human aqueous humor (AH) and mutations in the MYOC gene are the most common mutation observed in glaucoma patients. Human AH analyzed under non-reducing conditions suggests that MYOC is not normally found in a monomeric form, but rather is predominantly dimeric. Although MYOC was first reported almost 20 years ago, a technical challenge still faced by researchers is an inability to isolate full-length MYOC protein for experimental purposes. Herein we describe two methods by which to isolate sufficient quantities of human full-length MYOC protein from mammalian cells. One method involved identification of a cell line (HeLa S3) that would secrete full-length protein (15 mg/L) while the second method involved a purification approach from 293 cells requiring identification and modification of an internal MYOC cleavage site (Glu214/Leu215). MYOC protein yield from 293 cells was improved by mutation of two MYOC N-terminal cysteines (C47 and C61) to serines. Analytical size exclusion chromatography of our full-length MYOC protein purified from 293 cells indicated that it is predominantly dimeric and we propose a structure for the MYOC dimer. We hope that by providing methods to obtain MYOC protein, researchers will be able to utilize the protein to obtain new insights into MYOC biology. The ultimate goal of MYOC research is to better understand this target so we can help the patient that carries a MYOC mutation retain vision and maintain quality of life.

LINP1 facilitates DNA damage repair through non-homologous end joining (NHEJ) pathway and subsequently decreases the sensitivity of cervical cancer cells to ionizing radiation

ABSTRACTLncRNA in non-homologous end joining (NHEJ) pathway 1 (LINP1) is an lncRNA which promotes therapeutic resistance in triple-negative breast cancer (TNBC). However, the expression and function of LINP1 in cervical cancer is not yet well-understood. In this study, we evaluated the expression levels of LINP1 in tumor tissues and cell lines of cervical cancer. We found that LINP1 associates with NHEJ proteins (Ku80 and DNA-PKcs). LINP1 translocates from cytosol to nucleus in response to irradiation. In addition, LINP1 knockdown significantly increases the levels of cleaved caspase3 and PARP, leading to enhanced cell apoptosis after ionizing radiation (IR). LINP1-knockdown cells showed delayed repairs of DNA double-strand breaks (DSBs) after IR. Finally, LINP1 knockdown increases radiosensitivity of Hela S3 cells. These results suggest that LINP1 facilitates DSBs repair through NHEJ pathway and may thus serve as a prognostic marker and a potential target for the therapy of cervical cancer.

Simultaneous Proteoform Analysis of Histones H3 and H4 with a Simplified Middle-Down Proteomics Method.

Dynamic post-translational modifications of histones regulate transcriptional gene expression in eukaryotes. Unique combinations of modifications, almost exclusively displayed at the flexible N-terminal tails on histones, create distributions of proteoforms that need to be characterized in order to understand the complexity of gene regulation and how aberrant modification patterns influence disease. Although mass spectrometry is a preferred method for the analysis of histone modifications, information is lost when using conventional trypsin-based histone methods. Newer "middle-down" protocols may retain a greater fraction of the full proteoform distribution. We describe a strategy for the simultaneous characterization of histones H3 and H4 with near-complete retention of proteoform distributions, using a conventional proteomics liquid chromatography-tandem mass spectrometry (LC-MS/MS) configuration. The selective prolyl endoprotease neprosin generates convenient peptide lengths for retention and dispersion of modified H3 and H4 peptides on reversed-phase chromatography, offering an alternative to the hydrophilic interaction liquid chromatography typically used in middle-down methods. No chemical derivatizations are required, presenting a significant advantage over the trypsin-based protocol. Over 200 proteoforms can be readily profiled in a single analysis of histones from HeLa S3 cells. An in-gel digestion protocol provides additional options for effective histone analysis.

The roles of human MTH1, MTH2 and MTH3 proteins in maintaining genome stability under oxidative stress

The hydrolysis of nucleotides containing 8-oxo-7,8-dihydroguanine (8-oxoG) is important in the maintenance of genome stability. Human cells possess three types of proteins, MTH1 (NUDT1), MTH2 (NUDT15) and MTH3 (NUDT18), which have the potential to hydrolyze deoxyribonucleoside di- and triphosphates containing 8-oxoG to the monophosphate, the form of which is unusable for DNA synthesis. To elucidate the physiological roles of these enzymes, we constructed single knockout (KO) cell lines for each of the MTH1, MTH2 and MTH3 genes and MTH1 and MTH2-double KO cell lines from the human HeLa S3 line using CRISPR/Cas9. With the exception of MTH3-KO, all of the KO cell lines showed similar proliferation rates to the parental line, HeLa S3, indicating that the MTH1 and MTH2 functions are dispensable for cell growth. On the other hand, the MTH3-KO cells showed a significantly slower growth rate, suggesting that MTH3 has a definite role in cell growth in addition to the cleavage of 8-oxoG-containing deoxyribonucleotide. MTH1-KO, MTH2-KO and MTH1- MTH2-KO cells exhibited increased sensitivity to hydrogen peroxide, whereas MTH3-KO did not. MTH1-KO cells showed only a slight increase in mutant frequency in comparison to the parental HeLa S3 line. The overproduction of MTH1 and MTH2 suppressed the mutator phenotype of mutT-deficient E. coli cells, whereas the overproduction of MTH3 did not show such a suppressive effect. Our findings suggest that both MTH1 and MTH2 are involved in the maintaining genome stability in human cells against oxidative stress, while MTH3 may play some other role(s).

Cell Death Induced by Nanosecond Pulsed Electric Fields and its Dependence on Pulse Duration

SUMMARYWe discuss the death process of human cervical cancer cells HeLa S3 induced by two types of nonthermal intense electrical pulses of different durations and amplitudes, 120 ns‐long, 12.5 kV/cm and 5 ns‐long, 250 kV/cm. WST‐8 Caspase 3, ASK1 and BAX, were analyzed by using the antibody assay and flow cytometry. Apoptosis activity was evaluated by means of the TUNEL assay and flow cytometry. Also, Ca2+ dependence of the death process and the protein expression were explored by using the metal ion chelating agent, EDTA. Our experiments demonstrated that 5 ns and 120 ns pulses give quite different biological stresses leading to the different death process and the protein expressions. The cell death induced by 120 ns pulses are dependent on the presence of Ca2+ and accompanied by expression of proteins associated with apoptosis and stress response. Conversely, the protein expression induced by 5 ns pulses was independent of the presence of Ca2+, whereas the death was dependent on Ca2+.

Comparison between micro- and nanosized copper oxide and water soluble copper chloride: interrelationship between intracellular copper concentrations, oxidative stress and DNA damage response in human lung cells

BackgroundNano- and microscale copper oxide particles (CuO NP, CuO MP) are applied for manifold purposes, enhancing exposure and thus the potential risk of adverse health effects. Based on the pronounced in vitro cytotoxicity of CuO NP, systematic investigations on the mode of action are required. Therefore, the impact of CuO NP, CuO MP and CuCl2 on the DNA damage response on transcriptional level was investigated by quantitative gene expression profiling via high-throughput RT-qPCR. Cytotoxicity, copper uptake and the impact on the oxidative stress response, cell cycle regulation and apoptosis were further analysed on the functional level.ResultsCytotoxicity of CuO NP was more pronounced when compared to CuO MP and CuCl2 in human bronchial epithelial BEAS-2B cells. Uptake studies revealed an intracellular copper overload in the soluble fractions of both cytoplasm and nucleus, reaching up to millimolar concentrations in case of CuO NP and considerably lower levels in case of CuO MP and CuCl2. Moreover, CuCl2 caused copper accumulation in the nucleus only at cytotoxic concentrations. Gene expression analysis in BEAS-2B and A549 cells revealed a strong induction of uptake-related metallothionein genes, oxidative stress-sensitive and pro-inflammatory genes, anti-oxidative defense-associated genes as well as those coding for the cell cycle inhibitor p21 and the pro-apoptotic Noxa and DR5. While DNA damage inducible genes were activated, genes coding for distinct DNA repair factors were down-regulated. Modulation of gene expression was most pronounced in case of CuO NP as compared to CuO MP and CuCl2 and more distinct in BEAS-2B cells. GSH depletion and activation of Nrf2 in HeLa S3 cells confirmed oxidative stress induction, mainly restricted to CuO NP. Also, cell cycle arrest and apoptosis induction were most distinct for CuO NP.ConclusionsThe high cytotoxicity and marked impact on gene expression by CuO NP can be ascribed to the strong intracellular copper ion release, with subsequent copper accumulation in the cytoplasm and the nucleus. Modulation of gene expression by CuO NP appeared to be primarily oxidative stress-related and was more pronounced in redox-sensitive BEAS-2B cells. Regarding CuCl2, relevant modulations of gene expression were restricted to cytotoxic concentrations provoking impaired copper homoeostasis.

Chemical Constituents and Evaluation of Cytotoxic Activities of Curcuma zedoaria (Christm.) Roscoe Oils from Malaysia and Indonesia

The essential oils obtained by hydrodistillation of the rhizomes of Curcuma zedoaria (Christm.) Roscoe collected from Malaysia and Indonesia were analyzed by capillary GC and GC-MS. Both the oils were principally sesquiterpenic in nature and were characterized by zerumbone (17.14% and 12.07%, respectively). Camphor (17.56% and 19.69%, respectively) was also abundant in both oils. The cytotoxic effects of both the oils on human breast (MCF-7 and MDA-MB 231), lung (A549 and SK-LU-1) and cervical (HeLa S3 and SiHa) cancer cell lines were examined using the MTT assay. The Malaysian C. zedoaria rhizome oil was found to be cytotoxic against the MCF-7, SK-LU-1, HeLa S3 and SiHa cell lines, with IC50 values being less than 10 μg/mL. Our findings support the use of the Malaysian C. zedoaria rhizome oil as a potential chemopreventive agent towards a wide spectrum of cancer in particular cervical cancer.

Low concentrations of antimony impair DNA damage signaling and the repair of radiation-induced DSB in HeLa S3 cells.

Antimony is utilized in a large variety of industrial applications, leading to significant environmental and occupational exposure. Mainly based on animal experiments, the IARC and MAK Commission have classified antimony and its inorganic compounds as Group 2B or 2 carcinogens, respectively. However, the underlying mode(s) of action are still largely unknown. In the present study, we investigated the impact of non-cytotoxic up to cytotoxic concentrations of SbCl3 on DNA DSB repair and cell cycle control in HeLa S3 cells. We induced DSB by γ-irradiation and analyzed inhibitory actions of antimony on potential molecular targets of the DSB repair machinery. Antimony disturbed cell cycle control, affecting phosphorylation of Chk1. Furthermore, the repair of DSB was impaired in the presence of antimony, as monitored by pulsed-field gel electrophoresis and γH2AX foci formation of cells in G1 and G2 phase. Specifically, BRCA1 and RAD51 were identified as molecular targets. Our results point towards an interference with both non-homologous end-joining (NHEJ) and homologous recombination(HR), and inhibitory effects may be explained by interactions with critical cysteine groups; this needs to be further investigated. Altogether, the results provide further evidence for the impairment of DNA repair processes as one underlying mechanism in antimony-induced carcinogenicity.

Calcium‐dependent activation of transglutaminase 2 by nanosecond pulsed electric fields

Exposure of cultured human cells to nanosecond pulsed electric fields (nsPEFs) elicits various cellular events, including Ca2+ influx and cell death. Recently, nsPEFs have been regarded as a novel physical treatment useful for biology and medicine, but the underlying mechanism of action remains to be fully elucidated. In this study, we investigated the effect of nsPEFs on transglutaminases (TGs), enzymes that catalyze covalent protein modifications such as protein–protein crosslinking. Cellular TG activity was monitored by conjugation of cellular proteins with biotin‐cadaverine, a cell‐permeable pseudosubstrate for TGs. We applied nsPEFs to HeLa S3 cells and found that overall catalytic activity of cellular TGs was greatly increased in a Ca2+‐dependent manner. The Ca2+ ionophore ionomycin significantly augmented nsPEF‐induced TG activation, further supporting the importance of Ca2+. Among human TG family members, TG2 is known to be the most ubiquitously expressed, and its catalytic activity requires elevated intracellular Ca2+. Given the requirement of Ca2+ for TG activation by nsPEFs, we performed depletion of TG2 by RNA interference (RNAi). We observed that TG2 RNAi suppressed the nsPEF‐induced TG activation and partially alleviated the cytotoxic effects of nsPEFs. These findings demonstrate that TG2 activation is a Ca2+‐dependent event in nsPEF‐exposed cells and exerts negative effects on cell physiology.

Gap Junction Mediated Antigen Transport in Epithelium Promotes Amplification of CD8 T Cell Mediated Bystander Pathology Following Chlamydial Infection

Abstract Chlamydia-specific CD8 T cells play a significant role in oviduct pathology, not bacterial clearance. We hypothesized that gap junction mediated antigen transport (GMAT) from infected to uninfected cells could explain the apparent targeting of uninfected epithelial cells by Chlamydia-specific CD8 T cells. Since gap junction protein connexin 43 is highly expressed in oviduct epithelium, we generated Foxj1Cre-Cx43flox mice with a conditional deficiency of CX43 only in ciliated columnar epithelia in oviducts. Foxj1Cre-Cx43flox mice displayed similar chlamydial infection, but reduced splenic Ag-specific CD8 T cell response and oviduct pathology when compared to WT mice, suggesting that CX43-mediates chlamydial pathogenesis. To evaluate the role of GMAT via CX43 in pathogenesis, HeLa S3 cells were engineered to express functional CX43 or a closed channel point-mutant CX43 (T154A). Chlamydial peptides were observed in neighboring uninfected cells at mid-developmental cycle upon infection of HeLa-CX43, not HeLa S3 or HeLa-CX43-T154A cells. Naïve mouse antigen-presenting cells (APC) optimally activated Chlamydia-specific mouse CD8 T cells upon co-culture with Chlamydia-infected HeLa-CX43, when compared to HeLa S3 or HeLa-CX43-T154A cells, demonstrating that GMAT is involved in activation of CD8 T cells. Furthermore, naïve mouse APC optimally activated Chlamydia-specific mouse CD8 T cells upon co-culture with a 1:4 mixture of Chlamydia-infected HeLa-CX43 and uninfected HeLa-CX43 cells, when compared to mixtures of infected HeLa-CX43 with uninfected HeLa-S3 or HeLa-CX43-T154A cells. These results demonstrate that GMAT via uninfected epithelial cells amplifies pathogenic Chlamydia-specific CD8 T cell response.

Variable TERRA abundance and stability in cervical cancer cells.

Telomeres are transcribed into long non-coding RNA, referred to as telomeric repeat-containing RNA(TERRA), which plays important roles in maintaining telomere integrity and heterochromatin formation. TERRA has been well characterized in HeLa cells, a type of cervical cancer cell. However, TERRA abundance and stability have not been examined in other cervical cancer cells, at least to the best of our knowledge. Thus, in this study, we measured TERRA levels and stability, as well as telomere length in 6cervical cancer cell lines, HeLa, SiHa, CaSki, HeLaS3, C-33A and SNU-17. We also examined the association between the TERRA level and its stability and telomere length. We found that the TERRA level was several fold greater in the SiHa, CaSki, HeLaS3, C-33A and SNU-17 cells, than in the HeLa cells. An RNA stability assay of actinomycinD-treated cells revealed that TERRA had a short half-life of ~4h in HeLa cells, which was consistent with previous studies, but was more stable with a longer half-life(>8h) in the other 5cell lines. Telomere length varied from 4to 9kb in the cells and did not correlate significantly with the TERRA level. On the whole, our data indicate that TERRA abundance and stability vary between different types of cervical cancer cells. TERRA degrades rapidly in HeLa cells, but is maintained stably in other cervical cancer cells that accumulate higher levels of TERRA. TERRA abundance is associated with the stability of RNA in cervical cancer cells, but is unlikely associated with telomere length.

C‐Terminal 15kDa Fragment from β‐Actin Causes Apoptotic Cell Death

Truncated 15kDa‐β‐actin fragment was originally detected in apoptotic cancer cells treated with etoposide and its over‐expression of the fragment induced apoptotic morphological changes (Mashima, T. et al. Oncogene 18, 2423–2430, 1999). Our final goal is to reveal how the 15kDa‐β‐actin fragment induces the apoptotic morphological changes. To distinguish the transfected cells from non‐transfected ones, we have developed a new expression vector in which two coding sequences are able to be simultaneously expressed in a mammalian cell. This vector enabled to express a fluorescent protein with the 15kDa‐β‐actin in an identical cell. Using these co‐expression plasmids, we have investigated the effects of the 15kDa‐β‐actin on the apoptotic processes of HEK293, COS‐7 HeLa S3, and Jurkat cells.The over‐expression of 15kDa‐β‐actin fragment was seemed to cause the loss of adhesive properties of COS‐7 cells onto culture dish, since the GFP‐labeled cells were observed to detach from dishes by losing the adhesion ability. The numbers of detached cells from HEK293, COS‐7 and HeLa S3 transfected with the 15kDa‐β‐actin cDNA were determined by counting chamber. The numbers of detached cells were significantly increased by transfection of 15kDa‐β‐actin cDNA, but not by any mock transfections. To examine the relationship between the detachment and cell death, the detached cells were stained with Hoechst33342 and NucView488 caspase‐3 vital staining. The almost detached cells showed caspase‐3 activation and nuclear condensation in HEK293, COS‐7 and HeLa S3 cells. The cell death is not seemed to result from anoikis, since the transfection of the 15kDa‐β‐actin cDNA gave the similar effects on Jurkat cells. We also confirmed that the DNA fragmentations in HEK293, COS‐7, HeLa S3 and Jurkat cells by TUNEL assay.Cytoskeletal β‐actin has three targets of caspase‐3 motif but β‐actin has been so far considered to be a poor target of caspase‐3. We showed that the 15kDa‐β‐actin C‐terminal fragment actually activated caspase‐3. These results suggest that the cleaved molecule of cytoskeletal β‐actin is not an innocent bystander but an apoptogeneic inducer. Furthermore, we found that an actin binding protein, profilin suppressed the cell death by either 15kDa‐β‐actin or etoposide, VP‐16. The cleavage of cytoskeletal β‐actin would play a key role in the process of apoptotic cell death.