Anticancer drug action on poly(A) polymerase activity and isoforms during HeLa and WISH cell apoptosis

Poly(A) polymerase (PAP; EC 2.7.7.19) catalyzes mRNA polyadenylation. Its activity and isoform levels vary during cell cycle transformation and apoptosis. It has become widely accepted that cell death after DNA damage by anticancer agents is primarily the result of apoptosis and that cells able to evade apoptosis will be resistant to cell killing. The therapeutic agents interferon (IFN), 5-fluorouracil (5-FU) and tamoxifen (Tam) with different mechanisms of action mediate both partial dephosphorylation and inactivation of PAP, detected by immunoblotting analysis and PAP enzyme assay, respectively. We examined the apoptotic tendencies of HeLa and WISH cell lines caused by one of the drugs used, 5-FU. The trend in the cells examined, observed by DAPI and/or DNA fragmentation assay, was found to be accompanied by and reversibly related to PAP activity levels and PAP lower mobility phosphorylated forms of 106 and 100 kDa isoforms. Moreover, a cell type-modulated, differential response of HeLa (chemosensitive cells) versus WISH (drug-resistant diploid cells) has been revealed. This finding yields information on the possible use of PAP as a tumor marker involved in cell commitment and/or induction of apoptosis and may help to improve our understanding of tumor cell sensitivity to anticancer agents.     

Influence of interferons on the repair of UV-damaged DNA

The capacity for nucleotide excision repair of a normal (WISH) and three tumour (MCF-7, HeLa, Namalva) cell lines treated with human recombinant interferons (hrIFN-alpha and hrIFN-gamma) was compared by the host cell reactivation assay. The cells were transfected with in vitro UV-damaged plasmid DNA (pEGFP-N1). The repair capacity was determined by measuring the fluorescence intensity of the expressed marker protein in total cell lysates. The correlation between the interferon-induced NO content and the suppressive effect of interferons on DNA repair was shown. The decrease of repair activity and NO induction by hrIFN-alpha were greatest in WISH, followed by MCF-7, Namalva and HeLa cells, whereas hrIFN-gamma was the best NO inducer and inhibitor for the repair of Namalva, followed by WISH, MCF-7 and HeLa cells. Our data clearly show that the two types of interferon have a strong inhibitory effect on the repair of UV-damaged DNA and this effect is cell type-dependent.     

The interaction of Mengo virus ribonucleic acid with L, HeLa and human diploid cells: a comparative study

A study, designed to define the optimal conditions for infecting L, HeLa and human diploid cells with Mengo RNA, showed that the three cell types differ with respect to (a) the concentration of sucrose (in PBS) that produces maximum stimulation of infectious center (I.C.) formation (0.6 M, 1.2 M and 1.0 M for L, HeLa and human diploid cells respectively) and (b) the optimal combination of sucrose and dimethyl-sulfoxide (DMSO). With all three cell types, diethylaminoethyldextran (DEAE-D) was found to produce maximum stimulation of I.C. formation when present, in PBS, at a concentration of 100 μg/ml. Direct comparisons of I.C. formation in the three cell types using the sucrose, sucrose-DMSO and PBS-DEAE-D media optimal for each revealed that (a) the best medium for titration in L and HeLa cells is PBS containing 100 μg DEAE-D/ml, (b) DMSO produces a 4–8 fold increase in I. C. formation in L cells, but is only marginally effective in HeLa cells, and (c) the optimal medium for titration in human diploid cells is sucrose-DMSO. The results of studies of the “uptake” of ³H-Mengo RNA showed that there is no correlation between the amount of RNA that becomes cell associated and the number of I.C.'s that are produced in the cell population.     

5-氟尿嘧啶增强HeLa细胞对自然杀伤细胞敏感性研究

目的用5-氟尿嘧啶（5-fluorouracil,5-FU）处理HeLa细胞,检测其NKG2D配体MICA的表达及其对NK92细胞杀伤敏感性的变化。方法不同浓度的5-Fu处理HeLa细胞,在不同时间点用半定量PCR及流式细胞术检测HeLa细胞表面的NKG2D配体MICA在RNA及蛋白水平的表达变化情况,用MTT法检测NKG2D抗体封闭NK92细胞的NKG2D受体前后,NK92细胞对HeLa细胞的杀伤作用。结果不同浓度的5．Fu作用于HeLa细胞后,半定量RT—PCR结果显示MICA表达随5-Fu作用浓度增加逐渐增高。而且40μg／ml5．Fu作用于HeLa细胞后随着作用时间的延长（0、8、16、24h）MICA表达增加,流式细胞术检测结果表明,MICA表达的增加主要依赖于未凋亡细胞的MICA表达。在40μg／ml5-FU作用24h,效靶比为2．5：1,5：1,10：1,20：1时都检测到NK92细胞对HeLa细胞的杀伤增强,杀伤作用可部分被NKG2D抗体抑制。结论5-FU能够上调HeLa细胞表面NKG2D配体MICA的表达,增强HeLa细胞对NK92细胞的敏感性,提示化疗联合NK细胞免疫治疗宫颈癌可产生协同作用,提高治疗效果。     

The RNA exosome component hRrp6 is a target for 5-fluorouracil in human cells

The drug 5-fluorouracil (5-FU) is a widely used chemotherapeutic in the treatment of solid tumors. Recently, the essential 3'-5' exonucleolytic multisubunit RNA exosome was implicated as a target for 5-FU in yeast. Here, we show that this is also the case in human cells. HeLa cells depleted of the inessential exosome component hRrp6, also called PM/Scl100, are significantly growth impaired relative to control cells after 5-FU administration. The selective stabilization of bona fide hRrp6 RNA substrates on 5-FU treatment suggests that this exosome component is specifically targeted. Consistently, levels of hRrp6 substrates are increased in two 5-FU-sensitive cell lines. Interestingly, whereas down-regulation of all tested core exosome components results in decreased hRrp6 levels, depletion of hRrp6 leaves levels of other exosome components unchanged. Taken together, our data position hRrp6 as a promising target for antiproliferative intervention.     

Comparison of aldolase isozymes in placenta, HeLa cells, and human fibroblast cultures

The aldolase specific activity of the human carcinoma cell line, HeLa, against fructose 1,6-diphosphate as substrate is 4- to 5-fold greater than the specific activity of diploid human fibroblast cultures derived from skin and lung. HeLa aldolase is isozyme is predominantly the A type and its substrate preferences resemble human placenta. These findings provide further support for the oncofetal enzyme consitution of HeLa cells.     

Monoclonal antibodies to the antigens of human HeLa tumor cells

A panel of 6 hybridomas "XEJIMA" producing monoclonal antibodies specific to HeLa cells is prepared. Monoclonal antibodies do not bind to antigens of human diploid fibroblasts, human continuous B- and T-lymphocytes and animal cell lines. The specificity of monoclonal antibodies to cellular antigens of 5 HeLa-like cell lines and 6 human tumour cells lines, not contaminated with HeLa cells, is determined. Antibody containing ascitic fluid and culture media of hybridomas XEJIMA-3, -12, -13, and -22 significantly decrease the attachment of HeLa cells to the surface of culture flasks. Monoclonal antibodies XEJIMA-11, -12 and -13 block the multiplication of HeLa cells. The effect depends on serum concentration in the nutrient medium.     

Stability of glucose-6-phosphate dehydrogenase in human cells cultivated in vitro]

It was shown that the thermal stability of glucose-6-phosphate dehydrogenase in human diploid cells is much higher than in human heteroploid cell lines HeLa and T-9. The purified enzymes from human diploid cells and from HeLa and T-9 cells possess similar thermal stabilities. Mixing of T-9 extracts with the purified enzyme preparations revealed that the non-stability factors of the dehydrogenase are present in the T-9 extracts. An addition of NADP- and NADPH-containing buffers and crystalline NADP to the heteroploid cell extracts stabilizes the enzyme. The thermal stability of the enzyme from "in vitro" cultivated human cells depends on the concentration of the coenzyme. It was also demonstrated that glucose-6-phosphate dehydrogenase stability in HeLa and T-9 extracts is the same at low concentrations of the coenzyme and after addition of crystalline NADP. However, at NADP concentration of 10(-3) M the enzyme stability in HeLa and T-9 extracts is different. It is assumed that the destabilizing factors are the enzymes possessing the nucleotidases activity, which is different in various cell lines.     

Effect of the thymidylate synthase inhibitors on dUTP and TTP pool levels and the activities of DNA repair glycosylases on uracil and 5-fluorouracil in DNA

5-Fluorouracil (5-FU), 5-fluorodeoxyuridine (5-dUrd), and raltitrixed (RTX) are anticancer agents that target thymidylate synthase (TS), thereby blocking the conversion of dUMP into dTMP. In budding yeast, 5-FU promotes a large increase in the dUMP/dTMP ratio leading to massive polymerase-catalyzed incorporation of uracil (U) into genomic DNA, and to a lesser extent 5-FU, which are both excised by yeast uracil DNA glycosylase (UNG), leading to DNA fragmentation and cell death. In contrast, the toxicity of 5-FU and RTX in human and mouse cell lines does not involve UNG, but, instead, other DNA glycosylases that can excise uracil derivatives. To elucidate the basis for these divergent findings in yeast and human cells, we have investigated how these drugs perturb cellular dUTP and TTP pool levels and the relative abilities of three human DNA glycosylases (hUNG2, hSMUG1, and hTDG) to excise various TS drug-induced lesions in DNA. We found that 5-dUrd only modestly increases the dUTP and dTTP pool levels in asynchronous MEF, HeLa, and HT-29 human cell lines when growth occurs in standard culture media. In contrast, treatment of chicken DT40 B cells with 5-dUrd or RTX resulted in large increases in the dUTP/TTP ratio. Surprisingly, even though UNG is the only DNA glycosylase in DT40 cells that can act on U·A base pairs derived from dUTP incorporation, an isogenic ung(-/-) DT40 cell line showed little change in its sensitivity to RTX as compared to control cells. In vitro kinetic analyses of the purified human enzymes show that hUNG2 is the most powerful catalyst for excision of 5-FU and U regardless of whether it is found in base pairs with A or G or present in single-stranded DNA. Fully consistent with the in vitro activity assays, nuclear extracts isolated from human and chicken cell cultures show that hUNG2 is the overwhelming activity for removal of both U and 5-FU, despite its bystander status with respect to drug toxicity in these cell lines. The diverse outcomes of TS inhibition with respect to nucleotide pool levels, the nature of the resulting DNA lesion, and the DNA repair response are discussed.     

Comparative studies of the binding and growth-supportive ability of mammalian transferrins in human cells

The ability of human-derived cells in culture to bind, remove iron from, and grow in the presence of transferrins (Tf) isolated from the sera of species commonly included in tissue culture medium was investigated. Kinetic studies on HeLa cells reveal apparent first-order association rate constants of 0.43 min-1 for human Tf and 0.15 min-1 for equine Tf. Labeled chicken ovo-Tf and fetal bovine Tf were not recognized by the HeLa cells. Competition experiments with HeLa cells that use either isolated Tf or parent serum confirm these findings. Equilibrium binding experiments performed on HeLa cells at 37 degrees C in the presence of 2,4-dinitrophenol to prevent iron removal indicate 1 X 10(6) Tf bound/cell with a dissociation constant (K'D) of 28 nM for human Tf and 182 nM for equine Tf. Equilibrium binding performed at 0 degrees C to prevent endocytosis reveals 4.1-6.7 X 10(5) Tf binding sites/cell with a K'D of 8.3 nM for human Tf and 41.5 nM for equine Tf. Parallel experiments in normal human diploid fibroblast-like MRC-5 cells indicate expression of 0.82-2.78 X 10(5) Tf binding sites/cell with a K'D of 8.2 nM for human and 39.1 nM for equine Tf. Thus, the results of equilibrium binding studies of a more differentiated cell type are consistent with those found for HeLa cells. Fetal bovine Tf was found to compete weakly with labeled human Tf for human receptor on HeLa cells in a soluble receptor assay, with an approximately 500-fold excess needed to reduce binding to half maximal. Iron uptake experiments show an iron donating hierarchy where human greater than horse greater than calf, suggesting that the rate of iron uptake depends on the affinity of receptor for transferrin. Growth experiments involving HeLa cells in chemically defined serum-free medium demonstrate that bovine Tf will support growth as well as human Tf, but at concentrations much higher than are required of human Tf.     

Anticancer effects of the engineered stem cells transduced with therapeutic genes via a selective tumor tropism caused by vascular endothelial growth factor toward HeLa cervical cancer cells

The aim of the present study was to investigate the therapeutic efficacy of genetically engineered stem cells (GESTECs) expressing bacterial cytosine deaminase (CD) and/or human interferon-beta (IFN-β) gene against HeLa cervical cancer and the migration factors of the GESTECs toward the cancer cells. Anticancer effect of GESTECs was examined in a co-culture with HeLa cells using MTT assay to measure cell viability. A transwell migration assay was performed so as to assess the migration capability of the stem cells to cervical cancer cells. Next, several chemoattractant ligands and their receptors related to a selective migration of the stem cells toward HeLa cells were determined by real-time PCR. The cell viability of HeLa cells was decreased in response to 5-fluorocytosine (5-FC), a prodrug, indicating that 5-fluorouracil (5-FU), a toxic metabolite, was converted from 5-FC by CD gene and it caused the cell death in a co-culture system. When IFN-β was additionally expressed with CD gene by these GESTECs, the anticancer activity was significantly increased. In the migration assay, the GESTECs selectively migrated to HeLa cervical cancer cells. As results of real-time PCR, chemoattractant ligands such as MCP-1, SCF, and VEGF were expressed in HeLa cells, and several receptors such as uPAR, VEGFR2, and c-kit were produced by the GESTECs. These GESTECs transduced with CD gene and IFN-β may provide a potential of a novel gene therapy for anticervical cancer treatments via their selective tumor tropism derived from VEGF and VEGFR2 expressions between HeLa cells and the GESTECs.     

tRNA Modifying Enzymes,NSUN2 and METTL1, Determine Sensitivity to 5-Fluorouracil in HeLa Cells

Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.     

Decreased DNA repair capacity of UV-irradiated cells following interferon treatment

The aim of this study was to examine the effect of interferons (IFNs) on the recovery of UV-damaged cells by means of measuring cell viability rates. The influence of the recombinant human interferons IFN-alpha, IFN-beta and IFN-gamma on the repair capacity of the UV-irradiated human cell lines WISH and HeLa was studied. The ability of cells to repair UV-induced damage was determined by the comet assay and both short- and long-term survival assays in proliferating cell cultures. We found that INFs negatively regulated DNA repair in cells damaged by UV light. One day after treatment, in both cell lines tested, IFN-alpha had a stronger inhibitory effect than IFN-gamma. Combined treatment with different IFNs exhibited a stronger inhibitory effect on cell recovery than treatment with each of them. The protein kinase inhibitor wortmanin further aggravated the effect of IFNs on cell survival.     

Isolation and characterization of a human cDNA clone encoding a novel DNA topoisomerase II homologue from HeLa cells

We have isolated and sequenced 3 human DNA topoisomerase II (topo II) partial cDNA clones from a HeLa carcinoma cell cDNA library. Two clones were identical to an internal fragment of HeLa topo II cDNA. The third clone, CAA5, had a different and novel sequence which shared significant nucleotide (62%) and predicted peptide (70%) homologies with a region of the HeLa topo II cDNA. Our results suggest that HeLa cells express at least two homologous forms of DNA topoisomerase II. The new HeLa topo II homologue is discussed in relation to topo II isoenzymes recently described in a Burkitt lymphoma and other cell lines.     

Temporal and spatial regulation of the phosphatidate phosphatases lipin 1 and 2

Lipins are the founding members of a novel family of Mg(2+)-dependent phosphatidate phosphatases (PAP1 enzymes) that play key roles in fat metabolism and lipid biosynthesis. Despite their importance, there is still little information on how their activity is regulated. Here we demonstrate that the functions of lipin 1 and 2 are evolutionarily conserved from unicellular eukaryotes to mammals. The two lipins display distinct intracellular localization in HeLa M cells, with a pool of lipin 2 exhibiting a tight membrane association. Small interfering RNA-mediated silencing of lipin 1 leads to a dramatic decrease of the cellular PAP1 activity in HeLa M cells, whereas silencing of lipin 2 leads to an increase of lipin 1 levels and PAP1 activity. Consistent with their distinct functions in HeLa M cells, lipin 1 and 2 exhibit reciprocal patterns of protein expression in differentiating 3T3-L1 adipocytes. Lipin 2 levels increase in lipin 1-depleted 3T3-L1 cells without rescuing the adipogenic defects, whereas depletion of lipin 2 does not inhibit adipogenesis. Finally, we show that the PAP1 activity of both lipins is inhibited by phosphorylation during mitosis, leading to a decrease in the cellular PAP1 activity during cell division. We propose that distinct and non-redundant functions of lipin 1 and 2 regulate lipid production during the cell cycle and adipocyte differentiation.     

Proteomic analysis of antiproliferative effects by treatment of 5-fluorouracil in cervical cancer cells

The global effects of 5-fluorouracil (FU) on cervical carcinoma cells were analyzed using an efficient proteomic method. More than 50 proteins showed a significant change in 5-FU-treated cervical carcinoma cells compared to control cells. Among them, 34 proteins have been identified by employing two-dimensional gel electrophoresis and MALDI-TOF-MS using peptide mass fingerprinting. In results, 22 proteins were upregulated (CIDE-B [cell death-inducing DFFA-like effector B], caspase-3, caspase-8, Apo-1/CD95 (Fas), etc.) and 12 proteins were downregulated (mitotic checkpoint protein BUB3, myc proto-oncogene protein [c-myc], src substrate cortactin, transforming protein p21A, etc.) by 5-FU treatment in HeLa cervical carcinoma cells as determined by spot volume (P <0.05). Our experiments showed that 5-FU engaged the mitochondrial apoptotic pathway involving cytosolic cytochrome c release and subsequent activation of caspase-9 and caspase-3 as well as the membrane death receptor (DR)-mediated apoptotic pathway involving activation of caspase-8 with an Apo-1/CD95 (Fas)-dependent fashion. In addition, we could observe reduction of HPV-18 E6/E7 gene expression and activation of p53, pRb, and p21waf1 proteins by 5-FU treatment in HeLa cervical carcinoma cells. In conclusion, we suggest that 5-FU suppresses the growth of cervical cancer cells not only by antiproliferative effect but also antiviral regulation. Our findings may offer new insights into the mechanism of anticancer effect affected by 5-FU treatment in cervical cancer cells and its mode of action.     

Evaluation of cytotoxic activity of Vibrio cholerae non-01 culture filtrate on established cell lines and human diploid cells

The cell-destroying effect of cell free filtrates of 90 V. cholerae non-01 cultures was measured by titration method in 3 established cell lines: CHO, HeLa and Vero and in 3 human diploid cells cultures: MRC-5, WI-38 and PZ. The vibrio strains differed in the titre of toxic effect. Most sensitive was CHO cell line, least sensitive were human diploid cell cultures. It was found that bacterial strains produced different substances toxic for various cell lines. Among them NAG-ST toxin produced by 41% of examined strains was identified and hemolysins/cytolysins activity was evaluated. Both may play a role in the pathogenicity of those strains for humans.     

Induction of autophagy enhances apoptotic cell death via epidermal growth factor receptor inhibition by canertinib in cervical cancer cells

BackgroundIt has been known epidermal growth factor receptor (EGFR) frequently overexpressed in cervical cancer. High levels of EGFR expression in their tumors leads to a poor prognosis and inhibition frequently induces autophagy in cancer cells. This study aimed to investigate whether EGFR inhibition by canertinib induces autophagy and this induction influence the effect of Palladium (Pd) (II) complex and 5-fluorouracil (5-FU) especially in nontoxic doses.MethodsCytotoxicity was evaluated by using SRB assay. Apoptosis, autophagy, and EGFR key markers were determined by flow cytometry, fluorescence staining, and immunoblotting. Colony formation, invasion, and wound healing assays were performed to investigate cell proliferation, invasion, and migration, respectively.ResultsBlocking EGFR by the pan-ErbB tyrosine kinase inhibitor canertinib inhibited cell growth of HeLa cervical cancer cells in combination with Pd(II) complex and 5-FU. Combination of canertinib and Pd(II) complex promotes autophagy and apoptosis of HeLa cancer cells via blockade of the PI3K/AKT and MAPK/ERK pathway, which leads to cervical cancer cell death. ROS accumulation and DNA damage were increased after combinatorial treatment which causes depolarization of the mitochondrial inner membrane and leads to apoptotic cell death. Canertinib combined with Pd(II) complex leads to inhibition of migration and invasion.ConclusionInhibition of EGFR signaling by canertinib in combination with Pd(II) complex promotes apoptosis and autophagy via blockade of the PI3K/AKT and MAPK/ERK.General significanceThe cytotoxic activity of Pd(II) complex and 5-FU on HeLa cells is mediated by EGFR inhibition and autophagy induction, leading to activation of mitochondrial apoptotic cell death.     

5-Fluorouracil resistance in carrot cell cultures

Physiological studies of 5-fluorouracil (5-FU)-resistant cell line of wild carrot (Daucus carota L.), F5, showed that this variant is also resistant to 5-fluorouridine, but is as sensitive to 6-azauracil as the 5-FU-sensitive parent line, WOO1C. High levels of exogenous uracil, uridine, and thymine are slightly toxic to F5, but not to WOO1C. 5-FU sensitivity in WOO1C cannot be reversed by bases and nucleosides; bases like uracil and thymine even increase 5-FU toxicity. No substantial differences were found in the uptake, incorporation and degradation of WOO1C and F5. Carrot cultures seem to take up 5-FU by rapid diffusion, the kinetics being characteristic of non-saturable uptake, with infinite Km and zero Vmax. The rapid uptake of 5-FU and extensive degradation of bases and nucleosides are probably responsible for the inability of uracil and uridine to reverse the growth inhibition caused by 5-FU in carrot cells while, as shown earlier, phaseolotoxin ((N-phosphosulfamyl)ornithinylalanylhomoarginine), an inhibitor of the arginine biosynthetic enzyme, ornithine transcarbamylase was capable of reducing 5-FU toxicity. F5 callus contained less histidine and arginine than WOO1C. 5-FU increased the endogenous levels of arginine, histidine and aspartate in both lines. The aspartate transcarbamylase of F5 appears to be normal; it is as sensitive to uridine-monophosphate inhibition as that of WOO1C. The 5-FU resistance of F5 was stable in undifferentiated cells, but only 8 out of 50 calli reinitiated from the regenerated plantlets remained resistant to 5-FU. F5 is an aneuploid culture. Five 5-FU-sensitive reinitiated calli that were examined were all diploid whereas of the eight 5-FU-resistant reinitiated calli two became diploid and six remained as aneuploid.Abbreviation 5-FU 5-fluorouracil     

Early nuclear structural changes of transformed human amniotic cells (WISH) in presence of type IV collagen detected by the nuclear refringency assay

An early nuclear activation of transformed human amniotic epithelial (WISH) cells triggered by type IV collagen is visualized by a modification of the nuclear refringency obtained by mercury binding on condensed chromatin. This phenomenon is quantified by the nuclear refringency test. The nuclear activation of WISH cells by basement membrane collagen is also shown by the DNase I sensitivity of chromatin and by the measurement of mRNA synthesis. These nuclear phenomena are concomitant with WISH cell attachment and laminin synthesis. Reversible effects on nuclear refringency, cell attachment, and laminin synthesis are tested by the addition and removal of different metabolic inhibitors.