Regulation of the capacity for O6-methylguanine removal from DNA in human lymphoblastoid cells studied by cell hybridization.

Hybrids were made between a ouabain-resistant, thioguanine-resistant human lymphoma line able to remove O6-methylguanine from its DNA (Mex+) and human lymphoblastoid lines deficient in this capability (Mex-). The formation of hybrids was confirmed by chromosomal analysis. Hybrid cells had an O6-methylguanine removal capacity per mole of guanine about one third to one half that of the Mex+ parents, i.e., about the same per cell. Cell hybrids removed the same amount of the alkylation adduct 3-methyladenine as did their parents per mole of guanine, i.e., about twice as much per cell. Although the cell hybrids had intermediate resistance to the cytotoxic action of N-methyl-N'-nitro-N-nitrosoguanidine used to induce O6-methylguanine and 3-methyladenine, there is evidence that the ability to remove O6-methylguanine and resistance to the cytotoxic effect of N-methyl-N'-nitro-N-nitrosoguanidine are dissociable characteristics.     

The origin of O6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells transfected with human DNA

Transfection of Chinese hamster ovary (CHO) cells with human DNA has been shown in several laboratories to produce clones which stably express the DNA-repair protein, O6-methylguanine-DNA methyltransferase (MGMT), that is lacking in the parent cell lines (Mex- phenotype). We have investigated the genetic origin of the MGMT in a number of such MGMT-positive (Mex+) clones by using human MGMT cDNA and anti-human MGMT antibodies as probes. None of the five independently isolated Mex+ lines has human MGMT gene sequences. Immunoblot analysis confirmed the absence of the human protein in the extracts of these cells. The MGMT mRNA in the lines that express low levels of MGMT (0.6-1.4 x 10(4) molecules/cell) is of the same size (1.1 kb) as that present in hamster liver. One cell line, GC-1, with a much higher level of MGMT (4 x 10(4) molecules/cell) has two MGMT mRNAs, a major species of 1.3 kb and a minor species of 1.8 kb. It has also two MGMT polypeptides (32 and 28 kDa), both of which are larger than the 25 kDa MGMT present in hamster liver and other Mex+ transfectants. These results indicate that the MGMT in all Mex+ CHO cell clones is encoded by the endogenous gene. While spontaneous activation of the MGMT gene cannot be ruled out in the Mex+ cell clones, the intervention of human DNA sequences may be responsible for activation of the endogenous gene in the GC-1 line.     

Instability of Mex- phenotype in human lymphoblastoid cell lines

Three lymphoblastoid cell lines (LCLs) had extremely low activities of O6-alkylguanine-DNA alkyltransferase (O6-AGT), and were classified as Mex-. They were highly sensitive to cell killing by 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosoure a hydrochloride (ACNU), whereas NMO2, a Mex+ LCL with a high O6-AGT activity, was resistant to the agent. Small fractions of these Mex- LCLs survived the treatment with 10 micrograms/ml of ACNU for 24 h, and the surviving cells were found to be resistant to subsequent treatments with the agent. In addition, they contained O6-AGT activities comparable to that of NMO2 and were therefore regarded as Mex+. These results suggest that the Mex- phenotype in LCLs is unstable.     

Characterization of oxygen-tolerant Chinese hamster ovary cells: II. Energy metabolism and antioxidant status

Further characteristics of an oxygen-tolerant variant of Chinese hamster ovary cells (CHO-99) capable of stable proliferation at 99% O₂/1% CO₂, an O₂ level that is lethal to the parental line (CHO-20), are described. Previous work has revealed that CHO-99 cells have 2- to 4-fold increased activities of superoxide dismutases, catalase and glutathione peroxidase, and substantially increased relative volumes of mitochondria and peroxisomes. To document possible additional mechanisms of O₂ tolerance we compared CHO-20 cells growing at 20% O₂ (normoxia) and CHO-99 cells at 99% O₂ (normobaric hyperoxia). We show the following: (1) the estimated total (oxidative and glycolytic) ATP production in CHO-99 cells was 36% decreased. ATP production through oxidative phosphorylation was 52% lower in CHO-99 cells, while the relative contribution from glycolysis was increased from 6% to 30%. The ATP content was 29% lower in CHO-99 cells, the adenylate energy charge being also significantly decreased, indicating that energy production through oxidative phosphorylation is compromised in CHO-99 cells. Cyanide-resistant respiration was 4-fold higher in CHO-99 cells, probably reflecting, at least partly, the increased peroxisomal activity in these cells. (2) The level of reduced glutathione was several fold increased in CHO-99 cells, oxidized glutathione being unaltered; (NADPH + NADP⁺) levels were elevated 2.7-fold, while the ratio of NADPH to NADP⁺ was increased almost two-fold. These changes were associated with a 50% increased metabolism of glucose through the hexose monophosphate pathway. (3) No evidence was obtained for an increased steady-state level of endogenous lipid peroxidation in CHO-99 cells, in spite of a 50% increased content of polyunsaturated fatty acids in the phospholipid fraction.     

The monoclonal antibody CHO-131 identifies a subset of cutaneous lymphocyte-associated antigen T cells enriched in P-selectin-binding cells

T cells use the vascular adhesion molecules E- and P-selectin to enter inflamed skin. Previous studies have indicated the possibility for diversity in the synthesis of E- and P-selectin glycan ligands by activated T cells due to their different requirements for the O-glycan branching enzyme core 2 beta1,6-N-acetylglucosaminyltransferase I and its independent regulation. It is known that T cell staining by the mAb HECA-452 (referred to as cutaneous lymphocyte-associated Ag (CLA) T cells) correlates with E-selectin binding, yet whether these cells uniformly bind P-selectin is less clear. The mAb CHO-131 and P-selectin binding require a glycan moiety consisting of a sialylated and fucosylated oligosaccharide properly positioned on a core-2 O-glycan. Interestingly, CHO-131 stains a subset of CLA(+) T cells. A direct comparison of the selectin binding capacity of CHO-131(+) and CHO-131(-) CLA(+) T cells revealed a significantly greater P-selectin, but not E-selectin, binding activity by the former subset. Based on the expression of homing and central and effector memory cell markers, CHO-131(+) and CHO-131(-) CLA(+) T cells have an overlapping skin-tropic and memory phenotype. CHO-131(+) T cells were considerably enriched in psoriatic skin, yet, unlike the peripheral blood of healthy individuals, HECA-452 and CHO-131 stained a similar proportion of T cells in the cutaneous lesions, indicating an accumulation advantage by CHO-131(+) T cells. We conclude that the CHO-131(+)CLA(+) T cell subset is enriched in P-selectin binding cells. These findings should provide new insights into the regulation and function of skin homing T cells.     

High-frequency intracellular invasion of epithelial cells by serotype M1 group A streptococci: M1 protein-mediated invasion and cytoskeletal rearrangements

A clonal variant of serotype M1 group A streptococcus (designated M1inv+) has been linked to severe and invasive infections, including sepsis, necrotizing fasciitis and toxic shock. High frequency internalization of cultured epithelial cells by the M1inv+ strain 90-226 is dependent upon the M1 protein. Invasion of HeLa cells was blocked by an anti-M1 antibody, invasion by an M1- strain (90-226 emm1::km) was greatly reduced, and latex beads bound to M1 protein were readily internalized by HeLa cells. Beads coated with a truncated M1 protein were internalized far less frequently. Scanning electron microscopy indicated that streptococci invade by a zipper-like mechanism, that may be mediated by interactions with host cell microvilli. Initially, internalized streptococci and streptococci undergoing endocytosis are associated with polymerized actin. Later in the internalization process, streptococcal-containing vacuoles are associated with the lysosomal membrane glycoprotein, LAMP-1.     

Mismatch binding proteins and tolerance to alkylating agents in human cells

The Mex- (Mer-) phenotype of human cells is characterised by a sensitivity to agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU). The hypersensitivity of Mex- cells is a consequence of their failure to express the DNA-repair enzyme m6-Gua-DNA methyltransferase. Resistance to MNNG and MNU may be acquired by Mex- cells either by reexpression of a methyltransferase function or by an ill-defined process of tolerance in which the cytotoxic potential of m6-Gua is circumvented without the altered base being removed from DNA. It has been suggested that tolerance might involve an altered mismatch correcting function. We have investigated proteins which recognise and bind specifically to DNA fragments containing single-base mismatches. Cell-free extracts of a Burkitt's lymphoma cell line (Raji) contain two such mismatch binding activities. Neither protein appears to have a high affinity for m6-Gua-containing base pairs. The data indicate that m6-Gua-containing base pairs might be poor substrates for mismatch repair processes in human cells.     

Transfer of the E. coli O6-methylguanine methyltransferase gene into repair-deficient human cells and restoration of cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine

We have constructed a plasmid on which the E. coli O6-methylguanine-DNA methyltransferase (MT) gene (ada gene) was linked with an SV40 promoter sequence and a poly(A) site. After transferring this plasmid into Mer- HeLa MR cells by DNA transfection, effective expression of E. coli MT was observed. Isolated stable transformant clones showed higher resistance to N-methyl-N'-nitro-N-nitrosoguanidine in colony formation and sister-chromatid exchange induction than HeLa MR cells.     

Chromosomal instability in an oxygen-tolerant variant of Chinese hamster ovary cells

Background levels of chromosomal aberrations and sister-chromatid exchanges (SCEs) were determined in CHO-99 cells, an oxygen-tolerant variant substrain of Chinese hamster ovary (CHO-20) cells capable of stable proliferation under an atmosphere of 99% O2/1% CO2, a level of hyperoxia at which cultured mammalian cells normally cannot survive. The mean chromosomal aberration frequency in CHO-99 cells was as high as 1 aberration per cell (mainly chromatid and chromosome gaps and breaks) versus 0.05 aberration/cell in CHO-20 cells, while the SCE frequency was 1.7- to 2.1-fold increased. While most aberrations were apparently distributed at random over the chromosomes, up to 31% of the aberrations appeared to be involved in site-specific fragility at a homologous site in chromosomes Z3 and Z4. Immediately upon shifting CHO-99 cells to air-equilibrated conditions their SCE frequency decreased to the control level, whereas the aberration rate persisted at a still elevated level of 0.16-0.31 aberration per cell, even after a culture period of 14 weeks under normoxia. This indicates that at least part of the chromosomal instability is a constitutional property of the variant cells, i.e., not directly dependent upon hyperoxic stress. In CHO-99 X CHO-20 hybrids the occurrence of chromatid-type aberrations and fragile site but not that of chromosome-type aberrations was suppressed under normoxic conditions, suggesting that chromatid-type aberrations and fragile site expression on the one hand and chromosome-type aberrations on the other hand are mediated by different constitutional defects in CHO-99 cells. No gross alterations in (deoxy)ribonucleoside triphosphate pools were detected in CHO-99 cells that could be held responsible for their chromosomal instability. In addition, no increased level of DNA damage was detected by the technique of alkaline elution. The excessive chromosomal instability in CHO-99 cells, as observed under hyperoxic conditions, may originate from reactive intermediates giving rise to DNA double-strand breaks and/or a type of DNA lesion that is resistant to the conditions of the alkaline elution technique. However, alternative mechanisms based upon reactive species interfering with DNA replication/repair processes cannot be excluded.     

Differential potentiation of arachidonic acid release by rat alpha2 adrenergic receptor subtypes

CHO transfectants expressing the three subtypes of rat alpha2 adrenergic receptors (alpha2AR): alpha2D, alpha2B, alpha2C were studied to compare the transduction pathways leading to the receptor-mediated stimulation of phospholipase A2 (PLA2) in the corresponding cell lines CHO-2D, CHO-2B, CHO-2C. The alpha2B subtype stimulated the arachidonic acid (AA) release after incubation of the cells with 1 microM epinephrine, whereas alpha2D and alpha2C gave no stimulation. Calcium ionophore A23187 (1 microM) increased the release by a factor of 2-4 in the three strains. When cells were incubated with both epinephrine and Ca2+ ionophore, the AA release differed greatly between cell lines with strong potentiation in CHO-2B (2-3 times greater than Ca2+ ionophore alone), moderate potentiation in CHO-2D, and no potentiation in CHO-2C. The three cell lines each inhibited adenylylcyclase with similar efficiencies when 1 microM epinephrine was used as the agonist. The potentiation depended on both alpha2AR and Gi proteins since yohimbine and pertussis toxin inhibited the process. Pretreatment of CHO-2B cells with MAFP which inhibits both cytosolic and Ca2+-independent PLA2, reduced the release of AA induced by epinephrine+Ca2+ ionophore to basal value, whereas bromoenol lactone, a specific Ca2+-independent PLA2 inhibitor, had no effect. Preincubation of the cells with the intracellular calcium chelator BAPTA gave a dose-dependent inhibition of the arachidonic acid (AA) release. In CHO cells expressing the angiotensin II type 1 receptor, coupled to a Gq protein, the agonist (10-7 M) produced maximal AA release: there was no extra increase when angiotensin and Ca2+ ionophore were added together. There was no increase in the amount of inositol 1,4, 5-triphosphate following stimulation of CHO-2B, -2C, -2D cells with 1 microM epinephrine. Epinephrine led to greater phosphorylation of cPLA2, resulting in an electrophoretic mobility shift for all three cell lines, so inadequate p42/44 MAPKs stimulation was not responsible for the weaker stimulation of cPLA2 in CHO-2C cells. Therefore, the stimulation of cPLA2 by Gi proteins presumably involves another unknown mechanism. The differential stimulation of cPLA2 in these transfectants will be of value to study the actual involvement of the transduction pathways leading to maximal cPLA2 stimulation.     

The study of the Ca2+ role in cytotoxic response of human cells in culture to the action of xenobiotics

The role of Ca2+ in mechanisms of cell death, necrosis and apoptosis is diverse and generally recognized. The purpose of this work was to study Ca2+ participation in a cytotoxic response of human cultured cells in the presence of toxic concentrations of cationic antiseptic substance poly(hexamethylene guanidine), anionic surfactant SDS and monomeric methyl methacrylate (a component of bone cement applied in surgery). Human cell line U-937 grown in suspension was used for this study. A fluorescent probe chlortetracycline was used, as an indicator of Ca2+ transport through biologic membranes. Our results show that weakly toxic concentrations of xenobiotics under study, close to the minimum toxic doses, nearly always provoke a fair but statistically significant drop in Ca2+ binding by cells. At the same time, higher toxic doses lead to significant increase in Ca2+ influx. The latter event well compares with the majority of literary data, while the mentioned decrease in Ca2+ influx at low toxic concentrations of xenobiotics presumably correlates with the initial stage of acute cytotoxic response, accompanied by a metabolic activation and enhanced resistance of cells to injuring stimuli, demonstrated by the authors elsewhere. In parallel, a possible effect of Ca(2+)-channel antagonist nifedipine was explored under conditions of cytotoxic response of cell lines U-937, A-549 and human embryonic lung fibroblasts to poly(hexamethylene guanidine). Nifedipine (10 microM) was introduced in the incubation medium simultaneously with the toxic agent, and the cells were further maintained for 5 or 24 h in culture; their viability was monitored with the microtetrasolium test or by assessment of LDH leakage into the incubation medium. The effect of nifedipine proved to be dual, depending on the applied concentration of toxic agent: at low toxic concentrations the improvement of viability could be noticed, while at more pronounced toxic doses aggravation of viability was evident. From our point of view the explanation of this result could be the following. In weakly toxic conditions, as in intact cells, Ca2+ influx is brought about by specific mechanisms, mainly through Ca(2+)-channels, that is why nifedipine partly abolishes Ca(2+)-dependent cytotoxic response. At high concentrations, cell plasma membrane is directly damaged by toxic agent, Ca2+ enters cells mainly non-specifically, so that Ca2+ antagonist cannot protect cell injury. The reason of toxic effect aggravation by nifedipine in these conditions is still waiting for its explanation.     

Factors influencing the accumulation of tetraphenylphosphonium cation in HeLa cells.

Exposure of HeLa cells to tetraphenylphosphonium cation (TPP+) results in a rapid accumulation intracellularly, and a steady-state level is reached within 10 min. Accumulation of [3H]TPP+ in HeLa cells is reduced under the following conditions: (i) after preincubation of cells in buffered saline or in medium containing two- to fourfold higher concentrations of amino acids, (ii) exposure to the alkylating agent L-1-tosylamido-2-phenyl-ethylchloromethyl ketone, (iii) ouabain-mediated inhibition of the Na+, K+ ATPase, and (iv) high external K+ concentrations. In contrast, addition of serum increases the uptake of TPP+. In synchronized cells, intracellular levels of TPP+ differ at various stages of cell cycle and are lowest in mitosis.     

Conserved nuclear export sequences in Schizosaccharomyces pombe Mex67 and human TAP function in mRNA export by direct nuclear pore interactions

Mex67, the homolog of human TAP, is not an essential mRNA export factor in Schizosaccharomyces pombe. Here we show that S. pombe encodes a homolog of the TAP cofactor that we have also named p15, whose function in mRNA export is not essential. We have identified and characterized two distinct nuclear export activities, nuclear export signal (NES) I and NES II, within the region of amino acids 434-509 of Mex67. These residues map within the known NTF2-like fold of TAP (amino acids 371-551). We show that the homologs of these two NESs are present and are functionally conserved in TAP. The NES I, NES II, and NES I + II of TAP and Mex67 directly bind with -phenylalanine-glycine (-FG)-containing sequences of S. pombe Nup159 and Nup98 but not with human p62. Mutants of NES I or NES II of Mex67/TAP that do not bind -FG Nup159 and Nup98 in vitro are unable to mediate nuclear export of a heterologous protein in S. pombe and in HeLa cells. Fused with the RNA recognition motifs (RRMs) of Crp79 and green fluorescent protein (GFP) (RRM-NES-GFP), the NES I and NES II of Mex67 or TAP can suppress the mRNA export defect of the Deltap15 rae1-167 synthetic lethal S. pombe strain, suggesting that the NESs can function in the absence of p15. These novel nuclear export sequences may provide additional routes for delivering Mex67/TAP to the nuclear pore complex.     

Interaction of the frog brain kainate receptor expressed in Chinese hamster ovary cells with a GTP-binding protein.

A protein that binds kainate with high affinity has been purified and cloned from frog brain (Rana pipiens) and has approximately 35% sequence homology with mammalian non-N-methyl-D-aspartate glutamate receptors, some of which have been shown to be ligand-gated ion channels. Frog brain membranes and membranes from Chinese hamster ovary (CHO) cells transfected with the cDNA coding for the frog kainate-binding protein (CHO-4 cells) bound kainate with essentially identical affinity (KD values of 1.9 and 2.1 nM, respectively). In both tissues, the affinity for kainate decreased 9-fold in the presence of 100 microM GTP gamma S (guanosine 5'-O-(3-thio)triphosphate). No specific kainate binding to nontransfected CHO cell membranes was observed. GTP gamma S and GDP were effective inhibitors of kainate binding, while cGMP and adenosine 5'-O-(3-thio)triphosphate had no effect in either frog brain membranes or CHO-4 membranes. Pretreatment of CHO-4 cell membranes with pertussis toxin led to a 34% decrease in kainate binding. Kainate increased the binding of [3H]5'-guanylyl imidodiphosphate by 61%, and the rate of GTP hydrolysis by up to 5-fold. These results indicate that the kainate receptor cloned from frog brain can interact functionally with a G protein present in CHO-4 cell membranes.     

Stimulation of Ca2+ uptake and protein phosphorylation in tumor cells by fibronectin

Suspensions derived from attached HeLa cells transported 45Ca2+ considerably faster than those derived from spinner cultures grown in liquid medium. Incubation of spinner cells with fibronectin or cold-insoluble globulin in the presence of 5% calf serum at 37 degrees C for 1 to 2 h greatly increased the rate of Ca2+ flux into the cells. Suspensions of cells transformed by Rous sarcoma virus transported Ca2+ much more slowly than cell suspensions of the parent strain of normal rat kidney. Incubation of the transformed cells or Ehrlich ascites tumor cells with fibronectin increased the rate of Ca2+ uptake, while no effect was seen on Ca2+ transport by this treatment of normal kidney cells grown in tissue cultures. A 45,500-dalton protein was found to interact firmly with Ca2+ that entered into attached HeLa cells or fibronectin-treated spinner cells. This Ca2+-associated protein was detected by lithium dodecyl sulfate gel electrophoresis at 0 degrees C after 30 s of exposure to radioactive Ca2+. In tumor cells without fibronectin treatment, the radioactive band was not seen under the same conditions, even after 10 min incubation with 45Ca2+. In fibronectin-treated tumor cells, addition of Ca2+ to buffered solutions resulted in increased phosphorylation of a protein in the 45,000-dalton region. The phosphorylated protein band which appears to be associated with the cytoskeleton can be resolved by isoelectric focusing into four polypeptide chains. The relation of these observations to the cascade of protein kinases involved in the phosphorylation of the beta-subunit of the (Na+-K+)-ATPase is discussed.     

Ca2+ oscillations induced by histamine H1 receptor stimulation in HeLa cells: Fura-2 and patch clamp analysis

The response of HeLa cells to histamine H1 receptor stimulation is characterized by periodic increases in cytosolic free Ca2+ concentration. The mechanisms underlying this oscillatory behaviour are not well understood. Fura-2 and patch clamp experiments carried out on HeLa cells have previously shown: (a) that Ca2+ oscillations are not initially dependent on the presence of external Ca2+, that external Ca2+ is required to maintain the oscillatory activity; (b) that a depolarization of the cell membrane leads to an inhibition of Ca2+ oscillations during the external Ca2+ dependent phase of the process; and (c) that Ca2+ oscillations can be abolished during this latter phase by the exogenous addition of Ca2+ channel blocking agents, such as Co2+ or La3+. The contribution of the inositol phosphate pathway to Ca2+ oscillations was more recently investigated in whole cell experiments performed with patch pipettes containing IP3 or the non-hydrolysable GTP analogue GTP-gamma S. Clear periodic current fluctuations were recorded using both patch pipette solutions. Assuming that the intracellular IP3 level remained constant under these conditions, these findings provide direct evidence that the Ca2+ oscillations in HeLa cells do not arise from a periodic production of IP3. The effect of the internal and external cell pH on the oscillatory process was also investigated in Fura-2 and patch clamp experiments. It was found that an increase in intracellular pH from 7.4 to 7.7 during the external Ca2+ dependent phase of the histamine stimulation abolishes the appearance of Ca2+ spikes whereas, a cellular acidification to pH 7.2 maintains or stimulates the Ca2+ oscillatory activity. The former effect was observed in the absence of Ca2+ in the bathing medium, indicating that the inhibitory action of alkaline pH was not related to a reduced Ca2+ entry. An increase in extracellular pH from 7.3 to 9.0 in contrast elicited an intracellular Ca2+ accumulation which resulted in most cases in an inhibition of the oscillatory process. This effect was dependent on external Ca2+ and was observed in alkaline internal pH conditions (pH 7.7). These observations suggest: (a) that the net Ca2+ influx in HeLa cells is strongly dependent on the cell internal and external pH; and (b) that the magnitude of this Ca2+ influx controls to a large extent the oscillation frequency. Finally, an inhibition of the histamine induced Ca2+ oscillatory activity was observed following the addition of the Ca(2+)-induced Ca(2+)-release (CICR) inhibitor adenine to the external medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polycationic lipids translocate lipopolysaccharide into HeLa cells

We have investigated the ability of LIPOFECTAMINE, a polycationic lipid reagent used in DNA transfection, to translocate E. coli lipopolysaccharide (LPS) into HeLa cells. Although HeLa cells did not spontaneously take up fluorescein isothiocyanate-labelled LPS (FITC-LPS) from the culture medium, the cells that were co-incubated with greater than 1 g/mL FITC-LPS and LIPOFECTAMINE showed punctate fluorescence. Virtually all cells were loaded on incubation with 100 micrograms/mL FITC-LPS. Confocal scanning laser microscopy showed extensive FITC-LPS loading in the cytoplasm of HeLa cells, but no label was evident in the nuclear regions of these cells. Loading with LPS for up to six hours had no effect on the viability of HeLa cells, beyond the 30% reduction in live cells that is attributable to the toxic effect of LIPOFECTAMINE itself. In contrast to cells treated with etoposide for six hours, LPS-loaded cells did not display apoptotic bodies. Exposure of cells to 4 beta-phorbol 12-myristate 13-acetate led to the induction of the immediate early gene c-fos and resulted in an enhanced c-Fos signal, detected by Western blot analysis. In contrast, LPS loading did not alter the c-fos expression in HeLa cells. The loading of LPS into HeLa cells by means of polycationic lipids results in relatively low acute toxicity, as judged from cell viability, morphology and c-fos expression. Therefore, our method appears well suited to the study of acute actions of LPS in the intracellular compartment of mammalian cells.     

Adherence of Plasmodium falciparum infected erythrocytes to CHO-745 cells and inhibition of binding by protein A in the presence of human serum

Adhesion of erythrocytes infected with the malaria parasite Plasmodium falciparum to human host receptors is a process associated with severe malarial pathology. A number of in vitro cell lines are available as models for these adhesive processes, including Chinese hamster ovary (CHO) cells which express the placental adhesion receptor chondroitin-4-sulphate (CSA) on their surface. CHO-745 cells, a glycosaminoglycan-negative mutant CHO cell line lacking CSA and other reported P. falciparum adhesion receptors, are often used for recombinant expression of host receptors and for receptor binding studies. In this study we show that P. falciparum-infected erythrocytes can be easily selected for adhesion to an endogenous receptor on the surface of CHO-745 cells, bringing into question the validity of using these cells as a tool for P. falciparum adhesin expression studies. The adhesive interaction between CHO-745 cells and parasitized erythrocytes described here is not mediated by the known P. falciparum adhesion receptors CSA, CD36, or ICAM-1. However, we found that CHO-745-selected parasitized erythrocytes bind normal human IgM and that adhesion to CHO-745 cells is inhibited by protein A in the presence of serum, but not in its absence, indicating a non-specific inhibitory effect. Thus, protein A, which has been used as an inhibitor for a recently described interaction between infected erythrocytes and the placenta, may not be an appropriate in vitro inhibitor for understanding in vivo adhesive interactions.