Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells

Intact rabbit immunoglobulin G molecules (IgGs) and their papain or pepsin fragments were radio-iodinated and injected into HeLa cells. Whole IgGs, Fab2, and Fc fragments were degraded with half-lives of 60- 90 h, whereas half-lives of Fab fragments were 110 h. These results indicate that proteolytic cleavage in the hinge region of the IgG molecule is not the rate-limiting step in its intracellular degradation. The hingeless human myeloma protein, Mcg, was degraded at the same rate as bulk human IgG, providing further evidence that the proteolytically susceptible hinge region is not important for intracellular degradation of IgG molecules. SDS acrylamide gel analysis of injected rabbit IgG molecules revealed that heavy and light chains were degraded at the same rate. Injected rabbit IgGs and rabbit IgG fragments were also examined on isoelectric focusing gels. Fab, Fab2, and Fc fragments were degraded without any correlation with respect to isoelectric point. Positively charged rabbit IgGs disappeared more rapidly than their negative counterparts, contrary to the trend reported for normal intracellular proteins. The isoelectric points of two mouse monoclonal antibodies were essentially unchanged after injection into HeLa cells, suggesting that the altered isoelectric profile observed for intact rabbit IgG resulted from degradation and not protein modification. The intracellular distributions of IgG fragments and intact rabbit IgG molecules were determined by autoradiography of thin sections through injected cells. Intact IgG molecules were excluded from HeLa nuclei whereas both Fab and Fc fragments readily entered them. Thus, for some proteins, entry into the nuclear compartment is determined primarily by size.     

Sequence of lethal events in HeLa cells exposed to the G2 blocking cytolethal distending toxin

The bacterial cytolethal distending toxin (CDT) was previously shown to block the cell cycle of several cell lines at stage G2 through inactivation of the cyclin-dependent kinase Cdkl and without induction of DNA strand breaks. In the present study, we have analyzed, using various methods of analytical cytometry, the progressive transformation and delayed lethal events in the tumor-derived HeLa cell line temporarily exposed to CDT. The cell proliferation arrest induced by CDT was irreversible but, starting about two days after exposure, the G2 block released partially, concomitantly with a decline in the level of Cdkl phosphorylation. This partial release resulted in endoreduplication, leading to the emergence of a significant subpopulation of cells with a 8C DNA content, and by multipolar abortive mitosis which accounted for the mortality recorded 2 and 3 days after exposure. The other major lethal event was a micronucleation process which started to be significant about 3 days after exposure and amplified later on. Both multipolar abortive mitosis and micronucleation appeared topologically related to centrosomal amplification.     

Ultrastructural examinations of HeLa cells exposed to cadmium ions

Destructive changes in cadmium-treated HeLa cells affecting practically all the cell structures and organelles, were observed. Side by side with it compensation and adaptation responses of cells, which in definite period reduced cell pathological effect of this microelement were revealed.     

Reduced immunoglobulin G activates complement system with decreased cooperativity

Sheep erythrocytes sensitized with intact antibody or reduced and alkylated antibody were lysed by guinea-pig serum indicating that reduced and alkylated antibody bound and activated complement. Reduction of antibody caused erythrocyte lysis to exhibit pseudo-first-order kinetics, while the lysis kinetics of erythrocytes sensitized with intact antibody was sigmoidal. Analysis of erythrocyte lysis by complement according to the von Krogh equation showed that reduction of antibody diminished the von Krogh exponent $\text{n}$ from 2.8 to 1.3, while the value of $\text{K}$ remained unchanged at 0.17 (complement dilution). These observations suggested that the sole effect of the reduction of antibody inter-heavy-chain and heavy-light chain disulfide bonds was to diminish the cooperativity of antibody-complement interaction.     

Increased protein ADPribosylation in HeLa cells exposed to the anti-cancer drug methotrexate

DNA single-strand breaks (about 200-300 per genome) were transiently detected during the first hour when HeLa cells were incubated for up to 24 h with 100 microM methotrexate. There was an expected increase in ADPribosyltransferase activity, which reached a maximum 2-3-fold stimulation at 3 h but which was still greater than in control cells after 24 h. When hypoxanthine (25 microM) was present in the incubations together with the methotrexate the transferase was no longer activated, although basal, control levels of activity were still present. DNA strand breaks were reduced in number but were still just detectable under these conditions. Cellular NAD+ levels were mostly unaffected by the various drug treatments, except for a small transient decrease after 1 h, possibly as a result of the transferase activation. Methotrexate did not cause an increase in the rate of ADPribose degradation. Degradation of ADPribose residues labelled in a preincubation period in permeabilized cells was more extensive at pH 6.0 was a 50% loss of acid-insoluble radioactivity in 30 min at 26 degrees C. At pH 8.0 the loss did not exceed 30-35% even after 90 min incubation. The activation of the transferase is reflected in a general increase in protein ADPribosylation detected by autoradiography of 32P-labelled proteins in 6.25-18.25%T gradient acrylamide gels. There were three major acceptors with molecular masses of 17, 100 and over 100 kDa, which could be respectively a histone, a transferase-derived peptide fragment and the transferase itself. When ADPribosyltransferase was inhibited with 3-amino-benzamide DNA single-strand breaks were no longer detected. However, this had no observably signficant effect on the kinetics of loss of cell viability (from Trypan blue uptake), cell number or colony-forming ability. Similar results are observed in most cases when the activation of the transferase, resulting from the incubation of cells with methotrexate, is inhibited by hypoxanthine. We conclude from such observations that the enhanced protein ADPribosylation seen in the cells exposed to methotrexate is a direct consequence of drug-exposure, but does not have any significant influence over the course of events leading ultimately to cell death.     

Impaired angiogenic balance and suppression of tumorigenicity in HeLa cells chronically exposed to interferon-alpha

We have previously reported that IFNalpha-chronic treatment for 41 days induced a partial phenotype reversion on HeLa cells along with a down-regulation of HPV18 mRNA levels. However, tumorigenicity of these cells in nude mice was unchanged. Interestingly, after 1 year of IFNalpha-chronic exposition, HeLa cells failed to induce s.c. tumors when injected into nude mice. In such experimental conditions both HPV18 DNA integration pattern and viral DNA copy number present in HeLa cells remained intact in the nontumorigenic phenotype cells. As result of the treatment with IFNalpha, HeLa cells rendered more resistant to lysis mediated by activated natural killer cells in vitro. Furthermore, IFNalpha-chronic treatment was able to induce VEGF and decrease bFGF mRNA expression, suggesting a potential effect on the angiogenic behavior of these tumoral cells. Thus, long-term treatment of HeLa cells with IFNalpha can accomplish a reversion of the malignant phenotype by a sequential multistep mechanism, in which the antiangiogenic effect of IFNalpha could be one of the contributing events.     

Dimeric, trimeric and tetrameric complexes of immunoglobulin G fix complement.

The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability.     

Diffusion kinetics of tritiated uridine into HeLa cells previously exposed to hyperthermia

Hyperthermic treatment of HeLa cells at 42 degrees C for 60 min depressed the specific activity of these cells when incubated with 3H-uridine both during and post heating compared to cells maintained at 37 degrees C. These changes were unlikely to arise from increased leakage from the cells and may partially be attributed to membrane damage influencing facilitated diffusion. Diffusion kinetic data for incorporation of the radiolabel into the T.C.A. soluble and T.C.A. insoluble fractions of HeLa cells indicated that a significant depression of Vmax and a significant elevation of Km for incorporation of 3H UdR into RNA may possibly result from an isotope dilution effect attributed to degrading pre-ribosomal RNA under the effect of hyperthermia.     

Changes in the pattern of antioxidant enzymes in wheat exposed to water deficit and rewatering

Antioxidant defenses in two wheat cultivars differing in sensitivity to dehydration (YouJian (YJ-24) more sensitive than LongChun (LC-20) were analyzed during water deficit and rewatering. Resistant cultivar (LC-20) showed a higher relative water content than the sensitive cultivar (YJ-24) during the whole period of water withholding. In order to analyze the changes of antioxidant enzymes, native PAGE analysis of protein extract were performed. Wheat leaves had two isoforms of Mn-superoxide dismutase (SOD), two isoforms of Cu/Zn-SOD and one of Fe-SOD. Three catalase (CAT) isoforms were identified in the leaves of wheat. The activities of SOD and CAT isoforms were increased in two cultivars under water deficit. The intensities of SOD and CAT isoforms were slightly lower in LC-20 and increased continuously in YJ-24 after rewatering. Peroxidase (POD) isoforms were significantly increased during the whole dehydration-rehydration period. Three ascorbate peroxidase (APX) isoforms were present in gel. APX-1 and APX-3 were enhanced during water deficit and decreased during rewatering in LC-20. In YJ-24 only the activities of APX-2 were increased under water deficit. Seven isoforms of glutathione reductase (GR) were detected in the native gel. Activities of most of GR isoforms were higher in tolerant (LC-20) than in sensitive cultivar (YJ-24). Different isoforms of GR in two wheat cultivars behaved differently under water deficit and rewatering. These results collectively suggest that water deficit activates the SOD, CAT and ascorbate-glutathione cycle in wheat leaves. The response of enzyme isoforms to drought is not the same for all isoforms of antioxidant enzymes in two wheat cultivars.     

Mitochondrial changes during the apoptotic process of HeLa cells exposed to cisplatin.

HeLa cells undergo apoptosis after exposure to cisplatin. Since mitochondria have recently been proposed as a probable effector of this type of cell death, we performed an analysis using the fluorescent cation rhodamine 123, which is transported actively by this organelle. Cisplatin induces a decrease in the mitochondrial staining, as assessed by cytofluorometric analysis. Microscopic analysis demonstrated that this effect was accompanied by damage of the mitochondria. These features were not exclusive of cisplatin, as other antineoplasic agents (taxol, etoposide) elicited similar effects. These results point toward the notion of a general effect of antineoplasic drugs over the mitochondria during induction of apoptotic cell death.     

Immunoglobulin M and immunoglobulin G secretion by human B cells exposed to RU 41.740, a glycoprotein extract from Klebsiella pneumoniae

RU 41.740, a glycoprotein extract from Klebsiella pneumoniae, was seen to activate human B cells to immunoglobulin secretion in vitro. The effects of RU 41.740 on human B cells were compared to those induced by pokeweed mitogen, a T-cell-dependent polyclonal B-cell activator, and Epstein-Barr virus, a T-cell-independent polyclonal B-cell activator. Exposure of human B cells to all of these agents resulted in increased immunoglobulin M (IgM) and immunoglobulin G (IgG) secretion. IgM and IgG secretion induced by RU 41.740 appeared to be T cell dependent when B cells were isolated from human peripheral blood. However, this activity may have been T cell independent when B cells were isolated from human spleen. RU 41.740-induced IgM secretion by peripheral blood B cells was seen to peak after 6 days in culture; IgG secretion peaked after 7 days in culture. The optimal concentration of RU 41.740 for the induction of IgM and IgG secretion by human B cells in vitro was seen to be 200 micrograms/ml.     

Changes in the protein-synthesizing system of a HeLa cell culture exposed to a number of trace elements

The authors investigated the cytopathic action of maximal allowable concentrations (MAC) of zinc, nickel, cobalt, cadmium and fluorine on cell culture. The most significant changes in RNA synthesis were noticed after exposure to zinc MAC. After exposure to the MAC of zinc, nickel and fluorine considerable modifications of protein synthesis were recorded. It was established that the content of rRNA increased after incubation with all trace elements under study for 24 hours. The amount of protein experienced significant changes after nickel introduction into the incubation medium. It is concluded that exposure to some of trace elements entails considerable changes in cell metabolism.     

Immunoreactivity to antinucleoside antibodies persists during G2 arrest in X-irradiated HeLa cells

Arrest of HeLa cells in G2 after ionizing radiation is accompanied by persistent nuclear immunoreactivity to antinucleoside antibodies. The reactivity declined to the normal G2 level during escape from arrest and subsequent cell division.     

Changes in the electro-orientation of Escherichia coli cells exposed to disinfectants

Various disinfectants were shown to influence the frequency dependence of Escherichia coli electro-orientation. Cell inactivation by different agents was found to decrease the effect at high frequencies (5 X 10(5)-5 X 10(6) Hz). The decrease should be attributed to the fact that the barrier properties of membranes are disorganized and the equivalent electric conductivity of cells drops down. The microbiological control of the bactericidal action produced by disinfectants fits in well with changes in the electro-orientation of bacterial cells at these frequencies.     

Myeloperoxidase reduces the opsonizing activity of immunoglobulin G and complement component C3b

The effect of myeloperoxidase, hydrogen peroxide (H2O2) and a halide (Cl) on the opsonizing molecules in immunoglobulin G (IgG) and complement factor C3b was assayed. At concentrations of the enzyme (1 microgram/ml) that can be found in the extracellular fluid during inflammation, the myeloperoxidase-H2O2-Cl system inhibited the opsonizing effect of IgG and C3b measured as phagocytic uptake and superoxide generation. The effect was related to the enzymatic peroxidative activity of the protein. The presence of albumin (10 mg/ml) reduced the effect of myeloperoxidase with 10-20%. Taurine, which in the presence of myeloperoxidase-H2O2-Cl forms hydrophilic chloramines, and D-penicillamine, which scavenges HOCl, neutralize the inhibitory effect of myeloperoxidase. This suggests that either hypochlorous acid or lipophilic chloramines may exert its effect by oxidizing free sulphydryl groups exposed on the opsonizing ligands. Since the myeloperoxidase-H2O2-halide system also affects chemotactic factors, leukotrienes, proteinases and membrane receptors, the system may in several ways affect the development of the inflammatory response.