Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of particles in the smaller peak (6-11 microm diameter) closely correlated with the fractions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells.     

The effects of patchouli alcohol and combination with cisplatin on proliferation,apoptosis and migration in B16F10 melanoma cells

Melanoma is a highly metastatic cancer with a low incidence rate, but a high mortality rate. Patchouli alcohol (PA), a tricyclic sesquiterpene, is considered the main active component in Pogostemon cablin Benth, which improves wound healing and has anti-tumorigenic activity. However, the pharmacological action of PA on anti-melanoma remains unclear. Thus, the present study aimed to investigate the role of PA in the proliferation, cell cycle, apoptosis and migration of melanoma cells. These results indicated that PA selectively inhibited the proliferation of B16F10 cells in a dose- and time-dependent manner. It induced cell cycle arrest at the G₀/G₁ phase and typical morphological changes in apoptosis, such as chromatin condensation, DNA fragmentation and apoptotic bodies. In addition, PA reduced the migratory ability of B16F10 cells by upregulating E-cadherin and downregulating p-Smad2/3, vimentin, MMP-2 and MMP-9 expression. PA was also found to strongly suppress tumour growth in vivo. Furthermore, PA combined with cisplatin synergistically inhibited colony formation and migration of B16F10 cells and attenuated the development of resistance to treatment. Therefore, the results of this study indicate that PA may play a pivotal role in inducing apoptosis and reducing the migration of melanoma cells, and may thus be a potential candidate for melanoma treatment.     

In vivo analysis of chromosome condensation in Saccharomyces cerevisiae

Although chromosome condensation in the yeast Saccharomyces cerevisiae has been widely studied, visualization of this process in vivo has not been achieved. Using Lac operator sequences integrated at two loci on the right arm of chromosome IV and a Lac repressor-GFP fusion protein, we were able to visualize linear condensation of this chromosome arm during G2/M phase. As previously determined in fixed cells, condensation in yeast required the condensin complex. Not seen after fixation of cells, we found that topoisomerase II is required for linear condensation. Further analysis of perturbed mitoses unexpectedly revealed that condensation is a transient state that occurs before anaphase in budding yeast. Blocking anaphase progression by activation of the spindle assembly checkpoint caused a loss of condensation that was dependent on Mad2, followed by a delayed loss of cohesion between sister chromatids. Release of cells from spindle checkpoint arrest resulted in recondensation before anaphase onset. The loss of condensation in preanaphase-arrested cells was abrogated by overproduction of the aurora B kinase, Ipl1, whereas in ipl1-321 mutant cells condensation was prematurely lost in anaphase/telophase. In vivo analysis of chromosome condensation has therefore revealed unsuspected relationships between higher order chromatin structure and cell cycle control.     

Vanadate,an inhibitor of tyrosine phosphatases,induced premature anaphase in oocytes and aneuploidy and polyploidy in mouse bone marrow cells

Protein tyrosine phosphatases are needed for activating maturation promoting factor, meiotic spindle assembly and spindle checkpoint inactivation. The protein phosphatase inhibitor vanadate was used to upset the kinase-phosphatase equilibrium during oocyte maturation (OM) and the metaphase anaphase transition (MAT) prior to cytogenetic analyses of mouse oocytes and bone marrow cells. ICR females received pregnant mare serum gonadotrophin (PMSG) and 48h later received human chorionic gonadotrophin (hCG). Vanadate doses of 0, 5, 15, and 25mg/kg were administered intraperitoneally immediately after hCG and ovulated oocytes and bone marrow cells were processed for cytogenetic analyses 18h after hCG. Data were analyzed by Chi-square and Fisher's exact tests. Vanadate induced different cytogenetic abnormalities in oocytes and in bone marrow cells. The frequencies of oocytes exhibiting premature anaphase (spontaneous activation) in vanadate exposed mice were significantly (P<0.01) elevated over controls; whereas, in bone marrow cells, the levels of tetraploidy, hyperploidy and premature centromere separation were significantly (P<0.01) increased by vanadate treatment. These results suggest that alteration of the kinase-phosphatase equilibrium during OM and the MAT leads to cytogenetic abnormalities that differ between oocytes and bone marrow cells.     

Structures of glycosphingolipids isolated from human embryonal carcinoma cells. The presence of mono- and disialosyl glycolipids with blood group type 1 sequence

Structures of glycolipids present in the human embryonal carcinoma cell PA1, were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase digestion. PA1 cells contain globotriaosylceramide, sialosylgangliotriaosylceramide, sialylated and nonsialylated lacto-N-neotetraosylceramide, and the following glycolipids with a blood group type 1 sequence: (formula; see text) The two former glycolipids, lacto-N-tetraosylceramide and sialosyllacto-N-tetraosylceramide, reacted with monoclonal antibodies, K21 and K4, respectively. K21 and K4 antigens are present in many of the human embryonal carcinoma cells but not in a variety of other cell lines, suggesting that sialylated but not fucosylated blood group type 1 sequences are characteristic markers for human embryonal carcinoma cells and malignant teratocarcinomas.     

Characterization of a pluripotent stem cell line derived from a mouse embryo

A pluripotent, karyotypically normal, male culture line ESC-BLC 1 of embryonal stem cells was established from delayed mouse blastocysts of strain 129/ter Sv. The cell line was isolated after cultivation of inner cell mass cells on X-irradiated feeder layer of mouse embryonal fibroblasts. The pluripotent status of the cell line was confirmed by in vivo and in vitro differentiation. For in vivo differentiation, cells were injected subcutaneously into syngeneic mice. The resulting tumors contained various tissues, derivatives of all three primary germ layers. In vitro cultivated pluripotent stem cells differentiated into endoderm-like, neuronal-like and tubular structures. Determination of alkaline phosphatase in cell line ESC-BLC 1 yielded a high specific activity; G-banding of metaphases revealed a normal, male karyotype.     

Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. We next examined whether Draper is responsible for the phagocytosis of apoptotic cells in vivo using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells.     

Gene transfer of cyto-protective molecules in corneal endothelial cells and cultured corneas: analysis of protective effects in vitro and in vivo

The loss of corneal endothelial cells plays a critical role in many corneal diseases and is a common phenomenon following cornea transplantation. In addition, the non-regenerative capacity of human corneal endothelial cells (HCEC) ultimately requires appropriate protection of corneal tissues during ex vivo storage to ensure vitality of the cells. However, only 70% of donor corneas can be used for grafting because of endothelial deficiencies. Corneal endothelial cell loss during storage is mainly induced by apoptotic cell death. This study was undertaken, for proof of principle, to investigate whether over-expression of cyto-protective molecules Bcl-x(L), Bag-1, and HO-1 prevents the loss of corneal endothelial cells both in vitro and in vivo. We demonstrate that gene transfer of both Bcl-x(L) and HO-1 has cyto-protective effects on HCEC in vitro. However, gene transfer of a single cyto-protective molecule does not prevent its rejection upon transplantation in a MHC class I/II disparate rat model.     

Cytogenetic effects of low doses of radiation in mammalian cells: analysis of the hypersensitivity phenomenon and induced resistance

The induction of cytogenetic damage after irradiation of chinese hamster cells and human melanoma cells within a dose range 1-200 cGy was studied. The anaphase and metaphase analysis of chromosome damage and micronuclei test were applied. The hypersensitivity (HRS) at doses below 20 cGy and the increased radio-resistence at higher doses (IR) were shown with all cytogenetic critheria for both cell lines. The phenomenon of HRS/IR was reproduced in synchronic as well as in a synchronic population of chinese hamster cells. This fact shows that HRS was caused by high radiosensitivity of all cells and can not be explained by any differential sensitivity of cells in different phase of the cell cycle. So it was supposed that the increasing radio-resistence is determined by the inclusion of the inducible repair processes in all cells. This conclusion consents with the facts, that there was no evidence of HRS on dose-effect curves and that some parts of pre-existent damage was repaired after preliminary irradiation with low doses (1-20 cGy) which induce repair processes. It can be concluded that same inducible repair processes an analogous in mechanisms underlying in the base of HRS/IR phenomenon and adaptive response.     

In vivo phosphorylation of the 170-kDa form of eukaryotic DNA topoisomerase II. Cell cycle analysis

We have examined the level of incorporation of 32P into DNA topoisomerase II in vivo in chicken lymphoblastoid cells that were fractionated into the various cell cycle phases by centrifugal elutriation. We find that topoisomerase II is phosphorylated in vivo, with the level of incorporation being approximately 3.5-fold higher in the G2 + M fraction than earlier in the cell cycle. Our antibody studies have revealed that topoisomerase II antigen exists as a number of discrete polypeptide species in these cells. Of these, the 170-kDa intact polypeptide is phosphorylated approximately 4.5-fold more than several antigenic fragments that actually comprise the bulk of the topoisomerase II antigen in these cells at mitosis. Phosphorylation of the 170-kDa form of the enzyme may be involved in activation of the enzyme for its role in the disjunction of sister chromatids at anaphase.     

Possible mechanisms of the occurrence of chromosome restructurings. IV. Chromosome restructurings in spontaneous mutagenesis

An attempt was undertaken to modify the spontaneous mutation process by varying its conditions in somatic cells of different species and tissues. The rate of chromosome aberrations and their types were studied in anaphase and metaphase. Under normal conditions, chromosome breaks were only found to occur. Breakage of chromosomes occurs during interphase, and as a result, acentric fragments are located outside the equatorial plate during metaphase. This process of chromosome breakage leads to elimination of some genetic material, without concomitant exchanges, and therefore, it has been named "elimination" process. Spontaneous chromosome mutagenesis manifesting itself at cytogenetic level was concluded to be an elimination process directed to elimination of a portion of chromatin from chromosomes. When the conditions of spontaneous mutagenesis are altered, in particular, by cardiovascular diseases in man, by partial inhibition of DNA repair in mice and pea cells, by transformation of Chinese hamster cells, upon ageing of pea seeds-qualitative changes in the chromosomal aberrations are registered, connected with the appearance of chromosome exchanges and acentric fragments situated within the equatorial plate during metaphase. These two types of chromosome aberrations are proposed to be considered as new criteria of pathology. A system of processes was suggested to exist, preventing the appearance of aberrations during mitosis, and it is supposed to be one of the most significant homeostatic systems.     

In vitro apoptotic cell death during erythroid differentiation

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.     

p53-Independent Apoptosis and p53-Dependent Block of DNA Rereplication Following Mitotic Spindle Inhibition in Human Cells

We have studied the response of human transformed cells to mitotic spindle inhibition. Two paired cell lines, K562 and its parvovirus-resistant KS derivative clone, respectively nonexpressing and expressing p53, were continuously exposed to nocodazole. Apoptotic cells were observed in both lines, indicating that mitotic spindle impairment induced p53-independent apoptosis. After a transient mitotic delay, both cell lines exited mitosis, as revealed by flow-cytometric determination of MPM2 antigen and cyclin B1 expression, coupled to cytogenetic analysis of sister centromere separation. Both cell lines exited mitosis without chromatid segregation. K562 p53-deficient cells further resumed DNA synthesis, giving rise to cells with a DNA content above 4C, and reentered a polyploid cycle. In contrast, KS cells underwent a subsequent G1 arrest in the tetraploid state. Thus, G1 arrest in tetraploid cells requires p53 function in the rereplication checkpoint which prevents the G1/S transition following aberrant mitosis; in contrast, p53 expression is dispensable for triggering the apoptotic response in the absence of mitotic spindle.     

Nucleolar persistence in embryonal carcinoma cells

A series of embryonal carcinoma (EC) lines were found to have nucleoli which persisted through metaphase and anaphase in 60–88% of cells. When these cells differentiated, either spontaneously or upon chemical induction with retinoic acid or dimethylacetamide, the level of nucleolar persistence dropped to under 20%. Mouse embryo fibroblasts and mouse bone marrow cells had few persistent nucleoli. Persistent nucleoli in EC cells contained DNA, labeled with tritiated uridine and stained with ammoniacal silver, all of which support the hypothesis that rRNA synthesis continues in persistent nucleoli.     

Induction of cell proliferation and apoptosis: dependence on the dose of the inducer.

Protein A (PA) of Staphylococcus aureus is known as an immunomodulator. In a search of the molecular mechanism(s) of PA-induced immunocyte potentiation, we found dose-dependent binding of PA (0.01 to 100 microg/ml PA) to the mice splenic lymphocytes. Interestingly, treatment of 1 microg PA/20 g mice increased the splenic lymphocyte number approximately 5-fold over control but at a 10-microg dose the cell number was decreased compared with a 1-microg dose. Flow cytometric analysis of cell-cycle phase distribution of nuclear DNA in splenic lymphocytes showed that at a 1-microg dose, PA shifted the cell-cycle phases from G0/G1 to S and G2/M supporting the pro-proliferative role of PA. In contrast, the same inducer increased the sub-G1 cell population at a 10-microg dose indicating the breakdown of cellular DNA. These findings were supported by DNA ladder formation and nuclear breakdown at this higher dose. Further studies revealed that at a 1-microg dose, the level of the pro-proliferative/anti-apoptotic protein bcl-2 was increased in splenic lymphocytes whereas at a 10-microg dose it showed a decreasing trend. In contrast, concentrations of proapoptotic proteins, p53 and bax, were increased at a 10-microg dose. A search of the mechanism(s) of such differential action of PA at these two doses revealed that the lower dose of PA upregulated the production of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) to the extent which has already been reported by our laboratory to be beneficial to the host. However, at a larger dose, much higher release of TNF-alpha and interleukin-2 (IL-2) may account for the apoptosis of splenic cells. All these findings indicated that the cross-talk between all these pro- and anti-apoptotic factors may contribute to maintain a balance between growth and death of cells and may be one of the important factors deciding whether a cell would follow a proliferative pathway or an apoptotic pathway.     

Nuclear translocation of PDCD5 (TFAR19): an early signal for apoptosis?

The programmed cell death 5 (PDCD5) protein is a novel protein related to regulation of cell apoptosis. In this report, we demonstrate that the level of PDCD5 protein expressed in cells undergoing apoptosis is significantly increased compared with normal cells, then the protein translocates rapidly from the cytoplasm to the nucleus of cells. The appearance of PDCD5 in the nuclei of apoptotic cells precedes the externalization of phosphatidylserine and fragmentation of chromosome DNA. This phenomenon is parallel to the loss of mitochondrial membrane potential, independent of the feature of apoptosis-inducing stimuli and also independent of the cell types and the apoptosis modality. In conclusion, the nuclear translocation of PDCD5 is a universal earlier event of the apoptotic process, and may be a novel early marker for apoptosis.     

Effects of caspase inhibition on camptothecin-induced apoptosis of HL-60 cells

BACKGROUND: During camptothecin (CAM)-induced apoptosis of HL-60 cells, the external exposure of phosphatidylserine (PS) can either precede or follow DNA cleavage. The evidence suggests that cells in S-phase when CAM is added undergo rapid DNA, nuclear, and cellular disintegration before exposing PS on the outside of the plasma membrane, whereas cells moving from G1 into S-phase after CAM is added expose PS before they manifest the other phenomena. This study describes further investigations using the broad spectrum caspase inhibitor Z-VAD-FMK. The cells were cultured for a period long enough to ascertain whether a particular phenomenon was only delayed or was blocked completely. METHODS: Changes in cell light scatter, binding of annexin V-fluorescein isothiocyanate (FITC) to PS, uptake of propidium iodide (PI) as a measure of plasma membrane integrity, and DNA content after membrane fixation/permeabilization were monitored by flow cytometry during 24-h cultures. Fluorescence microscopy was used to examine cell morphology. RESULTS: Caspase inhibition blocked DNA cleavage, breakdown of the nuclear membrane, and formation of apoptotic bodies. It also revealed the existence of a CAM-activated early S-phase checkpoint. Cells arrested in early S-phase preceded the appearance of PS-positive cells. Caspase inhibition delayed both PS exposure and loss of plasma membrane integrity but did not prevent either. CONCLUSIONS: The results support the hypothesis that the sequence of apoptotic phenomena in an individual CAM-treated HL-60 cell depends on the stage of proliferation of that cell when it encounters the CAM. They are also consistent with the hypothesis that caspases are not required for PS exposure or the loss of plasma membrane integrity, but they are involved indirectly in promoting these phenomena.     

The breakage–fusion–bridge (BFB) cycle as a mechanism for generating genetic heterogeneity in osteosarcoma

Osteosarcoma (OS) is characterized by chromosomal instability and high copy number gene amplification. The breakage–fusion–bridge (BFB) cycle is a well-established mechanism of genome instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. To determine whether the BFB cycle could be increasing the de novo rate of formation of cytogenetic aberrations in OS, the frequency of anaphase bridge configurations and dicentric chromosomes in four OS cell lines was quantified. An increased level of anaphase bridges and dicentrics was observed in all the OS cell lines. There was also a strong association between the frequencies of anaphase bridges, dicentrics, centrosomal anomalies, and multipolar mitotic figures in all the OS cell lines, indicating a possible link in the mechanisms that led to the structural and numerical instabilities observed in OS. In summary, this study has provided strong support for the role of the BFB cycle in generating the extensive structural chromosome aberrations, as well as cell-to-cell cytogenetic variation observed in OS, thus conferring the genetic diversity for OS tumor progression.