Cell and nuclear enlargement of SW480 cells induced by a plant lignan, arctigenin: evaluation of cellular DNA content using fluorescence microscopy and flow cytometry

Arctigenin is a natural plant lignan previously shown to induce G(2)/M arrest in SW480 human colon cancer cells as well as AGS human gastric cancer cells, suggesting its use as a possible cancer chemopreventive agent. Changes in cell and nuclear size often correlate with the functionality of cancer-treating agents. Here, we report that arctigenin induces cell and nuclear enlargement of SW480 cells. Arctigenin clearly induced the formation of giant nuclear shapes in SW480, as demonstrated by fluorescence microscopic observation and quantitative determination of nuclear size. Cell and nuclear size were further assessed by flow cytometric analysis of light scattering and fluorescence pulse width after propidium iodide staining. FSC-H and FL2-W values (parameters referring to cell and nuclear size, respectively) significantly increased after arctigenin treatment; the mean values of FSC-H and FL2-W in arctigenin-treated SW480 cells were 572.6 and 275.1, respectively, whereas those of control cells were 482.0 and 220.7, respectively. Our approach may provide insights into the mechanism behind phytochemical-induced cell and nuclear enlargement as well as functional studies on cancer-treating agents.     

Doublet discrimination in DNA cell-cycle analysis.

Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used interchangeably without regard for the complexity in cell shapes and sizes of biological specimens. G(0/1) doublets were identified and quantitated using fluorescence height versus area and fluorescence width versus area pulse measurements, by enumerating the proportion of G(2) + M cells that lack cyclin B1 immunoreactivity, and modeled in the DNA histograms by software algorithms. These techniques were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents that induced cell-cycle arrest and were extended to human breast tumor specimens having DNA diploid patterns. G(0/1) doublets were easily discernible from G(2) + M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discrimination based on pulse processing or cyclin B1 measurements was nonconcordant in some nonspherical cell types and in cells following cell cycle arrest. Significant differences in G(0/1) doublet estimates were observed in breast tumor specimens (n = 50), with estimates based on pulse width twice those of pulse height and nearly five times greater than computer estimates. Differences between techniques are attributed to difficulties in the separation of the boundaries between G(0/1) doublets and G(2) + M singlet populations in biologically heterogeneous specimens. To improve reproducibility and enhance standardization among laboratories performing cell cycle analysis in experimental cell systems and in human breast tumors, doublet discrimination analysis should best be accomplished by computer modeling. Shape and size heterogeneity of tumor and arrested cells using pulse-processing can lead to errors and make interlaboratory comparison difficult.     

Etoposide-induced activation of c-jun N-terminal kinase (JNK) correlates with drug-induced apoptosis in salivary gland acinar cells.

We have examined the ability of etoposide to induce apoptosis in two recently established rat salivary acinar cell lines. Etoposide induced apoptosis in the parotid C5 cell line as evidenced by the appearance of cytoplasmic blebbing and nuclear condensation, DNA fragmentation and cleavage of PARP. Etoposide also induced activation of c-jun N-terminal kinase (JNK) in parotid C5 cells by 4 h after treatment, with maximal activation at 8 - 10 h. Coincident with activation of JNK, the amount of activated ERK1 and ERK2 decreased in etoposide-treated parotid C5 cells. In contrast to the parotid C5 cells, the vast majority of submandibular C6 cells appeared to be resistant to etoposide-induced apoptosis. Likewise, activation of JNKs was not observed in etoposide-treated submandibular C6 cells, and the amount of activated ERK1 and ERK2 decreased only slightly. Etoposide treatment of either cell line had no effect upon the activation of p38. Treatment of the parotid C5 cells with Z-VAD-FMK, a caspase inhibitor, inhibited etoposide-induced activation of JNK and DNA fragmentation. These data suggest that etoposide may induce apoptosis in parotid C5 cells by activating JNKs and suppressing the activation of ERKs, thus creating an imbalance in these two signaling pathways.     

Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

Differentiation of mitotic melanoma cells from G2 cells and their isolation by use of 5-bromo-2'-deoxyuridine and propidium iodide

This report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5-bromo-2'-deoxyuridine (BrdUrd) and double-stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 micrograms/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti-BrdUrd and PI. PGE1-treated cells monitored at 3-hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti-BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd-negative "S phase" cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move "backwards" in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to over-estimation of S phase cells in kinetic studies because of contaminating mitotic cells.     

Serum withdrawal and etoposide induce apoptosis in human lung carcinoma cell line A549 via distinct pathways

The molecular events associated with apoptosis induced by two distinct triggers (1) serum withdrawal and (2) etoposide treatment were investigated in the human lung carcinoma cell line A549. Although both serum withdrawal and etoposide treatment resulted in internucleosomal DNA fragmentation, the morphologic features were distinct. Serum deprived apoptotic cells appeared small, round and refractile, with little evidence of nuclear fragmentation; etoposide-induced apoptotic cells appeared enlarged and flattened and displayed prominent nuclear fragmentation. p53 and p21/waf1 protein levels were elevated in etoposide-treated cells, but not in cells subjected to serum with-drawal. Apoptosis induced by both treatments was accompanied by a significant reduction in Rb protein levels. However, etoposide treatment led to hypo-phosphorylation of Rb, while serum withdrawal did not alter the Rb phosphorylation pattern. Serum withdrawal-induced apoptosis was correlated with activation of JNK and suppression of ERK activities, while both JNK and ERK activities were slightly elevated during etoposid- induced apoptosis. Together, these results support the hypothesis that apoptosis induced by serum withdrawal and etoposide treatment occurs through different pathways and involves distinct mediators.     

The relationship between thrombomodulin expression and cell cycle stages in cultured rabbit endothelial cells

Cultured rabbit endothelial cells have significant but variable amounts of thrombomodulin (TM), both on their surface as well as inside the cell. To determine if variations in TM antigen is cell cycle related, cells with very high levels of TM antigen were identified and staged according to the intracellular distribution and relative amounts of the antigen, using immunofluorescence techniques. After staging, the nuclear DNA content of each of these cells was determined by measuring the propidium iodide (PI) fluorescence intensity cytophotometrically. Stages 1, 2, and 3, which exhibited TM immunofluorescence in the golgi area, clustered to the G1 phase of the cell cycle. Cells without discernible golgi fluorescence (stages 4 and 5) but with variable amounts of cytoplasmic and surface fluorescence appeared to have little or no relationship to the cell cycle.     

Midblastula transition (MBT) of the cell cycles in the yolk and pigment granule-free translucent blastomeres obtained from centrifuged Xenopus embryos

We obtained translucent blastomeres free of yolk and pigment granules from Xenopus embryos which had been centrifuged at the beginning of the 8-cell stage with cellular integrity. They divided synchronously regardless of their cell size until they had decreased to 37.5 microm in radius; those smaller than this critical size, however, divided asynchronously with cell cycle times inversely proportional to the square of the cell radius after midblastula transition (MBT). The length of the S phase was determined as the time during which nuclear DNA fluorescence increased in Hoechst-stained blastomeres. When the cell cycle time exceeded 45 min, S and M phases were lengthened; when the cell cycle times exceeded 70 min, the G2 phase appeared; and after cell cycle times became longer than 150 min, the G1 phase appeared. Lengths of G1, S and M phases increased linearly with increasing cell cycle time. Enhanced green fluorescent protein (EGFP)-tagged proliferating cell nuclear antigen (PCNA) expressed in the blastomeres appeared in the S phase nucleus, but suddenly dispersed into the cytoplasm at the M phase. The system developed in this study is useful for examining the cell cycle behavior of the cell cycle-regulating molecules in living Xenopus blastomeres by fluorescence microscopy in real time.     

Nuclear import of PKCdelta is required for apoptosis: identification of a novel nuclear import sequence

We have shown previously that protein kinase Cdelta (PKCdelta) is required for mitochondrial-dependent apoptosis. Here we show that PKCdelta is imported into the nucleus of etoposide-treated cells, that nuclear import is required for apoptosis and that it is mediated by a nuclear localization signal (NLS) in the C-terminus of PKCdelta. Mutation of the caspase cleavage site of PKCdelta inhibits nuclear accumulation in apoptotic cells, indicating that caspase cleavage facilitates this process. Expression of the PKCdelta catalytic fragment (CFdelta) in transfected cells results in nuclear localization and apoptosis. We show that the PKCdelta NLS is required for nuclear import of both full-length PKCdelta and CFdelta, and drives nuclear localization of a multimeric green fluorescent protein. Mutations within the NLS of CFdelta prevent nuclear accumulation and block apoptosis. Conversely, nuclear expression of a kinase-negative catalytic fragment (KN-CFdelta) protects cells from etoposide-induced apoptosis. Mutation of the NLS blocks the ability of KN-CFdelta to protect against etoposide-induced apoptosis. These results indicate that PKCdelta regulates an essential nuclear event(s) that is required for initiation of the apoptotic pathway.     

Simple discrimination of sub-cycling cells by propidium iodide flow cytometric assay in Jurkat cell samples with extensive DNA fragmentation

Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL3 plots, as well as decreased PI fluorescence, indicative of ongoing fragmentation and loss of nuclear DNA. By gating on these events, the resulting fraction of presumably sub-cycling cells (i.e. cells with cleaved DNA, counting sub-G₀/G₁, sub-S and sub-G₂/M cells altogether) was closely similar to the apoptotic rate assessed by Annexin V/PI labeling. Taken together, these findings suggest a possible way to recognize the entire population of cells undergoing apoptotic DNA cleavage and simultaneously determine the cell cycle distribution of non-apoptotic cells in PI-labeled cell samples with various degrees of DNA fragmentation, using a simple and reproducible multiparametric analysis of flow cytometric recordings.     

Both ERK1 and ERK2 kinases promote G2/M arrest in etoposide-treated MCF7 cells by facilitating ATM activation

The MEK–ERK pathway plays a role in DNA damage response (DDR). This has been thoroughly studied by modulating MEK activation. However, much less has been done to directly examine the contributions of ERK1 and ERK2 kinases to DDR. Etoposide induces G2/M arrest in a variety of cell lines, including MCF7 cells. DNA damage-induced G2/M arrest depends on the activation of the protein kinase ataxia-telangiectasia mutated (ATM). ATM subsequently activates CHK2 by phosphorylating CHK2 threonine 68 (T68) and CHK2 inactivates CDC25C via phosphorylation of its serine 216 (S216), resulting in G2/M arrest. To determine the contribution of ERK1 and ERK2 to etoposide-induced G2/M arrest, we individually knocked-down ERK1 and ERK2 in MCF7 cells using specific small interfering RNA (siRNA). Knockdown of either kinases significantly reduced ATM activation in response to etoposide treatment, and thereby attenuated phosphorylation of the ATM substrates, including the S139 of H2AX (γH2AX), p53 S15, and CHK2 T68. Consistent with these observations, knockdown of either ERK1 or ERK2 reduced etoposide-induced CDC25C S216 phosphorylation and significantly compromised etoposide-induced G2/M arrest in MCF7 cells. Taken together, we demonstrated that both ERK1 and ERK2 kinases play a role in etoposide-induced G2/M arrest by facilitating activation of the ATM pathway. These observations suggest that a cellular threshold level of ERK kinase activity is required for the proper checkpoint activation in MCF7 cells.     

Multivariate flow cytometry of epidermal regeneration provoked by a skin irritant and a tumor promoter.

The DNA content and the changes in cellular and nuclear size of isolated regenerating mouse epidermal basal cells were studied after topical application of the skin irritant cantharidin and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to the back skin of hairless mice. The DNA and protein contents of isolated basal cells were stained with propidium iodide and fluorescein isothiocyanate, respectively, and analysed by flow cytometry using the total protein fluorescence as an estimate of cell size and the DNA fluorescence pulse width as an estimate of nuclear size. Transmission electron microscopy was used to identify cells sorted from regions in the bivariate DNA/protein distributions. The results showed that both chemicals induced an increase in cellular as well as nuclear size of the basal cells. The increase in size was higher in TPA treated than in cantharidin treated animals, and the bivariate DNA/protein distributions of TPA treated cells differed from those of cantharidin treated cells in that two subpopulations of cycling keratinocytes could be identified. These deviations are probably related to the higher proliferative response observed after TPA treatment and the possibility that proliferative subpopulations in epidermis respond differently to TPA. It may reflect mechanisms providing for a growth advantage of initiated cells, important in tumor promotion. About 8% of the cells in the suspensions from treated animals were non-cycling non-keratinocytes, probably infiltrating leukocytes. The results indicate a strong correlation between rapid regenerative cell cycle progression, i.e., reduced G1 transit time and increased cellular and nuclear size.(ABSTRACT TRUNCATED AT 250 WORDS)     

血小板生成素诱导胎肝CD34^＋造血干／祖细胞增殖分化与周期蛋白表达的关系

为了解胚胎时期巨核细胞增殖分化特有的内在机制,本研究观察了在体外培养体系中,胎肝源CD34+造血干/祖细胞在血小板生成素(thrombopoietin,TPO)作用下增殖分化特征与相关周期蛋白B1、D1和D3表达及细胞内水平变化的关系。结果发现(1)经12d培养后,TPO使胎肝源CD34     

Caspase-independent autophagic cytotoxicity in etoposide-treated CaSki cervical carcinoma cells

We studied the in vitro mechanism of etoposide-induced cell death in cervical cancer cells. Etoposide is cytotoxic to these cells, causing cell death by both apoptosis and autophagy, which has recently been described as a possible mechanism for nonapoptotic cell death. Electron microscopy revealed that autophagosomes/autolysosomes exhibited an autophagic appearance in the presence of etoposide. When autophagy was blocked by inhibitors of autophagy, including 3-methyladenine, both the expression of beclin 1 protein and the antitumor effect of etoposide were suppressed. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor, reduced etoposide-induced cytotoxicity in CaSki cells. Hence, autophagy and apoptosis likely occur concurrently in etoposide-treated cervical cancer cells.     

Identification of G2/M phase transition by sequential nuclear and cytoplasmic changes and molecular markers in mice intestinal epithelial cells

Although the regulatory network of G2/M phase transition has been intensively studied in mammalian cell lines, the identification of morphological and molecular markers to identify G2/M phase transition in vivo remains elusive. In this study, we found no obvious morphological changes between the S phase and G2 phase in mice intestinal epithelial cells. The G2 phase could be identified by Brdu incorporation resistance, marginal and scattered foci of histone H3 phosphorylated at Ser10 (pHH3), and relatively intact Golgi ribbon. Prophase starts with nuclear transformation in situ, which was identified by a series of prophase markers including nuclear translocation of cyclinB1, fragmentation of the Golgi complex, and a significant increase in pHH3. The nucleus started to move upwards in the late prophase and finally rounded up at the apical surface. Then, metaphase was initiated as the level of pHH3 peaked. During anaphase and telophase, pHH3 sharply decreased, while Ki67 was obviously bound to chromosomes, and PCNA was distributed throughout the whole cell. Based on the aforementioned markers and Brdu pulse labeling, it was estimated to take about one hour for most crypt cells to go through the G2 phase and about two hours to go through the G2-M phase. It took much longer for crypt base columnar (CBC) stem cells to undergo G2-prophase than rapid transit amplifying cells. In summary, a series of sequentially presenting markers could be used to indicate the progress of G2/M events in intestinal epithelial cells and other epithelial systems in vivo.     

Direct and indirect flow cytometric enumeration of 6-thioguanine-resistant human peripheral blood lymphocytes

Methods based on flow cytometry and sorting, autoradiography, and cloning were used to evaluate the potential for the enumeration of 6-thioguanine-resistant human peripheral blood lymphocytes assumed to be deficient with respect to the enzyme hypoxanthine-guanine-phosphoribosyl-transferase. Flow cytometric sorting of proliferating cells in the late S- and the G2-stages by means of DNA content, as measured by propidium iodide fluorescence, enabled an enrichment of variant cells to about 99%. The main source of false events was contaminating doublets of G0/G1 cells appearing in the sorting region. Doublet discrimination measured as the difference between pulse height and area (Ortho-50) accomplished no further improvement. A combination of propidium iodide fluorescence and bromodeoxyuridine incorporation, measured by fluorescent anti-bromodeoxyuridine-DNA antibodies, allowed flow cytometric enrichment to about 99.99% of variant cells. By sorting of 3H-thymidine-labeled cell nuclei from the late S- and the G2-phases and subsequent autoradiographic evaluation, partly resistant variants could be discriminated; variant frequencies of the same magnitude as for the cell cloning methods were obtained.     

Cyclin-dependent protein kinase 2 activity is required for mitochondrial translocation of Bax and disruption of mitochondrial transmembrane potential during etoposide-induced apoptosis

Previous studies have suggested that upregulation of Cyclin A-dependent protein kinase 2 (Cdk2) activity is an essential event in apoptotic progression and the mitochondrial permeability transition in human cancer cells. Here, we show that upregulated Cyclin A/Cdk2 activity precedes the proteolytic cleavage of PARP and is correlated with the mitochondrial translocation of Bax and the loss of mitochondrial transmembrane potential (Δψm) during etoposide-induced apoptosis in human cervical adenocarcinoma (HeLa) cells. Etoposide-induced apoptotic cell death is efficiently prevented in cells that overexpress a dominant negative mutant of Cdk2 (Cdk2-dn) or p21^WAF1/CIP1, a specific Cdk inhibitor. Conversely, apoptotic cell death is promoted in Cyclin A-expressing cells. Disruption of the mitochondrial transmembrane potential in etoposide-induced cells is prevented in cells that overexpress Cdk2-dn or p21^WAF1/CIP1, while this transition is prominently promoted in Cyclin A-expressing cells. We screened for mitochondrial Cdk2 targets in the etoposide-induced cells and found that the mitochondrial level of Bax is elevated by more than three fold in etoposide-treated cells and this elevation is effectively prevented in cells expressing Cdk2-dn under the same conditions. Thus, we suggest that Cdk2 activity is involved in the mitochondrial translocation of Bax, which plays an important role in the mitochondrial membrane permeability transition during apoptotic progression.     

Flow cytometric discrimination of mitotic nuclei by right-angle light scatter

Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90 degree light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90 degree light scatter and protein content. M-phase nuclei can be distinguished from G2-phase nuclei on cytograms of 90 degree light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent (NP40) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTA/Tris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M-phase nuclei is reduced, and the nuclei are located in the late S/G2 region of the single-parameter histogram.     

Flow cytometric discrimination of mitotic cells: resolution of M, as well as G1, S, and G2 phase nuclei with mithramycin, propidium iodide, and ethidium bromide after fixation with formaldehyde

Cells in mitosis can be flow cytometrically discriminated from G1, S, and G2 cells by analysis of a nuclear suspension prepared with nonionic detergent, fixed with formaldehyde, and stained with mithramycin, propidium iodide, or ethidium bromide. With these DNA-fluorochromes, the fluorescence is quenched by formaldehyde less in mitotic nuclei than in interphase nuclei. Mitotic nuclei have a 20-40% increased mithramycin fluorescence and 30-60% decreased light scatter in comparison to those of G2 nuclei. There is a high correlation (r = 0.95; P less than 0.001) between microscope counts of mitotic figures in smear preparations of the initial cell suspension and the flow cytometrically estimated fraction of nuclei with increased mithramycin fluorescence. Flow sorting (FACS) demonstrates that the mitotic nuclei are confined to the peak of increased mithramycin fluorescence and decreased light scatter. The method has been applied to cultures of Yoshida ascites tumor cells, JB-1 reticulosarcoma cells, and PHA-stimulated human lymphocytes, incubated in the presence or absence of vinblastine for mitotic arrest. In a heteroploid mixture of fixed Yoshida (near-diploid) and JB-1 (hypotetraploid) nuclei, the mitotic fractions of the two cell lines could be estimated separately when analyzed with mithramycin fluorescence versus light scatter or with mithramycin fluorescence versus propidium iodide fluorescence.