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Cell Biology International (2005) 29, 3339 (Printed in Great Britain)
Analysis of 1H NMR-detectable mobile lipid domains for assessment of apoptosis induced by inhibitors of DNA synthesis and replication
V.M. Mikhailenko*, A.A. Philchenkov and M.P. Zavelevich
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, 45, Vasilkivska Street, Kyiv 03022, Ukraine
Cell membrane rearrangements coincident with apoptosis may contribute to the increase in the ratio of methylene (CH
We studied CH
Keywords: Apoptosis, 1H NMR, Mobile lipid domains.
Apoptotic cell death is coincident with changes in the cell membrane such as altered lipid packing of the lipid bilayer, membrane blebbing, decreased membrane microviscosity and a loss of membrane asymmetry with surface exposure of phosphatidylserine (Schlegel et al., 1993). Those changes were attributed to the increase in the ratio of the methylene (CH
Cellular origin of these resonances is related to lipid turnover and cell membrane structure. NMR signals from CH
1H NMR-visible mobile lipid domains (MLD) are reported as a peculiar feature of malignant cells in vitro and in vivo (Mountford et al., 1996) and also cells undergoing apoptosis (Blankenberg et al., 1997). 1H NMR spectral changes have been shown to be associated with apoptotic cell death in a variety of cell lines. The onset of apoptosis is associated with at least a 2 fold rise in the signal intensity of the membrane lipid methylene resonance (Blankenberg et al., 1996). The signal intensity of CH
In several NMR studies the models of cell death caused in vitro by chemotherapeutic drugs varying in mechanisms of apoptosis induction have been explored (Blankenberg et al., 1996). Nevertheless, it has not yet been known whether induction of apoptosis by different stimuli results in similar alterations in cell membrane structure revealed by NMR technique and to what extent the changes in cell membranes run in parallel with other manifestations of apoptosis.
Since the majority of anticancer drugs directly or indirectly alter DNA replication and synthesis it is worthwhile assessing by NMR technique the development of apoptosis induced by chemotherapeutic drugs of various classes affecting DNA. Apoptosis induced by these drugs is mediated by Fas-independent pathways that are far from being understood (Eischen et al., 1997). DNA topoisomerase inhibitors as well as metabolic inhibitors of DNA synthesis represent two different groups of widely used potent cytotoxic drugs known as inducers of the apoptosis in cancer cells of different genesis.
The cytotoxic and apoptotic effects of DNA topoisomerase II inhibitors seem to be associated with the stabilization of DNA/DNA-topoisomerase complexes finally resulting in DNA breaks (Stanulla et al., 1997). Nucleoside analogs are structurally and metabolically related agents affecting the structural integrity of DNA after incorporation during replication or DNA excision repair synthesis. In general, the intrinsic pathway of apoptosis triggered by DNA topoisomerase II inhibitors as well as by nucleotide analogs results in mitochondrial-dependent activation of effector caspases, which leads to the characteristic apoptotic phenotype. Nevertheless, the mechanisms of apoptosis as well as its kinetics are different for the drugs of these two groups. In particular, in contrast to early induction of apoptosis by DNA topoisomerase II inhibitors, some metabolic DNA inhibitors resulted in delayed apoptosis realized not earlier than in 72
Earlier we have studied the effects of the etoposide and fludarabine on the apoptotic cell death in human B-cell lymphoma cell line Namalwa (Philchenkov et al., 2001) which is relatively resistant to apoptosis induction partially due to the presence of antiapoptotic genes conferred by integrated EBV genome (Tarodi et al., 1994). Recently, it was shown that several bioflavonoids, in particular quercetin, possess the activity of natural DNA topoisomerase II inhibitors (Strick et al., 2000) and may induce apoptosis in malignant lymphoid cells (Liesveld et al., 2003). In present studies we have attempted to assess and compare the alterations in MLD by NMR technique in cultured human leukemia and lymphoma cell lines (MT4 and Namalwa) treated with chemotherapeutic agents – etoposide and fludarabine or bioflavonoid – quercetin.
Based on the ratio of the integrated areas of CH
2 Materials and methods
Deuterium oxide (D
2.2 Cell culture technique
Human B-cell lymphoma line Namalwa and T-ALL line MT4 were obtained from the National Collection of Cell Lines of Institute of Experimental Pathology, Oncology and Radiobiology (Kyiv, Ukraine). The cells were maintained at 37
MT4 cells were treated with etoposide (10
2.3 Cell morphologic analysis
For visualization of apoptotic cells with nuclear fragmentation Hoecht staining technique was used. The cells were washed in PBS, concentrated and incubated with 1
2.4 DNA fragmentation assay
Cell DNA was isolated by modified technique (Herrmann et al., 1994). Cells (5
2.5 Cell flow cytometry
Apoptotic cells were measured by flow cytometric analysis using a Becton Dickinson FACScan and CellQuest software (BD Biosciences, USA) according to the modified technique (Gougeon et al., 1996). Briefly, cell pellets were gently resuspended in hypotonic fluorochrome solution (5
Cells were harvested and washed with PBS then twice with PBS made with D
The human leukemia MT4 cells were treated with 10 Fig. 1 Fluorescence microphotograph of apoptotic MT4 cells. Cells were treated with 10 Fig. 2 Demonstration of apoptosis and necrosis in MT4 cells by flow cytometry. MT4 cells were treated with 10 Fig. 3 Demonstration of apoptosis and necrosis in MT4 cells by gel electrophoresis of cellular DNA. The same samples as described in Fig. 2 were analysed by the gel electrophoresis. 1 – DNA size markers; 2 – untreated cells; 3 – etoposide-treated cells; 4 – ethanol-treated cells. DNA fragmentation is evident in line 3, but completely absent in other lines. Fig. 4 1H NMR spectra (at 300
Fluorescence microphotograph of apoptotic MT4 cells. Cells were treated with 10
Demonstration of apoptosis and necrosis in MT4 cells by flow cytometry. MT4 cells were treated with 10
Demonstration of apoptosis and necrosis in MT4 cells by gel electrophoresis of cellular DNA. The same samples as described in Fig. 2 were analysed by the gel electrophoresis. 1 – DNA size markers; 2 – untreated cells; 3 – etoposide-treated cells; 4 – ethanol-treated cells. DNA fragmentation is evident in line 3, but completely absent in other lines.
1H NMR spectra (at 300
Cultured Namalwa cells were incubated with fludarabine phosphate either in concentration of 3
The incubation of Namalwa cells in continuous presence of quercetin resulted in 2 fold increase of the methylene protons signal intensity and the CH
In order to demonstrate that changes in the mobile membrane domains registered by 1H NMR are specific for apoptosis we have studied the CH
The apoptosis-associated changes in the MLD of T- and B-lymphoid malignant cell lines have been studied by 1H NMR spectroscopy. Research was conducted in vitro in Namalwa B-cell line originating from Burkitt's lymphoma that is relatively resistant to apoptosis induced by chemotherapeutic agents and in the MT4 T-cell line that is more sensitive to apoptosis-inducing agents.
The cells were treated by agents with direct or indirect DNA damaging properties: the inhibitor of topoisomerase II – etoposide, fludarabine phosphate and natural modulator of apoptosis with wide range of action – flavonoid quercetin. Nonapoptotic cell death was induced by treatment with ethyl alcohol and DMSO that cause necrosis of cells. 1H NMR detection of MLD in treated cells was based on estimation of the CH
1H NMR spectral changes associated with significant rise in the methylene signal intensity have been shown associated with apoptotic cell death in a variety of cell lines. The ratio of CH
Experiments were conducted on the cultured lines of human malignant lymphoid cells, incubated in the presence of various inducers of apoptosis. Previously it was shown that CH
The fludarabine phosphate is a nucleoside analog, acting as a metabolic inhibitor of DNA synthesis, and inducing apoptosis under certain conditions. The delayed apoptosis requires up to 72
The effect of bioflavonoid quercetin, as a factor, that in high concentrations is the inducer of apoptosis and natural inhibitor of DNA-topoisomerase II in malignant cells was studied in vitro in the cultured Namalwa cells at doses not resulting in the immediate apoptotic response. It was shown that permanent presence of quercetin in cultured medium slowed the rates of cells growth, morphologically showing separate apoptotic cells, on each passage the percent of the lost cells was about 15%. The 1H NMR analysis reveals the considerably elevated levels of CH
The induction of necrosis in Namalwa cells resulted in acute necrotic response, based on morphological criteria, in 95% and 60% of DMSO or ethanol-treated cells, respectively.
The results of the study demonstrate that upon the different apoptotic stimuli provided by the agents directly or indirectly affecting DNA structure or DNA synthesis and replication, the patterns of alterations in NMR visualized LMD are essentially the same irrespective of the specific mechanisms of apoptosis induction and DNA damage. This holds true both for acute exposure to the toxic doses of chemotherapeutics such as etoposide or fludarabine and for chronic exposure to quercetin as apoptosis-inducing agent possessing the activity of the natural DNA topoisomerase II inhibitor. The specified NMR visualized patterns are evident prior to the apoptotic cell death both in rapid and delayed evolution of apoptotic events. The signal intensity ratio of characteristic peaks is proportional to the number of apoptotic cells detected by other techniques. It is of importance that the technique was effective in recording rather small apoptotic percentage in the case of the continuous chronic exposure to non-toxic doses of quercetin. The complete patterns of lipid domain alterations need further analysis.
We thank Dr. N. Khranovskaya for her help with the flow cytometry. This work was in part supported by grants from: U.S. Civilian Research & Development Foundation (RESC 20-7; UR2-1028-KV-03) and the Fundamental Research Foundation of Ministry of Education and Science of Ukraine (F7/551-2001).
Barba I, Cabanas, ME, Arus, C. The relationship between nuclear magnetic resonance-visible lipids, lipid droplets, and cell proliferation in cultured C6 cells. Cancer Res 1999:59:1861-8
Blankenberg FG, Katsikis, PD, Storrs, RW, Beaulieu, C, Spielman, D, Chen, JY. Quantitative analysis of apoptotic cell death using proton nuclear magnetic resonance spectroscopy. Blood 1997:89:3778-85
Blankenberg FG, Storrs, RW, Naumovski, L, Goralski, T, Spielman, D. Detection of apoptotic cell death by proton nuclear magnetic resonance spectroscopy. Blood 1996:87:1951-6
Callies R, Sri-Pathmanathan, RM, Ferguson, DY, Brindle, KM. The appearance of neutral lipid signals in the 1H NMR spectra of a myeloma cell line correlates with the induced formation of cytoplasmic lipid droplets. Magn Reson Med 1993:29:546-50
Eischen CM, Kottke, TJ, Martins, LM, Basi, GB, Tung, JS, Earnshaw, WC. Comparison of apoptosis in wild-type and Fas-resistant cells: chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions. Blood 1997:90:935-43
Ferretti A, Knijn, A, Iorio, E, Pulciani, S, Giambenedetti, M, Molinari, A. Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts. Biochim Biophys Acta 1999:1438:329-48
Gougeon ML, Lecoeur, H, Dulioust, A, Enouf, MG, Crouvoiser, M, Goujard, C. Programmed cell death in peripheral lymphocytes from HIV-infected persons. Increased susceptibility to apoptosis of CD4 and CD8 cells correlates with lymphocyte activation and with disease progression. J Immunol 1996:156:3509-20
Hakumaki JM, Poptani, H, Sandmair, AM, Yla-Herttuala, S, Kauppinen, RA. 1H MRS detects polyunsaturated fatty acid accumulation during gene therapy of glioma: implications for the in vivo detection of apoptosis. Nat Med 1999:5:1323-7
Le Moyec L, Tatoud, R, Eugene, M, Gauville, C, Primot, I, Charlemagne, D. Cell and membrane lipid analysis by proton magnetic resonance spectroscopy in five breast cancer cell lines. Br J Cancer 1992:66:623-8
Philchenkov AA, Zavelevich, MP, Butenko, ZA. Apoptosis induction in human malignant lymphoid cells by DNA-damaging agents with different mechanisms of action. Exp Oncol 2001:23:170-4
Remy C, Fouilhe, N, Barba, I, Sam-Lai, E, Lahrech, H, Cucurella, MG. Evidence that mobile lipids detected in rat brain glioma by 1H nuclear magnetic resonance correspond to lipid droplets. Cancer Res 1997:57:407-14
Robertson LE, Chubb, S, Meyn, RE, Story, M, Ford, R, Hittelman, WN. Induction of apoptotic cell death in chronic lymphocytic leukemia by 2-chloro-2′-deoxyadenosine and 9-beta-
Rosi A, Luciani, AM, Matarrese, P, Arancia, G, Viti, V, Guidoni, L. 1H-MRS lipid signal modulation and morphological and ultrastructural changes related to tumor cell proliferation. Magn Reson Med 1999:42:248-57
Stanulla M, Wang, J, Chervinsky, DS, Thandla, S, Aplan, PD. DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis. Mol Cell Biol 1997:17:4070-9
Strick R, Strissel, PL, Borgers, S, Smith, SL, Rowley, JD. Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia. Proc Natl Acad Sci U S A 2000:97:4790-5
Tarodi B, Subramanian, T, Chinnadurai, G. Epstein-barr virus BHRF1 protein protects against cell death induced by DNA-damaging agents and heterologous viral infection. Virology 1994:201:404-7
Received 14 July 2004/2 November 2004; accepted 11 November 2004doi:10.1016/j.cellbi.2004.11.008