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Apoptosis-induced structural changes in leukemia cells identified by IR spectroscopy
Journal of Molecular Structure 565±566 (2001) 299±304
www.elsevier.nl/locate/molstruc
Apoptosis-induced structural changes in leukemia cells identi®ed by IR spectroscopy K.-Z. Liu, H.H. Mantsch* Institute for Biodiagnostics, National Research Council of Canada, 435 Ellice Avenue, Winnipeg, Man., Canada R3B 1Y6 Received 31 August 2000; accepted 29 September 2000
Abstract Apoptotic changes induced in the leukemia cell line CEM by treatment with the chemical etoposide were investigated by IR spectroscopy. Characteristic band alterations were identi®ed in the apoptotic cells arising from cellular protein, lipid and DNA. Besides general changes such as an increase in lipid content and a decrease in the amount of detectable DNA, there were speci®c changes that affected the secondary structure of proteins in the apoptotic leukemia cells, i.e. the dominant protein structure shifts from b-sheet in the control cells to unordered coil in the apoptotic cells. The student's t-test was applied to the spectral range 1500±1700 cm 21 in order to determine the signi®cant differences of protein structure between control and etoposide treated cells at various time points. A temporal relationship was found between the spectrally signi®cant differences of the protein structure in the apoptotic cells and the severity of apoptosis. The IR spectral changes of protein structure also correlate well with the activity of caspase-3, an important proteolytic enzyme in apoptosis. This preliminary study suggests that IR spectroscopy could possibly be used to monitor and quantitate apoptosis in leukemia cells. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Infrared spectroscopy; Apoptosis; Leukemic cells; Structural changes; Quantitative analysis
1. Introduction Whilst you read this, millions of your cells are dying. Relax, most are sacri®cing themselves to ensure your survival. Indeed, the health of all multicellular organisms, including humans, depends not only on the body's ability to produce new cells, but also on the ability of individual cells to self-distruct when they become super¯uous or undesirable. This process, referred to as apoptosis (from the Greek `dropping off'), is a genetically regulated natural form of cell death, as opposed to necrosis (Greek for * Corresponding author. Tel.: 11-204-984-4622; fax: 11-204984-5472. E-mail address: [email protected] (H.H. Mantsch).
`make dead'), which occurs when a cell is severely injured, e. g. by a physical blow or by oxygen deprivation. It is now clear that the cellular suicide mechanism of apoptosis plays a crucial role in many physiological and pathological processes including cancer, growth and development, ageing, AIDS, or immune suppression [1,2]. There are three steps in apoptosis, the initiation phase triggered by a stimulus received by the cell, the effector or decision phase during which the cell commits itself to live or to die, and the degradation phase when the cells acquire the morphological and biochemical hallmarks of apoptosis [3]. Common biochemical events include changes in cell membrane potential, altered levels of protein and RNA biosynthesis and degradation of DNA into small fragments [2,4]. Morphologically,
0022-2860/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0022-286 0(00)00817-6
300
K.-Z. Liu, H.H. Mantsch / Journal of Molecular Structure 565±566 (2001) 299±304
cells undergoing apoptosis display membrane blebbing, cell shrinkage, nuclear condensation, fragmentation and packaging of cellular material into apoptotic bodies. Clearly, it would be desirable to detect apoptosis at an early stage, i.e. before the phase that entails all the visible changes of apoptosis. Apoptosis can be initiated by various physiological, physical and chemical agents such as ionizing irradiation, deprivation of viability factors, and a variety of agents that include cytotoxic cancer chemotherapy compounds [5]. The study of in vitro effects of various drugs on apoptosis of malignant cells enables the prediction of their in vivo sensitivity and may represent a prognostic marker for the treatment response [6]. Herein we explore the potential of infrared (IR) spectroscopy as a means for the early detection of apoptosis, by using a common leukemia cell line treated with the etoposide, a chemical agent known to induce apoptosis.
2. Experimental 2.1. Induction of apoptosis The human T-lymphoblastic cell line CEM was used in this study. Cells were cultured at 378C in a 5% CO2 humidi®ed incubator [7] using a RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 25 mM HEPES, 2.0 mM l-glutamine, pH 7.4, penicillin (100 u/ml), streptomycin (100 mg/ml). After cells were harvested, they were re-suspended in fresh culture medium at a concentration of 1 £ 10 6/ml and apoptosis was induced by incubating with 10 mg/ml etoposide at different time points. 2.2. Assessment of apoptosis by ¯ow cytometry After treatment at different time points, cells were permeabilized with 70% ethanol and stained with 100 mg/ml propidium iodide. Propidium iodide ¯uorescence of the nuclei was measured by ¯ow cytometry. Cells with a DNA content less than G0/G1 were de®ned as apoptotic cells [8]. The apoptotic cell population reached 56.6% after 24 h treatment.
2.3. Measurement of caspase-3 activity Leukemic cells (5 £ 10 7) were incubated for 20 min on ice, then broken up with a glass homogenizer and the cytosol obtained by centrifugation at 15,000g for 30 min at 48C [9]. The reaction was initiated by the addition of Ac-Asp-Glu-Val-Asp-AMC, a ¯uorescent substrate of caspase-3, and stopped by addition of 50 ml of 1% sodium acetate trihydrate in 175 mM acetic acid. Fluorescence was measured at 380/ 460 nm. Caspase-3 activity was de®ned as mM AMC release per mg protein per hour. 2.4. Infrared spectroscopy IR measurements from three separate sample preparations were carried out as previously described [10]. At various time points, the cells treated with etoposide were washed by centrifugation (1500 rpm at 800g for 10 min) and re-suspended in saline. About 5 ml of the cell suspension was then deposited on an infrared-transparent barium ¯uoride window and dried down quickly as a thin circular ®lm of ,3 mm diameter. The coated windows were kept in a desiccator under mild vacuum for 5 min, then placed in a sample holder and the chamber covered with a second uncoated barium ¯uoride window. For each sample, three sets of absorption spectra were obtained using a blank barium ¯uoride window as background. Spectra were recorded on a Biorad FTS-60 IR spectrometer equipped with a nitrogen cooled mercury cadmium telluride detector at a nominal resolution of 2 cm 21 and an encoding interval of one wavenumber. For each spectrum, 256 interferograms were co-added and apodized with a triangular smoothing function before Fourier transformation. 2.5. Statistical analysis All IR spectra were baseline corrected and area normalized between 900 and 1800 cm 21 using the WIN-IR software. Second derivative spectra were then calculated from these normalized spectra. A student's t-test (Statistica5.1, StatSoft) was applied to these pre-processed spectra in order to assess the signi®cance of spectral differences at each spectral wavelength. Values with p , 0:05 are considered as statistically signi®cant.
K.-Z. Liu, H.H. Mantsch / Journal of Molecular Structure 565±566 (2001) 299±304
301
Fig. 1. Representative IR spectra of CEM cells before and after treatment (24 h) with etoposide (bottom), and IR difference spectrum between control and etoposide-treated CEM cells (top).
3. Results and discussion Representative IR spectra of CME cells before and 24 h after treatment with etoposide are shown in Fig. 1. To highlight possible differences, the top panel shows an IR difference spectrum, generated by subtracting the mean spectrum of etoposide treated CEM cells from that of control cells. There are several features in the IR difference spectrum that can be related to vibrations of proteins, lipid and DNA. For instance, the amide I band arising from the amide CyO stretching vibration of the peptide groups in all proteins, shifts from 1635 cm 21 in control cells to 1657 cm 21 in cells treated with etoposide. These amide I frequencies are compatible with the fact that the overall protein structure in the control cells consists primarily of parallel b-sheet constituents, whereas apoptotic leukemia cells have a relatively high proportion of unordered proteins [11,12]. The effect of apoptosis
on DNA can be identi®ed from IR bands associated with vibrations of various structural groups in DNA such as the band at 968 cm 21 (marked as C±C/C± O), a single bond stretching vibration characteristic of the deoxyribose sugar moiety in DNA, the bands at 1087 and 1240 cm 21 (marked as sPO22 and asPO22), due to vibrations of the phosphodiester groups in DNA, or the band at 1713 cm 21, characteristic of base-paired DNA strands [13]. The negative bands in the IR difference spectrum indicate that the DNA content has decreased, while the positive intensity of the lipid ester band at 1740 cm 21 and that of the lipid C±H stretching bands between 2800 and 3000 cm 21 (not shown in Fig.1) indicates that the lipid content has increased in the treated cells. Since apoptosis involves the modi®cation of existing proteins, as well as the synthesis of new proteins, we analysed the changes of protein structure upon etoposide treatment in more detail. Fig. 2 (top
302
K.-Z. Liu, H.H. Mantsch / Journal of Molecular Structure 565±566 (2001) 299±304
Fig. 2. Quantitation of protein changes during apoptosis. Top panel: mean spectra of control and etoposide-treated CEM cells as inverted second derivatives. Bottom panel: t-test between control cells and etoposide-treated cells. The shaded area comprises the P values that are less than 0.05, de®ned as signi®cantly different.
panel) shows the mean spectra of control and etoposide treated CEM cells in the region of the amide I (1600±1700 cm 21) and amide II (1500±1600 cm 21) bands. Spectra are shown as inverted second derivatives, a method commonly used for narrowing broad IR bands for better visualisation. The bottom panel in Fig. 2 is a plot of P values obtained from an unpaired t-test of control and etoposide-treated CME cells. The shaded section highlights the P values that are less than 0.05 and de®ned as statistically signi®cant. From this ®gure one can
observe that there are signi®cant changes in the region of the amide I band (shift from b-sheet to unordered, the amide II band (at ,1545 cm 21) and the band at 1517 cm 21 arising from tyrosine in protein side chains [14]. The amount (percentage) of spectral differences with statistical signi®cance between control and treated cells is time dependent, as shown in Table 1. The signi®cant protein changes increased with time and reached 23.4% after 24 h treatment, which is in agreement with the fact that CEM cells represent an apoptotic-sensitive cell line.
K.-Z. Liu, H.H. Mantsch / Journal of Molecular Structure 565±566 (2001) 299±304 Table 1 Quantitative determination of protein changes in the etoposidetreated CEM cells by IR spectroscopy. (P values were determined by an unpaired t-test in the range of 1470±1700 cm 21 n 5) Time of etoposide treatment (h)
Percentage of points with P , 0.05
2 4 6 8 24
2.8 4.4 10.6 16.1 23.4
One of the many important proteins in apoptosis is caspase-3. Caspase activation is a hallmark of apoptosis, its over-expression being suf®cient to induce apoptosis [15]. We therefore assayed for this cytosolic protein and found that its activity had already increased after treatment with etoposide for 4 h. In an attempt to understand the time dependence of the spectral changes after induction of apoptosis we correlated the activity of caspase-3 with the
303
signi®cant spectral changes described in Table 1. As shown in Fig. 3 there is a surprisingly good correlation (correlation coef®cient r 0:98; suggesting that the changes in protein structure detected by IR spectroscopy also re¯ect or include the modi®cation of this important protein in apoptosis. To conclude, we can state that IR spectroscopy is able to identify overall changes induced by apoptosis in a leukemic cell line. For instance, the amount of detectable DNA decreases and the total cellular lipid content increases after 24 h treatment with etoposide. In addition, the dominant protein secondary structure shifts from b-sheet to unordered, indicating an alterated protein pro®le in the apoptotic cells. Quantitatively, we found that the spectral differences between control and apoptotic cells are statistically signi®cant and that the signi®cant differences also exhibit a temporal relationship with the percentage of apoptotic cells. Furthermore, a correlation seems to exist between the signi®cant changes of protein structure in apoptotic cells and the activity of caspase-3, an important protease implicated in apoptosis.
Fig. 3. Temporal correlation between etoposide-induced activation of caspase-3 and protein changes obtained from the corresponding IR spectra.
304
K.-Z. Liu, H.H. Mantsch / Journal of Molecular Structure 565±566 (2001) 299±304
References [1] [2] [3] [4] [5] [6] [7]
A.H. Wylie, Nature 284 (1980) 555. M.C. Raff, Nature 356 (1992) 397. F. Malisa, R. Test, FEBS Lett. 452 (1999) 100. C.B. Thompson, Science 267 (1995) 1456. J.J. Cohen, Immunol. Today 14 (1993) 126. Y.A. Hannun, Blood 89 (1997) 1845. L. Jia, S.M. Kelsey, M.F. Grahn, A.C. Newland, Blood 87 (1996) 2401. [8] L. Jia, R.R. Doumashkin, P.D. Allen, A.B. Gray, A.C. Newland, Kelsey, S. M. Br. J. Haematol. 98 (1997) 673.
[9] L. Jia, K.Z. Liu, A.C. Newland, H.H. Mantsch, S.M. Kelsey, Br. J. Heamotol. 107 (1999) 861. [10] K.Z. Liu, C.P. Schultz, J.B. Johnston, H.H. Mantsch, Leuk. Res. 21 (1997) 1125. [11] W.K. Surewicz, H.H. Mantsch, Biochim. Biophys. Acta 952 (1988) 115. [12] M. Jackson, H.H. Mantsch, Crit. Rev. Biochem. Mol. Biol. 30 (1995) 95. [13] E. Taillandier, F. Liquier, Methods Enzymol. 211 (1992) 307. [14] J.L. Gal, H. Morjani, M. Manfait, Cancer Res. 53 (1993) 3681. [15] E.S. Alnemri, D.J. Livingston, D.W. Nicholson, et al., Cancer Res. 49 (1989) 4363.
www.elsevier.nl/locate/molstruc
Apoptosis-induced structural changes in leukemia cells identi®ed by IR spectroscopy K.-Z. Liu, H.H. Mantsch* Institute for Biodiagnostics, National Research Council of Canada, 435 Ellice Avenue, Winnipeg, Man., Canada R3B 1Y6 Received 31 August 2000; accepted 29 September 2000
Abstract Apoptotic changes induced in the leukemia cell line CEM by treatment with the chemical etoposide were investigated by IR spectroscopy. Characteristic band alterations were identi®ed in the apoptotic cells arising from cellular protein, lipid and DNA. Besides general changes such as an increase in lipid content and a decrease in the amount of detectable DNA, there were speci®c changes that affected the secondary structure of proteins in the apoptotic leukemia cells, i.e. the dominant protein structure shifts from b-sheet in the control cells to unordered coil in the apoptotic cells. The student's t-test was applied to the spectral range 1500±1700 cm 21 in order to determine the signi®cant differences of protein structure between control and etoposide treated cells at various time points. A temporal relationship was found between the spectrally signi®cant differences of the protein structure in the apoptotic cells and the severity of apoptosis. The IR spectral changes of protein structure also correlate well with the activity of caspase-3, an important proteolytic enzyme in apoptosis. This preliminary study suggests that IR spectroscopy could possibly be used to monitor and quantitate apoptosis in leukemia cells. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Infrared spectroscopy; Apoptosis; Leukemic cells; Structural changes; Quantitative analysis
1. Introduction Whilst you read this, millions of your cells are dying. Relax, most are sacri®cing themselves to ensure your survival. Indeed, the health of all multicellular organisms, including humans, depends not only on the body's ability to produce new cells, but also on the ability of individual cells to self-distruct when they become super¯uous or undesirable. This process, referred to as apoptosis (from the Greek `dropping off'), is a genetically regulated natural form of cell death, as opposed to necrosis (Greek for * Corresponding author. Tel.: 11-204-984-4622; fax: 11-204984-5472. E-mail address: [email protected] (H.H. Mantsch).
`make dead'), which occurs when a cell is severely injured, e. g. by a physical blow or by oxygen deprivation. It is now clear that the cellular suicide mechanism of apoptosis plays a crucial role in many physiological and pathological processes including cancer, growth and development, ageing, AIDS, or immune suppression [1,2]. There are three steps in apoptosis, the initiation phase triggered by a stimulus received by the cell, the effector or decision phase during which the cell commits itself to live or to die, and the degradation phase when the cells acquire the morphological and biochemical hallmarks of apoptosis [3]. Common biochemical events include changes in cell membrane potential, altered levels of protein and RNA biosynthesis and degradation of DNA into small fragments [2,4]. Morphologically,
0022-2860/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0022-286 0(00)00817-6
300
K.-Z. Liu, H.H. Mantsch / Journal of Molecular Structure 565±566 (2001) 299±304
cells undergoing apoptosis display membrane blebbing, cell shrinkage, nuclear condensation, fragmentation and packaging of cellular material into apoptotic bodies. Clearly, it would be desirable to detect apoptosis at an early stage, i.e. before the phase that entails all the visible changes of apoptosis. Apoptosis can be initiated by various physiological, physical and chemical agents such as ionizing irradiation, deprivation of viability factors, and a variety of agents that include cytotoxic cancer chemotherapy compounds [5]. The study of in vitro effects of various drugs on apoptosis of malignant cells enables the prediction of their in vivo sensitivity and may represent a prognostic marker for the treatment response [6]. Herein we explore the potential of infrared (IR) spectroscopy as a means for the early detection of apoptosis, by using a common leukemia cell line treated with the etoposide, a chemical agent known to induce apoptosis.
2. Experimental 2.1. Induction of apoptosis The human T-lymphoblastic cell line CEM was used in this study. Cells were cultured at 378C in a 5% CO2 humidi®ed incubator [7] using a RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 25 mM HEPES, 2.0 mM l-glutamine, pH 7.4, penicillin (100 u/ml), streptomycin (100 mg/ml). After cells were harvested, they were re-suspended in fresh culture medium at a concentration of 1 £ 10 6/ml and apoptosis was induced by incubating with 10 mg/ml etoposide at different time points. 2.2. Assessment of apoptosis by ¯ow cytometry After treatment at different time points, cells were permeabilized with 70% ethanol and stained with 100 mg/ml propidium iodide. Propidium iodide ¯uorescence of the nuclei was measured by ¯ow cytometry. Cells with a DNA content less than G0/G1 were de®ned as apoptotic cells [8]. The apoptotic cell population reached 56.6% after 24 h treatment.
2.3. Measurement of caspase-3 activity Leukemic cells (5 £ 10 7) were incubated for 20 min on ice, then broken up with a glass homogenizer and the cytosol obtained by centrifugation at 15,000g for 30 min at 48C [9]. The reaction was initiated by the addition of Ac-Asp-Glu-Val-Asp-AMC, a ¯uorescent substrate of caspase-3, and stopped by addition of 50 ml of 1% sodium acetate trihydrate in 175 mM acetic acid. Fluorescence was measured at 380/ 460 nm. Caspase-3 activity was de®ned as mM AMC release per mg protein per hour. 2.4. Infrared spectroscopy IR measurements from three separate sample preparations were carried out as previously described [10]. At various time points, the cells treated with etoposide were washed by centrifugation (1500 rpm at 800g for 10 min) and re-suspended in saline. About 5 ml of the cell suspension was then deposited on an infrared-transparent barium ¯uoride window and dried down quickly as a thin circular ®lm of ,3 mm diameter. The coated windows were kept in a desiccator under mild vacuum for 5 min, then placed in a sample holder and the chamber covered with a second uncoated barium ¯uoride window. For each sample, three sets of absorption spectra were obtained using a blank barium ¯uoride window as background. Spectra were recorded on a Biorad FTS-60 IR spectrometer equipped with a nitrogen cooled mercury cadmium telluride detector at a nominal resolution of 2 cm 21 and an encoding interval of one wavenumber. For each spectrum, 256 interferograms were co-added and apodized with a triangular smoothing function before Fourier transformation. 2.5. Statistical analysis All IR spectra were baseline corrected and area normalized between 900 and 1800 cm 21 using the WIN-IR software. Second derivative spectra were then calculated from these normalized spectra. A student's t-test (Statistica5.1, StatSoft) was applied to these pre-processed spectra in order to assess the signi®cance of spectral differences at each spectral wavelength. Values with p , 0:05 are considered as statistically signi®cant.
K.-Z. Liu, H.H. Mantsch / Journal of Molecular Structure 565±566 (2001) 299±304
301
Fig. 1. Representative IR spectra of CEM cells before and after treatment (24 h) with etoposide (bottom), and IR difference spectrum between control and etoposide-treated CEM cells (top).
3. Results and discussion Representative IR spectra of CME cells before and 24 h after treatment with etoposide are shown in Fig. 1. To highlight possible differences, the top panel shows an IR difference spectrum, generated by subtracting the mean spectrum of etoposide treated CEM cells from that of control cells. There are several features in the IR difference spectrum that can be related to vibrations of proteins, lipid and DNA. For instance, the amide I band arising from the amide CyO stretching vibration of the peptide groups in all proteins, shifts from 1635 cm 21 in control cells to 1657 cm 21 in cells treated with etoposide. These amide I frequencies are compatible with the fact that the overall protein structure in the control cells consists primarily of parallel b-sheet constituents, whereas apoptotic leukemia cells have a relatively high proportion of unordered proteins [11,12]. The effect of apoptosis
on DNA can be identi®ed from IR bands associated with vibrations of various structural groups in DNA such as the band at 968 cm 21 (marked as C±C/C± O), a single bond stretching vibration characteristic of the deoxyribose sugar moiety in DNA, the bands at 1087 and 1240 cm 21 (marked as sPO22 and asPO22), due to vibrations of the phosphodiester groups in DNA, or the band at 1713 cm 21, characteristic of base-paired DNA strands [13]. The negative bands in the IR difference spectrum indicate that the DNA content has decreased, while the positive intensity of the lipid ester band at 1740 cm 21 and that of the lipid C±H stretching bands between 2800 and 3000 cm 21 (not shown in Fig.1) indicates that the lipid content has increased in the treated cells. Since apoptosis involves the modi®cation of existing proteins, as well as the synthesis of new proteins, we analysed the changes of protein structure upon etoposide treatment in more detail. Fig. 2 (top
302
K.-Z. Liu, H.H. Mantsch / Journal of Molecular Structure 565±566 (2001) 299±304
Fig. 2. Quantitation of protein changes during apoptosis. Top panel: mean spectra of control and etoposide-treated CEM cells as inverted second derivatives. Bottom panel: t-test between control cells and etoposide-treated cells. The shaded area comprises the P values that are less than 0.05, de®ned as signi®cantly different.
panel) shows the mean spectra of control and etoposide treated CEM cells in the region of the amide I (1600±1700 cm 21) and amide II (1500±1600 cm 21) bands. Spectra are shown as inverted second derivatives, a method commonly used for narrowing broad IR bands for better visualisation. The bottom panel in Fig. 2 is a plot of P values obtained from an unpaired t-test of control and etoposide-treated CME cells. The shaded section highlights the P values that are less than 0.05 and de®ned as statistically signi®cant. From this ®gure one can
observe that there are signi®cant changes in the region of the amide I band (shift from b-sheet to unordered, the amide II band (at ,1545 cm 21) and the band at 1517 cm 21 arising from tyrosine in protein side chains [14]. The amount (percentage) of spectral differences with statistical signi®cance between control and treated cells is time dependent, as shown in Table 1. The signi®cant protein changes increased with time and reached 23.4% after 24 h treatment, which is in agreement with the fact that CEM cells represent an apoptotic-sensitive cell line.
K.-Z. Liu, H.H. Mantsch / Journal of Molecular Structure 565±566 (2001) 299±304 Table 1 Quantitative determination of protein changes in the etoposidetreated CEM cells by IR spectroscopy. (P values were determined by an unpaired t-test in the range of 1470±1700 cm 21 n 5) Time of etoposide treatment (h)
Percentage of points with P , 0.05
2 4 6 8 24
2.8 4.4 10.6 16.1 23.4
One of the many important proteins in apoptosis is caspase-3. Caspase activation is a hallmark of apoptosis, its over-expression being suf®cient to induce apoptosis [15]. We therefore assayed for this cytosolic protein and found that its activity had already increased after treatment with etoposide for 4 h. In an attempt to understand the time dependence of the spectral changes after induction of apoptosis we correlated the activity of caspase-3 with the
303
signi®cant spectral changes described in Table 1. As shown in Fig. 3 there is a surprisingly good correlation (correlation coef®cient r 0:98; suggesting that the changes in protein structure detected by IR spectroscopy also re¯ect or include the modi®cation of this important protein in apoptosis. To conclude, we can state that IR spectroscopy is able to identify overall changes induced by apoptosis in a leukemic cell line. For instance, the amount of detectable DNA decreases and the total cellular lipid content increases after 24 h treatment with etoposide. In addition, the dominant protein secondary structure shifts from b-sheet to unordered, indicating an alterated protein pro®le in the apoptotic cells. Quantitatively, we found that the spectral differences between control and apoptotic cells are statistically signi®cant and that the signi®cant differences also exhibit a temporal relationship with the percentage of apoptotic cells. Furthermore, a correlation seems to exist between the signi®cant changes of protein structure in apoptotic cells and the activity of caspase-3, an important protease implicated in apoptosis.
Fig. 3. Temporal correlation between etoposide-induced activation of caspase-3 and protein changes obtained from the corresponding IR spectra.
304
K.-Z. Liu, H.H. Mantsch / Journal of Molecular Structure 565±566 (2001) 299±304
References [1] [2] [3] [4] [5] [6] [7]
A.H. Wylie, Nature 284 (1980) 555. M.C. Raff, Nature 356 (1992) 397. F. Malisa, R. Test, FEBS Lett. 452 (1999) 100. C.B. Thompson, Science 267 (1995) 1456. J.J. Cohen, Immunol. Today 14 (1993) 126. Y.A. Hannun, Blood 89 (1997) 1845. L. Jia, S.M. Kelsey, M.F. Grahn, A.C. Newland, Blood 87 (1996) 2401. [8] L. Jia, R.R. Doumashkin, P.D. Allen, A.B. Gray, A.C. Newland, Kelsey, S. M. Br. J. Haematol. 98 (1997) 673.
[9] L. Jia, K.Z. Liu, A.C. Newland, H.H. Mantsch, S.M. Kelsey, Br. J. Heamotol. 107 (1999) 861. [10] K.Z. Liu, C.P. Schultz, J.B. Johnston, H.H. Mantsch, Leuk. Res. 21 (1997) 1125. [11] W.K. Surewicz, H.H. Mantsch, Biochim. Biophys. Acta 952 (1988) 115. [12] M. Jackson, H.H. Mantsch, Crit. Rev. Biochem. Mol. Biol. 30 (1995) 95. [13] E. Taillandier, F. Liquier, Methods Enzymol. 211 (1992) 307. [14] J.L. Gal, H. Morjani, M. Manfait, Cancer Res. 53 (1993) 3681. [15] E.S. Alnemri, D.J. Livingston, D.W. Nicholson, et al., Cancer Res. 49 (1989) 4363.