2.1.

Experimental Procedures

HeLa cells were maintained in Dulbecco's modified eagle's medium (DMEM) containing 5% fetal bovine serum, 3.7 g/L sodium bicarbonate, and 1% antibiotics. Cells treated with MNNG at two concentrations of 50 and 100 μM were maintained in the incubator at 37°C. An aliquot of 5 mM pyruvate and 10 mM glucose were added into fresh medium after the washout of 100 μM MNNG treatment for 30 min.

2.2.

Immunocytochemistry

Treated cells were fixed with 4% paraformaldehyde and then made permeable by 0.2% Triton X-100 at room temperature after being washed twice with phosphate buffer solution (PBS). Cells were blocked with 1% bovine serum albumin (BSA) at 37°C for 30 min and then incubated with an anti-poly(ADP-ribose) (pADPr) (10H, Santa Cruz, Delaware, California) antibody at room temperature in the dark for 2 h followed by extensive washing with PBS. Cells were incubated with fluorescein isothiocyanate-conjugated anti-mouse antibody (Novus Biologicals, Littleton, Colorado) at room temperature for 60 min. Cell images were taken on a microscope with the fluorescence imaging system (EPIPHOT200, Nikon, Melville, New York).

2.3.

Nicotinamide Adenine Dinucleotide/ Reduced Nicotinamide Adenine Dinucleotide Quantification Assay

The NAD⁺/NADH levels in the HeLa cells receiving different treatments were measured using a Bio-Vision NAD⁺/NADH quantification assay kit according to manufacture instructions (Biovision, Mountain View, California). A standard curve generated with known amounts of purified NADH was used to calculate the NAD⁺ and NADH concentrations, respectively, in the cells.

2.4.

Measurement of Intracellular Adenosine-5′-triphosphate Level

Intercellular ATP level was measured by the bioluminescent somatic cell ATP assay kit (Sigma-Aldrich, St. Louis, Missouri).²⁰ Cells were collected at different time points after MNNG treatment. An aliquot of 50 μl of viable cell suspension was mixed with 150 μl of somatic cell releasing buffer to release the intercellular ATP. Half of the mixture (i.e., 100 μl) was then transferred into a black OptiPlate-96F 96-well plate containing 100 μl of ATP assay mix in each well. The luminescence intensity was measured by using the Victor² 1420 Multilabel Counter (PerkinElmer, Waltham, Massachusetts). The luminescence intensity was normalized to the number of cells.

2.5.

Measurement of Mitochondrial Membrane Potential

The mitochondrial membrane potential (ΔΨ) of HeLa cells was determined by the fluorescence dye 5,5^′,6,6^′-tetrachloro-1,1^′;3,3^′-tetraethylbenzimidazolyl carbocyanine iodide (JC-1; Molecular Probes, Carlsbad, California) as our previous study and others.^{21, 22} Cell pellet was loaded with a fresh medium containing 2 μM JC-1 for 30 min at 37°C. Cells were then washed twice with PBS, resuspended in 200 μl PBS, and transferred into a black OptiPlate-96F 96-well plate to measure ΔΨ by the Victor² 1420 Multilabel Counter (PerkinElmer, Waltham, Massachusetts). J-monomer fluorescence was excited and emitted at 485 and 535 nm, respectively. J-aggregate fluorescence was excited and emitted at 544 and 590 nm, respectively. The ΔΨ was determined by the ratio of J-aggregate/J-monomer.²³

2.6.

Cell Viability Assay

The MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay was used for cell viability after different treatments. The treatment medium had been replaced with serum-free medium containing 0.2 mg/ml MTT, and the cells were incubated for 3 h at 37°C. After incubation, the MTT solution was discarded and 200 μl of DMSO was added. The extraction process was performed for 10 min at 37°C. The optical density (OD) was then recorded at 570 nm using the Varioskan Flash (Thermo Scientific, Waltham, Massachusetts). The mean OD of the control cells represents 100% of the viability, and the results are expressed as a percentage of the control.

2.7.

Measurement of the Respiration Rate

The cell respiration rate was measured by 782 Oxygen Meter (Mitocell MT200A, Strathelvin Instrument, Motherwell, Untied Kingdom) as previously reported.^{20, 24} An aliquot of 4×10⁵ cells was resuspended in 330 μl of assay buffer (125 mM sucrose, 65 mM KCl, 2 mM MgCl₂, 20 mM phosphate buffer, pH 7.2) and the cell suspension was transferred into the respiration chamber. An aliquot of 0.0004% digitonin was added to make the outer membrane of mitochondria permeable. The substrate-supported respiration rate was measured after 17 mM glutamate and 17 mM malate were injected into the chamber, and the State 3 respiration (the state of ATP synthesis) was then recorded after injection of 1.5 mM ADP. The acceptor control ratio (ACR) was calculated by dividing the State 3 respiration rate by the substrate-supported respiration rate. ACR differs from more commonly measured respiratory control ratio but can still reflect the mitochondrial coupling strength.²⁵

2.8.

Reduced Nicotinamide Adenine Dinucleotide Fluorescence Lifetime Image

NADH fluorescence lifetime images of Hela cells were obtained as previously described.^{18, 19} In brief, treated HeLa cells were imaged with a two-photon laser scanning microscope and with a 60×1.45 NA PlanApochromat oil objective lens (Olympus Corp., Tokyo, Japan). NADH fluorescence was excited at 740 nm by a Verdi pumped modelocked femtosecond Ti:sapphire laser (Coherent, Inc., Santa Clara, California) at 76 MHz and the emitted fluorescent light was detected at 447 ± 30 nm, the NADH fluorescence peaks by a bandpass filter (Semrock Inc., Rochester, New York). Fluorescence photons were detected by a photon-counting photomultiplier H7422P-40 (Hamamatsu Photonics K.K., Hamamatsu, Japan). Time-resolved detection was conducted by the time-correlated single photon counting SPC-830 board (Becker & Hickl GmbH, Berlin, Germany). Data were analyzed with the commercially available SPCImage v2.8 software (Becker & Hickl GmbH, Berlin, Germany) via a convolution of the two component exponential decay function, [TeX:] $F\left(t \right) = a_1 e^{ - {t / {\tau _1 }}} + a_2 e^{ - {t / {\tau _2 }}}$ $F\left(t\right)=a_1{e}^{-t/{\tau }_1}+a_2{e}^{-t/{\tau }_2}$ and the instrument response function (IRF), and were then fitted to the actual data to lifetime parameters τ₁, τ₂, a ₁, a ₂, and τ_m. τ_m is the mean lifetime defined as (a ₁τ₁+a ₂τ₂)/(a ₁+a ₂). IRF was measured using a second harmonic generated signal from a periodically poled lithium niobate crystal. HeLa cells were seeded onto a 24-mm diameter glass coverslips (Paul Marienfeld GmbH & Co., Lauda- Konigshofen, Germany) at 2×10⁴ cells/cm² 24 h before fluorescence lifetime measurement and drug treatment. The coverslip was washed twice using PBS and then transferred into an imaging chamber and added 1 ml aliquot of 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (5 mM KCl, 140 mM NaCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, pH 7.4) in the beginning of fluorescence lifetime imaging.

2.9.

Measurement of Reduced Nicotinamide Adenine Dinucleotide Fluorescence Intensity

NADH fluorescence intensity is measured by using a commercial fluorescence spectroscopy system (Victor² 1420 Multilabel Counter, PerkinElmer, Waltham, Massachusetts). Treated cells of ∼4×10⁵ were resuspended in 200 μl PBS and transferred into a black OptiPlate-96F 96-well plate (Packed Bioscience, PerkinElmer, Waltham, Massachusetts) to measure NADH fluorescence excited and emission at 355 and 460 nm. The process was done as soon as possible to prevent NADH degraded at room temperature and the measured value of the NADH fluorescence intensity was normalized to the cell number.

3.1.

N-methyl-N'-nitro-N-nitrosoguanidine Induced Significant Nicotinamide Adenine Dinucleotide Depletion but Less Reduced Nicotinamide Adenine Dinucleotide Depletion

MNNG induced cell death by activating PARP-1 that subsequently consumed NAD⁺ and adenosine 5^′-diphosphate (ADP) through the ADP-ribosylation.^{4, 6, 9, 26} We measured the effect of MNNG treatment on intracellular levels of NAD⁺ and NADH to confirm the depletion of NAD⁺ reported by several studies^{4, 6, 9} and to investigate if NADH is affected in a similar manner. As shown in Fig. 1a (open square), MNNG induced significantly rapid depletion of cellular NAD⁺, which was almost diminished 5 min after treatment with 100 μM MNNG. The cellular NADH level decreased less severely than NAD⁺ that NADH fell to 50% of the controls 5 min after MNNG induction from 88 ± 19 to 44 ± 2 pmol per 10⁶ cells [Fig. 1a, solid circle]. At 60 min after MNNG treatment, the cellular NADH further decreased to 31 ± 9 pmol per 10⁶ cells. We also performed the measurements of NADH fluorescence intensity [Fig. 1b], which has been used as the optical biomarker of mitochondrial NADH.²⁷ Figure 1c compares the results using both methods and the same time-dependent decrease of NADH was observed. This result indicates that the NADH fluorescence intensity was well correlated with the cellular NADH content. Finally, the dynamic PARP-1 activation shown in Fig. 1d indicates that massive expression of PAR protein immediately appeared in the nucleus of HeLa cells after MNNG treatment and then significantly decreased at 30 min and disappeared at 60 min. The immediate decrease of NAD⁺ and NADH 5 min after MNNG induction seems associated with this immediate PARP-1 activation at the same time point.

Fig. 1

Time course measurements of (A) cellular NAD⁺ and NADH content and (B) relative NADH flourescence intensity to the control after 100 μM MNNG treatment. (C) Comparison of (A) and (B) by normalizing (A) to the control value. (D) Immnuofluorescent images of anti-pADPr (a) before, and (b) 5, (c) 30, and (d) 60 min after 100 μM MNNG treatment. The scale bar in (c) is 50 μm.

3.2.

Reduced Nicotinamide Adenine Dinucleotide Fluorescence Lifetime Increased After Poly(adenosine-5′-diphosphate-ribose) polymerase-1 Induced Cell Death in a Dose-dependent Manner

Figure 2 shows the representative time course micrographs of NADH fluorescence lifetime from the same field of view (FOV) of HeLa cells before and after 100 μM MNNG treatment [Fig. 2a (a)–(e)] and the corresponding normalized lifetime histograms [Fig. 2a (f)]. Fluorescence signals in the cytoplasm display the punctuate perinuclear pattern, which is attributed to mitochondria-associated NADH.¹⁹ Each pixel represents the mean lifetime (τ_m) of the short (τ₁) and long (τ₂) lifetime components weighted by their relative contributions a ₁ and a ₂, respectively, such that τ_m = (a ₁τ₁ + a ₂τ₂)/(a ₁ + a ₂). The blue color in the color bar represents the longer lifetime (maximum 2000 ps) and the red color presents the shorter lifetime (minimum 500 ps). The color-coded lifetime images exhibit gradual color change from yellow to green and then blue from the image taken at the time point of controls to that taken 90 min after MNNG treatment. The normalized histograms of the mean lifetime (τ_m) from all pixels of the same FOV images taken at different time points show a peak shift from ∼950 to ∼1300 ps. The full-width-at-half-maximum is ∼300 ps at the time of the control but becomes ∼700 ps at 90 min after MNNG treatment indicating that the inhomogeneous lifetime distribution within cells varied greatly with time.

Fig. 2

(A) Two-photon fluorescence lifetime imaging (FLIM) micrographs of HeLa cells from the same sites (a) before, and (b) 5, (c) 30, (d) 60, and (e) 90 min after treatment with 100 μM MNNG. The normalized histograms of NADH fluorescence lifetime (f) over each 256×256 pixel lifetime image shown in (a) to (e) were plotted and coded with different colors. (B) Time course plots of the mean and standard deviation of NADH fluorescence lifetime over five fields of view of HeLa cells with 50 μM (open square) or 100 μM (solid diamond) MNNG treatment. Symbols * and # indicate the lifetime is significantly different from the value of the control with a p-value less than 0.05. (C) Relative cell survival to the control at 2 and 12 h after 30-min treatment with 50 μM (open square) or 100 μM (solid square) MNNG.

Similar results were repeatedly observed in a total of five samples at each time point and the average results are plotted in Fig. 2b. In Fig. 2b, the average results from cells treated with 50 μM MNNG are plotted to compare the MNNG dose effect. Figure 2b shows that NADH fluorescence lifetime increased up to 90 min after treatment with both MNNG doses. The increases at 30, 60, and 90 min are statistically significant (p-value < 0.05). The amplitude of the lifetime increase is dose-dependent that higher MNNG dose shows a higher increase in NADH fluorescence lifetime. Figure 2c plots the cell survival measured at 2 and 12 h after the treatment showing that 100 μM MNNG induced more cell death than did 50 μM. This dose dependent manner has been observed in our previous studies on STS-induced cell death of HeLa cells and cybrids with mitochondrial dysfunction, in which the faster the cell death proceeded, the higher NADH fluorescence lifetime increased.

3.3.

Reduced Nicotinamide Adenine Dinucleotide Fluorescence Lifetime Increased Before Mitochondrial Dysfunction

Because NADH fluorescence is mainly attributed by the mitochondrial NADH, we examined the time course mitochondrial functions during PARP-1-mediated cell death to understand the possible origin of NADH fluorescence lifetime change observed in Fig. 2. We measured the mitochondrial membrane potential (ΔΨ) for up to 8 h and the intracellular ATP level for up to 2 h after treatment with both 50 and 100 μM MNNG, respectively, as shown in Fig. 3. Similar to the previously published result in HeLa cells,²⁶ ΔΨ increased in the beginning of the MNNG treatment [Fig. 3a, solid square] due to short supply of ADP in mitochondrial State 4 respiration,²⁸ and ATP dropped to 10% or more after treatment with 100 μM MNNG for 30 min [Fig. 3b, solid square]. Both changes were dose-dependent as revealed by the observation that ATP was depleted more severely and faster and ΔΨ reached the plateau earlier by treating HeLa cells with 100 μM MNNG compared with 50 μM MNNG. Compared to Fig. 2, the increase of NADH fluorescence lifetime occurred in parallel with the ΔΨ raise and ATP depletion.

Fig. 3

Time course plots of (a) the relative mitochondrial membrane potential (ΔΨ) and (b) relative ATP level to the control before and after 50 μM (♦) or 100 μM (solid square) MNNG treatment.

We further investigated the mitochondrial respiration rate and the coupling strength. The upper panel of Table 1 shows the substrate-supported oxygen consumption rate and State 3 respiration of 100 μM MNNG-treated HeLa cells. Both the substrate-supported respiration and State 3 respiration significantly decreased after MNNG exposure for 30, 60, and 120 min as compared to the controls. The mitochondrial coupling strength, indicated by the so-called ACR which was the ratio between the rate of State 3 respiration and substrate-supported respiration rate,²⁵ was not observed at 30 and 60 min but was significantly decreased at 120 min after MNNG treatment (p-value = 0.007), indicating the compromise of mitochondrial respiration.

Table 1

The substrate-supported oxygen consumption rate and State 3 respiration rate of HeLa cells, which were treated by 100 μM MNNG for up to 120 min, or treated by metabolic substrate (i.e., glucose or pyruvate) or only culture medium after 100 μM MNNG treatment for 30 min, were measured at various time points by adding substrates of glutamate/malate and glutamate/malate plus ADP, respectively. The coupling effect was reflected via the ACR that was determined by the ratio of State 3 respiration rate divided by the substrate-supported oxygen consumption rate.

3.4.

N-methyl-N'-nitro-N-nitrosoguanidine-induced Cell Death is Rescued by Pyruvate but not Glucose

NAD⁺ and ADP/ATP depletion induced by PARP-1 hyperactivation inhibited glycolysis and mitochondrial respiration.^{9, 12, 26} After washout of 100 μM MNNG exposure for 30 min, we treated cells with pyruvate or glucose to investigate whether MNNG induced cell death was rescued. We compared the survival rate of HeLa cells treated with these two substrates with that of addition of DMEM, instead of metabolic substrates. As shown in Fig. 4a, the survival rate was not improved by addition of DMEM, which was 48 ± 3% and 27 ± 5% at 2 and 12 h, respectively, after treatment with 100 μM MNNG for 30 min. The survival rate was significantly increased by pyruvate to become 62 ± 8% (p-value = 0.007) and 36 ± 7% (p-value = 0.042) at 2 and 12 h time points, respectively. However, it was not changed by glucose (p-values = 0.071 and 0.172, respectively).

We then measured the mitochondrial functions, including ΔΨ, ATP level, and the rates of oxygen consumption of cells in DMEM, pyruvate, or glucose exposure for 30 min after the washout of 30 min MNNG exposure. The results are shown in Figs. 5a, 5b and the bottom panel of Table 1. In Fig. 5, the experiment to treat cells with MNNG only for 60 min was added so that all of the measurements were compared at the same time point (i.e., 60 min from the beginning of 100 μM MNNG treatment). Addition of pyruvate or glucose did prevent the increase of ΔΨ as compared with conditions of treating with MNNG for 60 min or treating with MNNG for 30 min plus DMEM for another 30 min that the relative membrane potentials to the controls were 1.54 ± 0.22 and 1.27 ± 0.08 (p-values < 0.05), respectively. In the measurement of the relative ATP level [Fig. 5b], pyruvate restored the relative ATP from 11.7 ± 1.3% to 19.4 ± 0.7% in cells exposed to 1-h MNNG treatment (p-value < 0.05) and from 16.6 ± 0.1% for 30 min MNNG plus 30 min DMEM exposure (p-value < 0.05). However, glucose was unable to prevent ATP depletion at all. In the measurement of the oxygen consumption rate (Table 1), addition of pyruvate significantly improved both substrate-supported and State 3 respiration rates. The substrate-supported respiration rate increased from 1.9 ± 0.2 nmole/min/10⁶ cells for 60 min MNNG treated cells or from 3.0 ± 0.3 nmole/min/10⁶ cells for 30 min MNNG plus 30 min DMEM treated cells to 4.4 ± 0.5 nmole/min/10⁶ cells. The State 3 respiration rate was increased from 3.0 ± 0.1 nmole/min/10⁶ cells for 60-min MNNG treatment only or 4.4 ± 0.6 nmole/min/10⁶ cells for 30 min-MNNG treatment plus 30 min DMEM to 6.4 ± 0.89 nmole/min/10⁶ cells. Similarly to the findings of ATP levels, glucose did not restore oxygen consumption rates.

Fig. 5

Change of (a) the mitocondrial membene potential (ΔΨ), (b) ATP level, (c) NADH floureascence intensity, and (d) NADH fluorescence lifetime after 60-min 100 μM MNNG treatment, or 30-min 100 μM MNNG treatment followed by either 30 min pyruvate, glucose, or DMEM only treatment. Symbols * and # indicate the value is significantly different (p-value < 0.05) from the value of the 60-min MNNG treatment and 30-min MNNG plus 30-min DMEM treatment, respectively. The dotted line shown in (d) indicates the NADH fluorescence lifetime of HeLa cells before any treatment (the control).

3.5.

Reduced Nicotinamide Adenine Dinucleotide Fluorescence Lifetime Responded to Pyruvate but not Glucose; Reduced Nicotinamide Adenine Dinucleotide Fluorescence Intensity Responded to Neither Pyruvate nor Glucose

Figures 5c, 5d show the NADH fluorescence intensity and lifetime, respectively, of HeLa cells treated with 30 min DMEM, pyruvate, or glucose after washout of 30 min MNNG exposure. An additional measurement at 60 min after MNNG treatment was included in both Figs. 5c, 5d for comparison. The NADH fluorescence intensity was expressed as the value relative to the controls. The mean value of NADH fluorescence lifetime of the controls shown in Fig. 2b (i.e., 993 ± 30 ps) is depicted by a dashed line in Fig. 5d. The NADH fluorescence intensity of HeLa cells with different treatments shows no difference. On the other hand, the NADH fluorescence lifetime, similar to the results in Fig. 2b, increased to 1215 ± 28 ps after cells had been exposed to MNNG for 30 min plus 30 min incubation in DMEM and increased to 1291 ± 102 ps after cells exposed to 60 min MNNG. Pyruvate significantly prevented the NADH fluorescence lifetime increase, and after pyruvate treatment the lifetime (i.e., 1142 ± 23 ps) was different from that without addition of pyruvate (i.e., p-values = 0.008 and 0.002 for 60 min-MNNG treatment and 30 min MNNG plus 30 min DMEM, respectively). Glucose did not alter the lifetime increase and the lifetime (1197 ± 21 ps) was not statistically different from that of the cells treated with MNNG for 30 min plus 30 min incubation in DMEM.