Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 5, 2001, pp. 438-445

Tsinghua Science and Technology, December 2001, 6(5), pp. 438-445

Lipid Peroxidation-Mediated Telomere Shortening in Hydroxyl Radical-Induced Apoptosis in HeLa Cells

REN Jianguo , CHEN Jing , DAI Yaoren

Department of Biological Sciences and Biotechnology, Tsinghua University , Beijing 100084, China

* To whom correspondence should be addressed

Received: 2001-02-08 revised: 2001-04-18

Code Number: ts01094

Abstract:

Many anti-cancer drugs have been found to trigger apoptosis in tumor cells through the production of reactive oxygen species (ROS) including hydroxyl radicals (·OH)  regardless of chemical types. At the same time, telomerase is found to be associated with malignancy and reduced apoptosis. However, little is known about the linkage between ROS (such as ·OH) and telomerase/telomere. The focus ofthis investigation was to examine the possible pathway of the apoptosis induced by  ·OH  production via Fe²⁺  and H₂O₂. Results of the present study demonstrated that after exposure of HeLa cells to Fe²⁺ -H₂O₂ system, an increase in lipid peroxidation and reduction of GSH was observed. These events proceeded and triggered apoptosis, resulting in DNA fragmentation. More interestingly, we did not observe any changes of telomerase activity. However, the telomere length in apoptotic cells shortened significantly. We also found that GSH rescued  ·OH- induced HeLa cell death and prevened telomere shortening, and that 3,3'-diethyoxadicarbocyanine (DODCB), a telomerase inhibitor, increased susceptbility of HeLa cells to  ·OH -induced apoptosis. Our results suggest that  ·OH -induced telomere shortening is not through telomerase inhibition but possibly a direct effect of  ·OH  on telomeres themselves via lipid peroxidation.

Key words: telomere ; telomerase; hydoxyl radicals; reactive oxygen species (ROS); lipid peroxidation; malondialdehyde

Introduction

Reactive oxygen species(ROS) generation has been found to be an important event in apoptotic tumor cell death induced by various anti-cancer agents such as captothecin, vinblastine, inostamycin and adrimycin[1] On the other hand, ROS scavengers such as N-acetyl-L-cysteine (NAC) and glutathione(GSH) have been found to inhibit apoptosis induced by TGFb-1, TNF-a and growth factor deprivation in IL-3-dependent murine pro-B lymphocyte, murine T cell hybridoma, human ovarian-carcinoma and Hit cells[2,3]. The ability of oxidative stress, which is an excessive production of ROS, to provoke apoptosis and necrotic cell death as a result of massive cellular damage, has been associated with lipid peroxidation, which is a free radical-related process, and alterations of protein and nuclei[4]. Some evidences have shown that lipid peroxidation plays a pivotal role during apoptosis. However, the mechanism involved in this activation is controversial; cell redox status[5]and direct H₂O₂-mediated apoptosis have both been proposed[6]. Hence, exploration of the mechanism underlying ROS-triggered apoptosis in tumor cells is crucial to our understanding of the chemotherapeutic effect of anti-cancer drugs.

At the same time, telomerase has become a potential target for anti-cancer therapeutic application based on the strong correlation between telomerase activity and malignancy[7]. Furthermore, it was found that inhibition of telomerase either by oligonucleotides against human telomerase RNA or via dominant-negative mutants of hTERT(human telomerase reverse transcriptase) in human tumor cells leads to progressive telomere shortening and apoptotic cell death[8].

It is thus intriguing to study the interrelation between ROS action and telomere/telomerase during apoptosis. To investigate the mechanism during anti-cancer agent-induced tumor cell death, HeLa cells were induced to undergo apoptosis with hydroxyl radicals (·OH)  generated directly via Fe²⁺ -mediated Fenton reaction and the changes in telomerase activity and telomere length during apoptosis were studied. We found that OH  caused lipid peroxidation and severe telomere shortening while with no effect on telomerase activity.

1 Materials and Methods

1.1 Materials

3,3'-Diethyloxadicarbocyanine, GSH, DAPI and propidium iodide were purchased from Sigma. 1,4-Dithiothreitol (DTT) was from Promega. Other substances were from commercial sources and of analytical grade. The telomere assay kit was a gift from Dr. T. Just (DAKO A/S, Denmark). The TUNEL kit and telomerase assay kit were purchased from Boehringer Mannheim. All cell culture reagents were purchased from GIBCO.

1.2 Cell culture

HeLa cells were seeded at 1x10⁵ cells per culture flask in 10 mL of DMEM. The complete culture media contained 10% fetal calf serum, 100 mg/mL of streptomycin, and 100 units/mL of penicillin. Cells were incubated at  37 °C in a humidified atmosphere containing 5% CO₂. Exponentially growing cells were used for experiments.

1.3  Induction of apoptosis and experiment treatments

·OH radicals were generated through the reaction of 0.1  mmol/L  FeSO₄ and 0.3, 0.6, or 0.9  mmol/L  H₂O₂, and were used for apoptosis induction for the indicated length of time (from 0 to 24 h). For telomerase inhibition, cells were preincubated with different concentrations of  3,3'-diethyoxadicarbocyanine  (DODCB) for 1 h before  ·OH  treatment and remained in the medium afterwards. In the experiments using GSH, cells were pretreated with  1-10 mmol/L  GSH for 1 h before apoptosis induction.

1.4  MTT test

The ·OH -induced inhibitory effect on cell proliferation was determined using tetrazolium dye colorimetric test (MTT test)[9]. The MTT absorbance was read using a plate reader(Bio-Rad, model 3550, Biorad, Hercules, CA, USA) at 595 nm.

1.5  Cell morphology

For phase-contrast microscopy, cells cultured on glass coverslips were washed with D-Hanks, and were observed and photographed under a phase-contrast microscope (Nikon DIAPHOT).

1.6  DAPI staining

Cells were seeded 24 h before FeSO₄-H₂O₂ treatment onto glass coverslips, precoated with 1 mg/mL of poly-L-lysine (Sigma, relative molecular mass  3.7 x10⁴) at the density of 3x10⁴ cells/cm² . The cells were rinsed in D-Hanks and fixed with methanol. After another rinse, cells were stained with 1 mg/mL  4',6-diamidin -2-phenylindole (DAPI, Sigma) in PBS for 30 min and counted. Apoptotic cells were determined by evaluating nuclear morphology using a fluorescence microscope (Nikon FLUOPHOT).

1.7  In situ detection of DNA cleavage by the TUNEL procedure

Apoptotic cells were also determined using a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) kit (Boehringer Mannheim) as described by Gavrieli et al.  [10]Briefly, cells were plated on glass coverslips and incubated for different periods of time in medium with or without FeSO₄-H₂O₂. Cells were then fixed in freshly prepared paraformaldehyde solution (4% in D-Hanks, pH 7.4) for 30 min at room temperature.  After being rinsed with D-Hanks, cells were permeabilized with  0.1%  Triton X-100 in 0.1% sodium citrate buffer and incubated for  1 h  at  37  °C with TdT and fluorescein isothiocyanate-dUTP to label the cleaved DNA. After that, coverslips were mounted in anti-fade mounting solution and observed under a fluorescence microscope (Nikon FLUOPHOT).

1.8  Measurement of sub-G1 cells

It is well established that DNA fragmentation during apoptosis may lead to extensive loss of DNA content and result in a distinct sub-G1 peak when cells are analyzed by flow cytometry. Flow cytometry measurements were carried out according to the methods described by Ishibashi et al. [11] About 1x10⁶ HeLa cells were fixed in 70% ice-cold ethanol for more than 2 h, and then incubated with freshly prepared staining buffer( 0.1 % Triton X-100 in phosphate-buffered saline, 200 mg/mL RNase A, and 20 mg/mL propidium iodide) at  37 °C for 30 min. The fluorescence intensity was measured using a Coulter Elite Flow Cytometer. For each sample, 20 000 cells were analyzed using the Coulter Elite workstation 4.0 software (Coulter Corp.).

1.9  Detection of telomerase activity

Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol (TRAP) ELISA kit (Boehringer Mannheim) according to the manufacturer's description with some modifications. In brief, approximately 1x10⁶cells were lysed in 200 mL lysis reagent and incubated on ice for 30 min. For conducting TRAP reaction, 2 mL of cell extract (containing 2 mg protein) was added to 25 mL of reaction mixture and then an appropriate amount of sterile water was added to make a final volume of 50 mL. Polymerase chain reaction (PCR) was performed in a PTC-100^TM  Programmable Thermal Controller (MJ Research, InC) as follows: primer elongation (30 min,  25  °C), telomerase inactivation (5 min,  94  °C), product amplification by repeat of 30 cycles ( 94  °C for 30 s,  50  °C for 30 s,  72  °C for  90 s ). Hybridization and ELISA reaction were carried out following the manufacturer's instruction. The extract from normal human fibroblasts served as negative controls. The extract of 293 cells was used as positive controls.

1.10  Measurement of telomere length

Fluorescence in situ hybridization was carried out following the procedure described by Ren et al.[12,13] The telomere fluorescence signal was defined as the mean fluorescence signal in cells after subtraction of the background fluorescence signal.

1.11  Lipid peroxidation measurement

The extent of lipid peroxidation was estimated in HeLa cells by measurement of malondialdehyde(MDA) formation using the thiobarbituric acid method. After treatment, cells were collected and washed twice with ice-cold D-Hanks. Then the MDA was measured and calculated according to the method of Nelson et al.[14] Protein was determined according to Bradfordusing bovine serum albumin as the standard.

1.12  Determination of intracellular GSH content

The determination of intracellular GSH was conducted as previously described[15]. The concentration of intracellular GSH was expressed as nanomoles per mg protein.

1.13  Statistical analysis

Data are expressed as means ± SD. Significance was assessed by two tailed t-test or one-way analysis of variance (ANOVA). All data represent at least three independent experiments performed in triplicates.

2  Results

2.1  Hydroxyl radicals induced apoptosis in human tumor cells

As shown in Fig.1, the proliferation of HeLa cells was significantly inhibited by hydroxyl radicals. After treatment with  ·OH , HeLa cells demonstrated typical morphological and biochemical changes of apoptotic cells. Cells treated with ·OH rounded up and lost their contact with surrounding cells and finally detached from the surface of culture flasks while control cells showed normal morphology (Fig. 2). DAPI staining showed disintegration and condensation in nuclei of apoptotic HeLa cells (Fig. 3(a)). Moreover, the TUNEL procedure which detects cleavage of DNA in situ (Fig. 3(b)) showed the apoptosis-specific DNA fragmentation in ·OH -induced human tumor cell death. The flow cytometric analysis provided similar results (Fig. 3(c)). Taken together, these results strongly suggest that exogenous ·OH induces typical apoptosis in HeLa cells.

2.2 ·OH-induced HeLa cell death was rescued by GSH

When HeLa cells were pre-incubated with GSH (3 mmol/L, 5 mmol/L), a well-known antioxidant which scavenges free radicals in both in vivo and in vitro systems, for 1 h, the induction of apoptosis by  ·OH  was almost completely eliminated as shown in Fig.4. The results thus provided convincing evidence for the involvement of ·OH in apoptosis induction.

2.3  ·OH-induced telomere shortening but had no effect on telomerase activity in human tumor cells

Telomere shortening has been found to account for the limitation of the numbers of divisions in normal human fibroblasts where telomerase activity is not detectable leading to cellular senescence. In this study, we investigated the interrelation between telomeres and hydroxyl radical-induced apoptosis and found that telomere length reduced significantly during ·OH-induced apoptosis in HeLa cells. Again, GSH protects telomere from shortening ( Fig.5 (a) ). Surprisingly, no inhibition in telomerase activity was observed throughout the ·OH-induced apoptosis in HeLa cells (Fig.5 (b)). The results are in accordance with a recent report regarding the cell killing by paclitaxol in murine melanoma cells,[16] and etoposide, cisplatin, irinotecan, mitomycin, and daunorubicin-mediated apoptosis in leukaemic-cells[17].

2.4  Inhibition of telomerase activity increased susceptibility of HeLa cells to ·OH -induced apoptosis

Although maintenance of telomerase activity does not protect tumor cells from ·OH-induced  cell killing in our systems, inhibition of telomerase with DODCB, a specific inhibitor of telomerase, caused an increased apoptosis at the same level of  ·OH . As shown in  Fig.6 (a) , 20 mmol/L DODCB caused a 90% decrease in telomerase activity. When HeLa cells were preincubated with 20 mmol/L DODCB before treated with 0.1  mmol/L  FeSO₄ and 0.3  mmol/L  H₂O₂, apoptosis was significantly higher than control as detected by the flow cytometric analysis (Fig. 6 (b)). The results suggest that inhibition of telomerase activity increased markedly the sensitivity of cells to apoptosis induction by  ·OH . The results implicate that maintenance of telomerase activity is required for prevention of DNA damage and maintenance of tumor cell viability. It is worth mentioning that DODCB alone does not cause cell death in HeLa cells and telomere shortening (data not shown). Our results suggest that telomere length but not telomerase activity plays a decisive role in apoptotic cell death under our experimental conditions.  

2.5 Lipid peroxidation increased in HeLa cells after treatment with ·OH

Henle et al.[18] and Oikawa et al.[19] have reported that ·OH can cleave telomeres preferentially in a chemical reaction system. However, ·OH is a kind of highly reactive radical, which can react with most molecules at their diffusion rate. Then how does exogenous ·OH enter into the nucleus and act upon telomeres? Some evidences have indicated that the free radical-related process, lipid peroxidation, can directly or indirectly through the generation of stable reactive compounds, cause damage to proteins and genetic materials in cells[20]. We assayed the MDA content, an index of lipid peroxidation, in HeLa cells after treatment with ·OH. As shown in Fig. 7, ·OH treatment led to an increase of MDA in a dose-dependent manner. The thiol antioxidant, GSH, could effectively prevent the formation of MDA. This may be the result of the actions of thiol on preventing Fe²⁺ -mediated radical formation.

2.6  Changes of intracellular GSH content

To measure the intracellular events promoted by Fe²⁺ -H₂O₂, we used glutathione measurement. Cellular GSH is an important compound, as it functions both as a substrate or cofactor of protective enzymes and as an efficient radical scavenger. As such, its concentration is an index of intracellular redox status. In this study, changes in intracellular GSH levels at the end of  ·OH  exposure are demonstrated in Fig.8. In the presence of Fe²⁺ -H₂O₂ system, we observed a decrease in intracellular total GSH which was dose-dependent and indicated an intacellular

3  Discussion

Reactive oxygen species have been implicated as potential modulators of apoptosis[21]. In addition to the involvement of ROS in anti-cancer agent-induced apoptotic tumor cell death, ROS are also reported to be involved in excitotoxic-, staurosporine-, and ceramide-induced neural cell death, ceramide-induced human leukemic U937 cell death, paraquat-induced death of endothelial cells, HIV-induced death of T cells, and other apoptotic events. There is currently much interest in the role of free radicals in apoptosis.

In this study we provide evidence that during ·OH (generated through 0.1 mmol/L FeSO₄/0.3-0.9 mmol/L H₂O₂) induced apoptosis in HeLa cells telomere shortening occurs without inhibition of telomerase activity. The results suggest that ROS induced telomere shortening is not through telomerase inhibition but possibly a direct effect of ROS on telomeres themselves. Using a chemical reaction system, Henle et al.[18]and Oikawa et al.[19] found that ·OH-mediated DNA oxidations has preferential cleavage sites which are at the nucleoside 5' to each of the dG moieties in the sequence RGGG, a sequence commonly found in telomeres. The results support the possibility that telomere may be a direct target of ·OH.

Some reports have shown that ·OH is the ultimate reactive species in DNA oxidation[22]. However, a large portion of H₂O₂-dependent DNA damage appears not to be due to diffusible hydroxyl radicals[20]. The nature of the ultimate oxidant responsible for DNA damage by ·OH is unclear. Detailed experiments have illustrated that a model of freely diffusible ·OH fails to account for the strikingly parallel dynamics of DNA strand scission in vitro. Oxidative DNA adducts may be formed indirectly; the lipid peroxidation results in various aldehyde breakdown products that are able to form covalent mutagenic adducts. Then how does exogenous  ·OH  enter into the nucleus and act upon telomeres since ·OH radical has a very short half-life and reacts with most molecules at diffusion rate? We suggest that the damage of  ·OH  to telomeres is realized through the lipid peroxidation. To test the hypothesis, we measured the changes of MDA, a product of lipid peroxidation, and intracellular GSH levels, the major intracellular antioxidant which can react with many aldyhede products produced from lipid peroxidation. Our results showed that lipid peroxidation increased dramatically and GSH levels decreased significantly. 5 mmol/L exogenous GSH can inhibit lipid peroxidation totally, and then prevent telomere shortening. These results indicated that ·OH did induce lipid peroxidation in HeLa cells, and then trigger telomere shortening. Then lipid peroxidation mediated-telomere shortening may play a pivotal role in the apoptosis induced by ·OH in HeLa cells.

Although telomerase inhibition is not required in  ·OH-induced  apoptotic tumor cell death, telomerase-inhibited tumor cells are more susceptible to ROS. As shown in Fig.6, when telomerase was inhibited by DODCB and the activity was only 12% of the original level, a four-fold increase in percentage of apoptosis was observed in ·OH-treated cells as compared with the control cells in which a normal telomerase activity was maintained. It is striking that DODCB alone without ·OH treatment caused very low level of apoptosis. This is in agreement with the data reported by Fu et al.[23] in PC12 cells. The results may have important implications for cancer chemotherapy since combinations of a telomerase inhibitor and an anti-cancer drug which induces ROS generation may be highly efficient in tumor cell killing.

In summary, our study provides evidence of lipid peroxidation on apoptosis induced by hydroxyl radicals in HeLa cells. Thus, lipid peroxidation may result in telomere shortening, which further trigger apoptosis. Our study may have some implications for the strategy of development of cocktails of chemotherapeutic agents. For example, combinations of telomerase inhibitors and anti-cancer agents that generate ROS and shorten telomeres would be highly effective for cancer treatment.

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