Nucleotide Excision Repair Is Not Required for the Antiapoptotic Function of Insulin-like Growth Factor 1

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

241, 458 – 466 (1998)

EX984087

Nucleotide Excision Repair Is Not Required for the Antiapoptotic Function of Insulin-like Growth Factor 1 Whaseon Lee-Kwon, Deokbae Park, and Michel Bernier1 Diabetes Section, Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224-6825

The expression of ERCC1, a member of the nucleotide excision repair (NER) family, is enhanced in cells transfected with insulin-like growth factor 1 (IGF-1) receptors. Of interest, an excellent concordance between ERCC1 expression and NER-mediated cell survival has been demonstrated. The two aims of the present study were to determine the signaling pathways used by IGF-1 to confer protection against apoptotic cell death in Chinese hamster ovary (CHO) cells and to assess the role of NER in this IGF-1 action. Experiments with pharmacological inhibitors indicated that phosphatidylinositol 3-kinase (PI 3-kinase) but not mitogen-activated protein kinase (ERK1/ ERK2) mediates IGF-1 antiapoptotic activity. Using two series of CHO cells that have altered expression of ERCC1 or XPB/ERCC3, we examined IGF-1’s ability to delay apoptotic death and reduction of mitochondrial oxidative function mediated by growth factor withdrawal. IGF-1 effectively blocked apoptosis, concomitant with increased MTT activity, in a pair of CHO cell lines expressing inactive ERCC1 (43-3B cells) and the transfected line of the mutant carrying the expressed human ERCC1 gene (83-G5 cells). Similarly, repairdeficient UV24 cells, which lack XPB/ERCC3, and their parental line AA8 were also responsive to the IGF-1’s antiapoptotic capacity. In the presence of IGF-1, these cell lines became resistant to the cleavage of poly(ADP-ribose) polymerase, a key player in DNA damage recognition and DNA repair. These results suggest that PI 3-kinase activation plays a determinant role in the antiapoptotic function of IGF-1, but that functional NER does not play a critical part in mediating this IGF-1 response. © 1998 Academic Press

INTRODUCTION

Apoptosis or programmed cell death plays a major role in normal turnover of cells. It is characterized by changes within the nucleus which include chromatin condensation and internucleosomal DNA cleavage [1; 1 To whom correspondence and reprint requests should be addressed. Fax: (410) 558-8381. E-mail: [email protected].

0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.

reviewed in Ref. 2]. DNA degradation occurs well before the loss of plasma membrane integrity and of mitochondrial oxidative function [2]. The process of apoptosis has been the subject of intense study over the past few years since the identification of gene products involved in prevention or induction of apoptosis [3]. Insulin-like growth factor-1 (IGF-1) is one of several major survival factors that prevent the onset of apoptosis [4 – 6]. Following ligand binding, the IGF-1 receptor undergoes activation of its intrinsic tyrosine kinase function and subsequent activation of at least two major signaling pathways; the first pathway involves phosphatidylinositol 3-kinase (PI 3-kinase) and p70 S6 kinase, whereas the second pathway consists of Ras/ Raf/mitogen-activated protein kinase (ERK1 and ERK2) [7]. The role of these pathways in the antiapoptotic function of IGF-1 has recently emerged [8 –11]. One factor that could influence cellular sensitivity to apoptosis is DNA damage recognition and DNA repair. Relatives of PI 3-kinase such as the product of the ataxia telangiectasia gene and the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) have been recently described to have key roles in components of cell cycle control, DNA repair, and DNA damage responses [12, 13]. DNA-PK phosphorylates p53 and replication protein A [14], the latter protein being involved in the recognition of DNA damage, a crucial step in nucleotide excision repair (NER). NER is the main repair system for the elimination of a wide range of DNA lesions. It is a complex process whereby lesions are located, a stretch of nucleotides containing the damage is excised, and a repair patch is synthesized [15]. NER genes including ERCC1 and XPB/ERCC3 are viewed as determinants of cellular sensitivity to a variety of lesions to DNA [16 –18]. The human excision repair cross-complementing 1 gene (ERCC1) is a mammalian DNA repair gene whose gene product has been shown to play a crucial role in the early excision step of damaged DNA by virtue of its intrinsic structure-specific nuclease activity [19 –21]. Because of its extensive N-terminal homology to the yeast RAD10 protein, ERCC1 may also participate in a repair mechanism involving homologous recombination [22]. The XPB/

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ERCC3 gene encodes a subunit of the basal transcription factor bTFIIH, which has been implicated in NER and in cell cycle regulation. Mutation in XPB/ERCC3 causes both a severe NER defect and a decrease in basal transcription activity [23], possibly due to alteration in the binding of the p53 tumor-suppressor protein to this helicase and other associated factors [24, 25]. A recent study has demonstrated that human primary fibroblasts with mutations in the XPB/ERCC3 or XPD/ERCC2 gene survive much better in response to p53-mediated apoptosis, as a consequence of the failure of p53 to bind to these constituents of the core TFIIH transcription-NER complex [26]. It follows that the loss of functional interactions between p53 and the TFIIH complex may reduce cellular apoptosis induced by genotoxic agents that cause DNA damage [27, 28]. However, Wang et al. [26] show that both cell types undergo a normal apoptotic response in the presence of inducers of p53-independent pathways which include gene products encoding proteases related to interleukin-converting enzyme (ICE). It has been found that IGF-1 protects cells from apoptosis induced by both p53-dependent and p53-independent pathways, including that induced by anticancer drugs [6, 29], overexpression of ICE protease [30], and after serum withdrawal [11]. In light of the fact that IGF-1 is involved in the regulation of the repair process of radiationinduced DNA breaks [31, 32], the question remains whether activation of the IGF-1 receptor may offer antiapoptotic protection as a result of its role in NER. Previous work from this laboratory [33] has shown that overexpression of the IGF-1 receptors induces an increase in ERCC-1 gene expression in Chinese hamster ovary (CHO) cells and that a correlation was established between protection from UV-induced cell death and ERCC-1 expression. In the present study, the goal was to examine the ability of IGF-1 to delay apoptosis induced by serum deprivation in wild-type and repair-deficient CHO cell lines with specific defects in ERCC1 or XPB/ERCC3 expression and to elucidate the antiapoptotic signal transduction pathways used by the IGF-1 receptor. We found that activation of the IGF-1 receptor exerted antiapoptotic protection in all the cell lines studied, supporting the notion that NER is not involved in the protective effect of the IGF-1 receptor. Selective inhibition of PI 3-kinase by wortmannin and LY294002 [34, 35] markedly reduced protection, whereas inhibition of ERK activity with PD98059 [36] did not influence the antiapoptotic capacity of the IGF-1 receptor. MATERIALS AND METHODS Cell lines and cell culture. CHO cell lines used in this study have been previously described [37–39]. These include: (i) CHO/K1 containing the neomycin resistance gene; (ii) UV24, a XPB/ERCC3-

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deficient cell, and its repair-competent parental cell line AA8, and; (iii) a pair of CHO cell lines expressing a defect in ERCC1 expression (43-3B) and the transfected line of the mutant carrying the expressed human ERCC1 gene (83-G5). These cell lines were kindly provided by Dr. Vilhelm A. Bohr (National Institute on Aging, NIH, Baltimore, MD). Cells were grown in a monolayer at 37°C in a humidified atmosphere of 95% air/5% CO2 in Ham’s F-12 medium (neo, AA8, UV24) or Dulbecco’s modified Eagle’s medium (43-3B, 83-G5) containing 100 U/ml penicillin, 100 mg/ml streptomycin, and 10% fetal bovine serum. Cell treatment. Confluent cells in serum-free medium were incubated in the absence or in the presence of a range of concentrations of recombinant IGF-1 [Upstate Biotechnology Inc. (UBI), Saranac Lake, NY] or 10% fetal bovine serum. Eighteen hours later, floating cells were collected by centrifugation, harvested, and combined with the cells remaining attached to the plate. In some experiments, pharmacological inhibitors were used. These include: wortmannin (Sigma Chemical, St. Louis, MO), LY294002 (Calbiochem, La Jolla, CA), and PD98059 (Calbiochem). Determination of apoptosis by DNA fragmentation analysis. Cells were lysed in lysis solution (Puregene, Gentra Systems, Inc., Minneapolis, MN) and incubated overnight at room temperature prior to the addition of 20 mg/ml RNase A for 1 h at 37°C. The samples were deproteinized, followed by DNA precipitation with isopropanol. The concentration and purity of DNA were determined spectrophotometrically by measuring UV adsorbance ratio at 260 over 280 nm. Equal amounts of DNA from each sample (1 mg) were 39-OH–labeled with 5 U Klenow fragment of DNA polymerase I (New England Biolabs, Beverly, MA) and 0.5 mCi [a-32P]dCTP ('3000 Ci/mmol, Amersham Corp., Arlington Heights, IL) in the presence of 10 mM Tris/HCl, pH 7.5, and 5 mM MgCl2. After a 10-min incubation at room temperature, the reaction was terminated by the addition of 10 mM EDTA. The samples were electrophoresed on 6% polyacrylamide gel. Following electrophoresis, the gel was fixed in a solution composed of 15% MeOH and 5% AcOH, dried, and analyzed by autoradiography using Kodak BioMax film and intensifying screens. The radioactivity associated with the DNA fragments was quantitated by electronic autoradiography with a Packard InstantImager (Meriden, CT). Determination of PI 3-kinase activity. PI 3-kinase activity was measured as described previously [40] with some minor modifications. Serum-deprived cells grown in 35-mm dishes were preincubated in the absence or presence of wortmannin (50 nM) or LY294002 (3 mM) for 30 min prior to the addition of IGF-1 (10 nM). Eighteen hours later, cells were lysed, and lysates were immunoprecipitated with a polyclonal anti-p85 antibody (UBI). l-a-Phosphatidylinositol (Sigma) was used as substrate in the kinase assay. Determination of MAP kinase activity. MAP kinase activity was measured in anti-ERK1/ERK2 immunoprecipitates as described by Kole et al. [40]. Western blot analysis. Confluent cells were lysed directly in Laemmli sample buffer [41] containing 5% 2-mercaptoethanol, and equal amounts of protein from each sample were subjected to onedimensional SDS–polyacrylamide gel electrophoresis (PAGE) under reducing conditions followed by electrotransfer to polyvinylidene difluoride (PVDF) membranes. ERCC1, XPB/ERCC3, and poly(ADPribose) polymerase (PARP) proteins were detected by Western immunoblotting using the ECL chemiluminescence detection system (Amersham). The polyclonal rabbit antiserum raised against ERCC1 was kindly provided by Dr. Lawrence C. Panasci [42], while polyclonal anti-XPB (S-19) and monoclonal anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and PharMingen (San Diego, CA), respectively. MTT assay. This measure of mitochondrial function was performed as described previously [11] with CHO cells plated on 24-well plates. Following treatments, the medium was removed from the wells, and 200 ml of MTT reagent (Sigma), at a concentration of 1

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FIG. 1. IGF-1 inhibits apoptosis induced by serum withdrawal. CHO/K1 cells were maintained in serum-free medium in the absence or in the presence of IGF-1 at the indicated concentrations. After an 18-h incubation, total DNA was extracted from adherent and detached cells and radiolabeled as described under Materials and Methods. Cells maintained in medium with 10% serum were used as controls. The data represent the means 6 SEM of three independent experiments, where the relative level of DNA fragmentation in the absence of trophic factors was arbitrarily set at 100%.

mg/ml in RPMI 1640 medium without phenol red, was added to each well. After a 1-h incubation at 37°C, the cells were lysed by addition of 1 vol of isoamyl alcohol and shaking for 20 min. Absorbance of converted dye was measured at a wavelength of 570 – 690 nm.

RESULTS

IGF-1 Prevents Apoptosis Induced by Trophic Factor Withdrawal in CHO Cells The ability of IGF-1 to block apoptosis in CHO cells was studied initially after incubation for 18 h in the absence or in the presence of a range of IGF-1 concentrations. CHO cells were used because they exhibit relatively high levels of receptor autophosphorylation and kinase activation upon IGF-1 stimulation [43]. Furthermore, a large number of excision repair mutants have been isolated from these rodent cells, allowing extensive characterization of the NER pathway [44]. In CHO/K1 cells maintained in 10% serum, there was very little DNA fragmentation detected (Fig. 1). The removal of serum led to a marked increase in apoptosis. The addition of IGF-1 (2.5 and 10 nM) at the time of trophic factor withdrawal delayed apoptosis very efficiently. For subsequent experiments, we chose to use IGF-1 at a concentration of 10 nM to detect the effects of agents which might enhance or inhibit the antiapoptotic action of IGF-1.

FIG. 2. Inhibition of PI 3-kinase activity blocks the antiapoptotic function of IGF-1. The effect of wortmannin or LY294002 on apoptosis was measured by adding these PI 3-kinase inhibitors to CHO/K1 cells at the time of serum withdrawal 30 min prior to an 18-h incubation in the absence (lanes 1, 4, and 6) or in the presence (lanes 3, 5, and 7) of 10 nM IGF-1. The collection and labeling of the DNA were performed as for Fig. 1. (A) Autoradiogram of a representative experiment. (B) The data represent the means 6 SEM of three independent experiments, where the relative level of fragmentation in the absence of trophic factors was arbitrarily set at 100%. (C) The stimulation of PI 3-kinase by IGF-1 is attenuated by wortmannin and LY294002. CHO/K1 cells were treated as indicated above, and the clarified lysates were immunoprecipitated with anti-p85 antibody. The immune pellets were analyzed for PI 3-kinase activity as described under Materials and Methods. The autoradiogram is representative of several independent experiments.

ANTIAPOPTOTIC ACTION OF IGF-1 AND DNA REPAIR

461

Activation of PI 3-Kinase, but Not ERK, Is Essential for the Antiapoptotic Function of IGF-1 Several tyrosine kinase receptors, including the IGF-1 receptor, increase the PI 3-kinase pathway, one of the most important intracellular mechanisms that are likely to be involved in antiapoptotic signaling [9, 45]. To evaluate the importance of PI 3-kinase in IGF-1’s protection against apoptosis, we examined the action of two chemically unrelated PI 3-kinase inhibitors. Cells were treated without or with wortmannin (50 nM) or LY294002 (3 mM) for 30 min at the time of serum withdrawal followed by an 18 h-incubation in the absence or presence of IGF-1. Figure 2A shows a representative autoradiogram. Treatment of CHO/K1 cells with either wortmannin or LY294002 in the absence of IGF-1 did not modify the degree of apoptosis detected (Fig. 2A, lanes 4 and 6 vs lane 1). However, these inhibitors markedly blocked IGF-1’s ability to reduce DNA laddering (lanes 5 and 7 vs lane 3). Quantitative analysis was performed where the relative levels of DNA fragmentation for each condition were normalized to the levels of DNA laddering in control, serum-starved cells (Fig. 2B). An immunoprecipitation/kinase assay of PI 3-kinase was carried out in the presence of phosphatidylinositol as the substrate, and the results indicated that the stimulatory effect of IGF-1 on PI 3-kinase was markedly attenuated by either wortmannin or LY294002 (Fig. 2C). Thus, these data suggest that the antiapoptotic function of IGF-1 involves activation of the PI 3-kinase pathway. In several cell lines, IGF-1 increases the extracellular signal-regulated kinase (ERK) activity via a Ras-dependent pathway involving sequential activation of Raf-1 and MEK1, the upstream regulator of ERK. ERK activation has been reported to be antiapoptotic in neuronal cells [11, 46]. To investigate the effect of MEK1 inhibition on apoptosis, CHO/K1 cells were treated with a maximally effective concentration of PD98059 (100 mM) [36] for 30 min at the time of serum withdrawal followed by an 18-h incubation in the absence or in the presence of IGF-1. A representative autoradiogram is shown in Fig. 3A. In the absence of IGF-1, the MEK1 inhibitor partially reduced DNA laddering but failed to attenuate the effect of IGF-1 on inhibition of apoptosis. Quantitative analysis was performed where the relative levels of DNA fragmentation for each condition were normalized to the levels of DNA laddering in unstimulated cells (Fig. 3B). An immunoprecipitation/kinase assay of ERK1/ERK2 was carried out with myelin basic protein as the substrate to establish whether IGF-1 can elicit ERK activation in these cells. While ERK activity was rapidly elevated following treatment with 10% serum, there was no significant in-

FIG. 3. PD98059 does not influence IGF-1’s ability to block apoptosis induced by serum withdrawal. At the time of trophic factor withdrawal, the MEK1 inhibitor PD98059 was added to CHO/K1 cells for 30 min prior to an 18-h incubation in the absence (lanes 1 and 4) or in the presence (lanes 3 and 5) of 10 nM IGF-1. (A) Autoradiogram of a representative experiment. (B) The data represent the means 6 SEM of three independent experiments, where the relative level of fragmentation in the absence of trophic factors was arbitrarily set at 100%. (C) Pharmacological inhibition of ERK1/ ERK2 activity by PD98059. Serum-starved cells were treated in the absence or in the presence of 50 mM PD98059 for 1 h followed by the addition of 10% serum (lanes 3, 4) or 10 nM IGF-1 (lanes 5 and 6). Ten minutes later, cells were lysed and ERK1/ERK2 activity was determined in an immunoprecipitation-based kinase assay using myelin basic protein as the substrate. The autoradiogram is representative of several independent experiments.

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FIG. 4. Analysis of DNA fragmentation in ERCC1-deficient CHO cells. ERCC1-deficient 43-3B cells (lanes 1–3) and transfectants 83-G5 containing the ERCC1 gene (lanes 4 – 6) were incubated in serum-free medium or in the presence of 10 nM IGF-1 or 10% serum. Eighteen hours later, DNA was collected and labeled. (A) Autoradiogram of a representative experiment. (B) The data represent the means 6 SEM of three independent experiments, where the relative level of fragmentation in the absence of trophic factors was arbitrarily set at 100%. (C) Western blot analysis of ERCC1 expression. Total cell extracts (30 mg) from 43-3B and 83-G5 cells were separated by SDS–PAGE, transferred to PVDF membrane, and immunoblotted using anti-ERCC1 antibody and the Amersham ECL detection system.

crease with IGF-1 (Fig. 3C). PD98059 blocked the enhancing effect of serum on ERK activity. Thus, these data suggest that the ERK pathway plays a minor role, if any, in the antiapoptotic function of IGF-1. Defect in Nucleotide Excision Repair System Does Not Affect IGF-1’s Antiapoptotic Protection Transfection of CHO cells with the human IGF-1 receptors results in enhanced expression of ERCC-1 [33], whose function has been implicated in the repair of various DNA lesions, concomitant with improved cell survival [15]. To assess the role of nucleotide excision repair system in the antiapoptotic function of IGF-1, two pairs of CHO cell mutants defective in NER were selected. Cells with complementation group 1 and 3 defects have impaired repair synthesis because each is missing a single component of the repair apparatus [15]. Figure 4A shows a representative experiment comparing the protection offered by IGF-1 in a UVsensitive line from complementation group 1, 43-3B, and the related cell line 83-G5. This line was derived by transfecting 43-3B with the human ERCC1 gene to restore UV and mitomycin resistance and normal repair capacity [47]. The withdrawal of serum from 43-3B cells increased DNA fragmentation. As previ-

ously observed with CHO/K1 cells, treatment of 43-3B cells with IGF-1 at the time of serum withdrawal efficiently blocked apoptosis. These experiments were repeated three times and quantitated (Fig. 4B). In 83-G5 cells, similar observations were made. Additional experiments were performed whereby ERCC1 protein expression was studied in total cell extracts (30 mg) that were fractionated by SDS–PAGE and transferred to PVDF membrane. Western blot analysis revealed that ERCC1 protein was indeed expressed in the transfectant 83-G5, while being undetectable in 43-3B cells (Fig. 4C). Thus, under conditions where ERCC1 expression was very low or missing, IGF-1’s antiapoptotic protection remained intact. We investigated next the protective role of IGF-1 against apoptotic death in the repair-proficient parental AA8 line and a complementation group 3 mutant, UV24. CHO/UV24 cells have a defect in the XPB/ ERCC3 gene which results in a combined transcription/repair deficiency [48]. In addition to their UV hypersensitivity, UV24 cells are highly sensitive to certain chemical agents that produce damage that is acted upon by the NER pathway. In the study herein, the removal of serum led to increased DNA laddering in both AA8 and UV24 cells (Fig. 5A). Similar to parental and ERCC1-deficient cells, IGF-1 greatly re-

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FIG. 5. Analysis of DNA fragmentation in XPB/ERCC3-deficient CHO cells. Parental AA8 cells (lanes 1–3) and UV24 cells (lanes 4 – 6) were incubated for 18 h in serum-free medium (lanes 1 and 4) or in the presence of 10 nM IGF-1 (lanes 2 and 5) or 10% serum (lanes 3 and 6). (A) Autoradiogram of a representative experiment. (B) The data represent the means 6 SEM of three independent experiments, where the relative level of fragmentation in the absence of trophic factors was arbitrarily set at 100%. (C) Western blot analysis of XPB/ERCC3 expression. Total cell extracts (30 mg) from AA8 cells (lanes 1–3) and UV24 cells (lane 4) were separated by SDS–PAGE, transferred to PVDF membrane, and immunoblotted using anti-XPB/ERCC3 antibody.

duced the level of apoptosis detected. These experiments were repeated three times and quantitated (Fig. 5B). Western blot analysis from total cellular extracts indicated a very low level of XPB/ERCC3 expression in UV24 cells when compared to AA8 cells (Fig. 5C, compare lanes 4 and 3). Of interest, densitometric readings of these blots revealed that the endogenous level of XPB/ERCC3 protein was increased 2.5- and 3.6-fold following incubation of AA8 cells with IGF-1 and 10% serum, respectively. This increase in XPB/ERCC3 expression by IGF-1 has not been investigated further. These results suggest that the degree of XPB/ERCC3 expression does not correlate with IGF-1-mediated protection. The ability of IGF-1 to maintain cell survival was studied using the MTT assay [11]. It was found that IGF-1 was able to prevent a reduction in MTT activity induced by serum withdrawal in all the CHO cell lines studied (Table 1). Thus, IGF-1 was capable of preventing apoptosis, concomitant with the maintenance of mitochondrial oxidative function.

IGF-1 Effect on PARP Integrity The poly(ADP-ribose) polymerase (PARP) protein has been implicated in the cellular defense against DNA damage [49]. Within minutes of the onset of apoptosis, PARP is cleaved by CPP32b, an ICE-related

TABLE 1 Loss of Mitochondrial Function in Various CHO Cell Lines Maintained in the Absence of Serum Cell type

10% serum

No serum

IGF-1 (10 nM)

K1 UV24 AA8 83-G5 43-3B

100 100 100 100 100

46.9 6 2.8 38.6 6 5.1 30.7 6 4.0 54.1 6 4.9 42.9 6 2.9

84.6 6 3.1 96.5 6 12.3 98.4 6 7.1 103.3 6 5.9 94.2 6 6.5

Note. MTT activity was measured in confluent cells that were maintained for 24 h in the absence or in the presence of 10 nM IGF-1 or 10% FBS. The values represent percent activity of serum-treated cells (mean 6 SD, n 5 4).

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FIG. 6. Detection of PARP by Western blot analysis. Total cell extracts (50 mg) from AA8 (lanes 1–3) and UV24 (lanes 4 – 6) cells were separated by SDS–PAGE, transferred to PVDF membrane, and immunoblotted using anti-PARP antibody.

protease [50, 51], which results in its inactivation and the cessation of DNA repair mechanisms [49]. The extent of PARP cleavage was examined following treatment of AA8 and UV24 cells with serum or IGF-1 using Western blot analysis (Fig. 6). The anti-PARP antibody recognized a single 116-kDa band in total cell lysates. In the absence of serum, there was a reduction in PARP integrity when compared to cells maintained in the presence of IGF-1 or 10% serum. This effect of IGF-1 was observed in both parental and XPB/ERCC3deficient cells. DISCUSSION

In the present study, we examined the ability of IGF-1 to exert antiapoptotic function in CHO cells and determined the importance of NER in this function. Toward this end, CHO cells expressing NER-impaired mutants were used. These mutant cell lines, which included the ERCC1-defective 43-3B cells and the XPB/ERCC3-deficient cells, have been previously characterized for their hypersensitivity to ultraviolet light and various chemical adducts [38]. A major effector for signaling by the IGF-1 receptor is PI 3-kinase. In the study herein, it is found that IGF-1 activates this heterodimeric enzyme in CHO cells. Inhibition of PI 3-kinase activity by wortmannin or LY294002 adversely affected IGF-1’s ability to block apoptosis induced by serum starvation. This finding is in agreement with several other reports of PI 3-kinasedependent protection from apoptosis [9, 11, 52]. Because wortmannin and LY294002 are chemically unrelated, it is most likely that the inhibition of the protection from apoptosis by IGF-1 is due to their common property of inhibiting PI 3-kinase instead of a shared nonspecific effect. Direct demonstration of a role for PI 3-kinase in the antiapoptotic signaling of IGF-1 has been provided with the expression of a dominant negative PI 3-kinase construct [10, 11]. Thus, IGF-1 activates a PI 3-kinase-dependent antiapoptotic pathway to permit cells to survive in the absence of trophic factors. Conflicting evidence that implicates MAP kinases as

survival signals to protect cells against apoptosis has been presented [46, 53, 54]. Inhibition of ERK activity by PD98059 blocks the protective capacity of IGF-1 in PC-12 cells [11], whereas IGF-1 antiapoptotic function remains intact under these conditions in Rat-1 fibroblasts [10]. The paradoxical ability of MAP kinases to protect from apoptosis in some cell lines and not in others indicates that cell type and the nature of inducers of apoptosis (e.g., serum deprivation, chemotherapeutic agents, UV irradiation) may define the antiapoptotic signaling pathway(s) used by any given survival factor. For these reasons, it was important to determine whether ERK activation participates in the protection by IGF-1 in CHO cells. The addition of PD98059, a selective inhibitor of MEK1 activation, had no detectable effect on IGF-1’s ability to delay apoptosis in these cells. In addition, our data demonstrate that IGF-1 failed to activate ERK, although 10% serum efficiently increased ERK activity. A possible interpretation for these findings is that the endogenously expressed IGF-1 receptor is inherently poorly coupled to the p21ras/ERK pathway in CHO cells. Our results corroborate earlier reports that supraphysiological amounts of insulin capable of binding to and activating the IGF-1 receptor failed to stimulate ERK despite activation of the PI 3-kinase/pp70 S6 kinase pathway in certain cell types [55, 56]. Thus, it can be concluded that apoptotic suppression mediated by the IGF-1 receptor is independent of ERK activation in CHO cells. We have considered the possibility that NER might play a determining part in the antiapoptotic function of IGF-1. Indeed, data from this laboratory have indicated that activation of the IGF-1 receptor enhances ERCC-1 [33] and XPD/ERCC3 expression (Fig. 5C). The XPB/ERCC3 helicase is one of at least six subunits that compose TFIIH. Both in transcription and in NER, TFIIH is thought to act by the local opening of the DNA helix where it is required for both the targeting of promoter-specific sequences and a number of other basal transcription factors [57] and the elimination of DNA lesions [15]. Our observation that mutant cell lines harboring defects in ERCC1 or XPB/ERCC3 gene products retained their responsiveness toward IGF-1 supports the notion that NER in general, and transcription-coupled repair in particular, plays only a minor role, if any, in IGF-1 antiapoptotic signaling. However, the possibility remains that other DNA lesion-repair mechanisms, including mismatch repair, O6-methyl guanine-DNA methyltransferase, and DNA glycosylases [58], may be important in determining cellular antiapoptotic capacity. Work along this line of research is ongoing in our laboratory. A recent study by Dudek et al. [59] indicated that IGF-1 is a potent activator of a downstream effector of PI 3-kinase, the serine/threonine protein kinase PKB (also termed c-Akt). This led the authors to

ANTIAPOPTOTIC ACTION OF IGF-1 AND DNA REPAIR

suggest that activation of this widely expressed kinase alone is sufficient to prevent apoptosis in cerebellar neurons upon growth factor withdrawal [59]. The ability of PKB to promote survival is likely to be dependent on its kinase activity [60]. As a consequence, phosphorylation of a specific subset of downstream effectors by PKB may result in inhibition of the activities of members of the caspase family of cysteine proteases [30, 61, 62], thereby preventing the degradation of chromosomal DNA. In support of this view, we observed that IGF-1 stimulated a marked reduction in the cleavage of PARP, a caspase-specific substrate. Furthermore, Teraoka et al. [63] reported that caspase cleaves the catalytic component of DNA-PK during apoptosis. DNA-PK is a homolog of the mammalian PI 3-kinase catalytic subunit that has been implicated in the doublestrand DNA break repair/recombination apparatus and whose activity is inhibited by wortmannin [64]. Taken together, these findings indicate that apoptosis may result from the cleavage of proteins essential for cellular repair which include DNA-PK and PARP [50, 65]. In summary, our work defines the signaling activities of the IGF-1 receptor which enable it to function as an inhibitor of apoptosis in CHO cells. In addition, this study provides evidence against NER playing a critical role in IGF-1 antiapoptotic function.

13.

We thank Dr. A. S. Balajee for fruitful discussions and comments on the manuscript. We also thank Lisa G. Adams and Sutapa Kole for their expert technical assistance.

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