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Endocrinology Vol. 138, No. 5 1829-1835
Copyright © 1997 by The Endocrine Society

Articles

Overexpression and Activation of the Insulin Receptor Enhances Expression of ERCC-1 Messenger Ribonucleic Acid in Cultured Cells

Riccardo Perfetti, Whaseon Lee-Kwon, Chahrzad Montrose-Rafizadeh and Michel Bernier

Diabetes Section, Laboratory of Clinical Physiology, National Institute on Aging, Baltimore, Maryland 21224

Address all correspondence and requests for reprints to: Dr. Michel Bernier, Diabetes Section, Gerontology Research Center, National Institute on Aging, 4940 Eastern Avenue, Baltimore, Maryland 21224. E-mail: Bernierm{at}vax.grc.nia.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, a partial hamster complementary DNA encoding ERCC-1, a member of the DNA excision repair gene family, has been cloned. The nucleic acid and amino acid sequences were highly homologous to those of human and mouse ERCC-1. The hamster ERCC-1 gene was expressed as a 1.2-kilobase message in cultured Chinese hamster ovary cells. Northern (RNA) blot analysis revealed that overexpression of the insulin receptor or various growth factor receptor tyrosine kinases in Chinese hamster ovary cells increased ERCC-1 messenger RNA (mRNA) levels. This effect did not occur in cells overexpressing mutated insulin receptors that are known to have impaired kinase-related signaling. Increased ERCC-1 expression correlated with resistance to UV exposure. Fluorescent-activated cell sorter analysis of confluent cell populations indicated no differences in cell cycle distribution. Furthermore, no significant relationship was demonstrated between the relative expression of ERCC-1 mRNA and the rate of glucose utilization. Insulin enhanced the accumulation of ERCC-1 mRNA in serum-deprived cells expressing wild-type insulin receptors. The potential role for activation of the insulin receptor and related growth factor receptors in ERCC-1 gene expression and function remains to be defined.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH MUCH work has focused on the early events in tyrosine kinase signaling and its associated metabolic and mitogenic responses, little is known about a possible enhancement of DNA repair pathways triggered by receptor tyrosine kinases in mammalian cells. Within the last several years, studies have clarified some of the mechanisms involved in the activation and regulation of these receptor-linked kinases. Activation of the intrinsic tyrosine kinase activity of the insulin receptor has been shown to augment phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2, and Shc proteins, leading to the rapid and substantial activation of cytoplasmic signal transduction elements that are known to contribute to transcriptional regulation of several genes. These include a group of signaling proteins containing Src homology-2 domains, Ras, Raf-1, and mitogen-activated protein kinases (1). Like insulin, several growth factors stimulate many of the same signaling intermediates (2).

One of the major DNA repair pathways in live cells is the nucleotide excision repair system. It is a complex process by which lesions are located, a stretch of nucleotides containing the damage is excised, and a repair patch is synthesized (3). A subset of nucleotide excision repair, termed transcription-coupled repair (4, 5), involves the rapid removal of DNA lesions located in the transcribed strand of active genes, and the sharing of basal transcription factor TFIIH between DNA repair and class II gene transcription (3). Reconstitution experiments with purified recombinant proteins and cellular fractions have suggested that a set of proteins, including the TFIIH complex (containing XPD) and a dimeric complex composed of ERCC-1 and XPF, is essential for efficient DNA repair (6, 7). By virtue of its intrinsic helicase activity, XPD has a dual role in transcription and nucleotide excision repair in general, including transcription-coupled repair (8). ERCC-1/XPF complex has structure-specific nuclease activity (9), whose role in transcription has not been shown. It appears that higher expression of ERCC-1 messenger RNA (mRNA) in cancer tissue confers resistance to chemotherapy (10), whereas cells deficient in ERCC-1 expression have an increase in DNA damage after treatment with many chemical adducts and UV irradiation (11). Analysis of mice deficient in ERCC-1 has revealed an elevated level of the p53 protein in several tissues, presumably resulting from an accumulation of DNA lesions (12). Further, these mice are small in size and die of liver failure before weaning, implying that ERCC-1 might also be involved in normal development.

A number of functional domains on the insulin receptor have been identified and have provided the tool for structure/function studies of the receptor using overexpression of wild-type and mutant insulin receptors in transfected cell models (13). When Tyr at position 9601 from the juxtamembrane region of the ß-subunit is mutated to Phe, autophosphorylation and exogenous kinase activity are the same as those for wild-type receptor (14); however, these receptors display a markedly reduced ability to modulate most insulin-regulated biological functions (14, 15). Of interest is the fact that two naturally occurring splice variants of the insulin receptor have been shown to exist, resulting in two distinct insulin receptor species (16). The two receptor splice variants have distinct tissue-specific expression and are functionally different (Refs. 17 and 18 and references therein). Thus, it is conceivable that regulation of specific genes may be elicited differentially by these two receptor variants because of the differences in their biological properties.

The present study was undertaken to investigate the role of receptor-linked protein tyrosine kinase on the accumulation of ERCC-1 mRNA and compare the response of cells transfected with both isoforms of the wild-type insulin receptor with that of signaling-deficient insulin receptor mutants. We provide evidence that expression of wild-type insulin receptors and their activation by insulin may regulate ERCC-1 mRNA levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
The Chinese hamster ovary (CHO) cell lines used in this study have been previously described (14, 19, 20, 21). These include the CHO cell lines stably cotransfected with a plasmid containing the neomycin resistance gene driven by simian virus 40 promoter and an expression plasmid encoding the A or B isoform of the human insulin receptor [exon 11+ (CHO/T); exon 11- (CHO/HIRc)] as well as CHO cell lines expressing mutated forms of the receptor. The mutant insulin receptors were as followed: receptor with a triple tyrosine to phenylalanine mutation in the autocatalytic domain (CHO/YF3) and receptor with a single tyrosine to phenylalanine mutation at position 960 near the juxtamembrane domain (CHO/F960). CHO/HIRc and CHO/F960 cells were gifts from Dr. Morris F. White (Joslin Diabetes Center, Boston, MA), CHO/T and CHO/YF3 cells were gifts from Dr. Richard A. Roth (Stanford University, Stanford, CA), CHO cells expressing large numbers of epidermal growth factor (EGF) receptors were gifts from Dr. Roger J. Davis (University of Massachusetts, Worcester, MA), and CHO cells overexpressing insulin-like growth factor I (IGF-I) receptors from Dr. Derek LeRoith (NIH, Bethesda, MD). CHO cells transfected with the neomycin resistance gene (CHO/neo and CHO/neoR) were used as the control. 3T3-L1 preadipocytes (American Type Culture Collection, Rockville, MD) were induced to differentiate into adipocytes essentially as previously described (22). The cells were grown on tissue culture plates in Ham’s F-12 medium (CHO cells lines) or DMEM (3T3-L1 adipocytes) containing 10% FBS and maintained in a humidified incubator with 5% CO2 at 37 C. The approximate number of the insulin, EGF, or IGF-I receptors in each cell line was determined by Scatchard analysis (19). All clones expressed between 140,000–350,000 receptors/cell, whereas control CHO/neo cells had about 20 times fewer receptors.

In some instances, CHO/HIRc cells and 3T3-L1 adipocytes were incubated for 3 h in culture medium without serum, followed by a 24-h incubation in the presence or absence of 100 nM insulin.

Oligonucleotide primers and probes
The synthesis of oligonucleotides was performed on an Applied Biosystems DNA synthesizer (Foster City, CA). Oligonucleotides were cleaved from the resin with concentrated ammonium hydroxide at room temperature and deprotected by heating to 55 C in ammonium hydroxide overnight. After purification with Nensorb Prep columns (New England Nuclear, Boston, MA), oligonucleotide(deoxythymidine)20 [oligo(dt)20; 5'-GCTGGATCCGATGGATCCTGCAGAAGCTTTTTTTTT-TTTTTTTT-3'] was used in the first step of the reverse transcription-PCR (RT-PCR) to obtain an ERCC-1 complementary DNA (cDNA) probe from CHO cells. ERCC-1 sense oligonucleotide primer was designed according to the published mouse cDNA sequence (5'-GCTCGAATTCTGTGCTGCTGGTTCAAGTGG-3') (23), whereas oligo(dt)20 acted as a downstream primer. An additional mouse oligonucleotide internal primer was synthesized (5'-GAAAAGCTGGAGCAGAACTTC-3') to be used as a probe for Southern blot analysis. Finally, a 24-base oligonucleotide (5'-ACGGTATCTGATCGTCTTCGAACC-3') complementary to 18S RNA was also synthesized.

RT-PCR and Southern blot analysis
Total cellular RNA from confluent cells was subjected to RT by using oligo(dt)20. RT was accomplished at 37 C in a final volume of 20 µl containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3; 25 C), 1.5 mM MgCl2, 0.01 mg/ml gelatin, 0.2 mM each of the deoxy (d)-NTPs, 40 U/tube RNasin (Promega, Madison, WI), 0.5 mM gene-specific downstream primer, and 7 U/tube avian myeloblastosis virus reverse transcriptase (Promega). The second strand was synthesized during the first cycle of PCR in which 5 µl of the RT reaction mixture (described above) were used in a final volume of 50 µl containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3; 25 C), 1.5 mM MgCl2, 0.01 mg/ml gelatin, 0.2 mM each of the dNTPs, 0.5 µM mouse ERCC-1 upstream primer, 0.5 µM oligo(dt)20 downstream primer, 1 U/tube Taq polymerase (Perkin-Elmer/Cetus, Norwalk, CT), and approximately 50 µl paraffin oil. Thirty-nine cycles of PCR were performed. Each cycle consisted of denaturation (1 min at 94 C), annealing (1 min at 42 C), and extension (1 min at 72 C), except for the first cycle, during which the denaturation time was increased to 5 min, and the last cycle, in which the extension time was increased to 10 min. RT-PCR products were subjected to electrophoresis on a composite gel consisting of 1% agarose (BRL, Gaithersburg, MD) and 2% Nusieve (FMC Bioproducts, Rockland, ME). The gel was then stained with 0.2 µg/ml ethidium bromide and photographed to verify that the amplified product corresponded to the predicted size.

Transfer of RT-PCR products onto a nitrocellulose membrane was performed according to the method of Sambrook et al. (24). The nitrocellulose was prehybridized at 37 C in the presence of 5 x SSPE, 5 x Denhardt’s solution, 1% SDS, and 100 µg/ml salmon sperm DNA. The membrane was then hybridized with 0.5–1 x 106 cpm 32P-radiolabeled oligonucleotide mouse ERCC-1 internal oligonucleotide probe. After an overnight incubation at 37 C, the blot was washed twice in 2 x SSPE-0.05% SDS for 15 min at 45 C, and autoradiography was performed by exposing the membrane to Kodak X-Omat films (Eastman Kodak, Rochester, NY) overnight at -80 C. Quantification of the autoradiograms was performed using ImageQuant software (version 3.3) on a Molecular Dynamic densitometer (Sunnyvale, CA).

Subcloning and DNA sequencing
A PCR-based cloning strategy was used to obtain a partial hamster ERCC-1 cDNA sequence.2 Hamster ERCC-1 PCR product was subcloned into the EcoRI and XbaI sites of Bluescript vector (Stratagene, La Jolla, CA) by direct ligation. Plasmid DNA was purified using the Q-Kiegen kit (Qiagen Inc., Chatsworth, CA). Purified plasmid DNA (1–3 µg) was subjected to DNA sequence analysis for both strands of DNA using a sequencing kit (U.S. Biochemical Corp., Cleveland, OH). Three different clones derived from ligation of different hamster ERCC-1 PCR products into the vectors were sequenced; they all confirmed the initial nucleotide sequence.

RNA isolation and Northern blot analysis
Total cellular RNA was extracted by homogenizing confluent cells in guanidinium isothiocyanate, followed by ultracentrifugation on a 5.7-M cesium chloride cushion (25). RNA was quantified spectrophotometrically at 260 nm. For Northern blot analysis, about 15 µg of total RNA of each sample were heated in the presence of 17% formaldehyde and 50% formamide to 65 C for 15 min and then applied to a 1.2% agarose gel containing 17% (vol/vol) formaldehyde. Ethidium bromide staining helped to assess the integrity of the RNA and to estimate loading efficiencies. RNA was transferred from the gel onto nylon membrane by capillary action [Schleicher and Schuell (Keene, NH) or Micron Separation, Westboro, MA] and fixed by baking in a vacuum oven at 80 C for 1.5 h. The Northern blots were hybridized with 1) a 400-bp hamster ERCC-1 cDNA probe, 2) an 800-bp PstI fragment of mouse ß-actin cDNA probe (gift from Dr. J. D. Gerhardt, Johns Hopkins University, Baltimore, MD), and 3) an 18S ribosomal subunit oligonucleotide probe (26). ERCC-1 and ß-actin probes were labeled with [32P]dCTP (Amersham) by the random priming procedure using either Sequenase (U.S. Biochemical Corp.) or Klenow fragments of DNA polymerase (BRL), whereas 18S probe was labeled with [32P]dATP (Amersham) by end labeling using polynucleotide kinase (BRL).

ERCC-1 hybridization was carried out at 42 C overnight in a solution of 50% (vol/vol) formamide, 0.1% SDS, 2.5 x Denhardt’s solution, and 5 x SSC (standard saline citrate) after prehybridization at 42 C for about 2 h. The membranes were then washed twice in 2 x SSC for 5 min at room temperature, followed by another two 30-min washes in 0.2 x SSC-0.5% SDS at 42 C. Blots were subsequently exposed to Kodak film at -80 C with intensifying screens. After quantitation, the blots were washed twice with 0.1% SDS at 80 C to remove the radiolabeled probes and to rehybridize the same membranes with the ß-actin probe. ß-Actin hybridization was carried out in the same buffer as that used for ERCC-1 hybridization, except that the final washes were performed twice for 30 min with 0.1 x SSC-0.5% SDS at 65 C. The blots were stripped again and rehybridized with an oligonucleotide specific for 18S RNA. The hybridization with 18S probe was carried out at 37 C overnight in 2.5 x Denhardt’s solution, 1% SDS, and 2.5 x SSPE, and the membranes were washed twice for 30 min each time with 2 x SSPE-0.5% SDS at 42 C. The ERCC-1 value in each lane was corrected by dividing by the 18S RNA value in the same lane.

Receptor tyrosine kinase activity in semipermeabilized cells
The determination of in vitro kinase activity in semipermeabilized cells was carried out as recently described (27).

Glucose measurements
CHO cells were grown to confluence, and the medium was replaced with Ham’s F-12 medium containing 10% FBS. Aliquots of medium were taken at regular intervals during the course of the experiments and stored at 4 C until analysis. Glucose levels were analyzed with a Glucose Analyzer II (Beckman, Palo Alto, CA).

UV survival
Exponentially growing cultures were trypsinized, plated on 60-mm dishes (2 x 105 cells/dish), and incubated overnight at 37 C. Cells were subsequently rinsed twice with PBS, incubated with 0.5 ml PBS, and then exposed to UV light (0–30 J/m2). After culture in Ham’s F-12 medium containing 10% FBS for 3 days, cells were trypsinized and counted using a Coulter counter (Hialeah, FL). For each UV dose, two dishes were counted in triplicate.

Cell cycle analysis
Confluent cell cultures were trypsinized, harvested, and washed once with PBS containing 2% FBS (wash buffer). Cells were resuspended in 100 µl wash buffer, fixed by adding 200 µl absolute ethanol while vortexing, and stored for at least 1 h at 4 C. After a series of washes, the cellular DNA was stained using 1 µg/ml 4',6-diamidino-2-phenylindole (Molecular Probes, Eugene, OR) in PBS containing 0.1% Triton X-100. Cells were incubated at 37 C for 15 min, and their DNA contents were analyzed on a FACStar Plus flow cytometer (Becton Dickinson, San Jose, CA). Excitation of stained cells was performed using a krypton laser with UV optics (350–356 nm). Emission signals were collected with a 424 bandpass filter. A total of 10,000 cells from each cell line were analyzed, and cell cycle analysis was performed using Multicycle software (Phoenix Flow Systems, San Diego, CA).

Statistical analysis
Student’s unpaired t test and Scheffe’s S ANOVA were used for the statistical analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of a partial hamster ERCC-1 cDNA
Total RNA from CHO/HIRc cells was prepared and subjected to RT-PCR, and the amplified products were separated by electrophoresis on agarose gel. Ethidium bromide staining of the gel showed the amplification of a 550-bp product, similar in size to the corresponding mouse ERCC-1 cDNA (data not shown). As for the mouse cDNA, digestion of the partial hamster cDNA with the restriction enzyme BamHI resulted in two DNA fragments of 200 and 300 bp. Furthermore, Southern blot analysis with a mouse internal oligonucleotide probe confirmed the specificity of the PCR-amplified product (data not shown). The sequence of this hamster 550-bp PCR product was established after its subcloning into Bluescript vector and DNA sequence analysis. The nucleotide sequence is given in Fig. 1AGo. The alignment of the deduced amino acid sequence of this amplification product with the carboxyl-terminal domain of previously reported mouse and human ERCC-1 is shown in Fig. 1BGo. It appears that the deduced amino acid sequence represents the hamster homolog of the mouse ERCC-1 mRNA. The overall homology between the hamster and human protein fragment is 91.3%, whereas more than 96% homology is found between the hamster and mouse ERCC-1 fragment.



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  		Figure 1. A, Partial nucleotide sequence of the hamster ERCC-1 cDNA. The polyadenylation signal AATAAA is underlined, and the TGA stop codon is in boldface. B, Alignment of hamster ERCC-1 amino acids (in one-letter code) with its mouse (m) and human (h) counterparts. From the hamster protein fragment, only differences from mouse and human ERCC-1 are depicted. The mouse and human ERCC-1 amino acid sequences and numbering on the right were described previously (23).

 
Enhanced expression of ERCC-1 mRNA levels in cells overexpressing functional receptor tyrosine kinases
CHO/neo cells and CHO cells expressing large numbers of human insulin receptors and receptors for EGF and IGF-I were maintained and grown to confluence in Ham’s F-12 medium in the presence of 10% FBS. Total RNA was prepared and analyzed by Northern blotting (Fig. 2Go, left panel), and mRNA levels for ERCC-1 were quantified (Fig. 2Go, right panel).



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  		Figure 2. ERCC-1 mRNA levels in CHO cells transfected with various tyrosine kinase receptors. CHO cells stably transfected with plasmids encoding the human insulin receptors (HIRc) and receptors for IGF-I or EGF were grown to confluence and maintained in medium containing 10% serum. Total RNA (15 µg) was analyzed by Northern hybridization for the presence of ERCC-1 and ß-actin mRNAs. Quantitative analysis is presented after normalization of the blots with an oligonucleotide specific for 18S RNA (right panel). Bars represent the mean ± SE of three to eight independent analysis. ***, P < 0.001; **, P < 0.01 (compared with CHO/neo cells).

 
ERCC-1 mRNA was increased nearly 3-fold in CHO/HIRc cells compared to that in control CHO/neo cells (P < 0.001; n = 8). Similarly, statistically significant 2.1- and 2.8-fold increases in the amount of ERCC-1 mRNA were observed in CHO cells overexpressing either the human IGF-I receptor or the EGF receptor (P < 0.001; n = 3). Hybridization of the same membranes with a ß-actin cDNA probe did not reveal any substantial difference among the various cell lines. That the ß-actin expression pattern did not change in cells overexpressing receptor-linked tyrosine kinases indicates that the changes in ERCC-1 expression are specific and not the result of a general mitogenic activation of the cells by receptor overexpression.

To investigate the contribution of insulin receptor kinase function in the expression of ERCC-1, we assessed the content of ERCC-1 mRNA in cells transfected with the exon 11- (HIRc) or exon 11+ (T) variant of the insulin receptor or with two signaling-deficient insulin receptor mutants, including a triple mutation in the tyrosine kinase core (YF3) and a point mutation within the juxtamembrane region of the ß-subunit (F960). Like CHO/HIRc cells, CHO/T cells showed enhanced levels of ERCC-1 mRNA (P < 0.05; n = 3; Fig. 3Go). In contrast, the abundance of ERCC-1 mRNA in cells expressing YF3 or F960 insulin receptor mutants was comparable to that in control CHO/neo cells. Our results indicate that intact tyrosine kinase activity is required for enhanced expression of the ERCC-1 gene. It is important to mention that the presence of 10% serum in the culture medium caused a constitutive activation of the insulin receptor kinase (data not shown). Therefore, the accumulation of ERCC-1 mRNA in CHO/HIRc and CHO/T cells was not the result of basal activation of the wild-type receptors, but, rather, was the product of a constitutive, yet significant, receptor kinase activity.



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  		Figure 3. Relative abundance of ERCC-1 mRNA in cells expressing various isoforms of the wild-type insulin receptors or receptor mutants. Northern blot analysis was performed on total RNA from confluent CHO cell lines maintained in culture medium with 10% serum. Quantitative analysis is presented after normalization of the blots with 18S probe. Bars represent the mean ± SE of two or three independent analyses from two different RNA preparations. **, P < 0.01; *, P < 0.05 [compared with CHO/neo(R) cells].

 
Overexpression of ERCC-1 affects cell killing by UV
Higher expression of DNA repair proteins correlates with increased resistance to DNA-damaging agents (28). Therefore, we examined whether the expression level of ERCC-1 mRNA correlates with cell survival after UV irradiation. CHO cells expressing wild-type and mutated insulin receptors were plated at low density and exposed to increasing doses of UV from 0–30 J/m2. Three days later, cell survival was determined in subconfluent cultures maintained in medium with 10% serum.

Under these conditions, there was a progressive decline in the number of CHO/neo cells in response to increasing doses of UV (Fig. 4AGo). An increase in the threshold level, as indicated by a shift to the right in the dose-response curve, was observed for CHO/HIRc cells. The dose-dependent UV effect on cell survival was very reproducible, with an IC50 of 13.05 ± 0.26 vs. 9.36 ± 0.90 J/m2 for CHO/HIRc cells vs. CHO/neo cells, respectively (P < 0.01; n = 3 independent experiments). To further document the relationship between ERCC-1 expression and cell survival, we evaluated the effect of UV irradiation on transfected cells expressing large number of exon 11- or exon 11+ isoforms of the wild-type insulin receptors and the YF3 or F960 mutant receptors. Similar to our results with CHO/HIRc cells, CHO/T cells survived better than CHO/neoR cells; however, cells overexpressing signaling-deficient receptor mutants showed sensitivity to UV comparable to that of control CHO/neo cells (data not shown). A significant correlation between the survival index (IC50) and the relative expression of ERCC-1 mRNA in various cell lines used in this study was obtained (Fig. 4BGo).



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  		Figure 4. Cell survival after UV treatment. Exponentially growing cells were irradiated with a range of UV intensities (0–30 J/m2) and allowed to recover for the next 3 days. A, The data plotted are expressed as the percentage of cells remaining 3 days after irradiation and represent the mean ± SD of a representative experiment that was repeated four times with comparable results. {circ}, Control CHO/neo cells; •, CHO/HIRc cells. B, Correlation between survival index (IC50) and cellular content of ERCC-1 mRNA among CHO cells transfected with either the wild-type or mutant insulin receptors. Each cell line is indicated by a data point that represents the mean ± SE from three or four separate experiments. Statistical analysis was performed by ANOVA.

 
Rate of glucose utilization
The production of free radicals and other reactive intermediates resulting from the metabolism of glucose significantly increases oxidatively damaged DNA and alters DNA repair (29). Therefore, we determined whether the expression of ERCC-1 mRNA was associated with higher glucose consumption in CHO/HIRc cells. Fresh medium containing 10 mM glucose was added to confluent CHO/neo and CHO/HIRc cells at the start of the experiment, and thereafter, samples of medium were taken at regular intervals for glucose measurements. In both cell lines, the rate of glucose disposal was slow (initial rate, 0.3 mM/h), with less than 50% of the glucose consumed within 24 h. We also performed experiments using CHO/T, CHO/YF3, and CHO/F960 cells and obtained similar results (data not shown).

Cell cycle analysis
To explore the possibility of a difference in cell cycle distribution between confluent populations of CHO/neo and CHO/HIRc cells, fluorescent-activated cell sorter analysis was performed (Table 1Go). Only marginal differences in cell cycle distribution were observed between the two cell lines. These results are in agreement with those of Petersen et al. (30), showing no change in the expression of repair enzymes during the cell cycle.


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  		Table 1. Cell cycle distribution of CHO/neo and CHO/HIRc cells

 
Effect of insulin on ERCC-1 mRNA levels
To evaluate the effect of insulin on ERCC-1 mRNA levels, the cells must first be incubated in the presence of medium without serum for 3–4 h before the addition of 100 nM insulin for 24 h. Total RNA (15 µg) was analyzed by Northern blotting, and mRNA levels for ERCC-1 were quantified (Fig. 5Go). Although a response in terms of insulin stimulation did occur in CHO/neo cells, the overexpression of insulin receptors allowed CHO/HIRc cells to respond with higher magnitude. CHO/HIRc cells produced a 2.6 ± 0.4-fold increase in ERCC-1 mRNA levels in response to a maximally stimulating dose of insulin compared to unstimulated cells (P < 0.001; n = 5). To assess the physiological relevance of this observation, the ability of 3T3-L1 adipocytes to exhibit increased expression of ERCC-1 mRNA by insulin was determined. Treatment with insulin for 24 h resulted in a ~2-fold increase in the levels of ERCC-1 mRNA compared to those in untreated adipocytes (data not shown).



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  		Figure 5. Regulation of ERCC-1 mRNA levels by insulin. Confluent CHO/neo and CHO/HIRc cells were incubated in serum-free medium for 3 h and then stimulated with (+) or without (-) 100 nM insulin for 24 h. Total RNA was extracted, and mRNA levels for ERCC-1 were quantitated by Northern blot analysis. The data are presented relative to unstimulated (-) CHO/neo cells and represent the average ± SE from five independent experiments. ***, P < 0.001 compared with cells incubated without insulin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Overexpression of wild-type insulin receptor, IGF-I receptor, and EGF receptor increases ERCC-1 mRNA levels, indicating that the mere presence of receptor-linked tyrosine kinases is responsible for this effect. Northern blot analysis of control CHO/neo cells reveals constitutive expression of ERCC-1 mRNA, which suggests a role for ERCC-1 in the repair of oxidative damage to DNA that may occur in all cells under normal physiological conditions (31). The addition of insulin to serum-depleted cells induces ERCC-1 mRNA expression to levels higher than those seen in cells without insulin stimulation. The fact that this insulin effect is also observed in an adipocyte cell line indicates the possible physiological relevance of the correlation between ERCC-1 mRNA levels and insulin receptor activation. Identification of the molecular mechanisms leading to enhanced expression of ERCC-1 mRNA (e.g. lowering of mRNA degradation and/or stimulation of gene transcription) requires further investigation.

This study provides novel data demonstrating a correlation between insulin receptor-mediated expression of ERCC-1 mRNA and resistance to UV-induced DNA damage in the cells. These results are in agreement with recent findings showing the involvement of receptor-linked tyrosine kinase in drug-induced DNA repair (32, 33) and in regulation of the response of mammalian cells to UV-induced cellular damage (34, 35). Previous studies have indicated that the resistance to cytotoxic agents seen in various tumor specimens is correlated with a 3- to 5-fold increase in ERCC-1 gene expression (36, 37). Whether the 2.5-fold increase in ERCC-1 levels reported here is meaningful will require some measure of in vitro DNA repair. Diabetes mellitus is characterized by chronic hyperglycemia resulting in the formation of reactive oxygen species that injure DNA (29). The marked impairment of insulin action during diabetes may contribute to oxidatively damaged DNA due to a defect in the synthesis of DNA repair enzymes. The importance of insulin receptor-mediated activation of ERCC-1 expression is uncertain and remains to be investigated.

Processing of DNA damage by the nucleotide excision repair pathway in eukaryotic cells is accomplished by multiprotein complexes (38). Therefore, it is conceivable that activation of receptor-linked tyrosine kinases may result in altered expression not only of ERCC-1 gene, but possibly other repair factors as well. Perturbation in the ratio between ERCC-1 and XPD, which have excision and helicase functions of the nucleotide excision repair, respectively, has been proposed to be a molecular characteristic of malignancy (39). Analysis of mRNA levels of ERCC-1 and XPD in glial tumors shows abnormalities in copy number for these genes (40). Interestingly, our preliminary experiments indicate increased XPD expression as a result of insulin stimulation of CHO/HIRc cells,3 suggesting a coordinate regulation of several components of DNA repair.

This study reports on the cloning of the carboxyl-terminal portion of the hamster ERCC-1 cDNA. Sequence analysis of genomic clones indicate that the hamster ERCC-1 gene is similar to the mouse and human genes. Comparison of hamster, mouse, and human amino acid sequences shows extensive homology within this region, indicating the importance of the carboxyl-terminal domain of ERCC-1 for its repair function. This conserved region appears to play a critical role in the interaction between ERCC-1 and XPA (41), a step required during processing by the nucleotide excision repair system (42, 43). Interestingly, studies of UV-sensitive yeast RAD mutants have revealed strong conservation of the components of nucleotide excision repair in eukaryotes. Rad 10 protein (ERCC-1 homolog in yeast) is needed for damage-specific incision during nucleotide excision repair and is also required for mitotic recombination events (44). In addition to its role in nucleotide excision repair, ERCC-1 might participate in recombinational repair pathway in mammalian cells (45, 46).

This report shows that overexpression of human insulin receptors with or without exon 11 is associated with increased accumulation of ERCC-1 mRNA. However, the comparison of the relative effectiveness of the two isoforms requires further study due to the limited number of cell clones overexpressing each of the receptor isoforms used. The signaling pathway(s) by which insulin affects ERCC-1 mRNA levels must still be resolved. The data presented here with an F960 insulin receptor mutant or kinase-impaired mutant indicate that activation of tyrosine kinase activity of the insulin receptor is required, but not sufficient, to induce this response. The importance of the juxtamembrane region of the insulin receptor ß-subunit has been shown where direct binding of adaptor molecules (e.g. IRS-1 and Shc) to this receptor domain provides a link to a variety of downstream signaling events (47, 48). It is likely that the response of activated IGF-I receptor or EGF receptor parallels that of the insulin receptor in this respect (2).


   Acknowledgments
 
We are grateful to Lisa G. Adams for expert technical assistance, and to Francis J. Chrest for assistance with the flow cytometric analysis. The critical reading of the manuscript by Drs. Vihelm A. Bohr and David Orren is acknowledged.


   Footnotes
 
1 According to exon 11 isoform. Back

2 The GenBank accession number for the partial hamster ERCC-1 cDNA sequence is U74491. Back

3 Perfetti, R., W. Lee-Kwon, and M. Bernier, unpublished results. Back

Received October 25, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. White MF, Kahn CR 1994 The insulin signalling system. J Biol Chem 269:1–4[Free Full Text]
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L. Heron-Milhavet and D. LeRoith
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