Evidence for Functionally Significant Polymorphism of Human Glutamate Cysteine Ligase Catalytic Subunit: Association with Glutathione Levels and Drug Resistance in the National Cancer Institute Tumor Cell Line Panel

Anne C. Walsh*,{dagger},1, J. Amelia Feulner*,2 and Andrew Reilly{ddagger},§

* Laboratory of Clinical and Experimental Endocrinology and Immunology, Wadsworth Center, Albany, New York 12201; {dagger} Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York 12222; {ddagger} Laboratory of Structural Pathology, Wadsworth Center, Albany, New York 12201; and § Department of Biometry and Statistics, School of Public Health, State University of New York, Albany, New York 12222

Received November 20, 2000; accepted January 28, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutamate-cysteine ligase (GCL) is the first and rate-limiting enzyme involved in the biosynthesis of glutathione (GSH). The GCL heterodimer is encoded by two genes: GLCLC, which directs synthesis of the catalytic subunit, and GLCLR, which encodes the regulatory subunit. We have previously identified a polymorphic GAG/CTC trinucleotide repeat within the 5' untranslated region of GLCLC. Here we report the further characterization of GLCLC polymorphism and the existence of five GLCLC alleles as defined by the trinucleotide repeat, which exhibits a range of 4 to 10 uninterrupted repeats. Significant variation in GLCLC allele frequencies was observed in four different ethnic populations examined. Interindividual variation in the capacity to produce GSH due to GLCLC polymorphism is hypothesized to influence the cellular response to environmental toxicants and chemotherapeutic agents. To test this hypothesis, the 60 tumor cell lines of the National Cancer Institute drug screening panel were genotyped for the GLCLC trinucleotide repeat, and the association of GLCLC genotype with GSH levels and drug sensitivity/resistance data was examined. Here we demonstrate an association between certain GLCLC alleles and GSH levels and/or drug sensitivity, providing evidence that suggests polymorphism of human GLCLC is functionally significant.

Key Words: glutamate-cysteine ligase; GCL; GLCLC; polymorphism; trinucleotide repeat; glutathione; drug resistance.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Glutathione (GSH) is an important and versatile intracellular antioxidant. This ubiquitous tripeptide represents the major nonprotein thiol of animal cells. GSH plays a role in metabolism, amino acid transport, and protection of the cell from reactive molecules and toxic compounds of endogenous and exogenous origin (Meister, 1988Go). Biosynthesis of GSH is catalyzed by two enzymes. The first and rate-limiting enzyme, glutamate cysteine ligase (GCL, also called {gamma}-glutamylcysteine synthetase; E.C. 6.3.2.2), catalyzes the ligation of glutamate to cysteine (Griffith and Mulcahy, 1999Go). GSH synthetase (E.C. 6.3.2.3) then promotes the addition of glycine, yielding L-{gamma}-glutamyl-L-cysteinylglycine. In mammalian species studied, GCL is composed of two subunits. The larger 73 kDa subunit, encoded by the gene GLCLC, possesses all of the catalytic activity and is feedback inhibited by GSH (Seelig et al., 1984Go). The smaller 31 kDa subunit, encoded by the gene GLCLR, is thought to have a regulatory function (Huang et al., 1993Go).

We have previously reported identification of a polymorphic GAG trinucleotide repeat (TNR) present just upstream of the human GLCLC coding region (Walsh et al., 1996Go). Examination of a Caucasian population revealed three alleles with 7, 8, or 9 repeats. Multiple transcriptional start sites are used during expression of GLCLC (Mulcahy and Gipp, 1995Go). The GAG repeat, located just 10 base pairs 5' to the start codon, is included in each of the four major transcripts produced. Therefore, variation in the size of this repeat, in theory, could influence transcription rate, translation efficiency, and/or mRNA stability. Additionally, the GLCLC alleles, defined by the TNR, may possess additional and potentially significant nucleotide differences in regulatory sequences or within the coding region.

Given the role that differences in the ability to synthesize GSH might play in interindividual variability in cellular responses to environmental chemicals and drugs and in susceptibility to various diseases associated with oxidative stress, it is important to determine whether polymorphism of GLCLC is functionally significant. The studies reported here further evaluate polymorphism of this locus and assess the association of GLCLC genotype with GSH levels and drug resistance in the National Cancer Institute (NCI) drug screening panel of human tumor cell lines.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Determination of GLCLC TNR allele frequencies in four populations.
Randomly chosen anonymous blood spots obtained from the New York State Newborn Screening Program were used to assess the frequencies of the five GLCLC TNR alleles. The blood spots were processed to obtain template genomic DNA according to the method of Caggana et al. (1998) with the following modification. After methanol treatment, each blood spot was dried, resuspended in 25 µl of water and boiled 10 min. Five microliters of the eluate was then removed to a new tube for PCR. The remaining eluate with the blood spot was stored at –20°C. TNR sizes were determined using incorporation of 32P-labeled dCTP into PCR-amplified fragments spanning the TNR as previously described (Walsh et al., 1996Go).

Determination of GLCLC TNR allele frequencies in the 60 NCI tumor cell line drug screening panel.
Frozen cell pellets (1 x 107 cells) for each of the 60 cell lines were obtained from the NCI, and genomic DNA was isolated using a Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). GLCLC GAG trinucleotide repeat lengths were determined as described above. The genotype of each cell line was verified at least once.

Statistical analyses.
An omnibus chi-square test was used to test the association between GLCLC allele frequencies and the four ethnic groups studied. Fisher's exact test (Fisher, 1935Go) was used for pairwise comparison of the frequency of each allele in two different ethnic groups.

The effect of GLCLC genotype on GSH levels was assessed with classical least squares regression (Draper and Smith, 1998Go) in which the effect of each allele was partitioned into hetero- and homozygotic effects (Liu, 1998Go). This analysis neglects the effects of additional uncontrolled covariates, i.e., variable levels of other gene products, which may significantly influence GSH levels. To address this, a stepwise covariate selection procedure was performed using two publicly available databases on gene expression in the NCI cell lines. The NCI's Developmental Therapeutics Program (DTP) molecular target database (MTD) was downloaded from ftp://dtpsearch.ncifcrf.gov/web_hooks_prim.zip. A second data set, ftp://dtpsearch.ncifcrf.gov/web_hooks_gc.zip, containing RNA levels determined using cDNA microarray analysis was also downloaded from DTP. This molecular target microarray data (MTMD) was collected by the Brown and Botstein group at Stanford University (Ross et al., 2000Go; Scherf et al., 2000Go). With the GLCLC genotype included in the analysis, the additional contribution of each of 161 potential covariates (molecular targets) from the MTD were individually assessed for their contribution in explaining variation in GSH levels. The best contributor was then added to the analysis and the contributions to the new analysis of each of the remaining 160 potential covariates was again individually tested. The procedure was repeated until none of the set of remaining molecular targets made a significant contribution. Correlations between early and late entry covariates alter the significance of the former. A backwards elimination procedure, deleting the least significant covariates one at a time until only the most important contributors remain, was implemented to eliminate this effect. The entire stepwise procedure was repeated on the 9703 targets in the MTMD data set. Finally, the backwards elimination procedure was run a third time starting with all covariates from the MTD and MTMD analyses to settle on a joint set of covariates. Because not all of the molecular targets were evaluated in all of the 60 NCI cell lines, average values were imputed for missing values using the mean for each data set. The final set of covariates was selected on the basis of having the optimal number of investigated imputations (three for the MTD database and five for MTMD database). Comparison of GSH in GLCLC homozygotes was by the Mann-Whitney U-test.

The DTP Human Tumor Cell Line Screen for anticancer activity has produced data on the sensitivities of 60 tumor cell lines to a standard set of 169 commonly used chemotherapeutic agents (Monks et al., 1991Go). This publicly available agent sensitivity data, August 1999 release, was downloaded from http://dtp.nci.nih.gov at ftp://dtpsearch.ncifcrf.gov/gi50_a99.bin. The influence of GLCLC genotype on tumor cell sensitivity to the growth inhibitory effects of these agents was estimated by classical least squares regression.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GLCLC Allele Frequencies in Four Ethnic Populations
In our initial report of polymorphism of human GLCLC, we described three alleles defined by the length of a polymorphic GAG trinucleotide repeat upstream of the coding region and determined the frequency of these alleles in 50 unrelated Caucasians. GLCLC alleles 1, 2, and 3 have 9, 8, and 7 GAG repeats, respectively. We now report the existence of two additional alleles that define the limits of the size of the repeat, which has now been observed to vary between 4 (allele 5) and 10 (allele 4) repeats. The frequencies of the GLCLC alleles in four different ethnic populations are shown in Figure 1Go. Allele 3 is the wild-type allele in all four populations, with its frequency ranging from 47.5% in blacks to 64.5% in whites. Allele 1 is the second most common allele, with a frequency of 20.5% in whites to 42.5% in blacks. The frequency of allele 2 ranges from 1.5% in blacks to 15.0% in whites. Allele 4 was observed only in the black (7.0% frequency) and Hispanic (1.0% frequency) populations. Allele 5 was present only in the black population (1.5% frequency). Statistically significant differences in allele frequencies (p < 0.003) were observed for allele 1 between blacks or Hispanics and whites; for allele 2 between Asians or blacks and whites and between blacks and Hispanics; for allele 3 between blacks or Hispanics and whites and between Asians and blacks; and for allele 4 between blacks and Hispanics.



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FIG. 1. The GLCLC allele frequencies in four populations is shown. Trinucleotide repeat lengths were determined by performing PCR across the repeat using blood spot genomic DNA, incorporating 32P-dCTP, and running the products on a DNA sequencing gel along with a DNA sequencing ladder. DNA from 100 individuals (200 chromosomes) was analyzed for each population.

 
Association of GLCLC Genotype with GSH Levels in the NCI Cell Lines
To address the question of the functional significance of GLCLC polymorphism, we determined the GLCLC genotypes of a panel of cell lines well characterized with respect to their GSH levels and GLCLC transcript levels (Tew et al., 1996Go), and their susceptibility to various chemotherapeutic drugs (using the agent sensitivity data downloaded from http://dtp.nci.nih.gov, as described in Materials and Methods). The results of this genotyping of the NCI 60 cell line drug screening panel is available upon request. The four most prevalent alleles are represented in this population of cell lines; however only one cell line possesses a copy of allele 4. Therefore, no conclusions about the functional significance of the more infrequent alleles (4 and 5) can be made. Data on the ethnic origin of many of the tumor cell donors are unavailable, and so this influence on allele frequency is not considered here.

Using data reported by Tew et al. (1996), we assessed the association of GLCLC genotype with total cellular GSH levels. Only 59 cell lines were used in this analysis, as GSH levels were not reported for one cell line. An initial regression analysis of GSH levels on GLCLC alleles explains a significant amount of the observed GSH variability (31%, p = 0.003). Alleles 1 and 2 are significantly associated with higher GSH levels (p < 0.0009 and 0.0139, respectively). Interestingly, this influence of allele 2 appears to be reversed in the homozygous condition, with the 2,2 genotype significantly associated (p < 0.0089) with decreased levels of GSH. Similarly, total GSH levels in cells homozygous for allele 3 are significantly lower than GSH levels in 1,1 homozygotes (p < 0.0034). A calculation of the contributions of individual alleles with respect to total GSH levels is represented in Figure 2Go. All GSH data were simultaneously used to estimate haploid effects and a single variance. Homozygote GSH levels were modeled by appropriate additions of haploid effect estimates. Although only two cell lines homozygous for GLCLC allele 2 exist, the significant p-value results from their large departure from the haploid prediction, which is based on 118 alleles.



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FIG. 2. GLCLC genotype-specific GSH levels in 59 NCI cell lines with fit decomposed into haploid effects. The GSH level of each tumor cell is indicated by a diamond. The calculated contribution of allele 1 is indicated with a crosshatch fill, allele 2 with a darker gray fill, and allele 3 with a lighter gray fill. (There is only one data point for allele 4 and this is shown for completeness, but no interpretation of the contribution of allele 4 can be made based on a single observation.) For homozygous genotypes, the observed GSH level is indicated by an asterisk and the predicted (from the sum of the estimated individual allele's contribution) is indicated with an arrow. Total GSH levels in these cell types was determined by Tew et al. (1996) in cells from logarithmically growing cells. The mean GSH level for these 59 cell lines is 14.32 nmol/mg protein.

 
The association of GLCLC genotype with total GSH levels was investigated further by analysis of covariance. The influence of variation in gene expression of approximately 8000 genes from two publicly available databases was considered. Fifteen of these were found to significantly influence the association of GSH with genotype (p < 0.05). When the influence of the expression of the covariants listed in Table 1Go is accounted for, the explained GSH variability increases to 92.4%, and the significance of the association of GLCLC alleles 1 and 2 with elevated GSH is strengthened (p < 0.0001 for both), as is the association of 2,2 homozygosity with decreased GSH (p < 0.0001).


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TABLE 1 Covariants That Significantly Influence the Association between GLCLC Genotype and GSH Levels
 
We did not find any correlation between GLCLC genotype and GLCLC transcript levels measured in log phase tumor cells as reported by Tew et al. (1996).

Association of GLCLC Genotype with Drug Resistance in the NCI Cell Lines
We next asked whether GLCLC genotype was significant with respect to drug susceptibility/resistance. The NCI's DTP in vitro anticancer drug screen assesses the growth-inhibitory effects of test agents on a panel of 60 tumor cell lines derived from leukemias, melanomas, and cancers of the breast, CNS, colon, lung, kidney, ovary, and prostate (Monks et al., 1991Go). Analysis of drug sensitivity is measured as the GI50 (Skehan et al., 1990Go). Over 70,000 agents have been screened and among these is a set of "standard agents" that includes most of the drugs approved for clinical use as chemotherapeutic agents. A publicly available database that contains information on the inhibitory effects of 169 standard agents was used to determine whether GLCLC genotype influences drug susceptibility. Sensitivity to 26 different agents was significantly influenced by GLCLC genotype, with allele 2 affecting sensitivity to the largest number of agents (Table 2Go). In 169 regressions, the occurrence of 19 agents negatively associated with a single allele is significant (p < 0.0002). The association of GLCLC allele 2 with increased sensitivity to these agents did not correlate with any single putative drug mechanism of action.


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TABLE 2 Agents for Which Tumor Cell Susceptibility Is Influenced by GLCLC Allele 2 (p < 0.05)
 
Several genes encoding glutathione S-transferases of the alpha class, GSTA1, GSTA2, GSTA3, and GSTA4, are located along with GLCLC on chromosome 6p12. Therefore, the possibility of a GSTA/GLCLC haplotype effect that could influence drug resistance was considered. No correlation was observed between GLCLC genotype and the expression of GSTA2, GSTA3, or GSTA4 in these cell lines as reported in the NCI/Stanford database of gene expression assessed by cDNA microarray analysis (http://discover.nci.nih.gov) (Ross et al., 2000Go; Scherf et al., 2000Go). Similarly, no association between GLCLC genotype and GST{alpha} mRNA or GSTA1 protein expression as reported by Tew et al. (1996) was observed.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this report, polymorphism of human GLCLC is further characterized. Depending on the population studied, three to five alleles of GLCLC are observed. We have examined over 500 individuals with respect to their GLCLC genotype and have observed no evidence of expansion of the GAG repeat beyond the 10 repeats of allele 4. Interestingly, no alleles with 5 or 6 GAG repeats were identified. The existence of five GLCLC alleles, at the frequencies reported, indicates that these alleles have been established for a considerable period of time. Therefore, in addition to the possible influence of the TNR itself on GLCLC expression, other genetic differences in the regulatory and/or coding region of this gene may exist in GLCLC alleles defined by the GAG TNR length. There is no association between any of the five GLCLC TNR alleles and either of two recently described GLCLC point mutations which independently, in the homozygous condition, lead to GCL deficiency (Beutler et al., 1999; Ristoff et al., 2000; data not shown).

In an effort to determine whether the described polymorphism of GLCLC is functionally significant, the 60 tumor cell lines of the NCI drug screening panel were genotyped and the results analyzed with respect to existing data on GLCLC transcript levels, GSH levels, and drug sensitivity/resistance. These studies indicate that GLCLC alleles 1 and 2 are significantly associated with increased levels of total GSH in the proliferating tumor cells. In contrast, allele 2 in the homozygous condition is associated with decreased levels of GSH in the two cell lines with this genotype. Likewise, GLCLC allele 3 homozygotes have decreased levels of GSH relative to GLCLC allele 1 homozygotes. A reasonable hypothesis would be that an allele producing low levels of GCL catalytic subunit or producing a catalytic subunit with decreased stability or activity would have minimal effects in the heterozygous condition, where the second allele could compensate for the "inadequate" allele. In the homozygous condition, this inadequate allele would be associated with a deficit of GCL activity leading to decreased levels of GSH. However, no correlation between reported GLCLC transcript levels and genotype was observed. Thus, it is not simply that alleles associated with lower GSH levels express lower levels of GLCLC mRNA.

Variation in the levels of expression of 15 genes, as assessed by cDNA microarray analysis performed by the DTP and the Stanford group, is shown to significantly influence the association between GLCLC genotype and GSH levels (Table 1Go). Both positive effects, increased levels of the covariant being associated with higher levels of GSH, and negative effects, increased levels of the covariant being associated with lower levels of GSH, were observed. The association, in some cell lines, of higher levels of GLCLC mRNA with increased cellular GSH is understandable. However, correction for this influence, which may be related to non-GLCLC effects such as the level of a transcription factor, tightens the association between GLCLC genotype and GSH levels. On the other hand, high levels of mRNA encoding the regulatory subunit, GLCLR, is associated with lower GSH levels, but again, eliminating this influence of GLCLR transcript levels strengthens the association between GLCLC genotype and GSH levels. It is unclear at this time what the connection is between expression of the other 13 genes and GSH levels. Among these genes are five encoding tyrosine kinases and one encoding a tyrosine phosphatase, possibly implicating signaling pathways involved in regulation of GSH synthesis, turnover, or export. Interestingly, 6 of the 15 genes having an effect (EIF1A, PTP4A2, LCK, TIE, EST H15054, and GLCLR) map to a less than 100 centimorgan (cM ) region on chromosome 1 (according to the GB4 map, http://www.ncbi.nlm.nih.gov/genemap). Based on a total genome size of 3488 cM (Broman et al., 1998Go), the likelihood of this occurring by chance is remote (p = 0.0003). Therefore, this clustering of genes influencing GSH levels may have biological significance. This covariance analysis illustrates a novel use of gene expression data obtained using cDNA microarrays. The inclusion of such data in the evaluation of allelic effects can significantly increase the sensitivity of the analysis by accounting for the influence of covariants.

Tew et al. (1996) have previously reported an analysis of these tumor cell lines with respect to GSH levels, expression of several GSH-related enzymes, and drug sensitivity. Their findings indicated that expression of GLCLC transcripts in the proliferating but untreated tumor cells was positively correlated with resistance to alkylating agents. GSH levels in these cells, however, were not observed to significantly correlate with drug sensitivity. In accordance with these findings, our data show that the increase in GSH levels associated with alleles 1 and 2 in untreated cells does not translate to increased protection of cells possessing these alleles upon exposure to the standard agents. In fact, allele 2 is significantly associated with increased sensitivity to certain chemotherapeutics, i.e., the concentration of drug required to result in 50% growth inhibition is decreased in those cells expressing GLCLC allele 2 in either the heterozygous or homozygous form (Table 2Go). These observations likely reflect a difference in regulation of GSH synthesis in proliferating, untreated cells versus in cells responding to exposure to a chemotherapeutic agent. In the latter case, compensation by a second GLCLC allele may not be adequate. GSH may interfere with the drug–molecular target interaction by conjugation with the drug, by participation in export of the drug by the MRP family of transporters, or by influencing DNA repair (Schroder et al., 1996Go). In each case, the ability to replenish intracellular GSH would be required for a continued contribution to drug resistance. Therefore, induced expression of GLCLC and GCL, which is rate-limiting for GSH biosynthesis, may be critical in the cellular drug response. Induction of GLCLC and MRP1 in response to exposure of cells to a number of agents has been demonstrated, and recent studies have shown that overexpression of GLCLC can lead to decreased sensitivity to drugs (Gomi et al., 1997Go; Ishida et al., 1997Go; Ishikawa et al., 1996Go; Mulcahy et al., 1997Go; Yamane et al., 1998Go). The association of GLCLC allele 2 with increased sensitivity to a number of agents suggests that allele 2 is impaired in its ability to direct expression of the GCL catalytic subunit in response to exposure to certain chemotherapeutic agents. It will be important to specifically determine how GLCLC genotype influences induction of GLCLC and/or GCL activity in cells exposed to drugs or oxidative stress.

The association of GLCLC allele 2 with sensitivity to those agents listed in Table 2Go suggests a cis effect of GLCLC polymorphism as opposed to variation in the levels of some trans-activating factor involved in regulation of GLCLC synthesis. An alternative explanation is that some other polymorphic locus in close physical linkage with GLCLC (with each of its alleles inherited in tandem with a particular GLCLC allele) is producing the effect. Because genes encoding the alpha class of GSTs map to the same region of chromosome 6p12 as GLCLC, and because polymorphism of GST{alpha}-encoding genes has been suggested (van Dyke et al., 1991Go), we examined the association between GLCLC genotype and expression of GST{alpha} mRNA and GSTA1 protein. No correlation was observed between GLCLC genotype and expression of the GST{alpha} genes. Therefore we do not believe the association between drug sensitivity and GLCLC genotype can be explained by the variable expression of linked GST{alpha} genes.

To our knowledge, this is the first reported evidence of functionally significant polymorphism of GLCLC. We propose that certain allelic forms of GLCLC may influence GSH synthesis and interindividual variation in response to drug or toxin exposure, and therefore suggest that GLCLC be considered a candidate susceptibility gene. Given the hypothesized involvement of oxidative stress and associated GSH deficiency in such far-ranging disease processes as aging, macular degeneration, chronic obstructive pulmonary disease, HIV infection, neurodegenerative disorders, and cancer, further analysis of GLCLC polymorphism is warranted. Studies to determine the mechanistic bases of functionally relevant GLCLC polymorphism are ongoing in our laboratory. In epidemiology studies designed to test the association of alleles of GLCLC with susceptibility to cellular damage upon chemical exposure or with risk of disease, consideration should be given to the fact that GLCLC allele frequencies can vary significantly depending on the ethnic origin of the population studied.


    ACKNOWLEDGMENTS
 
The authors acknowledge use of the Wadsworth Center Biochemistry, Molecular Genetics, Molecular Immunology, and Computational Molecular Biology and Statistics Core Facilities. Blood spots were generously provided by Dr. Michelle Caggana of the Wadsworth Laboratory of Genetic Epidemiology. We acknowledge the contribution of the Public Health Service in the development of the 60 cell line screening panel and are grateful for their willingness to provide cells for analysis. This work was supported by the Wadsworth Center and grant R29 ES08962 from the National Institute of Environmental Health Sciences, National Institutes of Health. Its contents are solely the responsibility of the authors and do not necessarily reflect the official views of the NIEHS.


    NOTES
 
1 To whom correspondence should be addressed at Wadsworth Center, Empire State Plaza, PO Box 509, Albany, NY 12201-0509. Fax: (518) 473-2849. E-mail: walsh{at}wadsworth.org. Back

2 Current address: Southwestern Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390. Back


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