XPD mRNA Research Articles

NOP2-mediated m5C methylation of XPD is associated with hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is a common malignant tumor with high mortality. Our previous study has confirmed that XPD acts as an anti-oncogene and is downregulated in HCC. The mechanism of XPD downregulation in HCC is unclear. In this work, we obtained the datasets related to HCC patients from GSE76427, LIRI-JP, and TCGA-LIHC cohorts. Among 15 m5C regulators (NSUN2, NSUN3, NSUN4, NSUN5, NSUN6, NSUN7, DNMT1, TRDMT1, DNMT3A, DNMT3B and NOP2, TET1, TET2, and TET3, ALYREF), 14 m5C regulators were upregulated in tumor tissues of HCC patients, except for TET2. HCC patients were divided into Cluster A and B with different m5C methylation patterns. Cluster B was enriched in metabolism-related signaling pathways, and Cluster A was prominently associated with the cell cycle signaling pathway. Moreover, XPD was positively correlated with NOP2. Cluster B exhibited upregulation of XPD and had an obvious survival advantage with respect to Cluster A. Additionally, NOP2 and XPD were downregulated in HCC tumors and cells. In vitro assays revealed that NOP2 overexpression enhanced XPD expression by elevating the m5C methylation of XPD, which contributed to inhibit proliferation, migration, and invasion of HCC cells. In conclusion, this work demonstrated that XPD mRNA stability was elevated by NOP2-mediated m5C methylation modification and then inhibited the malignant progression of HCC, suggesting that XPD may be a potential target for HCC treatment.

Imbalance in DNA repair machinery is associated with BRAFV600E mutation and tumor aggressiveness in papillary thyroid carcinoma.

The involvement of alterations in MLH1, an essential mismatch repair component, in BRAFV600E mutated papillary thyroid carcinoma (PTC) has been suggested to be associated with features of tumor aggressiveness. Thirty-two PTC and surrounding normal thyroid tissues were evaluated for 11 representative DNA repair genes expression. BRAFV600E mutational status assessment and clinicopathological correlations were evaluated for their gene and protein expression. BRAFV600E PTC is associated with lower levels of XPD and MLH1 gene expression. Decrease in MLH1 and XPD mRNA levels in BRAFV600E PTC (but not their protein products) are associated with predictors of poor patient outcomes. Considering the complete subset of patients, MGMT and XRCC2 genes were shown down and upregulated, respectively, in PTC tissues. Low expression of MGMT gene and weak XRCC2 protein expression were correlated with characteristics of tumor aggressiveness. These results suggest that an imbalance in DNA repair gene expression in PTC is associated with aggressive clinicopathological features and BRAFV600E mutation.

Xeroderma pigmentosum, complementation group D expression in H1299 lung cancer cells following benzo[a]pyrene exposure as well as in head and neck cancer patients

ABSTRACTDNA repair genes play critical roles in response to carcinogen-induced and anticancer therapy-induced DNA damage. Benzo[a]pyrene (BaP), the most carcinogenic polycyclic aromatic hydrocarbon (PAH), is classified as a group 1 carcinogen by International Agency for Research on Cancer. The aims of this study were to (1) evaluate the effects of BaP on DNA repair activity and expression of DNA repair genes in vitro and (2) examine the role of xeroderma pigmentosum, complementation group D (XPD) mRNA expression in human head and neck cancers. Host cell reactivation assay showed that BaP inhibited nucleotide excision repair in H1299 lung cancer cells. DNA repair through the non-homologous end-joining pathway was not affected by BaP. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blot demonstrated that XPD was downregulated by BaP treatment. BaP exposure did not apparently affect expression of another 11 DNA repair genes. BaP treatment increased the DNA damage marker γ-H2AX and ultraviolet (UV) sensitivity, supporting an impairment of DNA repair in BaP-treated cells. XPD expression was also examined by quantitative RT-PCR in 68 head and neck cancers, and a lower XPD mRNA level was found in smokers’ cancer specimens. Importantly, reduced XPD expression was correlated with patient 5-year overall survival rate (35 vs. 56%) and was an independent prognostic factor (hazard ratio: 2.27). Data demonstrated that XPD downregulation was correlated with BaP exposure and human head and neck cancer survival.

Functional and molecular genetic analyses of nine newly identified XPD‐deficient patients reveal a novel mutation resulting in TTD as well as in XP/CS complex phenotypes

The xeroderma pigmentosum (XP) group D protein is involved in nucleotide excision repair (NER) as well as in basal transcription. Determined by the type of XPD mutation, six different clinical entities have been distinguished: XP, XP with neurological symptoms, trichothiodystrophy (TTD), XP⁄TTD complex, XP⁄Cockayne syndrome (CS) complex or the cerebro-oculo-facio-skeletal syndrome (COFS). We identified nine new XPD-deficient patients. Their fibroblasts showed reduced post-UV cell survival, reduced NER capacity, normal XPD mRNA expression and partly reduced XPD protein expression. Six patients exhibited a XP phenotype in accordance with established XP-causing mutations (c.2079G>A, p.R683Q; c.2078G>T, p.R683W; c.1833G>T, p.R601L; c.1878G>C, p.R616P; c.1878G>A, p.R616Q). One TTD patient was homozygous for the known TTD-causing mutation p.R722W (c.2195C>T). Two patients were compound heterozygous for a TTD-causing mutation (c.366G>A, p.R112H) and a novel p.D681H (c.2072G>C) amino acid exchange, but exhibited different TTD and XP/CS complex phenotypes, respectively. Interestingly, the XP/CS patient's cells exhibited a reduced but well detectable XPD protein expression compared with hardly detectable XPD expression of the TTD patient's cells. Same mutations with different clinical outcomes in NER-defective patients demonstrate the complexity of phenotype-genotype correlations, for example relating to additional genetic variations (parental consanguinity), different allelic expression due to SNPs or differences in the methylation status.

Nucleotide Excision Repair Gene Subunit XPD is Highly Expressed in Cervical Squamous Cell Carcinoma

One of the subunits in the mammalian transcription factor II H complex, XPD (TFIIH p80), plays a significant role in the nucleotide excision repair pathway. Events such as abnormal DNA excision repair may be involved in the cervical carcinogenesis process. Expression of the human XPD protein was examined using immunohistochemistry in 84 normal cervical tissues and 148 cervical squamous cell carcinoma samples. Additionally, qRT-PCR was performed to analyse the XPD mRNA expression in 69 fresh normal cervical tissues and 110 cervical carcinoma samples. The relationships between XPD expression and various clinicopathological features (including age, FIGO stage, tumor size, stroma involvement, lymph node metastasis and histologic grade) were assessed. The XPD (TFIIH p80) protein was detected primarily in the cytoplasm. We found a statistically significant difference in XPD expression level in cervical carcinoma versus normal cervical tissue (Z = -7.302, P = 0.000). Notably, XPD mRNA was significantly over-expressed in cervical carcinoma tissues but not in normal cervix tissues (t = 6.942, P = 0.000). However, no statistically significant relationship was found regarding XPD expression and age, FIGO stage, tumor size, stroma involvement, lymph node metastasis or histologic grade (P = 0.089, 0.953, 0.809, 0.275, 0.421, 0.387 respectively). Our results showed that XPD was highly expressed in cervical squamous cell carcinoma tissues. A poorly understood change may occur during the XPD transcription process, resulting in the abnormal enrichment seen from mRNA to the protein level, thus leaving the protein unable to perform the normal function of excision repair. There is a need for further research in order to elucidate the specific mechanism involved.

1096 Molecular signatures of disseminated tumour cells in metastatic breast cancer patients

Gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) and cisplatin are commonly used in the treatment of many solid tumors, although the impact of chemotherapy is limited in metastatic non-small cell lung cancer. However, in clinical practice, there is a minority of patients who can attain long-term survival. Upregulation of mRNA transcripts has been linked to chemoresistance, and in some instances, mRNA expression has been correlated with polymorphisms. Cisplatin resistance is directly linked to the nucleotide excision repair system, specifically to the transcription-coupled nucleotide excision repair pathway that involves genes that are deficient in rare inborn disorders such as Cockayne syndrome and xeroderma pigmentosum. Overexpression of ERCC1 correlates with poor survival in gemcitabine/cisplatin-treated non-small cell lung cancer patients. At the preclinical level, ERCC1 and XPD mRNA expression correlate with each other, and overexpression of XPD causes selective cisplatin resistance in human tumor cell lines. XPD polymorphisms have been associated with lower DNA repair capacity. In our experience, time to progression is significantly higher in gemcitabine/cisplatin-treated patients with the Lys751Gln genotype (9.6 months) than in those with the Lys751Lys genotype (4.2 months; P = .03). Other polymorphisms involved in parallel DNA repair systems may well provide the same information, indicating a high degree of biologic redundancy. The overexpression of the subunit M1 of ribonucleotide reductase (RRM1) has been linked to gemcitabine resistance in our retrospective assessment. Preliminary findings that a subset of gemcitabine/cisplatin-treated patients with low ERCC1 and RRM1 mRNA levels show a significantly longer survival and highlight the possibilities of individually tailored chemotherapy.

Decreased expression and the Lys751Gln polymorphism of the XPD gene are associated with extreme longevity

Aging is associated with progressing genomic instability. The XPD gene encodes a DNA helicase involved in nucleotide excision repair and in transcription. We analyzed the common XPD polymorphisms that were previously shown to affect protein's DNA repair efficiency and to increase the risk of developing various cancers. Analysis was performed in 149 centenarians (mean age 101.1 years old) and in 413 young subjects (mean age 27.1 years old). We showed that the distribution of the Lys751Gln genotypes differed significantly between these groups (P = 0.017). In centenarians, the homozygous genotypes AA and CC were found less frequently than in young controls (29 vs. 36%, OR = 0.71, and 14 vs. 20%, OR = 0.652, respectively). The Arg156Arg and Asp312Asn were not significantly associated with extreme longevity. Analysis of the XPD mRNA level in blood mononuclear cells of people divided into three age groups (mean ages 28.7, 65.8 and 92.7 years old) showed that extreme longevity is associated with the decrease of the mean level of the specific mRNA; the differences between young or middle-aged vs. extremely old group were significant (P < 0.0001, P < 0.0001, respectively). In addition, the methylation pattern of the XPD promoter was analyzed in 30 people divided into three age groups (29.5, 65.9, and 101.4 years old). We showed that overall methylation of the XPD promoter is a rare event; however, aging is associated with the increase of methylation level upstream of the transcription start site. In summary, we showed for the first time that both the XPD polymorphic variants and the decreased level of its expression might be associated with aging.

Genotype-phenotype relationships in trichothiodystrophy patients with novel splicing mutations in theXPDgene

Trichothiodystrophy (TTD) is a rare, autosomal recessive neurodevelopmental disorder most commonly caused by mutations in ERCC2 (XPD), a gene that encodes a subunit of the transcription/repair factor IIH (TFIIH). Here, we describe two TTD cases in which detailed biochemical and molecular investigations offered a clue to explain their moderately affected phenotype. Patient TTD22PV showed new mutated XPD alleles: one contains a nonsense mutation (c.1984C>T) encoding a nonfunctional truncated product (p.Gln662X) whereas the second carries a genomic deletion (c.2191-18_c.2213del) that affects the splicing of intron 22 and generates multiple out-of-frame transcripts from codon 731. XPD mRNA from the second allele corresponds to 20% of the total. The predicted proteins, which are longer than normal, affect the cellular repair activity but only partially interfere with TFIIH stability, suggesting that the observed changes in the C-ter region of XPD cause minor structural changes that do not drastically compromise the transcriptional activity of TFIIH. Patient TTD24PV was compound heterozygous for a typical TTD allele (c.2164C>T, p.Arg722Trp) and for a new XPD allele with a mutation that partially affects intron 10 splicing, resulting in both mutated and normal XPD transcripts (that together represent 15% of the total XPD mRNA). Compared to the previously described TTD compound heterozygotes for the Arg722Trp change, Patient TTD24PV's cells show similar level of TFIIH but increased repair activity, suggesting that even low amounts of normal XPD subunits are able to partially rescue the functionality of TFIIH complexes.

Strict sun protection results in minimal skin changes in a patient with xeroderma pigmentosum and a novel c.2009delG mutation in XPD (ERCC2)

We examined the clinical, molecular and genetic features of a 16-year-old boy (XP2GO) with xeroderma pigmentosum (XP) and progressive neurological symptoms. The parents are not consanguineous. Increased sun sensitivity led to the diagnosis of XP at 2 years of age and a strict UV protection scheme was implemented. Besides recurrent conjunctivitis and bilateral pterygium, only mild freckling was present on his lips. He shows absent deep tendon reflexes, progressive sensorineural deafness and progressive mental retardation. MRI shows diffuse frontal cerebral atrophy and dilated ventricles. Symptoms of trichothiodystrophy (brittle hair with a tiger-tail banding pattern on polarized microscopy) or Cockayne syndrome (cachectic dwarfism, cataracts, pigmentary retinopathy and spasticity) were absent. XP2GO fibroblasts showed reduced post-UV cell survival (D(37) = 3.8 J/m(2)), reduced nucleotide excision repair, reduced expression of XPD mRNA and an undetectable level of XPD protein. Mutational analysis of the XPD gene in XP2GO revealed two different mutations: a common p.Arg683Trp amino acid change (c.2047C>T) known to be associated with XP and a novel frameshift mutation c.2009delG (p.Gly670Alafs*39). The latter mutation potentially behaves as a null allele. While not preventing neurological degeneration, early diagnosis and rigorous sun protection can result in minimal skin disease without cancer in XP patients.

Biological effects of SMMC-7721 hepatoma cells transfected by XPD in viro

Objective To investigate the role of wild-type XPD in SMMC-7721 hepatoma cells,its re-lationship with wild-type p53.CDK7 and c-myc.And the apoptosis mechanism of SMMC-7721 hepatoma cells.Methotis We inserted the human length XPD into the pEGFP-N2 plasmid vector which expresses the green fluorescence protein(GFP).And the pEGFP-N2 and pEGFP-N2-XPD were transfected into SMMC-7721 hepa-toma cell lines stably.Cel lines for omparison were matched on the sanle genetic background and passage.The expression of wild-type XPD,CDK7,p53,c-myc waft detected by RT-PCR,Westem blot.Cell growth wag detet-ed by MTT FCM wag employed for examining the cell cycle and apoptosis of the transfected SMMC-7721 hepa-toms cells.Results ①SMMC-7721-pEGFP-N2-xPD and SMMC-7721-pEGFP-N2 express the green fluores-cence protein which could be detected under the immunoffuorescence microscope.②RT-PCR The relative ex-\pression of p53 mRNA,XPD mRNA in the SMMC-7721,SMMC-7721-pEGFP-N2 and SMMC-7721-pEGFP-N2-XPD,the relative expression of p53 mRNA,XPD mRNA in the SMMC-7721-pEGFP-N2-XPD was signifiantly higher than other two oontrols(P 0.05). ③Western blot The relative expression of p53,XPD protein was signifiantly higher than other two oontrols (P 0.05).④MTT The ressuh showedthat the proliferative ability of SMMC-7721-pEGFP-N2-XPD was much more descreaged than other two oontrols (P 0.05).⑤FCM Wild-type XPD gene could change the cell8 and induce apoptitise in vitro though the result of FCM.Conclusions The pEGFP-N2 and pEGFP-N2-XPD were transfected into SMMC-7721hepatoma cell lines stably,wild-type XPD could decreaSe the expres-sion of CDK7,c-myc and increase the expression of p53.Wild-type.XPD could inhibit the activity of cell growth and change cell cycle,as well as induce cell apoptosis in SMMC-7721 hepatoma cell line in vitro. Key words: Carcinoma,hepatocellular; DNA repair

Platinum Resistance Related to a Functional NER Pathway

Tobacco carcinogens induce DNA adducts that are repaired by the nucleotide excision repair (NER) pathway.1 Inhaled combustion-derived particles, such as cigarette smoke, cause a local pulmonary inflammatory response that is characterized by the influx of neutrophils into the airways. On entering the lung, neutrophils are activated and release reactive oxygen species and an array of proteins, such as myeloperoxidase. A significant reduction of NER in human alveolar epithelial cells was observed when they were cocultured with activated neutrophils.2 NER, a highly versatile pathway for DNA damage removal, is often dysfunctional in non-small cell lung cancer (NSCLC) and could, therefore, be the Achilles heel for customizing chemotherapy. NER removes numerous types of DNA helix-distorting lesions, including cisplatinand ultraviolet-induced photo products.1 Inherited defects in the NER process cause serious repair disorders: xeroderma pigmentosum (XP), with extreme risk of ultraviolet-induced skin cancer, and Cockayne syndrome. NER functions by a “cutand-paste” mechanism in which cisplatin damage recognition, local opening of the DNA helix around the lesion, damage excision, and gap filling occur in successive steps1,3–5 (Figure 1). NER is composed of two subpathways: global genome NER (GG-NER) and transcription-coupled NER (TC-NER), which share the same core mechanism but differ in the way lesions are recognized.6 NER-defective XP is classified into seven complementation groups, XPA to XPG. XPC and XPE are specifically defective in GG-NER, which repairs the damage on the nontranscribed strand, whereas the other XP groups involve deficiencies in both TC-NER and GG-NER.7 The first step in GG-NER is damage recognition by the heterodimer XPC/hHR23B, which binds with higher affinity to helix-distorting DNA lesions than to nondamaged, doublestranded DNA1 (Figure 1A). Various NER factors, including transcription factor IIH (TFIIH, a general transcription factor for RNA polymerase II), XPA, replication protein A (RPA), and XPG work together to repair DNA damage (Figure 1B). TFIIH can be divided into two subcomplexes: the core TFIIH (composed of XPB, p62, p52, p44, p34, and p8) and a cdk-activating kinase subcomplex (containing cdk7, cyclin H, and MAT1).7 Both subcomplexes are bridged by XPD. In NER, TFIIH unwinds the duplex DNA around the lesion to allow the recruitment of the NER factors XPA, RPA, XPG, and excision repair cross-complementing 1 (ERCC1)/XPF (Figure 1B). TFIIH, with the two helicases XPB and XPD, opens an approximately 30-base-long DNA segment around the platinum damage. This open intermediate is stabilized by RPA and XPA (Figure 1C). The DNA strand that contains the damaged base(s) is excised by the two NER endonucleases XPG and ERCC1/XPF (Figure 1D). XPG cleaves the damaged DNA strand 3= from the lesion, and ERCC1/XPF cleaves the damaged strand 5= from the lesion (Figure 1D). The resulting gap is filled by DNA polymerase or in the presence of replication factors8 (Figure 1E). Importantly, the ERCC1/XPF structure-specific nuclease has an additional role in the repair of cisplatin adducts besides its function in NER: the recombination repair of interstrand cross-links.9 Moreover, colocalization of ERCC1 foci and RAD51 foci in response to cisplatin treatment has recently been found and may represent recruitment of ERCC1/XPF to sites of recombination repair.10 Nevertheless, in addition to NER, cisplatininduced cytotoxicity requires the interaction of components from several different DNA damage-processing systems.11

Single nucleotide polymorphisms of the DNA repair gene XPD/ERCC2 alter mRNA expression

Epidemiological studies documented associations between single nucleotide polymorphisms (SNPs) in the nucleotide excision repair gene XPD/ERCC2 and cancer risk. Little is known, however, about the underlying mechanisms for these associations. We explored a novel mechanism that could further explain the reported risk-modifying effect of these SNPs on disease susceptibility. Using quantitative real-time polymerase chain reaction, we examined the relationship between three SNPs in the XPD gene (R156R in exon 6, D312N in exon 10 and K751Q in exon 23) and mRNA levels as a potential mechanism by which these SNPs could alter DNA repair capacity and affect disease risk. To further investigate the mechanism(s) by which these SNPs alter mRNA transcription levels, we performed a localized Mfold structure analysis on the mRNA sequence surrounding the studied SNPs. All three SNPs studied, alone and in combination, significantly decreased constitutive XPD mRNA levels (P<0.003) in lymphocytes of healthy subjects. The decrease in mRNA levels was significantly greater in smokers and was exacerbated by smoking duration and intensity. The decrease was more pronounced in older than in younger subjects. The R156R and the K751Q polymorphisms were predicted to alter mRNA secondary structure, indicating that these SNPs potentially affect local folding and mRNA stability. Our results provide novel mechanistic explanations for epidemiological studies linking these SNPs to elevated cancer risk and emphasize the importance of comprehensively investigating the effect of both synonymous and nonsynonymous SNPs as risk modifiers by considering their potential effects on gene expression, protein translation and functions.

ERCC1, XPD and RAI mRNA levels in lymphocytes are not associated with lung cancer risk in a prospective study of Danes

Low DNA-repair capacity has been associated with increased risk of several types of cancer. mRNA levels of the nucleotide excision repair genes ERCC1 and XPD have been shown to correlate with the DNA-repair capacity. Likewise, mRNA levels of several DNA-repair genes including ERCC1 have been shown to be lower in lymphocytes from patients with lung cancer and head and neck cancer compared with healthy persons. In these studies, the low DNA-repair gene expression levels could be either a risk factor for disease or a consequence of the same. In this nested case–cohort study, which to our knowledge, is the first prospective study of DNA-repair gene mRNA levels as predictors of lung cancer, we have investigated the occurrence of lung cancer in relation to the mRNA level of the two DNA-repair genes ERCC1 and XPD and the NF κB inhibitor RAI in blood samples prior to disease. Among 54,220 members of a Danish prospective cohort study, 265 lung cancer cases were identified and a sub-cohort comprising 272 individuals was used for comparison. The expression levels of the three adjacent genes were found to be highly inter-correlated, to be higher in women compared to men and to be lower in older individuals. The incidence rate ratios for lung cancer in association with one log-unit increase (natural logarithm) in mRNA levels were 1.12 (CI = 0.89–1.41) for ERCC1, 1.00 (CI = 0.83–1.21) for XPD and 1.25 (0.89–1.74) for RAI. In conclusion, this study indicated no association between mRNA expression of the DNA-repair genes ERCC1 and XPD and risk of subsequent development of lung cancer.

ERCC1, RRM1 and XPD mRNA expression as a predictive marker of outcome in non-small-cell lung cancer (NSCLC) patients after platinum/gemcitabine induction chemotherapy followed by surgery

7142 Background: Nucleotide excision repair (NER) is the main DNA repair pathway. ERCC1, RRM1 and XPD are involved in the NER pathway, and tumor upregulation of these genes can lead to chemotherapy failure. Methods: We have analyzed ERCC1, RRM1 and XPD mRNA expression levels using real-time PCR in paraffin-embedded surgical specimens from 67 consecutive stage IIB-III NSCLC patients treated with induction platinum/gemcitabine followed by surgery between September 1998 and December 2002. Results: Radiographic response was observed in 57% of patients. Median survival was 38 months. Fifty-two patients with complete resection attained a 45 month median survival; while ten with incomplete resection had a 6.8 month median survival, and five unresectable patients had a 5.6 month median survival. There were no significant differences in survival according to ERCC1, RRM1 and XPD expression levels. Patients were divided into quartiles according to gene expression levels. For RRM1 levels, patients in the lowest quartile had a decreased risk of death compared to those in the top quartile (RR=0.30; P=0.033). Median survival for the 17 patients in the lowest quartile was 52 months, while for the 15 in the top quartile, it was 26 months (P=0.018). On comparing the characteristics of these 17 patients with the remaining 50, no differences in initial staging were observed. However, the 17 patients in the lowest quartile had more complete resections (94% vs 72%; P=0.03), and pathologic complete responses (29% vs 0%; P=0.00001). No significant differences were observed according to ERCC1 and XPD levels. Conclusions: Platinum-gemcitabine treated patients with low RRM1 levels have better outcomes. Based on these findings we will prospectively analyze the role of RRM1 expression in a neoadjuvant clinical trial. No significant financial relationships to disclose.

ERCC1, RRM1 and XPD mRNA expression as a predictive marker of outcome in non-small-cell lung cancer (NSCLC) patients after platinum/gemcitabine induction chemotherapy followed by surgery

7142 Background: Nucleotide excision repair (NER) is the main DNA repair pathway. ERCC1, RRM1 and XPD are involved in the NER pathway, and tumor upregulation of these genes can lead to chemotherapy...

Targeted therapy in combination with gemcitabine in non-small cell lung cancer

Gemcitabine (Gemzar; Eli Lilly and Company, Indianapolis, IN) and cisplatin are commonly used in the treatment of many solid tumors, although the impact of chemotherapy is limited in metastatic non-small cell lung cancer. However, in clinical practice, there is a minority of patients who can attain long-term survival. Upregulation of mRNA transcripts has been linked to chemoresistance, and in some instances, mRNA expression has been correlated with polymorphisms. Cisplatin resistance is directly linked to the nucleotide excision repair system, specifically to the transcription-coupled nucleotide excision repair pathway that involves genes that are deficient in rare inborn disorders such as Cockayne syndrome and xeroderma pigmentosum. Overexpression of ERCC1 correlates with poor survival in gemcitabine/cisplatin-treated non-small cell lung cancer patients. At the preclinical level, ERCC1 and XPD mRNA expression correlate with each other, and overexpression of XPD causes selective cisplatin resistance in human tumor cell lines. XPD polymorphisms have been associated with lower DNA repair capacity. In our experience, time to progression is significantly higher in gemcitabine/cisplatin-treated patients with the Lys751Gln genotype (9.6 months) than in those with the Lys751Lys genotype (4.2 months; P = .03). Other polymorphisms involved in parallel DNA repair systems may well provide the same information, indicating a high degree of biologic redundancy. The overexpression of the subunit M1 of ribonucleotide reductase (RRM1) has been linked to gemcitabine resistance in our retrospective assessment. Preliminary findings that a subset of gemcitabine/cisplatin-treated patients with low ERCC1 and RRM1 mRNA levels show a significantly longer survival and highlight the possibilities of individually tailored chemotherapy.

Influence of genetic markers on survival in non-small cell lung cancer.

Cisplatin or carboplatin is commonly used with gemcitabine, docetaxel, paclitaxel or vinorelbine as chemotherapy doublets in the treatment of advanced non-small cell lung cancer. Several randomized trials have failed to identify major differences in survival between any of these doublets. This lack of evidence for improvement in survival with any chemotherapy regimen has created a tabula rasa in which no more large randomized trials should be conducted with out including a genetic analysis. Patients see survival as their major concern, and other considerations, such as cost of treatment and qualify of life, are relegated to lower positions. Genetic alterations related to the transcription-coupled repair pathway of the nucleotide excision repair system (TC-NER) have revealed the subset of patients who are resistant to cisplatin. TC-NER involves genes that are deficient in rare inborn disorders such as Cockayne syndrome and xeroderma pigmentosum. For a long time, ERCC1 mRNA levels have been known to correlate with DNA repair capacity in various tissues. Levels of DNA cisplatin adducts in peripheral blood and buccal mucosa cells predict chemotherapy response, and high ERCC1 mRNA levels have been related to chemoresistance in ovarian cancer and in malignant lymphocytes from chronic lymphocytic leukemia. Moreover , in some instances, mRNA expression has been correlated with polymorphisms. Overexpression of ERCC1 correlates with poor survival gemcitabine/cisplatin-treated non-small cell lung cancer patients. An ongoing customized ERCC1-based chemotherapy trial has been established on this knowledge. Patients are randomized to the control arm of cisplatin/docetaxel is combined with cisplatin or gemcitabine according to ERCC1 levels. To date, 80 patients have been included. At the preclinical level, ERCC1 and XPD mRNA expression correlate with each other, and overexpression of XPD causes selective cisplatin resistance in human tumor cell lines. Some XPD polymorphisms have been associated with lower DNA repair capacity. In our experience, time to disease progression is significantly higher in gemcitabine/cisplatin-treated patients with the Lys751Gln genotype (9.6 months) than in those with the Lys751Lys genotype (4.2 months; p = 0.03). Other polymorphisms involved in parallel DNA repair systems may well provide the same information, indicating a high degree of biological redundancy. The overexpression of the subunit M1 of ribonucleotide reductase (RRM1) has been linked to gemcitabine resistance in our retrospective assessment. Preliminary findings indicate that a subset of gemcitabine/cisplatin-treated patients with low ERCC1 and RRM1 mRNA levels show a significantly longer survival. This highlights the possibilities of individually tailored chemotherapy. However, in patients treated with cisplatin/vinorelbine, the opposite effect has been observed. Patients with Lys751Lys had a longer time to progression. When docetaxel was added to gemcitabine/cisplatin, patients with Lys751Lys also had better survival. Our findings indicate that TC-NER status can help to decide between cisplatin/gemcitabine and docetaxel/ cisplatin. TC-NER-dependent activity is similar to other anticancer agents that cause DNA-binding enzymes to kill cells (topoisomerase inhibitors). At least 50% of non-small cell lung cancer patients harbor Lys751Lys and can benefit from docetaxel/ cisplatin treatment. Genes involved in spindle formation, centrosome functions and mRNA transport along the microtubule tracks should provide further information on potential markers of docetaxel resistance.

DNA repair capacity: inconsistency between effect of over-expression of five NER genes and the correlation to mRNA levels in primary lymphocytes

We have previously shown that high DNA repair capacity protects psoriasis patients against chemically induced basal cell carcinoma [Dybdahl et al. Mutat. Res. 433 (1999) 15–22]. We have used the same study persons to investigate the correlation between expression of eight genes involved in nucleotide excision repair and DNA repair capacity. mRNA levels of XPA, XPB, XPC, XPD, XPF, XPG, CSB and ERCC1 in primary lymphocytes from 33 individuals were quantified by dot-blots and normalized to β-actin. ERCC1 and XPD mRNA quantities were highly correlated (r=0.89; P<10−11) while XPA, XPB, XPC, XPG, XPFand CSB mRNAs were moderately correlated (r=0.2–0.7). Thus, the mRNA expressions seem to fall in at least two groups. There was a three to sevenfold variation in the expression levels of the mRNAs. This is in contrast to the more than a hundredfold variation in mRNA levels reported in cancer patients.DNA repair capacity was measured in a host cell reactivation assay, where primary lymphocytes were transfected with an UV-irradiated plasmid encoding firefly-luciferase. Only ERCC1 and XPD mRNA levels correlated with the DNA repair capacity (P<0.03). In order to see if ERCC1 or XPD activity was limiting for DNA repair, we cotransfected with plasmids encoding NER genes, thus over-expressing either XPB, XPC, XPD, CSB or ERCC1 in the host cell reactivation assay. Only XPB over-expression increased DNA repair capacity. Thus, there is no indication that neither XPD nor ERCC1 limits the DNA repair capacity. However, our results indicate that ERCC1 and XPD mRNA levels may be used as a proxy for DNA repair capacity in lymphocytes.

Signalling via receptor tyrosine kinase modulates the expression of the DNA repair enzyme XPD in cultured cells

Oxidative damage to DNA has been documented in cells isolated from subjects with diabetes. Herein, we evaluate the mechanism(s) that regulate the expression of the DNA repair enzyme XPD. CHO cells transfected with the human insulin receptor (CHO/HIRc) showed a threefold increase in the level of XPD mRNA when compared to control CHO/neo cells ( P<0.01). The addition of insulin to serum-starved cells led to an increase in XPD mRNA levels in both CHO/neo and CHO/HIRc cells, in a time and dose dependent fashion. Insulin acted primarily by inducing XPD transcription. Moreover, inhibition of protein synthesis by cyclohexamide induced a marked degradation of XPD mRNA levels in insulin treated cells. Site-directed mutagenesis of the tyrosine-kinase domain of the insulin receptor abolished the increase in XPD mRNA resulting from the transfection with wild type insulin receptors ( P<0.001). Western blot analysis of cell extracts from CHO/neo and CHO/HIRc cells revealed an increase in XPD counterpart protein was also induced by transfecting cells with the human insulin receptor. Evaluation of DNA damage by means of internucleosomal fragmentation showed a dramatic decrease in DNA fragmentation in CHO cells transfected with wild-type insulin receptor compared to control CHO/neo cells. DNA fragmentation was further decreased by the addition of insulin in the culture medium. In summary, our data indicates that activation of the insulin receptor plays an important role in the cellular response leading to repair of damaged DNA.