M. van Duin

The cloned human DNA excision repair gene ERCC-1 fails to correct xeroderma pigmentosum complementation groups A through I

The human DNA excision repair gene ERCC-1 complements the ultraviolet light (UV) and mitomycin C (MMC) sensitivity of CHO mutants of complementation group 1. We have investigated whether ERCC-1 is the mutated gene in cell lines from xeroderma pigmentosum (XP) complementation groups A through I by analyzing the endogenous gene in XP cells and by introduction of the gene followed by repair assays. Our studies show that ERCC-1 is not deleted or grossly rearranged in representative cell lines of 9 XP groups. Furthermore, Northern blot analysis revealed correct transcription of ERCC-1 in all groups. The cloned human ERCC-1 gene was introduced into immortalized XP cells by DNA transfection (groups A, C, D, E and F). The presence of the integrated transfected sequences was verified on Southern blots and by selection for 2 dominant marker genes that flank the ERCC-1 gene on the transfected cos43-34 DNA. ERCC-1 failed to confer a normal UV survival and UV-induced unscheduled DNA synthesis (UDS) to transfected populations. In the case of the remaining XP complementation groups (B, G, H and I), nuclear microinjection was used to introduce an ERCC-1 cDNA construct driven by an SV40 promoter into primary fibroblasts. Coinjection of the SV40 large T gene and analysis of its expression served as a control for the injection. The ERCC-1 cDNA failed to induce increased levels of UDS in the microinjected fibroblasts. We infer from these experiments that ERCC-1 is not the mutated gene in the 9 XP complementation groups examined. From a similar type of experiments we conclude that ERCC-1 is not the defective gene in UV-sensitive Cockaynes syndrome cells.

UV stimulation of DNA-mediated transformation of human cells.

Irradiation of dominant marker DNA with UV light (150 to 1,000 J/m2) was found to stimulate the transformation of human cells by this marker from two- to more than fourfold. This phenomenon is also displayed by xeroderma pigmentosum cells (complementation groups A and F), which are deficient in the excision repair of UV-induced pyrimidine dimers in the DNA. Also, exposure to UV of the transfected (xeroderma pigmentosum) cells enhanced the transfection efficiency. Removal of the pyrimidine dimers from the DNA by photoreactivating enzyme before transfection completely abolished the stimulatory effect, indicating that dimer lesions are mainly responsible for the observed enhancement. A similar stimulation of the transformation efficiency is exerted by 2-acetoxy-2-acetylaminofluorene modification of the DNA. No stimulation was found after damaging vector DNA by treatment with DNase or gamma rays. These findings suggest that lesions which are targets for the excision repair pathway induce the increase in transformation frequency. The stimulation was found to be independent of sequence homology between the irradiated DNA and the host chromosomal DNA. Therefore, the increase of the transformation frequency is not caused by a mechanism inducing homologous recombination between these two DNAs. UV treatment of DNA before transfection did not have a significant effect on the amount of DNA integrated into the xeroderma pigmentosum genome.

Conserved pattern of antisense overlapping transcription in the homologous human ERCC-1 and yeast RAD10 DNA repair gene regions.

We report that the genes for the homologous Saccharomyces cerevisiae RAD10 and human ERCC-1 DNA excision repair proteins harbor overlapping antisense transcription units in their 3 regions. Since naturally occurring antisense transcription is rare in S. cerevisiae and humans (this is the first example in human cells), our findings indicate that antisense transcription in the ERCC-1-RAD10 gene regions represents an evolutionarily conserved feature.

Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group-2 mutants

The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correction by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 43-34, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 43-34. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500-2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.

High-resolution array comparative genomic hybridization of chromosome 8q: evaluation of putative progression markers for gastroesophageal junction adenocarcinomas

Amplification of 8q is frequently found in gastroesophageal junction (GEJ) cancer. It is usually detected in high-grade, high-stage GEJ adenocarcinomas. Moreover, it has been implicated in tumor progression in other cancer types. In this study, a detailed genomic analysis of 8q was performed on a series of GEJ adenocarcinomas, including 22 primary adenocarcinomas, 13 cell lines and two xenografts, by array comparative genomic hybridization (aCGH) with a whole chromosome 8q contig array. Of the 37 specimens, 21 originated from the esophagus and 16 were derived from the gastric cardia. Commonly overrepresented regions were identified at distal 8q, i.e. 124–125 Mb (8q24.13), at 127–128 Mb (8q24.21), and at 141–142 Mb (8q24.3). From these regions six genes were selected with putative relevance to cancer: ANXA13, MTSS1, FAM84B (alias NSE2), MYC, C8orf17 (alias MOST-1) and PTK2 (alias FAK). In addition, the gene EXT1 was selected since it was found in a specific amplification in cell line SK-GT-5. Quantitative RT-PCR analysis of these seven genes was subsequently performed on a panel of 24 gastroesophageal samples, including 13 cell lines, two xenografts and nine normal stomach controls. Significant overexpression was found for MYC and EXT1 in GEJ adenocarcinoma cell lines and xenografts compared to normal controls. Expression of the genes MTSS1, FAM84B and C8orf17 was found to be significantly decreased in this set of cell lines and xenografts. We conclude that, firstly, there are other genes than MYC involved in the 8q amplification in GEJ cancer. Secondly, the differential expression of these genes contributes to unravel the biology of GEJ adenocarcinomas.

Molecular dissection of the chromosome band 7q21 amplicon in gastroesophageal junction adenocarcinomas identifies cyclin-dependent kinase 6 at both genomic and protein expression levels.

Amplification of chromosome band 7q21 has been frequently detected in various types of cancer including gastroesophageal junction (GEJ) adenocarcinomas. At present, no gene has been disclosed that can explain this frequent amplification of 7q21 in GEJ carcinomas. Therefore, a detailed genomic analysis of the 7q21 region was performed on a selected series of GEJ adenocarcinomas, i.e., 14 primary adenocarcinomas and 10 cell lines, by array comparative genomic hybridization (aCGH) with a 7q11.22‐q31.2 contig array. A distinct peak of amplification was identified at 92.1 Mb in 7q21.2, precisely comprising cyclin‐dependent kinase 6 (CDK6), a gene involved in cell cycle regulation. A smaller peak was seen at 116.2 Mb in 7q31.2, the locus of the MET proto‐oncogene. No distinct peak was detected for the hepatocyte growth factor (HGF) at 81.3 Mb in 7q21.11. An immunoprofile of HGF, CDK6 and MET revealed a strong correlation between aCGH and immunohistochemical protein expression for CDK6 (P = 0.002). Furthermore, immunohistochemistry did not show expression of CDK6 in Barretts dysplasia and carcinoma in situ, correlating expression of CDK6 with a malignant phenotype. We conclude that high‐resolution genomic analysis and immunoprofiling identify CDK6 as the main candidate target for the recurrent amplification of 7q21 in GEJ adenocarcinomas.

18F-fluoride-PET for dynamic in vivo monitoring of bone formation in multiple myeloma

BackgroundBone disease in multiple myeloma is characterized by reduced bone formation. The gold standard of bone formation is the mineral apposition rate (MAR), an invasive technique reflecting bone formation at a single site. We compared 18F-fluoride-PET with the MAR in myeloma patients.MethodsBone formation was measured before and after bortezomib treatment by determination of the MAR in iliac bone marrow biopsies and the measurement of 18F-uptake.ResultsThe inter- and intra-individual variations in 18F-uptake (SUVA50%) were pronounced as 33.50 (range 4.42 to 37.92) and 27.18 (range 4.00 to 31.18), respectively. A significant correlation between the MAR and 18F-uptake was found (ru2009=u20090.80, pu2009=u20090.017). There was a heterogeneous response after treatment varying from −2.20 to 4.53.ConclusionsIliac 18F-uptake was associated with the local MAR in myeloma patients. Furthermore, 18F-fluoride-PET demonstrated the heterogeneity of in vivo bone formation, enabling monitoring during treatment.

Erratum: A gene expression signature for high-risk multiple myeloma

Correction to: Leukemia (2012) 26, 2406–2413; doi:10.1038/leu.2012.127 Since the publication of this article, the authors have discovered an error in the script for calculating the IFM-15 risk scores. In their paper, they described the weights of all probe sets used in this signature to be positive,whereas four of these were actually negative, as published by Decaux et al.

Isolation and Characterization of Genes Involved in Mammalian Excision Repair

Repair of randomly occuring DNA injury in mammalian cells must require sophisticated and elaborative systems in view of the wide spectrum of different types of lesions that have to be recognized and removed, the enormous size of the mammalian genome and the complex chromatin structure, that should undergo reversible alterations for repair to take place. The finding of preferential repair of expressed genes as recently uncovered by Hanawalt and coworkers for the removal of pyrimidine dimers (Bohr et al., 1985, Mellon et al. 1986) elegantly illustrates how the cell deals with part of these problems: highest priority is given to repair of the most vital regions in the genome: namely those being used actively and of which transcription is hampered by damage in the template. The very recent discovery that the yeast repair gene RAD6 encodes a ubiquitin conjugating enzyme specific for histons 2A and 2B (Jentsch et al. 1987) and thought to be implicated in chromatin remodelling adds to the picture of tight interactions of repair events and chromatin structure and dynamics.