Synergistic interactions between XPC and p53 mutations in double-mutant mice: neural tube abnormalities and accelerated UV radiation-induced skin cancer

AbstractThe significance of DNA repair to human health has been well documented by studies on xeroderma pigmentosum (XP) patients, who suffer a dramatically increased risk of cancer in sun-exposed areas of their skin [1,2]. This autosomal recessive disorder has been directly associated with a defect in nucleotide excision–repair (NER) [1,2]. Like human XP individuals, mice carrying homozygous mutations in XP genes manifest a predisposition to skin carcinogenesis following exposure to ultraviolet (UV) radiation [3–5]. Recent studies have suggested that, in addition to roles in apoptosis [6] and cell-cycle checkpoint control [7] in response to DNA damage, p53 protein may modulate NER [8]. Mutations in the p53 gene have been observed in 50% of all human tumors [9] and have been implicated in both the early [10] and late [11] stages of skin cancer. To examine the consequences of a combined deficiency of the XPC and the p53 proteins in mice, we generated double-mutant animals. We document a spectrum of neural tube defects in XPC p53 mutant embryos. Additionally, we show that, following exposure to UV-B radiation, XPC p53 mutant mice have more severe solar keratosis and suffer accelerated skin cancer compared with XPC mutant mice that are wild-type with respect to p53

The DNA Damage Signal for Mdm2 Regulation, Trp53 Induction, and Sunburn Cell Formation In Vivo Originates from Actively Transcribed Genes

The stratum corneum and DNA repair do not completely protect keratinocytes from ultraviolet B. A third defense prevents cells with DNA photoproducts from becoming precancerous mutant cells: apoptosis of ultraviolet-damaged keratinocytes (“sunburn cells”). As signals for ultraviolet-induced apoptosis, some studies implicate DNA photoproducts in actively transcribed genes; other studies implicate non-nuclear signals. We traced and quantitated the in vivo DNA signal through several steps in the apoptosis-signaling pathway in haired mice. Homozygous inactivation of Xpa, Csb, or Xpc nucleotide excision repair genes directed the accumulation of DNA photoproducts to specific genome regions. Repair-defective Xpa−/− mice were 7–10-fold more sensitive to sunburn cell induction than wild-type mice, indicating that 86–90% of the ultraviolet B signal for keratinocyte apoptosis involved repairable photoproducts in DNA; the remainder involves unrepaired DNA lesions or nongenomic targets. Csb−/− mice, defective only in excising photoproducts from actively transcribed genes, were as sensitive as Xpa−/−, indicating that virtually all of the DNA signal originates from photoproducts in active genes. Conversely, Xpc−/− mice, defective in repairing the untranscribed majority of the genome, were as resistant to apoptosis as wild type. Sunburn cell formation requires the Trp53 tumor suppressor protein; 90–96% of the signal for its induction in vivo involved transcribed genes. Mdm2, which regulates the stability of Trp53 through degradation, was induced in vivo by low ultraviolet B doses but was suppressed at erythemal doses. DNA photoproducts in actively transcribed genes were involved in ≈ 89% of the Mdm2 response

Concerted Assembly and Cloning of Multiple DNA Segments Using In Vitro Site-Specific Recombination: Functional Analysis of Multi-Segment Expression Clones

The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame. The efficiency and flexibility of this approach enables collections of functional elements to be generated and mixed in a combinatorial fashion for the parallel assembly of numerous multi-segment constructs. The assembled constructs can be further manipulated by directing exchange of defined segments with alternate DNA segments. In this report, we demonstrate feasibility of the technology and application to the generation of fusion proteins, the linkage of promoters to genes, and the assembly of multiple protein domains. The technology has broad implications for cell and protein engineering, the expression of multidomain proteins, and gene function analysis

Feasibility of Genome-Scale Construction of Promoter::Reporter Gene Fusions for Expression in Caenorhabditis elegans Using a MultiSite Gateway Recombination System

The understanding of gene function increasingly requires the characterization of DNA segments containing promoters and their associated regulatory sequences. We describe a novel approach for linking multiple DNA segments, here applied to the generation of promoter::reporter fusions. Promoters from Caenorhabditis elegans genes were cloned using the MultiSite Gateway cloning technology. The capacity for using this system for efficient construction of chimeric genes was explored by constructing promoter::reporter gene fusions with a gfp reporter. The promoters were found to provide appropriate expression of GFP upon introduction into C. elegans, demonstrating that the short Gateway recombination site between the promoter and the reporter did not interfere with transcription or translation. The recombinational cloning involved in the Gateway system, which permits the highly efficient and precise transfer of DNA segments between plasmid vectors, makes this technology ideal for genomics research programs

XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas

DNA damage is a well-known initiator of tumorigenesis. Studies have shown that most cancer cells rely on aerobic glycolysis for their bioenergetics. We sought to identify a molecular link between genomic mutations and metabolic alterations in neoplastic transformation. We took advantage of the intrinsic genomic instability arising in xeroderma pigmentosum C (XPC). The XPC protein plays a key role in recognizing DNA damage in nucleotide excision repair, and patients with XPC deficiency have increased incidence of skin cancer and other malignancies. In cultured human keratinocytes, we showed that lentivirus-mediated knockdown of XPC reduced mitochondrial oxidative phosphorylation and increased glycolysis, recapitulating cancer cell metabolism. Accumulation of unrepaired DNA following XPC silencing increased DNA-dependent protein kinase activity, which subsequently activated AKT1 and NADPH oxidase-1 (NOX1), resulting in ROS production and accumulation of specific deletions in mitochondrial DNA (mtDNA) over time. Subcutaneous injection of XPC-deficient keratinocytes into immunodeficient mice led to squamous cell carcinoma formation, demonstrating the tumorigenic potential of transduced cells. Conversely, simultaneous knockdown of either NOX1 or AKT1 blocked the neoplastic transformation induced by XPC silencing. Our results demonstrate that genomic instability resulting from XPC silencing results in activation of AKT1 and subsequently NOX1 to induce ROS generation, mtDNA deletions, and neoplastic transformation in human keratinocytes