Historical aspects of xeroderma pigmentosum and nucleotide excision repair.

The discovery that xeroderma pigmentosum was a sun-sensitive hereditary human disease that was deficient in DNA repair was made when research into the fundamental mechanisms of nucleotide excision repair was in its infancy. The linkage between DNA damage, DNA repair and human cancer stimulated an enormous subsequent growth of the field of DNA repair and the identification of other repair deficient diseases and other repair pathways. This growth has established DNA repair as a central factor for maintaining genomic stability and preventing cancer, neurodegenerative disease and aging.

Clinical implications of the basic defects in Cockayne syndrome and xeroderma pigmentosum and the DNA lesions responsible for cancer, neurodegeneration and aging.

Cancer, aging, and neurodegeneration are all associated with DNA damage and repair in complex fashions. Aging appears to be a cell and tissue-wide process linked to the insulin-dependent pathway in several DNA repair deficient disorders, especially in mice. Cancer and neurodegeneration appear to have complementary relationships to DNA damage and repair.

Pol eta is required for DNA replication during nucleotide deprivation by hydroxyurea.

Hydroxyurea reduces DNA replication by nucleotide deprivation, whereas UV damage generates DNA photoproducts that directly block replication fork progression. We show that the low fidelity class Y polymerase Pol eta is recruited to proliferating cell nuclear antigen at replication forks both by hydroxyurea and UV light. Under nucleotide deprivation, Pol eta allows cells to accumulate at the G1/S boundary by facilitating slow S-phase progression and promotes apoptosis.

Contribution of Notch signaling activation to human glioblastoma multiforme.

Object: Because activation of Notch receptors has been suggested to be critical for Ras-mediated transformation, and because many gliomas exhibit deregulated Ras signaling, the authors measured Notch levels and activation in primary samples and cell lines derived from glioblastoma multiforme (GBM) as well as the contribution of Notch pathway activation to astrocytic transformation and growth.

Methods: Western blot analysis of Notch 1 expression and activation showed that Notch 1 protein was overexpressed and/or activated in Ras-transformed astrocytes, in three of four GBM cell lines, and in four of five primary GBM samples. Expansion of these studies to assess mRNA expression of components of the Notch signaling pathway by cDNA expression array showed that cDNAs encoding components of the Notch signaling pathway, including the Notch ligand Jagged-1, Notch 3, and the downstream targets of Notch (HES1 and HES2), were also overexpressed relative to non-neoplastic brain controls in 23, 71, and 51% of 35 primary GBMs, respectively.

The Mre11/Rad50/Nbs1 complex interacts with the mismatch repair system and contributes to temozolomide-induced G2 arrest and cytotoxicity.

The chemotherapeutic agent temozolomide produces O(6)-methylguanine (O6MG) in DNA, which triggers futile DNA mismatch repair, DNA double-strand breaks (DSB), G(2) arrest, and ultimately cell death. Because the protein complex consisting of Mre11/Rad50/Nbs1 (MRN complex) plays a key role in DNA damage detection and signaling, we asked if this complex also played a role in the cellular response to temozolomide. Temozolomide exposure triggered the assembly of MRN complex into chromatin-associated nuclear foci.

Cockayne syndrome exhibits dysregulation of p21 and other gene products that may be independent of transcription-coupled repair.

Cockayne syndrome (CS) is a progressive childhood neurodegenerative disorder associated with a DNA repair defect caused by mutations in either of two genes, CSA and CSB. These genes are involved in nucleotide excision repair (NER) of DNA damage from ultraviolet (UV) light, other bulky chemical adducts and reactive oxygen in transcriptionally active genes (transcription-coupled repair, TCR). For a long period it has been assumed that the symptoms of CS patients are all due to reduced TCR of endogenous DNA damage in the brain, together with unexplained unique sensitivity of specific neural cells in the cerebellum.

mTOR-independent translational control of the extrinsic cell death pathway by RalA.

Oncogenic potential is associated with translational regulation, and the prevailing view is that oncogenes use mTOR-dependent pathways to up-regulate the synthesis of proteins critical for transformation. In this study, we show that RalA, a key mediator of Ras transformation, is also linked to the translational machinery. At least part of this linkage, however, is independent of mTOR and acts through RalBP1 to suppress cdc42-mediated activation of S6 kinase and the translation of the antiapoptotic protein FLIP(S).

H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks.

The variant histone H2AX is phosphorylated in response to UV irradiation of primary human fibroblasts in a complex fashion that is radically different from that commonly reported after DNA double-strand breaks. H2AX phosphorylation after exposure to ionizing radiation produces foci, which are detectable by immunofluorescence microscopy and have been adopted as clear and consistent quantitative markers for DNA double-strand breaks. Here we show that in contrast to ionizing radiation, UV irradiation mainly induces H2AX phosphorylation as a diffuse, even, pan-nuclear staining.

Cancer in xeroderma pigmentosum and related disorders of DNA repair.

Nucleotide-excision repair diseases exhibit cancer, complex developmental disorders and neurodegeneration. Cancer is the hallmark of xeroderma pigmentosum (XP), and neurodegeneration and developmental disorders are the hallmarks of Cockayne syndrome and trichothiodystrophy. A distinguishing feature is that the DNA-repair or DNA-replication deficiencies of XP involve most of the genome, whereas the defects in CS are confined to actively transcribed genes.

Adenovirus overrides cellular checkpoints for protein translation.

mTOR is a critical regulator of protein translation, and plays an important role in controlling cellular replication. Recent studies indicate that nutrient and growth factor mediated activation of mTOR is deregulated in human cancer, and therefore represents an attractive tumor target. However, activation of mTOR is a complex process that is not yet fully understood.

Mending human genes: a job for a lifetime.

I have described a number of milestones along a career of nearly 40 years in DNA repair. Most important was the discovery that the human disease xeroderma pigmentosum represented mutations in various components of nucleotide excision repair. This ushered in a new field of research involving numerous investigators and which continues to expand and amaze.

Splitting hairs--discovery of a new DNA repair and transcription factor for the human disease trichothiodystrophy.

The gene responsible for the TTD-A group of the DNA repair deficient disease trichothiodystrophy has been identified as a small, 8 kDa, component of the transcription factor TFIIH which contributes to the stability and concentration of TFIIH in vivo.

DNA-PKcs function regulated specifically by protein phosphatase 5.

Unrepaired DNA double-strand breaks can lead to apoptosis or tumorigenesis. In mammals double-strand breaks are repaired mainly by nonhomologous end-joining mediated by the DNA-PK complex. The core protein of this complex, DNA-PKcs, is a DNA-dependent serine/threonine kinase that phosphorylates protein targets as well as itself.

A phase I and pharmacokinetic study of irinotecan in patients with hepatic or renal dysfunction or with prior pelvic radiation: CALGB 9863.

Background: To ascertain if hepatic or renal dysfunction or prior pelvic radiation (XRT) leads to increased toxicity at a given dose of irinotecan and to characterize the pharmacokinetics of irinotecan and its major metabolites in patients with hepatic or renal dysfunction.

Patients And Methods: Adults with tumors appropriate for irinotecan therapy and who had abnormal liver or renal function tests or had prior radiation to the pelvis were eligible. Patients were assigned to one of four treatment cohorts: I, aspartate aminotransferase (AST) > or = 3x upper limit of normal and direct bilirubin <1.

Excision repair--its bacterial beginnings.

Nucleotide excision repair was first reported in 1964 by Setlow and Carrier and by Boyce and Howard-Flanders. These two reports clearly defined the existence in bacteria of a repair process that physically removed damaged sites, pyrimidine dimers, from the DNA in the form of acid-soluble fragments. These reports were the starting point for subsequent development of the whole field of DNA excision point.

The p38 mitogen-activated protein kinase pathway links the DNA mismatch repair system to the G2 checkpoint and to resistance to chemotherapeutic DNA-methylating agents.

Although human cells exposed to DNA-methylating agents undergo mismatch repair (MMR)-dependent G(2) arrest, the basis for the linkage between MMR and the G(2) checkpoint is unclear. We noted that mitogen-activated protein kinase p38alpha was activated in MMR-proficient human glioma cells exposed to the chemotherapeutic methylating agent temozolomide (TMZ) but not in paired cells made MMR deficient by expression of a short inhibitory RNA (siRNA) targeted to the MMR protein Mlh1. Furthermore, activation of p38alpha in MMR-proficient cells was associated with nuclear inactivation of the cell cycle regulator Cdc25C phosphatase and its downstream target Cdc2 and with activation of the G(2) checkpoint, actions which were suppressed by the p38alpha/beta inhibitors SB203580 and SB202590 or by expression of a p38alpha siRNA.

Activation of the Wnt pathway in non small cell lung cancer: evidence of dishevelled overexpression.

Non small cell lung cancer (NSCLC) is the leading cause of cancer deaths in the United States and worldwide. Unfortunately, standard therapies remain inadequate. An increased understanding of the molecular biology of lung cancer biology is required to develop more effective new therapies.

Genome sequence and splice site analysis of low-fidelity DNA polymerases H and I involved in replication of damaged DNA.

POLH and POLI are paralogs encoding low-fidelity, class Y, DNA polymerases involved in replication of damaged DNA in the human disease xeroderma pigmentosum variant. Analysis of genomic regions for human and mouse homologs, employing the analytic tool Genome Cryptographer, detected low-repetitive or unique regions at exons and other potential control regions, especially within intron I of human POLH. The human and mouse homologs are structurally similar, but the paralogs have undergone evolutionary divergence.

DNA replication in the face of (In)surmountable odds.

We describe here a model for sequential recruitment of various enzymatic systems that maintain DNA replication fidelity in cells with damaged bases, especially those formed by ultraviolet (UV) irradiation. Systems of increasing complexity but decreasing fidelity are recruited to restore replication of damaged DNA. The first and most accurate response is nucleotide excision repair (NER) that is cell cycle-independent; next come various delaying cell cycle checkpoints that provide an extended time window for NER.

Mechanisms by which human cells bypass damaged bases during DNA replication after ultraviolet irradiation.

The replication of damaged DNA involves cascading mechanisms of increasing complexity but decreasing accuracy. The most accurate mechanism uses low-fidelity DNA polymerases, Pol H and Pol I, which have active sites sufficiently large to accommodate a pyrimidine dimer. Replicative bypass of DNA damage by these polymerases produces an accurately replicated, newly synthesized strand.

Photoreactivation.

Kelner and Dulbecco first reported in the 1940s and 1950s the reversal of ultraviolet damage in bacteria and phage by illumination with visible light. The first publications, reprinted here, represented the discovery of a widespread repair mechanism that was named "photoreactivation" (PHR), that directly reversed photoproducts to their individual pyrimidine components. Between them, these pioneers demonstrated that photoreactivation had a cellular basis, could be defined by wavelength optima indicating specific molecular photoreceptors, and had a widespread phyletic distribution except for its absence from placental mammals.

Ultraviolet photobiology: its early roots and insights into DNA repair.

This is the first of a series of commentaries on classic papers on DNA repair that highlight the birth of this discipline. The roots go deep, and in this first commentary, I describe some of the earliest discoveries of the mechanism of absorption of UV light in cells, and its lethal and mutagenic effects. Most remarkably, a discovery of DNA repair by the use of split doses of UV light was reported in 1919.

Ribozyme therapeutics.

Since their initial discovery, ribozymes have shown great promise not just as a tool in the manipulation of gene expression, but also as a novel therapeutic agent. This review discusses the promises and pitfalls of ribozyme technology, with a special emphasis on cancer-related applications, though relevance to skin disease will also be discussed.

Defined human cellular systems in the study of glioma development.

The creation and characterization of permanent cell lines derived from primary human gliomas in the 1960s gave scientists access to unlimited, renewable material in which to study the development of brain tumors. These cells, however, were already tumorigenic and selected for growth in culture, limiting the amount of information that could be gathered about the events that led to the formation of their tumors of origin. In response to these limitations, investigators moved to the study of primary tumors to identify in a correlative fashion the lesions important in tumor formation, and to the use of animal models to gain information about the transformation process.