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Biomedical subjects

James E Cleaver

Publications and source records attributed to James E Cleaver.

At least 19 recordsLinked to original sources
PubMed2006

Genes and pathways downstream of telomerase in melanoma metastasis.

Recent studies have demonstrated a role for telomerase in driving tumor progression, but its mechanism of action remains unclear. Here we show that stable, ribozyme-mediated suppression of mouse telomerase RNA reduced telomerase RNA expression, telomerase activity, and telomere length, which significantly reduced tumor invasion and metastatic potential. Our studies reveal that previously unidentified effects of telomerase may mediate its tumor-promoting effects. First, reducing telomerase activity induced a more dendritic morphology, accompanied by increased melanin content and increased expression of tyrosinase, a key enzyme in melanin biosynthesis. Second, gene expression profiling revealed that telomerase targeting down-regulated expression of several glycolytic pathway genes, with a corresponding decrease in glucose consumption and lactate production. Thus, telomerase activity controls the glycolytic pathway, potentially altering the energy state of tumor cells and thereby modulating tyrosinase activity and melanin production. These studies have important implications for understanding the mechanisms by which telomerase promotes tumor invasion and metastasis.

Animals↗
PubMed2006

Adenovirus mediated transduction of the human DNA polymerase eta cDNA.

Xeroderma pigmentosum (XP) is an autosomal recessive photosensitive disorder with an extremely high incidence of skin cancers. Seven complementation groups, corresponding to seven proteins involved in nucleotide excision repair (NER), are associated with this syndrome. However, in XP variant patients, the disorder is caused by defects in DNA polymerase eta; this error prone polymerase, encoded by POLH, is involved in translesion DNA synthesis (TLS) on DNA templates damaged by ultraviolet light (UV). We constructed a recombinant adenovirus carrying the human POLH cDNA linked to the EGFP reporter gene (AdXPV-EGFP) and infected skin fibroblasts from both XPV and XPA patients. Twenty-four hours after infection, the DNA polymerase eta-EGFP fusion protein was detected by Western blot analysis, demonstrating successful transduction by the adenoviral vector. Protein expression was accompanied by reduction in the high sensitivity of XPV cells to UV, as determined by cell survival and apoptosis-induction assays. Moreover, the pronounced UV-induced inhibition of DNA synthesis in XPV cells and their arrest in S phase were attenuated in AdXPV-EGFP infected cells, confirming that the transduced polymerase was functional. However, over-expression of polymerase eta mediated by AdXPV-EGFP infection did not result in enhancement of cell survival, prevention of apoptosis, or higher rate of nascent DNA strand growth in irradiated XPA cells. These results suggest that TLS by DNA polymerase eta is not a limiting factor for recovery from cellular responses induced by UV in excision-repair deficient fibroblasts.

Adenoviridae↗
PubMed2006

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. UV induced pan-nuclear phosphorylation of H2AX is present in all phases of the cell cycle and is highest in S phase. H2AX phosphorylation in G(1) cells depends on nucleotide excision repair factors that may expose the S-139 site to kinase activity, is not due to DNA double-strand breaks, and plays a larger role in UV-induced signal transduction than previously realized.

Cell Nucleus↗
PubMed2006

A novel role of DNA polymerase eta in modulating cellular sensitivity to chemotherapeutic agents.

Genetic defects in polymerase eta (pol eta; hRad30a gene) result in xeroderma pigmentosum variant syndrome (XP-V), and XP-V patients are sensitive to sunlight and highly prone to cancer development. Here, we show that pol eta plays a significant role in modulating cellular sensitivity to DNA-targeting anticancer agents. When compared with normal human fibroblast cells, pol eta-deficient cells derived from XP-V patients were 3-fold more sensitive to beta-d-arabinofuranosylcytosine, gemcitabine, or cis-diamminedichloroplatinum (cisplatin) single-agent treatments and at least 10-fold more sensitive to the gemcitabine/cisplatin combination treatment, a commonly used clinical regimen for treating a wide spectrum of cancers. Cellular and biochemical analyses strongly suggested that the higher sensitivity of XP-V cells to these agents was due to the inability of pol eta-deficient cells to help resume the DNA replication process paused by the gemcitabine/cisplatin-introduced DNA lesions. These results indicated that pol eta can play an important role in determining the cellular sensitivity to therapeutic agents. The findings not only illuminate pol eta as a potential pharmacologic target for developing new anticancer agents but also provide new directions for improving future chemotherapy regimen design considering the use of nucleoside analogues and cisplatin derivatives.

Antineoplastic Agents↗
PubMed2005

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.

DNA Damage↗
PubMed2005

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. Many of the proteins involved in repair are also components of dynamic multiprotein complexes, transcription factors, ubiquitylation cofactors and signal-transduction networks. Complex clinical phenotypes might therefore result from unanticipated effects on other genes and proteins.

Child↗
PubMed2005

Search for a prion-specific nucleic acid.

Diversity of prion strains was attributed to an elusive nucleic acid, yet a search spanning nearly two decades has failed to identify a prion-specific polynucleotide. In our search for a prion-specific nucleic acid, we analyzed nucleic acids in purified fractions from the brains of Syrian hamsters infected with Sc237 prions. Purification of Sc237 prions removed nucleic acids larger than 50 nucleotides as measured by return refocusing electrophoresis (RRGE). To determine the size of the largest polynucleotide present in purified fractions at an abundance of one molecule per infectious (ID50) unit, we measured prions present after inoculation. In order to account for the rapid clearance of prions after intracerebral inoculation, we determined the number of PrP(Sc) molecules and ID50 units of prions that were retained in brain. Factoring in clearance after inoculation, we estimate that the largest polynucleotide present in our purified fractions at one molecule per ID50 unit is approximately 25 nucleotides in length. In the same fractions, there were approximately 3,000 protease-resistant PrP(Sc) molecules per ID50 unit after accounting for clearance of PrP(Sc) following inoculation. We compared the resistance of Sc237 and 139H prions to inactivation by UV irradiation at 254 nm. Irradiation of homogenates and microsomes diminished prion infectivity by a factor of approximately 1,000 but did not alter the strain-specified properties of the Sc237 and 139H prions. The data reported here combined with the production of synthetic prions argue that the 25-mer polynucleotides found in purified prion preparations are likely to be host encoded and of variable sequence; additionally, these 25-mers are unlikely to be prion specific.

Animals↗
PubMed2004

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. Although the (auto)phosphorylation activity has been shown to be essential for repair of both random double-strand breaks and induced breaks at the immunoglobulin locus, the corresponding phosphatase has been elusive. In fact, to date, none of the putative phosphatases in DNA double-strand break repair has been identified. Here we show that protein phosphatase 5 interacts with DNA-PKcs and dephosphorylates with surprising specificity at least two functional sites. Cells with either hypo- or hyperphosphorylation of DNA-PKcs at these sites show increased radiation sensitivity.

Animals↗
PubMed2003

Rescue of Xrcc1 knockout mouse embryo lethality by transgene-complementation.

Xrcc1 knockout embryos show increased DNA breakage and apoptosis in tissues of the embryo proper prior to death at embryonic day E6.5. An additional deficiency in Trp53 allows Xrcc1(-/-) embryos to enlarge slightly and initiate gastrulation although ultimately death is delayed by less than 24h. Death presumably results from DNA damage that reaches toxic levels in the post-implantation mouse embryo. To investigate the level of XRCC1 protein needed for successful mouse development, we derived Xrcc1 transgene-complemented Xrcc1(-/-) mice that express Xrcc1 within the normal range or at a greatly reduced level (<10% normal). The greatly reduced XRCC1 protein level destabilized the XRCC1 partner protein DNA ligase III (LIG3) but still allowed for successful mouse development and healthy, fertile adults. Fibroblasts from these animals exhibited almost normal alkylation sensitivity measured by differential cytotoxicity. Thus, a large reduction of both XRCC1 and DNA ligase III has no observable effect on mouse embryogenesis and post-natal development, and no significant effect on cellular sensitivity to DNA alkylation. The presence of XRCC1, even at reduced levels of expression, is therefore capable of supporting mouse development and DNA repair.

Alkylation↗
PubMed2003

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.

Bacteria↗
PubMed2003

Recapitulation of the cellular xeroderma pigmentosum-variant phenotypes using short interfering RNA for DNA polymerase H.

The lesion-specific DNA polymerase POLH gene is mutated in xeroderma pigmentosum variant (XP-V) patients who exhibit an increased skin cancer incidence from UV exposure. Normal cells in which POLH expression was reduced using short interfering RNAs (siRNAs) were compared with the XP-V cellular phenotype that results from naturally occurring inactivating mutations. Stable clones expressing siRNA had partially reduced POLH protein levels, and intermediate levels of UV sensitivity and S phase checkpoint activation, but similar levels of Mre11 foci as in XP-V cells. Therefore, suppression of POLH expression levels by siRNA recapitulates most of the phenotypes seen in cells from XP-V patients with inactivating mutations in POLH.

Caffeine↗
PubMed2003

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.

Animals↗
PubMed2002

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. Later commentaries will take us sequentially through the early years of DNA repair.

Animals↗