Altered expression of the poly(ADP-ribosyl)ation enzymes in uveal melanoma and regulation of PARG gene expression by the transcription factor ERM.

PURPOSE: Poly(ADP-ribosyl)ation is a reversible post-translational modification that requires the contribution of the enzymes poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Our study explores expression and activity of PARP-1 and PARG in uveal melanoma cell lines with varying tumorigenic properties. METHODS: Gene profiling on microarrays was conducted using RNA prepared from the uveal melanoma cell lines T97, T98, T108, and T115. The activity of PARP-1 and PARG was monitored by enzymatic assays, whereas their expression was measured by Western blot and PCR. The PARG promoter was analyzed using promoter deletions and site-specific mutagenesis in transfection analyses. The transcription factors binding the PARG promoter were studied by electrophoretic mobility shift assay (EMSA) analyses. Suppression of PARP-1 and PARG expression was performed in T97 and T115 cells by RNAi, and their tumorigenic properties monitored by injections into athymic mice. RESULTS: Expression of PARP-1 was found to vary considerably between uveal melanoma cell lines with distinctive tumorigenic properties in vivo. Sp1 and the ETS protein ERM were shown to bind to the PARG gene promoter to ensure basal transcription in uveal melanoma. Importantly, suppression of PARG gene expression in T97 and T115 cells increased their capacity to form tumors in athymic mice, whereas suppression of PARP-1 significantly reduced or almost entirely abolished tumor formation. CONCLUSIONS: Our results suggest that while overexpression of PARP-1 may confer a proliferative advantage to aggressive uveal melanoma tumors, PARG may, on the other hand, support a tumor suppressor function in vivo.

Poly(ADP-ribose) metabolism analysis in the nematode Caenorhabditis elegans.

Poly(ADP-ribose) polymerases (PARPs) are a well-conserved family of enzymes found in many species. These enzymes catalyze poly(ADP-ribosyl)ation, a modification of proteins implicated in a variety of nuclear processes, such as DNA damage signaling and repair, cell death and survival, and transcription. Poly(ADP-ribose) glycohydrolase (PARG) is responsible for the specific hydrolysis of poly(ADP-ribose) (PAR), the product of poly(ADP-ribosyl)ation, and its action is required for the modified proteins to regain their original function in the cell. The metabolism of PAR can be studied in the nematode Caenorhabditis elegans as genes encoding PARP and PARG enzymes have been identified and characterized in its genome. We have shown the capacity of these PARPs to produce PAR as well as the capacity of the nematode to catabolize PAR into ADP-ribose units through the enzymatic activity of its PARGs. Therefore, C. elegans is a novel model to study PAR metabolism in eukaryotes that offers new avenues to investigate the role(s) of poly(ADP-ribosyl)ation in development as well as DNA repair, programmed cell death, and aging.

Photoactivated multivitamin preparation induces poly(ADP-ribosyl)ation, a DNA damage response in mammalian cells.

Multivitamin preparation (MVP) is part of total parenteral nutrition given to premature infants. Photoactivated MVP carries an important load in peroxides, but their cellular effects have not yet been determined. We hypothesized that these peroxides may elicit a DNA-damage response. We found that photoactivation of MVP and the resulting peroxide production were time-dependent and required the simultaneous presence of ascorbic acid and riboflavin. Cells treated with photoactivated MVP showed strongly stimulated poly(ADP-ribosyl)ation, an early DNA-damage response in mammals. Poly(ADP-ribosyl)ation stimulation was dependent on the presence of ascorbic acid and riboflavin in the photoactivated MVP. It did not occur in the presence of a specific PARP inhibitor nor in mouse fibroblasts deficient in PARP-1. Photoactivated MVP was able to induce single- and double-strand breaks in DNA, with a predominance of single-stand breaks. The presence of double-strand breaks was further confirmed using a 53PB1 focus analysis. Finally, photoactivated MVP was shown to be toxic to human cells and induced caspase-independent cell death. These results suggest that photoactivated MVP carries an important toxic load able to damage DNA and induce cell death. This study also emphasizes the importance of protecting MVP solution from light before use in preterm infants.

The DNA damage-inducible C. elegans tankyrase is a nuclear protein closely linked to chromosomes.

Tankyrases are protein members of the poly(ADP-ribose) polymerase family bearing several ankyrin domain and a WGR domain. They have functional role in telomere maintenance, are found at centrosome, and are associated with vesicular secretion. This diversity in localization and function makes it difficult to identify a unified role for tankyrases. We have shown that the C. elegans orthologue PME-5 is among the most transcriptionally up-regulated genes following ionizing radiations, linking a tankyrase with DNA damage response. Our analysis showed that the up-regulation of PME-5 is translated at the protein level, suggesting an effective role in DNA damage response or DNA repair. In order to gain more information on the potential role of PME-5 in DNA damage response, we analyzed its sub-cellular localization. Using immunostaining as well as gfp reporter assay, we have shown a nuclear localization for PME-5. Moreover, we showed that PME-5 is a ubiquitous nuclear protein expressed throughout the development of the worm and is closely linked to chromatin and condensed chromosomes. Taken together, our data suggest that C. elegans can be used to study the nuclear roles of tankyrase.

Caenorhabditis elegans LET-767 is able to metabolize androgens and estrogens and likely shares common ancestor with human types 3 and 12 17beta-hydroxysteroid dehydrogenases.

Mutations that inactivate LET-767 are shown to affect growth, reproduction, and development in Caenorhabditis elegans. Sequence analysis indicates that LET-767 shares the highest homology with human types 3 and 12 17beta-hydroxysteroid dehydrogenases (17beta-HSD3 and 12). Using LET-767 transiently transfected into human embryonic kidney-293 cells, we have found that the enzyme catalyzes the transformation of both 4-androstenedione into testosterone and estrone into estradiol, similar to that of mouse 17beta-HSD12 but different from human and primate enzymes that catalyze the transformation of estrone into estradiol. Previously, we have shown that amino acid F234 in human 17beta-HSD12 is responsible for the selectivity of the enzyme toward estrogens. To assess whether this amino acid position 234 in LET-767 could play a role in androgen-estrogen selectivity, we have changed the methionine M234 in LET-767 into F. The results show that the M234F change causes the loss of the ability to transform androstenedione into testosterone, while conserving the ability to transform estrone into estradiol, thus confirming the role of amino acid position 234 in substrate selectivity. To further analyze the structure-function relationship of this enzyme, we have changed the three amino acids corresponding to lethal mutations in let-767 gene. The data show that these mutations strongly affect the ability of LET-767 to convert estrone in to estradiol and abolish its ability to transform androstenedione into testosterone. The high conservation of the active site and amino acids responsible for enzymatic activity and substrate selectivity strongly suggests that LET-767 shares a common ancestor with human 17beta-HSD3 and 12.

Altered DNA damage response in Caenorhabditis elegans with impaired poly(ADP-ribose) glycohydrolases genes expression.

Poly(ADP-ribosyl)ation is one of the first cellular responses induced by DNA damage. Poly(ADP-ribose) is rapidly synthesized by nick-sensor poly(ADP-ribose) polymerases, which facilitate DNA repair enzymes to process DNA damage. ADP-ribose polymers are rapidly catabolized into free ADP-ribose units by poly(ADP-ribose) glycohydrolase (PARG). The metabolism of poly(ADP-ribose) is a well-defined biochemical process for which a physiological role in animals is just beginning to emerge. Two Caenorhabditis elegans PARGs, PME-3 and PME-4, have been cloned by our group. The pme-3 gene encodes an enzyme of 89kDa having less than 18% overall identity with human PARG but 42% identity with the PARG signature motif. The pme-4 gene codes for a PARG of 55kDa with approximately 22% overall identity with human PARG and 40% identity with the PARG signature motif. Two alternatively spliced forms of PME-3 were identified with an SL1 splice leader on both forms of the mRNA and were found to be expressed throughout the worm's life cycle. Similarly, pme-4 was shown to be expressed in all developmental stages of the worm. Recombinant enzymes that were expressed in bacteria displayed a PARG activity that may partly account for the PARG activity measured in the total worm extract. Reporter gene analysis of pme-3 and pme-4 using a GFP fusion construct showed that pme-3 and pme-4 are mainly expressed in nerve cells. PME-3 was shown to be nuclear while PME-4 localized to the cytoplasm. Worms with pme-3 and pme-4 expression knocked-down by RNAi showed a significant sensitivity toward ionizing radiations. Taken together, these data provide evidence for a physiological role for PARGs in DNA damage response and survival. It also shows that PARGs are evolutionarily conserved enzymes and that they are part of an ancient cellular response to DNA damage.

The Caenorhabditis elegans FancD2 ortholog is required for survival following DNA damage.

Fanconi anemia (FA) is an autosomal recessive disease characterized by bone-marrow failure, congenital abnormalities, and cancer susceptibility. There are 11 FA complementation groups in human where 8 genes have been identified. We found that FancD2 is conserved in evolution and present in the genome of the nematode Caenorhabditis elegans. The gene Y41E3.9 (CeFancD2) encodes a structural ortholog of human FANCD2 and is composed of 10 predicted exons. Our analysis showed that exons 6 and 7 were absent from a CeFancD2 EST suggesting the presence of a splice variant. In an attempt to characterize its role in DNA damage, we depleted worms of CeFANCD2 using RNAi. When the CeFANCD2(RNAi) worms were treated with a crosslinking agent, a significant drop in the progeny survival was noted. These worms were also sensitive, although to a lesser extent, to ionizing radiation (IR). Therefore, these data support an important role for CeFANCD2 in DNA damage response as for its human counterpart. The data also support the usefulness of C. elegans to study the Fanconi anemia pathway, and emphasize the biological importance of FANCD2 in DNA damage response throughout evolution.