Melanoma is the most fatal skin cancer, often highly resistant to chemotherapy. Here we show that treatment with an 11-base DNA oligonucleotide homologous to the telomere 3' overhang sequence (T-oligo) induces apoptosis of several established human melanoma cell lines, including the aggressive MM-AN line, whereas normal human melanocytes exposed to the same or higher T-oligo concentrations show only transient cell cycle arrest, implying that malignant cells are more sensitive to T-oligo effects. When MM-AN cells were briefly exposed to T-oligo in culture and injected into the flank or tail vein of SCID mice, eventual tumor volume and number of metastases were reduced 85-95% compared with control mice. Similarly, T-oligos administered intralesionally or systemically selectively inhibited the growth of previously established MM-AN tumor nodules in the flank and peritoneal cavity by 85 to 90% without detectable toxicity. We previously showed that T-oligos act through ATM, p95/Nbs1, E2F1, p16INK4A, p53, and the p53 homologue p73 to modulate downstream effectors and now additionally demonstrate striking down-regulation of the inhibitor of apoptosis protein livin/ML-IAP. We suggest that T-oligo mimics a physiologic DNA damage signal that is frequently masked in malignant cells and thereby activates innate cancer prevention responses. T-oligos may provide a novel therapeutic approach to melanoma.

PIF1 is a 59 to 39 DNA helicase that can unwind double-stranded DNA and disrupt nucleic acid-protein complexes. In Saccharomyces cerevisiae, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, we created pif1 null mutants in Drosophila melanogaster and assessed their phenotypes throughout development. We found that pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, experience reduced survival to adulthood. Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions with POL32 during times of replication stress but acts independently of POL32 to promote synthesis during double-strand gap repair. KEYWORDS break-induced replication; polymerase; homologous recombination; DNA damage; replication fork collapse T HE Pif1 family helicases are 59 to 39 superfamily 1 helicases that are highly conserved in most eukaryotes and some bacteria and are critical for DNA replication, recombination, and repair (Bochman et al. 2010; Chung 2014; Byrd and Raney 2017). Although Pif1 family helicases possess a conserved single-stranded DNA (ssDNA)-dependent helicase domain, their N-and C-terminal domains differ significantly in size and sequence between organisms (Supplemental Material, Figure S1) (Lahaye et al. 1991; Boulé and Zakian 2006). Furthermore, the cellular functions of PIF1 homologs in eukaryotes are variably conserved. Because human PIF1 appears to act as a tumor suppressor but is also required for the survival of cancer cells (Gagou et al. 2014), a better understanding of its contribution to genome stability in diverse cellular and organismal contexts is needed. Much of what is currently known about Pif1 structure and function comes from studies in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. S. cerevisiae possesses two Pif1 orthologs, ScPif1 and ScRrm3, which localize to both the mitochondria and nucleus (Ivessa et al. 2000; Bochman et al. 2010). ScPif1 was first discovered in a genetic screen where its deficiency resulted in reduced mitochondrial DNA (mtDNA) recombination (Foury and Kolodynski 1983). Further studies revealed that ScPif1 slows down replication progression in mtDNA to prevent double-strand breaks (DSBs) and cooperates with base excision ...

Cytoplasmic dynein is a microtubule-associated motor molecule involved in the retrograde transport of membrane-bound organelles. To determine whether the supranuclear melanin cap of transferred, phagocytosed melanosomes in keratinocytes is associated with cytoplasmic dynein, we performed immunofluorescent confocal microscopy on human keratinocytes in situ. We identified the intermediate chain of cytoplasmic dynein by immunoblotting and examined its distribution by confocal microscopy in relation to microtubules and melano-phagolysosomes in vitro. We also used antisense and sense oligonucleotides of the cytoplasmic dynein heavy chain 1 (Dyh1) and time-lapse and microscopy. The intermediate chain of cytoplasmic dynein was identified in extracts of human foreskin epidermis and in isolated human keratinocytes. The intermediate chain localized with the perinuclear melano-phagolysosomal aggregates in vitro and the supranuclear melanin cap in situ. Antisense oligonucleotides directed towards Dyh1 resulted in dispersal of the keratinocyte perinuclear melano-phagolysosomal aggregates after 24 to 48 h, whereas cells treated with diluent or sense oligonucleotides maintained tight perinuclear aggregates. Taken together, these findings indicate that in human keratinocytes, the retrograde microtubule motor cytoplasmic dynein mediates the perinuclear aggregation of phagocytosed melanosomes, participates in the formation of the supranuclear melanin cap or "microparasol" and serves as a mechanism to help protect the nucleus from ultraviolet-induced DNA damage.

The keratinocyte microparasol, composed of a perinuclear microtubular/melano-phagolysosomal complex, protects the nucleus from UV-induced DNA damage. We have previously demonstrated that cytoplasmic dynein is the motor involved in the perinuclear-directed aggregation of phagocytosed melanosomes. Dynactin, of which p150(Glued) is the major subunit, can link directly to microtubules and links organelles to dynein at different domains. To further define the mechanism of the microparasol, we transfected siRNA targeted against p150(Glued) into human keratinocytes cultured with 0.5 mm fluorescent microspheres and performed time-lapse analysis, confocal immunolocalization, and Western immunoblotting after 24 and 48 hours. Western blots revealed a significant knockdown of the p150(Glued) subunit. The knockdown decreased p150(Glued) colocalization with microtubules and decreased perinuclear positioning of the convergent microtubular framework. It also inhibited perinuclear aggregation of phagocytosed fluorescent microspheres and reduced mean centripetal microsphere displacement. The findings provide evidence that dynactin p150(Glued) plays an important role in the functional integrity of the keratinocyte microparasol.

PIF1 is a 5' to 3' DNA helicase that can unwind double-stranded DNA and disrupt nucleic acidprotein complexes. In Saccharomyces cerevisiae, Pif1 plays important roles in mitochondrial and nuclear genome maintenance, telomere length regulation, unwinding of G-quadruplex structures, and DNA synthesis during break-induced replication. Some, but not all, of these functions are shared with other eukaryotes. To gain insight into the evolutionarily conserved functions of PIF1, we created pif1 null mutants in Drosophila melanogaster and assessed their phenotypes throughout development. We found that pif1 mutant larvae exposed to high concentrations of hydroxyurea, but not other DNA damaging agents, experience reduced survival to adulthood.Embryos lacking PIF1 fail to segregate their chromosomes efficiently during early nuclear divisions, consistent with a defect in DNA replication. Furthermore, loss of the BRCA2 protein, which is required for homologous recombination and stabilization of stalled replication forks in metazoans, causes synthetic lethality in third instar larvae lacking either PIF1 or the polymerase delta subunit POL32. Interestingly, pif1 mutants have a reduced ability to synthesize DNA during repair of a double-stranded gap, but only in the absence of POL32. Together, these results support a model in which Drosophila PIF1 functions to promote efficient DNA synthesis during times of replication stress and acts independently of POL32 to promote synthesis during doublestrand gap repair.