UVA irradiation leads to photoaging including clinical features such as wrinkle formation, reduced recoil capacity and blister formation of the skin. Besides synthesis of the extracellular matrix, its regulated degradation by various matrix-metalloproteinases (MMPs) determines the amount and the composition of the extracellular matrix within the dermis and the basement membrane of the dermo-epidermal junction. In this study we therefore ascertained whether UV irradiation could modulate the synthesis of MMPs with substrate specificities for dermal (collagen I, III, V) and basement membrane compounds (collagen IV, VII, proteoglycans, laminin) and whether synthesis of the counteracting tissue inhibitor of metalloproteinases (TIMP-1) was also affected. Following UVA irradiation specific mRNAs of MMPs 1, 2 and 3 were induced concomitantly up to 5-fold compared to mock irradiated controls. In contrast, TIMP-1 mRNA levels remained unaltered. Immunoprecipitation indicated that after UVA irradiation synthesis and secretion of MMPs 1, 2 and 3 into the supernatant increased. Taken together, our data show that UVA irradiation coordinately induced MMPs 1, 2 and 3 implying similar mechanisms in their regulatory pathways, while TIMP-1 synthesis was not altered. Hence, unbalanced synthesis of MMPs potentially contributes to the dissolution of dermal and basement membrane compounds finally leading to blister formation and cutaneous photoaging.

Selective overexpression of Human epididymal secretory protein E4 (HE4) points to a role in ovarian cancer tumorigenesis but little is known about the role the HE4 gene or the gene product plays. Here we show that elevated HE4 serum levels correlate with chemoresistance and decreased survival rates in EOC patients. HE4 overexpression promoted xenograft tumor growth and chemoresistance against cisplatin in an animal model resulting in reduced survival rates. HE4 displayed responses to tumor microenvironment constituents and presented increased expression as well as nuclear translocation upon EGF, VEGF and Insulin treatment and nucleolar localization with Insulin treatment. HE4 interacts with EGFR, IGF1R, and transcription factor HIF1α. Constructs of antisense phosphorothio-oligonucleotides targeting HE4 arrested tumor growth in nude mice. Collectively these findings implicate increased HE4 expression as a molecular factor in ovarian cancer tumorigenesis. Selective targeting directed towards the HE4 protein demonstrates therapeutic benefits for the treatment of ovarian cancer.

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Recent studies on the 2'-O-methylation and pseudouridylation of U6 small nuclear RNA (snRNA) hypothesize that these posttranscriptional modifications might occur in the nucleolus. In this report, we present direct evidence for the nucleolar localization of U6 snRNA and analyze the kinetics of U6 nucleolar localization after injection of in vitro transcribed fluorescein-labeled transcripts into Xenopus laevis oocytes. In contrast to U3 small nucleolar RNA (snoRNA) which developed strong nucleolar labeling over 4 h and maintained strong nucleolar signals through 24 h, U6 snRNA localized to nucleoli immediately after injection, but nucleolar staining decreased after 4 h. By 24 h after injection of U6 snRNA, only weak nucleolar signals were observed. Unlike the time-dependent profile of strong nucleolar localization of U6 snRNA or U3 snoRNA, injection of fluorescein-labeled U2 snRNA gave weak nucleolar staining at all times throughout a 24-h period; U2 snRNA modifications are believed to occur outside of the nucleolus. The notion that the decrease of U6 signals over time was due to its trafficking out of nucleoli and not to transcript degradation was supported by the demonstration of U6 snRNA stability over time. Therefore, in contrast to snoRNAs like U3, U6 snRNA transiently passes through nucleoli.

Abstract. Cranberry extracts may provide beneficial health effects in the treatment of various diseases, including cancer. However, the underlying molecular mechanisms of antineoplastic properties are not understood. We report the effect of a proanthocyanidin (PAC)-rich isolate from cranberry (PAC-1) as a therapeutic agent with dual activity to target both ovarian cancer viability and angiogenesis in vitro. PAC-1 treatment of chemotherapy-resistant SKOV-3 cells blocked cell cycle progression through the G 2 /M phase, increased the generation of reactive oxygen species (ROS), and induced apoptosis through activation of intrinsic and extrinsic pathway components. Cytotoxicity of PAC-1 was partially based on ROS generation and could be blocked by co-treatment with antioxidant glutathione. PAC-1 reduced the cell viability of both SKOV-3 ovarian cancer cells and HUVEC endothelial cells in a dose-dependent manner and blocked the activation of the pro-survival factor AKT. Furthermore, PAC-1 blocked vascular endothelial growth factor (VEGF)-stimulated receptor phosphorylation in endothelial cells, which correlated with the inhibition of endothelial tube formation in vitro. Our findings suggest that PAC-1 exerts potent anticancer and anti-angiogenic properties and that highly purified PAC from cranberry can be further developed to treat ovarian cancer in combinational or single-agent therapy.

U8 small nucleolar RNA (snoRNA) is essential for metazoan ribosomal RNA (rRNA) processing in nucleoli. The sequences and structural features in Xenopus U8 snoRNA that are required for its nucleolar localization were analyzed. Fluorescein-labeled U8 snoRNA was injected into Xenopus oocyte nuclei, and fluorescence microscopy of nucleolar preparations revealed that wild-type Xenopus U8 snoRNA localized to nucleoli, regardless of the presence or nature of the 59 cap on the injected U8 snoRNA. Nucleolar localization was observed when loops or stems in the 59 portion of U8 that are critical for U8 snoRNA function in rRNA processing were mutated. Therefore, sites of interaction in U8 snoRNA that potentially tether it to pre-rRNA are not essential for nucleolar localization of U8. Boxes C and D are known to be nucleolar localization elements (NoLEs) for U8 snoRNA and other snoRNAs of the Box C/D family. However, the spatial relationship of Box C to Box D was not crucial for U8 nucleolar localization, as demonstrated here by deletion of sequences in the two stems that separate them. These U8 mutants can localize to nucleoli and function in rRNA processing as well. The single-stranded Cup region in U8, adjacent to evolutionarily conserved Box C, functions as a NoLE in addition to Boxes C and D. Cup is unique to U8 snoRNA and may help bind putative protein(s) needed for nucleolar localization. Alternatively, Cup may help to retain U8 snoRNA within the nucleolus.

Previously, we showed that spliceosomal U6 small nuclear RNA (snRNA) transiently passes through the nucleolus. Herein, we report that all individual snRNAs of the [U4/U6.U5] tri-snRNP localize to nucleoli, demonstrated by fluorescence microscopy of nucleolar preparations after injection of fluorescein-labeled snRNA into Xenopus oocyte nuclei. Nucleolar localization of U6 is independent from [U4/U6] snRNP formation since sites of direct interaction of U6 snRNA with U4 snRNA are not nucleolar localization elements. Among all regions in U6, the only one required for nucleolar localization is its 3Ј end, which associates with the La protein and subsequently during maturation of U6 is bound by Lsm proteins. This 3Ј-nucleolar localization element of U6 is both essential and sufficient for nucleolar localization and also required for localization to Cajal bodies. Conversion of the 3Ј hydroxyl of U6 snRNA to a 3Ј phosphate prevents association with the La protein but does not affect U6 localization to nucleoli or Cajal bodies. INTRODUCTIONThe nucleolus is the site of ribosome biogenesis (for review, see Gerbi et al., 2001). However, the nucleolus seems to be plurifunctional and contains RNA used for other events, such as the RNA component of RNase P, which catalyzes the 5Ј processing of pre-tRNA (Jacobson et al., 1997;Bertrand et al., 1998;Jarrous et al., 1999), signal recognition particle RNA that assembles with proteins in the nucleolus (Jacobson and Pederson, 1998;Politz et al., 2000) and telomerase RNA (Mitchell et al., 1999;Narayanan et al., 1999b).Recently, it has been reported that several of the small nuclear RNAs (snRNAs) pass through the nucleolus before their nucleoplasmic destination where splicing occurs. U6 snRNA transiently localizes to nucleoli (Lange and Gerbi, 2000) where it seems to undergo 2Ј-O-methylation and pseudouridylation of defined nucleotides, guided by small nucleolar RNAs (snoRNAs) (Tycowski et al., 1998;Ganot et al., 1999). Similarly, U2 snRNA is found in nucleoli (Lange and Gerbi, 2000) where it seems to be modified by guide snoRNAs, probably after reimport to the nucleus from the cytoplasm (Yu et al., 2001). In addition, guide snoRNAs for modification of several spliceosomal snRNAs have also been identified (Hü ttenhofer et al., 2001). In this report, we present direct evidence that two of the targets of modification, U4 and U5 snRNAs, localize to nucleoli. Therefore, the list of snRNAs associated with nucleoli is expanded to U6 (Lange and Gerbi, 2000), U2 (Lange and Gerbi, 2000;Yu et al., 2001), and U4 and U5 snRNAs (this report).The observation of nucleolar localization of U6 snRNA raises the question at which point of its life cycle U6 enters the nucleolus. Upon transcription by RNA polymerase III (Dahlberg and Lund, 1991), the first protein to associate with U6 snRNA is La (Rinke and Steitz, 1985;Kunkel et al., 1986), which generally binds to the 3Ј termini of nascent RNA polymerase III transcripts and a number of viral RNAs (Chang et al., 1994;Simons et al., 1996). Subsequently, the...

The Nucleolar Localization Elements (NoLEs) of Xenopus laevis U3 small nucleolar RNA (snoRNA) have been defined. Fluorescein-labeled wild-type U3 snoRNA injected into Xenopus oocyte nuclei localized specifically to nucleoli as shown by fluorescence microscopy. Injection of mutated U3 snoRNA revealed that the 5Ј region containing Boxes A and AЈ, known to be important for rRNA processing, is not essential for nucleolar localization. Nucleolar localization of U3 snoRNA was independent of the presence and nature of the 5Ј cap and the terminal stem. In contrast, Boxes C and D, common to the Box C/D snoRNA family, are critical elements for U3 localization. Mutation of the hinge region, Box B, or Box CЈ led to reduced U3 nucleolar localization. Results of competition experiments suggested that Boxes C and D act in a cooperative manner. It is proposed that Box B facilitates U3 snoRNA nucleolar localization by the primary NoLEs (Boxes C and D), with the hinge region of U3 subsequently base pairing to the external transcribed spacer of pre-rRNA, thus positioning U3 snoRNA for its roles in rRNA processing. INTRODUCTIONMany aspects of how macromolecules are targeted to their correct subcellular destination still need to be defined. Although principles governing RNA export to the cytoplasm and import into the nucleus are beginning to be understood, very little is known about signals that direct RNA within the nucleus to the nucleolus. The nucleolus contains a vast array of different RNA species involved in ribosome biogenesis: ribosomal RNA (rRNA) and its precursors and ϳ200 small nucleolar RNAs (snoRNAs). Unlike rRNA whose genes are located within the nucleolus, the other transcripts found in the nucleolus must travel there from their sites of synthesis in the nucleoplasm. What are the "zip codes" for targeting snoRNA to the nucleolus?To address this question, we have studied U3 snoRNA because it is the most abundant snoRNA in the nucleolus, has been sequenced from a large number of animals and plants (Gu and Reddy, 1997), and is well characterized in terms of secondary structure and function in rRNA processing. U3 snoRNA is synthesized in the nucleoplasm in the proximity of coiled bodies (Gao et al., 1997), and formation of its trimethylguanosine cap can occur in the nucleoplasm (Terns and Dahlberg, 1994;Terns et al., 1995), unlike spliceosomal snoRNAs that are exported to the cytoplasm for cap trimethylation (Mattaj, 1986). Once U3 snoRNA is transported to the nucleolus, it is found highly concentrated in the dense fibrillar component (Matera et al., 1994) where initial rRNA processing events are believed to occur, but up to half of the U3 snoRNA is also detected by electron microscopy to be diffuse in the granular component Puvion-Dutilleul et al., 1991Azum-Gélade et al., 1994) where later rRNA processing cleavages take place. On the basis of the localization of U3 snoRNA after various actinomycin D treatments (Puvion-Dutilleul et al., 1992;Rivera-Leó n and Gerbi, 1997), a model has been proposed in which U3 snoRNA trave...