Faculty Bibliography

Most metastatic melanoma patients fail to respond to available therapy, underscoring the need to develop more effective treatments. We screened 2,000 compounds from the Spectrum Library in human melanoma cell lines to identify compounds that enhanced the cytotoxic effect of temozolomide, a drug used to treat metastatic melanoma. Screening was done with the temozolomide-resistant melanoma cell line SK-MEL-19, and six compounds were identified that had little or no inherent cytotoxicity but significantly enhanced growth-inhibition by temozolomide. These compounds were tested in five additional melanoma cell lines. Cell proliferation and death assays were used to compare the efficacy of single agent temozolomide versus combination treatments. Effects of combination treatment on levels of DNA double-strand breaks, the DNA repair protein O(6)-methylguanine-DNA-methyltransferase, apoptosis [measured by cleaved caspase-3 and poly(ADP-ribose) polymerase], and cell cycle were examined. Pyrimethamine, an antiparasitic, sensitized melanoma cells to temozolomide. Temozolomide combined with Pyrimethamine synergistically inhibited cell proliferation in melanoma cells with combination index values of 0.7 or less. In addition, combination treatment induced cell cycle arrest and increased both DNA damage and apoptosis. The increase in cell death due to combination treatment was rescued by leucovorin. Other folate antagonists were also effective enhancers of temozolomide-induced cytotoxicity, and the effects of antifolates were also evident in gliomas. Our screening approach led to the identification of Pyrimethamine, an orally available drug that efficiently crosses the blood-brain barrier, as a potent enhancer of the efficacy of temozolomide as an antineoplastic agent via inhibition of folate metabolism

Lymphatic vessels play a key role in maintaining tissue-fluid homeostasis, immune surveillance and metastasis. The hyaluronan receptor, LYVE-1, is widely used as a molecular marker for adult and embryonic lymphatic endothelium, but its physiological functions have not yet been established in vivo. In agreement with a recent report, LYVE-1(-/-) mice, which are healthy and fertile, do not display any defects related to congenital abnormalities of the lymphatic system. One hypothesis for the absence of a phenotype in LYVE-1 null mice is that other hyaluronan receptors, such as CD44, may compensate for LYVE-1. To test this hypothesis, we created LYVE-1/CD44 double knockout mice with appropriate littermate controls. Lymphatic vessel structure and function, as determined by histological methods and intravital microscopy, show that LYVE-1(-/-), CD44(-/-) and LYVE-1(-/-)/CD44(-/-) mice are indistinguishable from wild-type mice under normal conditions. Furthermore, resolution of carrageenan-induced paw edema is comparable in all genotypes. However, LYVE-1(-/-)/CD44(-/-) mice exhibit increased edema formation in a carrageenan-induced paw inflammation model compared to wild-type mice, but not to LYVE(-/-) or CD44(-/-) mice. These data suggest that LYVE-1 and CD44 are not required for the formation or function of lymphatics, but do not rule out a role for LYVE-1 in inflammation

PURPOSE: To evaluate the capability of P947, a magnetic resonance (MR) imaging contrast agent that molecularly targets matrix metalloproteinases (MMPs), to aid detection and imaging of MMPs in atherosclerotic lesions in vivo; its specificity compared with that of P1135; expression and distribution of MMPs in atherosclerotic vessels; and in vivo distribution and molecular localization of fluorescent europium (Eu) P947. MATERIALS AND METHODS: The Animal Care and Use Committee approved all experiments. P947 was synthesized by attaching a gadolinium chelate (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) to a peptide that specifically binds MMPs. Scrambled form of P947 (P1135) was synthesized by replacing the targeting moiety of P947 with a scrambled peptide lacking the ability to bind MMPs. P947, P1135, and gadoterate meglumine were injected into atherosclerotic apolipoprotein E-deficient and wild-type mice. The aortic MR imaging enhancement produced by the contrast agents was measured at different times and was compared by using one-way analysis of variance. MMP expression was investigated in the aortas by using MMP immunostaining and in situ MMP zymography. A fluorescent form of P947 (Eu-P947) was synthesized to compare the in vivo distribution of the contrast agent (Eu-P947) with specific MMP immunofluorescent staining. RESULTS: MMP-targeted P947 facilitated a 93% increase (P < .001) in MR image signal intensity (contrast-to-noise ratio [CNR], 17.7 compared with 7.7; P < .001) of atherosclerotic lesions in vivo. Nontargeted P1135 (scrambled P947) provided 33% MR image enhancement (CNR, 10.8), whereas gadoterate meglumine provided 5% (CNR, 6.9). Confocal laser scanning microscopy demonstrated colocalization between fluorescent Eu-P947 and MMPs in atherosclerotic plaques. Eu-P947 was particularly present in the fibrous cap region of plaques. CONCLUSION: P947 improved MR imaging for atherosclerosis through MMP-specific targeting. The results were validated and provide support for further assessment of P947 as a potential tool for the identification of unstable atherosclerosis.

The objective of the paper is to evaluate the effect of acellular nerve allografts (ANA) seeded with Schwann cells to promote nerve regeneration after bridging the sciatic nerve defects of rats and to discuss its acting mechanisms. Schwann cells were isolated from neonatal Wistar rats. In vitro Schwann cells were microinjected into acellular nerve allografts and co-cultured. Twenty-four Wistar rats weighing 180-220 g were randomly divided into three groups with eight rats in each group: ANA seeded with Schwann cells (ANA + SCs), ANA group and autografts group. All the grafts were, respectively, served for bridging a 10-mm long surgically created sciatic nerve gap. Examinations of regeneration nerve were performed after 12 weeks by transmission electron microscope (TEM), scanning electron microscope (SEM), and electrophysiological methods, and then analyzed statistically. The results obtained indicated that in vitro Schwann cells displayed the feature of bipolar morphology with oval nuclei. Compared with ANA group, the conduction velocity of ANA + SCs group and autograft group was faster after 12 weeks, latent period was shorter, and wave amplitude was higher (P < 0.05). The difference between ANA + SCs group and autograft group is not significant (P > 0.05). Regeneration nerve myelinated fiber number, myelin sheath thickness, and myelinated fibers/total nerves (%) in both ANA + SCs group and autograft group are higher than that in ANA group; the difference is significant (P < 0.05). The difference between the former two is not significant (P > 0.05). In conclusion, ANA seeded with SCs could improve nerve regeneration and functional recovery after bridging the sciatic nerve gap of rats, which offers a novel approach for the repair peripheral nerve defect.

BACKGROUND: Apoptosis is a critical biological phenomenon, executed under the guidance of the Apoptotic Machinery (AM), which allows the physiologic elimination of terminally differentiated, senescent or diseased cells. Because of its relevance to BioMedicine, we have sought to obtain a detailed characterization of AM Omics in Homo sapiens, namely its Genomics and Evolution, Transcriptomics, Proteomics, Interactomics, Oncogenomics, and Pharmacogenomics. METHODS: This project exploited the methodology commonly used in Computational Biology (i.e., mining of many omics databases of the web) as well as the High Throughput biomolecular analytical techniques. RESULTS: In Homo sapiens AM is comprised of 342 protein-encoding genes (possessing either anti- or pro-apoptotic activity, or a regulatory function) and 110 MIR-encoding genes targeting them: some have a critical role within the system (core AM nodes), others perform tissue-, pathway-, or disease-specific functions (peripheral AM nodes). By overlapping the cancer type-specific AM mutation map in the fourteen most frequent cancers in western societies (breast, colon, kidney, leukaemia, liver, lung, neuroblastoma, ovary, pancreas, prostate, skin, stomach, thyroid, and uterus) to their transcriptome, proteome and interactome in the same tumour type, we have identified the most prominent AM molecular alterations within each class. The comparison of the fourteen mutated AM networks (both protein- as MIR-based) has allowed us to pinpoint the hubs with a general and critical role in tumour development and, conversely, in cell physiology: in particular, we found that some of these had already been used as targets for pharmacological anticancer therapy. For a better understanding of the relationship between AM molecular alterations and pharmacological induction of apoptosis in cancer, we examined the expression of AM genes in K562 and SH-SY5Y after anticancer treatment. CONCLUSION: We believe that our data on the Apoptotic Machinery will lead to the identification of new cancer genes and to the discovery of new biomarkers, which could then be used to profile cancers for diagnostic purposes and to pinpoint new targets for pharmacological therapy. This approach could pave the way for future studies and applications in molecular and clinical Medicine with important perspectives both for Oncology as for Regenerative Medicine

MicroRNAs are small regulatory RNAs that post-transcriptionally regulate gene expression and can be encoded by viral as well as cellular genomes. The functions of most viral miRNAs are unknown and few have been studied in an in vivo context. Here we show that the murine polyomavirus (PyV) encodes a precursor microRNA that is processed into two mature microRNAs, both of which are active at directing the cleavage of the early PyV mRNAs. Furthermore, we identify a deletion mutant of polyomavirus that is defective in encoding the microRNAs. This mutant replicates normally and transforms cultured cells with efficiencies comparable to wildtype PyV. The miRNA mutant is competent to establish a transient infection of mice following parenteral inoculation, and is cleared post infection at approximately the same rate as the wildtype virus. In addition, under these laboratory conditions, we observe no differences in anti-viral CD8 T cell responses. These results indicate that PyV miRNA expression is not essential for infection of cultured cells or experimentally inoculated mice, and raise the possibility that its role in natural infection might involve aspects of acquisition or spread that are not recapitulated by experimental inoculation.

MicroRNAs (miRNA) are short non-coding RNA molecules that regulate a variety of biological processes. The role of miRNAs in BMP2-mediated biological processes is of considerable interest. A comparative miRNA array led to the isolation of several BMP2-responsive miRNAs. Among them, miR-199a* is of particular interest, since it was reported to be specifically expressed in the skeletal system. Here we demonstrate that miR-199a* is an early responsive target of BMP2: its level was dramatically reduced at 5h, quickly increased at 24h and remained higher thereafter in the course of BMP2-triggered chondrogenesis of a micromass culture of pluripotent C3H10T1/2 stem cells. miR-199a* significantly inhibited early chondrogenesis, as revealed by the reduced expression of early marker genes for chondrogenesis such as COMP, type II collagen and Sox9, whereas anti- miR-199a* increased the expression of these chondrogenic marker genes. A computer-based prediction algorithm led to the identification of Smad1, a well-established downstream molecule of BMP-2 signaling, as a putative target of miR-199a*. The pattern of Smad1 mRNA expression exhibited the mirror opposite of miR-199a* expression following BMP-2 induction. Furthermore, miR-199a* demonstrated remarkable inhibition of both endogenous Smad1 as well as a reporter construct bearing the 3-UTR of Smad1 mRNA. In addition, mutation of miR-199a* binding sites in the 3'-UTR of Smad1 mRNA abolished miR-199a*-mediated repression of reporter gene activity. Mechanism studies revealed that miR-199a* inhibits Smad1/Smad4-mediated transactivation of target genes, and that overexpression of Smad1 completely corrects miR-199a*-mediated repression of early chondrogenesis. Taken together, miR-199a* is the first BMP2 responsive microRNA found to adversely regulate early chondrocyte differentiation via direct targeting of the Smad1 transcription factor

In this issue of Molecular Cell (Yanagitani et al., 2009) and recently published in Nature (Aragon et al., 2009) are reports on the importance of mRNA localization for unconventional splicing in the unfolded protein response, but the two papers suggest very different mechanisms in mammals and yeast

New and improved: The incorporation of a 6-chlorotryptophan (6-Cl-Trp) into a beta-peptide (M)-3(14) helix leads to a high-affinity hDM2 inhibitor, as demonstrated by fluorescence fluctuation analysis at single molecule resolution. When conjugated to penetratin, the newly derived hDM2 binder specifically inhibits tumour cell growth in vitro.

To ascertain the identities of cyclic nucleotide-binding proteins that mediate the insulin secretagogue action of cAMP, the possible contributions of the exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) were evaluated in a pancreatic beta-cell line (rat INS-1 cells). Assays of Rap1 activation, CREB phosphorylation, and PKA-dependent gene expression were performed in combination with live-cell imaging and high throughput screening of a FRET-based cAMP sensor (Epac1-camps) in order to validate the selectivities with which acetoxymethyl esters (AM-esters) of cAMP analogs preferentially activate Epac or PKA. Selective activation of Epac or PKA was achieved following exposure of INS-1 cells to 8-pCPT-2'-O-Me-cAMP-AM or db-cAMP-AM, respectively. Both cAMP analogs exerted dose-dependent and glucose metabolism-dependent actions to stimulate insulin secretion, and when each was co-administered with the other, a supra-additive effect was observed. Since 2.4-fold more insulin was secreted in response to a saturating concentration (10 micromolar) of db-cAMP-AM as compared to 8-pCPT-2'-O-Me-cAMP-AM, and because the action of db-cAMP-AM but not 8-pCPT-2'-O-Me-cAMP-AM was nearly abrogated by treatment with 3 micromolar of the PKA inhibitor H-89, it is concluded that for INS-1 cells, it is PKA that acts as the dominant cAMP-binding protein in support of insulin secretion. Unexpectedly, 10-100 micromolar of the non-AM-ester of 8-pCPT-2'-O-Me-cAMP failed to stimulate insulin secretion and was a weak activator of Rap1 in INS-1 cells. Moreover, 10 micromolar of the AM-ester of 8-pCPT-2'-O-Me-cAMP stimulated insulin secretion from mouse islets, whereas the non-AM ester did not. Thus, the membrane permeability of 8-pCPT-2'-O-Me-cAMP in insulin-secreting cells is so low as to limit its biological activity. It is concluded that prior reports documenting the failure of 8-pCPT-2'-O-Me-cAMP to act in beta cells, or other cell types, need to be re-evaluated through the use of the AM-ester of this cAMP analog