Cisplatin-conjugated gold nanoparticles as a theranostic agent for head and neck cancer.

BACKGROUND: The purpose of this study was to develop a nanoplatform, which simultaneously acts as radiosensitizer, drug carrier, and tumor imaging agent for head and neck cancer. METHODS: We synthesized 20 nm gold nanoparticles, coated with glucose and cisplatin (CG-GNPs). Their penetration into tumor cells and their cellular toxicity were evaluated in vitro. In vivo experiments were conducted to evaluate their impact on tumor growth and their imaging capabilities. RESULTS: The CG-GNPs showed efficient penetration into tumor cells and similar cellular toxicity as cisplatin alone. Combined with radiation, CG-GNPs led to greater tumor reduction than that of radiation alone and radiation with free cisplatin. The CG-GNPs also demonstrated efficient tumor imaging capabilities. CONCLUSION: Our CG-GNPs have a great potential to increase antitumor effect, overcome resistance to chemotherapeutics and radiation, and allow imaging-guided therapy.

Lighting up individual DNA damage sites by in vitro repair synthesis.

DNA damage and repair are linked to fundamental biological processes such as metabolism, disease, and aging. Single-strand lesions are the most abundant form of DNA damage; however, methods for characterizing these damage lesions are lacking. To avoid double-strand breaks and genomic instability, DNA damage is constantly repaired by efficient enzymatic machinery. We take advantage of this natural process and harness the repair capacity of a bacterial enzymatic cocktail to repair damaged DNA in vitro and incorporate fluorescent nucleotides into damage sites as part of the repair process. We use single-molecule imaging to detect individual damage sites in genomic DNA samples. When the labeled DNA is extended on a microscope slide, damage sites are visualized as fluorescent spots along the DNA contour, and the extent of damage is easily quantified. We demonstrate the ability to quantitatively follow the damage dose response to different damaging agents as well as repair dynamics in response to UV irradiation in several cell types. Finally, we show the modularity of this single-molecule approach by labeling DNA damage in conjunction with 5-hydroxymethylcytosine in genomic DNA extracted from mouse brain tissue.

The PIM-2 kinase is an essential component of the ultraviolet damage response that acts upstream to E2F-1 and ATM.

The oncogenic nature ascribed to the PIM-2 kinase relies mostly on phosphorylation of substrates that act as pro-survival/anti-apoptotic factors. Nevertheless, pro-survival effects can also result from activating DNA repair mechanisms following damage. In this study, we addressed the possibility that PIM-2 plays a role in the cellular response to UV damage, an issue that has never been addressed before. We found that in U2OS cells, PIM-2 expression and activity increased upon exposure to UVC radiation (2-50 mJ/cm(2)), and Pim-2-silenced cells were significantly more sensitive to UV radiation. Overexpression of PIM-2 accelerated removal of UV-induced DNA lesions over time, reduced γH2AX accumulation in damaged cells, and rendered these cells significantly more viable following UV radiation. The protective effect of PIM-2 was mediated by increased E2F-1 and activated ATM levels. Silencing E2F-1 reduced the protective effect of PIM-2, whereas inhibiting ATM activity abrogated this protective effect, irrespective of E2F-1 levels. The results obtained in this study place PIM-2 upstream to E2F-1 and ATM in the UV-induced DNA damage response.

Multiple signal transduction pathways are involved in G2/M growth arrest and apoptosis induced by the immunomodulator AS101 in multiple myeloma.

The organotellurium compound, AS101, induces G(2)/M growth arrest and apoptosis in multiple myeloma (MM) cell lines. To characterize the mechanism by which AS101 promotes these effects, an antibody microarray analysis was performed, comparing levels of proteins and phosphoproteins in untreated versus AS101-treated mouse 5T33 MM cells. We found that AS101 down-regulated Ilk-1, Cdc25C and phosphorylation of Plk-1 on Thr210, all of which can affect the onset of mitosis or cell survival. In addition, AS101 inhibited the activity of a high molecular weight matrix metalloproteinase complex corresponding to the MMP-9/NGAL complex. Another signaling pathway that was affected by AS101 involves p53 and p65/RelA. Levels of both proteins were elevated upon treatment with AS101. Thus, multiple signaling pathways are involved in the G(2)/M growth arrest and apoptosis induced by AS101 in multiple myeloma, suggesting that if one pathway becomes unresponsive, the therapeutic effect of AS101 might persist through alternative pathways.

Activation of cell cycle arrest and apoptosis by the proto-oncogene Pim-2.

Potent survival effects have been ascribed to the serine/threonine kinase proto-oncogene PIM-2. Elevated levels of PIM-2 are associated with various malignancies. In human cells, a single Pim-2 transcript gives rise mainly to two protein isoforms (34, 41 kDa) that share an identical catalytic site but differ at their N-terminus, due to in-frame alternative translation initiation sites. In this study we observed that the 34 kDa PIM-2 isoform has differential nuclear and cytoplasmic forms in all tested cell lines, suggesting a possible role for the balance between these forms for PIM-2's function. To further study the cellular role of the 34 kDa isoform of PIM-2, an N-terminally HA-tagged form of this isoform was transiently expressed in HeLa cells. Surprisingly, this resulted in increased level of G1 arrested cells, as well as of apoptotic cells. These effects could not be obtained by a Flag-tagged form of the 41 kDa isoform. The G1 arrest and apoptotic effects were associated with an increase in T14/Y15 phosphorylation of CDK2 and proteasom-dependent down-regulation of CDC25A, as well as with up-regulation of p57, E2F-1, and p73. No such effects were obtained upon over-expression of a kinase-dead form of the HA-tagged 34 kDa PIM-2. By either using a dominant negative form of p73, or by over-expressing the 34 kDa PIM-2 in p73-silenced cells, we demonstrated that these effects were p73-dependent. These results demonstrate that while PIM-2 can function as a potent survival factor, it can, under certain circumstances, exhibit pro-apoptotic effects as well.

Viral encephalitis of tilapia larvae: primary characterization of a novel herpes-like virus.

We report here an outbreak of an acute disease that caused high mortality rate in laboratory-reared tilapia larvae. The disease was initially observed in inbred gynogenetic line of blue tilapia larvae (Oreochromis aureus) and could be transmitted to larvae of other tilapia species. Based on the clinical manifestation (a whirling syndrome), we refer to the disease as viral encephalitis of tilapia larvae. The disease-associated DNA virus is described and accordingly designated tilapia larvae encephalitis virus (TLEV). A primary morphological, biophysical and molecular characterization of TLEV is presented. By virtue of these properties, the newly discovered virus is a herpes-like virus. Phylogenetic analysis, albeit limited, confirms this assumption and places TLEV within the family of Herpesviridae and distantly from the families Alloherpesviridae and Iridoviridae. By using PCR with virus-specific primers, diseased larvae and adult TLEV carriers were also identified in tilapia delivered from external hatcheries.

Expression and chromosomal organization of mouse meiotic genes.

Microarray technology which enables large scale analysis of gene expression and thus comparison between transcriptomes of different cell types, cells undergoing different treatments or cells at different developmental stages has also been used to study the transcriptome involved with spermatogenesis. Many new germ cell-specific genes were determined, and the resulting genes were classified according to different criteria. However, the biological significance of these classifications and their clustering according to developmental transcriptional patterns during spermatogenesis have not yet been addressed. In this study we utilized mouse testicular transcriptome analysis at five distinct post-natal ages (Days 7, 10, 12, 14, and 17), representing distinct meiotic stages, in an attempt to better understand the biological significance of genes clustered into similar expression patterns during this process. Among 790 sequences that showed an expression level change of twofold or more in any of the five key stages that were monitored, relative to the geometric average of all stages, about 40% peaked and about 30% were specifically suppressed at post-natal day 14 (representing the early pachytene stage of spermatocytes), reflecting tight transcriptional regulation at this stage. We also found that each of the six main transcription clusters that were determined was characterized by statistically significant representation of genes related to specific biological processes. Finally, our results indicated that genes important for meiosis are not randomly distributed along the mouse genome but rather preferentially located on specific chromosomes, suggesting for the first time that chromosomal location might be a regulating factor of meiotic gene expression.

Meig1 deficiency causes a severe defect in mouse spermatogenesis.

Meig1 is a mouse gene, abundantly expressed in the testis. It encodes two alternative transcripts that are expressed differentially in the somatic and germinal compartments of the testis. These transcripts share the same coding region but differ in their 5' un-translated regions, due to alternative promoters. Here we show that MEIG1 is a highly conserved short metazoan protein with a conserved core of 81 residues. It is present from chordates to radial symmetry animals, with an intriguing absence in insects and nematodes. It is also present in two earlier diverging protist lineages. To elucidate the role of MEIG1 during gamete production we established a knockout mouse line by eliminating the common coding region. Our results identified Meig1 as a critical spermatogenic gene, whose absence results in complete male infertility. Seminiferous tubules in Meig1-null males contained all early stages of spermatogenesis, up to elongating spermatids, but mature elongated spermatids were absent. Accordingly, the caudal epididymis was apparently missing spermatozoa, and the very few spermatozoa-like cells that were obtained were immotile and exhibited a wide range of severe morphological abnormalities. These results point at late spermiogenesis as the differentiative stage at which MEIG1's function is crucial. Nevertheless, delayed kinetics of earlier meiotic stages together with increased apoptosis of meiotic spermatocytes and haploid round spermadids in Meig1 knockout males, suggest involvement of MEIG1 in meiotic stages as well.

Male-specific protein (MSP): a new gene linked to sexual behavior and aggressiveness of tilapia males.

MSP is a male-specific protein initially identified in the serum of sexually active Sarotherodon galilaeus males, and is shown herein to be present in the serum of sexually mature males, but not females, of three other tilapia species. Cloning of the MSP cDNA and analysis of its predicted amino-acid sequence revealed that it is an outlier lipocalin that contains a signal peptide in its N-terminal region. The abundance of highly homologous sequences found in fish and the monophyletic relationship to tetrapod Alpha-1-acid glycoprotein (AGP) places it as a clade XII lipocalin. MSP was shown to undergo major N-glycosylation, characteristic of many lipocalins. The expression pattern of MSP, as determined at both the RNA and protein levels, points to the liver, head kidney and testis as production tissues, and resembles a pattern typical of some hormones. We found that MSP is secreted in urine and seminal fluids, and is present in the skin mucus of socially dominant males. Moreover, we discovered a positive correlation between MSP levels in the serum and the dominance and aggressive behavior displayed by socially dominant males. Based on these data, we suggest that MSP is a novel male-specific lipocalin that may function in intra and inter-sex communication.

The enigma of ATCE1, an acrosome-associated transcription factor.

Atce1 belongs to the CREB3/LZIP subtype of the ATF/CREB transcription factor gene family. Its transcription has previously been shown to be testis-specific and within the testis to be restricted to haploid spermatids. In this study, we characterized the protein's distribution in the testis and found that it accumulates in late round and in elongating spermatids, corresponding to developmental stages considered transcriptionally silent. ATCE1 accumulation is acrosome-specific and persists up to mature epididymal cells, at which stage the protein remained associated with the inner acrosome membrane even after acrosomal reaction. No nuclear localization was evident at any spermatogenic stage. Expression of full-length ATCE1 in various cell lines revealed ER and Golgi localization whereas truncation of the C-terminus allowed entrance into the nucleus. Potent transcriptional activation activity, from kB-containing regulatory elements (but not from CRE elements as one might expect), was observed using the C-terminally truncated nuclear form of ATCE1. These results raise the question of why would a transcription factor be specifically anchored to the acrosome inner membrane? An intriguing speculation that ATCE1 might be paternally delivered to the newly formed zygote is discussed.

The immunomodulator AS101 induces growth arrest and apoptosis in multiple myeloma: association with the Akt/survivin pathway.

Multiple Myeloma (MM) is a clonal B-cell malignancy affecting both the immune and the skeletal systems, and accounts for 10% of all hematological cancers. The immunomodulator ammonium trichloro (dioxoethylene-O,O') tellurate (AS101) is a non-toxic compound which has direct anti-tumoral properties in several tumor models. The present study examined the anti-tumoral activity of AS101 in MM by targeting the Akt/Survivin signaling pathway, crucial for survival. We showed that AS101 inhibites cell proliferation and colonies formation of MM cell lines, in a dose-dependent manner. AS101 induced G(2)/M growth arrest and increased both cyclin-dependent kinase inhibitor p21(waf1) protein levels and Cdk1 (p34(cdc2))-inhibitory phosphorylation. Longer incubation of MM cells with AS101 resulted in accumulation of apoptotic cell population and in increased caspase 9, 3 and 7 activities. We also showed that AS101 down-regulated Akt phosphorylation and decreased expression of the inhibitor of apoptosis, survivin. Since Akt and survivin are potentials targets for MM therapy, we suggest that AS101, currently being used in clinical studies, may have therapeutic implications in myeloma and other hematopoietic malignancies.

Aym1, a mouse meiotic gene identified by virtue of its ability to activate early meiotic genes in the yeast Saccharomyces cerevisiae.

Our understanding of the molecular mechanisms that operate during differentiation of mitotically dividing spermatogonia cells into spermatocytes lags way behind what is known about other differentiating systems. Given the evolutionary conservation of the meiotic process, we screened for mouse proteins that could specifically activate early meiotic promoters in Saccharomyces cerevisiae yeast cells, when fused to the Gal4 activation domain (Gal4AD). Our screen yielded the Aym1 gene that encodes a short peptide of 45 amino acids. We show that a Gal4AD-AYM1 fusion protein activates expression of reporter genes through the promoters of the early meiosis-specific genes IME2 and HOP1, and that this activation is dependent on the DNA-binding protein Ume6. Aym1 is transcribed predominantly in mouse primary spermatocytes and in gonads of female embryos undergoing the corresponding meiotic divisions. Aym1 immunolocalized to nuclei of primary spermatocytes and oocytes and to specific type A spermatogonia cells, suggesting it might play a role in the processes leading to meiotic competence. The potential functional relationship between AYM1 and yeast proteins that regulate expression of early meiotic genes is discussed.

Increased expression of the hPim-2 gene in human chronic lymphocytic leukemia and non-Hodgkin lymphoma.

Pim-1 and Pim-2 are murine proto-oncogenes implicated in lymphomagenesis. The aim of this study was to investigate whether the human Pim-2 (hPim-2) expression is altered in chronic lymphocytic leukemia (B-CLL) and non-Hodgkin's lymphomas (NHL). We analyzed hPim-2 expression in 48 patients with NHL and CLL by quantitative in-situ hybridization, quantitative RT-PCR and FACS analysis. In-situ hybridization revealed a 5.5 +/- 2.2 times higher expression of hPim-2 in NHL over normal lymphocytes (P < 0.001). Similarly, with quantitative RT-PCR, expression in NHL was 1.5 to 2.6 times higher in involved splenic foci compared to nearby uninvolved regions (n = 3). hPim-2 mRNA was increased 3-folds in B-CLL over normal B-cells (P < 0.006). The increased hPim-2 levels correlated with lymphocyte doubling time (DT), in that mRNA levels were two times greater in patients with rapid DT (P < 0.006). Moreover, a significant correlation was found between hPim-2 expression and the Binet staging system of CLL (P < 0.022). The hPIM-2-protein expression was also upregulated in CLL, as assessed by FACS analysis. Therefore, this report provides direct evidence for a linkage of hPim-2 upregulation to NHL and CLL in man. This relationship between hPim-2 and NHL and CLL raises a number of novel mechanistic options for the genesis and/or progression of some types of human lymphomas.

The expanding family of CREB/CREM transcription factors that are involved with spermatogenesis.

One of the molecular mechanisms shown to have played a major role in orchestrating the expression of the many genes with unique cellular and temporal specificity in spermatogenesis, is the cAMP-dependent signaling pathway. In this pathway, gene expression is mediated primarily by two members of the bZIP transcription factors-cAMP-response element binding protein (CREB) and cAMP-responsive element modulator (CREM). Both bind a specific cis element, cAMP-response element (CRE), in the promoter of target genes, both are activated by protein kinase A (PKA) phosphorylation that enables binding of CREB binding protein (CBP) and recruitment of the basal transcription machinery, and both are characterized by multiple alternatively spliced forms. Some of these alternatively spliced forms lack the transactivation domains and hence function as transcription suppressors. In Sertoli cells, CREB levels fluctuate in a cyclical manner that depends on the specific cell associations along the spermatogenic wave. Follicle stimulating hormone (FSH) activates the cAMP signaling pathway and consequently, CREB positively auto-regulates its own expression (by binding to a CRE like element in its promoter). Subsequently, activated CREB activates transcription of genes essential for proper germ cell differentiation. In addition, TNFalpha secreted by round spermatids, activates NF-kappaB dependent CREB expression in Sertoli cells and thus, contributes to the elevated CREB levels as long as these cells are intimately associated. Inducible cAMP early repressor (ICER), a suppressor isoform of CREM, also activated by CREB, down regulates CREB expression together with its own expression, resetting CREB to basal level that enables a new spermatogenic wave. In germ cells, antagonist forms of CREM (alpha, beta and gamma) are present in premeiotic cells and early prophase spermatocytes. A prominent switch to the CREMtau and CREMtheta; activating forms starts in pachytene spermatocytes leading to the activation of haploid genes important for spermiogenesis in round spermatids. Interestingly, in germ cells, CREM exerts activation of haploid genes independent of its phosphorylation state. It associates with activator of CREM in testis (ACT), that has an intrinsic transcriptional activity, rather than with CBP. These and other findings suggest that the expanding CREB/CREM proteins and potentially other members of the CREB family are key molecular regulators at all stages of spermatogenesis.

Atce1: a novel mouse cyclic adenosine 3',5'-monophosphate-responsive element-binding protein-like gene exclusively expressed in postmeiotic spermatids.

Members of the ATF/CREB family of transcription factors are involved in gene activation in various physiological systems ranging from metabolite homeostasis, through regulation of cell cycle, to learning and memory. Two members of this family, cAMP-responsive element binding protein (CREB), and cAMP-responsive element modulator (CREM) are active during mammalian spermatogenesis and are required for this process, as has been shown by knockout and dominant negative experiments. In an effort to identify mouse proteins that interact with the testis-specific protein Tctex2, a mouse testis expression library was screened via the two-hybrid system, using the carboxyl-terminal portion of this protein as bait. A clone containing two overlapping open reading frames, related by a frameshift of one nucleotide, was subsequently isolated. The peptide that interacted with Tctex2 does not initiate from a consensus AUG codon, and it is not clear whether it exists physiologically. However, the other reading frame, initiating from an AUG codon, encodes a 315-amino acid peptide with significant sequence homology to a subfamily of the CREB genes whose prototype is the mouse LZIP peptide. This novel CREB-like peptide, designated Atce1, is specifically expressed in the testis. A developmental study using Northern hybridization and in situ hybridization analyses revealed that Atce1 transcripts begin to accumulate in testes of mice 24 d after birth, reflecting expression in mid/late round spermatids. Interestingly, EMSAs revealed that in vitro translated Atce1 binds specifically to a nuclear factor-kappaB-binding element rather then to a CRE element. Potential roles for Atce1 are discussed.