Encapsulation of a TRPM8 Agonist, WS12, in Lipid Nanocapsules Potentiates PC3 Prostate Cancer Cell Migration Inhibition through Channel Activation.

In prostate carcinogenesis, expression and/or activation of the Transient Receptor Potential Melastatin 8 channel (TRPM8) was shown to block in vitro Prostate Cancer (PCa) cell migration. Because of their localization at the plasma membrane, ion channels, such as TRPM8 and other membrane receptors, are promising pharmacological targets. The aim of this study was thus to use nanocarriers encapsulating a TRPM8 agonist to efficiently activate the channel and therefore arrest PCa cell migration. To achieve this goal, the most efficient TRPM8 agonist, WS12, was encapsulated into Lipid NanoCapsules (LNC). The effect of the nanocarriers on channel activity and cellular physiological processes, such as cell viability and migration, were evaluated in vitro and in vivo. These results provide a proof-of-concept support for using TRPM8 channel-targeting nanotechnologies based on LNC to develop more effective methods inhibiting PCa cell migration in zebrafish xenograft.

NSD3, a new SET domain-containing gene, maps to 8p12 and is amplified in human breast cancer cell lines.

We describe the isolation and characterization of NSD3, the third member of a gene family including Nsd1 and NSD2. Murine Nsd1 was isolated in a search for proteins that interact with the ligand-binding domain of retinoic acid receptor alpha. NSD2 (also known as WHSC1 and MMSET) is located in the Wolf-Hirschhorn syndrome (WHS) critical region on 4p16.3 and is involved in multiple myeloma with t(4;14) translocations. The proteins Nsd1, NSD2, and NSD3 are highly similar within a block of about 700 amino acids. This block contains several conserved domains, such as the SET domain and the PHD finger, present in proteins involved in development and/or chromatin reorganization. The NSD3 gene consists of an 8.5-kb transcript composed of 23 coding exons and spans >90 kb of genomic DNA. NSD3 maps to chromosome band 8p12 and is amplified in several tumor cell lines and primary breast carcinomas.

Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances.

The probability that two sites on a linear DNA molecule will contact each other by looping depends on DNA flexibility. Although the flexibility of naked DNA in vitro is well characterized, looping in chromatin is poorly understood. By extending existing theory, we present a single equation that describes DNA looping over all distances. We also show that DNA looping in vitro can be measured accurately by FLP recombination between sites from 74 bp to 15 kb apart. In agreement with previous work, a persistence length of 50 nm was determined. FLP recombination of the same substrates in mammalian cells showed that chromatin increases the flexibility of DNA at short distances, giving an apparent persistence length of 27 nm.

Simplified generation of targeting constructs using ET recombination.

ET recombination is a way to engineer DNA in Escherichia coli using homologous recombination. Here we develop the potential of ET recombination in two ways relevant to complex engineering exercises such as building gene targeting constructs. First, a targeting construct was made in a single step. Second, ET recombination was used to place two unique restriction sites at precise positions in a large genomic clone. Subsequently a complex targeting construct was created by ligation with a multifunctional cassette.

Improved properties of FLP recombinase evolved by cycling mutagenesis.

The site-specific recombinases FLP and Cre are useful for genomic engineering in many living systems. Manipulation of their enzymatic properties offers a means to improve their applicability in different host organisms. We chose to manipulate the thermolability of FLP recombinase. A lacZ-based recombination assay in Escherichia coli was used for selection in a protein evolution strategy that relied on error-prone PCR and DNA shuffling. Improved FLP recombinases were identified through cycles of increasing stringency imposed by both raising temperature and reducing protein expression, combined with repetitive cycles of screening at the same stringency to enrich for clones with improved fitness. An eighth generation clone (termed FLPe) showed improved properties in E. coli, in vitro, in human 293- and mouse ES-cells.

Inducible expression based on regulated recombination: a single vector strategy for stable expression in cultured cells.

When fused to the ligand binding domain (LBD) of steroid hormone nuclear receptors, site-specific recombinases (SSRs) acquire a ligand-dependent activity. Here, we describe the use of SSR-LBD fusion proteins in an inducible expression system, introduced into cells in a single step. A single transgene contains a constitutively active, bi-directional enhancer/promoter, which directs expression, on one side, of an SSR-LBD fusion protein gene and, on the other, a selectable marker/inducible gene cassette. The selectable marker, the puromycin acetyltransferase (pac) gene, is used for stable genomic integration of the transgene and is flanked by recombination target sites. The inducible gene is not expressed because the pac gene lies between it and the promoter. Activation of the SSR-LBD by a ligand induces recombination and the pac gene is excised. The inducible gene is thus positioned next to the promoter and so is expressed. This describes a ligand-inducible expression strategy that relies on regulated recombination rather than regulated transcription. By inducible expression of diptheria toxin, evidence that this system permits inducible expression of very toxic proteins is presented. The combination of the complete regulatory circuit and inducible gene in one transgene relates expression of the selectable marker gene to expression from the bi-directional enhancer/promoter. We exploit this relationship to show that graded increases in selection pressure can be used to select for clones with different induction properties.

Temporally and spatially regulated somatic mutagenesis in mice.

In mice transgenesis through oocyte injection or DNA recombination in embryonal stem (ES) cells allows mutations to be introduced into the germline. However, the earliest phenotype of the introduced mutation can eclipse later effects. We show in mice that site-specific genomic recombination can be induced in a selected cell type, B lymphocytes, at a chosen time. This precision of somatic mutagenesis was accomplished by limiting expression of a Cre recombinase-estrogen receptor fusion protein to B lymphocytes by use of tissue-specific elements in the promoter of the transgene employed. The expressed fusion protein remained inactive until derepressed by systemic administration of an exogenous ligand for the estrogen receptor, 4-OH-tamoxifen. Upon derepression the Cre recombinase enzyme deleted specific DNA segments, flanked by loxP sites, in B lymphocytes only. The efficiency of recombination in cells expressing the fusion protein could be varied from low levels to >80%, depending on the dose of ligand administered. Our work presents a paradigm applicable to other uses of site-specific recombination in somatic mutagenesis where both temporal and spatial regulation are desired.

The Kw recombinase, an integrase from Kluyveromyces waltii.

Site-specific recombinases of the integrase family share limited amino-acid-sequence similarity, but use a common reaction mechanism to recombine distinct DNA target sites. Here we report the characterisation of the Kw site-specific recombinase, encoded on the 2 mu-like plasmid pKWS1 from the yeast Kluyveromyces waltii. Using in vitro-translated Kw recombinase, we show that the protein is able to bind and to recombine its putative DNA target site. Recombination is conservative and the Kw target site has a spacer of seven base pairs. We show that Kw recombinase is able to mediate recombination in a mammalian cell line, thus, it has potential for use as a tool for genomic manipulation in heterologous systems.

Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination.

Genomic manipulations using site-specific recombinases rely on their applied characteristics in living systems. To understand their applied properties so that they can be optimally deployed, we compared the recombinases FLP and Cre in two assays. In both Escherichia coli and in vitro, FLP shows a different temperature optimum than Cre. FLP is more thermolabile, having an optimum near 30 degrees C and little detectable activity above 39 degrees C. Cre is optimally efficient at 37 degrees C and above. Consistent with FLP thermolability, recombination in a mammalian cell line mediated by a ligand- regulated FLP-androgen receptor fusion protein is more efficient at 35 degrees C than at higher temperatures. We also document a mutation in a commercially available FLP plasmid (FLP-F70L) which renders this recombinase even more thermolabile. The different temperature optima of FLP, FLP-F70L and Cre influence their strategies of usage. Our results recommend the use of Cre for applications in mice that require efficient recombination. The thermolabilities of FLP and FLP-F70L can be usefully exploited for gain of function and cell culture applications.

A simple assay to determine the functionality of Cre or FLP recombination targets in genomic manipulation constructs.

We report the construction of two Escherichia coli strains (294-Cre and 294-FLP) which express either Cre- or FLP-recombinase. Plasmids containing authentic recognition targets for either recombinase (loxPs or FRTs) are recombined when propagated in the appropriate strain. 294-Cre and 294-FLP thus provide a simple test for the recombination competence of constructs that are designed for use in Cre- or FLP-mediated genomic manipulations.

Regulation of Cre recombinase activity by the synthetic steroid RU 486.

To create a strategy for inducible gene targeting we developed a Cre-lox recombination system which responds to the synthetic steroid RU 486. Several fusions between Cre recombinase and the hormone binding domain (HBD) of a mutated human progesterone receptor, which binds RU 486 but not progesterone, were constructed. When tested in transient expression assays recombination activities of all fusion proteins were responsive to RU 486, but not to the endogenous steroid progesterone. However, the observed induction of recombination activity by the synthetic steroid varied between the different fusion proteins. The fusion with the highest activity in the presence of RU 486 combined with low background activity in the absence of the steroid was tested after stable expression in fibroblast and embryonal stem (ES) cells. We could demonstrate that its recombination activity was highly dependent on RU 486. Since the RU 486 doses required to activate recombination were considerably lower than doses displaying anti-progesterone effects in mice, this system could be used as a valuable tool for inducible gene targeting.

Response of the phosphoenolpyruvate carboxykinase gene to glucocorticoids depends on the integrity of the cAMP pathway.

The phosphoenolpyruvate carboxykinase (PEPCK) gene is regulated at the transcriptional level by a variety of effectors in a tissue-specific fashion. In order to study the parameters involved in the tissue-specific hormonal regulation of the PEPCK gene, we have used a transient expression test in well-differentiated rat hepatoma cells as well as in dedifferentiated variants. In this test, the PEPCK promoter is induced by glucocorticoids in well-differentiated FGC4 cells, but not in H5 dedifferentiated variants, in spite of the presence in H5 cells of the glucocorticoid receptor. Study of the PEPCK promoter using electrophoretic mobility shift assays reveals binding sites for the liver-enriched transcription factors HNF1, vHNF1, HNF3, HNF4, and CAAT/enhancer binding protein members. Overexpression of the liver-enriched transcription factors absent in the dedifferentiated variants, such as HNF1 and HNF4, is not sufficient to restore glucocorticoid response of the PEPCK promoter in the variants. Moreover, systematic analysis of the PEPCK promoter reveals that the presence of a region covering a cAMP-responsive element (CRE1 at -80) and a CAAT box is necessary for full response of the PEPCK promoter to glucocorticoids in well-differentiated rat hepatoma cells. In a cotransfection test, overexpression of the regulatory subunit of protein kinase A (PKA), causing sequestering of PKA, abolishes the glucocorticoid response of the promoter in well-differentiated cells. On the other hand, in dedifferentiated variants, overexpression of the catalytic subunit of PKA restores the response to glucocorticoids. The action of PKA on the glucocorticoid response requires the presence of the CRE1 element and is promoter specific because it does not concern nonhepatic promoters such as the long terminal repeats of the mouse mammary tumor virus. These results suggest that the full response of the PEPCK promoter to glucocorticoids requires activation of another signal transduction pathway, the cAMP-mediated pathway.

HNF4 and HNF1 as well as a panel of hepatic functions are extinguished and reexpressed in parallel in chromosomally reduced rat hepatoma-human fibroblast hybrids.

Rat hepatoma-human fibroblast hybrids of two independent lineages containing only 8-11 human chromosomes show pleiotropic extinction of thirteen out of fifteen hepatic functions examined. Reexpression of the entire group of functions most often occurs in a block, and except for one discordant subclone, correlates with loss of human chromosome 2. The extinguished cells and their reexpressing derivatives have been examined for the expression of seven liver-enriched transcription factors. C/EBP, LAP, DBP, HNF3, and vHNF1 expression are not systematically extinguished in parallel with the hepatic functions. However, HNF1 and HNF4 show a perfect correlation with phenotype: these factors are expressed only in the cells showing pleiotropic reexpression. Since recent evidence indicates that HNF4 controls HNF1 expression, it can be proposed that the HNF4 gene is the primary target of the pleiotropic extinguisher.

Cell phenotype, binding affinity and promoter structure modulate transactivation by HNF1 and LAP.

To evaluate the importance of the transcription factors known to bind to the albumin promoter as well as the parameters involved in their activity, we have used cotransfections with an albumin promoter-cat plasmid combined with expression vectors driving the expression of cDNAs coding for liver-enriched factors known to interact with this promoter. We describe the characteristics of a set of clones of hepatic origin: well differentiated, partial variants or pleiotropic dedifferentiated variants. These lines have been characterized for the accumulation of RNAs corresponding to each of the albumin promoter-binding factors. Only HNF1, and to a lesser extent C/EBP, show differences depending upon the differentiation state of the cells. Overexpression of exogenous HNF1 in these cells reveals that this factor is able to transactivate the albumin promoter only in variant cells where the endogenous protein is limiting. By contrast, if the HNF1-binding site is of weak affinity, overexpression of exogenous HNF1 stimulates the albumin promoter even in the HNF1-rich differentiated cells. Overexpression of exogenous LAP strongly transactivates an artificial promoter containing one LAP-binding site, but surprisingly in all the cell lines, it has little effect upon the albumin promoter. These results demonstrate that the transactivation potential of a given transcription factor depends on the degree of differentiation of the recipient cells, on the promoter structure, and on the affinity of the binding site for this factor.

An exogenous albumin promoter can become silent in dedifferentiated hepatoma variants as well as intertypic hybrids.

In order to evaluate the ability of an exogenous tissue-specific promoter to undergo the same dynamic changes in activity as the endogenous one, a 400-base pair fragment of the rat albumin proximal promoter, upstream of the bacterial gpt gene, has been introduced into rat hepatoma cells. Four clones containing a single integrated copy of the construct and producing substantial amounts of albumin and of xanthine phosphoribosyltransferase were isolated. These clones were subjected to two treatments known to result in silencing of the albumin gene: selection for dedifferentiated variants, and fusion with L-cell fibroblasts. In most cases, the albumin-negative progeny obtained no longer expressed the gpt gene: the exogenous promoter of 400 base pairs must contain the sequences required to respond to the mechanisms that block activity of the endogenous gene. However, exceptions were observed: the albumin-deficient variants of one clone remained xanthine phosphoribosyltransferase positive, and some of the albumin-negative hybrids from a different clone continued to produce xanthine phosphoribosyltransferase. These cases of dissociation in expression of the endogenous and the exogenous genes indicate that the site of integration of the alb-gpt construct in one clone renders the sequences insensitive to the mechanisms responsible for albumin gene silencing in dedifferentiated variants, and in the other clone to the mechanism of extinction. Consequently, the mechanisms causing gene silencing in variants and in intertypic hybrids must be different.

Expression vectors for quantitating in vivo translational ambiguity: their potential use to analyse frameshifting at the HIV gag-pol junction.

Translational errors are necessary so as to allow gene expression in various organisms. In retroviruses, synthesis of pol gene products necessitates either readthrough of a stop codon or frameshifting. Here we present an experimental system that permits quantification of translational errors in vivo. It consists of a family of expression vectors carrying different mutated versions of the luc gene as reporter. Mutations include both an in-frame stop codon and 1-base-pair deletions that require readthrough or frameshift, respectively, to give rise to an active product. This system is sensitive enough to detect background errors in mammalian cells. In addition, one of the vectors contains two unique cloning sites that make it possible to insert any sequence of interest. This latter vector was used to analyse the effect of a DNA fragment, proposed to be the target of high level slippage at the gag-pol junction of HIV. The effect of paromomycin and kasugamycin, two antibiotics known to influence translational ambiguity, was also tested in cultured cells. The results indicate that paromomycin diversely affects readthrough and frameshifting, while kasugamycin had no effect. This family of vectors can be used to analyse the influence of structural and external factors on translational ambiguity in both mammalian cells and bacteria.