PI3 kinase integrates Akt and MAP kinase signaling pathways in the regulation of prostate cancer.

PI3 kinase (PI3K), Akt and MAP kinase (MAPK) pathways are central to many classical signaling cascades and are often de-regulated in many cancers. Due to this, inhibitors for a number of key signaling molecules in these pathways such as PI3K, Akt, mTOR, Raf and ERK are currently in clinical trials. In the current study, we investigated the effects of specific inhibition of these signaling molecules, alone or in combinations, on prostate cancer cells. Our study showed that integration of Akt-mTOR and MAPK signaling by PI3K was essential for the EGF-stimulated TRAMP cell migration, proliferation, survival and invasion as well as PC3 and LNCaP C4-2 (C4-2) colony/foci formation. Adenovirus-mediated expression of constitutively active Akt (Ad-myrAkt) in PC3 cells resulted in significant increase in number of foci. Even though PI3K inhibition significantly reduced foci formed by C4-2 cells, none of the Akt, ERK or mTOR inhibitors showed any significant inhibition. This indicated that functional redundancies and/or feed back loops between Akt-mTOR and MAPK signaling exist in prostate cancer. Further studies on cotargeting these signaling molecules revealed that combined inhibition of Akt (or mTOR) and ERK, but not Akt and mTOR, resulted in significant reduction in number of foci formed by the C4-2 cells. Overall, our study demonstrated that the effects of PI3K-mediated prostate cancer growth necessitates a synergism between the Akt and MAPK pathways and suggests cotargeting Akt (or mTOR) and MAPK as an effective method for prostate cancer therapeutic interventions.

The Drosophila RAD21 cohesin persists at the centromere region in mitosis.

'Cohesin' is a highly conserved multiprotein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes. Cohesin complexes in budding yeast hold sister chromatids together from S phase until anaphase, but in metazoans, cohesin proteins dissociate from chromosomes and redistribute into the whole cell volume during prophase, well before sister chromatids separate (reviewed in [1,2]). Here we address this apparent anomaly by investigating the cell-cycle dynamics of DRAD21, the Drosophila orthologue of the Xenopus XRAD21 and Saccharomyces cerevisiae Scc1p/Mcd1p cohesins [3]. Analysis of DRAD21 in S2 Drosophila tissue culture cells and live embryos expressing a DRAD21-green fluorescent protein (GFP) fusion revealed the presence of four distinct subcellular pools of DRAD21: a cytoplasmic pool; a chromosome-associated pool which dissociates from chromatin as chromosomes condense in prophase; a short-lived centrosome-associated pool present during metaphase-anaphase; and a centromere-proximal pool which remains bound to condensed chromosomes, is found along the junction of sister chromatids between kinetochores, and persists until the metaphase-anaphase transition. We conclude that in Drosophila, and possibly all metazoans, a minor pool of cohesin remains bound to centromere-proximal chromatin after prophase and maintains sister-chromatid cohesion until the metaphase-anaphase transition.

Drad21, a Drosophila rad21 homologue expressed in S-phase cells.

Cohesin is an evolutionarily conserved multiprotein complex required to establish and maintain sister chromatid cohesion. Here, we report the cloning and initial characterization of the Drosophila homologue of the fission yeast rad21 cohesin subunit, called Drad21. The Drad21 coding region was localized to centromeric heterochromatin and encodes a 715 amino acid (aa) protein with 42% aa identity to vertebrate Rad21p-homologues, 25% with Scc1p/Mcd1p (S. cerevisiae) and 28% with Rad21p (S. pombe). Sequences with similarity to the sites of proteolytic cleavage identified in Scc1p/Mcd1p are not evident in DRAD21. Northern blot and mRNA in-situ studies show that Drad21 is developmentally regulated, with high levels of expression in early embryogenesis, in S-phase cells of proliferating imaginal tissues, and in the early endocycling cells of the embryonic gut.

Integration specificity of the hobo element of Drosophila melanogaster is dependent on sequences flanking the integration site.

We analyzed the integration specificity of the hobo transposable element of Drosophila melanogaster. Our results indicate that hobo is similar to other transposable elements in that it can integrate into a large number of sites, but that some sites are preferred over others, with a few sites acting as integration hot spots. A comparison of DNA sequences from 112 hobo integration sites identified a consensus sequence of NTNNNNAC, but this consensus was insufficient to account for the observed integration specificity. To begin to define the parameters affecting hobo integration preferences, we analyzed sequences flanking a donor hobo element, as well as sequences flanking a hobo integration hot spot for their relative influence on hobo integration specificity. We demonstrate experimentally that sequences flanking a hobo donor element do not influence subsequent integration site preference, whereas, sequences contained within 31 base pairs flanking an integration hot spot have a significant effect on the frequency of integration into that site. However, sequence analysis of the DNA flanking several hot spots failed to identify any common sequence motif shared by these sites. This lack of primary sequence information suggests that higher order DNA structural characteristics of the DNA and/or chromatin may influence integration site selection by the hobo element.

Regulation of the germline immunoglobulin Cgamma1 promoter by CD40 ligand and IL-4: dual role for tandem NF-kappaB binding sites.

Transcription of germline Ig constant region genes and associated switch regions is an early and essential step in heavy chain class switch recombination. Transcription of the germline Cgamma1 and C epsilon Ig genes is induced by IL-4 via STAT6 activation; CD40 signaling can independently induce transcription of these genes and act in synergy with IL-4 to increase expression. In the present study, we investigated the role of three tandem NF-kappaB sites (site 1, -95; site 2, -71; site 3, -53) in the regulation of the germline Cgamma1 Ig promoter by CD40 Ligand (CD40L) and IL-4 in the mouse B lymphoma cell line, BCL1-3B3. Germline gamma1 transcripts are induced by CD40L and by IL-4 in BCL1-3B3 and the combination of signals is synergistic, as in normal B cells. EMSA with crude nuclear extracts demonstrated that stimulation with CD40L results in the induction of NF-kappaB complexes that bind to each of the three NF-kappaB sites and are composed mainly of p50 and RelB, but also include c-Rel and p65. Surprisingly, site-specific mutagenesis of the NF-kappaB sites did not reduce CD40-responsiveness of germline gamma1 promoter-luciferase reporter constructs transiently transfected into BCL1-3B3. Mutation in any one NF-kappaB site, however, significantly reduced overall transcriptional activity of the promoter, both basal and induced, suggesting a role in basal promoter function. In addition, activation of the promoter by IL-4 was blocked by mutation of all three NF-kappaB sites and similarly reduced by mutation of site 1, suggesting that NF-kappaB-STAT6 interactions may be necessary for STAT6-mediated transactivation of the germline gamma1 promoter. The results suggest that the three NF-kappaB sites may serve as a focus for formation of a higher-order transcription complex including STAT6, NF-kappaB and components of the basal transcription apparatus.

DNA methylation in the promoter region of the p16 (CDKN2/MTS-1/INK4A) gene in human breast tumours.

The p16 (CDKN2/MTS-1/INK4A) gene is one of several tumour-suppressor genes that have been shown to be inactivated by DNA methylation in various human cancers including breast tumours. We have used bisulphite genomic sequencing to examine the detailed sequence specificity of DNA methylation in the CpG island promoter/exon 1 region in the p16 gene in DNA from a series of human breast cancer specimens and normal human breast tissue (from reductive mammaplasty). The p16 region examined was unmethylated in the four normal human breast specimens and in four out of nine breast tumours. In the other five independent breast tumour specimens, a uniform pattern of DNA methylation was observed. Of the nine major sites of DNA methylation in the amplified region from these tumour DNAs, four were in non-CG sequences. This unusual concentration of non-CG methylation sites was not a general phenomenon present throughout the genome of these tumour cells because the methylated CpG island regions of interspersed L1 repeats had a pattern of (almost exclusively) CG methylation similar to that found in normal breast tissue DNA and in DNA from tumours with unmethylated p16 genes. These data suggest that DNA methylation of the p16 gene in some breast tumours could be the result of an active process that generates a discrete methylation pattern and, hence, could ultimately be amenable to therapeutic manipulation.

STAT6 is required for IL-4-induced germline Ig gene transcription and switch recombination.

Transcription of the germline C gamma1 and C epsilon Ig genes is believed to be a necessary prerequisite for isotype switching to IgG1 and IgE, respectively. IL-4 stimulation and ligation of CD40 can each independently induce low level germline gamma1 and epsilon transcription in murine B cells. Together these signals act synergistically to promote high level germline transcription and are normally required for T-dependent isotype switching to IgG1 and IgE. The STAT6 transcription factor has been suggested to play a critical role in IL-4-induced activation of germline C gamma1 and C epsilon genes. To directly assess the role of STAT6 in IL-4R- and CD40-mediated germline transcription and switching, we have analyzed these events in splenic B cells from STAT6-deficient mice. Our results demonstrate that IL-4 does not induce detectable levels of germline gamma1 or epsilon transcripts in STAT6-deficient B cells. Germline transcript expression induced by CD40 stimulation alone is unaffected, but synergism between CD40- and IL-4R-mediated signals is completely ablated. Switch recombination to S gamma1, as measured by digestion-circularization PCR, is dramatically reduced in STAT6-deficient B cells stimulated with CD40 ligand plus IL-4. Similarly, germline gamma1 transcript expression and switch recombination to S gamma1 are also impaired in STAT6-deficient B cells stimulated with IL-4, IL-5, and anti-IgD Abs conjugated to dextran, a model for T-independent type II responses. These results directly demonstrate a critical role for STAT6 in the IL-4-mediated activation of germline Ig gene transcription and switch recombination in nontransformed B cells.

DNA methylation in mouse A-repeats in DNA methyltransferase-knockout ES cells and in normal cells determined by bisulfite genomic sequencing.

Mouse ES cells with a null mutation of the known DNA methyltransferase retain some residual DNA methylation and can methylate foreign sequences de novo. We have used bisulfite genomic sequencing to examine the sequence specificity and distributions of methylation of a hypermethylated CG island sequence, mouse A-repeats. There were 13 CG dinucleotides in the region examined, 12 of which were methylated to variable extents in all DNAs. We found that: (1) there is considerable residual DNA methylation in ES cells lacking the known DNA methyltransferase (29% of normal methylation in the complete knockout ES DNA); (2) this other activity methylates at exactly the same CG sites as the major methyltransferase; and (3) differences in the distribution of methylated sites between A-repeats in these DNAs are consistent with this other activity methylating in a random de novo fashion. Also, the lack of any methylation in non-CG sites argues that, in other studies where non-CG methylation sites have been found by bisulfite sequencing, detection of such sites of non-CG methylation is not an inherent artifact in this methodology.

CD40 expression by gingival fibroblasts: correlation of phenotype with function.

CD40, a member of the tumor necrosis factor-alpha receptor family, is constitutively expressed by cells of hematopoietic and non-hematopoietic origin, including fibroblasts. Signaling through this receptor molecule regulates inflammatory cytokine secretion by many cell types. Based on the recently described cytokine secretory heterogeneity of fibroblast cell subsets, we hypothesized that secretion of inflammatory cytokines by gingival fibroblast cultures may be dictated by the existence of differential proportions of cytokine-secreting subpopulations which express high levels of CD40. After examining a large number of gingival fibroblast (GF) cultures we find that the frequency of IL-6- and IL-8-secreting cells mirrors the frequency of cells expressing high levels of CD40 in these cultures. In addition, we demonstrate a direct functional relationship between CD40 expression and IL-6 or IL-8 secretion by showing that ligation of this molecule on GF, and CD40+ fibroblast subsets in particular, up-regulates secretion of these cytokines in vitro.

Cloning and functional expression of human kynurenine 3-monooxygenase.

Kynurenine 3-monooxygenase, an NADPH-dependent flavin monooxygenase, catalyses the hydroxylation of L-kynurenine to L-3-hydroxykynurenine. By hybridization screening using a cDNA probe encoding the entire exon 2 of Drosophila melanogaster kynurenine 3-monooxygenase, we isolated a 2.0 kb cDNA clone coding for the corresponding human liver enzyme. The deduced amino acid sequence of the human protein consists of 486 amino acids with a predicted molecular mass of 55,762 Da. Transfection of the human cDNA in HEK-293 cells resulted in the functional expression of the enzyme with kinetic properties similar to those found for the native human protein. RNA blot analysis of human tissues revealed the presence of a major mRNA species of approximately 2.0 kb in liver, placenta and kidney.

Asymmetric methylation in the hypermethylated CpG promoter region of the human L1 retrotransposon.

We have investigated the function and sequence specificity of DNA methylation in the hypermethylated CpG island promoter region of the endogenous human LINE-1 (L1) retrotransposon family. In nontransformed human embryonic fibroblasts, inhibition of DNA methylation with 5-azadeoxycytidine induced a greater than 4-fold increase in transcription from potentially functional L1 elements without increasing the transcription level of the majority of degenerate elements, implicating hypermethylation in the repression of L1 activity. Using bisulfite genomic sequencing to assess the pattern of methylation in a subset of nondegenerate L1 elements, we found 29 sites within a 460-base pair region of the noncoding (top) DNA strand of the L1 promoter in which cytosine methylation was maintained with high efficiency. Of these, 25 were at CG dinucleotides and four were in non-CG sites. When the methylation sites were analyzed for the complementary (bottom) strand, the only highly conserved sites of methylation were in CG dinucleotides. Several of these sites of CG methylation in the bottom (coding) strand were at positions where top (noncoding) strand-derived sequences were unmethylated, suggesting that these sites might be maintained in a hemi-methylated state. Hence, there is a subset of human L1 elements in which methylation is efficiently maintained in asymmetric non-CG sites and further that this non-CG methylation may be part of a wider phenomenon involving hemi-methylation at CG dinucleotides. Maintenance of asymmetric methylation at non-CG sites (and possibly at hemi-methylated CG dinucleotides) could be through a novel DNA methyltransferase activity. Alternatively, the promoter region of L1 elements may be induced by factor binding to form some type of secondary structure that presents as a highly efficient substrate for de novo methylation.

Drosophila melanogaster contains both X-linked and autosomal homologues of the gene encoding calcineurin B.

A transcription unit was identified in the 43E polytene band region of the second chromosome of Drosophila melanogaster (Dm) whose putative translation product has 85% amino acid (aa) identity with the B subunit of the calcineurin protein (CnB) from humans. Unlike the previously described intronless Dm CnB gene homologue, which is located within the 4F band region of the X chromosome, the coding region of this second CnB is found to be interrupted by three introns. Conceptual translation of both Dm CnB genes predict proteins of identical size that are 98% identical in aa sequence. Northern blot analyses indicate that Dm pupae and adults express two different CnB-encoding transcripts that are differentially regulated.

Hermes, a functional non-Drosophilid insect gene vector from Musca domestica.

Hermes is a short inverted repeat-type transposable element from the house fly, Musca domestica. Using an extra-chromosomal transpositional recombination assay, we show that Hermes elements can accurately transpose in M. domestica embryos. To test the ability of Hermes to function in species distantly related to M. domestica we used a nonautonomous Hermes element containing the Drosophila melanogaster while (w+) gene and created D. melanogaster germline transformants. Transgenic G1 insects were recovered from 34.6% of the fertile G0 adults developing from microinjected w- embryos. This transformation rate is comparable with that observed using P or hobo vectors in D. melanogaster, however, many instances of multiple-element insertions and large clusters were observed. Genetic mapping, Southern blotting, polytene chromosome in situ hybridization and DNA sequence analyses confirmed that Hermes elements were chromosomally integrated in transgenic insects. Our data demonstrate that Hermes elements transpose at high rates in D. melanogaster and may be an effective gene vector and gene-tagging agent in this species and distantly related species of medical and agricultural importance.

Molecular characterization of the cinnabar region of Drosophila melanogaster: identification of the cinnabar transcription unit.

Early studies of eye pigmentation in Drosophila melanogaster provided compelling evidence that the cinnabar (cn) gene encodes the enzyme kynurenine 3-monooxygenase (EC 1.14.13.9). Here we report the cloning of approximately 60 kb of DNA encompassing the cn gene by chromosome walking in the 43E6-F1 region of chromosome 2. An indication of the position of cn within the cloned region was obtained by molecular analysis of mutants: 9 spontaneous cn mutants were found to have either DNA insertions or deletions within a 5 kb region. In addition, a 7.8 kb restriction fragment encompassing the region altered in the mutants was observed to induce transient cn function when microinjected into cn- embryos. The cn transcription unit was identified by Northern blotting and sequence analysis of cDNA and genomic clones from this region. The predicted cn protein contains several sequence motifs common to aromatic monooxygenases and is consistent with the assignment of cn as encoding the structural gene for kynurenine 3-monooxygenase.

Induction of germ-line gamma 1 and epsilon Ig gene expression in murine B cells. IL-4 and the CD40 ligand-CD40 interaction provide distinct but synergistic signals.

The interaction between B cell CD40 and its ligand (CD40L) on activated Th cells provides a critical signal necessary for T cell-dependent isotype switching. Previous studies suggest that this signal might be important in regulating isotype switching at the level of germ-line Ig transcription. To assess the effects of the CD40L-CD40 interaction on germ-line Ig transcript expression in murine B cells, a membrane-bound form of mouse CD40L was expressed in the baculovirus system. We show that stimulation of resting splenic B cells with CD40L-expressing Sf9 cells induces germ-line gamma 1 and epsilon transcripts independently of cytokines. The CD40-mediated induction cannot be blocked by anti-IL-4 Ab and is not mediated by other cytokines secreted endogenously in response to CD40 stimulation. Importantly, stimulation with CD40L and IL-4 together has a significant synergistic effect on germ-line transcript expression. Stimulation of CD40 does not activate the NF-IL-4-gamma 1 DNA binding factor believed to be required for IL-4-dependent germ-line gamma 1 transcription. Moreover, mutation of the NF-IL-4-gamma 1 DNA binding site in a germ-line gamma 1 promoter-luciferase reporter gene construct completely ablates IL-4 responsiveness but has no effect on responsiveness to CD40L in transient transfection assays. These results demonstrate that the CD40L-CD40 interaction and IL-4 activate germ-line Ig gene transcription by distinct but synergistic mechanisms and suggest that multiple signals may be required to induce sufficient germ-line transcription and/or germ-line transcript levels necessary to target switch recombination.

CD40-CD40 ligand interactions stimulate B cell antigen processing.

The interactions between B cell CD40 and T cell CD40 ligand (CD40L) have been shown recently to play an important role in T cell-dependent activation of B cells. Here, we show that the ligation of CD40 stimulates the processing of antigen by B cells. The activation of an antigen-specific T cell hybrid by B cells co-cultured with insect cells expressing recombinant CD40L or with a CD40-specific monoclonal antibody requires less antigen and fewer B cells compared to control cells. The augmentation was observed both for processing initiated by antigen binding to and cross-linking the surface immunoglobulin, and processing of antigen taken up by fluid-phase pinocytosis. CD40 appears to affect a step in the intracellular processing of antigen, as CD40 has no effect on the presentation of an antigenic peptide which does not require processing. In addition, the CD40-induced augmentation of processing is not attributable to the effect of CD40 ligation on the cell surface expression of B7, LFA-1 or CD23. CD40 ligation does not affect the biosynthesis of the class II EK molecules, and although ligation of CD40 induces B cell proliferation, the augmentation of processing does not require proliferation. The ability of CD40 to stimulate B cell antigen processing has the potential to influence significantly the outcome of antigen-dependent T cell-B cell interactions.

The transposable element mariner can excise in non-drosophilid insects.

Plasmid-based excision assays performed in embryos of two non-drosophilid species using the mariner transposable element from Drosophila mauritiana resulted in empty excision sites identical to those observed after the excision of mariner from D. mauritiana chromosomes. In the presence of the autonomous mariner element Mos1, excision products were recovered from D. melanogaster, D. mauritiana and the blowfly Lucilia cuprina. When a hsp82 heat shock promoter-Mos1 construct was used to supply mariner transposase, excision products were also recovered from the Queensland fruitfly Bactrocera tryoni. Analysis of DNA sequences at empty excision sites led us to hypothesise that the mariner excision/repair process involves the formation of a heteroduplex at the excision breakpoint. The success of these assays suggests that they will provide a valuable tool for assessing the ability of mariner and mariner-like elements to function in non-drosophilid insects and for investigating the basic mechanisms of mariner excision and repair.

Signaling via major histocompatibility complex class II molecules and antigen receptors enhances the B cell response to gp39/CD40 ligand.

Activated T cells induce proliferation and differentiation of resting B cells in vitro through their CD40 molecules and lymphokine receptors. However, despite constitutive B cell expression of CD40 and lymphokine receptors, widespread nonspecific polyclonal B cell activation by activated T cells is seldom observed in vivo. The present study was designed to test the hypothesis that signals delivered via the B cell antigen (Ag) receptor (membrane immunoglobulin, mIg) and major histocompatibility complex (MHC) class II molecules enhance B cell responsiveness to CD40-mediated signals, providing specificity to the Ag-nonspecific, MHC-unrestricted CD40 signal. To test this hypothesis, both an Ag-specific mouse B cell clone CH12.LX, and freshly isolated resting splenic B cells were cultured with either soluble or membrane-bound forms of the T cell ligand for CD40 (CD40L), in the presence or absence of additional signals provided by Ag or anti-IgM, interleukin-4, and class II-specific monoclonal antibody (mAb). Differentiation of CH12.LX cells and proliferation of splenic B cells in response to both forms of CD40L was greatly enhanced by exposure to mIg-mediated signals, with greatest enhancement seen when cells were cultured with Ag prior to receiving other signals. Response to CD40L was further enhanced by concurrent culture with class II-specific, but not class I-specific mAb. Enhancement was greatest at limiting concentrations of CD40L. The ability of class II MHC-mediated signals to enhance Ag-specific B cell responsiveness to CD40-mediated signaling may selectively promote the activation of B cell clones capable of cognate interactions with helper T cells.

The Hermes transposable element from the house fly, Musca domestica, is a short inverted repeat-type element of the hobo, Ac, and Tam3 (hAT) element family.

The genome of the house fly, Musca domestica, contains an active transposable element system, called Hermes. Using PCR and inverse PCR we amplified and sequenced overlapping segments of several Hermes elements and from these data we have constructed a 2749 bp consensus Hermes DNA sequence. Hermes termini are composed of 17 bp imperfect inverted repeats that are almost identical to the inverted terminal repeats of the hobo element of Drosophila melanogaster. Full length Hermes elements contain a single long ORF capable of encoding a protein of 612 amino acids which is 55% identical to the amino acid sequence of the hobo transposase. Comparison of the ends of the Hermes and hobo elements to those of the Ac element of Zea mays, and the Tam3 element of Antirrhinum majus, as well as several other plant and insect elements, revealed a conserved terminal sequence motif. Thus Hermes is clearly a member of the hobo, Ac and Tam3 (hAT) transposable element family, other members of which include the Tag1 element from Arabidopsis thaliana and the Bg element from Zea mays. The evolution of this class of transposable elements and the potential utility of Hermes as a genetic tool in M. domestica and related species are discussed.