Molecular targeting and gene delivery in bladder cancer therapy.

Urothelial carcinoma of the bladder is the second most common genitourinary malignancy and the second most common cause of genitourinary cancer-related deaths with a worldwide estimate of about 300,000 new cases diagnosed every year. A significant problem in this type of cancer is the high recurrence rate of non-invasive primary tumors, leading to a high percentage of tumor progression and to a very poor 5-year survival rate. Targeted and gene therapy are currently the two major efforts in cancer treatment. Targeted therapy refers to strategies against specific cellular molecules deregulated in tumors, whereas gene therapy focuses on the genetic modification of tumor cells, mainly for correcting gene defects, inducing selective tumor cell death or modulating host's immune response. Recent advances in our understanding of the pathogenesis of bladder cancer at the molecular level have provided a significant number of cellular targets for therapy and have shown the importance of individualized therapy according to the molecular profile exhibited by the tumor cells. While the major problems of both targeted and gene therapy are far from being solved yet, both lines of cancer therapy hold promising results. This article aims at providing a brief general overview of this broad subject.

Actin cytoskeleton reorganization of the apoptotic nurse cells during the late developmental stages of oogenesis in Dacus oleae.

In the present study, we demonstrate the actin cytoskeleton reorganization during nurse cells apoptosis of the olive fruit fly Dacus oleae. At the developmental stage 9A of oogenesis, the actin microfilaments are assembled in numerous ring canals and subcortically support all the nurse cells, as is shown by phalloidin-FITC staining. During the following stages, 9B and 10A, this structural pattern remains the same. The developmental stage 10B is characterized by actin microfilament rearrangement and formation of actin cables that are symmetrically organized around the nurse cell nuclei. At stage 11, when the dumping process begins, these actin cables seem to retain each nurse cell nucleus in the cell center, away from blocking the ring canals. The early stage 12 is characterized by an asynchronous nurse cell nuclear chromatin condensation, while at late stage 12 the actin cables become very thick, as adjacent ones overlap one another and traverse the disorganized apoptotic nurse cell nuclei that already have fragmented DNA, as is demonstrated by acridine orange staining and TUNEL assay. Finally, during stage 13, the apoptotic nuclear remnants are phagocytosed by the neighboring follicle cells. The data presented herein compared to previous reported results in Drosophila [Nezis et al., 2000: Eur J Cell Biol 79:610-620], demonstrate that actin cytoskeleton reorganization during nurse cell apoptosis is a developmentally regulated physiological mechanism, phylogenetically conserved in higher Dipteran.

Stage-specific apoptotic patterns during Drosophila oogenesis.

In the present study we demonstrate the existence of two apoptotic patterns in Drosophila nurse cells during oogenesis. One is developmentally regulated and normally occurs at stage 12 and the other is stage-specific and is sporadically observed at stages 7 and 8 of abnormally developed follicles. The apoptotic manifestation of the first pattern begins at stage 11 and is marked by a perinuclear rearrangement of the actin cytoskeleton and the development of extensive lobes and engulfments of the nurse cell nuclei located proximal to the oocyte. Consequently, at late stage 12 (12C), half of the nurse cell nuclei exhibit condensed chromatin, while at late stage 13 all the nuclei have fragmented DNA, as it is clearly shown by TUNEL assay. Finally, the apoptotic vesicles that are formed during stage 13, are phagocytosed by the neighboring follicle cells and at stage 14 the nurse cell nuclear remnants can be easily detected within the adjacent follicle cell phagosomes. In the second sporadic apoptotic pattern, all the nurse cell nuclei are highly condensed with fragmented DNA, accompanied by a completely disorganized actin cytoskeleton. When we induced apoptosis in Drosophila follicles through an etoposide and staurosporine in vitro treatment, we observed a similar pattern of stage-specific cell death at stages 7 and 8. These observations suggest a possible protective mechanism throughout Drosophila oogenesis that results in apoptosis of abnormal, damaged or spontaneously mutated follicles before they reach maturity.

A small amphipathic alpha-helical region is required for transcriptional activities and proteasome-dependent turnover of the tyrosine-phosphorylated Stat5.

Cytokines induce the tyrosine phosphorylation and associated activation of signal transducers and activators of transcription (Stat). The mechanisms by which this response is terminated are largely unknown. Among a variety of inhibitors examined, the proteasome inhibitors MG132 and lactacystin affected Stat4, Stat5 and Stat6 turnover by significantly stabilizing the tyrosine-phosphorylated form. However, these proteasome inhibitors did not affect downregulation of the tyrosine-phosphorylated Stat1, Stat2 and Stat3. With Stat5 isoforms, we have observed that tyrosine-phosphorylated carboxyl-truncated forms of Stat5 proteins were considerably more stable than phosphorylated wild-type forms of the protein. Also, the C-terminal region of Stat5 could confer proteasome-dependent downregulation to Stat1. With a series of C-terminal deletion mutants, we have defined a relatively small, potentially amphipathic alpha-helical region that is required for the rapid turnover of the phosphorylated Stat5 proteins. The region is also required for transcriptional activation, suggesting that the functions are linked. The results are consistent with a model in which the transcriptional activation domain of activated Stat5 is required for its transcriptional activity and downregulation through a proteasome-dependent pathway.

SOCS1 deficiency causes a lymphocyte-dependent perinatal lethality.

SOCS1 is an SH2-containing protein that is primarily expressed in thymocytes in a cytokine- and T cell receptor-independent manner. SOCS1 deletion causes perinatal lethality with death by 2-3 weeks. During this period thymic changes include a loss of cellularity and a switch from predominantly CD4+ CD8+ to single positive cells. Peripheral T cells express activation antigens and proliferate to IL-2 in the absence of anti-CD3. In addition, IFNgamma is present in the serum. Reconstitution of the lymphoid lineage of JAK3-deficient mice with SOCS1-deficient stem cells recapitulates the lethality and T cell alterations. Introducing a RAG2 or IFNgamma deficiency eliminates lethality. The results demonstrate that lymphocytes are critical to SOCS1-associated perinatal lethality and implicate SOCS1 in lymphocyte differentiation or regulation.

Identification of protein-tyrosine phosphatases in Archaea.

Protein-tyrosine dephosphorylation is a major mechanism in cellular regulation. A large number of protein-tyrosine phosphatases is known from Eukarya, and more recently bacterial homologues have also been identified. By employing conserved sequence patterns from both eukaryotic and bacterial protein-tyrosine phosphatases, we have identified three homologous sequences in two of the four complete archaeal genomes. Two hypothetical open reading frames in the genome of Methanococcus jannaschii (MJ0215 and MJECL20) and one in the genome of Pyrococcus horikoshii (PH1732) clearly bear all the conserved residues of this family. No homologues were found in the genomes of Archaeoglobus fulgidus and Methanobacterium thermoautotrophicum. This is the first report of protein-tyrosine phosphatase sequences in Archaea.

Stat5a and Stat5b proteins have essential and nonessential, or redundant, roles in cytokine responses.

A variety of cytokines mediate the activation of Janus protein tyrosine kinases (Jaks). The Jaks then phosphorylate cellular substrates, including members of the signal transducers and activators of transcription (Stat) family of transcription factors. Among the Stats, the two highly related proteins, Stat5a and Stat5b, are activated by a variety of cytokines. To assess the role of the Stat5 proteins, mutant mice were derived that have the genes deleted individually or together. The phenotypes of the mice demonstrate an essential, and often redundant, role for the two Stat5 proteins in a spectrum of physiological responses associated with growth hormone and prolactin. Conversely, the responses to a variety of cytokines that activate the Stat5 proteins, including erythropoietin, are largely unaffected.

Jak2 is essential for signaling through a variety of cytokine receptors.

A variety of cytokines activate receptor-associated members of the Janus family of protein tyrosine kinases (Jaks). To assess the role of Jak2, we have derived Jak2-deficient mice. The mutation causes an embryonic lethality due to the absence of definitive erythropoiesis. Fetal liver myeloid progenitors, although present based on the expression of lineage specific markers, fail to respond to erythropoietin, thrombopoietin, interleukin-3 (IL-3), or granulocyte/macrophage colony-stimulating factor. In contrast, the response to granulocyte specific colony-stimulating factor is unaffected. Jak2-deficient fibroblasts failed to respond to interferon gamma (IFNgamma), although the responses to IFNalpha/beta and IL-6 were unaffected. Lastly, reconstitution experiments demonstrate that Jak2 is not required for the generation of lymphoid progenitors, their amplification, or functional differentiation. Therefore, Jak2 plays a critical, nonredundant role in the function of a specific group of cytokines receptors.

Signaling by the cytokine receptor superfamily.

A variety of cytokines that regulate functions of multiple lineages share the utilization of receptors that are structurally and functionally related and are referred to as the cytokine receptor superfamily. These receptors associate with one or more of the four mammalian Janus kinases (Jaks) and ligand-induced receptor aggregation results in their activation. Critical roles for Jak3 and Jak2 are demonstrated by the phenotypes of mice that lack each gene. Among the substrates of the Jaks are one or more of the seven members of the signal transducers and activators of transcription (Stats). Each Stat family member plays a critical role in the biological functions of specific cytokines as demonstrated by the phenotype of mice lacking one or more of these genes.

Naturally occurring dominant negative variants of Stat5.

Stat5 was initially identified as a prolactin-induced member of the signal transducer and activator of transcription (Stat) family in sheep. However, Stat5 is also activated in the response to a variety of cytokines. In mice, and possibly in other species, there exist two Stat5 genes (Stat5a and Stat5b) that encode proteins of 92 and 94 kDa that are 95% identical. In the studies described here, we demonstrate that naturally occurring carboxyl-truncated, variant Stat5 proteins of 77 and 80 kDa exist and that these proteins are inducibly tyrosine phosphorylated in the response to several cytokines and form heterodimers with the full-length, wild-type proteins. Using expression constructs encoding truncated forms, we demonstrate that the truncated forms can be tyrosine phosphorylated and bind DNA. Surprisingly, the tyrosine phosphorylation of the carboxyl-truncated forms is considerably more stable than that of the wild-type proteins. Overexpression of a carboxyl-truncated Stat5a in cells resulted in the specific inhibition of the transcriptional activation by interleukin-3 of the genes for oncostatin M (Osm) and the cytokine-inducible, SH2 domain-containing gene (Cis), both of which have been shown to be normally regulated by Stat5. Although Stat5 dominantly suppressed the induction of these genes, no effects on cell proliferation were observed. Together, the results demonstrate the natural existence of potentially dominantly suppressive variants of Stat5 and implicate the carboxyl domain of Stats in transcriptional regulation and functions related to dephosphorylation.

Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for a mitogenic response.

The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a membrane-distal region that is dispensable for mitogenesis but is required for the recruitment and tyrosine phosphorylation of a variety of signaling proteins. The membrane-proximal region of 96 amino acids is necessary and sufficient for mitogenesis as well as Jak2 activation, induction of c-fos, c-myc, cis, the T-cell receptor gamma locus (TCR-gamma), and c-pim-1. The studies presented here demonstrate that this region is also necessary and sufficient for the activation of Stat5A and Stat5B. The membrane-proximal domain contains a single tyrosine, Y-343, which when mutated eliminates the ability of the receptor to couple Epo binding to the activation of Stat5. Furthermore, peptide competitions demonstrate that this site, when phosphorylated, can disrupt Stat5 DNA binding activity, consistent with a role of Y-343 as a site of recruitment to the receptor. Cells expressing the truncated, Y343F mutant (a mutant with a Y-to-F alteration at position 343) proliferate in response to Epo in a manner comparable to that of the controls. However, in these cells, Epo stimulation does not induce the appearance of transcripts for cis, TCR-gamma, or c-fos, suggesting a role for Stat5 in their regulation.

The MHC class II E beta promoter: a complex arrangement of positive and negative elements determines B cell and interferon-gamma (IFN-gamma) regulated expression.

The 5' proximal region of the E beta gene was studied with respect to B lymphoid expression and responsiveness to cytokines, revealing a complex array of general and cell type specific cis-elements and factors. Full lymphoid activity and response to interferon-gamma (IFN-gamma) is generated by the concerted action of the MHC boxes (H, X and Y) and additional elements. Combinatorial interactions between elements and their cognate factors are indicated by several lines of evidence. Thus, mutations within the X box in the promoter context are strongly deleterious to both B lymphoid activity and IFN-gamma regulation. However, the X box alone has minimal lymphoid activity upon heterologous promoters. Data from deletion, insertion and site directed mutagenesis demonstrate that sequences extending approximately 35 bp 5' of the X box (designated as Cytokine Response Sequence--CRS) have a dual role: they are required for cytokine-regulated expression as well as serving as an enhancer element for cell-specific constitutive expression. A region that carries X and CRS permits both lymphoid activity and IFN-gamma response. In contrast, sequences that include X and the downstream Y box are constitutively active in all cell types tested. Combination of the sequences both upstream and downstream of the X box results in a tissue-specific and cytokine-regulated enhancer of full strength. In vivo competition studies show that titratable trans-acting factors, shared by Class I and Class II promoters, mediate the CRS-dependent IFN-gamma response. We report here the identification of novel nuclear complexes that bind to the CRS and recognize sites which correlate with its negative or positive elements. One of these complexes is present in B lymphoid cells only. Three other CRS complexes that are upregulated by either IFN-alpha and IFN-gamma are competed by a non-Class II, IFN-alpha stimulated response element (ISRE), providing evidence for the functional interconnection of these cytokines.

One and two-level regulation patterns affecting NF-kappa B mRNA and nuclear NF-kappa B activity after treatment with TNF-alpha, IFN-gamma and IL-4.

The transactivating nuclear factor NF-kappa B is believed to be important in the pathophysiology of many cellular systems and mainly during HIV infection. kappa B activation has also been implicated in the process of differentiation as a cell progresses to a more mature and functional stage. As induction of differentiation equals growth retardation we undertook this study in order to establish the role of NF-kappa B in cell growth and maturity. Thus we employed the well described HL-60 cellular system that expresses constitutively basal amounts of NF-kappa B and is susceptible to NF-kappa B induction by various biological or chemical agents. We also used known inducers of differentiation like TNF-alpha, IFN-gamma and IL-4 that interact via their corresponding surface receptors found on HL-60 cells. We first studied by Northern analysis the possible correlation between c-myc and NF-kappa B precursor (p105) mRNA. We witnessed that all three cytokines were able to confer proliferative senescence and down-regulate concomitantly c-myc and NF-kappa B mRNA levels, events chronologically in accord with induction of differentiation as assessed by the induction of HLA-DR surface antigens. It is known that TNF-alpha is capable of inducing nuclear kappa B activity in HL-60 as the cells progress to a more mature stage. Therefore we examined whether the other two cytokines could do the same during the time they lead the cells to a differentiated phenotype. If this was the case, nuclear activation of NF-kappa B should be obtained by the same factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation of intracellular signals generated by gamma-interferon and IL-4 leading to the induction of class II antigen expression.

Signal transduction plays a vital role in cellular behaviour as cells respond to various stimuli in different ways and utilize diverse pathways for accomplishing their task. Determination of the pathway followed by various cytokines can be achieved using specific inhibitors which include theophylline (TPH), TMB-8 and W7 that hinder calmodulin binding to Ca(2+); sphingosine (SPH), H7 and staurosporine that inhibit protein kinase C (PKC) activation; and mevalonate (MEV) or the anti-p21(ras) antibody which block G-proteins. This study shows that the immunologically important class II antigens in human cells are up-regulated predominately via the same pathway after gamma-interferon (gamma-IFN) treatment, whereas murine cells are activated by other signalling routes. Thus, the calcium/calmodulin (Ca(2+)/Cam) pathway is preferentially selected for human cells whereas the PKC pathway is more often chosen for murine cells. These findings are firmly supported by other reports and show, in addition, a unique action exerted by gamma-IFN, since IL-4, another inducer of class II antigen expression, uses different pathways. This diversity of activation reveals the existence of a previously unknown complicated network of intracellular interactions able to regulate the same phenotype or cellular event. As major histocompatibility complex antigens (MHC) or human leukocyte antigens (HLA), are important in immune recognition and response, the results show that for human cells a more coherent method of HLA-DR antigen induction is followed after gamma-IFN administration, as calcium participation seems to be the first step in signal transduction. The same T-cell derived lymphokine, however, follows a totally different route when applied to murine cells.

A structural protein that plays an enzymatic role in the eggshell of Drosophila melanogaster.

E.S.P. is responsible for the hardening process of the egg-shell at the end of oogenesis (stage 14B) and constitutes a structural component. By immunoblotting, using polyclonal rabbit anti-HRP antibody and anti-rabbit IgG-HRP or Protein A-1251 as second antibody, one major band with MW 38KD on nitrocellulose filter showed positive reaction. We conclude that the E.S.P. is identical to the S38 chorionic protein. Morphological immunogold staining, using pre-embedding procedure, revealed positive reaction in the innermost chorionic layer (ICL) and the endochorion of the eggshell. In addition, electron probe X-ray microanalysis revealed the existence of 37% calcium (explained since the enzyme is Ca2(+)-activated) and 5% iron (explained due to the fact that it is a haemoprotein).