The Role of the Gallbladder, the Intestinal Barrier and the Gut Microbiota in the Development of Food Allergies and Other Disorders.

The microbiota present in the gastrointestinal tract is involved in the development or prevention of food allergies and autoimmune disorders; these bacteria can enter the gallbladder and, depending on the species involved, can either be benign or cause significant diseases. Occlusion of the gallbladder, usually due to the presence of calculi blocking the bile duct, facilitates microbial infection and inflammation, which can be serious enough to require life-saving surgery. In addition, the biliary salts are secreted into the intestine and can affect the gut microbiota. The interaction between the gut microbiota, pathogenic organisms, and the human immune system can create intestinal dysbiosis, generating a variety of syndromes including the development of food allergies and autoimmune disorders. The intestinal microbiota can aggravate certain food allergies, which become severe when the integrity of the intestinal barrier is affected, allowing bacteria, or their metabolites, to cross the intestinal barrier and invade the bloodstream, affecting distal body organs. This article deals with health conditions and severe diseases that are either influenced by the gut flora or caused by gallbladder obstruction and inflammation, as well as putative treatments for those illnesses.

The Interplay among Radiation Therapy, Antibiotics and the Microbiota: Impact on Cancer Treatment Outcomes.

Radiation therapy has been used for more than a century, either alone or in combination with other therapeutic modalities, to treat most types of cancer. On average, radiation therapy is included in the treatment plans for over 50% of all cancer patients, and it is estimated to contribute to about 40% of curative protocols, a success rate that may reach 90%, or higher, for certain tumor types, particularly on patients diagnosed at early disease stages. A growing body of research provides solid support for the existence of bidirectional interaction between radiation exposure and the human microbiota. Radiation treatment causes quantitative and qualitative changes in the gut microbiota composition, often leading to an increased abundance of potentially hazardous or pathogenic microbes and a concomitant decrease in commensal bacteria. In turn, the resulting dysbiotic microbiota becomes an important contributor to worsen the adverse events caused in patients by the inflammatory process triggered by the radiation treatment and a significant determinant of the radiation therapy anti-tumor effectiveness. Antibiotics, which are frequently included as prophylactic agents in cancer treatment protocols to prevent patient infections, may affect the radiation/microbiota interaction through mechanisms involving both their antimicrobial activity, as a mediator of microbiota imbalances, and their dual capacity to act as pro- or anti-tumorigenic effectors and, consequently, as critical determinants of radiation therapy outcomes. In this scenario, it becomes important to introduce the use of probiotics and/or other agents that may stabilize the healthy microbiota before patients are exposed to radiation. Ultimately, newly developed methodologies may facilitate performing personalized microbiota screenings on patients before radiation therapy as an accurate way to identify which antibiotics may be used, if needed, and to inform the overall treatment planning. This review examines currently available data on these issues from the perspective of improving radiation therapy outcomes.

The Use of Bacteriophages in Biotechnology and Recent Insights into Proteomics.

Phages have certain features, such as their ability to form protein-protein interactions, that make them good candidates for use in a variety of beneficial applications, such as in human or animal health, industry, food science, food safety, and agriculture. It is essential to identify and characterize the proteins produced by particular phages in order to use these viruses in a variety of functional processes, such as bacterial detection, as vehicles for drug delivery, in vaccine development, and to combat multidrug resistant bacterial infections. Furthermore, phages can also play a major role in the design of a variety of cheap and stable sensors as well as in diagnostic assays that can either specifically identify specific compounds or detect bacteria. This article reviews recently developed phage-based techniques, such as the use of recombinant tempered phages, phage display and phage amplification-based detection. It also encompasses the application of phages as capture elements, biosensors and bioreceptors, with a special emphasis on novel bacteriophage-based mass spectrometry (MS) applications.

Exome sequencing identifies novel somatic variants in African American esophageal squamous cell carcinoma.

Esophageal cancer has a strikingly low survival rate mainly due to the lack of diagnostic markers for early detection and effective therapies. In the U.S., 75% of individuals diagnosed with esophageal squamous cell carcinoma (ESCC) are of African descent. African American ESCC (AA ESCC) is particularly aggressive, and its biological underpinnings remain poorly understood. We sought to identify the genomic abnormalities by conducting whole exome sequencing of 10 pairs of matched AA esophageal squamous cell tumor and control tissues. Genomic analysis revealed diverse somatic mutations, copy number alterations (SCNAs), and potential cancer driver genes. Exome variants created two subgroups carrying either a high or low tumor mutation burden. Somatic mutational analysis based on the Catalog of Somatic Mutations in Cancer (COSMIC) detected SBS16 as the prominent signature in the high mutation rate group suggesting increased DNA damage. SBS26 was also detected, suggesting possible defects in mismatch repair and microsatellite instability. We found SCNAs in multiple chromosome segments, encoding MYC on 8q24.21, PIK3CA and SOX2 on 3q26, CCND1, SHANK2, CTTN on 11q13.3, and KRAS on 12p12. Amplifications of EGFRvIII and EGFRvIVa mutants were observed in two patients, representing a novel finding in ESCC that has potential clinical relevance. This present exome sequencing, which to our knowledge, represents the first comprehensive exome analysis exclusively in AA ESCC, and highlights novel mutated loci that might explain the aggressive nature of AA ESCC and lead to the development of diagnostic and prognostic markers as well as therapeutic targets.

Caveolin-1 promotes resistance to chemotherapy-induced apoptosis in Ewing's sarcoma cells by modulating PKCalpha phosphorylation.

Caveolin-1 (CAV1) has been implicated in the regulation of several signaling pathways and in oncogenesis. Previously, we identified CAV1 as a key determinant of the oncogenic phenotype and tumorigenic activity of cells from tumors of the Ewing's Sarcoma Family (ESFT). However, the possible CAV1 involvement in the chemotherapy resistance commonly presented by an ESFT subset has not been established to date. This report shows that CAV1 expression determines the sensitivity of ESFT cells to clinically relevant chemotherapeutic agents. Analyses of endogenous CAV1 levels in several ESFT cells and ectopic CAV1 expression into ESFT cells expressing low endogenous CAV1 showed that the higher the CAV1 levels, the greater their resistance to drug treatment. Moreover, results from antisense- and shRNA-mediated gene expression knockdown and protein re-expression experiments demonstrated that CAV1 increases the resistance of ESFT cells to doxorubicin (Dox)- and cisplatin (Cp)-induced apoptosis by a mechanism involving the activating phosphorylation of PKCalpha. CAV1 knockdown in ESFT cells led to decreased phospho(Thr(638))-PKCalpha levels and a concomitant sensitization to apoptosis, which were reversed by CAV1 re-expression. These results were recapitulated by PKCalpha knockdown and re-expression in ESFT cells in which CAV1 was previously knocked down, thus demonstrating that phospho(Thr(638))-PKCalpha acts downstream of CAV1 to determine the sensitivity of ESFT cells to chemotherapeutic drugs. These data, along with the finding that CAV1 and phospho(Thr(638))-PKCalpha are co-expressed in approximately 45% of ESFT specimens tested, imply that targeting CAV1 and/or PKCalpha may allow the development of new molecular therapeutic strategies to improve the treatment outcome for patients with ESFT.

A Significant Question in Cancer Risk and Therapy: Are Antibiotics Positive or Negative Effectors? Current Answers and Possible Alternatives.

Cancer is predominantly considered as an environmental disease caused by genetic or epigenetic alterations induced by exposure to extrinsic (e.g., carcinogens, pollutants, radiation) or intrinsic (e.g., metabolic, immune or genetic deficiencies). Over-exposure to antibiotics, which is favored by unregulated access as well as inappropriate prescriptions by physicians, is known to have led to serious health problems such as the rise of antibiotic resistance, in particular in poorly developed countries. In this review, the attention is focused on evaluating the effects of antibiotic exposure on cancer risk and on the outcome of cancer therapeutic protocols, either directly acting as extrinsic promoters, or indirectly, through interactions with the human gut microbiota. The preponderant evidence derived from information reported over the last 10 years confirms that antibiotic exposure tends to increase cancer risk and, unfortunately, that it reduces the efficacy of various forms of cancer therapy (e.g., chemo-, radio-, and immunotherapy alone or in combination). Alternatives to the current patterns of antibiotic use, such as introducing new antibiotics, bacteriophages or enzybiotics, and implementing dysbiosis-reducing microbiota modulatory strategies in oncology, are discussed. The information is in the end considered from the perspective of the most recent findings on the tumor-specific and intracellular location of the tumor microbiota, and of the most recent theories proposed to explain cancer etiology on the notion of regression of the eukaryotic cells and systems to stages characterized for a lack of coordination among their components of prokaryotic origin, which is promoted by injuries caused by environmental insults.

Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases.

Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.

Caveolin-1 (CAV1) is a target of EWS/FLI-1 and a key determinant of the oncogenic phenotype and tumorigenicity of Ewing's sarcoma cells.

Tumors of the Ewing's sarcoma family (ESFT), such as Ewing's sarcoma (EWS) and primitive neuroectodermal tumors (PNET), are highly aggressive malignancies predominantly affecting children and young adults. ESFT express chimeric transcription factors encoded by hybrid genes fusing the EWS gene with several ETS genes, most commonly FLI-1. EWS/FLI-1 proteins are responsible for the malignant phenotype of ESFT, but only few of their transcriptional targets are known. Using antisense and short hairpin RNA-mediated gene expression knockdown, array analyses, chromatin immunoprecipitation methods, and reexpression studies, we show that caveolin-1 (CAV1) is a new direct target of EWS/FLI-1 that is overexpressed in ESFT cell lines and tumor specimens and is necessary for ESFT tumorigenesis. CAV1 knockdown led to up-regulation of Snail and the concomitant loss of E-cadherin expression. Consistently, loss of CAV1 expression inhibited the anchorage-independent growth of EWS cells and markedly reduced the growth of EWS cell-derived tumors in nude mice xenografts, indicating that CAV1 promotes the malignant phenotype in EWS carcinogenesis. Reexpression of CAV1 or E-cadherin in CAV1 knockdown EWS cells rescued the oncogenic phenotype of the original EWS cells, showing that the CAV1/Snail/E-cadherin pathway plays a central role in the expression of the oncogenic transformation functions of EWS/FLI-1. Overall, these data identify CAV1 as a key determinant of the tumorigenicity of ESFT and imply that targeting CAV1 may allow the development of new molecular therapeutic strategies for ESFT patients.

Combined transcriptional and translational targeting of EWS/FLI-1 in Ewing's sarcoma.

PURPOSE: To show the efficacy of targeting EWS/FLI-1 expression with a combination of specific antisense oligonucleotides and rapamycin for the control of Ewing's sarcoma (EWS) cell proliferation in vitro and the treatment of mouse tumor xenografts in vivo. EXPERIMENTAL DESIGN: EWS cells were simultaneously exposed to EWS/FLI-1-specific antisense oligonucleotides and rapamycin for various time periods. After treatment, the following end points were monitored and evaluated: expression levels of the EWS/FLI-1 protein, cell proliferation, cell cycle distribution, apoptotic cell death, caspase activation, and tumor growth in EWS xenografts implanted in nude mice. RESULTS: Simultaneous exposure of EWS cells in culture to an EWS/FLI-1-targeted suppression therapy using specific antisense oligonucleotides and rapamycin resulted in the activation of a caspase-dependent apoptotic process that involved the restoration of the transforming growth factor-beta-induced proapoptotic pathway. In vivo, individual administration of either antisense oligonucleotides or rapamycin significantly delayed tumor development, and the combined treatment with antisense oligonucleotides and rapamycin caused a considerably stronger inhibition of tumor growth. CONCLUSIONS: Concurrent administration of EWS/FLI-1 antisense oligonucleotides and rapamycin efficiently induced the apoptotic death of EWS cells in culture through a process involving transforming growth factor-beta. In vivo experiments conclusively showed that the combined treatment with antisense oligonucleotides and rapamycin caused a significant inhibition of tumor growth in mice. These results provide proof of principle for further exploration of the potential of this combined therapeutic modality as a novel strategy for the treatment of tumors of the Ewing's sarcoma family.

Roscovitine is an effective inducer of apoptosis of Ewing's sarcoma family tumor cells in vitro and in vivo.

The Ewing's sarcoma family of tumors (ESFT) comprises several well-characterized malignant neoplasms with particularly aggressive behavior. Despite recent progress in the use of multimodal therapeutic approaches and aggressive local control measures, a substantial proportion of patients die because of disease progression. Furthermore, this outcome has not changed significantly over the last 15 to 20 years. Consequently, new, more effective therapeutic options are sorely needed for the treatment of ESFT. Because ESFT cells overexpress several cyclin-dependent kinases (CDK), we explored the efficacy against ESFT of roscovitine, a CDK inhibitor shown to be surprisingly safe for humans in clinical trials of their anticancer activity. Results showed that ESFT cell lines are uniformly sensitive to roscovitine. In addition to exerting comparatively minor cell cycle effects, roscovitine treatment concomitantly caused the up-regulation of the expression of the proapoptotic protein BAX and the down-regulation of both survivin and XIAP, thus resulting in caspase-dependent apoptosis. Furthermore, in vivo experiments showed that s.c. growth of ESFT xenografts was also significantly slowed by i.p. injection of roscovitine. These results strongly suggest that roscovitine may be an effective therapeutic agent against ESFT and recommend its evaluation against ESFT in clinical trials and its inclusion in future treatment protocols.

Rapamycin induces apoptosis of JN-DSRCT-1 cells by increasing the Bax : Bcl-xL ratio through concurrent mechanisms dependent and independent of its mTOR inhibitory activity.

Rapamycin, a complex macrolide and potent fungicide, immunosuppressant and anticancer agent, is a highly specific inhibitor of mammalian target of rapamycin (mTOR). Rapamycin has been shown to induce G1-phase cell cycle arrest in diverse tumor cell types, and its derivatives RAD001 and CCI-779 are currently in phase I and phase II clinical trials, respectively, as anticancer agents. In this study, we show that rapamycin induced the apoptotic death of JN-DSRCT-1 cells, the only available in vitro model for Desmoplastic Small Round Cell Tumors (DSRCT), while having only minor effects on their cell cycle. Rapamycin induced apoptosis by increasing the Bax : Bcl-xL ratio as a consequence of the concomitant downregulation of Bcl-xL and upregulation of Bax, both at the post-transcriptional level. Rapamycin also downregulated the levels of EWS/WT1, the fusion protein characteristic of DSRCT. Transient transfection studies using kinase-dead and rapamycin-resistant forms of mTOR demonstrated that only the downregulation of Bcl-xL was caused by the mTOR inhibitory action of rapamycin, which prevented cap-dependent translation initiation, whereas Bax upregulation was induced by rapamycin through a mechanism independent of its mTOR inhibitory activity. Moreover, rapamycin treatment downregulated the mRNA and protein levels of the 26S p44.5 proteasome subunit, suggesting the involvement of the proteasome complex in the mechanisms of rapamycin-induced apoptosis. Treatment of JN-DSRCT-1 cells with MG-132, a proteasome specific inhibitor, also resulted in the induction of apoptosis through a similar increase in the Bax : Bcl-xL ratio specifically caused by inhibiting Bax degradation and turnover. These results suggested that rapamycin induces apoptosis by preventing the degradation of the Bax protein by the proteasome, and that this process is independent of mTOR inhibition. Furthermore, these results strongly support the introduction of the use of rapamycin as a cytotoxic agent for the treatment of DSRCT.

PCPH expression is an early event in the development of testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) include various malignancies with distinct pathologies that share a common precursor lesion (intratubular germ cell neoplasia, unclassified, ITGCNU, or carcinoma in situ, CIS). TGCTs, as a whole, represent a highly curable tumor paradigm, with high sensitivity to radiotherapy and, especially, to cisplatin-based chemotherapy. However, a percentage of cases display therapeutic resistance, and the molecular mechanisms underlying such resistant phenotype remain to be elucidated. We put forward the notion that expression of oncogenic forms of the PCPH gene, which are known to confer resistance to radiation and chemotherapeutic drugs, including cisplatin, may be expressed in TGCTs, and thus contribute to the development of therapeutic resistance. To begin testing this concept, we studied PCPH expression in human TGCT cell lines and in 54 solid tumors by RT-PCR, western immunoblot and immunohistochemistry. The results demonstrated that: i) PCPH is expressed in TGCT cell lines and tumors, including CIS; ii) its expression levels vary among different TGCT pathologies, being generally higher in well differentiated regions and lower in areas of predominant proliferation; iii) PCPH expression is substantially increased in tumors relative to matched normal testicular tissue; iv) tumor samples express PCPH polypeptides of low molecular mass, consistent with the known size of the PCPH oncoprotein, that are either absent from, or markedly reduced in, matched normal tissue. Collectively, these results positively identify PCPH as a good early molecular marker for testicular neoplasms, and strongly indicate that immunodetection of truncated PCPH polypeptides may be a useful diagnostic tool for TGCT.

Interaction and stabilization of X-linked inhibitor of apoptosis by Raf-1 protein kinase.

Raf-1 serine/threonine protein kinase plays an important role in cell growth, differentiation and cell survival. Recent reports using c-raf-1 gene-knockouts have observed MEK/ERK independent functions of Raf-1 in cell survival and protection from apoptosis. Raf-1 has also been shown to be involved in counteracting specific apoptotic pathways by restraining caspase activation, although the precise mechanism is unknown. XIAP is a potent inhibitor of apoptosis that blocks both the mitochondria and death receptor mediated pathways of apoptosis by directly binding to and inhibiting the initiator and effector caspases. In our efforts to understand the mechanism by which Raf-1 inhibits caspase activation, we discovered a novel interaction between Raf-1 and XIAP. In this study, we describe the physical interaction between Raf-1 and XIAP in vitro and in vivo in mammalian cells. We also demonstrate that Raf-1 phosphorylates XIAP in vitro and in vivo. Additionally, Raf-1 prevents XIAP degradation in response to different apoptotic triggers. Our studies identify XIAP as a new substrate of Raf-1 and provide potentially important insight into mechanisms underlying Raf-1 effects on cell survival.

Partial depletion of intracellular ATP mediates the stress-survival function of the PCPH oncoprotein.

Promotion of cellular resistance to stressful stimuli, including ionizing radiation and chemotherapeutic drugs, contributes to the transforming activity of the PCPH oncogene. The mechanism of this action, however, has remained unknown. Consistent with its intrinsic ATP diphosphohydrolase activity, expression of the PCPH oncoprotein in cultured cells has now been shown to result in partial depletion of intracellular ATP and consequent inhibition of the c-JUN NH2-terminal kinase-mediated stress signaling pathway. Supplementation of cells expressing the PCPH oncoprotein with exogenous ATP restored both stress-response signaling and sensitivity to cisplatin-induced apoptosis. In contrast, overexpression of the wild-type PCPH protein had a minimal effect on stress-induced signaling and on the cellular ATP content and did not protect cells from apoptosis. These results suggest that the PCPH oncoprotein confers resistance to stressors by reducing the cellular ATP concentration to levels below those required for optimal stress-induced signaling and apoptosis. Treatment with adenosine or nucleoside analogues may thus enhance the response to radiation or chemotherapy of tumors that express the PCPH oncogene.

The PCPH oncoprotein antagonizes the proapoptotic role of the mammalian target of rapamycin in the response of normal fibroblasts to ionizing radiation.

Exposure of normal mouse fibroblasts (MEF3T3) to ionizing radiation (IR) resulted in a dose-dependent increase of mTOR mRNA and protein levels and the shuttling of the mTOR protein from its normal, predominantly mitochondrial location to the cell nucleus. The same IR doses that activated mTOR induced the phosphorylation of p53 on Ser(18) (mouse equivalent to human Ser(15)) and the subsequent transcriptional activation of PUMA, a known proapoptotic p53-target gene, and promoted apoptosis involving increased overall caspase activity, caspase-3 activation, cleavage of poly(ADP-ribose) polymerase (PARP) and classic protein kinase C (PKC) isoforms, and DNA fragmentation. The proapoptotic role of mTOR in this process was demonstrated by the fact that rapamycin, a mTOR inhibitor, blocked p53 Ser(18) phosphorylation, the induction of PUMA, and all other apoptosis events. Furthermore, the proapoptotic function of mTOR was also antagonized by the expression in MEF3T3 cells of the PCPH oncoprotein, known to enhance cell survival by causing partial ATP depletion. Tetracyclin (Tet)-regulated expression of oncogenic PCPH, or overexpression of normal PCPH, blocked both phosphorylation and nuclear shuttling of mTOR in response to IR. These results indicate that alterations in PCPH expression may render tumor cells resistant to IR, and perhaps other DNA-damaging agents, by preventing mTOR activation and signaling.

Rapamycin induces the fusion-type independent downregulation of the EWS/FLI-1 proteins and inhibits Ewing's sarcoma cell proliferation.

Ewing's sarcoma (ES) is the prototype of a family of tumors (ESFT) of neuroectodermal origin formed by small, round cells with limited neural differentiation, which arise most frequently within bones in children or adolescents. The proliferation of ESFT cells is highly dependent on the establishment of, and signaling through several growth factor-mediated autocrine loops. The mammalian target of rapamycin (mTOR) is a central regulator of translation and cell proliferation, involved in the cellular response to various nutritional, stress and mitogenic effectors. As mTOR has recently been associated with certain human cancers, we investigated the possibility that mTOR played a role in the regulation of ES cell proliferation. Results showed that ES cell lines carrying EWS/FLI-1 alleles of different types expressed different levels of total and phosphorylated mTOR protein. We demonstrate that rapamycin, an mTOR inhibitor, efficiently blocked the proliferation of all cell lines by promoting cell cycle arrest at the G1 phase. This was paralleled by the downregulation of the levels of the EWS/FLI-1 proteins, regardless of their fusion type, and the concomitant restoration of the expression of the TGF-beta type 2 receptor (TGFbeta RII), which is known to be repressed by several EWS-ETS fusion proteins. The expression of a rapamycin-resistant mTOR construct prevented both the proliferation blockade and the EWS/FLI-1 downregulation. These data demonstrate that mTOR signaling plays a central role in ES cell pathobiology and strongly suggest that the use of rapamycin as a cytostatic agent may be an efficient tool for the treatment of ES patients.

Differential regulation of the response to DNA damage in Ewing's sarcoma cells by ETS1 and EWS/FLI-1.

Ewing's sarcoma (EWS) cells contain levels of poly(ADP-ribose) polymerase (PARP) significantly higher than other eukaryotic cells. Previously, we cloned the PARP gene promoter region from EWS cells, showed that it contained multiple ETS-binding sites and demonstrated a positive regulation of PARP by ETS1. We now report that, contrary to ETS1, EWS/FLI-1, an aberrant ETS transcription factor present in most EWS cells, is a negative effector of PARP transcription. Because PARP levels have been associated with cellular resistance or sensitivity to genotoxic agents, we studied the effect of modifying PARP levels in EWS cells on their response to DNA damage by modulating the expression of ETS1 or EWS/FLI-1 using antisense methodology. Results show that stable down-regulation of ETS1 increases the resistance of EWS cells to various genotoxic agents, whereas down-regulation of EWS/FLI-1 has pro-apoptotic effects. Because down-regulation EWS/FLI-1 does not dramatically change PARP levels, these results suggest a direct effect for EWS/FLI-1 in the DNA damage response of EWS cells. Since expression of the aberrant fusion proteins by EWS cells is essential for maintaining their neoplastic phenotype, our results suggest that the use of antisense oligonucleotides in combination with chemotherapeutic agents or radiation may be doubly effective by causing both an increase in sensitivity to therapeutic agents and a simultaneous down-regulation, or reversion, of the neoplastic phenotype of EWS cells.

Deregulated expression of the PCPH proto-oncogene in human breast cancers.

We performed a study on the expression of the PCPH protein in samples corresponding to normal, pre-malignant and malignant stages of the human mammary gland by using protocols of immunohistochemistry and Western blot analysis with anti-PCPH specific antibodies. Results obtained from the immunohistochemical study showed that PCPH was undetectable in samples of normal breast and of benign diseases, with the exception of glands presenting apocrine metaplasia, in which an intense PCPH stain was observed both in the basal cytoplasm of the secretory cells and in the apocrine secretion. On the contrary, an intense labeling was observed in the cytoplasm of neoplastic cells in samples of both ductal and lobular carcinoma in situ, with this immunostaining increasing even further in samples of infiltrating carcinoma, both ductal and lobular. Western blot analyses of the same set of samples detected a 47 kDa form as the main PCPH polypeptide present in all cases studied. However, whereas this 47 kDa polypeptide was the only PCPH form detected in normal and pre-malignant samples, multiple forms could be detected in carcinoma samples, indicating the presence of altered PCPH polypeptides at these disease stages. These results were in agreement with those from the immunohistochemical study and together indicated that PCPH protein expression represents a good molecular marker to follow the process of human breast carcinogenesis. Furthermore, these results suggested that characterization of the pattern and level of PCPH expression may be a useful tool for early identification of breast cancers.

Role of an internal ribosome entry site in the translational control of the human transcription factor Sp3.

Sp1 and Sp3 are transcription factors involved in the regulation of numerous genes involved in oncogenesis. Sp3 is a bi-functional transcription factor with three different isoforms. Its bi-functional activity may in part be regulated by the relative expression of these isoforms. Northern blot analysis of Sp3 detects only a single transcript. Analysis of the known Sp3 cDNA sequence shows a high GC content and seven out-of-frame AUG codons located between the 5'-end of the mRNA and the two internal AUG initiation sites. This makes it highly unlikely that cap-recruited, translation initiation competent ribosomes could reach the internal start sites. A full human Sp3 expression construct was cloned. A bicistronic vector using Renilla and firefly luciferase showed internal ribosome entry site (IRES) activity in Sp3 RNA immediately 5' to the internal AUG sites. Also, the two smaller isoforms were translated more efficiently when full-length, uncapped transcripts were used, while the larger isoform was not translated. Mutants of Sp3 with AUG codons introduced 5' of the two internal start sites were generated. Results showed that they were unable to suppress the smaller isoforms in vitro. Furthermore, dual non-AUG to AUG mutations showed occlusion of the second introduced isoform (i.e., the isoform situated more 3') but not of the internally initiated isoforms. These experiments are consistent with IRES-mediated translation of the two smaller isoforms of Sp3. The presence of an IRES allows the possibility that Sp3 isoform ratios and activity are controlled at the translational level. This mechanism may allow cells to control the expression of numerous genes during mitosis and, thus, have profound effects on cell cycle regulation and tumorigenesis.

Characterization of gdp1+ as encoding a GDPase in the fission yeast Schizosaccharomyces pombe.

We have isolated the gdp1+ gene from Schizosaccharomyces pombe coding for a membrane protein with guanosine diphosphatase (GDPase) activity, which is highly homologous to Golgi GDPases isolated from other yeast species. The gdp1+ product, Gdp1p, displays both GDPase and uridine diphosphatase (UDPase) activities in vitro, with a strong dependence for calcium and manganese cations. The observation of a defect in N-glycosylation of invertase in S. pombe Deltagdp1 cells together with the ability of gdp1+ to functionally complement the defective O-mannosylation of chitinase in Saccharomyces cerevisiae cells disrupted in the GDA1 gene (gdp1+ homolog), suggests a main role for Gdp1p in protein glycosylation in fission yeast.