EDIL3 promotes epithelial-mesenchymal transition and paclitaxel resistance through its interaction with integrin αVß3 in cancer cells.

Epithelial-mesenchymal transition (EMT) has recently been associated with tumor progression, metastasis, and chemotherapy resistance in several tumor types. We performed a differential gene expression analysis comparing paclitaxel-resistant vs. paclitaxel-sensitive breast cancer cells that showed the upregulation of EDIL3 (EGF Like Repeats and Discoidin I Like Domains Protein 3). This gene codifies an extracellular matrix protein that has been identified as a novel regulator of EMT, so we studied its role in tumor progression and paclitaxel response. Our results demonstrated that EDIL3 expression levels were increased in paclitaxel-resistant breast and prostate cancer cells, and in subsets of high-grade breast and prostate tumors. Moreover, we observed that EDIL3 modulated the expression of EMT markers and this was impaired by cilengitide, which blocks the EDIL3-integrin αVß3 interaction. EDIL3 knockdown reverted EMT and sensitized cells to paclitaxel. In contrast, EDIL3 overexpression or the culture of cells in the presence of EDIL3-enriched medium induced EMT and paclitaxel resistance. Adding cilengitide resensitized these cells to paclitaxel treatment. In summary, EDIL3 may contribute to EMT and paclitaxel resistance through autocrine or paracrine signaling in cancer cells. Blockade of EDIL3-integrin αVß3 interaction by cilengitide restores sensitivity to paclitaxel and reverts EMT in paclitaxel-resistant cancer cells. Combinations of cilengitide and taxanes could be beneficial in the treatment of subsets of breast and prostate cancers.

Proliferative potential after DNA damage and non-homologous end joining are affected by loss of securin.

The faithful repair of DNA damage, especially chromosomal double-strand breaks (DSBs), is crucial for genomic integrity. We have previously shown that securin interacts with the Ku70/80 heterodimer of the DSB non-homologous DNA end-joining (NHEJ) repair machinery. Here we demonstrate that securin deficiency compromises cell survival and proliferation, but only after genotoxic stress. Securin(-/-) cells show a significant increase in gross chromosomal rearrangements and chromatid breaks after DNA damage, and also reveal an altered pattern of end resection in an NHEJ assay in comparison with securin(+/+) cells. These data suggest that securin has a key role in the maintenance of genomic stability after DNA damage, thereby providing a previously unknown mechanism for regulating tumour progression.

Human securin, hPTTG, is associated with Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase.

We have previously isolated the hpttg proto-oncogene, which is expressed in normal tissues containing proliferating cells and in several kinds of tumors. In fact, expression of hPTTG correlates with cell proliferation in a cell cycle-dependent manner. Recently it was reported that PTTG is a vertebrate analog of the yeast securins Pds1 and Cut2, which are involved in sister chromatid separation. Here we show that hPTTG binds to Ku, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). hPTTG and Ku associate both in vitro and in vivo and the DNA-PK catalytic subunit phosphorylates hPTTG in vitro. Furthermore, DNA double-strand breaks prevent hPTTG-Ku association and disrupt the hPTTG-Ku complexes, indicating that genome damaging events, which result in the induction of pathways that activate DNA repair mechanisms and halt cell cycle progression, might inhibit hPTTG-Ku interaction in vivo. We propose that hPTTG might connect DNA damage-response pathways with sister chromatid separation, delaying the onset of mitosis while DNA repair occurs.

The proline-rich region of Vav binds to Grb2 and Grb3-3.

Vav has structural features found in signaling proteins and is expressed only in hematopoietic cells. The recent development of mice Vav -/- has confirmed a major role of Vav in early blood cell development. We previously showed that Vav constitutively interacts with glutathione-S-transferase-Grb2. Coimmunoprecipitation experiments supported the idea of a complex formed by Vav-Grb2 in vivo. This complex is of potential interest in signaling of hematopoietic cells. In this work we localize the domains of Vav and Grb2 involved in this interaction. By the use of an in vivo genetic approach (the double hybrid system) and through in vitro experiments (glutathione-S-transferase fusion proteins) we furnish evidence that the interaction between Vav and Grb2 involves the C-SH3 domain of Grb2 and the proline-rich region located in the N-SH3 of Vav. Furthermore this was confirmed by the use of both Vav and Sos derived proline-rich peptides which blocked the binding. In addition we show that Vav also interacts with Grb3-3, a naturally occurring Grb2 isoform wich lacks functional SH2 domain.

hpttg is over-expressed in pituitary adenomas and other primary epithelial neoplasias.

The role of oncogenes in pituitary tumorigenesis remains elusive since few genetic changes have been identified so far in pituitary tumors. Pituitary tumor-transforming gene (pttg) has been recently cloned from rat GH4 pituitary tumor cells. We have previously isolated and characterized hpttg from human thymus. In the present study, we analyse the expression of hpttg mRNA in a series of human pituitary adenomas. We show that hpttg is highly expressed in the majority of pituitary adenomas while only very low levels of mRNA can be detected in normal pituitary gland by Northern blot analysis. hPTTG protein was immunolocalized mainly in the cytoplasm of adenoma cells. Other common extra-cranial malignant tumors were also analysed by immunohistochemistry. Interestingly, strong hPTTG immunoreactivity was detected in most adenocarcinomas of mammary and pulmonary origins.

hpttg, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG.

We have isolated a human cDNA clone encoding a novel protein of 22 kDa that is a human counterpart of the rat oncoprotein PTTG. We show that the corresponding gene (hpttg) is overexpressed in Jurkat cells (a human T lymphoma cell line) and in samples from patients with different kinds of hematopoietic malignancies. Analysis of the sequence showed that hPTTG has an amino-terminal basic domain and a carboxyl-terminal acidic domain, and that it is a proline-rich protein with several putative SH3-binding sites. Subcellular fractionation studies show that, although hPTTG is mainly a cytosolic protein, it is partially localized in the nucleus. In addition we demonstrate that the acidic carboxyl-terminal region of hPTTG acts as a transactivation domain when fused to a heterologous DNA binding domain, both in yeast and in mammalian cells.

Cell cycle regulated expression and phosphorylation of hpttg proto-oncogene product.

We recently isolated a cDNA for hpttg, the human homolog of rat pituitary tumor transforming gene. Now we have analysed the expression of hpttg as a function of cell proliferation. hPTTG protein level is up-regulated in rapidly proliferating cells, is down-regulated in response to serum starvation or cell confluence, and is regulated in a cell cycle-dependent manner, peaking in mitosis. In addition, we show that hPTTG is phosphorylated during mitosis. Immunodepletion and in vitro phosphorylation experiments, together with the use of a specific inhibitor, indicate that Cdc2 is the kinase that phosphorylates hPTTG. These results suggest that hpttg is induced by, and may have a role in, regulatory pathways involved in the control of cell proliferation.

Properties of a non-specific nucleotidase in the membrane of rabbit red cells.

A non-specific nucleotidase was found in rabbit red blood cell membrane and, using 5'-AMP as substrate, several kinetic parameters for the enzyme were determined. Rabbit red cell ghosts catalyzed the hydrolysis of a wide spectrum of nucleoside 5'-, 3'- and 2'-monophosphates and a limited number of non-nucleotide substrates. All these activities were heat inactivated at the same rate, suggesting that they are the result of catalysis by the same enzyme. The nucleotidase was not dependent on K+ or Mg2+ and was also insensitive to ouabain. Its specific activity and other kinetic parameters were identical in preparation of membranes from both reticulocytes and the mature erythrocytes.

Maturation dependent decline of adenylate cyclase in rabbit red blood cell membranes.

Adenylate cyclase (EC 4.6.1.1) was studied in membrane preparations of reticulocyte-rich blood obtained from phenylhydrazine-treated rabbits and compared to that of untreated animals. Basal and fluoride-stimulated activities were decreased 2- and 4-fold, respectively, during the process of maturation. Catalytic parameters such as time course, protein, ATP, Mg2+ concentration curves and Km have been determined and were found to be similar in the reticulocyte and the erythrocyte. Adenylate cyclase was sensitive to GTP, 5'-guanylyl imidodiphosphate, prostaglandin E1 and prostaglandin E2. Activation by prostaglandin E1 was higher than that produced by prostaglandin E2. Only additive effect was found when 5'-guanylyl imidodiphosphate or GTP was added to hormone-stimulated activity. The sensitivity of the enzyme to these effectors was decreased over the transition reticulocyte-erythrocyte. In either cell the enzyme was not activated by catecholamines (epinephrine, norepinephrine, isoproterenol).

Phosphorylation and dephosphorylation reactions by erythrocyte plasma membrane enzymes.

Human erythrocyte membranes contain a phosphoprotein phosphatase able to dephosphorylate membrane protein previously phosphorylated by the endogenous protein kinase. The level of dephosphorylation obtained after prolonged incubation is about one half of the phosphorylated residues. The characteristics of this enzyme are similar to those described for the cytoplasmic phosphoprotein phosphatase. In a membrane preparation the phosphorylation and dephosphorylation reactions can be repeated, at least twice, achieving similar levels of phosphate esterified or hydrolyzed. The coordination of these two enzyme systems might play a role in some of the functions attributed to the protein kinase system.

Studies of the plasma membrane during maturation of the mammalian erythrocyte.

Plasma membranes were obtained from a homogeneous population of rabbit red blood cells at different maturation periods. Minor modifications in membrane proteins and membrane phospholipids and significant decreases in membrane glycoproteins and total lipids were observed with the age of the cell. The ouabain inhibited (Na+K+)-ATPase and the adenylate cyclase decreased with maturation but acetylcholinesterase and 5'-nucleotidase remained almost unchanged. The apparent activation energy of the ATPase increased with maturation. The results indicate that structural and functional modifications of the plasma membrane occur concomitantly with the ageing processes of the red cell.

Protein kinase and adenylate cyclase of erythrocyte membrane from patients with Duchenne muscular dystrophy.

In freshly prepared erythrocyte membranes from normal individuals and from patients with Duchenne progressive muscular dystrophy the endogenous protein kinase and the cAMP stimulated phosphorylation was identical for the three main 32P proteins including spectrin (protein band II). Another enzyme, adenylate cyclase, was found unchanged. Altered protein kinase and adenylate cyclase has been reported in this disorder. We have no explanation for these discrepancies.

Properties of a membrane-bound phosphatase activity in normal and abnormal red blood cells.

A neutral, membrane-bound, phosphatase activity was characterized in normal red blood cells, using p-nitrophenylphosphate as substrate. Its specific activity was 1.59 nmol mg-1 min-1. The kinetics were of the Michaelis type: KM,app = 2.5 X 10(-3) M. It was stimulated by K+ and inhibited by ouabain, a behaviour reminiscent of (Na+ + K+)-ATPase. In 10 patients with homozygous sickle cell disease and in 11 patients with unidentified congenital hemolytic anemias, the specific activity was significantly increased. In general, the phosphatase retained Michaelis-Menten kinetics. However, in four patients from the same family with an unidentified hemolytic anemia, the kinetics yielded a biphasic curve instead of a rectangular hyperbola, a change consistent with the existence of an inhibition by substrate excess. From detailed analysis of the curve, the apparent inhibitor constant for pNPP was determined: Ki,app approx. 2.5 X 10(-2) M. This novel abnormality of the red cell membrane might be the distinctive feature of a given type of congenital hemolytic anemia.

Isolation of ntrA-like mutants of Azotobacter vinelandii.

A number of chlorate-resistant mutants of Azotobacter vinelandii affected in a general control of nitrogen metabolism were isolated. These mutants could not utilize dinitrogen, nitrate, or nitrite as a nitrogen source. The reason for this inability is that they were simultaneously deficient in nitrogenase and nitrate and nitrite reductase activities. They were complemented by a cosmid carrying a DNA fragment of A. vinelandii able to complement ntrA mutants of Escherichia coli, so they seemed to be ntrA-like mutants.

GnRH and hCG tests in healthy adolescents and adults.

With the aim of obtaining reference values of GnRH test and of hCG test in a Venezuelan population, the authors evaluated the response of the pituitary LH and FSH to the GnRH action in 48 adolescents and 43 adults. Also, in 22 adolescents and in 22 adults from the same sample they evaluated the hCG effect on the gonadal steroids secretion. The maximum LH increment was obtained 30 min after GnRH stimulation, independently from the patients' age and their sexual development. FSH maximum increment was obtained 60 min after GnRH in the adults; meanwhile, the FSH maximum increment was obtained 30 min after GnRH stimulation in the Tanner stage 4 adolescents, and 90 min post-GnRH in the Tanner 5 adolescents. The gonadal steroid secretion 2, 24, 48, and 72 h post-hCG was significantly lower in the Tanner 4 adolescents. The gonadal steroid response post-hGC stimulation in the Tanner 5 adolescent group was similar to the one obtained in the adults. In late puberty the LH response to GnRH stimulus is not related either to age or to sexual development, contrary to the FSH response obtained after GnRH and the gonadal steroid response after hCG stimulus, both of which are related to age and patients' sexual development.

nasST, two genes involved in the induction of the assimilatory nitrite-nitrate reductase operon (nasAB) of Azotobacter vinelandii.

An operon including two new genes (nasS and nasT) has been defined, cloned and sequenced. The deduced NASS protein is homologous to NRTA from Synechococcus sp. and to NASF from Klebsiella pneumoniae, two proteins involved in nitrate uptake. The predicted NAST polypeptide is homologous to the regulator proteins of the two-component regulatory systems. NASS plays a negative regulatory role in the synthesis of the nitrate and nitrite reductase. NAST is required for the expression of the nitrite-nitrate reductase operon (nasAB). Expression of the nasST operon is not under the control of the NTR system and is not regulated by the nitrogen source. A Phi(nasA-lacZ) fusion has been used to analyse expression of the nasAB operon in three different genetic backgrounds with altered nitrate reductase activity. Beta-galactosidase activity in two of them was independent of nitrate but in a mutant unable to reduce nitrate, nas-4, it was normally induced by nitrate.

An effective mutagenic method in Azotobacter vinelandii.

The acridine-like compound ICR-191 is an effective mutagenic agent in Azotobacter vinelandii. Selectable mutants, such as those resistant to chlorate, can be isolated without post-mutagenic segregation. Non selectable mutants, such as those unable to metabolize different sugars, can be easily isolated after twelve generations of post-mutagenic segregation.

Ammonium repression of the nitrite-nitrate (nasAB) assimilatory operon of Azotobacter vinelandii is enhanced in mutants expressing the nifO gene at high levels.

A number of Tn5 mutants were isolated which were unable to fix nitrogen and showed enhanced ammonium repression of the nitrate/nitrite assimilation genes. They also had reduced nitrate reductase activity under fully inducing conditions. Insertions were localized within the nifB gene, and inability to fix nitrogen was shown to be due to disruption of the nifB gene. However, enhanced ammonium repression proved to be the result of constitutive expression of the downstream nifO gene from an 'out' promoter present in Tn5. Our results suggest that molybdenum metabolism might function as a regulatory factor that acts through the nitrate reductase.

A chromosomal linkage map of Azotobacter vinelandii.

A chromosomal map of Azotobacter vinelandii strain UW was constructed. The map was based on measures of cotransfer of various markers mediated by plasmids R68.45 and pJB3JI, on results obtained from conjugal experiments with R-primes, and on recombinants obtained by chromosomal transfer mediated by RP4/Tn5-Mob.

Identification of an operon involved in the assimilatory nitrate-reducing system of Azotobacter vinelandii.

A number of mutants lacking nitrate reductase (Nas-) or nitrite reductase (Nis-) activities have been isolated and characterized. An operon including two new genes (nasA and nasB) has been defined and cloned from an Azotobacter vinelandii gene bank. nasA encodes for nitrite reductase apoenzyme, whereas nasB is specific for nitrate reductase activity. Nitrate reductase exerts a regulatory effect on nasAB.