Uri Nir

Single nucleotide polymorphisms in miRNA binding sites and miRNA genes as breast/ovarian cancer risk modifiers in Jewish high-risk women

We hypothesized that aberrant gene silencing by miRNA may affect mutant BRCA penetrance. To test this notion, frequency of single nucleotide polymorphisms (SNPs; n = 42) within predicted miRNA binding sites or miRNA precursors were determined and compared in 363 BRCA1 mutation carriers: asymptomatic (n = 160), breast cancer (n = 140) and ovarian cancer (n = 63) patients, and in 125 BRCA2 mutation carriers: asymptomatic (n = 48), breast cancer (n = 58) and ovarian cancer (n = 19) patients. Overall, 16 of 42 SNPs were polymorphic, 11 had a minor allele frequency greater than 5% and 9 of them maintained the Hardy‐Weinberg Equilibrium. Based on Cox regression and Kaplan–Meier analyses, statistically significant differences were noted in BRCA2 mutation carriers by health status in 3 SNPs: CC homozygosity at rs6505162 increased ovarian cancer risk (RR 2.77; p = 0.028; 95% CI, 1.11–6.9); heterozygote SNP carriers of rs11169571 had an ∼2 fold increased risk for developing breast/ovarian cancer, whereas heterozygotes of the rs895819 SNP had an ∼50% reduced risk for developing breast/ovarian cancer. This study provides preliminary evidence for another regulatory level of penetrance of deleterious mutations in cancer predisposition genes.

Deleterious Mutations in the Zinc-Finger 469 Gene Cause Brittle Cornea Syndrome

Brittle cornea syndrome (BCS) is an autosomal-recessive disorder characterized by a thin cornea that tends to perforate, causing progressive visual loss and blindness. Additional systemic symptoms such as joint hypermotility, hyperlaxity of the skin, and kyphoscoliosis place BCS among the connective-tissue disorders. Previously, we assigned the disease gene to a 4.7 Mb interval on chromosome 16q24. In order to clone the BCS gene, we first narrowed the disease locus to a 2.8 Mb interval and systematically sequenced genes expressed in connective tissue in this chromosomal segment. We have identified two frameshift mutations in the Zinc-Finger 469 gene (ZNF469). In five unrelated patients of Tunisian Jewish ancestry, we found a 1 bp deletion at position 5943 (5943 delA), and in an inbred Palestinian family we detected a single-nucleotide deletion at position 9527 (9527 delG). The function of ZNF469 is unknown. However, a 30% homology to a number of collagens suggests that it could act as a transcription factor involved in the synthesis and/or organization of collagen fibers.

TMF/ARA160 is a BC-box-containing protein that mediates the degradation of Stat3

TMF/ARA160 is a Golgi resident protein whose cellular functions have not been conclusively revealed. Herein we show that TMF/ARA160 can direct the proteasomal degradation of the key cell growth regulator – Stat3. TMF/ARA160 was dispersed in the cytoplasm of myogenic C2C12 cells that were grown under low-serum conditions. The cytoplasmic distribution of TMF/ARA160 was accompanied by its transient association with the tyrosine kinase Fer and with Stat3, which underwent proteasomal degradation under those conditions. Moreover, serum deprivation induced the association of ubiquitinated proteins, with the TMF/ARA160 complex. However, TMF/ARA160 did not bind Stat1, whose cellular levels were increased in serum-starved C2C12 cells. Amino-acid sequence analysis identified a BC-box element in TMF/ARA160 that mediated the binding of this protein to elongin C. Ectopic expression of TMF/ARA160 in serum-starved C2C12 cells drove the ubiquitination and proteasomal degradation of Stat3, an effect that was not caused by TMF/ARA160 devoid of the BC-box motif. Thus, the Golgi apparatus harbors a novel BC-box-containing protein that can direct Stat3 to proteasomal degradation. Interestingly, the level of TMF/ARA160 was significantly decreased in malignant brain tumors, implying a suppressive role of that protein in tumor progression.

Downregulation of Fer induces PP1 activation and cell-cycle arrest in malignant cells

Fer is a nuclear and cytoplasmic intracellular tyrosine kinase. Herein we show that Fer is required for cell-cycle progression in malignant cells. Decreasing the level of Fer using the RNA interference (RNAi) approach impeded the proliferation of prostate and breast carcinoma cells and led to their arrest at the G0/G1 phase. At the molecular level, knockdown of Fer resulted in the activation of the retinoblastoma protein (pRB), and this was reflected by profound hypo-phosphorylation of pRB on both cyclin-dependent kinase CDK4 and CDK2 phosphorylation sites. Dephosphorylation of pRB was not seen upon the direct targeting of either CDK4 or CDK2 expression, and was only partially achieved by the simultaneous depletion of these two kinases. Amino-acid sequence analysis revealed two protein phosphatase 1 (PP1) binding motifs in the kinase domain of Fer and the association of Fer with the pRB phosphatase PP1α was verified using co-immunoprecipitation analysis. Downregulation of Fer potentiated the activation of PP1α and overexpression of Fer decreased the enzymatic activity of that phosphatase. Our findings portray Fer as a regulator of cell-cycle progression in malignant cells and as a potential target for cancer intervention.

Analysis of the erythropoietin receptor gene in patients with myeloproliferative and myelodysplastic syndromes

The human erythropoietin receptor (EpoR) gene has been cloned and characterized. Very few EpoR genetic abnormalities have been reported so far. Polycythemia vera (PV) is characterized by low/normal serum erythropoietin (Epo) levels with proposed Epo hypersensitivity. Myelodysplastic syndromes (MDS) are characterized by refractory anemia with variable serum Epo levels. Several reports have suggested EpoR abnormalities in both types of stem cell disorders. We analyzed DNA obtained from peripheral blood mononuclear cells of seven healthy controls, 20 patients with myeloproliferative disorders (MPD, 11 patients with PV, five agnogenic myeloid metaplasia with myelofibrosis, four essential thrombocytosis) and eight patients with refractory anemia with ringed sideroblasts (RARS), an MDS variant. The DNA was digested with four restriction enzymes (BamHI, Bgl II, Sacl and HindIII), followed by Southern blot, using a 32P radiolabeled probe, containing 1.5 kb of the human EpoR cDNA. All 20 MPD patients and seven out of the eight MDS patients demonstrated a restriction pattern which was identical to the seven normal controls, as well as to the erythroid cell line K562, and also consistent with the expected restriction map, for all four enzymes tested. One RARS patient had a normal pattern with three enzymes but a different one with HindIII. The HindIII 12 kb large band was replaced by a faint 12 kb band and a new (about 9 kb) band appeared. The EpoR restriction map and the normal pattern obtained with the other three enzymes suggest that this patient has a 3 kb upstream deletion in one allelic EpoR gene. The same molecular pattern was detected in the patients sister, who suffers from anemia with mild bone marrow (BM) dyserythropoiesis and plasmacytosis. Northern blot analysis showed that the patients BM RNA carried normal EpoR message. This familial pattern may represent polymorphism. However, the patients very high serum Epo level, her resistance to treatment with recombinant Epo, and the abnormally low growth rate of in vitro erythroid cultures, suggesting poor response to Epo in this MDS patient as well as the hematological abnormalities in her sister, support the speculation that the different EpoR gene might serve as a genetic predisposing marker and potentially could be involved (probably via post-transcriptional mechanisms and by an interaction with other factors or cytokines) in the pathogenesis. Our data suggest that the EpoR is intact in MPD and in most patients with RARS. One RARS patient had a familial different genetic structure, which could represent polymorphism. However, we can speculate also that it might be involved in the pathogenesis of the disease.

Rat skeletal muscle in culture expresses the ?1 but not the ?2 protein subunit isoform of the Na+/K+ pump

Studies from this laboratory have shown that the physiological expression of the Na+/K+ pump in primary cultures of rat skeletal muscle increases with development. The molecular mechanisms underlying these changes are not known. Therefore, we have examined the expression of α and β subunits of the Na+/K+ pump at both the protein and mRNA levels during myogenesis of primary skeletal muscle cell cultures obtained from newborn rats. Protein isoforms were identified by Western blotting techniques with specific monoclonal and polyclonal antibodies and subunit mRNA was studied with specific cDNA probes. Freshly isolated skeletal muscle from newborn rats expressed both α1 and α2 protein subunits. From day 1 after plating, primary cultures expressed only the α1 protein isoform. In contrast, both β1 and β2 isoforms were expressed in freshly isolated muscle and in primary cultures, with β1 expression being stronger in both preparations. Studies on RNA expression showed that mRNA for α1, α2, β1, and β2 isoforms was identified both in freshly isolated muscle and after plating of cells in culture. These findings indicate that the lack of α2 protein expression in primary muscle cell cultures reflects a form of posttranscriptional regulation. There did not appear to be a quantitative difference in isoform expression as a function of age or of fusion in spite of developmental increases in Na+/K+ pump activity and its dependence on cell fusion. The lack of expression of the α2 subunit isoform suggests that the developmental changes in physiological expression of the Na+/K+ pump in primary cultures of skeletal muscle may be attributable either to the changes in activity of the α1 subunit or to differential activities of αβ complexes involving either of the β subunits. J. Cell. Physiol. 180:236–244, 1999.

Tyrosine phosphorylation of the TATA element modulatory factor by the FER nuclear tyrosine kinases

The FER locus in the mouse encodes two tyrosine kinases, p94fer and p51ferT. While p94fer accumulates in the cytoplasm and nucleus of most mammalian cells the expression of p51ferT is restricted to the nucleus of meiotic primary spermatocytes. The cellular function of the FER kinases is not understood, nor has a substrate for these enzymes been characterized. To identify putative substrates of p94fer and p51ferT, the two enzymes were used as ‘baits’ in the yeast two‐hybrid screening system. cDNAs encoding the mouse TATA element modulatory factor (TMF) were repeatedly isolated in this assay. TMF was previously shown to bind the TATA element in RNA polymerase II promoters and impaired their functioning in a cell free transcription system. Both p94fer and p51ferT phosphorylated the TMF protein in in vitro and in vivo kinase assays. Sequential deletions showed that the carboxy‐terminal region of TMF was essential for phosphorylation. In situ hybridization analysis revealed the preferential accumulation of TMF transcripts in meiotic spermatogenic and oogenic cells. p94fer and p51ferT may thus modulate the suppressive activity of TMF during cellular growth and in defined differentiation processes.

In vivo effects of porphyrins on bacterial DNA

The DNA damage in intact Staphylococcus aureus and E. coli cells induced by photosensitized deuteroporphyrin or hemin is described. Treatment of S. aureus cultures with hemin or photosensitized deuteroporphyrin (Dp) caused time-dependent changes in the plasmidial DNA profiles. The major observation was the disappearance of the plasmid supercoiled fraction. The chromosomal DNA was also affected by hemin and by photosensitized Dp, since its degradation products were detected after exposing the bacterial cells to the porphyrin drugs. Photosensitization of E. coli cells, pretreated with Dp and polymyxin B nonapeptide (PMBNP), also resulted in plasmidial damage. No such damage occurred when E. coli cultures were treated with hemin and PMBNP. The above results can be tightly correlated with the antimicrobial action of porphyrins. Their damage to the bacterial DNA seems to reflect one of the in vivo effects of these porphyrins.

TMF/ARA160: A key regulator of sperm development.

TMF/ARA160 is a Golgi-associated protein to which several cellular activities have been attributed. These include, trafficking of Golgi-derived vesicles and E3 ubiquitin ligase activity. Here we show that TMF/ARA160 is required for the onset of key processes which underlie the development of mature sperm in mammals. TMF/ARA160 is highly expressed in specific spermatogenic stages. While the protein is not detected in the spermatogenic progenitor cells - spermatogonia, it accumulates in the Golgi of spermatocytes and spermatids but then disappears and is absent from spermatozoa and epididymal sperm cells. Mice that are homozygous null for TMF develop normally are healthy and the females are fertile. However, the males are sterile and their spermatids suffer from several developmental defects. They lack homing of Golgi-derived proacrosomal vesicles to the perinuclear surface, resulting in spermatozoa and epididymal sperm cells which lack acrosome. In a later developmental stage, the cytoplasm is not properly removed, thus resulting in spermatids which bare the nucleus with tightly packed DNA, surrounded by a cytoplasm. Finally, the spermatozoa of TMF(-/-) mice also suffer from misshapen heads, tails coiling around the sperm heads, and lack of motility. Taken together our findings portray TMF/ARA160 as a key regulator which is essential for the onset of key events in the differentiation and maturation of mammalian sperm and whose absence severely compromises their ability to fertilize ova.

Rapamycin and curcumin induce apoptosis in primary resting B chronic lymphocytic leukemia cells

B chronic lymphocytic leukemia (B-CLL) cells exist in patients as slowly accumulating resting as well as proliferating B cells. In this study, we examined whether Rapamycin and Curcumin, two naturally occurring compounds shown to have apoptotic effects, could selectively induce apoptosis in resting B-CLL cells. Mononuclear cells isolated from patients with B-CLL were treated with these agents and analysed by AnnexinV/propidium iodide binding, caspase activity, and changes in bcl-2/Bax ratio. Rapamycin and curcumin significantly induced apoptosis in resting B-CLL cells obtained from patients with CLL. Furthermore, rapamycin and curcumin increased caspase 9, 3 and 7 activity, decreased anti-apoptotic bcl-2 levels, and increased the pro-apoptotic protein Bax. These data suggest rapamycin and curcumin may be an effective treatment for B-CLL and are of high clinical significance considering the growing population of patients and lack of efficient treatment for this malignant disease.