RESUMO
PIP4K2A is a lipid kinase that phosphorylates PtdIns5P, generating PtdIns4,5P2. Recently, PIP4K2A was identified as a potential target in acute myeloid leukemia cells. The objective of the present study was to investigate the PIP4K2A expression in hematological malignancies and verify the effects of PIP4K2A silencing on proliferation and survival of leukemia cell lines. PIP4K2A was found to be a cytoplasmic and nuclear protein with reduced levels in leukemia cell lines compared to normal leukocytes. PIP4K2A mRNA levels were significantly reduced in bone marrow cells from acute lymphoid leukemia (ALL) patients compared with healthy donors and in myelodysplastic syndromes (MDS) with ≥5% compared with <5% bone marrow blasts. Low PIP4K2A expression (lowest tertile versus 2 higher tertiles) negatively impacted overall survival of MDS patients by univariate analysis. PIP4K2A silencing did not modulate cell proliferation, clonogenicity and apoptosis of HEL and Namalwa leukemia cells. In summary, we characterized the expression of PIP4K2A in a cohort of patients with hematological malignancies and we found that PIP4K2A mRNA expression is downregulated in RAEB-1/RAEB-2 MDS and ALL cells, and PIP4K2A silencing does not modulate cell survival in HEL and Namalwa leukemia cells.
Assuntos
Neoplasias Hematológicas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Apoptose/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Neoplasias Hematológicas/patologia , Humanos , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Characterized for the first time in erythrocytes, phosphatidylinositol phosphate kinases (PIP kinases) belong to a family of enzymes that generate various lipid messengers and participate in several cellular processes, including gene expression regulation. Recently, the PIPKIIα gene was found to be differentially expressed in reticulocytes from two siblings with hemoglobin H disease, suggesting a possible relationship between PIPKIIα and the production of globins. Here, we investigated PIPKIIα gene and protein expression and protein localization in hematopoietic-derived cells during their differentiation, and the effects of PIPKIIα silencing on K562 cells. PIPKIIα silencing resulted in an increase in α and γ globins and a decrease in the proliferation of K562 cells without affecting cell cycle progression and apoptosis. In conclusion, using a cell line model, we showed that PIPKIIα is widely expressed in hematopoietic-derived cells, is localized in their cytoplasm and nucleus, and is upregulated during erythroid differentiation. We also showed that PIPKIIα silencing can induce α and γ globin expression and decrease cell proliferation in K562 cells.
Assuntos
Proliferação de Células/genética , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , alfa-Globinas/biossíntese , gama-Globulinas/biossíntese , Apoptose/genética , Regulação da Expressão Gênica , Inativação Gênica , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células K562 , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaRESUMO
BACKGROUND: The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1) and polyphosphate kinase (ppk) genes in bacteria in order to provide high mercury resistance and accumulation. RESULTS: In this study, bacterial transformation with transcriptional and translational enhanced vectors designed for the expression of metallothionein and polyphosphate kinase provided high transgene transcript levels independent of the gene being expressed. Expression of polyphosphate kinase and metallothionein in transgenic bacteria provided high resistance to mercury, up to 80 µM and 120 µM, respectively. Here we show for the first time that metallothionein can be efficiently expressed in bacteria without being fused to a carrier protein to enhance mercury bioremediation. Cold vapor atomic absorption spectrometry analyzes revealed that the mt-1 transgenic bacteria accumulated up to 100.2 ± 17.6 µM of mercury from media containing 120 µM Hg. The extent of mercury remediation was such that the contaminated media remediated by the mt-1 transgenic bacteria supported the growth of untransformed bacteria. Cell aggregation, precipitation and color changes were visually observed in mt-1 and ppk transgenic bacteria when these cells were grown in high mercury concentrations. CONCLUSION: The transgenic bacterial system described in this study presents a viable technology for mercury bioremediation from liquid matrices because it provides high mercury resistance and accumulation while inhibiting elemental mercury volatilization. This is the first report that shows that metallothionein expression provides mercury resistance and accumulation in recombinant bacteria. The high accumulation of mercury in the transgenic cells could present the possibility of retrieving the accumulated mercury for further industrial applications.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mercúrio/farmacocinética , Metalotioneína/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Biodegradação Ambiental , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Vetores Genéticos , Metalotioneína/biossíntese , Metalotioneína/genética , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/genética , TransgenesRESUMO
We are reporting here the results of differential gene expression experiments comparing two siblings, a 21-yr-old male and a 19-yr-old female, with the same alpha-thalassemia genotype (-alpha(3.7)/(--SEA)) and quite different levels of Hb H in the peripheral blood (18.7 and 5%, respectively). By using mRNA differential-display reverse-transcription-PCR and suppression subtractive hybridization, two main transcripts were selected in both procedures and validated by qRT-PCR, one corresponding to the phosphatidylinositol phosphate 4-kinase type II-alpha (PIP4KIIA) gene and the other to the beta-globin gene, both over expressed in the patient with the higher percentage of Hb H. Type II PIP kinases produce phosphatidylinositol 4,5 biphosphate, a critical and pleiotropic regulatory molecule involved in diverse cellular activities, including gene expression. Our results suggest that PIP4KIIA may be one of the factors related to the regulation of the beta-globin gene expression and the different levels of Hb H in alpha-thalassemic patients.
Assuntos
Regulação Enzimológica da Expressão Gênica , Hemoglobina H/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Reticulócitos/enzimologia , Irmãos , Talassemia alfa/enzimologia , Globinas beta/biossíntese , Feminino , Humanos , Masculino , Fosfatidilinositol 4,5-Difosfato/metabolismo , Adulto JovemRESUMO
When HeLa cells were selected for stable expression of a neo gene, linked either to mutated or wt c-H-ras genes, morphological examination of selected clones from several experiments revealed formation of giant multinucleated cells. These morphological alterations culminate in cell death, as a consequence of mitotic catastrophe (or mitotic death). Although clones expressing the mutated gene produced significantly larger numbers of these giant cells, those transfected with the normal allele were also found to produce significantly more giant multinucleated cells than non-transfected HeLa cells. Northern blot analysis of mRNA revealed overexpression of the normal H-ras gene in these clones. Chromatin structure analysis of these clones showed gross alterations, including the presence of micronuclei and heteroploid nuclei. Interestingly, odd numbers of nuclei were found in colonies of these giant cells. In addition, alterations in cell cycle parameters were observed, including the appearance of a subpopulation of cells with an abnormal content of DNA, probably representing dying cells. Our data support the notion that abnormal expression of H-ras contributes to mitotic catastrophe and death of a subpopulation of HeLa cells.