Knockdown of hypoxia-inducible factor-1alpha in breast carcinoma MCF-7 cells results in reduced tumor growth and increased sensitivity to methotrexate

The hypoxia-inducible factor (HIF) 1alpha is a key regulator of the cellular response to oxygen deprivation. Specific disruption of the HIF-1 pathway is important for exploring its role in tumor biology and developing more efficient weapons to treat cancer. In this study, we stably transfected human breast tumor MCF-7 cells with short hairpin RNA expression vectors targeting HIF-1alpha. After knockdown of HIF-1alpha, hypoxia-induced expression of its target genes such as vascular endothelial growth factor, Glut-1, phosphoglycerate kinase, and P-glycoprotein were markedly attenuated. Moreover, HIF-1alpha knockdown was found to suppress the shift from S-phase to G(1) induced by hypoxia and increase drug sensitivity to methotrexate. The growth rates of HIF1alpha-knockdown tumors were drastically retarded in both subcutaneous and orthotopic xenograft models, which were accompanied by decreased angiogenesis and reduced expression of glucose transporter in tissue sections. These data demonstrate that HIF-1alpha knockdown reduces tumorigenicity of MCF-7 cells and suggest a promising combination of both anti-HIF-1 strategy and traditional chemotherapy to improve cancer treatment.     

The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT(2A) serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTT proliferation assay. We have demonstrated that the 5-HT(2A) receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT(2A) receptor present in this cell line is identical to the 5-HT(2A) receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT(2A) receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT(2A) receptor subtype, which is fully expressed in this cell line.     

Propionate-regulated high-yield protein production in Escherichia coli

A new expression system containing the Salmonella enterica prpBCDE promoter (P(prpB)) responsible for expression of the propionate catabolic genes (prp BCDE) and prpR encoding the positive regulator of this promoter has been developed and tested. The main features of the expression system compared to those based on the bacteriophage T7 promoter are low background expression and high induced expression in Escherichia coli strains BL21, BL21(DE3), MG1655, and W3110. In addition, propionate is an inexpensive, simple-to-use, nontoxic inducer that is attractive for large-scale protein production. Hence, this new system is highly complementary to the widely used T7 promoter-driven expression systems.     

P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes

Contact of T lymphocytes with nicotinamide adenine dinucleotide (NAD) or ATP causes cell death that requires expression of purinergic receptor P2X(7) (P2X(7)R). T cell subsets differ in their responses to NAD and ATP, which awaits a mechanistic explanation. Here, we show that sensitivity to ATP correlates with P2X(7)R expression levels in CD4 cells, CD8 cells and CD4(+)CD25(+) cells from both C57BL/6 and BALB/c mice. But P2X(7)R ligands do not only induce cell death but also shedding of CD62L. It is shown here that in CD62L(high) T cells, CD62L shedding correlates with low expression of P2X(7)Rs and lower cell death, whereas in CD62L(low) cells P2X(7)R expression and death are higher. The possibility is therefore investigated that P2X(7)Rs induce T cell activation. Experiments show that spontaneous T cell proliferation is somewhat higher in cells expressing P2X(7)Rs, but this effect we suggest is caused by P2X(7)R expression on accessory cells.     

The murine TRAIL receptor signals caspase-independent cell death through ceramide

Death receptors such as the 55 kDa tumor necrosis factor (TNF) receptor (TNF-R55) or Fas can initiate both apoptotic (caspase-dependent) and caspase-independent routes to programmed cell death (PCD). Here, we demonstrate for the first time that the single murine receptor for (TNF)-related apoptosis-inducing ligand (mTRAIL-R2) can induce a caspase-independent form of PCD with necrosis-like features in addition to apoptosis. Analysis of morphological and cellular features of caspase-independent PCD in response to TRAIL and TNF suggests that mTRAIL-R2 and TNF-R55 elicit caspase-independent PCD through similar pathways, although without participation of cathepsins. Cells overexpressing acid ceramidase (AC), an enzyme that metabolizes the sphingolipid ceramide, show enhanced survival from TRAIL-induced caspase-independent PCD but not from apoptosis, implicating a function of ceramide as a key mediator in caspase-independent PCD (but not apoptosis) induced by mTRAIL-R2. In concert with the enhanced resistance of AC-overexpressing cells against caspase-independent PCD induced by TNF, our results suggest that ceramide acts as a common mediator of caspase-independent PCD caused by death receptors such as mTRAIL-R2 and TNF-R55.     

Plasmodium falciparum: biochemical characterization of the cysteine protease falcipain-2'

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 are hemoglobinases and potential antimalarial drug targets. The falcipain-2' gene was identified recently and is nearly identical in sequence to falcipain-2. The product of this gene has not been studied previously. The mature protease domain of falcipain-2' was expressed in Escherichia coli, purified, and refolded to active enzyme. Functional analysis revealed similar biochemical properties to those of falcipain-2, including pH optima (pH 5.5-7.0), reducing requirements, and substrate preference. Studies with cysteine protease inhibitors showed similar inhibition of falcipain-2 and falcipain-2', although specificities were not identical. Considering activity against the presumed biological substrate, both enzymes readily hydrolyzed hemoglobin. Our results confirm that falcipain-2' is an active hemoglobinase and suggest that falcipain-2 and falcipain-2' play similar roles in erythrocytic parasites but that, for promising cysteine protease inhibitors, it will be important to confirm activity against this additional target.     

Synergistic interaction between Gdf1 and Nodal during anterior axis development

Growth and Differentiation Factor 1 (GDF-1) has been implicated in left-right patterning of the mouse embryo but has no other known function. Here, we demonstrate a genetic interaction between Gdf1 and Nodal during anterior axis development. Gdf1-/-;Nodal+/- mutants displayed several abnormalities that were not present in either Gdf1-/- or Nodal+/- single mutants, including absence of notochord and prechordal plate, and malformation of the foregut; organizing centers implicated in the development of the anterior head and branchial arches, respectively. Consistent with these deficits, Gdf1-/-;Nodal+/- mutant embryos displayed a number of axial midline abnormalities, including holoprosencephaly, anterior head truncation, cleft lip, fused nasal cavity, and lack of jaws and tongue. The absence of these defects in single mutants indicated a synergistic interaction between Nodal and GDF-1 in the node, from which the axial mesendoderm that gives rise to the notochord, prechordal plate, and foregut endoderm originates, and where the two factors are co-expressed. This notion was supported by a severe downregulation of FoxA2 and goosecoid in the anterior primitive streak of double mutant embryos. Unlike that in the lateral plate mesoderm, Nodal expression in the node was independent of GDF-1, indicating that both factors act in parallel to control the development of mesendodermal precursors. Receptor reconstitution experiments indicated that GDF-1, like Nodal, can signal through the type I receptors ALK4 and ALK7. However, analysis of compound mutants indicated that ALK4, but not ALK7, was responsible for the effects of GDF-1 and Nodal during anterior axis development. These results indicate that GDF-1 and Nodal converge on ALK4 in the anterior primitive streak to control the formation of organizing centers that are necessary for normal forebrain and branchial arch development.     

Mice deficient in Dmrt7 show infertility with spermatogenic arrest at pachytene stage

Genes including DM domain regulate sexual development in diverse metazoan phyla. One of these genes, Dmrt7, was expressed only in testes of adult mice. To determine the role of Dmrt7 in mice, we generated Dmrt7-knockout mice (Dmrt7-/-). Although the Dmrt7-/- showed normal growth, null males were infertile. No sperm was detected in the epididymis of Dmrt7-/- adult males. Absence of spermatids in a histological analysis, decreased expression of Ccna1 mRNA and the accumulation of SCP3-positive spermatocytes showed the arrest of spermatogenesis at the pachytene stage in the Dmrt7-knockout mice.     

The effect of angiotensin 1-7 on tyrosine kinases activity in rat anterior pituitary

Angiotensin 1-7 (Ang 1-7) is a peptide originated from Ang II. It is known that in vessels Ang 1-7 shows opposite effects to Ang II. Ang 1-7 can modify processes of proliferation. However, Ang 1-7 action in pituitary gland cells was never studied. Moreover, the specific binding sites for Ang 1-7 are still unknown. The aim of this study was to examine the effects of Ang 1-7 on tyrosine kinases (PTKs) activity in the anterior pituitary. The reaction of phosphorylation was carrying out in presence of different concentration of Ang 1-7 and losartan (antagonist of AT1 receptor) and PD123319 (antagonist of AT2). Our results show that Ang 1-7 inhibited activity of PTK to 60% of basic activity. Losartan did not change the Ang 1-7-induced changes in PTKs activity. The presence of PD123319 together with Ang 1-7 caused stronger inhibition PTKs activity than Ang 1-7 alone. These observations suggest that Ang 1-7 binds to the novel, unknown, specific for this peptide receptor.