Roberta Di Pietro

In Vitro Mass Production of Human Erythroid Cells from the Blood of Normal Donors and of Thalassemic Patients

We describe a new two-step culture method for mass production in vitro of erythroid cells from either CD34+ (10(5) cells/mL) or light-density (10(6) cells/mL) cells purified from the blood of normal donors and thalassemic patients. The method includes (i) culture of the cells in the presence of dexamethasone and estradiol (10(-6) M each) and (ii) the growth factors SCF (50 ng/mL), IL-3 (1 ng/mL), and EPO (1 U/mL). In their proliferative phase, these cultures generated approximately 1.2 x 10(7) erythroblasts for each milliliter of blood collected from normal donors or thalassemic patients. They were composed mostly (90%) of CD45(low)/glycophorin (GPA)(neg)/CD71(1ow) cells at day 7, 50-60\% of which became CD45(neg)/GPA+/CD71high by days 15-20. However, when cells from days 7 to 12 of the proliferative phase were transferred in differentiation medium containing EPO and insulin, they progressed to mature erythroblasts (g90% benzidine(pos) and CD45(neg)/GPA+/CD71medium) in 4 days. Because of the high number of erythroid cells that are generated from modest volumes of blood, this method will prove useful in donor-specific studies of erythroid differentiation.

Emerging Non-Apoptotic Functions of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)/Apo2L

Tumor necrosis factor (TNF) is a cytokine that mediates tumor necrosis. To date, 20 different members of the TNF super‐family and 21 different receptors have been identified. All ligands of the TNF super‐family have been found to activate transcription factor NF‐κB and c‐Jun kinase. Members of this family have diverse biological effects, including induction of apoptosis, promotion of cell survival, and regulation of the immune system and hematopoiesis. The current review focuses on the biological effects of TNF‐related apoptosis‐inducing ligand (TRAIL), a TNF super‐family member which, a few years ago, generated considerable enthusiasm for its anticancer activity, not accompanied by general toxicity in most normal tissues and organs.

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (TRAIL) Sequentially Upregulates Nitric Oxide and Prostanoid Production in Primary Human Endothelial Cells

&NA; —Endothelial cells express tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) receptors, but the function of TRAIL in endothelial cells is not completely understood. We explored the role of TRAIL in regulation of key intracellular signal pathways in endothelial cells. The addition of TRAIL to primary human endothelial cells increased phosphorylation of endothelial nitric oxide synthase (eNOS), NOS activity, and NO synthesis. Moreover, TRAIL induced cell migration and cytoskeleton reorganization in an NO‐dependent manner. TRAIL did not activate the NF‐&kgr;B or COX‐2 pathways in endothelial cells. Instead, TRAIL increased prostanoid production (PGE2=PGI2>TXA2), which was preferentially inhibited by the COX‐1 inhibitor SC‐560. Because NO and prostanoids play a crucial role in the state of blood vessel vasodilatation and angiogenesis, our data suggest that TRAIL might play an important role in endothelial cell function. (Circ Res. 2003;92:732–740.)

PI-3K/Akt and NF-κB/IκBα pathways are activated in Jurkat T cells in response to TRAIL treatment†

The aim of this work was to evaluate the involvement of survival pathways in the response of Jurkat T leukaemic cells sensitive to the cytotoxic action of tumour necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL)/Apo2L. Jurkat T cells express TRAIL‐R2/DR5 and TRAIL‐R4/DcR2 receptors and start to die by apoptosis early (3 h) upon TRAIL administration reaching a dose‐dependent increase in the percentage of dead cells within 48 h (up to 85–90%). This increase in cell death is accompanied by a dose‐dependent significant (P < 0.05) increase in the G0/G1 phase of the cell cycle and reverted by the treatment with a broad inhibitor of caspases, z‐VAD‐fmk. Co‐treatment of the cells with inhibitors of PI‐3 kinase (LY294002) and nuclear factor kappa B (NF‐κB) (SN50) pathways leads to an earlier significantly increased cytotoxicity, respectively in the form of apoptosis and necrosis. Consistently with the data obtained with the pharmacological inhibitors, the activation and nuclear translocation of both PI‐3K and NF‐κB were observed. In summary, our results provide evidence that even in sensitive neoplastic cells TRAIL paradoxically activates pro‐survival pathways, which protect against TRAIL‐mediated death since their inhibition leads to an earlier and increased cytotoxicity.

Involvement of the pathway phosphatidylinositol-3-kinase/AKT-1 in the establishment of the survival response to ionizing radiation.

Ionizing radiation is one of the agents inducing activation of DNA repair, cell cycle arrest, apoptosis and cell death. Here we report evidence for an enhanced activity of DNA polymerase beta, one of the repair enzymes, concomitant to the activation of the pathway phosphatidylinositol-3-kinase/AKT-1 (PI-3-kinase/AKT-1), which delivers a survival signal in Friend erythroleukemia cells exposed to 15 Gy. Significantly, the preincubation of the cellls with PI-3-kinase inhibitors wortmannin and LY 294002, disactivating this pathway, sensitizes the cells to ionizing radiation by further reducing the rate of proliferation without substantial variations of the number of dead cells. Thus, we suggest a role for these enzymes in maintaining survival programs upon exposure to ionizing radiation and in giving to these cells a chance to recover from this stress.

PI3-K/Akt-dependent activation of cAMP-response element-binding (CREB) protein in Jurkat T leukemia cells treated with TRAIL

We recently demonstrated the activation of phosphatidylinositol 3‐kinase (PI3‐K/Akt) survival pathway in Jurkat T leukemia cells known for their sensitivity to the tumor necrosis factor (TNF)‐related apoptosis‐inducing ligand (TRAIL)/Apo2L cytotoxic action. The present investigation was done to elucidate the role of cAMP‐response element‐binding (CREB) protein in this system. Jurkat T cells were treated with 100–1,000 ng/ml TRAIL for time intervals up to 24 h in the presence or absence of selective pharmacologic inhibitors of PI3‐K/Akt (LY294002) or p38 MAPK (SB253580) pathways. Upon TRAIL treatment, a dose‐dependent increase in the percentage of apoptotic cells as well as in caspase‐3 activity was observed. A further enhancement of apoptotic cell death was obtained with the use of CREB1 siRNA technology, as demonstrated by flow cytometry. Western blot analysis showed a high constitutive level of CREB phosphorylation at Ser133 in Jurkat T cells under normal serum culture conditions. Under low serum culture conditions, an early (within 1 h) and transient increase in CREB phosphorylation was detected in response to both TRAIL doses and reduced upon pre‐treatment with LY294002 or SB253580, demonstrating the PI3‐K/Akt‐ and p38 MAPK‐dependency of this effect. The parallel analysis in immune fluorescence demonstrated the nuclear translocation of the phosphorylated form upon treatment with 100 ng/ml TRAIL, whereas the immune labeling was mainly detectable in the cytoplasm compartment upon the higher more cytotoxic dose. These results let us hypothesize that CREB activation can be an important player in the complex cross‐talk among pro‐ and anti‐apoptotic pathways in this peculiar cell model. J. Cell. Physiol. 214:192–200, 2008.

Nutlins and ionizing radiation in cancer therapy.

Radioresistance stands as a fundamental barrier that limits the effectiveness of radiotherapy in cancer treatment. Recent evidences suggest that radioresistance is due to tumour repopulation and involves several signalling pathways, including p53/MDM2 interaction. Ionizing radiation induces p53-dependent MDM2 gene transcription that, in turn, inhibits p53 transcriptional activity, favouring its nuclear export and stimulating its degradation. In light of the observation that in many human tumours the inadequate function of p53 is the result of MDM2 over-expression, several authors have considered as an attractive therapeutic strategy to activate p53 signalling in tumours by inhibiting MDM2 activities or p53/MDM2 interaction. We retain that, by preventing the interaction p53/MDM2 with Nutlin, a small molecule that bind at the interface between these two proteins, the effectiveness of ionizing radiation treatment could be improved. Promising results have recently emerged from in vitro studies performed on laryngeal, prostate and lung cancer cell lines treated with Nutlin in combination with ionizing radiation. Based on these findings, we believe that the combined approach Nutlin/ionizing radiation should be further investigated for efficacy on both solid tumours and lymphoproliferative disorders as well as for side effects on normal cells and tissues. Therefore, the purpose of this review is to report the first results obtained by using Nutlins alone or in combination with other therapeutic agents on primary tumour cells, in vitro cell lines or tumour xenografts and to present the most recent advances in the understanding of the molecular mechanisms underlining ionizing radiation cytotoxicity and resistance.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture

We have recently demonstrated that tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) increases endothelial nitric oxide synthase (eNOS) phosphorylation, NOS activity, and nitric oxide (NO) synthesis in cultured human umbilical vein endothelial cells (HUVEC), without inducing apoptotic cell death. Although an important factor that regulates eNOS activity is its localization within the cells, little is known about the role of TRAIL in the regulation of eNOS trafficking among cellular compartments and the cytoskeleton involvement in this machinery. Then, we did both quantitative and semi‐quantitative evaluations with biochemical assays and immune fluorescence microscopy in the presence of specific inhibitors of NOS activity as well as of cytoskeletal microtubule structures. In our cellular model, TRAIL treatment not only increased NO levels but also caused a time‐dependent NO migration of fluorescent spots from the plasma membrane to the inner part of the cells. In unstimulated cells, most of the eNOS was localized at the cell membranes. However, within 10 min following addition of TRAIL, nearly all the cells showed an increased cytoplasm localization of eNOS which appeared co‐localized with the Golgi apparatus at a higher extent than in unstimulated cells. These effects were associated to an increased formation of trans‐cytoplasm stress fibers with no significant changes of the microtubule network. Conversely, microtubule disruption and Golgi scattering induced with Nocodazole treatment inhibited TRAIL‐increased NOS activity, indicating that, on cultured HUVEC, TRAIL ability to affect NO production by regulating eNOS sub‐cellular distribution is mediated by cytoskeleton and Golgi complex modifications. J. Cell. Biochem. 97: 782–794, 2006.

Cyclic nucleotide response element binding (CREB) protein activation is involved in K562 erythroleukemia cells differentiation.

K562 are human erythroleukemia cells inducible to differentiate into megakaryocytic or erythroid lineage by different agents. Cyclic nucleotide Response Element Binding (CREB) protein, a nuclear transcription factor which mediates c‐AMP signaling, is a potential candidate involved in the occurrence of erythroid differentiation and adaptive response. Here we investigated signaling events in K562 cells induced with 30 µM hemin to undergo erythroid differentiation. CREB activation was detected early 1 h after hemin treatment and up to 4 and 6 days of treatment, when K562 terminal differentiation occurs together with caspase‐3 maximal activation and PARP degradation. It was interesting to note that after hemin treatment in the presence of SB203580, p38 MAP kinase specific inhibitor, a reduced rate of CREB phosphorylation as well as a lower percentage of CD71/Gly+ (Glycophorin A) cells were detectable, demonstrating the p38 MAP kinase dependency of these phenomena. All in all these results document a novel relationship between CREB activation and differentiation‐related apoptotic cell death and assign a role to p38 MAP kinase pathway in determining these events in K562 erythroleukemia cells. J. Cell. Biochem. 100: 1070–1079, 2007.

Phosphatidylinositol-3-kinase activation and atypical protein kinase C ζ phosphorylation characterize the DMSO signalling in erythroleukemia cells

Here we provide evidence for a role of phosphatidylinositol-3-kinase (PI-3-kinase) and for its product phosphatidylinositol-3,4, 5-triphosphate (PI3,4,5P3) in the occurrence of the metabolic differentiation state induced by DMSO in murine Friend erythroleukemia cells. Of note, the activation of PI-3-kinase correlated with the modulation of the activation of another enzyme, the atypical protein kinase C zeta (aPKC zeta). In particular, the expression of PI-3-kinase was substantially unaffected by DMSO treatment while its phosphorylation and the production of PI3,4,5P3 was strongly increased within 24 h of DMSO. Such a result was paralleled by an evident phosphorylation of a PKC zeta. Treatment of the cells with the two unrelated PI-3-kinase inhibitors wortmannin and LY 294002 impaired the recovery of the number of differentiated cells, therefore indicating that PI-3-kinase might be involved in the induction of erythroid differentiation, possibly engaging a protein kinase C zeta as downstream effector.