Biomedical subjects

U Testa

Publications and source records attributed to U Testa.

At least 73 records · Page 4Linked to original sources

PubMed1994

Coordinate expression and proliferative role of HOXB genes in activated adult T lymphocytes.

We investigated the expression of HOXB cluster genes in purified phytohemagglutinin (PHA)-activated T lymphocytes from normal adult peripheral blood by reverse transcription PCR and RNase protection. These genes are not expressed in quiescent T cells, except for barely detectable B1 RNA. After the PHA stimulus, HOXB gene activation initiates coordinately as a rapid induction wave in the 3'-->5' cluster direction (i.e., from HOXB1 through B9 genes). Thus, (i) expression of the foremost 3'-located B1 and B2 genes peaks 10 min after PHA addition and then rapidly declines, (ii) activation of B3, B4, and B5 begins 10 min after PHA addition and peaks at later times (i.e., at 120 min for B5), (iii) B6, B7, and B9 are expressed at a low level starting at later times (45 to 60 min), and (iv) B8 remains silent. Treatment of PHA-activated T lymphocytes with antisense oligonucleotides to B2 or B4 mRNA causes a drastic inhibition of T-cell proliferation and a decreased expression of T-cell activation markers (i.e., interleukin 2 and transferrin receptors). Similarly, treatment of CEM-CCRF, Peer, and SEZ627 T acute lymphocytic leukemia cell lines with anti-B4 oligomer markedly inhibits cell proliferation. Finally, T cells stimulated by a low dosage of PHA in the presence of 1 microM retinoic acid show a marked increase of both HOXB expression, particularly B2, and cell proliferation. These studies provide novel evidence on the role of HOX genes in adult cell proliferation. (i) Coordinate, early activation of HOXB genes from the 3'-->5' cluster side apparently underlies T-cell activation. (ii) The expression pattern in adult PHA-activated T cells is strikingly similar to that observed in retinoic acid-induced teratocarcinoma cells (A. Simeone, D. Acampora, L. Arcioni, P. W. Andres, E. Boncinelli, and F. Mavilio, Nature (London) 346:763-766, 1990), thus suggesting that molecular mechanisms underlying HOX gene expression in the earliest stages of development may also operate in activated adult T lymphocytes.

Adult↗

PubMed1994

High dose-intense chemotherapy alone or in combination with interleukin-2 for small cell lung cancer: a pilot study.

Given the antitumor activity of interleukin-2 (IL-2) against some drug-resistant cancer cells, 17 previously untreated patients with small cell lung cancer entered a pilot study to evaluate the feasibility, efficacy, and immunological effects of combining 12-week high dose-intense chemotherapy based on a modified Evans regimen (CAV/PE) with different IL-2 schedules (6-12 MU/m2 week as a 48-72-h infusion using the same cumulative dose, 72 MU/m2). Despite significant myelotoxicity, up to 70% of the intended dose intensity was delivered, showing no differences with regard to the IL-2 schedule used. Immunotherapy-induced toxicity was usually mild and manageable. No limiting effects were observed in patients receiving immunotherapy except for a very poor compliance to the 12-week IL-2 regimen. The low-dose 72-h infusion was the optimal IL-2 schedule. As given in this study, neither of the alternating CAV/PE regimens abrogated the effects of IL-2 on T-cell and NK-cell subsets, showing typical kinetics with rebound in lymphocytes following each discontinuation of the IL-2. While immunological changes cannot predict the antitumor effect of IL-2, they are consistent with those described for IL-2 alone, suggesting its compatibility with high dose-intense chemotherapy. Although no definite advantages have been demonstrated in this small pilot study with significant unbalanced prognostic factors (12% 2-year survival), both the preserved immunostimulatory effects and the lack of limiting overlapping toxicity make this combined approach promising and worthy of further clinical investigation.

Adult↗

PubMed1994

Cytoskeletal oxidative changes lead to alterations of specific cell surface receptors.

Electron acceptors in biological systems, termed "oxidants", have been associated with numerous cytopathic conditions. In particular, oxygen-derived free radicals have been shown to induce various subcellular oxidative injuries which can lead to different types of pathologies. It has also been suggested that oxidative stress induced by xenobiotics can determine specific subcellular alterations in in vitro models. An oxidative imbalance in cells seems in fact to affect intracellular functions including the expression of some growth factor surface receptors. In this work, we observed a specific early alteration of the microfilament system and the microtubular network induced in K562 cells by oxidative stress. In particular, we hypothesize that oxidative imbalance could lead to an impairment of the expression of these receptors, such as transferrin receptors, via a modification of the cytoskeleton. This could represent a general mechanism by which a modification of receptor regulation can lead to cell aging, injury or death.

Cell Membrane↗

PubMed1993

Enhanced production of LPS-induced cytokines during differentiation of human monocytes to macrophages. Role of LPS receptors.

In vitro cultivated human monocytes isolated from normal peripheral blood show a time-dependent differentiation into macrophages characterized by an increased expression of transferrin receptors, CD11/CD18, and CD14 Ag. We measured the secretion of TNF-alpha and IL-6 in freshly isolated monocytes and in differentiated macrophages after LPS treatment. Differentiated macrophages produced significantly higher amounts of TNF-alpha and IL-6 than freshly isolated monocytes. This increased secretion was not a result of an enhanced accumulation of TNF-alpha and IL-6 mRNA, as comparative levels of these transcripts were found in both cell types after LPS treatment. Furthermore, LPS did not induce an antiviral state to VSV3 in monocytes, but it reduced by 3 to 5 log10 the virus yield in differentiated macrophages. The addition of antibodies to IFN-beta completely inhibited the LPS-induced antiviral state to VSV, but antibodies to IFN-alpha, TNF-alpha, or IL-6 were ineffective. A marked accumulation of IFN-beta mRNA was found in both cell types after LPS treatment. Binding experiments with FITC-LPS revealed a slightly higher overall binding affinity for LPS in freshly explanted monocytes as compared with differentiated macrophages, even though the maximal binding was higher in macrophages. In both cell types, the LPS binding was partially inhibited by antibodies to CD14. These results demonstrate that: 1) in vitro differentiation of human monocytes to macrophages leads to an enhanced LPS response in terms of (a) progressive increase of IL-6/TNF-alpha production and (b) acquisition of an IFN-beta mediated antiviral state; 2) this enhanced response to LPS, largely CD14-independent, is not linked to any increased accumulation of cytokine mRNA, but is probably a result of an increased synthesis and/or secretion of these cytokines.

Adolescent↗

PubMed1993

The acute promyelocytic leukemia-specific PML-RAR alpha fusion protein inhibits differentiation and promotes survival of myeloid precursor cells.

Acute promyelocytic leukemia is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. The differentiation block can be reversed by retinoic acid, which induces blast maturation both in vitro and in vivo. Acute promyelocytic leukemia is characterized by a 15;17 chromosome translocation with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and the PML gene, which encodes a putative transcription factor, on 15. A PML-RAR alpha fusion protein is formed as a consequence of the translocation. We expressed the PML-RAR alpha protein in U937 myeloid precursor cells and showed that they lost the capacity to differentiate under the action of different stimuli (vitamin D3 and transforming growth factor beta 1), acquired enhanced sensitivity to retinoic acid, and exhibited a higher growth rate consequent to diminished apoptotic cell death. These results provide evidence of biological activity of PML-RAR alpha and recapitulate critical features of the promyelocytic leukemia phenotype.

Animals↗

PubMed1993

Autologous stem cell transplantation: sequential production of hematopoietic cytokines underlying granulocyte recovery.

We investigated the serum concentrations of a variety of cytokines [granulocyte-macrophage-colony-stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interleukin (IL) 1 alpha, IL-3, IL-6, IL-8, erythropoietin, tumor necrosis factor alpha, gamma-interferon in 10 patients with advanced ovarian cancer undergoing autologous peripheral blood stem cell (PBSC) harvesting followed by treatment with high-dose cisplatin, etoposide, and carboplatin and PBSC transplantation (chemotherapy was administered on days 1 through 3, PBSCT on day 6). Preliminary observations on cytokine serum levels were performed for 4 patients; on this basis, the kinetics of cytokines was then investigated in greater detail at closely sequential times in 6 further patients. We observed a consistent pattern of sequential GM-CSF, G-CSF, and IL-8 release after chemotherapy/PBSCT in all 10 cases, including the 6 patients monitored in detail: (a) at days 5-10 a GM-CSF peak; (b) at days 12-14 a pronounced release of both G-CSF and IL-8, which always preceded granulocyte recovery by approximately 7 days. At days 17-23, a second GM-CSF peak was monitored in 5 of the 6 patients analyzed in detail, as well as in the other 4 cases. Particularly relevant are the observations that: (a) the peak of G-CSF serum concentration and neutrophil number in the recovery phase are strikingly and directly correlated, thus indicating a key role for G-CSF in granulocyte rescue; (b) the time courses of G-CSF and IL-8 levels are strictly parallel, thereby suggesting a coordinate stimulus for production of granulocytes, mediated by G-CSF, and their activation/migration capacity, mediated by IL-8. Results were essentially negative for IL-3, tumor necrosis factor alpha, and gamma-interferon concentrations (except in one case for each cytokine). An early peak of IL-1 alpha was observed in all 3 analyzed patients, while an IL-6 peak was monitored at days 13-15 in all 4 patients analyzed in detail. The present results indicate a sequential coordinate pattern of cytokine release after ablative therapy and PBSCT and shed light on the mechanisms mediating the recovery of granulocytes, and more generally of hematopoiesis, after stem cell transplantation. Furthermore, these studies may contribute to the design of improved protocols for cytokine administration following myelosuppressive anticancer therapy, as well as to the prediction of granulocytic response.

Adult↗

PubMed1993

Transforming growth factor-beta potentiates vitamin D3-induced terminal monocytic differentiation of human leukemic cell lines.

We have investigated the effects of 1,25-dihydroxyvitamin D3 (D3) and/or transforming growth factor (TGF)-beta on one monocytic (U-937) and two human promyelocytic (HL-60 and AML-193) leukemic cell lines. D3 addition induces a partial monocytic maturation of the cell lines, whereas TGF-beta treatment is largely ineffective. Combined treatment with TGF-beta and D3 causes terminal monocytic maturation, as evaluated both by assessment of a large spectrum of membrane Ag and by functional assays. Furthermore, sequential addition of the two inducers showed that pretreatment with TGF-beta 1 followed by incubation with D3, but not vice versa, induces monocytic maturation as effectively as simultaneous treatment with both agents. In liquid culture the proliferative activity of these cell lines is slightly decreased by D3 and virtually unaffected by TGF-beta, whereas combined treatment with D3 and TGF-beta induces a markedly potentiated inhibitory effect. Furthermore, TGF-beta/D3 treatment (but not D3 alone) elicits the expression of membrane CD14, FcRI, FcRII, CD11a, CD11b, CD11c, ICAM-1, and PECAM-1 Ag at a level comparable to that observed on normal human monocytes. It is noteworthy that several of these Ag play an important role in monocyte physiology (e.g., CD14 Ag mediates the binding of bacterial LPS to monocytes). Treatment with both TGF-beta and D3 (but not D3 alone) induces superoxide anions and H2O2 production similar to that of circulating monocytes. In semisolid culture, D3 and TGF-beta alone cause, respectively, a marked and slight loss of cloning efficiency of the cell lines, whereas their combined addition synergistically results in a complete loss of the cloning capacity. These findings suggest a physiologic role for TGF-beta in monocyte maturation. Furthermore, they may pave the way to the design of clinical protocols combining D3 and TGF-beta in the differentiation therapy of acute promyelocytic/myelomonocytic leukemia.

Adjuvants, Immunologic↗

PubMed1993

Cascade transactivation of growth factor receptors in early human hematopoiesis.

Highly purified progenitors (including erythroid [BFU-E], granulo-monocytic [CFU-GM], multipotent [CFU-GEMM] progenitors, as well as multipotent progenitors with self-renewal capacity [CFU-B]) express high-affinity growth factor receptors (GFRs), with prevalent interleukin-3 receptors (IL-3Rs) (2,700/cell), a > or = 10-fold lower number of IL-6Rs (145/cell) and granulocyte-macrophage colony-stimulating factor receptors (GM-CSFRs) (300/cell), and a barely detectable level of erythropoietin (Ep) receptors (75/cell). Hematopoietic growth factor (HGF) dosages inducing peak clonogenetic effects are associated with partial/subtotal occupancy of the homologous HGF receptor (HGFR). Cross-reactivity between GFRs and heterologous GFs (including IL-6, IL-3, GM-CSF, Ep, and the kit ligand [KL]) was explored by competition experiments on purified progenitors with radiolabeled and excess cold HGFs at +4 degrees C. No cross-reaction was observed between IL-6R, IL-3R, EpR, and the heterologous GFs, whereas the GM-CSFR showed cross-reactivity with IL-3 and, to a lesser extent, KL. Modulation of GFRs was examined after 18 or 40 hours of incubation with GF(s) at 37 degrees C, followed by ligand-binding assay at 20 degrees C. IL-6, IL-3, GM-CSF, and Ep induce a marked down-modulation of their own receptors. Interestingly, each GF induces the transactivation of the R(s) for the "distal" GF(s): (1) IL-6 induces transactivation of IL-3R, but not of GM-CSFR/EpR; (2) IL-3 causes a rapid upmodulation of GM-CSFR/EpR ("pure" progenitors treated with IL-3 show upmodulation of GM-CSFR alpha-chain mRNA by reverse transcriptase-polymerase chain reaction); whereas (3) GM-CSF induces the transactivation of the EpR. This chain upmodulation of HGFRs may underlie the synergistic interactions between the HGFs in clonogenetic culture. It is emphasized that KL does not induce upmodulation of the other GFRs. Finally, Ep, GM-CSF, and IL-3 do not modulate the expression of the "proximal" HGFRs (ie, GM-CSFR/IL-3R/IL-6R, IL-3R/IL-6R, and IL-6R, respectively). These results allow insight into the cellular basis of hematopoiesis, ie, the complex and coordinate interactions between HGFs and their receptors. They are compatible with a model of cascade transactivation via upmodulation of GFRs in the initial key steps of hematopoietic differentiation, whereby the action of each GF enhances the effect of the distal GF(s) by a multistep chain-potentiation mechanism.

Adult↗

PubMed1993

Combined interleukin 1 beta/interleukin 2 treatment in mice: synergistic myelostimulatory activity and protection against cyclophosphamide-induced myelosuppression.

We have studied the effects of single and combined treatment with interleukin 1 beta (IL-1 beta) and interleukin 2 (IL-2) on spleen and bone marrow hematopoiesis in normal mice. Injection of IL-1 beta alone was followed by a significant increase in the number of granulocytes in spleen and progenitors (burst-forming units-erythroid and colony-forming units-granulomonocytic) in both spleen and bone marrow, s compared to control mice. In contrast, IL-2 alone induced only a slight increase in the number of marrow colony-forming units-granulomonocytic and had no significant effect on spleen progenitors. Repeated injections of both IL-1 beta and IL-2 resulted in a synergistic increase in spleen weight and splenocyte number, as compared to mice treated with the single cytokine regimen; in particular, the combined treatment induced a marked rise in the number of neutrophilic granulocytes and erythroblasts, whereas splenic lymphocytes were not affected. This regimen also caused a synergistic increase in the number of spleen and marrow progenitor cells: a time-course analysis showed an elevation in numbers of both burst-forming units-erythroid and colony-forming units-granulomonocytic, first in marrow (day 10) and subsequently in spleen (day 18). Combined IL-1 beta/IL-2 treatment dampened the decrease and accelerated the recovery of myeloid cells after cyclophosphamide injection, whereas the single cytokine regimen was not effective. Similarly, the rebound of WBC (especially neutrophilic granulocytes) after cyclophosphamide treatment was markedly enhanced by the combined treatment, whereas the single cytokine regimen was ineffective. These results, indicating a myelostimulatory effect by the combined cytokine regimen, together with our previous observations showing a synergistic antitumor activity by IL-1/IL-2 treatment in experimental mouse tumors (V. Ciolli et al., J. Exp. Med., 173: 313-322, 1991), may provide the basis for the development of new combination therapies with cytokines and antiblastic agents in the treatment of cancer patients.

Animals↗

PubMed1993

c-kit ligand reactivates fetal hemoglobin synthesis in serum-free culture of stringently purified normal adult burst-forming unit-erythroid.

We have analyzed the reactivation of fetal hemoglobin (HbF) synthesis under rigorous in vitro conditions, ie, in mature erythroblasts generated by erythroid burst-forming units (BFU-E) stringently purified from normal adult peripheral blood and grown in fetal calf serum(FCS)-free semisolid or liquid phase culture. In clonogenetic dishes, graded amounts of c-kit ligand (KL) were added together with saturating levels of erythropoietin (Ep) and variable amounts of interleukin-3 and granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF), ie, high or low level, or no IL-3/GM-CSF addition. In all conditions, KL induced a sharp, dose-dependent increase in the percentage of F cells and HbF content from nearly normal levels (< 10% and < 2.5%, respectively, at 0.1 and 1 ng/mL) up to 40% to 50% and 10% to 15% at 100 to 200 ng/mL. This increase was not associated with significant differences of burst number or stage of maturation at the time of analysis (as evaluated on the basis of percent mature erythroblasts and Hb content per cell). However, the KL-induced reactivation of HbF synthesis was strictly and directly correlated with a sharp increase of colony size, ie, cell number per burst. Addition of large amounts of IL-3 and GM-CSF (10 to 100 U and 1 to 10 ng/mL, respectively) significantly potentiated the KL-induced reactivation of HbF, as compared with low levels (0.1 U and 0.01 to 0.1 ng) or no addition of these growth factors: this increase was highly significant at low KL doses (ie, 1 to 10 ng/mL). Single-burst analysis showed that the KL-induced HbF reactivation occurs homogeneously in the erythroid colonies within each of these culture conditions. We have analyzed the effect of KL in liquid phase BFU-E culture treated with the IL-3/GM-CSF/Ep combination at sequential times until terminal erythroid maturation: KL causes a sharp increase in the percentage of F cells and HbF content in all stages of maturation, whereas the IL-3/GM-CSF/Ep combination alone has a markedly lower effect. These results suggest that KL plays a key role in the reactivation of HbF synthesis in adult life, whereas IL-3/GM-CSF potentiate this effect at low KL levels. The KL-induced HbF reactivation is seemingly related to an enhanced proliferation of erythroid progenitors in the erythropoietic differentiation pathway.

Adult↗

PubMed1993

Stringently purified human hematopoietic progenitors/stem cells: analysis of cellular/molecular mechanisms underlying early hematopoiesis.

Analysis of the cellular/molecular basis of the early steps of hematopoietic proliferation and differentiation is hindered by the rarity of hematopoietic progenitors and stem cells (HP/HSC). The intensive efforts devoted to the development of purification methods for early HP and HSC, although initially largely unsuccessful, have recently provided a high level of HP/HSC yield and/or recovery. The methodology developed by our group, recently improved, provides not only virtually complete purification, but also abundant recovery of early HP/HSC such as colony forming units granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM), burst forming units erythroid (BFU-E), CFU granulocyte/macrophage (CFU-GM)/CFU blast cells (CFU-B), and long-term culture initiating cells (LTC-IC) from adult peripheral and cord blood (CB). We have also developed a serum-free liquid suspension culture for unilineage erythroid (E), granulocytic (G) or monocytic (M) differentiation of stringently purified HP/HSC. These culture systems allow sequential collection and cellular/molecular analysis of discrete populations of hematopoietic cells at a homogenous stage of differentiation specifically along a unilineage pathway. These experimental tools have been utilized to investigate cellular/molecular mechanisms underlying early hematopoiesis. The transcription factor (TF) GATA-1 is considered to be the "master" gene of erythropoiesis. In highly purified HP/HSC undergoing E or GM differentiation, GATA-1 expression is characterized initially by proliferation-dependent activation and at later stages by sustained expression in the E pathway and suppression in the GM pathway. Hypothetically, similar on/off switches of lineage-restricted TF may underlie the binary fate decisions of early HP differentiation. The expression and modulation of hematopoietic growth factor receptors (HGFR) in early hematopoiesis have been extensively analyzed. The results suggest a model of transactivation cascade for HGFR such as interleukin 6 receptor (IL-6R), IL-3R, GM colony stimulating factor receptor (GM-CSFR), and erythropoietin receptor (EpR), whereby each HGF upmodulates the R(s) for distal-acting HGF(s). Finally, we have investigated the effect of HGF on reactivation of hemoglobin F (HbF) in clonogenic or liquid suspension serum-free culture of purified adult HP. The results suggest that c-kit ligand (KL) plays a key role in the reactivation of HbF synthesis in adult life, and IL-3/GM-CSF potentiate this effect at low KL level. The KL-induced HbF reactivation is seemingly related to an enhanced proliferation of early E progenitors in their differentiation pathway.

Animals↗

PubMed1993

Mannoprotein-induced anti-U937 cell cytotoxicity in peripheral blood mononuclear cells from uninfected or HIV-infected subjects: role of interferon-gamma and tumor necrosis factor-alpha.

A mannoprotein fraction (MP-F2: mannan, > 90%; protein, 4.5%) from the human commensal microorganism Candida albicans was as efficient as interleukin-2 (IL-2) in generating cytotoxicity against the uninfected or human immunodeficiency virus type-1 (HIV-1) persistently infected monocytoid U937 cell line in cultured peripheral blood mononuclear cells (PBMC) from healthy human subjects. MP-F2-activated killing of U937 cells (U937-MAK) decreased progressively with advancing stages of HIV-1 infection to virtually no killing effect in PBMC from advanced AIDS subjects (AIDS PBMC). This decrease paralleled a lowered susceptibility of U937 cells to natural killer cell activity. In contrast, IL-2-activated killing of U937 cells (U937-LAK) was not affected by the progression of HIV infection and persisted at high levels in AIDS PBMC. To shed light on the mechanisms of U937-MAK and its decrease during HIV infection, IL-1 beta, IL-6, TNF-alpha, GM-CSF, and IFN-gamma production was analyzed. Decreases in TNF-alpha, GM-CSF, and IFN-gamma, but not IL-1 beta or IL-6, levels were observed in MP-F2-stimulated PBMC from HIV-infected subjects, compared to healthy controls. Interestingly, these cytokine levels fell before the onset of AIDS. The greatest relative drop was that of IFN-gamma, from 4600 (+/- 600) to 290 (+/- 160) and 217 (+/- 110) mean pg/ml (+/- SE) in PBMC from healthy donors (11 subjects), CDC stages II + III (14 subjects), and CDC stage IV (10 subjects), respectively. The following observations suggest that decreased IFN-gamma production plays a role in the abrogation of U937-MAK activity: (i) addition of neutralizing anti-IFN-gamma antibodies abolished both IFN-gamma and U937-MAK activity in PBMC from healthy subjects; (ii) substantial levels of IFN-gamma were detected in supernatants of PBMC cultures stimulated by IL-2, in line with preserved U937-LAK activity. Interestingly, anti-IFN-gamma antibodies also abolished TNF-alpha production, and the anti-TNF-alpha antiserum effect was comparable to that of anti-IFN-gamma in U937-MAK inhibition. In contrast, anti-TNF-alpha antibodies abrogated TNF-alpha activity, but only partially reduced IFN-gamma production. Thus, in human PBMC, U937-MAK activity progressively decreases with advancing stages of HIV infection, whereas U937-LAK activity is sustained. Furthermore, the present results indicate a pivotal role for IFN-gamma in U937 MAK activity, possibly through activation of TNF-alpha production.

Adult↗

PubMed1993

Menadione-induced oxidative stress leads to a rapid down-modulation of transferrin receptor recycling.

It has been demonstrated that perturbation of oxidative balance plays an important role in numerous pathological states as well as in physiological modifications leading to aging. In order to evaluate the role of the oxidative state in cells, biochemical and ultrastructural studies were carried out on K562 and HL-60 cell cultures. Particular attention was given to the transferrin receptor, which plays an important role in cellular iron metabolism. In order to evaluate if oxidative stress influences the transferrin receptor regulation process, the free-radical inducer menadione was used. The results obtained seem to indicate that oxidative stress is capable of inducing a rapid and specific down-modulation of the membrane transferrin receptor due to a block of receptor recycling on the cell surface, without affecting ligand-binding affinity. These effects were observed in the early stages of menadione treatment and before any typical signs of subcellular damage, including surface blebbing, a well-known cytopathological marker of menadione-induced injury. The mechanisms underlying such phenomena appear to be related to cytoskeletal protein thiol group oxidation as well as to the perturbation of calcium homeostasis, both induced by menadione. It is thus hypothesized that the data reported here represent a specific example of a general mechanism by which cell surface receptor expression and recycling can be influenced by oxidative balance.

Actin Cytoskeleton↗

PubMed1993

Oxidative stress and transferrin receptor recycling.

Perturbation of the oxidative balance in biological systems plays an important role in numerous pathological states as well as in many physiological processes such as receptor activity. In order to evaluate if oxidative stress induced by menadione influences membrane receptor processes, a study was conducted on the transferrin receptor. Consequently, biochemical, biophysical and ultrastructural studies were carried out on different cell lines. The results obtained seem to indicate that oxidative stress is able of inducing a rapid and specific down-modulation of membrane transferrin receptor due to a block of receptor recycling on the cell surface without affecting binding affinity.

Biotechnology↗

PubMed1993

Isolation and characterization of a metastatic Eb-like tumor variant highly responsive to interleukin (IL)-2 and to combination cytokine therapy with IL-2/IL-1 beta and IL-1 beta/interferon-alpha/beta.

In this study, we describe the origin and characterization of a new metastatic tumor cell line (p11-R-Eb) obtained after i.p. passages of the nonmetastatic Eb lymphoma cells into DBA/2 mice. The p11-R-Eb cells exhibited the same morphology and in vitro growth properties and chromosome markers as the original Eb cells. FACS analysis of the p11-R-Eb cells also revealed a close similarity to the Eb cells. Moreover, the p11-R-Eb cells were specifically killed by anti-Eb cytotoxic lymphocytes. In spite of all these characteristics of the Eb line, p11-R-Eb cells metastasized to the liver when injected i.v. or s.c. in DBA/2 mice. Peritumoral interleukin (IL)-2 treatment resulted in a potent antitumor response in DBA/2 mice transplanted s.c. with p11-R-Eb cells. In contrast, the same IL-2 regimen did not significantly increase the survival time of mice transplanted with the highly metastatic ESb cell line. Combined IL-1/IL-2 treatments of established p11-R-Eb tumors resulted in a synergistic antitumor effect and in tumor regression in 70% of the injected mice. Similarly, combined peritumoral treatment with IL-1 and interferon-alpha/beta, which were poorly effective or ineffective as single cytokine therapy, resulted in a marked antitumor effect, and 30% of the mice were cured. Spleen cells from IL-1/IL-2-treated p11-R-Eb-cell-injected mice showed a marked antitumor activity when assayed in a Winn assay with homologous tumor cells. This antitumor activity was eliminated by preincubation of spleen cells with antibodies to CD4 and complement and markedly inhibited by anti-asialo GM1 antibodies. P11-R-Eb cells represent, therefore, a new tumor model which may be useful for investigating the relevant mechanisms which need to be activated to achieve a potent antitumor response to cytokine therapy in the DBA/2 mouse host.

Animals↗

PubMed1993

The transferrin receptor.

Transferrin receptor (TfR) is a membrane receptor involved in the control of iron supply to the cell through the binding of transferrin, the major iron-carrier protein. This receptor plays a key role in the control of cell proliferation because iron is essential for sustaining ribonucleotide reductase activity, and is the only enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides. Furthermore, TfR is overexpressed in several tumors. The synthesis of the receptor is controlled through an iron-dependent negative feedback in cells where the receptor is involved in the control of cell proliferation, but through a positive feedback in cell types involved in iron storage (monocytes-macrophages). Recent studies have shed light on the molecular basis by which iron regulates TfR expression. Thus, the 3' untranslated region of TfR mRNA contains a cis-acting RNA element, termed the iron-regulatory element (IRE), that interacts with an IRE-binding protein (IRE-BP). The high-affinity interaction between IRE-BP and IRE in the TfR mRNA leads to repression of mRNA degradation and thus to higher synthesis of TfR. Hence, the study of IRE-BP activity will elucidate the mechanisms that modulate TfR expression in normal and malignant cells.

Amino Acid Sequence↗

PubMed1992

Production of hemolymphopoietic cytokines (IL-6, IL-8, colony-stimulating factors) by normal human astrocytes in response to IL-1 beta and tumor necrosis factor-alpha.

Astrocyte-enriched populations were established from human embryonic brain analyzed for their ability to synthesize cytokines potentially relevant for mechanisms of inflammation and immunity in the brain. Unstimulated astrocytes did not secrete significant IL-6, IL-8, macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), or granulocyte-CSF (G-CSF), as determined by specific ELISA and/or bioassay. With the exception of M-CSF mRNA, transcripts for the above factors were not detected in unstimulated astrocytes. On exposure of human astrocytes to IL-1 beta, high levels of IL-6, IL-8, M-CSF, G-CSF, and GM-CSF mRNAs were detected; moreover, active secretion of all the above cytokines was demonstrated. TNF-alpha was also able to stimulate IL-6, IL-8, M-CSF, GM-CSF, and G-CSF synthesis and secretion, but was generally less potent than IL-1 beta. No IL-3 mRNA or protein was detected in unstimulated or cytokine-treated astrocytes. IL-1 alpha and IL-1 beta mRNAs and proteins were not detected in unstimulated astrocytes, but were present in very small amounts after stimulation with TNF-alpha/IL-1 beta. No IL-6, M-CSF, GM-CSF, G-CSF, or IL-8 were induced by IL-1 beta or TNF-alpha in early primary cultures, which mainly contain undifferentiated neuronal/glial progenitor cells. These studies demonstrate for the first time the production of multiple cytokines by normal human astrocytes stimulated in culture by IL-1 beta and TNF-alpha. The capacity of human astrocytes to synthesize and release cytokines active on hemolymphopoietic cells supports the concept that these cells play an important role in the regulation of inflammatory and immune responses in a variety of brain pathologies.

Astrocytes↗

PubMed1992

Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors.

The programmed activation/repression of transcription factors in early hematopoietic differentiation has not yet been explored. The DNA-binding protein GATA-1 is required for normal erythroid development and regulates erythroid-expressed genes in maturing erythroblasts. We analyzed GATA-1 expression in early human adult hematopoiesis by using an in vitro system in which "pure" early hematopoietic progenitors are induced to gradual and synchronized differentiation selectively along the erythroid or granulocyte-macrophage pathway by differential treatment with hematopoietic growth factors. The GATA-1 gene, though virtually silent in quiescent progenitors, is activated after entrance into the cell cycle upon stimulation with hematopoietic growth factors. Subsequently, increasing expression along the erythroid pathway contrasts with an abrupt downregulation in the granulocyte-macrophage lineage. These results suggest a microenvironment-directed, two-step model for GATA-1 expression in differentiating hematopoietic progenitors that involves (i) cycle-dependent initiation and (ii) lineage-dependent maintenance or suppression. Hypothetically, on/off switches of lineage-restricted transactivators may underlie the binary fate decisions of hematopoietic progenitors.

Antigens, Surface↗