Autologous stem cell transplantation improves microcirculation in systemic sclerosis.

BACKGROUND: In systemic sclerosis (SSc) reduced capillary density decreases blood flow and leads to tissue ischaemia and fingertip ulcers. Nail fold videocapillaroscopy (NVC) is a diagnostic and follow-up parameter useful to evaluate the severity, activity and the stage of SSc microvascular damage. Autologous haemopoietic stem cell transplantation (HSCT) is a new treatment for patients with severe diffuse cutaneous systemic sclerosis (dcSSc) refractory to conventional therapies. We aimed to evaluate the improvement of microvasculature after HSCT using NVC. METHODS: A total of 16 patients with severe dcSSc with a "late" videocapillaroscopy pattern underwent an immunesuppressive treatment: 6 were treated with HSCT and 10 with monthly pulse cyclophosphamide (CYC) 1 g for 6 months and then orally with 50 mg/day for further 6 months. NVC was performed before and after 3 months from the beginning of each treatment and then repeated every 3 months. RESULTS: In all patients, before HSCT NVC showed large avascular areas and ramified capillaries and vascular architectural disorganisation ("late" pattern). At 3 months after HSCT, the NVC pattern changed from "late" into "active", showing frequent giant capillaries (>6/mm) and haemorrhages, absence of avascular areas and angiogenesis phenomena; 1 year after HSCT, microvascular abnormalities were still in the "active" pattern. In patients treated with CYC, no NVC modifications were observed during 24 months of follow-up and the pattern always remained "late". CONCLUSIONS: These results indicate that HSCT with a high dose CYC regimen may foster vascular remodelling, while CYC at lower doses and with a chronic regimen does not influence the microvasculature.

Bosentan regulates the expression of adhesion molecules on circulating T cells and serum soluble adhesion molecules in systemic sclerosis-associated pulmonary arterial hypertension.

OBJECTIVES: To study the expression of adhesion molecules in patients with systemic sclerosis (SSc) with and without pulmonary arterial hypertension (PAH) and the effects of therapy with the endothelin-1 (ET-1) receptor antagonist, bosentan. METHODS: In all, 35 patients with SSc and 25 healthy donors (HD) were selected for this study. Of 35 patients, 10 had isolated PAH assessed by Doppler echocardiography and treated with bosentan. Peripheral blood (PB) lymphocytes were isolated by density gradient centrifugation, and the expression of lymphocyte function-associated antigen-1 (LFA-1), very late antigen-4 (VLA-4) and L-selectin on CD3 T cells was assessed by double immunofluorescence and flow-cytometry. As endothelial activation markers, serum soluble P-selectin, platelet/endothelial cell adhesion molecule (PECAM)-1, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1 and von Willebrand factor (vWF) antigen were assessed by ELISA. In patients with SSc-PAH, T cell subsets and soluble endothelial markers were assessed at baseline and after 6 and 12 months of bosentan therapy. RESULTS: In patients with SSc-PAH, serum soluble ICAM-1, VCAM-1, P-selectin and PECAM-1 levels were higher than in HD at baseline and fell to normal values after 12 months of bosentan therapy. CD3-LFA1 T cells were significantly higher in PAH-SSc at baseline than in HD or SSc and significantly decreased after therapy. CD3-L-selectin T cells were significantly lower in SSc-PAH at baseline than in HD or SSc and rose to normal levels after bosentan therapy. CONCLUSIONS: This study confirms that endothelial activation occurs in SSc, and suggests that changes in the T cell/endothelium interplay take place in SSc-associated PAH. Bosentan seems to be able to hamper these changes and restore T cell functions in these patients.

Cannabinoids reduce granuloma-associated angiogenesis in rats by controlling transcription and expression of mast cell protease-5.

BACKGROUND AND PURPOSE: Chronic inflammatory conditions, such as granulomas, are associated with angiogenesis. Mast cells represent the main cell type orchestrating angiogenesis, through the release of their granule content. Therefore, compounds able to modulate mast cell behaviour may be considered as a new pharmacological approach to treat angiogenesis-dependent events. Here, we tested the effect of selective cannabinoid (CB) receptor agonists in a model of angiogenesis-dependent granuloma formation induced by lambda-carrageenin in rats. EXPERIMENTAL APPROACH: Granulomas were induced by lambda-carrageenin-soaked sponges implanted subcutaneously on the back of male Wistar rats. After 96 h, implants were removed and granuloma formation was measured (wet weight); angiogenesis was evaluated by histological analysis and by the measurement of haemoglobin content. Mast cells in the granulomas were evaluated histologically and by RT-PCR and immunoblotting analysis for mast cell-derived proteins (rat mast cell protease-5 (rMCP-5) and nerve growth factor). Selective CB1 and CB2 receptor agonists(,) ACEA and JWH-015 (0.001-0.1 mg mL(-1)), were given locally only once, at the time of implantation. KEY RESULTS: The CB1 and CB2 receptor agonists decreased the weight and vascularization of granulomas after 96 h. This treatment also reduced mast cell number and activation in granulomatous tissue. Specifically, these compounds prevented the transcription and expression of rMCP-5, a protein involved in sprouting and advance of new blood vessels. CONCLUSION AND IMPLICATIONS: Modulation of mast cell function by cannabinoids reduced granuloma formation and associated angiogenesis. Therefore cannabinoid-related drugs may be useful in the management of granulomatous diseases accompanied by angiogenesis.

Object-Tool-Actor Interaction: Object Information Drives Intended Action.

Tool use is typically explored via actor-tool interactions. However, the target-object (that which is being acted on) may influence perceived action possibilities and thereby guide action. Three different tool-target-object pairings were tested (Experiment 1). The hammering action demonstrated the greatest sensitivity and therefore subsequently used to further investigate target-object pairings. The hammer was removed as an option and instructions were provided using pictorial (Experiment 2), written (Experiment 3), and both pictorial and written formats (Experiment 4). The designed tool is chosen when available (Experiment 1) and when removed as a choice (i.e., the hammer), participants perform the same action associated with the designed tool (i.e., hammering) regardless of instruction method (Experiments 2, 3, and 4).

Etrurians vs Greeks: May ACE I/D polymorphism still be considered as a marker of susceptibility to SSc?

OBJECTIVE: SSc is characterized by immune dysfunction and microvascular involvement. A different genetic background may determine a different polymorphic allele frequency between different populations, and data from literature reported conflicting results about the role of genetic components in predisposing to the disease. We carried out this study in order to compare the ACE I/D polymorphism genotype distribution and alleles frequency in two different populations from the Mediterranean area. METHODS: Forty-eight Italian and 41 Greek SSc patients compared with 112 Italian and 93 Greek controls, have been studied. The ACE I/D polymorphism has been analysed. RESULTS: The genotype distribution and allele frequency were in Hardy-Weinberg equilibrium for Italian and Greek SSc patients and controls. Among the Italian patients a significantly higher ACE D allele frequency than in the controls was found, whereas among the Greeks a higher prevalence was observed in the healthy subjects. A significant difference in ACE D allele frequency between Italian and Greek controls was observed (p = 0.04). ACE D allele was associated to the predisposition to SSc in Italians, but not in Greeks. CONCLUSION: We confirm that Italian SSc patients have a higher ACE D allele frequency that is not present in the Greek patients. Thus, the two populations living in different Mediterranean areas and resulting from the Mediterranean civilization, do not show the same ACE-gene related allele frequencies. Other populations of the Mediterranean area must be investigated by using unlinked genetic markers to verify the homogeneity of the genetic background, and to test for a "true" difference in their ethnic origin.

Rheumatoid synovial fibroblasts constitutively express the fibrinolytic pattern of invasive tumor-like cells.

OBJECTIVE: In rheumatoid arthritis (RA) the synovial membrane proliferates and invades the underlying tissues. The cell-associated fibrinolytic system (urokinase-type plasminogen activator, uPA; uPA receptor, uPAR; plasminogen activator inhibitor-type 1, PAI-1) is pivotal in cell invasion and proliferation. For this reason, the expression and the role of such enzymatic system was investigated in synovial fibroblasts (SF) of normal and RA patients. METHODS: In SF obtained from RA patients and control subjects, uPA, uPAR and PAI-1 were measured by ELISA of cell lysates and culture medium and by RT-PCR of mRNAs. uPA was also studied by zymography. Proliferation was measured by cell counting and cell invasion with the Boyden chamber. RESULTS: RA-SF over-express uPAR and PAI-1 and are more prone than the normal counterpart to spontaneous and uPA-challenged invasion and proliferation, which are counteracted by antagonists of the fibrinolytic system. CONCLUSIONS: RA-SF display the fibrinolytic pattern and behaviour of invasive tumor-like cells. Antagonists of the fibrinolytic system are able to revert growth and invasion of both normal and RA-SF.

The fibrinolytic system components are increased in systemic sclerosis and modulated by Alprostadil (alpha1 ciclodestryn).

OBJECTIVES: To evaluate urokinase plasminogen activator (u-PA), urokinase plasminogen activator soluble receptor (su-PAR), plasminogen activator inhibitor 1 (PAI-1) and tissue plasminogen activator (t-PA) plasma levels in SSc patients (pts) versus healthy controls and their modulation by intravenous alphacyclodestrine (Alprostadil). METHODS: Plasma levels of u-PA, su-PAR, PAI-1 and t-PA were measured in 40 SSc (34 lSSc and 6 dSSc) pts and in 30 healthy controls. In SSc, blood was drawn before and after 3 consecutive daily of Alprostadil infusion (60 mg in 250 cc NaCl 0.9%). RESULTS: In SSc su-PAR basal levels were higher than controls (7.48 +/- 2.5 vs 4.69 +/- 0.4 ng/ml; p = 0.001) and were significantly reduced by Alprostadil (5.93 +/- 1.7; p = 0.002), but remain higher than controls (p = 0.03). u-PA basal levels were higher than controls (3.78 +/- 1.5 vs 1.29 +/- 0.3 ng/ml; p < 0.001) and were reduced by Alprostadil (2.39 +/- 1.7; p < 0.001) to control levels. SSc PAI-1 basal levels were lower than controls (31.60 +/- 7.7 vs 48.30 +/- 6.8 ng/ml; p < 0.001) and increased by Alprostadil (34.66 +/- 5.4; p = 0.04), but lower than controls (p < 0.001). SSc t-PA basal levels were higher in respect to controls (1645.81 +/- 792.7 vs 571.95 +/- 75.5 pg/ml; p < 0.0001) and reduced by Alprostadil (1318.06 +/- 603.5; p = 0.04), but still higher than controls (p = 0.001). CONCLUSION: Fibrinolysis were increased in SSc. Infusions of Alprostadil modulate u-PA, su-PAR, PAI-1 and t-PA, restoring near normal levels. In SSc, fibrinolysis system may become a potential target for new therapies.

Acetylation and phosphorylation of the carboxy-terminal domain of p53: regulative significance.

Three fundamental domains are conventionally distinguished on the p53 molecule: an NH2 domain involved in transcription, a central core domain involved in specific DNA binding to the consensus sequences, and a carboxy-terminal domain of about 30 amino acids working as a basic regulatory domain, exhibiting aspecific DNA binding, tetramerization, and nuclearization. The presence of an unmodified carboxy-terminus does not allow the specific transactivation transcriptional function of the p53 protein. Therefore, for the activation of the protein function the carboxy-terminus must be modified. In the present commentary we discuss the role of two covalent modification events occurring at the carboxy-terminus, namely phosphorylation and acetylation, as well as the specific role of these events in the functional regulation of p53 molecule.

Apoptosis : molecular regulation of cell death and hematologic malignancies.

Apoptosis, or programmed cell death, represents in cell biology a functional program as important as cell growth or differentiation. Programmed cell death is of basic importance for the development of multicellular organisms and its basic mechanisms are conserved during the evolution of metazoa. Mammalian cells exhibit several different apoptotic pathways that converge to a common endpoint. Each pathway is triggered by a different stimulus: growth factor default, irradiation, induction of the p53 oncosuppressor protein, glucocorticoid hormones (in lymphocytes), ligand binding to Fas/APO (CD95), or tumor necrosis factor receptor (TNF-R), perforin secreted by cytotoxic T cells (reviewed by Hale et al. [1]). As opposed to necrosis, apoptosis is a "clean" process: as the cell shrinks, the cell membrane turns into the "apoptotic shell," the nucleus is condensed and reduced in volume, and eventually the cell disappears from the tissue, due to phagocytosis by neighboring cells or professional phagocytes, such as macrophages.

Role of p53 mutations in the radiosensitivity status of tumor cells.

Wild-type p53 is involved in cellular response to DNA damage including cell cycle control, DNA repair and activation of apoptosis. Accumulation of p53 protein following DNA damage may initiate the apoptotic process, resulting in cell death. DNA damage induced by radiation is an example of apoptotic stimulus involving p53. Regulation of apoptosis by p53 can occur through transcriptional regulation of pro-apoptotic (e.g. bax) and anti-apoptotic (e.g. bel-2) factors. Although wild-type p53 usually sensitizes cells to radiation therapy, p53 mutations have a variable effect on radiation response. For example p53 mutations in bone or breast tumors have been found to be associated with resistance to chemotherapeutic drugs or ionizing radiation. Mutated p53 has has been reported to increase sensitivity to radiation and drugs in colorectal and bladder tumors. The present brief commentary tries to find an explanation at molecular level of these conflicting results.

New insights in mast cell modulation by palmitoylethanolamide.

Since its discovery palmitoylethanolamide was considered as an endogenous compound able to negatively modulate the inflammatory process. Its effects have been extensively investigated in in vitro, in vivo and in clinical studies. Notwithstanding some discrepancy, nowadays the efficacy of palmitoylethanolamide in controlling mast cell behaviour, which likely accounts for its many anti-inflammatory, anti-angiogenic and analgesic effects, is well recognized. In view of their strategic localization at sites directly interfacing with the external environment, mast cells act as surveillance antennae against different types of injury and can undergo activation, thereby regulating both innate and adaptive immune reactions through the release of several preformed and newly synthesized mediators. Mast cells are now viewed as key players in orchestrating several disorders including both acute and chronic inflammatory processes, and have a role in angiogenesis and hyperalgesia. Since mast cells exert also important physiological, homeostatic functions, the most recent goal for pharmacologists is to control, rather than block, mast cell degranulation in order to modulate the pathological scenario. The aim of the present review is to summarise the evidence regarding the role played by palmitoylethanolamide in the control of mast cell activation, starting from in vitro studies, going through in vivo evidence in animal models of disease sustained by mast cell activation, and finally reviewing recent clinical studies using this molecule.

Morphology changes in human lung epithelial cells after exposure to diesel exhaust micron sub particles (PM1.0) and pollen allergens.

In the recent literature there has been an increased interest in the effects of particulate matter on the respiratory tract. The objective of this study was to use an in vitro model of type II lung epithelium (A549) to evaluate the cell ability to take up sub-micron PM(1.0) particles (PM(1.0)), Parietaria officinalis (ALL), and PM(1.0) + ALL together. Morphological analysis performed by Transmission Electron Microscope (TEM) showed that PM and ALL interacted with the cell surface, then penetrating into the cytoplasm. Each single treatment was able to point out a specific change in the morphology. The cells treated appear healthy and not apoptotic. The main effect was the increase of: multilamellar bodies, lysosomal enzymes, microvilli, and presence of vesicle/vacuoles containing particles. These observations demonstrate morphological and functional alterations related to the PM(1.0) and P. officinalis and confirm the induction of the inflammatory response in lung cells exposed to the inhalable particles.

Impairment of endothelial cell differentiation from bone marrow-derived mesenchymal stem cells: new insight into the pathogenesis of systemic sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a disorder characterized by vascular damage and fibrosis of the skin and internal organs. Despite marked tissue hypoxia, there is no evidence of compensatory angiogenesis. The ability of mesenchymal stem cells (MSCs) to differentiate into endothelial cells was recently demonstrated. The aim of this study was to determine whether impaired differentiation of MSCs into endothelial cells in SSc might contribute to disease pathogenesis by decreasing endothelial repair. METHODS: MSCs obtained from 7 SSc patients and 15 healthy controls were characterized. The number of colony-forming unit-fibroblastoid colonies was determined. After culture in endothelial-specific medium, the endothelial-like MSC (EL-MSC) phenotype was assessed according to the surface expression of vascular endothelial growth factor receptors (VEGFRs). Senescence, chemoinvasion, and capillary morphogenesis studies were also performed. RESULTS: MSCs from SSc patients displayed the same phenotype and clonogenic activity as those from controls. In SSc MSCs, a decreased percentage of VEGFR-2+, CXCR4+, VEGFR-2+/CXCR4+ cells and early senescence was detected. After culturing, SSc EL-MSCs showed increased expression of VEGFR-1, VEGFR-2, and CXCR4, did not express CD31 or annexin V, and showed significantly decreased migration after specific stimuli. Moreover, the addition of VEGF and stromal cell-derived factor 1 to cultured SSc EL-MSCs increased their angiogenic potential less than that in controls. CONCLUSION: Our data strongly suggest that endothelial repair may be affected in SSc. The possibility that endothelial progenitor cells could be used to increase vessel growth in chronic ischemic tissues may open up new avenues in the treatment of vascular damage caused by SSc.

Systemic sclerosis fibroblasts inhibit in vitro angiogenesis by MMP-12-dependent cleavage of the endothelial cell urokinase receptor.

Failure of endothelial cells to develop new vessels in response to hypoxia is a distinctive feature of systemic sclerosis (SSc) in the avascular phase. We have previously shown that SSc endothelial cells over-express matrix metalloproteinase-12 (MMP-12), which blocks angiogenesis by cleavage of the endothelial urokinase-type plasminogen activator receptor (uPAR). In the present study, we have investigated whether over-expression of MMP-12 and of angiostatic factors, or hypo-expression of angiogenic factors by SSc fibroblasts, contributes to impaired angiogenesis in SSc. Dermal fibroblasts were isolated from healthy subjects (N-Fb) and patients with diffuse SSc (SSc-Fb). Angiogenesis of target normal human microvascular endothelial cells (H-MVECs) was assayed by Matrigel invasion, cell proliferation, and capillary morphogenesis. uPAR cleavage and MMP-12 activity were evaluated by western blotting. We show that the over-expression of MMP-12 by SSc-Fb determines uPAR cleavage in H-MVECs. Conditioned medium from SSc-Fb impaired H-MVEC proliferation, invasion, and capillary morphogenesis. Anti-MMP-12 antibodies restored such impairment. Altered expression of angiostatic/angiogenic factors, including transforming growth factor beta1, did not account for SSc-Fb-dependent impairment of angiogenesis. The over-expression of MMP-12 by both SSc-Fb and SSc endothelial cells indicates that MMP-12 over-production may have a critical pathogenic role in SSc-associated vascular alterations.

Piascledine modulates the production of VEGF and TIMP-1 and reduces the invasiveness of rheumatoid arthritis synoviocytes.

BACKGROUND: In rheumatoid arthritis (RA), hypertrophy of the synovial membrane generates a tumour-like pannus that invades the joint cavity and erodes cartilage and bone. Invasion of the extracellular matrix (ECM) is accompanied by angiogenesis, in which vascular endothelial growth factor (VEGF) and tissue inhibitors of metalloproteinases (TIMPs), produced by synoviocytes lining the pannus, have a primary role. Piascledine (PSD) is used in the treatment of osteoarthritis and has anti-inflammatory effects in vitro. OBJECTIVE: To study the effects of PSD on levels of VEGF and TIMP-1 and chemoinvasion in RA synoviocytes and healthy controls. METHODS: The effects of PSD 5, 10, and 20 microg/mL were evaluated, with/without interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNFalpha) 20 ng/mL, on synoviocytes. The levels of VEGF and TIMP-1 were assayed in the culture medium by enzyme-linked immunosorbent assay (ELISA). Chemoinvasion was measured by the Boyden chamber invasion assay. RESULTS: RA synoviocytes treated with PSD showed, compared to basal, lower levels of VEGF (41080+/-830 vs. 79210+/-920 pg/106 cells, p<0.001) and increased levels of TIMP-1 (23540+/-93.2 vs. 12860+/-42.9 ng/106 cells, p<0.001). PSD decreased dose-dependently IL-1beta and TNFalpha induced migration. CONCLUSIONS: In RA synoviocytes, and also to a lesser extent in control cells, PSD modulates VEGF and TIMP-1 and decreases chemoinvasion. PSD might have a role in the treatment of RA synovitis controlling invasiveness.

Deflazacort modulates the fibrinolytic pattern and reduces uPA-dependent chemioinvasion and proliferation in rheumatoid arthritis synoviocytes.

OBJECTIVE: Extracellular fibrinolysis, controlled by the cell-associated fibrinolytic system (urokinase plasminogen activator, uPA; uPA receptor, uPAR; plasminogen activator inhibitor type-1, PAI-1), is involved in cartilage damage generation and in rheumatoid arthritis (RA) synovitis. Since steroids reduce the rate of radiological progression of RA, we planned to evaluate in healthy and RA synoviocytes the effects of the steroid deflazacort on uPA, uPAR and PAI-1 expression, and subsequent phenotypic modifications in terms of uPA/uPAR-dependent invasion and proliferation. METHODS: uPA, uPAR and PAI-1 levels were studied by ELISA, RT-PCR (uPAR) and zymography (uPA) in synoviocytes from four RA patients and four healthy controls. Chemoinvasion was assessed by the Boyden chamber invasion assay, using Matrigel as the invasion substrate. Proliferation was evaluated by cell counting. Both invasion and proliferation were measured upon treatment with deflazacort 5 muM with or without parallel stimulation with uPA 500 ng/ml or in the presence of monoclonal anti-uPA and anti-uPAR antibodies. RESULTS: Invasion and proliferation of RA synoviocytes require a proper functional balance of the fibrinolytic system. Both deflazacort and monoclonal antibodies against uPA and uPAR reduced expression and activity of the system, thus inhibiting invasion and proliferation. In RA synoviocytes, deflazacort induced higher PAI-1 and lower uPA and uPAR levels, as well as a decrease in uPA enzymatic activity. The levels of uPAR mRNA were concomitantly reduced, as was uPA-induced chemoinvasion. All these effects were also shown in controls, though to a lesser extent. CONCLUSIONS: Deflazacort might control RA synovial proliferation and invasion by differential modulation of single members of the fibrinolytic system.

Raloxifene reduces urokinase-type plasminogen activator-dependent proliferation of synoviocytes from patients with rheumatoid arthritis.

Extracellular fibrinolysis, controlled by the membrane-bound fibrinolytic system, is involved in cartilage damage and rheumatoid arthritis (RA) synovitis. Estrogen status and metabolism seem to be impaired in RA, and synoviocytes show receptors for estrogens. Our aims in this study were to evaluate in healthy and RA synoviocytes the effects of Raloxifene (RAL), a selective estrogen receptor modulator (SERM), on: proliferation; the components of the fibrinolytic system; and chemoinvasion. The effects of RAL were studied in vitro on synoviocytes from four RA patients and four controls. Proliferation was evaluated as cell number increase, and synoviocytes were treated with 0.5 microM and 1 microM RAL with and without urokinase-plasminogen activator (u-PA) and anti-u-PA/anti-u-PA receptor (u-PAR) antibodies. Fibrinolytic system components (u-PA, u-PAR and plasminogen activator inhibitor (PAI)-1) were assayed by ELISA with cells treated with 0.5 microM and 1 microM RAL for 48 h. u-PA activity was evaluated by zymography and a direct fibrinolytic assay. U-PAR/cell and its saturation were studied by radioiodination of u-PA and a u-PA binding assay. Chemoinvasion was measured using the Boyden chamber invasion assay. u-PA induced proliferation of RA synoviocytes was blocked by RAL (p < 0.05) and antagonized by antibodies alone. The inhibitory effect of RAL was not additive with u-PA/u-PAR antagonism. RA synoviocytes treated with RAL showed, compared to basal, higher levels of PAI-1 (10.75 +/- 0.26 versus 5.5 +/- 0.1 microg/10(6) cells, respectively; p < 0.01), lower levels of u-PA (1.04 +/- 0.05 versus 3.1 +/- 0.4 ng/10(6) cells, respectively; p < 0.001), and lower levels of u-PAR (11.28 +/- 0.22 versus 23.6 +/- 0.1 ng/10(6) cells, respectively; p < 0.001). RAL also significantly inhibited u-PA-induced migration. Similar effects were also shown, at least partially, in controls. RAL exerts anti-proliferative and anti-invasive effects on synoviocytes, mainly modulating u-PAR and, to a lesser extent, u-PA and PAI-1 levels, and inhibiting cell migration and proliferation.

Phorbol esters attenuate the expression of p53 in cells treated with doxorubicin and protect TS-P53/K562 from apoptosis.

The induction of p53 by doxorubicin in normal human fibroblasts was completely reverted by TPA, a phorbol ester. A ts-p53 mutant protein which is ineffective at 37 degrees C, but behaves in a wild-type fashion at 32 degrees C, was overexpressed in the p53-null human leukemia cell line K562. Wild-type-p53 overexpression induced apoptosis, whereas TPA protected K562 cells from this phenomena. By analogy with the observed human fibroblasts, TPA was found to decrease p53 amount. The TPA-dependent down-regulation of p53 could explain the chromosomal gross alterations typical of cells subjected to a chronic TPA treatment, alterations also found in cells defective for p53 function.