Differential effects of N-(4-hydroxyphenyl)retinamide and retinoic acid on neuroblastoma cells: apoptosis versus differentiation.

Retinoids exert various important biological effects in the control of normal growth, differentiation, and fetal development. While retinoic acid (RA) has entered clinical trials as a differentiation-promoting agent, it is only recently that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) has been shown to be of potential clinical interest in cancer chemoprevention and treatment. Since thus far no data exist on the effects of HPR on neural crest cell-derived tumors, we have examined its in vitro effects on neuroblastoma (NB) cell lines and found that at relevant pharmacological concentrations it induces a dose-dependent growth inhibition. The antiproliferative effects of HPR were, in six of six cell lines tested, drastically more potent that those induced by an equimolar dose of RA. Time course growth analysis showed that HPR at 3 x 10(-6) M induces a very rapid (24-72 h) fall in thymidine uptake (> 90%), whereas at 3 x 10(-7) M it exhibits cytostatic effects. In contrast to RA, HPR did not show morphological changes typical of NB cell maturation nor did it induce the expression of any cytoskeletal protein associated with neuronal differentiation. DNA flow cytofluorimetric analysis revealed that HPR did not induce an arrest in a specific phase of the cell cycle while triggering apoptosis. This phenomenon was evidenced both by the visualization of "DNA ladders" on gel electrophoresis and by a quantitative assay for evaluating programmed cell death based upon the labeling of DNA breaks with tritiated thymidine. With the latter method, apoptotic cells were detectable as early as 3-6 h after treatment of NB cells with 10(-5) M HPR, while more than 50% of cells were apoptotic by 24-72 h following exposure to 3 x 10(-6) M HPR. In contrast, RA induced a low rate of apoptosis in NB cells only after 3-5 days. Time lapse photomicroscopy showed that NB cells treated with HPR underwent a death process highly reminiscent of apoptosis, with progressive condensation of the cytoplasm around the nucleus and intense cell shrinkage. The cells then rounded up and detached from the plate. Furthermore, propidium iodide staining of the DNA showed that a high proportion of cells treated with HPR displayed a small and brightly staining nucleus; chromatin appeared aggregated into dense masses in the nuclear periphery, a typical feature of apoptotic cells. In conclusion, our study demonstrates that contrary to the differentiation-promoting activity of RA, HPR dramatically suppresses NB cell growth by inducing programmed cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization and tumorigenicity of human neuroblastoma cells transfected with the IL-2 gene.

Immunization of cancer patients with cytokine-engineered tumor cells is being currently tested in several trials. To test the feasibility of this approach in neuroblastoma (NB) patients we investigated the functional consequences of interleukin-2 (IL-2) gene transfer into NB cell lines. Two human NB cell lines were transfected with the plasmid expression vector RSV.5neo containing the human IL-2 cDNA, and their tumorigenicity was evaluated in a nude mice xenograft model after characterization of the growth patterns and phenotypic features in vitro. The combination of IL-2 gene transfection and the xenograft model in nude mice was chosen on the basis of the low or absent expression of HLA class I antigen in human NB tumors. Our aim was to evaluate the effectiveness of an immunization protocol that could elicit a nonspecific antitumor response. The IL-2 stable transfectants were morphologically identical to parental or vector-transfected cells but completely lost tumorigenicity and inhibited, through a bystander effect, the growth of parental cells injected simultaneously at the same site. Histologic and immunohistochemical analysis of the nodules showed extensive necrosis with severe endothelial damage. The infiltrating cells were mainly macrophages, while natural killer (NK) cells were scarce. However, depletion of NK cells by anti-CD122 monoclonal antibody indicated that the rejection process required NK cell activity. The relevance of these data for the development of therapeutic approaches using cytokine-engineered NB cell lines is discussed.

Left ventricular performance in the early stages of systemic hypertension. HARVEST Study Group. Hypertension and Ambulatory Recording Venetia Study.

To investigate whether and how frequently left ventricular (LV) systolic performance assessed with endocardial and midwall measurement is depressed in young subjects with mild systemic hypertension, we studied 722 borderline to mild hypertensive patients (mean age +/- SEM 33 +/- 0.3 years, mean office blood pressure (BP) 146 +/- 0.4/94 +/- 0.2 mm Hg) enrolled in the Hypertension and Ambulatory Recording Venetia Study and 50 normotensive controls with similar age and sex distribution. BP was measured with 24-hour ambulatory monitoring. LV dimensional and functional indexes were assessed by M-mode echocardiography and sympathetic activity from 24-hour urinary catecholamines. In 64 hypertensive subjects (8.9%) the LV midwall shortening-stress relation was < 95% of the confidence interval in 50 normotensive controls. Subjects with depressed LV myocardial function had age, duration of hypertension, and LV mass similar to those of hypertensives with normal performance, and greater relative wall thickness (0.42 vs 0.37, p < 0.001). Stroke volume and cardiac output were lower (p < 0.001) in the former group. Among these 64 subjects, endocardial performance was depressed in 35 (group 1) and normal in 29 (group 2). Group 2 subjects had greater posterior wall (10.0 vs 9.5 mm, p = 0.03), ventricular septum (10.6 vs 10.1 mm, p = 0.05), and relative wall (0.44 vs 0.40, p < 0.001) thicknesses than group 1 subjects. Urinary norepinephrine was 50% higher in group 2 subjects (106 vs 70 g/24 hours, p = 0.03). Stroke volume and cardiac output were similar in both groups. In conclusion, these results show that LV contractility may be depressed in young subjects with borderline to mild hypertension.

[Neuroblastoma as a model for the in-vitro study of differentiated therapy]. / Il neuroblastoma come modello di studio in vitro della terapia differenziativa.

Differentiation of human neuroblastoma (NB) cells is a very interesting biologic event, providing useful insights in both basic neurobiology and clinical management of this malignancy. Investigation of in vitro NB differentiation exploits several NB cell lines that can be induced to differentiate by an array of natural or synthetic chemicals, as well as biological factors such as some cytokines. The hallmarks of neuronal differentiation are represented by a partial or complete block of cell proliferation, morphological alterations and acquisition of biological features typical of mature neurons (for example, induced synthesis and storage of monoamines and neuropeptides, expression of peculiar cytoskeletal proteins and membrane antigens). The possibility to transfer the differentiative approach to the treatment of NB patients opens exciting therapeutic perspectives.

Break of reciprocity principle due to localized nonlinearities in concrete.

The effects of localized nonlinearities on the reciprocity principle in the context of ultrasounds and nonlinear elasticity are discussed in this paper. Experiments will be presented to prove that a localized crack in a concrete beam causes a break of reciprocity in the ultrasonic response to a mechanical excitation. The link between non-reciprocity and asymmetry in the nonlinear response will be demonstrated and discussed as a tool for NonDestructive Evaluation.

Lack of HLA-class I antigens in human neuroblastoma cells: analysis of its relationship to TAP and tapasin expression.

We studied the constitutive and the interferon (IFN)-gamma-induced expression of HLA class I antigen heavy chain, beta2-microglobulin (beta2m), TAP-1, TAP-2 and tapasin in a panel of eleven neuroblastoma cell lines. Surface expression of HLA class I antigens was low in eight out of eight neuroblastoma cell lines bearing MYC-N amplification and/or 1p deletion, while two out of three neuroblastoma cell lines lacking these genetic alterations showed normal expression. IFN-gamma treatment restored HLA class I antigen surface expression in all neuroblastoma cell lines. Eight out of 11 neuroblastoma cell lines did not express TAP-1 mRNA and three of them also lacked TAP-2 mRNA. beta2 m mRNA was barely detectable or absent in five neuroblastoma cell lines, while tapasin mRNA was always expressed. IFN-gamma upregulated the expression of HLA class I heavy chain, beta2 m, TAP-1, TAP-2 and tapasin, as detected at mRNA or protein level. Post-transcriptional events were involved in altered TAP-1 and beta2 m expression in one peculiar neuroblastoma cell line. These data indicate that multiple mechanisms play a role in the HLA class I antigen-deficient phenotype of human neuroblastoma.

Growth factors increase retroviral transduction but decrease clonogenic potential of umbilical cord blood CD34+ cells.

BACKGROUND AND OBJECTIVE: The feasibility of gene marking or gene therapy protocols making use of purified CD34+ cells greatly depends on the efficiency of their stable transduction. The great potential of umbilical cord blood as a source of CD34+ cells combined with the availability of advanced cell purification procedures prompted us to evaluate whether incubation with growth factors might influence the type of cells effectively transduced by retroviral vectors. DESIGN AND METHODS: Isolated, at least 95% pure, CD34+ cells were infected with the LXSN murine retrovirus carrying the neomycin-resistance gene. Different schedules of CD34+ cell infection were performed with or without incubation for different times in the presence of Interleukin-3 (IL-3), Interleukin-6 (IL-6) and stem cell factor (SCF). Efficiency of transduction was evaluated by clonogenic assays, semiquantitative PCR and RT-PCR analyses performed either immediately or after 7 day expansion of CD34+ cells in liquid culture in the presence of erythropoietin (EPO), IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). RESULTS: The results obtained indicated that the amount of transduced cells increased with the lenght of incubation with growth factors, either before or during infections. However, different types of cells were transduced depending on the duration of stimulation and infection. Thus, following one week culture of CD34+ cells in the presence of EPO, IL-3 and GM-CSF the clonogenic potential was affected dyshomogeneously. Precisely, with a single 3-hour infection performed after 12 hours of stimulation with growth factors, the clonogenic potential of the transduced cells greatly increased after one week in culture. In contrast, with a 48 hour infection, the transduced cells completely lost their clonogenic potential after one week in culture. INTERPRETATION AND CONCLUSIONS: These results demonstrate that a reasonably high transduction efficiency of purified CD34+ cells can be achieved with short schedules of incubation/infection in the absence of stroma or extracellular matrix.