Subcellular details of early events of differentiation induced by retinoic acid in human neuroblastoma cells detected by atomic force microscope.

Atomic force microscopy (AFM) is gaining ever-increasing attention in biology as it allows us to achieve very high resolution both in air and in liquid. Recently, this technique has been employed for the observation of dynamic phenomena of cells in their culture medium. We employed this technology for comparing different morphologies, neuronal and substrate-adherent type, of cell lines of human neuroblastoma, a tumor derived from neuroectodermal tissue. The AFM image allows to confirm and enrich the information given by optical microscopes adding further details especially on the neural protrusions. Furthermore, we took advantage of the possibility to perform the observation of the cells in their culture medium for studying the neuroblastoma cell differentiation. For the first time, we detected the very early events of the outgrowth of neurite-like structures induced by retinoic acid. A time-course experiment has showed that the acid induces changes in the cellular membrane and dramatic modifications in the cytoskeleton already within 2 h.

Images of cultured neurons: morphological and functional information.

The in vitro culture of neuronal cells is a useful tool for studying, in a controlled way, neurobiological and neuropharmacological phenomena. A first step towards the understanding of these phenomena is described. Effects of simulated excitatory/inhibitory synapses, artificially positioned along the digitized image of neural arborizations, are presented.

Early detection of cell metabolism with a silicon microsensor.

A silicon microsensor (ISFET--Ion Sensitive Field Effect Transistor) has been used to detect the metabolism of a cell population cultured on a coverslip and positioned close to the sensor surface. The system output is analyzed as a function of cell density.

Images of cultured neurons: Morphological and functional information.

The in vitro culture of neuronal cells is a useful tool for studying, in a controlled way, neurobiological and neuropharmacological phenomena. A first step towards the understanding of these phenomena is described. Effects of simulated excitatory/inhibitory synapses, artificially positioned along the digitized image of neural arborizations, are presented.

Early detection of cell metabolism with a silicon microsensor.

A silicon microsensor (ISFET - Ion Sensitive Field Effect Transistor) has been used to detect the metabolism of a cell population cultured on a coverslip and positioned close to the sensor surface. The system output is analyzed as a function of cell density.

Expression of protein kinase C-alpha (PKC-alpha) and MYCN mRNAs in human neuroblastoma cells and modulation during morphological differentiation induced by retinoic acid.

It is known that PKC is differently expressed in brain and the peripheral nervous system and is involved in cellular differentiation. We have analyzed 9 human neuronal-derived crest-cell lines for PKC-alpha mRNA. Seven out of nine expressed 9.0 kb and 4.0 kb PKC-alpha mRNAs, but three had high level of 9.0 kb transcription. The different expression of the two messenger RNAs may result from alternative splicing and a different degree of cell maturation. The same cell lines were studied for MYCN gene expression. A possible relation between the two genes is discussed. One cell line expressing high levels of both PKC-alpha mRNA was treated with 10(-5) M retinoic acid (RA). The expression of both messenger RNAs was suppressed when the cells achieved a morphological differentiation and showed neurite-like processes. A decrease of PKC-alpha gene expression was associated to down regulation of MYCN mRNA. These preliminary results suggest that PKC suppression of PKC-alpha mRNA is associated with reversion of the malignant phenotype.

Coordinate change of c-myc, transferrin receptor and H3 gene expression precedes induction of haemoglobin-producing cells of the leukaemia K562 cell line treated with cis-diamminedichloroplatinum (II).

Erythroid cell differentiation can be achieved in vitro by means of several chemical or natural agents. Cell differentiation is accompanied by biochemical and molecular changes which show that the inducer is active at different molecular levels. Among the chemical agents that are able to induce cell differentiation, the anticancer drugs are of great interest for the emerging study of in vitro and in vivo models for differentiation treatment. Cis-diamminedichloroplatinum II (CDDP), a crosslinking DNA compound, is usually employed at a high dosage in treating solid tumors. We have demonstrated that CDDP was also able to induce K562 cells towards erythroid differentiation. In our experiment 2.5 micrograms/ml of CDDP caused expression of alpha-globin chain gene and production of haemoglobulin. Continuous presence of the drug was not necessary to induce the cell to produce haemoglobin. Five days after CDDP treatment, the expression of c-myc oncogene had risen, while H3 histone mRNA expression fell to undetectable levels within 24 hours. Transferrin (Tf) receptor mRNA was reduced but later (about 48 hours) than c-myc and H3 messenger RNAs. The inhibition of cell proliferation was correlated both with the reduced expression of Tf receptor mRNA and low expression of the protein. However, expression of the cytoskeleton vimentin gene was only slightly affected during the time-course of the experiment. Data show that at least three phases were present in the irreversible CDDP induction of haemoglobin synthesis in the K562 cell. The erythroid differentiation was first preceded by a rise of c-myc mRNA, which probably precommits the cell towards the erythroid lineage. The mRNA levels of the cell-cycle dependent genes, H3 and Tf receptor, decreased and inhibition of DNA synthesis and loss of cell proliferation were detected. Finally, the alpha-globin chain gene was actively transcribed and the cell produced haemoglobin.

Cell metabolism measurements in culture via microelectronic biosensors.

Silicon-based H(+)-sensitive biosensors in proximity to a cell population detect variations in cell metabolism via local measurements of changes in pH. The feasibility of this approach is shown in the case of ISFET devices.

Cell metabolism measurements in culture via microelectronic biosensors.

Silicon-based H(+)-sensitive biosensors in proximity to a cell population detect variations in cell metabolism via local measurements of changes in pH. The feasibility of this approach is shown in the case of ISFET devices.

The specific inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), induces morphological change and cell differentiation of human neural crest-derived cell lineages.

It has been proved that inhibition of protein kinase C by 1-(5-isoquinolinylsulfonyl)-1-methylpiperazine (H7) induces morphological differentiation in murine neuroblastoma (nb) cell. Here we report that H7 is also active on human nb cell lines. The human nb cell had originally neuroblast-like (N) or intermediate (I) morphology. N and I type are thought to represent different stages of neuroblastoma differentiation. Neurite outgrowth was observed in N and I type morphology treating the cells with 7, 14 or 28 microM of H7. The results confirm previous observations and show that inhibition of PKC by H7 also promotes neuronal differentiation in human cell line variants.

Effects of gamma-interferon on the growth, morphology, and membrane and cytoskeletal proteins expression of Lan-1 cells.

The effects of gamma-interferon (gamma-IFN) on the growth, morphology, and phenotypic expression of the human neuroblastoma (NB) cell line, LAN-1, have been extensively tested. Low doses of gamma-IFN allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Cells exposed to gamma-IFN significantly decreased their growth rate, became smaller and poligonal, and sprouted long cellular processes with varicosities along their course, typical of the neurites seen in differentiated NB cells; morphological changes appeared within 48 h of culture with 1,000 U/ml gamma-IFN. The new morphological aspect reached the maximum expression after 6 days of culture, becoming more evident when fresh drug was added after 2 days of culture. A decrease in [3H]thymidine incorporation was also observed within 24 h; cell growth was completely inhibited at the 6th day. Membrane immunofluorescence showed several changes in NB-specific antigen expression after 6 days of treatment with gamma-IFN. At the same time gamma-IFN also modulated cytoskeletal proteins. These findings suggest that noncytotoxic doses of gamma-IFN do promote the differentiation of LAN-1 neuroblastoma cells which is associated with the reduced expression of the malignant phenotype.

Morphological change and cellular differentiation induced by cisplatin in human neuroblastoma cell lines.

In 1986 we reported on the capacity of cis-diaminedichloroplatinum(II) (cisplatin, CDDP) to induce erythroid cellular differentiation in the K562 cell. To continue our study of the differentiating activity of cisplatin, we treated two human neuroblastoma cell lines with different doses of the drug in vitro. Both cell lines showed changes in morphology; however, only one achieved a fully differentiated neuronal phenotype (cisplatin concentration 1 micrograms/ml). The differentiated neuroblastoma cells exhibited extensive neurite outgrowth that reached maximal elongation after 5 days of culture, forming several interconnections. Cisplatin could induce neuronal differentiation, as did retinoic acid, a neuroblastoma-differentiating agent. The results show that cisplatin should be a candidate for further in vitro and in vivo studies of induced differentiation.

[Immunosuppressive factors produced by a T cell line derived from acute lymphoblastic leukemia]. / Fattori immunosoppressivi prodotti da una linea cellulare T derivante da una leucemia linfoblastica acuta.

The supernatant of CD8+ cells, isolated from a permanent lymphoblastoid cell clone established from a long term culture of a T cell acute lymphoblastic leukemia, contained two distinct molecules with suppressive activity on PHA1-induced PBMC proliferation. This clone does not produce TNF-alpha, TNF-beta, alpha-IFN, gamma-IFN, IL-1, IL-2 and has not natural killer activity. In the attempt to purify and biochemically characterize the lymphoblastic cell line-derived T-cell SFs, a multi-step chromatographic separation has been used. Two different peaks of biologic activity have been separated by HPLC gel permeation in the range of 100-120 Kd and 75-85 Kd referred to as high molecular weight suppressor factor HMWSF, and low molecular weight suppressor factor LMWSF, respectively. These fractions were then concentrated, dialyzed and further purified by anion exchange HPLC. This chromatographic step allowed us to considerably purify the two SFs. The biologically active fractions derived from the previous chromatographic step were eventually subjected to hydrophobic interaction HPLC (HIC) for final purification. The highly purified material was characterized by polyacrylamide gel electrophoresis in sodium-dodecylsulfate (SDS-PAGE): a single band corresponding to 115 Kd was observed for HMWSF, while LMWSF yielded a single band at 80 Kd. The isoelectric points (pI) of the different SFs was determined by flat-bed isoelectric-focusing: the HMWSF yielded a single band at pI 7.4, while a much lower pI was observed for LMWSF, 3.5-3.6. Studies on temperature lability indicated that both proteins are stable for 3-4 hours at room temperature (RT), 24-36 hours at +4 degrees C and 7-10 days at -80 degrees C.

Cisplatin-induced erythroid differentiation in K562 cells: modulation of transferrin receptor.

The human erythroleukemia cell line K562 can be induced to erythroid terminal differentiation by several chemical agents. The property of inducing cellular differentiation in vitro has also been demonstrated for different antineoplastic agents commonly used in therapeutic protocols. The molecular events involved during erythroid differentiation are wholly unknown. Here we demonstrate that K562 cells, undergoing differentiation by the alkylating agent Cisplatin (cis-diamminedichloro platinum (II), CDDP) employed in clinical protocols for the treatment of solid tumors, are induced to produce haemoglobin. Down modulation of the transferrin receptor (Tfr), highly represented on the cell surface, is also observed during the early stages of differentiation. Furthermore, Tf receptor expression correlates with cell growth, is down regulated and lost during terminal differentiation. The Tfr down regulation is an early event, before haemoglobin production, during cell differentiation. The data suggest that CDDP can induce terminal differentiation in K562 cells in vitro, like other antineoplastic agents.

A human acute leukemia-derived T-cell line produces two inhibitor factors which suppress lymphocyte proliferation: characterization and purification of the molecules.

The crude supernatant of an CD1+/CD8+ T-cell clone (GI-CO-T-9) established from a long standing culture of an acute T-lymphoblastic leukemia (T-ALL) was shown to inhibit the responsiveness of normal PBMC to PHA. This clone does not produce TNF-alpha, TNF-beta, alpha-IFN, tau-IFN, IL-1, IL-2 and has no NK-like activity. The crude supernatant has been subjected to a multi-step chromatographic fractioning. Preparative gel permeation HPLC allowed us to recover two peaks of biologic activity in the range of 100-120 kDa and 75-85 kDa (referred to as "high molecular mass inhibitor factor", HMMIF, and "low molecular mass inhibitor factor", LMMIF, respectively). Both fractions were then subjected to anion exchange HPLC: HMMIF was recovered in fractions eluting at 0.04 M NaCl while LMMIF eluted at higher ionic strength (0.48 M NaCl). The fractions with biologic activity recovered from anion exchange HPLC have been subjected to hydrophobic interaction HPLC (HIC) for final purification. The highly purified material was characterized by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate (SDS) revealing single bands of 115 kDa and 80 kDa. The isoelectric points (pI), determined by flat-bed isoelectricfocusing, were 7.4 for HMMIF and 3.5-3.6 for LMMIF. Studies on temperature lability indicated that both proteins are stable for 3-4 hours at room temperature (RT), 24-36 hours at +4 degrees C and 7-10 days at -80 degrees C.

EBNA-negative polyclonal B cells derived from a long-term culture of unclassified acute lymphoblastic leukemia: IL-2-like activity of culture's supernatant.

A long-term culture of bone marrow lymphoblasts in a case of unclassified acute lymphoblastic leukemia is described. Cells lacking any lymphocytic marker in the early phase of the culture were gradually substituted by B cells showing a pattern of polyclonality. The culture supernatant contained high levels of immunoglobulins also showing interleukin 2 activity. Search for antigens related to the Epstein-Barr virus was negative. A clonal expansion of B cells versus spontaneous differentiation of unclassified leukemic cells is discussed; the long-term culture technique as a tool for a better evaluation of leukemic cells is suggested and discussed.

[Diminished in vitro colony forming capacity of bone marrow cells in a case of chromosome 8 trisomy (mosaicism): criteria for "high risk" pre-leukemia syndrome]. / Diminuita capacita' clonate in vitro dele cellule midollari in un caso di trisomia dell'otto (mosaico): criterio di "alto rischio" ? sindrome pre-leucemica?

A case has been described of Trisomy 8 mosaicism Syndrome. At onset the child presented with hyporegenerative anemia; the study of colony forming capacity in vitro (CFU) by Bone Marrow (B) and Peripheral Blood (PB) showed an abnormal colony formation by myeloid and erythroid progenitor cells. No immunological defects were discovered. The in vitro colony formation appears to have a definite role in the identification of patients who may be at higher risk of developing leukemia. The importance of 8 chromosome for hematopoiesis control is discussed.