Sven Påhlman

Differentiation and Survival Influences of Growth Factors in Human Neuroblastoma

Human neuroblastoma cell lines are established from high-stage, highly malignant tumours. Despite this and the fact that these tumours are arrested at an early, immature stage, many cell lines have the capacity to undergo neuronal differentiation under proper growth conditions. One such cell line is the noradrenergic SH-SY5Y cell line. These cells can be induced to mature by a variety of modalities, resulting in different mature phenotypes. The use of this cell system as a model to study the stem cell character of neuroblastoma is reviewed and discussed. In particular, we focus on growth factor dependencies in the SH-SY5Y system, and compare that to the normal situation, i.e. growth factor control of sympathetic neuronal and neuroendocrine differentiation during human and rat embryogenesis.

Hydrophobic interaction chromatography on noncharged sepharose® derivatives: Binding of a model protein, related to ionic strength, hydrophobicity of the substituent, and degree of substitution (determined by NMR)

A series of agarose gels, substituted with hydrophobic groups, has been synthesized and used for binding studies with the coloured model protein, phycoerythrin. The degrees of substitution for the derivatives can easily be estimated with proton magnetic resonance (NMR). It has been found that the capacity of the derivatives for phycoerythrin increases with increasing hydrophobicity of the substituent, degree of substitution and increasing ionic strength. For column experiments the degree of substitution should lie in the range 40-100 mmol substituent/mol galactose. When it is excessively high, the flow characteristics of the columns are unsatisfactory and difficulties to achieve complete desorption may arise.

Kinetics and concentration effects of TPA-induced differentiation of cultured human neuroblastoma cells

SH-SY5Y human neuroblastoma cells differentiate morphologically and biochemically in the presence of 12-0-tetradecanoylphorbol-13-acetate (TPA). The degree of differentiation, as demonstrated by the appearance of cell surface projections, growth inhibition and increase in noradrenalin concentration, was dependent on the TPA concentration and had an optimum at 1.6 X 10(-8) M of TPA. At that concentration neuron specific enolase (NSE) increased to a maximum level after 10 days of culture with no further changes in the NSE level during additional culture for 10 days. In contrast, the noradrenalin concentration reached a maximum after 4 days of TPA treatment and decreased during longer exposures to TPA. Based on the facts that the phorbolester induced differentiation shows stereo specificity and was optimal at the same concentration range as common polypeptide hormones, a putative TPA-hormone receptor interaction is discussed. An opposite effect of TPA on the SH-SY5Y cells, antagonizing the differentiation effect, is further suggested to explain the decrease in differentiation observed at TPA concentrations higher than 1.6 X 10(-8) M.

Muscarinic receptors in human SH-SY5Y neuroblastoma cell line: regulation by phorbol ester and retinoic acid-induced differentiation ☆

The specific binding of the muscarinic ligand [3H](-)quinuclidinyl benzilate [( 3H]QNB) to cell membranes of human SH-SY5Y neuroblastoma cells was studied. Saturation isotherms yielded a Kd = 0.28 +/- 0.06 nM and a Bmax of 337 +/- 47 pmol/g protein. Pirenzepine inhibited [3H]QNB binding; inhibition data showed best fit to a 2-site binding model revealing both a high affinity pirenzepine site (34%, KH = 10 nM) and a low affinity site (66%, KL = 1 microM). These results indicate that muscarinic receptors on SH-SY5Y cells may be subclassified as M1/M2 subtypes. Morphological and biochemical differentiation of these cells after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid (RA) resulted in a decrease and an increase in the number of muscarinic binding sites, respectively. Furthermore, TPA- and RA-treated cells showed a significant increase in acetylcholinesterase activity compared with non-treated cells. However, only RA-treated cells showed significant increase in choline acetyltransferase activity compared to non-treated cells. These findings demonstrate that TPA and RA can regulate both the number of muscarinic receptors and the acetylcholinesterase activity in human SH-SY5Y neuroblastoma cells.

Clinical and serologic markers of stage and prognosis in small cell lung cancer. A multivariate analysis

The respective pretreatment prognostic impacts of the following markers were evaluated in 125 patients with small cell lung cancer (SCLC): lactic dehydrogenase (LDH), serum thymidine kinase (S‐TK), carcinoembryonic antigen (CEA), neuronspecific enolase (NSE), and tissue polypeptide antigen (TPA). More traditional clinical and serologic markers were also evaluated. Univariate analysis showed that all of the biochemical markers mentioned above, the Karnofsky index (KI) and the patients sex were related to both the stage of disease (limited/extensive disease: LD/ED) and to survival. The strongest marker for the clinical stage was S‐TK, whereas TPA showed the strongest relationship with survival. Multivariate analyses produced a model consisting of S‐TK, CEA, NSE, and the patients sex for determining the clinical stage. To compare the prognostic capacity of easily determined biochemical and simple clinical variables to the more resource‐demanding variable of the clinical stage, three multivariate analyses in relation to survival were performed: (1) biochemical markers and simple clinical variables; (2) LD/ED and simple clinical variables; and (3) all available variables. The model obtained from the first analysis included TPA, KI, age, and the patients sex; the model from the second analyses included LD/ED, patients age, and KI; and the model from the third analysis, TPA, KI, age, sex, and LD/ED. Indices based on these three multivariate models were calculated for each patient and the prognostic capacity of these indices was compared. Pretreatment serum marker levels also had the capacity to predict both the grade and the duration of the response to therapy.

Divergent changes of chromogranin A/secretogranin II levels in differentiating human neuroblastoma cells

Human neuroblastoma cells were cultured either in the absence or presence of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) known to induce neuronal differentiation. This treatment led to a marked increase in the concentration of secretogranin II but to a decrease of chromogranin A. Analogous changes were observed for the respective mRNAs. Thus during differentiation of these cells the biosynthesis of two vesicle constituents of large dense core vesicles is differentially regulated as determined both at the mRNA and the protein level. Levels of both synaptin/synaptophysin and SV2 were also elevated but to a smaller degree than that of secretogranin II.

β-Adrenoceptor, vasoactive intestinal polypeptide (VIP) and neuropeptide tyrosine (NPY) receptors functionally coupled to adenylate cyclase in the human neuroblastoma SK-N-MC cell line

Neurotransmitter receptor coupling to adenylate cyclase (AC) was studied in the cultured human neuroblastoma SK-N-MC cell line. Activation of beta-adrenoceptors with isoprenaline (ISO) or vasoactive intestinal polypeptide (VIP) receptors, increased AC activity in a dose-dependent manner. Preincubation with ISO and VIP induced a ligand specific, i.e. homologous type of desensitization of the respective receptor. Neuropeptide tyrosine (NPY) was able to inhibit ISO as well as VIP induced AC activity. The effect of NPY was totally abolished in cells pretreated with pertussis toxin to inactivate inhibitory G-proteins. Thus, SK-N-MC cells possess functionally coupled beta-adrenoceptors, VIP and NPY receptors, and may be used to study interactions between ligands and receptors which couple to the AC system.

Neuron-specific enolase as a follow-up marker in small cell bronchial carcinoma: a prospective study in an unselected series

The value of measurement of serum neuron‐specific enolase (NSE) as a follow‐up marker was investigated in 88 patients with small cell bronchial carcinoma. Of these, 42 had extensive disease and 46 had limited disease. The mean NSE levels before treatment, at response, and at recurrence in extensive disease were 107, 10, and 52 ng/ml, respectively, and the corresponding levels in limited disease were 35, 10, and 19 ng/ml, respectively. All differences were statistically clearly significant. However, the sensitivity of NSE in serum at response was 66% and at recurrence, 38%. The predictive value of an NSE decrease at response was 88%, and at recurrence, 72%. It is concluded that NSE changes during follow‐up support the evaluation of the outcome but cannot be used as a monitoring agent in an individual patient.

Chromatographic purification of a mammalian histidine decarboxylase on charged and non-charged alkyl derivatives of agarose.

Histidine decarboxylase (EC 4.1.1.22) from a mouse mastocytoma has been purified by chromatography on charged and non-charged n-alkyl derivatives of agarose. The former was represented by the coupling product of CNBr-activated agarose and alkylmonoamines (alkylamino-agarose), the latter by the coupling of agarose and alkylglycidyl ehters (alkyl agarose). The choice of fractionation medium was restricted by the enzyme stability; excessively high ionic strength media could not be used. Under the conditions investigated, the best result was obtained with the non-charged ocytl agarose. The enzyme was adsorbed to this gel at a relatively high ionic strength, and on stepwise decrease in ionic stength of the eluting buffer it was desorbed with a total recovery of 80%. There was an approx. 10-fold increase in specific activity. The histidine decarboxylase, thus purified, retained 90-100% of its activity for 10 days or more at 6-8 degrees C. Some general comments on protein fractionation on charged and non-charged alkyl derivatives of agarose are given. The complexity of protein interaction with the charged alkyl derivatives is illustrated by experiments with a colored protein, phycoerythrin.

Mitogenically Uncoupled Insulin and IGF-I Receptors of Differentiated Human Neuroblastoma Cells Are Functional and Mediate Ligand-Induced Signals

The SH-SY5Y human neuroblastoma cell line is differentiated in vitro with nanomolar concentrations of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Untreated cells express insulin receptors, and both type I and type II insulin-like growth factor (IGF) receptors, as has been shown by agonist binding and immunoprecipitation studies. Via interaction with its own receptor and the IGF-I receptor, insulin induced a mitogenic response in these cells. IGF-I and IGF-II are also mitogens for SH-SY5Y cells, as shown by a transient increase of the c-fos mRNA level, ornithin decarboxylase activity, thymidine incorporation, and, finally, cell division. TPA-differentiated cells do not respond mitogenically to any of these factors, although insulin and IGF-I receptors are still present on the cell surface and remain functional, as demonstrated by ligand-stimulated autophosphorylation, actin reorganization, and c-fos induction. However, other prereplicative responses, i.e., increased ornithin decarboxylase activity and c-myc mRNA levels, cannot be induced. These phenomena, may be part of a receptor uncoupling mechanism(s). The findings are discussed in terms of differentiation stage-dependent signaling of growth factor receptors. We suggest that these receptors switch from controlling cell division in replicative neuronal cells to mediating externally controlled functions related to the differentiated neuronal phenotype.