Differentiation, Proliferation and Adhesion of Human Neuroblastoma Cells After Treatment with Retinoic Acid

Differentiation, Proliferation and Adhesion of Human Neuroblastoma Cells After Treatment with RetinoFull Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=icac20
Cell Adhesion and Communication
Differentiation, Proliferation and Adhesion of Human Neuroblastoma Cells After Treatment with Retinoic Acid
Astrid Voigt, Peter Hartmann & Felix Zintl
To cite this article: Astrid Voigt, Peter Hartmann & Felix Zintl (2000) Differentiation, Proliferation and Adhesion of Human Neuroblastoma Cells After Treatment with Retinoic Acid, Cell Adhesion and Communication, 7:5, 423-440, DOI: 10.3109/15419060009109023
To link to this article: https://doi.org/10.3109/15419060009109023
Published online: 05 Aug 2009.
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Differentiation, Proliferation and Adhesion of Human Neuroblastoma Cells After Treatment with Retinoic Acid
ASTRID VOIGT*, PETER HARTMANN and FELIX ZINTL
Department qf Pediatrics, Uiiivcvsitj~ of Jena, Kochstra/h 2, 0-07740 Jeira, Gernianj,
(Received 10 Sc~pieniher 1999; Ri~\~iseri 10 November 1999; Iti .fkul,forri? 15 Nowinher 1999)
Because of the known property of spontaneous regression in stage IVS of neuroblastoma all attempts are made to elucidate whether differentiation inducers possibly could be applied for neuroblastoma therapy. Here we examined the influence of retinoic acid (RA) in vitro on differentiation, proliferation and adhesion of 10 permanent and 4 primary cell lines as well as of several SCID-mouse tumour transplants. In general, after RA treatment morphologically different cell types which are characteristic for neuroblastoma cells have changed. N (neurona1)-type cells prolonged their neuronal processes, whereas S (epithelial, substrate-adherent, Schwann cell-like)-type cells lost their adherence to substratum and became apoptotic. Additionally, the reactions of all neuroblastoma cell lines with monoclonal antibodies against P-tubulin (for neuronal cells) and glial fibrillary acidic protein (for epithelial cells) were determined. The anti-proliferative effect of all- trans-RA as well as I3-cis-RA was more profound in S-type cells (up to 40% in primary cell lines). T o elucidate the role of adhesion molecules during neuronal cell differentiation, we have analysed the adhesion of neuroblastoma cells on poly-D-lysin-precoated plates under RA influence. While N-type cells displayed an increased adhesion, all S-type cell lines as well as all primary cell lines exhibited a reduced adhesion (IMR-5 and IMR-32:p < 0.001: JW, SR and PM: p < 0.05). RA treatment increased predominantly the tested antigens (HCAM, ICAM-1, NCAM, PECAM-1, VCAM-1, cadherin, FGF-R, IGF-R, NGF-R, TGF-PII, NF200, NF160, NF68, NSE, HLA-ABC) in all cell lines independently of their phenotypes (TGF-PIl: p < 0.001; NF68: p < 0.01: PECAM-I and NGF-R: p < 0.05). In recultured SCID-mouse-passaged tumour cells antigens were down-regulated (FGF-R: p < 0.01), but increased again after RA influence (TGF-/?/I: p < 0.05). In summary, the RA differentiation model demonstrates the possibility to interfere in cell adhesion and to diminish growth potential both in N-type as well as S-type neuroblastoma cells.
Keyvords: Neuroblastoma, retinoic acid, differentiation, proliferation, adhesion
*Corresponding author. Tel.: +49 3641 938437. Fax: +49 3641 938470. E-mail: Astrid.Voigt@;ined.uni-jena.de
423
INTRODUCTION
In the last decade numerous results have been pub- lished, which present the invasion and metastasis of tumour cells as an extremely complex process. Neoblastic cells can detach themselves from the primary tumour. They penetrate the extracellular matrix, migrate in the organism and invade into capillaries through both the basement membrane and the connective tissue at the place of future metastasis (Bussemakers and Schalken, 1996). Con- sequently, tumour cells must exhibit a considerable flexibility in their adhesive interactions that is reflected in a complex and dynamic expression pattern of cell adhesion molecules (CAMs). CAMs, obviously associated with cell differentiation, play an important role in tumour progression and metastasis. Changes in the expression and function of adhesion molecules can supply important char- acteristics in tumour development. Therefore, they can be used as prognostic factors or as targets in diagnosis and therapy (Streit et al., 1996).
Neuroblastoma, a tumour of the peripheral sympathic nervous system, is not only from the clinical but also from the biological perspective one of the fascinating and most mysterious children’s tumour (Brodeur and Nakagawara, 1992). Neuro- blastoma represents a very useful model in the creation of new diagnostic and therapeutic meth- ods, especially for the investigation of fundamental processes of cancer development. Additionally, spontaneous regression in patients under 1 year of age with a localised primary tumour with dissemi- nation limited to skin, liver, and/or bone marrow (stage IVS, “special”) can be observed, whereby the malignant neuroblastoma may differentiate into a benign and well operable ganglioneuroma. This phenomenon can also be imitated in vitro by means of chemical substances that can induce the differ- entiation process. Retinoic acid (RA), as a product of the vitamin A-metabolism, belongs to the best known differentiation inducers causing different morphological and biochemical alterations in this kind of tumour. In vitro aggressive and dissemina- ting tumour cells could be transformed to matured
and non-proliferating cells (Barletta et al., 1997). This is also recognisable by a change in certain adhesion and differentiation antigens. Human neuroblastoma cell lines frequently exhibit diverse morphological phenotypes, showing transdifferen- tiation between morphologically and biochemically distinct cell types (Hartley et al., 1996). Neuroblas- toma cell lines, but not all, respond to RA with an enhanced expression of more differentiated fea- tures. Morphologically, RA has distinctive effects on neuroblastoma cell lines which contain cells expressing three phenotypes: N-type cells (neuro- nal), S-type cells (epithelial, substrate-adherent, Schwann cell-like) and I-type cells (intermediate) (Ciccarone et al., 1989). In general, after RA treatment N-type cells prolong their neuronal processes that are reminiscent of axons or dendrites (Gudas et al., 1994), whereas S-type cells lose their adherence to substratum and become apoptotic (Rozzo et al., 1997). Further examples of RA effects on the differentiation program include changes in ion channels (Morton et al., 1992), receptors (Adem et al., 1987) and activation of DNA binding (Bourget, 1995). The oncogene N-myc exhibits reduced expression in response of RA (Wada et al., 1997), whereas RA treatment increased expression of membrane proteins like ICAM-1 in the neuroblastoma cell line SK-N-SH (Bouillon et al., 1991) and stimulated an increase in IGF-I1 mRNA in LAN-115 (Matsumoto et al., 1992). All these effects of RA are evidences for maturation and differentiation of neuroblastoma tumour cells which are mediated primarily through two classes of nuclear receptors, RA receptors (RARs) and retinoic X receptors (RXRs), with three distinct receptors in each class (a, p and 7) (Rosati et al., 1998). While all-trans-RA only binds to RARs with high affinity, 13-cis-RA additionally attach to the RXRs receptors so that a better ligand/ receptor proportion for the latter is assumed.
The purpose of this study was to examine the influence of RA on differentiation, proliferation and adhesion of a panel of 10 permanent neuro- blastoma cell lines, 4 primary cell lines from tumours of patients with neuroblastoma stage IV
NEUROBLASTOMA AND RETINOIC ACID 425
and several SCID-mouse tumour transplants Establishment of Primary Cell Lines from covering the broadly variable phenotype of neuro- Fresh Clinical Specimens blastoma cells included all phenotypic character- istics from neuroblastic to Schwann cell-like features. Additionally, the cell lines represented different stages of neuronal maturation and showed a different spectrum of adhesion characteristics to extracellular matrix proteins. Furthermore, the influence of pretreatment of the tumour cells with anti-adhesion antibodies (ICAM, cadherin, NCAM and 15/7) was studied. Assuming a change of cell-to-cell or cell-to-substratum adhe- sion, this “anti-adhesion-therapy” could be an alternative approach to the treatment and preven- tion of cancer since evolving lesions or tumour cell invasion could be reversed or suppressed (Hakamori, 1996).
MATERIALS AND METHODS
Permanent Cell Lines
A panel of human neuroblastoma cell lines was evaluated to represent the genetic, morphological and biochemical variability found in neuroblas- toma tumours. The permanent cell lines SK-N-FI,
IMR-5, IMR-32, SK-N-MC, SK-N-LO and SK- N-AS, kindly provided by G. Bruchelt (Department of Pediatrics, University of Tubingen, Germany), were grown as monolayers in neuroblastoma medium (NB medium) consisting of a 1 : 1 mixture of Iscove’s modified Dulbecco’s medium (IM DM) and Ham’s F12 medium (both from Biochrom, Berlin, Germany). To this medium 10% fetal calf serum (FCS), 0.02 mM N-acetyl-L-alanyl-L-gluta- mine and 50 mM gentamicin were added. The cells were cultured at 37°C in a humidified environment with 5% C02 atmosphere. As the cells approached confluence, adherent cells were split following treatment with 2 mM ethylenediaminotetraacetate (EDTA) in phosphate-buffered saline (PBS) con- taining 1% bovine serum albumin (BSA) and replated in a completely fresh medium.
SK-N-SH, SH-SYSY, CHP-134, SK-PN-DW,
The primary cell lines US, JW and PM were established in our laboratory from tumour material of patients admitted to our hospital with advanced neuroblastoma stage IV. Solid tumour samples were taken surgically and cut into small pieces with scissors, homogenised in a Potter glass homogeniser and passed through a cell strainer with a pore size of 70 pm (Becton Dickinson, Heidelberg, Germany) to obtain a single cell suspension. The cells were cultured in NB medium as described above.
The cell line SR was established from a morpho- logically tumour cell free bone marrow prepared for autologous bone marrow transplantation after stem cell selection with the anti-CD34 monoclonal anti- body (moAb) 12.8 on the CellPro column (CellPro, Bothell, WA, USA) (Voigt et al., 1997a).
The supernatant from growing tumour cells was removed each week and fresh medium was added. In general, three-dimensional outgrowth of neuro- blastoma cells occurred after 3-4 weeks. Then the tumour cells were removed from the stroma layer by repeated detachment of cells with 2 m M EDTA/ BSAjPBS and subcultured for 6-8 weeks to initiate stable growth. Primary cell lines were defined as established after more than 8 weeks of growth following cloning by limiting dilution and a mini- mum of 3 passages. All experiments with primary cell lines were done with cells from passages 10 to 15.
SCID-Mouse Passage
For tumour implantation, a minimum of 1 x lo7 viable neuroblastoma cells were harvested from permanent cell lines during their exponential growth phase and suspended into isotonic sodium chloride solution (NaCI). These cells were implanted subcutaneously into the flanks of 6 weeks old female SCID-mice. After 2-3 weeks of growth, solid tumours were resected from mice, and single cell suspensions were obtained similarly to that from patient material as described above. Some- times, cells from the first mouse passage (passage I)
426 A. VOIGT e / al.
were implanted into a SCID-mouse for a second passage (passage 11) and the resected tumour was investigated again.
Retinoic Acid Treatment
Cells were harvested by EDTA/BSA/PBS treat- ment from logarithmically growing cell cultures and seeded either into 96-well microtiterplates for cell proliferation measurement or into 25 cm2- tissue culture flasks (both from Greiner GmbH, Frickenhausen, Germany) for assessment of mor- phological differentiation or cell adhesion. RA in form of all-trans-RA or 13-cis-RA (both from ICN Biomedicals GmbH, Eschwege, Germany) was added at 3-day intervals, beginning on the first day after plating (3 total doses) at final concen- trations of lop5, and 10-7M according to known pharmacologic dosages used in phase I trials of RA administered orally to neuroblastoma patients (Villablanca et al., 1995). The retinoids were first dissolved in absolute ethanol and then supplemented with distilled water to achieve con- centrations of and lO-’M in 70% ethanol. These stock solutions were kept at -21°C. For each experiment, RA was diluted from the stock solutions directly into the growth medium. Cells cultured in medium containing 0.1 % ethanol were used as control.
Morphological Differentiation
Cells were harvested by EDTAiBSAjPBS treat- ment, seeded with a concentration of 2 x lo4 cells/ ml inculture flasks and treated with all-trans-RA or 13-cis-RA with the described schedule. Morpholog- ical differentiation was examined on the 7th day by assessing neurite formation and adherence behav- iour of the cells with a phase-contrast microscope.
Cell Proliferation
Cell proliferation was measured by the MTT cell proliferation assay. Briefly, one day after the last RA treatment described above, the cells were
incubated for 4 h in microtiter plates with 5 mg/ml MTT-solution (3-(4,5-dimethylthiazol-2-yl)diphe- nyl tetrazolium bromide)) in PBS. During this incu- bation period, water-insoluble formazan crystals were formed. After overnight solubilisation with 2% dodecylsulfate (SDS; Boehringer Ingelheim, Heidelberg, Germany) the formazan dye was quan- tified at 570nm using a microtiter plate reader. The absorbance (optical density, OD) revealed directly correlates to the cell number. The experi- ment was done in triplicate with 6 parallels. Results were expressed as mean + SEM (standard error of the mean).
Cell-Substratum Adhesion Assay
To confirm the best adhesion of neuroblastoma cells, cell attachment was pretested on ready-to-use 6-well plates separately coated with the components of the extracellular matrix collagen I , poly-D-lysin (PDL), fibronectin and laminin (Falcon, Becton/ Dickinson, Heidelberg, Germany). Neuroblastoma cells harvested from stock culture by EDTA/BSA/ PBS treatment were seeded with a cell concentration of 2 x lo4 cells/ml. Because proliferation and adhe- sion had proved to be optimal on PDL in this experi- ment (data not shown), all further adhesion tests were done on coated plastic surfaces prepared in our laboratory by layering aliquots of 2 pg/cm2 PDL ( Boehringer Ingelheim, Heidelberg, Germany) solution in PBS on a 96-well microtiter plate for 30 min at 37°C. This was later followed by incuba- tion with 0.1 YO BSA in PBS for 30 min to block non- specific cell attachment sites.
To determine the influence of RA on the adhesion of the neuroblastoma cells, cells were detached from RA-treated culture with EDTA/BSA/PBS, washed and then resuspended in medium to a final density of 4 x 10’ cells/ml. Aliquots of 100 pl cell suspension were added to each of the precoated wells. The cells were allowed to adhere for 1 h at 37°C; unattached cells were discarded and the plate was washed with PBS. The remaining cells were fixed for 15 min with 100 pl/well of formalin solution (10% in PBS). Then this solution was exchanged with 1% Toluidine
NEUROBLASTOMA AND RETINOIC ACID 427
blue in 10% formalin for incubation overnight at room temperature. The plate was washed two times with distilled water, then air dried. Stained cells were lysed with 1OOpl of 2 % SDS for 10min on a plate shaker. The absorbance was read as OD at 620 nm with a microtiter plate reader, and the results were expressed as the percentage of attached cells to the adhesion of untreated cells. The experiment was done in triplicate with 6 parallels. Results were expressed as mean + SEM.
Cell Attachment Inhibition Assay
For testing the inhibitory effect of the anti-adhesion moAbs ICAM (10 pg/ml), cadherin (20 pg/ml), NCAM (3 pg/ml) and 15/7 (50 pg/ml) (see Table I),
4 x lo5 cells/ml were incubated with the moAbs in Eppendorf-tubes on a shaker for 1 h at room temperature. One hundred p1 of this cell and antibody mixture were added to a PDL-coated microtiter plate and incubated for another 1 h. After washing out the non-adherent cells, the test has to be continued by fixing and staining the cells identically to the attachment assay. Cells without antibody treatment were used as control. The experiment was done in duplicate with 6 parallels.
Expression of Cellular Antigens Involved in Adhesion
Cellular molecules involved in adhesion were immu- nohistochemically detected on primary, permanent
TABLE I Monoclonal antibodies used in this study
Antibody Antigen CD Subclass Clone Source
P-tubulin GFAP
Cadherin HCAM
NF 200 NF 160 N F 68 NSE HLA-ABC 1517
P-tubulin Glia fibrillaric acidic
molecule Intracellular adhesion
molecule (125 kD) Platelet endothelial cell
adhesion molecule-1 ( 100,130 kD)
Fibroblast growth factor receptor-] (1 10,120 kD)
Insulin-like growth factor- receptor (90,130 kD)
Nerve growth factor receptor p75NGFR
protein (52 kD)
~.
Endoglin as component of the transforming growth factor Pal receptor system
C D 105
Neurofilaments (200 kD) Neurofilaments (160 kD) Neurofilaments (68 kD) Neuron specific enolase HLA class I molecules Antigen involved in differentiation,
until uncharacterised
IgG2b IgGl
IgGl IgG2a
IgG 1
KMX-1 DP46.10
VI-HI4 W6/32
"See Ref.: Voigt ef al. (1993).
428 A. VOIGT et al.
and SCID-mouse passaged neuroblastoma cell lines, with and without RA treatment, using the APAAP method. Neuroblastoma cell lines were stained with moAbs against adhesion molecules (cadherin, HCAM, ICAM, NCAM, PECAM and VCAM), growth factor receptors (FGF-R, IGF-R, NGF-R and TGF), neuronal differentiation mark- ers (@-tubulin, GFAP, NF200, NF160, NF68 and 15/7) and miscellaneous antigens (NSE and HLA-ABC) which can alter during neuroblastoma differentiation (Table I). Permanent and primary cell lines were seeded on multiwell-slides (Falcon, Becton/Dickinson, Heidelberg, Germany) and treated with RA according to the scheme described above. The cells remained for fixation on these slides so that the neurites, typical in their length for the various cell lines, were preserved. Cells from mouse-passaged tumours used as control cells were attached to slides using a cytocentrifuge. For RA treatment they were cultured again on multiwell- slides.
Both multiwell- and cytocentifuged slides were fixed with icycold acetone for 2min, allowed to drain for some minutes, and stored at -21°C. After thawing, the samples were blocked with 1% BSA in PBS for 30min, and the moAbs were added in concentrations recommended by the manufacturers for overnight incubation at 4°C. The reactivities of the appropriate antigens were detected with an APAAP- Kit (DAKO Diagnostika GmbH, Hamburg, Germany) according to the manufac- turer’s instructions. The evaluation of stained cells was performed by two independent examiners. The immunoreactivity of about 500 cells in each case was assessed microscopically based on a semiquantita- tive scoring system comparing RA-treated cells with untreated cells as control. The staining results were determined classifying the intensity of antigen expression into one of five grades: 0 = unstained, + =4-20% positive, ++ = 21-50% positive, +++ = 51-75% positive, and ++++ = 76- 100% positive. The results from staining with anti-GFAP and anti-0-tubulin were transformed into numerals and presented in a graph. All experiments were done in duplicate.
Statistical Analysis
For statistical analysis of the experiments the calculation of SEM (standard error of the mean) and unpaired Students’ t-test were used.
RESULTS
Morphological Differentiation and Cell Proliferation After Retinoic Acid Treatment
The differentiation of neuroblastoma cells within a single line need not always result in neurite sprout- ing since many lines are composed of heterogeneous cell elements with different phenotypic potential (Abemayor and Sidell, 1989). Generally, the N- phenotype is more neuronally programmed and the S-type cells are programmed to undergo apoptosis after induction of differentiation (Melino, 1994). In our experiments, cultivation for 7 days with lop5, lop6 and 1 OP7 M all-trans-RA or 13-cis-RA induced morphological alterations of cells in proportion to their phenotypes, whereby no significant differences between the two retinoid isoforms could be seen. The treatment of the cell lines SK-N-FI, SK-N-SH and SH-SY5Y with RA resulted in a marked differentiation toward the neuronal phenotype (N- type cells), inducing the cells to be mainly polar with longer and out-branched neurites, but remaining substrate-adherent. In contrast, the cell lines IMR- 5, IMR-32, SK-N-MC, SK-N-LO and SK-N-AS underwent a death process with progressive con- densation of the cytoplasm and cell shrinkage. During this process, termed apoptosis (Piacentini et al., 1992), the cells rounded up and predomi- nantly detached from the substratum. These cell lines were accounted to the S-type cells. In the cell lines CHP- 134 and SK-PN-DW, phenotypic char- acteristics ofboth N- and S-cells after RA treatment were observed. These intermediate (I) cell lines could be found with long neurites as well as with detached cells. According to Ciccarone et al. (1989) and Ross et al. ( 1995) I-type cells appear to present multipotent precursor cells with biochemical attri- butes of both N- and S-cells. A similar situation was
NEUROBLASTOMA AND RETINOIC ACID 429
found by culturing primary cell lines. While US cells acquired the ability to extend long and branched neurites,…