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Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression.

Messi E, Florian MC, Caccia C, Zanisi M, Maggi R - BMC Cancer (2008)

Bottom Line: Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line.Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression.However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin. a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness;b) Retinoic acid treatment reduces migration and invasiveness of the more aggressive cell components of SK-N-SH cells;c) The cells that after retinoic acid exposure show migration and invasive capability may be identified on the basis of doublecortin expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Endocrinology, Centre of Oncological Endocrinology, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. elio.messi@unimi.it

ABSTRACT

Background: Neuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem.Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment.

Methods: We analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration) and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively.

Results: Doublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness. Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin.

Conclusion: a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness;b) Retinoic acid treatment reduces migration and invasiveness of the more aggressive cell components of SK-N-SH cells;c) The cells that after retinoic acid exposure show migration and invasive capability may be identified on the basis of doublecortin expression.

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DCX and vimentin are expressed by different SK-N-SH neuroblastoma cell subtypes. The sets of figures A-E and F-J are representative of a series of images taken from SK-N-SH cells cultured in control conditions. PC analysis (panels A and F) clearly shows the heterogeneity of the cell population. The triple IF for DCX (B), LIS1 (C) and TUB (D) shows an immunoreactive signal for DCX only in the N-type and in some I-type cells. The anti-LIS1 antibody stains all the cell types. When co-expressed by the same cell, TUB and DCX colocalize both in the cell body and along the neuritic processes (see inset 1 of panel E). The triple IF for DCX (G), NF-68 (H) and VIM (I) shows that none of the DCX+ cells is immunoreactive for VIM. Vimentin is present merely in the S-type and in some I-type DCX- cells. Some other I-type cells do express neither DCX nor VIM (arrow in the inset 2 of panel J). The NF-68 protein is expressed by all the cell subtypes.
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Figure 1: DCX and vimentin are expressed by different SK-N-SH neuroblastoma cell subtypes. The sets of figures A-E and F-J are representative of a series of images taken from SK-N-SH cells cultured in control conditions. PC analysis (panels A and F) clearly shows the heterogeneity of the cell population. The triple IF for DCX (B), LIS1 (C) and TUB (D) shows an immunoreactive signal for DCX only in the N-type and in some I-type cells. The anti-LIS1 antibody stains all the cell types. When co-expressed by the same cell, TUB and DCX colocalize both in the cell body and along the neuritic processes (see inset 1 of panel E). The triple IF for DCX (G), NF-68 (H) and VIM (I) shows that none of the DCX+ cells is immunoreactive for VIM. Vimentin is present merely in the S-type and in some I-type DCX- cells. Some other I-type cells do express neither DCX nor VIM (arrow in the inset 2 of panel J). The NF-68 protein is expressed by all the cell subtypes.

Mentions: As already reported [2,3] the three different cell types composing SK-N-SH cell line were distinguishable by phase contrast (PC) microscopy analysis (Fig. 1A and 1F).


Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression.

Messi E, Florian MC, Caccia C, Zanisi M, Maggi R - BMC Cancer (2008)

DCX and vimentin are expressed by different SK-N-SH neuroblastoma cell subtypes. The sets of figures A-E and F-J are representative of a series of images taken from SK-N-SH cells cultured in control conditions. PC analysis (panels A and F) clearly shows the heterogeneity of the cell population. The triple IF for DCX (B), LIS1 (C) and TUB (D) shows an immunoreactive signal for DCX only in the N-type and in some I-type cells. The anti-LIS1 antibody stains all the cell types. When co-expressed by the same cell, TUB and DCX colocalize both in the cell body and along the neuritic processes (see inset 1 of panel E). The triple IF for DCX (G), NF-68 (H) and VIM (I) shows that none of the DCX+ cells is immunoreactive for VIM. Vimentin is present merely in the S-type and in some I-type DCX- cells. Some other I-type cells do express neither DCX nor VIM (arrow in the inset 2 of panel J). The NF-68 protein is expressed by all the cell subtypes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254429&req=5

Figure 1: DCX and vimentin are expressed by different SK-N-SH neuroblastoma cell subtypes. The sets of figures A-E and F-J are representative of a series of images taken from SK-N-SH cells cultured in control conditions. PC analysis (panels A and F) clearly shows the heterogeneity of the cell population. The triple IF for DCX (B), LIS1 (C) and TUB (D) shows an immunoreactive signal for DCX only in the N-type and in some I-type cells. The anti-LIS1 antibody stains all the cell types. When co-expressed by the same cell, TUB and DCX colocalize both in the cell body and along the neuritic processes (see inset 1 of panel E). The triple IF for DCX (G), NF-68 (H) and VIM (I) shows that none of the DCX+ cells is immunoreactive for VIM. Vimentin is present merely in the S-type and in some I-type DCX- cells. Some other I-type cells do express neither DCX nor VIM (arrow in the inset 2 of panel J). The NF-68 protein is expressed by all the cell subtypes.
Mentions: As already reported [2,3] the three different cell types composing SK-N-SH cell line were distinguishable by phase contrast (PC) microscopy analysis (Fig. 1A and 1F).

Bottom Line: Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line.Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression.However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin. a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness;b) Retinoic acid treatment reduces migration and invasiveness of the more aggressive cell components of SK-N-SH cells;c) The cells that after retinoic acid exposure show migration and invasive capability may be identified on the basis of doublecortin expression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Endocrinology, Centre of Oncological Endocrinology, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. elio.messi@unimi.it

ABSTRACT

Background: Neuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem.Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment.

Methods: We analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration) and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively.

Results: Doublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness. Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin.

Conclusion: a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness;b) Retinoic acid treatment reduces migration and invasiveness of the more aggressive cell components of SK-N-SH cells;c) The cells that after retinoic acid exposure show migration and invasive capability may be identified on the basis of doublecortin expression.

Show MeSH
Related in: MedlinePlus