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Characterization of cells recovered from the xenotransplanted NG97 human-derived glioma cell line subcultured in a long-term in vitro.

Machado CM, Ikemori RY, Zorzeto TQ, Nogueira AC, Barbosa SD, Savino W, Schenka AA, Vassallo J, Heinrich JK, Boetcher-Luiz F, Verinaud L - BMC Cancer (2008)

Bottom Line: Results showed that NG97(ht) had an decrease in doubling time through sub cultivation, which was characterized by a converse modulation between the expression of glial fibrillary acidic protein (GFAP) and vimentin.In the latter case, these presumably chromosomes were originated as fusions between murine stroma cells and NG97 cell lineage in the xenotransplant.Our results emphasize important queries about astrocytomas tumor progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Institute of Biology, UNICAMP - Campinas, São Paulo, Brazil. camilalongomachado@gmail.com

ABSTRACT

Background: In order to elucidate tumoral progression and drug resistance, cultured cell lines are valuable tools applied on tumor related assays provided they are well established and characterized. Our laboratory settled the NG97 cell line derived from a human astrocytoma grade III, which started to develop and express important phenotypical characteristics of an astrocytoma grade IV after injection in the flank of nude mice. Astrocytomas are extremely aggressive malignancies of the Central Nervous System (CNS) and account for 46% of all primary malignant brain tumors. Progression to worse prognosis occurs in 85% of the cases possibly due to changes in cell tumor microenvironment and through biological pathways that are still unclear.

Methods: This work focused on characterizing the NG97 cell line specifically after being recovered from the xenotransplant, who maintained their undifferentiated characteristics along the following 60th passages in vitro. These cells were subcultivated to evaluate the possible contribution of these undifferentiated characteristics to the malignant progression phenotype. These characteristics were the expression of molecules involved in the processes of migration, dedifferentiation and chromosomal instability.

Results: Results showed that NG97(ht) had an decrease in doubling time through sub cultivation, which was characterized by a converse modulation between the expression of glial fibrillary acidic protein (GFAP) and vimentin. In addition, beta1 integrins were present in intermediate levels while alpha5 integrins had a high expression profile as well as fibronectin and laminin. Cytogenetic analysis of NG97(ht) revealed several chromosomal abnormalities, 89% of the cells showed to be hyperdiploid and the modal number was assigned to be 63. Several acrocentric chromosomes were visualized and at least 30 figures were attributed to be murine. These findings suggest a possible fusion between the original NG97 cells with stromal murine cells in the xenotransplant.

Conclusion: In this study the NG97(ht) cells were characterized to embryonic recovery patterns of intermediate filaments, adhesion molecules expression, chromosomal imbalances and murine chromosomes. In the latter case, these presumably chromosomes were originated as fusions between murine stroma cells and NG97 cell lineage in the xenotransplant. Our results emphasize important queries about astrocytomas tumor progression.

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GFAP and vimentin immunocytochemistry through cell subcultivation. Notice the negative modulatory expression pattern to GFAP (A – D) concomitant to the positive modulatory pattern to vimentin (E-H) in NG97(ht). The GFAP appears intensely marked in 100% of the 19th cell passage cytoplasm (A), being presented in approximately 85% of the 30th (B) and, finally, totally absent in 51st (C) and 80th (D) cell passages. Nonetheless, the vimentin were absent at 21st (E) cell passage, presented in weakly areas in the 40th (F) and in 54% of the 59th (G) cell passage and, finally, markedly positive at 83rd (H) cell passage (Original magnification: A – C and E – G = 200× and D and H = 1000×) (Positive and negative controls were assayed but were not displayed here).
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Figure 4: GFAP and vimentin immunocytochemistry through cell subcultivation. Notice the negative modulatory expression pattern to GFAP (A – D) concomitant to the positive modulatory pattern to vimentin (E-H) in NG97(ht). The GFAP appears intensely marked in 100% of the 19th cell passage cytoplasm (A), being presented in approximately 85% of the 30th (B) and, finally, totally absent in 51st (C) and 80th (D) cell passages. Nonetheless, the vimentin were absent at 21st (E) cell passage, presented in weakly areas in the 40th (F) and in 54% of the 59th (G) cell passage and, finally, markedly positive at 83rd (H) cell passage (Original magnification: A – C and E – G = 200× and D and H = 1000×) (Positive and negative controls were assayed but were not displayed here).

Mentions: This observed GFAP detection did not represent the functional molecular polymerized expression. These GFAP and vimentin cytoskeletons conformational arrangements were demonstrated by immunocytochemistry and suggested a modulation pattern through cell subcultivation. An intense cytoplasmatic staining for GFAP was observed in 100% of cells in the 19th cell passage (fig. 4A). The same pattern was present in approximately 85% of the NG97(ht) cells in the 30th (fig. 4B) and, finally, totally absent in 51st (fig. 4C) and 80th (fig. 4D) cell passages. Nevertheless, it was shown an absence of staining for cytoplasmatic vimentin in the 21st cell passage (fig. 4E), followed by weakly stained areas in the 40th NG97(ht) cell passage (fig. 4F). Approximately 54% of NG97(ht) cells in the 59th passage (fig. 4G) and, finally, a markedly positive staining in the 83rd cell passage (fig. 4H).


Characterization of cells recovered from the xenotransplanted NG97 human-derived glioma cell line subcultured in a long-term in vitro.

Machado CM, Ikemori RY, Zorzeto TQ, Nogueira AC, Barbosa SD, Savino W, Schenka AA, Vassallo J, Heinrich JK, Boetcher-Luiz F, Verinaud L - BMC Cancer (2008)

GFAP and vimentin immunocytochemistry through cell subcultivation. Notice the negative modulatory expression pattern to GFAP (A – D) concomitant to the positive modulatory pattern to vimentin (E-H) in NG97(ht). The GFAP appears intensely marked in 100% of the 19th cell passage cytoplasm (A), being presented in approximately 85% of the 30th (B) and, finally, totally absent in 51st (C) and 80th (D) cell passages. Nonetheless, the vimentin were absent at 21st (E) cell passage, presented in weakly areas in the 40th (F) and in 54% of the 59th (G) cell passage and, finally, markedly positive at 83rd (H) cell passage (Original magnification: A – C and E – G = 200× and D and H = 1000×) (Positive and negative controls were assayed but were not displayed here).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572634&req=5

Figure 4: GFAP and vimentin immunocytochemistry through cell subcultivation. Notice the negative modulatory expression pattern to GFAP (A – D) concomitant to the positive modulatory pattern to vimentin (E-H) in NG97(ht). The GFAP appears intensely marked in 100% of the 19th cell passage cytoplasm (A), being presented in approximately 85% of the 30th (B) and, finally, totally absent in 51st (C) and 80th (D) cell passages. Nonetheless, the vimentin were absent at 21st (E) cell passage, presented in weakly areas in the 40th (F) and in 54% of the 59th (G) cell passage and, finally, markedly positive at 83rd (H) cell passage (Original magnification: A – C and E – G = 200× and D and H = 1000×) (Positive and negative controls were assayed but were not displayed here).
Mentions: This observed GFAP detection did not represent the functional molecular polymerized expression. These GFAP and vimentin cytoskeletons conformational arrangements were demonstrated by immunocytochemistry and suggested a modulation pattern through cell subcultivation. An intense cytoplasmatic staining for GFAP was observed in 100% of cells in the 19th cell passage (fig. 4A). The same pattern was present in approximately 85% of the NG97(ht) cells in the 30th (fig. 4B) and, finally, totally absent in 51st (fig. 4C) and 80th (fig. 4D) cell passages. Nevertheless, it was shown an absence of staining for cytoplasmatic vimentin in the 21st cell passage (fig. 4E), followed by weakly stained areas in the 40th NG97(ht) cell passage (fig. 4F). Approximately 54% of NG97(ht) cells in the 59th passage (fig. 4G) and, finally, a markedly positive staining in the 83rd cell passage (fig. 4H).

Bottom Line: Results showed that NG97(ht) had an decrease in doubling time through sub cultivation, which was characterized by a converse modulation between the expression of glial fibrillary acidic protein (GFAP) and vimentin.In the latter case, these presumably chromosomes were originated as fusions between murine stroma cells and NG97 cell lineage in the xenotransplant.Our results emphasize important queries about astrocytomas tumor progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Institute of Biology, UNICAMP - Campinas, São Paulo, Brazil. camilalongomachado@gmail.com

ABSTRACT

Background: In order to elucidate tumoral progression and drug resistance, cultured cell lines are valuable tools applied on tumor related assays provided they are well established and characterized. Our laboratory settled the NG97 cell line derived from a human astrocytoma grade III, which started to develop and express important phenotypical characteristics of an astrocytoma grade IV after injection in the flank of nude mice. Astrocytomas are extremely aggressive malignancies of the Central Nervous System (CNS) and account for 46% of all primary malignant brain tumors. Progression to worse prognosis occurs in 85% of the cases possibly due to changes in cell tumor microenvironment and through biological pathways that are still unclear.

Methods: This work focused on characterizing the NG97 cell line specifically after being recovered from the xenotransplant, who maintained their undifferentiated characteristics along the following 60th passages in vitro. These cells were subcultivated to evaluate the possible contribution of these undifferentiated characteristics to the malignant progression phenotype. These characteristics were the expression of molecules involved in the processes of migration, dedifferentiation and chromosomal instability.

Results: Results showed that NG97(ht) had an decrease in doubling time through sub cultivation, which was characterized by a converse modulation between the expression of glial fibrillary acidic protein (GFAP) and vimentin. In addition, beta1 integrins were present in intermediate levels while alpha5 integrins had a high expression profile as well as fibronectin and laminin. Cytogenetic analysis of NG97(ht) revealed several chromosomal abnormalities, 89% of the cells showed to be hyperdiploid and the modal number was assigned to be 63. Several acrocentric chromosomes were visualized and at least 30 figures were attributed to be murine. These findings suggest a possible fusion between the original NG97 cells with stromal murine cells in the xenotransplant.

Conclusion: In this study the NG97(ht) cells were characterized to embryonic recovery patterns of intermediate filaments, adhesion molecules expression, chromosomal imbalances and murine chromosomes. In the latter case, these presumably chromosomes were originated as fusions between murine stroma cells and NG97 cell lineage in the xenotransplant. Our results emphasize important queries about astrocytomas tumor progression.

Show MeSH
Related in: MedlinePlus