Chromosomal heterogeneity and instability characterize pediatric medulloblastoma cell lines and affect neoplastic phenotype

Abstract

Chromosomal heterogeneity is a hallmark of most tumors and it can drive critical events as growth advantages, survival advantages, progression and karyotypic evolution. Medulloblastoma (MB) is the most common malignant central nervous system tumor in children. This work attempted to investigate chromosomal heterogeneity and instability profiles of two MB pediatric cell lines and their relationship with cell phenotype. We performed GTG-banding and cytokinesis-block micronucleus cytome assays, as well as morphological characterization, cell population doubling time, colony-forming efficiency, and chemo-sensitivity assays in two pediatric MB cell lines (UW402 and UW473). Both MB cells showed a high chromosomal heterogeneity. UW473 cells showed ~2 fold higher both clonal- and non-clonal chromosomal alterations than UW402 cells. Besides, UW473 showed two clonal-groups well-differentiated by ploidy level (<2n> and <4n>) and also presented a significantly higher number of chromosomal instability biomarkers. These results were associated with high morphological heterogeneity and survival advantages for UW473 and proliferation advantages for UW402 cells. Moreover, UW473 was significantly more sensitive to methotrexate, temozolomide and cisplatin while UW402 cells were more sensitive to doxorubicin. These data suggest that distinct different degrees of karyotypic heterogeneity and instability may affect neoplasic phenotype of MB cells. These findings bring new insights into cell and tumor biology.

Fig. 1

Karyotypic analysis of MB cell lines. a G-banded karyotype of the hyperdiploid MB cell line UW402, showing both complex structural and numerical changes. b–c Representative karyotypes of the MB cell line UW473 showing b hyperdiploid and c near-tetraploid karyotypic populations. Arrows point to main chromosomal alterations. M marker chromosome

Fig. 2

Determination of proliferative status and chromosomal instability phenotype of MB cells by CBMN-Cyt assay. a Frequencies of mono-, bi-, tri-, tetra-nucleated and apoptotic MB cells (per 500 cells scored). b Frequencies of binucleated MB cells with at least an MN, NPB, or NBUD (per 1,000 cells scored). Data represent mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. MN micronucleus, NPB nucleoplasmic bridge, NBUD nuclear bud

Fig. 3

Dominant presence of instability biomarkers in MB cells identified by CBMN-Cyt assay. a, f Binucleated cells presenting one MN, the main instability biomarker found in both MB cell lines. b, g Binucleated cells containing many NPBs. c, h Presence of NBUDs indicating extrusion of amplified DNA and/or DNA repair complex. d, i Simultaneous presence of MNi, NPBs, and NBUDs indicating a dominant chromosomal instability phenotype in a trinucleated UW402 cell and a binucleated UW473 cell. e, j Aberrant mitotic figures with anaphase bridges are identified in an apparent tetranucleated UW402 cell and a multipolar mitosis of UW473. In addition to anaphase bridges, lagging chromosomes are shown in the panels e and j. In all cases an ×100 oil objective and Giemsa stain were used. CBMN-Cyt cytokinesis-block micronucleus cytome assay, MNi micronuclei, NPBs nucleoplasmic bridges, NBUDs nuclear buds

Fig. 4

Cell morphology, proliferation and cell cycle distribution of MB cells. a UW402 showed a spindle-shaped population with long nervous extensions. b UW473 presented an heterogeneous morphology with groups of anaplastic and elongated smooth cells. Both cells lines showed a population characterized by multinucleated pyramidal shape cells. An ×100 oil objective and Giemsa stain were used. c, d Cell growth curves. e, f Histograms showing the percentages of cells in G1, S or G2/M phases

Fig. 5

Chemosensitivity differences between both MB cell lines. Cell lines were exposed to increasing concentrations of TMZ, CDDP, MTX and DX for 72 h and the proliferation was determined by XTT assay. The IC₅₀ value was defined as concentration required by each drug for a reduction of 50 % in the cell proliferation. Data represent mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. TMZ temozolomide, CDDP cisplatin, MTX methotrexate, DOX doxorubicin