Protection of Ewing’s sarcoma family tumor (ESFT) cell line SK-N-MC from betulinic acid induced apoptosis by α-dl-tocopherol

ABSTRACT The chimeric fusion gene EWS/FLI-1 is detected in most cases of Ewing's sarcoma (ES), the second most common malignant bone tumor of childhood. Although 80% of ES tumors develop in skeletal sites, the remainder can arise in almost any soft tissue location. The lineage of the cell developing the EWS/FLI-1 gene fusion has not been fully characterized but is generally considered to be of either mesenchymal or neural crest origin. To study this oncogene in a conceptually relevant target cell, EWS/FLI-1 was introduced into the murine cell line C2C12, a myoblast cell line capable of differentiation into muscle, bone, or fat. In this cellular context, EWS/FLI-1 profoundly inhibited the myogenic differentiation program. The block in C2C12 myogenic differentiation required the nuclear localization and DNA-binding functions of EWS/FLI-1 and was mediated by transcriptional and posttranscriptional suppression of the myogenic transcription factors MyoD and myogenin. Interestingly, C2C12-EWS/FLI-1 cells constitutively expressed alkaline phosphatase, a bone lineage marker, and were alkaline phosphatase positive by histochemistry but showed no other evidence of bone lineage commitment. Consistent with recent findings in human ES tumor cell lines, C2C12-EWS/FLI-1 cells constitutively expressed cyclin D1 and demonstrated decreased expression of the cell cycle regulator p21cip1, even under differentiation conditions and at confluent density. This C2C12-EWS/FLI-1 cell model may assist in the identification of novel differentially expressed genes relevant to ES and provide further insight into the cell(s) of origin developing ES-associated genetic fusions.

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Dual Transforming Activities of the FUS (TLS)-ERG Leukemia Fusion Protein Conferred by Two N-Terminal Domains of FUS (TLS)

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7639 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7639-7650 ◽

Cited By ~ 23

Author(s):

Hitoshi Ichikawa ◽

Kimiko Shimizu ◽

Rieko Katsu ◽

Misao Ohki

Keyword(s):

Amino Acids ◽

Cell Line ◽

Ewing’S Sarcoma ◽

Ewing's Sarcoma ◽

Ectopic Expression ◽

Mouse Fibroblast ◽

Chimeric Proteins ◽

Dependent Manner ◽

Transforming Activity ◽

Nih 3T3

ABSTRACT The FUS (TLS)-ERG chimeric protein associated with t(16;21)(p11;q22) acute myeloid leukemia is structurally similar to the Ewing’s sarcoma chimeric transcription factor EWS-ERG. We found that both FUS-ERG and EWS-ERG could induce anchorage-independent proliferation of the mouse fibroblast cell line NIH 3T3. However, only FUS-ERG was able to inhibit the differentiation into neutrophils of a mouse myeloid precursor cell line L-G and induce its granulocyte colony-stimulating factor-dependent growth. We constructed several deletion mutants of FUS-ERG lacking a part of the N-terminal FUS region. A deletion mutant lacking the region between amino acids 1 and 173 (exons 1 to 5) lost the NIH 3T3-transforming activity but retained the L-G-transforming activity. On the other hand, a mutant lacking the region between amino acids 174 and 265 (exons 6 and 7) lost the L-G-transforming activity but retained the NIH 3T3-transforming activity. These results indicate that the N-terminal region of FUS contains two independent functional domains required for the NIH 3T3 and L-G transformation, which we named TR1 and TR2, respectively. Although EWS intrinsically possessed the TR2 domain, the EWS-ERG construct employed lacked the EWS sequence containing this domain. Since the TR2 domain is always found in chimeric proteins identified from t(16;21) leukemia patients but not in chimeric proteins from Ewing’s sarcoma patients, it seems that the TR2 function is required only for the leukemogenic potential. In addition, we identified three cellular genes whose expression was altered by ectopic expression of FUS-ERG and found that these are regulated in either a TR1-dependent or a TR2-dependent manner. These results suggest that FUS-ERG may activate two independent oncogenic pathways during the leukemogenic process by modulating the expression of two different groups of genes simultaneously.

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