In vitro transformation of mesenchymal stem cells by oncogenic H-rasVal12

Abstract

Tissue stem cells may serve as progenitors for malignant tumors derived from the same tissue. Here, we report the establishment of immortalized human mesenchymal stem cells (ihMSC) and tested the feasibility of using ihMSC as presarcomatous cells. Immortalization was achieved by introducing the genes for human telomerase reverse transcriptase and Bmi1. ihMSC retained the potential for multi-directional differentiation of the original MSC. To transform ihMSC, we introduced an oncogenic H-ras(Val12) gene, and established the cell line ihMSC-ras. ihMSC-ras had the phenotype of fully transformed cells and retained adipogenic and chondrogenic, but not osteogenic, potential. Interestingly, ihMSC-ras demonstrated morphological features of autophagy, and inhibition of the ERK pathway suppressed the production of autophagosomes, indicating that ras/ERK signaling is responsible for the induction of autophagy. Thus ihMSC will serve as a material with which to analyze the tumorigenic and differentiation-modifying effects of candidate oncogenes involved in the development of sarcomas.

Deficiency in p53 but not Retinoblastoma Induces the Transformation of Mesenchymal Stem Cells In vitro and Initiates Leiomyosarcoma In vivo

“…This observation suggests that the loss of accurate regulation of the cell cycle may be key in the transformation process. In fact, alterations in p16, p53, and p21 have been detected in transformed MSCs (23,24,42). We have previously reported that the loss of heterozygosity of p53 induced tumoral transformation in p21 −/− MSCs, and that this process was accompanied by karyotypic instability and the loss of p16 (26).…”

Modeling sarcomagenesis using multipotent mesenchymal stem cells

“…In one of these models, the process of transformation of hMSCs is associated with a gradual increase in genomic hypomethylation, although this phenomenon is not necessary for transformation [43]. Using an alternative approach, another group succeeded in transforming hMSCs through ectopic expression of hTERT, H-RAS and BMI-1, which inhibits the expression of genes controlled by polycomb response elements including p16 INK4A [44]. It was also reported that some hTERT-transduced hMSC lines lose contact inhibition, acquire anchorage-independent growth and form tumors in mice after long-term in vitro culture [45].…”

“…This transformation process was associated with the deletion of the Ink4a/ARF locus and with the acquisition of an activating mutation in K-RAS. Overall, in vivo tumors originated from most of these transformed hMSCs were classified as undifferentiated spindle cell sarcomas [41,42,44].…”

“…For instance, SOX2 has been reported to play a key role in the development of Ewing's sarcoma [62], whereas BMI1, which has been previously shown to be involved in sarcomagenesis [44], has also very recently been shown to be relevant in induced pluripotency [86].…”

Identification of AFAP1L1 as a prognostic marker for spindle cell sarcomas

“…ANOS, YaFuSS and an immortalized hMSC (ihMSC) line were established in our laboratory as described previously (Aoyama et al, 2004;Ishibe et al, 2005;Shima et al, 2007) Production of anti-AFAP1L1 polyclonal antibody The polyclonal antibody for AFAP1L1 was raised by immunizing rabbits with glutathione S-transferase-fused polypeptides corresponding to codon 35-113 of the human AFAP1L1 gene and purified with standard protocols using affinity columns.…”