Inappropriate activation of the T-cell leukemia homeobox 1 (TLX1) gene by chromosomal translocation is a recurrent event in human T-cell acute lymphoblastic leukemia (T-ALL). Enforced expression of TLX1 in murine hematopoietic progenitor cells using a conventional retroviral vector yields immortalized cell lines and induced T-cell tumors in mice. We showed that in contrast to the cell lines established by insertional mutagenesis with empty vectors, none of the TLX1-immortalized cell lines contained insertions in Evi1 or Prdm16. The observations suggest that TLX1 and Evi1/Prdm16 subvert overlapping growth and survival pathways leading to cellular immortalization. Furthermore, we incorporated the TLX1 gene into a safety-modified self-inactivating retroviral vector to virtually eliminate insertional genotoxicity during transgene delivery. Post transduction, TLX1-expressing bone marrow cells had a growth/survival advantage, and readily gave rise to immortalized cell lines. These results confirm and extend our previous findings suggesting that constitutive TLX1 expression is sufficient to initiate the series of events culminating in progenitor cell immortalization. In addition, we demonstrated that continued TLX1 expression is necessary for the maintenance and survival of these cell lines by TLX1 shRNA knockdown experiments. We further showed that the TLX1-expressing cell lines have intact transcripts for p15/INK4b, p16/INK4a, and p19/ARF and encode wild-type p53. These results suggest the possibility of direct or indirect mechanisms of inactivation of these tumor suppressors by TLX1. We also found that diploid TLX1-expressing cells treated with a mitotic inhibitor bypass the mitotic checkpoint and progress toward aneuploidy, thereby supporting a role for TLX1 in the dysregulation of mitotic checkpoint control. Furthermore, under culture conditions for T-cell differentiation TLX1-immortalized progenitor cells exhibited T-lineage potential but were unable to upregulate Bcl11b expression and transit the DN2 stage. These results support other studies indicating that TLX1 oncogenic activity is partially due to its ability to inhibit T-cell differentiation. The pathogenesis of T-ALL is a multistep process involving the regulation of a variety of genes involved in differentiation, proliferation, and survival. This study investigated the molecular mechanism of TLX1-mediated cellular immortalization in an in vitro model in attempt to provide a better understanding of the early stages of TLX1-associated T-ALL development.
|Advisor:||Hawley, Robert G.|
|Commitee:||Lee, Norman H., Leitenberg, David, McCaffrey, Timothy A., Moody, Sally A.|
|School:||The George Washington University|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 72/06, Dissertation Abstracts International|
|Subjects:||Molecular biology, Oncology|
|Keywords:||Hematopoiesis, Immortalization, Leukemia, Oncogene, Stem cell, Tlx1|
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