Title

E-cadherin Mediated Homotypic Cell–cell Interaction Confers Cytokine Independence in Human Leukemia Ut-7 Cell Line

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Date of Award

Fall 2017

Abstract

Acute myeloid leukemia (AML) is an aggressive hematopoietic stem cell cancer and 10,590 deaths from AML are estimated in 2017. The lack of knowledge regarding pathways that lead to cytokine independence of leukemia cell progression results in poor prognosis. Since cytokine signals parental leukemogenesis, we hypothesized that cytokine independence confers survival and proliferative advantages to AML progenitor cells. To test this hypothesis, we established a novel erythropoietin (Epo) independent UT-7/EN cell line from UT-7/Epo, an Epo-dependent megakaryocytic leukemia cell line. The UT-7/EN cells survived and proliferated without Epo whereas Epo was indispensable for the survival of parental UT-7/Epo cells (p < 0.0001). Moreover, UT-7/EN cells formed cell aggregates as they proliferated, suggesting the formation of cell-cell adhesion. The gene expression profiles of UT-7/Epo and UT-7/EN cells were compared using HumanHT-12 v4 BeadChip array and by validation with qRT-PCR. Validation of E-cadherin (CDH1) upregulation at both the transcriptional level by RT-qPCR and the translation level by immunoblot (3.7 fold) and immunostaining (p < 0.01) suggested E-cadherin mediated cell-cell interactions in UT-7/EN cells. Furthermore, UT-7/EN lost the characteristic of cell aggregates in calcium stripped media using EDTA (5mM) relative to untreated UT-7/EN, suggesting E-cadherin induced cell-cell interactions. Stabilization of cell adhesion complex by co-localization of β-catenin with E-cadherin was supported by immuno co-staining of β-catenin and E-cadherin. Additionally, Epo supplementation suppressed the E-cadherin gene expression significantly (p < 0.001) in UT-7/EN at the transcription level while concurrently upregulating CDH1 transcriptional repressor SNAIL2 (p < 0.01), whereas β-catenin gene expression remained non-significant at 24, 48, and 72 hours relative to un-supplemented UT-7/EN cells. Epo supplementation also significantly upregulated vimentin gene expression (p < 0.001) relative to Epo un-supplemented UT-7/EN cells. In summary, the results indicate that E-cadherin induced cell-cell interactions confer cytokine independence in UT-7/EN in the absence of Epo.

Advisor

Venu Cheriyath

Subject Categories

Biochemistry, Biophysics, and Structural Biology | Biology | Life Sciences

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