Presentation Title

Role of NF-kappaB pathway in RCAN1-dependent apoptosis of human leukemia cells

Faculty Mentor

Rheem D. Medh

Start Date

18-11-2017 12:30 PM

End Date

18-11-2017 1:30 PM

Location

BSC-Ursa Minor 83

Session

Poster 2

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Our laboratory is studying the molecular basis of glucocorticoid (GC) induced apoptosis and GC resistance in human leukemic CCRF-CEM sister cell lines. Upregulation of RCAN1 correlates with sensitivity to Dexamethasone, a synthetic GC used as an anti-leukemic agent. Studies suggest that RCAN1 interacts with the NFkB pathway to modulate its activity. NFkB, known to antagonize apoptosis, promotes cell survival and proliferation. To understand the role of RCAN1 in lymphoid cell apoptosis, and its effect on NFkB, we are employing RCAN1 knockdown/knockout leukemia cell lines along their wild-type counterparts. CRISPR-Cas9-mediated genome editing of GC-sensitive CEM C7-14 cells yielded heterozygous knockout in two distinct clones. Western blotting using specific antibodies for intermediates of the NFkB pathway including NFkB1, IkB and NIK evaluated correlation of their expression to that of RCAN1 in wild-type and knockout lines. Our data suggest that RCAN1 knockdown decreased the abundance of IkB, suggesting that RCAN1 may mediate IkB abundance via promoting its phosphorylation and subsequent degradation. NFkB1 (p50) abundance is also reduced in knockout cells, suggesting additional levels of RCAN1-mediated regulation of the NFkB pathway. Different dimeric combinations of p50 with other NFkB family proteins modulate transcriptional activation or repression; hence, studies are underway to understand the molecular crosstalk between the two pathways. To evaluate the effect of NFkB activity on RCAN1 expression, HEK293FT cells are being transfected with a construct harboring 1400bp of the RCAN1 promoter upstream of the Gussia Luciferase reporter, with Secreted Alkaline Phosphatase, an internal reference. Two inhibitors of the NFkB pathway, BV6 and Cardamonin, and an activator of NFkB, Betulinic acid are being used to modulate the NFkB pathway and study its effect on GC-dependent and independent RCAN1 promoter activity. Our data suggest that RCAN1 and NFkB pathways crosstalk to modulate each others’ activity, which may affect GC-evoked apoptosis of leukemia cells.

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Nov 18th, 12:30 PM Nov 18th, 1:30 PM

Role of NF-kappaB pathway in RCAN1-dependent apoptosis of human leukemia cells

BSC-Ursa Minor 83

Our laboratory is studying the molecular basis of glucocorticoid (GC) induced apoptosis and GC resistance in human leukemic CCRF-CEM sister cell lines. Upregulation of RCAN1 correlates with sensitivity to Dexamethasone, a synthetic GC used as an anti-leukemic agent. Studies suggest that RCAN1 interacts with the NFkB pathway to modulate its activity. NFkB, known to antagonize apoptosis, promotes cell survival and proliferation. To understand the role of RCAN1 in lymphoid cell apoptosis, and its effect on NFkB, we are employing RCAN1 knockdown/knockout leukemia cell lines along their wild-type counterparts. CRISPR-Cas9-mediated genome editing of GC-sensitive CEM C7-14 cells yielded heterozygous knockout in two distinct clones. Western blotting using specific antibodies for intermediates of the NFkB pathway including NFkB1, IkB and NIK evaluated correlation of their expression to that of RCAN1 in wild-type and knockout lines. Our data suggest that RCAN1 knockdown decreased the abundance of IkB, suggesting that RCAN1 may mediate IkB abundance via promoting its phosphorylation and subsequent degradation. NFkB1 (p50) abundance is also reduced in knockout cells, suggesting additional levels of RCAN1-mediated regulation of the NFkB pathway. Different dimeric combinations of p50 with other NFkB family proteins modulate transcriptional activation or repression; hence, studies are underway to understand the molecular crosstalk between the two pathways. To evaluate the effect of NFkB activity on RCAN1 expression, HEK293FT cells are being transfected with a construct harboring 1400bp of the RCAN1 promoter upstream of the Gussia Luciferase reporter, with Secreted Alkaline Phosphatase, an internal reference. Two inhibitors of the NFkB pathway, BV6 and Cardamonin, and an activator of NFkB, Betulinic acid are being used to modulate the NFkB pathway and study its effect on GC-dependent and independent RCAN1 promoter activity. Our data suggest that RCAN1 and NFkB pathways crosstalk to modulate each others’ activity, which may affect GC-evoked apoptosis of leukemia cells.