Role of BTG2/TIS21/PC3 in differentiation of myeloid leukemia cells and its regulation under stress conditions

Mouse TIS21 (12-O-tetradecanoyl phorbol-13-acetate inducible sequence 21), an ortholog of human BTG2 (B-cell translocation gene 2) and rat PC3, is a tumor suppressor that belongs to antiproliferative gene family, and is implicated in a variety of biological processes. Deregulation of c-Myc transcription factor is common in leukemia and lymphomas; the tumors are highly proliferative and often blocked at an earlier phase than the terminal stage of differentiation. The interrelation and the functional interplay of these two different proteins are not defined yet. We have shown here that the tumor suppressor TIS21 negatively regulated c-Myc expression during all-trans-retinoic acid (ATRA)-induced differentiation that accelerated differentiation and reduced proliferation of acute promyelocytic leukemia HL-60 cells. TIS21 downregulated c-Myc mRNA and additionally decreased c-Myc protein stability by increasing its phosphorylation at S62 and T58 residues via activation of Erk1/2 and inhibition of PI3K/Akt along with subsequent activation of GSK-3β in response to low dose ATRA treatment. HL-60 cells treated with GSK-3β or proteosome inhibitors revealed marked accumulation of c-Myc both in the presence and absence of ATRA and TIS21, confirming ATRA plus TIS21 mediated c-Myc phosphorylation and its consequent degradation in proteosome. Immunoprecipitation assay revealed that TIS21 hindered interaction of p-Erk1/2 with Akt, thus directly regulating MAPK and Akt activities without interaction with c-Myc. These findings exhibit anticarcinogenic potential of TIS21 via downregulation of c-Myc expression during ATRA induced differentiation of HL-60 cells involving activation and deactivation of two major c-Myc regulators Erk1/2 and Akt, respectively. Recently, we have reported transient induction of Btg2 expression in response to oxidative damage; however, the regulatory mechanism was not explored. In the present study we revealed NF-κB as the upstream mediator involved in Btg2 transcription in response to cell stress challenges such as serum deprivation and oxidative stress i.e. H2O2, or doxorubicin treatments. We observed close interrelation between generation of reactive oxygen species (ROS), enhanced IκBα degradation, nuclear translocation of NF-κB(p65/RelA) and the significant increase of Btg2 expression independent of p53 status. ChIP analysis revealed an enrichment of RelA (p65) bound to the κB response element on Btg2 promoter in response to the cell stress challenges. Employing various inhibitors led to cytoplasmic accumulation of IκBα, decreased p65 nuclear translocation along with significant reduction of Btg2 expression. Generation of ROS was the common event mediating NF-κB activation and Btg2 transcription. Furthermore, PKC activation was also found to be a critical factor mediating ROS-mediated signals to NF-κB pathway that culminate on Btg2 regulation, and specifically PKC-δ was responsible for this regulation under oxidative stress. Serum deprivation-associated ROS generation bypassed PKC activation, however, regulated NF-κB-Btg2 cascade via MAPK activation. The present data imply that oxidative stress upregulates Btg2 expression via ROS-PKC-NFκB or ROSMAPK-NFκB cascade, independent of p53 status that in turn could be involved in mediating various biological phenotypes depending on the cellular context. NF-κB plays crucial roles in inflammation and immunity and its activation is an important event for macrophage differentiation both in vivo and in vitro. In contrast alltrans- retinoic acid (ATRA) induces granulocytic differentiation independent of NF-κB involvement. Here we report that NF-κB activation enhanced and switched ATRA inducedgranulocytic programme to macrophages. Serum withdrawal and LPS treatment dampened IκBα expression via MAPK activation and ATRA treatment further corroborated this effect in HL-60 cells. The data revealed that NF-κB activation diverted ATRA induced granulocytic differentiation to macrophages as confirmed by microscopic examination and assessing the macrophages specific markers CD68 and MMP9 along with high level of Btg2. Employing various inhibitors attenuated NF-κB associated enhanced cells maturation and differentiation switch thus suggesting that NF-κB determines the lineage specificity of ATRA induced differentiation of myeloid leukemia cells. MAPK activation is important both for granulocytic and macrophage differentiation and the data revealed that MAPK- NF-κB signaling was an important event in differentiation switch. The study shows that NF-κB plays an important role in determining lineage specificity of ATRA induced differentiation of myeloid leukemia cells.

Author dc:creator

• MUHAMMAD, IMRAN

Contributors dc:contributor

• 임, 인경
• 대학원 의생명과학과
• 200824427

• ATRA, all-trans-retinoic acid
• Btg2, B-Cell translocation gene 2
• GSK3, glycogen synthase kinase 3
• DCF, dichlorofluorescin diacetate
• LPS, Lipopolysaccharide
• NAC, N-acetyl cysteine
• PC3, pheochromocytoma cell-3
• ROS, reactive oxygen species
• TIS21(12-O-tetradecanoyl phorbol-13-acetate inducible sequence 21)

Language dc:language

en

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000016480
000000016480

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/10859