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"Mechanisms of 1Ą, 25-Dihydroxyvitamin D3 -induced cell differentiation in human leukemia cells"

Jing Zhang
Molecular Pathology & Immunology Program
B.S. 2002, Sichuan University, P.R. China

Thesis Advisor: George P. Studzinski, M.D., Ph.D.
Department of Pathology and Laboratory Medicine

Thursday, September 23, 2010
MSB C-555, 9:30 A.M.


Exposure of acute myeloid leukemia (AML) cells to 1£,25-dihydroxyvitamin D3 (1,25D) or its analogs can mediate normalization of malignant cell phenotype to cells that resemble mature monocytes. Better understanding of the intrinsic molecular events of this cell maturation should be helpful to potential clinical applications. We now show that the enhancement of 1,25D-induced differentiation by a selective p38MAPK alpha/beta inhibitor SB202190 (SB) leads to increased expression of p38MAPK isoforms gamma and delta in AML cell lines and patients¡¦ blasts. Although the activating phosphorylations of p38MAPK are also increased as a result of exposure of the cells to SB, the kinase activity of p38MAPK alpha is blocked. A positive role of p38MAPKs in 1,25D-induced differentiation is shown by the inhibition of differentiation by antisense oligonucleotides to all p38MAPK isoforms. An early (6h) activation of MEK/ERK by SB exposure, followed by activation of JNK1/2 pathway and enhanced expression and/or activation of PU.1, ATF-2 differentiation-related transcription factors (TFs) are also revealed by this study. Their activation appears to result from the removal of feedback inhibition of an upstream regulator of those pathways, when p38MAPK alpha and beta are inhibited by SB.
The well-known limitation to the therapeutic use of 1,25D is its hypercalcemic effect. One approach that may abrogate this limitation is to combine 1,25D, or its higher potency but less calcemic analogs, with other agents such as plant-derived antioxidants (PAOX) that potentiate differentiation of low, non-toxic concentrations of deltanoids. We found that in four established human myeloid leukemia cell lines, HL60, U937, THP-1, NB4, one of the deltanoids JK-1624F2-2 (JKF), although a little less potent than 1,25D, when supplemented with an antioxidant, carnosic acid (CA) and SB increases the differentiation efficiency of JKF to a level similar to that observed when 1,25D is used in such combinations. This suggests that JKF is likely to provide a therapeutic advantage over 1,25D, used as a sole agent in regimens for myeloid leukemias. We also found that SB inhibits the phosphorylation of MAPKAPK-2 and Hsp27, downstream targets of p38MAPK, in at least two cell lines, but upregulates the phosphorylation of the isoform of JNK (p46 JNK1) and of c-jun, in all four cell lines studied. This indicates that the JNK1 pathway is positively associated with monocytic differentiation of several subtypes of myeloid leukemia cells arrested at different developmental stages.
To increase the significance of these studies, we translated the findings obtained using cell lines to primary cultures of AML cells from leukemic peripheral blood. We incubated freshly obtained blood cells from AML patients with another PAOX silibinin (SIL), alone or together with a vitamin D derivative. In 70% of the 20 patients with AML available for this study, SIL (60 £gM) either induced differentiation, or enhanced differentiation induced by deltanoids, or both, which was confirmed by qRT-PCR. Interestingly, SIL acting alone induced differentiation only in cases in which chromosome aberrations could not be detected. Western blot analysis demonstrated that SIL upregulated TFs such as members of jun and C/EBP families in deltanoid-induced monocytic differentiation.
In conclusion, it is shown here that p38MAPK pathway contributes to 1,25D-induced differentiation of AML cells, and that SB potentiates the effect of 1,25D by enhancing expression of p38MAPK isoforms as well as activity of JNK pathway and differentiation-related TFs. SIL can potentially serve as a differentiation enabling factor for blasts obtained from a large proportion of patients with AML.

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