This is a proposal to study the mechanism by which the chemopreventive agent, retinoic acid (RA) which is a form of vitamin A, regulates cell growth and differentiation to reverse a transformed phenotype to a differentiated, growth arrested phenotype. The proposed studies utilize an immature, uncommitted precursor cell, HL-60 human myeloblastic leukemia cells, which undergoes G0 arrest and either myeloid or monocytic differentiation when treated with RA or vitamin D3 (D3) respectively. RA causes activation of MAPK signaling in the process of inducing terminal myeloid differentiation and G0 arrest. The RA-induced MAPK pathway activation diverges from that of typical peptide hormone mitogenic RAF/MEK/ERK signaling in that activation is slow and long lived. Enhancing MAPK signaling enhances RA-induced differentiation while blocking it prevents RA-induced differentiation. MAPK signaling is thus necessary and rate limiting in RA-induced progressive phenotypic changes leading to terminal differentiation. An early response to RA is the induced expression of the BLR1 membrane receptor. BLR1 over expression by stable transfection causes RAF signaling and accelerates RA-induced myeloid differentiation and G0 arrest. In contrast, the BLR1 knock out by bialleilic homologous recombination fails to activate RAF, terminally differentiate or G0 arrest in response to RA. Ectopic expression of activated RAF in the knock out cells rescues their ability to differentiate and arrest in response to RA. BLR1 and its attendant RAF signaling are thus necessary and rate limiting for RA to induce differentiation and arrest. The hypothesis is that there is a BLR1 directed differentiation control point that acts at a certain point during progression of differentiation to control progress beyond it through RAF driven MAPK signaling to activate needed transcription factors and effect phenotypic conversion.
The specific aims are:
AIM 1 : Identify the BLR1 differentiation control point during progression of myeloid differentiation induced by RA by determining the differentiation markers that define it.
AIM 2 : Determine the ensemble of transcription factors that the BLR1 differentiation control point activates to cause progression of myeloid differentiation.
AIM 3 : Determine the specific RAF phosphorylation sites that the BLR1 differentiation control point uses to MAPK signal and cause the needed transcription factor and phenotypic changes. The results for HL-60 will be confirmed in U937 and NB4 as well. This will determine where RA-induced progressive phenotypic conversion depends on BLR1, the transcription factors controlled there, and the specific RAF phosphorylations directing these effects. This is the prototype of a paradigm where RA induces differentiation via hyperactive RAF signaling.
Chemoprevention of cancer by dietary factors can have a significant effect on dietary recommendations to reduce cancer incidence and also on cancer treatment. Retinoic acid is an active form of vitamin A that has chemopreventive effects, but its mechanism of action now appears to depend on cellular signaling processes customarily attributed to cancer promotion. The proposed research will show that manipulation of that signal by retinoic acid uses other hormones to change it from one that promotes neoplastic transformation to one that reverses transformation to a growth arrested, normal like cell, thereby providing a new way to enhance its chemopreventive effects.
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