1,25-dihydroxyvitamin D(3) regulation of c-myc expression and HL-60 cell differentiation: A role for PKC-beta and HOXB4

The hormone, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D 3], has been established as an important regulator of cellular proliferation and differentiation in various target tissues. HL-60 promyelocytic leukemia cells are terminally differentiated into mature monocytes/macrophages by 1,25-(OH) 2D3. Promotion of HL-60 cell differentiation involves programmed changes in the transcription of numerous genes, including protein kinase Cbeta (PKCbeta) and c-myc protooncogene. 1,25-(OH)2D 3 maximally increased PKCbeta levels and promoted HL-60 cell differentiation within 48--72 hours of continuous treatment. When PKCbeta levels and cell differentiation were assayed at 72 hours, treatment with 1,25-(OH) 2D3 for the initial 6 hours significantly increased PKCbeta levels but had little effect on cell differentiation. Therefore, continuous treatment with 1,25-(OH)2D3 is required for differentiation. Cell differentiation promoted by ionomycin, a calcium ionophore and activator of PKCbeta, in 1,25-(OH)2D3 pretreated cells was similar to differentiation promoted by continuous 1,25-(OH)2D3 treatment. The data suggest that the hormone must activate PKCbeta as well as increase its expression to promote cell differentiation fully. Additionally, the results reveal that 1,25-(OH)2D3 has increased sufficient de novo synthesis of PKCbeta after 6 hours to promote differentiation fully if activation cofactors, calcium and diacylglycerol, are increased. Next, the physiological relevance of three putative regulatory c-myc intron element protein binding sites (MIE1, MIE2, and MIE3) was determined. 1,25-(OH)2D3 enhanced nuclear proteins to bind to MIE1, MIE2, and MIE3. With a wildtype c-myc promoter construct containing MIE1, MIE2, and MIE3, 1,25-(OH)2D3 reduced CAT reporter expression. The ability of 1,25-(OH)2D3 to inhibit CAT activity was significantly decreased with a MIE1 deletion construct and completely abolished with a MIE1, MIE2, and MIE3 deletion construct. Chelerythrine chloride, a PKC activity inhibitor, and a PKCbeta antisense oligonucleotide, prevented the binding of nuclear proteins to MIE1, MIE2, and MIE3. The predominant MIE1 binding protein was purified, sequenced, and found to be HOXB4. A HOXB4 antisense oligonucleotide was able to significantly inhibit the induction of HOXB4 levels by 1,25-(OH)2D3. Moreover, this HOXB4 antisense was able to block 1,25-(OH)2D3 from decreasing c- myc levels. These results shows that HOXB4, a nuclear phosphoprotein, is expressed and activated by 1,25-(OH)2D3 leading to a block in c-myc transcription and HL-60 cell differentiation. In summary, this dissertation brings a better understanding of the mechanisms involved in terminal differentiation of a cancer cell and may provide the basis for novel therapeutic approaches to combat cancer.