Search

Mature human lymphocytes are unique targets of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in that vitamin D receptors (VDR) are not constitutively expressed, and specific cellular activation signals are required for both the up-regulation of VDR and establishment of reactivity to the lipophilic ligand. Treatment of B lymphocytes with the cytokine IL-4 (IL-4), in the absence of prior activation, induces a weak up-regulation of VDR expression but fails to generate vitamin D-responsive element (VDRE)-reactive nuclear protein complexes or to initiate the genomic transcription of 25-hydroxyvitamin D3 24-hydroxylase. Stimulation of B lymphocytes by either ligation of CD40 Ag or cross-linking the Ig receptor is also insufficient to render B lymphocytes responsive to 1 alpha,25(OH)2D3. However, this apparent lack of response to the secosterol can be overcome by stimulation of B lymphocytes with a combination of these cellular activation signals, which are sufficient to lead to G1 cell cycle progression. In the presence of 1 alpha,25(OH)2D3, cellular activation associated with stimulation of such a progression appears to be sufficient for the up-regulation of VDR message and protein and necessary for the establishment of VDRE binding complexes and the induction of 24-hydroxylase message. Furthermore, biologic functions are modulated, in that the hormone inhibits proliferation in a subset of the activated B cells. These observations suggest that reactivity to 1 alpha,25(OH)2D3 is tightly regulated in B lymphocytes, requiring specific signals for its initiation.

1,25-Dihydroxyvitamin D3 inhibits the proliferation of the chronic myelogenous leukemia cell line RWLeu-4 but not the resistant variant, JMRD3. Although these cells exhibit no detectable differences in the vitamin D receptor, alterations in the interaction of nuclear extracts with the osteocalcin-1,25-dihydroxyvitamin D3-response element are noted. It is shown herein that the 1,25-dihydroxyvitamin D3 receptor binds to the osteocalcin-1,25-dihydroxyvitamin D3-response element along with activator protein-1 (AP-1) complexes and that the DNA binding activities of members of the Jun and Fos proto-oncogene families, which make up the AP-1 transcription factor, are differentially regulated by 1,25-dihydroxyvitamin D3. It is shown that JunD DNA binding activity is enhanced by 1,25-dihydroxyvitamin D3 during cell cycle arrest in the sensitive cells but is decreased in the resistant cells. These results suggest that the level of JunD DNA binding activity may be a critical factor in the regulation of proliferation.

A variant of the chronic myelogenous leukemia cell line, RWLeu-4, that is resistant to the antiproliferative effects of vitamin D3 was established. Although RWLeu-4 proliferation is inhibited by 1 nmol/L vitamin D3, the resistant cells (JMRD3) continue to proliferate in the presence of 100 nmol/L vitamin D3. Both cells express similar patterns of differentiation-specific antigens after treatment with vitamin D3, and both express the retinoblastoma gene product (p110Rb). Vitamin D3 treatment of the sensitive RWLeu-4 cells decreased the level of the p110Rb protein, as well as its phosphorylation. In contrast, vitamin D3 treatment of JMRD3 had no effect on p110Rb expression or phosphorylation. Both RWLeu-4 and JMRD3 express similar vitamin D3 receptors and vitamin D3-inducible enzyme activities. Differences were detected in the DNA binding characteristics of the vitamin D3 receptors as determined by electrophoretic mobility shift studies. However, sequence analysis of the DNA-binding domain and immunoblot analysis showed no differences in the receptors. We conclude that some process subsequent to vitamin D3 receptor activation is altered in JMRD3 that partially separates vitamin D3-induced inhibition of proliferation from the induction of differentiation.

We evaluated the proliferative and differentiative effects of analogs of 1,25(OH)2 vitamin D3 [1,25(OH)2D3] on a chronic myelogenous leukemia cell line, RWLeu-4, which is growth-inhibited and differentiates in response to 1,25(OH)2D3 (ED50 = 3-10 nM). Side-chain-fluorinated analogs were more potent (ED50 = 0.7-2 nM) while most of those with altered saturation of the D ring or side-chain carbon-carbon bonds were equally or less effective than 1,25(OH)2D3. However, the two analogs with either two additional double bonds or an extra double and triple bond in the D ring had greater antiproliferative activity [1,25(OH)2-16,23-diene D3 (ED50 = 2.7 nM) and 1,25(OH)2-16-ene-23-yne D3 (ED50 = 0.7 nM)]. Since the latter of these has been reported to be less potent at mobilizing calcium than 1,25(OH)2D3, it (or a similar compound) may be a candidate for clinical use as an antineoplastic agent.

The effects of 1 alpha, 25-dihydroxyvitamin D3 (VD3) on proliferation, differentiation, and macromolecular synthesis in the new Philadelphia chromosome-positive chronic myelogenous leukemia cell line, RWLeu-4, were investigated. Binding of [3H]VD3 was saturable, with approximately 2000-3000 sites/cell, and half-maximal binding occurring at 0.21-0.33 nM. Treatment of RWLeu-4 cells with VD3 induced 24R-hydroxylase activity, a marker of vitamin D3 responsiveness in many tissues. Exposure of RWLeu-4 cells to VD3 also inhibited proliferation and DNA synthesis with a 50% effective dose of 3.5-10 nM within 72 h; in addition, protein and RNA synthesis were inhibited by VD3 treatment. Exposure of RWLeu-4 cells to 5 nM VD3 for 72 h caused 50% of the cells to differentiate into macrophage/monocyte type cells as judged by nitroblue tetrazolium staining and adherence to plastic. Progressive expression of cell surface maturation-specific antigens of the monocyte/macrophage lineage was induced by treatment of RWLeu-4 cells with VD3 for 24 to 72 h at doses that inhibited cellular proliferation. c-myc RNA, which is constitutively expressed in RWLeu-4 cells, increased after 0.5 h of treatment with 50 nM VD3 and then rapidly decreased to barely detectable levels after 4 h of treatment. Finally, the in vitro tyrosine kinase activity associated with the p210bcr-abl oncogene product was decreased approximately 50% by VD3 treatment. Because of the presence of a functional receptor-effector system for VD3 and multiple biological responses to the hormone, these cells provide a unique model system with which to probe the specific effects of VD3 on cell growth and differentiation in chronic myelogenous leukemia.