H P Koeffler

The active metabolite of vitamin D known as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is a major physiologic regulator of mineral metabolism in man. The compound is also a potent inducer of differentiation of a human promyelocytic leukemia cell line known as HL-60. The induction of differentiation of myeloid leukemia cells to functional end cells offers an appealing therapeutic prospect. We investigated the ability of 1,25(OH)2D3 both to induce in vitro the differentiation of blast cells taken from patients with acute myelogenous leukemia and to improve hematopoiesis in vivo in patients with the myelodysplastic syndromes (preleukemia). We found that high concentrations (10-6 M) of 1,25(OH)2D3 significantly induced the in vitro differentiation of blast cells as measured by morphology, phagocytosis, and superoxide production. A concentration of 10-9 M 1,25(OH)2D3 had no effect on blast cell differentiation. We gave 2 microgram/day of 1,25(OH)2D3 to 18 patients with myelodysplastic syndrome (preleukemia) in an attempt to improve their hematopoiesis. During therapy, their peak peripheral blood granulocyte, platelet, and macrophage concentrations were slightly elevated as compared to their baseline, starting levels. Eight patients had a partial or minor peripheral blood response to the compound during the administration of 1,25(OH)2D3. However, no patient showed significant improvement of peripheral blood cell or marrow blast cell counts by the end of the study (greater than or equal to 12 weeks) as compared to their starting levels. Seven of the patients developed leukemia before or by 12 weeks of treatment. Nine of the 18 patients developed hypercalcemia. Taken together, the study shows that high concentrations (10-6M) of 1,25(OH)2D3 can induce differentiation of leukemia blast cells in vitro, but the administration of 1,25(OH)2D3 to patients with the myelodysplastic syndromes (preleukemia) does not have an enduring therapeutic effect. Hypercalcemia prevented administering greater amounts of 1,25(OH)2D3. In the future, the use of new vitamin D analogs that induce hematopoietic cell differentiation without inducing hypercalcemia might allow the achievement of higher blood levels of the inducing compound and might be medically useful for selected preleukemic and leukemic patients.

We have synthesized and studied the ability of a series of seven novel 1 alpha,25(OH)2 vitamin D3 analogues to inhibit clonal growth of prostate cancer cells (LNCaP, PC-3 and DU-145). Addition of double and triple bonds to the C/D ring (C-16) and side chain (C-22 and C-23) as well as lengthening of the side chain were important for enhanced activity against LNCaP and PC-3. Reorientation of the side chain in the 20-epi configuration resulted in analogues that were extremely potent only against LNCaP (ED50 approximately 5 x 10(-11) M). Compounds with six fluorines on the end of the side chain were very active against both PC-3 and LNCaP (ED50 approximately 2 x 10(-8) M). DU-145 cells were relatively resistant to compounds with all of these modifications, but removal of C-19 (e.g. 1,25(OH)2-16-ene-23-yne-26,27-F6-19-nor-D3) resulted in an analogue that was inhibitory against all three prostate cell lines. Further analysis showed that pulse exposure (3 days, 10(-7) M) to this analogue was enough to inhibit clonal growth of PC-3 cells by 50%. The same exposure also induced cell cycle arrest of all three cell lines, accompanied by upregulated protein expression of the cyclin-dependent kinase inhibitor (CDKI) known as p21waf1 in all three cell lines, and the CDKI known as p27kip1 in LNCaP cells. Associated with upregulation of these CDKIs, partial differentiation occurred as measured by increased expression of both prostate-specific antigen by LNCaP cells and E-cadherin, a cell adhesion protein that may act as a putative tumour suppressor (LNCaP and PC-3 cells). In summary, this is the first report of a potent series of 19-nor-vitamin D3 analogues with the ability to inhibit proliferation of LNCaP, PC-3 and DU-145 prostate cancer cell lines. These compounds may mediate their potent anti-proliferative activities through a cell cycle arrest pathway.

The p27/Kip1 protein belongs to the recently identified family of proteins called cyclin-dependent kinase inhibitors. These proteins play an important role as negative regulators of cell cycle-dependent kinase activity during progression of the cell cycle. Since cyclin-dependent kinase inhibitors can inhibit cell proliferation, they may have a role as tumor suppressor genes. To determine whether p27 alterations may be involved in tumorigenesis, we examined its mutational status in 36 primary breast carcinomas and 9 breast cancer cell lines using PCR-single-strand conformational polymorphism, direct DNA sequencing, and Southern blot analysis. Southern blot analysis showed no homozygous deletions of the p27 gene in either the clinical samples or cell lines. Two point mutations were found in primary tumors. One represents a previously undescribed polymorphism at codon 142; another is a nonsense mutation at codon 104. The latter mutation was absent in the normal matched control sample, and, in addition, it was accompanied with the loss of heterozygosity (LOH) of a microsatellite marker in the vicinity of the p27 gene on chromosome 12p13. These data indicate that p27 mutations are a rare event in breast cancer, but may play an important role in the development of a minority of these cancers. Furthermore, LOH analysis of the 12p13 locus revealed that an additional four of six matched DNA samples had LOH at 12p13 but did not have an alteration of the p27 gene, suggesting that another tumor suppressor gene is located on the short arm of human chromosome 12 which may be frequently involved in the pathogenesis of breast cancers.