JOURNAL OF BONE AND MINERAL RESEARCH Volume 6. Number 8, 1991 Mary Ann Liebert, Inc., Publishers
Nonhypercalcemic 1,25-(OH),D, Analogs Potently Induce the Human Osteocalcin Gene Promoter Stably Transfected into Rat Osteosarcoma Cells (ROSCO-2) NIGEL A. MORRISON and JOHN A. EISMAN
ABSTRACT 1,25-Dihydroxyvitamin D, [ 1,25-(OH),D,] is the active hormonal form of vitamin D, and has potent effects on bone and calcium regulation. Over the past decade it has become apparent that 1,25-(OH),D, has other effects on cellular proliferation that potentially could be developed for therapy in human malignancy. Since the hypercalcemic effects of 1,25-(OH),D, have limited that use in the human, novel nonhypercalcemic analogs of 1,25-(OH),D3 have been synthesized. The molecular mechanism of this divergence in these antiproliferative and calcium-regulating actions is unexplained. We have previously examined the human bone-specific gene osteocalcin as a model of the molecular mechanisms of vitamin D action in bone and have shown that induction of the osteocalcin gene by 1,25-(OH),D3 is mediated through an unique and complex palindromic region of the promoter similar to but distinct from those of other steroid hormone-responsive elements. Using an osteosarcoma cell line permanently transfected with the vitamin D-responsive promoter of the human osteocalcin gene linked to a “reporter” gene, we have shown that there is a dose-dependent induction of CAT activity by 1,25-(OH),D3 and that the potencies of vitamin D metabolites and analogs are comparable to those found in other vitamin D bioassays. Furthermore, vitamin D analogs, including MC-903, 22-oxa-1,25-(OH),D3, and A22-1,25S,26-trihydroxyvitaminD,, which effect cellular differentiation but lack hypercalcemic activity in vivo, exhibit osteocalcin promoter inductive actions virtually identical to those of 1,25-(OH),D,. Consideration of these and other data support the hypothesis that the divergent effects of such analogs on differentiation and calcium homeostasis reflect pharmacokinetic differences in vivo rather than distinct 1,25-(OH),D,-sensitive pathways.
INTRODUCTION HE ACTIVE HORMONAL FORM of vitamin D,, 1,25-dihydroxyvitamin D, [ 1,25-(OH),D,], has long been recognized as an important mediator of calcium and bone homeostasis. More recently it has become clear, however, that it has far more extensive effects on gene expression in a wide variety of tissues. For example, a little over a decade ago receptors for this hormone were reported in human breast cancer cells,‘1,21and subsequently receptors have been reported in a variety of human and animal cancer cell line^,(^-^) as reviewed in Ref. 10. Shortly thereafter, the effects of 1,25-(OH)*D, on inhibition of cellular repli-
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cation and induction of differentiation in vitro were reported.‘4-L3)In vivo inhibitory effects of 1,25-(OH),D3 or the la-hydroxyvitamin D, analog were reported on MI leukemic cells in syngeneic mice and on human colon cancer and melanoma cell xenografts in immune-suppressed mice.(14,151 The mechanisms of these effects on cell cycle kinetics(16-18) and at the cellular and molecular leve1‘19-231 have been studied. Although these active vitamin D, metabolites raise serum calcium levels at the doses effective in limiting cancer cell proliferation, this is not necessary for their effect. In studies in xenografted mice, the serum calcium levels were kept normal with a strict low-calcium diet and the inhibitory effect on xenograft growth paralleled
Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia,
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the 1,25-(OH),D3 receptor levels in the tumor cells.(1s1 Nevertheless, the limiting factor in terms of translating these potential anticancer effects into human therapy has been the potential toxic hypercalcemic effect of the active vitamin D metabolites at the dose levels required to mediate these effects in the human. In view of this limitation, considerable effort has been directed to developing nonhypercalcemic analogs of 1,25-(OH)’D,, and several new compounds have been shown to have a potent differentiating activity in ~ i t r o ~ ” - ’ ~ at) ;least one is active in vivo without significant hypercalcemic The mechanism of this divergence of effect on differentiation and calcium regulation has been the subject of considerable debate. Potential mechanisms have included differences in the molecular mechanisms of action of 1,25-(OH),D, and of these analogs in differentiation and calcium regulatory pathways. Inherent in this hypothesis is an insensitivity of calcium regulatory events to these analogs. We have used the human osteocalcin gene promoter as a model gene in calcium regulatory pathways because it is a bone-specific gene regulated by active vitamin D, metabolites through a specific vitamin D-responsive element (VDRE) in the gene promoter region.(291 In transient transfection this construct is half-maximally induced by 1,25-(OH)’D3 at M and saturated at lo-’ M, consistent with the dissociation constant K d of the vitamin D receptor. The specificity of this vitamin D-responsive element reflects the distinct role of this hormone in bone and calcium metabolism. It is a useful tool in examining the molecular mechanisms of action of vitamin D metabolites and analogs in a typical calcium regulatory gene. We established a permanently transfected rat osteosarcoma cell line containing an osteocalcin promoter-reporter construct and have found that the sensitivity of this promoter to various vitamin D, metabolites and analogs, including various fluorinated analogs, correlates with their biologic activity in vivo. Using this model we were then able to examine the ability of the nonhypercalcemic analogs to activate a vitamin D-responsive gene.
MATERIALS AND METHODS The rat osteoblastic sarcoma cell line, ROS 17/2.8, which has a osteoblast phenotype and expresses osteocalcin and mineralizes in ~ i t r o , ‘was ~ ~ kindly ’ supplied by Dr. S. Rodan (Merck, Sharpe and Dohme Bone Biology and Osteoporosis Research, West Point, PA). Various vitamin D, metabolites, fluorinated analogs, and the nonhypercalcemic analog A22-1,2SS,26-trihydroxyvitaminD3,(24)were generously supplied by Dr. M. Uskokovic (Hoffmann La Roche, Nutley, NJ). Calcipotriol (MC903)(25)was kindly supplied by Dr. L. Binderup (Leo Pharmaceuticals, Ballerup, Denmark) and Oxacalcitriol (22-oxa-1,25(OH)2D3]”6)was kindly supplied by Drs. J. Abe and Y. Nishii (Chugai Pharmaceuticals, Tokyo, Japan). The promoter expression construct (pOSCAT2) of the human osteocalcin gene, as previously described, lz9) was used in cotransfection with the neomycin resistance gene (pSV2 n e ~ ) ( ~into l ) ROS 17/23 cells. Transfections were done by the standard calcium phosphate coprecipitation
techniq~e‘~’] with the following modifications. Cells were cultured in Ham’s F12 medium (Flow Laboratories) with 5% fetal calf serum (FCS) to a density of 2 x 10’ cells in 150 cm’ flasks. The medium was changed 2 h before transfection to Dulbecco’s modified Eagle’s medium (DMEM) buffered with 25 mM HEPES, pH 7.2, and supplemented with 5% FCS. DNA precipitate (50 pg in 2 ml) was added to the cells, and 6 h later the cells exposed for 1 minute to 15% glycerol in DMEM containing 5% FCS. Transfected cells were replated after 24 h and left to grow in Ham’s F12 with 5% FCS for 3 days before Geneticin selection was applied with 500 p g h l of active Geneticin ((3418, GIBCO Laboratories, Sydney). Transfected cells were refed selective medium every 3 days and replated into smaller flasks. After 2 weeks the number of surviving cells had decreased to a level permitting replating into six-well trays, with large numbers of dead cells evident. After 4 weeks Geneticin selection no longer reduced cellular replication or plating efficiency, and this pool of Geneticin-resistant cells gave levels of chloramphenicol acetyltransferase (CAT) activity inducible by 1,25-(OH),D,. After a further 4 weeks to ensure that wildtype cells were excluded, Geneticin was no longer included in the culture medium. These cells, which were designated ROSCO-2, have been cultured for 2 years in the absence of Geneticin and have maintained the same level of CAT activity. ROSCO-2 CAT activity is induced by 1,25-(OH),D3 and retinoids and repressed by glucocorticoids in precisely the same manner as we have previously reported by transient transfection with pOSCAT2 in ROS 17/2.8 cell^.^'^^ The neomycin-resistant cells were subcultured and replated into six-well trays (10 cm’ per well) at 2 x lo5 cells per well in HEPES-buffered DMEM with 2% dextran charcoal-stripped fetal calf serum (ChFCS) for hormone and analog sensitivity studies. After plating down, cells were treated in triplicate with either ethanol vehicle (control) or metabolite or analog. After 24 h the cells were harvested by a brief trypsinization, washed in normal saline, and resuspended in 100 pl of 0.25 M Tris-HCI, pH 7.8, before lysis by three cycles of freeze-thaw in liquid nitrogen. CAT activity was measured by a sensitive nonchromatographic CAT assay as previously described.(291 The activity is expressed as the transfer to chloramphenicol (2 mM) of “C-labeled acetyl groups from “C-acetyl CoA, 2 x lo5 dpm per 100 pl (52 Ci/mol, Amersham), and 80 pM unlabeled acetyl CoA at 37°C. The basal CAT in ROSCO-2 cells of about 2 nmol per lo5 cells per h is not affected by estrogen, progesterone, or thyroid hormones.
RESULTS In the permanently transfected ROSCO-2 cells, various vitamin D, metabolites induced CAT activity with a range of potencies (Fig. 1). 1,25-(oH)’D3 was the most active metabolite tested. The induction by 1,25-(OH)D, was reproducibly detected at lo-” M and maximal at 10-9-19-0 M, with a decline at lo-’ M presumably due to cytotoxicity. The results of several different experiments are normalized to the basal (uninduced) level and combined to examine the relative potencies of other metabolites and ana-
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FIG. 3. Regulation of the osteocalcin promoter expression vector by nonhypercalcemic analogs of 1 ,25-(OH),D, in ROSCO-2 cells. The nonhypercalcemic analogs MC903, 22-oxa-1,25-(OH),D, (22-oxa-1,25-D3), and A22-1 ,2S-(OH),D, (D-22-1,25-D3) were tested over a dose range from lo-” to lo-’ M. All three analogs are approximately equipotent with I,2S-(OH),D3 (1,25-D3) both in terms of dose response and maximal response. Values are means f SEM of activity, expressed relative to basal (uninduced) activity, from three to eight separate wells from two to four experiments.
relates to their resistance to catabolism by side-chain-diAn alternative possibility for the divergence of the calrected oxidative reactions. (34) cium regulatory and differentiating functions is that the inThe full potency of the nonhypercalcemic analogs of teraction of these agents with intestinal receptor may be 1,25-(OH),D3 in terms of activating the osteocalcin pro- subtly different to their interaction with bone vitamin D moter cannot be explained by any receptor or postreceptor receptor. However, any difference would need to be a interaction in bone cells. 1 ,25-(OH),D3 induces its own me- major change to explain their virtual lack of calcium mobitabolism and changes the half-life of its r e ~ e p t o r . ‘ ~ ’ , lizing ~ ~ ) activity even 3 orders of magnitude higher doses However, these mechanisms generally limit the activity of than for 1,25-(OH),D,. This seems most unlikely given the the native hormone, and if they played any such role in the strong similarities between the activity of various vitamin lack of activity of these analogs, this should be seen in this D, metabolites and analogs in binding to the chick intestiintact cell system. It is possible that some postreceptor nal receptor binding and regulation of human mechanism, such as stabilization of mRNA, may be modi- and animal cancer cell lines,(10.19.21) and now the human fied by receptor activated by 1,25-(OH),D,, which the ana- osteocalcin gene promoter activation system reported here. logs cannot reproduce. However, this would need to be Thus at the present time there have been no data suggestspecific since the present system would be subject to any ing any difference either in the receptors in calcium regulageneral mechanism. A mechanism acting in vivo on spe- tory pathways, such as bone and intestine, or in differenticific mRNA species, such as the native osteocalcin mRNA, ation-related pathways. As yet there is no comparable might not be detectable in this in vitro system. However, gene-cell system to evaluate the action of these analogs on other groups have reported in abstract form that one of a calcium-related gene in intestinal cells in vitro. Morethese analogs, Calcipotriol, is approximately equipotent over, Oxacalcitriol in vitro acts in another calcium regulawith 1,25-(OH),D, in the stimulation of osteocalcin syn- tory pathway in that it suppresses rat parathyroid hormone thesis by human osteosarcoma cells in More- synthesis and secretion equipotently with 1 ,25-(OH),D3‘421 over, in a recent study on osteocalcin synthesis in human and continuous infusions are able to elevate calcium in roosteoblasts, MC903 and 1,25-(OH),D, were equipotent dent models (Mawer, personal communication). over 24-28 h i n c ~ b a t i o n s . ‘ ~ At~ )longer times, however, A potentially important mechanism relates to major when catabolism could be a factor, MC903 was less active pharmacokinetic differences between 1 ,25-(OH),D, and on osteocalcin induction, yet the two compounds were the nonhypercalcemic analogs. Since both Oxacalcitriol equally active in stimulation of DNA synthesis. These data and Calcipotriol have relatively low affinity for human support the concept that distinct effects on cell replication serum vitamin D binding protein, they achieve higher peak and calcium regulation could not be operating through a free levels and have markedly shortened serum half-lives postreceptor mechanism in these in vitro bone cell systems. (less than 1 h) compared with 1,25-(OH)2D3.(43,44) Because
NONHYPERCALCEMIC CALCITRlOL ANALOGS INDUCE OSTEOCALCIN the nonhypercalcemic analogs achieve high free levels only transiently, they may be unable to sustain activation of calcium-regulatory pathways, which by their nature are continuously variable responses. A reduced in vitro half-life mechanism for a lower effect on calcium regulatory pathways has been demonstrated for 24-epi- 1,25-dihydroxyergocalciferol. ( 4 4 . 4 5 ) In contrast with the nonhypercalcemic analogs, 1,25-(OH),D3 achieves more sustained but lower free levels and thus may be able to maintain the induction of genes responsible for calcium regulation, such as calcium transport in the intestine, which are inherently reversible processes. On the other hand, cell replication and differentiation are essentially irreversible steps in the biology of a cell, quite distinct from the reversible modulation of a physiologic function of a differentiated cell. In this model the transient but high free level of a 1,25-(OH)2D3analog could initiate an irreversible differentiation step or “switch” in a cell’s biology yet be unable to maintain activation of the inherently reversible calcium regulatory pathways. Irrespective of the mechanism of these differences, it is clear that the nonhypercalcemic analogs we have evaluated, which have potential for antiproliferative actions in vivo, d o not have abnormal interactions with the 1,25(OH),D3 receptor or abnormal postreceptor mechanisms in a well-characterized gene closely regulated in relation to bone and calcium homeostasis. At present there is no evidence of any difference in vitamin D receptors for the differentiation and calcium regulatory pathways. The data presented here complement previous data in this area but focus on the molecular mechanisms involved. Thus at least two of the nonhypercalcemic analogs analyzed here have the in vivo pharmacokinetics described, and all have low affinity to the serum vitamin D binding protein (R. Bouillon, personal communication). All three nonhypercalcemic analogs studied here have virtually identical activating potency on the stably transfected human osteocalcin gene promoter. Pharmacokinetic differences, which can explain these differential effects, may be an important component of the design and evaluation of these and other agents, such as altered side-chain-length analogs of 1,25(OH)2D3.(46-48) Success of the effort to develop analogs with differential effects in various calcium and differentiation pathways depends in part on adequate understanding of their molecular mechanisms of action. The ROSCO-2 cells can be a valuable tool for investigating these molecular mechanisms.
ACKNOWLEDGMENTS The valuable technical contributions of Rama Vedam, Judith Flanagan, and Susan Gillies are acknowledged. This work was supported by a Centre Grant to the Garvan Institute from the National Health and Medical Research Council and by the New South Wales State Cancer Council. This work was presented in part at the Sydney International Bone Symposium (April 1990) and the American Society for Bone and Mineral Research meeting, Atlanta, Georgia (September 1990).
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REFERENCES 1. Eisman JA, Martin T J , Maclntyre I , Moseley JM 1979 1,25Dihydroxyvitamin D receptors in breast cancer cells. Lancet 2: 1335- 1336. 2. Eisman JA, Martin T J , Maclntyre I , Frampton RJ, Moseley JM, Whitehead R 1980 1,25-Dihydroxyvitamin D, receptor in a cultured human breast cancer cell line (MCF-7). Biochem Biophys Res Commun 93:9-15. 3. Findlay DM, Michelangeli VP, Eisman JA. Frampton RJ, Moseley JM, Maclntyre I , Whitehead R, Martin TJ 1980 Calcitonin and 1,25-dihydroxyvitamin D, receptors on human breast cancer cell lines. Cancer Res 40:4764-4769. 4. Frampton RJ, Suva LJ, Eisman JA, Findlay DM, Moore GE, Moseley JM, Martin TJ 1982 Presence of 1,25 dihydroxyvitamin D, in established human cancer cell lines in culture. Cancer Res 42:1l16-1119. 5. Colston K, Colston MJ, Feldman D 1981 1.25-Dihydroxyvitamin D, and malignant melanoma: The presence of receptors and inhibition of cell growth in culture. Endocrinology 108: 1083-1086. 6. Freake HC, Marcocci C , Iwasaki J , Maclntyre I 1981 1.25-Dihydroxyvitamin D, specifically binds to a human breast cancer cell line (T47D) and stimulates growth. Biochem Biophys Res Commun 101:1131-1138. 7. Abe E, Miyaura C , Sakagami H, Takeda M, Konno K , Yamazaki T, Yoshiki S, Suda T 1981 Differentiation of mouse myeloid leukaemia cells induced by la-25-dihydroxyvitamin D,. Proc Natl Acad Sci USA 78:4990-4994. 8. Bhalla AK, Amento AP, Clemens TL, Hollick MF, Krane SM 1983 Specific high-affinity receptors for 1,25 dihydroxyvitamin D, in human peripheral blood mononuclear cells: Presence in monocytes and induction in T lymphocytes following activation. J Clin Endocrinol Metab 57:1308. 9. Brehier A, Thomasset M 1988 Human colon cell line HT-29: Characterisation of 1,25-dihydroxyvitamin D, receptor and induction of differentiation by the hormone. J Steroid Biochem 29:265-270. 10. Eisman J A 1983 1.25-Dihydroxyvitamin D, receptor and role of 1,25-dihydroxyvitamin D, in human cancer cells. In: Kumar R (ed.) Vitamin D Metabolism: Basic and Clinical Aspects, Martinus Nijhoff, The Hague, pp. 365-385. 1. Miyaura C , Abe E, Kuribayashi T , Tanaka H , Konno H , Nishii Y, Suda T 1981 1,25-Dihydroxyvitamin D, induced differentiation of human myeloid leukaemia cells. Biochem Biophys Res Commun 102:937-943. 2. Tanaka H , Abe E, Miyaura C, Kuribayashi T, Konno K, Nishii Y, Suda T 1982 1,25-Dihydroxycholecalciferoland a human myeloid leukaemia cell line (HL-60): The presence of a cytosol receptor and induction of differentiation. Biochem J 204~713-719. 13. Frampton RJ, Omond SA, Eisman J A 1983 Inhibition of human cancer cell growth by 1,25 dihydroxyvitamin D, metabolites. Cancer Res 43:4443-4447. 14. Honma Y, Hozumi M, Abe E, Konno K, Fukushima M, Hata S, Nishil Y, DeLuca HF, Suda T 1983 1 Alpha, 25 dihydroxyvitamin D, and 1 alpha hydroxyvitamin D, prolong survival time of mice inoculated with myeloid leukemia cells. Proc Natl Acad Sci USA 80:201-204. 15. Eisman JA, Barkla DH, Tutton PJM 1987 Suppression of in vivo growth of human cancer solid tumor xenografts by 1,25(OH),D,. Cancer Res 47:21-25. 16. Rigby WF, Noelle RJ, Krause K, Fanger MW 1985 The effects of 1,25-dihydroxyvitamin D, on human T lymphocyte activation and proliferation: A cell cycle analysis. J lmmunol 135:2279-2286.
898 17. Studzinski GP, Bhandal AK, Brevli ZS 1985 Cell cycle sensitivity of HL-60 cells to the differentiation-inducing effects of 1-alpha, 25-dihydroxyvitamin D,. Cancer Res 45:3898-3905. 18. Eisman JA, Sutherland RL, McMenemy ML, Fragonas J-C, Musgrove EA, pang GYN 1989 Effect of 1.25-dihydroxyvitamin D, on cell-cycle kinetics of T 47D human breast cancer cells. J Cell Physiol 138:611-616. 19. Reitsma PH, Rothberg PG, Astrin SM, Trial J, Bar-Shavit 2, Hall A, Teitelbaum SL, Kahn AJ 1983 Regulation of myc gene expression in HL-60 leukaemia cells by a vitamin D metabolite. Nature 306:492-494. 20. Koga M,Eisman JA, Sutherland RL 1988 Regulation of epidermal growth factor receptor levels by 1,25-dihydroxyvitamin D, in human breast cancer cells. Cancer Res 48:27342739. 21. Ravid A, Koren R, Novorodsky A, Liberman UA 1984 1,25Dihydroxyvitamin D, inhibits selectively the mitogenic stimulation of mouse medullary thymocytes. Biochem Biophys Res Commun 123:163. 22. Lemire JM, Adams JS, Sakai R, Jordon SC 1984 1 Alpha, 25 dihydroxyvitamin D, suppresses proliferation and immunoglobulin production by normal human peripheral blood mononuclear cells. J Clin Invest 74:657. 23. Tsoukas CD, Provvedini DM, Manolagas SC 1984 1.25-Dihydroxyvitamin D,: A novel immunoregulatory hormone. Science 224:1438. 24. Wovkulich PM, Batcho AD, Baggiolini EG, Boris A, Truitt G, Uskokovic MR 1985 Synthesis of 1,25S,26-trihydroxy-A22-cholecalciferol, a potent inducer of cell differentiation. In: Norman AW, Schaefer K, Grigoleit H-G, von Herrath D (eds.) Vitamin D, Chemical Biochemical and Clinical Update. Walter de Gruyter, Berlin, pp. 755-764. 25. Binderup L, Bramm E 1988 Effects of a novel vitamin D analogue MC903 on cell proliferation and differentiation in vitro and on calcium metabolism in vivo. Biochem PharmacOI 37:889-895. 26. Abe J, Takita Y, Nakano T, Miyaura C, Suda T, Nishii Y 1989 A synthetic analogue of vitamin D,, 22-oxa-I-alpha,25dihydroxyvitamin D,, is a potent modulator of in vivo immunoregulating activity without inducing hypercalcemia in mice. Endocrinology 124:2645-2647. 27. Kragballe K, Beck HI, SBgaard H 1988 Improvement of psoriasis by a topical vitamin D, analogue (MC 903) in a doubleblind study. Br J Dermatol 119:223-230. 28. Staberg B, Roed-Petersen J, Menne T 1989 Efficacy of topical treatment in psoriasis with MC903, a new vitamin D analogue. Acta Derm Venereol (Stockh) 69:147-150. 29. Morrison NA, Shine J, Fragonas JC, Verkest V, McMenemy LM, Eisman JA 1989 1,25-Dihydroxyvitamin D responsive element and glucocorticoid repression in the human osteocalcin gene. Science 246:1158-1161. 30. Rodan GA, Rodan SG 1984 Expression of the osteoblastic phenoype. In: WA Peck (ed.) Advances in Bone and Mineral Research, Annual 11. Amsterdam: Excerpta Medica, pp. 244-285. 31. Southern PJ, Berg P 1982 Transformation of mammalian cells to antibiotic resistance with bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1:327341. 32. Wigler M, Perucho M, Kurtz D, Dana S, Pellicer A, Axel R, Silverstein S 1980 Transformation of mammalian cells with an amplifiable dominant-acting gene. Proc Natl Acad Sci
MORRISON AND EISMAN USA 77:3567-3570. 33. Eisman JA, DeLuca HF 1977 Intestinal 1,25-dihydroxyvitamin D, binding protein: Specificity of binding protein. Steroids 30:245-257. 34. Eisman JA, Frampton RJ, McLean FJ 1986 Biochemical significance of enhanced activity of fluorinated 1,25-dihydroxyvitamin D, in human cultured cell lines. Cell Biochem Func 4: 115-121. 35. Inaba M, Okuno S, lnoue A, Nishizawa Y, Morii H, DeLuca HF 1989 DNA binding property of vitamin D, receptors associated with 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D,. Arch Biochem Biophys 268:35-39. 36. Yukioka K, Otani S, Matsui-Yuasa I, Goto H, Morisawa S, Okuno S, Inaba M, Nishizawa Y, Morii H 1988 Biological activity of 26,26,26,27,27,27-hexafluorinatedanalogs of vitamin D, in inhibiting interleukin-2 production by peripheral blood mononuclear cells stimulated by phytohemagglutinin. Arch Biochem Biophys Mo:45-50. 37. Eisman JA, Fragonas JC, McMenemy ML 1988 Rapid turnover of the 1,25-dihydroxyvitamin D, receptor in human target cells. Endocrinology 122:1613-1621. 38. Reinhardt TA, Horst RL 1989 Self-induction of 1,25-dihydroxyvitamin D, metabolism limits receptor occupancy and target tissue responsiveness. J Biol Chem 264:15917-15921. 39. Valaja T, Mahonen A, Pirskanaen A, Maenpaa PH 1990 Affinity of a novel vitamin D analog MC903 for vitamin D receptor and effects of the analog on the synthesis of osteocalcin by human osteosarcoma cells (abstract 732). J Bone Miner Res 4:S300. 40. Evans DB, Thavarajah M, Binderup L, Kanis JA 1990 Comparison of the action of MC903 and 1,25-(OH),D, on human osteosarcoma cells in vitro (abstract 868). J Bone Miner Res 45334. 41. Marie PJ, Connes D, Hott M, Miravet L 1990 Comparative effects of a novel vitamin D analogue MC-903 and 1,25-dihydroxyvitamin D, on alkaline phosphatase activity, osteocalcin and DNA synthesis by human osteoblastic cells in culture. Bone 11:171-179. 42. Brown AJ, Ritter CR, Finch JL, Morrissey J, Martin KJ, Murayama E, Nishii Y, Slatopolsky E 1989 The noncalcemic analogue of vitamin D, 22-oxacalcitriol, suppresses parathyroid hormone synthesis and secretion. J Clin Invest 84:728732. 43. Sorenson H, Binderup L, Calverley MJ, Hoffmeyer L, Andersen NR 1990 In vitro metabolism of Calcipotriol (MC903). a vitamin D analogue. Biochem Pharmacol 39:391-393. 44. Nishii Y, Okano T, Tsugawa N, Masuda S, Takeuchi A, Kobayashi T, Abe J, Yakita Y, Nakano T 1990 Recent advances in 22-oxacalcitriol (OCT) and a reason why OCT is different from multifunctional vitamin D, calcitriol (abstract). Proceedings of the Sydney Bone Symposium, Fifth lnternational Symposium on Bone, Structure, Function and Disease, April 1990, p. 32. Bone 10. 45. DeLuca HF, Sicinski RR, Tanaka Y, Stern PH, Smith CM 1988 Biological activity of 1,25-dihydroxyvitarnin D, and 24epi-l,25-dihydroxyvitaminD,. Am J Physiol 254:E402-406. 46. Reinhardt TA, Ramberg CF, Horst RL 1989 Comparison of receptor binding, biological activity, and in vivo tracer kinetics for 1,25-dihydroxyvitamin D,, 1,25-dihydroxyvitamin D,, and its 24 epimer. Arch Biochem Biophys 273:64-71. 47. DeLuca HF, Ostrem VK 1988 Analogs of the hormonal form
NONHYPERCALCEMIC CALCITRIOL ANALOGS INDUCE OSTEOCALCIN of vitamin D and their possible use in leukemia. Prog Clin Biol Res 259:41-55. 48. Gill HS, Londowski JM, Corradino RA, Zinsmeister AR, Kumar R 1988 The synthesis and biological activity of 25-hydroxy-26,27-dimethylvitaminD, and 1,25-dihydroxy-26,27dimethylvitamin D,: Highly potent novel analogs of vitamin D,. J Steroid Biochem 31:147-160.
Address reprint requests to: J.A. Eisman Garvan Institute of Medical Research St. Vincent’s Hospital Sydney, New South Wales 2010, Australia Received for publication December 3, 1990; in revised form February 7, 1991; accepted February 9, 1991.
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Nonhypercalcemic 1,25-(OH),D, Analogs Potently Induce the Human Osteocalcin Gene Promoter Stably Transfected into Rat Osteosarcoma Cells (ROSCO-2) NIGEL A. MORRISON and JOHN A. EISMAN
ABSTRACT 1,25-Dihydroxyvitamin D, [ 1,25-(OH),D,] is the active hormonal form of vitamin D, and has potent effects on bone and calcium regulation. Over the past decade it has become apparent that 1,25-(OH),D, has other effects on cellular proliferation that potentially could be developed for therapy in human malignancy. Since the hypercalcemic effects of 1,25-(OH),D, have limited that use in the human, novel nonhypercalcemic analogs of 1,25-(OH),D3 have been synthesized. The molecular mechanism of this divergence in these antiproliferative and calcium-regulating actions is unexplained. We have previously examined the human bone-specific gene osteocalcin as a model of the molecular mechanisms of vitamin D action in bone and have shown that induction of the osteocalcin gene by 1,25-(OH),D3 is mediated through an unique and complex palindromic region of the promoter similar to but distinct from those of other steroid hormone-responsive elements. Using an osteosarcoma cell line permanently transfected with the vitamin D-responsive promoter of the human osteocalcin gene linked to a “reporter” gene, we have shown that there is a dose-dependent induction of CAT activity by 1,25-(OH),D3 and that the potencies of vitamin D metabolites and analogs are comparable to those found in other vitamin D bioassays. Furthermore, vitamin D analogs, including MC-903, 22-oxa-1,25-(OH),D3, and A22-1,25S,26-trihydroxyvitaminD,, which effect cellular differentiation but lack hypercalcemic activity in vivo, exhibit osteocalcin promoter inductive actions virtually identical to those of 1,25-(OH),D,. Consideration of these and other data support the hypothesis that the divergent effects of such analogs on differentiation and calcium homeostasis reflect pharmacokinetic differences in vivo rather than distinct 1,25-(OH),D,-sensitive pathways.
INTRODUCTION HE ACTIVE HORMONAL FORM of vitamin D,, 1,25-dihydroxyvitamin D, [ 1,25-(OH),D,], has long been recognized as an important mediator of calcium and bone homeostasis. More recently it has become clear, however, that it has far more extensive effects on gene expression in a wide variety of tissues. For example, a little over a decade ago receptors for this hormone were reported in human breast cancer cells,‘1,21and subsequently receptors have been reported in a variety of human and animal cancer cell line^,(^-^) as reviewed in Ref. 10. Shortly thereafter, the effects of 1,25-(OH)*D, on inhibition of cellular repli-
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cation and induction of differentiation in vitro were reported.‘4-L3)In vivo inhibitory effects of 1,25-(OH),D3 or the la-hydroxyvitamin D, analog were reported on MI leukemic cells in syngeneic mice and on human colon cancer and melanoma cell xenografts in immune-suppressed mice.(14,151 The mechanisms of these effects on cell cycle kinetics(16-18) and at the cellular and molecular leve1‘19-231 have been studied. Although these active vitamin D, metabolites raise serum calcium levels at the doses effective in limiting cancer cell proliferation, this is not necessary for their effect. In studies in xenografted mice, the serum calcium levels were kept normal with a strict low-calcium diet and the inhibitory effect on xenograft growth paralleled
Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia,
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the 1,25-(OH),D3 receptor levels in the tumor cells.(1s1 Nevertheless, the limiting factor in terms of translating these potential anticancer effects into human therapy has been the potential toxic hypercalcemic effect of the active vitamin D metabolites at the dose levels required to mediate these effects in the human. In view of this limitation, considerable effort has been directed to developing nonhypercalcemic analogs of 1,25-(OH)’D,, and several new compounds have been shown to have a potent differentiating activity in ~ i t r o ~ ” - ’ ~ at) ;least one is active in vivo without significant hypercalcemic The mechanism of this divergence of effect on differentiation and calcium regulation has been the subject of considerable debate. Potential mechanisms have included differences in the molecular mechanisms of action of 1,25-(OH),D, and of these analogs in differentiation and calcium regulatory pathways. Inherent in this hypothesis is an insensitivity of calcium regulatory events to these analogs. We have used the human osteocalcin gene promoter as a model gene in calcium regulatory pathways because it is a bone-specific gene regulated by active vitamin D, metabolites through a specific vitamin D-responsive element (VDRE) in the gene promoter region.(291 In transient transfection this construct is half-maximally induced by 1,25-(OH)’D3 at M and saturated at lo-’ M, consistent with the dissociation constant K d of the vitamin D receptor. The specificity of this vitamin D-responsive element reflects the distinct role of this hormone in bone and calcium metabolism. It is a useful tool in examining the molecular mechanisms of action of vitamin D metabolites and analogs in a typical calcium regulatory gene. We established a permanently transfected rat osteosarcoma cell line containing an osteocalcin promoter-reporter construct and have found that the sensitivity of this promoter to various vitamin D, metabolites and analogs, including various fluorinated analogs, correlates with their biologic activity in vivo. Using this model we were then able to examine the ability of the nonhypercalcemic analogs to activate a vitamin D-responsive gene.
MATERIALS AND METHODS The rat osteoblastic sarcoma cell line, ROS 17/2.8, which has a osteoblast phenotype and expresses osteocalcin and mineralizes in ~ i t r o , ‘was ~ ~ kindly ’ supplied by Dr. S. Rodan (Merck, Sharpe and Dohme Bone Biology and Osteoporosis Research, West Point, PA). Various vitamin D, metabolites, fluorinated analogs, and the nonhypercalcemic analog A22-1,2SS,26-trihydroxyvitaminD3,(24)were generously supplied by Dr. M. Uskokovic (Hoffmann La Roche, Nutley, NJ). Calcipotriol (MC903)(25)was kindly supplied by Dr. L. Binderup (Leo Pharmaceuticals, Ballerup, Denmark) and Oxacalcitriol (22-oxa-1,25(OH)2D3]”6)was kindly supplied by Drs. J. Abe and Y. Nishii (Chugai Pharmaceuticals, Tokyo, Japan). The promoter expression construct (pOSCAT2) of the human osteocalcin gene, as previously described, lz9) was used in cotransfection with the neomycin resistance gene (pSV2 n e ~ ) ( ~into l ) ROS 17/23 cells. Transfections were done by the standard calcium phosphate coprecipitation
techniq~e‘~’] with the following modifications. Cells were cultured in Ham’s F12 medium (Flow Laboratories) with 5% fetal calf serum (FCS) to a density of 2 x 10’ cells in 150 cm’ flasks. The medium was changed 2 h before transfection to Dulbecco’s modified Eagle’s medium (DMEM) buffered with 25 mM HEPES, pH 7.2, and supplemented with 5% FCS. DNA precipitate (50 pg in 2 ml) was added to the cells, and 6 h later the cells exposed for 1 minute to 15% glycerol in DMEM containing 5% FCS. Transfected cells were replated after 24 h and left to grow in Ham’s F12 with 5% FCS for 3 days before Geneticin selection was applied with 500 p g h l of active Geneticin ((3418, GIBCO Laboratories, Sydney). Transfected cells were refed selective medium every 3 days and replated into smaller flasks. After 2 weeks the number of surviving cells had decreased to a level permitting replating into six-well trays, with large numbers of dead cells evident. After 4 weeks Geneticin selection no longer reduced cellular replication or plating efficiency, and this pool of Geneticin-resistant cells gave levels of chloramphenicol acetyltransferase (CAT) activity inducible by 1,25-(OH),D,. After a further 4 weeks to ensure that wildtype cells were excluded, Geneticin was no longer included in the culture medium. These cells, which were designated ROSCO-2, have been cultured for 2 years in the absence of Geneticin and have maintained the same level of CAT activity. ROSCO-2 CAT activity is induced by 1,25-(OH),D3 and retinoids and repressed by glucocorticoids in precisely the same manner as we have previously reported by transient transfection with pOSCAT2 in ROS 17/2.8 cell^.^'^^ The neomycin-resistant cells were subcultured and replated into six-well trays (10 cm’ per well) at 2 x lo5 cells per well in HEPES-buffered DMEM with 2% dextran charcoal-stripped fetal calf serum (ChFCS) for hormone and analog sensitivity studies. After plating down, cells were treated in triplicate with either ethanol vehicle (control) or metabolite or analog. After 24 h the cells were harvested by a brief trypsinization, washed in normal saline, and resuspended in 100 pl of 0.25 M Tris-HCI, pH 7.8, before lysis by three cycles of freeze-thaw in liquid nitrogen. CAT activity was measured by a sensitive nonchromatographic CAT assay as previously described.(291 The activity is expressed as the transfer to chloramphenicol (2 mM) of “C-labeled acetyl groups from “C-acetyl CoA, 2 x lo5 dpm per 100 pl (52 Ci/mol, Amersham), and 80 pM unlabeled acetyl CoA at 37°C. The basal CAT in ROSCO-2 cells of about 2 nmol per lo5 cells per h is not affected by estrogen, progesterone, or thyroid hormones.
RESULTS In the permanently transfected ROSCO-2 cells, various vitamin D, metabolites induced CAT activity with a range of potencies (Fig. 1). 1,25-(oH)’D3 was the most active metabolite tested. The induction by 1,25-(OH)D, was reproducibly detected at lo-” M and maximal at 10-9-19-0 M, with a decline at lo-’ M presumably due to cytotoxicity. The results of several different experiments are normalized to the basal (uninduced) level and combined to examine the relative potencies of other metabolites and ana-
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NONHYPERCALCEMIC CALCITRIOL ANALOGS INDUCE OSTEOCALCIN
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FIG. 3. Regulation of the osteocalcin promoter expression vector by nonhypercalcemic analogs of 1 ,25-(OH),D, in ROSCO-2 cells. The nonhypercalcemic analogs MC903, 22-oxa-1,25-(OH),D, (22-oxa-1,25-D3), and A22-1 ,2S-(OH),D, (D-22-1,25-D3) were tested over a dose range from lo-” to lo-’ M. All three analogs are approximately equipotent with I,2S-(OH),D3 (1,25-D3) both in terms of dose response and maximal response. Values are means f SEM of activity, expressed relative to basal (uninduced) activity, from three to eight separate wells from two to four experiments.
relates to their resistance to catabolism by side-chain-diAn alternative possibility for the divergence of the calrected oxidative reactions. (34) cium regulatory and differentiating functions is that the inThe full potency of the nonhypercalcemic analogs of teraction of these agents with intestinal receptor may be 1,25-(OH),D3 in terms of activating the osteocalcin pro- subtly different to their interaction with bone vitamin D moter cannot be explained by any receptor or postreceptor receptor. However, any difference would need to be a interaction in bone cells. 1 ,25-(OH),D3 induces its own me- major change to explain their virtual lack of calcium mobitabolism and changes the half-life of its r e ~ e p t o r . ‘ ~ ’ , lizing ~ ~ ) activity even 3 orders of magnitude higher doses However, these mechanisms generally limit the activity of than for 1,25-(OH),D,. This seems most unlikely given the the native hormone, and if they played any such role in the strong similarities between the activity of various vitamin lack of activity of these analogs, this should be seen in this D, metabolites and analogs in binding to the chick intestiintact cell system. It is possible that some postreceptor nal receptor binding and regulation of human mechanism, such as stabilization of mRNA, may be modi- and animal cancer cell lines,(10.19.21) and now the human fied by receptor activated by 1,25-(OH),D,, which the ana- osteocalcin gene promoter activation system reported here. logs cannot reproduce. However, this would need to be Thus at the present time there have been no data suggestspecific since the present system would be subject to any ing any difference either in the receptors in calcium regulageneral mechanism. A mechanism acting in vivo on spe- tory pathways, such as bone and intestine, or in differenticific mRNA species, such as the native osteocalcin mRNA, ation-related pathways. As yet there is no comparable might not be detectable in this in vitro system. However, gene-cell system to evaluate the action of these analogs on other groups have reported in abstract form that one of a calcium-related gene in intestinal cells in vitro. Morethese analogs, Calcipotriol, is approximately equipotent over, Oxacalcitriol in vitro acts in another calcium regulawith 1,25-(OH),D, in the stimulation of osteocalcin syn- tory pathway in that it suppresses rat parathyroid hormone thesis by human osteosarcoma cells in More- synthesis and secretion equipotently with 1 ,25-(OH),D3‘421 over, in a recent study on osteocalcin synthesis in human and continuous infusions are able to elevate calcium in roosteoblasts, MC903 and 1,25-(OH),D, were equipotent dent models (Mawer, personal communication). over 24-28 h i n c ~ b a t i o n s . ‘ ~ At~ )longer times, however, A potentially important mechanism relates to major when catabolism could be a factor, MC903 was less active pharmacokinetic differences between 1 ,25-(OH),D, and on osteocalcin induction, yet the two compounds were the nonhypercalcemic analogs. Since both Oxacalcitriol equally active in stimulation of DNA synthesis. These data and Calcipotriol have relatively low affinity for human support the concept that distinct effects on cell replication serum vitamin D binding protein, they achieve higher peak and calcium regulation could not be operating through a free levels and have markedly shortened serum half-lives postreceptor mechanism in these in vitro bone cell systems. (less than 1 h) compared with 1,25-(OH)2D3.(43,44) Because
NONHYPERCALCEMIC CALCITRlOL ANALOGS INDUCE OSTEOCALCIN the nonhypercalcemic analogs achieve high free levels only transiently, they may be unable to sustain activation of calcium-regulatory pathways, which by their nature are continuously variable responses. A reduced in vitro half-life mechanism for a lower effect on calcium regulatory pathways has been demonstrated for 24-epi- 1,25-dihydroxyergocalciferol. ( 4 4 . 4 5 ) In contrast with the nonhypercalcemic analogs, 1,25-(OH),D3 achieves more sustained but lower free levels and thus may be able to maintain the induction of genes responsible for calcium regulation, such as calcium transport in the intestine, which are inherently reversible processes. On the other hand, cell replication and differentiation are essentially irreversible steps in the biology of a cell, quite distinct from the reversible modulation of a physiologic function of a differentiated cell. In this model the transient but high free level of a 1,25-(OH)2D3analog could initiate an irreversible differentiation step or “switch” in a cell’s biology yet be unable to maintain activation of the inherently reversible calcium regulatory pathways. Irrespective of the mechanism of these differences, it is clear that the nonhypercalcemic analogs we have evaluated, which have potential for antiproliferative actions in vivo, d o not have abnormal interactions with the 1,25(OH),D3 receptor or abnormal postreceptor mechanisms in a well-characterized gene closely regulated in relation to bone and calcium homeostasis. At present there is no evidence of any difference in vitamin D receptors for the differentiation and calcium regulatory pathways. The data presented here complement previous data in this area but focus on the molecular mechanisms involved. Thus at least two of the nonhypercalcemic analogs analyzed here have the in vivo pharmacokinetics described, and all have low affinity to the serum vitamin D binding protein (R. Bouillon, personal communication). All three nonhypercalcemic analogs studied here have virtually identical activating potency on the stably transfected human osteocalcin gene promoter. Pharmacokinetic differences, which can explain these differential effects, may be an important component of the design and evaluation of these and other agents, such as altered side-chain-length analogs of 1,25(OH)2D3.(46-48) Success of the effort to develop analogs with differential effects in various calcium and differentiation pathways depends in part on adequate understanding of their molecular mechanisms of action. The ROSCO-2 cells can be a valuable tool for investigating these molecular mechanisms.
ACKNOWLEDGMENTS The valuable technical contributions of Rama Vedam, Judith Flanagan, and Susan Gillies are acknowledged. This work was supported by a Centre Grant to the Garvan Institute from the National Health and Medical Research Council and by the New South Wales State Cancer Council. This work was presented in part at the Sydney International Bone Symposium (April 1990) and the American Society for Bone and Mineral Research meeting, Atlanta, Georgia (September 1990).
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898 17. Studzinski GP, Bhandal AK, Brevli ZS 1985 Cell cycle sensitivity of HL-60 cells to the differentiation-inducing effects of 1-alpha, 25-dihydroxyvitamin D,. Cancer Res 45:3898-3905. 18. Eisman JA, Sutherland RL, McMenemy ML, Fragonas J-C, Musgrove EA, pang GYN 1989 Effect of 1.25-dihydroxyvitamin D, on cell-cycle kinetics of T 47D human breast cancer cells. J Cell Physiol 138:611-616. 19. Reitsma PH, Rothberg PG, Astrin SM, Trial J, Bar-Shavit 2, Hall A, Teitelbaum SL, Kahn AJ 1983 Regulation of myc gene expression in HL-60 leukaemia cells by a vitamin D metabolite. Nature 306:492-494. 20. Koga M,Eisman JA, Sutherland RL 1988 Regulation of epidermal growth factor receptor levels by 1,25-dihydroxyvitamin D, in human breast cancer cells. Cancer Res 48:27342739. 21. Ravid A, Koren R, Novorodsky A, Liberman UA 1984 1,25Dihydroxyvitamin D, inhibits selectively the mitogenic stimulation of mouse medullary thymocytes. Biochem Biophys Res Commun 123:163. 22. Lemire JM, Adams JS, Sakai R, Jordon SC 1984 1 Alpha, 25 dihydroxyvitamin D, suppresses proliferation and immunoglobulin production by normal human peripheral blood mononuclear cells. J Clin Invest 74:657. 23. Tsoukas CD, Provvedini DM, Manolagas SC 1984 1.25-Dihydroxyvitamin D,: A novel immunoregulatory hormone. Science 224:1438. 24. Wovkulich PM, Batcho AD, Baggiolini EG, Boris A, Truitt G, Uskokovic MR 1985 Synthesis of 1,25S,26-trihydroxy-A22-cholecalciferol, a potent inducer of cell differentiation. In: Norman AW, Schaefer K, Grigoleit H-G, von Herrath D (eds.) Vitamin D, Chemical Biochemical and Clinical Update. Walter de Gruyter, Berlin, pp. 755-764. 25. Binderup L, Bramm E 1988 Effects of a novel vitamin D analogue MC903 on cell proliferation and differentiation in vitro and on calcium metabolism in vivo. Biochem PharmacOI 37:889-895. 26. Abe J, Takita Y, Nakano T, Miyaura C, Suda T, Nishii Y 1989 A synthetic analogue of vitamin D,, 22-oxa-I-alpha,25dihydroxyvitamin D,, is a potent modulator of in vivo immunoregulating activity without inducing hypercalcemia in mice. Endocrinology 124:2645-2647. 27. Kragballe K, Beck HI, SBgaard H 1988 Improvement of psoriasis by a topical vitamin D, analogue (MC 903) in a doubleblind study. Br J Dermatol 119:223-230. 28. Staberg B, Roed-Petersen J, Menne T 1989 Efficacy of topical treatment in psoriasis with MC903, a new vitamin D analogue. Acta Derm Venereol (Stockh) 69:147-150. 29. Morrison NA, Shine J, Fragonas JC, Verkest V, McMenemy LM, Eisman JA 1989 1,25-Dihydroxyvitamin D responsive element and glucocorticoid repression in the human osteocalcin gene. Science 246:1158-1161. 30. Rodan GA, Rodan SG 1984 Expression of the osteoblastic phenoype. In: WA Peck (ed.) Advances in Bone and Mineral Research, Annual 11. Amsterdam: Excerpta Medica, pp. 244-285. 31. Southern PJ, Berg P 1982 Transformation of mammalian cells to antibiotic resistance with bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet 1:327341. 32. Wigler M, Perucho M, Kurtz D, Dana S, Pellicer A, Axel R, Silverstein S 1980 Transformation of mammalian cells with an amplifiable dominant-acting gene. Proc Natl Acad Sci
MORRISON AND EISMAN USA 77:3567-3570. 33. Eisman JA, DeLuca HF 1977 Intestinal 1,25-dihydroxyvitamin D, binding protein: Specificity of binding protein. Steroids 30:245-257. 34. Eisman JA, Frampton RJ, McLean FJ 1986 Biochemical significance of enhanced activity of fluorinated 1,25-dihydroxyvitamin D, in human cultured cell lines. Cell Biochem Func 4: 115-121. 35. Inaba M, Okuno S, lnoue A, Nishizawa Y, Morii H, DeLuca HF 1989 DNA binding property of vitamin D, receptors associated with 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D,. Arch Biochem Biophys 268:35-39. 36. Yukioka K, Otani S, Matsui-Yuasa I, Goto H, Morisawa S, Okuno S, Inaba M, Nishizawa Y, Morii H 1988 Biological activity of 26,26,26,27,27,27-hexafluorinatedanalogs of vitamin D, in inhibiting interleukin-2 production by peripheral blood mononuclear cells stimulated by phytohemagglutinin. Arch Biochem Biophys Mo:45-50. 37. Eisman JA, Fragonas JC, McMenemy ML 1988 Rapid turnover of the 1,25-dihydroxyvitamin D, receptor in human target cells. Endocrinology 122:1613-1621. 38. Reinhardt TA, Horst RL 1989 Self-induction of 1,25-dihydroxyvitamin D, metabolism limits receptor occupancy and target tissue responsiveness. J Biol Chem 264:15917-15921. 39. Valaja T, Mahonen A, Pirskanaen A, Maenpaa PH 1990 Affinity of a novel vitamin D analog MC903 for vitamin D receptor and effects of the analog on the synthesis of osteocalcin by human osteosarcoma cells (abstract 732). J Bone Miner Res 4:S300. 40. Evans DB, Thavarajah M, Binderup L, Kanis JA 1990 Comparison of the action of MC903 and 1,25-(OH),D, on human osteosarcoma cells in vitro (abstract 868). J Bone Miner Res 45334. 41. Marie PJ, Connes D, Hott M, Miravet L 1990 Comparative effects of a novel vitamin D analogue MC-903 and 1,25-dihydroxyvitamin D, on alkaline phosphatase activity, osteocalcin and DNA synthesis by human osteoblastic cells in culture. Bone 11:171-179. 42. Brown AJ, Ritter CR, Finch JL, Morrissey J, Martin KJ, Murayama E, Nishii Y, Slatopolsky E 1989 The noncalcemic analogue of vitamin D, 22-oxacalcitriol, suppresses parathyroid hormone synthesis and secretion. J Clin Invest 84:728732. 43. Sorenson H, Binderup L, Calverley MJ, Hoffmeyer L, Andersen NR 1990 In vitro metabolism of Calcipotriol (MC903). a vitamin D analogue. Biochem Pharmacol 39:391-393. 44. Nishii Y, Okano T, Tsugawa N, Masuda S, Takeuchi A, Kobayashi T, Abe J, Yakita Y, Nakano T 1990 Recent advances in 22-oxacalcitriol (OCT) and a reason why OCT is different from multifunctional vitamin D, calcitriol (abstract). Proceedings of the Sydney Bone Symposium, Fifth lnternational Symposium on Bone, Structure, Function and Disease, April 1990, p. 32. Bone 10. 45. DeLuca HF, Sicinski RR, Tanaka Y, Stern PH, Smith CM 1988 Biological activity of 1,25-dihydroxyvitarnin D, and 24epi-l,25-dihydroxyvitaminD,. Am J Physiol 254:E402-406. 46. Reinhardt TA, Ramberg CF, Horst RL 1989 Comparison of receptor binding, biological activity, and in vivo tracer kinetics for 1,25-dihydroxyvitamin D,, 1,25-dihydroxyvitamin D,, and its 24 epimer. Arch Biochem Biophys 273:64-71. 47. DeLuca HF, Ostrem VK 1988 Analogs of the hormonal form
NONHYPERCALCEMIC CALCITRIOL ANALOGS INDUCE OSTEOCALCIN of vitamin D and their possible use in leukemia. Prog Clin Biol Res 259:41-55. 48. Gill HS, Londowski JM, Corradino RA, Zinsmeister AR, Kumar R 1988 The synthesis and biological activity of 25-hydroxy-26,27-dimethylvitaminD, and 1,25-dihydroxy-26,27dimethylvitamin D,: Highly potent novel analogs of vitamin D,. J Steroid Biochem 31:147-160.
Address reprint requests to: J.A. Eisman Garvan Institute of Medical Research St. Vincent’s Hospital Sydney, New South Wales 2010, Australia Received for publication December 3, 1990; in revised form February 7, 1991; accepted February 9, 1991.
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