[Studies on the biosynthesis of sterol side chain in higher plants].

Campesterol (3) and dihydrobrassicasterol (4), typical C28-sterols in higher plants, are biosynthesized from a steroidal 24-ene precursor (desmosterol 1) via 24-methylenecholesterol (2) and 24-methyldesmosterol (5). A typical plant C29-sterol, sitosterol (6), is produced from 24-methylenecholesterol via isofucosterol (7) and 24-ethyldesmosterol (8). The biosynthetic mechanism, focussing stereochemical features, of these side-chain transformations has been studied in detail by feeding regio- and stereoselectively 13C- or 2H-labeled steroidal substrates to cell cultures of higher plants such as Oryza sativa, Catharanthus roseus and Morus alba. These studies allowed to correlate the metabolic origin of C-26 and C-27 of the intermediate sterols. It has been established that the 1st methylation leading to 24-methylenecholesterol from desmosterol involves a Re-face hydrogen migration from C-24 to C-25 based on unambiguous assignment of the isopropyl pro-R-Me and pro-S-Me of 24-methylenecholesterol. The 2nd methylation leading to isofucosterol was revealed to proceed in a trans-mechanism in which addition of the methyl group and elimination of the C-28 hydrogen occur on opposite faces of the original delta 24(28) plane. The double bond isomerization from delta 24(28) to delta 24(25) was found to proceed in a syn-SE2' mechanism with the pro-S-methyl group of isofucosterol becoming the (E)-methyl of 24-ethyldesmosterol. Finally, feeding studies of [E-Me-13C]- and [Z-Me-13C]-24-methyldesmosterols established that an anti-mode of hydrogen addition is operating in the conversion of 24-methyldesmosterol to campesterol and dihydrobrassicasterol. Similar studies established that 24-ethyldesmosterol is converted to sitosterol in an anti-mode of hydrogen addition. In addition, the mechanism of sterol side-chain formation in hairy roots of Ajuga reptans var. atropurpurea is briefly described.

Inhibition of vitamin D3-induced cell differentiation by interferon-gamma in HL-60 cells determined by a nitroblue tetrazolium reduction test.

The combined effects of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] and interferon-gamma (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) on cell differentiation in HL-60 human promyelocytic leukemia cells were examined by a nitroblue tetrazolium (NBT) reduction test. 1,25(OH)2D3 at the concentrations of 7-70 nM induced NBT-positive cells, which was used as a criterion of cell differentiation. IFN-gamma itself showed little effect on induction of NBT-positive cells or on cell growth at a concentration up to 1000 U/ml. However, in a combination of 1,25(OH)2D3 with IFN-gamma (100 and 300 U/ml), cell differentiation was strongly inhibited and was accompanied by growth inhibition. Treatment with a combination of 1,25(OH)2D3 and TNF-alpha or IFN-gamma and TNF-alpha showed an additive effect on cell differentiation. IFN-gamma seems to act as a specific inhibitor for 1,25(OH)2D3-induced cell differentiation. To elucidate the cause of the inhibition of cell differentiation by IFN-gamma, the ability of the cells to produce superoxide (O2-) was examined after culture for 5 days in the presence of 1,25(OH)2D3 and IFN-gamma. The results indicated that the inhibition of IFN-gamma was caused by a reduction in the ability of the cells to produce O2- in response to stimulation by 12-O-tetradecanoylphorbol-13-acetate (TPA).

Elongation of the side chain of analogs of 1 alpha,25-dihydroxyvitamin D3 prevents osteopenia in a rat model.

Five analogs of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] [1], 26,27-dimethyl-1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2(Me)2D3] [2], 26,27-dimethyl-1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2(Et)2D3] [3], 26,27-dipropyl-1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2(Pr)2D3] [4], 26,27-dimethyl-24,24-difluoro-1 alpha,25-dihydroxyvitamin D3 [24F2-1,25(OH)2(Me)2D3], and [5] 24a-homo-24,24-difluoro-1 alpha,25- dihydroxyvitamin D3 [24aF2-homo-1,25(OH)2D3] were investigated to clarify the possibility that prevents osteopenia induced in rats by ovariectomy and sciatic neurotomy. The objective of our studies was to determine whether these analogs may be effective for treatment of subjects with osteoporosis. 1,25(OH)2(Me)2D3, 24F2-1,25(OH)2(Me)2D3, and 24aF2-homo-1,25(OH)2D3 prevented decreases in bone mineral density (BMD) of the femur, as measured by dual energy X-ray absorptiometry (DXA). The potency of 1,25(OH)2(Me)2D3 in this test was higher than that of 1,25(OH)2D3. The potencies of 24F2-1,25(OH)2(Me)2D3 and 24aF2-homo-1,25(OH)2D3 were similar to that of 1,25(OH)2D3. On the other hand, though 1,25(OH)2(Et)2D3 and 1,25(OH)2(Pr)2D3 had a preventive effect on the decrease in BMD, the potency of two analogs was lower than that of 1,25(OH)2D3. Decreases in cortical and trabecular bone areas of the femur were prevented by three analogs of 1,25(OH)2D3, 1,25(OH)2(Me)2D3, 24F2-1,25(OH)2(Me)2D3, and 24aF2-homo-1,25(OH)2D3. Serum calcium (Ca) concentration was elevated at the last administration of three analogs of 1,25(OH)2D3, 1,25(OH)2(Me)2D3, 24F2-1,25(OH)2-(Me)2D3 and 24aF2-homo-1,25(OH)2D3.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of HT-29 human colon cancer growth under the renal capsule of severe combined immunodeficient mice by an analogue of 1,25-dihydroxyvitamin D3, DD-003.

An analogue of 1,25-dihydroxyvitamin D3, 22(S)-24-homo-26,26,26,27,27,27-hexafluoro-1 alpha,22,25-trihydroxyvitamin D3 (DD-003), showed 10-fold greater inhibiting effect than 1,25-dihydroxyvitamin D3 on the growth of HT-29 human colonic adenocarcinoma cells in culture. To examine the anticancer activity of DD-003 in vivo, a fibrin clot of HT-29 cells was prepared with fibrinogen and thrombin and implanted under the renal capsule of the severe combined immunodeficient mouse. Starting 7 days after implantation of HT-29 tumor, mice were given 3 micrograms/kg body weight of DD-003 or the vehicle i.p. every other day for 5 times. The HT-29 tumor grew rapidly in control mice; malignant growth was clearly observed with mitosis, massive tumor angiogenesis, and invasion into normal kidney tissue. Tumors in DD-003 treated mice were smaller with less invasion compared to the control. Administration of DD-003 inhibited growth of HT-29 tumor by 63%. Serum calcium concentrations and body weights of the treated mice were similar to those of the control. DD-003 inhibited growth of HT-29 tumor in a dose-dependent manner over the range of 0.1-10 micrograms/kg body weight, with no increase of serum calcium concentration observed at any dose level. When DD-003 was withdrawn after 2 weeks of treatment, tumor growth resumed. Since chemosensitivity tested by the subrenal capsule assay correlates well with clinical response, DD-003 may be clinically applicable in procedures such as postsurgical chemotherapy of colon cancer.

Sex pheromones in marine polychaetes: steroids from ripe Nereis succinea.

A number of neutral marine steroids such as desmosterol, campesterol, brassicasterol, gorgosterol, and other trace steroids were isolated from the coelomic fluid of ripe Nereis succinea and checked for biological activity as sex pheromones on swarming specimens of Platynereis dumerilii and Nereis succinea. No significant influence of synthetic gorgosterol or a natural extract of gorgosterol or the other identified steroids on the swarming behavior was observed.

Manipulation of thromboxane synthesis by novel 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 in human promyelocytic leukemia (HL-60) cells.

Using new steroidal side-chain-lengthened 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], we manipulated the synthesis of thromboxane and thromboxane-producing enzymes, cyclo-oxygenase and thromboxane synthase, in human promyelocytic leukemia (HL-60) cells in serum-free culture. The order of potency of the analogues for stimulating thromboxane B2 synthetic activity from arachidonic acid (reflecting combined cyclo-oxygenase activity and thromboxane synthase activity) and from prostaglandin H2 (thromboxane synthase activity only) as well as for cyclo-oxygenase induction was 1 alpha,25-(OH)2D3 > or = 1 alpha,25-(OH)2-26,27-CH3)2D3 > 1 alpha,25-(OH)2-26,27-(C2H5)2D3 >> 1 alpha,25-(OH)2-26,27-(C3H7)2D3. These results suggest that there are functional and structural limits to the chain length of C-26 and C-27 dialkyl groups flanking the C-25-OH group in the 1 alpha,25-(OH)2D3 molecule for expressing thromboxane synthetic activity in HL-60 cells. Removal of the C-1 alpha-OH group from 1 alpha,25-(OH)2D3 led to markedly decreased thromboxane synthetic activity in HL-60 cells. These structure-activity relationships indicate that both the C-25-OH and C-1 alpha-OH groups in the 1 alpha,25-(OH)2D3 molecule are essential for expressing thromboxane synthesis in HL-60 cells. Also, the rank order for stimulating thromboxane synthesis correlated well with the binding affinity of these dialkyl analogues for the 1 alpha,25-(OH)2D3 receptor of HL-60 cells, suggesting a 1 alpha,25-(OH)2D3 receptor-mediated induction mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of hexafluoro-1,25-dihydroxyvitamin D3 and sodium butyrate combination on differentiation and proliferation of HL-60 leukemia cells.

We have investigated the combined effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and its fluoroanalog 26,26,26,27,27,27-hexafluoro-1,25-(OH)2D3 [F6-1,25-(OH)2D3] with sodium butyrate (NaB) on growth and differentiation of HL-60 human promyelocytic leukemia cells. F6-1,25-(OH)2D3 was 10-fold more active than 1,25-(OH)2D3 for induction of cell differentiation in HL-60 cells. Exposure to suboptimal concentration of F6-1,25-(OH)2D3 and NaB had synergistic effects compared to that of F6-1,25-(OH)2D3 or NaB alone and in the presence of 0.1-0.3 mM NaB, the dosage of F6-1,25-(OH)2D3 required to inhibit cell growth and colony formation and to induce cell differentiation was significantly reduced. The mechanism for the synergistic effect is probably that NaB increases cytoplasm content and nuclear binding of 1,25-(OH)2D3.

Calcium regulating activity of 26,27-dialkyl analogs of 1 alpha,25-dihydroxyvitamin D3.

A series of analogs of 1 alpha,25-dihydroxyvitamin D3[1,25(OH)2D3] with alkyl substitutions in 26- and 27-positions were tested for calcium (Ca) regulating activity. The potencies of dialkyl analogs in stimulating bone resorption in neonatal mouse calvaria cultures were the highest in 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3[1,25(OH)2-(Me)2D3], followed by 1,25(OH)2D3, 1 alpha,25-dihydroxy-26,27-diethylvitamin D3[1,25(OH)2(Et)2D3], and 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3[1,25(OH)2(Pr)2D3] in that order. A similar order of potential regarding formation of osteoclast-like cells in mouse bone marrow cell cultures and on bone Ca mobilization with long-term vitamin D-deficient rats was observed in the same series. The relative potencies of 1,25(OH)2D3, 1,25(OH)2(Me)2D3, 1,25(OH)2(Et)2D3, and 1,25(OH)2(Pr)2D3 in competing with 1,25(OH)2D3 for binding to chick intestinal cytosol receptors were 1:1:0.16:0.036. A similar order of potential in case of intestinal Ca transport in situ was observed in the same series. The potencies of dialkyl analogs in competing with 25-hydroxy-vitamin D3 for binding to rat serum vitamin D binding protein were much lower than that of 1,25(OH)2D3. Effect of 1,25(OH)2(Me)2D3 on osteopenia in rats induced by ovariectomy and right sciatic neurotomy was higher than that of 1,25(OH)2D3. From these results, the lengthening by one carbon at 26- and 27-positions was shown to maintain the Ca regulatory activity of 1,25(OH)2D3.

Effect of 22R-hydroxycholesterol on the action of sphingomyelinase from Bacillus cereus toward bovine erythrocytes.

The incorporation of 22R-hydroxycholesterol [(22R)-5-cholestene-3 beta,22-diol] into the bovine erythrocyte membranes remarkably enhanced the degradation of sphingomyelin in erythrocyte membranes by the action of sphingomyelinase from Bacillus cereus, causing much faster hemolysis of erythrocytes. The stimulative effect of 22R-hydroxycholesterol on the breakdown of sphingomyelin was maximal in the presence of Mg2+. On the other hand, in spite of the presence of 22R-hydroxycholesterol, the breakdown of sphingomyelin was inhibited by increasing concentrations of Ca2+. Also, the incorporation of 22R-hydroxycholesterol into the erythrocyte membranes facilitated the specific adsorption of the enzyme onto the surface of the erythrocyte membranes. The specific adsorption of sphingomyelinase amounted to 20-40% of the total activity in the presence of Mg2+ and the absence of divalent metal ions. In the presence of Ca2+, the incorporation of 22R-hydroxycholesterol enhanced the enzyme adsorption, exceeding more than 90% of the total activity. Therefore, the incorporation of 22R-hydroxycholesterol into bovine erythrocyte membranes remarkably accelerates the breakdown of sphingomyelin in the presence of Mg2+, and the specific adsorption of sphingomyelinase onto erythrocytes in the presence of Ca2+.

Effects on cultured neonatal mouse calvaria of 1 alpha,25-dihydroxyvitamin D3, 26,26,26,27,27,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 and 26,26,26,27,27,27-hexafluoro-1 alpha,23S,25-trihydroxyvitamin D3.

The potency of 26,26,26,27,27,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 (26,27-F6-1,25(OH)2D3) to enhance bone calcium (Ca) mobilization in vitro was higher than that of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3). In a 48-h organ culture system using fetal rat limb bones, effects of 26,27-F6-1,25(OH)2D3 on bone resorption were similar to those of 1,25(OH)2D3. Thus, we attempted to clarify whether or not the potency of 26,27-F6-1,25(OH)2D3 in bone resorption in vitro would be higher than that of 1,25(OH)2D3. Calvarial bones from neonatal mice were used as explants and the culture period was extended to 144 from 48 h. In cultures of 0 to 48 h, both drugs increased the release of prelabeled 45Ca from cultured calvariae, in a dose-dependent manner. In cultures of 48 to 144 h, 26,27-F6-1,25(OH)2D3 was much more effective than 1,25(OH)2D3 regarding indices of bone resorption. 26,26,26,27,27,27-F6-1 alpha,23S,25-trihydroxyvitamin D3 (26,27-F6-1,23,25(OH)3D3), a main metabolite of 26,27-F6-1,25(OH)2D3 in rats, stimulated 45Ca release equipotently to 1,25(OH)2D3. In mouse bone marrow cells cultured for 7 days, the potency of 26,27-F6-1,25(OH)2D3 on osteoclast formation was much higher than 1,25(OH)2D3. These results suggest that the higher potency of 26,27-F6-1,25(OH)2D3 in bone resorption may be due at least in part to both the higher potency of 26,27-F6-1,25(OH)2D3 in osteoclast formation and the larger retention of 26,27-F6-1,23,25(OH)3D3 in calvariae.

Effects of novel 26,27-dialkyl analogs of 1 alpha,25-dihydroxyvitamin D3 on differentiation-inducing activity of human promyelocytic leukemia (HL-60) cells in serum-supplemented or serum-free culture.

Monocytic differentiation-inducing activity of steroidal side chain-lengthened 26,27-dialkyl analogs of 1 alpha,25-dihydroxyvitamin D3 was examined in human promyelocytic leukemia (HL-60) cells in serum-supplemented or serum-free culture. The order of in vitro potency for reducing nitroblue tetrazolium was 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3 greater than or equal to 1 alpha,25-dihydroxy-26,27-diethylvitamin D3 much greater than 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3 under serum-free culture conditions. Analysis by sucrose density-gradient centrifugation or polyethylene glycol precipitation technique showed that the potency order for differentiation-inducing activity correlated well with binding affinity of these analogs for vitamin D3 receptor of HL-60 cells. Under serum-supplemented culture conditions, the lack of correlation between biologic activity and analog-binding affinity for receptor was caused by differences in binding affinity of these analogs for serum vitamin D-binding proteins. These results suggest that serum vitamin D-binding proteins apparently modulate monocytic differentiation of HL-60 cells by these analogs under serum-supplemented culture conditions.

Biological activity of 1 alpha, 25-dihydroxyvitamin D derivatives--24-epi-1 alpha, 25-dihydroxyvitamin D-2 and 1 alpha,25-dihydroxyvitamin D-7.

Biological activity of 24-epi-1 alpha,25-dihydroxyvitamin D-2 (24-epi-1,25(OH)2D2) and 1 alpha,25-dihydroxyvitamin D-7 (1,25(OH)2D7), the 22,23-dihydro derivative of the former compound, was investigated. Both of the vitamin D derivatives stimulated intestinal calcium transport and calcium mobilization from bones in rats; however, the effect was about 50% of that of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)2D3). On the other hand, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 inducement of HL-60 human leukemia cell differentiation was comparable to that of 1,25(OH)2D3. Accordingly, the differentiation-inducing activity of 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 was much greater than their ability to stimulate calcium metabolism. In contrast to 1,25(OH)2D3, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 exerted little hypercalcemic activity in mice. These results suggest that both vitamin D derivatives will be useful as anti-tumor agents.

Effects of vitamin D2 analogs on calcium metabolism in vitamin D-deficient rats and in MC3T3-E1 osteoblastic cells.

The effects of 1 alpha-hydroxyvitamin D2 on calcium metabolism in vivo and of 1 alpha, 25-dihydroxyvitamin D2, which is an active metabolite of 1 alpha-hydroxyvitamin D2, on bone metabolism in vitro was studied and compared with that of 1 alpha-hydroxyvitamin D3 or 1 alpha,25-dihydroxyvitamin D3. 1 alpha-Hydroxyvitamin D2 and 1 alpha-hydroxyvitamin D3 was equally potent in stimulating intestinal calcium transport by using the everted sac method and of calcium mobilization from bone in vitamin D-deficient rats. On the other hand, the hypercalcemic activity of 1 alpha-hydroxyvitamin D2 was much lower than that of 1 alpha-hydroxyvitamin D3 in normal mice and rats. 1 alpha,25-Dihydroxyvitamin D2 and 1 alpha,25-dihydroxyvitamin D3 stimulated alkaline phosphatase activity in osteoblastic MC3T3-E1 cells and bone resorption in newborn mouse calvaria maintained in organ culture. These results show that 1 alpha-hydroxyvitamin D2 as well as 1 alpha-hydroxyvitamin D3 promote calcium absorption and may accelerate bone remodelling via direct action on osteoblasts. In addition, they suggest that 1 alpha-hydroxyvitamin D2 may be more useful than 1 alpha-hydroxyvitamin D3 for the treatment of senile osteoporosis, because hypercalcemia is one of the major side effects of 1 alpha-hydroxyvitamin D3.

26,27-Hexafluoro-1,25-dihydroxyvitamin D3 (F6-1,25(OH)2D3) prevents osteoporosis induced by immobilization combined with ovariectomy in the rat.

The effect of 26,27-hexafluoro-1,25-dihydroxyvitamin D3 (F6-1,25(OH)2D3) on experimental osteoporosis in the rat induced by a combination of immobilization and ovariectomy was evaluated. F6-1,25(OH)2D3 increased the femur score and the photo-density. The administration of F6-1,25(OH)2D3 also significantly increased the dry weight, the ash weight and the ash content of the bone. Both F6-1,25(OH)2D3 and 1 alpha(OH)D3 showed a nearly dose-dependent effect and significant inhibition of the decrease of bone mass. Histomorphometry revealed a significant decrease of resorption by the administration of F6-1,25(OH)2D3. Bone formation rate in the F6-1,25(OH)2D3 treated group significantly decreased compared with the vehicle group. In conclusion, the pharmacological effective dose of F6-1,25(OH)2D3 was considered to prevent the osteoporotic decrease of bone mass by suppressing the elevated bone turnover.

Effect on carbon lengthening at the side chain terminal of 1 alpha,25-dihydroxyvitamin D3 for calcium regulating activity.

A series of analogs of 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3 (1)] with alkyl substitutions in 26- and 27-positions have been tested for their activity 1) in competing with 1,25-(OH)2D3 for binding to chick intestinal cytosol receptor, 2) in ability for formation of multinucleated cells (MNC) with various osteoclastic cell characteristics from blast cells, and 3) in stimulating bone calcium mobilization in vitamin D-deficient rats. The relative potencies of 1,25-(OH)2D3, 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3 (2), 1 alpha,25-dihydroxy-26,27-diethylvitamin D3 (3), and 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3 (4) in competing for intestinal cytosolic binding were 1:1.1:0.25:0.05. The similar order of the abilities on formation of the multinucleated cells in the same series was observed. In a bone calcium mobilization test with vitamin D-deficient rats, 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3 showed slightly less activity than 1,25-(OH)2D3 at 12 h after administration, but long lasting activity was observed during time course experiments. 1 alpha,25-Dihydroxy-26,27-diethylvitamin D3, and 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3 were found to be much less active than 1,25-(OH)2D3 in a bone calcium mobilization test.

Synthesis and biological activity of (22E,25R)- and (22,25S)-22-dehydro- 1 alpha,25-dihydroxy-26-methylvitamin D3.

Both 25-epimers of (22E)-22-dehydro-1 alpha,25-dihydroxy-26-methylvitamin D3 [22-dehydro-26-methyl-1,25-(OH)2D3] were synthesized. The biological activity of these compounds was tested in binding affinity to chick intestinal receptor protein of 1 alpha,25-dihydroxy-vitamin D3 [1,25-(OH)2D3] and in stimulating for intestinal calcium transport and bone calcium mobilization with vitamin D-deficient rats. The relative potency of (25R)- and (25S)-22-dehydro-26-homo-1,25-(OH)2D3 and 1,25-(OH)2D3 in competing for the intestinal cytosolic binding was 1.7:1.5:1. A similar order of activity was observed on intestinal calcium transport and bone calcium mobilization. In the ability for stimulation of intestinal calcium transport, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were about 3.6 and 2.1 times as active as 1,25-(OH)2D3, respectively. In bone calcium mobilization tests, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were estimated to be 2.2 and 1.6 times as potent as 1,25-(OH)2D3, respectively.

Effects of 1,25-dihydroxyvitamin D3 and its analogs on butyrate-induced differentiation of HT-29 human colonic carcinoma cells and on the reversal of the differentiated phenotype.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) greatly enhances sodium butyrate (NaB)-induced enterocyte differentiation of HT-29 human colonic carcinoma cells while 1,25-(OH)2D3 alone induces growth restriction without associated differentiation. In the present study, the efficacies of various analogs of 1,25-(OH)2D3 to enhance NaB-induced HT-29 differentiation and to prolong the reversal of the differentiated phenotype under NaB-free growth conditions were subsequently examined. Extent of HT-29 differentiation was assessed by measurement of alkaline phosphatase (AP) activity, appearance of mucin-producing cells, changes in morphological characteristics, and expression of differentiation-associated cytokeratin proteins. Among active analogs of 1,25-(OH)2D3, 26,26,26,27,27,27-hexafluoro-1,25-(OH)2D3 (F6-1,25-(OH)2D3), 24,24-difluoro-24-homo-1,25-(OH)2D3, and 26,27-dimethyl-1,25-(OH)2D3 were 100-, 10-, and 5-fold, respectively, more effective than 1,25-(OH)2D3 in enhancing NaB-induced mucin production. Combined use of NaB and F6-1,25-(OH)2D3 (10(-9) M) also induced HT-29 cells to form highly differentiated goblet-like enterocytes, and increased both cellular AP enzymatic activity and tissue-type cytokeratin content. This differentiated state was qualitatively more advanced than that achieved by a combination of NaB and 10(-7) M 1,25-(OH)2D3. NaB-mediated HT-29 differentiation (in short-term inductions) was found to be reversible following a return to NaB-free medium. HT-29 cells differentiated by combined use of NaB and 1,25-(OH)2D3 or its analogs exhibited a significant prolonged reversal time relative to cells differentiated with NaB alone. The most prominent effect was achieved using cells differentiated with NaB and 10(-9) M F6-1,25-(OH)2D3 which exhibited a 7-fold prolonged reversal time over colonocytes differentiated by NaB alone. Our data suggest that a combined use of NaB and 1,25-(OH)2D3 or its derivatives may provide a convenient in vitro model system to probe molecular events associated with steroid-target tissue interactions in a differentiating cell system as commonly occurs in vivo. Such an analysis might lend itself to design of a rational combination differentiation-based therapy for the clinical management of colon cancer.