Intestinal response to 1 alpha, 25-dihydroxycholecalciferol. I. RNA polymerase, alkaline phosphatase, calcium and phosphorus uptake in vitro, and in vivo calcium transport and accumulation

The dynamics of intestinal response in rachitic chicks to 1alpha,25-dihydroxycholecalciferol were evaluated by various biochemical parameters. The following observations were made: 1. The earliest detected intestinal response to 1alpha,25-dihydroxycholecalciferol was increased in vitro calcium uptake and in vivo calcium transport, occurring by 2 h and 2.5 h respectively. 2. Increased RNA polymerase activity was observed by 4 h after 1alpha,25-dihydroxycholecalciferol treatment. 3. Calcium binding protein was detected by 5 h, but could not be detected 2.5 h after 1alpha,25-dihydroxycholecalciferol treatment. 4. Increased alkaline phosphatase activity and in vitro accumulation of inorganic phosphate were first demonstrable 6 h after 1alpha,25-dihydroxycholecalciferol treatment. 5. In vivo duodenal calcium accumulation in the mucosa was elevated after 5 h, peaked at 6.5 h, and then began to decrease at 9 h. In vitro duodenal calcium accumulation was elevated at 2 h, peaked at 12 h, and decreased to control level by 18 h. Our data emphasize the lack of correlation between the appearance of calcium binding protein or increased alkaline phosphatase activity and the transport rate of calcium across the duodenum after treatment with 1alpha,25-dihydroxycholecalciferol. The data suggest a correlation between duodenal calcium accumulation and the appearance of calcium binding protein or increased alkaline phosphatase activity.     

Multifaceted regulation of cell cycle progression by estrogen: regulation of Cdk inhibitors and Cdc25A independent of cyclin D1-Cdk4 function

Estrogens induce proliferation of estrogen receptor (ER)-positive MCF-7 breast cancer cells by stimulating G(1)/S transition associated with increased cyclin D1 expression, activation of cyclin-dependent kinases (Cdks), and phosphorylation of the retinoblastoma protein (pRb). We have utilized blockade of cyclin D1-Cdk4 complex formation through adenovirus-mediated expression of p16(INK4a) to demonstrate that estrogen regulates Cdk inhibitor expression and expression of the Cdk-activating phosphatase Cdc25A independent of cyclin D1-Cdk4 function and cell cycle progression. Expression of p16(INK4a) inhibited G(1)/S transition induced in MCF-7 cells by 17-beta-estradiol (E(2)) with associated inhibition of both Cdk4- and Cdk2-associated kinase activities. Inhibition of Cdk2 activity was associated with delayed removal of Cdk-inhibitory activity in early G(1) and decreased cyclin A expression. Cdk-inhibitory activity and expression of both p21(Cip1) and p27(Kip1) was decreased, however, in both control and p16(INK4a)-expressing cells 20 h after estrogen treatment. Expression of Cdc25A mRNA and protein was induced by E(2) in control and p16(INK4a)-expressing MCF-7 cells; however, functional activity of Cdc25A was inhibited in cells expressing p16(INK4a). Inhibition of Cdc25A activity in p16(INK4a)-expressing cells was associated with depressed Cdk2 activity and was reversed in vivo and in vitro by active Cdk2. Transfection of MCF-7 cells with a dominant-negative Cdk2 construct inhibited the E(2)-dependent activation of ectopic Cdc25A. Supporting a role for Cdc25A in estrogen action, antisense CDC25A oligonucleotides inhibited estrogen-induced Cdk2 activation and DNA synthesis. In addition, inactive cyclin E-Cdk2 complexes from p16(INK4a)-expressing, estrogen-treated cells were activated in vitro by treatment with recombinant Cdc25A and in vivo in cells overexpressing Cdc25A. The results demonstrate that functional association of cyclin D1-Cdk4 complexes is required for Cdk2 activation in MCF-7 cells and that Cdk2 activity is, in turn, required for the in vivo activation of Cdc25A. These studies establish Cdc25A as a growth-promoting target of estrogen action and further indicate that estrogens independently regulate multiple components of the cell cycle machinery, including expression of p21(Cip1) and p27(Kip1).     

Increased intestinal chromatin template activity. Influence of 1alpha,25-dihydroxyvitamin D3 and hormone-receptor complexes.

1alpha,25-Dihydroxyvitamin D3 administration to rachitic chicks results in an increase in the chromatin template activity of intestinal target tissue assayed in vitro using Escherichia coli RNA polymerase. The maximum stimulation of template capacity was 12 to 20% over control values and occurred 2 hours after administration of the sterol. This rapid effect preceded the biologic response to 1alpha,25-dihydroxyvitamin D3 in the intestine and was not observed in other tissues such as liver or kidney. The in vivo enhancement of intestinal chromatin template activity was specific for the 1alpha,25-dihydroxyvitamin D3 hormone in that equivalent doses of 25-hydroxyvitamin D3 or vitamin D3 did not elicit a response in 2 to 3 hours. Only 1alpha-hydroxyvitamin D3, a synthetic sterol which is very rapidly metabolized to the 1alpha,25-dihydroxyvitamin D3 form, was able to minic the natural hormone in vivo. To further elucidate the nuclear mechanism of action of 1alpha,25-dihydroxyvitamin D3, the hormone was preincubated at 0 degrees with intestinal cytosol to form hormone-receptor complexes. After addition of the hormone-receptor complexes to purified intestinal mucosa nuclei and incubation for 1 hour at 25 degrees, chromatin isolated from this reconstituted system displayed a significant increase in template activity as compared to chromatin prepared from similar in vitro incubations not containing hormone. This stimulation was 12 to 24% over control values and exhibited an absolute requirement for intestinal cell cytosol. The response was specific for physiologic levels of 1alpha,25-dihydroxyvitamin D3, but occurred with pharmacologic doses of 25-hydroxyvitamin D3. It is concluded that a stimulation of the chromatin template activity of intestinal target tissue by 1alpha,25-dihydroxyvitamin D3 may be an integral part of the ultimate physiologic response of enhanced calcium transport.     

A rapid and sensitive in vitro assay of 25-hydroxyvitamin D3-1 alpha-hydroxylase and 24-hydroxylase using rat kidney homogenates

A sensitive and rapid in vitro assay of 25-hydroxyvitamin D3 [25-(OH)D3]-1 alpha- and 24-hydroxylase activities was developed using rat kidney homogenates. A potent inhibitor of the enzymes in rat plasma was removed by thoroughly perfusing rats with saline. Kidney homogenates prepared from vitamin D-deficient rats preferentially produced tritiated 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] from 25(OH) [3H]D3. Addition of 10 microliter or more of rat plasma to 3 ml of 10% kidney homogenates suppressed 1 alpha-hydroxylase activity dose-dependently. Thyroparathyroidectomy (TPTX) of vitamin D-deficient rats greatly abolished 1 alpha-hydroxylase activity. Administration of parathyroid hormone to the TPTX rats increased 1 alpha-hydroxylase activity and that of 1 alpha,25(OH)2D3 enhanced 24-hydroxylase markedly. Since this assay is technically simple, rapid and sensitive, it will be useful in studying the regulatory mechanism in the renal metabolism of 25(OH)D3 in mammals.     

Species difference exists in the effects of 1alpha,25(OH)(2)D(3) and its analogue 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD) on osteoblastic cells

The direct effect of 1alpha,25(OH)(2)D(3) on osteoblasts remains unclear. In this study, we evaluated the in vitro effects of 1alpha,25(OH)(2)D(3) and its analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D(3) (2MD), on osteoblasts from three different species, i.e. bone marrow stromal cells from the Sprague-Dawley (SD) rat, from the C57BL/6 mouse, as well as human osteoblast NHOst cells and human osteosarcoma derived MG-63 cells. We found that in rat cells, both compounds increased cell proliferation, inhibited cell apoptosis and increased alkaline phosphatase (ALP) activity. In mouse cells, however, both compounds initiated cell apoptosis and inhibited ALP activity. In human cells, although cell proliferation was inhibited by both compounds, cell apoptosis was inhibited and ALP activity was enhanced. In each species, 2MD was much more potent than 1alpha,25(OH)(2)D(3). To summarize, species differences should be taken into account in studies of vitamin D effects. However, in all tested species - rat, mouse and human - 2MD is considerably more potent in its effects on osteoblastic cells in vitro than 1alpha,25(OH)(2)D(3).     

Methyl substitution of the 25-hydroxy group on 2-methylene-19-nor-1alpha,25-dihydroxyvitamin D3 (2MD) reduces potency but allows bone selectivity

The discovery of 2-methylene-19-nor-1alpha,25-dihydroxyvitamin D3 (2MD) as a bone selective and bone anabolic form of vitamin D has stimulated an investigation of structure/function of bone selectivity. Four new 2-substituted-19-norvitamin D analogs 3-6 have been developed to study the structure-activity relationship at C-25. As predicted, removing the 25-hydroxy group (compound 3) from the very potent analog 2MD and its 2-methyl derivatives (5 and 6) dramatically reduces in vitro activities, but biological potency is nearly fully restored in vivo likely due to in vivo 25-hydroxylation. The introduction of a methyl group at C-25 (compound 4) that blocks in vivo 25-hydroxylation reduces biological activity both in vitro and in vivo. However, analog 4 retains bone selectivity making it interesting as a possible therapeutic for bone loss diseases.     

Synthesis, antimalarial, antileishmanial, and antimicrobial activities of some 8-quinolinamine analogues

In the present communication, newly synthesized 8-quinolinamines (25-27) related to previously reported 2-tert-butylprimaquine (2) were evaluated for their in vitro antimalarial activity against chloroquine sensitive and resistant Plasmodium falciparum strains, in vivo antimalarial activity against P. berghei infected mice, in vitro antileishmanial activity against Leishmania donovani, in vitro antimicrobial activity against various fungi and bacteria, and cytotoxicity in a panel of mammalian cell lines. No promising cytotoxicities were observed for compounds reported herein. Analogue 25 was found to exhibit curative antimalarial activity at a dose of 25 mg/kg/dayx4 in a P. berghei infected mice model, and produced suppressive activity at a lower dose of 10 mg/kg/dayx4. In vitro antileishmanial activities (IC50 and IC90) comparable to standard drug pentamidine were exhibited by all synthesized 8-quinolinamines 25-27. At the same time, promising antibacterial and antifungal activities were also observed for synthesized compounds against a panel consisting of several bacteria and fungi.     

Synthesis and biological evaluation of new 6-s-cis locked 1,2,25-trihydroxyprevitamin D3 analogues

An efficient synthesis of several diastereomers of 2-hydroxy substituted 1alpha,25-dihydroxyprevitamin D3 derivatives was accomplished utilizing a practical route to the A-ring synthon. The biological activity of the analogues was evaluated in vitro. All the synthesized derivatives demonstrated low affinity for the vitamin D receptor and vitamin D-binding protein compared with 1alpha,25-dihydroxyvitamin D3, the natural hormone. 1alpha,2beta,25-trihydroxy-19-nor-pre-D3 was the most potent of the analogues in inhibiting proliferation of MCF-7 cells but requires higher EC50 concentrations than 1alpha,25-dihydroxyvitamin D3.     

Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity

Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.     

The unique action for bone metabolism of 1 alpha,25-(OH)2D3-26,23-lactone

[23 (S), 25 (R)]-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone [( 23 (S),25 (R)]-1 alpha,25-(OH) 2D3-26,23-lactone) increased dose-dependently alkaline phosphatase activity in osteoblastic cells, clone MC3T3-E1, in medium containing 0.1% bovine serum albumin. The maximal stimulated enzyme activity per mg protein was 1.6-fold over that of control cultures at 250 pg/ml. The metabolite also increased collagen synthesis in a dose-related fashion. On the other hand, [23 (S),25 (R)]-1 alpha,25-(OH)2D3-26,23-lactone decreased slightly but significantly 45Ca mobilization, and blocked the resorptive action of 1 alpha,25-dihydroxyvitamin D3 but not that of parathyroid hormone, in mouse calvaria in organ culture. These results indicate that [23 (S),25 (R)]-1 alpha, 25-(OH)2D3-26,23-lactone stimulates the differentiation of osteoblasts and inhibits bone resorption in vitro.     

1 alpha, 25-Dihydroxyvitamin D3 and analogues of vitamin D3 induce alkaline phosphatase activity in osteoblastic cells derived from newborn mouse calvaria

In order to study the effects of vitamin D metabolites on bone metabolism, clone MC3T3-E1 cells, which have retained osteoblastic activity, were cultured with various concentrations of the hormone, 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3]. A physiological concentration of 1 alpha, 25 (OH)2D3 stimulated alkaline phosphatase (ALP) activity in the cells. Other metabolites--1 alpha, 24-dihydroxyvitamin D3 [1 alpha, 24 (OH)2D3], 1 alpha-hydroxyvitamin D3 [1 alpha (OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25 (OH)2D3]--also induced increases in ALP activity in a dose-dependent fashion. However, their effective concentrations were 100 or 1,000 times greater than that of 1 alpha, 25 (OH)2D3. Hormone-induced and native ALP activities in the cells were of the same type as that found in newborn mouse calvaria; that is, they were heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive (liver-bone-kidney type). These results show that vitamin D metabolites stimulate bone formation in vitro and that they may be involved in bone formation in vivo as well.     

Sulfamic and succinic acid derivatives of 25-OH-PPD and their activities to MCF-7, A-549, HCT-116, and BGC-823 cell lines

In the search for new anti-tumor agents with higher potency than our previously identified compound 1 (25-OH-PPD, 25-hydroxyprotopanaxadiol), 12 novel sulfamic and succinic acid derivatives that could improve water solubility and contribute to good drug potency and pharmacokinetic profiles were designed and synthesized. Their in vitro anti-tumor activities in MCF-7, A-549, HCT-116, and BGC-823 cell lines and one normal cell line were tested by standard MTT assay. Results showed that compared with compound 1, compounds 2, 3, and 7 exhibited higher cytotoxic activity on A-549 and BGC-823 cell lines, together with lower toxicity in the normal cell. In particular, compound 2 exhibited the best anti-tumor activity in the in vitro assays, which may provide valuable data for the research and development of new anti-tumor agents.     

Binding and catalytic properties of the Cdc2 and Crp proteins of Dictyostelium.

Dictyostelium expresses at least two proteins of the cyclin-dependent kinase (Cdk) family, Cdc2 and Crp. Cdc2 levels remain relatively constant during differentiation, whereas the levels of Crp increase dramatically as differentiation progresses. Crp is highly related to the mammalian Cdk5, and p25 (a truncated form of p35, the activating subunit of Cdk5 from mammalian brain) stimulates the histone H1 kinase activity of GST-Crp by several fold. In contrast, p25 does not stimulate the histone H1 kinase activity of GST-Cdc2 or the Cdc2 activity present in cell extracts from vegetative Dictyostelium cells. GST-Cdc2, in vitro translated Cdc2 and Cdc2 from all stages of differentiation bind to p13suc1. In contrast, GST-Crp, in vitro translated Crp and the Crp protein present in cell extracts do not bind to p13suc1. We have confirmed a previous report by Arakane and Maeda [J. Plant Res. (1997) 110, 81-85] that there is a peak of p13suc1 bound histone H1 kinase activity during late development, but we found that there was no corresponding peak of p13suc1 bound Cdc2 protein that corresponds to this activity. Taken together, these data suggest that neither Cdc2 nor Crp is responsible for the late developmental peak of histone H1 kinase activity that binds to p13suc1.     

A Cdc25A antagonizing K vitamin inhibits hepatocyte DNA synthesis in vitro and in vivo

Thioalkyl containing K vitamin analogs have been shown to be potent inhibitors of hepatoma cell growth and antagonizers of protein tyrosine phosphatase activity. We now show that they inhibit the activity of specific protein tyrosine phosphatases (PTP) in cell-free conditions in vitro, particularly the dual specificity phosphatase Cdc25A. Using primary cultures of adult rat hepatocytes that are in G0/G1 phase until stimulated into DNA synthesis by epidermal growth factor, we found that 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5) inhibited hepatocyte DNA synthesis and PTP activity in cell culture and in vivo after a two-thirds partial hepatectomy. We found a selective inhibition of Cdc25A activity in vitro, using both synthetic substrates and authentic cellular substrate, immunoprecipitated phospho-Cdk4. Intact Cpd 5-treated cells had decreased cellular Cdc25A activity and increased tyrosine phosphorylation of Cdk4, resulting in decreased phosphorylation of retinoblastoma (Rb). Loss of Cdk4 activity was confirmed using Cdk4 immunoprecipitates from either Cpd 5-treated or untreated cells and measuring its kinase activity using GST-Rb as target. We found a similar order of activity for inhibition of growth and Cdc25A activity using several thiol-containing analogs. Cdc25A inhibitors may thus be useful for defining biochemical pathways involving protein tyrosine phosphorylation that mediate cell growth inhibition.     

Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase

We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.     

B-type cyclins regulate G1 progression in fission yeast in opposition to the p25rum1 cdk inhibitor.

The onset of S phase in fission yeast is regulated at Start, the point of commitment to the mitotic cell cycle. The p34cdc2 kinase is essential for G1 progression past Start, but until now its regulation has been poorly understood. Here we show that the cig2/cyc17 B-type cyclin has an important role in G1 progression, and demonstrate that p34cdc2 kinase activity is periodically associated with cig2 in G1. Cells lacking cig2 are defective in G1 progression, and this is particularly clear in small cells that must regulate Start with respect to cell size. We also find that the cig1 B-type cyclin can promote G1 progression. Whilst p25rum1 can inhibit cig2/cdc2 activity in vitro, and may transiently inhibit this complex in vivo, cig1 is regulated independently of p25rum1. Since cig1/cdc2 kinase activity peaks in mitotic cells, and decreases after mitosis with similar kinetics to cdc13-associated kinase activity, we suggest that cig2 is likely to be the principal fission yeast G1 cyclin. cig2 protein levels accumulate in G1 cells, and we propose that p25rum1 may transiently inhibit cig2-associated p34cdc2 activity until the critical cell size required for Start is reached.     

The calcium ionophore A23187 is a potent stimulator of the vitamin D3-25 hydroxylase in hepatocytes isolated from normocalcaemic vitamin D-depleted rats.

The role played by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and/or by calcium on the C-25 hydroxylation of vitamin D3 (D3) was studied in hepatocytes isolated from D-depleted rats which were divided into four treatment groups: Group 1 served as controls, Group 2 received calcium gluconate, Groups 3 and 4 were infused with 1,25(OH)2D3 at 7 and 65 pmol/24 h x 7 days respectively. The treatments normalized serum calcium in all but the controls which remained hypocalcaemic, while serum 1,25(OH)2D3 remained low in Groups 1 and 2 but increased to physiologic and supraphysiologic levels in Groups 3 and 4. The data show that basal D3-25 hydroxylase activities were not significantly affected by any of the treatments. Addition of CaCl2, EGTA, or Quin-2 in vitro revealed that relative to basal values, EGTA strongly inhibited the enzyme activity in all groups (P less than 0.0001), except in G 1; Quin-2 and CaCl2 had no significant effect on the activity of the enzyme in any of the groups. Addition of 1,25(OH)2D3 or A23187 in vitro in the presence of CaCl2 revealed that 1,25(OH)2D3 did not significantly affect enzyme activity, while A23187 was found to stimulate its activity in vitamin D-depleted animals, but most specifically in Group 2 (P less than 0.001); low serum calcium (Group 1) dampened (P less than 0.01), and 1,25(OH)2D3 treatment in vivo totally blunted (P less than 0.001) the response to A23187. The data suggest that 1,25(OH)2D3 supplementation in vivo has per se little or no effect on the basal D3-25 hydroxylase activity. The data show, however, that the magnitude of the response to various challenges in vitro is greatly influenced by the conditioning in vivo of the animals. They also show that A23187 can be a potent stimulator of the enzyme activity, which allowed us to demonstrate a significant reserve for the C-25 hydroxylation of D3 which is well expressed in hepatocytes obtained from D-depleted calcium-supplemented rats.     

Synthesis and anti-inflammatory activity of some new 4,5-dihydro-1,5-diaryl-1H-pyrazole-3-substituted-heteroazole derivatives

A series of 4,5-dihydro-1,5-diaryl-1H-pyrazole-3-substituted-heteroazoles were designed and synthesized in order to obtain new compounds with potential anti-inflammatory activity. The title compounds were screened for in vivo anti-inflammatory activity by using Carrageenan induced rat paw edema method. Diclofenac sodium was used as a standard drug for comparison. Out of the 30 compounds tested, compound 19a, 19b, 25a, 25b exhibited significant anti-inflammatory activity. Selected compounds were also screened for in vitro COX-2 inhibition assay and analgesic activity in the acetic acid induced writhing model.