Effects of 1,25(OH)2D3, 25OHD3, and EB1089 on cell growth and Vitamin D receptor mRNA and 1alpha-hydroxylase mRNA expression in primary cultures of the canine prostate

The aim of this study was to investigate effects of 1,25(OH)(2)D(3) (calcitriol), 25OHD(3), and EB1089 on cell growth and on Vitamin D receptor (VDR) mRNA and 1alpha-hydroxylase (1alpha-OHase) mRNA expression in normal canine prostatic primary cultures. Canine prostatic epithelial cells were isolated, cultured, and treated with vehicle (ethanol), calcitriol, 25OHD(3), and EB1089 at 10(-9) and 10(-7)M. The VDR was present in epithelial and stromal cells of the canine prostate gland. 1,25(OH)(2)D(3), 25OHD(3), and EB1089 inhibited epithelial cell growth at 10(-7)M compared to vehicle-treated controls [calcitriol (P < 0.01), EB1089 (P < 0.01), and 25OHD(3) (P < 0.05)]. Epithelial cells treated with calcitriol and EB1089 at 10(-7)M had slightly increased VDR mRNA expression (0.2-0.3-fold) at 6 and 12h compared to controls. There was no difference in 1alpha-OHase mRNA expression in epithelial cells treated with these three compounds. 1,25(OH)(2)D(3) and its analogs may be effective antiproliferative agents of epithelial cells in certain types of prostate cancer.     

Effects of 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962)--a mechanism-based irreversible inhibitor of aromatase--in cultured human foreskin fibroblasts

In male subjects, peripheral aromatization of androgens accounts for most of the estrogen production, and skin is an important site of such enzymatic activity. We have studied the effects of a mechanism-based, irreversible aromatase inhibitor, 10-(2-propynyl)-estr-4-ene-3,17-dione (MDL 18,962) on androgen action and metabolism in cultured human foreskin fibroblasts. Cells were incubated simultaneously in the presence of substrate, androstenedione, and inhibitor, MDL 18,962. Aromatase activity was linear with time up to 3 h of incubation at 37 degrees C in the absence and presence of 1.0-10 nM inhibitor. The IC50 for four different cell strains ranged from 4.0 to 8.6 nM MDL 18,962. Kinetic analysis of competitive inhibition by the Eadie-Hofstee method yielded an apparent Ki of 2.75 nM for the inhibitor. Preincubation of cells with MDL 18,962 resulted in irreversible inhibition of aromatase activity which was time- and concentration-dependent. We calculated a Ki of 7.6 nM for MDL 18,962. Preincubation of cells with 25 nM MDL 18,962 suppressed enzyme activity for up to 6 h following removal of the inhibitor, before a return of enzyme activity due to synthesis of new enzyme. MDL 18,962 (0.2-20 microM) did not influence the 5 alpha-reduction of testosterone (200 nM). In addition, binding of dihydrotestosterone (2 nM) to androgen receptors was not affected by MDL 18,962 (25-1000 nM). In summary, MDL 18,962 is a specific, high potency inhibitor of aromatase. By virtue of its high binding affinity to the enzyme active site, it competes very effectively with substrate, resulting in irreversible inactivation of aromatase.     

Synthesis and evaluation of unsaturated caprolactams as interleukin-1beta converting enzyme (ICE) inhibitors

Peptidomimetic compounds possessing a caprolactam ring constraint were prepared and evaluated as interleukin-1beta converting enzyme (ICE) inhibitors. The caprolactam ring was used to constrain the P3 region of our inhibitors. This strategy proved to be effective for the synthesis of ICE inhibitors, maintaining key hydrogen bond interactions with the enzyme and invoking a preferred conformation for binding. Several compounds exhibited IC(50) values less than 10nM in a caspase-1 enzyme assay and less than 100nM in a THP-1 whole cell assay measuring IL-1beta production. Two compounds, 13c and 13j, were found to have good oral bioavailability (>50%) in rats when administered as prodrugs.     

Regulation of natural killer cytotoxicity by 1,25-dihydroxyvitamin D3

The steroid hormone 1 alpha, 25-dihydroxyvitamin D3, calcitriol, is crucial in calcium homeostasis. Calcium plays a central role in T, B, and NK cell functions, and calcitriol is a known inhibitor of T cell proliferation and immunoglobulin production. We have analyzed here the immunoregulatory effects of calcitriol on NK cell function. We show that calcitriol specifically specifically inhibits, in a time- and dose-dependent fashion, the generation of cytotoxic activity from cultured CD16+ peripheral blood NK cells. It also suppresses, at similar molar concentrations (1-10 nM), interleukin 2 (IL-2) production by PHA-activated peripheral blood lymphocytes. Calcitriol does not interfere with the cytotoxic function of NK cells, whether fresh or generated in vitro, placing the inhibition at the level of NK cell activation. Interestingly enough, exogenous IL-2 can completely reverse the suppressive effect. These findings suggest that modulation of NK cell activation by control of the internal level of IL-2 may reflect an additional paracrine calcitriol-dependent circuit with immunoregulatory consequences.     

Steroid inhibition of calcitriol-induced prolactin production in GH4C1 cells. Specificity and sensitivity.

The induction of prolactin (PRL)-gene expression by calcitriol (1,25-dihydroxyvitamin D3, 1,25-dihydroxycholecalciferol) in clonal rat pituitary tumour (GH4C1) cells was selectively inhibited by cortisol [IC50 (concentration causing 50% inhibition) = 3.2-4.1 nM]. The steroid specificity of this effect was investigated and various steroids were found to inhibit calcitriol-stimulated PRL production with the following relative potencies: cortisol, 1; dexamethasone, 8; 11-deoxycortisol, 0.5; corticosterone, 0.4; aldosterone, 0.07; testosterone and oestradiol, less than 0.003. The steroid antagonist RU 38486 did not affect basal or calcitriol-stimulated PRL production, but antagonized the effect of 10 nM-cortisol in a concentration-dependent manner. Neither progesterone nor 11-deoxycortisol antagonized the effect of 10 nM-cortisol. Calcitriol-induced PRL production was 14 times more sensitive to dexamethasone inhibition than was non-stimulated PRL production. Growth-hormone production was stimulated by dexamethasone, in the presence or absence of calcitriol, with a concentration-dependence similar to that of dexamethasone inhibition of basal PRL production. These data indicate that steroid inhibition of calcitriol-stimulated PRL production is a specific glucocorticoid effect. The sensitivity of calcitriol-stimulated PRL production to dexamethasone was 14-26-fold greater than that of other measured responses in the same cells. Two of the possible explanations for this selectively increased sensitivity to glucocorticoids are: amplification of the glucocorticoid effect via an induced mediator; and the presence of very-high-affinity glucocorticoid-receptor-binding sites on DNA.     

Stimulation of interleukin-6 production by corticotropin-releasing factor.

Based on the immune-modulating properties of corticotropin-releasing factor (CRF), the effect of this peptide for interleukin-6 (IL-6) production was investigated. Using human peripheral blood mononuclear cells (MNC), the amount of bioactive IL-6 produced was significantly (P less than or equal to 0.05) increased by CRF (10(-10) to 10(-7) M range). However, the IL-6 production of lipopolysaccharide-treated MNC cultures was not modified. At concentrations of greater than or equal to 10 nM, CRF and two analogous peptides (Tyr-CRF and alpha-helical CRF) elicited 16- to 21-fold stimulation of IL-6 production by MNC. Purified monocytes, but not purified lymphocytes, were the cells that responded to CRF action exhibiting nearly 19-fold stimulation at 100 nM concentration. The CRF-induced production of IL-6 cytokine by peripheral blood MNC may suggest a messenger role for this neurohormone in the feedback control of neuroendocrine-immune circuitry.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1-34 (PTH1-34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [3H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylase activity appeared to follow Michaelis-Menten kinetics, and 1,25-dihydroxyvitamin D-3 treatment increased the Vmax of 24-hydroxylase from 33 to 95 pmol/h per 10(6) cells without affecting the apparent Km value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 X 10(-10) M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 microM cycloheximide. Treatment of the cells with PTH1-34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 X 10(-9) M PTH1-34. When 2.4 X 10(-9) M PTH1-34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to 70.7 +/- 2.9% of control. Higher concentrations of PTH1-34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH1-34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the Vmax of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH1-34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

Effect of interleukin-6/interferon-beta 2 on glucocorticoid action in rat hepatoma cells

Reuber hepatoma cells (RHC) were treated 4 h with dexamethasone (dex), with and without simultaneous fibroblast-conditioned medium (cIL-6). A cytosol fraction, prepared in the presence of molybdate and dithiothreitol, was analyzed for [3H]dex (20 nM) binding in the presence and absence of 1 microM dex at 4 degrees C. Receptor levels declined from 76.0 fmol/mg at zero dex to 28.8 fmol/mg at 10 nM dex, and to 11.8 fmol/mg at 1 microM dex (P less than or equal to 0.05). cIL-6 plus 10 nM dex lowered binding to 18.3 fmol/mg (P less than or equal to 0.05), and treatment with cIL-6 alone diminished binding to 9.8 fmol/mg (P less than or equal to 0.05). Thus, cIL-6 diminished the number of available glucocorticoid receptors.     

Calcitriol Modulates the CD46 Pathway in T Cells

The complement regulator CD46 is a costimulatory molecule for human T cells that induces a regulatory Tr1 phenotype, characterized by large amounts of IL-10 secretion. Secretion of IL-10 upon CD46 costimulation is largely impaired in T cells from patients with multiple sclerosis (MS). Vitamin D can exert a direct effect on T cells, and may be beneficial in several pathologies, including MS. In this pilot study, we examined whether active vitamin D (1,25(OH)₂D₃ or calcitriol) could modulate the CD46 pathway and restore IL-10 production by CD46-costimulated CD4+ T cells from patients with MS. In healthy T cells, calcitriol profoundly affects the phenotype of CD46-costimulated CD4+ T cells, by increasing the expression of CD28, CD25, CTLA-4 and Foxp3 while it concomitantly decreased CD46 expression. Similar trends were observed in MS CD4+ T cells except for CD25 for which a striking opposite effect was observed: while CD25 was normally induced on MS T cells by CD46 costimulation, addition of calcitriol consistently inhibited its induction. Despite the aberrant effect on CD25 expression, calcitriol increased the IL-10:IFNγ ratio, characteristic of the CD46-induced Tr1 phenotype, in both T cells from healthy donors and patients with MS. Hence, we show that calcitriol affects the CD46 pathway, and that it promotes anti-inflammatory responses mediated by CD46. Moreover, it might be beneficial for T cell responses in MS.     

Expression of 25 hydroxyvitamin D3-1alpha-hydroxylase in human endometrial tissue

1,25(OH)(2)D(3) (calcitriol) has been shown to play an important role in cell proliferation, differentiation and immune responsiveness. The enzyme responsible for calcitriol synthesis 25 hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-OHase) has been reported in many human tissues. The aim of this study was to investigate the expression of 1alpha-OHase in gynaecological tissues. Using a highly specific nested touchdown PCR we examined the expression of 1alpha-OHase in normal and malignant endometrial tissue and in human endometrial Ishikawa cells. In addition, we analyzed the protein expression of 1alpha-OHase by Western blot. The expression of 1alpha-OHase in normal and malignant endometrial tissue and Ishikawa cells was detected and splice variants of the enzyme in Ishikawa cells were identified. These data suggest an alternative splicing of 1alpha-OHase in malignant endometrial tissue and cells. We postulate that the expression of 1alpha-OHase gene variants may contribute to the antiproliferative effects of calcitriol. In conclusion, the modulation of the 1alpha-OHase opens up a new target for vitamin D(3) related therapies in endometrial cancer.     

Metabolism of vitamin D in the human choriocarcinoma cell line JEG-3

Calcitriol is an antiproliferative prodifferentiating secosteroid that exerts a protective role for some kinds of cancer. Alterations in 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1) activity have been found in some tumor cells, but there are no studies performed in human choriocarcinoma. In the present work, calcitriol production and CYP27B1 gene regulation were studied in the human choriocarcinoma cell line JEG-3, and compared with normal human syncytiotrophoblasts (hS) in culture. In JEG-3 cells, secretion of [(3)H]calcitriol was significantly less (P<0.001) than in hS (45+/-17fmol/mg protein versus 174+/-87fmol/mg protein, respectively; n=8). CYP27B1 mRNA was similar in both JEG-3 and hS cells; but the protein was detected only in hS extracts. In contrast to the hS, JEG-3 CYP27B1 gene expression was not regulated by calcitriol or by a cAMP analogue. Our results indicate that in JEG-3 cells calcitriol production is diminished due to CYP27B1 dysregulation and low protein content, and suggest that hyperproliferation could be a consequence of these alterations.     

Suppression of PTH by the vitamin D analog eldecalcitol is modulated by its high affinity for the serum vitamin D-binding protein and resistance to metabolism

Eldecalcitol [1α,25‐dihydroxy‐2β‐(3‐hydroxypropyloxy)vitamin D₃], a vitamin D analog with enhanced efficacy for treatment of osteoporosis, has been found to be less potent than 1,25‐dihydroxyvitamin D₃ (calcitriol) in suppressing PTH in vivo. To define the mechanism for the latter observation, we compared the effects of eldecalcitol and calcitriol on PTH secretion by bovine parathyroid cells. While the two compounds showed similar potency when the cells were cultured in medium containing 15% newborn calf serum, eldecalcitol was 100 times more potent than calcitriol in the absence of serum. Eldecalcitol has a higher affinity for the serum vitamin D‐binding protein (DBP), and therefore binding to DBP, and possibly other serum components, appears to limit the uptake and activity of eldecalcitol in parathyroid cells, providing an explanation for the lower PTH suppressing activity in vivo (100% serum). However, the 100‐fold higher activity of eldecalcitol in the absence of serum was unexpected since the VDR affinity for eldecalcitol is eightfold lower than for calcitriol. The enhanced activity was not due to preferential uptake, but to a resistance to metabolism. While 1 nM [³H]calcitriol was completely degraded within 24 h, [³H]eldecalcitol was not metabolized, despite the induction of the vitamin D catabolic enzyme, 24‐hydroxylase (CYP24A). The resistance to metabolism is the likely explanation for the higher potency of eldecalcitol in suppressing PTH in cell culture lacking serum. Thus, the unique properties of eldecalcitol in vivo can be attributed, at least in part, to its high‐DBP affinity which increases the half‐life, but limits the uptake of eldecalcitol, and to its reduced metabolism, which prolongs the activity of this analog in target tissues. J. Cell. Biochem. 112: 1348–1352, 2011. © 2011 Wiley‐Liss, Inc.     

Cyclooxygenase-2 directly regulates gene expression of P450 Cyp19 aromatase promoter regions pII, pI.3 and pI.7 and estradiol production in human breast tumor cells

The present studies evaluated the direct effects of the presence of human cyclooxygenase-2 (Cox-2) on gene expression of specific promoter regions of the P450 Cyp19 enzyme aromatase enzyme and its product, estradiol, in Cox-2 null estrogen-dependent MCF-7 breast tumor cells and in a stable clone of MCF-7 cells containing transfected Cox-2 cDNA, designated as MCF-7/Cox-2 Clone 10. Clone 10 human breast tumor cells have significantly increased gene expression of total mRNA of the P450 Cyp19 enzyme aromatase, with high levels of gene expression of specific aromatase promoter (p) regions pII, pI.3, and p1.7, with no significant change in mRNA levels of p1.4. Clone 10 human breast tumor cells produced significantly increased amounts of both prostaglandin E2 (PGE2) derived from Cox-2 enzyme activity and estradiol derived from aromatase enzyme activity (p<0.01), compared to MCF-7/vector control cells. The greatest inhibition of PGE2 or estradiol production was observed by the combination of the selective Cox-2 inhibitor celecoxib (25 microM) and the aromatase inhibitor, formestane (10nM) (p<0.01). The greatest anti-proliferative effect in Cox-2 null MCF-7/vector control cells was observed with the combination of 25 microM celecoxib and 10nM formestane but not with 10 microM celecoxib, suggesting that there are Cox-2-independent mechanisms involved in the anti-proliferative effect of this agent at doses greater than 10 microM. Celecoxib (25 microM) also significantly inhibited proliferation of MCF-7/Cox-2 Clone 10 human breast tumor cells, with no further anti-proliferative activity with the addition of 10 nM formestane observed at either 24 or 48 h of treatment. These studies demonstrate that Cox-2 directly regulates gene expression of specific aromatase promoter regions and regulates aromatase enzyme activity. Agents that inhibit Cox-2 or block the biological effects of PGE2 may be useful in significantly limiting aromatase activity and proliferation of human breast tumor cells regardless of the presence of Cox-2. In addition, the unique human breast tumor cell model used in these studies may be a useful tool in identifying the spectrum of activities of agents that block the biological effects of PGE2 and estradiol.     

Epidermal growth factor receptors in porcine endometrium: binding characteristics and the regulation of prostaglandin E and F2 alpha production.

Epidermal growth factor (EGF) and its receptor have been implicated in the control of uterine cell growth and differentiation. The objectives of this study were to determine EGF binding characteristics and effects of EGF on prostaglandin (PG) production in vitro by glandular and stromal cells from porcine endometrium. Endometrial tissues were taken from 10 sows on Day 13 of pregnancy (first day of estrus = Day 0). Glandular and stromal cells were separated by enzymatic dispersion and sieve filtration and cultured for 3 days. EGF-binding assay was carried out at 20 degrees C in the presence of 0.2 nM 125I-EGF with increasing concentrations of unlabeled EGF (0-12 nM). Scatchard analyses revealed one class of high-affinity binding sites in each cell type with apparent equilibrium dissociation constants (n = 6) of 2.96 +/- 0.60 nM and 2.48 +/- 0.50 nM for stromal and glandular cells, respectively. The apparent binding capacities were 199.3 +/- 34.8 fmol/10(6) cells for stromal cells and 40.7 +/- 6.5 fmol/10(6) cells for glandular cells. Effects of EGF on PG production were determined by including 1, 5, 10, or 20 ng/ml EGF in the medium for the final 24 h of the 72-h culture. EGF increased PGE (p less than 0.01) and PGF2 alpha (p less than 0.05) secretion by stromal cells. The highest concentration (20 ng/ml) of EGF increased secretion of PGE and PGF2 alpha by 133% and 64%, respectively, over controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of LHRH effect on testosterone production by Leydig cells from fetal mouse testis

The effect of LHRH and one of its agonist (des-gly10 (D-Ala6)-LHRH-ethylamide) on the functional activity (testosterone and progesterone production) of purified fetal mouse Leydig cells was examined in short-term primary culture and under dynamic conditions. The continuous presence of increasing concentrations of LHRH (10(-10) to 10(-6) M) for 3 days was unable to affect the hCG-stimulated testosterone production on any day of culture. Stimulated testosterone production progressively decreased from day 1 to day 3 of culture (P less than 0.001). Progesterone accumulation increased in both basal and hCG stimulated conditions during the same period (P less than 0.001) and was not altered by the presence of LHRH at all three concentrations tested. There was no effect of LHRH pretreatment either on the basal production or on the acute hCG stimulation studied during a subsequent 6 h incubation. Exposure of cells to hCG for 120 min enhanced testosterone accumulation. No change in kinetic characteristics was observed when LHRH (10(-6) M) was continuously present in the medium. These results show that LHRH does not have any detectable effect on the fetal population of Leydig cell in the mouse.     

Inhibin production by macaque granulosa cells from pre- and periovulatory follicles: regulation by gonadotropins and prostaglandin E2.

Although inhibin (IN) is secreted by granulosa cells (GC) of preovulatory follicles, the major source of immunoreactive IN circulating during the primate ovarian cycle is the corpus luteum. The aims of this study were (1) to investigate culture conditions for optimal IN production by luteinized GC (LGC) from rhesus monkeys and (2) to compare IN and progesterone (P) production by nonluteinized GC (NGC) and LGC in response to putative agonists. Animals were treated for up to 9 days with human menopausal gonadotropins to promote the development of multiple preovulatory follicles. GC were obtained from large follicles before (NGC) or 27 h after (LGC) an ovulatory injection of hCG. For Aim 1, cells were cultured in Hams F-10 medium +/- hCG (100 ng/ml) with or without the addition of insulin/transferrin/selenium, 10% fetal bovine serum, or 10% Serum-Plus (JRH Biosciences, Lenexa, KS). Medium was changed on Days 1, 2, 4, 6, and 8, and IN and P concentrations were determined by RIA. Basal (unstimulated) IN production by LGC was enhanced and maintained for 6-8 days in the presence of serum, but rapidly declined in the absence of serum. In contrast, basal P secretion declined regardless of exposure to serum. Human CG consistently increased (p less than 0.05) IN production only in the presence of serum but stimulated (p less than 0.05) P production under all conditions. For Aim 2, cells were cultured for 4 days in Ham's F-10 medium + 10% macaque serum +/- hCG (100 ng/ml), hFSH (100 ng/ml), prostaglandin E2(PGE2; 14 microns), or dibutyryl(db)-cAMP (5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Vitamin D insensitive prostate cancer cells

The antitumor effects of 1,25-dihydroxyvitamin D₃ (calcitriol) are being exploited for prevention and treatment of prostate cancer (CaP). These studies examined the antiproliferative effects of calcitriol in primary cell cultures derived from transgenic adenocarcinoma of mouse prostate (TRAMP) mice chronically treated with calcitriol (20 μg/kg) or vehicle 3×/week from 4 weeks-of-age until palpable tumors developed. This is a report on the response of two representative control (Vitamin D naïve, naïve) and calcitriol-treated (Vitamin D insensitive, VDI) cells to calcitriol. VDI cells were less sensitive to calcitriol based on less cell growth inhibition and less inhibition of DNA synthesis as measured by MTT and BrdU incorporation assays. Similarly, VDI cells were less sensitive to growth inhibition by the vitamin analog, 19-nor-1,25-dihydroxyvitamin D₂ (paricalcitol). There was no change in apoptosis following treatment of naïve and VDI cells with calcitriol. Vitamin D receptor (VDR) expression was up-regulated by calcitriol in both naïve and VDI cells. In addition, calcitriol induced the Vitamin D metabolizing enzyme, 24-hydroxylase (cyp24) mRNA and enzyme activity similarly in naïve and VDI cells as measured by RT-PCR and HPLC, respectively. In summary, VDI cells are less responsive to the antiproliferative effects of calcitriol. Understanding Vitamin D insensitivity will further clinical development of Vitamin D compounds for prevention and treatment of CaP.     

Expression of splice variants of 1alpha-hydroxylase in mcf-7 breast cancer cells

1,25-Dihydroxyvitamin D(3) (calcitriol) is the most active natural metabolite of Vitamin D(3). It has strong antiproliferative and differentiating effects on various cell types including breast cancer cells. 25-Hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase, CYP27B1) is one of the key enzymes in the formation of calcitriol. It has been found in breast cancer cells suggesting an autocrine regulation of formation of calcitriol in these cells. Alternative splicing of the encoding genes for this enzyme can possibly play a role in regulating the enzyme level and can explain tissue specific variations of 1alpha-hydroxylase activity. Splice variants containing intron 1 may encode for truncated proteins with deletion of protein domains which are essential for its enzymatic activity. In order to obtain more information on the abundance of 1alpha-hydroxylase splice variants, we performed a highly specific nested touchdown PCR in MCF-7 cells. The full-length sequence of 1alpha-hydroxylase and two different splice variants of this enzyme containing intron 1 were isolated. By Western blot technique we then confirmed the protein products of the full-length enzyme and its splice variants. We hypothesize that that the expression of splice variants can lead to a quantitatively lower expression of the mRNA of the full-length enzyme. The abundance of less active 1alpha-hydroxylase protein variants can alter the local synthesis of calcitriol in the cells and may explain variations of enzymatic activity in different cells and tissues.     

Purification and characterization of selenomethionyl thymidylate synthase from Escherichia coli: comparison with the wild-type enzyme

Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques [Yang et al. (1990) Science (Washington, D.C.) 249, 1398-1405]. The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet. We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E. coli Rue10. An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E. coli in 90% yield in no more than 8 h. The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values. Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h). The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen. Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes. In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme.