William S. Mellon

Effect of Molecular Structure on the Performance of Triarylmethane Dyes as Therapeutic Agents for Photochemical Purging of Autologous Bone Marrow Grafts from Residual Tumor Cells

Extensively conjugated cationic molecules with appropriate structural features naturally accumulate into the mitochondria of living cells, a phenomenon typically more prominent in tumor than in normal cells. Because a variety of tumor cells also retain pertinent cationic structures for longer periods of time compared with normal cells, mitochondrial targeting has been proposed as a selective therapeutic strategy of relevance for both chemotherapy and photochemotherapy of neoplastic diseases. Here we report that the triarylmethane dye crystal violet stains cell mitochondria with efficiency and selectivity, and is a promising candidate for photochemotherapy applications. Crystal violet exhibits pronounced phototoxicity toward L1210 leukemia cells but comparatively small toxic effects toward normal hematopoietic cells (murine granulocyte-macrophage progenitors, CFU-GM). On the basis of a comparative examination of chemical, photochemical, and phototoxic properties of crystal violet and other triarylmethane dyes, we have identified interdependencies between molecular structure, and selective phototoxicity toward tumor cells. These structure-activity relationships represent useful guidelines for the development of novel purging protocols to promote selective elimination of residual tumor cells from autologous bone marrow grafts with minimum toxicity to normal hematopoietic stem cells.

An equilibrium and kinetic study of 1,25-dihydroxyvitamin D3 binding to chicken intestinal cytosol employing high specific activity 1,25-dihydroxy[3H-26, 27]vitamin D3

Abstract A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D3 binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[ 3 H-26,27 ]vitamin D3 (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (Kd) of 7.1 × 10−11 m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 103 receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D3 binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 108 m − min− and 7.1 × 10−3 min−, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[ 3 H-26,27 ] vitamin D3 a truly high affinity binding site.

Evaluation of Urothelial Stretch-Induced Cyclooxygenase-2 Expression in Novel Human Cell Culture and Porcine in Vivo Ureteral Obstruction Models

Obstruction and stretch induce cyclooxygenase (COX)-2 expression and prostanoid synthesis in urinary tissues, causing pain, inflammation, hypercontractility, and cell proliferation. Our objective was to characterize acute COX-2 induction during in vivo ureteral obstruction, establish a cell culture model of urothelial stretch-induced COX-2 expression, and evaluate whether mechanotransduction could alter transcriptional and post-transcriptional regulation of COX-2. We performed laparoscopic unilateral ureteral ligation in pigs and allowed progression for 1, 2, 6, 24, or 48 h. We evaluated COX-2 expression with reverse transcriptase (RT)-polymerase chain reaction (PCR) and immunoblotting. We cultured primary human urothelial cells on stretch plates, applied stretch for up to 48 h, and measured COX-2 expression by RT-PCR and immunoblotting, transcription with run-on assays, and mRNA stability with actinomycin mRNA decay assays. In vivo ureteral obstruction induced COX-2 expression 4-fold within 6 h, maintaining induction for 24 h. In cell culture, stretch induced COX-2 steady-state mRNA and protein within the first 3 h of stretch, maintaining this induction for over 6 h. Three hours of stretch doubled COX-2 transcription relative to unstretched controls and increased COX-2 mRNA half-life 3-fold. This is the first report to characterize in vivo temporal stretch-induced COX-2 expression in the urothelium and establish a primary urothelial cell culture model for the study of stretch-induced COX-2 mechanisms. This is also the first report to identify alterations in steady-state COX-2 mRNA having components of both transcriptional and post-transcriptional regulation of stretch-regulated COX-2. Future elucidation of COX-2 signaling may identify novel therapeutic targets for treating stretch and distension of urinary tissues.

An in vitro study of the stability of the chicken intestinal cytosol 1,25-dihydroxyvitamin D3-specific receptor☆

Abstract The stability of the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 has been examined using radiological binding studies and sucrose density gradient ultracentrifugation. Specific binding of 1,25-dihydroxyvitamin D3 to the 3.7 S binding protein decreases in crude cytosol in a time- and temperature-dependent manner. Increased receptor instability is also observed outside a pH range of 6 to 10. Ionic strength does not seem to be a critical factor in preventing loss of specific 1,25-dihydroxyvitamin D3 binding activity. However, when KCl is present at a concentration of 300 m m during cytosol preparation, quantitatively more specific binding per unit protein was obtained. Consistent with the idea that loss of specific binding might be due to enzymatic degradation or inactivation of receptor, dilution of cytosol was found to slow the rate of loss of specific 1,25-dihydroxyvitamin D3 binding. The importance of maintaining a reducing environment for the 1,25-dihydroxyvitamin D3 binding protein is demonstrated by the destruction of binding activity by n -ethylmaleimide and by the increased stability in the presence of 5.0 m m dithiothreitol. Likewise, 5.0 m m monothioglycerol was partially effective in preventing the loss of specific 1,25-dihydroxyvitamin D3 binding during in vitro incubation. Several protease inhibitors were not able to exert a stabilizing influence on receptor integrity during in vitro incubations. Albeit, both tosylamide-phenylethylchloromethyl ketone and p-hydroxymercuribenzoate actually decreased specific 1,25-dihydroxyvitamin D3 binding. This inhibition appeared to be reversible if samples were subsequently incubated in the presence of dithiothreitol. These results clearly demonstrate that the aporeceptor is extremely unstable and the integrity of sulfhydryl constituents is of primary importance.

1,25-Dihydroxyvitamin D-3 destabilizes c-myc mRNA in HL-60 leukemic cells

The differentiation process is accompanied by alterations in the expression of a variety of genes. Monocytic maturation of hematopoietic cells (HL-60) induced by 1,25-dihydroxyvitamin D-3 (1,25(OH)2D3), results in a decrease in steady state c-myc mRNA levels. To elucidate the mechanism by which 1,25(OH)2D3 regulates c-myc mRNA expression, transcriptional and post-transcriptional modes of regulation were investigated. No transcriptional regulation was identified, however, 1,25(OH)2D3 appeared to decrease steady state c-myc mRNA levels by increasing its turnover rate. Using actinomycin D to block transcription, the half-life of c-myc mRNA was shown to decrease from 20 min in the absence of 1,25(OH)2D3 to < 5 min in the presence of 1,25(OH)2D3. Cycloheximide reversed the instability induced by 1,25(OH)2D3, prolonging the half-life of c-myc mRNA in both uninduced and 1,25(OH)2D3-induced HL-60 cells to > 60 min, indicating a translational requirement for the destabilization process. Additionally, the c-myc mRNA instability induced by 1,25(OH)2D3 in HL-60 appears to be a specific result of this agent, as indicated by the inability of other monocytic and granulocytic differentiation inducing agents to destabilize c-myc mRNA.

Evidence for the metabolism of 24R-hydroxy-25-fluorovitamin D3 and 1α-hydroxy-25-fluorovitamin D3 to 1α,24R-dihydroxy-25-fluorovitamin D3

Abstract High-pressure liquid chromatography capable of resolving all known vitamin D metabolites and a sensitive competitive binding protein assay specific for 1α,25-dihydroxyvitamin D 3 were used to assay the blood of rats dosed with ethanol, 1α-hydroxyvitamin D 3 , 24 R -hydroxy-25-fluorovitamin D 3 , or 1α-hydroxy-25-fluorovitamin D 3 . Compared to the ethanoldosed animals, the blood of rats dosed with 1α-hydroxyvitamin D 3 had increased levels of 1α,25-dihydroxyvitamin D 3 ; but those dosed with the fluorinated vitamins did not. Instead, their blood contained a compound that cochromatographs with 1α,24 R -dihydroxyvitamin D 3 on high-pressure liquid chromatography and binds to the 1,25-dihydroxyvitamin D 3 receptor proteins. 1α,24 R -Dihydroxyvitamin D 3 binds as well as 1α, 25-dihydroxyvitamin D 3 to the chick-intestinal cytosol receptor protein for 1α,25-dihydroxyvitamin D 3 ; whereas 1α,24 S -dihydroxyvitamin D 3 binds only one-tenth as well as 1α,25-dihydroxyvitamin D 3 . Thus it appears that in vivo , the fluorinated vitamin D compounds are converted to a compound likely to be 1α,24 R -dihydroxy-25-fluorovitamin D 3 and that may rival the potency of 1α,25-dihydroxyvitamin D 3 .

1,25-Dihydroxyvitamin D3 selectively translocates PKCα to nuclei in ROS 17/2.8 cells

We have investigated protein kinase C (PKC) regulation by 1,25-(OH)2D3 in the rat osteosarcoma cell line ROS 17/2.8 since previous reports have implicated PKC in the 1,25-(OH)2D3-mediated regulation of osteocalcin gene expression (J. Biol. Chem. 267 (1992) 12562; Endocrinology 136 (1995) 5685). Here we report that 1,25-(OH)2D3 increased PKCalpha, but not PKCbetaI, epsilon or zeta, levels in the nuclear fraction in a time-dependent manner. Unlike PMA, 1,25-(OH)2D3 did not alter the association of any of the expressed PKC isoenzymes with the plasma membrane. Treatment with 20 nM 1,25-(OH)2D3 for 15 min, 30 min, 1 h and 24 h increased PKCalpha levels in the nuclear fraction by 2.3- to 2.6-fold. Nuclear PKCalpha expression was also increased with doses of 1,25-(OH)2D3 as low as a 0.05 nM. 1,25-(OH)2D3-mediated stabilization of osteocalcin mRNA (Arch. Biochem. Biophys. 332 (1996) 142) was inhibited with bisindolylmaleimide treatment, suggesting that PKCalpha may be involved in the 1,25-(OH)2D3-mediated regulation of osteocalcin gene expression.

Ring A-stereoisomers of 1-hydroxyvitamin D3 and their relative binding affinities for the intestinal 1α,25-dihydroxyvitamin D3 receptor protein

Abstract A set of eight 1-hydroxyvitamin D 3 compounds comprising the four possible (5 Z )-1,3-diol stereoisomers and the corresponding (5 E )-double bond isomers, has been prepared in order to assess the effect of 1,3-diol stereochemistry and 5,6-double bond geometry on binding affinity for the intestinal 1,25-(OH) 2 D 3 -receptor protein. The compounds were synthesized from either vitamin D 3 or 3-epivitamin D 3 via 3,5-cyclovitamin D intermediates. Competitive receptor binding assays establish that all changes from the natural ring A-configuration (1 S , 3 R , 5 Z ) lead to decreased binding affinity, and confirm the importance of the 1-hydroxy function since the conversion of stereochemistry at that center from 1 α ( S ) to 1 β ( R ) has the most pronounced effect on binding affinity (attenuation by more than three orders of magnitude). Other modifications (i.e., conversion at C-3, or cis to trans isomerization of the 5,6-double bond) decrease binding affinity by more moderate (ca. 10-fold) but cumulative factors.

Induction of monocytic differentiation by calcitriol (1,25-dihydroxyvitamin D3) in the human promyelocytic leukemic cell line (HL-60) in serum-free medium.

The effect of calcitriol on the induction of differentiation in human promyelocytic leukemic cell line (HL-60) cultured in serum-free chemically defined medium (SFM) was investigated. The utilization of SFM containing RPMI-1640 basal medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), sodium selenite (5 ng/ml), and bovine serum albumin (0.5 micrograms/ml), transferrin examination of the cellular/molecular mechanism of calcitriols action in HL-60 cell differentiation without interference of components present in serum. HL-60 cells grown in SFM were induced to differentiate into monocytes/macrophages by calcitriol as indicated by induction of differentiation-associated biological and biochemical parameters: chemiluminescent (CL) responsiveness, lysozyme activity, nonspecific esterase, expression of cell surface antigens, and reduced proliferation. The exposure of HL-60 cells in SFM to calcitriol (from 10(-10) to 10(-8)M) resulted in dose-dependent induction of these parameters, which was similar to those obtained with cells grown in 10% fetal calf serum containing medium (10% SCM). However, calcitriol was 5-fold more potent for HL-60 cells cultured in SFM than those cultured in 10% SCM as indicated by shifts in dose-response curves for induction of CL responsiveness and lysozyme activity. The effect of calcitriol on the proliferation and acquisition of several monocyte-associated cell surface antigens was also more sensitive for HL-60 cells cultured in SFM than for cells grown in 10% SCM. We characterized and quantitated calcitriol receptors in HL-60 cells cultured in SFM in comparison to those in 10% SCM after exposing intact cells to radiolabeled calcitriol. Cells cultured in either SFM or 10% SCM exhibited calcitriol receptors that migrated at 3.4S as a single peak on sucrose gradients and elicited inherent DNA binding ability. There was essentially no difference in the apparent dissociation constants (Kd) nor in the number of calcitriol binding sites per HL-60 cell, that is approximately 6.0 X 10(-11) M and approximately 3000 binding sites/cell respectively. It is concluded that culturing HL-60 cells in SFM results in full expression of calcitriol-induced phenotypic changes excluding the possibility that such changes result from the indirect effect of calcitriol mediated by identified and/or unidentified components present in serum.

Characterization of 1,25-dihydroxyvitamin D3-receptor complex interactions with DNA by a competitive assay☆

Abstract To identify and assess the specificity of the 1,25-dihydroxyvitamin D3 chick intestinal cytoplasmic receptors nucleotide binding site, a competitive DNA-cellulose binding assay was utilized. Unlike other steroid hormone receptors, the 1,25-dihydroxyvitamin D3-receptor complex binds homologous DNA at 4 °C and does not appear to undergo thermal- or salt-induced activation. Data are presented which suggest that receptor binding discriminates between double-stranded DNA and RNA but is not specific with respect to DNA base sequences. However, DNA base sequence selectivity by 1,25-dihydroxyvitamin D3-receptor complexes is observed using synthetic polydeoxyribonucleotides, particularly, poly(dA-dT) · poly(dA-dT) and poly(dA) · poly(dT). Preference for double-stranded over single-stranded DNA was also observed. Consistent with this finding, both actinomycin D and ethidium bromide caused a dose-dependent inhibition of receptor binding to DNA-cellulose. It is concluded that the 1,25-dihydroxyvitamin D3-receptor complex has specificity for AT-rich segments of double-stranded DNA and that this interaction is not merely electrostatic, but also involves hydrophobic interaction with the major and/or minor grooves of the DNA helix.