Identification and cloning of a human urea transporter HUT11, which is downregulated during adipogenesis of explant cultures of human bone.

Bipotential cells in human trabecular bone explant cultures that express osteoblast characteristics are able to undergo adipogenesis in the presence of 3-isobutyl-1-methylxanthine plus dexamethasone (Nuttall et al. [1998] J Bone Miner Res 13:371-382). The initial studies of these bipotential cells in explant cultures have been extended to examine differential gene expression during osteoblast/adipocyte transdifferentiation. Using differential display, we have identified a gene expressed in trabecular bone explant cultures that is downregulated as these cells differentiate from an osteoblast to an adipocyte phenotype. Homology searching identified this gene as the human urea transporter HUT11. The expression and downregulation of HUT11 have been observed in multiple patient bone explant cultures. The size of the bone explant-derived HUT11 mRNA is approximately 4.4 kb, which is identical to the largest splice variant reported. In this article, we report the cloning and sequencing of this gene from primary human osteoblasts. In addition, we report tissue distribution for the bone explant-derived form of HUT11 mRNA and show a reciprocal relationship between the expression of HUT11 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma 2, which is a marker of adipocyte differentiation. Because the control of osteoblast/adipocyte transdifferentiation is unknown, selective downregulation of HUT11 during adipogenesis suggests that HUT11 expression may be a marker of the switch from an osteoblast to an adipocyte phenotype. Understanding the role of HUT11 in osteoblasts may provide insights into the mechanism controlling osteoblast and adipocyte differentiation.

Screening for high-affinity ligands to the Src SH2 domain using capillary isoelectric focusing-electrospray ionization ion trap mass spectrometry.

A solution-based microscale approach for determination of high-affinity noncovalent complexes from mixtures of compounds is presented, based on capillary isoelectric focusing coupled on-line with electrospray ionization ion trap mass spectrometry. The studies are performed using the src homology 2 domain and tyrosine-phosphorylated peptide ligands as a model system. Tight complexes are formed in solution, preconcentrated up to 2 orders of magnitude and separated on the basis of their isoelectric points. The complexes are then dissociated in the mass spectrometer and the freed ligands identified. Picomole or less amounts of protein reagent are consumed per experiment. Structural information for the ligands involved in tight complex formation may be obtained using the MSn capabilities of the ion trap. The methodology can potentially be used to screen rapidly combinatorial mixtures of compounds for high-affinity ligands.

Tumor-initiating activity of the 9,10-dihydrodiol- and 9,10-dihydrodiol-7,8-epoxide of 3-methylcholanthrene in SENCAR mice.

The skin tumor-initiating activities of several bay-region metabolites of 3-methylcholanthrene (3-MCA) were determined in SENCAR mice. 3-MCA-anti-9,10-diol-7,8-epoxide possessed weak tumor-initiating activity when tested at 100 and 200 nmol/mouse doses (0.27 and 0.67 papillomas per mouse after 18 weeks of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA)). 3-MCA-trans-9,10-diol at initiating doses of 50 and 100 nmol/mouse was as active as 3-MCA. 3-MCA-trans-9,10-diol was also tested for mutagenic activity toward V79 cells in cell-mediated assays and found to be approximately 2-times more potent than 3-MCA. The data suggest that 3-MCA-trans-9,10-diol is a proximate carcinogen for mouse skin.

Mechanism of mouse skin tumor promotion by chrysarobin.

The skin tumor-promoting ability of 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin) was compared with that of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,8-dihydroxy-9-anthrone (anthralin) in SENCAR mice. Although dose-response comparisons indicated that chrysarobin was several orders of magnitude less potent than TPA for promoting papilloma formation, this anthrone was 1.5 to 2 times more potent than anthralin. Maximal papilloma responses were achieved by 15 weeks of promotion with TPA whereas at least 25 weeks of promotion were necessary to achieve maximal papilloma responses with chrysarobin or anthralin indicating marked differences in tumor latency between the two classes of compounds. Interestingly, at optimal promoting doses, chrysarobin gave a carcinoma response (22% with 0.3 carcinomas per mouse at 45 weeks) similar to that of TPA suggesting that this compound may be more efficient at promoting carcinomas than papillomas. In two-stage promotion experiments, chrysarobin was incapable of functioning independently as a Stage I or II promoter despite its complete promoting activity. Chrysarobin and TPA were compared at optimal promoting doses for their ability to induce: (a) skin edema, (b) epidermal hyperplasia, and (c) epidermal ornithine decarboxylase. In each case, distinct differences were noted between the two compounds. When taken together, the data support the hypothesis that anthracene-derived skin tumor promoters work at least in part by a mechanism different from the phorbol esters.

Formation and disappearance of benzo[a]pyrene DNA-adducts in mouse epidermis.

The rates of formation and disappearance of benzo[a]pyrene (B[a]P) DNA-adducts were analyzed in the epidermis of SENCAR mice over a 21-day time course. Mice were treated topically with 200 nmol/mouse of [3H]B[a]P at various times prior to sacrifice. The formation and disappearance of total adducts as well as individual adducts was determined and in addition, the rate of DNA turnover was monitored concurrently so that adduct disappearance could be expressed as a function of epidermal cell turnover. Under these experimental conditions, covalent binding of B[a]P to epidermal DNA reached a peak 24 h after treatment. Interestingly, between 24-48 h after application of the hydrocarbon there was a very rapid drop in the level of bound B[a]P to value approximately 50% of the maximum level at 24 h. Thereafter, the level of bound B[a]P disappeared at a much slower rate. In dual-label experiments, where the epidermal DNA was pre-labeled with [14C]thymidine, [3H]B[a]P DNA-adduct disappearance between 24-48 h was clearly more rapid than could be explained on the basis of epidermal DNA turnover. By 72 h and beyond, however, [3H]B[a]P DNA-adduct disappearance approximately paralleled DNA turnover. Examination of the rate of formation and disappearance of individual B[a]P DNA-adducts (nine individual adducts) suggested that some deoxyadenosine adducts were removed more rapidly than deoxyguanosine adducts. The results indicate that at least some epidermal cells have the capacity to repair B[a]P DNA-adducts. The data are discussed in relation to the process of tumor initiation in mouse skin.

Covalent binding of 7,12-dimethylbenz(a)anthracene and 10-fluoro-7,12-dimethylbenz(a)anthracene to mouse epidermal DNA and its relationship to tumor-initiating activity.

10-Fluoro-7,12-dimethylbenz(a)anthracene (10-F-DMBA) is a more potent skin tumor initiator in SENCAR mice when compared with the parent hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA). To elucidate the mechanism for this difference, the covalent binding of these two hydrocarbons to the DNA of mouse epidermal cells in vivo and in vitro was compared. The quantity of 10-F-DMBA covalently bound to mouse epidermal DNA in vivo was greater than that of DMBA at all doses tested over the range of 4 to 200 nmol/mouse. The magnitude of this binding difference between 10-F-DMBA and DMBA was greater at the higher doses (e.g., 1.5-fold at 4 nmol/mouse versus 3.4-fold at 200 nmol/mouse). These results correlated closely with the dose-response relationships for tumor initiation by the two hydrocarbons. Analysis of the isolated DNA samples by Servacel DHB chromatography revealed the relative proportion of syn-diol-epoxide:DNA adducts derived from DMBA increased dramatically as a function of dose (approximately 30% at 4 nmol/mouse versus approximately 55% at 200 nmol/mouse). Conversely, the relative proportion of syn-diol-epoxide adducts derived from 10-F-DMBA was low and remained essentially constant over the same dose range. High-pressure liquid chromatographic analyses of the DNA adducts derived from DMBA- and 10-F-DMBA-treated mice revealed qualitatively similar profiles. However, as expected, there was a marked reduction in the relative proportion of syn-diol-epoxide:DNA adducts in the profiles of epidermal samples from 10-F-DMBA-treated mice. The major syn-diol-epoxide:deoxy-adenosine adduct was present at a level only 30% that found in high-pressure liquid chromatographic profiles of DMBA samples. Similar results were obtained when primary cultures of mouse epidermal cells were treated with the hydrocarbons. The results suggest that the increased total binding and possibly the decreased proportion of syn-diol-epoxide:DNA adducts confer greater tumor-initiating potency on 10-F-DMBA.

DBA/2 mice are as sensitive as SENCAR mice to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

Mice of the inbred strain DBA/2 responded to a two-stage, initiation-promotion tumorigenesis protocol when high initiating doses (400 nmol/mouse) of 7,12-dimethylbenz[a]anthracene were utilized. They also responded when N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was used as the initiating agent. The tumor response in both cases was characterized by a rapid rate of tumor development with the maximal tumor responses reached on or before the 15th week of promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). When DBA/2 mice were compared with SENCAR mice for promotion sensitivity following initiation with MNNG, the two mouse stocks responded with a nearly identical tumor response. C57BL/6 mice were essentially resistant to TPA promotion regardless of the initiator or the dose of initiator used. A preliminary study was conducted to determine how susceptibility to tumor promotion by TPA was inherited in F1 mice derived from DBA/2 (sensitive) and C57BL/6 (resistant) parents. The B6D2F1 mice were as sensitive as the DBA/2 parent, suggesting that susceptibility in these two inbred mouse strains is inherited as an autosomal dominant trait. The results show that these two inbred mouse strains may provide a model system for studying genetic factors controlling susceptibility to phorbol ester skin tumor promotion.