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In chronic uremia, the requirement of supraphysiological doses of serum 25-hydroxyvitamin D3 [25(OH)D3] for the normalization of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels has been attributed to impaired substrate availability to renal 1 alpha-hydroxylase. Because serum 1,25(OH)2D3 can also be corrected by 25(OH)D3 supplementation in bilaterally nephrectomized patients, we examined the role of substrate availability on 1,25(OH)2D3 production by peripheral blood monocytes (PBM). In hemodialysis patients (HP), 25(OH)D3 uptake was 50% lower than normal, and the maximal velocity (Vmax) and apparent Michaelis constant (Km) for 25(OH)D3 of 1 alpha-hydroxylase were 2.7- and 4-fold above normal, respectively. When serum 1,25(OH)2D3 of HP was corrected by intravenous 1,25(OH)2D3, 25(OH)D3 uptake, Km, and Vmax returned to normal values. The effect of 25(OH)D3 supplementation was also examined. In normal adults, 25(OH)D3 administration had no effect on serum 1,25(OH)2D3 levels nor on the Km or the Vmax of PBM 1 alpha-hydroxylase but caused a 11-fold increase in serum 24R,25-dihydroxyvitamin D3[24R, 25(OH)2D3]. In HP, 25(OH)D3 therapy raised serum 1,25(OH)2D3 and reduced the Km and Vmax of PBM 1 alpha-hydroxylase, which correlated negatively with serum 1,25(OH)2D3. However, serum 24R,25(OH)2D3 only increased slightly above basal. These results demonstrate that, in HP, 1) impaired uptake of 25(OH)D3 and low affinity for substrate determine the need for high 25(OH)D3 levels to normalize serum 1,25(OH)2D3, despite higher enzymatic activity; 2) 1,25(OH)2D3 deficiency plays a role in enhanced 1,25(OH)2D3 synthesis and impaired access of 25(OH)D3 to PBM 1 alpha hydroxylase; and 3) abnormal 25(OH)D3 delivery also affects 24-hydroxylation.

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It has been suggested recently that hormone-free glucocorticoid receptors are located predominantly in the cytoplasm, and, after the addition of steroid, they are rapidly translocated to the nucleus (3-6). The transfer of glucocorticoid receptor into the nucleus involves translocation along microtubules as revealed by immunofluorescent studies (7) in a process that is driven by tubulin-associated dynein motors (8).In the case of vitamin D, there is some controversy as to whether apoVDRs reside only in the nucleus like the thyroid hormone receptor (9) or whether they can undergo ligand-dependent translocation like the glucocorticoid receptor (10, 11). Using a recently developed fluorescent ligand, Barsony et al. (12) were able to demonstrate the cytoplasmic localization of the VDR in viable human skin fibroblasts, porcine kidney epithelial cells, human breast cancer cells, and rat osteosarcoma cells, supporting previous immunocytochemical findings in fixed human fibroblasts (13) and osteoblasts (14). Although immunocytology has shown that cytoplasmic VDR co-localizes with tubulin and that disruption of microtubular assembly blocks the translocation of the 1,25(OH) 2 D 3 -VDR complex into the nucleus (15) in microwave fixed fibroblasts, the role of microtubules on VDR transport in viable cells has never been evaluated. We hypothesized that if this intracellular transport system is of physiological relevance, the genomic response to 1,25(OH) 2 D 3 should be impaired with alterations in the structure or function of the microtubule network. We tested this hypothesis in normal human monocytes.Human monocytes express receptors for 1,25(OH) 2 D 3 that are indistinguishable from those described in classical 1,25(OH) 2 D 3 target tissues (16), and the interactions of 1,25(OH) 2 D 3 with monocytes-macrophages have critical implications for the regulation of immune responses (17)(18)(19)(20). Our laboratory has demonstrated that peripheral blood monocytes from normal individuals constitutively express 1␣-hydroxylase, the enzyme responsible for the conversion of 25-hydroxyvitamin D 3 (25(OH)D 3 ) to 1,25(OH) 2 D 3 (21). We have also shown that when peripheral blood monocytes were exposed to physiological concentrations of 1,25(OH) 2 D 3 , 1␣-hydroxylase activity is markedly suppressed. In addition, exogenous 1,25(OH) 2 D 3 promotes an induction of vitamin D catabolism by increasing 24-hydroxylase mRNA 2 and activity (22). Because both effects of the sterol require at least 2 h of exposure to 1,25(OH) 2 D 3 (22), it is likely that the inhibition of 1,25(OH) 2 D 3 production by 1,25(OH) 2 D 3 also involves a genomic mechanism. In the present studies, we used this human monocyte model to assess the physiological relevance of microtubule integrity in the response to 1,25(OH) 2 D 3 . This report demonstrates for the first time that integrity of the microtubule network is critical for a normal genomic response to 1,25(OH) 2 D 3 and that an intracel-* This work was supported in part by United States Public Health Service NIDDK, N...

The aim of this study is to address the role of mdm-2-gene amplification in the tumorigenesis of uterine sarcomas. Differential PCR with DNA from formalin-fixed paraffin-embedded specimens was employed in 12 patients with uterine sarcomas. We detected mdm-2-gene amplification in 4 out of 12 uterine sarcomas. The estimated copy number of the mdm-2 gene ranged from 4 to 13. Positive cases included 1 leiomyosarcoma and 3 carcinosarcomas, however, there was no correlation between mdm-2-gene amplification and clinicopathological characteristics. Over-expression of p53 protein was also immunohistochemically studied in the same series of patients: 4 out of 8 carcinosarcomas displayed p53 immunoreactivity. Taking these results together, only one carcinosarcoma was found to have both mdm-2-gene amplification and p53 over-expression. In contrast, half of the patients were found to have alterations either of mdm-2 or of p53. These findings support the notion that mdm-2-gene amplification might be an alternative mechanism for escaping from the regulatory pathway of p53 to suppress cell growth. Int. J. Cancer 73:33-37, 1997. Wiley-Liss, Inc.The p53 tumor-suppressor gene is altered in a wide variety of human malignancies. In fact, mutations or deletions at the p53 locus are the single most common genetic alteration in human neoplasms (Levine et al., 1994). It was initially thought that p53 was a dominant oncoprotein, however, it is now apparent that wild-type p53 is a growth-suppressor protein that inhibits the growth both of normal and of transformed cells . Furthermore, it has been revealed that p53 is a transcriptional regulatory factor with a cell-cycle-checkpoint function . A gene named WAF1, whose induction was directly associated with wild-type p53-gene expression, was identified as an important mediator of p53-dependent tumor-growth suppression (El-Deiry et al., 1993). Therefore, functional loss of p53 leads to increased proliferation and genetic instability (Livingstone et al., 1992;Yin et al., 1992).It is widely known that viral proteins (the SV 40 T, the adenovirus E1B or the human papillomavirus E6) or cellular proteins (the 70-kDa heat-shock protein) can bind to p53 protein and inactivate the function of its putative checkpoint controls Vousden, 1993). A cellular protein, the murine double minute 2 (mdm-2) oncogene product (p90) also forms a tight complex with both wild-type and mutant p53 protein, and over-expression of p90 leads to inhibition of p53-mediated transactivation (Momand et al., 1992) and to enhanced tumorigenicity of cells (Fakharzadeh et al., 1991). This gene was originally isolated from a tumorigenic mouse fibroblast cell line containing double minutes, a cytogenetic hallmark of gene amplification (CahillySnyder et al., 1987). These observations led to the hypothesis that over-expression of mdm-2 might be an alternative mechanism of loss of function of p53. In fact, the mdm-2 gene was found to be amplified in a number of human sarcomas including liposarcoma, osteosarcoma and malignant fibrous hist...