Mark Van Camp

NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization

Here, we report on the identification of nucleolar spindle–associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.

The Effects of 1α,25‐Dihydroxyvitamin D3 on the Expression of DNA Replication Genes

To identify key genes in the antiproliferative action of 1,25(OH)2D3, MC3T3‐E1 mouse osteoblasts were subjected to cDNA microarray analyses. Eleven E2F‐driven DNA replication genes were downregulated by 1,25(OH)2D3. These results were confirmed by quantitative RT‐PCR in different cell types, showing the general nature of this action of 1,25(OH)2D3.

Interaction of Two Novel 14-Epivitamin D3 Analogs with Vitamin D3 Receptor-Retinoid X Receptor Heterodimers on Vitamin D3 Responsive Elements

This study provides a detailed and exact evaluation of the interactions between vitamin D3 receptor (VDR), retinoid X receptor (RXR), and vitamin D3 responsive elements (VDREs) mediated by two novel 14‐epianalogs of 1,25‐dihydroxyvitamin D [1,25(OH)2D3], 19‐nor‐14‐epi‐23‐yne‐1,25(OH)2D3 (TX 522) and 19‐nor‐14,20‐bisepi‐23‐yne‐1,25(OH)2D3 (TX 527). Both analogs were more potent (14‐ and 75‐fold, respectively) than 1,25(OH)2D3 in inhibiting cell proliferation and inducing cell differentiation. However, DNA‐independent experiments indicated that both analogs had a lower affinity to VDR and that the stability of the induced VDR conformation, as measured by limited protease digestion assays, was similar (TX 527) or even weaker (TX 522) than that induced by the parent compound. However, DNA‐dependent assays such as gel shift experiments revealed that those analogs were slightly more potent (3–7 times) than 1,25(OH)2D3 in enhancing binding of VDR‐RXR heterodimers to a direct repeat spaced by three nucleotides (DR3) type VDRE. The functional consequences of the ligand‐VDR‐RXR‐VDRE interactions observed in vitro were subsequently evaluated in transfection experiments. Both 14‐epianalogs enhanced transcription of VDRE containing reporter constructs more efficiently than 1,25(OH)2D3 in COS‐1 and MCF‐7 cells regardless of the presence of ketoconazole. Transactivation activity is suggested to be a cell‐specific process because maximal transcriptional induction and the half‐maximal transactivation concentration for each reporter construct were different in both cell lines. The superagonistic transactivation activity closely resembled the biological potency of these analogs on the inhibition of MCF‐7 cell proliferation. These data clearly indicate that superagonistic activity starts beyond the binding of the ligand‐heterodimer (VDR‐RXR) complex to VDRE and thus probably involves coactivator/corepressor molecules.

Characterization of the condensin component Cnap1 and protein kinase Melk as novel E2F target genes down-regulated by 1,25-dihydroxyvitamin D3

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has potent antiproliferative effects characterized by a hampered G1/S transition. cDNA microarrays were used to monitor expression of 21,492 genes in MC3T3-E1 mouse osteoblasts at 1, 6, 12, 24, and 36 h after treatment with 1,25(OH)2D3. Statistical analysis revealed a cluster of genes that were strongly down-regulated by 1,25(OH)2D3 and which not only function in cell cycle regulation and DNA replication but also mediate checkpoint control, DNA repair, chromosome modifications, and mitosis. Because many of these genes were shown earlier to be regulated by the transcriptional repressor E2F4, the intergenic regions of these 1,25(OH)2D3-down-regulated genes were searched for the presence of E2F binding sites. This led to the characterization of two novel E2F target genes, chromosome condensation-related SMC-associated protein 1 (Cnap1) and maternal embryonic leucine zipper kinase (Melk). Transfection studies and site-directed mutagenesis confirmed Cnap1 and Melk to be bona fide E2F targets. Repression of Cnap1 and Melk by 1,25(OH)2D3 was confirmed not only in MC3T3-E1 cells but also in several other bone-unrelated cell types. This down-regulation as well as the antiproliferative effect of 1,25(OH)2D3 depended on the pocket proteins p107 and p130 because 1,25(OH)2D3 failed to repress these E2F target genes and lost its antiproliferative action in p107–/–;p130–/– cells but not in pRb–/– cells.

Microarray analysis of 1α,25-dihydroxyvitamin D3-treated MC3T3-E1 cells☆

The active form of Vitamin D, 1α,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ], demonstrates potent antiproliferative actions on normal as well as on malignant cell types by blocking the transition from the G1- to the S-phase of the cell cycle. Key target genes for 1,25-(OH) 2 D 3 in this non-classic effect remain largely unknown. Therefore, this study aims to identify genes that, through changes in expression after 1,25-(OH) 2 D 3 treatment, contribute to the observed antiproliferative effect. cDNA microarrays containing 4600 genes were used to investigate changes in gene expression in MC3T3-E 1 mouse osteoblasts at 6 and at 12 h after treatment with 1,25-(OH) 2 D 3 (10 -8 M), preceding (6 h) or coinciding with (12 h) the G1/S block in these cells. Approximately one fifth of the genes that were significantly down-regulated after a 12 h incubation period with 1,25-(OH) 2 D 3 were genes involved in the DNA replication process, a basic process for cell growth that starts at the end of G 1-phase and continues in S-phase. Down-regulation of these genes by 1,25-(OH) 2 D 3 was confirmed by quantitative RT-PCR in MC3T3-E1. In conclusion, cDNA microarrays revealed that treatment of MC3T3-E1 cells with 1,25-(OH) 2 D 3 resulted in the down-regulation of DNA replication genes in parallel with the observed Gl/S-arrest.

1α,25-Dihydroxyvitamin D3-induced down-regulation of the checkpoint proteins, Chk1 and Claspin, is mediated by the pocket proteins p107 and p130

A previous cDNA microarray analysis in murine MC3T3-E1 osteoblasts revealed a cluster of genes involved in cell cycle progression that was significantly down-regulated after a single treatment with 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] [L. Verlinden, G. Eelen, I. Beullens, M. Van Camp, P. Van Hummelen, K. Engelen, R. Van Hellemont, K. Marchal, B. De Moor, F. Foijer, H. Te Riele, M. Beullens, M. Bollen, C. Mathieu, R. Bouillon, A. Verstuyf, Characterization of the condensin component Cnap1 and protein kinase Melk as novel E2F target genes down-regulated by 1,25-dihydroxyvitamin D3, J. Biol. Chem. 280 (45) (2005) 37319-37330]. Among those genes were the DNA replication and DNA damage checkpoint proteins, Chk1 and Claspin, of which the human homologues were recently shown to be E2F-responsive. Quantitative real-time PCR experiments in 1,25(OH)(2)D(3)-treated MC3T3-E1 cells confirmed the down-regulation observed in the microarray experiment. Moreover, Chk1 and Claspin promoter activities were also reduced after incubation with 1,25(OH)(2)D(3), and this reduction was mediated through the E2F recognition motifs within their promoters because mutation of these motifs almost completely abolished the repressive effect of 1,25(OH)(2)D(3). The antiproliferative effect of 1,25(OH)(2)D(3) as well as its potential to down-regulate the expression of Chk1 and Claspin depended on the pocket proteins p107 and p130 because 1,25(OH)(2)D(3) lost its antiproliferative action and failed to repress these E2F-target genes in p107(-/-);p130(-/-)-cells, but not in pRb(-/-)-cells.

Altered Vitamin D receptor–coactivator interactions reflect superagonism of Vitamin D analogs

The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], has potent antiproliferative actions on various normal and malignant cells. Calcemic effects, however, hamper therapeutic application of 1,25-(OH)(2)D(3) in hyperproliferative diseases. Two 14-epi-analogs of 1,25-(OH)(2)D(3) namely 19-nor-14-epi-23-yne-1,25-(OH)(2)D(3) (TX522) and 19-nor-14,20-bisepi-23-yne-1,25-(OH)(2)D(3) (TX527), display reduced calcemic effects coupled to an (at least 10-fold) increased antiproliferative potency when compared with 1,25-(OH)(2)D(3). Altered cofactor recruitment by the Vitamin D receptor (VDR) might underlie the superagonism of these 14-epi-analogs. Therefore, this study aims to evaluate their effects at the level of VDR-coactivator interactions. Mammalian two-hybrid assays with VDR and the coactivators TIF2 and DRIP205 showed the 14-epi-analogs to be more potent inducers of VDR-coactivator interactions than 1,25-(OH)(2)D(3). TX522 and TX527 require 30- and 40-fold lower doses to obtain the VDR-DRIP205 interaction induced by 1,25-(OH)(2)D(3) at 10(-8)M. Evaluation of additional 1,25-(OH)(2)D(3)-analogs and their impact on VDR-coactivator interactions revealed a strong correlation between the antiproliferative potency of an analog and its ability to induce VDR-coactivator interactions. In conclusion, these data show that altered coactivator binding by the VDR is one possible explanation for the superagonistic action of the two 14-epi-analogs TX522 and TX527.