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The involvement of membrane-bound solute carriers (SLCs) in neoplastic transdifferentiation processes is poorly defined. Here, we examined changes in the SLC landscape during epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. We show that two SLCs from the organic anion/cation transporter family, SLC22A10 and SLC22A15, favor EMT via interferon (IFN) α and γ signaling activation of receptor tyrosine kinase-like orphan receptor 1 (ROR1) expression. In addition, SLC22A10 and SLC22A15 allow tumor cell accumulation of glutathione to support EMT via the IFNα/γ-ROR1 axis. Moreover, a pan-SLC22A inhibitor lesinurad reduces EMT-induced metastasis and gemcitabine chemoresistance to prolong survival in mouse models of pancreatic cancer, thus identifying new vulnerabilities for human PDAC.

The sodium iodide symporter (NIS) functions to transport iodide and is critical for successful radioiodide ablation of cancer cells. Approaches to bolster NIS function and diminish recurrence post-radioiodide therapy are impeded by oncogenic pathways that suppress NIS, as well as the inherent complexity of NIS regulation. Here, we utilised NIS in high-throughput drug screening and undertook rigorous evaluation of lead compounds to identify and target key processes underpinning NIS function. We find that multiple proteostasis pathways, including proteasomal degradation and autophagy, are central to the cellular processing of NIS. Utilizing inhibitors targeting distinct molecular processes, we pinpoint combinatorial drug strategies giving robust >5-fold increases in radioiodide uptake. We also reveal significant dysregulation of core proteostasis genes in human tumors, identifying a 13-gene riskscore classifier as an independent predictor of recurrence in radioiodide-treated patients. We thus propose and discuss a new model for targetable steps of intracellular processing of NIS function.

The sodium iodide symporter (NIS) functions to transport iodide and is critical for successful radioiodide ablation of cancer cells. Approaches to bolster NIS function and diminish recurrence post-radioiodide therapy are impeded by oncogenic pathways that suppress NIS, as well as the inherent complexity of NIS regulation. Here, we utilize NIS in high-throughput drug screening and undertake rigorous evaluation of lead compounds to identify and target key processes underpinning NIS function. We find that multiple proteostasis pathways, including proteasomal degradation and autophagy, are central to the cellular processing of NIS. Utilizing inhibitors targeting distinct molecular processes, we pinpoint combinatorial drug strategies giving robust >5-fold increases in radioiodide uptake. We also reveal significant dysregulation of core proteostasis genes in human tumors, identifying a 13-gene risk score classifier as an independent predictor of recurrence in radioiodide-treated patients. We thus propose and discuss a model for targetable steps of intracellular processing of NIS function.

The vitamin D receptor (VDR) binds the secosteroid hormone 1,25(OH)2D3 with high affinity and regulates gene programs that control a serum calcium levels, as well as cell proliferation and differentiation. A significant focus has been to exploit the VDR in cancer settings. Although preclinical studies have been strongly encouraging, to date clinical trials have delivered equivocal findings that have paused the clinical translation of these compounds. However, it is entirely possible that mining of genomic data will help to refine precisely what are the key anticancer actions of vitamin D compounds and where these can be used most effectively.

Recently, we undertook a pan-cancer analyses of the nuclear hormone receptor (NR) superfamily in The Cancer Genome Atlas (TCGA), and revealed that the vitamin D receptor (NR1I1/VDR) was commonly and significantly down-regulated specifically in colon adenocarcinoma cohort (COAD). To examine the consequence of down-regulated VDR expression we re-analyzed VDR chromatin immunoprecipitation sequencing (ChIP-Seq) data from LS180 colon cancer cells (GSE31939). This analysis identified 1809 loci that displayed significant (p.adj

Background The aldo-keto reductase 1C3 (AKR1C3) has been heavily implicated in the propagation of prostate malignancy. AKR1C3 protein is elevated within prostate cancer tissue, it contributes to the formation of androgens and downstream stimulation of the androgen receptor (AR). Elevated expression of AKR1C3 is also reported in acute myeloid leukemia but the target nuclear receptors have been identified as members of the peroxisome-proliferator activated receptor (PPARs) subfamily. Thus, AKR1C3 cancer biology is likely to be tissue dependent and hormonally linked to the availability of ligands for both the steroidogenic and non-steroidogenic nuclear receptors. Methods In the current study we investigated the potential for AKR1C3 to regulate the availability of prostaglandin-derived ligands for PPARg mainly, prostaglandin J2 (PGJ2). Using prostate cancer cell lines with stably reduced AKR1C3 levels we examined the impact of AKR1C3 upon proliferation mediated by PPAR ligands. Results These studies revealed knockdown of AKR1C3 had no effect upon the sensitivity of androgen receptor independent prostate cancer cells towards PPAR ligands. However, the reduction of levels of AKR1C3 was accompanied by a significantly reduced mRNA expression of a range of HDACs, transcriptional co-regulators, and increased sensitivity towards SAHA, a clinically approved histone deacetylase inhibitor. Conclusions These results suggest a hitherto unidentified link between AKR1C3 levels and the epigenetic status in prostate cancer cells. This raises an interesting possibility of a novel rational to target AKR1C3, the utilization of AKRIC3 selective inhibitors in combination with HDAC inhibition as part of novel epigenetic therapies in androgen deprivation therapy recurrent prostate cancer.

Bioinformatics applies unbiased approaches to develop statistically-robust insight into health and disease. At the global, or "20,000 foot" view bioinformatic analyses of vitamin D receptor (NR1I1/VDR) signaling can measure where the VDR gene or protein exerts a genome-wide significant impact on biology; VDR is significantly implicated in bone biology and immune systems, but not in cancer. With a more VDR-centric, or "2000 foot" view, bioinformatic approaches can interrogate events downstream of VDR activity. Integrative approaches can combine VDR ChIP-Seq in cell systems where significant volumes of publically available data are available. For example, VDR ChIP-Seq studies can be combined with genome-wide association studies to reveal significant associations to immune phenotypes. Similarly, VDR ChIP-Seq can be combined with data from Cancer Genome Atlas (TCGA) to infer the impact of VDR target genes in cancer progression. Therefore, bioinformatic approaches can reveal what aspects of VDR downstream networks are significantly related to disease or phenotype.

The current study aimed to examine the gene specific mechanisms by which the actions of the vitamin D receptor (VDR) are distorted in prostate cancer. Transcriptional responses toward the VDR ligand, 1α,25(OH)2D3, were examined in non-malignant prostate epithelial cells (RWPE-1) and compared to the 1α,25(OH)2D3-recalcitrant prostate cancer cells (PC-3). Time resolved transcriptional studies for two VDR target genes revealed selective attenuation and repression of VDR transcriptional responses in PC-3 cells. For example, responses in PC-3 cells revealed suppressed responsiveness of IGFBP3 and G0S2. Furthermore, Chromatin Immunoprecipitation (ChIP) assays revealed that suppressed transcriptional responses in PC-3 cells of IGFBP3 and G0S2 were associated with selective VDR-induced NCOR1 enrichment at VDR-binding regions on target-gene promoter regions. We propose that VDR inappropriately recruits co-repressors in prostate cancer cells. Subsequent direct and indirect mechanisms may induce local DNA methylation and stable transcriptional silencing. Thus a transient epigenetic process mediated by co-repressor binding, namely, the control of H3K9 acetylation, is distorted to favor a more stable epigenetic event, namely DNA methylation.This article is part of a Special Issue entitled ‘Vitamin D Workshop’.

The main physiological actions of the biologically most active metabolite of vitamin D, 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)), are calcium and phosphorus uptake and transport and thereby controlling bone formation. Other emergent areas of 1α,25(OH)(2)D(3) action are in the control of immune functions, cellular growth and differentiation. All genomic actions of 1α,25(OH)(2)D(3) are mediated by the transcription factor vitamin D receptor (VDR) that has been the subject of intense study since the 1980's. Thus, vitamin D signaling primarily implies the molecular actions of the VDR. In this review, we present different perspectives on the VDR that incorporate its role as transcription factor and member of the nuclear receptor superfamily, its dynamic changes in genome-wide locations and DNA binding modes, its interaction with chromatin components and its primary protein-coding and non-protein coding target genes and finally how these aspects are united in regulatory networks. By comparing the actions of the VDR, a relatively well-understood and characterized protein, with those of other transcription factors, we aim to build a realistic positioning of vitamin D signaling in the context of other intracellular signaling systems.

Induction of growth arrest and differentiation by 1alpha,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) occurs in non-malignant cell types but is often reduced in cancer cells. For example, androgen-independent prostate cancer cells, DU-145 and PC-3, are relatively insensitive to the anti-proliferative action of 1,25-(OH)(2)D(3). This appears to be due to increased 1,25-(OH)(2)D(3)-metabolism, as a result of CYP24 enzyme-induction, which in turn leads to decreased anti-proliferative efficacy. In the in vitro rat kidney mitochondria assay, the 2-(4-hydroxybenzyl)-6-methoxy-3,4-dihydro-2H-naphthalen-1-one (4) was found to be a potent inhibitor of Vitamin D(3) metabolising enzymes (IC(50) 3.5 microM), and was shown to be a more potent inhibitor than the broad spectrum P450 inhibitor ketoconazole (IC(50) 20 microM). The combination of the inhibitor and 1,25-(OH)(2)D(3) caused a greater inhibition of proliferation in DU-145 cells than when treated with both agents alone. Examination of the regulation of VDR target gene mRNA in DU-145 cells revealed that co-treatment of 1,25-(OH)(2)D(3) plus inhibitor of Vitamin D(3) metabolising enzymes co-ordinately upregulated CYP24, p21(waf1/cip1) and GADD45alpha.

The Vitamin D-activating enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-hydroxylase) is now known to be expressed in a much wider range of tissues that previously thought, suggesting a role for 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), which is more in keeping with a cytokine than a hormone. In this capacity, the function of 1alpha-hydroxylase in tumors is far from clear. Studies from several groups including ours have shown altered expression of 1alpha-hydroxylase in different types of neoplasm including breast, prostate and colon cancers. However, functional analysis of Vitamin D metabolism in cancer is complicated by the heterogenous composition of tumors. Immunohistochemical analysis of breast tumors has shown that 1alpha-hydroxylase is expressed by both epithelial cells and by tumor-infiltrating macrophages, suggesting an immunomodulatory component to 1,25(OH)(2)D(3) production in some types of cancer. The demonstration of 1alpha-hydroxylase activity in tumors and their equivalent normal tissues has implications for both the treatment and prevention of cancers. For example, in tumors chemotherapy options may include the use of non-1alpha-hydroxylated Vitamin D analogs to increase local concentrations of active metabolites without systemic side-effects. The role of 1alpha-hydroxylase in protection against cancer is likely to be more complicated and may involve anti-tumor immune responses.

Normal prostate epithelial cells are acutely sensitive to the antiproliferative action of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), whilst prostate cancer cell lines and primary cultures display a range of sensitivities. We hypothesised that key antiproliferative target genes of the Vitamin D receptor (VDR) were repressed by an epigenetic mechanism in 1alpha,25(OH)(2)D(3)-insensitive cells. Supportively, we found elevated nuclear receptor co-repressor and reduced VDR expression correlated with reduced sensitivity to the antiproliferative action of 1alpha,25(OH)(2)D(3). Furthermore, the growth suppressive actions of 1alpha,25(OH)(2)D(3) can be restored by co-treatment with low doses of histone deacetylation inhibitors, such as trichostatin A (TSA) to induce apoptosis. Examination of the regulation of VDR target genes revealed that co-treatment of 1alpha,25(OH)(2)D(3) plus TSA co-operatively upregulated GADD45alpha. Similarly in a primary cancer cell culture, the regulation of appeared GADD45alpha repressed. These data demonstrate that prostate cancer cells utilise a mechanism involving deacetylation to suppress the responsiveness of VDR target genes and thus ablate the antiproliferative action of 1alpha,25(OH)(2)D(3).

Proliferation of the non-malignant breast epithelial cell line, MCF-12A, is sensitively and completely inhibited by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) (ED90 = 70 nM), We used real time RT-PCR to demonstrate that the relative resistance to 1alpha,25(OH)(2)D(3) of MDA-MB-231 cells (ED50100 nM) correlated with significantly reduced Vitamin D receptor (VDR) and increased NCoR1 nuclear receptor co-repressor mRNA (0.1-fold reduction in VDR and 1.7-fold increase in NCoR1 relative to MCF-12A (P0.05)). This molecular lesion can be targeted by co-treating cells with 1alpha,25(OH)(2)D(3) or potent analogs and the histone deacetylation inhibitor trichostatin A (TSA). For example, the co-treatment of 1,25-dihydroxy-16,23,Z-diene-26,27-hexafluoro-19-nor Vitamin D(3) (RO-26-2198) (100 nM) plus TSA results in strong additive antiproliferative effects in MDA-MB-231 cells. This may represent novel chemotherapeutic regime for hormone insensitive breast cancer.

Induction of growth arrest and differentiation of some cancer cells by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], and its potent analogs, is well characterized. However, aggressive cancer cell lines are often either insensitive to the antiproliferative effects of 1alpha,25(OH)(2)D(3) or require toxic concentrations to recapitulate them which has, to-date, precluded its use in anticancer therapy. Therefore we are interested in mechanisms by which 1alpha,25(OH)(2)D(3) signaling has become deregulated in malignant cells in order to identify novel therapeutic targets. We observed previously that 1alpha,25(OH)(2)D(3) and its metabolites, generated via the C-24 oxidation pathway, drive simultaneous differentiation and hyper-proliferation within the same cell population. Thus we have proposed that metabolism of 1alpha,25(OH)(2)D(3) via the C-24 oxidation pathway represents a novel-signaling pathway, which integrates proliferation with differentiation. In the current study we examined further the role of this pathway and demonstrated that these effects are not restricted to leukemic cells but are observed also in both normal myeloid progenitors and breast cancer cell lines. Intriguingly, stable transfection of MCF-7 breast cancer cells with antisense vitamin D(3) receptor (VDR) reduced antiproliferative sensitivity to 1alpha,25(OH)(2)D(3) but significantly enhanced growth stimulation, which, in turn, was blocked by inhibiting metabolism of 1alpha,25(OH)(2)D(3) via C-24 oxidation pathway with ketoconazole. Taken together, these studies indicate that metabolism of 1alpha,25(OH)(2)D(3) via C-24 oxidation pathway gives rise to ligands with different biologic effects. We propose that this mechanism may allow the co-ordination of population expansion and cell maturation during differentiation. Cancer cells appear to corrupt this process during malignant transformation, by only responding to the pro-proliferative signals, thereby deriving a clonal advantage.

The secosteroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1alpha,25(OH)(2)D(3) and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1alpha,25(OH)(2)D(3) is metabolized in its target tissues via modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway initiated by hydroxylation at C-24 leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways initiated by hydroxylations at C-23 and C-26 respectively together lead to the formation of the end product, 1alpha,25(OH)(2)D(3)-lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 to form 1alpha,25-dihydroxy-3-epi-vitamin-D(3). We performed the present study first to examine in detail the metabolism of 1alpha,25(OH)(2)D(3) in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1alpha,25(OH)(2)D(3) in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1beta-(3)H] 1alpha,25(OH)(2)D(3) and demonstrated that these cells metabolize 1alpha,25(OH)(2)D(3) mainly via the C-24 oxidation pathway and to a lesser extent via the C-23 oxidation pathway, but not via the C-3-epimerization pathway. Three of the natural intermediary metabolites of 1alpha,25(OH)(2)D(3) derived via the C-24 oxidation pathway namely, 1alpha,24(R),25-trihydroxyvitamin D(3), 1alpha,25-dihydroxy-24-oxovitamin D(3) and 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S),25(OH)(3)-24-oxo-D(3)] were almost as potent as 1alpha,25(OH)(2)D(3) in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1alpha,23(S),25(OH)(3)-24-oxo-D(3) which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1alpha,25(OH)(2)D(3) to examine further its effects on these cells. Our results indicated that 1alpha,23(S),25(OH)(3)-24-oxo-D(3) was also equipotent to its parent in inhibiting clonal growth of HL-60 cells and in inducing expression of CD11b protein. In summary, we report that 1alpha,25(OH)(2)D(3) is metabolized in HL-60 cells into several intermediary metabolites derived via both the C-24 and C-23 oxidation pathways but not via the C-3 epimerization pathway. Some of the intermediary metabolites derived via the C-24 oxidation pathway are found to be almost equipotent to 1alpha,25(OH)(2)D(3) in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1alpha,25(OH)(2)D(3) were found to be less calcemic. Thus, the findings of our study suggest that some of the natural metabolites of 1alpha,25(OH)(2)D(3) may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1alpha,25(OH)(2)D(3).

In a previous report, we indicated that 1alpha,23(S), 25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S), 25(OH)(3)-24-oxo-D(3)], a natural metabolite of 1alpha, 25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is almost equipotent to 1alpha,25(OH)(2)D(3) in suppressing parathyroid hormone (PTH) secretion (Lee et al., 1997. Biochemistry 36, 9429-9437). Also, 1alpha,23(S),25(OH)(3)-24-oxo-D(3) has been shown to possess only weak in vivo calcemic actions. Thus, vitamin D(3) analogs structurally related to 1alpha,23(S),25(OH)(3)-24-oxo-D(3) may have therapeutic value. Furthermore, biologic activity studies of various synthetic analogs of 1alpha,25(OH)(2)D(3) showed that the removal of carbon-19 (C-19) reduces the calcemic activity of 1alpha, 25(OH)(2)D(3.) Therefore, in an attempt to produce vitamin D(3) analogs with a better therapeutic index, we synthesized C(23) epimers of 1alpha,23,25(OH)(3)-24-oxo-19-nor-vitamin D(3) [1alpha,23, 25(OH)(3)-24-oxo-19-nor-D(3)]. The two epimers were compared to 1alpha,25(OH)(2)-19-nor-D(3) and 1alpha,25(OH)(2)D(3) in their ability to generate biologic activities in several in vitro assay systems. In the assay measuring the suppression of parathyroid hormone (PTH) secretion in bovine parathyroid cells, 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3) was as potent as 1alpha, 25(OH)(2)-19-nor-D(3) but was less potent than 1alpha,25(OH)(2)D(3). In the same assay 1alpha,23(R),25(OH)(3)-24-oxo-19-nor-D(3) exhibited greater potency than 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3). In the assays measuring the ability of vitamin D compounds to inhibit clonal growth and to induce differentiation of human promyelocytic leukemia (HL-60) cells, 1alpha,23(S),25(OH)(3)-24-oxo-19-nor-D(3) was less potent than 1alpha,25(OH)(2)-19-nor-D(3) but was equipotent to 1alpha, 25(OH)(2)D(3). More importantly, in the same assays, 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3) was more potent than 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3) and was equipotent to 1alpha, 25(OH)(2)-19-nor-D(3). Also, the vitamin D receptor-mediated transcriptional activity of 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3) was almost equal to that of 1alpha, 25(OH)(2)-19-nor-D(3), but higher than that of 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3). This finding explains in part the greater in vitro biologic activities of 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3). In summary, our results indicate that 1alpha,23(R),25(OH)(3)-24-oxo-19-nor-D(3) and to a lesser extent 1alpha,23(S),25(OH)(3)-24-oxo-19-nor-D(3) are potent 19-nor vitamin D(3) analogs, which suppress PTH secretion in bovine parathyroid cells and strongly inhibit clonal growth and induce differentiation of HL-60 cells in vitro.

Induction of growth arrest and monocyte differentiation of HL-60 leukemia cells by 1alpha,25 dihydroxyvitamin D3 (1alpha,25(OH)2D3) is well established. By contrast, we have observed, that 1alpha,25(OH)2D3 and its metabolites play separate roles in clonal expansion and survival of differentiating HL-60 cells. Cells that had differentiated by 48 h (CD14 positive) grew slower than control cells, whereas CD14 negative cells were growing faster at this time point. Inhibiting 1alpha,25(OH)2D3 or 1alpha,25(OH)2-20-epi-D3 metabolism, by the 25(OH)D3-24-hydroxylase inhibitor ketoconazole, abolished hyperproliferation of CD14 negative cells. Instead, both the onset of differentiation and subsequent apoptosis were enhanced. These events were associated with immediate up-regulation of the cyclin-dependent kinase inhibitor p21(waf1) and a lack of sustained expression, respectively. Stimulation and inhibition of growth by vitamin D3-related compounds was observed to be concentration and metabolite specific. Low amounts of 1alpha,25(OH)2-20-epi-D3 and 1alpha,24,25(OH)3-20-epi-D3 stimulated HL-60 cell growth. At higher concentrations, 1alpha,25(OH)2-20-epi-D3 was a more potent inducer than 1alpha,24,25(OH)3-20-epi-D3 of HL-60 differentiation; 1alpha,25(OH)2-20-epi-24-oxo-D3 was exclusively pro-differentiative at all concentrations. 1alpha,25(OH)2-20-epi-D3 and 1alpha,24,25(OH)3-20-epi-D3 stimulated proliferation of KG-1a leukemia cells, but neither of these compounds nor 1alpha,25(OH)3-20-epi-24-oxo-D3 exerted pro-differentiative effects on these cells. These findings shed new light on the pro- and anti-proliferative effects of 1alpha,25(OH)2D3 and lead to the postulate that metabolism of 1alpha,25(OH)2D3 and its 20-epi analog regulates different subsets of genes so as to co-ordinate population expansion and the differentiation process. Furthermore, 1alpha,25(OH)2D3 metabolism and/or sensitivity to the effects of metabolites may be altered in transformed cells to derive a clonal advantage.

Allelic variations of the vitamin D receptor (VDR) gene have been associated with the risk of developing prostate cancer in men and osteoporosis in postmenopausal women. Three RFLP's (TaqI, ApaI, BsmI) define two common haplotypes: BAt and baT. None of these polymorphisms change the translated protein. Since sequence variations in the 3′ UTR of VDR have been linked to the different haplotypes, investigators have proposed that the stability of VDR mRNA is influenced by allelic variations. Indirect evidence suggested that allele T is less stable than allele t. In this study, we used a RT-PCR based approach to compare the stability of the big T and small t allele in normal heterozygous lymphocytes and the heterozygous cell lines NB4 (myeloid leukemia) and PC-3 and DU 145 (prostate cancers). In all three cases, we did not find a significant difference in stability. Interestingly, we consistently observed 30% less RT-PCR product derived from the small t allele mRNA in steady state, a finding which also speaks against a higher stability of the small t allele mRNA. These results indicate a variation in transcriptional regulation rather than mRNA stability between the alleles. We hypothesize that an unknown gene or genes in linkage with the polymorphisms is (are) responsible for the relationship between risk of prostate cancer and VDR polymorphisms.

Activation of cyclin-dependent kinases (CDKs) by interaction with cyclins regulates progression through cell cycle checkpoints. This process is counterbalanced by CDK inhibitors (CDKIs), which can inhibit progression through the cell cycle. Because CDKI expression acts to inhibit cellular proliferation, CDKIs may have a role as tumor suppressors. One class of CDKIs, characterized by the presence of ankyrin repeats, has at least four members (p15INK4B), p16INK4, p18, and p19). Two of these, p15INK4B, p16INK4, have been mapped to chromosome 9p21, a region of frequent loss in a wide variety of cancers. Alterations of p16INK4 have been detected in various tumors and cell lines. We analyzed p15INK4B, p16INK4, and p18 alterations in 52 osteosarcomas (including 11 explants), and 23 other various sarcomas. Single-stranded conformation polymorphism analysis [polymerase chain reaction (PCR-SSCP)] of the coding regions of these CDKI genes detected a missense mutation of p16INK4 exon 1 in one soft tissue sarcoma. Southern blotting detected complete deletion of p15INK4B and p16INK4 genes in osteosarcomas from 2 patients and a soft tissue sarcoma from another individual. Loss of heterozygosity (LOH) at chromosome 9p21 was observed with a microsatellite probe closely linked to the INK4 genes in the latter case. Deletions of both p15INK4B and p16INK4 genes were detected in five of eight osteosarcoma cell lines. By contrast, no alterations of p18 were detected in any sample. Together these data suggest that alterations of the p15INK4B and p16INK4 genes, but not p18, may occur in approximately 5% of sarcomas. However, deletions of the p15INK4B and P16INK4 genes are frequent in osteosarcoma cell lines and probably have a role in tumor cell growth in culture. Notably, all seven detectable deletions involved both p15INK4B and p16INK4 genes, suggesting that both contribute individual tumor suppressor activity.

One of the advantages of ultrafiltration for the bioseparation of proteins is the comparatively mild condition used. A key operational parameter in such a system is the temperature utilised for the process. There are conflicting demands when selecting an operating temperature between the fluid dynamics and the biochemical properties of the system. An analysis of this conflict, in order to establish the optimal temperature, involved a model crossflow filtration system, where shear rate, volume of protein containing solution and protein concentration can be kept constant and only the temperature varied. In such studies denaturation of the permeate was observed whereas the processing efficiency did not appear to have a temperature optimum.