The Pim family of proto-oncogenes encodes a distinct class of serine/threonine kinases consisting of PIM1, PIM2, and PIM3. Although the Pim genes are evolutionarily highly conserved, the contribution of PIM proteins to mammalian development is unclear. PIM1-deficient mice were previously described but showed only minor phenotypic aberrations. To assess the role of PIM proteins in mammalian physiology, compound Pim knockout mice were generated. Mice lacking expression of Pim1, Pim2, and Pim3 are viable and fertile. However, PIM-deficient mice show a profound reduction in body size at birth and throughout postnatal life. In addition, the in vitro response of distinct hematopoietic cell populations to growth factors is severely impaired. In particular, PIM proteins are required for the efficient proliferation of peripheral T lymphocytes mediated by synergistic T-cell receptor and interleukin-2 signaling. These results indicate that members of the PIM family of proteins are important but dispensable factors for growth factor signaling.
To survive under hypoxic conditions, cancer cells remodel glucose metabolism to support tumor progression. HIF transcription factor is essential for cellular response to hypoxia. The underlying mechanism how HIF is constitutively activated in cancer cells remains elusive. In the present study, we characterized a regulatory feedback loop between HIF-1α and PIM2 in HepG2 cells. Serine/threonine kinase proto-oncogene PIM2 level was induced upon hypoxia in a HIF-1α-mediated manner in cancer cells. HIF-1α induced PIM2 expression via binding to the hypoxia-responsive elements (HREs) of the PIM2 promoter. In turn, PIM2 interacted with HIF-1α, especially a transactivation domain of HIF-1α. PIM2 as a co-factor but not an upstream kinase of HIF-1α, enhanced HIF-1α effect in response to hypoxia. The positive feedback loop between PIM2 and HIF-1α was correlated with glucose metabolism as well as cell survival in HepG2 cells. Such a regulatory mode may be important for the adaptive responses of cancer cells in antagonizing hypoxia during cancer progression.
The genomic action of calcitriol (1,25-dihydroxy-vitamin D3) is mediated through the interaction of the calcitriol receptor (VDR) with vitamin D response elements (VDREs). Although renal failure is associated with resistance to the action of calcitriol, the mechanism of this resistance is not well understood. Therefore, we used the electrophoretic mobility shift assay to compare the ability of VDRs from normal and renal failure rats to bind to the osteocalcin gene VDRE. The results indicate that VDRs from renal failure rats have only half the DNA binding capacity as VDRs from control rats, despite identical calcitriol binding. Furthermore, incubation of normal VDRs with a uremic plasma ultrafiltrate resulted in a loss of > 50% of the binding sites for the osteocalcin VDRE. When VDRs bound to DNA as heterodimers with retinoid X receptors, the inhibitory effect of the uremic ultrafiltrate was due to a specific interaction with the VDR, not retinoid X receptors. In addition, uremic ultrafiltrate blocked calcitriol-induced reporter gene activity in transfected JEG-3 cells. Taken together, the results indicate that an inhibitory effect of a uremic toxin(s) on VDR-VDRE binding could underlie the calcitriol resistance of renal failure.
The serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2.
Here we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases.
The structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors.
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Pim kinases, belong to a distinctive serine/threonine protein-kinase family and are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Human Pim-1 kinase has been cloned, expressed and crystallized
Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1–313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14–313) by thrombin digestion during purification. The Pim-1 (14–313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 Å resolution and belong to space group P65, with unit-cell parameters a = b = 95.9, c = 80.0 Å, β = 120° and one molecule per asymmetric unit.
Potent survival effects have been ascribed to the serine/threonine kinase proto-oncogene PIM-2. Elevated levels of PIM-2 are associated with various malignancies. In human cells, a single Pim-2 transcript gives rise mainly to two protein isoforms (34, 41 kDa) that share an identical catalytic site but differ at their N-terminus, due to in-frame alternative translation initiation sites. In this study we observed that the 34 kDa PIM-2 isoform has differential nuclear and cytoplasmic forms in all tested cell lines, suggesting a possible role for the balance between these forms for PIM-2's function. To further study the cellular role of the 34 kDa isoform of PIM-2, an N-terminally HA-tagged form of this isoform was transiently expressed in HeLa cells. Surprisingly, this resulted in increased level of G1 arrested cells, as well as of apoptotic cells. These effects could not be obtained by a Flag-tagged form of the 41 kDa isoform. The G1 arrest and apoptotic effects were associated with an increase in T14/Y15 phosphorylation of CDK2 and proteasom-dependent down-regulation of CDC25A, as well as with up-regulation of p57, E2F-1, and p73. No such effects were obtained upon over-expression of a kinase-dead form of the HA-tagged 34 kDa PIM-2. By either using a dominant negative form of p73, or by over-expressing the 34 kDa PIM-2 in p73-silenced cells, we demonstrated that these effects were p73-dependent. These results demonstrate that while PIM-2 can function as a potent survival factor, it can, under certain circumstances, exhibit pro-apoptotic effects as well.
Pim (proviral insertion in murine lymphoma) kinases are a small family of constitutively active, highly conservative serine/threonine oncogenic kinases and have 3 members: Pim1, Pim2, and Pim3. Pim kinases are also implicated in the regulation of B- and T- cell responses to cytokines and hematopoietic growth factors. The roles of Pim kinases in the regulation of primitive hematopoietic stem cells (HSCs) are largely unknown.
In the current study, Pim1−/−2−/−3−/− triple knockout (TKO) mice were used to determine the role of Pim kinases in hematopoiesis. Peripheral blood hematological parameters were measured in Pim TKO mice and age-matched wild-type (WT) controls. Primary, secondary, and competitive transplantations were performed to assay the long-term repopulating HSCs in Pim TKO mice. In vivo BrdU incorporation assay and ex vivo Ki67 staining and caspase 3 labeling were performed to evaluate the proliferation and apoptosis of HSCs in Pim TKO mice.
Compared to age-matched WT controls, Pim TKO mice had lower peripheral blood platelet count and exhibited erythrocyte hypochromic microcytosis. The bone marrow cells from Pim TKO mice demonstrated decreased hematopoietic progenitor colony-forming ability. Importantly, Pim TKO bone marrow cells had significantly impaired capacity in rescuing lethally irradiated mice and reconstituting hematopoiesis in primary, secondary and competitive transplant models. In vivo BrdU incorporation in long-term HSCs was reduced in Pim TKO mice. Finally, cultured HSCs from Pim TKO mice showed reduced proliferation evaluated by Ki67 staining and higher rate of apoptosis via caspase 3 activation.
Pim kinases are not only essential in the hematopoietic lineage cell development, but also important in HSC expansion, self-renewal, and long-term repopulation.
Serine/threonine kinase; Pim kinase; Hematopoietic stem cells; Hematopoietic stem cell transplantation; Proliferation; Apoptosis; Knockout mouse
Upon ligand binding the 1α,25-dihydroxy vitamin D3 receptor (VDR) undergoes a conformational change that allows interaction with coactivator proteins including p160/SRC family members and the multimeric DRIP complex through the DRIP205 subunit. Casein kinase II (CKII) phosphorylates VDR both in vitro and in vivo at serine 208 within the hinge domain. This phosphorylation does not affect the ability of VDR to bind DNA, but increases its ability to transactivate target promoters. Here, we have analyzed whether phosphorylation of VDR by CKII modulates the ability of VDR to interact with coactivators in vitro. We find that both mutation of serine 208 to aspartic acid (VDRS208D) or phosphorylation of VDR by CKII enhance the interaction of VDR with DRIP205 in the presence of 1α,25-dihydroxy vitamin D3. We also find that the mutation VDRS208D neither affects the ability of this protein to bind DNA nor to interact with SRC-1 and RXRα. Together, our results indicate that phosphorylation of VDR at serine 208 contributes to modulate the affinity of VDR for the DRIP complex and therefore may have a role in vivo regulating VDR-mediated transcriptional enhancement.
1α; 25-dihydroxy vitamin D3 receptor; Coactivators; Transcription
Human Pim1 kinase is a serine/threonine protein kinase that plays important biological roles in cell survival, apoptosis, proliferation, and differentiation. Moreover, Pim1 is up-regulated in various hematopoietic malignancies and solid tumors. Thus, Pim1 is an attractive target for cancer therapeutics, and there has been growing interest in developing small molecule inhibitors for Pim1. Here, we describe the crystal structure of Pim1 in complex with a newly developed pyrido[4,3-d]pyrimidine-derivative inhibitor (SKI-O-068). Our inhibitor exhibits a half maximum inhibitory concentration (IC50) of 123 (±14) nM and has an unusual binding mode in complex with Pim1 kinase. The interactions between SKI-O-068 and the Pim1 active site pocket residue are different from those of other scaffold inhibitor-bound structures. The binding mode analysis suggests that the SKI-O-068 inhibitor can be improved by introducing functional groups that facilitate direct interaction with Lys67, which aid in the design of an optimized inhibitor.
The serine/threonine kinase, PIM1, is involved in promoting cell survival in part by phosphorylation and inhibition of proapoptotic proteins. ASK1, a mitogen-activated protein kinase kinase kinase (MAPKKK), is involved in the so-called stress-activated pathways that contribute to apoptotic cell death. Here we show that PIM1 phosphorylates ASK1 specifically on serine residue 83 (Ser83) both in vitro and in vivo and that PIM1 binds to ASK1 in cells by co-immunoprecipitation. Using H1299 cells, our results further demonstrate that PIM1 phosphorylation of ASK1 decreases its kinase activity induced by oxidative stress. PIM1 phosphorylation of ASK1 on Ser83 inhibited ASK1-mediated c-Jun N-terminal kinase (JNK) phosphorylation as well as phosphorylation of p38 kinase. Under H2O2-induced stress conditions that normally lead to apoptosis, these phosphorylation events were associated with inhibition of caspase-3 activation and resulted in reduced cell death. Moreover, knockdown of PIM1 in H1299 cells decreased phosphorylation of endogenous Ser83 of ASK1 and was associated with a decrease in cell viability after H2O2 treatment. Taken together, these data reveal a novel mechanism by which PIM1 promotes cell survival that involves negative regulation of the stress-activated kinase, ASK1.
PIM1; phosphorylation; ASK1; kinase activity; apoptosis; cell survival
Tumor necrosis factor-α (TNF-α) plays an important role in pathological angiogenesis associated with inflammatory response. Pim-3 kinase belonging to serine/threonine protein kinases is a potent suppressor of myc-induced apoptosis. We have recently demonstrated that Pim-3 plays an essential role in endothelial cell (EC) spreading and migration. In this study, we showed that TNF-α transiently increased Pim-3 mRNA expression, and this was mediated through Tumor necrosis factor-α receptor-1 (TNFR1) pathway in ECs. TNF-α could promote stabilization of Pim- 3 mRNA in ECs. Small-interfering RNA (siRNA)-mediated gene knockdown of Pim-3 significantly impaired TNF-α- induced formation of EC membrane protrusions in vitro. Furthermore, Pim-3 silencing inhibited EC sprouting in subcutaneous Matrigel in vivo. eNOS mRNA abundance was lower in Pim-3 siRNA transfected ECs compared with the control ECs. These observations suggest that Pim-3 plays a role in TNF-α-induced angiogenesis.
angiogenesis; endothelial cell; Pim-3; TNF-α
Pim-1 is a serine-threonine kinase which promotes early transformation, cell proliferation and cell survival during tumorigenesis. Several studies have demonstrated that Pim-1 kinase play a role in different cancer types, however, the function of Pim-1 in bladder cancer is poorly understood.
Expression and localization of Pim-1 in human normal and malignant bladder specimens were examined by Immunohistochemistry and Pim-1 staining score was compared with several clinicopathologic parameters. To further demonstrate the biological function of Pim-1 in bladder cancer, its expression was validated in five bladder cancer cell lines by western blot and immunohistochemistry analyses. Subsequent knockdown of Pim-1 was achieved by lentivirus encoding small interfering RNA, and the effect of Pim-1 on bladder cell survival and drug sensitivity were further assessed by colony formation and cell proliferation assays.
When compared with normal epithelium, Pim-1 was overexpressed in bladder cancer epithelium, and the expression level was higher in invasive bladder cancer than Non-invasive bladder cancer specimens. Pim-1 was also detected in all the bladder cancer cell lines examined in our study. Moreover, the knockdown of Pim-1 significantly inhibited bladder cancer cell growth and also sensitized cells to chemotherapeutic drugs in vitro.
Our results in this study suggest that Pim-1 may play a role in bladder cancer initiation and progression. Since Pim-1 is also involved in bladder cancer cell survival and drug resistance, Pim-1 is a potential candidate for targeted therapy in bladder cancer.
Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogs, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogs cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a potent agonist analog, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D3 [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of co-activators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and 12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of co-activators.
The hormone 1,25 dihydroxyvitamin D (1,25(OH)2D) binds to the nuclear vitamin D receptor (nVDR), which heterodimerizes with retinoid X receptor α (RXRα), and this complex interacts with specific response elements [vitamin D response elements (VDREs)] to regulate gene transcription. Previous results show a significant reduction in 1,25(OH)2D-induced nVDR transcriptional activity in fibroblast (C3H10T1/2) cells transfected with the Harvey ras gene (ras cells) compared with parental cells. The purpose of this study was to investigate the mechanisms by which the H-ras gene interferes with nVDR transcriptional activity. Similar to the ras cells, transcriptional activity of the nVDR was reduced following induction of the H-ras gene for 9 days. The ras cells expressed similar protein levels of RXRα with the parent cells, and overexpression of the wild-type RXRα plasmid did not restore 1,25(OH)2D-mediated nVDR activity in ras cells. Inhibiting activation of extracellular signal-regulated kinase (ERK1/2) had no effect on nVDR activity in ras cells. Furthermore, the binding of nVDR to VDREs was reduced in 1,25(OH)2D-treated ras cells. In addition, neither treatment of ras cells with an inhibitor (ketoconazole) of the 1,25(OH)2D degradative enzyme, 24-hydroxylase, nor the protein kinase C inhibitors, bisindoylmaleimide I and Gö 6976, had an effect on nVDR activity. In contrast, inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 resulted in a 1.6-fold significant increase in the nVDR activity in the ras cells. Taken together, these results indicate that PI3K may, at least in part, mediate the suppression of the 1,25 (OH)2D regulation of nVDR transcriptional activity by the H-ras gene, leading to reduced ability to associate with response elements.
1,25-Dihydroxyvitamin D; Vitamin D; Vitamin D receptor; ras; Phosphoinositide 3-kinases
The cytokine gamma interferon (IFN-γ) and the calcitropic steroid hormone 1,25-dihydroxyvitamin D (1,25D) are activators of macrophage immune function. In sarcoidosis, tuberculosis, and several granulomatoses, IFN-γ induces 1,25D synthesis by macrophages and inhibits 1,25D induction of 24-hydroxylase, a key enzyme in 1,25D inactivation, causing high levels of 1,25D in serum and hypercalcemia. This study delineates IFN-γ-1,25D cross talk in human monocytes-macrophages. Nuclear accumulation of Stat1 and vitamin D receptor (VDR) by IFN-γ and 1,25D promotes protein-protein interactions between Stat1 and the DNA binding domain of the VDR. This prevents VDR-retinoid X receptor (RXR) binding to the vitamin D-responsive element, thus diverting the VDR from its normal genomic target on the 24-hydroxylase promoter and antagonizing 1,25D-VDR transactivation of this gene. In contrast, 1,25D enhances IFN-γ action. Stat1-VDR interactions, by preventing Stat1 deactivation by tyrosine dephosphorylation, cooperate with IFN-γ/Stat1-induced transcription. This novel 1,25D-IFN-γ cross talk explains the pathogenesis of abnormal 1,25D homeostasis in granulomatous processes and provides new insights into 1,25D immunomodulatory properties.
Provirus integration site for Moloney murine leukemia virus (PIM1) is a proto-oncogene that encodes a serine/threonine kinase with multiple cellular functions. Overexpression of PIM-1 plays a critical role in progression of prostatic and hematopoietic malignancies. Here we describe the generation of a mAb specific for GST–PIM-1, which reacted strongly with most human and mouse cancer tissues and cell lines of prostate, breast, and colon origin but only weakly (if at all) with normal tissues. The mAb binds to PIM-1 in the cytosol and nucleus as well as to PIM-1 on the surface of human and murine cancer cells. Treatment of human and mouse prostate cancer cell lines with the PIM-1–specific mAb resulted in disruption of PIM-1/Hsp90 complexes, decreased PIM-1 and Hsp90 levels, reduced Akt phosphorylation at Ser473, reduced phosphorylation of Bad at Ser112 and Ser136, and increased cleavage of caspase-9, an indicator of activation of the mitochondrial cell death pathway. The mAb induced cancer cell apoptosis and synergistically enhanced antitumor activity when used in combination with cisplatin and epirubicin. In tumor models, the PIM-1–specific mAb substantially inhibited growth of the human prostate cancer cell line DU145 in SCID mice and the mouse prostate cancer cell TRAMP-C1 in C57BL/6 mice. These findings are important because they provide what we believe to be the first in vivo evidence that treatment of prostate cancer may be possible by targeting PIM-1 using an Ab-based therapy.
The provirus integration site for Moloney murine leukemia virus (Pim) 1 kinase is an oncogenic serine/threonine kinase implicated in cytokine-induced cell signaling, whereas Runt-related transcription factor (Runx) has been implicated in the regulation of T-cell differentiation. The interaction of Pim1 kinase and Runx3 in the pathogenesis of peanut allergy has not been defined.
We sought to determine the effects of Pim1 kinase modulation on Runx3 expression and TH2 and TH17 cell function in an experimental model of peanut allergy. Methods: A Pim1 kinase inhibitor was administered to peanut-sensitized and challenged wild-type and Runx3+/− mice. Symptoms, intestinal inflammation, and Pim1 kinase and Runx3 mRNA expression and protein levels were assessed. The effects of Pim1 kinase inhibition on TH1, TH2, and TH17 differentiation in vivo and in vitro were also determined.
Peanut sensitization and challenge resulted in accumulation of inflammatory cells and goblet cell metaplasia and increased levels of Pim1 kinase and TH2 and TH17 cytokine production but decreased levels of Runx3 mRNA and protein in the small intestines of wild-type mice. All of these findings were normalized with Pim1 kinase inhibition. In sensitized and challenged Runx3+/− mice, inhibition of Pim1 kinase had less effect on the development of the full spectrum of intestinal allergic responses. In vitro inhibition of Pim1 kinase attenuated TH2 and TH17 cell differentiation and expansion while maintaining Runx3 expression in T-cell cultures from wild-type mice; these effects were reduced in T-cell cultures from Runx3+/− mice.
These data support a novel regulatory axis involving Pim1 kinase and Runx3 in the control of food-induced allergic reactions through the regulation of TH2 and TH17 differentiation.
Pim1 kinase; Runx3; peanut; intestinal allergy; TH2; TH17
We have identified and characterized two mutations in the hormone binding domain of the vitamin D receptor (VDR) in patients with hereditary vitamin D-resistant rickets. One patient was found to have a premature stop mutation (CAG to TAG) in the hinge region affecting amino acid 149 (Q149X) and the other demonstrated a missense mutation (CGC to CTC) resulting in the substitution of arginine 271 by leucine (R271L) in the steroid binding domain. Eukaryotic expression analyses in CV-1 cells showed the inability of both patients' VDR to induce transcription from the osteocalcin hormone gene response element at 10(-7) M 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Normal transcription levels could, however, be elicited by the missense mutated VDR (R271L) in the presence of 1,000-fold higher 1,25-(OH)2D3 concentrations than needed for the wild-type receptor. This shows that Arg 271 directly affects the affinity of the VDR for its ligand and its conversion to leucine decreases its affinity for 1,25(OH)2D3 by a factor of 1,000. Arg 271 is located immediately 3-prime to a 30 amino acid segment (VDR amino acids 241-270) that is conserved among members of the steroid/thyroid/retinoid hormone receptor superfamily. These results represent the first missense mutation identified in the hormone binding domain of VDR and further define the structure-function relationship of 1,25(OH)2D3 ligand binding to its nuclear receptor.
We previously described a control element in the granulocyte-macrophage colony-stimulating factor (GM-CSF) enhancer that is necessary and sufficient to mediate both transcriptional activation in response to T-cell stimuli and transcriptional repression by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] through the vitamin D3 receptor (VDR). This DNA element is a composite site that is recognized by both Fos-Jun and NFAT1; it is directly bound by VDR in the absence of a retinoid X receptor as an apparent monomer, and it is bound in a unique tertiary conformation. We describe here the mechanism by which VDR elicits its transcriptional inhibitory effect. Firstly, VDR outcompetes NFAT1 for binding to the composite site. Overexpression of NFAT1 in vivo by transient transfection is able to relieve the 1,25(OH)2D3-dependent repression. Secondly, VDR stabilizes the binding of a Jun-Fos heterodimer to the adjacent AP-1 portion of the element. This appears to occur through a direct interaction between VDR and c-Jun, as demonstrated in vitro by direct glutathione S-transferase coprecipitation assays. In vivo, overexpression of c-Jun, but not c-Fos, leads to a rescue of the 1,25(OH)2D3-mediated repression. Transfected FLAG-VDR bound to the NFAT1–AP-1 DNA binding element can be selectively precipitated from nuclear extracts that are made from cells treated with activating agents in the presence of 1,25(OH)2D3. VDR is not detected in the complex in the absence of the ligand. Thus, VDR acts selectively on the two components required for activation of this promoter/enhancer: it competes with NFAT1 for binding to the composite site, positioning itself adjacent to Jun-Fos on the DNA. Co-occupancy apparently leads to an inhibitory effect on c-Jun’s transactivation function. These two events mediated by VDR effectively block the NFAT1–AP-1 activation complex, resulting in an attenuation of activated GM-CSF transcription.
Pim-1 is an oncogenic serine/threonine kinase with poorly defined function in epithelial cancers. In this study, we determined 1) associations of Pim-1 expression with clinicopathological parameters including responsiveness to irradiation in squamocellular cancers of head and neck and 2) how Pim-1 expression is controlled subsequent to irradiation. Moderate to high expression of Pim-1 correlated to poor response to radiation therapy (P = .003). It is also associated to the expression of epidermal growth factor receptor (EGFR, P < .0001), which has been shown to be activated by irradiation. In radioresistant tumors, irradiation promoted nuclear translocation of Pim-1 (P < .005). When directly testing EGFR dependence of Pim-1 expression, up-regulation and nuclear translocation of Pim-1 could be induced through stimulation of EGFR with its ligands EGF or transforming growth factor α. Both ligand- and irradiation-induced changes in Pim-1 expression and localization could be inhibited by the monoclonal anti-EGFR antibody cetuximab and by the tyrosine kinase inhibitor gefitinib also targeting EGFR. These results suggest that irradiation-induced activation of EGFR upregulates Pim-1, and Pim-1 may be used as a novel predictive marker of radiation response in patients with squamocellular cancers of head and neck.
The incidence and death rate of prostate cancer is increasing rapidly. In addition, the low sensitivity of prostate cancer to chemotherapy makes it difficult to treat this condition. The serine/threonine kinase Pim-1 plays an important role in cell cycle progression and apoptosis inhibition, resulting in prostate tumorigenesis. Therefore, Pim-1 inhibition has been expected to be an attractive target for developing new anti-cancer drugs. However, no small compounds targeting Pim-1 have progressed to clinical use because of their lack of specificity. Here, we have reported a new cell-permeable Pim-1 inhibitory p27Kip1 peptide that could interfere with the binding of Pim-1 to its substrates and act as an anti-cancer drug. The peptide could bind to Pim-1 and inhibit phosphorylation of endogenous p27Kip1 and Bad by Pim-1. Treatment of prostate cancer with the peptide induces G1 arrest and subsequently apoptosis in vitro. However, the peptide showed almost no growth inhibitory or apoptosis-inducing effects in normal cells. The peptide could inhibit tumor growth in in vivo prostate cancer xenograft models. Moreover, the peptide treatment could overcome resistance to taxol, one of the first line chemotherapeutic agents for prostate cancer, and a combination of the peptide with taxol synergistically inhibited prostate cancer growth in vivo. These results indicate that a Pim-1 inhibitory p27Kip1 peptide could be developed as an anti-cancer drug against prostate cancer.
Anticancer Drug; Cancer Therapy; Crystal Structure; Peptide Interactions; Serine Threonine Protein Kinase; Pim-1; p27Kip1; Prostate Cancer
Pim‐1 is a serine/threonine kinase that has been shown to play an integral role in the development of a number of human cancers, such as haematolymphoid malignancies. Recently, evidence has shown Pim‐1 to be important in prostatic carcinogenesis. In order to further our understanding of its role in prostate cancer, we investigated Pim‐1 expression in normal, premalignant, and malignant prostate tissue.
Using immunohistochemistry, Pim‐1 expression was analysed in prostate tissue from 120 radical prostatectomy specimens. In each case, Pim‐1 staining was evaluated in benign prostatic epithelium, high grade prostatic intraepithelial neoplasia (PIN), and prostatic adenocarcinoma. The number of positively staining cells was estimated, and the intensity of staining was scored on a scale of 0 to 3+.
Pim‐1 immunoreactivity was identified in 120 cases (100%) of adenocarcinoma, 120 cases (100%) of high grade PIN, and 62 cases (52%) of benign glands. The number of cells staining in benign epithelium (mean 34%) was much lower than that in high grade PIN (mean 80%; p<0.0001) or adenocarcinoma (mean, 84%; p<0.0001). There was no significant difference between high grade PIN and adenocarcinoma in the percentage of cells staining positively for Pim‐1 (p = 0.34). The staining intensity for Pim‐1 was significantly lower in benign prostatic epithelium than in PIN and adenocarcinoma (p<0.001). There was no statistically significant correlation between the level of Pim‐1 expression and Gleason score, patient age, tumour stage, lymph node metastasis, perineural invasion, vascular invasion, surgical margin status, extraprostatic extension, or seminal vesicle invasion.
Pim‐1 expression is elevated in PIN and prostatic adenocarcinoma compared with benign prostatic epithelium. This finding suggests that upregulation of Pim‐1 may play a role in prostatic neoplasia.
prostate; prostatic intraepithelial neoplasia; adenocarcinoma; Pim‐1; biomarkers; progression; carcinogenesis
The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.
Hereditary Vitamin D Resistant Rickets (HVDRR) is a rare disease caused by mutations in the vitamin D receptor (VDR). The consequence of defective VDR is the inability to absorb calcium normally in the intestine. This leads to a constellation of metabolic abnormalities including hypocalcemia, secondary hyperparathyroidism and hypophosphatemia that cause the development of rickets at an early age in affected children. An interesting additional abnormality is the presence of alopecia in some children depending on the nature of the VDR mutation. The data indicate that VDR mutations that cause defects in DNA binding, RXR heterodimerization or absence of the VDR cause alopecia while mutations that alter VDR affinity for 1,25(OH)2D3 or disrupt coactivator interactions do not cause alopecia. The cumulative findings indicate that hair follicle cycling is dependent on unliganded actions of the VDR. Further research is ongoing to elucidate the role of the VDR in hair growth and differentiation.
Prior studies have given no evidence for regulation of vitamin D receptor (VDR) compartmentalization or subcellular organization. Microwave fixation (9-15 s) and an indirect immunodetection system of avidin-biotin enhancement and phycoerythrin fluorophore resulted in sufficient spatial and temporal resolution to allow analysis of these processes. We studied cultured fibroblasts from normals or from patients with four different types of hereditary defect compromising VDR function (mutant cells). Compartmentalization of VDRs in the absence of 1,25-dihydroxyvitamin D3 (calcitriol) was regulated by serum or estrogen. VDRs were mainly cytoplasmic in cells cultured without serum and phenol red, but VDRs were mainly intranuclear after addition of serum or an estrogen to cells for at least 18 h (slow regulation). Calcitriol initiated a rapid and multistep process (rapid regulation) of reorganization in a portion of VDRs: clumping within 15-45 s, alignment of clumps along fibrils within 30-45 s, perinuclear accumulation of clumps within 45-90 s, and intranuclear accumulation of clumps within 1-3 min. We found similar rapid effects of calcitriol on VDRs in various other types of cultured cells. These sequential VDR pattern changes showed calcitriol dose dependency and calcitriol analogue specificity characteristic for the VDR. In mutant fibroblasts VDR pattern changes after calcitriol were absent or severely disturbed at selected steps. Treatment of normal cells with wheat germ agglutinin, which blocks protein transport through nuclear pores, also blocked calcitriol-dependent translocation of VDRs. We conclude that immunocytology after microwave fixation provides evidence for regulation of VDR organization and localization.