Exaptation of an ancient Alu short interspersed element provides a highly conserved vitamin D-mediated innate immune response in humans and primates.

BACKGROUND: About 45% of the human genome is comprised of mobile transposable elements or "junk DNA". The exaptation or co-option of these elements to provide important cellular functions is hypothesized to have played a powerful force in evolution; however, proven examples are rare. An ancient primate-specific Alu short interspersed element (SINE) put the human CAMP gene under the regulation of the vitamin D pathway by providing a perfect vitamin D receptor binding element (VDRE) in its promoter. Subsequent studies demonstrated that the vitamin D-cathelicidin pathway may be a key component of a novel innate immune response of human to infection. The lack of evolutionary conservation in non-primate mammals suggested that this is a primate-specific adaptation. Evidence for evolutionary conservation of this regulation in additional primate lineages would provide strong evidence that the TLR2/1-vitamin D-cathelicidin pathway evolved as a biologically important immune response mechanism protecting human and non-human primates against infection. RESULTS: PCR-based amplification of the Alu SINE from human and non-human primate genomic DNA and subsequent sequence analysis, revealed perfect structural conservation of the VDRE in all primates examined. Reporter gene studies and induction of the endogenous CAMP gene in Rhesus macaque peripheral blood mononuclear cells demonstrated that the VDREs were conserved functionally. In addition, New World monkeys (NWMs) have maintained additional, functional steroid-hormone receptor binding sites in the AluSx SINE that confer retinoic acid responsiveness and provide potential thyroid hormone receptor binding sites. These sites were less well-conserved during human, ape and Old World monkey (OWM) evolution and the human CAMP gene does not respond to either retinoic acid or thyroid hormone. CONCLUSION: We demonstrated that the VDRE in the CAMP gene originated from the exaptation of an AluSx SINE in the lineage leading to humans, apes, OWMs and NWMs and remained under purifying selection for the last 55-60 million years. We present convincing evidence of an evolutionarily fixed, Alu-mediated divergence in steroid hormone nuclear receptor gene regulation between humans/primates and other mammals. Evolutionary selection to place the primate CAMP gene under regulation of the vitamin D pathway potentiates the innate immune response and may counter the anti-inflammatory properties of vitamin D.

Novel Gemini vitamin D(3) analogs have potent antitumor activity.

The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], modulates proliferation and induces differentiation of many cancer cells. A new class of analogs of vitamin D(3) has been synthesized, having two side-chains attached to carbon-20 (Gemini) and deuterium substituted on one side-chain. We have examined six of these analogs for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP, PC-3, DU145), lung (H520), colon (HT-29), and breast (MCF-7) cancer cell lines. Dose-response clonogenic studies showed that all six analogs had greater antiproliferative activities against cancer cells than 1,25(OH)(2)D(3). Although they had similar potency, the most active of these analogs was BXL-01-0120. BXL-01-0120 was 529-fold more potent than 1,25(OH)(2)D(3) in causing 50% clonal growth inhibition (ED(50)) of HL-60 cells. Pulse-exposure studies demonstrated that exposure to BXL-01-120 (10(-9)M, 48h) resulted in 85% clonal inhibition of HL-60 growth. BXL-01-0120 (10(-11)M, 4 days) induced the differentiation marker, CD11b. Also, morphologically differentiation was more prominent compared to 1,25(OH)(2)D(3). Annexin V assay showed that BXL-01-0120 (10(-10)M, 4 days) induced significantly (p<0.05) more apoptosis than 1,25(OH)(2)D(3). In summary, these analogs have a unique structure resulting in extremely potent inhibition of clonal proliferation of various types of cancer cells, especially HL-60 cells.

Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on lung cancer: roles of cyclooxygenase-2.

We examined the effects of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the lung cancer cell lines PC-9, LA-1 and A549. In addition, we examined if the effects of the cytokines on the cell lines are mediated by activation of cyclooxygenase (COX)-2. The three cell lines did not constitutively produce either G-CSF or GM-CSF. G-CSF did not influence cell growth in the three cell lines, while GM-CSF increased cell growth in the A549 and LA-1 lines. G-CSF and GM-CSF dose-dependently decreased cell death in the three cell lines. RT-PCR demonstrated GM-CSF receptor expression in the three lung cancer cell lines, whereas the G-CSF receptor exists only in the PC-9 line. We suggest that G-CSF might rescue the tumor cells from cytotoxicity due to serum deprivation through cellular pathways independent of the G-CSF receptor. G-CSF and GM-CSF increased cyclooxygenase-2 (COX-2) expression in PC-9 and LA-1 cells whereas they decreased COX-2 expression in A549 cells. The COX-2 inhibitor NS-398 increased cell death in PC-9 and LA-1 cells, whereas it decreased cell death in A549 cells. PC-9 and LA-1 clones transfected with sense G-CSF- or GM-CSF showed an increase in COX-2 expression, while COX-2 expression was decreased in transfected A549 clones. COX-2 expression was increased in anti-sense G-CSF- and GM-CSF-transfected A549 clones. Thus, although COX-2 activation seems to induce different biological behavior depending on the cell type, we propose that G-CSF and GM-CSF might accelerate tumor progression by directly regulating COX-2 expression, independently of an autocrine mechanism.

Regulation of the CAMP gene by 1,25(OH)2D3 in various tissues.

The induction of antimicrobial peptides such as the human cathelicidin, CAMP/hCAP18, by 1,25(OH)(2)D(3) provides a very exciting therapeutic approach in boosting immunity against infectious diseases. To explore the range of cell types and expand the number of cell models for studying the regulation of CAMP gene expression by 1,25(OH)(2)D(3), we treated cell lines from various tissue types and determined CAMP gene expression. Also, we tested additional compounds together with 1,25(OH)(2)D(3) to look for possible cooperative activation of the gene. We identified 1,25(OH)(2)D(3)-mediated induction of the CAMP gene in B-cell lymphomas, prostate and endometrial cancer lines and found cooperative activation with the histone deacetylase inhibitor sodium butyrate. The data suggest that regulation of CAMP by 1,25(OH)(2)D(3) is potentially important in a wide range of tissues.

Effects of GM-CSF and M-CSF on tumor progression of lung cancer: roles of MEK1/ERK and AKT/PKB pathways.

Several studies have demonstrated that colony-stimulating factors (CSFs) are closely associated with tumor progression, metastasis and invasion through autocrine or paracrine mechanism in lung cancer. However, biologic roles of CSFs are still unknown. Elucidating the biologic roles of CSFs and the regulatory mechanisms of tumor-specific behavior by CSFs raises the possibility of having a new therapeutic approach for lung cancer. We previously established two adenocarcinoma cell lines, A924 and A964 and a large cell carcinoma cell line MI-4. MI-4 and A924 constitutively produced an abundant dose of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). We examined the effects of GM-CSF and M-CSF on tumor growth, death, and invasion in CSF-producing (A924 and MI-4) and non-producing lung cancer cells (A549 and A964). These cell lines demonstrated both GM-CSF and M-CSF receptor mRNA expression. In our study, GM-CSF seemed to have advantage for tumor proliferation and invasion in lung cancer cells. M-CSF seemed to have advantage for tumor invasion, but not proliferation. The tumor-specific phenotypes (proliferation, invasion and survival) up-regulated by GM-CSF and M-CSF were mediated through MEK/ERK and PI3k/Akt pathways. However, when MEK/ERK was activated by transfection of active form of MEK1 cDNA, the tumor-specific behavior was promoted in CSF-non-producing cells, whereas inhibited in CSF-producing cells though MEK/ERK activation increased constitutive GM-CSF production. MEK/ERK signaling regulated differently tumor-specific behavior between CSF-producing cells and CSF-non-producing cells.

Role of protein kinase C in expression of granulocyte-colony stimulating factor and granulocyte macrophage-colony stimulating factor in lung cancer cells.

We examine the role of protein kinase C (PKC) pathways in the constitutive expression of granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF) in lung cancer cells. Two cell lines, OKa-C-1 and MI-4, constitutively produce an abundant dose of G-CSF and GM-CSF. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated the production of GM-CSF in a dose-dependent manner and reduced G-CSF in the cell lines. The PKC inhibitor staurosporine had effects opposite to those of PMA in the cell lines. Another PKC activator (4beta-phorbol 12, 13-dibutyrate) and six specific PKC inhibitors (bisindolylmaleimide I, calphostin C, chelerythrine chloride, Gö 6976, PKC inhibitor 19-27, and Ro-32-0432) also worked as well as PMA and staurosporine, respectively. The induction of GM-CSF expression via PKC activation was mediated by the activation of nuclear factor-kappaB. The induction of G-CSF expression via PKC inhibition was mediated by p44/42 mitogen-activated protein kinase and c-Jun N-terminal kinase pathway signaling. GM-CSF may accelerate cell growth and inhibit cell death via PKC activation in the cell lines. G-CSF also seems to reverse growth suppression and cell death induced by PKC inhibition.

Oridonin, a diterpenoid purified from Rabdosia rubescens, inhibits the proliferation of cells from lymphoid malignancies in association with blockade of the NF-kappa B signal pathways.

This study found that oridonin, a natural diterpenoid purified from Rabdosia rubescens, inhibited growth of multiple myeloma (MM; U266, RPMI8226), acute lymphoblastic T-cell leukemia (Jurkat), and adult T-cell leukemia (MT-1) cells with an effective dose that inhibited 50% of target cells (ED50) ranging from 0.75 to 2.7 microg/mL. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining showed that oridonin caused apoptosis of MT-1 cells in a time-dependent manner. We explored effects of oridonin on antiapoptotic Bcl-2 family members and found that it down-regulated levels of Mcl-1 and BCL-x(L), but not Bcl-2 protein, in both MT-1 and RPMI8226 cells. Further studies found that oridonin inhibited nuclear factor-kappa B (NF-kappa B) DNA-binding activity in these cells as measured by luciferase reporter gene, ELISA-based, and electrophoretic mobility shift assays. Oridonin also blocked tumor necrosis factor-alpha- and lipopolysaccharide-stimulated NF-kappa B activity in Jurkat cells as well as RAW264.7 murine macrophages. Of note, oridonin decreased survival of freshly isolated adult T-cell leukemia (three samples), acute lymphoblastic leukemia (one sample), chronic lymphocytic leukemia (one sample), non-Hodgkin's lymphoma (three samples), and MM (four samples) cells from patients in association with inhibition of NF-kappa B DNA-binding activity. On the other hand, oridonin did not affect survival of normal lymphoid cells from healthy volunteers. Taken together, oridonin might be useful as adjunctive therapy for individuals with lymphoid malignancies, including the lethal disease adult T-cell leukemia.