Network-guided modeling allows tumor-type independent prediction of sensitivity to all-trans-retinoic acid.

Background: All-trans-retinoic acid (ATRA) is a differentiating agent used in the treatment of acute-promyelocytic-leukemia (APL) and it is under-exploited in other malignancies despite its low systemic toxicity. A rational/personalized use of ATRA requires the development of predictive tools allowing identification of sensitive cancer types and responsive individuals. Materials and methods: RNA-sequencing data for 10 080 patients and 33 different tumor types were derived from the TCGA and Leucegene datasets and completely re-processed. The study was carried out using machine learning methods and network analysis. Results: We profiled a large panel of breast-cancer cell-lines for in vitro sensitivity to ATRA and exploited the associated basal gene-expression data to initially generate a model predicting ATRA-sensitivity in this disease. Starting from these results and using a network-guided approach, we developed a generalized model (ATRA-21) whose validity extends to tumor types other than breast cancer. ATRA-21 predictions correlate with experimentally determined sensitivity in a large panel of cell-lines representative of numerous tumor types. In patients, ATRA-21 correctly identifies APL as the most sensitive acute-myelogenous-leukemia subtype and indicates that uveal-melanoma and low-grade glioma are top-ranking diseases as for average predicted responsiveness to ATRA. There is a consistent number of tumor types for which higher ATRA-21 predictions are associated with better outcomes. Conclusions: In summary, we generated a tumor-type independent ATRA-sensitivity predictor which consists of a restricted number of genes and has the potential to be applied in the clinics. Identification of the tumor types that are likely to be generally sensitive to the action of ATRA paves the way to the design of clinical studies in the context of these diseases. In addition, ATRA-21 may represent an important diagnostic tool for the selection of individual patients who may benefit from ATRA-based therapeutic strategies also in tumors characterized by lower average sensitivity.

Diminished regulatory T cells in cutaneous lesions of thymoma-associated multi-organ autoimmunity: a newly described paraneoplastic autoimmune disorder with fatal clinical course.

Thymoma-associated multi-organ autoimmunity is a rare, autoimmune disease that causes colitis, liver dysfunction and cutaneous graft-versus-host (GVH)-like skin damage. This paraneoplastic autoimmune disorder may be due to inadequate T cell selection in the tumour environment of the thymus. Although sporadic case reports have revealed its clinical features, little is known about its pathological mechanism. By comparing the skin-infiltrating T cell subsets with those of GVH disease (GVHD) and other inflammatory skin diseases, we sought to elucidate the pathological mechanism of thymoma-associated multi-organ autoimmunity. Histopathological and immunohistochemical analysis of skin biopsies was performed for three patients with thymoma-associated multi-organ autoimmunity. Histopathological findings of thymoma-associated multi-organ autoimmunity were indistinguishable from those of patients with acute GVHD, although the aetiologies of these diseases are completely different. The frequency of regulatory T cells (T(regs)) is reduced in cutaneous lesions and CD8+ cytotoxic T lymphocytes that massively infiltrate into the epidermis of patients with thymoma-associated multi-organ autoimmunity. Additionally, the ratio of T helper type 17 (Th17) cells to CD4+ cells in patients with thymoma-associated multi-organ autoimmunity and acute GVHD was higher than that in healthy controls, but similar to that in psoriasis vulgaris patients. Similarity of the skin-infiltrating T cell subsets with those of acute GVHD suggested that skin damage in patients with thymoma-associated multi-organ autoimmunity might be induced by self-reactive cytotoxic T lymphocytes under the diminished suppressive capacity of T(regs).

Effects of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor and low-density lipoprotein on proliferation and migration of keratinocytes.

BACKGROUND: Keratinocytes can obtain cholesterol either by de novo synthesis or by extraction, primarily from low-density lipoprotein (LDL). LDL is internalized following binding to the LDL receptor (LDLR). Because LDLR is expressed at a higher level in the cells of the basal layer of the epidermis, it might be assumed that LDLR upregulation is associated with keratinocyte proliferation. However, the effect of LDLR stimulation on keratinocyte function remains unclear. OBJECTIVES: To investigate the effects and mechanism of action of pitavastatin and effects of LDL on proliferation and migration of keratinocytes. METHODS: Pitavastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, was used to induce upregulation of LDLR. LDLR expression was evaluated by immunofluorescence staining, fluorescence-activated cell sorting, immunohistochemical staining and real-time polymerase chain reaction (PCR). HaCaT cells and normal human keratinocytes (NHKs) were used for evaluation of migration. 5-Bromo-2'-deoxyuridine incorporation was used to evaluate keratinocyte proliferation and differentiation. C57BL6 mice were used for in vivo evaluation of the effect of topical pitavastatin or lovastatin. RESULTS: Pitavastatin was most effective in LDLR induction at a concentration of 1 micromol L(-1) in NHKs. Real-time PCR showed that pitavastatin significantly increased LDLR and liver X receptor (LXR) beta mRNA expression in these cells. Similar results were obtained in vivo. However, pitavastatin had no effect on the migration of NHKs. After the addition of LDL and/or mevalonate concomitantly with pitavastatin to NHK cultures, or topical application of pitavastatin on mouse skin, keratinocyte proliferation was significantly increased. CONCLUSIONS: Pitavastatin significantly upregulates LDLR in both NHKs and C57BL6 mouse skin, resulting in increased keratinocyte proliferation. LXRbeta may be involved in the pitavastatin-induced keratinocyte proliferation.

Cellular DNA rearrangements and early developmental arrest caused by DNA insertion in transgenic mouse embryos.

Insertional mutagenesis was investigated in a transgenic mouse strain (HUGH/4) derived from a fertilized egg injected with plasmid DNA containing the human growth hormone gene. Lethality occurred in homozygous embryos and was traced to the egg cylinder stage on days 4 to 5 of gestation, shortly after implantation. The mutation is on chromosome 12 and is distinct in location and integration pattern from another mutation also leading to lethality of homozygotes in the egg cylinder stage. Based on this and other evidence, relatively many genes may be recruited to activity near the time of implantation and may therefore present a large target of vulnerability to mutagenesis. The single insert in HUGH/4, consisting of approximately three tandem copies of plasmid sequences, is flanked by mouse cellular sequences that have undergone rearrangements, including a probable deletion. The data suggest the hypothesis that DNA rearrangements, which appear to be commonplace in transgenic mice, may arise because the initial insertional complex is unstable; stepwise changes may then be generated until a more stable conformation is achieved.

Isolation and characterization of the gene coding for human cytidine deaminase.

The human gene coding for cytidine deaminase (CD), the enzyme which catalyzes the deamination of cytidine and deoxycytidine to uridine and deoxyuridine, was isolated and structurally characterized. CD is a single copy gene with a length of 31 kb and consists of four exons. Exon-intron junctions do not bracket functional domains of the encoded protein as the boundary between exons 2 and 3 interrupts the catalytically important zinc-finger domain, which is well conserved along phylogenesis. 5'-RACE and RNase mapping experiments identify one major and multiple other minor transcription initiation sites, which are present in placenta as well as in the myeloid cell lines, HL-60 and U937. The 5'-flanking region of the gene contains an orientation-dependent functional promoter and is characterized by the presence of several potential sites for the binding of known transcriptional factors.

The mouse aldehyde oxidase gene: molecular cloning, chromosomal mapping and functional characterization of the 5'-flanking region.

In this article, we report on the chromosome mapping and molecular cloning of the genetic locus encoding the mouse molybdo-iron/sulfur-flavoprotein aldehyde oxidase. The aldehyde oxidase locus maps to mouse chromosome 1 band C1-C2, as determined by fluorescence in situ hybridization experiments conducted on metaphase chromosomes. The gene is approximately 83 kb long and consists of 35 exons. The exon/intron boundaries are perfectly conserved relative to the corresponding human homolog and almost completely conserved relative to the mouse xanthine oxidoreductase gene. This further supports the concept that the aldehyde oxidase and xanthine oxidoreductase loci evolved from the same ancestral precursor by a gene duplication event. The position of a major transcription start site was defined by primer extension and RNase mapping analysis. The 5'-flanking region of the mouse aldehyde oxidase gene contains a functional and orientation-dependent promoter as well as several putative binding sites for known cell-specific and general transcription factors. Deletion analysis of the 5'-flanking region defines an approximately 470 bp DNA stretch which is necessary and sufficient for the transcription of the mouse aldehyde oxidase gene.

Retinoid-dependent growth inhibition, differentiation and apoptosis in acute promyelocytic leukemia cells. Expression and activation of caspases.

In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARalpha-selective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARbeta agonist and an anti-AP1 retinoid have very limited activity, ATRA- and AM580-dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARalpha. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD.

Retinoic acid and methylation cis-regulatory elements control the mouse tissue non-specific alkaline phosphatase gene expression.

To understand the mechanisms regulating the tissue non-specific alkaline phosphatase (TNAP) activity during development, we characterized cis-transcriptional regulatory elements. In embryonic cells and tissues, TNAP expression was driven preferentially by the exon 1A (E1A) promoter, one of the two promoters previously defined. Transcriptional activity of E1A promoter was up-regulated by retinoic acid (RA) through a putative RA-responsive element. Transgenic mice analysis with lacZ reporter constructs revealed negative regulatory elements within 8.5 kb of E1A promoter. Promoter sequences of endogenous TNAP in non-expressing tissues and those carried by the 8.5 kb-lacZ transgene were found to be highly methylated. A 1 kb fragment of E1A promoter increased the methylation level of lacZ and promoter sequences. The role of RA and DNA methylation in defining the embryonic expression pattern of TNAP is discussed.

Cytodifferentiation: a novel approach to cancer treatment and prevention.

Cytodifferentiation therapy promises to control cancer growth and progression with less serious side effects than cytotoxic chemotherapy. Despite recent progress, the molecular mechanisms regulating the differentiation of many cell types are still obscure and the number of active cytodifferentiating agents is limited. Rational ways to develop these types of agents are necessary.

Inhibition of melanogenesis by BMY-28565, a novel compound depressing tyrosinase activity in B16 melanoma cells.

The mechanism of a novel melanin synthesis inhibitor, BMY-28565, was studied using mouse B16 melanoma cells. This compound was active in depressing the intracellular accumulation of melanin with an IC50 of 5 microM. At dose levels causing no cytotoxicity, the melanolytic effect of this compound was correlated strongly with depression of the enzymatic activity of tyrosinase (monophenol oxygenase, EC 1.14.18.1), the key enzyme in the melanin synthesis pathway. Transcription of the tyrosinase gene was not inhibited by BMY-28565, as determined by RNA blotting analysis. BMY-28565 and three other active derivatives of this compound caused increased glycosylation of proteins in B16 melanoma cells, as assessed by radioactive mannose incorporation. It is, thus, suggested that the mechanism of inhibition of tyrosinase might be related to modifications of the sugar moiety of this enzyme or of a protein(s) that is essential for the expression of its enzymatic activity.

Molecular mechanisms of retinoid action in acute promyelocytic leukemia (Review).

Acute promyelocytic leukemia (APL) is, at present, the first and only example of leukemia which can be induced into remission with a single cyto-differentiating agent. This is due to the fact that APL is exquisitely sensitive to the differentiating action of all-trans retinoic acid (ATRA). Thus, the APL model offers a unique opportunity to study the cyto-differentiating action of ATRA and synthetic retinoids in a clinically relevant setting. This review article summarizes the work relating to the molecular mechanisms underlying the action of retinoic acid and retinoids in APL cells, and focuses on: a) genes which are expressed and regulated by ATRA; b) synthetic retinoids as cyto-differentiating agents; c) rational combinations between retinoids and cytokines or other cyto-differentiating agents; d) cellular paradigms of retinoic acid resistance. It is our aim to give an updated, about nonexhaustive, account of some of the most recent development regarding the pharmacological action of retinoic acid and its derivatives in APL cells.

Selective localization of mouse aldehyde oxidase mRNA in the choroid plexus and motor neurons.

Aldehyde oxidase (AO), a protein involved in the catabolism of catecholamines, is the product of a gene potentially responsible for one of the familial forms of the motor neuron disease, amyotrophic lateral sclerosis (ALS). Here, we report on the cloning of a partial cDNA coding for the mouse enzyme. Using this cDNA as a probe, we demonstrate that the AO transcript is expressed in the epithelial component of the choroid plexus. More importantly, in the gray matter, the mRNA is selectively localized in the large motor neurons of the nuclei of facial, motor trigemini and hypoglossus nerves and in the motor neurons of the anterior horns of the spinal cord. This localization is consistent with a possible role of AO in the pathogenesis of ALS.

Selective inhibition by ketanserin and spiroperidol of 5-HT-induced myometrial contraction.

The inhibitory effects of ketanserin and spiroperidol (a neuroleptic drug) on the contractile response of isolated rat uterus to serotonin (5-HT) were investigated. Ketanserin caused non-competitive inhibition of the contractile response to 5-HT and showed more selective inhibition than the other 5-HT antagonists tested. The inhibitory effect of spiroperidol was comparable with the effects of classical 5-HT antagonists. These results suggest that ketanserin and spiroperidol selectively inhibit the contractile response of isolated rat uterus to 5-HT.

Study of platelet activating factor and its antagonists on rat colon strip with a new method avoiding tachyphylaxis.

Platelet activating factor induced slow, but sustained contraction of isolated rat colon in a dose-dependent manner. The contraction persisted even when the strips were washed several times with Tyrode solution. However, the addition of high concentrations of bovine serum albumin to the washing solution led to the rapid relaxation of the strips to the basal level, possibly by removing platelet activating factor tightly bound to rat colon. These strips then responded normally to a second addition of platelet activating factor. Neither the cholinergic nervous system nor release of histamine, serotonin, prostaglandins or leukotriene D4 were significantly involved in the contraction induced by platelet activating factor. FR-900452 and CV-3988, recently found to be antagonists of platelet activating factor, selectively blocked the contraction of rat colon induced by the active phospholipid, indicating that it induced contraction by a direct stimulatory effect on the smooth muscle of rat colon in a receptor-mediated manner.

Cross-talk between retinoic acid and interferons: molecular mechanisms of interaction in acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) and interferons (IFNs) are active anticancer agents. ATRA is capable of inducing complete remission in acute promyelocytic leukemia (APL) patients, whereas IFNalpha is successfully used in the treatment of the stable phase of chronic myeloid leukemia. ATRA and IFNs have shown synergistic interactions in various experimental conditions and represent a potentially useful therapeutic combination in the treatment of various types of leukemias and solid tumors. The molecular basis of these interactions are poorly understood and need to be elucidated. In this review, we summarize a series of recent observations concerning the molecular mechanisms underlying the cross-talk between the intracellular pathways activated by ATRA and IFNs in APL cells. In APL blasts, IFNs regulate the expression of retinoic acid receptors, and ATRA, in turn, modulates the levels and the state of activation of members of the Jak-STAT second messenger pathway. This demonstrates a two-way interaction between ATRA and IFNs, which leads to cross-modulation of genes normally under the control of the retinoid and the cytokine. These data may be relevant in the context of a rational use of the combination between ATRA and IFNs in the clinical management of myeloid leukemias.

Placental form of membrane-bound neutral arylamidase found in renal cell carcinoma.

A placental form of membrane-bound neutral arylamidase was found in the tissue of renal cell carcinoma. Membrane-bound neutral arylamidases from renal cell carcinoma, an intact part of the same kidney and placenta, had the same molecular weight (240 000) and were identical with respect to KM value and effect of inhibition by chelators or amino acids. Membrane-bound neutral arylamidase from renal carcinoma had the same electrophoretic mobility and heat stability as placental membrane-bound neutral arylamidase, but differed from kidney membrane-bound neutral arylamidase with respect to electrophoretic mobility, heat stability and susceptibility to urea inactivation. Results suggest the carcinoplacental alterations in membrane-bound neutral arylamidase isoenzyme in renal cell carcinoma.

Membrane-bound arylamidase from human hepatoma cells having the same electrophoretic mobility as placental membrane-bound arylamidase.

A membrane-bound arylamidase from well-differentiated hepatocellular carcinoma having the same electrophoretic mobility as placental membrane-bound arylamidase was found. The enzyme was found to be a sialoprotein and was activated by Co2+. Hepatoma membrane-bound arylamidase had a similar molecular weight (240 000) and kinetic properties to normal liver membrane-bound arylamidase, but differed from the liver enzyme with respect to electrophoretic mobility, heat stability and urea inactivation.

Comparison of human membrane-bound neutral arylamidases from small intestine, lung, kidney, liver and placenta.

Human membrane-bound neutral arylamidases were solubilized from small intestinal mucosa, lung, kidney, liver and placenta with trypsin. These five membrane-bound neutral arylamidases were identified by polyacrylamide gel-disc electrophoresis. The heat sensitivity of each enzyme was in the order, liver and placenta greater than kidney greater than lung greater than small intestine. This order correlates with that of electrophoretic mobility, except for the placental membrane-bound neutral arylamidase. Five membrane-bound neutral arylamidases have the same molecular weight, 240 000, as estimated by Sephadex G-200 gel filtration. The five membrane-bound neutral arylamidase have very similar KM values (8.7 x 10(-5) M towards L-alanyl-beta-naphthylamide), optimal pH values, hydrolysis ratios towards L-alanyl-beta-naphthylamide and L-leucyl-beta-naphthylamide, and sensitivities of inhibition by chelators or amino acids. These results suggest that the multiple forms of membrane-bound neutral arylamidase found in five different human organs are organ-specific isoenzymes.

A placental type of membrane-bound arylamidase in serum and ascites from a patient with renal cell carcinoma.

A placental type of membrane-bound arylamidase was detected in serum and ascites from a patient with renal cell carcinoma by isoelectric focusing. The enzyme had the same molecular weight, electrophoretic mobility on polyacrylamide gel, isoelectric point and KM value as placental membrane-bound arylamidase. The new type of membrane-bound arylamidase was named Shiba-isoenzyme after the patient's name.