Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by protease mapping.

The pleiotropic effects of retinoic acid on cell differentiation and proliferation are mediated by two subfamilies of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Recently the synthetic retinoid Ro 41-5253 was identified as a selective RAR alpha antagonist. As demonstrated by gel retardation assays, Ro 41-5253 and two related new RAR alpha antagonists do not influence RAR alpha/RXR alpha heterodimerization and DNA binding. In a limited trypsin digestion assay, complexation of RAR alpha with retinoic acid or several other agonistic retinoids altered the degradation of the receptor such that a 30-kDa proteolytic fragment became resistant to proteolysis. This suggests a ligand-induced conformational change, which may be necessary for the interaction of the DNA-bound RAR alpha/RXR alpha heterodimer with other transcription factors. Our results demonstrate that antagonists compete with agonists for binding to RAR alpha and may induce a different structural alteration, suggested by the tryptic resistance of a shorter 25-kDa protein fragment in the digestion assay. This RAR alpha conformation seems to allow RAR alpha/RXR alpha binding to DNA but not the subsequent transactivation of target genes. Protease mapping with C-terminally truncated receptors revealed that the proposed conformational changes mainly occur in the DE regions of RAR alpha. Complexation of RAR beta, RAR gamma, and RXR alpha, as well as the vitamin D3 receptor, with their natural ligands resulted in a similar resistance of fragments to proteolytic digestion. This could mean that ligand-induced conformational changes are a general feature in the hormonal activation of vitamin D3 and retinoid receptors.

DNA damage induced in human diploid cells by decay of incorporated radionuclides.

Alkaline and neutral elution techniques were used to characterize the production of single- and double-strand DNA breaks in human diploid fibroblasts by incorporated radionuclides. 125I was incorporated in DNA as [125I]iododeoxyuridine, 3H as [3H]-thymidine, and 14C as [14C]thymidine. Under frozen conditions, 125I was 3 times as efficient as 3H per decay in inducing single-strand breaks and 6 times as efficient as 14C. For double-strand break production, however, 125I was 6 times as efficient per decay as 3H. It was calculated that, on the average, each 125I decay produces about one double-strand break in the frozen state. Under nonfrozen conditions, 125I and external X-rays were roughly 5-fold and 3H about 3-fold more efficient in double-strand break induction than under frozen conditions.

Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies.

Notch receptors have been implicated as oncogenic drivers in several cancers, the most notable example being NOTCH1 in T-cell acute lymphoblastic leukemia (T-ALL). To characterize the role of activated NOTCH3 in cancer, we generated an antibody that detects the neo-epitope created upon gamma-secretase cleavage of NOTCH3 to release its intracellular domain (ICD3), and sequenced the negative regulatory region (NRR) and PEST (proline, glutamate, serine, threonine) domain coding regions of NOTCH3 in a panel of cell lines. We also characterize NOTCH3 tumor-associated mutations that result in activation of signaling and report new inhibitory antibodies. We determined the structural basis for receptor inhibition by obtaining the first co-crystal structure of a NOTCH3 antibody with the NRR protein and defined two distinct epitopes for NRR antibodies. The antibodies exhibit potent anti-leukemic activity in cell lines and tumor xenografts harboring NOTCH3 activating mutations. Screening of primary T-ALL samples reveals that 2 of 40 tumors examined show active NOTCH3 signaling. We also identified evidence of NOTCH3 activation in 12 of 24 patient-derived orthotopic xenograft models, 2 of which exhibit activation of NOTCH3 without activation of NOTCH1. Our studies provide additional insights into NOTCH3 activation and offer a path forward for identification of cancers that are likely to respond to therapy with NOTCH3 selective inhibitory antibodies.

Characterization of synthetic retinoids with selectivity for retinoic acid or retinoid X nuclear receptors.

The broad spectrum of physiological activities of retinoids is mediate d by two types of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Though they have 9-cis retinoic acid as a common ligand, the amino acid sequence of their ligand binding domains is only distantly related (27%). This fact makes it probable that the ligand binding pockets of RARs and RXRs differ significantly with respect to their three dimensional structure. Therefore, one can expect that selective ligands for these receptor subclasses do exit. A clear example of a naturally existing RAR-selective retinoid is all-trans retinoic acid. Here we report on two synthetic retinoids which are very closely related to retinoic acid in structure yet show good receptor subclass selectivity. These compounds have a saturated double bond in the polyene side chain between either the 7, 8 or 9, 10 carbon atoms and are highly RAR or RXR selective, respectively (as shown by receptor binding, transactivation activity and the ability to induce RXR homodimer formation). In addition, we present compounds of the synthetic arotinoid class which are highly RAR selective. Interestingly the corresponding '9-cis analogs' are not able to bind or activate RXR alpha and show greatly reduced activity on the RARs.

Rapid and efficient generation of PCR-derived riboprobe templates for in situ hybridization histochemistry.

In situ hybridization histochemistry (ISH) using cRNA probes (riboprobes) has become a powerful technique for the examination of gene expression in tissue sections. The construction of plasmid templates for the synthesis of riboprobes with phage RNA polymerases is often a difficult and time-consuming step. We have therefore developed a rapid, efficient, and flexible method to generate totally artificial riboprobe templates by the polymerase chain reaction (PCR). We have made riboprobe templates using self-priming oligonucleotide primers spanning 146 BP of the 3' end of the human cytokeratin 1 (K1) gene coding region flanked by T7 and T3 promoters. These PCR-derived riboprobe templates were used to synthesize 35S-labeled anti-sense riboprobes as well as sense riboprobes as negative controls. The riboprobes were then applied in ISH to human skin sections made from routinely fixed and paraffin-embedded clinical biopsy material. Consistent with published results, we observed strong expression of K1 mRNA in the suprabasal cell layers of the epidermis but only weak to undetectable signals in the basal and cornified cell layers and in the dermis. With this experimental procedure we see no decrease in probe efficiency or quality compared to conventional methods. The use of PCR-derived riboprobe templates for ISH makes it possible to detect expression of any desired gene of known sequence rapidly and efficiently.

A comparison of the lethal effects of intracellular radionuclides in human and rodent cells.

The relative sensitivities of a human diploid fibroblast strain (AG1522) and an established line of contact-inhibited mouse embryo fibroblasts (10T1/2) were compared after incorporation of [3H]thymidine or [125I]iododeoxyuridine into the cellular DNA and growth at 37 degrees C. Whereas these cells show similar sensitivities to acute X irradiation, the D0 for clonal survival of the mouse fibroblasts was 5- to 10-fold higher than the D0 for the human fibroblasts for both 3H and 125I. A large difference in sensitivity between these two cell types was also seen after protracted irradiation from 3H2O at 37 degrees C, but this difference was markedly reduced when cells were exposed to 3H2O at 0 degree C to simulate an "acute" irradiation. An established human tumor cell line was similarly hypersensitive to killing by 3H2O at 37 degrees C as compared with early passage diploid hamster embryo cells. These results indicate that human cells are more radiosensitive than rodent cells when a radiation dose is protracted over several days rather than given as an "acute" exposure, and are consistent with the hypothesis that human cells are hypersensitive to low-dose-rate irradiation as compared with rodent cells.

Differential expression of CRABP II, psoriasin and cytokeratin 1 mRNA in human skin diseases.

In order to confirm and further explore the significance of the overexpression of the CRABP II (cellular retinoic acid binding protein type II) and psoriasin genes in psoriatic versus normal skin, we examined the mRNA expression levels of these two genes by in situ hybridization in skin samples from psoriatic plaques and in one case from the border between a psoriatic plaque and uninvolved skin. Both genes were markedly upregulated in lesional skin, with a shift from low to high expression in the transitional zone of the plaque. Expression of the cytokeratin 1 (K1) gene was, in contrast, high in normal skin and decreased in the transition from uninvolved skin to psoriatic plaque, Examination of mRNA levels of CRABP II and psoriasin in other hyperproliferative and inflammatory skin diseases showed high expression of psoriasin, and in some cases also of CRABP II, in atopic dermatitis, mycosis fungoides, Darier's disease and inflammatory lichen sclerosus et atrophicus. In atrophic lesions of lichen sclerosus et atrophicus that lacked an inflammatory infiltrate, these changes were only weakly expressed. These findings demonstrate that altered epidermal gene expression of K1, psoriasin and CRABP II is not disease-specific and may reflect instead an altered state of epidermal differentiation and/or may be linked to the inflammation and cellular infiltration common to all the conditions studied.

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells.

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters, [125I]iododeoxyuridine ([125I]dUrd) and [3H]thymidine ([3H]TdR), incorporated into cellular DNA. [125I]dUrd was more effective than [3H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TGR). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D0 for cell killing for [125I]dUrd was 28 dpc and for [3H]TdR was 385 dpc. The D0 for X-rays was 48 rad at 37 degrees C. The slopes of the mutation curves were approximately 75 x 10(-8) 6TGR mutants per cell per disintegration for [125I]dUrd and 2 x 10(-8) for [3H]TdR. X-Rays induced 8 x 10(-8) 6TGR mutants per cell per rad. Normalizing for survival, [125I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37 degrees C or while frozen at -70 degrees C yielded no difference in cytotoxicity or mutation for [125]dUrd or [3H]TdR, whereas X-rays were 6 times less effective in killing cells at -70 degrees C. Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TGR locus is 1000-3000 base pairs, then the mutagenic efficiency of [125I]dUrd is 1.0-3.0 and of [3H]TdR is 0.02-0.06 total genomic mutations per cell per disintegration. 125I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D0) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0-3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.

Induction and autoregulation of ada, a positively acting element regulating the response of Escherichia coli K-12 to methylating agents.

The ada gene of Escherichia coli K-12, the regulatory locus for the adaptive response to methylating agents, coded for a 39,000-dalton protein. An adjacent gene coding for a 27,000-dalton protein was coregulated with ada. The Ada protein was strongly induced upon exposure of cells to methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine. An analysis of ada regulation with an ada-lacZ operon fusion showed that ada+ function was required for induction of ada transcription. Derivatives of the ada gene truncated from the 3' end produced proteins which were more potent in stimulating transcription than the product of the intact ada gene, indicating that the transcription-activating function of the Ada protein resided in its amino terminus. The sequence of the ada-regulatory region and the identification of the start site of ada transcription are also presented.

Alteration of the carboxyl-terminal domain of Ada protein influences its inducibility, specificity, and strength as a transcriptional activator.

The ada gene of Escherichia coli K-12 encodes the regulatory protein for the adaptive response to alkylating agents. A set of plasmids carrying ordered deletions from the 3' end of the ada gene were isolated and characterized. These ada deletions encode fusion proteins that derive their amino termini from ada and their carboxyl termini from the downstream vector sequence that occurs before an in-frame stop codon. Several of these ada deletions encode Ada derivatives that constitutively activate ada transcription to very high levels. A second class of ada deletions encode Ada derivatives that are dominant inhibitors of the inducible transcription of ada but are inducible activators of alkA transcription. In addition, we found that two Ada derivatives containing the same ada sequences but fused to different vector-derived tails have strikingly different properties. One Ada derivative constitutively activates both ada and alkA expression to very high levels. In contrast, the other Ada derivative is an inducible activator of ada expression, like the wild-type Ada protein, but is not an inducible activator of alkA transcription. Our data suggest that the carboxyl terminus of the Ada protein plays a key role in modulating the ability of the Ada protein to function as a transcriptional activator.

Malignant transformation induced by incorporated radionuclides in BALB/3T3 mouse embryo fibroblasts.

The induction of lethality and malignant transformation by 5-[125I]iododeoxyuridine and [3H]thymidine incorporated into cellular DNA and by x-irradiation was studied in vitro in BALB/3T3 cells. Under these conditions, 125I radiation is highly localized to small regions of the DNA at the site of each decay and produces DNA double-strand breaks with high efficiency. Incorporated 125I was found to be 12-16 times as lethal per decay as incorporated 3H. For the induction of malignant transformation, however, 125I was approximately 25 times as effective per decay as 3H. When the frequencies of transformation induced at various levels of survival by 125I, 3H, and x-rays were compared, lethality was found to correlate closely with transformation at doses that yielded significant cell killing. An exception occurred at low doses, where 125I appeared much more efficient than x-irradiation in inducing transformation; transformation frequencies equal to those induced by 3-5 Gy of x-rays resulted from 125I exposures that yielded little or no cell killing.

Phytanic acid is a retinoid X receptor ligand.

Metabolic defects in phytanic acid catabolism have been shown to be connected with a number of human diseases which can lead to lethal defects of the nervous system and other organs. These effects are probably a result of the very high accumulation of phytanic acid in tissues throughout the body, due to defects in phytanic acid oxidation, the peroxisome being a major site for this process. The nuclear hormone receptors peroxisome proliferator-activated receptor and retinoid X receptor (RXR) have been shown to function as transcription factors in the control of the peroxisomal enzyme expression. Known activators of peroxisome proliferator-activated receptor include polyunsaturated fatty acids and, for RXR, the 9-cis isomer of retinoic acid. Here we report that phytanic acid is also a natural ligand for RXR alpha, being able to activate a RXR-responsive promoter. We present evidence that phytanic acid binds to RXR alpha, promotes formation of an RXR alpha/RXR response element complex (as detected by gel retardation), and induces a RXR alpha conformational change similar to that induced by 9-cis-retinoic acid (as detected by protease sensitivity). These results suggest an involvement of RXR alpha in the control of fatty acid metabolism and could imply that RXRs have a role in the disease effects resulting from defective phytanic acid catabolism.

The specificities of Sex combs reduced and Antennapedia are defined by a distinct portion of each protein that includes the homeodomain.

The sequence requirements for distinguishing the functional specificities of two homeodomain proteins, Antennapedia and Sex combs reduced, involved in the specification of segmental identities in Drosophila, have been determined. A series of deletions and hybrid proteins was generated and assayed for their function in vivo after heat shock-induced ectopic expression during development. A distinct portion of each protein, including the residues within and adjacent to both ends of the homeodomain, has been found to almost entirely determine its functional specificity as measured by diagnostic cuticular transformations of embryonic and adult head structures. The remaining sequences contribute to the potency with which the proteins act in different cells and are to a limited extent functionally transferable from one protein to the other.

Enhancement of HL-60 differentiation by a new class of retinoids with selective activity on retinoid X receptor.

Cellular responsiveness to retinoic acid and its metabolites is conferred through two distinct families of receptors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Herein, we report on the identification and characterization of several conformationally restricted retinoids, which selectively bind and activate RX receptors. Under the influence of retinoids, HL-60 myelocytic leukemia cells differentiate into granulocytes. This effect is mediated by RAR alpha, as has been demonstrated through the use of a selective RAR alpha antagonist (Apfel, C., Bauer, F., Crettaz, M., Forni, L., Kamber, M., Kaufmann, F., LeMotte, P., Pirson, W., and Klaus, M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7129-7133). Here, we show that conformationally restricted RXR-specific retinoids, at doses that are per se inactive, are able to potentiate by up to one order of magnitude the pro-differentiating effects of all-trans retinoic acid and an RAR alpha-selective synthetic retinoid. We also present evidence that these RXR-selective ligands are able to bind to a DNA RXR.RAR heterodimer complex. This finding demonstrates that agonists for RARs and RXRs can synergistically promote HL-60 differentiation, which could be mediated through a heterodimer of these receptors.

RXR-dependent and RXR-independent transactivation by retinoic acid receptors.

The binding affinity of retinoic acid receptors (RARs) to their response elements is strongly enhanced in vitro by the formation of heterodimers with retinoid X receptors (RXRs) suggesting that heterodimerization with RXR may be a prerequisite for a RAR-mediated transcriptional response. We found that in Drosophila SL-3 cells that are devoid of endogenous RARs and RXRs the presence of RAR is sufficient to confer a response to all-trans retinoic acid (RA). The transfection of both RAR and RXR and stimulation with their respective ligands all-trans and 9-cis RA leads to a synergistic response. On point mutations of the RAR beta 2 gene promoter RA response element (RARE) the stimulation by RARs showed distinct differences in the absence and presence of RXR. The same differences in transcriptional activity are observed, if mammalian cells containing endogenous RARs and RXRs are stimulated with all-trans RA only or additionally with 9-cis RA. This establishes an RXR-independent and an RXR-dependent pathway of all-trans RA action in Drosophila SL-3 cells as well as in mammalian cells. The presence or absence of 9-cis RA determines by which of the two pathways a response to all-trans RA is mediated.

Marked resistance of RAR gamma-deficient mice to the toxic effects of retinoic acid.

Excessive intake of retinol or of retinoic acid causes a syndrome of characteristic toxic effects known as hypervitaminosis A. To test the role of the nuclear retinoic acid receptor (RAR gamma) in this process we produced mice with a targeted disruption of the RAR gamma gene and examined toxic effects of repeated doses of retinoic acid and two other synthetic retinoids, Ro 15-1570 and Ro 40-6055. Surprisingly, homozygous mutant mice were resistant to fourfold higher doses of retinoic acid than wild-type mice as well as to elevated doses of the synthetic retinoids, indicating that RAR gamma may have a major role in mediating retinoid toxicity, a finding that possibly has practical implications for reducing the toxicity of synthetic retinoids in clinical use.

The homeotic gene Sex Combs Reduced of Drosophila: gene structure and embryonic expression.

The homeotic gene Sex Combs Reduced (Scr) of Drosophila is required during embryogenesis for labial and first thoracic segment development. We define the Scr gene structure, showing that the major embryonic transcript is proximal to the fushi tarazu gene, and report the sequence of the transcript, which encodes a 413-amino acid, homeodomain-containing protein. We describe Scr protein distribution throughout embryogenesis. Expression begins at gastrulation and is eventually apparent in three tissues, epidermis, nervous system and visceral mesoderm, though there are clear contrasts in the domains of expression in these three tissues.

A crystallizable form of RIIbeta regulatory domain obtained by limited proteolysis.

The type RIIbeta regulatory subunit of protein kinase A is primarily expressed in adipose tissue and brain. Knockout mice suggest a role for RIIbeta in regulating energy balance and adipose-tissue content, thus making it a potential target for therapeutic intervention in obesity. A truncated version of the RIalpha subunit has been used in a crystallographic study and was used here to design an analogous RIIbeta construct. Despite substantial screening, conditions were not found for the crystallization of the truncated RIIbeta subunit. However, limited proteolysis of the full-length RIIbeta subunit identified boundaries of the 'hinge' region and a fragment containing the two cAMP-binding domains which did crystallize. A recombinant version of the fragment was expressed and crystallized for X-ray diffraction studies. The crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 91.6, b = 105.9, c = 85.8 A, and diffracted to at least 2.3 A.

In situ hybridization analysis of CRABP II expression in sebaceous follicles from 13-cis retinoic acid-treated acne patients.

The aim of this study was to investigate the effects of 13-cis retinoic acid treatment on cellular retinoic acid binding protein II (CRABP II) mRNA expression in sebaceous follicles from acne patients, using in situ hybridization. Biopsies were taken from uninvolved skin areas in close juxtaposition to inflamed comedos before therapy, and at 2-4 or 14-16 weeks of treatment. Paraffin sections were used for in situ hybridization study with riboprobes transcribed from human CRABP II cDNA. After oral treatment with 13-cis retinoic acid, sebaceous glands were reduced in size and atrophic, and the ratio of sebum-free to fully differentiated (sebum-producing) sebocytes was dramatically increased. The CRABP II expression in the sebaceous gland, and to some extent in infundibular structures, was strongly increased compared with the level of expression in the epidermis. The maximum signal was always found in layers of suprabasal sebocytes lacking lipid droplets, but never in the basal layers. These findings indicate a selective activity of 13-cis retinoic acid on CRABP II mRNA expression in the sebaceous glands of acne patients.

A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects.

Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RAR alpha, RAR beta, and RAR gamma. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation. Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions.