RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

Hsp90 nuclear accumulation in quiescence is linked to chaperone function and spore development in yeast.

The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Delta cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the alpha/beta importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.

ANKRD1, the gene encoding cardiac ankyrin repeat protein, is a novel dilated cardiomyopathy gene.

OBJECTIVES: We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients. BACKGROUND: CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction. METHODS: In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP. CONCLUSIONS: ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.

Reduced vascular endothelial growth factor expression in contusive spinal cord injury.

Vascular endothelial growth factor (VEGF) is being investigated as a potential interventional therapy for spinal cord injury (SCI). In the current study, we examined SCI-induced changes in VEGF protein levels using Western blot analysis around the epicenter of injury. Our results indicate a significant decrease in the levels of VEGF(165) and other VEGF isoforms at the lesion epicenter 1 day after injury, which was maintained up to 1 month after injury. We also examined if robust VEGF(165) decrease in injured spinal cords affects neuronal survival, given that a number of reported studies show neuroprotective effect of this VEGF isoform. However, exogenously administered VEGF(165) at the time of injury did not affect the number of sparred neurons. In contrast, exogenous administration of VEGF antibody that inhibits actions of not only VEGF(165) but also of several other VEGF isoforms, significantly decreased number of sparred neurons after SCI. Together these results indicate a general reduction of VEGF isoforms following SCI and that isoforms other than VEGF(165) (e.g., VEGF(121) and/or VEGF(189)) provide neuroprotection, suggesting that VEGF(165) isoform is likely involved in other pathophysiological process after SCI.

Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina.

PURPOSE: To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. METHODS: CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. RESULTS: CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. CONCLUSIONS: The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.

Traumatic brain injury stimulates hippocampal catechol-O-methyl transferase expression in microglia.

Outcome following traumatic brain injury (TBI) is in large part determined by the combined action of multiple processes. In order to better understand the response of the central nervous system to injury, we utilized an antibody array to simultaneously screen 507 proteins for altered expression in the injured hippocampus, a structure critical for memory formation. Array analysis indicated 41 candidate proteins have altered expression levels 24h after TBI. Of particular interest was catechol-O-methyl transferase (COMT), an enzyme involved in metabolizing catecholamines released following neuronal activity. Altered catecholamine signaling has been observed after brain injury, and may contribute to the cognitive dysfunctions and behavioral deficits often experienced after TBI. Our data shows that COMT expression in the injured ipsilateral hippocampus was elevated for at least 14 d after controlled cortical impact injury. We found strong co-localization of COMT immunoreactivity with the microglia marker Iba1 near the injury site. Since dopamine transporter expression has been reported to be down-regulated after brain injury, COMT-mediated catecholamine metabolism may play a more prominent role in terminating catecholamine signaling in injured areas.

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.

Expression and Regulation of Human Cytochrome P450 4F Isoforms in Tissue Samples and Under TNF-Alpha Challenges

CYP4F (Cytochrome P4504F) enzymes metabolize endogenous molecules including leukotrienes, prostaglandins and arachidonic acid. The involvement of these endogenous compounds in inflammation has led to the hypothesis that changes in the inflamed tissue environment may affect the expression of CYP4Fs during the pro-inflammatory state, which in turn may modulate inflammatory conditions during the anti-inflammatory state. We demonstrated that inflamed tissues have different levels of CYP4F isoform expression profiles in a number of human samples when compared to the average population. The CYP4F isoform expression levels change with the degree of inflammation present in tissue. Further investigation in cell culture studies revealed that inflammatory cytokines, in particular TNF-α, play a role in regulating the expression of the CYP4F family. One of the isoforms, CYP4F11, had different characteristics than that of the other five CYP4F family members. CYP4F11 metabolizes xenobiotics while the other isoforms metabolize endogenous compounds with higher affinity. CYP4F11 also was expressed at high quantities in the brain, and was up-regulated by TNF-α, while the other isoforms were not expressed at high quantities in the brain and were down-regulated by TNF-α. We identified the AP-1 protein of the JNK pathway as the signaling protein that causes significant increase in CYP4F11 expression. Since TNF-α stimulation causes a simultaneous activation of both JNK pathway and NF-κB signaling, we investigated further the role that NF-κB plays on expression of the CYP4F11 gene. We concluded that although there is a significant increase in CYP4F11 expression in the presence of TNF-α, the activation of NF-κB signaling inhibits CYP4F11 expression in a time dependent manner. The expression of CYP4F11 is only significantly increased after 24 hours of treatment with TNF-α; at shorter time points NF-κB signaling overpowers the JNK pathway activation. We believe that these findings may in the future lead to improved drug design for modulating inflammation.

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis

Enforced expression of Tbx1 in fetal thymic epithelial cells antagonizes thymus organogenesis Kim T. Cardenas The thymus and parathyroid glands originate from organ-specific domains of 3rd pharyngeal pouch (PP) endoderm. At embryonic day 11.5 (E11.5), the ventral thymus and dorsal parathyroid domains can be identified by Foxn1 and Gcm2 expression respectively. Neural crest cells, (NCCs) play a role in regulating patterning of 3rd PP endoderm. In addition, pharyngeal endoderm influences fate determination via secretion of Sonic hedgehog (Shh), a morphogen required for Gcm2 expression and generation of the parathyroid domain. Gcm2 is a downstream target of the transcription factor Tbx1, which in turn is positively regulated by Shh. Although initially expressed throughout pharyngeal pouch endoderm, Tbx1 expression is excluded from the thymus-specific domain of the 3rd PP by E10.5, but persists in the parathyroid domain. Based on these observations, we hypothesized that Tbx1 expression is non-permissive for thymus fate specification and that enforced expression of Tbx1 in the fetal thymus would impair thymus development. To test this hypothesis, we generated knock-in mice containing a Cre-inducible allele that allows for tissue-specific Tbx1 expression. Expression of the R26iTbx1 allele in fetal and adult thymus using Foxn1Cre resulted in severe thymus hypoplasia throughout ontogeny that persisted in the adult. Thymic epithelial cell (TEC) development was impaired as determined by immunohistochemical and FACS analysis of various differentiation markers. The relative level of Foxn1 expression in fetal TECs was significantly reduced. TECs in R26iTbx1/+ thymi assumed an almost universal expression of Plet-1, a marker associated with a TEC stem/progenitor cell fate. In addition, embryonic R26iTbx1/+ mice develop a perithymic mesechymal capsule that appears expanded compared to control littermates. Interestingly, thymi from neonatal and adult R26iTbx1/+ but not R26+/+ mice were encased in adipose tissue. This thymic phenotype also correlated with a decrease in thymocyte cellularity and aberrant thymocyte differentiation. The results to date support the conclusion that enforced expression of Tbx1 in TECs antagonizes their differentiation and prevents normal organogenesis via both direct and indirect effects.

REGULATION OF HGF EXPRESSION BY ΔEGFR-MEDIATED C-MET ACTIVATION IN GLIOBLASTOMA CELLS

Overexpression of the hepatocyte growth factor receptor (c-Met) and its ligand, the hepatocyte growth factor (HGF), and a constitutively active mutant of the epidermal growth factor receptor (∆EGFR/EGFRvIII), occur frequently in glioblastoma. c-Met is activated in a ligand-dependent manner by HGF or in a ligand-independent manner by ∆EGFR. Dysregulated c-Met signaling contributes to the aggressive phenotype of glioblastoma, yet the mechanisms underlying the production of HGF in glioblastoma are poorly understood. We found a positive correlation between HGF and c-Met expression in glioblastoma, suggesting that they are coregulated. This is supported by the finding that in a c-Met/HGF axis-dependent glioblastoma cell line, shRNA-mediated silencing of c-Met, or treatment with the c-Met inhibitor SU11274, attenuated HGF expression. Biologically, c-Met knockdown decreased anchorage-independent colony formation and the tumorigenicity of intracranial xenografts. Building on prior findings that ∆EGFR enhanced c-Met activation, we found that ∆EGFR also led to increased HGF expression, which was reversed upon ∆EGFR inhibition with AG1478. ∆EGFR required c-Met to maintain elevated HGF expression, colony formation of glioblastoma cells, and the tumorigenicity of orthotopic xenografts. An unbiased mass spectrometry-based approach identified phosphotyrosine-related signaling changes that occurred with c-Met knockdown in a glioblastoma cell line expressing ΔEGFR and in parental cells. Notably, phosphorylation of STAT3, a master regulator of the mesenchymal GBM subtype and a known target of ∆EGFR, also decreased when c-Met was silenced in these cells, suggesting that the signals from these receptors converge on STAT3. Using a STAT3 inhibitor, WP1193, we showed that STAT3 inhibition decreased HGF mRNA expression in ΔEGFR-expressing glioblastoma cells. Consistent with these findings, constitutively active STAT3 partially restored HGF expression and anchorage-independent growth of c-Met knockdown glioblastoma cells that overexpressed ΔEGFR. We found that higher levels of HGF and c-Met expression associated with the mesenchymal GBM subtype. Taken together, these results suggest that the activity of c-Met regulates the expression of HGF in glioblastoma cells, that ∆EGFR feeds positively into this autocrine loop, that signaling of the two receptors together modulate HGF expression via STAT3, and that the HGF/c-Met axis may therefore be a good additional target for therapy of mesenchymal GBM tumors.

THE EXPRESSION PROFILE OF A GWAS DETECTED GENE (NINJ2) IN THE BRAIN OF A MOUSE MODEL OF ISCHEMIC STROKE

Stroke is the third leading cause of death and a major debilitating disease in the United States. Multiple factors, including genetic factors, contribute to the development of the disease. Genome-wide association studies (GWAS) have contributed to the identification of genetic loci influencing risk for complex diseases, such as stroke. In 2010, a GWAS of incident stroke was performed in four large prospective cohorts from the USA and Europe and identified an association of two Single Nucleotide Polymorphisms (SNPs) on chromosome 12p13 with a greater risk of ischemic stroke in individuals of European and African-American ancestry. These SNPs are located 11 Kb upstream of the nerve injury-induced gene 2, Ninjurin2 (NINJ2), suggesting that this gene may be involved in stroke pathogenesis. NINJ2 is a cell adhesion molecule induced in the distal glial cells from a sciatic-nerve injury at 7-days after injury. In an effort to ascribe a possible role of NINJ2 in stroke, we have assessed changes in the level of gene and protein expression of NINJ2 following a time-course from a transiently induced middle cerebral artery ischemic stroke in mice brains. We report an increase in the gene expression of NINJ2 in the ischemic and peri-infarct (ipsilateral) cortical tissues at 7 and 14-days after stroke. We also report an increase in the protein expression of NINJ2 in the cortex of both the ipsilateral and contralateral cortical tissues at the same time-points. We conclude that the expression of NINJ2 is regulated by an ischemic stroke in the cortex and is consistent with NINJ2 being involved in the recovery time-points of stroke.

Prognostic significance of xCT polymorphisms and expression in patients with advanced pancreatic cancer treated with chemotherapy

The plasma membrane xc- cystine/glutamate transporter mediates cellular uptake of cystine in exchange for intracellular glutamate and is highly expressed by pancreatic cancer cells. The xCT gene, encoding the cystine-specific xCT protein subunit of xc-, is important in regulating intracellular glutathione (GSH) levels, critical for cancer cell protection against oxidative stress, tumor growth and resistance to chemotherapeutic agents including platinum. We examined 4 single nucleotide polymorphisms (SNPs) of the xCT gene in 269 advanced pancreatic cancer patients who received first line gemcitabine with or without cisplatin or oxaliplatin. Genotyping was performed using Taqman real-time PCR assays. A statistically significant correlation was noted between the 3' untranslated region (UTR) xCT SNP rs7674870 and overall survival (OS): Median survival time (MST) was 10.9 and 13.6 months, respectively, for the TT and TC/CC genotypes (p = 0.027). Stratified analysis showed the genotype effect was significant in patients receiving gemcitabine in combination with platinum therapy (n = 145): MST was 10.5 versus 14.1 months for the TT and TC/CC genotypes, respectively (p = 0.013). The 3' UTR xCT SNP rs7674870 may correlate with OS in pancreatic cancer patients receiving gemcitabine and platinum combination therapy. Paraffin-embedded core and surgical biopsy tumor specimens from 98 patients with metastatic pancreatic adenocarcinoma were analyzed by immunohistochemistry using an xCT specific antibody. xCT protein IHC expression scores were analyzed in relation to overall survival in 86 patients and genotype in 12 patients and no statistically significant association was found between the level of xCT IHC expression score and overall survival (p = 0.514). When xCT expression was analyzed in terms of treatment response, no statistically significant associations could be determined (p = 0.908). These data suggest that polymorphic variants of xCT may have predictive value, and that the xc- transporter may represent an important target for therapy in pancreatic cancer.

EXPRESSION AND IMMUNOLOGICAL CHARACTERIZATION OF HERV-K TRANSMEMBRANE ENVELOPE PROTEIN IN HUMAN BREAST CANCER

The human endogenous retrovirus K (HERV-K) env gene encodes envelope protein comprising surface (SU) and transmembrane (TM) domains. Having shown the exclusive expression of SU in human breast cancer and the stimulation of SU-specific immune responses in patients with breast cancer, our research here confirmed and extended the data by investigating the expression of HERV-K TM envelope domain and the induction of specific immune responses against TM in breast cancer patients. We found HERV-K TM mRNA and protein expression only in human breast cancer cells but not in normal controls. The specific immune responses against TM domain were induced in mice determined by enzyme-linked immunosorbent assay (ELISA) and IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay. Furthermore, ELISA detected higher titers of anti-HERV-K TM Env IgG antibodies in sera of breast cancer patients. In addition, the magnitude of the anti-HERV TM B cell response was correlated with the disease stage. Peripheral blood mononuclear cells (PBMCs) before and after in vitro stimulation (IVS) with HERV-K TM from patients with breast cancer as well as healthy controls were tested for T cell responses against HERV-K TM domain by ELISPOT assay. Breast cancer patients (n=21) had stronger HERV-K TM-specific cellular responses than healthy controls (n=12) (P < 0.05). These findings suggest, for the first time, that HERV-K TM expression was enhanced in human breast cancer cells and was able to induce specific B cell and T cell immune responses in breast cancer patients. This study provides support for HERV-K TM as a promising source of antigen for anti-tumor immunotherapy, prevention, diagnosis, and prognosis.

The molecular mechanism of retinoic acid action: Regulation of tissue transglutaminase gene expression

Retinoic acid is a small lipophilic molecule that exerts profound effects on the growth and differentiation of both normal and transformed cells. It is also a natural morphogen that is critical in the development of embryonic structures. The molecular effects of retinoic acid involve alterations in the expression of several proteins and these changes are presumably mediated in part by alterations in gene expression. For instance, retinoic acid causes a rapid induction of tissue transglutaminase, an enzyme involved in protein cross-linking. The molecular mechanisms responsible for the effects of retinoic acid on gene expression have not been characterized. To approach this question, I have isolated and characterized tissue transglutaminase of cDNA clones. The deduced amino acid sequences of tissue transglutaminase and of factor XIIIa showed a relatively high degree of homology in their putative calcium binding domains.^ To explore the mechanism of induction of this enzyme, both primary (macrophages) and cultured cells (Swiss 3T3-C2 and CHO fibroblasts) were used. I found that retinoic acid is a general inducer of tissue transglutaminase mRNA in these cells. In murine peritoneal macrophages retinoic acid causes a rapid accumulation of this mRNA and this effect is independent of concurrent protein synthesis. The retinoic acid effect is not mediated by a post-transcriptional increase in the stability of the tissue transglutaminase mRNA, but appears to involve an increase in the transcription rate of the tissue transglutaminase gene. This provides the first example of regulation by retinoic acid of a specific gene, supporting the hypothesis that these molecules act by directly regulating the transcriptional activity of specific genes. A molecular model for the effects of retinoic acid on the expression of genes linked to cellular proliferation and differentiation is proposed. ^

Structure-function relation in retinoid regulated gene expression

Retinoids are known to inhibit proliferation of and induce terminal differentiation of many normal and transformed cells. It has been postulated that retinoids exert their effect by altering gene expression. HL-60 cells and macrophages both respond to retinoic acid action by the rapid induction of the enzyme tissue transglutaminase. The induction has been shown to be due to increased transcription of the transglutaminase gene. The first part of the dissertation studied the structure-function relationship of retinoid-regulated transglutaminase induction, differentiation and proliferation in HL-60 cells using retinoid analogs. The results indicated strict structural constraints and a strong structure-function correlation between transglutaminase induction and differentiation; those retinoids that induced transglutaminase also induced differentiation, those analogs that did not induce transglutaminase could not induce differentiation. The ability of the retinoids to induce transglutaminase in HL-60 cells was paralleled in macrophages. However, the antiproliferative effect of the retinoids displayed less stringent structural constraints than their differentiation- and transglutaminase-inducing properties. Specifically all the retinoids were able to inhibit proliferation to varying extents. It is concluded that the induction of transglutaminase and of differentiation by retinoids is mediated by receptors. While receptor mediation cannot be entirely ruled out, with the current data no definitive statement can be made about the antiproliferative activity of retinoids. Also, the concordance in the ability of the retinoids to induce transglutaminase and the ability to induce differentiation of HL-60 cells suggests that the former is an early response of the cells to retinoids and differentiation a later consequence on the same pathway. Using the induction of transglutaminase as an index of the direct, or primary, effect of retinoids on gene expression, the second part of the dissertation investigates, by 2D gel electrophoresis, the alteration in the rates of synthesis of other proteins in macrophages and HL-60 cells in response to short incubations with retinoic acid. Any changes in parallel with transglutaminase were taken to indicate proteins directly under the control of retinoic acid. It is concluded that retinoic acid regulates the expression of a circumscribed set of genes in a cell-specific manner. The results support the hypothesis that retinoids exert their multiple effects on myeloid cells, in part, by receptor-mediated alternations in gene expression. ^