Activated STING in a vascular and pulmonary syndrome.

BACKGROUND: The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS: We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-ß, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS: We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mononuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibroblasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients' lymphocytes was reduced by JAK inhibitors. CONCLUSIONS: STING-associated vasculopathy with onset in infancy (SAVI) is an autoinflammatory disease caused by gain-of-function mutations in TMEM173. (Funded by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases; ClinicalTrials.gov number, NCT00059748.).

Patient characteristics support unfavorable psychiatric outcome after treatment of unruptured intracranial aneurysms.

INTRODUCTION: Previous studies demonstrated an unfavorable psychological outcome after treatment of unruptured intracranial aneurysms despite an objectively favorable clinical and radiological outcome. The current study was therefore designed to analyze the psychiatric vulnerability of this specific patient collective. MATERIALS AND METHODS: Patients treated for a WHO grade I meningioma and incidental intracranial aneurysms in two German neurosurgical centers between 2007 and 2013 were screened for exclusion criteria including malignant/chronic diseases, recurrence of the tumor/aneurysm after more than 12 months and focal neurological deficits, among others. Seventy-five meningioma patients (M) and 56 incidental aneurysm patients (iA) met the inclusion criteria. The past medical psychiatric history, post-morbid personality characters and coping strategies were determined by questionnaires mailed to the patients in a printed version (Brief COPE, Big Five Personality Test). RESULTS: Fifty-eight M and 45 iA patients returned the questionnaires. Patients with iA demonstrated significantly higher pre-interventional rates of depressive episodes (p = 0.002) and psychological supervision (p = 0.038). These findings were especially aggravated in iA patients who received their cranial imaging for unspecific symptoms such as dizziness, headaches or tinnitus (n = 33, history of depressions: 39.4%; previous psychological supervision: 33.3%). Furthermore, the analysis of the Big Five personality traits revealed remarkably elevated neuroticism scores in the iA collective. CONCLUSION: The current study demonstrates an increased rate of positive pre-interventional psychiatric histories in the iA collective. Although those patients represent only a small subgroup, they still may play an important role concerning the overall outcome after iA treatment. Early detection and psychological support in this subgroup might help to improve the overall outcome. Further studies are needed to evaluate the influence of this new aspect on the multifactorial etiology of unfavorable psychiatric outcome after treatment of iA.

[Effects of GLP-1 (glucagon-like peptide 1) on liver]. / Úcinky GLP-1 (glucagonlike peptide 1) na játra.

Effects of glucagonlike peptide 1 (GLP1) on liver cells are very intensively studied. In the metabolism of saccharides GLP1 stimulates synthesis of glycogen and reduces glucose production -  thus acting like insulin. In the lipid metabolism it enhances fatty acid oxidation and lipid transport from hepatocytes while reducing de novo lipogenesis -  effects more similar to glucagon action. Some studies suggest beneficial effects of GLP1 on oxidative stress, endoplasmic reticulum stress, production of inflammatory mediators and dysfunction of biliary secretion. Current results suggest that drugs affecting incretin system could be used in the treatment of certain liver diseases (e.g. NAFLD and NASH) in the future. In the following article we mention the known effects of GLP 1 on liver functions and liver metabolism and we point out its possible future therapeutic use in the treatment of liver diseases.

Modulation of brain dead induced inflammation by vagus nerve stimulation.

Because the vagus nerve is implicated in control of inflammation, we investigated if brain death (BD) causes impairment of the parasympathetic nervous system, thereby contributing to inflammation. BD was induced in rats. Anaesthetised ventilated rats (NBD) served as control. Heart rate variability (HRV) was assessed by ECG. The vagus nerve was electrically stimulated (BD + STIM) during BD. Intestine, kidney, heart and liver were recovered after 6 hours. Affymetrix chip-analysis was performed on intestinal RNA. Quantitative PCR was performed on all organs. Serum was collected to assess TNFalpha concentrations. Renal transplantations were performed to address the influence of vagus nerve stimulation on graft outcome. HRV was significantly lower in BD animals. Vagus nerve stimulation inhibited the increase in serum TNFalpha concentrations and resulted in down-regulation of a multiplicity of pro-inflammatory genes in intestinal tissue. In renal tissue vagal stimulation significantly decreased the expression of E-selectin, IL1beta and ITGA6. Renal function was significantly better in recipients that received a graft from a BD + STIM donor. Our study demonstrates impairment of the parasympathetic nervous system during BD and inhibition of serum TNFalpha through vagal stimulation. Vagus nerve stimulation variably affected gene expression in donor organs and improved renal function in recipients.

Assessing analytical variability of measurement of vitamin A in corn-soy blend.

Two multilaboratory investigations were conducted by SUSTAIN to assess variability in the measurement of vitamin A, the marker used to verify levels of vitamin premix addition to enriched/fortified food aid products, including the widely distributed corn-soy blend (CSB). CSB specifications identify AACC Approved Method 86-06 or equivalent methods for vitamin A analysis, however there is no requirement to demonstrate equivalency. CSB samples with known and blinded levels of vitamin A and a reference standard were analyzed by 16 laboratories using their respective methods. Calculated coefficients of variation across all laboratories and methods for unknown samples and reference standard were 35 and 7.1%, respectively, suggesting the largest source of variation is the vitamin extraction procedure. Laboratories generally overestimated low levels and underestimated high levels of vitamin A within the range of 6000 and 16 000 IU/lb. Only two laboratories demonstrated excellent internal precision (+/- 300 IU vitamin A/lb) and reported values within 95% confidence interval for all blinded samples. Results of this study have implications both for quality control in food aid products (due to the use of vitamin A as a marker) and for regulatory oversight of vitamin A content in commercial food products.

Chemical control of Loxosceles intermedia (Araneae: Sicariidae) with pyrethroids: field and laboratory evaluation.

Bites from the recluse or brown spiders (genus Loxosceles) can cause necrotic lesions and systemic effects in humans throughout the world. In the state of Paraná, Brazil, loxoscelism is considered a serious public health problem, and Loxosceles intermedia Mello-Leitão (Araneae: Sicariidae) is associated with the majority of reported accidents. In the present research we evaluated the susceptibility of L. intermedia to pyrethroid insecticides currently used for the control of spiders in both field and laboratory conditions. In laboratory tests, the most active pesticides in descending order were microencapsulated lambda-cyhalothrin (LC50 = 0.023 mg/kg), nonmicroencapsulated lambda-cyhalothrin (LC50 = 0.047 mg/kg), deltamethrin (LC50 = 0.26 mg/kg), and cypermethrin (LC50 = 1.38 mg/kg). Cockroaches, Phoetalia circumvagans (Burmeister) (n = 30), killed with microencapsulated lambdacyalothrin, were offered to the spiders. L. intermedia fed on 63.3% of the dead cockroaches during the first 6 h of experiment; none of the spiders died during the subsequent 15 d. Microencapsulated lambdacyalothrin was chosen for application in two contiguous houses. The mean volume applied was 22.8 mg (AI)/m2. Dead spiders were found during all the inspections up to 60 d after the initial application. In total, 297 dead spiders were collected; 65.7% in the attic shared by the two homes, 10.8% inside the house that had most cracks and crevices sealed and 23.6% in the control house. The use of lambda-cyhalothrin-based products for L. intermedia control is discussed.

Metabolic cooperation in the ovarian follicle.

Granulosa cells (GCs) are somatic cells essential for establishing and maintaining bi-directional communication with the oocytes. This connection has a profound importance for the delivery of energy substrates, structural components and ions to the maturing oocyte through gap junctions. Cumulus cells, group of closely associated GCs, surround the oocyte and can diminished the effect of harmful environmental insults. Both GCs and oocytes prefer different energy substrates in their cellular metabolism: GCs are more glycolytic, whereas oocytes rely more on oxidative phosphorylation pathway. The interconnection of these cells is emphasized by the fact that GCs supply oocytes with intermediates produced in glycolysis. The number of GCs surrounding the oocyte and their age affect the energy status of oocytes. This review summarises available studies collaboration of cellular types in the ovarian follicle from the point of view of energy metabolism, signaling and protection of toxic insults. A deeper knowledge of the underlying mechanisms is crucial for better methods to prevent and treat infertility and to improve the technology of in vitro fertilization.

Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3.

TR3, an immediate-early response gene and an orphan member of the steroid-thyroid hormone-retinoid receptor superfamily of transcription factors, regulates apoptosis through an unknown mechanism. In response to apoptotic stimuli, TR3 translocates from the nucleus to mitochondria to induce cytochrome c release and apoptosis. Mitochondrial targeting of TR3, but not its DNA binding and transactivation, is essential for its proapoptotic effect. Our results reveal a mechanism by which a nuclear transcription factor translocates to mitochondria to initiate apoptosis.

Molecular determinants of AHPN (CD437)-induced growth arrest and apoptosis in human lung cancer cell lines.

6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437), originally identified as a retinoic acid receptor gamma-selective retinoid, was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study, we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis, a process that is accompanied by rapid induction of c-Jun, nur77, and p21(WAF1/CIP1). In addition, we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein, a dominant-negative inhibitor of c-Jun, in A549 cells, nur77 expression and apoptosis induction by AHPN/CD437 were impaired, whereas p21(WAF1/CIP1) induction and G0/G1 arrest were not affected. Furthermore, overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus, expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together, our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer.

Glucagon-like peptide-1 analogues exenatide and liraglutide exert inhibitory effect on the early phase of liver regeneration after partial hepatectomy in rats.

Glucagon-like peptide-1 (GLP-1) is an incretin known for proliferative and antiapoptotic effects on various tissues. Exenatide and Liraglutide are GLP-1 analogues used in clinical practice as antidiabetic drugs. Since GLP-1 and its analogues exert significant effect on liver metabolism and since changes in intermediary metabolism play an important role in the process of liver regeneration, we decided to determine the effect of Exenatide and Liraglutide on the early phase of liver regeneration and selected metabolic parameters in a model of 2/3 partial hepatectomy (PHx) in rats. Animals were submitted either to PHx or laparotomy and received 3 doses of either GLP-1 analogues (Exenatide - 42 microg/kg b.w., Liraglutide - 0.75 mg/kg b.w.) or saline intraperitoneally. We analyzed body and liver weight, liver bromodeoxyuridine incorporation, liver content of DNA, triacylglycerols and cholesterol and biochemical serum parameters. Bromodeoxyuridine labeling was significantly lower in hepatectomized rats receiving either type of GLP-1 analogues when compared to hepatectomized controls. This effect was more pronounced in the Liraglutide group compared to Exenatide (p<0.001). In addition, liver DNA content was lower in hepatectomized rats receiving Liraglutide than in hepatectomized control rats (p<0.001). In conclusion, GLP-1 analogues Exenatide and Liraglutide significantly inhibited an early phase of liver regeneration after PHx in rats. This inhibitory effect was more pronounced in rats receiving Liraglutide.

Impact of Steroids on the Inflammatory Response after Ischemic Acute Kidney Injury in Rats.

Inflammation plays a crucial role in acute kidney injury (AKI). The current study was designed to analyze the influence of prednisolone treatment on the inflammatory reaction during the first 96 h after AKI induction in a rat model. AKI was induced by unilateral clipping of the renal vessels. The treatment group received prednisolone 5 mg/kg s.c. daily. Infiltration rates of macrophages, leukocytes, and T-cells (24, 96 h) as well as plasma concentrations of the inflammatory markers intercellular adhesion molecule, interleukin-1 beta (IL-1ß), IL-18, IL-6, and tumor necrosis factor-alpha (0, 6, 24, 96 h) were determined by fluorescence-activated cell sorting (FACS) analysis only. Ninety-six hours after AKI induction, the prednisolone group demonstrated significantly lower creatinine concentrations compared to the control group (P < 0.05). Twenty-four hours after induction of AKI, a significantly higher rate of infiltrating leukocytes was detectable with FACS analysis in the control group (P < 0.01) with a corresponding significantly higher rate of macrophages after 96 h (P < 0.01). IL-6 and IL-1ß demonstrated a peak after 6 h with a significantly higher release in the control group (IL-6: P < 0.01; IL-1ß: P < 0.05). In contrast to the control group, the prednisolone group demonstrated no further incline of IL-18 after 24 h. The results demonstrate the importance of stretching the observation period in an ischemia-reperfusion-induced AKI setting beyond the first 24 h. Despite the demonstrated protective effects of a continuous prednisolone application, it seems that this single anti-inflammatory agent will not be able to completely suppress the inflammatory response after an ischemia-reperfusion-induced AKI.

Post-transcriptional regulation of the DNA damage-inducible gadd45 gene in human breast carcinoma cells exposed to a novel retinoid CD437.

The biologically active synthetic retinoid CD437 (6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene, AHPN) and different human breast carcinoma (HBC) cell lines were used to examine the possible mechanism(s) of gadd45 induction. Northern blot analysis of mRNA isolated from MCF-7, MDA-MB-468 and MDA-MB-231 HBC cell lines demonstrated a progressive increase in the 1.4 kb gadd45 transcript after exposure to 1 microM CD437. Western blot analysis showed increased gadd45 protein levels in MDA-MB-468 HBC cells following exposure to CD437. CD437 increased gadd45 mRNA levels by approximately 20-fold in MDA-MB-468 cells, however, the transcriptional activity was increased approximately 2-3-fold as demonstrated by the human gadd45 promoter-luciferase reporter construct and nuclear run-off assays. Sublines of MDA-MB-468 HBC cells expressing stably integrated GADD45 cDNA fragments were obtained and CD437-dependent induction of GADD45 analyzed. We report that approximately 300 nt located in the 5"-untranslated region (5"-UTR) of gadd45 mRNA are involved in the CD437-dependent 4-fold enhanced stability of gadd45 transcripts. MDA-MB-468 cells were stably transfected with either a plasmid having a CMV promoter-driven rabbit beta-globin gene or plasmids having a CMV promoter-driven chimeric gadd45 5"-UTR-rabbit beta-globin gene, where the entire gadd45 5"-UTR (from +1 to +298) or a 45 bp subfragment of the gadd45 5"-UTR (from +10 to +55) was positioned at the 5"-end of the rabbit beta-globin gene. CD437 was found to up-regulate expression of both the chimeric gadd45 -rabbit beta-globin transcripts, suggesting that cis element(s) involved in the CD437-dependent enhanced stability of gadd45 mRNA are contained in the 45 nt of the 5"-UTR of the gadd45 mRNA.

Inhibition of human mammary carcinoma cell proliferation by retinoids and intracellular cAMP-elevating compounds.

Retinoids and cAMP-elevating agents markedly inhibited the proliferation of human mammary tumor cells. Their response has been previously correlated with the presence of estrogen receptor (ER) positivity. MDA-MB-231 cells were ER negative and insensitive to the antiproliferative effects of retinoids. However, their growth was markedly inhibited by agents that elevated intracellular cAMP levels, i.e., 8-bromo-cAMP, cholera toxin (CT), forskolin, and the phosphodiesterase inhibitor papaverine. The CT and forskolin inhibition of the ER-positive cells (MCF-7) was associated with an elevation of adenylate cyclase activity and intracellular cAMP levels; however, similar elevations in intracellular cAMP levels were not observed following CT or forskolin inhibition of MDA-MB-231 cells but only following the addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine.

Responses to retinoic acid of tamoxifen-sensitive and -resistant sublines of human breast cancer cell line MCF-7.

Growth of the human breast cancer cell line MCF-7 is known to be inhibited both by antiestrogens such as 4-hydroxytamoxifen (OHTAM) and by retinoic acid (RA). Uncloned MCF-7 cells (UNC) and two cloned sublines, one sensitive to antiestrogens (E-3) and the other resistant to them (RR), were used in this study. Growth of UNC and E-3 was inhibited by either OHTAM (10(-7) M) or RA (10(-6) M), and this inhibition could not be overcome by the simultaneous addition of estradiol. Subline RR, which was originally selected for resistance to tamoxifen, was resistant to both OHTAM and RA as measured by either growth in culture or colony forming ability. RR was resistant to RA at all concentrations tested between 10(-9) M and 10(-6) M. The inhibition of uncloned MCF-7 cells by RA was dose dependent between 10(-9) M and 10(-6) M. Subline E-3, however, exhibited a mixed response to RA. At 10(-9) M and 10(-8) M, growth was stimulated, but at 10(-7) M and 10(-6) M it was inhibited. The level of estrogen receptor was measured in the same experiment by using a whole cell assay. In the uncloned MCF-7 cultures and in both the RR and E-3 sublines the level of estrogen receptor was increased between 50 and 200% by RA. The production of plasminogen activator by MCF-7 cells is stimulated by estrogen. RA had a dual effect on plasminogen activator production. In the absence of estrogen, RA inhibited production below the unstimulated level, but in cells stimulated by estrogen, RA increased plasminogen activator production. The results reported here support possible interactions between the mechanisms by which cells respond to estrogen, antiestrogens, and retinoids.

Retinoic acid inhibition of human breast carcinoma proliferation is accompanied by inhibition of the synthesis of a Mr 39,000 protein.

Retinoic acid (RA) inhibits proliferation of numerous breast carcinoma cells and prevents estrogen stimulation of growth of several estrogen receptor (ER)-positive cell lines. RA inhibition of human breast carcinoma cell proliferation is associated with marked inhibition of the synthesis of a Mr 39,000 protein in the ER-positive human breast carcinoma cell lines investigated. Inhibition of the synthesis of the Mr 39,000 protein occurred within 24 h of RA addition and coincides with the onset of inhibition of cellular proliferation. Increasing the dose of RA results in increasing inhibition of Mr 39,000 synthesis. RA does not inhibit the proliferation of the ER-negative human breast carcinoma cell line MDA MB-231; synthesis and inhibition of the Mr 39,000 protein is not noted in this cell line. Tamoxifen, which inhibits ER-positive breast carcinoma proliferation, moderately inhibits Mr 39,000 synthesis, while a concentration of difluoromethylornithine which inhibits cellular proliferation by greater than 50% does not affect Mr 39,000 protein synthesis. Thus, inhibition of the Mr 39,000 protein appears not to be simply related to the cessation of cellular proliferation.

The p53-binding protein MDM2 gene is differentially expressed in human breast carcinoma.

The human p53-binding protein murine double minute 2 (MDM2) is believed to function as a negative regulator of p53. The MDM2 gene was cloned and sequenced only recently and was found to be amplified in a variety of sarcomas. Although mutations in the p53 gene have been shown to occur in human breast carcinoma (HBC), no information is available on MDM2 gene expression in HBC. In this study we report for the first time that the MDM2 gene is differentially expressed in HBC. Our results demonstrate a correlation between the estrogen receptor (ER) status and the MDM2 mRNA levels. In contrast to the ER-negative cell lines, all the ER-positive cell lines were found to express higher levels of MDM2 mRNA. ER-positive ZR-75 cells express 30-fold higher levels of MDM2 mRNA than does the ER-negative cell line Hs578T. Estrogen enhanced albeit modestly the MDM2 mRNA levels in ER-positive MCF-7 cells. Estrogen enhancement of MDM2 mRNA levels was also observed in ER-negative MDA-MB-231 cells transfected with functional ERs. Our data thus suggest that estrogen may play an important role in HBC growth stimulation by modulating the expression of MDM2, which in turn may inactivate the p53 function.

Retinoid antagonism of estrogen-responsive transforming growth factor alpha and pS2 gene expression in breast carcinoma cells.

Exposure of MCF-7 breast carcinoma cells to estradiol results in an increase in transforming growth factor alpha (TGF-alpha) synthesis and secretion. Since TGF-alpha is a potent inducer of proliferation in MCF-7 cells, the increase in TGF-alpha production by estradiol is thought to play an important role in the estrogen stimulation of growth of these cells. Retinoic acid inhibits the proliferation of MCF-7 cells and antagonizes the estrogen stimulation of growth. Addition of retinoic acid resulted in a greater than 70% inhibition of estradiol-induced TGF-alpha synthesis and secretion in MCF-7 cells. The increase in TGF-alpha mRNA expression by estradiol was also inhibited by exposure of the cells to retinoic acid. Pretreatment of the cells with retinoic acid for 24 or 72 h caused more than 50 and 90% inhibition, respectively, of the estradiol-enhanced expression of TGF-alpha mRNA. Expression of pS2 mRNA in MCF-7 cells was stimulated approximately 8-fold by estradiol. Retinoic acid treatment suppressed by greater than 80% both the basal and estradiol-induced pS2 mRNA expression. Retinoic acid modulation of the estrogen receptor gene mRNA was not responsible for the retinoic acid inhibition of the stimulation of pS2 and TGF-alpha gene expression by estradiol, since estrogen receptor gene expression was increased rather than decreased in the presence of retinoic acid. The nuclear retinoic acid receptors alpha and gamma mRNA were expressed in MCF-7 cells and its retinoic acid-resistant derivative RROI. Addition of estradiol to MCF-7 cells resulted in a decreased expression of retinoic acid receptor gamma mRNA; this reduction is prevented by the presence of retinoic acid. These results indicate that retinoic acid can inhibit estradiol-induced TGF-alpha and pS2 mRNA expression in MCF-7 cells. The suppression of TGF-alpha expression may represent one possible mechanism by which retinoic acid antagonizes the stimulation of MCF-7 proliferation by estradiol.

Changes in the translational activity of polyadenylated messenger RNA of HL60 promyelocytic leukemia cells associated with myeloid or macrophage differentiation.

In order to characterize the events which commit the HL60 human promyelocytic leukemia cell line to differentiate into macrophages or mature myeloid cells, we have analyzed the in vitro [35S]methionine-labeled translational products obtained from polyadenylated messenger RNA of the HL60 cells before and after exposure to: (a) dimethylformamide (DMF), an inducer of myeloid differentiation; (b) 12-O-tetradecanylphorbol-13-acetate (TPA), an inducer of macrophage differentiation; or (c) a combination of the two inducers. Exposure of the HL60 cells to either TPA or DMF results in decreases in the relative abundancy of translational products with molecular weights of 20,000, 17,000, and 15,000. Exposure of the HL60 cells so as to generate macrophage differentiation results in elevations of translational products with molecular weights of 60,000, 47,000, 42,000, 32,000, 27,000, 14,000, and 12,300, while DMF-induced myeloid differentiation is associated with increases in the abundancy of translational products with molecular weights of 60,000, 42,000, 35,000, 32,000, 27,000, 13,000 and 12,300. The addition of the macrophage inducer TPA to HL60 cells previously exposed to the myeloid inducer DMF results in changes in the relative abundance of several translational products, yielding a pattern which differs quantitatively from that obtained from cells treated with DMF or TPA alone. These changes in the relative abundancies of the HL60 translational products suggest that the steady state levels of several different populations of mRNA or the ability of these mRNAs to be translated are being modified during the induction of myeloid or macrophage differentiation in the HL60 promyelocytic leukemia cell line.

Retinoid modulation of estradiol-stimulated growth and of protein synthesis and secretion in human breast carcinoma cells.

All-trans-retinoic acid (RA) has been demonstrated to inhibit the growth of numerous breast carcinoma cell lines. In this study we demonstrated that RA inhibits estradiol stimulation of proliferation of the estrogen-dependent breast carcinoma cell lines MCF-7 and ZR-75. RA inhibition of MCF-7 breast carcinoma cell proliferation is associated with the marked stimulation of the synthesis and secretion of a 75,000 molecular weight protein. Minor stimulation of the synthesis and secretion of other proteins was also noted, including 160,000, 95,000, 89,000, 82,000, 52,000 and 46,000 molecular weight proteins. Increased synthesis and secretion of the Mr 75,000 protein was noted with as little as 10 nM RA. Stimulation of the synthesis and secretion of this protein occurred within 24 h of adding the retinoid and at the time of RA inhibition of cellular proliferation. RA stimulated the production and secretion of this protein both in the absence and presence of estradiol. Stimulation of the secretion and synthesis was not noted in the presence of difluoromethyl-ornithine or tamoxifen, both of which significantly inhibited cellular proliferation. The estrogen-dependent breast carcinoma cell line ZR-75 when exposed to RA also expressed increased synthesis of the Mr 75,000 and 46,000 proteins but increased synthesis was not noted in the RA-resistant cell line MDA-MB-231. Stimulation of the synthesis and secretion of the Mr 75,000 and/or 46,000 proteins may be intimately involved in RA inhibition of cellular proliferation.

Correlation between the induction of leukemic cell differentiation by various retinoids and modulation of protein kinases.

During retinoic acid induced differentiation of the human promyelocyte leukemia cell line HL60 and the human myeloblast cell line RDFD along the myeloid pathway, there is marked modulation of both the cyclic adenosine 3':5'-monophosphate dependent protein kinase and the phospholipid-sensitive calcium dependent protein kinase. In order to further assess whether these kinases are intimately associated with the differentiation process, we have correlated the modulation of these enzymes and phosphorylations of their substrates with the extent of differentiation induced by various retinoid derivatives. We observed that there was a direct relationship between the degree of differentiation of the two leukemic cell lines and the elevation of the cyclic adenosine 3':5'-monophosphate dependent protein kinase and phospholipid sensitive calcium dependent protein kinase activities. In addition, the increased phosphorylation of the various substrates of these enzymes also correlates with the degree of differentiation. These observations support the hypothesis that modulation of these protein kinases and phosphorylation of their substrates are necessary steps in the differentiation process.