Enhancement of fractionated-dose irradiation by retinoic acid plus interferon

PURPOSE: To evaluate the relative cytotoxicity of fractionated-dose radiation in the presence and absence of 13-cis-retinoic acid (RA) plus alpha-2a-interferon (IFN), as a function of overall treatment time. METHODS AND MATERIALS: Studies were performed with the human squamous cell carcinoma line FaDu, in vitro. Attached exponential phase cells were treated with RA + IFN for 8-10 h and then exposed to single graded doses of radiation, or 1 to 6 doses of radiation at 2 Gy per dose, or to 5 doses of radiation at 2 Gy/dose with a time interval of 4-24 h between treatments. Following irradiation, the cells were incubated with drugs present throughout colony formation, and the fraction of survivors in the presence and absence of the combined drugs was calculated. RESULTS: For single graded-dose irradiation, the surviving fraction ratio at 2 Gy in the absence vs. presence of drugs was 1.27 +/- 0.19 in 3 repeat experiments. Following administration of 6 doses of radiation at 2 Gy/fraction with a 5-h time interval between treatments and, after correcting for cell proliferation between treatments, the surviving fractions differed by a factor of 3.25, again indicating an average difference in survival of 1.26 after each of the 6 2-Gy/fractions. Treatment with 5 2-Gy doses of irradiation with 24 vs. 4 h elapsing between doses, resulted in a 3-fold greater decrease in survival in the presence of drugs vs. no drug. The relatively greater cell kill due to 24 vs. 4 h between treatments was due to drug inhibition of cell proliferation between the more prolonged treatments. CONCLUSIONS: The results of this study indicate that retinoic acid plus interferon both sensitizes and inhibits cell proliferation during treatment. These results suggest that this combination of radiation and drugs, when used concurrently, may be effective for inhibiting tumor cell proliferation or accelerated repopulation during clinical fractionated radiotherapy.     

Inhibition of herpes simplex virus replication by retinoic acid

Superior laryngeal nerve (SLN) stimulation can activate the brainstem swallowing mechanism to produce a complete swallowing sequence consisting of oropharyngeal, oesophageal and lower oesophageal sphincter (LOS) components. However, little is known of the effect of SLN stimulation (peripheral-sensory input from the pharynx) on the characteristics of oesophageal motor activity, especially in the smooth muscle portion. The present study examined the effect of varying stimulus train length and frequency on each of the three components of the reflex. Acute studies were performed in urethane anaesthetized cats. Oesophageal motility was monitored using conventional manometric techniques, and oropharyngeal swallowing by the mylohyoid electromyogram. SLN stimulus train length (1-10 sec) and frequency (5-30 Hz) were varied independently. Increased train length or frequency resulted in (1) an increase in oropharyngeal swallowing and incidence of the complete swallowing response, (2) an increase in latency to onset of the oesophageal peristaltic wave, (3) reduction of the amplitude of the evoked peristaltic contraction in the smooth muscle portion, without altering its velocity, (4) increased LOS relaxation, and increased LOS after-contraction. The LOS contraction was abolished by atropine (100 micrograms kg-1). Therefore, increased SLN stimulation not only results in excitation of the central swallowing program and the oropharyngeal stage of swallowing, but has major effects on the oesophageal and LOS stages of swallowing. Afferent SLN stimuli can impact on the control mechanisms for each stage, to inhibit or excite the stages in different ways.     

Teratogenesis by retinoic acid analogs positively correlates with elevation of retinoic acid receptor-beta 2 mRNA levels in treated embryos

The concentration/effect relationship of bunazosin, a selective alpha-1-adrenoceptor antagonist of the quinazoline class, was established in hypertensive patients after a first dose of bunazosin and after 8 weeks of chronic dosing. The study was part of a placebo controlled, multicenter dose-finding trial with parallel group design. After a wash-out period and a 2-week placebo run-in phase, each 4 patients received either placebo, 3 mg, 6 mg or 12 mg bunazosin o.d. in a slow-release formulation. Concentration/effect analysis based on a sigmoidal Emax-model and the drop in the mean arterial blood pressure application of this model was feasible in 9/11 patients. The EC50 after the first dose was 4.6 +/_ 2.0 ng/ml and increased to 9.1 +/- 7.3 ng/ml at steady-state. The decrease in the average of the responsiveness parameter was mainly due to about three-fold, right-shift in the concentration/effect curves at steady-state in 3 patients, the remaining patients showed no or considerably lesser changes of the EC50. Whereas a significant correlation was observed between the pretreatment mean arterial blood pressure and the height of the maximal effect under bunazosin, no relationship appears between the bunazosin dose, the resulting plasma levels at trough and the corresponding effect.     

Altered retinoic acid receptors

Structurally and functionally altered retinoic acid receptors have been associated with rare human neoplasms: acute promyelocytic leukemia and hepatocellular carcinoma. Whereas the retinoic acid receptor beta (RARbeta) rearrangement in hepatocellular carcinoma is unique, in acute promyelocytic leukemia (APL), RARalpha fusion to the promyelocytic leukemia (PML) gene by the t(15;17) translocation is a general feature of the disease. APL is an important model in cancer biology because retinoic acid induces complete remissions in this malignancy, providing the first example of differentiation therapy and of an antineoplastic drug directly targeted at the underlying genetic lesion. The molecular basis of PML/RARalpha fusion leukemogenesis is discussed with respect to dominant negative inhibition of nuclear receptor and PML functions.     

Stimulation of tissue-type plasminogen activator expression by retinoic acid in human endothelial cells requires retinoic acid receptor beta 2 induction

We previously showed the involvement of retinoic acid receptor alpha (RAR alpha) in the induction of tissue-type plasminogen activator (t-PA) synthesis by RA in human umbilical vein endothelial cells (HUVECs). However, the rather slow onset of this induction of t-PA synthesis suggested an indirect role of RAR alpha. Here, we show that the protein synthesis inhibitor, cycloheximide completely blocks the induction of t-PA by RA, which points to the need of an intermediary protein in t-PA stimulation. This intermediary protein is likely to be RAR beta 2 on the basis of the following findings: (1) the induction of RAR beta by RA exactly precedes that of t-PA; (2) HUVECs with elevated RAR beta mRNA levels show an undelayed t-PA induction on stimulation with RA, and this response can be almost completely inhibited with an RAR antagonist; and (3) an antisense oligodeoxynucleotide against the translation initiation site of RAR beta 2 mRNA greatly reduces the t-PA induction by RA. Thus, induction of t-PA by RA in HUVECs involves a 2-step mechanism requiring induction of RAR beta 2 via RAR alpha, followed by induction of t-PA synthesis via RAR beta 2. Each of these steps is shown to have a different activation profile with RA and 9 cis RA.     

Altered wound arginine metabolism by corticosterone and retinoic acid

The objective of this study is to study the pharmacokinetics of imipenem-cilastatin in patients with anuria related to multiorgan failure during continuous venovenous hemodialysis (CVVHD) at a fixed blood flow rate of 60 ml/min, fixed dialysate flow rate of 20 ml/min, and drainage flow rate of 1-3 mil/min. A prospective open label study was designed in an intensive care unit in a university hospital. Six patients who required mechanical ventilation and inotropic agents and exhibited acute anuric renal failure were examined. Intravenous imipenem-cilastatin, 500/500 mg, was administered over 30 mins. Blood samples were obtained from the inlet and the outlet of the dialyzer and dialysate samples from the outlet before and at 0.0, 0.5, 1.0, 2.0, 3.0, 6.0, 9.0, and 12.0 hrs after infusion ended. The peak and trough concentrations of imipenem were 32.47 +/- 6.69 micrograms/ml and 1.12 +/- 0.46 micrograms/ml, respectively, whereas those of cilastatin were 50.05 micrograms/ml +/- 14.80 and 9.53 +/- 3.25 micrograms/ml, respectively. The half life was 2.79 +/- 0.3 hr for imipenem, and 6.67 +/- 0.93 hr for cilastatin. Total body clearance of imipenem was 89.4 +/- 17.5 ml/min, including the clearance by CVVHD of 18.7 +/- 1.2 ml/min, and corresponding values for cilastatin were 31.7 +/- 9.0 ml/min and 13.7 +/- 3.4 ml/min. In conclusion, in patients with anuria, the elimination of imipenem-cilastatin was constant during CVVHD. The recommended regimen in patients with anuria who receive CVVHD in this setting was estimated as intravenous imipenem-cilastatin, 500/500 mg, every 12 hrs.     

Regulation of IRF and STAT gene expression by retinoic acid

Ex vivo production of cytokines as determined by whole blood stimulation and supernatant ELISA is partly determined by heritability. To assess the ability of this system to distinguish between high and low producers the laboratory error and individual variation were investigated. Whole blood samples from healthy volunteers were collected using endotoxin-free tubes and were incubated with 0 to 1000 ng/ml lipopolysaccharide concentrations for 4 and 24 h, and subsequently centrifuged. In the supernatants, TNF-alpha and IL10 were measured by ELISA. Coefficients of variation for the day-to-day variation in the blood sampling, transport and stimulation as well as in the whole blood stimulation per se ranged from 7.5% to 12.3%. The intra-individual variation was 15% (TNF-alpha) and 19% (IL10) in contrast to the inter-individual variation of, on average, 35%. No interchanging of ranks between high and low producers was observed after repeating the whole blood stimulation on distinct days. The whole blood stimulation system is able to distinguish high and low producers of TNF-alpha and IL10.     

The regulation of retinoic acid formation

Rat liver microsomal as well as cytosolic retinol dehydrogenases from cellular retinal-binding protein (CRBP)-retinal from CRBP-retinol. A cytosolic dehydrogenase transforms retinal into retinoic acid. Apo-CRBP and ethanol inhibit the cytosolic but not the microsomal retinol dehydrogenase, the latter being the primary enzyme in the rate-limiting step in retinoic acid synthesis.     

Arg278, but not Lys229 or Lys236, plays an important role in the binding of retinoic acid by retinoic acid receptor gamma

The diverse biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, beta and gamma) and retinoid X receptors (RXR alpha, beta, and gamma). Although the ligand-binding domains of RARs share the same novel folding pattern, many RAR subtype-specific retinoids have been synthesized indicating that the ligand-binding pocket of each RAR subtype has unique features. Previously we have demonstrated the importance for RA binding and RA-dependent transactivation of Arg276 of RARalpha alone and in RARbeta Arg269 in conjunction with Lys220. In this study, we have examined the role of the homologous amino acid residues (Lys229 and Arg278) in RARgamma for these activities. Like RARalpha but dissimilar to RARbeta, Arg278 in RARgamma alone was found to play an important role in RA binding and RA-dependent transactivation. Since Lys236 in RARgamma was suggested from the crystal structure of holo-RARgamma to interact with RA, we also examined its role and that of its homologs in RARalpha and RARbeta. Despite the suggestion from the crystal structure, neither Lys236 nor its homologs in RARalpha and RARbeta play a role in the binding of RA or RA-dependent transactivation. It is likely that Lys236 in RARgamma and its homologs in RARalpha and RARbeta are solvent exposed rather than pointing into the RA-binding pocket.     

The effect of retinoic acid on amino acid uptake and protein synthesis by lung fibroblasts

The effect of retinoic acid (RA) on the uptake and utilization of extracellular amino acids by fetal lung fibroblasts was examined. RA decreased the incorporation of [3H]proline into collagen and other proteins. The effect was maximal at a RA concentration of 10(-5) M; smaller decreases were observed at a RA concentration of 10(-6) M. This decrease in collagen formation was associated with a large decrease in intracellular [3H] proline. The decrease in intracellular [3H]proline was first observed at 2 h following the addition of RA to cell cultures. Transport studies employing radiolabeled amino acids revealed that RA decreased the uptake of proline, 2-aminoisobutyric acid, and 2-(methylamino)isobutyric acid but not leucine or methionine. Kinetic analysis of 2-aminoisobutyric acid uptake indicated that this effect was mediated primarily by an increase in apparent Km, with a lesser decrease in Vmax, RA-induced inhibition of proline uptake was not abolished by the presence of cycloheximide nor by pretreatment with indomethacin. Na+,K(+)-ATPase activity was not affected by RA treatment. These results suggest that RA modulates protein production in fibroblasts by altering the function of the Na(+)-dependent A transport system for amino acid uptake.     

Caudalization by the amphibian organizer: brachyury, convergent extension and retinoic acid

Caudalization, which is proposed to be one of two functions of the amphibian organizer, initiates posterior pathways of neural development in the dorsalized ectoderm. In the absence of caudalization, dorsalized ectoderm only expresses the most anterior (archencephalic) differentiation. In the presence of caudalization, dorsalized ectorderm develops various levels of posterior neural tissues, depending on the extent of caudalization. A series of induction experiments have shown that caudalization is mediated by convergent extension: cell motility that is based on directed cell intercalation, and is essential for the morphogenesis of posterior axial tissues. During amphibian development, convergent extension is first expressed all-over the mesoderm and, after mesoderm involution, it becomes localized to the posterior mid-dorsal mesoderm, which produces notochord. This expression pattern of specific down regulation of convergent extension is also followed by the expression of the brachyury homolog. Furthermore, mouse brachyury has been implicated in the regulation of tissue elongation on the one hand, and in the control of posterior differentiation on the other. These observations suggest that protein encoded by the brachyury homolog controls the expression of convergent extension in the mesoderm. The idea is fully corroborated by a genetic study of mouse brachyury, which demonstrates that the gene product produces elongation of the posterior embryonic axis. However, there exists evidence for the induction of posterior dorsal mesodermal tissues, if brachyury homolog protein is expressed in the ectoderm. In both cases the brachyury homolog contributes to caudalization. A number of other genes appear to be involved in caudalization. The most important of these is pintavallis, which contains a fork-head DNA binding domain. It is first expressed in the marginal zone. After mesoderm involution, it is present not only in the presumptive notochord, but also in the floor plate. This is in contrast to the brachyury homolog, whose expression is restricted to mesoderm. The morphogenetic effects of exogenous RA on anteroposterior specification during amphibian embryogenesis are reviewed. The agent inhibits archencephalic differentiation and enhances differentiation of deuterencephalic and trunk levels. Thus the effect of exogenous RA on morphogenesis of CNS is very similar to that of caudalization, which is proposed to occur through the normal action of the organizer. According to a detailed analysis of the effect of lithium on morphogenesis induced by the Cynops organizer, lithium has a caudalizing effect closely comparable with that of RA. Furthermore, lithium induces convergent extension in the prechordal plate, which normally does not show cell motility.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid

Osteogenic protein-1 (OP-1), a member of the TGF-beta family of proteins, induces endochondral bone formation. Here we studied the effect of OP-1 on the development of primary cultures of avian growth plate (GP) chondrocytes in either serum-free or serum-containing medium, in the absence or presence of retinoic acid (RA). OP-1 was added on day 7 of culture and continued for 7 days, or until the cultures were harvested, typically on day 21. Alone, OP-1 caused approximately 2-fold increase in proteoglycan synthesis into both the medium and the cell:matrix layer. Additionally, OP-1 caused a dosage-dependent increase in alkaline phosphatase (ALP) activity, and an increase in protein, when given from days 7-14 and examined on day 14. This stimulation was greater in cells grown in serum-free than in serum-containing media (3-5-fold vs. 2-3-fold increase in ALP; approximately 40% vs. approximately 20% increase in protein). Such stimulation of ALP activity and proteoglycan (PG) synthesis in cultured GP cells indicates that OP-1 elicits differentiation of chondrocytes. OP-1 minimally affected cell division (DNA content); however, a slight increase was seen when examined early in the culture. Alone, OP-1 increased mineral (Ca and Pi) content of the cultures by approximately 2-fold in both types of media. As early as day 14, clusters of mineral encircled many of the OP-1 treated cells. Thus, as in vivo, OP-1 strongly promoted mineral formation by the cultured GP chondrocytes. When present together, OP-1 and RA generally blocked the action of the other. Separately OP-1 and RA each stimulated protein synthesis, ALP activity, and Ca2+ deposition; together they were inhibitory to each. Also, RA blocked the stimulation of PG synthesis induced by OP-1; whereas OP-1 decreased cell division engendered by RA. Thus, this GP chondrocyte culture system is a good model for studying factors that influence differentiation and mineral deposition during bone growth in vivo.     

Regulation of neurotrophin receptor expression by retinoic acid in mouse sympathetic neuroblasts

We have studied the effect of retinoic acid on the expression of the neurotrophin receptors trkA, trkC, and p75 by neuroblasts and neurons at different axial levels along the embryonic mouse paravertebral sympathetic chain. In dissociated cultures of sympathetic neuroblasts, retinoic acid inhibited the developmental increase in trkA mRNA expression and the developmental decrease in trkC mRNA expression that normally occurs in these cells but did not affect p75 mRNA expression. At higher concentrations, retinoic acid also increased the proliferation of sympathetic neuroblasts. After sympathetic neuroblasts became postmitotic, retinoic acid no longer affected receptor expression. Studies with retinoic acid receptor agonists and antagonists indicated that the effects of retinoic acid on neurotrophin receptor expression were mediated mainly by alpha retinoic acid receptors, not beta or gamma receptors. The observation that alpha-antagonists increased trkA mRNA expression in intact sympathetic ganglion explants suggests that endogenous retinoic acid is a physiological regulator of trkA receptor expression.     

Retinoic acid receptors regulate expression of retinoic acid 4-hydroxylase that specifically inactivates all-trans retinoic acid in human keratinocyte HaCaT cells

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), 7beta-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-Ibeta and/or tumor necrosis factor (TNF)-alpha secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7beta-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1beta secretion. TNF-alpha secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the alpha- or beta-hydroxyl radical position plays a key role in apoptosis and IL-1beta secretion.     

Retinoic acid receptor-gamma in human epidermis preferentially traps all-trans retinoic acid as its ligand rather than 9-cis retinoic acid

Brain injury in the premature infant is an extremely important problem, in part because of the large absolute number of infants affected yearly. The 2 principal brain lesions that underlie the neurological manifestations subsequently observed in premature infants are periventricular hemorrhagic infarction and periventricular leukomalacia. The emphases of this article are the neuropathological features, pathogenesis, and potential means of prevention of these 2 lesions. Recent work suggests that the ultimate goal, prevention of the lesions, is potentially achievable. Periventricular hemorrhagic infarction may be avoidable by prevention of germinal matrix-intraventricular hemorrhage, and periventricular leukomalacia by detection of impaired cerebrovascular autoregulation, prevention of impaired cerebral blood flow, and interruption of the cascade to oligodendroglial cell death by such agents as free radical scavengers.