Induction of chromosomal aberrations, sister-chromatid exchanges and cell-cycle delay in cow peripheral blood lymphocytes treatment with two N-nitroso compounds in vitro

We investigated the effect of NDMA and DNSGU on the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs), as well as the influence of the former compound on cell-cycle kinetics in cultured cow peripheral lymphocytes. A clastogenic effect was observed in treated cell cultures at 6 or 12 × 10⁻⁵ M concentrations of NDMA and DNSGU, respectively, but no increase of chromosomal breaks was seen at the lowest dose. NDMA at 6 × 10⁻⁴ M was toxic to cow lymphocytes. NDMA and DNSGU induced statistical increases of SCEs at the test doses (6 or 12 × 10⁻⁶ and 6 or 12 × 10⁻⁵ M, respectively). In addition, treatment with NDMA at a dose of 6 × 10⁻⁵ M revealed significant heterogeneity of the first, second and third metaphases between treated and untreated groups. A reduction of the proliferation index and proliferation delay per cycle was shown too.     

Vagally induced release of gastrin, somatostatin and bombesin-like immunoreactivity from perfused rat stomach. Effect of stimulation frequency and cholinergic mechanisms

The isolated stomach of rats was vascularly perfused to measure the secretion of gastrin, somatostatin (SLI) and bombesin-like immunoreactivity (BLI). The gastric lumen was perfused with saline pH 7 or pH 2, and electrical vagal stimulation was performed with 1 ms, 10 V and 2, 5 or 10 Hz, respectively. Atropine was added in concentrations of 10⁻⁹ or 10⁻⁷ M to evaluate the role of cholinergic mechanisms. In control experiments, vagal stimulation during luminal pH 2 elicited a significant increase of BLI secretion only at 10 Hz but not at 2 and 5 Hz. Somatostatin release was inhibited independent of the stimulation frequency employed. Gastrin secretion at 2 Hz was twice the secretion rates observed at 5 and 10 Hz, respectively. At luminal pH 7 BLI rose significantly at 5 and 10 Hz. SLI secrtion was decreased by all frequencies. Gastrin secretion at 2 and 5 Hz was twice as high as during stimulation with 10 Hz. Atropine at doses of 10⁻⁹, 10⁻⁸, 10⁻⁷ and 10⁻⁶ M had no effect on basal secretion of BLI, SLI and gastrin. At luminal pH 2, atropine increased dose-dependently the BLI response at 2 and 5 but not at 10 Hz. The decrease of SLI during 2 and 5 Hz but not 10 Hz was abolished by atropine 10⁻⁹ M. SLI was reversed to stimulation during atropine 10⁻⁷ M at all frequencies. The rise of gastrin at 2 Hz was reduced by 50%. At luminal pH 7, atropine had comparable effects with a few differences: the BLI response at 10 Hz was augmented and the gastrin response to 2 and 5 Hz was reduced. In conclusion the present data demonstrate a frequency and pH-dependent stimulation of BLI and gastrin release. The stimulation of BLI is predominantly due to atropine-insensitive mechanisms while muscarinic cholinergic mechanisms exert an inhibitory effect on BLI release during lower stimulation frequencies (2 and 5 Hz) independent of the intragastric pH and also during higher frequencies at neutral pH. Both, atropine sensitive and insensitive mechanisms are activated frequency dependent. The atropine-sensitive cholinergic mechanisms but not the noncholinergic mechanisms involved in regulation of G-cell function are pH and frequency dependent. Somatostatin is regulated largely independent of stimulation frequency and pH by at least two pathways involving cholinergic mechanisms of different sensitivity to atropine. These data suggest a highly differentiated regulation of BLI, gastrin and SLI secretion and the interaction between these systems awaits further elucidation.     

Orexins stimulate corticosterone secretion of rat adrenocortical cells, through the activation of the adenylate cyclase-dependent signaling cascade

Orexins-A and B are two novel hypothalamic peptides, which, like leptin and neuropeptide-Y (NPY), are involved in the central regulation of feeding. Since leptin and NPY were found to modulate adrenal function, we have examined whether orexins are able to directly affect rat adrenal steroid secretion. Both orexin-A and orexin-B raised basal corticosterone secretion of dispersed rat zona fasciculata–reticularis (ZF/R) cells, their maximal effective concentration being 10⁻⁸ M. In contrast, orexins did not affect either maximally ACTH (10⁻⁹ M)-stimulated corticosterone production by ZF/R cells or the basal and agonist-stimulated aldosterone secretion of dispersed zona glomerulosa cells. The ACTH-receptor antagonist corticotropin-inhibiting peptide (10⁻⁶ M) annulled corticosterone response of ZF/R cells to ACTH (10⁻⁹ M), but not to orexins (10⁻⁸ M). Orexins (10⁻⁸ M) enhanced cyclic-AMP release by ZF/R cells, and the selective inhibitor of protein-kinase A (PKA) H-89 (10⁻⁵ M) abolished corticosterone responses to both ACTH (10⁻⁹ M) and orexins (10⁻⁸ M). A subcutaneous injection of both orexins (5 or 10 nmol/kg) evoked a clear-cut increase in the plasma concentration of corticosterone (but not aldosterone), the effect of orexin-A being significantly more intense than that of orexin-B. Collectively, these findings suggest that orexins exert a selective and direct glucocorticoid secretagogue action on the rat adrenals, acting through a receptor-mediated activation of the adenylate cyclase/PKA-dependent signaling pathway.     

Angiotensin II and angiotensin-(1-7) inhibit the inner cortex Na+ -ATPase activity through AT2 receptor

In the present paper, the modulation of the basolateral membrane (BLM) Na⁺-ATPase activity of inner cortex from pig kidney by angiotensin II (Ang II) and angiotensin-(1–7) (Ang-(1–7)) was evaluated. Ang II and Ang-(1–7) inhibit the Na⁺-ATPase activity in a dose-dependent manner (from 10⁻¹¹ to 10⁻⁵ M), with maximal effect obtained at 10⁻⁷ M for both peptides. Pharmacological evidences demonstrate that the inhibitory effects of Ang II and Ang-(1–7) are mediated by AT₂ receptor: The effect of both polypeptides is completely reversed by 10⁻⁸ M PD 123319, a selective AT₂ receptor antagonist, but is not affected by either (10⁻¹²–10⁻⁵ M) losartan or (10⁻¹⁰–10⁻⁷ M) A779, selective antagonists for AT₁ and AT_(1–7) receptors, respectively. The following results suggest that a PTX-insensitive, cholera toxin (CTX)-sensitive G protein/adenosine 3′,5′-cyclic monophosphate (cAMP)/PKA pathway is involved in this process: (1) the inhibitory effect of both peptides is completely reversed by 10⁻⁹ M guanosine 5′-O-(2-thiodiphosphate) (GDPβS; an inhibitor of the G protein activity), and mimicked by 10⁻¹⁰ M guanosine 5′-O-(3-thiotriphosphate) (GTPγS; an activator of the G protein activity); (2) the effects of both peptides are mimicked by CTX but are not affected by PTX; (3) Western blot analysis reveals the presence of the G_s protein in the isolated basolateral membrane fraction; (4) (10⁻¹⁰–10⁻⁶ M) cAMP has a similar and non-additive effect to Ang II and Ang-(1–7); (5) PKA inhibitory peptide abolishes the effects of Ang II and Ang-(1–7); and (6) both angiotensins stimulate PKA activity.     

Stimulation of pancreatic amylase release is associated with a parallel sustained increase of cytoplasmic calcium

The kinetics of the changes in the cytoplasmic Ca²⁺ concentration (Ca_i²⁺) and amylase release were measured in fura-2-loaded pancreatic acinar cells and perifused pancreatic acini, respectively. Cholecystokinin octapeptide (CCK-8) and its amphibian analogue caerulein induced similar dose-related increases of Ca_i²⁺ and amylase secretion with threshold concentrations of 2–6·10⁻¹² M, and maximal effects at 2·10⁻¹⁰ M. The action of CCK/caerulein on Ca_i²⁺ was complex and similar to that of carbachol and bombesin with a prompt several-fold increase within seconds followed by a gradual decline over more than 5 min to a new sustained suprabasal level. The kinetics of amylase release in response to CCK and carbachol correlated with the changes in Ca_i²⁺. Additions of the antagonists N²,O²-dibutyrylguanosine 3′:5′-cyclic monophosphate and atropine after 30 min of CCK-8 and carbachol stimulation, respectively, were associated with prompt lowerings of Ca_i²⁺ and inhibitions of amylase secretion. The patterns observed with substance P (SP) and eledoisin were different with high concentrations (10⁻⁸–10⁻⁷ M) giving monophasic increases of Ca_i²⁺ and amylase release. An initial stimulation of cells with a high dose of CCK eliminated the Ca_i²⁺ response to further stimulation with CCK, carbachol, bombesin and SP, whereas cells subjected to initial stimulation with SP responded to subsequent exposure to CCK with prolonged elevation of Ca_i²⁺. The data indicate that stimulation with CCK, carbachol and bombesin may be associated with intracellular mobilization of calcium from more than one pool, and that an increase of Ca_i²⁺ is involved even in threshold stimulation of amylase release.     

Vasopressin and oxytocin modulation of melatonin secretion from rat pineal glands

The rat pineal gland is known to release melatonin in response to noradrenergic stimulation. Since vasopressin (VP)- and oxytocin (OT)-containing fibers innervate the pineal gland, the effects of VP and OT on melatonin release from perifused rat pineal glands were investigated. VP (10⁻⁷ M) and OT (10⁻⁶ M) decreased the basal melatonin secretion. No dose-dependent effect was observed. At high concentrations (10⁻⁵) these peptides potentiated the isoproterenol-induced increase of melatonin secretion. Below 10⁻⁵ M no potentiation was observed. Fragments of VP {[pGlu⁴,Cys⁶]VP(4–9)} and OT {[pGlu⁴,Cys⁶]OT(4–9)} did not display any effect on the isoproterenol-induced melatonin secretion.     

Highly potent and specific inhibitors of human renin

We have designed and synthesized a series of small peptides containing a perfluoroalkyl ketone group at the C-terminal position of the angiotensin I sequence as inhibitors of human renin. From this series of compounds, 8 and 10 showed strong inhibition of human renin (IC₅₀ = 3 × 10⁻⁹, 7 × 10⁻⁹ M, respectively). Compound 10 did not inhibit pepsin and cathepsin D at 10⁻⁴ M. Comparison of the IC₅₀ of compound 8 and compound 11 (8.7 × 10⁻⁷ M) demonstrated the marked effect of the perfluoropropyl group on the potency of inhibition on renin, presumably due to the strong electron-withdrawing effect causing the ketone in 8 to exist predominantly as the hydrate — thus mimicking the tetrahedral transition state during hydrolysis of the scissile Leu¹⁰—Val¹¹ amide bond.     

Vasoactive intestinal peptide receptor activity in human fetal enterocytes

Functional and specific receptors for vasoactive intestinal peptide (VIP) (determined by their capacity to bind ¹²⁵I-VIP and activate adenylate cyclase) and cyclic AMP-dependent phosphodiesterase activities were characterized in enterocytes of human fetal small intestine between 18 and 23 weeks of gestation. Half-maximal stimulation of the cyclase and inhibition of ¹²⁵I-VIP binding in membrane preparations were respectively observed at 1.4 and 5 × 10⁻¹⁰ M VIP. The peptides structurally related to VIP activated the cyclic AMP generating system at pharmacological doses (10⁻⁷M and above) in the following order of potency: VIP> PHI> GRF> secretin. Other peptides or test substances, including GIP, pancreatic glucagon, somatostatin-14, gastrin, CCK, neurotensin, pancreatic polypeptide, PYY, substance P, histamine and isoproterenol are inactive in this system, while the ubiquitous adenylate cyclase activators NaF, forskolin and prostaglandins were effective. These results, combined with the appearance of intestinal VIP in nerve fibers at 8 weeks and with the morphological and enzymatic maturation at 9–12 weeks of the intestinal mucosa, indicate that this neuropeptide may regulate either the differentiation or function of enterocytes during the early development of human intestinal mucosa.     

Stimulatory effect of vasoactive intestinal peptide (VIP) on the secretory activity of dispersed rat adrenocortical cells. Evidence for the interaction of VIP with ACTH receptors

VIP dose-dependently increased basal, but not submaximally ACTH (10⁻¹⁰ M)-stimulated, aldosterone (ALDO) and corticosterone (B) secretion of dispersed rat capsular and inner adrenocortical cells, respectively. The maximal stimulatory effect (60–70% rise) was obtained with a VIP concentration of 10⁻⁸ M. [4-Cl-D-Phe⁶,Leu¹⁷]-VIP, a VIP-receptor antagonist (VIP-A), and corticotropin inhibiting peptide (CIP), an ACTH receptor antagonist (both 10⁻⁶ M), completely annulled VIP (10⁻⁸M)-evoked rises in basal ALDO and corticosterone secretions. The ACTH (10⁻¹⁰ M)-enhanced (about 5-fold) production of both hormones was completely reversed by CIP (10⁻⁶ M) and only partially reduced (about −30%) by VIP-A (10⁻⁶ M). The hypothesis is advanced that the weak secretagogue effect of VIP on dispersed rat capsular and inner adrenocortical cells may be due to its positive interaction with ACTH receptors.     

Synthetic peptide analogs to barnacle settlement pheromone

Barnacle pheromone enhances the rate of settlement and metamorphosis of larvae of Balanus amphitrite Darwin. Analogs to the heterogeneous pheromone peptides were sought. Settlement assays were used to assess both the pheromone and the potential analogs. The pheromone has a lower threshold of activity at a concentration of 0.2 μg BSA protein equivalence l⁻¹. Treatment with carboxypeptidase eliminates biological activity. Series of dipeptides were tested to determine if dipeptides could promote settlement. Combinations of acidic, neutral, and basic amino acids in dipeptides were examined. Specific small peptides can mimic barnacle pheromone. Only peptides with a basic carboxy-terminal amino acid and either a neutral or a basic amino-terminal amino acid enhance settlement. Six peptides were shown to mimic pheromone activity at concentrations comparable to the native molecule. Some peptides were more potent than others. The most effective peptides were L-leucyl-L-arginine and L-histidyl-L-lysine which had a lower threshold of settlement enhancement of 2.0×10⁻¹⁰ M and caused a 130% increase in settlement rate at 2.0×10⁻⁸ M. Glycyl-glycyl-L-arginine, glycyl-L-histidyl-L-lysine, L-leucyl-glycyl-L-arginine and L-tyrosyl-L-arginine had thresholds between 2.0×10⁻⁸ M and 2.0×10⁻⁹ M. Peptide pheromone analogs should be useful in determining the nature and mechanism of barnacle pheromone receptor interactions.     

Interactions between vasotocin and other corticotropic factors on the frog adrenal gland

The adrenocortical cells of the amphibian interrenal (adrenal) gland are controlled by multiple factors including neuropeptides and classical neurotransmitters. In particular, it has recently been shown that vasotocin (AVT), the amphibian counterpart of vasopressin, is a potent stimulator of frog corticosteroidogenesis. In the present study, we have investigated the possible interactions between AVT and other regulatory factors on frog interrenal tissue. When AVT (10⁻⁹ M) and serotonin (10⁻⁶ M) were infused together, a strict addition of the individual effects was observed. Similar results were obtained with concomitant infusion of AVT and vasoactive intestinal peptide or AVT and ACTH. In contrast, when AVT (10⁻⁹ M) and acetylcholine (5 × 10⁻⁵ M) were added together, the increase in corticosteroid secretion was less than additive. Dopamine induced a significant reduction of AVT-evoked stimulation of corticosterone production. These results indicate that regulatory peptides or classical neurotransmitters which participate in the control of adrenal steroidogenesis may interact on their target cell to modulate the activity of their congeners.     

Glutathione-proline activation of feeding behavior in the zoanthid Palythoa psammophilia Walsh & Bowers

Palythoa psammophilia Walsh & Bowers has a well coordinated, stereotyped feeding response, the culminating step of which is ingestion; this may be elicited by the synergistic effect of the tripeptide glutathione and the -imino acid, proline. Either activator acting separately causes responses only at high concentrations (above 10⁻⁵ M for glutathione; above 10⁻⁴ M for proline) in a reduced number of animals and at a low rate (5.00 ± 1.73 min in 5 × 10⁻³ M solutions of glutathione; 11.10±3.74 min in 5 × 10⁻³ M solutions of proline). Highest percentages of response were obtained in combinations where glutathione was at a concentration of 5 × 10⁻³ M and proline at 5 × 10⁻⁴ M or in combinations of glutathione at concentrations 5 × 10⁻⁶ M and proline at 5 × 10⁻⁵ M. The speed of ingestion is considerably enhanced when these activators are combined (1.17±1.18 min).     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Cortisol and testosterone: Inhibitory agents of the mineralocorticoid pathway. Is there a physiopathological meaning in adrenal tumors?

The authors incubated adrenal mitochondria to study the in vitro action of cortisol and testosterone on the transformation of corticosterone and 18-hydroxycorticosterone into aldosterone. The results show that cortisol at concentrations of 5 × 10⁻⁶ and 10⁻⁴ M inhibit the conversion of corticosterone into aldosterone by 23.6 to 90%; testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibit the reaction by 78.4 and 87.2%, respectively. The inhibition of the conversion of 18-hydroxycorticosterone into aldosterone is 12.5 to 91% by cortisol with concentrations ranging from 5 × 10⁻⁷ to 5 × 10⁻⁵ M and testosterone 5 × 10⁻⁵ and 10⁻⁴ M inhibits the reaction by 87.3 and 91%, respectively. Aldosterone (10⁻⁸ and 10⁻⁶ M) does not inhibit aldosterone biosynthesis from corticosterone or 18-hydroxycorticosterone. It thus appears that cortisol and testosterone have an effect on the aldosterone biosynthesis pathways in mitochondria. This action may be located at the binding site of the cytochrome P450 11β, which catalyzes all hydroxylation steps in the mineralocorticoid biosynthesis pathway. Because cortisol and testosterone may interfere with aldosterone biosynthesis, and since functional zonation is expected in adrenal carcinomas, the presence of these steroids in substantial amounts could explain the very low plasma aldosterone level usually observed, in adrenal carcinomas studies in our laboratory.     

Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2+3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (<0.049×10⁻⁷). The frequency of HPRT exons 2+3 deletions was 0.10×10⁻⁷ per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%–70%) and previous results using the T-cell cloning assay (2–3×10⁻⁷ per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7×10⁸ leukocytes analyzed), and which occurred at frequencies from <1×10⁻⁷ (undetected) to 1.6×10⁻⁴ among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2+3 deletions in the leukocytes of the newborns, from 0.07×10⁻⁷ to 0.23×10⁻⁷. We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2+3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.     

ACTH(1–24) stimulates the migration of human monocytes in vitro

In view of the increasing evidence that a variety of stresses can influence immune responses, the direct effect of adrenocorticotropic hormone on the migration of human monocytes was studied in vitro. ACTH(1–24) significantly increased the number of migrating cells when placed in the same or the opposite compartment of the chemotaxis chamber, maximum activity being obtained at 10⁻¹⁴ and 10⁻⁸ M. The results indicate that ACTH(1–24) directly and potently stimulates the migration of human monocytes by means of a chemokinetic effect.     

Characterization of muscarinic acetylcholine receptors on the rat pancreatic gastrin-producing cell line B6 RIN

The mechanisms of cholinergic stimulation of gastrin cells were studied in the rat pancreatic cell line B6 RIN. Carbachol induced an increase in intracellular Ca²⁺ and stimulated gastrin release in a dose-dependent manner over the range 10⁻⁵-10⁻³ M. These effects were completely abolished by atropine, suggesting the implication of muscarinic cholinergic receptors. The binding properties of these receptors were investigated. [N-Methyl-³H]scopolamine ([³h]nms) binding on cell homogenates was time-dependent, saturable and consistent with a single high-affinity binding class (K_d = 39.5 pM, and B_max = 7.9 fmol/mg DNA). Carbachol competitively inhibited [³H]NMS binding. The potency of inhibition of [³H]NMS binding by subtype selective antagonists was hexahydrodifenidol> pirenzepine> AF-DX 116. These results suggest the M₃, muscarinic receptors may be involved in the carbachol-induced gastrin release from B6 RIN cells.     

Bradykinin counteracts the stimulatory effect of angiotensin-(1-7) on the proximal tubule Na+ -ATPase activity through B2 receptor

Recently, we demonstrated that angiotensin-(1–7) (Ang-(1–7)) stimulates the Na⁺-ATPase activity through a losartan-sensitive angiotensin receptor, whereas bradykinin inhibits the enzyme activity through the B₂ receptor [Regul. Pept. 91 (2000) 45; Pharmacol. Rev. 32 (1980) 1]. In the present paper, the effect of bradykinin (BK) on Ang-(1–7)-stimulated Na⁺-ATPase activity was evaluated. Preincubation of Na⁺-ATPase with 10⁻⁹ M Ang-(1–7) increases enzyme activity from 7.9±0.9 to 14.1±1.5 nmol Pi mg⁻¹ min⁻¹, corresponding to an increase of 79% (p<0.05). This effect is reverted by bradykinin in a dose-dependent manner (10⁻¹⁴–10⁻⁸ M), reaching maximal inhibitory effect at 10⁻⁹ M. Des-Arg⁹ bradykinin (DABK), an agonist of B₁ receptor, at the concentrations of 10⁻⁹–10⁻⁷ M, does not mimic the BK inhibitory effect, and des-Arg⁹-[Leu⁸]-BK (DALBK), a B₁ receptor antagonist, at the concentrations of 10⁻¹⁰–10⁻⁷ M, does not prevent the inhibitory effect of BK on Ang-(1–7)-stimulated enzyme. On the other hand, HOE 140, an antagonist of B₂ receptor, abolishes the inhibitory effect of BK on the Ang-(1–7)-stimulated enzyme in a dose-dependent manner, reaching maximal effect at 10⁻⁷ M. Taken together, these data indicate that stimulation of B₂ receptors by BK can counteract the stimulatory effect of Ang-(1–7) on the proximal tubule Na⁺-ATPase activity.     

Differentiation induction of human leukemia cells (HL60) by a combination of 1,25-dihydroxyvitamin D3 and retinoic acid (all trans or 9-cis)

1,25(OH)₂D₃ and two stereoisomers of retinoic acid, all trans and 9-cis retinoic acid, are regulators of cell proliferation and differentiation. The aim of this study was to evaluate the effects of a combination of 1,25(OH)₂D₃ and retinoic acid (all trans or 9-cis) on proliferation and cell differentiation of the human promyelocytic leukemia cell line HL60, and to test the reversibility of the induced differentiation. Cell proliferation was inhibited as expected by 1,25(OH)₂D₃ and all trans retinoic acid alone (IC₅₀ of cell survival was 4 × 10⁻⁷ M, 9 × 10⁻⁶ M and 9 × 10⁻⁷ M for 1,25(OH)₂D₃, all trans and 9-cis retinoic acid, respectively). Combination of 1,25(OH)₂D₃ and either form of retinoic acid resulted in a partially additive decrease in cell proliferation. 1,25(OH)₂D₃ induced a monocytic differentiation (100% CD14+ cells with 10⁻⁷ M 1,25(OH)₂D₃), while retinoic acid led to a predominantly granulocytic differentiation (36 and 42% CD67+ cells with 10⁻⁶ M all trans and 9-cis retinoic acid, respectively). Additive effects on differentiation were observed upon combination of subtherapeutical doses of the drugs, achieving a mainly monocytic population, demonstrating the dominant role of 1,25(OH)₂D₃ in determining the direction of differentiation. The effects on proliferation and differentiation of the solitary drugs were reversible, while the proliferation arrest and differentiation induced by the combination persisted and even progressed after withdrawal of the drugs. We conclude that 1,25(OH)₂D₃ and retinoic acid (all trans or 9-cis) exert additive effects on inhibition of proliferation and induction of cell differentiation of HL60 cells, leading to a persistent differentiation, even after drug withdrawal.