Retinoylation (covalent modification by retinoic acid) of Rho-GDIß in the human myeloid leukemia cell line HL60 and its functional significance.

Retinoic acid (RA) has a variety of biological effects in mammalian cells and tissues. It is well known that RA induces differentiation of human acute promyelocytic leukemia (APL) HL60 cells, fresh human APL cells, and clinical remission in patients with APL. Retinoylation (acylation of proteins by RA) is a possible pathway for RA action. However, an understanding of the role that retinoylation plays in the actions of RA is lacking. In the current study, several retinoylated proteins were detected in RA-treated HL60 fractions following Mono Q anion exchange chromatography and analysis using two-dimensional polyacrylamide gel electrophoresis. One of the retinoylated proteins was identified as Rho-GDIß (28kDa) by TOF-MS and co-migration with Rho-GDIß (28kDa). Truncated Rho-GDIß (23kDa, N∆19), a product of cleavage by caspase-3, was not retinoylated. RA covalently bound to the Thr2 residue in Rho-GDIß (5kDa), which is the second product resulting from the cleavage of Rho-GDIß (28kDa) by caspase-3. RA treatment increased the level of Rho-GDIß (28kDa) and decreased the level of Rho-GDIß (23kDa). RA did not induce caspase-3 activity or Rho-GDIß mRNA expression. It is likely that retinoylation of Rho-GDIß increases its metabolic stability.

Protein kinase A activation by retinoic acid in the nuclei of HL60 cells.

BACKGROUND: Activation of protein kinase A (PKA) occurs during retinoic acid (RA)-induced granulocytic differentiation of human promyelocytic leukemia HL60 cells. It is known that the RIIα regulatory subunit of PKA, is modified by RA (retinoylated) in the early stages of differentiation. We have investigated the effects of RA on PKA during cell differentiation in order to understand the potential significance of this process in the retinoylation of RIIα subunits. METHODS: Immunoblotting, immunoprecipitation, confocal microscopy, PCR, and PKA activity assays were employed for characterizing the effects of RA on PKA. RESULTS: We found that RA induces intracellular mobility of RIIα and the activation of PKA in HL60 cells. Increases in RIIα levels were observed in RA-treated HL60 cells. RA treatment altered intracellular localization of the PKA subunits, RIIα and Cα, and increased their protein levels in the nuclei as detected by both immunoblotting and immunostaining analyses. Coincident with the increase in nuclear Cα, RA-treated HL60 cells showed increases in both the protein phosphorylation activity of PKA and the levels of phosphorylated proteins in nuclear fractions as compared to control cells. In addition, RIIα protein was stabilized in RA-treated HL60 cells as compared to control cells. CONCLUSIONS: These results suggest that RA stabilizes RIIα protein and activates PKA in the nucleus, with a resultant increase in the phosphorylation of nuclear proteins. GENERAL SIGNIFICANCE: Our evidence suggests that retinoylation of PKA might contribute to its stabilization and activation and that this could potentially participate in RA's ability to induce granulocytic differentiation of HL60 cells.

Demonstration of basic proteins that bind retinoic acid in the human myeloid leukemia cell line HL60.

Retinoic acid (RA) has a variety of biological effects in mammalian cells and tissues. It is well known that RA is a potent anticancer agent that induces differentiation of leukemia cells as well as inhibiting cell growth. The current study examined HL60 proteins using anti-RA monoclonal antibodies (ARMAs) and found that some RA-binding proteins may be histones. These proteins eluted in the void volume fractions following Mono Q anion exchange chromatography and immunostained with ARMAs. These ARMAs showed specific binding to the proteins in a saturable manner that depended on antibody concentration. Certain of these proteins co-migrated with histones on one-dimensional polyacrylamide gel electrophoresis. It was also found that histones isolated from HL60 cells treated with RA also immunostained with ARMAs. These results indicate that basic proteins, including histones, may be bound to RA covalently in HL60 cells and that RA-binding histones may play significant roles in the transcriptional regulation of genes by RA.

p-Dodecylaminophenol derived from the synthetic retinoid, fenretinide: antitumor efficacy in vitro and in vivo against human prostate cancer and mechanism of action.

Fenretinide, N-(4-hydroxyphenyl)retinamide (4-HPR) is an aminophenol-containing synthetic retinoid derivative of all-trans-retinoic acid, which is a potent chemopreventive and antiproliferative agent against various cancers. Clinical studies of 4-HPR have shown side effects consisting of night blindness and ocular toxicity. To maintain potent anticancer activity without side effects, p-dodecylaminophenol (p-DDAP) was designed based on structure-activity relationships of 4-HPR. In our study, we investigate whether p-DDAP shows anticancer activity against human prostate cancer cell line PC-3 when compared with 4-HPR. p-DDAP inhibited PC-3 cell growth progressively from low to high concentration in a dose-dependent manner. p-DDAP was the most potent antiproliferative agent in vitro among 6 p-alkylaminophenols and 3 4-hydroxyphenyl analogs examined including 4-HPR. Cells treated with p-DDAP were shown to undergo apoptosis, based on condensation nuclei, cytofluorimetric analysis, propidium iodide staining and the expression of bcl-2 and caspase 3. p-DDAP arrested the S phase of the cell cycle, while 4-HPR arrested the G(0)/G(1) phase. In addition, both the i.v. and i.p. administration of p-DDAP suppressed tumor growth in PC-3-implanted mice in vivo. p-DDAP showed no effects on blood retinol concentrations, in contrast to reductions after 4-HPR administration. These results indicate that p-DDAP exhibits excellent anticancer efficacy against hormonal independent prostate cancer in vitro and in vivo, and it may have great potential for clinical use in the treatment of prostate cancer with reduced side effects.

Proteins in human myeloid leukemia cell line HL60 reacting with retinoic acid monoclonal antibodies.

The vitamin A derivative, retinoic acid (RA) has various biological effects in mammalian cells and tissues. It is well known that RA induces differentiation of leukemia cells and inhibits cell growth. There are two pathways for RA action; one via RA nuclear receptors (RARs), and one via acylation of proteins by RA (retinoylation). However, an understanding of which actions of RA occur via RARs and which occur via retinoylation is lacking. Thus, we undertook the examination of HL60 proteins using anti-RA monoclonal antibodies (ARMAs). These ARMAs showed specific binding to proteins in a saturable manner depending on protein and antibody concentration. Proteins eluted by Mono Q anion exchange chromatography and separated using two-dimensional polyacrylamide gel electrophoresis were detected by ARMAs. One of these ARMA-bound proteins in HL60 cells was identified as alpha-actinin. These results indicate that retinoylated proteins in HL60 cells can be recognized by ARMAs and that alpha-actinin modified by RA may play a significant role in RA-induced differentiation, including the promotion of cytomorphology changes.

Formation of retinoylated proteins from retinoyl-CoA in rat tissues.

Retinoylation (acylation of proteins by retinoic acid) is considered as one mechanism of retinoic acid (RA) action occurring in cells in vitro and in vivo. Previously, our studies showed that in rat tissues the formation of retinoyl-CoA from RA, the first step of retinoylation, required ATP, CoA and MgCl(2). In the current study, we examined whether the transfer of retinoyl-CoA into proteins, the second step of retinoylation, occurs in rat tissues. [(3)H]-Labeled-retinoyl-CoA bound covalently to proteins in rat liver, kidney, testis, and brain. The levels of incorporation of retinoyl-CoA into proteins were higher in vitamin A-deficient rats than in normal ones. The formation of retinoylated proteins depended on the incubation time, and the concentrations of retinoyl-CoA and homogenate. The reaction was suppressed by fatty acyl-CoAs and palmitic acid, but not by arachidonic acid. The Vmax and Km values for retinoyl-CoA in the formation of retinoylated proteins using a crude liver extract were estimated to be 2,597.3 pmol/min/mg protein and 9.5 x 10(-5) M, respectively. Retinoylated proteins formed from retinoyl-CoA, including a 17 kDa protein exhibiting high radioactivity, disappeared in the presence of 2-mercaptoethanol, indicating that RA was linked to the proteins through a thioester bond. These results demonstrate that retinoylation in rat tissues occurs via retinoyl-CoA formed from RA. This process may play a significant physiological role in cells.

Biological activity of p-methylaminophenol, an essential structural component of N-(4-hydroxyphenyl)retinamide, fenretinide.

Fenretinide, N-(4-hydroxyphenyl)retinamide (4-HPR), is a synthetic amide of all-trans-retinoic acid (RA), which inhibits cell growth, induces apoptosis, and is an antioxidant, and cancer chemopreventive and antiproliferative agent. These findings led us to investigate which structural component of 4-HPR contributes to these potent activities. Our approach was to examine 4-aminophenol (4-AP), p-methylaminophenol (p-MAP), and p-acetaminophen (p-AAP). It was found that vitamin E, 4-AP and p-MAP scavenge alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radicals in a 1:2 ratio, in contrast to 4-HPR and p-AAP, for which 1:1 and 1:0.5 ratios were observed relative to DPPH radicals. However, RA was inactive. Lipid peroxidation in rat liver microsomes was reduced by compounds (RA > p-MAP = 4-HPR > 4-AP) in a dose-dependent manner, while p-AAP was inactive. In addition, both p-MAP and 4-HPR are potent inhibitors of cell growth and inducers of apoptosis in HL60 cells. p-MAP exhibits the same level of antiproliferative activity as 4-HPR against HL60R cells, which are a resistant clone against RA, and it inhibits the growth of various cancer cell lines (MCF-7, MCF-7/Adr(R), HepG2, and DU-145) to an extent greater than 4-AP and p-AAP, but is less potent than 4-HPR. Thus, although the antioxidant activity of p-MAP is more potent than that of 4-HPR, p-MAP is less potent than 4-HPR in anticancer activity. These results suggest that both the anticancer and antioxidative activities shown by 4-HPR are due to the structure of p-MAP. The retinoyl residue or long alkyl chain substituent attached to an aminophenol may be significant for anticancer properties.

The anti-tumor agent, p-DDAP potently suppresses proliferation through apoptosis in human neuroblastoma NB-39-nu cells.

Retinoic acid (RA) is a chemotherapeutic agent used to induce neuronal cellular differentiation of neuroblastoma. However, because treatment with RA is associated with the side-effect of nyctalopia, efforts have been underway to identify new compounds that could potentially overcome these drawbacks. As part of these studies we have examined anti-cancer effects on the neuroblastoma NB-39-nu cells of p-dodecylaminophenol (p-DDAP), a novel derivative of N-(4-hydroxyphenyl) retinamide (4-HPR). p-DDAP suppresses proliferation, and induces G(0)/G(1) arrest and apoptosis to a greater extent than RA and 4-HPR. Neuronal differentiation was not detected in p-DDAP-treated cells. Since p-DDAP is not toxic and does not reduce blood retinol levels, p-DDAP might be a useful anti-neuroblastoma drug having reduced side-effects.

Potent anticancer activities of novel aminophenol analogues against various cancer cell lines.

Novel aminophenol analogues were synthesized based on the structure of fenretinide (N-(4-hydroxyphenyl)retinamide, 5), which is a potent anticancer agent. Our findings showed that the anticancer activities of 5 were due to the side chain attached to the aminophenol moiety. A p-octylaminophenol (p-OAP) provided the most potent anticancer activity among p-alkylaminophenols examined. In this study, we investigated anticancer activities against various cancer cell lines by the new aminophenols, p-dodecylaminophenol (1), p-decylaminophenol (2), N-(4-hydroxyphenyl)dodecananamide (3), and N-(4-hydroxyphenyl)decananamide (4), which exhibits a side chain as long as 5. Cell growth of breast cancer (MCF-7, MCF-7/Adr(R)), prostate cancer (DU-145), and leukemia (HL60) cells was suppressed by 1 and 2 in a fashion dependent on the length of the alkyl chain attached to the aminophenol. In contrast, 3 and 4 were extremely weak. Compound 5 was less potent than 1. Cell growth of liver cancer (HepG2) was not markedly affected by these compounds. In addition, apoptosis of HL60 cells was induced by 1 and 2 in a chain length-dependent manner, but not by 3 and 4. Incorporation of compounds into HL60 cells was in the order 1>2=3>4. These results indicated that anticancer activities for 1 and 2 are correlated with their incorporation into cancer cells and their capability to induce apoptosis, but not for 3 and 4. Compound 1, a potent anticancer agent with potency strikingly greater than 5, may potentially be useful in clinic.

Anticancer and superoxide scavenging activities of p-alkylaminophenols having various length alkyl chains.

A series of p-alkylaminophenols including 3, p-butylaminophenol; 4, p-hexylaminophenol; 5, p-octylaminophenol; and 6, N-(p-methoxybenzyl)aminophenol were synthesized based on the structure of fenretinide, N-(4-hydroxyphenyl)retinamide (1). This latter agent is a synthetic amide of all-trans-retinoic acid (RA), which is a cancer chemopreventive and antiproliferative agent. It was found that elongation of the alkyl chain length in these compounds increased antioxidative activity and inhibition of lipid peroxidation. These findings led us to investigate whether antiproliferative activity against cancer cells was effected by the length of alkyl chains linked to the aminophenol residue. All p-alkylaminophenols inhibited growth of HL60 and HL60R cells in a dose-dependent manners. The HL60R line is a resistant clone against RA. Growth of various cancer cell lines (HL60, HL60R, MCF-7, MCF-7/Adr(R), HepG2, and DU-145) was suppressed by p-alkylaminophenols in a fashion dependent on the aminophenol alkyl chain length (5>4>3>p-methylaminophenol (2)), with 5 being the most potent inhibitor of cell growth against HL60R, MCF-7/Adr(R), and DU-145 cells among p-alkylaminophenols tested, including 1. In particular, with the exception of compound 2, antiproliferative activity against DU-145 cells by these p-alkylaminophenols was greater than by 1. In HL60 cells, growth inhibition was associated with apoptosis. On the other hand, elongation of the alkyl chain length reduced superoxide trapping capability (2>3>4>5) in contrast to the effects on inhibition of lipid peroxidation. These results indicate that anticancer activity of p-alkylaminophenols correlated with the inhibitory activity of lipid peroxidation, but not with the superoxide scavenging activity.

Antioxidant and anticancer activities of novel p-alkylaminophenols and p-acylaminophenols (aminophenol analogues).

Novel compounds were designed based on fenretinide, N-(4-hydroxyphenyl)retinamide (2), which is a synthetic amide of all-trans-retinoic acid (1) that is a potent antioxidant and anticancer agent. Our recent findings indicated that antioxidant and anticancer activities were due to p-methylaminophenol moiety (8) in 2, and that p-octylaminophenol (7), which has an elongated alkyl chain, was more potent than 8. This finding lets us to investigate whether compounds containing alkyl or acyl chains linked to an aminophenol residue as long as 2 and 1, would show activities greater than 2. For this purpose, we prepared p-dodecanoylaminophenol (3), p-decanoylaminophenol (4), p-dodecylaminophenol (5), and p-decylaminophenol (6). The p-alkylaminophenols, 5 and 6, exhibited superoxide scavenging activities, but not p-acylaminophenols, 3 and 4. Elongation of the alkyl chain length reduced superoxide trapping capability (8>7>6>5). In contrast, lipid peroxidation in rat liver microsomes was reduced by 5 and 6 in dose-dependent manner. Compounds 3 and 4 were poor lipid peroxidation inhibitors, being approximately 400- to 1300-fold lower than 5 and 6. In addition, all compounds inhibited cell growth of human leukemia cell lines, HL60 and HL60R, in dose-dependent manners (5>6>3=4). The HL60R cell line is resistant against 1. Growth of both cell lines was suppressed by 5 and 6 in a fashion dependent on the length of the aminophenol alkyl chain, but not by 3 and 4. These results indicate that 5, a potent anticancer agent greater than 2, may potentially have clinical utility, and that its anticancer activity is correlated with inhibitory potency against lipid peroxidation, but not with superoxide scavenging activity.