Cyclic AMP-dependent protein kinase isoenzymes in human myeloid leukemia (HL60) and breast tumor (MCF-7) cells.

Combinations of retinoic acid (RA) and cAMP mediate many biological responses in a large variety of cell types. While the basis for the apparent synergistic effects of RA and cAMP are not clearly defined, it is likely that activation of PKA by cAMP is involved. However, literature reports concerning the identity of PKA isoforms in HL60 and MCF-7 cells are conflicting. The purpose of the present investigation is to identify PKA isoforms in HL60 and MCF-7 cells. Utilization of high-performance anion-exchange liquid chromatography, immunoblotting, and 8-azido-cAMP photoaffinity binding resulted in the finding that HL60 cells contain PKA types I alpha and II alpha, while MCF-7 cells contain PKA types I alpha, II alpha, and II beta. PKA type I alpha in both HL60 and MCF-7 cells eluted from columns as two well-separated peaks. One peak eluted at a low salt concentration in agreement with previous reports. The second HL60 PKA type I alpha peak eluted at a salt concentration intermediate between that eluting the first peak and that eluting PKA type II alpha and contained approximately 62% of the total RI alpha protein. However, the second MCF-7 PKA type I alpha peak contained approximately 66% of the total RI alpha protein and co-eluted with PKA types II alpha and II beta. This "contamination" of PKA type II fractions with PKA type I has led, in some cases, to interpretations that may need reevaluation.

Potentiation of retinoic acid-induced differentiation of human acute promyelocytic leukemia NB4 cells by butyric acid, tributyrin, and hexamethylene bisacetamide.

Cytodifferentiation therapy by all-trans-retinoic acid (RA) for acute promyelocytic leukemia patients is encouraging in spite of several limitations preventing better clinical outcomes. Most patients in complete remission induced by RA experience relapse and resist further treatment with RA. This resistance primarily is due to a systemic self-induced catabolism of RA, which interferes with the maintenance of effective plasma levels of RA. In this report we explored the possibility that treatment with combinations of RA and other differentiation agents may induce differentiation at lower RA concentrations, which in turn may produce diminished levels of resistance. We found that although n-butyric acid (BA), tributyrin (TB) (a prodrug of BA), or hexamethylene bisacetamide (HMBA) were inactive as sole agents they potentiated RA-induced differentiation of human acute promyelocytic NB4 cells. A measure of the effectiveness of these combinations was that the concentrations of RA in combination with BA and HMBA inducing half-maximal differentiation were 20- to 40-fold lower than those needed with RA alone. Furthermore, the concentrations of BA and HMBA in these combinations were at achievable plasma levels. Therefore, these combinations may have clinical utility for treatment of a variety of malignancies that are sensitive to RA alone.

Conformationally defined retinoic acid analogues. 4. Potential new agents for acute promyelocytic and juvenile myelomonocytic leukemias.

We recently synthesized several conformationally constrained retinoic acid (RA) analogues [8-(2'-cyclohexen-1'-ylidene)-3, 7-dimethyl-2,4,6-octatrienoic acids with different alkyl substituents at 2' (R1) and 3' (R2) positions on the cyclohexene ring] (Muccio et al. J. Med. Chem. 1996, 39, 3625) as cancer chemopreventive agents. UAB8 (R1 = Et; R2 = iPr), which contains sufficient steric bulk at the terminal end of the polyene chain to mimic the trimethylcyclohexenyl ring of RA, displayed biological properties similar to those of RA. To explore the efficacy of this retinoid in acute promyelocytic leukemia (APL) and juvenile myelomonocytic leukemia (JMML), we evaluated UAB8 isomers in in vitro assays which measure the capacity of retinoids to inhibit aberrant myeloid colony growth from blood or bone marrow cells obtained from human JMML patients and in assays measuring the potential of retinoids to differentiate NB4 cells (an APL cell line). Both (all-E)- and (13Z)-UAB8 were 2-fold more active than RA in the NB4 cell differentiation assay; however, only (all-E)-UAB8 had comparable activity to the natural retinoids in the JMML cell assays. These results were compared to the biological effectiveness of a new retinoid, UAB30 [8-(3', 4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4, 6-octatrienoic acid], which had different nuclear receptor binding and transactivational properties than UAB8. Relative to (all-E)-RA and (all-E)-UAB8, (all-E)-UAB30 bound well to RARalpha but did not activate transcription-mediated RARalpha homodimers, even though it was effective in RARbeta- and RARgamma-mediated transactivational assays. In APL assays, this retinoid had much reduced activity and was only moderately effective in JMML assays and in cancer chemoprevention assays.

Decreased retinoylation in NIH 3T3 cells transformed with activated Ha-ras.

Retinoylation (retinoic acid acylation) is a post-translation modification of proteins occurring in a variety of mammalian cell lines and in vivo. To gain further knowledge of the role of retinoylation we studied it in NIH 3T3 cells and NIH 3T3 cells transformed by an activated Ha-ras oncogene (NIH Ha-ras-3T3 cells). In serum-free medium retinoic acid (RA) inhibited growth of NIH 3T3 cells but did not inhibit growth of NIH Ha-ras-3T3 cells. After incubation with [3H]RA, the level of retinoylated protein in NIH 3T3 cells was about 1.5-fold greater than in NIH Ha-ras-3T3 cells. On one-dimensional polyacrylamide gel electrophoresis, both the rate and the extent of retinoylation were greater in NIH 3T3 cells. We detected about 40 retinoylated proteins in NIH 3T3 cells by two-dimensional polyacrylamide gel electrophoresis. Only about 15 proteins were retinoylated, but at reduced levels, in NIH Ha-ras-3T3 cells. These results suggest that the activated ras oncogene inhibits retinoylation. This inhibition may in turn be related to the loss of other RA responses of NIH 3T3 cells, including growth inhibition, retinoic acid catabolism, down-regulation of fibronectin biosynthesis, and induction of tissue-type transglutaminase, which are not seen to the same extent in NIH Ha-ras-3T3 cells.

N-4-hydroxyphenylretinamide enhances retinoic acid-induced differentiation and retinoylation of proteins in the human acute promyelocytic leukemia cell line, NB4, by a mechanism that may involve inhibition of retinoic acid catabolism.

All-trans-retinoic acid (RA) induces differentiation of acute promyelocytic leukemia cells both in vitro and in vivo and is an alternative to cytotoxic chemotherapy in the treatment of acute promyelocytic leukemia. However, despite a complete remission rate of about 90%, most patients relapse and are resistant to further treatment with RA. This resistance primarily is due to an increased systemic catabolism of RA. In this study we examined the catabolism of RA by the human acute promyelocytic leukemia cell line NB4 and the human myeloid leukemia cell line HL60. NB4 cells converted RA to 4-hydroxy-RA, 4-oxo-RA and more polar unidentified retinoids at a much greater rate than HL60 cells. Exposure of NB4 cells to RA induced RA catabolism. We found that 4-hydroxyphenylretinamide (4-HPR) inhibited the catabolism of RA. This inhibition was dosedependent and greater, on a molar basis, than the inhibition seen with the cytochrome P450 inhibitor, ketoconazole, or two synthetic retinoids, Ch55 and Am80. 4-HPR alone was a poor inducer of differentiation of NB4 cells. However, it markedly enhanced RA-induced differentiation and increased the level of retinoylation, the covalent binding of RA to proteins. These results suggest some retinoid analogs, including 4-HPR, may have clinical utility because of their ability to increase the biological half-life of RA.

Growth, differentiation, and death of retinoic acid-treated human acute promyelocytic leukemia NB4 cells.

Published reports vary markedly on some characteristics of retinoic acid (RA) effects on cell growth and differentiation of the human acute promyelocytic leukemia cell line NB4. We explored possible reasons for this variability and found that the initial cell density of the experimental culture and the stage of growth of the cells used to inoculate experimental cultures were critical parameters for obtaining reproducible growth and differentiation responses of NB4 cells. Thus, the time to reach 50% differentiation in the presence of 1 microM RA and various initial concentrations of cells was 1.9 days with 2 x 10(6) cells/ml, 3.5 days with 2 x 10(5) cells/ml, and 4.7 days with 2 x 10(4) cells/ml. With an initial concentration of 2 x 10(5) cells/ml we saw time- and dose-dependent differentiation with RA concentrations >250 nM. However, in the presence of 25-250 nM RA, differentiation reached a maximum of about 20% on either Day 1 or Day 2 and then declined with time. The catabolism of RA appeared to be responsible for the decline in differentiation. In addition, large decreases in viability occurred after NB4 cultures, growing without or with RA, reached a density of only 1 x 10(6) cells/ml. The decreases in viability were greater at intermediate concentrations of RA with a nadir at about 100 nM. Loss of viability was associated with DNA fragmentation and changes in morphology typical of apoptosis and necrosis. The loss of viability occurring in control cultures necessitates caution when these cultures are used to seed experimental cultures.

Tunicamycin in combination with retinoic acid synergistically inhibits cell growth while decreasing palmitoylation and enhancing retinoylation of proteins in the human breast cancer cell line MCF-7.

All-trans-Retinoic acid (RA) induces differentiation and inhibits growth of many tumor types. Whereas the RA nuclear receptors mediate genomic effects of RA, there also are many nongenomic effects that do not have defined mechanisms. Some nongenomic effects of RA may involve retinoylation (RA acylation), a posttranslational modification of proteins occurring in many eukaryotic cell lines including the human breast cancer cell line MCF-7. To gain further knowledge of the role(s) of retinoylation, we studied the effects of tunicamycin (TM), an inhibitor of both protein N-glycosylation and palmitoylation, on growth and retinoylation in MCF-7 cells. We found that RA or TM alone inhibited growth of MCF-7 cells. Combinations of RA and TM inhibited growth synergistically. TM increased retinoylation and decreased palmitoylation. These results suggest that increased retinoylation and decreased glycosylation and palmitoylation may play a role in the synergistic inhibition of cell growth by combinations of TM and RA in MCF-7 cells. Furthermore, our results suggest that combinations of TM and RA may have clinical utility.

Effects of prostaglandin E2 and all-trans retinoic acid combination on induced differentiation of human acute promyelocytic leukemia NB4 cells.

OBJECTIVE: A human promyelocytic leukemia (APL) cell line NB4 was used to demonstrate the synergistic effects between all-trans retinoic acid (ATRA) and prostaglandin E2 (PGE2) on growth inhibition and cytodifferentiation induction. METHODS: NB4 cells were cultured in the presence of either ATRA or PGE2 as a single agent or in combinations at various ratio. Cell growth was measured and myeloid differentiation was tested on consecutive days over the whole course of culture. RESULTS: PGE2 and ATRA synergistically induced the myeloid differentiation of NB4 cells. In comparison with ATRA alone, the combination of PGE2 with ATRA caused an almost 20-fold decrease of effective concentration of ATRA. CONCLUSION: The combination of ATRA and PGE2 at an appropriate ratio may provide a convenient oral regimen of combined differentiation therapy for a better clinical outcome in APL patients.

Retinoylation of proteins in rat liver, kidney, and lung in vivo.

Retinoylation (retinoic acylation) is a posttranslational modification of proteins occurring in a variety of cell types in vitro. This study was done to examine whether retinoylation occurs in vivo. We found that in retinol-deficient rats, radiolabeled retinol or retinoic acid was incorporated into the liver, kidney, and lung in a form that was not removed by extraction with CHCl3:CH3OH. About 98% of the radiolabeled retinoid was acid-soluble after digestion with proteinase K indicating that it was covalently bound to protein. About 50% of the retinoid covalently bound to liver and kidney protein was removed by mild hydrolysis with CH3OH-KOH. Methyl retinoate, all-trans-retinoic acid, and polar metabolites of retinoic acid accounted for essentially all of the retinoids released. We conclude that retinoylation of protein occurs in vivo primarily via the formation of an ester bond.

N-(4-hydroxyphenyl)retinamide (Fenretinide) in combination with retinoic acid enhances differentiation and retinoylation of proteins.

The synthetic retinoid, N-(4-hydroxyphenyl)retinamide (4-HPR; Fenretinide), is a cancer chemopreventive and antiproliferative agent whose mechanism of action is unknown. 4-HPR alone is a poor inducer of differentiation of HL-60 cells compared to all-trans-retinoic acid (RA). Here, we found that combinations of 4-HPR and RA synergistically induced differentiation of HL-60 cells. In addition, 4-HPR increased the level of retinoylation, the covalent binding of RA to proteins. Retinoylation occurs in many eukaryotic cell lines and may be involved in RA-induced differentiation. These results suggest that 4-HPR may be a member of a class of retinoids that are active because they displace RA from extracellular and intracellular sites or because they inhibit RA catabolism. On the basis of these proposed mechanisms, retinoids that do not cause differentiation as sole agents may have utility in the clinic in combination with RA.

Potential applications of cytodifferentiation therapy in hematologic malignancies.

Retinoids, including retinoic acid (RA), are naturally occurring and synthetic analogs of vitamin A that inhibit cell growth and induce cell differentiation in many experimental tumor models. Differentiation of the human myelogenous leukemia cell line HL-60 by RA led to the finding that cells from patients with acute promyelocytic leukemia (APL) are terminally differentiated by RA. One mechanism for the activity of RA in a variety of cell types involves the RA nuclear receptors (RA receptors [RARs] and retinoid X receptors), which have specific high-affinity binding sites for RA and some of its metabolites. Other mechanisms may also be involved in RA-induced differentiation. Recent studies suggest that RA acylation (retinoylation) may be involved in the RA induction of differentiation in leukemia cells. Combinations of RA with cyclic adenosine monophosphate (cAMP)-elevating agents led to synergistically induced differentiation of HL-60 cells. The lower doses of RA needed in combination therapy are unlikely to lead to RA resistance, a major limitation of RA therapy in APL. In vitro studies suggest that combinations of RA with either PGE or the butyric acid (BA) prodrug tributyrin (TB) may be useful in differentiation therapy for APL and other malignancies. This is a US government work. There are no restrictions on its use.

Induction of differentiation and covalent binding to proteins by the synthetic retinoids Ch55 and Am80.

all-trans-Retinoic acid (RA) is a potent inducer in vitro of the differentiation of the human acute myeloid leukemia cell line HL60. A mechanism for RA-induced differentiation of HL60 cells may involve retinoylation (RA acylation) which is a post-translational modification of proteins occurring in many eukaryotic cell lines. Here, we found that differentiation by the synthetic retinoid (E)4-[3-(3,5-di-tert-butylphenyl)-3-oxo-1-propenyl]-benzoic acid (Ch55) was dose-dependent in serum-free medium. The synthetic retinoid 4(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenylcarbamoyl) benzoic acid (Am80) did not induce differentiation. Ch55 bound covalently to proteins of HL60 cells. In contrast, covalent binding of Am80 to HL60 proteins was much lower. Two-dimensional gel electrophoresis patterns of proteins labeled covalently by RA and Ch55 were different with few proteins labeled by both retinoids. The level of retinoylation was increased by Am80 and combinations of RA with either Ch55 or Am80 synergistically induced differentiation of HL60 cells. These results suggest that covalent modification of proteins by a retinoid may play a role in inducing differentiation of HL60 cells. In addition, the synergy seen with combinations of RA and either Ch55 or Am80 suggests that some synthetic retinoids may be active because they displace RA from intracellular sites or because they inhibit RA catabolism.

Unassembled (soluble) vimentin in human myeloid leukemia cell line HL60.

The intermediate filament proteins which include vimentin, desmin, and the keratins are one of three major classes of cytoskeletal proteins in eukaryotic cells. In this study we found that most of the vimentin of undifferentiated HL60 and cells induced to differentiate either along the monocytoid pathway by 12-O-tetradecanoylphorbol-13-acetate (TPA) or along the granulocytic pathway by retinoic acid was soluble in a buffer containing 1% Triton X-100/0.6 mol/l KCl in which the intermediate filament proteins usually are not soluble. HL60 vimentin separated on polyacrylamide gel electrophoresis into two proteins of Mr 55,000 and 54,000 that we detected by immunoblotting. The Mr 55,000 species was the major form in undifferentiated HL60 cells and cells induced by retinoic acid. The distribution of both forms of vimentin changed during induction of differentiation by TPA and after 24 h the Mr 54,000 species was predominant. After an additional 24 h exposure to TPA the relative levels of the two forms of vimentin approached equivalence and a high level of vimentin degradation products was seen. These results suggest that TPA may increase vimentin degradation along a pathway that has a Mr 54,000 intermediate. In addition, the high levels of soluble vimentin in HL60 cells suggests that these cells may be a good model for studying components involved in vimentin assembly.

Tributyrin: a prodrug of butyric acid for potential clinical application in differentiation therapy.

Butyric acid (BA) induces cytodifferentiation in vitro of a wide variety of neoplastic cells. The potential clinical utility of BA is limited by the apparent difficulty of achieving effective concentrations because of its rapid metabolism and short plasma half-life. In this study we addressed two approaches that may achieve effective concentrations of BA in vivo. One strategy is to use BA derivatives as prodrugs that can be metabolized to yield effective BA concentrations in vivo over a sustained period of time. Another strategy is to define agents that are synergistic with BA so that the desired effect can be achieved at lower concentrations of BA. In this study monobutyrin (MB) and tributyrin (TB) were studied in vitro for their effects on inducing differentiation of human myeloid leukemia HL60 cells and murine erythroleukemia cells. On a molar basis TB was about 4-fold more potent than either BA or MB for inducing differentiation of HL60 cells. BA, MB, or TB induced erythroid differentiation of murine erythroleukemia cells. On a molar basis TB was 3- to 4-fold more potent than BA, whereas MB was much less potent than BA. Combinations of all-trans-retinoic acid with either BA, MB, or TB induced myeloid differentiation of HL60 cells synergistically. We saw marked reductions in the doses of each agent that were needed in combination to achieve the same effect as single agents. For example, 130 microM TB, 110 nM all-trans-retinoic acid, and a combination of 13 microM TB plus 13 nM all-trans-retinoic acid all induced half-maximal differentiation of HL60 cells. Our results suggest that the readily available TB may be an effective prodrug of BA and may be useful either as a sole agent or in combination with other agents for cytodifferentiation therapy of human malignancies.

Retinoylation of vimentin in the human myeloid leukemia cell line HL60.

Retinoylation (retinoic acid acylation) is a posttranslational modification of proteins occurring in many eukaryotic cell lines. The widespread occurrence of retinoylation suggests that it may play a role in many effects of retinoic acid (RA) on cells. The regulatory subunits of cyclic AMP-dependent protein kinase are retinoylated in the human myeloid leukemia cell line HL60 (Takahashi, N., Liapi, C., Anderson, W. B., and Breitman, T. R. (1991) Arch. Biochem. Biophys. 290, 293-302), and cytokeratins are retinoylated in normal human keratinocytes (Takahashi, N., Jetten, A. M., and Breitman, T. R. (1991) Biochem. Biophys. Res. Commun. 180, 393-400). We show, in this study, that the intermediate filament protein vimentin is retinoylated in HL60 cells during a 24-h exposure to 100 nM [3H]RA. We found that a retinoylated protein of M(r) 55,000 coeluted on anion exchange chromatography and comigrated on either one- or two-dimensional polyacrylamide gel electrophoresis with a protein that also was stained on immunoblots by an anti-vimentin antibody. About 50% of the [3H]RA was released from this M(r) 55,000 retinoylated protein after hydrolysis with either NH2OH (1 M, pH 10) or CH3OH, 0.1 M KOH. These results indicated that a large fraction of the RA was bound to vimentin by an ester bond. Both the M(r) 55,000 retinoylated protein and immunoreactive vimentin were associated with cell nuclei isolated by two procedures. They were detached during exposure to a nonionic detergent buffer, suggesting that they are bound to the nuclear envelope. These results indicate that retinoylation is a new modification of vimentin that may be an early event in RA-induced differentiation of HL60 cells.

Covalent modification of proteins by ligands of steroid hormone receptors.

Retinoylation, acylation with retinoic acid (RA), is a covalent modification of proteins occurring in a variety of eukaryotic cell lines. In this study, we found that proteins in HL-60 cells were labeled by 17 beta-[3H]estradiol (E2), [3H]progesterone (Pg), 1 alpha,25-dihydroxy[3H]vitamin D3 [1,25(OH)2D3], [125I]triiodothyronine (T3), [125I]thyroxine (T4), and [3H]prostaglandin E2 (PGE2). All of these hormones, except PGE2, are ligands of the steroid hormone receptor family. Addition to the growth medium of 5 microM ketoconazole, an inhibitor of cytochrome P450-dependent enzymes, increased about 2-fold the labeling of proteins by T3, T4, 1,25(OH)2D3, and PGE2. In contrast, ketoconazole did not change markedly the extent of labeling by RA, E2, or Pg. Alkaline methanolysis, which cleaves ester bonds, released variable percentages of the radioactive ligands bound to protein. These values were about 80% for RA and PGE2; 50% for T3, T4, and Pg; and 20% for E2 and 1,25(OH)2D3. Treatment with thioether-cleavage reagents, iodomethane or Raney nickel catalyst, released < 2% of the covalently bound ligands. Two-dimensional polyacrylamide gel electrophoresis patterns of labeled proteins were unique for each ligand. Proteins of M(r) 47,000 and 51,000 were labeled by RA, E2, T3, and T4. These proteins had the same mobilities as RI and RII, the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases. 1,25(OH)2D3 also bound to proteins of M(r) 47,000 and 51,000. However, these proteins had pI values different from those of RI or RII. These results suggest that some activities of ligands of the steroid hormone receptor family and of PGE2 may be mediated by their covalent modification of proteins.

The covalent labeling of proteins by 17 beta-estradiol, retinoic acid, and progesterone in the human breast cancer cell lines MCF-7 and MCF-7/AdrR.

In this study we analyzed the covalent binding to proteins of 17 beta-estradiol (E2), retinoic acid (RA), and progesterone in MCF-7 and MCF-7/AdrR cells. MCF-7 cells have receptors for E2 and progesterone. MCF-7/AdrR cells do not have these receptors. After a 1-day incubation period with either [3H]E2, [3H]progesterone, or [3H]RA the levels of covalently bound radioactivity was between 1.4- to 2-fold greater in MCF-7 cells than in MCF-7/AdrR cells. We analyzed the labeled proteins with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. About 40 proteins were labeled by E2 in MCF-7 cells and about 10 of these proteins were the only proteins labeled by E2 in MCF-7/AdrR cells. We saw that the same 8 proteins were labeled by RA in both cell lines. Progesterone labeled 2 proteins with M(r) values of 37,000 and 20,000 in MCF-7 cells. These 2 proteins had mobilities that were the same as proteins that were labeled by either E2 or RA in both MCF-7 and MCF-7/AdrR cells. Besides these 2 proteins, we saw proteins of M(r) 51,000 (p51) and 55,000 that were covalently labeled by E2 in MCF-7 cells and by RA in both MCF-7 and MCF-7/AdrR cells. The p51 had the same mobility on 2D-PAGE as an 8-azido-[32P]cAMP-labeled protein. This protein is probably RII alpha, the type II cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase. These results suggest that the estrogen receptor, while not obligatory, might still modulate the covalent linkage of E2 to protein. In addition, our results raise the possibility that some effects of some ligands of the thyroid/steroid hormone receptor family may involve the covalent linking of these hormones to proteins, including RII alpha.

Retinoylation of the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases in HL60 cells.

Retinoylation (retinoic acid acylation) is a post-translational modification of proteins occurring in a variety of eukaryotic cell lines. There are at least 20 retinoylated proteins in the human myeloid leukemia cell line HL60 (N. Takahashi and T.R. Breitman (1990) J. Biol. Chem. 265, 19, 158-19, 162). Here we found that some retinoylated proteins may be cAMP-binding proteins. Five proteins, covalently labeled by 8-azido-[32P]cAMP which specifically reacts with the regulatory subunits of cAMP-dependent protein kinase, comigrated on two-dimensional polyacrylamide gel electrophoresis with retinoylated proteins of Mr 37,000 (p37RA), 47,000 (p47RA), and 51,000 (p51RA) labeled by [3H]retinoic acid treatment of intact cells. Furthermore, p47RA coeluted on Mono Q anion exchange chromatography with the type I cAMP-dependent protein kinase holoenzyme and p51RA coeluted on Mono Q anion exchange chromatography with the type II cAMP-dependent protein kinase holoenzyme. An antiserum specific to RI, the cAMP-binding regulatory subunit of type I cAMP-dependent protein kinase, immunoprecipitated p47RA. An antiserum specific to RII, the cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase, immunoprecipitated p51RA. These results indicate that both the RI and the RII regulatory subunits of cAMP-dependent protein kinase are retinoylated. Thus, an early event in RA-induced differentiation of HL60 cells may be the retinoylation of subpopulations of both RI and RII.

Retinoylation of cytokeratins in normal human epidermal keratinocytes.

Retinoylation (retinoic acid acylation) is a covalent modification of proteins occurring in a variety of eukaryotic cell lines. In this study, we found that proteins in undifferentiated and squamous-differentiated normal human epidermal keratinocytes were retinoylated after treatment with [3H]retinoic acid. The major retinoylated proteins were identified as cytokeratins based on their profile in two-dimensional gel electrophoresis and their immunoreactivity with anti-keratin monoclonal antibodies. The covalently bound [3H]retinoic acid was not removed by mild hydrolysis with methanolic-KOH indicating that it is not linked to the cytokeratins by a thioester bond. The results raise the possibility that retinoylation of cytokeratins is involved in some of the effects of retinoic acid on keratinocytes.