Abstract

Trans retinoic acid (RA) has proven to be a potent therapeutic agent in the treatment of acute promyelocytic leukemia. Unfortunately, other subtypes of acute myelogenous leukemia are resistant to the antiproliferative and differentiating effects of RA. In this report, we describe a novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN; CD437) that not only totally inhibits the proliferation of RA-resistant leukemic cell lines HL-60R and K562 but also induces apoptosis in these cells. Exposure of HL-60R to CD437 results in the rapid (within 30 minutes) increase of the cyclin-dependent kinase inhibitor p21waf1/cip1 as well as GADD45 mRNA. Manifestations of CD437-mediated programmed cell death are noted within 2 hours, as indicated by both the cleavage and activation of the CPP32 protease and cleavage of poly (ADP-ribose) polymerase. This is followed by cleavage of bcl-2 and internucleosomal DNA degradation. HL-60R cells do not express the retinoid nuclear receptor RARβ and RARγ and express a truncated RARα. Thus, CD437 induction of p21waf1/cip1 and GADD45 mRNAs and apoptosis occurs through a unique mechanism not involving the retinoid nuclear receptors. CD437 represents a unique retinoid with therapeutic potential in the treatment of myeloid leukemia.

RETINOIDS HAVE BEEN found to inhibit the growth and induce differentiation of a number of leukemic cells and to inhibit the proliferation of a wide variety of both normal and malignant cell types.1-4 Retinoic acid (RA) has been found to inhibit the growth of primarily estrogen receptor (ER)-positive breast carcinoma cell lines.5-8 Retinoids mediate their antiproliferative action as well as their ability to induce differentiation through their binding and activation of specific nuclear receptors, ie, RARs or RXRs (Giguere,9 Gudas,10 and Mangelsdorf and Evans11 and the references within those reports). These activated nuclear receptors, in turn, bind to specific DNA sequences referred to as consensus sequences that are located in the regulatory portions of genes and thus, in turn, modulate gene activity.9-11 These liganded nuclear receptors can also inhibit gene expression through their ability to complex with transcription factors and thus inhibit AP-1–mediated gene transcription.12,13 

We have previously described a novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN; CD437) that induces G1 arrest and apoptosis in human breast carcinoma cells.14 CD437, although displaying extremely poor binding and transactivation of RARα and RARβ, binds and transactivates RARγ in CV-1 cells at a concentration resulting in half-maximal activation ED50 that is approximately three times higher than that found with RA.15 In addition, CD437 does not bind to or transactivate the RXRs. ER-positive breast carcinoma cells express RARα and RARγ, whereas ER-negative cells in general express only RARγ and are refractory to the antiproliferative effects of RA.6-8 We have found that CD437 is a much poorer transactivator of the endogenous RARs and RXRs than RA in ER-negative breast carcinoma cells that are sensitive to CD437-mediated G1 arrest and apoptosis but resistant to RA-mediated growth arrest.14 These results suggest that CD437 mediates its action independent of RARγ expression.14 In addition, contrary to RA, CD437 does not display anti–AP-1 activity in breast carcinoma cells.14 

To further document that CD437 exerts its effects through an RAR/RXR-independent pathway and independent of RARγ expression, we investigated the ability of CD437 to inhibit the growth and induce apoptosis in HL-60R cells. HL-60R cells do not express RARγ or RARβ, as shown by RNase protection assays, and express a truncated RARα that displays a 10-fold higher kd in terms of RA binding than the normal RARα receptor.16,17 We have found that HL-60R cells are exquisitely sensitive to CD437-mediated growth arrest and apoptosis. In addition, CD437 modulates the expression of the GADD45 and p21waf1/cip1 genes in these cells. These results strongly suggest that CD437 mediates its effects independently of RARγ expression and through a RAR/RXR-independent pathway. More importantly, we also show that CD437 is a potent inducer of apoptosis in both RA-sensitive and -resistant leukemic cell lines as well as fresh leukemic cells obtained from patients.

MATERIALS AND METHODS

Material.RPMI 1640 medium and fetal bovine serum were obtained from GIBCO-BRL (Grand Island, NY). α[32P] dCTP (3,000 Ci/mmol−1) and the ECL Western blotting system were obtained from Amersham (Arlington Heights, IL). The antihuman bcl-2 antibodies were obtained from Genosys Biotechnology (The Woodlands, TX). The antihuman actin antibody was purchased from Sigma (St Louis, MO). The mouse antihuman poly (ADP-ribose) polymerase (PARP) antibody has been previously described.18 All trans retinoic acid (RA) was purchased from Sigma Chemicals. 6-[3-(1-adamantly)-4 hydroxyphenyl]-2-naphthalene carboxylic acid was synthesized as described by Charpentier et al.19 

cDNA probes.The full-length human WAF1 cDNA20 probe was kindly provided by Drs K. Kinzler and B. Vogelstein (Johns Hopkins University, Baltimore, MD). A nearly full-length 1.4-kb human GADD45 cDNA probe21 was provided by Dr Michael Kastan (Johns Hopkins University). The 36B4 cDNA probe has been previously described22 and was kindly given by Dr Beth Martin (National Institutes of Health, Bethesda, MD). The plasmid clone HH CSA 65 containing the 1.5-kb cDNA insert specific for the human 18S ribosomal RNA was obtained from American Type Culture Collection (Rockville, MD). The cDNA probes were labeled using the method of Feinberg and Vogelstein.23 

Cell lines and growth conditions.HL-60R was kindly supplied by Dr Steve Collins (University of Washington, Seattle, WA). HL-60 and K562 cells were obtained from Dr Douglas Ross (University of Maryland at Baltimore, Baltimore, MD). Cells were grown in RPMI 1640 medium supplemented with 5% heat-inactivated fetal bovine serum and gentamicin (25 μg/mL) in a 5% CO2 /95% air and 100% humidity atmosphere. Leukemic blasts from patients were isolated using Ficoll hypaque. Cells were collected at the interface between plasma and red blood cells and diluted with sterile phosphate-buffered saline (PBS). The cell suspension was then layered over Ficoll hypaque (1.077 density) and the leukemic blasts isolated at the interface. The sample represented greater than 80% blasts.

Northern blots.RNA was isolated using the single-step method of Chomczyski and Sacchi.24 Electrophoresis, transfer, washing conditions, and hybridizations were performed as previously described.25 Equivalent loading was confirmed by hybridization to the housekeeping gene 36B4 or the 18S ribosomal RNA gene.22 The bands were quantified using a Molecular Dynamics Densitometer (Sunnyvale, CA).

Western blots.Western blots were performed essentially as we have described.26 Logarithmically growing cells were treated with CD437 for various times and cells were harvested and lysed in Laemmli lysis buffer (500 mmol/L Tris-HCl, pH 6.8, 2 mmol/L EDTA, 10% glycerol, 10% sodium dodecyl sulfate, and 5% β-mercaptoethanol). Protein lysates (150 μg/lane) were electrophoresed on 12% sodium dodecyl sulfate-polyacrlyalamide gels and transferred to nitrocellulose membranes. The filters were blocked with 5% nonfat dried milk in 1× PBS/0.5% Tween 20 and then incubated with the appropriate antibodies.

Horseradish peroxidase-conjugated rabbit antimouse IgG (Biorad Laboratories, Hercules, CA) was used as the secondary antibody and the bands were developed using the Amersham ECL nonradioactive method per the manufacturer's instructions.

Apoptosis quantification.Apoptotic cell staining was performed as previously described.27 In brief, after exposure to CD437, cells were harvested, washed with PBS, and resuspended at 1 × 106 cells/mL. Fifty microliters of cell suspension was stained with 5 mL of a solution of acridine orange (100 mg/mL in PBS) in the dark. DNA isolation and electrophoresis were performed as we have previously described.14 

RESULTS

CD437 inhibition of HL-60R growth and induction of apoptosis.Although HL-60R cells are resistant to RA-mediated inhibition of growth,16,17 they are exquisitely sensitive to CD437-mediated inhibition of growth (Fig 1A) with as little as 25 nmol/L CD437 inhibiting growth by approximately 50% (Fig 1B). Similar inhibition of growth was noted in the parental HL-60 cell line (Fig 1C), as well as the RA-resistant K562 leukemia cell line28 (Fig 1D). As evidenced in Fig 1A, there was not only a total inhibition of cellular proliferation in the presence of CD437 but an actual cell loss. We therefore examined whether CD437 induced programmed cell death (apoptosis) in the HL-60R cells. That CD437 indeed induced apoptosis in the HL-60R cells was documented using three independent parameters. (A) HL-60R cells after exposure to 1 μmol/L CD437 displayed marked nuclear fragmentation and chromatin condensation, with the nuclear and cytoplasmic membranes remaining intact (Fig 2), a morphology indicative of apoptosis.29 (B) Incubation of HL-60R cells with 1 μmol/L CD437 resulted in internucleosomal cleavage and laddering of the DNA on gel electrophoresis (Fig 3), a hallmark of apoptosis.29 (C) A number of studies have suggested a critical role for cysteine proteases in the apoptotic process.30-37 Numerous substrates for this family of proteases have now been identified.37-41 PARP, which plays an important role in both DNA synthesis and repair, is cleaved early in the apoptotic process.37,38 

Fig. 1.

(A) CD437 inhibition of HL-60R proliferation. HL-60R cells were seeded in RPMI 1640 at a cell density of 5 × 104 cells/mL. Cells were incubated overnight, after which vehicle alone (dimethyl sulfoxide at 0.1% final concentration) or CD437 (1 μmol/L final concentration) were added. Cells were harvested at various times and cell number determined using a hemocytometer. (B) HL-60R cells were grown as described above in the presence of various concentrations of CD437 for 48 hours. (C) HL-60 cells grown in the presence and absence of 1 μmol/L CD437, as described above. (D) K562 cells were grown as described above in the absence or presence of CD437 (1 μmol/L).

Fig. 1.

(A) CD437 inhibition of HL-60R proliferation. HL-60R cells were seeded in RPMI 1640 at a cell density of 5 × 104 cells/mL. Cells were incubated overnight, after which vehicle alone (dimethyl sulfoxide at 0.1% final concentration) or CD437 (1 μmol/L final concentration) were added. Cells were harvested at various times and cell number determined using a hemocytometer. (B) HL-60R cells were grown as described above in the presence of various concentrations of CD437 for 48 hours. (C) HL-60 cells grown in the presence and absence of 1 μmol/L CD437, as described above. (D) K562 cells were grown as described above in the absence or presence of CD437 (1 μmol/L).

Fig. 2.

CD437-induced apoptosis in HL-60R cells. HL-60R cells were grown as described in the Materials and Methods and exposed to either vehicle alone or CD437 (1 μmol/L) for 24 hours and then stained with aciridine orange. (A) Cells exposed to only vehicle. (B) Cells exposed to CD437 (1 μmol/L).

Fig. 2.

CD437-induced apoptosis in HL-60R cells. HL-60R cells were grown as described in the Materials and Methods and exposed to either vehicle alone or CD437 (1 μmol/L) for 24 hours and then stained with aciridine orange. (A) Cells exposed to only vehicle. (B) Cells exposed to CD437 (1 μmol/L).

Fig. 3.

DNA fragmentation induced by CD437 in HL-60R cells. HL-60R cells were grown as described in the Materials and Methods. CD437 was added to a final concentration of 1 μmol/L and cells were harvested. DNA was extracted and fractionated by gel electrophoresis as described in the Materials and Methods.

Fig. 3.

DNA fragmentation induced by CD437 in HL-60R cells. HL-60R cells were grown as described in the Materials and Methods. CD437 was added to a final concentration of 1 μmol/L and cells were harvested. DNA was extracted and fractionated by gel electrophoresis as described in the Materials and Methods.

As shown in Fig 4, exposure of HL-60R cells to CD437 for 2 hours results in the cleavage of the 116-kD PARP to the 85-kD fragment noted in many forms of programmed cell death.42 Numerous members of the interleukin-1β–converting enzyme (ICE) family of cysteine proteases have been implicated in this cleavage of PARP.37,38,42 Recent evidence suggests that YAMA/CPP32 is responsible for this cleavage.42 We therefore examined YAMA/CPP32 expression in CD437-treated HL-60R cells and found that YAMA/CPP32 is highly expressed in HL-60 cells (Fig 5; data not shown). YAMA/CPP32 is also rapidly cleaved in HL-60 cells after exposure to 1 μmol/L CD437 (Fig 5). This cleavage most likely represents activation of the proform, because, whereas most cells contain an abundance of the precursors of the various cysteine proteases, these precursors must be cleaved at aspartate residues and assembled into enzymatically active heterotetramers.37 

Fig. 4.

CD437-mediated PARP cleavage. HL-60R cells were grown as described in the Materials and Methods and exposed to 1 μmol/L CD437. Cells were harvested at various times and Western blots were performed as described in the Materials and Methods.

Fig. 4.

CD437-mediated PARP cleavage. HL-60R cells were grown as described in the Materials and Methods and exposed to 1 μmol/L CD437. Cells were harvested at various times and Western blots were performed as described in the Materials and Methods.

Fig. 5.

CD437-mediated CPP32 cleavage. HL-60R cells were grown, exposed to 1 μmol/L CD437, and harvested and Western blots were performed as described in the legend to Fig 4.

Fig. 5.

CD437-mediated CPP32 cleavage. HL-60R cells were grown, exposed to 1 μmol/L CD437, and harvested and Western blots were performed as described in the legend to Fig 4.

A number of antagonists and promoters of apoptosis have recently been described. The bcl-2 family, which now consists of 7 different proteins encoded by 7 different genes, has now been characterized (Vaux and Strasser37 and the references contained within). Bcl-2 and bcl-XL are expressed in numerous cell types and are potent antagonists of apoptosis. We were unable to detect bcl-XL expression in HL-60R or HL-60 cells as has previously been described,43 but found that bcl-2 is highly expressed in these cells and is also cleaved during CD437-mediated apoptosis (Fig 6). Phosphorylation of bcl-2 has been previously described as a mechanism for bcl-2 inactivation during the apoptotic process.44 Whether cleavage of bcl-2 represents another mechanism by which bcl-2 is inactivated remains to be determined. That this cleavage of PARP, CPP32, and bcl-2 does not simply represent random cleavage of a number of proteins is suggested by our inability to detect cleavage of lamin B (data not shown), which is often found cleaved in a variety of forms of programmed cell death.39,41 CD437 also markedly inhibited growth of the K562 cells (Fig 1C) but contrary to HL-60R cells apoptosis was not noted until 144 hours of exposure to CD437, as indicated by internucleosomal degradation demonstrated by gel electrophoresis (Fig 7). We also examined the ability of CD437 to induce PARP cleavage and apoptosis in primary leukemic blasts obtained from a patient with acute myelogenous leukemia. As shown in Fig 8C, enhanced PARP cleavage was noted within 24 hours after exposure to 1 μmol/L CD437, with the apoptotic morphology noted at 48 hours of exposure (Fig 8A and B).

Fig. 6.

Bcl-2 cleavage during CD437-mediated apoptosis. HL-60R cells were grown in the presence of 1 μmol/L CD437 for various periods of time, the cells were harvested, and Western blots were performed as described in the Materials and Methods.

Fig. 6.

Bcl-2 cleavage during CD437-mediated apoptosis. HL-60R cells were grown in the presence of 1 μmol/L CD437 for various periods of time, the cells were harvested, and Western blots were performed as described in the Materials and Methods.

Fig. 7.

DNA fragmentation induced by CD437 in K562 cells. K562 cells were grown as described in the Materials and Methods. CD437 was added to a final concentration of 1 μmol/L, cells were harvested, and DNA was extracted and fractionated by gel electrophoresis as described in the Materials and Methods.

Fig. 7.

DNA fragmentation induced by CD437 in K562 cells. K562 cells were grown as described in the Materials and Methods. CD437 was added to a final concentration of 1 μmol/L, cells were harvested, and DNA was extracted and fractionated by gel electrophoresis as described in the Materials and Methods.

Fig. 8.

CD437-mediated apoptosis in primary leukemia cells. Leukemic blasts were isolated as described in the Materials and Methods. Cells were exposed to 1 μmol/L CD437 for various periods of time and PARP cleavage was assessed as previously described. (A) Leukemic cells exposed only to vehicle for 48 hours. (B) Leukemic cells exposed to 1 μmol/L CD437 for 48 hours. (C) CD437-mediated PARP cleavage. Lanes 1 and 3, cells exposed to vehicle only for 24 and 48 hours, respectively. Lanes 2 and 4, cells exposed to 1 μmol/L CD437 for 24 and 48 hours, respectively.

Fig. 8.

CD437-mediated apoptosis in primary leukemia cells. Leukemic blasts were isolated as described in the Materials and Methods. Cells were exposed to 1 μmol/L CD437 for various periods of time and PARP cleavage was assessed as previously described. (A) Leukemic cells exposed only to vehicle for 48 hours. (B) Leukemic cells exposed to 1 μmol/L CD437 for 48 hours. (C) CD437-mediated PARP cleavage. Lanes 1 and 3, cells exposed to vehicle only for 24 and 48 hours, respectively. Lanes 2 and 4, cells exposed to 1 μmol/L CD437 for 24 and 48 hours, respectively.

CD437 induction of p21WAF1/CIP1 and GADD 45 mRNA expression.Cell cycle progression is regulated through a number of checkpoints. p21, also referred to as p21waf1/cipI and SDI,45-48 forms a quaternary complex with cyclins, cyclin-dependent kinases, and the proliferating cell nuclear antigen,48 resulting in their inhibition and cessation of cell cycle progression.48 The addition of CD437 to breast carcinoma cells results in the rapid increase in p21waf1/cip1 in a p53-independent manner.14 We therefore examined whether CD437 also modulated p21waf1/cip1 in these cells. As shown in Fig 9A, the addition of CD437 resulted in a rapid increase in p21waf1/cip1 mRNA expression. A 10-fold increase in p21waf1/cip1 was noted within 30 minutes of exposure to CD437, with a maximum increase of 80-fold noted at 2 hours (Fig 9B). This elevation in p21waf1/cip1 occurs significantly before the onset of apoptosis. Because HL-60R cells are p53 null,49 CD437 modulates p21waf1/cip1 in a p53-independent fashion. CD437 modulation of p21waf1/cip1 mRNA expression was also examined in K562 cells (Fig 10A). A twofold increase in p21 mRNA expression was noted at 24 hours after the addition of CD437 and significantly before the onset of apoptosis (Fig 10B).

Fig. 9.

CD437-enhanced p21WAF1/CIP1 mRNA expression in HL-60R cells. Exponentially growing HL-60R cells were exposed to 1 μmol/L CD437 for varying periods of time. Cells were harvested and Northern blots were performed as described in the Materials and Methods. The intensity of each band was scanned using laser densitometry and values were normalized with respect to 18S mRNA signals. (A) A representative Northern blot from two separate determinations. (B) Quantification of a representative experiment.

Fig. 9.

CD437-enhanced p21WAF1/CIP1 mRNA expression in HL-60R cells. Exponentially growing HL-60R cells were exposed to 1 μmol/L CD437 for varying periods of time. Cells were harvested and Northern blots were performed as described in the Materials and Methods. The intensity of each band was scanned using laser densitometry and values were normalized with respect to 18S mRNA signals. (A) A representative Northern blot from two separate determinations. (B) Quantification of a representative experiment.

Fig. 10.

CD437-enhanced p21WAF1/CIP1 mRNA expression in K562 cells. Exponentially growing HL-60R cells were exposed to 1 μmol/L CD437 for varying periods of time. Cells were harvested and Northern blots were performed as described in the Materials and Methods. The intensity of each band was scanned using laser densitometry and the values were normalized with respect to 18S mRNA signals. (A) A representative Northern blot from two separate determinations. (B) Quantification of a representative experiment.

Fig. 10.

CD437-enhanced p21WAF1/CIP1 mRNA expression in K562 cells. Exponentially growing HL-60R cells were exposed to 1 μmol/L CD437 for varying periods of time. Cells were harvested and Northern blots were performed as described in the Materials and Methods. The intensity of each band was scanned using laser densitometry and the values were normalized with respect to 18S mRNA signals. (A) A representative Northern blot from two separate determinations. (B) Quantification of a representative experiment.

The GADD (growth arrest and DNA damage inducible) genes are also noted to be induced by a number of DNA-damaging agents as well as by agents inducting growth arrest (Kersey et al50 and Zhan et al51 and the references contained within). GADD 45 mRNA expression is markedly enhanced after growth arrest (Kersey et al50 and Zhan et al51 and the references contained within). Exposure of HL-60R cells to CD437 also resulted in a rapid (within 1 hour) increase in GADD 45 mRNA expression (Fig 11A and B) before the onset of apoptosis. CD437 modulation of GADD 45 mRNA expression was not noted in K562 cells (data not shown).

Fig. 11.

CD437-enhanced GADD 45 mRNA expression. Exponentially growing HL-60R cells were exposed to 1 μmol/L CD437 for varying periods of time. Cells were harvested and Northern blots were performed as described in the Materials and Methods. The intensity of each band was scanned using a laser densitometer and values were normalized with respect to 18S mRNA signals. (A) A representative Northern blot assay from two separate determinations. (B) Quantification of a representative experiment.

Fig. 11.

CD437-enhanced GADD 45 mRNA expression. Exponentially growing HL-60R cells were exposed to 1 μmol/L CD437 for varying periods of time. Cells were harvested and Northern blots were performed as described in the Materials and Methods. The intensity of each band was scanned using a laser densitometer and values were normalized with respect to 18S mRNA signals. (A) A representative Northern blot assay from two separate determinations. (B) Quantification of a representative experiment.

DISCUSSION

The differentiation of the HL-60 leukemia cell line and acute promyelocytic leukemia cell lines exposed to RA in vitro has been documented by a number of investigators (Chomienne et al52 and the references contained within). The recent observation that the vast majority of patients with acute promyelocytic leukemia achieve a complete remission upon treatment with RA has generated a great deal of enthusiasm and interest in the use of retinoids as a treatment for this disease.53-55 This interest has been further accentuated by the two observations that the RA-mediated remission is not associated with aplasia but a continual differentiation of the leukemic cells into mature granulocytes and that the leukemic blasts possess a unique PML-RARα gene fusion as a result of a 15-17 translocation.55,56 Numerous mechanisms have been proposed by which PML-RARα expression interferes with normal myeloid differentiation and by which ligand binding to the PML-RARα fusion product results in the progression of normal differentiation.57-59 Unfortunately, the other subtypes of acute myelogenous leukemia (AML) are refractory to RA-mediated differentiation both in vivo as well as in vitro.60 In addition, the RA-induced remission in patients with acute promyelocytic leukemia is only transient, with a mean duration of approximately 6 months.61 The observation that CD437 is able to induce apoptosis in a number of RA-sensitive and -resistant leukemia cell lines would suggest that CD437 may be used therapeutically in the treatment of this disease.

CD437 induces apoptosis and not differentiation in the leukemic cells, as evidenced by the lack of acquisition by the cells of those phenotypic makers associated with myeloid maturation (data not shown). That CD437 rapidly induced apoptosis in these cells was indicated by multiple parameters. Incubation of the cells with CD437 resulted in the rapid cleavage of CPP32 and PARP, the onset of morphologic changes indicative of apoptosis, and internucleosomal degradation of the DNA. These markers of apoptosis are rapidly induced in these cells by CD437 with CPP32 and PARP cleavage noted within 2 hours. In addition, the time course associated with CD437-mediated apoptosis in these cells is significantly different from RA-mediated differentiation of HL-60 cells, in which no evidence of apoptosis is noted within the first 24 hours of exposure to RA.62 The mechanism by which CD437 induces apoptosis in the leukemic cells is unclear. A large number of intermediaries have been implicated in the signalling of the apoptotic process. These intermediaries have included tyrosine kinases, steroid receptors, ceramides, inositol phosphates, and a variety of cytokine receptors (Vaux and Strasser37 and the references contained within). Perhaps the best described have been the ceramide and FAS pathways. Whether any of these pathways are involved in CD437-mediated apoptosis remains to be defined. However, it is clear that signalling through the retinoid nuclear receptor pathways does not appear to be involved because, as indicated previously, HL-60R cells do not express RARβ or RARγ, as indicated by RNase protection assay, and express a truncated RARα receptor. The HL-60R truncated RARα possesses a 14-fold higher kd in terms of RA binding and 70% reduction in receptor number, rendering it inactive to RA-mediated antiproliferation and differentiation.16,17,63 CD437 possesses a 300-fold increase in kd in terms of binding to the wild-type RARα and a 70-fold increase in the required concentration for 50% maximal transactivation of the wild-type RARα nuclear receptor when compared with RA.15 Thus, it appears very unlikely that CD437 would bind and transactivate the truncated RARα receptor found in HL-60R cells that cannot be activated by RA.

CD437 rapidly induces p21waf1/cip1 expression in HL-60R cells. Increased p21waf1/cip1 expression is noted within 30 minutes, significantly before the onset of apoptosis. We have previously shown that CD437 rapidly induces p21waf1/cip1 expression in breast carcinoma cells through a p53-independent process.14 In addition, p21waf1/cip1 expression occurs through the unique method of stabilization of message.64 Akaski et al65 have previously documented a similar mechanism in irradiation-induced p21waf1/cip1 message in KG-1 leukemic cells. We have previously shown that the CD437 increased expression of p21waf1/cip1 in breast carcinoma cells is responsible for the observed G1 cell cycle arrest that occurs significantly before apoptosis.14 We have found that fibroblasts lacking p21waf1/cip1 will not undergo G1 arrest upon exposure to CD437, whereas fibroblast possessing p21waf1/cip1 readily undergo G1 cell cycle arrest; both cell types undergo apoptosis (unpublished data). Whether the CD437-mediated increase in p21waf1/cip1 results in G1 arrest in HL-60R cells is presently being determined. Numerous studies have examined the role of p21waf1/cip1 in the apoptotic process.62,66-68 The majority of studies have suggested that p21waf1/cip1 plays no role in p53-mediated apoptosis.62,66,68 Deng et al66 have found that thymocytes lacking p21waf1/cip1 still undergo apoptosis after irradiation. However, controversy still exists regarding the role of p21waf1/cip1 in apoptosis since Sheikh et al67 found that high expression of p21waf1/cip1 in breast cancer cells results in apoptosis. We also noted a rapid increase in GADD45 mRNA expression HL-60R cells after incubation with CD437. GADD45 mRNA expression is enhanced by a large variety of stimuli, including hypoxia, X irradiation, and genotoxic drugs (Kearsey et al69 and the references contained within). Modulation of GADD45 massage can occur through both p53-dependent and -independent pathways as well as stabilization of message (Kearsey et al69 and the references contained within). The function of GADD45 remains unclear. Overexpression of GADD45 has resulted in growth arrest in some cells and apoptosis in others (Kearsey et al69 and Zhan et al70 and the references contained within). Whether the CD437-mediated increased expression of GADD45 plays a role in CD437-mediated apoptosis remains to be defined.

Chemotherapy has been highly effective in inducing remission in acute myelogenous leukemia.71 Unfortunately, despite intensive chemotherapy, the vast majority of patients still relapse and succumb to their disease.71 Therefore, new drugs with different mechanisms of actions need to be developed that can be used alone or in conjunction with chemotherapy to treat this disease. RA has certainly proven to be a powerful tool in the treatment of acute promyelocytic leukemia.52-54 Whether CD437 will similarly prove to be therapeutically useful in the treatment of other forms of leukemia remains to be determined.

Supported in part by the Medical Research Services of the Department of Veterans Affairs (J.A.F.) and National Institutes of Health Grants No. CA63335 (J.A.F.) and CA51993 (M.I.D.).

Address reprint requests to Joseph A. Fontana, MD, PhD, 655 W Baltimore St, Bressler Research Bldg, Room 9-031, Baltimore, MD 21201.

REFERENCES

REFERENCES
1
Sporn
MB
Roberts
AB
Interaction of retinoids and transforming growth factor-B in the regulation of cell differentiation and proliferation.
Mol Endocrinology
5
1991
37
2
Moon RC, Itri LM: Retinoids and Cancer, in Sporn MB, Roberts AB, Goodman DS (eds): The Retinoids, vol 2. New York, NY, Academic, 1980, p 327
3
Lotan
R
Effects of vitamin A and its analogs (retinoids) on normal and neoplastic cells.
Biochem Biophys Acta
605
1980
33
4
Breitman
TR
Selonick
SE
Collins
SJ
Induction of differentiation of the human promeylocytic leukemia cell line (HL-60) by retinoic acid.
Proc Natl Acad Sci USA
77
1980
2936
5
Fontana
JA
Interaction of retinoids and tanoxifen in the inhibition of human mammory carcinoma cell proliferation.
Exp Cell Biol
55
1987
136
6
Van der Burg
B
Van der Leede
BJM
Kwakkenbos
Isbrucker L
Salverda
S
de Laat
SW
Van der Saag
PT
Retinoic acid resistance of estradiol-independent breast cancer cells coincides with diminished retinoic acid receptor function.
Mol Cell Endocrinol
91
1993
149
7
Roman
SD
Ormandy
CJ
Manning
DL
Blamey
RW
Nicholson
RI
Sutherland
RL
Clarke
CL
Estradiol induction of retinoic acid receptors in human breast cancer cells.
Cancer Res
53
1993
5970
8
Shiekh
MS
Shao
Z-M
Chen
JC
Hussain
A
Jetten
AM
Fontana
JA
Estrogen receptor-negative breast cancer cells transfected with the estrogen receptor exhibit increased RARα gene expression and sensitivity to growth inhibition by retinoic acid.
J Cell Biochem
53
1993
394
9
Giguere
V
Retinoic acid receptors and cellular retinoid binding proteins: Complex interplay in retinoid signalling.
Endocrine Rev
15
1994
61
10
Gudas
L
Retinoids and vertebrae development.
J Biol Chem
269
1994
15399
11
Mangelsdorf
DJ
Evans
RM
The RXR heterodimers and orphan receptors.
Cell
83
1995
841
12
Schule
R
Rangarajan
P
Yang
N
Kliewer
S
Ransone
LJ
Bolado
J
Verma
IM
Evans
RM
Retinoic acid is a negative regulator of AP-1 responsive genes.
Proc Natl Acad Sci USA
88
1991
6092
13
Kamei
Y
Xu
L
Heinzel
T
Torchia
J
Kurokawa
R
Gloss
B
Lin
S-C
Heyman
RA
Rose
DW
Glass
CK
Rosenfeld
MG
A CBP Integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors.
Cell
85
1996
403
14
Shao
Z-M
Dawson
MI
Li
X-S
Rishi
A-K
Shiekh
MS
Han
Q-X
Ordonez
JV
Shroot
B
Fontana
JA
p53 independent G0 /G1 arrest and apoptosis induced by a novel retinoid in human breast cancer cells.
Oncogene
11
1995
493
15
Bernard
BA
Bernardon
JM
Delesclose
C
Martin
B
Lenoir
MC
Maignan
J
Charpentier
B
Pilgrim
WR
Reichert
U
Shroot
B
Identification of synthetic retinoids with selectivity for human nuclear retinoic acid receptor γ.
Biochem Biophys Res Commun
186
1992
977
16
Robertson
KA
Emami
B
Collins
SJ
Retinoic acid resistant HL-60R cells harbor a point mutation in the retinoic acid receptor ligand-binding domain that confers dominant negative activity.
Blood
80
1992
1881
17
Nagy
L
Thomazy
VA
Shipley
GL
Fesus
L
Lamph
W
Heyman
RA
Chandraratina
RAS
Davies
PJA
Acitvation of retinoid X receptors induces apoptosis in HL-60 cell lines.
Mol Cell Biol
15
1995
3570
18
Kaufmann
SH
Desnoyers
S
Ottavino
Y
Davidson
NE
Poirer
GG
Specific proteolytic cleavage of poly (ADP-ribose) polymerase: An early marker of chemotherapy-induced apoptosis.
Cancer Res
53
1993
3976
19
Charpentier
B
Bernardon
JM
Eustache
J
Millois
C
Martin
B
Michel
S
Shroot
B
Syntheisis, structure affinity relationships and biological activities of ligands binding to retinoic acid receptor subtyes.
J Med Chem
38
1995
4993
20
El-Diery
WS
Tokino
T
Vielculescu
VE
Levy
DB
Parsons
R
Trent
JM
Lin
D
Mercer
WE
Kinzler
KW
Vogelstein
B
WAF1 a potential mediator of p53 tumor suppression.
Cell
75
1993
817
21
Zhan
Q
Bae
I
Kastan
MB
Fornace
AJ Jr
The p53 dependent γ ray response of GADD45.
Cancer Res
54
1994
2755
22
Laborda
J
36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein.
Nucleic Acids Res
19
1991
3998
23
Feinberg
AP
Vogelstein
B
A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity.
Anal Biochem
132
1983
6
24
Chomczynski
P
Sacchi
N
Single-step method of RNA isolation by acid quanidinium thiocyanate-phenol chloroform extraction.
Anal Biochem
162
1987
156
25
Li
X-S
Chen
JC
Sheikh
MS
Shao
ZM
Fontana
JA
Retinoic acid inhibition of insulin-like growth factor I stimulation of C-Fos mRNA levels in a breast carcinoma cell line.
Exp Cell Res
211
1994
68
26
Sheikh
MS
Li
X-S
Chen
JC
Shao
ZM
Ordonez
JV
Fontana
JA
Mechanism of regulation of WAF1/Cip1 gene expression in human breast carcinoma. Role of p53-dependent and independent signal transduction pathways.
Oncogene
9
1994
3407
27
Whitacre
CM
Hashimato
H
Tsai
ML Chatterjee S
Berger
SJ
Berger
NA
Involvement of NAD poly (ADP-ribose) metabolism in p53 regulation and its consequences.
Cancer Res
55
1995
3697
28
Robertson
KA
Mueller
J
Collins
SJ
Retinoic acid receptors in myeloid leukemia. Characterization of receptors in retinoic acid-resistant K-562 cells.
Blood
77
1991
340
29
Issacs
JT
Advances and controversies in the study of programmed cell death/apoptosis in the development of and therapy for cancer.
Curr Opin Oncol
6
1994
82
30
Yuan
J
Shaham
S
Ledoux
S
Ellis
HM
Horvitz
HR
The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1β-converting enzyme.
Cell
75
1993
641
31
Kuman
S
Kinoshita
M
Noda
M
Copeland
NG
Jenkins
NA
Induction of apoptosis by the mouse Nedd 2 gene which encodes a protein similar to the product of the Caenor habditis elegans cell death gene ced-3 and the mammalian IL-1β-converting enzyme.
Genes Dev
8
1994
1613
32
Wang
L
Miura
M
Bergerson
L
Zhu
H
Yuan
J
Ich-1 an Ice/ced-3 related gene encodes both positive and negative regulators of programmed cell death.
Cell
78
1994
739
33
Faucheu
C
Diu
A
Chan
AWE
Blanchet
AM
Miossec
C
Herve
F
Collard-Dutilloul
V
Guy
Y
Aldape
RA
Lippke
JA
Rocher
C
Su
MS-S
Livingston
DJ
Hercend
T
Lalanne
JL
A novel human protease similar to the interleukin-1β converting enzyme induces apoptosis in transfected cells.
EMBO J
14
1995
1917
34
Kamens
J
Paskind
M
Huguinin
M
Talanian
RV
Allen
H
Banach
D
Bump
N
Hackett
M
Johnston
CG
Li
P
Mankouitch
JA
Terranova
M
Ghayur
T
Identification and characterization of Ich-2, a novel member of the interleukin-1β converting enzyme family of cysteine proteases.
J Biol Chem
270
1995
15250
35
Munday
NA
Vaillancort
JP
Ali
A
Casano
FJ
Miller
DK
Molineau
SM
Yamin
TT
Yu
VL
Nicholson
DW
Molecular cloning and pro-apoptotic activity of ICE rel-II and ICE rel III, members of the ICE/CED-3 family of cysteine proteases.
J Bio Chem
270
1995
15250
36
Fernandes-Alnermiri
T
Litwack
G
Alnemri
ES
Mch 2a new member of the apoptotic Ced-3/ICE cysteine protease gene family.
Cancer Res
55
1995
2737
37
Vaux
DL
Strasser
A
The molecular biology of apoptosis.
Proc Natl Acad Sci USA
93
1996
2239
38
Lazebnik
YA
Kaufman
SH
Desnoyers
S
Poirier
GG
Earnshaw
WC
Cleavage of poly (ADP-ribose)polymerase by a proteinase with properties like ICE.
Nature
371
1994
346
39
Neamati
N
Fernandez
A
Wright
S
Kiefer
J
McConkey
DJ
Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei.
J Immunol
154
1994
1693
40
Nicholson
DW
Ali
A
Thornberry
NA
Vaillancort
JJP
Ding
CK
Gallant
M
Gareau
Y
Griffin
PR
Labelle
M
Lazebnik
YA
Munday
NA
Raju
SM
Smulson
ME
Yamin
TT
Yu
VL
Miller
DK
Identification and inhibition of the ICE/ced-3 protease necessary for mammalian apoptosis.
Nature
376
1995
37
41
Oberhammer
FA
Hochegger
K
Froschi
G
Tiefenbacher
R
Pauelka
M
Chromatin condensation during apoptosis is accompanied by degradation of lamin A + B without enhanced activation of cdc2 kinase.
J Cell Biol
126
1994
827
42
Tewari
M
Quan
LT
O'Rourke
K
Desnoyers
S
Zeng
Z
Beidler
DR
Poirer
GG
Salvesen
GS
Dixit
VM
Yama/CPP32 B. A mammalian homolog of CED-3 is a CRM A-inhibitable protease that cleaves the death substrate poly (ADP-Ribose) polymerase.
Cell
81
1995
801
43
Han
Z
Chatterjee
D
Early
J
Pantazis
P
Herdrickson
EA
Wyche
JH
Isolation and characterization of an apoptosis-resistant variant of human leukemia HL-60 cells that has switched expression from bcl-2 to bcl-XL.
Cancer Res
56
1996
1621
44
Haldar
S
Jena
H
Croce
CM
Inactivation of bcl-2 by phosphorylation.
Proc Natl Acad Sci USA
92
1995
4507
45
El-Deiry
W
Harper
JW
O'Connor
PM
Velculescu
VE
Canman
CE
Jackman
J
Peitenpol
JA
Burrell
M
Hill
DE
Wang
Y
Wiman
KG
Mercer
WE
Kastan
MB
Kohn
KW
Elledge
SJ
Kinszler
KW
Vogelstein
B
WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis.
Cancer Res
54
1994
1169
46
Harper
JW
Adami
GR
Wei
N
Keyomarsi
K
Elledge
SJ
The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases.
Cell
75
1993
805
47
Xiong
Y
Hannon
GJ
Zhang
H
Casso
D
Kobayashi
R
Beach
D
P21 is a universal inhibitor of cyclin kinases.
Nature
366
1993
701
48
Macleod
KF
Sherry
N
Hannon
G
Beach
D
Torino
T
Kinzler
K
Vogelstein
B
Jacks
T
p53-dependent and independent expression of p21 during cell growth differentiation and DNA damage.
Genes Dev
9
1995
935
49
Kastan
MB
Radin
AI
Kuergitz
SJ
Onyekwere
O
Walkow
CA
Civin
CI
Stone
KD
Woo
T
Ravindravath
Y
Craig
RW
Levels of p53 protein increase with maturation in human hematopoietic cells.
Cancer Res
51
1991
4279
50
Kersey
JM
Coates
PJ
Prescott
AR
Warbrick
E
Hall
PA
Gadd45 is a nuclear cell cycle regulated protein which interacts with p21cip1.
Oncogene
11
1995
1675
51
Zhan
Q
Lord
KA
Alano
I Jr
Hallander
MC
Carrier
R
Ron
D
Kohn
KW
Hoffman
B
Liebermann
DA
Fornace
AJ Jr
The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth.
Mol Cell Biol
14
1994
2361
52
Chomienne
C
Ballerini
P
Balitrand
N
Daniel
MT
Fenaux
P
Castaigne
S
Degos
L
All-trans retinoic acid in acute promyelocytic leukemias II. In vitro studies: Structure-function relationship.
Blood
76
1990
1710
53
Huang
ME
Chen
Y
Shu-rong C
Jin-ren C
Jia-Xiang
L
Long-Jun
G
Zhen-yi W
Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia.
Blood
72
1988
567
54
Castaigne
S
Chomienne
C
Daniel
MT
Ballerini
P
Berger
R
Fenaux
P
Degos
L
All trans-retinoic acid as a differentiation therapy for acute promyelocytic leukemia. I. Clinical Results.
Blood
76
1990
1707
55
Kakizuka
A
Miller
WH
Umesono
K
Warrell
RP Jr
Frankel
SR
Murty
VVVS
Dimitrovsky
E
Evans
RM
Chromosomal translocation t(15:17) in human acute promeylocytic leukemia fuses RARα with a novel putative transcription factor, PML.
Cell
66
1991
663
56
deThe
H
Lavau
C
Marchio
A
Chomienne
C
Degos
L
Dejean
A
The PML-RARα fusion mRNA generated by the t(15:17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR.
Cell
66
1991
675
57
Weis
K
Rambaud
S
Levau
C
Jansend
J
Carvalho
T
Carmo-Fonseca
M
Lamond
A
Dejean
A
Retinoic acid regulates aberrant nuclear localization of PML-RARα in acute promyelocytic leukemia cells.
Cell
76
1994
345
58
Dyck
JA
Maul
GG
Miller
WH Jr
Chen
D
Kakizuka
A
Evans
RM
A novel macromolecular structure is a target of the promyelocyte-retinoic acid receptor oncoprotein.
Cell
76
1994
333
59
Doucas
V
Brockes
JP
Yaniv
M
deThe
H
DeJean
A
The PML-retinoic acid receptor α translocation converts the receptor from an inhibitor to a retinoic acid dependent activator of transcription factor AP-1.
Proc Natl Acad Sci USA
90
1993
9345
60
Smith
MA
Parkinson
DR
Cheson
BD
Friedman
MA
Retinoids in cancer therapy.
J Clin Oncol
10
1992
7839
61
Degos
L
Dombert
H
Chomienne
C
Daniek
MT
Miclea
JM
Chastang
C
All trans-retinoic acid as a differentiating agent in the treatment of acute promyelocytic leukemia.
Blood
85
1995
2673
62
Steinman
RA
Hoffman
B
Iro
A
Guillouf
Lieberman DA
EL-Houseini
ME
Induction of p21(WAF1-CIP1) during differentiation.
Oncogene
9
1994
3389
63
Robertson
KA
Emami
B
Mueller
L
Collins
SJ
Multiple members of the retinoic acid receptor family are capable of mediating granulocytic differentiation of HL-60 cells.
Mol Cell Biol
12
1992
3473
64
Li
X-S
Rishi
AK
Shao
ZM
Dawson
MI
Jong
L
Shroot
B
Reichert
U
Ordenez
J
Fontana
JA
Post transcriptional regulation of p21waf1/cip1 expression in human breast carcinoma cells.
Cancer Res
56
1996
5055
65
Akaski
M
Hachiya
M
Osawa
Y
Spirin
K
Suzudi
G
Koeffler
HP
Irradiation induces WAF1 expression through a p53-independent pathway in KG-1 cells.
J Biol Chem
270
1995
19181
66
Deng
C
Zhang
P
Harper
JW
Elledge
S
Leder
P
Mice lacking p21waf1/cip1 undergo normal development but are defective in G1 checkpoint control.
Cell
82
1995
675
67
Sheikh
MS
Rochefort
H
Garcia
M
Overexpression of p21waf1/cip1 induces growth arrest, giant cell formation and apoptosis in human breast carcinoma cell lines.
Oncogene
11
1995
1899
68
Caducei
D
Wersto
R
Cowan
KH
Seth
P
Effects of a recombinant adenovirus expressing WAF1/CIP1 on cell growth, cell cycle and apoptosis.
Cell Growth Diff
6
1995
1207
69
Kearsey
JM
Coates
PJ
Prescott
AR
Warbrick
E
Hall
PA
Gadd45, is a nuclear cell cycle regulated protein which interacts with p21cip1.
Oncogene
11
1995
1675
70
Zhan
Q
Lord
KA
Alamo
I Jr
Hollander
MC
Carrier
R
Ron
D
Kohn
KW
Hoffman
B
Lieberman
DA
Fornace
AJ Jr
The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth.
Mol Cell Biol
4
1994
2361
71
Mayer
RJ
Davis
RB
Schiffer
CA
Bery
DT
Powell
BL
Schulman
P
Frei
E III
Comparative evaluation of intensive post-remission therapy with different dose schedules of Ara C in adults with acute myeloid leukemia (AML). Initial results of a CALGB phase III study.
Proc Am Soc Clin Oncol
11
1992
261