WO2001015722A2 - Methods useful in affecting apoptosis - Google Patents

Methods useful in affecting apoptosis Download PDF

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WO2001015722A2
WO2001015722A2 PCT/US2000/040738 US0040738W WO0115722A2 WO 2001015722 A2 WO2001015722 A2 WO 2001015722A2 US 0040738 W US0040738 W US 0040738W WO 0115722 A2 WO0115722 A2 WO 0115722A2
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acid glycoprotein
geranylgeraniol
agp
cell
apoptosis
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PCT/US2000/040738
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French (fr)
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WO2001015722A3 (en
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Glenn D. Prestwich
Li Feng
Hubert Wojtasek
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University Of Utah Research Foundation
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Publication of WO2001015722A3 publication Critical patent/WO2001015722A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates to the field of molecular biology, in particular to apoptosis.
  • compositions and methods useful to alter apoptosis which compositions and methods enable modification of pathological conditions, such as cancer.
  • Apoptosis a signal-dependent mode of programmed cell death in multicellular organisms, is crucial for (i) the remodeling of tissues in embryonic development, (ii) clonal selection of specifically reactive populations of lymphocytes, (iii) elimination of damaged cells during hematopoiesis, and (iv) normal tissue turnover. Absence of apoptotic death is often associated with the transformation from a normal to a malignant phenotype.
  • Apoptosis is characterized by morphological and biochemical changes in the cell: shrinkage of cell volume, condensation of chromatin, fragmentation of DNA, and a relatively high degree of preservation of plasma membrane and cytoplasmic organelles.
  • Cells undergoing apoptosis are fragmented into apoptotic bodies, which are then phagocytosed and degraded by neighboring cells and macrophages.
  • the roles of specific genes and changes in intracellular signals have provided considerable insight on the mechanisms of apoptosis (1- 4).
  • caspase-1 ICE/Ced-3
  • CPP32 caspase-3
  • apoptotic signals are complex and dependent on cell-types and growth states.
  • extracellular macromolecular factors or physical stressors e.g., tumor necrosis factor ⁇ (TNF- ⁇ ), Fas ligand, UV and ⁇ -irradiation
  • TNF- ⁇ tumor necrosis factor ⁇
  • Fas ligand Fas ligand
  • UV and ⁇ -irradiation a number of lipids have been identified as potent inducers of apoptosis.
  • Ceramide and sphingosine (6,7) mediate apoptosis initiated by TNF- ⁇ , Fas ligand, and X-ray irradiation (8).
  • retinoids (9,10), farnesol
  • Isoprenoids are essential compounds for cell proliferation and differentiation. Post- translational prenylation of nuclear lamins, G-proteins and small GTP-binding proteins is mediated by farnesyl protein transferase and geranylgeranyl protein transferase. Interestingly, the inhibition of isoprenoid synthesis (24-26) blocks cell growth and induces apoptosis.
  • GGA can be produced from GGOH in rat liver, and is envisaged to be derived from pyrophosphatase-mediated hydrolysis of geranylgeranyl pyrophosphate or by degradation of isoprenylated proteins (27).
  • the present invention provides methods to inhibit isoprenoid-induced apoptosis of at least one cell, comprising introducing at least one ⁇ l-acid glycoprotein to an isoprenoid-containing cellular milieu.
  • said cell is a human cell.
  • said ⁇ l- acid glycoprotein is a human ⁇ l-acid glycoprotein.
  • Preferred are those methods, wherein said ⁇ l-acid glycoprotein is a 50 kDa serum protein capable of binding geranylgeraniol.
  • isoprenoid comprises at least three isoprene units are preferred, with those wherein said isoprenoid comprises four isoprene units being more preferred.
  • said ⁇ l-acid glycoprotein is a human ⁇ l-acid glycoprotein.
  • methods to inhibit geranylgeraniol-induced hypodiploid cell proliferation comprising introducing at least one ⁇ l-acid glycoprotein to a geranylgeraniol-containing cellular milieu.
  • there are provided methods wherein said ⁇ l-acid glycoprotein is a human ⁇ l-acid glycoprotein.
  • ⁇ l-acid glycoprotein is a human ⁇ l-acid glycoprotein.
  • Isoprenoid is meant to refer to any naturally-occurring isoprenoid, or a functional analogue thereof, including synthetic compounds described in the present disclosure.
  • FIG. 1 Photoaffinity labeling of FBS (top panels) and human serum (bottom panels) by [ 3 H]EBDA. Lanes 1-8 (left, Coomassie-blue stained) and l'-8' (fluorogram) of FBS (top panels) and human serum (bottom panels) by [ 3 H]EBDA. Lanes 1-8 (left, Coomassie-blue stained) and l'-8' (fluorogram) of FBS (top panels) and human serum (bottom panels) by [ 3 H]EBDA. Lanes 1-8 (left, Coomassie-blue stained) and l'-8' (fluorogram) of
  • Panel A IEF purified p50 was labeled by [ H]EBDA with GGOH as a competitor at 0, 10, 30, 50, 100, 200, 300, 500 and 1000-fold molar excess.
  • Panel B IEF purified p50 was labeled by
  • FIG 4 Photoaffinity labeling of p50 by photolabile benzophenone analogs of GGOH. IEF purified p50 was labeled by [ 3 H]EBDA (overexposed), lm, lp, 2m, and 2p respectively. Left: Coomassie-blue stained gel; right: fluorogram.
  • Figure 5 conserveed sequences of p50 identify it as bovine AGP. Amino acid sequences of the six Edman-degradation sequenced p50 fragments were aligned with AGPs from three other species. conserveed amino acids are highlighted in red.
  • FIG. 6 AGP inhibits GGOH-induced activation of caspase 3.
  • HL-60 cells were incubated for 1 h in the presence of increasing amounts of GGOH (30, 40, 50, 60, and 70 ⁇ M) in either the absence (open circles O) or presence ( closed circles it) of 40 ⁇ M AGP.
  • Caspase activity was measured by the generation of fluorescent cleavage products. All values were normalized to protein concentration in samples, and represent averages of three independent measurements; error bars show standard deviation, while absence of error bars indicates that the standard deviation is less than 5 units.
  • Figure 7 AGP protects cells from GGOH-induced apoptosis.
  • HL-60 cells were incubated for 3 h in the presence of increasing amounts of GGOH (50, 60, and 70 ⁇ M) in either the absence (A) or presence (B) of 30 ⁇ M AGP.
  • the induction of apoptosis was measured at the creation of a hypodiploid population of cells.
  • the DNA content of the cells was determined by propidium iodide staining and by FACS analysis.
  • the percentage values in each of the histograms indicate the fraction of the cells that contain a Sub-Gl DNA content. The estimate was generated using ModFit Software, version 2.0.
  • FIG. 8 Model for the role of AGP in modulating apoptosis.
  • GGOH is released from dying cells when GGPP, a key precursor in sterol biogenesis and in geranylgeranylation of signaling proteins, is hydrolyzed.
  • AGP may bind and sequester GGOH, thereby protecting neighboring cells from this apoptotic signal originating from cells undergoing programmed or necrotic cell death.
  • the present invention provides methods to inhibit isoprenoid-induced apoptosis of at least one cell, comprising introducing at least one ⁇ l-acid glycoprotein to an isoprenoid-containing cellular milieu.
  • said cell is a human cell.
  • said ⁇ l- acid glycoprotein is a human ⁇ l-acid glycoprotein.
  • Preferred are those methods, wherein said ⁇ l-acid glycoprotein is a 50 kDa serum protein capable of binding geranylgeraniol.
  • isoprenoid comprises at least three isoprene units are preferred, with those wherein said isoprenoid comprises four isoprene units being more preferred.
  • said ⁇ l-acid glycoprotein is a human ⁇ l-acid glycoprotein.
  • methods to inhibit geranylgeraniol-induced hypodiploid cell proliferation comprising introducing at least one ⁇ l-acid glycoprotein to a geranylgeraniol-containing cellular milieu.
  • there are provided methods wherein said ⁇ l-acid glycoprotein is a human ⁇ l-acid glycoprotein.
  • said ⁇ l-acid glycoprotein is a human ⁇ l-acid glycoprotein.
  • AGP isoprenoid binding protein
  • AGP is a serum protein, which is synthesized mainly by the liver and is also found in lymphocytes. The level of AGP rises dramatically with inflammation, trauma, and HIV infection. In addition, as an acute-phase protein, AGP reaches peak serum levels in mice 12-48 h after turpentine or IL-1 injection, and can confer resistance to Gram negative infections such as Klebsiella pneumoniae (41). AGP binds a variety of administered drugs and is a target protein in pharmacokinetic studies of drug clearance (42). Although AGP has been reported to be involved in immune responses, the exact biological function of AGP is unknown.
  • AGP has high affinity, selective binding to isoprenoids, with GGOH as the most likely candidate for the principal endogenous ligand.
  • AGP will bind isoprenoids with three isoprene units (sesquiterpenes), but it preferentially binds the diterpenes, which have four isoprene units.
  • This interaction is highly AGP-specific.
  • [3H]EBDA we found that the active form of AGP seemed quite labile. Repeated freezing and thawing reduced the selectivity between AGP and BSA labeling by [3HJEBDA .
  • AGP was structurally similar to serum retinol binding protein and its relatives (43).
  • the strong binding of isoprenoids by AGP is consistent with its well-characterized ability to bind lipophilic drugs (44,45) and may be a reflection of its true biological role.
  • AGP and ⁇ l-antitrypsin are reported to protect mice from tumor necrosis factor (TNF)-induced death (46-48).
  • TNF tumor necrosis factor
  • antitrypsin is conveyed by specifically inhibiting the TNF-induced apoptosis of hepatocytes in vivo (49). The inhibition is thought to be indirect because AGP and antitrypsin did not protect hepatoma cell-lines from apoptosis in vitro.
  • the inventor's findings further support the proposition that AGP functions to protect cells from apoptosis.
  • GGOH and GGA are known apoptosis inducers.
  • Our data demonstrate that AGP binds these apoptosis mediators, and that binding appears to inhibit the induction of apoptosis (Figure 6 and 7).
  • the location of AGP in serum would seem to limit its potential interaction with intracellular isoprenols ligands.
  • intracellular isoprenols in vivo.
  • intracellular FOH levels increase and FOH is secreted (50-52).
  • There is currently no evidence for the existence of extracellular GGOH and GGA but it is plausible for one to speculate that an analogous mechanism exists.
  • the intracellular isoprenols may also be released when a cell becomes fragmented during necrosis or as proteins and or co-factors are degraded during apoptosis.
  • the free isoprenols could induce unregulated apoptosis in neighboring cells.
  • AGP may function to protect the neighboring cells from excess GGOH secreted by pre-apoptotic cells, or by escaping from dying cells.
  • FIG. 8 A working model to explain the function of AGP as an important regulator of apoptosis is shown in Figure 8. Initially, extracellular signals induce apoptosis or injury produces cellular necrosis. Intracellular isoprenols produced from hydrolysis of FPP or
  • GGPP escape to the surrounding cells.
  • the isoprenols In the absence of a protective sesquestrant, the isoprenols have the potential to initiate a localized apoptotic cascade.
  • apoptosis is contained by binding these apoptotic extracellular lipid signals and preventing the unregulated spread of apoptotic signals.
  • the radioactive JH I analog f H]EBDA was synthesized (28,29) by Dr. I. Ujvary (The University at Stony Brook, Stony Brook, NY) and the GGOH analogs were prepared as described (30,31). Sera were purchased from Life Technologies (Gaithersburg, MD). N-terminal sequencing was performed by Dr. R. Schackmann (The University of Utah, Salt
  • Fetal bovine serum (FBS, 450 ml) was freed of paniculate matter using a disposable filter (Corning, 115 ml, 0.45 ⁇ m) and dialyzed overnight (Spectra por, 3000 MWCO) against buffer A (20 mM phosphate buffer, pH 7.1).
  • buffer A (20 mM phosphate buffer, pH 7.1).
  • BSA bovine serum albumin
  • the fractions that contained p50 were pooled, lyophilized, and resuspended in 300 ⁇ l of gel filtration buffer (50 mM phosphate buffer, 150 mM sodium chloride, pH 7.0). It was then purified on a gel filtration column (Tosohaas TSK 3000 SW) and the fractions were monitored by UV and liquid scintillation counting (LSC).
  • gel filtration buffer 50 mM phosphate buffer, 150 mM sodium chloride, pH 7.0
  • the wet PVDF membrane was rinsed several times with TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) and blocked with 5% nonfat milk in TBST for 1.5 h. The membrane was then incubated with an anti-AGP polyclonal antibody (1: 1000 dilution in TBST) for 1 h. The PVDF membrane was washed with TBST three times (10 min each). Alkaline phosphatase (AP) coupled with anti rabbit-IgG was then incubated with the membrane for 1 h and washed with TBS three times (10 min each). The membrane was finally developed using an AP conjugate substrate kit and protocol (Bio- Rad).
  • TBST 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20
  • the bands of p50 were cut from the PVDF membrane and minced into small pieces.
  • 50 ⁇ l of digestion buffer 1% reduced Triton X-100, 10% acetonitrile, 100 mM Tris-HCl, pH 8.0
  • the mixture was vortexed for 10-20 sec and incubated for 30 min at it.
  • Proteolytic enzyme Lys C (enzyme/substrate, 1:10) was added and the reaction was incubated for 24 h at 37 oC.
  • the mixture was then vortexed for 5-10 sec, sonicated for 5 min and centrifuged (Fisher Scientific) for 2 min at 4,000 rpm. The supernatant was stored.
  • the cell-line, HL-60 was maintained in suspension culture in RPMI 1640 (GibcoBRL Rockville, MD) supplemented with 10% FBS (Hyclone, Logan, UT), 100 units penicillin (Sigma), 100 ⁇ g/ml streptomycin (Sigma), 200 ⁇ M glutamate (GibcoBRL), and 5% CO2.
  • HL-60 cells were obtained in cells collected by centrifugation (100 x g) for 5 min at it. Media was aspirated and cells were resuspended in RPMI 1640 (GibcoBRL), supplemented with 1% BSA (Sigma, cat #A2153), 100 units penicillin (Sigma), 100 ⁇ g/ml streptomycin (Sigma), and 200 ⁇ M glutamate (GibcoBRL) with or without 1.5mg/ml AGP (Sigma, cat. #G9885) at 37 °C.
  • Cells were collected in 1.5 ml microcentrifuge tubes by centrifugation (100 x g) for 5 min at it, and the media was aspirated. Cells were fixed and permeabilized overnight in 1 ml of MeOH at -20°C . Fixed cells were centrifuged (100 x g) for 5 min at it and the MeOH was removed by aspiration. Cells were resuspended in PBS with 5 ⁇ g/ml propidium iodide and the DNA content of cells was determined by fluorescence-activated cell sorting using a Beckton Dickson FACS Scan instrument operated with CellQuest Software. The fraction of the cell population that was hypodiploid and apoptotic was estimated using ModFitLT V2.0 (Win32).
  • Caspase 3 Activity Assay Cell samples were prepared as described above for the DNA fragmention assays. The exceptions were that AGP was added to the resuspending media at 2.0 mg/ml, and the cells were incubated in the 24-well plate for 1 h at 37 °C, 5% CO2. After the incubation cells were collected in 1.5 ml microcentrifuge tubes by centrifugation (100 x g) for 5 min at it and the media was aspirated. The cell pellet was washed once with 1 ml PBS at 4 °C. The PBS was aspirated and cells were resuspended in cold Caspase Activity Buffer (CAB), 25mM HEPES, 5 mM EDTA, 0.1% CHAPS, pH 7.5,
  • CAB Cold Caspase Activity Buffer
  • Spectrofluorimeter with excitation at 360 ⁇ 40 nm and emission detection at 515 ⁇ 40 nm. Samples were measured in triplicate and normalized by protein concentration. Protein concentrations were determined using a BCA protein assay (Pierce, Rockford, IL) following precipitation in 10% trichloroacetic acid.
  • bovine p50 To identify bovine p50, a large scale protein purification and photoaffinity labeling was performed. FBS (450 ml) was dialyzed and filtered to remove particulate matter, and then purified by Affi-Gel blue gel chromatography by elution with 1.5 M NaSCN, 20 mM phosphate. The flow-through and the eluted fractions were tested by photoaffinity labeling with [3H]EBDA. The flow-through contained all of the p50 (data not shown), and was concentrated by ultrafiltration and further purified by IEF. Each fraction from the IEF was analyzed for pH and subjected to further dialysis.
  • the p50 fractions were photoaffinity labeled with [ 3 H]EBDA and further purified by RP-HPLC and gel filtration. Peak fractions from the gel filtration purification were identified by LSC and fluorography. RP-HPLC, but not gel filtration, provided a substantial increase in purity. In order to obtain protein sequence data, the peaks were separated by 8% SDS-PAGE and transferred to a PVDF membrane. Edman degradation failed to provide any sequence for the intact p50, suggesting a blocked N-terminus. In order to get internal sequence information, p50 was digested by Lys C directly on the PVDF membrane. The Lys C digests of p50 were applied to an RP C18 column and six single-peak fractions were sequenced.
  • GGOH is capable of inducing apoptosis in a variety of cell-lines (12,14,21,37-39). We therefore tested whether the association of AGP with GGOH altered the apoptosis- inducing activity of GGOH. The two most common cellular characteristics associated with
  • GGOH-induced apoptosis are DNA fragmentation and caspase 3 activation (12,14,21,37- 39).
  • DNA fragmentation ( Figure 6) was consistent with induced apoptosis.
  • the addition of GGOH to the cells created a large population of hypodiploid cells (39).
  • the inclusion of AGP during incubation with GGOH substantially decreased the ability of GGOH to induce the hypodiploid cell population.
  • the inclusion of AGP inhibited GGOH-induced caspase 3 activation ( Figure 7). While caspase 3 could be activated in the presence of AGP, activation required significantly higher doses of GGOH.

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Abstract

The present invention relates to the field of molecular biology, in particular to apoptosis. The inventors herein disclose compositions and methods useful to alter apoptosis, which compositions and methods enable modification of pathological conditions, such as cancer.

Description

Methods Useful in Affecting Apoptosis
This work was supported by grants to G.D.P. from the National Institutes of Health (Grant GM 44836), the U.S. Government may have certain rights in the invention.
This application claims priority to U. Provisional Patent Application
Serial Number 60/151,032, filed August 27, 1999.
FIELD OF THE INVENTION
The present invention relates to the field of molecular biology, in particular to apoptosis.
The inventors herein disclose compositions and methods useful to alter apoptosis, which compositions and methods enable modification of pathological conditions, such as cancer.
BACKGROUND OF THE INVENTION
Apoptosis, a signal-dependent mode of programmed cell death in multicellular organisms, is crucial for (i) the remodeling of tissues in embryonic development, (ii) clonal selection of specifically reactive populations of lymphocytes, (iii) elimination of damaged cells during hematopoiesis, and (iv) normal tissue turnover. Absence of apoptotic death is often associated with the transformation from a normal to a malignant phenotype.
Apoptosis is characterized by morphological and biochemical changes in the cell: shrinkage of cell volume, condensation of chromatin, fragmentation of DNA, and a relatively high degree of preservation of plasma membrane and cytoplasmic organelles. Cells undergoing apoptosis are fragmented into apoptotic bodies, which are then phagocytosed and degraded by neighboring cells and macrophages. The roles of specific genes and changes in intracellular signals have provided considerable insight on the mechanisms of apoptosis (1- 4). In particular, several members of a cysteine protease family including caspase-1 (ICE/Ced-3) and caspase-3 (CPP32) have been implicated in the apoptotic process (5). The signal transduction and determination processes initiated by apoptotic signals are complex and dependent on cell-types and growth states. In addition to a variety of extracellular macromolecular factors or physical stressors (e.g., tumor necrosis factor α (TNF-α), Fas ligand, UV and γ-irradiation) a number of lipids have been identified as potent inducers of apoptosis. Ceramide and sphingosine (6,7) mediate apoptosis initiated by TNF-α, Fas ligand, and X-ray irradiation (8). Recently, a new family of isoprenoid apoptotic inducers has been described: retinoids (9,10), farnesol
(FOH) (11-13), geranylgeraniol (GGOH) (12,14), geranylgeranoic acid (GGA) (15,16), dolichyl phosphate (17,18), and other polyprenol monophosphates (19). The discovery of these endogenous lipid inducers is important in understanding the apoptotic signal transduction pathway. Substantial progress has been made toward understanding the mechanism of isoprenoid-induced apoptosis. Consistent with known apoptotic events, mitochondrial membrane potential is rapidly lost during GGA-induced apoptosis (20), and dihydroheptaprenyl, dihydrodecaprenyl monophosphate (19), and GGOH-induced caspase- 3-like activity (21). Additionally, it has been observed that inhibition of geranylgeranylation promotes apoptosis in pulmonary vascular smooth muscle (22) and that activation of protein kinase C is required to induce apoptosis with GGOH in human HL-60 cells (23).
Isoprenoids are essential compounds for cell proliferation and differentiation. Post- translational prenylation of nuclear lamins, G-proteins and small GTP-binding proteins is mediated by farnesyl protein transferase and geranylgeranyl protein transferase. Interestingly, the inhibition of isoprenoid synthesis (24-26) blocks cell growth and induces apoptosis. GGA can be produced from GGOH in rat liver, and is envisaged to be derived from pyrophosphatase-mediated hydrolysis of geranylgeranyl pyrophosphate or by degradation of isoprenylated proteins (27).
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. For example, in some instances above, the publication was less than one year before the filing date of this patent application. All statements as to the date or representation as to the contents of these documents is based on subjective characterization of information available to the applicant at the time of filing, and does not constitute an admission as to the accuracy of the dates or contents of these documents.
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Pitt, B., Hamilton, A. D., Sebti, S. M., and Davies, P. (1998) Am. J. Physiol. 275, L55-63 23. Masuda, Y., Yoda, M., Ohizumi, H., Aiuchi, T., Watabe, M., Nakajo, S., and Nakaya, K. (1997) Int. J. Cancer 71, 691-697 24. Jones, K. D., Couldwell, W. T., Hinton, D. R., Su, Y., He, S., Anker, L., and
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SUMMARY OF THE INVENTION
The present invention provides methods to inhibit isoprenoid-induced apoptosis of at least one cell, comprising introducing at least one αl-acid glycoprotein to an isoprenoid-containing cellular milieu. In one such embodiment, there are provided methods, wherein said cell is a human cell. Also provided are methods, wherein said αl- acid glycoprotein is a human αl-acid glycoprotein. Preferred are those methods, wherein said αl-acid glycoprotein is a 50 kDa serum protein capable of binding geranylgeraniol. Methods wherein isoprenoid comprises at least three isoprene units are preferred, with those wherein said isoprenoid comprises four isoprene units being more preferred.
In particular, those methods wherein said isoprenoid is selected from the group consisting of farnesol, geranylgeraniol, and geranylgeranoic acid are provided, with geranylgeraniol being most preferred. Methods as described, wherein said αl-acid glycoprotein is a 50kDa human serum protein are more preferred, preferrably those which further comprises clusterin as an adjuvant.
Also provided are methods to inhibit geranylgeraniol-induced DNA fragmentation of at least one cell, comprising introducing at least one αl-acid glycoprotein to a geranylgeraniol-containing cellular milieu. In one such embodiment, there are provided methods wherein said αl-acid glycoprotein is a human αl-acid glycoprotein. Also provided are methods to inhibit geranylgeraniol-induced hypodiploid cell proliferation, comprising introducing at least one α l-acid glycoprotein to a geranylgeraniol-containing cellular milieu. In one such embodiment, there are provided methods wherein said αl-acid glycoprotein is a human αl-acid glycoprotein.
Also provided are methods to inhibit geranylgeraniol-induced caspase 3 activation, comprising introducing at least one αl-acid glycoprotein to a geranylgeraniol-containing cellular milieu. In one such embodiment, there are provided methods wherein said αl-acid glycoprotein is a human αl-acid glycoprotein. Definitions:
For the purposes of the present application, the following terms have the following meanings. All other terms have the meaning as generally recognized in the art.
"Isoprenoid" is meant to refer to any naturally-occurring isoprenoid, or a functional analogue thereof, including synthetic compounds described in the present disclosure.
Brief Description of the Figures
Figure 1 Potential ligands of p50 and their photolabile analogs. Legend: T = 3H or
1H; X = CH2OH (GGOH) or CO2H (GGA).
Figure 2 Photoaffinity labeling of FBS (top panels) and human serum (bottom panels) by [3H]EBDA. Lanes 1-8 (left, Coomassie-blue stained) and l'-8' (fluorogram) of
FBS labeled by [3H]EBDA competed by no competitor (1,1'), and with competitors JHI (2,2'), methyl farnesoate (3, 3'), farnesol (4,4') , linoleic acid (5,5'), methylprene (6,6'), retinol (7,7') and cholesterol (8,8'), respectively. Lanes a - c (Coomassie-blue) and a1 - c' (fluorogram). Lanes a and a1 show FBS labeled by [3H]EBDA for comparison; human serum labeled by [3H]EBDA in the absence (b,b') and presence (c,c') of excess JH I.
Figure 3 Concentration-dependent displacement of by [3H]EBDA labeling of p50 by
GGOH and GGA. Left: Coomassie-blue stained gels; right, fluorograms. Panel A: IEF purified p50 was labeled by [ H]EBDA with GGOH as a competitor at 0, 10, 30, 50, 100, 200, 300, 500 and 1000-fold molar excess. Panel B: IEF purified p50 was labeled by
[3HJEBDA with GGA as a competitor at 0, 10, 30, 50, 100, 200, 300, 1000-fold molar excess.
Figure 4 Photoaffinity labeling of p50 by photolabile benzophenone analogs of GGOH. IEF purified p50 was labeled by [3H]EBDA (overexposed), lm, lp, 2m, and 2p respectively. Left: Coomassie-blue stained gel; right: fluorogram. Figure 5 Conserved sequences of p50 identify it as bovine AGP. Amino acid sequences of the six Edman-degradation sequenced p50 fragments were aligned with AGPs from three other species. Conserved amino acids are highlighted in red.
Figure 6 AGP inhibits GGOH-induced activation of caspase 3. HL-60 cells were incubated for 1 h in the presence of increasing amounts of GGOH (30, 40, 50, 60, and 70 μM) in either the absence (open circles O) or presence ( closed circles it) of 40 μM AGP. Caspase activity was measured by the generation of fluorescent cleavage products. All values were normalized to protein concentration in samples, and represent averages of three independent measurements; error bars show standard deviation, while absence of error bars indicates that the standard deviation is less than 5 units.
Figure 7 AGP protects cells from GGOH-induced apoptosis. HL-60 cells were incubated for 3 h in the presence of increasing amounts of GGOH (50, 60, and 70 μM) in either the absence (A) or presence (B) of 30 μM AGP. The induction of apoptosis was measured at the creation of a hypodiploid population of cells. The DNA content of the cells was determined by propidium iodide staining and by FACS analysis. The percentage values in each of the histograms indicate the fraction of the cells that contain a Sub-Gl DNA content. The estimate was generated using ModFit Software, version 2.0.
Figure 8 Model for the role of AGP in modulating apoptosis. GGOH is released from dying cells when GGPP, a key precursor in sterol biogenesis and in geranylgeranylation of signaling proteins, is hydrolyzed. AGP may bind and sequester GGOH, thereby protecting neighboring cells from this apoptotic signal originating from cells undergoing programmed or necrotic cell death.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods to inhibit isoprenoid-induced apoptosis of at least one cell, comprising introducing at least one αl-acid glycoprotein to an isoprenoid-containing cellular milieu. In one such embodiment, there are provided methods, wherein said cell is a human cell. Also provided are methods, wherein said αl- acid glycoprotein is a human αl-acid glycoprotein. Preferred are those methods, wherein said αl-acid glycoprotein is a 50 kDa serum protein capable of binding geranylgeraniol. Methods wherein isoprenoid comprises at least three isoprene units are preferred, with those wherein said isoprenoid comprises four isoprene units being more preferred.
In particular, those methods wherein said isoprenoid is selected from the group consisting of farnesol, geranylgeraniol, and geranylgeranoic acid are provided, with geranylgeraniol being most preferred. Methods as described, wherein said αl-acid glycoprotein is a 50kDa human serum protein are more preferred, preferrably those which further comprises clusterin as an adjuvant.
Also provided are methods to inhibit geranylgeraniol-induced DNA fragmentation of at least one cell, comprising introducing at least one αl-acid glycoprotein to a geranylgeraniol-containing cellular milieu. In one such embodiment, there are provided methods wherein said αl-acid glycoprotein is a human αl-acid glycoprotein. Also provided are methods to inhibit geranylgeraniol-induced hypodiploid cell proliferation, comprising introducing at least one αl-acid glycoprotein to a geranylgeraniol-containing cellular milieu. In one such embodiment, there are provided methods wherein said αl-acid glycoprotein is a human αl-acid glycoprotein.
Also provided are methods to inhibit geranylgeraniol-induced caspase 3 activation, comprising introducing at least one αl-acid glycoprotein to a geranylgeraniol-containing cellular milieu. In one such embodiment, there are provided methods wherein said αl-acid glycoprotein is a human αl-acid glycoprotein. During production of a recombinant insect JH I receptor (35), the inventors discovered a new isoprenoid binding protein, AGP, in vertebrate serum. Isoprenoids, particularly FOH and GGOH, are degradation products of the essential protein prenylation precursors FPP and GGPP, which are required to maintain normal cell function. The recent discovery that FOH, GGOH and GGA can induce apoptosis further emphasizes their importance in cell physiology. Several mechanisms have been proposed regarding isoprenoid-induced apoptosis. One hypothesis is that GGOH or GGA compete with GGPP and inhibit prenylation and could thereby indirectly induce apoptosis. Inhibition of protein prenylation has been reported to induce apoptosis (22). The other possibility is that GGA and GGOH are intracellular messengers themselves and directly stimulate apoptosis (40).
AGP is a serum protein, which is synthesized mainly by the liver and is also found in lymphocytes. The level of AGP rises dramatically with inflammation, trauma, and HIV infection. In addition, as an acute-phase protein, AGP reaches peak serum levels in mice 12-48 h after turpentine or IL-1 injection, and can confer resistance to Gram negative infections such as Klebsiella pneumoniae (41). AGP binds a variety of administered drugs and is a target protein in pharmacokinetic studies of drug clearance (42). Although AGP has been reported to be involved in immune responses, the exact biological function of AGP is unknown.
The data presented herein demonstrate that AGP has high affinity, selective binding to isoprenoids, with GGOH as the most likely candidate for the principal endogenous ligand. AGP will bind isoprenoids with three isoprene units (sesquiterpenes), but it preferentially binds the diterpenes, which have four isoprene units. This interaction is highly AGP-specific. Even at undetectable levels of AGP (by Coomassie-Blue staining) and in the presence of over 1000-fold excess of the nonspecific lipid binding protein BSA, we can still detect specific photoaffinity labeling AGP by [3H]EBDA. Importantly, we found that the active form of AGP seemed quite labile. Repeated freezing and thawing reduced the selectivity between AGP and BSA labeling by [3HJEBDA .
The specific binding between AGP and the JH I analog suggested a possible biological function. Homology studies revealed that AGP was structurally similar to serum retinol binding protein and its relatives (43). The strong binding of isoprenoids by AGP is consistent with its well-characterized ability to bind lipophilic drugs (44,45) and may be a reflection of its true biological role. AGP and αl-antitrypsin are reported to protect mice from tumor necrosis factor (TNF)-induced death (46-48). The protection by AGP and antitrypsin is conveyed by specifically inhibiting the TNF-induced apoptosis of hepatocytes in vivo (49). The inhibition is thought to be indirect because AGP and antitrypsin did not protect hepatoma cell-lines from apoptosis in vitro.
The inventor's findings further support the proposition that AGP functions to protect cells from apoptosis. GGOH and GGA are known apoptosis inducers. Our data demonstrate that AGP binds these apoptosis mediators, and that binding appears to inhibit the induction of apoptosis (Figure 6 and 7). The location of AGP in serum would seem to limit its potential interaction with intracellular isoprenols ligands. However, there is evidence for production of extracellular isoprenols in vivo. Under certain circumstances, intracellular FOH levels increase and FOH is secreted (50-52). There is currently no evidence for the existence of extracellular GGOH and GGA, but it is tempting for one to speculate that an analogous mechanism exists. The intracellular isoprenols may also be released when a cell becomes fragmented during necrosis or as proteins and or co-factors are degraded during apoptosis. The free isoprenols could induce unregulated apoptosis in neighboring cells. AGP may function to protect the neighboring cells from excess GGOH secreted by pre-apoptotic cells, or by escaping from dying cells. There is also limited evidence for the existence of specific receptors for AGP (53). The investigation of a lectin- like AGP receptor and isoprenoid-bound AGP could give useful information on the potential implications for AGP in apoptosis.
A working model to explain the function of AGP as an important regulator of apoptosis is shown in Figure 8. Initially, extracellular signals induce apoptosis or injury produces cellular necrosis. Intracellular isoprenols produced from hydrolysis of FPP or
GGPP escape to the surrounding cells. In the absence of a protective sesquestrant, the isoprenols have the potential to initiate a localized apoptotic cascade. However, in the presence of AGP, apoptosis is contained by binding these apoptotic extracellular lipid signals and preventing the unregulated spread of apoptotic signals.
Examples
The radioactive JH I analog f H]EBDA was synthesized (28,29) by Dr. I. Ujvary (The University at Stony Brook, Stony Brook, NY) and the GGOH analogs were prepared as described (30,31). Sera were purchased from Life Technologies (Gaithersburg, MD). N-terminal sequencing was performed by Dr. R. Schackmann (The University of Utah, Salt
Lake City, UT). All solutions were made with nanopure water (ultrafiltered, distilled and deionized). Proteases (sequencing grade) were obtained from Boehringer Mannheim (Indianapolis, EN). En3Hance and XAR X-ray films were obtained from NEN-Dupont (Boston, MA). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). Affi-gel Blue gel column chromatography
Fetal bovine serum (FBS, 450 ml) was freed of paniculate matter using a disposable filter (Corning, 115 ml, 0.45 μm) and dialyzed overnight (Spectra por, 3000 MWCO) against buffer A (20 mM phosphate buffer, pH 7.1). Affi-gel Blue gel (Bio-Rad
Laboratories, Richmond, CA) chromatography was used to remove most of the bovine serum albumin (BSA). The column (3.0 cm x 45 cm) was first equilibrated with 400 ml of Buffer A; then, an aliquot (50 ml) of filtered, dialyzed FBS was pumped onto the column. Flow-through of 400 ml was collected. The column was then regenerated with 400 ml of Buffer B (1.5 M NaSCN, 20 mM phosphate buffer).
Isoelectric focusing
The flow-through from the Affi-gel Blue gel column was pooled and concentrated by ultrafiltration (Amicon, YM 3) to 300 ml. This partially-purified serum (50 ml) with 1.8% of Ampholytes (Bio-Rad, Biolyte 3/10) was further purified by isoelectric focusing (IEF,
Rotofor™, Bio-Rad). The focusing typically lasted for 4-6 h, with a final voltage of 1000 V (12 W fixed power). The fractions were harvested by house vacuum, analyzed for pH, and dialyzed against 10 mM Tris, pH 7.4 overnight.
HPLC purification of p50
Combined IEF fractions (3-ml aliquots), which had been labeled with 1.0 μCi of [3H]EBDA, were applied to a VYDAC semi-preparative C8 reversed-phase (RP) column on a Rainin Dynamix HPLC system (model SD-200). The proteins were eluted at a mean gradient over 1 h (eluent A: 0.1% TFA in water, eluent B: 70% acetonitrile, 0.085% TFA in water). The fractions were collected by an automatic collector and monitored by UV at 210 nm (model UV-D II). The fractions that contained p50 were pooled, lyophilized, and resuspended in 300 μl of gel filtration buffer (50 mM phosphate buffer, 150 mM sodium chloride, pH 7.0). It was then purified on a gel filtration column (Tosohaas TSK 3000 SW) and the fractions were monitored by UV and liquid scintillation counting (LSC).
Photoaffinity labeling(32.33
To a quartz tube, 1 μl of [3H]EBDA (0.38 μCi in heptane/toluene, 10:1, 58 Ci/mmol, 0.13 μM ) was added and rinsed in with 10 μl of hexane. The solvent was briefly removed by a SpeedVac concentrator (Savant Corporation, Holbrook, NY) and the radioactive probe was redissolved in 3 μl of ethanol. Total protein of 10-15 μg was used for labeling and buffer
(10 mM Tris, pH 7.4) was added to bring the total volume to 50 μl. The mixture was vigorously vortexed, incubated for 1 h at 4 oC and then exposed to UV (254 nm) for 25 sec. The covalently-modified proteins were then analyzed by SDS-PAGE and fluorography. For photoaffinity labeling by benzophenone-containing probes (34), 10-15 μg of total protein was mixed with 0.4 μCi of the radioactive probes (42.5 Ci/mmol, 0.20 μM) . The mixture was incubated at 4 oC for 15 min and then subjected to UV light (RMR-3600, 350 nm, 1900 μW/ci ) for 45 min at 4 oC.
Electrophoresis and western blot Electrophoresis, fluorography and electroblotting were carried outby standard protocols.
For western blot, the wet PVDF membrane was rinsed several times with TBST (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) and blocked with 5% nonfat milk in TBST for 1.5 h. The membrane was then incubated with an anti-AGP polyclonal antibody (1: 1000 dilution in TBST) for 1 h. The PVDF membrane was washed with TBST three times (10 min each). Alkaline phosphatase (AP) coupled with anti rabbit-IgG was then incubated with the membrane for 1 h and washed with TBS three times (10 min each). The membrane was finally developed using an AP conjugate substrate kit and protocol (Bio- Rad).
Digestion of p50 on PVDF membrane
The bands of p50 were cut from the PVDF membrane and minced into small pieces. In a 1.5 ml conical tube, 50 μl of digestion buffer (1% reduced Triton X-100, 10% acetonitrile, 100 mM Tris-HCl, pH 8.0) was added to the PVDF membrane. The mixture was vortexed for 10-20 sec and incubated for 30 min at it. Proteolytic enzyme Lys C (enzyme/substrate, 1:10) was added and the reaction was incubated for 24 h at 37 oC. The mixture was then vortexed for 5-10 sec, sonicated for 5 min and centrifuged (Fisher Scientific) for 2 min at 4,000 rpm. The supernatant was stored. Another 50 μl of digestion buffer was added and the vortexing, sonication, and centrifugation steps were repeated. The last wash of the membrane was performed using 0.1% TFA and again the supernatant was stored. The combined supernatant was injected onto an RP column C18 (VYDAC, 4.6 x 250 mm) on a Rainin Dynamix HPLC system and eluted in a gradient of acetonitrile (0-10 min, 0% B, 10- 70 min, 40% B, 70-100 min, 80% B, 100-115 min, 100% B, 115-120 min, 100% B, 120- 121 min, 0% B; eluent A: 0.06% TFA in water; eluent B: 0.056% TFA, 80% acetonitrile in water) at 0.6 ml/min.
Cell Culture
The cell-line, HL-60, was maintained in suspension culture in RPMI 1640 (GibcoBRL Rockville, MD) supplemented with 10% FBS (Hyclone, Logan, UT), 100 units penicillin (Sigma), 100 μg/ml streptomycin (Sigma), 200 μM glutamate (GibcoBRL), and 5% CO2.
Cell cultures were split to approximately 5 x 105 cells/ml 24 h prior to starting any experiment. Cell numbers and culture densities were determined with a hemocytometer, and cell viability identified by negative trypan blue (Sigma) staining at 0.2% v/v.
Apoptosis Assays
DNA fragmentation and FACS Scan Analysis: HL-60 cells were obtained in cells collected by centrifugation (100 x g) for 5 min at it. Media was aspirated and cells were resuspended in RPMI 1640 (GibcoBRL), supplemented with 1% BSA (Sigma, cat #A2153), 100 units penicillin (Sigma), 100 μg/ml streptomycin (Sigma), and 200 μM glutamate (GibcoBRL) with or without 1.5mg/ml AGP (Sigma, cat. #G9885) at 37 °C.
When AGP was present in the resuspending media, it was added to the media just prior to each use. Cells were resuspended to a concentration of 1 x 106 cells per ml. Aliquots (1- ml) of cell samples were transferred to each well of a 24- well tissue culture plate (Costar, Corning, NY). Next, stock solutions of GGOH (Echelon Research Laboratories Inc., Salt Lake City, UT) at 10 mM in EtOH were transferred to appropriate wells and mixed with cells by pipeting. Samples were incubated at 37 °C, 5% CO2 for 3 h. Cells were collected in 1.5 ml microcentrifuge tubes by centrifugation (100 x g) for 5 min at it, and the media was aspirated. Cells were fixed and permeabilized overnight in 1 ml of MeOH at -20°C . Fixed cells were centrifuged (100 x g) for 5 min at it and the MeOH was removed by aspiration. Cells were resuspended in PBS with 5 μg/ml propidium iodide and the DNA content of cells was determined by fluorescence-activated cell sorting using a Beckton Dickson FACS Scan instrument operated with CellQuest Software. The fraction of the cell population that was hypodiploid and apoptotic was estimated using ModFitLT V2.0 (Win32).
Caspase 3 Activity Assay: Cell samples were prepared as described above for the DNA fragmention assays. The exceptions were that AGP was added to the resuspending media at 2.0 mg/ml, and the cells were incubated in the 24-well plate for 1 h at 37 °C, 5% CO2. After the incubation cells were collected in 1.5 ml microcentrifuge tubes by centrifugation (100 x g) for 5 min at it and the media was aspirated. The cell pellet was washed once with 1 ml PBS at 4 °C. The PBS was aspirated and cells were resuspended in cold Caspase Activity Buffer (CAB), 25mM HEPES, 5 mM EDTA, 0.1% CHAPS, pH 7.5,
2mM DTT. Cells were bath-sonicated for 10 sec and snap frozen. For the enzyme assay, 45-μl aliquots of cell extract were transferred to a 96-well microtiter plate (Costar) on ice. Then, 5 μl of fluorogenic caspase 3 substrate DEVD (Peptide Institute, Inc., Osaka, Japan), freshly diluted 1:20 in CAB, was added to each sample. The microtiter plate was incubated at 37 °C for 1 h, and the fluorescence in each well was measured with a TECAN Polarion
Spectrofluorimeter with excitation at 360 ±40 nm and emission detection at 515 ±40 nm. Samples were measured in triplicate and normalized by protein concentration. Protein concentrations were determined using a BCA protein assay (Pierce, Rockford, IL) following precipitation in 10% trichloroacetic acid.
Results
Earlier studies of a recombinant 29 kDa juvenile hormone (JH) binding protein cloned from the caterpillar Manduca sexta included expression of the recombinant protein in baculovirus-infected Sf9 cells(35). We serendipitously discovered that [3H]EBDA (Figure 1), a photoaffinity analogue of JH I (32), also covalently modified a previously unidentified 50 kDa protein, called p50 (36). Surprisingly, the source of the p50 was neither the vector nor the parent insect cell expression system, but rather the FBS supplement employed in the cell culture. The labeling of p50 by [3H]EBDA was efficiently displaced by JH I (Figure 2), and partially displaced by 100- to 500-fold molar excess of the sesquiterpenoids farnesol (FOH), methoprene, and methyl farnesoate (32). Photoaffinity labeling of human serum (Figure 2) also showed specific labeling of a protein of similar molecular size, suggesting a human homolog of p50.
To identify bovine p50, a large scale protein purification and photoaffinity labeling was performed. FBS (450 ml) was dialyzed and filtered to remove particulate matter, and then purified by Affi-Gel blue gel chromatography by elution with 1.5 M NaSCN, 20 mM phosphate. The flow-through and the eluted fractions were tested by photoaffinity labeling with [3H]EBDA. The flow-through contained all of the p50 (data not shown), and was concentrated by ultrafiltration and further purified by IEF. Each fraction from the IEF was analyzed for pH and subjected to further dialysis. Ampholytes were removed by addition of solid NaCl to each sample to give a 2 M NaCl solution prior to dialysis overnight against buffer A. Protein concentration was measured and an aliquot of each fraction was photoaffinity labeled with [3HJEBDA. Peak fractions were combined and either applied to the competition studies or subjected to further purification. The photoaffinity labeling was essential at each stage of purification to preclude isolation of a co-eluting protein of similar size that was not the target of the photoactivatable probe.
Using the IEF-purified p50, competitive displacement experiments were performed to screen for potential endogenous ligands of p50. A number of terpenoids and fatty acids (geranylgeraniol, GGA, FOH, linalool, methyl farnesoate, linoleic acid, retinol and cholesterol) were examined as competitors for [3H]EBDA photoaffinity labeling. The linear diterpenes, GGOH and GGA were the best competitors (Figure 3). Using photoactivatable benzophenone analogs of FOH and GGOH (30) (lp, lm, 2p and 2m, Figure 4) we directly tested for binding of GGOH-like molecules to p50. Both GGOH analogs [3HJBZ-/.-C10-OH (2p) and [3H]BZ- -C10OH (2m) specifically labeled p50. Compound 2m, which best mimicked the extended form of GGOH, appeared to have a stronger association. The tritium-labeled benzophenone analogs of FOH (lp, lm) did not show detectable labeling of p50. In confirmatory cross-over experiments, unlabeled compounds 2p and 2m were also used as competitors in the labeling of p50 with [3H]EBDA. Again, compound 2m showed more potent competition than 2p (data not shown). In order to identify p50, we further purified the IEF peak fractions. The p50 fractions were photoaffinity labeled with [3H]EBDA and further purified by RP-HPLC and gel filtration. Peak fractions from the gel filtration purification were identified by LSC and fluorography. RP-HPLC, but not gel filtration, provided a substantial increase in purity. In order to obtain protein sequence data, the peaks were separated by 8% SDS-PAGE and transferred to a PVDF membrane. Edman degradation failed to provide any sequence for the intact p50, suggesting a blocked N-terminus. In order to get internal sequence information, p50 was digested by Lys C directly on the PVDF membrane. The Lys C digests of p50 were applied to an RP C18 column and six single-peak fractions were sequenced. The six sequence fragments corresponded well to conserved sequences with AGP (Figure 5). The full-length amino acid sequence of bovine AGP is not currently available. To determine whether the 50 kDa protein is indeed AGP, we performed a western blot. We found that anti-human AGP recognized bovine p50. Two additional results substantiate the identification of p50 as AGP. First, anti-AGP antagonized the labeling of p50 by [3H]EBDA. Second, both commercially obtained bovine and human AGP were effectively photoaffinity labeled by [3H]EBDA (data not shown).
GGOH is capable of inducing apoptosis in a variety of cell-lines (12,14,21,37-39). We therefore tested whether the association of AGP with GGOH altered the apoptosis- inducing activity of GGOH. The two most common cellular characteristics associated with
GGOH-induced apoptosis are DNA fragmentation and caspase 3 activation (12,14,21,37- 39). Thus, we examined HL-60 cells for these cellular responses in the presence and absence of AGP. First, DNA fragmentation (Figure 6) was consistent with induced apoptosis. The addition of GGOH to the cells created a large population of hypodiploid cells (39). The inclusion of AGP during incubation with GGOH substantially decreased the ability of GGOH to induce the hypodiploid cell population. Similarly, the inclusion of AGP inhibited GGOH-induced caspase 3 activation (Figure 7). While caspase 3 could be activated in the presence of AGP, activation required significantly higher doses of GGOH. Taken together, these data demonstrate that the binding of AGP to GGOH reduced GGOH- induced apoptosis.
Although the present invention has been fully described herein, it is to be noted that various changes and modifications are apparent to those skilled in the art. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:
I . A method to inhibit isoprenoid-induced apoptosis of at least one cell, comprising introducing at least one αl-acid glycoprotein to an isoprenoid- containing cellular milieu.
2. A method of claim 1, wherein said cell is a human cell.
3. A method of claim 1, wherein said αl-acid glycoprotein is a human αl- acid glycoprotein.
4. A method of claim 3, wherein said αl-acid glycoprotein is a 50 kDa serum protein capable of binding geranylgeraniol.
5. A method of claim 1, wherein isoprenoid comprises at least three isoprene units.
6. A method of claim 5, wherein said isoprenoid comprises four isoprene units.
7. A method of claim 5, wherein said isoprenoid is selected from the group consisting of farnesol, geranylgeraniol, and geranylgeranoic acid.
8. A method of claim 1 wherein said isoprenoid is geranylgeraniol.
9. A method of claim 8, wherein said αl-acid glycoprotein is a 50kDa human serum protein.
10. A method of claim 9, which further comprises clusterin as an adjuvant.
I I. A method to inhibit geranylgeraniol-induced DNA fragmentation of at least one cell, comprising introducing at least one αl-acid glycoprotein to a geranylgeraniol-containing cellular milieu.
12. A method of claim 11 , wherein said αl-acid glycoprotein is a human αl- acid glycoprotein.
13. A method to inhibit geranylgeraniol-induced hypodiploid cell proliferation, comprising introducing at least one αl-acid glycoprotein to a geranylgeraniol-containing cellular milieu.
14. A method of claim 13, wherein said αl-acid glycoprotein is a human αl- acid glycoprotein.
15. A method to inhibit geranylgeraniol-induced caspase 3 activation, comprising introducing at least one αl-acid glycoprotein to a geranylgeraniol-containing cellular milieu.
16. A method of claim 15, wherein said αl-acid glycoprotein is a human αl- acid glycoprotein.
PCT/US2000/040738 1999-08-27 2000-08-25 Methods useful in affecting apoptosis WO2001015722A2 (en)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
W. VAN MOLLE ET AL.: "Activation of caspases in lethal experimental hepatitis and prevention by acute phase proteins" J. IMMUNOL., vol. 163, 1999, pages 5235-5241, XP002164997 *
W. VAN MOLLE ET AL.: "Alpha1-acid glycoprotein and alpha1-antitrypsin inhibit TNF-induced but not anti-Fas inducedapoptosis of hepatocytes in mice" J. IMMUNOL., vol. 159, 1997, pages 3555-3564, XP002164996 cited in the application *

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