[go: up one dir, main page]

WO1999031135A1 - Proteines destinees a provoquer l'apoptose specifique de cellules cancereuses et procede pour isoler ces proteines - Google Patents

Proteines destinees a provoquer l'apoptose specifique de cellules cancereuses et procede pour isoler ces proteines Download PDF

Info

Publication number
WO1999031135A1
WO1999031135A1 PCT/US1998/027108 US9827108W WO9931135A1 WO 1999031135 A1 WO1999031135 A1 WO 1999031135A1 US 9827108 W US9827108 W US 9827108W WO 9931135 A1 WO9931135 A1 WO 9931135A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
apogen
protein
apoptosis
conditioned medium
Prior art date
Application number
PCT/US1998/027108
Other languages
English (en)
Inventor
David Tsai
Jenny Yu
Original Assignee
David Tsai
Jenny Yu
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by David Tsai, Jenny Yu filed Critical David Tsai
Priority to KR1020007006696A priority Critical patent/KR20010033260A/ko
Priority to AU19330/99A priority patent/AU1933099A/en
Priority to JP2000539058A priority patent/JP2002516821A/ja
Priority to CA002325368A priority patent/CA2325368A1/fr
Priority to IL13685098A priority patent/IL136850A0/xx
Priority to EP98964142A priority patent/EP1037919A1/fr
Priority to MXPA00005954A priority patent/MXPA00005954A/es
Publication of WO1999031135A1 publication Critical patent/WO1999031135A1/fr
Priority to NO20003099A priority patent/NO20003099L/no

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Apoptosis is also called “programmed cell death” or “cell suicide”.
  • Apoptosis is also called “programmed cell death” or “cell suicide”.
  • apoptosis is an active process of gene-directed, cellular self-destruction and that it serves a biologically meaningful function.
  • apoptosis is an active process of gene-directed, cellular self-destruction and that it serves a biologically meaningful function.
  • the cause or the result can be one of a number of diseases, including: cancer, viral infections, auto-immune disease/allergies, neurodegeneration or cardiovascular diseases.
  • apoptosis is becoming a prominent buzzword in the pharmaceutical research field. Huge amounts of time and money are being spent in an attempt to understand how it works, how it can be encouraged or Inhibited and what this means for practical medicine.
  • a handful of companies have been formed with the prime direction of turning work in this nascent field into marketable pharmaceutical products.
  • the emergence of a core of innovative young companies combined with the tentative steps being taken by established industrial players are certain to make apoptosis research one of the fastest-growing and most promising areas of medical study of the 1990s.
  • cancer may be caused by insufficient apoptosis merged only recently.
  • This idea opens a door for a new concept in cancer therapy — Cancer cells may be killed by encouraging apoptosis.
  • Apoptosis modulation based on the processes present in normal development, is a potential mechanism for controlling the growth of tumor cells. Restoring apoptosis in tumor cells is an attractive approach because, at least in theory, it would teach the cells to commit suicide.
  • Apogen P-l was isolated from the conditioned medium of a cell line called XC which was derived from rat tumor and is purchased from American Type Culture Collection (ATCC). XC cells were first grown in Dulbecco's Modification of Eagle's Medium (DMEM) containing 10 % Fetal bovine serum (FBS) for 3 days. XC cells were then washed with PBS (3X100 ml) to remove serum and then grown in DMEM containing no FBS for 4 days. From this serum free conditioned medium, we detected an activity inducing apoptosis in a prostate cancer cell line called LNCAP. On the other hand, normal human lung fibroblast cell line (CCD 39 Lu) and breast cancer cells (MCF-7) is not affected by this activity.
  • DMEM Dulbecco's Modification of Eagle's Medium
  • FBS Fetal bovine serum
  • the activity of the crude conditioned medium of XC cells was tested on the following cell lines: JEG-3 (Choriocarcinoma), G401 (Wilm's tumor) LNCAP (Prostate cancer), T84 (colon cancer), HL-60 (leukemia), breast cancer cells (MCF-7), and CCD 39 Lu (normal lung fibroblast).
  • JEG-3 Chocarcinoma
  • G401 Wang tumor
  • LNCAP Prostate cancer
  • T84 colon cancer
  • HL-60 leukemia
  • MCF-7 breast cancer cells
  • CCD 39 Lu normal lung fibroblast
  • Apoptosis is a distinct type of cell death that differs fundamentally from degenerative death or necrosis in its nature and biological significance.
  • a cell undergoing apoptosis is distinct from a cell undergoing necrosis both morphologically and biochemically.
  • the XC conditioned medium contains activity inducing apoptosis
  • LNCAP cells were incubated with control medium or the conditioned medium treated as described as above for 15 hr and then stained with Hoechst dye for 2 hours.
  • the nuclei of the LNCAP cells that have been incubated with control medium are normal and healthy (A).
  • the nuclei of the LNCAP cells that have been incubated with the conditioned medium (X20, exchanged to RPMI) shown the characteristic of apoptosis (Fig. 1 (B)).
  • the conditioned medium causes the condensation of nucleus, demonstrated by the more intense fluorescence light compared with the control nucleus in Fig. 1 (A).
  • the nucleus condensation is accompanied by the fragmentation of DNA, demonstrated by the breakage of nucleus as shown in Fig. 1 (B).
  • the nucleus condensation and DNA fragmentation are the morphological characteristic of cells under apoptosis.
  • the partially purified Apogen P-lb (Q2 anionic exchanger chromatography step) isolated as described below was recently found to contain an activity other than inducing apoptosis.
  • Apogen P-lb have the activity to repel cells away. This activity is opposite to that of growth factors; many growth factors such as Platelet Derived Growth Factor (PDGF), Epidermal Growth factor (EGF), Fibroblast Growth factor (FGF) or Transforming Growth factor (TGF) function as a "chemoattractant”— which means that these growth factors attract cells toward them.
  • PDGF Platelet Derived Growth Factor
  • EGF Epidermal Growth factor
  • FGF Fibroblast Growth factor
  • TGF Transforming Growth factor
  • Apogen P-lb isolated in this invention plays opposite biological functions as that of growth factors. For example, growth factors induce cell growth and attract cells, whereas Apogen P-lb induces cell death and repel cells. Apogen P-lb is the first "chemorepellent" found in the field of modern biology.
  • a tissue culture device called Transwell Insert purchased from Costar (Cambridge,
  • MA was used to discover the chemorepellent activity of Apogen P-lb.
  • This device which has been widely used for the studies of cell migration/invasion, contains an upper chamber and a lower chamber. Between these two chambers is a polyester microporous membrane with 3.0 um pore size which allows cell to migrate through the membrane. Tested cells are grown on the upper chamber and tested compound is placed in the lower chamber. If this tested compound is a chemoattractant, we should see more cells migrate through the membrane than the control sample.
  • Hep G2 (100,000 cells) cells which have cell size 3-4 times as big as the membrane pore size were grown in the upper chamber for 2 hours and then the partially purified Apogen-lb (30 ⁇ l) isolated by ammonium sulfate precipitation and Q2 HPLC chromatography as described above was placed in the lower chamber. After 15 hours, cells that have migrated through the membrane were collected by treating the membrane with 0.2 ml of trypsin solution for 30 rnin. Cells in ten microliters of the trypsin solution were counted in a Hemacytometer. In several experiments, we found that the partially purified Apogen-lb contained an activity decreasing the number of cells going through the membrane.
  • the Apogen P-l present in the conditioned medium was isolated by the following steps:
  • Step 1 Ammonium sulfate precipitation
  • Apogen P-l was precipitated by 80% saturated of ammonium sulfate by adding 561g of ammonium sulfate per liter of conditioned medium. Pellet was collected by centrifugation and the proteins were dissolved in 10 mM Tris-HCI (pH 7.4). After removal of ammonium sulfate by dialysis, the dissolved proteins were separated by a Q2 HPLC column.
  • the dissolved proteins isolated by ammonium sulfate precipitation were concentrated and loaded on to a Q2 column (Bio-Rad )which is further developed by a linear gradient constructed by buffer A (10 mM Tris-HCI, pH 7.4) and buffer B (1 0 mM Tris-HCI, pH 7.4)
  • Step 3 Reverse phase chromatography.
  • Apogen P-la, Apogen P-lb and Apogen P-lc were separately concentrated to 1.5 ml.
  • One ml of methanol containing 0.05% trifluoracetic acid was added.
  • large amount of proteins were precipitated by this treatment.
  • the apoptosis inducing activity remained in supernatant.
  • the supernatant was then applied to a reverse phase RP-4 column (Micra Scientific Inc) and developed by a linear gradient constructed by solution A (H2O, 0.05% TFA) and solution B ( Methanol, 0.05% TFA).
  • the linear gradient was constructed by increasing solution B from 0% to 100 % in solution A within 10 min (20 milliliter elution volume and thereafter the column was eluted with 100% solution B for 5 min.)
  • Apogen lc isolated by anion exchange chromatography was purified by both Reverse phase chromatography (step 3) and Preparative Electrophoresis by a MiniPrep Gel electrophoresis (Bio-Rad)
  • the reverse phase chromatogram of Apogen P- la is shown in Fig. 4(a). fractions 12-13 have activity inducing 80 % cell death in LNCAP cells at 10 hr.
  • the reverse phase chromatogram of Apogen P-lb is shown in Fig. 4(b). fractions 14 and 15 have activity inducing 45 % cell death in LNCAP cells at 18 hr.
  • the purity of the isolated Apogen P-la, Apogen P-lb and Apogen P-lc were checked with SDS-polyacrylamide gel electrophoresis stained with silver staining.
  • Apogen P-lc The purification of Apogen lc by Reverse Phase chromatography leads to the isolation of a 70 KD protein whereas the purification of Apogen- lc by preparative electrophoresis leads to the purification of a 57 KD protein. As shown in Fig.6(A), a major protein band with molecular weight of 70 KD was obtained by Reverse Phase chromatography. A 57 KD protein, on the other hand, was isolated by preparative electrophoresis. (Fig. 6B).
  • Apogen P-2 was isolated from the conditioned medium of a cell line called C3H 1OT1/2 which was derived from mouse embryo cells and is purchased from American Type Culture Collection (ATCC). C3H 1OT1/2 cells were first grown in alpha Modification of Eagle's Medium (alpha-MEM) containing 10 % Fetal bovine serum (FBS) for 3 days. Cells were then washed with PBS (3X1OO ml) to remove serum and then grown in alpha-MEM containing no FBS for 4 days. From this serum free conditioned medium, we detected an activity inducing apoptosis in a prostate cancer cell line called LNCAP. On the other hand, normal human lung fibroblast cell line (CCD 39 Lu) is not affected by this activity. (2) Activity of Apogen P-2
  • the nuclei of the LNCAP cells that have been incubated with control medium are normal and healthy(A).
  • the nuclei of the LNCAP cells that have been incubated with the conditioned medium shown the characteristic of apoptosis (Fig.7B).
  • the conditioned medium causes the condensation of nucleus, demonstrated by the more intense fluorescent light compared with the control nucleus in Fig.7A.
  • the nucleus condensation is accompanied by the fragmentation of DNA, demonstrated by the breakage of nucleus as shown in Fig.7B.
  • the nucleus condensation and DNA fragmentation are the morphological characteristic of cells under apoptosis.
  • C3H1OT1/2 cells contains an activity inducing apoptosis in LNCAP and MCF-7 cells.
  • the conditioned medium fails to induce apoptosis in normal human lung fibroblast (CCD 39 Lu cells).
  • CCD 39 Lu cells normal human lung fibroblast
  • Tested cells were grown on the upper chamber and tested compound (Apogen P-2) is placed in the lower chamber.
  • HL-60 100,000 cells
  • Apogen P-2 partially purified Apogen P-2 (30 ⁇ l) isolated by ammonium sulfate precipitation, hydroxylapatite and Heparin agarose as described above was placed in the lower chamber.
  • Apogen P-2 30 ⁇ l
  • cells that have migrated through the membrane were collected from the lower chamber, the medium in lower chamber (0.6 ml) was centrifuged for 10 min and the HL-60 cells that went through the membrane were collected and resuspended in 80 ⁇ l of PBS.
  • the Apogen P-2 present in the conditioned medium was isolated by the following steps:
  • Step 1 Ammonium sulfate precipitation
  • Apogen P-2 was precipitated by 80% saturated of ammonium sulfate by adding 561 g of ammonium sulfate per liter of conditioned medium. Pellet was collected by centrifugation and the proteins were dissolved in 10 mM Tris-HCI (pH 7.4). Step 2: Hydroxylapatite treatment.
  • step 4 Reverse phase chromatography.
  • Apogen P-2 presents in the supernatant of Heparin agarose in step 3 was further purified by a reverse phase chromatography. Apogen P-2 was concentrated to 1 ml. One milliliter of methanol containing 0.05% Trifluoracetic acid was added. Large amount of proteins were precipitated by this treatment. Whereas, the apoptosis inducing activity (P-2) remained in supernatant. The supernatant was then applied to a reverse phase RP-4 column
  • Apogen L was isolated from the conditioned medium of XC cell line purchased from American Type Culture Collection (ATCC). XC cells were grown in Dulbecco's Modification of Eagle's Medium (DMEM) containing 10 % Fetal bovine serum (FBS) for 4 days. From this conditioned medium, we detected an activity inducing apoptosis in a leukemia cell line called HL-60. On the other hand, normal human lung fibroblast cell line
  • Step 1 DE52 absorption
  • the conditioned medium was incubated with the anion exchanger, DE 52
  • Step 2 Heparin agarose absorption Apogen L isolated as described in step 1 was further absorbed by Heparin agarose
  • HL-60 leukemia
  • CCD 39 Lu normal lung fibroblast
  • Apogen L causes the condensation of nucleus, demonstrated by the more intense fluorescent light compared with the control nucleus in Fig. 13A.
  • the nucleus condensation is accompanied by the fragmentation of DNA, demonstrated by the breakage of nucleus as shown in Fig. 13B.
  • the nucleus condensation and DNA fragmentation are the two morphological characteristic of cells under apoptosis.
  • Fig. 1 Induction of apoptosis in prostate cancer cells (LNCAP) by the conditioned medium of XC cells.
  • LNCAP cells were incubated with control medium or the conditioned medium for 15 hr and then stained with Hoechst dye for 2 hours.
  • the nuclei of the LNCAP cells that have been incubated with control medium are normal and healthy (Fig. 1 A).
  • the nuclei of the LNCAP cells that have been incubated with the conditioned medium X20, exchanged to RPMI
  • Fig. 1 (B) the conditioned medium causes the condensation of nucleus, demonstrated by the more intense fluorescent light compared with the control nucleus in Fig. 1 (A).
  • the nucleus condensation is accompanied by the fragmentation of DNA, demonstrated by the breakage of nucleus as shown in Fig. I (B).
  • the nucleus condensation and DNA fragmentation are the two morphological characteristic of cells under apoptosis.
  • Fig. 2 The XC conditioned medium fails to induce apoptosis in normal lung fibroblast (CCD 39 Lu) cells.
  • CCD 39 Lu cells were incubated with control medium or the conditioned medium for 15 hr and then stained with Hoechst dye for 2 hours as described in Fig. 1. Cells looked normal and healthy; the nuclei of CCD 39 Lu cells remain the same with or without incubating with the conditioned medium of XC cells (Fig 2 (A) and Fig. 2 (B)). This results suggest that the XC conditioned medium fails to induce apoptosis in normal lung fibroblast (CCD 39 Lu) cells.
  • Fig. 3 Isolation of Apogen P-ls by Anion (Q2) exchange chromatography.
  • the dissolved proteins isolated by ammonium sulfate precipitation were concentrated and loaded onto a Q2 column (Bio Rad )which is further developed by a linear gradient constructed by buffer A (10 mM Tris-HCI, pH 7.4) and buffer B (10 mM Tris-HCI, pH 7.4. 0.55 M NaCI) using BioRad's BioLogic HPLC system.
  • the linear gradient was constructed by increasing buffer B from 0% to 100 % in buffer A within 10 min (20 milliliter elution volume and thereafter the column was eluted with 100% buffer B for 5 min.
  • the Apogen P-l activity was assayed by the induction of apoptosis in LNCAP cells.. We found that there are three activity peaks across the chromatogram profile.
  • Fig. 4 Isolation of Apogen P-l by Reverse Phase chromatography.
  • Apogen P-la, Apogen P-lb and Apogen P-lc were separately concentrated to 1.5 ml.
  • One milliliter of methanol containing 0.05% Trifluoracetic acid was added.
  • large amount of proteins were precipitated by this treatment.
  • the apoptosis inducing activity remained in supernatant.
  • the supernatant was then applied to a reverse phase RP-4 column (Micra Scientific Inc) and developed by a linear gradient constructed by solution A (H2O, 0.05% TFA) and solution B (Methanol, 0.05% TFA).
  • the linear gradient was constructed by increasing solution B from 0% to 100 % in solution A within 10 min (20 milliliter elution volume and thereafter the column was eluted with 100% solution B for 5 min.
  • the reverse phase chromatogram of Apogen P-lb is shown in Fig. 4(b). fractions 14- 15 have activity inducing 45 % cell death in LNCAP cells at 18 hr.
  • the reverse phase chromatogram of Apogen P-lc is shown in Fig. 4(c). fraction No. 5 have activity inducing 52 % cell death in LNCAP cells at 18 hr.
  • the purity of the isolated Apogen P-la, Apogen P-lb and Apogen P-lc were checked with SDS-polyacrylamide gel electrophoresis and stained with silver staining.
  • Fig. 5 Apogen la isolated by anion exchange chromatography and reverse phase chromatography was concentrated and subjected to electrophoresis under denaturing conditions through a 4-20% gradient Tris-Gly SDS-Polyacrylamide gel. The gel was silver stained. A major protein band with molecular weight of 70 KD was obtained. This result suggest the nearly successful purification of Apogen p-lc which have molecular weight of 70 KD on SDS-PAGE.
  • Fig. 6 Apogen lc isolated by anion exchange chromatography and reverse phase chromatography and preparative electrophoresis were concentrated and subjected to electrophoresis under non-denaturing conditions through a 10% resolving gel and 4% stacking gel on SDS Polyacrylamide electrophoresis.
  • Fig. 7 Induction of apoptosis in prostate cancer cells (LNCAP) by the conditioned medium of C3H 10T1/2 cells.
  • LNCAP cells were incubated with control medium or the conditioned medium for 15 hr and then stained with Hoechst dye for 2 hours.
  • the nuclei of the LNCAP cells that have been incubated with control medium are normal and healthy(A).
  • the nuclei of the LNCAP cells that have been incubated with the conditioned medium shown the characteristic of apoptosis (Fig.7(B)).
  • the conditioned medium causes the condensation of nucleus, demonstrated by the more intense fluorescent light compared with the control nucleus in Fig.
  • nucleus condensation is accompanied by the fragmentation of DNA, demonstrated by the breakage of nucleus as shown in Fig.7(B).
  • nucleus condensation and DNA fragmentation are the morphological characteristic of cells under apoptosis.
  • Fig. 8 The C3H 1OT1/2 conditioned medium fails to induce apoptosis in normal lung fibroblast (CCD 39 Lu) cells.
  • CCD 39 Lu cells were incubated with control medium or the conditioned medium for 15 hr and then stained with Hoechst dye for 2 hours as described in Fig. 1. Cells looked normal and healthy; the nuclei of CCD 39 Lu cells remain the same with or without incubating with the conditioned medium of C3H 1OT1/2 cells (Fig 8(A) and Fig. 8(B)). This results suggest that the C3H 1OT1/2 conditioned medium fails to induce apoptosis in normal lung fibroblast (CCD 39 Lu) cells.
  • Fig. 9 Reverse phase chromatography of Apogen P-2.
  • Apogen P-2 that has been purified by DE52 cellulose, hydroxylapatite and heparin agarose was further purified by a reverse phase chromatography.
  • Apogen P-2 was concentrated to 1 ml.
  • One milliliter of methanol containing 0.05% trifluoracetic acid was added. Large amount of proteins were precipitated by this treatment.
  • the apoptosis inducing activity (P-2) remained in supernatant.
  • the supernatant was then applied to a reverse phase RP-4 column (Micra Scientific Inc) and developed by a linear gradient constructed by solution A (TFJO, 0.05% TFA) and solution B (Methanol, 0.05% TFA).
  • the linear gradient was constructed by increasing solution B from 0% to 100 % in solution A within 10 min (20 milliliter elution volume and thereafter the column was eluted with 100% solution B for 5 min.
  • Fig. 10 Apogen P-2 isolated by anion exchange chromatography and reverse phase chromatography was concentrated and subjected to electrophoresis under denaturing conditions through a 4-20% gradient SDS-Polyacrylamide gel. The gel was silver stained. A protein band with molecular weight of 65 KD was obtained. This result suggest the successful purification of Apogen p-2 which have molecular weight of 65 KD on SDSPAGE.
  • Fig. 11 Apogen L isolated by anion exchange chromatography and reverse phase chromatography was concentrated and subjected to electrophoresis under denaturing conditions through a 4-20% gradient SDS-Polyacrylamide gel. The gel was silver stained. A protein band with molecular weight of 55 KD was obtained.
  • Fig. 12 Anion exchange chromatography of Apogen L.
  • Apogen L isolated by DE 52 cellulose, heparin agarose was concentrated and loaded onto a Q2 column (Bio-Rad )which is further developed by a linear gradient constructed by buffer A (10 mM Tris-HCI, pH 7.4) and buffer B (10 mM Tris-HCI, pH 7.4. 0.5 M NaCI) using Bio-Rad's BioLogic HPLC system.
  • the linear gradient was constructed by increasing buffer B from 0 % to 100 % in buffer A within 10 min.
  • Fractions 7 and 8 contain activity inducing apoptosis in HL-60 cells.
  • Fig. 13 induction of apoptosis in HL-60 cells by Apogen L.
  • the activity of Apogen L isolated by DE 52 cellulose, heparin agarose and anion exchange chromatography was tested on the following cell lines: HL-60 (leukemia) and CCD 39 Lu (normal lung fibroblast).
  • HL-60 leukemia
  • CCD 39 Lu normal lung fibroblast
  • HL-60 cells were incubated with Apogen L isolated as described as above for 15 hr and then stained with Hoechst dye for 2 hours.
  • the nuclei of the HL-60 cells that have been incubated with control medium are normal and healthy(A).
  • the nuclei of the HL-60 cells that have been incubated with Apogen L shown the characteristic of apoptosis
  • Fig. 13B shows that Apogen L causes the condensation of nucleus, demonstrated by the more intense fluorescent light compared with the control nucleus in Fig. 13 A.
  • the nucleus condensation is accompanied by the fragmentation of DNA, demonstrated by the breakage of nucleus as shown in Fig. 13B.
  • the nucleus condensation and DNA fragmentation are the two morphological characteristic of cells under apoptosis.
  • Fig. 14 induction of apoptosis in MCF-7 cells by Apogen L.
  • the activity of Apogen L isolated by DE 52 cellulose, heparin agarose and anion exchange chromatography was tested on the following cell lines: MCF-7 (breast cancer cells).
  • MCF-7 breast cancer cells
  • Apogen L contains activity inducing apoptosis
  • MCF-7 cells were incubated with Apogen L isolated as described as above for 15 hr.
  • the nuclei of the MCF-7 cells that have been incubated with control medium are normal and healthy(A).
  • the nuclei of the MCF-7 cells that have been incubated with Apogen L shown the characteristic of apoptosis (Fig. 14B).
  • Fig. 15 Apogen L fails to induce apoptosis in normal breast cells (Hs578 Bst) cells in MEM-insulin- 10% serum medium. Hs578 Bst cells were incubated with control medium or the conditioned medium for 15 hr. Cells looked normal and healthy; the nuclei of Hs578 Bst cells remain the same with or without incubating with Apogen L. These results suggest that Apogen L fails to induce apoptosis in normal breast cells (Hs578 Bst).
  • XC condition medium for isolation of Apogen p- 1.
  • Apogen P-l was isolated from the conditioned medium of a cell line called XC which was derived from rat tumor and is purchased from American Type Culture Collection (ATCC).
  • DMEM Dulbecco's Modification of Eagle's Medium
  • FBS fetal bovine serum
  • DMEM fetal calf serum
  • C3H 1OT1/2 condition medium for isolation of Apogen P-2.
  • Apogen P-2 was isolated from the conditioned medium of a cell line called C3H1OT 1/2 which was derived from mouse embryo and is purchased from American Type Culture
  • alpha-MEM alpha Modification of Eagle's Medium
  • FBS Fetal bovine serum
  • penicillin and streptomycin penicillin and streptomycin
  • Prostate cancer cell line LNCAP was routinely used for the isolation of Apogen P-l and Apogen P-2, whereas leukemia cell line HL-60 was used for the isolation of Apogen L.
  • the methods of assays are as following: LNCAP or HL-60 (1,000 cells) was seeded in 10 microliters RPMI containing 15% or 20% Fetal bovine serum, penicillin and streptomycin at 37 degree, 5% CO2 in Microtray plates (25 ⁇ l wells, Robbins Scientific Corp.). Tested sample (10 ⁇ l) was added 3-4 hours after cells were seeded. After incubation of the tested sample with cells for 15 hours, two microliters of Hoechst dye (0.03 ng/ml in PBS) was added.
  • % Apoptotic cells Number of cells with DNA condensation and fragmentation
  • Hep G2 cells are chosen for the study of cell repelling activity.
  • Hep G2 cells are not sensitive to Apogen P-l in inducing apoptosis.
  • the cell size of Hep G2 cell is about 3-4 times as big as the pore size of the membrane on the Transwell Insert, which is a good cell size for cell migration/invasion study.
  • a tissue culture device called Transwell Insert purchased from Costar (Cambridge, MA) was used to discover the chemorepellent activity of Apogen P-lb. This device, which has been widely used for the studies of cell migration/invasion, contains an upper chamber and a lower chamber.
  • Apogen P-l was precipitated by 80 % saturated of ammonium sulfate by adding 561 g of ammonium sulfate per liter of XC conditioned medium. Pellet was collected by centrifugation and the proteins were dissolved in 10 mM Tris-HCI (pH 7.4). After removal of ammonium sulfate by dialysis, the dissolved proteins were separated by a Q2 HPLC column.
  • the dissolved proteins isolated by ammonium sulfate precipitation were concentrated and loaded onto a Q2 column (Bio Rad )which is further developed by a linear gradient constructed by buffer A (10 mM Tris-HCI, pH 7.4) and buffer B (10 mM Tris-HCI, pH 7.4. 0.55 M NaCI) using BioRad's BioLogic HPLC system.
  • the linear gradient was constructed by increasing buffer B from 0% to 100 % in buffer A within 10 min (20 milliliter elution volume) and thereafter the column was eluted with 100% buffer B for 5 min.
  • the Apogen P- 1 activity was assayed by the induction of apoptosis in LNCAP cells.. We found that there are three activity peaks across the chromatogram profile. Fraction 5 to 7 cause 70 % cell death, fraction 8-10 cause 65% cell death and fraction 11-14 caused 90 % cell death in 18 Hr.
  • Step 3 Reverse phase chromatography. Apogen P-la, Apogen P-lb and Apogen P-lc were separately concentrated to 1.5 ml.
  • Step 4 Preparative electrophoresis Apogen lc isolated by anion exchange chromatography was purified by both Reverse phase chromatography (step 3) and Preparative Electrophoresis by a MiniPrep Gel electrophoresis (Bio-Rad). The reverse phase chromatogram of Apogen P-la is shown in Fig. 4(a). fractions 12-13 have activity inducing 80 % cell death in LNCAP cells at 10 hr.
  • the purity of the isolated Apogen P-la, Apogen P-lb and Apogen P-lc were checked with SDS-polyacrylamide gel electrophoresis stained with silver staining.
  • Apogen P-lb A single faint protein band with molecular weight of 55 KD was obtained. This result suggest the successful purification of Apogen P-lb which have molecular weight of 55 KD on SDS-PAGE.
  • Apogen P-lc The purification of Apogen lc by Reverse Phase chromatography leads to the Isolation of a 70 KD protein whereas the purification of Apogen lc by preparative electrophoresis leads to the purification of a 57 KD protein. As shown in Fig.6(A), a major protein band with molecular weight of 70 KD was obtained by Reverse Phase chromatography. A 57 KD protein, on the other hand, was isolated by preparative electrophoresis. (Fig. 6B). B . Isolation of Apogen P-2
  • Step 1 Ammonium sulfate precipitation
  • Apogen P-2 was precipitated by 80 % saturated of ammonium sulfate by adding 56 lg of ammonium sulfate per liter of conditioned medium. Pellet was collected by centrifugation and the proteins were dissolved in 10 mM Tris-HCI (pH 7.4). Step 2: Hydroxylapatite treatment.
  • Step 3 Heparin agarose treatment.
  • step 4 Reverse phase chromatography.
  • Apogen P-2 presents in the supernatant of Heparin agarose in step 3 was further purified by a reverse phase chromatography.
  • Apogen P-2 was concentrated to 1 ml.
  • One milliliter of methanol containing 0.05% trifluoacetic acid was added. Large amount of proteins were precipitated by this treatment.
  • the apoptosis inducing activity (P-2) remained in supernatant.
  • the supernatant was then applied to a reverse phase RP-4 column (Micra Scientific Inc) and developed by a linear gradient constructed by solution A (H2O, 0.05% TFA) and solution B Methanol, 0.05% TFA).
  • the linear gradient was constructed by increasing solution B from 0 % to 100 % in solution A within 10 min (20 milliliter elution volume and thereafter the column was eluted with 100% solution B for 5 min.
  • the reverse phase chromatogram of Apogen P-2 is shown in Fig. 9. Fractions 12-14 have activity inducing 80 % cell death in LNCAP cells at 12 hr.
  • the purity of the isolated Apogen P-2 was checked with SDS polyacrylamide gel electrophoresis stained with silver staining. A single protein band with molecular weight of 65 Kd was obtained (Fig. 10) C. Isolation of Apogen L
  • the conditioned medium was incubated with the anion exchanger, DE 5 2 (Diethylaminoethyl cellulose, Whatman) for 1 hr.
  • the incubation mixture was centrifuged and DE 5 2 which binds Apogen L was collected and washed with 10 mM Tris-HCI (pH 7.5) containing 0. 15 M NaCl.
  • Apogen L was then eluted from DE 5 2 cellulose by 10 mM Tris- HCI (pH 7.5) containing 0.5 M NaCl.
  • Step 2 Heparin agarose absorption
  • step 1 Apogen L isolated as described in step 1 was further absorbed by Heparin agarose (Sigma) by incubating Apogen L with Heparin agarose for I hr. Heparin agarose was collected by centrifugation and was washed with 10 mM Tris-HCI (pH 7.5). Apogen L absorbed in Heparin agarose was then eluted by 2 M NaCl. Step 3: O2 HPLC chromatography
  • the linear gradient was constructed by increasing buffer B from 0% to 100% in buffer A in 10 min.
  • the chromatogram is shown in Fig. 10.
  • the purity of the isolated Apogen L was checked with SDSpolyacrylamide gel electrophoresis stained with silver staining. A single protein band have activity with molecular weight of approximately 55 Kd was obtained (Fig. 11).
  • This invention describes the methods for the isolation of five proteins (Apogen P-la, Apogen P-lb, Apogen P-lc, Apogen P-2 and Apogen L) that are able to induce apoptosis in prostate cancer cells (Apogen P-l's), in prostate cancer cells and breast cancer cells (Apogen P-2), and leukemia and breast cancer cells (Apogen L).
  • TNF Tumor Necrosis Factor
  • TGF-Beta Transforming Growth Factor
  • Fas ligand and TRAIL are the proteins reported to induce apoptosis in certain cell lines.
  • TRAIL and Fas are membrane bound proteins, (Wiley, S. R. et al. Immunity 3, 673. 1995. Tomei, et al. "Apoptosis”: The molecular Basis of Cell Death. Cold Spring Harbor Laboratory Press. PP. 87. 1991 ) whereas the Apogen P-la, Apogen P-lb, Apogen
  • P-lc, Apogen P-2 and Apogen L are all soluble (non-membrane bound) proteins.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention se rapporte à des procédés pour isoler les protéines qui provoquent expressément l'apoptose (la mort cellulaire contrôlée) des cellules du cancer de la prostate (LNCAP), de celles de la leucémie (HL-60) et de celles du cancer du sein (MCF-70) sans pour autant affecter les fibroblastes normaux des poumons chez l'humain (CCD 39 Lu). P-1 n'a aucun effet sur les cellules du cancer du sein. Selon cette invention, on a isolé cinq protéines à partir d'un milieu conditionné de cellules de culture: (1) Apogène P-1, protéines Apogène P-1a, Apogène P-1b et Apogène P-1c isolées à partir d'un milieu conditionné de cellules XC et capables de provoquer l'apoptose des cellules du cancer de la prostate (LNCAP) mais n'affectant pas les fibroblastes normaux des poumons chez l'humain (CCD 39 Lu) ni les cellules du cancer du côlon (T84), du cancer du sein (MCF-7) ou de la leucémie (HL-60); (2) Apogène P-2, protéine isolée à partir du milieu conditionné de cellules C3HlOTl/2 et capable de provoquer l'apoptose des cellules du cancer de la prostate (LNCAP) et des cellules du cancer du sein (MCF-7) sans pour autant affecter ni les fibroblastes normaux des poumons chez l'humain (CCD 39 Lu) ni les cellules du cancer du côlon (T84) et; (3) Apogène L, protéine isolée à partir d'un milieu conditionné de cellules XC et capable de provoquer l'apoptose des cellules de la leucémie (HL-60) et celles du cancer du sein (MCF-7) sans affecter ni les fibroblastes normaux des poumons chez l'humain (CCD 39 Lu) ni les cellules du cancer du côlon (T84) ni celles du cancer de la prostate (LNCAP). L'invention pourrait déboucher sur la découverte d'une nouvelle classe de médicaments anticancéreux qui sont dirigés contre les cancers de la prostate et du sein, la leucémie ou d'autres cancers et dont l'action consiste à provoquer l'apoptose des cellules cancéreuses sans affecter les cellules normales.
PCT/US1998/027108 1997-12-18 1998-12-18 Proteines destinees a provoquer l'apoptose specifique de cellules cancereuses et procede pour isoler ces proteines WO1999031135A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
KR1020007006696A KR20010033260A (ko) 1997-12-18 1998-12-18 암 세포의 아팝토시스를 유도하는 단백질 및 이를분리하는 방법
AU19330/99A AU1933099A (en) 1997-12-18 1998-12-18 Proteins for cancer cell specific induction of apoptosis and method for isolation thereof
JP2000539058A JP2002516821A (ja) 1997-12-18 1998-12-18 アポトーシスをがん細胞特異的に誘導するためのタンパク質およびその単離方法
CA002325368A CA2325368A1 (fr) 1997-12-18 1998-12-18 Proteines destinees a provoquer l'apoptose specifique de cellules cancereuses et procede pour isoler ces proteines
IL13685098A IL136850A0 (en) 1997-12-18 1998-12-18 Proteins for cancer cell specific induction of cell death
EP98964142A EP1037919A1 (fr) 1997-12-18 1998-12-18 Proteines destinees a provoquer l'apoptose specifique de cellules cancereuses et procede pour isoler ces proteines
MXPA00005954A MXPA00005954A (es) 1997-12-18 1998-12-18 Proteinas para la induccion especifica de apoptosis de celulas cancerosas y metodo para su aislamiento.
NO20003099A NO20003099L (no) 1997-12-18 2000-06-16 Proteiner for kreftcellespesifikk induksjon av apoptosis, og fremgangsmÕte til isolering derav

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US99343297A 1997-12-18 1997-12-18
US08/993,432 1997-12-18

Publications (1)

Publication Number Publication Date
WO1999031135A1 true WO1999031135A1 (fr) 1999-06-24

Family

ID=25539541

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/027108 WO1999031135A1 (fr) 1997-12-18 1998-12-18 Proteines destinees a provoquer l'apoptose specifique de cellules cancereuses et procede pour isoler ces proteines

Country Status (11)

Country Link
EP (1) EP1037919A1 (fr)
JP (1) JP2002516821A (fr)
KR (1) KR20010033260A (fr)
CN (1) CN1314914A (fr)
AU (1) AU1933099A (fr)
CA (1) CA2325368A1 (fr)
IL (1) IL136850A0 (fr)
MX (1) MXPA00005954A (fr)
NO (1) NO20003099L (fr)
PL (1) PL343247A1 (fr)
WO (1) WO1999031135A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997031107A2 (fr) * 1996-02-20 1997-08-28 Coles John G Apoptose induite par des lectines de serum humain et procede de detection d'apoptose

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997031107A2 (fr) * 1996-02-20 1997-08-28 Coles John G Apoptose induite par des lectines de serum humain et procede de detection d'apoptose

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SU X, ET AL.: "DEPHOSPHORYLATION OF A 65 KD PROTEIN DELIVERS SIGNALS FOR FAS-MEDIATED APOPTOSIS", THE FASEB JOURNAL, FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY, US, vol. 08, no. 04, 1 March 1994 (1994-03-01), US, pages A218, XP002918300, ISSN: 0892-6638 *
WANG H, ET AL.: "BRCA1 PROTEINS ARE TRANSPORTED TO THE NUCLEUS IN THE ABSENCE OF SERUM AND SPLICE VARIANTS BRCA1A, BRCA1B ARE TYROSINE PHOSPHOPROTEINS THAT ASSOCIATE WITH E2F, CYCLINS AND CYCLIN DEPENDENT KINASES", ONCOGENE, NATURE PUBLISHING GROUP, GB, vol. 15, 1 January 1997 (1997-01-01), GB, pages 143 - 157, XP002918299, ISSN: 0950-9232, DOI: 10.1038/sj.onc.1201252 *

Also Published As

Publication number Publication date
CN1314914A (zh) 2001-09-26
IL136850A0 (en) 2001-06-14
JP2002516821A (ja) 2002-06-11
NO20003099D0 (no) 2000-06-16
PL343247A1 (en) 2001-07-30
MXPA00005954A (es) 2002-09-18
EP1037919A1 (fr) 2000-09-27
NO20003099L (no) 2000-08-17
CA2325368A1 (fr) 1999-06-24
KR20010033260A (ko) 2001-04-25
AU1933099A (en) 1999-07-05

Similar Documents

Publication Publication Date Title
US5994298A (en) Proteins for cancer cell specific induction of apoptosis and method for isolation thereof
Bridgen et al. Human lymphoblastoid interferon. Large scale production and partial purification.
US7341724B2 (en) Polypeptide for the treatment of cancer and a method for preparation thereof
Bosso et al. Exploiting the human peptidome for novel antimicrobial and anticancer agents
US6258779B1 (en) Method of using fetuin to induce apoptosis in cancer cells
EA010291B1 (ru) Способы защиты млекопитающего от радиации
US6737402B2 (en) Method of preparing fetuin to induce apoptosis
US7238662B2 (en) Alpha 2HS glycoprotein for treatment of cancer and a method for preparation thereof
WO1999031135A1 (fr) Proteines destinees a provoquer l'apoptose specifique de cellules cancereuses et procede pour isoler ces proteines
US8247380B2 (en) Alpha 1-acid glycoprotein, alpha 2-HS glycoprotein, alpha 1-antitrypsin, and fragments thereof induce apoptosis in cancer cell lines
Damme et al. Identification by sequence analysis of chemotactic factors for monocytes produced by normal and transformed cells stimulated with virus, double‐stranded RNA or cytokine
WO2023092463A1 (fr) Application de chlorhydrate de doxorubicine dans inhibition de mcl-1
TWI280248B (en) Polypeptide for the treatment of cancer and a method for preparation thereof
STURM et al. Expression of p53 after sv40 virus-infection of quiescent cells
CN116173049A (zh) 盐酸阿霉素在抑制Mcl-1中的应用
WO2023092465A1 (fr) Application du brazil hématoxyline dans la préparation d'un inhibiteur de la protéine anti-apoptotique mcl-1
WO2023092462A1 (fr) Utilisation de dipyridamole dans l'inhibition de mcl-1
CN116173035A (zh) 双嘧达莫在抑制Mcl-1中的应用
CN116173005A (zh) 巴西苏木素在制备抗凋亡蛋白Mcl-1抑制剂中的应用
Pani et al. Extraction of Lectin FRIL from Leguminous Plant Lablab

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 136850

Country of ref document: IL

Ref document number: 98813660.0

Country of ref document: CN

AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA CN IL JP KR MX NO NZ PL

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 2325368

Country of ref document: CA

Ref document number: 2325368

Country of ref document: CA

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: PA/A/2000/005954

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 1020007006696

Country of ref document: KR

ENP Entry into the national phase

Ref document number: 2000 539058

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 1998964142

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 505819

Country of ref document: NZ

Ref document number: 19330/99

Country of ref document: AU

WWP Wipo information: published in national office

Ref document number: 1998964142

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1020007006696

Country of ref document: KR

WWW Wipo information: withdrawn in national office

Ref document number: 1998964142

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1020007006696

Country of ref document: KR