Proteins for cancer cell specific induction of apoptosis and method for isolation thereof

The present invention provides the methods to isolate the proteins specifically induced apoptosis (programmed cell death) in prostate cancer cells (LNCAP), leukemia cells (HL-60), and breast cancer cells (MCF-70), but without effect in normal human lung fibroblast cells (CCD 39 Lu). P-1 has no effect on breast cancer cells. Five proteins have been isolated from the conditioned media of culture cells: (1) Apogen P-1: the proteins (Apogen P-1a, Apogen P-1b and Apogen P-1c) isolated from the conditioned medium of XC cells are able to induce apoptosis in prostate cancer cells (LNCAP) without effect in normal human lung fibroblast (CCD 39 Lu), colon cancer (T84), breast cancer (MCF-7) and leukemia (HL-60) cells. (2) Apogen P-2: the protein isolated from the conditioned medium of C3H10T1/2 cells is able to induce apoptosis in prostate cancer cells (LNCAP) and breast cancer (MCF-7) without effect in normal human lung fibroblast (CCD 39 Lu) and colon cancer (T84) cells. (3) Apogen L: the protein isolated from the conditioned medium of XC cells is able to induce apoptosis in leukemia cells (HL-60), and breast cancer (MCF-7) without effect in normal human lung fibroblast (CCD 39 Lu), colon cancer (T84) and prostate cancer (LNCAP) cells. The isolated protein Apogen P-2 is at least in part comprised of bovine fetuin. When properly prepared, fetuin is able to induce apoptosis in leukemia cells (HL-60), prostate cancer (LNCaP and PC-3) cells, colon cancer (Colo 205) cells, breast cancer (MCF-7) cells, and lung cancer (Calu-1) cells. The invention may lead to the discovery of a novel class of anticancer drug that aims at prostate cancer, breast cancer, leukemia and other cancers by inducing apoptosis in cancer cells without affecting normal cells.

BACKGROUND OF THE INVENTION 
Human beings have had a long battle against cancer. Because the disease is 
so widespread, manifests itself in so many different ways and is so 
relentless, the potential market for effective cancer therapies is 
enormous. It is estimated that 10 million people in the U.S. either have 
or have had cancer. The National Cancer Institute (NCI) projects that in 
1995, some 1.2 million new cases of cancer will be diagnosed in the United 
States, and that 538,000 people will die of the disease. Cancer is 
currently treated, with a low degree of success, with combinations of 
surgery, chemotherapy and radiation. The reason of the low degrees of 
success in cancer chemotherapy is as the following: Current 
chemotherapeutic approaches target rapidly dividing tumor cells. This 
approach is ineffective when the cancer is dormant or growing slowly. Such 
treatments also affect other, noncancerous cells that divide rapidly, 
causing harmful side effects. 
Only in the last several years has a new approach emerged in the battle 
against cancer. This approach is based on the newly discovered biological 
phenomenon called "Apoptosis". Apoptosis is also called "programmed cell 
death" or "cell suicide". (Krammer, et al., "Apoptosis in the APO-1 
System", Apoptosis: The molecular Basis of Cell Death, pp. 87-99 Cold 
Spring Harbor Laboratory Press, 1991). In contrast to the cell death 
caused by cell injury, apoptosis is an active process of gene-directed, 
cellular self-destruction and that it serves a biologically meaningful 
function. (Kerr, J. F. R and J. Searle J. Pathol. 107:41, 1971). One of 
the examples of the biologically meaningful functions of apoptosis is the 
morphogenesis of embryo. (Michaelson, J. Biol. Rev. 62:115, 1987). Just 
like the sculpturing of a sculpture, which needs the addition as well as 
removal of clay, the organ formation (Morphogenesis) of an embryo relies 
on cell growth (addition of clay) as well as cell death (removal of clay). 
As a matter of fact, apoptosis plays a key role in the human body from the 
early stages of embryonic development through to the inevitable decline 
associated with old age. (Wyllie, A. H. Int. Rev. Cytol. 68:251, 1980). 
The normal function of the immune, gastrointestinal and hematopoietic 
system relies on the normal function of apoptosis. When the normal 
function of apoptosis goes awry, 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. Because 
of the versatility of apoptosis involved in human 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. 
The idea that cancer may be caused by insufficient apoptosis merged only 
recently (Cope, F. O and Wille, J. j, "Apoptosis": The Molecular Basis of 
Cell Death, Cold Spring Harbor Laboratory Press, p. 61, 1991). This idea 
however, 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. Nevertheless, since the objective of cancer 
treatment is to kill cancer cells without killing the host, although 
apoptosis may open a new door for cancer therapy by inducing apoptosis in 
tumor cells, the success of this treatment is still dependent on the 
availability of drugs that can selectively induce apoptosis in tumor cells 
without affecting normal cells. In this patent application, we described 
the methods for the Isolation of proteins that specifically induce 
apoptosis in cancer cells without effect in normal cells. These proteins 
may present a new class of anticancer drugs that induce apoptosis in 
cancer cells which may offer a breakthrough in cancer therapy. 
DETAILED DESCRIPTION OF THE INVENTION 
This patent application describes the isolation of five proteins named: 
Apogen P-1a, Apogen 1b, Apogen 1c, Apogen P-2 and Apogen L. 
(A) Isolation of Apogen P-1 
(1) Source of Apogen P-1 
Apogen P-1 was isolated from the conditioned medium of a cell line called 
XC which was derived from rat tumor (ATCC CCL 165). 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 
(3.times.100 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. 
(2) Activity of Apogen P-1 
(a) Apoptosis Inducing Activity 
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). When 10 folds 
concentrated conditioned medium was incubated for 18 hours with the above 
cell lines in the presence of 5% serum, the conditioned medium induced 
apoptosis in JEG-3 cells (35%), G 401 cell (27%), LNCaP (100%) and without 
activity in CCD 39 Lu (0%), T84 (0%), MCF-7 (0%) and HL-60 (0%). 
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. Morphologically, the earliest 
definitive changes in apoptosis that have been detected with the electron 
microscope are compaction of the nuclear chromatin into sharply 
circumscribed, uniformly dense masses about the nuclear envelop and 
condensation of the cytoplasms. Phase-contrast microscope of cells under 
apoptosis shows the condensation and the fragmentation of DNA and the 
budding of cell to form apoptotic body. 
To morphologically demonstrated that 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. As shown in FIG. 1A, the 
nuclei of the LNCAP cells that have been incubated with control medium are 
normal and healthy(A). However, 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)). First, the conditioned medium 
causes the condensation of nucleus, demonstrated by the more intense 
fluorescence light compared with the control nucleus in FIG. 1(A). 
Secondly, the nucleus condensation is accompanied by the fragmentation of 
DNA, demonstrated by the breakage of nucleus as shown in FIG. 1(B). As we 
have mentioned above, the nucleus condensation and DNA fragmentation are 
the morphological characteristic of cells under apoptosis. These results 
suggest that the conditioned medium from XC cells contains an activity 
inducing apoptosis in LNCAP cells. On the other hand, the conditioned 
medium fails to induce apoptosis in normal human lung fibroblast (CCD 39 
Lu cells) and breast cancer cells (MCF-7). As shown in FIG. 2, 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)). 
(b) Cell Repelling Activity 
The partially purified Apogen P-1b (Q2 anionic exchanger chromatography 
step) isolated as described below was recently found to contain an 
activity other than inducing apoptosis. We found that Apogen P-1b 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. (Grotendorst, 
G. R. et al., Proc. Natl. Acad. Sci. 78:3669, 1981; Grant, M. B. et al 
Invest. Ophthal. Visual Science. 33:3292, 1992). This finding suggests 
that Apogen P-1b 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-1b induces cell death and 
repel cells. Apogen P-1b is the first "chemorepellent" found in the field 
of modern biology. 
A tissue culture device called Transwell Insert purchased from Costar 
(Cambridge, Mass.) was used to discover the chemorepellent activity of 
Apogen P-1b. 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. In our 
experiments, 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-1b (30 .mu.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 min. Cells in ten microliters of the 
trypsin solution were counted in a Hemacytometer. In several experiments, 
we found that the partially purified Apogen-1b contained an activity 
decreasing the number of cells going through the membrane. For example, in 
one experiment, in the presence of the partially purified Apogen P-1b, the 
cells number in 10 microliters trypsin solution (which are the cells go 
through membrane) is 24+-4, whereas the cells number that go through 
membrane in the control experiment is 82+-27. This result suggests that 
the partially purified Apogen P-1b prevents Hep G2 cells migrating through 
membrane. To unequivocally shown that Apogen P-1b repel cells, an inverted 
experiment was installed, instead of placing Apogen P-1b in the lower 
chamber, we placed Apogen P-1b in the upper chamber, after 12 hours, we 
found that 56+-19 cells went through membrane compared with control 
experiment of 30+-1.7 cells per 10 microliters of trypsin solution. The 
statistically significant increase or decrease in cell number going 
through the membrane by alternatively placing Apogen P-1b in the upper or 
lower chamber of this tissue culture device strongly suggests that Apogen 
P-1b repels cells. 
(3) Isolation of Apogen P-1 from XC Conditioned Medium 
The Apogen P-1 present in the conditioned medium was isolated by the 
following steps: 
Step 1: Ammonium Sulfate Precipitation 
Apogen P-1 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). After removal of ammonium sulfate by dialysis, the 
dissolved proteins were separated by a Q2 HPLC column. 
Step 2: Q2 HPLC Chromatography 
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 (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 chromatogram is shown in FIG. 3. 
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. We 
collected fractions 5-7 and named it Apogen P-1a, fractions 8-10 is named 
Apogen P-1b and fractions 11 to 14 is named Apogen P-1c. These three 
Apogen P-1's were further purified by a reverse phase column. 
Step 3: Reverse Phase Chromatography 
Apogen P-1a, Apogen P-1b and Apogen P-1c were separately concentrated to 
1.5 ml. One ml of methanol containing 0.05% trifluoracetic acid was added. 
In each samples, large amount of proteins were precipitated by this 
treatment. Whereas, the apoptosis inducing activity remained in 
supernatant. The supernatant was then applied to a reverse phase RP4 
column (Micra Scientific Inc) and developed by a linear gradient 
constructed by solution A (H.sub.2 O, 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.) 
Step 4: Preparative Electrophoresis 
Apogen 1c 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-1a 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-1b is shown in FIG. 4(b). fractions 14 and 15 have activity 
inducing 45% cell death in LNCAP cells at 18 hr. 
The reverse phase chromatogram of Apogen P-1c 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-1a, Apogen P-1b and Apogen P-1c were 
checked with SDS-polyacrylamide gel electrophoresis stained with silver 
staining. 
(1) Apogen P-1a: As shown in FIG. 5, a protein band with molecular weight 
of 70 KD was obtained. This result suggest the nearly successful 
purification of Apogen P-1a which have molecular weight of 70 KD on 
SDSPAGE. 
(2) Apogen P-1b: A single faint protein band with molecular weight of 55 KD 
was obtained. This result suggest the successful purification of Apogen 
P-1b which have molecular weight of 55 KD on SDS-PAGE. (Data not shown) 
(3) Apogen P-1c: The purification of Apogen 1c by Reverse Phase 
chromatography leads to the isolation of a 70 KD protein whereas the 
purification of Apogen-1c 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). 
Our next step, obviously, will be put our entire efforts on obtaining 
enough protein band for amino acid sequence. 
(B) Isolation of Apogen P-2 
(1) Source of Apogen P-2 
Apogen P-2 was isolated from the conditioned medium of a cell line called 
C3H 10T1/2 which was derived from mouse embryo cells (ATCC CCL 226). C3H 
10T1/2 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 (3.times.100 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 
(a) Apoptosis Inducing Activity 
The activity of the crude conditioned medium of C3H 10T1/2 cells was tested 
on the following cell lines: LNCAP (Prostate cancer), breast cancer cells 
(MCF-7), and CCD 39 Lu (normal lung fibroblast). When 10 folds 
concentrated conditioned medium was incubated for 18 hours with the above 
cell lines in the presence of 5% serum, the conditioned medium induced 
apoptosis in LNCaP (100%) and without activity in CCD 39 Lu (0%). To 
morphologically demonstrated that the C3H 10T1/2 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. As shown in FIG. 7A, 
the nuclei of the LNCAP cells that have been incubated with control medium 
are normal and healthy(A). However, the nuclei of the LNCAP cells that 
have been incubated with the conditioned medium shown the characteristic 
of apoptosis (FIG. 7B). First, the conditioned medium causes the 
condensation of nucleus, demonstrated by the more intense fluorescent 
light compared with the control nucleus in FIG. 7A. Secondly, the nucleus 
condensation is accompanied by the fragmentation of DNA, demonstrated by 
the breakage of nucleus as shown in FIG. 7B. As we have mentioned above, 
the nucleus condensation and DNA fragmentation are the morphological 
characteristic of cells under apoptosis. The same held true of breast 
cancer cells (MCF-7) in which 85% apoptotic effect was observed after 18 
hours of exposure to P-2. These results suggest that the conditioned 
medium from C3H10T1/2 cells contains an activity inducing apoptosis in 
LNCAP and MCF-7 cells. On the other hand, the conditioned medium fails to 
induce apoptosis in normal human lung fibroblast (CCD 39 Lu cells). As 
shown in FIG. 8, the nuclei of CCD 39 Lu cells remain the same with or 
without incubating with the conditioned medium of C3H10T1/2 cells (FIG. 8A 
and FIG. 8B). 
(b) Cell Repelling Activity 
The partially purified Apogen P-2 isolated by ammonium sulfate 
precipitation, hydroxylapatite and heparin treatment as described above 
was recently found to contain an activity other than inducing apoptosis. 
Similar to Apogen P-1b, Apogen P-2 have the activity to repel cells away. 
Transwell Insert purchased from Costar (Cambridge, Mass.) was used to 
discover the chemorepellent activity of Apogen P-2. 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 .mu.m pore size which allows cell 
to migrate through the membrane. Tested cells (HL-60) were grown on the 
upper chamber and tested compound (Apogen P-2) is placed in the lower 
chamber. In our experiments, HL-60 (100,000 cells) cells, which have cell 
size 2-3 times as big as the membrane pore size were grown in the upper 
chamber for 2 hours and then the partially purified Apogen P-2 (30 .mu.l) 
isolated by ammonium sulfate precipitation, hydroxylapatite and Heparin 
agarose as described above was placed in the lower chamber. After 6 hours, 
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 .mu.l of PBS. Cells in ten microliters of the PBS 
solution were counted in a Hemacytometer. In several experiments, we found 
that the partially purified Apogen P-2 contained an activity decreasing 
the number of cells going through the membrane. For example, in one 
experiment, in the presence of the partially purified Apogen P-2, the 
cells number in 10 microliters PBS solution (which are the cells go 
through membrane) is 47+-5.6, whereas the cells number that go through 
membrane in the control experiment is 213+-40. At this moment, no 
apoptosis was observed in HL-60 cells present in the upper chamber. This 
result suggests that the partially purified Apogen P-2 prevents HL-60 
cells migrating through membrane. 
(3) Isolation of Apogen P-2 from C3H10T1/2 Conditioned Medium 
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 
After removal of ammonium sulfate by dialysis in 10 mM Tris-HCI (pH 7.5), 
the dissolved proteins were incubated with Hydroxylapatite gel (Bio-Gel 
HTP gel, Bio-Rad) for 1 Hr. After remove HTP gel by centrifugation, the 
activity inducing apoptosis in LNCAP cells was found to present in the 
supernatant which was then further treated with Heparin agarose gel. 
Step 3: Heparin Agarose Treatment 
The supernatant from step 2 was further incubated with Heparin agarose 
(Sigma) for 1 Hr. After remove HTP gel by centrifugation, the activity 
inducing apoptosis in LNCAP cells was found to be present in the 
supernatant. 
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 (Micra Scientific Inc) and developed by a linear gradient 
constructed by solution A (H.sub.2 O, 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 in 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 
(1) Source of Apozen L 
Apogen L was isolated from the conditioned medium of XC cell line (ATCC CCL 
165). 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 (CCD 39 Lu) is not affected by this activity. 
(2) Isolation of Apozen L from XC Conditioned Medium 
The Apogen L present in the conditioned medium was isolated by the 
following steps: 
Step 1: DE52 Absorption 
The conditioned medium was incubated with the anion exchanger, DE 52 
(Diethylaminoethyl cellulose, Whatman) for 1 hr. The incubation mixture 
was centrifuged and DE 52 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 52 cellulose by 10 mM Tris-HCI (pH 7.5) containing 0.5 M 
NaCI. 
Step 2: Heparin Agarose Absorption 
Apogen L isolated as described in step 1 was further absorbed by Heparin 
agarose (Sigma) by incubating Apogen L with Heparin agarose for 1 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 NaCI. 
Step 3: Q2 HPLC Chromatography 
Apogen L isolated as described above 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 00/o to 100% 
in buffer A within 10 min. The chromatogram is shown in FIG. 12. The 
purity of the isolated Apogen L was checked with SDS polyacrylamide gel 
electrophoresis stained with silver staining. A single protein band with 
molecular weight of 55 Kd was obtained (FIG. 11) 
(3) Activity of Apogen L 
The activity of Apogen L isolated as described above was tested on the 
following cell lines: HL-60 (leukemia) and CCD 39 Lu (normal lung 
fibroblast). To morphologically demonstrated that Apogen L contains 
activity inducing apoptosis, 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. As shown in FIG. 13A, the nuclei of the HL-60 cells that have 
been incubated with control medium are normal and healthy (FIG. 13A). 
However, the nuclei of the HL-60 cells that have been incubated with 
Apogen L shown the characteristic of apoptosis (FIG. 13B). First, Apogen L 
causes the condensation of nucleus, demonstrated by the more intense 
fluorescent light compared with the control nucleus in FIG. 13A. Secondly, 
the nucleus condensation is accompanied by the fragmentation of DNA, 
demonstrated by the breakage of nucleus as shown in FIG. 13B. As we have 
mentioned above, the nucleus condensation and DNA fragmentation are the 
two morphological characteristic of cells under apoptosis. These results 
suggest that the isolated Apogen L contains an activity inducing apoptosis 
in HL-60 cells. Apogen L also induces apoptosis in MCF-7 (breast cancer) 
cells). On the other hand, the conditioned medium fails to induce 
apoptosis in normal human lung fibroblast (CCD 39 Lu cells).

EXAMPLES 
A. Methods 
1. Preparation of Condition Media 
A. Preparation of XC Condition Medium for Isolation of Apogen p-1 
Apogen P-1 was isolated from the conditioned medium of a cell line called 
XC which was derived from rat tumor (ATCC CCL 165). XC cells were first 
seeded in roller bottle (Polystyrene, area surface=850 Cm.sup.2, Coming) 
in Dulbecco's Modification of Eagle's Medium (DMEM) containing CO.sub.2, 
10% fetal bovine serum (FBS), non-essential amino acids, penicillin and 
streptomycin for 3 days. XC cells were then washed with PBS (3.times.100 
ml) to remove serum and then grown in 100 ml of DMEM containing no FBS 
(with CO.sub.2), non-essential amino acids, penicillin and streptomycin) 
for 4 days. The conditioned medium was collected and clarified by 
centrifugation. 
B. Preparation of C3H 10T1/2 Condition Medium for Isolation of Apogen P-2 
Apogen P-2 was isolated from the conditioned medium of a cell line called 
C3H10T1/2 which was derived from mouse embryo and is purchased from 
American Type Culture Collection (ATCC CCL 226). C3H 10T1/2 cells were 
first seeded in roller bottle (Polystyrene, area surface=850 Cm.sup.2, 
Coming) in alpha Modification of Eagle's Medium (alpha-MEM) containing 
CO.sub.2, 10% Fetal bovine serum (FBS), penicillin and streptomycin for 3 
days. C3H 10T1/2 cells were then washed with PBS (3.times.100 ml) to 
remove serum and then grown in 100 ml of alpha MEM containing no FBS (with 
CO.sub.2, penicillin and streptomycin) for 4 days. The conditioned medium 
was collected and clarified by centrifugation. 
C. Preparation of XC Condition Medium for Isolation of Apogen L 
Apogen L was isolated from the conditioned medium of a cell line called XC 
which was derived from rat tumor (ATCC CCL 165). XC cells were first 
seeded in roller bottle (Polystyrene, area surface=850 Cm.sup.2, Corning) 
in Dulbecco's Modification of Eagle's Medium (DMEM) containing penicillin, 
streptomycin, CO.sub.2, non-essential amino acids and 10% Fetal bovine 
serum (FBS) for 4 days. The conditioned medium was collected and clarified 
by centrifugation. 
2. Assays 
(a) Cell Death (Apoptosis) Assay 
Prostate cancer cell line LNCAP was routinely used for the isolation of 
Apogen P-1 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% 
CO.sub.2 in Microtray plates (25 .mu.l wells, Robbins Scientific Corp.). 
Tested sample (10 .mu.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. Two hours later, 
cells that were stained with Hoechst dye were examined under fluorescence 
microscope. The nuclei of apoptotic cells showed DNA condensation and 
fragmentation are easily be identified by Hoechst dye staining. The 
percentage of apoptotic cells was calculated by the following equation: 
EQU % Apoptotic cells=Number of cells with DNA condensation and 
fragmentation/Total cell number 
(b) Cell Repelling Assay 
There are two reasons that Hep G2 cells are chosen for the study of cell 
repelling activity. First, Hep G2 cells are not sensitive to Apogen P-1 in 
inducing apoptosis. Secondly, 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, 
Mass.) was used to discover the chemorepellent activity of Apogen P-1b. 
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 were 
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 membrane than the control sample. In our 
experiments, 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 (Minimum 
Essential Medium Eagle containing 10% FBS, PS and nonessential amino acid, 
0.1 ml) for 2 hours and then the partially purified Apogen-1b (30 .mu.l) 
isolated by ammonium sulfate precipitation and Q2 HPLC chromatography as 
described above was placed in the lower chamber which contains 0.6 ml of 
the same growth medium for Hep G2 cells. 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 min. Cells in ten microliters of the 
trypsin solution were counted in a Hemacytometer. 
3. Protein Isolation 
A. Isolation of Apogen P-1 
Step 1: Ammonium Sulfate Precipitation 
Apogen P-1 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. 
Step 2: Q2 HPLC 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-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. We 
collected fractions 5-7 and named it Apogen P-1a, fractions 8-10 is named 
Apogen P-1b and fractions 11-14 is named Apogen P-1c. These three Apogen 
P-1's were further purified by a reverse phase column. 
Step 3: Reverse Phase Chromatography 
Apogen P-1a, Apogen P-1b and Apogen P-1c were separately concentrated to 
1.5 ml. One ml of methanol containing 0.05% Trifluoracetic acid was added. 
In each samples, large amount of proteins were precipitated by this 
treatment. Whereas, 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 (H.sub.2 O, 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. 
Step 4: Preparative Electrophoresis 
Apogen 1c 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-1a 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-1b is shown in FIG. 4(b). 
fractions 14 and 15 have activity inducing 45% cell death in LNCAP cells 
at 18 hr. 
The reverse phase chromatogram of Apogen P-1c 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-1a, Apogen P-1b and Apogen P-1c were 
checked with SDS-polyacrylamide gel electrophoresis stained with silver 
staining. 
(1) Apogen P-1a: As shown in FIG. 5, a protein band with molecular weight 
of 70 KD was obtained. This result suggest the nearly successful 
purification of Apogen p-1a which have molecular weight of 70 KD on 
SDSPAGE. 
(2) Apogen P-1b: A single faint protein band with molecular weight of 55 KD 
was obtained. This result suggest the successful purification of Apogen 
P-1b which have molecular weight of 55 KD on SDS-PAGE. 
(3) Apogen P-1c: The purification of Apogen 1c by Reverse Phase 
chromatography leads to the Isolation of a 70 KD protein whereas the 
purification of Apogen 1c 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 
The Apogen P-2 present in C3H10T1/2 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 
After removal of ammonium sulfate by dialysis in 10 mM Tris-HCI (pH 7.5), 
the dissolved proteins were incubated with Hydroxylapatite gel (Bio-Gel 
HTP gel, Bio-Rad) for 1 hr. After remove HTP gel by centrifugation, the 
activity inducing apoptosis in LNCAP cells was found to be present in the 
supernatant which was then further treated with Heparin agarose gel. 
Step 3: Heparin Agarose Treatment 
The supernatant from step 2 was further incubated with Heparin agarose 
(Sigma) for 1 Hr. After remove HTP gel by centrifugation, the activity 
inducing apoptosis in LNCAP cells was found to be present in the 
supernatant. 
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. Whereas, 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 (H.sub.2 O, 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 Apogen L present in the conditioned medium was isolated by the 
following steps: 
Step 1: DE52 Absorption 
The conditioned medium was incubated with the anion exchanger, DE 52 
(Diethylaminoethyl cellulose, Whatman) for 1 hr. The incubation mixture 
was centrifuged and DE 52 which binds Apogen L was collected and washed 
with 10 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl. Apogen L was then 
eluted from DE 52 cellulose by 10 mM Tris-HCl (pH 7.5) containing 0.5 M 
NaCl. 
Step 2: Heparin Agarose Absorption 
Apogen L isolated as described in step 1 was further absorbed by Heparin 
agarose (Sigma) by incubating Apogen L with Heparin agarose for 1 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 NaCI. 
Step 3: Q2 HPLC Chromatography 
Apogen L isolated as described above 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-HCl, pH 7.4) and buffer B (10 mM 22 
Tris-HCI, pH 7.4. 0.5 M NaCl) using Bio-Rad's BioLogic HPLC system. 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 SDS polyacrylamide gel 
electrophoresis stained with silver staining. A single protein band have 
activity with molecular weight of approximately 55 Kd was obtained (FIG. 
11). 
4. Isolation of Bovine Fetuin as Component of Protein P-2 and Apoptotic 
Effect Thereof in Tumor Cell Lines 
The observation that Apogen P-1a, P-1b, P-1c, P-2 and L were isolated from 
embryonic cell lines led us to speculate that new born or embryonic tissue 
may secrete "Apogen" that may selectively induce apoptosis in tumor cell 
lines. Due to this speculation, a protein named "Fetuin" has thus raised 
our attention based on the following reasons: (1) Fetuin is mainly a fetal 
protein, in the sense that the highest concentrations are found in serum 
and body fluids of embryos and fetuses. For example, the concentration of 
fetuin in bovine serum drastically decreases, probably within a few days 
after birth, to 1-2% of the fetal level (Yang, et al., Biochim. Biophy. 
Acta. 1130, 149-156 1992) (2) A histochemical study has shown that fetuin 
may control tissue remodelling and physiological cell death during 
embryonic development (Von Bulow, et al., Histochemistry 99:13-22, 1993). 
This result raises the possibility that fetuin may contain activity 
inducing cell death (apoptosis). 
Additionally, a protein with an amino acid sequence identical to Fetuin was 
isolated from the preparation of Apogen P-2. Thus, the composition of 
Apogen P-2 consists at least in part of fetuin. 
We therefore prepare/obtain fetuin and test in our apoptosis assay. 
Interestingly, we found that only bovine fetuin that is prepared by a 
special method is able to induce apoptosis in tumor cell lines. The 
commercial fetuin that is prepared by ammonium sulfate precipitation and 
EDTA treatment was found to contain very low activity in inducing 
apoptosis in tumor cells. 
A. Preparation of Bovine Fetuin 
Bovine fetuin was prepared by the modified Spiro method (Spiro R. G. 
Journal of Biological Chemistry 235, 10: 2860, 1960) according to the 
following steps: 
1. One hundred milliliters of Fetal Bovine Serum (FBS). 
2. Add two hundred milliliters of 0.05 M Zinc Acetate containing 30% (V/V) 
ethanol, adjust to pH 6.4 by 1M NH.sub.4 OH--NH.sub.4 Cl, let stand 15 
hours at -5.degree. C. 
3. Collect the supernatant by centrifugation, add 1.0 M Barium Acetate and 
95% ethanol to give 0.03 M Barium Acetate, 25% ethanol. Let stand 2 hours 
at -5.degree. C. 
4. Collect the supernatant by centrifugation, add 95% ethanol to give 40% 
ethanol. Let stand 15 hours at -10.degree. C. 
5. Collect the precipitate. Dissolve the pellet by Phosphate buffer saline. 
The purified fetuin showed a single protein band with apparent molecular 
weight of 63 Kd on SDS-PAGE. 
B. Induction of Apoptosis in Tumor Cell Lines Using Bovine Fetuin 
Fetuin purified from fetal bovine serum by the procedure described above 
was dissolved in phosphate buffer saline (PBS). The free Zinc Acetate and 
Barium Acetate were removed by repetitive concentration. Fetuin was tested 
in LNCaP and HL-60 cells. LNCAP or HL-60 (1,000 cells) was seeded 10 
microliters RPMI containing 15% or 20% Fetal bovine serum, penicillin and 
streptomycin at 37 degree, 5% CO.sub.2 in microtray plates (25 .mu.l 
wells, Robbins Scientific Corp.). Fetuin (in 10 .mu.l PBS) at 
concentration of 100 ng/ml 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. Two hours later, 
cells that were stained with Hoechst dye were examined under fluorescence 
microscope. The nuclei of apoptotic cells showed DNA condensation and 
fragmentation can be easily identified by Hoechst dye staining. The 
percentage of apoptotic cells was calculated by the following equation: 
EQU % Apoptotic cells=Number of cells with DNA condensation and 
fragmentation/Total cell number 
As shown in FIG. 16(A), the nuclei of the LNCaP cells that have been 
incubated with control sample (PBS) are normal and healthy (A). However, 
the nuclei of the LNCAP cells that have been incubated with fetuin (100 
ng/ml in PBS) show the characteristics of apoptosis (FIG. 16(B)). First, 
the cells in the presence of fetuin showed the condensation of nucleus, 
demonstrated by the more intense fluorescent light compared with the 
control nucleus in FIG. 16(A). Secondly, the nucleus condensation is 
accompanied by the fragmentation of DNA, demonstrated by the breakage of 
nucleus as shown in FIG. 16(B). As the nucleus condensation and DNA 
fragmentation are the two morphological characteristics of cells under 
apoptosis. These results suggest that fetuin contains an activity inducing 
apoptosis in LNCAP cells. As shown in FIG. 17(A), the nuclei of the HL-60 
cells that have been incubated with control buffer (PBS) are normal and 
healthy(A). However, the nuclei of the HL-60 cells that have been 
incubated with fetuin show the characteristics of apoptosis (FIG. 17(B)). 
Fetuin causes the condensation of nucleus, demonstrated by the more 
intense fluorescent light compared with the control nucleus in FIG. 17(A). 
Secondly, the nucleus condensation is accompanied by the fragmentation of 
DNA, demonstrated by the breakage of nucleus as shown in FIG. 17(B). As we 
have mentioned above, the nucleus condensation and DNA fragmentation are 
the two morphological characteristics of cells under apoptosis. These 
results suggest that fetuin contains an activity inducing apoptosis in 
HL-60 cells. 
(C) Bovine Fetuin Selectively Induces Apoptosis in Cancer Without Having 
Effect on Normal Cell Lines 
We compared the effect of fetuin on the induction of apoptosis in various 
cell lines. As shown in FIG. 18, at concentration 50 .mu.g/ml, fetuin 
prepared as described above strongly induced apoptosis in tumor cell lines 
such as: LNCAP (prostate cancer), PC-3 (prostate cancer), HL-60 
(leukemia), MCF-7 (breast cancer), Colo 205 (colon cancer), Calu-1 (lung 
cancer). Normal lung fibroblast (CCD 39 Lu) on the other hand, is not 
affected by fetuin. 
Fetuin was found to be inactive in inducing apoptosis in CCD 39 Lu cells 
(normal lung fibroblast) at the concentration (25 .mu.g/ml) that highly 
induced apoptosis in LNCAP (prostate cancer) or HL-60 cells (leukemia). 
Fetuin (25 .mu.g/ml) prepared as described above was incubated with CCD 39 
Lu cells grown in MEM in microtray plate for 15 hours. As shown in FIG. 
19, the CCD 39 Lu cells remained morphologically unchanged in the presence 
of fetuin (FIG. 19B). At this concentration (25 .mu.g/ml) of fetuin, as 
shown in FIG. 20, in the presence of fetuin, less MCF-7 cell remaining, 
due to cell death and cell shrinkage, was observed (FIG. 20(B)) 
(D) Only Fetuin Prepared by the Method Described Above is Able to Induce 
Apoptosis in Tumor Cell Lines 
We found that fetuin purchased from Sigma have very low activity in 
inducing apoptosis in LNCAP cells. However, fetuin (25 .mu.g/ml) prepared 
in our laboratory by the method described in Section 4.A above induce 
apoptosis in LNCaP cells by up to 90% in 4 hours. For the fetuin purchased 
from Sigma, apoptosis inducing activity was observed only at a very high 
concentration (&gt;250 .mu.g/ml) and at long incubation time (2 days). We 
estimated that the activity of fetuin prepared in our laboratory is more 
than fifty thousand folds higher than that of fetuin prepared by other 
methods. 
We have examined the preparation method for Sigma's fetuin and found that 
these fetuins are prepared by methods including ammonium sulfate 
precipitation and EDTA treatment. Both treatments may cause the 
deprivation of Zinc ion from the protein which may cause the irreversible 
loss of the protein activity. 
DISCUSSION 
This invention describes the methods for the isolation of five proteins 
(Apogen P-1a, Apogen P-1b, Apogen P-1c, Apogen P-2 and Apogen L) that are 
able to induce apoptosis in prostate cancer cells (Apogen P-1's), in 
prostate cancer cells and breast cancer cells (Apogen P-2), and leukemia 
and breast cancer cells (Apogen L) , as well as the identification of 
fetuin as a component of Apogen P-2. The following evidence lead us to 
believe that these apoptosis-inducing proteins are novel and that they 
have never been found before: Tumor Necrosis Factor (TNF), Transforming 
Growth Factor (TGF-Beta), Fas ligand and TRAIL are the proteins reported 
to induce apoptosis in certain cell lines. (Lin, J. K. et al., Cancer 
Research 52:385, 1992. Kawakawi, et al., J. of Cellular Physiology 138:1, 
1989; Wiley, S. R. et al., Immunity 3:673, 1995; Krammer, et al. 
"Apoptosis in the APO-1 System", Apoptosis: The molecular Basis of Cell 
Death, Cold Spring Harbor Laboratory Press p. 87, 1991). Evidences 
suggested that these five proteins are different from any of these known 
proteins inducing apoptosis as described below: 
(1) The activities are different. In our assays, TNF and TGF induced 
apoptosis in liver cancer cells without effects in prostate cancer (LNCAP 
cells) even a very high dose (100 ng/ml) are used. Whereas Apogen P-1's 
and Apogen P-2 induced apoptosis in prostate cancer rather than in cancer 
liver cells. 
(2) TRAIL and Fas are membrane bound proteins, (Wiley, S. R. et al. 
Immunity 3:673, 1995; Krammer, et al., "Apoptosis in the APO-1 System", 
Apoptosis: The molecular Basis of Cell Death, Cold Spring Harbor 
Laboratory Press, p. 87, 1991) whereas the Apogen P-1a, Apogen P-1b, 
Apogen P-1c, Apogen P-2 and Apogen L are all soluble (non-membrane bound) 
proteins. 
(3) The molecular weights of TNF, TGF and Fas ligand TRAIL are around 17-40 
Kd (TNF=17 KD, TGF=24 KD, TRAIL=32 KD, Fas ligand=43 KD) (McGrath, M. H. 
Clinics in Plastic surgery 17:421, 1993; Wiley, S. R. et al., Immunity 
3:673, 1995; Krammer, et al., "Apoptosis in the APO-1 System", Apoptosis: 
The molecular Basis of Cell Death, Cold Spring Harbor Laboratory Press, p. 
87, 1991) whereas the molecular weight of Apogen P-1a, Apogen P-1b, Apogen 
P-1c, Apogen P-2 and Apogen L are between 55-70 Kd.