U.S. patent application number 10/801207 was filed with the patent office on 2004-09-09 for reagents and methods for identifying and modulating expression of genes regulated by p21.
Invention is credited to Chang, Bey-Dih, Roninson, Igor.
Application Number | 20040175748 10/801207 |
Document ID | / |
Family ID | 23784727 |
Filed Date | 2004-09-09 |
United States Patent
Application |
20040175748 |
Kind Code |
A1 |
Chang, Bey-Dih ; et
al. |
September 9, 2004 |
Reagents and methods for identifying and modulating expression of
genes regulated by p21
Abstract
This invention provides methods and reagents for identifying
genes involved in cell cycle progression, growth promotion,
modulation of apoptosis, cellular senescence and aging, and methods
for identifying compounds that inhibit or potentiate cellular
senescence.
Inventors: |
Chang, Bey-Dih; (Lombard,
IL) ; Roninson, Igor; (Wilmette, IL) |
Correspondence
Address: |
McDonnell Boehnen Hulbert & Berghoff LLP
32nd Floor
300 S. Wacker Drive
Chicago
IL
60606
US
|
Family ID: |
23784727 |
Appl. No.: |
10/801207 |
Filed: |
March 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10801207 |
Mar 16, 2004 |
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09449589 |
Nov 29, 1999 |
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6706491 |
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60128676 |
Apr 9, 1999 |
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Current U.S.
Class: |
435/5 ; 435/6.14;
435/7.2; 800/12 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 14/4738 20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/006 ;
435/007.2; 800/012 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567; A01K 067/00 |
Claims
We claim:
1. A method for identifying a compound that inhibits
senescence-associated changes in cellular gene expression in a
mammalian cell, the method comprising the steps of: (a) treating
the mammalian cell in the presence and absence of the compound with
an agent that induces senescence or culturing the mammalian cell
under conditions that induce senescence; (b) assaying the mammalian
cell for repression or induction of a cellular gene that is
repressed or induced by p21 gene expression; and (c) identifying
the compound as an inhibitor of senescence-associated changes in
cellular gene expression if the gene that is repressed by p21 is
repressed to lesser extent, or the gene that is induced by p21 is
induced to a lesser extent, in the presence of the compound than in
the absence of the compound.
2. The method of claim 1, wherein the mammalian cell is assayed for
a cellular gene that is induced by p21.
3. The method of claim 2, wherein the gene is Fibronectin 1 (Acc.
No. X02761), Plasminogen activator inhibitor, type I (Acc. No.
M14083), Plasminogen activator, tissue type (Acc. No. M15518),
Laminin .beta.2 (Acc. No. X79683), Desmocollin 2a/bb (Acc. No.
X56807), Podocalyxin-like protein (Acc. No. U97519), Activin A
(inhibin .beta.A) (Acc. No. J03634), Galectin 3 (Mac-2) (Acc. No.
AB006780), Mac-2 binding protein (Acc. No. L13210), Prosaposin
(Acc. No. J03077), CTGF (connective tissue growth factor) (Acc. No.
M92934), Granulin/epithelin (Acc. No. AF055008), Cathepsin B (Acc.
No. L04288), Tissue transglutaminase (Acc. No. M55153), P37NB (slit
homolog) (Acc. No. U32907), Serum amyloid A protein precursor (Acc.
No. M26152), Alzheimer's disease amyloid A4 protein precursor (Acc.
No. D87675), Complement C3 precursor (Acc. No. K02765), Testican
(Acc. No. X73608), Integrin .beta.3 (Acc. No. M35999),
N-acetylgalactosamine-6-- sulfate sulfatase (Acc. No. U06088), Acid
alpha-glucosidase (Acc. No. X55079), Acid lipase A (cholesterol
esterase) (Acc. No. X76488), Lysosomal pepstatin-insensitive
protease (CLN2) (Acc. No. AF017456), Superoxide dismutase 2 (Acc.
No. X07834), Metaxin (Acc. No. J03060), 2,4-dienoyl-CoA reductase
(Acc. No. U78302), Ubiquitin-conjugating enzyme (UbcH8) (Acc. No.
AF031141), Ubiquitin-specific protease 8 (Acc. No. D29956),
RTP/Cap43/Drg1/Ndr1(Inducible by nickel, retinoids, homocysteine
and ER stress) (Acc. No. D87953), C-193 muscle ankyrin-repeat
nuclear protein (cytokine-inducible) (Acc. No. X83703), LRP major
vault protein associated with multidrug resistance (Acc. No.
X79882), .beta.-arrestin related HHCPA78 homolog (upregulated by
vitamin D3) (Acc. No. S73591), R-RAS (Acc. No. M14949), RAB 13
small GTPase (Acc. No. X75593), P66 SHC (ski oncogene) (Acc. No.
U73377), MK-STYX (MAP kinase phosphatase-like protein) (Acc. No.
N75168), H73 nuclear antigen/MA-3 apoptosis-related/TIS
(topoisomerase-inhibitor suppressed) (Acc. No. U96628), Natural
killer cells protein 4 (Acc. No. M59807), TXK tyrosine kinase
(T-cell specific) (Acc. No. L27071), X-linked PEST-containing
transporter (Acc. No. U05321), AMP deaminase 2 (Acc. No. M91029),
FIP2/HYPL huntingtin-interacting protein (Acc. No. AF061034), DNASE
I homolog (Acc. No. X90392), Transcription factor 11 (Acc. No.
X77366), Histone H2A.2 (Acc. No. L19779), Histone H2B (Acc. No.
AL021807), 23808 (Acc. No. AF038192), CGI-147 (Acc. No. AA307912),
EST (Acc. No. W89120), EST (Acc. No. A1026140), EST (Acc. No.
AA218982), or EST (Acc. No. W63684).
4. The method of claim 1, wherein the mammalian cell is assayed for
a cellular gene that is repressed by p21.
5. The method of claim 4, wherein the gene is CDC2 (Acc. No.
X05360), CKsHs1 (CDC2 kinase) (Acc. No. X54941), PLK1 (polo-like
kinase) (Acc. No. U01038), XCAP-H condensin homolog (Acc. No.
D38553), CENP-A (centromere protein A) (Acc. No. U14518), CENP-F
(centromere protein F) (Acc. No. U30872), MAD2 (Acc. No. U65410),
BUBR1 (Acc. No. AF053306), MCAK (mitotic centromere-associated
kinesin) (Acc. No. U63743), HSET kinesin-like protein (Acc. No.
AL021366), CHL1 helicase (Acc. No. U75968), AIK-1
(aurora/IPL1-related kinase) (Acc. No. D84212), AIM-1 (AIK-2;
aurora/IPL1-related kinase) (Acc. No. AF004022), PRC1 (protein
regulating cytokinesis 1) (Acc. No. AF044588), Citron kinase (Acc.
No. H10809), Lamin B1 (Acc. No. L37747), Lamin B2 (Acc. No.
M94362), LAP-2 (lamin-associated protein 2) (Acc. No. U18271), MPP2
(M phase phosphoprotein 2) (Acc. No. U74612), MPP5 (M phase
phosphoprotein 5) (Acc. No. X98261), Thymidine kinase 1 (Acc. No.
K02581), Thymidylate synthase (Acc. No. X02308), Uridine
phosphorylase (Acc. No. X90858), Ribonucleotide reductase M1 (Acc.
No. X59543), Ribonucleotide reductase M2 (Acc. No. X59618), CDC47
homolog (MCM7) (Acc. No. D55716), CDC21 homolog (MCM4) (Acc. No.
X74794), CDC45 homolog (Porc-PI) (Acc. No. AJ223728), HsORC1
(origin recognition complex 1) (Acc. No. U40152), DNA polymerase
.alpha. (Acc. No. X06745), Replication factor C (37-kD subunit)
(Acc. No. M87339), B-MYB (Acc. No. X13293), HPV16 E1 protein
binding protein (Acc. No. U96131), Topoisomerase II.alpha. (Acc.
No. J04088), Chromatin assembly factor-I (p60 subunit) (Acc. No.
U20980), High-mobility group chromosomal protein 2 (Acc. No.
X62534), High-mobility group chromosomal protein 1 (Acc. No.
D63874), Histone H2A.F/Z variant (Acc. No. AA203494), XRCC9 (Acc.
No. U70310), RAD54 homolog (Acc. No. X97795), HEX1 5'-3'exonuclease
(RAD2 homolog) (Acc. No. AF042282), ATP-dependent DNA ligase I
(Acc. No. M36067), RAD21 homolog (Acc. No. D38551), Putative
transcription factor CA150 (Acc. No. AF017789), Transcriptional
coactivator ALY (Acc. No. AF047002), WHSC1/MMSET (SET domain
protein) (Acc. No. AA401245), NN8-4AG (SET domain protein) (Acc.
No. U50383), EZH2 (enhancer of zeste homolog 2) (Acc. No. U61145),
PTB-associated splicing factor (Acc. No. X70944), AU-rich element
RNA-binding protein AUF1 (Acc. No. U02019), U-snRNP-associated
cyclophilin (Acc. No. AF016371), 3-phosphoglycerate dehydrogenase
(Acc. No. AF006043), L-type amino acid transporter, subunit LAT1
(Acc. No. M80244), Hyaluronan-mediated motility receptor (Acc. No.
U29343), Phorbolin I (PKC-inducible) (Acc. No. U03891), PSD-95
binding family protein (Acc. No. D13633), HTRIP (TNF receptor
component) (Acc. No. U77845), NAD-dependent
methylenetetrahydrofolate dehydrogenase (Acc. No. X16396), Membrane
glycoprotein 4F2 antigen heavy chain (Acc. No. J02939), Mucin-like
protein (Acc. No. 079992), MAC30 (differentially expressed in
meningiomas) (Acc. No. L19183), P52rIPK (regulator of
interferon-induced protein kinase) (Acc. No. AF007393), Putative
phosphoserine aminotransferase (Acc. No. AA192483), Glucose
6-phosphate translocase (Acc. No. Y15409), Calcyclin binding
protein (Acc. No. AF057356), Ornithine decarboxylase 1 (Acc. No.
X16277), Trophinin assisting protein (tastin) (Acc. No. U04810),
Acyl-coenzyme A cholesterol acyltransferase(Acc. No. L21934),
Pinin/SDK3 (Acc. No. Y10351), EST (Acc. No. AA975298), EST (Acc.
No. AA034414), EST (Acc. No. AA482549), Cyclin A1 (Acc. No.
U66838), Cyclin B1 (Acc. No. M25753), CDC25A (Acc. No.
NM.sub.--001789), Dihydrofolate reductase (Acc. No. J00140), or
ING1 (Acc. No. NM.sub.--005537).
6. The method of claim 1, wherein expression of the cellular gene
is detected using an immunological reagent.
7. The method of claim 1, wherein expression of the cellular gene
is detected by assaying for an activity of the cellular gene
product.
8. The method of claim 1, where expression of the cellular gene is
detected by hybridization to a complementary nucleic acid.
9. A method for identifying a compound that promotes
senescence-associated changes in cellular gene expression in a
mammalian cell, the method comprising the steps of: (d) treating
the mammalian cell with an agent that induces senescence or
culturing the mammalian cell under conditions that induce
senescence in the presence and absence of the compound; (e)
assaying the mammalian cell for repression or induction of a
cellular gene that is repressed or induced by p21 gene expression;
and (f) identifying a compound that promotes senescence-associated
changes in cellular gene expression if a gene that is repressed by
p21 is repressed in the presence of the compound, or a gene that is
induced by p21 is induced in the presence of the compound.
10. The method of claim 9, wherein the mammalian cell is assayed
for a cellular gene that is induced by p21.
11. The method of claim 10, wherein the gene is Fibronectin 1 (Acc.
No. X02761), Plasminogen activator inhibitor, type I (Acc. No.
M14083), Plasminogen activator, tissue type (Acc. No. M15518),
Laminin .beta.2 (Acc. No. X79683), Desmocollin 2a/bb (Acc. No.
X56807), Podocalyxin-like protein (Acc. No. U97519), Activin A
(inhibin .beta.A) (Acc. No. J03634), Galectin 3 (Mac-2) (Acc. No.
AB006780), Mac-2 binding protein (Acc. No. L13210), Prosaposin
(Acc. No. J03077), CTGF (connective tissue growth factor) (Acc. No.
M92934), Granulin/epithelin (Acc. No. AF055008), Cathepsin B (Acc.
No. L04288), Tissue transglutaminase (Acc. No. M55153), P37NB (slit
homolog) (Acc. No. U32907), Serum amyloid A protein precursor (Acc.
No. M26152), Alzheimer's disease amyloid A4 protein precursor (Acc.
No. D87675), Complement C3 precursor (Acc. No. K02765), Testican
(Acc. No. X73608), Integrin .beta.3 (Acc. No. M35999),
N-acetylgalactosamine-6-- sulfate sulfatase (Acc. No. U06088), Acid
alpha-glucosidase (Acc. No. X55079), Acid lipase A (cholesterol
esterase) (Acc. No. X76488), Lysosomal pepstatin-insensitive
protease (CLN2) (Acc. No. AF017456), Superoxide dismutase 2 (Acc.
No. X07834), Metaxin (Acc. No. J03060), 2,4-dienoyl-CoA reductase
(Acc. No. U78302), Ubiquitin-conjugating enzyme (UbcH8) (Acc. No.
AF031141), Ubiquitin-specific protease 8 (Acc. No. D29956),
RTP/Cap43/Drg1/Ndr1(Inducible by nickel, retinoids, homocysteine
and ER stress) (Acc. No. D87953), C-193 muscle ankyrin-repeat
nuclear protein (cytokine-inducible) (Acc. No. X83703), LRP major
vault protein associated with multidrug resistance (Acc. No.
X79882), .beta.-arrestin related HHCPA78 homolog (upregulated by
vitamin D3) (Acc. No. S73591), R-RAS (Acc. No. M14949), RAB 13
small GTPase (Acc. No. X75593), P66 SHC (ski oncogene) (Acc. No.
U73377), MK-STYX (MAP kinase phosphatase-like protein) (Acc. No.
N75168), H73 nuclear antigen/MA-3 apoptosis-related/TIS
(topoisomerase-inhibitor suppressed) (Acc. No. U96628), Natural
killer cells protein 4 (Acc. No. M59807), TXK tyrosine kinase
(T-cell specific) (Acc. No. L27071), X-linked PEST-containing
transporter (Acc. No. U05321), AMP deaminase 2 (Acc. No. M91029),
FIP2/HYPL huntingtin-interacting protein (Acc. No. AF061034), DNASE
I homolog (Acc. No. X90392), Transcription factor 11 (Acc. No.
X77366), Histone H2A.2 (Acc. No. L19779), Histone H2B (Acc. No.
AL021807), 23808 (Acc. No. AF038192), CGI-147 (Acc. No. AA307912),
EST (Acc. No. W89120), EST (Acc. No. AI026140), EST (Acc. No.
AA218982), or EST (Acc. No. W63684).
12. The method of claim 9, wherein the mammalian cell is assayed
for a cellular gene that is repressed by p21.
13. The method of claim 12, wherein the gene is CDC2(Acc. No.
X05360), CKsHs1 (CDC2 kinase) (Acc. No. X54941), PLK1 (polo-like
kinase) (Acc. No. U01038), XCAP-H condensin homolog (Acc. No.
D38553), CENP-A (centromere protein A) (Acc. No. U14518), CENP-F
(centromere protein F) (Acc. No. U30872), MAD2 (Acc. No. U65410),
BUBR1 (Acc. No. AF053306), MCAK (mitotic centromere-associated
kinesin) (Acc. No. U63743), HSET kinesin-like protein (Acc. No.
AL021366), CHL1 helicase (Acc. No. U75968), AIK-1
(aurora/IPL1-related kinase) (Acc. No. D84212), AIM-1 (AIK-2;
aurora/IPL1-related kinase) (Acc. No. AF004022), PRC1 (protein
regulating cytokinesis 1) (Acc. No. AF044588), Citron kinase (Acc.
No. H10809), Lamin B1 (Acc. No. L37747), Lamin B2 (Acc. No.
M94362), LAP-2 (lamin-associated protein 2) (Acc. No. U18271), MPP2
(M phase phosphoprotein 2) (Acc. No. U74612), MPP5 (M phase
phosphoprotein 5) (Acc. No. X98261), Thymidine kinase 1 (Acc. No.
K02581), Thymidylate synthase (Acc. No. X02308), Uridine
phosphorylase (Acc. No. X90858), Ribonucleotide reductase M1 (Acc.
No. X59543), Ribonucleotide reductase M2 (Acc. No. X59618), CDC47
homolog (MCM7) (Acc. No. D55716), CDC21 homolog (MCM4) (Acc. No.
X74794), CDC45 homolog (Porc-PI) (Acc. No. AJ223728), HsORC1
(origin recognition complex 1) (Acc. No. U40152), DNA polymerase
.alpha. (Acc. No. X06745), Replication factor C (37-kD subunit)
(Acc. No. M87339), B-MYB (Acc. No. X13293), HPV16 E1 protein
binding protein (Acc. No. U96131), Topoisomerase II.alpha. (Acc.
No. J04088), Chromatin assembly factor-I (p60 subunit) (Acc. No.
U20980), High-mobility group chromosomal protein 2 (Acc. No.
X62534), High-mobility group chromosomal protein 1 (Acc. No.
D63874), Histone H2A.F/Z variant (Acc. No. AA203494), XRCC9 (Acc.
No. U70310), RAD54 homolog (Acc. No. X97795), HEX1 5'-3'exonuclease
(RAD2 homolog) (Acc. No. AF042282), ATP-dependent DNA ligase I
(Acc. No. M36067), RAD21 homolog (Acc. No. D38551), Putative
transcription factor CA150 (Acc. No. AF017789), Transcriptional
coactivator ALY (Acc. No. AF047002), WHSC1/MMSET (SET domain
protein) (Acc. No. AA401245), NN8-4AG (SET domain protein) (Acc.
No. U50383), EZH2 (enhancer of zeste homolog 2) (Acc. No. U61145),
PTB-associated splicing factor (Acc. No. X70944), AU-rich element
RNA-binding protein AUF1 (Acc. No. U02019), U-snRNP-associated
cyclophilin (Acc. No. AF016371), 3-phosphoglycerate dehydrogenase
(Acc. No. AF006043), L-type amino acid transporter, subunit LAT1
(Acc. No. M80244), Hyaluronan-mediated motility receptor (Acc. No.
U29343), Phorbolin I (PKC-inducible) (Acc. No. U03891), PSD-95
binding family protein (Acc. No. D13633), HTRIP (TNF receptor
component) (Acc. No. U77845), NAD-dependent
methylenetetrahydrofolate dehydrogenase (Acc. No. X16396), Membrane
glycoprotein 4F2 antigen heavy chain (Acc. No. J02939), Mucin-like
protein (Acc. No. 079992), MAC30 (differentially expressed in
meningiomas) (Acc. No. L19183), P52rIPK (regulator of
interferon-induced protein kinase) (Acc. No. AF007393), Putative
phosphoserine aminotransferase (Acc. No. AA192483), Glucose
6-phosphate translocase (Acc. No. Y15409), Calcyclin binding
protein (Acc. No. AF057356), Ornithine decarboxylase 1 (Acc. No.
X16277), Trophinin assisting protein (tastin) (Acc. No. U04810),
Acyl-coenzyme A cholesterol acyltransferase (Acc. No. L21934),
Pinin/SDK3 (Acc. No. Y10351), EST (Acc. No. AA975298), EST (Acc.
No. AA034414), EST (Acc. No. AA482549), Cyclin A1 (Acc. No.
U66838), Cyclin B1 (Acc. No. M25753), CDC25A (Acc. No.
NM.sub.--001789), Dihydrofolate reductase (Acc. No. J00140), or
ING1 (Acc. No. NM.sub.--005537).
14. The method of claim 9, wherein expression of the cellular gene
is detected using an immunological reagent.
15. The method of claim 9, wherein expression of the cellular gene
is detected by assaying for an activity of the cellular gene
product.
16. The method of claim 9, where expression of the cellular gene is
detected by hybridization to a complementary nucleic acid.
17. A method for identifying a compound that induces
senescence-associated changes in cellular gene expression in a
mammalian cell, the method comprising the steps of: (a) assaying a
mammalian cell in the presence and absence of the compound for
expression of a gene whose expression is modulated by p21; and (b)
identifying a compound that induces senescence-associated changes
in cellular gene expression if expression of a gene that is
repressed by p21 is repressed in the cell, or expression of a gene
that is induced by p21 is increased in the cell, to a greater
extent in the presence than in the absence of the compound.
18. The method of claim 17, wherein the mammalian cell is assayed
for a cellular gene that is induced by p21.
19. The method of claim 18, wherein the gene is Fibronectin 1 (Acc.
No. X02761), Plasminogen activator inhibitor, type I (Acc. No.
M14083), Plasminogen activator, tissue type (Acc. No. M15518),
Laminin .beta.2 (Acc. No. X79683), Desmocollin 2a/bb (Acc. No.
X56807), Podocalyxin-like protein (Acc. No. U97519), Activin A
(inhibin .beta.A) (Acc. No. J03634), Galectin 3 (Mac-2) (Acc. No.
AB006780), Mac-2 binding protein (Acc. No. L13210), Prosaposin
(Acc. No. J03077), CTGF (connective tissue growth factor) (Acc. No.
M92934), Granulin/epithelin (Acc. No. AF055008), Cathepsin B (Acc.
No. L04288), Tissue transglutaminase (Acc. No. M55153), P37NB (slit
homolog) (Acc. No. U32907), Serum amyloid A protein precursor (Acc.
No. M26152), Alzheimer's disease amyloid A4 protein precursor (Acc.
No. D87675), Complement C3 precursor (Acc. No. K02765), Testican
(Acc. No. X73608), Integrin .beta.3 (Acc. No. M35999),
N-acetylgalactosamine-6-- sulfate sulfatase (Acc. No. U06088), Acid
alpha-glucosidase (Acc. No. X55079), Acid lipase A (cholesterol
esterase) (Acc. No. X76488), Lysosomal pepstatin-insensitive
protease (CLN2) (Acc. No. AF017456), Superoxide dismutase 2 (Acc.
No. X07834), Metaxin (Acc. No. J03060), 2,4-dienoyl-CoA reductase
(Acc. No. U78302), Ubiquitin-conjugating enzyme (UbcH8) (Acc. No.
AF031141), Ubiquitin-specific protease 8 (Acc. No. D29956),
RTP/Cap43/Drg1/Ndr1 (Inducible by nickel, retinoids, homocysteine
and ER stress) (Acc. No. D87953), C-193 muscle ankyrin-repeat
nuclear protein (cytokine-inducible) (Acc. No. X83703), LRP major
vault protein associated with multidrug resistance (Acc. No.
X79882), .beta.-arrestin related HHCPA78 homolog (upregulated by
vitamin D3) (Acc. No. S73591), R-RAS (Acc. No. M14949), RAB 13
small GTPase (Acc. No. X75593), P66 SHC (ski oncogene) (Acc. No.
U73377), MK-STYX (MAP kinase phosphatase-like protein) (Acc. No.
N75168), H73 nuclear antigen/MA-3 apoptosis-related/TIS
(topoisomerase-inhibitor suppressed) (Acc. No. U96628), Natural
killer cells protein 4 (Acc. No. M59807), TXK tyrosine kinase
(T-cell specific) (Acc. No. L27071), X-linked PEST-containing
transporter (Acc. No. U05321), AMP deaminase 2 (Acc. No. M91029),
FIP2/HYPL huntingtin-interacting protein (Acc. No. AF061034), DNASE
I homolog (Acc. No. X90392), Transcription factor 11 (Acc. No.
X77366), Histone H2A.2 (Acc. No. L19779), Histone H2B (Acc. No.
AL021807), 23808 (Acc. No. AF038192), CGI-147 (Acc. No. AA307912),
EST (Acc. No. W89120), EST (Acc. No. A1026140), EST (Acc. No.
AA218982), or EST (Acc. No. W63684).
20. The method of claim 17, wherein the mammalian cell is assayed
for a cellular gene that is repressed by p21.
21. The method of claim 20, wherein the gene is CDC2 (Acc. No.
X05360), CKsHs1 (CDC2 kinase) (Acc. No. X54941), PLK1 (polo-like
kinase) (Acc. No. U01038), XCAP-H condensin homolog (Acc. No.
D38553), CENP-A (centromere protein A) (Acc. No. U14518), CENP-F
(centromere protein F) (Acc. No. U30872), MAD2 (Acc. No. U65410),
BUBR1 (Acc. No. AF053306), MCAK (mitotic centromere-associated
kinesin) (Acc. No. U63743), HSET kinesin-like protein (Acc. No.
AL021366), CHL1 helicase (Acc. No. U75968), AIK-1
(aurora/IPL1-related kinase) (Acc. No. D84212), AIM-1 (AIK-2;
aurora/IPL1-related kinase) (Acc. No. AF004022), PRC1 (protein
regulating cytokinesis 1) (Acc. No. AF044588), Citron kinase (Acc.
No. H10809), Lamin B1 (Acc. No. L37747), Lamin B2 (Acc. No.
M94362), LAP-2 (lamin-associated protein 2) (Acc. No. U18271), MPP2
(M phase phosphoprotein 2) (Acc. No. U74612), MPP5 (M phase
phosphoprotein 5) (Acc. No. X98261), Thymidine kinase 1 (Acc. No.
K02581), Thymidylate synthase (Acc. No. X02308), Uridine
phosphorylase (Acc. No. X90858), Ribonucleotide reductase M1 (Acc.
No. X59543), Ribonucleotide reductase M2 (Acc. No. X59618), CDC47
homolog (MCM7) (Acc. No. D55716), CDC21 homolog (MCM4) (Acc. No.
X74794), CDC45 homolog (Porc-PI) (Acc. No. AJ223728), HsORC1
(origin recognition complex 1) (Acc. No. U40152), DNA polymerase
.alpha. (Acc. No. X06745), Replication factor C (37-kD subunit)
(Acc. No. M87339), B-MYB (Acc. No. X13293), HPV16 E1 protein
binding protein (Acc. No. U96131), Topoisomerase II.alpha. (Acc.
No. J04088), Chromatin assembly factor-I (p60 subunit) (Acc. No.
U20980), High-mobility group chromosomal protein 2 (Acc. No.
X62534), High-mobility group chromosomal protein 1 (Acc. No.
D63874), Histone H2A.F/Z variant (Acc. No. AA203494), XRCC9 (Acc.
No. U70310), RAD54 homolog (Acc. No. X97795), HEX1 5'-3'exonuclease
(RAD2 homolog) (Acc. No. AF042282), ATP-dependent DNA ligase I
(Acc. No. M36067), RAD21 homolog (Acc. No. D38551), Putative
transcription factor CA150 (Acc. No. AF017789), Transcriptional
coactivator ALY (Acc. No. AF047002), WHSC1/MMSET (SET domain
protein) (Acc. No. AA401245), NN8-4AG (SET domain protein) (Acc.
No. U50383), EZH2 (enhancer of zeste homolog 2) (Acc. No. U61145),
PTB-associated splicing factor (Acc. No. X70944), AU-rich element
RNA-binding protein AUF1 (Acc. No. U02019), U-snRNP-associated
cyclophilin (Acc. No. AF016371), 3-phosphoglycerate dehydrogenase
(Acc. No. AF006043), L-type amino acid transporter, subunit LAT1
(Acc. No. M80244), Hyaluronan-mediated motility receptor (Acc. No.
U29343), Phorbolin I (PKC-inducible) (Acc. No. U03891), PSD-95
binding family protein (Acc. No. D13633), HTRIP (TNF receptor
component) (Acc. No. U77845), NAD-dependent
methylenetetrahydrofolate dehydrogenase (Acc. No. X16396), Membrane
glycoprotein 4F2 antigen heavy chain (Acc. No. J02939), Mucin-like
protein (Acc. No. 079992), MAC30 (differentially expressed in
meningiomas) (Acc. No. L19183), P52rIPK (regulator of
interferon-induced protein kinase) (Acc. No. AF007393), Putative
phosphoserine aminotransferase (Acc. No. AA192483), Glucose
6-phosphate translocase (Acc. No. Y15409), Calcyclin binding
protein (Acc. No. AF057356), Ornithine decarboxylase 1 (Acc. No.
X16277), Trophinin assisting protein (tastin) (Acc. No. U04810),
Acyl-coenzyme A cholesterol acyltransferase(Acc. No. L21934),
Pinin/SDK3 (Acc. No. Y10351), EST (Acc. No. AA975298), EST (Acc.
No. AA034414), EST (Acc. No. AA482549), Cyclin A1 (Acc. No.
U66838), Cyclin B1 (Acc. No. M25753), CDC25A (Acc. No.
NM.sub.--001789), Dihydrofolate reductase (Acc. No. J00140), or
ING1 (Acc. No. NM.sub.--005537).
22. The method of claim 17, wherein expression of the gene is
detected using an immunological reagent.
23. The method of claim 17, wherein expression of the gene is
detected by assaying for an activity of the cellular gene
product.
24. The method of claim 17, where expression of the gene is
detected by hybridization to a complementary nucleic acid.
25. The method of claim 17, wherein the mammalian cell comprises an
inducible p21 gene, and wherein the assay in subpart (a) is
performed in the presence of an amount of an agent that induces p21
expression whereby the extent of p21 induction is insufficient for
complete inhibition of genes inhibited by p21.
Description
[0001] This is a continuation of U.S. patent application Ser. No.
60/128,676, filed Apr. 9, 1999, the disclosure of which is
explicitly incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention is related to cellular senescence and changes
in cellular gene expression that accompany senescence. In
particular, the invention is related to the identification of genes
the expression of which is modulated by a cellular gene product,
p21, induced in cells at the onset of senescence. More
specifically, the invention provides markers of cellular senescence
that are genes whose expression in induced or repressed by p21. The
invention provides methods for identifying compounds that inhibit
or potentiate cellular senescence by detecting inhibition of
repression or induction of these marker genes. Also provided are
reagents that are recombinant mammalian cells containing a
recombinant expression construct encoding p21 that is
experimentally-inducible, and recombinant mammalian cells
containing a recombinant expression construct that expresses a
reporter gene under the transcriptional control of a promoter for a
gene that is regulated by p21.
[0004] 2. Summary of the Related Art
[0005] p21.sup.WAF1/CIP1/SDI1 is an important mediator of growth
arrest and senescence in mammalian cells. p21 has been
independently identified by several groups as a protein that binds
and inhibits cyclin-dependent kinases (CDK) (Harper et al., 1993,
Cell 75: 805-816), as a gene upregulated by wild-type p53 (el-Deiry
et al., 1993, Cancer Res. 55: 2910-2919), and as a
growth-inhibitory gene overexpressed in senescent fibroblasts (Noda
et al., 1994, Exp. Cell. Res. 211: 90-98). Because of its pivotal
role in p53-regulated growth arrest, p21 is usually regarded as a
tumor suppressor. Nevertheless, p21 mutations in human cancer are
rare (Hall & Peters, 1996, Adv. Cancer Res. 68: 67-108), and
p21 knockout mice develop normally and do not show all increased
rate of tumorigenesis (Deng et al., 1995, Cell 82: 675-684).
[0006] Cellular levels of p21 are increased in response to a
variety of stimuli. including DNA-damaging and differentiating
agents. Some of these responses are mediated through
transcriptional activation of the p21 gene by p53, but p21 is also
regulated by a variety of p53-independent factors (reviewed in
Gartel & Tyner, 1999, Exp. Cell Res. 227: 171-181). Increased
p21 expression leads to cell growth arrest (Noda et al., 1994,
ibid.), which occurs in both G1 and G2 (Niculescu et al., 1998,
Mol. Cell. Biol. 18: 629-643) and is accompanied by the development
of morphologic and phenotypic markers of senescence (Vogt et al.,
1998, Cell Growth Differ. 9: 139-146; McConnell et al., 1998, Curr.
Biol. 8: 351-354; Bates et al., 1998, Oncogene 17: 1691-1703; Fang
et al., 1999, Oncogene 18: 2789-2797).
[0007] Transient induction of p21 mediates different forms of
damage-induced growth arrest, including transient arrest that
allows cell to repair DNA damage, as well as permanent growth
arrest (also termed "accelerated senescence"), which is induced in
normal fibroblasts (DiLeonardo et al., 1994, Genes Develop. 8:
2540-255 1; Robles & Adami, 1998, Oncogene 16: 1113-1123) and
tumor cells (Chang et al., 1999, Cancer Res. 59: 3761-3767) by DNA
damage or introduction of oncogenic RAS (Serrano et al., 1997, Cell
88: 593-602). A surge of p21 expression also coincides with the
onset of terminal growth arrest during replicative senescence of
aging fibroblasts (Noda et al., 1994, ibid.; Alcorta et al., 1996,
Proc. Natl. Acad. Sci USA 93: 13742-13747; Stein et al., 1999, Mol.
Cell. Biol. 19: 2109-2117) and terminal differentiation of
postmitotic cells (El-Deiry et al., 1995, ibid.; Gartel et al.,
1996, Exp. Cell Res. 246: 280-289). Analysis of cells that cannot
express p21 (p21-/- homozygotes) demonstrated the requirement of
p21 in transient G1 and G2 arrest (Deng et al., 1995, ibid.;
Waldman et al., 1995, Cancer Res. 55: 5187-5190; Bunz et al., 1998,
Science 282: 1497-1501), in replicative senescence of normal
fibroblasts (Brown et al., 1997, Science 277: 831-834), and in
accelerated senescence of tumor cells (Chang et al., 1999, Oncogene
18: 4808-4818).
[0008] While p21 is not a transcription factor per se, it has
indirect effects on gene expression that may play a role in its
cellular functions. The best-known biochemical function of p21 is
the inhibition of CDK complexes that regulate transitions between
different phases of the cell cycle (reviewed in Gartel & Tyner,
1998, "The growth-regulatory role of p21 (WAF1/CIP1)," in
INHIBITORS OF CELL GROWTH, PROGESS IN MOLECULAR AND SUBCELLULAR
BIOLOGY, Vol. 20 (A. Macieir-Coelho, ed.), Springer-Verlag: Berlin
Heidelberg, pp. 43-71.). One of the consequences of CDK inhibition
is dephosphorylation of Rb, which in turn inhibits E2F
transcription factors that regulate many genes involved in DNA
replication and cell cycle progression (Nevins, 1998, Cell Growth
Differ. 9: 585-593). A comparison of p21-expressing cells (p21+/+)
and p21-nonexpressing cells (p21-/-) has implicated p21 in
radiation-induced inhibition of several E2F-regulated cellular
genes (de Toledo et al., 1998, Cell Growth Differ. 9: 887-896).
Another result of CDK inhibition by p21 is stimulation of
transcription cofactor p300 that augments NF.kappa.B (Perkins et
al., 1988, Science 275: 523-527 ). Activation of histone
acetyltransferase p300, that enhances many inducible transcription
factors, may have a pleiotropic effect on gene expression (Snowden
& Perkins. 1988. Biochem. Pharmacol. 55: 1947-1954). p21 may
also affect gene expression through its interactions with proteins
other than CDK. For example, p21 has been found to inhibit the
expression of keratinocyte differentiation markers; this effect was
dependent on the C-terminal portion of p21, which is not required
for CDK inhibition but is known to bind the proliferating cell
nuclear antigen (Di Cunto et al., 1998, Science 280: 1069-1072).
p21 was also reported to bind JNK kinases (Shim et al., 1996,
Nature 381: 804-807), apoptosis signal-regulating kinase I (Asada
et al., 1999, EMBO J. 18: 1223-1234), and Gadd45 (Kearsey et al.,
1995, Oncogene 11: 1675-1683); these interactions may affect the
expression of genes regulated by the corresponding pathways.
[0009] There remains a need in this art to identify genes whose
expression is modulated by induction of p21 gene expression. There
is also a need in this art to develop targets for assessing the
effects of compounds on cellular senescence, carcinogenesis and
age-related diseases.
SUMMARY OF THE INVENTION
[0010] This invention provides reagents and methods for identifying
genes whose expression is modulated by induction of p21 gene
expression. The invention also provides reagents and methods for
identifying compounds that inhibit or potentiate the effects of p21
on cellular gene expression, as a first step in rational drug
design for preventing cellular senescence, carcinogenesis and
age-related diseases or for increasing the efficacy of anticancer
therapies.
[0011] In a first aspect, the invention provides a mammalian cell
containing an inducible p21 gene. In preferred embodiments, the
mammalian cell is a recombinant mammalian cell comprising a
recombinant expression construct encoding an inducible p21 gene.
More preferably, the construct comprises a nucleotide sequence
encoding p21, most preferably human p21, under the transcriptional
control of an inducible promoter. In alternative embodiments, the
construct comprises a nucleotide sequence encoding the
amino-terminal portion of p21 comprising the CDK binding domain,
more preferably comprising amino acids 1 through 78 of the p21
amino acid sequence. In more preferred embodiments, the inducible
promoter can be induced by contacting the cells with an inducing
agent, most preferably a physiologically-neutral inducing agent,
that induces transcription from the inducible promoter or by
removing an agent that inhibits transcription from such promoter.
In a preferred embodiment, the mammalian cell is a fibrosarcoma
cell.
[0012] In another embodiment of the first aspect of the invention
are provided recombinant mammalian cells comprising a recombinant
expression construct in which a reporter gene is under the
transcriptional control of a promoter derived from a cellular gene
whose expression is modulated by p21. In a preferred embodiment,
the promoter is derived from a cellular gene whose expression is
repressed by p21. In these embodiments, the promoter is most
preferably derived from a gene identified in Table I. Most
preferably, the promoter is derived from ORC1, PRC1, XRCC9, CDC2,
cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1,
thymopoietin .alpha., MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA
ligase I, DNA polymerase .alpha., Rad54, exonuclease HEX1/RAD2, or
citron kinase. In other preferred embodiments, the promoter is
derived from a cellular gene whose expression is induced by p21. In
these embodiments, the promoter is most preferably derived from a
gene identified in Table II. Most preferably, the promoter is
derived from serum amyloid A, complement C3, connective tissue
growth factor, integrin .beta.-3, activin A, natural killer cell
protein 4, prosaposin, Mac2 binding protein, galectin-3, superoxide
dismutase 2, or cathepsin B. Preferred reporter genes comprising
the recombinant expression constructs of the invention include
firefly luciferase, chloramphenicol acetyltransferase,
beta-galactosidase, green fluorescent protein, or alkaline
phosphatase.
[0013] In additional preferred embodiments, the invention provides
a mammalian cell comprising a first recombinant expression
construct encoding a reporter gene under the transcriptional
control of a promoter for a mammalian gene whose expression is
modulated by p21, and a second recombinant expression construct
encoding a mammalian p21 gene, wherein expression of p21 is
experimentally-induced in the mammalian cell thereby. In preferred
embodiments, the recombinant expression construct encoding a
mammalian p21 gene is under the transcriptional control of an
inducible heterologous promoter, wherein expression of p21 from the
recombinant expression construct is mediated by contacting the
recombinant cell with an inducing agent that induces transcription
from the inducible promoter or by removing an agent that inhibits
transcription from such promoter. Preferably, the construct
comprises a nucleotide sequence encoding p21, most preferably human
p21. In alternative embodiments, the construct comprises a
nucleotide sequence encoding the amino-terminal portion of p21
comprising the CDK binding domain, more preferably comprising amino
acids 1 through 78 of the p21 amino acid sequence. In a preferred
embodiment, the promoter is derived from a cellular gene whose
expression is repressed by p21. In these embodiments, the promoter
is most preferably derived from a gene identified in Table I. In
other preferred embodiments, the promoter is derived from a
cellular gene whose expression is induced by p21. In these
embodiments, the promoter is most preferably derived from a gene
identified in Table II. Preferred reporter genes comprising the
recombinant expression constructs of the invention include firefly
luciferase, chloramphenicol acetyltransferase, beta-galactosidase,
green fluorescent protein, or alkaline phosphatase. In a preferred
embodiment, the mammalian cell is a fibrosarcoma cell.
[0014] In a second aspect, the invention provides a conditioned
cell culture medium wherein the medium is conditioned by cells
expressing p21. A method for producing said conditioned medium,
comprising the step of culturing p21-expressing cells in a
mammalian cell culture medium is also provided.
[0015] In a third aspect, the invention provides methods for
identifying compounds that inhibit p21-mediated modulation of
cellular gene expression. These methods comprise the steps of
inducing or otherwise producing p21 in a mammalian cell; assaying
the cell in the presence of the compound for changes in expression
of cellular genes whose expression is modulated by p21; and
identifying compounds that inhibit p21-mediated modulation of
cellular gene expression if expression of the cellular genes is
changed to a lesser extent in the presence of the compound than in
the absence of the compound. In preferred embodiments, the cellular
genes are repressed by p21, and inhibitors are detected by
detecting expression of the genes at levels greater than those
detected when p21 is expressed in the absence of the compound. In
preferred embodiments, the genes are identified in Table I. In
alternative preferred embodiments, the cellular genes are induced
by p21, and inhibitors are detected by detecting expression of the
genes at levels less than those detected when p21 is expressed in
the absence of the compound. In preferred embodiments, the genes
are identified in Table II. In further alternative embodiments, the
method is performed using a recombinant mammalian cell comprising a
reporter gene under the transcriptional control of a promoter
derived from a gene whose expression is modulated by p21. In these
embodiments using constructs comprising promoters derived from
genes repressed by p21, the reporter gene product is produced at
greater levels in the presence than in the absence of the compound
when the compound is an inhibitor of p21 gene expression
modulation. In these embodiments, the promoter is most preferably
derived from a gene identified in Table I. Most preferably, the
promoter is derived from ORC1, PRC1, XRCC9, CDC2, cyclin B1, AIK1,
CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1, thymopoietin
.alpha., MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA ligase I, DNA
polymerase .alpha., Rad54, exonuclease HEX1/RAD2, or citron kinase.
When using constructs comprising promoters derived from genes
induced by p21, the reporter gene product is produced at lesser
levels in the presence than the absence of the compound when the
compound is an inhibitor of p21 gene expression modulation. In
these embodiments, the promoter is most preferably derived from a
gene identified in Table II. Most preferably, the promoter is
derived from serum amyloid A, complement C3, connective tissue
growth factor, integrin .beta.-3, activin A, natural killer cell
protein 4, prosaposin, Mac2 binding protein, galectin-3, superoxide
dismutase 2, or cathepsin B. Preferred reporter genes comprising
the recombinant expression constructs of the invention include
firefly luciferase, chloramphenicol acetyltransferase,
beta-galactosidase, green fluorescent protein, or alkaline
phosphatase. In other preferred embodiments, the cell comprises a
first recombinant expression construct encoding a reporter gene
under the transcriptional control of a promoter for a mammalian
gene whose expression is modulated by p21, and a second recombinant
expression construct encoding a mammalian p21 gene, wherein
expression of p21 is experimentally-induced in the mammalian cell
thereby. The product of the reporter gene or the endogenous gene
that is induced or repressed by p21 is detected using an
immunological reagent, by assaying for an activity of the gene
product, or by hybridization to a complementary nucleic acid.
[0016] In a fourth aspect, the invention provides methods for
identifying compounds that inhibit senescence in a mammalian cell.
These methods comprise the steps of treating the mammalian cell in
the presence of the compound with an agent or culturing the
mammalian cell under conditions that induce senescence; assaying
the mammalian cell for repression or induction of genes that are
repressed or induced by p21 gene expression; and identifying the
compound as an inhibitor of senescence if genes that are repressed
by p21 are not repressed, or genes that are induced by p21 are not
induced, in the presence of the compound. In preferred embodiments,
the cellular genes are repressed by p21, and senescence inhibitors
are identified by detecting expression of the genes at levels
greater than those detected when p21 is expressed in the absence of
the compound. In preferred embodiments, the genes are identified in
Table I. In alternative preferred embodiments, the cellular genes
are induced by p21, and senescence inhibitors are detected by
detecting expression of the genes at levels less than those
detected when p21 is expressed in the absence of the compound. In
preferred embodiments, the genes are identified in Table II. In
further alternative embodiments, the method is performed using a
recombinant mammalian cell comprising a reporter gene under the
transcriptional control of a promoter derived from a gene whose
expression is modulated by p21. In these embodiments, production of
the product of the reporter gene at greater levels in the presence
than in the absence of the compound when using constructs
comprising promoters derived from genes repressed by p21, or at
lesser levels in the presence than the absence of the compound when
using constructs comprising promoter derived from genes induced by
p21, is detected when the compound is an inhibitor of senescence.
The promoters are preferably derived from genes identified in Table
I (for genes repressed by p21) or Table II (for genes induced by
p21). For p21-repressed genes, the promoter is most preferably
derived from ORC1, PRC1, XRCC9, CDC2, cyclin B1, AIK1, CENP-A,
CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1, thymopoietin .alpha., MPP2,
MPP5, CDC47/MCM7, CDC21/MCM4, DNA ligase I, DNA polymerase .alpha.,
Rad54, exonuclease HEX1/RAD2, or citron kinase. For p21-induced
genes, the promoter most preferably is derived from serum amyloid
A, complement C3, connective tissue growth factor, integrin
.beta.-3, activin A, natural killer cell protein 4, prosaposin,
Mac2 binding protein, galectin-3, superoxide dismutase 2, or
cathepsin B. In other preferred embodiments, the cell comprises a
first recombinant expression construct encoding a reporter gene
under the transcriptional control of a promoter for a mammalian
gene whose expression is modulated by p21, and a second recombinant
expression construct encoding a mammalian p21 gene, wherein
expression of p21 is experimentally-induced in the mammalian cell
thereby. The product of the reporter gene or the endogenous gene
that is induced or repressed by p21 is detected using an
immunological reagent, by assaying for an activity of the gene
product, or by hybridization to a complementary nucleic acid.
[0017] In a fifth aspect, the invention provides methods for
inhibiting cellular senescence, age-related diseases or
age-associated gene products, the method comprising the steps of
contacting the cell with a compound that inhibits senescence as
determined using the methods provided in the aforesaid aspects of
the invention.
[0018] In a sixth aspect, the invention provides methods for
identifying compounds that potentiate senescence in a mammalian
cell. These methods comprise the steps of inducing p21 in the
mammalian cell in the presence and absence of the compound;
assaying the mammalian tumor cell for repression or induction of
genes that are repressed or induced by p21 gene expression; and
identifying the compound as a potentiator of senescence if genes
that are repressed by p21 are repressed to a greater extent, or
genes that are induced by p21 are induced to a greater extent, in
the presence of the compound. In preferred embodiments, the
cellular genes are repressed by p21, and potentiators are detected
by detecting expression of the cellular gene at levels less than
those detected when p21 is expressed in the absence of the
compound. In preferred embodiments, the genes are identified in
Table I. In alternative preferred embodiments, the cellular genes
are induced by p21, and potentiators are detected by detecting
expression of the cellular gene at levels greater than those
detected when p21 is expressed in the absence of the compound. In
preferred embodiments, the genes are identified in Table II. In
further alternative embodiments, the method is performed using
recombinant mammalian cells comprising a reporter gene under the
transcriptional control of a promoter derived from a gene whose
expression is modulated by p21, wherein the cells comprise
constructs having the reporter gene under the transcriptional
control of promoters from genes whose expression is modulated by
p21. In these embodiments, production of the product of the
reporter gene at lower levels in the presence than in the absence
of the compound when using constructs comprising promoters derived
from genes repressed by p21, or at greater levels in the presence
than the absence of the compound when using constructs comprising
promoter derived from genes induced by p21, is detected when the
compound is a potentiator of senescence. In preferred embodiments,
the promoters are derived from genes whose expression is repressed
by p21, most preferably genes identified in Table I. Most
preferably, the promoter is derived from ORC1, PRC1, XRCC9, CDC2,
cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1,
thymopoietin .alpha., MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA
ligase I, DNA polymerase .alpha., Rad54, exonuclease HEX1/RAD2, or
citron kinase. In alternative preferred embodiments, the promoters
are derived from genes whose expression is induced by p21, most
preferably genes identified in Table II. Most preferably, the
promoter is derived from serum amyloid A, complement C3, connective
tissue growth factor, integrin .beta.-3, activin A, natural killer
cell protein 4, prosaposin, Mac2 binding protein, galectin-3,
superoxide dismutase 2, or cathepsin B. In other preferred
embodiments, the cell comprises a first recombinant expression
construct encoding a reporter gene under the transcriptional
control of a promoter for a mammalian gene whose expression is
modulated by p21, and a second recombinant expression construct
encoding a mammalian p21 gene, wherein expression of p21 is
experimentally-induced in the mammalian cell thereby. The product
of the reporter gene or the endogenous gene that is induced or
repressed by p21 is detected using an immunological reagent, by
assaying for an activity of the gene product, or by hybridization
to a complementary nucleic acid.
[0019] In a seventh aspect, the invention provides methods for
promoting or potentiating cellular senescence in tumor cells,
hyperplastic cells or any cell type that is pathological or
disease-causing due to excessive proliferation, the method
comprising the steps of contacting the cell with a compound that
potentiates senescence as determined using the methods provided in
the aforesaid aspect of the invention.
[0020] In a eighth aspect, the invention provides compounds that
are identified using any of the methods of the invention as
disclosed herein.
[0021] In a ninth aspect, the invention provides methods for
obtaining a plurality of nucleic acid species enriched for genes
involved in cell cycle progression. These methods comprise the
steps of inducing the expression of p21 in a mammalian cell;
obtaining cellular mRNA from a mammalian cell before p21 induction
and after p21 is induced and cell growth is stopped; and obtaining
the plurality of nucleic acid species enriched for genes involved
in cell cycle progression. In a preferred embodiment, the plurality
of nucleic acid species enriched for cell cycle progression genes
is obtained by subtractive hybridization methods known in the art,
whereby nucleic acid species underrepresented in cells expressing
p21 are selectively enriched.
[0022] In a tenth aspect, the invention provides methods for
obtaining a plurality of nucleic acid species enriched for genes
that encode secreted proteins with paracrine functions and proteins
involved in senescence and age-related diseases. These methods
comprise the steps of inducing expression of p21 in a mammalian
cell; obtaining cellular mRNA from a mammalian cell before and
after p21 is induced; and obtaining the plurality of nucleic acid
species enriched for genes whose expression is increased in the
cell after p21 is induced. In preferred embodiments, the paracrine
functions of the proteins are mitogenic and anti-apoptotic effects.
In a preferred embodiment, the plurality of nucleic acid species
enriched for genes that encode secreted proteins with paracrine
functions and proteins involved in senescence and age-related
diseases is obtained by subtractive hybridization methods known in
the art, whereby nucleic acid species overrepresented in cells
expressing p21 are selectively enriched.
[0023] In an eleventh aspect, the invention provides a method for
identifying genes that are markers of cellular senescence, the
method comprising the steps of inducing senescence by producing p21
expression in a first population of mammalian cells and inducing
quiescence in a second population of mammalian cells; obtaining
mRNA from each population of cells; comparing the pattern of gene
expression in cells before and after production or p21 in the cells
with the pattern of gene expression in cells before and after the
cells became quiescent; comparing the plurality of genes strongly
induced in the cells after p21 is produced with the plurality of
genes strongly induced in quiescent cells; and identifying the
genes strongly induced in cells producing p21 that are not strongly
induced in quiescent cells.
[0024] In a twelfth aspect, the invention provides methods for
detecting senescence in a mammalian cell. These methods comprise
the step of detecting expression of a gene that is a marker for
senescence. In preferred embodiments, preferred markers of
senescence include connective tissue growth factor (CTGF), serum
amyloid A, integrin .beta.-3, activin A, natural killer cell
protein 4, Mac2 binding protein, or tissue transglutaminase.
[0025] In a thirteenth aspect, the invention provides methods for
identifying compounds that promote induction of senescence in a
mammalian cell. These methods comprise the steps of treating the
mammalian cell with an agent or culturing the mammalian cell under
conditions that induce senescence in the presence of the compound;
assaying the mammalian tumor cell for repression or induction of
genes that are repressed or induced by p21 gene expression; and
identifying the compound as a potentiator of senescence if genes
that are repressed by p21 are further repressed, i.e., to a greater
extent, or genes that are induced by p21 are further induced, i.e.,
to a greater extent, in the presence of the compound. In preferred
embodiments, the cellular genes are repressed by p21, and compounds
that promote induction of senescence are detected by detecting
expression of the cellular gene at levels less than those detected
when p21 is expressed in the absence of the compound. In preferred
embodiments, the genes are identified in Table I. In alternative
preferred embodiments, the cellular genes are induced by p21, and
compounds that promote induction of senescence are detected by
detecting expression of the cellular gene at levels greater than
those detected when p21 is expressed in the absence of the
compound. In preferred embodiments, the genes are identified in
Table II. In further alternative embodiments, the method is
performed using recombinant mammalian cells comprising a reporter
gene under the transcriptional control of a promoter derived from a
gene whose expression is modulated by p21, wherein the cells
comprise constructs having the reporter gene under the
transcriptional control of promoters from genes whose expression is
modulated by p21. In these embodiments, production of the product
of the reporter gene at lower levels in the presence than in the
absence of the compound when using constructs comprising promoters
derived from genes repressed by p21, or at greater levels in the
presence than the absence of the compound when using constructs
comprising promoter derived from genes induced by p21, is detected
when the compound promotes induction of senescence. In preferred
embodiments, the promoters are derived from genes whose expression
is repressed by p21, most preferably genes identified in Table I.
Most preferably, the promoter is derived from ORC1, PRC1, XRCC9,
CDC2, cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET,
CHL1, thymopoietin .alpha., MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA
ligase I, DNA polymerase .alpha., Rad54, exonuclease HEX1/RAD2, or
citron kinase. In alternative preferred embodiments, the promoters
are derived from genes whose expression is induced by p21 most
preferably genes identified in Table II. Most preferably, the
promoter is derived from serum amyloid A, complement C3, connective
tissue growth factor, integrin .beta.-3, activin A, natural killer
cell protein 4, prosaposin, Mac2 binding protein, galectin-3,
superoxide dismutase 2, or cathepsin B. In other preferred
embodiments, the cell comprises a first recombinant expression
construct encoding a reporter gene under the transcriptional
control of a promoter for a mammalian gene whose expression is
modulated by p21, and a second recombinant expression construct
encoding a mammalian p21 gene, wherein expression of p21 is
experimentally-induced in the mammalian cell thereby. The product
of the reporter gene or the endogenous gene that is induced or
repressed by p21 is detected using an immunological reagent, by
assaying for an activity of the gene product, or by hybridization
to a complementary nucleic acid.
[0026] In a fourteenth aspect, the invention provides methods for
identifying compounds that induce senescence in a mammalian cell.
These methods comprise the steps of assaying a mammalian cell in
the presence and absence of the compound for repression or
induction of genes that are repressed or induced by p21 gene
expression; and identifying compounds that induce senescence if
genes that are repressed by p21 are repressed, or genes that are
induced by p21 are induced, in the presence of the compound. In
preferred embodiments, the cellular genes are repressed by p21, and
compounds that induce senescence are detected by detecting
expression of the cellular gene at levels less than those detected
in the absence of the compound. In preferred embodiments, the genes
are identified in Table I. In alternative preferred embodiments,
the cellular genes are induced by p21, and compounds that induce
senescence are detected by detecting expression of the cellular
gene at levels greater than those detected in the absence of the
compound. In preferred embodiments, the genes are identified in
Table II. In further alternative embodiments, the method is
performed using recombinant mammalian cells comprising a reporter
gene under the transcriptional control of a promoter derived from a
gene whose expression is modulated by p21, wherein the cells
comprise constructs having the reporter gene under the
transcriptional control of promoters from genes whose expression is
modulated by p21. In these embodiments, production of the product
of the reporter gene at lower levels in the presence than in the
absence of the compound when using constructs comprising promoters
derived from genes repressed by p21, or at greater levels in the
presence than the absence of the compound when using constructs
comprising promoter derived from genes induced by p21, is detected
when the compound induces senescence. In preferred embodiments, the
promoters are derived from genes whose expression is repressed by
p21, most preferably genes identified in Table I. Most preferably,
the promoter is derived from ORC1, PRC1, XRCC9, CDC2, cyclin B1,
AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1, thymopoietin
.alpha., MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA ligase I, DNA
polymerase .alpha., Rad54, exonuclease HEX1/RAD2, or citron kinase.
In alternative preferred embodiments, the promoters are derived
from genes whose expression is induced by p21, most preferably
genes identified in Table II. Most preferably, the promoter is
derived from serum amyloid A, complement C3, connective tissue
growth factor, integrin .beta.-3, activin A, natural killer cell
protein 4, prosaposin, Mac2 binding protein, galectin-3, superoxide
dismutase 2, or cathepsin B. The product of the reporter gene or
the endogenous gene that is induced or repressed by p21 is detected
using an immunological reagent, by assaying for an activity of the
gene product, or by hybridization to a complementary nucleic
acid.
[0027] Specific preferred embodiments of the present invention will
become evident from the following more detailed description of
certain preferred embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a schematic diagram of the IPTG-regulated
retroviral vector LNp21CO3 used to produce the human HT1080
fibrosarcoma cell line variant p21-9.
[0029] FIG. 2A is a graph of the time course of p21 induction after
the addition of 50 .mu.M IPTG, where p21 levels were determined by
ELISA.
[0030] FIG. 2B is a graph of the time course of p21 decay after
removal of IPTG.
[0031] FIG. 3A is a graph of the time course of changes in
.sup.3H-thymidine labeling index (as determined by autoradiography)
after the addition of 50 .mu.M IPTG.
[0032] FIG. 3B is a graph of the time course of changes in mitotic
index (as determined by microscopy after DAPI staining) after the
addition of 50 .mu.M IPTG.
[0033] FIG. 3C is a graph of the time course of changes in cell
cycle distribution (determined by fluorescence-activated cell
sorting (FACS) analysis following propidium iodide) after the
addition of 50 .mu.M IPTG; -.circle-solid.-: cells in G1 phase of
the cell cycle; -.largecircle.-: cells in G2/M phase of the cell
cycle; -.tangle-soliddn.-: cells in S phase of the cell cycle.
[0034] FIG. 4A is a graph showing the effects of the duration of
treatment with different doses of IPTG on colony formation by p21-9
cells; -.circle-solid.-: 0.5 .mu.M IPTG; -.largecircle.-: 5 .mu.M
IPTG; -.tangle-soliddn.-: 50 .mu.M IPTG.
[0035] FIG. 4B is a graph of the time course of changes in
.sup.3H-thymidine labeling index (determined by autoradiography)
after the removal of 50 .mu.M IPTG; -.circle-solid.-: 1 day;
-.largecircle.-: 5 days.
[0036] FIG. 4C is a graph of the time course of changes in mitotic
index (determined by microscopy) after the removal of 50 .mu.M
IPTG; -.circle-solid.-: 1 day; -.largecircle.-: 5 days.
[0037] FIG. 4D is a graph of the time courses of changes in the
percentage of floating cells after the removal of 50 .mu.M IPTG
following one day or three days of treatment; -.circle-solid.-:
untreated; -.largecircle.-: 1 day; -.tangle-soliddn.-: 3 days.
[0038] FIG. 5A is a histogram showing changes in PKH2 fluorescence
profiles of untreated cells (left) and cells treated for 5 days
with 50 .mu.M IPTG and released in IPTG-free media (right), as
determined by FACS.
[0039] FIG. 5B is a graphical representation of FACS profiles of
DNA content of PKH2.sup.loSS.sup.lo (thin line) and
PKH2.sup.hiSS.sup.hi (thick line) cell populations isolated by FACS
after 5-day treatment with 50 .mu.M IPTG, PKH2 labeling, and 6-day
growth without IPTG.
[0040] FIG. 5C is a graphical representation of FACS profiles of
DNA content of floating cells, collected 48 hrs after release from
3-day treatment with 50 .mu.M IPTG (left) and from untreated cells
(right).
[0041] FIG. 5D is a graphical representation of FACS profiles of
DNA content of attached cells at 0 h, 12 h, 24 h, 28 h, 36 h and 48
h after release from 1-day IPTG treatment.
[0042] FIG. 6 are photomicrographs illustrating examples of normal
(left) and abnormal (right) mitotic figures observed 1-2 days after
release from IPTG (DAPI staining; photographed at 1,000.times.
magnification.
[0043] FIG. 7A are photographs of gel electrophoresis patterns of
RT-PCR experiments (left), northern blot analysis of cellular mRNA
expression (middle) and immunoblotting assays for IPTG-induced
changes in expression of the denoted genes; C: control untreated
p21-9 cells: 1: cells treated for 3 days with 50 .mu.M IPTG.
.beta.2-microglobuln (.beta.2-M) was used as a normalization
control for RT-PCR and S14 ribosomal protein gene for northern
hybridization.
[0044] FIG. 7B are photographs of gel electrophoresis of RT-PCR
experiments (left) and immunoblotting analysis (right) showing the
time course of changes in the expression of the denoted
p21-inhibited genes upon IPTG addition and release.
[0045] FIG. 7C are photographs of gel electrophoresis patterns of
RT-PCR experiments (left) and northern hybridization analysis
(right) of the time course of changes in the expression of the
denoted p21-induced genes upon IPTG addition.
[0046] FIG. 7D is a comparison of gene expression in untreated
control p21-9 cells (C), serum-starved quiescent cells (Q) and
IPTG-treated senescent cells (I).
[0047] FIG. 8A is a histogram showing the effects of fresh media
(F), conditioned media from IPTG-treated (I) or untreated p21-9
cells (U), and 1:1 mixtures of conditioned and fresh media (I/F and
U/F), supplemented with 1% or 2% serum, on .sup.3H-thymidine
incorporation by HS 15.T cells.
[0048] FIG. 8B are graphical representations of FACS profiles of
the DNA content of combined attached and floating C8 cells after 24
hr or 48 hr incubation in 10% serum (control), in low-serum fresh
media (F) or in conditioned media from IPTG-treated (I) or
untreated (U) p21-9 cells. Relative numbers of attached cells (as
determined by methylene blue staining) after 48 hr incubation in
the same media are listed beneath each set of histograms.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0049] This invention provides reagents and methods for identifying
genes involved in mediating p21-induced cellular senescence, and
compounds capable of inhibiting or potentiating senescence or
quiescence in mammalian cells.
[0050] For the purposes of this invention, reference to "a cell" or
"cells" is intended to be equivalent, and particularly encompasses
in vitro cultures of mammalian cells grown and maintained as known
in the art.
[0051] For the purposes of this invention, reference to "cellular
genes" in the plural is intended to encompass a single gene as well
as two or more genes. It will also be understood by those with
skill in the art that effects of modulation of cellular gene
expression, or reporter constructs under the transcriptional
control of promoters derived from cellular genes, can be detected
in a first gene and then the effect replicated by testing a second
or any number of additional genes or reporter gene constructs.
Alternatively, expression of two or more genes or reporter gene
constructs can be assayed simultaneously within the scope of this
invention.
[0052] For the purposes of this invention, the term "quiescence"
will be understood to encompass temporary cessation of cell growth
and DNA replication such as occurs in cultured mammalian cells
under conditions of serum starvation.
[0053] For the purposes of this invention, the term "senescence"
will be understood to include permanent cessation of DNA
replication and cell growth not reversible by growth factors, such
as occurs at the end of the proliferative lifespan of normal cells
or in normal or tumor cells in response to cytotoxic drugs, DNA
damage or other cellular insult.
[0054] Senescence can be induced in a mammalian cell in a number of
ways. The first is a natural consequence of normal cell growth,
either in vivo or in vitro: there are a limited number of cell
divisions, passages or generations that a normal cell can undergo
before it becomes senescent. The precise number varies with cell
type and species of origin (Hayflick & Moorhead, 1961, Exp.
Cell Res. 25: 585-621). Another method for inducing senescence in
any cell type is treatment with cytotoxic drugs such as most
anticancer drugs, radiation, and cellular differentiating agents.
See, Chang et al., 1999, Cancer, Res. 59: 3761-3767. Senescence
also can be rapidly induced in an mammalian cell by transducing
into that cell a tumor suppressor gene (such as p53, p21, p16 or
Rb) and expressing the gene therein. See, Sugrue et al., 1997,
Proc. Natl. Acad. Sci. USA 94: 9648-9653; Uhrbom et al., 1997,
Oncogene 15: 505-514; Xu et al., 1997, Oncogene 15: 2589-2596; Vogt
et al., 1998, Cell Growth Differ. 9: 139-146
[0055] The reagents of the present invention include any mammalian
cell, preferably a rodent or primate cell, more preferably a mouse
cell and most preferably a human cell, that can induce expression
of the p21 gene, wherein such gene is either the endogenous gene or
all exogenous gene introduced by genetic engineering. Although the
Examples disclose recombinant mammalian cells comprising
recombinant expression constructs encoding such an inducible p21
gene, it will be understood that these embodiments are merely a
matter of experimental design choice and convenience, and that the
invention fully encompasses induction of endogenous p21.
[0056] In preferred embodiments, the invention provides mammalian
cells containing a recombinant expression construct encoding an
inducible mammalian p21 gene. In preferred embodiments, the p21
gene is human p21 having nucleotide and amino acid sequences as set
forth in U.S. Pat. No. 5,424,400, incorporated by reference herein.
In alternative embodiments, the p21 gene is an amino-terminal
portion of the human p21 gene, preferably comprising amino acid
residues 1 through 78 of the native human p21 protein (as disclosed
in U.S. Pat. No. 5,807,692, incorporated by reference) and more
preferably comprising the CDK binding domain comprising amino acids
21-71 of the native human p21 protein (Nakanishi et al., 1995, EMBO
J. 14: 555-563). Preferred host cells include mammalian cells,
preferably rodent or primate cells, and more preferably mouse or
human cells. A particularly preferred embodiment are fibrosarcoma
cells, more preferably human fibrosarcoma cells and most preferably
human HT1080 fibrosarcoma cell line and derivatives thereof.
[0057] Recombinant expression constructs can be introduced into
appropriate mammalian cells as understood by those with skill in
the art. Preferred embodiments of said constructs are produced in
transmissible vectors, more preferably viral vectors and most
preferably retrovirus vectors, adenovirus vectors, adeno-associated
virus vectors, and vaccinia virus vectors, as known in the art.
See, generally, MAMMALIAN CELL BIOTECHNOLOGY: A PRACTICAL APPROACH,
(Butler, ed.). Oxford University Press: New York, 1991, pp.
57-84.
[0058] In additionally preferred embodiments, the recombinant cells
of the invention contain a construct encoding an inducible p21
gene, wherein the gene is under the transcriptional control of an
inducible promoter. In more preferred embodiments, the inducible
promoter is responsive to a trans-acting factor whose effects can
be modulated by an inducing agent. The inducing agent can be any
factor that can be manipulated experimentally, including
temperature and most preferably the presence or absence of an
inducing agent. Preferably, the inducing agent is a chemical
compound, most preferably a physiologically-neutral compound that
is specific for the trans-acting factor. In the use of constructs
comprising inducible promoters as disclosed herein, expression of
p21 from the recombinant expression construct is mediated by
contacting the recombinant cell with an inducing agent that induces
transcription from the inducible promoter or by removing an agent
that inhibits transcription from such promoter. A variety of
inducible promoters and cognate trans-acting factors are known in
the prior art, including heat shock promoters than can be activated
by increasing the temperature of the cell culture, and more
preferably promoter/factor pairs such as the tet promoter and
fusions thereof with mammalian transcription factors (as are
disclosed in U.S. Pat. Nos. 5,654,168, 5,851,796, and 5,968,773),
and the bacterial lac promoter of the lactose operon and its
cognate lacI repressor protein. In a preferred embodiment, the
recombinant cell expresses the lacI repressor protein and a
recombinant expression construct encoding human p21 under the
control of a promoter comprising one or a multiplicity of
lac-responsive elements, wherein expression of p21 can be induced
by contacting the cells with the physiologically-neutral inducing
agent, isopropylthio-.beta.-galactoside. In this preferred
embodiment, the lacI repressor is encoded by a recombinant
expression construct identified as 3'SS (commercially available
from Stratagene. LaJolla, Calif.).
[0059] The invention also provides recombinant expression
constructs wherein a reporter gene is under the transcriptional
control of a promoter of a gene whose expression is modulated by
p21. These include genes whose expression is induced by p21 and
genes whose expression is repressed by p21. In preferred
embodiments, the promoters are derived from genes whose expression
is repressed by p21, and are identified in Table I. Most
preferably, the promoter is derived from ORC1, PRC1, XRCC9, CDC2,
cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1,
thymopoietin .alpha., MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA
ligase I, DNA polymerase .alpha., Rad54, exonuclease HEX1/RAD2, or
citron kinase. In additional preferred embodiments, the promoters
are derived from genes whose expression is induced or otherwise
increased by p21, and are identified in Table II. Most preferably,
the promoter is derived from serum amyloid A, complement C3,
connective tissue growth factor, integrin .beta.-3, activin A,
natural killer cell protein 4, prosaposin, Mac2 binding protein,
galectin-3, superoxide dismutase 2, or cathepsin B. These reporter
genes are then used as sensitive and convenient indicators of the
effects of p21 induction, and enable compounds that inhibit or
potentiate the effects of p21 expression in mammalian cells to be
easily identified. Host cells for these constructs include any cell
in which p21 gene expression can be induced, and preferably include
cells also containing recombinant expression constructs containing
an inducible p21 gene as described above. Reporter genes useful in
the practice of this aspect of the invention include but are not
limited to firefly luciferase, chloramphenicol acetyltransferase,
beta-galactosidase, green fluorescent protein, and alkaline
phosphatase.
[0060] In preferred embodiments, cells according to the invention
comprise both a first recombinant expression construct encoding a
reporter gene under the transcriptional control of a promoter for a
mammalian gene whose expression is modulated by p21, and a second
recombinant expression construct encoding a mammalian p21 gene,
wherein p21 expression is experimentally-inducible thereby in the
mammalian cell.
[0061] In alternative embodiments, the invention provides a
mammalian cell comprising a recombinant expression construct
encoding a reporter gene under the transcriptional control of a
promoter for a mammalian gene whose expression is repressed by p21,
wherein the promoter is from the gene ORC1, PRC1, XRCC9, CDC2,
cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1,
thymopoietin .alpha., MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA
ligase I, DNA polymerase .alpha., Rad54, exonuclease HEX1/RAD2, or
citron kinase. In further alternative embodiments, the invention
provides a mammalian cell comprising a recombinant expression
construct encoding a reporter gene under the transcriptional
control of a promoter for a mammalian gene whose expression is
induced by p21, wherein the promoter is from the gene connective
tissue growth factor, serum amyloid A, complement C3, integrin
.beta.-3, activin A, natural killer cell protein 4, prosaposin,
Mac2 binding protein, galectin-3, superoxide dismutase 2, or
cathepsin B.
[0062] The invention provides methods for identifying compounds
that inhibit or promote senescence, whereby the effects of the
compound are assayed by determining whether the compounds inhibit
or potentiate induction or repression of genes whose expression is
modulated by p21. In the practice of the methods of the invention,
cultured mammalian cells in which p21 can be induced are treated to
induce p21, for example, by radiation treatment or treatment with
cytotoxic drugs, or transduced with a transmissible vector encoding
p21. More preferably, p21-9 cells are used in which p21 can be
induced by contacting the cells with IPTG. Typically, cells are
grown in appropriate culture media (e.g., DMEM supplemented with
10% fetal calf serum (FCS) for p21-9 cells). p21 gene expression is
induced in p21-9 cells by adding IPTG to the culture media at a
concentration of about 50 .mu.M. Typically, p21 is induced in these
cells in the presence or absence of the compound to be tested
according to the methods of the invention. mRNA is then isolated
from cells in which p21 is induced, and expression of genes that
are regulated by p21 is analyzed. Expression is compared in cells
in which p21 is induced in the presence of the compound with
expression induced in the absence of the compound, and the
differences used to identify compounds that affect cellular gene
expression according co the methods set forth herein. In certain
embodiments, cellular gene expression is analyzed using microarrays
of oligonucleotides or cellular cDNAs such as are commercially
available (for example, from Genome Systems, Inc., St. Louis, Mo.).
In alternative embodiments, genes known to be induced or repressed
by p21 are assayed. Gene expression can be assayed either by
analyzing cellular mRNA or protein for one or a plurality of
p21-modulated genes. Most preferably, the genes used in these
assays are genes identified in Tables I and II.
[0063] In alternative embodiments, such compounds are identified
independently of p21-directed experimental manipulation. In such
assays, cells are treated to induce senescence in any of the ways
disclosed above, including but not limited to treatment with
cytotoxic drugs, radiation or cellular differentiating agents, or
introduction of a tumor suppressor gene. Expression of genes that
are repressed or induced by p21 is analyzed in the presence or
absence of the test compound. Most preferably, the genes used in
these assays are genes identified in Tables I and II, using the
types of mRNA and protein assays discussed above for gene
expression analysis.
[0064] In alternative embodiments, the cells in which p21 is
induced further comprise a recombinant expression construct
encoding a reporter gene under the transcriptional control of a
promoter of a cellular gene that is induced or repressed by p21. In
preferred embodiments the cellular gene is a gene that is repressed
by p21, and the promoter is derived from a gene identified in Table
I. Examples of known promoters for such genes include ORC1, PRC1,
XRCC9, CDC2, cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK,
HSET, CHL1, thymopoietin .alpha., MPP2, MPP5, CDC47/MCM7,
CDC21/MCM4, DNA ligase I, DNA polymerase .alpha., RAD54, HEX1/RAD2,
or citron kinase. In preferred embodiments, the cellular gene is a
gene that is induced by p21, and the promoter is derived from a
gene identified in Table II. Examples of known promoters for such
genes include connective tissue growth factor, serum amyloid A,
complement C3, integrin .beta.-3, activin A, natural killer cell
protein 4, prosaposin, Mac2 binding protein, galectin-3, superoxide
dismutase 2, or cathepsin B. Preferred reporter genes include but
are not limited to firefly luciferase, .beta.-galactosidase,
alkaline phosphatase and green fluorescent protein, all of which
are commercially available.
[0065] The invention also provides methods for identifying genes
mediating the effects of p21-induced cellular senescence. Induction
of p21 turns out to be an integral part of cell growth arrest
associated with senescence, terminal differentiation and response
to cellular damage. As described in the Examples below, cDNA array
hybridization was used to investigate whether these effects were
due to p21-induced changes in gene expression. This analysis showed
that p21 selectively inhibited multiple genes involved in the
control of mitosis, DNA replication, segregation and repair. Many
proteins that were induced by p21 in these experiments have been
associated with senescence and aging or implicated in age-related
diseases, including atherosclerosis, Alzheimer's disease,
amyloidosis and arthritis. These findings suggest that cumulative
effects of p21 induction may contribute to the pathogenesis of
cancer and age-related diseases. In addition, a number of
p21-activated genes encode secreted proteins with potential
paracrine effects on cell growth and apoptosis. In agreement with
this observation, conditioned media from p21-induced cells showed
mitogenic and anti-apoptotic activity.
[0066] The analyses disclosed in the Examples below showed that
inhibition of cell cycle progression genes was not simply a
consequence of p21-induced growth arrest. Shutoff of some of these
genes occurred together with cell growth arrest, and the
re-expression of all the tested genes upon release from p21
preceded the re-entry of cells into cell cycle. The nature of the
immediate-response genes, such as ORC1 (required for the initiation
of DNA replication), topoisomerase II (which is central to DNA
segregation in G2), and PLK1 (involved in the initiation of
mitosis), suggested that inhibition of their expression may in fact
play a causal role in the induction of growth arrest by p21. These
observations formed the basis for one aspect of the methods of the
invention, which provide methods for identifying genes involved in
cell cycle progression in mammalian cells.
[0067] Furthermore, the biological functions of both immediate- and
early-response genes indicate that their shutoff serves to maintain
p21-induced growth arrest. The use of the reagents and methods of
the present invention has demonstrated that release from
p21-induced growth arrest results in endoreduplication and mitotic
abnormalities. DNA replication and mitosis did not resume after
release from IPTG until all the p21-inhibited genes were
re-expressed, and DNA replication resumed considerably before
mitosis. The results disclosed in the Examples below indicate that
prolonged p21 induction leads to the decay of many proteins
involved in cell cycle progression, including a number of proteins
involved in the "quality control" of replication or mitosis. As a
result of failing to regenerate the pools of such proteins by the
time cells re-entered the cell cycle after release from p21,
abnormal replication and abnormal mitosis ensued. For example, the
production of polypolid cells was observed after release from
prolonged p21-induced cell growth arrest. Endoreduplication, the
process that leads to polyploidization of the cells, may be a
consequence of the abrogation of mitotic checkpoint control (Hixon
et al., 1998, Mol. Cell Biol. 18: 6224-37), which could result from
a lack of p21-inhibited checkpoint control proteins, such as MAD2
and BUBR1. Furthermore, polyploid cells may arise due to a failure
of cytokinesis that can be triggered by a lack of
cytokinesis-associated proteins Prc1, Aim1 and citron kinase, which
we found to be inhibited by p21.
[0068] Different mitotic abnormalities that were observed after
release from p21 have been previously found to result from mutation
or inhibition of proteins that control proper chromosome alignment
and segregation. including the products of such p21-inhibited genes
as MAD2, BUBR1, PLK1, AIK-1, CENP-A, CHL1 and MCAK (Li &
Benezra, 1996, Proc. Natl. Acad. Sci. USA 93: 10436-10440; Glover
et al., 1998, Genes Develop. 12: 3777-3787; Chan et al., 1999, J.
Cell Biol. 146: 941-954 ). The role of such proteins in p21-induced
mitotic abnormalities is supported by the analysis of the time
course of decay and resynthesis of mitosis control proteins. Thus,
at the time of resumed mitosis (36 hrs after release), the pools of
Cdc2 and Plk1, which are required for the initiation of mitosis,
are regenerated to levels comparable to untreated cells (as shown
in FIG. 7B). In contrast, MAD2, the function of which is to prevent
anaphase unless chromosomes are properly attached to the mitotic
spindle, is resynthesized much less efficiently (FIG. 7B).
Furthermore, MAD2 levels that remain after one day of IPTG
treatment are much higher than after 3 or more days (FIG. 7B),
which agrees with a lower frequency of abnormal mitosis in cells
that are released after one day of p21 induction.
[0069] p21 overexpression has been reported to inhibit DNA repair
(Pan et al., 1995, J. Biol. Chem. 270: 22008-22016; Umar et al.,
1996, Cell 87: 65-73). In light of our results, this effect of p21
can be attributed to the inhibition of DNA repair genes, such as
XRCC9, RAD54, HEX1/RAD2, RAD21 homolog and DNA ligase I. Inhibition
of DNA repair is also likely to increase the frequency of mutations
in cells that recover from p21-induced growth arrest, contributing
to the overall genetic instability of such cells.
[0070] p21-induced genetic destabilization in normal cells may also
have a potential carcinogenic effect. Growth arrest of senescent
cells is triggered by transient p21 induction, while another CDK
inhibitor, p16, appears to be responsible for maintaining the
growth arrest after the decay of p21 (Alcorta et al., 1996, Proc.
Natl. Acad. Sci. USA 93: 13742-13747). p16(in striking contrast to
p21) is frequently mutated in human tumors (Hall & Peters,
1996, Adv. Cancer. Res. 68: 67-108), including HT1080 fibrosarcoma
used in the present study. If the primary carcinogenic effect of
p16 mutations is abortive senescence, then cells expressing mutated
p16 would experience prolonged p21 induction. Consistent with the
results disclosed herein, re-entry into the cell cycle under these
conditions would be expected to result in the development of
karyotypic abnormalities. Unlike p16, p21 would act more as an
oncogene than as a tumor suppressor in this process, which can
explain the rarity of p21 mutations in cancer.
[0071] Thus, the invention provides methods for identifying
compounds having an anticarcinogenic effect by inhibiting
p21-induced cell cycle arrest. The compounds produced by these
methods would be expected to be able to minimize the development of
cells having karyotypic abnormalities, which in turn would be
expected to reduce the likelihood that such cells would develop
into malignant disease.
[0072] The invention also provides methods for identifying
compounds that induce or promote senescence. In this aspect, the
invention provides compounds that increase inhibition of genes
inhibited by p21 expression. Inhibition of cell division and cell
cycle progression control genes is shown herein to prevent cells
from re-entering the cell cycle after p21 induction and result in
irreversible growth arrest. Thus, compounds that induce or
potentiate p21-induced repression of such genes are effective in
promoting cell senescence and terminal growth arrest. Thus, the
invention provides methods for identifying compounds that inhibit
cellular genes that control cell cycle progression, most preferably
genes identified in Table I. In preferred embodiments, the
compounds are used to promote senescence of mammalian cells, most
preferably tumor cells, hyperplastic cells or any cell type that is
pathological or disease-causing due to excessive proliferation. In
preferred embodiments of this aspect of the invention, the methods
comprise the steps of inducing p21 in a mammalian cell in the
presence or absence of the compound; assaying the cell for
expression of genes repressed by p21; and identifying the compound
as a potentiator of senescence if the genes are repressed to a
greater extent in the presence of the compound than in the absence
of the compound. In other aspects of the methods of the invention,
compounds that promote senescence in a mammalian cell are
identified independently of p21-directed experimental manipulation,
for example, by inducing senescence in the cells in any of the ways
disclosed above. It is known in the art that senescence can be
induced even in p21-deficient cells (Chang et al., 1999, Oncogene
18: 4808-4818 and Pantoja et al., 1999, Oncogene 18: 4974-4982) and
that some senescence-inducing treatments, such as the treatment of
MCF-7 cells with all-trans retinoic acid (Chang et al., 1999,
Cancer Res. 59: 3761-3767), are associated with a decrease rather
than an increase in the cellular levels of p21 (Zhu et al., 1997,
Exp. Cell Res. 234: 293-299).
[0073] The invention also provides methods for potentiating
senescence in a mammalian cell, comprising the step of contacting
the cell with a compound identified by the methods of the
invention. In preferred embodiments, the mammalian cells are tumor
cells. hyperplastic cells or any cell type that is pathological or
disease-causing due to excessive proliferation. In alternative
embodiments, the methods comprise the additional step of contacting
the cells with radiation or anticancer, cytotoxic or
antiproliferative drugs.
[0074] The observed effects of p21 induction on gene expression
show numerous correlations with the changes that have been
associated with cell senescence and organism aging. Some of these
correlations come from the analysis of p21-inhibited genes. Thus,
senescent fibroblasts were reported to express lower levels of Rb
(Stein et al., 1999, Mol. Cell. Biol. 19: 2109-2117), as we have
also observed upon p21 induction. It is also interesting that three
p21-inhibited genes, CHL1, CDC21 and RAD54 encode members of the
helicase family. A deficiency in another protein of the helicase
group has been identified as the cause of Werner syndrome, a
clinical condition associated with premature aging and , at the
cellular level, accelerated senescence of cells in culture (Gray et
al., 1997, Nature Genet. 17: 100-103).
[0075] The strongest correlations with the senescent phenotype,
however, come from identification of p21-induced genes, many of
which are known to increase their levels during replicative
senescence or organism aging. Overexpression of ECM proteins is a
known hallmark of replicative senescence, and two p21-induced genes
in this group, fibronectin 1 and plasminogen activator inhibitor 1
(PAI-1), have been frequently associated with cellular senescence
(reviewed in Crisofalo & Pignolo, 1996, Exp. Gerontol. 31:
111-123). Other p21-induced genes that were also reported to be
overexpressed in senescent fibroblasts include tissue-type
plasminogen activator (t-PA) (West et al., 1996, Exp. Gerontol. 31:
175-193), cathepsin B (diPaolo et al., 1992, Exp. Cell Res. 201:
500-505), integrin .beta.3 (Hashimoto et al., 1997, Biochem.
Biophys. Res. Commun. 240: 88-92) and APP (Adler et al., 1991,
Proc. Natl. Acad. Sci. USA 88: 16-20). Expression of several
p21-induced proteins was shown to correlate with organism aging,
including t-PA and PAI-1 (Hashimoto et (al., 1987, Thromb. Res. 46:
625-633), cathepsin B (Bernstein et al., 1990, Brain Res. Bull. 24:
43-549) activin A (Loria et al., 1998, Eur. J. Endocrinol. 139:
487-492), prosaposin (Mathur et al., 1994, Biochem. Mol. Biol. Int.
34: 1063-1071), APP (Ogomori et al., 1988, J. Gerontol. 43:
B157-B162), SAA (Rosenthal & Franklin, 1975, J. Clin. Invest.
55: 746-753) and t-TGase (Singhal et al., 1997, J. Investig. Med.
45: 567-575).
[0076] The most commonly used marker of cell senescence is the
SA-.beta.-gal activity (Dimri et al., 1995, Proc. Natl. Acad. Sci.
USA 92: 9363-9367). This gene is strongly elevated in IPTG-treated
p21-9 cells (Chang et al., 1999, Oncogene 18: 4808-4818).
SA-.beta.-gal was suggested to represent increased activity and
altered localization of the lysosomal .beta.-galactosidase (Dimri
et al., 1995, ibid.), and other studies have described elevated
lysosome activities in senescent cells (Cristofalo & Kabakijan,
1975, Mech. Aging Dev. 4: 19-28). Five lysosomal enzymes appear in
Table 2, including N-acetylgalactosamine-6-sulfate sulfatase
(GALNS), cathepsin B, acid .alpha.-glucosidase, acid lipase A and
lysomal pepstatin-insensitive protease. p21 also upregulated genes
for mitochondrial proteins SOD2, metazin and 2,4-dienoyl-CoA
reductase, which correlates with reports of different mitochondrial
genes overexpressed in senescent cells (Doggett et al., 1992, Mech.
Aging Dev. 65: 239-255; Kodama et al., 1995, Exp. Cell Res. 219:
82-86; Kumazaki et al., 1998, Mech. Aging Dev. 101: 91-99).
[0077] As disclosed in the following Examples, there are many
similarities between the effects of p21 induction in p21-9 cells
and changes associated with senescence in normal fibroblasts.
Senescent cells, in particular, were shown to overproduce different
growth factors and ECM proteins that may promote metastasis
(Campisi et al., 1998, J. Investig. Dermatol. Symp. Proc. 3: 1-5).
Several growth factors and growth factor receptors have also been
identified among the genes that are induced by irradiation in a
p53-dependent manner, under the conditions of strong p21 induction
(Komarova et al., 1998, Oncogene 17: 1089-1096). Interestingly,
most of these genes did not contain p53-binding sites in their
promoters. Our results suggest that induction of growth factors by
p53 may be an indirect effect, mediated through p21 induction.
[0078] Thus, the invention provides methods for identifying genes
associated with cellular senescence, particularly genes that are
induced during senescence, and particularly by p21 expression. The
invention also provides methods for identifying compounds that can
inhibit p21-mediated induction of such genes. Such compounds would
be expected to exhibit the capacity to reduce, repress or reverse
cellular senescence by their effects on p21-mediated induction of
gene expressions.
[0079] Strikingly, products of many genes that we found to be
induced by p21 have been linked to age-related diseases, including
Alzheimer's disease, amyloidosis, atherosclerosis and arthritis.
Thus, APP gives rise to .beta.-amyloid peptide, the main component
of Alzheimer's amyloid plaques. Complement C3 (Veerhuis et al.,
1995, Virchows Arch. 426: 603-610) and AMP deaminase (Sims et al.,
1998, Neurobiol. Aging 19: 385-391) were also suggested to play a
role in Alzheimer's. It is especially interesting that t-TGase,
which is most rapidly induced by p21 and which has been described
as a pleiotropic mediator of cell differentiation, carcinogenesis.
apoptosis and aging (Park et al., 1999, J. Gerontol. A Biol. Sci.
54: B78-B83), is involved in the formation of plaques associated
with both Alzheimer's disease and amyloidosis (Dudek & Johnson,
1994, Brain Res. 651: 129-133). The latter disease is due to the
deposition of another p21-induced gene product, SAA, which has also
been implicated in atherosclerosis, osteoarthritis and rheumatoid
arthritis (Jensen & Whitehead, 1998, Biochem. J. 334: 489-503).
Two other p21-upregulated secreted proteins, connective tissue
growth factor (CTGF) and galectin 3 are involved in atherosclerosis
(Oemar et al., 1997, Circulation 95: 831-839; Nachtigal et al.,
1998, Am. J. Pathol. 152: 1199-1208). In addition, cathepsin B
(Howie et al., 1985, J. Pathol. 145: 307-314), PAI-1 (Cerinic et
al., 1998, Life Sci. 63: 441-453), fibronectin (Chevalier, 1993,
Semin. Arthritis Rheum. 22: 307-318), GALNS and Mac-2 binding
protein (Seki et al., 1998, Arthritis Rheum. 41: 1356-1364) have
been associated with osteoarthritis and/or rheumatoid arthritis.
Furthermore, senescence-related changes in ECM proteins, such as
increased PAI-1 expression, were proposed to result in age-specific
deterioration in the structure of skin and other tissues (Campisi,
1998, J. Investig. Dermatol. Symp. Proc. 3: 1-5). Increased
fibronectin production by aging cells was also suggested to
increase the density of the fibronectin network in ECM, which may
contribute to slower would healing in aged individuals (Albini et
al., 1988, Coll. Relat. Res. 8: 23-37).
[0080] The results disclosed herein indicate that p21 induction
affects cellular gene expression in a way that may increase the
probability of the development of cancer or age-related diseases. A
surge of p21 expression occurs not only in normal replicative
senescence but also in response to cellular damage; in both cases,
the undesirable effects of p21 induction would be expected to
accumulate in an age-dependent manner. Elucidation of specific
molecular interactions and regulatory pathways that are responsible
for these effects of p21 on gene expression may suggest new
approaches to the prevention of cancer and age-related
diseases.
[0081] Thus, the invention provides methods for identifying genes
associated with age-related diseases. The invention also provides
methods for identifying compounds that can inhibit p21-mediated
induction of such genes. Such compounds would be expected to
exhibit therapeutic capacity to prevent, retard or reverse
age-related diseases.
[0082] The methods of the invention directed towards identifying
genes whose expression is modulated by p21 take advantage of the
ability to experimentally induce p21 expression. Cells as provided
by the invention containing inducible p21 genes can be used to
isolate cellular mRNA reflecting the expression status of genes
induced, repressed and unchanged by p21 expression. Naive cells in
which p21 is not induced provide a comparative, control source of
cellular mRNA. A plurality of nucleic acids, most preferably cDNA
copies of cellular mRNA, can be obtained that is specific for
either induced or repressed genes by constructing differential cDNA
libraries using subtractive hybridization methods. See, for
example, Diatchenko et al., 1996, Proc. Natl. Acad. Sci. USA 93:
6025-6030. mRNA or cDNA isolated before and after p21 induction can
also be used as probes for hybridization analysis, either using
arrayed or non-arrayed cDNA libraries, and differentially-expressed
genes can be identified from such hybridization. See, generally,
Sambrook et al., 1990, MOLECULAR CLONING: A LABORATORY MANUAL, Cold
Spring Harbor Laboratory Press; New York. Alternatively,
differential display of the subtracted cDNA population can be
performed to yield sets of genes that are either upregulated or
downregulated by p21 expression.
[0083] In additional embodiments, genes that are upregulated or
downregulated by p21 expression can be isolated using molecular
cloning techniques well known in the art. Sambrook et al., ibid.
Differential cDNA libraries produced as described above can be
screened with probes specific for genes induced or repressed by
p21, using subtractive hybridization methods that enrich the probes
for the appropriate cDNA population. Alternatively, such probes can
be used to screen conventionally-prepared cDNA libraries
constructed to maximize the percentage of colonies comprising
full-length or close to full length cDNAs, to facilitate cloning of
p21-modulated genes particularly novel genes identified using the
methods of the invention. Said genes are also intended to fall
within the scope of this invention.
[0084] The following Examples are intended to further illustrate
certain preferred embodiments of the invention and are not limiting
in nature.
EXAMPLE 1
Production of a Mammalian Cell Comprising an Inducible p21 Gene
[0085] A recombinant derivative of human fibrosarcoma cell line
HT1080, p21-9, was produced essentially according to Chang et al.
(1999, Oncogene 18: 4808-4818, incorporated by reference herein).
This cell line contained a p21 coding sequence under the
transcriptional control of a promoter regulated by
isopropyl-.beta.-thiogalactoside (IPTG). Expression of p21 can be
induced by culturing these cells in the presence of a sufficient
amount of IPTG, thereby permitting the sequellae of p21 expression
to be studied in the absence of any additional effects that
induction of the endogenous p21 gene might provoke. This cell line
has been deposited in the American Type Culture Collection
(A.T.C.C.), Manassas, Va. and given Accession Number ______.
[0086] Briefly, a subline of HT1080 expressing a murine ecotropic
retrovirus receptor and a modified bacterial lacI repressor encoded
by the plasmid 3'SS (Stratagene) (described in Chang &
Roninson, 1996, Gene 33: 703-709, incorporated by reference) was
infected with retroviral particles containing recombinant
retrovirus LNp21CO3, the structure of which is shown in FIG. 1.
This retroviral vector contains the bacterial neomycin resistance
gene (neo) under the transcriptional control of the retroviral long
terminal repeat promoter. p21-encoding sequences are cloned in the
opposite orientation to the transcriptional direction of the neo
gene and under the control of a modified human cytomegalovirus
promoter. Specifically, the CMV promoter contains a three-fold
repeat of bacterial lac operator sequences that make expression
from the promoter sensitive to the lacI repressor expressed in the
cell. LNp21CO3 was constructed by cloning a 492 bp fragment of DNA
comprising the p21 coding sequence into the NotI and BglII sites of
the parent vector, LNXCO3 (disclosed in Chang & Roninson,
ibid.).
[0087] After infection, cells infected with the LNp21CO3X vector
were selected by culturing the cells in the presence of 400
.mu.g/mL G418 (obtained from BRL-GIBCO, Gaithersburg, Md.). Clonal
line p21-9 was derived from LNp21CO3 transduced, G418-resistant
cell lines by end-point dilution until a clonal cell line was
obtained.
EXAMPLE 2
Cell Growth Assays
[0088] p21-9 cells produced as described in Example 1 were used in
cell growth assays to determine what changes in cell growth
occurred when p21 was expressed in the cell.
[0089] p21 expression from the LNp21CO3 vector in p21-9 cells was
induced by culturing the cells in DMEM medium containing 10% fetal
calf serum (Hyclone, Logan, Utah) and IPTG. Results of these assays
are shown in FIGS. 2A and 2B. FIG. 2A shows the time course of p21
protein production in cells cultured in the presence of 50 .mu.M
IPTG. p21 gene expression increased between 6 and 12 hours after
introduction of IPTG into the growth media, which expression peaked
at about 24 hours post-induction. Upon removing the cells from
IPTG-containing media, p21 expression fell about as rapidly as it
had risen, returning to pre-induction levels at about 24 hours
after IPTG was removed, (FIG. 2B).
[0090] Cell growth in the presence of IPTG was assayed in three
ways: measuring .sup.3H-thymidine incorporation (termed the
"labeling index"); observing the number of mitotic cells in the
culture by microscopy (termed the "mitotic index") and determining,
the distribution of the culture cells in different portions of the
cell cycle (termed the "cell cycle distribution"). These results
are shown in FIGS. 3A through 3C.
[0091] .sup.3H-thymidine incorporation assays were performed
substantially as described by Dimri et al. (1995, Proc. Natl. Acad.
Sci. USA 92: 9363-9367). Cells were cultured in the presence of
.sup.3H-thymidine for 3 h, and then analyzed by autoradiography.
DNA replication as determined by autoradiography ceased entirely by
9 hours after addition of IPTG to the culture media (FIG. 3A).
[0092] The mitogenic index was determined by observing cells
microscopically and calculating the number of cells in mitosis
after staining with 5 .mu.g/mL 4,6-diamino-2-phenylindole (DAPI),
and images were collected using a Leica DMIRB fluorescence
microscope and Vaytek (Fairfield, Iowa) imaging system.
Microscopically-detectable mitotic cells disappeared from these
cultures in the presence of IPTG in two stages: the first occurring
between 0-4 hours after IPTG addition (wherein the mitotic index
dropped from about 15% in untreated cells to about 5% in
IPTG-treated cells) and then again between about 10-14 hours after
IPTG addition (wherein the mitotic index dropped to zero at about
13 hours after IPTG addition (FIG. 3B).
[0093] Cell cycle distribution was determined using FACS analysis
of DNA content after staining with propidium iodide as described by
Jordan et al. (1996, Cancer Res. 56: 816-825) using Becton
Dickinson FACSort. Cell cycle distribution stabilized after 24 hrs
of IPTG treatment (shown in FIG. 3C). By this time, 42-43% of
IPTG-treated cells were arrested in G1 and G2, respectively, and
about 15% of the cells were arrested with S-phase DNA content.
[0094] The effects of p21 expression were also investigated by
releasing cells from the effects of p21 by removing IPTG from the
cell culture media. It was known that IPTG-treated p21-9 cells
displayed morphological senescence markers (Chang et al., 1999,
ibid.). As shown in FIG. 2B, p21 gene expression levels in p21-9
cells reverted to basal levels within 24 hours after removal of
IPTG. Here, it was determined whether IPTG-treated p21-9 cells show
any loss of clonogenic capacity after removal of IPTG. The results
of these experiments are shown in FIGS. 4A through 4D.
[0095] Colony assays for recovery from IPTG treatment were
performed by plating about 2,000 p21-9 cells per 10 cm culture dish
in DMEM/10% FCS and the presence or absence of IPTG. Cells were
allowed to form colonies for 10 days before their clonogenic
capacity was determined. p21-9 cells were treated with three
concentrations of IPTG: 0.5 .mu.M, 5 .mu.M and 50 .mu.M. These
treatments induced, respectively, no measurable increase over basal
p21 levels (0.5 .mu.M), half-maximal (5 .mu.M) or maximal increase
(50 .mu.M) in p21 gene expression. As shown in FIG. 4A, treatment
of p21-9 cells with 0.5 .mu.M IPTG did not inhibit colony
formation. In contrast, continuous exposure of the cells to 5 .mu.M
or 50 .mu.M IPTG reduced the clonogenicity of p2l-9 cells by 80%
and 100%, respectively. When IPTG was removed after 12 or 14 hours,
cells treated with 5 .mu.M IPTG showed substantially undiminished
colony formation. However, the 50 .mu.M IPTG-treated cells showed a
decrease in clonogenicity of 58-63%. After 3-5 days treatment,
cells cultured in 5 .mu.M IPTG showed a decreased clonogenicity of
55-58%, and cells cultured in 50 .mu.M IPTG showed a decreased
clonogenicity of 95-99%. These results indicated that the ability
of cells to recover after p21 gene expression decayed was inversely
correlated with the level of induced p21 and with the duration of
p21 induction. This result was consistent with results obtained by
others in other cell culture systems (Fang et al., 1999, Oncogene
18: 2789-2797).
[0096] The causes of the loss of clonogenicity were investigated as
follows. Resumption of DNA replication was first detected about 20
hours after release from IPTG using the .sup.3H-thymidine
incorporation assay as described above. These results are shown in
FIG. 4B. Resumption of mitosis in these cells was first detected
about 30 hours after IPTG release, as determined from the mitotic
index as described above. These results are shown in FIG. 4C. The
percentages of cells entering the S or M phase of the cell cycle
were higher in cells that were treated with IPTG for one day than
for five days (compare the curves for each in FIGS. 4B and 4C) but
the differences were not significant enough to account for the
corresponding difference in clonogenic recovery as shown in FIG.
4A.
[0097] Microscopic examination of culture plates from the
clonogenic assays showed that plates treated with 50 .mu.M IPTG for
three or more days contained numerous single cells and small cell
clusters that failed to develop into colonies. In addition, release
from IPTG was associated with the appearance of floating cells
during the first two days after IPTG release, and the number of
such cells was much higher when cells were released after three
days of IPTG induction than after one day (as shown in FIG. 4D).
Most of these floating cells were dead, as indicated by trypan blue
staining and a 100-1,000-fold decrease in clonogenicity.
[0098] The effect of p21 induction in these cells was further
studied by examining the DNA content of growth-retarded and dead
cells that appeared after release from prolonged IPTG treatment.
Growth-retarded cells were isolated using FACS on the basis of
increased retention of PKH2, a lipophilic fluorophore that stably
incorporates into the cell membrane and is evenly divided between
daughter cells; this leads to a proportional decrease in cellular
fluorescence with each round of cell division, and no decrease in
non-dividing or dead cells (Horan & Slezak, 1989, Nature 340:
167-168). These assays were performed as described in Chang et al.
(1999, Cancer Res. 59: 3761-3767). Untreated p21-9 cells and cells
treated with 50 .mu.m IPTG for five days were labeled with PKH2,
plated in IPTG-free medium, and their PKH2 fluorescence was
analyzed on consecutive days. As shown in FIG. 5A, IPTG-treated
cells started dividing later than the control cells and developed a
heterogeneous PKH2 profile, with an emerging peak of proliferating
cells and a shoulder of growth-retarded cells with high PKH2
fluorescence. The growth-retarded cells also showed elevated side
scatter which is characteristic for senescent cells (Chang et al.,
1999, ibid.). The proliferating (PKH2.sup.loSS.sup.lo) and
growth-retarded (PKH2.sup.hiSS.sup.hi) cell populations were
separated by FACS six days after release from IPTG, and their DNA
content was analyzed by PI staining. The growth-retarded fraction
differed from the proliferating cells in having a higher G2/M
fraction and a large number of cells with greater than 4C DNA
content (shown in FIG. 5B). The polyploid nature of the latter
cells was confirmed by fluorescence in situ hybridization (FISH) of
interphase nuclei with specific probes for chromosomes 18 and 21;
these experiments were performed as described in Chang et al.
(1999, ibid.). High polyploid and G2/M fractions were also observed
among floating dead cells collected after release from IPTG (shown
in FIG. 5C); microscopic analysis indicated that many of these dead
cells were in mitosis.
[0099] To investigate the origin of polyploid cells, the time
course of changes in DNA content of the entire cell population
after release from IPTG was determined. The number of polyploid
cells greatly increased 24-28 hrs after release (as shown in FIG.
5D), concurrently with the resumption of DNA synthesis (compare,
FIG. 4B). This result indicated that many of the released cells
were undergoing endoreduplication, an unscheduled round of DNA
replication. The time course and magnitude of endoreduplication
were very similar, however, between cells released after one day
(FIG. 5D) or after 3-5 days of IPTG inhibition.
[0100] A major difference between cells that were inhibited by IPTG
for one day compared with those inhibited for 5 days emerged,
however, when the morphology of attached mitotic cells arising 1-2
days after release from IPTG was examined. These results are shown
in FIG. 6. While an overwhelming majority of mitotic figures in
untreated cells appeared morphologically normal (FIG. 6, left),
most of the mitotic figures in cells released after IPTG treatment
showed numerous abnormalities, including multicentric mitosis,
uneven chromosome distribution and prophase arrest (FIG. 6, right).
The percentage of normal mitoses in 1-day and 5-day IPTG treated
cells were 45% and 2%, respectively, which is close to the
corresponding values for clonogenic recovery (38% and 1%). These
results suggest that abnormal mitosis, together with
endoreduplication, are responsible for the loss of clonogenicity
after release from p21.
[0101] These results indicated that induced expression of p21 has
profound effects not only on the cells while the gene is expressed,
but effects that linger and interfere with normal recovery of the
cells into the cell cycle and growth.
EXAMPLE 3
Analysis of Gene Expression Modulated by p21 Gene Expression
[0102] The results disclosed in Example 3 suggested that the
morphological and cell cycle consequences of p21 induction could be
the result of repression of genes that control cell cycle
progression. The effects of p21 induction on cellular gene
expression were examined as follows.
[0103] Reverse transcription-polymerase chain reaction (RT-PCR)
analysis was performed to investigate expression of genes known to
be involved in the control of cell cycle checkpoint progression.
Preliminary RT-PCR analysis of 27 genes involved in cell cycle
control and DNA replication revealed that eight of these genes were
inhibited by IPTG in p21-9 cells. Total RNA was extracted from
p21-9 cells collected at different time points during IPTG
treatment and release. RT-PCR analysis of changes in gene
expression for downregulated genes was carried out essentially as
described by Noonan et al. (1990, Proc. Natl. Acad. Sci. USA 87:
7160-7164).
[0104] A more comprehensive analysis was performed by isolating
poly(A).sup.- RNA from untreated p21-9 cells and from cells that
were treated for 3 days with 50 .mu.m IPTG. cDNA was prepared from
the poly(A).sup.- RNA and used as probes for differential
hybridization with the Human UniGEM V cDNA microarray (as performed
by Genome Systems, Inc., St. Louis, Mo.), which contains over 4,000
sequence-verified known human genes and 3,000 ESTs. More than 2,500
genes and ESTs showed measurable hybridization signals with probes
from both untreated and IPTG-treated p21-9 cells. Genes that were
downregulated with balanced differential expression.gtoreq.2.5 or
upregulated with balanced differential expression.gtoreq.2.0 are
listed in Tables 1 and 2, respectively.
[0105] Expression of 69 of these genes was individually tested by
RT-PCR or northern hybridization with probes derived from inserts
of the cDNA clones present in the microarray; these cDNAs were
obtained from Genome Systems, Inc. In addition, enzyme-linked
immunosorbent assay (ELISA) measurement of p21 protein was carried
out using WAF1 ELISA kit (obtained from Oncogene Science,
Uniondale, N.Y.) as described (Chang et al., 1999, Oncogene 18:
4808-4818). The following primary antibodies were used for
immunoblotting: mouse monoclonal antibodies against Cdc2 (Santa
Cruz), cyclin A (NeoMarkers), Plk 1 (Zymed) and Rb (PharMingen):
rabbit polyclonal antibodies against MAD2 (BadCo), p107 (Santa
Cruz), CTGF (Fisp-12; a gift of Dr. L. Lau). Prc 1 (a gift of Drs.
W. Jiang and T. Hunter), and topoisomerase II.alpha. (Ab0284; a
gift of Dr. W. T. Beck), and sheep polyclonal antibody against SOD2
(Calbiochem). Horse radish peroxidase (HRP)-conjugated secondary
antibodies used were goat anti-mouse and goat anti-rabbit IgG
(Santa Cruz) and rabbit anti-sheep IgG (KPL). Protein
concentrations in all samples were equalized after measurement with
BioRad protein assay kit. Immunoblotting was carried out by
standard procedures, and the signal was detected by
chemiluminescence using LumiGlo (KPL).
[0106] These results are shown in FIGS. 7A through 7C. The changes
in gene expression predicted by the microarray assays described
above were confirmed for 38/39 downregulated and 27/30 upregulated
genes. The observed signal differences in northern hybridization or
RT-PCR for most of the tested genes (FIGS. 7A through 7C) appeared
to be higher than the values of balanced differential expression
determined from the cDNA array (Tables 1 and 2), suggesting that
cDNA array hybridization tends to underestimate the magnitude of
p21 effects on gene expression. Changes in the expression of 6
downregulated and 4 upregulated genes were also tested at the
protein level by immunoblotting (FIG. 7B) or zymography (not shown)
and were confirmed in all cases tested.
[0107] It was recognized that p21-mediated changes in gene
expression were comprised of near-term effects and longer-term
effects that followed p21-induced cell growth arrest. For this
purpose, the time course of changes in the RNA levels of a subset
of p21-inhibited (FIG. 7B) and p21-induced genes (FIG. 7C) after
the addition and removal of IPTG was determined. Immunoblotting was
used to analyze the time course of p21-induced changes in Rb
phosphorylation (as indicated by electrophoretic mobility) and in
the cellular levels of Rb and several proteins that were inhibited
by p21 according to the cDNA array; these results are shown in FIG.
7B. Rb was found to become dephosphorylated as early as 6 hrs after
the addition of IPTG. Furthermore, Rb protein levels decreased
sharply between 12-24 hrs (shown in FIG. 7B), but no significant
changes were detected in RB mRNA levels (data not shown). A similar
decrease was observed for a Rb-related protein p107 (shown in FIG.
7A).
[0108] A. Gene Expression Inhibited by p21.
[0109] All the tested p21-inhibited genes showed a rapid response
to p21 induction and release. Five of these genes (topoisomerase
IIa. ORC1, PLK1, PRC1 and XRCC9) showed significant inhibition at
both RNA and protein levels between 4 and 8 hrs after the addition
of IPTG (FIG. 7B). This pattern has been termed an "immediate
response," which parallels the kinetics of cell growth arrest and
Rb dephosphorylation. Other p21-inhibited genes (such as CDC2 or
DHFR) showed an "early response" pattern that lags slightly behind
the cessation of DNA replication and mitosis, with a major decrease
in mRNA levels detectable only 12 hrs after the addition of IPTG.
All p21-inhibited genes, however, resumed their expression 12-16
hrs after the removal of IPTG, when the cells were still
growth-arrested and before the resumption of DNA replication and
mitosis (FIG. 7B). This analysis indicated that changes in the
expression of p21-inhibited genes were near-term effects of p21
induction and release and were not a consequence of cell growth
arrest and recovery.
[0110] In summary, 69 genes and 3 ESTs were identified by the cDNA
microarray as downregulated in p21-induced cells, with balanced
differential expression of 2.5-12.6 (Table 1A); 5 additional genes
identified by our earlier assays as downregulated in IPTG-treated
cells are listed in Table 1B. A strikingly high fraction of
downregulated genes identified by the cDNA array (43 of 69) were
associated with mitosis, DNA replication, segregation and repair
and chromatin assembly, indicating a highly selective nature of
p21-mediated inhibition of gene expression.
[0111] The largest group of p21-downregulated genes are that have
been implicated in the signaling, execution and control of mitosis.
These genes include CDC2 and cyclin B1 that form the
mitosis-initiating complex, polo-like kinase (PLK1) that plays a
role in the onset of mitosis, mitotic checkpoint control and
cytokinesis (Glover et al., 1998, Genes Develop. 12: 3777-3787) and
CDC2-interacting protein CKsHs1, a target of mitotic checkpoint
control (Hixon et al., 1998, Mol. Cell Biol. 18: 6224-37). Other
genes in this group encode a homolog of Xenopus condensin protein
XCAP-H, a homolog of Rad21 repair protein involved in sister
chromatid cohesion (Losada et al., 1998, Genes Develop. 12:
1986-1997) and mitotic recombination (McKay et al., 1996, Genomics
36: 305-315), a centrosome-associated kinase AIK1 involved in
spindle formation (Kimura et al., 1997, J. Biol. Chem. 272:
13766-13771), centromere proteins CENP-A and CENP-F, as well as
MAD2 and BUBR1 proteins that play a central role in the spindle
checkpoint control (Li and Benezra, 1996, Science 274: 246-248;
Chan et al., 1999, J. Cell Biol. 146: 941-954), mitotic
centromere-associated kinesin (MCAK), kinesin-like protein HSET
located at the interphase centrosome and mitotic spindle, CHL1
helicase (a homolog of a yeast protein that plays a role in proper
chromosome distribution during mitosis; Gerring et al., 1990, EMBO
J. 9: 4347-4358), and three proteins involved in cytokinesis, Prc1,
Aim1/Aik2 and citron kinase (Jiang et al., 1998, Mol. Cell 2:
877-885; Terada et al., 1998, EMBO J. 17: 667-676; Madaule et al.,
1988, Nature 394: 491-494). p21 also inhibits genes that encode
nuclear envelope proteins lamin B1 and lamin B2, lamin-associated
polypeptides .alpha. (thymopoietin .alpha.) involved in nuclear
assembly, and M-phase phosphoproteins MPP2 aMPP5. Deficiencies in
many of the above proteins are known to result in abnormal
chromosome segregation and polyploidization, the same events that
we observed in p21-9 cells after release from IPTG.
[0112] Many p21-inhibited genes are involved in DNA replication and
segregation, chromatin assembly and DNA repair. Some of these genes
encode enzymes involved in nucleotide biosynthesis, including
ribonucleotide reductase subunits M1 and M2, thymidine kinase,
thymidylate synthase, uridine phosphorylase and dihydrofolate
reductase. Other proteins are involved in DNA replication,
including components of the replication licensing factor
Cdc47/Mcm4, Cdc45 homolog, Orc1 protein of the origin recognition
complex, DNA polymerase a, B-Myb, 37-kD subunit of replication
factor C, and DNA ligase I. This group also includes genes involved
in the segregation of replicated DNA (topoisomerase IIa),
inheritance of epigenetically determined chromosomal states (p60
subunit of chromatin assembly factor-I), and other chromatin
components, such as high mobility group proteins 1 and 2. Several
p21-inhibited genes are associated with DNA repair, including
XRCC9, which may be involved in DNA post-replication repair or cell
cycle checkpoint control (deWinter et al., 1998, Nat Genet. 20:
281-283), Rad54 recombination repair protein, exonuclease
Hex1/Rad2, and the above Rad21 homolog and DNA ligase I.
[0113] Over 60% of p21-inhibited genes in the cDNA array are
involved in mitosis. DNA replication, segregation and repair. Such
biological selectivity is unprecedented in large-scale expression
profiling studies. A corollary to this observation is that
p21-inhibited genes whose function is presently unknown are likely
to play a role in cell cycle progression. Indeed, six p21-inhibited
genes were originally listed in the cDNA array as ESTs or genes
with unknown function, but a database search has linked three of
their products to cell division of DNA repair. In one case, the
originally identified EST was found to map in a genomic clone 3' to
the coding sequence of citron kinase; inhibition of the citron
kinase gene by p21 was then demonstrated by RT-PCR based on its
coding sequence. Cloning of additional p21-inhibited genes is
likely to yield novel genes that play a role in mammalian cell
division.
[0114] These results also suggest further opportunities for
discovering components of the cellular program of p21-induced
senescence that would be targets for therapeutic intervention. It
has been suggested that p21-mediated inhibition of gene expression
is a result of E2F inhibition (de Toledo et al., 1998, Cell Growth
Differ. 9: 887-896). In agreement with this interpretation, a
subset of our p21-inhibited genes (e.g. CDC2, ORC1, DHFR, cyclin
A1) contain E2F sites in their promoters. On the other hand, no E2F
sites could be found in the promoters of some p21-inhibited genes
(e.g. cyclin B1), and some E2F-dependent genes (e.g. cyclin E) were
unaffected by p21 induction (data not shown). Some as yet
unidentified regulatory factors, in addition to E2F, may therefore
be involved in p21-mediated inhibition of gene expression. Such
additional factors represent targets for novel pharmaceuticals, the
existence and identity of said targets being available for
elucidation using the methods and reagents provided by the instant
invention.
[0115] B. Gene Expression Induced by p21
[0116] In addition to genes repressed by p21 expression, the assays
described above detected genes induced by p21. The pattern of gene
expression of p21-induced genes is shown in FIG. 7C. In contrast to
p21-inhibited genes, p21-upregulated genes increased their
expression only 48 hrs after the addition of IPTG, i.e. after the
onset of growth arrest in all cells. Only one tested gene, tissue
transglutaminase (t-TGase), showed a detectable increase 12 hrs
after the addition of IPTG, but its expression reached a maximum
only by 48 hrs (as shown in FIG. 7C). Furthermore, elevated
expression of all the tested genes (except for t-TGase) persisted
for at least three days after release from IPTG, well after
resumption of the cell cycle (not shown). This "late response"
kinetics indicated that p21 induction of such genes was a delayed
effect relative to p21-mediated growth arrest.
[0117] 48 known genes and 6 ESTs or genes with unknown functions
were identified as upregulated in p21-induced cells, with balanced
differential expression of 2.0-7.8 (Table 2). A very high fraction
(20/48) of identifiable genes in this group encode extracellular
matrix (ECM) components (e.g. fibronectin 1, laminin b2, Mac-2
binding protein), other secreted proteins (e.g. activin A,
connective tissue growth factor, serum amyloid A), or ECM receptors
(such as integrin b3). Several of these secreted proteins, as well
as a large group of p21-induced intracellular proteins (Table 2),
are known to be induced in different forms of stress response or to
play a role in stress-associated signal transduction. Remarkably,
many genes that we found to be induced by p21 are also upregulated
in cellular senescence, organism aging, or different age-related
diseases.
[0118] In contrast to p21-inhibited genes, none of the genes found
to be induced by p21 have any known functions that may trigger cell
growth arrest. Furthermore, the induction of such genes is a late
response that lags far behind the onset of growth arrest.
Interestingly, several p21-induced genes are positively regulated
by NF.kappa.B, including superoxide dismutase 2 (SOD2) (Jones et
al., 1997, Mol. Cell. Biol. 17: 6970-6981), t-TGase (Mirza et al.,
1997, Amer. J. Physiol. 272: G281-G288). Alzheimer's .beta.-amyloid
precursor protein (APP) (Grilli et al., 1996. J. Biol. Chem. 271:
15002-15007) and the inflammatory protein serum amyloid A (SAA)
(Jensen and Whitehead, 1998, Biochem J. 334: 489-503). Since p21
activates NF.kappa.B-dependent transcription through its effect on
the transcription cofactor p300 (Perkins et al., 1997, Science 275:
523-527), it is possible that activation of p300 or related
transcription cofactors may be responsible for the effect of p21 on
some of the upregulated genes. The delayed kinetics of p21-mediated
induction of gene expression suggest, however, that this induction
occurs far downstream of the immediate effects of p21.
[0119] These results, and the nature of the genes set forth in
Table 2, indicate that expression of these genes is not involved in
the growth arrest function of p21. However, the abundance of
secreted proteins that we found among the products of p21-activated
genes has important physiological consequences. As disclosed in
Example 5 below, conditioned media from p21-induced cells exhibits
two biological effects predicted by the nature of p21-upregulated
genes: stimulation of cell growth and suppression of apoptosis.
This finding, taken with the above discussed genetic
destabilization in p21-induced cells, suggests that "paracrine"
effects of p21 may contribute to carcinogenesis through a
tumor-promoting effect on neighboring cells. This raises the
possibility that suppression of p21-mediated gene induction may
provide a way to achieve an anti-carcinogenic effect, and that
p21-mediated gene induction pathways are targets of rational drug
design for a new generation of cancer-preventing drugs.
[0120] The observed paracrine, anti-apoptotic effect of p21
induction agrees with the reported activities of prosaposin and
galectin-3, secreted proteins that we found to be induced by p21
(Table 2). Anti-apoptotic activity has also been associated with
p21-induced intracellular proteins SOD2 (Manna et al., 1998, J.
Biol. Chem. 273: 13245-13254) and R-Ras (Suzuki et al., 1998, FEBS
Lett. 437: 112-116). Paradoxically, p21-induced t-TGase and
cathepsin B (Singhal et al., 1997, J. Investig. Med. 45: 567-575)
have been ascribed a pro-apoptotic function. There are conflicting
reports in the literature regarding the effects of p21 on
apoptosis. In some systems, p21 overexpression induced apoptosis
(Prabhu et al., 1996, Clin. Cancer Res. 2: 1221-1229; Tsao et al.,
1999, J. Virol. 73: 4983-4990), but in other studies p21 protected
cells from apoptosis induced by several types of treatment (Gorospe
et al., 1997, Oncogene 14: 929-935; Lu et al., 1998, Oncogene 16:
705-712; Bissonnette & Hunting, 1998, Oncogene 16: 3461-3469).
The results disclosed herein that p21 induces both anti-apoptotic
and pro-apoptotic genes may explain the contradictory reports on
the effects of p21 on apoptosis.
EXAMPLE 4
[0121] Identifying the Specificity of p21 Induction by Comparing
IPTG-Treated and Serum-Starved p21-9 Cells
[0122] The identity of p21-induced changes in cellular gene
expression that are likely to be a consequence of cell growth
arrest was determined as follows. Analogous experiments were
performed using a truncated form of p21 (comprising amino acids
1-90) and identical results were obtained.
[0123] Growth arrest (quiescence) was induced in p21-9 cells by
serum starvation produced by culturing the cells in serum-free
media for 4 days. In serum-starved cells, unlike IPTG-treated p21-9
cells, the cells did not develop a senescent morphology and showed
only very weak SA-.beta.-gal expression. p21 levels in
serum-starved cells were increased only about 2-fold, as opposed to
the 15-20 fold increase seen in IPTG-treated cells. FIG. 9 shows
RT-PCR analysis performed as described above of the expression of a
group of p21-inhibited and p21-induced genes in p21-9 cells that
were growth-arrested after 4 days in serum-free media or 3 days in
the presence of 50 .mu.M IPTG. Genes that were completely inhibited
in p21-9 cells when the culture media contained 50 .mu.M IPTG were
also inhibited in serum-starved cells, but most of these genes were
inhibited to a lesser extent than in IPTG-treated cells.
[0124] Genes whose expression is induced by p21 showed three
distinct patterns. The first group are genes whose expression is
induced as strongly in quiescent cells as in senescent cells. These
include galectin-3, superoxide dismutase 2, complement C3 and
prosaposin, indicating that their induction was a consequence of
cell growth arrest or that such genes were exquisitely sensitive to
slightly elevated p21 levels. The second group are genes that were
up-regulated in quiescent cells but not as strongly as in senescent
cells. These genes include fibronectin-1, Mac2 binding protein and
the Alzheimer precursor protein serum amyloid A. The third group
are genes that are not detectably induced in quiescent cells but
are strongly induced in senescent cells. These genes include CTGF,
plasminogen activator inhibitor 1, tissue transglutaminase or
natural killer cell marker protein NK4, integrin beta 3 and activin
A.
[0125] The difference between the response of certain genes to
induction of quiescence by serum starvation and cellular senescence
through IPTG-induced overexpression of p21 identified these genes
as diagnostic markers of senescence. Furthermore, novel senescence
markers can now be identified by comparing their expression between
p21-expressing and quiescent cells.
EXAMPLE 5
Production of Conditioned Media Containing Mitogenic Factors and
Mitogenic Activity Assays
[0126] Several p21-upregulated secreted proteins act as growth
factors, including CTGF (Bradham et al., 1991, J. Cell Biol. 114:
1285-1294), activin A (Sakurai et al., 1994, J. Biol. Chem.
269:14118-14122), epithelin/granulin (Shoyab et at., 1990, Proc.
Natl. Acad. Sci. USA 87: 7912-7916) and galectin-3 (Inohara et al.,
1998, Exp Cell Res. 245: 294-302), suggesting that p21 induction
may cause paracrine mitogenic effects. In addition, galectin-3
(Akahani et al., 1997, Cancer Res. 57:5272-5276) and prosaposin
(Hiraiwa et al., 1997, Proc. Natl. Acad. Sci. USA 94: 4778-4781)
were shown to have anti-apoptotic activity. Conditioned media from
IPTG-treated p21-9 cells was tested to investigate whether it would
have an effect on cell growth and apoptosis.
[0127] In these experiments, conditioned media were prepared by
plating 10.sup.6 p21-9 cells per 15 cm plate in the presence of
DMEM/10% FCS. The next day, IPTG was added to a final concentration
of 50 .mu.M, and this media was replaced three days later with DMEM
supplemented with 0.5% FCS and 50 .mu.M IPTG. Two days later (days
3-5 of IPTG treatment), this conditioned media was collected and
stored at 4.degree. C. Up to 15 days before use. Control media were
prepared by adding IPTG-free DMEM/0.5% FCS to untreated cells grown
to the same density as IPTG-treated cells and collecting the media
two days thereafter.
[0128] The slow-growing human fibrosarcoma cell line HS 15.T was
used to detect mitogenic activity in these conditioned media. For
mitogenic activity assays, both types of conditioned media, as well
as fresh media and 1:1 mixtures of conditioned media and fresh
media were used to test mitogenic activity. In these experiments,
the conditioned media were supplemented with 1% or 2% FCS. Briefly,
HS 15.T cells were plated in 12-well plates at 15,000 cells per
well. Two days later, these cells were cultured in different types
of media. The cells were grown in conditioned media for 60 hr, and
the .sup.3H-thymidine at a concentration of 3.13 .mu.Ci/mL was
added and incubated for 24 hrs. Cells were then collected and their
.sup.3H-thymidine incorporation determined as described by Mosca et
al. (1992, Mol. Cell. Biol. 12: 4375-4383).
[0129] The addition of IPTG to fresh media had no effect in this
assay (not shown). As shown in FIG. 8A, there was no significant
difference between cell growth in fresh media and in conditioned
media from untreated p21-9 cells. In contrast, conditioned media
from IPTG-treated cells increased .sup.3H-thymidine incorporation
up to three-fold (FIG. 8A). Growth stimulation of HS 15.T by
conditioned media from IPTG-treated cells was also detectable by
methylene blue staining (data not shown).
[0130] The effect of this conditioned media on apoptosis was also
determined. These experiments used a mouse embryo fibroblast line
C8, immortalized by EIA. This cell line is highly susceptible to
apoptosis induced by different stimuli (Lowe et al., 1994, Science
266: 807-810; Nikiforov et al., 1996, Oncogene 13: 1709-1719),
including serum starvation (Lowe et al., 1994, Proc. Natl. Acad.
Sci. USA 91: 2026-2030). Apoptosis was analyzed by plating
3.times.10.sup.5 CS cells per 6-cm plate, and replacing the media
on the following day with fresh media supplemented with 0.4% serum
or with conditioned media (no fresh serum added). DNA content
analysis and DAPI staining were carried out after 24 hrs and 48
hrs, and relative cell numbers were measured by methylene blue
staining (Perry et al., 1992, Mutat. Res. 276: 189-197) after 48
hrs in low-serum media.
[0131] The addition of low-serum fresh media or conditioned media
from IPTG-treated or untreated cells rapidly induced apoptosis in
C8 cells, as evidenced by cell detachment and apoptotic morphology
detectable in the majority of cells after DAPI staining (not
shown). Conditioned media from IPTG-treated cells, however,
strongly increased cell survival relative to fresh media and
conditioned media from untreated cells, as measured by methylene
blue staining of cells that remained attached after 48 hrs (as
shown in FIG. 8B). The effect of the conditioned media from
p21-induced cells was even more apparent in FACS analysis of
cellular DNA content, which was carried out on combined attached
and floating C8 cells 24 hrs and 48 hrs after media change (FIG.
8B). Unlike many other cell lines, apoptosis of C8 cells produces
only a few cells with decreased (sub-G1) amount of DNA, and it is
characterized by selective disappearance of cells with G2/M DNA
content (Nikiforov et al., 1996, ibid.). Serum-starved cells in
conditioned media from IPTG-treated cells retained the G2/M
fraction and showed cell cycle profiles that resembled control
cells growing in serum-rich media (FIG. 8B). The addition of IPTG
by itself had no effect on apoptosis in C8 cells (not shown). Thus,
p21 induction in HT1080 cells results in the secretion of mitogenic
and anti-apoptotic factors, as predicted by the nature of
p21-unregulated genes.
[0132] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims.
1TABLE 1 Genes downregulated by p21 induction A. p21-inhibited
genes identified by UniGemV array: Balanced Diff. Confirmed Genes
Accession No. Expr. by.sup.a Associated with mitosis: CDC2 X05360
2.5 R, W CKsHs1 (CDC2 kinase) X54941 5.5 R PLK1 (polo-like kinase)
U01038 5.1 R, W XCAP-H condensin homolog D38553 6 R CENP-A
(centromere protein A) U14518 5.3 R CENP-F (centromere protein F)
U30872 2.5 R MAD2 U65410 6.6 R, W BUBR1 AF053306 5.9 R MCAK
(mitotic centromere-associated kinesin) U63743 3.8 R HSET
kinesin-like protein AL021366 3.6 R CHL1 helicase U75968 3.3 R
AIK-1 (aurora/IPL1-related kinase) D84212 4.6 R AIM-1 (AIK-2;
aurora/IPL1-related kinase) AF004022 10.2 R PRC1 (protein
regulating cytokinesis 1) AF044588 12.6 R, W Citron kinase H10809
2.7 R Lamin B1 L37747$$ 7 Lamin B2 M94362 2.7 LAP-2
(lamin-associated protein 2) U18271 4.6 R MPP2 (M phase
phosphoprotein 2) U74612 3.7 R MPP5 (M phase phosphoprotein 5)
X98261 3.7 Associated with DNA replication, segregation and
chromatin assembly: Thymidine kinase 1 K02581 2.9 R Thymidylate
synthase X02308 3.9 R Uridine phosphorylase X90858 2.5
Ribonucleotide reductase M1 X59543 4.6 R Ribonucleotide reductase
M2 X59618 10.7 R CDC47 homolog (MCM7) D55716 9.6 R CDC21 homolog
(MCM4) X74794 2.7 R CDC45 homolog (Porc-Pl) AJ223728 4.1 R HsORC1
(origin recognition complex 1) U40152 2.7 R DNA polymerase .alpha.
X06745 2.8 R Replication factor C (37-kD subunit) M87339 2.6 B-MYB
X13293 9.1 HPV16 E1 protein binding protein U96131 3.7
Topoisomerase II.alpha. J04088 8.6 R Chromatin assembly factor-I
(p60 subunit) U20980 2.7 R High-mobility group chromosomal protein
2 X62534 3.7 R High-mobility group chromosomal protein 1 D63874 3.6
R Histone H2A.F/Z variant AA203494 2.8 Associated with DNA repair:
XRCC9 U70310 3.6 R RAD54 homolog X97795 5.4 R HEX1 5'-3'
exonuclease (RAD2 homolog) AF042282 5.2 R ATP-dependent DNA ligase
I M36067 2.5 R RAD21 homolog D38551 2.9 R Associated with
transcription and RNA processing: Putative transcription factor
CA150 AF017789 2.8 Transcriptional coactivator ALY AF047002 3.3
WHSC1/MMSET (SET domain protein) AA401245 2.9 NN8-4AG (SET domain
protein) U50383 2.8 EZH2 (enhancer of zeste homolog 2) U61145 2.8
PTB-associated splicing factor X70944 2.5 AU-rich element
RNA-binding protein AUF1 U02019 2.8 U-snRNP-associated cyclophilin
AF016371 2.8 Other genes: 3-phosphoglycerate dehydrogenase AF006043
4.8 L-type amino acid transporter, subunit LAT1 M80244 4.1 R
Hyaluronan-mediated motility receptor U29343 4 Phorbolin I
(PKC-inducible) U03891 3.9 PSD-95 binding family protein D13633 3.7
R HTRIP (TNF receptor component) U77845 3.6 NAD-dependent
methylenetetrahydrofolate dehydrogenase X16396 3.4 Membrane
glycoprotein 4F2 antigen heavy chain J02939 3.2 Mucin-like protein
D79992 3.2 MAC30 (differentially expressed in meningiomas) L19183
2.9 P52rlPK (regulator of interferon-induced protein kinase)
AF007393 2.8 Putative phosphoserine aminotransferase AA192483 2.8
Glucose 6-phosphate translocase Y15409 2.7 Calcyclin binding
protein AF057356 2.6 Ornithine decarboxylase 1 X16277 2.6 R
Trophinin assisting protein (tastin) U04810 2.5 Acyl-coenzyme A
cholesterol acyltransferase L21934 2.5 Pinin/SDK3 Y10351 2.5 Genes
with unknown function: EST AA975298 2.7 EST AA034414 2.5 EST
AA482549 2.5 B. p21-inhibited genes identified by RT-PCR: Genes
Accession No. UniGemV result.sup.b Cyclin A1 U66838 IS Cyclin B1
M25753 IS CDC25A NM_001789 A Dihydrofolate reductase J00140 1.5
ING1 NM_005537 A .sup.aAbbreviations: R, RT-PCR; W, western
blotting .sup.bAbbreviations: IS, insufficient signal; A, absent
from the array
[0133]
2TABLE 2 Genes upregulated by p21 induction Accession Balanced Diff
Genes No Expr Confirmed by.sup.a Secreted proteins and proteins
associated with extracellular matrix: Fibronectin 1 X02761 5.7 R
Plasminogen activator inhibitor, type I M14083 3.7 R, N Plasminogen
activator, tissue type M15518 2.8 Z Laminin .beta.2 X79683 2.1
Desmocollin 2a/bb X56807 3.5 Podocalyxin-like protein U97519 2
Activin A (inhibin .beta.A) J03634 2 R Galectin 3 (Mac-2) AB006780
2.4 N Mac-2 binding protein L13210 2 R, N Prosaposin J03077 2.9 N
CTGF (connective tissue growth factor) M92934 3.3 N
Granulin/epithelin AF055008 2.1 N Cathepsin B L04288 2.4 N Tissue
transglutaminase M55153 2.5 R, N, W P37NB (slit homolog) U32907 2.1
Serum amyloid A protein precursor M26152 4 R, N, W Alzheimer's
disease amyloid A4 protein precursor D87675 2 R, N Complement C3
precursor K02765 5.9 R, N Testican X73608 2.1 N Integrin .beta.3
M35999 2.1 R, N Lysosomal proteins: N-acetylgalactosamine-6-sulfate
sulfatase U06088 2.3 N Acid alpha-glucosidase X55079 2.4 N Acid
lipase A (cholesterol esterase) X76488 2.1 N Lysosomal
pepstatin-insensitive protease (CLN2) AF017456 2.5 Mitochondrial
proteins: Superoxide dismutase 2 X07834 3.5 R, N, W Metaxin J03060
3.4 2,4-dienoyl-CoA reductase U78302 2 Other genes associated with
stress response and signal transduction: Ubiquitin-conjugating
enzyme (UbcH8) AF031141 2 Ubiquitin-specific protease 8 D29956 2
RTP/Cap43/Drg1/Ndr1 (Inducible by nickel, retinoids, D87953 2.5
homocysteine and ER stress) C-193 muscle ankyrin-repeat nuclear
protein (cytokine- X83703 3 inducible) LRP major vault protein
associated with multidrug resistance X79882 2.2 N .beta.-arrestin
related HHCPA78 homolog (upregulated by S73591 4.1 N vitamin D3)
R-RAS M14949 2.4 RAB 13 small GTPase X75593 2.2 P66 SHC (ski
oncogene) U73377 2 N MK-STYX (MAP kinase phosphatase-like protein)
N75168 2 H73 nuclear antigen/MA-3 apoptosis-related/TIS U96628 2.4
(topoisomerase-inhibitor suppressed) Other genes: Natural killer
cells protein 4 M59807 4.4 R TXK tyrosine kinase (T-cell specific)
L27071 3.8 X-linked PEST-containing transporter U05321 2.1 AMP
deaminase 2 M91029 2 N FIP2/HYPL huntingtin-interacting protein
AF061034 2 DNASE I homolog X90392 2.5 N Transcription factor 11
X77366 2 Histone H2A.2 L19779 2.8 Histone H2B AL021807 2.4 Genes
with unknown function: 23808 AF038192 2.1 CGI-147 AA307912 2.1 N
EST W89120 2.8 EST AI026140 2.5 EST AA218982 2.4 EST W63684 2
.sup.aAbbreviations: R, RT-PCR; N, northern hybridization; W,
western blotting; Z, zymography
* * * * *