U.S. patent application number 12/815125 was filed with the patent office on 2011-04-14 for cellular targets for treatment of retroviral infection.
This patent application is currently assigned to Fox Chase Cancer Center. Invention is credited to Rene Daniel, Gary D. Kao, Giuseppe Nunnari, Roger J. Pomerantz, Anna Marie Skalka.
Application Number | 20110086423 12/815125 |
Document ID | / |
Family ID | 35732723 |
Filed Date | 2011-04-14 |
United States Patent
Application |
20110086423 |
Kind Code |
A1 |
Daniel; Rene ; et
al. |
April 14, 2011 |
Cellular Targets for Treatment of Retroviral Infection
Abstract
Cellular targets for anti-retroviral drug development are
disclosed. The cellular targets comprise ATR kinase and its
relevant substrates, based on the identification of the ATR kinase
as required for the final step of retroviral DNA integration.
Assays for identifying modulators of retroviral integration via the
ATR kinase pathway are disclosed, as well as modulators identified
by such assays. Pharmaceutical preparations and methods of their
use in treating retroviral infection are also disclosed.
Inventors: |
Daniel; Rene; (Ambler,
PA) ; Skalka; Anna Marie; (Princeton, NJ) ;
Kao; Gary D.; (Wynnewood, PA) ; Nunnari;
Giuseppe; (Cantania, IT) ; Pomerantz; Roger J.;
(Chalfont, PA) |
Assignee: |
Fox Chase Cancer Center
|
Family ID: |
35732723 |
Appl. No.: |
12/815125 |
Filed: |
June 14, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11093692 |
Mar 30, 2005 |
7736848 |
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12815125 |
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PCT/US03/31164 |
Sep 30, 2003 |
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11093692 |
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60414791 |
Sep 30, 2002 |
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Current U.S.
Class: |
435/366 ;
435/325 |
Current CPC
Class: |
C12Q 1/18 20130101; C12Q
1/485 20130101 |
Class at
Publication: |
435/366 ;
435/325 |
International
Class: |
C12N 5/071 20100101
C12N005/071 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This application was made with government support under
National Institute of Health Grants AI40385, CA71515, CA06927 and
CA98090. The government has certain rights in the invention.
Claims
1-7. (canceled)
8. A method of interfering with retroviral replication in a host
cell infected with the retrovirus, comprising inhibiting an
constituent of the host cell needed to repair nucleic acids during
retroviral DNA integration into the host cell genome, wherein the
constituent is ATR kinase or a downstream target of ATR kinase.
9. The method of claim 8, wherein the host cell is a human
cell.
10. The method of claim 8, wherein the host cell is a non-human
cell.
11. The method of claim 8, wherein the inhibiting is accomplished
by exposing the cell to an agent that inhibits production or
activity of ATR kinase or a downstream target of ATR kinase.
12. The method of claim 11, wherein the agent is a
methylxanthine
13. The method of claim 12, wherein the methylxanthine is caffeine,
paraxanthine, theobromine, theophylline, or metabolites or
derivatives thereof.
14. The method of claim 8, wherein the retrovirus is human
immunodeficiency virus (HIV) or human T-cell leukemia virus
(HTLV).
15-25. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/093,692, filed Mar. 30, 2005 which is a
continuation-in-part of PCT Application No. PCT/US03/31164, filed
Sep. 30, 2003, which claims priority under 35 U.S.C. .sctn.119(e)
to U.S. Provisional Patent Application No. 60/414,791, filed Sep.
30, 2002, all of which are hereby incorporated by reference in
their entirety herein.
FIELD OF THE INVENTION
[0003] This invention relates to the field of retroviruses and
pathological conditions caused by retroviruses. In particular, this
invention provides novel cellular targets for anti-retroviral drug
development.
BACKGROUND OF THE INVENTION
[0004] Retroviruses cause several diseases and pathological
conditions, including a variety of tumors and leukemias. For
instance, human immunodeficiency virus (HIV) is the causative agent
of acquired immunodeficiency syndrome (AIDS) in humans. Another
significant disorder, adult T-cell leukemia-lymphoma, is caused by
the retrovirus HTLV I (human t-cell leukemia virus type I). HTLV I
also has been associated with other diseases, such as tropical
spastic paraparesis and HTLV I-associated myelopathy. Moreover,
many animal diseases of agricultural and veterinary importance are
known to be caused by retroviruses. These include avian sarcoma
leukosis virus (ASLV), feline leukemia virus (FeLV), bovine
immunodeficiency virus (BIV) and equine infectious anemia virus
(EIAV), among others.
[0005] Retroviruses encode several enzymes that are assembled into
the virus particle and whose activities catalyze essential steps in
the infectious cycle: (a) protease (PR), (b) the polymerase and (c)
ribonuclease H (RNAse H) activities of reverse transcriptase (RT),
and (d) integrase (IN). Following attachment and penetration of the
retrovirus into the host cell, the RT activities catalyze reverse
transcription of the viral RNA into DNA, and IN catalyzes the
integration of the retroviral DNA into the host cell DNA. The
cellular transcription and translation machinery is used to produce
viral RNA from the integrated viral DNA and thereafter to produce
the viral proteins.
[0006] Retroviral DNA integration proceeds in three distinct steps,
the first two of which have been reconstituted in vitro with the
purified retroviral enzyme, integrase. In the first step of
integration, denoted processing, retroviral integrase removes two
nucleotides from 3'-ends of the viral DNA, In the second step,
joining, these newly created ends are joined to staggered
phosphates in the host DNA in a concerted cleavage and ligation
reaction. This process creates an integration intermediate that
leaves gaps in the flanking host DNA sequence (FIG. 1). In the last
step of integration, these gaps are repaired, creating a stably
integrated provirus. This repair reaction can also be reconstituted
in vitro using combinations of various polymerases, ligases, and an
endonuclease (Brin et al., J. Biol. Chem, 275: 39287-39295, 2000;
Yoder & Bushman, J. Virol. 74: 11191-11200, 2000), but the
identity and mechanism of action of proteins responsible for these
reactions in vivo are not yet known. Retroviral DNA integration is
an essential step in retroviral replication and the integrase
protein is an attractive target for antiviral therapy. However, the
integrase gene is virus-encoded and therefore subject to a high
mutation rate, leading to drug resistance. This rapid evolution of
resistance should not occur with drugs that target cellular
functions necessary for integration, but not cell viability.
[0007] As reported in WO 00/17386 and by Daniel et al. (Science
284: 644-647), retroviral infection induces programmed death in
scid lymphocytes that are deficient in the DNA repair protein,
DNA-PK (DNA-dependent protein kinase). Furthermore, this response
to infection requires an active integrase. In addition to
retrovirus-induced scid cell death, it was also observed that
stable transduction by retroviral vectors, a measure of successful
DNA integration, is reduced in cells deficient in DNA-PK and other
components of the non-homologous end-joining (NHEJ) pathway. These
findings indicate that retroviral DNA integration is sensed as DNA
damage by the host cell, and that DNA repair proteins, such as
DNA-PK, may be recruited to facilitate stable integration into the
host genome. Components of the DNA damage response pathway(s) that
are required for this process therefore present advantageous
targets for anti-retroviral therapy.
[0008] DNA-PK belongs to a family of large, PI-3K-related protein
kinases, that also includes ATM (ataxia telangiectasia mutated) and
ATR (ATM and Rad3 related) kinases. The ATM and AIR kinases seem to
have a broader role than DNA-PK in response to DNA damage,
including regulation of cell cycle checkpoints. Detection of
aberrant DNA and chromosome structures by these proteins
coordinately triggers checkpoint pathways and DNA repair systems
(Zhou & Elledge, Nature 408: 433-439, 2000). Activation of a
DNA damage checkpoint results in cell cycle arrest, allowing time
for DNA repair or, in its absence, cell death.
[0009] Cells derived from A-T patients and from atm knockout mice
display sensitivity to ionizing radiation, chromosomal instability,
and defects in cell cycle checkpoints. The regulation of
checkpoints by ATM has been studied extensively and a number of ATM
substrates have been identified (Shiloh, Curr. Opin. Genet, Devel.
11: 71-77, 2001; Durocher & Jackson, Curr. Opin. Cell Biol. 13:
225-231, 2001). ATM also appears to play a direct role in DNA
repair at the sites of DNA damage (Durocher & Jackson, 2001,
supra). This ATM function may be mediated by modification of repair
proteins, such as phosphorylation of BRCA1, which is induced by
ionizing radiation (Cortez et al., Science 286: 1162-1166,
1999).
[0010] Studies with an inducible, transdominant-negative ATR mutant
have also implicated the ATR protein in cell cycle checkpoint
control (Cliby et al., EMBO J. 17: 159-169, 1998). Furthermore ATR,
like ATM, can respond to DNA damage induced by ionizing radiation,
and some data suggest that phosphorylation is sequential, with ATM
kinase being activated first (Durocher & Jackson, 2001, supra).
ATR and ATM seem therefore to be operating in similar or
overlapping pathways (Shiloh et al., 2001, supra). However, the
kinase activities of these proteins also have distinct
functions--for example, BRCA1 is phosphorylated by ATR, but not
ATM, in response to damage induced by UV light and stalled DNA
replication forks (Tibbetts et al., Genes Dev. 14: 2989-3002,
2000).
[0011] In addition to phosphorylation of checkpoint and DNA repair
proteins, ATM and ATR also share in vitro and in vivo sensitivity
to the radiosensitizing agent caffeine (Zhou et al., J. Biol. Chem.
275: 10342-10348, 2000; Blasina et al., Curr. Biol. 9: 1135-1138,
1999; Hall-Jackson et al., Oncogene 18: 6707-6713, 1999). The IC5Os
for ATM and ATR kinase inhibition are similar, and fall in the
range of 1-2 mM in vitro (Sarkaria et al., Cancer Res. 59:
4375-4382, 1999). Although ATM function is required for the
residual retroviral transduction that occurs in cells that are
deficient in NHEJ proteins such as DNA-PK, retroviral transduction
is normal in ATM-deficient cells (WO 00/17386; Daniel et al., Mal.
Cell. Biol. 21: 1164-1172, 2001). Further, DNA-PK is not sensitive
to caffeine (Sarkaria et al., 1999, supra).
SUMMARY OF THE INVENTION
[0012] It has been discovered in accordance with the present
invention that the cellular ATR kinase is required for efficient
retroviral integration. Accordingly, the present invention features
both in vitro and in vivo methods of identifying agents that
modulate this novel cellular target for retroviral therapy, and
resultant agents identified by such methods. Such in vitro methods
comprise combining a test compound suspected of inhibiting
production or activity of ATR kinase with ATR kinase and a
downstream target of ATR kinase activity, and then measuring the
effect of the test compound on the product of ATR kinase reactivity
with the downstream target as compared to appropriate controls.
Such in vivo methods comprise combining a test compound suspected
of inhibiting production or activity or ATR kinase with cultured
transducible host cells and a retroviral vector encoding a
detectable gene product under conditions wherein transduction of
the host cells with the retroviral vector results in production of
the detectable gene products, and then measuring the amount of
detectable gene product produced as compared to appropriate
controls.
[0013] The invention also features a method of inhibiting
retroviral replication in a host cell, which comprises inhibiting
the production or activity of cellular ATR kinase or downstream
targets of ATR kinase. The invention also provides pharmaceutical
preparations for treating retroviral infection, which utilize as
their active ingredient agents that modulate ATR kinase or
downstream targets of ATR kinase. Such agents include
methylxanthines such as caffeine, paraxanthine, theobromine, and
theophylline, as well as their respective derivatives and
metabolites. The invention further provides methods of inhibiting
retroviral infection in subjects infected with a retrovirus
comprising administering the pharmaceutical preparations of the
invention to the subjects in an amount and for a time effective for
inhibiting retroviral replication.
[0014] Various features and advantages of the present invention
will be understood by reference to the drawings, detailed
description and examples that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1. Diagram showing steps in the pathway of stable
retroviral DNA integration.
[0016] FIG. 2. Effect of caffeine on transduction by HIV-1- and
ASV-based vectors. (A) HeLa cells were infected with the HIV-1-lacZ
vector and exposed to caffeine for 24 hr, Five days post infection,
cells were stained using a .beta.-galactosidase assay and blue
colonies were counted. (B) HeLa cells were infected with the ASV
neo.sup.r vector and exposed caffeine for 24 hr. After removal of
caffeine, G418 was added to a final concentration of 1
G418-resistant colonies were counted 6 days post infection.
[0017] FIG. 3. Effect of caffeine on the growth and colony
formation of uninfected HeLa cells and on expression of the
neo.sup.r reporter. (A) Effect on cell growth. Cells were plated at
a density of 10.sup.5 cells per 60 mm dish in the presence of
increasing concentrations caffeine. The drug was applied to the
cells for 24 hr and then removed. At indicated intervals, cells
were harvested and viable cells counted. Open circles, no caffeine
added; crosses, 1 mM caffeine; squares, 2 mM caffeine; triangles, 4
mM caffeine, (B) Effect on colony formation. 10.sup.3 HeLa cells
were plated on 60 mm dishes in the presence of caffeine;
drug-containing medium was removed after 24 hr and colonies
(triangles) were counted 9 days later. A population of ASV
transduced, G418-resistant HeLa cells was plated at 10.sup.3 per 60
mm dishes, in the presence of both caffeine and G418. Caffeine was
removed after 24 hr, and G418 was retained in the medium
continuously. Colonies (circles) were counted 8 days later. (C)
Expression of the neo.sup.r reporter. A population of HeLa cells
that had been stably transduced with the ASV vector carrying the
neo.sup.r reporter gene was exposed to increasing concentrations of
caffeine for 24 hr. The cells were then lysed and neomycin
phosphotransferase II was detected by Western blot analysis. Lane
1, non-transduced HeLa cells, lanes 2-5, stably transduced HeLa
cells.
[0018] FIG. 4. Effect of caffeine on the synthesis of viral DNA,
its nuclear import, and the yield of host-viral junction DNA. HeLa
cells were infected with the ASV vector and treated with caffeine
for 24 hr at which time cells were harvested. (A) Quantitation of
viral DNA. Real time PCR was used to determine the amount of viral
DNA relative to mitochondrial DNA, Cells were infected at m.o.i.
0.1 and the average of two independent experiments analyzed in
duplicate are shown, UN, uninfected cells; HI, cells infected with
the heat-inactivated virus; 1:10, cells infected with 1:10 dilution
of the virus. (B) Nuclear import of viral DNA determined by
formation of LTR-LTR circle junctions. The expected band is
indicated by an arrow. Band intensity at 1 .mu.l DNA was analyzed
by a phosphorimager and is plotted in (D) as % intensity of circle
junctions in the absence of caffeine. (C) Retroviral DNA
integration. Detection of host-viral junction DNA. Covalent joining
of viral and cell DNA in cells infected at m.o.i. 0.01 was
evaluated by Alu-PCR. NP, no Alu primer in the first round of PCR
(infection in the absence of caffeine); +, positive control (HeLa
cells stably transduced by the ASV vector). Results were analyzed
using a phosphorimager and are plotted in D as % intensity in the
absence of caffeine. (D) Comparison of the affects of caffeine on
the relative amounts of total, nuclear, and host-viral junction DNA
in cells infected with the ASV vector based on quantitation of data
in A, B, and C. Open circles and top curve, total viral DNA; open
triangles and middle curve, nuclear viral DNA (circle junctions);
filled circles and bottom curve, host-viral DNA junctions. The
integrated DNA percentages represent an average of two experiments,
one of which is shown in (C).
[0019] FIG. 5. Effect of caffeine on retroviral integrases in
vitro. Processing and joining activities of ASV and HIV-1
integrases were assayed in vitro. Open triangles, ASV integrase
joining activity; full circles, ASV processing activity; filled
triangles, HIV-1 integrase processing activity.
[0020] FIG. 6. Inhibition of ATR function leads to a reduction in
the number of retroviral transductants and the yield of host-viral
junction DNA. (A) Effect of expression of a dominant-negative ATR
gene (ATRkd) on transduction by the HIV-1 vector. GM847/ATRkd cells
were treated with doxycycline and infected. Two days after
infection, cells were stained using the .beta.-galactosidase assay
and blue cells counted. Grey columns, cells infected with 10-2
dilution of the virus (m.o.i. <0.01); black columns, cells
infected with 10-3 dilution of the virus. (B) ATRkd protein
detected after treatment of GM847/ATRkd cells with 1 or 5 .mu.g/ml
doxycycline for 24 hr. Cells were harvested and Western blot
analysis was performed with an ATR antibody. (C) Growth of cells
expressing ATRkd. GM847/ATRkd cells were plated at a density of
10.sup.5 cells per 60 mm dish in the presence of doxycycline, which
was left on the cells for 48 hr and then removed. At indicated
intervals, cells were harvested and viable cells counted. Open
circles, cells grown in the absence of doxycycline; crosses, cells
treated with 1 .mu.g/ml doxycycline; squares, cells treated with 5
.mu.g/ml doxycycline. (D) Integration of ASV DNA in cells
expressing ATRkd. Detection of host-viral junction DNA in cells
that overexpress ATRkd. GM847/ATR-Kd cells were treated with
doxycycline and infected with the ASV vector. ALu-PCR was performed
24 hr postinfection. NP, no Ale primer in the first round of PCR
(cells infected in the absence of doxycycline); UN, no virus
(uninfected cells).
[0021] FIG. 7. Induction of apoptosis by retroviral infection of
cells induced to over-express ATRkd. Doxycycline was added or not
to GM847/ATRkd cells, and the following day the cells were infected
at multiplicity of infection (moi) 10 with either avian sarcoma
virus (ASV) vector (IN+) or the integrase-deficient (IN-) vector,
or treated with 50 .mu.M etoposide (ETP), in the continued presence
or absence of doxycycline. Twenty-four hours later, cells were
stained using the TUNEL assay, and analyzed as described in
Experimental Methods, infra. The percentage of cells that fell
within the apoptotic window is indicated. Solid lines show results
with cells treated with doxycycline, dashed lines are results with
uninduced cells. The bracket in each plot shows the apoptotic
fraction used for the percentages shown in Table 2.
[0022] FIG. 8. Association of ASV IN and Atr with viral DNA. HeLa
cells were infected and chromatin prepared at the indicated times
(A) or at 6 hours (B) post-infection (pi). In (B) chromatin from
cells infected with IN+ the IN- (D64E) mutant was analyzed. 0=no
virus. Chromatin immunoprecipitation was performed using antibodies
(Ab) against ASV IN or ATR. Viral DNA was detected using nested PCR
with primers targeting viral LTR sequences.
[0023] FIG. 9. Chromatin immunoprecipitation (ChIP) analysis using
antibodies specific for human Wm and Brcal proteins. The graph
shows the percentage of viral DNA that is associated with each CUP,
corrected for the immunoprecipitation efficiency of each
antibody.
[0024] FIG. 10. Effect of caffeine on HIV-1 transduction of
nocodazole-arrested cells. (A) Exponentially dividing 293T cells
were infected with the same aliquots of HIV-1-based vectors and
exposed to the indicated concentrations of caffeine for 24 h. Two
days post infection, cells were stained in a .beta.-galactosidase
assay and blue cells were counted. (B) 293T cells were infected and
treated with caffeine as in (A), except they were arrested with
nocodazole 24 hrs prior to addition of the vectors. (C) Amount of
PCNA in dividing and nocodazole-treated 293T cells. C ells were
treated with nocodazole as in (B), for 24 hrs, at which time they
were harvested and Western blot analysis was performed. (D) Ser
10-phosphorylated historic H3 in dividing and nocodazole-treated
cells. "wt"--HIV-1-based vector containing Vpr and wild-type
integrase, MAV--multiply attenuated HIV-1-based vector,
IN.sup.--HIV-1-based vector carrying a D64V substitution in
retroviral integrase.
[0025] FIG. 11. Effect of caffeine on transduction of
contact-inhibited MEFs. (A) Exponentially dividing mouse embryo
fibroblasts were infected with the HIV-1-base vector carrying Vpr
and exposed to caffeine for 24 hrs. Two days post-infection, cells
were stained with a .beta.-galactosidase assay and blue cells were
counted. (B) MEFs were distributed in 96-well plates as in (A) and
infected at the point of confluency. Caffeine was added as in (A).
(C) PCNA in dividing and confluent MEFs, Cells were treated as in
(A) and (B) and Western blot analysis was performed at the time
when MEFs would be infected. (D) Ser 10-phosphorylated histone H3
in dividing and confluent MEFs,
[0026] FIG. 12. Effect of caffeine on transduction of terminally
differentiated neurons and macrophages. (A) Terminally
differentiated hNT-2 neurons were infected with the HIV-1-based
vector carrying Vpr and exposed to caffeine for 24 hrs. Two days
post-infection, cells were stained in a .beta.-galactosidase assay
and blue cells were counted. (B) PCNA in terminally differentiated
neurons. Cells were treated as in (A) and Western blot analysis was
performed at the time when cells would be infected, with
2.times.10.sup.5 cells per lane. (C) Effect of caffeine on
transduction of terminally differentiated macrophages. Terminally
differentiated macrophages were infected with the HIV-1-based
vector and exposed to caffeine for 24 h. Two days post-infection,
cells were stained in a .beta.-galactosidase assay and blue cells
were counted.
[0027] FIG. 13. Effect of overexpression of dominant negative,
kinase-dead ATRkd protein on transduction of nocodazole-arrested
cells. (A) Exponentially dividing GM847/ATRkd cells were exposed to
doxycycline and infected with the HIV-1-based vectors. Two days
post-infection, cells were stained with a .beta.-galactosidase
assay and blue cells were counted. (B) GM847/ATRkd cells were
infected and doxycycline-treated as in (A), except they were growth
arrested with nocodazole 24 hrs prior to addition of the viruses.
(C) PCNA in dividing and nocodzaole-treated GM847/ATRkd cells,
Cells were treated as in (A) and Western blot analysis was
performed at the time when cells would be infected. (D) Ser
10-phosphorylated histone H3 in dividing and nocodazole-treated
GM847/ATRkd cells. "wt"--HIV-1-based vector containing Vpr protein
and wild-type integrase, MAV--multiply attenuated HIV-1-based
vector, IN.sup.--HIV-1-based vector carrying a D64V substitution in
retroviral integrase.
[0028] FIG. 14. Effect of caffeine on HIV-1 transduction of
ATR-deficient cells. (A) Exponentially dividing GM847/ATRkd cells
were exposed to doxycycline (5 .mu.g/ml), infected with the same
aliquots of HIV-1-based vectors and treated with the indicated
concentrations of caffeine. Two days post-infection, cells were
stained in a .beta.-galactosidase assay and transduced cells were
counted. (B) Caffeine effect expressed as relative transduction
efficiency. 100%--number of transduced cells in absence of
doxycycline.
[0029] FIG. 15. Caffeine and related methylxanthines suppress
replication by HIV-1 NL4-3 and ADA strains. (FIG. 15A) Peripheral
blood mononuclear cells (PBMCs) were infected with the HIV-1 strain
NL4-3 and treated with metyhylxanthines (4 nM concentration). The
level of p24 antigen in the culture supernatant was measured at 3,
7 and 12 days post-infection. Open circles--culture infected with
NL4-3 virus in the absence of methylxanthines, open
squares--uninfected cells, filled diamonds--culture infected with
NL4-3 and treated with theobromine, filled circles--culture
infected with NL4-3 and treated with paraxanthine, filled
triangles--culture infected with NL4-3 and treated with caffeine,
filled squares--culture infected with NL4-3 and treated with
theophylline. PBMCs were infected with the NL4-3 (FIG. 15B) and ADA
(FIG. 15C) strains and treated with methylxanthines. The level of
HIV-1 p24 antigen in the culture supernatant was measured at 6 days
post-infection. FIG. 15B--NL4-3 infected culture, FIG.
15C--ADA-infected culture. CF--cells infected with a given HIV-1
strain and treated with caffeine, TP--cells infected with a
specific HIV-1 strain and treated with theophylline, TB--cells
infected with a specific HIV-1 strain and treated with theobromine.
V--cells infected with a specific HIV-1 strain, no methylxanthine
added, U--uninfected cells. Black columns--a methylxanthine was
added to culture, white columns--no methylxanthine was added.
[0030] FIG. 16. Effect of caffeine on early steps of the HIV-1
life-cycle. PBMCs were infected with the NL4-3 strain and treated
with methylxanthines. Twenty-four hours post-infection, cells were
harvested and analyzed. The relative intensity of bands was
determined by Phospholmager and densitometry analysis and compared
to the intensity of sample infected with the NL4-3 virus, but
untreated with any of the methylxanthines (left panels). (A) Level
of gag HIV-1-specific DNA in infected and methylxanthine-treated
cultures. (B) Level of HW-1 2-LTR DNA circles. (C) Level of
viral-host DNA joining as evaluated by Alu-PCR. (D) Level of gag
HIV-1 RNA. (E) Control comparison of beta-globin DNA level in
analyzed cells. CF--cells infected with NL4-3 and treated with
caffeine, TP--cells infected with NL4-3 and treated with
theophylline, TB--cells infected with NL4-3 and treated with
theobromine, PX--cells infected with NL4-3 and treated with
paraxanthine, V--cells infected with NL4-3, no methylxanthine
added, U--uninfected cells, AC--ACH-2 cells, NA--ACH-2 cells, no
Alu primer in the first round of PCR.
[0031] FIG. 17. Effects of caffeine on late steps of the HIV-1
life-cycle. ACH-2.cells were stimulated with PMA or left
unstimulated, and treated with methylxanthines at given
concentrations. Twenty-Tour hours after addition of PMA and
methylxanthines, cultures and cells were analyzed. (A) HIV-1 p24
antigen level in the culture supernatant, (B) Intracellular HIV-1
p24 antigen level. M--mock, no methylxanthine was added,
CF--caffeine-treated cells, TP--theophylline-treated cells.
[0032] FIG. 18. Caffeine suppresses ATR- and ATM-mediated
phosphorylation. PBMCs were infected with the NL4-3 virus and
treated with caffeine and theophylline, and harvested 24 hrs later.
Cell lysates were subjected to Western blotting analysis to detect
the BRCA1 phosphorylated on serine 1423 and the p53 protein
phosphorylated on serine 15. The level of Ku 86 protein served as a
loading control. M--mock, no methylxanthine added,
CF--caffeine-treated cells, TP--theophylline-treated cells.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0033] The present invention features assays, active agents,
pharmaceutical preparations and methods of treating retroviral
infection, arising from the inventors' identification of ATR kinase
as an integral component of the retroviral lifecycle, required for
stable retroviral DNA integration into a host genome. Without
intending to be limited by any mechanism as to how ATR kinase is
involved in retroviral integration, the inventors offer the
following discussion related to the role of ATR kinase in the
retroviral lifecycle.
[0034] As described in detail in the examples below, the inventors
have shown that methylxanthines such as caffeine inhibit stable
transduction by HIV-1- and ASV-based retroviral vectors at doses
that have little or no effect on early steps that precede
integration, the synthesis of viral DNA and its nuclear import. In
addition, methylxanthines do not inhibit integrase activities in
vitro and have no effect on LTR-driven expression of a selectable
reporter gene in vivo. However, methylxanthines inhibit stable
retroviral DNA integration, as evaluated by Alu-PCR. It has now
been demonstrated that the ATR kinase, a major target of
methylxanthines such as caffeine, is required for stable retroviral
DNA integration and transduction, whereas absence or inhibition of
the related kinases, ATM and mTOR has no effect on transduction
efficiency under the conditions tested.
[0035] ATR is a major component in cellular DNA repair. Its kinase
activity is required in cellular response to ionizing and UV
radiation, and collapsed replication forks. The finding that
retroviral DNA integration can be inhibited by caffeine and other
methylxanthines, and requires ATR, points to ATR as an advantageous
cellular target for anti-retroviral therapy. One feature such an
approach should be that the inhibition of cellular proteins does
not have a detrimental effect on cellular function. As the observed
inhibition of integration occurs at drug concentrations below those
that affect growth of cultured cells, it is believed that
inhibitors of ATR or other components of the DNA damage response
can also serve as inhibitors of retroviral infection,
[0036] What aspect of retroviral DNA integration requires the ATR
function? The product of the first two steps of retroviral DNA
integration is an intermediate comprising the viral DNA flanked by
gaps in host DNA sequence (FIG. 1). These DNA gaps, and perhaps
discontinuities in the viral DNA, need to be repaired to create a
stable integrated provirus. Furthermore, unrepaired gaps might
become double-strand breaks when a cellular replication fork
encounters such a lesion. On the basis on comparisons of
experimental data with computer simulations, it is believed that
this, or a related event, triggers the apoptotic response in
NHEJ-deficient scid lymphocytes (Katz et al., J. Biol. Chem, 276:
34213-34220, 2001). In this context, integration-incurred damage
may be similar to other replication fork catastrophes in which ATR
has been proposed to play a pivotal role (Tibbetts et al., 2000,
supra). Without being bound to any particular theory or mechanism
of action, it may be that signaling through ATR-mediated
phosphorylation of various targets leads to a transient cell cycle
arrest, and recruitment or activation of the proteins necessary for
repair. Failure in either or both processes could therefore render
the integration intermediate unstable and could also lead to cell
death. In this model, the observed reduction in integration
efficiency may reflect the loss of infected cells from the
population. However, as discussed herein, HIV-1 transduction is
effectively reduced in non-dividing cells as well as dividing
cells. For this reason, it may be that ATR kinase is directly
involved in postintegration repair at sites of retroviral DNA
integration, through either recruitment or modification of the
necessary nucleic acid repair proteins.
[0037] Thus, the present invention features ATR kinase as a novel
cellular target for inhibition of retroviral replication. Though
all retroviral replication may be targeted in this manner, of
particular interest is the inhibition of HIV replication, as a new
therapy for AIDS. Current anti-HIV therapies target HIV proteins,
predominantly HIV reverse transcriptase and protease. However,
rapid emergence of resistance to these drugs is well documented,
due to the ability of the virus to rapidly mutate. ATR kinases are
encoded by a cellular gene, which does not mutate with the
frequency of HIV or any other retrovirus. Accordingly, inhibitors
of ATR kinase could impede or interrupt retroviral replication, and
the probability of resistance developing to such inhibitors would
be comparatively low as compared to inhibitors of viral genes.
Moreover, the effect of ATR kinase on retroviral DNA integration
can be inhibited under conditions that do not result in significant
inhibition of cell viability, as demonstrated by studies on the ATR
kinase inhibitor, caffeine. This enables the option of delivering
an ATR inhibitor systemically, without incurring overall cellular
toxicity. Alternatively, targeting of ATR inhibitors to cells
typically subject to retroviral infection may be employed.
[0038] The identification of ATR kinase as required for the final
step of retroviral DNA integration also implicates one or more
downstream substrates of ATR kinase in the retroviral integration
process. Accordingly, compounds that modulate one or more of these
downstream substrates are also considered useful in the present
invention. Several substrates of ATR kinase have been identified
and characterized. These include: (1) ATRIP (Guntuku et al.,
Science 294:1713-1716, 2001); (2) Bloom syndrome protein
(Franchitto & Pichierri, J Cell Biol. 157:19-30, 2002); (3)
Rad17 (Zou et al., Genes Dev. 16:198-208, 2002; Weng et al., Proc
Natl Acad Sci USA 98:13102-13107, 2001); (4) Histone H2AX (Ward
& Chen, J Biol Chem. 276:47759-47762, 2001); (5) Chk1 (Liu et
al., Genes Dev. 14:1448-1459, 2000); (6) p53 (Lakin et al.,
Oncogene 18:3989-3995, 1999; Tibbetts et al., Genes Dev.
13:152-157, 1999; and (7) BRCA1 (Tibbetts et al., Genes Dev.
14:2989-3002, 2000). However, any downstream target of ATR kinase
may be implicated in control of retroviral integration, and
therefore is contemplated as a cellular target for development of
anti-retroviral agents. Substrates and downstream targets of ATR
kinase, whether directly or indirectly acted upon by ATR kinase,
may be considered part of an ATR kinase cascade.
[0039] One aspect of this invention features screening assays for
identifying compounds that inhibit retroviral replication by
modulating ATR kinase or its relevant substrates. Preferably, the
ATR kinase is a human ATR kinase, and the retrovirus is one that
infects humans, e.g., HIV and HTLV. However, inasmuch as animal
diseases of agricultural and veterinary importance are known to be
caused by retroviruses (e.g., ASLV, FeLV, BIV, EJAV), other
embodiments of the invention target mammalian or avian ATR kinases,
for treatment of retroviral infections of agronomic and veterinary
significance.
[0040] A preferred type of assay to identify modulators of ATR
kinase activity is one that can measure ATR kinase activity in
vitro, in the presence or absence of a test compound suspected of
being able to regulate ATR activity. For instance, one of skill in
the art would appreciate that a cell- free ATR kinase activity
assay could be utilized, combining ATR with one of its
phosphorylation substrates, e.g., BRCA1, and measuring
incorporation of labeled phosphate into the substrate.
Alternatively or in addition, assays of more relevance to
retroviral integration may be utilized. For example, as described
in the examples to follow and in the art (e.g., Daniel et al.,
1999, supra; WO 00/17386), stable transduction of cultured cells by
a retroviral-based vector expressing a detectable gene product may
be used to assess retroviral integration in the presence or absence
of a test compound suspected of regulating ATR kinase activity or
the activity of one of its substrates that may be involved in the
integration process. Detectable gene products include, but are not
limited to, Beta-galactosidase, placental alkaline phosphatase,
secreted embryonic alkaline phosphatase, luciferase,
chloramphenicol acetyltransferase, Beta-glucuronidase, green
fluorescent protein, red fluorescent protein, yellow fluorescent
protein, cyan fluorescent protein, or cerianthus orange fluorescent
protein. Detectable gene products also include proteins that confer
drug resistance, e.g., resistance to neomycin, tetracycline,
hygromycin, and the like, as would be appreciated by one of skill
in the art.
[0041] In a more specific assay, the integration of such vectors
into genomes of cultured cells may be monitored by Alu-PCR. As a
control to ensure that a test compound is affecting a cellular DNA
repair enzyme rather that the retroviral integrase, integrase
assays known in the art also may be carried out in the presence or
absence of the test compound. As another control to focus on ATR
activity in the cultured cells, assays may be performed in the
presence of known inhibitors of other DNA repair enzymes. For
instance, it is known that rapamycin inhibits mTOR (Sarkaria et
al., 1999, supra), and DNA PK.sub.CS can be inhibited by single
stranded DNA, pyrophosphate, 6-dimethyl-aminopurine and the
pyridone derivative, OK-1035 (Take et al., Biochem. Biophys. Res.
Comm. 215: 41-47, 1995).
[0042] It will be appreciated by those of skill in the art that
regulation of expression of ATR kinase-encoding genes is also a
means by which the kinase can be modulated in the treatment of
retroviral infection. Further, as an integral component of cell
cycle regulation, expression of ATR kinase genes is likely
controlled by at least one transactivating protein. Accordingly,
assays for agents capable of inhibiting or otherwise modulating
such activation or induction of expression should identify
additional useful compounds for the treatment of retroviral
infection. Such assays are familiar to the skilled practitioner.
Furthermore, such assays may be employed to identify compounds that
modulate expression of genes encoding downstream substrates of ATR
kinase.
[0043] Another aspect of the present invention features compounds
identified by any of the foregoing methods. Such compounds may
include, but are not limited to, antibodies and antibody fragments
that bind the ATR kinase at an active site or other epitope that
interferes with enzyme activity, antisense or other agents that
modulate expression of genes encoding the ATR kinase, and
inhibitors, antagonists, or reverse agonists of the ATR kinase,
including, but not limited to, proteins, nucleic acids, or other
organic molecules. Also included in this aspect of the invention
are compounds identified by the above assays using downstream
targets of ATR kinase as the cellular target.
[0044] Caffeine is a compound already known in the art to inhibit
the ATM and ATR kinases. Moreover, as discussed herein, caffeine
inhibits retroviral transduction of both dividing and non-dividing
cells at concentrations that do not substantively affect cell
growth. Caffeine is 1,3,7-trimethylxanthine and some of
caffeine-related methylxanthines are products of caffeine
metabolism in vivo (e.g., paraxanthine, theobromine and
theophylline). Theophylline is in clinical use for the treatment of
asthma. It has also been demonstrated that certain methylxanthines
exhibit caffeine-like effects on DNA damage response (Bohm, L et
al., 2003, Toxicology 193:153-160; Sarkaria, et al., 2001, Semin.
Radiat. Oncol. 11: 316-327). Accordingly, another aspect of the
invention comprises interfering with or inhibiting retroviral
transduction or replication by using an agent that acts upon and
interferes with or inhibits the ATR kinase-mediated host cell
machinery needed to repair nucleic acids during retroviral DNA
integration into the host cell genome. The agent may be a
methylxanthine. Methylxanthines include caffeine, paraxanthine,
pentoxyfylline, theobromine, theophylline, and their respective
metabolites or derivatives, from whatever source derived. The agent
may function in a dividing or nondividing cell. The host cell
machinery may be ATR kinase or a substrate or downstream target
activated directly or indirectly by ATR kinase, such as those in
the ATR kinase cascade.
[0045] Also provided in accordance with the present invention are
pharmaceutical preparations for treating a patient for retroviral
infections. These preparations contain active ingredients that act
by inhibiting the final step of retroviral integration through the
modulation of ATR kinase or its downstream substrates that are
required for this process. The term "patient" as used herein refers
to human or animal subjects (animals being particularly useful as
models for clinical efficacy of a particular composition).
Selection of a suitable pharmaceutical preparation depends upon the
method of administration chosen, and may be made according to
protocols well known to medicinal chemists.
[0046] The pharmaceutical preparations of the invention comprising
ATR kinase-modulating active agents or agents that modulate the
activity of ATR substrates that affect retroviral DNA integration
are conveniently formulated for administration with a acceptable
medium such as water, buffered saline, ethanol, polyol (for
example, glycerol, propylene glycol, liquid polyethylene glycol and
the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending
agents or suitable mixtures thereof. The concentration of a
particular active ingredient in the chosen medium will depend on
the hydrophobic or hydrophilic nature of the medium, in combination
with the specific properties of the delivery vehicle and active
agents disposed therein. Solubility limits may be easily determined
by one skilled in the art.
[0047] As used herein, "biologically acceptable medium" includes
any and all solvents, dispersion media and the like which may be
appropriate for the desired route of administration of the
pharmaceutical preparation, as exemplified in the preceding
paragraph. The use of such media for pharmaceutically active
substances is known in the art. Except insofar as any conventional
media or agent is incompatible with the compositions to be
administered, its use in the pharmaceutical preparation is
contemplated.
[0048] The term "chemical derivative" describes a molecule that
contains additional chemical moieties that are not normally a part
of the base molecule. Such moieties may improve the solubility,
half-life, absorption, etc. of the base molecule. Alternatively the
moieties may attenuate undesirable side effects of the base
molecule or decrease the toxicity of the base molecule. Examples of
such moieties are described in a variety of texts, such as
Remington's Pharmaceutical Sciences,
[0049] According to another aspect of the invention, methods of
treating acute or latent retroviral infection are provided,
comprising administering the aforementioned pharmaceutical
preparation under conditions effective to reduce or eliminate
retroviral infection, or to reduce or eliminate pathological
conditions associated with such infection. The pharmaceutical
preparation is formulated in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form, as used
herein, refers to a physically discrete unit of the pharmaceutical
preparation appropriate for the patient undergoing treatment. Each
dosage should contain a quantity of the active ingredient(s)
calculated to produce the desired retroviral-inhibitory effect in
association with the selected pharmaceutical carrier. Procedures
for determining the appropriate dosage unit are well known to those
skilled in the art. The pharmaceutical compositions may be provided
to the individual by a variety of routes such as subcutaneous,
topical, oral, intranasal and intramuscular.
[0050] ATR kinase-modulating compounds or agents that modulate the
activity of ATR kinase substrates that affect retroviral DNA
integration identified according to the methods disclosed herein
may be used alone at appropriate dosages defined by routine testing
in order to obtain optimal inhibition of a selected ATR kinase or
its relevant substrates while minimizing any potential toxicity. In
addition, co-administration or sequential administration of other
anti-retroviral agents may be desirable.
[0051] The daily dosage of the products may be varied over a wide
range from 0.01 to 1,000 mg per patient, per day. For oral
administration, pharmaceutical preparations are preferably provided
in the form of scored or un-scored tablets containing 0.01, 0.05,
0.1, 0.5, 1.0, 2.5, 5:0, 10.0, 15.0, 25.0, and 50.0 milligrams of
the active ingredient for the symptomatic adjustment of the dosage
to the patient to be treated. An effective amount of the drug is
ordinarily supplied at a dosage level of from about 0.0001 mg/kg to
about 100 mg/kg of body weight per day. The range is more
particularly from about 0.001 mg/kg to 10 mg/kg of body weight per
day. The dosages are adjusted when combined to achieve desired
effects. On the other hand, dosages of these various agents may be
independently optimized and combined to achieve a synergistic
result wherein the pathology is reduced more than it would be if
any single agent were used alone.
[0052] The dosage regimen utilizing the compounds or modulators of
the present invention is selected in accordance with a variety of
factors including type, species, age, weight, sex and medical
condition of the patient; the severity of the condition to be
treated; the route of administration; the renal and hepatic
function of the patient; and the particular compound thereof
employed. A physician or veterinarian of ordinary skill can readily
determine and prescribe the effective amount of the drug required
to prevent, counter or arrest the progress of the condition.
Optimal precision in achieving concentrations of drug within the
range that yields efficacy without toxicity requires a regimen
based on the kinetics of the drug's availability to target sites.
This involves a consideration of the distribution, equilibrium, and
elimination of a drug.
[0053] In the methods of the present invention, the compounds or
modulators of ATR kinase or its relevant substrates are typically
administered in admixture with biologically compatible diluents as
described above, excipients or carriers (collectively referred to
herein as "carrier" materials) suitably selected with respect to
the intended form of administration, that is, oral tablets,
capsules, elixirs, syrups and the like, and consistent with
conventional pharmaceutical practices.
[0054] For instance, for oral administration in the form of a
tablet or capsule, the active drug component can be combined with
an oral, non-toxic pharmaceutically acceptable inert carrier such
as ethanol, glycerol, water and the like. Moreover, when desired or
necessary, suitable binders, lubricants, disintegrating agents and
coloring agents can also be incorporated into the mixture. Suitable
binders include, without limitation, starch, gelatin, natural
sugars, such as glucose or beta-lactose, corn sweeteners, natural
and synthetic gums, such as acacia, tragacanth or sodium alginate,
carboxymethylcellulose, polyethylene glycol, waxes and the like.
Lubricants used in these dosage forms include, without limitation,
sodium oleate, sodium stearate, magnesium stearate, sodium
benzoate, sodium acetate, sodium chloride and the like.
Disintegrators include starch, methylcellulose, agar, bentonite,
xanthan gum and the like.
[0055] For liquid forms, the active drug component can be combined
in suitably flavored suspending or dispersing agents, such as the
synthetic and natural gums, for example, tragacanth, acacia,
methyl-cellulose and the like, Other dispersing agents that may be
employed include glycerin and similar substances. For parenteral
administration, sterile suspensions and solutions are desired.
Isotonic preparations that generally contain suitable preservatives
are employed when intravenous administration is desired.
[0056] Topical preparations containing the active drug component
can be admixed with a variety of carrier materials well known in
the art, such as, e.g., alcohols, aloe vera gel, allantoin,
glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl
propionate, and the like,
[0057] The compounds or modulators of the present invention can
also be administered in the form of liposome delivery systems, such
as small unilamellar vesicles, large unilamellar vesicles and
multilamellar vesicles. Liposomes can be formed from a variety of
phospholipids, such as cholesterol, stearylamine or
phosphatidylcholines.
[0058] Compounds of the present invention may also be delivered by
the use of antibodies as individual carriers to which the compound
molecules are coupled. The compounds or modulators of the present
invention may also be coupled with soluble polymers as targetable
drug carriers. Such polymers can include polyvinylpyrrolidone,
pyran copolymer, polyhydroxypropylmethacryl-amidephenol,
polyhydroxy-ethylaspartamidephenol, or polyethyleneoxidepolylysine
substituted with palmitoyl residues. Furthermore, the compounds or
modulators of the present invention may be coupled to a class of
biodegradable polymers useful in achieving controlled release of a
drug, for example, polylactic acid, polyepsilon caprolactone,
polyhydroxy butyric acid, polyorthoesters, polyacetals,
polydihydro-pyrans, polycyanoacrylates and cross-linked or
amphipathic block copolymers of hydrogels.
[0059] The compounds or modulators may alternatively be
administered parenterally via injection of a formulation consisting
of the active ingredient dissolved in an inert liquid carrier.
Injection may be either intramuscular, intra-ruminal,
intratracheal, or subcutaneous. The injectable formulation consists
of the active ingredient mixed with an appropriate inert liquid
carrier. Acceptable liquid carriers include the vegetable oils,
such as peanut oil, cottonseed oil, sesame oil and the like as well
as organic solvents such as solketal, glycerol formal and the like.
As an alternative, aqueous parenteral formulations may also be
used. The vegetable oils are the preferred liquid carriers. The
formulations are prepared by dissolving or suspending the active
ingredient in the liquid carrier such that the final formulation
contains from 0.005 to 10% by weight of the active ingredient.
[0060] The following examples are provided to describe the
invention in greater detail. These examples are intended to
illustrate, not to limit, the invention.
EXAMPLE 1
Experimental Methods
[0061] The following materials and methods were employed in the
examples to follow, unless otherwise described.
[0062] Cells and Viruses. GM847/ATRkd is a simian virus
40-transformed human fibroblast cell line stably transfected with a
mutant ATR gene under the control of a tet-inducible promoter
(Cliby, W. A. , et al., (1998) EMBO J. 17, 159-169). Upon addition
of doxycycline (1-5 .mu.g/ml) the cells overproduce a protein
containing a D2475A substitution that inactivates the kinase
activity. HeLa, 293T, and GM847/ATRkd cells were maintained in DMEM
supplemented with 10% fetal bovine serum (FBS) and Pen/Strep. The
HIV-1 vector carrying the lacZ reporter was prepared as follows:
293T cells were plated on 100 mm dishes at a density approximately
10.sup.6 cells per dish. The following day, cells were transfected
using the 3-plasmid system described previously (Naldini et al.,
Science 272: 263-267, 1996). 25 .mu.g lacZ plasmid, 50 .mu.g
backbone plasmid, 5 .mu.g VSV G plasmid were used per dish; the
Profection kit (calcium phosphate) (Promega) was employed. The
following day, spent medium was replaced. The second day after
transfection, supernatant from the transfected 293T cells was
harvested, passed through a 0.45 .mu.M filter, and subjected to
centrifugation at 4.degree. C., 25,000 rpm, for 30 min. The
virus-containing pellet was dissolved in medium. Preparation of the
avian sarcoma virus (ASV) neo.sup.r vector was described previously
(Daniel et al., 1999, supra). Titers of infectious units of HIV and
ASV vectors were determined by transduction assays. The titer of
the ASV (IN-) vector was calculated by measuring reverse
transcriptase activity in viral particles and comparing the results
with activity in ASV (IN+) particles of known titer. AT221JE-T
cells and derivative lines were maintained as described (Daniel et
al., Mol. Cell Biol. 21: 1164-1172, 2001).
[0063] Chemicals. Caffeine, thoebromine, theophylline, and
paraxanthine (purchased from Sigma Chemical Co.) were prepared in
100 mM stock concentrations: Caffeine was dissolved in water,
whereas theobromine, theophylline, and paraxanthine were dissolved
in 0.1 N NaOH. Doxycycline (Clontech) was dissolved in water at 10
mg/ml. Etoposide (ETP) was obtained from Sigma and dissolved in
DMSO (stock concentration 30 mg/ml). Stocks were stored at
-20.degree. C.
[0064] Colony Assays and Cell Growth in the Presence of Caffeine
and Doxycycline. To determine the effect of caffeine on cell
growth, HeLa cells were plated at a density of 10.sup.5 cells per
60 mm dish in the presence of caffeine. The drug was kept on cells
for 24 hr and then removed. At indicated intervals, cells were
harvested and viable cells were counted. To determine the effect of
caffeine on colony formation, 10.sup.3 HeLa cells were plated on
each 60 mm dish in the presence of caffeine; the drug-containing
medium was removed after 24 hr and colonies counted 9 days later.
To determine whether G418 treatment potentiates caffeine toxicity,
G418-resistant HeLa cells were plated as above, in the presence of
caffeine and 1 mg/ml G4178. Medium was replaced after 24 hr, with
medium containing only G418 and colonies counted 8 days later. To
determine if doxycycline treatment affects the growth of
GM847/ATRkd cells, 10.sup.5 cells were plated per 60 mm dish in the
presence of the drug. After 48 hr doxycycline-containing medium was
replaced and viable cells counted at the indicated intervals.
[0065] Infection of HeLa Cells. HeLa cells were plated at 10.sup.5
per 60 mm dish (FIG. 2A) and infected the following day with either
the HIV-1- or ASV-based vectors for 2 hr in the presence of 10
.mu.g/ml DEAE dextran. The virus-containing medium was then
replaced with fresh medium. Two to seven days after infection with
the HIV-1 vector (as indicated) cells were stained using a
.beta.-galactosidase assay (Stratagene). Cells were selected for
G418 resistance one day after infection with the ASV vector by
addition of 1 mg/ml G418.
[0066] Caffeine was added to cells together with the infecting
virus and maintained in the medium until the following day (24 hr),
at which time caffeine-containing medium was replaced with fresh
medium. After infection with ASV, G418 was also added at this
time.
[0067] Infection of ATR-deficient Cells. GM847/ATRkd cells were
plated at 10.sup.5 per 60 mm dish and infected the following day
with either the HIV-1- or ASV vector for 2 hr in the presence of 10
.mu.g/ml DEAE dextran. Doxycyclihe was added at the time of plating
and kept on cells for 24 hr after addition of the virus, To
determine the transduction efficiency of the HIV-1 vector,
ATR-deficient cells were stained 48 hr post infection using a
.beta.-galactosidase assay and following the Stratagene
protocol.
[0068] Infection of ATM-deficient Cells. ATM-deficient AT22IJE-T
cells and AT22IJE-T cells containing a vector encoding ATM or an
empty vector (Cortez et al., 1999, supra) were plated at
5.times.10.sup.4 per well of a 24 well plate, 2 wells each point
and infected the following day with the HIV-1 vector for 2 hr in
the presence of 10 .mu.ml DEAE dextran. Caffeine was added to cells
together with the HIV-1 vector, and maintained in the medium until
the following day (24 hr). Cells were stained three days post
infection using the .beta.-galactosidase assay.
[0069] Western Blot Analyses. For Western blot analysis of
nea.sup.r expression, the polyclonal population of G418-resistant
HeLa cells (ca. 1000 clones) was exposed to caffeine for 24 hr.
Cells were then harvested, lysed, and lysates were subjected to
electrophoresis in a 10% SDS polyacrylamide gel. The separated
proteins were then transferred to a PVDF membrane and the filter
was treated with neomycin phosphotransferase II antibody (Upstate
Biotech). Bands were detected using a chemi-luminiscence assay.
[0070] For detection of ATRkd, GM847/ATRkd cells were exposed to
doxycycline (1 or 5 .mu.g/ml) for 24 hr. Cells were then harvested
and Western blot analysis was performed as described above, except
samples were resolved by electrophoresis in a 6% SDS-polyacrylamide
gel, and the filter was exposed to anti-ATR antibody (Ab-2,
Oncogene Science).
[0071] Real Time PCR. Extrachromosomal DNA from infected and
uninfected cells was prepared by HIRT extraction. Real-Time PCR
amplification, data-acquisition and analysis were preformed with
the Cephid Smart Cycler. The Biochemistry and Biotechnology
Facility at the Fox Chase Cancer Center prepared the primers using
BHQ-1 non-fluorogenic quencher (Biosearch Technologies). Viral
sequence primers directed against ASV poi were selected using
Primer Express.TM. (Applied Biosystems) and had the following
sequences: Forward primer, 5'-TCA GCG ATA GTC GTA ACT CAG CAT-3'
(SEQ ID NO:1); Reverse primer, 5'-AGC CGT GGC CCA ATG AT-3' (SEQ ID
NO:2); Probe, 5'-(FAM)CC GTG TTA CAT COG TTG CTG CAC AA (BHQ)-3'
(SEQ ID NO:3), Results were normalized with those from primers
against mitochondrial DNA (Asaad et al., Oncogene 19: 5788-5800,
2000). Each reaction contained 1.times. Reaction Buffer (20 mM
Tris-HCl, pH 8.4), 50 mM KCl), 0.25 mM each dNTP, 2.5 mM
MgCl.sub.2, 400 nM primers, 200 nM probe and 2.5,U Platinum Taq
Polymerase (Invitrogen). Relative quantitation was calculated with
the Comparative Cycle Threshold Method using the untreated samples
as the reference (User, B. ABI Prism 7700 Sequence Detection
System; the Perkin Elmer Corporation, P/N 4303859 Rev. A. Stock No.
77802-001, 1997). This method was validated with a five-fold
dilution curve of the untreated sample (slope was -0.0234). Similar
results were obtained with calculations using a standard curve
generated from a viral plasmid. Relative quantitation was averaged
and standard deviations were determined between independent
experiments.
[0072] PCR Detection of Circle Junctions In Vivo. 10.sup.6 HeLa
cells were plated per dish and infected the following day with 1 ml
of the undiluted ASV vector (titer ca 1.times.10.sup.6 GFU
(G418-resistant colony-forming units)/ml), for 2 hr, in the
presence of 10 .mu.g/ml DEAE dextran. Caffeine-containing medium
was replaced after 24 hr. Cells were harvested the following day
and extrachromosomal DNA extracted using the HIRT method. DNA was
dissolved in 50 .mu.l per sample and 1-10 .mu.l were used for PCR.
Circle junctions were amplified using primers comprising ASV LTR
sequences. The sequence of the upstream primer was 5'-ACC AAT GTG
GTG AAT GGT CAA-3' (SEQ ID NO:4), and the sequence of the
downstream was 5'-CTA CGA GCA CCT GCA TGA AGC-3' (SEQ ID NO:5). PCR
was run for 45 cycles, 94.degree. C. 30 see, 55.degree. C. 30 sec,
and 72.degree. C. 30 sec. PCR products were analyzed on 1.5%
agarose gels.
[0073] In Vitra Assays of Integrase Activities. Reactions contained
2 .mu.M ASV IN, 15 .mu.M U3 18/18 substrate in 55 mM Hepes, pH 8.2,
50 mM NaCl, 2 mM 2-mercaptoethanol, 0.1% thiodiglycol, 200 .mu.g/ml
BSA, 10 .mu.M EDTA, 4% glycerol, and 10 mM MnCl.sub.2, in a final
volume of 10 .mu.l. Caffeine stocks were prepared in water, and
added to the reaction as a 1/10 add, for final concentrations of 1
.mu.M to 10 mM, as indicated. The order of additions was as
follows: IN, water, 10.times. reaction buffer and MnCl.sub.2 were
combined on ice, then caffeine was added to the reactions and they
were preincubated for 30 min at 30.degree. C. Reactions were then
placed on ice and the DNA substrate added. After substrate
addition, the reactions were incubated at 37.degree. C. for 15 min,
and then stopped with 20 .mu.l Maxam and Gilbert loading buffer.
Samples were then analyzed by electrophoresis in a 20% sequencing
gel, and the radioactive bands were detected using a Fuji
Bio-Imaging Analyzer.
[0074] For HIV-1 integrase, reactions contained 1.0 .mu.M HIV IN,
1.0 .mu.M U5 21/21 substrate (Katz & Skalka, Ann. Rev. Biochem.
63: 133-173, 1994; Coffin et al., Retroviruses, Cold Spring Harbor
Laboratory, 1997) in 22 mM Hepes, pH 7.5, 5.1 mM DTT, 6% glycerol,
6.66% DMSO, 50 mM KCl and 10 mM MnCl.sub.2. Conditions were as
described for ASV IN, except that the final incubation time was 60
min.
[0075] Alu-PCR. HeLa cells were treated with caffeine and
GM847/ATRkd cells stimulated with doxycycline as described above.
Infection was performed at an m.o.i, 0.01. The inventors
established first that this m.o.i, falls in the linear range for
detection of viral DNA, Cells were harvested 24 hr after infection
and chromosomal DNA was extracted. DNA concentrations of all
samples were normalized by UV absorbance. PCR reactions (50 .mu.L)
contained 50 mM KCl, 20 mM Tris-HCl buffer (pH 8.4), 5 mM
MgCl.sub.2, 200 uM dNTP, 0.5 U Taq-polymerase (GibcoBRL). 100 ng of
chromosomal DNA was used in the first round of PCR with Alu-primer
5'-GCC TCC CAA AGT GCT GGG ATT ACA G-3' (SEQ ID NO:6) and ASV virus
primer 5'-GGC TTC GGT TGT ACG CGG TTA GGA GT-3' (SEQ ID NO:7),
Samples were denatured at 92.degree. C. for 3 min, and then
subjected to 20 PCR cycles of 92.degree. C. for 40 s, 65.degree. C.
for 40 s, and 72.degree. C. for 1 min 30 s, Products of the first
round were diluted 1/1000 and used in the 25-cycle second round
(nested) with viral LTR primers: 5'-AGG TGC ACA CCA ATG TGG TG-3'
(SEQ ID NO:8) and 5'-AAA AGC ACC GTG CAT GC-3' (SEQ ID NO:9).
Second round PCR was cycled as follows: 92.degree. C. for 3 min; 25
cycles of 92.degree. C. for 40 s, 58.degree. C. for 40 s,
72.degree. C. for 40 s. All PCR reactions were performed in a
"Genius" TECHNE system. Products (10 .mu.L) of the PCR reactions
were loaded on the 2% agarose gel and transferred (Vacuum Blotter,
BioRad) to Zeta-Probe GT Blotting Membranes (BioRad). After UV
crosslinking (Stratalinker, Stratagene) membranes were subjected to
Southern hybridization procedure as suggested by the membrane
manufacturer. The Southern probe was amplified from a plasmid with
a cloned ASV genome using LTR primers 5'-CAA ATG GCG TTT ATT GTA
TCG-3' (SEQ ID NO:10) and 5'-GAT TGG TGG AAG TAA GGT GG-3' (SEQ ID
NO:11) and labeled with [.alpha.-.sup.32P]dATP (ICN). Radioactive
bands were detected overnight by exposure with Kodak BioMax MR
film.
[0076] Apoptosis Assay with ATRkd-Expressing Cells.
[0077] GM847/ATRkd cells were treated with 5 .mu.g/ml doxycycline,
and the next day aliquots were either mock-infected or infected
with the ASV IN+ or IN vectors [multiplicity of infection (moi) 10]
in the presence of 5 .mu.g/ml DEAE dextran. A fourth aliquot of
these cells was treated with 50 .mu.M ETP. Doxycycline was
maintained in the medium after infection and ETP treatment. As a
control, aliquots of GM847/ATRkd cells to which no doxycycline was
added were treated in the same way. After 24 h, cells were
harvested and stained with a terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)
assay (In Situ Death Detection kit, Fluorescein, Roche
Diagnostics). Stained cells were then quantitated by flow
cytometry.
Chromatin Immunoprecipitation
[0078] Chromatin immunoprecipitation (ChIP) was performed as
described (Boyd, K. E. & Farnham, P. J., Mol. Cell. Biol.
19:8393-8399, 1999; Orlando, V. TIBS 25:99-104, 2000.). Briefly, at
defined time points, infected and uninfected cells were treated
with 1% formaldehyde to crosslink DNA and proteins. For Brcal and
Wrn analyses, nuclei were prepared and Brcal and Wm were
immunoprecipitated from nuclear extracts with corresponding
antibodies. DNA was then eluted from immunoprecipitates and amounts
of total viral DNA (vDNA) in the nuclear extracts and vDNA
immunoprecipitated with Brca1 and Wrn were determined by subjecting
samples to standardized PCR with viral LTR probes, followed by
agarose gel electrophoresis and Southern blotting with
radioactively-labeled DNA probes. Filters were exposed to X ray
film and results analyzed by a phosphorimager. The human Wm
antibody was a kind gift from Dr. Hiro Furiuichi and the Brca1
antibody was purchased from Santa Cruz Biotechnology, Inc.
[0079] For ChIP analyses, PCR can be performed with the following
primers: 5'-AGC TCC AGG GCC CGG AGC GAC-3' (SEQ ID NO:12) and
5'-CTT CAA TGC CCC CAA AAC CAA-3' (SEQ ID NO:13).
EXAMPLE 2
Stable Transduction of HeLa Cells by HIV-1-based and ASV-based
Vectors is Inhibited by Caffeine
[0080] To determine if caffeine can inhibit stable transduction by
HIV-1, HeLa cells were infected with an HIV-1-based vector that
expresses a lacZ reporter gene (Naldini et al., 1996, supra), in
the presence of a range of concentration of the drug. Caffeine was
added at the time of infection, and removed after 24 hr.
Transduction was measured five to six days later by the presence of
.beta.-galactosidase positive colonies. The results showed a
reduction in the number of cells stably transduced by the vector,
with an IC.sub.50 for caffeine estimated at 1.5 mM (FIG. 2A).
[0081] To determine if caffeine would inhibit stable transduction
by another retrovirus, the protocol was repeated with an ASV-based
vector (Daniel et al., 1999, supra) that carries a neo.sup.r
reporter, and selected stable transductants after treatment with
G418. As with HIV-1, a reduction in the number of cells stably
transduced by the ASV vector was observed, with IC.sub.50
approximately 0.8 mM (FIG. 2B).
EXAMPLE 3
Lack of Effect of Caffeine on Inhibition of HeLa Cell Growth,
Colony Formation and Expression of the Transduced Reporter Gene
Under Conditions in Which Retroviral Transduction is Inhibited
[0082] Caffeine has been reported to have no detectable effect on
cell growth at 0.5-10 mM concentration (Zhu et al., J, Virol. 73:
3309-3316, 1999). Similar results were observed at 2 mM or 4 mM
concentration (FIG. 3A). To test the effect of caffeine on colony
formation, uninfected HeLa cells were plated at a low density and
exposed them to the drug for 24 hr. The results showed that such
treatment with caffeine had no significant effect on either colony
number or colony size (not shown) at up to 4 mM concentration (FIG.
3B, triangles). Finally, the combined effects of G418 and caffeine
treatment on colony formation by HeLa cells were examined. An
ASV-neo.sup.r-transduced, G418-resistant population of HeLa cells
was treated with the concentration of G418 used to select resistant
colonies, together with the indicated concentrations of caffeine.
No effect on colony formation by HeLa cells under these conditions
were observed (FIG. 3B, circles). Thus, inhibition of stable
transduction by the ASV vector cannot be attributed to an effect on
colony formation by the HeLa cells.
[0083] The effect of caffeine on stable transduction by expression
of a retrovirus-transduced reporter in infected cells (FIG. 2),
suggested the possibility that the observed reduction might be due
to suppression of such expression following retroviral DNA
integration. Therefore, the inventors investigated the effect of
caffeine on production of the neomycin phosphotransferase protein
in HeLa cells that had been stably transduced by the ASV vector.
The results showed no reduction in the amounts of reporter protein
after treatment of these cells for 24 hr with up to 4 mM caffeine
(FIG. 3C).
EXAMPLE 4
Effect of Caffeine on Viral DNA Synthesis, Nuclear Import, and
Integration
[0084] To determine if caffeine affects the synthesis of viral DNA
by reverse transcriptase, or other steps preceding reverse
transcription, HeLa cells were infected with the ASV vector in the
presence of caffeine and extracted extrachromosomal DNA at 24 hr
post-infection. Synthesis of viral DNA was detected by quantitative
PCR. Addition of caffeine at up to 4 mM final concentration had
only a modest effect on the amount of viral DNA detected (FIG. 4A),
which could not account for the observed reduction in transduction
efficiency. Consistent with this result, similar concentrations of
caffeine had no detectable effect on the activity of reverse
transcriptase in permeabilized ASV particles.
[0085] Caffeine may also affect the nuclear import of
pre-integration complexes, a step that immediately precedes
retroviral DNA integration. To test this possibility, the inventors
determined the effect of the drug on formation of viral DNA circle
junctions, which serve as a marker for nuclear import of
pre-integration complexes: As shown in FIG. 4B, formation of circle
junctions by the ASV vector was not significantly affected by 2 mM
caffeine, and only a modest decrease, similar to that seen with
total viral DNA, was observed at 4 mM caffeine.
[0086] To determine whether caffeine affects the formation or
stability of viral-host DNA junctions in viva, HeLa cells were
infected with the ASV vector, treated with caffeine, and
integration was monitored 24 hr later using an Alu-PCR-based method
(see Example 1). This method detects the covalent joining of viral
and host DNA, irrespective of whether such junctions are repaired
or unrepaired. The results showed a dose-dependent reduction in
integration, with almost ten-fold (88%) inhibition in 4 mM caffeine
(FIG. 4C, D), consistent with the observed reduction in the number
of transductants shown in FIG. 2. Comparison of the relative
amounts of total, nuclear, and integrated viral DNA, based on
quantitation of the data in FIGS. 4A-C, is shown in FIG. 4D. The
results are consistent with a selective inhibition of integration
in the presence of caffeine and indicate either that the
integrase-catalyzed processing or joining reactions (FIG. 1) are
affected by caffeine, or that the integration intermediate is
unstable in the presence of this DNA damage-sensitizing drug. Based
on observations with non-homologous end-joining deficient cells
(Daniel, R., Katz, R. A. & Skalka, A. M. (1999) Science 284,
644-647; Daniel, R., Katz, R. A., Merkel, G., Hittle, J. C., Yen,
T. J. & Skalka, A. M. (2001) Mol. Cell. Biol. 21, 1164-1172),
it may also be the case that unrepaired integration intermediates
induce cell death, with a concomitant loss of these cells from the
population.
EXAMPLE 5
Lack of Effect of Caffeine on the Activities of ASV and HIV-1
Integrases In Vitro
[0087] It has been reported that retroviral integrase can be
inhibited by treatment with caffeine-related compounds, such as
dicaffeoylquinic acids (Pommier & Neamati, Antiviral Res. 52:
427-458, 1999; de Klein et al., Curr. Biol. 10: 479-482, 2000).
These compounds also inhibit HIV-1 infection in cell culture
(Pommier & Neamati, 1999, supra). To determine if caffeine
inhibition of stable retroviral transduction is a consequence of
its effect on retroviral integrases, the activities of HIV-1 and
ASV integrases in vitro were assayed in the presence of this drug.
As shown in FIG. 5, the processing and joining activities of ASV
integrase and processing activity of HIV-1 integrase were
unaffected by caffeine at up to 10 mM concentration. From these
results it seems unlikely that the inhibitory effect of caffeine on
stable integration and transduction can be explained by a direct
effect on retroviral integrase.
EXAMPLE 6
Stable Transduction of ATM-Deficient and ATM-Proficient Cells is
Inhibited by Caffeine
[0088] The major cellular targets of caffeine related to the DNA
damage response are the ATM and ATR kinases (Zhou et al., 2000,
supra; Blasina et al., 1999, supra; Hall-Jackson et al., 1999,
supra; Sarkaria et al., 1999, supra). Because retroviral DNA
transduction is normal in ATM-deficient cells (Daniel et al., 2001,
supra), the data suggested that the caffeine effects observed were
unlikely to reflect a requirement for ATM. To test this, the effect
of caffeine on HIV-1 transduction of ATM-deficient and
ATM-proficient cells was examined (Table 1). An ataxia
telangiectasia (A-T) cell line was used in which ATM function was
restored by stable transfection with an ATM-expressing plasmid; A-T
cells stably transfected with an empty vector served as a
control.
TABLE-US-00001 TABLE 1 Effect of caffeine on transduction of A-T
cells that express an empty vector or a vector encoding ATM No. of
lacZ-transduced (blue) colonies at caffeine concentration (mM) of:
Cell line 0 1 2 4 AT22IJE-T (ATM-) 284 171 (60)* 88 (31) 40 (14)
AT22IJE-T/ATM (ATM+) 347 262 (76) 144 (41) 64 (18) AT22IJE-T/empty
(ATM-) 313 234 (75) 132 (42) 46 (14) *Numbers in parenthesis are
percentages of colonies observed in absence of caffeine. Cells were
infected with the HIV-1 (lacZ) vector and Lac+ (blue) colonies
detected as described in Experimental Methods, supra; colony counts
from two plates were averaged for each datum point.
[0089] As the inventors had observed previously, deficiency of ATM
had no detectable effect on the efficiency of transduction by HIV-1
in the absence of caffeine. Furthermore, retroviral transduction
was inhibited similarly by caffeine, regardless of the presence or
absence of ATM.
[0090] Caffeine also inhibits a related kinase, mTOR, in vitro, at
concentrations similar to those which inhibit ATM and ATR (Sarkaria
et al., 1999, supra). To determine if mTOR is involved in
retroviral transduction, the inventors infected HeLa cells and
treated them with the mTOR-specific inhibitor, rapamycin. As no
effect of this inhibitor on retroviral transduction was observed,
it appears that mTOR is not required for efficient retroviral
transduction. These data are consistent with the notion that it is
the inhibition of ATR, and not ATM or mTOR, that leads to a
reduction in the number or stability of integrated viral genomes
after treatment with caffeine.
EXAMPLE 7
Reduction in Number of Stable Transductants Resulting from
Overexpression of a Dominant-Negative ATR Mutant in Cells
[0091] To examine the role of ATR more directly, the inventors
examined the efficiency of retroviral DNA integration and
transduction in cells defective in ATR function. Knockout of ATR is
embryonic lethal in mice, and cells lacking ATR die in culture.
Cells that synthesize the dominant-negative ATRkd protein
(doxycycline-inducible expression) are viable, but show
deficiencies in DNA repair that are distinct from those in cells
deficient in ATM. As illustrated in FIG. 6A, cells expressing the
ATRkd showed a dramatic, doxycycline-dependent reduction in the
percentage of stable HIV-1 transductants with two concentrations of
virus that differed ten-fold; similar results were observed with
the ASV vector. These effects cannot be attributed to doxycycline
alone, as treatment of parental cells with this drug had no effect
on stable transduction (not shown). Furthermore, the reduction in
transduction efficiency was correlated with the amount of the
dominant-negative ATRkd protein present (FIG. 6B). As shown in FIG.
6C, expression of ATRkd had no significant effect on the cell
growth rate. Thus, reduction in transduction efficiency by this
vector could not be due to a general inhibition of cell growth.
Finally, it was observed that, as with caffeine treatment, the
relative amount of host-junction DNA, measured by Alu-PCR, was
reduced upon overexpression of ATRkd (FIG. 6D), even though the
amount of viral DNA was similar to that in the uninduced control
(not shown). Thus, the caffeine target, ATR kinase, appears to be
required for stable integration and efficient transduction by
retroviral vectors. It may be the case that the integration process
cannot be completed in the absence of normal ATR function and that
all or some cells that contain unrepaired integration intermediates
fail to survive the infection and are lost from the population.
EXAMPLE 8
Infection of Cells That Express Dominant-Negative ATRkd Leads to
Apoptosis
[0092] The inventors examined the viability of cells that
overexpress ATRkd after viral infection, GM857/ATRkd cells were
treated for 24 h with doxycycline to induce ATRkd synthesis, and
samples were then mock-infected or infected with the ASV vector
(IN+) or an integrase-defective derivative (IN-). The IN- virus is
competent for all early steps of viral replication, but defective
for the integrase-mediated steps in the integration process (FIG.
1). A fourth sample was treated with etoposide (ETP), a DNA
topoisomerase II poison thought to generate DNA double-strand
breaks throughout the cell cycle and shown to reduce the viability
of ATRkd overexpressing cells (Cliby, W. A. Lewis, K. A., Lilly, K.
K. & Kaufmann, S. H. (2002) J. Biol. Chem. 277, 1599-1606).
Samples of uninduced cells were treated similarly as a control. The
cultures were harvested 24 h later and the percentage of apoptotic
cells was determined by terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling and
flow cytometry. The results (Table 2 and FIG. 7) show significant
increases in the percentage of apoptotic cells after infection with
the IN+ ASV and ETP treatment, when the cells were induced to
overexpress ATRkd. In contrast, less or no increase in the
apoptotic fraction was detected in the induced cells that were
mock-infected or infected with the IN virus. These results support
the notion that formation of the retroviral integration
intermediate triggers a cellular DNA damage response. They also
suggest that the yields of host-viral junction DNA and stable
transductants are reduced in ATRkd overexpressing cells because,
lacking normal ATR function, a significant fraction of such cells
do not survive the damage induced by formation of the integration
intermediate.
TABLE-US-00002 TABLE 2 Induction of apoptosis by retroviral
infection of cells induced to overexpress ATRkd Mock- ETP-
ASV(IN.sup.-)- ASV(IN.sup.+)- infected treated infected infected
Uninduced 10.0 10.0 9.6 2.3 (-doxycycline) Induced 17.6 25.5 7.1
36.1 (+doxycycline) Difference 7.6 15.5 <0 33.8 Doxycycline was
added or not to GM847/ATRkd cells, and the following day the cells
were infected at moi 10 with either ASV vector (IN.sup.+) or with
the integrase-deficient (IN.sup.-) vector, or treated with 50 .mu.M
etoposide, in the continued presence or absence of doxycycline.
Twenty-four hours later, cells were stained using the TUNEL assay
and analyzed as described in Experimental Methods. The percentage
of cells that fell within the apoptotic window (see FIG. 7) is
indicated.
EXAMPLE 9
Association of ASV IN and ATR with Viral DNA
[0093] HeLa cells were infected and chromatin prepared at the
indicated times (FIG. 8 A) or at 6 hours (FIG. 8B) post-infection
(pi). In (FIG. 8B) chromatin from cells infected with TN+ the IN-
(D64E) mutant was analyzed. 0=no virus. Chromatin
immunoprecipitation was performed as described in Boyd, K. E. &
Farnham, P. J., Mol. Cell. Biol. 19:8393-8399, 1999; Orlando, V.
TIBS 25:99-104, 2000, using antibodies (Ab) against ASV IN or ATR
and viral DNA was detected using nested PCR with primers targeting
viral LTR sequences.
EXAMPLE 10
Evidence that Brcal is Recruited to Sites of Retroviral DNA
Integration
[0094] FIG. 9 shows results of a chromatin precipitation (ChIP)
analysis using antibodies specific for human Wm and Brcal proteins.
The graph shows the percentage of viral DNA that is associated with
each ChIP, corrected for the immunoprecipitation efficiency of each
antibody. The association of Wrn helicase with viral DNA peaks at 6
hours post-infection, This finding indicates that Wrn is recruited
to integration sites very rapidly. The present data also show that
the ATR substrate Breal also associates with viral DNA, but only
after about 8-10 hours post-infection. This ChIP data suggests
different temporal patterns of association of repair proteins with
sites of integration. Preliminary results from genetic assays also
suggest that Brcal, or some component of a Brcal-dependent complex,
may be required for formation or survival of stable
transductants.
EXAMPLE 11
Transduction of Nocodazole-Arrested 293T Cells is Sensitive to
Caffeine
[0095] To determine if caffeine has any effect on HIV-1
transduction of non-dividing cells, exponentially dividing and
nocodazole-arrested 293T cells were infected with an HIV-1-based
vector. To arrest cells in M phase, cells were treated with
nocodazole (1 .mu.g/ml) for 24 hrs prior to addition of the virus.
Nocodazole was maintained in the cell culture medium during and
after infection. Cells were distributed in a 24-well plate at a
density of 5.times.10.sup.4 cells per well and nocodazole was added
to a final concentration of 1 .mu.g/ml. Cells were infected 24 hrs
later with the HIV-1 based vectors in the presence of 5 .mu.g/ml
DEAE dextran. Caffeine was added to cells along with the vector and
maintained on cells for 24 hrs. Two days post-infection, cells were
stained using a .beta.-galactosidase assay (Stratagene according to
manufacturer's protocol) and blue cells counted. To control for a
possible caffeine contamination, 293T cells were also treated with
caffeine from different sources (Upstate, USB). The results
obtained were consistent with those observed with caffeine from
Sigma.
[0096] As shown in FIGS. 10A and 10B, caffeine inhibited HIV-1
transduction of the dividing (A) and nocodazole-arrested cells (B)
in the same dose-dependent manner. Similar results were obtained
with HeLa cells (data not shown). Caffeine also inhibited
transduction by a multiply attenuated HIV-1-based vector which
lacked the vpr, vif, vpu and nef genes FIGS. 10A and 10B. No
caffeine cytotoxicity was observed under these conditions. To
determine if the transduced lacZ gene was expressed from integrated
vector DNA, 293T cells were infected with a vector carrying an
inactivating D64V substitution in HIV-1 integrase. As shown in
FIGS. 10A and 10B, this vector transduced 293T cells with about
ten-fold lower efficiency than the vector carrying wild-type
integrase.
[0097] To determine the efficiency of the nocodazole arrest, cells
were assayed for expression of the proliferating-cell nuclear
antigen (PCNA) by Western blotting with an anti-PCNA antibody. PCNA
accumulates in cells as they enter S phase, but is rapidly degraded
in other phases of the cell cycle. (Takase, K et al. Reversible G1
arrest induced by dimethyl sulfoxide in human lymphoid cell lines:
kinetics of the arrest and expression of the cell cycle marker
proliferating cell nuclear antigen in Raji cells. Cell Growth
Differ 1992, 3:515-521). FIG. 10C shows that the amount of PCNA in
nocodazole-treated cells is only about 5% or less of that detected
in exponentially dividing cells, indicating an efficient
nocodazole-mediated growth arrest.
[0098] The phosphorylation of histone H3 on serine 10, which is
tightly associated with mitosis, was also evaluated by Western
blotting, using an anti-phosphorylated histone H3 (Ser 10) antibody
(sc-8656-R; Santa Cruz). FIG. 10D reveals increased histone H3
phosphorylation on serine 10 of nocodazole-treated cells,
consistent with the nocodazole-mediated mitotic arrest. To
determine if the observed HIV-1 transduction occurred in the few
cells that still divided, nocodazole-arrested 293T cells were
infected with a high-titer vector, which resulted in transduction
of approximately of the 25% cells in the absence of caffeine.
However, caffeine reduced HIV-1 transduction efficiency even under
these conditions (data not shown).
EXAMPLE 12
Caffeine Reduces Transduction of Contact-Inhibited Mouse Embryo
Fibroblasts
[0099] Because nocodazole inhibits cellular passage through the M
phase, whether caffeine reduces transduction of cells arrested in
G1 phase was investigated. Because agents that arrest cells in
G1/S, such as aphidicolin and hydroxyurea, also trigger an
ATR-dependent DNA damage response, mouse embryo fibroblasts (MEFs),
which are very sensitive to contact inhibition, were used. MEFs
were distributed in a 96-well plate at a density of
5.times.10.sup.4 cells per well to prepare confluent cells, or at a
density of 1.times.10 .sup.4 cells per well to obtain exponentially
growing cells. The following day, the cultures were infected with
the HIV-1-based vectors in the presence of 5 .mu.g/ml DEAE dextran.
Caffeine was added to cells at the same time as the vector and
maintained in the medium for 24 hrs, Two days post-infection, cells
were stained using a .beta.-galactosidase assay (Stratagene
protocol) and blue cells counted.
[0100] Contact inhibition of MEFs led to a substantial reduction in
the absolute numbers of HIV-1-transduced cells (compare FIGS. 11A
and 11B; cells were infected with the same amount of virus at the
same time). However, transduction of both exponentially growing (A)
and contact-inhibited (B) cells was further reduced by treatment
with caffeine in a similar, dose-dependent manner. In each case, no
caffeine-associated cytotoxicity was observed at the concentrations
utilized. To confirm that contact inhibition of MBFs resulted in an
efficient growth arrest, the level of the PCNA protein and
phosphorylation (Ser 10) of histone H3 was evaluated by Western
blotting. FIG. 11C shows that the amount of PCNA in
contact-inhibited MEFs is only about 2% of that in exponentially
dividing MEFs. A reduction in the amount of phosphorylated histone
H3 was also observed (FIG. 11D), consistent with the MEF growth
arrest.
EXAMPLE 13
Caffeine Inhibits Transduction of Terminally Differentiated
Neuronal Cells and Macrophages
[0101] The effect of caffeine on transduction of naturally arrested
human cells was investigated. Human peripheral blood mononuclear
cells (PBMC) were isolated by centrifugation in Ficoll-Hypaque
(Sigma, St. Louis, Mo., USA) from buffy coats of HIV-1 seronegative
individuals. Monocyte-derived macrophages (MDM) were obtained from
PBMC by adherence to plastic for 12 hours in DMEM supplemented with
10% human serum (Cellgro, Herndon, Va., USA), washed, and cultured
in the same medium in the presence of macrophage colony-stimulating
factor (MCSF, 2 ng/ml; Sigma, St. Louis, Mo., USA) for another 7-10
days, allowing cells to differentiate before infection. The medium
was replaced twice during the incubation period. The primary cells
were kept at 37.degree. C. in a humidified incubator with 5%
CO.sub.2.
[0102] NT-2 neuronal precursor cells were purchased from Stratagene
(Stratagene cloning system, La Jolla, Calif.), cultured, and
differentiated into mature human neurons (over 95%) after treatment
with retinoic acid, as described in Patel, C. A., et al. Lentiviral
expression of HIV-1 Vpr induces apoptosis in human neurons. J
Neurovirol 2002, 8:86-99; Patel, C. A., et al. Human
immunodeficiency virus type 1 Vpr induces apoptosis in human
neuronal cells. J Viral 2000,74:9717-26; Pleasure, S. J., et al.
NTera 2 cells: a human cell line which displays characteristics
expected of a human committed neuronal progenitor cell. J Neurosci
Res 1993, 35:585-602; and, Pleasure, S. J., et al. Pure,
postrnitotic, polarized human neurons derived from NTera 2 cells
provide a system for expressing exogenous proteins in terminally
differentiated neurons. J Neurosci 1992, 12:1802-15, each of which
is herein incorporated by reference. Mature neurons generated by
differentiating NT-2 cells were characterized by immunostaining for
expression of ubiquitous neuronal markers (such as MAP2.beta. and
.tau., as well as phenotypically elaborating extensive neuritic
processes identifiable as axons and dendrites.
[0103] To the human macrophages and neurons, caffeine was added
along with the HIV-1-based vector in the presence of 5 .mu.g/ml
DEAE dextran. Caffeine was maintained on the cells for 24 hrs
(macrophages) or 48 hrs (neurons) and .beta.-galactosidase staining
was performed two days post-infection.
[0104] Terminally differentiated, post-mitotic neurons were
infected with the HIV-1 vector. As shown in FIG. 12A, caffeine also
reduced the efficiency of transduction of these cells with the
HIV-1-based vector. To verify that the cells were not cycling, the
amount of PCNA protein was measured. FIG. 12B shows that PCNA
expression was not detected in these differentiated neuronal cells.
Lastly, the effect of caffeine on transduction of terminally
differentiated primary human macrophages was examined. As with the
neurons, caffeine was found to inhibit transduction of these cells,
under conditions that showed no visible cytotoxicity (FIG.
12C).
EXAMPLE 14
Transduction of Nocodazole-Arrested Cells is Inhibited by
Expression of the Dominant-Negative, Kinase-Dead ATR, ATRkd
[0105] ATR is an essential gene; its knockout is embryonic lethal
in mice and cultured cells die rapidly after the ATR gene is
excised. (de Klein, A et al. Targeted disruption of the cell-cycle
checkpoint gene ATR leads to early embryonic lethality in mice.
Curr. Biol. 2000, 10:479-482). However, cells that express a
dominant-negative, kinase-dead ATR protein (GM847/ATRkd) are
viable, although they have deficiencies in DNA repair and/or
checkpoint regulation (Cliby W A et al. Overexpression of a
kinase-inactive ATR protein causes sensitivity to DNA-damaging
agents and defects in cell cycle checkpoints. EMBO J 1998,
17:159-169). In the cells used for these studies, the ATRkd gene is
under control of a doxycycline-inducible promoter.
[0106] GM847/ATRkd cells were plated at a density of
2.times.10.sup.4 cells per well of 24-well plate, in the presence
or absence of doxycycline (5 .mu.g/ml) and nocodazole (1 .mu.g/ml).
The following day, they were infected with the HIV-1-based vectors,
again in the presence or absence of doxycycline and nocodazole,
and, in the experiments described in Example 19 in the presence of
caffeine. Doxycycline and caffeine were removed 24 hrs later, while
nocodazole was maintained on the cells until two days
post-infections, when the cultures were stained using the
.beta.-galactosidase assay.
[0107] As shown in FIG. 13A, there is a doxycycline-dependent
reduction in the percentage of dividing cells that are transduced
by the HIV-1-based vector. Doxycline had no effect on transduction
of parental GM847 cells (data not shown). Because ATR was also
implicated in the regulation of Vpr-induced G2IM arrest,
transduction of ATRkd-expressing cells by the multiply attenuated
HIV-1-based vector was also evaluated. Reduced transduction of
cells expressing the dominant negative ATRkd protein was observed,
similar to that observed with the Vpr-containing HIV-1 vector (FIG.
10A).
[0108] To examine the role of ATR in growth-arrested cells, the
GM847/ATRkd cells were treated with nocodazole in addition to
doxycycline. As shown in FIG. 13B, nocodazole-treated,
ATRkd-expressing cells are transduced with HIV-1-based vectors at a
reduced level, when compared to control nocodazole-arrested cells.
As was the case with 293T cells, FIG. 13C shows that the amount of
PCNA in nocodazole-treated cells is only about 10-20% of that in
exponentially dividing cells, indicating an efficient
nocodazole-mediated growth arrest. FIG. 13D shows an increase in
histone H3 phosphorylation on serine 10 in nocodazole-treated
cells, consistent with the nocodazole-mediated mitotic arrest.
EXAMPLE 15
Residual HIV-1 Transduction of ATRkd-Expressing Cells is Relatively
Resistant to Caffeine
[0109] ATM and ATR kinases are reported to be two major cellular
targets of caffeine and the HIV-1 transduction of ATM-deficient
cells is inhibited by caffeine with the same efficiency as
transduction of ATM-proficient cells. To determine if the residual
transduction of ATRkd-expressing cells can be inhibited by
caffeine, GM847/ATRkd cells were treated with doxycycline, infected
with the HIV-1-based vector, and treated with caffeine. As shown in
FIG. 14, caffeine inhibited HIV-1 transduction of GM847/ATRkd cells
in the absence of doxycycline as efficiently as it inhibited
transduction of 293T cells (FIG. 10). In the presence of
doxycycline, the effect of caffeine was markedly different.
Addition of 0.5 mM caffeine led to a 40% drop in transduction
efficiency, regardless of the presence or absence of doxycycline.
However, further increase in caffeine concentrations had little
effect on transduction efficiency of doxycycline-treated,
ATRkd-expressing cells. At the highest caffeine concentration, 4
mM, the transduction efficiency of doxycycline-treated cells was
reduced only twofold when compared to control cells infected in the
absence of caffeine. In contrast, addition of 4 mM caffeine led to
a 9-fold reduction in transduction efficiency of GM847/ATRkd cells
infected in the absence of doxycycline.
EXAMPLE 16
Suppression of Replication of Different HIV-1 Strains by
Methylxanthines
[0110] Human peripheral blood mononuclear cells (PBMCs) were
isolated from HIV-1-seronegative individuals, and depleted of
monocytes and CD8+ lymphocytes. CD8+ T-Iymphocyte/monocyte-depleted
PBMCs were cultured and infected with the T-cell-line-tropic HIV-1
NL4-3 (.times.4) strain, and simultaneously treated with the
methylxanthines caffeine, theobromine, theophylline and
paraxanthine obtained from Sigma Chemical Co. (St. Louis, Mo.).
HIV-1 p24 antigen levels were assessed by ELISA at. 3, 7 and 12
days post-infection, and positive cultures were defined as those
demonstrating at least 30pg/ml of HIV-1 p24 antigen in the cell
culture supernatant. All procedures were performed under level P3
biosafety conditions to minimize the possibility of
cross-contamination.
[0111] A 9-fold decrease in HIV-1 p24 antigen values was observed
in samples treated with caffeine and theophylline
(1,3-dimethylxanthine), when compared to control samples infected
with NL4-3 alone (FIG. 15A). A three- to four-fold drop in HIV-1
p24 antigen levels was still observed at 7 days post-infection in
samples treated with caffeine and theophylline, respectively. P24
antigen levels in caffeine- and theophylline-treated samples
reached the control level at 12 days post-infection. A suppression
of HIV-1 replication with paraxanthine (1,7-dimethylxanthine) and
theobromine (3,7-dimethylxanthine) was also observed, however,
these compounds suppressed HIV-1 replication only 2 to 3- fold at
day 3 post-infection when compared with the untreated,
NL4-3-infected control (FIG. 15A), Caffeine, theophylline and
paraxanthine did not show any significant cellular cytotoxicity
when evaluated by the XTT assay; only theobromine inhibited the
PBMC growth by 40% at the utilized concentration (data not
shown).
[0112] To determine if the effects of methylxanthines extend to
different HIV-1 strains, a similar experiment with the
macrophage-tropic (R5) HIV-1 strain, ADA, and the NL4-3 strain as a
control was performed in parallel (FIG. 15B). Samples treated with
lower methylxanthine concentrations, including 100 microM, which is
a concentration close to that reached in the plasma of
theophylline-treated patients [28-110 .mu.M, (Ohnishi et al., 2003,
Drugs Aging 20:71-84)], were also tested, Methylxanthines were
added to the medium at the time of infection and maintained in the
media until 6 days post-infection, when HIV-1 p24 antigen levels
were analyzed. As shown in FIG. 150A, methylxanthines inhibited
replication of the rapidly-growing NL4-3 strain at 1 and 4 mM, but
not at 100 microM (FIG. 15B, left panel). Caffeine and theophylline
inhibited NL4-3 growth approximately 9-fold and 5-fold,
respectively, at a concentration of 4 mM, whereas the effect of
theobromine was much weaker (about a 2-fold inhibition), consistent
with the data presented in FIG. 15A. Replication of the HIV-1 ADA
strain was inhibited even at lower concentrations, with an
IC.sub.50 close to 60 microM (theophylline) and 100 microM
(caffeine and theobromine) (FIG. 15B, right panel). The most
efficient inhibitor of the HIV-1 ADA strain appeared to be
theophylline, followed by caffeine and theobromine (FIG. 1513,
right panel).
EXAMPLE 17
Effect of Methylxanthines on Early Steps of the HIV-1
Life-Cycle
[0113] To determine which steps of the HIV-1 life-cycle are
targeted by methylxanthines, the levels of HIV-1 DNA, HIV-1 nuclear
import and HIV-1 DNA integration in infected, caffeine-treated
cells were analyzed. To determine whether caffeine inhibits HIV-1
DNA synthesis or preceding stages of the HIV-1 life cycle, the
levels of HIV-1 DNA were first determined (Nunnari et al., 2002,
Aids 16:39-45; Otero et al., 2003, AIDS Res Hum Retroviruses
19:9237-41). PBMCs were infected with the HIV-1 strain NL4-3 and
caffeine was added at the time of infection. Twenty-four hours
after addition of the virus, cells were harvested and early steps
of the HIV-1-life cycle analyzed by Southern blotting. As shown in
FIG. 16A, methylxanthines, even at the highest utilized
concentration (4 mM), did not have any significant negative effect
on the level of HIV-1 DNA in infected and caffeine-treated
cells.
[0114] To determine if methylxanthines altered the nuclear import
of HIV-1 pre-integration complexes (PICs), we analyzed the level of
2-LTR DNA junctions in infected cells by a quantitative DNA-PCR
technique. The following sense and anti-sense primers were
utilized: 5'-GTAACTAGAGA TACCCTCAAC-3' (SEQ ID NO: 14) and
5'-CAGATCTGGTCTAACCAGAGA-3' (SEQ ID NO: 15). A specific primer that
is contained in the amplicon of the 2-LTR DNA circle junction,
5'-AGTGGCGAGCCCTC AGATGCTGC-3' (SEQ ID NO: 16) was labeled with
.sup.32P for Southern blotting analysis. To normalize the cell
number, the human beta-globin gene was used as a standard (Nunnari
et al., 2002, supra; Otero et al., 2003, supra). No significant
inhibitory effect of caffeine on the level of 2-LTR junctions was
demonstrated (FIG. 16B, data not shown). Then, the level of
integrated HIV-1 DNA in these samples was determined by Alu-PCR in
a PE 9700 system with 45 amplification cycles under conditions
described in Example 1. As a positive control, ACH-2 cells, which
carry one copy of latent, integrated proviral DNA in the cellular
genome, were used. Comparison of caffeine-treated and control NL4-3
samples, revealed approximately 4- to 50-fold less integrated viral
DNA in methylxanthine-treated samples (FIG. 16C).
[0115] The level of unspliced HIV-1-specific RNA in these cells was
also investigated. Without being bound to any particular theory, it
is believed that since integration begins to occur approximately
4-8 hrs post-infection, most HIV-1-specific RNA at the 24 hour
time-point is likely due to expression of the integrated provirus.
Intracellular unspliced viral RNA was analyzed utilizing a
super-sensitive reverse transcriptase-polymerase chain reaction
(RT-PCR) and Southern blotting assays for HIV-1 gag sequences.
Southern blotting was utilized to visualize the specific bands and
compared with a serially-diluted standard curve to quantitate viral
unspliced RNA to 5 copies, with detection but not quantification
between 1 to 5 copies, as described previously (Nunnari et al.,
2002, supra). A decreased unspliced HIV-1-specific RNA level in
cells treated with methylxanthines was observed, which correlates
with the decreased levels of integrated HIV-I DNA in these samples
(FIG. 16D).
EXAMPLE 18
Effect of Methylxanthines on Late Steps of the HIV-1 Life-Cycle
[0116] To determine if methylxanthines affect the late,
post-integrative steps of the HIV-1 life-cycle, the ACH-2 line,
which produces HIV-1 particles upon stimulation with phorbol
myristate acetate (PMA), was used. Methylxanthines did not affect
either production of HIV-1 virion particles in the culture
supernatant, nor p24 antigen expression in unstimulated ACH-2 cells
(FIGS. 17A and 17B). Likewise, methylxanthines had no effect on
production of HIV-1 particles and intracellular p24 antigen levels
when ACH-2 cells were stimulated with PMA (FIGS. 17A and 17B).
EXAMPLE 19
Effect of Caffeine and Theophylline on Phosphorylation of ATM and
ATR Substrates
[0117] The cellular targets of caffeine are the ATM and ATR
kinases. To establish if HIV-1 infection triggers the DNA damage
response that is controlled by these kinases, human PBMCs were
infected with the HIV-1 NL4-3 strain and analyzed by Western
blotting to determine the levels of phosphorylation by ATM and ATR
in these cells. Western blotting was sequentially performed with an
anti pBRCA1 (Bethyl Laboratories, Montgomery, Tex., cat #
A300-008A) or an anti-Ku86 antibody (Santa Cruz, sc-9034, Santa
Cruz, Calif.). For detection of the p53 phosphorylated on serine 15
residue, Western blotting was performed with a rabbit polyclonal
antibody from Cell Signaling Technology, Beverly, Mass. (cat #
9284S). As a positive control, dsDNA breaks were induced with the
drug etoposide (ETP). Increased levels of p53 phosphorylated on
serine 15 residue in infected cells, which is a well-established
target of the ATM and ATR kinases (FIG. 18A) were observed.
Similarly, increased phosphorylation of the BRCA1 protein on serine
1423 residue, which is also an ATR and ATM substrate, were observed
(FIG. 18). p53 and BRCA1 phosphorylation were suppressed by
addition of caffeine and also with theophylline (FIG. 18). These
drugs failed to suppress a basal phosphorylation of BRCA1 on serine
1423 (FIG. 18, left lanes), suggesting a role of caffeine-resistant
kinase in this process.
[0118] The contents of each patent, patent application and
publication cited or described in this document are hereby
incorporated herein by reference, in their entirety.
[0119] The present invention is not limited to the embodiments
described and exemplified above, but is capable of variation and
modification without departure from the scope of the appended
claims.
Sequence CWU 1
1
16124DNAArtificial SequenceOligonucleotide primer 1tcagcgatag
tcgtaactca gcat 24217DNAArtificial SequenceOligonucleotide primer
2agccgtggcc caatgat 17325DNAArtificial SequenceProbe 3ccgtgttaca
tcggttgctg cacaa 25421DNAArtificial SequenceOligonucleotide primer
4accaatgtgg tgaatggtca a 21521DNAArtificial SequenceOligonucleotide
primer 5ctacgagcac ctgcatgaag c 21625DNAArtificial
SequenceOligonucleotide primer 6gcctcccaaa gtgctgggat tacag
25726DNAArtificial SequenceOligonucleotide primer 7ggcttcggtt
gtacgcggtt aggagt 26820DNAArtificial SequenceOligonucleotide primer
8aggtgcacac caatgtggtg 20917DNAArtificial SequenceOligonucleotide
primer 9aaaagcaccg tgcatgc 171021DNAArtificial
SequenceOligonucleotide primer 10caaatggcgt ttattgtatc g
211120DNAArtificial SequenceOligonucleotide primer 11gattggtgga
agtaaggtgg 201221DNAArtificial SequenceOligonucleotide primer
12agctccaggg cccggagcga c 211321DNAArtificial
SequenceOligonucleotide primer 13cttcaatgcc cccaaaacca a
211421DNAArtificialOligonucleotide primer 14gtaactagag ataccctcaa c
211521DNAArtificialOligonucleotide primer 15cagatctggt ctaaccagag a
211623DNAArtificialOligonucleotide primer 16agtggcgagc cctcagatgc
tgc 23
* * * * *