U.S. patent application number 14/085309 was filed with the patent office on 2014-10-30 for use of a rock inhibitor to sustain primary human keratinocytes in a proliferative state.
This patent application is currently assigned to The United States of America as Represented by the Secretary, Department of Health and Human Service. The applicant listed for this patent is Georgetown University, The United States of America as Represented by the Secretary, Department of Health and Human Service. Invention is credited to Sandra Chapman, Xuefeng Liu, Alison McBride, Richard Schlegel.
Application Number | 20140322809 14/085309 |
Document ID | / |
Family ID | 42233901 |
Filed Date | 2014-10-30 |
United States Patent
Application |
20140322809 |
Kind Code |
A1 |
McBride; Alison ; et
al. |
October 30, 2014 |
Use of a Rock Inhibitor to Sustain Primary Human Keratinocytes in a
Proliferative State
Abstract
Disclosed herein is the finding that treatment with a ROCK
inhibitor increases proliferation and induces immortalization of
primary keratinocytes. Accordingly, provided is a method of
immortalizing primary keratinocytes by exposure to a ROCK
inhibitor. Also provided are immortalized primary keratinocytes
produced by the described method, as well as organotypic tissue
equivalents and cell cultures comprising the immortalized primary
keratinocytes. Furthermore, ROCK inhibitor-treated cells show a
greatly increased ability to support viral DNA replication of both
"low risk" and "high risk" HPV genomes, indicating that ROCK
inhibitors will be useful for studying the life cycles of a wide
range of HPVs.
Inventors: |
McBride; Alison; (Bethesda,
MD) ; Chapman; Sandra; (Washington, DC) ;
Schlegel; Richard; (Rockville, MD) ; Liu;
Xuefeng; (Gaithersburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America as Represented by the Secretary,
Department of Health and Human Service
Georgetown University |
Bethesda
Washington |
MD
DC |
US
US |
|
|
Assignee: |
The United States of America as
Represented by the Secretary, Department of Health and Human
Service
Bethesda
MD
Georgetown University
Washington
DC
|
Family ID: |
42233901 |
Appl. No.: |
14/085309 |
Filed: |
November 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13132391 |
Jun 2, 2011 |
8637310 |
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PCT/US2009/066844 |
Dec 4, 2009 |
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14085309 |
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61120272 |
Dec 5, 2008 |
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Current U.S.
Class: |
435/373 |
Current CPC
Class: |
A61P 17/00 20180101;
A61P 17/02 20180101; C12N 5/0629 20130101; C12N 2501/70 20130101;
A61K 35/12 20130101 |
Class at
Publication: |
435/373 |
International
Class: |
C12N 5/071 20060101
C12N005/071 |
Claims
1-26. (canceled)
27. A method of immortalizing primary keratinocytes, comprising (i)
culturing the primary keratinocytes in the presence of fibroblast
feeder cells and in media containing an effective amount of a ROCK
inhibitor for a period of time sufficient to allow immortalization
of the primary keratinocytes; and (ii) continuing to culture the
immortalized keratinocytes in media lacking the ROCK inhibitor,
wherein the immortalized keratinocytes retain the capacity to
differentiate when cultured in media lacking the ROCK
inhibitor.
28. The method of claim 27, wherein continuing to culture the
immortalized keratinocytes comprises culturing the immortalized
keratinocytes until they form an organotypic tissue equivalent.
29. The method of claim 27, wherein the primary keratinocytes are
foreskin keratinocytes, vaginal keratinocytes or cervical
keratinocytes.
30. The method of claim 27, wherein the ROCK inhibitor is
Y-27632.
31. The method of claim 30, wherein the effective amount of Y-27632
is about 1 to about 100 .mu.M.
32. The method of claim 30, wherein the effective amount of Y-27632
is about 5 to 25 .mu.M.
33. The method of claim 27, wherein the primary keratinocytes are
cultured in media containing the ROCK inhibitor for at least 15
days.
34. The method of claim 30, wherein the effective amount of Y-27632
is about 10 .mu.M.
35. The method of claim 27, wherein the ROCK inhibitor is a small
molecule inhibitor.
36. The method of claim 35, wherein the effective amount of the
ROCK inhibitor is about 1 to about 100 .mu.M.
37. The method of claim 35, wherein the effective amount of the
ROCK inhibitor is about 5 to about 25 .mu.M.
38. The method of claim 35, wherein the effective amount of the
ROCK inhibitor is about 10 .mu.M.
39. The method of claim 27, wherein the ROCK inhibitor is
fasudil.
40. The method of claim 27, wherein the primary keratinocytes are
cultured in media containing the ROCK inhibitor for at least 20
days.
41. The method of claim 27, wherein the primary keratinocytes are
cultures in media containing the ROCK inhibitor for at least 40
days.
42. The method of claim 27, wherein the primary keratinocytes are
cultured in media containing the ROCK inhibitor for at least 60
days.
43. The method of claim 27, wherein the primary keratinocytes are
cultured in media containing the ROCK inhibitor for at least 100
days.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/120,272, filed Dec. 5, 2008, which is herein
incorporated by reference in its entirety.
SEQUENCE LISTING SUBMISSION VIA EFS-WEB
[0002] A computer readable text file, entitled
"036681-5028-01-SequenceListing.txt" created on or about Feb. 7,
2013 with a file size of about 71 kb contains the sequence listing
for this application and is hereby incorporated by reference in its
entirety.
FIELD
[0003] This disclosure relates to the use of Rho-associated kinase
(ROCK) inhibitors to increase the proliferative capacity and induce
immortalization of primary keratinocytes.
BACKGROUND
[0004] Somatic cells have a limited lifespan and gradually slow in
growth and stop dividing, a process known as cellular senescence.
This process is thought to limit the vulnerability of aging cells
to disease. Human keratinocytes are invaluable for the study of
skin biology and the pathogenesis of skin-related diseases, but
their short lifespan in culture is a limitation.
[0005] The life cycle of human papillomavirus (HPV) is best studied
in primary human keratinocytes, the natural host cells of HPV.
Papillomaviruses infect the mitotically active cells of the basal
layer of the epithelium, but viral progeny are only produced when
these infected precursor cells differentiate. Papillomavirus
infections are persistent and the viral genome is maintained in the
continuously dividing basal cells for long time periods. The HPV
genome can be transfected into isolated keratinocytes where it
becomes established as an extrachromosomally replicating element.
These infected cells can be induced to differentiate and stratify
and support the productive cycle of HPVs (Frattini et al., Proc.
Natl. Acad. Sci, USA 93:3062-3067, 1996; Meyers et al., Science
257:971-973, 1992).
[0006] A subset of about 15 papillomaviruses from the alpha genera
is associated with cancer, primarily of the uterine cervix (Smith
et al., Int. J. Cancer 121:621-632, 2007). Almost 100% of cervical
carcinomas contain a "high-risk" HPV. These "high-risk" viruses are
also associated with a subset of head and neck cancers (Gillison
and Shah, Curr. Opin. Oncol. 13:183-188, 2001). These
cancer-associated HPVs are also able to immortalize primary human
keratinocytes in culture (Hawley-Nelson et al., EMBO J.
8:3905-3910, 1989; Munger et al., J. Virol. 63:4417-4421, 1989).
Under the appropriate culture conditions, the viral genome is
maintained as an extrachromosomal element and the functions of the
viral E6 and E7 oncoproteins provide a selective growth advantage
for these cells (Goodwin et al., Proc. Natl. Acad. Sci. USA 97:
10978-10983, 2000). These cells can be further cultured as an
organotypic raft where progeny virus can be produced (Frattini et
al., Proc. Natl. Acad. Sci, USA 93:3062-3067, 1996).
[0007] Much less is known about the "low-risk" HPVs that are not
associated with malignant carcinomas. The genomes of these viruses
can be introduced into primary cells, but the E6 and E7 proteins do
not provide a growth advantage to the cells which will often
senesce before extensive studies can be carried out or before they
can be cultured in an organotypic raft. These "low-risk" viruses
are, however, the causative agents of a wide range of benign,
proliferative lesions that can cause intractable disease. These
viruses can be studied in immortalized keratinocyte cell lines, but
these lines have been shown to have genetic abnormalities that
could interfere with functional analyses of the virus (Lehman et
al., Carcinogenesis 14:833-839, 1993). Thus, a need exists to
increase the proliferative capacity of human primary keratinocytes
and to develop an efficient means to induce immortalization of
these cells. Such methods are desirable not only for studies of HPV
replication, but for a variety of therapeutic purposes.
SUMMARY
[0008] It is disclosed herein that treatment of primary
keratinocytes with a ROCK inhibitor increases proliferation and
leads to immortalization of these cells. The immortalized
keratinocytes have a normal karyotype, an intact DNA damage
response and are able to differentiate into stratified epithelium.
In addition, primary keratinocytes treated with a ROCK inhibitor
support viral replication of both low-risk and high risk human
papillomaviruses (HPVs).
[0009] Thus, provided herein is a method of immortalizing primary
keratinocytes by culturing the primary keratinocytes in the
presence of an effective amount of a ROCK inhibitor for a period of
time sufficient to allow immortalization of the primary
keratinocytes. In some embodiments, the method further includes
continuing to culture the immortalized keratinocytes in the absence
of the ROCK inhibitor. The immortalized keratinocytes retain the
capacity to differentiate when cultured in the absence of the ROCK
inhibitor. Isolated immortalized primary keratinocytes produced by
the disclosed methods, organotypic tissue equivalents comprising
the immortalized primary keratinocytes, and cell cultures
comprising the immortalized primary keratinocytes, are also
provided herein.
[0010] Also provided herein is a method of increasing proliferation
of primary keratinocytes by culturing the primary keratinocytes in
the presence of an effective amount of a ROCK inhibitor. In some
embodiments, the method comprises culturing the primary
keratinocytes in the presence of the ROCK inhibitor for a period of
time sufficient to allow immortalization of the primary
keratinocytes.
[0011] Further provided are organotypic tissue equivalents
comprised of immortalized primary keratinocytes. The primary
keratinocytes are immortalized by culturing the primary
keratinocytes in the presence of an effective amount of a ROCK
inhibitor for a period of time sufficient to allow immortalization
of the primary keratinocytes. In some embodiments, the immortalized
keratinocytes are further cultured in the absence of the ROCK
inhibitor, allowing the keratinocytes to differentiate and form the
organotypic tissue equivalent.
[0012] Also provided are compositions including an isolated
immortalized primary keratinocyte. The primary keratinocyte is
immortalized by culturing in the presence of an effective amount of
a ROCK inhibitor for a period of time sufficient to allow
immortalization of the primary keratinocyte.
[0013] A method of promoting HPV replication in primary
keratinocytes is also provided. The method includes infecting the
primary keratinocytes with HPV or transfecting the primary
keratinocytes with an HPV genome, and culturing the primary
keratinocytes in the presence of an effective amount of a ROCK
inhibitor.
[0014] Further provided is a method of preparing an organotypic
tissue equivalent including obtaining isolated primary
keratinocytes, culturing the primary keratinocytes in the presence
of an effective amount of a ROCK inhibitor for a period of time
sufficient to allow for proliferation of the primary keratinocytes,
and continuing to culture the primary keratinocytes in the absence
of the ROCK inhibitor, thereby allowing the primary keratinocytes
to differentiate and form an organotypic tissue equivalent. A
method of treating a wound or skin disease in a subject by treating
the subject with an organotypic tissue equivalent prepared
according to the disclosed method is also provided.
[0015] Any molecule that inhibits expression or activity of ROCK is
contemplated for use with the provided compositions and methods.
For example, the ROCK inhibitor can be a small molecule, an
antibody, a negative regulator or an antisense compound. In
particular examples, the inhibitor is a small molecule ROCK
inhibitor, such as Y-27632.
[0016] The foregoing and other objects, features, and advantages of
the invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1: The kinase activity of ROCK is auto-inhibited by an
intramolecular interaction in which the C-terminal PH (Plekstrin
homology) domain and the rho binding region of the CC (coiled coil)
domain interact with and inhibits the kinase domain. Binding of
GTP-bound rho disrupts this interaction and activates the ROCK
kinase.
[0018] FIG. 2: A schematic of the chemical structure of
Y-27632.
[0019] FIG. 3: Morphology of untreated human foreskin keratinocytes
at p13 and Y-27632-treated cells at p81.
[0020] FIG. 4: Growth curves of primary human keratinocytes
cultured in the presence or absence of 10 .mu.M Y-27632. After 48
passages, Y-27632 was removed from the culture of treated cells and
the resulting growth is indicated.
[0021] FIG. 5: Replication of HPV18 and HPV31 viral genomes in the
presence of 10 .mu.m Y-27632 at two days and five days
post-transfection.
[0022] FIG. 6: Long-term replication assay of "low-risk" HPV6 and
"high-risk" HPV18 in the presence or absence of 10 .mu.M Y-27632.
The pass number of the cells post-electroporation at which DNA was
isolated is shown along the bottom of the figure and the
corresponding number of days at the top of each image.
[0023] FIG. 7: Growth curves of the immortalized CIN1 patient
derived cell lines, W12 and CIN612 9E, containing
extrachromosomally replicating HPV DNA in the presence or absence
of 10 .mu.M Y-27632.
[0024] FIG. 8: Effect of 10 .mu.M Y-27632 treatment on the genome
copy number of HPV16 in the CIN1-derived line W12, and of HPV31 in
the CIN1-derived line CIN612 9E.
[0025] FIG. 9: Y-27632 stabilizes the growth rate of primary
keratinocytes. Shown is the growth rate of human keratinocytes from
foreskin (HFK strain c), ectocervix (HCK) and vaginal tissue (HVK)
cultured in the presence (solid squares) or absence (open diamonds)
of 10 .mu.M Y-27634. The arrows indicate that these cells lines
continued to divide indefinitely. Growth rate is measured as
population doubling time.
[0026] FIG. 10: Morphology of Y-27632-immortalized cells resembles
early pass primary keratinocytes. The left column shows images of
human foreskin keratinocytes (HFK), ectocervical keratinocytes
(HCK), and vaginal keratinocytes (HVK) at pass P1. The middle
column shows keratinocytes near senescence (HFK P15, HCK P9 and HVK
P5). The right column shows keratinocytes immortalized by 10 .mu.M
Y-27632 (HFK P100, HCK P29 and HVK P26). Scale bar, 10 .mu.m.
[0027] FIG. 11A is a graph showing the relative levels of hTERT
mRNA from HFK strain a, cultured in the absence or presence of 10
.mu.M Y-27632, at the pass indicated, as quantitated by real-time
PCR. FIG. 11B is a graph showing the relative length of telomeres
in HFK strain a cultured in the absence or presence of 10 .mu.M
Y-27632, at the pass indicated, as quantitated by real-time
PCR.
[0028] FIG. 12A shows an immunoblot analysis of Myc and p16
proteins in cells cultured in HFK strain c, HVK, and HCK cells in
the absence or presence of 10 .mu.M Y-27632, collected at the pass
indicated. Cells containing oncogenic HPV31 and HPV 18 viruses are
included as controls. .alpha.-tubulin is included as a loading
control. FIG. 12B is an immunoblot showing that DNA damage was
induced by treatment of cells with 0.5 nM actinomycin D. The
response was measured by immunoblot analysis of p53 protein levels
and its downstream target p21. HFKs grown in the absence of Y-27632
were used at P4, and the Y-27632-treated HFKs were used at
P122.
[0029] FIGS. 13A-13D: Keratinocytes are still able to differentiate
in organotypic raft culture after long term culture with Y-27632.
Shown are H&E stained histological sections of primary
keratinocytes grown in the absence of drug at P1 (A) or
Y-27632-immortalized cells at P18 (B) in Y-27632, cultured in
organotypic raft culture for 17 days, and P1 primary keratinocytes
grown in organotypic raft culture for 14 days in raft media without
(C) or with 10 .mu.M Y-27632 (D).
[0030] FIG. 14: Treatment with Y-27632 immortalizes primary
keratinocytes. Human foreskin keratinocytes (HFK strain a) were
cultured in the presence (solid squares) or absence (open diamonds)
of 10 .mu.M Y-27632. Cells were passed when confluent and split at
a ratio 1:10, with a few exceptions when cells were split 1:20.
[0031] FIG. 15: Telomerase expression in Y-27632-treated cells
increases with passage. Relative levels of hTERT mRNA in HFK strain
c, HCK and HVK cells cultured in the absence or presence of
Y-27632, as quantitated by real-time PCR.
[0032] FIG. 16: Expression level of Myc is upregulated in Y-27632
immortalized cells at a late passage. Shown is an immunoblot
analysis of Myc protein in HFK strain a P2 or after 107 passages in
10 .mu.M Y-27632. Cells containing the oncogenic HPV31 are included
as a comparison. .alpha.-tubulin is included as a loading
control.
SEQUENCE LISTING
[0033] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand. In
the accompanying sequence listing:
[0034] SEQ ID NO: 1 is the nucleotide sequence of human ROCK1
(GenBank Accession No. NM.sub.--005406).
[0035] SEQ ID NO: 2 is the amino acid sequence of human ROCK1
(GenBank Accession No. NM.sub.--005406).
[0036] SEQ ID NO: 3 is the nucleotide sequence of human ROCK2
(Genbank Accession No. NM.sub.--004850).
[0037] SEQ ID NO: 4 is the amino acid sequence of human ROCK2
(Genbank Accession No. NM.sub.--004850).
[0038] SEQ ID NOs: 5-8 are the nucleotide sequences of primers used
in the telomere length assay.
DETAILED DESCRIPTION
I. Introduction
[0039] It is disclosed herein that inhibition of Rho-associated
kinase (ROCK) significantly increases the proliferation of primary
keratinocytes and enables these cells to bypass senescence. This
disclosure provides the first description of a defined chemical
compound that mediates efficient cell immortalization.
[0040] Mammalian cells encode two Rho kinases, ROCK1 and ROCK2.
These kinases are activated by binding to an active, GTP-bound Rho
GTPase (see FIG. 1). ROCK phosphorylates a number of substrates on
serine or threonine residues. These substrates are involved in a
wide range of cell behavior. For example, myosin light chain
phosphatase, involved in stress fiber formation and contractility;
LIM kinase, involved in actin stabilization; NHE1 involved in focal
adhesions and actin; and PTEN and Ezrin, involved in apoptosis
(Mueller et al., Nat. Rev. Drug Discov. 4:387-398, 2005; Riento et
al., Nat. Rev. Mol. Cell Biol. 4:446-456, 2003). ROCK inhibitors
such as Y-27632 (see FIG. 2) and fasudil bind to the catalytic site
in the kinase domain and displace ATP (Jacobs et al., J. Biol.
Chem. 281:260-268, 2006). These inhibitors have been found to have
diverse and profound effects on cell behavior and have great
therapeutic promise in many areas of disease.
[0041] The results described herein show that primary keratinocytes
treated with a ROCK inhibitor have greatly increased proliferation,
become immortalized, retain the ability to differentiate, and can
very efficiently support HPV DNA replication. As disclosed herein,
treatment with a ROCK inhibitor resulted in bypass of senescence
and immortalization of different types of keratinocytes from human
foreskin, and vaginal and cervical epithelium. Efficient
immortalization occurred in the presence of fibroblast feeder
cells. As demonstrated herein, keratinocytes immortalized using a
ROCK inhibitor are functionally equivalent to normal cells; they
have a normal karyotype, an intact DNA damage response and are able
to form a stratified epithelium in organotypic culture. The
immortalized keratinocytes exhibit upregulated telomerase mRNA
levels and have telomeres that are shortened, but remain at a
stable length. Myc mRNA levels also are increased in ROCK inhibitor
immortalized keratinocytes.
[0042] Thus, these cells are very useful for a variety of
therapeutic and research purposes. For example, immortalized
keratinocytes are useful for studying the pathogenesis of many
different skin-related diseases. Furthermore, since these cells can
form organotypic skin equivalents in culture, they can be used as
epidermal autographs for wound repair of burns or chronic ulcers.
The immortalized keratinocytes disclosed herein are also useful for
studying various aspects of the HPV life cycle. They provide the
ideal host cell for the long term study of `low-risk` HPVs that are
unable to immortalize keratinocytes. Furthermore, inhibition of the
ROCK pathway also increases the replication efficiency of `high
risk` HPVs, most likely because of the increase in cell
proliferation and plating efficiency.
[0043] The greatly extended proliferative capacity of primary human
keratinocytes treated with Rho kinase inhibitors is also invaluable
for research of many aspects of keratinocyte biology or
keratinocyte associated therapeutics.
II. Abbreviations
[0044] EGF Epidermal growth factor
[0045] FBS Fetal bovine serum
[0046] H&E Hematoxylin & Eosin
[0047] HCK Human cervical keratinocytes
[0048] HFK Human foreskin keratinocytes
[0049] HPV Human papilloma virus
[0050] hTERT Human telomerase reverse transcriptase
[0051] HVK Human vaginal keratinocytes
[0052] PCR Polymerase chain reaction
[0053] PH Plekstrin homology
[0054] QRT-PCR Quantitative reverse transcription-PCR
[0055] ROCK Rho-associated kinase
[0056] SDS Sodium dodecyl sulfate
III. Terms and Methods
[0057] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0058] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0059] Antibody: A protein (or protein complex) that includes one
or more polypeptides substantially encoded by immunoglobulin genes
or fragments of immunoglobulin genes. The recognized immunoglobulin
genes include the kappa, lambda, alpha, gamma, delta, epsilon and
mu constant region genes, as well as the myriad immunoglobulin
variable region genes. Light chains are classified as either kappa
or lambda. Heavy chains are classified as gamma, mu, alpha, delta,
or epsilon, which in turn define the immunoglobulin classes, IgG,
IgM, IgA, IgD and IgE, respectively. As used herein, the term
antibody includes intact immunoglobulins as well as a number of
well-characterized fragments produced by digestion with various
peptidases, or genetically engineered artificial antibodies.
Antibodies for use in the methods and compositions of this
disclosure can be monoclonal or polyclonal. Merely by way of
example, monoclonal antibodies can be prepared from murine
hybridomas according to the classical method of Kohler and Milstein
(Nature 256:495-497, 1975) or derivative methods thereof. Detailed
procedures for monoclonal antibody production are described in
Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York,
1988).
[0060] Also specifically contemplated are human antibodies (arising
from human genes) and humanized antibodies, either of which is
suitable for administration to humans without engendering an
adverse immune response by the human against the administered
immunoglobulin. Humanized forms of antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab).sub.2 or other antigen-binding subsequences
of antibodies) that are principally comprised of the sequence of a
human immunoglobulin, and contain minimal sequence derived from a
non-human immunoglobulin. Humanization can be performed following
methods known in the art, such as by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human antibody
(see, for instance, U.S. Pat. No. 5,225,539; Jones et al., Nature
321(6069):522-525, 1986; Riechmann et al., J Mol Biol.
203(3):825-828, 1988; Verhoeyen et al., Science
239(4847):1534-1536, 1988; Riechmann et al., Nature
332(6162):323-327 1988; or Verhoeyen et al., Bioessays 8(2):74-78,
1988). Antibodies specific for IaI are known in the art (see, for
example, U.S. Pat. No. 6,660,482; U.S. Patent Application
Publication No. 2007/0297982; and Lim et al., J. Infect. Dis.
188:919-926, 2003).
[0061] Antisense compound: Refers to an oligomeric compound that is
at least partially complementary to the region of a target nucleic
acid molecule to which it hybridizes. As used herein, an antisense
compound that is "specific for" a target nucleic acid molecule is
one which specifically hybridizes with and modulates expression of
the target nucleic acid molecule. As used herein, a "target"
nucleic acid is a nucleic acid molecule to which an antisense
compound is designed to specifically hybridize and modulate
expression. In one embodiment, the target nucleic acid molecule is
ROCK1. In another embodiment, the target nucleic acid molecule is
ROCK2. Nonlimiting examples of antisense compounds include primers,
probes, antisense oligonucleotides, siRNAs, miRNAs, shRNAs and
ribozymes. As such, these compounds can be introduced as
single-stranded, double-stranded, circular, branched or hairpin
compounds and can contain structural elements such as internal or
terminal bulges or loops. Double-stranded antisense compounds can
be two strands hybridized to form double-stranded compounds or a
single strand with sufficient self complementarity to allow for
hybridization and formation of a fully or partially double-stranded
compound.
[0062] Antisense oligonucleotide: As used herein, an "antisense
oligonucleotide" is a single-stranded antisense compound that is a
nucleic acid-based oligomer. An antisense oligonucleotide can
include one or more chemical modifications to the sugar, base,
and/or internucleoside linkages. Generally, antisense
oligonucleotides are "DNA-like" such that when the antisense
oligonucleotide hybridizes to a target mRNA, the duplex is
recognized by RNase H (an enzyme that recognizes DNA:RNA duplexes),
resulting in cleavage of the mRNA.
[0063] Differentiate or differentiation: Refers to the process by
which a pluripotent cell becomes distinct in form and function
(i.e., develops into a specialized cell, such as a skin cell). For
example, an embryonic stem cell can differentiate into an
epithelial cell, such as a keratinocyte. Differentiate can also
refer to the process a specific cell type undergoes to become more
specialized. For example, a keratinocyte can differentiate from a
basal keratinocyte to more specialized keratinocytes in a
stratified squamous epithelium.
[0064] Effective amount: As used herein, an "effective amount of a
ROCK inhibitor" is the amount of inhibitor required to inhibit
expression of ROCK or inhibit activity of ROCK. For example, when
the ROCK inhibitor is a small molecule, antibody or negative
regulator of ROCK, an effective amount is the concentration
required to partially or completely eliminate ROCK activity, such
as its kinase activity. In some examples, the ROCK inhibitor is
Y-27632. In one embodiment, an effective amount of Y-27632 is at
least 1 .mu.M. In another embodiment, an effective amount of
Y-27632 is at least 5 .mu.M. In another embodiment, an effective
amount of Y-27632 is at least 10 .mu.M. In some embodiments, the
effective amount of ROCK inhibitor is about 1 to about 100 .mu.M,
about 5 to about 25 .mu.M, or about 10 .mu.M. In another example,
the ROCK inhibitor is an antisense compound. An effective amount of
an antisense compound specific for ROCK is an amount required to
inhibit ROCK mRNA level by at least about 10%, at least about 20%,
at least about 30%, at least about 40% or at least about 50%. An
effective amount of a ROCK inhibitor can also refer to the amount
required to achieve a particular effect, such as immortalization of
a primary keratinocyte.
[0065] Epithelial cells: Cells that line the exterior of a organism
(e.g., skin, cornea), body lumens (e.g., gastrointestinal tract,
urinary tract, reproductive tract, lungs) and mucous membranes
(e.g., oesophagus, mouth and rectum). Epithelial cells also make up
exocrine and endocrine glands.
[0066] Expand: A process by which the number or amount of cells in
a cell culture is increased due to cell division. Similarly, the
terms "expansion" or "expanded" refers to this process. The terms
"proliferate," "proliferation," "proliferated" or "outgrowth" may
be used interchangeably with the words "expand," "expansion," or
"expanded."
[0067] Expose: To bring into contact with. As used herein, exposing
cells to an inhibitor generally refers to culturing or incubating
the cells in the presence of the inhibitor.
[0068] Feeder cells: Cells that are used in culture with other
types of cells to assist in their growth. Feeder cells are growth
arrested (such as by irradiation), but viable and form a substratum
on which other cells can growth. Feeder cell layers provide an
intact and functional extracellular matrix and typically secrete
factors into the medium, such as matrix-associated factors and
cytokines, which can assist in the growth of other cells. In some
embodiments, feeder cells are fibroblast cells, such as a
fibroblast cell line. In particular examples, the feeder cells are
irradiated 3T3 J2 cells. In other examples, the feeder cells are
murine embryonic fibroblasts.
[0069] Fibroblast: A type of cell that synthesizes the
extracellular matrix and collagen, the structural framework
(stroma) for animal tissues, and plays a critical role in wound
healing. Fibroblasts are the most common cells of connective tissue
in animals.
[0070] Human papillomavirus (HPV): A type of virus that infects the
skin and mucous membranes of humans. HPVs are phylogenetically
categorized into five genera: alpha, beta, gamma, mu and nu.
Approximately 130 HPV types have been identified, some of which
have been shown to cause warts (verrucae) or cancer (such as
cervical cancer). Papillomaviruses are DNA viruses with a
non-enveloped viron having icosahedral symmetry. The
double-stranded, circular HPV DNA genome contains one coding region
for late genes, one coding region for early genes, and a non-coding
upstream regulatory region with binding sites for the various
transcription factors controlling expression of early and late
genes. Two separate open reading frames in the late gene coding
region encode viral capsid proteins L1 and L2. Capsid protein L1 is
the major capsid protein that is highly conserved among different
HPV types. Eight open reading frames in the early gene coding
region encode eight viral early proteins, designated E1, E2, E3,
E4, E5, E6, E7, and E8. Early proteins E6 and E7 are oncoproteins
critical for host cell immortalization and transformation, as well
as for long term viral replication and survival.
[0071] "High-risk" HPV includes HPV types that are associated with
malignant cancers, such as cervical carcinoma and head and neck
cancers. High-risk HPVs are capable of immortalizing primary
keratinocytes in culture. Examples of high-risk HPVs include, but
are not limited to, HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, 59 and 68.
[0072] "Low-risk" HPV includes HPV types that are not associated
with malignant cancers, but are known to cause a wide range of
benign, hyperproliferative conditions such as genital warts,
cutaneous warts and respiratory papillomatosis. The E6 and E7
proteins of low-risk HPVs are not capable (in the absence of other
factors) of immortalizing primary cells in culture. Examples of
low-risk HPVs include, but are not limited to, HPV types 1, 2, 3,
4, 6, 7, 11, 42, 43, 44 and 55.
[0073] Immortalized cell: A cell that has bypassed senescence and
is capable of continuous growth in culture.
[0074] Inhibitor of ROCK: As used herein, a ROCK inhibitor is a
protein, nucleic acid, small molecule, antibody or other agent that
prevents expression of ROCK or down-regulates ROCK activity, such
as its kinase activity. Examples of ROCK inhibitors are disclosed
herein. ROCK inhibitors include, but are not limited to, small
molecules, antibodies, antisense compounds and negative regulators
of ROCK. ROCK inhibitors include inhibitors of ROCK-1, ROCK-2 or
both. In some examples, the ROCK inhibitor is Y-27632.
[0075] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein or cell) has been substantially
separated or purified away from other biological components in the
cell or tissue from which the component naturally occurs. As used
herein, an "isolated" cell is a cell that has been substantially
separated from the tissue from which it is derived.
[0076] Keratinocyte: A cell found in the epidermis that produces
keratin. Keratinocytes make up about 90% of epidermal cells.
Keratinocytes are produced by keratinocyte stem cells in the basal
layer of the epidermis. As used herein, "primary keratinocytes" are
keratinocytes isolated from tissue and grown in culture, but are
not immortalized. In the context of the present disclosure, an
"immortalized primary keratinocyte" is a primary keratinocyte that
has become immortalized, such as by culturing the cell in the
presence of a ROCK inhibitor. In some embodiments, the primary
keratinocyte is a foreskin keratinocyte, a vaginal keratinocyte, a
cervical keratinocyte, an oral keratinocyte or a cutaneous
keratinocyte.
[0077] MicroRNA (miRNA): Single-stranded RNA molecules that
regulate gene expression. miRNAs are generally 21-23 nucleotides in
length. miRNAs are processed from primary transcripts known as
pri-miRNA to short stem-loop structures called pre-miRNA and
finally to functional miRNA. Mature miRNA molecules are partially
complementary to one or more messenger RNA molecules, and their
primary function is to down-regulate gene expression. MicroRNAs
regulate gene expression through the RNAi pathway.
[0078] Organotypic tissue equivalent: A cell culture characterized
by the organized growth of the cells in a form resembling a tissue
(also referred to herein as an "organotypic cell culture"). In some
embodiments, the organotypic tissue equivalent is an organotypic
skin equivalent.
[0079] Percent identity: The similarity between amino acid or
nucleic acid sequences is expressed in terms of the similarity
between the sequences, otherwise referred to as sequence identity.
Sequence identity is frequently measured in terms of percentage
identity (or similarity or homology); the higher the percentage,
the more similar the two sequences are. Methods of alignment of
sequences for comparison are well known in the art. Various
programs and alignment algorithms are described in: Smith and
Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.
Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.
USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988;
Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic
Acids Res. 16:10881-10890, 1988; Pearson and Lipman, Proc. Natl.
Acad. Sci. USA 85:2444, 1988; and Altschul et al., Nature Genet.
6:119-129, 1994. The NCBI Basic Local Alignment Search Tool
(BLAST.TM.) (Altschul et al., J. Mol. Biol. 215:403-410, 1990) is
available from several sources, including the National Center for
Biotechnology Information (NCBI, Bethesda, Md.) and on the
Internet, for use in connection with the sequence analysis programs
blastp, blastn, blastx, tblastn and tblastx.
[0080] Pharmaceutically acceptable carrier: The pharmaceutically
acceptable carriers (vehicles) useful in this disclosure are
conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of one or more therapeutic compounds or molecules, such as one or
more nucleic acid molecules, proteins, antibodies, cells or small
molecules, and additional pharmaceutical agents.
[0081] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0082] Preventing, treating or ameliorating a disease: "Preventing"
a disease refers to inhibiting the full development of a disease.
"Treating" refers to a therapeutic intervention that ameliorates a
sign or symptom of a disease or pathological condition after it has
begun to develop. "Ameliorating" refers to the reduction in the
number or severity of signs or symptoms of a disease.
[0083] Primary cell: A non-immortalized cell taken from a living
organism or tissue source.
[0084] Prolonging viability: As used herein, "prolonging viability"
of a cell, such as a primary cell, refers to extending the duration
of time the cell is capable of normal growth and/or survival.
[0085] Rho-associated kinase (ROCK): Also known as Rho-associated
coiled-coil kinase and Rho kinase. The ROCK family includes ROCK1
(also called ROK.beta. or p160ROCK) and ROCK2 (also called
ROK.alpha.). ROCK proteins are serine-threonine kinases that
interact with Rho GTPases. Nucleotide and amino acid sequence of
exemplary human ROCK1 and ROCK2 are set forth herein as SEQ ID NOs:
1-4.
[0086] Ribozyme: A catalytic RNA molecule. In some cases, ribozymes
can bind to specific sites on other RNA molecules and catalyze the
hydrolysis of phosphodiester bonds in the RNA molecules.
[0087] Senescence: Refers to the point at which a cell is no longer
capable of undergoing mitosis (cell division).
[0088] Short hairpin RNA (shRNA): A sequence of RNA that makes a
tight hairpin turn and can be used to silence gene expression via
the RNAi pathway. The shRNA hairpin structure is cleaved by the
cellular machinery into siRNA.
[0089] Small interfering RNA (siRNA): A double-stranded nucleic
acid molecule that modulates gene expression through the RNAi
pathway. siRNA molecules are generally 20-25 nucleotides in length
with 2-nucleotide overhangs on each 3' end. However, siRNAs can
also be blunt ended. Generally, one strand of a siRNA molecule is
at least partially complementary to a target nucleic acid, such as
a target mRNA. siRNAs are also referred to as "small inhibitory
RNAs."
[0090] Small molecule inhibitor: A molecule, typically with a
molecular weight less than about 1000 Daltons, or in some
embodiments, less than about 500 Daltons, wherein the molecule is
capable of modulating, to some measurable extent, an activity of a
target molecule.
[0091] Subject: Living multi-cellular vertebrate organisms, a
category that includes both human and non-human mammals.
[0092] Therapeutically effective amount: A quantity of a specified
agent sufficient to achieve a desired effect in a subject, cell or
culture being treated with that agent.
[0093] Y-27632: A small molecule inhibitor that selectively
inhibits activity of Rho-associated kinase. Also known as
(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide.
Y-27632 is disclosed in U.S. Pat. No. 4,997,834 and PCT Publication
No. WO 98/06433.
[0094] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this invention belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Hence "comprising A or B" means including A,
or B, or A and B. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present invention, suitable
methods and materials are described below. All publications, patent
applications, patents, GenBank accession numbers and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
explanations of terms, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting.
IV. Overview of Several Embodiments
[0095] Described herein is the finding that treatment of primary
keratinocytes with a ROCK inhibitor increases their proliferative
capacity and induces immortalization of these cells. The
immortalized keratinocytes exhibit characteristics typical of
normal primary keratinocytes, including having a normal karyotype
and an intact DNA damage response. In addition, primary
keratinocytes immortalized by exposure to a ROCK inhibitor retain
the capacity to differentiate into stratified epithelium upon
removal of the ROCK inhibitor. Further disclosed herein is the
finding that primary keratinocytes treated with a ROCK inhibitor
support viral replication of both low-risk and high-risk human
papillomaviruses.
[0096] Thus, disclosed herein is a method of immortalizing primary
keratinocytes, comprising culturing the primary keratinocytes in
the presence of an effective amount of a ROCK inhibitor for a
period of time sufficient to allow immortalization of the primary
keratinocytes. In some embodiments, the method further comprises
continuing to culture the immortalized keratinocytes in the absence
of the ROCK inhibitor. As disclosed herein, the immortalized
keratinocytes retain the capacity to differentiate when cultured in
the absence of the ROCK inhibitor.
[0097] As used herein, culturing in the absence of a ROCK inhibitor
does not require the complete absence of a ROCK inhibitor. For
example, low or trace levels of a ROCK inhibitor may be present in
the culture medium such that the level is below the threshold
required to enhance proliferation, induce differentiation and/or
inhibit differentiation of a primary keratinocyte. In some
embodiments, culturing primary keratinocytes in the absence of a
ROCK inhibitor comprises culturing the primary keratinocytes in the
presence of less than about 1 .mu.M, less than about 0.1 .mu.M,
less than about 0.01 .mu.M, or less than about 0.001 .mu.M ROCK
inhibitor. In some embodiments, the primary keratinocytes are
cultured in the complete absence of a ROCK inhibitor. Culturing in
the absence of a ROCK inhibitor refers both to the original ROCK
inhibitor present in the culture, as well as other types of ROCK
inhibitor.
[0098] Culturing primary keratinocytes in the absence of ROCK
inhibitor can be achieved by any one of a number of suitable means,
such as by replacing ROCK inhibitor-containing media with fresh
media lacking ROCK inhibitor. Alternatively, ROCK inhibitor can be
removed from the existing media, such as by dialyzing the
media.
[0099] In some embodiments of the method, continuing to culture the
immortalized keratinocytes comprises culturing the immortalized
keratinocytes until they form an organotypic tissue equivalent. In
some embodiments, culturing the primary keratinocytes comprises
culturing the primary keratinocytes in the presence of fibroblast
feeder cells.
[0100] Also provided herein is a method of increasing proliferation
of primary keratinocytes by culturing the primary keratinocytes in
the presence of an effective amount of a ROCK inhibitor. In some
embodiments, the method comprises culturing the primary
keratinocytes in the presence of the ROCK inhibitor for a period of
time sufficient to allow immortalization of the primary
keratinocytes.
[0101] The primary keratinocytes can be any type of primary
keratinocyte. In some examples, the primary keratinocyte is a
foreskin keratinocyte, vaginal keratinocyte or cervical
keratinocyte.
[0102] The ROCK inhibitor can be any type of molecule that inhibits
expression or activity of ROCK, such as a small molecule inhibitor,
antibody, antisense compound or negative regulator. Suitable ROCK
inhibitors are discussed in greater detail below. In some
embodiments, the ROCK inhibitor is a small molecule inhibitor. In
particular examples, the ROCK inhibitor is Y-27632.
[0103] In some embodiments, when the ROCK inhibitor is Y-27632, the
effective amount of the ROCK inhibitor is about 1 to about 100
.mu.M, or about 5 to about 25 .mu.M, or about 10 .mu.M.
[0104] The ROCK inhibitor can also be a negative regulator of ROCK,
such as, but not limited to small GTP-binding proteins such as Gem,
RhoE and Rad. In other examples, the ROCK inhibitor is an antibody
that specifically binds ROCK1 or ROCK2 or both isoforms. In one
example, the antibody specifically binds ROCK1 (SEQ ID NO: 2). In
another example, the antibody specifically binds ROCK2 (SEQ ID NO:
4).
[0105] In other examples, the ROCK inhibitor is an antisense
compound. Antisense compounds include, but are not limited to,
antisense oligonucleotides, siRNA, miRNA, shRNA and ribozymes.
Antisense compounds specifically target ROCK nucleic acids. In one
example, a ROCK antisense compound specifically hybridizes with
ROCK1 (SEQ ID NO: 1). In another example, a ROCK antisense compound
specifically hybridizes with ROCK2 (SEQ ID NO: 3).
[0106] As described herein, the primary keratinocytes are cultured
in the presence of the ROCK inhibitor for a period of time
sufficient to allow immortalization. In some embodiments, the
primary keratinocytes are cultured in the presence of the ROCK
inhibitor for at least 15 days, at least 20 days, at least 40 days,
at least 60 days, at least 100 days, at least 150 days, at least
200 days, at least 250 days, at least 300 days, at least 350 days,
at least 400 days, at least 450 days, or at least 500 days.
[0107] Also provided herein are isolated immortalized primary
keratinocytes produced by the disclosed method.
[0108] Further provided are cell cultures comprising isolated
immortalized primary keratinocytes produced by the disclosed
method.
[0109] Organotypic tissue equivalents comprising immortalized
primary keratinocytes produced by the disclosed method are also
provided.
[0110] Further provided are organotypic tissue equivalents
comprising immortalized primary keratinocytes. The primary
keratinocytes are immortalized by culturing the primary
keratinocytes in the presence of an effective amount of a ROCK
inhibitor for a period of time sufficient to allow immortalization
of the primary keratinocytes and are further cultured in the
absence of the ROCK inhibitor. When cultured in the absence of the
ROCK inhibitor, the immortalized keratinocytes differentiate to
form the organotypic tissue equivalent.
[0111] In some examples, the organotypic tissue equivalents
described herein comprise primary keratinocytes that have been
cultured in the presence of a ROCK inhibitor to increase
proliferation of these cells, but the cells are not yet
immortalized. Thus, also provided are organotypic tissue
equivalents comprising primary keratinocytes that have been
cultured in the presence of a ROCK inhibitor for a period of time
sufficient to increase proliferation of the primary
keratinocytes.
[0112] In some embodiments, culturing the primary keratinocytes
comprises culturing the primary keratinocytes in the presence of
fibroblast feeder cells.
[0113] The organotypic tissue equivalent can be comprised of any
type of primary keratinocyte. In some embodiments, the primary
keratinocytes are foreskin keratinocytes, vaginal keratinocytes,
cervical keratinocytes, oral keratinocytes or cutaneous
keratinocytes.
[0114] Also provided are compositions comprising an isolated
immortalized primary keratinocyte. The compositions include a
primary keratinocyte immortalized by culturing the primary
keratinocyte in the presence of an effective amount of a ROCK
inhibitor for a period of time sufficient to allow immortalization
of the primary keratinocyte. In some embodiments, the immortalized
keratinocyte is further cultured in the absence of the ROCK
inhibitor. The immortalized keratinocyte retains the capacity to
differentiate when cultured in the absence of the ROCK inhibitor.
In some embodiments, culturing the primary keratinocytes comprises
culturing the primary keratinocytes in the presence of fibroblast
feeder cells.
[0115] In some embodiments, the composition further comprises a
pharmaceutically acceptable carrier. In some embodiments, the
immortalized primary keratinocyte is part of an organotypic tissue
equivalent. In some embodiments, the composition is suitable for
application to human skin. For example, the composition can include
an ointment or viscous material suitable for application to and
retention on the skin. Such pharmaceutically acceptable carriers
are known in the art.
[0116] Further provided is a method of promoting human papilloma
virus (HPV) replication in primary keratinocytes. In some
embodiments, the method comprises infecting the primary
keratinocytes with HPV or transfecting the primary keratinocytes
with an HPV genome, and culturing the primary keratinocytes in the
presence of an effective amount of a ROCK inhibitor, thereby
promoting HPV replication in primary keratinocytes.
[0117] In some embodiments, the method further comprises culturing
the primary keratinocytes in the presence of the ROCK inhibitor
prior to infection with HPV or transfection with the HPV genome. In
some embodiments, the primary keratinocytes are cultured in the
presence of fibroblast feeder cells.
[0118] The primary keratinocytes can be any type of keratinocyte
suitable for propagation of HPV. In particular examples, the
primary keratinocytes are foreskin keratinocytes, vaginal
keratinocytes or cervical keratinocytes.
[0119] The ROCK inhibitor can be any type of ROCK inhibitor
(discussed in greater detail below). In some embodiments, the ROCK
inhibitor is a small molecule inhibitor. In particular examples,
the ROCK inhibitor is Y-27632.
[0120] In some embodiments, when the ROCK inhibitor is Y-27632, the
effective amount of the ROCK inhibitor is about 1 to about 100
.mu.M, about 5 to about 25 .mu.M, or about 10 .mu.M. The primary
keratinocytes are cultured in the presence of the ROCK inhibitor
for any suitable period of time to allow for an enhancement in HPV
replication. In some embodiments, the primary keratinocytes are
cultured in the presence of the ROCK inhibitor for at least 15
days, at least 20 days, at least 40 days, at least 60 days, or at
least 100 days. The primary keratinocytes can be cultured in the
presence of the ROCK inhibitor before or during, or both before and
during infection with HPV.
[0121] The HPV can be from any genus (alpha, beta, gamma, mu or
nu). In some embodiments, the HPV is an alpha, beta or gamma HPV.
The HPV can be a low-risk HPV or a high-risk HPV. In some
embodiments, the HPV is a low-risk HPV, such as HPV type 1, 2, 3,
4, 6, 7, 11, 42, 43, 44 and 55. In particular examples, the
low-risk HPV is HPV6. In some embodiments, the HPV is high-risk,
such as HPV type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or
68. In particular examples, the high-risk HPV is HPV18.
[0122] Also provided is a method of preparing an organotypic tissue
equivalent. The method includes obtaining isolated primary
keratinocytes; culturing the primary keratinocytes in the presence
of an effective amount of a ROCK inhibitor for a period of time
sufficient to allow for proliferation of the primary keratinocytes;
and continuing to culture the primary keratinocytes in the absence
of the ROCK inhibitor, thereby allowing the primary keratinocytes
to differentiate and form an organotypic tissue equivalent.
[0123] In some embodiments, the primary keratinocytes are obtained
by a tissue biopsy. In some examples, the tissue biopsy is taken
from the skin (e.g., the cutaneous and/or mucosal squamous
epithelium).
[0124] Further provided is a method of treating a wound or skin
disease in a subject, comprising treating the subject with an
organotypic tissue equivalent prepared according to the method
disclosed herein. In some embodiments, the wound is a burn or
ulcer. In some embodiments, the primary keratinocytes are obtained
by a tissue biopsy of the subject to be treated with the
organotypic tissue equivalent (thus, the organotypic tissue
equivalent is an autograft). In particular examples, the tissue is
skin.
V. Keratinocyte Proliferation, Immortalization and
Differentiation
[0125] Somatic cells have a limited lifespan and gradually slow in
growth and stop dividing, a process known as cellular senescence.
This process is thought to limit the vulnerability of aging cells
to disease. Human keratinocytes are invaluable for the study of
skin biology and the pathogenesis of skin-related diseases, but
their short lifespan in culture is a limitation. Different
conditions have been developed to optimize the culture of
keratinocytes; the presence of fibroblast feeder cells increases
the proliferative capacity of primary keratinocytes from
approximately 20 to 40-60 population doublings (Ramirez et al.
Genes Dev. 15: 398-403, 2001).
[0126] Spontaneous immortalization of human cells is rare. Primary
keratinocytes can only proliferate for a limited number of cell
divisions before they undergo replicative senescence (Ben-Porath
and Weinberg, Cell Biol. 37:961-976, 2005; Liu et al., J. Virol.
82: 11568-11576, 2008). Several viral oncogenes can efficiently
immortalize cells in culture, such as the E6 and E7 proteins from
oncogenic types of HPV, T-antigen from SV40, and E1A and E1B from
adenovirus. Retinal pigment epithelial cells and foreskin
fibroblasts can be efficiently immortalized by exogenous expression
of the catalytic subunit of human telomerase (Bodnar et al.,
Science 279: 349-352, 1998). However, most studies find that
expression of hTERT is not sufficient for keratinocyte
immortalization and additional changes, such as disruption of
p16.sup.INK4a function, are also required (Kiyono et al., Nature
396: 84-88, 1998; Dickson et al., Mol. Cell Biol. 20: 1436-1447,
2000) Immortalization of human keratinocytes is very rare and only
a few immortalized cell lines exist. These cell lines have genetic
abnormalities such as p53 mutations in HaCaT cells (Lehman et al.,
Carcinogenesis 14:833-839, 1993) or an extra isochromosome of the
long arm of chromosome 8 in the NIKs cell line (Allen-Hoffmann et
al., J. Invest Dermatol. 114:444-455, 2000).
[0127] The events leading to senescence of human keratinocytes are
well known. Cells in culture demonstrate increasing levels of the
p16.sup.ink4 cyclin dependent kinase inhibitor until the cells
reach senescence and cease to proliferate. This is concomitant with
a gradual erosion of telomeres which also results in a growth
crisis. The high risk HPVs are able to abrogate this process. The
E7 protein inactivates and degrades the pRb retinoblastoma protein
and induces G1/S phase progression of the cell cycle (Wise-Draper
and Wells, Front Biosci. 13:1003-1017, 2008). This process
increases the levels of p16.sup.ink4 but the inactivation of the
pRb pathway renders it functionless (Kiyono et al., Nature
396:84-88, 1998). The E6 protein inactivates the p53 protein that
is induced in response to the usurpation of the pRb pathway and
activates telomerase which abrogates the erosion of the telomeres
and allows the cells to proliferate beyond senescence.
[0128] This disclosure describes the effect of a ROCK inhibitor,
Y-27632, on keratinocyte proliferation and immortalization and
subsequent effects on HPV DNA replication. These observations could
have far-reaching implications for the study and treatment of HPV
disease. The greatly improved culture and extended lifespan of
keratinocytes is also invaluable for both research and therapeutic
purposes.
[0129] It is also important to determine whether ROCK inhibition
changes the ability of the keratinocytes to differentiate. Studies
of the complete viral life cycle require that cells containing the
replicating viral genome can differentiate to switch the life cycle
into the late stage necessary for the production of progeny
virions. Studies on the effect of Y-27632 on keratinocyte
differentiation have been controversial. Y-27632 enhances the
survival rate of human embryonic stem cells following
cryopreservation and the resulting treated cells are able to fully
differentiate into all three germ layers after long term culture
(Li et al., Stem Cells Dev 6:1079-1085, 2008). However, ROCK
inhibition has also been reported to abrogate suspension-induced
differentiation in keratinocytes (McMullan et al., Current Biology
13:2185-21 89, 2003), but another study showed that differentiation
is negatively regulated by Rho signaling (Grossi et al., Proc.
Natl. Acad. Sci. USA 102:11313-11318, 2005). It is disclosed herein
that ROCK inhibitor immortalized keratinocytes retain the ability
to differentiate and express appropriate differentiation
markers.
VI. Use of ROCK Inhibitor Immortalized Keratinocytes
[0130] A. HPV-Related Studies
[0131] ROCK-inhibited keratinocytes are useful for studying all
modes of HPV replication. It is shown herein that the efficiency of
initial replication and maintenance replication is greatly
increased in the presence of a ROCK inhibitor. An increase in
virion production will be of great research benefit and can provide
useful reagents for serological studies, for testing vaccines and
for identifying receptors using authentic viral particles.
[0132] Efficient replication of "low-risk" HPV genomes will allow a
much greater understanding of the life cycle of these less well
understood viruses. Although not associated with cancer, these
viruses are responsible for a great burden of recalcitrant disease
such as genital warts, respiratory papillomatosis and cutaneous
warts. These lesions can be especially problematic in individuals
who are immunocompromised by HIV infection or organ transplant.
Efficient means of studying the viral life cycle will allow testing
of anti-viral therapies in a system that closely reflects the in
vivo situation.
[0133] Most HPV studies use keratinocytes derived from neonatal
foreskins because of the availability of this tissue from routine
circumcision. However, these keratinocytes might not be the best
host for papillomavirus studies as HPV infection of the foreskin is
mostly clinically unapparent. A more appropriate cell type for the
study of the cancer associated HPVs is that of the uterine cervix
and in particular cells from the transformation zone between the
ectocervix and the endocervix. Because of the difficulties in
obtaining such tissue, only a few HPV studies have used cervical
tissue. Different HPVs have a very specific tropism for different
regions of epithelia. ROCK inhibitor treatment and expansion of
small numbers of keratinocytes derived from different types of
epithelia could greatly increase the understanding of HPV
biology.
[0134] Remarkably, cervical carcinoma cell lines that have been in
culture for decades still rely on the function of the E6 and E7
oncoproteins for continued proliferation and survival.
Down-regulation of E6 and E7 expression results in immediate
senescence of these cell lines (Goodwin et al., Proc. Natl. Acad.
Sci. USA 97: 10978-10983, 2000). Disclosed herein is the finding
that treatment of cells with Y-27632 removes the growth advantage
conferred by the E6 and E7 oncoproteins (FIG. 6). In the absence of
this selection, there is no need to maintain the viral genomes and
they are gradually lost. This observation could be the basis of a
therapy for persistent HPV infections. A combination of ROCK
inhibitor treatment and a therapy to interfere with
extrachromosomal viral DNA replication could clear infection from
persistently infected cells.
[0135] The studies described herein have focused on replication of
papillomaviruses in ROCK inhibitor-treated keratinocytes but the
same approach can be used for other epitheliotropic viruses. For
example, a range of herpesviruses and poxviruses have been shown to
infect and replicate in keratinocyte organotypic raft cultures and
these are used to test anti-viral therapies in a system that
closely resembles natural infection (Jeon et al., J. Virol.
69:2989-2997, 1995).
[0136] B. Therapeutic Uses
[0137] The Rho/ROCK pathway has been shown to function in the
cardiovascular system, central nervous system, cancers, and
embryonic development (Shi et al., Arch. Immunol. Ther. Exp.
(Warsz.) 55:61-75, 2007). This pathway is an important therapeutic
target and one ROCK inhibitor (fasudil) is already marketed for
cerebral vasospasm after surgery (Shihuya et al., Acta Neurochir.
Suppl 77:201-204, 2001) and is currently being tested for the
treatment of angina pectoris, acute cerebral thrombosis and other
vascular diseases. Studies in animal models suggest widespread
therapeutic benefits in the treatment of inflammation, fibrosis and
neurological disorders (Kubo et al., Ther. Clin. Risk Manag.
4:605-615, 2008; Olson, Curr. Opin, Cell Biol. 20:242-248,
2008).
[0138] Large scale production of keratinocytes from a small number
of cells, especially from individual hosts, is very valuable
therapeutically. A small number of keratinocytes from a biopsy can
be isolated, expanded in monolayer culture and developed into
organotypic skin equivalents (Phillips, Arch. Dermatol.
135:977-978, 1999). These tissue sheets are very useful for
epidermal replacement of regions of ulcers and burns.
[0139] Increased cell proliferation due to ROCK inhibition would
greatly increase the amount of tissue generated and decrease the
time required. It could also allow the use of donor graft tissue
from more appropriate regions of the epithelium to be expanded.
[0140] Keratinocyte-mediated gene therapy is an intensively studied
topic of research (Therrien et al., Toxicol. Pathol. 36:104-111,
2008). Autologous keratinocytes can be isolated and transfected or
transduced with a vehicle expressing a therapeutic gene. Increased
cellular proliferation and immortalization due to ROCK inhibition
could greatly enhance the efficiency of this process. Notably, it
has already been observed that Y-27632 can greatly increase the
survival of embryonic stem cells (Watanabe et al., Nature
Biotechnology 25:681-686, 2007).
[0141] In the dawn of the era of personalized medicine, it is
becoming more and more important to test therapies directly on
cells derived from the patient for which the therapy is eventually
intended. Isolation of host keratinocytes and expansion in culture
under conditions of ROCK inhibition could greatly increase the
efficiency and time frame of this process. Patient-derived
keratinocytes or tissue engineered skin equivalents could be used
to test specific therapies to determine the outcome on the host.
Large quantities of keratinocytes could be expanded from a small
tissue biopsy of patients with specific diseases for research
purposes. The ability to greatly expand these keratinocytes and
derive tissue engineered skin from them will be of great research
and therapeutic benefit.
[0142] Transplantation of apparently immortalized human
keratinocytes onto human hosts raises concerns of uncontrolled
growth and tumorogenicity. However, the experiment shown in FIG. 4
demonstrated that keratinocyte growth rate slows after withdrawal
of Y-27632. In addition, it is also disclosed herein that ROCK
inhibited keratinocytes have a normal karyotype and a normal DNA
damage response. Thus, primary keratinocytes immortalized by
exposure to a ROCK inhibitor would not be tumorigenic.
[0143] C. Organotypic Cultures and Tissue Equivalents
[0144] Preparation and use of organotypic cell cultures and tissue
equivalents, including organotypic skin equivalents, have been
described (see, for example, U.S. Patent Application Publication
Nos. 2009/0280095, 2009/04228, 2006/0292126 and 2005/0079578; Stark
et al., Biological Procedures Online 6: 55-60, 2004). An
"organotypic culture" refers to a culture of cells that associate
in a way that as closely as possible replicates the biochemical and
physiological properties of the organ from which the cells are
derived.
[0145] An organotypic cell culture is a cell culture characterized
by the organized growth of the cells in a form resembling a tissue
(also referred to herein as an "organotypic tissue equivalent"). As
an example of an organotypic tissue equivalent, human primary
keratinocytes are seeded onto a fibroblast-embedded (murine or
human fibroblasts) collagen matrix and grown exposed to air. Within
about 10 days keratinocytes resemble a stratified epithelia with
the characteristic epidermal structure of human skin. These
skin-equivalents have already been evaluated in clinical trials
(Bell et al., Science 211:1052-1054, 1981; Greenberg et al.,
Methods Mol. Biol. 289:425-430, 2005).
[0146] Example 2 below describes a method for generating stratified
epithelium using primary keratinocytes immortalized by exposure to
a ROCK inhibitor. Similar methods can be employed to prepare an
organotypic tissue equivalent for therapeutic purposes. As
described herein, organotypic tissue equivalents are useful for the
treatment of a variety or skin diseases or wounds.
[0147] Chronic wounds disrupt the integrity of the skin by tearing,
cutting, piercing or breaking the tissue. The causes may be
structural, such as injury, or physiological, such as an underlying
disease. The most frequently occurring skin wounds are venous
ulcers, pressure ulcers and diabetic foot ulcers.
[0148] Chronic wounds occur in individuals with underlying diseases
of various types whose medical conditions compromise the body's
ability to repair injured tissue on its own. Despite the use of a
variety of medical and surgical treatments, chronic wounds can take
months or even years to heal and frequently recur. These wounds are
often large and unsightly and may be painful in some patients.
[0149] Chronic wounds are of three major types: venous stasis
ulcers, diabetic ulcers and pressure ulcers. A venous ulcer is an
ulceration that develops on the ankle or lower leg in patients with
chronic vascular disease. In these patients, blood flow in the
lower extremities is impaired, leading to edema (swelling) and mild
redness and scaling of the skin that gradually progress to
ulceration. Venous ulcers are a condition affecting 500,000-700,000
patients in the U.S. and 1.3 million people in the industrialized
world.
[0150] A diabetic ulcer is a chronic wound that occurs in patients
with diabetes. While the actual cause of the ulcer in these
patients is an injury such as a callus, blister or foreign body
such as a pebble or splinter, it is the patient's underlying
disease that places him or her at high risk for developing an
ulcer. Important risk factors include: inadequate local blood
supply, which impairs their ability to repair injured tissue and
ward off infection, and reduced sensation in the extremities, which
causes the initial injury to go unrecognized until it becomes a
serious, chronic wound. Diabetic ulcers are a condition affecting
just under 500,000 patients in the US and 1.2 million people in the
industrialized world.
[0151] A pressure ulcer is defined as any lesion caused by
unrelieved pressure on tissues that are located over a bony
prominence on the body. Pressure ulcers were formerly referred to
as bedsores or decubitus ulcers. Pressure ulcers develop in
immobile patients whose tissues are subjected to continuous
pressure from bones on the interior and hard surfaces such as beds
or chairs on the exterior. In addition to their immobility,
patients at risk for the development of pressure ulcers typically
have poor nutritional status, inadequate hydration, and other
underlying medical conditions that compromise their ability to heal
injuries. Pressure ulcers affect over 1.6 million people in the US
and 4.1 million people in the industrialized world.
[0152] Organotypic tissue equivalents comprising primary
keratinocytes immortalized by exposure to a ROCK inhibitor can be
prepared using any technique known in the art. An exemplary
procedure is described below (see U.S. Patent Application
Publication No. 2006/0292126).
[0153] Organotypic tissue cultivation is generally performed in
inserts with microporous membranes, which contain homologous or
autologous human dermal fibroblasts (HDF), especially postmitotic
HDF at their undersurface. HDF secrete factors that condition the
medium in order to get a better growth of the epidermal
equivalents. The HDF layer can be formed from between about
5.times.10.sup.3 to 1.times.10.sup.5 cells/cm.sup.2, and in some
cases approximately 1.times.10.sup.4 to 5.times.10.sup.4
cells/cm.sup.2. The HDF are preferably postmitotic, but earlier
passage cells can be used if they are irradiated, treated with
mitomycin-C, or otherwise treated to inhibit their proliferation
but maintain their metabolism (for example, by reduction of serum
concentration).
[0154] Microporous membranes are suitable as a culture substrate
because they allow substances to diffuse from one side to the
other, but work as a barrier for cells. The pore size of the
membrane should be adequate so as to allow diffusion of proteins of
up to 100,000 Daltons molecular weight, and preferably of up to
70,000 Daltons molecular weight. The membrane should at least allow
diffusion of small hormones such as insulin, and allow passage of
proteins of up to 15,000 Daltons molecular weight. Other means than
a microporous membrane for performing the function of allowing
diffusion of soluble factors to the primary keratinocytes, while
preventing mixing of the keratinocytes with the HDF can also be
used.
[0155] Microporous membranes typical in the art can be used.
However, membranes fabricated from a biodegradable material (e.g.,
polyhyaluronic acid or polylactic acid) can also be used. When a
biodegradable microporous membrane is employed, the entire culture,
including the differentiated keratinocytes, the microporous
membrane and the HDF, can be transplanted into the skin defect.
Thus, in this alternative embodiment, the HDF grown on the
underside of the membrane need not be post-mitotic or treated to
preclude proliferation. While HDF tend to be less immunogenic than
keratinocytes, it is preferable that when this embodiment is
employed, the HDF be allogeneic cells, preferably autologous
cells.
[0156] In some cases, the thickness of mesh graft can range from
30-300 microns. In some examples, the mesh graft thickness ranges
from 0.5-0.75 mm. A graft of tissue (for example, dermal collagen
plus fibroblasts overlaid with keratinocytes tissue) that is too
thick can result in a too rapid ischemic cell death, especially for
the keratinocyte layer residing above the dermal fibroblast
collagen layer. By contrast, this mesh graft tissue can take in
wound sites.
[0157] To improve the stability of the organotypic tissue
equivalents, a carrier membrane can be placed on top. As an
adhesive, fibrin glue is can be used, or alternatives include
extracellular matrix components such as collagen, fibronectin,
proteoglycans (e.g., hyaluronic acid, chondroitin sulfate, and the
like), or basement membrane zone components (e.g., laminin,
Matrigel.TM., or L-polylysine), or similar tissue glues, may also
be utilized.
[0158] The carriers used with the organotypic tissue equivalents
can consist of a synthetic membrane, made from one or more of
polyester, PTFE or polyurethane; from one or more biodegradable
polymers (e.g., hyaluronic acid, polylactic acid or collagen); or a
silicone or vaseline gauze dressing, or any other material suitable
for wound dressing. These materials that are suitable for wound
dressing allow the carrier to remain in place to immobilize the
implanted tissue equivalents for several days, rather than
requiring the carrier to be removed immediately after the tissue
equivalents are transplanted. Thus, the carrier not only enhances
stability and improves handling, but it also serves as a protective
coat against physical damage as well as the proteolytic milieu and
bacteria in the wound. Moreover, it serves for orientation of the
graft.
[0159] The organotypic tissue equivalents can transplanted by
simply placing them in the bed of the wound or other skin defect.
The tissue equivalents are then immobilized. In some cases, the
method for immobilization is by use of a biodegradable material,
such as by using a tissue glue or adequate bandage.
VII. Rho Family Members and their Role in Cell Fate
[0160] Rho GTPase family proteins, which include Rho, Rac1 and
Cdc42, control a wide variety of cellular processes, such as cell
adhesion, motility, proliferation, differentiation and apoptosis
(Etienne-Manneville and Hall, Nature 420:629-635, 2002; Hagerty et
al., J. Biol. Chem. 282:4884-4893, 2007; Van Aelst and
D'Souza-Schorey, Genes Dev. 11:2295-2322, 1997). One of the best
characterized effectors of Rho is Rho-associated coiled-coil
protein kinase (ROCK).
[0161] ROCK proteins are serine/threonine kinases that bind Rho.
The catalytic kinase domain of ROCK, which comprises conserved
motifs characteristic of serine/threonine kinases, is found at the
N-terminus ROCK proteins also have a central coiled-coil domain,
which includes a Rho-binding domain (RBD). The C-terminus is made
up of a pleckstrin-homology (PH) domain with an internal
cysteine-rich domain. The coiled-coil domain is thought to interact
with other .alpha.-helical proteins. The RBD, located within the
coiled-coil domain, interacts only with activated Rho GTPases,
including RhoA, RhoB, and RhoC. The PH domain is thought to
interact with lipid mediators such as arachidonic acid and
sphingosylphosphorylcholine, and may play a role in protein
localization. Interaction of the PH domain and RBD with the kinase
domain results in an auto-inhibitory loop. In addition, the kinase
domain is involved in binding to RhoE, which is a negative
regulator of ROCK activity (Shi and Wei, Arch. Immunol. Ther. Exp.
55:61-75, 2007).
[0162] The ROCK family consists of two members, ROCK1 (also known
as ROK.beta. or p160ROCK) and ROCK2 (also known as ROK.alpha.).
ROCK1 (1354 amino acids; SEQ ID NO: 2) and ROCK2 (1388 amino acids;
SEQ ID NO: 4) share 65% overall identity and 92% identity in the
kinase domain.
[0163] Although both ROCK isoforms are ubiquitously expressed in
tissues, they exhibit differing intensities in some tissues. For
example, ROCK2 is more prevalent in brain and skeletal muscle,
while ROCK1 is more abundant in liver, testes and kidney. Both
isoforms are expressed in vascular smooth muscle and heart. In the
resting state, both ROCK1 and ROCK2 are primarily cytosolic, but
are translocated to the membrane upon Rho activation (Shi and Wei,
Arch. Immunol. Ther. Exp. 55:61-75, 2007).
[0164] ROCK activity is regulated by several different mechanisms.
As a result, Rho-dependent ROCK activation is highly cell-type
dependent, ranging from changes in contractility, cell
permeability, migration and proliferation to apoptosis. At least 20
ROCK substrates have been identified (Hu and Lee, Expert Opin.
Ther. Targets 9:715-736, 2005; Loirand et al., Cir. Res.
98:322-334, 2006; Riento and Ridley, Nat. Rev. Mol. Cell Biol.
4:446-456, 2003), several of which are involved in apoptosis.
[0165] The role of ROCK in regulating apoptotic signaling is highly
cell-type dependent and stimulus dependent. For example, several
studies have demonstrated that Rho/ROCK activation is required for
endothelial cell death elicited by cytokine or drug treatment. ROCK
also appears to play a pro-apoptotic role in a number of other cell
types, including primary thymocytes, embryonic fibroblasts and HeLa
cells. In vivo, inhibition of ROCK results in protective effects in
a variety of animal models. The protective effects of ROCK
inhibition are often accompanied by a reduced inflammatory response
(Shi and Wei, Arch. Immunol. Ther. Exp. 55:61-75, 2007).
[0166] In contrast, ROCK has also been associated with mediating
cell-survival signals in vitro and in vivo. A ROCK-mediated
pro-survival effect has been reported in epithelial cells, cancer
cells and endothelial cells, as well as in other cell types. In
airway epithelial cells, inhibition with Y-27632 or HA 1077 (also
known as fasudil) induces membrane ruffling, loss of actin stress
fibers and apoptosis (Moore et al., Am. J. Respir. Cell Mol. Biol.
30:379-387, 2004). Rho/ROCK activation also plays a pro-survival
role during oxidative stress-induced intestinal epithelial cell
injury (Song et al., Am. J. Physiol. Cell Physiol. 290:C1469-1476,
2006). ROCK has also been associated with pro-survival events in
thyroid cancer cells (Zhong et al., Endocrinology 144:3852-3859,
2003), glioma cells (Rattan et al., J. Neurosci. Res. 83:243-255,
2006), human umbilical vein endothelial cells (Li et al., J. Biol.
Chem. 277:15309-15316, 2002), hepatic stelate cells (Ikeda et al.,
Am. J. Physiol. Gastrointest. Liver Physiol. 285:G880-886, 2003)
and human neuroblastoma cells (De Sarno et al., Brain Res.
1041:112-115, 2005). Evidence of ROCK playing a pro-survival role
has also been reported in vivo, for example in vascular smooth
muscle cells (Shibata et al., Circulation 103:284-289, 2001) and
spinal motor neurons (Kobayashi et al., J. Neurosci. 24:3480-3488,
2004).
VIII. Rho-Associated Kinase (ROCK) Inhibitors
[0167] In one embodiment, the ROCK inhibitor is a small molecule.
Exemplary small molecule ROCK inhibitors include Y-27632 (U.S. Pat.
No. 4,997,834) and fasudil (also known as HA 1077; Asano et al., J.
Pharmacol. Exp. Ther. 241:1033-1040, 1987). These inhibitors bind
to the kinase domain to inhibit ROCK enzymatic activity. Other
small molecules reported to specifically inhibit ROCK include
H-1152
((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine,
Ikenoya et al., J. Neurochem. 81:9, 2002; Sasaki et al., Pharmacol.
Ther. 93:225, 2002); N-(4-Pyridyl)-N'-(2,4,6-trichlorophenyl)urea
(Takami et al., Bioorg. Med. Chem. 12:2115, 2004); and
3-(4-Pyridyl)-1H-indole (Yarrow et al., Chem. Biol. 12:385,
2005).
[0168] Additional small molecule Rho kinase inhibitors include
those described in PCT Publication Nos. WO 03/059913, WO 03/064397,
WO 05/003101, WO 04/112719, WO 03/062225 and WO 03/062227; U.S.
Pat. Nos. 7,217,722 and 7,199,147; and U.S. Patent Application
Publication Nos. 2003/0220357, 2006/0241127, 2005/0182040 and
2005/0197328.
[0169] In another embodiment, the ROCK inhibitor is a negative
regulator of ROCK activity. Negative regulators of ROCK activation
include small GTP-binding proteins such as Gem, RhoE, and Rad,
which can attenuate ROCK activity. Auto-inhibitory activity of ROCK
has also been demonstrated upon interaction of the carboxyl
terminus with the kinase domain to reduce kinase activity.
[0170] In another embodiment, the ROCK inhibitor can be an antibody
that specifically binds ROCK1 or ROCK2 or both isoforms. In one
example, the antibody specifically binds ROCK1 (SEQ ID NO: 2). In
another example, the antibody specifically binds ROCK2 (SEQ ID NO:
4). By way of example and not limitation, an antibody specific for
a ROCK protein can interfere with binding of ROCK to Rho or other
binding partners, or the antibody can directly disrupt kinase
activity of ROCK.
[0171] In another embodiment, the ROCK inhibitor is an antisense
compound. Generally, the principle behind antisense technology is
that an antisense compound hybridizes to a target nucleic acid and
effects the modulation of gene expression activity, or function,
such as transcription, translation or splicing. The modulation of
gene expression can be achieved by, for example, target RNA
degradation or occupancy-based inhibition. An example of modulation
of target RNA function by degradation is RNase H-based degradation
of the target RNA upon hybridization with a DNA-like antisense
compound, such as an antisense oligonucleotide. Antisense
oligonucleotides can also be used to modulate gene expression, such
as splicing, by occupancy-based inhibition, such as by blocking
access to splice sites.
[0172] Another example of modulation of gene expression by target
degradation is RNA interference (RNAi) using small interfering RNAs
(siRNAs). RNAi is a form of antisense-mediated gene silencing
involving the introduction of double stranded (ds)RNA-like
oligonucleotides leading to the sequence-specific reduction of
targeted endogenous mRNA levels. Another type of antisense compound
that utilizes the RNAi pathway is a microRNA. MicroRNAs are
naturally occurring RNAs involved in the regulation of gene
expression. However, these compounds can be synthesized to regulate
gene expression via the RNAi pathway. Similarly, shRNAs are RNA
molecules that form a tight hairpin turn and can be used to silence
gene expression via the RNAi pathway. The shRNA hairpin structure
is cleaved by the cellular machinery into siRNA.
[0173] Other compounds that are often classified as antisense
compounds are ribozymes. Ribozymes are catalytic RNA molecules that
can bind to specific sites on other RNA molecules and catalyze the
hydrolysis of phosphodiester bonds in the RNA molecules. Ribozymes
modulate gene expression by direct cleavage of a target nucleic
acid, such as a messenger RNA.
[0174] Each of the above-described antisense compounds provides
sequence-specific target gene regulation. This sequence-specificity
makes antisense compounds effective tools for the selective
modulation of a target nucleic acid of interest. In one embodiment,
the target nucleic acid is ROCK1 (SEQ ID NO: 1; Genbank Accession
No. NM.sub.--005406). In another embodiment, the target nucleic
acid is ROCK2 (SEQ ID NO: 3; Genbank Accession No.
NM.sub.--004850). However, other known ROCK sequences can be used
to design antisense compounds.
[0175] Methods of designing, preparing and using antisense
compounds that specifically target ROCK are within the abilities of
one of skill in the art. Examples of ROCK antisense
oligonucleotides are described in U.S. Patent Application No.
2004/0115641.
[0176] Antisense compounds specifically targeting ROCK1 or ROCK2
can be prepared by designing compounds that are complementary to a
ROCK1 or ROCK2 nucleotide sequence. Antisense compounds targeting
ROCK1 or ROCK2 need not be 100% complementary to ROCK1 or ROCK2 to
specifically hybridize and regulate expression of the target gene.
For example, the antisense compound, or antisense strand of the
compound if a double-stranded compound, can be at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 99%
or 100% complementary to the selected ROCK1 or ROCK2 nucleic acid
sequence. Methods of screening antisense compounds for specificity
are well known in the art (see, for example, U.S. Patent
Application No. 2003-0228689). Antisense compounds can contain one
or more modifications to enhance nuclease resistance and/or
increase activity of the compound. Modified antisense compounds
include those comprising modified internucleoside linkages,
modified sugar moieties and/or modified nucleosides.
[0177] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the disclosure to the particular features or
embodiments described.
EXAMPLES
Example 1
A ROCK Inhibitor Promotes Keratinocyte Proliferation and
Differentiation Experimental Procedures
Plasmids
[0178] The HPV6 genome was cloned into the BamHI site of pBR322
(Schwarz et al., EMBO J. 2:2341-2348, 1983); the HPV11 genome was
cloned into the pML2d plasmid (derivative of pBR322) (Dartmann et
al., Virology 151: J 24-130, 1986); the HPV18 genome was cloned
into the EcoRI site of pBR322 (Boshart et al., EMBO J. 3:1151-1157,
1984); and the HPV31 genome was cloned into the EcoRI site of
pT713.
Cell Lines and Culture
[0179] Primary human foreskin keratinocytes (HFKs) were isolated
and pooled from neonatal human foreskins from seven donors and
grown in F medium (3:1(v/v) F-12 Nutrient Mixture: DMEM, 5% FBS,
0.4 .mu.g/ml hydrocortisone, 5 .mu.g/ml insulin, 8.4 ng/ml cholera
toxin, 10 ng/ml EGF, and 24 .mu.g/ml adenine) (Jeon et al., Proc.
Natl. Acad. Sci. USA 92:1654-1658, 1995) in the presence of
irradiated J2 feeder cells. HFKs were divided into 10 cm plates,
containing F-media with or without the addition of 10 .mu.M Y-27632
(Alexis Biochemicals, San Diego, Calif.).
[0180] The 9E and W12 (20863 clone) cell lines were cultured in
F-media on irradiated J2 feeder cells. For the Y-27632 experiments
they were cultured in F-medium with or without 10 .mu.M Y-27632 and
continually passed for the times indicated.
Replication Assay
[0181] Cloned viral genomes were cleaved with restriction enzymes
to separate viral and vector sequences. The genomes were religated
at a concentration of 5 .mu.g/ml to favor intramolecular
circularization. Prior to electroporation, J2 feeder cells were
removed from the keratinocytes by versene treatment and the HFKs
were collected by trypsinization. HFKs (1.times.10.sup.6) were
electroporated with 2 .mu.g of re-circularized viral genomic DNA
using the Amaxa Nucleofector II electroporator and the Human
Keratinocyte Nucleofector Kit. The Amaxa program optimized for cell
survival was used according to the manufacturer's specifications.
Cells were removed from the cuvettes immediately following
electroporation, and seeded onto 10 cm plates containing irradiated
feeder cells and F-medium, in the presence or absence of 10 .mu.M
Y-27632, as indicated. At various times post-transfection, low
molecular weight DNA was isolated by a modified Hirt extraction
procedure (Ustav et al., EMBO J. 10:449-457, 1991). For Southern
blot analysis, the isolated DNA was linearized with EcoRI or BamHI,
depending on cloning site for each viral genome, and DpnI, to
digest any unreplicated input DNA. DNA fragments were separated by
agarose gel electrophoresis followed by Southern blot
hybridization. A .sup.32P-labeled probe was synthesized from the
isolated viral genomic DNA (purified from the vector sequence) by
the random prime method and used in Southern hybridizations.
Results
[0182] Inhibition of the Rho associated kinase ROCK by the
inhibitor Y-27632 has been shown to increase survival of human
embryonic stem cells (Watanabe et al., Nature Biotechnology
25:681-686, 2007) and to increase the colony forming ability of
primary human foreskin keratinocytes. To determine whether
inhibition of this pathway would increase the ability of
keratinocytes to support HPV replication and the viral life-cycle,
primary human foreskin keratinocytes were cultured in the presence
or absence of 10 .mu.M Y-27632. The treated cells proliferated at a
rate that exceeded the untreated cells almost immediately. After
about 15 passages (120 days in culture), the untreated cells ceased
dividing and formed only abortive colonies containing flat cells
that appeared senescent. The Y-27632-treated cells continued to
divide without any decrease in growth rate. The treated cells were
small and cuboidal and grew in closely packed colonies
characteristic of basal cells (see FIG. 3). The cells were passed
at least 82 times and were in continuous culture for over 10
months. Thus, they can be considered immortal. The growth curves of
these cells are shown in FIG. 4. To determine whether the
continuous presence of Y-27632 was required for this immortalized
state, Y-27632 treatment was withdrawn at passage 48 (p48), after
196 days in culture. These cells continued to grow unchecked with
only a gradual slowing in the rate of population doubling.
[0183] To determine whether the greatly improved culture conditions
of the primary human keratinocytes would benefit HPV DNA
replication, viral genomes from the "high-risk" viruses HPV 18 and
HPV31 were transfected into early passage primary foreskin
keratinocytes using nucleofection. Transfected cultures were
maintained in the presence or absence of Y-276328 and low molecular
weight extrachromosomal DNA was isolated at two days and five days
post-electroporation. The isolated DNA was cleaved with a
restriction enzyme that linearized the viral genome and with DpnI
which will only cleave unreplicated input DNA that harbors
methylation from propagation in bacteria. The levels of replicated
DNA were further analyzed by Southern blot analysis. As shown in
FIG. 5, treatment with Y-27632 greatly increased the efficiency of
HPV replication up to five days.
[0184] The great increase in efficiency of "high-risk" HPV DNA
replication in the presence of Y-27632 prompted examination of the
replication of "low-risk" HPV6. Replication assays are much more
difficult with the low risk viruses because the E6 and E7 viral
proteins do not provide the cells with a selective growth
advantage. FIG. 6 shows a replication experiment with the "low
risk" HPV6 and the "high-risk" HPV 18 viral genomes. Both genomes
were electroporated into primary human keratinocytes and were
passed in the presence or absence of Y-27632 for up to 12 passages.
In the absence of Y-27632, the keratinocytes containing HPV6 DNA
had only reached passage 4 by day 49 after electroporation. The
viral DNA was detectable at days five and ten but by the next pass,
at day 32, was undetectable. Treatment with Y-27632 greatly
increased the efficiency of replication and robust levels of viral
DNA were present after three passes. After this, however, the
levels of viral DNA declined. This is likely because there is no
selective advantage to having the viral genome present.
Co-transfection of "low risk" HPV DNA with a drug selectable marker
in addition to Y-27632 treatment should enable maintenance of the
viral genome for much greater time periods. At early times,
replication of the "high risk" HPV18 was greater in the presence of
Y-27632 (FIGS. 5 and 6). However, after day 10 there appeared to be
greater levels of replication in the absence of Y-27632. HPV18
expresses oncogenic E6 and E7 proteins that give the cells a
selective growth advantage. It is most likely that inhibition of
the Rho kinase pathway by Y-27632 has negated the selective growth
advantage conferred by the "high-risk" E6 and E7 oncoproteins.
Again, co-transfection with a drug-selectable marker should
increase apparent replication levels by selecting for the
transfected cells. It has been reported that HPV replication of
"high risk" HPVs requires E6 and E7 functions (Park et al., J.
Virol. 76: 11359-11364, 2002; Thomas et al., Proc. Natl. Acad, Sci.
USA 96:8449-8454, 1999). It will be of interest to determine
whether these functions are required in the presence of
Y-27632.
[0185] Only two cell lines that harbor extrachromosomally
replicating HPV DNA have been successfully cultured from patients
with early dysplastic cervical lesions. W12 cells (Stanley et al.,
Int. J. Cancer 43:672-676, 1989) and CIN612 9E cells (Rader et al.,
Oncogene 5:571-576, 1990) were isolated from CIN1 lesions
containing HPV16 and HPV31, respectively. Both CIN612 9E cells and
the 20863 clone (Jeon et al., J. Virol. 69:2989-2997, 1995) of W12
cells were cultured in the presence or absence of Y-27632 for 44
days (approximately 35 population doublings) to determine any
effects on cell growth and HPV replication levels. As shown in FIG.
7, no differences in growth rate were observed in either cell line
in the presence of Y-27632. Low molecular weight DNA was isolated
and analyzed for HPV DNA at various time points. As shown in FIG.
8, Y-27632 had somewhat different effects on each cell line. The
HPV31 copy number remained constant in the 9E cells, showing that
Y-27632 had no direct effect on HPV replication. The copy number of
HPV16 declined gradually in the W12 cells, both in the presence and
absence of Y-27632. This is likely explained by the previous
observation that extrachromosomal maintenance of the viral genome
in W12 cells is somewhat unstable and the genomes have the
propensity to integrate upon prolonged culture, giving the cells
with integrated genomes a selected growth advantage (Jeon et al.,
J. Virol. 69:2989-2997, 1995). Y-27632 partially rescued the
decline in copy number and this is likely due to removing the
selective advantage provided by integration of the viral
genome.
Example 2
Immortalization of Human Keratinocytes Using a Rock Inhibitor
Materials and Methods
Cell Culture
[0186] Human foreskin keratinocytes (HFKs) were isolated from pools
of at least seven neonatal foreskins and grown in 154 medium
supplemented with Human Keratinocyte Growth Supplement and
Gentamicin/Amphotericin (Invitrogen, Carlsbad, Calif.) or in F
medium [3:1 (v/v) F-12 (Ham)-DMEM, 5% FBS, 0.4 .mu.g/ml
hydrocortisone, 5 .mu.g/ml insulin, 8.4 ng/ml cholera toxin, 10
ng/ml EGF, 24 .mu.g/ml adenine, 100 U penicillin, 100 .mu.g/ml
streptomycin (Invitrogen, cat no. 15140-148)] in the presence of
irradiated 3T3 J2 feeder cells (Jeon et al., J. Virol. 69:
2989-2997, 1995).
[0187] The HPV 18 cell line was established by introducing the HPV
18 genome into primary HFKs using the Amaxa human keratinocyte
nucleofection system. Primary human cervical keratinocytes (HCKs),
human vaginal keratinocytes (HVKs), the HPV18 cell lines and the
HPV31 positive cell line (CIN-612 9E) were grown in the presence or
absence of 10 .mu.M Y-27632 (Alexis Biochemicals), as indicated.
Cells were subcultured by removing the fibroblast feeder cells with
versene and keratinocytes were collected by trypsinization. At each
pass, 2.times.10.sup.5 cells were plated on a 10 cm plate of J2
feeder cells. Population doubling was calculated as: PD=3.32(log(#
cells harvested/# cells seeded)).
Immunodetection
[0188] Proteins were extracted in 2% sodium dodecyl sulfate (SDS),
50 mM Tris-HCl (pH 6.8), 10% glycerol supplemented with inhibitors
Complete and PhosphoSTOP (Roche, Indianapolis, Ind.). Protein
samples were resolved on NuPage gels, electrotransferred to
Immobilon-P membrane (Millipore, Billerica, Mass.), and probed with
the relevant antibodies before detection by chemiluminescence.
Monoclonal antibody against p53 (DO-1) was obtained from Santa Cruz
Biotechnology (Santa Cruz, Calif.). Monoclonal antibody against
.alpha.-tubulin (B-5-1-2) was obtained from Sigma-Aldrich.
Polyclonal antibodies against Myc (N-262), p21 (C-19), and p16
(C-20) were obtained from Santa Cruz Biotechnology.
Real Time QRT-PCR
[0189] Total cellular RNA was isolated with TRIZol.TM. reagent
(Invitrogen, Carlsbad, Calif.) and treated with DNA-free kit
(Ambion, Austin, Tex.). First-strand cDNA was synthesized from 8
.mu.g of total cellular RNA using the Superscript III First-Strand
Synthesis System (Invitrogen, Carlsbad, Calif.). RNase H-treated
cDNA from 20 ng RNA was amplified by quantitative real time PCR
using the Taqman Gene Expression Assay for hTERT (Assay ID:
Hs00972646 ml, Applied Biosystems) spanning exons 14 and 15 and
human RPLPO (large ribosomal protein) endogenous control, VIC/MGB
Probe, primer limited (Applied Biosystems). All samples were run in
triplicate using the ABI 7900HT system and the amount of product
was calculated with reference to standard curves generated by
4-fold serial dilutions of a mixed set of cDNAs with high
telomerase expression. Values were adjusted relative to the level
of RPO transcripts.
Telomere Length Assay
[0190] Genomic DNA was extracted from cells and the average
telomere length was assessed by a modified method of the real-time
PCR-based telomere assay described previously (Cawthon et al.,
Nucleic Acids Res. 30: e47, 2002; Cawthon, Nucleic Acids Res. 37:
e21, 2009). Briefly, the telomere repeat copy number to single gene
copy number (T/S) ratio was determined using a Bio-Rad IQ5
thermocycler. Genomic DNA (5 ng) was subjected to PCR reactions and
detected with SYBR Green Super Mix (Bio-Rad). The primers for
telomere length and HBG1 (a single copy gene) were as follows.
TABLE-US-00001 Tel-1: (SEQ ID NO: 5)
CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT Tel-2: (SEQ ID NO: 6)
GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT HBG1: (SEQ ID NO: 7)
TGTGCTGGCCCATCACTTTG HBG2: (SEQ ID NO: 8)
ACCAGCCACCACTTTCTGATAGG
[0191] Reaction conditions were: 1 cycle, 95.degree. C., 5 minutes;
41 cycles, 95.degree. C., 15 seconds; 1 cycle, 60.degree. C. for 45
seconds. All reactions were carried out in triplicate and compared
to a standard curve of 0, 0.2, 1, 5, 25 and 125 ng genomic DNA
(telomere length 10.4 kb) from Roche Telo-kit. The T/S ratio (dCt)
for each sample was calculated by normalizing the average HBG Ct
value to the average telomere Ct value.
Karyotype Analysis
[0192] This was conducted by Molecular Diagnostic Services, Inc.
San Diego, Calif. Metaphase spreads were prepared and stained to
observe chromosomal G bands.
[0193] Twenty metaphase spreads of each cell line was analyzed and
five complete karyotypes were prepared from each.
DNA Genotype Analysis
[0194] Cellular DNAs were analyzed using the PowerPlex 1.2 STR
genotyping kit (Promega) by Molecular Diagnostic Services, Inc.,
San Diego, Calif.
Organotypic Raft Culture
[0195] Organotypic cultures were generated as described previously
with modifications. (Banerjee et al., Methods Mol. Med. 119:
187-202, 2005). Briefly, 1.times.10.sup.5 keratinocytes were seeded
onto a rat tail type 1 collagen dermal equivalent containing
1-2.times.10.sup.6 J2 3T3 feeder cells. The rafts were lifted onto
stainless steel grids and were fed by diffusion from below with
raft medium [3:1 (v/v) DMEM-F-12 (Ham), 10% FBS, 0.4 .mu.g/ml
hydrocortisone, 0.1 nM cholera toxin, and 5 .mu.g/ml transferring].
Raft cultures were allowed to stratify and differentiate for 11-17
days. The collagen-epithelial rafts were fixed in formalin for 4
hours, paraffin embedded, sectioned and stained with hematoxylin
and eosin (H&E) or by immunofluorescence as described in Pei et
al. (Methods Mol. Med. 119: 49-59, 2005). Monoclonal antibody
against anti-keratin 14 (K14) (Ab-1) was from Thermo Fisher
Scientific, Fremont, Calif. Goat polyclonal antiserum against
Filaggrin (N-20) and rabbit polyclonal antiserum against involucrin
(H-120) were from Santa Cruz Biotechnology.
Growth Arrest Assay
[0196] Keratinocytes (1-2.times.10.sup.6) were seeded on a 10 cm
plate. Forty-eight hours later they were treated with 0.5 nM
actinomycin D for 24 hours. Protein extracts were prepared, as
described above, and analyzed for p53 and p21 protein levels.
Results
Y-27632 Immortalizes Primary Human Keratinocytes
[0197] Rho kinase inhibition has been reported to affect
keratinocyte proliferation and differentiation (Terunuma et al.,
Tissue Eng Part A, Nov. 15, 2009 [Epub]; McMullan et al., Current
Biology 13: 2185-2189, 2003). To further explore the effect of Rho
kinase inhibition on the long term growth of keratinocytes, human
neonatal foreskin keratinocytes and adult vaginal and ectocervical
keratinocytes were cultured in the presence or absence of 10 .mu.M
Y-27632, a well-characterized inhibitor of the Rho-associated
kinase, ROCK. As shown in FIG. 9, the growth rate of all three
keratinocyte types slowed with time and senescence was observed at
approximately population doubling 20-40, depending on the specific
cell type. In the presence of Y-27632, a dramatic increase in
cellular proliferation of all three types of keratinocytes was
observed within days and continued indefinitely. Y-27632-treated
cells had a constant and steady growth rate, as indicated by the
constant slope of population doublings against time. All three
types of keratinocytes efficiently bypassed senescence with no
observed decline in growth rate. As shown in Table 1, efficient
keratinocyte immortalization was observed at least eight times with
three different donor pools of foreskin keratinocytes (strains a,
b, and c) and twice each with ectocervical and vaginal
keratinocytes. Foreskin keratinocytes have been cultured for up to
150 passages for a period of 500 days and can be considered
immortal (see FIG. 14). Occasionally, spontaneously immortalized
cells grew out from quiescent cells that were close to senescence
in the absence of Y-27632, but this only occurred after a long lag
period suggesting that individual cells had picked up rare
mutations allowing them to escape senescence. In contrast,
Y-27632-treated cells grew steadily at all times.
TABLE-US-00002 TABLE 1 Keratinocyte immortalization in the presence
and absence of Y-27632 Keratinocyte strain +Y-27632.sup.1
-Y-27632.sup.2 HFK a PD195 PD62 PD193 PD51 PD177 PD42 PD183 PD29
PD145 PD29 b PD199 PD34 c PD150 PD69 PD105 PD41 HCK PD93 PD28 PD67
PD22 HVK PD80 PD17 PD66 PD15 .sup.1Cells were cultured to the
population doubling (PD) shown and were considered to be immortal.
.sup.2Cells were determined to be senescent at the population
doubling shown. Senescence was defined as growth rate (population
doubling/day) less than or equal to 0.2 within the time period of
one month. HFK: human foreskin keratinocyte; HCK: human cervical
keratinocytes; HVK: human vaginal keratinocyte
[0198] Genetic analysis was carried out on two of the immortalized
foreskin keratinocyte strains (a and b) to ensure that they were
identical to the original donor cells. Short tandem repeat
analysis, a method used to distinguish individuals based on the
highly polymorphic nature of certain regions of chromosomes, showed
that the immortalized cells were genetically indistinguishable from
the original keratinocytes, eliminating the possibility of
contamination by an immortalized cell line.
Immortalization by Y-27632 is Dependent on Co-Culture with
Fibroblasts
[0199] Culturing keratinocytes in the presence of fibroblast feeder
cells increases the lifespan of keratinocytes (Fu et al., Cancer
Res. 63: 7815-7824, 2003; Rheinwald et al., Mol. Cell Biol. 22:
5157-5172, 2002) and might contribute to the observed
immortalization by Y-27632. Therefore, the effect of Y-27632 on
foreskin keratinocytes grown in the absence of fibroblast feeder
cells, cultured on plastic and in serum-free medium was analyzed.
Y-27632 treatment resulted in somewhat increased proliferation but
this was not as pronounced as in the presence of feeders.
Furthermore, in repeated experiments, these cells did not bypass
senescence. Therefore, co-culture with feeder fibroblasts is
required in concert with ROCK inhibition to immortalize
keratinocytes.
Morphology of Y-27632 Immortalized Keratinocytes Resembles Early
Passage, Basal-Like Keratinocytes
[0200] At early passages, primary keratinocytes are actively
dividing and are small, cuboidal and homogeneous in shape (FIG.
10). When cultured with fibroblast feeder cells, they grow in
tightly packed colonies and resemble basal keratinocytes. As they
approach senescence, their morphology changes and they becomes
flat, and heterogeneous with enlarged cytoplasmic volume. The
morphology of the Y-27632 immortalized cells was similar to early
passage keratinocytes and is typical of actively dividing
cells.
The Karyotype of Y-27632 Immortalized Cells is Normal
[0201] Immortalization of primary human keratinocytes is rare and
the resulting cell lines have genetic changes and abnormal
karyotypes. The karyotype was analyzed for one of the foreskin
keratinocyte lines (strain a) that had been cultured in the
presence of Y-27632 for 95 passages. The karyotype of the
immortalized cells was identical to that of the donor cells with
the correct number of chromosomes with no apparent
abnormalities.
Telomerase is Upregulated in Y-27632-Immortalized Cells
[0202] Human telomerase verse transcriptase (hTERT) is a subunit of
telomerase, which maintains the telomere caps throughout the
multiple cell divisions of development. hTERT expression is turned
off in most somatic cells and so the telomere ends become
progressively shorter over multiple cell divisions. Replicative
senescence is triggered when these protective ends become
critically short (Harley et al, Nature 345: 458-460, 1990). To
overcome this constraint, most tumor-derived or immortal cell lines
have reactivated hTERT expression to maintain the telomere ends.
Quantitative RT-PCR analysis showed that the level of hTERT mRNA
increased with passage of foreskin keratinocytes in the presence of
Y-27632 (FIG. 11A). As a comparison, hTERT mRNA levels were also
determined in keratinocytes immortalized with HPV 18. The "high
risk" HPV E6 protein directly upregulates hTERT transcription as
part of the immortalization process (Klingelhutz et al., Nature
380: 79-82, 1996). By passage 34 in Y-27632, hTERT mRNA levels were
comparable to those in HPV 18-immortalized keratinocytes. A similar
induction of hTERT mRNA was observed in vaginal and cervical
keratinocytes immortalized by Y-27632, as well as in another strain
of foreskin keratinocytes (FIG. 15).
The Lengths of Telomeres Shorten, but are Stabilized, in
Keratinocytes Immortalized by Y-27632
[0203] In HPV immortalized cells, telomere ends erode despite
telomerase induction, but the shortened length becomes stable
(Stoppler et al., J. Biol. Chem. 272: 13332-13337, 1997). A similar
phenomenon was observed in Y-27632 immortalized cells. The relative
length of telomeres was measured using a quantitative PCR assay.
Despite increased levels of telomerase expression, the length of
the telomeres in cells cultured with Y-27632 became progressively
shorter with passage (FIG. 11B). However, the length became stable
from passage 50 to 120 and was similar to the length of telomeres
in HPV18 immortalized cells.
P16.sup.INK4a is Expressed in Y-27632-Immortalized Cells
[0204] Unlike the situation for fibroblasts, telomerase expression
is not sufficient for immortalization of human keratinocytes and
the pRB/p16.sup.INK4a pathway must also be inactivated (Dickson et
al., Mol. Cell Biol. 20: 1436-1447, 2000). p16.sup.INK4a mRNA and
protein levels were examined in keratinocytes during long term
culture with Y-27632. P16.sup.INK4a mRNA and protein expression
were still observed, albeit at a low level, after long-term culture
with Y-27632 (see FIG. 12A). However, at this point it is unknown
whether the observed p16.sup.INK4a is functional. In contrast, the
level of p16.sup.INK4a in HPV immortalized cells is very high, but
non-functional because of inactivation of the pRb pathway. Y-27632
treatment had no effect on the p16.sup.INK4a levels in these
cells.
c-Myc is Upregulated in Y-27632-Immortalized Cells
[0205] The Myc protein binds to the E-boxes of the hTERT promoter
to induce transcription (Wang et al., Genes Dev. 12: 1769-1774,
1998) and HPV E6 requires Myc for cellular immortalization (Liu, et
al., J. Virol. 82: 11568-11576, 2008). As shown in FIG. 12A,
Y-27632 has both short term and long term effects on Myc expression
in all three keratinocyte types. Myc protein levels are induced
transiently immediately after culture with Y-27632 (compare p4 for
HFK and P2s for HCK and HVK). After this initial induction there is
a decrease in Myc expression but then a general increase over time
in all three cell types. At very late passages (p107), the level of
Myc is equivalent to that of an HPV31-containing cell line (FIG.
16A). The long term increase in Myc levels is similar to the
increase in hTERT expression implying that increased telomerase
expression might be due to Myc induction.
[0206] The Tumor Suppressor Gene p.53 is Expressed in Y-27632
Immortalized Cells and can Mediate a Normal DNA Damage Response
[0207] The tumor suppressor gene, p53, prevents aberrant
proliferation and arrests the growth of cells that have sustained
genetic damage. In most cancer-derived or immortalized cell lines
the p53 pathway is either mutated or suppressed to allow cells to
proliferate in conditions of aberrant growth regulation. p53
protein levels gradually increase in keratinocytes cultured with
Y-27632, but this does not appear to be inhibitory to cell growth
and p21 is not induced. To test whether the p53 pathway was
functional in the Y-27632 immortalized cells, the response of the
cells to p53-induced growth arrest mediated by DNA damage was
analyzed. Normal cells exhibit growth arrest when exposed to a
mutagen but this arrest is abrogated in cells immortalized by the
human papillomavirus E6 and E7 oncoproteins (Foster et al., J Virol
68: 5698-5705, 1994). Keratinocytes were treated with 0.5 nM
actinomycin D, which induces DNA strand breaks and induces a
p53-mediated growth arrest (Abrams et al., Cell Immunol. 182:
137-151, 1997). Early passage keratinocytes and Y-27632-treated
keratinocytes exhibited a normal DNA damage response; both p53 and
the p53-responsive protein, p21, were upregulated (see FIG. 12B).
In contrast, the HPV31 immortalized cell line CIN612, as well as
HPV18 immortalized cells, did not induce p53 levels or the p53
pathway in response to the DNA damage. Therefore, Y27632
immortalized keratinocytes retain a normal DNA damage response.
[0208] Y-27632-Treated Cells Retain the Ability to
Differentiate
[0209] McMullan et al. (Current Biology 13: 2185-2189, 2003) have
shown that blocking ROCK function inhibits suspension induced
differentiation of human keratinocytes. To determine whether
keratinocytes grown in the presence of Y-27632 retain their
differentiation potential, their ability to form a stratified
epithelium in organotypic raft culture was assayed. Early passage
HFKs and Y-27632-treated keratinocytes were seeded onto a
fibroblast-collagen matrix, and cultured as a "raft" at the
liquid-air interphase for 17 days in the absence of Y-27632 in the
raft media. As shown in FIG. 13 (panel a), this induces primary
keratinocytes to produce a stratified epithelial tissue. Similarly,
keratinocytes cultured in the presence of Y-27632 for 18 passages
(FIG. 13 panel b) and late pass Y-27632 immortalized cells could
also produce a stratified epithelium in organotypic culture.
However, when 10 .mu.M Y-27632 was added to the organotypic raft
culture medium, no differentiation or stratification were observed
(compare FIG. 13 panel c and panel d), confirming the findings of
McMullan et al. in a different differentiation system.
[0210] To further demonstrate that the stratified epithelial tissue
grown from Y-27632 immortalized cells expressed appropriate
differentiation markers, the expression of keratin 14 (expressed in
the basal layer), involucrin (upper spinous layer) and filaggrin
(granular/cornified layer) was analyzed by immunofluorescence on
fixed tissue sections. The results demonstrated that raft tissue
grown from either untreated or Y-27632-treated cells expressed
these differentiation markers in the appropriate layer (Pommerencke
et al., BMC Bioinformatics. 9: 473, 2008). Thus, epithelial tissue
generated from Y-27632 treated keratinocytes retains the ability to
differentiate normally.
Example 3
Treatment of a Chronic Wound with an Organotypic Tissue
Equivalent
[0211] Organotypic tissue equivalents comprised of primary
keratinocytes that have been exposed to a ROCK inhibitor to
increase their proliferation (and induce immortalization if
cultured for a sufficient period of time) can be used to treat a
subject with a chronic wound, such as a venous statis ulcer,
diabetic ulcer or pressure ulcer. As described herein, primary
keratinocytes are obtained from a donor. In some cases, the donor
is the subject to be treated. The primary keratinocytes are
obtained by skin biopsy and expanded in a monolayer culture
containing an effective amount of a ROCK inhibitor, such as 10
.mu.M Y-27632, to allow for expansion of the primary keratinocytes.
The expanded primary keratinocytes are developed into an
organotypic tissue equivalent by seeding onto a suitable matrix,
such as a fibroblast-embedded collagen matrix and growing the cells
exposed to air. After culturing for approximately, 7-14 days, the
keratinocytes resemble a stratified epithelium with the
characteristic epidermal structure of the human skin. The
organotypic tissue equivalent is transplanted directly onto the
ulcer and immobilized using a suitable bandage.
[0212] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only preferred
examples of the invention and should not be taken as limiting the
scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
Sequence CWU 1
1
816650DNAHomo sapiensCDS(942)..(5006) 1gctggttccc cttccgagcg
tccgcgcccc gcatgcgcag tctgccccgg cggtctccgt 60ttgtttgaac aggaaggcgg
acatattagt ccctctcagc ccccctcgcc ccacccccca 120ggcattcgcc
gccgcgactc gccctttccc cggctgggac cgcagcccct cccagaagct
180cccccatcag cagccgccgg gacccaacta tcgtcttcct cttcgcccgc
tctccagcct 240ttcctctgct aagtctccat cgggcatcga cctcgccctg
ccccaccgga caccgtagca 300gcagccccag cagcgacggg acaaaatggg
agagtgaggc tgtcctgcgt ggaccagctc 360gtggccgaga ctgatcggtg
cgtcgggccg ggccgagtag agccggggac gcggggctag 420accgtctaca
gcgcctctga gcggagcggg cccggcccgt ggcccgagcg gcggccgcag
480ctggcacagc tcctcacccg ccctttgctt tcgcctttcc tcttctccct
cccttgttgc 540ccggagggag tctccaccct gcttctcttt ctctacccgc
tcctgcccat ctcgggacgg 600ggacccctcc atggcgacgg cggccggggc
ccgctagact gaagcacctc gccggagcga 660cgaggctggt ggcgacggcg
ctgtcggctg tcgtgagggg ctgccgggtg ggatgcgact 720ttgggcgtcc
gagcggctgt gggtcgctgt tgcccccggc ccggggtctg gagagcggag
780gtcccctcag tgaggggaag acgggggaac cgggcgcacc tggtgaccct
gaggttccgg 840ctcctccgcc ccgcggctgc gaacccaccg cggaggaagt
tggttgaaat tgctttccgc 900tgctggtgct ggtaagaggg cattgtcaca
gcagcagcaa c atg tcg act ggg gac 956 Met Ser Thr Gly Asp 1 5 agt
ttt gag act cga ttt gaa aaa atg gac aac ctg ctg cgg gat ccc 1004Ser
Phe Glu Thr Arg Phe Glu Lys Met Asp Asn Leu Leu Arg Asp Pro 10 15
20 aaa tcg gaa gtg aat tcg gat tgt ttg ctg gat gga ttg gat gct ttg
1052Lys Ser Glu Val Asn Ser Asp Cys Leu Leu Asp Gly Leu Asp Ala Leu
25 30 35 gta tat gat ttg gat ttt cct gcc tta aga aaa aac aaa aat
att gac 1100Val Tyr Asp Leu Asp Phe Pro Ala Leu Arg Lys Asn Lys Asn
Ile Asp 40 45 50 aac ttt tta agc aga tat aaa gac aca ata aat aaa
atc aga gat tta 1148Asn Phe Leu Ser Arg Tyr Lys Asp Thr Ile Asn Lys
Ile Arg Asp Leu 55 60 65 cga atg aaa gct gaa gat tat gaa gta gtg
aag gtg att ggt aga ggt 1196Arg Met Lys Ala Glu Asp Tyr Glu Val Val
Lys Val Ile Gly Arg Gly 70 75 80 85 gca ttt gga gaa gtt caa ttg gta
agg cat aaa tcc acc agg aag gta 1244Ala Phe Gly Glu Val Gln Leu Val
Arg His Lys Ser Thr Arg Lys Val 90 95 100 tat gct atg aag ctt ctc
agc aaa ttt gaa atg ata aag aga tct gat 1292Tyr Ala Met Lys Leu Leu
Ser Lys Phe Glu Met Ile Lys Arg Ser Asp 105 110 115 tct gct ttt ttc
tgg gaa gaa agg gac atc atg gct ttt gcc aac agt 1340Ser Ala Phe Phe
Trp Glu Glu Arg Asp Ile Met Ala Phe Ala Asn Ser 120 125 130 cct tgg
gtt gtt cag ctt ttt tat gca ttc caa gat gat cgt tat ctc 1388Pro Trp
Val Val Gln Leu Phe Tyr Ala Phe Gln Asp Asp Arg Tyr Leu 135 140 145
tac atg gtg atg gaa tac atg cct ggt gga gat ctt gta aac tta atg
1436Tyr Met Val Met Glu Tyr Met Pro Gly Gly Asp Leu Val Asn Leu Met
150 155 160 165 agc aac tat gat gtg cct gaa aaa tgg gca cga ttc tat
act gca gaa 1484Ser Asn Tyr Asp Val Pro Glu Lys Trp Ala Arg Phe Tyr
Thr Ala Glu 170 175 180 gta gtt ctt gca ttg gat gca atc cat tcc atg
ggt ttt att cac aga 1532Val Val Leu Ala Leu Asp Ala Ile His Ser Met
Gly Phe Ile His Arg 185 190 195 gat gtg aag cct gat aac atg ctg ctg
gat aaa tct gga cat ttg aag 1580Asp Val Lys Pro Asp Asn Met Leu Leu
Asp Lys Ser Gly His Leu Lys 200 205 210 tta gca gat ttt ggt act tgt
atg aag atg aat aag gaa ggc atg gta 1628Leu Ala Asp Phe Gly Thr Cys
Met Lys Met Asn Lys Glu Gly Met Val 215 220 225 cga tgt gat aca gcg
gtt gga aca cct gat tat att tcc cct gaa gta 1676Arg Cys Asp Thr Ala
Val Gly Thr Pro Asp Tyr Ile Ser Pro Glu Val 230 235 240 245 tta aaa
tcc caa ggt ggt gat ggt tat tat gga aga gaa tgt gac tgg 1724Leu Lys
Ser Gln Gly Gly Asp Gly Tyr Tyr Gly Arg Glu Cys Asp Trp 250 255 260
tgg tcg gtt ggg gta ttt tta tac gaa atg ctt gta ggt gat aca cct
1772Trp Ser Val Gly Val Phe Leu Tyr Glu Met Leu Val Gly Asp Thr Pro
265 270 275 ttt tat gca gat tct ttg gtt gga act tac agt aaa att atg
aac cat 1820Phe Tyr Ala Asp Ser Leu Val Gly Thr Tyr Ser Lys Ile Met
Asn His 280 285 290 aaa aat tca ctt acc ttt cct gat gat aat gac ata
tca aaa gaa gca 1868Lys Asn Ser Leu Thr Phe Pro Asp Asp Asn Asp Ile
Ser Lys Glu Ala 295 300 305 aaa aac ctt att tgt gcc ttc ctt act gac
agg gaa gtg agg tta ggg 1916Lys Asn Leu Ile Cys Ala Phe Leu Thr Asp
Arg Glu Val Arg Leu Gly 310 315 320 325 cga aat ggt gta gaa gaa atc
aaa cga cat ctc ttc ttc aaa aat gac 1964Arg Asn Gly Val Glu Glu Ile
Lys Arg His Leu Phe Phe Lys Asn Asp 330 335 340 cag tgg gct tgg gaa
acg ctc cga gac act gta gca cca gtt gta ccc 2012Gln Trp Ala Trp Glu
Thr Leu Arg Asp Thr Val Ala Pro Val Val Pro 345 350 355 gat tta agt
agt gac att gat act agt aat ttt gat gac ttg gaa gaa 2060Asp Leu Ser
Ser Asp Ile Asp Thr Ser Asn Phe Asp Asp Leu Glu Glu 360 365 370 gat
aaa gga gag gaa gaa aca ttc cct att cct aaa gct ttc gtt ggc 2108Asp
Lys Gly Glu Glu Glu Thr Phe Pro Ile Pro Lys Ala Phe Val Gly 375 380
385 aat caa cta cct ttt gta gga ttt aca tat tat agc aat cgt aga tac
2156Asn Gln Leu Pro Phe Val Gly Phe Thr Tyr Tyr Ser Asn Arg Arg Tyr
390 395 400 405 tta tct tca gca aat cct aat gat aac aga act agc tcc
aat gca gat 2204Leu Ser Ser Ala Asn Pro Asn Asp Asn Arg Thr Ser Ser
Asn Ala Asp 410 415 420 aaa agc ttg cag gaa agt ttg caa aaa aca atc
tat aag ctg gaa gaa 2252Lys Ser Leu Gln Glu Ser Leu Gln Lys Thr Ile
Tyr Lys Leu Glu Glu 425 430 435 cag ctg cat aat gaa atg cag tta aaa
gat gaa atg gag cag aag tgc 2300Gln Leu His Asn Glu Met Gln Leu Lys
Asp Glu Met Glu Gln Lys Cys 440 445 450 aga acc tca aac ata aaa cta
gac aag ata atg aaa gaa ttg gat gaa 2348Arg Thr Ser Asn Ile Lys Leu
Asp Lys Ile Met Lys Glu Leu Asp Glu 455 460 465 gag gga aat caa aga
aga aat cta gaa tct aca gtg tct cag att gag 2396Glu Gly Asn Gln Arg
Arg Asn Leu Glu Ser Thr Val Ser Gln Ile Glu 470 475 480 485 aag gag
aaa atg ttg cta cag cat aga att aat gag tac caa aga aaa 2444Lys Glu
Lys Met Leu Leu Gln His Arg Ile Asn Glu Tyr Gln Arg Lys 490 495 500
gct gaa cag gaa aat gag aag aga aga aat gta gaa aat gaa gtt tct
2492Ala Glu Gln Glu Asn Glu Lys Arg Arg Asn Val Glu Asn Glu Val Ser
505 510 515 aca tta aag gat cag ttg gaa gac tta aag aaa gtc agt cag
aat tca 2540Thr Leu Lys Asp Gln Leu Glu Asp Leu Lys Lys Val Ser Gln
Asn Ser 520 525 530 cag ctt gct aat gag aag ctg tcc cag tta caa aag
cag cta gaa gaa 2588Gln Leu Ala Asn Glu Lys Leu Ser Gln Leu Gln Lys
Gln Leu Glu Glu 535 540 545 gcc aat gac tta ctt agg aca gaa tcg gac
aca gct gta aga ttg agg 2636Ala Asn Asp Leu Leu Arg Thr Glu Ser Asp
Thr Ala Val Arg Leu Arg 550 555 560 565 aag agt cac aca gag atg agc
aag tca att agt cag tta gag tcc ctg 2684Lys Ser His Thr Glu Met Ser
Lys Ser Ile Ser Gln Leu Glu Ser Leu 570 575 580 aac aga gag ttg caa
gag aga aat cga att tta gag aat tct aag tca 2732Asn Arg Glu Leu Gln
Glu Arg Asn Arg Ile Leu Glu Asn Ser Lys Ser 585 590 595 caa aca gac
aaa gat tat tac cag ctg caa gct ata tta gaa gct gaa 2780Gln Thr Asp
Lys Asp Tyr Tyr Gln Leu Gln Ala Ile Leu Glu Ala Glu 600 605 610 cga
aga gac aga ggt cat gat tct gag atg att gga gac ctt caa gct 2828Arg
Arg Asp Arg Gly His Asp Ser Glu Met Ile Gly Asp Leu Gln Ala 615 620
625 cga att aca tct tta caa gag gag gtg aag cat ctc aaa cat aat ctc
2876Arg Ile Thr Ser Leu Gln Glu Glu Val Lys His Leu Lys His Asn Leu
630 635 640 645 gaa aaa gtg gaa gga gaa aga aaa gag gct caa gac atg
ctt aat cac 2924Glu Lys Val Glu Gly Glu Arg Lys Glu Ala Gln Asp Met
Leu Asn His 650 655 660 tca gaa aag gaa aag aat aat tta gag ata gat
tta aac tac aaa ctt 2972Ser Glu Lys Glu Lys Asn Asn Leu Glu Ile Asp
Leu Asn Tyr Lys Leu 665 670 675 aaa tca tta caa caa cgg tta gaa caa
gag gta aat gaa cac aaa gta 3020Lys Ser Leu Gln Gln Arg Leu Glu Gln
Glu Val Asn Glu His Lys Val 680 685 690 acc aaa gct cgt tta act gac
aaa cat caa tct att gaa gag gca aag 3068Thr Lys Ala Arg Leu Thr Asp
Lys His Gln Ser Ile Glu Glu Ala Lys 695 700 705 tct gtg gca atg tgt
gag atg gaa aaa aag ctg aaa gaa gaa aga gaa 3116Ser Val Ala Met Cys
Glu Met Glu Lys Lys Leu Lys Glu Glu Arg Glu 710 715 720 725 gct cga
gag aag gct gaa aat cgg gtt gtt cag att gag aaa cag tgt 3164Ala Arg
Glu Lys Ala Glu Asn Arg Val Val Gln Ile Glu Lys Gln Cys 730 735 740
tcc atg cta gac gtt gat ctg aag caa tct cag cag aaa cta gaa cat
3212Ser Met Leu Asp Val Asp Leu Lys Gln Ser Gln Gln Lys Leu Glu His
745 750 755 ttg act gga aat aaa gaa agg atg gag gat gaa gtt aag aat
cta acc 3260Leu Thr Gly Asn Lys Glu Arg Met Glu Asp Glu Val Lys Asn
Leu Thr 760 765 770 ctg caa ctg gag cag gaa tca aat aag cgg ctg ttg
tta caa aat gaa 3308Leu Gln Leu Glu Gln Glu Ser Asn Lys Arg Leu Leu
Leu Gln Asn Glu 775 780 785 ttg aag act caa gca ttt gag gca gac aat
tta aaa ggt tta gaa aag 3356Leu Lys Thr Gln Ala Phe Glu Ala Asp Asn
Leu Lys Gly Leu Glu Lys 790 795 800 805 cag atg aaa cag gaa ata aat
act tta ttg gaa gca aag aga tta tta 3404Gln Met Lys Gln Glu Ile Asn
Thr Leu Leu Glu Ala Lys Arg Leu Leu 810 815 820 gaa ttt gag tta gct
cag ctt acg aaa cag tat aga gga aat gaa gga 3452Glu Phe Glu Leu Ala
Gln Leu Thr Lys Gln Tyr Arg Gly Asn Glu Gly 825 830 835 cag atg cgg
gag cta caa gat cag ctt gaa gct gag caa tat ttc tcg 3500Gln Met Arg
Glu Leu Gln Asp Gln Leu Glu Ala Glu Gln Tyr Phe Ser 840 845 850 aca
ctt tat aaa acc cag gta aag gaa ctt aaa gaa gaa att gaa gaa 3548Thr
Leu Tyr Lys Thr Gln Val Lys Glu Leu Lys Glu Glu Ile Glu Glu 855 860
865 aaa aac aga gaa aat tta aag aaa ata cag gaa cta caa aat gaa aaa
3596Lys Asn Arg Glu Asn Leu Lys Lys Ile Gln Glu Leu Gln Asn Glu Lys
870 875 880 885 gaa act ctt gct act cag ttg gat cta gca gaa aca aaa
gct gag tct 3644Glu Thr Leu Ala Thr Gln Leu Asp Leu Ala Glu Thr Lys
Ala Glu Ser 890 895 900 gag cag ttg gcg cga ggc ctt ctg gaa gaa cag
tat ttt gaa ttg acg 3692Glu Gln Leu Ala Arg Gly Leu Leu Glu Glu Gln
Tyr Phe Glu Leu Thr 905 910 915 caa gaa agc aag aaa gct gct tca aga
aat aga caa gag att aca gat 3740Gln Glu Ser Lys Lys Ala Ala Ser Arg
Asn Arg Gln Glu Ile Thr Asp 920 925 930 aaa gat cac act gtt agt cgg
ctt gaa gaa gca aac agc atg cta acc 3788Lys Asp His Thr Val Ser Arg
Leu Glu Glu Ala Asn Ser Met Leu Thr 935 940 945 aaa gat att gaa ata
tta aga aga gag aat gaa gag cta aca gag aaa 3836Lys Asp Ile Glu Ile
Leu Arg Arg Glu Asn Glu Glu Leu Thr Glu Lys 950 955 960 965 atg aag
aag gca gag gaa gaa tat aaa ctg gag aag gag gag gag atc 3884Met Lys
Lys Ala Glu Glu Glu Tyr Lys Leu Glu Lys Glu Glu Glu Ile 970 975 980
agt aat ctt aag gct gcc ttt gaa aag aat atc aac act gaa cga acc
3932Ser Asn Leu Lys Ala Ala Phe Glu Lys Asn Ile Asn Thr Glu Arg Thr
985 990 995 ctt aaa aca cag gct gtt aac aaa ttg gca gaa ata atg aat
cga 3977Leu Lys Thr Gln Ala Val Asn Lys Leu Ala Glu Ile Met Asn Arg
1000 1005 1010 aaa gat ttt aaa att gat aga aag aaa gct aat aca caa
gat ttg 4022Lys Asp Phe Lys Ile Asp Arg Lys Lys Ala Asn Thr Gln Asp
Leu 1015 1020 1025 aga aag aaa gaa aag gaa aat cga aag ctg caa ctg
gaa ctc aac 4067Arg Lys Lys Glu Lys Glu Asn Arg Lys Leu Gln Leu Glu
Leu Asn 1030 1035 1040 caa gaa aga gag aaa ttc aac cag atg gta gtg
aaa cat cag aag 4112Gln Glu Arg Glu Lys Phe Asn Gln Met Val Val Lys
His Gln Lys 1045 1050 1055 gaa ctg aat gac atg caa gcg caa ttg gta
gaa gaa tgt gca cat 4157Glu Leu Asn Asp Met Gln Ala Gln Leu Val Glu
Glu Cys Ala His 1060 1065 1070 agg aat gag ctt cag atg cag ttg gcc
agc aaa gag agt gat att 4202Arg Asn Glu Leu Gln Met Gln Leu Ala Ser
Lys Glu Ser Asp Ile 1075 1080 1085 gag caa ttg cgt gct aaa ctt ttg
gac ctc tcg gat tct aca agt 4247Glu Gln Leu Arg Ala Lys Leu Leu Asp
Leu Ser Asp Ser Thr Ser 1090 1095 1100 gtt gct agt ttt cct agt gct
gat gaa act gat ggt aac ctc cca 4292Val Ala Ser Phe Pro Ser Ala Asp
Glu Thr Asp Gly Asn Leu Pro 1105 1110 1115 gag tca aga att gaa ggt
tgg ctt tca gta cca aat aga gga aat 4337Glu Ser Arg Ile Glu Gly Trp
Leu Ser Val Pro Asn Arg Gly Asn 1120 1125 1130 atc aaa cga tat ggc
tgg aag aaa cag tat gtt gtg gta agc agc 4382Ile Lys Arg Tyr Gly Trp
Lys Lys Gln Tyr Val Val Val Ser Ser 1135 1140 1145 aaa aaa att ttg
ttc tat aat gac gaa caa gat aag gag caa tcc 4427Lys Lys Ile Leu Phe
Tyr Asn Asp Glu Gln Asp Lys Glu Gln Ser 1150 1155 1160 aat cca tct
atg gta ttg gac ata gat aaa ctg ttt cac gtt aga 4472Asn Pro Ser Met
Val Leu Asp Ile Asp Lys Leu Phe His Val Arg 1165 1170 1175 cct gta
acc caa gga gat gtg tat aga gct gaa act gaa gaa att 4517Pro Val Thr
Gln Gly Asp Val Tyr Arg Ala Glu Thr Glu Glu Ile 1180 1185 1190 cct
aaa ata ttc cag ata cta tat gca aat gaa ggt gaa tgt aga 4562Pro Lys
Ile Phe Gln Ile Leu Tyr Ala Asn Glu Gly Glu Cys Arg 1195 1200 1205
aaa gat gta gag atg gaa cca gta caa caa gct gaa aaa act aat 4607Lys
Asp Val Glu Met Glu Pro Val Gln Gln Ala Glu Lys Thr Asn 1210 1215
1220 ttc caa aat cac aaa ggc cat gag ttt att cct aca ctc tac cac
4652Phe Gln Asn His Lys Gly His Glu Phe Ile Pro Thr Leu Tyr His
1225 1230 1235 ttt cct gcc aat tgt gat gcc tgt gcc aaa cct ctc tgg
cat gtt 4697Phe Pro Ala Asn Cys Asp Ala Cys Ala Lys Pro Leu Trp His
Val 1240 1245 1250 ttt aag cca ccc cct gcc cta gag tgt cga aga tgc
cat gtt aag 4742Phe Lys Pro Pro Pro Ala Leu Glu Cys Arg Arg Cys His
Val Lys 1255 1260 1265 tgc cac aga gat cac tta gat aag aaa gag gac
tta att tgt cca 4787Cys His Arg Asp His Leu Asp Lys Lys Glu Asp Leu
Ile Cys Pro 1270 1275 1280 tgt aaa gta agt tat gat gta aca tca
gca
aga gat atg ctg ctg 4832Cys Lys Val Ser Tyr Asp Val Thr Ser Ala Arg
Asp Met Leu Leu 1285 1290 1295 tta gca tgt tct cag gat gaa caa aaa
aaa tgg gta act cat tta 4877Leu Ala Cys Ser Gln Asp Glu Gln Lys Lys
Trp Val Thr His Leu 1300 1305 1310 gta aag aaa atc cct aag aat cca
cca tct ggt ttt gtt cgt gct 4922Val Lys Lys Ile Pro Lys Asn Pro Pro
Ser Gly Phe Val Arg Ala 1315 1320 1325 tcc cct cga acg ctt tct aca
aga tcc act gca aat cag tct ttc 4967Ser Pro Arg Thr Leu Ser Thr Arg
Ser Thr Ala Asn Gln Ser Phe 1330 1335 1340 cgg aaa gtg gtc aaa aat
aca tct gga aaa act agt taa ccatgtgact 5016Arg Lys Val Val Lys Asn
Thr Ser Gly Lys Thr Ser 1345 1350 gagtgccctg tggaatcgtg tgggatgcta
cctgataaac caggcttctt taaccatgca 5076gagcagacag gctgtttctt
tgacacaaat atcacaggct tcagggttaa gattgctgtt 5136tttctgtcct
tgctttggca caacacactg agggtttttt ttattgcggg tttgcctaca
5196ggtagattag attaattatt actatgtaat gcaagtacag ttgggggaaa
gcttaggtag 5256atatattttt tttaaaaggt gctgcctttt tggatttata
agaaaatgcc tgtcagtcgt 5316gatagaacag agttttcctc atatgagtaa
gaggaaggga ctttcacttt caagtggaac 5376agccatcact atcaagatca
gctcatggaa ggagtaaaga aaatatctca aaatgagaca 5436aactgaagtt
ttgttttttt tttaatgact taagtttttg tgctcttgca agactataca
5496aaactatttt aagaaagcag tgatatcact tgaacttcag tgccctcact
gtagaattta 5556aaagccttac tgttgattgc ccatgttgga cttgatggag
aaattaaata tctttcatta 5616tgctttacaa aatactgtat atgtttcagc
aagtttgggg aatgggagag gacaaaaaaa 5676agttacattt aatctatgca
tttttgccaa gccatattga gttattttac tactagagac 5736attaggaaac
taactgtaca aaagaaccaa gtttaaaagc attttgtggg gtacatcatt
5796tctataattg tataatgtat ttctttgtgg ttttaaatga taaagacatt
aagttaacaa 5856acatataaga aatgtatgca ctgtttgaaa tgtaaattat
tcttagaaca ctttcaatgg 5916gggttgcatt gtccttttag tgccttaatt
tgagataatt attttactgc catgagtaag 5976tatagaaatt tcaaaaaatg
tattttcaaa aaattatgtg tgtcagtgag tttttcattg 6036ataattggtt
taatttaaaa tatttagagg tttgttggac tttcataaat tgagtacaat
6096ctttgcatca aactacctgc tacaataatg actttataaa actgcaaaaa
atgtagaagg 6156ttgcaccaac ataaaaagga aatatggcaa tacatccatg
atgttttcca gttaacatag 6216gaattaccag ataaatactg ttaaactctt
gtccagtaac aagagttgat tcatatggac 6276agtatgattt attgtttatt
tttttaacca aatacctcct cagtaattta taatggcttt 6336gcagtaatgt
gtatcagata agaagcactg gaaaaccgat cgtctctagg atgatatgca
6396tgtttcaagt ggtattgaaa gccgcactga tggatatgta ataataaaca
tatctgttat 6456taatatacta atgactctgt gctcatttaa tgagaaataa
aagtaattta tggatgggta 6516tctttaattt ttactgcaat gtgttttctc
atggctgaaa tgaatggaaa acatacttca 6576aattagtctc tgattgtata
taaatgtttg tgaaattcca tggttagatt aaagtgtatt 6636tttaaaagat aaaa
665021354PRTHomo sapiens 2Met Ser Thr Gly Asp Ser Phe Glu Thr Arg
Phe Glu Lys Met Asp Asn 1 5 10 15 Leu Leu Arg Asp Pro Lys Ser Glu
Val Asn Ser Asp Cys Leu Leu Asp 20 25 30 Gly Leu Asp Ala Leu Val
Tyr Asp Leu Asp Phe Pro Ala Leu Arg Lys 35 40 45 Asn Lys Asn Ile
Asp Asn Phe Leu Ser Arg Tyr Lys Asp Thr Ile Asn 50 55 60 Lys Ile
Arg Asp Leu Arg Met Lys Ala Glu Asp Tyr Glu Val Val Lys 65 70 75 80
Val Ile Gly Arg Gly Ala Phe Gly Glu Val Gln Leu Val Arg His Lys 85
90 95 Ser Thr Arg Lys Val Tyr Ala Met Lys Leu Leu Ser Lys Phe Glu
Met 100 105 110 Ile Lys Arg Ser Asp Ser Ala Phe Phe Trp Glu Glu Arg
Asp Ile Met 115 120 125 Ala Phe Ala Asn Ser Pro Trp Val Val Gln Leu
Phe Tyr Ala Phe Gln 130 135 140 Asp Asp Arg Tyr Leu Tyr Met Val Met
Glu Tyr Met Pro Gly Gly Asp 145 150 155 160 Leu Val Asn Leu Met Ser
Asn Tyr Asp Val Pro Glu Lys Trp Ala Arg 165 170 175 Phe Tyr Thr Ala
Glu Val Val Leu Ala Leu Asp Ala Ile His Ser Met 180 185 190 Gly Phe
Ile His Arg Asp Val Lys Pro Asp Asn Met Leu Leu Asp Lys 195 200 205
Ser Gly His Leu Lys Leu Ala Asp Phe Gly Thr Cys Met Lys Met Asn 210
215 220 Lys Glu Gly Met Val Arg Cys Asp Thr Ala Val Gly Thr Pro Asp
Tyr 225 230 235 240 Ile Ser Pro Glu Val Leu Lys Ser Gln Gly Gly Asp
Gly Tyr Tyr Gly 245 250 255 Arg Glu Cys Asp Trp Trp Ser Val Gly Val
Phe Leu Tyr Glu Met Leu 260 265 270 Val Gly Asp Thr Pro Phe Tyr Ala
Asp Ser Leu Val Gly Thr Tyr Ser 275 280 285 Lys Ile Met Asn His Lys
Asn Ser Leu Thr Phe Pro Asp Asp Asn Asp 290 295 300 Ile Ser Lys Glu
Ala Lys Asn Leu Ile Cys Ala Phe Leu Thr Asp Arg 305 310 315 320 Glu
Val Arg Leu Gly Arg Asn Gly Val Glu Glu Ile Lys Arg His Leu 325 330
335 Phe Phe Lys Asn Asp Gln Trp Ala Trp Glu Thr Leu Arg Asp Thr Val
340 345 350 Ala Pro Val Val Pro Asp Leu Ser Ser Asp Ile Asp Thr Ser
Asn Phe 355 360 365 Asp Asp Leu Glu Glu Asp Lys Gly Glu Glu Glu Thr
Phe Pro Ile Pro 370 375 380 Lys Ala Phe Val Gly Asn Gln Leu Pro Phe
Val Gly Phe Thr Tyr Tyr 385 390 395 400 Ser Asn Arg Arg Tyr Leu Ser
Ser Ala Asn Pro Asn Asp Asn Arg Thr 405 410 415 Ser Ser Asn Ala Asp
Lys Ser Leu Gln Glu Ser Leu Gln Lys Thr Ile 420 425 430 Tyr Lys Leu
Glu Glu Gln Leu His Asn Glu Met Gln Leu Lys Asp Glu 435 440 445 Met
Glu Gln Lys Cys Arg Thr Ser Asn Ile Lys Leu Asp Lys Ile Met 450 455
460 Lys Glu Leu Asp Glu Glu Gly Asn Gln Arg Arg Asn Leu Glu Ser Thr
465 470 475 480 Val Ser Gln Ile Glu Lys Glu Lys Met Leu Leu Gln His
Arg Ile Asn 485 490 495 Glu Tyr Gln Arg Lys Ala Glu Gln Glu Asn Glu
Lys Arg Arg Asn Val 500 505 510 Glu Asn Glu Val Ser Thr Leu Lys Asp
Gln Leu Glu Asp Leu Lys Lys 515 520 525 Val Ser Gln Asn Ser Gln Leu
Ala Asn Glu Lys Leu Ser Gln Leu Gln 530 535 540 Lys Gln Leu Glu Glu
Ala Asn Asp Leu Leu Arg Thr Glu Ser Asp Thr 545 550 555 560 Ala Val
Arg Leu Arg Lys Ser His Thr Glu Met Ser Lys Ser Ile Ser 565 570 575
Gln Leu Glu Ser Leu Asn Arg Glu Leu Gln Glu Arg Asn Arg Ile Leu 580
585 590 Glu Asn Ser Lys Ser Gln Thr Asp Lys Asp Tyr Tyr Gln Leu Gln
Ala 595 600 605 Ile Leu Glu Ala Glu Arg Arg Asp Arg Gly His Asp Ser
Glu Met Ile 610 615 620 Gly Asp Leu Gln Ala Arg Ile Thr Ser Leu Gln
Glu Glu Val Lys His 625 630 635 640 Leu Lys His Asn Leu Glu Lys Val
Glu Gly Glu Arg Lys Glu Ala Gln 645 650 655 Asp Met Leu Asn His Ser
Glu Lys Glu Lys Asn Asn Leu Glu Ile Asp 660 665 670 Leu Asn Tyr Lys
Leu Lys Ser Leu Gln Gln Arg Leu Glu Gln Glu Val 675 680 685 Asn Glu
His Lys Val Thr Lys Ala Arg Leu Thr Asp Lys His Gln Ser 690 695 700
Ile Glu Glu Ala Lys Ser Val Ala Met Cys Glu Met Glu Lys Lys Leu 705
710 715 720 Lys Glu Glu Arg Glu Ala Arg Glu Lys Ala Glu Asn Arg Val
Val Gln 725 730 735 Ile Glu Lys Gln Cys Ser Met Leu Asp Val Asp Leu
Lys Gln Ser Gln 740 745 750 Gln Lys Leu Glu His Leu Thr Gly Asn Lys
Glu Arg Met Glu Asp Glu 755 760 765 Val Lys Asn Leu Thr Leu Gln Leu
Glu Gln Glu Ser Asn Lys Arg Leu 770 775 780 Leu Leu Gln Asn Glu Leu
Lys Thr Gln Ala Phe Glu Ala Asp Asn Leu 785 790 795 800 Lys Gly Leu
Glu Lys Gln Met Lys Gln Glu Ile Asn Thr Leu Leu Glu 805 810 815 Ala
Lys Arg Leu Leu Glu Phe Glu Leu Ala Gln Leu Thr Lys Gln Tyr 820 825
830 Arg Gly Asn Glu Gly Gln Met Arg Glu Leu Gln Asp Gln Leu Glu Ala
835 840 845 Glu Gln Tyr Phe Ser Thr Leu Tyr Lys Thr Gln Val Lys Glu
Leu Lys 850 855 860 Glu Glu Ile Glu Glu Lys Asn Arg Glu Asn Leu Lys
Lys Ile Gln Glu 865 870 875 880 Leu Gln Asn Glu Lys Glu Thr Leu Ala
Thr Gln Leu Asp Leu Ala Glu 885 890 895 Thr Lys Ala Glu Ser Glu Gln
Leu Ala Arg Gly Leu Leu Glu Glu Gln 900 905 910 Tyr Phe Glu Leu Thr
Gln Glu Ser Lys Lys Ala Ala Ser Arg Asn Arg 915 920 925 Gln Glu Ile
Thr Asp Lys Asp His Thr Val Ser Arg Leu Glu Glu Ala 930 935 940 Asn
Ser Met Leu Thr Lys Asp Ile Glu Ile Leu Arg Arg Glu Asn Glu 945 950
955 960 Glu Leu Thr Glu Lys Met Lys Lys Ala Glu Glu Glu Tyr Lys Leu
Glu 965 970 975 Lys Glu Glu Glu Ile Ser Asn Leu Lys Ala Ala Phe Glu
Lys Asn Ile 980 985 990 Asn Thr Glu Arg Thr Leu Lys Thr Gln Ala Val
Asn Lys Leu Ala Glu 995 1000 1005 Ile Met Asn Arg Lys Asp Phe Lys
Ile Asp Arg Lys Lys Ala Asn 1010 1015 1020 Thr Gln Asp Leu Arg Lys
Lys Glu Lys Glu Asn Arg Lys Leu Gln 1025 1030 1035 Leu Glu Leu Asn
Gln Glu Arg Glu Lys Phe Asn Gln Met Val Val 1040 1045 1050 Lys His
Gln Lys Glu Leu Asn Asp Met Gln Ala Gln Leu Val Glu 1055 1060 1065
Glu Cys Ala His Arg Asn Glu Leu Gln Met Gln Leu Ala Ser Lys 1070
1075 1080 Glu Ser Asp Ile Glu Gln Leu Arg Ala Lys Leu Leu Asp Leu
Ser 1085 1090 1095 Asp Ser Thr Ser Val Ala Ser Phe Pro Ser Ala Asp
Glu Thr Asp 1100 1105 1110 Gly Asn Leu Pro Glu Ser Arg Ile Glu Gly
Trp Leu Ser Val Pro 1115 1120 1125 Asn Arg Gly Asn Ile Lys Arg Tyr
Gly Trp Lys Lys Gln Tyr Val 1130 1135 1140 Val Val Ser Ser Lys Lys
Ile Leu Phe Tyr Asn Asp Glu Gln Asp 1145 1150 1155 Lys Glu Gln Ser
Asn Pro Ser Met Val Leu Asp Ile Asp Lys Leu 1160 1165 1170 Phe His
Val Arg Pro Val Thr Gln Gly Asp Val Tyr Arg Ala Glu 1175 1180 1185
Thr Glu Glu Ile Pro Lys Ile Phe Gln Ile Leu Tyr Ala Asn Glu 1190
1195 1200 Gly Glu Cys Arg Lys Asp Val Glu Met Glu Pro Val Gln Gln
Ala 1205 1210 1215 Glu Lys Thr Asn Phe Gln Asn His Lys Gly His Glu
Phe Ile Pro 1220 1225 1230 Thr Leu Tyr His Phe Pro Ala Asn Cys Asp
Ala Cys Ala Lys Pro 1235 1240 1245 Leu Trp His Val Phe Lys Pro Pro
Pro Ala Leu Glu Cys Arg Arg 1250 1255 1260 Cys His Val Lys Cys His
Arg Asp His Leu Asp Lys Lys Glu Asp 1265 1270 1275 Leu Ile Cys Pro
Cys Lys Val Ser Tyr Asp Val Thr Ser Ala Arg 1280 1285 1290 Asp Met
Leu Leu Leu Ala Cys Ser Gln Asp Glu Gln Lys Lys Trp 1295 1300 1305
Val Thr His Leu Val Lys Lys Ile Pro Lys Asn Pro Pro Ser Gly 1310
1315 1320 Phe Val Arg Ala Ser Pro Arg Thr Leu Ser Thr Arg Ser Thr
Ala 1325 1330 1335 Asn Gln Ser Phe Arg Lys Val Val Lys Asn Thr Ser
Gly Lys Thr 1340 1345 1350 Ser 36401DNAHomo sapiensCDS(450)..(4616)
3caaggcggcc ggcggcgacc atggcagcgg gccggcggcg gccgtagtgg cccaggcctg
60ggcttcagcc tcccggggcc ccagagggcg gggcggtccg ggccgcggcg gtggcggcgc
120cacttccctg ctcccgcccg aggactcctg cgggcactcg ctgaggacca
gcggaccggc 180ggcgcgaatc tgactgaggg gcggggacgc cgtctgttcc
ccgccgctcc cggcagggcc 240gggccgggct gggccgggct gggccgggcg
ggcccctggg agcagccccc aggcggggga 300ccgccttgga gacccgaagc
cggagctaga ggcaggcggt gggcccgggt ggagtcccgg 360ccggagctgg
tggttcgggg gcggtgctag gccccgaggc tgcgggacct gagcgcgagg
420agcctgagtg cgggtccagc ggtggcggc atg agc cgg ccc ccg ccg acg ggg
473 Met Ser Arg Pro Pro Pro Thr Gly 1 5 aaa atg ccc ggc gcc ccc gag
acc gcg ccg ggg gac ggg gca ggc gcg 521Lys Met Pro Gly Ala Pro Glu
Thr Ala Pro Gly Asp Gly Ala Gly Ala 10 15 20 agc cgc cag agg aag
ctg gag gcg ctg atc cga gac cct cgc tcc ccc 569Ser Arg Gln Arg Lys
Leu Glu Ala Leu Ile Arg Asp Pro Arg Ser Pro 25 30 35 40 atc aac gtg
gag agc ttg ctg gat ggc tta aat tcc ttg gtc ctt gat 617Ile Asn Val
Glu Ser Leu Leu Asp Gly Leu Asn Ser Leu Val Leu Asp 45 50 55 tta
gat ttt cct gct ttg agg aaa aac aag aac ata gat aat ttc tta 665Leu
Asp Phe Pro Ala Leu Arg Lys Asn Lys Asn Ile Asp Asn Phe Leu 60 65
70 aat aga tat gag aaa att gtg aaa aaa atc aga ggt cta cag atg aag
713Asn Arg Tyr Glu Lys Ile Val Lys Lys Ile Arg Gly Leu Gln Met Lys
75 80 85 gca gaa gac tat gat gtt gta aaa gtt att gga aga ggt gct
ttt ggt 761Ala Glu Asp Tyr Asp Val Val Lys Val Ile Gly Arg Gly Ala
Phe Gly 90 95 100 gaa gtg cag ttg gtt cgt cac aag gca tcg cag aag
gtt tat gct atg 809Glu Val Gln Leu Val Arg His Lys Ala Ser Gln Lys
Val Tyr Ala Met 105 110 115 120 aag ctt ctt agt aag ttt gaa atg ata
aaa aga tca gat tct gcc ttt 857Lys Leu Leu Ser Lys Phe Glu Met Ile
Lys Arg Ser Asp Ser Ala Phe 125 130 135 ttt tgg gaa gaa aga gat att
atg gcc ttt gcc aat agc ccc tgg gtg 905Phe Trp Glu Glu Arg Asp Ile
Met Ala Phe Ala Asn Ser Pro Trp Val 140 145 150 gtt cag ctt ttt tat
gcc ttt caa gat gat agg tat ctg tac atg gta 953Val Gln Leu Phe Tyr
Ala Phe Gln Asp Asp Arg Tyr Leu Tyr Met Val 155 160 165 atg gag tac
atg cct ggt gga gac ctt gta aac ctt atg agt aat tat 1001Met Glu Tyr
Met Pro Gly Gly Asp Leu Val Asn Leu Met Ser Asn Tyr 170 175 180 gat
gtg cct gaa aaa tgg gcc aaa ttt tac act gct gaa gtt gtt ctt 1049Asp
Val Pro Glu Lys Trp Ala Lys Phe Tyr Thr Ala Glu Val Val Leu 185 190
195 200 gct ctg gat gca ata cac tcc atg ggt tta ata cac aga gat gtg
aag 1097Ala Leu Asp Ala Ile His Ser Met Gly Leu Ile His Arg Asp Val
Lys 205 210 215 cct gac aac atg ctc ttg gat aaa cat gga cat cta aaa
tta gca gat 1145Pro Asp Asn Met Leu Leu Asp Lys His Gly His Leu Lys
Leu Ala Asp 220 225 230 ttt ggc acg tgt atg aag atg gat gaa aca ggc
atg gta cat tgt gat 1193Phe Gly Thr Cys Met Lys Met Asp Glu Thr Gly
Met Val His Cys Asp 235 240 245 aca gca gtt gga aca ccg gat tat ata
tca cct gag gtt ctg aaa tca 1241Thr Ala Val Gly Thr Pro Asp Tyr Ile
Ser Pro Glu Val Leu Lys Ser 250 255 260 caa ggg ggt gat ggt ttc tat
ggg cga gaa tgt gat tgg tgg tct gta 1289Gln Gly Gly Asp Gly Phe Tyr
Gly Arg Glu Cys Asp Trp Trp Ser Val 265 270 275 280 ggt gtt ttc ctt
tat gag atg cta gtg ggg gat act cca ttt tat gcg 1337Gly Val Phe Leu
Tyr
Glu Met Leu Val Gly Asp Thr Pro Phe Tyr Ala 285 290 295 gat tca ctt
gta gga aca tat agc aaa att atg gat cat aag aat tca 1385Asp Ser Leu
Val Gly Thr Tyr Ser Lys Ile Met Asp His Lys Asn Ser 300 305 310 ctg
tgt ttc cct gaa gat gca gaa att tcc aaa cat gca aag aat ctc 1433Leu
Cys Phe Pro Glu Asp Ala Glu Ile Ser Lys His Ala Lys Asn Leu 315 320
325 atc tgt gct ttc tta aca gat agg gag gta cga ctt ggg aga aat ggg
1481Ile Cys Ala Phe Leu Thr Asp Arg Glu Val Arg Leu Gly Arg Asn Gly
330 335 340 gtg gaa gaa atc aga cag cat cct ttc ttt aag aat gat cag
tgg cat 1529Val Glu Glu Ile Arg Gln His Pro Phe Phe Lys Asn Asp Gln
Trp His 345 350 355 360 tgg gat aac ata aga gaa acg gca gct cct gta
gta cct gaa ctc agc 1577Trp Asp Asn Ile Arg Glu Thr Ala Ala Pro Val
Val Pro Glu Leu Ser 365 370 375 agt gac ata gac agc agc aat ttc gat
gac att gaa gat gac aaa gga 1625Ser Asp Ile Asp Ser Ser Asn Phe Asp
Asp Ile Glu Asp Asp Lys Gly 380 385 390 gat gta gaa acc ttc cca att
cct aaa gct ttt gtt gga aat cag ctg 1673Asp Val Glu Thr Phe Pro Ile
Pro Lys Ala Phe Val Gly Asn Gln Leu 395 400 405 cct ttc atc gga ttt
acc tac tat aga gaa aat tta tta tta agt gac 1721Pro Phe Ile Gly Phe
Thr Tyr Tyr Arg Glu Asn Leu Leu Leu Ser Asp 410 415 420 tct cca tct
tgt aga gaa act gat tcc ata caa tca agg aaa aat gaa 1769Ser Pro Ser
Cys Arg Glu Thr Asp Ser Ile Gln Ser Arg Lys Asn Glu 425 430 435 440
gaa agt caa gag att cag aaa aaa ctg tat aca tta gaa gaa cat ctt
1817Glu Ser Gln Glu Ile Gln Lys Lys Leu Tyr Thr Leu Glu Glu His Leu
445 450 455 agc aat gag atg caa gcc aaa gag gaa ctg gaa cag aag tgc
aaa tct 1865Ser Asn Glu Met Gln Ala Lys Glu Glu Leu Glu Gln Lys Cys
Lys Ser 460 465 470 gtt aat act cgc cta gaa aaa aca gca aag gag cta
gaa gag gag att 1913Val Asn Thr Arg Leu Glu Lys Thr Ala Lys Glu Leu
Glu Glu Glu Ile 475 480 485 acc tta cgg aaa agt gtg gaa tca gca tta
aga cag tta gaa aga gaa 1961Thr Leu Arg Lys Ser Val Glu Ser Ala Leu
Arg Gln Leu Glu Arg Glu 490 495 500 aag gcg ctt ctt cag cac aaa aat
gca gaa tat cag agg aaa gct gat 2009Lys Ala Leu Leu Gln His Lys Asn
Ala Glu Tyr Gln Arg Lys Ala Asp 505 510 515 520 cat gaa gca gac aaa
aaa cga aat ttg gaa aat gat gtt aac agc tta 2057His Glu Ala Asp Lys
Lys Arg Asn Leu Glu Asn Asp Val Asn Ser Leu 525 530 535 aaa gat caa
ctt gaa gat ttg aaa aaa aga aat caa aac tct caa ata 2105Lys Asp Gln
Leu Glu Asp Leu Lys Lys Arg Asn Gln Asn Ser Gln Ile 540 545 550 tcc
act gag aaa gtg aat caa ctc cag aga caa ctg gat gaa acc aat 2153Ser
Thr Glu Lys Val Asn Gln Leu Gln Arg Gln Leu Asp Glu Thr Asn 555 560
565 gct tta ctg cga aca gag tct gat act gca gcc cgg tta agg aaa acc
2201Ala Leu Leu Arg Thr Glu Ser Asp Thr Ala Ala Arg Leu Arg Lys Thr
570 575 580 cag gca gaa agt tca aaa cag att cag cag ctg gaa tct aac
aat aga 2249Gln Ala Glu Ser Ser Lys Gln Ile Gln Gln Leu Glu Ser Asn
Asn Arg 585 590 595 600 gat cta caa gat aaa aac tgc ctg ctg gag act
gcc aag tta aaa ctt 2297Asp Leu Gln Asp Lys Asn Cys Leu Leu Glu Thr
Ala Lys Leu Lys Leu 605 610 615 gaa aag gaa ttt atc aat ctt cag tca
gct cta gaa tct gaa agg agg 2345Glu Lys Glu Phe Ile Asn Leu Gln Ser
Ala Leu Glu Ser Glu Arg Arg 620 625 630 gat cga acc cat gga tca gag
ata att aat gat tta caa ggt aga ata 2393Asp Arg Thr His Gly Ser Glu
Ile Ile Asn Asp Leu Gln Gly Arg Ile 635 640 645 tgt ggc cta gaa gaa
gat tta aag aac ggc aaa atc tta cta gcg aaa 2441Cys Gly Leu Glu Glu
Asp Leu Lys Asn Gly Lys Ile Leu Leu Ala Lys 650 655 660 gta gaa ctg
gag aag aga caa ctt cag gag aga ttt act gat ttg gaa 2489Val Glu Leu
Glu Lys Arg Gln Leu Gln Glu Arg Phe Thr Asp Leu Glu 665 670 675 680
aag gaa aaa agc aac atg gaa ata gat atg aca tac caa cta aaa gtt
2537Lys Glu Lys Ser Asn Met Glu Ile Asp Met Thr Tyr Gln Leu Lys Val
685 690 695 ata cag cag agc cta gaa caa gaa gaa gct gaa cat aag gcc
aca aag 2585Ile Gln Gln Ser Leu Glu Gln Glu Glu Ala Glu His Lys Ala
Thr Lys 700 705 710 gca cga cta gca gat aaa aat aag atc tat gag tcc
atc gaa gaa gcc 2633Ala Arg Leu Ala Asp Lys Asn Lys Ile Tyr Glu Ser
Ile Glu Glu Ala 715 720 725 aaa tca gaa gcc atg aaa gaa atg gag aag
aag ctc ttg gag gaa aga 2681Lys Ser Glu Ala Met Lys Glu Met Glu Lys
Lys Leu Leu Glu Glu Arg 730 735 740 act tta aaa cag aaa gtg gag aac
cta ttg cta gaa gct gag aaa aga 2729Thr Leu Lys Gln Lys Val Glu Asn
Leu Leu Leu Glu Ala Glu Lys Arg 745 750 755 760 tgt tct cta tta gac
tgt gac ctc aaa cag tca cag cag aaa ata aat 2777Cys Ser Leu Leu Asp
Cys Asp Leu Lys Gln Ser Gln Gln Lys Ile Asn 765 770 775 gag ctc ctt
aaa cag aaa gat gtg cta aat gag gat gtt aga aac ctg 2825Glu Leu Leu
Lys Gln Lys Asp Val Leu Asn Glu Asp Val Arg Asn Leu 780 785 790 aca
tta aaa ata gag caa gaa act cag aag cgc tgc ctt aca caa aat 2873Thr
Leu Lys Ile Glu Gln Glu Thr Gln Lys Arg Cys Leu Thr Gln Asn 795 800
805 gac ctg aag atg caa aca caa cag gtt aac aca cta aaa atg tca gaa
2921Asp Leu Lys Met Gln Thr Gln Gln Val Asn Thr Leu Lys Met Ser Glu
810 815 820 aag cag tta aag caa gaa aat aac cat ctc atg gaa atg aaa
atg aac 2969Lys Gln Leu Lys Gln Glu Asn Asn His Leu Met Glu Met Lys
Met Asn 825 830 835 840 ttg gaa aaa caa aat gct gaa ctt cga aaa gaa
cgt cag gat gca gat 3017Leu Glu Lys Gln Asn Ala Glu Leu Arg Lys Glu
Arg Gln Asp Ala Asp 845 850 855 ggg caa atg aaa gag ctc cag gat cag
ctc gaa gca gaa cag tat ttc 3065Gly Gln Met Lys Glu Leu Gln Asp Gln
Leu Glu Ala Glu Gln Tyr Phe 860 865 870 tca acc ctt tat aaa aca caa
gtt agg gag ctt aaa gaa gaa tgt gaa 3113Ser Thr Leu Tyr Lys Thr Gln
Val Arg Glu Leu Lys Glu Glu Cys Glu 875 880 885 gaa aag acc aaa ctt
ggt aaa gaa ttg cag cag aag aaa cag gaa tta 3161Glu Lys Thr Lys Leu
Gly Lys Glu Leu Gln Gln Lys Lys Gln Glu Leu 890 895 900 cag gat gaa
cgg gac tct ttg gct gcc caa ctg gag atc acc ttg acc 3209Gln Asp Glu
Arg Asp Ser Leu Ala Ala Gln Leu Glu Ile Thr Leu Thr 905 910 915 920
aaa gca gat tct gag caa ctg gct cgt tca att gct gaa gaa caa tat
3257Lys Ala Asp Ser Glu Gln Leu Ala Arg Ser Ile Ala Glu Glu Gln Tyr
925 930 935 tct gat ttg gaa aaa gag aag atc atg aaa gag ctg gag atc
aaa gag 3305Ser Asp Leu Glu Lys Glu Lys Ile Met Lys Glu Leu Glu Ile
Lys Glu 940 945 950 atg atg gct aga cac aaa cag gaa ctt acg gaa aaa
gat gct aca att 3353Met Met Ala Arg His Lys Gln Glu Leu Thr Glu Lys
Asp Ala Thr Ile 955 960 965 gct tct ctt gag gaa act aat agg aca cta
act agt gat gtt gcc aat 3401Ala Ser Leu Glu Glu Thr Asn Arg Thr Leu
Thr Ser Asp Val Ala Asn 970 975 980 ctt gca aat gag aaa gaa gaa tta
aat aac aaa ttg aaa gat gtt caa 3449Leu Ala Asn Glu Lys Glu Glu Leu
Asn Asn Lys Leu Lys Asp Val Gln 985 990 995 1000 gag caa ctg tca
aga ttg aaa gat gaa gaa ata agc gca gca gct 3494Glu Gln Leu Ser Arg
Leu Lys Asp Glu Glu Ile Ser Ala Ala Ala 1005 1010 1015 att aaa gca
cag ttt gag aag cag cta tta aca gaa aga aca ctc 3539Ile Lys Ala Gln
Phe Glu Lys Gln Leu Leu Thr Glu Arg Thr Leu 1020 1025 1030 aaa act
caa gct gtg aat aag ttg gct gag atc atg aat cga aaa 3584Lys Thr Gln
Ala Val Asn Lys Leu Ala Glu Ile Met Asn Arg Lys 1035 1040 1045 gaa
cct gtc aag cgt ggt aat gac aca gat gtg cgg aga aaa gag 3629Glu Pro
Val Lys Arg Gly Asn Asp Thr Asp Val Arg Arg Lys Glu 1050 1055 1060
aag gag aat aga aag cta cat atg gag ctt aaa tct gaa cgt gag 3674Lys
Glu Asn Arg Lys Leu His Met Glu Leu Lys Ser Glu Arg Glu 1065 1070
1075 aaa ttg acc cag cag atg atc aag tat cag aaa gaa ctg aat gaa
3719Lys Leu Thr Gln Gln Met Ile Lys Tyr Gln Lys Glu Leu Asn Glu
1080 1085 1090 atg cag gca caa ata gct gaa gag agc cag att cga att
gaa ctg 3764Met Gln Ala Gln Ile Ala Glu Glu Ser Gln Ile Arg Ile Glu
Leu 1095 1100 1105 cag atg aca ttg gac agt aaa gac agt gac att gag
cag ctg cgg 3809Gln Met Thr Leu Asp Ser Lys Asp Ser Asp Ile Glu Gln
Leu Arg 1110 1115 1120 tca caa ctc caa gcc ttg cat att ggt ctg gat
agt tcc agt ata 3854Ser Gln Leu Gln Ala Leu His Ile Gly Leu Asp Ser
Ser Ser Ile 1125 1130 1135 ggc agt gga cca ggg gat gct gag gca gat
gat ggg ttt cca gaa 3899Gly Ser Gly Pro Gly Asp Ala Glu Ala Asp Asp
Gly Phe Pro Glu 1140 1145 1150 tca aga tta gaa gga tgg ctt tca ttg
cct gta cga aac aac act 3944Ser Arg Leu Glu Gly Trp Leu Ser Leu Pro
Val Arg Asn Asn Thr 1155 1160 1165 aag aaa ttt gga tgg gtt aaa aag
tat gtg att gta agc agt aag 3989Lys Lys Phe Gly Trp Val Lys Lys Tyr
Val Ile Val Ser Ser Lys 1170 1175 1180 aag att ctt ttc tat gac agt
gaa caa gat aaa gaa caa tcc aat 4034Lys Ile Leu Phe Tyr Asp Ser Glu
Gln Asp Lys Glu Gln Ser Asn 1185 1190 1195 cct tac atg gtt tta gat
ata gac aag tta ttt cat gtc cga cca 4079Pro Tyr Met Val Leu Asp Ile
Asp Lys Leu Phe His Val Arg Pro 1200 1205 1210 gtt aca cag aca gat
gtg tat aga gca gat gct aaa gaa att cca 4124Val Thr Gln Thr Asp Val
Tyr Arg Ala Asp Ala Lys Glu Ile Pro 1215 1220 1225 agg ata ttc cag
att ctg tat gcc aat gaa gga gaa agt aag aag 4169Arg Ile Phe Gln Ile
Leu Tyr Ala Asn Glu Gly Glu Ser Lys Lys 1230 1235 1240 gaa caa gaa
ttt cca gtg gag cca gtt gga gaa aaa tct aat tat 4214Glu Gln Glu Phe
Pro Val Glu Pro Val Gly Glu Lys Ser Asn Tyr 1245 1250 1255 att tgc
cac aag gga cat gag ttt att cct act ctt tat cat ttc 4259Ile Cys His
Lys Gly His Glu Phe Ile Pro Thr Leu Tyr His Phe 1260 1265 1270 cca
acc aac tgt gag gct tgt atg aag ccc ctg tgg cac atg ttt 4304Pro Thr
Asn Cys Glu Ala Cys Met Lys Pro Leu Trp His Met Phe 1275 1280 1285
aag cct cct cct gct ttg gag tgc cgc cgt tgc cat att aag tgt 4349Lys
Pro Pro Pro Ala Leu Glu Cys Arg Arg Cys His Ile Lys Cys 1290 1295
1300 cat aaa gat cat atg gac aaa aag gag gag att ata gca cct tgc
4394His Lys Asp His Met Asp Lys Lys Glu Glu Ile Ile Ala Pro Cys
1305 1310 1315 aaa gta tat tat gat att tca acg gca aag aat ctg tta
tta cta 4439Lys Val Tyr Tyr Asp Ile Ser Thr Ala Lys Asn Leu Leu Leu
Leu 1320 1325 1330 gca aat tct aca gaa gag cag cag aag tgg gtt agt
cgg ttg gtg 4484Ala Asn Ser Thr Glu Glu Gln Gln Lys Trp Val Ser Arg
Leu Val 1335 1340 1345 aaa aag ata cct aaa aag ccc cca gct cca gac
cct ttt gcc cga 4529Lys Lys Ile Pro Lys Lys Pro Pro Ala Pro Asp Pro
Phe Ala Arg 1350 1355 1360 tca tct cct aga act tca atg aag ata cag
caa aac cag tct att 4574Ser Ser Pro Arg Thr Ser Met Lys Ile Gln Gln
Asn Gln Ser Ile 1365 1370 1375 aga cgg cca agt cga cag ctt gcc cca
aac aaa cct agc taa 4616Arg Arg Pro Ser Arg Gln Leu Ala Pro Asn Lys
Pro Ser 1380 1385 ctgccttcta tgaaagcagt cattattcaa ggtgatcgta
ttcttccagt gaaaacaaga 4676ctgaaatatg atggcccaaa atttattaaa
aagctatatt ttcctgagag actgatacat 4736acactcatac atatatgtgt
tccccttttc cctgtaatat aaattacaaa tctgggctcc 4796tttgaagcaa
caggttgaac caacaatgat tggttgatag actaaggata tatgcaactc
4856ttccagactt ttccataaag ctctctcggc agtcgctcac actacaatgc
acacaaggat 4916tgagaagagt taaaggctaa agaaaacatc ttttctagct
tcaacagaga ggtttcacca 4976gcacatttac cagaagaatc tgggaatgga
ttccactaca gtgatattga ctgcatcttt 5036aagaagtgac cattatactg
tgtatatata tataaacaca cacacatata tatatatata 5096tatagtactc
taatactgca agaaggtttt ttaaacttcc cactttattt tttatacaca
5156ttaatcagat atcattactt gctgcagttg caactatgca cttgtataaa
gccataatgt 5216tggagtttat atcactcatt cctgtgtacc tgatggaagt
tgcatgttca tgtttaagca 5276gttactgtaa caagaagttt aaagttaatt
atatcagttt cctaatgctt catgataggc 5336aactttaccc attttgaatg
ccttaattta atttttttca aagtctcagc cctgtctgta 5396ttaaaaaaca
aaaaaagcgt ttaccagctc ttaggatgta aactagcttt gtggaagata
5456aatcgtgcac tatttttaca cataaatagt tatatcaatg tcagcctatt
ttgattaaca 5516aatgttttta aagtattatt ggttatagaa acaataatgg
atggtgttgg aactaatata 5576tccttgatgt ctgtctatta ttcattcaac
tctttttaca gacctcagta ttagtctgtg 5636actacaaaat attttatttg
ctttaaattt gctggctacc ctagatgtgt ttttattcct 5696ggtaaagaca
tttgtgatta cattttcaca cttaagattc aaaatttttc ccaaatataa
5756agaaaactaa gacagactgt agatgcattt taaatattta aatatgatcc
tcagacatgc 5816agctgtgtgt ggcagtattt tagtaccggg ttaagaaaac
tggcaactgg gaagaagtgg 5876cctcaaaggc acttaatttg atttttattt
tttaaatgct gtcaaagtta cagtttacgc 5936aggacattct tgccgtattc
tcatgatccc agataagtgt gtgttttata ctgcaacaat 5996atgcagcaat
ggtaagcgta aagttttttt tttgtttttg ttttttttta tattatgaag
6056tcttttaaca gtctctcttt atataaatac acagagtttg gtatgatatt
taaatacatc 6116atctggccag gcatggtggc ttacgcctgt aatcctagca
ctttgggagg ccaagacggg 6176cggatcacct gaggtgagga gttcaagacc
agcctgccca acatagtgaa actccgtctc 6236taccaatata caaaaattag
ccgggcatga tggtggtggc ctgtaatccc agctacttgg 6296gaggctgaga
caggagaatc gcttgaaccc aggagacggt ggttgcagtg agcgaagatc
6356gagccactgc actccagcct gggcagctga acaagactcc gtctc
640141388PRTHomo sapiens 4Met Ser Arg Pro Pro Pro Thr Gly Lys Met
Pro Gly Ala Pro Glu Thr 1 5 10 15 Ala Pro Gly Asp Gly Ala Gly Ala
Ser Arg Gln Arg Lys Leu Glu Ala 20 25 30 Leu Ile Arg Asp Pro Arg
Ser Pro Ile Asn Val Glu Ser Leu Leu Asp 35 40 45 Gly Leu Asn Ser
Leu Val Leu Asp Leu Asp Phe Pro Ala Leu Arg Lys 50 55 60 Asn Lys
Asn Ile Asp Asn Phe Leu Asn Arg Tyr Glu Lys Ile Val Lys 65 70 75 80
Lys Ile Arg Gly Leu Gln Met Lys Ala Glu Asp Tyr Asp Val Val Lys 85
90 95 Val Ile Gly Arg Gly Ala Phe Gly Glu Val Gln Leu Val Arg His
Lys 100 105 110 Ala Ser Gln Lys Val Tyr Ala Met Lys Leu Leu Ser Lys
Phe Glu Met 115 120 125 Ile Lys Arg Ser Asp Ser Ala Phe Phe Trp Glu
Glu Arg Asp Ile Met 130 135 140 Ala Phe Ala Asn Ser Pro Trp Val Val
Gln Leu Phe Tyr Ala Phe Gln 145 150 155 160 Asp Asp Arg Tyr Leu Tyr
Met Val Met Glu Tyr Met Pro Gly Gly Asp 165 170 175 Leu Val Asn Leu
Met Ser Asn Tyr Asp Val Pro Glu Lys Trp Ala Lys 180 185 190 Phe Tyr
Thr Ala Glu Val Val Leu
Ala Leu Asp Ala Ile His Ser Met 195 200 205 Gly Leu Ile His Arg Asp
Val Lys Pro Asp Asn Met Leu Leu Asp Lys 210 215 220 His Gly His Leu
Lys Leu Ala Asp Phe Gly Thr Cys Met Lys Met Asp 225 230 235 240 Glu
Thr Gly Met Val His Cys Asp Thr Ala Val Gly Thr Pro Asp Tyr 245 250
255 Ile Ser Pro Glu Val Leu Lys Ser Gln Gly Gly Asp Gly Phe Tyr Gly
260 265 270 Arg Glu Cys Asp Trp Trp Ser Val Gly Val Phe Leu Tyr Glu
Met Leu 275 280 285 Val Gly Asp Thr Pro Phe Tyr Ala Asp Ser Leu Val
Gly Thr Tyr Ser 290 295 300 Lys Ile Met Asp His Lys Asn Ser Leu Cys
Phe Pro Glu Asp Ala Glu 305 310 315 320 Ile Ser Lys His Ala Lys Asn
Leu Ile Cys Ala Phe Leu Thr Asp Arg 325 330 335 Glu Val Arg Leu Gly
Arg Asn Gly Val Glu Glu Ile Arg Gln His Pro 340 345 350 Phe Phe Lys
Asn Asp Gln Trp His Trp Asp Asn Ile Arg Glu Thr Ala 355 360 365 Ala
Pro Val Val Pro Glu Leu Ser Ser Asp Ile Asp Ser Ser Asn Phe 370 375
380 Asp Asp Ile Glu Asp Asp Lys Gly Asp Val Glu Thr Phe Pro Ile Pro
385 390 395 400 Lys Ala Phe Val Gly Asn Gln Leu Pro Phe Ile Gly Phe
Thr Tyr Tyr 405 410 415 Arg Glu Asn Leu Leu Leu Ser Asp Ser Pro Ser
Cys Arg Glu Thr Asp 420 425 430 Ser Ile Gln Ser Arg Lys Asn Glu Glu
Ser Gln Glu Ile Gln Lys Lys 435 440 445 Leu Tyr Thr Leu Glu Glu His
Leu Ser Asn Glu Met Gln Ala Lys Glu 450 455 460 Glu Leu Glu Gln Lys
Cys Lys Ser Val Asn Thr Arg Leu Glu Lys Thr 465 470 475 480 Ala Lys
Glu Leu Glu Glu Glu Ile Thr Leu Arg Lys Ser Val Glu Ser 485 490 495
Ala Leu Arg Gln Leu Glu Arg Glu Lys Ala Leu Leu Gln His Lys Asn 500
505 510 Ala Glu Tyr Gln Arg Lys Ala Asp His Glu Ala Asp Lys Lys Arg
Asn 515 520 525 Leu Glu Asn Asp Val Asn Ser Leu Lys Asp Gln Leu Glu
Asp Leu Lys 530 535 540 Lys Arg Asn Gln Asn Ser Gln Ile Ser Thr Glu
Lys Val Asn Gln Leu 545 550 555 560 Gln Arg Gln Leu Asp Glu Thr Asn
Ala Leu Leu Arg Thr Glu Ser Asp 565 570 575 Thr Ala Ala Arg Leu Arg
Lys Thr Gln Ala Glu Ser Ser Lys Gln Ile 580 585 590 Gln Gln Leu Glu
Ser Asn Asn Arg Asp Leu Gln Asp Lys Asn Cys Leu 595 600 605 Leu Glu
Thr Ala Lys Leu Lys Leu Glu Lys Glu Phe Ile Asn Leu Gln 610 615 620
Ser Ala Leu Glu Ser Glu Arg Arg Asp Arg Thr His Gly Ser Glu Ile 625
630 635 640 Ile Asn Asp Leu Gln Gly Arg Ile Cys Gly Leu Glu Glu Asp
Leu Lys 645 650 655 Asn Gly Lys Ile Leu Leu Ala Lys Val Glu Leu Glu
Lys Arg Gln Leu 660 665 670 Gln Glu Arg Phe Thr Asp Leu Glu Lys Glu
Lys Ser Asn Met Glu Ile 675 680 685 Asp Met Thr Tyr Gln Leu Lys Val
Ile Gln Gln Ser Leu Glu Gln Glu 690 695 700 Glu Ala Glu His Lys Ala
Thr Lys Ala Arg Leu Ala Asp Lys Asn Lys 705 710 715 720 Ile Tyr Glu
Ser Ile Glu Glu Ala Lys Ser Glu Ala Met Lys Glu Met 725 730 735 Glu
Lys Lys Leu Leu Glu Glu Arg Thr Leu Lys Gln Lys Val Glu Asn 740 745
750 Leu Leu Leu Glu Ala Glu Lys Arg Cys Ser Leu Leu Asp Cys Asp Leu
755 760 765 Lys Gln Ser Gln Gln Lys Ile Asn Glu Leu Leu Lys Gln Lys
Asp Val 770 775 780 Leu Asn Glu Asp Val Arg Asn Leu Thr Leu Lys Ile
Glu Gln Glu Thr 785 790 795 800 Gln Lys Arg Cys Leu Thr Gln Asn Asp
Leu Lys Met Gln Thr Gln Gln 805 810 815 Val Asn Thr Leu Lys Met Ser
Glu Lys Gln Leu Lys Gln Glu Asn Asn 820 825 830 His Leu Met Glu Met
Lys Met Asn Leu Glu Lys Gln Asn Ala Glu Leu 835 840 845 Arg Lys Glu
Arg Gln Asp Ala Asp Gly Gln Met Lys Glu Leu Gln Asp 850 855 860 Gln
Leu Glu Ala Glu Gln Tyr Phe Ser Thr Leu Tyr Lys Thr Gln Val 865 870
875 880 Arg Glu Leu Lys Glu Glu Cys Glu Glu Lys Thr Lys Leu Gly Lys
Glu 885 890 895 Leu Gln Gln Lys Lys Gln Glu Leu Gln Asp Glu Arg Asp
Ser Leu Ala 900 905 910 Ala Gln Leu Glu Ile Thr Leu Thr Lys Ala Asp
Ser Glu Gln Leu Ala 915 920 925 Arg Ser Ile Ala Glu Glu Gln Tyr Ser
Asp Leu Glu Lys Glu Lys Ile 930 935 940 Met Lys Glu Leu Glu Ile Lys
Glu Met Met Ala Arg His Lys Gln Glu 945 950 955 960 Leu Thr Glu Lys
Asp Ala Thr Ile Ala Ser Leu Glu Glu Thr Asn Arg 965 970 975 Thr Leu
Thr Ser Asp Val Ala Asn Leu Ala Asn Glu Lys Glu Glu Leu 980 985 990
Asn Asn Lys Leu Lys Asp Val Gln Glu Gln Leu Ser Arg Leu Lys Asp 995
1000 1005 Glu Glu Ile Ser Ala Ala Ala Ile Lys Ala Gln Phe Glu Lys
Gln 1010 1015 1020 Leu Leu Thr Glu Arg Thr Leu Lys Thr Gln Ala Val
Asn Lys Leu 1025 1030 1035 Ala Glu Ile Met Asn Arg Lys Glu Pro Val
Lys Arg Gly Asn Asp 1040 1045 1050 Thr Asp Val Arg Arg Lys Glu Lys
Glu Asn Arg Lys Leu His Met 1055 1060 1065 Glu Leu Lys Ser Glu Arg
Glu Lys Leu Thr Gln Gln Met Ile Lys 1070 1075 1080 Tyr Gln Lys Glu
Leu Asn Glu Met Gln Ala Gln Ile Ala Glu Glu 1085 1090 1095 Ser Gln
Ile Arg Ile Glu Leu Gln Met Thr Leu Asp Ser Lys Asp 1100 1105 1110
Ser Asp Ile Glu Gln Leu Arg Ser Gln Leu Gln Ala Leu His Ile 1115
1120 1125 Gly Leu Asp Ser Ser Ser Ile Gly Ser Gly Pro Gly Asp Ala
Glu 1130 1135 1140 Ala Asp Asp Gly Phe Pro Glu Ser Arg Leu Glu Gly
Trp Leu Ser 1145 1150 1155 Leu Pro Val Arg Asn Asn Thr Lys Lys Phe
Gly Trp Val Lys Lys 1160 1165 1170 Tyr Val Ile Val Ser Ser Lys Lys
Ile Leu Phe Tyr Asp Ser Glu 1175 1180 1185 Gln Asp Lys Glu Gln Ser
Asn Pro Tyr Met Val Leu Asp Ile Asp 1190 1195 1200 Lys Leu Phe His
Val Arg Pro Val Thr Gln Thr Asp Val Tyr Arg 1205 1210 1215 Ala Asp
Ala Lys Glu Ile Pro Arg Ile Phe Gln Ile Leu Tyr Ala 1220 1225 1230
Asn Glu Gly Glu Ser Lys Lys Glu Gln Glu Phe Pro Val Glu Pro 1235
1240 1245 Val Gly Glu Lys Ser Asn Tyr Ile Cys His Lys Gly His Glu
Phe 1250 1255 1260 Ile Pro Thr Leu Tyr His Phe Pro Thr Asn Cys Glu
Ala Cys Met 1265 1270 1275 Lys Pro Leu Trp His Met Phe Lys Pro Pro
Pro Ala Leu Glu Cys 1280 1285 1290 Arg Arg Cys His Ile Lys Cys His
Lys Asp His Met Asp Lys Lys 1295 1300 1305 Glu Glu Ile Ile Ala Pro
Cys Lys Val Tyr Tyr Asp Ile Ser Thr 1310 1315 1320 Ala Lys Asn Leu
Leu Leu Leu Ala Asn Ser Thr Glu Glu Gln Gln 1325 1330 1335 Lys Trp
Val Ser Arg Leu Val Lys Lys Ile Pro Lys Lys Pro Pro 1340 1345 1350
Ala Pro Asp Pro Phe Ala Arg Ser Ser Pro Arg Thr Ser Met Lys 1355
1360 1365 Ile Gln Gln Asn Gln Ser Ile Arg Arg Pro Ser Arg Gln Leu
Ala 1370 1375 1380 Pro Asn Lys Pro Ser 1385 539DNAArtificial
SequenceSynthetic oligonucleotide 5cggtttgttt gggtttgggt ttgggtttgg
gtttgggtt 39639DNAArtificial SequenceSynthetic oligonucleotide
6ggcttgcctt acccttaccc ttacccttac ccttaccct 39720DNAArtificial
SequenceSynthetic oligonucleotide 7tgtgctggcc catcactttg
20823DNAArtificial SequenceSynthetic oligonucleotide 8accagccacc
actttctgat agg 23
* * * * *