U.S. patent application number 11/927246 was filed with the patent office on 2009-01-22 for method for extracting quantitative information relating to an influence on a cellular response.
This patent application is currently assigned to FISHER BIOIMAGE APS. Invention is credited to Kasper Almholt, Sara Petersen Bjorn, Kurt Scudder, Ole Thastrup, Soren Tullin.
Application Number | 20090023598 11/927246 |
Document ID | / |
Family ID | 8092994 |
Filed Date | 2009-01-22 |
United States Patent
Application |
20090023598 |
Kind Code |
A1 |
Thastrup; Ole ; et
al. |
January 22, 2009 |
METHOD FOR EXTRACTING QUANTITATIVE INFORMATION RELATING TO AN
INFLUENCE ON A CELLULAR RESPONSE
Abstract
A method for screening a library of compounds to detect a
biologically active compound that modulates intracellular
translocation of a subunit of a component of an intracellular
pathway affecting intracellular processes includes: culturing one
or more cells containing a nucleotide sequence coding for a hybrid
polypeptide comprising a luminophore linked to the subunit of the
component; introducing a compound of the library of compounds into
the cell culture; screening the compound to determine whether the
compound modulates the intracellular translocation of the subunit
of the component; measuring light emitted from the luminophore to
determine a first distribution; measuring light emitted from the
luminophore to determine a second distribution; computing a
variation between the first distribution and the second
distribution by processing the measured light, any variation is
indicative that the compound is biologically active. The method is
also performed with a library of compounds.
Inventors: |
Thastrup; Ole; (Birkerod,
DK) ; Bjorn; Sara Petersen; (Lyngby, DK) ;
Tullin; Soren; (Soborg, DK) ; Almholt; Kasper;
(Copenhagen S, DK) ; Scudder; Kurt; (Virum,
DK) |
Correspondence
Address: |
Workman Nydegger;1000 Eagle Gate Tower
60 East South Temple
Salt Lake City
UT
84111
US
|
Assignee: |
FISHER BIOIMAGE APS
Soborg
DK
|
Family ID: |
8092994 |
Appl. No.: |
11/927246 |
Filed: |
October 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10072036 |
Feb 5, 2002 |
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11927246 |
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09417197 |
Oct 7, 1999 |
6518021 |
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10072036 |
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PCT/DK98/00145 |
Apr 7, 1998 |
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09417197 |
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Current U.S.
Class: |
506/10 |
Current CPC
Class: |
G01N 33/533 20130101;
G01N 33/5014 20130101; G01N 33/5035 20130101; G01N 33/5008
20130101; G01N 33/5005 20130101; G01N 33/582 20130101; G01N
2333/9121 20130101; G01N 2333/43595 20130101; G01N 33/5041
20130101 |
Class at
Publication: |
506/10 |
International
Class: |
C40B 30/06 20060101
C40B030/06 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 17, 1997 |
DK |
PA 00392 |
Claims
1. A method for screening a library of compounds to detect a
biologically active compound that modulates intracellular
translocation of at least a subunit of a component of an
intracellular pathway affecting intracellular processes, the method
comprising: (a) culturing one or more cells of at least one cell
culture containing a nucleotide sequence coding for a hybrid
polypeptide comprising a luminophore linked to said at least a
subunit of the component under conditions that permit expression of
the nucleotide sequence, said at least one cell culture being
substantially devoid of a compound of the library of compounds to
be screened for biological function or biological effect on said at
least a subunit of the component in the one or more cells, wherein
said at least a subunit of the component exhibits a biological
activity of the component; (b) introducing said compound of the
library of compounds into said at least one cell culture; (c)
screening said compound of the library of compounds to determine
whether said compound modulates the intracellular translocation of
said at least a subunit of the component in the one or more cells;
(d) measuring light emitted from the luminophore in the one or more
cells to determine a first distribution of said at least a subunit
of the component in the one or more cells; (e) measuring light
emitted from the luminophore in the one or more cells to determine
a second distribution of said at least a subunit of the component
in the one or more cells in response to said compound; (f)
computing a variation between the first distribution and the second
distribution by processing the measured light of the first
distribution and the second distribution, said variation being
indicative of the translocation of said at least a subunit of the
component in said one or more cells and said translocation being
indicative that said compound of the library of compounds is
biologically active; and (g) repeating at least steps (b)-(f) with
a plurality of compounds of the library of compounds.
2. A method according to claim 1, wherein the method is
characterized by at least one of the following: at least the
measuring is performed with an automated system; at least the
introducing is performed with an automated system; or at least the
processing and computing is performed with a computing system.
3. A method according to claim 1, further comprising at least one
of the following: fixing the one or more cells; or selecting the
one or more cells to be stable cells that are stably transformed
with the nucleotide sequence coding for the hybrid polypeptide.
4. A method according to claim 1, further comprising at least one
of the following: processing measured light data obtained from
measuring light emitted from the luminophore through an algorithm;
recording a plurality of digital images of the light emitted from
the luminophore; implementing a digital filtering method on a
plurality of digital images of the light emitted from the
luminophore, said filtering method being selected from the group
consisting of smoothing, sharpening, edge detection, and
combinations thereof, or implementing a spatial frequency method on
the plurality of digital images of the light emitted from the
luminophore, said spatial frequency method being selected from
Fourier filtering, image cross-correlation, image autocorrelation,
object finding, object classification, color space manipulation for
visualization, and combinations thereof.
5. A method according to claim 1, further comprising individually
screening the plurality of compounds of the library of compounds
alone in the one or more cells to determine whether each compound
of the plurality of compounds modulates the intracellular
translocation of said at least a subunit of the component in the
one or more cells.
6. A method according to claim 1, further comprising screening at
least one combination of compounds of the library of compounds to
determine whether the combination of compounds modulates the
intracellular translocation of said at least a subunit of the
component in the one or more cells.
7. A method according to claim 1, wherein the component is a
protein.
8. A method as in claim 7, wherein said at least a subunit of the
component is substantially the entire protein.
9. A method for screening a library of compounds to detect a
biologically active compound that modulates intracellular
translocation of at least a subunit of a component of an
intracellular pathway affecting intracellular processes, the method
comprising: (a) culturing one or more cells of at least one cell
culture containing a nucleotide sequence coding for a hybrid
polypeptide comprising a luminophore linked to said at least a
subunit of the component under conditions that permit expression of
the nucleotide sequence, said cell culture being substantially
devoid of a compound of the library of compounds to be screened for
biological function or biological effect on said at least a subunit
of the component in the one or more cells, wherein said at least a
subunit of the component exhibits a biological activity of the
component; (b) introducing said compound of the library of
compounds into said at least one cell culture; (c) screening said
compound of the library of compounds to determine whether said
compound modulates the intracellular translocation of said at least
a subunit of the component in the one or more cells; (d) measuring
light emitted from the luminophore in the one or more cells at a
first time point and a second time point; (e) processing the
measured light of the first time point and the second time point;
and (f) computing a variation in distribution of said at least a
subunit of the component in the one or more cells from the first
time point to the second time point, said variation in distribution
of said at least a subunit of the component being in response to
said compound and being indicative of the translocation of said at
least a subunit of the component in said one or more cells and said
translocation being indicative that said compound of the library of
compounds is biologically active; and (g) repeating at least steps
(b)-(f) with a plurality of compounds of the library of
compounds.
10. A method according to claim 9, wherein the method is
characterized by at least one of the following: at least the
measuring is performed with an automated system; at least the
introducing is performed with an automated system; or at least the
processing and computing is performed with a computing system.
11. A method according to claim 9, further comprising at least one
of the following: fixing the one or more cells; or selecting the
one or more cells to be stable cells that are stably transformed
with the nucleotide sequence coding for the hybrid polypeptide.
12. A method according to claim 9, further comprising at least one
of the following: processing measured light data obtained from
measuring light emitted from the luminophore through an algorithm;
recording a plurality of digital images of the light emitted from
the luminophore; implementing a digital filtering method on a
plurality of digital images of the light emitted from the
luminophore, said filtering method being selected from the group
consisting of smoothing, sharpening, edge detection, and
combinations thereof, or implementing a spatial frequency method on
the plurality of digital images of the light emitted from the
luminophore, said spatial frequency method being selected from
Fourier filtering, image cross-correlation, image autocorrelation,
object finding, object classification, color space manipulation for
visualization, and combinations thereof.
13. A method according to claim 9, further comprising individually
screening a plurality of compounds of the library of compounds
alone in the one or more cells to determine whether each compound
of the plurality of compounds modulates the intracellular
translocation of said at least a subunit of the component in the
one or more cells.
14. A method according to claim 9, further comprising screening at
least one combination of compounds of the library of compounds to
determine whether the combination of compounds modulates the
intracellular translocation of said at least a subunit of the
component in the one or more cells.
15. A method according to claim 9, wherein the component is a
protein.
16. A method as in claim 15, wherein said at least a subunit of the
component is substantially the entire protein.
17. A method for screening a library of compounds to detect a
biologically active compound that modulates intracellular
translocation of at least a subunit of a component of an
intracellular pathway affecting intracellular processes, the method
comprising: (a) culturing one or more cells of at least one cell
culture containing a nucleotide sequence coding for a hybrid
polypeptide comprising a luminophore linked to said at least a
subunit of the component under conditions that permit expression of
the nucleotide sequence and that are substantially devoid of a
compound of the library of compounds to be screened for biological
function or biological effect on said at least a subunit of the
component in the one or more cells, wherein said at least a subunit
of the component exhibits a biological activity of the component;
(b) introducing said compound of the library of compounds into said
at least one cell culture; (c) screening said compound of the
library of compounds to determine whether said compound modulates
the intracellular translocation of said at least a subunit of the
component in the one or more cells; (d) measuring light emitted
from the luminophore in the one or more cells to determine a first
distribution of said at least a subunit of the component when said
at least one cell culture is substantially devoid of said compound
of the library of compounds to be screened; (e) measuring light
emitted from the luminophore in the incubated one or more cells to
determine a second distribution of said at least a subunit of the
component in the one or more cells in response to said compound;
(f) processing distribution data obtained from the first
distribution and the second distribution through an algorithm; and
(g) computing a variation of said at least a subunit of the
component from the first distribution to the second distribution,
such variation being indicative of the translocation of said at
least a subunit of the component in said one or more cells and said
translocation being indicative that said compound of the library of
compounds is biologically active; and (h) repeating at least steps
(b)-(h) with a plurality of compounds of the library of
compounds.
18. A method according to claim 17, wherein the method is
characterized by at least one of the following: at least the
measuring is performed with an automated system; at least the
introducing is performed with an automated system; or at least the
processing and computing is performed with a computing system.
19. A method according to claim 17, further comprising at least one
of the following: fixing the one or more cells; or selecting the
one or more cells to be stable cells that are stably transformed
with the nucleotide sequence coding for the hybrid polypeptide.
20. A method according to claim 17, further comprising at least one
of the following: processing measured light data obtained from
measuring light emitted from the luminophore through an algorithm;
recording a plurality of digital images of the light emitted from
the luminophore; implementing a digital filtering method on a
plurality of digital images of the light emitted from the
luminophore, said filtering method being selected from the group
consisting of smoothing, sharpening, edge detection, and
combinations thereof, or implementing a spatial frequency method on
the plurality of digital images of the light emitted from the
luminophore, said spatial frequency method being selected from
Fourier filtering, image cross-correlation, image autocorrelation,
object finding, object classification, color space manipulation for
visualization, and combinations thereof.
21. A method according to claim 17, further comprising individually
screening a plurality of compounds of the library of compounds
alone in the one or more cells to determine whether each compound
of the plurality of compounds modulates the intracellular
translocation of said at least a subunit of the component in the
one or more cells.
22. A method according to claim 17, further comprising screening at
least one combination of compounds of the library of compounds to
determine whether the combination of compounds modulates the
intracellular translocation of said at least a subunit of the
component in the one or more cells.
23. A method according to claim 17, wherein the component is a
protein.
24. A method according to claim 23, wherein said at least a subunit
of the component is substantially the entire protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This United States patent application is a divisional of
U.S. patent application Ser. No. 10/072,036, filed on Feb. 5, 2002,
which is a divisional of U.S. patent application Ser. No.
09/417,197, filed on Oct. 7, 1999, which is now U.S. Pat. No.
6,518,021, which is a continuation of PCT Patent Application Number
PCT/DK98/00145, filed on Apr. 7, 1998, which claims benefit and
priority to Danish Patent Application number 0392/97, filed Apr. 7,
1997, wherein each of the foregoing patents and applications is
incorporated herein in its entirety by specific reference.
BACKGROUND OF THE INVENTION
[0002] 1. The Field of the Invention
[0003] The present invention relates to a method and tools for
extracting quantitative information relating to an influence, on a
cellular response, in particular an influence caused by contacting
or incubating the cell with a substance influencing a cellular
response, where the cellular response is manifested in
redistribution of at least one component in the cell. In
particular, the invention relates to a method for extracting
quantitative information relating to an influence on an
intracellular pathway involving redistribution of at least one
component associated with the pathway. The method of the invention
may be used as a very efficient procedure for testing or
discovering the influence of a substance on a physiological
process, for example in connection with screening for new drugs,
testing of substances for toxicity, identifying drug targets for
known or novel drugs Other valuable uses of the method and
technology of the invention will be apparent to the skilled person
on the basis of the following disclosure. In a particular
embodiment of the invention, the present invention relates to a
method of detecting intracellular translocation or redistribution
of biologically active polypeptides, preferably an enzyme,
affecting intracellular processes, and a DNA construct and a cell
for use in the method.
[0004] 2. The Related Technology
[0005] Intracellular pathways are tightly regulated by a cascade of
components that undergo modulation in a temporally and spatially
characteristic manner. Several disease states can be attributed to
altered activity of individual signaling components (i.e. protein
kinases, protein phosphatases, transcription factors). These
components therefore render themselves as attractive targets for
therapeutic intervention. Protein kinases and phosphatases are well
described components of several intracellular signaling pathways.
The catalytic activity of protein kinases and phosphatases are
assumed to play a role in virtually all regulatable cellular
processes. Although the involvement of protein kinases in cellular
signaling and regulation have been subjected to extensive studies,
detailed knowledge on e.g. the exact timing and spatial
characteristics of signaling events is often difficult to obtain
due to lack of a convenient technology.
[0006] Novel ways of monitoring specific modulation of
intracellular pathways in intact, living cells is assumed to
provide new opportunities in drug discovery, functional genomics,
toxicology, patient monitoring etc.
[0007] The spatial orchestration of protein kinase activity is
likely to be essential for the high degree of specificity of
individual protein kinases. The phosphorylation mediated by protein
kinases is balanced by phosphatase activity. Also within the family
of phosphatases translocation has been observed, e.g. translocation
of PTP2C to membrane ruffles [(Cossette et al. 1996)], and likewise
is likely to be indicative of phosphatase activity.
[0008] Protein kinases often show a specific intracellular
distribution before, during and after activation. Monitoring the
translocation processes and/or redistribution of individual protein
kinases or subunits thereof is thus likely to be indicative of
their functional activity. A connection between translocation and
catalytic activation has been shown for protein kinases like the
diacyl glycerol (DAG)-dependent protein kinase C (PKC), the
cAMP-dependent protein kinase (PKA) [(DeBernardi et al. 1996)] and
the mitogen-activated-protein kinase Erk-1 [(Sano et al.
1995)].
[0009] Commonly used methods of detection of intracellular
localization/activity of protein kinases and phosphatases are
immunoprecipitation, Western blotting and immunocytochemical
detection.
[0010] Taking the family of diacyl glycerol (DAG)-dependent protein
kinase Cs (PKCS) as an example, it has been shown that individual
PKC isoforms that are distributed among different tissues and cells
have different activator requirements and undergo differential
translocation in response to activation. Catalytically inactive
DAG-dependent PKCs are generally distributed throughout the
cytoplasm, whereas they upon activation translocate to become
associated with different cellular components, e.g. plasma membrane
[(Farese, 1992), (Fulop Jr. et al. 1995)] nucleus [(Khalil et
a1.1992)], cytoskeleton [(Blobe et al. 1996)]. The translocation
phenomenon being indicative of PKC activation has been monitored
using different approaches: a) immunocytochemistry where the
localization of individual isoforms can be detected after
permeabilisation and fixation of the cells [(Khalil et a1.1992)];
and b) tagging all DAG-dependent PKC isoforms with a fluorescently
labeled phorbol myristate acetate (PMA) [(Godson et al. 1996)]; and
c) chemical tagging PKC b1 with the fluorophore Cy3 [(Bastiaens
& Jovin 1996)] and d) genetic tagging of PKC.alpha. ([Schmidt
et al. 1997]) and of PKC.gamma. and PKC.epsilon. ([Sakai et al.
1996]). The first method does not provide dynamic information
whereas the latter methods will. Tagging PKC with fluorescently
labeled phorbol myristate acetate cannot distinguish between
different DAG-dependent isoforms of PKC but will label and show
movement of all isoforms. Chemical and genetic labeling of specific
DAG-dependent PKCs confirmed that they in an isoform specific
manner upon activation move to cell periphery or nucleus.
[0011] In an alternative method, protein kinase A activity has been
measured in living cells by chemical labeling one of the kinase's
subunit (Adams et al. 1991). The basis of the methodology is that
the regulatory and catalytic subunit of purified protein kinase A
is labeled with fluorescein and rhodamine, respectively. At low
cAMP levels protein kinase A is assembled in a heterotetrameric
form which enables fluorescence resonance energy transfer between
the two fluorescent dyes. Activation of protein kinase A leads to
dissociation of the complex, thereby eliminating the energy
transfer. A disadvantage of this technology is that the labeled
protein kinase A has to be microinjected into the cells of
interest. This highly invasive technique is cumbersome and not
applicable to large scale screening of biologically active
substances. A further disadvantage of this technique as compared to
the presented invention is that the labeled protein kinase A cannot
be inserted into organisms/animals as a transgene.
[0012] Recently it was discovered that Green Fluorescent Protein
(GFP) expressed in many different cell types, including mammalian
cells, became highly fluorescent [(Chalfie et al. 1994)].
WO95/07463 describes a cell capable of expressing GFP and a method
for detecting a protein of interest in a cell based on introducing
into a cell a DNA molecule having DNA sequence encoding the protein
of interest linked to DNA sequence encoding a GFP such that the
protein produced by the DNA molecule will have the protein of
interest fused to the GFP, then culturing the cells in conditions
permitting expression of the fused protein and detecting the
location of the fluorescence in the cell, thereby localizing the
protein of interest in the cell. However, examples of such fused
proteins are not provided, and the use of fusion proteins with GFP
for detection or quantitation of translocation or redistribution of
biologically active polypeptides affecting intracellular processes
upon activation, such as proteins involved in signaling pathways,
e.g. protein kinases or phosphatases, has not been suggested. WO
95/07463 further describes cells useful for the detection of
molecules, such as hormones or heavy metals, in a biological
sample, by operatively linking a regulatory element of the gene
which is affected by the molecule of interest to a GFP, the
presence of the molecules will affect the regulatory element which
in turn will affect the expression of the GFP. In this way the gene
encoding GFP is used as a reporter gene in a cell which is
constructed for monitoring the presence of a specific molecular
identity. Green Fluorescent Protein has been used in an assay for
the detection of translocation of the glucocorticoid receptor (GR)
[Carey, K L et al., The Journal of Cell Biology, Vol. 133, No. 5,
p. 985-996 (1996)]. A GR-S65TGFP fusion has been used to study the
mechanisms involved in translocation of the glucocorticoid receptor
(GR) in response to the agonist dexamethasone from the cytosol,
where it is present in the absence of a ligand, through the nuclear
pore to the nucleus where it remains after ligand binding. The use
of a GR-GFP fusion enables real-time imaging and quantitation of
nuclear/cytoplasmic ratios of the fluorescence signal.
[0013] Many currently used screening programs designed to find
compounds that affect protein kinase activity are based on
measurements of kinase phosphorylation of artificial or natural
substrates, receptor binding and/or reporter gene expression.
BRIEF SUMMARY OF THE INVENTION
[0014] Generally, cells are genetically modified to express a
luminophore, e.g., a modified (F64L, S65T, Y66H) Green Fluorescent
Protein (GFP, EGFP) coupled to a component of an intracellular
signaling pathway such as a transcription factor, a cGMP- or
cAMP-dependent protein kinase, a cyclin-, calmodulin- or
phospholipid-dependent or mitogen-activated serine/threonin protein
kinase, a tyrosine protein kinase, or a protein phosphatase (e.g.
PKA, PKC, Erk, Smad, VASP, actin, p38, Jnk1, PKG, IkappaB, CDK2,
Grk5, Zap70, p85, protein-tyrosine phosphatase 1C, Stat5, NFAT,
NFkappaB, RhoA, PKB). An influence modulates the intracellular
signaling pathway in such a way that the luminophore is being
redistributed or translocated with the component in living cells in
a manner experimentally determined to be correlated to the degree
of the influence. Measurement of redistribution is performed by
recording of light intensity, fluorescence lifetime, polarization,
wavelength shift, resonance energy transfer, or other properties by
an apparatus consisting of e.g. a fluorescence microscope and a CCD
camera. Data stored as digital images are processed to numbers
representing the degree of redistribution. The method can be used
as a screening program for identifying a compound that modulates a
component and is capable of treating a disease related to the
function of the component.
[0015] In one embodiment, the present invention provides a method
for screening a library of compounds to detect a biologically
active compound that modulates intracellular translocation of at
least a subunit of a component of an intracellular pathway
affecting intracellular processes. Such a method includes the
following: (a) culturing one or more cells of at least one cell
culture containing a nucleotide sequence coding for a hybrid
polypeptide comprising a luminophore linked to the subunit of the
component under conditions that permit expression of the nucleotide
sequence, wherein the cell culture is substantially devoid of a
compound of the library of compounds to be screened for biological
function or biological effect on the subunit of the component in
the one or more cells, and wherein said at least a subunit of the
component exhibits a biological activity of the component; (b)
introducing a compound of the library of compounds into said at
least one cell culture; (c) screening the compound to determine
whether the compound modulates the intracellular translocation of
the subunit of the component in the one or more cells; (d)
measuring light emitted from the luminophore in the one or more
cells to determine a first distribution of the subunit of the
component in the one or more cells; (e) measuring light emitted
from the luminophore in the one or more cells to determine a second
distribution of the subunit of the component in the one or more
cells in response to said compound; (f) computing a variation
between the first distribution and the second distribution by
processing the measured light of the first distribution and the
second distribution, said variation being indicative of the
translocation of said at least a subunit of the component in said
one or more cells and said translocation being indicative that said
compound of the library of compounds is biologically active; and
(g) repeating at least steps (b)-(f) with a plurality of compounds
of the library of compounds.
[0016] In one embodiment, the present invention provides another
method for screening a library of compounds to detect a
biologically active compound that modulates intracellular
translocation of at least a subunit of a component of an
intracellular pathway affecting intracellular processes. Such a
method includes the following: (a) culturing one or more cells of
at least one cell culture containing a nucleotide sequence coding
for a hybrid polypeptide comprising a luminophore linked to the
subunit of the component under conditions that permit expression of
the nucleotide sequence, wherein the cell culture is substantially
devoid of a compound of the library of compounds to be screened for
biological function or biological effect on the subunit of the
component in the one or more cells, and wherein the subunit of the
component exhibits a biological activity of the component; (b)
introducing a compound of the library of compounds into said at
least one cell culture; (c) screening the compound of the library
of compounds to determine whether the compound modulates the
intracellular translocation of the subunit of the component in the
one or more cells; (d) measuring light emitted from the luminophore
in the one or more cells at a first time point and a second time
point; (e) processing the measured light of the first time point
and the second time point; (f) computing a variation in
distribution of the subunit of the component in the one or more
cells from the first time point to the second time point, said
variation in distribution of the subunit of the component being in
response to the compound and being indicative of the translocation
of the subunit of the component in the one or more cells and the
translocation is indicative that the compound of the library of
compounds is biologically active; and (g) repeating at least steps
(b)-(f) with a plurality of compounds of the library of
compounds.
[0017] In one embodiment, the present invention provides another
method for screening a library of compounds to detect a
biologically active compound that modulates intracellular
translocation of at least a subunit of a component of an
intracellular pathway affecting intracellular processes. Such a
method includes the following: (a) culturing one or more cells of
at least one cell culture containing a nucleotide sequence coding
for a hybrid polypeptide comprising a luminophore linked to the
subunit of the component under conditions that permit expression of
the nucleotide sequence and that are substantially devoid of a
compound of the library of compounds to be screened for biological
function or biological effect on the subunit of the component in
the one or more cells, and wherein the subunit of the component
exhibits a biological activity of the component; (b) introducing a
compound of the library of compounds into said at least one cell
culture; (c) screening the compound of the library of compounds to
determine whether the compound modulates the intracellular
translocation of the subunit of the component in the one or more
cells; (d) measuring light emitted from the luminophore in the one
or more cells to determine a first distribution of the subunit of
the component when the cell culture is substantially devoid of a
compound of the library of compounds to be screened; (e) measuring
light emitted from the luminophore in the one or more cells to
determine a second distribution of the subunit of the component in
the one or more cells in response to the compound; (f) processing
distribution data obtained from the first distribution and the
second distribution through an algorithm; (g) computing a variation
of the subunit of the component from the first distribution to the
second distribution, wherein such a variation is indicative of the
translocation of the subunit of the component in the one or more
cells, and such a translocation is indicative that the compound of
the library of compounds is biologically active; and (h) repeating
at least steps (b)-(h) with a plurality of compounds of the library
of compounds.
[0018] In one embodiment, the method is characterized by at least
one of the following: at least the measuring is performed with an
automated system; at least the introducing is performed with an
automated system; or at least the processing and computing is
performed with a computing system.
[0019] In one embodiment, the method includes at least one of the
following: fixing the one or more cells; or selecting the one or
more cells to be stable cells that are stably transformed with the
nucleotide sequence coding for the hybrid polypeptide.
[0020] In one embodiment, the method includes at least one of the
following: processing measured light data obtained from measuring
light emitted from the luminophore through an algorithm; recording
a plurality of digital images of the light emitted from the
luminophore; implementing a digital filtering method on a plurality
of digital images of the light emitted from the luminophore, said
filtering method being selected from the group consisting of
smoothing, sharpening, edge detection, and combinations thereof, or
implementing a spatial frequency method on the plurality of digital
images of the light emitted from the luminophore, said spatial
frequency method being selected from Fourier filtering, image
cross-correlation, image autocorrelation, object finding, object
classification, color space manipulation for visualization, and
combinations thereof.
[0021] In one embodiment, the method includes individually
screening the plurality of compounds of the library of compounds
alone in the one or more cells to determine whether each compound
of the plurality of compounds modulates the intracellular
translocation of the subunit of the component in the one or more
cells.
[0022] In one embodiment, the method includes screening at least
one combination of compounds of the library of compounds to
determine whether the combination of compounds modulates the
intracellular translocation of the subunit of the component in the
one or more cells.
[0023] In one embodiment, the component is a protein. In another
embodiment, the subunit of the component is substantially the
entire protein.
[0024] These and other embodiments and features of the present
invention will become more fully apparent from the following
description and appended claims, or may be learned by the practice
of the invention as set forth hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] To further clarify the above and other advantages and
features of the present invention, a more particular description of
the invention will be rendered by reference to specific embodiments
thereof which are illustrated in the appended drawings. It is
appreciated that these drawings depict only typical embodiments of
the invention and are therefore not to be considered limiting of
its scope. The invention will be described and explained with
additional specificity and detail through the use of the
accompanying drawings in which:
[0026] FIG. 1. CHO cells expressing the PKAc-F64L-S65T-GFP hybrid
protein have been treated in HAM's F12 medium with 50 mM forskolin
at 37 degree C. The images of the GFP fluorescence in these cells
have been taken at different time intervals after treatment, which
were: a) 40 seconds b) 60 seconds c) 70 seconds d) 80 seconds. The
fluorescence changes from a punctate to a more even distribution
within the (non-nuclear) cytoplasm.
[0027] FIG. 2. Time-lapse analysis of forskolin induced
PKAc-F64L-S65T-GFP redistribution. CHO cells, expressing the
PKAc-F64L-S65T-GFP fusion protein were analyzed by time-lapse
fluorescence microscopy. Fluorescence micrographs were acquired at
regular intervals from 2 min before to 8 min after the addition of
agonist. The cells were challenged with 1 mM forskolin immediately
after the upper left image was acquired (t=0). Frames were
collected at the following times: i) 0, ii) 1, iii) 2, iv) 3, v) 4
and vi) 5 minutes. Scale bar 10 mm.
[0028] FIG. 3. Time-lapse analyses of PKAc-F64L-S65T-GFP
redistribution in response to various agonists. The effects of 1 mM
forskolin (A), 50 mM forskolin (B), 1 mM dbcAMP (C) and 100 mM IBMX
(D) (additions indicated by open arrows) on the localization of the
PKAc-F64L-S65T-GFP fusion protein were analyzed by time-lapse
fluorescence microscopy of CHO/PKAc-F64L-S65T-GFP cells. The effect
of addition of 10 mM forskolin (open arrow), followed shortly by
repeated washing with buffer (solid arrow), on the localization of
the PKAc-F64L-S65T-GFP fusion protein was analyzed in the same
cells (E). In a parallel experiment, the effect of adding 10 mM
forskolin and 100 mM IBMX (open arrow) followed by repeated washing
with buffer containing 100 mM IBMX (solid arrow) was analyzed (F).
Removing forskolin caused PKAc-F64L-S65T-GFP fusion protein to
return to the cytoplasmic aggregates while this is prevented by the
continued presence of IBMX (F). The effect of 100 nM glucagon (FIG.
3G, open arrow) on the localization of the PKAc-F64L-S65T-GFP
fusion protein is also shown for BIK/GR, PKAc-F64L-S65T-GFP cells.
The effect of 10 mM norepinephrine (H), solid arrow, on the
localization of the PKAc-F64L-S65T-GFP fusion protein was analyzed
similarly, in transiently transfected CHO, PKAc-F64L-S65T-GFP
cells, pretreated with 10 mM forskolin, open arrow, to increase
[cAMP]i N. B. in FIG. 3H the x-axis counts the image numbers, with
12 seconds between images. The raw data of each experiment
consisted of 60 fluorescence micrographs acquired at regular
intervals including several images acquired before the addition of
buffer or agonist. The charts (A-G) each show a quantification of
the response seen through all the 60 images, performed as described
in analysis method 2. The change in total area of the highly
fluorescent aggregates, relative to the initial area of fluorescent
aggregates is plotted as the ordinate in all graphs in FIG. 3,
versus time for each experiment. Scale bar 10 mm.
[0029] FIG. 4. Dose response curve (two experiments) for
forskolin-induced redistribution of the PKAc-F64L-S65T-GFP
fusion.
[0030] FIG. 5. Time from initiation of a response to half maximal
(t.sub.1/2max) and maximal (t.sub.max) PKAc-F64L-S65T-GFP
redistribution. The data was extracted from curves such as that
shown in "FIG. 2." All t.sub.1/2max and t.sub.max values are given
as mean.+-.SD and are based on a total of 26-30 cells from 2-3
independent experiments for each forskolin concentration. Since the
observed redistribution is sustained over time, the t.sub.max
values were taken as the earliest time point at which complete
redistribution is reached. Note that the values do not relate to
the degree of redistribution.
[0031] FIG. 6. Parallel dose response analyses of forskolin induced
cAMP elevation and PKAc-F64L-S65T-GFP redistribution. The effects
of buffer or 5 increasing concentrations of forskolin on the
localization of the PKAc-F64L-S65T-GFP fusion protein in
CHO/PKAc-F64L-S65T-GFP cells, grown in a 96 well plate, were
analyzed as described above. Computing the ratio of the SD's of
fluorescence micrographs taken of the same field of cells, prior to
and 30 min after the addition of forskolin, gave a reproducible
measure of PKAc-F64L-S65T-GFP redistribution. The graph shows the
individual 48 measurements and a trace of their mean.+-.s.e.m at
each forskolin concentration. For comparison, the effects of buffer
or 8 increasing concentrations of forskolin on [cAMP]i was analyzed
by a scintillation proximity assay of cells grown under the same
conditions. The graph shows a trace of the mean.+-.s.e.m of 4
experiments expressed in arbitrary units.
[0032] FIG. 7. BHK cells stably transfected with the human
muscarinic (hM1) receptor and the PKCa-F64L-S65T-GFP fusion.
Carbachol (100 mM added at 1.0 second) induced a transient
redistribution of PKCa-F64L-S65T-GFP from the cytoplasm to the
plasma membrane. Images were taken at the following times: a)
1-second before carbachol addition, b) 8.8 seconds after addition
and c) 52.8 seconds after addition.
[0033] FIG. 8. BHK cells stably transfected with the hM1 receptor
and PKCa-F64L-S65T-GFP fusion were treated with carbachol (1 mM, 10
mM, 100 mM). In single cells intracellular [Ca.sup.2+] was
monitored simultaneously with the redistribution of
PKCa-F64L-S65T-GFP. Dashed line indicates the addition times of
carbachol. The top panel shows changes in the intracellular
Ca.sup.2+ concentration of individual cells with time for each
treatment. The middle panel shows changes in the average
cytoplasmic GFP fluorescence for individual cells against time for
each treatment. The bottom panel shows changes in the fluorescence
of the periphery of single cells, within regions that specifically
include the circumferential edge of a cell as seen in normal
projection, the regions which offers best chance to monitor changes
in the fluorescence intensity of the plasma membrane.
[0034] FIG. 9. a) The hERK1-F64L-S65T-GFP fusion expressed in
HEK293 cells treated with 100 mM of the MEK1 inhibitor PD98059 in
HAM F-12 (without serum) for 30 minutes at 37 degree C. The nuclei
empty of fluorescence during this treatment.
[0035] FIG. 9. b) The same cells as in (a) following treatment with
10% foetal calf serum for 15 minutes at 37 degree C.
[0036] FIG. 9. c) Time profiles for the redistribution of GFP
fluorescence in HEK293 cells following treatment with various
concentrations of EGF in Hepes buffer (HAM F-12 replaced with Hepes
buffer directly before the experiment). Redistribution of
fluorescence is expressed as the change in the ratio value between
areas in nucleus and cytoplasm of single cells. Each time profile
is the mean for the changes seen in six single cells.
[0037] FIG. 9. d) Bar chart for the end-point measurements, 600
seconds after start of EGF treatments, of fluorescence change
(nucleus:cytoplasm) following various concentrations of EGF.
[0038] FIG. 10. a) The SMAD2-EGFP fusion expressed in HEK293 cells
starved of serum overnight in HAM F-12. HAM F-12 was then replaced
with Hepes buffer pH 7.2 immediately before the experiment. Scale
bar is 10 mm.
[0039] FIG. 10. b) BEK 293 cells expressing the SMAD2-EGFP fusion
were treated with various concentration of TGF-beta as indicated,
and the redistribution of fluorescence monitored against time. The
time profile plots represent increases in fluorescence within the
nucleus, normalized to starting values in each cell measured. Each
trace is the time profile for a single cell nucleus.
[0040] FIG. 10. c) A bar chart representing the end-point change in
fluorescence within nuclei (after 850 seconds of treatment) for
different concentrations of TGF-beta. Each bar is the value for a
single nucleus in each treatment.
[0041] FIG. 11. The VASP-F64L-S65T-GFP fusion in CHO cells stably
transfected with the human insulin receptor. The cells were starved
for two hours in HAM F-12 without serum, then treated with 10%
foetal calf serum. The image shows the resulting redistribution of
fluorescence after 15 minutes of treatment. GFP fluorescence
becomes localized in structures identified as focal adhesions along
the length of actin stress fibers.
[0042] FIG. 12. Time lapse recording GLUT4-GFP redistribution in
CHO-HIR cells. Time indicates minutes after the addition of 100 nM
insulin.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] The present invention provides an important new dimension in
the investigation of cellular systems involving redistribution in
that the invention provides quantification of the redistribution
responses or events caused by an influence, typically contact with
a chemical substance or mixture of chemical substances, but also
changes in the physical environment. The quantification makes it
possible to set up meaningful relationships, expressed numerically,
or as curves or graphs, between the influences (or the degree of
influences) on cellular systems and the redistribution response.
This is highly advantageous because, as has been found, the
quantification can be achieved in both a fast and reproducible
manner, and--what is perhaps even more important--the systems which
become quantifiable utilizing the method of the invention are
systems from which enormous amounts of new information and insight
can be derived.
[0044] The present screening assays have the distinct advantage
over other screening assays, e.g., receptor binding assays,
enzymatic assays, and reporter gene assays, in providing a system
in which biologically active substances with completely novel modes
of action, e.g. inhibition or promotion of
redistribution/translocation of a biologically active polypeptide
as a way of regulating its action rather than inhibition/activation
of enzymatic activity, can be identified in a way that insures very
high selectivity to the particular isoform of the biologically
active polypeptide and further development of compound selectivity
versus other isoforms of the same biologically active polypeptide
or other components of the same signaling pathway.
[0045] In its broadest aspect, the invention relates to a method
for extracting quantitative information relating to an influence on
a cellular response, the method comprising recording variation,
caused by the influence on a mechanically intact living cell or
mechanically intact living cells, in spatially distributed light
emitted from a luminophore, the luminophore being present in the
cell or cells and being capable of being redistributed in a manner
which is related with the degree of the influence, and/or of being
modulated by a component which is capable of being redistributed in
a manner which is related to the degree of the influence, the
association resulting in a modulation of the luminescence
characteristics of the luminophore, detecting and recording the
spatially distributed light from the luminophore, and processing
the recorded variation in the spatially distributed light to
provide quantitative information correlating the spatial
distribution or change in the spatial distribution to the degree of
the influence. In a preferred embodiment of the invention the
luminophore, which is present in the cell or cells, is capable of
being redistributed by modulation of an intracellular pathway, in a
manner which is related to the redistribution of at least one
component of the intracellular pathway. In another preferred
embodiment of the invention, the luminophore is a fluorophore.
I. The Cells
[0046] In the invention the cell and/or cells are mechanically
intact and alive throughout the experiment. In another embodiment
of the invention, the cell or cells is/are fixed at a point in time
after the application of the influence at which the response has
been predetermined to be significant, and the recording is made at
an arbitrary later time.
[0047] The mechanically intact living cell or cells could be
selected from the group consisting of fungal cell or cells, such as
a yeast cell or cells; invertebrate cell or cells including insect
cell or cells; and vertebrate cell or cells, such as mammalian cell
or cells. This cell or these cells is/are incubated at a
temperature of 30 degree C. or above, preferably at a temperature
of from 32 degree C. to 39 degree C., more preferably at a
temperature of from 35 degree C. to 38 degree C. and most
preferably at a temperature of about 37 degree C. during the time
period over which the influence is observed. In one aspect of the
invention the mechanically intact living cell is part of a matrix
of identical or non-identical cells.
[0048] A cell used in the present invention should contain a
nucleic acid construct encoding a fusion polypeptide as defined
herein and be capable of expressing the sequence encoded by the
construct. The cell is a eukaryotic cell selected from the group
consisting of fungal cells, such as yeast cells; invertebrate cells
including insect cells; vertebrate cells such as mammalian cells.
The preferred cells are mammalian cells.
[0049] In another aspect of the invention the cells could be from
an organism carrying in at least one of its component cells a
nucleic acid sequence encoding a fusion polypeptide as defined
herein and be capable of expressing said nucleic acid sequence. The
organism is selected from the group consisting of unicellular and
multicellular organisms, such as a mammal.
II. The Luminophore
[0050] The luminophore is the component which allows the
redistribution to be visualized and/or recorded by emitting light
in a spatial distribution related to the degree of influence. In
one embodiment of the invention, the luminophore is capable of
being redistributed in a manner which is physiologically relevant
to the degree of the influence. In another embodiment, the
luminophore is capable of associating with a component which is
capable of being redistributed in a manner which is physiologically
relevant to the degree of the influence. In another embodiment, the
luminophore correlation between the redistribution of the
luminophore and the degree of the influence could be determined
experimentally. In a preferred aspect of the invention, the
luminophore is capable of being redistributed in substantially the
same manner as the at least one component of an intracellular
pathway. In yet another embodiment of the invention, the
luminophore is capable of being quenched upon spatial association
with a component which is redistributed by modulation of the
pathway, the quenching being measured as a change in the intensity
of the luminescence.
[0051] The luminophore could be a fluorophore. In a preferred
embodiment of the invention, the luminophore could be a polypeptide
encoded by and expressed from a nucleotide sequence harbored in the
cell or cells. The luminophore could be a hybrid polypeptide
comprising a fusion of at least a portion of each of two
polypeptides one of which comprises a luminescent polypeptide and
the other one of which comprises a biologically active polypeptide,
as defined herein.
[0052] The luminescent polypeptide could be a GFP as defined herein
or could be selected from the group consisting of green fluorescent
proteins having the F64L mutation as defined herein such as
F64L-GFP, F64L-Y66H-GFP, F64L-S65T-GFP, and EGFP. The GFP could be
N- or C-terminally tagged, optionally via a peptide linker, to the
biologically active polypeptide or a part or a subunit thereof. The
fluorescent probe could be a component of a intracellular signaling
pathway. The probe is coded for by a nucleic acid construct.
[0053] The pathway of investigation in the present invention could
be an intracellular signaling pathway.
III. The Influence
[0054] In a preferred embodiment of the invention, the influence
could be contact between the mechanically intact living cell or the
group of mechanically intact living cells with a chemical substance
and/or incubation of the mechanically intact living cell or the
group of mechanically intact living cells with a chemical
substance. The influence will modulate the intracellular processes.
In one aspect the modulation could be an activation of the
intracellular processes. In another aspect the modulation could be
an deactivation of the intracellular processes. In yet another
aspect, the influence could inhibit or promote the redistribution
without directly affecting the metabolic activity of the component
of the intracellular processes.
[0055] In one embodiment the invention is used as a basis for a
screening program, where the effect of unknown influences such as a
compound library, can be compared to influence of known reference
compounds under standardized conditions.
IV. The Recording
[0056] In addition to the intensity, there are several parameters
of fluorescence or luminescence which can be modulated by the
effect of the influence on the underlying cellular phenomena, and
can therefore be used in the invention. Some examples are resonance
energy transfer, fluorescence lifetime, polarization, wavelength
shift. Each of these methods requires a particular kind of filter
in the emission light path to select the component of the light
desired and reject other components. The recording of property of
light could be in the form of an ordered array of values such as a
CCD array or a vacuum tube device such as a vidicon tube.
[0057] In one embodiment of the invention, the spatially
distributed light emitted by a luminophore could be detected by a
change in the resonance energy transfer between the luminophore and
another luminescent entity capable of delivering energy to the
luminophore, each of which has been selected or engineered to
become part of, bound to or associated with particular components
of the intracellular pathway. In this embodiment, either the
luminophore or the luminescent entity capable of delivering energy
to the luminophore undergoes redistribution in response to an
influence. The resonance energy transfer would be measured as a
change in the intensity of emission from the luminophore,
preferably sensed by a single channel photodetector which responds
only to the average intensity of the luminophore in a non-spatially
resolved fashion.
[0058] In one embodiment of the invention, the recording of the
spatially distributed light could be made at a single point in time
after the application of the influence. In another embodiment, the
recording could be made at two points in time, one point being
before, and the other point being after the application of the
influence. The result or variation is determined from the change in
fluorescence compared to the fluorescence measured prior to the
influence or modulation. In another embodiment of the invention,
the recording could be performed at a series of points in time, in
which the application of the influence occurs at some time after
the first time point in the series of recordings, the recording
being performed, e.g., with a predetermined time spacing of from
0.1 seconds to 1 hour, preferably from 1 to 60 seconds, more
preferably from 1 to 30 seconds, in particular from 1 to 10
seconds, over a time span of from 1 second to 12 hours, such as
from 10 seconds to 12 hours, e.g., from 10 seconds to one hour,
such as from 60 seconds to 30 minutes or 20 minutes. The result or
variation is determined from the change in fluorescence over time.
The result or variation could also be determined as a change in the
spatial distribution of the fluorescence over time.
V. Apparatus
[0059] The recording of spatially distributed luminescence emitted
from the luminophore is performed by an apparatus for measuring the
distribution of fluorescence in the cell or cells, and thereby any
change in the distribution of fluorescence in the cell or cells,
which includes at a minimum the following component parts: (a) a
light source, (b) a method for selecting the wavelength(s) of light
from the source which will excite the fluorescence of the protein,
(c) a device which can rapidly block or pass the excitation light
into the rest of the system, (d) a series of optical elements for
conveying the excitation light to the specimen, collecting the
emitted fluorescence in a spatially resolved fashion, and forming
an image from this fluorescence emission, (e) a bench or stand
which holds the container of the cells being measured in a
predetermined geometry with respect to the series of optical
elements, (f) a detector to record the spatially resolved
fluorescence in the form of an image, (g) a computer or electronic
system and associated software to acquire and store the recorded
images, and to compute the degree of redistribution from the
recorded images.
[0060] In a preferred embodiment of the invention the apparatus
system is automated. In one embodiment the components in d and e
mentioned above comprise a fluorescence microscope. In one
embodiment the component in f mentioned above is a CCD camera.
[0061] In one embodiment the image is formed and recorded by an
optical scanning system.
[0062] In one embodiment a liquid addition system is used to add a
known or unknown compound to any or all of the cells in the cell
holder at a time determined in advance. Preferably, the liquid
addition system is under the control of the computer or electronic
system. Such an automated system can be used for a screening
program due to its ability to generate results from a larger number
of test compounds than a human operator could generate using the
apparatus in a manual fashion.
VI. Quantitation of the Influence
[0063] The recording of the variation or result with respect to
light emitted from the luminophore is performed by recording the
spatially distributed light as one or more digital images, and the
processing of the recorded variation to reduce it to one or more
numbers representative of the degree of redistribution comprises a
digital image processing procedure or combination of digital image
processing procedures. The quantitative information which is
indicative of the degree of the cellular response to the influence
or the result of the influence on the intracellular pathway is
extracted from the recording or recordings according to a
predetermined calibration based on responses or results, recorded
in the same manner, to known degrees of a relevant specific
influence. This calibration procedure is developed according to
principles described below (Developing an image-based Assay
Technique). Specific descriptions of the procedures for particular
assays are given in the examples.
[0064] While the stepwise procedure necessary to reduce the image
or images to the value representative of the is particular to each
assay, the individual steps are generally well-known methods of
image processing. Some examples of the individual steps are point
operations such as subtraction, ratioing, and thresholding, digital
filtering methods such as smoothing, sharpening, and edge
detection, spatial frequency methods such as Fourier filtering,
image cross-correlation and image autocorrelation, object finding
and classification (blob analysis), and color space manipulations
for visualization. In addition to the algorithmic procedures,
heuristic methods such as neural networks may also be used.
VII. Nucleic Acid Constructs
[0065] The nucleic acid constructs used in the present invention
encode in their nucleic acid sequences fusion polypeptides
comprising a biologically active polypeptide that is a component of
an intracellular signaling pathway, or a part thereof, and a GFP,
preferably an F64L mutant of GFP, N- or C-terminally fused,
optionally via a peptide linker, to the biologically active
polypeptide or part thereof.
[0066] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a protein kinase or a
phosphatase.
[0067] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a transcription factor or
a part thereof which changes cellular localization upon
activation.
[0068] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a protein, or a part
thereof, which is associated with the cytoskeletal network and
which changes cellular localization upon activation.
[0069] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a protein kinase or a part
thereof which changes cellular localization upon activation.
[0070] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a serine/threonine protein
kinase or a part thereof capable of changing intracellular
localization upon activation.
[0071] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a tyrosine protein kinase
or a part thereof capable of changing intracellular localization
upon activation.
[0072] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a phospholipid-dependent
serine/threonine protein kinase or a part thereof capable of
changing intracellular localization upon activation.
[0073] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a cAMP-dependent protein
kinase or a part thereof capable of changing cellular localization
upon activation. In a preferred embodiment the biologically active
polypeptide encoded by the nucleic acid construct is a
PKAc-F64L-S65T-GFP fusion.
[0074] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a cGMP-dependent protein
kinase or a part thereof capable of changing cellular localization
upon activation.
[0075] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a calmodulin-dependent
serine/threonine protein kinase or a part thereof capable of
changing cellular localization upon activation.
[0076] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a mitogen-activated
serine/threonine protein kinase or a part thereof capable of
changing cellular localization upon activation. In preferred
embodiments the biologically active polypeptide encoded by the
nucleic acid constructs are an ERK1-F64L-S65T-GFP fusion or an
EGFP-ERK1 fusion.
[0077] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct is a cyclin-dependent
serine/threonine protein kinase or a part thereof capable of
changing cellular localization upon activation.
[0078] In one embodiment the biologically active polypeptide
encoded by the nucleic add construct is a protein phosphatase or a
part thereof capable of changing cellular localization upon
activation.
[0079] In one preferred embodiment of the invention the nucleic
acid constructs may be DNA constructs.
[0080] In one embodiment the biologically active polypeptide
encoded by the nucleic acid construct In one embodiment the gene
encoding GFP in the nucleic acid construct is derived from Aequorea
victoria. In a preferred embodiment the gene encoding GFP in the
nucleic acid construct is EGFP or a GFP variant selected from
F64L-GFP, F64L-Y66H-GFP and F64L-S65T-GFP. In preferred embodiments
of the invention the DNA constructs which can be identified by any
of the DNA sequences shown in SEQ ID NO: 38, 40, 42, 44, 46, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 108,
110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,
136, 138, 140, 142 or are variants of these sequences capable of
encoding the same fusion polypeptide or a fusion polypeptide which
is biologically equivalent thereto, e.g. an isoform, or a splice
variant or a homologue from another species.
VIII. Screening Program
[0081] The present invention describes a method that may be used to
establish a screening program for the identification of
biologically active substances that directly or indirectly affects
intracellular signaling pathways and because of this property are
potentially useful as medicaments. Based on measurements in living
cells of the redistribution of spatially resolved luminescence from
luminophores which undergo a change in distribution upon activation
or deactivation of an intracellular signaling pathway the result of
the individual measurement of each substance being screened
indicates its potential biological activity.
[0082] In one embodiment of the invention the screening program is
used for the identification of a biologically toxic substance as
defined herein that exerts its toxic effect by interfering with an
intracellular signaling pathway. Based on measurements in living
cells of the redistribution of spatially resolved luminescence from
luminophores which undergo a change in distribution upon activation
or deactivation of an intracellular signaling pathway the result of
the individual measurement of each substance being screened
indicates its potential biologically toxic activity. In one
embodiment of a screening program a compound that modulates a
component of an intracellular pathway as defined herein, can be
found and the therapeutic amount of the compound estimated by a
method according to the method of the invention. In a preferred
embodiment the present invention leads to the discovery of a new
way of treating a condition or disease related to the intracellular
function of a biologically active polypeptide comprising
administration to a patient suffering from said condition or
disease of an effective amount of a compound which has been
discovered by any method according to the invention. In another
preferred embodiment of the invention a method is established for
identification of a new drug target or several new drug targets
among the group of biologically active polypeptides which are
components of intracellular signaling pathways.
[0083] In another embodiment of the invention an individual
treatment regimen is established for the selective treatment of a
selected patient suffering from an ailment where the available
medicaments used for treatment of the ailment are tested on a
relevant primary cell or cells obtained from said patient from one
or several tissues, using a method comprising transfecting the cell
or cells with at least one DNA sequence encoding a fluorescent
probe according to the invention, transferring the transfected cell
or cells back the said patient, or culturing the cell or cells
under conditions permitting the expression of said probes and
exposing it to an array of the available medicaments, then
comparing changes in fluorescence patterns or redistribution
patterns of the fluorescent probes in the intact living cell or
cells to detect the cellular response to the specific medicaments
(obtaining a cellular action profile), then selecting one or more
medicament or medicaments based on the desired activity and
acceptable level of side effects and administering an effective
amount of these medicaments to the selected patient.
IX. Back-Tracking of a Signal Transduction Pathway
[0084] The present invention describes a method that may be used to
establish a screening program for back-tracking signal transduction
pathways as defined herein. In one embodiment the screening program
is used to establish more precisely at which level one or several
compounds affect a specific signal transduction pathway by
successively or in parallel testing the influence of the compound
or compounds on the redistribution of spatially resolved
luminescence from several of the luminophores which undergo a
change in distribution upon activation or deactivation of the
intracellular signaling pathway under study.
X. Construction and Testing of Probes
[0085] In general, a probe, i.e. a "GeneX"-GFP fusion or a
GFP-"GeneX" fusion, is constructed using PCR with "GeneX"-specific
primers followed by a cloning step to fuse "GeneX" in frame with
GFP. The fusion may contain a short vector derived sequence between
"GeneX" and GFP (e.g. part of a multiple cloning site region in the
plasmid) resulting in a peptide linker between "GeneX" and GFP in
the resulting fusion protein.
XI. Detailed Stepwise Procedure
[0086] Identifying the sequence of the gene. This is most readily
done by searching a depository of genetic information, e.g. the
GenBank Sequence Database, which is widely available and routinely
used by molecular biologists. In the specific examples below the
GenBank Accession number of the gene in question is provided.
[0087] Design of gene-specific primers. Inspection of the sequence
of the gene allows design of gene-specific primers to be used in a
PCR reaction. Typically, the top-strand primer encompasses the ATG
start codon of the gene and the following ca. 20 nucleotides, while
the bottom-strand primer encompasses the stop codon and the ca. 20
preceding nucleotides, if the gene is to be fused behind GFP, i.e.
a GFP-"GeneX" fusion. If the gene is to be fused in front of GFP,
i.e. a "GeneX"-GFP fusion, a stop codon must be avoided.
Optionally, the full length sequence of GeneX may not be used in
the fusion, but merely the part which localizes and redistributes
like GeneX in response to a signal.
[0088] In addition to gene-specific sequences, the primers contain
at least one recognition sequence for a restriction enzyme, to
allow subsequent cloning of the PCR product. The sites are chosen
so that they are unique in the PCR product and compatible with
sites in the cloning vector. Furthermore, it may be necessary to
include an exact number of nucleotides between the restriction
enzyme site and the gene-specific sequence in order to establish
the correct reading frame of the fusion gene and/or a translation
initiation consensus sequence. Lastly, the primers always contain a
few nucleotides in front of the restriction enzyme site to allow
efficient digestion with the enzyme.
[0089] Identifying a source of the gene to be amplified. In order
for a PCR reaction to produce a product with gene-specific primers,
the gene-sequence must initially be present in the reaction, e.g.
in the form of cDNA. Information in GenBank or the scientific
literature will usually indicate in which tissue(s) the gene is
expressed, and cDNA libraries from a great variety of tissues or
cell types from various species are commercially available, e.g.
from Clontech (Palo Alto), Stratagene (La Jolla) and Invitrogen
(San Diego). Many genes are also available in cloned form from The
American Type Tissue Collection (Virginia).
[0090] Optimizing the PCR reaction. Several factors are known to
influence the efficiency and specificity of a PCR reaction,
including the annealing temperature of the primers, the
concentration of ions, notably Mg.sup.2+ and K.sup.+, present in
the reaction, as well as pH of the reaction. If the result of a PCR
reaction is deemed unsatisfactory, it might be because the
parameters mentioned above are not optimal. Various annealing
temperatures should be tested, e.g. in a PCR machine with a
built-in temperature gradient, available from e.g. Stratagene (La
Jolla), and/or various buffer compositions should be tried, e.g.
the OptiPrime buffer system from Stratagene (La Jolla).
[0091] Cloning the PCR product. The vector into which the amplified
gene product will be cloned and fused with GFP will already have
been taken into consideration when the primers were designed. When
choosing a vector, one should at least consider in which cell types
the probe subsequently will be expressed, so that the promoter
controlling expression of the probe is compatible with the cells.
Most expression vectors also contain one or more selective markers,
e.g. conferring resistance to a drug, which is a useful feature
when one wants to make stable transfectants. The selective marker
should also be compatible with the cells to be used.
[0092] The actual cloning of the PCR product should present no
difficulty as it typically will be a one-step cloning of a fragment
digested with two different restriction enzymes into a vector
digested with the same two enzymes. If the cloning proves to be
problematic, it may be because the restriction enzymes did not work
well with the PCR fragment. In this case one could add longer
extensions to the end of the primers to overcome a possible
difficulty of digestion close to a fragment end, or one could
introduce an intermediate cloning step not based on restriction
enzyme digestion. Several companies offer systems for this
approach, e.g. Invitrogen (San Diego) and Clontech (Palo Alto).
[0093] Once the gene has been cloned and, in the process, fused
with the GFP gene, the resulting product, usually a plasmid, should
be carefully checked to make sure it is as expected. The most exact
test would be to obtain the nucleotide sequence of the
fusion-gene.
XII. Testing the Probe
[0094] Once a DNA construct for a probe has been generated, its
functionality and usefulness may be tested by subjecting it to the
following tests:
[0095] Transfecting it into cells capable of expressing the probe.
The fluorescence of the cell is inspected soon after, typically the
next day. At this point, two features of cellular fluorescence are
noted: the intensity and the sub-cellular localization.
[0096] The intensity should usually be at least as strong as that
of unfused GFP in the cells. If it is not, the sequence or quality
of the probe-DNA might be faulty, and should be carefully
checked.
[0097] The sub-cellular localization is an indication of whether
the probe is likely to perform well. If it localizes as expected
for the gene in question, e.g. is excluded from the nucleus, it can
immediately go on to a functional test. If the probe is not
localized soon after the transfection procedure, it may be because
of overexpression at this point in time, as the cell typically will
have taken of very many copies of the plasmid, and localization
will occur in time, e.g. within a few weeks, as plasmid copy number
and expression level decreases. If localization does not occur
after prolonged time, it may be because the fusion to GFP has
destroyed a localization function, e.g. masked a protein sequence
essential for interaction with its normal cellular anchor-protein.
In this case the opposite fusion might work, e.g. if GeneX-GFP does
not work, GFP-GeneX might, as two different parts of GeneX will be
affected by the proximity to GFP. If this does not work, the
proximity of GFP at either end might be a problem, and it could be
attempted to increase the distance by incorporating a longer linker
between GeneX and GFP in the DNA construct.
[0098] If there is no prior knowledge of localization, and no
localization is observed, it may be because the probe should not be
localized at this point, because such is the nature of the protein
fused to GFP. It should then be subjected to a functional test.
[0099] In a functional test, the cells expressing the probe are
treated with at least one compound known to perturb, usually by
activating, the signaling pathway on which the probe is expected to
report by redistributing itself within the cell. If the
redistribution is as expected, e.g. if prior knowledge tell that it
should translocate from location X to location Y, it has passed the
first critical test. In this case it can go on to further
characterization and quantification of the response.
[0100] If it does not perform as expected, it may be because the
cell lacks at least one component of the signaling pathway, e.g. a
cell surface receptor, or there is species incompatibility, e.g. if
the probe is modeled on sequence information of a human gene
product, and the cell is of hamster origin. In both instances one
should identify other cell types for the testing process where
these potential problems would not apply.
[0101] If there is no prior knowledge about the pattern of
redistribution, the analysis of the redistribution will have to be
done in greater depth to identify what the essential and indicative
features are, and when this is clear, it can go on to further
characterization and quantification of the response. If no feature
of redistribution can be identified, the problem might be as
mentioned above, and the probe should be retested under more
optimal cellular conditions.
[0102] If the probe does not perform under optimal cellular
conditions it's back to the drawing board.
XIII. Developing an Image-Based Assay Technique
[0103] The process of developing an image-based redistribution
assay begins with either the unplanned experimental observation
that a redistribution phenomenon can be visualized, or the design
of a probe specifically to follow a redistribution phenomenon
already known to occur. In either event, the first and best
exploratory technique is for a trained scientist or technician to
observe the phenomenon. Even with the rapid advances in computing
technology, the human eye-brain combination is still the most
powerful pattern recognition system known, and requires no advance
knowledge of the system in order to detect potentially interesting
and useful patterns in raw data. This is especially if those data
are presented in the form of images, which are the natural "data
type" for human visual processing. Because human visual processing
operates most effectively in a relatively narrow frequency range,
i.e., we cannot see either very fast or very slow changes in our
visual field, it may be necessary to record the data and play it
back with either time dilation or time compression.
[0104] Some luminescence phenomena cannot be seen directly by the
human eye. Examples include polarization and fluorescence lifetime.
However, with suitable filters or detectors, these signals can be
recorded as images or sequences of images and displayed to the
human in the fashion just described. In this way, patterns can be
detected and the same methods can be applied.
[0105] Once the redistribution has been determined to be a
reproducible phenomenon, one or more data sets are generated for
the purpose of developing a procedure for extracting the
quantitative information from the data. In parallel, the biological
and optical conditions are determined which will give the best
quality raw data for the assay. This can become an iterative
process; it may be necessary to develop a quantitative procedure in
order to assess the effect on the assay of manipulating the assay
conditions.
[0106] The data sets are examined by a person or persons with
knowledge of the biological phenomenon and skill in the application
of image processing techniques. The goal of this exercise is to
determine or at least propose a method which will reduce the image
or sequence of images constituting the record of a "response" to a
value corresponding to the degree of the response. Using either
interactive image processing software or an image processing
toolbox and a programming language, the method Is encoded as a
procedure or algorithm which takes the image or images as input and
generates the degree of response (in any units) as its output. Some
of the criteria for evaluating the validity of a particular
procedure are:
[0107] Does the degree of the response vary in a biologically
significant fashion, i.e., does it show the known or putative
dependence on the concentration of the stimulating agent or
condition?
[0108] Is the degree of response reproducible, i.e., does the same
concentration or level of stimulating agent or condition give the
same response with an acceptable variance?
[0109] Is the dynamic range of the response sufficient for the
purpose of the assay? If not, can a change in the procedure or one
of its parameters improve the dynamic range?
[0110] Does the procedure exhibit any clear "pathologies", i.e.,
does it give ridiculous values for the response if there are
commonly occurring imperfections in the imaging process? Can these
pathologies be eliminated, controlled, or accounted for?
[0111] Can the procedure deal with the normal variation in the
number and/or size of cells in an image?
[0112] In some cases the method may be obvious; in others, a number
of possible procedures may suggest themselves. Even if one method
appears clearly superior to others, optimization of parameters may
be required. The various procedures are applied to the data set and
the criteria suggested above are determined, or the single
procedure is applied repeatedly with adjustment of the parameter or
parameters until the most satisfactory combination of signal,
noise, range, etc. are arrived at. This is equivalent to the
calibration of any type of single-channel sensor.
[0113] The number of ways of extracting a single value from an
image are extremely large, and thus an intelligent approach must be
taken to the initial step of reducing this number to a small,
finite number of possible procedures. This is not to say that the
procedure arrived at is necessarily the best procedure--but a
global search for the best procedure is simply out of the question
due to the sheer number of possibilities involved.
[0114] Image-based assays are no different than other assay
techniques in that their usefulness is characterized by parameters
such as the specificity for the desired component of the sample,
the dynamic range, the variance, the sensitivity, the concentration
range over which the assay will work, and other such parameters.
While it is not necessary to characterize each and every one of
these before using the assay, they represent the only way to
compare one assay with another.
XIV. Example: Developing a Quantitative Assay for GLUT4
Translocation
[0115] GLUT4 is a member of the class of glucose transporter
molecules which are important in cellular glucose uptake. It is
known to translocate to the plasma membrane under some conditions
of stimulation of glucose uptake. The ability to visualize the
glucose uptake response noninvasively, without actually measuring
glucose uptake, would be a very useful assay for anyone looking
for, for example, treatments for type II diabetes.
[0116] A CHO cell line which stably expressed the human insulin
receptor was used as the basis for a new cell line which stably
expressed a fusion between GLUT4 and GFP. This cell line was
expected to show translocation of GLUT4 to the plasma membrane as
visualized by the movement of the GFP. The translocation could
definitely be seen in the form of the appearance of local increases
in the fluorescence in regions of the plasma membrane which had a
characteristic shape or pattern. This is shown in FIG. 12.
[0117] These objects became known as "snircles", and the phenomenon
of their appearance as "snircling". In order to quantitate their
appearance, a method had to be found to isolate them as objects in
the image field, and then enumerate them, measure their area, or
determine some parameter about them which correlated in a
dose-dependent fashion with the concentration of insulin to which
the cells had been exposed. In order to separate the snircles, a
binarization procedure was applied in which one copy of the image
smoothed with a relatively severe gaussian kernel (sigma=2.5) was
subtracted from another copy to which only a relatively light
gaussian smooth had been applied (sigma=0.5). The resultant image
was rescaled to its min/max range, and an automatic threshold was
applied to divide the image into two levels. The thresholded image
contains a background of one value all found object with another
value. The found objects were first filtered through a filter to
remove objects far too large and far too small to be snircles. The
remaining objects, which represent snircles and other artifacts
from the image with approximately the same size and intensity
characteristics as snircles, are passed into a classification
procedure which has been previously trained with many images of
snircles to recognize snircles and exclude the other artifacts. The
result of this procedure is a binary image which shows only the
found snircles to the degree to which the classification procedure
can accurately identify them. The total area of the snircles is
then summed and this value is the quantitative measure of the
degree of snircling for that image.
XV. Definitions
[0118] In the present specification and claims, the term "an
influence" covers any influence to which the cellular response
comprises a redistribution. Thus, e.g., heating, cooling, high
pressure, low pressure, humidifying, or drying are influences on
the cellular response on which the resulting redistribution can be
quantified, but as mentioned above, perhaps the most important
influences are the influences of contacting or incubating the cell
or cells with substances which are known or suspected to exert and
influence on the cellular response involving a redistribution
contribution. In another embodiment of the invention the influence
could be substances from a compound drug library.
[0119] In the present context, the term "green fluorescent protein"
is intended to indicate a protein which, when expressed by a cell,
emits fluorescence upon exposure to light of the correct excitation
wavelength (cf [(Chalfie et al. 1994)]). In the following, GFP in
which one or more amino acids have been substituted, inserted or
deleted is most often termed "modified GFP". "GFP" as used herein
includes wild-type GFP derived from the jelly fish Aequorea
victoria and modifications of GFP, such as the blue fluorescent
variant of GFP disclosed by Heim et al. (1994). Proc. Natl. Acad.
Sci. 91:12501, and other modifications that change the spectral
properties of the GFP fluorescence, or modifications that exhibit
increased fluorescence when expressed in cells at a temperature
above about 30 degree C. described in PCT/DK96/00051, published as
WO 97/11094 on Mar. 27, 1997 and hereby incorporated by reference,
and which comprises a fluorescent protein derived from Aequorea
Green Fluorescent Protein (GFP) or any functional analogue thereof,
wherein the amino acid in position 1 upstream from the chromophore
has been mutated to provide an increase of fluorescence intensity
when the fluorescent protein of the invention is expressed in
cells. Preferred GFP variants are F64L-GFP, F64L-Y66H-GFP and
F64L-S65T-GFP. An especially preferred variant of GFP for use in
all the aspects of this invention is EGFP (DNA encoding EGFP which
is a F64L-S65T variant with codons optimized for expression in
mammalian cells is available from Clontech, Palo Alto, plasmids
containing the EGFP DNA sequence, cf. GenBank Acc. Nos. U55762,
U55763).
[0120] The term "intracellular signaling pathway" and "signal
transduction pathway" are intended to indicate the coordinated
intracellular processes whereby a living cell transduce an external
or internal signal into cellular responses. Said signal
transduction will involve an enzymatic reaction said enzymes
include but are not limited to protein kinases, GTPases, ATPases,
protein phosphatases, phospholipases. The cellular responses
include but are not limited to gene transcription, secretion,
proliferation, mechanical activity, metabolic activity, cell
death.
[0121] The term "second messenger" is used to indicate a low
molecular weight component involved in the early events of
intracellular signal transduction pathways.
[0122] The term "luminophore" is used to indicate a chemical
substance which has the property of emitting light either
inherently or upon stimulation with chemical or physical means.
This includes but is not limited to fluorescence, bioluminescence,
phosphorescence, chemiluminescence.
[0123] The term "mechanically intact living cell" is used to
indicate a cell which is considered living according to standard
criteria for that particular type of cell such as maintenance of
normal membrane potential, energy metabolism, proliferative
capability, and has not experienced any physically invasive
treatment designed to introduce external substances into the cell
such as microinjection.
[0124] The term "physiologically relevant" when applied to an
experimentally determined redistribution of an intracellular
component, as measured by a change in the luminescence properties
or distribution, is used to indicate that said redistribution can
be explained in terms of the underlying biological phenomenon which
gives rise to the redistribution.
[0125] The terms "image processing" and "image analysis" are used
to describe a large family of digital data analysis techniques or
combination of such techniques which reduce ordered arrays of
numbers (images) to quantitative information describing those
ordered arrays of numbers. When said ordered arrays of numbers
represent measured values from a physical process, the quantitative
information derived is therefore a measure of the physical
process.
[0126] The term "fluorescent probe" is used to indicate a
fluorescent fusion polypeptide comprising a GFP or any functional
part thereof which is N- or C-terminally fused to a biologically
active polypeptide as defined herein, optionally via a peptide
linker consisting of one or more amino acid residues, where the
size of the linker peptide in itself is not critical as long as the
desired functionality of the fluorescent probe is maintained. A
fluorescent probe according to the invention is expressed in a cell
and basically mimics the physiological behavior of the biologically
active polypeptide moiety of the fusion polypeptide.
[0127] The term "mammalian cell" is intended to indicate any living
cell of mammalian origin. The cell may be an established cell line,
many of which are available from The American Type Culture
Collection (ATCC, Virginia, USA) or a primary cell with a limited
life span derived from a mammalian tissue, including tissues
derived from a transgenic animal, or a newly established immortal
cell line derived from a mammalian tissue including transgenic
tissues, or a hybrid cell or cell line derived by fusing different
cell types of mammalian origin e.g. hybridoma cell lines. The cells
may optionally express one or more non-native gene products, e.g.
receptors, enzymes, enzyme substrates, prior to or in addition to
the fluorescent probe. Preferred cell lines include but are not
limited to those of fibroblast origin, e.g. BHK, CHO, BALB, or of
endothelial origin, e.g. HUVEC, BAE (bovine artery endothelial),
CPAE (cow pulmonary artery endothelial) or of pancreatic origin,
e.g. RIN, INS-1, MIN6, bTC3, aTC6, bTC6, HIT, or of hematopoietic
origin, e.g. adipocyte origin, e.g. 3T3-L1, neuronal/neuroendocrine
origin, e.g. AtT20, PC12, GH3, muscle origin, e.g. SKMC, A10,
C2C12, renal origin, e.g. BEK 293, LLC-PK1.
[0128] The term "hybrid polypeptide" is intended to indicate a
polypeptide which is a fusion of at least a portion of each of two
proteins, in this case at least a portion of the green fluorescent
protein, and at least a portion of a catalytic and/or regulatory
domain of a protein kinase. Furthermore a hybrid polypeptide is
intended to indicate a fusion polypeptide comprising a GFP or at
least a portion of the green fluorescent protein that contains a
functional fluorophore, and at least a portion of a biologically
active polypeptide as defined herein provided that said fusion is
not the PKC.alpha.-GFP, PKC.gamma.-GFP, and PKC.epsilon.-GFP
disclosed by Schmidt et al. and Sakai et al., respectively. Thus,
GFP may be N- or C-terminally tagged to a biologically active
polypeptide, optionally via a linker portion or linker peptide
consisting of a sequence of one or more amino acids. The hybrid
polypeptide or fusion polypeptide may act as a fluorescent probe in
intact living cells carrying a DNA sequence encoding the hybrid
polypeptide under conditions permitting expression of said hybrid
polypeptide.
[0129] The term "kinase" is intended to indicate an enzyme that is
capable of phosphorylating a cellular component.
[0130] The term "protein kinase" is intended to indicate an enzyme
that is capable of phosphorylating serine and/or threonine and/or
tyrosine in peptides and/or proteins.
[0131] The term "phosphatase" is intended to indicate an enzyme
that is capable of dephosphorylating phosphoserine and/or
phosphothreonine and/or phosphotyrosine in peptides and/or
proteins.
[0132] In the present context, the term "biologically active
polypeptide" is intended to indicate a polypeptide affecting
intracellular processes upon activation, such as an enzyme which is
active in intracellular processes or a portion thereof comprising a
desired amino acid sequence which has a biological function or
exerts a biological effect in a cellular system. In the polypeptide
one or several amino acids may have been deleted, inserted or
replaced to alter its biological function, e.g. by rendering a
catalytic site inactive. Preferably, the biologically active
polypeptide is selected from the group consisting of proteins
taking part in an intracellular signaling pathway, such as enzymes
involved in the intracellular phosphorylation and dephosphorylation
processes including kinases, protein kinases and phosphorylases as
defined herein, but also proteins making up the cytoskeleton play
important roles in intracellular signal transduction and are
therefore included in the meaning of "biologically active
polypeptide" herein. More preferably, the biologically active
polypeptide is a protein which according to its state as activated
or non-activated changes localization within the cell, preferably
as an intermediary component in a signal transduction pathway.
Included in this preferred group of biologically active
polypeptides are cAMP dependent protein kinase A.
[0133] The term "a substance having biological activity" is
intended to indicate any sample which has a biological function or
exerts a biological effect in a cellular system. The sample may be
a sample of a biological material such as a sample of a body fluid
including blood, plasma, saliva, milk, urine, or a microbial or
plant extract, an environmental sample containing pollutants
including heavy metals or toxins, or it may be a sample containing
a compound or mixture of compounds prepared by organic synthesis or
genetic techniques.
[0134] The phrase "any change in fluorescence" means any change in
absorption properties, such as wavelength and intensity, or any
change in spectral properties of the emitted light, such as a
change of wavelength, fluorescence lifetime, intensity or
polarization, or any change in the intracellular localization of
the fluorophore. It may thus be localized to a specific cellular
component (e.g. organelle, membrane, cytoskeleton, molecular
structure) or it may be evenly distributed throughout the cell or
parts of the cell.
[0135] The term "organism" as used herein indicates any unicellular
or multicellular organism preferably originating from the animal
kingdom including protozoans, but also organisms that are members
of the plant kingdoms, such as algae, fungi, bryophytes, and
vascular plants are included in this definition.
[0136] The term "nucleic acid" is intended to indicate any type of
poly- or oligonucleic acid sequence, such as a DNA sequence, a cDNA
sequence, or an RNA sequence.
[0137] The term "biologically equivalent" as it relates to proteins
is intended to mean that a first protein is equivalent to a second
protein if the cellular functions of the two proteins may
substitute for each other, e.g. if the two proteins are closely
related isoforms encoded by different genes, if they are splicing
variants, or allelic variants derived from the same gene, if they
perform identical cellular functions in different cell types, or in
different species. The term "biologically equivalent" as it relates
to DNA is intended to mean that a first DNA sequence encoding a
polypeptide is equivalent to a second DNA sequence encoding a
polypeptide if the functional proteins encoded by the two genes are
biologically equivalent.
[0138] The phrase "back-tracking of a signal transduction pathway"
is intended to indicate a process for defining more precisely at
what level a signal transduction pathway is affected, either by the
influence of chemical compounds or a disease state in an organism.
Consider a specific signal transduction pathway represented by the
bioactive polypeptides A-B-C-D, with signal transduction from A
towards D. When investigating all components of this signal
transduction pathway compounds or disease states that influence the
activity or redistribution of only D can be considered to act on C
or downstream of C whereas compounds or disease states that
influence the activity or redistribution of C and D, but not of A
and B can be considered to act downstream of B.
[0139] The term "fixed cells" is used to mean cells treated with a
cytological fixative such as glutaraldehyde or formaldehyde,
treatments which serve to chemically cross-link and stabilize
soluble and insoluble proteins within the structure of the cell.
Once in this state, such proteins cannot be lost from the structure
of the now-dead cell.
EXAMPLES
Example 1
Construction, Testing and Implementation of an Assay for cAMP Based
on PKA Activation in Real Time within Living Cells
[0140] Useful for monitoring the activity of signaling pathways
which lead to altered concentrations of cAMP, e.g. activation of
G-protein coupled receptors which couple to G-proteins of the
G.sub.s or G.sub.I class.
[0141] The catalytic subunit of the murine cAMP dependent protein
kinase (PKAc) was fused C-terminally to a F64L-S65T derivative of
GFP. The resulting fusion (PKAc-F64L-S65T-GFP) was used for
monitoring in vivo the translocation and thereby the activation of
PKA.
[0142] Construction of the PKAc-F64L-S65T-GFP Fusion
[0143] Convenient restriction endonuclease sites were introduced
into the cDNAs encoding murine PKAc (Gen Bank Accession number:
M12303) and F64L-S65T-GFP (sequence disclosed in WO 97/11094) by
polymerase chain reaction (PCR). The PCR reactions were performed
according to standard protocols with the following primers:
TABLE-US-00001 5'PKAc: (SEQ ID NO:3)
TTggACACAAgCTTTggACACCCTCAggATATgggCAACgCCgCCgCCgC CAAg, 3'PKAc:
(SEQ ID NO:4) gTCATCTTCTCgAgTCTTTCAggCgCgCCCAAACTCAg-TAAACTCCTT gC
CACAC, 5'GFP: (SEQ ID NO:1)
TTggACACAAgCTTTggACACggCgCgCCATgAgTAAAggAgAAgAACTT TTC, 3'GFP: (SEQ
ID NO:2) gTCATCTTCTCgAgTCTTACTCCTgAggTTTgTATAgT-TCATCCATgC CA
TgT.
[0144] The PKAc amplification product was then digested with
HindIII+AscI and the F64L-S65T-GFP product with AscI+XhoI. The two
digested PCR products were subsequently ligated with a HindIII+XhoI
digested plasmid (pZeoSV.RTM. mammalian expression vector,
Invitrogen, San Diego, Calif., USA). The resulting fusion construct
(SEQ ID NO: 68 & 69) was under control of the SV40
promoter.
[0145] Transfection and Cell Culture Conditions
[0146] Chinese hamster ovary cells (CHO), were transfected with the
plasmid containing the PKAc-F64L-S65T-GFP fusion using the calcium
phosphate precipitate method in HEPES-buffered saline (Sambrook et
al., 1989). Stable transfectants were selected using 1000 mg
Zeocin/ml (Invitrogen) in the growth medium (DMEM with 1000 mg
glucose/I, 10% fetal bovine serum (FBS), 100 mg
penicillin-streptomycin mixture ml.sup.-1, 2 mM L-glutamine
purchased from Life Technologies Inc., Gaithersburg, Md., USA).
Untransfected CHO cells were used as the control. To assess the
effect of glucagon on fusion protein translocation, the
PKAc-F64L-S65T-GFP fusion was stably expressed in baby hamster
kidney cells overexpressing the human glucagon receptor (BIK/GR
cells) Untransfected BIK/GR cells were used as the control.
Expression of GR was maintained with 500 mg G418/ml (Neo marker)
and PKAc-F64L-S65T-GFP was maintained with 500 mg Zeocin/ml (Sh ble
marker). CHO cells were also simultaneously co-transfected with
vectors containing the PKAc-F64L-S65T-GFP fusion and the human a2a
adrenoceptor (hARa2a).
[0147] For fluorescence microscopy, cells were allowed to adhere to
Lab-Tek chambered cover-glasses (Nalge Nunc Int., Naperville, Ill.,
USA) for at least 24 hours and cultured to about 80% confluence.
Prior to experiments, the cells were cultured over night without
selection pressure in HAM F-12 medium with glutamax (Life
Technologies), 100 mg penicillin-streptomycin mixture ml.sup.-1 and
0.3% FBS. This medium has low autofluorescence enabling
fluorescence microscopy of cells straight from the incubator.
[0148] Monitoring Activity of PKA Activity in Real Time
[0149] Image acquisition of live cells were gathered using a Zeiss
Axiovert 135M fluorescence microscope fitted with a Fluar 40x, NA:
1.3 oil immersion objective and coupled to a Photometrics CH250
charged coupled device (CCD) camera. The cells were illuminated
with a 100 W HBO arc lamp. In the light path was a 470.+-.20 nm
excitation filter, a 510 nm dichroic mirror and a 515.+-.15 nm
emission filter for minimal image background. The cells were kept
and monitored to be at 37 degree C. with a custom built stage
heater.
[0150] Images were processed and analyzed in the following
manner:
[0151] Method 1: Stepwise Procedure for Quantitation of
Translocation of PKA:
[0152] 1. The image was corrected for dark current by performing a
pixel-by-pixel subtraction of a dark image (an image taken under
the same conditions as the actual image, except the camera shutter
is not allowed to open).
[0153] 2. The image was corrected for non-uniformity of the
illumination by performing a pixel-by-pixel ratio with a flat field
correction image (an image taken under the same conditions as the
actual image of a uniformly fluorescent specimen).
[0154] 3. The image histogram, i.e., the frequency of occurrence of
each intensity value in the image, was calculated.
[0155] 4. A smoothed, second derivative of the histogram was
calculated and the second zero is determined. This zero corresponds
to the inflection point of the histogram on the high side of the
main peak representing the bulk of the image pixel values.
[0156] 5. The value determined in step 4 was subtracted from the
image. All negative values were discarded.
[0157] 6. The variance (square of the standard deviation) of the
remaining pixel values was determined. This value represents the
"response" for that image.
[0158] 7. Scintillation proximity assay (SPA) for independent
quantitation of cAMP.
[0159] Method 2: Alternative Method for Quantitation of PKA
Redistribution:
[0160] 1. The fluorescent aggregates are segmented from each image
using an automatically found threshold based on the maximization of
the information measure between the object and background. The a
priori entropy of the image histogram is used as the information
measure.
[0161] 2. The area of each image occupied by the aggregates is
calculated by counting pixels in the segmented areas.
[0162] 3. The value obtained in step 2 for each image in a series,
or treatment pair, is normalized to the value found for the first
(unstimulated) image collected. A value of zero (0) indicates no
redistribution of fluorescence from the starting condition. A value
of one (1) by this method equals full redistribution.
[0163] Cells were cultured in HAM F-12 medium as described above,
but in 96-well plates. The medium was exchanged with Ca.sup.2+-EPES
buffer including 100 mM IBMX and the cells were stimulated with
different concentrations of forskolin for 10 min. Reactions were
stopped with addition of NaOH to 0.14 M and the amount of cAMP
produced was measured with the cAMP-SPA kit, RPA538 (Amersham) as
described by the manufacturer.
[0164] Manipulating intracellular levels of cAMP to test the
PKAc-F64L-S65T-GFP fusion.
[0165] The following compounds were used to vary cAMP levels:
Forskolin, an activator of adenylate cyclase; dbcAMP, a membrane
permeable cAMP analog which is not degraded by phosphodiesterase;
IBMX, an inhibitor of phosphodiesterase.
[0166] CHO cells stably expressing the PKAc-F64L-S65T-GFP, showed a
dramatic translocation of the fusion protein from a punctate
distribution to an even distribution throughout the cytoplasm
following stimulation with 1 mM forskolin (n=3), 10 mM forskolin
(n=4) and 50 mM forskolin (n=4) (FIG. 1), or dbcAMP at 1 mM
(n=6).
[0167] FIG. 2 shows the progression of response in time following
treatment with 1 mM forskolin.
[0168] FIG. 3 gives a comparison of the average temporal profiles
of fusion protein redistribution and a measure of the extent of
each response to the three forskolin concentrations (FIGS. 3A, E,
B), and to 1 mM dbcAMP (FIG. 3C) which caused a similar but slower
response, and to addition of 100 mM IBMX (n=4, FIG. 3D) which also
caused a slow response, even in the absence of adenylate cyclase
stimulation. Addition of buffer (n=2) had no effect (data not
shown).
[0169] As a control for the behavior of the fusion protein,
F64L-S65T-GFP alone was expressed in CHO cells and these were also
given 50 mM forskolin (n=5); the uniform diffuse distribution
characteristic of GFP in these cells was unaffected by such
treatment (data not shown).
[0170] The forskolin induced translocation of PKAc-F64L-S65T-GFP
showed a dose-response relationship (FIGS. 4 and 6), see
quantitative procedures above.
[0171] Reversibility of PKAc-F64L-S65T-GFP Translocation.
[0172] The release of the PKAc probe from its cytoplasmic anchoring
hotspots was reversible. Washing the cells repeatedly (5-8 times)
with buffer after 10 uM forskolin treatment completely restored the
punctate pattern within 2-5 min (n=2, FIG. 3E). In fact the fusion
protein returned to a pattern of fluorescent cytoplasmic aggregates
virtually indistinguishable from that observed before forskolin
stimulation.
[0173] To test whether the return of fusion protein to the
cytoplasmic aggregates reflected a decreased [cAMP]i, cells were
treated with a combination of 10 mM forskolin and 100 mM IBMX (n=2)
then washed repeatedly (5-8 times) with buffer containing 100 mM
IBMX (FIG. 3F). In these experiments, the fusion protein did not
return to its prestimulatory localization after removal of
forskolin.
[0174] Testing the PKA-F64L-S65T-GFP Probe with Physiologically
Relevant Agents.
[0175] To test the probe's response to receptor activation of
adenylate cyclase, BHK cells stably transfected with the glucagon
receptor and the PKA-F64L-S65T-GFP probe were exposed to glucagon
stimulation. The glucagon receptor is coupled to a Gs protein which
activates adenylate cyclase, thereby increasing the cAMP level. In
these cells, addition of 100 nM glucagon (n=2) caused the release
of the PKA-F64L-S65T-GFP probe from the cytoplasmic aggregates and
a resulting translocation of the fusion protein to a more even
cytoplasmic distribution within 2-3 min (FIG. 3G). Similar but less
pronounced effects were seen at lower glucagon concentrations (n=2,
data not shown). Addition of buffer (n=2) had no effect over time
(data not shown).
[0176] Transiently transfected CHO cells expressing hARa2a and the
PKA-F64L-S65T-GFP probe were treated with 10 mM forskolin for 7.5
minutes, then, in the continued presence of forskolin, exposed to
10 mM norepinephrine to stimulate the exogenous adrenoreceptors,
which couple to a G.sub.I protein, which inhibit adenylate cyclase.
This treatment led to reappearance of fluorescence in the
cytoplasmic aggregates indicative of a decrease in [cAMP]i (FIG.
3H).
[0177] Fusion Protein Translocation Correlated with [cAMP]i
[0178] As described above, the time it took for a response to come
to completion was dependent on the forskolin dose (FIG. 5). In
addition the degree of responses was also dose dependent. To test
the PKA-F64L-S65T-GFP fusion protein translocation in a semi high
through-put system, CHO cells stably transfected with the
PKA-F64L-S65T-GFP fusion was stimulated with buffer and 5
increasing doses of forskolin (n=8). Using the image analysis
algorithm described above (Method 1), a dose response relationship
was observed in the range from 0.01-50 mM forskolin (FIG. 6). A
half maximal stimulation was observed at about 2 mM forskolin. In
parallel, cells were stimulated with buffer and 8 increasing
concentrations of forskolin (n=4) in the range 0.01-50 mM. The
amount of cAMP produced was measured in an SPA assay. A steep
increase was observed between 1 and 5 mM forskolin coincident with
the steepest part of the curve for fusion protein translocation
(also FIG. 6)
Example 2
Quantitation of Redistribution in Real-Time within Living Cells
[0179] Probe for Detection of PKC Activity in Real Time within
Living Cells:
[0180] Construction of PKC-GFP Fusion:
[0181] The probe was constructed by ligating two restriction enzyme
treated polymerase chain reaction (PCR) amplification products of
the cDNA for murine PKC.alpha. (GenBank Accession number: M25811)
and F64L-S6ST-GFP (sequence disclosed in WO 97/11094) respectively.
Taq.RTM. polymerase and the following oligonucleotide primers were
used for PCR;
TABLE-US-00002 5'mPKCa: (SEQ ID NO:5)
TTggACACAAgCTTTggACACCCTCAggATATggCTgACgTTTACCCggC CAACg, 3'mPKCa:
(SEQ ID NO:6) gTCATCTTCTCgAgTCTTTCAggCgCgCCCTACTgCAC-TTTgCAAgAT Tg
ggTgC, 5'F64L-S65T-GFP: (SEQ ID NO:1)
TTggACACAAgCTTTggACACggCgCgCCATgAgTAAAggAgAAgAACTT TTC,
3'F64L-S65T-GFP: (SEQ ID NO:2)
gTCATCTTCTCgAgTCTTACTCCTgAggTTTgTATAgT-TCATCCATgC CA TgT.
[0182] The hybrid DNA strand was inserted into the pZeoSV.RTM.
mammalian expression vector as a HindIII-XhoI cassette as described
in example 1.
[0183] Cell Culture:
[0184] BHK cells expressing the human M1 receptor under the control
of the inducible metallothionine promoter and maintained with the
dihydrofolate reductase marker were transfected with the
PKC.alpha.-F64L-S65T-GFP probe using the calcium phosphate
precipitate method in HEPES buffered saline (HBS [pH 7.10]). Stable
transfectants were selected using 1000 ug Zeocin.RTM./ml in the
growth medium (DMEM with 1000 mg glucose/l, 10% foetal bovine serum
(FBS), 100 mg penicillin-streptomycin mixture ml-1, 2 mM
1-glutamine). The hM1 receptor and PKC.alpha.-F64L-S65T-GFP fusion
protein were maintained with 500 nM methotrexate and 500 ug
Zeocin.RTM./ml respectively. 24 hours prior to any experiment, the
cells were transferred to HAM F-12 medium with glutamax, 100 ug
penicillin-streptomycin mixture ml.sup.-1 and 0.3% FBS. This medium
relieves selection pressure, gives a low induction of signal
transduction pathways and has a low autofluorescence at the
relevant wavelength enabling fluorescence microscopy of cells
straight from the incubator.
[0185] Monitoring the PKC Activity in Real Time:
[0186] Digital images of live cells were gathered using a Zeiss
Axiovert 135M fluorescence microscope fitted with a 40x, NA: 1.3
oil immersion objective and coupled to a Photometrics CH250 charged
coupled device (CCD) camera. The cells were illuminated with a 100
W arc lamp. In the light path was a 470.+-.20 nm excitation filter,
a 510 nm dichroic mirror and a 515.+-.15 nm emission filter for
minimal image background. The cells were kept and monitored to be
at 37 degree C. with a custom built stage heater.
[0187] Images were analyzed using the IPLab software package for
Macintosh.
[0188] Upon stimulation of the M1-BHK cells, stably expressing the
PKC alpha-F64L-S65T-GFP fusion, with carbachol we observed a
dose-dependent transient translocation from the cytoplasm to the
plasma membrane (FIGS. 7a,b,c). Simultaneous measurement of the
cytosolic free calcium concentration shows that the
carbachol-induced calcium mobilization precedes the translocation
(FIG. 8).
[0189] Stepwise Procedure for Quantitation of Translocation of
PKC:
[0190] 1. The image was corrected for dark current by performing a
pixel-by-pixel subtraction of a dark image (an image taken under
the same conditions as the actual image, except the camera shutter
is not allowed to open).
[0191] 2. The image was corrected for non-uniformity of the
illumination by performing a pixel-by-pixel ratio with a flat field
correction image (an image taken under the same conditions as the
actual image of a uniformly fluorescent specimen).
[0192] 3. A copy of the image was made in which the edges are
identified. The edges in the image are found by a standard
edge-detection procedure--convolving the image with a kernel which
removes any large-scale unchanging components (i.e., background)
and accentuates any small-scale changes (i.e., sharp edges). This
image was then converted to a binary image by threshholding.
Objects in the binary image which are too small to represent the
edges of cells were discarded. A dilation of the binary image was
performed to close any gaps in the image edges. Any edge objects in
the image which were in contact with the borders of the image are
discarded. This binary image represents the edge mask.
[0193] 4. Another copy of image was made via the procedure in step
3. This copy was further processed to detect objects which enclose
"holes" and setting all pixels inside the holes to the binary value
of the edge, i.e., one. This image represents the whole cell
mask.
[0194] 5. The original image was masked with the edge mask from
step 3 and the sum total of all pixel values is determined.
[0195] 6. The original image was masked with the whole cell mask
from step 4 and the sum total of all pixel values was
determined.
[0196] 7. The value from step 5 was divided by the value from step
6 to give the final result, the fraction of fluorescence intensity
in the cells which was localized in the edges.
Example 3
[0197] Probes for Detection of Mitogen Activated Protein Kinase
Erk1 Redistribution.
[0198] Useful for monitoring signaling pathways involving MAPK,
e.g. to identify compounds which modulate the activity of the
pathway in living cells.
[0199] Erk1, a serine/threonine protein kinase, is a component of a
signaling pathway which is activated by e.g. many growth
factors.
[0200] Probes for Detection of ERK-1 Activity in Real Time within
Living Cells.
[0201] The extracellular signal regulated kinase (ERK-1, a mitogen
activated protein kinase, MAPK) is fused N- or C-terminally to a
derivative of GFP. The resulting fusions expressed in different
mammalian cells are used for monitoring in vivo the nuclear
translocation, and thereby the activation, of ERK1 in response to
stimuli that activate the MAPK pathway.
[0202] a) Construction of Murine ERK1-F64L-S65T-GFP Fusion:
[0203] Convenient restriction endonuclease sites are introduced
into the cDNAs encoding murine ERK1 (GenBank Accession number:
Z14249) and F64L-S65T-GFP (sequence disclosed in WO 97/11094) by
polymerase chain reaction (PCR). The PCR reactions are performed
according to standard protocols with the following primers:
TABLE-US-00003 5'ERK1: (SEQ ID NO:7)
TTggACACAAgCTTTggACACCCTCAggATATggCggCggCggCggCggC TCCggggggCgggg,
3'ERK1: (SEQ ID NO:8) gTCATCTTCTCgAgTCTTTCAggCgCgCCCggggCCCT-
CTggCgCCCC Tg gCTgg, 5'F64L-S65T-GFP: (SEQ ID NO:1)
TTggACACAAgCTTTggACACggCgCgCCATgAgTAAAggAgAAgAACTT TTC
3'F64L-S65T-GFP: (SEQ ID NO:2)
gTCATCTTCTCgAgTCTTACTCCTgAggTTTgTATAgT- TCATCCATgC CA TgT
[0204] To generate the mERK1-F64L-S65T-GFP (SEQ ID NO: 56 & 57)
fusion the ERK1 amplification product is digested with HindIII+AscI
and the F64L-S65T-GFP product with AscI+XhoI. To generate the
F64L-S65T-GFP-mERK1 fusion the ERK1 amplification product is then
digested with HindIII+Bsu36I and the F64L-S65T-GFP product with
Bsu36I+XhoI. The two pairs of digested PCR products are
subsequently ligated with a HindIII+XhoI digested plasmid
(pZeoSV.RTM.) mammalian expression vector, Invitrogen, San Diego,
Calif., USA). The resulting fusion constructs are under control of
the SV40 promoter.
[0205] b) The human Erk1 gene (GenBank Accession number: X60188)
was amplified using PCR according to standard protocols with
primers Erk1-top (SEQ ID NO: 9) and Erk1-bottom/+stop (SEQ ID NO:
10). The PCR product was digested with restriction enzymes EcoR1
and BamH1, and ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank
Accession number U55763) digested with EcoR1 and BamH1. This
produces an EGFP-Erk1 fusion (SEQ ID NO:38 &39) under the
control of a CMV promoter.
[0206] The plasmid containing the EGFP-Erk1 fusion was transfected
into HEK293 cells employing the FUGENE transfection reagent
(Boehringer Mannheim). Prior to experiments the cells were grown to
80%-90% confluency 8 well chambers in DMEM with 10% FCS. The cells
were washed in plain HAM F-12 medium (without FCS), and then
incubated for 30-60 minutes in plain HAM F-12 (without FCS) with
100 micromolar PD98059, an inhibitor of MEK1, a kinase which
activates Erk1; this step effectively empties the nucleus of
EGFP-Erk1. Just before starting the experiment, the HAM F-12 was
replaced with Hepes buffer following a wash with Hepes buffer. This
removes the PD98059 inhibitor; if blocking of MEK1 is still wanted
(e.g. in control experiments), the inhibitor is included in the
Hepes buffer.
[0207] The experimental setup of the microscope was as described in
example 1.
[0208] 60 images were collected with 10 seconds between each, and
with the test compound added after image number 10.
[0209] Addition of EGF (1-100 nM) caused within minutes a
redistribution of EGFP-Erk1 from the cytoplasm into the nucleus
(FIGS. 9a,b).
[0210] The response was quantitated as described below and a
dose-dependent relationship between EGF concentration and nuclear
translocation of EGFP-Erk1 was found (FIGS. 9c,d). Reditribution of
GFP fluorescence is expressed in this example as the change in the
ratio value between areas in nuclear versus cytoplasmic
compartments of the cell. Each time profile is the average of
nuclear to cytoplasmic ratios from six cells in each treatment.
Example 4
Probes for Detection of Erk2 Redistribution
[0211] Useful for monitoring signaling pathways involving MAPK,
e.g. to identify compounds which modulate the activity of the
pathway in living cells.
[0212] Erk2, a serine/threonine protein kinase, is closely related
to Erk1 but not identical; it is a component of a signaling pathway
which is activated by e.g. many growth factors.
[0213] a) The rat Erk2 gene (GenBank Accession number: M64300) was
amplified using PCR according to standard protocols with primers
Erk2-top (SEQ ID NO: 11) and Erk2-bottom/+stop (SEQ ID NO: 13) The
PCR product was digested with restriction enzymes Xho1 and BamH1,
and ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank Accession
number U55763) digested with Xho1 and BamH1. This produces an
EGFP-Erk2 fusion (SEQ ID NO:40 &41) under the control of a CMV
promoter.
[0214] b) The rat Erk2 gene (GenBank Accession number: M64300) was
amplified using PCR according to standard protocols with primers
(SEQ ID NO: 11) Erk2-top and Erk2-bottom/-stop (SEQ ID NO: 12). The
PCR product was digested with restriction enzymes Xho1 and BamH1,
and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank Accession
number U55762) digested with Xho1 and BamH1. This produces an
Erk2-EGFP fusion (SEQ ID NO:58 &59) under the control of a CMV
promoter.
[0215] The resulting plasmids were transfected into CHO cells and
BHK cells. The cells were grown under standard conditions. Prior to
experiments, the cells were starved in medium without serum for
48-72 hours. This led to a predominantly cytoplasmic localization
of both probes, especially in BHK cells. 10% fetal calf serum was
added to the cells and the fluorescence of the cells was recorded
as explained in example 3. Addition of serum caused the probes to
redistribute into the nucleus within minutes of addition of
serum.
Example 5
Probes for Detection of Smad2 Redistribution
[0216] Useful for monitoring signaling pathways activated by some
members of the transforming growth factor-beta family, e.g. to
identify compounds which modulate the activity of the pathway in
living cells.
[0217] Smad 2, a signal transducer, is a component of a signaling
pathway which is induced by some members of the TGFbeta family of
cytokines.
[0218] a) The human Smad2 gene (GenBank Accession number: AF027964)
was amplified using PCR according to standard protocols with
primers Smad2-top (SEQ ID NO:24) and Smad2-bottom/+stop (SEQ ID
NO:26). The PCR product was digested with restriction enzymes EcoR1
and Acc65I, and ligated into pEGFP-C1 (Clontech; Palo Alto; GenBank
Accession number U55763) digested with EcoR1 and Acc65I. This
produces an EGFP-Smad2 fusion (SEQ ID NO: 50 & 51) under the
control of a CMV promoter.
[0219] b) The human Smad2 gene (GenBank Accession number: AF027964)
was amplified using PCR according to standard protocols with
primers Smad2-top (SEQ ID NO:24) and Smad2-bottom/-stop (SEQ ID
NO:25). The PCR product was digested with restriction enzymes EcoR1
and Acc65I, and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank
Accession number U55762) digested with EcoR1 and Acc65I. This
produces a Smad2-EGFP fusion (SEQ ID NO:74 &75) under the
control of a CMV promoter.
[0220] The plasmid containing the EGFP-Smad2 fusion was transfected
into cells, where it showed a cytoplasmic distribution. Prior to
experiments the cells were grown in 8 well Nunc chambers in DMEM
with 10% FCS to 80% confluency and starved overnight in HAM F-12
medium without FCS.
[0221] For experiments, the HAM F-12 medium was replaced with Hepes
buffer pH 7.2.
[0222] The experimental setup of the microscope was as described in
example 1.
[0223] 90 images were collected with 10 seconds between each, and
with the test compound added after image number 5.
[0224] After serum starvation of cells, each nucleus contains less
GFP fluorescence than the surrounding cytoplasm (FIG. 10a).
Addition of TGFbeta caused within minutes a redistribution of
EGFP-Smad2 from the cytoplasm into the nucleus (FIG. 10b).
[0225] The redistribution of fluorescence within the treated cells
was quantified simply as the fractional increase in nuclear
fluorescence normalized to the starting value of GFP fluorescence
in the nucleus of each unstimulated cell.
Example 6
Probe for Detection of VASP Redistribution
[0226] Useful for monitoring signaling pathways involving
rearrangement of cytoskeletal elements, e.g. to identify compounds
which modulate the activity of the pathway in living cells.
[0227] VASP, a phosphoprotein, is a component of cytoskeletal
structures, which redistributes in response to signals which affect
focal adhesions.
[0228] a) The human VASP gene (GenBank Accession number: Z46389)
was amplified using PCR according to standard protocols with
primers VASP-top (SEQ ID NO:94) and VASP-bottom/+stop (SEQ ID
NO:95). The PCR product was digested with restriction enzymes Hind3
and BamH1, and ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank
Accession number U55763) digested with Hind3 and BamH1. This
produces an EGFP-VASP fusion (SEQ ID NO: 124 & 125) under the
control of a CMV promoter.
[0229] The resulting plasmid was transfected into CHO cells
expressing the human insulin receptor using the calcium-phosphate
transfection method. Prior to experiments, cells were grown in 8
well Nunc chambers and starved overnight in medium without FCS.
[0230] Experiments are performed in a microscope setup as described
in example 1.
[0231] 10% FCS was added to the cells and images were collected.
The EGFP-VASP fusion was redistributed from a somewhat even
distribution near the periphery into more localized structures,
identified as focal adhesion points (FIG. 11).
[0232] A large number of further GFP fusions have been made or are
in the process of being made, as apparent from the following
Examples 7-22 which also suggest suitable host cells and substances
for activation of the cellular signaling pathways to be monitored
and analyzed.
Example 7
Probe for Detection of Actin Redistribution
[0233] Useful for monitoring signaling pathways involving
rearrangement or formation of actin filaments, e.g. to identify
compounds which modulate the activity of pathways leading to
cytoskeletal rearrangements in living cells.
[0234] Actin is a component of cytoskeletal structures, which
redistributes in response to very many cellular signals.
[0235] The actin binding domain of the human alpha-actinin gene
(GenBank Accession number: X15804) was amplified using PCR
according to standard protocols with primers ABD-top (SEQ ID NO:90)
and ABD-bottom/-stop (SEQ ID NO:91). The PCR product was digested
with restriction enzymes Hind3 and BamH1, and ligated into pEGFP-N1
(Clontech, Palo Alto; GenBank Accession number U55762) digested
with Hind3 and BamH1. This produced an actin-binding-domain-EGFP
fusion (SEQ ID NO: 128 &129) under the control of a CMV
promoter.
[0236] The resulting plasmid was transfected into CHO cells
expressing the human insulin receptor. Cells were stimulated with
insulin which caused the actin binding domain-EGFP probe to become
redistributed into morphologically distinct membrane-associated
structures.
Example 8
Probes for Detection of p38 Redistribution
[0237] Useful for monitoring signaling pathways responding to
various cellular stress situations, e.g. to identify compounds
which modulate the activity of the pathway in living cells, or as a
counterscreen.
[0238] p38, a serine/thronine protein kinase, is a component of a
stress-induced signaling pathway which is activated by many types
of cellular stress, e.g. TNFalpha, anisomycin, UV and mitomycin
C.
[0239] a) The human p38 gene (GenBank Accession number: L35253) was
amplified using PCR according to standard protocols with primers
p38-top (SEQ ID NO:14) and p38-bottom/+stop (SEQ ID NO: 16). The
PCR product was digested with restriction enzymes Xho1 and BamH1,
and ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank Accession
number U55763) digested with Xho1 and BamH1. This produced an
EGFP-p38 fusion (SEQ ID NO:46 &47) under the control of a CMV
promoter.
[0240] b) The human p38 gene (GenBank Accession number: L35253) was
amplified using PCR according to standard protocols with primers
p38-top (SEQ ID NO:13) and p38-bottom/-stop (SEQ ID NO:15). The PCR
product was digested with restriction enzymes Xho1 and BamH1, and
ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank Accession
number U55762) digested with Xho1 and BamH1. This produced a
p38-EGFP fusion (SEQ ID NO:64 & 65) under the control of a CMV
promoter.
[0241] The resulting plasmids are transfected into a suitable cell
line, e.g. HEK293, in which the EGFP-p38 probe and/or the p38-EGFP
probe should change its cellular distribution from predominantly
cytoplasmic to nuclear within minutes in response to activation of
the signaling pathway with e.g. anisomycin.
Example 9
Probes for Detection of Jnk1 Redistribution
[0242] Useful for monitoring signaling pathways responding to
various cellular stress situations, e.g. to identify compounds
which modulate the activity of the pathway in living cells, or as a
counterscreen.
[0243] Jnk1, a serine/threonine protein kinase, is a component of a
stress-induced signaling pathway different from the p38 described
above, though it also is activated by many types of cellular
stress, e.g. TNFalpha, anisomycin and UV.
[0244] a) The human Jnk1 gene (GenBank Accession number: L26318)
was amplified using PCR according to standard protocols with
primers Jnk-top (SEQ ID NO: 17) and Jnk-bottom/+stop (SEQ ID NO:
19). The PCR product was digested with restriction enzymes Xho1 and
BamH1, and ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank
Accession number U55763) digested with Xho1 and BamH1. This
produced an EGFP-Jnk1 fusion (SEQ ID NO:44 & 45) under the
control of a CMV promoter.
[0245] b) The human Jnk1 gene (GenBank Accession number: L26318)
was amplified using PCR according to standard protocols with
primers Jnk-top (SEQ ID NO:17) and Jnk-bottom/-stop (SEQ ID NO:18).
The PCR product was digested with restriction enzymes Xho1 and
BamH1, and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank
Accession number U55762) digested with Xho1 and BamH1. This
produced a Jnk1-EGFP fusion (SEQ ID NO:62 & 63) under the
control of a CMV promoter.
[0246] The resulting plasmids are transfected into a suitable cell
line, e.g. HEK293, in which the EGFP-Jnk1 probe and/or the
Jnk1-EGFP probe should change its cellular distribution from
predominantly cytoplasmic to nuclear in response to activation of
the signaling pathway with e.g. anisomycin.
Example 10
Probes for Detection of PKG Redistribution
[0247] Useful for monitoring signaling pathways involving changes
in cyclic GMP levels, e.g. to identify compounds which modulate the
activity of the pathway in living cells.
[0248] PGK, a cGMP-dependent serine/threonine protein kinase,
mediates the guanylylcyclase/cGMP signal.
[0249] a) The human PKG gene (GenBank Accession number: Y07512) is
amplified using PCR according to standard protocols with primers
PKG-top (SEQ ID NO:81) and PKG-bottom/+stop (SEQ ID NO:83). The PCR
product is digested with restriction enzymes Xho1 and BamH1, and
ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank Accession
number U55763) digested with Xho1 and BamH1. This produces an
EGFP-PKG fusion (SEQ ID NO: 134 & 135) under the control of a
CMV promoter.
[0250] b) The human PKG gene (GenBank Accession number: Y07512) is
amplified using PCR according to standard protocols with primers
PKG-top (SEQ ID NO:81) and PKG-bottom/-stop (SEQ ID NO: 82). The
PCR product is digested with restriction enzymes Xho1 and BamH1,
and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank Accession
number U55762) digested with Xho1 and BamH1. This produces a
PKG-EGFP fusion (SEQ ID NO:136 & 137) under the control of a
CMV promoter.
[0251] The resulting plasmids are transfected into a suitable cell
line, e.g. A10, in which the EGFP-PKG probe and/or the PKG-EGFP
probe should change its cellular distribution from cytoplasmic to
one associated with cytoskeletal elements within minutes in
response to treatment with agents which raise nitric oxide (NO)
levels.
Example 11
Probes for Detection of IkappaB Kinase Redistribution
[0252] Useful for monitoring signaling pathways leading to NFkappaB
activation, e.g. to identify compounds which modulate the activity
of the pathway in living cells.
[0253] IkappaB kinase, a serine/threonine kinase, is a component of
a signaling pathway which is activated by a variety of inducers
including cytokines, lymphokines, growth factors and stress.
[0254] a) The alpha subunit of the human IkappaB kinase gene
(GenBank Accession number: AF009225) is amplified using PCR
according to standard protocols with primers IKK-top (SEQ ID NO:96)
and IKK-bottom/+stop (SEQ ID NO:98). The PCR product is digested
with restriction enzymes EcoR1 and Acc65I, and ligated into
pEGFP-C1 (Clontech, Palo Alto; GenBank Accession number U55763)
digested with EcoR1 and Acc65I. This produces an
EGFP-IkappaB-kinase fusion (SEQ ID NO: 120 & 121) under the
control of a CMV promoter.
[0255] b) The alpha subunit of the human IkappaB kinase gene
(GenBank Accession number: AF009225) is amplified using PCR
according to, standard protocols with primers IKK-top (SEQ ID
NO:96) and IKK-bottom/-stop (SEQ ID NO:97). The PCR product is
digested with restriction enzymes EcoR1 and Acc65I, and ligated
into pEGFP-N1 (Clontech, Palo Alto; GenBank Accession number
U55762) digested with EcoR1 and Acc65I. This produces an
IkappaB-kinase-EGFP fusion (SEQ ID NO: 122 & 123) under the
control of a CMV promoter.
[0256] The resulting plasmids are transfected into a suitable cell
line, e.g. Jurkat, in which the EGFP-IkappaB-kinase probe and/or
the IkappaB-kinase-EGFP probe should achieve a more cytoplasmic
distribution within seconds following stimulation with e.g.
TNFalpha.
Example 12
Probes for Detection of CDK2 Redistribution
[0257] Useful for monitoring signaling pathways of the cell cycle,
e.g. to identify compounds which modulate the activity of the
pathway in living cells.
[0258] CDK2, a cyclin-dependent serine/threonine kinase, is a
component of the signaling system which regulates the cell
cycle.
[0259] a) The human CDK2 gene (GenBank Accession number: X61622) is
amplified using PCR according to standard protocols with primers
CDK2-top (SEQ ID NO:102) and CDK2-bottom/+stop (SEQ ID NO: 104).
The PCR product is digested with restriction enzymes Xho1 and
BamH1, and ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank
Accession number U55763) digested with Xho1 and BamH1. This
produces an EGFP-CDK2 fusion (SEQ ID NO: 114 & 115) under the
control of a CMV promoter.
[0260] b) The human CDK2 gene (GenBank Accession number: X61622) is
amplified using PCR according to standard protocols with primers
CDK2-top (SEQ ID NO: 102) and CDK2-bottom/-stop (SEQ ID NO: 103).
The PCR product is digested with restriction enzymes Xho1 and
BamH1, and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank
Accession number U55762) digested with Xho1 and BamH1. This
produces a CDK2-EGFP fusion (SEQ ID NO: 112 & 113) under the
control of a CMV promoter.
[0261] The resulting plasmids are transfected into a suitable cell
line, e.g. HEK293 in which the EGFP-CDK2 probe and/or the CDK2-EGFP
probe should change its cellular distribution from cytoplasmic in
contact-inhibited cells, to nuclear location in response to
activation with a number of growth factors, e.g. IGF.
Example 13
Probes for Detection of Grk5 Redistribution
[0262] Useful for monitoring signaling pathways involving
desensitization of G-protein coupled receptors, e.g. to identify
compounds which modulate the activity of the pathway in living
cells.
[0263] Grk5, a G-protein coupled receptor kinase, is a component of
signaling pathways involving membrane bound G-protein coupled
receptors.
[0264] a) The human Grk5 gene (GenBank Accession number: L15388) is
amplified using PCR according to standard protocols with primers
Grk5-top (SEQ ID NO:27) and Grk5-bottom/+stop (SEQ ID NO:29). The
PCR product is digested with restriction enzymes EcoR1 and BamH1,
and ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank Accession
number U55763) digested with EcoR1 and BamH1. This produces an
EGFP-Grk5 fusion (SEQ ID NO:42 & 43) under the control of a CMV
promoter.
[0265] b) The human Grk5 gene (GenBank Accession number: L15388) is
amplified using PCR according to standard protocols with primers
Grk5-top (SEQ ID NO:27) and Grk5-bottom/-stop (SEQ ID NO:28). The
PCR product is digested with restriction enzymes EcoR1 and BamH1,
and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank Accession
number U55762) digested with EcoR1 and BamH1. This produces a
Grk5-EGFP fusion (SEQ ID NO:60 & 61) under the control of a CMV
promoter.
[0266] The resulting plasmids are transfected into a suitable cell
line, e.g. HEK293 expressing a rat dopamine DIA receptor, in which
the EGFP-Grk5 probe and/or the Grk5-EGFP probe should change its
cellular distribution from predominantly cytoplasmic to peripheral
in response to activation of the signaling pathway with e.g.
dopamine.
Example 14
[0267] Probes for Detection of Zap70 Redistribution
[0268] Useful for monitoring signaling pathways involving the T
cell receptor, e.g. to identify compounds which modulate the
activity of the pathway in living cells.
[0269] Zap70, a tyrosine kinase, is a component of a signaling
pathway which is active in e.g. T-cell differentiation.
[0270] a) The human Zap70 gene (GenBank Accession number: L05148)
is amplified using PCR according to standard protocols with primers
Zap70-top (SEQ ID NO: 105) and Zap70-bottom/+stop (SEQ ID NO: 107).
The PCR product is digested with restriction enzymes EcoR1 and
BamH1, and ligated into pEGFP-C1 (GenBank Accession number U55763)
digested with EcoR1 and BamH1. This produces an EGFP-Zap70 fusion
(SEQ ID NO: 108 & 109) under the control of a CMV promoter.
[0271] b) The human Zap70 gene (GenBank Accession number: L05148)
is amplified using PCR according to standard protocols with primers
Zap70-top (SEQ ID NO: 105) and Zap70-bottom/-stop (SEQ ID NO: 106).
The PCR product is digested with restriction enzymes EcoR1 and
BamH1, and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank
Accession number U55762) digested with EcoR1 and BamH1. This
produces a Zap70-EGFP fusion (SEQ ID NO: 110 & 111) under the
control of a CMV promoter.
[0272] The resulting plasmids are transfected into a suitable cell
line, e.g. Jurkat, in which the EGFP-Zap70 probe and/or the
Zap70-EGFP probe should change its cellular distribution from
cytoplasmic to membrane-associated within seconds in response to
activation of the T cell receptor signaling pathway with e.g.
antibodies to CD3epsilon.
Example 15
Probes for Detection of p85 Redistribution
[0273] Useful for monitoring signaling pathways involving PI-3
kinase, e.g. to identify compounds which modulate the activity of
the pathway in living cells.
[0274] p85alpha is the regulatory subunit of PI3-kinase which is a
component of many pathways involving membrane-bound tyrosine kinase
receptors and G-protein-coupled receptors.
[0275] a) The human p85alpha gene (GenBank Accession number M61906)
was amplified using PCR according to standard protocols with
primers p85-top-C (SEQ ID NO:22) and p85-bottom/+stop (SEQ ID
NO:23). The PCR product was digested with restriction enzymes BgI2
and BamH1, and ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank
Accession number U55763) digested with BgI2 and BamH1. This
produced an EGFP-p85alpha fusion (SEQ ID NO:48 & 49) under the
control of a CMV promoter.
[0276] b) The human p85alpha gene (GenBank Accession number:
M61906) was amplified using PCR according to standard protocols
with primers p85-top-N (SEQ ID NO:20) and p85-bottom/-stop (SEQ ID
NO:21). The PCR product was digested with restriction enzymes EcoR1
and BamH1, and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank
Accession number U55762) digested with EcoR1 and BamH1. This
produced a p85alpha-EGFP fusion (SEQ ID NO:66 & 67) under the
control of a CMV promoter.
[0277] The resulting plasmids are transfected into a suitable cell
line, e.g. CHO expressing the human insulin receptor, in which the
EGFP-p85 probe and/or the p85-EGFP probe may change its cellular
distribution from cytoplasmic to membrane-associated within minutes
in response to activation of the receptor with insulin.
Example 16
Probes for Detection of Protein-Tyrosine Phosphatase
Redistribution
[0278] Useful for monitoring signaling pathways involving tyrosine
kinases, e.g. to identify compounds which modulate the activity of
the pathway in living cells.
[0279] Protein-tyrosine phosphatase1C, a tyrosine-specific
phosphatase, is an inhibitory component in signaling pathways
involving e.g. some growth factors.
[0280] a) The human protein-tyrosine phosphatase 1C gene (GenBank
Accession number: X62055) is amplified using PCR according to
standard protocols with primers PTP-top (SEQ ID NO:99) and
PTP-bottom/+stop (SEQ ID NO:101). The PCR product is digested with
restriction enzymes Xho1 and EcoR1, and ligated into pEGFP-C1
(Clontech, Palo Alto; GenBank Accession number U55763) digested
with Xho1 and EcoR1. This produces an EGFP-PTP fusion (SEQ ID NO:
116 & 117) under the control of a CMV promoter.
[0281] b) The human protein-tyrosine phosphatase 1C gene (GenBank
Accession number: X62055) is amplified using PCR according to
standard protocols with primers PTP-top (SEQ ID NO:99) and
PTP-bottom/-stop (SEQ ID NO:100). The PCR product is digested with
restriction enzymes Xho1 and EcoR1, and ligated into pEGFP-N1
(Clontech, Palo Alto; GenBank Accession number U55762) digested
with Xho1 and EcoR1. This produces a PTP-EGFP fusion (SEQ ID NO:
118 & 119) under the control of a CMV promoter.
[0282] The resulting plasmids are transfected into a suitable cell
line, e.g. MCF-7 in which the EGFP-PTP probe and/or the PTP-EGFP
probe should change its cellular distribution from cytoplasm to the
plasma membrane within minutes in response to activation of the
growth inhibitory signaling pathway with e.g. somatostatin.
Example 17
Probes for Detection of Smad4 Redistribution
[0283] Useful for monitoring signaling pathways involving most
members of the transforming growth factor-beta family, e.g. to
identify compounds which modulate the activity of the pathway in
living cells.
[0284] Smad4, a signal transducer, is a common component of
signaling pathways induced by various members of the TGFbeta family
of cytokines.
[0285] a) The human Smad4 gene (GenBank Accession number: U44378)
was amplified using PCR according to standard protocols with
primers Smad4-top and Smad4-bottom/+stop (SEQ ID NO:35). The PCR
product was digested with restriction enzymes EcoR1 and BamH1, and
ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank Accession
number U55763) digested with EcoR1 and BamH1. This produce an
EGFP-Smad4 fusion (SEQ ID NO:52 & 53) under the control of a
CMV promoter.
[0286] b) The human Smad4 gene (GenBank Accession number: U44378)
was amplified using PCR according to standard protocols with
primers Smad4-top (SEQ ID NO:33) and Smad4-bottom/-stop (SEQ ID
NO:34). The PCR product was digested with restriction enzymes EcoR1
and BamH1, and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank
Accession number U55762) digested with EcoR1 and BamH1. This
produced a Smad4-EGFP fusion (SEQ ID NO:76 & 77) under the
control of a CMV promoter.
[0287] The resulting plasmids are transfected into a cell line,
e.g. HEK293 in which the EGFP-Smad4 probe and/or the Smad4-EGFP
probe should change its cellular distribution within minutes from
cytoplasmic to nuclear in response to activation of the signaling
pathway with e.g. TGFbeta.
Example 18
Probes for Detection of Stat5 Redistribution
[0288] Useful for monitoring signaling pathways involving the
activation of tyrosine kinases of the Jak family, e.g. to identify
compounds which modulate the activity of the pathway in living
cells.
[0289] Stat5, signal transducer and activator of transcription, is
a component of signaling pathways which are induced by e.g. many
cytokines and growth factors.
[0290] a) The human Stat5 gene (GenBank Accession number: L41142)
was amplified using PCR according to standard protocols with
primers Stat5-top (SEQ ID NO:30) and Stat5-bottom/+stop (SEQ ID
NO:32). The PCR product was digested with restriction enzymes BgI2
and Acc65I, and ligated into pEGFP-C1 (Clontech; Palo Alto; GenBank
Accession number U55763) digested with BgI2 and Acc65I. This
produced an EGFP-Stat5 fusion (SEQ ID NO:54 & 55) under the
control of a CMV promoter.
[0291] b) The human Stat5 gene (GenBank Accession number: L41142)
was amplified using PCR according to standard protocols with
primers Stat5-top (SEQ ID NO:30) and Stat5-bottom/-stop (SEQ ID
NO:331). The PCR product was digested with restriction enzymes BgI2
and Acc65I, and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank
Accession number U55762) digested with BgI2 and Acc65I. This
produced a Stat5-EGFP fusion (SEQ ID NO:78 & 79) under the
control of a CMV promoter.
[0292] The resulting plasmids are transfected into a suitable cell
line, e.g. MIN6 in which the EGFP-Stat5 probe and/or the Stat5-EGFP
probe should change its cellular distribution from cytoplasmic to
nuclear within minutes in response to activation signaling pathway
with e.g. prolactin.
Example 19
Probes for Detection of NFAT Redistribution
[0293] Useful for monitoring signaling pathways involving
activation of NFAT, e.g. to identify compounds which modulate the
activity of the pathway in living cells.
[0294] NFAT, an activator of transcription, is a component of
signaling pathways which is involved in e.g. immune responses.
[0295] a) The human NFAT1 gene (GenBank Accession number: U43342)
is amplified using PCR according to standard protocols with primers
NFAT-top (SEQ ID NO:84) and NFAT-bottom/+stop (SEQ ID NO:86). The
PCR product is digested with restriction enzymes Xho1 and EcoR1,
and ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank Accession
number U55763) digested with Xho1 and EcoR1. This produces an
EGFP-NFAT fusion (SEQ ID NO: 130 & 131) under the control of a
CMV promoter.
[0296] b) The human NFAT gene (GenBank Accession number: U43342) is
amplified using PCR according to standard protocols with primers
NFAT-top (SEQ ID NO:84) and NFAT-bottom/-stop (SEQ ID NO:85). The
PCR product is digested with restriction enzymes Xho1 and Ecor1,
and ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank Accession
number U55762) digested with Xho1 and EcoR1. This produces an
NFAT-EGFP fusion (SEQ ID NO: 132 & 133) under the control of a
CMV promoter.
[0297] The resulting plasmids are transfected into a suitable cell
line, e.g. Jurkat, in which the EGFP-NFAT probe and/or the
NFAT-EGFP probe should change its cellular distribution from
cytoplasmic to nuclear within minutes in response to activation of
the signaling pathway with e.g. antibodies to CD3epsilon.
Example 20
Probes for Detection of NFkappaB Redistribution
[0298] Useful for monitoring signaling pathways leading to
activation of NFkappaB, e.g. to identify compounds which modulate
the activity of the pathway in living cells.
[0299] NFkappaB, an activator of transcription, is a component of
signaling pathways which are responsive to a variety of inducers
including cytokines, lymphokines, some immunosuppressive
agents.
[0300] a) The human NFkappaB p65 subunit gene (GenBank Accession
number: M62399) is amplified using PCR according to standard
protocols with primers NFkappaB-top (SEQ ID NO:87) and
NFkappaB-bottom/+stop (SEQ ID NO:89). The PCR product is digested
with restriction enzymes Xho1 and BamH1, and ligated into pEGFP-C1
(Clontech, Palo Alto; GenBank Accession number U55763) digested
with Xho1 and BamH1. This produces an EGFP-NFkappaB fusion (SEQ ID
NO: 142 & 143) under the control of a CMV promoter.
[0301] b) The human NFkappaB p65 subunit gene (GenBank Accession
number: M62399) is amplified using PCR according to standard
protocols with primers NFkappaB-top (SEQ ID NO:87) and
NFkappaB-bottom/-stop (SEQ ID NO:88). The PCR product is digested
with restriction enzymes Xho1 and BamH1, and ligated into pEGFP-N1
(Clontech, Palo Alto; GenBank Accession number U55762) digested
with Xho1 and BamH1. This produces an NFkappaB-EGFP fusion (SEQ ID
NO:140 & 141) under the control of a CMV promoter.
[0302] The resulting plasmids are transfected into a suitable cell
line, e.g. Jurkat, in which the EGFP-NFkappaB probe and/or the
NFkappaB-EGFP probe should change its cellular distribution from
cytoplasmic to nuclear in response to activation of the signaling
pathway with e.g. TNFalpha.
Example 21
Probe for Detection of RhoA Redistribution
[0303] Useful for monitoring signaling pathways involving RhoA,
e.g. to identify compounds which modulate the activity of the
pathway in living cells.
[0304] RhoA, a small GTPase, is a component of many signaling
pathways, e.g. LPA induced cytoskeletal rearrangements.
[0305] The human RhoA gene (GenBank Accession number: L25080) was
amplified using PCR according to standard protocols with primers
RhoA-top (SEQ ID NO:92) and RhoA-bottom/+stop (SEQ ID NO:93). The
PCR product was digested with restriction enzymes Hind3 and BamH1,
and ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank Accession
number U55763) digested with Hind3 and BamH1. This produced an
EGFP-RhoA fusion (SEQ ID NO: 126 & 127) under the control of a
CMV promoter.
[0306] The resulting plasmid is transfected into a suitable cell
line, e.g. Swiss3T3, in which the EGFP-RhoA probe should change its
cellular distribution from a reasonably homogenous to a peripheral
distribution within minutes of activation of the signaling pathway
with e.g. LPA.
Example 22
Probes for Detection of PKB Redistribution
[0307] Useful for monitoring signaling pathways involving PKB e.g.
to identify compounds which modulate the activity of the pathway in
living cells.
[0308] PKB, a serine/threonine kinase, is a component in various
signaling pathways, many of which are activated by growth
factors.
[0309] a) The human PKB gene (GenBank Accession number: M63167) is
amplified using PCR according to standard protocols with primers
PKB-top (SEQ ID NO:36) and PKB-bottom/+stop (SEQ ID NO:80). The PCR
product is digested with restriction enzymes Xho1 and BamH1, and
ligated into pEGFP-C1 (Clontech, Palo Alto; GenBank Accession
number U55763) digested with Xho1 and BamH1. This produces an
EGFP-PKB fusion (SEQ ID NO: 138 & 139) under the control of a
CMV promoter.
[0310] b) The human PKB gene (GenBank Accession number M63167) was
amplified using PCR according to standard protocols with primers
PKB-top (SEQ ID NO:36) and PKB-bottom/-stop (SEQ ID NO:37). The PCR
product was digested with restriction enzymes Xho1 and BamH1, and
ligated into pEGFP-N1 (Clontech, Palo Alto; GenBank Accession
number U55762) digested with Xho1 and BamH1. This produced a
PKB-EGFP fusion (SEQ ID NO:70 & 71) under the control of a CMV
promoter.
[0311] The resulting plasmids are transfected into a suitable cell
line, e.g. CHO expressing the human insulin receptor, in which the
EGFP-PKB probe and/or the PKB-EGFP probe cycles between cytoplasmic
and membrane locations during the activation-deactivation process
following addition of insulin. The transition should be apparent
within minutes.
REFERENCES
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Biochem. Biophys. Acta 1313, 63-71 [0320] Khalil, R. A., Lajoie,
C., Resnick, M. S. & Morgan, K. G. (1992) American Physiol.
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(1995) Brain Res. 688, 213-218 [0322] Bastiaens, P. I. H. &
Jovin, T. M. (1996) Proc. Natl. Acad. Sci. USA 93, [0323] Schmidt,
D. J., Ikebe, M., Kitamura, K., & Fay, F. S. (1997) FASEB J.
11, 2924 (Abstract) [0324] Sakai, N., Sasaki, K., Hasegawa, C.,
Ohkura, M., Suminka, K., Shirai, Y. & Saito, N. (1996) Soc.
Neuroscience 22, 69P (Abstract) [0325] Sakai, N., Sakai, K.
Hasegawa, C., Ohkura, M., Sumioka, Shirai, Y., & Naoaki, S.
(1997) Japanese Journal of Pharmacology 73, 69P (Abstract of a
meeting held 22-23 March)
[0326] The present invention may be embodied in other specific
forms without departing from its spirit or essential
characteristics. The described embodiments are to be considered in
all respects only as illustrative and not restrictive. The scope of
the invention is, therefore, indicated by the appended claims
rather than by the foregoing description. All changes which come
within the meaning and range of equivalency of the claims are to be
embraced within their scope.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090023598A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090023598A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References