WO1998041090A1 - Methods and compositions for stimulating apoptosis and cell death or for inhibiting cell growth and cell attachment - Google Patents

Abstract

The present invention relates generally to methods of modulating the rate and/or amount of a cellular process selected from the group consisting of cell growth, cell detachment and cell migration, and cellular apoptosis, said method comprising altering the RECEPTOR/PTK-STAT pathway of a cell. More particularly, the present invention relates to methods wherein the RECEPTOR/PTK-STAT pathway is altered by increasing or decreasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell. The present invention also relates to the identification of agents that either promote or inhibit the phosphorylation of RECEPTOR/PTK-STAT proteins, as well as to the agents themselves and to the methods which utilize such identified agents. The methods of the present invention are useful for treating mammalian diseases, including, but not limited to, cancer, autoimmune diseases, viral susceptibility, degenerative disorders, ischemic injuries, and conditions of obesity.

Description

METHODS AND COMPOSIΗONS FOR SΗMULAΗNG APOPTOSIS AND CELL DEATH OR FOR INHIBITING CELL GROWTH AND CELL ATTACHMENT

This application is based on U.S. provisional application 60/041,401, which is incorporated in its entirety by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH

The present invention was developed in part using government funds. The

government has certain rights to the present invention. The underlying research was

supported by grants from the NIH (RO1 Al 34522).

FIELD OF THE INVENTION

The present invention pertains, in general, to the fields of cell death (apoptosis), cell

growth control, and cell attachment. In particular, the present invention pertains to methods

and compositions for increasing cell death or apoptosis, and methods and compositions for

reducing cell growth or cell adhesion based on the phosphorylation, activation and expression

of cellular proteins.

BACKGROUND OF THE INVENTION

All publications and patent applications herein are incorporated by reference to the

same extent as if each individual publication or patent application was specifically and

individually indicated to be incorporated by reference.

Apoptosis or programmed cell death is an active process that is essential for normal development and homeostasis in multicellular organisms and provides a defense against viral

invasion and oncogenesis (Wyllie et al, 1980; Ellis et al, 1991; Raff, 1992; Steller, 1995;

Martin and Green, 1995; White, 1996). It is known that there are a number of independent

pathways to apoptosis. For example, p53 is involved in apoptosis in response to DNA

damage and other cellular damages (Clarke et al, 1993; Lowe et al, 1993; White, 1996).

Certain growth inhibitory cytokines are capable of inducing apoptosis independent of p53.

Tumor necrosis factors (TNFs) and Fas can both trigger a cell death (Nagata and Golstein,

1995; Cleveland and Ihle, 1995; Fraser and Evan, 1996). It has been recently shown that

TNF/Fas may induce a cascade of proteolytic signaling pathways to mediate apoptosis

(Tartaglia et al, 1993; Hsu et al, 1995; Muzio et al, 1996; Boldin et al, 1996; Cleveland

and Ihle, 1995; Fraser and Evan, 1996). The key mediators of apoptosis are the ICE

(interleukin- lb-converting enzyme) family cysteine proteases (recently renamed as Caspase,

see Alnemri et al, 1996), which are related to the C. elegans programmed cell death gene

ced-3 (Yuan et al, 1993). The mammalian ICE protease family comprises at least eleven

members (Stanger et al, 1995; Fraser and Evan, 1996; Salvesen, 1997). It is possible that

these different ICE family members may function in response to the different apoptosis

pathways.

In contrast to TNF/Fas, many other growth factors or cytokines can activate receptor

protein tyrosine kinase and/or receptor-associated tyrosine kinases (presented here as

Receptor/PTK) signaling pathways (Schlessinger and Ullrich, 1992; van der Geer et al. ,

1994; Ihle and Kerr, 1995). The Receptor PTK pathways are believed to mediate cell growth and to protect cells from apoptosis (Cleveland and Ihle, 1995; Thompson, 1995). For

instance, many kinds of cells can not survive unless the necessary growth factors or cytokines

are provided. Thus, growth factors, such as insulin-like growth factor (IGF) -1, EGF and

PDGF, which normally induce mitogenic responses, act as survival factors (Bennett et al,

1994; Harrington et al, 1994; Englert et al, 1995; Jung et al, 1996; Thompson, 1995).

It is well-established that growth factors such as EGF can activate a signaling

cascade. This cascade links the growth factor receptor tyrosine kinase or receptor associated

tyrosine kinases to the Ras protein, then to downstream serine/threonine kinases, such as the

members of the MAP kinase family (Schlessinger and Ullrich, 1992; van der Geer et al,

1994). The kinases may translocate to the nucleus and phosphorylate transcription factors

such as c-Jun and TCF. However, it is not understood in detail how the cell survival or

apoptosis is regulated through this cascade pathway.

Parallel to this kinase cascade signaling pathway, a direct signaling pathway has also

been revealed in the past few years. In this pathway, signal transduction is mediated by the

protein tyrosine kinases, and their specific substrates: SH2 (Src homology region) containing

STAT proteins (Fu, 1992; Schindler et al, 1992; Velazquez et al, 1992; Lamer et al, 1993;

Muller et al, 1993; Darnell et al, 1994; Fu, 1995a; Ihle and Kerr, 1995). Although this

signaling pathway was first revealed in the interferon system, it has been further

demonstrated that most cytokines and growth factors, including EGF, PDGF, CSF-1, insulin,

IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, and IL-10 etc., can activate the direct STAT signaling

pathway (Fu and Zhang, 1993; Larner et al, 1993; Ruff- Jamison et al, 1995; Sadowski et al, 1993; Silvennoinen et al, 1993) (reviewed in Darnell et al, 1994; Fu, 1995; Ihle, 1996).

So far more than six members of the STAT protein family have been identified in higher

eukaryotes, and these STAT proteins are believed to respond specifically to different

cytokine signals (Ihle, 1996). Antibodies and other entities that are specific to the functional

domain of a STAT protein and that could possibly be used to selectively modulate the

activity of a STAT protein have been identified (Danell et. al, WO 96/20954 (published 11

July 1996)).

The Jak family of tyrosine kinases were initially recognized as activators of STAT

proteins (Ihle, 1995; Muller et al, 1993; Velazquez et al, 1992). However, a variety of

tyrosine kinases, such as EGF receptor tyrosine kinase and Src kinase have been shown to

activate STAT proteins directly and independently of JAK kinases (Fu and Zhang, 1993;

Quelle et al, 1995; Yu et al, 1995). It has been further shown that focal adhesion kinase

(FAK), FGF receptor tyrosine kinase, and many other tyrosine kinases can also directly

activate STAT proteins (Su et al, 1997; inventors' unpublished). Therefore, this is a common

pathway from cell surface receptors with intrinsic, and/or associated with, protein tyrosine

kinases (these two kinds are presented here as Receptor/PTKs) to STAT proteins

(Receptor/PTK- STAT pathway). However, the physiological and cellular functions of the

Receptor/PTK-STAT pathway were not well understood. It has been proposed that the

JAK-STAT pathway is involved in cell proliferation and transformation (reviewed in

Leonard and O'Shea, 1998).

Mammalian cell proliferation is controlled by cytokines and other polypeptide ligands which may produce positive or negative growth signals. For example, epidermal growth

factor (EGF) can stimulate proliferation of many cell types (Carpenter and Cohen, 1979;

Cross and Dexter, 1991). In contrast, interferons (IFNs) often inhibit cell proliferation (De

Maeyer and De Maeyer-Guignard, 1988). Thus many cytokines or growth factors have

traditionally been classified into one of these categories of growth stimulator or inhibitor.

However, many cytokines have been shown to stimulate growth in one cell type, while

inhibiting growth or inducing differentiation in the other cell types (Sporn and Roberts,

1988). A431 cells, a classical system for the study of EGF receptor function for the past

decade, were growth-inhibited by EGF (Gill and Lazar, 1981; Bravo et al, 1985).

Interleukin-4 (IL-4) is a well-known growth factor for B-cells, but it can evoke strong

growth suppression in many tumor cells (Tepper et al, 1989; Toi et al, 1992; and Lahm et

al, 1994). The molecular mechanisms for such cell specific responses to cytokines are not

well-defined.

In the recent years, the machinery of cell proliferation and the molecular mechanisms

of cell cycle control have been analyzed in detail. The cell cycle is controlled by a family of

cyclin-dependent kinases (CDKs) which can be negatively regulated by families of CDK

inhibitors (Hunter and Pines, 1994; Sherr and Roberts, 1995). One of the well-studied CDK

inhibitors is p21 (WAFl/Cipl/CAPl) which, upon binding to CDKs, blocks their activity

and causes cell cycle arrest (El-Deiry et al, 1993; Gu et al, 1993; Harper et al, 1993; Xiong

et al, 1993; Noda et al, 1994). p21 is induced by the transcriptional activating function of

the tumor suppresser protein p53, suggesting its inhibitory role in p53-mediated Gl check-point control (El-Deiry et al, 1993; 1994). However, p21 is also induced by the

proliferative signal in T lymphocytes and other growing cells (Firpo et al, 1994; Li et al,

1994; Sheikh et al, 1994). Detailed biochemical analysis has shown that p21 may exist in

both active and inactive CDK cyclin quaternary complexes. The increase of the ratio of p21

to CDK cyclin may convert the active complex into inactive complexes (Zhang et al, 1994;

Harper et al, 1995; reviewed in Hunter and Pines, 1994; Sherr and Roberts, 1995). It is of

interest to determine whether any cytokines may play a role in regulation of p21 expression,

which may shift the p21: CDK cyclin ratio, resulting in proliferative or anti-pro liferative

effects.

It is believed that some of the genes that control the cell cycle are regulated by

cytokine-induced signals. However, pathways from cytokine-induced signal transduction to

control of cell growth are largely undefined. For example, it is not understood how signals

from cytokine receptors are transduced to specific transcription factors regulating expression

of genes encoding cell cycle regulators such as p21. It is well-established that growth factors

can activate a protein kinase cascade (reviewed in Cantley et al, 1991; Schlessinger and

Ullrich, 1992). This cascade links the growth factor receptor associated tyrosine kinase to the

Ras protein, then to downstream serine/threonine kinases, such as the members of MAP

kinase family. The kinases may translocate to the nucleus and phosphorylate transcription

factors such as c-Jun and TCF (Hill and Treisman, 1995; Karin and Hunter, 1995).

However, it is not known how the cell cycle machinery is regulated through this cascade

pathway. The properties and functions of a living cell are tightly regulated by extracellular

matrix (ECM) proteins and soluble cytokines. A variety of transmembrane receptors, which

can specifically interact with these ECM proteins and cytokines, transduce signals into the

cell causing cellular effects, such as induction of gene expression. Integrins that are

heterodimeric transmembrane receptors, bind the ECM proteins including fibronectin and

other cell adhesion molecules (Clark and Brugge, 1995; Hynes, 1992; Schwartz, et al,

1995). Similarly to the signal transduction induced by cytokine:receptor binding, interaction

of integrins with the ECM proteins can induce tyrosine phosphorylation of many intracellular

proteins. Among them, the focal adhesion kinase (FAK) has been shown to be tyrosine

phosphorylated during some integrin-mediated cell adhesion and is believed to play

important roles in integrin signal transduction (Guan and Shalloway, 1992; Hanks, et al,

1992; Schaller, et al, 1992). Like receptor tyrosine kinases, FAK interacts with a pool of

signaling intracellular proteins, including c-Src, phosphatidylinositol-3 (PB)-kinase, Grb2

and pl30^CAS (Schaller, et al, 1994; Schlaepfer, et al, 1994; Chen and Guan, 1994; Cobb, et

al, 1994; Polte and Hanks, 1995; Frisch, et al, 1996). Consistent with functions of these

signal proteins, recent studies have shown that FAK may be involved in cell survival (Frisch,

et al, 1996, Hanks and Polte, 1997).

SUMMARY OF THE INVENTION

This invention comprises methods of modulating the rate and/or amount of a cellular

process selected from the group consisting of cell growth, cell detachment and cell migration, and cellular apoptosis, said method comprising altering the RECEPTOR PTK-STAT

pathway of a cell. More specifically, the present invention provides methods wherein the

RECEPTOR/PTK-STAT pathway is altered by increasing or decreasing the amount of

phosphorylated RECEPTOPJPTK-STAT proteins present in a cell.

The present invention provides methods wherein the amount of phosphorylated

RECEPTOR/PTK-STAT proteins present in the cell is increased or decreased by introducing

into the cell a sense or antisense nucleic acid molecule that encodes a tyrosine kinase and/or a

RECEPTOPJPTK-STAT protein.

The present invention comprises altering the RECEPTOR/PTK-STAT pathway by,

among other possible methods, increasing or decreasing the expression and/or activation of a

RECEPTOR in the pathway; increasing or decreasing the amount of STAT in a cell;

increasing or decreasing the amount of kinase present in a cell; altering the interaction of

STAT with a RECEPTOR in the pathway; altering the interaction of STAT with a PTK; and

by altering the interaction among or between the RECEPTORS, the PTKs, and the STATs in

the pathway.

The present invention also provides methods of identifying agents which inhibit

apoptosis in a cell through the mechanism of blocking the phosphorylation of

RECEPTOR/PTK-STAT by a tyrosine kinase comprising the steps of:

a) incubating STAT, or a fragment thereof, and a tyrosine kinase, or a fragment

thereof, with an agent to be tested,;

b) determining whether said agent blocks the phosphorylation of STAT, or a fragment thereof by said tyrosine kinase,

wherein the inhibition of STAT phosphorylation indicates the potential to inhibit

apoptosis.

In addition, the present invention provides methods of identifying agents which

stimulate or promote apoptosis in a cell through the mechanism of stimulating the

phosphorylation of RECEPTOR/PTK-STAT by a tyrosine kinase comprising the steps of:

a) incubating STAT, or a fragment thereof, and a tyrosine kinase, or a fragment

thereof, with an agent to be tested, and

b) determining whether said agent stimulates the phosphorylation of STAT, or a

fragment thereof by said tyrosine kinase,

wherein the promoting of STAT phosphorylation indicates the potential to stimulate

or promote apoptosis.

The presents invention also provides methods for determining whether a

RECEPTOR/PTK-STAT protein is phosphorylated as well as for correlating apoptosis with

the presence and degree of said RECEPTOR/PTK-STAT phosphorylation, wherein an

increase of RECEPTOR/PTK-STAT phosphorylation indicates STAT-mediated apoptosis.

The presence of elevated levels of RECEPTOR PTK-STAT proteins is a diagnostic marker

of a number of mammalian diseases, including, but not limited to, Thanatophoric Dysplasia

Type II, FGF-receptor associated diseases, cancer, metastasis of cancer cells, autoimmune

disorders, diabetes, degenerative diseases, aging, and inflammation.

The present invention provides methods for treating mammalian diseases or developmental defects caused by abnormal cell death induction wherein the methods

comprise promoting or inhibiting apoptosis by altering the RECEPTOR/PTK-STAT

pathway. The present invention further provides methods of treating mammalian diseases or

developmental defects caused by abnormal cell death induction wherein the method

comprises inhibiting apoptosis by altering the RECEPTOR/PTK-STAT pathway. The

present invention provides methods of treating mammalian diseases or developmental defects

caused by abnormal cell proliferation wherein the method comprises inhibiting abnormal cell

growth by altering the RECEPTOR PTK-STAT pathway. The present invention provides

methods of treating mammalian diseases or developmental defects caused by cell growth

retardation wherein the method comprises promoting cell growth by altering the

RECEPTOR/PTK-STAT pathway. The present invention also provides methods of treating

mammalian diseases or developmental defects caused by abnormal cell detachment wherein

the method comprises promoting cell attachment by altering the RECEPTOR/PTK-STAT

pathway. The present invention further provides methods of treating mammalian diseases or

developmental defects caused by abnormal cell detachment wherein the method comprises

inhibiting cell attachment by altering the RECEPTOR/PTK-STAT pathway.

The present invention provides a method for identifying diagnostic agents for

measuring RECEPTOR PTK-STAT activities in order to determine physiological and

pathological conditions, wherein the method comprises the steps of:

a) measuring the activity of a RECEPTOR/PTK-STAT protein,

b) determining whether the activity of the RECEPTOR/PTK-STAT protein is associated with a specific phenotype or a specific disease, and

c). examining cellular localization of STAT protein to determine activation of

STATs.

The present invention also provides clones that produce exogenous levels of STAT

protein in an amount significantly greater than the parental cell lines from which the clones

were developed. The invention further provides clones which exhibit significantly faster cell

death following serum withdrawal than the cell death of the parental cell line under the same

conditions.

The invention also provides a method for identifying agents that block the

phosphorylation of RECEPTOPJPTK-STAT comprising the steps of:

a) growing a clone that over-produces STAT proteins in a serum-based growth

media,

b) removing the serum from the growing media and concurrently adding the

agent of interest,

c) determining whether said agent blocks the phosphorylation of

RECEPTOPJPTK-STAT by observing clone cell viability over time.

The invention also provides a method of diagnosing abnormal STAT activation

related to mammalian diseases comprising the steps of:

a) isolating and growing test cells from an individual of interest;

b) conducting nuclear staining of the test cells using anti-STAT antibodies;

c) examining the stained nuclei of the test cells to determine whether or not 4

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STAT has been translocated into the nuclei of the test cells; and,

d) comparing the extent of STAT translocation into the nuclei of the test cells to

that of normal control cells stained in the same manner.

The invention also provides methods of determining the amount of phosphorylated

STAT proteins wherein the methods comprise using anti-phospho-tyrosine STAT, such as

anti-phospho-tyrosine STATl.

One skilled in the art can easily make any necessary adjustments in accordance with

the necessities of the particular situation.

Further objects and advantages of the present invention will be clear from the

description and examples which follow.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 . Activation of STATl during Integrin-Mediated Cell Adhesion and by FAK.

A. Activation of STATl in A431 cells after plated on fibronectin.

B. STATl activation was observed in cells transfected with FAK and STATl.

Fi ure 2. Expression of FAK can Cause Cell Apoptosis through Activation of STATl .

A. Dramatic morphological changes in transfected cells seemed to parallel with

STATl activation by FAK.

B. A large portion of cells showed bright white spots representing apparent DNA

condensation, caused by FAK-STATl activation, indicating that induction of apoptosis.

C. A quantitative measurement of apoptotic cells in various transfected cells. Figure . STATl is Essential for Induction of Apoptosis by FAK.

A-B. The wild type (STATl +/+) fibroblasts, but not STATl null (-/-) fibroblasts,

undergo apoptosis, or apoptosis induced when STATl was re-introduced with FAK.

C-D. U3A-pSG5 cell line which is STATl defective, and U3A-STAT1 cells, in

which STATl has been stably reintroduced were examined for FAK- induced apoptosis,

showing that FAK-STATl activation is necessary for the induction of apoptosis.

Fi ure 4. Both Integrin Signaling and STATl are Necessary for Promotion of Apoptosis

Under Physiological Conditions Caused by Serum Withdrawal.

A-B. STATl +/+ or STATl -/- embryonic fibroblasts were plated on fibronectin (FN)

with media containing no serum. STATl positive cells were dying faster.

C-D. STATl null and wild type cells were dying at the same rate when they were

plated on BSA.

Figure 5. co-expression of each member of HER receptor family, with each member of

STAT proteins causes induction of apoptosis.

A. Joint actions of HER1 and each STAT proteins cause cell death.

B. Joint actions of HER2 and each STAT proteins cause cell death.

C. Joint actions of HER3 and each STAT proteins cause cell death.

D. Joint actions of HER4 and each STAT proteins cause cell death.

Figure 6. STAT activation induced by EGF causes apoptosis.

A. Comparison of STAT activation in HeLa cells vs. A431 cells in response to EGF.

B. Apoptosis induction of these two cell lines correlates with STAT activation. C. EGF receptor autophosphorylation and activation of the Ras-MAP kinase

pathway are normal in both A431 and HeLa cells.

D. Correlation between Receptor/PTK-STAT activation and apoptosis in MDA-MB-

468 cells, a breast cancer cell line and A431-R, an A431 variant.

Figure 7. ICE Expression Correlated with EGF-STAT activation and induced Apoptosis.

A-B-C. EGF induced ICE gene expression in both A431 and MDA-MB-468 cells, but

not in HeLa cells (A) which was correlated with STAT activation.

Figure 8. Jakl is necessary for induction of apoptosis in response to IFN-γ.

A. DNA binding activity of STAT activation was absent as determined by EMSAs in

E2A4 cells.

B. The strong induction of ICE mRNA normally seen upon IFN-γ treatment in the

parental HeLa cells was completely abolished in this JAK1 deficient cell line.

C. Bis-benzimide staining showed that E2A4 cells did not apoptose in the presence of

IFN-γ .

Figure 9. Analysis of apoptosis induction in U3A cells, a STATl -defective cell line and the

parental cell line 2fTGH, and STATl reintroduced U3A-S1-2 cells in response to IFN-γ.

A-B. IFN-γ activate STAT, induce ICE mRNA expression, or lead to apoptosis in

2fTGH and the U3A-S1-2, but not U3A cells in response to IFN-γ .

C. The condensed and/or fragmented nuclei were observed in 2fTGH and U3A-S1-2,

but not U3A cells treated with IFN-γ.

Figure 1 . ICE Gene Is necessary for IFN-γ-Induced Apoptosis A. Normally activated STATl in response to IFN-γ in both ICE'- and ICE^{+ +} cells.

B. Induced DNA fragmentation was significantly reduced in ICE'" cells compared

with that in ICE^{+ +} cells.

Fi ure 1 1 . A General Pathway to Induction of Apoptosis through the Joint Actions of a

variety of different Receptor/PTKs and ST ATs.

A. Apoptosis induction through joint actions of TrkA, a nerve trophin receptor, and

each of STAT proteins.

B. Apoptosis induction through joint actions of TrkB, a nerve trophin receptor, and

each of STAT proteins.

C. Apoptosis induction through joint actions of a EPH protein, a nerve trophin

receptor involved in neuron differentiation, and each of STAT proteins.

D. Apoptosis induction through joint actions of Tie2, a receptor involved in

angiogenesis and early development etc., and each of STAT proteins; STAT5A was

especially active in causing apoptosis.

E. Apoptosis induction through joint actions of FGFR2, a receptor involved in

development and angiogenesis etc., and each of STAT proteins.

F. Apoptosis induction through joint actions of FGFR3, a receptor involved in

development and angiogenesis etc., and each of STAT proteins.

G. Apoptosis induction through joint actions of Src, a cytoplasmic tyrosin kinase

involved in bone development and tumor transformation etc., and each of STAT proteins.

H. Apoptosis induction through joint actions of Lck, a cytoplasmic tyrosin kinase involved in lymphocytes development and function etc., and each of STAT proteins.

I. Apoptosis induction through joint actions of Itk, a cytoplasmic tyrosin kinase

involved in lymphocytes development and function etc., and each of STAT proteins.

The mock was the vector alone transfected cells.

Figures 12-14. The STAT proteins control the apoptosis induction by default after growth

factor withdrawal.

Figure 12. Expression of the STATl protein in mouse embryonic fibroblasts promotes

apoptosis by default after serum withdrawal while deficiency of STATl protein reduces

apoptosis after serum withdrawal

Figure 1 . Expression of the STATl protein in Ba/F3, a cell line derived from pro-B cells,

promotes apoptosis after serum or growth factor (IL-3) withdrawal.

Figure 14. Expression of the STAT3 protein in Ba/F3, mouse embryonic fibroblasts

promotes apoptosis after serum withdrawal.

Figure 15. A negative and positive signaling model is proposed to explain the molecular

basis responsible for the dual functions of cytokines.

Figure 1 . Receptor/PTK- STAT activation is a broad molecular signal mediating induction

of apoptosis, and represent a mechanism of apoptosis induction by default.

Fi ure 17. STAT Activation Induced by EGF and IFN-γ is Correlated with Cell Growth

Arrest.

Figure 18. The p21/WAFl Expression by STATs in Response to EGF and IFN-γ

A. p21-SIEs are regulatory sites of STAT proteins in the p21 gene. B. p21 Gene Expression is Correlated with STAT Activation in Response to EGF.

Figure 1 . STATl is Essential for Induced Cell Growth Arrest. U3A/Control cells which

were deficient in STATl were not inhibited by IFN-γ but U3A/STATlα cells were inhibited

by IFN-γ.

Figure 20. STATl Activation induced by expression of a mutant TDII FGFR3 receptor.

A. Kinase activities were assessed by an in vitro autophosphorylation for wild type

and the TDII mutant FGFR3.

B. Wild type and the TDII mutant FGFR3 were at similar levels.

C. STAT activation assayed using EMSA. Mutant TDII, but not wild type FGFR3

could induce a STATl complex.

Figure 21 . STATl nuclear translocation, p21/WAFl induction and cell growth arrest in TDII

transfected cells and in chondrocytes from TDII patients.

a. The FGFR3 protein was expressed on the cell surface (brown color).

b. Expression of TDII receptor on the membrane, and localization of STATl in the

nucleus.

c. The nuclei in the TDII receptor-transfected cells were counter-stained (dark brown,

indicated by arrows.

Figure 22. STATl activation by the expression of TDII receptor would induce expression

ofp21.

a. the p21 mRNA level was particularly enhanced in TDII-transfected cells compared

with other transfected cells. b. p21 protein was enriched in the nuclei in TDII transfected cells as demonstrated by

an immunocytochemical stain with anti-p21 antibody (indicated by arrows).

c. The nuclei in the TDII receptor- transfected cells were counter- stained (dark brown,

indicated by arrows.

Figure 23. STATl translocation in chondrocytes from TDII affected, but not other control

individuals.

a. STATl was expressed at a low level in the chondrocytes from a normal control

individual, and STATl protein was found in the cytoplasm (brown staining of STATl was

indicated by arrows; the nuclei were counter stained in blue)

b. STATl was translocated into the nuclei, and exclusively stained in the nuclei in

many chondrocytes from three TDII-affected individuals

c-d. The nuclear staining by the anti-STATl antibody in chondrocytes of the TDII

affected patient (c ) was completely abolished by the specific competitor (d ).

Figure 24. p21 expression in the same TDII-affected chondrocytes.

a. p21 protein was undetectable with an anti-p21 antibody (no brown stain) in normal

chondrocytes.

b. p21 expression was clearly observed in the TDII chondrocytes as indicated by

brown or darker nuclear stain by the anti-p21 antibodies (indicated by arrows). There were

vacuole-like structures in these cells indicating the cell degeneration or apoptosis.

Figure 25. Activation loop K650E for TDII of FGFR3 and other tyrosine kinase mutations

that may be involved in STAT activation. Figure 26. STATl can interact with FAK in the transfected cells.

The FAK protein was co-immunoprecipitated with the anti-STATl antibody. The

identity of the HA-tagged FAK was confirmed further by blotting with an anti-FAK

antibody. The expression levels of STATl protein were also assayed (lower panel).

Figure 27. STAT:FAK interactions in untransfected cells.

A. The co-immunoprecipitated STATl from 293T cells was protein phosphorylated

after they had interacted with FAK.

B. A similar observation was also made in A431 cells.

Figure 28. The specificity of activation of STATl by FAK was confirmed by using

various STATl and FAK mutants. Mutations of the SH2 domain (STAT1-SH2RQ) and of

the tyrosine 701 (STATl-CYF) in STATl prevented its activation when co-transfected with

FAK.

Figure 29. STATl and FAK co-expression causes cell detachment.

Co-expression of FAK and STATl in 293T cells greatly inhibited the cell adhesion

on fibronectin. Expression of either STATl or mock expression of β-galactosidase, or

STAT1-SH2RQ mutant, had less effect.

Figure 30. STATl is required for cell detachment.

A. Re-introduction of STATl protein to U3A cells significantly reduced cell

attachment to fibronectin.

B. Embryonic fibroblasts derived from STATl deficient or from wild type mice were

compared. STATl null (-/-) cells attach better than STATl +/+ cells at different concentrations of plated fibronectin.

C. A picture showing STATl wild-type fibroblast cells were detached and aggregated

on the plating to FN, whereas STATl -/- cells could attach well at the same conditions.

Figure 31. STATl promotes cell migration. STATl -/- and STATl +/+ fibroblasts were

further analyzed using Boyden chamber assay for their migration ability. It was found

STATl positive cells migrate significantly faster than STATl negative cells.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same

meaning as commonly understood by one of ordinary skill in the art to which this invention

belongs. Although any methods and materials similar or equivalent to those described herein

can be used in the practice or testing of the present invention, the preferred methods and

materials are described.

The terms "RECEPTOR PTK-STAT" and "tyrosine kinase-STAT", and other variants

which may be used interchangeably, and have been used throughout this application and

claims to refer to expression and/or activation of STAT proteins (including STATl, STAT3,

STAT4, STAT5A/B, STAT6) by kinases, including receptor tyrosin kinases, such as EPH,

HER and FGFR families, and cytoplasmic tyrosine kinases, such as FAK, Itk, TIE, and Src

families, in response to stimulations caused by a number of polypeptides and their receptors,

such as fibronectin/integrin, EGF and FGF families and their receptors. In the other words,

the term RECEPTOR PTK-STAT is used for description of the collective actions and the signaling pathways from these ligands/receptors to protein tyrosine kinases and to STAT

proteins and their target genes.

The current inventions in this application are in the fields of cellular functions of

RECEPTOR/PTK-STAT signaling pathways in cell death or survival, cell growth retardation

or over-proliferation, cell adhesion or detachment, and cell migration.

The methods of the present invention for increasing or decreasing the amount of

phosphorylated RECEPTOR/PTK-STAT proteins present in a cell can be performed by: 1)

increasing or decreasing the amount of STATs or kinases or receptors expressed and/or

activated in the cell; 2) by altering the interaction of STAT with a receptor or a PTK; or, 3)

increasing or decreasing the amount of kinase present in a cell. The methods of the present

invention are particularly useful in diagnosis and treatments of cancer, metastasis of cancer

cells, autoimmune disorders, Thanatophoric Dysplasia Type II (TDII) and other FGFR-

associated growth retardation disorders, diabetes, degenerative diseases, aging and

inflammation and those listed in items 4)-6) below.

The present invention further provides methods for identifying agents for use in

modulating STAT mediated biological and pathological processes. A skilled artisan can

readily use the information disclosed herein, particularly the Examples, to develop assays to

identify agents for use in modulating STAT mediated activity. Such agents can increase

STAT activity or can be used to decrease STAT activity. For example, an agent that blocks

the phosphorylation of STAT by a tyrosine kinase can be identified by: a) incubating STAT,

or a fragment thereof, and a tyrosine kinase, or a fragment thereof, with an agent to be tested, and b) determining whether said agent blocks the phosphorylation of STAT by said tyrosine

kinase. Such methods can be used to identify agents for use in stimulating or blocking

apoptosis, cell adhesion/detachment, cell differentiation, and cell growth and is particularly

useful as a diagnostic marker of TDII.

Utilizing the results provided below, a skilled artisan can readily practice and develop

the diagnostic, screening and therapeutic methods outlined above and in the claims.

The Examples provide detailed scientific results that can be used by a skilled artisan

to 1) develop assay methods for identifying agents that modulate RECEPTOR PTK-STAT

mediated biological and pathological processes; 2) develop diagnostic assays to identify

RECEPTOR/PTK-STAT mediated biological and pathological processes; and, 3) act as a

target for therapeutic agents for use in modulating RECEPTOR/PTK-STAT mediated

biological and pathological processes. Specifically, the Examples provide a basis of

therapeutic and diagnostic methods for identifying and treating conditions involving

abnormal cell apoptosis/survival, cell growth/retardation, and cell attachment/detachment

and migration. Techniques and methods which can be used for the above purposes are

described in the literature, such as "Current Protocols in Molecular Biology ' John Wiley &

Sons, Inc. 1994 and updated versions; "Current Protocols in Immunology," John Wiley &

Sons, Inc. 1994 and updated versions; "Current Protocols in Neural Sciences," John Wiley &

Sons, Inc. 1994 and updated versions, etc.

In detail, as listed in items 1)-10) below, the present invention provides methods for

modulating the rate and amount of a cell growth, cell adhesion/detachment and cell migration, and cellular apoptosis. The methods of the present invention are based on the

unexpected observation that RECEPTOR/PTK-STAT signaling pathways, in contrast to

many other signaling pathways, can act in a negative fashion (see Figure 15 and Figure 16

for summary). Specifically, the Examples show that activation of the STAT proteins,

including STATl, STAT3, STAT4, STAT5A/B, STAT6, mediated by the phosphorylation

by a cellular kinase, receptor tyrosine kinases and/or cytoplasmic tyrosine kinases and/or

other kinases, such as but not limited to, EGFR (Her family), FGFR family, FAK, JAK, Src,

Lck, Itk, TIE2, c-kit, RET, INRK, PDGFR-B and other members of the tyrosine kinase

family of proteins, in response to polypeptide ligands or by co-expression of PTKs and

STATs, cause cell apoptosis, decreases the rate and extent of cell growth and promotes cell

detachment and migration. Accordingly, cell survival, proliferation and cell adhesion can be

stimulated by blocking the RECEPTOR/PTK-STAT signaling pathways, and cell survival

and cell growth and cell adhesion can be reduced by increasing the phosphorylation and

activation of RECEPTOR/PTK-STAT signaling pathways. Similarly, the activities of

RECEPTOR/PTK-STAT proteins can be used as indicators or markers for detection,

measurement, diagnostic analysis of status, potentials and commitment of apoptosis, cell

proliferation, and cell adhesion/detachment and cell migration.

The following items 1)-10) further explain various aspects of the present invention:

1) The first aspect of this invention is based on the unexpected discovery that

activation of RECEPTOR/PTK-STAT causes apoptosis. This invention provides general

methods, compositions and procedures for development of diagnostic agents for detection and assaying of apoptosis through measuring activities and/or expression of

RECEPTOR/PTK-STAT; and for development of therapeutic agents for either inhibiting or

stimulating, induction of apoptosis through interfering with the RECEPTOR/PTK-STAT

signaling pathways.

In particular, it is the object of this invention to provides a pharmaceutical and gene-

therapeutic methods and composition for treating mammalian diseases or developmental

defects caused by abnormal cell death induction or reduction through either promoting or

inhibiting apoptosis through interfering with the RECEPTOR/PTK-STAT signaling

pathways.

Furthermore, this invention provides methods of utilizing RECEPTOR/PTK-STAT

proteins, for developing and designing screening protocols for pharmaceutical (a natural or

synthetically produced) and gene-therapeutic agents that can affect activities of STATs and

Receptor PTKs to control apoptosis, and provide diagnosis and treatment of apoptosis-

related mammalian diseases or developmental defects through interfering with the

RECEPTOR/PTK-STAT signaling pathways.

2) A second aspect of this invention is based on the unexpected discovery that

activation of RECEPTOR/PTK-STAT causes cell growth arrest and inhibition of cell

proliferation. This invention provide general methods, compositions and procedures for

development of diagnostic agents for detection and assaying of cell proliferation or growth

retardation through measuring activities and/or expression of RECEPTOR/PTK-STAT; and

for development of therapeutic agents for either inhibiting or stimulating, induction of cell proliferation or growth retardation through interfering with the RECEPTOPJPTK-STAT

signaling pathways.

In particular, it is the object of this invention to provide a pharmaceutical and gene-

therapeutic methods and composition for treating mammalian diseases or developmental

defects caused by abnormal cell proliferation or growth retardation, with induction or

reduction abnormal cell growth through interfering with the RECEPTOR PTK-STAT

signaling pathways.

Furthermore, this invention provides methods of utilizing RECEPTOR/PTK-STAT

proteins, for developing and designing screening protocols for pharmaceutical (a natural or

synthetically produced) and gene-therapeutic agents that can affect activities of STATs and

Receptor/PTKs to control cell proliferation, and provide diagnosis and treatment of cell

proliferation-related mammalian diseases or developmental defects through interfering with

the RECEPTOR/PTK-STAT signaling pathways.

3) A third aspect of this invention is based on the unexpected discovery that

activation of RECEPTOR PTK-STAT causes cell detachment and cell migration

enhancement. This invention provides general methods, compositions and procedures for

development of diagnostic agents for detection and assaying of cell detachment or adhesion

and cell migration through measuring activities and/or expression of RECEPTOR/PTK-

STAT; and for development of therapeutic agents for either inhibiting or stimulating,

induction of cell detachment or adhesion and cell migration through interfering with the

RECEPTOR PTK-STAT signaling pathways. In particular, it is the object of this invention to provide a pharmaceutical and gene-

therapeutic methods and composition for treating mammalian diseases or developmental

defects caused by abnormal cell detachment or adhesion and cell migration, with induction or

reduction of abnormal cell adhesion/detachment and/or cell migration through interfering

with the RECEPTOPJPTK-STAT signaling pathways.

Furthermore, this invention provides methods of utilizing RECEPTOR/PTK-STAT

proteins, for developing and designing screening protocols for pharmaceutical (a natural or

synthetically produced) and gene-therapeutic agents that can affect activities of STATs and

Receptor/PTKs to control cell attachment and cell migration, and provide diagnosis and

treatment of mammalian diseases or developmental defects caused by abnormal cell adhesion

or detachment and/or cell migration through interfering with the RECEPTOR/PTK-STAT

signaling pathways.

4) The inventions in this application provide diagnostic and therapeutic methods

for studies and treatments of the diseases and abnormalities that may be associated with

inhibition or reduction of apoptosis include, but are not limited to, the following (partly

reviewed in Thompson, Science, 267, 1456-1462):

i. Cancer, such as breast, prostate, ovarian and colon cancer; leukemia, such as acute

leukemia, follicular lymphophomas; carcinoma with p53 mutations.

ii. Autoimmune diseases with overactive, abnormally produced and increased number

of lymphocytes due to less apoptosis, such as arthritis, diabetes, multiple sclerosis and

asthma etc, due to over-active lymphocytes, lupus erythematosus, glomerulonephritis. iii. Less resistance and restriction to viral (including herpes viruses, poxviruses,

adenoviruses etc.) infections due to less apoptosis.

iv. Conditions of obesity caused by increased number of adipocytes and loss of

feedback control; cardiovascular diseases due to less apoptosis such as atherosclerosis.

In the above conditions and diseases, treatments can be provided by induction of

apoptosis by introducing and activating RECEPTOR/PTK-STAT proteins. Additionally, it is

desirable to provide expression of the proteins with an agent which targets the target cells,

such as an antibody specific for a surface protein on the target cell, a ligand for a receptor on

the target cell, etc. Furthermore, pharmaceutical (a natural or synthetically produced) and

gene- therapeutic agents that can enhance activities of STATs and Receptor/PTKs to induce

apoptosis can be searched, screened, developed, and assayed (see below).

5) The inventions in this application provide diagnostic and therapeutic methods

and compositions for studies and treatments of the diseases and abnormalities that may be

associated with increased apoptosis include but not limited to the following:

i. Degenerative disorders, in particular, the neurological abnormalities, developmental

defects, and aging which are due to cell death resulting from overly active

RECEPTOR/PTK-STAT pathways, and/or by default after survival signal reduction and/or

deprivation (examples and their mechanisms are presented and discussed in Figures 12 to 16

above). These may include but not limited to: Alzheimer's disease, Parkinson's disease,

cerebellar degeneration, neuronal damage in multiple sclerosis, diabetes mellitus type I,

cartilage destruction such as in rheumatoid arthritis, sepsis and septic shock such as adult respiratory distress syndrome)

ii Ischemic injuries, such as stroke, myocardial infarction, and other related

cardiovascular disorders due to too much apoptosis.

iii. Viral infection induced cell death, such as AIDS by HIV, causing elimination of

special lymphocytes.

iv. Cell apoptosis during inflammatory responses, due to overly-active

RECEPTOR/PTK-STAT signaling pathways, such as those cell death caused by cytokines,

antigen receptors, and other cell surface receptors. Cell apoptosis due to cachexia associated

with chronic disease, and to Mycobacterium tuberculosis, gastritis, and Helicobacter pylori,

etc. Cell apoptosis after toxic stress, such as alcohol, generation of reactive oxygen species,

radiation, chemotherapeutical compounds, and other apoptosis inducing or activating agents.

In the above conditions and disorders, diagnosis can be provided by assaying the

activities of RECEPTOR/PTK-STAT proteins; and treatments can be provided by

prohibition of apoptosis by reducing and/or inactivating RECEPTOR/PTK-STAT proteins.

Additionally, it is desirable to provide expression of the proteins with an agent which targets

the target cells, such as an antibody specific for a surface protein on the target cell, a ligand

for a receptor on the target cell, etc. Furthermore, pharmaceutical (a natural or synthetically

produced) and gene-therapeutic agents that can inhibit activities of STATs and

Receptor/PTKs to prevent apoptosis can be searched, screened, developed, and assayed (see

below).

6) The inventions in this application provide diagnostic and therapeutic methods for studies and treatments of the diseases and abnormalities that may be associated with

increased cell proliferation, and cell detachment and cell migration such as a variety cancers,

tumor cell metastasis, and invasion during the later stages of cancer development. For these

abnormalities, diagnosis and treatments can be provided by induction of apoptosis by

introducing and activating RECEPTOR/PTK-STAT proteins. Furthermore, pharmaceutical

(a natural or synthetically produced) and gene-therapeutic agents that can enhance activities

of STATs and Receptor/PTKs to induce apoptosis can be searched, screened, developed, and

assayed (see below).

7) The inventions in this application provide methods and compositions for

enhancing the RECEPTOR/PTK-STAT signaling pathways in their functions during cell

apoptosis, growth arrest, and cell detachment which include but not limited to the following:

Generating and expressing functional RECEPTOR/PTK-STAT proteins are as

described in trade books such as Molecular Cloning, A Laboratory Manual (2nd Ed.,

Sambrook, Fritsch and Maniatis, Cold Spring Harbor), and Current Protocols in Molecular

Biology (Wiley-Interscience, NY, N.Y., 1996). Currently available systems include, but are

not limited to, expression in bacteria such as E. coli and eukaryotes such as yeast,

baculovirus, or mammalian cell-based expression systems such as using CHO cells, etc.; in

vivo delivery systems include but not limited to retrovirus or other viral delivery systems,

such as modified and specially engineered viral vectors derived from adenovirus, herpes

simplex virus, avipox virus etc.; electroporation and lyposome, such as lipofectin {Life-

Sciences) mediated fusion, CaPO4 and DEAE-Dextran transfections etc.. Various other delivery techniques can be used for providing the subject compositions at the site of interest,

such as injection, use of catheters, trocars, projectiles, pluronic gel, stents, sustained drug

release polymers or other device which make local and internal access. These expression and

delivery systems provide methods to introduce RECEPTOR/PTK-STAT proteins in vitro

into tissue culture cells and in vivo into mammals, for therapeutic purposes.

Additionally, constitutively activated RECEPTOR/PTK-STAT proteins, such as TDII

FGFR receptor in Figures 20-22, and the kinases listed in Figure 25. and constitutive

activated STAT proteins can be introduced into cells and mammals using the methods

described above.

Furthermore, RECEPTOR/PTK-STAT protein-expression vectors may be

incorporated into recombinant cells for expression and screening, cell lines and transgenic

animals for functional studies (e.g. the efficacy of candidate compounds and other agents and

their effects on disease- and/or functional-associated RECEPTOR/PTK-STAT protein

activities as regards cell apoptosis/survival, proliferation/retardation, adhesion/detachment

and migration). Some of these examples are shown in Figures 2-5, Figure 11, Figures 13-14.

Figures 20-22, Figure 26, Figures 28-30A, etc. These expression systems also provide

methods for searching for partner proteins or antagonist proteins or molecules which may

enhance RECEPTOR/PTK-STAT functions, which include the yeast two hybrid system,

GST-fusion proteins, in vitro translation assays, and co-immunoprecipatation assays (Figure

26-27) etc..

8) The inventions in this application provide diagnostic and therapeutic methods and compositions for inhibiting the RECEPTOR/PTK-STAT signaling pathways in their

functions during cell apoptosis, growth arrest, and cell detachment which include but not

limited to the following:

Expression of antisense molecules of RECEPTOR/PTK-STAT proteins; using

dominant negative constructs such as STATl-CYF used in Figure 28, kinase dead mutant

proteins, and intra-cellular-domain truncated receptors etc. These antagonist molecules can

be expressed and delivered into in vitro and in vivo cell and mammal systems using methods

described above in item 7. Furthermore, peptides selected from combinatorial peptide

libraries and/or represent the interaction domains of the RECEPTOR/PTK-STAT

interactions, tyrosine phosphorylated peptides binding to SH2 domains of STATs, and

antagonists for STAT DNA binding, such as SIE-like oligonucleotides which have high

affinity with STAT DNA binding domain, and ST AT-inhibitor proteins or other molecules

etc, which can be used as antagonists for inhibition of RECEPTOR/PTK-STAT signaling

pathway and its function in induction of apoptosis, growth arrest, and cell detachment.

9) The inventions in this application provide methods and compositions for in

vitro and in vivo systems and methods for screening compounds and other agents which can

affect, inhibiting or stimulating, the RECEPTOR/PTK-STAT signaling pathways causing

either negative or positive effects on cell survival, proliferation, and adhesion/detachment.

The inventions and cell systems listed in examples and discussed above provides

efficient methods of identifying pharmacological agents or lead compounds for agents active

in interfering with the RECEPTOPJPTK-STAT signaling and their effects on cells. The SH2 domains of STATs, interactive domains of receptor with STAT or PTKs, STAT-DNA

binding site etc. can be targets of these agents. The methods are upgraded to automated,

cost-effective high throughput drug screening and should have immediate applications to

drug discovery. Target therapeutic indications can be provided by cellular function changes

of the RECEPTOR/PTK-STAT signaling and alterations of target genes (such as those

observed in Figure 7 and Figure 18). The readouts can be as the expression of target genes or

DNA binding to their regulatory DNA elements, such as CASPASES or p21 and the SIE

identified in their gene promoters (see Figure 18), and/or using the reporter constructs (such

as gene encoding luciferase) linked with their promoters of the targeted genes.

The cellular readout for effective compounds or other agents are rates of cell death

induction, cell proliferation and cell attachment. Altered resistant or sensitive cells are

isolated by feeding the cells with these agents.

The systems for screening antagonist agents or other negative or positive effectors

may use full proteins or key domains of proteins of RECEPTOR/PTK-STAT and their

partners in signaling and target gene induction. The candidate agents can be mixtures which

include a nucleic acid comprising a sequence which shares sufficient sequence similarity with

a gene or gene regulatory region to which it may produce negative or positive effects on

RECEPTOR/PTK-STAT functions and their interactions among each other or with other

partners. The assay mixture may also comprise a candidate gene therapeutical and

pharmacological agent. Typically a plurality of assay mixtures are analyzed in parallel with

different agents with a variety of testing concentrations to obtain a differential responses to the various readout systems (see above). Candidate pharmaceutical agents include numerous

chemical classes, such as organic compounds; preferably small organic compounds. Small

organic compounds have a molecular weight of more than 50 yet less than about 2,500.

Agents with chemical groups necessary for structural interactions with proteins and/or DNA

include but not limited to an amine, carbonyl, hydroxyl or carboxyl group, and their

derivatives, preferably with at least two of the functional chemical groups, more preferably

at least three. The candidate agents often comprise cyclical carbon or heterocyclic structures

and/or aromatic or polyaromatic structures substituted with one or more of the

aforementioned functional groups. Other kinds of agents include but not limited to

biomolecules including peptides, saccharides, fatty acids, sterols, isoprenoids, purines,

pyrimidines, derivatives, structural analogs or combinations thereof, and so on. Candidate

agents can be found and screened from a wide variety of natural or synthetic sources

including libraries of synthetic compounds, expression of randomized oligonucleotides, or

natural compounds selected from bacterial, fungal, plant and animal extracts. Natural and

synthetically selected libraries and compounds are readily modified through conventional

chemical, physical, and biochemical means, such as acylation, alkylation, esterification,

amidification, etc., to produce more effective structural analogs.

Other components of mixtures include reagents like salts, buffers, neutral proteins,

detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-

nucleic acid binding and/or reduce non-specific or background interactions, etc. Also,

reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. are also considered and can be selectively used.

10) The inventions in this application provide methods and compositions for the

development and discovery of diagnostic agents for measuring RECEPTOR/PTK-STAT

activities in order to determine physiological and pathological conditions associated with a

phenotype or specific diseases. Examples are shown in Figure 20-24. Diseases and

abnormalities listed above in item 4), item 5), and item 6) can be diagnosed by measuring the

activities of the RECEPTOR/PTK-STAT proteins. The assays, in vivo and in vitro, for

detection, and measurement of activities of receptor, PTK and STATs are provided such as

EMSA, kinase phosphorylation, in vitro and in vivo protein-protein binding assays, target

gene expression, cellular location of STAT, phosphorylation statues as assayed by anti-

phosphotyrosine-STAT antibodies (Figure 27), antibodies against RECEPTORs. PTKs,

STATs, and target proteins presented in Figures and examples in this application, and by

using epitope tagged, such as Flu-HA tag, Myc tag, Flag-tag (Kodak) and all kinds of

commercial available green fluorescent protein tags, etc. and corresponding reagents and

assay systems to detect these tagged proteins.

EXAMPLE 1

In Example 1, in contrast to the conventional view that the RECEPTOR/PTK are

promote cell proliferation, the unexpected observations and evidence are presented showing

that the STAT pathway initiated by Receptor/PTK activation induces apoptosis. It was

demonstrated that expression of a member of a variety of tyrosine kinases, in combination

with each of six different STAT proteins, can cause apoptosis both in cultured cells and/or in ligand- stimulated cells. These observations and experiments provide methods and

compositions for identifying and developing therapeutic agents for use in modulating

Receptor/PTK-STAT mediated physiological and pathological processes. Materials and Methods

Cell Culture, Extracts, Antibodies and Mobility Gel Shift Assay. For integrin/FAK

related experiments, tissue culture plates were coated overnight with lOug/ml human plasma

fibronectin (Gibco) in PBS, washed twice with PBS and then incubated with 2 mg/ml

heat-inactivated (1 hr at 70 °C) BSA in PBS for 2 hrs at 37 °C. Cells were harvested by brief

trypsinization and washed twice with PBS containing 0.5mg/ml soybean trypsin inhibitor

(Sigma). The cells were resuspended in DMEM without serum and added to coated plates

(100mm) at 8 x 10⁶. After various times of incubation at 37 °C, cells were washed twice with

cold PBS and lysed in whole-cell-extract (WCE) buffer (15mM Hepes, pH 7.9, 400mM

NaCl, 0.5% NP-40, 10% Glycerol, and ImM EDTA) containing a cocktail of protease and

phosphatase inhibitors (0.5mM PMSF, 1 mg/ml leupeptin, lmg/ml aprotinin, lmg/ml

pepstatin, ImM vanadate, lOmM NaF, and ImM DTT), left on ice for 45 min., centrifuged

for 10 min. at 4 °C. WCE containing the same amount of total proteins were subjected to

EMS A with 10 fmol of ³²P-labeled high-affinity SIE probe.

(5'-AGCTTCATTTCCCGTAAATCCCTAAAGCT-3') (SEQ ID NO. 1) (Chin, et al. 1996)

A431, MDA-MB-468 and HeLa cells (ATCC) were grown in monolayer at 37°C in

Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum

(FBS) or calf serum. 2fTGH and U3 A cells, obtained from Dr. G. Stark lab, were grown in DMEM supplemented with 10% FBS and 400 μg/ml hygromycine. U3A-S1-2 cells were

grown in DMEM supplemented with 10% FBS and 400 μg/ml G418. Whole cell extracts

were prepared as described previously (Chin et al, 1996). Briefly, cells were starved

overnight and treated with 50 - 200 ngml ¹ EGF (Gibco) or 10 ng-mr' IFN-γ (Genzyme) for

30 min. PBS rinsed cells were lysed in 20 mM Hepes (pH 7.9) buffer containing 0.2%

NP-40, 400 mM NaCl, 0.1 mM EDTA, 10% glycerol, 1 mM dithiothreitol (DTT), 1 mM

sodium vanadate, 0.5 mM phenylmethylsulphonyl fluoride, 1 μg/ml each of aprotinin,

leupeptin and pepstatin. After 30 min gently agitation at 4°C, the supematants were collected

by centrifugation. For all electrophoretic mobility shift assays (EMSAs), M67-SIE was used

as the probe (10). DNA-protein binding reactions (15 μl) were performed by incubation of

the whole cell extracts in 10 mM Hepes (pH 7.9), 50 mM NaCl, 0.1 mM EDTA, 5%

glycerol, 50 μg/ml poly(dl-dC) (Pharmacia), 0.5 mM DTT, and 0.01% NP-40 for 10 min at

room temperature, followed by an additional 30 min incubation with ³²P-end-labeled M67

SIE probe (0.1 ng) at room temperature. DNA-protein complexes were separated on 6%

non-denaturing acrylamide gels in 0.5XTBE and detected by autoradiography. Anti-EGF

receptor antibody was purchased from Gibco and a purified anti-phosphotyrosine polyclonal

antibody was a generous gift from Jun-Lin Guan (Cornell University). The EGF receptor

immunoprecipitation (IP) and phosphotyrosine antibody blotting were performed as

previously described (Fu and Zhang, 1993). Anti-MAP kinase (ERK-2) and anti-ICE (plO)

antibodies were from Santa Cruz Biotech, Inc. and the Western blot assays using these two

antibodies were performed according to the manufacture's protocol. Analysis of Apoptotic Cells.

1) Morphological changes in the nuclear chromatin of cells undergoing apoptosis

were detected by staining with the DNA-binding fluorochrome bis-benzimide (Hoechst

33258; Sigma). Briefly, monolayer cells (3 - 6 x 10⁵) were grown in 6-well plates, and

treated with or without EGF (100 - 200 ng/ml) or IFN-γ (80 - 160 ng/ml) in the presence of

1 % calf serum or fetal bovine serum for different times. After treatment, cells were collected

and pelleted at 300 x g for 5 min. and washed once with PBS. Cells were resuspended in 100

μl of 3% paraformaldehyde in PBS and incubated for 15 min. at room temperature. After

fixation, the cells were washed once with PBS and were stained with 15 μl of bis-benzimide

(16 μg/ml) in PBS. Following 15 min. incubation at room temperature, a 5 μl aliquot of

cells was placed on a glass slide, and the average number of nuclei per field was scored for

the incidence of apoptotic chromatin changes under a fluorescence microscope. Cells with

three or more condensed chromatin fragments were considered apoptotic.

2) X-gal analysis and Apoptosis assays: Forty eight hrs after transfection, cells were

fixed by 1% glutaraldehyde (in PBS) in 37°C for 15 min. Cells were stained with 0.2%

X-gal (Amersham) (Buffer: 10 mM Na₃PO₄(pH 7.0), 150 mM NaCl, 1 mM MgCl₂, 3.3 mM

K₄Fe(CN)₆, 3.3 mM K3Fe(CN)6) for lhr. Wash with 70% ethanol, then cover cells with

PBS.

3) For TUNEL assay, cover-slip coated with fibronectin (lOug/ml) in 6-well plate in

4°C overnight. 1.7xl0⁵ cells were seeded for overnight. After 48 hrs of transfection, cells

were fixed with 3% paraformaldehyde for 10 min. in room temperature. ApopTag Kit (ONCOR) was used for in situ apoptosis detection according to the company's instructions.

Northern Blot Analysis. Total RNA was prepared with an RNA isolation kit from

Gibco-Life Science. RNA (40 ug) was analyzed by electrophoresis in a 1.2 %

agarose-formaldehyde gel and transferred to a nylon membrane (Zeta-Probe, Bio-Rad).

Hybridization was performed at 65°C overnight in 0.25 M Na₂PO₄ (pH 7.2), 7% SDS, ImM

EDTA. The wash was performed at 65°C in 0.04 M Na₂PO₄ (pH 7.2), 1% SDS. The probes

(ICE cDNA and CPP32 cDNA) were labeled with a random primed DNA labeling kit

(Boehringer-Manheim) .

Primary cell preparation, cell viability and DNA fragmentation assay. Mouse

(ICE+/+ or ICE-/-) spleens washed with PBS twice and chopped up with a sterilized blade.

The chopped spleen cells were then treated with lx trypsin in EDTA at 37 C for 10 min. The

trypsinized spleen cells were then suspended in RPMI medium supplemented with 10% FBS,

100 U of penicillin per ml, and 100 ng of streptomycin sulfate per ml. After standing in a

15-ml tube for 1 - 2 min., the suspended single cells were collected and maintained at a

concentration of approximately 5xl0⁶ cells per ml. Cell viability was determined by trypan

blue exclusion after 48 hrs treated with or without IFN-γ .To examine DNA fragmentation,

approximately 1 x 10⁷ cells were seeded in a 100-mm dish and treated with IFN-γ- (50U/ml)

or untreated. After 48 hrs treatment, cells were harvested, washed with cold PBS twice and

used for DNA isolation. 0.6 ml lysis buffer (10 mM TrisHCl, pH 7.5, 10 mM EDTA, 0.2%

Triton-XlOO) was added to the cells and the lysis was allowed to proceed at room

temperature for 15 min. and then centrifuged for 10 min. at 12,000 rpm. The supernatant was collected and mixed with equal volume of phenol and centrifuged for 10 min. at 12,000

rpm. The supernatant was adjusted to 300 mM NaCl and added with 2 volume of ethanol to

precipitate DNA. After centrifugation for 10 min. at 12,000 rpm, the DNA pellet was

resuspended in 20 1 TE buffer and digested with 0.2 g RNAse at 37°C for 30 min. The

fragmented DNA was analyzed by running a 2% agarose gel staining with ethedium bromide.

Results

Activation of STATl during Tntegrin-Mediated Cell Adhesion and by FAK. It was

found that low levels of STATl activity were immediately and transiently enhanced in

human A431 cells after plating on fibronectin, a ligand for integrin (Figure 1A). Activation

of STATl after plated on fibronectin was clearly observed at the time point of 0.5 hour. The

nature of STATl in this complex was confirmed when this induced complex was recognized

by a STATl specific antibody, generating a suppershifted complex (indicated by SS). The

STATl activity was significantly reduced after 4 hours when cells became attached. These

results suggested that STAT proteins may be activated through integrin-activated tyrosine

kinase(s) in this condition in vivo.

Since focal adhesion kinase (FAK) is a major tyrosine kinase activated during

integrin signaling, 293T cells that were transfected with vectors expressing FAK and STATl,

separately or in combination were further examined (Figure IB). STATl activation was

observed in cells transfected with FAK. but not in mock transfected cells, suggesting that

FAK activated endogenous STATl in vivo in these cells. Transfection of a HA-tagged

STATl(Fu and Zhang, 1993) also generated a weak STATl complex, which migrated slightly slower than endogenous STATl complex possibly due to the added HA-tag in the

protein. However, in cells co-transfected with FAK and STATl, STATl was strongly

activated. This STATl complex was recognized by an anti-STATl antibody, forming a

supershifted complex (SS) in the EMSA.

Expression of FAK can Cause Cell Apoptosis through Activation of STATl . It was

observed that dramatic morphological changes in transfected cells seemed to parallel with

STATl activation by FAK (Figure 2A). Since these cells were cotransfected with a vector

that expressed b-galactosidase, transfectants could be specifically recognized by the blue

color after X-gal staining. Cells that were mock transfected or transfected with STATl alone,

had little change on cell morphology. However, cells that had been co-transfected with FAK

and STATl, clearly lost cell spreading and were detached from the plate. For the cells

transfected with FAK alone, a portion of transfected cells also underwent the similar

morphological alterations which might result from the endogenous STATl activity induced

by FAK.

To confirm that cell morphological alterations caused by FAK-STATl activation may

induce apoptosis, cells were fixed with paraformaldehyde, then stained with DNA-specific

fluorochrome bis-benzimide, and examined by fluorescence microscopy (Figure 2B). A large

portion of cells showed bright white spots representing apparent DNA condensation, a

hallmark of apoptosis. These condensed DNA spots coincided with DNA ladders assayed on

an agarose gel electrophoresis (data not shown). However, the induction of DNA

condensation and fragmentation was not observed in cells transfected with mock or STATl alone or mutant FAK with STAT. Consistent with morphological changes, a portion of

FAK-alone transfected cells were also apoptotic.

A quantitative measurement of apoptotic cells in various transfected cells (only those

cells stained blue due to co-expression of b-galactosidase were counted) based on cell

morphology was shown (Figure 2C). The results were derived from three repeated

experiments.

STAT1 is Essential for Induction of Apoptosis by FAK. To confirm the role of

STATl in FAK-induced apoptosis, the embryonic fibroblasts isolated from STATl null or

control mice (Durbin, et al, 1996) were subjected to the further analysis. Consistent with the

above results with 293T cells, exogenously expressing FAK with STATl or FAK alone

resulted in dramatic morphological changes, indicating possible apoptosis, which was further

confirmed by the TUNEL assay, in the wild type (STATl +/+) fibroblasts. In contrast,

expression of STATl alone or in mock transfected cells, had no effect. However, in STATl

null (-/-) fibroblasts, little morphological change and apoptosis were observed in FAK alone

transfected cells. Furthermore, these cells could undergo apoptosis when STATl was

re-introduced with FAK.

The results of quantitative measurement of apoptotic cells by the morphological

examination (Figure 3A), or measured by the TUNEL assay (Figure 3B) were consistent.

Please be noted that in the calculation, only transfected cells which were stained blue in the

total STATl deficient or wild-type cells were measured. Similarly, the relative numbers of

apoptotic cells observed in each field were counted and compared. The results were derived from three repeated experiments.

In addition to using these STATl null fibroblasts, U3A-pSG5 cell line was also used,

which is STATl defective, and U3A-STAT1 cells, in which STATl has been stably

reintroduced (Chin, et al, 1996), to further determine whether introduction of STATl to the

STATl defective cells can confer FAK-induced apoptosis. As anticipated, transfection by

FAK alone induced significantly more apoptotic cells in STATl positive cells than in STATl

defective cells as determined by both cell morphology (Figure 3C) and TUNEL assay (Figure

3D). These results suggest that FAK-STATl activation is necessary for the induction of

apoptosis in these transfectants. Both Tntegrin Signaling and STATl are Necessary for Promotion of Apoptosis under

Physiological Conditions Caused by Semm Withdrawal. Previous studies have indicated that

a role of FAK is to prevent apoptosis under certain conditions. This might be due to the fact

that FAK activates survival signals (RAS, PI3 kinase etc.) in parallel. Moreover, the cell

culture media contain growth factors which provided additional survival signals. Thus the

activation of STAT and the apoptosis signal might be negated or covered.

Embryonic fibroblasts, derived from either STATl null or wild type mice, were

plated on fibronectin (FN) in a culture media containing no semm. If STATl contributes to

the induction of apoptosis in response to the integrin-FAK signaling after cells are plated on

fibronectin, then STATl wild type fibroblasts will undergo apoptosis faster than STATl null

fibroblasts under this stringent condition. The results supported this hypothesis: STATl

positive cells plated on FN or tissue culture dish (on which the cells are able to secrete matrix proteins) were dying significantly faster than those STATl null cells under the same

condition (Figure 4 A and 4B).

As an important control, these cells were also plated on bovine semm albumins

(BSA), which does not activate integrin signaling. In contrast to the faster cell death rate for

STATl wild type cells plated on FN (Figure 4A), it was found that STATl null and wild

type cells were dying at the same rate when they were plated on BSA (Figure 4C).

These experiments have further shown that STATl protein can promote apoptosis,

and this promotion of apoptosis through STATl is dependent on the integrin signaling which

is triggered by adhesion to FN, but not to BSA. These results have further implicated that

integrin-induced STAT activation can promote apoptosis under the physiological conditions

when the survival signals are weakened, such as after semm or growth factor withdrawal.

Moreover, no overexpression of STAT or FAK proteins was involved under the experimental

conditions above.

Induction of apoptosis by cn-expression of the HER receptor family and STAT

proteins. Similar to apoptosis induction through expressing FAK and STAT, co-expression

of EGF receptor (HER-1) or each of other members of HER receptor family, with each

member of STAT proteins (except STAT2) can cause induction of apoptosis (Figure 5).

Vectors expressing each member of the HER family and vectors expressing each member of

STAT proteins were co-transfected into 293T cells. The apoptotic cells were identified by

cell morphological changes and trypan blue exclusion. The results were derived from three

repeated experiments. Joint actions of HER1 and each STAT proteins cause cell death (Figure 5 A); joint actions of HER2 and each STAT proteins cause cell death (Figure 5B);

joint actions of HER3 and each STAT proteins cause cell death (Figure 5C); and, joint

actions of HER4 and each STAT proteins cause cell death (Figure 5D).

STAT activation induced by EGF causes apoptosis. Comparison of STAT activation

in HeLa cells vs. A431 cells in response to EGF (Figure 6A). Cells were treated with EGF

for 30 min, and protein extracts were prepared. Electrophoretic mobility shift assays

(EMSAs) using M67SIE as the probe showed that in A431 cells treated with EGF,

DNA-bound STAT dimers were formed (SIF-A: STAT3 homodimer, SIF-C: STATl

homodimer, and SIF-B: STAT1/STAT3 heterodimer). In contrast, no obvious STAT

activities were detected in HeLa cells treated with EGF under the same conditions.

Apoptosis induction of these two cell lines correlates with STAT activation in

response to EGF treatments (Figure 6B). In A431 cells, EGF treatment induced cell

apoptosis. In contrast, no apoptosis was observed in EGF -treated HeLa cells. The apoptotic

cells were clearly identified by altered nuclear stmcture with condensed chromatin fragments

seen under fluorescence microscopy after staining with fluorochrome bis-benzimide. EGF-

induced apoptosis was further confirmed by DNA fragmentation assays (data not shown).

Since EGF elicited very different response in these two cell lines, EGF receptor

autophosphorylation and MAP kinase activity in A431 and HeLa cells were examined

(Figure 6C). EGF treatment lead to EGF receptor autophosphorylation in both A431 and

HeLa cells. The same protein extracts were also probed with anti-MAP kinase antibody in

Western blot assays. MAP kinase (ERK-2) was phosphorylated (slowed mobility) and therefore activated after EGF treatment in both A431 and HeLa cells. These data indicate

that failure of EGF to activate STAT proteins in HeLa cells was not due to an EGF receptor

defect. The data also indicate that EGF-induced apoptosis in A431 cells was not due to

inactivation of the Ras-MAP kinase pathway.

Additional evidence for the correlation between Receptor/PTK-STAT activation and

apoptosis was obtained from the studies of MDA-MB-468 cells, a breast cancer cell line and

A431-R, an A431 variant (Figure 6D). It was found that STAT was not activated by EGF,

consequently, no apoptosis was induced by EGF in A431-R cells. In contrast, in

MDA-MB-468 cells, apoptotic cells were induced which was apparently caused by STAT

activation (right panel).

TCE Expression Correlated with EGF-STAT activation and induced Apoptosis.

Since the ICE protease family play important roles in apoptosis, the gene expression patterns

of most members of these two apoptosis gene families were examined by Northern blot

analysis (Figures 7A, 7B, and 7C). Among the genes tested, ICE (Caspase-1) expression was

upregulated in a STAT-dependent manner. EGF induced ICE gene expression in both A431

and MDA-MB-468 cells, but not in HeLa cells which was correlated with STAT activation in

these cells. In addition, in A431-R cells, which are defective in STAT activation, ICE mRNA

expression was uninducible (data not shown).

To confirm that ICE induction at the protein level, Western blot analysis of

whole-cell protein extracts from A431, HeLa, and MDA-MB-468 cells, which were treated

with or without EGF, revealed that ICE protein levels increased following EGF treatments. A proteolytically cleaved form of ICE, plO, was clearly observed in A431 cells after EGF

treatment. To obtain further evidence for the involvement of ICE in the EGF-induced

apoptosis, it was examined whether ZVAD, an irreversible inhibitor of ICE family proteases

can block EGF-induced apoptosis. ZVAD effectively blocked either EGF- or IFN-γ -induced

apoptosis in cells tested.

Taking together these data, It was concluded that induction of ICE mRNA and

protein are due to STAT activation in these cells, indicating that ICE protease may be

involved in EGF-induced apoptosis.

Jakl is necessary for induction of apoptosis in response to IFN-γ. Like many other

cytokines, interferons may activate multiple pathways including the STAT and the Ras-MAP

kinase pathways (David et al, 1995; Xia et al, 1996). Both ICE mRNA and apoptosis

induction in JAK- and STAT-deficient cell lines in response to IFN-γ treatment were

investigated.

E2A4 is a JAKl kinase-deficient cell line derived from HeLa cells (Loh et al, 1994).

DNA binding activity of STAT activation was absent as determined by EMSAs in E2A4

cells (Figure 8A). The strong induction of ICE mRNA normally seen upon IFN-γ treatment

in the parental HeLa cells was completely abolished in this JAKl deficient cell line (Figure

8B).Moreover, bis-benzimide staining showed that E2A4 cells did not apoptose in the

presence of IFN-γ (Figure 8C). These results (A-C) suggest that JAKl kinase is essential for

the induction of both ICE expression and apoptosis by IFN-γ.

Apoptosis induction in response to IFN-γ. Analysis of apoptosis induction in U3 A cells, a STATl -defective cell line (McKendry et al, 1991). and the parental cell line 2fTGH,

and STATl reintroduced U3A-S1-2 cells in response to IFN-γ. IFN-γ failed to activate

STAT, induce ICE mRNA expression, or lead to apoptosis in U3A cells. In contrast, IFN-γ

was able to induce STAT-DNA binding activity, ICE mRNA, and apoptosis in both the U3A

parental cell line 2fTGH cells and the U3 A-S 1 -2 cells, in which STAT 1 had been

reintroduced (Figures 9A and 9B). As a control, U3A cells that were stably transfected with

the vector alone were unable to respond to IFN-γ (Chin et al., 1997).

Bis-benzimide staining of parental 2fTGH cells, STATl defective U3A, and STATl

reintroduced U3A-S1-2 cells. The condensed and/or fragmented nuclei were observed in

2fTGH and U3 A-S 1 -2, but not U3 A cells treated with IFN-γ (Figure 9C).

ICE Gene Is necessary for IFN-γ-Induced Apoptosis. To further confirm that ICE

expression induced by IFN-γ is critical to provoke apoptosis, the primary spleen cells from

ICE'- and ICE^{+ +} mice (Kuida et al, 1995) were isolated and their responses to IFN-γ

treatment were compared. Although STATl can be activated by IFN-γ in both ICE'- and

ICE^{+ +} cells (Figure 10A), IFN-γ-induced DNA fragmentation was significantly reduced in

ICE'- cells compared with that in ICE^{+ +} cells (Figure 10B). The cell viability assays (trypan

blue exclusion) showed that IFN-γ triggered much more apoptosis in ICE^+/+ cells than in

ICE'- cells, in a dose-dependent manner (Chin et al, 1997). Thus, ICE expression plays an

important role in IFN-γ-induced apoptosis.

A General Pathway to Induction of Apoptosis through the Joint Actions of a variety

of different Receptor PTKs and STATs. Besides the above well-characterized examples of apoptosis induction through activation of STAT proteins by the family of EGF receptor

kinases, FAK, and Jak kinases, apoptosis induction through joint actions of a variety of

Receptor/PTKs with each of the STAT proteins after they are pairly transfected into 293T

cells, have been further analyzed:

Figure 11 A shows apoptosis induction through joint actions of TrkA, a nerve trophin

receptor, and each of the STAT proteins;

Figure 1 IB shows apoptosis induction through joint actions of TrkB, a nerve trophin

receptor, and each of the STAT proteins;

Figure 1 IC shows apoptosis induction through joint actions of a EPH protein, a nerve

trophin receptor involved in neuron differentiation, and each of the STAT proteins;

Figure 11D shows apoptosis induction through joint actions of Tie2, a receptor

involved in angiogenesis and early development etc., and each of the STAT proteins;

STAT5A was especially active in causing apoptosis;

Figure 1 IE shows apoptosis induction through joint actions of FGFR2, a receptor

involved in development and angiogenesis etc., and each of the STAT proteins;

Figure 1 IF shows apoptosis induction through joint actions of FGFR3, a receptor

involved in development and angiogenesis etc., and each of the STAT proteins;

Figure 11G shows apoptosis induction through joint actions of Src, a cytoplasmic

tyrosin kinase involved in bone development and tumor transformation etc., and each of the

STAT proteins;

Figure 11H shows apoptosis induction through joint actions of Lck, a cytoplasmic tyrosin kinase involved in lymphocytes development and function etc., and each of the STAT

proteins;

Figure 111 shows apoptosis induction through joint actions of Itk, a cytoplasmic

tyrosin kinase involved in lymphocytes development and function etc., and each of the STAT

proteins.

The results of quantitative measurement of apoptotic cells are obtained by the

morphological examination, and by the trypan-blue exclusion assay, and only transfected

cells were accounted which were stained blue dye to co-transfection with a vector that

expressed b-galactosidase, thus transfectants could be specifically recognized by the blue

color after X-gal staining. The mock was the vector alone transfected cells.

The STAT proteins control the apoptosis induction by default after growth factor

withdrawal. Cultured mammalian cells will die through apoptosis when the necessary

cytokines, growth factors or semm are deprived. This induction of apoptosis after growth

factor or semm withdrawal has been believed to be due to a "default" mechanism (Raff,

1992). According to this notion, cells can only survive when growth factors are provided to

suppress this mechanism to die. It is not known what is the molecular basis of this default

mechanism. It is unclear either whether this induction of apoptosis by default can be

regulated and affected by signaling pathways. Nothing is known about the mediators that

carry out this apoptosis by default.

The importance of cell death induced by growth factor deprivation, or by default is

not only limited in cultured cells. This kind of cell death occurs commonly during certain critical developmental stages. It is required for organogenesis and maintenance of

homeostasis of whole body. Furthermore, a variety of degenerative diseases should be caused

by apoptosis through reduction or deprivation of survival factors, an event resembling cell

death triggered by growth factor withdrawal in culture cells (Thompson, 1995). It was shown

that STAT proteins control the induction of apoptosis caused by growth factor withdrawal. In

other words, induction of apoptosis by default after survival factor deprivation, which is a

mechanism involving many kinds of degenerative diseases and developmental processes, can

be controlled by STAT-regulated expression of apoptosis genes.

Expression of the STATl protein in mouse embryonic fibroblasts promotes apoptosis

by default after semm withdrawal while deficiency of STATl protein reduces apoptosis after

semm withdrawal. The comparison of apoptosis induction after serum reduced to 0.02%.

STATl deficient cells undergo apoptosis slower that STATl positive cells under these

conditions (Figure 12A). The comparison of apoptosis induction after semm withdrawal

(0.0%>). STATl deficient cells undergo apoptosis slower that STATl positive cells under

these conditions (Figure 12B).

Expression of the STATl protein in Ba/F3, a cell line derived from pro-B cells,

promotes apoptosis after semm or growth factor (IL-3) withdrawal. STATl protein

expression is higher in a Ba/F3+STAT1 cell clone that expresses exogenous STATl than its

parental cells (Figure 13 A). STATl SIE-DNA activities were higher in the Ba/F3+STAT1

cells than that of the parental Ba/F3 cells (Figure 13B). Higher activities of STATl in

Ba/F3+STAT1 cells cause faster cell death after semm withdrawal (Figure 13C). Expression of the STAT3 protein in Ba/F3, mouse embryonic fibroblasts promotes

apoptosis after semm withdrawal. STAT3 protein expression is higher in two independent

cell clones of Ba/F3+STAT3 (STAT3, wt3, and STAT3, wtlO), that express exogenous

STAT3, than its parental Ba/F3 cells (Figure 14A). Higher expression of STAT3 protein in

these two clonal cells cause faster cell death after semm withdrawal (Figure 14B).

Discussion

In this Example, the present inventors have discovered, for the first time, new

methods and compositions to regulate and modulate induction of apoptosis. They have shown

the Receptor/PTK-STAT signaling pathway is cmcial in the induction of apoptosis. This

finding contrasts with the general concept that protein tyrosine kinase- activating cytokines

usually act to stimulate growth and survival factors in a cell. However, the present inventors'

finding supports the notion that many cytokines may have dual functions in cell growth

control, generating either positive or negative growth signals depending on the cell types.

Activation of STAT is one of these negative signals induced by receptor-associated tyrosine

kinases.

As shown in Figure 15, a negative and positive signaling model is proposed to

explain the molecular basis responsible for the dual functions of cytokines. It is proposed that

a cytokine, by binding to its receptor, can turn on at least two separate signaling pathways:

activation of the Ras-MAP kinase pathway (or other pathways such as PI3 kinase pathway)

for cell growth/survival and activation of the STAT pathway for cell arrest/death. The

intracellular homeostasis requires a balance between growth/survival and arrest/death signaling events. Different cells may have different dynamic states and hence different

phenotypic outputs. In HeLa cells, the STAT pathway is not very active, EGF mainly

triggers the MAP kinase pathway, so cells proliferate and survive. While in A431 or

MDA-MB-468 cells, the STAT pathway is more sensitive in response to EGF, the caspase

can be induced and cells arrest and die. Therefore, EGF can activate a negative signaling

pathway through STAT activation and expression of caspases.

Therefore, the polypeptide ligand-activated Receptor/PTK signaling, could not only

transduce the proliferative and surviving signals, as most commonly observed, but also, in

some conditions, generate anti-proliferative and cell death signals which are mediated by the

STAT proteins. This conclusion may provide a solid explanation for the dual functions of

many cytokines and growth factors. The discoveries made in this invention showing that the

Receptor/PTK- STAT signaling can lead to apoptosis which will provide methods and

compositions for finding agents to interfere apoptosis during development and apoptosis

during development of a variety of mammalian degenerative diseases which are triggered by

survival factor deprivation.

Several important issues concerning the conclusions in this invention should be

further clarified here. First, the greatly reduced apoptotic response in ICE ' cells (see above)

constitutes definitive evidence for the requirement for ICE in IFN-γ-induced cell death.

While ICE expression is involved in the EGF- and IFN-γ-induced apoptosis as demonstrated

here, it could be a general mechanism that regulation of caspases by each of the STAT

proteins is the molecular basis in many other conditions, such as in cell matrix-related, in particular, integrin-induced apoptosis shown above. Additionally, caspase- 1 is the first, but

may not be the only caspase That can be regulated by the Receptor/PTK-STAT signaling.

Other members of caspases may also be targets of regulation. For example, ICH-1/caspase 2

could also be up-regulated by STAT activation (data not shown). In some cells, BAX and

FAS may also be up-regulated by the STAT pathway (inventors' unpublished results).

Therefore, it must have multiple target genes in STAT-mediated apoptosis induction.

Therefore the regulation of caspases by the STAT pathway can be regarded as the first model

system.

Furthermore, some diseases resulting from inappropriate cell arrest and apoptosis may

be due to an overactive Receptor/PTK-STAT pathway. It has been shown that in this

invention that a variety of Receptor/PTKs, activate different STAT proteins and induce

apoptosis. For example, the Receptor/PTK-STAT can be strongly activated and the induction

of caspases is high after cytokine treatment. Therefore, the survival signals from

Receptor/PTK are overcome by the effects of STAT activation and upregulated caspases,

causing apoptosis.

Importantly, The Receptor/PTK-STAT signaling pathway and its regulated gene

expression may provide a molecular mechanism of the apoptosis induction by DEFAULT.

There must be certain mediators or gene products to carry out the death execution after

survival factor deprivation, based on the view that apoptosis is an active process. It is known

that for different types of cells there are significant differences in their sensitivities or

thresholds for apoptosis induction by default (Thompson, 1995). The Receptor/PTK-STAT pathway can mediate induction of apoptosis through the regulation of caspases and/or other

pro-apoptotic proteins. However, in normal cell culture conditions without large amount of

cytokines, STAT proteins may only be activated at a lower level (possibly due to growth

factors in the semm supplied, or matrix/integrin interactions etc.). Coordinately, a lower and

constitutive level of caspases or other pro-apoptotic gene expression is maintained which is

not strong enough to overcome the survival signals. Thus these cells survive. However, when

the survival signals are deprived, such as when growth factors are withdrawal, the balance

between death signals (STAT and caspase activities etc.) and survival signals may shift

towards death. Thus apoptosis may occur. Evidence was presented in this invention to

support this important conclusion. For example, it is shown above that STAT activation

through integrin signaling could promote apoptosis when semm were deprived.

Additionally, since caspase activities are required for most, if not all, apoptosis execution,

STAT-regulated caspase expression should also affect many kinds apoptosis induction

through different signaling pathways, which may include apoptosis induction caused by

radiation, TNF/FAS, Myc expression, and many other apoptosis-inducing agents. This

conclusion is shown in the Figure 16.

Thus, according to principles presented in Figure 16, the hard- wired, intrinsic

executioner or mediator (THE DEFAULT) of apoptosis is the low level of STAT activities

and STAT-regulated caspase or other pro-apoptotic protein activities. The differences in the

thresholds for induction of apoptosis may be determined by the different levels of STAT and

STAT-regulated caspase or other pro-apoptotic protein activities. This conclusion may be correct for many situations in which apoptosis is induced due to weakened survival signals.

These are significant and fundamental issues, since apoptosis induction by this default

mechanism after survival signal withdrawal may be responsible for many important

situations of apoptosis induction during stages of development or in the pathogenesis of auto

immune disorders, leukemia, and some degenerative diseases, and for many other types of

"spontaneous" apoptosis. Moreover, although some caspases such as CPP32/caspase-3 may

not be regulated by STAT proteins, its enzymatic activities may be activated by other

caspases, such as caspase- 1. It has been shown most STAT proteins can well play a role in

induction of apoptosis (see Figures above). The possible redundant functions of different

members of STATs and caspases may provide an explanation to the observation that a single

null mutation of one member of STATs or caspases may not cause significant defects in

development (Durbin et al, 1996; Kuida et al, 1995).

In summary, Receptor/PTK-STAT activation is a broad molecular signal mediating

induction of apoptosis, and modulating Receptor/PTK-STAT activities can provide an

important diagnostic and therapeutical tools and treatment methods for a variety of

apoptosis-related diseases.

EXAMPLE ?.

Many growth factors and cytokines, such as EGF, PDGF, FGF, IL-3 and IL-6 etc.,

have been shown to play critical roles in cell proliferation, differentiation and development.

Moreover, many mammalian diseases are associated with abnormal functions of growth

factors. It is reasonable to speculate that as major functional mediators of these cytokines, STAT proteins may be involved in regulation of cell proliferation, development and in

pathogenesis of some developmental disorders. However, it has been unclear until recently

whether STAT proteins play any decisive roles in these important biological processes.

In the following studies, evidence was presented showing that STAT activation will

induce immediate gene expression of CDK inhibitor p21 , which may play key roles in cell

growth arrest in response to cytokines and also may be involved in the regulation of some

critical developmental steps. In particular, it was shown that growth defects in the bone

development observed in mutant FGFR3 patients are probably caused by abnormal functions

of Receptor/PTK-STAT signal transduction and induction of CDK inhibitor ρ21/WAFl.

Thus, it was discovered that the Receptor/PTK-STAT pathway may play a decisive role in

the negative regulation of cell growth. Materials and Methods

Site-directed mutagenesis and plasmid construction. Chameleon Double- Stranded,

Site-Directed Mutagenesis Kit (Stratagene) was used to engineer the TDII type mutation on a

mouse FGFR3 expression vector ρMo/mFR3/S V (Omitz et al. ( 1992)) . An Oligonucleotide

5'-GGACTACTACAAGGAGACCACAAACGGCCGGCTACC-3' (K644E in mouse) (SEQ

ID NO. 2) and the Afl III primer from the manufacturer were used for the reaction. The

successful mutagenesis was verified by sequencing. The mutated and wild-type cDNAs

cloned into pEF-BOS vector were used for transfections otherwise mentioned (Muzushima et

al. (1990).

Tmmunocytochemistry and immunohistochemistry. Two days after the transfection, 293T cells were harvested and smeared on glass slides. Cells were fixed and permeabilized

in cold methanol/acetone (1:1) solution at -20 C for 10 minutes, followed by incubation with

normal horse or goat semm to block the nonspecific binding sites. Paraffin sections were

de-paraffmized by the successive treatment with xylene, 95% ethanol and water. After

treatment with FICIN (Zymed) enzyme to retrieve antigens, sections were incubated in 2.0%

hydrogen peroxide and 0.1 % sodium azide in methanol for 10 minutes to inactivate

endogenous peroxidase. Antibodies against STATl (monoclonal for the cultured cells and

polyclonal for the tissue sections, Transduction Laboratory), p21^{WAF1 clp}' (Santa-Cmz), and

FGFR3 (Santa-Cmz) and Vectastain Elite ABC Kit (Vector Laboratory) were used to stain

the cells. 3,3'-Diaminobenzidine (DAB) with or without nickel chloride were used as

substrate of peroxidase to give a black or brown color, respectively .

Northern blotting. Total RNAs (5mg) from 293T cells transfected with the FGFR3

expression vectors were subjected to Northern blot analysis with the p21 cDNA used as a

probe as previously described (Chin et al, 1996).

Growth assays. Twenty- four hours after the transfection, 293T cells were replated

into 24-well culture plates at a density of 1.5x10⁴ cells/well and cultured for additional 24

hours. Then the cells were labeled with [³H]-thymidine (1.5mCi/ml) for 4 hours, followed

by washing with PBS twice, and in ice-cold 10% trichloroacetic acid three times. The

incorporated radioactivity was extracted by incubation in 3% perchloric acid at 95 C for 40

minutes and measured by liquid scintillation. Clonogenic assay was performed with 293T

cells after the transient transfection as previously described (Chin (1996)), except that the concentration of the cells was reduced to 5 x 10³ cells per plate. Colonies were counted 10

days after plating.

Results

STAT Activation Induced by EGF and IFN-γ is Correlated with Cell Growth Arrest.

Using M67-SIE as the probe in an EMSA, many cell lines were analyzed for STAT

activation in response to EGF and found no or very poor STAT activation by EGF in most

cells except A431 cells. The results from two representative cell lines, HT29 and WiDr,

which are derived from human colon adenocarcinoma, are shown in Figure 17 A. In contrast

to A431 cells in which STAT proteins (SIF-A: STAT3, SIF-C: STATl, SIF-B:STAT1 and

STAT3 heterodimer) were activated and cell growth was inhibited in response to EGF

treatment, no detectable STAT activation was observed after EGF treatment of HT29 and

WiDr cells, and these cells grew normally in the presence of EGF (Figures 17C and 17D).

However, all these cells, including A431, were responsive to IFN-γ, producing activated

STATl (SIF-C) as shown above. As expected, growth of all these cells was inhibited by

IFN-γ treatment (Figures 17B, 17C and 17D). Additionally, results from ³H-thymidine

incorporation assay were consistent with the growth curves (data not shown).

These results suggest that activation of STATs, STATl in particular, in A431 cells

by EGF, as well as STATl activation in all these cells by IFN-γ, might be a key event

leading to the inhibition of cell growth .

The p21/WAFl Expression by STATs in Response to EGF and IFN-γ. Vogelstein

and his colleagues have cloned the promoter region of the p21 gene and mapped the p53 regulatory sites (El-Deiry et al, 1995).

After a careful examination of this promoter, It was found that there are three

sequences in this promoter that contain potential STAT interacting site (SIE) which contain a

palindrome sequence TIC (N3) GΔA_(see Chin et al, 1996) (Figure 18A). It has been

further shown that these p21-SIEs are regulatory sites of STAT proteins (see Chin et al,

1996), raising the possibility that the p21 gene may be up-regulated by STAT proteins.

It has been found that p21 Gene Expression is Correlated with STAT Activation and

Growth Suppression in Response to EGF and IFN-γ. One example is shown in which p21

mRNA was induced after EGF treatment (Figure 18B). These SIE sites are possible targets

for agents that block STAT-DNA interaction.

STATl is Essential for Induced Cell Growth Arrest IF STATl is essential for

growth inhibition, STATl deficient U3A cells will not be inhibited by IFN-γ. On the other

hand, reintroducing functional STATl protein into U3A cells may restore its responsiveness,

including cell growth arrest, to IFN-γ. U3A cells were restored by stable transfection with a

STATla expression vector pSG91 (Fu and Zhang, 1993). These STATla expressing U3A

cells were named as U3A/STATl . After these initial analyses, the rates of DNA synthesis

by ³H-thymidine incorporation in U3 A/Control and U3A/STATla cells in response to IFN-γ

treatment were compared (Figure 19).

As shown in above, U3 A/Control cells which were deficient in STATl were not

inhibited by IFN-γ even at high doses of IFN-γ, whereas the U3 A/ST ATI cells were

dramatically inhibited by IFN-γ at relatively low doses of IFN-γ. To further analyze the role of STATl in growth suppression, It was also found that U3 A/ST AT la cells were greatly

inhibited by IFN-γ for its ability to form colonies (about four fold fewer colonies) as

compared with U3 A/Control cells that were not affected by IFN-γ (Chin et al, 1996). These

results have convincingly demonstrated that STATl was essential for the growth inhibition

induced by IFN-γ in these cells. Consistently, p21 mRNA was expressed and induced at a

much higher level in U3A/STATia than in U3A/Control cells (data not shown). Similarly, It

was further shown that STAT activation is also required for EGF induced p21 induction and

cell growth arrest in A431 cells and other EGF inhibitory cells such as breast cancer

MDA-MB-468 cells (data not shown). In summary of the above results, it has been clearly

shown that the activation of STAT proteins is essential for growth suppression in response to

IFN-γ and EGF, probably through induction of CDK inhibitors like p21.

STATl Activation induced by expression of a mutant TDTT FOFR receptor. It has

been shown that the FGFR3 mutants have a gain-of-function nature leading to the abnormal

and excessive repression of bone growth. Although FGF is known for its proliferative effects,

the phenotypes of mutant FGFR3 related disorders suggest that the biological effect of these

FGFR3 mutants at the cellular level is inhibition or retardation of chondrocyte proliferation

in the cartilaginous growth plates. For example, the achondroplasia class of

chondrodysplasias is comprised of the most common genetic forms of dwarfism in human.

Its member, thanatophoric dysplasia types II (TDII), is caused by a distinct mutation at the

kinase domain of FGFR3 which retard skeletal growth and development. However, there is

no clue about the mediators of this mutant FGFR3-related growth abnormalities in development.

To study the effects of the TDII mutation on FGFR3 activity for downstream signal

transduction, the expression vectors of FGFR3 with the TDII mutation (Lys650Glu) was

constmcted. After transient transfection into 293T cells, the FGFR3 proteins were

immunoprecipitated from transfected cells, and their kinase activities were assessed by an in

vitro autophosphorylation (Figure 20 A). The amount of wild type and the TDII mutant

FGFR3 in the precipitate were at similar levels (Figure 20B). However, TDII mutant FGFR3

showed a greatly elevated intrinsic kinase activity in comparison with the wild-type

receptors. STAT activation in these different FGFR3 transfected cells was assayed using the

electrophoresis mobility shift assay (EMS A). The expression of mutant TDII FGFR3 could

induce a distinct protein complex that specifically bound to the high-affinity STAT

interacting site (M67-SIE) in a labeled DNA probe (Figure 20C).This complex co-migrated

with IFN-γ induced STATl complex (lane 1), and was further "supershifted" by a specific

anti-STATl antibody, but not by a control antibody, indicating that expression of this

mutant TDII FGFR3 resulted in STATl activation. These results demonstrate that the

expression of mutant TDII FGFR3 can constitutively activate STATl. The wild-type

receptor could also activate STATl, especially when STATl protein was co-expressed (data

not shown). However, TDII mutant receptor activated STATl much more strongly than

wild-type FGFR3, and this pattern of different levels of STAT activation might correlate

with the constitutive kinase activities of these receptors. Moreover, STATl activation by the

TDII FGFR3 could be further enhanced by FGF1, a ligand for FGFR3 (Omitz and Leder, 1992) (data not shown).

The status of MAP kinase in these transfected cells was analyzed. No detectable

differences in the forms of phosphorylated and unphosphorylated MAP kinases between

TDII and wild-type or other mutant FGFR3 transfected cells was found, indicating that in

contrast to STATl protein, MAP kinase might not be involved in the abnormal function of

the TDII receptor.

STAT1 nuclear translocation, p?1 /WAF1 induction and cell growth arrest in TDTI

transfected cells and in chondrocytes from TDII patients. To confirm that the TDII receptor

was expressed properly in the transfected cells, and to demonstrate the expression of this

TDII receptor-induced STAT activation at the cellular level, intracellular localization of

STATl in response to the expression of FGFR3 was examined. It is established that latent

STATl protein was located in the cytoplasm, whereas activated STATl could translocate

into the nucleus (Schindler, 1992b; Fu and Zhang, 1993). Therefore, translocation of STATl

into the nucleus is an indication of STATl activation. Although not illustrated further at this

point, one skilled in the art can recognize that an assay utilizing this observation could be

readily developed. For example, a method of diagnosing abnormal STAT activation could

involve nuclear staining of test cells using anti-STAT antibodies and the examination of the

stained nuclei for evidence of STAT protein translocation into the nuclei.

As shown in cells transfected with the wild-type FGFR3, the STATl protein

(visualized by black color) was barely recognizable, possibly due to the fact that STATl was

expressed at a low level and normally spread out in the cytoplasm, while the FGFR3 protein was expressed on the cell surface (brown color), as indicated by double staining of the cells

with an immunocytochemical method using specific anti-STATl and anti-FGFR3 antibodies

(Figure 21 A, indicated by arrows). However, the cells transfected with the TDII constmct

showed the expression of TDII receptor on the membrane, and many cells showed

concentrated localization of STATl in the nucleus (Figure 21B, indicated by arrows),

suggesting STATl activation in these cells. To confirm the nuclear localization of STATl,

the nuclei were counter stained by blue color with hematoxylin, while the anti-STATl

antibody-recognized proteins were visualized by brown color, showing the overlap stainings

of STATl and the nuclei in the TDII receptor-transfected cells (dark brown, indicated by

arrows in Figure 21C). These results further confirmed that expression of TDII mutant

receptor can activate STAT, resulting in STATl translocation to the nucleus.

Relationship between STATl activation by expression of TDTT receptor and

expression p21. It was further determined whether STATl activation by the expression of

TDII receptor would induce expression of p21. As anticipated, the p21 mRNA level was

particularly enhanced in TDII-transfected cells compared with other transfected cells (Figure

22 A). Probably due to the calcium mediated transfection, p21 mRNA levels were

non-specifically higher in transfected cells than in untransfected cells (Mock, Figure 22A,

lane 3). EGF induced p21 mRNA in A431 cells served as a positive control of p21 induction.

Consistent with p21 mRNA expression, p21 protein was enriched in the nuclei in TDII

transfected cells as demonstrated by an immunocytochemical stain with anti-p21 antibody

(indicated by arrows in Figures 22D and 22D). Since 293T cells express adenovims ElB and SV40 large T antigen (Pear et al, 1993), which are capable of inactivating p53, this

observed p21 induction in TDII transfected cells may not involve p53 (Ko and Prives,

1996). It was further shown that the rate of DNA synthesis and cell growth were significantly

reduced in TDII-transfected cells, whereas expression of the wild- type receptor did not

inhibit DNA synthesis or colony formation (data not shown).

Analysis of chondrocytes in situ in growth plates from TDTT affected individuals or

other control individuals for possible STATl activation and p21 expression, were further

examined using immunohistochemistry. Heterozygosity for the Lys650Glu mutation at the

DNA level was previously confirmed in two TDII patients (data not shown).

As shown in Figure 23, STATl was expressed at a low level in the chondrocytes

from a normal control individual, and STATl protein was found in the cytoplasm (Figure

23 A, brown staining of STATl was indicated by arrows; the nuclei were counter stained in

blue), indicating STATl is not normally activated in these cells. Strikingly, STATl was

found to be translocated into the nuclei, and exclusively stained in the nuclei in many

chondrocytes at the growth plates of femur from three TDII-affected individuals (Figure 23B

indicated by arrows), indicating STATl activation in these TDII chondrocytes. The

arrowheads in the low magnification view on the left of each panel showed the area where

the higher magnification view (on the right) was taken (indicated by arrowheads). To

confirm that this nuclear stain by anti-STATl antibody was specifically caused by STATl

protein, the staining procedure with or without the purified recombinant STATl protein as a

competitor was repeated. This nuclear staining by the anti-STATl antibody in chondrocytes of the TDII affected patient (Figure 23C ) was completely abolished by the specific

competitor (Figure 23D ), confirming specificity of STATl staining and the solidity of

observation of nuclear localization of STATl in these TDII chondrocytes. This nuclear

STATl staining pattern in the growth plate was consistent with the staining pattern of

FGFR3 (data not shown), suggesting this STAT translocation was probably caused by actions

of the mutant TDII receptor. p? 1 expression in the same TDTT-affected chondrocytes was further examined ■ As

shown in Figure 24, p21 protein was undetectable with an anti-p21 antibody (no brown stain)

in normal chondrocytes (Figure 24 A), however, p21 expression was clearly observed in the

TDII chondrocytes as indicated by brown or darker nuclear stain by the anti-p21 antibodies

(Figure 24B, indicated by arrows). p21 expression might cause growth retardation of these

TDII chondro-cytes, resulting in anomalies in bone development. Furthermore, as shown in

Figure 24B, the p21 staining is mostly in the nucleus (arrows). However, in some cells, the

p21 staining looked to be in the cytoplasm (arrowhead). Intriguingly, there were vacuole-like

stmctures in these cells. This vacuole-like structure indicates the cell degeneration which was

frequently observed in the TDII chondrocytes. Thus it was further shown that expression of

TDII mutant FGFR3 would cause cell death, especially the programmed cell death or apoptosis. Discussion

Cytokines or growth factors, such as EGF, FGF, IFN-γ, and many interleukins, can

stimulate a number of different and parallel signaling pathways. The Ras-MAP kinase and PI3 kinase signaling pathways have been implicated in mitogenic responses and cell survival.

However, many cytokines have been shown to stimulate growth in one cell type, while

inhibiting growth or inducing differentiation in the other cell types (Chin et al, 1996). In

contrast, the STAT signaling pathway may mediate negative control of cell growth or induce

apoptosis in response to extracellular stimuli as shown above. Thus, it is possible that

cytokines simultaneously initiate mitogenic pathways, possibly involving the RAS protein,

MAP kinases, PI3 kinase or other signaling proteins, and the STAT signaling pathway, that

may negatively regulate cell growth and survival by inducing expression of cell cycle

inhibitors such as p21, in addition to the apoptotic mediator ICE.

Thus whether a cytokine promotes or inhibits growth is determined by the relative

strengths of the positive and negative signals that may be induced simultaneously by the

same ligand (such as EGF)-bound receptor. Additionally, the relative strength of these

positive or negative signals may also vary in different cell types in according to specific cell

content. For example, EGF has been shown to activate the MAP kinase pathway, but not the

STAT pathway in many types of cells (see above). In those cells, in the presence of the

predominant positive signals to the nucleus, EGF acts as a survival or growth factor.

However, in A431 and MDA-MB-468 cells (Chin et al, 1997), although EGF treatment

could activate the EGF receptor, MAP kinase and other downstream signaling proteins, the

STAT proteins were also strongly activated by EGF in these cells, generating a negative

signal, including induction of cyclin inhibitors such as p21, which overrides the positive

growth signal, causing net inhibition of cell growth. Similarly, in normal development, STAT activation may be required for proper negative control to balance actions of the

mitogenic pathway. Thus, cells can grow or differentiate by joint actions of both pathways.

However, during abnormal pathological situations, such as TDII FGFR3, this receptor

experienced abnormally higher kinase activity, causing stronger STATl activation, p21

induction and cell growth arrest in chondrocytes of long bone and other tissues where the

FGFR3 is expressed (Peters et al., 1993). Thus, normal growth and development are

disrupted.

It is well known that p53 can mediate induction of ρ21. However, p53 is probably not

involved in p21 induction in responses to cytokines as shown here, because the level of p53

during the p21 induction by IFN-γ and EGF was not altered (data not shown) and the p53

protein in A431 cell was mutated at the codon 273 and probably nonfunctional (Kwok et al,

1994). It has recently been shown that in addition to the CDK inhibitor p27^κipl, p21 is also

induced in a p53-independent pathway in some types of cells treated with TGF-b (Datto et

al, 1995). Therefore, the p21 gene may act as a common mediator of the negative growth

signals for the genotoxic stimulation of the cell, such as radiation, as well as from actions of

cytokine receptors, although the promoter elements involved in each event may be different.

One of the striking examples observed is growth inhibition mediated by the FGF

receptor. Fibroblast growth factor (FGF) and its receptors (FGFRs) have cmcial functions in

differentiation, cell migration and development. At least nine members of FGFs have been

identified. The four FGFRs also have been known to be encoded by unlinked genes

{FGFR1-4 ) (Johnson and Williams, 1993; Mason, 1994). All these FGFRs have an extracellular region with three distinct domains which exhibit stmcture similarities to

immunoglobulins (Ig-like), a transmembrane domain and a split cytoplasmic tyrosine kinase

domain (see Figure 1). All these domains are necessary for functions of FGFRs. Genetic

mutations at these various domains could have great biological consequences (see below).

FGFs binds to these FGFRs causing activation of the intrinsic tyrosine kinase and

autophosphorylation of the receptors (Bellot et al, 1991; Coughlin et al., 1988; Mohammadi

et al, 1996). The tyrosine phosphorylated receptor exhibits elevated tyrosine kinase activity

which can further phosphorylate intracellular signal proteins that interact with the

autophosphorylated receptor (Mason, 1994). The tyrosine phosphorylation of the receptors is

clearly essential for the biological functions of FGFs. Besides these high affinity FGFRs,

FGFs also bind with low affinity to cell surface proteoglycans, such as heparin, which are

required for generating a full biological response (Schlessinger et al, 1995).

One of the most notable functions of FGFs and FGFRs is thought to be promotion of

cell growth; or in other words, FGFs can act as growth factors (Mason, 1994; Wang et al,

1994). Furthermore, some FGFs, such as FGF4, have been identified as oncogenes in a

transformation assay of NIH 3T3 cells (Mason, 1994). The expression of a constitutively

activated mutant FGFR3 has been shown to stimulate growth of a type of cell (Naski et al,

1996). Consistent with these observations, FGF-FGFR interaction can activate the

RAS-MAP kinase pathway which may initiate mitogenic responses (MacNicol et al, 1993;

Mason, 1994; Shaoul et al, 1995). PLC-g, a cellular signaling protein, also can be activated

by FGF (Antonelli-Orlidge et al, 1989; Jaye et al, 1992). In addition, FGFs also can modulate cell differentiation, migration and survival (Mason, 1994). However, the

mechanisms of how these downstream signal proteins can exert the variety of functions of

FGFs are largely unknown. It is possible that additional signaling pathways may be required

for these FGF functions.

Mutations in FGFRs have been shown to cause dominantly inherited human skeletal

bnormalities (Erlebacher et al, 1995; Muenke and Schell, 1995). For example, the

hondroplasia class of chondrodysplasias is comprised of the most common genetic forms

of dwarfism in human. Its members, achondroplasia (ACH), hypochondroplasia (HCH) and

thanatophoric dysplasia types I and II (TDI and TDII), are caused by distinct mutations of

fibroblast growth factor receptor 3 (FGFR3) which retard skeletal growth and development

(Figure 1, see below). However, there is no reported connection about the mediators of

these mutant FGFR3 -related growth abnormalities in development.

TDII is a lethal form of dwarfism that results from a recurrent point mutation

(Lys650Glu) within the activation loop of tyrosine kinase domain (Tavormina et al, 1995).

Its phenotype is characterized by severe shortening of limbs, thin vertebral bodies, and skull

anomalies and its histopathology by dismpted proliferation and organization of the

cartilaginous growth plates of long bones (Gorlin et al, 1990; Sillence et al, 1979). ACH is

associated with a Gly380Arg substitution in the transmembrane domain of FGFR3 (Rousseau

et al, 1994; Shiang et al, 1994), and HCH is related to a recurrent mutation in a distinct

region of tyrosine kinase domain (Bellus et al, 1995). ACH and HCH are not fatal diseases;

they represent similar, but milder phenotypes than those of TDII in the retardation of bone growth.

Mutant FGFR3, such as ACH, HCH, and TDII, can negatively regulate chondrocyte

proliferation. More importantly, the negative effects are not caused by loss of the signaling

function of FGFR3. On the contrary, all these FGFR3 mutations are gain-of- function

mutations which produce a dominant activating effect, especially the constitutively activated

tyrosine kinase activities (Muenke and Schell, 1995; Naski et al, 1996; Webster and

Donoghue, 1996). In support of the notion of negative regulation of cell proliferation by

FGFR3, homozygous dismption of FGFR3 in mice causes severe and progressive bone

dysplasia with enhanced growth, further indicating that FGFR3 is a negative regulator of

bone growth (Colvin et al, 1996; Deng et al, 1996). On the basis of these in vivo data, the

logical explanation for phenotypes of the chondrodysplasias is that the negative regulation of

cell growth by FGFR3 is abnormally elevated in the chondrocytes that express the

gain-of- function mutant FGFR3, such as ACH, HCH, TDI and TDII, causing retardation in

bone growth.

Thus, one of the biological effects of these FGFR3 mutants at the cellular level is

inhibition or retardation of chondrocyte proliferation in the cartilaginous growth plates. To

understand the molecular pathogenesis of these genetic diseases, the identify of the mediators

of this negative control through mutant FGFR3 signal transduction during bone growth must

be determined. In Example 2, the mediator of the activity of mutant FGFR3 was shown to be

activated STAT protein.

Furthermore, as shown in Figure 25, a number of other important tyrosine kinases are well conserved in the activation loop of the kinase domain. The present inventors predict

that mutations at the conserved Lys or Arg residues (boxed) results in constitutive activation

of the kinases. One possible substrate(s) may be STATl or other STAT proteins. This mode

of activation and the downstream signaling are involved in the pathogenesis of several

mammalian diseases, and can therefore serve as a target for both diagnosing the disorder as

well as a therapeutic target for agent development.

The present inventors found that there is no significant difference in the state of

phosphorylation of MAP kinases among the 293T cells transfected with the TDII or other

vectors (unpublished result). Intriguingly, the initial studies of STATl null mice showed no

overt defect in the development (Meraz et al. 1996; Durbin et al. (1996). This might be due

to the redundancy in the STAT family proteins or to the possibility that STATl can be

activated only when the FGFR3 has the constitutively activating mutation. In this context, it

is noteworthy that either the ACH or HCH receptor showed a weak activation of STATl in

the transfection assay, which may correlate with milder phenotypes of ACH and HCH than

TDII (unpublished result). This is the first example of human genetic disease which involves

an abnormality in the tyrosine kinase-STAT pathway. This observation can therefor form a

basis of developing diagnostic and therapeutic agents for use in TDII and other FGFR

associated disease (see Muenke et al. 1995).

On the basis of the observations provided in these Examples, the present inventors

suggest that their recently discovered STAT signaling pathway may be involved in negative

regulation of cell growth (Chin et al, 1996). This invention provides methods for developing agents that can be used to diagnosis or treat growth abnormalities, such as TDII,

that are regulated by the way of STAT activation.

EXAMPLES

In parallel to the known mitogenic pathways mediated by signal proteins such as

Grb2, many cytokines can also activate STAT proteins which mediate direct signal

transduction and gene expression. However, it is not known whether STAT proteins can be

activated by FAK.

The data presented herein indicate that FAK may activate in vivo a negative signaling

pathway involving STAT. Strikingly, it was found that in contrast to many other signaling

pathways initiated by FAK, the FAK-STAT signaling pathway may negatively regulate cell

attachment.

STATl can interact with FAK in the transfected cells. STAT proteins can bind

directly to phosphorylated receptor-tyrosine kinase complexes (Greenlund et al. (1994)). To

determine how FAK can activate STATl, the present inventors examined interactions

between STATl and FAK. STATl was immunoprecipitated by an anti-STATl antibody

from lysates of 293T cells co-transfected with STATl and FAK. The

co-immunoprecipitated proteins were analyzed by a Western blot with an anti-HA-tag

antibody that could recognize both exogenously expressed STAT and FAK (both proteins

were HA-tagged) (Figure 26). The FAK protein was co-immunoprecipitated with the

anti-STATl antibody. The identity of the HA-tagged FAK was confirmed further by

blotting with an anti-FAK antibody . The expression levels of STATl protein were also assayed (Figure 26, lower panel). These results suggest that STATl and FAK are associated

with each other in these STATl and FAK co-transfected cells.

STAT-FAK interactions in untransfected cells were examined. Complementary to the

above co-immunoprecipitation with an anti-STATl antibody described above, an anti-FAK

antibody was used to perform immunoprecipitation in untransfected 293T cells. Then the

immunoprecipitated complexes were further examined using an anti-STATl antibody in a

Western blot. In this assay, STATl was clearly co-immunoprecipitated with anti-FAK

antibody. The co-immunoprecipitated STATl migrated slightly slower than the major

STATl band (indicated as STATl, Figure 27 A, lane 1-4), immunoprecipitated by the

anti-STATl antibody (Figure 27 A, lane 5). The more slowly migrating STATl bands

resulted from protein phosphorylation after they had interacted with FAK. This notion was

confirmed by Western blot with an antibody that specifically recognizes tyrosine

phosphorylated, but not unphosphorylated, STATl (New England Biolab). Only these

slower migrating bands were recognized by this anti-phospho-tyrosine STATl (STATlp)

antibody (Figure 27 A, lanes 6-10), while the major unphosphorylated STATl band shown in

lane 6, was not recognized by this antibody (Figure 27 A, lane 10). Intriguingly, it appeared

that only tyrosine-phosphorylated STATl was co-immunoprecipitated with FAK, and this

FAK-STAT association transiently reached the maximal level when cells were just attached

to fibronectin (at 0.5 hour time point, lanes 2 and 7), a ligand for the integrin receptor which

could activate FAK during cell adhesion. With the progression of cell attachment, the

amount of STATl associated with FAK was significantly reduced. These results suggest that STAT1 can associate transiently with FAK at the beginning of cell adhesion when FAK is

activated.

A similar observation was also made in A431 cells. Consistent with the transient

STATl association with FAK and STATl tyrosine phosphorylation, a specific STATl DNA

binding activity was observed at the beginning of the cell attachment and this STATl

activity was reduced gradually as the cell attachment proceeded (Figure 27B). These data

suggest that STATl can be transiently activated during cell adhesion.

The above observations provides a target for developing agents that stimulate or

block cell adhesion by interfering with STAT-FAK interaction (see below). The specificity of activation of STATl by FAK was confirmed by using various

STAT1 and FAK mutants. Mutations of the SH2 domain (STAT1-SH2RQ) and of the

tyrosine 701 (STATl-CYF) in STATl prevented its activation when co-transfected with

FAK (Figure 28). Almost equal STATl protein levels in various transfected cells were

verified by Western blotting (Figure 28, lower panel). Similarly, expression of wild-type

STATl in these cells occasionally resulted in a low level of STATl activation; however,

expression of SH2 mutants (STAT1-SH2RQ, and -CYF) alone did not generate this STATl

activation. These results indicate that the SH2 domain and tyrosine 701 are essential for

STATl activation by FAK.

STATl and FAK co-expression causes cell detachment. The present inventors were

further interested in the possible cellular effects of STATl activation by FAK. Intriguingly,

dramatic morphological changes in transfected cells that seemed to increase with the increased level of STATl activation by FAK were observed (Figure 29). In contrast to mock

transfected cells, many cells which had been transfected with FAK and STAT were detached

from the plate or aggregated. In cells transfected with STATl alone, there was little effect

on cell morphology, whereas in cells transfected with FAK alone, there was a certain degree

of similar morphological alterations, which may correlate with the endogenous STATl

activity induced by FAK. These results suggest that the joint action by FAK and STATl

have significant effects on cell morphology.

One possible mechanism for these morphological changes is that STATl activation

by FAK could negatively affect cell adhesion, thus providing a target for developing agents

that modulate cell adhesion. Although FAK has been suggested to be involved in focal

adhesion in normal conditions (Richardson et al. 1996), STAT activation may trigger another

signaling pathway causing negative effects on cell adhesion, similar to the negative effects on

cell growth induced by EGF through STAT activation.

To test this possibility, the present inventors determined the ability of these

transfected cells to adhere to fibronectin, the ligand for integrin that can activate FAK

(Figure 29). To accomplish this, Fibronectin (1 ug) was adsorbed onto plastic 96-well tissue

culture plates. After using 0.5% BSA to block the plates in 37°C, certain numbers of cells

were plated and incubated at 37°C for 3 hours. Plates were washed with PBS twice and cells

fixed with 3% paraformaldehyde (pH7.4) for 30 min. at 4 C. Cells were stained with 0.5%

crystal violet and incubated overnight at room temp. The relative extent of cell adherence

was determined by OD630. Co-expression of FAK and STATl in 293T cells greatly inhibited the cell adhesion

on fibronectin. Expression of either STATl or mock expression of b-galactosidase, or

STAT1-SH2RQ mutant, had less effect. Thus, although FAK alone has certain negative

effects on cell attachment, both STAT and FAK are required for the maximal inhibition of

cell adhesion in this cell transfection system. While not explained further at this point, one

of ordinary skill in the art can readily envision assay methods based on agents which either

inhibit or promote STAT and FAK activity and expression, either separately or together.

These assay methods can be used to identify agents which either increase or decrease the

inhibition of cell adhesion by altering STAT and FAK levels and activities. STATl is required for cell detachment. Endogenous FAK-STAT can be activated by

cell attachment to FN. To confirm that the STAT protein is involved in detachment of cells,

U3A cells which are defective in STATl protein, and three U3A derived cell clones that

expressed reintroduced STATl protein (U3A-Statl, clones 2.1, 2.3, 2.9) were analyzed for

cell adherence on fibronectin (FN). The re-introduction of STATl protein to U3A cells

significantly reduced cell attachment to fibronectin (Figure 30A). The results presented were

an average of three repeated experiments.

Embryonic fibroblasts derived from STATl deficient mice or from wild type mice

from the same litter also were compared. STATl null (-/-) cells attached better than STATl

+/+ cells at different concentrations of plated fibronectin (Figure 30B).

Figure 30C shows that STATl wild-type fibroblast cells were detached and

aggregated on a plate coasted with FN, whereas STATl -/- cells could attach well at the same conditions.

STATl promotes cell migratioru STATl -/- and STATl +/+ fibroblasts were further

analyzed using a Boyden chamber assay for their migration ability. It was found that STATl

positive cells migrate significantly faster than STATl negative cells, indicating that STATl

can promote cell migration (Figure 31).

In summary, cell adhesion and anchorage are necessary for growth and survival of

most types of cells (Fang et al. (1996); Meredith et al. (1993); Frisch et al. (1994); and

Rouslahts et al. (1994)). However, the present invention demonstrates the surprising results

that the STAT signaling pathway can also be activated by FAK, resulting in a negative effect

on cell attachment and enhancement of cell migration. The above observations may be

relevant to the pathogenesis of many diseases that are caused by abnormal cell detachment

and migration. The above experiments in this invention provide tools and methods for

developing assay systems and screening for agents that modulate STAT:FAK activation and

subsequently, cell attachment and migration.

EXAMPLE 4

Methods to identify agents that block or stimulate the phosphorylation of

RECEPTOR/PTK-STAT may utilize any commonly available tyrosine kinase assays to assay

for the modulation of phosphorylation. Such assays are widely available such as those

disclosed by Ruksen et al, "Nonradioactive Assays of Protein-Tyrosine Kinase Activity

Using Anti-phosphotyrosine Antibodies" Methods in Enzymology 200:98-107 (1991) or Sahal et al, "Solid-Phase Protein-Tyrosine Kinase Assay" Methods in Enzymology

200:90-97 (1991),

For instance, a STAT protein or fragment thereof is incubated with a tyrosine kinase

such as FAK, Itk, TIE or Src tyrosine kinases and ³²P-labeled gamma- ATP in the presence

and absence of the agent to be tested. The samples are then contacted with anti-STAT

antibodies immobilized on a column; the column is washed; and the bound STAT protein or

fragment is eluted with 0.1M glycine, pH 2.5. The eluant is then subjected to fractionation to

separate the resulting radiolabeled STAT protein or fragment from the free radioactivity in

the sample using any conventional technique, such as precipitation in 5-10% trichloroacetic

acid. Following fractionation, the amount of radioactivity incorporated into the STAT

protein or fragment is counted. Comparing the amount of radioactivity incorporated into the

STAT protein or fragment in the presence and absence of the agent to be tested identifies an

agent which modulates, blocks or stimulates the phosphorylation of RECEPTOR/PTK-

STAT. Inhibition of STAT phosphorylation indicates the potential to inhibit apoptosis while

the promotion of STAT phosphorylation indicates the potential to stimulate or promote

apoptosis.

In an alternative assay format, 100 ng of a STAT protein or fragment is added to 1 ml

buffered solution containing a tyrosine kinase such as a FAK, Itk, TIE or Src tyrosine

kinases, together with 30 μC ³²P-gamma ATP in the presence or absence of the agent to be

tested. Following incubation, the mixture is heated to 100° C in a solution containing sodium

lauryl sulfate (SDS) and beta-mercaptoethanol. Aliquots are electrophoresed on 10-15%) gradient SDS polyacrylamide gels and exposed to X-Omat X-ray film to

identify radioactive STAT protein or fragments. Comparing the amount of radioactivity

incorporated into the STAT protein or fragment in the presence and absence of the agent to

be tested identifies an agent which modulates, blocks or stimulates the phosphorylation of

RECEPTOPJPTK-STAT. Inhibition of STAT phosphorylation indicates the potential to

inhibit apoptosis while the promotion of STAT phosphorylation indicates the potential to

stimulate or promote apoptosis.

EXAMPLE 5. Uses for Agents which Modulate STAT-Mediated Apoptosis.

As discussed herein, RECEPTOR-PTK-STAT pathways play an important role in a

wide variety of intracellular events, disease processes, cell morphology, and intracellular

interactions including cellular attachment, cellular aggregation and cellular migration. In

addition, these RECEPTOR-PTK-STAT pathways play an important role in the apoptosis

process. Agents that modulate, reduce or block the interactions of a receptor with its

associated phosphotyrosine kinases or block the sequential interactions with members of the

STAT family of proteins can be used to modulate biological and pathologic processes

associated with the RECEPTOR-PTK-STAT pathways.

The data presented herein demonstrates the involvement of tyrosine kinases with a

variety of receptors that participate in modulating the STAT-mediated apoptotic process.

Agents that modulate, e.g., cell mortality, cell migration and/or intercellular interactions may

therefore, also control pathologic cell growth migration such as tumor metastasis or chronic inflammation.

As used herein, a subject can be any mammal, so long as the mammal is in need of

modulation of a pathological or biological process effected by the cascade of intracellular

events that follow the various RECEPTOR-PTK-STAT pathways. The term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in

the treatment of human subjects.

As used herein, a biological or pathological process mediated by STAT proteins and

involving the various RECEPTOR-PTK-STAT pathways refers to the wide variety of cellular events in which a STAT protein modulates the apoptosis process or other observable or

detectable intracellular events, including morphological changes, and various other biological

processes. These latter processes include, but are not limited to, cellular attachment or

adhesion to substrates and other cells, cellular aggregation, cellular migration, cell

proliferation, and cell differentiation.

As used herein, the phrases "pathological state" or "pathological condition" in reference

to events that are mediated by STAT proteins and involving the various

RECEPTOR-PTK-STAT pathways includes, but is not limited to Thanatophoric Dysplasia Type

II, cancer, metastasis of cancer cells, autoimmune disorders, diabetes, degenerative diseases,

aging, and inflammation. A variety of cardiac and circulatory diseases also may involve such

STAT proteins and the various RECEPTOR-PTK-STAT pathways including thrombosis,

inflammation, angiogenesis, wound healing (including cutaneous wounds such as burn wounds,

donor site wounds from skin transplants and cutaneous, decubitis, venous stasis and diabetic ulcers), acute coronary syndrome, myocardial infarction, unstable angina, refractory angina,

occlusive coronary thrombus occurring post-thrombolytic therapy or post-coronary angioplasty,

a thrombotically mediated cerebrovascular syndrome, embolic stroke, thrombotic stroke,

transient ischemic attacks, venous thrombosis, deep venous thrombosis, pulmonary embolus,

coagulopathy, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura,

thromboangiitis obliterans, thrombotic disease associated with heparin-induced

thrombocytopenia, thrombotic complications associated with extracorporeal circulation,

thrombotic complications associated with instrumentation such as cardiac or other intravascular catheterization, intra-aortic balloon pump, coronary stent or cardiac valve, and conditions requiring the fitting of prosthetic devices.

Pathological processes refer to a category of biological processes which produce a

deleterious effect. For example, thrombosis is the deleterious attachment and aggregation of

platelets while metastasis is the deleterious migration and proliferation of tumor cells. These

pathological processes can be modulated using agents which reduce or block the intracellular

effects of STAT proteins and/or the various RECEPTOR-PTK-STAT pathways.

As used herein, an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process. For example, an agent is said to modulate

thrombosis when the agent reduces the attachment or aggregation of platelets. Methods of Treating Pathological Conditions.

The agents of the present invention can be provided alone, or in combination with other

agents that modulate a particular pathological process. For example, an agent of the present invention that modulates or increases apoptosis of an abnormal cell by increasing the amount

of phosphorylated RECEPTOR/PTK-STAT proteins or by facilitating their translocation into the cell nucleus can be administered in combination with other therapeutic agents. As used

herein, two agents are said to be administered in combination when the two agents are

administered simultaneously or are administered independently in a fashion such that the agents

will act at the same time.

The agents of the present invention can be administered via parenteral, subcutaneous,

intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be

dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if

any, frequency of treatment, and the nature of the effect desired.

The present invention further provides compositions containing one or more agents

which either increase or decrease the rate or extent of apoptosis in a treated cell, tissue or

subject. While individual needs vary, determination of optimal ranges of effective amounts

of each component is within the skill of the art. Typical dosages comprise about 0.1 to 100

μg/kg body wt. The preferred dosages comprise about 0.1 to 10 μg/kg body wt. The most

preferred dosages comprise about 0.1 to 1 μg/kg body wt. However, these dosage ranges will

vary according to the chemical class and overall activity of the agents to be administered, as will be appreciated by one skilled in the art.

In addition to the pharmacologically active agent, the compositions of the present

invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be

used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral

administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include

fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or

triglycerides. Aqueous injection suspensions may contain substances which increase the

viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol,

and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also

be used to encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according to the invention

may be formulated for enteral, parenteral or topical administration. Indeed, all three types of

formulations may be used simultaneously to achieve systemic administration of the active ingredient. Suitable formulations for oral administration include hard or soft gelatin capsules,

pills, tablets, including coated tablets, elixirs, suspensions, syraps or inhalations and controlled

release forms thereof.

In practicing the methods of this invention, the compounds of this invention may be

used alone or in combination, or in combination with other therapeutic or diagnostic agents.

In certain preferred embodiments, the compounds of this invention may be coadministered

along with other compounds typically prescribed for these conditions according to generally accepted medical practice, such as anticancer agents. The compounds of this invention can be

utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs,

cats, rats and mice, or in vitro.

It should be understood that the foregoing discussion and examples present merely

present a detailed description of certain preferred embodiments. It therefore should be apparent

to those of ordinary skill in the art that various modifications and equivalents can be made

without departing from the spirit and scope of the invention. All articles, patents and patent

applications that are identified above are incorporated by reference in their entirety.

While the invention has been described in connection with specific embodiments thereof,

it will be understood that it is capable of further modifications and this application is intended

to cover any variations, uses, or adaptations of the invention following, in general, the principles

of the invention and including such departures from the present disclosure as come within known

or customary practice within the art to which the invention pertains and as may be applied to the

essential features hereinbefore set forth and as follows in the scope of the appended claims.

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Claims

CLAIMSWHAT IS CLAIMED:

1. A method of modulating the rate and/or amount of a cellular process selected from the

group consisting of cell growth, cell detachment and cell migration, and cellular apoptosis, said

method comprising altering the RECEPTOPJPTK-STAT pathway of a cell.

2. The method of claim 1 wherein the RECEPTOPJPTK-STAT pathway is altered by

increasing or decreasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins

present in a cell.

3. The method of claim 2 wherein the amount of phosphorylated RECEPTOPJPTK-STAT

proteins present in the cell is increased by introducing into the cell a nucleic acid molecule that

encodes a tyrosine kinase.

4. The method of claim 2 wherein the amount of phosphorylated RECEPTOPJPTK-STAT

proteins present in the cell is increased by introducing into the cell a nucleic acid molecules that

encodes RECEPTOPJPTK-STAT proteins.

5. The method of claim 1 wherein the RECEPTOPJPTK-STAT pathway is altered by

increasing or decreasing the expression and/or activation of a RECEPTOR in the pathway.

6. The method of claim 1 wherein the RECEPTOPJPTK-STAT pathway is altered by

increasing or decreasing the amount of STAT in a cell.

7. The method of claim 6 wherein the STAT is selected from the group consisting of

STATl, STAT3, STAT4, STAT5A/B and STAT6.

8. The method of claim 1 wherein the RECEPTOPJPTK-STAT pathway is altered by

altering the interaction of STAT with a RECEPTOR in the pathway.

9. The method of claim 1 wherein the RECEPTOPJPTK-STAT pathway is altered by

altering the interaction of STAT with a PTK.

10. The method of claim 9 wherein the PTK is selected from the group consisting of a

cellular kinase, a receptor tyrosine kinase, and a cytoplasmic tyrosine kinase.

11. The method of claim 9 wherein the PTK is selected from the group consisting of EGFR,

FGFR, FAK, JAK, Src, Lck, TIE2, c-kit, RET, INRK, EPH, TRKA, TRKB, Itk and PDGFR-B.

12. The method of claim 11 wherein the EGFR is selected from the group consisting of

HER1 , HER2, HER3 and HER4.

13. The method of claim 1 wherein the RECEPTOPJPTK-STAT pathway is altered by

increasing or decreasing the amount of kinase present in a cell.

14. The method of claim 13 wherein the kinase is selected from the group consisting of a

cellular kinase, a receptor tyrosine kinase, or a cytoplasmic tyrosine kinase.

15. The method of claim 13 wherein the kinase is selected from the group consisting of

EGFR, FGFR, FAK, JAK, Src, Lck, TIE2, c-kit, RET, INRK, EPH, TRKA, TRKB, Itk and

PDGFR-B.

16. The method of claim 15 wherein the EGFR is selected from the group consisting of

HER1, HER2, HER3 and HER4.

17. The method of claim 1 wherein the rate and/or amount of cell growth is decreased.

18. The method of claim 17 wherein the RECEPTOPJPTK-STAT pathway is altered by

increasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

19. The method of claim 1 wherein the rate and/or amount of cell growth is increased.

20. The method of claim 19 wherein the RECEPTOPJPTK-STAT pathway is altered by decreasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

21. The method of claim 1 wherein the rate and/or amount of cell detachment and cell

migration is decreased.

22. The method of claim 21 wherein the RECEPTOPJPTK-STAT pathway is altered by

decreasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

23. The method of claim 1 wherein the rate and/or amount of cell detachment and cell

migration is increased.

24. The method of claim 23 wherein the RECEPTOPJPTK-STAT pathway is altered by

increasing the amount of phosphorylated RECEPTOPJPTK-STAT proteins present in a cell.

25. The method of claim 1 wherein the rate and/or amount of cellular apoptosis is decreased.

26. The method of claim 25 wherein the RECEPTOPJPTK-STAT pathway is altered by

decreasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

27. The method of claim 1 wherein the rate and/or amount of cellular apoptosis is increased.

28. The method of claim 27 wherein the RECEPTOPJPTK-STAT path way is altered by

increasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

29. A method of identifying agents which inhibit apoptosis in a cell through the mechanism

of blocking the phosphorylation of RECEPTOPJPTK-STAT by a tyrosine kinase comprising

the steps of:

a) incubating STAT, or a fragment thereof, and a tyrosine kinase, or a fragment

thereof, with an agent to be tested,;

b) determining whether said agent blocks the phosphorylation of STAT, or a

fragment thereof by said tyrosine kinase,

wherein the inhibition of STAT phosphorylation indicates the potential to inhibit

apoptosis.

30. A method of identifying agents which stimulate or promote apoptosis in a cell through

the mechanism of stimulating the phosphorylation of RECEPTOR/PTK-STAT by a tyrosine

kinase comprising the steps of:

a) incubating STAT, or a fragment thereof, and a tyrosine kinase, or a fragment

thereof, with an agent to be tested, and

b) determining whether said agent stimulates the phosphorylation of STAT, or a

fragment thereof by said tyrosine kinase,

wherein the promoting of STAT phosphorylation indicates the potential to stimulate or promote apoptosis.

31. A method to assay for ST AT -mediated apoptosis comprising the steps of determining

whether a RECEPTOR/PTK-STAT protein is phosphorylated and correlating said apoptosis with

the presence and degree of said RECEPTOR/PTK-STAT phosphorylation, wherein an increase

of RECEPTOPJPTK-STAT phosphorylation indicates STAT-mediated apoptosis.

32. The method of claim 31 wherein the presence of elevated levels of

RECEPTOPJPTK-STAT proteins is a diagnostic marker of Thanatophoric Dysplasia Type II,

FGF-receptor associated diseases, cancer, metastasis of cancer cells, autoimmune disorders,

diabetes, degenerative diseases, aging, and inflammation.

33. The method of claim 31 further comprising the steps of:

a) preparing an extract of a cell,

b) examining the proteins of said cell extract to determine the presence of a

phosphorylated RECEPTOPJPTK-STAT protein, and

c) examining cellular localization of STAT protein to determine activation of

STATs.

34. The method of claim 31 further comprising the steps of:

a) preparing an extract of a cell b) examining the mRNA of said cell extract to determine the presence of a FGFR3

encoding mRNA, and

c) examining cellular localization of STAT protein to determine activation of

STATs.

35. A method of treating mammalian diseases or developmental defects caused by abnormal

cell death induction wherein the method comprises promoting apoptosis by altering the

RECEPTOPJPTK-STAT pathway.

36. The method of claim 35 wherein the RECEPTOPJPTK-STAT pathway is altered by

increasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

37. The method of claim 36 wherein the treatment comprises administering an agent that

increases the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

38. The method of claim 36 wherein the treatment consists of a gene-therapeutic method that

increases the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

39. The method of claim 35 wherein the mammalian disease or developmental defect is

selected from the group consisting of cancer, autoimmune disease, viral susceptibility, and

conditions of obesity.

40. A method of treating mammalian diseases or developmental defects caused by abnormal

cell death induction wherein the method comprises inhibiting apoptosis by altering the

RECEPTOPJPTK-STAT pathway.

41. The method of claim 40 wherein the RECEPTOPJPTK-STAT pathway is altered by

decreasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

42. The method of claim 41 wherein the treatment consists of administering an agent that

decreases the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

43. The method of claim 41 wherein the treatment consists of a gene- therapeutic method that

decreases the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

44. The method of claim 40 wherein the mammalian disease or developmental defect is

selected from the group consisting of degenerative disorders, ischemic injuries, viral infection

induced by cell death and cell apoptosis during inflammatory responses.

45. A method of treating mammalian diseases or developmental defects caused by abnormal

cell proliferation wherein the method comprises inhibiting abnormal cell growth by altering the

RECEPTOPJPTK-STAT pathway.

46. The method of claim 45 wherein the RECEPTOPJPTK-STAT pathway is altered by

increasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

47. The method of claim 46 wherein the treatment consists of administering an agent that

increases the amount of phosphorylated RECEPTOPJPTK-STAT proteins present in a cell.

48. The method of claim 46 wherein the treatment consists of a gene-therapeutic method that

increases the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

49. The method of claim 45 wherein the mammalian disease or developmental defect is

selected from the group consisting of cancer and tumor cell metastasis.

50. A method of treating mammalian diseases or developmental defects caused by cell

growth retardation wherein the method comprises promoting cell growth by altering the

RECEPTOPJPTK-STAT pathway.

51. The method of claim 50 wherein the RECEPTOPJPTK-STAT pathway is altered by

decreasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

52. The method of claim 51 wherein the treatment consists of administering an agent that

decreases the amount of phosphorylated RECEPTOPJPTK-STAT proteins present in a cell.

53. The method of claim 51 wherein the treatment consists of a gene-therapeutic method that

decreases the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

54. A method of treating mammalian diseases or developmental defects caused by abnormal

cell detachment wherein the method comprises promoting cell attachment by altering the

RECEPTOPJPTK-STAT pathway.

55. The method of claim 54 wherein the RECEPTOPJPTK-STAT pathway is altered by

decreasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

56. The method of claim 55 wherein the treatment consists of administering an agent that

decreases the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

57. The method of claim 55 wherein the treatment consists of a gene-therapeutic method that

decreases the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

58. A method of treating mammalian diseases or developmental defects caused by abnormal

cell detachment wherein the method comprises inhibiting cell attachment by altering the

RECEPTOPJPTK-STAT pathway.

59. The method of claim 58 wherein the RECEPTOPJPTK-STAT pathway is altered by increasing the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

60. The method of claim 59 wherein the treatment consists of administering an agent that

increases the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

61. The method of claim 59 wherein the treatment consists of a gene- therapeutic method that

increases the amount of phosphorylated RECEPTOR/PTK-STAT proteins present in a cell.

62. A method for identifying diagnostic agents for measuring RECEPTOR/PTK-STAT

activities in order to determine physiological and pathological conditions, wherein the method

comprises the steps of:

a) measuring the activity of a RECEPTOPJPTK-STAT protein,

b) determining whether the activity of the RECEPTOPJPTK-STAT protein is

associated with a specific phenotype or a specific disease, and

c). examining cellular localization of STAT protein to determine activation of

STATs.

63. The method of claim 62 wherein the method is selected from the group consisting of in

vivo assays and in vitro assays.

64. The method of claim 62 wherein the method is selected from the group consisting of measuring the activities of the RECEPTOR proteins, measuring the activities of the PTK

proteins, and measuring the activities of the STAT proteins.

65. A clone that produces an exogenous level of STAT protein in an amount significantly

greater than the parental cell line from which the clone was developed.

66. The clone of claim 65 wherein the STAT protein is selected from the group consisting

of STATl, STAT3, STAT4, STAT5A/B and STAT6.

67. The clone of claim 65 wherein the clone is selected from the group consisting of

Ba/F3+STAT1, Ba/F3+STAT3 wt3, Ba/F3+STAT3 wtlO, and U3A-STAT1.

68. The clone of claim 65 wherein the clone exhibits significantly faster cell death following

semm withdrawal than the cell death of the parental cell line under the same conditions.

69. A method for identifying agents that block the phosphorylation of

RECEPTOPJPTK-STAT comprising the steps of:

a) growing the clone of claim 65 in a serum-based growth media,

b) removing the semm from the growing media and concurrently adding the agent

of interest,

c) determining whether said agent blocks the phosphorylation of RECEPTOPJPTK-STAT by observing clone cell viability over time.

70. A method of diagnosing abnormal STAT activation related to mammalian diseases

comprising the steps of:

a) isolating and growing test cells from an individual of interest;

b) conducting nuclear staining of the test cells using anti-STAT antibodies;

c) examining the stained nuclei of the test cells to determine whether or not STAT

has been translocated into the nuclei of the test cells; and,

d) comparing the extent of STAT translocation into the nuclei of the test cells to that

of normal control cells stained in the same manner.

71. The method of claim 70 wherein the anti-STAT antibody is anti-STAT 1.

72. The method of claim 70 wherein the mammalian disease is selected from the group

consisting of Thanatophoric Dysplasia Type II, FGF-receptor associated diseases, cancer,

metastasis of cancer cells, autoimmune disorders, diabetes, degenerative diseases, aging, and

inflammation.

73. A method of determining the amount of phosphorylated STAT proteins wherein the

method comprises using anti-phospho-tyrosine STAT.

74. The method of claim 73 wherein the anti-phospho-tyrosine STAT is anti-phospho-

tyrosine STATl.

75. The method of claim 73 wherein the method further comprising using Westem blot

analysis.

76. The method of claim 1 wherein the RECEPTOPJPTK-STAT pathway is altered by

altering the interaction of STAT with a STAT DNA binding element.

77. The method of claim 1 wherein the RECEPTOPJPTK-STAT pathway is altered by

altering the interaction among the RECEPTORs, the PTKs, and the STATs in the pathway.

78. The method of claim 1 wherein the RECEPTOPJPTK-STAT pathway is altered by

altering the expression and activation of RECEPTOR, PTK, and STAT, either individually or

in combination.

79. The method of claim 1 wherein the RECEPTOPJPTK-STAT pathway is altered by

changing the RECEPTOR in the pathway.