WO1998044908A1 - Method of regulating epithelial growth - Google Patents

Abstract

Methods of treating epithelial hyperproliferative disorders and accelerating wound healing, by modulation of NF-λB activity in the epithelium, are disclosed. Also disclosed are methods of screening for compounds effective to treat epithelial hyperproliferative disorders or enhance wound healing.

Description

METHOD OF REGULATING EPITHELIAL GROWTH

FIELD OF THE INVENTION

The present invention relates to methods of promoting or inhibiting cell proliferation in the epithelium by modulation of NF-_KB activity. More particularly, the invention relates to the treatment of epithelial hyperproliferative diseases, methods of promoting wound healing, and methods of screening for compounds effective for these applications.

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BACKGROUND OF THE INVENTION

NF-_KB/Rel proteins are potent inducible gene regulatory factors expressed in a wide variety of tissues. NF-_KB subunits function as DNA-binding transcription factors, with mammalian family members that include RelA (p65), RelB, c-Rel, p50 and p52. NF-_KB activity is controlled at a number of levels, prominent among these being the regulation of its transition from an inactive preexisting cytoplasmic form to an active nuclear protein.

NF-_KB gene regulatory proteins are activated in a range of conditions involving cellular stress and injury (Baeuerle et al. 1996, Verma et al, Baldwin et al). Studies of NF-_KB in lymphoid tissues have revealed potent effects in stimulating proliferation, preventing apoptosis, activating the immune response, and triggering cellular stress response genes.

Stratified epithelial tissues must respond to such frequent environmental stresses while maintaining a precise balance between cellular proliferation and cell loss via desquamation. In stratified epithelium, proliferative basal cells adherent to the underlying basement membrane undergo cell cycle arrest associated with outward migration and activation of terminal differentiation genes (Jones et al). Abnormalities in this process disrupt epithelial homeostasis and are characteristic of cutaneous neoplasms as well as a wide array of inflammatory skin diseases.

In addition, normal somatic cells do not replicate indefinitely but ultimately undergo cellular senescence, which is characterized by an irreversible withdrawal from the cell cycle that is resistant to mitogenic stimuli. Recent evidence suggests that neoplastic transformation may require mechanisms that, in addition to avoiding apoptosis, also bypass cellular senescence

(Brown et al.; Serrano et al; Yeager et al).

The gene regulatory transcription factors mediating control of epithelial growth and differentiation are not fully known. In particular, little has been reported about the expression and function of NF-_KB proteins in epithelia.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of inhibiting cellular proliferation in the epithelium. According to the method, a therapeutically effective amount of an activator of NF-_KB activity, or of an NF-_KB protein subunit, is administered to a subject in need of such treatment, and is effective to inhibit said proliferation. Examples of such activators include tumor necrosis factor alpha (TNF_α), phorbol 12-myristate 13-acetate (PMA), interleukin-1 (IL-1), interleukin-2 (IL-2), and bacterial lipopolysaccharide (LPS). Preferred NF-_KB protein subunits include the p50 and the p65 subunit. In one embodiment, where the method is used to treat a hyperproliferative skin disorder, the activator is administered, preferably topically, to the epidermis.

In another aspect, the invention provides a method of promoting cellular proliferation in the epithelium. According to the method, a therapeutically effective amount of an inhibitor of NF-_KB activity is administered to a subject in need of such treatment, and is effective to promote said proliferation. Examples of such inhibitors include I_κBβ, IκBα, pyrrolidine dithiocarbamate (PDTC), dimethyl sulfoxide (DMSO), an _α-amido-substituted cyclic imide, 2- (2,6-dioxo-3-piperidinyl)-4-azaisoindoline-l,3-dione, serine protease inhibitors, glucocorticoids, and NF-_KB antisense compounds,. In one embodiment, where the method is used to accelerate or enhance wound healing, the inhibitor is administered, preferably topically, to the site of the wound.

Also included in the invention is a method of identifying compounds effective to treat an epithelial hyperproliferation disorder. The method includes the steps of measuring the activity of NF-_KB in the presence and absence of a test compound, and identifying the test compound as effective if it results in an upregulation or promotion of NF-_KB activity. In a related aspect, the invention includes a method of identifying compounds useful for promoting wound healing. The method includes the steps measuring the activity of NF-_KB in the presence and absence of a test compound, and identifying the test compound as useful if it results in a downregulation or inhibition of NF-_KB activity.

These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1A, IB, 1C, ID are schematics of retroviral expression vectors used in experiments assessing effects of modulating NF-_KB activity. Figures 2A and 2B are computer-generated images of Western Blots showing expression of

NF-_KB activating (Fig. 2A) and inhibitory (Fig. 2B) proteins.

Figs. 3A-3B show the effects of the above expression vectors on NF-_KB directed gene expression of a reporter gene in epithelial cells.

Figs. 4A-H are computer-generated images of the epidermal architecture of human skin transfected with the indicated vectors and grown in vivo on SCID mice. Vector CN.50 was used in Figs. 4C and 4D. Vector Iι B_αM was used in Figs. 4B, 4E, 4F, 4G and 4H. Fig. 4A shows skin transduced with lacZ control vector.

Figs. 5A-5B summarize the histological results from images such as are shown in Figs. 4A- H. Fig. 4A shows the % sections with deep hyperplasia, and Fig. 4B shows the % sections with epidermis < 0.03 mm.

Fig. 6 shows schematics of K14-I_κB_αM and K14-p50 transgenes used for targeted expression to murine epidermis.

Figs. 7A-7B show the impact of altering NF-_KB function on epithelial cell growth in vitro.

Fig. 8 shows DNA synthetic activity as a function of NF-_KB subunit expression, expressed as the number of cells incorporating BrdU as a percentage of total.

Fig. 9 shows a cell cycle analysis of NF-_KB transduced cells versus control, giving the relative percentage of cells in different cell cycle phases.

Fig. 10 shows the lack of effect of added growth factors on NF-_KB induced growth arrest, vs. p50 and lacZ control-transduced cells. Fig. 11 shows the proportion of transduced and control cells demonstrating senescence- associated β-galactosidase staining.

Fig. 12 shows the proportions of cells transduced with expression vectors for p50, p65,

I_κB_αM and lacZ control, respectively, expressing nuclear p21^ciP¹.

Fig. 13 shows percentages of cells transduced with a retroviral expression vector for p21 ipl and GFP control, respectively, demonstrating senescence-associated β-galactosidase expression.

Fig. 14 shows cell cycle distribution for cells transduced as for Fig. 13. DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

"Cellular proliferation in the epithelium" refers to cell growth in the mitotically active basal layer. In normal cells, this proliferation ultimately ceases, and cells undergo a transition to growth-arrest and terminal differentiation, associated with outward migration to suprabasal layers.

The term "significant", when used with reference to, e.g., "significantly different", "significantly inhibits" or "significantly increases", refers to a difference in a quantifiable parameter between the two groups being compared that is statistically significant using standard statistical tests. For example, the degree of binding in a protein binding assay may be quantified using standard methods, and the degree of binding under different conditions can be compared for statistically significant differences.

The term "therapeutically effective amount" refers to an amount of compound that is of sufficient quantity to ameliorate a selected disorder, such as a hyperproliferative disorder of the skin. The term "ameliorate" refers to a lessening of the detrimental effect of the disorder in the patient receiving the therapy.

"Treating" a disease refers to administering a therapeutic substance effective to reduce the symptoms of the disease and/or lessen the severity of the disease.

II. Effects of NF-_rB Modulation in Epithelium

Several studies reported to date have proposed that inhibition of NF-_KB activity can be used to treat a variety of inflammatory and hyperproliferative diseases (see, e.g., Muller et al; Ghosh, 1997; Narayananof et al). For example, induction of NF-_KB DNA-binding activity has been associated with the GO to Gl transition in murine fibroblasts (Baldwin et al). In ras- transformed NIH 3T3 fibroblasts, antisense inhibition of p65 expression decreased tumor cell growth in vivo (Higgins et al), and, in a separate study with 3T3 fibroblasts, the blockade of I_κB_α increased neoplastic growth in vivo (Beauparlant et al, 1994). A similar NF-_KB growth- promoting role has been suggested in lymphocytes, as in the cases of lymphoid cells transformed by HTLV-1 (Kitajima et al). Results disclosed herein, however, demonstrate the opposite effect in epithelium, i.e. that stimulation of NF-_KB results in a decrease in the proliferation of epithelial cells, while inhibition of NF-_KB increases such proliferation. Specifically, activation of NF-_KB activity, e.g., through the expression of a constitutively nuclear p50 NF-_KB, led to inhibition of epithelial cell proliferation. Conversely, blockade of NF-^B function, e.g., using a trans-dominant I_κB_α mutant, led to stimulation of epithelial cell proliferation. These effects were manifested, for example, in transgenic human tissue and transgenic mice as atrophic epithelium and massive epithelial hyperplasia, respectively.

Further experiments showed that blockade of NF-_KB function prevents nuclear expression of the cyclin-dependent kinase inhibitor p21/CIP-l/WAF-l, while dominantly active p50 augments the proportion of epithelial cells with nuclear localized p21. The latter effect results in inhibition of cell cycle progression and premature cellular senescence, as discussed below.

Taken together, the data described below indicate that activators or stimulators of NF-_KB may be used to reduce or inhibit undesirable epithelial cell proliferation, such as occurs during various epithelial hyperproliferative disorders. Inhibitors or antagonists of NF-_KB may be used to upregulate or increase desirable epithelial cell proliferation, such as occurs during wound healing.

III. Inhibitors and Activators of NF-_KB Activity

Compounds effective to inhibit or promote the activity of NF-_KB are known in the art, and progress continues in this field. See, for example, recent reviews by Beauparlant et al, 1996; Barnes; and Okamoto et al.

Presently known inhibitors include, for example, proteinaceous inhibitors, such as I_κBβ, described in U.S. Patent 5,597,898 (Ghosh, 1997) and I_κB_α, described herein and in Hiscott et al. Also effective are a class of non-polypeptide cyclic imides disclosed as inhibitors of TNF-_α in U.S. Patent 5,605,914 (Muller, 1997) and in Corral et al. An exemplary compound of this group is 2-(2,6-dioxo-3-piperidinyl)-4-azaisoindoline-l,3-dione. Other NF-_KB inhibitors known in the art include antisense inhibitors, as described in U.S. Patent 5,591,840 (Narayananof , 1997) and Sharma et al, serine proteinase inhibitors such as N-tosyl-L-phenylalanine cloromethyl ketone and N-_α-p-tosyl-L-lysine chloromethyl ketone (see e.g. Jeong et al), sesquiterpene lactones (Bork et al) and glucocorticoids (Barnes). Pharmacological inhibitors include dimethyl sulfoxide (Essani et al.) and pyrrolidine diothiocarbamate.

Known activators of NF-_KB include subunit proteins, such as the p50 and p65 proteins, as described herein. Other activators known in the art include tumor necrosis factor alpha (TNF- _α), phorbol 12-myristate 13-acetate (PMA), hydrogen peroxide (see e.g. Kaul et al), interleukin-1 (IL-1), interleukin-2 (IL-2), neuropeptide substance P (Lieb et al.) and bacterial lipopolysaccharide (LPS).

Although exemplary inhibitors and activators are noted above, it is understood that other compounds known to have such activity may also be used in the methods disclosed herein, and that additional suitable compounds may be determined by use of the screening methods described in Section VII, below. III. Experimental Results

A. Site of Expression of NF-_KB Proteins within the Epithelium

To characterize the expression of NF-_KB proteins in different layers of stratified epithelium, frozen tissue sections of non-sun exposed adult human abdominal skin were double immunostained with antibodies to p50 and the activated form of RelA/p65, then analyzed via laser confocal microscopy. Results demonstrated that, in cells of the basal epithelial layer, pl05/p50 is strongly expressed in the cytoplasm. In contrast, in the non-proliferative cells of the suprabasal layers, this cytoplasmic expression is entirely absent and cells demonstrate nuclear expression. Given the well characterized regulation of NF-_KB via control of nuclear translocation (see e.g. Baeuerle et al, 1994, 1996; Verma et al; Ghosh et al. 1990), these results are consistent with a key role of NF-_KB in the control of epithelial growth and differentiation.

B. Preparation and Expression in vitro of Vectors Encoding NF-_KB-Activating and - Inhibiting Proteins

Vectors encoding proteins which exert either activating (constitutively nuclear p50 and p65/RelA) or inhibitory (trans-dominant mutant I_κB_αM repressor) effects on NF-_KB function were constructed as described in Example 1. These vectors, shown schematically in Figs. 1A- D, were used to directly assess the role of NF-_KB in the control of epithelial growth and differentiation. Cells transduced with a particular construct effectively express the protein(s) encoded by that construct, as verified via Western blot (Figs. 2A-2B; see Example 1).

The vectors were expressed in primary cultures of human keratinocyte epithelial cells, and the cells were assessed for expression of the proteins encoded by the transducing vectors, as described in Example 3, below. Data is shown in Figs. 3A-3B, reported as fold induction in reporter gene activity. The LZRS lacZ vector served as a control. As shown in Fig. 3A, p50 combined with p65, as well as either subunit alone, produced consistent activation of NF_KB- directed gene expression. I_κB_αM, in contrast, blocks phorbol ester induced NF-_κB-directed reporter gene expression in a dose-dependent manner (Fig. 3B).

To assess the cellular localization of the p50 NF-_KB subunit after gene transfer, the transduced keratinocytes were immunostained with an antibody to the p50/105 NF-_KB subunit and visualized by laser confocal fluorescence microscopy. Results showed that expression of the NF-_KB activating and inhibitory proteins altered the subcellular distribution pattern of NF-_KB subunits. Specifically, expression of NF-_KB activating proteins, encoded by vector CN.p50, resulted in nuclear localization of NF-_KB immunoreactivity, while expression of NF-_KB inhibiting proteins, encoded by vector I_κB_αM, resulted in cytoplasmic localization of NF-_KB immunoreactivity.

C. Effect of Altering NF-_KB Function in Human Epithelial Tissue in vivo. Keratinocytes expressing p50, Iκ;B_αM and lacZ control were used to regenerate human epidermis on SCID mice, as described in Example 4. Results are shown in Figs. 4A-4H. Figs. 4C and 4D show skin transduced with CN.50 (Fig. 1A), and Figs. 4B, 4E, 4F, 4G and 4H show skin transduced with I_κB_αM (Fig. IB). Control skin is shown in Fig. 4A. Immunostaining of each tissue section was performed prior to hematoxylin and eosin staining to confirm human tissue origin using species -specific antibody to a panel of human proteins. The results reveal striking effects of altering NF-_KB function in vivo. Tissue transgenic for constitutively nuclear p50 consistently produced markedly atrophic epithelium (Figs. 4C, 4D) while inhibition of NF-_KB function was consistently associated with massive epithelial hyperplasia (Figs. 4B, 4E, 4F, 4G and 4H). This hyperplasia was characterized by extensive epidermal thickening as well as formation of invaginations and cystic structures penetrating deeper in the dermis. These changes in tissue architecture in vivo provide strong support for the regulation of epithelial growth by NF-_KB. Other vectors had no effect on the respective characteristics.

The frequency of histologic abnormalities in the above-described transgenic human epidermis samples was quantitated, as described in Example 4. Atrophic changes were defined as less than 30% thickness of viable epidermis as compared to normal value of 0.1 mm found in lacZ transgenic and unengineered control, as measured with a micrometer. Hyperplastic proliferations were defined as epithelial tissue islands penetrating into underlying dermis to a depth of at least 3 times the thickness of surrounding epidermis. The results are shown in Figs. 5A and 5B, expressed as the percent of individual tissue sections displaying the given histologic abnormality. As can be appreciated from the figures, 89% of sections of skin transfected with CN.p50 had an epidermis thickness of <0.03 mm (Fig. 5B), while 100% of sections of skin transfected with I_κB_αM had deep hyperplasia.

D. Transgenic Mice with Alterations in Epidermal NF-_KB Function

Transgenic mice were generated expressing proteins for dominant-negative inhibition, as well as for constitutive activation of NF-_KB function. For blockade of epidermal NF-_KB fiinction, expression of the dominant negative IκB_αM mutant was targeted to the epidermis via the keratin 14 promoter, as described in Vassar et al. (See schematic in Fig. 6.). I_KB_OM contains substitutions at serines 32 and 36 of I_KB a along with deletion of COOH-terminal PEST sequences and has been shown to abolish nuclear DNA binding activity by any of the 5 NF_KB subunits in a range of mammalian cells (Baeuerle et al. 1996; Van Antwerp et al). Fourteen I_κB_αM[+] transgenic mice were generated, with transgene integration confirmed by Southern analysis and polymerase chain reaction. The mice expressed IκB_αM on Western analysis of epidermal tissue extracts, as detected by antibodies to I_κB_α that recognize both the wild type and mutant proteins. I^BαMI-l-] mice also demonstrated markedly increased immunostaining with these antibodies throughout all layers of transgenic epidermis, consistent with the expected resistance to degradation by this mutant protein. I_κB_αM[+] transgenic epidermis displays a complete absence of nuclear NF-_KB subunit expression in the suprabasal layers.

All I_κB_αM[-t-] transgenic mice developed epidermal hyperplasia clinically and histologically within 2 days after birth. This hyperplasia appeared to be due to an increase in the thickness of the suprabasal squamous layer (to approx. 200 μM, as compared to approx.

60μM in control mice). In addition, these mice lacked both clinical and histologic evidence of normal hair formation, exhibited growth retardation and died within 5 to 7 days. No abnormalities in any internal organs were seen on either histologic or macroscopic evaluation.

Transgenic mice expressing constitutively nuclear NF-_KB subunits in the basal layer of the epidermis were also generated, using the p50 construct of Fig. 6. Constitutive nuclear expression of p50 in transgenic skin, confirmed by immunohistochemistry, resulted in epidermal hypoplasia at the clinical and histologic levels. The markedly thin epidermis (approx. 25 μM) of the p50[+] mice consisted of as little as 2 viable cell layers, and the mice failed to gain weight normally and died within 5 days after birth. The most severely affected mice demonstrated open eyes at birth accompanied by extreme skin fragility and death within hours after being born.

These results suggest that nuclear translocation of NF-_KB subunits to cells of the non- proliferative compartment of the tissue is necessary for proper growth control in stratified epithelium (I_κB_αM[+] mice), and that induced activation of NF-_KB in the proliferative basal compartment of the epidermis leads to premature growth inhibition (p50[+] mice).

E. Growth Characteristics of Murine Epidermis Transgenic for Alterations in NF-_KB

Function

To follow epithelial growth characteristics beyond the time of perinatal mortality observed in both I_κB_αM[+] and P50[+] mice, skin from transgenic mice and nontransgenic littermate controls was grafted onto immune deficient mice, according to a procedure recently shown to preserve growth characteristics of donor skin (Oro et al).

At 14 days postgrafting, grafted IκB_αM[+] epidermis demonstrated pronounced epidermal hyperplasia clinically and histologically (approx. 150 μM in thickness, vs. approx. 65 μM for control), with epidermal invaginations penetrating deeply into the underlying dermis. The p50[+] epidermis, in contrast, remained hypoplastic clinically and histologically (approx. 20 μM in thickness).

To further examine the basis for these alterations in epidermal homeostasis, DNA synthetic activity was studied in transgenic and control epidermis by BrdU incorporation. CB.17 scid/scid mice bearing I^BαM^], p50[+] and non-transgenic control skin were injected with BrdU (250 mg/kg of body weight) intraperitoneally. Skin biopsy specimens were taken 2 hours later and tissue sections stained with antibody to BrdU. Immunofluorescence micrographs showed increased labeling activity in I_κB_αM[+] skin, extending into cells of the suprabasal layers, and a marked decrease in labeling activity in p50[+] skin, compared with the control. I_κB_αM[+] epidermis thus demonstrated a marked increase in the proportion of cells actively synthesizing DNA. BrdU positive cells in I_κB_αM[ + ] transgenic epidermis included those well above the basal layer, indicating a possible failure of the cell cycle arrest that is normally associated with outward migration. p50[ + ] epidermis, in contrast, demonstrated a near absence of cells incorporating BrdU over the 2 hour time period analyzed, consistent with inhibition of proliferation. These findings further suggest that functional loss of NF-_KB leads to hyperproliferation in stratified epithelium, due to failure of growth arrest, and that premature expression of activated NF_KB subunits in mitotically active basal epithelial cells leads to growth inhibition.

F. Effect of a Pharmacologic Inhibitor of NF-_KB on Normal Murine Skin.

In an alternative approach to alter NF-_KB function in epidermis, a pharmacologic inhibitor of NF-_KB, pyrrolidine dithiocarbamate (PDTC) (Schreck et al), was applied topically in a PBS solution to the skin of normal adult C57BL/6J mice. A 0.01 % SDS solution in PBS was applied under occlusion for the same period as an additional control for non-specific reactive hyperplasia to irritant stimuli.

Application of 10 mM PDTC twice daily for one week induced significant epidermal thickening (to approx. 225 μM) over the controls (approx. 70 μM for SDS and 30 μM for the PBS control). These results indicate that topical application of an agent that blocks NF-_KB function to normal intact adult skin is associated with epidermal hyperplasia.

G. Effect of NF-_KB on Epithelial Morphology and Proliferation in vitro.

In this study, normal human keratinocytes were transduced with the vectors described above (Figs. 1A and IB, plus a lac Z control), incubated in SFM/154 growth media, and assayed for number of viable cells every 24 hours for 4 days. As shown in Figs. 7A-7B, proliferation in cells expressing I_κB_αM was comparable to that of control cells, but was significantly inhibited in cells expressing CN.p50.

Control-transduced cells and cells expressing molecules that are dominant-negative for NF- _KB function, including I_κB_αM and ΛSP, exhibited the normal polygonal shaped cell morphology and colony growth pattern. In contrast, NF-_KB subunits produced cell morphologic changes as early as 24 hours after gene transfer. Cells became flat and enlarged with a vacuolated cytoplasm and lost the pattern of growth in colonies. These morphologic changes are consistent with those seen in epithelial cells undergoing replicative senescence (Saunders et al). Nuclear stains failed to reveal morphologic changes characteristic of apoptosis, and non-viable cells comprised <5% of cells in each vector group at all time points, indicating that these findings were not due to increased cell death.

Cellular DNA synthesis and cell cycle distribution was also examined two days post- transduction with p50, p65 and I_κB M retroviral expression vectors. Expression of active p50 and p65 subunits decreased the proportion of cells actively synthesizing DNA in vitro (Fig. 8), as measured by incorporation of bromodeoxyuridine (BrdU). Consistent with this result, cell cycle distribution analysis demonstrated that activated NF-_KB subunits produced a greater than

50% decrease in epithelial cells in S-phase (Fig. 9).

H. Effect of Growth Factors on NF-_KB Subunit Transduced Cells. The NF-_KB subunit expressing cells described above could be maintained for up to 4 weeks displaying the same morphology and apparent growth arrest. Because cellular senescence is characterized by irreversible growth arrest that is resistant to growth factor growth stimulation

(Goldstein; Phillips et al), the cells were next grown either in minimal media lacking growth factors or in media containing both epidermal growth factor (EGF) and keratinocyte growth factor (KGF). Under appropriate conditions, these factors can serve as epithelial cell mitogens in vitro (Gilchrest; Rheinwald et al).

Control cells, transduced with lacZ, grew slowly in minimal media, but proliferate exponentially in the presence of growth factors, as expected (Fig. 10). Growth factors, however, did not overcome the growth arrest of NF-_KB subunit expressing cells (Fig. 10), indicating that NF-_KB rendered these cells resistant to these mitogenic stimuli.

An additional feature of cells that have undergone senescence is the induction of a senescence-associated β.galactosidase (SA-β-gal) that can be specifically detected in vitro at pH

6.0 (Dimri et al). Accordingly, SA-_β-gal was observed in NF-_KB expressing cells as soon as 3 days after gene transfer, and the percentage of SA-β-gal positive cells consistently increases over the following 4 days (Fig. 11).

I. Effects of NF-_rB on the Cvclin-Dependent Kinase (CDK Inhibitor Protein p21^cipl

Two families of cyclin-dependent kinase (cdk) inhibitors, Ink4 proteins and Cipl/Kipl proteins, interact with cyclin/cdk targets, by different mechanisms (Sherr et al). Only members of the latter group are upregulated during epithelial differentiation (Missero et al; el-Deiry et al, 1993; Harper et al; Xiong et al). Nuclear p21^Cιpl expression is seen in cells undergoing terminal differentiation (Gartel et al), and p21, like NF-_KB, is expressed in the nuclei of suprabasal cells in normal stratified epithelial tissues, including skin and gastrointestinal tract

(el-Deiry et al, 1995; Inohara et al).

To further elucidate the basis for NF-_KB inhibition of cellular growth, the effect of NF-_KB on expression of cyclin-dependent kinase inhibitors (CKIs) was studied. Cells were transduced with retroviral expression vectors for NF-_KB subunits p50 and p65. Western blot analysis of cell extracts were prepared at 9 and 18 hours post transduction. The analysis showed that F-_KB subunit-expressing cells induced high levels of p21^ciP¹ protein (Fig. 12). Such induction was not observed in cells transduced with the transcriptionally inactive ΛSP p50 deletion mutant, lacZ or I_κB_αM

To analyze this effect at the level of individual cells, immunofluorescence staining was performed with antibodies to p21 ^lP¹ with cells expressing p50, p65 or lacZ control. Expression of active NF-_KB subunits was associated with an augmented proportion of cells with nuclear p21 ⁱP¹. NF-_KB induction of p21^c,P¹ appeared to be selective in that it was not accompanied by changes in the levels of other CKIs, including p27^κiP¹ or the INK4 family proteins pl5^INK4B or pl6^INK4A . The induction also occurred without an increase in p53 expression, suggesting a p53-independent mechanism.

This increase in nuclear p21^ClP¹ expression is consistent with a role for p21^ciP¹ in NF-_KB- induced epithelial growth arrest. This was confirmed by demonstrating that p21^ClP¹ triggers growth inhibitory and senescence features induced by NF- B. An amphotropic retroviral vector for constitutive p21^ciP¹ expression was produced. After confirming expression by the vector of full length p21^ciP¹ protein and >98% gene transfer efficiency by immunofluorescence, using antibody to p21^ClP¹, transduced cells were analyzed for proliferation kinetics and appearance of SA-β-gal. Similar to NF-_KB, p21^ClP¹-expressing cells demonstrated induction of SA-_β-gal (Fig. 13). In addition, p21^ClP¹ caused cell cycle arrest, with cell cycle distribution similar to that induced by NF-_KB subunits (Fig. 14).

The above results demonstrate that activation of NF-_KB inhibits cell cycle progression, and can trigger cell cycle arrest and cellular senescence in association with induction of the cell cycle inhibitor p21Cipi.

V. Therapeutic Applications

A. Hyperproliferative disorders

Neoplastic and non-neoplastic hyperproliferative skin disorders present an ever-increasing burden to health care providers. Increased UV exposure of skin has contributed to a significant increase in the incidence of premalignant lesions (e.g., actinic keratoses). Specifically, the number of cases in the U.S. of superficial squamous and basal cell carcinoma now exceeds

700,000 per year. Further, other localized hyperproliferative conditions, such as warts and psoriasis, are extremely prevalent.

Presently available treatments for hyperproliferative skin disorders limit the options available to the clinician. For example, many current treatments involve the application of cytotoxic agents (e.g., bleomycin and 5-fluorouracil) that are not selective for the hyperproliferative tissue and have significant side effects, including irreparable damage to surrounding skin and systemic absorption. Further, these methods are not always curative. The present invention provides an alternative to the above-described treatments. Specifically, in one aspect, the invention includes a method of treating a hyperproliferative disorder of the skin, by administering to the subject a therapeutically effective amount of an activator of NF-_KB. The term "hyperproliferative disorder of the skin" refers to malignant as well as non-malignant cell populations which morphologically differ from the surrounding tissue due to excessive growth and/or proliferation of epithelial cells. Hyperproliferative disorders thus include most skin diseases wherein the growth control mechanisms have been disrupted. Examples of hyperproliferative skin disorders include, but are not limited to, human papilloma virus (HPV) infected cells commonly associated with warts, superficial neoplasias of the skin such as melanomas, pre-malignant and malignant carcinomas, actinic keratosis, and psoriasis. Additional conditions amenable to treatment using methods of the invention include atopical dermatitis, contact dermatitis and further eczematous dermatitises, seborrhoeis dermatitis, pemphigus, lichen planus, lupus erythematosus, bullous pemphigoid, angioedemas, vasculitides, epidermolysis bullosa, urticaria, erythema, cutaneous eosinophilia, acne and alopecia areata. It is contemplated that diseases of epithelial tissues other than the skin (e.g. endotfielium, mesothelium) may also be treated using the methods of the invention. Examples include reversible obstructive airway disease, which includes conditions such as asthma (e.g., bronchial asthma, allergic asthma, dust asthma, intrinsic asthma, and extrinsic asthma), certain chronic or inveterate asthma (e.g., late asthma and airway hyper-responsiveness), bronchitis and the like. Further, various eye diseases may be treated, including conical cornea, dystrophia epithelialis corneae, keratoconjunctivitis, vernal conjunctivitis, keratitis, herpetic keratitis, sarcoidosis, corneal ϊeukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' ophthalmopathy, and Vogt-Koyanagi-Harada syndrome. The methods may also be used to treat hyperproliferative vascular diseases such as intimal smooth muscle cell hyperplasia, restenosis and vascular occlusion.

In addition, as noted above, recent evidence suggests that neoplastic transformation may require mechanisms that, in addition to avoiding apoptosis, also bypass cellular senescence.

Activation of NF-_KB has been shown, as described above, to induce the cell cycle inhibitor p21^ciP¹ and promote premature senescence. Accordingly, the method may also be used to inhibit neoplastic growth.

B. Promotion of Cell Proliferation

In one aspect, inhibition of NF-_KB activity, as described herein, may be used to promote wound healing. Wound healing involves the repair of injured tissue, the regeneration of specialized tissue, and reorganization of new tissue. It consists of three major phases: i) an inflammation phase lasting up to about three days, ii) a cellular proliferation phase lasting from about three to about 12 days, and (c) a remodeling phase lasting from about three days to six months. During the inflammation phase, clotting factors and platelet aggregation act to form a matrix, trapping plasma proteins and blood cells. New connective or granulation tissue, as well as blood vessels, form during the cellular proliferation phase. The granulation tissue is replaced by a network of collagen and elastic fibers (leading to the formation of scar tissue) during the remodeling phase.

It will be appreciated that wound healing can be facilitated if any of the above-described phases can be accelerated. In this context, it is an object of the present invention to provide a method of accelerating or enhancing the healing of wounds to the epithelium by stimulating epithelial cell proliferation. The method includes administering to the subject a therapeutically effective amount of an activator of NF-_KB.

In transgenic mouse epidermis overexpressing p-50 (Section E above), a increase in hair growth was also observed. Because the activation of NF-_KB has been shown herein to promote cell cycle arrest, and thus accelerate terminal differentiation, in epithelial cells, this would be consistent with increased production of hair, a terminally differentiated epidermal structure.

Accordingly, these preliminary results suggest that administration of an NF-_KB activator, as described herein, could promote hair growth in the epidermis.

VI. Administration of Compounds Effective to Alter NF- B Activity

In accordance with the method, a therapeutically effective amount of an activator or inhibitor of NF-_KB, in a pharmaceutically acceptable carrier, is administered to a subject in which it is desired to promote or inhited, respectively, NF-_KB activity. Any conventional method for delivery of a biologically active compound may be used to deliver a therapeutically effective compound according to the methods of the present invention. The preferred dosage and formulation typically depends on the type of compound to be delivered.

For example, in the case of NF-_KB antisense oligomer compositions useful for wound healing applications, the oligomer may be delivered as described, e.g., in Narayananof et al. (1997). The oligomer may be delivered alone, or in composition with a suitable pharmaceutical carrier or coupled with carriers. Examples of suitable carriers include peptides, immunoglobulins and their fragments, liposomes, receptor molecules, ligand molecules such as hormones, enzymes, and any conventional compounds for pharmaceutical administration.

In the case of proteinaceous compounds, such as IL-1, IL-2, and I_κBβ, the compound can be, for example, encapsulated in microspheres or proteinoids. Such compounds may also be delivered transdermally by iontophoresis or transdermal electroporation. Methods for the preparation and administration of therapeutically-active proteins are known to one of skill in the art (see, for example, Banga, 1995).

Delivery of an effective amount of a therapeutic compound may be oral, parenteral, intravenous, transdermal, or by any conventional pharmaceutical route. As activating and inhibiting compounds have been shown herein to be effectively expressed in vivo, administration via gene therapy is also contemplated.

In a preferred embodiment, the compound is applied topically to the site of the hyperproliferative disorder, wound, or site of desired hair growth, to minimize systemic activity of the compound. Such topical applications typically involve suspending the therapeutic compound in a solution, emulsion, cream or ointment with a pharmaceutically acceptable carrier.

For transdermal delivery, the use of a transdermal patch allows for continuous delivery of compound to a selected skin region. Examples of transdermal patch delivery systems are provided by U.S. Patent 4,655,766 (fluid-imbibing osmotically driven system), and U.S. Patent 5,004,610 (rate controlled transdermal delivery system). If desired, permeation enhancing substances, such as fat soluble substances (e.g., aliphatic carboxylic acids, aliphatic alcohols), or water soluble substances (e.g., alkane polyols such as ethylene glycol, 1,3-propanediol, glycerol, propylene glycol, and the like) may be included. In addition, as described in U.S. Patent 5,362,497, a "super water-absorbent resin" may be added to transdermal formulations to further enhance transdermal delivery. Examples of such resins include, but are not limited to, polyacrylates, saponified vinyl acetate-acrylic acid ester copolymers, cross-linked polyvinyl alcohol-maleic anhydride copolymers, saponified polyacrylonitrile graft polymers, starch acrylic acid graft polymers, and the like. Such formulations may be provided as occluded dressings to the region of interest, or may be provided in one or more of the transdermal patch configurations described above. For delayed release, the activator or inhibitor may be included in a pharmaceutical composition formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

The dosage of therapeutic compound administered is determined in accord with clinical practice, and will vary depending upon such factors as the patient's age, previous medical history, and general medical condition. The dose is determined in part based on the pharmacokinetics of clearance of the administered compound, using standard pharmacokinetics principles known in the art (Gennaro, 1990; Gilman et al, 1995).

IVII. Screening Applications

The present invention also includes methods of identifying compounds effective to treat an epithelial hyperproliferation disorder. One such method includes the steps measuring the activity of NF-_KB in the presence and absence of a test compound, and identifying the test compound as effective in the treatment if it results in an upregulation of NF-_KB activity. Any of a number of screens of NF-_KB activity can be employed by one of skill in the art. An exemplary assay of NF-_KB activity is the reporter gene assay described in Example 2, herein.

In another aspect, the invention includes a method of identifying compounds useful for promoting wound healing. The method includes the steps measuring the activity of NF-_KB in the presence and absence of a test compound, and identifying the test compound as useful if it results in a downregulation of NF-_KB activity. As described above, any of a number of NF-_KB activity assays known to those skilled in the art may be employed in such a screen.

A variety of different compounds may be screened using methods of the present invention. They include peptides, macromolecules, small molecules, chemical and/or biological mixtures, and fungal, bacterial, or algal extracts. Such compounds, or molecules, may be either biological, synthetic organic, or even inorganic compounds, and may be obtained from a number of sources, including pharmaceutical companies and specialty suppliers of libraries (e.g., combinatorial libraries) of compounds.

The following examples illustrate but are not intended to limit the present invention.

MATERIALS AND METHODS

Unless otherwise indicated, restriction enzymes and DNA modifying enzymes were obtained from New England Biolabs (Beverly, MA) or Boehringer Mannheim (Indianapolis, IN). Nitrocellulose paper was obtained from Schleicher and Schuell (Keene, NH). Materials for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Bio-Rad Laboratories (Hercules, CA). Other chemicals were purchased from Sigma (St. Louis, MO) or United States

Biochemical (Cleveland, OH).

Production of transgenic animals and skin tissue Sequences encoding the I_κB_α ^M mutant (Van Antwerp et al.) and the constitutively nuclear p50 Xbal mutant (Blank et al.) were subcloned downstream of a 2075 bp human keratin 14 promoter (Vassar et al) construct containing a 5' intron from the D-globin gene and used to produce transgenic mice. Transgene integration was confirmed both by Southern analysis and by polymerase chain reaction of genomic DNA. For the latter, primers specific for the 3' end of the K14 promoter and the 5' end of the expressed cDNA, either the IκB_αM mutant and the constitutively nuclear p50 Xbal mutant, were used to amplify an 850 base pair fragment. Fourteen I^αMf-l-] and thirteen CN.p50[+]mice were characterized.

Genetically engineered human epidermis was regenerated on CB.17 scid/scid mice from early passage keratinocytes after high efficiency retroviral gene transfer by previously described methods (Choate et al, 1996a; Medalie et al). Briefly, transduced keratinocytes were plated on devitalized human dermis and grown in vitro with growth media for 7 to 10 days followed by grafting to the backs of CB.17 scid/scid mice. Analysis of grafted human tissue was performed 3 weeks post-grafting.

Immunoblotting and Immunohistochemistry

Whole cell extracts were prepared from cells grown in vitro and immunoblotted as described (Choate et al, 1996a,b) after separation by SDS-PAGE on 8%, 10% or 15% gel. Approximately 20μg of protein, as determined by Bradford (Biorad, Hercules, CA), were loaded per lane. Equal loading conditions were confirmed by Coomassie blue staining. Antibodies to p21Cipi, _P27Ki_Pι, _P15^INK4B, pl6^INK4A, p50, p65, p50/105 and I_κB_α were obtained from Santa Cruz Biotech. Blots were incubated simultaneously with polyclonal antiserum to BRG1 (Khavari et al), a constitutively expressed 205 kDa protein that served to control for cell extract quality and protein transfer efficiency. Blots were visualized using the ECL-detection system (Amersham, Arlington Heights, IL). Immunoblots were performed on transgenic skin tissue extracts as an additional confirmation of transgene expression. Following skin biopsy, tissue was incubated for 1 hour at 37°C with dispase (Becton-Dickinson) (25U/ml) to separate the epidermis from underlying dermis, then epidermal extracts were prepared and analyzed as above.

Immunohistochemical analysis was performed as previously described (Choate et al._} 1996a,b) using antibodies to NF- B subunits and to p21^ClP¹ (Santa Cruz Biotech) as well as to BrdU

(Becton-Dickinson). Prior to immunostaining, cells were rinsed with PBS, fixed for 10 minutes in acetone at room temperature, air-dried and blocked with 5% normal goat serum. For staining, slides were incubated with primary antibodies for 30 minutes, followed by PBS washing and incubation with FITC-conjugated secondary antibodies (Sigma, St. Louis, MO) and mounted with Vectashield mounting media (Vector Inc., Burlingame, CA). Immunohistochemical staining after retroviral transduction of cells grown in vitro was performed as noted below. Where indicated, cells were counterstained 15 seconds with propidium iodide (20μg/ml in PBS) to visualize all nuclei. Slides were then analyzed by fluorescence microscopy. For senescence-associated β-gal (SA-β-gal) staining, cells were washed in PBS, fixed with 2% formaldehyde/ 0.2% glutaraldehyde for 5 minutes at room temperature and stained for β-gal at pH 6.0 as described. (Dimri et al., 1995)

Cell culture and gene transfer

Normal human epithelial cells were isolated from human skin as described (Rheinwald et al.) .

Cells were grown in a 1:1 mixture of SFM (Gibco) and 154 media (Cascade Biologies), optimal conditions for proliferation. Retroviral expression vectors for ΔSP, IκBαM, p50 and p65 were constructed as described (Seitz et al.. 1998) . cDNA sequences corresponding to the coding regions of human p50 (amino acids 1-502, Xbal truncation) (Blank et al), p65 (Nolan et al.) and the dominant-negative mutants IκB_αM (Van Antwerp et al.) and ^SP (Logeat et al.) were subcloned into the EcoRI site of the LZRS retroviral vector (Kinsella et al). The p21^ciP¹ vector was produced by subcloning the full length p21^ciP¹ cDNA into the EcoRI site of the LZRS backbone vector (Kinsella et al.) after removal of the EcoRI fragment containing the lacZ gene. Amphotropic retrovirus production and gene transfer with test and lacZ and GFP control vectors was performed as previously described (Choate et al.. 1996a,b; Kinsella et al.) ^■ >95% gene transfer efficiency was confirmed for each vector by immunofluorescence staining with antibodies to NF- B subunits, IκB and p2lCipl. Transient transfections were performed by the modified polybrene shock method, as previously described (Freiberg et al). Briefly, 30% confluent normal human keratinocytes were transfected using 2 μg of p50, p65, IκB_αM or ^SP expression plasmid, 2 μg of NF-_κB-luciferase reporter plasmid and 1 μ of RSV-CAT internal control. For assessment of I_κB_αM and ^SP dominant negative effects, NF-_KB activity was induced for 4 hours with 30ng/ml of PMA prior to reporter gene analysis (Khavari et al).

Analysis of mitotic activity and cell cycle distribution

For analysis of cellular proliferation, cells were transduced in triplicate 35 mm plates for each vector as previously described (Choate et al.. 1996a,b). 48 hours following gene transfer, cells were re-plated at low densities of 10⁴ cells/35 mm plate. Following this, cells were harvested and counted in triplicate at 24 hour intervals. Cell morphology was determined at each time point by phase contrast microscopy, and nuclear morphology was evaluated by fluorescence microscopy after staining with propidium iodide. Cell viability was determined by trypan blue exclusion. For mitogenic stimulation, cells were grown in the presence of epidermal growth factor (EGF) and keratinocyte growth factor (KGF), both at a concentration of approximately 10 ng/ml.

For BrdU labeling in vitro, cells were grown on glass cover slides and incubated for 2 hours with 10 μM BrdU (Boehringer, Indianapolis, IN), then rinsed with PBS, fixed for 30 minutes in

70% ethanol and air dried. After treatment with 0.07 N NaOH for 2 minutes, the slides were thoroughly rinsed in PBS and stained with anti-BrdU monoclonal antibody (Becton Dickinson, San Jose, CA). Cells were then counterstained with propidium iodide (20μg/ml) for 15 seconds to visualize all cells in a given field. For in vivo BrdU labeling, mice were injected intraperitoneally with BrdU (250mg/kg body weight), then sacrificed 2 hours later and tissue sections subjected to immunofluorescence staining with FITC-conjugated antibody to BrdU.

For cell cycle analysis, cells were stained with propidium iodide 72 hours after transduction, then subjected to flow cytometry. Briefly, cells were trypsinized, washed in PBS and incubated for

20 minutes in a solution containing 0.1% sodium citrate (pH 7.8), 0.1% Triton X, 50μg ml propidium iodide, and lmg/ml RNAse. Then an equal volume of a solution containing 0.376M

NaCl, 0.1% Triton X, 50μg/ml propidium iodide was added and kept at 4°C until subjected to flow cytometry. Data was analyzed using ModFit software as previously described (Missero et al.) .

EXAMPLE 1 : Construction and Expression of Retroviral Vectors for Activating or Inhibiting

NF-_KB

Retroviral expression vectors encoding proteins exerting either activating or inhibitory effects on NF-_KB function were generated using standard cloning techniques (Ausubel et al, Sambrook et al, 1989). The vectors were made using the MFG-based LZRS retroviral expression vector (Kinsella and Nolan, 1996) and amphotropic retrovirus produced in modified

293 packaging cells as described (Choate et al, 1996a,b; Kinsella et al).

Schematics of the vectors are shown in Figs 1A-D. ψ4- represents extended retroviral packaging sequence. Vector CN.p50 (Fig. 1A) contains cDNA sequences encoding constitutively nuclear p50 (pl05 amino acids 1-502) (Blank et al), while vector IκB_αM (Fig.

IB) contains cDNA sequences encoding a trans-dominant mutant I_κB_αM repressor (Van

Antwerp et al, 1996). A lacZ vector (Fig. 1C; Kinsella et al), a p50 deletion construct (Fig.

ID), as well as mock transduction, served as controls.

The above-described vectors were effectively expressed in primary cultures of human keratinocyte epithelial cells (Rheinwald et al.) using a high efficiency gene transfer approach (Choate et al, 1996a,b). Forty eight hours following transduction, cell extracts were prepared, separated via SDS-PAGE on an 8% polyacrylamide gel, and immunoblotted with antibodies to p50 and I_κB_α- [-] = untransduced.

The results are shown in Figs. 2 A and 2B. The blot in Fig. 2 A was stained with an anti- p50 antibody. The lanes were as follows: lane 1 - untransduced control, lane 2 - mutant dominant negative p50 transduced, and lane 3 - CN.pSO transduced. The blot in Fig. 2B was stained with an anti-I_κB_α antibody. The lanes were as follows: lane 1 - untransduced control, and lane 2 - IκB_αM transduced. The results indicate that cells transduced with the indicated constructs effectively express the proteins encoded by those constructs.

EXAMPLE 2: Assessment of NF- B Activity in Transduced Cells

Additional experiments were performed to assess the cellular localization of the p50 NF-_KB subunit after gene transfer. After retroviral transduction with the panel of expression vectors described above, normal human keratinocytes were immunostained with an antibody to the p50/105 NF-_KB subunit and visualized by laser confocal fluorescence microscopy. Results, as described above, showed that expression of NF-_KB activating proteins (encoded by vector CN.p50) resulted in nuclear localization of NF-_KB immunoreactivity, whereas expression of NF-_KB inhibiting proteins (encoded by vector I_κB_αM) resulted in cytoplasmic localization of NF-_KB immunoreactivity. To determine effect of expression of the NF-_KB activating and inhibitory proteins on

NF-_KB directed reporter gene expression in these cells, keratinocytes were transfected with a plasmid containing 3 copies of NF-_KB DNA consensus binding sites driving expression of the luciferase reporter gene (Freiberg et al), along with a CMV-CAT plasmid that served as an internal control of transfection efficiency. Reporter gene activity, summarized in Fig. 3, was assessed 48 hours after transduction with the indicated vectors, normalized for transfection efficiency using a cotransfected RSV-CAT internal control. The results illustrate that I_KB_aM significantly inhibits the ability of NF-_KB to induce expression of the reporter gene, while CN.50 causes a slight but insignificant increase in reporter gene expression.

EXAMPLE 3: Effects of Modulating NF-_KB Activity In Vivo in Human Skin

Primary human keratinocytes transduced with the indicated vectors, as described above, were used to regenerate transgenic human skin on SCID mice in vivo as described in Choate et al, 1996a,b, and Medalie et al, 1996. Transduced cells were seeded on devitalized human dermal substrate in vitro and left to grow for 7 days prior to direct grafting onto the fasia of SCID mice recipients. Six mice were grafted for each vector in 2 separate sets of experiments. Four weeks after grafting, dressings were removed and the tissue was analyzed. Exemplary results of histological analyses are shown in Figs. 4A-4H.

Expression of terminal differentiation markers was normal in human skin transgenic for either activating or inhibitory NF-_KB subunits and lacZ control. Double immunostaining was performed with species-specific antibodies to human involucrin (Murphy et al.) and BPAG2, a basement membrane zone protein to highlight the inferior boundary of the basal epidermal layer

(Fairley et al). The results showed that epidermal differentiation markers including keratin 10, involucrin, keratinocyte transglutaminase and filaggrin, however, were expressed in normal suprabasal distribution in transgenic skin of all vector groups.

The frequency of histologic abnormalities in the above-described transgenic human epidermis samples was quantitated as follows. Multiple 5 μM sections were obtained in a stepwise fashion through tissue biopsies that spanned the full 1.5 cm thickness of each transgenic and control regenerated human graft, and representative sections were analyzed. After confirmation of human species origin via immunostaining with antibody to involucrin (Murphy et al, 1984), histologic appearance was analyzed. Atrophic changes were defined as less than 30% thickness of viable epidermis as compared to normal value of 0.1 mm found in lacZ transgenic and unengineered control, as measured with a micrometer. Hyperplastic proliferations were defined as epithelial tissue islands penetrating into underlying dermis to a depth of at least 3 times d e thickness of surrounding epidermis.

The results are shown in Figs. 5 A and 5B. A total of 9 representative tissue sections were analyzed from all grafted mice for constitutively nuclear p50 subunit (CN.p50), 12 for I_κB_αM, and 6 for lacZ and unengineered control. The data are expressed as the % of individual tissue sections displaying the given histologic abnormality. As can be appreciated from the figures,

89% of sections of skin transfected with CN.p50 had an epidermis thickness of < 0.03 mm

(Fig. 5B), while 100% of sections of skin transfected with Iκ_B_αM had deep hyperplasia.

EXAMPLE 4: Effects of NF-_KB on Epithelial Proliferation

Normal keratinocytes were transduced in 6 parallel transductions for each vector. The cells were incubated in SFM/154 growth media as described (Choate et al, 1996a,b) and replicate transductions for each vector harvested by trypsinization at 24 hour intervals for 4 days. Cells were stained with trypan blue and counted using phase contrast microscopy. The proportion of viable cells for all vectors was > 95% at all timepoints.

The results are shown in Fig. 7. Cells expressing IκB_αM proliferated at rates similar to control cells, whereas proliferation of cells expressing constitutively nuclear p50 was significantly inhibited. In additional experiments, asynchronously dividing primary keratinocytes were incubated with BrdU for 3 hours in growth media and stained with monoclonal antibody to BrdU as described in Missero et al, 1996. Counterstaining with propidium iodide was used to identify all cells present in a given field. Three independent transductions were evaluated for each vector; at least 1000 cells were counted in all cases.

These results demonstrate that constitutively nuclear p50 NF-_KB is associated with a decreased proportion of cells actively synthesizing DNA.

Cell cycle distribution was assessed by transducing normal primary human keratinocytes with activating and inhibitory subunits for NF-_KB function and staining with propidium iodide.

The cells were analyzed by flow cytometry, and fraction of cells in G_, S and G₂/M was calculated using "MODFIT LT" software, as described in Missero et al. The results showed that blockade of NF-_KB function was associated with an increase in proportion of cells in S phase.

The above-described changes correlated with the proportion of basal keratinocytes expressing the Ki-67 marker of cellular proliferation in vivo. Constitutively nuclear p50 was associated with a decrease as opposed to the augmented numbers of Ki-67(+) cells that were seen in IκB_αM transgenic epidermis compared to lacZ controls.

While the invention has been described with reference to specific methods and embodiments, it is appreciated that various modifications and changes may be made without departing from the invention.

Claims

It is claimed:

1. A method of inhibiting cellular proliferation in the epithelium, comprising administering to a subject in need of such treatment an amount of an activator of NF-_KB activity, or an NF-_KB protein subunit, which is effective to inhibit said proliferation.

2. The method of claim 1, wherein said protein subunit is a p50 or p65 subunit.

3. The method of claim 1, wherein said activator is selected from die group consisting of tumor necrosis factor alpha (TNF_╬▒), phorbol 12-myristate 13-acetate (PMA), interleukin-1

(IL-1), interleukin-2 (IL-2), and bacterial lipopolysaccharide (LPS).

4. The method of claim 1, for use in treating a hyperproliferative skin disorder, wherein said activator is administered to the epidermis.

5. The method of claim 4, wherein said activator is administered topically.

6. A method of promoting cellular proliferation in the epithelium, comprising administering to a subject in need of such treatment an amount of an inhibitor of NF-_KB activity which is effective to promote said proliferation.

7. The method of claim 6, wherein said inhibitor is selected from the group consisting of

Ii B╬▓, I╬║^B╬▒, pyrrolidine dithiocarbamate (PDTC), 2-(2,6-dioxo-3-piperidinyl)-4-azaisoindoline- 1,3-dione, a glucocorticoid, a serine protease inhibitor, and an NF-_KB antisense compound.

8. The method of claim 6, for use in accelerating or enhancing wound healing, wherein said inhibitor is administered to the site of the wound.

9. The method of claim 8, wherein said inhibitor is administered topically.

10. A method of identifying compounds effective to treat an epithelial hyperproliferation disorder, comprising measuring the activity of NF-^B in the presence and absence of a test compound, and identifying the test compound as effective if it results in an upregulation or promotion of NF-_KB activity.

11. A method of identifying compounds useful for promoting wound healing, comprising measuring the activity of NF-_KB in the presence and absence of a test compound, and identifying the test compound as useful if it results in a downregulation or inhibition of NF-_KB activity.