WO1996005850A1

WO1996005850A1 - Cytokine regulation of cellular senescence

Info

Publication number: WO1996005850A1
Application number: PCT/US1995/010457
Authority: WO
Inventors: Bharat B. Aggarwal
Original assignee: Research Development Foundation
Priority date: 1994-08-18
Filing date: 1995-08-17
Publication date: 1996-02-29
Also published as: IL114744A0; AU3407795A; ZA956748B

Abstract

The present invention provides a novel method of decreasing cellular senescence comprising the step of administering a pharmacologically effective dose of basic fibroblast growth factor to an animal. Also provided are pharmaceutical compositions for use in the methods of the present invention.

Description

CYTOKINE REGULATION OF CELLULAR 8ENESCENCE

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to the fields of immunology and protein chemistry. More specifically, the present invention relates to the novel use of a cytokine to inhibit cellular senescence. Description of the Related Art Loss of proliferative potential of normal human cells in culture, initially described in normal human diploid fibroblasts, is widely used as a reliable in vitro model of aging. This model of cellular senescence has now been reproduced with a variety of differentiated cell types including vascular endothelial cells, lymphocytes, and adrenocortical cells.

Why cells stop dividing after certain number of population doublings is unknown. Cell fusion experiments have suggested that senescence is a dominant phenotype and that immortal cell lines result from an inactivation of key cell senescence related genes. The latter have been assigned to four distinct complementation groups. Cellular senescence is accompanied by several changes including: (1) a decrease of cell cycle related enzyme ornithine decarboxylase and thymidine kinase activities; (2) a decrease in protein synthesis and degradation; (3) a decrease in the length of telomeres and of 5-methylcytosine content in DNA; and (4) the presence of higher frequency of nuclear and chromosomal aberrations. Phenotypically, senescent cells have been shown to be larger and grow to a lower saturation density as compared to young counterparts. The age-dependent loss of cellular proliferation has been shown to be due to the inability to phosphorylate retinoblastoma gene product, failure to express cell cycle- dependent genes such as c-fos, cdc 2 or cyclins and acquisitions of factors such as senescence-associated genes (SAG) , terminin and sdi (senescence derived inhibitor) that prevent proliferation. Thus, it is possible that cellular senescence is not due to down-regulation of proliferative factors but is instead due to up-regulation of antiproliferative factors. During the last decade, several different novel proliferative and antiproliferative growth factors have been identified which control the replicative potential of cells. Most of these factors, also referred as cytokines, are polypeptide hormones which differentially regulate cell growth in a cell type specific manner. Among the cytokines, it has been shown that basic fibroblast growth factor, tumor necrosis factor and interleukin-1 could induce the proliferation of young normal human diploid fibroblasts, an in vitro model commonly used for aging. Recently, these cytokines were shown to induce proliferation of young cells and have no effect on senescent human diploid fibroblasts. How various growth factors modulate the limited in vitro long-term life span of these cells, is not known.

The prior art is deficient in the lack of effective means of inhibiting cellular senescence. The prior art is also deficient in the lack of varied and diverse pharmacological tools to treat non-malignant hyperproliferative diseases. The present invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION The present invention illustrates the effect of ten different cytokines on the age-dependent proliferation of normal human diploid fibroblasts (HDF) cells in culture. As disclosed herein, certain cytokines can increase the life span of fibroblasts and postpone the senescence process. As also disclosed herein, certain other cytokines are very effective in significantly decreasing fibroblast proliferation. In one embodiment of the present invention, there is provided a method of decreasing cellular senescence comprising the step of administering a pharmacologically effective dose of basic fibroblast growth factor to an animal.

In another embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of cellular senescence comprising a pharmacologically effective dose of basic fibroblast growth factor and a pharmaceutically acceptable carrier.

In yet another embodiment of the present invention, there is provided a method of a method of increasing endogenous levels of insulin growth factor-1 (IGF-1) in animals comprising the administration of a pharmacologically effective dose of basic fibroblast growth factor to an animal.

In still yet another embodiment of the present invention, there is provided a method of decreasing endogenous levels of senescence inducing protein in animals comprising the administration of a pharmacologically effective dose of basic fibroblast growth factor to an animal.

In another embodiment of the present invention, there is provided a method of treating a pathophysiological state characterized by undesirable fibroblast proliferation, comprising the administration of a pharmacologically effective dose of a fibroblast-inhibiting cytokine to an animal having said state.

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

Figure 1 shows the effect of interferon-alpha on the life span of human diploid fibroblasts. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with interferon-alpha at 37°C for

7 days and then cells were trypsinized and viable cell number determined by trypan blue exclusion. Figure 2 shows the effect of interleukin-6 on the life span of human diploid fibroblasts. 4 X10⁴ cells (2 ml) in 24- well plates were incubated with interleukin-6 at 37°C for 7 days and then cells were trypsinized and viable cell number determined by trypan blue exclusion.

Figure 3 shows the effect of interleukin-4 on the life span of human diploid fibroblasts. 4 X10⁴ cells (2 ml) in 24- well plates were incubated with interleukin-4 at 37°C for 7 days and then cells were trypsinized and viable cell number determined by trypan blue exclusion.

Figure 4 shows the effect of leukemia inhibitory factor (LIF) on the life span of human diploid fibroblasts. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with leukemia inhibitory factor at 37°C for 7 days and then cells were trypsinized and viable cell number determined by trypan blue exclusion.

Figure 5 shows the effect of tumor necrosis factor

(TNF) on the life span of human diploid fibroblasts. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with tumor necrosis factor at 37°C for 7 days and then cells were trypsinized and viable cell number determined by trypan blue exclusion.

Figure 6 shows the effect of transforming growth factor-beta on the life span of human diploid fibroblasts. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with transforming growth factor-beta at 37°C for 7 days and then cells were trypsinized and viable cell number determined by trypan blue exclusion.

Figure 7 shows the effect of interferon-gamma on the life span of human diploid fibroblasts. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with interferon-gamma at 37°C for

7 days and then cells were trypsinized and viable cell number determined by trypan blue exclusion.

Figure 8 shows the effect of interleukinl-beta on the life span of human diploid fibroblasts. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with interleukinl-beta at 37°C for

7 days and then cells were trypsinized and viable cell number determined by trypan blue exclusion. Figure 9 shows the effect of interferon-beta on the life span of human diploid fibroblasts. 4 X10⁴ cells (2 ml) in

24-well plates were incubated with interferon-beta at 37°C for

Figure 10 shows the effect of basic fibroblast growth factor on the life span of human diploid fibroblasts. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with interferon- beta at 37°C for 7 days and then cells were trypsinized and viable cell number determined by trypan blue exclusion.

Figure 11 shows the effect of basic fibroblast growth factor on the replicative potential of human diploid fibroblasts at different population doublings. 4 X10³ cells (0.2 ml) in 24- well plates were incubated with the cytokine at 37°C for 7 days, then trypsinized and viable cell number determined by trypan blue exclusion.

Figure 12 shows the effect of different concentrations of tumor necrosis factor (Figure 12A) , interleukin-1 (Figure 12B) and basic fibroblast growth factor (Figure 12C) on young and senescent fibroblast cells. 8 X10³ cells (0.2 ml) in 96-well plates were incubated with the cytokine at 37°C for 5 days and then thymidine uptake was determined during last 24 hours. All determinations were made in triplicate.

Figure 13 shows the effect of human interferon-alpha on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13 and 14 weeks. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings. Figure 14 shows the effect of human interleukin-4 on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 8 and 13 weeks. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings.

Figure 15 shows the effect of human interleukin-6 on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13, 14 and 18 weeks. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings. Figure 16 shows the effect of human leukemia inhibitory factor on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13 and 14 weeks. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings.

Figure 17 shows the effect of human interleukin-lB on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 8 and 9 weeks. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings.

Figure 18 shows the effect of human tumor necrosis factor on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 6, 8 and 9 weeks. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings.

Figure 19 shows the effect of human interferon-γ on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13 and 14 weeks. 4 X10⁴ cells (2 ml) in 24- well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings.

Figure 20 shows the effect of human interferon-3 on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13 and 14 weeks. 4 X10⁴ cells (2 ml) in 24- well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings. Figure 21 shows the effect of human transforming growth factor-jS on the morphology of human diploid fibroblasts at different population doublings, i.e., 4 and 6 weeks. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings.

Figure 22 shows the effect of human basic fibroblast growth factor on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13, 14 and 17 weeks. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings. Figure 23 shows the effect of aging on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13 and 17 weeks. 4 X10⁴ cells (2 ml) in 24-well plates were incubated with different cytokine at 37°C for 7 days and then cells were photographed at different population doublings. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of decreasing cellular senescence comprising the step of administering a pharmacologically effective dose of basic fibroblast growth factor to an animal. Generally, the methods of the present invention are equally advantageous and of desirable use in treating various animals, including mammals. Most preferably, the methods of the present invention would be most useful in a human.

The present invention is also directed to a method of increasing endogenous levels of insulin growth factor-1 (IGF-1) in animals comprising the administration of a pharmacologically effective dose of basic fibroblast growth factor to an animal.

In a separate embodiment, the present invention pertains to a method of decreasing endogenous levels of senescence inducing proteins in animals comprising the administration of a pharmacologically effective dose of basic fibroblast growth factor to an animal. Most preferably, the senescence inducing protein is sdi or senescence derived inhibitor. A pharmaceutical composition, comprising basic fibroblast growth factor and a pharmaceutically acceptable carrier is also provided. The pharmaceutical compositions of the present invention are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science , 249:1527-1533 (1990). Methods for preparing administrable compounds will be known or apparent to those skilled in the art and are described in more detail, for example, in Remington's Pharmaceutical Science , 17th ed. , Mack Publishing Company, Easton, PA (1988) . A person having ordinary skill in this art would readily recognize the most appropriate route of administration and dosages for fibroblast growth factor. Preferably, basic fibroblast growth factor is administered in a daily amount of from about 1 mg/kg to about 100 mg/kg.

Accordingly, the present invention provides a pharmaceutical composition for the treatment of cellular senescence comprising a pharmacologically effective dose of basic fibroblast growth factor and a pharmaceutically acceptable carrier.

Administration of basic fibroblast growth factor in the methods of the present invention may be by topical, parenteral, oral, intranasal, intravenous, intramuscular, subcutaneous, or any other suitable means. The preferred method of administration for treatment of skin cell proliferative diseases is by topical application or subcutaneous injection.

The present invention also provides a method of treating a pathophysiological state characterized by undesirable fibroblast proliferation, comprising the administration of a pharmacologically effective dose of a fibroblast-inhibiting cytokine to an animal having said state. Representative examples of relevant pathophysiological state include psoriasis, arthritis, restenosis, benign proliferative skin diseases, ichthyosis, papilloma, basal cell carcinoma, squamous cell carcinoma, scleroderma and hemangioma. As is discussed more fully below, the fibroblast-inhibiting cytokine useful in treating pathophysiological states characterized by uncontrollable and/or undesirable fibroblast proliferation include tumor necrosis factor, transforming growth factor-_/?, interferon-jS, interferon-gamma and interleukin-1/3. As shown in the present invention, these specific cytokines may be useful in regulating a wide variety of conditions where control of fibroblast proliferation is beneficial or advantageous. A person having ordinary skill in this art would readily recognize or be able to determine without undue experimentation, the most appropriate doses of tumor necrosis factor, transforming growth factor-/!?, interferon-/5, interferon-gamma and interleukin-13 as these cytokines have become useful for various other medical conditions.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1 Materials

Bacteria-derived recombinant human TNF, IL-lb, interferons α and 7 (IFN-o, IFN-7) and transforming growth factor-/? (TGF-/3) were provided by Genentech Inc., South San Francisco, CA; human fibroblast interferon (IFN-S) was obtained from Toray Company, Kamakura, Japan. IL-4 was a gift of Schering-Plough Co., NJ. IL-6 was a gift of Sandoz Pharmaceutical Corp., East Hanover, NJ. Leukemia inhibitory factor (LIF) was a gift of Amgen, Thousand Oaks, CA. Basic fibroblast growth factor (b-FGF) was provided by Chiron Corp., Emeryville, CA. All cytokines were highly purified recombinant human proteins. Other chemicals were obtained from Sigma Chemical Co., St. Louis, MO. Dulbeco's modified Eagle's medium (DMEM) was obtained from Whittaker MA Bioproducts (Walkersville, MD) . RPMI 1640 medium, fetal bovine serum and gentamicin were obtained from GIBCO, Grand Island, NY. 12-well plates were obtained from Becton Dickinson and Co. (Oxnard, CA; Lincoln Park, NJ) . Primary human foreskin fibroblasts at early passages were supplied by Dr. Olivia Smith of Baylor College of Medicine, Houston, TX.

EXAMPLE 2 Cells Human foreskin fibroblasts cultures were maintained in continuous exponential growth by weekly passage. Cells were routinely grown in RPMI 1640 medium supplemented with glutamine (2 mM) , gentamicin (50 μg/ml) , and fetal bovine serum (10%) i a humidified incubator in 5% C0₂ in air.

EXAMPLE 3 Fibroblast Proliferation Assays Young human foreskin fibroblasts at seeding populati doublings of 22.6 (4 x 10⁴ cells) were plated into 12 well plat in 2 ml RPMI 1640 media containing 10% FBS. The next day, t media was removed and fresh media containing different cytokin (20 ng/ l) was added into the wells. After 7 days, t confluent cells were trypsinized, counted by trypan bl exclusion method and the population doublings calculated a described below:

(N_H = cell harvest number, N, = cell inoculum number and X = the number of population doublings) . The increase i population doubling level (PDL) was added to the previou population doubling level to arrive at the current cumulati population doubling level. Each week 0.04 x 10⁶ cells in 12-wel plates were cultured in the presence of fresh cytokine-containin media. The cells were examined for mycoplasma every six month by DAPI staining method. DAPI or 4-6-diamine-2-phenyl-indol dihydrochloride is a fluorescent dye which selectively binds t DNA and strongly forms fluorescent DNA-DAPI complexes Mycoplasmas present in the cytoplasm appear as strongl fluorescent areas. The cytochemical detection of mycoplasma wit DAPI was performed according to the manufacturer's, Boehringe Mannheim, suggestions.

EXAMPLE 4 Determination of Interleukin-6 Human diploid fibroblasts (0.04 x 10⁶ cells in 2 ml were incubated with different cytokines (20 ng/ml) for 7 days an then the supernatants were collected. The presence of IL-6 wa determined by using the IL-6 dependent murine B cell hybridoma B-9 cell line described by Schmidt, et al., J. Immunol . , 128:217 (1982). Briefly, 2 x 10³ cells were incubated in 0.2 ml of PRMI 1640 containing 10% FBS in 96-well plates with differen concentrations of either IL-6 or IL-6 containing supernatants fo 96 hours. During the last 6 hours, cells were pulsed with 0.5 μCi of tritiated thymidine, harvested and cell associated radioactivity counted as described for cell proliferation assay. Fifty per cent of the maximum thymidine incorporation was defined as one unit which was obtained with one picogram of IL-6.

EXAMPLE 5 Effect of Cvtokines on the Life Span of HDF

To illustrate the effect of different cytokines on the total life span of human diploid fibroblasts, cells were grown continuously in the presence various cytokines, subcultivated every week, cell number counted and same number replated, i.e., the number of cells that were plated in the last immediate passage. As shown in Figures 1-4, Interferon-α, Interleukin-4, Interleukin-6 and Leukemia Inhibitory Factor has no effect on the life span of human diploid fibroblasts as compared to the control, media alone. Normal human diploid fibroblasts are known to secrete Interleukin-6. However, the present invention shows that Interleukin-6 does not effect the long-term growth of human diploid fibroblasts. The effect of cytokines, tumor necrosis factor, transforming growth factor-/3, interferon-/3, interferon-gamma and interleukin-13, was to reduce the life span of human diploid fibroblasts as compared to the control (Figures 5-9) , thus resulting in premature aging. Since normal human diploid fibroblasts are known to express Transforming Growth Factor-_/?, Interferon-3 and Interleukin-ljS, these cytokines can reduce the life span of the cells in an autocrine manner. The present invention suggests that antibodies to Transforming Growth Factor-/?, Interferon-j8 and Interleukin-1/3 may enhance the life span of these cells.

Surprisingly, human TGF-3 was found to be the most potent inhibitor of human diploid fibroblasts proliferation, since ming lung fibroblasts have been shown to proliferate in response to this cytokine. The effects seen in the present invention with tumor necrosis factor are unexpected because in short-term culture tumor necrosis factor enhances the proliferation of human diploid fibroblasts. In contrast to all the other cytokines tested, basic fibroblast growth factor was found to enhance the total life spa of human diploid fibroblasts (Figure 10) . As compared to th control, an approximately 60% increase in population doubling was observed when cells were grown in the presence of huma diploid fibroblasts. No significant proliferation of untreate cells was observed after 9 weeks whereas basic fibroblast growt factor-treated cells (even after 14 weeks) had not developed classical senescent morphology characterized by increase in cel size and sparse growth. When analyzed for replicative potential basic fibroblast growth factor- treated cells have highe replicative potential as compared to the control cells at al passage levels (Figure 11) . At week 11, the control cell reached complete senescence whereas basic fibroblast growt factor-treated cells had a weekly replicative potential of 3.6. Thus, the present invention clearly indicates that basi fibroblast growth factor has a highly significant effect on th life span of human diploid fibroblasts. An extracellular facto that increases programmed cellular senescence of fibroblasts ha heretobefore never been described.

EXAMPLE 6 Continuous Presence of Cytokines is Needed

The need of human diploid fibroblasts to be exposed t cytokines was evaluated while cells were young or whethe senescent cells can undergo proliferation when exposed t cytokines. As shown in Figure 3, tumor necrosis factor (Figur 3A) , basic-fibroblast growth factor (Figure 3B) and IL-lS (Figur 3C) induce the proliferation of young human diploid fibroblasts However, these cytokines had no effect on senescent human diploi fibroblasts. Thus, cytokines must be present at early stages i order to display their effects on long-term growth.

EXAMPLE 7 Cytokine Induced Morphological Alterations

It is known that young human diploid fibroblasts gro densely and are spindle-shaped. In contrast, the senescent huma diploid fibroblasts exhibit flattish and sparse morphology an they are bigger in size than young cells. As the cells advanc toward the senescent morphology, they first stop dividing and then there is a change in phenotype.

Figure 13 shows the effect of human interferon-alpha on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13 and 14 weeks.

Figure 14 shows the effect of human interleukin-4 on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 8 and 13 weeks.

Figure 15 shows the effect of human interleukin-6 on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13, 14 and 18 weeks.

Figure 16 shows the effect of human leukemia inhibitory factor on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13 and 14 weeks. Figures 17-21 illustrate the effect of cytokines which decrease fibroblast proliferation. Figure 17 shows the effect of human interleukin-lB on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 8 and 9 week. Figure 18 shows the effect of human tumor necrosis factor on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 6, 8 and 9 weeks. Figure 19 shows the effect of human interferon-7 on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13 and 14 weeks. Figure 20 shows the effect of human interferon-_/5 on the morphology of human diploid fibroblasts at different population doublings, i.e., 4, 13 and 14 weeks. Figure 21 shows the effect of human transforming growth factor-_/? on the morphology of human diploid fibroblasts at different population doublings, i.e., 4 and 6 weeks. As can be noted in Figure 22, the basic-fibroblast growth factor treated cells even at week 14 with a population doubling of 71.2 display the morphology of control cells at week 4 with population doubling of 32.5. Between week 14 and 17, the basic- fibroblast growth factor treated cells do not divide significantly but they acquire senescent phenotype. Thus, it is clear that basic- fibroblast growth factor treatment of human diploid fibroblasts improves the morphological characteristics of human diploid fibroblasts as well.

EXAMPLE 8 Expression of IL-6 gene is not related to cellular senescence Since fibroblasts are known to produce IL-6, the effect of basic-fibroblast growth factor and other cytokines on the expression of IL-6 was examined at different population doublings by bioassay and ELISA. 4x 10⁴ cells (2 ml) were incubated with and without cytokines (20 ng/ml) at 37°C for seven days and then cell supernatants were harvested for IL-6 determination. IL-6 levels in the human diploid fibroblasts were determined by bioassay as well as by ELISA (shown in parenthesis) . All determinations were made in duplicate. As can be seen from TABLE I, as population doublings increase, there is an increased production of IL-6 which declines at later population doublings. In addition, tumor necrosis factor and Interleukin-1 induced large amounts of Interleukin-6 in these cells, whereas other cytokines including basic-fibroblast growth factor had no significant effects. Thus, it is clear that the extension of lifespan of cells by basic-fibroblast growth factor is not due to its effect on the expression of IL-6.

TABLE I

Effect of Cytokines on the Production of Interleukin- -6 by HDF

Treatment Interleukin-6 levels (ng/ml)

Weeks in Culture

1 1 8 11

Control 6(8) 12(10) 24(14) 12(12) bFGF 6(5) 12(14) 48(26) 6(6)

TNF 120(280) 480(440) 60(50) 0.3(0.3)

IL-1B 3840(3600) 3840(3200) 120(100) 0.3(0.5)

IL-4 6(14) 24(32) 12(14) 0.3(0.6)

IFN-a 3(8) 12(10) 6(10) 6(5)

IFN-B 3(8) 12(14) 6(6) 0.3(0)

IFN-g 6(8) 6(6) 24(12) 6(6)

TGF-b 12(14) 6(5) NS NS

LIF 6(6) 12(8) 6(7) 6(5) EXAMPLE 9 Effect of bFGF on senescence-inducing genes

Human diploid fibroblasts at both young (population doubling of 23) and senescent (population doubling of 52) are treated with either basic fibroblast growth factor or served as controls. Subsequently, these cells are analyzed for expression by northern blot analysis of the genes which play a functional role in senescence. The northern blot analysis of mRNA is as follows: Total cellular RNA is extracted from cells according to procedures well known to one with ordinary skill in this art. For northern blot analysis, RNA samples (20 μg) are denatured with formaldehyde and formamide and electrophoresed in 0.8% agarose gel containing 0.67 M formaldehyde at 75 V for approximately 3 hours. RNA is alkali-transferred to Hybound N⁺ nylon membranes (Amersham Corp., Arlington Heights, IL) . After alkali transfer, the membranes are rinsed with 2X SSC (IX SSC; 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0). The sample is prehybridized at 65°C for one hour in a buffer containing 7% SDS, 0.5 M sodium phosphate, 1 mM EDTA, pH 7.2 (hybridization buffer) . The membranes are then hybridized for

16-20 hours with ³²P-labeled cDNA probes for various genes

(approximate specific activity 5-10 x 10⁸ cpm/ug DNA) in a hybridization buffer containing denatured salmon sperm DNA (0.2 mg/ml) . After hybridization, membranes are washed twice with 2X SSPE (1 x SSPE: 0.18 M NaCl, 0.01 M sodium phosphate and 0.001 M EDTA) containing 0.1% SDS at room temperature, once with 1 X SSPE, 0.1% SDS at 65°C and finally with 0.1 X SSPE, 0.1% SDS at 65°C. The blots are then exposed to phosphoimager screen and the images are recorded and quantitated using "Image Quant" software. To demonstrate the equal loading of lanes, the probes were stripped off by washing the filters twice for 30 minutes each at 95°C with 0.5% SDS and rehybridized with labeled cDNA probes for glyceraldehyde 3-phosphate dehydrogenase GAPDH) . A person having ordinary skill in this art would readily be able to obtain the necessary information concerning the sdi gene (See, e.g., Noda et al.. Cloning of a senescent cell derived inhibitor of DNA synthesis using an expression screen. Exp. Cell Res. 211: 90-98 Res. 211: 90-98 (1994) or the insulin growth factor-1 gene (See, e.g., Ferber, et al.. Failure of Senscent Human Fibroblasts to Express the Insulin-like Growth Factor-l Gene. J. Biol . Chem . 268 : 17883-17888 (1993). In the present invention, the effect of different cytokines on the replicative potential of human diploid fibroblasts was illustrated. The present invention demonstrated an increase in replicative potential, population doubling and total life span of human diploid fibroblasts by basic fibroblast growth factor, while a decrease in these parameters occurred with tumor necrosis factor, interferon-/S, interferon-gamma, interleukin-1/S and transforming growth factor-_/?; whereas interleukins-4 and -6, leukemia inhibitory factor and interferon- alpha had no effect. A continuous presence of cytokines was needed since short-term treatment of human diploid fibroblasts with cytokines provided results that had no relationship with long-term that obtained with human diploid fibroblasts in long term culture.

Lack of responsiveness of cells to certain cytokines could be in part due to the inability of senescent cells to express the IGF-1 gene, which could act as an autocrine as well paracrine growth factor. Basic fibroblast growth factor induction of the insulin growth factor -1 gene in human diploid fibroblasts may result in cellular proliferation. The effect of different growth factors on the proliferation of fibroblasts have been examined, but only in short term culture. The present invention indicates that several cytokines independently enhance the proliferation of normal human diploid fibroblasts at an early stage in the in vitro lifespan but not when the cells become senescent.

Why senescent fibroblast fail to proliferate in response to growth factors is still not understood. Senescent fibroblasts are growth-arrested predominantly at the Gl/S boundary of the cell cycle analogous to terminal differentiation. Several cell cycle-dependent genes (4F1, c-myc, JE-3, 2F1, ornithine decarboxylase, 2A9, p53, c-Ha-ras, _/?-actin, thymidylate kinase and histone H3) have been shown to be equally expressed in young and senescent fibroblasts following serum stimulation. However other growth regulatory genes are repressed in senescent human diploid fibroblasts cells, including c-fos, cdc2, cyclin A and cyclin B. Senescent cells also underexpress the tissue inhibitor of metalloproteases and L7 ribosomal protein genes. Interestingly, several genes are overexpressed in senescent cells compared with young cells, including collagenase, αl-procollagen and cathepsin B. Recently, a gene termed the senescent derived inhibitor (SDI) was described and is overexpressed in senescent fibroblasts and is responsible for inhibition of DNA synthesis. The failure of senescent fibroblasts to enter S-phase has also been ascribed to its inability to phosphorylate retinoblastoma gene product. The shortening of telomeres, i.e., the terminal guanine rich sequence of chromosomes, are other characteristics of senescent human diploid fibroblasts. Whether any of these mechanisms are responsible for the lack of response of senescent cells to tumor necrosis factor is not clear.

Recently it was reported that tumor necrosis factor induces CDC2 and CDK2 gene expression in young, quiescent WI-38 fibroblasts but not in senescent fibroblasts. Since both of these protein kinases are essential to enable cells to enter into the S-phase; this may explain the lack of effect of tumor necrosis factor and other growth factors on the proliferation of senescent human diploid fibroblasts. Alternatively, senescent cells may also be unable to respond to tumor necrosis factor because of overexpression of SDI which has been shown to be a potent inhibitor of CDK2. However, this is unlikely because quiescent human diploid fibroblasts which also overexpress SDI, were found to be sensitive to proliferative effects of tumor necrosis factor. In contrast to senescent human diploid fibroblasts, tumor necrosis factor-induced proliferation of quiescent human diploid fibroblasts may be due to its ability to downmodulate SDI expression.

Treatment of different cell types, including human diploid fibroblasts by tumor necrosis factor causes the activation of a nuclear transcriptional factor NF-kB. The activation of NF-kB is an early event in TNF signal transduction and is necessary for TNF-mediated induction of various cytokin genes including the interleukins, IL-2, IL-6, IL-8, TNF, lymphotoxin, interferon, GM-colony stimulating factor (GM-CSF) , G-CSF, IL-2R, and human immunodeficiency virus-I (HIV-I) genes. The present invention demonstrates that the tumor necrosi factor-mediated activation of NF-kB occurs equally in young an senescent human diploid fibroblasts. Thus, the inability o senescent cells to proliferate or their reduced ability t produce interleukins in response to tumor necrosis factor appear to not be due to lack of activation of NF-kB.

Several reports have recently shown that there is age related modulation of activation and production of cytokine from lymphocytes. The present invention demonstrates that ther is a decline in the production of cytokines from senescen fibroblasts. Both the interleukins examined play an importan role in inflammation.

All patents and publications mentioned in thi specification are indicative of the levels of those skilled i the art to which the invention pertains. Any patents an publications mentioned herein are herein incorporated b reference to the same extent as if each individual publicatio was specifically and individually indicated to be incorporate by reference.

One skilled in the art will readily appreciate that th present invention is well adapted to carry out the objects an obtain the ends and advantages mentioned, as well as thos inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compound described herein are presently representative of preferre embodiments, are exemplary, and are not intended as limitation on the scope of the invention. Changes therein and other use will occur to those skilled in the art which are encompasse within the spirit of the invention as defined by the scope of th claims.

WHAT IS CLAIMED IS:

Claims

1. A method of decreasing cellular senescence comprising the step of administering a pharmacologically effective dose of basic fibroblast growth factor to an animal.

2. The method of claim 1, wherein said animal is a human.

3. The method of claim 1, wherein said basic fibroblast growth factor is administered in a daily amount of from about 1 mg/kg to about 100 mg/kg.

4. A pharmaceutical composition for the inhibition of cellular senescence comprising a pharmacologically effective dose of basic fibroblast growth factor and a pharmaceutically acceptable carrier.

5. A method of increasing endogenous levels of IGF-1 in animals comprising the administration of a pharmacologically effective dose of basic fibroblast growth factor to an animal.

6. A method of decreasing endogenous levels of senescence inducing proteins in animals comprising the administration of a pharmacologically effective dose of basic fibroblast growth factor to an animal.

7. The method of claim 6, wherein said senescence inducing protein is sdi.

8. A method of treating a pathophysiological state characterized by undesirable fibroblast proliferation, comprising the administration of a pharmacologically effective dose of a fibroblast-inhibiting cytokine to an animal having said state.

9. The method of claim 8, wherein said pathophysiological state is selected from the group selected from psoriasis, arthritis, restenosis, benign proliferative skin diseases, ichthyosis, papilloma, basal cell carcinoma, squamous cell carcinoma, scleroderma and hemangioma.

10. The method of claim 8, wherein said animal is a human.

11. The method of claim 8, wherein said fibroblast- inhibiting cytokine is selected from the group consisting of tumor necrosis factor, transforming growth factor-_/?, interferon-/?, interferon-gamma and interleukin-1?.