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WO1998003195A9 - Procedes et compositions de modulation du taux de croissance - Google Patents

Procedes et compositions de modulation du taux de croissance

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Publication number
WO1998003195A9
WO1998003195A9 PCT/US1997/012498 US9712498W WO9803195A9 WO 1998003195 A9 WO1998003195 A9 WO 1998003195A9 US 9712498 W US9712498 W US 9712498W WO 9803195 A9 WO9803195 A9 WO 9803195A9
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WO
WIPO (PCT)
Prior art keywords
cell
abl
cells
nucleic acid
tumor
Prior art date
Application number
PCT/US1997/012498
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English (en)
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WO1998003195A1 (fr
Filing date
Publication date
Application filed filed Critical
Priority to AU37313/97A priority Critical patent/AU3731397A/en
Publication of WO1998003195A1 publication Critical patent/WO1998003195A1/fr
Publication of WO1998003195A9 publication Critical patent/WO1998003195A9/fr

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  • the present invention relates generally to the fields of cancer therapy and control of cell proliferation. More particularly, it concerns the use of the c-Abl and p300 genes, either alone or in combination with p53, to induce programmed cell death or decreased cell proliferation.
  • Certain cancer treatment methods involve damaging the DNA of the cancer cell.
  • the cellular response to DNA damage includes activation of DNA repair, cell cycle arrest and lethality (Hall, 1988).
  • the signaling events responsible for the regulation of these events remain unclear.
  • EGR early growth response
  • Mitomycin C is an antitumor antibiotic isolated from Streptomyces caespitosus that covalently binds to DNA (Tomasz et al, 1988) This agent induces both monofunctional and bifunctional DNA lesions (Carrano et al, 1979) Other studies have demonstrated that MMC stimulates the formation of hydroxyl radicals (Dusre et al, 1989) Although the precise mechanism of action of this agent is unclear, MMC-induced cytotoxicity has been attributed to DNA alkylation and the formation of interstrand cross-links (Carrano et al. , 1979, Dusre et al. , 1989, Tomasz et al.
  • Protein tyrosine kinases can be divided into receptor-type and nonreceptor- type (Src-like) kinases (Cantley et al, 1991; Hanks et al, 1988; Bonni et al, 1993; Larner et al, 1993; Ruff- Jamison et al, 1993).
  • Several protein tyrosine kinases have been purified from the cytosolic fractions of various tissues (Nakamura et al, 1988; Wong & Goldberg, 1984;
  • the Src-like kinases which can associate with receptors at the plasma membrane, induce rapid tyrosine phosphorylation and/or activation of effectors such as phospholipase C- ⁇ l (PLC ⁇ l) (Carter et al, 1991), PLC ⁇ 2 (Hempel et al, 1992), mitogen-activated protein
  • MAP MAP kinase
  • GAP GTPase activating protein
  • PI3-K phosphatidylinositol 3 -kinase
  • the present invention in one embodiment, provides a method for increasing p53-mediated apoptosis in a tumor cell comprising the step of increasing the activity level of at least one of c-Abl and p300 in said tumor cell.
  • the tumor cell may be derived from various tissues including brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cells, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood tissue
  • the activity levels of both c-Abl and p300 are increased in said tumor cell
  • Increasing activities may be accomplished (i) by providing at least one of a c-Abl polypeptide and a p300 polypeptide to said tumor cell in an amount effective to increase p53-mediated apoptosis, (ii) by providing at least one of a nucleic acid encoding c-Abl and a nucleic acid encoding p300 to said tumor cell, wherein said nucleic acid is operably linked to a promoter active in eukaryotic cells in an amount effective to increase p53-mediated apoptosis, or (iii) by providing an agent to said cell that increases the expression or stability of at least one of c-Abl and p300 in an amount effective to increase p53-mediated apoptosis
  • nucleic acids contained in viral expression vectors, via infection of target cells by encapsulated viral vectors also is contemplated
  • viral vectors include herpesvirus, adenovirus, vaccinia virus, retrovirus and adeno-associated virus
  • the method may further comprise determining the p53 activity of the target cell Where the target cell is defective in p53, and the method further comprises providing to said tumor cell a nucleic acid encoding a wild-type p53 operably linked to a promoter active in eukaryotic cells
  • the p53 nucleic acid preferably is contained in an expression vector, and more preferably in a viral vector
  • target cells may be subjected, either before or after the methods described above, to ionizing radiation or to a chemotherapeutic agent
  • the ionizing radiation may be x-irradiation or ⁇ -irradiation
  • the chemotherapeutic agent may be mitomycin C, etoposide, genistin, cisplatin, 5-FU, adriamycin, doxorubicin, actinomycin D, verapamil, nitrosourea, ara-C and camptothecin
  • a method for treating a patient having a tumor comprising the step of increasing the activity level of at least one of c-Abl and p300 in cells of said tumor Again, the activity level may be increased by providing at least one of a c-Abl polypeptide and a p300 polypeptide to said tumor in an amount effective to increase p53-mediated apoptosis in cells thereof
  • the method may further comprising screening the tumor cells to determine the p53 status thereof
  • the method may further comprise providing to said tumor cells a nucleic acid encoding a wild-type p53 operably linked to a promoter active in eukaryotic cells
  • the method may further take advantage of combined therapies, for example, by additionally treating the tumor with ionizing radiation or a chemotherapeutic agent
  • a method of screening a candidate substance for p53 -stimulatory activity comprising the steps of (i) providing a eukaryotic cell expressing a functional p300 polypeptide, (ii) contacting said cell with said candidate substance, and (iii) determining the effect of said candidate substance on the p300 level of said cell, wherein an increase in the p300 level in said cell, as compared to an untreated cell, indicates that said candidate substance increases p53 activity
  • a similar method is provided where the effect of the candidate substance on c-Abl is determined
  • the p300 or c-Abl level may be measured by Western blot or ELISA
  • contacting comprises transferring said nucleic acid into said cell
  • Methods of transferring include transfection, lipofection, protoplast fusion, bombardment, electroporation or viral infection
  • a use of at least one of a p300 and a c-Abl polypeptide for the preparation of a pharmaceutical composition effective to increase p53-mediated apoptosis in a cell (ii) a use of at least one of a nucleic acid encoding a p300 polypeptide and a nucleic acid encoding a c-Abl polypeptide for the preparation of a pharmaceutical composition effective to increase apoptosis in a cell, (iii) a use of a p300 polypeptide or gene coding therefor for the preparation of a pharmaceutical composition for the treatment of cancer, and (iv) a use of a p300 polypeptide or gene coding therefor for the preparation of a pharmaceutical composition for the treatment of cancer
  • kits for use in killing malignant cells or reducing their growth are kits for use in cancer treatment
  • the kits of the invention will generally comprise, in suitable container means, pharmaceutical formulations of a p53 polypeptides or gene construct, c-Abl polypeptides or gene constructs and p300 polypeptides or gene constructs
  • p53 polypeptides or gene construct a p53 polypeptides or gene construct
  • c-Abl polypeptides or gene constructs p300 polypeptides or gene constructs
  • kits for use in killing malignant cells or reducing their growth will generally comprise, in suitable container means, pharmaceutical formulations of a p53 polypeptides or gene construct, c-Abl polypeptides or gene constructs and p300 polypeptides or gene constructs
  • These agents may be present within a single container, or these components may be provided in distinct or separate container means
  • Classical chemotherapeutic pharmaceutical preparations also are contemplated for use with the therapeutic compositions of the present invention
  • FIGS. lA-C Overexpression of kinase active c-Abl downregulates Cdk activity
  • FIG 1A MCF-7 cells were transfected with 2 ⁇ g p53-enhancer-luciferase plasmid (mdm2NA-Luc) and, (1) 8 ⁇ g control vector pSRaMSVtkNeo, (2) 5 and 8 ⁇ g c-abl vector, (3) 5 and 8 ⁇ g c-Aabl (K-R) vector, and (4) 5 and 8 ⁇ g c-abl ⁇ Pro 4 vector
  • Cells were also transfected with 2 ⁇ g SV40-promoter-luciferase plasmid (pGL2-control vector, Promega) and 8 ⁇ g c-Abl vector Luciferase activity was measured and normalized for protein concentration to that for control vector, D, MCF-7 cells were transfected with 8 ⁇ g control, c-abl, c-abl (K-R) of c-abl ⁇
  • FIGS. 2A-D c-Abl kinase activity regulates irradiation (IR)-induced inhibition of Cdk2
  • FIG 2A MCF-7 cells stably transfected with null pSR vector or c-abl (K-R) were treated with 5 Gy IR and collected after 3h Nuclei were isolated and nuclear proteins immunoprecipitated with anti-Abl (Ab-3, Oncogene Science) as described Immune- complex kinase was analyzed using a glutathione S transferase (GST)-Crk (120-225) fusion protein as substrate (top) And Abl immunoprecipitates were also analyzed by immunoblotting with anti-Abl (bottom)
  • FIG 2B Lysates were immunoblotted with anti- Abl, anti-p53 (Ab-6, Oncogene Science), anti-p21, anti-GADD45 (AT-26, Santa Cruz Biotechnology) and anti-c-Myc (9E10, Santa Cruz Biotechnology)
  • FIG 2C Lys
  • FIGS. 3A-D c-Abl kinase activity regulates IR-induced growth arrest
  • FIG 3 A Representative two-dimensional FACS analysis of MCF-7/pSR and MCF-7/c-Abl (K-R) cells after exposure to 0 or 5 Gy IR Synchronized cells were irradiated and percentage of cells in S phase was assessed at 24h Boxes labeled Rl, R2 and R3 represent S, Gl and G2/M phase cells, respectively
  • FIG 3B Percentage of cells entering S phase after IR relative to control unirradiated cells Results are expressed as the mean ⁇ s e of 6 experiments for each of two (a,b) independently selected clones
  • FIG 3C C57BL6 wild- type (ab h ) and abl' MEFs were exposed to 0 or 5 Gy and collected at 3 h Cell lysates were immunoblotted with anti-Abl and anti-PCNA antibodies
  • FIGS. 4A-D c-Abl kinase downregulates Cdk2 activity by a p53-dependent. p21 -independent mechanism
  • FIG 4A Lysates were immunoprecipitated with anti-Abl (left) or anti-p53 (right) and the precipitates analyzed by immunoblotting with the indicated antibodies
  • FIG 4B MEFs (p59 7 , p53 +/+ ) n were transfected with the c-abl or c-abl (K-R) vectors and collected at 48 h
  • FIG 4C MEFs (abt' ab ' ) 11 were exposed to D or 5 Gy and collected at the indicated times
  • FIG 4D MEFs (p2T , p21 +/+ were transfected with the c-Abl or c-Abl (K-R) vectors and collected at 48 h
  • Cell lysates were immunoprecipitated with anti-Cdk2 and the precipitates assayed for histone HI
  • the c-Abl protein tyrosine kinase is activated by certain DNA-damaging agents, and its overexpression causes arrest in the Gl phase of the cell cycle by the tumor- suppressor protein p53 (Sawyers et al, 1994, Mattioni et al., 1995, Goga et al, 1995)
  • the present investigators studied the role of c-Abl in growth arrest induced by DNA damage Transient transfection experiments using wild-type or inactivated c-Abl show that both induce expression of p21, an effector of p53, but only wild-type c-Abl down regulates the activity of the cyclin-dependent kinase Cdk2 and causes growth arrest
  • Exposure to ionizing radiation of cells that stably express active or inactive c-Abl is associated with induction of c-Abl/p53 complexes and p21 expression.
  • cells expressing the dominant-negative c-Abl mutant and cells lacking the c-abl gene are impaired in their ability to down-regulate Cdk2 or undergo Gl arrest in response to ionizing radiation.
  • p300 is a nuclear protein originally identified for its ability to interact with adenovirus El A protein It is a regulator of transcription and interacts with a variety of cellular as well as viral proteins p300 possesses histone acetyltransferase activity resulting from either intrinsic activity and/or from an assocaited protein, P/CAF Bannister et al, 1996, Ogryzko et al, 1996, Yang et al, 1996 The acetylation of histones in thought to be involved in destabilization and restructuring of nucleosomes, which is likely a crucial event for accessibility of transcription factors to DNA templates
  • p300 A role for p300 in control of cellular growth has been proposed on the basis of the functional behavior of adenovirus El A and SV40 Tag proteins, which lose the ability to bind p300 Moran, 1993, Avantaggiati et al, 1996, Eckner et al, 1996a Such mutants are defective in the induction of cellular DNA synthesis and, in several instances, of transformation
  • p300 is required for the activation of muscle-specific genes and for cell cycle arrest during differentiation of muscle cells. Eckner et al, 1996b, Puri et al, 1997.
  • mutations or translocation of the p300 gene has been described in human tumors.
  • p300 mutations in colorectal carcinomas are somatic and coupled to deletion of the second allele of the gene, suggesting that p300 is consequently inactivated It also is induced upon a cell's exposure to radiation On the basis of this evidence, p300 is envisioned to be a negative regulator of cell growth
  • p300 apparently is responsible for several p53-mediated functions, such as inhibition of API -regulated enhancers, transcriptional activation, apoptosis and cell cycle progression
  • the present inventors have determined that p300 is activated by c-Abl More specifically, p300 is phosphorylated by a tyrosine kinase function of c-Abl This phosphorylation causes p300 interaction with p53, and the aforementioned activities
  • the present invention exploits signaling pathways that are involved in the correction of DNA damage, in cell cycle progression and in apoptosis
  • the methods involve the use of the c-Abl, p300 and p53 gene products to effect cellular responses to various external stimuli
  • the present invention provides a means of increasing the levels of wild-type p53 expression in tumor cells, thereby allowing for an increased apoptosis or reduction in cellular proliferation
  • c-Abl and p300 can increase the expression of wild-type p53
  • cancers that express normal p53 can be treated with c-Abl genes or the c-Abl gene product, or with p300 or the corresponding p300 gene
  • the compositions of the present invention can be used to augment conventional gene therapy, where wild- type p53 is introduced into tumor cells concomitantly with c-Abl and/or p300 to increase the level of p53 so that programmed cell death is triggered
  • the present application is drawn, in one embodiment, to methods of screening cells for their ability to express wild-type p53 protein expression of cancer cells and to concomitantly induce apoptosis in said cells
  • To augment the expression of p53 one would then add the c-Abl gene product and/or p300 to a cell If wild-type p53 is lacking in a cell, wild-type p53 and c-Abl are administered to a cell in combination to induce the apoptotic response
  • Effective amounts are those amounts of compositions effective at reproducibly increasing wild-type p53 expression in cancer cells in comparison to their normal levels Compounds that achieve significant appropriate changes in activity will be used A significant increase in wild-type p53 expression, e.g., as measured using for example,
  • Western blot analysis are represented by an increase in wild-type p53 levels of at least about 30%-40%, and most preferably, by increases of at least about 50%, with higher values of course being possible Assays that measure p53 content and expression in cells are well known in the art and may be conducted in vitro or in vivo
  • MTT assay it may be desirable simply to measure inhibition of growth of cancer cells, for example, by measuring growth according to the MTT assay
  • a significant inhibition in growth is represented by decreases of at least about 30%-40% as compared to uninhibited, and most preferably, of at least about 50%, with more significant decreases also being possible.
  • Growth assays as measured by the MTT assay are well known in the art Assays may be conducted as described by Mosmann et al, 1983; Rubinstein et al, 1990 (incorporated herein by reference) Therefore, if a candidate substance exhibited inhibition of growth of cancer cells in this type of study, it would likely be a suitable compound for use in the present invention.
  • vectors that incorporate nucleic acid sequences that encode the c-Abl and p300 sequences It is contemplated that these vectors may either be transiently incorporated into the host cell, or may be stably integrated into the host cell genome This expression preferably occurs in a mammalian cell, and even more preferably, the mammalian cell is a human cell.
  • suitable vectors for use within the scope of the present invention include, but are not limited to, adenovirus, adeno-associated virus, retrovirus or herpes simplex virus 1.
  • the present invention contemplates the preparation of nucleic acid molecules that comprise a coding region that contains regions complementary to and capable of hybridizing with a c-Abl gene sequence.
  • the preferred nucleic acid molecules will be DNA sequences arranged in a vector, such as a virus or plasmid, and positioned under the control of an appropriate promoter.
  • the antisense RNA molecule may itself be an appropriate nucleic acid, such as retrovirus RNA into which the appropriate coding sequences have been incorporated.
  • the nucleic acids may be introduced into cells by means of liposomes, or the like.
  • a target cell with a c-Abl and/or p300 construct in a combined amount effective to kill the cell. This process may involve contacting the cells with the p53 construct and c-Abl/p300 construct(s) at the same time.
  • compositions or pharmacological formulations that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the p53 construct and the other composition includes the c-Abl construct
  • target cell where lacking a functional p53 molecule, may be first exposed to a p53 expression construct and then contacted with a c-Abl or p300 construct, or vice versa
  • contacted and “exposed”, when applied to a cell are used herein to describe the process by which a p53 or c-Abl construct are delivered to a target cell or are placed in direct juxtaposition with the target cell
  • either one or both agents are delivered to a cell in a combined amount effective to kill the cell, i.e , to induce programmed cell death or apoptosis
  • killing i.e , to induce programmed cell death or apoptosis
  • apoptosis are used interchangeably in the present text to describe a series of intracellular events that lead to target cell death Reduced cell growth refers to a lower rate of cellular proliferation than observed for the untreated cells
  • p53 As a Tumor Suppressor p53 is currently recognized as a tumor suppressor gene (Montenarh, 1992) High levels have been found in many cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses, including SV40 The p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and is already documented to be the most frequently-mutated gene in common human cancers (Mercer, 1992) It is mutated in over 50% of human NSCLC (Hollestein et al, 1991) and in a wide spectrum of other tumors
  • the p53 gene encodes a 393-amino-acid phosphoprotein that can form complexes with host proteins such as large-T antigen and E1B
  • the protein is found in normal tissues and cells, but at concentrations which are minute by comparison with transformed cells or tumor tissue Interestingly, wild-type p53 appears to be important in regulating cell growth and division Overexpression of wild-type p53 has been shown in some cases to be anti-proliferative in human tumor cell lines Thus p53 can act as a negative regulator of cell growth (Weinberg, 1991) and may directly suppress uncontrolled cell growth or indirectly activate genes that suppress this growth Thus, absence or inactivation of wild type p53 may contribute to transformation However, some studies indicate that the presence of mutant p53 may be necessary for full expression of the transforming potential of the gene
  • Wild-type p53 is recognized as an important growth regulator in many cell types Mis-sense mutations are common for the p53 gene and are essential for the transforming ability of the oncogene
  • a single genetic change prompted by point mutations can create carcinogenic p53 Unlike other oncogenes, however, p53 point mutations are known to occur in at least 30 distinct codons, often creating dominant alleles that produce shifts in cell phenotype without a reduction to homozygosity Additionally, many of these dominant negative alleles appear to be tolerated in the organism and passed on in the germ line Various mutant alleles appear to range from minimally dysfunctional to strongly penetrant, dominant negative alleles (Weinberg, 1991)
  • a patient presenting a p53-positive tumor that expresses wild-type p53 will be treated with c-Abl and/or p300 based therapies
  • the p53 status of the tumor cells will be determined using any conventional methods, examples of which are described below
  • Patients may, but need not, have received previous chemo-, radio- or gene therapies
  • patients will have adequate bone marrow function (defined as peripheral absolute granulocyte count of > 2,000/mm 3 and platelet count of 100,000/mm 3 ), adequate liver function (bilirubin ⁇ 1 5 mg/dl) and adequate renal function (creatinine ⁇ 1 5 mg/dl)
  • the patient will be treated with a pharmaceutically acceptable form of the c-Abl and/or p300 gene products, or will be treated by gene therapy techniques that will allow the in vivo expression of the c-Abl or p300 gene products Alternatively, the patient may be treated with a substance that augments the expressionon c-Abl or p300 normally produced by the target cell Treatment regimens normally last between about 3 and 6 weeks, and involve repeat dosings In order to know whether a cell will respond to provision of c-Abl and/or p300, it must first be determined whether the cell expresses an active p53 molecule Thus, screening for p53 expression is an important first step
  • detection methods can be employed in the present invention to detect the p53 status of a cell
  • any assay that utilizes antibodies for detection for example, ELISAs, Western Blotting, immunoassay techniques etc
  • assays that employ nucleotide probes may be used to identify the presence/absence/status of p53, for example, Southern blotting,
  • Immunoassays encompassed by the present invention include, but are not limited to those described in U S Patent No 4,367,110 (double monoclonal antibody sandwich assay) and U S Patent No 4,452,901 (Western blot) Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo
  • Immunoassays in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art Immunohistochemical detection using tissue sections is also particularly useful.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • anti-p53 antibodies are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate Then, a test composition suspected of containing the desired antigen, such as a clinical sample, is added to the wells After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected Detection is generally achieved by the addition of another antibody, specific for the desired antigen, that is linked to a detectable label This type of ELISA is a simple "sandwich ELISA" Detection may also be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label
  • Varaitions of ELISA techniques are know to those of skill in the art
  • the samples suspected of containing the desired antigen are immobilized onto the well surface and then contacted with the antibodies of the invention After binding and appropriate washing, the bound immune complexes are detected
  • the immune complexes may be detected directly
  • the immune complexes may be detected using a second antibody that has binding affinity for the first antigen specific antibody, with the second antibody being linked to a detectable label
  • Competition ELISAs are also possible in which test samples compete for binding with known amounts of labeled antigens or antibodies
  • the amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation ⁇ with coated wells
  • the presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes These are described as below
  • Antigen or antibodies may also be linked to a solid support, such as in the form of plate, beads, dipstick, membrane or column matrix, and the sample to be analyzed applied to the immobilized antigen or antibody
  • a solid support such as in the form of plate, beads, dipstick, membrane or column matrix
  • the sample to be analyzed applied to the immobilized antigen or antibody
  • a plate with either antigen or antibody one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period The wells of the plate will then be washed to remove incompletely adsorbed material Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera These include bovine serum albumin (BSA), casein and solutions of milk powder
  • BSA bovine serum albumin
  • casein casein
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of anti
  • the immobilizing surface is contacted with the clinical or biological sample to be tested under conditions effective to allow immune complex
  • Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween These added agents also tend to assist in the reduction of nonspecific background
  • the suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25° to 27°C, or may be overnight at about 4°C or so
  • washing often includes washing with a solution of
  • the second or third antibody will have an associated label to allow detection
  • this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate
  • a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation, e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween.
  • the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and H 2 O , in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer
  • the label may be a chemiluminescent one.
  • Assays for the p53 radiation status of the cell can determine normal/abnormal tissue distribution for diagnostic purposes
  • Methods for in vitro and in situ analysis are well known and involve assessing binding of antigen-specific antibodies to tissues, cells or cell extracts These are conventional techniques well within the grasp of those skilled in the art
  • the antibodies to p53 may be used in conjunction with both fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC) Each tissue block may consist of 50 mg of residual "pulverized" tumor
  • IHC immunohistochemistry
  • frozen-sections may be prepared by rehydrating 50 ng of frozen pulverized tumor at room temperature in PBS in small plastic capsules, pelleting the particles by centrifugation, resuspending them in a viscous embedding medium (OCT), inverting the capsule and pelleting again by centrifugation, snap-freezing in -70°C isopentane, cutting the plastic capsule and removing the frozen cylinder of tissue, securing the tissue cylinder on a cryostat microtome chuck, and cutting 25-50 serial sections containing an average of about 500 remarkably intact tumor cells
  • OCT viscous embedding medium
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube, pelleting, resuspending in 10% formalin for 4 hours fixation, washing/pelleting; resuspending in warm 2 5% agar, pelleting, cooling in ice water to harden the agar; removing the tissue/agar block from the tube, infiltrating and embedding the block in paraffin, and cutting up to 50 serial permanent sections
  • nucleic acids may be isolated from cells according to standard methodologies (Sambrook et al, 1989)
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA Where mRNA is used, it may be desired to convert the mRNA to a complementary DNA
  • the RNA is whole cell RNA, in another, it is poly-A RNA Normally, the nucleic acid is amplified
  • the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification
  • the identified product is detected
  • the detection may be performed by visual means (e.g., ethidium bromide staining of a gel)
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals
  • alterations should be read as including deletions, insertions, point mutations and duplications
  • Point mutations result in stop codons, frameshift mutations or amino acid substitutions
  • Somatic mutations are those occurring in non-germline tissues
  • Germ-line tissue can occur in any tissue and are inherited Mutations in and outside the coding region also may affect the amount of p53 produced, both by altering the transcription of the gene or in destabilizing or otherwise altering the processing of either the transcript (mRNA) or protein
  • FISH fluorescent in situ hybridization
  • PFGE direct DNA sequencing
  • SSCA single-stranded conformation analysis
  • ASO allele-specific oligonucleotide
  • dot blot analysis denaturing gradient gel electrophoresis, RFLP and PCR-SSCP
  • primer as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process
  • primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed
  • Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred
  • Probes are defined differently, although they may act as primers
  • Probes while perhaps capable of priming, are designed to binding to the target DNA or RNA and need not be used in an amplification process
  • the probes or primers are labeled with radioactive species ( 32 P, 14 C, 35 S, 3 H, or other label), with a fluorophore (rhodamine, fluorescein) or a chemillumiscent (luciferase)
  • radioactive species 32 P, 14 C, 35 S, 3 H, or other label
  • fluorophore rhodamine, fluorescein
  • chemillumiscent luciferase
  • PCRTM polymerase chain reaction
  • PCR two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence
  • An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g. , Taq polymerase
  • a DNA polymerase e.g. , Taq polymerase
  • the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases These methods are described in WO 90/07641 filed December 21, 1990 Polymerase chain reaction methodologies are well known in the art
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No PCT US87/00880, may also be used as still another amplification method in the present invention
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase The polymerase will copy the replicative sequence that can then be detected
  • restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha- thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al, (1992)
  • SDA Strand Displacement Amplification
  • RCR cyclic probe reaction
  • DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion The original template is annealed to another cycling probe and the reaction is repeated Still another amplification methods described in GB Application No 2 202 328, and in PCT Application No PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention In the former application, "modified" primers are used in a PCR-like, template- and enzyme- dependent synthesis The primers may be modified by labeling with a capture moiety
  • an excess of labeled probes are added to a sample
  • the probe binds and is cleaved catalytically After cleavage, the target sequence is released intact to be bound by excess probe Cleavage of the labeled probe signals the presence of the target sequence
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989, Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference in their entirety)
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR 3SR
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA
  • amplification techniques involve annealing a primer which has target specific sequences Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded
  • DNA molecules are heat denatured again In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6 In an isothermal cyclic reaction, the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded
  • RNA polymerase such as T7 or SP6
  • ssRNA single- stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase)
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA)
  • RNase H an RNase specific for RNA in duplex with either DNA or RNA
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template This primer is then extended by DNA polymerase (exemplified by the large "K
  • dsDNA double-stranded DNA
  • dsDNA double-stranded DNA
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA These copies can then re-enter the cycle leading to very swift amplification With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA
  • a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose
  • the different species should be spatially separated to facilitate analysis This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter
  • the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization Because the probe is designed to base pair with the target, the probe will binding a portion of the target sequence under renaturing conditions Unbound probe is then removed, and detection is accomplished as described above
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods See Sambrook et al, 1989
  • chromatographic techniques may be employed to effect separation
  • adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography
  • Products may be visualized in order to confirm amplification of the marker sequences
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation
  • ⁇ visualization is achieved indirectly
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence
  • the probe preferably is conjugated to a chromophore but may be radiolabeled
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety
  • detection is by a labeled probe
  • chromophore or radiolabel probes or primers identify the target during or following amplification
  • oligonucleotide primers may be designed to permit the amplification of sequences throughout the p53 gene that may then be analyzed by direct sequencing
  • kits This generally will comprise preselected primers and probes Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, SequenaseTM, etc ), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification
  • enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, SequenaseTM, etc ), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification
  • kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe
  • chip-based DNA technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996)
  • a c-Abl and optionally or alternatively, p300 polypeptide or a gene construct that comprises a c-Abl sequence, and optionally or alternatively, a p300 sequence are used in combination with a recombinant vector that comprises a nucleic acid sequence capable of expressing the desired gene sequence in the cell.
  • the vector typically is introduced into the cell in a manner that allows expression of the encoded gene sequence at a level sufficient to effect cell function and cause an apoptotic response or to reduce cell proliferation.
  • expression construct is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed
  • the transcript may be translated into a protein, but it need not be
  • expression includes both transcription of a p53, p300 or c-Abl gene and translation of p53, p300 or c-Abl mRNA into the proper protein product
  • promoter refers to a DNA sequence recognized by the synthetic machinery of the host cell, or introduced synthetic machinery, that is required to initiate the specific transcription of a gene
  • under transcriptional control means that the promoter is in the correct location in relation to the polynucleotide to control RNA polymerase initiation and expression of the polynucleotide
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units
  • At least one module in each promoter functions to position the start site for RNA synthesis
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation
  • promoter elements regulate the frequency of transcriptional initiation Typically, these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription
  • the particular promoter that is employed to control the expression of ap53 or c- Abl polynucleotide is not believed to be critical, so long as it is capable of expressing the polynucleotide in the targeted cell
  • a promoter that is capable of being expressed in a human cell
  • such a promoter might include either a human or viral promoter
  • the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter and the Rous sarcoma virus long terminal repeat can be used to obtain high-level expression of the p53 or c-Abl polynucleotide
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of polynucleotides is contemplated as well, provided that the levels of expression are sufficient to produce a growth inhibitory effect
  • a promoter which is active in specific cells, such as tyrosinase (melanoma), alpha-fetoprotein and albumin (liver tumors), CC10 (lung tumor) and prostate-specific antigen (prostate tumor) will permit tissue-specific expression of p53 or c-Abl constructs
  • Table 1 lists several elements/promoters which may be employed, in the context of the present invention, to regulate the expression o ⁇ p53 or c-Abl constructs This list is not intended to be exhaustive of all the possible elements involved in the promotion of p53 or c-Abl expression but, merely, to be exemplary thereof
  • Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins
  • enhancers are operational An enhancer region as a whole must be able to stimulate transcription at a distance, this need not be true of a promoter region or its component elements On the other hand, a promoter must have one or. more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization
  • Eukaryotic Promoter Data Base EPDB any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of a p53, p300 or c-Abl construct
  • a T3, T7 or SP6 cytoplasmic expression system Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment Eukaryotic cells can support cytoplasmic transcription from certain bacteriophage promoters if the appropriate bacteriophage polymerase is provided, either as part of the delivery complex or as an additional genetic expression vector
  • NCAM Neural Cell Adhesion Molecule
  • SAA Human Serum Amyloid A
  • a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the p53 construct.
  • expression is inducible by tumor necrosis factor.
  • Table 2 illustrates several promoter/inducer combinations:
  • the delivery of an expression vector in a cell may be identified in vitro or in vivo by including a marker in the expression vector
  • the marker would result in an identifiable change to the transfected cell permitting easy identification of expression
  • a drug selection marker aids in cloning and in the selection of transformants
  • enzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic) or chloramphenicol acetyltransferase (CAT) (prokaryotic)
  • CAT chloramphenicol acetyltransferase
  • Immunologic markers also can be employed
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed along with the polynucleotide encoding p53 Further examples of selectable markers are well known to one of skill in the art
  • polyadenylation signal to effect proper polyadenylation of the transcript
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed
  • the inventor has employed the SV40 polyadenylation signal in that it was convenient and known to function well in the target cells employed Also contemplated as an element of the expression construct is a terminator These elements can serve to enhance message levels and to minimize read through from the construct into other sequences
  • the cells contain nucleic acid constructs of the present invention
  • a cell may be identified in vitro or in vivo by including a marker in the expression construct
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct
  • a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers
  • enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed
  • Immunologic markers also can be employed The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product Further examples of selectable markers are well known to one of skill in the art
  • IRES elements internal ribosome binding sites
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites
  • IRES elements from two members of the picanovirus family polio and encephalomyocarditis
  • IRES elements from two members of the picanovirus family Polio and encephalomyocarditis
  • IRES elements can be linked to heterologous open reading frames
  • Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages
  • each open reading frame is accessible to ribosomes for efficient translation
  • Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message
  • Any heterologous open reading frame can be linked to IRES elements This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker
  • the expression construct comprises a virus or engineered construct derived from a viral genome
  • a virus or engineered construct derived from a viral genome
  • expression vectors need not be viral but, instead, may be any plasmid, cosmid or phage construct that is capable of supporting expression of encoded genes in mammalian cells, such as pUC or BluescriptTM plasmid series
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990)
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants
  • the retroviral genome contains three genes - gag, pol, and env - that code for capsid proteins, polymerase enzyme, and envelope components, respectively
  • a sequence found upstream from the gag gene, termed ⁇ functions as a signal for packaging of the genome into virions
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990)
  • a nucleic acid encoding a p53 is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective
  • a packaging cell line containing the gag, pol and env genes but without the LTR and ⁇ components is constructed (Mann et al, 1983)
  • the ⁇ sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988, Temin, 1986, Mann et al, 1983)
  • the media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer Retroviral vectors are able to infect a broad variety of cell types
  • Retroviral vectors are able to infect a broad variety of cell types
  • adenoviruses are double-stranded DNA tumor viruses with genome sizes of approximate 36 kb (Tooza, 1981)
  • adenoviruses As a model system for eukaryotic gene expression, adenoviruses have been widely studied and well characterized, which makes them an attractive system for development of adenovirus as a gene transfer system
  • This group of viruses is easy to grow and manipulate, and exhibit a broad host range in vitro and in vivo
  • adenoviruses are capable of shutting off host protein synthesis, directing cellular machineries to synthesize large quantities of viral proteins, and producing copious amounts of virus
  • the El region of the genome includes El A and E1B which encode proteins responsible for transcription regulation of the viral genome, as well as a few cellular genes E2 expression, including E2A and E2B, allows synthesis of viral replicative functions, e.g. DNA-binding protein, DNA polymerase, and a terminal protein that primes replication E3 gene products prevent cytolysis by cytotoxic T cells and tumor necrosis factor and appear to be important for viral propagation Functions associated with the E4 proteins include DNA replication, late gene expression, and host cell shutoff
  • the late gene products include most of the virion capsid proteins, and these are expressed only after most of the processing of a single primary transcript from the major late promoter has occurred
  • the major late promoter exhibits high efficiency during the late phase of the infection (Stratford-Perricaudet and Perricaudet, 1991)
  • adenovirus-derived vectors offer excellent potential for the substitution of large DNA fragments when used in connection with cell lines such as 293 cells
  • Ad 5 -transformed human embryonic kidney cell lines have been developed to provide the essential viral proteins in trans. The inventor thus reasoned that the characteristics of adenoviruses rendered them good candidates for use in targeting cancer cells in vivo (Grunhaus & Horwitz, 1992)
  • an adenovirus system for delivering foreign proteins to a cell include (i) the ability to substitute relatively large pieces of viral DNA by foreign DNA, (ii) the structural stability of recombinant adenoviruses, (iii) the safety of adenoviral administration to humans, £nd (iv) lack of any known association of adenoviral infection with cancer or malignancies, (v) the ability to obtain high titers of the recombinant virus, and (vi) the high infectivity of Adenovirus
  • adenovirus vectors over retroviruses include the higher levels of gene expression Additionally, adenovirus replication is independent of host gene replication, unlike retroviral sequences Because adenovirus transforming genes in the El region can be readily deleted and still provide efficient expression vectors, oncogenic risk from adenovirus vectors is thought to be negligible (Grunhaus & Horwitz, 1992)
  • adenovirus gene transfer systems are based upon recombinant, engineered adenovirus which is rendered replication-incompetent by deletion of a portion of its genome, such as El, and yet still retains its competency for infection Sequences encoding relatively large foreign proteins can be expressed when additional deletions are made in the adenovirus genome For example, adenoviruses deleted in both El and E3 regions are capable of carrying up to 10 Kb of foreign DNA and can be grown to high titers in 293 cells (Stratford-Perricaudet and Perricaudet, 1991) Surprisingly persistent expression of transgenes following adenoviral infection has also been reported
  • Adenovirus-mediated gene transfer has recently been investigated as a means of mediating gene transfer into eukaryotic cells and into whole animals
  • OTC rare recessive genetic disorder ornithine transcarbamylase
  • viral vectors may be employed as expression constructs in the present invention.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpes viruses may be employed. These viruses offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden,
  • CAT chloramphenicol acetyltransferase
  • the expression vector In order to effect expression of therapeutic constructs according to the present invention, the expression vector must be delivered into a cell As described above, one mechanism for delivery is via viral infection where the expression vector is encapsidated in an infectious virus particle
  • Another embodiment of the invention for transferring a naked DNA expression vector into cells may involve particle bombardment This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987) Several devices for accelerating small particles have been developed One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990) The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads
  • Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al, 1990, Zelenin et al, 1991) This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ DNA encoding a p53, p300 or c-Abl construct may be delivered via this method
  • the expression vector may be entrapped in a liposome
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium Multilamellar liposomes have multiple lipid layers separated by aqueous medium Liposomes form spontaneously when phospholipids are suspended in an excess of aqueous solution The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991) Also contemplated are lipofectamine-DNA complexes
  • Liposome-mediated polynucleotide delivery and expression of foreign DNA in vitro has been very successful Wong et al. (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo,
  • the liposome may be complexed with a hemagglutinating virus (HVJ) This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989)
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991)
  • HMG-1 nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1
  • expression vectors have been successfully employed in transfer and expression of a polynucleotide in vitro and in vivo, then they are applicable for the present invention
  • a bacteriophage promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacteriophage polymerase
  • Receptor-mediated gene targeting vehicles generally consist of two components a cell receptor-specific ligand and a DNA-binding agent Several ligands have been used for receptor-mediated gene transfer The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1993) Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993, Perales et al, 1994) and epidermaj growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085)
  • the delivery vehicle may comprise a ligand and a liposome
  • a ligand and a liposome For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes
  • an adenoviral expression vector also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems, with or without liposomes
  • epidermal growth factor (EGF) may be used as the receptor for mediated delivery of p53, p300 or cAbl construct in many tumor cells that exhibit upregulation of EGF receptor Mannose can be used to target the mannose receptor on liver cells
  • antibodies to CD 5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties
  • gene transfer may more easily be performed under ex vivo conditions
  • Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a polynucleotide into the cells, in vitro, and then the return of the modified cells back into an animal This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues Anderson et al. , U S Patent 5,399,346, and incorporated herein in its entirety, disclose ex vivo therapeutic methods During ex vivo culture, the expression vector can express the p53 construct Finally, the cells may be reintroduced into the original animal, or administered into a distinct animal, in a pharmaceutically acceptable form by any of the means described below
  • a patient presenting a tumor that does not express wild-type p53 will be treated with a combination of p53 and c-Abl and/or p300
  • the patient will be treated with a pharmaceutically acceptable form of the c-Abl and/or p300 genes or gene products, as described above, and in addition, will be treated with a p53 gene or gene product
  • a typical treatment regimen will involve repeated administrations over a six week period During this time, the tumor will be monitored for absence of tumor progression, response or toxicity and the doses adjusted accordingly
  • compositions of the present invention To kill tumor cells or reduce their proliferation, using the methods and compositions of the present invention, one would generally contact a "target" cell with (i) radiation or a chemotherapeutic and (ii) a therapeutic protein or gene These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell
  • This process may involve contacting the cells with c-Abl, p300 and p53 protein or gene, at the same time This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes c-Abl protein or gene and/or p300, and the other includes the p53 protein or gene
  • the protein or gene therapy treatment may precede or follow the standard chemo- or radiotherapy treatment by intervals ranging from minutes to weeks
  • the other agent and an expression construct are applied separately to the cell, one generally would ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell
  • both agents are delivered to a cell in a combined amount effective to kill the cell
  • Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell
  • Such agents and factors include radiation and waves that induce DNA damage such as, ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like
  • chemotherapeutic agents function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein
  • Chemotherapeutic agents contemplated to be of use include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide
  • the invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide
  • the use of cisplatin in combination with a c-Abl or p300 expression construct is particularly preferred as this compound
  • the tumor cells In treating cancer according to the invention, one would contact the tumor cells with an agent in addition to the expression construct This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, ⁇ -rays or even microwaves Alternatively, the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin
  • the agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with an expression construct, as described above
  • Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m 2 for 5 days every three weeks for a total of three courses Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally
  • chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m 2 at 21 day intervals for adriamycin, to 35-50 mg/m 2 for etoposide intravenously or double the intravenous dose orally
  • agents that have undergone extensive testing and are readily available
  • agents such as 5-fluorouracil (5-FU)
  • 5-FU 5-fluorouracil
  • 5- FU is preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells
  • 5- FU is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used
  • ⁇ -rays X-rays
  • UV-irradiation UV-irradiation
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells
  • Aqueous compositions of the present invention will have an effective amount of a compound that increases the expression of wild-type p53, for example p300 or c-Abl gene products or p300 or c-Abl gene constructs. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Also included in various embodiments of the present invention are standard chemotherapeutics in their various pharmaceutical forms.
  • phrases "pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or human, as appropriate.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in the therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-cancer agents, can also be incorporated into the compositions.
  • other pharmaceutically acceptable forms include, e.g. , tablets or other solids for oral administration; time release capsules; and any other form currently used, including cremes, lotions, mouthwashes, inhalants and the like.
  • the active compounds of the present invention will often be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • the preparation of an aqueous composition that contains a compound or compounds that increase the expression of wild-type p53 will be known to those of skill in the art in light of the present disclosure.
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared, and the preparations can also be emulsified
  • compounds may be injected directly into a tumor
  • the route of delivery is by a slow drip into the circulatory system of the patient
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, formulations including sesame oil, peanut oil or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions
  • the form must be sterile and must be fluid to the extent that easy syringability exists It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi
  • the active compounds may be formulated into a composition in a neutral or salt form
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the therapeutic formulations of the invention could also be prepared in forms suitable for topical administration, such as in cremes and lotions. These forms may be used for treating skin-associated diseases, such as various sarcomas.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, with even drug release capsules and the like being employable.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure
  • one dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580)
  • kits All the essential materials and reagents required for determining wild-type p53 in a sample or for increasing the expression of wild-type p53 using c-Abl and p300 in tumor cells may be assembled together in a kit
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred
  • the kit may contain materials for PCR analyses, such primers, buffers and appropriate solvents Alternatively, if the detection is via immunologic means, the kit may contain antibodies directed to the p53, secondary antibodies that binding primary antibodies, labels or signal generating compounds (either conjugated or unconjugated) and various reagents for the generation and detection of signals
  • an inducer of wild-type p53 expression alone or in combination with, a expression vectors may be formulated into a single or separate pharmaceutically acceptable syringeable composition
  • the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit
  • the components of these kits may also be provided in dried or lyophilized forms When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent It is envisioned that the solvent also may be provided in another container means
  • the kits of the invention may also include an instruction sheet defining administration of wild-type p53 and c-Abl gene therapy agents, or explaining the assays for determining p53 levels in samples
  • kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained Irrespective of the number or type of containers, the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal
  • an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle
  • Other instrumentation includes devices that permit the reading or monitoring of reactions in vitro
  • c-Abl The product of the c-Abl gene is a non-receptor tyrosine kinase that is localized to the nucleus and cytoplasm Ionizing radiation (IR) activates c-Abl Similar results were obtained with the alkylating agents cisplatinum and mitomycin C Cells deficient in c-Abl fail to activate Jun kinase (JNK/SAP kinase) following IR or alkylating agent exposure and that reconstitution of c-Abl in these cells restores that response In contrast, the stress response to tumor necrosis factor is stimulated by a c-Abl-independent mechanism
  • c-Abl contains actin binding and DNA binding domains
  • Rh retinoblastoma
  • MCF-7 cells were co- transfected with a construct containing the luciferase gene driven by a p53 enhancer from the MDM2 promoter (Barak et al, 1994), and vectors expressing wild-type c-Abl, a kinase-inactive K(290)R mutant (Sawyers et al, 1994), or a kinase-active mutant, designated ⁇ Pro 4 which is deleted at the p53-binding domain (Goga et al, 1995)
  • Co- transfections of the reporter with wild-type or kinase-inactive c-Abl (K-R), but not c- Abl ⁇ Pro 4 resulted in induction of luciferase activity (FIG 1A)
  • c-Abl expression had no detectable effect on activation of an SV40-luciferase construct (FIG la) As transcription of p21 is regulated by
  • MCF-7 cells were prepared to stably express the dominant-negative c-Abl (K-R) 2 , which effectively inhibits the increase in c-Abl kinase activity induced by ionizing radiation control cells (FIG 2A)
  • Irradiation of MCF-7/pSR or MCF-7/c-Abl (K-R) cells caused an increase in p53, p21 and GADD45, but not in c-Myc (which is not dependent on p53) (FIG 2B)
  • FIG 2B Irradiation of MCF-7/pSR or MCF-7/c-Abl (K-R) cells caused an increase in p53, p21 and GADD45, but not in c-Myc (which is not dependent on p53) (FIG 2B)
  • FIG. 3C Wild-type MEFs after 5 Gy radiation had an S-phase population that was 45% of that for untreated cells (FIG 3D) By contrast, irradiated abl ' MEFs had more than 70%> of cells in S phase compared with controls (FIG 3D) Exposure of the wild-type and abl' ' MEFs to 20 Gy radiation partially inhibited arrest in Gl in the abl ' cells, indicating that the c-Abl kinase function is necessary for radiation-induced Gl arrest
  • mutant c-Abl may prevent Gl arrest in response to DNA damage by inactivating Rb But as irradiated cells expressing c-Abl (K-R) do not arrest in Gl because Cdk2 is not inhibited, the effect probably occurs upstream of Rb
  • K-R irradiated cells expressing c-Abl
  • the present inventors have demonstrated that c-Abl functions in the cellular response to DNA damage through p53-dependent pathways, confirmed by the failure of the c-Abl ⁇ Pro 4 mutant to bind p53 and downregulate Cdk2 and arrest growth It has also been shown that the kinase-defective c-Abl (K-R) mutant induces p53 transactivation but no Gl arrest although
  • RESULTS Cell Culture - MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (Sigma) containing 10%> heat-inactivated fetal bovine serum (Sigma), 2 mM L-glutamine, 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin Null pSR ⁇ MSVtKneo, pSR MSVc-AblK(290)-RtKNeo (Sawyers et al, 1994), or E6Neo (Scheffner et al, 1990) vectors were stably introduced into cells by LipofectAMINE (Life
  • Immunoprec ⁇ itations and Immunoblot Analysis were prepared as described (Yuan et al, 1996) and incubated with rabbit anti-Cdk2 (sc-163, Santa Cruz Biotechnology, San Diego, CA), mouse anti-c-Abl (Ab-3, Oncogene Science, Cambridge, MA), mouse anti-p53 (Ab-6, Oncogene Science) antibodies for 6-12 h at 4°C and then for 60 min with protein A-Sepharose
  • 10 ⁇ g/sample of rabbit anti-mouse IgG was added and incubated for 60 min before the addition of protein A beads
  • the immune complexes were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters and analyzed by immunoblotting using an ECL (Amersham Corp ) detection system
  • recombinant SHPTP1 protein 100 ⁇ g/ml was used as substrate (Kharbanda et al, 1996), and the reaction was incubated for 30 min at 30°C
  • ara-C induces an interaction between c-Abl and p53 in vivo
  • the inventors also prepared cells that express the human papillomavirus E6 protein to promote degradation of p53 (Scheffner et al, 1990)
  • the MCF-7/E6 cells responded to ara-C with induction of c-Abl activity
  • MMS exposure resulted in increased c-Abl activity in the MCF-7/pSR and MCF-7/E6 cells, but not the MCF-7/c-Abl(K-R) cells
  • MMS increased binding of c-Abl and p53 in MCF-7/pSR and MCF-7/c-Abl(K-R), but not MCF-7/E6, cells
  • MMS also induced the expression of p53 and p21 in only the MCF-7/pSR and MCF-7/c-Abl(K-R) cells
  • the MMS-induced increases in p21 levels were associated with binding of p21 to Cdk2
  • Cdk2 activity was down-regulated in MMS-treated MCF-7/pSR cells, there was little effect of MMS on Cdk2 activity in the MCF-7/c-Abl(K-R) cells
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention.
  • Gazit, A Yaish, P , etal, JMedChem, 32(10) 2344-52, 1989
  • Temin "Retrovirus vectors for gene transfer Efficient integration into and expression of exogenous DNA in vertebrate cell genome," In. Kucherlapati R, ed Gene transfer

Abstract

La présente invention, qui concerne des thérapies anticancéreuses, concerne plus particulièrement l'induction d'une apoptose des cellules cancéreuses à la suite d'une thérapie par c-Abl et p300 permettant de contrôler le niveau des p53 de type sauvage. L'invention concerne également un traitement associé consistant à apporter, aux cellules déficientes en p53, des c-Abl, p300 et p53 de type sauvage de façon à induire des réponses apoptotiques.
PCT/US1997/012498 1996-07-18 1997-07-18 Procedes et compositions de modulation du taux de croissance WO1998003195A1 (fr)

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