WO2007134142A2 - Procédés et compositions d'identification et d'utilisation de protéines régulées par p21 - Google Patents

Procédés et compositions d'identification et d'utilisation de protéines régulées par p21 Download PDF

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WO2007134142A2
WO2007134142A2 PCT/US2007/068619 US2007068619W WO2007134142A2 WO 2007134142 A2 WO2007134142 A2 WO 2007134142A2 US 2007068619 W US2007068619 W US 2007068619W WO 2007134142 A2 WO2007134142 A2 WO 2007134142A2
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cell
regulated protein
protein
cancer
assessing
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WO2007134142A3 (fr
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William Gallagher
Darran O'connor
Caroline Currid
Igor Roninson
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University College Dublin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity

Definitions

  • the present disclosure relates to compositions and methods useful for modulating cellular senescence.
  • Cellular senescence is the phenomenon where cells lose the ability to divide. It can be activated spontaneously in stressful cellular environments or induced during therapeutic intervention. The response of tumor cells to this senescent event is complex, poorly understood and can have significant consequences for prognosis and subsequent cancer treatment [I].
  • Cells displaying a senescent phenotype are not responsive to mitogens, but remain in a dynamic state, serving as pools of secreted factors with tumor-inhibiting and promoting activities [2, 3, 4]. Whether these reservoirs promote a favorable environment for tumor progression is thought to be mediated in part by the expression of cyclin-dependent kinase inhibitors, such as p 2l Wafl/CiP1/Sdi I (hereon referred to as ⁇ 21).
  • p21 plays a key role in preserving the integrity of the genome. However, p21 may also mediate oncogenic effects and as such contribute to tumor progression and promote carcinogenesis [3], Such paradoxical duality in terms of activity is not without precedence among central regulators of the cell cycle.
  • E2F regulates the expression of proliferation- dependant genes, thereby acting as an oncogene; however, it also displays noted pro- apoptotic activity [5], p21 classically acts downstream of p53 during the DNA damage response as a broadspecificity inhibitor of cyclin-dependent kinases (CDKs) [6].
  • CDKs cyclin-dependent kinases
  • p21 Inhibition of CDKs by p21 results in the hypophosphorylation of Rb, which inhibits the activity of E2F transcription factor complexes and leads to cell cycle arrest [7].
  • p21 can also bind and sequester PCNA, which is required by DNA polymerase ⁇ , thus blocking DNA replication and affecting growth arrest [8].
  • p21 can bind directly to c-Myc, STAT3 and E2F1, negatively modulating their activity [9, 10, 11].
  • p21 is also found in transcription complexes positively influencing the transcriptional activity of NF- ⁇ B and C/EBP [12, 13], both of which are implicated in cell growth and differentiation.
  • p21 also selectively controls the transcriptional activity of Estrogen receptor ⁇ in a histone acetyltransferase-dependent manner [14] and stimulates transactivation by p300 and CBP through the inhibition of a potent transcriptional repression domain (CRD 1 ) present in both proteins [15].
  • p21 has also been implicated in mediating the proliferative effect of IGF-I and Ets-1 [16, 17] and increased cytosolic p21 has been shown to result in cell cycle progression in vascular smooth muscle cells [18]. Indeed, within the p21 -expressing cell itself, subcellular localization is a key determinant of function.
  • Akt Phosphorylation by Akt causes the stabilization and cytoplasmic accumulation of p21 [19, 20], leading to a proposed "gain of function" anti- apoptotic activity when retained in the cytoplasm.
  • p21 was found to be predominantly cytoplasmic in many breast tumors, a feature associated with poor prognosis [21].
  • p21/cyclin/CDK complexes that are catalytically active have been identified [22] and p21 inhibition with antisense oligonucleotides in vascular smooth muscle cells results in loss of assembly of cyclin D complexes and inhibition of growth factor- stimulated proliferation [23], Also, the mTor inhibitor, RADOOl, has been shown to sensitize tumor cells to cisplatin-mediated cell death through the inhibition of p21, further supporting an anti-apoptotic function [24]. Finally, p21 overexpression is an early event in the genesis of some cancers [25] and also occurs in response to mitogenic stimulation [26].
  • p21 inhibits the expression of a wide variety of genes associated with mitosis, DNA replication and proliferation, but also induces the expression of genes implicated in a number of age-related disorders, including cancer, Alzheimer's disease and amyloidosis [27].
  • Several p21-induced genes encode secreted proteins with known paracrine effects on mitosis and apoptosis.
  • conditioned medium from cells in which p21 was induced was found to have anti-apoptotic and mitogenic activity [27].
  • p21 An anti-apoptotic function for p21 is consistent with the observation that it is cleaved by caspase 3 at the onset of apoptosis [28], indeed p21 can form a complex with procaspase 3, an interaction that provides resistance to Fas-mediated cell death [29].
  • p21 -deficient tumors also display increased radiosensitivity [30] and p21 -disrupted colon cancer cells have a higher sensitivity to cisplatin and nitrogen mustard [31].
  • p21 inhibits pro- apoptotic signalling kinases, such as SAPK and ASKl [32, 33], as well as its inhibitory effect on transcription factors involved in stimulating programmed cell death, such as E2F and Myc [11, 34].
  • alterations in the levels of some p21w a ⁇ -inducible genes has been found to be mediated, in part, at the level of transcription, as the inhibitory and stimulatory effects of p21wa ⁇ can be reproduced through the use of promoter-reporter constructs (Zhu et al, 2002; Poole et al, 2004).
  • the inhibitory effects on gene transcription of p21wafi expression have shown to be mediated through E2F, c-myc and STAT complexes [34]. Indeed, many of the genes inhibited by p21wa ⁇ -induction [27] were found to have E2F sites in their promoters.
  • the invention provides a method for inhibiting apoptosis in a cell (e.g., a human or mammalian cell) by administering to said cell, an effective amount of a p21 -regulated protein.
  • a cell e.g., a human or mammalian cell
  • Suitable methods for administering a p21 -regulated protein to a cell include, for example, contacting the cell with a p21 -regulated protein and introducing into the cell, or neighboring cells (and subsequently inducing the transcription thereof) a nucleic acid encoding a p21 -regulated protein, wherein the nucleic acid is operably linked to a transcriptional promoter.
  • the cells are at increased risk for undergoing apoptosis.
  • the increased risk for apoptosis may be a result of any cause including, for example, exposure to ionizing radiation, a chemotherapeutic or radiomimetic agent, a toxin, hypoxia, traumatic injury, neurodegenerative disease, or an immunological reaction.
  • the invention provides a method for inducing apoptosis in a cell, said method comprising reducing the biological activity of a p21 -regulated protein in the cell, or neighboring cells.
  • Suitable methods for reducing p21 -regulated protein biological activity include, for example, administering, or immunodepletion with, an antibody that specifically binds to the p21-regulated protein, or introducing into the cell, or neighboring cells (and subsequently inducing the transcription thereof) a nucleic acid encoding a sequence complementary to the nucleic acid sequence of a p21 -regulated protein (e.g. antisense or RNAi), wherein said nucleic acid is operably linked to a transcriptional promoter.
  • the nucleic acid comprises at least 12 nucleotides, although other amounts are contemplated, by way of non-limiting example, greater than 50 is contemplated within the scope of the present disclosure.
  • the cell is a cancer cell including, for example cells of a breast cancer, ovarian cancer, skin cancer, lung cancer, cervical cancer, colon cancer, bladder cancer, prostate cancer and other known cancers affecting the human body.
  • the invention provides a method for assessing the effectiveness and outcomes of an anti-neoplastic treatment in a patient.
  • the method comprises the steps of: (i) assessing the level of a p21 -regulated protein prior to an anti-neoplastic treatment regimen; (ii) assessing the level of the p21 -regulated protein following the anti-neoplastic treatment regimen; and (iii) comparing the results of the assessing step (U) with the results of the assessing step (i), wherein a decrease in the level of the p21 -regulated protein is indicative of efficacy, and an increase in the level of the p21 -regulated protein is indicative of residual cellular senescence and the potential for tumor recurrence.
  • Suitable methods for assessing the levels of the p21 -regulated protein include, for example, assessing the RNA levels and directly assessing the protein levels.
  • Particularly useful p21 -regulated proteins for any of the foregoing methods include, for example, ⁇ -2-microglobulin, cystat ⁇ n C, and pro-platelet basic protein.
  • FIG. 3 Enrichment and identification scheme. Optimal conditions from the profiling study carried out previously were used to enrich for the peaks found at 10.2, 11.7 and 13.4 kD in the p21 -induced cells. Following Q Hyper D fractionation samples were run subject to IDPAGE where enrichment allowed subsequent enzymatic digestion and passive elution for identification. Subsequent to identification, immunoassay and western blotting provided reconf ⁇ rmation.
  • FIG. 4 Results of enrichment process.
  • the top spectrum of (a) shows the 72hr induced sample pre-Hyper Q fractionation where 11.7 and 13.4 peaks are evident.
  • the lower spectrum of (a) shows this sample post-Q fractionation. Many contaminant peaks have been removed and the 11.7 and 13.4 peaks have been enriched (as measured by an increase in intensity).
  • This sample was further purified on reverse-phase beads with (b) clearly showing the enrichment of the 3 peaks, (c) Reverse-phase bead fraction was run on ID SDS-PAGE gel (Lane 1). Molecular weights are indicated in Lane 2.
  • MS/MS and SELDI-TOF identification (a) MS/MS analysis of ion with m/z of 1724 which was identified as a tryptic fragment of PPBP. This peptide was sequenced and matched pro-platelet basic factor, (b) The ion was fragmented by collision-induced dissociation and the MS/MS spectrum was submitted to MASCOT search tool for identification. The ion was identified as a fragment of pro-platelet basic factor with probability-based Mowse score of 47.5 (>30 indicates identity), (c) SELDI-TOF tryptic digest of 11.7 band in Figure 4 (c). (d) (i) Amino acid sequence of ⁇ -2 -microglobulin.
  • On-chip immunoassay demonstrates accumulation in induced CCM compared to control CCM.
  • Polyclonal antibodies were coupled to pre-activated PS20 arrays with subsequent blocking, uninduced and induced CCM was incubated followed by washing and application of SPA.
  • Arrays were read in PBS-IIc with laser settings of 180, detector sensitivity 8.
  • Figure 8 Increased expression of extracellular and intracellular Cystatin C in p21- induced cells. 15 ⁇ g of total protein within conditioned medium (a) and whole cell lysates (b) obtained from induced and non-induced HT1080 p21-9 cells was subjected to SDS-PAGE and immobilised to PVDF and probed with a 1:500 dilution of mouse monoclonal anti- Cystatin C antibody. An anti-BSA antibody and anti-actin antibody were used as loading controls for CCM and cell lysates respectively.
  • Figure 9 Increased expression of extracellular and intracellular ⁇ -2- microglobulin in p21-induced cells. Immunoblot analysis of extracellular and intracellular ⁇ -2-microglobulin.
  • Figure 10 Increased expression of intracellular PPBP in p21-induced human HT1080 fibrosarcoma cells. 15 ⁇ g of total protein in whole cell lysates obtained from induced and non-induced HT1080 p21-9 cells was subjected to SDS-PAGE, and then probed with 1:1000 mouse monoclonal anti-PPBP antibody. An anti-actin antibody (AbCam) was used as loading controls for cell lysates.
  • AbCam anti-actin antibody
  • CMlO, QlO and IMAC surfaces were increased or decreased in the CCM of p21 -induced cells when compared to equivalent uninduced cells.
  • Three of the peaks that were reproducibly increased in the p21 -induced CCM were identified by SELDI-MS fingerprinting and tandem MS analysis and their identity was validated by onchip immunoassay and Western blot analysis.
  • ⁇ -2-microblobulin and cystatin C have been previously implicated in amyloid fibril formation [42, 43].
  • cystatin C is an endogenous cysteine proteinase inhibitor.
  • Monomeric cystatin C is ubiquituous in human body fluids, being particularly abundant in cerebrospinal fluid, seminal plasma, and milk, and has been previously implicated as a modulator of proliferation.
  • the glycosylated form, CCg acts as an autocrine/paracrine factor required for the mitogenic activity of fibroblast growth factor 2 (FGF-2) on neural stem cells [44].
  • FGF-2 fibroblast growth factor 2
  • Treatment with chicken cystatin C results in growth stimulation of 3T3 fibroblasts as measured by radiolabeled thymidine incorporation [45] and secretion of cystatin C by rat glomerular megengial cells creates an autocrine feedback loop with in vitro growth promoting activity [46].
  • Cy sC -/- homozygous null mice also show decreased metastatic spread of B 16-Fl 0 melanoma cells [47]. Compared with wild type mice, CysC -/- mice showed a seven-fold decrease in metastatic colonies. ⁇ -2-microglobulin is constitutively secreted and found in normal serum. Its levels are elevated in urine in renal tubular disfunction and it has been implicated in a number of angiopathies. It is the most widely studied low-molecular-weight protein in end-stage renal disease and has been shown to cause dialysis-related amyloidosis, through its retention when renal function fails, its deposition in tissues, its aggregation into fibrils, and its ability to become glycosylated [48].
  • ⁇ -2-microglobulin levels in the CCM of human prostate cancer cell lines and primary cultures derived from metastasis were higher than normal, through increased shedding from MHC I molecules [49].
  • ⁇ - 2-microglobulin has been found to induce apoptosis in leukemia cell lines [50] and it suppresses the growth of isolated myeloma tumors and myeloma cell lines through inducing apoptosis and cell cycle arrest [51].
  • Pro-platelet basic protein (“PPBP”; also known as NAP-2 or CXCL7) is a member of the ELR+ CXC family of chemokines and is known as a major granular protein of platelets [52]. It is maintained as an inactive precursor and requires proteolytic cleavage to obtain receptor affinity [53]. PPBP (upon activation) binds to the CXCR2 receptor and is a powerful inducer of neutrophil adhesion and transmigration [54]. ELR+ CXC chemokines have been shown to be potent mediators of angiogenesis, whereas CXC chemokines lacking the ELR motif were found to be angiostatic [55].
  • RT-PCR analysis also indicated that the levels of cystatin C mRNA increase in response to p21 induction (data not shown), and based on earlier transcriptomic studies it would appear that there is a degree of de novo protein synthesis involved in the accumulation of these three proteins. However, there may also be intracellular accumulation from earlier time points that we have not examined yet.
  • p21 -induction leads to a secretory phenotype whereby neighboring cell growth is affected, is based on the evidence that the CCM taken from p21-overexpressing cells had mitogenic and anti-angiogenic activity on 2 cell line models.
  • CCM from induced cells increased radiolabeled thymidine incorporation three-fold in HS 15T fibroblast fibrosarcoma cells [27].
  • induced CCM decreased apoptosis in the mouse embryo fibroblast cell line C8 [27].
  • the three secreted factors that we have identified in this study have all been shown previously to have growth promoting effects and as such may contribute to this observed anti-apoptotic and mitogenic paracrine activity of p21 -expressing cells.
  • Table I summarizes the number of peaks identified by profiling CCM on the three different ProteinChip arrays (CMlO, QlO and IMAC).
  • Samples profiled on the ion-exchange array surfaces (CMlO and QlO) produced the highest numbers of peaks, with a total of 340 peaks either increased or decreased in the CCM from p21 -induced cells.
  • CMlO and QlO ion-exchange array surfaces
  • IMAC surfaces metal affinity arrays
  • Two metal ions were loaded on the IMAC surfaces (nickel and copper). Proteins with exposed histidine, tryptophan, or cysteine typically bind to metals immobilised on this array surface.
  • Nickel is mainly used to bind proteins with multiple histidine residues [35], while copper is known to bind proteins that have a mitogenic effect [36].
  • Profiling the CCM on copper surfaces produced more peaks than the nickeladsorbed arrays (92 peaks as compared to 52 peaks, Table I).
  • Figure Ia shows a representative spectrum obtained on a CMlO array, binding at pH4, for these three putative p21 -responsive proteins. Also shown is a gel representation view of their expression levels in CCM from induced versus non-induced cells ( Figure Ib) and the Biomarker Wizard (Ciphergen Biosystems) determination of their statistical significance ( Figure Ic).
  • Spectra were analyzed individually in the mid- and high mass ranges. Prior to statistical analysis spectra were manipulated as follows: Using Ciphergen ProteinChip software the baseline was subtracted, noise values were calculated from the minimum of 2500Da (medium mass range) and lOOOODa (high mass region) to 100% spectrum size. All spectra were then normalized using total ion current normalization with any outliers removed before proceeding to statistical analysis.
  • Samples were then assigned the sample groups of "non-induced” or "induced”, regardless of 48 or 72 hr collection interval i.e. 4 non-induced and 4 induced samples.
  • Example 2 Enrichment and Identification of the 10.2, 11.7, 13.4 kDa Proteins.
  • the enriched 3 -day induced CCM sample was separated by ID-PAGE and bands at 10.2, 11.7 and 13.4 kDa were excised and subject to enzymatic digestion.
  • Identification of the 10.2 kDa peak was carried out by sequencing tryptic digest peptides prepared as above, by interfacing the SELDI source to a hybrid quadrupole-TOF (Q-STAR) MS.
  • the MS/MS spectrum was submitted to the Mascot search tool which returned proplatelet basic factor (UniProt entry P02775), with a probability-based Mowse score of 47.5 (>54 indicates identity) ( Figures 5b, c, d).
  • the HT- 1080 human fibrosarcoma cell line p21-9 that carries p21 in an isopropy- ⁇ - D-thiogalactoside (IPTG)-inducible stably integrated transgene [27] was propagated in DMEM supplemented with 10% FC2 serum (HyClone, Logan, UT, USA).
  • CCM was prepared by plating 1x106 HT-1080 p21-9 cells per 15cm plate, adding 50 ⁇ M IPTG the following day and replacing the medium 3 days later with medium containing IPTG and 0.5% FC2. Conditioned medium was then collected at 2 and 3 days post-replacement. Control IPTG-free conditioned medium containing 0.5% FC2 was collected from untreated cells grown to the same density as the IPTG-treated cells.
  • IMAC30 immobilised metal ion chromatography surface a nitriloacetic acid surface for transition metal binding
  • CMlO carboxymethyl weak cation exchange
  • QlO strong anion exchanger
  • CMlO arrays were equilibrated with 10OmM sodium acetate buffers of varying pH range (pH 3-7) with 0.5% Triton X-100; QlO arrays with 10OmM Tris-HCl of varying pH range (pH6-9) with 0.5% Triton X-100; IMAC arrays with 10OmM copper sulphate/nickel sulphate buffers, in a bioprocessor module (Ciphergen Biosystems) on a Micromix orbital shaker.
  • CMlO arrays were equilibrated with 10OmM sodium acetate buffers of varying pH range (pH 3-7) with 0.5% Triton X-100; QlO arrays with 10OmM Tris-HCl of varying pH range (pH6-9) with 0.5% Triton X-100; IMAC arrays with 10OmM copper sulphate/nickel sulphate buffers, in a bioprocessor module (Ciphergen Biosystem
  • Ions were extracted using a 3kV ion extraction pulse and accelerated to a final velocity using 2OkV of acceleration potential.
  • Time-lag focusing delay times were set to 750ns for mid-mass scans and 1420ns for high-mass scans.
  • CCM from induced and non-induced HT1080 p21-9 cells was adjusted with IM Tris- HCl to give a final concentration of 10OmM Tris-HCl, pH 9.0 and then bound to Q Ceramic HyperD F columns (Ciphergen Biosystems) (pre-equilibrated in above buffer). Flow- through fractions were collected at pH 9.0, followed by elutions at pH 4.0 and an organic acetonitrile wash. pH 9.0 fractions were purified further on reverse phase beads (Polymer Labs Ltd., Shropshire, UK) followed by elution of the enriched biomarkers using a discontinuous gradient (10%, 50%, 60%) of acetonitrile. The latter fractions were combined and speed vacuum dried.
  • Identification of PPBP was carried out by sequencing tryptic digest peptides prepared as above by interfacing the SELDI source to a hybrid quadrupole-TOF (QSTAR) MS (Applied Biosystems/MDS SCIEX, Foster City, CA, USA).
  • gel pieces were washed with 50% acetonitri.e/50mM ammonium bicarbonate then dehydrated with 100% acetonitrile.
  • the gel pieces were heated to 5O 0 C before addition of a 45% formic acid/30% acetonitrile/ 10% isopropanol solution and incubation in a sonicating waterbath for 30 min at room temperature. Gel pieces were then incubated for 3hr at room temperature.
  • the passively eluted material was then spotted on an NP-20 array followed by addition of saturated SPA.
  • mice monoclonal anti-human ⁇ -2-microgIobuIin (1 mg/ml; 2 ⁇ l per spot; R & D Systems Inc) in PBS pH 8.0 were added to the spots of a pre- activated PS-20 array.
  • the extent of non-specific binding was tested using a bovine IgG antibody (Ciphergen Biosystems) instead of the specific antibody.
  • the arrays were incubated in a humidity chamber for lhr at room temperature to allow covalent binding of the antibody.
  • Antibody solution was removed and ethanolamine (0.5M; 4 ⁇ l per spot) at pH 8.0 was added to block remaining activated coupling sites and incubated for 20min at room temperature.
  • the whole array was then washed twice in 10ml PBS for 5min and then transferred to a bioprocessor module.
  • Antigen solution 75 ⁇ g conditioned medium from induced and noninduced HT 1080 ⁇ 21-9 cells and 50 ⁇ l PBS
  • the solution was removed and the spots washed with PBS pH 7.2/0.05% Triton X-IOO, PBS without Triton X-100 and then finally with doubly distilled water.
  • SPA was applied to each spot, the array was air-dried and then analyzed by SELDI-MS.
  • CCM and whole cell lysates (15 ⁇ g of each sample in both cases) from induced and noninduced HT 1080 p21-9 cells were loaded on 10% NuPage Bis-Tris gels (Invitrogen) and run under non-reducing conditions in 2-morpholinoethanesulfonic acid (MES) SDS buffer. Resolved proteins were transferred to polyvinylidenedifluoride (PVDF) membrane (BioRad, Hurcules, CA, USA) using a Mini-Protean II blotting system. Proteins were detected by incubation with primary antibodies in 5% non-fat dried milk in TBS containing 0.1% Tween-20, followed by incubation with a 1:7500 dilution of appropriate secondary antibody (Promega).
  • PVDF polyvinylidenedifluoride

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Abstract

La présente invention concerne un procédé d'identification de protéines régulées par p21 pouvant être utilisées pour induire des effets cellulaires anti-apoptotiques et/ou mitogènes, paracrines et dans la cellule d'expression de p21 elle-même. Trois protéines libérées à partir des cellules de fibrosarcome humaines HT-1800 présentant une expression inductible du gène p21 ont été identifiées: la β 2 microglobuline, la cystatine C, et la protéine basique d'activation plaquettaire. L'invention concerne également des compositions thérapeutiques, des procédés d'utilisation, et des procédés d'identification de protéines régulées par p21.
PCT/US2007/068619 2006-05-11 2007-05-10 Procédés et compositions d'identification et d'utilisation de protéines régulées par p21 Ceased WO2007134142A2 (fr)

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