WO2009080852A1 - Rplp1-based antitumour compounds - Google Patents

Abstract

The invention relates to identifying the ability of the ribosomal protein Rplp1, a member of the P group of ribosomal proteins, to avoid the replicative senescence of primary cells. The invention relates to the use of Rplp1 for the treatment of diseases associated with an unwanted cellular senescence, as well as Rplp1 inhibitor compounds (antisense oligonucleotides, ribozymes and similar) for the treatment of diseases associated with an unwanted cell proliferation.

Description

 RPLPL-BASED ANTITUMORIAL COMPOUNDS

TECHNICAL FIELD OF THE INVENTION

The invention falls within the field of cell proliferation and, more specifically, in the field of compounds capable of promoting cell proliferation or preventing the entry of a cell into replicative senescence as well as in the field of compounds capable of inhibiting proliferation. unwanted cell phone

BACKGROUND OF THE INVENTION

Somatic cells in primary culture show a very limited proliferative capacity (Hayflick, L. and Moorhead, PS (1961) Exp. CeIl Res. 25, 585-621) which manifests itself in said cells, after a limited number of cycles of division, irreversibly stop their growth by entering what is known as a state of replicative senescence.

Cellular senescence seems to be involved in multiple pathological conditions in the elderly that could be relieved by extending the half-life of the cells in situ. Examples of such conditions include skin atrophy due to loss of homeostasis of the extracellular matrix in fibroblasts of the dermis, degeneration of the macula caused by the accumulation of lipofuscin and decreased expression of a neuronal survival factor in epithelial cells of the retina and atherosclerosis caused by the loss of the proliferative capacity of endothelial cells and the overexpression of hypertensive and prothrombotic factors in said cells.

Achieving an increase in cell half-life may also have in vitro applications. Thus, normal diploid cells could be used instead of tumor cell lines established in biochemical and pathological studies on growth and differentiation. Normal cells with an increased half-life could be used for the production of products of therapeutic utility and expanded cell populations Normal or genetically modified rejuvenated could be used for gene therapy anthologous or allogeneic for. Therefore, the ability to extend the half-life of cells maintained their diploid character, the growth characteristics and the pattern of normal cell expression has important implications for research in biology, the pharmaceutical industry and medicine.

Therefore, there is an interest in the identification of compounds that allow to inhibit cell senescence or promote the proliferation of cells that would normally reach replicative senescence.

Thus, a whole series of methods have been described in which the stimulation of cell proliferation is achieved by the activation of telomerase. For example, WO0031238 describes methods to increase cell proliferative capacity by using a combination of a compound that activates telomerase reversibly and a compound that reversibly inactivates the Rb / INK4 pathway or the p53 pathway; WO9935243 describes methods to increase cell proliferative capacity by ectopic expression of EST2, the catalytic subunit of telomerase; WO06066247 describes methods to increase cellular replicative capacity by increasing hTERT activity, WO05000245 describes the ability of astragenol glycosides to promote cell proliferation by activating telomerase activity; and WO0061617A describes compositions capable of promoting cell proliferation through the use of fusion proteins formed by the catalytic subunit of telomerase and a peptide capable of promoting translocation through biological membranes of compounds that are bound to it ( for example herpesvirus VP22 and TAT).

Other methods to promote cell proliferation or to inhibit replicative senescence not based on directly promoting telomerase activity include the use of migratory inhibition factor (MIF), as described in WO9942578, which is capable of increasing the proliferative capacity of a cell. since it is able to avoid the stop of the cell cycle mediated by p53. Other methods to prevent the entry of cells into replicative senescence include the use of specific culture media such as those described in WO07115223 and WO07107303, the use of SIRTl protein inhibitors as described in US2007160586, or by the increased intracellular expression of MOT-2, as described in US2003170783.

On the other hand, methods for inhibiting the replicative senescence of keratinocytes by using compositions comprising retonoic acid including all trans retinoic acid, 3,4-didehydroretinoic acid and 9-cis retinoic acid have been described in US6566399. Additionally, in WO2004007720 methods have been described to inhibit the proliferative capacity of tumor cells by the use of inhibitors of the expression and / or activity of glycolytic enzyme inhibitors.

Recently it has been revealed that ribosomal proteins may also be involved in modulating cell proliferation processes. The biogenesis of ribosomes and translation control are essential cellular processes, which are governed at various levels. After receiving the mitogenic signals, the cells activate transcription factors to ultimately induce proliferation. The ribosome is decisive for this process, since it is the factory where protein production takes place. Ribosomal proteins are central proteins that assemble with the rRNA in the nucleolus to form the small and large subunits, 4OS and 6OS, of the ribosome (8). More than 80 proteins actively cooperate in this process by stabilizing both subunits of the ribosome and fixing the appropriate ribosomal function. The specific role of each ribosomal protein during ribosome biogenesis and protein synthesis is, however, uncertain, and the possible functions that they may have in other cellular processes have not been fully defined.

In addition, it has been suggested that the ribosome could transduce cancer because alterations in ribosomal proteins appear to be consistent with oncogenesis (Ruggero D and Pandolfi PP, Nat. Rev. Cancer 2003; 3: 179-192 and Welsh JB et al. , Proc. Nati. Acad. Sci USA 2001; 98: 1176-1181). The increase in several ribosomal proteins in oncogenic cell lines and human cancers detected at mRNA level in large-scale DNA chip analysis (Chiao PJ et al., Mol. Carcinog. 1992; 5: 219-231 and Zhang L. et al., Science 1997, 276: 1268-1272). Some reports have correlated this increase with a poor prognosis and an advanced state of the disease (Bee, A. et al, Clin Cancer Res., 2006; 12: 2061-2065; Huang XP. Et al. Gene 2006, 366: 161 -168 and Kobayashi T, Int. J. Mol. Med. 2006; 18: 161-170). Therefore, ribosomal proteins are markers for general cell transformation, they are overexpressed as individual proteins and multiple groups of proteins not only in solid tumors but also in leukemia cells (Ferrari S. et al., Cancer Res 1992; 52: 11-16. And Uechi T. et al., Genomics 2001; 72: 223-230). A widely held notion that ribosomal proteins increase as a result of cell proliferation has been recognized for decades. However, in recent years an increasing number of reports have described additional functions for ribosomal proteins not related to ribosome, including cell proliferation, drug resistance, and apoptosis. However, so far only alterations have been identified in which an expression of ribosomal proteins is observed as a result of cell transformation.

To date, they have only been described in a limited number of occasions in which the expression of ribosomal proteins seems to be directly related to the degree of cellular transformation. On the one hand, Wang, H. et al. (BMC Cancer, 2006, 6:91) have described the overexpression of RpI 15 in gastric cancer tissues as well as the ability of RpI-specific siRNA to cause proliferation decrease in a cell line derived from gastric cancer. Williams, G.T. et al. {Gene Ther. Mol Biol., 2006, 12: 255-262) have described the ability of Rpllp to suppress apoptosis induced by treatment with PHA in the thymoma cell line W7.2. Finally, Chen, A. et al., Cancer Research, 2002, 62: 1036-1044 have described that treatment with GnRH of a cell line derived from a breast tumor results in a decrease in the proliferation of said cells accompanied by a decrease in the expression levels of RpIp 1.

However, there is a need in the art for additional proteins capable of modulating cell proliferation. Functional genetic screening using retroviral distribution of highly complex cDNA libraries is valuable for investigating the new genes involved in the phenotypic characteristics of the oncogenic process. Thus, Hannon, GJ et al (Science, 1999; 283: 1129-1130) have described a method for the identification of genes capable of promoting cell transformation using cDNA libraries obtained from tumor cell lines and which are introduced into a population of cells with limited proliferative capacity using a retrovirus-mediated integration system. Berns, K. et al. (Nature 2004, 428: 431-437) have described a method for the identification of genes required for p53-mediated cell arrest through the use of siRNA libraries.

In WO2004007720 a method has been described for the identification of polypeptides capable of promoting cell proliferation based on the transformation of cells of primary cultures and, therefore, are intended to achieve replicative senescence after a limited number of replication cycles, with a cDNA library obtained from poly A ⁺ RNA derived from 7 and 15 day embryos, day 15 and brain embryo heads and adult mouse testicles. However, this method has proven to be limited since it has only allowed the identification of glycolytic pathway proteins. Therefore, there is a need in the art for alternative methods of greater sensitivity for the identification of proteins capable of promoting cell proliferation.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to RpI Ip, a functionally equivalent variant thereof or a polynucleotide encoding RpIp 1 or a functionally equivalent variant for use in medicine.

In a second aspect, the invention relates to RpI Ip, a functionally equivalent variant thereof, a polynucleotide encoding RpIp 1 or a Functionally equivalent variant thereof for the treatment of a disease related to unwanted cell senescence.

In a third aspect, the invention relates to a compound capable of inhibiting the expression and / or activity of RpIp 1, with a pharmaceutical composition comprising said inhibitor, with the use of said inhibitor in medicine and with the inhibitor compound for treatment of diseases associated with unwanted cell proliferation.

In another aspect, the invention relates to an in vitro method for the identification of proteins capable of activating cell proliferation or preventing replicative senescence comprising

(i) contacting a cell of limited proliferative capacity with a trunk cell cDNA library.

(ii) select those cells in which cell proliferation or inhibition of replicative senescence is observed and

(iii) characterize the library member of the selected cells in (ii)

In another aspect, the invention relates to an in vitro method for activating cell proliferation or for preventing replicative senescence comprising the introduction into a cell of RpIp 1, of a functionally equivalent variant thereof or of an expression vector comprising a polynucleotide sequence encoding the RpIp 1 protein or a functionally equivalent variant thereof.

In another aspect, the invention relates to an in vitro method for transforming a cell of a primary culture comprising the introduction into a cell of a first expression vector comprising a polynucleotide sequence encoding the RpIp 1 protein or a functionally variant equivalent thereto and a second expression vector comprising a sequence encoding a pro-oncogenic variant of Ras. In another aspect, the invention relates to an in vitro method for the identification of compounds capable of blocking cell proliferation or blocking the avoidance of replicative senescence comprising the steps of:

(i) contacting a cell in which RpIp 1 protein is overexpressed with a candidate compound and

(ii) identify those compounds that block cell proliferation induced by RpIp 1 or that block the avoidance of replicative senescence induced by RpIp 1.

In another aspect, the invention relates to an in vitro method for the identification of compounds capable of blocking cell transformation comprising the steps of:

(i) contacting a cell in which RpIp 1 protein is overexpressed and in which a pro-oncogenic variant of Ras is expressed with a candidate compound and

(ii) identify those compounds that block the cellular transformation induced by RpIp 1 and the pro-oncogenic variant of Ras.

In another aspect, the invention relates to a method for diagnosing a metastasis in a subject affected by cancer and / or prognosis to develop a metastasis in a subject affected by cancer comprising:

(i) quantify the expression of RpIp 1 in a biological sample of said subject

(ii) comparing the level of expression of RpIp 1 previously obtained with that of a biological control sample where an increase of said level in relation to that of the biological control sample is indicative of a positive diagnosis of metastasis or a greater propensity of the subject to develop a metastasis

In another aspect, the invention relates to a method for differentiating between an adenoma and a malignant tumor in a subject comprising: (i) quantify the expression of RpIp 1 in a biological sample of said subject (ii) compare said level of expression of RpIp 1 previously obtained with that of a biological control sample, where an increase of said expression level of RpIp 1 in relation to with that of the biological control sample it is indicative that the subject has a malignant tumor.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Rplpl prevents replicative senescence.

Growth curve of ES CGR8 cells against lung cancer cells (DV90) or primary MEFs cells (Figure la).

Phenotypic aspect of MEFs infected with Rplpl against cells infected with GFP and p53175H (Figure Ib). Growth curves of MEFs infected with Rplpl against the L34 ribosomal protein (Figure Ic). The data were representative of three independent experiments. Expression of mRNA and Rplpl protein in RpIp 1-MEFs versus GFP-MEFs (Figure Id). The data were representative of three independent experiments.

Figure 2. Rplpl activates a promoter transcriptionally dependent on E2F1.

Luciferase indicator assay of a vector with an E2Fl-luciferase promoter in the presence of E2F1-WT in MEFs and H1299 cells (Figure 2a).

Cyclin E mRNA by quantitative RT-PCR in RpIp 1-MEFs versus GFP-MEFs (Figure 2b). Data were reproduced in three independent experiments. Rplpl mRNA levels in spontaneously immortalized MEFs by RT-PCR (Figure 2c).

Figure 3. Rplpl cooperates with (ras ^Val12 ) oncogenic in the transformation of NIH3T3 cells. Growth curve of MEFs cells that coexpress GFP, Rplpl and p53175H plus ^Va112 flush against an empty vector designated S (Figure 3a). Colony formation assay of MEFs infected with RpIp 1-ra, GFP-ras and p53175H-ras against the same cells infected with an empty vector (S), seeded at low density and stained with violet crystal after 20 days (Figure 3b). Soft agar assay in NIH3T3 cells where Rplpl cooperates with ras (Figure 3c). Focal formation in spontaneously immortalized MEFs in pass 15 infected with the indicated genes (Figure 3d).

Figure 4. Rplpl in human cancer

Normal and tumor tissue mRNA of the same patient was analyzed by quantitative RT-PCR (Figure 4a) in 22 cases. White = normal tissue; black = tumor tissue.

Protein level of the Rplpl and p53 proteins of two patients in which the Rplpl mRNA was increased (Figure 4b).

Figure 5. Immunoblotting of the Rplpl protein in MEFs and NIH3T3 cells after expression of p53175H against GFP infected cells. ES cells are also shown as a positive control of Rplpl expression in immunoblot.

Figure 6. Rplpl in metastases The expression of Rplpl in colon tumor tissue of 14 patients diagnosed with absence of metastasis and 8 patients diagnosed with metastasis was analyzed by quantitative RT-PCR. Rplpl mRNA expression values are given as relative values. Rplpl expression levels appear increased in colon cancer patients diagnosed with metastases in relation to patients without metastases.

Figure 7. Rplpl in adenomas

The expression of Rplpl was analyzed by quantitative RT-PCR in tumor tissue of 6 patients diagnosed with adenomas and carcinomas (malignant tumor tissue) in the colon tissue. The values obtained from the tumor tissue (T) were compared with those of the tissue of the control sample (N) and tissue from an adenoma (A) (all of them from the same patient). The expression level of Rplpl is twice as high in cancerous tissue (T) if compared with control tissue (N) and tissue from adenoma (A). The expression levels of RpIp 1 in the adenoma and the control tissue are similar. The relative mRNA values of RpIp 1 are represented on the ordinate axis.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention, using a genetic screening method based on the ability of genes that are expressed in embryonic stem cells to inhibit cellular replicative senescence in primary fibroblast cultures, have shown that, surprisingly, overexpression of a ribosomal protein RpIp 1 is able to avoid replicative senescence in primary MEFs. The RpIp 1 protein belongs to the group of phosphoribosomal proteins that in cooperation with Rplp2 and RpIpO form the ribosomal stem (7). Without wishing to be bound by any theory, it is thought that the ability of RpIp 1 to prevent replicative senescence is associated with an increase in the transcriptional activity of an E2F1-dependent promoter.

Thus, in a first aspect, the invention relates to RpI Ip, a functionally equivalent variant thereof or a polynucleotide encoding RpIp 1 or a functionally equivalent variant thereof for use in medicine. In another aspect, the invention relates to RpIp 1, a functionally equivalent variant thereof or a polynucleotide encoding RpIp 1 or a functionally equivalent variant thereof for the treatment of a disease related to an unwanted cellular senescence.

RpIp 1 means the human protein also known as 6OS PI acidic protein and whose sequence is defined by SEQ ID NO: 1 (accession number Q6FG99). By "functionally equivalent variant" is meant all those proteins derived from the sequence of SEQ ID NO: 1 by modification, insertion and / or deletion of one or more nucleotides, provided that the inhibitory function of the replicative senescence is substantially maintained. The function of the variant can be determined using tests widely known in the state of the art such as determination. of the ability of a variant to promote the survival of MEF when they are transfected with a vector expressing said protein, as demonstrated in example 1. Alternatively, it is possible to detect the function of RpIp 1 by the co-transformation of MEFs with a first vector that encodes the variant whose RpI Ip activity is to be studied and with a second vector comprising a reporter gene operatively coupled to an E2F promoter. As demonstrated in example 2, the cells in which activity of the reporter gene is observed will be those in which the protein encoded by the first vector is a functionally equivalent variant of RpI Ip. The expert will appreciate that the invention does not only contemplate the use of RpIp 1 of human origin but that it is possible to use homologues of other species such as mouse (SEQ ID NO: 2 - UniProt P47955), rat (SEQ ID NO: 3, P19944 ), pig (SEQ ID NO: 4, A1XQU7), bovine (SEQ ID NO: 5, Q56K14), chicken (SEQ ID NO: 6, P 18660) and the like.

In the event that the RpIp 1 protein or a functionally equivalent variant thereof is used, the invention contemplates the use of pharmaceutical compositions comprising the RpIp 1 protein or a functionally equivalent variant thereof accompanied by a pharmaceutically acceptable excipient. For use in medicine, the compounds and combinations of compounds of the invention can be formulated together with an excipient that is pharmaceutically acceptable. Preferred excipients for use in the present invention include sugars, starches, celluloses, gums and proteins. In a particular embodiment, the pharmaceutical composition of the invention will be formulated in a pharmaceutical form of solid administration (e.g., tablets, capsules, dragees, granules, suppositories, etc.) or liquid (e.g., solutions, suspensions , emulsions, etc.). In another particular embodiment, the pharmaceutical compositions of the invention can be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intrarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteric, topical, sublingual or rectal. A review of the different forms of administration of active ingredients, the excipients to be used and their manufacturing procedures can be found in the Treaty of Farmacia Galenica, C. Faulí i Trillo, Luzán 5, SA de Ediciones, 1993. Alternatively, it is possible to put in contact with the cell with a variant of the protein that has been modified with a peptide that is capable of promoting translocation of the protein into the cell, such as the Tat peptide derived from the HIV-I TAT protein, the third helix of the homeodomain of the Antennapedia protein from D.melanogaster, the VP22 protein from herpes simplex virus and arginine oligomers (Lindgren, A. et al., 2000, Trends Pharmacol. Sci, 21: 99-103, Schwarze, SR et al. , 2000, Trends Pharmacol. Sci., 21: 45-48, Lundberg, M et al., 2003, Mol. Therapy 8: 143-150 and Snyder, EL and Dowdy, SF, 2004, Pharm. Res. 21: 389 -393).

Alternatively, when the active ingredient comprises a polynucleotide encoding RpIp 1 or a functionally equivalent variant thereof, the pharmaceutical composition of the invention can be formulated in the form of a composition intended for use in gene therapy; by way of illustration, not limitation, in this case, the pharmaceutical composition of the invention may contain a vector, viral or non-viral, comprising a polynucleotide of the invention or a gene construct of the invention. By way of illustration, not limitation, said vectors may be viral vectors, for example, based on retro viruses, adenoviruses, etc., or non-viral such as DNA-liposome complexes, DNA-polymer, DNA-polymer-liposome, etc. . [see "Nonviral Vectors for Gene Therapy", edited by Huang, Hung and Wagner, Academic Press (1999)]. Such vectors, which contain a polynucleotide or a gene construct of the invention can be administered directly to the human or animal body by conventional methods. Alternatively, said vectors can be used to transform, transfect or infect cells, for example, mammalian cells, including man, ex vivo, and then implant them in the human or animal body to obtain the desired therapeutic effect. For administration to the human or animal body, said cells will be formulated in a suitable medium that does not adversely affect the viability of said cells.

"Disease associated with an unwanted senescence" means, in the context of the present invention, all those diseases in which a disappearance of cells in a certain tissue caused by an abnormal senescence occurs from the same. By way of illustration, not limitation, the invention contemplates the use of RpIp 1 and functionally equivalent variants thereof in the treatment of:

a) diseases related to unwanted senescence in the central nervous system, which affects cells of the central nervous system including neurons, glial cells (eg astrocytes), endothelial cells and fibroblasts such as Alzheimer's disease where produces neuronal degeneration in the central nervous system, Parkinson's disease, Huntington's disease and stroke,

b) age-related skin diseases where there is an unwanted senescence of fibroblasts, sebaceous gland cells, melanocytes, keratinocytes, Langerhans cells, microvascular endothelial cells and hair follicle cells. Examples of such diseases are dermal atrophy and thinning, elastolysis and wrinkle formation, sebaceous gland hyperplasia, senile lentigo and other pigmentation disorders, encanecimento and alopecia and chronic skin ulcers.

c) degenerative diseases of the joints in which senescence of the articular cartilage cells and synovial and lacunal fibroblasts occurs.

d) osteoporosis and other degenerative conditions of the skeletal system caused by the degeneration of osteoblasts, bone marrow cells or mesenchymal cells, progenitor cells of bone tissue.

e) diseases of the vascular system associated with age and stress in which unwanted degeneration of heart and vascular system cells such as endothelial cells, smooth muscle cells and adventitial fibroblasts occur. Examples of these diseases include atherosclerosis, calcification, thrombosis and aneurysms. f) age-related macular degeneration caused by the senescence of eye cells, including pigmented epithelial cells and vascular endothelial cells.

g) AIDS, characterized by the early senescence of cytotoxic T lymphocytes that are responsible for destroying infected CD4 + cells and

h) immunosuppression caused by an unwanted degeneration of lymphoid, myeloid and erythroid, monocyte, circulating and specialized macrophages, neutrophils, eosinophils, basophils, NK cells and their respective progenitors such as those associated with aging, stress, chemotherapy, acute or chronic diseases and certain genetic alterations in which degeneration occurs in immune system cells such as Down syndrome, where lymphocyte degeneration occurs,

i) diseases related to poor healing of wounds, burns, abrasions, surgical incisions, implant sites, lesions caused by infectious agents and chronic conditions such as chronic venous ulcer, diabetic ulcer, compression ulcer, as well as epithelial lesions caused by a persistent inflammatory condition or infection.

Compositions containing RpI Ip or a functionally equivalent variant thereof can also be used ex vivo on cells that have been obtained from a patient in order to prolong the survival of said cells during their in vitro expansion before reimplantation in the individual. For example, muscle satellite cells can be used for the treatment of muscular dystrophy, osteoblasts for the treatment of osteoporosis, pigmented epithelial cells of the retina for the treatment of age-related macular degeneration, chondrocytes for the treatment of osteoarthritis , etc. The effect of the compounds of the invention on cell proliferation or on the inhibition of replicative senescence can be determined by: (i) determination of telomerase activity

The enzymatic activity of telomerase can be determined by any method known in the art. For example, telomerase activity can be determined by determining the elongation rate of a certain repetitive sequence containing 2, 3 or more repeats of the telomeric unit sequence (Yegorov, YE et al., 1997, Mol.Biol., 31: 130-136). For the measurement of such activity, cytoplasmic extracts, nuclear extracts, Cellular used or intact cells can be used. By "increase" in telomerase activity is meant that the absolute level of telomerase activity in a particular cell is increased compared to normal cells in the same individual or compared to normal cells in subjects not suffering from the condition. Examples of such conditions include cancer or conditions related to the presence in a subject of cells that are not usually present in said individual such as infections by microorganisms that require telomerase for replication.

(ii) determination of telomere length

Methods for the determination of telomere length have been widely described in the art by Harley, C. B., et al. (Nature, 1990, 345: 458-460); Levy, M. Z. et al., (J. Mol. Biol., 1992, 225: 951-960); Lindsey, J.et al., (Mutat. Res., 1991, 256: 45-48) and Allsopp, RCet al., (Proc. Nati. Acad. Sci. USA, 1992, 89: 10114-10118), among others.

Conventionally, restriction endonucleases that do not fragment the telomeric DNA are used to then separate the fragments obtained by their molecular weight and detection of the telomeres by hybridization using specific probes for the sequence of the telomeres.

(iii) determination of cell proliferation The determination of cell proliferation can be carried out by methods widely known to the person skilled in the art including the determination of cell doubling time as described by Harley et al. (Nature, 1990, 345: 458- 460). The cell proliferation rate can be determined by determining the incorporation of tritiated uridine into the cell or colorimetric assays employed BrdU.

(iv) determination of cell senescence

As the cells progress towards senescence they show a lengthening of the Gl phase of the cell cycle, resulting in an increase in the total time in which a cell cycle passes (Hayflick, L. and Moorhead, PS, Exp. CeIl Res 1961, 25: 585-621 (1961) and Hayflick, L., Exp. CeIl Res. 1965, 37: 614-636). As the cell progresses from a mitotically active state to a state of senescence, the cells lose the ability to respond to mitotic signals and remain in Fl. This inability is one of the main characteristics between young and old cells, however, to Unlike old cells, young cells enter quiescence when they enter the GO phase, but they can be subsequently induced to re-enter the cell cycle and divide. However, senescent cells, while remaining viable and metabolically active, are unable to re-enter the cell cycle.

Cells that have stopped in the Gl phase of the cell cycle contain a double set of chromosomes. These cells show a characteristic morphology that can be detected under a microscope and that allows cells to be detected in a state of senescence. Another difference in replicative senescence is that the cells show changes in the pattern of gene expression as the cells progress to senescence. These changes are reflected in the expression of specific genes from young cells and specific from old cells. Genes that are differentially expressed in senescent cells include beta-galactosidase, collagenase, interferon- gamma, collagen I, collagen III, elastase, elastin, TIMP3 and IL-Ia. Other genes that are differentially expressed in senescent cells have been described in international application WO 96/13610.

The authors of the present invention have identified a mechanism by which replicative senescence can be inhibited in cells. Similarly, the use of RpIp 1 inhibitors will have the effect of promoting cell senescence. Thus, in another aspect, the invention relates to a compound capable of inhibiting the expression and / or activity of RpIp 1.

Compounds capable of inhibiting the expression and / or activity of RpIp 1 include antisense oligonucleotides, ribozymes, siRNAs, inhibitory antibodies, aptamers, and spiegemers.

SiRNA of the invention

Small interfering RNAs or siRNAs (SiRNAs) are agents that are capable of inhibiting the expression of a target gene through RNA interference. An siRNA can be chemically synthesized, can be obtained by in vitro transcription or can be synthesized in vivo in the target cell. Typically, siRNAs consist of a double strand of RNA between 15 and 40 nucleotides in length and which may contain a 3 'and / or 5' protruding region of 1 to 6 nucleotides. The length of the protuberant region is independent of the total length of the siRNA molecule. SiRNAs act through degradation or post-transcriptional silencing of the target messenger.

The siRNAs can be called shRNA (short hairpin RNA) characterized in that the antiparallel chains that form the siRNA are connected by a loop or hairpin region. These siRNAs are composed of a short antisense sequence (from 19 to 25 nucleotides), followed by a loop of between 5 and 9 nucleotides followed by the sense chain. The shRNAs can be encoded by plasmids or viruses, particularly retroviruses and, more particularly, retroviruses and being under the control of promoters such as the U6 promoter of RNA polymerase III.

The siRNAs of the invention are substantially homologous to the mRNA of RpIp 1 or to the genomic sequence encoding said protein. By "substantially homologous" is meant that they have a sequence that is sufficiently complementary or similar to the target mRNA so that the siRNA is capable of

'cause degradation of this by RNA interference. Suitable siRNAs for causing such interference include siRNAs formed by RNAs, as well as siRNAs containing various chemically modified such as:

- siRNA in which the bonds between nucleotides are different from those that appear in nature, such as phosphorothioate bonds.

- conjugates of the RNA chain with a functional reagent, such as a fluorophore. - Modifications of the ends of the RNA chains, in particular the end

3 'by modification with different hydroxyl functional groups in position 2'.

- Nucleotides with modified sugars such as O-alkylated moieties in 2 'position such as 2'-O-methylribose p 2'-O-fluorosibose. - Nucleotides with modified bases such as halogenated bases (for example

5-bromouracil and 5-iodouracil), alkylated bases (for example 7- methylguanosine).

The siRNAs and siRNAs of the invention can be obtained using a series of techniques known to the person skilled in the art. For example, the siRNA can be chemically synthesized from ribonucleosides protected with forsphoramidites in a conventional DNA / RNA synthesizer. Alternatively, the siRNAs can be recombinantly produced from plasmid and viral vectors in which case the region encoding the chain or chains that form the siRNAs are under operational control of RNA polymerase III promoters.

In cells, the Dicer RNAse processes the shRNAs in functional siRNAs. The region of RpIp 1 that is taken as a basis for designing the siRNAs is not limiting and may contain a region of the coding sequence (between the initiation codon and the termination codon) or, alternatively, may contain sequences from the non-translated region 5 'or 3'. it is preferably between 25 and 50 nucleotides in length and in any position in 3 'position with respect to the initiation codon.

One way to design an siRNA involves identifying the AA (N ^) TT motifs where N can be any nucleotide in the RpIp 1 sequence and selecting those that have a high G / C content. If not found this reason, it is possible to identify the reason NA (N ₂ O, where N may be any nucleotide.

Antisense oligonucleotides of the invention

In another embodiment, the invention relates to antisense oligonucleotides specific for RpIp 1, that is, molecules whose sequence is complementary to the mRNA encoding RpIp 1, that is, complementary to the cDNA coding chain. The antisense oligonucleotide may be complementary to the entire coding region or to a region thereof that includes both the coding region and the 5 'and 3 untranslated regions. Antisense oligonucleotides may consist of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. Antisense oligonucleotides can be obtained by chemical synthesis or by enzymatic ligation reactions widely known to those skilled in the art. For example, an antisense oligonucleotide may also contain modified nucleotides that increase their biological stability, or the stability of DNA-double stranded complexes. RNA formed between the antisense oligonucleotide and the target polynucleotide, such as phosphorothioate derivatives, peptide nucleic acids and acridine substituted oligonucleotides. Modified oligonucleotides that can be used to prepare antisense nucleic acids include 5-fluoruracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methylladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D- Mannosylkeosine, 5'-methoxycarboxymethyluracil, 5-methoxuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4- thiouracil, 5-methyluracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, 5-methyl-2- thiouracil, 3- (3-amino-3-N-2- carboxypropyl) uracil, and 2 , 6- diaminopurine. Alternately, the antisense nucleic acid can be produced biologically using an expression vector in which the nucleic acid has been cloned in antisense orientation.

Ribozymes of the invention

Another group of compounds contemplated in the present invention are the catalytically active nucleic acids known as ribozymes. Ribozymes comprise a catalytic region and a second region whose sequence is complementary to the target nucleic acid and which confers substrate specificity to the ribozyme. After the interaction between ribozyme and its substrate by hybridization and pairing between complementary regions of the target nucleic acid and ribozyme, activation of the catalytic region occurs causing inter- or intramolecular breakdown of the target nucleic acid. Ribozymes and basic considerations for their design are widely known to the person skilled in the art (see for example Doherty and Doudna (Annu. Ref. Biophys. Biomolstruct. 2000; 30: 457-75).

Inhibitor antibodies of the invention

By "inhibitor antibody" is meant in the context of the present invention any antibody that is capable of binding to RpIp 1 causing the inhibition of its pro-proliferative activity. Antibodies can be prepared using any of the methods that are known to the person skilled in the art. Thus, polyclonal antibodies are prepared by immunization of an animal with the protein to be inhibited. Monoclonal antibodies are prepared using the method described by Kohler, Milstein et al. (Nature, 1975, 256: 495). Once antibodies with RpIp 1 binding capacity have been identified, those that are capable of inhibiting the activity of this protein will be selected using the aforementioned assays for the determination of RpIp 1 activity.

Suitable antibodies in the context of the present invention include intact antibodies comprising a variable region of antigen binding and a constant region, "Fab", "F (ab ') 2" and "Fab"' fragments, Fv, scFv, diabodies and bispecific antibodies.

Other compounds inhibiting the activity of RpIp 1 of the invention

Other compounds capable of inhibiting the expression of RpIp 1 include aptamers and spiegemers, which are single or double stranded D or L nucleic acids that specifically bind to the protein resulting in a modification of its biological activity. The aptamers and spiegemers have a length of between 15 and 80 nucleotides and, preferably, between 20 and 50 nucleotides.

The compounds of the invention (siRNA, antisense oligonucleotides, ribozymes, aptamers and spiegemers) can be administered to the cells in which you wish to inhibit the expression of RpIp 1 by microinjection of a composition comprising the agent or by contacting the cell with a composition comprising the inhibitory agent. Alternatively, a virus-mediated method of administration can be used both in vitro and in vivo. Other methods of nucleic acid administration capable of inhibiting RpIp 1 include co-precipitation in the presence of calcium phosphate or calcium chloride, transfection mediated by DEAE-dextran, lipofection, electroporation as well as a whole series of transfection kits based on prior art. and that are commercially available. The effectiveness of the transfection will depend on a series of factors that include the type of cell, the number of passes, the state of confluence as well as the conditions (time, form of preparation of the liposomes or precipitates, etc.) of the transfection All these parameters can be determined and adjusted by routine experimentation. In another aspect, the invention relates to a pharmaceutical composition comprising an inhibitor of the expression or activity of RpIp 1 according to the invention. For use in medicine, the compounds and combinations of compounds of the invention can be formulated together with an excipient that is pharmaceutically acceptable. Preferred excipients for use in the present invention include sugars, starches, celluloses, gums and proteins. In a particular embodiment, the pharmaceutical composition of the invention will be formulated in a pharmaceutical form of solid administration (e.g., tablets, capsules, dragees, granules, suppositories, etc.) or liquid (e.g., solutions, suspensions , emulsions, etc.). In another particular embodiment, the pharmaceutical compositions of the invention can be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intrarterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteric, topical, sublingual or rectal. A review of the different forms of administration of active ingredients, of the excipients to be used and their manufacturing procedures can be found in the Treaty of Farmacia Galenica, C. Faulí i Trillo, Luzán 5, SA de Ediciones, 1993.

In another particular embodiment, when the active ingredient comprises a polynucleotide of the invention, the pharmaceutical composition of the invention can be formulated in the form of a composition intended for use in gene therapy; by way of illustration, not limitation, in this case, the pharmaceutical composition of the invention may contain a vector, viral or non-viral, comprising a polynucleotide of the invention or a gene construct of the invention. By way of illustration, not limitation, said vectors may be viral vectors, for example, based on retroviruses, adenoviruses, etc., or non-viral such as DNA-liposome, DNA-polymer, DNA-polymer-liposome complexes, etc. [see "Nonviral Vectors for Gene Therapy", edited by Huang, Hung and Wagner, Academic Press (1999)].

Such vectors, which contain a polynucleotide or a gene construct of the invention can be administered directly to the human or animal body by conventional methods. Alternatively, said vectors can be used to transform, transfect or infect cells, for example, mammalian cells, including man, ex vivo, and then implant them in the human or animal body to obtain the desired therapeutic effect. For administration to the human or animal body, said cells will be formulated in a suitable medium that does not adversely affect the viability of said cells.

In another aspect, the invention relates to compounds that inhibit the activity and / or expression of RpIp 1 for use in medicine. In another aspect, the invention relates to compounds that inhibit the activity and / or expression of RpIp 1 for the treatment of diseases associated with unwanted cell proliferation. Diseases related to unwanted cell proliferation include the growth, progression and metastasis of cancer and tumors.

The terms "cancer" and "tumor" refer to the physiological condition in mammals characterized by deregulated cell growth. The RpIp 1 activity and / or expression inhibitor compounds of the invention have utility for the treatment of tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain , pancreas, skin, bone, bone marrow, blood, thymus, uterus, testicles and liver. In particular, tumors that can be treated with the compounds of the invention include adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma retinoblastoma, rhabdomyosarcoma, sarcoma and teratoma. In particular, the tumor / cancer is selected from the group of lentiginous acral melanoma, actinic adenocarcinoma keratosis, cystic adenoidal carcinoma, adenomas, adenosarcoma, adenoescamosal carcinoma, astrocytic tumors, Bartholin gland carcinoma, basal cell carcinoma, branchial gland carcinoma , capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometroid adenocarcinoma, ependial sarcoma, Swing sarcoma, focal nodular hyperplasia, gastromeoma, germline tumors, glioblastoma , hemagioblastoma, hemagioendothelioma, hemagioma, hepatic adenoma, Hepatic adenomastosis, hepatocellular carcinoma, insulinite, intraepithelial neoplasia, interepithelial squamous cell neoplasm, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, melanoma, malignant melonoma, mesothelial malignant tumor, medullobastoma, medulloepheryngeal carcinoma , nodular melanoma, osteosacrcoma, papillary serous adenocarcinoma, pituitary tumors, plasmacytoma, pseudosarcoma, pulmonary blastema, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, small cell carcinoma, carcinoma of soft tissues, somatostatin secretory tumor, carcinogen squamous, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, warty carcinoa, vipoma, WiIm tumor. Even more preferably, the tumor / cancer to be treated with the compounds of the invention includes intracerebral cancer, head and neck cancer, rectal cancer, astrocytoma, preferably grade II, III or IV astrocytoma, glioblastoma, preferably glioblastoma multiforme, cancer of small cells, and non-small cell cancer, preferably non-small cell lung cancer, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, breast cancer and colon cancer.

In a preferred embodiment, compounds with inhibitory capacity for the expression / activity of RpIp 1 are used for the treatment of diseases associated with an unwanted cell proliferation caused by a mutation in the Ras gene, including any of the three members of the Ras family (N-, K- and liras). Approximately 20% of cancer is associated with a mutation in the Ras gene. Thus, the methods of the invention have utility for the treatment of the types of cancer indicated in Table 1 of Bos, J. (Cancer Research, 1989, 49: 4682-4689), the content of which is incorporated by reference in its entirety. In particular, tumors in which ras mutations are frequently observed include tumors of the exocrine pancreas, in colon carcinomas, in follicular and undifferentiated thyroid carcinomas, in non-small cell lung carcinoma, melanomas, carcinoma pancreatic and myeloid leukemias including myelodysplastic syndrome or MDS and acute myelogenous leukemia or AML, malignant lymphoid diseases including acute lymphoblastic leukemia or ALL, chronic lymphocytic leukemia or CLL and non-Hogdkin lymphoma or NHL.

Ras mutations that can be treated with the compounds of the present invention include mutations in the GTP binding domain (codons 12 and 13) and mutations in the GTPase domain (codon 61). In a preferred embodiment, the Ras mutation is Ras ^Va112 .

The authors of the present invention have shown that, interestingly, the expression of RpIp 1 in primary fibroblasts is capable of causing a two-fold increase in the expression of genes that are under the operational control of the E2F1 promoter, both in the presence as in the absence of E2F1.

The activation of E2F transcription factors, through the disruption of the suppressor gene of retinoblastoma tumors (Keyomarsi, K. et al., 2002,

N.Eng.J. Med., 347: 1566-1575) is a key fact in the development of many human cancers (Zhang, SY et al., 2000, Cancer Res., 60: 5972-5976, Zhang, SY et al., 2000, Epidemiol Cancer Biomarkers Prev., 9: 395-401). The E2F family of genes controls progression through the cell cycle by regulating the transcription of genes that are essential for DNA synthesis and cell cycle progression.

(Nevins, J.R., 1998, CeIl Growth Differ., 9: 585-593). Several members of E2F, such as E2F1, behave like oncogenes due to their ability to induce quiescent cells to enter the cell cycle, ignore several growth stop signals and transform primary cells (Davis, JN et al., 2006, Cancer Res., 66: 11897-11906). The authors of the present invention have shown, for the first time, that the expression of RpIp 1 alone can transactivate an E2F-dependent promoter twice in cells with a natural genetic endowment. This effect was observed with and without the exogenous expression of E2F1-WT. Such activation was not observed in other oncogenic cell lines, including SAOS2, H 1299, HEK and NIH3T3 cells. The fact that RpIp 1 induces transcriptional activation of

E2F1 in primary cells suggests that RpIp 1 can transactivate E2F1 target genes, including at least partially induction of cyclin E as the target E2F main. On the contrary, in immortalized cells, RpIp 1 would activate other genes than the E2F target genes in their contribution to the transformation.

Therefore, in another aspect, the invention relates to the use of compounds capable of inhibiting the expression / activity of RpIp 1 for the treatment of diseases associated with an associated cell proliferation caused by an increase in the expression of target genes of E2F1. In a preferred embodiment, the E2F1 target gene is cyclin E.

By "E2F1" is meant, in the context of the present invention, the transcription factor that is inactively found in the cell by forming a complex with the retinoblastoma protein when it is dephosphorylated. When the retinoblastoma protein is phosphorylated by the cyclin D / CDK 4 and 6 complex and by cyclin E / CDK2, the complex dissociates allowing the association of E2F1 with a protein from the dimerization partner (DP) family thus generating an active complex capable of promoting the transcription of a series of genes that are regulated by E2F elements and that allow the entry of the S-phase cell.

"E2F1 targets" means, in the context of the present invention, genes whose promoter region contains binding sites for dimers formed by E2F1 and its dimerization partner and whose transcription is activated in response to the binding of said dimers to the promoter region. E2F1 target genes include cyclin E, cyclin A and PCNA. Cyclin E

By "cyclin E" is meant, in the context of the present invention, the protein that by forming a complex with Cdk2 induces the entry of the S-phase cell. Cancerous diseases in which an increase in E2F1 activity has been detected include metastases derived from prostate cancer. In breast cancer, overexpression of cyclin E correlates with a worse prognosis of the patient.

Method for the identification of proteins capable of activating cell proliferation according to the invention.

The authors of the present invention have shown that the immortal capacity observed in ES cells and cancer cells allows genetic screening to infect primary mouse embryonic fibroblasts (MEFs) with a cDNA library of mouse ES cells in order of identifying genes that confer a selective advantage for cell proliferation. The authors of the invention have shown that said genetic screening allows the identification of genes whose involvement in cell proliferation was unknown until now.

Thus, in another aspect, the invention relates to an in vitro method for the identification of proteins capable of activating cell proliferation or preventing replicative senescence comprising (i) contacting a cell of limited proliferative capacity with a library of Stem cell cDNA, (ii) select those cells in which cell proliferation or inhibition of replicative senescence is observed and

(iii) characterize the library member of the selected cells in (ii).

By "cell of limited proliferative capacity" is understood in the context of the present invention, a cell that after a limited number of division cycles stops proliferating reaching a state known as replicative senescence. Normally, cells of limited proliferative capacity are somatic cells in primary culture. By "trunk cell" is meant, in the context of the present invention, undifferentiated or poorly differentiated cells, with the ability to divide indefinitely without losing their properties and produce specialized cells when grown under certain conditions. Trunk cells include totipotent cells, capable of growing and forming a complete organism, pluripotent, capable of forming any cell of the three embryonic lineages (mesoderm, endoderm and ectoderm), multipotent cells, capable of generating cells of their own embryonic lineage of origin and unipotent cells, capable of forming a single particular cell type. Pluripotent stem cells can come from a blastocyst (embryos of 4 to 5 days), in which case they are called embryonic stem cells or from the gonads of a 5-10 week fetus, in which case they are called embryonic germ cells, from the. Multipotent stem cells can come from fetal tissue (fetal stem cells) or from adult animal tissues (adult stem cells).

In a first phase, the protein identification method capable of activating the cell proliferation of the invention comprises contacting a cell of limited proliferative capacity, normally forming part of a culture, with a cDNA library of a stem cell. Trunk cell cDNA libraries are obtained using any of the methods widely known to those skilled in the art, widely described in Sambrook, J. et al. (Molecular Cloning: A Laboratory Manual, 2nd ed. CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY) and Ausubel, F (Current Protocols in Molecular Biology). Alternatively, the use of commercial stem cell cDNA libraries is possible. In a preferred embodiment, the library used in the context of the present invention is derived from embryonic stem cells. Preferably, embryonic stem cells are non-human cells. Even more preferably, stem cells are derived from murine embryos.

The library of stem cell DNAs is contacted with the cell or cell population using any of the methods known to those skilled in the art to introduce polynucleotides into the cell. Methods Suitable include the transfer mediated by a vector (for example, by infection with a recombinant virus or by transfection with a viral vector), methods in which DNA complexes to be introduced into the cells with proteins, lipids or other polymers are used (lipofection, DEAE-dextran, polibreon) and methods that allow the introduction of naked DNA into the cell

(electroporation, through biolistic methods, microinjection).

In the second phase, the cells in which cell proliferation is observed or cells that have been able to avoid replicative senescence are selected from those cells that have been transfected. Methods for the selection of cells that avoid replicative senescence include the identification of colonies on soft agar plates, increased saturation density of the cultures, loss of serum requirement, ability to grow on agar or regardless of substrate adhesion , ability to form tumors when introduced into animals, loss of movement contact inhibition, ability to grow in monolayer cultured cells, increased lectin agglutination, changes in the glycoprotein and glycolipid profile, loss of hermetic intercellular junctions , expression of lethal antigens, alteration of staining patterns, increase in metabolic rate, increase in secretion of proteases, cytoskeleton alterations, alteration in the levels of certain signaling molecules (cyclic nucleotides, phosphoinositols).

In the third phase, the member of the library of the selected cells in the second phase is characterized. For this, it is necessary to carry out a preparation of nucleic acids from the selected cell to carry out from said preparation a PCR reaction using primers based on the known sequences of the vector regions of the library flanking the cDNA. By selecting suitable primers, it is possible to obtain a PCR fragment that can be digested with restriction endonucleases allowing the cloning of active cDNAs in previously linearized vectors and having cohesive ends compatible with the ends generated in the PCR products. If the cDNA library is a library based on viral vectors, the cDNA rescue It is carried out by contacting the cell or organism with a vector that provides all those functionalities of the virus that are not present in the viral vector of the library.

The process described above may result in the isolation of a single clone or the isolation of a subpopulation of clones from the original population. If this is the case, the process can be repeated successively using the subpopulation of clones obtained after the first isolation process as the starting material. After one or more additional purification cycles, a single clone or a reduced clone population will be obtained with the cDNA with the desired function. Once the gene encoding the ANDc responsible for proliferative capacity has been recovered, the region of the insert is sequenced. Sequencing can be carried out using methods widely known to those skilled in the art such as the Sanger method using dideoxynucleotides, sequencing by hybridization (sequencing-by-hybridization or SBH), nanopore sequencing, sequencing by synthesis (sequencing-by- syntehsis or SBS), pyrosequencing and the like. Suitable primers for the sequencing reaction can be obtained from the sequences flanking the cDNA.

In case the selected vector contains only a partial cDNA sequence, it is possible to analyze sequence databases to identify the complete nucleic acid. Preferably, the identification of the complete nucleic acid is carried out using the algorithms known to those skilled in the art, such as the BLAST algorithm (Altschul et al., Nuc. Acids Res., 25: 3389-3402 (1977)), BLAST 2.0 (Altschul et al., J. Mol. Biol., 215: 403-410 (1990) or FASTA (WR

Pearson and DJ Lipman (1988), Proc.Natl.Acad.Sci. USA 85: 2444-2448). Alternatively, if the selected vector contains only a partial cDNA sequence, said partial sequence can be used to construct probes or primers that allow isolation of the entire target mRNA. Thus, the partial sequence can be used for the preparation of radioactively labeled probes for screening cDNA and genomic libraries. Alternatively, the partial sequence can be used to synthesize primers that They can be used to amplify cDNAs from a cDNA or genomic library using techniques such as 5'-RACE or 3'-RACE.

In vitro method for activating cell proliferation of the invention

The characterization by the authors of the present invention of the ability of the RpIp 1 protein to prevent replicative senescence in primary MEFs when it is overexpressed allows the development of in vitro methods to activate cell proliferation, to avoid senescence replicative of a cell or to transform a cell by expressing RpIp 1.

Thus, in another aspect, the invention relates to an in vitro method for activating cell proliferation or for preventing replicative senescence comprising the introduction into a cell of RpIp 1, of a functionally equivalent variant thereof or of an expression vector comprising a polynucleotide sequence encoding the RpIp 1 protein or a functionally equivalent variant thereof.

In another aspect, the invention relates to an in vitro method for transforming a cell of a primary culture comprising the introduction into a cell of a first expression vector comprising a polynucleotide sequence encoding the RpIp 1 protein or a functionally variant equivalent thereof and a second expression vector comprising a sequence encoding a pro-oncogenic variant of Ras. In a preferred embodiment, the oncogenic variant of ras is Ras ^VaI12 .

In vitro methods to activate cell proliferation, to prevent replicative senescence or to transform a cell from a primary culture involve bringing a cell into contact with a composition capable of causing an increase in intracellular levels of RpIp 1. In a embodiment, the cell is contacted with a variant of the protein that has been modified with a peptide capable of promoting the translocation of the protein into the cell interior, such as the Tat peptide derived from the TAT protein of HIV-I, the third helix of the home domain of the Antennapedia protein of D.melanogaster, the VP22 protein of herpes simplex virus and arginine oligomers (Lindgren, A. et al, 2000, Trenas Pharmacol .Sci, 21: 99-103, Schwarze, SR et al., 2000, Trends Pharmacol. Sci., 21: 45-48, Lundberg, M et al., 2003, Mol. Therapy 8: 143-150 and Snyder, EL Y

Dowdy, S.F., 2004, Pharm. Res. 21: 389-393). In another embodiment, the cell is contacted with a polynucleotide encoding RpIp 1 in the presence of reagents capable of promoting translocation of the polynucleotide into the cell interior. The polynucleotide is introduced into the cells using any of the transfection methods known to those skilled in the art (see sections 9.1 to 9.5 in

Ausubel, F.M. et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc; ringbou edition, 2003). In particular, cells can be transfected by co-precipitation of DNA with calcium phosphate, DEAE-dextran, polibreon, electroporation, microinjection, liposome-mediated fusion, lipofection, retro virus infection and biolistic transfection.

In vitro method for the identification of compounds capable of blocking cell proliferation according to the invention.

The characterization by the authors of the present invention of the ability of the RpIp 1 protein to prevent replicative senescence in primary MEFs when over-expressed allows the development of in vitro methods for the identification of compounds capable of blocking activation. of cell proliferation or blocking the avoidance of replicative senescence.

Thus, in another aspect, the invention relates to an in vitro method for the identification of compounds capable of blocking cell proliferation or blocking the avoidance of replicative senescence comprising the steps of: (a) contacting a cell in which RpIp 1 protein is overexpressed with a candidate compound and (b) identify those compounds that block cell proliferation induced by RpIp 1 or that block the avoidance of replicative senescence induced by RpIp 1.

In another aspect, the invention relates to an in vitro method for the identification of compounds capable of blocking cell transformation comprising the steps of:

(a) contacting a cell in which RpIp 1 protein is overexpressed and in which a pro-oncogenic variant of Ras is expressed with a candidate compound and

(b) identify those compounds that block the cellular transformation induced by RpIp 1 and the Ras variant.

The methods of the invention to identify compounds capable of blocking cell proliferation, preventing entry into replicative senescence or blocking transformation require a first step in which a cell expressing RpIp 1 or a cell expressing RpIp 1 and a variant Ras pro-oncogenic with a candidate compound.

By "contacting" a cell with the candidate compound, according to the present invention, any possible way of bringing the candidate compound into the cell expressing RpIp 1 or RpIp 1 and pro-oncogenic variant of Ras is included. Thus, in case the candidate compound is a low molecular weight molecule, it is sufficient to add said molecule to the culture medium. In case the candidate compound is a high molecular weight molecule (for example, biological polymers such as a nucleic acid or a protein), it is necessary to provide the means for that molecule to access the cellular interior. In case the candidate molecule is a nucleic acid, conventional methods for transfection can be used, as described above for the introduction of the DNA construct. In case the candidate compound is a protein, the cell can contact both the protein directly and the nucleic acid that encodes it coupled to elements that allow its transcription / translation once they are inside the cell. For this, any of the methods mentioned above can be used to allow entry into the cell interior. Alternatively, it is possible to contact the cell with a variant of the protein to be studied that has been modified with a peptide that is capable of promoting translocation of the protein into the cell, such as the Tat peptide derived from the TAT protein of HFV-I, the third helix of the home domain of the Antennapedia protein of D.melanogaster, the VP22 protein of herpes simplex virus and arginine oligomers (Lindgren, A. et al., 2000, Trends Pharmacol. Sci, 21:99 -103, Schwarze, SR et al., 2000, Trenas Pharmacol. ScL, 21: 45-48, Lundberg, M et al., 2003, Mol. Therapy 8: 143-150 and

Snyder, E.L. and Dowdy, S.F., 2004, Pharm. Res. 21: 389-393).

Preferably, the compound to be tested is not isolated but is part of a more or less complex mixture either derived from a natural source or part of a library of compounds. Examples of libraries of compounds that can be tested according to the method of the present invention include, without limitation, peptide libraries including both peptides and peptide analogs comprising D-amino acids or peptides comprising non-peptide bonds, nucleic acid libraries including acids Nuclei with non-phosphodiester bonds of the phosphorothioate type or peptide nucleic acids, libraries of antibodies, carbohydrates, low molecular weight compounds, preferably organic molecules, peptidomimetics, and the like. In the case that a library of low molecular weight organic compounds is used, the library may have been preselected to contain compounds that can access the cell interior more easily. Thus, the compounds can be selected based on certain parameters such as size, lipophilicity, hydrophilicity, ability to form hydrogen bonds.

In a second stage, the method comprises the identification of those cells in which RpIp 1-induced cell proliferation has been blocked, in which RpIp 1-induced replicative senescence has been prevented or in which the transformation has been blocked. cell induced by RpIp 1 and the Ras variant. Any of the parameters indicative of cell senescence, proliferation or cell transformation indicated above can be used to identify compounds that inhibit the activity of RpIp 1. Alternatively, the detection of inhibition of the pro-proliferative capacity of RpIp 1 can be carried out. carried out by means of a reporter gene system that is under the control of an E2F promoter, where the decrease in the activity of the reporter gene in the presence of the compound studied with respect to the activity of the reporter gene in the absence of said compound is indicative that the compound inhibits RpIp 1. Reporter genes suitable for use in the present invention include luciferase, green fluorescent protein and variants thereof that emit fluorescence at different wavelengths (eg, DS-Red or red fluorescent protein), chloramphenicol acetyltransferase , β-galactosidase, alkaline phosphatase and horseradish peroxidase.

If the candidate compound is part of a mixture of greater or lesser complexity, the invention further comprises one or more stages (iii) of fractionation of said mixture and the repetition of steps (i), (ii) and (iii) of the method of the invention a variable number of times until the compound of the mixture responsible for the promoter activity of transcription is isolated. Methods for fractionation of compounds present in a mixture include chromatography (thin-film, gas or gel molecular exclusion, affinity), crystallization, distillation, filtration, precipitation, sublimation, extraction, evaporation, centrifugation, mass spectrometry, adsorption and the like.

In a preferred embodiment, the pro-oncogenic variant of Ras that is expressed together with RpIp 1 includes mutations in the GTP binding domain (codons 12 and 13) and mutations in the GTPase domain (codon 61) in any of the three members of the Ras family (N-, K- and H-ras). In an even more preferred embodiment, the Ras mutation is Ras ^Va112 . The method of the invention for the identification of compounds capable of blocking cell transformation has a particular interest in the case of cells in which the transformation is related to an alteration of the activity of the p53 protein. The authors of the present invention have shown that the expression of p53175H increases Rplpl protein levels in vitro. On the other hand, it has also been observed that in six patients in which Rplpl mRNA was increased, overexpression of the p53 protein was observed (possibly indicating the presence of mutations in p53), suggesting that the increase in Rplpl may explain at least in some cases the presence of mutations in p53 in human cancers. The fact that in three patients in which the mRNA of

Rplpl was increased, no increase in Rplpl protein was observed, it could be influenced by the genetic history of the tumor. Without wishing to be bound by any theory, it is hypothesized that the activation of some ribosomal proteins act as "speed-limiting proteins" in proliferation. The possibility that these ribosomal proteins were those that are targets of p53 mimics is a tempting speculation.

So. In a preferred embodiment, the method of the invention for the identification of compounds capable of blocking cell proliferation, blocking the avoidance of replicative senescence or blocking cell transformation is carried out in cells in which there is an envelope -expression of Rplpl and where the activity of p53 is inhibited.

Inhibition of p53 activity may be due to deletion of the p53 gene, by phosphorylation of the serines at positions 15 and / or 392 or by mutation to give rise to negative dominant variants of the p53 protein. In a preferred embodiment, the inhibition of p53 takes place by expression in the cell of a dominant dominant mutant of p53.

By "negative dominant imitant" is meant, in the context of the present invention, a mutant that adversely affects the function of the native form of the protein in the same cell. Typically, the dominant negative mimics they interact with the same functional effectors as the native product, thus blocking some of its functions. Illustratively, not limitation, dominant negative imitations of p53 that can be employed in the context of the invention include mutations 127P, 132N, 135C, 151R, 151H, 158P, 175H, 176R, 179N, 233D, 241F, 242F, 244D, 244S, 245S, 245R, 245D, 2461, 246R,

248W, 249S, 2521, 257P, 257Q, 259Y, 265P, 273P, 277Y, 278H, 278S, 279E, 280T, 280S, 281G, 281Y, Δ175-180, Δ176-181, Δ177-182, Δ216, Δ217, Δ218, Δ252-254, Δ251-253 and the like. In an even more preferred embodiment, the negative dominant imitant of p53 is 175H.

Method for the diagnosis and / or the propensity of a subject to suffer a metastasis according to the invention.

The characterization by the authors of the present invention of the correlation of the overexpression of the RpIp 1 protein in tumor tissue samples from patients with metastases compared to tumor samples from patients without metastases and from non-cancerous tissue samples , allows the development of in vitro methods for the identification of metastasis.

"Metastasis" is the spread to organs distant from an infection or from a primary malignant tumor or cancer, which usually occurs by blood or lymph. Approximately 98% of deaths due to undetected cancers are due to metastasization. Cancers are able to spread through the body thanks to two mechanisms: invasion and metastasis. Invasion is migration and direct penetration by cancer cells into neighboring tissues. Metastasis is the ability of cancer cells to penetrate the blood and lymph vessels, circulate through the bloodstream, and then grow into a new focus (metastasis) in normal tissues of another part of the body.

Thus, in another aspect, the invention relates to an in vitro method for diagnosing a metastasis in a subject affected by cancer and / or predicting the propensity to develop a metastasis in a subject affected by cancer comprising: (i) quantify the expression of RpIp 1 in a biological sample of said subject, and (ii) compare said level of expression of RpIp 1 previously obtained with that of a biological control sample, where an increase in said expression level of RpIp 1 in relation to that of the biological control sample, it is indicative of a positive diagnosis of metastasis or a greater propensity of the subject to develop a metastasis.

In a first stage, the quantification of the expression of RpIp 1 in a biological sample is performed.

By "biological sample" is meant any biological tissue or fluid taken from the subject. In a particular embodiment, said biological sample is a biological fluid such as blood, urine or serum; In another particular and preferred embodiment, said biological sample is a tumor tissue sample, e.g., a tumor tissue biopsy.

The expression of RpIp 1 can be determined by a wide variety of conventional methods, for example, by detecting the expression of the transcription molecule or the corresponding protein.

In a particular embodiment, the expression level of RpIp 1 is determined by the detection and quantification of the corresponding transcription molecule. In this case, additionally, the method of the invention may include performing an extraction step in order to obtain the total RNA, which can be done by conventional techniques.

Virtually any conventional method can be used within the framework of the invention to detect and quantify mRNA levels encoded by the RpIp 1 gene and its corresponding cDNA. By way of illustration, not limitation, the levels of mRNA encoded by said gene can be quantified by the use of conventional methods, for example, methods comprising the amplification of the mRNA and quantification of the product of the amplification of said mRNA, such as electrophoresis and staining, or alternatively, by Southern blot and use of appropriate probes, northern blot and use of specific mRNA probes of the genes of interest or their Corresponding cDNA, mapping with Sl nuclease, RT-LCR, hybridization, microarrays, etc., preferably, by real-time quantitative PCR using appropriate probe sets and primers. Similarly, the level of cDNA corresponding to the mRNA encoded by the RpIp 1 gene can also be quantified using conventional techniques; in this case, the method of the invention includes a step of synthesis of the corresponding cDNA by reverse transcription

(RT) of the corresponding mRNA followed by amplification and quantification of the amplification product of said cDNA. Conventional methods of quantifying expression levels can be found, for example, in Sambrook et al, 2001. "Molecular cloning: a Laboratory Manual.", 3 ^rd ed, Cold Spring Harbor Laboratory Press, NY, VoI.. 1 -3.

In a preferred embodiment of the invention, quantification of the expression level of the RpIp 1 gene is performed by a polymerase chain reaction (PCR), in any of its variants, or by an array of DNA or RNA.

In another particular embodiment, the determination of the expression levels of the RpIp 1 gene is performed by quantifying the level of the protein encoded by said gene, that is, the RpIp 1 protein.

The expression level of RpIp 1 can be quantified by any conventional method that allows detecting and quantifying said protein in a sample of a subject. By way of illustration, not limitation, the levels of RpIp 1 can be quantified, for example, by the use of antibodies capable of binding RpIp 1 (or fragments thereof containing an antigenic determinant) and the subsequent quantification of the complexes formed. The antibodies used in these assays may or may not be labeled. Illustrative examples of markers to be They can use include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, etc. There is a wide variety of known assays that can be used in the present invention, which use unlabeled antibodies (primary antibody) and labeled antibodies (secondary antibody); These techniques include Western blotting or Western blotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (sandwich ELISA with double antibody), immunocytochemical and immunohistochemical techniques , techniques based on the use of biochips or microarrays of proteins that include specific antibodies or tests based on colloidal precipitation in formats such as dipsticks. Other ways to detect and quantify said RpIp 1 protein, include affinity chromatography techniques, ligand binding assays, etc.

In a particular embodiment, the quantification of the levels of the protein encoded by the RpIp 1 gene is performed by western blot, ELISA, immunohistochemistry or an array of proteins.

In a second stage, the method comprises comparing the level of RpIp 1 obtained in the test sample with that of a biological control sample.

"Control biological sample" is defined as the biological sample that is obtained from subjects who are well documented clinically and who do not have the disease. In said samples (reference or control) the reference or control concentrations of RpIp 1 will be determined, for example by giving an average value of the concentrations of the reference population. For the determination of the reference concentration of RpIp 1, a series of considerations must be taken. Among these considerations are the type of sample, sex, weight, age, physical condition of the patient and others similar. For example, an equal number of samples from a group of at least 2, at least 10, at least 100 to preferably 1000 subjects, preferably arranged according to the characteristics of the subject indicated above, will be taken as the reference group. The person skilled in the art understands that the detection, diagnosis and evolution will be carried out by detecting the increase (over-expression) or the decrease in the expression of RpIp 1 with respect to the reference value. It is considered that there is a rise in the expression in relation to the reference value of RpIp 1 when the difference between the sample under examination and the reference (control sample) is at least 1.1, 1.5, 5, 10 -, 20-, 30-, 40, 50, 60, 70, 80, 90, 100 times or more.

In a preferred embodiment, the detection of RpIp 1 is performed on a sample of a patient with colon cancer, the biological control sample being a sample of the same non-cancerous tissue patient, in a more preferred form the control sample will be a set of samples from a well-characterized reference control population.

Method to differentiate between the presence of adenoma and the presence of a malignant tumor in a subject.

The study by the inventors of the expression of RpIp 1 in samples of benign tumors (adenomas) with respect to samples of non-cancerous tissue (control biological samples) and of cancerous tissue, allows the development of methods for the differentiation between a adenoma (benign tumor) and a malignant tumor.

In another aspect, the invention relates to a method for differentiating between the presence of an adenoma and a malignant tumor in a subject comprising:

(i) quantify the expression of RpIp 1 in a biological sample of said subject, and (ii) compare said level of expression of RpIp 1 previously obtained with that of a biological control sample, where an increase in said expression level of RpIp 1 in relation to that of the biological control sample it is indicative of malignant tumor. In a first stage, the method comprises quantifying the expression of RpIp 1 in a cancerous sample of said subject.

In a second stage the method comprises comparing the level of expression with a biological control sample, the diagnosis of a malignant tumor being, if the expression level of RpIp 1 is higher than that of the biological control sample. In the opposite case, if the level of Rplpl expression is similar between the tumor tissue and the biological control sample, it can be concluded that the tumor tissue is a colon adenoma,

The quantification of the expression of Rplpl, both in the biological test sample and in the biological control sample, can be carried out by any of the previously mentioned methods. Biological sample and biological control sample have been previously defined.

An "adenoma" is a benign epithelial tumor whose internal structure is similar to that of a gland.

There are many different kinds of adenomas depending on the location such as: Thyroid adenoma, adrenal adenoma, bronchial adenoma, colon adenoma, prostate adenoma, pleomorphic adenoma (frequently appears in the salivary glands), liver adenoma, pituitary adenoma, breast adenoma, adenoma of pancreas, parathyroid adenoma, testicular adenoma and renal adenoma, among others.

In a preferred embodiment, the detection of Rplpl is performed in a tumor sample of the colon, the control biological sample being a sample of the same non-cancerous tissue patient, in a more preferred form the control sample will be a set of samples from a well-characterized reference control population. In the event that the level of Rplpl expression is similar between the tumor tissue and the biological control sample, it can be concluded that the tumor tissue is a colon adenoma, in the opposite case, where the Rplpl expression levels of the tumor tissue are increased with respect to levels of the biological control sample, the tumor tissue would be considered a malignant tumor.

The invention is described below by means of the following examples that should be considered as merely illustrative and not limiting thereof.

EXAMPLES

EXAMPLE 1

Materials and methods

Construction of a cDNA library

Two types of murine ES cells (CGR.8 and Rl) were used for RNA extraction. The cDNA of the two cell lines was cut with restriction enzymes

EcoRI and Xhol and was inserted into a p-pureMaRx retroviral vector previously linearized with these enzymes (Promega, Madison, WI), as described (25). The resulting cDNA library contained 6x10 ⁶ different clones.

Cell culture and infections

Mouse embryonic fibroblasts were isolated from a strain of pregnant mice (CDI) at 13.5 days gestation. The LinXE packaging cell line was used to produce the retroviruses (4). The cells were grown in DMEM (Lonza, Basel, Switzerland) with 10% SFT (Lonza). MEFs were infected in pass 4 (P4) with the cDNA library. In parallel, the positive and negative proliferation controls, the GFP and p53175H genes, respectively, were included to validate that the clones found in the genetic screening were really positive. After puromycin selection, the cells were seeded at low density and grown for more than a month. Small clones of cells that had prevented replicative senescence were expended in culture, and mRNA was extracted to detect clones inserted by RT-PCR with specific primers of the MaRx retroviral vector. The amplification product of RpIp 1 PCR was subcloned into the retro viral vector pII-hygro-MaRx using the EcoRI and Xhol enzymes for the 5 'and 3' ends, respectively. Sequencing indicated that the clone corresponded to the full-length cDNA of RpIp 1.

Growth curves

Mouse embryonic fibroblasts were infected with GFP, RpIp 1 and p53175H, and after selection with hygromycin, they were seeded at low density. After 20 days, the plates were stained with violet crystal to observe the colony formation assay and then stained with acetic acid to determine the number of cells. MEFs infected with the indicated oncogenes were seeded at 1x10 ⁵ cells per 10 cm plate; every three days, the cells were counted and seeded at the same density as indicated in a 3T3 protocol.

For soft agar assays, MEFs cells were infected with GFP, RpIp 1 and p53175H plus an empty vector against the ^Va112 cDNA. After 14 days of selection, the cells were counted and seeded at a density of 1x10 ⁵ cells per well in a 6-well plate in standard medium with 0.2% agarose. The culture medium of the cells was changed every 3 days and the colonies were counted after 20-30 days in culture. Infected cells were selected in hygromycin (50 μg / mL) or puromycin (2 μg / mL) as appropriate for both

MEFs as for NIH3T3 cells.

The foci formation was done by growing the infected MEFs in the P14 pass with GFP-ras, RpIp 1 -ras and p53175H-ras in confluent plates without antibiotic selection. In this pass, the cells were already immortalized. The medium was changed every 3 days and the foci were counted after 15 to 20 days.

Immunoblot

For immunoblotting, Cells were prepared as described (26) and resolved in polyacrylamide gels. Immunoblotting was done using antibodies against p53 (DO7 of DAKO, Denmark), b-actin

(A5316 from Sigma, St. Louis, MO), pio (sc-468 from Santa Cruz Biotechnology, CA), pl9 (ab80 from abcam, RU), SSEA-I (MC-480 from Developmental Studies Hybridoma Bank, Iowa, IA) and the mouse monoclonal antibody against RpIp 1 that was kindly donated by Dr. JP García-Ballesta.

Luciferase Assay

Mouse embryonic fibroblasts, 1299 H cells, and HEK293T cells were seeded in triplicate in 12 wells at a density of 50-10 x ⁵ cells per well. The cells were transfected with JetPei (Genycell, Granada, Spain) using the following plasmids: pGL3basic, pGL3basic with the promoter indicator of p21 -luciferase designated pGL3basicp21-Luc, p53WT, mdm2, Rplpl and an empty vector designated Va (per vector alone, vector only). For the E2F1 luciferase assay the following plasmids were used: pGL3basic, E2F1-WT, and pGL3basic with the E2F1 -luciferase promoter indicator designated pGL3basicE2Fl-Pm-Luc kindly provided by Dr. M. Serrano. The cells were used 48 hours later and a fifth of the sample was tested following the manufacturer's instructions (Promega).

Real-time PCR

Real-time PCR reactions were carried out in triplicate using the Assays-on-Demand Taqman Gene Expression Assays of Applied Biosystems. Probes for murine Rplpl (Mn02601846_gl) and human Rplpl (HsO1653O88_gl) were used. For the endogenous human genes, the following probes were used: b-actin (Hs99999903_ml), POL2A (HsOOl 72187_ml), and for mouse b-actin (Mn00607939_sl), and 18S RNA (4310893E) for both species.

Human subjects

RNA from normal and tumor tissue was extracted from each patient (Quiagen, Hilden, Germany) and cDNA synthesis was performed as described (27). Samples from six patients were used for protein extraction to immunoblot with antibodies against p53 (DO-7) and Rplpl. All the procedures of this study have been approved by the Ethics Committee of the Valí d'Hebron Hospital. EXAMPLE 2

RpIp 1 is able to avoid replicative senescence in primary cells. The proliferation capacity of ES cells against primary cells and the lung cancer cell line DV90 was studied by plotting growth curves (Figure la). ES CGR8 and R1 cells were fully characterized by the presence of the specific cell surface marker, SSEA-I (data not shown) (28). Based on the observation of the proliferation capacity of ES cells, MEFs were infected with an ES cDNA library. Surviving clones were selected based on the phenotypic appearance of proliferating cells. A clone with very high proliferation was identified as the Rplpl wild ribosomal protein cDNA. While GFP infected cells underwent replicative senescence, RpIp 1- MEFs had an extended morphology similar to that of p53 ^175H- MEFs (Figure Ib). The cDNA encoding human p53 ^{175H was used} as the positive proliferation control (6).

The function of Rplpl was evaluated by plotting growth curves of MEFs infected with the GFP, Rplpl and p53 ^{175H genes} , carrying out an experiment in 3T3 (Supplementary Figure 1).

To determine whether the proliferation effect provided by Rplpl was common to all ribosomal proteins as a general mechanism, primary MEFs were also infected with the L34 ribosomal protein cDNA. L34 also belongs to the large subunit of the ribosome, but not to the P group. Unlike MEFs infected with Rplpl, MEFs with L34 entered senescence (Figure Ic). Therefore, the proliferation effect observed for Rplpl was specific and not a common property of all ribosomal proteins.

Rplpl expression was tested at the level of mRNA and protein (Figure Id).

EXAMPLE 3 Effects of Rplpl on the p53 and Rb pathways

To explain the supposed role of Rplpl in avoiding replicative senescence, the role of Rplpl in a p53-dependent transcriptional activation construct was examined. A p21wafl-luciferase indicator vector was arranged as a transcriptional target of p53. No effect of Rplpl was observed on the p21 promoter in primary MEFs (Supplementary Figure 2) or in human cells without p53, as in SAOS2 or H 1299 cells against the empty vector (supplementary figure 2 and data not shown).

To study the Rb pathway, it was analyzed whether overexpression of Rplpl had any effect on an E2F1-dependent promoter. MEFs were co-transfected with human E2F1-WT, a luciferase indicator with synthetic E2F promoter pGL3basicE2Fl-Pm-Luc, and Rplpl. Interestingly, a two-fold increase was observed in MEFs transfected with Rplpl against the parental vector under the same conditions, in the presence or absence of E2F1 (Figure 2a). Other oncogenic cell lines were also studied: HEK 293T, NIH3T3, SAOS2, and H 1299 cells. With the exception of H 1299 cells, in which a small increase was detected, there were no significant differences in any of them (supplementary figure 3 and data not shown). To determine if one of the well-recognized E2F1 targets in the RpIp 1-MEFs was affected, the cyclin E mRNA was studied by quantitative RT-PCR (Figure 2b).

To analyze whether Rplpl mRNA levels changed during the spontaneous immortalization of the MEFs, MEFs were immortalized by carrying out a 3T3 protocol. Cell extracts were collected from several countries. MRNA of

Rplpl showed a two-fold decrease during senescence, and when immortalization occurred, Rplpl mRNA increased to levels similar to those of primary MEFs (Figure 2c), indicating that Rplpl expression was associated with cell proliferation.

The pio and pl9 proteins were analyzed in RpIp 1-MEFs by immunoblot. There were no differences in the expression of these proteins in RpIp 1 -MEFs compared to GFP-MEFs (data not shown), indicating that RpIp 1 prevents replicative senescence without affecting two classic genes whose state affects the senescence process.

EXAMPLE 4

RpIp 1 cooperates with ras ^Va112 in the transformation.

To assess whether RpIp 1 had any transformation capacity in MEFs or NIH3T3 cells, the growth curves of MEFs infected with GFP + ras ^{Va112 were plotted} against Rplpl + ras ^Va112 , using as positive controls p53175H + ras ^Va112 or Ela + ras ^Va112 (Figure 3a). Soft agar assays were performed after infection and cell selection with the different oncogenes, including cooperation with ras ^Va112 (Figure 3b). Colony formation of NIH3T3 cells that co-express Rplpl and oncogenic ras ^Va112 growing on soft agar was observed (Figure 3 c). The foci formation was seen in spontaneously immortalized MEFs in the P14 pass (Figure 3d). In contrast to immortalized cells, Rplpl did not contribute to the transformation when it was coexpressed with oncogenic ^Va112 flush in primary MEFs (data not shown).

From these data, the consequence is that Rplpl contributes to the transformation of immortalized cells when other genetic events occur, such as oncogenic ^Va112 ras.

EXAMPLE 5

Expression of Rplpl in human tumors The final purpose of this study was to determine whether the gene identified by genetic screening had any relevance in vivo in human tumors. For this purpose, mRNA levels in normal versus tumor tissue of colon cancer patients were determined by quantitative RT-PCR. This type of cancer was chosen for this study because it was found that Rplpl was augmented in a colon cancer DNA chip screen (18). In 13 of 22 cases, the mRNA of

Rplpl was increased in tumor tissue compared to normal (Figure 4a). Six cases were chosen from the 13 that showed increased mRNA in the tumor tissue for a immunoblot study; in three cases it was found that the Rplpl protein was increased (50%) (Figure 4b). These 6 cases were analyzed by immunoblot for p53 protein expression and a clear overexpression was verified. An example of 2 patients is shown (Figure 4b).

EXAMPLE 6

Increase in Rplpl expression after p53-175H expression. It was also determined whether Rplpl was affected by the expression of a p53 mimicking gene, p53175H. The Rplpl protein was increased in both NIH3T3 and MEFs expressing p53175H (Figure 5a).

To verify that Rplpl was not induced by wild p53 (p53WT), NIH3T3 cells were infected with p53WT. After proper selection, the levels of the Rplpl protein did not change when compared to control cells infected with the GFP vector (data not shown).

EXAMPLE 7

Rplpl expression in metastasis

To assess whether Rplpl had any relevance in vivo in the development of metastases in cancer patients, Rplpl mRNA levels in colon cancer patients diagnosed with metastases and patients where no metastasis had been detected were determined. This type of cancer was chosen for this study because it was found that Rplpl was augmented in a colon cancer DNA chip screen (18).

Relative levels of mRNA were detected using RT-PCR. Rplpl was detected using specific Taqman probes. Normalization of the values obtained from Rplpl mRNA was performed using the detection of endogenous genes. Rplpl expression levels were studied in a series of 22 patients diagnosed with colon cancer. Of these patients, 8 were positive for the diagnosis of metastasis, while 14 of the patients were diagnosed as absence of metastases. As you can see in Figure 6, after performing analysis Statistical (Wilcoxon test using SPSS), a significant increase in the expression of RpIp 1 was observed in the tumor tissue samples of patients diagnosed with metastases compared to that of patients without metastases (p = 0.026).

EXAMPLE 8

Differentiation between adenoma and malignant colon tumor

To study whether the increase in the level of RpIp 1 mRNA observed in cancer tissue appears during progression from a non-cancerous tissue to a cancerous tissue, 6 patients diagnosed with adenomas and carcinomas in the colon tissue were studied. The graph in Figure 7 represents the means of the results of the 6 patients with their corresponding standard deviation. In these patients, an increase in the level of RpIp 1 was observed twice in the cancerous tissue (T) in relation to the control tissue (N) and the tissue from the adenoma (A).

Claims

1. RpI Ip, a functionally equivalent variant thereof or a polynucleotide encoding RpIp 1 or a functionally equivalent variant for use in medicine.

2. RpI Ip, a functionally equivalent variant thereof, a polynucleotide encoding RpIp 1 or a functionally equivalent variant thereof for the treatment of a disease related to an unwanted cellular senescence.

3. A compound capable of inhibiting the expression and / or activity of RpIp 1.

4. A compound according to claim 3 wherein the compound is selected from the group of siRNAs, antisense oligonucleotides, specific ribozymes and specific inhibitors of RpIp 1

5. A pharmaceutical composition comprising an inhibitor according to claims 3 or 4.

6. A compound according to claims 3 or 4 for use in medicine.

7. A compound according to claims 3 or 4 for the treatment of diseases associated with an unwanted cell proliferation.

8. A compound according to claim 7 wherein the disease associated with an unwanted cell proliferation is caused by a Ras mutation.

9. A compound according to claim 8 wherein the Ras mutation is

Ras ^Va112 .

10. A compound according to any of claims 7 to 9 wherein the disease associated with an unwanted cell proliferation is caused by an increase in the expression of E2F1 target genes.

11. A compound according to claim 10 wherein the E2F1 target gene is the cyclin E encoding gene.

12. An in vitro method for the identification of proteins capable of activating cell proliferation or preventing replicative senescence comprising (i) contacting a cell of limited proliferative capacity with a trunk cell cDNA library, (ii) selecting those cells in which cell proliferation or inhibition of replicative senescence is observed and

(iii) characterize the library member of the selected cells in (ii)

13. Method according to claim 12 wherein the stem cells are of embryonic origin.

14. In vitro method for activating cell proliferation or for preventing replicative senescence comprising the introduction into a RpIp 1 cell, of a functionally equivalent variant thereof or of an expression vector comprising a polynucleotide sequence encoding the protein RpIp 1 or a functionally equivalent variant thereof.

15. In vitro method for transforming a cell of a primary culture comprising the introduction into a cell of a first expression vector comprising a polynucleotide sequence encoding the RpIp 1 protein or a functionally equivalent variant thereof and of a second expression vector comprising a sequence encoding a pro-oncogenic variant of Ras.

16. The method according to claim 15 wherein the pro-oncogenic variant of Ras is Ras ^Va112 .

17. In vitro method for the identification of compounds capable of blocking cell proliferation or blocking the avoidance of replicative senescence comprising the steps of:

(i) contacting a cell in which RpIp 1 protein is overexpressed with a candidate compound and

(ii) identify those compounds that block cell proliferation induced by RpIp 1 or that block the avoidance of replicative senescence induced by RpIp 1.

18. In vitro method for the identification of compounds capable of blocking cell transformation comprising the steps of: (i) contacting a cell in which RpIp 1 protein is overexpressed and in which a pro variant is expressed -onchogenic Ras with a candidate compound and

(ii) identify those compounds that block the cellular transformation induced by RpIp 1 and the pro-oncogenic variant of Ras.

19. Method according to claim 18 wherein the pro-oncogenic variant of Ras is Ras ^Va112 .

20. Method according to any of claims 17 to 19 wherein the cell additionally contains a construct comprising a reporter gene under the control of an E2F promoter.

21. Method according to any of claims 17 to 19 wherein the cell expresses a negative dominant mutant of p53.

22. Method according to claim 18 wherein the negative dominant mutant of p53 is p53 ^175H .

23. Method for diagnosing a metastasis in a subject affected by cancer and / or to predict the propensity to develop a metastasis in a subject affected by cancer comprising: (i) quantifying the expression of RpIp 1 in a biological sample of said subject, and (ii) compare the level of expression of RpIp 1 previously obtained with that of a control biological sample. where an increase of said level in relation to that of the biological control sample is indicative of a positive diagnosis of metastasis or a greater propensity for metastasis.

24. Method according to claim 23, wherein the biological control sample is a sample of a control subject or control group and / or a sample of a non-cancerous tissue of the same subject to be diagnosed.

25. Method according to any of claims 23-24, wherein the cancer is a colon cancer.

26. Method for differentiating between an adenoma and a malignant tumor in a subject comprising:

(i) quantify the expression of RpIp 1 in a biological sample of said subject, and

(ii) comparing said level of expression of RpIp 1 previously obtained with that of a control biological sample, where an increase of said level of expression of RpIp 1 in relation to that of the biological control sample is indicative that the subject has a tumor evil one.

27. A method according to claim 28 wherein the adenoma is a colon adenoma and the malignant tumor is a colon cancer.