WO2010043713A2

WO2010043713A2 - Use of cyclin o in a medical setting

Info

Publication number: WO2010043713A2
Application number: PCT/EP2009/063609
Authority: WO
Inventors: Gabriel GIL GÓMEZ
Original assignee: Institut Municipal D'investigació Mèdica (Imim)
Priority date: 2008-10-16
Filing date: 2009-10-16
Publication date: 2010-04-22
Also published as: WO2010043713A3

Abstract

The present invention relates to a method for identifying responders for adjuvant therapy. Furthermore, a mutant Cyclin O is provided. Moreover, the present invention relates to the use of Cyclin O or mutant Cyclin O in a medical setting. Corresponding pharmaceutical compositions are provided.

Description

Use of Cyclin O in a medical setting

The role of Cyclms and Cdks in cell cycle regulation is well known in the art, based on compelling data from genetic and biochemical approaches¹. However, their role in apoptosis is, due to the lack of clear genetic evidence, not well defined.

Thymocytes are a good model to study the connection between apoptosis and cell cycle, since about 90% of the cells of the normal thymus are quiescent and do not progress through the cell cycle in vitro. As a consequence, any activation of cell cycle-related kinases is not necessarily related to cell cycle progression. Activation of Cdk2 has been shown to be obligatory for the apoptosis of quiescent cells such as thymocytes as well as endothelial cells dying by trophic factor withdrawal⁴. This activation of Cdk2 is specific to apoptosis and is not an abortive attempt to re-enter the cell cycle^J.

It is known in the art⁵ that Cdk2 activation is an early step in cell death that precedes both loss of plasma membrane asymmetry and activation of apical Caspases. This implies that Cdk2 activation is upstream of the translocation of the Bcl2 family members Bax and Bid to the mitochondria and the loss of mitochondrial function that finally results in the release of Cytochrome c into the cytoplasm. Activation of Cdk2 during thymocyte apoptosis does not seem to be a consequence of binding to Cyclin A or Cyclin E, the canonical Cdk2 activating Cyclins in its cell cycle regulatory function³.

Previously, a new member of the Cyclin family has been identified and characterized. This member has been termed "Cyclin O"; see PhD thesis of Maurici Brunet Roig (2006), Departament tie Ciencies Experimentalis i de Ia Salut, Universitat Pompeu Fabra, Barcelona. Spain,

The known human A and B Cyclins have an amino acid similarity of only about 30 % with human Cyclin O. Members of the Cyclin family share a homologous region of about 100 amino acids called "Cyclin box". The "Cyclin box" contains structural motifs necessary for interaction with Cdks. Due to its presence the newly found protein described in Roig (2006) was belived to be associated with the Cyclin family. However, the functional role of Cyclin O and whether it might be involved in cell cycle regulation at all remained elusive in the art.

The generic Cyclin O locus encodes two transcripts that arise from the use of the alternative promoter Pl (human/mouse Cyclin Oa (Cyclin O alpha) and the alternatively spliced product Cyclin Oβ (Cyclin O beta). The sequence of the alpha form of Cyclin O has been detected in all the vertebrate genomes sequenced up to now. It is a highly conserved gene in its middle and C-terminal part, where the Cyclin Box is located, while the first 100 amino acid residues are less conserved. It is not present in plants, invertebrates, fungi or prokaryotes. The structure of the Cyclin O alpha gene is always formed by three exons separated by two small introns. The alternatively spliced transcript of the beta form of Cyclin O encodes a protein which lacks part of the Cyclin box. Its expression has been confirmed both in murine and human tissues.

Thus, the technical problem underlying the present invention is the provision of means and methods for the medical intervention of proliferative diseases. Moreover, the technical problem is the finding of a use for Cyclin O, in particular a therapeutic application for Cyclin O.

The technical problem is solved by provision of the embodiments characterized in the claims.

The advantages of the present invention can be summarized as follows:

- The method of identification of a responder to adjuvant therapy allows selecting a patient group which shows a higher survival rate when treated with adjuvant therapy. This patients/patient group is suspected to suffer from or being prone to suffer from a proliferative, in particular a cancerous disease such as colorectal carcinoma. - Furthermore, a novel mutant of Cyclin O alpha is provided which has a particularly strong proapoptotic activity. This mutant or other Cyclin O transcript variants (in particular Cyclin O-beta) can be used in the treatment of diseases, e.g. by inducing cell death in diseased cells. Without being bound by theory it is believed that cells can neither adapt to the expression of the Cyclin O-alpha mutant nor of Cyclin O-beta due to their high proapoptotic activity. As documented in the appended examples, Cyclin O beta is expressed in most mouse and human tissues at extremely low levels, which again may be due to its potent proapoptotic role.

In one embodiment, the present invention relates to a method for the identification of a responder for or a patient sensitive to adjuvant therapy, said method comprising the following steps:

(a) obtaining a sample from a patient suspected to suffer from or being prone to a colorectal carcinoma, whereby said sample is histopathologically categorized as normal

(healthy) and said colorectal carcinomais associated with a stage II tumor;

(b) determining in said sample obtained in (a) the expression level of Cyclin O ;

(c) comparing the expression level of Cyclin O of Cyclin O determined in (a) with a reference expression level of said nucleic acid molecule. wherein the extent of difference between said expression level determined in (b) and said reference expression level is indicative for a responding patient or is indicative for a sensitivity of said patient to adjuvant therapy.

As pointed out above, there is a need in the art for means and methods for the treatment of proliferative diseases and evaluation of biological samples. In particular markers are needed, which can predict the outcome of an anti-cancer therapy prior to and during treatment with adjuvant therapy. There is a need for stratification of patients who are to be subjected to or are being subjected to said adjuvant, anti-cancer therapy and distinguishing between "responders'^" and ''non-responder" patients.

It was surprisingly found that an unexpected group of patients that is characterized by a high expression or overexpression of Cyclin O in a histopathologically normal (healthy) sample, whereby said patient group is suspected to suffer from or being prone to suffer from a proliferative disease (in particular colocrectal carcinoma) is highly responsive to adjuvant therapy; see Example 4. "High expression^*' or "overexpression" in this context means that the expression level of Cyclin O is higher (preferably statistically significant) in the herein described histopathologically normal sample of a patient than in a corresponding sample of a healthy person. Preferably, the expression level is 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, more preferably, 10-fold, 15-fold, even more preferably 20-fold and more, higher than in such a sample from a healthy person (which can be considered in this context as a reference or control level).

The "extent of difference" used in this context reflects, accordingly, the higher expression in the sample obtained from a patient suspected to suffer from or being prone to suffer from a proliferative disease.

As illustrated in the appended examples, Cyclin O is overexpressed in histopathologically normal (healthy) samples taken from the margins of surgically resected colorectal cancer specimens. The overexpression of Cyclin O (in particular Cyclin O alpha and/or Cyclin O beta) in these samples is used for the identification of patients who will have shorter survival times and thus, would particularly benefit from adjuvant therapy. The expression level of Cyclin O is, thus, used herein for the identification of patients who can benefit from adjuvant therapy. The term "colorectal carcinoma" is well known in the art. As used herein, it refers to a type of colorectal cancer which is characterized in that the origin of the neoplastic cells is epithelial, in the case of colorectal cancer the gut epithelium. Also the term "colorectal cancer" ( also called colon cancer or large bowel cancer) is well known in the art. Colorectal cancer is a malignant neoplastic disease of the large intestine which includes cancerous growths in the colon, rectum and appendix. A subject/person may be prone to suffer from a colorectal carcinoma when it is suspected that it suffers from this disease, however, the diagnosis needs, for example, to be confirmed. A final confirmation may sometimes only be made after resection and analysis of the tumour. The term "suspected to suffer from" reflects a relatively higher degree of certainty that a patient/subject indeed suffers from colorectal cancer.

Preferably, the colon mucosa sample obtained from a patient suspected to suffer from or being prone to suffer from colorectal carcinoma is a colon carcinoma sample. Preferably, the sample is taken from the vicinity or margin of the herein described colorectal tumor. In other words, it is preferred that the sample contains or consists of tissue or cells of said vicinity or margin. It is preferred thai the tissue or cells of the vicinity/margin of the colorectal tumor are, in contrast to the "tumourous tissue" (tissue or cells of the colorectal tumor) substantially histopathologically normal (healthy). Though the sample of the vicinity or margin may contain tumour cells or tissue, it is preferred herein that the content of the tumour cells or tissue in this sample is neglible, preferably below 10 %, more preferably, below 5 %, even more preferably, below 1 % or even lower. Most preferably, the sample does not contain detectable levels of tumour cells or tissue, i.e. tumour cells or tissue can neither be detected histopathologically nor are they visible to the naked eye. If the sample contains tumour cells it is preferred that the content of these cells in the sample is adapted to levels as described above by appropriate means prior to the analysis of Cyclin O expression. Preferably, the tumour cells or tissue is completely removed or at least adapted to a non-detectable level as defined above. However, should it not be feasible to remove the tumour cells or tissue or adapt the level of a skilled person is aware of means and measures for taking this into account in the analysis of the expression level of Cyclin O in the sample. For example, an artisan will be in the position to calculate the expression level of Cyclin O of normal/healthy cells in the sample depending on the percentage of tumour cells in the sample.

A skilled person will be aware that the spatial extension of the "vicinity of the colorectal tumor" or the "margin of the colorectal tumor" will depend on various factors, such as the tumour location, tumour spread, and the like. However, an artisan is, based on his general knowledge and the teaching provided herein, easily in the position to identify and resect cells or tissue from the above mentioned "vicinty" of "margin".

In the following, an exemplary procedure for identifying such cells or tissue(s) is described: Surgical removal of the involved segment of colon (colectomy) along with its blood supply and regional lymph nodes is the primary therapy for colon cancer. Usually, the partial colectomies are separated into right, left, transverse, or sigmoid sections based on the blood supply. The removal of the blood supply at its origin along with the regional lymph nodes that accompany it ensures an adequate margin of normal colon on either side of the primary tumor. When the cancer lies in a position such that the blood supply and lymph drainage between two of the major vessels, both vessels are taken to assure complete radical resection or removal (extended radical right or left colectomy). If the primary tumor penetrates through the bowel wall, any tissue adjacent to the tumor extension is also taken if feasible.Usually the surgeon can distinguish between the tumour and the healthy parts and decide which area should be removed. However, if there is niicroinfiltration of the healthy tissue by the tumour, this may only be discovered after histopathological analysis. Again, here applies the above explanation, for example, thai the sample should, preferably, contain non- detectable level of tumour cells. Nonetheless, a skilled person is usually in the position to resect the tumour (histopathologically classified as tumourous tissue) with appropriate margins (histopathologically classified as normal or healthy tissue). In appended example 3. it is shown that the margin may extend up to 8 cm from the colorectal tumour.

However, in contrast to the colorectal tumour cells or tissue, the cells or tissue of the above described colon mucosa sample is histopathologically categorized as normal (healthy). Notwithstanding the above explanation that the sample may contain tumour cells in minor amounts, it is to be understood that the expression level of Cyclin O of histopathologically normal (healthy) cells or tissue of this sample is primarily to be assessed. As also explained herein above and below, the expression level of Cyclin O in histopathologically normal (healthy) of the sample is indicative for a responsiveness to adjuvant therapy.

The sample is described in more detail further below.

It has been surprisingly found herein that overexpression of Cyclin O, in particular Cyclin O alpha, correlates with the presence of certain proliferative diseases as defined and described herein above. Example 3 provides experimental data which show that Cyclin O is overexpressed in adenocarcinomas (colorectal carcinoma). Further carcinomas found to overexpress Cyclin O are shown in the Table of Example 3. item I. In contrast, Cyclin O expression is typically indetectable (or at most detectable at only very low levels) in the normal epithelium (e.g. colon mucosa, bladder tissue, lung tissue) in healthy persons, whereas it is highly expressed in the tumour.

However, it has been surprisingly been found herein that in colorectal carcinoma Cyclin O (in particular Cyclin O-alpha) is both overexpressed in the tumour (i.e. in tissue histopathologically categorized as tumorous tissue) and also in the normal epithelium (i.e. tissue that may be found in close vicinity to the tumour, but categorized histopathologically as healthy). Most importantly, it has been found in respect of colorectal carcinoma that high expression of Cyclin O in histopathologically normal epithelium is indicative for an increased likelihood to respond to adjuvant therapy.

As demonstrated in the appended examples, the expression of Cyclin O in normal (i.e. healthy) colon mucosa in healthy persons (i.e. persons not suspected of suffering from or being prone to suffering from colorectal carcinoma) is very low. Cyclin O is overexpressed when the epithelial cells acquire the malignant phenotype. As shown herein, Cyclin O is overexpressed in more than 90% of the adenoma and adenocarcinoma samples (i.e. "malignant" lesions). In benign lesions (polyps), only 60% of the samples are positive, whereas in the samples taken from the margins of the surgically excised tumours that the pathologist diagnosed as tumour free (histopathologically categorized as "normal" (healthy) colon mucosa), surprisingly a 40% of Cyclin O positive samples has been found. It is of note that these 40% of histopathologically "normal" samples but Cyclin O positive samples are indicative for a decreased survival rate of patients.

In sum, there herein provided data demonstate that patients suffering from stage II colorectal carcinoma who show overexpression of Cyclin O in the samples taken from the margins of the tumour that were diagnosed as morphologically "normal" by a pathologist have a long term survival shorter than patients with Cyclin O negative tumour margins. The patients identified herein profit particularly from adjuvant therapy. As stated herein above, there is up to now, no consensus in oncology whether stage II colorectal cancer patients who undergo surgery should, subsequent to surgery, be treated with a second therapy (adjuvant therapy). In contrast, all stage I patients are usually cured only by surgery and in stage III and IV tumours the patients will receive adjuvant therapy in any case since the disease has spread out. In view of the severe side effects associated with adjuvant therapy (such as chemotherapy etc.), it is highly desirable to only subject patients to this therapy which are likely to respond. The herein provided method allows for the first time the identification of stage II colorectal cancer patients which are responsive to adjuvant therapy.

As also shown herein, the presence of Cyclin O alpha and/or beta sensitizes cells to adjuvant therapy. For example, in vitro experiments in cell lines transfected with anti-Cyclin O shRNAs (i.e. downregulation of Cyclin O) leads to resistance Io chemo and radiotherapy.

As described herein below in more detail, assessment of the expression level of Cyclin O may be performed by evaluating the protein expression level of Cyclin O or the mRNA level individually or by performing two or three types of evaluation after one another or in parallel. The terms "responder for adjuvant therapy^"' means in the context of the present invention that a patient suspected to suffer from or being prone to suffer from a colorectal carcinoma shows a response to a treatment to adjuvant therapy. An artisan will readily be in the position to determine whether a person shows a response to treatment with adjuvant therapy. The term "'adjuvant therapy" as used herein refers to treatment after (a first) surgery. Chemotherapy, radiotherapy and second surgery are non-limiting examples of said adjuvant therapy. For example, a response to adjuvant therapy may be reflected in a decreased suffering from a proliferative disease, in particular colorectal carcinoma, such as the prevention of the growth of a tumour (prevention of recurrence of the tumor) after adjuvant therapy, a diminished and/or halted growth of a tumor that recurred after adjuvant therapy and/or a reduction of the size of a tumor that recurred after adjuvant therapy, the prevention of the formation of metastases or a reduction of number or size of metastases after adjuvant therapy. The above explanations also apply in cases where the tumor has not been completely resected by surgery, i.e. a part of the tumor tissue (though perhaps not visible to the naked eye) has remained in the body.

Similarly, the term "'patient sensitive to a adjuvant therapy" refers in the context of the present invention to a patient which shows in some way a positive reaction when treated. This reaction of the patient may be less pronounced when compared to a responder as described herein above. For example, the patient may experience less suffering from colorectal carcinoma though no reduction in tumor growth may be measured.

The reaction (response/sensitivity) of the patient to adjuvant therapy may also be only of a transient nature, i.e. growth of (a) tumor and/or (a) metastasis(es) may only be temporarily reduced or halted. However, it is preferred that a responder for adjuvant therapy will not suffer from colorectal carcinoma after treatment with adjuvant therapy. Preferably, (a) tumor(s) and/or (a) metastasis(es) which has been resected will not recur within 1 year after termination of the treatment of the responder, more preferably within 2 years, 3 years, 4 years, 5 years. 10 years or, most preferably within 15 year after termination of the treatment.

As mentioned above, adjuvant treatment is a given after the primary treatment (surgery) to increase the chances of a cure. Adjuvant therapy may include chemotherapy, radiation therapy, hormone therapy, or biological therapy. In contrast, neoadjuvant therapy is known as treatment given before the primary treatment, e.g. chemotherapy prior to surgery. Examples of neoadjuvant therapy include chemotherapy, radiation therapy, and hormone therapy. Uusually it is given to first reduce the size of the primary tumour in order to increase the chances of complete removal by surgery. Determining Cyclin O levels in a biopsy of the tumour can also be useful to predict the efficacy of neoadjuvant therapy. In this context, a sample may be taken prior to surgery and the expression level of Cyclin O be determined. A high expression level of Cyclin O in histopathologically normal cells or tissue from the margin/vicinity of the tumour is indicative for a responsiveness to neoadjuvant therapy. All explanations and definitions given herein in context of adjuvant therapy apply, mutatis mutandis to neoadjuvant therapy.

Determining the determination of the responsiveness to neoadjuvant therapy may be particularly relevant in selecting patients suffering from (or being prone to suffering from) colorectal carcinoma of the annus and/or of the rectum. In such cases radiotherapy is often given as neoadjuvant therapy prior to surgery.

Non-limiting examples of cbemotherapeutic agents to be used in adjuvant or neoadjuvant therapy are antimetabolites (5-fluorouracil, methotrexate, etc); antibiotics (adriamycm, bleomycin, etc); alkylating agents (cyclophosphamide, nitrosoureas, etc); plant alkaloids (taxanes; vinca alkaloids, etc); topoisomerase inhibitors (etoposide; camptothecins, etc); monoclonal antibodies (herceptin. avastin, etc); hormonal therapy (tamoxifen, finasteride, etc).

There are three main divisions of radiotherapy to be used in adjuvant/neo adjuvant therapy external beam radiotherapy (EBRT or XBRT) or teletherapy, brachytherapy or sealed source radiotherapy and unsealed source radiotherapy. The differences relate to the position of the radiation source; external is outside the body, while sealed and unsealed source radiotherapy has radioactive material delivered internally. Brachytherapy sealed sources are usually extracted later, while unsealed sources may be administered by injection or ingestion. Proton therapy is a special case of external beam radiotherapy where the particles are protons. Introperative radiotherapy is a special type of radiotherapy that is delivered immediately after surgical removal of the cancer. This method has been employed in breast cancer (TARGeted Introperative radioTherapy), brain tumours and rectal cancers. In general, colorectal carcinoma is quite radioresistant. However, colorectal carcinoma of the annus and rectum, are relatively radiosensitive, and readiotherapy is particularly useful in such a context.

Also monoclonal antibodies against EGFR (Epidermal Growth Factor Receptor) are used in adjuvant/neoadjuvant therapy. Preferably, anti-EGFR antibodies are used in combination with chemotherapy in context of metastatic colorectal cancer. Also inhibitors of the tyrosine kinase activity of this receptor (small molecules) may be useful in this context. It is known that EGF is a growth-promoting factor produced by many cells and one of its receptors

(EGFR) is overexpressed by many gastrointestinal tumours, particularly in a subset of colorectal tumours. The anti-EGFR antibodies block EGFR action and the cells die by apoptosis. Another example of hormonal (or anti-hormonal treatment) is the use or anti-IGFR monoclonal antibodies, which are now on clinical trials for colon cancer. These antibodies block the binding or the growth factor IGF (Insulin-like Growth Factor) to its receptor

(IGFR). which is also overexpressed in cancer cells, avoiding its proliferative actions. An example of biological therapy may be the use of colon cancer vaccines to induce immunity directed against proteins made by the tumour such as CEA (carcinoembrionary antigen) or using cells from the tumour as antigens.

It is preferred herein that the sample is obtained from the patient prior to surgery or from the surgically removed tumour and that the responder to adjuvant therapy identified in accordance with the present invention may. accordingly, be subjected to said adjuvant therapy.

Tumors, in particular, colorectal tumors are classified according to the following scheme:

The '"pT^"' parameter measures the degree of invasion of the normal colon structures by the tumour, as determined by the pathologist (the "p^*! prefix denotes pathologic (rather than clinical) assessment). The staging of the tumour is a result of the combination of the "pT" parameter and two additional parameters, namely the "N" parameter, which determines whether the tumour has spread to the local and regional lymph nodes (one or more than one positive lymph node) and the "M" parameter, which indicates the presence or absence of distant metastatic disease. Overall they form the so called TNM classification system. TNM is a well known term in the art and is an abbreviation of "tumour/lymph nodes/distant metastasis". The TNM classification system is well known in the art and routinely applied by practioners and pathologists.

Herein below follows the colorectal staging classification according to TNM:

"T" in the above table denotes the "pT" parameter, "N" denotes the "N" parameter and "M" denotes the "M" parameter.

It is apparent from the above table that a "stage II" tumor is assessed when the parameter "pT" is T3, "N" is NO and "M" is MO (Stage II A); a "stage II" tumour is also assessed when the parameter "pT" is T4, "N" is NO and "M" is MO (Stage II B).

In the following explanations of the various levels of parameters "pT", "N" and "M" are given. pTl : Tumours which invade into but not through the submucosa pT2: Tumours which invade into but not through the muscularis propia pT3: Tumours which invade through the muscularis propia into the subserosa or into non peritonealised pericolic or perirectal tissue pT4: Tumours which invade other organs or structures or perforate the visceral peritoneum. pNO: all lymph nodes examined are negative for tumour cells pNl : Tumours with metastasis in one to three regional lymph nodes pN2: Tumours with metastasis in four or more regional lymph nodes

Only two "M" categories exist: MO: no evidence of distant metastasis Ml : distant metastasis present

In case the "M" parameter cannot be determined in a sample pathologists sometimes use the subindex "x" (for example Mx). .

Tissue and/or cells colorectal tumours ("tumour cells") can easily be distinguised by a skilled person from "normal" tissue and/or cells. For example, the practicioner may distinguish these tissues with the naked eye and/or by histopathological assays. Corresponding means and methods are well known in the art and also shown in the appended examples. Accordingly, the skilled person can easily distinguish a sample (and tissue/cells contained therein) as described herein (e.g. a sample from the margin/vicinity of a colorectal tumor) which is categorized normal from other samples (for example, colorectal tumour tissue/tumour cells).

The following relates to the measurement of expression levels of Cyclin O, in particular of Cyclin O alpha and/or Cyclin O beta.

As mentioned, a person skilled in the art will be aware of corresponding means and methods for detecting and evaluating the expression level. Exemplary methods to be used include but are not limited to molecular assessments such as Western Blots, Northern Blots, Real-Time PCR and the like. Usually the activity of the Cyclin O protein is reflected by the expression level, i.e. the activity correlates with the expression level. For example, the kinase activity of the Cyclin O-aipha isoforra is believed to correlate with the expression level of Cyclin O- alpha.

Tf the gene product is an RNA, in particular an mRNA (e.g. unspliced, partially spliced or spliced mRNA), determination can be performed by taking advantage of northern blotting techniques, hybridization on microarrays or DNA chips equipped with one or more probes or probe sets specific for mRNA transcripts or PCR techniques referred to above, like, for example, quantitative PCR techniques, such as Real time PCR. These and other suitable methods for binding (specific) mRNA are well known in the art and are. for example, described in Sambrook and Russell (2001, loc. cit). A skilled person is capable of determining the amount of the component. In particular said gene products, by taking advantage of a correlation, preferably a linear correlation, between the intensity of a detection signal and the amount of the gene product to be determined.

In case the component is a polypeptide/protein, quantification can be performed by taking advantage of the techniques referred to above, in particular Western blotting techniques. Generally, the skilled person is aware of methods for the quantitation of (a) polypeptide(s)/protein(s). Amounts of purified polypeptide in solution can be determined by physical methods, e.g. photometry. Methods of quantifying a particular polypeptide in a mixture rely on specific binding, e.g of antibodies. Antibodies specifically binding to Cyclin O (either to both Cyclin O-alpha and Cyclin O-beta or capable of specifically binding to only Cyclin O-alpha or Cyclin O-beta are also disclosed herein and illustrated in the appended examples. Specific detection and quantitation methods exploiting the specificity of antibodies comprise for example immunohistochemistry (in situ). Western blotting combines separation of a mixture of proteins by electrophoresis and specific detection with antibodies. Electrophoresis may be multi -dimensional such as 2D electrophoresis. Usually, polypeptides are separated in 2D electrophoresis by their apparent molecular weight along one dimension and by their isoelectric point along the other direction. Alternatively, protein quantitation methods may involve but are not limited to mass spectrometry or enzyme-linked immunosorbant assay methods.

It may be that an aberrant (high) activity of Cyclin O, in particular Cyclin O-alpha, is determined (in a sample obtained) from a patient. This activity may, for example, be an aberrant (high) kinase activity of Cyclin O protein and may be measured by corresponding methods known in the art. Without being bound by theory, an aberrant activity of Cyclin O activity, in particular kinase activity of Cyclin O, may be caused by oncogenic point mutations which increase the activity of the Cdk/Cyclin complexes or which change the biochemical properties of Cyclin O.

The following relates to samples in general (e.g. colon mucosa samples) obtained from a patient/subject suspected to suffer from or being prone to suffering from a proliferative disease. As mentioned above, sample is preferably obtained derived from a human being suffering from a proliferative disease, e.g. adenocarcinoma or transitional bladder carcinoma. In context of this invention particular useful samples are, accordingly, human cells. These cells can be obtained from e.g. biopsies or from biological samples but the term "'cell" also relates to in vitro cultured cells.

As used herein, the term "'cell, tissue and cell culture^*' is not only limited to isolated cells, tissues and cell cultures but also comprises the use of samples, i.e. biological, medical or pathological samples that consist of fluids that comprise such cells, tissues or cell cultures. Such a fluid may be a body fluid or also excrements and may also be a culture sample, like the culture medium from cultured cells or cultured tissues. The body fluids may comprise, but are not limited to blood, serum, plasma, urine, saliva, synovial fluid, spinal fluid, cerebrospinal fluid, tears, stool and the like.

The meaning of the terms "cell(s)^"', "tissue(s)" and "cell culture(s)'^" is well known in the ait and may, for example, be deduced from "The Cell" (Garland Publishing, Inc., third edition). Generally, the term "cell(s) used herein refers to a single cell or a plurality of cells. The term "plurality of cells'^" means in the context of the present invention a group of cells comprising more than a single cell. Thereby, the cells out of said group of cells may have a similar function. Said cells may be connected ceils and/or separate cells. The term "tissue" in the context of the present invention particularly means a group of cells that perform a similar function. The term "cell culture(s)'^" means in context of the present invention cells as defined herein above which are grown/cultured under controlled conditions. Cell culture(s) comprise in particular cells (derived/obtained) from multicellular eukaryotes, preferably (transgenic) animals as defined elsewhere herein. It is to be understood that the term "cell culture(s)" as used herein refers also "tissue culture (s)" and/or "organ culfure(s)^"', an "organ" being a group of tissues which perform the some function.

The present invention also relates to a kit for carrying out the methods of this invention. For example, said kit comprises (a) compound(s) for specifically determining the expression level of Cyclin O, in particular Cyclin O alpha and/or Cyclin O beta. . On the basis of the teaching of this invention, the skilled person knows which compound(s) is(are) required for specifically determining the expression level of Cyclin O as defined herein. Particularly, such compound(s) may be (a) (nucleotide) probe(s), (a) primer(s) (pair(s)), (an) antibody(ies) and/or (an) aptamer(s) specific for Cyclin O, in particular Cyclin O alpha and/or Cyclin O beta as described herein.,

In a particularly preferred embodiment of the present invention, the kit (to be prepared in context) of this invention or the methods and uses of the invention may further comprise or be provided with (an) instruction manual(s). For example, said instruction manual(s) may guide the skilled person (how) to determine the (reference) expression level of (a) marker gene(s) described herein, i.e. (how) to diagnose a disease or a susceptibility thereto, (how) Io monitor the efficacy of a treatment of a disease or a susceptibility thereto or (how) to predict the efficacy of a treatment of a disease disease or a susceptibility thereto in accordance with the present invention. Particularly, said instruction manual(s) may comprise guidance to use or apply the herein provided methods or uses.

The kit (to be prepared in context) of this invention may further comprise substances/chemicals and/or equipment suitable/required for carrying out the methods and uses of this invention. For example, such substances/chemicals and/or equipment are solvents, diluents and/or buffers for stabilizing and/or storing (a) compound(s) required for specifically determining the expression level of Cyclin O, in particular Cyclin O alpha and/or Cyclin O beta as defined herein.

The following relates to the Cyclin O to be used in accordance with the present invention. Generally, the term '"Cyclin O" used herein refers to any gene product, in particular an amino acid sequence, having (partial) Cyclin O activity as described herein and nucleic acid sequence(s) encoding such (an) amino acid sequence(s). The term "Cyclin O" as used herein refers, in particular in context of "expression of Cyclin 0". to a nucleic acid molecule encoding a Cyclin O gene product (e.g. mRNA, RNA transcript, protein). In context of ''activity of Cyclin O" the term "Cyclin 0'^' refers in particular to the gene product, e.g. Cyclin O protein. The term Cyclin O as used herein refers to various transcript variants of Cyclin O₅ namely Cyclin O-alpha, Cyclin O-beta, Cyclin 0-gamma and Cyclin O-delta. Preferably, the Cyclin O transcript variant to be used herein is Cyclin O-alpha or Cyclin O-beta. The terms ^■'Cyclin O-alpha" and "Cyclin O-α", "Cyclin O-alpha'^" and "Cyclin O-β'\ -'Cyclin 0-gamma" and "Cyclin O-γ", and "Cyclin O-delta^"' and "Cyclin O-δ" (and grammatical variants thereof), respectively, are used interchangeably herein.

The nucleotide sequences and amino acid sequences of the above-mentioned Cyclin O transcript variants are depicted in SEQ ID NOs: 1 to 8, 11 and 12. It is of note that only

Cyclin O-alpha and Cyclin O-alpha transcript variants encode a protein, whereas Cyclin O- gamma and Cyclin O-delta encode an RNA transcript. The nucleotide sequences and amino acid sequences of human and murine Cyclin O-alpha and Cyclin O-beta are depicted in SEQ

ID NOs 1 to 8, respectively. SEQ ID NOs: 11 and 12 show the nucleotide sequence of human Cyclin 0-gamma and murine Cyclin O-delta, respectively. The transcript variant Cyclin O- gamma has so far only be isolated and identified in humans/human tissue whereas transcript variant Cyclin O-delta has merely been isolated and identified in mice/murine tissue.

In a further, independent embodiment, the present invention relates to a mutant of the Cyclin O-alpha transcript variant is provided in the present invention. The mutant has been surprisingly found to be highly proapoptotic. This mutant is also termed "L3A mutant",

"'Cyclin O-alpha mutant". "Cyclin O-alpha L3A mutant^" or "mutant Cyclin O" herein.

Generation and biochemical/hi stochemical characterization of said mutant is given in

Example 5. The mutations performed are also shown in Figure 30. In particular, Leucines 92, 95 and 97 present in the conserved motif LxxLxL of murine Cyclin O alpha were replaced by alanines.

Yet. it is believed that the replacement of the first leucine of the LxxLxL motif may not be essential for the highly apoptotic activity of the mutant protein. Accordingly, also a mutant where the first leucine is not replaced or mutated is envisaged herein. Moreover, this first leucine is also not conserved in all species. For example, in the human Cyclin O alpha, a valine residue is at the corresponding position. In other words, it is believed that the essential residues are the two leucines from the LxL motif, which are also invariant in all the species Accordingly, it is preferred herein that the leucines of the LxxLxL motif are replaced where applicable. More preferably, at least one of the two leucines (preferably both) from the LxL motif are replaced in the mutant proteins. It is preferred that one leucine is replaced by one alanine though other amino acids (preferably having same or similar biochemical properties as alanine may under certain circumstances be used). Since these leucines are, as mentioned, conserved in Cyclin O orthologs, a person skilled in the art is, based on his general knowledge and the teaching provided in the present invention, easily in the position to provide correspondingly mutated Cyclin O orthologs, such as a human Cyclin O-alpha L3A mutant. As described herein below, a skilled person is in the position to identify further Cyclin O orthologs, in particular Cyclin O-alpha orthologs, and can, accordingly, provide further mutant Cyclin O orthologs, for example a porcine Cyclin O- alpha L3A mutant, a fish Cyclin O-alpha L3A mutant and the like. The mutated murine nucleotide and amino acid sequence of the Cyclin O-alpha L3A mutant is depicted in SEQ ID NOs 9 and 10, respectively. The correspondingly mutated human nucleotide and amino acid sequence of the Cyclin O-alpha L3A mutant is depicted in SEQ ID NOs 13 and 14, respectively

The one (or three) letter code used for annotating the above-mentioned mutations in the amino acid sequences of the (mutated) Cyclin O gene product, is well known in the ait and may be deduced from standard text books (such as "The Cell", Garland Publishing, Inc. third edition). A comprehensive list of the specific list of amino acids and their respective abbreviations using the one (or three) letter code is given herein below:

Accordingly, the present invention relates in one embodiment to a nucleic acid molecule selected from the group consisting of

(a) a nucleic acid molecule having a nucleic acid sequence as depicted in SEQ ID NO 9 or 13;

(b) a nucleic acid molecule having a nucleic acid sequence encoding a polypeptide as depicted in SEQ ID NO 10 or 14;

(c) a nucleic acid sequence which is capable of hybridizing to the complementary strand of the nucleic acid sequence of (a) or (b) and encoding a functional mutant Cyclin O- alpha or a functional fragment thereof;

(d) a nucleic acid molecule having a nucleic acid sequence having at least 60 % homology to the nucleic acid sequence of (a) or (b) and encoding a functional mutant Cyclin O-alpha or a functional fragment thereof;

(e) a nucleic acid molecule having a nucleic acid sequence being degenerate as a result of the genetic code to the nucleic acid sequence as defined in any one of (a), (b), (c) and

(d); and

(f) the complementary strand of the nucleic acid molecule of any one of (a) to (e).

Furthermore, the present invention relates to a polypeptide selected from the group consisting of

(a) a pol^ypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO 9 or 13;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO 10 or 14;

(c) a polypeptide comprising an amino acid encoded by a nucleic acid molecule encoding a peptide having an amino having an amino acid sequence as depicted in SEQ ID NO

9 or 13; (d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional mutant Cyclin O-alpha or a functional fragment thereof; (e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), and encoding a functional mutant Cyclin O-alpha or a functional fragment thereof; and

(f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having a nucleic acid sequence which is degenerate as a result of the genetic code to the nucleic acid sequence of a nucleic acid molecule as defined in (a), (c). (d) and (e).

As mentioned above, the Cyclin O-beta variant has been identified herein as having a particularly high proapoptotic activity which may, without being bound by theory, be due to the fact that it is involved in a completely different signalling pathway, namely the induction of ER stress, compared to the alpha variant. Cyclin O beta is expressed in most mouse and human tissues at extremely low levels, most likely because of its potent proapoptotic activity. Prima facie, the beta form lacks two α-helixes of the Cyclin-box domain which are essential for the interaction with Cdkl/2. Accordingly, it is believed that the mechanism by which the Cyclin O-beta variant may exert its highly proapoptotic activity is Cdk independent.

As shown in the appended examples, the beta variant is located in the endoplasmic reticulum (ER) but it also colocalizes with markers of stress granules such as TIA-I . Cyclin O beta can not bind Cdks, although it is highly proapoptotic, using a signalling pathway completely different to the one used by the alpha form, namely the induction of ER stress. Cyclin O beta is expressed in most mouse and human tissues at extremely low levels. Without being bound by theory this low level may be due to its potent proapoptotic role as documented in the experimental part of the present application.

The prior art has not been successful in obtaining cells which overexpress the beta form. suggesting that its overexpression leads to cell death. Surprisingly, the mutant of the alpha variant (the L3A mutant) provided herein has similar characteristics as the beta variant, for example, regarding subcellular localisation and cell death. Again, it is impossible to get cells stably overexpressing the Cyclin O alpha L3A mutant. The findings provided in the experimental part suggest that cells can adapt to overexpression of Cyclin O alpha (which may be related to its overexpression in tumours as described herein above), but cannot adapt to overexpression of Cyclin O beta or the L3A mutant. Accordingly, the L3A mutant or the beta valiant may be particularly useful in the treatment of (proliferative) diseases. Exemplary (proliferative) diseases have been described elsewhere herein, for example in context of diagnosing methods or pharmaceutical compositions.

The proapoptotic properties of the mutant of the Cyclin O alpha protein (L3 A mutant) may not necessarily correlate with an increased expression level of the protein, but rather with an increased kinase activity of the complexes with Cdks together with new biochemical properties that resemble those of the beta form. Without being bound by theory, the L3A mutant may show enhanced "'alpha-like" signalling plus newly acquired ''beta-like'^" apoptotic inducing properties. The L3A mutant is a triple point mutant that substitutes 3 leucine for alanine residues (L92A, L95A and L97A) in the mouse Cyclin O alpha sequence. However, the Cyclin box sequence remains intact, so L3A mutant may interact with and activate Cdkl/2. This sequence is conserved in all the known Cyclin O orthologs, from fish to man and seems to be involved in the subcellular localisation of the alpha/beta proteins. Then, both variants are located at the endoplasmic reticulum (ER), but the beta and the L3A mutant proteins form stress granules, whereas the alpha foπii does not. Stress granules are dense aggregations located in the endoplasmic reticulum composed of proteins and RKAs that appear when the cell is under stress. The RNA molecules stored are called translation pre- initiation complexes - failed attempts to make protein from mRNA. They are usually associated to apoptoεis induction by agents triggering ER stress by the unfolded protein response (UPR, accumulation of unfolded proteins in the ER) such as hypoxia, heat shock or oxidative stress.

The present invention relates also to antibodies specifically binding to the novel Cyclin O- alpha mutant as described and defined herein. Such antibodies can be produced by methods known in the art.

A cDNA highly homologous to human Cyclin Oγ locus was reported to encode a protein with uracil-DNA glycosylase activity¹⁷. However, the correct sequence of both the genomic locus and several ESTs show that this transcript does not encode the reported protein. Neither purified recombinant Cyclin Oa nor cell extracts obtained after its transient transfection into HEK293 cells show detectable uracil DNA-glycosy3ase activity as a consequence of its overexpression as measured by an enzymatic assay (Figure 46).

The following relates to Cyclin O transcript variants (and mutant Cyclin O) and the mechanism which may, without wishing to be bound by theory, underly their activity. Furthermore and without wishing to be bound by theory, it is believed that (proliferative) diseases characterized by an (over)expression of Cyclin O show, in particular, an (over)expression of Cyclin O-alpha and/or Cyclin O-beta, wherein Cyclin O-alpha shows a higher expression level than Cyclin O-beta. This relates to the fact that cells can be become tolerant to the overexpression of the alpha form but not to the expression of the beta form. Accordingly, a tumor overexpressing the beta form would be killed and, thus, it is believed that the Cyclin O-alpha transcript variant is the predominant variant in (proliferative) diseases characterized by an overexpression of Cyclin O. This is explained in the following in more detail, in particular in context of the mutant Cyclin O-alpha which shows a surprisingly high proapoptotic activity similar to the Cyclin O-beta variant.

The appended Examples, in particular Example 1 , show in depth characterisation of the function of human and mouse Cyclin O-alpha. Although its function is not directly related to cell cycle regulation, it is demonstrated that Cyclin O-alpha associates with Cdk2 and Cdkl and activates them. Neither the recombinant protein nor the native protein expressed in HEK293 cells have Uracil-DNA glycosylase activity as reported in some prior art documents; see also Figure 46.

In Example 1 , the isolation and characterization of a Cdk2 and Cdkl activating Cyclin O variant specific to apoptosis is shown. This Cyclin O variant is able to bind and activate Cdk2 in response to intrinsic apoptotic stimuli such as glucocorticoids or DNA damaging agents.

The de novo synthesis of Cyclin O precedes apoptosis induction. Downregulation of the expression of Cyclin O abrogates DNA damage and dexamefhasone-induced apoptosis. whereas CD95-induced apoptosis remains intact. This lack of apoptotic response to DNA damage and glucocorticoids is due to a failure to activate a supramitochondrial step leading to the activation of apical Caspases and not to defective signaling to these intrinsic stimuli. It has surprisingly been found herein that Cyclin O can induce apoptosis in lymphoid cells in concert with Cdk2. Previously, it has been speculated that a protein different to Cyclins A and E may be responsible for Cdk2 activation during apoptosis of thymocytes, as the levels of the canonical Cdk2 cyclins (Cyclins A and E) are barely detectable by Western blotting and the Cdk2 kinase activity detected is not associated with Cyclins A or E . Evidence is provided in the appended examples that Cyclin O as the most likely candidate to be the so-called Apoptosis Related Cdk2 Activator.

Cyclin O binds to Cdkl and Cdk2, leading to their activation (Figure 3). In wild type mouse fibroblasts where both kinases are expressed. Cdk2 is the main kinase, forming complexes with Cyclin O. However, in the absence of Cdk2_f Cdkl binds and gets activated by Cyclin O, which further confirms the observations of the redundancy of Cdk2 as found in Cdk2 KO mice¹¹. Binding of a Cyclin to different Cdks is not a surprising finding in itself, given the well characterized high degree of redundancy in the Cdk family¹⁹.

As shown in appended Example 1, the expression of Cyclin O is rapidly elevated in the thymus and spleen of the mouse and in WEHI7.2 cells after irradiation or dexamethasone treatment. This pattern, especially in the thymus, is compatible with expression of the Cyclin preceding apoptosis induction. Thus, the kinetics of Cyclin O expression after apoptosis induction is compatible with a causal role in apoptosis, a role demonstrated by the use of RNA interference both in WEHI7.2 cells (Figures 5A and 6A), in EL-4 lymphoid cells, mouse fibroblasts and in U2OS human osteosarcoma cells (data not shown).

It has been shown previously in different experimental models , that CD95 is an example of extrinsic stimuli which is independent of Cdk2 activation, and that anti-CD95-induced apoptosis cannot be blocked by Cdk2 chemical inhibitors. We show that in WEHI7.2 cells,

CD95-induced apoptosis is completely independent of protein synthesis. In fact, it is necessary to add the protein synthesis inhibitor, cycloheximide, in order to uncover the proapoptotic action of anti-CD95 antibody. Inhibiting protein synthesis blocks Cyclin O production and so (Figure 7), CD95-induced apoptosis is indistinguishable between a control clone (shGl) and a Cyclin O-interfered clone (3.7). Previously, the activation of Cdk2 has been positioned in DNA damage and glucocorticoid- induced thymocyte apoptosis as an early step preceeding phosphatidylserine exposure, mitochondrial dysfunction and Caspase activation^''. The appended example provides evidence that Cyclin O downregulation leads to a complete apoptosis block that is characterized by a lack of phosphatidylserine exposure, mitochondrial dysfunction and a total absence of activation of the apical Caspases, Caspase-8 and -9 and the executory Caspase-3. In addition to this, the induction of Cyclin O mRNA after γ-radiation treatment is independent of Bax or Bcl2, indicating that its action lies upstream of the mitochondrial action of the Bcl2 family proteins. However, the magnitude of induction of the Cyclin mRNA is increased by transgenic expression of Bax and diminished by Bcl2, perhaps reflecting a regulation of the apoptosis amplification feedback loop²⁰.

The Cyclin O shRNA clone 3.7 shows normal Bak translocation to the mitochondria, while Bax is not translocated. However, genetic evidences show that tBid generation is necessary to oligomerizate Bak or Bax and induce Cytochrome c release²¹. Due to the defect in Caspase-8 activation, clone 3.7 is unable to generate tBid and. therefore, to induce Cytochrome c release, in spite of the normal translocation of Bak to the mitochondria (Figure 17). Defective Cytochrome c release to the cytosol explains the lack of Caspase-9 and Caspase-3 activation due to the inability to assemble the apoptosome¹⁶.

Our findings support the notion that Cyclin O is the Apoptosis-Related Cdk2 Activator protein predicted for thymocytes to undergo apoptosis by intrinsic stimuli. The evidence provided herein also suggests that the only role of Cyclin O-Cdk complexes in lymphoid cells is the regulation of apoptosis through the control of activation of apical Caspases such as Caspase-8. It has been previously described that de novo gene expression is required in thymocytes downstream of Cdk2 activation and before Caspase-8 cleavage\ These results would be compatible with Cyclin O-Cdk2 complexes activating latent transcription factor(s) resulting in the transcription of genes necessary for the activation of the apical Caspase.

The fact that Cyclin O-Cdkl/2 complexes are involved in the activation of Caspases, opens the possibility that they may also regulate their activation during other non-apoptotic functions, such as tissue differentiation, cell proliferation or maturation of proinflamatory cytokines (reviewed in ). Induction of apoptosis by unscheduled activation or expression of cell cycle regulators such as Cyclins or Cdks has been extensively described (see²⁴ for a recent review). Inappropriate activation of a Cdk such as Cdkl perturbs the normal cell cycle progression leading to checkpoint activation and apoptosis induction²⁵. Expression of a Cyclin in a quiescent tissue in the presence of a mitogenic stimulus such as serum, has been shown to lead to an abortive attempt to re-enter cell cycle, leading to apoptosis²⁶. However, these cases are based solely on the forced over-expression of components of the normal cell cycle machinery in altered conditions. The physiological function of the Cyclin characterized in the appended examples is to participate in the signalling of the intrinsic apoptotic stimuli at least in lymphoid cells. Its normal expression precedes apoptosis induction and if it is downregulated, apoptosis is blocked. Accordingly, Cyclin O can be considered as a novel apoptosis-specific regulator of Cdk activity.

The nucleic acid sequences of Cyclin O of other mammalian or non-mammalian species (in particular rat, mouse, pig, guinea pig, chimpanzee, macaque) than the herein provided sequences for human and murine Cyclin O can be identified by the skilled person using methods known in the ait, e.g. by nucleic acid sequencing or using hybridization assays or by using alignments, either manually or by using computer programs such as those mentioned herein below in connection with the definition of the term "hybridization" and degrees of homology. In one embodiment, the nucleic acid sequence encoding for orthologs of human or murine Cyclin O is at least 40% homologous to the nucleic acid sequences as shown in SEQ ID NOs: 1, 3, 5. 7, 11 ,12, 13 and 14. More preferably, the nucleic acid sequence encoding for orthologs of murine or human Cyclin O is at least 45%, 50%, 55%, 60%, 65%, 70%, 72 %, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% homologous to the nucleic acid sequence as shown in SEQ ID NOs. 1, 3, 5, 7, 1 1 12, 13 and 14, wherein the higher values are preferred. Most preferably, the nucleic acid sequence encoding for orthologs of human or murine Cyclin O is at least 99% homologous to the nucleic acid sequence as shown in SEQ ID NOs. 1, 3, 5, 7, 11 12, 13 and 14.

Hybridization assays for the characterization of orthologs of known nucleic acid sequences are well known in the art; see e.g. Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N. Y. (2001); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N. Y, (1989). The term "hybridization¹¹ or "hybridizes" as used herein may relate to hybridizations under stringent or non- stringent conditions. If not further specified, the conditions are preferably non- stringent. Said hybridization conditions may be established according to conventional protocols described, e.g., in Sambrook (2001) loc. cit.; Ausubel (1989) loc. cit, or Higgins and Hames (Eds.) "Nucleic acid hybridization, a practical approach" IRL Press Oxford. Washington DC, (1985). The setting of conditions is well within the skill of the artisan and can be determined according to protocols described in the art. Thus, the detection of only specifically hybridizing sequences will usually require stringent hybridization and washing conditions such as. for example, the highly stringent hybridization conditions of 0.1 x SSC. 0.1% SDS at 65⁰C or 2 x SSC. 60⁰C, 0.1 % SDS. Low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6 x SSC, 1% SDS at 65⁰C. As is well known, the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditi ons.

In accordance with the present invention, the terms "homology" or "percent homology" or "identical" or "percent identity" or '"percentage identity" or ''sequence identity" in the context of two or more nucleic acid sequences refers to two or more sequences or subsequences that are the same, or that have a specified percentage of nucleotides that are the same (preferably at least 40% identity, more preferably at least 45%, 50%. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity, most preferably at least 99% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 75% to 90% or greater sequence identity may be considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Preferably the described identity exists over a region that is at least about 15 to 25 nucleotides in length, more preferably, over a region that is at least about 50 to 100 nucleotides in length and most preferably, over a region that is at least about 800 to 1200 nucleotides in length. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245). as known in the ait.

The definitions given herein in respect of "homology" or "percent homology" or "identical" or "percent identity" or "percentage identity" or "sequence identity^"' of nucleic acid sequences also apply, mutatis mutandis, to amino acid sequences. A person skilled in the art is easily in the position to adapt the teaching given herein in respect of the identification of homologous nucleic acid sequences to homologous amino acid sequences.

Although the FASTDB algorithm typically does not consider internal non-matching deletions or additions in sequences, i.e.. gaps, in its calculation, this can be corrected manually to avoid an overestimation of the % identity. CLUSTALW, however, does take sequence gaps into account in its identity calculations. Also available to those having skill in this art are the BLAST and BLAST 2.0 algorithms (Altschul, (1997) Nucl. Acids Res. 25:3389-3402; Altschul (1993) J. MoI. Evol. 36:290-300; Altschul (1990) J. MoI. Biol. 215:403-410). The BLASTN program for nucleic acid sequences uses as defaults a word length (W) of 11. an expectation (E) of 10, M=5, N=4. and a comparison of both strands. The BLOSUM62 scoring matrix (Henikoff (1989) PNAS 89: 10915) uses alignments (B) of 50. expectation (E) of 10, M=5, N=4. and a comparison of both strands.

In order to determine whether a nucleotide residue in a nucleic acid sequence corresponds to a certain position in the nucleotide sequence of e.g. SEQ ID NOs: 1, 3, 5, 7, 11. 12, 13 and 14, the skilled person can use means and methods well-known in the art. e.g., alignments, either manually or by using computer programs such as those mentioned herein. For example, BLAST 2.0, which stands for Basic Local Alignment Search Tool BLAST (Altschul (1997), loc. cit.; Altschul (1993). loc. cit.; Altschul (1990), loc. cit), can be used to search for local sequence alignments. BLAST, as discussed above, produces alignments of nucleotide sequences to determine sequence similarity. Because of the local nature of the alignments. BLAST is especially useful in determining exact matches or in identifying similar sequences. The fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP). An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cut-off score set by the user. The BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches, which satisfy the user-selected threshold of significance. The parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output.

Analogous computer techniques using BLAST (Altschul (1997), loc. cit; Altschul (1993). loc. cit.; Altschul (1990), loc. cit.) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL. This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score which is defined as:

% sequence identity x %, maximum BLAST score

100

and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1-2% error; and at 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules. Another example for a program capable of generating sequence alignments is the CLUSTALW computer program (Thompson (1994) Nucl. Acids Res. 2:4673-4680) or FASTDB (Brutlag (1990) Comp. App. Biosci. 6:237-245), as known in the ait.

The definitions and explanations given herein above in respect of Cyclin O orthologs apply, mutatis mutandis, in respect of mutant Cyclin O as described herein and further mutant Cyclin O orthologs provided in accordance with the present invention.

In further embodiments, the present invention relates to a nucleic acid construct comprising a nucleic acid molecule as defined herein above in context of mutant Cyclin O or encoding a polypeptide as defined in this context. Also provided is a vector comprising this nucleic acid construct. Preferably, the vector comprises a nucleic acid molecule which is a regulatory molecule operably linked to said nucleic acid molecule as defined and described above. It is preferred that the vector is an expression vector.

Moreover, the present invention relates to a host cell genetically engineered with the nucleic acid molecule described above or comprising the nucleic acid construct described above or the vector described above. In one embodiment, the present invention relates Io a process for the production of a nucleic acid molecule as described above or a polypeptide provided herein, said process comprising culturing a above-described host cell under conditions allowing the production of the nucleic acid molecule and/or the polypeptide and recovering the nucleic acid molecule and/or the polypeptide from the culture.

Furthermore, the present invention relates to a pharmaceutical composition comprising Cyclin O and/or an agonist of Cyclin O. Preferably, the Cyclin O is Cyclin O-alpha, Cyclin O-beta and/or the mutant Cyclin O -alpha as described herein above.

Surprisingly, it has been found herein that high levels of Cyclin O-alpha, and in particular of Cyclin O-beta and/or the Cyclin O-alpha L3A mutant, are highly apoptotic. Cyclin O-alpha. Cyclin O-beta and/or the Cyclin O-alpha L3A mutant can, therefore, be used to kill cells due to their potent proapoptotic activity. This is particularly advantageous in the treatment of diseases where naturally occurring apoptosis is reduced or where diseases cells are highly proliferative. Cyclin O-alpha, Cyclin O-beta and/or the Cyclin O-alpha L3A mutant can be used in such diseases to induce apoptosis in diseased cells, thereby eliminating and deleting these cells or tissues containing these cells. Accordingly. Cyclin O-alpha, Cyclin O-beta and/or the Cyclin O-alpha L3A mutant are particularly useful in proliferative diseases, especially in cancerous diseases.

As shown in the appended examples (see, for example, Fig. 31), Cyclin O-alpha. Cyclin O- beta and/or the Cyclin O-alpha L3A mutant are capable of killing tumour cells. In these examples, the efficacious proapoptotic activity of Cyclin O-alpha. Cyclin O-beta and/or the Cyclin O-alpha L3A mutant is demonstrated in cell lines which are representative for cancerous diseases like adenocarcinoma (e.g. pancreatic adenocarcinoma) or sarcoma (e.g. osteosarcoma or fibrosarcoma). For example, the U2OS cell is a human osteosarcoma cell line (i.e. it is a cell line derived from a human osteosarcoma patient) and is thus representative for osteosarcomas. Phenotypically it is similar to the cell line Saos-2, another osteosarcoma- derived cell line. Both cell lines are also distributed as osteosarcoma cell lines by the American Type Culture Collection, the main cell line repository (WWW.ATCC.org). It is shown in Fig. 31 that Cyclin O-alpha, Cyclin O-beta and/or the Cyclin O-alpha L3A mutant can efficaciously induce apoptosis in U2OS cells. The colony transfection assay shown in Fig. 32 demonstrates that the constitutive expression in particular of Cyclin O-beta and of the Cyclin O-alpha mutant is not compatible with cell survival; i.e. cells cannot adapt to the expression of Cyclin O-beta and/or of the Cyclin O-alpha mutant.

The AR42 J cell line is derived from a rat pancreatic exocrine adenocarcinoma (WWW.ATCC.org). As shown for example in Figure 36 B, Cyclin O alpha, Cyclin O-beta and the Cyclin O-alpha mutant induce the transcription factor CHOP. The expression of CHOP is a generally recognized marker of apoptosis, in particular apoptosis induced by stress of the endoplasmic reticulum. In other words, its induced expression shows that Cyclin O alpha, Cyclin O-beta and the Cyclin O-alpha mutant are capable of inducing apoptosis in adenocarcinoma cells, in particular in pancreatic adenocarcinoma cells.

It is well known that fibroblasts give rise to fibrosarcomas when becoming malignant. Figure 36 C shows a clonogenic assay (=colonies surviving after transfection of expression plasmids) in mouse embryonic fibroblasts (wild type and PERK deficient) after overexpression of Cyclin O alpha and Cyclin O-beta. Fig. 36C clearly shows that expression of Cyclin O alpha and, in particular, Cyclin O-beta leads to decreased transformation efficiency and, thus, to reduced viability of fibroblasts.

In sum, the appended examples demonstrate that Cyclin O-alpha, Cyclin O-beta and/or the Cyclin O-alpha L3A mutant can successfully be used in the treatment of proliferative diseases, and in particular cancerous diseases such as adenocarcinoma (e.g. pancreatic adenocarcinoma) and sarcoma (e.g. osteosarcoma or fibrosarcoma) and the like.

It has been described above that Cyclin O, especially Cyclin O-alpha and, in fewer cases and at low levels also Cyclin O-beta, is expressed in a variety of tumours; see also the table in Example 3. However, this does not preclude Cyclin O having a proapoptotic activity, especially when overexpressed in cancerous or tumor cells. As described above and illustrated in the appended examples, the overexpression of Cyclin O-alpha, Cyclin O-beta and/or the Cyclin O-alpha L3A mutant results in cell death of the transfected cells.

^•'Overexpression" in this context generally means that Cyclin O-alpha on the one hand and Cyclin O-beta and/or the Cyclin O-alpha L3A on the other hand are expressed at high levels compared to control or reference levels of Cyclin O-alpha and Cyclin O-beta, respectively. Depending on the circumstances, the reference level may be determined in healthy tissue and/or in diseased tissue. Preferably, the Cyclin O variants are expressed at least at the same level, more preferably, at higher levels compared to the control or reference levels, e.g. levels found in diseased cells (tumour cells and the like). For example, if a patient suffers from a particular cancer, the Cyclin O variants are to be expressed at a higher level than typically found in a respective tumour. The reference level may also be determined by means and methods disclosed herein, for example, in a biopsy from the person/subject to be treated. The biopsy sample, may, e.g. be or be obtained from the resected tumour. It is preferred that the overexpression level is significantly higher than the reference level, A skilled person will know how to determine the appropriate expression level of the Cyclin O variants to be used herein. Generally, it is preferred that the expression level of Cyclin O variants is at least twice as high as the reference level (e.g. levels found in diseased ceils (inter alia, tumour cells), i.e. if the reference level is set as "100 %", the overall expression level should be at least "200 %^'' (2-fold expression level). However, also a 3-fold, 4-fold. 5-fold or even higher expression level is envisaged.

For example, levels of expression achieved by appropriate vectors known in gene therapy (e.g. Ientiviral vectors and the like) will usually lead to a high enough expression level of Cyclin O variants as defined and described herein. As demonstrated herein, infecting AR42J cells with Ientiviral vectors at a multiplicity of infection of 1 leads to the expression of CHOP after 24 hours. The expression levels of the protein coming from the viral vector are in the range of expression of the endogenous protein in the tumor.

Due to the high potency of Cyclin O beta and/or mutant Cyclin O, the relative expression level of these proteins may be lower than that of Cyclin O alpha. For example, the expression level of Cyclin 0 beta and/or mutant Cyclin O. respectively, may be 90 %, 80 %, 70 %, 60 % or 50 % (or even lower) of the expression level of Cyclin O alpha. Nonetheless, it is believed that Cyclin O beta and/or mutant Cyclin O are, even at such lower levels highly potent. Also a combination therapy of Cyclin O-alpha, Cyclin O beta and/or mutant Cyclin O is envisaged herein; for example, Cyclin O-alpha may be combined either with Cyclin O beta or mutant Cyclin O or with both. The combined use of Cyclin O beta and mutant Cyclin O is also envisaged. The use of Cyclin O. alpha may be particularly indicated when cells/tissue have not yet adapted to Cyclin O alpha overexpression, while combined therapy with Cyclin O beta and/or mutant Cyclin O may be indicated when cells/tissue(s) have at least partially adapted to Cyclin O overexpression. Therapy only with Cyclin O beta and/or mutant Cyclin O may be indicated when cells/tissue(s) have adapted to Cyclin O alpha overexpression. Therapy only with Cyclin O beta and/or mutant Cyclin O may also be useful when relatively lower overexpression levels of Cyclin O are desirable.

It is of note that the expression of Cyclin O observed in tumours in the human or animal body does not imply that Cyclin O leads to an increased apoptosis in these tumour cells. It is of note that an apoptotic activity of Cyclin O (in particular of Cyclin O-alpha, Cyclin O-beta and/or the Cyclin O-alpha mutant) has not been described in the art. In the present invention it was surprisingly found that that higher expression levels of Cyclin O-alpha, Cyclin O-beta and/or the Cyclin O-alpha mutant than usually found in tumours are necessary to induce or enhance apoptosis. The herein disclosed successful treatment of proliferative diseases, particularly cancerous diseases like adenocarcinoma (e.g. pancreatic adenocarcinoma) or sarcoma (e.g. osteosarcoma or fibrosarcoma) with Cycϋn O-alpha, Cyclin O-beta and/or the Cyclin O-alpha mutant represent the first use of Cyclin O (in particular Cyclin O-alpha, Cyclin O-beta and/or the Cyclin O-alpha mutant) in medicine.

The term "agonist" as used herein is known in the art and relates to a compound/substance capable of fully or partially stimulating the physiologic activity of (a) specific receptor(s). In the context of the present invention said agonist, therefore, may stimulate the physiological activity Cyclin O (in particular Cyclin O alpha, Cyclin O-beta and/or the mutant Cyclin O- alpha) upon binding of said compound/substance to said Cyclin O. As used herein the term "agonist" also encompasses partial agonists or co-agonists/co-activators. ϊn addition thereto, however, an "agonist" or "activator" of Cyclin O in the context of the present invention may also be capable of stimulating the function of a Cyclin O by inducing/enhancing the expression of the nucleic acid molecule encoding for said Cyclin O. Thus, an agonist/activator of Cyclin O may lead to an increased expression level of Cyclin O (e.g. increased level of Cyclin O mRNA, Cyclin O protein) which may be reflected in an increased activity of Cyclin O. This increased activity can be measured/detected by the herein described methods. An activator of Cyclin O in the context of the present invention, accordingly, may also encompass transcriptional activators of Cyclin O expression that are capable of enhancing Cyclin O function. The term "agonist" comprises partial agonists. As partial agonists the art defines candidate molecules that behave like agonists, but that, even at high concentrations, cannot activate Cyclin O to the same extend as a full agonist.

For example, it is shown herein that expression of Cyclin O can be strongly increased by the agonist dexamethasone; see Figure 10. This strong expression of Cyclin O leads to cell death e.g. in WEHI7.2 cells. As explained herein, these cells are representative for T cell lymphoma. Accordingly, it is particularly envisaged that T cell lymphoma can be treated with Cyclin O (and also Cyclin O agonists). However, also overexpression of Cyclin O as it is described herein and illustrated in the appended examples, can be understood as "agonizing'^' Cyclin O, i.e. Cyclin O would, then, be its own agonist.

Without being bound by theory, it is believed that cells, such as (tumour) cells found in human or animal tumors, can adapt to the expression of Cyclin O-alpha, in particular to expression levels in naturally occurring tumour cells, thus masking the proapoptotic activity of Cyclin O-alpha. As shown in the appended examples, cells cannot adapt to high levels of Cyclin O-beta or the Cyclin O-alpha mutant. It is also believed that this particularly high proapoptotic activity of Cyclin O-beta may be one reason for the fact that Cyclin O-beta is expressed at very low levels in tumour cells and in the human body. Without being bound by theory, it is thought that naturally occurring tumour cells tolerate only low levels of Cyclin O- beta. As illustrated in the appended examples, tumour cells can, however, not endure high levels of Cyclin O-beta and/or the mutant Cyclin O-alpha.

The following describes a mechanism that may, without wishing to be bound by theory, underly the apoptotic activity of Cyclin O (in particular Cyclin O-alpha. Cyclin O-beta and/or the mutant Cyclin O-alpha) and, hence, underly its use in the treatment of proliferative disease, especially cancerous diseases. It is generally acknowledged in the art that tumours develop from a group of cells which accumulate oncogenic mutations, activate the DNΛ damage response (which tries to repair the DNA damage or to kill by apoptosis the damaged cells) but succeeded in accumulating mutations that rendered them resistant to apoptosis induction by the DNA damage response (reviewed in Halazonetis et al. (2008), Science 319:1352-1355). Without being bound by theory, Cyclin O may be expressed as a consequence of the activation of the DNA damage response. In this context, it is of note that an expression of Cyclin O in tumour cells, let alone a role in apoptosis, has neither been described nor proposed in the art.

As illustrated in the appended examples. Cyclin O is expressed after DNA is damaged by gamma-radiation or radiomimetic drugs such as etoposide: see e.g. Figures 1C, 2, 5 A, 8. 1OA, 11, 15, 16 and 18. The adaptation to the DNA damage response described above might imply that cancer cells become "tolerant" to apoptosis induced by Cyclin O expression. In other words, Cyclin O might be expressed at relatively higher levels compared to "normal" (healthy) cells and apoptosis might still not be induced in the tumour cells. Again, there was no hint in the prior art that Cyclin O is involved in apoptosis. However, it has been surprisingly found herein and successfully shown that further increasing Cyclin Oa levels or, especially, Cyclin Oβ and/or the L3A mutant (Cyclin O alpha mutant) (i.e. increasing the expression levels above levels usually found in naturally occurring tumors) efficaciously kills the cells: see. for example the killing of T-cell lymphoma cells in Figure 16 (where cells positive for active Caspase-3 and Cyclin O appear, activation of Caspase-3 being generally accepted as one of the last steps in the apoptosis pathways), of pancreas adenocarcinoma cells in Figure 36B and of mouse fibroblasts in Figure 36C. As shown in appended Example 6. the underlying mechanism of the high apoptotic activity of Cyclin O may be its capacitiy to induce ER stress. It is believed herein that the induction of ER stress eventually leads to apoptosis of the cell. As demonstrated in the appended examples, Cyclin O beta and the Cyclin O alpha mutant are particularly potent inducers of ER stress.

For example, the pharmaceutical composition may comprise a Cyclin O alpha polypeptide selected from the group consisting of

(a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO 1 or 5; (b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO 2 or 6;

(c) a polypeptide comprising an amino acid encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO 2 or 6;

(d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional Cyclin O-alpha or a functional fragment thereof;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d). and encoding a functional Cyclin O-alpha or a functional fragment thereof; and

(f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having a nucleic acid sequence which is degenerate as a result of the genetic code to the nucleic acid sequence of a nucleic acid molecule as defined in (a), (c), (d) and (e).

Furthermore, the pharmaceutical composition may comprise a Cyclin O beta polypeptide selected from the group consisting of

(a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO 3 or 7;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO 4 or 8; (c) a polypeptide comprising an amino acid encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO 4 or 8;

(d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional Cyclin O-beta or a functional fragment thereof;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), and encoding a functional Cyclin O-beta or a functional fragment thereof; and

The terms "functional Cyclin O-alpha" or "functional Cyclin O-beta" used in context of the present invention refer to a polypeptide having at least 60 % homology to a polypeptide as defined in section (a) to (d) of the above-described specific aspects of the present invention which has essentially the same biological activity as a polypeptide having 100 % homology to a polypeptide as indicated in section (a) Io (d). Preferably, a "functional Cyclin O-alpha" or "functional Cyclin O-beta". has the same biological activity as a polypeptide shown in SEQ ID NO 2 or 6 or in SEQ ID NO 4 or 8, respectively. Methods for determining the activity Cyclin O alpha or Cyclin O beta (such as the pro-apoptotic (apoptosis-inducing) activity) are described herein and also illustrated in the appended examples. Further methods for determining the activity of Cyclin O alpha or Cyclin O beta are well known in the art and may, for example, be deduced from standard text books, such as Bioanalytik (Lottspeich/Zorbas (eds.), 1998, Spektruni Akademischer Verlag).

The explanations given herein above in respect of determining the activity of Cyclin O alpha or Cyclin O beta and "functional Cyclin O-alpha'^' or "functional Cyclin O-beta", respectively, also apply, mutatis mutandis, to a "'functional fragment of Cyclin O-alpha" or "functional fragment of Cyclin O-beta", respectively'^*. In other words, a functional fragment of Cyclin O- alpha or Cyclin O-beta has essentially the same activity as defined herein above as functional Cyclin O-alpha or functional Cyclin O-beta, respectively. As mentioned, methods/assays for determining the activity of the above described polypeptides and functional fragments thereof are well known in the art and also described herein above. There herein above provided explanations and definitions in respect of "functional Cyclin O-alpha" or "functional Cyclin O-beta", and a '"functional fragment of Cyclin O-alpha" or "functional fragment of Cyclin O- beta". respectively"apply, mutatis mutandis, to the herein described mutant Cyclin O alpha (Cyclin O L3A mutant).

Further embodiments of the present invention relate to a pharmaceutical composition, which comprises, in addition or in the alternative to Cyclin O alpha and/or Cyclin O beta, the herein defined Cyclin O alpha mutant. Accordingly, the pharmaceutical composition may comprise a nucleic acid molecule encoding a Cyclin O-alpha polypeptide as defined herein, a Cyclin O- beta polypeptide as defined herein and/or a nucleic acid molecule encoding the Cyclin O alpha mutant as defined herein or as produced by the herein above described process.

Moreoer, the herein provided pharmaceutical composition may comprise a nucleic acid construct comprising the nucleic acid molecule as defined above, a vector which comprises the nucleic acid construct, and/or a host cell which comprises the vector. The pharmaceutical composition may further comprise, optionally, suitable formulations of earner, stabilizers and/or excipients.

The proliferative disease to be treated in accordance with the present invention is preferably a cancerous disease and may, inter alia, be adenocarcinoma, sarcoma, transitional bladder carcinoma, primitive neuroepithelial tumor and squamous skin carcinoma. Non-limiting examples of adenocarcinoma are pancreas carcinoma, colorectal carcinoma, lung adenocarcinoma, well differentiated squamous lung carcinoma, stomach carcinoma, endometroid adenocarcinoma and breast adenocarcinoma. The term "pancreas carcinoma'^" as used herein refers particularly to pancreatic adenocarcinoma. Sarcomas may. inter alia, be osteosarcoma, fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, chondrosarcoma, and histiocytoma. The use of Cyclin O-beta and/or Cyclin O-alpha mutant in the treatment sarcoma and adenocarcinoma is particularly preferred herein, whereby osteosarcoma is a particularly preferred sarcoma to be treated. Also preferred herein is the treatment of fibrosarcoma. A specific adenocarcinoma preferably to be treated in accordance with the present invention is pancreatic adenocarcinoma. Furthermore, Cyclin O, in particular Cyclin O-beta, may be used in the treatment of diseases which are characterized by an insufficient apoptosis, such as autoimmune disorders or viral infections.

It is also envisaged in this context that not only proliferative diseases but also diseases characterized by pathologically elevated apoptosis (e.g. AIDS, neurodegenerative diseases/disorders such as Alzheimer^'s disease, Parkinson's disease, Amyotrophic lateral sclerosis, retinitis pigmentosa, cerebral degeneration), myelodysplatic syndromes, aplastic anaemia, post-ischaemic cell death (such as myocardial infarction, stroke, reperfusion injury) and postischaemic cell death, which are. preferably, characterized by Cyclin O (over)expression may be treated or prevented. In this context, use of antagonists/inhbitors of Cyclin O (in particular of Cyclin O-alpha and/or Cyclin O-beta) is envisaged. As demonstrated herein, Cyclin O (Cylin O-alpha and/or Cyclin O-beta) is expressed predominantly in diseased cells; thus antagonists against Cyclin O will specifically be efficacious and target these diseased cells characterized by Cyclin O expression. As mentioned above, it has been found herein that the Cyclin O variants Cyclin O-alpha, -beta and the alpha mutant are located at the endoplasmic reticulum (ER); however, the beta and the L3A mutant proteins form stress granules, whereas the alpha form does not. The RNA molecules stored in these stress granules are usually associated with the induction of apoptosis. Inter alia, the unfolded protein response (UPR, accumulation of unfolded proteins in the ER) which may be associated with hypoxia, heat shock or oxidative stress agents may trigger ER stress,

In the appended examples it is demonstrated that Cyclin O causes ER stress. In particular, Cyclin O-beta is a potent inducer of ER stress. Accordingly, antagonists/inhbitors of Cyclin O (in particular of Cyclin O-alpha and/or Cyclin O-beta) are useful in the treatment or prevention of diseases induced by ER stress, which are, preferably, characterized by Cyclin O (over)expression. As documented in the appended examples, dowrrregulation of Cyclin O expression with the shRNA leads to abrogation of apoptosis induced by drugs that induce ER stress (Figure 34B). This is a crucial finding which supports that inhibition of Cyclin O expression or the activity of its complexes can be used to treat diseases in which an exacerbated apoptosis (via ER stress or other Cyclin Odependent pathways) leads to pathologically elevated cell death.

The explanations given herein in respect of pharmaceutical compositions comprising Cyclin O, modes of administration of Cyclin O and so on also apply, mutatis mutandis, to the administration of antagonists of Cyclin O.

In the following, the phenomenon of ER stress and diseases induced by ER stress are described.

The lumen of the endoplasmic reticulum (ER) is the cellular site where most secreted and transmembrane proteins fold and mature. The folding process is monitored by a sensitive mechanism that avoids accumulation of misfolded proteins and their transit to the secretory pathway. When the folding capacity of the ER is overtaken, stress arises from the aberrant accumulation or processing of proteins. Proteins synthesized in the ER are properly folded with the assistance of ER chaperones. Malfolded proteins are disposed by ER-associated protein degradation (ERAD, reviewed in Malhotra (2007). Semin Cell Dev Biol 18:716-731). The ER stress response induces expression of ER chaperones and ERAD components and transiently attenuates protein synthesis to decrease the burden on the ER. A malfunction of the ER stress response caused by aging, genetic mutations or environmental factors can result in various diseases such as diabetes, inflammation and neurodegenerative disorders including Alzheimer^'s disease, Parkinson's disease and bipolar disorder, which are collectively known as "conformational diseases" (Yoshida (2007) FEBS J. 274:630-658).

Unfolded or malfolded proteins form aggregates in the ER and in the cytosol, which are highly toxic as they impair the ubiquitin proteasome pathway and sequester transcription factors. The diseases caused by the malfolding of cellular proteins are collectively called "conformational diseases" or ''folding diseases". Since malfolded proteins and protein aggregates can evoke ER stress, it is believed that ER stress is involved in most conformational diseases, although it is unclear whether it is the major cause of these diseases.

The table provided in figure 47 gives a non-limiting list of ER-stress induced diseases which can be treated in accordance with the present invention with antagonists/inhibitors against Cyclin O (Cyclin O alpha and, in particular Cyclin O-beta).

The term "inhibitor of Cyclin O" means in this context a compound capable of inhibiting the expression and/or activity of "Cyclin O" defined herein above. A Cyclin O inhibitor may, for example, interfere with transcription of a Cyclin O gene, processing (e.g. splicing, export from the nucleus and the like) of the gene product (e.g. unspliced or partially spliced mRNA) and/or translation of the gene product (e.g. mature mRNA). The Cyclin O inhibitor may also interfere with further modification (like glycosylation or phosphorylation) of the polypeptide/protein encoded by the Cyclin O gene and thus completely or partially inhibit the activity of the Cyclin O protein as described herein above. Furthermore, the Cyclin O inhibitor may interfere with interactions of the Cyclin O protein with other proteins (thus, for example, interfering with the activity of complexes involving Cyclin O protein(s)) or, in general, with its synthesis, e.g. by interfering with upstream steps of Cyclin O expression.

Also siRNAs/RNAis, antisense molecules and ribozymes directed against nucleic acid molecules encoding Cyclin O are envisaged as (a) Cyclin O mhibitor(s) for the use and the method of the present invention. The above-mentioned antagonist/inhibitor of Cyclin O may also be a co-suppressive nucleic acid. An siRNA approach is. for example, dislosed in Eibashir ((2001), Nature 411, 494-498)). It is also envisaged in accordance with this invention that for example short hairpin RNAs (shRNAs) are employed in accordance with this invention as pharmaceutical composition. The shRNA approach for gene silencing is well known in the art and may comprise the use of st (small temporal) RNAs; see, inter alia, Paddison (2002) Genes Dev. 16, 948-958.

As mentioned above, approaches for gene silencing are known in the art and comprise "RNA"'-approaches like RNAi (iRNA) or siRNA, Successful use of such approaches has been shown in Paddison (2002) loc. cit, Eibashir (2002) Methods 26, 199-213; Novina (2002) Mat. Med. June 3, 2002; Donze (2002) Nucl. Acids Res. 30, e46; Paul (2002) Nat. Biotech 20, 505-508; Lee (2002) Nat. Biotech. 20, 500-505; Miyagashi (2002) Nat. Biotech. 20, 497-500; Yu (2002) PNAS 99, 6047-6052 or Brummeikamp (2002), Science 296, 550-553. These approaches may be vector-based, e.g. the pSUPER vector, or RNA polIII vectors may be employed as illustrated, inter alia, in Yu (2002) loc. cit.; Miyagishi (2002) loc. cit. or Brummeikamp (2002) loc. cit.

In the appended examples the efficacy of specific short RNAs in the down regulation of Cyclin O is shown. In these examples, WEHI7.2 cells are used. This cell line is derived from a "T-cell lymphoma" from the thymus of a mouse and are, accordingly, representative for T- cell lymphoma. It is also referred to in the art as a cell line representative for "lymphocytic leukaemia" or ''thymoma'^". However, the denomination "thymoma^"' is presently not accepted in the art since this refers to a tumour of epithelial origin originated in the thymus. Also murine T-cell lymphoma lines 679Thy and EL4 which again are representative for T-cell lymphoma.

The specific shRNAs used in this context can generally be applied as antagonists of Cyclin O in accordance with the present invention and are. accordingly, useful in the treatment of proliferative diseases, especially cancerous diseases. The following oligos can, therefore, be used to make shRNA constructs, for example, based on the pSuper¹³ plasmid system: shEx3: 5^"- GCGCCCACCATC AACTTC-3' and shC5: S'-GCTCTAGAGGCTCAAACCC-S'.

For example, Figure 5 A shows that anti-Cyclin O shRNA inhibits apoptosis induced by DNA damaging drugs (etoposide in this case) and glucocorticoids (Figure 6A) in lymphoid cells.. Figures 10 and 11 show that anti-Cyclin O shRNA inhibits apoptosis induced by DNA damaging drugs and glucocorticoids in lymphoid cells. Moreover, Figure 18 shows that p53 inhibition avoids Cyclin O expression in mouse embryonic fibroblasts. Given the fact that Cyclin O is a p53 target gene, also available p53 inhibitors (for example pifithrin-alpha) can be used in the inhibition of Cyclin O (expression). Figure 34B shows that anti-Cyclin O shRNA inhibits apoplosis induced by drugs that induce stress of the endoplasmic reticulum (thapsigargin, DTT, tunicamycin) in lymphoid cells. All these data illustrate that antagonists against Cyclin O can efficaciously be used in the treatment of the above-mentioned diseases.

As demonstrated in the appended examples, siRNAs work very effectively in mouse cells after transfection of a plasmid expression vector and corresponding constructs targeting the human Cyclin O alpha and/or Cyclin O-beta can readily be constructed by the skilled artisan and used in accordance with the present invention. In respect of gene therapy (also described herein below) using either viral vectors to direct the expression to the target organs or modified siRNA oligos is preferred.

Besides siRNAs, also further inhibitors/antagonists of Cyclin O can be used herein. For example, inhibitors of the kinase activity of Cyciin O alpha complexes can be used. A non- limiting example of an inhibitor of Cyclin O is roscovitine. a molecule that inhibits Cdkl, Cdk2 and Cdk5 complexes. Roscovitine is commercially available and under clinical trials as antitumoral agent. It is patented for many applications as roscovitine or its R-enantiomer. also called R-roscovitine, CYC202, or Seliciclib. It was developed by Dr. Laurent Meijer at the CNRS in Ro scoff, France as a Cdk inhibitor and is described e.g. in Meijer et al. (2006) "(R)- Roscovitine (CYC202, Seliciclib)" in Inhibitors of Cyclin-dependεnt Kinases as Anti-tumor Agents. CRC Press. Taylor & Francis Group Boca Raton London New York. In Figure 9C it is demonstrated that Cyclin O alpha complexes can be inhibited by roscovitine. Besides roscovitine. furtherCdk2 inhibitors are able to inhibit (non selectively) Cdk2 and Cdkl - containing compounds. It is of note that there is. a close correlation between efficiency of inhibition of the Cdkl/2 complexes and apoptosis inhibition (e.g. in thymocytes), which is Cyclin O/Cdk2 dependent. For example, butyrolactone I may be used. Other compounds may taken. Non-limiting exemplary compounds are described in Mclnnes C. (2008) "Progress in the evaluation of CDK inhibitors as anti-tumor agents'^* Drug Discov Today. 19-20:875-81. In one embodiment, the present invention relates to a method for preventing or treating a proliferative disease as defined herein above. Exemplary diseases to be treated are cancerous diseases like, adenocarcinoma (e.g. pancreatic adenocarcinoma), sarcoma (e.g. osteosarcoma or fibrosarcoma) and the like. The method comprises the administration of an effective amount of Cyclin O and/or an agonist of Cyclin O, the nucleic acid molecule as defined above, the Cyclin O polypeptide as defined above, the nucleic acid construct as defined above, the vector as defined above, and/or the host cell as defined above to a subject in need of such a prevention or treatment.. It is preferred that the subject is a human.

Also envisaged herein is the use of an Cyclin O and/or an agonist of Cyclin O as defined herein above for the preparation of a pharmaceutical composition for the prevention, treatment of a proliferative disease. An exemplary proliferative disease is a cancerous disease like adenocarcinoma (e.g. pancreatic adenocarcinoma), sarcoma (e.g. osteosarcoma or fibrosarcoma) and the like. Also envisaged in this context is the use of the herein above described a nucleic acid molecule(s), polypeptide(s), a nucleic acid construct(s), vector(s) and/or host cell(s). In a further embodiment the present invention relates to a pharmaceutical composition as described herein above, an inhibitor of Cyclin O, an agonist of Cyclin O, a nucleic acid molecule, a polypeptide, a nucleic acid construct, a vector, and/or a host cell for use in preventing, treating or ameliorating a proliferative disease as defined herein above. The nucleic acid molecule, polypeptide, nucleic acid construct, vector and host cell to be used have been described herein above, in particular in context of the pharmaceutical composition.

The present invention provides also a kit comprising a nucleic acid molecule, a polypeptide, a nucleic acid construct, a vector, and/or the host cell. The nucleic acid molecule, polypeptide, nucleic acid construct, vector and host cell have been described herein above, in particular in context of the pharmaceutical composition.

The dosage regimen/administration mode of Cyclin O and/or the, agonist of Cyclin O as defined herein above (and also of the herein disclosed and defined nucleic acid molecule(s), polypeptide(s), nucleic acid construct(s), vector(s), and/or host cell(s)) to be employed/used herein, comprised in the herein provided pharmaceutical composition will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and drugs being administered concurrently, A person skilled in the art is aware of or able to determine suitable doses of the above-mentioned compounds of the present invention. The pharmaceutical composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual subject, the site of delivery of the pharmaceutical composition, the method of administration, the scheduling of administration, and other factors known to practitioners. The "effective amount" of the pharmaceutical composition for purposes herein is thus determined by such considerations.

The skilled person knows that the effective amount of pharmaceutical composition administered to a subject will, inter alia, depend on the nature of the compound. For example, if said compound is a (poly)peptide or protein the total pharmaceutically effective amount of the pharmaceutic composition administered parenteral Iy per dose can. for example, be in the range of about 1 μg protein/kg to 10 mg protein/kg of patient body weight. More preferably, this dose is at least 0.01 mg protein/kg, and most preferably for humans between about 0.01 and 1 mg protein/kg. If given continuously, the pharmaceutical composition can, for example, be administered at a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, for example by continuous subcutaneous infusions, using, for example, a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect. The particular amounts may be determined by conventional tests which are well known to the person skilled in the art.

Pharmaceutical compositions of the invention may be administered orally, rectally, parenterally, intracisternally, inlravaginally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.

Pharmaceutical compositions of the invention preferably comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The pharmaceutical composition is also suitably administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained release pharmaceutical compositions also include liposomally entrapped compound. Liposomes containing the pharmaceutical composition are prepared by methods known per se: DE 3,218,121 ; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641 : Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal therapy.

For parenteral administration, the pharmaceutical composition is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is nontoxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

Generally, the formulations are prepared by contacting the components of the pharmaceutical composition uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) (poly)peptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The components of the pharmaceutical composition are preferably sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 or 0.4 micron membranes). Components of the pharmaceutical composition generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The components of the pharmaceutical composition ordinarily will be stored in unit or multi- dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution, As an example of a lyophilized formulation, 10-mI vials are filled with 5 ml of sterile- filtered 1% (w/v) aqueous solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized cornρound(s) using bacteriostatic Water-for-Injection.

In a further preferred aspect of the present invention, the pharmaceutical composition of the present invention may further comprise (an) instruction manual(s) which guide the skilled person how to diagnose a disease as defined herein. Particularly, said instruction manual(s) may comprise guidance how to use or apply the methods of diagnosing (or in context of a pharmaceutical composition treating, preventing or ameliorating) a disease described herein.

Also the use of the herein described nucleic acid molecules having sequences encoding Cyclin O alpha, Cyclin O-beta and/or Cyclin O mutant, and the corresponding nucleic acid constructs, vectors and/or host cells in gene therapy is envisaged. For example, nucleic acid molecules having sequences encoding Cyclin O alpha, Cyclin O- beta and/or Cyclin O mutant are administered to treat or prevent a proliferative disease, in particular a cancerous disease like adenocarcinoma (e.g. pancreatic adenocarcinoma) or sarcoma (e.g. osteosarcoma or fibrosarcoma) by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel et ah, Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); May, TIBTECH 1 1(5): 155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

ϊn a preferred aspect, a composition of the invention comprises, or alternatively consists of, nucleic acid molecules having a sequence encoding Cyclin O alpha, Cyclin O-beta and/or Cyclin O mutant, said nucleic acids being part of an expression vector. The vector expresses Cyclin O alpha, Cyclin O-beta and/or Cyclin O mutant in a suitable host. In particular, such nucleic acids have promoters, preferably heterologous promoters, operably linked to the coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the Cyclin O alpha, Cyclin O-beta and/or Cyclin O mutant coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al, Nature 342:435-438 (1989). Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target ceil types specifically expressing the receptors), etc. In another embodiment, nucleic acid-Iigand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06 180; WO 92/22715; W092/203 16; W093/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al, Nature 342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acid sequences encoding an antibody of the invention or fragments or variants thereof are used. For example, a retroviral vector can be used (see Miller et al, Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen el al, Biotherapy 6:29 1-302 ( 1994), which describes the use of a retroviral vector to deliver the mdr 1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al, J. Clin. Invest. 93:644-651(1994); Klein et al, Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); and Grossman and Wilson, Cuπ\ Opin. in Genetics and Devel. 3: 110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et at, Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al, Science 252:431-434 (1991); Rosenfeld et al, Cell 68: 143- 155 (1992); Mastrangeli et al, J. Clin. Invest. 91 :225-234 (1993); PCT Publication W094/12649; and Wang, et al, Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used. Adeno- associated vims (AAV) has also been proposed for use in gene therapy (Walsh et al, Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofecύ^'on, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcellmediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the ait for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-718 (1993); Cohen et at, Meth. Enzymol. 217:718-644 (1993); Clin. Pharma. Ther. 29:69-92m (1985)) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the ait. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc. In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

Figure 1. Cyclin O locus and expression. (A) The Cyclin O locus encodes four transcripts that arise from the use of three alternative promoters: Pl (human/mouse Cyclin Oa and the alternatively spliced product human/mouse Cyclin Oβ), P2 (human Cyclin Oγ) and P3 (mouse Cyclin Oδ). Black boxes denote coding regions. White boxes denote 5' and 3' non-coding regions. Grey boxes denote non-coding transcripts.

(B) Expression of Cyclin Oa, β and δ transcripts were measured by quantitative RT-PCR in normal mouse thymus (white bars) and spleen (black bars) of the same mouse, using HPRT levels as control. RNA levels were measured in triplicate and the mean values ± the SEM are shown. (C) Human Cyclin O was detected by Western blotting in cytosol-enriched extracts (SNl and SN2) or in nuclear protein-enriched extracts (P) of the human colon adenocarcinoma cell line HT-29 (left panel) using the Nl antibody or in mouse fibroblast extracts (MEF) using the C2 antibody (right panel). In both cases, SuperSignal West Pico chemilumini scent substrate was used. The membranes were stripped and reprobed with antibodies against Lamin Bl as a nuclear marker and α-tubulin as a cytosolic marker.

Mice transgenic for (D) Bax and non-lransgenic littermates (wild type) and (E) Bcl2 and non- transgenic littermates (wild type) were irradiated as described in the material and methods section and thymuses isolated and processed for RNA isolation. The levels of mCyclin Oa mRNA were analyzed by quantitative RT-PCR. As a loading control, the rnRNA levels of HPRT were determined in the same samples. The experiment was repeated twice and the result of a representative measure of the Cyclin O done in triplicate is shown (mean plus standard deviation) as fold of induction over the levels of the non-transgenic mice at time 0. Eiτor bars show the calculated error range for two independent experiments. Figure 2. Mouse Cyclic O is induced during thymocyte apoptosis in vivo and precedes Caspase-3 activation.

(A) Swiss CD-I mice were irradiated as described in the Material and Methods section, At the indicated times, mice were sacrificed and the thymuses processed for Cyciin O (mCyclin O) or active Caspase-3 (aCaspase-3) detection by immunohistochemistry. Samples were counterstained with haematoxylin. A section of the same samples was stained with haematoxylin-eosin (H&E) as a control. In the case of the active Caspase-3, no En Vision™ signal enhancing system was used, c: cortex; m: medulla. The region magnified in the active Caspase-3 staining is indicated by a rectangle. (B) Swiss CD-I mice were irradiated or injected with dexamethasone as described in the Material and Methods section, the thymuses collected at the indicated times and processed for immunofluorescence detection of Cyclin O using the biotinylated Nl antibody revealed by streptavidin-fluorescein and the active Caspase-3 using the 5Al antibody followed by an anti- rabbit antibody labelled with Cy3. Fluorescence was detected using a Leica SP2 confocal microscope.

Figure 3. Cyclin O binds preferentially Cdk2.

(A) Protein extracts of HEK293 cells transiently transfected with an expression plasmid for myc-tagged mCyclin O were immunoprecipitated with a control rabbit IgG (IgG) or with a monoclonal antibody against the myc-tag (9El 0) covalently bound to Protein G-Sepharose beads. After extensive washing, the proteins bound to the beads were first analyzed by Western blotting with antibodies against Cdkl, Cdk2 and Cdk4 (upper panel) and, after stripping of the membranes, with antibodies against mCyclin O (C2, middle panel). As a control. 2.5% of the input was run in the gel and processed. To run out the possibility that the co-immunoprecipitations were due to the endogenous c-Myc protein immunoprecipitated by the 9E10 antibody, cell extracts from mock transfected HEK293 cells were immunoprecipitated with the 9E10 antibody and probed for the presence of Cdkl , Cdk2 and Cdk4 (lower panel). (B) Extracts from Cdk2 wild type (⁴V+) and Cdk2 knockout (-/-) mouse fibroblasts were immunoprecipitated with rabbit immunoglobulins (rlgG), and the anti-Cyclin O antibody C2 covalently bound to Protein G-Sepharose beads. The presence of Cdkl and Cdk2 in the immunoprecipitates were detected by Western blotting. As a control, the membrane was stripped and mCyclin O was detected by Western blotting with the antibody C2. (C) Recombinant MBP-Cyclin O bound to amylose beads was incubated with whole cell extracts from mouse fibroblasts Cdk2 (+/+) or (-/-). After washing, the Cyclin O complexes were eluted with maltose and immunoprecipitated with normal rabbit IgGs or antibodies against Cdkl or Cdk2. The immunoprecipitates, an aliquot of the beads before elution (Beads) and 5 μL of the eluate were processed for Histone Hl kinase assay. All the experiments were performed at least three times and a representative result is shown.

Figure 4. Mouse Cyclin O overexpression induces apoptosϊs. (A) The plasmids pcDNA3 or pcDNAS-HAmCyclin O were linearised with Ahdl. and transfected into U2OS osteosarcoma cells by calcium phosphate transfection. After two weeks of selection colonies were stained with methylene blue and counted. Represented are the mean percentage of variation in the number of colonies after transfection of the pcDNA3- HAmCyclin O plasmid (Cyclin O vector), respect to the transfection of the pcDNA3 empty vector ± the SEM obtained from three independent experiments.

(B) The plasmids pcDlSlA3 or pcDNA3 -HAm Cyclin O were transiently transfected into HEK293 cells and 72 hours after the transfection, the cells floating in the culture medium or attached to the culture plate were counted. Data averaged from three independent experiments are shown. Error bars show the calculated SEM.

(C) The attached and floating cells from (B) were stained with propidium iodide and the cell cycle analyzed by flow cytometry. Represented are the mean values from three independent experiments and the calculated SEM.

(D) HEK293 cells were transiently transfected with expression plasmids for myc-tagged Cyclin O and human Cyclins A2 and Dl. After 48 hours, floating and attached cells were harvested and the presence of apoptosis assessed by the detection of the processing of the proCaspase-3 and the appearance of active Caspase-3 (aCaspase-3) by Western blotting. Transfected myc-Cyclin O and Cyclins A2 and Dl were detected by Western blotting as controls. Tubulin levels were measured as a loading control.

Figure 5. Dowϊϊregulation of Cyclin O abrogates apoptosis induced by intrinsic but not extrinsic stimuli.

(A) WEHI7.2 cells were stably transfected with empty pSUPERpuro vector (clone V3), with pSUPERpuro expressing a shRNA against GFP (clone shGl), or two different shRNAs directed against mCyclin O Exon 3 (clone 3.7 and clone 5.8) and single cell clones isolated. The selected clones were treated with lOμM etoposide. Samples were harvested at the indicated times and the percentage of apoptotic cells measured as sub-Gl DNA peak by propidium iodide staining by flow cytometry. Measures were done in triplicate. Represented are the mean FACS values from three independent experiments. Bars correspond to the mean ± the SEM.

(B) The same clones were treated with 2 μM dexamethasone and processed as in the preceeding section. Samples were harvested at the indicated times and the percentage of apoptotic cells measured as sub-Gl DNA peak by propidium iodide staining by flow cytometry. Measures were done in triplicate. Represented are the mean FACS values from three independent experiments.Bars correspond to the mean -= the SEM. (C) To check the response of the clones to the apoptotic effect of the CD95 pathway, a control clone (shGl) and an shRNA clone (3.7) were cultured 0 and 24 hours in the absence (Oh, 24h) or presence of 0.5 μg/mL of the agonistic antibody Jo2 (αFas), 30 μg/mL of cycloheximide (CHX) or the combination of both treatments (αFas+CHX). Apoptosis was measured as described and the figure shows the relative increase in apoptosis of each condition relative to the value at 0 hours (percentage). For comparative purposes, the response of the same cells to γ-radiation is also included. Error bars show the calculated error range for two independent experiments.

Figure 6. Cyclin O acts at a premitochondrial step prior to the loss of plasma membrane assymetry in the signalling of intrinsic apoptotic stimuli.

(A) Clones shGl (white bars) and 3.7 (black bars) were treated with 10 Gy of γ-radiation using a ¹³⁷Cs irradiator and the percentage of cells exposing the phospholipid phosphatidyl serine at the outer plasma membrane measured by means of their ability to bind to (fluorescein-labeled) annexin-V and quantitation by flow cytometry in triplicate. Represented are the mean ± the SEM of the percentage of annexin-V positive cells.

(B) The same experiment was performed but the dissipation of the mitochondrial transmembrane potential was measured by means of the quantitation by flow cytometry of the percentage of cells with low fluorescence for the mitochondrial potential-sensitive dye CMX- Ros (ΔΨm^i0W) in triplicate. Represented are the mean ± the SEM of the percentage of ΔΨm^0W cells.

(C) Clones shGl and 3.7 were treated with 10 Gy of γ-radiation and at the indicated times cells were harvested and subcellular fractions obtained. Bax, Bak, Bid and Cytochrome c abundance were measured by Western blotting in cytosol (supernatant. SN) and mitochondria- (pellet, P) enriched fractions. plόBak is the product an alternatively spliced Bak mRNA. As a control of the subcellular fractionation, Pyruvate kinase was used as a cytosolic marker and Tom20 as a mitochondrial membrane marker.

Figure 7. Downregulatioii of Cyclin O abrogates apoptosis induced by intrinsic stimuli due to a failure to activate Caspsses. (A) Similarly to the case of γ-raciiation-treated cells (Figure 6A), Cyclin O downregulation leads to a complete lack of activation of apical Caspases -8 (cleaved Caspase-8, cCaspase-8) and Caspase-9 (active Caspase-9, aCaspase-9) and executory Caspase-3 (active Caspase-3, aCaspase-3) upon dexamethasone treatment. As a control, the inactive, proforms of the Caspases are shown.

(B) Clones shGl and 3.7 were treated with 10 Gy of γ-radiation using a ^lj7Cs irradiator (left panel) or treated with 2 μM dexamethasone (right panel) and cultured for 6h. Cell extracts were obtained at Oh and 6h and the DEVDase activity measured in triplicate using the fluorogenic substrate Ac-DEVD-AFC. Represented are the mean ± the SEM of the fluorescence units generated per minute and per microgram of protein.

(C) Clones shGl and 3.7 were treated with 2 μM dexamethasone and cells harvested at the indicated time points. Cell extracts were analyzed by Western blotting for PARP full length (pi 16) and cleaved (p85) expression.

(D) Clones shGl and 3.7 were cultured 24 hours in the absence (PBS) or presence of 0.5μg/mL of the agonistic antibody Jo2 (αFas), 30 μg/mL of cycloheximide (CHX) or the combination of both treatments (αFas^-CHX). Whole cell extracts were analyzed by Western blotting for the presence of cleaved Caspase-8 (plO), downregulation of full length (FL) p24 Bid and the appearance of the cleaved forms of Bid, tBid pl3/pl5.

Figure 8. Mouse spleen expresses Cyclin O after γ-radiation.

(A) Swiss CD-I mice were treated with 10 Gy of gamma radiation using a ^lj7Cs irradiator and for the times indicated, mice were sacrificed and the spleens removed and immediately fixed in paraformaldehyde. Spleen sections were processed for mCyclin O and active Caspase-3 (aCaspase-3) detection by immunohistochemislry. Samples were counterstained with haematoxylin. A section of the same samples was stained with haematoxylin-eosin (H&E) as control. mCyclin O was detected by using the Nl antibody and the EnVision system (Dako, Denmark) with the colorimetric substrate DAB. wp: white pulp; rp: red pulp; mk: megakaryocyte,

(B) Swiss CD-I mice were irradiated (upper panel) or injected with dexamethasone (lower panel) as described in the Material and Methods section, the thymuses collected at the indicated times and processed for immunofluorescence detection of Cyclin O using the Nl antibody revealed by anti-rabbit antibody labelled with Cy2 (green) and the active Caspase-3 using the 5Al antibody followed by an anti-rabbit antibody labelled with Cy3 (red). To avoid cross-recognition of the two rabbit primary antibodies by the anti-rabbit secondary antibodies, after incubating the samples with the first antibody (anti-active Caspase-3) and its secondary reagent (Cy3-labeled anti-rabbit IgGs), the samples were fixed with 4% paraformaldehyde for 15 minutes at room temperature, washed, incubated with the second antibody and finally with the Cy2-labeled anti-rabbit IgGs. Due to the very high green and red aulofluorescen.ee of erythrocytes, samples were co-stained with TOPRO-3 (shown in blue) to visualise the nuclear DNA. Only nucleated cells positive for Cyclin O and active Caspase-3 are considered apoptotic (arrows). There is a direct correlation between the positivity for both proteins and an abnormal morphology of the nucleus, compatible with condensed chromatin. Fluorescence was detected using a Leica SP2 confocal microscope, wp: white pulp; rp: red pulp; mk: megakaryocyte; e: erythrocyte.

Figure 9. Biochemical properties of Cyclin O-containing complexes.

(A) Cyclin O-Cdk2 recombinant complexes are active. Complexes of recombinant MBP- Cyclin O and either GST-Cdk2 WT or GST-Cdk2 DN (a mutant that can bind Cyclins but lacks kinase activity) purified from E.coli, were assembled in vitro and the (1 :1) stochiometry assessed by SDS-PAGE and silver staining (upper panel). Asterisks denote degradation bands of MBP-Cyclin O. The activity of the complexes was measured by a kinase assay using Hi stone Hl (HHl) as a substrate (lower panel). (B) Cyclin O-containing complexes preferentially phosphorylate Histone Hl over RNA Polymerase Il C-terminal domain. HA-tagged mCyclin Oa was immunoprecipitated from HEK293 cells transiently transfected with an expression vector using the 12CA5 anti-HA tag monoclonal antibody and the immunoprecipitates assayed for Histone Hl (HHl) and RNA polymerase II C-terminai domain (GST-CTD) kinase activity (upper panel). The same experiment was performed with immunoprecipitates against Cdk7 using a polyclonal antibody against endogenous Cdk7 (lower panel).

(C) Cyclin O-containing complexes are sensitive to roscovitine. HEK293 cells were transiently transfected with an expression vector for myc-tagged mCyclin Oa and 48 hours after trail sfecti on, whole cell extracts were immunoprecipitated with antibodies against Cdkl, Cdk2 and anti-myc tag. The immunoprecipitated kinase activity was measured in the presence of the indicated concentrations of roscovitine or equivalent amounts of the solvent DMSO.

Figure 10. Validation of the WEHI7.2 model. (A) WEHI7.2 mouse lymphoid cells were treated with 10μM etoposide or with 2 μM dexameihasone and samples collected at the indicated times, RNA was extracted from the samples and the levels of mCyclin O analyzed by semi quantitative RT-PCR. As a loading control, the niRNA levels of HPRT were semiquantitated in the same samples. (B) WEH17.2 cells were cultured in the presence of 10μM etoposide or the equivalent volume of the solvent DMSO. At the indicated times, aliquots of the cultures were taken from the culture, stained with annexin-V and analyzed by flow cytometry in triplicate. Represented are the mean percentage of arniexin-V positive cells from three independent experiments. Bars correspond to the mean ± the SEM of the measure. (C) WEHI7.2 cells were cultured in the presence of 2μM dexamethasone or the equivalent volume of the solvent ethanol (EtOH) and processed as in (B).

Figure 11. Absence of immuϊϊoreactivity to anti-Cyclin O antibodies in etoposide-treated Clones 3.7 and 5.8 and etoposide-induced Hrstorae Hl kinase activity in anti-Cycϋn O immuπoprecipitates of protein extracts of Clone 3.7.

(A) Clones V3, shGl . 3.7 and 5,8 were treated with lOμM etoposide, samples were harvested at 0 and 6h and processed for mCyclin O detection by immunohislochemistry. Both control clones V3 and shGl show very low, cytoplasmic, punctate expression of Cyclin O in untreated cultures, whereas Cyciin O levels rise dramatically after 6 hours of etoposide treatment, showing a clear punctate cytoplasmic staining. In the case of clones 3.7 and 5.8, almost no staining is detected in the untreated cultures whereas only a faint, punctate staining is seen in the etoposide-treated cultures. Bar, 50 μm.

(B) Clones shGl and 3.7 were harvested 0, 3, 6 and 9 hours after treatment with etoposide (lOμM). Cell extracts were obtained and immunoprecipitated with the C2 antibody that preserves the activity of the Cyclin O associated kinase(s). Etoposide treatment induces time dependent, Cyciin O-associated Histone Hl (HHl) kinase activity, whereas this effect is strongly diminished in the cells expressing the Cyclin O shRNA vector (clone 3.7).

Figure 12. Cyciin O dowϊiregϊilatioti does not affect either the DNA damage response- checkpoint activation or the antiproliferative action of glucocorticoids.

(A) The integrity of the DNA damage response was evaluated in the control clone shGl and the Cyclin O deficient clone 3.7 by measuring by Western blotting the kinetics of phosphorylation of ATM and Chkl, the checkpoint regulatory proteins Cdc25A and C and the activation by phosphorylation in Serl5 of p53 and the expression of its target gene p21^CφI. As a loading control, the membranes were probed with an antibody against α-Tubulin.

(B) DNA profile of the control clone shGl and the Cyclin O shRNA clone 3.7 cells after γ- radiation. Only the shGl clone shows a sub-Gl DNA peak, but the cell cycle distribution of the live cells looks similar, indicating the integrity of the cell cycle effector mechanisms of the checkpoint in the absence of Cyclin O.

(C) Time course of the DNA profile of the control clone shGl and the Cyclin O sliRNA clone 3.7 cells after treatment with dexamethasone (2 μM). No significative differences in the kinetics of the glucocorticoid- induced cell cycle arrest are observed. (D) Time course of induction of BimEL. Dexamethasone treatment of clones shGl and 3.7 led to a similar induction of BimEL, a BH3-only Bcl-2 family member target of the glucocorticoid receptor, indicating a normal transcriptional response to the hormone.

Figure 13. Cyclin O downregulation leads to a defect in apical Caspases -8 and -9 and executory Caspase-3 activation in response to intrinsic apoptotic stimuli.

(A) Activation of apical Caspases -8 (cleaved Caspase-8, cCaspase-8) and Caspase-9 (active Caspase-9, aCaspase-9) and executory Caspase-3 (active Caspase-3, aCaspase-3) was detected by Western blotting in extracts of shGl and 3.7 cells treated with γ-radiation (5Gy) and collected at the indicated times. As a control, the inactive, preforms of the Caspases are shown.

(B) GFP shRNA transfected control clone shG2 and clones isolated after transfection of shRNA constructs "3" (clone 3.4) and "5" (clones 5.1 and 5.8) were cultured in the presence of 10 μM etoposide (left panel) or 2 μM dexamethasone (right panel) and at the indicated times, the percentage of apoptotic cells of the cultures was determined in triplicate by mesuring the sub-Gl population of cells by propidium iodide staining and analysis by flow cytometry. Represented are the mean ± the SEM of the percentage of sub-Gl (apoptotic) cells in three independent experiments for each clone.

(C) Cultures of the clones described in (A) were treated with 5 Gy of γ-radiation (upper panel) or with 2 μM dexamethasone (lower panel), aliquots taken at the indicated times and the inactive (proCaspase) and active (aCaspase) forms of Caspases -9 and -3 detected by Western blotting.

Figure 14. Specificity tests for the Nl and C2 antibodies. Competitive inhibition of the ELISA. Affinity purified antibodies Nl (A) and C2 (B) were incubated with the peptides pNl, pCl, pC2, (pCl is a pNl and pC2 non-related peptide derived from the middle part of the protein), the proteins MBP, MBP-Cyclin O, GST or GST- Cyclin O at a final concentration of 300μg/mL or diluted in the same volume of PBS (samples indicated in the key of the figure). After one hour of incubation at room temperature, serial dilutions of the mixture were assayed by ELISA to measure the titre of the antibody not bound to antigen.

(C) Specificity of the Nl antibody in immunohistochemistry. Swiss CD-I mice were treated with 10Gy of gamma radiation using a ^1J7CS irradiator. 6 hours after irradiation, mice were sacrificed and the thymuses isolated, fixated in paraformaldehyde and processed for immunohistochemistry analysis of Cyclin O expression. Samples were counterstained with haematoxylin. mCyclin O was detected by using the Nl antibody and the En Vision™ system (Dako. Denmark) with the colorimetric substrate DAB. Bar, 50 μm. Cyclin O expression using the Nl antibody (PBS). If the Nl antibody is preincubated with the specific pNl peptide (pepNl ) but not with a non-relevant peptide (unpublished data), all at 1.6 mg/mL, the signal of the antibody disappears, proving its specificity.

Figure 15. Two-dimensional cytofliiorogram of Cyclin O and active Caspase-3 expression in thymus. Thymuses from mice treated with γ-radiation (upper panel) or dexamethasone (lower panel) for the indicated times were processed for confocal microscopy and stained with biotinylated anti-Cyclm O antibody followed by streptavidin- fluorescein (green) plus anti-active Caspase- 3 followed by Cy3-labelled anti-rabbit IgGs (red). Colocalized pixels (located inside the square of the two-dimensional dot plot) are visualized in white. The green fluorescence detected at Oh is due to autofluorescence of the tissue because of the paraformaldehyde fixative.

Figure 16. Coexpression of Cyclin O and active Caspase-3 in shGl ceils treated with DNA damaging agents. Clone shGl was treated with etoposide (10 μM), cells harvested at the indicated times, fixed in paraformaldehyde, processed for confocal microscopy and stained with biotinylated anti- Cyclin O antibody followed by streptavidine- fluorescein (green) plus anti-active Caspase-3 followed by Cy3 -labelled anti-rabbit IgGs (red). The colocalization of the active Caspase-3 positive cells with Cyclin O and their nuclear staining is most likely due to the breakage of the nucleocytoplasmic barrier at late stages of apoptosis as a consequence of Caspase activation.

Figure 17, Cyclin O downregulaiion leads to defective Bax translocation to mitochondria and Cytochrome c release as a response to glucocorticoid treatment.

Clones shGl and 3.7 were treated with 2 μM of dexamethasone, harvested at the indicated times and subcellular fractions obtained. Bax, Bale, Bid and Cytochrome c abundance were measured by Western blotting in cytosol (supernatant, SN) and mitochondria- (pellet, P) enriched fractions. As a control of the subcellular fractionation, Pyruvate Kinase was used as a cytosolic marker and Tom20 as a mitochondrial membrane marker. There is relatively high cytosolic contamination of the pellet fraction in the case of the clone 3.7 extracts in this particular experiment.

Figure 18. p53 dependence of the γ-radiation induction of Cyclin O in MEFs.

Passage 4 mouse embryonic fibroblasts (MEF) from p53 wild type (+/+), heterozygous (⁴V-) or homozygous (-/-) knockout embryos were treated with γ-radiation (10 Gy), at the times indicated the RNA extracted and the levels of Cyclin O mRNA semiquantitated by RT-PCR using HPRT as a loading control.

Figure 19. Surgica! specimens from TMA 1 and TMA 2 informative for Cyclin O expression and absolute number of positive and negative expression amongst the tissue samples derived from the surgical pieces. The number of tumours (specimens) used in TMAl and TMA2 are indicated . Tissue samples indicate the type and number of the different samples taken from the surgical specimens.

Figure 20. Percentage of Cyclin O negative (white bars) and positive (black bars) tissue samples obtained from the surgical specimens.

The type and percentage of Cyclin O negative (white columns) or positive (black columns) tissue samples analyzed in TMAl and TMA2 is represented.

Figure 21. Expression of Cycϊin O in tissue samples from colon surgical specimens. (A) Cyclin O negative, normal colon mucosa; (B) Cyclin O positive, normal colon mucosa; (C) Cyclin O positive polyp; (D) Cyclin O positive adenoma; (E), (F) Cyclin O positive colon adenocarcinomas.

Figure 22. Analysis of the surgical specimens from TMA 1 with aπti-Cyclin O antibodies recognizing alpha and beta isoforms (Nl), only the alpha isoform (αl) or only the beta isoform (βl).

85% of the human colorectal carcinomas analyzed show overexpression of the alpha isoform, whereas about 20% show beta overexpression. From the Cyclin O positive samples, about 60% show only alpha overexpression, 10% only beta overexpression and 30% alpha + beta overexpression (data not included in previous analysis). This confirms that Cyclin O alpha is the predominant isofrom expressed in human colorectal carcinoma.

Figure 23. Dysplasic colon crypt from an ulcerative colitis patient. Hypercellularity, multilayered hyperchromatic nuclei and the lack of mucus are of note. Cyclin O staining is positive.

Figure 24. Cyeiin O expression in resected adenomas from Familial Adenomatous Polyposis (FAP) patients. Staining is positive in the normal crypts (N), and strongly positive in the dysplasic (D) and adenomatous (A) crypts.

Figure 25. Cyclin O expression in the primary tumour and metastasis of a colon adenocarcinoma. (A) Expression in normal colon mucosa from the margins of the surgical specimen (B) Expression in peritumoral, histologically normal mucosa and in the primary tumour (C) Expression in the primary tumour (D) Expression in a regional lymph node metastasis (E) Expression in a liver metastasis (F) High magnification view of Cyclin O expression in the primary tumour.

Figure 26. Relationship between Cyclin O expression in normal colon mucosa and survival of colorectal cancer patients. If the normal colon mucosas are sorted according to the tumour stage, no statistically significance is reached in stages I₅ III and IV.

Figure 27. Relationship between CycHn O expression in normal colon mucosa from colorectal tumours stage II and survival.

Stage II turnout's wherein normal mucosa expresses Cyclin O significantally correlate with worse prognosis.

Figure 28. Correlation between pT and Cyclin O expression in normal mucosa areas from colorectal tumours.

Statistic analysis of the correlation between the local tumour invasion (pT) and Cyclin O expression.

Figure 29. Expression of Cyclin O in adenomas resected by colonoscopy. The expression of Cyclin O in two adenomas resected by colonoscopy was determined by immunohistochemistry .

Figure 30. Schematic characterization of Cyclin Oa, Cyclin Oβ and the Cydin Oa L3A mutant. Cyclin Oβ is an alternatively spliced form coming from the Cyclin O locus which lacks exon 2 (splicing exonl-exon3). The reading frame is maintained, so the N- and C- terminal parts of the protein are identical to Cyclin Oa. The mutant L3A was made starting from mouse Cyclin Oa by site directed mutagenesis, changing Leucines 92, 95 and 97 encoded by exon 1 (indicated by a triangle) to alanines. This sequence is also present in Cyclin Oβ. The LxxLxL sequence is conserved in all the known Cyclin O orthologs, from fish to man and may be involved in the subcellular localisation of the alpha/beta proteins.

Figure 31. Overexpression of Cyclin Oa, Cyclin Qβ and the Cyclin Oa L3A mutant are proapoptotic. The figure shows that overexpression of Cyclin Oa, Cyclin Oβ and the Cyclin Oa L3A mutant confers a proapoptotic effect The plasmids pcDNA3 (empty), pcDNA3Myc-mCyclin Oa, pcDNA3Myc-mCyclin Oβ and pcDNA3Myc-L3A were linearised with Ahdl and transfected into U2OS osteosarcoma cells by the PEI method. After two weeks of selection colonies were stained with methylene blue and counted. Represented are the mean percentage of variation in the number of colonies after transfection of the Cyclin O expression plasmids, respect to the transfection of the pcDNA3 empty vector ± the SEM obtained from five independent experiments.

Figure 32. Constitutive expression of Cyclin Oa, Cyclin Oβ or the Cyciin Oa L3A mutant.

The figure shows that constitutive expression of Cyclin Oa but not Cyclin Oβ or the Cyclin Oa L3A mutant is compatible with cell survival.

Figure 33. Interaction of Cyclin Oa and β isoforms with Cdk2.

(A) Extracts from HEK293 cells transfected with empty vector or expression vectors for myc- tagged Cyclin Oa wild type or the L3A mutant and Cyclin Oβ were immunoprecipitated with purified normal mouse IgGs or with the anti-myc tag antibody 9E10 bound to Protein-G Sepharose beads. Immunoprecipitated proteins were analysed for the presence of Cdk2 by western blotting. The membrane was stripped and reprobed with the anti-Cyclin O antibody C2 to detect the immunoprecipitated Cyclin Oa wild type or the L3A mutant and Cyclin Oβ. IP: immunoprecipitation; WB: western blotting; IgL: Immunoglobulin light chain. (B) Extracts from HEK293 cells transfected with empty vector or expression vectors for HA- tagged Cyclin Oa wild type or the L3A mutant and Cyclin Oβ were immunoprecipitated with the anti-HA tag antibody 12CA5 bound to Protein- A Sepharose beads. Immunoprecipitates were incubated with [V-³²P]ATP and the exogenous substrate Histone Hl and analyzed by SDS-PAGE and autoradiography. (C) The U20S osteosarcoma cell line was transfected with either empty pcDNA3 plasmid, or with the same plasmid driving the expression of myc-tagged Cyclin Oa wild type or the L3A mutant and Cyclin Oβ. After G418 selection, colonies were stained and counted. Transfection efficiency between the different plasmids was normalized by cotransfection with an EGFP expression plasmid and quantitation by FACS of GFP positive cells 24 hours after transfection. The ratio between the number of colonies obtained for each plasmid divided by the number of colonies obtained by transfection of the empty vector (clonogenic capacity) is represented. The results shown are the mean ± the SEM of at least four independent experiments. Asterisks indicate statistically significant measures as determined by the Student's t-test.

Figure 34. Cyclin O α and β are upregulated by ER stress and are necessary for ER stress-induced apoptosis.

(A) WEHI7.2 cells were treated with 0.1 μM thapsigargin or 4 niM DTT and RNA purified from samples taken at the indicated times. The abundance of Cyclin Oa (black bars) and β (white bars) mRNAs was measured by quantitative RT-PCR.

(B) WEHI7.2 cells stably transfected with a control (white squares) or a Cyclin O -specific (black squares) shRNA expression vectors (Roig et al , 2009) were treated with 0.1 μM thapsigargin, 4mM DTT or 5 μg/mL of tunicamycin, samples taken at the indicated times and processed for measuring the cellular DNA content by flow cytometry. Percentage of apoptosis (indicating the percentage of cells with subdiploid DNA content) is indicated. In (A) and (C). samples were analyzed in triplicate and the results shown are the mean ± the SEM of at least three independent experiments. Asterisks indicate statistically significant measures as determined by the Student^'s t-test.

(C) WEHI7.2 cells stably transfected with a control (shGFP) or a Cyclin O-specific (shCyclin O) shRNA expression vectors were treated with 0.1 μM thapsigargin or 5 μg/mL of tunicamycin and samples taken at the indicated times. The levels of phospho-Ser51 eIF2α CHOP and total eIF2α (as a control) were measured by western blotting.

Figure 35. Cyciin O signals ER stress by selective activation of the PERK/CHOP pathway.

(A) Mouse fibroblasts were treated with 0.1 μM thapsigargin (thapsi) or infected with control (empty) or Cyclin Oa wild type or the L3A mutant or Cyclin Oβ lentiviruses at a MOI of 1, harvested at the indicated times after infection and RNA isolated. The IREl -dependent splicing of Xbp-1 was assessed by RT-PCR and detection of the unspliced product (Xbp-lu) of 254bp and the spliced product (Xbp-is) of 229 bp (upper panel). Expression of the two isoforms of Cyclin O encoded by the lentivirus (eCyclin O) was assessed in the same RNA samples by RT-PCR (lower panel). Expression of CHOP (B) and its target gene Carbonic Anhydrase 6 (CA6, C) were measured in different aliquots of the same RNA samples by quantitative RT-PCR. Measures were done in triplicate. Represented are the mean values from at least three independent experiments. Bars correspond to the mean ± the SEM. Asterisks indicate statistically significant measures as determined by the Student's t-test.

(D) AR42J cells were treated with 0.1 μM thapsigargin (thapsi) or infected with control (empty) or Cyclin Oa wild type or the L3A mutant or Cyclin Oβ lentiviruses at a MOI of 1, harvested at the indicated times after infection and the levels of full length ATF6α p90 measured by western blotting. The membrane was reprobed with anti-Tubulin antibodies as a loading control.

Figure 36. Cyclin O overexpression leads to the activation of the PERK/CHOP pathway.

(A) Subcellular localization of endogenous Cyclin O and PERK of mouse fibroblasts were determined by laser-scanning confocal microscopy using specific antibodies detected by a Cy3 -labeled donkey anti-rabbit secondary antibody (to visualize the PERK antibody, red signal) and streptavidin-FITC (to detect the anti-Cyclin O antibody which was biotin labeled). Colocalization is shown in white pseudocolor. In the upper right corner of the dot plot is shown the percentage of colocalization as calculated by the software ImageJ after analyzing three independent fields of the preparation. (B) AR42J cells were infected with control (empty) or myc-tagged Cyclin Oa wild type or L3A mutant or Cyclin Oβ lentiviruses at a MOI of 1, harvested at the indicated times after infection and the levels of total and phospho-Thr980 PERK, phospho-Ser51 eIF2α and CHOP measured by western blotting. As a control of ER stress, cells infected with the empty lentivirus were treated with 0.1 μM thapsigargin 48 hours post-infection, harvested at the indicated times and processed as the rest of the samples. The levels of expression of the proteins encoded by the lentivirus (myc-tagged Cyclin Oa wild type and L3A mutant and Cyclin Oβ) were detected using the anti-myc tag antibody 9E10. As a control of the efficiency of the lenti viral infection, the levels of GFP expressed from the IRES-GFP cassette encoded by the lentiviral vector, were measured by western blotting. (C) Immortalized MEFs derived from PERK knockout (KO) mice or wild type (WT) controls were transfected with either empty pB ABEpuro plasmid, with pBABEpuro-Cyclin Oa or with pBABEpuro-Cyclin Oβ. After puromycin selection, colonies were stained and counted. Transfection efficiency between the two cell lines was normalized by cotransfection of each plasmid with an EGFP expression plasmid and quantitation by FACS of GFP positive cells 24 hours after transfection. The ratio between the number of colonies obtained in each case by the number of colonies obtained by transfection of the empty vector (clonogenic capacity) is represented. Measures were done in triplicate. Represented are the mean values from at least three independent experiments.

Bars correspond to the mean ± the SEM. Asterisks indicate statistically significant measures as determined by the Student's t-test.

Figure 37, Cycliii O isoforms are located in the stress granules.

(A) Colocalization of Cyclin O isoforms α and β with the stress granules and P-bodies marker TlA-I was determined by laser- scanning confocal microscopy in control or thapsigargin- treated AR42J cells. Cyclin Oa (upper panel) or anti-Cyclin Oβ (lower panel) were detected using isoform specific antibodies and a Cy3-labelled anti-rabbit secondary antibody (red signal). TIA-I was detected with a specific antibody detected with an Alexa 488-labelled anti- goat secondary antibody (green signal). DNA was stained with TQPRO-3 (blue pseudocolor). Colocalization is shown in white pseudocolor. In the upper right comer of each figure is shown the percentage of colocalization as calculated by the software ImageJ analyzing three independent fields for each preparation.

Figure 38. Cyclm O-depeκdeπt induction of CHOP by γ-radiation. WEHI7.2 cells stably transfected with a control (shGFP) or a Cyclin O-specific (shCyclin O) shRNA expression vectors were treated with 5 Gy of γ-radiation and samples taken at the indicated times. The levels of CHOP, cleaved (active) Caspase-3 (aCaspase-3) and phospho- Ser51 eIF2α were measured by western blotting.

Figure 39. Characterization of anti-Cyclin Oa and β specific antibodies.

(A) Location of the peptides used to rise anti Cyclin Oa and β (Nl and C2_? Roig et al, 2009) serum, anti-Cyclin Oa (al) and anti-CycHn Oβ (βl) sera. The black boxes indicate the position of the peptides in the protein. The structure of the Cyclin Oa and β proteins is represented in the upper diagrams, while the exons encoding them are depicted in the lower diagrams. The light and dark grey boxes (positioned in exon 2 and part of exon 3) denote the Cyclin Box, which is incomplete in the case of Cyclin Oβ.

(B) Extracts from HEK293 cells transfected with empty vector or expression vectors for myc- tagged Cyclins Oa or β were analysed by western blotting using the anti-Cyclin O C2 antibody and the isoform specific alpha- 1 and beta-1 antibodies. The membrane was reprobed with anti-Tubulin antibodies as a loading control. In the case of the betal antibody, the film was overexposed to rule out crossreactivity with Cyclin Oa protein (right panel).

(C) HEK293 cells transfected with empty vector or expression vectors for myc-tagged Cyclins Oa or β were analysed by immunofluorescence and confocal microscopy using the anti-Cyclin O Nl antibody and the isoform specific alpha-1 and beta-1 antibodies.

Figure 40. Subcellular distribution of Cyclin Oa and β isoforms.

The subcellular localization of Cyclin Oa and β isoforms was determined in AR42J rat pancreas adenocarcinoma cells (left panels) and in mouse fibroblasts (right panels) by laser- scanning confocal microscopy using Calreticulin as a marker for the endoplasmic reticulum. Cyclin Oa (upper panel) or anti-Cyclin Oβ (lower panel) were detected using isoform specific antibodies and a Cy2-labelled anti-rabbit secondary antibody (green signal). Calreticulin was detected with a specific antibody detected with an Alexa 555-labelled anti-mouse secondary antibody (red signal). DNA was stained with TOPRO-3 (blue pseudocolor). Colocalization is shown in white pseudocolor. In the upper right corner of each figure is shown the percentage of colocalization as calculated by the software ImageJ after analyzing three independent fields for each preparation.

Figure 41. Transient transfection of Cyclin O-alpha and Cyclin O-beta fused to fluorescent proteins.

By transient transfection of the alpha and beta forms fused to fluorescent proteins into human tumour cells, we have observed that the alpha form shows a cytoplasmic (red, see below) and nuclear (pink) localisation, whereas the beta protein forms cytoplasmic aggregates (green) which correspond to stress granules, based on their colocalization with the marker TIA-I (not shown).

Figure 42. Transient cotransfection of Cycϋn O-alpha and Cyclin O-beta fused to fluorescent proteins. Cotransfection of both proteins relocalises the alpha protein (red) to the stress granules, where the beta form is localised (green), showing full colocalisation (yellow dots). DNA is shown in blue in all the pictures.

Figure 43. Coexpression of wild type Cyclin O-alpha and L3A mutant fused to fluorescent proteins.

L3A mutant transfection (central pannel) leads to the same cytoplasmic punctate distribution than the beta form (not shown), whereas cotransfection of the wild type Cyclin O alpha (red/pink) and the L3 A mutant (green), leads to the same relocalisation phenomenon (yellow).

Figure 44. Cyclin Oa and Cyclin Oβ have different cellular localisation. U2OS cells were transiently transfected with expression plasmids encoding Cyclin Oa fused to Red Fluorescent Protein (RFP) and the Cyclin Oβ or the L3A mutant fused to Green Fluorescent Protein (GFP), the DNA stained with TOPRO-3 (shown in blue false colour) and analysed by confocal microscopy, Cyclin Oa shows nuclear and homogeneous cytoplasmic localisation, whereas Cyclin Oβ and the L3A mutant form dense cytoplasmic aggregates.

Figure 45. Cyclin Oa forms stress granules after heat shock or oxidative stress.

The identity of the cytoplasmic agregates formed by the L3A mutant and Cyclin Oβ consitutively and Cyclin Oa when coexpressed with them was assessed by cotransfection with markers of different cytoplasmic granules and confocal colocalisation studies. We obtained positive results with the stress granule marker TIA-I . After induction of oxidative stress or heat shock. Cyclin Oa colocalises with TIA-I in the cytoplasmic granules, confirming their identity as stress granules. The beta and L3A mutant forms also colocalise with TIA-I, indicating that they also form stress granules consitutively (not shown).

Figure 46. Cyclin O is not a Uracil-DNA glycosyϊase.

(A) HEK293 cells were transiently transfected with expression vectors for the nuclear isoform of mouse Uracil-DNA glycosylase (nUDG, (Pearl, 2000)). untagged mouse Cyclin Oa (CycO) or empty pcDNA3.1 plasmid (mock). Forty-eight hours after transfection. cells were harvested and Uracil-DNA glycosylase (UDG) activity measured in whole cell extracts of the transfected as well as the endogenous activity of the non-transfected HEK293 cells (293).

(B) Expression of transfected, myc-tagged nUDG was detected by western blotting with the 9E10 monoclonal antibody. Transfected mCyclin O was detected with the C5 antibody and, as a loading control, the levels of Cyclin A2 were measured.

Figure 47. The table provided in figure 47 gives a non-limiting list of ER-stress induced diseases.

The Examples illustrate the invention.

Example 1: Identification and Characterization of Cyclin O transcript variants

Materials and methods

In silico Cyclin O identification and cloning

Human Cyclin A2 N-terminal Cyclin box domain (amino acids 200 to 360 from gi 85396865) were used to identify new cyclins in silico by screening the first draft of the human genome using the tblastn program (http://www.ncbi.nlm.nih.gov/Blast) with default stringency parameters given by the program. We detected two previously undescribed Cyclin-like sequences in chromosomes 5 and 19. For the sequence in chromosome 5, later on named Cyclin O⁷, we could complete the structure of the gene with the help of ESTs present in the Genbank data base. We then searched the first draft of the mouse genome and found the ortholog gene in mouse chromosome 13. The Cyclin-like sequence of chromosome 19 was not further characterized.

The mCyclin O coding region was amplified by PCR using as a template DNA from a cDNA library constructed with RNA purified from apoptotic thymocytes and using the oligonucleotides S'-GGAATTCCATGGTTACCCCTTGCCCTGCCAGCCC-S 'and 5'- GCGAATTCGCTTATTGCCAACTCTGGGGCAGGCTGC-3'. The PCR products amplified (corresponding to the alpha and beta forms) were gel purified, digested with EcoRl and cloned in suitable vectors. The GenclD from Cyclin O in the mouse genome corresponds to 218630 and in the human genome to 10309.

Mouse Cyclin Oδ locus transcript is represented by the EST gi:58060079, Rat Cyclin Oδ is represented by the EST gi:49903553. Two representative human ESTs that encode the Cyclin Oγ specific exon Ely are gi: 14569066 and gi: 14817125.

Antibodies

Anti-Cyclin O antibodies. The Nl and C2 antibodies were generated against the peptides H- LRAPVKKSRRPC-NH₂ (Nl) coupled with glutaraldehyde and H-SSLPRILPPQIWERC-NH₂ (C2) coupled with 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS, Sigma- Aldrich, St. Louis, MO, USA) to keyhole lympet haemocyanine (KLH). The production of the specific antibody in the different batches of sera was confirmed and titrated by ELISA. The N 1 and C2 sera were affinity purified using the peptide bound to an EAH-sepharose column according to the manufacturer (GE Healthcare, USA). Affinity purified Nl antibody was biotynilaled using biotin N-hydroxysuccinimide ester. The affinity purified antibodies were checked for different applications and their specificity carefully determined by several independent tests (Figure 1 for Western blotting. Figures 4 and 8 for immunohistochemisiry, immunoprecipitation-kinase and ELISA). All the antibody techniques were done as described²⁷.

New Zealand Rabbits were used for immunisation and were kept at the animal facility of the Facultat de Farmacia, Universitat de Barcelona.

Other antibodies were from commercial suppliers: Lamin Bl (AM6375) was from Abeam (Cambridge, UK); anti-Bak (Ab-2) was from Calbiochcm; Cdk2 (M2), Cdc25A (F-6). Cdc25C (C-20), Bax (N-20), Tom20 (FL- 145) and p21Cipl (C-19) were from Santa Cruz (Santa Cruz Biotechnology, CA, USA); monoclonal antibody against Cdk2 (clone 55, BD Biosciences, San Jose, CA, USA); monoclonal antibody anti-rnyc tag 9E10 was from a hybridoma from the American Type Culture Collection (ATCC, Rockville, MD, USA): cleaved Caspase-3 (Asp 175, 5Al rabbit mAb). Caspase-9 (C9 mAb), phospho-ATM (Serl981, 10H11.E12 mAb), phospho-Chkl (Ser345, 133D3 rabbit mAb) and phospho-p53 (Serl5 rabbit antibody) were from Cell Signal (Cell Signalling Technology, Boston, MA₅ USA), anti-Pyruvate kinase was from Chemicon International (Temecula, CA): anti-Bim (14A8) was from Alexis (Lausane, Switzerland), mouse anti α-Tubulin clone DMlA was from Sigma (St. Louis, MO, USA), rabbit anti-Caspase-8 (Ab-4) was from Neomarkers (Fremont, CA, USA) and the anti-Cdkl (A-17), anti-Cytochrome c (7H8.2C12), anti-Bid (559681) and the CD95 agonistic antibody Jo2 were from Beckton-Dickinson (BD Biosciences, San Jose, CA, USA). Monoclonal antibodies against PARP were kindly provided by Dr. Jose Yelamos (IMIM₅ Barcelona).

Cell fractionation and immunoblotting.

Cell fractionation was performed essentially as described⁵. SNl is the result of the first centrifugation and is highly enriched in cytosol. SN2 is the supernatant of the second centrifugation and contains diluted cytosolic extract. The pellet (P) contains mainly nuclei, membranes and mitochondria.

Semiquantitative and quantitative RT-PCR

RNA was prepared from mouse thymus and spleen using Trizol reagent (Invitrogen, Carlsbad. CA, USA) following the manufacturer instructions.

Forward primers used for quantitative RT-PCRs were: for mCyclin O α 5'- CGCTTG

CAAGCAGGTAGAGG-3' and for mCyclin O β S'-CGCGCCAGCCACAAGTAGAGG-S^'.

For both isoforms, the reverse primer was: 5'- TGA GTG A AGTGCTCC AGGA A G- 3'. For mCyclin O δ, the forward primer was: S^'-CCCATGGCTCCCCTAGGTG-S', and the reverse 5' -GCTGCCCGGGCCGCCTTC AGC-S^". In the case of HPRT, the primers used were: 5'-

GGCCAGACTTTGTTGGATTTG-3' and S'-TGCGCTCATCTTAGGCTTTGT-S'.

Quantitative RT-PCR was performed using QuantiTect Sybr Green reagent (Qiagen) according to the manufacturer^'s protocol and the data were analysed using SDS2.1 software

(Applied Biosystems).

Immυnohistochemistry Immunohistochemical analyses were performed using 3 μm sections of parafonnaldehyde- fixed paraffin-embedded mouse tissue blocks or cultured cell pellets. Antigen retrieval was done by boiling the slides in 10 mM sodium citrate pH 6 for 10 min. Slides were blocked with filtered 5% non fat milk dissolved in PBS. Affinity purified antibody against Cyclin O was incubated for 90 min at 27⁰C. As secondary antibody, the EnVision^R anti-rabbit system was applied (DakoCytomation, Goldstrup, Denmark). Sections were counterstained with haematoxylin, dehydrated and mounted.

Cell culture and transfections

The human HEK293 and U2OS cell lines were obtained from ATCC and maintained in Dulbecco^'s modified Eagle's medium supplemented with antibiotics and 10% foetal calf serum. The mouse T cell lymphoma cell line WEHI7.2 (obtained from Dr. Roger Miesfeld, University of Arizona, Tucson, Arizona, USA) was grown in low glucose (1 g/L) Dulbecco's modified Eagle^'s medium supplemented with antibiotics and 10% foetal calf serum. U2OS and HEK293 cells were transfected using the calcium phosphate method as described .The total cell counting by flow cytometry was carried out using the Cell Count Fluorospheres method according to the manufacturer (Beckman Coulter, Fullerton, CA. USA). WEHI7.2 cells were transfected by electroporation as described .

Pulldown-eluiion-IP -Kinase assay

Purified Maltose Binding Protein (MBP)-mCyclin O (10 μg) was bound to 75 μl of amylose beads (New England Biolabs, Ipswich, MA, USA) and incubated with a cell extract of HEK293 cells at 4⁰C. After 4 hours of incubation, the beads were extensively washed and the MBP-Cyclin O together with associated proteins was eluted by incubation with 15 mM maltose. After 30 min, aliquots of the eluate were immunoprecipitated with 1 μg of normal rabbit IgGs (Sigma, St. Louis, MO, USA), 2μl of anti-Cdkl or 2 μ] of anti-Cdk2 (M2) and kinase assay performed in the immunoprecipitates using 1 μg of Histone Hl as exogenous substrate. As controls, an aliquot of the amylose beads before elutioii (Beads) or from the eluate were processed directly by the kinase assay³⁰.

Cyclin O-Cdk2 coimmunoprecipUation Cell extracts were prepared from HEK293 cells transiently transfected with a myc-tagged Cyclin O expression vector and then was immunoprecipitated with 50 μL of the supernatant of the 9E10 hybridoma (monoclonal antibody directed against the anti-myc epitope) bound to Protein G-Sepharose beads (GE Healthcare, USA). After washing steps, myc-Cyclin O and associated proteins (Cdk2) were detected by Western blotting of the beads (M2 antibody). As a negative control, an immunoprecipitation with the corresponding amounts of purified nonimmune rabbit immunoglobulins was carried out in both cases.

Mice and treatments

CD2Baxα (Baxl 8 line³¹), EμBcl2 (Eμ-bd-2-25 line³²) transgenics bred to FVB/N wild type mice were kept in SPF conditions in the mouse facility of PRBB with free access to water and food during all the procedures. Mice used in the experiments came from one or two litters from the same parents and only non-transgenic littermates were used as controls. In case two different litters were used, for each time point, the control and transgenic mice came from the same litter.

Irradiation procedure: mice were treated with 10Gy of gamma radiation using a ¹³⁷Cs irradiator and kept with free access to water and food during the time course of the experiment. At the indicated times, mice were sacrificed, the organs removed and either fixated in paraformaldehyde for immunohistochemistry or immediately frozen in liquid nitrogen for RNA isolation.

Dexamethasone treatment: mice were intraperitoneally injected with 2 mg of dexamethasone (dexamethasone phosphate, Fortecortm^R) and processed as indicated for the irradiation procedure.

Colony forming assays

U2OS osteosarcoma cells (5x10^" per well) were plated in a 6-well plate. Next day 3 wells were transfected with pcDNA3 empty vector (ϊnvitrogen, Carlsbad, CA, USA) and 3 more with pcDNA3-HAmCyclin O (2 μg per well). All the plasmids used were previously linearised with Ahdl. 48h after transfection cells were trypsinised, each well was diluted 1 /100 and plated in triplicate in 6 well plates in the presence of lmg/ml of G418. Selection media was replaced every 3 days. Two weeks after transfection, cells were fixed with 4% paraformaldehyde, stained with Coomassie Blue and counted.

ShRNA cell clones

To downregulate mCyclin O in WEHI7.2 cells, we used the following oligos to make shRNA constructs based on the pSuper¹³ plasmid system (provided by Reuven Agami, The Netherlands Cancer Institute, Amsterdam, The Netherlands): shEx3 : 5'- GCGCCC ACC ATC AACTTC-3' and shC5: 5 '-GCTCTAGAGGCTCAAACCC-S ' ; shRNA against GFP : 5 '-GCTGACCCTGAAGTTCATC-3 ' .

WEHI7.2 cells were transfected by electroporation with the pSuper-based shRNA constructs described above and single cell clones isolated. After selection with 1 μg/mL of puromycin (Sigma, St. Louis, MO, USA), apoptosis was induced in the selected clones with etoposide (10 μM), dexamethasone (2μM) or with the CD95-agonistic antibody Jo2 (0.5 μg/mL plus 30 μg/mL of cycloheximide). Immunoprecipitation and kinase assay were performed as described⁵ using the C2 antiserum. Apoptosis was measured by quantitation of the sub-Gl cells population by flow cytometry after propidium iodide staining of ethanol fixed cells.

Statistical analysis

Except where indicated, data were expressed as the mean ± Standard Error of the Mean (SEM) of the values from the number of experiments as indicated in the corresponding figures. Data were evaluated statistically by using the Student's t test. p<0.05 was the minimum significance level.

Results

Cloning of mouse and human Cyclin O

Our previous results suggested the possibility that an unidentified Cyclin-like protein could be responsible for the activation of Cdk2 during the apoptosis of quiescent cells in response to intrinsic but not extrinsic stimuli in thymocytes^{3 6}. Using in silico analysis of the human genome we identified several non-characterized Cyclins, from which preliminary results encouraged us to further characterize one of them located on human chromosome 5 (on mouse chromosome 13), later designated Cyclin O⁷.

The generic Cyclin O locus encodes four transcripts that arise from the use of three alternative promoters (Figure IA): Pl (Cyclin Oa, and the alternatively spliced product Cyclin Oβ, both expressed at least at the mRNA level in human and mouse cells), P2 (Cyclin Oγ, expressed only in human cells) and P3 (Cyclin Oδ, expressed only in mouse cells). About two thirds of the Cyclin Oa (hereforth Cyclin O) sequence encompasses a highly conserved Cyciin box, sharing about 28% of identity with human Cyclins A2 and Bl. Neither Cyclin Oγ nor Cyclin Oδ are protein coding and their function is unknown. However, they may have a regulatory function.

Transcript Cyclin Oδ has been detected only by PCR in mouse tissues. Similarly to human Exon lγ, Cyclin Oδ is a non-coding transcript product of an alternative promoter located upstream of the first exon of the gene. A few mouse and rat ESTs are present in the Genbank EST database (i.e. gi:58060079 for mouse and gi:49903553 for rat), and the corresponding human genomic region is not conserved. Again, it seems to be a non-coding RNA, this time rodent-specific.

Expression, regulation and subcellular localization of mouse Cyclin O (mCyclin O)

The expression of the different transcripts coming from mcyclin O gene were analyzed by quantitative RT-PCR in mRNA coming from thymus and splenocytes. Expression of Cyclin Oa, β and δ was detected in both samples at low levels (Figure IB).

The Cyclin O protein is detected by immunohistochemistry as a cytosolic protein (see below). The use of cyto sol- enriched samples from cell lines such as colon adenocarcinoma cell line HT-29 and immortalised mouse embryonic fibroblasts allowed us to detect Cyclin O by Western blotting. Cyclin O migrates as a diffuse group of bands of around 40 kDa in SDS- PAGE gels, suggesting postranslational modifications (Figure 1 C) and exclusively in cytosol enriched extracts (SN). Whole body irradiation of mice with 10Gy of γ-radiation from a ¹³⁷Cs source results in a time- dependent induction of mCyclin O mRNA expression (Figure ID, E). To determine if mCyclin O mRNA expression is regulated by Bcl2 family members as is Cdk2 kinase activity in irradiated thymocytes³, we measured the kinetics of induction of its mRNA after γ- irradiation in mice transgenic for Bax or Bcl2 and compared it with non-transgenic littermates. The induction of mCyclin O mRNA after γ-radiation treatment was independent of both Bax and Bcl2. but they can modulate its levels at late time points: Bax transgenic mice show significantly elevated levels of expression of mCyclin O mRNA with respect to non- transgenic controls, whereas in Bcl2 transgenic thymocytes its expression lags behind significantly (Figure ID and IE).

Cyclin O activation correlates with apoptosis induction in vivo

We analyzed the expression of mCyclin O and active Caspase-3 as an apoptosis marker in the thymus of mice treated with 10Gy of γ-radiation 2, 3, 6 and 8 hours after the treatment. Both Cyclin O and active Caspase-3 staining are negative at time 0 (Figure 2A). Radiation treatment leads to a time-dependent increase in the expression of Cyclin O in the cytoplasm of the thymocytes located mainly in the cortex of the thymus, in parallel with increased number of apoptotic cells positive for active Caspase~3 and showing pyknotic nuclei in the H&E staining. To demonstrate that thymocytes that upregulate Cyclin O are commited to apoptosis. we performed double immunofluorescence staining for Cyclin O and active Caspase-3 (Figure 2B). Two hours after total body radiation double positive cells start to appear in the cortex of the thymus. The number of cells staining simultaneously for Cyclin O and active Caspase-3 increases with time, although the kinetics is slower in the case of dexamethasone treated mice, so the time course was extended from 4 to 14 hours (Figure 2B. lower panel).

Similarly, both total body irradiation and dexamethasone treatment induced Cyclin O expression and Caspase-3 activation in the spleen of the mice (Figure 8). Again, both proteins colocalized in the cytosol of apoptotic cells. However, apoptotic cells do not accumulate, most likely due to the presence of high amounts of phagocytic cells in the spleen.

These data suggest that Cyclin O expression precedes or is concomitant with apoptosis induction. Cyclin O binds and activates preferentially Cdk2

MBP-Cyclin O can bind stochiometrically and activate the Histone Hl kinase activity of Glutathione- S -Transferase (GSThCdk2 (Figure 9A), but not the activity of the inactive mutant D145N (Cdk2 DN), which can bind Cyclins but lacks kinase activity . To study the substrate preferences of Cyclin O-containing complexes, HA-tagged Cyclin O was immunoprecipitated from whole cell extracts of transiently transfected HEK293 cells and a kinase assay of the immunoprecipitates was carried out using either purified Histone Hl or GST-RNA polymerase II C-terminal domain (GST-CTD) as exogenous substrates. The Cyclin O immunocomplexes efficiently phosphorylate Histone Hl but not GST-CTD. Conversely, immunoprecipitation of Cdk7. a bona fide RNA polymerase II kinase, lead to veiy efficient phosphorylation of GST-CTD but not of Histone Hl (Figure 9B).

To find out which Cdk is the preferred partner of Cyclin O we took into account the fact that active Cdk2 complexes immunoprecipitated from apoptotic thymocytes are inhibited by the ATP analogue roscovitine⁵. Cyclin O complexes immunoprecipitated from transfected HEK293 cells were as sensitive to roscovitine in vitro as Cdkl or Cdk2 complexes (Figure 9C). From all the Cdks tested, only Cdkl, Cdk2, CdIo, Cdk7 and Cdk9 are known to be efficiently inhibited in vitro by roscovitine⁹, so they were the best candidates to be Cyclin O partners. Given the fact that the immunoprecipitated Cyclin O complexes poorly phosphorylate GST-CTD. this excludes Cdk7 and Cdk9. as they are efficient CTD kinases¹ . In addition, pulldown experiments show that they fail to interact significatively with Cyclin O in vitro (data not shown).

Cdkl and Cdk2 were efficiently coimmunoprecipitated in vivo from transiently transfected HEK293 cells, whereas no interaction can be detected with Cdk4, a roscovitine insensitive Cdk⁹ (Figure 3A). To detect the interaction of endogenous Cyclin O with Cdk2 and Cdkl . whole cell extracts from fibroblasts derived either from wild type or Cdk2 knockout mice¹¹ were immunoprecipitated with the anti-mCyclin O C2 antibody (C2). The immunoprecipitates were then analyzed by Western blotting against Cdkl and Cdk2 (Figure 3B). The membranes were stripped and probed with the anti-Cyclin O antibody as a control. Endogenous Cyclin O could be co-immunoprecipitated with Cdk2 only from extracts from wild type mouse fibroblasts, while the complexes with Cdkl could be recovered from extracts of either wild type or Cdk2 knockout mouse fibroblasts. Therefore, we can conclude that endogenous Cyclin O forms stable complexes in vivo with Cdkl and Cdk2.

To investigate if Cyclin O-Cdkl and Cyclin O-Cdk2 complexes are active, we incubated the MBP-mCyclin O fusion protein bound to amylose beads with whole cell extracts made from wild type mouse fibroblasts (Figure 3 C, lower panel) and from Cdk2 KO fibroblasts (upper panel). Following incubation, the beads were extensively washed and the MBP-Cyclin O fusion protein together with the associated proteins were eluted in native conditions. Aliquots of the eluate were immunoprecipitated using normal rabbit IgGs or antibodies against Cdkl or Cdk2 and a kinase assay was performed in the immunoprecipitates using Histone Hl as exogenous substrate. Only the antibodies against Cdk2 immunoprecipitated significant amounts of kinase activity from wild type fibroblasts. However, if Cdk2 is not present (Cdk2 KO fibroblasts), then the kinase activity is recovered in the anti-Cdkl immunoprecipitates. This experiment strongly suggests that, at least in vitro, Cdk2 is the preferred binding partner for Cyclin O, that Cdkl can substitute for Cdk2, and that Cyclin O binds and activates Cdkl and Cdk2.

Cyclin O overexpression induces apoptosis

We looked for direct evidence that Cyclin O induces apoptosis by overexpressing it in cell lines both stably and transiently. The poor transfectability of the lymphoid cell lines precluded most of the experiments directed to demonstrate a pro-apoptotic role of Cyclin O expression. We therefore used the highly transfectable cell lines HEK293 and U2OS to characterize the effect.

U2OS cells were transfected with a mCyclin Oa expression vector including neomycin resistance gene and then selected in G418 for two weeks. Colonies were fixed, stained with Coomassie Blue and counted. Around five fold more colonies were consistently obtained in the empty vector transfected cells versus the Cyclin O transfected cells (Figure 4A).

HEK293 cells were transiently transfected by the calcium phosphate method with empty expression plasmid or a mCyclin O expression plasmid (CycO, Figure 4B). Cells floating in the medium or adhered to the plate were quantitated 72 hours after transfeclion by a flow cytometry based total cell count. The number of adherent cells is about the same in both conditions, whereas the number of floating cells is much higher in cultures transfected with mCyclin O expression plasmid (Figure 4B). It is likely that the cells that die and detach from the plate are replaced following division of the remaining cells on the plate. Analysis of the DNA content shows that the adherent cells contain a significantly higher number of cells with subdiploid DNA content in the culture transfected with the mCyclin O expression vector (Figure 4C). In the floating cells, a high percentage of sub-Gl nuclei were detected, No obvious differences were observed in the cell cycle distribution of live cells regardless of the expression of Cycϊin O. suggesting a lack of effect on the cell cycle.

To confirm that Cyclin O overexpression induces apoptosis and its specificity versus other Cyclins also reported to induce apoptosis, HEK293 cells transiently transfected with Cyclins Oj A2 and Dl were analyzed for activation of Caspase-3 by Western blotting. Only Cyclin O overexpression led to activation of this executor Caspase (Figure 4D).

These data demonstrate that mCyclin Oa overexpression can induce Caspase-dependent apoptosis in cell lines of non lymphoid origin.

Cyclin O expression is necessary for apoptosis induced by intrinsic stimuli

Thymocytes are quiescent, short lived cells in culture that cannot be transfected. The WEHI7.2 cell line is derived from a mouse T cell lymphoma that is sensitive to glucocorticoids and DNA damaging agents as well as to extrinsic stimuli such as CD95 agonistic antibodies¹². This cell line expresses Cyclin O at very low levels in basal conditions but dramatically upregulates its expression in a time-dependent manner in response to DNA damage or the glucocorticoid dexamethasone similarly to its regulation in thymocytes (Figure 10).

To determine whether Cyclin O induction is necessary for apoptosis to take place in WEHI7.2 cells, we downregulated its expression level by shRNA. To avoid off-target effects, we designed two different shRNA constructs directed against exon 3 of the gene and obtained stable clones expressing each one. As a negative control, we transfected the empty pSuperpuro vector and the same vector containing a shRNA construct against GFP. After puromycin selection we obtained single cell clones and analyzed the effect of the shRNA on apoptotic response. Given the impossibility of measuring directly the levels of Cyclin O by Western blotting, the effectivity of the shRNA was assessed by two independent methods: checking the dissappearance of the immunoreactivity by immunohislochemistry and abrogation of the induction of Cyclin O-associated kinase activity of the Cyclin O shRNA clones after etoposide treatment (Figure 11). Clones containing empty vector ("V'clones) or the GFP shRNA clones ("shG" clones) behaved indistinguishably from the parental cells in response to apoptotic stimuli. Clones containing the different shRNAs directed against exon 3 of the gene were designated "3"clones and "5"clones according to the Cyclin O shRNA construct transfected and followed by the number of the clone. To rule out possible clonal effects, three to five independent clones from each group were characterized for their response to apoptosis and a representative clone of each group was chosen to show the results of the tests performed. Both shRNA constructs behaved very similarly.

In contrast to the control clones V3 and shGΪ, clones 3.7 and 5.8 were refractory to etoposide and to dexamethasone-induced apoptosis (Figure 5A and 5B). To investigate if downregulation of Cyclin O expression had any effect on the signalling of the extrinsic stimuli, we studied the response of the clone 3.7 to the CD95 agonistic antibody Jo2 in the presence of cycloheximide. The apoptotic response to anti-CD95 remains intact in the presence of downregulated Cyclin O expression (Figure 5C).

The failure of the cells with downregulated Cyclin O to undergo DNA damage-induced apoptosis could be due to a defect in the signal transduction pathway that detects DNA lesions. However, the DNA damage response parameters analyzed are not significatively changed as a result of the downregulation of Cyclin O (Figure 12A). In agreement with this, the DNA profile of the cells 9 hours after treatment with γ-radiation show a profile similar to the shGl clone cells, indicating a GL intra-S and G2 arrest, most likely as a consequence of checkpoint activation (Figure 12B).

A characteristic response of lymphoid cells to glucocorticoids is a cell cycle arrest upon treatment previous to apoptosis induction, reflecting the antiproliferative action of these hormones¹⁴. Dexamethasone treatment of clones shGl and 3.7 leads to a time-dependent arrest mainly in G0/G1 , that is almost complete in clone 3.7 after 48 hours, while in clone shGl appears the sub-Gl apoptotic DNA peak superimposed to the cell cycle arrest (Figure 12C). Glucocorticoid dependent transcription also seems to be unaffected, since the kinetics of induction of the proapoptotic BH3-only Bcl-2 family member BimEL, a known glucocorticoid receptor target¹ . is unaltered (Figure 12D).

To attempt to identify the reason why the shRNA clones show defective apoptosis, we measured other apoptotic parameters in a control clone (clone shGl) and in a Cyclin O shRNA clone (clone 3.7). Cyclin O downregulation avoided plasma membrane phosphatidylserine exposure and mitochondrial transmembrane potential dissipation in response to DNA damage, In agreement with this last observation, Bax and Bid (but not Bak) translocation from cytosol to mitochondria after v-radiation (data not shown) and dexamethasone (Figure 17) treatments are impaired in clone 3.7. As a consequence, Cytochrome c was released to the cytosol in the case of the shGl control clone but not in the clone with downregulated Cyclin O.

All these observations indicate that Cyclin O downregulation leads to an early, premitochondrial block of the signalling of intrinsic stimuli.

Cyclin O downregulation leads to a block in Caspase activation

The lack of Cytochrome c release from mitochondria precludes the formation of the apoptosome, the multiprotein complex responsible of activation of Caspase-9¹⁶. The treatment of clone shGl with dexamethasone (Figure 7A) or γ-radiation (Figure 13A) induces activation of the apical Caspases -8 and -9, as well as Caspase-3. However, the activation of all these Caspases are completely abolished in the 3.7 clone, indicating defective apoptosis. The same phenotype is found in independent clones isolated in the original screening (Figures 13B and ] 3C), ruling out a clonal effect.

To demonstrate that the extrinsic apoptosis pathway is intact, we measured Caspase-8 activation, full length Bid cleavage and generation of tBid in the shGl control and 3.7 Cyclin O shRNA clones (Figure 7). As expected, no significant differences were detected. These experiments demonstrate that Cyclin O expression is necessary for DNA-damage and glucocorticoid induced apoptosis, but not for CD95 signalling. All these results strongly suggest that, in agreement to previous data from thymocytes using Cdk2 chemical inhibitors, lack of activation of the Cyclin O-Cdk2 complexes during DNA damage or glucocorticoid-induced apoptosis results in the absence of apical and executory Caspase activation and, hence, a specific lack of the apoptotic response of the lymphoid cells.

Example 2: Cyciin O expression is induced by chemotherapeutic agents

Material and methods used in this Example are described in Example 1.

In normal and neoplastic T and B lymphocytes, the levels of Cyclin O mRNA and protein are very low, measured by RT-PCR and Q-PCR or by immunofluorescence, respectively (Figures 1C, 2BC, 8, 11. 15 and 16). However, treatment with γ -radiation (Figures 1C, 2, 8, 15 and 18) and a wide variety of chemotherapeutic agents like the etoposide (Figures 10A₅ 1 1 and 16) highly induce the levels of both Cyclin O mRNA and protein . This increase is dependent on the expression of p53 in the case of the DNA damaging agents such as γ -radiation or etoposide (Figure 18). However, other drugs used against lymphomas and leukaemias such as the glucocorticoid dexamethasone can induce Cyclin O levels in a p53 independent manner both in normal thymocytes and in lymphoma cells (Figures 10 and 15)

After apoptosis induction in mouse thymuses or murine lymphoid cell lines by glucocorticoids or DNA damaging agents, Cyclin O is upregulated preceeding activation of Caspase-3. After its activation, all the apoptotic cells show colocalisation of both proteins in the cytosol (Figures 2, 8, 15 and 16).

The downregulation of Cyclin O blocks the glucocorticoid-induced apoptotic signal at a premitochondrial level (Figure 17).

Primary MEFs show a vigorous Cyclin O induction in response to γ-radiation. This response is blunted in established fibroblast cell lines.

Example 3: Cyclin O is overexpressed in colon adenocarcinomas

Material and Methods Tissue specimens

Patients and tumour samples

We selected 174 patients with colorectal adenocarcinoma who underwent surgery of the primary tumour between January 1995 and December 2001 at the Hospital del Mar, Barcelona. Clinical data and follow-up were obtained from the review of the patient's medical records and from the Tumour Registry. Postoperative adjuvant chemotherapy with 5- fluorouracil was performed for all stage III and IV patients. All patients were followed under the same protocol for at least eight years after surgery. Microscopic confirmation of diagnosis, tumour type and histological grade was carried out by pathologists of Servei de Patologia, Hospital del Mar. Patient staging was classified according to the International Union against Cancer tumour-node metastasis criteria. The analysis of the samples was approved by the Ethical Committee for Clinical Experimentation of the IMAS (Barcelona, Spain). Specific survival was calculated from time of surgery for the primary tumour to patient death secondary to its colorectal cancer.

Tissue microarray construction

In order to prepare the tissue microarray, formalin-fixed, paraffin-embedded tissue blocks of colorectal tumours were retrieved from the archives of the Servei de Patologia from Hospital del Mar. Multiple areas of invasive carcinoma and different histological patterns of those tumours (cribiform, mucinous, poorly-differentiated, "signet ring cell"), adenomatous lesions from the same surgical sample, and normal mucosa, far from the infiltrating tumour, were identified on corresponding haematoxylin-eosin-stained slides, and the tissue blocks were transferred to a recipient "master" block using a Tissue Microarrayer. Each core is O.ό-mrn wide spaced 0.7-0.8 mm apart. Two different tissue microarrays (TMA) were made with the tumours selected:

TMAl Surgical specimens (n=100) were collected from patients undergoing elective large bowel resection for colorectal carcinoma. Samples of normal mucosa distant from tumour (>8cm), benign polyps, peri tumoral adenomas and samples from superficial and deep parts of the tumour were fixed with 4% neutral paraformaldehyde and embedded in paraffin. Representative paraffin- embebbed samples containing normal mucosa, benign polyps, adenomas and superficial or deep tumour tissue were used to construct the tissue microarray.

TMA2

Surgical specimens (n=74) were collected from patients undergoing programmed large bowel resection for colorectal carcinoma. Samples of normal mucosa distant from tumour (>8cm), benign polyps and samples from superficial and deep parts of the tumour were fixed with 4% neutral paraformaldehyde and embedded in paraffin. Representative paraffin- embebbed samples containing normal mucosa, benign polyps and superficial or deep tumour tissue were used to construct the tissue microarray.

Statistical analysis

Survival data were analyzed according to the method of Kaplan-Meier and tested for significance between the groups with the log rank test. A p value lower than 0.05 was considered significant. The associations between Cyclin O expression and other clinicopathologic variables were assessed by the chi-square test using with categorical variables. All statistical analyses were carried out using StatView for Windows version 5.0 (SAS Institute Inc).

Except where indicated, data were expressed as the mean ± Standard Error of the Mean (SEM) of the values from the number of experiments as indicated in the corresponding figures. Data were evaluated statistically by using the Student^'s t test. p<0.05 was the minimum significance level.

Antibodies Anti-Cyclin O antibodies. The Nl, αl (anti-Cyclin O alpha) and β l (anti-Cyclin O beta) antibodies were generated against the peptides H-LRAPVKKSRRPC-NH_2, H- ESRSKLLS WLIPVHRQFGLSC-NH2 (alpha- 1) and H-SLARQPQ VEVHPPRC-NH2 (beta- I)₅ respectively, coupled with 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS, Sigma-Aldrich, St. Louis, MO, USA) glutaraldehyde to keyhole lympet haemocyanine (KLH). The production of the specific antibody in the different batches of sera was confirmed and titrated by ELISA. The Nl₅ alpha- 1 and beta-1 sera were affinity purified using the peptide bound to an EAH-sepharose column according to the manufacturer (GE Healthcare, USA). Previous to the affinity chromatography step, the beta-1 sera was preadsorbed to a column containing GST-Cyclin Oa covalently bound to Sepharose to remove Cyclin Oa crossreacting antibodies. Affinity purified Nl antibody was biotinilated using biotin N- hydroxysuccinimide ester. The affinity purified antibody was checked for different applications and their specificity carefully determined by several independent tests. New Zealand Rabbits were used for immunisation and were kept at the animal facility of the Facultat de F armada, Universitat de Barcelona.

Immunohistochemistry

Immunohistochemical analyses were performed using 3 μm sections of paraformaldehyde- fixed paraffin-embedded mouse tissue blocks or cultured cell pellets. Antigen retrieval was done by boiling the slides in 10 mM sodium citrate pH 6 for 10 min. Slides were blocked with filtered 5% non fat milk dissolved in PBS. Affinity purified antibody against Cyclin O was incubated for 90 min at 27⁰C. As secondary antibody, the EnVision^R anti-rabbit system was applied (DakoCytomation. Goldstrup, Denmark). Sections were counterstained with haematoxylin, dehydrated and mounted.

Semiquantitative and quantitative RT-PCR

RNA was prepared from mouse thymus and spleen using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer instructions.

Forward primers used for quantitative RT-PCRs were: for mCyclin Oa 5'- CGCTTG CAAGCAGGTAGAGG-3' (forward primer): the reverse primer was: 5'- TGAGTGAAGTGCTCCAGGAAG-S '. In the case of HPRT₅ the primers used were: 5^'- GGCC AGACTTTGTTGGATTTG-S ' and 5'-TGCGCTCATCTTAGGCTTTGT-S ' . Quantitative RT-PCR was performed using QuantiTect Sybr Green reagent (Qiagen) according to the manufacturer's protocol and the data were analysed using SDS2.1 software (Applied Biosystems).

Cell culture

The mouse T cell lymphoma cell line WEHI7.2 (obtained from Dr. Roger Miesfeld, University of Arizona, Tucson, Arizona, USA) was grown in low glucose (1 g/L) Dulbecco's modified Eagle's medium supplemented with antibiotics and 10% foetal calf serum.

Mice and treatments

Swiss CD-I wild type mice were kept in SPF conditions in the mouse facility of PRBB with free access to water and food during all the procedures. Mice used in the experiments came from one or two litters from the same parents.

Irradiation procedure: mice were treated with 10Gy of gamma radiation using a ^lj Cs irradiator and kept with free access to water and food during the time course of the experiment. At the indicated times, mice were sacrificed, the organs removed and either fixated in paraformaldehyde for immunohistochemistry or immediately frozen in liquid nitrogen for RNA isolation.

Dexamethasone treatment: mice were intraperitoneally injected with 2 mg of dexamethasone (dexamethasone phosphate, Fortecortin^R) and processed as indicated for the irradiation procedure.

Colon adenocarcinomas

We have analyzed the expression of Cyclin O in tissue samples obtained from patients undergoing elective or programmed large bowel resection for colorectal carcinoma. From the surgical specimen, several areas from each tumour were sampled and analysed: mucosa distant from tumour (>8cm, "normal colon"), benign hyperplasic polyps, peritumoral adenomas and samples from the superficial and deep parts of the tumour area. In total, 174 specimens were informative for Cyclin O expression (Figure 19), having obtained information of Cyclin O expression in different parts of the surgical piece.

Expression of Cyclin O was variable amongst the different parts of the surgical specimen. As it is shown in Figure 19, about 60% of the normal colon mucosa samples were negative for Cyclin O expression. The percentage of positivity increased in the polyps (about 60%), whereas adenomas and adenocarcinomas had around 90% of Cyclin O positive cells. No significant difference in staining was found between the superficial and deep parts of the tumour.

It is remarkable that the percentage of Cyclin O positive samples increases with the severity of the lesion, being relatively low in the mucosa of the margins of the surgical piece, increases in the benign polyps while it reaches its maximum in adenomas, a lesion that precedes the development of the adenocarcinoma (Figure 20).

The Cyclin O gene encodes two coding transcripts (producing Cyclin Oa and Cyclin Oβ proteins) arising by alternative splicing of exon 2 of the gene (Roig el al.. 2009). The results presented in Figure 19 and 20 have been obtained by using the polyclonal antibody Nl, which recognizes an epitope present in exon 1, common to both isoforms. To confirm that it is the Cyclin Oalpha form which is predominantly overexpressed in human tumours, isoform specific antibodies were developed. These antibodies were named αl (detecting only Cyclin Oa) and βl (detecting only Cyclin Oβ).

The affinity purified αl and βl antibodies were used in the analysis of the tumour samples of TMAl and compared the results with the analysis done with the Nl antibody (Figure 22).

As shown in Figure 22, the results of the analysis with the Nl antibody are, as expected, comparable to the results with αl. In the case of the immunodetection of the beta isoform. only 21% of the samples were positive, showing a very faint staining pattern, predominantly nuclear, indicating low expression levels. This is in agreement with the relative abundance of the mRNA of Cyclin Oβ respect to Cyclin Oa as measured by quantitative RT-PCR (Roig et al, 2009) This observation substantiates the finding herein that cells cannot adapt to overexpression of Cyclin O-beta; as shown herein, it is impossible to generate cell lines overexpressing Cyclin Oβ (Figure 32), which makes Cyclin O a particular potent pro-apoptotic agent. In the case of the αl antibody, the staining pattern was very similar to the one obtained with the Nl antibody, showing in most of the cases a cytoplasmic staining. These results demonstrate again that Cyclin O-alpha is overexpressed in human colorectal carcinomas and that the results obtained with the Nl antibody are equivalent to using the alpha specific antibody αl .

The results suggest that Cyclin O overexpression would be an early event in the molecular chain of events leading to the development of a full blown colon tumour. To explore this possibility, we studied Cyclin O expression in the earliest lesions detectable in the colon mucosa that may lead to the development of an adenocarcinoma. Immuno staining of surgical samples from patients affected of ulcerative colitis (Figure 23) or biopsies from adenomas arising in FAP (Familial Adenomatous Polyposis) patients (Figure 24) showed that dysplasic crypts found in these samples are already positive for Cyclin O expression.

All these results confirm that Cyclin O overexpression is a very early event in colon epithelial cell neoplasic trans formation.

On the other hand, analysis of lymph nodes, liver and lung metastasis of colon adenocarcinomas and comparison with the primary tumour shows that Cyclin O expression is maintained even after the cancer cells have spread out of the primary tumour and grown at distant sites (Figure 25).

Cyclin O expression in other tumours.

We have investigated the expression of Cyclin O in other tumours. Although only a very limited number of cases have been analysed, we have found that CycJin O is overexpressed in adenocarcinomas from diverse origin and in transitional cell carcinomas of the bladder, while it is not overexpressed in soft tissue tumours, squamous cell carcinomas, Merkel tumours and melanomas; see Table below..

Table showing expression of Cyclin O in human tumours. I. Tumours with Cyclin O overexpression: l .Lung adenocarcinomas (well differentiated): 2/2

2.WeII differentiated areas of squamous lung carcinomas (gland-forming)

3. Stomach adenocarcinomas: 2/2

4. Pancreas adenocarcinomas: 2/2

5. Colon adenocarcinomas* ό.Endometroid adenocarcinomas: 3/4

7. Breast adenocarcinomas: 7/7

8. Transitional cell carcinomas of the bladder: 8/8

9.Primitive neuroepithelial tumour: 1/1

II. Tumours positive for Cyclin O, without overexpression:

1. Rhabdomyosarcomas: 2/2

2. Leiomyosarcoma: 3/3

3.Liposarcomas: 3/3

4.Chondrosarcomas: 3/3

5. Fibrosarcomas: 3/3 ό.Histiocytomas: 2/2

7. Well-differentiated squamous skin carcinomas, keratinising areas: 7/8

8. Poorly differentiated squamous skin carcinomas: 13/23

HI. Tumours negative for Cyclin O:

1. Melanomas: 0/10

2. Squamous lung carcinomas: 0/6

3. Poor Iy differentiated lung adenocarcinomas: 0/1

4.Merkel cell tumours: 0/35

S.Dermatofibrosarcomas protubens: 0/3 δ.Lymphomas: 0/4

All the results presented before strongly suggest that Cyclin O may be a gene involved in the genesis or progression of most human cancers and then a good candidate both as an early cancer marker (as an extension of the results found in colorectal cancer) or as an anticancer target.

Example 4: Cyclin O overexpression correlates with survival of patients

Material and methods used in this Example are described in Example 3.

In the following it is shown that Cyclin O can be used as a prognostic factor in cancer. We have tried to establish correlations between Cyclin O expression in the analysed surgical specimens, the histopathological features of the tumours and the clinical outcome of the patients.

No correlation was found between Cyclin O expression in polyps, adenomas or adenocarcinomas and the age and sex of the patients, the anatomical localisation of the tumour, its TNM classification, histological subtype, degree of differentiation and survival after surgery and response to chemo/radiotherapy. However, unexpectedly, the samples taken from the surgical specimen and categorized as "'normal colon mucosa'^" by the pathologists showed a heterogeneous staining for Cyclin O. Hence, a substantial proportion (40%) of colon mucosa samples taken distally from the tumour and histopathologically categorized as "normal^" show Cyclin O overexpression. Although the statistical significance is not reached, there is a trend to correlate Cyclin O positive '"normal" colon mucosa with a shorter survival after surgery (Figure 26). If the normal colon mucosas are sorted according to the tumour stage, again no statistically significance is reached in stages I, III and IV. However, in stage II tumours which normal mucosa expresses Cyclin O. significatively correlate with worse prognosis (Figure 27).

In agreement with the raise in the percentage of Cyclin O positive staining in increasingly malignant lesions (Figure 20). we have found a positive correlation between the extension of the tumour at the time of surgery (pT) and the expression of Cyclin O in normal mucosa. As it is shown in Figure 28, Cyclin O expression is associated with the extent of the tumour at the time of surgery, being positive in pT3 and pT4 tumours. Conversely, pTl and pT2 tumours are mostly negative for its expression.

The identification of expression of Cyclin O in otherwise normal colon mucosa would indicate that even these morphology normal cells, the peritumoral epithelium and even areas as far as 8 cm apart from the tumour already carry preneoplasic mutations. Should the margins of the surgical specimen contain Cyclin O positive colon mucosa, would indicate that residual disease has been left behind by the surgeon, with the thread of developing a local recurrence by the resumption of the tumorogenic process by the apparently normal colon epithelial cells. In this context, the analysis of the tumour margins only by conventional haematoxylin and eosin staining and morphological analysis would be insufficient to ensure complete removal of the neoplasia. Immunohistochemistry analysis using anti-Cyclin O antibodies, would demonstrate whether the epithelial cells are truly normal (i.e. Cyclin O negative) and help in reducing the number of patients suffering recurrences. Should the staining be positive for Cyclin O₅ the oncologist would have to consider further surgery to extend the amount of intestine resected until finding Cyclin O negative borders or. alternatively, instaurate/intensify chemo/radiotherapy and the surveillance of the patient. This strategy would be especially useful in the case of colorectal carcinoma stage II, where the benefit of adjuvant chemotherapy after surgery is controversial. This group of patients accounts for about 30% of the total of newly diagnosed colorectal cancers and is considered the most challenging stage since no consensus therapeutic protocol after surgery has been fully accepted yet. Only patients with completely resected stage II colon cancer seem to benefit from adjuvant therapy (Figueredo et a!., 2008 Cochrane Database Syst Rev., 3: CD005390), but the criteria to assign the patients to the adjuvant therapy group are not standardized. Usually high risk patients (presenting obstruction, perforation, inadequate lymph node sampling or T4 disease) receive further therapy. However, our findings show that more patients would benefit of potentially curative second surgery or chemo/radiotherapy by applying a simple irrmiunohistochemical test for Cyclin O to the normal mucosa of the margins of the surgical specimen.

We have also demonstrated that the Cyclin O test can be easily applied to samples as small as biopsies obtained by colonoscopy. In Figure 24 we show the results of the analysis of polyps resected by colonoscopy from FAP patients, while in Figure 29 the results of the analysis of adenomas found in a routine colonoscopy of a healthy patient are shown. Positive staining for Cyclin O in intestinal preneoplasic lesions or histologically normal mucosa may indicate progression towards full neoplasic transformation and it may help the digestologist/oncologist in the follow up of high risk patients or indicate further supervision in the case of Cyclin O positive lesions.

Example 5: Characterization of the apoptotic properties of Cyciin Oa (wild type), the Cyclin Oa L3A mutant and Cyclin Oβ Figure 30 shows the origin of Cyclin Oa, Cyclin Oβ and the Cyclin Oa L3A mutant. Cyclin Oβ is an alternatively spliced form coming from the Cyclin O locus which lacks exon 2 (splicing exonl-exon3). The reading frame is maintained, so the N- and C- terminal parts of the protein are identical to Cyclin Oa. The mutant L3A was made starting from mouse Cyclin Oa by site directed mutagenesis, changing Leucines 92, 95 and 97 encoded by exon 1 (located inside the box and in bold type) to alanines. This sequence is present in the Cyclin Oβ but we have not made the corresponding mutation. The LxxLxL sequence is conserved in all the known Cyclin O orthologs. being the second and third Leucines invariant from fish Io man and preliminary evidences of our laboratory show that it may be involved in the subcellular localisation of the alpha/beta proteins.

Overexpressed Cyclin Oa. Cyclin Oβ and the Cyclin Oa L3A mutant have a proapoptotic effect; see Figure 31. The plasmids pcDNA3 (empty), pcDNA3Myc-mCyclin Oa, pcDNA3Myc-mCyclin Oβ and pcDNA3Myc-L3A were linearised with Ahdl and transfected into U2OS osteosarcoma cells by the PEl method. After two weeks of selection colonies were stained with methylene blue and counted. Represented are the mean percentage of variation in the number of colonies after transfection of the Cyclin O expression plasmids. respect to the transfection of the pcDNA3 empty vector ± the SEM obtained from five independent experiments.

Surprisingly, it was found that constitutive expression only of Cyclin Oa but not Cyclin Oβ or the Cyclin Oa L3A mutant is compatible with cell survival: see Figure 32. Colonies growing in the experiments described in the preceeding section were isolated, expanded and analysed by western blotting for expression of Cyclin Oa, Cyclin Oβ, and the L3A mutant by using the anti-Myc tag antibody. Expression of Cyclin Oa was detected in 6 out of 7 colonies tested. No expression of Cyclin Oβ (0/4 colonies) or from the L3A mutant (0/4 colonies) was found.

The results of these experiments suggest that in spite of the similar apoptosis-inducing properties of the three proteins, it is possible to isolate cells adapted to expression of Cyclin Oa, but expression of Cyclin Oβ or the L3A mutant is incompatible with cell survival. Only colonies which have downregulated expression of the transfected plasmid are able to grow. Example 6: Cyclin Oa, Cyclin Oβ and the Cyclin Oa L3A mutant can signal apoptosis by triggering stress of the endoplasmic reticulum

Abbreviations

ATF6: Activating Factor 6; CA6: Carbonic Anhydrase 6; CHOP: C/EBP-Homologous Protein; DTT: 1,4-Dithiothreitol: EMCV: Encephalomyocarditis virus; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; IREl: Inositol -Requiring Protein- 1 ;IRES: internal ribosome entry site; L3A: mouse Cyclin Oa mutant L92A/L95A/L97A; MOI: multiplicity of infection; P bodies: processing bodies; PERK: Protein Kinase RNA (PKR)-like ER kinase; SG: stress granules; TIA-I : cytotoxic granule-associated RNA binding protein 1: UPR: unfolded protein response; Xbp-lu: X-box binding protein I₅ unspliced; Xbp- Is: X-box binding protein 1 , spliced

Material atϊd methods

Cell culture and transections

The mouse T cell lymphoma cell line WEHI7.2 and the clones shGl (shGtP) and 3.7 (shCyclin O) were grown in low glucose (1 g/L) Duibecco's modified Eagle's medium supplemented with antibiotics and 10% foetal calf serum as described (Roig et ah. 2009).

Semiquantitative and quantitative RT-PCR

RNA was prepared from WEHI7.2 cell line and derivatives using Trizol reagent (Invitrogen, Carlsbad. CA, USA) following the manufacturer instructions.

Forward primers used for quantitative RT-PCRs were: for mCyclinOα 5^"- CGCTTG CAAGCAGGTAGAGG-3' and for mCycIinOβ5^'-CGCGCCAGCCACAAGTAGAGG-3\ For both isoforms. the reverse primer was: 5"- TGAGTGAAGTGCTCCAGGAAG-3^". In the case of HPRT, the primers used were: 5'- GGCCAGACTTTGTTGGATTTG-3^* and 5^'- TGCGCTC ATCTTAGGCTTTGT-3'. Detection of the mRNAs expressed from the lentiviruses was done using a forward oligo from Cyclin O and a reverse oligo from the EMCV IRES sequence from the lentiviral pWPI vector. To detect Xbp-1 mRNA splicing, the oligos mXBP1.39S 5'- GGCCTTGTGGTTGAGAACC AGGAG-3^: and mXBP1 .14AS 5'-

GAATGCCC AAAAGGATATC AGACTC-3' were used. The unspliced band is 254 bp and the band from the spliced mRNA 229 bp. The primers used to quantitate CHOP and CA6 mRNA were

Quantitative RT-PCR was performed using QuantiTect Sybr Green reagent (Qiagen) according to the manufacturer's protocol and the data were analysed using SDS2.1 software (Applied Biosystems).

Antibodies

Anti-Cyclin O antibodies. The αl and βl antibodies were generated against the peptides H- ESRSKLLSWLIPVHRQFGLSC-NH2 (alpha-1) and H-SLARQPQVEVHPPRC-NH2 (beta- 1) coupled with 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS, Sigma- Aldrich, St. Louis, MO₅ USA) to keyhole lympet haemocyanine (KLH). The production of the specific antibody in the different batches of sera was confirmed and titrated by ELISA. The αl and βl sera were affinity purified using the peptide bound to an EAH-sepharose column according to the manufacturer (GE Healthcare, USA). Previous to the affinity chromatography step, the βl sera was preadsorbed to a column containing GST-Cyclin Oa covalently bound to Sepharose to remove Cyclin Oa crossreacting antibodies. The affinity purified antibodies were checked for different applications and their specificity carefully determined by several independent tests (Figure 39). All the antibody techniques were done as described (Harlow et α/. , 1999).

Other antibodies were from commercial suppliers: anti-GADD153 (F- 168), ATFόα (H-280), PERK (H-300), phospho-Thr981 PERK (sc-32577), TIA-I (C-20) and Calreticulin (FMC75) were from Santa Cruz (Santa Cruz Biotechnology, CA, USA); anti-CHOP mouse monoclonal 9C8 (Affinity Bioreagents, Thermo Fisher Scientific, Rockford, IL, USA); anti-GFP was from Clontech (Takara BIO INC. Shiga, Japan); phosρho-Ser51 eIF2α (119Al 1 rabbit monoclonal antibody) were from Cell Signal (Cell Signalling Technology, Boston, MA, USA); monoclonal antibody anti-myc tag 9E10 was from a hybridoma from the American Type Culture Collection (ATCC, Rockville, MD, USA); anli-Pyruvate kinase was from Chemicon International (Temecula, CA); mouse anti α-Tubulin clone DMlA was from Sigma (St. Louis, MO₅ USA).

Recombinant Lentivirus generation and infection Lentiviral particles encoding myc-tagged wildtype mouse Cyclin Oa, Cyclin Oβ, and the mutant L3A were generated by transient cotransfection of the bicistronic vector pWPl, the psPAX2 and the VSV-G envelope encoding vector in HEK293T cells (reviewed in Wiznerowicz and Trono. 2005). Lentiviral particles were titrated on HEK293T cells by measuring the number of GFP positive cells by flow cytometry. Viral supernatants were adjusted to give a MOI of 1 to avoid superinfection of the cells. After 16 hours of infection, the viral suspension was removed and new media added to the infected cells.

Colony forming assays

Immortalized PERK wild type or knockout MEFs (5x10^ per well) were plated in a 6- well plate. Next day 3 wells were transfected with pBABEpuro empty vector and 3 more with pBABEpuro-myc-mCyclin O (2 μg per w^rell). All the plasmids used were previously linearised with ^Mt. 48h after transfection cells were trypsinised, each well was diluted 1/100 and plated in triplicate in 6 well plates in the presence of 1 μg/ml of puromycin. Selection media was replaced every 3 days. Two weeks after transfection, cells were fixed with 4% paraformaldehyde, stained with Coomassie Blue and counted.

Statistical analysis

Results

Both Cyclin Oa and the L3A mutant interact with Cdk2 and show kinase activity. However, Cyclin Oβ is proapoptotic in spite of its lack of association to kinases (Figure 31).

Cyclin O participates in the ER stress pathway Preliminary results of our laboratory indicated that Cyclin Oa and its splicing variant Cyclin Oβ (Roig et a!., 2009) were located in the cytosol and endoplasmic reticulum. To investigate whether the Cyclin O gene is regulated by stimuli causing ER stress, the lymphoid cell line WEHI7.2 was treated either with thapsigargin, an inhibitor of SERCA (sarco / endoplasmic reticulum Ca2+ ATPase) leading to a raise in cytoplasmic calcium concentrations or with DTT. a reducing agent which leads to protein denaturation by reduction of disulfide bridges. Both drugs caused a time dependent increase in the levels of Cyclin Oa and β mRNAs as measured by quantitative RT-PCR (Figure 34A).

To determine the role of Cyclin O in ER stress, we compared the apoptosis rates induced by ER stress stimuli in WEHI7.2-derived cell lines transfected with a control shRNA construct or an shRNA directed against Cyclin Oa and β (Roig et ai, 2009). In addition to thapsigargin and DTT. the apoptotic response to tunicamycin, an antibiotic inter ferring with protein glycosylation, was assessed. As it is shown in Figure 34B. All the drugs caused time- dependent apoptosis in the shRNA control-transfected cell line, whereas downregulation of Cyclin O leaded to an almost complete abrogation of the apoptotic effect.

These data suggested that the Cyclin O gene is a target of the ER stress response and that Cyclins Oa and/or β are needed for the apoptotic branch of the pathway.

As a first approach to understand the biochemical mechanisms linking Cyclin O to the ER stress response, we compared the kinetics of phosphorylation of the translation factor eiF2α in response to ER stress in cells proficient or deficient in Cyclin O. As it is shown in Figure 34C, a vigorous time-dependent phosphorylarion of eIF2α takes place in the control cell line after treatment with thapsigargin or tunicamycin. However, this response lags behind and is significatively blunted in cells deficient in Cyclin O. In agreement with this, the product of the ER stress target gene CHOP is detected 180 minutes after inducing stress in control cells whereas it is barely detectable at the same time point in shCyclin O transfected cells (Figure 34C, middle panel).

Cyclin O signals ER stress by inducing PERK Thr980 phosphorylation PERK is a Ser/Thr protein kinase located at the membrane of the ER which couples ER stress to protein synthesis inhibition through phosphorylation of eIF2α. Its catalytic domain is located in the cytoplasm while its lumenal domain upon binding to unfolded proteins triggers oligomerization and activation of the kinase domain, resulting in phosphorylation of the protein in different residues (reviewed in Ron and Walter, 2007). To investigate a putative connection between Cyclin O and PERK and to determine its subcellular localization, we performed colocalization studies by confocal microscopy in the rat pancreas adenocarcinoma cell line AR42J which has a well developed endoplasmic reticulum. As it can be seen in Figure 36A, PERK shows a cytoplasmic punctate pattern consistent with its location in the ER. In the case of Cyclin O, a mixture of fine and coarse granular cytoplasmic staining is observed. A significant fraction of the Cyclin O signal colocalizes with the dotted PERK staining. These results indicate that a fraction of the Cyclin O is located at the ER and the rest most likely in the cytoplasm.

To investigate the consequences of the overexpression of each of the two isoforms of Cyclin O in the ER homeostasis, we infected AR42J cells with lenti viruses encoding myc-tagged versions of Cyclin Oa wild type, the L3A mutant and β. As it can be seen in Figure 36B, expression of either form of the Cyclin O leaded to the instauration of ER stress as assessed by phosphorylation of PERK Thr980. phosphorylation of eIF2α SerSl and the induction of the synthesis of CFIOP. No detectable ER stress as assessed by the abovementioned markers was induced by infection of the cells with a control lentivirus (made with an empty vector) 48 hours post-infection, the longest time analysed after infection with all the viruses. Moreover, thapsigargin induced an energic response within 30 minutes in cells infected with the control lentivirus for 48 hours. Similar results were obtained by expression of Cyclin Oa WT, L3A or β in mouse fibroblasts (data not shown).

These results indicate that overexpression of Cyclin Oa or Cyclin Oβ induces ER stress by promoting PERK activation.

Although the variability in the expression levels of Cyclin Oa and β obtained by lenlivirai infection of AR42J cell or mouse fibroblasts do not allow quantitative analysis, we have consistently observed a trend to obtain higher levels of PERK activation by autophosphorylation when Cyclin Oβ is overexpressed. To gain further insight into a putative differential action of the Cyclin O isoforms, we performed clonogenic assays in PERK wild type and knockout fibroblasts. As it can be seen in Figure 36C, infection of PERK wild type fibroblasts with lentiviruses encoding Cyclin Oa leads to a decrease in the clonogenic capacity of the cells which is not significatively different in PERK deficient fibroblasts. However, Cyclin Oβ is more potent in decreasing the clonogenic capacity upon lentiviral expression in wild type fibroblasts, but this effect is significatively blunted in PERK knockout fibroblasts.

Thus, PERK is necessary for Cyclin Oβ to fully induce apoptosis as a consequence of ER stress.

Cyclin O signals ER stress through activation of the PERK/CHOP pathway

In response to ER stress, three ER-localized transmembrane signal transducers are activated: the protein kinases Inositol Requiring Kinase 1 (IREl) and double stranded RNA-activated protein kinase-like ER kinase (PERK), and the transcription factor Activating Transcription Factor 6 (ΛTF6), which are constitutively expressed in all the cells (reviewed in Malhotra and Kaufman, 2007). To investigate which ER stress transducers are activated by overexpression of Cyclin α or β, we checked especific indicators of the activation of each of the three pathways.

In the case of IREl, the best characterized outcome of its activation is the IREl -mediated splicing of the Xbp-lu mRNA. which encodes a labile protein able to repress UPR target genes. IREl endoribonuclease activity removes a 26 bp intron from the precursor RNA yielding Xbp-ls, now encoding a stable protein, potent activator of UPR target genes (reviewed in Ron and Walter, 2007). As it is shown in Figure 35A₅ thapsigargin treatment of mouse fibroblasts rapidly leads to the splicing of Xbp-lu mRNA. However, in spite of the vigorous activation of the ER stress pathway induced by overexpression of Cyclins Oa WT, L3A and β in AR42J and mouse fibroblasts (Figure 35 and data not shown), no splicing of Xbp-lu could be detected by RT-PCR, even in non-linear amplification conditions. As a control of expression of Cyclins Oa and β upon lentivirus infection, the exogenous mRNA encoded by the lentiviral vector was especifically amplified by RT-PCR again in non-linear amplification conditions (Figure 35A).

To confirm that the overexpression of Cyclins Oa and β in mouse fibroblasts leads to activation of the PERK/CHOP pathway, CHOP mRNA levels were measured by quantitative RT-PCR in mouse fibroblasts infected with an empty retrovirus or a retrovirus encoding Cyclins Oa WT. L3A or β. As a positive control, non-infected fibroblasts were treated with 0.1 μM thapsigargin and the mRNA analysed. As it can be seen in Figure 35B, thapsigargin treatment induced a time-dependent induction of CHOP mRNA levels. Lentiviral infection with a control (empty) virus did not change significatively the levels of CHOP mRNA. However, infection of the fibroblasts with either Cyclin Oa or β leaded to a time-dependent induction in the levels of CHOP comparable to the effects observed after thapsigargin treatment, indicating instauration of ER stress, most likely through the PERK pathway. Moreover, as it is shown in Figure 35C, the upregulation of CHOP correlated with the synthesis of Carbonic Anhydrase 6 (CA6) mRNA, one of its target genes (Sok el al, 1999).

The activation of ATF6, the third signal transducer of ER stress, we analyzed the processing of fall length ATFόα (ρ90) into its active form ρ50. ATF6 is an ER transmembrane protein in unstressed cells which upon induction of ER stress traffics to the Golgi complex where it is proteolitically cleaved generating a 50 kDa fragment which goes to the nucleus where it is active as a transcriptional activator of UPR genes (reviewed in Malhotra and Kaufman, 2007). To study whether activation of ER stress by overexpression of Cyclins Oa and β leaded to ATF6 activation, we measured by western blotting the levels of ATFόα p90 and p50 in AR42J cells infected with a control (empty) lentivirus or lentiviruses encoding Cyclins Oa WT, L3A and β. As a positive control we treated the cells with 0.1 μM thapsigargin. As it is shown in Figure 35D, thapsigargin treatment of AR42J cells leaded to a time-dependent decrease in the levels of ATFόα p90, whereas no significant change was observed in cells infected with the control lentiviruses or the Cyclin Oa or β encoding viruses.

All these data indicate that induction of ER stress by Cyclins Oa and β is a consequence of signalling exclusively through the PERK/CHOP pathway. Cyclin Oa and β are part of TIA-I -containing cytoplasmic granules

To start understanding the molecular details of Cyclin O action, we determined the subcellular localization of the protein. Confocal microscopy experiments using the antibody Nl which recognizes both Cyclin Oa and β (Figure 39) showed that in rat tumour cells, mouse fibroblasts (Figure 40) and human U2OS osteosarcoma cells (data not shown), Cyclin O is present mostly in the cytoplasm. Biochemical fractionation and western blotting experiments confirmed this observation (Roig et a , 2009). The staining showed a dotted pattern, being more intense around the nucleus. The dots were heterogeneus, being the biggest around 300 run in size. Confocal microscopy studies using ER markers such as the chaperone Calreticulin showed partial co localization, indicating that Cyclin O is present in two cytoplasmic pools, one associated to the ER and another present in the cytosol. Minor amounts of Cyclin O were also detected in the nucleus, specially in cells expressing high levels of the protein such as the pancreas adenocarcinoma cell line AR42J (data not shown). The availability of isoform- especific antibodies allowed us to study the subcellular distribution of each of the two Cyclin O proteins. Thus, the most abundant isoform. Cyclin Oa, shows a fine dotted staining pattern which colocalizes with Calreticulin (Figure 40) independently of the induction of ER stress (not shown). Λ minor fraction of Cyclin Oa is detected in the nucleus. Cyclin Oβ is a very low abundance protein which is most likely responsible for the coarse dots detected by the Nl antibody. It is located in the nucleus and the cytoplasm and shows low percentages of colocalization with the ER marker Calreticulin in AR42J cells and negligible colocalization in mouse fibroblasts.

In an attempt to identify the cytoplasmic granules where Cyclin Oβ is located, we performed colocalization experiments using Cyclin Oa or β-especific antibodies and the marker of Stress

Granules (SG) and Processing (P) bodies TIA-I. As it can be seen in Figure 37A, both Cyclin

Oa and β show significant colocalization with TIA-I in untreated cells. Induction of ER stress with thapsigargin leads to an increase in the staining intensities of both Cyclin O (in agreement with the mRNA regulation shown in Figure 34A) and TIA-I, together with a relocalization from nucleus to cytoplasm in the case of TIA-I, but the percentage of colocalization does not increase. These experiments suggest that Cyclin Oa is located mainly in the ER and in the cytosol, whereas Cyclin Oβ forms granules present in the ER, cytosol and nucleus. The colocalization with TiA-I indicates that both isoforms can be part of either Stress Granules or P bodies.

The above data shows that expression of the beta or the L3A forms in mammalian cells induces endoplasmic reticulum stress by the unfolded protein response (UPR), characterised by phosphorylation of the PERK protein kinase, phosphorylation of the eIF2α translation factor and induction of the target transcription factor CHOP, and which ends with the induction of apoptosis of the cells. Without being bound by theory, it is believed that this is most likely the mechanism of apoptosis induction of the Cyclin O beta form, since it does not activate Cdkl/2 kinase activity.

The present invention refers to the following nucleotide and amino acid sequences:

The sequences provided herein are available in the NCBI database and can be retrieved from www.ncbi.nlm.nih. gov/sites/entrez?db=gene; Theses sequences also relate to annotated and modified sequences. The present invention also provides techniques and methods wherein homologous sequences, and variants of the concise sequences provided herein are used. Preferably, such '"variants" are genetic variants.

SEQ ID No. 1 :

Nucleotide sequence encoding Homo sapiens Cyclin O-alpha. Coding region of Homo sapiens Cyclin O-alpha ranges from nucleotide 259 to nucleotide 1308 of SEQ ID NO.1.

CTCCCCCCGGCGGGCCGCGGAGGCGACGCGCTGCCTGCGCCCTGAGGCGA GTGGTTCTCCAGCCGGAAAATCGCGGCTCCGGTGCCGCTCTGGGCTCAGC CAGTGAGAGGCCGGGGGTGTGGTAGAGAGAAGGAGCCGGCCGGGTACTTA GGACCGCGCCAGCCTCTGAGCCCGGCCTCCTTCGCACTTTCGAGTGCGCG TTTGCTGCCCGCCGCAGCCCGGGCGTTGAAGGTAGTAAAGCGGCCACCCG GCCGCATCATGGTGACCCCCTGTCCCACCAGCCCCTCGAGCCCCGCCGCC CGAGCGGGGAGGCGGGACAACGACCAGAACCTTCGCGCCCCGGTGAAGAA GAGCAGGCGTCCGCGCCTCCGGAGGAAGCAGCCGCTGCATCCCCTGAACC CGTGCCCGCTCCCGGGAGACTCCGGCATTTGCGACCTGTTCGAGTCCCCC AGCTCCGGCTCAGACGGCGCAGAGAGCCCCTCTGCGGCGCGGGGTGGTAG CCCCCTGCCCGGCCCGGCCCAGCCCGTGGCGCAGCTAGATCTACAGACCT TCCGCGACTACGGCCAGAGCTGCTACGCCTTCCGCAAGGCGCAGGAGAGC CACTTCCACCCGCGGGAGGCGCTGGCACGGCAGCCACAAGTGACGGCGGA ATCCCGCTGTAAGCTGCTCAGCTGGCTGATCCCGGTGCACCGCCAATTCG GCCTCTCCTTCGAGTCGCTGTGCCTGACGGTGAACACTCTGGACCGCTTC CTCACCACCACGCCGGTGGCTGCAGACTGCTTCCAGCTGCTTGGGGTCAC CTCCTTGCTCATCGCTTGCAAACAGGTGGAGGTGCACCCGCCGCGCGTGA AGCAGCTTCTGGCCCTCTGCTGCGGCGCCTTCTCCCGGCAGCAGCTCTGC AACCTCGAGTGCATCGTGCTGCACAAGCTGCACTTCACCCTGGGTGCGCC CACCATTAGCTTCTTCCTGGAGCATTTCACGCACGCTCGCGTGGAGGCGG GGCAGGCTGAGGCCTCCGAAGCTCTGGAAGCGCAAGCCCTGGCGCGGGGG GTGGCAGAGCTGAGTCTGGCCGACTATGCCTTCACCAGCTACTCCCCTTC CCTCCTGGCGATCTGCTGCCTGGCGCTGGCGGACCGCATGCTGCGGGTCT CGCGGCCCGTGGACTTGCGACTGGGAGACCACCCGGAGGCGGCGCTGGAG GACTGTATGGGCAAGTTGCAGCTGCTGGTGGCCATAAACAGTACTTCCTT GACTCACATGCTGCCCGTTCAGATCTGCGAGAAGTGCAGCCTGCCCCCGA GCTCGAAATAAAACAGATCCTTCGTTTCCTTTTAGTCCCTGGCCCGGCTG CTGGACCTCTCCCGTAGCCTCAGAAGAGTGCAGTACTGGTCCACAGAGAA GGCTTCAGGACCTGCTTGGTCAGCTGCAGGTTGTAAATAGTGTACGATAC TAGCATCTGGTATTTTATTTATTTTGCAGCGAGCACATGAGGAAGCTGAG TCTTTCACCAATAAACAGTTGTGGTTTGTCT

SEQ ID No. 2:

Amino acid sequence of Homo sapiens Cyclin O-alpha.

MVTPCPTSPSSPAARAGRRDNDQNLRAPVKKSRRPRLRRKQPLHPLNPCP LPGDSGICDLFESPSSGSDGAESPSAARGGSPLPGPAQPVAQLDLQTFRD YGQSCYAFRKAQESHFHPREALARQPQVT AESRCKLLSWLIPVHRQFGLS FESLCLTVNTLDRFLTTTPV AADCFQLLGVTSLLIACKQVEVHPPRVKQL LALCCGAFSRQQLCNLECIVLHKLHFTLGAPTISFFLEHFTHARVGAGQA EASEALEAQAL ARGV AELSLADY AFTSYSPSLLAICCLALADRMLRVSRP VDLRLGDHPEAALEDCMGKLQLLV AINSTSLTHMLPVQICEKCSLPPSSK

SEQ ID No. 3:

Nucleotide sequence encoding Homo sapiens Cyclin O-beta. Coding region of Homo sapiens Cyclin O-beta ranges from nucleotide 259 to nucleotide 1 122 of SEQ ID NO.3.

CTCCCCCCGGCGGGCCGCGGAGGCGACGCGCTGCCTGCGCCCTGAGGCGA

GTGGTTCTCCAGCCGGAAAATCGCGGCTCCGGTGCCGCTCTGGGCTCAGC

CAGTGAGAGGCCGGGGGTGTGGTAGAGAGAAGGAGCCGGCCGGGTACTTA GGACCGCGCCAGCCTCTGAGCCCGGCCTCCTTCGCACTTTCGAGTGCGCG TTTGCTGCCCGCCGCAGCCCGGGCGTTGAAGGTAGTAAAGCGGCCACCCG GCCGCATCATGGTGACCCCCTGTCCCACCAGCCCCTCGAGCCCCGCCGCC CGAGCGGGGAGGCGGGACAACGACCAGAACCTTCGCGCCCCGGTGAAGAA GAGCAGGCGTCCGCGCCTCCGGAGGAAGCAGCCGCTGCATCCCCTGAACC CGTGCCCGCTCCCGGGAGACTCCGGCATTTGCGACCTGTTCGAGTCCCCC AGCTCCGGCTCAGACGGCGCAGAGAGCCCCTCTGCGGCGCGGGGTGGTAG CCCCCTGCCCGGCCCGGCCCAGCCCGTGGCGCAGCTAGATCTACAGACCT TCCGCGACTACGGCCAGAGCTGCTACGCCTTCCGCAAGGCGCAGGAGAGC CACTTCCACCCGCGGGAGGCGCTGGCACGGCAGCCACAAGTGGAGGTGCA CCCGCCGCGCGTGAAGCAGCTTCTGGCCCTCTGCTGCGGCGCCTTCTCCC GGCAGCAGCTCTGCAACCTCGAGTGCATCGTGCTGCACAAGCTGCACTTC ACCCTGGGTGCGCCCACCATTAGCTTCTTCCTGGAGCATTTCACGCACGC TCGCGTGGAGGCGGGGCAGGCTGAGGCCTCCGAAGCTCTGGAAGCGCAAG CCCTGGCGCGGGGGGTGGCAGAGCTGAGTCTGGCCGACTATGCCTTCACC AGCTACTCCCCTTCCCTCCTGGCGATCTGCTGCCTGGCGCTGGCGGACCG CATGCTGCGGGTCTCGCGGCCCGTGGACTTGCGACTGGGAGACCACCCGG AGGCGGCGCTGGAGGACTGTATGGGCAAGTTGCAGCTGCTGGTGGCCATA AACAGTACTTCCTTGACTCACATGCTGCCCGTTCAGATCTGCGAGAAGTG CAGCCTGCCCCCGAGCTCGAAATAAAACAGATCCTTCGTTTCCTTTTAGT CCCTGGCCCGGCTGCTGGACCTCTCCCGTAGCCTCAGAAGAGTGCAGTAC TGGTCCACAGAGAAGGCTTCAGGACCTGCTTGGTCAGCTGCAGGTTGTAA ATAGTGTACGATACTAGCATCTGGTATTTTATTTATTTTGCAGCGAGCAC ATGAGGAAGCTGAGTCTTTCACCAATAAACAGTTGTGGTTTGTCT SEQ ID No. 4:

Amino acid sequence of Homo sapiens Cyclin O -beta.

MVTPCPTSPSSPAARAGRRDNDQNLRAPVKKSRRPRLRRKQPLHPLNPCP LPGDSGICDLFESPSSGSDGAESPSAARGGSPLPGP AQPV AQLDLQTFRD YGQSCYAFRKAQESHFHPREALARQPQVEVHPPRVKQLLALCCGAFSRQQ

LCNLECIVLHKLHFTLGAPTISFFLEHFTHARVEAGQ AEASEALEAQALA RGVAELSLADYAFTSYSPSLLAICCLALADRMLRVSRPVDLRLGDHPEAA LEDCMGKLQLLVAINSTSLTHMLPVQICEKCSLPPSSK

SEQ ID No. 5:

Nucleotide sequence encoding murine Cyclin O-alpha. Coding region of murine Cyclin O- alpha ranges from nucleotide 95 to nucleotide 1150 of SEQ ID NO.5.

AGAGCTGCGCCGCCAGCGGGACCGGATCTTCCTCGCAGCTCTCGCGCCGA CGCTCCGGGCGTCGTAGACGCTAGGAGCGCTACCCGATTGTAACATGGTT ACCCCTTGCCCTGCCAGCCCGGGCAGCCCCGCCGCGGGAGCCGGGAGGCG AGACAGCCACCAGAACCTCCGCGCCCCAGTGAAGAAGAGCAGACGCCCGT GCCTCCGGAGAAAGAAGCCGCTGCGGCCGCTGAACGCGTGTTCGCTCCCG GGAGACTCGGGCGTCTGTGACCTTTTCGAGTCCCCCAGCTCCAGCTCGGA CGGCGCCGACAGCCCCGCAGTGTCTGCGGCGCGGGACTGCAGCTCCCTAC TCAACCCTGCCCAGCCCTTGACAGCGCTGGATCTCCAGACCTTCCGAGAA TACGGCCAGAGCTGCTACGACTTCCGAAAGGCGCAGGAGAATCTTTTCCA CCCGCGGGAGTCGCTGGCGCGCCAGCCACAAGTGACTGCCGAATCGCGCT GTAAACTGCTCAGCTGGCTCCTGCAAGTCCACCGCCAGTTCGGCCTCTCT TTCGAGTCGCTGTGTCTGACCGTGAATACTCTGGACCGTTTTCTCCTCAC GACCCCTGTTGCTGCAGACTGCTTCCAGCTGCTCGGGGTCACCTGTCTGC TCATCGCTTGCAAGCAGGTAGAGGTGCACCCACCTCGCTTGAAACAGCTC CTGGCCCTGTGCGGCGGTGCGTTCTCCCGGCAGCAGCTGTGCAACCTGGA GTGCATCGTGCTGCATAAGCTCCACTTCAGCCTGGGCGCGCCCACCATCA ACTTCTTCCTGGAGCACTTCACTCAGTGGCGCATGGAAGCCGGCCAGGCT GAGGTCACCGAAGCTCTAGAGGCTCAAACCCTGGCCCGGGGAGTGGCGGA GCTGAGCCTAACGGATTACGCGTTCACTACCTACACGCCGTCCCTGATGG CTATCTGCTGCCTGGCGCTGGCTGACGGATTGCTGCAGCACCAGCACGAG ATGGACCTGCGCCTGGGAGAGCACCCAGAGGCCACACTGCAAGACTGTCT GGGCAAGCTGCAGACGCTAGTGTCCATCAATAGCAGCTCCCTGCCTCGCA TTCTGCCTCCCCAGATCTGGGAGAGGTGCAGCCTGCCCCAGAGTTGGCAA TAAAAAGCACCCCCTTGAGCTCCTTTTTAAAATTCCCTGGTCCCTGCTCC GTGCCTTAAGGAGAATATGCAGTACAGATCCAAAGAGAAGTTGAGGTCTC CTACCTGTAAATACTGTACAATAACGGATTCTTTATTTATTTTGTAGTAC ACAAGGGCAGGGGTGACAGTTCTGGCTTGTCTAAATGCTGCTGTTGGCAT GCCTGACTGTCTGCTTTTCCCAGGCGAAGTCCATCACGAGGTAGCCAATT CCGGAGGCTCTTCCCTGCTCCCACAGTTTTACTTTGTAGTTAGCTCTGTG AAAACAACCTGGCAAGATTTTATATGCCACTGTTCCAGAAAGCAGACAAG TCAAGGTGGCTTGTCCGTATCTTAACCCCCTCCAGTCAGGAGGCTGAGTT CCCTCCCCCATGCCAGTGGCCCAAGGAGAATGAGACAGGGTTAGAAGTCA GAGTGGTCTGTGCTGTCCGGAAAGGCAGTGGCTGCACTAACTGATGTGGA GCTGGGAAGCTTTGGGGACCGGGCAGCCATACTAGCAGTGGGTGAAGCAA GAGACAGTCCCTGGTGCAACACAGGAAATGCTGGAATGCCTTTTGCGTAT TGTTATAGCTGTTGCAACCTGGGTCATTTCCTCTGCAGCTACACTCATGG TTCAGTTTATACTGCAGCATGAGCCCAGGGTAATTCTGGTACGGCAGTAA TAAATATTATACTATTTGCAACAT

SEQ lD No. 6:

Amino acid sequence of murine Cyclin O-alpha.

MVTTCPASPGSPAAGAGRRDSHQNLRAPVK-KSRRPCLRRKKPLRPLNACS LPGDSGVCDLFESPSSSSDGADSPAVSAARDCSSLLNPAQPLTALDLQTF REYGQSCYDFRKAQENLFHPRESLARQPQVTAESRCKLLSWLLQVHRQFG

LSFESLCLTVNTLDRFLLTTPVAADCFQLLGVTCLLIACKQVEVHPPRLK QLLALCGGAFSRQQLCNLECIVLHKLHFSLGAPTINFFLEHFTQWRMEAG QAEVTEALEAQTLARGV AELS LTD Y AFTTYTPSLMAICCLALADGLLQHQ HEMDLRLGEHPEATLQDCLGKLQTLVSINSSSLPRILPPQIWERCSLPQS WQ SEQ ID No. 7:

Nucleotide sequence encoding murine Cyclin O-beta. Coding region of murine Cyclin O-beta ranges from nucleotide 95 to nucleotide 964 of SEQ ID NO.7.

AGAGCTGCGCCGCCAGCGGGACCGGATCTTCCTCGCAGCTCTCGCGCCGA CGCTCCGGGCGTCGTAGACGCTAGGAGCGCTACCCGATTGTAACATGGTT ACCCCTTGCCCTGCCAGCCCGGGCAGCCCCGCCGCGGGAGCCGGGAGGCG AGACAGCCACCAGAACCTCCGCGCCCCAGTGAAGAAGAGCAGACGCCCGT GCCTCCGGAGAAAGAAGCCGCTGCGGCCGCTGAACGCGTGTTCGCTCCCG GGAGACTCGGGCGTCTGTGACCTTTTCGAGTCCCCCAGCTCCAGCTCGGA CGGCGCCGACAGCCCCGCAGTGTCTGCGGCGCGGGACTGCAGCTCCCTAC TCAACCCTGCCCAGCCCTTGACAGCGCTGGATCTCCAGACCTTCCGAGAA TACGGCCAGAGCTGCTACGACTTCCGAAAGGCGCAGGAGAATCTTTTCCA CCCGCGGGAGTCGCTGGCGCGCCAGCCACAAGTAGAGGTGCACCCACCTC GCTTGAAACAGCTCCTGGCCCTGTGCGGCGGTGCGTTCTCCCGGCAGCAG CTGTGCAACCTGGAGTGCATCGTGCTGCATAAGCTCCACTTCAGCCTGGG CGCGCCCACCATCAACTTCTTCCTGGAGCACTTCACTCAGTGGCGCATGG AAGCCGGCCAGGCTGAGGTCACCGAAGCTCTAGAGGCTCAAACCCTGGCC CGGGGAGTGGCGGAGCTGAGCCTAACGGATTACGCGTTCACTACCTACAC GCCGTCCCTGATGGCTATCTGCTGCCTGGCGCTGGCTGACGGATTGCTGC AGCACCAGCACGAGATGGACCTGCGCCTGGGAGAGCACCCAGAGGCCACA CTGCAAGACTGTCTGGGCAAGCTGCAGACGCTAGTGTCCATCAATAGCAG CTCCCTGCCTCGCATTCTGCCTCCCCAGATCTGGGAGAGGTGCAGCCTGC CCCAGAGTTGGCAATAAAAAGCACCCCCTTGAGCTCCTTTTTAAAATTCC CTGGTCCCTGCTCCGTGCCTTAAGGAGAATATGCAGTACAGATCCAAAGA GAAGTTGAGGTCTCCTACCTGTAAATACTGTACAATAACGGATTCTTTAT TTATTTTGTAGTACACAAGGGCAGGGGTGACAGTTCTGGCTTGTCTAAAT GCTGCTGTTGGCATGCCTGACTGTCTGCTTTTCCCAGGCGAAGTCCATCA CGAGGTAGCCAATTCCGGAGGCTCTTCCCTGCTCCCACAGTTTTACTTTG TAGTTAGCTCTGTGAAAACAACCTGGCAAGATTTTATATGCCACTGTTCC AGAAAGCAGACAAGTCAAGGTGGCTTGTCCGTATCTTAACCCCCTCCAGT CAGGAGGCTGAGTTCCCTCCCCCATGCCAGTGGCCCAAGGAGAATGAGAC AGGGTTAGAAGTCAGAGTGGTCTGTGCTGTCCGGAAAGGCAGTGGCTGCA CTAACTGATGTGGAGCTGGGAAGCTTTGGGGACCGGGCAGCCATACTAGC

AGTGGGTGAAGCAAGAGACAGTCCCTGGTGCAACACAGGAAATGCTGGAA TGCCTTTTGCGTATTGTTATAGCTGTTGCAACCTGGGTCATTTCCTCTGC AGCTACACTCATGGTTCAGTTTATACTGCAGCATGAGCCCAGGGTAATTC TGGTACGGCAGTAATAAATATTATACTATTTGCAACAT

SEQ ID No. 8:

Amino acid sequence of murine Cyclin O-beta.

MVTPCP ASPGSPAAGAGRRDSHQNLRAPVKKSRRPCLRRKKPLRPLNACS LPGDSGVCDLFESPSSSSDGADSPAVSAARDCSSLLNPAQPLTALDLQTF REYGQSCYDFRKAQENLFHPRESLARQPQVEVHPPRLKQLLALCGGAFSR QQLCNLECIVLHKLHFSLGAPTINFFLEHFTQWRMΈAGQ AEVTEALEAQT LARGV AELSLTD YAFTTYTPSLMAICCLALADGLLQHQHEMDLRLGEHPE ATLQDCLGKLQTLVSINSSSLPRILPPQIWERCSLPQSWQ

SEQ ID No. 9:

Nucleotide sequence encoding murine Cyclin O-alpha L3A mutant. Coding region of murine

Cyclin O-alpha L3A mutant ranges from nucleotide 95 to nucleotide 1150 of SEQ ID NO.9.

AGAGCTGCGCCGCCAGCGGGACCGGATCTTCCTCGCAGCTCTCGCGCCGACGCTC CGGGCGTCGTAGACGCTAGGAGCGCTACCCGATTGTAACATGGTTACCCCTTGCC CTGCCAGCCCGGGCAGCCCCGCCGCGGGAGCCGGGAGGCGAGACAGCCACCAGA ACCTCCGCGCCCCAGTGAAGAAGAGCAGACGCCCGTGCCTCCGGAGAAAGAAGC CGCTGCGGCCGCTGAACGCGTGTTCGCTCCCGGGAGACTCGGGCGTCTGTGACCT TTTCGAGTCCCCCAGCTCCAGCTCGGACGGCGCCGACAGCCCCGCAGTGTCTGCG GCGCGGGACTGCAGCTCCCTACTCAACCCTGCCCAGCCCGCGACAGCGGCGGATG CCCAGACCTTCCGAGAATACGGCCAGAGCTGCTACGACTTCCGAAAGGCGCAGG AGAATCTTTTCCACCCGCGGGAGTCGCTGGCGCGCCAGCCACAAGTGACTGCCGA ATCGCGCTGTAAACTGCTCAGCTGGCTCCTGCAAGTCCACCGCCAGTTCGGCCTC TCTTTCGAGTCGCTGTGTCTGACCGTGAATACTCTGGACCGTTTTCTCCTCACGAC CCCTGTTGCTGCAGACTGCTTCCAGCTGCTCGGGGTCACCTGTCTGCTCATCGCTT GCAAGCAGGTAGAGGTGCACCCACCTCGCTTGAAACAGCTCCTGGCCCTGTGCGG CGGTGCGTTCTCCCGGCAGCAGCTGTGCAACCTGGAGTGCATCGTGCTGCATAAG CTCCACTTCAGCCTGGGCGCGCCCACCATCAACTTCTTCCTGGAGCACTTCACTCA GTGGCGCATGGAAGCCGGCCAGGCTGAGGTCACCGAAGCTCTAGAGGCTCAAAC CCTGGCCCGGGGAGTGGCGGAGCTGAGCCTAACGGATTACGCGTTCACTACCTAC ACGCCGTCCCTGATGGCTATCTGCTGCCTGGCGCTGGCTGACGGATTGCTGCAGC ACCAGCACGAGATGGACCTGCGCCTGGGAGAGCACCCAGAGGCCACACTGCAAG ACTGTCTGGGCAAGCTGCAGACGCTAGTGTCCATCAATAGCAGCTCCCTGCCTCG CATTCTGCCTCCCCAGATCTGGGAGAGGTGCAGCCTGCCCCAGAGTTGGCAATAA AAAGCACCCCCTTGAGCTCCTTTTTAAAATTCCCTGGTCCCTGCTCCGTGCCTTAA GGAGAATATGCAGTACAGATCCAAAGAGAAGTTGAGGTCTCCTACCTGTAAATA CTGTACAATAACGGATTCTTTATTTATTTTGTAGTACACAAGGGCAGGGGTGACA GTTCTGGCTTGTCTAAATGCTGCTGTTGGCATGCCTGACTGTCTGCTTTTCCCAGG CGAAGTCCATCACGAGGTAGCCAATTCCGGAGGCTCTTCCCTGCTCCCACAGTTT TACTTTGTAGTTAGCTCTGTGAAAACAACCTGGCAAGATTTTATATGCCACTGTTC CAGAAAGCAGACAAGTCAAGGTGGCTTGTCCGTATCTTAACCCCCTCCAGTCAGG AGGCTGAGTTCCCTCCCCCATGCCAGTGGCCCAAGGAGAATGAGACAGGGTTAG AAGTCAGAGTGGTCTGTGCTGTCCGGAAAGGCAGTGGCTGCACTAACTGATGTGG AGCTGGGAAGCTTTGGGGACCGGGCAGCCATACTAGCAGTGGGTGAAGCAAGAG ACAGTCCCTGGTGCAACACAGGAAATGCTGGAATGCCTTTTGCGTATTGTTATAG CTGTTGCAACCTGGGTCATTTCCTCTGCAGCTACACTCATGGTTCAGTTTATACTG CAGCATGAGCCCAGGGTAATTCTGGTACGGCAGTAATAAATATTATACTATTTGC AACAT

SEQ ID No. 10: Amino acid sequence of murine Cyclin O-alpha L3 A mutant.

MVTPCPASPGSP AAGAGRRDSHQNLRAPVKKSRRPCLRRKKPLRPLNACSLPGDSGV CDLFESPSSSSDGADSP AVSAARDCSSLLNP AQP ATAADAQTFREYGQSCYDFRKAQ

ENLFHPRESLARQPQVTAESRCKLLSWLLQ VHRQFGLSFESLCLTVNTLDRFLLTTPV AADCFQLLGVTCLLIACKQVEVHPPRLKQLLALCGGAFSRQQLCNLECIVLHKLHFSL GAPTINFFLEHFTQWRMEAGQAEVTEALEAQTLARGVAELSLTDYAFTTYTPSLMAI CCLALADGLLQHQHEMDLRLGEHPEATLQDCLGKLQTLVSTNSSSLPRILPPQIWERC

SLPQSWQ SEQ ID No. 11 :

Nucleotide sequence encoding Homo sapiens Cyclin 0-gamma RNA transcript. Human Cyclin O-gamma RNA transcript is not translated into polypeptides/proteins.

CACCGGCCTCATTTTACAGATGGAGAAAAGTGGGGCGTAGACGGCTTGAG GGACTTGCCCCGTCCCAAGCTGGCTACTGGGGCACGGTAGAGAGGAATTC GTTTCCGGGTTGAGTCTGGACTCCTACCGAACTCGGGCAAGTTATTTAAC ACCCCTGAGCTTCTTTCTTCATCTGCAAAATGGAACCCCTCCCCTTCCTT TGAATTGCTGAGCCCATTACGCGAGGTAATGTGTACGACGCTCTCTGCAC AGCGCCAGGTCCCAATCCCGGTGTTCCGGCTCAGCACGGCCCACGCGTTT CCCAGTTTCCATTGCGTTTCTCTTTCTGAAATCTTCTTGCCCCTCTCCTG GGAAGCTCCCCGATTCTTCTTGGCCCTTCCTTCCCTTCCACAGTCCCTCT CCACCCTAAGCCCAGCGGGCCCGCCTCCCCCCTCCTTCCCGGCAGGTGAC GGCGGAATCCCGCTGTAAGCTGCTCAGCTGGCTGATCCCGGTGCACCGCC AATTCGGCCTCTCCTTCGAGTCGCTGTGCCTGACGGTGAACACTCTGGAC CGCTTCCTCACCACCACGCCGGTGGCTGCAGACTGCTTCCAGCTGCTTGG GGTCACCTCCTTGCTCATCGCTTGCAAACAGGTGGAGGTGCACCCGCCGC GCGTGAAGCAGCTTCTGGCCCTCTGCTGCGGCGCCTTCTCCCGGCAGCAG CTCTGCAACCTCGAGTGCATCGTGCTGCACAAGCTGCACTTCACCCTGGG TGCGCCCACCATTAGCTTCTTCCTGGAGCATTTCACGCACGCTCGCGTGG AGGCGGGGCAGGCTGAGGCCTCCGAAGCTCTGGAAGCGCAAGCCCTGGCG CGGGGGGTGGCAGAGCTGAGTCTGGCCGACTATGCCTTCACCAGCTACTC CCCTTCCCTCCTGGCGATCTGCTGCCTGGCGCTGGCGGACCGCATGCTGC GGGTCTCGCGGCCCGTGGACTTGCGACTGGGAGACCACCCGGAGGCGGCG CTGGAGGACTGTATGGGCAAGTTGCAGCTGCTGGTGGCCATAAACAGTAC TTCCTTGACTCACATGCTGCCCGTTCAGATCTGCGAGAAGTGCAGCCTGC CCCCGΛGCTCGAAATAAAACAGATCCTTCGTTTCCTTTTAGTCCCTGGCC CGGCTGCTGGACCTCTCCCGTAGCCTCAGAAGAGTGCAGT ACTGGTCCAC AGAGAAGGCTTCAGGACCTGCTTGGTCAGCTGCAGGTTGTAAATAGTGTA CGATACTAGCATCTGGTATTTTATTTATTTTGCAGCGAGCACATGAGGAA GCTGAGTCTTTCACCAATAAACAGTTGTGGTTTGTCTA SEQ lD No. 12:

Nucleotide sequence encoding murine Cyclin O-delta RNA transcript. Murine Cyclin O-delta

RNA transcript is not translated into polypeptides/proteins.

TTGTGCGACGGTCATTTGAAGTAACTTCTTACGTTTTTGGATCACAAATA CCTGTCTGACAGGACGGGCTCCCACATACTTAACAGACGTTAGCGACGCG CCAAAGTTCTCCTTCCCTGGGCCAGCGGAATGAAATAAAATAGTCGCAAA TCAAGTCCTTTTATGGGTCACTGAAGAAAACCTAGCCTGATCTTGGTCCG GAGCTCAGAGGCGGACCCACCTGGATGAGGGTTGGCATAAAAGATGATCA CACCAGGCCAGACAATCTAACCAAGGACCAGGTTCCCCGGAGCAGCCTTT CCCTTTTTCCGGGGGCGGCTGAACCGCTCAGACGGCCCATGGCTCCCCTA GGTGGCCAGTGATCTTTACGCTCTTCCACCGAGCTCAAGCAGCTGGCGCC AGGACCCGGGCTGCGGTTGCCACGGTGACGACAGACGCGCGGAGCCTGCA GCTGAAGGCGGCCCGGGCAGCCCCGCCGCGGGAGCCGGGAGGCGAGACAG CCACCAGAACCTCCGCGCCCCAGTGAAGAAGAGCAGACGCCCGTGCCTCC GGAGAAAGAAGCCGCTG

SEQ ID No. 13:

Nucleotide sequence encoding human Cyclin O-alpha L3A mutant. Changed nucleotides are indicated in bold font. Coding region of Homo sapiens Cyclin O-alpha L3A mutant ranges from nucleotide 259 to nucleotide 1308 of SEQ ID NO.13.

CTCCCCCCGGCGGGCCGCGGAGGCGACGCGCTGCCTGCGCCCTGAGGCGA GTGGTTCTCCAGCCGGAAAATCGCGGCTCCGGTGCCGCTCTGGGCTCAGC CAGTGAGAGGCCGGGGGTGTGGTAGAGAGAAGGAGCCGGCCGGGTACTTA GGACCGCGCCAGCCTCTGAGCCCGGCCTCCTTCGCACTTTCGAGTGCGCG TTTGCTGCCCGCCGCAGCCCGGGCGTTGAAGGTAGTAAAGCGGCCACCCG GCCGCATCATGGTGACCCCCTGTCCCACCAGCCCCTCGAGCCCCGCCGCC CGAGCGGGGAGGCGGGACAACGACCAGAACCTTCGCGCCCCGGTGAAGAA GAGCAGGCGTCCGCGCCTCCGGAGGAAGCAGCCGCTGCATCCCCTGAACC CGTGCCCGCTCCCGGGAGACTCCGGCATTTGCGACCTGTTCGAGTCCCCC AGCTCCGGCTCAGACGGCGCAGAGAGCCCCTCTGCGGCGCGGGGTGGTAG CCCCCTGCCCGGCCCGGCCCAGCCCGCGGCGCAGGCAGATGCACAGACCT TCCGCGACTACGGCCAGAGCTGCTACGCCTTCCGCAAGGCGCAGGAGAGC CACTTCCACCCGCGGGAGGCGCTGGCACGGCAGCCACAAGTGACGGCGGA ATCCCGCTGTAAGCTGCTCAGCTGGCTGATCCCGGTGCACCGCCAATTCG GCCTCTCCTTCGAGTCGCTGTGCCTGACGGTGAACACTCTGGACCGCTTC CTCACCACCACGCCGGTGGCTGCAGACTGCTTCCAGCTGCTTGGGGTCAC CTCCTTGCTCATCGCTTGCAAACAGGTGGAGGTGCACCCGCCGCGCGTGA AGCAGCTTCTGGCCCTCTGCTGCGGCGCCTTCTCCCGGCAGCAGCTCTGC AACCTCGAGTGCATCGTGCTGCACAAGCTGCACTTCACCCTGGGTGCGCC CACCATTAGCTTCTTCCTGGAGCATTTCACGCACGCTCGCGTGGAGGCGG GGCAGGCTGAGGCCTCCGAAGCTCTGGAAGCGCAAGCCCTGGCGCGGGGG GTGGCAGAGCTGAGTCTGGCCGACTATGCCTTCACCAGCTACTCCCCTTC CCTCCTGGCGATCTGCTGCCTGGCGCTGGCGGACCGCATGCTGCGGGTCT CGCGGCCCGTGGACTTGCGACTGGGAGACCACCCGGAGGCGGCGCTGGAG GACTGTATGGGCAAGTTGCAGCTGCTGGTGGCCATAAACAGTACTTCCTT GACTCACATGCTGCCCGTTCAGATCTGCGAGAAGTGCAGCCTGCCCCCGA GCTCGAAATAAAACAGATCCTTCGTTTCCTTTTAGTCCCTGGCCCGGCTG CTGGACCTCTCCCGTAGCCTCAGAAGAGTGCAGTACTGGTCCACAGAGAA GGCTTCAGGACCTGCTTGGTCAGCTGCAGGTTGTAAATAGTGTACGATAC TAGCATCTGGTATTTTATTTATTTTGCAGCGAGCACATGAGGAAGCTGAG TCTTTCACCAATAAACAGTTGTGGTTTGTCT

SEQ ID No. 14:

Amino acid sequence of human Cyclin O-alpha L3A mutant. Changed amino acid residues are indicated in bold font. It is of note that the conserved motif in the human sequence is VxxLxL.

MVTPCPTSPSSPAARAGRRDNDQNLRAPVKKSRRPRLRRKQPLHPLNPCP LPGDSGICDLFESPSSGSDGAESPSAARGGSPLPGPAQPAAQADAQTFRD YGQSCYAFRKAQESHFHPREALARQPQVTAESRCKLLSWLIPVHRQFGLS FESLCLTVNTLDRFLTTTPVAADCFQLLGVTSLLIACKQVEVHPPRVKQL LALCCGAFSRQQLCNLECIVLHKLHFTLGAPTISFFLEHFTHARVGAGQA EASEALEAQALARGVAELSLADYAFTSYSPSLLAICCLALADRMLRVSRP VDLRLGDHPEAALEDCMGKLQLL V AINSTSLTHMLPVQICEKCSLPPSSK References cited herein:

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Claims

1. Method for the identification of a responder for or a patient sensitive to adjuvant therapy, said method comprising the following steps:

(a) obtaining a sample from a patient suspected to suffer from or being prone to a colorectal carcinoma, whereby said sample is histopathologically categorized as normal (healthy) and said colorectal carcinomais associated with a stage II tumor; (b) determining in said sample obtained in (a) the expression level of Cyclin O ;

(c) comparing the expression level of Cyclin O of Cyclin O determined in (a) with a reference expression level of said nucleic acid molecule, wherein the extent of difference between said expression level determined in (b) and said reference expression level is indicative for a responding patient or is indicative for a sensitivity of said patient to adjuvant therapy.

2. The method according to claim 1, wherein said sample is obtained prior to surgery,

3. The method according to claim 1 or 2, wherein said adjuvant therapy is selected from the group consisting of chemotherapy, radiotherapy and second surgery.

4. The method according to any one of claims 1 to 3, wherein said sample is a histopathologically normal colon mucosa sample.

5. The method according to any one of claims 1 to 4, wherein said Cyclin O is Cyclin O- alpha and/or Cyclin O-beta.

6. Kit for carrying out the method according to any one of claims 1 to 5, said kit comprising (a) compound(s) for specifically determining the expression level of Cyclin O.

7. A nucleic acid molecule selected from the group consisting of

(a) a nucleic acid molecule having a nucleic acid sequence as depicted in SEQ ID NO 9 or 13:

(c) a nucleic acid sequence which is capable of hybridizing to the complementary strand of the nucleic acid sequence of (a) or (b) and encoding a functional mutant Cyclin O-alpha or a functional fragment thereof;

(d) a nucleic acid molecule having a nucleic acid sequence having at least 60 % homology to the nucleic acid sequence of (a) or (b) and encoding a functional mutant Cyclin O-alpha or a functional fragment thereof; (e) a nucleic acid molecule having a nucleic acid sequence being degenerate as a result of the genetic code to the nucleic acid sequence as defined in any one of (a), (b), (c) and (d): and (f) the complementary strand of the nucleic acid molecule of any one of (a) to (e).

A polypeptide selected from the group consisting of

(a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO 9 or 13;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO 10 or 14; (c) a polypeptide comprising an amino acid encoded by a nucleic acid molecule encoding a peptide having an amino having an amino acid sequence as depicted in SEQ ID NO 9 or 13;

(d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional mutant Cyclin

O-alpha or a functional fragment thereof;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), and encoding a functional mutant Cyclin O-alpha or a functional fragment thereof; and (f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having a nucleic acid sequence which is degenerate as a result of the genetic code to the nucleic acid sequence of a nucleic acid molecule as defined in (a), (C), (d) and (e).

9. A nucleic acid construct comprising a nucleic acid molecule as defined in claim 7 or encoding the polypeptide as defined in claim 8.

10. A vector comprising the nucleic acid construct of claim 9.

1 1. The vector according to claim 10, which further comprises a nucleic acid molecule which is a regulatory molecule operably linked to said nucleic acid molecule.

12. The vector according to claim 11, wherein said vector is an expression vector.

13. A host cell genetically engineered with the nucleic acid molecule of claim 7 or comprising the nucleic acid construct of claim 9 or the vector of any one of claims 10 to 12.

14. A process for the production of a nucleic acid molecule of claim 7 or a polypeptide of claim 8, said process comprising culturing a host cell of claim 13 under conditions allowing the production of the nucleic acid molecule and/or the polypeptide and recovering the nucleic acid molecule and/or the polypeptide from the culture.

15. A pharmaceutical composition comprising Cyclin O or an agonist of Cyclin O.

16. The pharmaceutical composition according to claim 15, wherein said Cyclin O is Cyclin O-alpha, Cyclin O-beta and/or the mutant Cyclin O-alpha as defined in claim 7 or 8 or as produced by the process of claim 14.

17. The pharmaceutical composition according to claim 16, wherein said Cyclin O alpha is a polypeptide selected from the group consisting of

(a) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule having the nucleic acid sequence as depicted in SEQ ID NO 1 or 5;

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO 2 or

6;

(d) a polypeptide comprising an amino acid encoded by a nucleic acid molecule hybridizing under stringent conditions to the complementary strand of nucleic acid molecules as defined in (a) or (c) and encoding a functional Cyclin O- alpha or a functional fragment thereof;

(e) a polypeptide having at least 60 % homology to the polypeptide of any one of (a) to (d), and encoding a functional Cyclin Oalpha or a functional fragment thereof; and (f) a polypeptide comprising an amino acid encoded by a nucleic acid molecule having a nucleic acid sequence which is degenerate as a result of the genetic code to the nucleic acid sequence of a nucleic acid molecule as defined in (a), (C)₅ (d) and (e).

18. The pharmaceutical composition according to claim 16 or 17, wherein said Cyclin O beta is a polypeptide selected from the group consisting of

(b) a polypeptide having an amino acid sequence as depicted in SEQ ID NO 4 or 8;

(c) a polypeptide comprising an amino acid encoded by a nucleic acid molecule encoding a peptide having an amino acid sequence as depicted in SEQ ID NO 4 or 8;

19. A pharmaceutical composition comprising a nucleic acid molecule encoding Cyclin O-alpha as defined in claim 17, Cyclin O-beta as defined in claim 18 and/or a nucleic acid molecule as defined in claim 7 or as produced by the process of claim 14.

20. A pharmaceutical composition comprising a nucleic acid construct comprising the nucleic acid molecule as defined in claim 19.

21. A pharmaceutical composition comprising a vector which comprises the nucleic acid construct as defined in claim 20.

22. A pharmaceutical composition comprising a host cell which comprises the vector as defined in claim 21.

23. The pharmaceutical composition according to any of 15 to 22, further comprising, optionally, suitable formulations of carrier, stabilizers and/or excipients.

24. The pharmaceutical composition according to any of claims 15 to 23 for preventing or treating a proliferative disease.

25. Method for preventing or treating a proliferative disease comprising the administration of an effective amount of an agonist of Cyclin O, a Cyclin O polypeptide as defined in any of claims 16 to 18, the nucleic acid molecule as defined in claim 19, the nucleic acid construct as defined in claim 20, the vector as defined in claim 21. and/or the host cell as defined in claim 22 to a subject in need of such a prevention, or treatment.

26. The method of claim 25, wherein said subject is a human.

27. Kit comprising the nucleic acid molecule as defined in claim 19, the polypeptide as defined in any of claims 16 to 18, the nucleic acid construct as defined in claim 20, the vector as defined in claim 21, and/or the host cell as defined in claim 22.

28. The pharmaceutical composition according to claim 24 or the method according to claim 25, wherein said proliferative disease is a cancerous disease.

29. The pharmaceutical composition according to claim 28 or the method according to claim 28, wherein said cancerous disease is selected from the group consisting of adenocarcinoma, sarcoma, transitional bladder carcinoma, primitive neuroepithelial tumor, and squamous skin carcinoma.

30. The pharmaceutical composition according to claim 29 or the method according to claim 29, wherein said adenocarcinoma is selected from the group consisting of pancreas carcinoma, colorectal carcinoma, lung adenocarcinoma, well differentiated squamous lung carcinoma, stomach carcinoma, , endometroid adenocarcinoma and breast adenocarcinoma.

31. The pharmaceutical composition according to claim 29 or the method of claim 29, wherein said sarcoma is selected from the group consisting of osteosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, chondrosarcoma, fibrosarcoma, and histiocytoma.