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WO2007022588A1 - Procédé d’évaluation d’une réponse à un agent antiproliférant - Google Patents

Procédé d’évaluation d’une réponse à un agent antiproliférant Download PDF

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Publication number
WO2007022588A1
WO2007022588A1 PCT/AU2006/001230 AU2006001230W WO2007022588A1 WO 2007022588 A1 WO2007022588 A1 WO 2007022588A1 AU 2006001230 W AU2006001230 W AU 2006001230W WO 2007022588 A1 WO2007022588 A1 WO 2007022588A1
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Prior art keywords
bim
cell
level
responsiveness
bad
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Inventor
Junya Kuroda
Hamsa Puthalakath
Mark Cragg
Philippe Bouillet
Andrew Roberts
Priscilla Kelly
David Huang
Andreas Strasser
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Walter and Eliza Hall Institute of Medical Research
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Walter and Eliza Hall Institute of Medical Research
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates generally to a method of diagnosing, predicting and/or monitoring cellular responsiveness to antiproliferative agents. More particularly, the present invention relates to a method of diagnosing, predicting and/or monitoring reduced cellular responsiveness to antiproliferative agents by screening for modulation in the levels of functional Bim and/or Bad in the subject cell. The present invention further provides a method for predicting, diagnosing and/or monitoring the responsiveness of a condition characterised by unwanted cellular proliferation to treatment with an antiproliferative agent. Also provided are diagnostic agents useful for detecting functional Bim and/or Bad expression levels.
  • Malignant tumours, or cancers grow in an uncontrolled manner, invade normal tissues, and often metastasize and grow at sites distant from the tissue of origin.
  • cancers are derived from one or only a few normal cells that have undergone a poorly understood process called malignant transformation. Cancers can arise from almost any tissue in the body. Those derived from epithelial cells, called carcinomas, are the most common kinds of cancers.
  • Sarcomas are malignant tumours of mesenchymal tissues, arising from cells such as fibroblasts, muscle cells, and fat cells. Solid malignant tumours of lymphoid tissues are called lymphomas, and marrow and blood-borne malignant tumours of lymphocytes and other hematopoietic cells are called leukemias.
  • Cancer is one of the three leading causes of death in industrialised countries. As treatments for infectious diseases and the prevention of cardiovascular disease continues to improve, and the average life expectancy increases, cancer is likely to become the most common fatal disease in these countries. Therefore, successfully treating cancer requires that all the malignant cells be removed or destroyed without killing the patient. An ideal way to achieve this would be to induce an immune response against the tumour that would discriminate between the cells of the tumour and their normal cellular counterparts. However, immunological approaches to the treatment of cancer have been attempted for over a century with unsustainable results.
  • tumours may contain a proportion of apoptotic cells and even areas of necrosis before anti-cancer treatment is given, an increased number of apoptotic cells and larger areas of necrosis are anticipated in tumours that respond to the anti-cancer treatment.
  • cytotoxic chemotherapeutic agents are used for the treatment of advanced cancer, the degree of cell kill and thus the response of the tumour to the first treatment is frequently difficult to assess.
  • the term "derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of "a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.
  • nucleotide sequence information prepared using the programme Patentln Version 3.1, presented herein after the bibliography.
  • Each nucleotide sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (eg. ⁇ 210>l, ⁇ 210>2, etc).
  • the length, type of sequence (DNA, etc) and source organism for each nucleotide sequence is indicated by information provided in the numeric indicator fields ⁇ 211>, ⁇ 212> and ⁇ 213>, respectively.
  • Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO: 1 , SEQ ID NO:2, etc.).
  • sequence identifier referred to in the specification correlates to the information provided in numeric indicator field ⁇ 400> in the sequence listing, which is followed by the sequence identifier (eg. ⁇ 400>l, ⁇ 400>2, etc). That is SEQ ID NO:1 as detailed in the specification correlates to the sequence indicated as ⁇ 400>l in the sequence listing.
  • One aspect of the present invention is directed to a method for assessing the responsiveness of a cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness .
  • Another aspect of the present invention therefore provides a method for assessing the responsiveness of a neoplastic cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • Yet another aspect of the present invention is directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • Still another aspect of the present invention is directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces death, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • Yet still another aspect of the present invention provides a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces apoptosis, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • Still yet another aspect of the present invention is directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces apoptosis, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein the absence of said protein and/or gene expression is indicative of unresponsiveness.
  • the present invention is directed to a method for assessing the responsiveness to an antiproliferative agent of a cell in a mammal or in a biological cell derived from said mammal, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • Another further aspect of the present invention relates to a method for monitoring the responsiveness of a cellular population to an antiproliferative agent, said method comprising screening for modulation of the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression in the subject cellular population relative to a previously obtained level or a normal level is indicative of the maintenance or worsening of responsiveness and an increase in said level is indicative of an improvement in said responsiveness.
  • Yet another aspect of the present invention is directed to a method for assessing the actual or likely responsiveness to an antiproliferative agent of a condition characterised by unwanted cellular proliferation, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • the present invention relates to a method for monitoring the responsiveness to an antiproliferative agent of a condition characterised by unwanted cellular proliferation, said method comprising screening for modulation of the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression in the subject cellular population relative to a previously obtained level or a normal level is indicative of the maintenance or worsening of responsiveness and an increase in said level is indicative of an improvement in said responsiveness.
  • Yet another aspect of the present invention is directed to assessing and/or monitoring the effectiveness of an antiproliferative treatment regime in a mammal, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression by a cellular population of interest wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • Another aspect of the present invention provides a diagnostic kit comprising an agent for detecting Bim and/or Bad or a nucleic acid molecule encoding Bim and/or Bad and reagents useful for facilitating the detection by said agent.
  • the present invention further contemplates the use of an interactive molecule directed to Bim and/or Bad in the manufacture of a quantitative or semi-quantitative diagnostic kit to assess cellular responsiveness to an antiproliferative agent.
  • the kit may come with instructions for use and may be automated or semi-automated or in a form which is compatible with an automated machine or software.
  • FIG. 1 Effect of Bcl-2 over-expression or dominant negative FADD on the proliferation inhibitory and pro-apoptotic effects of imatinib.
  • K562 and BV173 human Ph 1 + leukemia- derived cells were treated with the indicated concentrations of imatinib for 48 h.
  • A Cell proliferation was measured using the MTS assay.
  • B Staining with PI followed by flow cytometric analysis was used to discriminate between live and dead cells (PI + ). Results represent mean ⁇ SD of three experiments.
  • C Western blotting was performed to determine the levels of anti-apoptotic Bcl-2 family members before and after treatment with imatinib in K562 and BV 173 cells.
  • FIG. 1 Treatment with imatinib causes increased expression of Bim and Bim de- phosphorylation in Ph 1 + human leukemic cells.
  • A K562 cells were treated for the indicated amount of time with 0.5 ⁇ M or 1 ⁇ M imatinib
  • B BV173 cells were treated for the indicated amount of time with 1.5 ⁇ M or 3 ⁇ M imatinib.
  • Western blotting was performed with an anti-Bim antibody and as a loading control with an antibody to HSP70 or ⁇ -actin.
  • C K562 cells were treated for 3 or 6 h with 1 ⁇ M imatinib. Semi-quantitative PCR was performed to determine levels of bim mRNA.
  • RNAi -mediated knock-down protects K562 and BVl 73 cells against imatinib.
  • A Western blot analysis using antibodies to Bim and ⁇ -actin (loading control) documents the levels of Bim expression in parental (ctl: control) and RNAi Bim knock-down subclones of K562 and BV173 cells.
  • B, C Parental and RNAi Bim knock-down subclones of K562 and BVl 73 cells were treated for the indicated amount of time with 1.5 ⁇ M (B) or 3 ⁇ M (C) imatinib respectively. Cell viability was determined by staining with PI followed by blow cytometric analysis.
  • Figure 4 Effect of imatinib on expression of Bim, Bad and Bmf and phosphorylation of Bad in control and RNAi Bim knock-down K562 and BV173 cells.
  • Parental as well as RNAi Bim knock-down K562 and BVl 73 cells were treated for the indicated periods of time with 1.5 ⁇ M or 3 ⁇ M imatinib, respectively.
  • Western blotting was performed using antibodies to Bim, Bad, phosphorylated Bad, Bmf and ⁇ -actin (loading control).
  • FIG. 6 Effect of imatinib on expression of Bim, Bad, Bmf, Bcl-2, Bcl-xL and Bax as well as phosphorylation of Bad in bcr-c-abl transformed wt, bim '1' and bad 1" fetal liver cells.
  • A Cells were treated for 0, 24 or 48 h with 3 ⁇ M imatinib. Western blotting was performed with antibodies specific to Bim, Bad (total), phosphorylated Bad, Bmf, Bcl-2, BCI-X L , Bax or ⁇ -actin (loading control).
  • B Cells were treated for 3, 6, 12 or 24 h with 5 ⁇ M imatinib. Semi-quantitative RT-PCR analysis was performed to determine the levels of bmf, puma and ⁇ -actin (loading control) mRNA
  • Bim expression is induced in primary Ph 1 + leukemia cells from good responders after ex vivo imatinib treatment. Cells were cultured for O 5 12 or 24 h with 3 ⁇ M imatinib. Western blotting was performed with antibodies to Bim or ⁇ -actin. Clinical response and, where known, mutational status of bcr-abl are indicated.
  • Figure 8 Cell viability was quantified by flow cytometric analysis with PI/FITC- conjugated Annexin V counterstaining.
  • K562, BV173 and v ⁇ .bcr-c-abt FLCs were treated for 48 h with the indicated concentration of imatinib.
  • X-axis represents staining with FITC labelled Annexin V
  • Y-axis represents staining with PI.
  • FIG. 10 Western Blot analysis for Bim expression in K562 cells treated with imatinib with or without the broad spectrum caspase inhibitor ZVAD-fmk.
  • FIG 11 Effect of the expression of a control RNAi construct on K562 cells: (A)Bcr/Abl expression level, and (B) the response to imatinib were examined.
  • the present invention is predicated, in part, on the determination that the loss of Bim and/or Bad functionality, for example due to loss of expression, serves as a highly sensitive indicator of the reduced sensitivity of a cell to the actions of an antiproliferative agent. Accordingly, the detection of reduced levels of functional Bim and/or Bad relative to normal levels provides a convenient and precise mechanism for assessing potential or actual cellular responsiveness to antiproliferative agents, in particular in the context of predicting or monitoring the responsiveness of a neoplastic condition to chemotherapy.
  • one aspect of the present invention is directed to a method for assessing the responsiveness of a cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • the subject cells may have been freshly isolated from an individual (such as an individual who may be the subject of treatment) or they may have been sourced from a non-fresh source, such as from a culture (for example, where cell numbers were expanded and/or the cells were cultured so as to render them receptive to differentiative signals) or a frozen stock of cells (for example, an established cell line), which had been isolated at some earlier time point either from an individual or from another source. It should also be understood that the subject cells, prior to undergoing analysis, may have undergone some other form of treatment or manipulation, such as but not limited to enrichment or purification, modification of cell cycle status or the formation of a cell line.
  • the subject cell may be a primary cell or a secondary cell.
  • a primary cell is one which has been isolated from an individual.
  • a secondary cell is one which, following its isolation, has undergone some form of in vitro manipulation such as the preparation of a cell line, prior to the application of the method of the invention.
  • the subject cell is an abnormal cell and even more preferably a neoplastic cell.
  • the present invention therefore more particularly provides a method for assessing the responsiveness of a neoplastic cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • neoplastic cell should be understood as a reference to a cell exhibiting abnormal growth.
  • growth should be understood in its broadest sense and includes reference to proliferation.
  • abnormal growth in this context is intended as a reference to cell growth which, relative to normal cell growth, exhibits one or more of an increase in the rate of cell division, an increase in the number of cell divisions, a decrease in the length of the period of cell division, an increase in the frequency of periods of cell division or uncontrolled proliferation and evasion of apoptosis.
  • the common medical meaning of the term “neoplasia” refers to "new cell growth” that results as a loss of responsiveness to normal growth controls, eg. to neoplastic cell growth. Neoplasias include “tumours" which may be either benign, pre-malignant or malignant.
  • the term “neoplasm” should be understood as a reference to a lesion, tumour or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells.
  • Neoplasia refers to "new cell growth” that results as a loss of responsiveness to normal growth controls, e.g. to neoplastic cell growth.
  • a “hyperplasia” refers to cells undergoing an abnormally high rate of growth.
  • the terms “neoplasia” and “hyperplasia” can be used interchangeably, referring generally to cells experiencing abnormal cell growth rates.
  • Neoplasias and hyperplasias include “tumours” which may be either benign, pre-malignant or malignant.
  • the term “neoplasm” should be understood as a reference to a lesion, tumour or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells.
  • hypoproliferative and “neoplastic” are used interchangeably and refer to those cells in an abnormal state or condition characterized by rapid proliferation or neoplasm. The terms are meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs irrespective of histopathologic type or state of invasiveness. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumour growth.
  • nuclear in the context of the present invention should be understood to include reference to all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly or non-malignantly transformed cells, tissues or organs irrespective of histopathologic type or state of invasiveness.
  • carcinoma is recognised by those skilled in the art and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine system carcinomas and melanomas. Exemplary carcinomas include those forming from tissue of the breast.
  • the term also includes carcinosarcomas, e.g. which include malignant tumours composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumour cells form recognisable glandular structures.
  • neoplastic cells comprising the neoplasm may be any cell type, derived from any tissue, such as an epithelial or non-epithelial cell.
  • neoplasm should be understood as a reference to a lesion, tumour or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells.
  • the neoplastic cells comprising the neoplasm may be any cell type, derived from any tissue, such as an epithelial or non-epithelial cell.
  • Examples of neoplasms and neoplastic cells encompassed by the present invention include, but are not limited to central nervous system tumours, retinoblastoma, neuroblastoma and other paediatric tumours, head and neck cancers (e.g.
  • squamous cell cancers squamous cell cancers
  • breast and prostate cancers lung cancer (both small and non-small cell lung cancer), kidney cancers (e.g. renal cell adenocarcinoma), oesophagogastric cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g. adenocarcinomas and islet cell tumours), colorectal cancer, cervical and anal cancers, uterine and other reproductive tract cancers, urinary tract cancers (e.g. of ureter and bladder), germ cell tumours (e.g. testicular germ cell tumours or ovarian germ cell tumours), ovarian cancer (e.g.
  • ovarian epithelial cancers carcinomas of unknown primary, human immunodeficiency associated malignancies (e.g. Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroid gland), mesothelioma and other pleural or peritoneal tumours, neuroendocrine tumours and carcinoid tumours.
  • human immunodeficiency associated malignancies e.g. Kaposi's sarcoma
  • lymphomas e.g., leukemias, malignant melanomas, sarcomas
  • endocrine tumours e.g. of thyroid gland
  • mesothelioma and other pleural or peritoneal tumours e.g. of thyroid gland
  • neuroendocrine tumours e.g. of carcinoid tumours.
  • the present invention is therefore preferably directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • an "antiproliferative agent” should be understood as a reference to any agent which acts to reduce the rate or level of cellular proliferation.
  • the subject agent may function by any suitable mechanism including, but not limited to, inducing apoptosis, modulating cellular microtubule structure (eg. promoting microtubule polymerisation), inhibiting tyrosine kinase mediated signalling, antagonising cell surface receptor binding (eg. EGFR & VEGFR inhibitors), modulating glucocorticoid receptor functioning, downregulating angiogenesis (eg. inhibiting VEGF functioning) or otherwise inducing cell death or reducing cellular proliferation events.
  • Gleevec also called imatinib
  • imatinib is an example of a tyrosine kinase inhibitor. It works efficiently in the treatment of CML, cases of AML that have a bcr-abl chromosomal translocation and in certain (rare) cases of gastric lymphoma.
  • Other tyrosine kinase inhibitors include gefitinib, used for treatment of certain lung cancers, while still others are being developed for the treatment of lymphomas, leukemias, colon cancer, brain tumours etc. It is understood that Bim is critical for tumour cell killing using these drugs.
  • Glucocorticoids such as dexamethasone
  • Glucocorticoids bind to the glucocorticoid receptor, which is a transcription factor, and thereby cause upregulation of certain genes and downregulation of others.
  • Glucocorticoids are used (in combination with other chemotherapeutic drugs) in the treatment of certain lymphomas.
  • Bim is essential for killing by dexamethasone.
  • Taxol promotes excessive polymerisation of microtubules. It is used in the treatment of a range of cancers (usually in combination with other chemotherapeutic drugs). Bim is critical for Taxol-induced killing of at least certain types of cells.
  • Avastin and related drugs inhibit VEGF and/or its receptor
  • VEFG-R blood vessels
  • angiogenesis blood vessels
  • the subject antiproliferative agent is one which mediates its functional objective via the Bim signalling pathway. This may be effected in either a direct or an indirect manner. For example, the agent may directly act on either Bim or upstream signalling molecules. Alternatively, the agent may act on some other aspect of cellular functioning which itself acts to modulate Bim mediated signalling.
  • the present invention is directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces death, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • said cell death is apoptosis.
  • the present invention provides a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces apoptosis, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • Bim and “Bad” should be understood as a reference to all forms of these molecules and to fragments, mutants or variants thereof. It should also be understood to include reference to any isoforms which may arise from alternative splicing of Bim or Bad mRNA or mutant or polymorphic forms of Bim or Bad.
  • isoforms of human Bim these being Bini L and BUTIEL
  • isoforms of murine Bim these being Binis, BiniL and Bini EL -
  • Bim is known as a "BH3-only" protein since the only Bcl-2 homology region which it encompasses is BH3.
  • Bcl-2 related BH3-only pro-apoptotic group which also comprises, for example, Bik/Nbk, Bid and Hrk.
  • Bim is the only BH3-only protein for which splice variants exist, thereby resulting in the expression of a variety of isoforms.
  • the BH3-only proteins share with each other and the Bcl-2 family at large only the 9-16 amino acid BH3 region and they are essential for initiation of apoptosis signalling [Huang, 2000].
  • BH3-only proteins are regulated by a range of transcriptional and post-translational mechanisms [Puthalakath et al. Cell Death Differ.
  • Bim and Bad being “functional” should be understood as a reference to the capacity of these protein molecules to perform their normal range of activities. To this end, changes to the level of functional Bim or Bad can be caused in two ways, as follows:
  • the changes to Bim or Bad levels which one is screening for are changes to the absolute intracellular concentrations of these molecules.
  • the present invention is predicated on the finding that reduced intracellular levels of functional Bim and/or Bad expression result in reduced responsiveness to the actions of antiproliferative agents.
  • responsiveness is meant the capacity of the subject cell to undergo a slowing or cessation of its proliferative activity. Accordingly, it should be understood that although the present invention is exemplified in terms of cells which produce no Bim and have been found to be completely resistant to the effects of apoptosis inducing agents such as imatinib, in other situations a cell may produce lower than normal levels of Bim and/or Bad, thereby correlating to a reduced level of responsiveness, but not necessarily the complete absence of any responsiveness. The method of the present invention is nevertheless very useful in this situation since determination of the existence of sub-optimal potential to respond to a particular antiproliferative agent may indicate that consideration should be given to alternative treatment options.
  • the present invention is directed to a method for assessing the responsiveness of a malignant neoplastic cell to an antiproliferative agent, which agent induces apoptosis, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein the absence of said protein and/or gene expression is indicative of unresponsiveness.
  • the method of the present invention is predicated on the correlation of functional Bim and/or Bad levels in a cellular population with normal levels of these molecules.
  • the "normal level” is the level of Bim and/or Bad in a corresponding cellular sample which does not exhibit reduced responsiveness to the actions of an antiproliferative agent.
  • the present invention is directed to screening for changes to Bim and/or Bad levels relative either to a normal reference level (or normal reference level range) or to an earlier Bim and/or Bad level result determined from a particular in vitro biological sample or subject.
  • a normal reference level is the Bim and/or Bad level from a relevant biological sample of an in vitro source or a sample from a subject or group of subjects who are not exhibiting reduced responsiveness.
  • said normal reference level is the level determined from one or more subjects of a relevant cohort to that of the subject being screened by the method of the invention.
  • relevant cohort is meant a cohort characterised by one or more features which are also characteristic of the subject who is the subject of screening. These features include, but are not limited to, age, gender, ethnicity or health status, for example.
  • This reference level may be a discrete figure or may be a range of figures.
  • the reference level may vary between individual classes of Bim and/or Bad molecules (such as the differentially spliced forms of Bim).
  • the method of the invention should be understood to encompass all suitable forms of analysis such as the analysis of test results relative to a standard result which reflects individual or collective results.
  • the present method may be performed in the context of analysing the responsiveness of an in vitro cellular population, such as a cell line, or the responsiveness of a cellular population which has been harvested from a mammal.
  • an in vitro cellular population such as a cell line
  • the present invention will likely have its most significant application in terms of assessing the neoplastic cells of a patient, it is also conceivable that one might analyse a cellular population, such as a cell line, for the purpose of analysing the phenomenon of the existence of resistance or development of resistance to the apoptotic effects of a chemotherapy agent.
  • the present invention is directed to a method for assessing the responsiveness to an antiproliferative agent of a cell in a mammal or in a biological sample derived from said mammal, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression in said cell wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • said cell is a neoplastic cell and even more preferably a malignant neoplastic cell.
  • said antiproliferative agent induces cell death, in particular apoptosis.
  • mammal includes humans, primates, livestock animals (eg. horses, cattle, sheep, pigs, donkeys), laboratory test animals (eg. mice, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animal (eg. kangaroos, deer, foxes).
  • livestock animals eg. horses, cattle, sheep, pigs, donkeys
  • laboratory test animals eg. mice, rats, guinea pigs
  • companion animals eg. dogs, cats
  • captive wild animal eg. kangaroos, deer, foxes.
  • the mammal is a human or a laboratory test animal. Even more preferably, the mammal is a human.
  • Bim is thought to function as the critical protein for facilitating the apoptotic effects of antiproliferative agents such as imatinib.
  • antiproliferative agents such as imatinib.
  • variation can and does exist in terms of the level and extent of unresponsiveness across a neoplastic cell population.
  • the method of the present invention has application in relation to a broad range of situations including, but not limited to:
  • the method of the present invention can be applied to assess the actual responsiveness of a cell to an antiproliferative agent, that is, after the cell has been exposed to the agent. Alternatively, it can be applied to assess the likely or potential response which may occur if that cell was exposed to the agent.
  • the preferred method is to detect a decrease in Bim and/or Bad levels in order to diagnose the onset or predisposition to the onset of resistance to the effects of an antiproliferative agent
  • the detection of an increase in the levels of these molecules may be desired under certain circumstances.
  • to monitor for a reversal of chemoresistance such as in the circumstance where a patient exhibiting chemoresistance is subjected to a treatment regime directed to upregulating or inducing functional Bim and/or Bad expression or otherwise increasing the intracellular levels of these molecules in resistant cells.
  • This aspect of the present invention also enables one to monitor the progression of a condition characterised by unwanted cellular proliferation, such as a neoplastic condition.
  • progression is meant the ongoing nature of the condition in terms of the existence or emergence of resistance to treatment with an antiproliferative agent.
  • progression is meant the ongoing nature of the condition in terms of the existence or emergence of resistance to treatment with an antiproliferative agent.
  • applications of the present technology lie with other conditions characterised by some form of unwanted cellular proliferation, such as autoimmunity.
  • another aspect of the present invention relates to a method for monitoring the responsiveness of a cellular population to an antiproliferative agent, said method comprising screening for modulation of the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression in the subject cellular population relative to a previously obtained level or a normal level is indicative of the maintenance or worsening of responsiveness and an increase in said level is indicative of an improvement in said responsiveness.
  • said cellular population is a neoplastic population and more preferably a malignant neoplastic population.
  • said antiproliferative agent induces cell death, in particular apoptosis.
  • Yet another aspect of the present invention is directed to a method for assessing the actual or likely responsiveness to an antiproliferative agent of a condition characterised by unwanted cellular proliferation to an antiproliferative agent, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • said condition is a neoplastic condition and more preferably a malignant neoplastic condition.
  • said antiproliferative agent induces cell death, in particular apoptosis.
  • the present invention relates to a method for monitoring the responsiveness to an antiproliferative agent of a condition characterised by unwanted cellular proliferation, said method comprising screening for modulation of the level of functional Bim and/or Bad protein and/or gene expression wherein a decrease in the level of said protein and/or gene expression in the subject cellular population relative to a previously obtained level or a normal level is indicative of the maintenance or worsening of responsiveness and an increase in said level is indicative of an improvement in said responsiveness.
  • said condition is a neoplastic condition and more preferably a malignant neoplastic condition.
  • said antiproliferative agent induces cell death, in particular apoptosis.
  • Yet another aspect of the present invention is directed to assessing and/or monitoring the effectiveness of an antiproliferative treatment regime in a mammal, said method comprising screening for the level of functional Bim and/or Bad protein and/or gene expression by a cellular population of interest wherein a decrease in the level of said protein and/or gene expression relative to normal levels is indicative of reduced responsiveness.
  • an antiproliferative treatment regime will generally be assessed in terms of the responsiveness of the unwanted cellular population to the antiproliferative agent, one may also seek to determine the level of responsiveness of normal cells in order to make an assessment of the nature and extent of likely cell death related side effects which may be experienced by the patient.
  • said antiproliferative treatment regime is preferably targeted to a neoplastic condition and still more preferably a malignant neoplastic condition. Still more preferably, said antiproliferative agent induces cell death, in particular apoptosis.
  • the present invention therefore provides means for assessing both the existence and extent of cellular populations which exhibit reduced or changed responsiveness to an antiproliferative agent.
  • said neoplastic condition is characterised by a neoplasm of the central nervous system tumours, retinoblastoma, neuroblastoma and other paediatric tumours, head and neck cancers (e.g. squamous cell cancers), breast and prostate cancers, lung cancer (both small and non-small cell lung cancer), kidney cancers (e.g. renal cell adenocarcinoma), oesophagogastric cancers, hepatocellular carcinoma, pancreaticobiliary neoplasias (e.g.
  • adenocarcinomas and islet cell tumours adenocarcinomas and islet cell tumours
  • colorectal cancer cervical and anal cancers
  • uterine and other reproductive tract cancers urinary tract cancers (e.g. of ureter and bladder), germ cell tumours (e.g. testicular germ cell tumours or ovarian germ cell tumours), ovarian cancer (e.g. ovarian epithelial cancers), carcinomas of unknown primary, human immunodeficiency associated malignancies (e.g. Kaposi's sarcoma), lymphomas, leukemias, malignant melanomas, sarcomas, endocrine tumours (e.g. of thyroid gland), mesothelioma and other pleural or peritoneal tumours, neuroendocrine tumours and carcinoid tumours.
  • human immunodeficiency associated malignancies e.g. Kaposi's sarcoma
  • lymphomas leukemias
  • biological sample should be understood as a reference to any sample of biological material derived from an animal such as, but not limited to, cellular material, biofluids (eg. blood) which contain cellular material, faeces, tissue biopsy specimens or surgical specimens.
  • the biological sample which is tested according to the method of the present invention may be tested directly or may require some form of treatment prior to testing.
  • a biopsy or surgical sample may require homogenisation prior to testing or it may require sectioning for in situ testing.
  • the cell sample may require permeabilisation prior to testing.
  • the biological sample is not in liquid form, (if such form is required for testing) it may require the addition of a reagent, such as a buffer, to mobilise the sample.
  • the biological sample may be directly tested or else all or some of the nucleic acid material present in the biological sample may be isolated prior to testing (assuming that one is screening for mRNA levels, for example, or genomic DNA mutations, such as in promoters or other regulatory regions which may affect Bim or Bad expression).
  • the sample may be partially purified or otherwise enriched prior to analysis.
  • a biological sample comprises a very diverse cell population, it may be desirable to select out a sub-population of particular interest, such as enriching for the cell population of which the neoplastic cell forms part.
  • the target cell population or molecules derived therefrom pretreated prior to testing, for example, inactivation of live virus or being run on a gel. It should also be understood that the biological sample may be freshly harvested or it may have been stored (for example by freezing) prior to testing or otherwise treated prior to testing (such as by undergoing culturing).
  • Means for screening for changes in Bim and/or Bad levels in a subject, a biological sample derived therefrom or other form of cellular population (such as a cell line), can be achieved by any suitable method, which would be well known to the person of skill in the art. Briefly, one may seek to detect Bim and/or Bad protein in a biological sample. Anti-Bim or anti-Bad immunointeractive molecules may be used to identify the protein directly. In terms of in vivo analyses, anti-Bim and/or anti-Bad immunointeractive molecules could be coupled to medical imaging agents in order to visualise specific binding to the cellular population of interest, in particular following the administration of anti-cancer treatments.
  • these methods include, but are not limited to: (i) In vivo detection of Bim and/or Bad.
  • Molecular Imaging may be used following administration of imaging probes or reagents capable of disclosing altered expression levels of the Bim and/or Bad mRNA or protein expression product in the biological sample.
  • Molecular imaging [Moore et ⁇ l., Nature Medicine, (5:351-355, 2000] is the in vivo imaging of molecular expression that correlates with the macro-features currently visualized using "classical" diagnostic imaging techniques such as X-Ray, computed tomography (CT), MRI, Positron Emission Tomography (PET) or endoscopy.
  • CT computed tomography
  • PET Positron Emission Tomography
  • FISH Fluorescent In Situ Hybridization
  • QRTPCR Quantitative Reverse Transcriptase Polymerase Chain Reaction
  • a labelled polynucleotide encoding Bim and/or Bad may be utilized as a probe in a Northern blot of an RNA extract obtained from a biological sample.
  • a nucleic acid extract from the subject is utilized in concert with oligonucleotide primers corresponding to sense and antisense sequences of a polynucleotide encoding Bim and/or Bad, or flanking sequences thereof, in a nucleic acid amplification reaction such as RT PCR, real time PCR or SAGE.
  • a nucleic acid amplification reaction such as RT PCR, real time PCR or SAGE.
  • VLSIP STM very large scale immobilized primer arrays
  • RNA is isolated from a suitable cellular sample.
  • RNA can be isolated by methods known in the art, e.g. using TRIZOLTM reagent (GIBCO-BRL/Life Technologies, Gaithersburg, Md.).
  • Oligo-dT, or random-sequence oligonucleotides, as well as sequence-specific oligonucleotides can be employed as a primer in a reverse transcriptase reaction to prepare first-strand cDNAs from the isolated RNA.
  • Resultant first-strand cDNAs are then amplified with sequence-specific oligonucleotides in PCR reactions to yield an amplified product.
  • PCR Polymerase chain reaction
  • RNA and/or DNA are amplified as described in U.S. Patent No. 4,683,195.
  • sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers. These primers will be identical or similar in sequence to opposite strands of the template to be amplified.
  • PCR can be used to amplify specific RNA sequences and cDNA transcribed from total cellular RNA. See generally Mullis et ah, 1987; Erlich, 1989.
  • amplification of specific nucleic acid sequences by PCR relies upon oligonucleotides or "primers" having conserved nucleotide sequences wherein the conserved sequences are deduced from alignments of related gene or protein sequences.
  • one primer is prepared which is predicted to anneal to the antisense strand and another primer prepared which is predicted to anneal to the sense strand of a cDNA molecule which encodes Bim and/or Bad.
  • the reaction mixture is typically subjected to agarose gel electrophoresis or other convenient separation technique and the relative presence of Bim and/or Bad specific amplified nucleic acid detected.
  • the Bim and/or Bad amplified nucleic acid may be detected using Southern hybridization with a specific oligonucleotide probe or comparing its electrophoretic mobility with nucleic acid standards of known molecular weight.
  • Isolation, purification and characterization of the amplified Bim and/or Bad nucleic acid may be accomplished by excising or eluting the fragment from the gel (for example, see references Lawn et ah, 1981; Goeddel et ah, 1980), cloning the amplif ⁇ ed product into a cloning site of a suitable vector, such as the pCRII vector (Invitrogen), sequencing the cloned insert and comparing the sequence to the known sequence of Bim and/or Bad. The relative amounts of Bim and/or Bad mRNA and cDNA can then be determined.
  • an antibody according to the invention having a reporter molecule associated therewith, may be utilized in immunoassays.
  • immunoassays include but are not limited to radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic techniques (ICTs), Western blotting which are well known to those of skill in the art.
  • RIAs radioimmunoassays
  • ELISAs enzyme-linked immunosorbent assays
  • ICTs immunochromatographic techniques
  • Western blotting which are well known to those of skill in the art.
  • Immunoassays may include competitive assays. It will be understood that the present invention encompasses qualitative and quantitative immunoassays.
  • Suitable immunoassay techniques are described, for example, in U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two-site assays of the non-competitive types, as well as the traditional competitive binding assays. These assays also include direct binding of a labelled antigen-binding molecule to a target antigen.
  • the antigen in this case is Bim and/or Bad or a fragment thereof.
  • a two-site assay is used.
  • an unlabelled antigen-binding molecule such as an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • another antigen-binding molecule suitably a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observation of a signal produced by the reporter molecule.
  • results may be either qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of antigen.
  • Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody.
  • a first antibody having specificity for the antigen or antigenic parts thereof is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample.
  • the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient and under suitable conditions to allow binding of any antigen present to the antibody.
  • the antigen-antibody complex is washed and dried and incubated with a second antibody specific for a portion of the antigen.
  • the second antibody has generally a reporter molecule associated therewith that is used to indicate the binding of the second antibody to the antigen.
  • the amount of labelled antibody that binds, as determined by the associated reporter molecule, is proportional to the amount of antigen bound to the immobilized first antibody.
  • An alternative method involves immobilizing the antigen in the biological sample and then exposing the immobilized antigen to specific antibody that may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound antigen may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • the reporter molecule associated with the antigen-binding molecule may include the following :-
  • the reporter molecule may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorochrome, a chemiluminescent molecule, a paramagnetic ion, a lanthanide ion such as Europium (Eu 34 ), a radioisotope including other nuclear tags and a direct visual label.
  • a colloidal metallic or non- metallic particle a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • Suitable enzymes suitable for use as reporter molecules is disclosed in U.S. Patent Nos. U.S. 4,366,241, U.S. 4,843,000, and U.S. 4,849,338.
  • Suitable enzymes useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, ⁇ -galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like.
  • the enzymes may be used alone or in combination with a second enzyme that is in solution.
  • Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • RPE R-Phycoerythrin
  • Texas Red Texas Red
  • Other exemplary fluorochromes include those discussed by Dower et al., International Publication No. WO 93/06121. Reference also may be made to the fluorochromes described in U.S. Patent Nos. 5,573,909 (Singer et al), 5,326,692
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • the substrates to be used with the specific enzymes are generally chosen for the production of, upon hydrolysis by the corresponding enzyme, a detectable colour change. Examples of suitable enzymes include those described supra. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody- antigen complex, allowed to bind, and then the excess reagent washed away.
  • a solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody.
  • the substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of antigen which was present in the sample.
  • fluorescent compounds such as fluorescein, rhodamine and the lanthanide, europium (EU) may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent- labelled antibody is allowed to bind to the first antibody-antigen complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to light of an appropriate wavelength. The fluorescence observed indicates the presence of the antigen of interest.
  • Immunofluorometric assays IFMA
  • IFMA Immunofluorometric assays
  • other reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules may also be employed.
  • Another aspect of the present invention provides a diagnostic kit comprising an agent for detecting Bim and/or Bad or a nucleic acid molecule encoding Bim and/or Bad and reagents useful for facilitating the detection by said agent.
  • the agent may be an antibody or other suitable detection molecule.
  • the present invention further contemplates the use of an interactive molecule directed to Bim and/or Bad in the manufacture of a quantitative or semi-quantitative diagnostic kit to assess cellular responsiveness to an antiproliferative agent.
  • the kit may come with instructions for use and may be automated or semi-automated or in a form which is compatible with an automated machine or software.
  • PRO-APOPTOTIC BH3-ONLY PROTEIMS BIM AND BAD MEDIATE IMATINIB MESYLATE-INDUCED KILLING OF BCR/ABL-POSITIVE LEUKEMIC CELLS IN CULTURE AND IN PATIENTS
  • K562 is an erythroleukemia
  • BVl 73 is a pre-B cell leukemia
  • MEG-Ol is a megakaryoblastic leukemia cell lines, all derived from Ph 1 + CML patients in blast-crisis phase.
  • Cells were maintained as suspension cultures in RPMI- 1640 with 10% or 15% heat-inactivated foetal calf serum (FCS) and 2mM L-glutamate.
  • FCS foetal calf serum
  • FADD-DN FADD/M0RT1
  • a Gene-Pulser BioRad Laboratories, Hercules, CA was used for electroporation and selection was performed with 5 ⁇ g/mL puromycin. Cells were single cell cloned by limiting dilution. FLAG-tagged proteins were detected by cytoplasmic immunofluorescence staining with anti-FLAG antibody (Sigma) and flow cytometric analysis in a FACScan (Becton Dickinson, Franklin Lakes, NJ).
  • RNA construct cloned into pSUPER vector with the neomycin-resistant gene was described elsewhere.
  • mice All experiments with mice were performed according to the guidelines of the Melbourne Directorate Animal Ethics Committee. The bim ' ⁇ [Bouillet et al, 1999, supra], bad' ' [Ranger et al, Proc Natl Acad Sci USA. 2003; 100:9324-9329] and vav-bcl-2 transgenic mice have all been described. These animals were either generated on a C57BL/6 background or had been backcrossed with C57BL/6 mice for >8 generations. The him 1' bad' ' mice were produced by intercrossing Mm "1" and bad 1' mice.
  • Fetal liver cells were harvested from 14.5 day old embryos and pre-cultured in DMEM containing 20% FCS, 2mM L-glutamate, 50 ⁇ M 2-mercaptoethanol and cytokines for 24h (50 ng/mL stem cell factor, SCF, 50 ng/mL thrombopoietin: TPO, 500 ng/mL fetal liver kinase-ligand, FIkL and 100 LVmL interleukin-6, IL-6; all gifts of Dr. W Alexander, Walter and Eliza Hall Institute).
  • the pMPZen 50 ng/mL stem cell factor, SCF, 50 ng/mL thrombopoietin: TPO, 500 ng/mL fetal liver kinase-ligand, FIkL and 100 LVmL interleukin-6, IL-6; all gifts of Dr. W Alexander, Walter and Eliza Hall Institute).
  • the pMPZen 50 ng/mL stem cell factor, SCF
  • bcr-c-abl expression vector has been described [Hariharan et al, Oncogene Res 1988;3:387-399] and was transfected to packaging Phoenix cells using FuGENE ⁇ (Roche, Basel, Switzerland). Pre-cultured FLCs were infected by co-cultivation on Retronectin (Takara, Kusatsu, Japan)-coated plates in virus containing medium supplemented with cytokines (see above). Infected cells expressing Bcr/Abl were selected by growth in the absence of cytokines.
  • Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and then electroblotted onto a Hybond-nitrocellulose membrane (Amersham Biosciences, Upsula, Sweden).
  • the membranes were saturated with 5% (wt/vol) non-fat dry milk in PBS with 0.1% (vol/vol) Tween 20 (Sigma).
  • 2-D protein electrophoresis was performed using the IPGphor isoelectric focusing (IEF) system (Amersham Pharmacia Biotech, Piscataway, NJ). Protein lysates were loaded onto IPG gels, rehydrated at 20 0 C for 12 h, and subjected to IEF for at least 12,000 volt x h. After the equilibration with SDS/PAGE buffer for 10 minutes at room temperature, the IPG gel was subjected to second-dimensional SDS/PAGE. Transfer, immunoblotting and visualization were performed as described above (Western blotting).
  • RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
  • Primers were designed as follows: murine bmf: forward (fwd) 5'-cgagtgttcaccctgga-3' (SEQ ID NO:1), reverse (rev) 5'- ccccttccctgttttcttgt -3' (SEQ ID NO:2), human puma: fwd 5'-gacctcaacgcacagta-3' (SEQ ID NO:3), rev 5'-ctaattgggctccatct-3' (SEQ ID NO:4), murine puma: fwd 5'- gcccagcagcacttagagtc-3' (SEQ ID NO:5), rev 5'-tgtcgatgctgctcttcttg-3' (SEQ ID NO:6). Primers for bim, bcr/abl and human ⁇ m/were utilized as described, ⁇ -actin was used as a control for the
  • BM samples were harvested from four Ph 1 + leukemia patients before and after treatment by imatinib with informed consent according to the Declaration of Helsinki.
  • BM mononuclear cells were isolated by centrifugation with Ficoll-Hypaque, resuspended in RPMI1640 containing 10% FCS at 2.0 x 10 5 cells/mL and exposed to 3.0 ⁇ M imatinib for 12 or 24h. Protein extraction and Western blotting were performed as described.
  • Imatinib Activates the Bcl-2-Regulated Apoptotic Pathway in Ph 1 + Leukemia Cell Lines
  • Ph 1 + human leukemia lines K562 and BVl 73 were chosen for initial studies on the effects of imatinib. Imatinib inhibited cell proliferation with similar efficiency in both cell lines ( Figure IA), but BV 173 cells were less susceptible to imatinib-induced cell death than K562 cells (Figure IB, Figure 8). Western blot analysis showed clear differences in Bcl-2 expression between K562 and BVl 73 cells. Upon treatment with imatinib, Bcl-2 levels dropped significantly in K562 cells but were maintained or even increased slightly in BVl 73 cells ( Figure 1C).
  • K562 cells are insensitive to FasL and TNF; so the function of FADD-DN in these cells could not be demonstrated, but expression of this vector at similar levels was previously shown to protect several human lymphoma lines from FasL [Huang, 1999].
  • Imatinib Increases Bim Expression in Ph 1 + Leukemia Cell Lines
  • BiniEL from imatinib-treated K562 cells migrated more rapidly in SDS-PAGE than Bi ⁇ i E L from untreated cells ( Figure 2A and Figure 10), indicating that BCR-ABL inactivation might cause a change in Bini EL phosphorylation. Accordingly, 2-D gel electrophoresis and Western blotting were used to examine the phosphorylation status of Bim before and after imatinib treatment.
  • Figure 2D shows that BiniEL proteins from imatinib-treated K562 cells migrated more closely to the anode than Bim EL proteins from untreated cells, indicating the accumulation of lesser phosphorylated forms of Bim.
  • Bim Is the Critical but not the only BH3-Only Protein Mediating Imatinib-induced Killing of Ph + Leukemia Lines
  • Imatinib inhibited proliferation and induced death in wt.bcr-c-abt cells in a time- and dose-dependent manner:
  • the bim ' ' ' bcr- c-abt and to a somewhat lesser extent also the bad " bcr-c- ⁇ bt cells were more resistant to imatinib-induced cell death than their wt counterparts, showing on average 2- to 3-fold higher viability ( Figure 5B).
  • Many of the bcr-c-abl transformed bim ⁇ ' ⁇ and bad 1' cells did, however, eventually die after treatment with imatinib, indicating that these two BH3-only proteins might have overlapping function.
  • BM samples were obtained from two Ph 1 + CML and two Ph 1 + ALL patients both before imatinib treatment and at relapse/disease progression after imatinib treatment.
  • cytogenetic studies >90% of BM cells were identified to be leukemic (data not shown).
  • Imatinib was provided as the initial treatment for the two CML patients, while given as the alternative treatment for the two chemoresistant Ph 1 + ALL patients.
  • the primary imatinib efficacy was assessed by peripheral blood and BM cell counts and systemic evaluation: good responders were defined as sustained complete haematologic response, complete BM response and no extramedullar ⁇ involvement that lasted for at least 4 weeks [Ottmann et al, Blood, 2002; 100:1965-1971].
  • the causes of resistance to imatinib were assessed in two patients at relapse and were found to be an E255K mutation in Bcr-Abl in Case 3 and a T3151 mutation in Case 4, respectively.
  • BM cells were cultured for 12 h in medium alone (control) or in the presence of 3.0 ⁇ M imatinib.
  • Elefanty AG Hariharan IK, Cory S. bcr-abl, the hallmark of chronic myeloid leukaemia in man, induces multiple haemopoietic neoplasms in mice.

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Abstract

La présente invention concerne généralement un procédé de diagnostic, prédiction et/ou surveillance de la sensibilité cellulaire à des agents antiproliférants. Plus particulièrement, la présente invention a trait à un procédé de diagnostic, prédiction et/ou surveillance de la sensibilité cellulaire réduite à des agents antiproliférants en effectuant un dépistage de la modulation des niveaux de Bim et/ou de Bad fonctionnels dans la cellule du sujet. La présente invention fournit en outre un procédé de prédiction, diagnostic et/ou surveillance de la sensibilité d’une condition caractérisée par la prolifération cellulaire indésirable face à un traitement avec un agent antiproliférant. Sont également fournis des agents de diagnostics utiles pour détecter des niveaux d’expression de Bim et/ou Bad fonctionnels.
PCT/AU2006/001230 2005-08-24 2006-08-24 Procédé d’évaluation d’une réponse à un agent antiproliférant Ceased WO2007022588A1 (fr)

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WO2010106285A1 (fr) * 2009-03-17 2010-09-23 Centre National De La Recherche Scientifique - Cnrs - Procedes de mesure de la quantite intracellulaire de molecules d'interet intrinsequement fluorescentes par cytometrie en flux et leurs applications
EP2106451A4 (fr) * 2006-12-04 2010-12-15 Abbott Lab Tests de diagnostic d'accompagnement pour un traitement du cancer
WO2012082074A1 (fr) * 2010-12-14 2012-06-21 National University Of Singapore Procédé de détection de la résistance vis-à-vis d'une thérapie anticancéreuse

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2106451A4 (fr) * 2006-12-04 2010-12-15 Abbott Lab Tests de diagnostic d'accompagnement pour un traitement du cancer
WO2010106285A1 (fr) * 2009-03-17 2010-09-23 Centre National De La Recherche Scientifique - Cnrs - Procedes de mesure de la quantite intracellulaire de molecules d'interet intrinsequement fluorescentes par cytometrie en flux et leurs applications
FR2943418A1 (fr) * 2009-03-17 2010-09-24 Centre Nat Rech Scient Procedes de mesure de la quantite intracellulaire de molecules d'interet intrinsequement fluorescentes par cytometrie en flux et leurs applications
WO2012082074A1 (fr) * 2010-12-14 2012-06-21 National University Of Singapore Procédé de détection de la résistance vis-à-vis d'une thérapie anticancéreuse

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