WO2005065723A1 - Methodes de criblage rapides pour un p53 fonctionnel, faisant appel aux motifs de la reactivite au trioxyde d'arsenic - Google Patents

Methodes de criblage rapides pour un p53 fonctionnel, faisant appel aux motifs de la reactivite au trioxyde d'arsenic Download PDF

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WO2005065723A1
WO2005065723A1 PCT/US2004/039922 US2004039922W WO2005065723A1 WO 2005065723 A1 WO2005065723 A1 WO 2005065723A1 US 2004039922 W US2004039922 W US 2004039922W WO 2005065723 A1 WO2005065723 A1 WO 2005065723A1
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cells
cell population
cell
status
ato
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PCT/US2004/039922
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Yair Gazitt
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Board Of Regents, The University Of Texas System
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/36—Arsenic; Compounds thereof

Definitions

  • the present invention relates generally to the fields of biochemistry and cancer.
  • the invention provides rapid screening methods to identify the functional status of the p53 tumor suppressor protein in cells, and thus provides improved methods and kits for use in selecting suitable anti-cancer agents for use in treating a given patient.
  • the methods and kits of the invention analyze the cell cycle status of cells in response to arsenic trioxide.
  • cancer As a result, there have been significant advances in cancer therapy, including in the treatment of solid tumors, such as breast, colon and prostate cancer, and in the treatment of lymphomas, leukemias and myelomas. Despite such advances, cancer remains a significant worldwide health problem and is one of the leading killers in the western world.
  • One particular problem remaining in the treatment of cancer is the selection of the most suitable treatment regimen for a given patient, such as the most suitable class of anti- cancer drug and/or particular chemotherapeutic agent. It is known, for example, that a given class of drugs or certain chemotherapeutic agent will not be equally effective in the treatment of cancers of a particular type, such as breast cancer. Accordingly, there remains in the art a need for methods to better predict the response of cancer cells to different categories of anti- cancer drugs and chemotherapeutic agents, thus facilitating the selection of effective treatment regimens and particular drugs for use in a given patient.
  • the present invention addresses the foregoing long-felt need in the art by providing improved methods to predict the response of cancer cells to different categories of anti- cancer drugs and chemotherapeutic agents, for use in selecting suitable anti-cancer agents for treating a given patient.
  • the invention particularly provides rapid screening methods to identify the functional status of the p53 tumor suppressor protein in cells, thus improving the selection of suitable anti-cancer agents for use in treatment.
  • the methods of the invention involve analyzing the cell cycle status of cells in response to arsenic trioxide, whereupon the p53 functional status of the cells is determined.
  • the invention provides methods for identifying a cell with a null, mutant or non-functional p53 tumor suppressor protein or status, generally comprising contacting a cell, or contacting a cell in vitro, with an effective amount of arsenic trioxide and analyzing the cell cycle status of the cell, wherein a cell in the G2/M stage of the cell cycle is indicative of a cell with a null, mutant or non-functional p53 tumor suppressor protein or status.
  • mutant or non-functional p53 tumor suppressor protein or status means a cell, cell population, animal, human subject or patient in which the p53 tumor suppressor protein is either substantially absent or substantially non-functional.
  • mutant means “a substantially non-functional mutant", i.e., a mutant in which the biological functions have been substantially impaired, removed, reduced, inhibited or otherwise attenuated.
  • mutant as may be used succinctly herein without further qualification or explanation, excludes mutations in a p53 tumor suppressor protein that are "permissive for function”. That is, the term “mutant”, as ordinarily used herein, excludes mutants in which the biological functions are substantially maintained, preserved or unimpaired, and further excludes any mutant in which the biological functions are increased or prolonged.
  • the invention further provides methods for identifying a cell population with null, mutant or non-functional p53 tumor suppressor protein or status, generally comprising contacting the cell population, or contacting the cell population in vitro, with an effective amount of arsenic trioxide and analyzing the cell cycle status of cells in the cell population, wherein arrest of cells in the G2/M stage of the cell cycle is indicative of a cell population with null, mutant or non-functional p53 tumor suppressor protein status. Further methods of the invention are those for determining the p53 status of a cell.
  • the term "p53 status” means "functional p53 status", i.e., the functional status of a p53 tumor suppressor protein in a cell, cell population, animal, human subject or patient. Accordingly, the invention provides methods for determining the p53 status or the functional status of a p53 tumor suppressor protein in a cell, which generally comprise contacting a cell with an effective amount of arsenic trioxide and analyzing the cell cycle status of the cell, wherein a cell in the G2/M stage of the cell cycle is indicative of a cell with a null, mutant or non-functional p53 rumor suppressor protein or status and a cell in the Gl stage of the cell cycle is indicative of a cell with wild-type or functional p53 tumor suppressor protein status.
  • wild-type or functional p53 tumor suppressor protein or status means a cell, cell population, animal, human subject or patient in which the p53 tumor suppressor protein is wild-type or substantially retains wild-type functions. Accordingly, as will be understood by those of ordinary skill in the art, the terms “wild-type” and “functional”, as may be used succinctly herein, mean “substantially wild-type and substantially normally functioning", i.e., in which the biological functions are substantially retained, maintained, preserved, unimpaired or otherwise intact. Thus, the terms “wild-type” and “functional”, as may be used succinctly herein without further qualification or explanation, include mutations in a p53 tumor suppressor protein that are "permissive for function". That is, the terms “wild-type” and “functional”, as ordinarily used herein, include both unmodified and non-mutant forms as well as “silent mutations” in which the biological functions are substantially retained, maintained, preserved, unimpaired or otherwise intact.
  • the invention in this context, preferably provides methods for determining the p53 status or the p53 tumor suppressor protein functional status of a cell population, generally comprising contacting the cell population, or contacting the cell population in vitro, with an effective amount of arsenic trioxide and analyzing the cell cycle status of cells in the cell population, wherein arrest of cells in the G2/M stage of the cell cycle is indicative of a cell population with a null, mutant or non-functional p53 tumor suppressor protein status and arrest of cells in the Gl stage of the cell cycle is indicative of a cell population with wild-type or functional p53 tumor suppressor protein status.
  • the "arrest of cells in the G2/M stage of the cell cycle" which is indicative of a cell population with a null, mutant or non-functional p53 tumor suppressor protein status, is a "significant or substantial arrest" of cells in the G2/M stage of the cell cycle.
  • arsenic trioxide induces time- and dose-dependent apoptosis and arrest of null, mutant and non-functional p53 cells in the G2/M stage of the cell cycle.
  • G2/M stage of the cell cycle is exemplified by an arrest in accordance with the arrest of RPMI8226 cells in G2/M as reported in FIG. 2A and/or an arrest in accordance with the arrest of ARP-1 and/or U266 cells in G2/M as reported in FIG. 3B.
  • certain particular, non- limiting examples are arrest of at least about 50% of cells at G2/M following 38 hours of exposure to 6 ⁇ M arsenic trioxide; arrest of at least about 60% of cells at G2/M following 38 hours of exposure to 6 ⁇ M arsenic trioxide; and preferably, arrest of at least about 50% of cells at G2/M following 16 hours of exposure to 2-4 ⁇ M arsenic trioxide.
  • a "significant or substantial arrest" of cells in the G2/M stage of the cell cycle is also defined as a "rapid induction of apoptosis and arrest of cells in G2/M in response to low concentrations of arsenic trioxide".
  • apoptosis of at least about 50% of cells correlating with the % of cells arrested in G2/M or concomitant with arrest of cells in G2/M, in response to exposure to arsenic trioxide at concentrations of between about 2 ⁇ M and about 4 ⁇ M for a period of time of less than about 16 hours, or preferably, for a period of time of between about 8 hours and about 16 hours.
  • certain methods of the invention are those for determining the p53 status or the p53 tumor suppressor protein functional status of a cell population, generally comprising contacting the cell population, or contacting the cell population in vitro, with an effective amount of arsenic trioxide and analyzing the cell cycle status of cells in the cell population, wherein a significant or substantial arrest of cells in the G2/M stage of the cell cycle is indicative of a cell population with a null, mutant or non-functional p53 tumor suppressor protein status and arrest of cells in the Gl stage of the cell cycle is indicative of a cell population with wild-type or functional p53 tumor suppressor protein status.
  • the "arrest of cells in the Gl stage of the cell cycle" which is indicative of a cell population with wild-type or functional p53 tumor suppressor protein status, is a "moderate" of cells in the Gl stage of the cell cycle.
  • Arsenic trioxide induces time- and dose-dependent arrest of wild-type or functional p53 cells in the Gl stage of the cell cycle, as disclosed herein. Therefore, those of ordinary skill in the art will understand that the "moderate" arrest of wild-type or functional p53 cells in the Gl stage of the cell cycle is to be determined using exposure to "effective doses" of arsenic trioxide for "effective times". Thus, a "moderate arrest" of cells in the Gl stage of the cell cycle is exemplified by an arrest in accordance with the arrest of HS-sultan and/or IM9 cells in Gl as reported in FIG. 3B. Many effective combinations of doses and times, and the resultant moderate arrest in
  • Gl can be derived from the teachings of the present invention, as exemplified in the working examples and figures.
  • certain particular, non-limiting examples are an increase in the proportion of cells arrested at Gl of between about 15% and about 20% following exposure to arsenic trioxide at a concentration of between about 3 ⁇ M and about 6 ⁇ M for a period of time of between about 30 hours and 38 hours; as further exemplified by an increase in the proportion of cells arrested at Gl of at least about 15% following 38 hours of exposure to 6 ⁇ M arsenic trioxide; an increase in the proportion of cells arrested at Gl of at least about 20% following 38 hours of exposure to 6 ⁇ M arsenic trioxide; and an increase in the proportion of cells arrested at Gl of at least about 15% following 30 hours of exposure to
  • a “moderate arrest” of cells in the Gl stage of the cell cycle is also defined as a "significant resistance to arsenic trioxide following a long exposure to a high concentration of arsenic trioxide". For example, an induction of apoptosis of only between about 5% and about 10% of cells, correlating with the % of cells arrested in Gl or concomitant with arrest of cells in Gl, in response to exposure to arsenic trioxide at concentrations of between about 6 ⁇ M and about lO ⁇ M for a period of time of about 16 hours; and also, an induction of apoptosis of less than about 35%, preferably of only between about 5% and about 35% of cells, correlating with the % of cells arrested in Gl or concomitant with arrest of cells in Gl, in response to exposure to arsenic trioxide at concentrations of between about 6 ⁇ M and about lO ⁇ M for a period of time of about 48 hours, or preferably, for a period
  • the terms “arrest of cells in the Gl stage of the cell cycle” and “moderate arrest of cells in the Gl, stage of the cell cycle” also mean “substantial absence of cells arrested or present in the G2/M stage of the cell cycle”.
  • the important differentiating factor between null, mutant or non-functional p53 tumor suppressor protein status and wild-type or functional p53 tumor suppressor protein status is the presence or absence of cells arrested in the G2/M stage of the cell cycle.
  • the inventor typically chooses to herein define "wild-type or functional p53 tumor suppressor protein status" in the positive recitation of "arrest and moderate arrest of cells in the Gl stage of the cell cycle", which does not exclude the equivalent definition of a substantial absence of cells arrested in the G2/M stage of the cell cycle.
  • the invention thus further provides methods for determining the p53 status or the p53 tumor suppressor protein functional status of a cell population, generally comprising contacting the cell population, or contacting the cell population in vitro, with an effective amount of arsenic trioxide and analyzing the cell cycle status of cells in the cell population, wherein a significant or substantial arrest of cells in the G2/M stage of the cell cycle is indicative of a cell population with a null, mutant or non-functional p53 tumor suppressor protein status and moderate arrest of cells in the Gl stage of the cell cycle is indicative of a cell population with wild-type or functional p53 tumor suppressor protein status.
  • Also provided are methods for determining the p53 status or the p53 tumor suppressor protein functional status of a cell population generally comprising contacting the cell population, or contacting the cell population in vitro, with an effective amount of arsenic trioxide and analyzing the cell cycle status of cells in the cell population, wherein a significant or substantial arrest of cells in the G2/M stage of the cell cycle is indicative of a cell population with a null, mutant or non-functional p53 tumor suppressor protein status and wherein a significant or substantial absence of cells arrested in the G2/M stage of the cell cycle is indicative of a cell population with wild-type or functional p53 tumor suppressor protein status.
  • an "effective amount" of arsenic trioxide is an amount of arsenic trioxide effective to induce arrest, and preferably a significant or substantial arrest, of cells in the G2/M stage of the cell cycle when effectively provided to a population of cells with null, mutant or non- functional p53 tumor suppressor protein status.
  • an "effective amount" of arsenic trioxide is also an amount of arsenic trioxide effective to induce arrest, and preferably a moderate arrest, of cells in the Gl stage of the cell cycle when effectively provided to a population of cells with wild-type or functional p53 tumor suppressor protein status.
  • an "effective amount" of arsenic trioxide is an amount of arsenic trioxide effective to induce arrest of cells in the G2/M stage of the cell cycle when effectively provided to a population of cells with null, mutant or non-functional p53 tumor suppressor protein status, which same amount is also effective to induce arrest of cells in the Gl stage of the cell cycle when effectively provided to a population of cells with wild-type or functional p53 tumor suppressor protein status.
  • an "effective amount" of arsenic trioxide is an amount of arsenic trioxide effective to induce a significant or substantial arrest of cells in the G2/M stage of the cell cycle when effectively provided to a population of cells with null, mutant or non-functional p53 tumor suppressor protein status, which same amount is also effective to induce a moderate arrest of cells in the Gl stage of the cell cycle when effectively provided to a population of cells with wild-type or functional p53 tumor suppressor protein status.
  • Exemplary effective amounts range, for example, from concentrations of between about 0.1 ⁇ M and about 50 ⁇ M; preferably, of between about 0.25 ⁇ M and about 20 ⁇ M; and more preferably, of between about 0.5 ⁇ M and about lO ⁇ M. Further exemplary effective amounts include between about 2 ⁇ M and about 4 ⁇ M; between about 3 ⁇ M and about 6 ⁇ M; between about 2 ⁇ M and about 6 ⁇ M; between about 2 ⁇ M and about lO ⁇ M; between about 3 ⁇ M and about 6 ⁇ M; and between about 3 ⁇ M and about lO ⁇ M. Exemplary low, but effective concentrations of arsenic trioxide include those between about 2 ⁇ M and about 4 ⁇ M; about 2 ⁇ M; and about 4 ⁇ M. Exemplary high and effective concentrations of arsenic trioxide include those between about 6 ⁇ M and about lO ⁇ M; about 6 ⁇ M; and about lO ⁇ M.
  • the term "effective amount” naturally includes contact with arsenic trioxide in an amount "and for a period of time effective”.
  • the meaning of "effectively provided”, as used herein, means “in an amount and for a period of time effective”.
  • an "effective amount and time” is thus an amount and for a period of time effective to induce arrest, and preferably a significant or substantial arrest, of cells in the G2/M stage of the cell cycle when provided to a population of cells with null, mutant or non-functional p53 tumor suppressor protein status; in an amount and for a period of time effective to induce arrest, and preferably a moderate arrest, of cells in the Gl stage of the cell cycle when effectively provided to a population of cells with wild-type or functional p53 tumor suppressor protein status; and in an amount and for a period of time effective to induce arrest, and preferably a significant or substantial arrest, of cells in the G2/M stage of the cell cycle when provided to a population of cells with null, mutant or non-functional p53 tumor suppressor protein status, which same amount and same period of time is also effective to induce arrest, and preferably a moderate arrest, of cells in the Gl stage of the cell cycle when provided to a population of cells with wild-type or functional p53 tumor suppressor protein status
  • Exemplary effective times range, for example, from between about 2 hours to about 60 hours; preferably, from between about 4 hours to about 56 hours; and more preferably, from between about 8 hours to about 48 hours. Exemplary effective times range, for example, from about 4 hours to about 8, about 16, about 24, about 30, about 38 or about
  • 48 hours from about 8 hours to about 16, about 24, about 30, about 38 or about 48 hours; from about 16 hours to about 24, about 30, about 38 or about 48 hours; from about 24 hours to about 30, about 38 or about 48 hours; and from about 30 to about 38 or about 48 hours.
  • exemplary effective times include those of between about 8 hours to about 16 hours; of about 8 hours; and of about 16 hours.
  • Exemplary long and effective times of exposure to arsenic trioxide include those of between about 30 hours and about 48 hours; between about 30 hours and about
  • any method for analyzing the cell cycle status of cells and cells in a cell population may be used in the present invention.
  • the cell cycle status of cells and cells in a cell population may be analyzed by staining the cell population with propidium iodide and subjecting the cell population to flow cytometry.
  • the cells for analysis in the invention may be from any source in which it is useful or desirable to determine the functional status of a p53 tumor suppressor protein. This includes animal, mammalian and human cells, e.g., those for use in drug discovery and/or pre-clinical testing in a variety of embodiments. Thus, the invention may be used in conjunction with cells and cell lines maintained in vitro.
  • the invention may also be used in conjunction with animal, mammalian and human cells obtained, isolated or purified from an animal, mammal or human subject or patient.
  • Such cells and cell populations include animal, mammalian and human cells in various states of purification, such as from tissue and fluid samples, including biopsies and urine, sputum, blood, serum and plasma samples and such like.
  • the invention may be used in conjunction with cells freshly isolated or purified from in vivo sources. Determining the functional status of a p53 tumor suppressor protein in such embodiments may be used to provide diagnostic and/or prognostic information, including to select a mode of treatment and/or to monitor the progress of therapy.
  • the methods of the invention are particularly well suited for analyzing cancer cell populations, including cells and cell lines maintained in vitro and cells obtained, isolated or purified from an animal, mammal or human subject or patient.
  • the range of such cells for analysis in the invention includes any potentially or overtly malignant or neoplastic cell or cell population, such as cells from an animal, subject or patient that has or is suspected to have myeloma, leukemia or any solid tumor.
  • these include carcinomas of the lung, breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, squamous cell carcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas, neuroblastomas, and the like.
  • the methods of the invention thus comprise identifying a cancer cell or cancer cell population with wild-type or functional p53 status and treating the cancer cell or cancer cell population with an anti-cancer drug that is p53-sensitive or p53 -dependent, such as a chemotherapeutic drug.
  • a "p53 -sensitive drug” is a drug or agent that requires at least some, and preferably a substantial amount, of functional p53 in a cell for significant or maximal effectiveness against that cell, or a population, animal or human containing such cells.
  • Effectiveeness in this context, means effective to kill, induce apoptosis or otherwise exert a beneficial anti-cancer effect against the cell, population, animal or human.
  • a "p53 -dependent drug or agent”, as used herein, is a drug or agent that requires a substantial amount of substantially functional p53, preferably approaching a wild-type amount of p53 with wild-type function, in a cell for significant or maximal effectiveness against that cell, or a population, animal or human containing such cells.
  • a "p53 -dependent” drug or agent will therefore exert little, inadequate or no detectable effect against cells or cell populations with null, mutant or non-functional p53 tumor suppressor protein status, or against an animal or human containing such cells.
  • p53-sensitive drugs and agents are often "chemotherapeutic" drugs and agents.
  • the present invention also comprises the counterpart methods, i.e., methods for identifying a cancer cell or cancer cell population with null, mutant or non-functional p53 tumor suppressor protein status and treating the cancer cell or cancer cell population with an anti-cancer drug that is p53 -insensitive or p53-independent.
  • p53-independent drug or agent is a drug or agent that does not require a substantial amount of substantially functional p53 in a cell for significant or maximal effectiveness against that cell, or a population, animal or human containing such cells. Typically, the presence of functional p53 does not impair the effectiveness of p53 -independent drugs and agents, but it is not required. Therefore, a "p53 -independent drug or agent” is able to exert significant, and even maximal, effectiveness against cells and cell populations with null, mutant and/or non-functional p53 tumor suppressor protein status, and against animals and humans containing such cells. p53 -independent drugs and agents are often termed "biological" drugs and agents, such as immunomodulators and antibodies.
  • a further particular embodiment made possible by the present invention is the selection of cancer cells, cancer cell populations, and animals and humans containing such cells, with null, mutant or non-functional p53 tumor suppressor protein status and treating the cancer cells, population, animal or human to restore p53 functionality.
  • treating "to restore p53 functionality” concerns the provision of an amount of wild-type or functional p53 effective to provide a significant amount of wild-type or functional p53 in the cells, preferably, to substantially restore the p53 functions to normal.
  • the p53 functionality may be restored by any effective means of "provision", J. e. , any method effective to provide wild-type or functional p53 to the cells in need thereof.
  • preferred methods of providing wild-type or functional p53 are the use of a recombinant vector, viral vector or virus, preferably a recombinant adenovirus ("Adp53"), engineered to express an effective amount of wild-type or functional p53 when provided to cells in need thereof, such as by administration to animal or human, particularly an animal or human with cancer.
  • Adp53 a recombinant adenovirus
  • the invention further provides general methods for predicting the sensitivity of a cancer cell population to chemotherapeutic drugs.
  • the methods generally comprise contacting the cancer cell population, or contacting the cancer cell population in vitro, with an effective amount of arsenic trioxide and analyzing the cell cycle status of cells in the cancer cell population, wherein arrest of cells in the G2/M stage of the cell cycle is indicative of a cancer cell population with a null or non-functional p53 tumor suppressor protein status and is predictive of a cancer cell population with low sensitivity to chemotherapeutic drugs.
  • these methods comprise contacting a cancer cell population, or contacting the cancer cell population in vitro, with an effective amount of arsenic trioxide and analyzing the cell cycle status of cells in the cancer cell population, wherein arrest of cells in the G2/M stage of the cell cycle is indicative of a cancer cell population with a null or non-functional p53 tumor suppressor protein status and predicts the cancer cell population as having low sensitivity to the selected, p53-sensitive or p53-dependent drug.
  • the invention concerns methods for predicting the sensitivity of a cancer cell population to chemotherapeutic drugs, comprising contacting the cell population, or contacting the cell population in vitro, with an effective amount of arsenic trioxide and analyzing the cell cycle status of cells in the cell population, wherein:
  • a substantial arrest of cells in the G2/M stage of the cell cycle is indicative of a cell population with a null or non-functional p53 tumor suppressor protein status and is predictive of a cancer cell population with low sensitivity to chemotherapeutic drugs;
  • a moderate arrest of cells in the Gl stage of the cell cycle is indicative of a cell population with wild-type or functional p53 tumor suppressor protein status and is predictive of a cancer cell population with high sensitivity to chemotherapeutic drugs.
  • the present invention further provides a range of kits.
  • a kit for identifying a cell with a null or non-functional p53 tumor suppressor protein comprising:
  • a further example is a kit for determining the functional status of a p53 tumor suppressor protein in a cell, comprising:
  • kits for identifying a cell population with null or non-functional p53 tumor suppressor protein status comprising:
  • stage of the cell cycle is indicative of a cell population with null or non- functional p53 tumor suppressor protein status.
  • kits may further be used for determining the p53 tumor suppressor protein functional status of a cell population, and may comprise: (a) an effective amount of arsenic trioxide; and
  • kits of the present invention are kits comprising:
  • arsenic trioxide preferably an effective amount of arsenic trioxide
  • propidium iodide preferably an effective amount of propidium iodide.
  • kits of the invention may further include instructions for use.
  • the kits may be defined as kits for identifying a cell population with null or non-functional p53 tumor suppressor protein status, comprising:
  • instructions for using the arsenic trioxide and the propidium iodide to identify a cell population with null or non-functional p53 tumor suppressor protein status preferably by analyzing the cell cycle status of cells in the cell population, wherein arrest of cells in the G2/M stage of the cell cycle is indicative of a cell population with null or non-functional p53 tumor suppressor protein status.
  • kits of the invention are those for determining the p53 tumor suppressor protein functional status of a cell population, comprising:
  • such instructions may comprise:
  • kits for identifying a cell population with null or non-functional p53 tumor suppressor protein status may comprise: (a) an effective amount of arsenic trioxide;
  • the arsenic trioxide is applied to the cell population to influence the cell cycle status of cells in the cell population dependent on the p53 tumor suppressor protein status of the cells; and (ii) the propidium iodide is subsequently applied to the cell population to stain cells before subjecting the cell population to flow cytometry.
  • kits for determining the p53 tumor suppressor protein functional status of a cell population may comprise:
  • the arsenic trioxide is applied to the cell population to influence the cell cycle status of cells in the cell population dependent on the p53 tumor suppressor protein status of the cells; and (ii) the propidium iodide is subsequently applied to the cell population to stain cells before subjecting the cell population to flow cytometry.
  • kits of the invention may be described as those for identifying a cell population with null or non-functional p53 tumor suppressor protein status, comprising:
  • the present invention further provides a range of uses and medicaments.
  • An example is use of arsenic trioxide in the manufacture of a formulation or medicament for use in identifying or diagnosing a cell with a null or non-functional p53 tumor suppressor protein.
  • arsenic trioxide in the manufacture of a formulation or medicament for use in identifying or diagnosing a cell with a null or non-functional p53 tumor suppressor protein by analyzing the cell cycle status of the cell, wherein a cell in the G2/M stage of the cell cycle is indicative of a cell with a null or non-functional p53 tumor suppressor protein.
  • such as use is for the prediction or diagnosis of the sensitivity a cell or a cancer cell to chemotherapeutic drugs, such as wherein a cell or a cancer cell with a null or non- functional p53 tumor suppressor protein is predictive of a cell or a cancer cell with low sensitivity to chemotherapeutic drugs.
  • a further use is the use of arsenic trioxide in the manufacture of a formulation or medicament for use in identifying or diagnosing a cell population with null or non-functional p53 tumor suppressor protein status.
  • arsenic trioxide in the manufacture of a formulation or medicament for use in identifying or diagnosing a cell population with null or non-functional p53 tumor suppressor protein status by analyzing the cell cycle status of cells in the cell population, wherein arrest of cells in the G2/M stage of the cell cycle is indicative of a cell population with null or non-functional p53 tumor suppressor protein status.
  • such as use is for the prediction or diagnosis of the sensitivity a cell population or a cancer cell population to chemotherapeutic drugs, e.g., wherein a cell population or a cancer cell population with a null or non-functional p53 tumor suppressor protein status is predictive of a cell population or a cancer cell population with low sensitivity to chemotherapeutic drugs.
  • arsenic trioxide in the manufacture of a formulation or medicament for use in identifying or diagnosing the functional status of a p53 tumor suppressor protein in a cell.
  • use of arsenic trioxide in the manufacture of a formulation or medicament for use in identifying or diagnosing the functional status of a p53 tumor suppressor protein in a cell by analyzing the cell cycle status of the cell, wherein a cell in the G2/M stage of the cell cycle is indicative of a cell with a null or non-functional p53 tumor suppressor protein status and a cell in the Gl stage of the cell cycle is indicative of a cell with wild-type or functional p53 tumor suppressor protein status.
  • such as use is for the prediction or diagnosis of the sensitivity a cell or a cancer cell to chemotherapeutic drags, such as wherein a cell or a cancer cell with a null or non-functional p53 tumor suppressor protein is predictive of a cell or a cancer cell with low sensitivity to chemotherapeutic drags and a cell or a cancer cell with wild-type or functional p53 tumor suppressor protein is predictive of a cell or a cancer cell with high sensitivity to chemotherapeutic drugs.
  • the invention includes the use of arsenic trioxide in the manufacture of a formulation or medicament for use in identifying or diagnosing the p53 tumor suppressor protein functional status of a cell population.
  • use of arsenic trioxide in the manufacture of a formulation or medicament for use in identifying or diagnosing the p53 tumor suppressor protein functional status of a cell population by analyzing the cell cycle status of cells in the cell population, wherein arrest of cells in the G2/M stage of the cell cycle is indicative of a cell population with a null or non-functional p53 tumor suppressor protein status and arrest of cells in the Gl stage of the cell cycle is indicative of a cell population with wild-type or functional p53 tumor suppressor protein status.
  • such as use is for the prediction or diagnosis of the sensitivity a cell population or a cancer cell population to chemotherapeutic drugs, e.g., wherein a cell population or a cancer cell population with a null or non-functional p53 tumor suppressor protein is predictive of a cell population or a cancer cell population with low sensitivity to chemotherapeutic drags and a cell population or a cancer cell population with wild-type or functional p53 tumor suppressor protein is predictive of a cell population or a cancer cell population with high sensitivity to chemotherapeutic drugs.
  • kits and/or uses to cancer cell population(s) obtained from one or more cancer patients, the invention thus has important uses in the selection or design of cancer therapy for a particular patient or patient population.
  • the identifying of a cancer cell population or patient with wild-type or functional p53 tumor suppressor protein status would be followed by treating the cell population or patient with a p53-sensitive or p53-dependent drug such as a chemotherapeutic drug.
  • the identifying of a cancer cell population or patient with null or non-functional p53 tumor suppressor protein status could be effectively followed by treating the cell population or patient to restore p53 functionality, such as by providing recombinant wild-type or functional p53, e.g., using a recombinant adenovirus other type of virus that expresses wild-type or functional p53, or using any therapy based on manipulation of p53.
  • the invention particularly provides methods for selecting and designing cancer therapy, and for treating a particular patient or patient population, which generally comprise: (a) contacting a population of cancer cells from a patient or patient population with an effective amount of arsenic trioxide;
  • an arrest of cells in the Gl stage of the cell cycle is indicative of a cancer patient or patient population with wild-type or functional p53 tumor suppressor protein status
  • a substantial arrest of cells in the G2/M stage of the cell cycle is indicative of a cancer patient or patient population with null or nonfunctional p53 tumor suppressor protein status; and (c) treating a cancer patient or patient population with wild-type or functional p53 tumor suppressor protein status by administering a p53-sensitive or p53 -dependent anti-cancer drag, such as a chemotherapeutic drag; and treating a cancer patient or patient population with null or non-functional p53 tumor suppressor protein status with a treatment other than administering a substantially p53-dependent chemotherapeutic drug, such as by administering a biological anti-cancer drag, e.g., an immunomodulatory agent, a p53-expressing recombinant virus or adenovirus, and/or by treating with surgery.
  • a biological anti-cancer drag e.g., an immunomodulatory agent, a p53-expressing recombinant virus or adenovirus, and/or by treating with surgery.
  • FIG. 1A and FIG. IB ATO-induced apoptosis in myeloma cells expressing mutated
  • FIG. 1A or w.t. (FIG. IB) p53.
  • Cells were cultured for 2 days with 0-10 ⁇ M ATO. Apoptosis was determined following 48 hours of treatment by the annexin N method. ⁇ B4 cells were used as a reference for an ATO sensitive cell line. Bars are + S.D. of at least 3 studies. Note the clear difference in sensitivity to apoptosis between cells expressing w.t. or mutated p53.
  • FIG. 2A and FIG. 2B Effect of ATO on the cell cycle (FIG. 2A) and apoptosis (FIG. 2B) of 8226 cells.
  • RPMI 8226 cells, expressing mutated p53 were cultured for 48 hours with 0-4 ⁇ M ATO.
  • Cell cycle distribution was determined by the propidium iodide staining method and stained cells were analyzed by flow cytometry using the ModFit software. Ten thousand cells were analyzed.
  • Apoptosis was determined by the Annexin V method. Note the time and dose dependent arrest of cells in G2/M and the parallel kinetics of cell cycle arrest and extent of apoptosis. At least 3 studies were performed and 1 representative study is shown.
  • FIG. 3A and FIG. 3B ATO-induced apoptosis (FIG. 3A) and cell cycle arrest (FIG. 3B) in IM 9, HS-Sultan, U266, ARP-1 cells. Cells were cultured for 0, 4, 8, 16, 24, 30 and 38 hours with 0-6 ⁇ M ATO. Apoptosis was determined by the Annexin V method and cell cycle distribution by the propidium iodide staining method. Representative results from at least 3 different studies are shown.
  • FIG. 4 ATO induces upregulation of p21 expression in cells expressing w.t. p53.
  • Cells were cultured as above for 0, 16, 24, 32 and 48 hours with 6 ⁇ M ATO and aliquots were taken for Western immunoblotting. 50 ⁇ g of protein extract was loaded onto each lane. Loading control was performed according to the results obtained from a pre run of gels stained for protein by the Coomassie blue and quantification of protein bands by densitometry.
  • FIG. 5 A and FIG. 5B Differential blocking of apoptosis by caspases specific blocking peptides in myeloma cells expressing mutated p53 (FIG. 5A) or w.t. p53 (FIG. 5B).
  • FIG. 5B were cultured for 48 hours with 7.5 ⁇ M ATO, with or without 2 ⁇ M of each caspase inhibitory peptide, or control peptide (FA-FMK).
  • FFA-FMK control peptide
  • CP was around 5%.
  • the toxicity of the blocking peptides was 2-5% above untreated, control cells.
  • Apoptosis was determined by the annexin V method.
  • Ar is ATO alone;
  • Ar+1 is ATO plus caspase 1 inhibitor;
  • Ar+2 is ATO plus caspase 2 inhibitor, etc. Bars are ⁇ S.D. of at least 3 studies. Note the differential blocking of apoptosis by caspase 9 and caspase 8 and 10, depending on the status of p53.
  • Caspase 8 is the dominant activated caspase in cells with mutated p53.
  • RPMI8226 cells were cultured for 0, 16, 24 and 48 hours with or without 7.5 ⁇ M ATO followed by 1 hour incubation with FITC-tagged caspase peptides specific for caspase 3, 8 and 9 as recommended by the manufacturer.
  • FITC-caspase substrate peptides had 5%-10% background staining (0 H).
  • apoptosis was determined in separate tubes by the FITC-annexin V method. Fluorescence was quantitated by flow cytometry. Ten thousand cells were analyzed for each sample. Representative results from at least 3 different studies are shown. Note the similar kinetics between apoptosis and caspase activity. Note also the preferential activation of caspase 8 over caspase 9 in RPMI8226 cells.
  • FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D Differential activation of caspase 8 and 9 in myeloma cells expressing mutated or w.t. p53.
  • ARP-1, p53 null cells (FIG. 7A); U266 cells expressing mutated p53 (FIG. 7B); IM9 cells expressing w.t. p53 (FIG. 7C); and HS-Sultan cells expressing w.t. p53 (FIG. 7D) were cultured with ATO and assayed for apoptosis and for caspase activity using FITC-tagged caspase-specific substrate peptides. Bars are ⁇ S.D.
  • FIG. 8 Western immunoblot analysis of ATO-induced PARP, caspase 3, 8 and 9 activation in w.t. and mutated p53 expressing myeloma cells.
  • ARP-1, IM9, U266 and HS- Sultan cells were cultured for 0, 16, 24 and 48 hours with or without 7.5 ⁇ M ATO. Representative results from at least 3 different studies are shown. For loading controls, membranes were striped and reprobed for ⁇ -actin.
  • FIG. 9A, FIG. 9B, FIG. 9C and FIG. 9D ATO sVnergizes with APO2/TRAIL in the induction of apoptosis in partially-resistant myeloma cells.
  • HS-Sultan FIG. 9A
  • ARP-1 ARP-1
  • FIG. 9B IM9 (FIG. 9C) and U266 (FIG. 9D) and cells were cultured for 2 days with 0, 2.5, 5 and lO ⁇ M ATO, with or without 25, 50 and lOOng/ml of APO2/TRAIL.
  • Apoptosis was determined by the annexin N method. Bars represent S.D. from at least 3 independent studies. Note the clear synergy between ATO and APO2/TRAIL in IM9 and HS-Sultan cells expressing w.t. p53.
  • FIG. 10 Kinetics of ATO-induced upregulation of RI and R2 APO2/TRAIL receptors in HS-Sultan cells.
  • HS-Sultan cells were cultured (0.4x10 cells/ml) in RPMI medium plus 15% FCS for 0, 6 and 12 hours with 4 ⁇ M ATO.
  • Surface expression of APO2/TRAIL receptors was determined by indirect staining with monoclonal antibodies specific for RI, R2, R3 and R4 APO2/TRAIL receptors.
  • the thin dashed line is the immunoglobulin isotype-matched control (C); other lines represented staining of cells with the corresponding antibody (RI to R4) in cells harvested following 0 hours (0); at 6 hours (6) and 12 hours (12) of treatment with 4 ⁇ M ATO.
  • Ten thousand cells were analyzed on the live cells gating by light scatter.
  • ATO upregulates the expression of RI and R2 APO2/TRAIL receptors as early as 6-12 hours.
  • FIG. 11 A and FIG. 1 IB Treatment with ATO (FIG. 1 IB) enhances the expression of RI and R2 APO2/TRAIL receptors in IM9 cells as opposed to untreated cells (FIG. 11 A).
  • IM9 cells were cultured for 24 hours with 4 ⁇ M ATO (FIG. 11B).
  • APO2/TRAIL receptors was determined by indirect staining with monoclonal antibodies specific for RI, R2, R3 and R4 APO2/TRAIL receptors.
  • the thick line is the corresponding antibody and the thin line is the corresponding immunoglobulin isotype control.
  • ATO upregulates the expression of RI and R2 APO2/TRAIL receptors and downregulates the expression of R3 and R4 decoy receptors, resulting with a net increase in the number functional TRAIL receptors.
  • FIG. 12A, FIG. 12B and FIG. 12C Bcl-2 does not block ATO-induced apoptosis in RPMI 8226 cells.
  • RPMI 8226 cells FIG. 12B
  • Bcl-2 overexpressing RPMI8226 cells express mutated ⁇ 53.
  • Normal T-cells blasts FIG. 12A
  • FIG. 12A Normal T-cells blasts
  • FIG. 12A Normal T-cells blasts
  • FIG. 12A were prepared from peripheral blood of a normal donor. Cells were cultured for up to 40 hours with 0-6 ⁇ M ATO.
  • Apoptosis was determined by the Annexin V method. Ten thousand cells were analyzed. Bar represent ⁇ SD derived from at least 3 studies. Note that overexpression of Bcl-2 did not protect from ATO-induced apoptosis.
  • FIG. 13 ATO-induced cytochrome c release in HS-Sultan, IM9 and U266 cells.
  • Cells were cultured for 24 hours with 5 ⁇ M ATO. Staining for cytochrome c and confocal imaging were performed. Representative results from at least 3 different studies are shown. Micrographs are magnified x600.
  • FIG. 14A ATO induces a rapid depolarization of MMP preceding apoptosis and cytochrome c release in RPMI 8226 cells.
  • RPMI 8226 cells mutated p53
  • TMRE mitochondrial membrane depolarization
  • HS-Sultan cells (w.t. p53) were cultured with 5 ⁇ M ATO for up to 24 hours and aliquots were taken for determination of apoptosis; cytochrome c release; generation of hydrogen peroxide by HE and mitochondrial membrane depolarization (MMP) by TMRE.
  • MMP mitochondrial membrane depolarization
  • FIG. 15 A Effect of ATO on apoptosis, cytochrome c release, generation of hydrogen peroxide and MMP depolarization in RPMI 8226, U266 and HS-Sultan cells.
  • Cells were cultured with 5 ⁇ M ATO for up to 40 hours and aliquots were taken for determination of apoptosis; cytochrome c release; generation of hydrogen peroxide by HE and mitochondrial membrane depolarization (MMP) by TMRE.
  • MMP mitochondrial membrane depolarization
  • depolarization of MMP, as measured by TMRE uptake is the earliest apoptotic event in RPMI8226 and U266 cells (mutated p53) compared to HS-Sultan cells (w.t. p53).
  • Ten thousand cells were analyzed. Bar represent
  • FIG. 15B Effect of ATO on apoptosis, cytochrome c release, generation of hydrogen peroxide and MMP depolarization in ARH-77, ARP-1 and IM9 cells.
  • Cells were cultured with 5 ⁇ M ATO for up to 40 hours and aliquots were taken for determination of apoptosis; cytochrome c release; generation of hydrogen peroxide by HE and mitochondrial membrane depolarization (MMP) by TMRE.
  • MMP mitochondrial membrane depolarization
  • depolarization of MMP as measured by TMRE uptake is the earliest apoptotic event in ARH-77 and ARP-1 cells (mutated p53) compared to IM9 cells (w.t. p53).
  • Ten thousand cells were analyzed. Bar represent ⁇ SD derived from at least 3 studies.
  • AIF is the major proapoptotic protein released from mitochondria in ATO- induced apoptosis of cells expressing mutated p53.
  • Cells were cultured for 0, 16, 24 and 48 hours with 5 ⁇ M ATO.
  • Cell fractionation and isolation of the cytosolic fraction were carried out. SDS-PAGE, immunoblotting and detection of specific protein bands were performed. 20 ⁇ g of protein was loaded onto each lane. Representative results from at least 3 different studies are shown. For loading controls, membranes were stripped and reprobed for cytosolic ⁇ -actin. Note that AIF is the major proapoptotic protein released from mitochondria in cells expressing mutated p53 whereas cytochrome c in the major proapoptotic protein released from mitochondria in cells expressing w.t. p53.
  • FIG. 17 ATO induces BID activation in U266 and 8226 and ARH-77 cells expressing mutated p53.
  • Cells were cultured for 0, 16, 24 and 48 hours with 5 ⁇ M ATO. Extraction of total cellular protein, SDS-PAGE, immunoblotting and detection of specific protein bands were performed. 50 ⁇ g of protein was loaded onto each lane. Representative results from at least 3 different studies are shown. For loading controls, membranes were stripped and reprobed for ⁇ -actin. Note the differential effect of ATO on BID cleavage in cells with mutated (U266, 8226 and ARH-77) vs. w.t. ⁇ 53 (IM9, HS-Sultan).
  • FIG. 18A and FIG. 18B APO2/TRAIL and TRAIL receptors are induced in myeloma cells with mutated (FIG. 18A) and w.t. p53 (FIG. 18B).
  • U266 cells mutated p53; FIG. 18 A
  • HS-Sultan cells w.t. p53; FIG. 18B
  • Results from the viable cell fraction gated by light scatter are shown.
  • Cells were stained for surface expression of APO2/TRAIL and RI R2 TRAIL receptors.
  • TRAIL Ten thousand cells were analyzed. One representative study out of 3 studies is shown.
  • FIG. 19 A. Effect of ATO on MMP depolarization, apoptosis, expression of
  • U266 cells (mutated p53) were cultured with 5 ⁇ M ATO for up to 16 hours and aliquots were taken for determination of apoptosis; surface expression of APO2/TRAIL; depolarization of mitochondrial membrane potential (MMP) by TMRE and translocation of AIF to the nucleus.
  • MMP mitochondrial membrane potential
  • FIG. 19B Effect of ATO on MMP depolarization, apoptosis, expression of APO2/TRAIL and translocation of AIF to the nucleus in HS-Sultan cells.
  • HS-Sultan cells w.t. p53
  • 5 ⁇ M ATO 5 ⁇ M ATO for up to 16 hours and aliquots were taken for determination of apoptosis; surface expression of APO2/TRAIL; depolarization of mitochondrial membrane potential (MMP) by TMRE and translocation of AIF to the nucleus.
  • MMP mitochondrial membrane potential
  • FIG. 20A and FIG. 20B A model for ATO-induced apoptosis in cells expressing mutated p53 (FIG. 20A) and w.t. p53 (FIG. 20B). Based on the results reported herein, in the absence of functional p53 (FIG. 20A), ATO induces the expression of APO2/TRAIL and TRAIL receptors and thereby engaging the caspase 8/BID/Bax pathway resulting in a rapid depolarization of MMP, release of AIF followed by activation of caspase 3 and nuclear/cell apoptosis. According to this model, release of cytochrome c, SMAC and activation of caspase 9 are secondary apoptotic events of dying cells.
  • ATO induces primarily expression p21 and arrest of cells in Gl of the cell cycle.
  • p53 can also activate Bax (with no BID cleavage) and Bax translocates to the mitochondria. These events lead to the release of cytochrome-c, SMAC and activation of the caspase 9-dependent apoptosome/mitochondrial pathway.
  • TRAIL receptors and caspase 8/BID pathway are not engaged and the observed depolarization of MMP and release of small amounts of AIF are secondary apoptotic events of dying cells.
  • cells expressing wild-type p53 will be more sensitive to chemotherapeutic drugs compared to cells expressing non-functional p53 mutants or cells that are p53 null.
  • Examples of this include the findings that mutations in the p53 gene confer resistance to anthracyclines in various tumors. Aas et al. (1996) also reported that mutations in the p53 gene were linked to primary resistance to doxorubicin therapy and early relapse in breast cancer patients.
  • p53 null cells p53 gene knockout in human colon cancer cells resulted in increased resistance to 5-fluorouracil treatment (Bunz et al, 1999).
  • Overexpression of the p53 protein in most cases, is due to a mutated gene; the concordance between p53 detection immunohistochemically and gene mutation has been reported to be 79% for breast cancer specimens (Soong et al, 1996). Overexpression of mutant p53, in this context, has also been reported to be associated with resistance to paclitaxel in the treatment of patients with metastatic breast cancer (Schmidt et al, 2003) and with lack of response to cisplatin-based chemotherapy in non-small cell lung cancer.
  • p53 Whilst wild-type p53 is generally required for beneficial effects of chemotherapeutic agents, other studies have identified classes of agents in which the therapeutic effects are relatively independent of p53 status. These are typically non-chemotherapeutic agents (becoming known as "biologicals").
  • biologicals One particular example is the use of antibodies to Her-2/neu, which induce apoptosis in Her-2/neu overexpressing breast cancer cells independently from p53 status (Brodowicz et al, 2001).
  • the status of p53 in the malignant cells of a cancer patient is important in selecting the most effective therapy for use in treating that patient.
  • the currently available methods for analyzing p53 all suffer from various drawbacks.
  • immunocytochemical methods to detect mutated p53 using "overexpression" of protein are subjective, inaccurate, non-standard and oftentimes do not reflect p53 function.
  • the cytoplasmic vs. nuclear p53 localization methods require indirect inferences, are even more subjective and do not reflect p53 function.
  • Testing for point mutations by DNA analyses whilst technically more routine, does not provide a reliable method for predicting function, as mutations can be "permissive", resulting in functionally indistinguishable p53.
  • the present invention is ideally placed to meet these objectives and solve the problems in the art by providing rapid and effective methods to identify the p53 functional status of cancer cells, thus providing improved methods for use in selecting suitable anti- cancer agents treating a given patient.
  • the methods of the invention particularly analyze the cell cycle status of cells in response to arsenic trioxide. When treated with arsenic trioxide, arrest of cells in the G2/M stage of the cell cycle is indicative of null, mutant or nonfunctional p53 status, whereas arrest of cells in the Gl stage of the cell cycle is indicative of wild-type or functional p53 status.
  • Cancer cells with null, mutant or non-functional p53 status should preferably be treated with a non-chemotherapeutic agent that does not rely on the p53 status of the cell.
  • Trastuzumab Herceptin
  • Cancer cells wild-type or functional p53 status can be effectively treated with chemotherapeutic agents that depend on the p53 status of the cell.
  • Screening for p53 function using the present invention also provides an important tool for use in the selection of patients for treatment with recombinant p53, particularly Adp53. These treatment modalities kill tumors cells with mutant p53; it has been shown that treatment with Adp53 is not effective in patients with w.t.
  • p53 (Liu and Gazitt, 2000), including in clinical trials with Adp53 in head and neck as well as in lung cancer patients.
  • U.S. Patent Nos. 6,143,290 and 6,410,010 are specifically incorporated herein by reference for purposes of even further describing and enabling compositions and methods for treating cancer using adenoviral delivery of p53, as is U.S. Patent No. 6,262,032, which concerns partic ⁇ lar combination therapies.
  • Various methods are available for analyzing the cell cycle status of cells to determine whether they are in the G2/M or Gl stage of the cell cycle. Certain preferred methods involve staining cells with propidium iodide and subjecting them to flow cytometry.
  • Example I The method employed in Example I (Gazitt et al, 1978; specifically incorporated herein by reference) is designed for use with unfixed cells.
  • the tubes are stable for 2-3 days at 4°C, if protected from light. This method works best with lymphoid cells and less with adherent cells (e.g. epithelial or fibroblast type of cells) and preserves membrane/nuclear staining.
  • adherent cells e.g. epithelial or fibroblast type of cells
  • Additional methods that may be used in the present invention include those based upon staining with Hoechst 33342, rhodamine 123, Brdu and 7-amino-actinomycin D (7-AAD), which are each known to those of ordinary skill in the art (e.g., see Juan et al,
  • MM myeloma
  • MM myeloma
  • Arsenic trioxide like all-trans retinoic acid (ATRA), is a potent drug in the treatment of acute promyelocytic leukemia (APL) (Soiget et al, 1998).
  • ATO has been shown to induce differentiation and apoptosis of APL cells, in vitro and in vivo in animal models (Cai et al, 2000; Hu et al, 1999; Perkins, et al, 2000; Zhang et al, 1999).
  • ATO is very effective in the treatment of APL patients with very little toxicity (Hu et al, 1999; Ora et al, 2000; Akao et al, 1999).
  • ATO induction of differentiation and apoptosis in APL cells is concomitant with down regulation of the PML-RAR ⁇ fusion protein, a product of the t(15:17) translocation characteristic to APL Ieukemic cells.
  • ATO is also a potent inducer of apoptosis in a number of other cancer cells lacking the t(15:17) translocation, including AML (Perkins et al, 2000), gastric cancer (Zhang et al, 1999), neuroblastoma (Ora et al, 2000; Akao et al, 1999) and in MC/CAR cells (Park et al, 2000), as well as in a number of other cell types lacking the PML-RAR ⁇ fusion protein (Larochette et al, 1999; Warrell, 1999; Kinjo et al, 2000; Kitamura et al, 2000; Rego et al, 2000; Shen et al, 2000; Jiang et al, 2001; Park
  • ATO is effective in APL patients resistant to ATRA (Kinjo et al, 2000; Rego et al, 2000), and is cytotoxic to cells lacking the PML-RAR ⁇ fusion protein, other mechanisms of action have also been attributed to ATO in cells lacking the t(15:17) translocation (Larochette et al,
  • caspases such as caspase-3 (Akao et al, 1999; Jiang et al, 2001), caspase 9 (Seol et al, 2001) and caspase 8 (Kitamura et al, 2000) were also reported for ATO-induced apoptosis. Depletion of glutathione (Dai et al, 1999), production of superoxide (Iwama et al, 2001) and immunomodulation (Baj et al, 2002; Hayashi et al. , 2002) have further been proposed as possible mechanisms.
  • APO2/TRAIL TNF-related, apoptosis inducing ligand belongs to the large family of TNF-like signal-inducing proteins, and their corresponding receptors belong to the large family of TNF-like signal transduction receptor proteins (Wiley et al, 1995; Pitti et al, 1996; Marsters et al, 1996).
  • APO2/TRAIL induces a death signal following binding to the RI or
  • R2 APO2/TRAIL-receptors (Wiley et al, 1995; Pitti et al, 1996; Marsters et al, 1996; Pan et al, 1997; Sheridan et al, 1997; Ashkenazi and Dixit; 1998; Griffith et al, 1998; Sheikh et al, 1999).
  • Normal cells escape APO2/TRAIL-induced apoptosis by virtue of co-expressing decoy receptor molecules such as R3 or R4, which are capable of binding of APO2/TRAIL but lack the intracellular death domains that transmit down stream cell death signals through activation of caspase 8 (Ashkenazi and Dixit, 1998).
  • Tumor cells generally do not express these decoy receptor molecules (Pan et al, 1997; Sheridan et al, 1997; Ashkenazi and Dixit, 1998; Griffith et al, 1998; Sheikh et al, 1999).
  • Other mechanisms for APO2/TRAIL resistance include mutations in caspase-8, a caspase involved in APO2/TRAIL death signaling upstream from caspase-3 (Kim et al, 2000; Sprick et al, 2000; Kischkel et ⁇ /., 2000).
  • Ad-p53 adenoviras-mediated delivery of p53
  • Ad-p53 adenoviras-mediated delivery of p53
  • APO2/TRAIL receptors APO2/TRAIL decoy receptors.
  • APO2/TRAIL has been shown to exert an anti tumor effect in vivo in different xenograft models of cancer and exhibited very limited toxicity in monkeys (Ashkenazi et al, 1999).
  • ATO-induced apoptosis was conducted using seven myeloma cell lines with different p53 status. Two distinct pathways are described for ATO-induced apoptosis, in terms of the effect on cell cycle and involvement of -initiator caspases, depending on p53 status.
  • ATO induced rapid and extensive (> 90%) apoptosis in a time/dose dependent manner concomitant with arrest of cells in G2/M phase of the cell cycle.
  • p53 are relatively resistant to ATO, with maximal apoptosis of about 40% concomitant with partial arrest of cells in Gl and upregulation of p21 (Example I; Liu et al, 2003).
  • caspase blocking peptides, fluorescence-tagged caspase-specif ⁇ c substrate peptides and Western immunoblotting confirmed the involvement of primarily caspase 8 and 3 in ATO-induced apoptosis in myeloma cells with mutated p53 and primarily caspase 9 and 3 in cells expressing w.t. p53 (Example II; Liu et al, 2003).
  • Upregulation by ATO of RI and R2 APO2/TRAIL receptors was also observed.
  • the pro-apoptotic Bcl-2 family member, BID is a 'BH3-only' protein (Wang et al, 1998; Korsmeyer et al, 1999; Gross et al, 1999; Korsmeyer et al, 2000). BID is cleaved by active caspase 8 followed by post-translational myristoylation. The modified BID translocates to the mitochondria, where the truncated pl5tBID inserts into the membrane
  • BID is a protein that connects activation of the extrinsic death receptor pathway to activation of the intrinsic pathways and thereby increases chemosensitivity (Roth and Reed, 2002).
  • Mitochondria constitute the primary target for apoptosis through the release of proapoptotic proteins, in addition to caspases.
  • Several apoptotic signals converge in mitochondria to induce depolarization of mitochondrial membrane potential (MMP), which can be blocked by the Bcl-2 family of proteins (Kroemer and Reed; 2000).
  • MMP mitochondrial membrane potential
  • Depolarization of MMP can result from primary caspase activation (e.g., caspase 8) or from the caspase- independent pathway (Green and Kroemer, 1998).
  • cytochrome c In caspase-dependent apoptosis, depolarization of MMP leads to the release of cytochrome c, which triggers the proteolytic maturation of caspases within the apoptosome, the complex of cytochrome c, Apaf-1, and caspase 9 (Budihardjo et al, 1999).
  • AIFs apoptosis inducing factors
  • AIF is capable of translocating to the nucleus and can bind directly to chromatin and induce chromatin condensation and partial DNA degradation (Cande et al. , 2002).
  • cytoplasmic extracts from dying cells can trigger the breakdown of isolated nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for AIF, suggesting that AIF is involved in DNA degradation during cell death (Cande et al, 2002).
  • AIF In addition to its apoptotic activity, AIF plays an important role in redox activity through its enzymatic NADH-like activity (Lipton et al, 2002).
  • p53 can induce apoptosis by activation the translocation of Bax from the cytosol to the mitochondria resulting in the release of cytochrome c and activation of caspase 9 through Apaf-1 (Schuler and Green, 2002).
  • p53 can regulate the synthesis and release of the proapoptotic protein SMAC/DIABLO to induce indirect activation of caspase 3 and apoptosis through its binding of anti-ap ⁇ ptotic proteins, such as XI AP and cIAP-1, which are normally bound to caspase 3 and block its proteolytic activity (Henry etal, 2002; Du, 2000).
  • p53 can regulate the BID expression and thereby p53 can actually link the extrinsic and intrinsic apoptotic pathways (Roth and Reed, 2002; Sax et al, 2002).
  • HP hydrogen peroxide
  • cytochrome c and AIF release of cytochrome c and AIF from mitochondria in myeloma cells expressing mutated or w.t. p53
  • MMP and generation of HP occur early on, before release of cytochrome c and apoptosis as measured by Annexin N.
  • An early induction by ATO of APO2/TRAIL was also observed in these cells, in addition to APO2/TRAIL receptors and a concomitant cleavage of BID and early release of AIF and early apoptosis.
  • APO2/TRAIL is not induced, BID is not cleaved and depolarization of MMP occurs concurrently with release of cytochrome c and apoptosis.
  • Example III further explain the greater sensitivity to ATO of cells with mutated p53 and also suggest a general mechanism for ATO-induced apoptosis (see also, Akay and Gazitt, 2003).
  • paclitaxel paclitaxel
  • a plethora of studies on the anti-cancer effects of paclitaxel (taxol) have identified a number of cellular and molecular targets, such as induction of cytokines and tumor- suppressor genes (e.g., p53); indirect cytotoxicity due to secretion of tumor necrosis factor; induction of mitotic arrest; and activation of various signal-transduction pathways (Blagosklonny and Fojo, 1999).
  • taxol induces stabilization of microtubule; mitotic arrest in metaphase; protein-serine phosphorylation, including hyper-phosphorylation of Bcl-2 and Bcl-xL; activation of cyclin B; and apoptosis (Blagosklonny and Fojo, 1999; Haider et al, 1996; Srivastava et al, 1998; Blagosklonny et al, 1999).
  • Taxol is also involved in upregulation of surface expression of RI and R3 Apo2/TRAIL receptors and synergizes with TRAIL in the induction of apoptosis ( ⁇ agane et al, 2000; Lacour et al, 2001; ⁇ immanapalli et al, 2001).
  • taxol- induced apoptosis involves the mitochondrial intrinsic pathway engaging cytochrome c release and activation of caspase 9 and caspase 3 (Pan et al, 2001; Ofir et al, 2002; Yuan et al. , 2002) in a Bcl-2 dependent fashion in various cell types, including myeloma cells (Gazitt et al, 1998).
  • Bcl-2 is a potent anti apoptotic protein and was shown to protect cells from a great variety of apoptotic insults (Adams and Cory, 1998; Wang et al, 1998).
  • Apo2/TRAIL-induced apoptosis in "type I” cells is independent of mitochondria and not affected by overexpression of Bcl-2 (Walczak et al, 2000), apoptosis in "type II” cells can be blocked by overexpression of Bcl-2 (Lamothe and Aggarwal, 2002).
  • Bcl-2 plays an important role in drag resistance in myeloma cells and blocks apoptosis induced by dexamethasone and adenovirus delivery of p53 (Tian and Gazitt, 1996; Tian et al, 1996; Liu and Gazitt, 2000) and that pretreatment with Bcl-2 anti-sense oligonucleotides results with sensitization to dexamethasone, taxol and Ad-p53 in myeloma cells expressing high levels of Bcl-2 (Liu and Gazitt, 2003).
  • Example II and Example III the present studies also compare the effect of ATO and taxol on MM depolarization, induction of TRAIL, activation of caspases, release of mitochondrial proapoptotic proteins, chromatin condensation and nuclear fragmentation in myeloma cells expressing varying levels of Bcl-2.
  • the results indicate distinct apoptotic pathways for ATO and taxol (Example IN; Akay et al, 2004).
  • ATO-induced apoptosis involved rapid expression of Apo2/TRAIL, activation of caspase 8, cleavage of BID, depolarization of MM and release of AIF from mitochondria in a Bcl-2 independent fashion.
  • Apoptosis was associated with early formation of ring-like perinuclear condensed chromatin co-localized with AIF. In contrast, taxol-induced apoptosis and MM depolarization was observed only in low Bcl-2 expressing cells and apoptosis involved cytochrome c release and activation of caspase 9. Apoptosis was associated with a random chromatin condensation and nuclear fragmentation with no early involvement of AIF.
  • ATO likely had a differential effect on cell cycle in cells expressing w.t. vs. mutated p53.
  • the effect of varying doses of ATO on the cell cycle and apoptosis in cells with mutated or w.t. p53 was therefore examined.
  • Myeloma cell lines with different p53 status were used in order to determine the role of p53 in ATO-induced apoptosis.
  • Table 1 outlines the myeloma cell lines used in this study.
  • U266, RPMI8226 and ARH-77 cells express mutated p53 (Mazars et al, 1992), whereas IM9, MC-CAR and HS-Sultan express w.t. p53 (Liu et al, 2001).
  • ARP-1 cells are p53 null cells (Tian and Gazitt, 1996; Tian et al, 1996).
  • a time-dose titration of ATO was first performed to determine preferred conditions for maximal apoptosis in each cell line.
  • the results obtained from U266 and RPMI 8226, myeloma cell lines expressing mutated p53, and the NB4 cells (acute promyelocytic leukemia cells) are depicted in FIG. 1 A.
  • a time and dose dependent apoptosis was observed between 1 and 10 ⁇ M ATO, with apoptosis of >85% observed after 48 hours treatment with
  • FIG. 1A ATO-sensitive target cells
  • HS-Sultan, IM9 and MC-CAR cells all expressing w.t. p53
  • maximal apoptosis was only 35% following 48 hours exposure to lO ⁇ M ATO (FIG. IB).
  • FIG. 2A apoptosis
  • FIG. 2B apoptosis of RPMI8226 cells expressing mutated p53 was first tested.
  • ATO induced a dose-dependent apoptosis (8.8% to 53%) concomitant with arrest of cells in G2/M of the cell cycle (7.5% to 50% of cells in G2/M).
  • Similar results were obtained for other myeloma cells with mutated p53.
  • the combined results obtained from U266 and ARP-1 cells (mutated and null p53, respectively) and IM9 and HS-Sultan cells (w.t. p53) are depicted in FIG. 3A (apoptosis) and FIG. 3B (cell cycle).
  • ATO induces apoptosis in two distinct pathways, depending on p53 status.
  • myeloma cells with mutated p53 or p53 null cells U266, ARH-77, 8226, ARP-1
  • low concentrations of ATO 2-4 ⁇ M
  • Percent apoptosis closely correlated with the percent of cells arrested in the G2/M phase of the cell cycle, in a time/dose dependent fashion.
  • myeloma cells expressing mutated p53 myeloma cells expressing w.t. p53, such as MC-CAR, IM9 and HS-Sultan, demonstrated partial or full resistance to ATO following long exposures (48 hours) to high doses of ATO (6-1 O ⁇ M), with apoptosis ranging between 5%-10% at 16 hours and ⁇ 35% at 48 hours.
  • ATO acts like a DNA damaging agent, e.g., UN and ionizing radiation, most likely inducing D ⁇ A breaks that trigger the p53 -dependent D ⁇ A repair apparatus, involving upregulation of gadd45, p21 and blocking of Gl cyclins followed by Gl arrest and eventually leading to differentiation and/or apoptosis (Cai et al, 2000; Woods and Mattden, 2001; Bargonetti and Manfredi, 2002).
  • a DNA damaging agent e.g., UN and ionizing radiation
  • Apoptosis can be induced in these cells as a result of p53 -dependent transactivation of the apoptosis inducing protein, p53AIPl leading to mitochondrial damage and apoptosis via the intrinsic mitochondrial pathway (Oda et al, 2000).
  • This apoptotic pathway could be activated if ATO, directly or indirectly, induces phosphorylation of ser-46 on the p53 molecule (Oda et ⁇ ., 2000).
  • D ⁇ A damage can result in a G2 arrest independent of p53, but involving other D ⁇ A-damage sensing proteins, such as Atm and Atr, through a downstream activation of Chkl and Chk2 kinases, which phosphorylate the Cdc25 phosphatase and thus block Cdc25-regulated dephosphorylation of Cdc2.
  • This can lead to blocking of the formation of the mitotic cyclin B/Cdc2 complex, effectively blocking cells in G2 (Taylor and Stark, 2001).
  • a p53 -independent differential blocking of cyclins indeed occurs in cells undergoing apoptosis by ATO and in cells expressing non-functional p53.
  • the effects of ATO in vitro shown in the present application could also explain the low toxicity observed in patients undergoing treatment with ATO, since Gl block, unlike G2 block, is less toxic to the cells and can be reversible.
  • V-FITC Bio Vision, Palo Alto, CA
  • Stained cells were analyzed by flow cytometry (FACScalibur, BDIS, San Jose, CA). Quantification of apoptosis was done by the CellQuest program. Ten thousand cells were analyzed (Liu and
  • Antibodies (l ⁇ g/10 6 cells) were incubated for 30 min. at 4°C, after which unbound antibody was washed out. Secondary antibody, goat-anti-mouse-FITC (Jackson
  • caspase-specific blocking peptides (2 ⁇ M) were used (FMK derivatives, BioVision, Palo Alto, CA) as follows: YVAD (caspase 1 inhibitor); VDVAD (caspase 2 inhibitor); DEVD (caspase 3 inhibitor); LEVD (caspase 4 inhibitor); WEHD (caspase 5 inhibitor); VEID (caspase 6 inhibitor); IETD (caspase 8 inhibitor); LEHD (caspase 9 inhibitor); AEVD (caspase 10 inhibitor) and VAD (pan-caspase inhibitor).
  • YVAD caspase 1 inhibitor
  • VDVAD caspase 2 inhibitor
  • DEVD caspase 3 inhibitor
  • LEVD caspase 4 inhibitor
  • WEHD caspase 5 inhibitor
  • VEID caspase 6 inhibitor
  • IETD caspase 8 inhibitor
  • LEHD caspase 9 inhibitor
  • AEVD caspase 10 inhibitor
  • VAD pan-caspase inhibitor
  • Cells (4xl0 5 /ml) were cultured in 1ml of FCS-medium, in a 24-well plate.
  • ATO was added at concentration of 7.5 ⁇ M, with or without the blocking peptide, or control (FA- FMK). Cultures were harvested after 48 hours and were tested for apoptosis by the Annexin V binding method.
  • ATO was added at concentration of 7.5 ⁇ M and cultures continued for 0, 16, 24 and 48 hours.
  • caspase activity For measurement of caspase activity, cells were further cultured for 1 hour at 37°C with fluorescence-tagged caspase specific substrate peptide, specific for caspase 3, 8 and 9 and the fluorescence generated due to the hydrolysis of the caspase substrate peptide was analyzed by flow cytometry as determined above for annexin V,
  • Mouse monoclonal antibodies to p21/WAF-l (clone 187), caspase-8 (clone H-134), caspase 3 (clone E8) and rabbit polyclonal anti-caspase-9 (clone H-83) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
  • a low molecular weight ladder of biotinylated protein markers (Biorad, Hercules, CA) was run for each gel.
  • the p21/WAF-l, pro caspase 3, 8 and 9 were tentatively identified according to their migration on the blot.
  • Caspase activation was first assayed by caspase specific blocking peptides.
  • U266 cells mutated p53
  • HS-Sultan cells w.t. p53
  • apoptosis was estimated by the annexin N method.
  • Ten different caspase blocking peptides were employed. The results obtained with caspase blocking peptides are depicted in FIG. 5.
  • Caspase 3 and the pan caspase blocking peptide, Z-VAD substantially blocked ATO- induced apoptosis in both cell lines.
  • caspase 8 and caspase 10 blocking peptide completely blocked apoptosis
  • caspase 9 blocking peptide only partially blocked apoptosis (25%) in these cells.
  • caspase 9 completely blocked apoptosis in HS-sultan cells
  • caspase 8 and 10 blocking peptide had minimal effect (FIG. 5).
  • caspase blocking peptides In order to confirm the results obtained from using caspase blocking peptides, fluorescence tagged caspase-specific substrate peptides were employed. In this assay, activated caspases are capable of degrading the fluorescence-tagged substrate peptide, thereby generating a fluorescent hydrolysis product.
  • RPMI8226 cells mutated p53
  • FIG. 6 An example of the flow cytometry histograms obtained for apoptosis and for caspase 3, 8 and 9 activity are depicted in FIG. 6.
  • a time-dependent activation of caspase 3 and 8 was observed coinciding with apoptosis.
  • Caspase 8 activation in RPMI8226 cells was evident early, following 16 hours of treatment with ATO.
  • caspase 9 was less active in each time point tested, with percent cells expressing active caspase 9 lagged behind the percent of cells undergoing apoptosis by Annexin V.
  • caspase 9 activity was much lower in these cells, with mean fluorescence intensity (MFI) of 63 units compared to a MFI of 210 units for caspase 8 (FIG. 6).
  • MFI mean fluorescence intensity
  • FIG. 7 The combined results obtained from ARP-1 cells (p53 null), U266 cells (mutated p53) and from the w.t. p53-expressing IM9 and HS-Sultan cells are presented in FIG. 7. Whereas caspase 3 was activated in all cell lines, differential activation of caspase 8 was observed for ARP-1 and U266 cells expressing non-functional p53. In contrast, differential activation of caspase 9 was observed in IM9 and HS-Sultan cells expressing w.t. p53. Caspase 9 activation in these cells was higher and occurred faster than caspase 8 and preceded apoptosis, despite of low levels of apoptosis. A similar trend was observed when the MFI of these two caspases was compared.
  • ARP-1 and U266 cells which are more sensitive to ATO than IM9 and HS-Sultan cells, exhibited extensive cleavage of caspase 8, 24 hours after onset of treatment, whereas procaspase 9 was very low in these cells.
  • IM9 and HS-Sultan cells expressed relatively high levels of procaspase 9 and activation occurred late, 48 hours after onset of ATO treatment, correlating well with the extent of apoptosis (FIG. 8, FIG. 7). Cleavage of caspase 8, on the other hand was very minimal in these cells.
  • ATO resulted with a maximum of 97% apoptosis with both drags, compared to a maximum of 40% and 59% with Apo2/TRAIL or ATO alone, respectively, at lO ⁇ M ATO and lOOng/ml of APO2/TRAIL (FIG. 9).
  • APO2/TRAIL results with a synergy between the two drugs.
  • IM9 and HS-Sultan (w.t. p53), and U266 and ARP-1 (mutated p53), was performed according to the model of Laska et al. (1994). The results indicate clear synergy in all 4 cell lines, with p values of more than 0.0001, 0.0005, 0.0008 and 0.001 for HS-Sultan, IM9, ARP-1 and U266, respectively.
  • TRAIL was present throughout the studies. Evidence indicates that the increase in the expression of RI and R2 TRAIL receptors is not permanent and a substantial internalization occurs 36-48 hours after removal of TRAIL.
  • TRAIL decoy receptors as early as 6-12 hour after addition of ATO.
  • FIG. 11A and FIG. 11B depict surface expression in IM9 cells of RI and R2 agonist APO2/TRAIL receptors and R3 and R4 APO2/TRAIL-decoy receptors before (FIG. 11 A) and following 24 hours treatment with 4 ⁇ M ATO (FIG. 1 IB). Before treatment, both RI and R2 agonist APO2/TRAIL receptors and R3 and R4 APO2/TRAIL-decoy receptors before (FIG. 11 A) and following 24 hours treatment with 4 ⁇ M ATO (FIG. 1 IB). Before treatment, both RI and
  • R2 APO2/TRAIL receptors were expressed on the surface of IM9 cells, with relatively low expression of R3 and R4 APO2/TRAIL decoy receptors.
  • treatment with ATO resulted with enhanced expression of R1/R2 APO2/TRAIL receptors from 46% to 82%, and from 21% to 58% for RI and R2 TRAIL receptors, respectively.
  • Concomitant with the observed increase in the expression of RI and R2 TRAIL receptors a decrease in the expression of R3/R4 decoy receptors was observed, from 22% and 28% to 3% and 2% for R3 and R4 TRAIL decoy receptors, respectively. Therefore, the synergy between ATO and APO2/TRAIL is indeed the result of modulation of APO2/TRAIL receptors.
  • myeloma cells were isolated from the bone marrow of six MM patients by flow- sorting and myeloma cells with >95% purity were obtained (Gazitt, 1999). Freshly isolated myeloma cells were cultured for 2 days with 4 ⁇ M ATO, lOOng/ml of APO2 TRAIL, or both, and apoptosis was scored after 24 and 48 hours. The results are depicted in Table 2.
  • M1-M6 represent myeloma cells sorted from the bone marrow of six MM patients. Background apoptosis in non-treated cells was ⁇ 10%. ⁇ , numbers in parentheses represent % myeloma cells in bone marrow before sorting. %, numbers represent % apoptotic cells
  • Example I indicating a p53 -dependent apoptosis in cells treated with ATO, are further supported by the results obtained from studies of caspase cascade activation by ATO in cells expressing w.t. vs. mutated p53.
  • caspase blocking peptides caspase-specific substrate peptides
  • Western immunoblotting it is clearly shown that in cells expressing functional p53, the initiator caspase 9 is the principal caspase activated by ATO, leading to caspase 3 activation and apoptosis.
  • caspase 8 and 10 are the principal caspases activated by ATO leading to caspase 3 activation and apoptosis.
  • Seol et al. 2001
  • Kitamura et al. (2000) reported activation of caspase 8 in ATO-induced apoptosis in gastric cancer cells.
  • the present results explain the seemingly conflicting reports by these two groups for caspase activation by ATO, presuming the difference between these two cell lines is the status of p53.
  • Caspase 8 and 10 are the primary caspases involved in the well characterized extrinsic apoptosis pathways attributed to the fasL and TRAIL (Ashkenazi and Dixit, 1998; Lacour et al, 2001; Mitsiades et al, 2002), whereas caspase 9 is the primary caspase involved in the intrinsic mitochondrial pathway (Adams and Cory, 1998).
  • the two apoptotic pathways could be linked through activation by caspase 8 of the proapoptotic protein, Bid, resulting in its translocation to the mitochondria and apoptosis via mitochondrial damage (Roth and Reed, 2002). It is possible that ATO might be involved in triggering both apoptotic pathways, since depolarization of mitochondrial membrane potential occurs early in cells expressing mutated p53, preceding apoptosis as measured by
  • Upregulation of R1/R2 APO2/TRAIL receptors was also reported by Sun et al. (2000) for ATRA and, similar to the present results, ATRA synergized with APO2/TRAIL in the induction of apoptosis in lung cancer cells (Sun et al. , 2000).
  • This effect of ATO on surface APO2/TRAIL receptors is similar to the effect reported for various other chemotherapeutic drugs (Nagane et al, 2000; Gliniak and Le, 1999; Chinnaiyan et al, 2000) and for adenovirus delivery of p53 (Kim et al, 2001; Liu et al, 2001).
  • ATO is a potent inducer of apoptosis in myeloma, particularly in cells expressing mutated p53, and synergizes with APO2/TRAIL in the induction of apoptosis.
  • the fact that freshly-isolated myeloma cells have increased susceptibility to the combination of ATO and TRAIL indicate that these findings are clinically relevant and that these two drugs will work in a similar way in vivo.
  • EXAMPLE III ATO-induced and Mitochondrial Apoptosis Pathways The present example shows that ATO selectively induces early and extensive apoptosis via the APO2/caspase 8 pathway in myeloma cells with mutant p53 and engages the mitochondrial pathway.
  • A. Materials and Methods Cell Lines and Cell Culture The list of myeloma cell lines used in this study, their source, p53 status and bcl-2 status is depicted in Table 3.
  • U266, RPMI 8226, IM9, HS-Sultan and ARH-77 cell lines were obtained from the ATCC.
  • ARP-1 cells were isolated at University of Arkansas (Tian et al, 1996).
  • the ARP-l/bcl-2 and 8226/bcl-2 are bcl-2-overexpressing ARP-1 and RPMI 8226 cells, respectively, prepared by stable transfection with a bcl-2 expressing vector (Tian et al, 1996). Cells ⁇ ransfected with empty vector were used as a control throughout this study.
  • the relative expression of Bcl-2 in all the cell lines used in this study has been published recently (Tian and Gazitt, 1996; Liu and Gazitt, 2003).
  • Bcl-2 and mock-transfected cells (neo) 8226 cells were generated and maintained in G418 as described (Tian et al, 1996).
  • the Bcl-2 expression plasmid (obtained from Dr. S. Korsmeyer) contained the LTR-SV-NEO construct, driven by an SV-40 promoter and hu Bcl-2 cDNA and a neomycin selection gene (Tian et al., 1996) in a partial Blue Script plasmid (Stratagene, La JoUa, CA).
  • Apoptosis was determined by staining of exposed phosphatidylserine with Annexin V-FITC (BioVision, Palo Alto, CA) as recommended by the manufacturer. Stained cells were analyzed by flow cytometry (FACScalibur, BDIS, and San Jose, CA). Quantification of apoptosis was done by the CellQuest program. Ten thousand cells were analyzed (Liu and
  • TMRE Tetramethylrhodamine ethyl ester
  • MMP mitochondrial membrane depolarization
  • HP hydrogen peroxide
  • HP catalyzes the conversion HE (non-fluorescent form) to ethidium bromide (EB; fluorescent at 520nm). Briefly, cells (4xl05/ml) were cultured in 1ml of FCS-medium in a 24-well plate. At the time points indicated, 5 ⁇ M ATO was added.
  • MMP depolarization or generation of HP For measurement of MMP depolarization or generation of HP, cells were washed and resuspended in FACS-buffer (PBS+ 1%BSA) and TMRE (150nM) or HE (20 ⁇ M) was added and cells were further cultured for 20 min. at 37°C. Cells were then washed with FACS- buffer and TMRE uptake or generation of EB was determined by flow cytometry as recommended by the manufacturer. Quantification of MMP depolarization was performed by measurement of the left shift in TMRE fluorescence of depolarized mitochondria and HE conversion to EB was determined by measurement of the right shift at 520nm of newly formed EB. Analysis of the results was performed by the CellQuest program. Ten thousand cells were analyzed.
  • cytosolic cell fraction was prepared by resuspending the cell pellets in mitochondria/cytosol buffer (BioVision Palo Alto, CA) and passing 300 ⁇ l of cell suspension (1x107 cells/ml) through a syringe with a 27 gauge needle several times at 4°C. This was followed by centrifugation at 10,000g for 20 min. at 4°C. About 75% of the cells were homogenized routinely by this procedure with 10%- 15% loss of viability of mitochondria, as determined by uptake of TMRE.
  • APO2/TRAIL receptors cells were cultured for 24 hours with 3 ⁇ M ATO, at which time apoptosis was ⁇ 40%. Cells were then harvested for immunofluorescence staining and for determination of apoptosis. Indirect immunofluorescence staining for surface APO2/TRAIL receptors and surface APO2/TRAIL was performed as described (Liu et al, 2001). Primary antibodies to APO2/TRAIL RI and R2 were obtained from Immunex Corporation (Seattle,
  • APO2/TRAIL Antibodies to APO2/TRAIL were from R&D Systems (Minneapolis, MN). Stained cells were analyzed by flow cytometry (FACScalibur, BDIS, San Jose, CA). Quantification of apoptosis was done by the CellQuest program. Ten thousand cells were analyzed.
  • a time and dose dependent apoptosis was observed between 1.5 and 6 ⁇ M ATO, with apoptosis of >85% observed after 48 hours of treatment with 6 ⁇ M ATO in RPMI 8226 cells.
  • a fixation/permeation method was first developed to selectively stain cytosolic cytochrome c.
  • An example is shown in a composite Confocal Imaging micrograph in
  • FIG. 13 U266 (mutated p53); IM9 and HS-Sultan cells (w.t. p53) were treated for 24 hours with 5 ⁇ M ATO and stained with FITC-anti cytochrome c antibodies. Control cells without ATO (CON) exhibited only small background staining, whereas ATO-treated IM9 and HS-
  • Activated T-cells were practically resistant to doses of up to 20 ⁇ M ATO. Similar to the results obtained with SH-Sultan cells, activated normal T-cells had relatively low levels of apoptosis and low percentage of cells with depolarized MMP and cytosolic cytochrome c. In these cells, like HS-Sultan cells (w.t. p53), a small increase in the proportion of cells arrested in Gl was observed.
  • FIG. 15A and FIG. 15B The compiled data from three studies are shown in FIG. 15A and FIG. 15B for RPMI 8226, U266, ARH-77 and APR-1 cells (mutated p53) and HS-Sultan and IM9 cells (w.t. p53) treated with 5 ⁇ M ATO for 0-40 hours.
  • mutated p53 exhibited a rapid MMP depolarization and generation of HP (8-16 hours) preceding cytochrome c release and apoptosis by annexin V.
  • MMP depolarization and generation of HP were concurrent or occurred after cytochrome c release and apoptosis (FIG. 15A and FIG. 15B).
  • FIG. 17 Indeed, BID was cleaved in RPMI 8226, U266 and ARH-77 cells expressing mutated p53. In contrast, in cells expressing w.t. p53, cleavage of BID was very little, if any.
  • caspase 8 is the major caspase activated in these cells (Example II; Liu et al, 2003).
  • APO2/TRAIL receptors was tested in cells undergoing apoptosis (30% to 50%) by ATO. Examples from a representative study with U266 (mutated p53) and HS-Sultan (w.t. p53) are presented in FIG. 18A and FIG. 18B, respectively. Indeed, in U266 cells, a significant increase was observed in percent cells expressing
  • R2 APO2/TRAIL receptor solid thin line
  • APO2/TRAIL thin solid line
  • MFI mean fluorescence intensity
  • APO2/TRAIL induction was significant (-40%) as early as 8 hours post induction, where MMP depolarization was evident in about 70% of the cells, at which time apoptosis by annexin V reached 31%.
  • AIF translocation to the nucleus occurred in 56% of the cells, 8 hours post induction of apoptosis, reaching 84% by 16 hours, at which time 94% of the cells exhibited depolarization of MMP. Therefore, in these cells, the APO2/TRAIL/TRAIL receptor/BID/AIF pathway is engaged by ATO, preceding apoptosis by annexin V and coinciding with depolarization of MMP (FIG. 19A).
  • APO2/TRAIL and APO2/TRAIL receptors are induced concomitant with BID cleavage in the absence of p53, although overexpression of p53 can induce the expression of APO2/TRAIL receptor, in myeloma cells (Liu et al, 2001).
  • caspase 8 is the primary caspase involved in ATO-induced apoptosis in cells expressing mutated p53 (Example II)
  • these results indicate that ATO engages the extrinsic apoptotic pathway in these cells resulting with a rapid release of AIF, which leads to nuclear apoptosis.
  • AIF is localized to the nucleus as early as 8-16 hours after induction of apoptosis and cytochrome c release to the cytosol is relatively small in these cells compared to the rapid and substantial release of AIF.
  • both pathways are engaged and caspase 3 is also cleaved and contributed to apoptosis.
  • ATO-induced apoptosis in cells with mutated p53 are not restricted to myeloma cells, since the p53 mutated Jurkat, T-ALL cells (Blagosklonny, 2002) were also sensitive to ATO-induced apoptosis, engaging APO2/TRAIL/TRAIL receptor pathway, cleavage of BID, early depolarization of MMP, release of AIF to the cytosol and arrest of cells in the G2/M.
  • Over expression of Bcl-2 does not protect against ATO-induced apoptosis and the partial resistance of HS-sultan and IM9 cells correlates with p53 status and not on the expression of bcl-2.
  • the primary apoptotic events included early release of cytochrome c to the cytosol and activation of caspase 3 and caspase 9 (Example I; Example II; Liu et al, 2003).
  • induction of APO2/TRAIL cleavage of BID or substantial release of AIF to the cytosol and its translocation to the nucleus was not observed.
  • the APO2/TRAIL pathway is not the primary apoptotic pathway in these cells and the observed partial and delayed depolarization of MMP and slight release of AIF could be secondary to apoptosis. Instead, in cells expressing w.t.
  • p53 the primary apoptotic pathway engaged by ATO is the p53-mediated Bax translocation to the mitochondria, cytochrome c release to the cytosol and activation of caspase 9 (Example I; Example II; Liu et al. , 2003).
  • the p53 regulated proapoptotic regulatory protein (Henry et al, 2002; Moll and Zaika, 2001) could also be involved to some extent in ATO-induced apoptosis in these cells.
  • ATO activates p53-dependent cell cycle regulation through activation of p21 and arrest of the cells in Gl of the cell cycle (Example I; Example II; Liu et al, 2003).
  • ATO acts like DNA damaging agent, eventually leading to differentiation and/or apoptosis (Example I (see Discussion); Cai et al, 2000; Woods and Vousden, 2001 ; Bargonetti and Manfredi, 2002).
  • the fact that activated normal T-cells were relatively resistant to ATO and that the pattern of apoptosis in these cells was similar to the one observed in myeloma cells expressing w.t. p53 suggest a general mechanism for ATO- induced apoptosis in cells with w.t. p53. Based on the results of the present invention, a model for ATO-induced apoptosis in cells expressing w.t.
  • p53 is summarized in FIG. 20B.
  • the effect of ATO in vitro could explain the relative low hematological toxicity observed in patients undergoing treatment with ATO, since Gl block, unlike G2 block, is less toxic to the cells and can be reversible.
  • ATO has been used in myeloma patients in phase I-II clinical trials as a single agent, or in combination with ascorbic acid or thalidomide.
  • prescreening for p53 mutations should increase the efficacy of ATO in multiple myeloma patients.
  • a rapid, simple and cost-effective screening for the p53 status of cells using the response to ATO is provided by the present invention.
  • ATO Induces p53-Dependent Apoptotic Signals in Temperature-Sensitive p53 Mutants
  • ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53.
  • both the intrinsic and extrinsic apoptotic pathways are engaged.
  • This example further establishes the differential effect of ATO in relation to p53 status, using the effect of temperature shift in temperature-sensitive (Ts) p53 mutant cells.
  • BRK Baby Rat Kidney
  • results in the present example further substantiate the role of p53 in ATO- induced apoptosis in myeloma and other cancer cell types, as set forth in the earlier examples.
  • ATO and Taxol Induce Apoptosis by Different Mechanisms ATO and taxol (paclitaxel) are effective in the treatment of various types of cancers. Since both ATO and taxol induce arrest of cells in G2/M, it was postulated the ATO belongs to the anti-mitotic family of drags such as the taxenes and vinca alkaloids.
  • the present example shows that ATO and taxol induce apoptosis involving mitochondrial membrane depolarization and G2/M cell cycle block, but differ in the pattern of caspase activation, release of mitochondrial proapoptotic proteins, chromatin condensation, nuclear fragmentation and protection by overexpression of Bcl-2. Thus, there are clear differences in the mode of action of ATO and taxol and these two drags induce apoptosis by different mechanisms.
  • U266 and RPMI 8226 cell lines were obtained from the ATCC.
  • the cell culture and induction of apoptosis by ATO were conducted as described above in Example III.
  • Staining with Annexin V and detection of apoptotic cells were ALSO conducted as described above in Example III.
  • TMRE mitochondrial membrane depolarization
  • TMRE TMRE-stained cells were then stained with annexin V, also as described above in Example III. Analysis of the results was performed by the CellQuest program. Ten thousand cells were analyzed. A portion of the TMRE/annexin V-stained cells was viewed by confocal microscopy as described below. For confocal microscopy, cells were treated as above with ATO or taxol and were stained according to the described. About 100,000 cells in 20 ⁇ l of anti-fade were layered on a slide and cells viewed using the Olympus FV500 Confocal Imaging System. Images were taken with x60 plan APO (1.4 aperture) lens and x40 UAPO 340 (1.53 aperture) lens as described above in Example III.
  • Example III The determination by flow cytometry of AIF release from mitochondria and translocation to the nucleus was performed as described above in Example III. To determine the effect of ATO on surface expression of Apo2/TRAIL and Apo2/TRAIL receptors, the immunofluorescence staining techniques set forth above in Example III were employed.
  • Example III The cell fractionation and preparation of the cytosolic cell fraction were conducted as described above in Example III. The determination by western immunoblotting of cleavage of BID and release of cytochrome c and AIF to the cytosol was also performed as described above in Example III.
  • Bcl-2-transfected RPMI8226 cells were more resistant to taxol-induced apoptosis at all time points and at all doses tested, with maximal apoptosis of only 25% following 48 hours of treatment with 0.6 ⁇ M taxol.
  • taxol-induced apoptosis could be blocked by overexpression of Bcl-2.
  • ATO mitochondrial membrane potential
  • MM mitochondrial membrane potential
  • apoptosis 94% vs. 63%) in the range of concentrations used (6 ⁇ M ATO; 0.6 ⁇ M taxol).
  • Both drags induced apoptosis concomitant with MM depolarization and arrest of cells at G2/M of the cell cycle; however, in ATO- treated cells, MM depolarization and apoptosis preceded G2/M arrest. In contrast, in cells treated with taxol, cell cycle arrest at G2/M preceded MM depolarization and apoptosis.
  • ARP/Bcl-2 myeloma cells over-expressing Bcl-2 were also tested and shown to be resistant to taxol-induced apoptosis and depolarization of MM, despite cell cycle arrest at
  • TMRE was used to mark mitochondria with active mitochondrial membrane, permitting detection via red fluorescence.
  • Cells undergoing apoptosis were detected by staining for exposed phosphatidylserine using annexin V-FITC, permitting detection via green fluorescence.
  • RPMI8226 cells were tested after undergoing apoptosis for up to 24 hours by contact with 6 ⁇ M ATO. Cells were viewed by confocal microscopy immediately following staining with annexin V.
  • ATO induces a time-dependent exposure of phosphatidylserine (increased number of cells with green fluorescence) with a concomitant decrease in cells with active mitochondrial membrane (decreased TMRE+ cells with bright red fluorescence).
  • a comparison between the cells stained in green (apoptosis) and the cells stained in red (active mitochondrial membrane) show that these two indicators are mutually exclusive; namely, cells with active MM (red cells) are not the cells undergoing apoptosis (green cells) and vice versa. Similar results were observed in RPMI8226 cells undergoing apoptosis with taxol. Quantitative results were also obtained.
  • RPMI8226 cells were treated with 6 ⁇ M ATO or 0.6 ⁇ M taxol for up to 24 hours and stained for mitochondrial membrane by TMRE (red fluorescence) and for apoptosis by annexin V-FITC (green fluorescence) and the results were analyzed by flow cytometry.
  • TMRE red fluorescence
  • annexin V-FITC green fluorescence
  • RPMI8226 cells and the isogenic Bcl-2-overexpressing 8226/Bcl-2 cells were induced to undergo apoptosis by contact with either ATO or taxol.
  • Treatment of RPMI8226 cells with 6 ⁇ M ATO for 16 hours resulted with the formation of perinuclear, ring-like apoptotic bodies in both isogenic cell types.
  • ATO induced-apoptosis involves early translocation of AIF from mitochondria to the nucleus in myeloma cells expressing mutant p53 (see foregoing examples). Unlike ATO, it was hypothesized that taxol-induced apoptosis does not involve translocation of AIF to the nucleus, but rather involves cytochrome c release and caspase 9 activation.
  • AIF antibody and with PI to visualize nuclei were viewed by confocal microscopy.
  • ATO-induced apoptosis was accompanied with progressive appearance of apoptotic bodies in PI stained cells (red fluorescence), reaching a maximum at 24 hours post induction of apoptosis.
  • AIF appeared as perinuclear, ring-like, punctated (green fluorescence) staining pattern, with a gradual increase in the percentage of stained cells in cells undergoing apoptosis with a change to a diffuse nuclear staining of AIF in cells with visible apoptotic nuclei, 24 hours post-induction of apoptosis.
  • ATO-induced apoptosis in myeloma cells expressing mutated p53 involves early induction of surface Apo2/TRAIL (see foregoing examples). Since taxol-induced apoptosis engages the intrinsic and not the extrinsic apoptotic pathway, the hypothesis that taxol- induced apoptosis does not involve induction of surface Apo2/TRAIL was tested. It is clear that ATO induces early expression of Apo2/TRAIL (up to 48%) in RPMI8226 cells, whereas treatment with taxol practically did not result in an increase in surface expression of Apo2/TRAIL.
  • ATO-induced apoptosis As shown in the previous examples, ATO engages both the intrinsic and extrinsic apoptotic pathway in cells with mutant p53, whereas it is widely accepted taxol-induced apoptosis involves the extrinsic apoptotic pathway (Pan et al, 2001; Ofir et al, 2002; Yuan et al, 2002).
  • taxol-induced apoptosis involves the mitochondrial intrinsic pathway engaging cytochrome c release and activation of caspase 9 and caspase 3 (Pan et al. , 2001; Ofir et al, 2002; Yuan et al, 2002) and that overexpression of Bcl-2 blocks taxol- induced apoptosis in myeloma cells (Gazitt et al, 1998).
  • the present results are consistent, showing an early MM depolarization, release of cytochrome c and activation of caspase 9 concomitant with mitotic arrest in myeloma cells undergoing apoptosis by taxol.
  • ATO-treated cells In contrast to taxol-treated cells, ATO-treated cells exhibited BID cleavage and translocation of AIF from mitochondria to the nucleus in cells undergoing apoptosis, regardless of intracellular levels of Bcl-2. Furthermore, whereas in cells treated with ATO, both Apo2/TRAIL and its receptors, RI and R2, were upregulated; in taxol-treated cells,
  • Apo2/TRAIL was not upregulated.
  • upregulation of Apo2/TRAIL receptors by taxol and other drags has been reported (Nagane et al, 2000; Lacour et al, 2001; Nimmanapalli et al, 2001; Gazitt, 1999; Liu et al, 2003).
  • Apo2/TRAIL activation between the two drags is important in understanding the differences in the ability of Bcl-2 to protect against taxol and ATO-induced apoptosis, since the inventor and others have shown that Apo2/TRAIL-induced apoptosis in myeloma cells could not be abrogated by overexpression of Bcl-2 (Gazitt, 1999).
  • the present results show striking differences in the mode of chromatin condensation and nuclear fragmentation between the two drags in cells undergoing nuclear apoptosis. While ATO-treated cells exhibited an early ring-like distribution of chromatin (stage I chromatin condensation) and nuclear fragmentation (stage II chromatin condensation), cells treated with taxol exhibited a random pattern of chromatin condensation and nuclear fragmentation. The pattern of chromatin condensation observed here for ATO is consistent with that reported for staurosporin-induced apoptosis (Susin et al, 1999; 2000).
  • a similar pattern of localization of AIF in the nucleus was reported in cells treated with staurosporin (Susin et al, Ye et al, 2002) and in serum-starved cells (Ravagnan et al, 2001).
  • cells treated with taxol did not exhibit the early ring-like distribution of AIF in the early stages of nuclear apoptosis (stage I) and nuclear AIF was visible only in cells at late stages of nuclear apoptosis (stage II).
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods, and in the steps and/or in the sequence of steps of the methods described herein, without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. REFERENCES The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
  • Blagosklonny and Fojo "Molecular effects of paclitaxolel: myths and reality (a critical review),” Int. J. Cancer, 83:151-156, 1999.
  • Blagosklonny, Chuman, Bergan, Fojo "Mitogen-activated protein kinase pathway is dispensable for microtubule-active drag-induced Raf-1 /Bcl-2 phosphorylation and apoptosis in leukemia cells," Leukemia, 13(7): 1028-36, 1999.
  • Blagosklomiy "Paradox of Bcl-2 (and p53): why may apoptosis-regulating proteins be irrelevant to cell death?" Bioessays, 23:947-53, 2001.
  • Gazitt "APO2/TRAIL is a potent inducer of apoptosis in myeloma cells derived from multiple myeloma patients and is not cytotoxic to hematopoietic stem cells," Leukemia, 13:1817-1824, 1999. Gazitt, Shaughnessy, Montgomery, "Apoptosis induced by APO2/TRAIL and TNF ⁇ in human multiple myeloma cells is not blocked by bcl-2," Cytokine, 11:1010-1019, 1999.
  • Lamothe and Aggarwal "Ectopic expression of Bcl-2 and Bcl-xL inhibits apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) through suppression of caspases- 8, 7, and 3 and BID cleavage in human acute myelogenous leukemia cell line HL-60," J. Interferon Cytokine Res., 22(2):269-79, 2002.
  • TRAIL TNF-related apoptosis-inducing ligand
  • Mitsiades Mitsiades, Poulaki et al. , "Infracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human multiple myeloma cells," Blood, 99:2162-2171, 2002.

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Abstract

L'invention concerne des méthodes de criblage rapides et faciles destinées à identifier l'état fonctionnel de la protéine de suppression tumorale p53 dans des cellules, notamment dans des cellules cancéreuses. L'invention concerne également des trousses associées. Les méthodes et les trousses de l'invention testent la réactivité, en particulier l'état de cycle cellulaire, de cellules exposées à du trioxyde d'arsenic. Les méthodes et les trousses de l'invention sont utiles pour élaborer une thérapie anticancéreuse destinée à traiter plus efficacement des patients particuliers.
PCT/US2004/039922 2003-12-19 2004-11-30 Methodes de criblage rapides pour un p53 fonctionnel, faisant appel aux motifs de la reactivite au trioxyde d'arsenic WO2005065723A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110250291A1 (en) * 2009-11-12 2011-10-13 Yuan Zhi-Min Use of arsenic for cancer therapy protection
EP2474624A1 (fr) * 2011-01-05 2012-07-11 Daniela Kandioler Prédiction de la réponse dans le traitement du cancer (thérapie du cancer adaptée p53)
WO2012093152A1 (fr) 2011-01-05 2012-07-12 Daniela Kandioler Prédiction d'une réponse dans le traitement du cancer
WO2012145908A1 (fr) * 2011-04-28 2012-11-01 Tongji University Molécules p21 et associées à p21 comme biomarqueurs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG T. ET AL.: "Opposite biological effects of arsenic trioxide and arsacetin involve a different regulation of signaling in human gastric cancer MGC-803 cells", PHARMACOLOGY, vol. 64, 2002, pages 160 - 168 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110250291A1 (en) * 2009-11-12 2011-10-13 Yuan Zhi-Min Use of arsenic for cancer therapy protection
US8795738B2 (en) * 2009-11-12 2014-08-05 Board Of Regents Of The University Of Texas System Use of arsenic for cancer therapy protection
EP2474624A1 (fr) * 2011-01-05 2012-07-11 Daniela Kandioler Prédiction de la réponse dans le traitement du cancer (thérapie du cancer adaptée p53)
WO2012093155A1 (fr) 2011-01-05 2012-07-12 Daniela Kandioler Prédiction d'une réponse dans le traitement du cancer (thérapie anticancéreuse adaptée à p53)
WO2012093152A1 (fr) 2011-01-05 2012-07-12 Daniela Kandioler Prédiction d'une réponse dans le traitement du cancer
WO2012145908A1 (fr) * 2011-04-28 2012-11-01 Tongji University Molécules p21 et associées à p21 comme biomarqueurs

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