WO2002079504A9 - Neotic and endoapoptotic cells and detection of neosis and endoapoptosis - Google Patents

Abstract

Described herein are methods for isolating cells involved in neosis, cells or tissue compositions having an enriched populations of cells involved in neosis, methods of identifying compounds that modulates neosis, and tumor progression diagnostic methods.

Description

NEOTIC AND ENDOAPOPTOTIC CELLS AND DETECTION OF NEOSIS AND ENDOAPOPTOSIS

Background of the Invention

The invention relates to cells involved in the process of neosis, and methods for isolating or enriching for such cells. The invention also relates to methods of identifying compounds that modulate neosis, and tumor progression diagnostic methods .

Meiosis and mitosis are the two classical forms of cell division known in mammals that give rise to populations of cells that are genotypically similar to the mother cell. Meiosis, or reduction division, occurs in the gonads and results in the formation of haploid gamete cells with new combinations of alleles due to the cross-over phenomenon. Mitosis involves the division of one diploid mother cell giving rise to two daughter diploid cells. While the former is involved in sexual reproduction, the latter is primarily responsible for the somatic growth of multicellular organisms; therefore, it is important that somatic cells faithfully duplicate and distribute genetic material equally to the daughter cells during mitotic division. Normal itotic cell cycle progression through Gl, S, and G2/M phases is under strict genetic control, and several genes are involved in the regulation of cell cycle checkpoint controls that have evolved to maintain genetic fidelity and stability through successive generations of somatic cells- Therefore, the two mitotic daughter cells will display a genotype and phenotype identical to the mother cell .

In vi tro studies with human and rodent cell model systems have defined the various steps involved in cellular aging (senescence) , immortalization (preneoplastic) , and neoplastic transformation. Primary diploid cells have ^"a^~~ finite population doubling potential, after which the cells do not undergo mitotic division. These cells become senescent and eventually die. For example, human cells undergo about 100 +/- 10 population doublings in vi tro, after which they become senescent, while rodent cells at the end of their mitotic life span (about 15-30 population doublings) may often gain immortality after going through a mitotic crisis period.

Classic experiments aimed at describing the process of tumorigenesis in vi tro have shown that when normal and established "normal" rodent cells are exposed to genotoxins, such as ionizing radiation and chemical carcinogens, a given frequency of cells give rise to transformed colonies over the course of 8 weeks. The cells isolated from these colonies are tumorigenic. This transformed foci formation has been shown to be an in vi tro correlate of in vivo tumorigenesis. Unlike normal cells, neoplastic cells inherit genomic instability due to loss of checkpoint control (s) and by carrying mutant genes (oncogenes) that contribute to their unlimited division potential. Accumulation of further mutations may lead to tumor progression and malignancy.

Summary of the Invention

The present invention relates to methods and compositions for the enrichment, isolation, and molecular screening of neoplastic and pre-neoplastic mammalian cells that are responsible for transformed foci formation in vi tro, or tumorigenesis in vivo . In particular, the methods relate to enriching or isolating cell fractions from a heterogenous neoplastic and pre-neoplastic mammalian cell population that are involved in different stages of transformed foci formation in vitro or tumorigenesis in vivo . The invention also relates to molecular screening methods, including differential display, that identify nucleic acid molecules involved in regulating or executing the processes responsible for the transformed foci formation in vi tro, or tumorigenesis in vivo . In addition, the invention features methods for diagnosing the presence of a proliferative disease, and methods for identifying compounds that inhibit tumor progression.

Accordingly, in a first aspect, the invention features a method for isolating an endoapoptotic cell, involving identifying the endoapoptotic cell in a population of cells, and separating at least one endoapoptotic cell from the non-endoapoptotic cells in the population of cells.

In a second aspect, the invention features a method for isolating a neosis mother cell, involving identifying the neosis mother cell in a population of cells, and separating at least one neosis mother cell from the cells that are not neosis mother cells in the population of cells.

In a third aspect, the invention features a method for isolating a Raju cell, involving identifying the Raju cell in a population of cells, and separating at least one Raju cell from the cells that are not Raju cells in the population of cells.

In preferred embodiments of any of the above aspects of the invention, separation of the cell from the population of cells involves the use of flow cytometry, micro coverslip capture, the use of a Percoll or other density gradient, or laser capture microdissection.

In a fourth aspect, the invention features an isolated endoapoptotic cell . In a fifth aspect, the invention features an isolated neosis mother cell.

In a sixth aspect, the invention features an isolated Raju cell.

In preferred embodiments of any of the fourth, fifth, or sixth aspects of the invention, the cell is mammalian or avian cell, a fruit fly or related insect cell, a nematode cell, a fish cell, or a yeast cell.

In a seventh aspect, the invention features a cell or tissue composition having an enriched population of endoapoptotic cells.

In an eighth aspect, the invention features a cell or tissue composition having an enriched population of neosis mother cells.

In a ninth aspect, the invention features a cell or tissue composition having an enriched population of Raju cells .

In preferred embodiments of any of the seventh, eighth, or ninth aspects of the invention, the cell or tissue composition is mammalian or avian, or of fruit fly or related insect, fish, or nematode origin. The cell or tissue composition may be a tissue biopsy from a mammal or avian species, or from a fruit fly or related insect, a fish, or a nematode. In another embodiment, the cell or tissue composition of any of the seventh, eighth, or ninth aspects includes a cell culture line isolated from any of the above tissues. Preferably the cell line is selected from the group consisting of C3H10T1/2 cells, NIH3T3 cells, A10 cells, HTB11 cells, HeLa cells, mouse embryo cells, rat embryo cells, hamster embryo cells, rodent cells, avian cells, fish cells, Chinese hamster ovary cells, normal human cells, human neoplastic cells, cells from fruit flies or related insects, nematodes, or yeast cells. The mouse embryo cell line may be a p53-/- mouse embryo cell line, an RB-/- mouse embryo cell line, or any other gene knockout cell line, where the gene is involved in differentiation, cell cycle regulation, or apoptosis. The rodent cell, avian cell, or human cell may be a normal cell or a neoplastic cell.

In a tenth aspect, the invention features a secondary cell or tissue composition that is obtained by culturing the cell or tissue composition of any of the seventh, eighth, or ninth aspects of the invention.

In preferred embodiments, the secondary cell or tissue composition contains a mononucleate giant neosis mother cell, a multinucleate giant neosis mother cell, or a Raju cell .

In an eleventh aspect, the invention features a method of identifying a nucleic acid molecule that modulates neosis, involving: (a) isolating nucleic acid molecules from a neosis mother cell, Raju cell, or endoapoptotic cell; (b) isolating nucleic acid molecules from a control cell (a cell that is not a neosis mother cell, Raju cell, or endoapoptotic cell) ; (c) comparing the expression of the nucleic acid molecules of step (a) with the expression of the nucleic acid molecules of step (b) , where a nucleic acid that is differentially expressed between the nucleic acid molecule samples identifies a gene that modulates neosis.

In preferred embodiments of the eleventh aspect of the invention, the neosis mother cell, Raju cell, or endoapoptotic cell and the control cell are the same cell type or different cell types. In other preferred embodiments, the comparison of nucleic acid molecule expression between the nucleic acid molecules of step (a) and the nucleic acid molecules of step (b) involves the use of differential display, nucleic acid library hybridization, cDNA icroarray hybridization, or oligonucleotide microarray hybridization.

In a twelfth aspect, the invention features a method of diagnosing a mammal for the presence of a neoplastic or proliferative disease, or an increased likelihood of developing a neoplastic or proliferative disease. The method involves isolating a biological sample from the mammal and determining the presence or absence of a neosis mother cell, Raju cell, or endoapoptotic cell. The presence of the cell being an indication of the presence of a neoplastic or proliferative disease, or an increased likelihood of developing a neoplastic or proliferative disease. In one embodiment, the biological sample is a tissue biopsy or a blood sample. In another embodiment, the biological sample is a tumor biopsy.

In a thirteenth aspect, the invention features a method of monitoring tumor progression in a mammal, involving isolating a tumor sample from the mammal and determining the frequency of neosis mother cells, Raju cells, or endoapoptotic cells, over a time interval, where an increase in the number of neosis mother cells, Raju cells, or endoapoptotic cells over time indicates tumor progression.

In one embodiment of the twelfth or thirteenth aspects of the invention, the neosis mother cells, Raju cells, or endoapoptotic cells are detected using a biochemical stain, or are morphologically detected. In a fourteenth aspect, the invention features a method of identifying a compound that inhibits tumor progression, involving the steps of: (a) providing a neosis mother cell; (b) contacting the neosis mother cell with a candidate compound; and (c) determining the viability or proliferation of the neosis mother cell . A candidate compound that decreases the viability or proliferation of the neosis mother cell, relative to the level of viability of proliferation of a neosis mother cell not contacted with the candidate compound, is a compound that inhibits tumor progression.

In a fifteenth aspect, the invention features a method of identifying a compound that inhibits tumor progression, involving the steps of: (a) providing a Raju cell; (b) contacting the Raju cell with a candidate compound; and (c) determining the viability or proliferation of the Raju cell. A candidate compound that decreases the viability or proliferation of the cell, relative to the level of viability or proliferation of a Raju cell not contacted with the candidate compound, is a compound that inhibits tumor progression.

In a sixteenth aspect, the invention features a method of identifying a compound that inhibits tumor progression, involving the steps of: (a) providing an endoapoptotic cell; (b) contacting the endoapoptotic cell with a candidate compound; and (c) determining the viability or proliferation of the endoapoptotic cell, wherein a candidate compound that decreases the viability or proliferation of the cell, relative to the level of viability or proliferation of an endoapoptotic cell not contacted with the candidate compound, is a compound that inhibits tumor progression. In preferred embodiments of any of the fourteenth, fifteenth, or sixteenth aspects of the invention, the cell is from a biological sample of a mammal or from a cultured cell line.

In a seventeenth aspect, the invention features a nucleic acid library derived from an endoapoptotic cell.

In a eighteenth aspect, the invention features a nucleic acid library derived from a neotic cell .

In a preferred embodiment of the eighteenth aspect of the invention, the neotic cell is a neosis mother cell or a Raju cell. In preferred embodiments of either the seventeenth or eighteenth aspects of the invention, the library is a cDNA library or a genomic library.

By "endoapoptosis" is meant the intracellular self destruction of a daughter genome within the cytoplasmic boundaries of the mother cell, wherein the mother cell is a cell that does not normally undergo meiosis before the completion of the mitotic cell division processes karyokinesis (nuclear division) or cytokinesis (cytoplasmic division) that yields two individual daughter cells. By "intracellular self- destruction" is meant within the cytoplasmic membrane boundaries of the mother cell . The occurrence of endoapoptosis can be determined visually, using, for example, phase contrast microscopy at 200X or higher magnification. Preferably a daughter genome that has undergone endoapoptosis had nucleic acid that is a replica of the DNA equivalent or almost equivalent to that of the surviving viable nuclei in the same cell to begin with, but had lost DNA gradually due to DNA fragmentation during the progression of endoapoptosis, which can be assayed using standard staining for DNA, such as

Feulgen stain followed by icrospectrophotometry. The appearance and destruction of endoapoptotic bodies (or non-viable genomes) varies depending on the stage of mitosis at which endoapoptosis occurs (Fig. 1) . For example, initiation of endoapoptosis of a non-viable nascent genome before the homologous chromosome pairs are separated by telophase during mitosis results in an endoapoptotic body that is trapped within the viable sister nucleus (intranuclear endoapoptotic body; Fig. 1A) . Initiation of endoapoptosis of the non-viable nascent genome after karyokinesis is completed, but before cytokinesis is initiated, results in the appearance and destruction of a nucleus (genome) within the cytoplasm of the mother cell (nucleocentric endoapoptotic body; Fig. IB) . In addition, initiation of endoapoptosis of a non-viable nascent genome after the completion of karyokinesis and an asymmetric intracellular cytokinesis results in a dying "mini cell" having a non-viable nucleus and a thin film of cytoplasm within the cytoplasm of the mother cell . The dying mini cell is found very close to the surviving daughter cell nucleus, or is accommodated in a cup-shaped depression in the viable sister nucleus, which looks crescent shaped (Fig. IC) . These various types of endoapoptotic bodies may be visually identified using phase contrast microscopy at 20OX or higher magnification or after staining with HO/PI using fluorescence microscopy at a magnification of 200X or higher.

By an "endoapoptotic cell" is meant a cell displaying endoapoptosis within its cytoplasm. An endoapoptotic cell can be identified according to the above- described characteristics of a cell undergoing endoapoptosis, or according to any of the characteristics described in U.S.S.N. 08/759,295, incorporated herein by reference.

"Neosis" is a novel form of cell division other than the classical forms of cell division known as mitosis and meiosis. Neosis is characterized by at least two of the following features: (a) induction by spontaneous or induced genetic and/or epigenetic changes; (b) karyokinesis occurring via nuclear budding or other related processes without the dissolution of the nuclear envelope, which can be determined using visual inspection and phase contrast microscopy at 20OX or higher magnification; and (c) cytokinesis occurring via a unique asymmetric division (uneven partitioning of the cytoplasm with a daughter nucleus or genome during cytokinesis, as assayed by phase contrast microscopy or by fluorescence microscopy after Hoechst 33342/Propidium Iodide (HO/PI) staining) giving rise to one to several immature daughter cells (Raju cells) . The nascent Raju cell may detach itself from the neosis mother cell via contractile motion or "birth dance" or via budding. Preferably a cell undergoing neosis is characterized by all three of the above characteristics. The occurrence of neosis is preceded by a neosis DNA synthesis phase leading to polyploidization or multinucleation, which can be assessed using phase contrast microscopy at 200X or higher magnification or by Feulgen staining followed by microspectrophotometer. Endoapoptosis and the formation or Raju cells are two events that may occur during neosis.

By "neosis mother cell" is meant a cell that gives rise to a Raju cell through the process of neosis. An endoapoptotic cell may undergo mitotic or neotic division.

By "Raju cell" is meant a nascent daughter cell produced via neosis that is morphologically different from a mitotic daughter cell due to the asymmetric nature of cytokinesis (an uneven partitioning of the cytoplasm) that occurs during neosis. A Raju cell is characterized by at least two of the following: (a) its small size (8-15 μm in diameter at birth) ; (b) a true nuclear to cytoplasmic (T-N/C) ratio greater than 1, as determined by the ratio between the area of the nucleus and the total area of the cell minus the area of the nucleus, or a classical nuclear cytoplasmic (N/C) ratio of 0.7 or above but always less than 1.0, as determined by the ratio between the area of the nucleus and that of the whole cell (Figs. 18A and 18B) ; (c) no distinguishable nuclear envelope, as observed by phase contrast microscopy at 20OX magnification; (d), highly condensed chromatin, as determined by the difference in fluorescence intensity between the nuclei of the Raju cell and a mitotic progeny, which is measurable by densitometric tracing or by flow cytometry; (e) the absence of a visible nucleolus at birth, as observed by phase contrast microscopy at 200X or 400X magnification; and (f) differential staining with Giemsa stain, with the nascent Raju cells exhibiting little or no staining, while the maturing and mature Raju cells (neoplastic cells) show increased amounts of staining, which are measurable by densitometric tracing. Preferably a Raju cell is characterized by at least three of the above characteristics, more preferably by four or five of the above characteristics, and most preferably by all six of the above characteristics.

Neosis mother cells produce one to an indefinite number of Raju cells through the process of neosis, and these Raju cells are further characterized by variable degrees of genomic instability, in which the genome of the cells undergoes random variations through successive mitotic generations, and indefinite mitotic division potential, which can be assayed by measuring total cell counts (using, for example, a cell counter) . Raju cells display a genotypic alteration from their mother cell, identifiable by performing standard molecular biology assays to compare the gene expression profiles of the Raju cells and their neosis mother cells; they may also differ phenotypically from their neosis mother cells, which is discernible using phase contrast microscopy at a magnification of 20OX or more. In addition, Raju cells may grow in size by gaining cell mass, which can be visually determined, and are potentially tumorigenic (as assessed using, for example, standard anchorage independent growth studies or in vivo tumorigenicity when injected into an appropriate host, e.g., a nude mouse) .

By "neotic cell" is meant any cell involved in the process of neosis. Examples of neotic cells include, but are not limited to, neosis mother cells and Raju cells. An endoapoptotic cell may undergo mitotic division or neotic division.

By "neosis mother cell" or "NMC" is meant a cell that undergoes neosis, giving rise to a Raju cell; a non- viable Raju cell may undergo endoapoptosis before birth, or apoptosis after birth, while a viable Raju cell undergoes repeated mitotic division. Potential neosis mother cells are characterized as being polyploid/multinucleate giant cells, and by morphologically exhibiting genomic instability-induced abnormal nuclear morphology, including micronuclei formation, multinucleation, dicentric bridges linking two nuclei, or pulverized or lobed nuclei formation (all of which can be visualized using phase contrast microscopy or fluorescence DNA staining techniques) . Neosis mother cells are generally 80- 150 μm in diameter.

As used herein, by "isolated" is meant a cell that is physically separated from a population of cells. Preferably, the isolated cell is a neosis mother cell, a Raju cell, or an endoapoptotic cell. Methods for isolating such cells are described herein. As used herein, by "enriched population" is meant a group of cells with an above normal frequency of a desired cell type, for example, endoapoptotic cells, neotic mother cells, or Raju cells. A population of cells may be enriched on the basis of cell size (using, for example, centrifugal elutriation, cell sorting techniques, or Percoll or other density gradient centrifugation) and morphological features (for example, using laser capture microdissection [LCM] or micro coverslip capture [MCC] ) .

By "micro coverslip capture" or "MCC" is meant the use of small micro coverslips for the culture and isolation of individual or small groups of cells from a larger culture vessel .

By "primary neosis" is meant a normal cell undergoing neosis for the first time; by "secondary/tertiary neosis" is meant the mitotic product of a cell derived from primary neosis undergoing additional rounds of neosis due to genomic instability. The cell with genomic instability may be, for example, a neotic cell or the progeny of the neotic cell.

By "leaky neosis" is meant the slow and sequential production of one Raju cell after another (e.g., secondary/tertiary neosis in tumor-derived HTB11 cells) as opposed to "burst neosis" by which is meant production of Raju cells in rapid succession (e.g., during the initial stages of transformed foci formation of irradiated C3H10T1/2) .

By the "birth dance" is meant the contractile activity of the unborn Raju cell during the cleavage process, as indicated by rapid change in cell shape and size during the course of several hours (observed, for example, using digital video time lapse imaging, Fig. 7B) . It is thought that such contractile activity is related to the loading of the nascent nucleus and other cellular organelles or their precursors into the protruding plasma membrane destined to become the daughter Raju cell.

By "giant neosis mother cell" is meant a cell an abnormally large neosis mother cell that is rich in cytoplasm with a large polyploid nucleus .

By "multinucleate giant neosis mother cell" is meant a cell that is two or more times larger than the majority of the cells in a give population, and that contains two or more nuclei .

As used herein, by "nucleic acid molecule" is meant a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides or ribonucleotides . The nucleic acid may be free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the gene. The term, therefore, includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

By "modulates" is meant either increasing or decreasing the number of cells that undergo neosis or endoapoptosis in a given cell population. Preferably, the cell population is selected from a group originating from different cell types, including cancer cells (e.g., ovarian cancer cells, breast cancer cells, pancreatic cancer cells) , leukemic cells, lymphoma cells, T cells, neuronal cells, fibroblasts, or any other cell line known to proliferate in a laboratory setting. It will be appreciated that the degree of neosis or endoapoptosis modulation, provided by an neosis- or endoapoptosis-modulating compound in a given assay will vary, but that one skilled in the art can determine the statistically significant change in the level of neosis or endoapoptosis that identifies a compound, for example, a nucleic acid molecule, that increases or decreases neosis or endoapoptosis. Preferably neosis or endoapoptosis is decreased by at least 20%, more preferably, by at least, 40%, 50%, or 75%, and, most preferably, by at least 90%, relative to a control sample that was not administered a neosis- or endoapoptosis-modulating test compound. Also as used herein, preferably neosis or endoapoptosis is increased by at least 1.5-fold to 2-fold, more preferably, by at least 3-fold, and most preferably, by at least 5-fold, relative to a control sample that was not administered a neosis- or endoapoptosis- modulating test compound.

By "biological sample" is meant a tissue biopsy, cells, blood, serum, urine, stool, or other specimen obtained from a patient or test subject. The sample is analyzed to detect the presence of neosis mother cells, Raju cells, or endoapoptotic cells, by methods described herein.

By a "compound," "test compound," or "candidate compound" is meant a chemical molecule, be it naturally- occurring or artificially-derived, and includes, for example, peptides, proteins, synthetic organic molecules, naturally- occurring organic molecules, synthetic or naturally-occurring non-organic molecules, nucleic acid molecules, and components thereof . By "inhibiting tumor progression" is meant decreasing the rate at which a tumor grows and/or metastasizes, or decreasing the rate or likelihood of initial tumor occurrence or of tumor reoccurrence, for example, after tumor therapy. Preferably the rate of tumor progression is decreased by at least 20%, more preferably, by at least, 40%, 50%, or 75%, and, most preferably, by at least 90% when the tumor is contacted with a candidate compound that inhibits tumor progression, compared to a tumor that is not contacted with the candidate compound. Tumor progression can be measured, for example, by measuring the presence of neosis mother cells, Raju cells, or endoapoptotic cells, in a biological sample, wherein a decrease in the number of neosis mother cells, Raju cells, or endoapoptotic cells in the sample, for example, in response to exposure to a candidate compound indicates inhibition of tumor progression.

By "proliferative disease" is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of cell death, or both. For example, cancers such as lymphoma, leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, and lung cancer are all examples of proliferative disease. Another example of a proliferative disease is a myeloproliferative disorder.

By "neoplastic disease" is meant a condition characterized by abnormal cell growth that can occur even in the absence of a stimulus for cell growth. Tumors are examples of neoplastic diseases.

Brief Description of the Drawings

Figs. 1A-1C are schematic representations of endoapoptotic bodies as a result of the initiation of intracellular destruction at different times during mitosis. Dark nuclei represent the dying genomes, while the viable genomes are represented by the stippled nuclei.

Fig. 2 is a schematic representation of the events that occur during genotoxin-induced transformation of a normal cell and its progression towards malignancy by neosis.

Fig. 3A is a schematic representation of how a normal cell undergoes primary and secondary/tertiary neosis to contribute to tumorigenesis and tumor progression.

Fig. 3B is a schematic representation of how cell fate after genetic damage contributes to tumorigenesis and tumor progression through secondary/tertiary neosis.

Fig. 3C is a flow chart of neosis as a paradigm for multistep tumor progression.

Fig. 4A is a light microscope image of a multinucleate giant cell (MuNGC) on day 15 after exposure to etoposide (20 μg/ml for 48 h) , giving rise to two post-crisis mononucleate cells.

Fig. 4B is a fluorescence microscope image of a C3H10T1/2 MuNGC, stained with HO and PI on day 30 after etoposide treatment (20 μg/ml for 48 h) , giving rise to at least 7 small Raju cells. The arrow points to a nascent Raju cell in the process of performing the "birth dance" or the contractile activity aimed at detachment from the neosis mother cell.

Figs. 4C-4F are fluorescence microscope images of the spontaneous transformation of p53-/-MEF/MGB (mixed genetic background) cells. Fig. 5A is a series of video images of the spontaneous transformation of p53-/- MEF/MGB cells.

Fig. 5B is a series of video images showing the synchronous cytokinesis of the entire cytoplasm of a p53-/- MEF/MGB MuNGC giving rise to more than 70 cells within one day.

Fig. 6A is a drawing of a neosis mother cell and its progeny on post-irradiation day 17 at 9:00 a.m.

Fig. 6B is a video image ^rof the cell of Fig. 6A on the same day at 12:54 p.m.

Fig. 6C is a series of images from a video time lapse study of the cell of Fig. 6B, showing subsequent events.

Fig. 6D is a pedigree analysis of the cell of Figs. 6B and 6C.

Fig. 7A is a series of video images of HTB-11 cells undergoing neosis.

Fig. 7B is a series of video images of HTB-11 cells undergoing the birth dance .

Fig. 7C is a drawing of the birth dance process depicted in Fig. 7B. The emerging Raju cell around the peri- nuclear area in Fig. 7B has been traced and shown in line drawings, highlighting morphological changes of the unborn Raju cell during cytokinesis. Red lines indicate the plasma membrane boundaries of the neosis mother cell; the green lines indicate the ever-changing plasma membrane boundaries of the unborn Raju cell (arrows) ; and the blue line indicates the nucleus of the neosis mother cell . The green dotted domain corresponds to the cytoplasm of the unborn Raju cells. Fig. 7D is a set of video images showing a Raju cell undergoing mitosis.

Fig. 7E is a set of video images showing Raju cells gaining cell mass.

Fig. 8 is a series of images showing morphological variations and differences in the frequency of spontaneous multinucleate giant cell formation of four different etoposide-induced (20 μg/ml for 48 h) neotic colonies.

Fig. 9 is a series of images showing the clonogenic potential of Raju cells.

Fig. 10 is a set of fluorescence microscopy images of various stages of HTB-11 cells undergoing neosis. Figs. 10A, 10D, and 10G are photographs of the cells as viewed by light microscopy; Figs. 10B, 10E, and 10H were viewed by light and fluorescence microscopy; and Figs. 10C, 10F, and 101 were viewed by fluorescence microscopy alone. These cells were viewed using Filter No. 02.

Fig. 11 is a set of fluorescence microscopy images of various stages of HTB-11 cells undergoing neosis. Figs. 11A, 11D, and 11G are images of the cells as viewed by light microscopy; Figs. 11B, HE, and 11H were viewed by light and fluorescence microscopy; and Figs. 11C, HF, and HI were viewed by fluorescence microscopy alone. These cells were viewed using Filter No. 02.

Fig. 12 is a set of fluorescence microscopy images of various stages of p53-/- MEF/129B cells undergoing leaky neosis. Figs. 12A, 12D, and 12G are photographs of the cells as viewed by light microscopy; Figs. 12B, 12E, and 12H were viewed by light and fluorescence microscopy; and Figs. 12C, 12F, and 121 were viewed by fluorescence microscopy alone. All cells, except those shown in 121, were viewed using Filter No. 02. The cells of Fig. 121 were viewed using Filter No. 09.

Fig. 13A is a photograph of human metastasizing neuroblastoma-derived HTB-11 cells undergoing neosis (40X magnification) .

Fig. 13B is a photograph of a magnified view (100X magnification) of human metastasizing neuroblastoma-derived HTB-11 cells undergoing neosis.

Fig. 13C is a photograph of a micro coverslip with

Raju cells (lower left) adjacent to another micro coverslip (upper micro coverslip) with mitotic cells on it (40X magnification) .

Fig. 13D is a photograph of an isolated coverslip from Fig. 13C containing Raju cells (100X magnification) .

Figs. 14A-14I are images of HTB-11 cells before capture using laser capture microdissection (LCM) (Figs. 14A, 14B, and 14C) and after capture using LCM (Figs. 14D, 14E, and 14F) , while Figs. 14G, 14H, and 141 are images of the captured cells on the cap.

Fig. 15 is a dot plot of the distribution of HTB-11 cells sorted by a cell sorter.

Fig. 16A is an image of HTB-11 cells before cell sorting.

Fig. 16B is an image of Raju cells immediately after sorting of the HTB-11 cells.

Fig. 16C is an image of the Raju cells of Fig. 16B 18 h after growing in culture. Fig. 16D is an image of HTB-11 larger cells (potential neosis mother cells) immediately after sorting.

Fig. 16E is an image of the HTB-11 larger cells of Fig. 16D 18 h after growing in culture.

Figs. 17A-17C are scanned images of autoradiograms of agarose gels after RT-PCR differential display (DD-RT-PCR) analysis of C3H10T1/2 cells before and during exposure to etoposide, as well as the neotic progeny 1ET1 cells, derived from the etoposide-treated C3H10T1/2 cells. RNA from these cells was used in three different DD-RT-PCR analyses with three different primer sets TI and P2 (Fig. 17A) ; T4 and P4 (Fig. 17B) ; and T2 and P4 (Fig. 17C) . Lane 1, nucleic acids from C3H10T1/2 cells before exposure to etoposide; Lane 2, nucleic acids from C3H10T1/2 cells at the end of 48 h exposure to etoposide; Lane 3, nucleic acids from C3H10T1/2 cells on Day 4 after exposure to etoposide;. Lane 4, nucleic acids from C3H10T1/2 cells on Day 8 after exposure to etoposide; Lane 5, nucleic acids from C3H10T1/2 cells on Day 12 after exposure to etoposide; Lane 6, nucleic acids from etoposide-transformed neotic clone 1ET1 cells. Arrows point to the prominent alterations in the gene expression profiles during and after exposure to etoposide and in the etoposide-induced neotic progeny: UR, upregulation; DR, down regulation; M, missing bands; N, new bands.

Fig. 18A is a schematic representation of methods of calculating classical nucleus/cytoplasmic ratios (N/C ratios) and true nucleus/cytoplasmic ratios (T-N/C ratios) . Cells were stained with HO/PI and examined with a fluorescence microscope. The cells of interest were photographed using a digital camera operated by an ISee Imaging software (Inovision Corporation, Raleigh, N.C.). Using the same software, the areas of the nucleus (N) and the entire cell (C) were measured as the number of pixels in a selected area. A survey of literature indicated that the classical mode of calculation nucleus/cytoplasmic ratio (N/C ratio) equals the area or volume of the nucleus, divided by the area or volume of the entire cell, such that the value will never be more than one. In other words, the classical N/C ratio is actually the nucleus/cell ratio and not a true nucleus/cytoplasm ratio (Hamilton et al . , Histopathol . 11:901-911, 1987; Allen et al . , Histopathol. 11:913-926, 1987; Tsuda et al . , Br J Cancer

75:1519-1524, 1997; Longchampt et al . , Acta Cytol . 44:515-523, 2000) . Therefore, we proceeded to compute true nucleus/cytoplasmic (T-N/C) ratios using the formula: true nucleus/cytoplasmic ratio equals the area or volume of the nucleus divided by the entire cell volume minus the nuclear volume (T-N/C = N/C-N) .

Figs. 18B and 18C are histograms and tables with absolute numbers showing data on the classical N/C ratio and the T-N/C ratio for nascent Raju cells, maturing Raju cells, normal (Fig. 18B) or neoplastic cells (Fig. 18C) and neosis mother cells and for two cell systems: NIH3T3, a "normal' established cell line undergoing neosis after exposure to N- methyl N-nitroso Guanidine (MNNG) (Fig. 18B) and human metastasizing neuroblastoma derived HTB11 cells undergoing spontaneous neosis (Fig. 18C) . The rate of maturation of Raju cells, as well as the final cell size after maturation may vary between the progenies from different neosis mother cells as well as between different tumor cell types. Overall, the Raju cell N/C ratio is very high, on the order of 0,7 by the classical formula, and above the value of 1.0 by the T-N/C ratio computed by us. The value of the classical N/C ratio or

T-N/C ratio should be viewed in conjunction with the cell size

(e.g., 8-15 μM in diameter) . The N/C ratio or T-N/C ratio by itself should not be viewed as a characteristic of the Raju cells in isolation, since this may overlap the respective ratios of mitotic cells immediately after the completion of mitosis .

Figs. 19A-19G show images of cells stained with

5BUdR. Fig. 19A is an image of a cell showing 5BUdR incorporation in the viable nucleus (arrow 1) , while the other nucleus is undergoing endoapoptosis (arrow 3) . Arrow 2 points to 5BUdR incorporation in the endoapoptotic nuclear fragments. Figs. 19B-19E are images of cells labeled with 5BUdR undergoing metaphase (Fig. 19B) , early anaphase (Fig. 19C) , and telophase (Figs. 19D and E) , respectively. Figs. 19D and 19E are the same cell in light/fluorescence microscopy and in fluorescence microscopy alone, respectively. Fig. 19F is an image of HeLa cells that were irradiated with 9 Gy X- radiation. Three days later, these cells are multinucleate (DNA fluorescing blue after DAPI staining) and display endoapoptotic nuclei (those without blue fluorescence) . Fig. 19G is an image of a multinucleate giant HeLa cell undergoing neosis.

Figs. 20A-20D are images of HeLa cells stained for expression of cyclin dependent kinase inhibitors (CKIs) as negative markers for neosis. Fig. 20A is an image of a multinucleate HeLa cell formed 5 days post irradiation. The CKI, pl6^{I K4a}, is expressed specifically in a dying

(endoapoptotic) nucleus of the multinucleate HeLa cell. Fig. 20B is an image of cell a HeLa cell showing that occasionally pl6^INKa is expressed in the cytoplasm of live cells (arrow 1) . Expression of pl6^IN4a is also occurring in a dead cell (apoptosis) (arrow 2) . Fig. 20C is an image of dead mini cells produced by a giant HTB11 cell formed 4 days after exposure to 8 Gy X-radiation and stained with DAPI stain. Fig. 20D is an image of the expression of p2ι^WAF1/CIP1 i_{n a} non- viable nucleus of a multinucleate giant HeLa cell formed after exposure to 9 Gy X-radiation.

Figs. 21A-21H show the pattern of SA-β-gal stain in multinucleate giant cells (potential neosis mother cells) spontaneously occurring in X-REF cells (an established rat embryo fibroblast cell line) . Fig. 21A is an image of a multinucleate giant cell showing that the cytoplasm all around the four nuclei are stained positively for SA-/3-gal, indicating that this probably is early stage expression of SA- jβ-gal, and may eventually lead to a situation similar to that seen in Figs. 21B and 21C, with more intense SA- -gal staining. Fig. 21D is an image of a giant multinucleate cell undergoing apoptosis and staining positively for SA- -gal. Figs. 21E-21H are images of multinucleate giant cells showing SA-jS-gal expression restricted to one or two nuclei, while other nuclei in the same cell are negative for SA-/3-gal.

Figs. 22A-22C are Schematic representations of the mode of mitotic spindle formation during yeast mitosis, mammalian neosis, and mammalian cell mitosis.

Detailed Description of the Invention

By following cellular events during transformed foci formation, we have discovered that when normal or neoplastic cells are under genetic duress, such as exposure to genotoxins, they undergo a novel type of cell division leading to tumorigenesis (Fig. 2) . Similar non-mitotic cell division occurs spontaneously in tumor cell populations, due to the genomic instability-induced genetic damage, and also occurs after exposure of tumor cell populations to genotoxins, such as etoposide and X-radiation. After exposure of cells to genotoxins, some cells die by apoptosis; some cells may undergo terminal proliferation arrest (or cytostasis) ; and some cells slowly enter S phase and get arrested in G2/M phase, unable to complete mitosis, and then become polyploid/multinucleate giant cells. Eventually a fraction of these giant cells undergoes neosis, which, unlike mitosis, is characterized by a special kind of karyokinesis involving nuclear budding without dissolution of the nuclear envelope. This is followed by a novel type of intracellular asymmetric cytokinesis involving cytoplasmic budding and cleavage by prolonged contractile motion of the emerging daughter cell, termed the "birth dance." This novel type of cell division is called neosis.

Neosis gives rise to neoplastic population of mononuclear cells with altered genotype and phenotype.

Natural selection ensures survival of the fit. Increasing genetic instability through mitosis triggers the next round of neosis and natural selection leading to heterogeneity and tumor progression.

Unlike mitosis, neosis gives rise to immature daughter cells, termed Raju cells. Raju cells appear to be immature at birth, as evidenced by (a) their small size (8-15 μm in diameter) ; (b) a very thin film of cytoplasm; (c) no distinguishable nuclear envelope in phase contrast microscope; (d) highly condensed chromatin; (e) the absence of a visible nucleolus at birth; and (f) their ability to be histologically stained by HO/PI stains, but displaying differential staining (less readily stainable compared to other cells in the population) with Giemsa stain due to the condensed nature of the chromatin in the cells. Raju mother cells, or neosis mother cells (NMC) , produce one to an indefinite number of Raju cells through neosis, and these Raju cells are further characterized by genomic instability, and indefinite mitotic division potential. Raju cells also display a genotype and phenotype different from the mother cell, grow in size by gaining cell mass, and are potentially tumorigenic. Since Raju cells are produced by a genetically damaged neosis mother cell that is not viable, the chromatin is repackaged to yield viable Raju cells and their mitotic derivatives, with altered gene expression profiles, compared to the neosis mother cell.

While primary neosis (the first neosis of a normal cell) may be the first step in the long process of tumorigenesis, secondary or tertiary (S/T) neosis contributes to tumor progression, as shown in Figs. 3A, 3B, and 3C. Fig. 3A shows the steps involved in secondary and tertiary neosis, depicted schematically in Fig. 3B. The neotic progenies from different neosis mother cells have different phenotypes and genotypes since often the genetic damage occurs in a random fashion. The neotic progeny inherits genomic instability and, depending upon the degree of genomic instability, the descendants of each neosis mother may cell undergo secondary/tertiary neosis sooner or later. Neosis results in genotype and phenotype heterogeneity of the tumor cell population, and also leads to natural selection due to the microenvironment { in vitro or in vivo) , thus contributing to tumor progression. In such circumstances, the cell population that successfully competes due to its mitotic division potential is selected for.

Fig. 3C is a flow chart depicting neosis as a paradigm for multistep tumor progression. This flow chart shows the various cellular events that occur during primary and secondary/tertiary neosis. Each occurrence of neosis extends the mitotic life span of the offspring, and thus, the cells are immortal. Since these cells inherit genomic instability, this immortality will inevitably result in the accumulation of random genetic defects leading to neosis and repackaging of the chromatin to produce the next generation of neotic progeny.

Tumor cells can also undergo spontaneous neosis during tumor progression or can be induced to undergo neosis after exposure to genotoxin-based anti-tumor therapy, resulting in tumor recurrence. This has been observed in many unrelated cell types (Table 1) . Thus, neosis plays a significant role in tumorigenesis, tumor progression, and recurrence of tumor growth after anti-tumor therapy.

Table 1. Cell types that are capable of undergoing neosis

Neoplastic cell lines and fresh tumor biopsies also display a population of cells that undergo a novel form of apoptosis without cell death, termed endoapoptosis. During the process of endoapoptosis, one or more non-viable daughter nuclei, surrounded by a thin film of cytoplasm, are intracellularly destroyed by a process that shares many features with classical apoptosis. Neosis is characterized not only by the birth of reproductively viable cells that are genotypically different from the mother cell, as described above, but is often accompanied by the generation of endoapoptotic cells. As opposed to classical apoptosis, which results in the death and degradation of the cell, endoapoptosis of a non-viable genome favors the survival and propagation of the viable genome either via mitosis or neosis. Endoapoptosis conserves living matter and restores some degree of genomic stability to the surviving nucleus (genome) after spontaneous or genotoxin-induced perturbations in the genome dynamics of cells, and helps the cell escape from the termination of the cell lineage by apoptosis.

The present invention overcomes the problem of characterizing the cellular mechanism for the origin of transformed colonies, and is based upon the discovery of neosis, as well as endoapoptotic and Raju cell production. Described below are methods for isolating endoapoptotic cells, neosis mother cells, and Raju cells, and for enriching cell populations for these types of cells. The present invention further describes molecular methods for the characterization of the nucleic acid molecules responsible for endoapoptosis, neosis, and Raju cell production, as a means for identifying gene(s), gene product (s), modulator binding targets, or other molecular regulatory mechanisms for these novel cell types and cellular phenomena.

The present invention is a useful means for discovering and developing drugs to suppress endoapoptosis and neosis as a novel approach to anti-tumor therapy, and to avoid tumor recurrence after genotoxin-based anti-tumor therapy in a subject. The present invention can also be used for novel gene therapy, and for the development of other anti-cancer therapeutic drugs and treatment modalities. The cell isolation methods of the present invention can be employed in a variety of drug candidate screening assays to identify compounds that modulate neosis or endoapoptosis. The nucleic acid molecule characterization methods of the present invention can be employed in a variety of molecular target characterization and target validation techniques against which candidate modulation drugs can be designed and/or tested. It should also be recognized by one skilled in the art of cancer treatment modalities, that there are other useful applications of the present invention in a broader field of cell biology including, but not limited to cell cycle control, cell proliferation, cell differentiation, cell death, infection, inhibition of the origin of antibiotic-resistant bacteria, inflammation, and immunity.

The following examples are to illustrate the invention. They are not meant to limit the invention in any way.

Example 1: Neosis occurs during etoposide-induced transformation of C3H10T1/2 cells

C3H10T1/2 cells were treated with etoposide (20 μg/ml for 48 h) . After etoposide exposure, the cells were cultured. On Day 15 after etoposide exposure, a C3H10T1/2 cell had become a multinucleate giant cell (MuNGC) giving rise to two post-crisis mononucleate Raju cells (Fig. 4A) . On Day 30 after etoposide treatment, another C3H10T1/2 MuNGC gave rise to at least half a dozen small Raju cells (Fig. 4B) . Example 2: Spontaneous transformation of p53-/-mouse embryo fibrόblasts

p53-/-mouse embryo fibroblasts (MEF) /MGB (mixed genetic background, obtained from Dr. Tyler Jacks, Massachusetts Institute of Technology) were cultured using standard culture conditions. As shown in Fig. 4C, MuNGCs formed spontaneously in these p53-/- MEF/MGB cells, giving rise to a post-neotic population of mononucleate Raju cells, some of which underwent endoapoptosis (maroon fluorescence due to PI stain, black arrow head) , while others remained viable (blue fluorescence due to HO stain) . Some cells also developed into atypical multinucleate cells, some of which show pulverized nuclei (white arrow; Fig. 4C) . Figures 4D and 4E show fluorescence and light/fluorescence images of the same MuNGC in different magnifications displaying the emergence of five post-crisis mononucleate Raju cells, of which one is undergoing endoapoptosis (maroon fluorescence) , and the others are viable (blue fluorescence) . Figure 4F shows a MuNGC giving rise to a cluster of mononucleate Raju cells via neosis.

Example 3: Video analysis of the spontaneous transformation of p53-/- MEF/MGB cells

The spontaneous transformation of p53-/- MEF/MGB cells was also videotaped (Fig. 5) . During spontaneous transformation, the cells became extremely large and multinucleate, and these neosis mother cells eventually gave rise to one to an indefinite number of tiny immature neosis daughter cells (Raju cells) .

Frame 1 of Fig. 5A is a video image of a p53-/- MEF/MGB MuNGC at 12:18 p.m., giving rise to a post-crisis population of small mononuclear Raju cells. The arrow on the left poififts to the asymmetric cytokinesis resulting in the surface emergence of a small mononuclear cell. In addition, another small cell is emerging just above the small mononuclear cell. The arrow on the right in Frame 1 of Fig. 5A points to a mini cell (Raju cell) undergoing endoapoptosis. The line drawings in the corners of Frame 1 depict a diagrammatic sagittal view of the viable small cell (Raju cell) during cytokinesis (lower left) and of the endoapoptotic non-viable Raju cell (upper right) .

Frame 2 of Fig. 5A is an image of the same cell in

Frame 1, at 8:58 a.m. the next day, producing more mini cells. Frames 3 through 7 of Fig. 5, show images of a time lapse sequence of the surface emergence of a small mononucleate cell from a MuNGC. The arrows point to the same area at different times from where the nascent cell in Frame 7 emerged. The times of the recordings of Frames 3 through 7 are: 10:35 a.m.; 12:34 p.m.; 1:04 p.m.; 1:22 p.m.; and 2:12 p.m., respectively, on the same day, indicating the emergence of a Raju cell took about 4 to 6 h. In a time lapse video such as this, it can be seen that the process of cytokinesis during Raju cell emergence is accompanied by violent contractile motions of the cell trying to detach itself from the cytoplasm of the giant cell; this phenomenon may also be related to the loading of the nascent nucleus and cellular organelles or their precursors into the new cell in the making and has been termed the birth dance.

Frames 8 through 11 (Fig. 5B) are a series of video images of the temporal sequence of events showing the synchronous cytokinesis of the entire cytoplasm of a p53-/- MEF/MGB giving rise to more than 70 Raju cells within one day. This particular MuNGC was about one third of the average size of MuNGCs in the petri plate, implying that an average size MuNGC is potentially capable of producing about 200 post- crisis mini (Raju) cells by repeated endomitosis, followed by karyokinesis through nuclear budding, and synchronized asymmetric cytokinesis or cellularization. In Frame 11 of Fig. 5B, the newly emerged post-crisis population of Raju cells are seen moving away from the multinucleate neosis mother cell in all directions, leaving the multinucleate cell with a highly reduced amount of cytoplasm. The nascent mini cells (Raju cells) do not show well defined nuclear envelopes until several hours later.

Example 4 : Neosis occurs during X-ray-induced transformation of C3H10T1/2 cells

C3H10T1/2 cells were exposed to 8 Gy of X-radiation and plated at a density of 1500 cells/10 cm tissue culture plate with DMEM plus 10% fetal calf serum (Gibco/BRL) and incubated in a humidified atmosphere at 37.5°C with 7% C0₂- The growth medium was changed every four days. About 30-40% of cells died by apoptosis by about days 4 to 6. By day 10 post-irradiation, the surviving cells were distributed far apart, and had become giant cells with one or more large polyploid nuclei.

Figure 6A is a line drawing of a neosis mother cell and its progeny on post-irradiation day 17 (9:00 a.m.). At this time, one giant multinucleate cell undergoing neosis, generating at least 4 mini daughter cells (cells numbered 1-4) with well defined nuclear envelopes, and four Raju cells showing no well defined nuclear envelope (cells numbered 5-8) . Another Raju cell that emerged later on (cell numbered 9) and three prospective daughter cells (cells numbered 10-12) are not completely separated from the neosis mother cell. Figure 6B shows a video image of the cell depicted in Fig. 6A at 12:54 p.m. the same day. Figure 6C is a series of images from a video time lapse study of the cells shown in Figs. 6A and 6B (beginning at 12:54 p.m. of the same day) to follow subsequent events. Frame 1 is the first partial image of the giant cell recorded at the beginning of the studies. Each frame was recorded every ten minutes and the numbers denoted at the lower left hand corner is an indication of time elapsed since the beginning of the study (e.g., 69 = 69-1x10 = 680 minutes) .

The giant multinucleate neosis mother cell of Fig. 6C actually shows two large polyploid nuclei connected by an isthmus, due to dicentric chromosome formation. This indicates that the cell has unsuccessfully attempted to undergo mitosis. According to the understanding prior to the present invention, this cell should undergo death by mitotic catastrophe and should not reproduce any further. It can be seen however, that this cell has undergone additional cycles of DNA synthesis (neotic DNA synthesis) and has produced several daughter cells (Raju cells) by a process other than mitosis, of which only three Raju cells (numbered 5 through 7 of Fig. 6A) survived and eventually multiplied. Frames 69, 84, and 94 show the shrinking of the giant cell and its eventual death around frame 180, leaving only three viable Raju daughter cells (cells numbered 5 through 7 of Fig. 6A) .

Frames 179 through 181 of Fig. 6C show that one of the daughter cells (cell number 5) has undergone mitosis producing two daughter cells (cells numbered 5.1 and 5.2), and in frame 226, another daughter cell (cell numbered 7) has divided by mitosis into cells numbered 7.1 and 7.2, yielding a clone of five cells. While the genome (nuclei) of the giant cell is not reproductively viable, the genomes of the neotic progeny are reproductively viable, indicating that these cells have inherited an altered genotype and phenotype, including repackaging (chromatin remodeling) of the genetically damaged (mutated) genome to yield a reproductively viable genome with mitotic division potential.

Figure 6D is a pedigree analysis of the of the above-described video time lapse study. This analysis shows the number of Raju cells originating from the neosis mother cell and their fate during the course of observation.

Example 5: Neosis occurs spontaneously in the human metastasizing neuroblastoma-derived HTB11 cell line:

Figures 7A-7E depict the process of neosis occurring spontaneously in HTB-11 metastasizing neuroblastoma cells. In Fig. 7A, video images of HTB-11 cells were recorded every 10 min starting at the day and time shown in Frame 1. The cell aligned north to south in the center of the field gives rise to a neotic progeny (Raju cell; arrow) at Frame 141. In Frame 174, the Raju cell has moved away, from the mother cell. Note that the Raju cell nucleus is not clearly visible in the early stages of its birth.

Figure 7B is a series of video images of an HTB-11 neosis mother cell showing the birth dance of a Raju cell after the emergence of two other Raju cells. In Frame 200 (at 10 x 200 min after the start of recording) , contractile activity can be seen in the peri-nuclear region; the tiny arrows in all the frames shown here point to the area of contractile activity in the neosis mother cell (NMC) from where at least two Raju cells emerged. The larger arrow in Frame 404 points to the newly emerged Raju cell, which was not present in previous Frame 403 recorded 10 minutes earlier.

The larger arrow shown in Frame 406 points to the second Raju cell that has emerged close by, and to the right of the first- emerged Raju cell, shown in Frame 404. The Raju cells shown in Frame 404 and 406 have moved away from the neosis mother cell on their own accord, as seen in Frame 550, unlike the cell debris that is lying static to the right of the Raju cell in Frame 404, which is persistent at the same location through the entire experiment. After the emergence of the two Raju cells (Frame 404 and 406) , the peri-nuclear region is still exhibiting active contractile movement through the end of the experiment (through Frame 595, arrows) indicating that this neosis mother cell may have produced more Raju cells through neosis, had the experiment been continued further.

Figure 7C is a schematic representation of the birth dance displayed by an emerging Raju cell in the peri-nuclear area of the neosis mother cell in Fig. 7B. The neosis mother cell shown in Fig. 7B has been traced and shown in line drawings, highlighting morphological changes of the unborn Raju cell during cytokinesis. Red lines indicate the plasma membrane boundaries of the neosis mother cell; the green lines indicate the plasma membrane boundaries of the unborn Raju cell; and the blue line indicates the nucleus of the neosis mother cell. The green dotted domain corresponds to the cytoplasm of the unborn Raju cells. This figure clearly shows the changing boundaries of the unborn Raju cell due to the contractile movement during asymmetric cytokinesis ("birth dance" ) .

Figure 7D is a series of video images displaying the mitotic potential of Raju cells. The tiny Raju cell at the lower left corner (arrow) rounds off and undergoes mitotic division into two daughter cells over a period of 1 hour and 40 minutes. The cell is very small compared to the neosis mother cell. Figure 7E is a series of video images of events that demonstrate that Raju cells eventually gain cell mass and increase in size over a duration of approximately 24 h.

Example 6: Morphological variations and differences in the frequency of spontaneous multinucleate giant cells formation of etoposide-induced neotic colonies

Morphological variations and differences in the frequency of spontaneous multinucleate giant cell formation of different etoposide-induced (20 μg/ml for 48 h) neotic colonies exist. We have isolated 30 such colonies (1ET1 through 1ET30), arising from different neosis mother cells.

Four of these colonies are shown in Fig. 8. When one of these colonies (1ET1) was exposed to etoposide again, some cells survived at a dose of 500 μg/ml for 48 h (the parental C3H10T1/2 cells did not survive at this concentration) , and underwent secondary neosis yielding several individual neotic clones (1ET1.1, 1ET1.2, etc...). The fact that 1ET1 cells were about 25 times more resistant to etoposide than the parent cells suggests a role for neosis as a mechanism for the recurrence of resistant tumor growth from a tumor tissue that was originally susceptible to a given anti-tumor therapeutic regimen.

Example 7: Raju cells have clonogenic potential

Raju cells of variable sizes (due to differences in age) gain cell mass and increase in cell size. In addition, most Raju cells are able to undergo mitotic division. Figure 9 is a series of digital video time lapse images of a group of Raju cells displaying clonogenic potential. Each image follows at least 10 such cells (numbered 1 to 10 in each of the frames shown) that increase in cell size and also undergo mitotic division, giving rise to a colony of cells. For example, cell number 4 divides two times and its mitotic daughter cells (identified as cell numbers 4.1 and 4.2) and the granddaughter cells as (identified as cell numbers 4.1.1 and 4.1.2) are shown in the appropriate frames. These results demonstrate that Raju cells have clonogenic potential.

Example 8: Use of fluorescent dyes to evaluate neosis

To more fully elucidate the cellular processes that occur during neosis, HTB-11 cells undergoing spontaneous neosis (tumor progression) and p53-/- MEF/129B cells (129 genetic background; obtained from Dr. Tyler Jacks, Massachusetts Institute of Technology) were seeded on glass cover slips and stained live with FITC-Annexin-V (AV) , Hoechst 33342 (HO), and Propidium Iodide (PI) (AV/HO/PI stain) and photographed under visible light alone, visible and UV light, or UV light, alone using a Zeiss Axioskop fluorescence microscope. This AV/HO/PI stain is extremely useful for quantitatively examining the plasma membranes of early apoptotic cells, and for differentiating between multinucleate neosis mother cells and mononucleate Raju cells.

AV stains the surface membrane of a cell if the cell is dead or in the process of dying. One of the early changes during cell death is the externalization of phosphatidylserine in the plasma membrane, which in live cells is distributed only in the inner leaflet of the plasma membrane. Annexin-V has a high affinity for phophatidylserine. When viewed using immunofluorescence microscopy with Filter No. 09, dead and dying cell plasma membranes fluoresce green. HO is a vital dye that penetrates live cells and intercalates into the DNA. When viewed under UV light with a long pass Filter No. 02, the nucleus fluoresces blue. PI is a dye that penetrates the nuclear envelope after it has lost its integrity and intercalates in the DNA. When viewed under UV light with a long pass Filter No. 02, dead nuclei fluoresce red.

In the presence of both HO and PI, dead cell nuclei fluoresce maroon under UV light with a long pass Filter No. 02, due to the presence of both blue and red colors. The nucleus of a cell in the early stages of cell death, however, exhibits more HO staining, and less PI staining; under these circumstances, the nucleus fluoresces white, and eventually turns maroon as more PI enters the nuclei.

HTB-11 cells undergoing spontaneous neosis (tumor progression) were stained with AV/HO/PI stain (Fig. 10) . Figs. 10A, 10D, and 10G are photographs of cells viewed using light microscopy; Figs. 10B, 10E, and 10H are photographs of the same cells viewed under both visible light and UV light; and Figs. 10C, 10F, and 101, are photographs of the same cells viewed under fluorescent light.

The cell in Fig. 10A has two nuclei, each displaying one prominent nucleolus. The upper nucleus has a nuclear protrusion or budding, as indicated by the blue fluorescence of DNA in 10B and 10C, but is not distinguishable in Fig. 10A using phase contrast microscopy. DNA also extends laterally to the left beyond the boundaries of the nuclear envelope visible in 10A. Thus, DNA in the bud-like structure is still within an envelope that is not visible under the phase contrast microscope, but is visible under UV light after staining the DNA with HO. Since these cells are still alive, PI did not enter the nuclei. These structures are thought to represent the early stages of unique karyokinesis, which is not accompanied by dissolution of the nuclear membrane, an important characteristic of neosis. Figures 10D-10F show a prospective neotic progeny undergoing the initial stages of cytoplasmic cleavage (arrows) after the completion of karyokinesis (nuclear division) via budding. The nucleus of the prospective daughter cell is fluorescing brightly due to the highly condensed nature of DNA. In addition, the nucleolus is conspicuous by its absence, suggesting that the nascent unborn Raju cell is transcriptionally inactive.

Figures 10G-10I show (arrows) the emergence of another Raju cell (note the highly condensed chromatin and no visible nucleolus) from the neosis mother cell via karyokinesis. The chromatin of the nascent would-be Raju cell is still contiguous with the chromatin of the neosis mother cell. Note the highly condensed chromatin in the Raju cell nucleus compared to that of the neosis mother cell.

Additional photographs of HTB-11 cells undergoing spontaneous neosis, and stained with AV/HO/PI stain are shown in Figs. 11A-11I. Figures 11A, 11D, and 11G are photographs of cells viewed using light microscopy; Figs. 11B, HE, and 11H are photographs of the same cells viewed under both visible light and UV light; and Figs. 11C and HF are photographs of cells viewed under fluorescent light, while Fig. HI was photographed in the dark field without UV light.

In Figs. 11A-11C, two neosis mother cells are displaying the emergence of Raju cells via cytoplasmic cleavage (arrows) . The chromatin (DNA) of the nascent Raju cell (upper arrow) is still contiguous with the DNA of the mother neosis cell nucleus. This indicates that the cytoplasmic cleavage process has been initiated before the completion of karyokinesis via nuclear budding (see Figs. 11B and 11C) . The daughter nucleus in the lower cell (lower arrow) has completed karyokinesis (see Fig. 11C) , but cytokinesis is still in progress (Fig. HA and 11B) . Note that this cytokinesis is unusual in that it is highly asymmetric such that the neosis daughter cells (Raju cells) are much smaller than the neosis mother cells.

Figures HD-llF show an unborn Raju cell in the process of the birth dance (arrow) . The emergence of the Raju cell occurs through nuclear budding followed by asymmetric intracellular cytokinesis, which is often attended by severe contractile motion of the nascent Raju cell termed the "birth dance."

Figures HG-llI show a group of approximately six Raju cells (small arrows) and two neosis mother cells (fat arrows) . The Raju cells are much smaller than the neosis mother cells at birth.

Figures 12A-12I show P53-/- MEF/129B cells with a 129 genetic background undergoing leaky neosis. These cells were stained with AV/HO/PI as described above. Figures 12A, 12D, and 12G are photographs of cells viewed using light microscopy; Figs. 12B, 12E, and 12H are photographs of the same cells viewed under both visible light and UV light; and Figs. 12C, 12F, and 121, are photographs of the same cells viewed under fluorescent light, except that 121 is a photograph taken with filter No. 09 to look at the green fluorescence of FITC-AV if the plasma membrane has lost is integrity.

Figures 12A-12C show a cell displaying nuclear budding, where the large nucleus has a lobed nature (arrow) .

Figures 12D-12F show a cell displaying an emerging bud that contains DNA (upper arrow pointing to the right) . In addition, what appears to look like a small cell (lower arrow pointing to the right) is also emerging from the large mother cell; staining with AV/HO/PI indicates that this possible cell does not have any nucleus or DNA, and therefore is a cytoplast.

Figures 12G-12I show a multinucleate giant cell undergoing mitotic catastrophe (cell death due to unsuccessful mitosis) . Note the two large nuclei, probably bridged via a dicentric chromosome (upper arrow) , are dying and taking in PI on top of HO, resulting in a white fluorescence (Fig. 12H) . When examined with Filter No. 09 to detect externalized phosphatidylserine with FITC-Annexin V (AV) , no green fluorescence is detected in the cell membrane, but fluorescence in the nucleus indicates PI penetrating the dying nucleus (Fig. 121) . Note that the white fluorescent patches in Fig. 12H are overlapping the red fluorescence of PI in Fig. 121). This indicates: (1) that PI penetrates the intact live cell plasma membrane, but not the intact nuclear membrane; (2) that this giant cell is in the process of dying; but (3) that this cell has produced one Raju cell with a viable nucleus that fluoresces blue with UV (Fig. 12G and 12H, lower arrows) and contains a viable nucleus, since PI did not enter the nucleus of the cell (Fig. 121) . It is also important to note that the emerging Raju cell is produced by the multinucleate giant cell before its death, thus ensuring the continuity of cell lineage. Example 9: Isolation of Raju cells through the use of a micro coverslip capture (MCC) technique

In vitro cell cultures of several types of normal or neoplastic cells of heterogeneous phenotype were obtained following exposure to genotoxins, and Raju cell populations were enriched or isolated using the following MCC techniques.

The established mouse embryo fibroblast cell line C3H10T1/2, human tumor derived cell lines ACHN (renal adenocarcinoma) , rat vascular smooth muscle cell line A10, HTB-11 (SK-N-SH, neuroblastoma, metastasis to bone) , HeLa (epithelioid carcinoma of the cervix) (all obtained from American Type Culture Collection ) , p53-/- MEF/MGB (mixed genetic background) and p53-/-MEF/129B (129 genetic background) (both obtained from Dr. Tyler Jacks of MIT) , X- REF23 (established rat embryo fibroblast; obtained from Dr.

D.L. Guernsey, Dalhousie University, Halifax, NS, Canada), and primary cultures of human foreskin fibroblasts ( (Hfsk) ; obtained from Dr. S.H.S. Lee, Dalhousie University, Halifax, NS, Canada) were used in these micro coverslip capture studies. All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with high glucose supplemented with 10% fetal calf serum (Gibco/BRL) , or new born calf serum (Gibco/BRL) without antibiotics and antimycotics, in a humidified water jacketed incubator at 37.5°C with 7% C0₂.

To prepare micro coverslips, glass coverslips of 1-2 mm² were cut using a diamond knife and washed with agitation thoroughly in distilled water to remove the adhering minute glass particles. The micro cover slips were rinsed 3 times with 80% ethanol and sterilized by autoclaving at 120°C and 15 PSI of pressure for 45 min in a glass petri plate wrapped in aluminum foil. The sterilized micro cover slips were evenly and aseptically spread in 10 cm bacterial plates and a known number of cells were seeded using 10 ml DMEM supplemented with 10% FBS. The cells were allowed to attach to the glass micro cover slips by incubating the plates in a water jacketed cell culture incubator maintained at 37°C and 7% C0₂. The cells attached on the micro cover slips were monitored every day until the desired Raju cell phenotype was formed. Those micro cover slips having a single cell of the Raju cell phenotype were carefully lifted using sharp forceps and placed in the well of a 48 well tissue culture plate with growth medium for multiplication of the cell and further characterization, if so desired. For downstream molecular characterization, as described in detail below, the micro cover slips with desired phenotypes were directly used for extraction of RNA followed by gene expression analysis. Since the desired cell types were selected and isolated for further characterization from a heterogenous cell population using micro cover slips, this method is called micro cover slip capture (MCC) .

While some cells (e.g., those that are genomically unstable, such as HTB-11 cells, ACHN cells, and p53-/- mouse embryo fibroblasts) undergo spontaneous neosis, all cells can be induced to undergo neosis upon exposure to X-rays or chemical compounds, for example, etoposide or N-methyl-N- nitro-N-nitrosoguanidine (MNNG) . To initiate neosis, an external stimulus was applied according to the following methods. Cells seeded in 10 cm petri plates or on glass cover slips were exposed during their exponential phase of growth to 7-9 Gy units of X-rays, at a rate of 3.5 cGys/s, with a Siemens X-ray unit or with a Theratron X-ray unit.

Immediately after irradiation, the medium was changed, the cells were rinsed two times with DMEM, and fresh media was then added. In other experiments, etoposide was used as an alternative genotoxin. Cells seeded as described above were exposed to 20 μg/ml etoposide (dissolved in dimethylsulfoxide) in the growth medium for 48 h and then grown in regular culture medium; preliminary experiments using carrier controls did not show any tumorigenic potential at the concentration used (0.01%) .

Figures 13A-13C show human metastasizing neuroblastoma derived HTB-11 cells undergoing neosis. The cells were seeded on sterile glass coverslips and allowed to multiply under standard culture conditions. After 1 week, the glass coverslips were examined under the microscope for the status of cell growth and for the presence of neotic progenies (Raju cells; Fig. 13D) and photographs were taken using a Coolpix digital camera.

Figure 13A shows the HTB-11 cells growing on a typical glass micro coverslip (G) photographed with 40X magnification, and Fig. 13B shows a magnified view of the same cells photographed at 100X magnification. Figure 13C (40X magnification) shows a micro coverslip with Raju cells (lower left) adjacent to another micro coverslip (upper micro coverslip) with mitotic cells on it.

Figure 13D shows an isolated coverslip from Fig. 13C containing Raju cells photographed with 100X magnification. These micro coverslips are then transferred to a separate culture vessel for subsequent growth and expansion of the isolated cell.

Example 10: Use of laser capture microdissection (LCM)

To prepare cells for isolation of Raju cells by laser capture microdissection (LCM) , HTB-11 cells undergoing neosis were seeded on flame-sterilized microslides in 10 cm bacterial plates, using DMEM supplemented with 10% FBS. The microslides having the desired Raju cells (based on cell size and morphology) were rinsed 3 times with serum free DMEM and the cells were fixed with 70% ethanol for 5 min at room temperature. Subsequently, the cells were dehydrated with 80%, 95%, and 100% ethanol (30 seconds each) followed by exposure to Xylene 1 (2 times 1 minute) . The cells were then air dried for at least 10 min before laser capture microdissection.

Raju cells were captured onto CapSure™ (Arcturus Engineering Inc., Mountain View, CA) capture film using a PixCell II laser capture microdissection system (Arcturus Engineering Inc., Mountain View, CA) . Parameters for LCM were a 7.5 μm laser spot size, 80 mW power, and 3.5 ms duration. The images of cells before capture (Figs. 14A, 14B, and 14C) and after capture (Figs. 14D, 14E, and 14F) are shown along with the captured cell on the cap (Figs. 14G, 14H, and 141).

The isolated Raju cells are then used for RNA isolation for the molecular characterization of Raju cells, if so desired.

Example 11: Isolation of Raju cells through the use of a Percoll gradient

Raju cells are harvested with 0.25% trypsin, rinsed three times with DMEM supplemented with 10% FBS, and resuspended in 2 ml of HBSS containing 1% FBS. The cell suspension is passed through a 21 gauge needle to break the cell clumps .

Percoll (Pharmacia) having a density of 1.129 g/ml and an osmolality of 12 mOs/Kg H₂0 is made into 90% Percoll by mixing it with 10% Hanks balanced salt solution (HBSS) containing 10% FBS. This 90% Percoll is used to make 60%, 50%, 40%, 30%, and 20% Percoll solutions by mixing it with the required amount of HBSS plus 1% FBS. A discontinuous gradient is prepared by layering 2 ml of each of the above solutions in 15 ml clear centrifuge tubes. A prepared single cell suspension (2 ml) is layered over the top layer of Percoll and centrifuged at 10,000 rpm for 5 min using a swinging bucket rotor. At the end of centrifugation, the different layers of cells from the tube are drawn out using a long syringe, and rinsed 3 times with HBSS plus 1% FBS to remove Percoll. The rinsed cells are separately seeded in 10 cm culture plates and cultured for further observation of Raju cell phenotypes. Part of the rinsed cells are used to prepare cytocentrifuge slides, which are stained with a mixture of FITC-Annexin-V

(AV) (Roche) , Hoechst 33342 (HO) (Sigma) and propidium iodide (PI) (Sigma), as described above. Once a Raju cells is identified, it is isolated according to the above-described methods .

Example 12: Enrichment of Raju cells through the use of flow cytometry

HTB-11 cells undergoing neosis were grown in large tissue culture flasks (185 cm²) containing DMEM growth medium supplemented with 10% fetal calf serum (FCS) . One hour prior to cell sorting, the cells were harvested using 0.25% trypsin, washed three times with growth medium, and resuspended at 200,000 cells per ml of warm growth medium (37°C) previously gassed with C0₂. The cells were then sorted using Hank's Balanced Salt Solution (HBSS) as sheath fluid in a FACSort cell sorter (Beckton Dickinson) . The gates in the cell sorter were adjusted to capture Raju cells that are typically of between 5 and 15 μm in diameter; larger cells were rejected. Sorted cells were concentrated by centrifugation, washed three times with growth medium, and reseeded on sterile microslides. The cells were then allowed to attach overnight under normal growth conditions.

Figure 15 shows a dot plot of the distribution of

HTB-11 cells in a linear scale, the gate R2 to select Raju cells, and the gate R3 to isolate an enriched population of neosis mother cells. The parameters used to select the larger neosis mother cells vary according to the cell type, with the larger cells usually falling at the upper right hand corner of the dot plot.

Figures 16A-16E show HTB-11 cells before sorting (Fig. 16A) , Raju cells immediately after sorting (Fig. 16B) and 18 h after sorting (Fig. 16C) , HTB-11 larger cells (potential neosis mother cells) immediately after sorting

(Fig. 16D) and 18 h after growing in culture (Fig. 16E) . In this system, the neosis mother cells are not of uniform size; therefore, further identification of neosis mother cells may be required. The potential neosis mother cells can be identified by staining with HO/PI and looking for lobed nuclei (Fig. 11) or by further incubation of these cells to verify the frequency of neotic colony formation.

If desired, the population of enriched Raju cells are used to isolate Raju cells according to the isolation methods described above.

Example 13: Isolation or enrichment of neosis mother cells

Neosis mother cells are isolated or enriched using MCC or LCM techniques, flow cytometry, or Percoll gradients, according to the above-described methods. Preferably neosis mother cells are isolated by capturing them by MCC or LCM during the process of Raju cell emergence.

Example 14: Isolation or enrichment of endoapoptotic cells

Endoapoptotic cells are isolated or enriched using either the MCC technique or the LCM procedure, according to the above-described methods, based on the presence of the endoapoptotic body within a given cell, as described above (Fig. 1) .

Example 15: Generation of genomic or cDNA libraries from neosis mother cells, Raju cells, or endoapoptotic cells

Genomic DNA libraries or cDNA libraries can be generated from neosis mother cells, Raju cells, or endoapoptotic cells. Methods for generating such libraries are well known to one skilled in the art (see, for example, Ausubel et al . , Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1990; Sambrook & Russell, Molecular Cloning, A Laboratory Manual , Vol 1-3, Cold Spring Harbor Laboratory Press, 3rd Edition, 2001) . These libraries are useful for the molecular characterization of neosis mother cells, Raju cells, or endoapoptotic cells, and for identifying nucleic acids that modulate neosis.

Example 16: Molecular characterization of neosis mother cells, Raju cells, and endoapoptotic cells using differential display techniques

C3H10T1/2 mouse embryo fibroblasts were used to study the differential gene expression between the untreated control mitotic population of C3H10T1/2 cells and the neotic offspring that emerged after exposure to etoposide (20 μM in culture medium for 48 h) . Total RNA was extracted from the above population of cells using Trizol reagent (Gibco BRL) and RNA concentrations were determined spectrophotometrically. RNA samples were then treated with DNAse I enzyme to remove any contaminating DNA.

Reverse transcription reactions were carried out by combining 2 μg of RNA and 1 μl of 1 μM oligo dT (12-18 mer) in an RNAse-free microfuge tube. The sample was heat denatured at 70°C for 3 min and then chilled on ice for 2 min. A master mix containing 2 μl of 5X first strand cDNA buffer, 2 μl of 5 mM dNTPs, and 1 μl of M-MLV reverse transcriptase (Gibco BRL) was added to the heat denatured RNA/oligo dT mixture and the contents were incubated at 42°C for 60 min. The reaction was stopped by heat inactivating the reverse transcriptase enzyme at 80°C for 5 min. The single stranded cDNA obtained from the reverse transcription reaction was diluted to a final volume of 100 μl and stored at -20°C until the differential display reactions were carried out.

The differential display reactions were carried out as follows. A master mix was prepared by combining 3.3 μl of ddH₂0; 0.5 μl of 10X Klen Taq buffer (Clontech) ; 0.05 μl of 5 mM dNTPs; 0.05 μl of [ -³³P]dATP (2000 Ci/mmol) ; 0.25 μl of 20 μM 5' primer; 0.25 μl of 20 μM 3' primer; and 0.1 μl of 50X Advantage™ Klen Taq polymerase mix (Clontech) . Four and one- half microliters of this master mix was added to PCR tubes containing 0.5 μl of the diluted single-stranded cDNA. PCR was carried out using the following conditions: 94°C for 5 min; 40°C for 5 min; 68°C for 5 min; (94°C for 2 min; 40°C for 5 min; and 68°C for 5 min, repeated two times); (94°C for 1 min; 60°C for 1 min; 68°C for 2 min plus 4 sec/cycle, repeated twenty six times) ; 68°C for 7 min; and finally to 4°C, using a Lab-Line Programmable Thermal Block II thermal cycler. Gel electrophoresis of the amplified cDNA was carried out in a Genomix LR sequencer using a Tris-Boric acid- EDTA (TBE) buffer gradient and a 6% acrylamide, 8M urea gel at 50°C, 3000 V, and 100 W for 5 h. Four μl of heat denatured PCR product was run in each well until the xylene cyanol ran off the bottom of the sequencing gel. The urea in the gel was crystalized by directly drying the gel on the glass plate, and then the gel was rinsed until it became transparent and rehydrated. The gel was then transferred to Whatman 3M paper and dried to completion. The dried gel was exposed to X-ray film at room temperature overnight.

Figures 17A-17C show autoradiograms of RT-PCR- differential display analysis of untreated C3H10T1/2 parent cells before and after exposure to etoposide (20 μM for 48 h) , and of the neotic 1ET1 progeny cells derived from etoposide- treated C3H10T1/2 cells, showing the, differential expression of genes. RNA from these cells was used in three different DD-RT-PCR analyses with three different primer sets TI and P2 (Fig. 17A) , T4 and P4 (Fig. 17B) , and T2 and P4 (Fig. 17C) . Lane 1 shows nucleic acids from C3H10T1/2 cells before exposure to etoposide; Lane 2 shows nucleic acids from C3H10T1/2 cells at the end of 48 h exposure to etoposide; Lane 3, shows nucleic acids from C3H10T1/2 cells on Day 4 after exposure to etoposide; Lane 4, shows nucleic acids from C3H10T1/2 cells on Day 8 after exposure to etoposide; Lane 5, shows nucleic acids from C3H10T1/2 cells on Day 12 after exposure to etoposide; and Lane 6, show nucleic acids from etoposide-transformed neotic clone 1ET1 cells.

There were quantitative and qualitative changes in gene expression between the original C3H10T1/2 cells and the 1ET1 neotic derivative cells. Some genes were up regulated, some were down regulated, some were not expressed, and some new genes were expressed in the 1ET1 cells. The gene expression pattern in 1ET1 cells did not change even after several subcultures, indicating that in addition to gene mutations induced by the genotoxin (etoposide) , the genome has been subjected to epigenetic changes, probably via chromatin remodeling. This is consistent with the concept that epigenetic changes are caused by covalent modifications of the DNA, which result in stable but potentially reversible alterations of gene activity. Reversion may be induced due to genomic instability or environmental factors that might lead to further chromatin remodelling, contributing to tumor progression (Denko et al., Clin. Cancer Res. 6:480-487, 2000; Sugimura and Ushijima, Mut. Res. 462:235-246, 2000; Jones and Laird, Nat. Genet. 21:163-167, 2999; Heppner and Miller, Int. Rev. Cytol. 177:1-56, 1998).

Differentially expressed nucleic acid molecules can be recovered from the gel according to methods described, for example, by Ausubel et al. (supra).

This differential display technique is also used to identify nucleic acid sequences that are differentially expressed in endoapoptotic cells compared to other cells involved in neosis or to any other desired cell type.

Example 17: Molecular characterization of neosis mother cells, Raju cells, and endoapoptotic cells using gene array techniques

Gene array, cDNA microarray, or oligonucleotide microarray technologies are used to identify nucleic acids that modulate neosis. Nucleic acids from cells involved in neosis, for example, neosis mother cells, Raju cells, or endoapoptotic cells are isolated and labeled for comparison to labeled nucleic acids isolated from a different population of cells involved in neosis, or to normal cells that have not undergone neosis. Using gene array technology, the expression levels of many different nucleic acids are simultaneously examined and compared. Examples of methods for such expression analysis are described by Marrack et al. (Current Opinions in Immunology 12:206-209, 2000); Harkin (Oncologist 5:501-507, 2000); Pelizzari et al. (Nucleic Acids Res. 28:4577-4581, 2000); and Marx (Science 289:1670-1672, 2000).

Example 18: Molecular characterization of neosis mother cells, Raju cells, or endoapoptotic cells using a ribozyme library approach

Nucleic acids that modulate neosis are also identified using a library-based inverse genomics approach as described, for example, by Beger et al. (Proc. Natl. Acad. Sci. U.S.A. 98:130-135, 2001). The gene expression profiles of these cells are characterized in order to identify the gene(s) involved in the regulation and/or execution of neosis and endoapoptosis.

Example 19: Molecular characterization of neosis mother cells, Raju cells, or endoapoptotic cells using nucleic acid hybridization approaches

Comparative gene expression between two cell types is also studied by subtraction hybridization procedures, where the two cell types to be compared are termed tracer and driver cells, and mRNAs expressed in the tracer, but not the driver are isolated, for example, as described by Ausubel et al (supra) . cDNAs or mRNAs from one population are allowed to hybridize with an excess of complementary driver nucleic acid from a second cell population to ensure that a high percentage of the tracer forms hybrids. Hybrids that form include sequences that are common in both populations of cells. The unhybridized fraction is enriched for sequences that are preferentially expressed in the tracer cell population. Reciprocal subtractive hybridizations can be performed in order to obtain nucleic acid sequences that are unique to each population. After each subtraction, cDNA is PCR amplified, to¹ achieve maximal enrichment of differentially expression genes in both cell populations. Modifications of this principle, for example, SDD, as described by Wang and Uhl (Mol. Brain Res., 53:344-347, 1998); RDA, as described by Hubank and Schatz (Nucleic Acids Res. 22:5640-5648, 1994; and O'Neil and Sinclair, Nucleic Acid Res., 25:2681-2682, 1997); and SSH (Suppression Subtractive Hybridization) , as described by Lee et al. (Biochem. Biophys. Res. Comm. 277:680-685, 2000) are also incorporated into the above-described nucleic acid hybridization approaches to molecular characterization of neosis mother cells, Raju cells, or endoapoptotic cells.

Other techniques that are used to characterize neosis mother cells, Raju cells, or endoapoptotic cells at the molecular level include, but are not limited to RAGE, (Rapid Analysis of Gene Expression) (Wang et al., Nucleic Acids Research 27:4609-4618, 1999; Welsh and McClelland, Nucleic Acids Res. 18, 7213-7218, 1990; and Welsh et al., Nucleic Acids Res 20:4965-4970, 1992); TEA PCR, as described by Dixon et al. (Nucleic Acids Res. 20:4426-4431, 1998); SAGE (Serial Analysis of Gene Expression), Velculescu et al., Science

270:484-487, 1995); SAGE in combination with cDNA microarray (Nacht et al., Cancer Res., 59: 5464-5470, 1999); MicroSAGE (Datson et al . , Nucleic Acids Res. 27:1300-1307, 1999 (Raju cells) ) ; and a combination of various procedures (Wang and Rowley Proc. Natl. Acad. Sci. U.S.A. 95:11909-11914, 1998). Example 20: Markers for cells involved in neosis

Markers for multinucleate neosis mother cells:

Neosis mother cells are usually very large (80-150 μm) multinucleate or polyploid cells (based on the abnormal size of the nucleus compared to the size of the nuclei in the general population) , while the nascent offspring of neosis are usually on the order of 8-15 μm in diameter. In order to further study the genetic profile of these cells, and differentiate them from other cells in the population by specific markers, 5BϋdR pulse chase studies were employed to distinguish proliferative multinucleate cells undergoing DNA synthesis from non-proliferative, non-DNA synthesizing multinucleate cells.

5BUdR pulse chase studies were used to analyze multinucleate giant cells formed after exposure to X-radiation of HeLa cells, with HeLa cells usually being mononucleate (having 2-3% spontaneous multinucleate cells) . 5BUdR incorporation in a neosis mother cell and its progeny was detected by immunofluorescence using FITC-anti-5BϋdR. Figures 19A-19D are images of 5BUdR incorporation into A10 cells, demonstrating that A10 cells undergoing endoapoptosis are capable of division. Figures 19A-19G show that the multinucleate giant cells that did incorporate 5BUdR were also producing neotic progeny of mononucleate small cells. For example, Fig. 19A is an image of a cell showing 5BUdR incorporation in the viable nucleus (arrow 1) , while the other nucleus is undergoing endoapoptosis (arrow 3) . Arrow 2 points to 5BUdR incorporation in the endoapoptotic nuclear fragments. Figures 19B-19E are images of cells labeled with 5BUdR undergoing metaphase (Fig. 19B) , early anaphase (Fig. 19C) , and telophase (Fig. 19D and E) , respectively. Figures 19D and 19E show the same cells in light/fluorescence microscopy and in fluorescence microscopy alone, respectively. The residual endoapoptotic body in both daughter cells incorporates 5BUdR. Figure 19F is an image of HeLa cells irradiated with 9 Gy X- radiation 3 days earlier, that are now multinucleate (DNA fluorescing blue after DAPI staining) and display endoapoptotic nuclei (those without blue fluorescence) . And Fig. 19G is an image of a multinucleate giant HeLa cell undergoing neosis.

Similarly, markers for dead or dying nuclei

(measuring expression of pl6^INK4a, p21^ΛF1/CIP1, and SA-β-gal) indicated that in a multinucleate giant cell, there could be one nucleus (genome) in the process of endoapoptosis, while the other nucleus could be viable. For example, Figs. 20A-20D are HeLa cells stained for expression of cyclin dependent kinase inhibitors (CKIs) as negative markers for neosis. Fig 20A is an image of a multinucleate HeLa cell formed 5 days post-irradiation. The CKI pl6^INK4a is expressed specifically in a dying (endoapoptotic) nucleus of the multinucleate giant cell. Fig. 20B is an image of cell a HeLa cell showing that occasionally pl6^INKa is expressed in the cytoplasm of live cells (arrow 1) . Expression of pl6^INK4a is also occurring in a dead cell (apoptosis) (arrow 2) . Fig. 20C is an image of dead mini cells produced by a giant HTBll cell formed 4 days after exposure to 8 Gy X-radiation and stained with DAPI stain.

Fig. 20D is an image of the expression of p21^{WAF1 CIP1} in a non- viable nucleus of a multinucleate giant HeLa cell formed after exposure to 9 Gy X-radiation. These observations confirm that _pl6iNKa^ _p21AFiciPi_{^ and probably} other CKIs are markers for a non-viable nucleus in a multinucleate cell.

Figs. 21A-21H show the pattern of SA-β-gal stain in multinucleate giant cells (potential neosis mother cells) that spontaneously occur in X-REF cells (an established rat embryo fibroblast cell line) . Fig 21A is an image of a multinucleate giant cell showing that the cytoplasm all around the four nuclei is stained positively for SA-β-gal, indicating that this probably is early stage expression of SA-β-gal, and may eventually lead to a situation similar to that seen in Figs. 21B and 21C, with more intense SA-β-gal staining. Fig. 21D is an image of a giant multinucleate cell undergoing apoptosis and staining positively for SA-β-gal. Figs. 21E-21H are images of multinucleate giant cells showing SA-β-gal expression restricted to one or two nuclei, while other nuclei in the same cell are negative for SA-β-gal. This is analogous to CKI expression in a dying cell or nucleus, while the SA-β- gal-positive nucleus in the same cell might be reproductively viable, as shown by the 5BUdR studies above. Such cells with at least one viable nucleus are potential candidates for neosis mother cells. Similarly, giant cells with a viable polyploid nucleus (negative for SA-β-gal or CKI expression are potential candidates for neosis mother cells.

The findings herein demonstrate a method of differentiating multinucleate giant cells on the basis of the viability of their genomes (i.e. their nuclei). The link between the expression of CKIs, such as pl6^INK4a and _p2l^AF1/CIP1 and cell cycle arrest and cell death has been well established. Here, these markers have been used to differentiate between the viable and non-viable genomes (nuclei) in multinucleate giant cells, which are otherwise considered by those in the art to have arrived at a terminal non-proliferative state. Similarly, SA-β-gal expression, which is considered to be expressed by a senescent non- proliferative population of cells, has also been used to identify viable and non-viable genomes within the multiple nuclei of a giant cell. These markers are useful for differentiating between potential neosis mother cells and those cells that are truly senescent or have reached a terminal, non-proliferative state, leading eventually to the demise of the cell, and thus posing no harm by producing potentially tumorigenic Raju cells via neosis.

In addition, viable multinucleate neosis mother cells are distinguished from the non-viable cells by differential expression of the anti-apoptotic Bcl-2 gene product, as well as by differential expression of the proliferation specific ornithine decarboxylase (ODC) gene product. The expression of both these markers is tested by immunofluorescence procedures using a primary antibody and secondary antibody conjugated to a fluorochrome (Bcl-2: Conus et al., 2000. EMBO J. 19:1534-44; Baines et al . , 2000. Eur. J. Haematol. 64:211-9; Shihab et al., 1999. Kidney Int. 56:2147-59; Banasiak et al., 1999. Brain Res. Mol. Brain. Res. 72:214-25; ODC: Pomidor et al., 1999. Mol. Biol. Cell. 10:4299-310; Banan et al . , 1998. Am. J. Physiol. 274:G879-85; Shore et al., 1997. J. Biol. Che . 272:12536-43; Shayovits & Bacharach, 1994. J. Histochem. Cytochem. 42:807-11; and Lowkwist et al., 1987. Cell Tissue Res. 247:75-84).

Markers for polyploid neosis mother cells :

The above markers are also useful for the identification of potential NMCs that are polyploid and not multinucleate. Alternatively, identification of potential NMCs that are polyploid and not multinucleate features the following strategy. The process of neosis, by definition, does not involve dissolution of the nuclear membrane. The genetic material is duplicated and distributed to the two daughter nuclei without the dissolution of the nuclear membrane by nuclear budding, and is very similar to a process commonly observed in the budding yeast Saccharomyces cerevisiae (Byers & Goetsch, 1975. J. Bacteriol. 124:511-523; and Winey & O'Toole, 2001. Nat. Sci. 112:2313-21).

Fig. 22A is a schematic representation of the mode of mitotic spindle formation during yeast mitosis, and the events occurring during karyokinesis in yeast cells. This cell division does not involve dissolution of the nuclear envelope and is often called c-mitosis or closed mitosis. Stage 1 shows a yeast cell in Gl phase of the cell cycle, with one spindle pole body in the nuclear envelope. Stage 2 shows a yeast cell in G2 phase of the cell cycle, with two spindle pole bodies. Stages 3 and 4 show the initiation and growth of the budding of the yeast cell wall. Stages 5 and 6 show karyokinesis without dissolution of the nuclear envelope, and the distribution of the two prospective nuclei into the two prospective daughter cells.

Fig. 22B shows proposed events taking place during karyokinesis in a neosis mother cell, drawn from the events occurring during secondary/tertiary neosis in HTBll cells (Fig. 7) . Since during neosis, the nuclear membrane is not dismantled, neotic karyokinesis resembles that of yeast cell karyokinesis. Based on this observation, the centrosome involved in the formation of the mitotic spindle in mammalian mitotic cells is replaced by or becomes incorporated in the nuclear membrane in the neotic mother cell and favors karyokinesis without the dissolution of the nuclear envelope, similar to the events in yeast cell mitosis. Stage 1 shows a neoplastic cell or a normal cell with genetic damage in Gl phase of the cell cycle exhibiting an extranuclear centrosome, the mitotic spindle organizer, in the cytoplasm. In stage 2, the same cell is gaining cell mass and increasing in size, but the extra nuclear centrosome is replaced by a spindle pole body embedded in the nuclear envelope. In stages 3 and 4, the cell is increasing in ploidy by continuous DNA synthesis in a diploid or tetraploid state, respectively, as indicated by the number of spindle pole bodies. In stage 5, initiation of a nuclear bud aids the distribution of a copy of the genome to the budding nucleus, with the nuclear envelope intact, as occurs in yeast cell karyokinesis. In stage 6, the nascent nucleus (genome) detaches into the cytoplasm, and a vesicular structure is positioned adjacent to it. The appearance of this vesicular structure is the beginning of this novel type of asymmetric intracellular cytokinesis. In stages 7 and 8, the vesicle progresses into a furrow during the advance of cytokinesis and completely encircles the prospective Raju cell nucleus. The neosis mother cell may produce another Raju cell nucleus. Meanwhile, the product of the first karyokinesis performs the "birth dance" during the whole processes of cytokinesis, trying to detach itself from the neosis mother cell, and eventually succeeds in its effort in stage 9. Finally, in stage 10, the nascent immature Raju cell (8-15 μm in diameter) may gain cell mass and reach the average size (30-50 or more μm in diameter) . During the process of maturation, the spindle pole body is replaced by the extranuclear centrosome in the cytoplasm, which is characteristic of mammalian somatic cells that divide by mitosis.

Fig. 22C shows the role of the centrosome in the formation of the mitotic spindle apparatus that is accompanied by the dismantling of the nuclear envelope during the mitotic phase and is reorganized during the telophase of the mitotic division of a mammalian cell. Stage 1 shows a somatic cell in Gl phase of the cell cycle, with one centrosome. Stage 2 shows the same cell in G2 phase of the cell cycle, showing two centrosomes. In stage 3, the two centrosomes move to opposite poles during prophase, at which time the spindle apparatus is organized and the nuclear envelope begins to dismantle itself. Stage 4 shows the spindle apparatus with chromosomes aligned at the equatorial plane in the mitotic phase. Stages 5 and 6 show the late telophase stage of mitosis, the reorganization of the nuclear envelope, and the imitation of equatorial cytokinesis, which when completed yields two identical daughter cells.

As shown in Fig. 22A, in the absence of the dissolution of the nuclear membrane in budding yeast, the function of spindle formation is performed by specialized regions of the nuclear membrane called spindle pole bodies. Each yeast cell receives a single spindle pole body after a mitotic division (Gl phase) , and this is replicated to give rise to two spindle pole bodies during the G2 phase. These two spindle pole bodies migrate to opposite poles and become the nucleation center for the formation of the mitotic spindle, which is attached to the equatorial region of the nucleus during the mitotic phase. Thus, it appears that neotic cell division in mammalian cells under genetic duress represents a reversion to a primitive cell division process resembling that seen in yeast (Fig. 22B) . In the mammalian somatic cell system, each daughter cell of a mitotic division inherits a single centrosome, which remains outside the nuclear membrane; this centrosome divides into two centrosomes taking position at opposite poles during G2 phase of the cell cycle.' The nuclear membrane is dismantled when the microtubules radiate from the two centrosomes from opposite poles and meet at the equatorial region making contact with condensed chromosomes during the mitotic phase (Fig. 22C) .

Since the nuclear membrane remains intact during neosis, a structure similar to a spindle pole body must perform the function of the centrosome. Accordingly, the cells that divide by mitosis should display centrosomes outside the nuclear membrane, while the potential neosis mother cells should display spindle pole bodies embedded in the nuclear membrane and no extranuclear centrosomes. Since the nuclei in the neosis mother cells are often polyploid, those nuclei might show two, four, or more spindle pole bodies in each nucleus. Using antibodies to known antigens common for the centrosomes and spindle pole bodies, the cells that are destined to undergo mitosis and those cells that are destined to undergo neosis are distinguished.

Examples of antibodies to be tested include, for example, calmodulin (Geiser et al., 1993. Mol. Cell. Biol. 13:7913-24; Moser et al . , 1997, J. Cell. Sci. 110:1805-12; Sun et al., 1992. J Cell Biol. 119:1625-39; and Erent et al., 1999. Mol. Cell. Biol. Res. Commun. 1:209-215), γ-tubulin (gamma-tubulin) (Oakley et al., 1990. Cell 61:1289-301; and Nigg, 2001. Nat. Rev. Mol. Cell. Biol. 2:21-32), and cdclδ (Cerutti & Simanis, 1999. J. Cell. Sci. 112:2313-21; Zachariae et al., 1996. Science 274:1201-4; Lamb et al., 1994. EMBO J. 13:4321-8; and Tugendreich et al., 1995. Cell 81:261- 8) . The nascent Raju cells will display a single spindle pole body in the nuclear membrane, which will disappear during Raju cell maturation and a centrosome will be formed.

Markers for Raju cells :

Raju cells maybe identified using the markers described above for neosis mother cells. In addition, other Raju cell specific markers may be ^λpublic' (common to all the Raju cells irrespective of the cell type or tumor type) or ^Λprivate' (specific to a given cell type, e.g., lung cell, kidney cell, epithelial cell, etc.) For example, it is likely that such marker antigens are encoded by novel genes involved in embryonic development and are not expressed in somatic cells. Such markers can be identified by electrophoresing the proteins expressed by Raju cells on a two dimensional SDS PAGE gel, and comparing the results to those obtained from electrophoresis of their respective mitotic cells. Novel Raju cell-specific antigens are identified, and monoclonal antibodies against these antigens are prepared. As mentioned above, some of these antigens may be ^λpublic' and others may be ^private' .

The Raju cell specific antibodies prepared against primary Raju cells, as described above, may be potentially different from Raju cell specific antibodies raised against Raju cells emanating from a neosis mother cell in a malignant tumor (e.g., metastasizing human neuroblastoma derived HTBll cells) . For example, the latter may display differential gene expression related to the degree of malignancy, e.g., an absence of metastasis suppressor genes or the presence of metastasis related genes. Thus, marker-based antisera against potentially non-malignant and malignant Raju cells, can be developed for diagnostic, prognostic, and therapeutic applications.

Example 21: Diagnostic/prognostic reagents

The findings herein demonstrate that neosis is a unique process of cell division involved in tumorigenesis and occurs as a result, in part, of genetic damage. Neosis is a marker of tumorigenesis, carcinogenesis, and tumor progression. A higher frequency of neosis or endoapoptosis indicates a higher rate of tumor progression toward malignancy, while a lower frequency of neosis indicates non- malignant neoplastic growth. Successive mitotic divisions in post-neotic progeny will lead to genomic instability-induced mitotic crisis and secondary/tertiary rounds of spontaneous neosis, culminating in genetic heterogeneity in the tumor cell population, upon which natural selection leads to the survival of the fittest, highly malignant cells. When tumor cells are exposed to genotoxin-based anti-tumor therapy, a minor fraction of neoplastic cells escape termination of cell lineage by undergoing neosis and producing cells with extended mitotic potential. Such cells, contribute to the recurrence of tumor growth, which may be relatively resistant to the given genotoxin.

The findings herein demonstrate that neosis underlies and is a diagnostic and/or prognostic marker of tumorigenesis, tumor progression, and tumor recurrence after anti-tumor therapy.

Neosis mother cells, Raju cells, and endoapoptotic cells find diagnostic and prognostic use in the detection or monitoring of tumorigenesis, tumor progression, tumor recurrence, or the presence of a neoplastic or proliferative disease. For example, the presence of, or an increased frequency of the number of neosis mother cells, Raju cells, or endoapoptotic cells in a biological sample obtained from a subject indicates the presence of a neoplastic or proliferative disease or an increased likelihood of developing a neoplastic or proliferative disease. Similarly, increased frequency of neosis mother cells, Raju cells, or endoapoptotic cells in a biological sample obtained from a subject indicates tumor progression or recurrence. Conversely, the absence of, or a decreased frequency of neosis mother cells, Raju cells, or endoapoptotic cells in a biological sample obtained from a subject indicates the absence of a neoplastic or proliferative disease, or a decreased likelihood of developing a neoplastic or proliferative disease, or indicates that a tumor in the subject is not progressing or recurring. The presence or absence of neosis mother cells, Raju cells, or endoapoptotic cells in a biological sample, for example, a tissue or tumor biopsy is detected using methods as described herein.

The presence or absence of neosis mother cells, Raju cells, or endoapoptotic cells, or an increased or decreased frequency of neosis mother cells, Raju cells, or endoapoptotic cells is compared to biological samples which are absent of neosis mother cells, Raju cells, or endoapoptotic cells, if detection of the presence or an increased frequency of neosis mother cells, Raju cells, or endoapoptotic cells is desired, or to biological samples that contain neosis mother cells, Raju cells, or endoapoptotic cells, if detection of the absence or a decreased frequency of neosis mother cells, Raju cells, or endoapoptotic cells is desired (controls samples) . The biological sample may be from the same subject as the sample to be tested. Alternatively, the control sample may be from a different subject, or it may be a cultured tissue culture sample, for example, those described herein. A decrease in the presence or frequency of neosis mother cells, Raju cells, or endoapoptotic cells, relative to a control cell, indicates the absence of a neoplastic or proliferative disease or a decreased likelihood of developing a neoplastic or proliferative disease, or indicates that a tumor in the subject is not progressing or recurring. An increase in the presence of, or an increased frequency of neosis mother cells, Raju cells, or endoapoptotic cells in the biological sample compared to the control indicates the presence of a neoplastic or proliferative disease or an increased likelihood of developing a neoplastic or proliferative disease or increased tumor progression or recurrence. Example 22: Identification of compounds that inhibit tumor progression

Methods for identifying compounds that inhibit tumor progression are also a feature of the present invention. In one approach, a candidate compound is added, in varying concentrations, to the culture medium of cells capable of undergoing neosis or endoapoptosis. Such cells include, but are not limited to the cells described in Table 1. The presence of neosis mother cells, Raju cells, or endoapoptotic cells, or an increased frequency of neosis mother cells, Raju cells, or endoapoptotic cells is then detected in the sample, using methods described herein. The number of neosis mother cells, Raju cells, or endoapoptotic cells in the sample in the presence of the candidate compound is compared to the number of neosis mother cells, Raju cells, or endoapoptotic cells in the sample in the absence of the candidate compound, all other factors (e.g., cell type and culture conditions) being equal. A candidate compound inhibits tumor progression when the number of neosis mother cells, Raju cells, or endoapoptotic cells in the sample in the presence of the candidate compound decreased compared to the number of neosis mother cells, Raju cells, or endoapoptotic cells in sample in the absence of the candidate compound.

Compounds that inhibit tumor progression may be purified, or substantially purified, or may be one component of a mixture of compounds, such as an extract or supernatant obtained from cells (Ausubel et al., supra) . In an assay of a mixture of compounds, inhibition of tumor progression in the cell sample is tested against progressively smaller subsets of the compound pool (e.g., produced by standard purification techniques such as HPLC or FPLC) until a single compound or minimal number of effective compounds is demonstrated to inhibit tumor progression.

Compounds that function to inhibit tumor progression include, for example, peptide and non-peptide molecules, such as those present in cell extracts, mammalian serum, or growth medium in which cells have been cultured. Compounds also include small molecules from combinatorial libraries, for example, those generated by ChemBridge Corporation (San Diego, CA) or Nanoscale Combinatorial Synthesis, Inc. (Mountain View, CA) .

Example 23: Administration of inhibitors of tumor progression

A compound that inhibits tumor progression is administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering a neoplastic or proliferative disease, for example, a tumor. Administration may begin before the patient is symptomatic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found, for example, in "Remington's Pharmaceutical Sciences." Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene- polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for inhibitors of tumor progression include ethylene-vinyl acetate copolymer particles, osmotic pumps, i plantable infusion systems, and liposo es. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

If desired, treatment with a compound that inhibits tumor progression may be combined with more traditional therapies, such as surgery, radiation therapy, or chemotherapy.

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

All publications and patents mentioned in this speci ication are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMS :

1. A method for isolating an endoapoptotic cell, said method comprising identifying said endoapoptotic cell in a population of cells, and separating at least one endoapoptotic cell from the non-endoapoptotic cell in said population of cells.

2. A method for isolating a neosis mother cell, said method comprising identifying said neosis mother cell in a population of cells, and separating at least one neosis mother cell from cells that are not neosis mothe cells in said population of cells.

3. A method for isolating a Raju cell, said method comprising identifying said Raju cell in a population of cells, and separating at least one Raju cell from cells that are not Raju cells in said population of cells.

4. The method of claim 1, 2, or 3, wherein separation of said cell from said population of cells comprises the use of flow cytometry.

5. The method of claim 1, 2, or 3, wherein separation of said cell from said population of cells comprises the use of micro coverslip capture.

6. The method of claim 1, 2, or 3, wherein separation of said cell from said population of cells comprises the use of a Percoll gradient.

7. The method of claim 1, 2, or 3, wherein separation of said cell from said population of cells comprises the use of laser capture microdissection.

8. An isolated endoapoptotic cell.

9. An isolated neosis mother cell.

10. An isolated Raju cell.

11. The cell of any of claims 8, 9, or 10, wherein said cell is a mammalian, yeast, fruit fly, nematode, fish, or avian cell.

12. A cell or tissue composition having an enriched population of endoapoptotic cells.

13. A cell or tissue composition having an enriched population of neosis mother cells.

14. A cell or tissue composition having an enriched population of Raju cells.

15. The cell or tissue composition of claim 12, 14, or

14. wherein said cell or tissue composition is a mammalian, avian, fruit fly, yeast, fish, or nematode cell or tissue composition.

16. The cell or tissue composition of claim 15, wherein said cell or tissue composition comprises a tissue biopsy from a mammal, avian species, fruit fly, fish, yeast, or nematode.

17. The cell or tissue composition of claim 12, 13, or 14, wherein said cell or tissue composition comprises a cell culture line, said cell culture line isolated from any tissue.

18. The cell or tissue composition of claim 17, wherein said cell line is selected from the group consisting of C3H10T1/2 cells, A10 cells, mouse embryo cells, rat embryo cells, hamster embryo cells, rodent cells, avian cells, nematodes cells, fruit fly cells, yeast cells, Chinese hamster ovary cells, and human cells.

19. The cell or tissue composition of claim 18, wherein said mouse embryo cell line is a p53-/- or an Rb-/- mouse embryo cell line.

20. The cell or tissue composition of claim 18, wherein said rodent cell is a normal rodent cell or a neoplastic rodent cell.

21. The cell or tissue composition of claim 18, wherein said avian cell is a normal avian cell or a neoplastic avian cell.

22. The cell or tissue composition of claim 18, wherein said human cell is a normal human cell or a neoplastic human cell.

23. The cell or tissue composition of claim 22, wherein said human neoplastic cell line is a HeLa cell line.

24. A secondary neotic cell or tissue composition, wherein said cell or tissue composition is obtained by culturing the cell or tissue composition of any of claims 12, 13, or 14.

25. The secondary cell or tissue composition of claim 24, wherein said cell or tissue composition comprises a mononucleate giant neosis cell.

26. The secondary cell or tissue composition of claim

24, wherein said cell or tissue composition comprises a multinucleate giant neosis cell.

27. A method of identifying a nucleic acid molecule that modulates neosis, said method comprising the steps of:

(a) isolating nucleic acid molecules from a neosis mother cell, Raju cell, or endoapoptotic cell; (b) isolating nucleic acid molecules from a control cell, said control cell not being a neosis mother cell, Raju cell, or endoapoptotic cell and;

(c) comparing the expression of the nucleic acid molecules of step (a) with the expression of the nucleic acid molecules of step (b) , wherein a nucleic acid that is differentially expressed between the nucleic acid molecule samples identifies a gene that modulates neosis.

28. The method of claim 27, wherein said neosis mother cell, Raju cell, or endoapoptotic cell and said control cell are the same cell type.

29. The method of claim 27, wherein said neosis mother cell, Raju cell, or endoapoptotic cell and said control cell are different cell types.

30. The method of claim 27, wherein said comparison of nucleic acid molecule expression between the nucleic acid molecules of step (a) and the nucleic acid molecules of step (b) comprises the use of differential display.

31. The method of claim 27, wherein said comparison of nucleic acid molecule expression between the nucleic acid molecules of step (a) and the nucleic acid molecules of step (b) comprises the use of nucleic acid library hybridization.

32. The method of claim 27, wherein said comparison of nucleic acid molecule expression between the nucleic acid molecules of step (a) and the nucleic acid molecules of step (b) comprises the use of DNA microarray hybridization or oligonucleotide microarray hybridization.

33. A method of diagnosing a mammal for the presence of a neoplastic or proliferative disease, or an increased likelihood of developing a neoplastic or proliferative disease, said method comprising isolating a biological sample from said mammal and determining the presence or absence of a neosis mother cell, Raju cell, or endoapoptotic cell, the presence of said cell being an indication of the presence of a neoplastic or proliferative disease or an increased likelihood of developing a neoplastic or proliferative disease.

34. The method of claim 33, wherein said biological sample is a tissue biopsy or a blood sample.

35. The method of claim 34, wherein said biological sample is a tumor biopsy.

36. A method of monitoring tumor progression in a mammal, said method comprising isolating a tumor sample from said mammal and determining the frequency of neosis mother cells, Raju cells, or endoapoptotic cells, over a time interval, wherein an increase in the number of neosis mother cells, Raju cells, or endoapoptotic cells over time indicates tumor progression.

37. The method of claim 33 or 36, wherein said cell is detected using a biochemical stain or is detected morphologically.

38. A method of identifying a compound that inhibits tumor progression, said method comprising the steps of:

(a) providing a neosis mother cell;

(b) contacting said neosis mother cell with a candidate compound; and

(c) determining the viability or proliferation of said neosis mother cell, wherein a candidate compound that decreases said viability or proliferation of said neosis mother cell, relative to the level of viability of proliferation of a neosis mother cell not contacted with said candidate compound, is a compound that inhibits tumor progression.

39. A method of identifying a compound that inhibits tumor progression, said method comprising the steps of:

(a) providing a Raju cell;

(b) contacting said Raju cell with a candidate compound; and

(c) determining the viability or proliferation of said Raju cell, wherein a candidate compound that decreases said viability or proliferation of said Raju cell, relative to the level of viability or proliferation of a Raju cell not, contacted with said candidate compound, is a compound that inhibits tumor progression.

40. A method of identifying a compound that inhibits tumor progression, said method comprising the steps of:

(a) providing an endoapoptotic cell;

(b) contacting said endoapoptotic cell with a candidate compound; and

(c) determining the viability or proliferation of said endoapoptotic cell, wherein a candidate compound that decreases said viability or proliferation of said endoapoptotic cell, relative to the level of viability or proliferation of an endoapoptotic cell not contacted with said candidate compound, is a compound that inhibits tumor progression.

41. The method of any of claims 38, 39, or 40, wherein said cell is from a biological sample of a mammal.

42. The method of any of claims 38, 39, or 40, wherein said cell is from a cultured cell line.