Patent Application
20060051739
The replication and subsequent faithful segregation of duplicated chromosomes are crucial for the proper transmission of the cellular genome to daughter cells. In higher eukaryotes, the nuclear membrane breaks down at the beginning of mitosis, and subsequently spindle microtubules attach to centromeric kinetochores to assure the even distribution of sister chromatids. At the end of mitosis, the nuclear membrane reassembles around each group of chromosomes to form two daughter nuclei. Therefore, it would be reasonable to assume that acentric DNA molecules should not be maintained stably in nuclei as they do not attach to microtubules, and that acentric DNA molecules should be dispersed throughout the cytoplasm subsequent to nuclear membrane breakdown.
In many cases, however, acentric DNA molecules, lacking functional centromeres, exhibit a surprisingly high stability in dividing cells. Examples of such stably-transmitted acentric DNA molecules in human cells include cellular acentric chromosomes called double minute chromosomes (DMs) and extrachromosomally replicating viral DNAs. DMs are cancer specific genomic anomalies known to harbor amplified oncogenes and drug resistance genes (Alitalo and Schwab, 1986; Hahn, 1993; Wahl, 1989). They are autonomously replicating, acentric, atelomeric, circular chromatin bodies, and usually 1-2 megabase pairs in size. Although they apparently lack functional centromeres (Barker and Hsu, 1978; Levan and Levan, 1978), their segregation efficiency is much higher than expected (Kimmel et al., 1992; Pauletti et al., 1990). Clues to the mechanisms underlying the efficient segregation came from light and electron microscopic observations showing that DMs frequently associated with mitotic chromosomes (Barker and Hsu, 1978; Hamkalo et al., 1985; Jack et al., 1987; Levan and Levan, 1978). These observations were extended using a fusion protein of human histone H2B and Aequorea victoria green fluorescent protein (H2B-GFP) to reveal DM clusters tethered to segregating daughter chromosomes in living cancer cells (Kanda et al., 1998). Time-lapse microscopy demonstrated that DMs could ‘hitchhike’ on segregating chromosomes from anaphase to telophase, indicating how chromosome tethering could contribute to increased segregation efficiency.
There is a continuing need for improved visualization of viral and cellular acentric extrachromosomal molecules that tether to chromosomes. There is a further need to identify compounds that effectively interfere with the chromosomal tethering of these viral and cellular acentric extrachromosomal molecules.
Abbreviations: Epstein-Barr virus (EBV); lac operator (lacO); Epstein-Barr nuclear antigen-1 (EBNA-1); green fluorescent protein (GFP); yellow fluorescent protein (YFP); red fluorescent protein (RFP); cyan fluorescent protein (CFP); Chinese hamster ovary cells (CHO); double minute chromosome (DM); lac repressor (lacR); fluorescent in situ hybridization (FISH); ampicillin (Amp); family of repeats (FR).
The invention provides a preselected nucleic acid molecule comprising an extrachromosomal molecule operably linked to a tag. The extrachromosomal molecule can be any molecule that segregates with cellular chromosomes during cell division. For example, the extrachromosomal molecule may be a double minute chromosome or a viral nucleic acid sequence from a DNA or RNA virus. The viral genome may be maintained as an episome within a cell. Examples of sources of the viral nucleic acid include, but are not limited to Flaviviridae, Retroviridae, Hepadnaviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, a Hepatitis C virus, a Papillomavirus, an Epstein-Barr virus, an Influenza virus or a Polyomavirus. Alternatively, the extrachromosomal molecule may be an oncogene, such as sis, erbB, fins, sea, kit, ros, mpl, eyk, erbA, H-ras, K-ras, crk, src, abl, fps, fes, fgr, yes, mos, raf, mil, akt, jun, fos, myc, myb, ets, rel, maf, ski or qin.
The tag includes any nucleic acid sequence that encodes a selection marker (such as a blasticidin resistance gene (bsr), ampicillin, rifampicin, chloramphenicol or other art recognized drug resistance selection marker gene). Such selection markers may be driven by any operably linked promoter, such as the SRα promoter. The tag may be a reporter gene (such as green fluorescent protein, yellow fluorescent protein, red fluorescent protein or cyan fluorescent protein) that allows the extrachromosomal molecule to be detected. The tag may also be a binding site for a detectable trans-acting element that binds to the tag and thereby allows detection of the extrachromosomal molecule. The tag can be operably linked to the extrachromosomal molecule through integration of an integrating vector, into the extrachromosomal molecule.
The preselected nucleic acid may further contain an integrating vector that specifically labels double-minute chromosomes (DMs). The vector may contain Epstein-Barr virus (EBV), bovine papillomavirus (BPV), or Kaposi's sarcoma associated herpesvirus (KSHV) sequences. The vector may contain an EBNA-1 gene and an oriP sequence, wherein the oriP sequence has a plurality of EBNA-1 binding sites in two distinct regions. The vector may contain a plurality of tandem repeats of a lac operator (lacO). The vector may contain a nucleotide sequence that interferes with chromosomal tethering such as an antisense message to a tethering protein, such as the Epstein-barr nuclear antigen. The vector may encode a fusion protein that binds to an extrachromosomal molecule such as a lac repressor (lacR)-GFP fusion protein that binds with high affinity to the lacO that may be integrated into the extrachromosomal molecule.
Examples of the present invention include, but are not limited to, a plasmid vector containing a retroviral vector, a gene encoding GFP fused to lac repressor-nuclear localization signal, or a vector containing a gene encoding YFP fused to lac repressor-nuclear localization signal, or a histone H2B gene fused to a CFP gene.
The present invention further provides a chromosomal tethering polypeptide. The polypeptide can operably link a cellular chromatid and an extracellular molecule. Such an extracellular molecule may be an oriP-containing vector. This polypeptide may be of cellular or viral origin. The tethering protein may also be a peptidomimetic or a fusion polypeptide having a chromatid binding domain and a domain that binds to an extrachromosomal molecule.
The present invention further provides a method of visualizing chromosomal tethering of extrachromosomal molecules, such as viral acentric extrachromosomal molecules (DAE) or double-minute chromosomes (DM), by contacting a vector of the invention with a cell suspected of containing a DM or a DAE. The extrachromosomal molecule, such as the DM or DAE, may associate with a cellular chromatid through the action of a tethering polypeptide.
The present invention provides a method of interfering with chromosomal tethering of extrachromosomal molecules, such as viral or cellular acentric extrachromosomal molecules, by administering a vector that specifically labels the extrachromosomal molecule, such as a double-minute chromosome (DM). Additionally, the vector may produce a product that inhibits the expression or function of a tethering polypeptide, such as an antisense message to the Epstein-Barr nuclear antigen or the herpesvirus latent nuclear antigen.
The present invention also provides a method to identify an agent that modulates segregation of extrachromosomal molecules into daughter cells following division of a parent cell. Such methods can be used to identify agents that are useful for treating cancer, viral infections or other afflictions that involve extrachromosomal molecules.
The present invention also provides a method of treating cancer or viral infections by administering a pharmaceutical composition containing a compound that inhibits the tethering of viral or cellular acentric extrachromosomal molecules to a chromosome.
The present invention further comprises a chromosomally integrating vector that specifically labels DMs. The vector may be an Epstein-Barr virus (EBV), bovine papillomavirus (BPV), or Kaposi's sarcoma associated herpesvirus (KSHV) vector sequences. The vector may contain an EBNA-1 gene and an oriP sequence, wherein the oriP sequence has a plurality of EBNA-1 binding sites in two distinct regions. Further, the vector may contain a plurality of tandem repeats of a lac operator (lacO). The vector may contain a reporter gene, such as GFP or YFP, and/or may contain a selection marker, such as a blasticidin resistance gene (bsr) driven by SR promoter. The vector may further contain a nucleotide sequence that interferes with chromosomal tethering. The vector may contain a lac repressor (lacR)-GFP fusion protein that binds with high affinity to the lacO. The vector may be a modified virus, such as a modified animal virus. It may be a DNA virus, such as a member of the Herpesviridae, Papovaviridae or Adenoviridae.
The present invention provides a plasmid vector comprising retroviral vector, a gene encoding GFP fused to lac repressor-nuclear localization signal. It also provides a plasmid vector comprising retroviral vector, a gene encoding YFP fused to lac repressor-nuclear localization signal. Moreover, the present invention provides a plasmid vector comprising a histone H2B gene and a CFP gene.
Recent studies revealed that chromosome tethering may be a common mechanism for enhancing the transmission of extrachromosomally replicating viruses into daughter nuclei (Bastien and McBride, 2000; Ilves et al., 1999; Lehman and Botchan, 1998; Marechal et al., 1999; Skiadopoulos and McBride, 1998). One of the best characterized episomal vectors is based on the Epstein-Barr virus (EBV) replicon, which utilizes the cis-acting oriP sequence and the virally-encoded EBNA-1 protein (Mackey and Sugden, 1999). OriP is composed of two clusters of EBNA-1 binding sites referred to as the family of repeats and the dyad symmetry element (Reisman et al., 1985). It has been shown that EBNA-1 both enables autonomous replication of oriP-containing plasmids in human cells (Yates et al., 1985) and mediates the nuclear retention of the plasmids (Krysan et al., 1989; Middleton and Sugden, 1994). Since EBNA-1 protein localizes on mitotic chromosomes (Grogan et al., 1983; Marechal et al., 1999; Petti et al., 1990), it is reasonable to infer that EBNA-1 could recruit oriP plasmids to mitotic chromosomes. Consistent with this, fluorescence in situ hybridization (FISH) previously demonstrated that EBV vectors did associate with mitotic chromosomes (Simpson et al., 1996; Westphal et al., 1998). Such chromosome tethering should facilitate the efficient segregation of EBV vectors into daughter nuclei when the nuclear membrane reforms at the end of mitosis.
The observation of viral association with host chromosomes, “hitchhiking,” raised the intriguing question of whether DMs achieve efficient segregation by a similar mechanism. This possibility was explored by introducing EBV vectors into DM-harboring cells. Unexpectedly, EBV vectors frequently integrated into DMs subsequent to DNA transfection and selection. This observation enabled a method to preferentially integrate exogenous DNA into DMs. Cell lines with DM-EBV chimeras were derived in which lac operator (lacO) repeats were introduced as part of the EBV vector. These lacO-tagged DMs were readily detected using a fusion protein between the lac repressor (lacR) and green fluorescent protein (GFP), as previously demonstrated for visualizing homogeneously stained regions in CHO cells (Robinett et al., 1996). This provided a powerful tool for analyzing the mitotic behavior of DMs. Different distributions of free EBV vectors and DM-EBV chimeras were found, although they both hitchhiked onto mitotic chromosomes. Possible molecular mechanisms governing the behavior of these acentric molecules are discussed and described herein.
A “cis-acting” element or sequence refer to DNA or RNA sequences whose function require them to be on the same molecule. An example of a cis-acting element is an origin of replication.
An “extrachromosomal molecule” of the invention includes nucleic acid molecules which segregate during cell division through interaction with cellular chromatin. Examples of such extrachromosomal molecules include, but are not limited to, viral acentric extrachromosomal molecules, viral genomes and double-minute chromosomes. Extrachromosomal molecules also include nucleic acid constructs containing a nucleic acid sequence that allows interaction of the nucleic acid construct with cellular chromatin and provides for segregation of the nucleic acid construct with the cellular chromatin during cell division. Such nucleic acid sequences include origins of replication, such as oriP and the like, as well as fragments of such origins such as the FR element of oriP. It is well within the skill in the art to identify such sequences and link such sequences into nucleic acid constructs of the invention. Such constructs can also include retroviral vectors, plasmids, phagemids, yeast artificial chromosomes, bacterial artificial chromosomes and the like.
The term “modulate” or “modulates” means an increase or decrease in the occurrence of an event. For example, an agent that modulates the segregation of an extrachromosomal molecule in a cell will either increase or decrease the efficiency of extrachromosomal molecule segregation to sister cells following division of a cell treated with the agent.
“Operably-linked” refers to the association of nucleic acid sequences on single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
A “polypeptide” of the invention includes proteins found in nature as well as poly-amino acids that are not found in nature. Such polypeptides include fusion proteins as well as poly-amino acids that contain non-natural amino acids. Persons of skill in the art realize that many amino acid derivatives exist that can be linked into a chain of amino acids in a desired manner and that fusion proteins can be designed through recombinant techniques. The terms “polypeptide,” “protein” and “peptide” are used synonymously herein.
A “reporter gene” is a nucleic acid that expresses a detectable polypeptide. Examples of such reporter genes include those that encode for green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, or other polypeptides that may be detected.
A “selection marker” is a nucleic acid sequence that confers resistance to a chemical, such as a drug, to a cell. Examples of such chemicals include chloramphenicol, ampicillin, rifampicin, blasticidin or the like.
A “tag” of the invention includes a marker that is associated with an extrachromosomal molecule and that allows detection of an extrachromosomal molecule. Examples of a tag include, but are not limited to, a reporter gene that expresses a polypeptide that can be detected, such as green fluorescent protein, yellow fluorescent protein, red fluorescent protein, cyan fluorescent protein, or other detectable polypeptides. A tag can also be a nucleic acid sequence that encodes a selection marker such as chloramphenicol, ampicillin, rifampicin, blasticidin resistance or the like. Such nucleic acid sequences are well known in the art. A tag can also be a cis-acting element that is recognized by a trans-acting element. An example of a cis-acting element is a nucleic acid sequence that encodes a repressor binging site, such as the lac-repressor binding site found within the lac operator (lacO). This cis-acting element can be integrated into an extrachromosomal molecule and bound by a detectable trans-acting element that allows detection of an extrachromosomal molecule. An example of such a trans-acting element is a fusion polypeptide of a repressor polypeptide, such as the lac repressor (lacR), and a detectable polypeptide, such as green fluorescent protein. Such trans-acting factors may also include a nuclear localization signal that will cause the fusion polypeptide to localize to the nucleus. It will be recognized that there are many combinations of such cis-acting and trans-acting elements that are within the scope of the invention.
A “tethering polypeptide” is a polypeptide that provides an association of an extrachromosomal molecule with a cellular chromatid. A tethering polypeptide may comprise a cellular protein or a viral protein. Examples of such a tethering polypeptide are the Epstein-Barr Nuclear Antigen-1 protein (EBNA-1) and the Herpesvirus late nuclear antigen (LANA).
A “recombinant tethering polypeptide” is a tethering polypeptide produced through recombinant methods. An example of such a recombinant polypeptide is a protein having the N-terminal cellular chromatid binding domain of EBNA-1 linked to the C-terminal oriP binding domain of EBNA-1 through an recombinantly produced amino acid linkage. Alternatively, such a polypeptide can have the N-terminal cellular chromatid binding domain of EBNA-1 fused to a domain that would bind to a recombinantly designed cis-acting element within an extrachromosomal molecule. An example of such a recombinant polypeptide is a protein having the N-terminal cellular chromatid binding domain of EBNA-1 linked to the lac repressor binding domain of the lac repressor protein.
A “trans-acting” element refers to a polypeptide or nucleic acid whose function requires them not to be directly linked to the same molecule. An example includes a polypeptide that binds to a cis-acting element that is operably linked to an extrachromosomal molecule.
I. A Preselected Nucleic Acid Molecule Comprising an Extrachromosomal Molecule Operably Linked to a Tag.
The invention provides a preselected nucleic acid molecule comprising an extrachromosomal molecule operably linked to a tag. It is contemplated that the extrachromosomal molecule is any molecule that is tethered to a cellular chromatid during cellular division. Such tethering allows the extrachromosomal molecule to be segregated into progeny cells upon division of the parent cell. The invention provides a method to tag the extrachromosomal molecule which allows the extrachromsomal molecule to be detected during chromosome segregation and cell division. The tag may become operably linked to the extrachromosomal molecule through integration of an integrating vector, which contains the tag, into the extrachromosomal molecule. Thus, the invention provides integrating vectors that are able to integrate into an extrachromosomal molecule and operably link a tag into the extrachromosomal molecule.
Extrachromosomal molecules: The extrachromosomal molecule can be any molecule that segregates with cellular chromosomes during cell division. Examples of such extrachromosomal molecules include, but are not limited to, double minute chromosomes, viral acentric extrachromosomal molecules, cellular acentric extrachromosomal molecules and recombinantly engineered extrachromosomal molecules.
Double minute chromosomes, viral acentric extrachromosomal molecules and cellular acentric extrachromosomal molecules are well known in the art. Recombinantly engineered extrachromosomal molecules include, but are not limited to, plasmid and viral based vectors into which a nucleic acid sequence has been inserted that allows the extrachromosomal molecule to associate with a cellular chromatid and be segregated into daughter cells during cell division. Nucleic acid sequences that may be operably linked into extrachromosomal molecules include, but are not limited to, an origin of replication such as EBV-oriP, or nucleic acid sequences that are able to interact with tethering proteins, such as the FR element of EBV-oriP. A recombinantly engineered extrachromosomal molecule can also contain nucleic acid sequences that allow the molecule to be detected within a cell. Such nucleic acid sequences include reporter genes that encode fluorescent proteins or selection markers that confer chemical, i.e. drug, resistance to a cell. It is contemplated that a recombinantly engineered extrachromosomal molecule may be introduced into a cell such that it is segregated into progeny cells during division of the parent cell. It is further contemplated that such extrachromosomal molecules may be constructed to associate with recombinantly engineered tethering polypeptides, described below. This allows for the production of recombinantly engineered extrachromosomal molecules and tethering polypeptides that specifically associate with each other and allow specific interactions of the recombinant extrachromosomal molecules with chromatids to be assessed.
Tag: A tag includes any nucleic acid sequence which provides for detection of an operably linked extrachromosomal molecule. A tag may confer a detectable trait onto an extrachromosomal molecule. Examples of traits that may be conferred include selection markers and reporter genes. Selection markers include genes that confer resistance to a chemical or drug such as blasticidin, ampicillin, rifampicin or chloramphenicol. Reporter genes encode a detectable polypeptide such as a fluorescent protein as described herein. Additionally, a tag may function as a cis-acting binding site for a detectable trans-acting element that binds to the tag when the tag has integrated into an extrachromosomal molecule. Binding of the detectable trans-acting element to the cis-acting tag, that is operably linked to the extrachromosomal molecule, allows the extrachromosomal molecule to be detected. Binding sites for trans-acting elements include nucleic acid sequences to which a detectable trans-acting element will bind and thereby tag the extrachromosomal molecule containing the binding site. There are many combinations of binding sites and trans-acting elements that are well known in the art. These combinations are exemplified by the lac repressor binding site (lacO) and the lac repressor (lacR). Thus, a cis-acting lac repressor binding site may be operably linked to an extrachromosomal molecule through integration of an integrating vector, which contains the tag, into the extrachromosomal molecule. A trans-acting fusion protein having the lac repressor fused to a fluorescent protein can bind to the cis-acting lac repressor binding site and allow the extrachromosomal molecule to be detected. Such methods are within the scope of the invention and are well known to those of skill in the art.
Integrating vector: The invention provides vectors that can integrate into the extrachromosomal molecules discussed above. The vectors of the invention may be derived from a plasmid, virus, retrovirus, phagemid or other cloning vehicles and vectors known in the art. Such vectors may include a nucleic acid sequence that promotes the insertion of the vector into an extrachromosomal molecule. In one embodiment, the vectors of the invention include Epstein-Barr nucleic acid sequences, like that encoding the origin of replication (oriP). Optionally, such vectors may encode the ENBA-1 protein, lac operator repeats and lac repressor-fluorescent protein fusion protein individually or in combination. One example of a vector of the invention is the EBV-lacO vector (
Chromosomal tethering polypeptide: The invention provides chromosomal tethering polypeptides. A tethering polypeptide of the invention includes those that promote the association of an extrachromosomal molecule with a cellular chromatid such that the extracellular molecule is segregated with the cellular chromatid during cell division.
Tethering polypeptides can be isolated according to the methods of the invention or be created through recombinant DNA technology according to methods known in the art. Such recombinant molecules include, but are not limited to, a fusion polypeptide having a chromosome binding domain linked to an extrachromosomal molecule binding domain. An example of a tethering polypeptide is a polypeptide having the N-terminal chromosome binding domain of EBNA-1 linked to the C-terminal oriP binding domain of EBNA-1. In another example, a tethering polypeptide can have the N-terminal chromosome binding domain of EBNA-1 linked to any domain that is able to bind to a site within an extrachromosomal molecule. This type of tethering polypeptide is exemplified by a fusion protein having the N-terminal chromosome binding domain of EBNA-1 fused to a lac repressor.
Cells: The invention provides cells that contain an extrachromosomal molecule that has an operably linked tag as described above. The cells of the invention include, but are not limited to, mammalian cells, insect cells, bacteria, yeast and any other cell which can contain an extrachromosomal molecule that segregates with a cellular chromatid during cell division. Examples of cells which may be used include 3T3, BHK21, MDCK, HeLa, PtK1, L6, PC12, COLO320DM and SP2 cells. Additional cells may be obtained from the American Type Culture Collection. (Hay et al., eds., American Type Culture Collection Catalogue of Cell Lines and Hybridomas, 6th ed. Rockville, Md.: American Type Culture Collection, 1988). These cells may be grown under any condition that allows them to divide. Conditions for cell and tissue culture are well known in the art. Ham, Proc. Natl. Acad. Sci. USA 53:288 (1965); Loo et al., Science, 236:200 (1987); Sato et al., eds. Growth of Cells in Hormonally Defined Media. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1982).
II. A Method to Visualize Chromosomal Tethering of an Extrachromosomal Molecule to a Cellular Chromatid.
The invention provides a method to visualize tethering of an extrachromosomal molecule to a cellular chromatid. The method involves tagging the extrachromosomal molecule and visually detecting the tag in order to determine whether the extrachromosomal molecule is tethered to a cellular chromatid. The extrachromosomal molecule may be tagged by contacting the molecule with a vector of the invention such that the vector integrates into the molecule. Integration of the vector into the extrachromsomal molecule incorporates a cis-acting element into the extrachromosomal molecule. Such cis-acting elements may include, but are not limited to, nucleic acid sequences to which trans-acting elements bind and thereby tag the extrachromosomal molecule. In one embodiment, the extrachromosomal molecules are tagged with a fluorescent polypeptide. The fluorescent polypeptide may then be visualized through use of a microscope as described herein.
The present method may be used to screen for agents that modulate chromosomal tethering and segregation of extrachromosomal molecules as described herein. Alternatively, the above method may be used as a diagnosis tool to determine if cells contain extrachromosomal molecules that are segregated during cell division. During such a diagnosis scheme, cells could be obtained through biopsy of tissue suspected of containing extrachromosomal molecules. These cells would then be contacted with a vector of the invention that would integrate into and tag extrachromosomal molecules contained within the suspect cells. The cells could then be cultured and the presence of extrachromosomal molecules could then be determined. Such a method would be useful because extrachromosomal molecules are known to encode oncogenes and drug resistance genes that cause proliferative disorders.
III. A Method to Interfere with Tethering of Extrachromosomal Molecules to a Cellular Chromatid.
The present invention also provides a method for treating a test cell that contains a tagged extrachromosomal molecule with a candidate agent and then determining if the candidate agent inhibits tethering of the extrachromosomal molecule to a cellular chromomatid. In one embodiment, the method involves use of a cell that contains a fluorescently tagged extrachromosomal molecule. Such molecules can be produced according to the methods described herein. A control cell is used that has the tagged extrachromosomal molecule but that is not treated with a candidate agent. A test cell has a tagged extrachromosomal molecule and is treated with a candidate agent to produce a treated cell. The control cell and the treated cell are allowed to begin cell division and the linkage of the extrachromosomal molecule in the test cell and the control cell is then determined and compared. If a candidate agent interferes with tethering, the extrachromosomal molecule will not be associated with a cellular chromatid through cell division. In one embodiment, the extrachromosomal molecule may be fluorescently tagged according to the methods described herein and the effects of a candidate agent on tethering can be determined through use of fluorescent microscopy as described herein.
It is understood that any trait that can be integrated into an extrachromosomal molecule and detected in order to determine the tethering of an extrachromosomal molecule to a cellular chromatid is within the scope of the invention.
IV. A Method to Identify an Agent that Modulates Segregation of Extrachromosomal Molecules into Daughter Cells during Division of a Parent Cell.
The method involves treating a test cell that contains a tagged extrachromosomal molecule with a candidate agent and then determining if the candidate agent increases or decreases segregation of the extrachromosomal molecule during cell division when compared to a control cell. In one embodiment, the method involves use of a cell having a fluorescently tagged extrachromosomal molecule. Methods to prepare a fluorescently tagged extrachromosomal molecule are disclosed herein. Segregation of the fluorescently tagged extrachromosomal molecule can then be followed through use of fluorescence microscopy. Alternatively, fluorescent activated cell sorting (FACS) can be used to determine segregation efficiency. Through use of FACS, cells containing an extrachromosomal molecule can be sorted from those that do not. This method provides an automated procedure to rapidly screen numerous candidate agents for their ability to inhibit or increase segregation of extrachromosomal molecules.
Alternatively, an extrachromosomal molecule having a selection marker may be used within the method of the invention. In this embodiment, a cell having an extrachromosomal molecule that encodes a selective marker is treated with a test agent to produce a test cell. The test cell is allowed to undergo cell division and is then subjected to selection according to the selection marker. A untreated control cell having an extrachromosomal molecule that encodes the selective marker is allowed to divide in parallel and then is subjected to selection according to the selection marker. The number of resistant cells of the test cell and the control cell is then compared to determine if the candidate agent increased or decreased segregation of the extrachromosomal molecule.
It is understood that any trait that can be integrated into an extrachromosomal molecule and detected in order to determine segregation efficiency of the extrachromosomal molecule is within the scope of the invention.
V. A method of Treating Cancer or Viral Infection.
The invention provides a method of treating cancer or viral infection. The method involves contacting cells with an agent that interferes with the tethering of extrachromosomal molecules to a cellular chromatid which causes loss of the extrachromosomal molecule upon cell division. Such agents can be identified according to the methods disclosed herein and administered to an animal in need thereof according to methods well known in the pharmaceutical arts.
The agents of the invention may be formulated into a variety of acceptable compositions. In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
Pharmaceutically acceptable salts are obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids also are made.
The compounds may be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient that are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Examples of useful dermatological compositions that can be used to deliver the compounds of the present invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
Useful dosages of the compounds of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
Generally, the concentration of the compound(s) of the present invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.
The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
The compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about 30 μM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).
The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
Plasmids: EBV oriP and EBNA-1 coding sequences derived from pCEP4 (Invitrogen) and a blasticidin resistance gene (Izumi et al., 1991) derived from pYN3215-bsr (kindly provided by Dr. Fumio Hanaoka, Osaka University) were subcloned into pMBL19 to make pMBL19-EBVbsr. pMBL19, which has a bacterial p15A ori, was chosen for its ability to subclone unstable inserts (Nakano et al., 1995). Lac operator (lacO) repeats (256 direct repeats) derived from pSV2-dhfr 8.32 (Robinett et al., 1996) were subcloned into the pMBL19-EBVbsr to make EBV-lacO vector using STBL2 competent cells (Life Technologies, Grand Island, N.Y.) (Belmont et al., 1999).
pCLMFG-lacR-GFP was constructed by subcloning a gene encoding EGFP (Clontech, Palo Alto, Calif.) fused to lac repressor-nuclear localization signal (p3′SSdimerClonEGFP) (Robinett et al., 1996) into a splicing retroviral vector pCLMFG-MCS (kindly provided by Dr. Nikunj Somia, Salk Institute), a derivative of the pMFG vector (Dranoff et al., 1993). pCLMFG-lacR-YFP was constructed in the same way using EYFP gene (Clontech).
A histone H2B-CFP fusion gene was made by swapping the GFP gene of H2B-GFPN1 (Kanda et al., 1998) with the ECFP gene (Clontech). The H2B-CFP gene was subcloned into a pCLNRX vector (Naviaux et al., 1996).
Production of VSV-G pseudotyped retroviruses was performed by co-transfection of each retroviral vector and pMD.G (the plasmid encoding the envelope protein VSV-G) into 293 gp/bsr cells as described (Miyoshi et al., 1997).
Establishing cell lines with lacO-tagged DMs: COLO320DM cells harboring DMs containing an amplified c-myc gene were grown as described (Kanda et al., 1998). Exponentially growing cells (1×107) were transfected with 5 μg of the EBV-lacO vector using electroporation (BioRad, Hercules, Calif.), resuspended in 10 ml of culture medium, and plated into two 10 cm dishes (8 ml, 2 ml for each dish). Blasticidin (15 μg/ml, Calbiochem, San Diego, Calif.) was added to the transfected cells 24 hours after transfection, and cells were selected for 14 days. Drug resistant cells were further grown under reduced blasticidin concentration (5 μg/ml). Blasticidin resistant colonies were isolated 4 weeks after transfection and then re-plated into 48 well dishes. Cells were expanded in media containing blasticidin (5 μg/ml) and 12 fast growing clones were selected for infection with the lacR-GFP retrovirus. Punctate staining in nuclei was observed in all clones analyzed, and three independent clones which exhibited the brightest fluorescent dots by lacR-GFP staining were chosen for further FISH analyses as these clones were expected to contain the highest number of EBV-lacO vectors. This strategy was repeated independently three times to confirm the reproducibility of the experimental data.
Fluorescence in situ hybridization (FISH): Cells were treated with colcemid (100 μg/ml) for 50 minutes, and chromosome spreads were prepared by conventional fixation. For dual color FISH, c-myc cosmid DNA was labelled with biotin, while the lacO repeat (SalI-XhoI fragment of pSV2-dhfr 8.32) (Robinett et al., 1996) was labelled with digoxigenin using random prime labeling. Denaturation, hybridization, and washing were performed as previously described (Shimizu et al., 1996). Signals were detected using FITC-avidin (10 μg/ml, Vector Laboratories, Burlingame, Calif.) and rhodamine-conjugated sheep anti-digoxigenin antibody (4 μg/ml, Boehringer Mannheim, Indianapolis, Ind.). Chromosomes were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, 1 μg/ml) in VectaShield (Vector).
Chromatin fibers were prepared on slide glasses as previously described for fiber-FISH (Parra and Windle, 1993). Signals were detected using 3 sequential steps of a signal amplification protocol as follows: (1) FITC-avidin (5 μg/ml) and anti-digoxigenin monoclonal antibody (1 μg/ml, Boehringer Mannheim); (2) biotinylated goat anti-avidin (1 μg/ml, Vector) and digoxigenin-labelled sheep anti-mouse IgG (2 μg/ml, Boehringer Mannheim); (3) FITC-avidin (5 μg/ml) and rhodamine-conjugated sheep anti-digoxigenin antibody (4 μg/ml).
Visualizing DM-integrated and free EBV vectors by in vivo lacR-GFP staining: One of the established cell lines, containing DMs tagged with lacO repeat, was infected with lacR-GFP retrovirus. The infected cells were subcloned by limited dilution to obtain sublines in which DMs are more uniformly labelled with lacR-GFP. For time lapse imaging, the same cell line was infected with H2B-CFP and lacR-YFP viruses simultaneously, and the double-labelled cells were subcloned by limited dilution.
For visualizing EBV vectors in transiently transfected cells, a subline of COLO320DM cells stably expressing lacR-GFP protein was established by retrovirus infection and subcloning of lacR-GFP positive cells. The established cells were transfected with the EBV-lacO vector by electroporation, and the transfected cells were harvested for immunofluorescence analyses at 3 days posttransfec
Immunofluorescence staining: Cells were harvested by gentle pipetting, attached to slide glasses by cytospin (500 rpm, 1 minute, Shandon, Pittsburgh, Pa.), and fixed with 3.7% formaldehyde for 10 minutes. When indicated, cells were treated with either taxol (10 μM paclitaxel, Sigma Chemical Co., St. Louis, Mo.) or vinblastine (10 μg/ml, Sigma) for 3 hours prior to harvesting them in order to disrupt microtubules. Slides were washed with PBS three times, and treated with blocking buffer (2.5% BSA, 0.2M glycine, 0.1% TritonX-100) for 30 minutes.
Primary and secondary antibodies were diluted in the blocking buffer. Primary antibodies were rabbit anti EBNA-1 serum K67-3 (1:1000, kindly provided by Dr. Jaap Middeldorp, Free University Hospital, Amsterdam, the Netherlands), human CREST autoantiserum hACA-M (for detecting centromeres, 1:2000 dilution) (Sullivan et al., 1994), and monoclonal anti α-tubulin (for detecting microtubules, 1:2000 dilution, Sigma). Following incubation for 60 minutes at room temperature, glass slides were washed three times with PBS. Secondary antibodies were Texas-Red conjugated anti-rabbit IgG (1:500, Jackson ImmunoResearch, West Grove, Pa.), Cy5 conjugated goat anti-human IgG (1:1000, Amersham, Piscataway, N.J.), and rhodamine conjugated anti mouse IgG (1:1000, Boehringer Mannheim), respectively. Following incubation for 60 minutes at room temperature, slides were washed three times with PBS, and chromosomes were counterstained with DAPI (1 μg/ml). Fluorescence of lacR-GFP was preserved well by this protocol.
Microscopy: All images appearing in the Figures were collected using a DeltaVision microscope system (Applied Precision Inc. Issaquah, Wash.) with either a 63×/NA 1.4 or a 100×/NA 1.35 oil immersion objective. For fixed specimens (except for the images of
For observation of living specimens, cells were grown on 40 mm cover slips pretreated with fibronectin (25 μg/ml in PBS) and placed in an FCS2 chamber system (Bioptechs, Butler, Pa.) with prewarmed medium (containing 20 mM HEPES pH 7.3). Special filter sets required for CFP and YFP detection (Ellenberg et al., 1999) were installed into the DeltaVision microscope system. Single slice images were collected every 2 minutes using 100× objective equipped with an objective heater (Bioptechs). Pixel size was 0.1338 μm and a binning factor of 2 was used to minimize the total exposure time during observation. Pseudo-color images were created using Adobe Photoshop (Adobe Systems, San Jose, Calif.).
FISH analyses revealed that stably transfected EBV vectors frequently co-localized with DMs. One explanation of this finding was that EBV vectors integrated into DMs. To facilitate the tracking of transfected EBV vectors, two hundred and fifty-six direct repeats of the lac operator (lacO) were added to the EBV vector (EBV-lacO) (
Direct evidence was obtained that EBV-lacO vectors integrated into DMs using FISH analysis of stretched chromatin fibers (Parra and Windle, 1993). This analysis revealed that arrays of lacO signals were detected on the same DNA fibers exhibiting c-myc signals (
The established cell lines containing the lacO-integrated DMs were infected with retrovirus expressing the lacR-GFP fusion protein. Retroviral infection resulted in readily detectable lacR-GFP protein expression in approximately 80% of the recipient cells, and approximately 20% of which exhibited punctate fluorescent dots representing DMs. The lacR-GFP expressing populations were subcloned to yield clones in which 60% of the cells had punctate fluorescent DMs.
The distribution of EBV-lacO/DM chimeras in prometaphase cells, in which chromosomes aggregate briefly into a single, wheel-shaped ring called a chromosome rosette (Nagele et al., 1995) was first focused on. It was found that the fluorescent dots always attached to the periphery of the chromosome rosette (
The fluorescent labeling strategy for DMs described above enables visualization of DMs, centromeres, and microtubules simultaneously in various phases of mitosis. Centromeres and spindle microtubules were detected by indirect immunofluorescence while preserving the fluorescence of lacR-GFP. This analysis confirmed that DMs lack centromeric antigens (Barker and Hsu, 1978; Levan and Levan, 1978) (
Real Time Observation of DM Behavior using Dual-Color Fluorescent Protein Labeling
The above results with fixed cells could mask movements occurring in living cells, and it does not provide a dynamic view of DM behavior. These concerns were addressed by taking advantage of the in vivo expression of distinguishable fluorescent fusion proteins (Ellenberg et al., 1999) in order to analyze the dynamics of DMs and chromosomes in living mitotic cells. Cells with lacO-labelled DMs were simultaneously infected with two different retroviruses expressing either H2B-CFP or lacR-YFP. H2B-CFP should label chromosomes and DMs, as described previously (Kanda et al., 1998), while lacR-YFP should label only DMs as descried above. Chromosomes and DMs in the same cell were observed with minimal spectral overlap (
Representative time lapse images demonstrating the behavior of DMs at the metaphase-anaphase transition are shown (
The ability to readily visualize DMs without using FISH protocol has enabled the investigation of the mechanisms that contribute to their behavior during mitosis. The possibility that DMs are held away from the spindle poles by the microtubule-mediated antipolar force, to which normal chromosome arms are also known to be subjected (Fuller, 1995; Rieder et al., 1986) was examined. DM-labelled cells were treated with either a microtubule stabilizer (taxol) or destabilizer (vinblastine). DMs and microtubules were visualized by lacR-GFP and immunofluorescence staining, respectively. Microtubules of taxol-treated cells showed multiple aster-like structures (
The mechanisms underlying the precision of chromosome segregation are being elucidated with increasing detail. It now appears that some autonomously replicating DNA viruses achieve high efficiency segregation not merely by their high copy number, but rather by having devised strategies to associate with chromosomes (Bastien and McBride, 2000; Ilves et al., 1999; Lehman and Botchan, 1998; Marechal et al., 1999; Skiadopoulos and McBride, 1998). The data herein highlights the role of chromosomes as “cargo ships” on which both viral replicons and cellular DMs are loaded to enable their efficient transmission to daughter nuclei.
It was determined that EBV vectors integrate into DMs at high frequency. This targeted integration of EBV vectors into DMs was totally unexpected since EBV vectors containing oriP and the EBNA-1 gene are usually maintained as extrachromosomal elements without integrating into chromosomes (Yates et al., 1985). It was found that EBV vectors randomly associated with mitotic chromosomes as well as DMs after transient transfection (
The molecular basis of preferential integration into DMs remains a mystery. It is possible that DMs and normal chromosomes have different tendencies to undergo recombination. Another possibility is that EBV replicons and DMs share the same replication machinery during S phase of the cell cycle, which increases the probability of recombination between replication intermediates. This would be consistent with the observed requirements for both oriP and EBNA-1 to recombine with DMs. The heterogeneity in the number and fraction of DMs containing integrated EBV-lacO sequences (
The chimeric extrachromosomal molecules of DMs and EBV-lacO vectors appear to exhibit the same behavior as native (unlabelled) DMs. The visualization strategy involving lacR-GFP can be used in combination with immunofluorescence, as it does not require harsh denaturation of DNA, and it preserves chromosomal fine structures far better than FISH (Robinett et al., 1996). This sensitive methodology enabled the visualization of DMs together with centromeres and microtubules (
A model is proposed in which DMs are subject to two “forces” in mitotic cells (
The second “force” acting on DMs keeps them attached to mitotic chromosomes (
The ability of viral replicons and DMs to interact with chromosomes provides a simple solution to the problem of high efficiency segregation of acentric DNA molecules. Interfering with the molecular interactions between viral replicons/DMs and mitotic chromosomes would increase the mitotic loss rate of latently infected viruses, or DMs that are providing survival or selective advantage to cancer cells. Therefore, understanding the molecular interactions that mediate such associations suggests new molecular targets for anti-viral and anti-cancer therapy.
The invention is described with reference to various specific and preferred embodiments and techniques. It should be understood, however, that many variations and modifications may be made while remaining within the scope of the invention. All referenced publications, patents, and patent documents are intended to be incorporated by reference, as though individually incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US01/28130 | 9/7/2001 | WO | 8/20/2004 |
| Number | Date | Country | |
|---|---|---|---|
| 60230730 | Sep 2000 | US |
