WO1994017182A1 - Region du promoteur de l-plastine et ses utilisations - Google Patents

Region du promoteur de l-plastine et ses utilisations Download PDF

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WO1994017182A1
WO1994017182A1 PCT/US1994/000436 US9400436W WO9417182A1 WO 1994017182 A1 WO1994017182 A1 WO 1994017182A1 US 9400436 W US9400436 W US 9400436W WO 9417182 A1 WO9417182 A1 WO 9417182A1
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plastin
promoter
cells
gene
expression
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John C. Leavitt
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Palo Alto Medical Research Foundation
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Palo Alto Medical Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • This invention relates to the nucleotide sequences corresponding to the L-plastin promoter region and their uses.
  • Plastin was first noted as a polypeptide which appeared to be induced abundantly accompanying tumorigenic transformation of human cells. Plastins are a family of highly conserved actin-binding proteins approximately 70 kd in size. In yeast, a plastin homolog was found to be required for actin organization and morphogenesis.
  • SV40-mediated transformation can also lead to activation of the L-plastin gene as it has been shown that SV40-transformed MRC fibroblasts express L-plastin while the parental normal MRC fibroblasts do not express L-plastin. Since L-plastin expression is a frequent event in human solid tumor formation, understanding the mechanism by which the L-plastin gene is activated may shed light on a fundamental aspect of human tumorigenesis.
  • the L-plastin gene promoter and regulatory region is provided for use as a transcriptional and translational vector of other genes for expression in mammalian hosts.
  • the L-plastin promoter with a limited portion of the regulatory region functions as a strong promoter.
  • the promoter together with the upstream regulatory region is active in hemopoietic cells and in transformed cells of solid tissues which are of non-hemopoietic origin and is inducible in response to estrogen and progesterone.
  • the invention comprises a nucleotide sequence of not more than about 5.0 kilobases comprising a nucleotide sequence corresponding to the sequence of the L-plastin promoter, preferably including at least a portion of the upstream regulatory region.
  • An expression construct comprising a nucleotide sequence corresponding to the sequence of the L-plastin promoter and a foreign gene is also provided.
  • the construct preferably includes sequences corresponding to at least about two kilobases of the upstream regulatory sequences, more preferably about five kilobases of the upstream regulatory sequences.
  • the invention also provides a method for inducing steroid-responsive production of RNA in a cell which is estrogen-responsive or progesterone-responsive comprising engineering the cell with an expression vector comprising a nucleotide sequence coding for said RNA sequence and a nucleotide sequence corresponding to the L-plastin promoter and a portion of the upstream regulatory region containing a progesterone responsive element or the estrogen responsive element.
  • the nucleotide sequence is preferably a DNA sequence encoding a protein.
  • the invention provides a method for production of RNA in a hemopoietic cell comprising engineering said cell with an expression vector of this invention.
  • the invention comprises a method for determining whether an agent is cancer-causing.
  • the method comprises contacting a non-malignant tissue cell engineered with an expression vector comprising the L-plastin promoter and a reporter gene with said agent and observing the cell for the expression of the reporter gene, the expression of said reporter gene indicating that the agent is cancer causing.
  • an expression vector of this invention can be used to express a protein only in cancer cells which are present in a mixed population of cancer cells and normal tissue cells.
  • the L-plastin promoter and regulatory region, expression constructs containing the L-plastin promoter (or the L-plastin promoter and regulatory region) and methods employing the L-plastin promoter are provided for the expression of RNA and polypeptides in mammalian cells, in particular hemopoietic cells and cancer cells of hemopoietic and non-hemopoietic origin.
  • the sequences can be employed for expression of RNA and L-plastin or other polypeptides, usually other polypeptides.
  • the sequences are not active in cells which do not express L-plastin. In particular, the sequences are not active in a normal tissue cell, but are active when the cell is transformed, if the endogenous L-plastin gene has been activated by the transforming event.
  • the constructs when the constructs include a reporter gene, the constructs can be used to identify transformed cells and to identify agents that induce transformation.
  • the L-plastin regulatory region contains an active estrogen receptor element and one or two active progesterone receptor elements. Therefore, the L-plastin promoter and a portion of the regulatory region can be used to provide hormonally regulated expression of a protein (or production of RNA) , in a cell with the appropriate receptors.
  • the L-plastin promoter region refers to the L-plastin promoter and at least a portion of the upstream regulatory region.
  • the L-plastin promoter and its regulation in normal and transformed cells was characterized. Genomic DNA spanning the promoter region of the gene was sequenced. Table 1 illustrates genomic fragments of the promoter and gene region obtained by use of various restriction enzymes. The nucleotide numbers in the table refer to the location of the restriction sites with 1 being the transcription initiation site nearest the TATAAA box. The length of the promoter region does not include coding region sequences that may be present in the fragment.
  • nucleotide 1 represents the transcription initiation site
  • TATAAA box bases -15 to -10
  • 3 1 end of the first exon is indicated by an asterisk ( * ) .
  • Upstream sequences homologous to various transcription factor binding motifs are underlined and indicated as follows: Ets-1 ; PR, progesterone responsive
  • API 10 -585 TTAAAGAGAT CCCTAGCACA TAGATGTTCT ATAAATAAAA GAATGAGTAA ATAATCTAGT 1740
  • the promoter region contained multiple transcription start sites which were mapped using standard primer extension and SI nuclease mapping methods.
  • several potential cis-acting regulatory elements were identified flanking the TATAAA box.
  • the L-plastin promoter was flanked by progesterone and estrogen responsive elements. This finding was surprising since this gene encodes a ubiquitous and abundant hemopoietic cell architectural protein.
  • the promoter and regulatory elements of the L-plastin gene were characterized. Transcription initiation from this promoter was found to occur at multiple sites and as near as 10 bp from the 3' side of the TATAAA box.
  • the promoter and its flanking DNA was cloned and sequenced to identify potential regulatory elements that participate in the induction of the L-plastin gene in neoplastic cells. Examination of upstream sequences revealed the existence of two progesterone, one estrogen, and four Ets-1 responsive elements flanking the promoter. A 315 bp fragment spanning the TATAAA box, an Ets-1 binding site, an estrogen responsive element, and an Spl binding site exhibited maximum promoter activity using CAT (chloramphenicol acetyltransferase) as a reporter while longer promoter fragments extending into upstream flanking sequences spanning the two progesterone responsive elements API site and 3 potential Ets-1 sites exhibited reduced promoter activity.
  • CAT chloramphenicol acetyltransferase
  • the L-plastin gene promoter has a classic TATAAA box, which usually directs transcription initiation at a single site about 30 bp downstream, transcription initiation occurs at multiple sites. In addition, transcription initiation can occur as close as 10 bp from the TATAAA box. Nevertheless, the presence of a perfect TATAAA box and an adjacent Spl binding site in the L-plastin promoter indicates its potency in promoting highly efficient transcription, as demonstrated in the Examples.
  • the L-plastin promoter region contains upstream regulatory sequences that both inhibit the activity of the promoter and contain regulatory elements that make the activity responsive to estrogen and/or progesterone.
  • the L-plastin promoter region sequence spanning the TATAAA box the Spl site, and the proximal Ets -1 site is preferred (fragments 1 and 4 in
  • Table 1 contain these sequences) .
  • the region from the PvuII site through the Seal site (Fragment No. 1 in Table 1) , which is about 315 nt is used.
  • This portion of the L-plastin promoter region which functions as a strong promoter can be used for expression in hemopoietic cells or non- hemopoietic cell.
  • sequences corresponding to the native sequence use of sequences corresponding to the native sequence.
  • the phrase "corresponding thereto" means that the sequences can contain nucleotides that are not identical to those of the L-plastin promoter region sequence. Those non-identical sequences can be substitutions in the sequence, insertions or deletions. Numerous changes can be made in the native sequence that preserve the ability of the corresponding sequence to be active as a promoter and be regulated by the desired cis acting elements. For example, a modified sequence in which the estrogen responsive element or one or both progesterone responsive elements has been deleted or mutated may be preferable.
  • changes in the sequence can be made and preserve the activity of the sequence as a promoter.
  • any alterations preserve the activity of the promoter and the regulatory elements.
  • the sequences are identical to that of the native promoter with any engineered changes in the promoter that are desired, such as deletion of a selected regulatory element.
  • a sequence corresponding to the sequence from the transcription initiation region through the proximal Ets-1 site (-111) , more preferably through the distal Ets-1 site, most preferably at least about the first two kilobases (kb) of the L-plastin promoter region is used.
  • kb first two kilobases
  • the region extending from the first transcription initiation site through the first progesterone responsive element, preferably through the second progesterone responsive element can be used for hormone responsive expression of a structural gene in a cell with an estrogen and a progesterone receptor.
  • the L-plastin promoter region can be used in a cell with only the estrogen or the progesterone receptor.
  • the sequences corresponding to the regulatory region should extend through the estrogen responsive element but not extend through the proximal progesterone responsive element.
  • the sequences corresponding to the estrogen responsive element should be mutated so that the estrogen responsive element is no longer active.
  • a region starting at a restriction site upstream from the estrogen responsive element through a downstream restriction site can be amplified using a primer that starts at the closest restriction site and spans the estrogen responsive element.
  • the primer is sufficiently homologous to bind to and amplify the region, but contains sufficient mismatches to ensure that the amplified region contains an inactive estrogen responsive element.
  • the mutated amplified sequence can be ligated into the remainder of the promoter region sequence so that the modified sequence is identical to the native sequence except at the selected sites in the mutated estrogen responsive element region.
  • the portion of the L-plastin promoter region through the estrogen responsive element for estrogen- dependent expression in a cell with estrogen receptors is contemplated.
  • the region of the L-plastin promoter region surrounding the estrogen responsive element can be removed or mutated so that the region is not functional. Techniques for selectively mutating or excising a portion of a nucleotide sequence are well known.
  • the sequences corresponding to the regulatory region should extend through the estrogen responsive element but not extend through the proximal progesterone responsive element.
  • the sequences corresponding to the estrogen responsive element should be mutated so that the estrogen responsive element is no longer active.
  • the region a restriction site upstream from the estrogen responsive element through a downstream restriction site can be amplified using a primer that starts at the closest restriction site and spans the estrogen responsive element. The primer is sufficiently homologous to bind to and amplify the region, but contains sufficient mismatches to ensure that the amplified region contains an inactive estrogen responsive element.
  • the mutated amplified sequence can be ligated into the remainder of the promoter region sequence so that the modified sequence is identical to the native sequence except at the selected sites in the mutated estrogen responsive element region.
  • the strength of this promoter was demonstrated using truncated genomic fragments. Fragments which included the regulatory region sequences 180 bp upstream from the TATAAA box showed the strongest promoter activity, which was nearly equal to the activity of the 0-actin promoter, a well known strong promoter. However, this activity of the promoter alters as upstream negative control elements affect its activity.
  • An expression vector of this invention comprises the L-plastin promoter transcription initiation region together with either L-plastin or a foreign gene, usually a foreign gene.
  • a transcription initiation region can be used to express a protein or produce RNA (as for production of antisense sequences) in a mammalian cell.
  • the regulatory region can be added to provide the described regulation. The portions of the L-plastin promoter region used for production of RNA in various types of cells and under various types of regulation have been described previously.
  • L-plastin or a foreign protein may be achieved in a variety of ways in mammalian host cells.
  • the expression construct involves the L-plastin promoter region and the structural gene present as a contiguous entity or as exons separated by one or more introns.
  • the expression construct may be joined to an appropriate vector, if desired.
  • a vector is intended a replication system utilized by the intended host.
  • the expression construct includes one or more markers to ensure the stable maintenance of the DNA construct in the host.
  • the construct contains a marker gene to determine presence of the construct in the cell and a reporter gene to monitor promoter activity.
  • Various replication systems include bacterial and viral replication systems, such as retroviruses, simian virus, bovine papilloma virus, or the like.
  • a gene which allows for selection in a host. This gene can complement an auxotrophic host or provide protection from a biocide.
  • Illustrative genes include thymidine kinase, dihydrofolate reductase, which provides protection from methotrexate, or the like.
  • markers can provide resistance to a biocide, e.g. , G418, methotrexate, etc.; resistance to a heavy metal, e.g., copper; prototropy to an auxotroph; or the like.
  • Suitable genes for selection of a host cell include thymidine kinase, dihydrofolate reductase, metallothionein, and the like.
  • marker genes can express a detectable protein to determine activation of the promoter in a host.
  • Reporter genes are well known and include CAT and, preferably, jS-galactosidase.
  • the subject gene or antisense sequence to be expressed may be joined to an amplifiable gene, so that multiple copies of the sequence of interest may be made.
  • the gene may be maintained on an extrachromosomal element or be integrated into the host genome.
  • the foreign gene may come from a wide variety of sources such as prokaryotes, eukaryotes, pathogens, fungi, plants, mammals, including primates, particularly humans, or the like.
  • proteins may include hormones, lymphokines, enzymes, capsid proteins, membrane proteins, structural proteins, growth factors and inhibitors, blood proteins, immunoglobulins, etc.
  • proteins to be expressed in cells which are responsive to estrogen or progesterone, in hemopoietic cells or cancer cells.
  • the constructs can be used to produce therapeutic proteins, viral resistance proteins, and proteins involved in repair of genetic defects.
  • the constructs can be used to produce antisense RNA and antisense ribozymes.
  • the manner in which an individual DNA sequence coding for a protein or antisense sequence of interest is obtained, divided into individual exons, and joined to the transcriptional and translational regulatory signals of the L-plastin gene will depend upon each individual polypeptide of interest, as well as the information available concerning the DNA sequence coding for such polypeptide.
  • the L-plastin promoter or transcription system including the promoter may be used for the regulation of expression of other genes by regulating transcription of RNA complementary to another mRNA or portion thereof. In effect, the L-plastin promoter would regulate transcription of the nonsense strand or portion thereof of the gene whose expression is to be inhibited.
  • Such inhibition may find use in making an auxotrophic host, inhibiting one pathway in favor of another metabolic pathway, reversing or enhancing oncogenic characteristics of a cell, or the like.
  • Introduction of the DNA into the host will vary depending upon the particular construction. Introduction can be achieved by any suitable gene transfer technique such as transfection, transformation, transduction, or the like, as amply described in scientific literature.
  • the host cells will normally be immortalized cells, that is, cells that can be continuously passaged in culture.
  • these cells will be normal and may be any convenient mammalian cell, which is able to express the desired polypeptide, and where necessary or desirable, process the polypeptide, so as to provide a mature polypeptide.
  • Processing the polypeptide can include glycosylation, methylation, terminal acylation, e.g., formylation or acetylation, cleavage, or the like.
  • the host should be able to recognize the leader sequence and the processing signal for peptidase cleavage and removal of the leader.
  • the constructs can be used or expression of proteins in fertilized eggs for development of transgenic animals or in human cells removed from the body, engineered and put back into the body (e.g.; lymphocytes).
  • lymphocytes e.g., lymphocytes
  • L-plastin promoter finds particular application in genetic engineering of hemopoietic cells where constitutive expression is desired and in tissue cells where gene expression in response to progesterone or estrogen is desired.
  • portion of the L-plastin promoter region can be used so that the L-plastin promoter region is active only in hemopoietic cells.
  • the promoter can be present in an expression vector for expression limited to hemopoietic cells. In this way, one can engineer expression of a protein (or production of an antisense sequence) only in the hemopoietic cells of a mixed population of cells.
  • the L-plastin promoter region can be used for expression of proteins in lymphocytes.
  • the protein included in the construct is expressed at high levels, similar to the level of expression of L-plastin in hemopoietic cells.
  • the engineered protein or RNA can be used for HIV therapy in lymphocytes.
  • the selected coding region is placed under the transcriptional control of the portion of the L-plastin promoter region described previously.
  • the engineered cells are then subject to hormonal control.
  • the cells can be used for expression of a selected protein in response to the environment in female reproductive tissues.
  • a protein which is genetically defective and leads to loss of pregnancy can be engineered into the cells and expressed in response to the changing hormonal levels during pregnancy.
  • engineered cells facilitate study of the levels of expression of various proteins during the course of pregnancy.
  • the L-plastin gene is normally expressed only in hemopoietic cells. However, the gene is activated in other cell types of solid tissues accompanying tumorigenesis. Engineered cells containing an expression vector of this invention can be used to evaluate whether an agent or condition is cancer causing.
  • the expression vector will contain a reporter gene in addition to the L-plastin promoter. Suitable reporter genes are well known and were described previously.
  • the reporter gene is the E. coli ⁇ -galactosidase gene or neomycin resistance gene (neo) which confers resistance to the drug G418.
  • the Examples demonstrate that the 5.1 kb promoter fragment of the L-plastin gene contains control elements which participate in the suppression of its activity in normal cells and its activation in human tumor cells.
  • the demonstrated ability of the ⁇ -galactosidase assay to discriminate between normal and neoplastic cells that do not have an active endogenous L-plastin gene and the tumor cells that exhibit activated expression of L-plastin demonstrates that cells engineered with an expression vector of this invention can be used for early detection of nascent in vitro neoplastic cells that also have activated expression of the endogenous the L-plastin gene.
  • a reporter gene By putting 3-galactosidase or other reporter genes under the control of the 5.1 kb L-plastin promoter fragment, a reporter gene can be inserted into normal cells which remains silent until transformation by a mechanism leading to activation of the endogenous L-plastin gene.
  • the reporter gene is activated at the time of some transformation events which catalyze activation of the endogenous L-plastin gene and expression of the reporter gene signals the onset of the development of the neoplastic or tumorigenic state.
  • growth selectable markers like neomycin- resistance (G418-resistance) can be put under the control of this promoter to select nascent neoplastic cells from a normal cell population within several population doublings after transformation which is accompanied by activation of the endogenous L-plastin gene.
  • G418-resistance growth selectable markers like neomycin- resistance
  • Such a selection system allows early selection of transformed cells in vitro.
  • nascent cancer cells may be selectively inhibited in growth in vitro compared to normal nontransformed cells
  • use of a growth selectable marker such as G418-resistance under the control of the L-plastin promoter will facilitate identification, isolation, and propagation of the nascent cancer cell for further evaluation and characterization.
  • the L-plastin promoter is specifically and constitutively active in many cancer cell types and in normal leukocytes. Therefore, a recombinant gene comprising a gene encoding a cytotoxic product (hereinafter "toxin gene") under the control of the L-plastin promoter is specifically active in these target cell types and can be used to kill these cell types specifically.
  • Cells that can be targeted by a recombinant toxin gene of this invention include any cancer cell in which the L-plastin promoter is active.
  • Leukocytes that are neoplastic or infected with a pathogenic virus such as HIV or HTLV are examples of hemopoietic cells that are suitable targets for a recombinant toxin gene of this invention.
  • Undesirable leukocytes or other selected cells can be targeted using a ligand that binds to a receptor that is specific for the intended target cell type.
  • the encoded cytotoxic product can be a toxin which generally kills cells such as diphtheria toxin or ricin or a toxin that kills either cancer cells or leukocytes specifically.
  • the choice of the encoded toxin for a particular target cell population depends on the sensitivity of the selected target cells to the toxin. More specifically, some toxins kill certain cancer cells but do not kill leukocytes, and vice versa.
  • certain cytokines such as tumor necrosis factor and interferon, have inhibitory effects on the growth of cancer cells. These inhibitory effects are not apparent on normal eukaryotic cells, including leukocytes.
  • the gene for the encoded toxin of choice that is placed under the control of the L-plastin promoter encodes a toxin that provides specificity toward killing only the intended target cell type.
  • a method for producing the toxin in cancer cells and/or leukocytes comprises transfecting these eukaryotic cells with a vector encoding a toxin gene, the toxin gene being under the control of an L-plastin promoter.
  • the recombinant toxin gene can be used to transfect a mixed population of cells which population includes both normal, non-target cells and target cells where it is desired that the recombinant toxin gene is expressed selectively in the target cells under the control of the L-plastin promoter.
  • Such mixed populations include normal tissue that contains cancerous cells.
  • general toxins like diphtheria toxin or ricin whose synthesis is placed under the control of the L-plastin promoter can be specifically directed to the intended target cell through the use of drug delivery systems.
  • drug delivery systems include use of liposomes or retroviruses having an antibody combining site, a receptor, or the like which directs the liposome or retrovirus encapsulated drug to the target cells. These drug delivery systems are well known and do not constitute part of this invention.
  • toxins can be genetically modified to kill only the cell in which they are synthesized, thus preventing the spread of the toxin from the intended target cell to surrounding cells that are not the intended targets.
  • diphtheria toxin mutants have been developed in vitro that cannot enter eukaryotic cells but retain toxic activity if synthesized inside the target cells.
  • Example 8 illustrates use of the L-plastin promoter to express a foreign gene (neo) in transformed cells transfected with a construct comprising the promoter and the foreign gene. This example demonstrates that foreign genes can be expressed using the L-plastin promoter in cancer cells which endogenously express L-plastin.
  • Example 7 demonstrates that most, if not all neoplastic human cells exhibit some degree of activation of the L-plastin gene.
  • the example study demonstrates that a foreign gene, such as neo, attached to the L-plastin promoter was expressed in cells having endogenous activation of the L-plastin gene.
  • the cells that are the intended targets for gene therapy may be useful or necessary to recover the cells that are the intended targets for gene therapy as described above.
  • recovery of these cells facilitates the characterization of properties of the cells such as chromosomal ploidy, cellular protein synthesis, and oncogene activation.
  • recovery of target cells allows monitoring of the efficacy and safety of L-plastin therapy. This recovery can be accomplished by in vivo or ex vivo delivery of a recombinant drug resistance gene under the control of the L-plastin promoter.
  • a method for recovering the intended target cells comprises transfecting a population of cells containing the target cells with a vector encoding a selectable gene that is under the control of the L-plastin promoter.
  • the selectable gene can be any suitable drug resistance gene, i.e., any gene that encodes a protein which confers resistance to a drug, such as the antibiotic neomycin (G418 analogue) .
  • the cell population can be transfected either in vivo or ex vivo.
  • a transfected target cell can be selectively cultured and replicated in vitro from the tissue biopsy or body fluids (blood, mucus, urine amniotic fluid, etc.) because the transfected cell is capable of activating the L-plastin promoter.
  • the target cell can be isolated in the presence of an overwhelming majority of other cells which either lack an active L-plastin gene, lack the ability to activate the recombinant L-plastin promoter, or have not been targeted by the drug delivery system to receive the recombinant selectable gene under the control of the L-plastin promoter. Growth of the intended target cell out of the biopsy or body fluid requires culturing of those cells under selective conditions.
  • the cells are cultured in the presence of a suitable concentration of the appropriate drug such that the untransfected cells or transfected cells lacking the ability to activate the recombinant L-plastin promoter are killed by the drug or are unable to replicate in the presence of the drug, while the cells that synthesize the product of the drug resistance gene are not killed and can replicate in the presence of the drug. After a sufficient culturing time, all untransfected cells will be killed. The remaining replicating cells in the culture are the target cells. These cells can then be examined to determine their relevant properties.
  • This invention is further illustrated by the following specific but non-limiting examples. Temperatures are given in degrees Centigrade and concentrations as weight percent unless otherwise specified. Procedures which are constructively reduced to practice are described in the present tense, and procedures which have been carried out in the laboratory are set forth in the past tense.
  • Cell cultures were cell lines HuT-12, HuT-14, HOS, HT1080, MG63, RD, Wi-38VA13, Wi-26VA4, and rat-2. Those cell lines have been described in the literature and are available commercially from Leavitt et al., Hoi . Cell Biol . 7:2457 (1987). All cell types were cultured in MEM- ⁇ medium (Sigma) supplemented with 10% fetal calf serum and antibiotics.
  • CAT plasmid construction and CAT assay L-plastin genomic DNA fragments attached with Xmal linkers were cloned into the Xmal site of PUMSVOCAT, which was described by Salier and Kurachi Biotechniejue ⁇ , 7:30 (1989).
  • a 4.3 kb EcoRI-Hindlll fragment containing the 0-actin promoter was derived from the plasmid Ph ⁇ Apr-1-neo Gunning et al., Proc. Nat 'l Acad. Science USA 84:4831 (1987) and cloned into PUMSVOCAT.
  • Transfection of plasmid DNA into HuT-14 cells was performed by the calcium phosphate precipitation method Ng et al., Nucleic Acids Res . 17:601 (1989).
  • Cells grown in a 100 mm dish were harvested at 80-90% confluency, centrifuged, resuspended in 200 ml PBS, and lysed by freeze-thaw. After removing the insoluble cell debris by centrifugation, each cell lysate was measured for protein concentration by the protein assay kit of Bio-Rad.
  • CAT assay was then performed with the CAT ELISA kit purchased from 5 Prime—3 Prime, Inc. (West Chester, P.A.).
  • Primer extension A 25-mer oligonucleotide complimentary to the mRNA and corresponding to the end of the first exon 5 (bases 125 to 149; see Table 2) was labeled at the 5 end with ⁇ P-ATP. After labeling, the oligonucleotide (0.1 ⁇ g) was precipitated with ethanol and resuspended in 20 ⁇ l of distilled water. A 10X dilution was made 1 ⁇ l of which was annealed to 16 ⁇ g of cellular RNA in a 10 ⁇ l solution
  • the annealing mixture was composed of 48 ⁇ l (4.8 ⁇ g) of M13 DNA, 16 ⁇ l of annealing buffer (0.1 M Tris, pH 8.5, 50 mM MgCl 2 ) , and 16 ⁇ l of the 10X diluted labeled oligonucleotide. After incubation at 56°C for 1 hour, the annealing mixture was further mixed with 10 ⁇ l of 0.1 M DTT, 9
  • the pellet was resuspended in 25 ⁇ l of sequencing dye solution, heated at 90°C for 10 minutes, and loaded onto a 5% polyacrylamide sequencing gel. After electrophoresis, a DNA band was detected by autoradiography and eluted from the gel by the "crush and soak" method.
  • the single-stranded probe prepared above was resuspended in distilled water, and an aliquot was re-precipitated with 28 ⁇ g of each test cellular RNA.
  • the pellet was resuspended in 30 ⁇ l of hybridization buffer (40 mM PIPES, pH 6.4, 1 mM EDTA, 0.4 M NaCl, 80% formamide) , and incubated at 30°C for 15 hour.
  • Anchored PCR The procedure used for cloning the 5' ends of plastin cDNAs with the anchored PCR method is described in Lin et al., Mol . Cell Biol . 10:1818 (1990). That procedure was used with the following modifications. (i) For reverse transcription, the procedure as described above in primer extension was used. (ii) For PCR, a downstream primer closer to the 5' ends of the L-plastin mRNA (247 bp from the 5' end of the existing cDNA clone as described in ref. Lin et al. , Mol . Cell Biol . 10:1818 (1990) was used.
  • HuT-14 L-plastin 1 19/53 35.8 (f ibrosarcoma) 2 9/35 25.7 3 25/96 26.0 4 23/79 29.1 total 76/263 28.9
  • Wi-26VA4 embryonic L-plastin 1 3/2893 0.01 lung 2 7/3172 0.02 fibroblast 3 9/3043 0.03
  • This L-plastin genomic fragment contained 4.2 kb of 5'-flanking sequence, the first exon, and 0.8 kb of the first intron.
  • lacZ E. coli 3-galactosidase
  • 0-actin promoter expression vector pH ⁇ APr-1-neo, to generate the plasmid pH0APr-/3gal-neo.
  • transfected diploid cells were then incubated in medium containing 600 ⁇ g G418 (Gibco-BRL) per ml for six to seven days after which the drug was omitted for up to 12 days after transfection initiation at which time colonies were assayed
  • transfected cells were trypsinized and divided into two or three 100 mm dishes per each transfection. After 18 to 24 hours, the culture medium was adjusted to 800 ⁇ g G418 per ml
  • the /3-galactosidase product (120,000 M,, pi 5.2) was identified in subclonal HuT cells by the following criteria: its co-migration in the 2-D gel with purified unlabeled E. coli 3-galactosidase and by its binding of anti-E. coli jS-galactosidase antibody in a two dimensional gel Western blot. Identical results were obtained with rat-2 colonial cells expressing 3-galactosidase.
  • the growth-selectable neomycin-resistance gene in pSV2-neo and pHSApr-1-neo was used to select mammalian cell colonies that co-expressed a recombinant gene driven by the jS-actin promoter.
  • the protein product of the native human mutant 0-actin gene was co-expressed in 4 out of 9 (44%) of the colonies produced by G418-resistant diploid human fibroblasts and 12 out of 17 (71%) of the colonies produced by G418-resistant HuT-12 fibrosarcoma cell line.
  • tubulin and actin antisense RNA transcripts were detected in 3 out of 5 (60%) and 4 out of 8 (50%) of the colonies produced by G418-resistant HuT-12.
  • expression of a recombinant human tissue plasminogen activator (tpa) gene was observed in 4 out of 8 (50%) of the colonies produced by G418-resistant diploid human fibroblasts and 12 out of 17 (71%) of the colonies produced by G418-resistant HuT-12.
  • Ets-1 core motif AGGAAG nearest the L-plastin promoter (-104 bp, or -88 bp upstream from the TATAAA box) was found to be homologous to Ets-1 motifs next to the promoters of other genes that encoded hemopoietic-specific proteins such as Ets-1, interleukins 2, 3, 4, and 6, G-CSF, GM-CSF, the T-cell receptor a and ⁇ chains.
  • this core motif was found in the LTR sequences of the lymphotropic viruses HIV-1 and HTLV-1.
  • AGAACAGTTTGGTTT Two regions related to the progesterone responsive element at -1127 bp (AGAACAGTTTGGTTT; Table 2 , indicated as PR) and at -1660 bp (AGAACACTGTGCTTT; Table 2 , indicated as PR) which are half-palindromes of the consensus progesterone responsive element (AGAACAN 3 TGTTCT) and one region related to the estrogen responsive element at -73 bp (ATTTCACTGTGACCT; Table 2, indicated as ER) which is a half-palindrome of the consensus estrogen responsive element (AGGTCAN 3 TGACCT) were also found.
  • the estrogen binding motif was flanked by the Ets-1 motif 16 bp upstream and the Spl motif 24 bp downstream.
  • Both the estrogen responsive element and the progesterone responsive element are functional since these hormones induce L-plastin expression in cell cultures of hormonally responsive reproductive tissues.
  • PCR with primers homologous to these estrogen responsive element and progesterone responsive element sequences established that these specific elements are unique to the L-plastin gene because only a single amplification product homologous to the L-plastin sequence between the estrogen responsive element and the upstream progesterone responsive element could be amplified.
  • the two fragments (PS and PH) having the shortest sequence (180 bp) upstream from the TATAAA box showed the strongest promoter activity, which was nearly equal to the activity of the -actin promoter. These two fragments differed only by the presence of additional 29 bp of the first exon and 717 bp of the first intron in the longer fragment PH. These additional sequences apparently had no effect on the promoter activity.
  • Fragments SS and CS differed from fragment PS by having longer upstream sequences, which appeared to account for a 63-65% reduction in promoter activity.
  • the longest fragment (EH) differed from fragment PH only by having a 4 kb additional upstream sequence, which resulted in a reduction of promoter activity by approximately 88%. This lowest level of L-plastin promoter activity was consistent with the difference in the levels of protein synthesis between L-plastin and jS-actin in HuT-14 cells.
  • L-plastin gene contains a very potent transcriptional promoter which is attenuated by upstream negative regulatory elements.
  • the high activity of the smallest promoter fragment indicates that in some instances during transfection the recombinant promoter became truncated leading to removal of upstream elements that attenuate the activity of the promoter in these L-plastin-negative cell lines.
  • L-plastin The finding of steroid hormone response elements near the L-plastin promoter indicates that expression of L-plastin in hemopoietic cells may, under some circumstances, be subject to hormonal control either locally (in reproductive tissues that synthesize and secrete steroid hormones) or systemically by the circulating levels of these hormones.
  • progesterone receptors have been detected in a small sub- population of peripheral blood lymphocytes of non-pregnant women and the abundance of this hemopoietic sub-population has been shown to increase as much as 30-fold in the late stages of pregnancy when progesterone levels become greatly elevated.
  • L-plastin a fundamental hemopoietic architectural protein such as L-plastin demonstrates the role of hormones in control of immune system by their regulation of hemopoietic-specific gene expression during pregnancy. Therefore, the L-plastin promoter region can be used to engineer steroid hormone- regulated expression of beneficial proteins and RNA in steroid hormone-responsive tissues and in steroid hormone-responsive hemopoietic cells during pregnancy.
  • Ets-l binding motifs upstream from the TATAAA box is consistent with the hemopoietic specificity of L-plastin gene expression in normal cells because the Ets-l binding motif is found near the promoters of many other genes that are expressed in a hemopoietic cell-specific manner.
  • the presence of the four Ets-l elements explains the high stable rate of constitutive expression observed in hemopoietic cells and the stringent repression of the L-plastin promoter observed in non-hemopoietic cells.
  • the transcription start site(s) were determined by primer extension and SI (mung bean nuclease) mapping methods.
  • SI mung bean nuclease mapping methods.
  • a 25-nucleotide primer corresponding to the 3' end of exon 1 (Table 2) was used.
  • the primer could only be extended with RNA templates prepared from the two cell lines that expressed L-plastin, i.e. HuT-14 fibrosarcoma cells and CEM lymphoblastoid cells, but the primer could not be extended with RNA from diploid KD fibroblasts which do not express L-plastin.
  • the extension products were of multiple lengths, indicating that the L-plastin gene had multiple transcription start sites.
  • One start site appeared to be 10 nucleotides downstream from the TATAAA box (nucleotide +1) .
  • the genomic probe was specifically protected by RNAs prepared from the two cell lines that expressed L-plastin, i.e. HuT-14 fibrosarcoma cells and CEM lymphoblastoid cells, but the genomic probe could not be protected with RNA from diploid KD fibroblasts which do not express L-plastin.
  • the protection products were also of multiple lengths. However, there were some differences in the banding patterns between the primer extension and the nuclease mapping products. These differences may result from local DNA secondary structure which may influence the movement of reverse transcriptase along the template.
  • transcription initiation occurs at multiple sites between 10 bp and 111 bp downstream from the TATAAA box, (ii) the most frequently used transcription initiation sites are located 91 bp to 111 downstream from the TATAAA box, and (iii) patterns of transcription initiation are virtually identical in CEM lymphoblastoid cells and in HuT fibrosarcoma cells.
  • EXAMPLE 5 • ecomJ inaiit L-plastin Promoter Activity in Normal and Neoplastic Fibroblasts
  • E. coli /3-galactosidase expression from a recombinant E. coli lacZ gene as a reporter was used to measure the ability of various cell lines and strains to support constitutive activation of the L-plastin promoter.
  • This assay permits assessment of the percentage of cells in individual G418-resistant colonies that co-express jS-galactosidase and assessment of uniformity of expression under the control of a mammalian gene promoter.
  • An L-plastin promoter expression vector, pHLPPr-1-neo was constructed using the largest promoter fragment characterized in Table 4 because this fragment contained potential cis-acting regulatory elements that were greater than 1 kb upstream from the TATAAA box.
  • the jS-galactosidase reporter gene (lacZ) was inserted into this plasmid at the 3' end HinDIII site of the promoter (Table 4) to create the plasmid pHLPPr-Sgal-neo.
  • the ⁇ -actin promoter was chosen for comparison with the L-plastin promoter because this promoter is a strong constitutive promoter which is active in all replicating cell types; thus, the 3-galactosidase reporter gene was also inserted into the plasmid pH/?APr-l-neo at the HinDIII site of its polylinker to create the plasmid pH ⁇ APr- ⁇ gal-neo (as described in Example 1) .
  • Table 5 compares the co-expression frequencies of these two promoters in driving ⁇ -galactosidase expression in various human tumor-derived cell lines, a diploid human fibroblast strain, and two SV40-transformed cell lines.
  • relative activity is the activity of L-plastin divided by the activity of 3-actin.
  • the color reaction catalyzed by ⁇ -galactosidase developed faster and produced a darker blue color when the 0-actin promoter was used indicating that the jS-actin promoter was the stronger of the two promoters as suggested by their relative activities in supporting CAT expression (Table 4) .
  • the extent of the color reaction produced by the two promoters can be seen by comparison of the darker 3-galactosidase-positive colonies and cells produced with the 0-actin promoter with those colonies and cells produced with the L-plastin promoter.
  • the lower colonial frequency of jS-galactosidase co-expression with the L-plastin promoter is due to the lower level of constitutive activity (and thus lower sensitivity of the assay) compared to the 0-actin promoter.
  • HOS which expresses a relatively low level of L-plastin protein and mRNA constitutively, exhibited a 5.5% jS-galactosidase expression frequency when expression was under the control of the L-plastin promoter and a 35% expression frequency when expression was under the control of the 0-actin promoter (Table 5) .
  • MG63 and RD which do not express detectable L-plastin protein and mRNA, exhibited only one 0-galactosidase-positive colony (1%) and no positive colonies ( ⁇ 0.2%), respectively, when expression was under the control of the L-plastin promoter in contrast to a 25% and 31% expression frequency, respectively, when expression was under the control of the /3-actin promoter (Table 5) .
  • Two SV40-transformed human fibroblast cell lines were also tested for their ability to utilize the L-plastin promoter constitutively. These cell lines, which do not express endogenous L-plastin gene (Wi-38VA13, Wi-26VA4) , exhibited a low percentage of colonies that expressed 3-galactosidase at low constitutive levels under the control of the L-plastin promoter, ranging from 0.01 to 0.5. In contrast, ⁇ -galactosidase expression under the control of the 0-actin promoter ranged from 24% to 38% in these three cell lines (Table 5) .
  • the rare Wi-38VA13 and Wi-26VA4 colonies that expressed 0-galactosidase with the L-plastin promoter were weakly stained and could only be detected by microscopic examination indicating low levels of 0-galactosidase expression.
  • Table 5 the relative frequency of G418-resistant colonies that expressed jS-galactosidase under the control of the L-plastin promoter was compared among the seven immortal cell lines and normal diploid BC fibroblasts after normalization to the frequency of G418-resistant colonies that expressed ⁇ -galactosidase under the control of the j8-actin promoter. This calculation takes into account the finding that differing cell strains express the co-selected gene at different frequencies and allows the comparative rating of the differing cell types for their ability to support activation of the recombinant L-plastin promoter.
  • the two fibrosarcoma cell lines which expressed the highest levels of L-plastin (HuT-14 and HT1080; Lin et al. , Mol . Cell Biol . 8:4659 (1988) also exhibited the highest relative activity of the recombinant L-plastin promoter which was 0.50 to 0.53 as active as the recombinant 3-actin promoter.
  • HOS cells which expressed a lower, but detectable, level of L-plastin exhibited reduced activity of the recombinant L-plastin promoter which was 0.16 as active as the ⁇ -actin promoter.
  • the four other immortal cell lines (MG63, RD, Wi-38VA13, and Wi-26VA4) which did not express detectable levels of L-plastin polypeptide or mRNA exhibited very low relative activity of the recombinant L-plastin promoter ranging from 0.04 down to less than 0.001 as active as the 0-actin promoter.
  • the 0.04 rating for MG63 was based on the observation of only one positive colony out of 103 G418-resistant colonies examined while the other three cell lines gave much lower ratings based upon examination of much larger numbers of colonies.
  • diploid BC fibroblasts which exhibited no expression of j8-galactosidase under the control of the L-plastin promoter was rated by this method at less than 0.023.
  • EXAMPLE 6 Use of the L-plastin Promoter to Determine Neoplastic Transformation
  • An expression vector, designated pHLPPr-1-neo was constructed using a 5.1 kb genomic fragment containing the L-plastin promoter and flanking sequences, as described in detail in the Examples.
  • ⁇ -galactosidase as a reporter of activation of the L-plastin promoter
  • the activity of L-plastin promoter was evaluated in a panel of fibroblastoid cell types which included a normal fibroblast strain that did not express detectable L-plastin, tumor-derived fibrosarcoma strains which did or did not express L-plastin, and SV40- transformed human fibroblasts which did not express L-plastin.
  • a second expression vector designated pHLPPr-neo was constructed using the same 5.1 kb genomic fragment containing the L-plastin promoter and flanking sequences as described in detail in the Examples. Using the neomycin resistance gene (neo) as the reporter of activation of the L-plastin promoter, the activity of the L-plastin promoter was evaluated in a panel of fibroblastoid cell types.
  • This example demonstrates the utility of the recombinant promoter in investigation of mechanism(s) of neoplastic transformation leading to activation of the L-plastin promoter region, and the potential role of hormonal regulation in activation of the L-plastin gene in tumorigenesis.
  • EXAMPLE 7 Evaluation of Activation of the L-plastin Gene Since L-plastin is a common marker of human cancer cells, the frequency and nature of this gene activation event were of great interest. Therefore, to determine whether activation of the L-plastin gene was more wide-spread than previously thought, the sensitive Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used to examine L-plastin SV40- transformed fibroblast cell lines, tumor derived cell lines, and in a variety of diploid cell types for low level activation of the L-plastin gene which were L-plastin negative by less sensitive methods.
  • RT-PCR Reverse Transcriptase Polymerase Chain Reaction
  • RT-PCR analysis was performed as follows for detection of latent L-plastin expression.
  • Cellular RNA was isolated from cultured cells by the guanidine hydrochloride phenol-chloroform extraction method (Chomczynski et al, Anal . Biochem . 162: 156-159 (1987)).
  • 50 ng of random hexamers was annealed to 5 ⁇ g sample RNA and extended by Moloney Murine Leukemia Virus RNase H-Reverse Transcriptase (Gibco BRL; Gaithersberg, MD) in a reaction volume of 10 ⁇ l.
  • a portion (1 ⁇ l) of the cDNA was used in PCR which included 25 ng of L-plastin oligomer (TGAAAGAACAATCAACAAA) as the upstream primer and 25 ng of L-plastin oligomer (TTAATGGAACCTGGTTGG) as the downstream primer in a reaction volume of 50 ⁇ l containing 1.25 units of taq DNA polymerase (Gibco BRL; Gaithersberg, MD) and buffer supplemented with 2 mM of MgCl 2 and 0.1 mM deoxynucleotide triphosphates.
  • the PCR was run in an Ericomp thermocycler for 35 cycles with each cycle consisting of 94°C, 30 seconds; 45°C, 40 seconds; 72°C, 40 seconds. After an additional 10 minute incubation at 72°C, 10 ⁇ l of the PCR reaction solution was electrophoresed in a 1.5 % agarose gel. After staining and photography, the DNA was transferred to a Duralose-UV membrane (Stratagene; La Jolla, CA) and hybridized with a 32 P-labeled L-plastin probe.
  • a Duralose-UV membrane Stratagene; La Jolla, CA
  • RT-PCR was performed as described above on RNA isolated from eight diploid human cell types (four fibroblast strains, mammary epithelial cells, skin keratinocytes, umbilical vein endothelial cells, and aortic smooth muscle cells) , four SV40-transformed human fibroblast strains (Wi38-VA13, Wi26-VA4, GM3022, and M1SV; Lin et al, J. Biol . Chem . 268:2781-2792 (1993)), and 12 tumor derived cell lines in which L-plastin expression was undetectable using two dimensional gel protein profiling or Northern blotting (Lin et al, J. Biol . Chem . 268:2781-2792 (1993)).
  • RT-PCR amplification products were electrophoretically resolved in a 1% agarose gel, trans- blotted onto nylon membranes and hybridized with an L-plastin cDNA probe to confirm their identities as amplified sequences from the L-plastin mRNA for analysis by Southern blots as described above.
  • These Southern blots were designated Blot 1 (described below) , and the results of the full survey of L-plastin expression are summarized in Table 6 (below) which includes the additional cell types that were found previously to express L-plastin more abundantly (Lin et al, J. Biol . Chem . 268:2781-2792 (1993)).
  • Blot 1 was a Southern blot of RT-PCR amplification products from normal and neoplastic human cells. The samples in the blot were as follows:
  • Wi26-VA4 SV40 transformed fibroblasts 14 HuVEC umbilical endothelial cells
  • L-plastin expressions are as follows: “none” means not detectable by RT-PCR; “trace” means detectable by RT-PCR only; “low” means detectable by two dimensional gel or Northern blot analysis (Lin et al, J “ . Biol . Chem. 268:2781-2792 (1993)); “high” means abundant expression (Lin et al, J. Biol . Chem.
  • RNA isolated from the diploid cell types was uniformly negative for the L-plastin mRNA/RT-PCR product (Blot 1, samples 10 and 14-18) , while RNA from all four SV40- transformed fibroblasts supported the significant amplification of two L-plastin mRNA/RT-PCR products, one at
  • the smaller amplification product may be generated by primer annealing to an alternative sequence in the duplicated actin binding domain of L-plastin (de Arruda et al, J. Cell Biol .
  • RNA from diploid MCR5 fibroblasts (Blot 1, sample 16) , the parent strain of MRC5-SV2 which synthesizes abundant levels of L-plastin (Lin et al, J. Biol . Chem. 268:2781-2792 (1993); Celis et al, Electrophoresis 11:1072-1113 (1990)), and
  • RNA from diploid Wi38 fibroblasts (Blot 1, sample 10) , the parent strain of Wi38-VA13 (Blot 1, sample 12) , exhibited no L-plastin mRNA transcripts detectable by RT-PCR.
  • 8 out of 12 of the human tumor-derived cell lines that were thought to be negative for L-plastin expression (Lin et al, J.
  • pHuLPPr-neo All plasmids were grown in E. coli host XL-blue, and recombinant plasmid DNA was extracted by a standard alkaline lysate procedure.
  • the plasmid, pHjSAPr-1 which contains human / 8-actin gene promoter has been described in Gunning et al, Proc . Natl . Acad . Sci . 84: 4831-4835 (1987).
  • the plasmid, pNEO which contains 1.5 kb neo-gene fragment was purchased from Pharmacia (Alameda, CA) . DNA restriction fragments separated on agarose gels were purified using Gene Clean kit (Bio 101) . All restriction enzymes were from New England BioLabs (Beverly, MA) , and T4 ligase was from Gibco BRL (Gaithersburg, MD) .
  • the 4.3 kb EcoRI-Hindlll fragment of the j8-actin gene promoter was excised from pH ⁇ APr-l and replaced with the 5.1 kb, EcoRI-Hindlll fragment containing the L-plastin gene promoter, 4.2 kb of the 5'-flanking sequence, the first exon, and 0.8 kb of the first intron (Lin et al, J. Biol . Chem . 268:2793-2801 (1993)).
  • the 1.5 kb neo cDNA HinDIII-BamHl fragment was excised from pNEO, and inserted at the Hindlll and BamHI sites in the plasmid polylinker adjacent to the 3' end L-plastin promoter fragment to produce pHuLPPr-neo.
  • the plasmid, pH/SAPr-1-neo which contains an SV40-neo gene (Gunning et al, Proc. Natl . Acad. Sci . 84: 4831-4835 (1987)) was used for control transfections.
  • the frequency of colony formation in pHuLPPr-neo transfected cells was divided by the frequency achieved with pH0APr-l-neo conducted in parallel. G418-resistant colonies were isolated by trypsinization, and transferred to replicate 15 mm wells of 24 well culture dishes for 35 S-methionine labeling and further culturing.
  • Sub- confluent cell monolayers were labeled in 24 well tissue culture dishes with 35 S-methionine for 6-7 hours. Samples were prepared and two dimensional gels were analyzed as described in Leavitt et al, Molec . Cell . Biol . 6: 2721-2726 (1986) and Leavitt et al, Molec . Cell . Biol . 7: 2467-2476 (1987).
  • the osteosarcoma-derived MG63 cell line produced no G418-resistant colonies from 5.2xl0 5 transfectedcells in three independent transfection trials with the HuLPPr-neo gene while HuT-14 fibrosarcoma cell line (the L-plastin positive variant cell line of the 8387 fibrosarcoma) produced on average 568 colonies from 3.4x10 s transfected cells.
  • HuT-14 fibrosarcoma cell line the L-plastin positive variant cell line of the 8387 fibrosarcoma
  • the rhabdomyosarcoma- derived RD cell line produced an average of 41 G418-resistant colonies from 4.6x10 s transfected cells, and the SV40-transformed fibroblast cell line, Wi38-VA13, produced an average of 101 G418-resistant colonies from 7.4x10 s transfected cells.
  • the cells of these G418-resistant colonies produced with HuLPPr-neo and the SV40-neo gene were examined for L-plastin expression by trypsinization of the primary colony and duplicate plating of individual colonial cells which provided one culture for immediate labeling with 35 S-methionine and the second culture for further propagation of the subclonal cell line and characterization of its properties.
  • the clonal cells were estimated to have achieved about 14 population doublings from the start of colony development through to protein labeling (expansion of 1 cell to 32,000 cells).
  • Polypeptides x and T were used in each gel as reference polypeptides for the location of L-plastin in the gel pattern.
  • Gel 1 was a protein profile which showed that one Wi38-VA13 subclone D-1.4 exhibited no apparent synthesis of L-plastin because it lacked a polypeptide species at the L-plastin electrophoretic position. By contrast, gels showing the proteins synthesized by the four other clonal strains
  • L-plastin mRNA and protein had not been previously detected in either of these cell lines by conventional Northern blotting and repeated two dimensional gel protein profiling (Lin et al, J. Biol . Chem . 268:2781-2792 (1993))
  • 9 out of 12 of the Wi38-VA13 subclones tested and 18 out of 19 of the RD subclones clones tested exhibited a trace level of L-plastin synthesis by the appearance of a polypeptide at the discrete electrophoretic position of L-plastin. This barely detectable level of L-plastin synthesis was too low to be confirmed by Western blotting with anti-plastin antibody.
  • expression of L-plastin from the endogenous genes of other cells (Lin et al, J.
  • L-plastin polypeptide synthesis was examined in the two stable Wi38-VA13 subclones which were expanded in cell number through at least 10 additional population doublings (the estimated number of doublings from a confluent 15-mm culture well to confluence in two 100 mm petri dishes) .
  • Subclone D-2.8 exhibited a low, but stable level of L-plastin synthesis, while subclone C-2.3 exhibited no trace of L-plastin expression like the parent Wi38-VA13 strain (Lin et al, J. Biol . Chem . 268:2781-2792 (1993)).
  • a Wi38-VA13 G418-resistant subclone which was transfected with a cDNA gene placed under the control of the 0-actin promoter was also analyzed.
  • the electrophoretic position of this recombinant form of L-plastin was identical to the electrophoretic species in D-2.8 tentatively identified as L-plastin (this can be judged by comparing the relative positions of polypeptides x and C with L-plastin) .
  • the electrophoretic position of L-plastin in MRC5-SV2 (Lin et al, J. Biol . Chem .
  • L-plastin in the MRC5-SV2 strain was also identical to the protein tentatively identified as L-plastin in D-2.8.
  • the level of synthesis of L-plastin in D-2.8 was too low to be confirmed by Western blotting which was used to confirm the identity of L-plastin in MRC5-SV2 (Lin et al, J. Biol . Chem . 268:2781-2792 (1993)).
  • Blot 2 was a Northern blot analysis of L-plastin mRNA levels in HuLPPr-neo-selected subclones.
  • the upper panel of the blot was hybridized to the L-plastin specific probe and the lower panel was the same blot in which the L-plastin probe was stripped off and re-hybridized with a T-plastin probe (Lin et al, Mol . Cell . Biol . 8:4659-4668 (1988)).
  • the sample RNA of the blot was from the following cell lines:
  • Wi38-VA13 subclone C-2.3 cultured in the continuous presence of G418 Wi38-VA13 subclone C-2.3 cultured in the absence of G418 for one passage (approximately 3 population doublings)
  • Blot 2 demonstrated the presence and elevation of L-plastin mRNA (3.7 kb) in D-2.8 grown in the absence or continuous presence of G418 (Blot 2, samples 8 and 9, respectively) and the absence of detectable L-plastin mRNA in C-2.3 also grown in the absence or continuous presence of G418 (Blot 2, samples 10 and 11, respectively).
  • L-plastin mRNA in C-2.3 was consistent with its failure to synthesize the L-plastin-like polypeptide species.
  • the level of L-plastin RNA in D-2.8 was lower than the level of L-plastin mRNA in HuT-13 fibrosarcoma cells that express L-plastin abundantly (Blot 2, sample 1; Leavitt et al, Molec . Cell . Biol . 6:2721-2726 (1986)).
  • Even lower levels of L-plastin mRNA were detected in two additional subclonal strains B-3.2 (Blot 2, sample 2) and C-3.10 (Blot 2, sample
  • exogenous L-plastin promoter fragments that had integrated into the genomic DNA of these Wi38-VA13 subclones were examined by Southern blotting using a DNA probe that spanned the entire length of the genomic L-plastin promoter fragment to determine the amount of the exogenous L-plastin promoter DNA in the transfected strains.
  • Blot 3 was a Southern genomic blot performed as follows using a genomic Hindlll fragment probe that was homologous and hybridized to the 6 kb Hindlll genomic fragment containing the L-plastin promoter (Lin et al, J. Biol . Chem. 268:2781-2792 (1993); Lin et al, J. Biol . Chem . 268:2793-2801 (1993)).
  • 10 ⁇ g Hindlll digested genomic DNA Feinberg et al, Anal. Biochem .
  • Wi38-VA13 subclone C-2.3 While the parent Wi38-VA13 cell line exhibited only the expected 6 kb Hindlll genomic hybrid (Blot 3, sample 1) corresponding to the endogenous gene, transfected subclones exhibited the exogenous promoter sequence (a 5.1 kb
  • D-2.8 (Blot 3, sample 4) exhibited approximately 2 diploid genomic equivalents of exogenous promoter DNA determined by comparison of the hybridization signal of the larger exogenous restriction fragments with the intensity of the hybridization signal from the native genomic band (6 kb Hindlll fragment).
  • C-2.3 (Blot 3, sample 5) exhibited a more intense hybridization signal for the exogenous promoter fragment suggesting the possibility of its amplification.
  • Two other clones, B-3.4 and D-2.1 (Blot 3, samples 2 and 3, respectively) exhibited similar levels of exogenous L-plastin gene sequences (Blot 3, samples 2 and 5). Thus, the exogenous L-plastin promoter fragment was not further fragmented or truncated during the transfection process.

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Abstract

La région de régulation et du promoteur du gène L-plastine est prévue pour être utilisée comme un vecteur transcriptionnel et traductionnel d'autres gènes d'expression chez des hôtes mammifères. Le promoteur L-plastine associé à une partie limitée de la region de régulation agit comme un promoteur fort. Le promoteur associé à la région de régulation amont est actif spécifiquement dans les cellules hémopoïétiques et dans les cellules transformées des tissus solides qui sont d'origine non hémopoïétique, et peut être induit en réponse à l´÷strogène et la progestérone dans les cellules sensibles à ces hormones.
PCT/US1994/000436 1993-01-26 1994-01-25 Region du promoteur de l-plastine et ses utilisations Ceased WO1994017182A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU60876/94A AU6087694A (en) 1993-01-26 1994-01-25 The l-plastin promoter region and its uses

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US916793A 1993-01-26 1993-01-26
US009,167 1993-01-26

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000049147A1 (fr) * 1999-02-19 2000-08-24 Octagene Gmbh Couples hormone-recepteur hormonal, constructions d'acides nucleiques, et utilisation de ceux-ci en therapie genique
EP1135022A4 (fr) * 1998-12-04 2003-07-09 Univ Yale Therapie genique dirigee par le promoteur de plastine
US8445660B2 (en) 2001-04-05 2013-05-21 The Johns Hopkins University Chimeric vaccines

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5002870A (en) * 1988-06-07 1991-03-26 California Institute For Medical Research Plastin isoforms and their use

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5002870A (en) * 1988-06-07 1991-03-26 California Institute For Medical Research Plastin isoforms and their use

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIOCHIMICA ET BIOPHYSICA ACTA, Volume 951, issued 1988, B. WASYLYK, "Enhancers and Transcription Factors in the Control of Gene Expression", pages 17-35. *
CELL, Volume 58, issued 1989, W.S. DYNAN, "Modularity in Promoters and Enhancers", pages 1-4. *
NATURE, Volume 301, issued 17 February 1983, TAVERNIER et al., "Deletion Mapping of the Inducible Promoter of Human IFN-Beta Gene", pages 634-636. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 84, issued July 1987, GUNNING et al., "A Human Beta-Actin Expression Vector System Directs High-Level Accumulation of Antisense Transcripts", pages 4831-4835. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 85, issued August 1988, VAN ZONNEVELD et al., "Type 1 Plasminogen Activator Inhibitor Gene: Functional Analysis and Glucocorticoid Regulation of its Promoter", pages 5525-5529. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 261, Number 13, issued 05 May 1986, EPSTEIN et al., "Isolation of a Rat Parvalbumin Gene and Full Length cDNA", pages 5886-5891. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1135022A4 (fr) * 1998-12-04 2003-07-09 Univ Yale Therapie genique dirigee par le promoteur de plastine
AU777479B2 (en) * 1998-12-04 2004-10-21 Yale University Plastin promoter directed gene therapy
WO2000049147A1 (fr) * 1999-02-19 2000-08-24 Octagene Gmbh Couples hormone-recepteur hormonal, constructions d'acides nucleiques, et utilisation de ceux-ci en therapie genique
US8445660B2 (en) 2001-04-05 2013-05-21 The Johns Hopkins University Chimeric vaccines
US9499589B2 (en) 2001-04-05 2016-11-22 The Johns Hopkins University Chimeric vaccines
US9993546B2 (en) 2001-04-05 2018-06-12 The Johns Hopkins University Lysosomal targeting of antigens employing nucleic acids encoding lysosomal membrane polypeptide/antigen chimeras
US11110164B2 (en) 2001-04-05 2021-09-07 The Johns Hopkins University Lysosomal targeting of antigens employing nucleic acids encoding lysosomal membrane polypeptide/antigen chimeras

Also Published As

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