WO2001063292A2 - Compositions et utilisations de het, un nouveau modulateur de l'action oestrogenique - Google Patents

Compositions et utilisations de het, un nouveau modulateur de l'action oestrogenique Download PDF

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WO2001063292A2
WO2001063292A2 PCT/US2001/006135 US0106135W WO0163292A2 WO 2001063292 A2 WO2001063292 A2 WO 2001063292A2 US 0106135 W US0106135 W US 0106135W WO 0163292 A2 WO0163292 A2 WO 0163292A2
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het
cells
cell
saf
dna
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PCT/US2001/006135
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WO2001063292A3 (fr
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Steffi Oesterreich
C. Kent Osborne
Adrian V. Lee
Suzanne A.W. Fuqua
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Board Of Regents, The University Of Texas System
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Definitions

  • the present invention relates generally to cancer biology. In particular, it concerns novel methods and compositions for modulating estrogen actions.
  • the present invention further relates to detection, diagnosis and prognosis of breast cancer and the identification of tamoxifen-resistant breast cancers.
  • Another aspect of the present invention relates to gene therapy for altering the phenotype of tumor cells. More particularly, it concerns use of expression vectors comprising an HET gene to increase the sensitivity of the tumor cell to estrogen antagonists, or to decrease the sensitivity of the tumor cell to estrogen and estrogen agonists.
  • Nuclear receptors are divided into three groups: steroid receptors, retinoic acid/thyroid receptors, and orphan receptors.
  • a fundamental difference between steroid receptors and retinoic acid/thyroid receptors is that the latter are DNA-bound active repressors in the absence of ligand.
  • An active role for corepressors such as N- CoR (Shibata et al, 1997; Horlein et al, 1995) and SMRT (Chen and Evans, 1995) for the silencing activity of unliganded retinoic acid/thyroid receptors has been well established.
  • steroid receptors In contrast to retinoid/thyroid receptors, steroid receptors show little DNA- binding activity in the absence of ligand and thus are thought to have no silencing ability. However, recently it has become clear that steroid receptors are also found in repressor complexes, particularly when the receptor is bound to antagonists, and that the antagonist function may in part be mediated by corepressors (Montano et al, 1999; Jackson et al, 1997; Lavinsky et al, 1998).
  • the estrogen receptor (ER) is a steroid receptor that is a member of a superfamily of nuclear transcription factors.
  • ER action can be altered by: 1) interaction with other nuclear transcription factors such as API (Webb et al, 1995), SP1 (Rishi et al, 1995; Porter et al, 1997; Porter et al, 1996, and members of the basal transcription machinery (Tsai and O'Malley, 1994; Smith and Toft, 1993) cross-talk with growth factor systems (Kato et al, 1998; Webb et al, 1995) associations with nuclear receptor coactivators and corepressors (Shibata et ⁇ /., 1997).
  • API Webb et al, 1995
  • SP1 Rhi et al, 1995
  • Porter et al, 1997 members of the basal transcription machinery
  • cofactors that can regulate the transcriptional activity of nuclear hormone receptors was first suggested by transcriptional squelching between ER and progesterone receptor (PgR) (Conneely et al, 1989; Bocquel et al, 1989).
  • PgR progesterone receptor
  • coactivators A number of cofactors capable of increasing hormone receptor action, termed coactivators, have been identified (reviewed in Shibata et al, 1997; Glass et al, 1997).
  • the family of corepressors is smaller, the best characterized being nuclear receptor corepressor (N-CoR) (Shibata et al, 1997; Horlein et al, 1995) and silencing mediator of retinoid and thyroid receptors (SMRT) (Sande and Privalsky, 1996; Chen and Evans, 1995).
  • N-CoR nuclear receptor corepressor
  • SMRT silencing mediator of retinoid and thyroid receptors
  • REA a corepressor termed REA, which is specific for ER.
  • Many cofactors seem to regulate receptor activity by modulating chromatin structure.
  • Coactivators such as p300/CBP (Ogryzko et al, 1996; Bannister and Kouzarides, 1996), PCAF (Yang et al, 1996; Chen et al, 1997), and SRC-1 (Spencer et al, 1997) have intrinsic histone acetyltransferase activity, which results in the destabilization of nucleosomes, creating a permissive state for promoter activation.
  • HET/SAF-B mediated repression can be released by using inhibitors of histone deacetylase, such as trichostatin A (Oelabor et al, 2000), which suggests that chromatin remodeling is involved in the action of HET/SAF-B as a repressor.
  • Another modulator of hormone action is the nuclear matrix, which is a dynamic structure involved in DNA replication, transcription, repair, and RNA processing (Bird et al; 1997).
  • a role for the nuclear matrix in hormone receptor action was postulated many years ago (Getzenberg and Coffey, 1990; Barrack and Coffey, 1980; Barrack and Coffey, 1982; Rue et al, 1996; Getzenberg et al, 1990), but only recently have specific nuclear matrix proteins been characterized which directly bind to hormone receptors and modulate their activity (Barrett and Spelsberg, 1999).
  • the glucocorticoid receptor interacting protein GRIP 120 has been identified as the nuclear matrix protein hnRNPU (Eggert et al, 1997).
  • HET was originally cloned as a nuclear matrix protein binding to the promoter of the estrogen-regulated heat shock protein hsp27 (Oelabor et al, 1997; Renz and Fackelmayer, 1996; USSN 09/201,523). Renz (1996). cloned the same protein based on its ability to bind to scaffold/matrix attachment regions (S/MAR's), and hence called it scaffold attachment factor B (SAF-B). Scaffold attachment factors are a specific subset of nuclear matrix proteins that are thought to mediate the attachment of chromatin to nuclear protein structures (Udvardy, 1999, Bode et al, 1995).
  • HET/SAF-B has recently been shown to bind to the C-terminal domain of RNA polymerase II (RNA pol II) and to a subset of serine-/arginine-rich RNA processing factors (SR proteins) (Nayler et al, 1998). This suggests that HET/SAF-B is involved in the formation of a "transcriptosomal" complex, bringing transcription and pre- mRNA processing together. These macromolecular complexes have previously been shown to be associated with the nuclear matrix (Mortillaro et al, 1996; Blencothe inventors et al, 1994).
  • Hsp27 plays a role in both growth and drug resistance of human breast cancer cells in culture (Oeteil et al, 1993). Hsp27 has been found to contribute to increased drug resistance in CHO cells (Lavoie et al, 1993), colon cancer cells
  • Hsp 27 is not an independent prognostic marker for breast cancer (Oelius et ah, 1996b). However, hsp27 predicts a significantly worse outcome in a subset of ER-positive/untreated breast cancer patients (Oe faux et al, 1996b). Expression of hsp27 is strongly correlated with the expression of ER in breast tumors (Oe faux et al, 1996b). Several groups have tried to decrease the expression of heat shock proteins in order to circumvent drug resistance in tumors.
  • the antiestrogen toremifene (Mahvi et al, 1996) and the bioflavonoid quercetin (Sliutz et al, 1996) both decrease hsp expression and have been used as chemosensitizers in tissue culture.
  • N-CoR or SMRT can inhibit Tam's agonist activity (Jackson et al, 1997; Lavinsky et al, 1998; Smith et al, 1997). Reduction of N-CoR by microinjection of N-CoR specific antibodies can convert Tarn into a full ER agonist displaying activity similar to estrogen (Lavinsky et al, 1998). However, it is unknown whether N-CoR or SMRT are responsible or necessary for the antagonist activity of Tarn. REA can potentiate the antiestrogenic effect of Tarn, but when overexpressed at high levels also inhibits estrogen activation of the ER.
  • SARMs selective estrogen receptor modulators
  • breast cancers are antiestrogen resistant, and it is not unusual for resistance to develop following antiestrogen therapy.
  • a method of converting antiestrogen-resistant tumors to antiestrogen- sensitive tumors would be of great benefit for treatment of breast cancer.
  • compositions and methods of use in converting antiestrogen- resistant to antiestrogen-sensitive tumors by administering expression vectors comprising an HET coding sequence.
  • Specific examples include compositions and methods of use in differentiating antiestrogen-resistant and antiestrogen-sensitive tumors and in converting antiestrogen-resistant to antiestrogen-sensitive tumors.
  • Specific antiestrogens that are within the context of the invention include the nonsteroidal compounds Tamoxifen, Toremifene, Idoxifene, Droloxifene, TAT-59, Zindoxifene, Trioxifene, and Raloxifene as well as the steroidal antiestrogens ICI 182,780 (FASLODEXTM) and EM-800.
  • Tamoxifen is a particularly well-known estrogen antagonist that exhibits efficacy for treatment of breast cancer.
  • Some of the other nonsteroidal compounds, e.g. TAT-59 are metabolized into an active metabolite of Tamoxifen or are analogues of Tamoxifen, e.g. Toremifene. (See Minton, 1999, incorporated herein by reference.)
  • the skilled artisan will realize that the present invention is not limited to effects on tamoxifen, but encompasses any estrogen antagonist or antiestrogen.
  • HET/SAF-B does not interact exclusively with ER. HET/SAF-B also inhibits the activity of other members of the steroid receptor as well as the retinoic acid/thyroid receptor families. Activities of progesterone, glucocorticoid and thyroid receptors are inhibited upon cotransfection of cells with HET/SAF-B. The inhibition is not a general, non-specific inhibition since other promoters such as API, and SV240 were not affected. Rather, HET/SAF-B is a regulator of all steroid hormone receptors.
  • the present invention is not limited to embodiments regarding the inhibition of ER by HET/SAF-B, but, rather, encompasses the exploitation of the inventor's discovery that HET/SAF-B inhibits these other receptors.
  • HET/SAF-B inhibits these other receptors.
  • HET/SAF-B binding ER in both the DBD and the hinge region.
  • the association of ER with HET/SAF-B occurs in the absence of ligand, but is increased by the antiestrogen tamoxifen (Tam).
  • HET/SAF-B can enhance the antiestrogenic effect of Tarn, but when overexpressed at high levels can also repress both estrogen and tamoxifen agonist activity on the ER.
  • the ER DBD is critical for the repressive activity of
  • HET/SAF-B on ER as HET/SAF-B does not repress activity of an ER-GAL4DBD chimera, and can cause transcriptional repression of an ER DBD fused to a heterologous transcription factor.
  • the invention relates to methods for detecting resistance to modulators of steroid receptor activity, comprising: a) obtaining a sample suspected of containing cells resistant to modulators of steroid receptor activity; b) measuring the amount of HET gene product in said sample, wherein said HET gene product is a molecule of RNA; and c) comparing the amount of HET gene product present in said sample with the amount of HET gene product in normal cells, wherein a decrease in the amount of HET gene product in said sample compared with the amount in normal cells indicates resistance to modulators of steroid receptor activity.
  • the invention embodies methods for altering the phenotype of a cell comprising contacting the cell with a nucleic acid comprising (i) a DNA sequence encoding a HET protein and (ii) a promoter active in said cell, wherein said promoter is operably linked to the region encoding said protein, under conditions effective for the uptake and expression of said nucleic acid by said tumor cell, wherein said cell is converted from a phenotype displaying normal steroid hormone receptor activity to one displaying reduced steroid hormone receptor activity.
  • the steroid hormone receptor activity is progesterone receptor activity, glucocorticoid receptor activity, thyroid receptor activity, or estrogen receptor activity.
  • breast cancer entails the diagnosing and treatment of breast cancer.
  • exemplary forms of breast cancer that may be diagnosed and/or treated according to the invention include infiltrating duct carcinoma, lobular carcinoma, medullary carcinoma, mucinous carcinoma, tubular carcinoma, ductal carcinoma in situ and lobular carcinoma in situ.
  • the invention relates to methods for detecting resistance to antiestrogens in breast cancer cells, comprising: a) obtaining a sample suspected of containing breast cancer cells; b) contacting said sample with an antibody that specifically binds to an HET polypeptide under conditions effective to bind said antibody and form a complex; c) measuring the amount of HET polypeptide present in said sample by quantitating the amount of said complex; and d) comparing the amount of HET polypeptide present in said sample with the amount of HET polypeptide in normal cells, wherein a decrease in the amount of HET polypeptide in said sample compared with the amount in normal cells indicates presence of antiestrogen resistance.
  • Exemplary measuring techniques in the embodiments of the invention discussed herein include Western blotting, ELISA. Northern blotting, slot blotting, dot blotting and/or DNA chip assay
  • Alternative embodiments include methods for predicting antiestrogen resistance in breast cancer cells, comprising: a) measuring the amount of HET gene product in a sample containing breast cancer cells; and b) comparing the amount of HET gene product present in said sample with the amount of HET gene product in samples selected from patients with antiestrogen-resistant and antiestrogen-sensitive breast cancers.
  • Exemplary antiestrogens can be selected from the group consisting of Tamoxifen, Toremifene, Idoxifene, Droloxifene, TAT-59, Zindoxifene, Trioxifene, Raloxifene, ICI 182,780 and EM-800.
  • the antiestrogen is Tamoxifen.
  • the gene product for example, may be a polypeptide or protein or an RNA molecule.
  • the invention also relates to method for predicting antiestrogen resistance in breast cancer cells, comprising: a) obtaining a breast cancer cell sample and a normal cell sample from the same individual; b) amplifying chromosomal DNA from said breast cancer and normal cell samples using primers selected to amplify a chromosomal locus comprising the HET gene; and c) comparing the amplification products from said breast cancer and normal cells, wherein loss of heterozygosity (LOH) at said locus indicated by an amplification product present in the normal cell and missing in the breast cancer cell is indicative of antiestrogen resistance in said breast cancer cell.
  • exemplary primers include primers comprising the sequences of SEQ ID NO: 7 and SEQ ID NO: 8.
  • the antiestrogen can be selected from the group consisting of Tamoxifen, Toremifene, Idoxifene, Droloxifene, TAT-59, Zindoxifene, Trioxifene, Raloxifene, ICI 182,780 and EM-800, with Tamoxifen being a presently preferred embodiment.
  • the invention anticipates methods for detecting antiestrogen resistance in breast cancer cells, comprising: a) obtaining a sample suspected of containing breast cancer cells; b) measuring the amount of HET gene product in said sample, wherein said HET gene product is a molecule of RNA; and c) comparing the amount of HET gene product present in said sample with the amount of HET gene product in normal cells, wherein a decrease in the amount of HET gene product in said sample compared with the amount in normal cells indicates anti-estrogen resistance of breast cancer cells.
  • the invention further encompasses methods of malignant breast cancer diagnosis, comprising determining loss of heterozygosity (LOH) at a chromosomal locus comprising the HET gene, wherein LOH at said locus is indicative of antiestrogen resistance in breast cancer cells.
  • the invention encompasses methods of determining likelihood of survival for a breast tumor subject, comprising determining loss of heterozygosity (LOH) at a chromosomal locus comprising the HET gene in a breast tumor cell sample from said subject, wherein LOH at said locus is associated with a decreased probability of survival.
  • the invention further contemplates methods for altering the phenotype of a breast tumor cell comprising contacting the cell with a nucleic acid comprising (i) a DNA sequence encoding a HET protein and (ii) a promoter active in said breast tumor cell, wherein said promoter is operably linked to the region encoding said protein, under conditions effective for the uptake and expression of said nucleic acid by said tumor cell.
  • a nucleic acid comprising (i) a DNA sequence encoding a HET protein and (ii) a promoter active in said breast tumor cell, wherein said promoter is operably linked to the region encoding said protein, under conditions effective for the uptake and expression of said nucleic acid by said tumor cell.
  • the HET protein has the amino acid sequence of SEQ ID NO:2.
  • the breast tumor cell may be converted from a phenotype resistant to antiestrogen to a phenotype sensitive to antiestrogen.
  • the antiestrogen may be any antiestrogen known to those of skill in the art, including the exemplary antiestrogens listed above.
  • the phenotypic alteration in some embodiments, comprises an increased sensitivity to estrogen antagonists.
  • the phenotypic alteration may comprise a decreased sensitivity to estrogen agonists.
  • FIG. 1A, FIG. IB, and FIG. IB HET/SAF-B directly interacts with ER in vitro (GST-pulldown studies).
  • FIG. 1A ER was labeled with 35S-methionine by in vitro transcription translation and tested for interaction with GST alone and GST- HET/SAF-B in the absence of ligands, or in the presence of 10 "6 M E2 or 10 "6 M Tam (as indicated). The input lane contains 20% input of the in vitro transcribed/translated ER.
  • FIG. IB Schematic presentation of GST-tagged ER domains. The numbers indicate amino acids in ER's open reading frame.
  • FIG. lC Schematic presentation of GST-tagged ER domains. The numbers indicate amino acids in ER's open reading frame.
  • HET/SAF-B was labeled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST alone and a number of GST-ER domain fusion proteins (as indicated).
  • the input lane contains 20% input of the in vitro transcribed/translated HET/SAF-B.
  • FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, and FIG. 2E HET/SAF-B and ER interact in cells (co-immunoprecipitations).
  • FIG. 2A COS-7 cells were transiently transfected with expression constructs for HA-tagged ER (ER/HA) and HET/SAF-B as indicated, and lysed in low stringency buffer. The cell lysates were immunoprecipitated with an anti-HET/SAF-B antibody, subjected to electrophoresis, and immunoblotted with an anti-HA antibody (left panel).
  • FIG. 2B MCF-7 cells were lysed in low stringency (LS) and high stringency (HS) buffer, the lysates from duplicate plates were immunoprecipitated with a anti-HET/SAF-B antibody, and the immunoprecipitates were subjected to electrophoresis. For the immunoblot anti- HET/SAF-B and anti-ER antibody were used.
  • FIG. 2C MCF-7 cells were lysed in low stringency (LS) and high stringency (HS) buffer, the lysates from duplicate plates were immunoprecipitated with a anti-HET/SAF-B antibody, and the immunoprecipitates were subjected to electrophoresis. For the immunoblot anti- HET/SAF-B and anti-ER antibody were used.
  • FIG. 2C MCF-7 cells were lysed in low stringency (LS) and high stringency (HS) buffer, the lysates from duplicate plates were immunoprecipitated with a anti-HET/SAF-B antibody, and the
  • MCF-7 LS lysates were immunoprecipitated with an HET/SAF-B antibody, with HET/SAF-B antibodies preincubated with HET/SAF-B peptide, or without antibody.
  • the immunoblots were performed with HET/SAF-B antibody.
  • FIG. 2D MCF-7 cells were incubated without ligand, with 10 "9 M E2, or with 10 "9 M Tam for 24 hours.
  • the immunoblots were performed with HET/SAF-B antibody (left top panel) and ER antibody (left bottom panel).
  • the lysates were also immunoblotted with an ER antibody (right top panel).
  • the bar graph represents intensity ratios of immunoprecipitated ER to immunoblotted ER in the lysates.
  • COS-7 cells were transfected with expression plasmids for HET/SAF-B and ER-HA. Treatment of the cells, immunoprecipitation and immunoblotting were performed as described in FIG. 2C with the exception that HA antibody was used instead of ER antibody.
  • FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D Overexpression of HET/SAF-B inhibits ER activity.
  • FIG. 3 A Cells were transiently transfected with ER (25 ng) and 10 ng of pcDNAI or HET/SAF-B plasmids, and incubated in the presence of 10 "9 M E2 and increasing amounts of Tam, as indicated (* p ⁇ 0.05, t-test).
  • FIG. 3 A Cells were transiently transfected with ER (25 ng) and 10 ng of pcDNAI or HET/SAF-B plasmids, and incubated in the presence of 10 "9 M E2 and increasing amounts of
  • FIG. 3B Cells were transfected with ER and HET/SAF-B plasmids as indicated. Open bars represent incubation in the absence of E2, and black bars represent incubation in the presence of 10 "9 M E2.
  • FIG. 3C Cells were transiently transfected with ER (25 ng) and HET/SAF-B (100 and 250 ng) plasmids, and incubated in the presence of 10 "9 M E2 and increasing amounts of Tam, as indicated.
  • FIG. 3D Cells were incubated in the absence of ligand (open bar) and in the presence of 10 "8 M Tam (black bars), and transfected with ER (25 ng) and the indicated amounts of HET/SAF-B.
  • FIG. 4A and FIG. 4B HET/SAF-B-mediated repression of ER's transcriptional activity is not the result of a general repression mechanism.
  • FIG. 4A HepG2 cells were transfected with expression plasmids for ER and HET/SAF-B, as indicated, and with the reporter constructs tk-luc and ERE-tk-luc, respectively. Cells were incubated in the presence of 10-9 M E2.
  • FIG. 4B ⁇ -gal values measured from the experiment shown in FIG. 4A.
  • FIG. 5A, FIG. 5B and FIG. 5C ER-DBD is involved in HET/SAF-B 's mediated repression of ER's activity.
  • HepG2 cells were transiently transfected with expression vectors as indicated. Bars are the mean + SEM of triplicate wells and the graphs are representative of at least three experiments each.
  • FIG. 5A Cells were transiently transfected with various ER constructs (25 ng) and HET/SAF-B (200 ng), as indicated, and ERE-tk-luc and the Gal4-responsive promoter construct Gal4-luc, respectively.
  • the right panel shows the result from an immunoprecipitation using lysates from COS-7 cells which were transiently transfected with expression plasmids for HET/SAF-B and ER-Gal4DBD.
  • the cell lysates were immunoprecipitated with an anti-HET/SAF-B antibody, subjected to electrophoresis, and immunoblotted with an anti-ER antibody.
  • FIG. 5B shows the result from an immunoprecipitation using lysates from COS-7 cells which were transiently transfected with expression plasmids for HET/SAF-B and ER-Gal4DBD.
  • the cell lysates were immunoprecipitated with an anti-HET/SAF-B antibody, subjected to electrophoresis, and immunoblotted with an anti-ER antibody.
  • FIG. 5B shows the result from an immunoprecipitation using lysates from COS-7 cells which were transiently transfected with expression plasmids
  • FIG. 5C Cells were transiently transfected with HET/SAF-B (as indicated), with 25 ng VP16-gal4DBD and VP16-ERDBD, and with the Gal4-responsive promoter construct Gal4-luc and ERE-tk-luc, respectively.
  • FIG. 5C Cells were transfected with 1 ⁇ g CMV-ERE- CAT, and ER, and HET/SAF-B as indicated. Cells were incubated in the absence of ligand (white bars), in the presence of 10 "9 M E2 (gray bars), and 10 '7 M Tam (black bars).
  • FIG. 6A and FIG. 6B HET/SAF-B expression is decreased in antiestrogen- resistant xenograft tumors.
  • FIG. 7 illustrates a human metaphase spread with the HET PI probe fiuorescently labeling both chromosome 19 homologs at 19pl3.2— >pl3.3
  • FIG. 8 shows an LOH analysis at human chromosomal locus 19pl3 of breast tumor specimens.
  • Breast biopsy DNA normal and tumor
  • the resulting PCRTM products were separated on a high resolution sequencing gel, dried and exposed to a Molecular Dynamics (MD) storage phosphor screen.
  • MD Molecular Dynamics
  • FIG. 9 illustrates HET expression in primary breast cancers. Frozen tumor powder was homogenized in 5% SDS, and 25 ⁇ g protein was resolved on 7.5% PAGE. After transferring onto nitrocellulose, HET was detected by Western blot using an HET- specific polyclonal antibody.
  • FIG. 10A and FIG. 10B show that HET inhibits the growth of NIH3T3 cells.
  • FIG. 11 shows that transient transfection of antisense HET into 293 cancer cells causes an increased rate of cell division, as measured by [ 3 H]-thymidine incorporation into DNA.
  • Cells were transfected with 0.02, 0.2 or 2.0 ⁇ g of HET/pcDNAl antisense vector.
  • Control cells were transfected with pcDNAl plasmid alone. After 24 h recovery, the rate of [ 3 H] thymidine incorporation was determined by scintillation counting and the numbers were corrected for protein concentration.
  • FIG. 12A demonstrates that total cellular levels of HET decrease progressively when MCF-7/MG cells were stimulated with 5% and 10% serum, respectively, compared to cells in serum-free medium (SFM). HET levels increased in MCF-7 cells exposed to 50 nM TPA, which has been shown to cause growth inhibition of these cells. These results are consistent with an inverse correlation between HET expression and cell proliferation.
  • FIG. 12B shows the effect of expression of HET on colony growth in MCF-7/MG cells.
  • Cells transfected with an HET expression construct (HET sense) showed a dramatic reduction in number of colonies formed, compared with control vector alone (PCDNA1) or an HET antisense construct (HET antisense).
  • HET HET protein
  • HET Hsp27-ERE-TATA-binding protein
  • HET is a nuclear matrix protein affecting the transcriptional activity of the hsp27 promoter. It is not identical to the pi 14 kDa matrix attachment region binding protein of Yanigasawa et al (1996). "Biologically active" forms of HET gene product and HET are capable of binding to the hsp27 promoter and suppressing expression of hsp27.
  • HET and “HET gene product” refer to proteins having amino acid sequences which are substantially identical to the native amino acid sequence in SEQ ID NO:2.
  • HET gene product also includes analogs of HET molecules which exhibit at least some biological activity in common with native HET.
  • HET analogs may be constructed wherein certain amino acid residues recited in SEQ ID NO:2 are substituted by other amino acid residues of similar charge, size, shape and hydrophobicity.
  • mutagenesis will appreciate that such analogs of HET may be generated by well- known techniques such as site-directed mutagenesis, as described below.
  • HET or wild-type activity it is meant that the molecule in question has the ability to inhibit cell transformation, or to prevent metastasis or invasive tumor growth.
  • Other phenotypes that may be regulated by the normal HET gene product are angiogenesis, cell adhesion, migration, cell-to-cell signaling, cell growth, cell proliferation, density-dependent growth, anchorage- dependent growth, and others. Molecules possessing this activity may be identified using assays familiar to those of skill in the art. For example, transfer of genes encoding HET, or variants thereof, into cells that do not have a functional HET product, and hence exhibit impaired growth control, will identify, by virtue of growth suppression, those molecules having HET function.
  • tumor suppressor is well-known to those of skill in the art. Examples of other tumors suppressors are p53, Rb and pl6, to name a few. While these molecules are structurally distinct, they form a group of functionally-related molecules, of which HET is a member. The uses for which these other tumor suppressors now are being exploited are equally applicable here.
  • HET gene refers to any DNA sequence that is substantially identical to a DNA sequence encoding an HET gene product as defined above. Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, usually at least about 60%, more usually about 10%, most usually about 80%, preferably at least about 90%, and most preferably about 95% of nucleotides that are identical to the nucleotides of SEQ ID NO:l will be sequences that are "as set forth in SEQ ID NO:l.” Sequences that are substantially identical or "essentially the same” as those set forth in SEQ ID NO:l also may be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment containing the complement of SEQ ID NO:l under conditions of relatively high stringency.
  • Such conditions are typically relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C. Such selective conditions tolerate little, if any, mismatch between the complementary stands and the template or target strand.
  • HET gene is also intended to include RNA, or antisense sequences compatible with the nucleic acid sequence set forth in SEQ ID NO:l. Any HET gene sequences may also comprise associated control sequences.
  • substantially identical when used to define either a HET amino acid sequence or a HET gene nucleic acid sequence, means that a particular subject sequence, for example, a mutant sequence, varies from the sequence of natural HET or
  • HET gene by one or more substitutions, deletions, or additions, the net effect of which is to retain at least some biological activity of the HET protein or gene.
  • DNA analog sequences are "substantially identical" to specific DNA sequences disclosed herein if: (a) the DNA analog sequence is derived from coding regions of the natural HET gene; or (b) the DNA analog sequence is capable of hybridization of DNA sequences of (a) under moderately stringent conditions and which encode biologically active HET; or (c) DNA sequences which are degenerative as a result of the genetic code to the DNA analog sequences defined in (a) or (b).
  • the HET gene is a tumor suppressor gene. HET has been mapped to chromosomal locus 19pl3.2-pl3.3. Using LOH technology, it was found that this locus is lost in 50-60% of breast cancer patients, which is higher than the LOH described for any other tumor suppressor gene described to date ⁇ e.g., p53, Rb).
  • the present invention also relates to fragments of the polypeptide that may or may not retain the tumor suppressing (or other) activity of HET. Fragments including the N-terminus of the molecule may be
  • fragments may include contiguous residues of the HET amino acid sequence given in SEQ ID NO:2 of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 200, 300, 400, or more amino acids in length.
  • fragments may be purified according to known methods, such as precipitation (e.g., ammonium sulfate), HPLC, ion exchange chromatography, affinity chromatography (including immunoaf ⁇ inity chromatography), or various size separations ⁇ e.g., sedimentation, gel electrophoresis, gel filtration).
  • precipitation e.g., ammonium sulfate
  • HPLC high-density chromatography
  • affinity chromatography including immunoaf ⁇ inity chromatography
  • size separations e.g., sedimentation, gel electrophoresis, gel filtration.
  • Substantially identical analog proteins will be greater than about 80% similar to the corresponding sequence of the native protein. Sequences having lesser degrees of similarity but comparable biological activity are considered to be equivalents.
  • all subject nucleic acid sequences capable of encoding substantially similar amino acid sequences are considered to be substantially similar to a reference nucleic acid sequence, regardless of differences in codon sequence.
  • HET Amino acid sequence variants of the HET polypeptide can be substitutional, insertional or deletion variants.
  • Deletion variants lack one or more residues of the native protein which are not essential for function or immunogenic activity.
  • Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell.
  • Insertional variants typically involve the addition of material at a non-terminal point in the polypeptide. This may include the insertion of an immunoreactive epitope or simply a single residue. Terminal additions, called fusion proteins, are discussed below.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • amino acids of a protein may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological and functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 1 shows the codons that encode particular amino acids. In making such changes, the hydropathic index of amino acids may be considered.
  • hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • Patent 4,554,101 the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine -0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • Mimetics are peptide-containing molecules that mimic elements of protein secondary structure. See, for example, Johnson et al (1993).
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • a specialized kind of insertional variant is the fusion protein.
  • This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C- terminus, to all or a portion of a second polypeptide.
  • fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
  • Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification.
  • Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity).
  • Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, irnmunoaffinity chromatography and isoelectric focusing.
  • a particularly efficient method of purifying peptides is fast protein liquid chromatography (FPLC) or even HPLC.
  • Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide.
  • the term "purified protein or peptide" as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
  • a purified protein or peptide therefore, also refers to a protein or peptide free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity therein, assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification, and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Various techniques suitable for use in protein purification will be well known to those of skill in the art.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of min, or at most an h. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography, or molecular sieve chromatography is a special type of partition chromatography that is based on molecular size.
  • gel chromatography The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size of the pores. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. Thus the elution volume is related in a simple matter to molecular weight.
  • Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind to. This is a receptor-ligand type of interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur ⁇ e.g., altered pH, ionic strength, temperature, etc.).
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below.
  • the present invention also describes smaller HET-related peptides for use in various embodiments of the present invention. Because of their relatively small size, the peptides of the invention can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tam et al, (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference.
  • Short peptide sequences or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to selected regions of the HET protein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultivated under conditions suitable for expression.
  • the present invention also provides for the use of HET proteins or peptides as antigens for the immunization of animals relating to the production of antibodies. It is envisioned that either HET, or portions thereof, will be coupled, bonded, bound, conjugated, or chemically-linked to one or more agents via linkers, polylinkers, or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i.e., pharmaceutically acceptable. Preferred agents are the carriers are keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the present invention also provides, in another embodiment, genes encoding HET.
  • the present invention is not limited to the specific nucleic acids disclosed herein as SEQ ID NO:l.
  • a "HET gene” may contain a variety of different bases and yet still produce a corresponding polypeptide that is indistinguishable functionally, and in some cases structurally, from the genes disclosed herein.
  • any reference to a nucleic acid should be read as encompassing a host cell containing that nucleic acid and, in some cases, capable of expressing the product of that nucleic acid.
  • cells expressing nucleic acids of the present invention may prove useful in the context of screening for agents that induce, repress, inhibit, augment, interfere with, block, abrogate, stimulate, or enhance the function of HET.
  • Nucleic acids according to the present invention may encode an entire HET gene, a domain of HET that expresses a tumor suppressing function, or any other fragment of the HET sequences set forth herein.
  • the nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA). Also contemplated is a cD A plus a natural intron or an intron derived from another gene; such engineered molecules are sometime referred to as "mini- genes. " At a minimum, these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis.
  • cDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
  • mRNA messenger RNA
  • HET may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein (see Table 1 below).
  • a nucleic acid encoding a HET refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid.
  • the invention concerns a nucleic acid sequence essentially as set forth in SEQ ID NO:l.
  • the term “as set forth in SEQ ID NO:l” means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:l.
  • the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine (Table 1, below), and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.
  • Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
  • DNA segments of the present invention include those encoding biologically functional equivalent HET proteins and peptides, as described above. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site- directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.
  • a search of the GenBank nucleic acid sequence database identified the scaffold attachment factor SAF-B (GenBank accession number L43631) as the sequence most homologous to HET.
  • SAF-B recently was cloned from a HeLa cell cDNA library (Renz and Fackelmayer, 1996) based on its ability to bind to scaffold/matrix attachment regions (S/MAR).
  • S/MAR scaffold/matrix attachment regions
  • the 5' end of the HET gene is missing from the published SAF-B sequence (Renz and Fackelmayer, 1996).
  • the HET cDNA disclosed herein has 205 bp at the 5' end which are not present in the SAF-B sequence, of which 152 bp code for amino acids.
  • the reported N-terminal amino acid sequences of SAF-B and HET differ substantially (Renz and Fackelmayer, 1996).
  • nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementary rules.
  • complementary sequences means nucleic acid sequences that are complementary to the extent that they are capable of hybridizing to the nucleic acid segment of SEQ ID NO:l under relatively stringent conditions such as those described herein. Such sequences may encode the entire HET protein or functional or nonfunctional fragments thereof. Alternatively, the hybridizing segments may be shorter oligonucleotides.
  • Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter o ⁇ gomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more base pairs will be used, although others are contemplated.
  • oligonucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000, or 3040 bases and longer are contemplated as well. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.
  • Suitable hybridization conditions will be well known to those of skill in the art. In certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl, at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C.
  • hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3 mM MgCl 2 , 10 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 ⁇ M MgCl 2 , at temperatures ranging from approximately 40°C to about 72°C.
  • Formamide and SDS sodium dodecylsulphate also may be used to alter the hybridization conditions.
  • One method of using probes and primers of the present invention is in the search for genes related to HET.
  • the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules.
  • screening may involve analysis of RNA molecules.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.
  • the technique typically employs a bacteriophage vector that exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage.
  • Double stranded plasmids are also routinely employed in site directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid.
  • site-directed mutagenesis is performed by first obtaining a single- stranded vector, or by melting two strands of a double stranded vector, which includes within its sequence a DNA sequence encoding the desired protein.
  • An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared.
  • This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment.
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained.
  • recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • mutant tumor suppressors may not be non-functional. Rather, they may have aberrant functions that cannot be overcome by replacement gene therapy, even where the "wild-type” molecule is expressed in amounts in excess of the mutant polypeptide. Antisense treatments are one way of addressing this situation. Antisense technology also may be used to "knock-out" function of HET in the development of cell lines or transgenic mice for research, diagnostic and screening purposes.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
  • complementary it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A.T) in the case of DNA, or adenine paired with uracil (A.U) in the case of RNA.
  • ds double-stranded DNA
  • RNA RNA will lead to double-helix formation.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNAs may be employed to inhibit gene transcription, or translation, or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns, or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within about 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region ⁇ e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • a genomic clone will need to be used.
  • the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Gerlach et al, 1987; Forster and Symons, 1987).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992).
  • This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
  • IGS internal guide sequence
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al, 1981).
  • U.S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme- mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990).
  • ribozymes elicited genetic changes in some cell lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
  • expression vectors are employed to express the HET polypeptide product, which can then be purified and, for example, be used to vaccinate animals to generate antisera or monoclonal antibody with which further studies may be conducted.
  • the expression vectors are used in gene therapy.
  • Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.
  • regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.
  • Elements designed to optimize messenger RNA stability and translatability in host cells also are defined.
  • the conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
  • expression construct is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid coding sequence is capable of being transcribed.
  • the transcript may be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.
  • the nucleic acid encoding a gene product is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene .
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase ⁇ tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box.
  • a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline.
  • individual elements can function either co- operatively or independently to activate transcription.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent and under the control of a promoter that is capable of being expressed in a human cell.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalovirus
  • S V40 early promoter the Rous sarcoma virus long terminal repeat
  • rat insulin promoter the glyceraldehyde-3 -phosphate dehydrogenase promoter
  • glyceraldehyde-3 -phosphate dehydrogenase promoter can be used to obtain high-level expression of the coding sequence of interest.
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.
  • Tables 2 and 3 list several elements/promoters which may be employed, in the context of the present invention, to regulate the expression of the gene of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression but, merely, to be exemplary thereof.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
  • enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • Eukaryotic promoters can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • NCAM Neural Cell Adhesion Molecule
  • SAA Human Serum Amyloid A
  • a cDNA insert typically one will typically include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed, such as human growth hormone and SV40 polyadenylation signals.
  • a terminator also contemplated as an element of the expression construct. These elements can serve to enhance message levels and to minimize read through from the construct into other sequences.
  • the cells containing nucleic acid constructs of the present invention may be identified in vitro or in vivo by including a marker in the expression construct.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
  • a drug selection marker aids in cloning and in the selection of transformants.
  • genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers.
  • enzymes such as herpes simplex virus thymidine kinase ⁇ tk) or chloramphenicol acetyltransferase (CAT) may be employed.
  • Immunologic markers also can be employed.
  • the selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • Preferred gene therapy vectors are generally viral vectors.
  • viruses that can accept foreign genetic material are limited in the number of nucleotides they can accommodate and in the range of cells they infect, these viruses have been demonstrated to successfully effect gene expression.
  • adenoviruses do not integrate their genetic material into the host genome and therefore do not require host replication for gene expression making them ideally suited for rapid, efficient, heterologous gene expression.
  • Techniques for preparing replication infective viruses are well known in the art. Of course in using viral delivery systems, one will desire to purify the virion sufficiently to render it essentially free of undesirable contaminants, such as defective interfering viral particles or endotoxins and other pyrogens such that it will not cause any untoward reactions in the cell, animal or individual receiving the vector construct.
  • a preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.
  • Viruses used as gene vectors such as DNA viruses may include the papovaviruses ⁇ e.g., simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein.
  • the expression vector comprises a genetically engineered form of adenovirus.
  • retroviral infection the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
  • MLP major late promoter
  • TPL 5 '-tripartite leader
  • recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
  • adenovirus vectors which are replication deficient depend on a unique helper cell line, designated 293, which is transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the E3, or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al, 1987), providing capacity for about 2 extra kb of DNA.
  • the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).
  • Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g. , Vero cells or other monkey embryonic mesenchymal or epithelial cells. As discussed, the preferred helper cell line is 293.
  • Racher et al (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
  • natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.
  • Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) are employed as follows.
  • a cell innoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h.
  • the medium is then replaced with 50 ml of fresh medium and shaking is initiated.
  • cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking is commenced for another 72 h.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • a typical vector applicable to practicing the present invention is replication defective and will not have an adenovirus El region.
  • the position of insertion of the construct within the adenovirus sequences is not critical.
  • the polynucleotide encoding the HET gene may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al, (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.
  • Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 9 -10 ⁇ plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al, 1990; Rich et al, 1993).
  • retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse- transcription (Coffin, 1990).
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes, gag, pol, and env. that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences, and also are required for integration in the host cell genome (Coffin, 1990).
  • a nucleic acid encoding a HET gene is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • Retroviral vectors are capable of infecting a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
  • retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al, 1981).
  • retrovirus vectors Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This may result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.
  • viral vectors may be employed as expression constructs.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988), adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984), and herpes viruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
  • the expression construct To effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
  • the nucleic acid encoding the HET gene may be positioned and expressed at different sites.
  • the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
  • Dubensky et al (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes.
  • DNA encoding a HET gene may also be transferred in a similar manner in vivo and express the gene product.
  • a naked DNA expression construct into cells may involve particle bombardment.
  • This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987).
  • Several devices for accelerating small particles have been developed.
  • One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990).
  • the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution, The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
  • Wong et al, (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells.
  • Nicolau et al, (1987) accomplished successful liposome- mediated gene transfer in rats after intravenous injection.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989).
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991).
  • HMG-1 nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
  • a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a HET gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferring (Wagner et al, 1990).
  • ASOR asialoorosomucoid
  • transferring Wang and Wu, 1990
  • the delivery vehicle may comprise a ligand and a liposome.
  • a ligand and a liposome For example, Nicolau et al, (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a nucleic acid encoding a HET gene also may be specifically delivered into a cell type such as lung, epithelial, or tumor cells, by any number of receptor-ligand systems with or without liposomes.
  • epidermal growth factor may be used as the receptor for mediated delivery of a nucleic acid encoding a gene in many tumor cells that exhibit upregulation of EGF receptor.
  • Mannose can be used to target the mannose receptor on liver cells.
  • CD25 T-cell leukemia
  • MAA melanoma
  • gene transfer may more easily be performed under ex vivo conditions.
  • Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.
  • Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture ⁇ techniques are well documented and are disclosed herein by reference (Fresliner, 1992).
  • Examples of useful mammalian host cell lines are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NIH3T3, RIN, and MDCK cells.
  • a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the manner desired. Such modifications ⁇ e.g., glycosylation) and processing ⁇ e.g., cleavage), of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the foreign protein expressed.
  • a number of selection systems may be used including, but not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively.
  • anti- metabolite resistance can be used as the basis of selection for dhfr: that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; and hygro, that confers resistance to hygromycin.
  • Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation ⁇ i.e., a monolayer type of cell growth).
  • Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products.
  • suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent T-cells.
  • Large scale suspension culture of mammalian cells in stirred tanks is a common method for production of recombinant proteins.
  • Two suspension culture reactor designs are in wide use - the stirred reactor and the airlift reactor.
  • the stirred design has been used successfully on an 8000 liter capacity for the production of interferon.
  • Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1 : 1 to 3:1.
  • the culture usually is mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.
  • the airlift reactor also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture. The gas stream enters a riser section of the reactor and drives circulation.
  • the present invention contemplates an antibody that is immunoreactive with an HET molecule of the present invention, or any portion thereof.
  • An antibody can be a polyclonal or a monoclonal antibody.
  • an antibody is a monoclonal antibody.
  • Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988). Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera.
  • an animal used for production of anti-antisera is a non-human animal, for example, rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • Antibodies both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • a composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention.
  • Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
  • the antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELIS A and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to HET-related antigen epitopes.
  • the antibodies of the present invention are also useful for the isolation of HET polypeptides by immunoprecipitation.
  • Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein.
  • For the isolation of membrane proteins cells must be solubilized into detergent micelles.
  • Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
  • Antibodies are and their uses are discussed further, below.
  • both polyclonal and monoclonal antibodies against HET may be used in a variety of embodiments.
  • they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding other isoforms of HET or related proteins. They also may be used in inhibition studies to analyze the effects of HET-related peptides in cells or animals.
  • Anti-HET antibodies also will be useful in immunolocalization studies to analyze the distribution of HET during various cellular events, for example, to determine the cellular or tissue-specific distribution of HET polypeptides under different points in the cell cycle.
  • a particularly useful application of such antibodies is in purifying native or recombinant HET, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure. Means for preparing and characterizing antibodies are well known in the art
  • compositions may vary in its immunogenicity. It is often necessary, therefore, to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin also can be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization.
  • a variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection also may be given. The process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate monoclonal antibodies.
  • Monoclonal antibodies may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified HET protein, polypeptide, or peptide or a cell expressing high levels of HET.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Cells from rodents such as mice and rats are preferred, however, the use of rabbit, sheep or frog cells is also possible.
  • the use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol.
  • B-cells B-lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 10 8 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984).
  • the immunized animal is a mouse
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG have been described by Gefter et al, (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).
  • Fusion procedures usually produce viable hybrids at low frequencies, around 1 x 10 "6 to 1 x 10 "8 . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfiised cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two wk. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells. This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • hybridomas typically are performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three wk) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
  • the individual cell lines also could be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • mAbs produced by either means may be further purified, if desired, using filtration, centrifugation, and various chromatographic methods such as HPLC or affinity chromatography.
  • HET 4.4 Diagnosing Cancers Involving HET
  • HET and the corresponding gene may be employed as a diagnostic or prognostic indicator of cancer. More specifically, point mutations, deletions, insertions, allelic loss, or regulatory perturbations relating to HET may cause cancer or promote cancer development, cause or promote tumor progression at a primary site, and/or cause or promote metastasis. Other phenomena associated with malignancy that may be affected by HET expression include angiogenesis and tissue invasion.
  • Tamoxifen resistance is associated with decreased levels of HET gene products in breast cancer cells.
  • Determination of HET expression levels, by assay of HET mRNA or protein, may be used to distinguish tumors that are resistant to estrogen antagonists (such as tamoxifen) from tumors that are sensitive to estrogen antagonists.
  • LOH assay may be used to identify tumors that have lost an allele of the HET gene. Such tumors are expected to show a decreased expression of HET gene product.
  • One embodiment of the instant invention comprises a method for detecting variation in the expression of HET. This may comprise determining the level of expression of HET or determining specific alterations in the expressed product in a biological sample.
  • the present invention relates to the diagnosis or prognosis of breast cancer.
  • the nucleic acid used in the disclosed methods is isolated from cells contained in a biological sample, according to standard methodologies (Sambrook et al, 1989).
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desirable to convert the RNA to a complementary DNA.
  • the RNA is whole cell RNA; in another embodiment, it is poly-A RNA. Normally, the nucleic acid is amplified.
  • the specific nucleic acid of interest is identified directly in the sample using amplification or by hybridization with a second, known nucleic acid following amplification.
  • the identified product is detected.
  • the detection may be performed by visual means ⁇ e.g., ethidium bromide staining of a gel).
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label, or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).
  • HET-related pathologies such as malignant breast tumors.
  • HET-related pathologies such as malignant breast tumors.
  • HET detected for example, mutant or truncated HET polypeptides
  • different levels of progression of breast cancer may be identified.
  • the sensitivity of tumors to estrogen antagonists, such as tamoxifen may be determined.
  • alterations should be read as including deletions, insertions, point mutations and duplications. Point mutations result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those occurring in non-germline tissues. Germ-line mutations can occur in reproductive tissue and are inherited. Mutations in and outside the coding region also may affect the amount of HET produced, both by altering the transcription of the gene or in destabilizing or otherwise altering the processing of either the transcript
  • FISH fluorescent in situ hybridization
  • PFGE direct DNA sequencing
  • SSCP single-stranded conformation polymorphism
  • ASO allele-specific oligonucleotide
  • dot blot analysis denaturing gradient gel electrophoresis, RFLP and PCRTM-SSCP.
  • An alternative method for detection of mutations in the HET sequence involves the recently developed protein truncation assay (PTT) to detect mutations affecting the length of the protein.
  • PTT protein truncation assay
  • This method is based on RT-PCRTM using an upstream PCRTM primer containing a RNA polymerase promoter and a eukaryotic translation initiation signal.
  • Approximately 200 ng of the PCRTM product is used directly for the coupled in vitro transcription/translation reaction (coupled TNT T7 reticulocyte system, Promega) which is substituted with 35 S methionine.
  • the TNT system has been used successfully to analyze the inventors' HET cDNA clones (Oerios et al, 1997).
  • genomic DNA may be used as a template instead of total RNA to perform PCRTM using primers covering the appropriate HET exon.
  • the amplified oligonucleotide products may be sequenced by standard techniques known to those skilled in the art. 4.4.1.1 Primers and Probes
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double- stranded or single-stranded form, although the single-stranded form is preferred.
  • Probes are defined differently, although they may act as primers. Probes, while perhaps capable of priming, are designed to bind to the target DNA or RNA and need not.be used in an amplification process.
  • the probes or primers are labeled with radioactive species ( P, C, S, H, or other label), with a fluorophore (rhodamine, fluorescein), or a chemilluminescent moiety (luciferase).
  • radioactive species P, C, S, H, or other label
  • fluorophore rhodamine, fluorescein
  • luciferase a chemilluminescent moiety
  • PCRTM polymerase chain reaction
  • the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCRTM amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641 filed December 21, 1990. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, may also be used as still another amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence that can then be detected.
  • restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]-triphosphates in one strand of a restriction site also may be useful in the amplification of nucleic acids in the present invention, Walker et al, (1992).
  • Strand Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • a similar method called Repair Chain Reaction (RCR)
  • RCR Repair Chain Reaction
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • Target specific sequences also can be detected using a cyclic probe reaction (CPR).
  • CPR a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample.
  • the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion.
  • the original template is annealed to another cycling probe and the reaction is repeated.
  • modified primers are used in a PCRTM-like, template- and enzyme-dependent synthesis.
  • the primers may be modified by labeling with a capture moiety ⁇ e.g., biotin) and/or a detector moiety ⁇ e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes is added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989; Gingeras et al, PCT Application WO
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al, 1989; Gingeras et al, PCT Application WO
  • the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer, and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA.
  • amplification techniques involve annealing a primer which has target specific sequences.
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • an RNA polymerase such as T7 or SP6.
  • the RNA's are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule having a sequence identical to that of the original RNA between the primers, and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al, PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and "one-sided PCRTM" (Frohman, 1990; Ohara et al, 1989; each herein incorporated by reference in their entirety).
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. (See Sambrook et al, 1989)
  • chromatographic techniques may be employed to effect separation.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adsorption, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography (Freifelder, 1982).
  • Products may be visualized in order to confirm amplification of the marker sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to X-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
  • detection is by a labeled probe.
  • the techniques involved are well known to those of skill in the art and can be found in many standard books on molecular protocols. (See Sambrook et al, 1989) For example, chromophore or radiolabel probes or primers identify the target during or following amplification.
  • chromophore or radiolabel probes or primers identify the target during or following amplification.
  • U.S. Patent No. 5,279,721 incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • amplification products described above may be subjected to sequence analysis to identify specific kinds of variations using standard sequence analysis techniques.
  • General techniques for determination of the DNA sequence of amplification products are well known in the art and include standard dideoxy sequencing by the Sanger technique (See Sambrook et al, 1989). Within certain methods, exhaustive analysis of genes is carried out by sequence analysis using primer sets designed for optimal sequencing (Pignon et al, 1994).
  • oligonucleotide primers such as those exemplified by SEQ ID NO: 10 and SEQ ID NO: 11, may be designed to permit the amplification of sequences throughout the HET gene that may then be analyzed by direct sequencing. The amplified sequences may then be identified and quantitated. The presence of the
  • HET gene or mutants thereof may be used in the methods disclosed herein to determine degree of malignancy, cell tumorigenicity, and potential prognosis/diagnosis of cancers such as breast cancers.
  • Blotting techniques are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting RNA species. Briefly, a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by transfer of the separated nucleic acids ("blotting") on to the filter.
  • the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and the labeled probe detected using standard techniques known to those skilled in the art.
  • a probe usually labeled
  • kits This generally will comprise preselected primers and probes. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, SequenaseTM etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • RT polymerases
  • Taq Taq
  • SequenaseTM deoxynucleotides
  • buffers to provide the necessary reaction mixture for amplification.
  • kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.
  • LOH loss of heterozygosity
  • kit form Materials and reagents for detecting loss of heterozygosity (LOH) may also be assembled in kit form. This will comprise primers selected to amplify a polymorphic marker at chromosomal locus 19pl3, for example at 19pl3.2-13.3. Primers for additional loci are contemplated within the scope of the instant invention, so long as they are capable of detecting LOH at 19p 13. Additional components as described in the preceding paragraph may also be included.
  • chip-based DNA technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al (1994); Fodor et al. (1991). 4.4.2 Immunodiagnosis
  • Antibodies of the present invention can be used in characterizing the HET content of healthy and diseased tissues, through techniques such as ELISA and
  • Western blotting This may provide a screen for the presence or absence of malignancy, as a predictor of future cancer, or to distinguish tamoxifen-resistant from tamoxifen-sensitive tumors.
  • anti-HET antibodies are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a non-specific protein that is known to be antigenically neutral with regard to the test antisera, such as bovine serum albumin (BSA), casein or solutions of powdered milk.
  • BSA bovine serum albumin
  • casein casein
  • the immobilizing surface is contacted with the sample to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • the occurrence and even amount of immunocomplex formation may be determined by subjecting the same to a second antibody having specificity for HET that differs from that of the first antibody.
  • Appropriate conditions preferably include diluting the sample with diluents such as BSA, bovine gamma globulin (BGG), and phosphate buffered saline (PBS)/Tween ® .
  • the layered antisera is then allowed to incubate for from about 2 to about 4 h, at temperatures preferably on the order of about 25° to about 27°C. Following incubation, the antisera-contacted surface is washed so as to remove non- immunocomplexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween ® or borate buffer.
  • the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • one will desire to contact and incubate the second antibody-bound surface with a urease or peroxidase-conjugated anti-IgG for a period of time and under conditions which favor the development of immunocomplex formation ⁇ e.g., incubation for 2 h at room temperature in a PBS- containing solution such as PBS/Tween ® ).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H O , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H O , in the case of peroxidase as the enzyme label.
  • Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • the preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody.
  • the antibody compositions of the present invention will find great use in immunoblot or Western blot analysis.
  • the antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof.
  • a solid support matrix such as nitrocellulose, nylon or combinations thereof.
  • immunoprecipitation followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background.
  • Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against HET protein or the primary antibody.
  • the present invention also contemplates the use of HET and active fragments, and nucleic acids coding therefor, in the screening of compounds for activity in either stimulating HET activity, overcoming the lack of HET or blocking the effect of a mutant HET molecule.
  • These assays may make use of a variety of different formats and may depend on the kind of "activity" for which the screen is being conducted.
  • Contemplated functional "read-outs” include binding to a compound, inhibition of binding to a substrate, ligand, receptor or other binding partner by a compound, phosphatase activity, anti-phosphatase activity, phosphorylation of HET, dephosphorylation of HET, or inhibition or stimulation of cell-to-cell signaling, growth, metastasis, cell division, cell migration, soft agar colony formation, contact inhibition, invasiveness, angiogenesis, apoptosis, tumor progression or other malignant phenotype.
  • Preferred embodiments include assay of cell replication by incorporation of radiolabeled thymidine or colony formation.
  • a preferred ligand for HET-binding activity comprises the hsp27 promoter. The functional activity of different forms of HET protein may be determined by assaying their effects on transcription of reporter genes functionally linked to an hsp27 promoter.
  • the invention is to be applied for the screening of compounds that bind to the HET molecule or a fragment thereof.
  • the polypeptide or fragment may be either free in solution, fixed to a support, or expressed in or on the surface of a cell. Either the polypeptide or the compound may be labeled, thereby permitting the determination of binding.
  • the assay may measure the inhibition of binding of HET to a natural or artificial substrate or binding partner.
  • Competitive binding assays can be performed in which one of the agents (HET, binding partner or compound) is labeled.
  • the polypeptide will be the labeled species.
  • One may measure the amount of free label versus bound label to determine binding or inhibition of binding.
  • WO 84/03564 Another technique for high throughput screening of compounds is described in WO 84/03564, the contents of which are incorporated herein by reference.
  • Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with HET and washed. Bound polypeptide is detected by various methods.
  • Purified HET can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase.
  • fusion proteins containing a reactive region may be used to link the HET active region to a solid phase.
  • Various cell lines containing wild-type or natural or engineered mutations in HET can be used to study various functional attributes of HET and how a candidate compound affects these attributes.
  • Methods for engineering mutations are described elsewhere in this document, as are naturally-occurring mutations in HET that lead to, contribute to and/or otherwise cause malignancy.
  • the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell.
  • culture may be required.
  • the cell may then be examined by virtue of a number of different physiologic assays.
  • molecular analysis may be performed in which the function of HET, or related pathways, may be explored. This may involve assays such as those for protein expression, enzyme function, substrate utilization, phosphorylation states of various molecules including HET, cAMP levels, mRNA expression (including differential display of whole cell or polyA RNA) and others.
  • the present invention also encompasses the use of various animal models.
  • By developing or isolating mutant cells lines that fail to express normal HET one can generate cancer models in mice that will be predictive of cancers in humans and other mammals.
  • These models may employ the orthotopic or systemic administration of tumor cells to mimic primary and/or metastatic cancers.
  • one may induce cancers in animals by providing agents known to be responsible for certain events associated with malignant transformation and/or tumor progression.
  • transgenic animals discussed below
  • Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal.
  • Administration will be by any route that could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, rectal, vaginal or topical.
  • administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • intratracheal instillation bronchial instillation
  • intradermal subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • systemic intravenous injection regional administration via blood or lymph supply and intratumoral injection.
  • Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Such criteria include, but are not limited to, survival, reduction of tumor burden or mass, arrest or slowing of tumor progression, elimination of tumors, inhibition or prevention of metastasis, increased activity level, improvement in immune effector function and improved food intake.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides or compounds with which they interact (agonists, antagonists, inhibitors, binding partners, etc.). By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules.
  • sufficient amounts of HET can be produced to perform crystallographic studies.
  • knowledge of the polypeptide sequences permits computer employed predictions of structure-function relationships.
  • an "alanine scan” involves the random replacement of residues throughout molecule with alanine, followed by determining the resulting effect(s) on protein function.
  • HET sequence By virtue of the availability of the cloned HET sequence, sufficient amounts of HET can be produced to perform crystallographic studies. In addition, knowledge of the polypeptide sequences permits computer employed predictions of structure-function relationships. It also is possible to isolate a HET specific antibody, selected by a functional assay, and then solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody.
  • an anti-idiotype antibody As a mirror image of a mirror image, the binding site of an anti-idiotype antibody would be expected to be an analog of the original antigen.
  • the anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore.
  • Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
  • the present invention also contemplates, in another embodiment, the treatment of cancer.
  • the types of cancer that may be treated, according to the present invention are limited only by the involvement of HET. By involvement is meant that, it is not even a requirement that HET be mutated or abnormal - the overexpression of this tumor suppressor may actually overcome other lesions within the cell. Thus, it is contemplated that a wide variety of tumors may be treated using HET therapy.
  • the tumor cell be killed or induced to undergo normal cell death or "apoptosis.” Rather, to accomplish a meaningful treatment, all that is required is that the tumor growth be slowed to some degree. It may be that the tumor growth is completely blocked, however, or that some tumor regression is achieved. Clinical terminology such as “remission” and “reduction of tumor” burden also are contemplated given their normal usage.
  • the treatment of cancer with HET therapy may be directed towards malignancies that are or are likely to become resistant to therapeutic compounds.
  • HET therapy may be used to treat cancer cells that have become resistant to compounds that inhibit steroid receptors.
  • HET therapy may be used to treat cells that have become resistant to antiestrogen compounds, both non-steroidal and steroidal.
  • HET therapy may be used to treat malignant cells resistant to Tamoxifen.
  • One of the therapeutic embodiments contemplated by the present inventors is the intervention, at the molecular level, in the events involved in the tumorigenesis of some cancers.
  • the present inventors intend to provide, to a cancer cell, an expression construct capable of providing HET to that cell. Any of the gene sequence variants discussed above which would encode the same, or a biologically equivalent polypeptide could be used.
  • the lengthy discussion of expression vectors and the genetic elements employed therein is incorporated into this section by reference.
  • Particularly preferred expression vectors are viral vectors such as adenovirus, adeno- associated virus, herpes virus, vaccinia virus and retrovirus. Also preferred is liposomally-encapsulated expression vector.
  • routes are contemplated for various tumor types. The section below on routes contains an extensive list of possible routes. For practically any tumor, systemic delivery is contemplated. This will prove especially important for attacking microscopic or metastatic cancer. Where discrete tumor mass may be identified, a variety of direct, local and regional approaches may be taken. For example, the tumor may be injected directly with the expression vector. A tumor bed may be treated prior to, during or after resection. Following resection, one generally will deliver the vector by a catheter left in place following surgery. One may utilize the tumor vasculature to introduce the vector into the tumor by injecting a supporting vein or artery. A more distal blood supply route also may be utilized. In a different embodiment, ex vivo gene therapy is contemplated.
  • cells from the patient are removed and maintained outside the body for at least some period of time. During this period, a therapy is delivered, after which the cells are reintroduced into the patient. Preferably, any tumor cells in the sample have been killed.
  • ABMT Autologous bone marrow transplant
  • the concept underlying ABMT is that the patient will serve as his or her own bone marrow donor.
  • a normally lethal dose of irradiation or chemotherapeutic may be delivered to the patient to kill tumor cells, and the bone marrow repopulated with the patient's own cells that have been maintained (and perhaps expanded) ex vivo.
  • bone marrow is often contaminated with tumor cells, it is desirable to purge the bone marrow of these cells.
  • HET may be utilized according to the present invention.
  • Immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • native or wild type HET may not be a likely target for an immune effector given that (i) it has not been shown to be expressed on the surface of the cell and (ii) the presence, not absence, of HET is associated with the normal state.
  • particular mutant forms of HET may be targeted by immunotherapy, either using antibodies, antibody conjugates, or immune effector cells.
  • tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA,.
  • compositions of the present invention To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of tumor cells, using the methods and compositions of the present invention, one would generally contact a "target" cell with an HET expression construct and at least one other agent. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the expression construct and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations simultaneously, wherein one composition includes the expression construct and the other includes the agent.
  • the gene therapy treatment may precede or follow the other agent treatment by intervals ranging from min to wk.
  • the other agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined ⁇ e.g., synergistic) effect on the cell.
  • HET HET
  • B the other agent
  • both agents are delivered to a cell in a combined amount effective to kill the cell.
  • Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell.
  • agents and factors include radiation and waves that induce DNA damage such as ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like.
  • Chemotherapeutic agents contemplated to be of use include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide.
  • the invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.
  • HET expression construct is particularly preferred.
  • the tumor cells In treating cancer according to the invention, one would contact the tumor cells with an agent in addition to the expression construct. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, ⁇ -rays or even microwaves.
  • the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin.
  • the agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a HET expression construct, as described above.
  • Agents that directly cross-link nucleic acids, specifically DNA, are envisaged to facilitate DNA damage leading to a synergistic, antineoplastic combination with HET.
  • Agents such as cisplatin, and other DNA alkylating agents may be used.
  • Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m 2 for 5 days every three wk for a total of three courses. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
  • Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation.
  • chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered intravenously through bolus injections at doses ranging from 25-75 mg/m 2 at 21 day intervals for adriamycin, to 35-50 mgm 2 for etoposide intravenously or double the intravenous dose orally. Agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits also lead to DNA damage.
  • nucleic acid precursors A number of nucleic acid precursors have been developed for this purpose. Particularly useful are agents that have undergone extensive testing and are readily available, such as 5-fluorouracil (5-FU). Although quite toxic, 5-FU is applicable in a wide range of carriers, including topical. However intravenous administration with doses ranging from 3 to 15 mg/kg/day is commonly used.
  • 5-FU 5-fluorouracil
  • DNA damaging factors also are contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage to DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • BRCA2 mutations may produce an improved anti-cancer treatment.
  • Any other tumor- related gene conceivably can be targeted in this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, pl6, FHIT, WT-1, MEN-I, MEN-II, VHL, FCC, MCC, ras, myc, neu, raf, erb, src,ftns,jun, trk, ret, gsp, hst, bcl and abl 4.6.4 Formulations and Routes for Administration to Patients
  • compositions - expression vectors, virus stocks, proteins, antibodies and drugs - in a form appropriate for the intended application.
  • this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as innocula.
  • pharmaceutically or pharmacologically acceptable refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • the active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions normally would be administered as pharmaceutically acceptable compositions, described supra.
  • the active compounds also may be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, 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 forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars 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 compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the polypeptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • ACTACGCCATGACCATGACCCTCCAC-3' (SEQ ID NO:3) comprising the HA- tag, and nucleotides 1-18 coding for the ER
  • the antisense primer was 5'- GATGAATTCCTCAGACTGTGGCAGGGAA-3' (SEQ ID NO:4) comprising nucleotides 1770-1789 of the ER.
  • the PCRTM product was cloned into pcDNA3.1/V5/His-TOPO (Invitrogen, Carlsbad, CA).
  • GST-ER fusion proteins containing the AF1, DBD/Hinge, DBD, Hinge, and AF2/Hinge domains were generated by performing ligation reactions with the appropriate PCRTM products and EcoRI BamHI digested pGEX-2TK.
  • the positions of the PCRTM primers (linked to Eco RI or BamHI sites) within the ER cDNA (Greene et al, 1986 are: AF1 - sense (1-20) and antisense (519-540), DBD/Hinge - sense (519-540) and antisense (849-869), DBD - sense (513-533) and antisense (730-746),
  • Hinge - sense (750-771) and antisense (844-863), AF2/Hinge - sense (756-775) and antisense (1769-1788).
  • the AF1/DBD construct was obtained from Dr. S. Kato (Kumar et al, 1987; Kato etal, 1995).
  • the gal4-luc construct (pfrluc) was purchased from Stratagene (La Jolla, CA). Constructs containing the activation domains (AF-1 and AF-2) of the ER fused to the gal4 DNA binding domain (DBD) (amino acids 1-94) have been previously described (Castles et al, 1997). Briefly, the AF-1 domain of the ER was cloned upstream of the gaWDBD in pABgal94 (Baniahmad et al, 1995) to create AF- lgal4.
  • the AF-2 domain of ER was cloned downstream of the gal4DBD to create AF2-gal4. Finally the AF-1 and AF-2 domains were cloned upstream and downstream respectively of the gaMDBD to create ER-gal4DBD.
  • a construct containing a chimeric activator with the ER DBD and the activation region of VP16 was provided by Dr. P. Chambon, and has been previously described (Tora et al, 1989).
  • the ER-dependent promoter interference reporter plasmid was provided by Dr. B. Katzenellenbogen, and has been described in Reese and Katzenellenbogen 1992).
  • the antiestrogen 4-hydroxytamoxifen (Tam) was a gift from Zeneca Pharmaceuticals, Macclesfield, UK. All other chemicals were purchased from Sigma (St. Louis, MO) unless stated otherwise.
  • SFM serum-free medium
  • GIBCO phenol red free BVIEM + lOmM HEPES pH7.4 + 1 ⁇ g/ml fibronectin (GIBCO) + trace elements (Biofluids) + 1 ⁇ g/ml transferrin (GIBCO).
  • Cotransfections were performed using 1 ⁇ g reporter plasmid, 100 ng ⁇ -galactosidase ( ⁇ -gal) expression vector, and HET/SAF-B and ER plasmids as indicated in the Figure legends for each study.
  • luciferase activity was measured using the Luciferase kit from Promega (Madison, WI). ⁇ -gal activity was measured as described (Oeteil et al, 1997), and the luciferase activities were normalized by dividing by the ⁇ -gal activity to give relative luciferase units (RLU).
  • RLU relative luciferase units
  • the inventors utilized a CAT ELISA from Roche (Indianapolis, IN), and followed the manufacturer's instructions. Values were corrected for protein concentrations, and are presented as relative CAT activity.
  • COS-7 cells were plated at 0.6x106 into 10 cm dishes, and transiently transfected with 5 ⁇ g expression plasmids for HET/SAF-B and ER-HA. Twelve hours later the medium was replaced with phenol red-free IMEM + 5% charcoal stripped serum and ligands as indicated in the FIGure legends. The cells were lysed 24 hours later.
  • ER expression plasmids 10 ⁇ l of lysates were incubated with 60 ⁇ l equivalent amounts of GST proteins (as assessed by Coomassie staining) at 4°C for 1 hr. The beads were washed three times with IPAB buffer without BSA. Bound proteins were eluted in SDS sample buffer, resolved by SDS-PAGE, and visualized by fluorography.
  • the peptide used for generation of a monoclonal antibody (mAb) to HET-SAF/B was PEARDSKEDGRKF (SEQ ID NO:9).
  • the mAb was generated at the UTHSCSA Institutional Hybridoma Facility following methods described in Kohler (1979) and Oi and Herzenberg (1980). Briefly, spleen cells from two Balb/c female mice immunized subcutaneously three times with 50 ⁇ g KLH-coupled peptide in Freund's adjuvant were fused with NS-1 myeloma cell line. A 50% PEG solution was added drop-wise.
  • the subsequent dilution was performed in selection media (HAT), and 10 days later supernatants were screened for relevant antibody using the A156 HET/SAF-B peptide coupled to an alternative carrier (BSA).
  • BSA selection media
  • Culture supernatant from clone 6F7 was purified using the ImmunoPure (A/G) IgG purification kit (Pierce, Rockford, D ).
  • MCF-7 cells were plated at 2x10 6 cells in 10 cm dishes. The next day the media was changed to media containing 5% charcoal stripped , FCS and ligand as indicated in the FIGure legends. Twenty-four hours later the cells were lysed in low stringency (LS) buffer (PBS, 0.1 %> NP40, protease inhibitors), and high stringency (HS) buffer (20 mM Tris PH7.4, 50 mM NaCl, 1 mM EDTA, 0.5% NP40, 0.5% SDS, 0.5%) deoxycholate, and protease inhibitors), followed by sonication.
  • LS low stringency
  • HS high stringency
  • Sodium tetrathionate (50 ⁇ M) was added to the lysis buffer since it is known to selectively stabilize interactions between hormone receptors and the nuclear matrix (Kaufmann et al, 1986), however its addition is not essential for co- immunoprecipitation of ER and HET/SAF-B.
  • the lysate was precleared with 50 ⁇ l protein G-agarose for 30 min at 4°C, and than incubated overnight with 7 ⁇ l HET/SAF-B mAb at 4°C. Protein G agarose was added for another 4 hours, the beads pelleted, and washed three times with the indicated buffer.
  • the inventors precleared the lysates with 20 ⁇ l protein A-agarose, incubated with 5 ⁇ l HA antibodies (Babco,
  • the membrane was incubated with HRP -linked anti-mouse IgG at 1:1000 (Amersham Pharmacia Biotech, Piscataway, NJ) in PBST + 5% milk, washed 6 x 5 mins, and the signal was developed using enhanced chemiluminesence according to the manufacturers instructions (Pierce, Rockford, DL).
  • FIG. 1 In vitro transcribed and translated ER was incubated with full length GST-HET/SAF-B bound to glutathione-sepharose beads (FIG. 1A). There was no signal when ER was incubated with GST only, in the absence of hormone or in the presence of estradiol (E2). In contrast, ER interacted with GST-HET/SAF-B in the absence of hormone, in the presence of E2, and especially in the presence of Tam. Consistently increased binding of ER to HET/SAF-B was observed in the presence of Tam compared to no ligand.
  • HET/SAF-B The ability of HET/SAF-B to interact with different domains of ER (represented graphically in FIG. IB) was examined in GST-pulldown assays.
  • the different GST-ER domain fusion proteins were separated on SDS-PAGE and
  • HET/SAF-B potentially interacts with multiple regions within ER.
  • HET/SAF-B To determine whether an interaction occurs between HET/SAF-B and ER within cells, COS-7 cells were transiently transfected with expression plasmids for HET/SAF-B and HA-tagged ER. Immunoprecipitation of HET/SAF-B followed by immunoblotting for HA revealed a band with the molecular weight of ER (-68 kDa) that was only seen when cells were transfected with both ER and HET/SAF-B, but not in cells transfected with HET/SAF-B only (FIG. 2A - left panel). A similar experiment, but in a reciprocal manner, was performed using an HA-antibody to immunoprecipitate, and the HET/SAF-B antibody for immunoblotting.
  • HET/SAF-B immunoprecipitation was repeated with HET/SAF-B antibodies preincubated with HET/SAF-B peptide, or without antibody. As shown in FIG. 2C, only the immunoprecipitation with HET/SAF-B antibody resulted in a detectable band at the molecular weight of HET/SAF-B (-130 kDa), whereas no bands were detected using a peptide-preincubated antibody or no antibody.
  • endogenous HET/SAF-B and ER interact, and this interaction can be detected when the cells are lysed under low stringency conditions.
  • MCF-7 cells were incubated in the absence of ligand and in the presence of E2 or Tam, and lysed in LS buffer. After immunoprecipitation with HET/SAF-B antibodies, the membrane was immunoblotted with HET/SAF-B antibodies (FIG. 2D, left top panel) and ER antibodies (FIG. 2D, left bottom panel). While HET/SAF-B levels remained constant, co-immunoprecipitated ER levels changed. ER was detectable in the absence of ligand, low levels were detectable in the presence of E2, but much higher levels of ER were co-immunoprecipitated in the presence of Tam.
  • ER itself is known to be downregulated by E2 via ubiquitin-mediated degradation (Nawaz et al, 1999; Alarid et al, 1999), as a control ER levels were measured in the lysate (FIG. 2D, right top panel).
  • HET/SAF-B which did not change with E2 and Tam treatment
  • ER levels decreased dramatically after E2 treatment but were unaffected by Tam.
  • the amount of ER was measured in the immunoprecipitate and the lysate by densitometry. The results are presented as the ratio of ER levels immunoprecipiated with HET/SAF-B antibodies to ER levels in the lysate (FIG.
  • EXAMPLE 2 HET/SAF-B Overexpression Decreases ER Activity As shown in FIG. 2, the association between HET/SAF-B and ER is stronger in the presence of Tam as compared with no ligand.
  • the effect of HET/SAF-B on the antagonist activity of Tam was examined by transient transfection assays in ER- negative HepG2 cells, using a single ERE-tk-luciferase construct as the reporter gene (FIG. 3 A).
  • the results in FIG. 3 A represent the effect of HET/SAF-B on Tam acting as an antagonist of E2-occupied ER; i.e. cells were incubated in the presence of both E2 and Tam.
  • HET/SAF-B The effect of overexpression of HET/SAF-B on the transcriptional activity of E2-occupied ER was examined. In the absence of ER, the addition of E2 did not result in a significant change in basal activity of the construct, and HET/SAF-B had no effect on this basal activity. Transfection of ER led to an approximately 6-fold increase of transcriptional activity in the presence of E2. The coexpression of increasing amounts of HET/SAF-B (0-150 ng) led to a significant dose-dependent decrease in ER activity (FIG. 3B). A similar HET/SAF-B-mediated repression was also seen in Saos-2 cells transfected with ER. Increasing concentrations of HET/SAF- B (100 and 250 ng) were also able to further enhance the antagonist activity of Tam, as shown in FIG. 3C.
  • FIG. 3D shows that Tam (10 "8 M) caused a 2-2.5 fold increase in ER activity.
  • FIG. 4 To exclude a non-specific repressor effect of HET/SAF-B, a series of control experiments was performed (FIG. 4). As shown in FIG. 3A and 3B, basal activity of the ERE-tk-promoter was not inhibited by overexpression of HET/SAF-B. The deletion of the ERE sequence led to a tk-promoter construct which was also not affected by HET/SAF-B overexpression (FIG. 4A, left bars). In the same experiment the ERE-tk-promoter was inhibited by overexpression of HET/SAF-B (FIG. 4A, right bars), as shown previously (FIG. 3).
  • luciferase values were corrected for the values of a second cotransfected gene, an SV40-promoter driven ⁇ -galactosidase ( ⁇ -gal) construct.
  • ⁇ -gal SV40-promoter driven ⁇ -galactosidase
  • This example concerns the inhibition of steroid receptors generally by HET/SAF-B.
  • the effect of HET/SAF-B on the activity of members of the steroid hormone receptor family may be assessed by transient transfection assays in MCF-7 or HepG2 cells, using a response-element and luciferase construct as the reporter gene in a manner analogous to the reporter system described above. Briefly, a response element specific to a particular steroid hormone receptor may be inserted in the reporter construct in place of the ERE. Further, non-specific response elements, such as API and SV40, may be used in constructs to control for non-specific inhibition of the reporter system by HET/SAF-B. Co-transfection of the reporter with HET/SAF-B allows measurement of the response through quantification of luciferase expression as described.
  • the response to HET may be measured in cells co-transfected with the reporter construct lacking a response element, reporter constructs with non-specific response elements, and constructs containing response elements individually specific to progesterone, glucocorticoid and thyroid receptors.
  • a wide variety of response elements specific to the steroid hormone receptor family generally are included so that the general inhibitory effects of HET upon steroid receptors may be measured.
  • Further control and precision may be obtained by utilizing other reporter constructs.
  • a useful negative control may utilize an SV40-promoter driven ⁇ -galactosidase ( ⁇ -gal) construct.
  • the cells may be incubated in the presence of compounds antagonistic or agonistic to steroid receptors.
  • the effects of these compounds upon receptor activity may be measured and the effects of HET expression upon the receptor activity in the presence of the compounds may also be determined.
  • a Zn- finger DNA-binding domain (DBD) is flanked by an N-terminal region which displays a constitutive activator function domain 1 (AF-1) and the C-terminus containing the ligand-binding domain, heterodimerization domain, and ligand-dependent activation function domain 2 (AF-2).
  • AF-1 constitutive activator function domain 1
  • AF-2 ligand-dependent activation function domain 2
  • chimeric constructs in which the ER-DBD (aa 178-257) was replaced by a GAL4-DBD. Reporter activity was tested on 4 copies of a gal4 responsive element upstream of luciferase (gal4-luc).
  • GAL4DBD alone (GAL4DBD) was included as a negative control. Wildtype ER, HET/SAF-B, and the ERE-tk-luc were transfected as a positive control. All constructs were cotransfected with HET/SAF-B into HepG2 cells.
  • E2 increased transcriptional activity from the ERE-tk-luc reporter construct, and the induction was repressed by co-expression of HET/SAF-B (FIG. 5A).
  • Co-expression of the gal4DBD with the gal4-luc reporter construct resulted in basal activity that was not affected by E2 treatment and was also not affected by coexpression of HET/SAF-B.
  • Expression of both the AF-1 and the AF-2 domains fused to GAL4DBD (ER-GAL4DBD) behaved like wildtype ER with strong E2 inducibility.
  • HET/SAF-B did not repress activity while it did repress wildtype ER activity.
  • HET/SAF-B was not able to repress activity of either AF-1 fused to GAL4DBD, or AF-2 fused to GAL4DBD.
  • HET/SAF-B was not able to repress activity of the ER-GAL4DBD chimera, HET/SAF-B was still able to bind to this chimera as shown by coimmunoprecipitation (FIG. 5 A, right panel).
  • the binding of HET/SAF-B to ER GAL4DBD substantiates the in vitro GST binding experiments indicating that HET/SAF-B can bind ER not only in the DBD, but also in the Hinge/ AF2 region.
  • HET/SAF-B can bind ER-GAL4DBD, it cannot repress its activity, indicating that the ERE-DBD is required for transcriptional repression.
  • HET/SAF-B-mediated repression To directly assess the importance of the ER-DBD in HET/SAF-B-mediated repression, the effect of HET/SAF-B on the ER-DBD fused with a heterologous transcription factor (VP16) was examined. As a control the effect of HET/SAF-B on VP-16 with a GAL4-DBD was examined. As shown in FIG. 5B, the addition of increasing amounts of HET/SAF-B (50, 100, 250 ng) did not affect the activity of VP16-GAL4 DBD on a GAL4 reporter construct. In contrast, HET/SAF-B caused a dose-dependent decrease of VP16-ER-DBD activity on a ERE-Luc reporter construct.
  • HET/SAF-B caused a dose-dependent decrease of VP16-ER-DBD activity on a ERE-Luc reporter construct.
  • ER-DBD can mediate the HET/SAF-B transcriptional repression effect.
  • a simple explanation for the repressive effect of HET/SAF-B would be if HET/SAF-B bound to the ERE-DBD and blocked ER binding to DNA. To examine this possibility, it was determined whether HET/SAF-B had an effect on the DNA- binding properties of ER. It was confirmed that HET/SAF-B could not bind directly to an ERE-sequence using gel-shift assays and in vitro transcribed and translated HET/SAF-B. To examine if HET/SAF-B could inhibit ER binding to DNA, a promoter interference assay originally described by Reese et al (1992) was used.
  • an ERE is inserted between the CMV promoter (containing the TATA box) and the start site of transcription of the chloramphenicol acetyl transferase reporter gene (CMV-ERE-CAT) (FIG. 5C).
  • HET/SAF-B Expression is Decreased in Antiestrogen- Resistant Xenograft Tumors
  • HET/SAF-B expression was analyzed in a mouse model in which a breast tumor becomes Tam resistant (Osborne et al, 1991)
  • a loss of HET/SAF-B was detected in the resistant tumor (FIG. 6).
  • HET/SAF-B may be used as a marker or predictor for response to tamoxifen or other antiestrogens.
  • HET/SAF-B is consistent with HET/SAF-B being an ER corepressor.
  • HET/SAF-B interacted with ER in the absence of ligand, although the association was increased by Tam.
  • Co-immunoprecipitation experiments confirmed that the interaction between HET/SAF-B and ER was stronger in the presence of Tam.
  • the ability of Tam to recruit HET/SAF-B, as well as N-CoR and REA, to ER suggests an active co- repression mechanism.
  • HET/SAF-B has been shown to bind to the C-terminal domain of RNA pol II (Nayler et al, 1998). It is conceivable that HET/SAF-B locks the transcriptional initiation complex into a nonfunctional state. HET/SAF-B-mediated repression might also involve changes in histone acetylation, since treatment with the HDAC inhibitor trichostatin A (Yoshida et al, 1995) relieves HET/SAF-B-mediated repression (Oerios et /.,2000).
  • RNA-binding domain of HET/SAF-B may be involved in repression since the estrogen receptor interacting domains in HET/SAF-B are near the RNA-binding motif, (unpublished results).
  • HET/SAF-B other RNA-binding proteins and more recently RNA itself have been described as coregulators of nuclear hormone receptor action, such as L7/SPA (Jackson et al, 1997), hnRNP U (Oelabor et al, 1997), and SRA (Lanz et al, 1999).
  • HET/SAF-B shares some of the characteristics of other coregulators, it is possible that its repressive action results from its ability to associate with the nuclear matrix.
  • acceptor proteins specific binding sites for ER, called "acceptor proteins", in the nuclear matrix was postulated following in vitro reconstitution experiments showing binding of the ER to the nuclear matrix to be saturable and of high affinity (Belisle et al, 1989; Metzger and Korach, 1990).
  • HET/SAF-B is associated with the nuclear matrix in biochemical fractionations (Oerioset al, 1997). HET/SAF-B and ER can both be cross-linked to scaffold attachment regions (Samuel et al, 1998). ER can associate with the nuclear matrix as shown by direct visualization with a GFP-tagged
  • HET/SAF-B is a nuclear matrix protein/scaffold attachment factor that associates with the ER.
  • HET/SAF-B- mediated repression is lost when the ER-DBD is substituted by a Gal4DBD.
  • the ER- DBD is sufficient for HET/SAF-B-mediated repression.
  • the repression is not a result of inhibiting ER' s ability to bind to DNA.
  • MCF-7/MG The human breast cancer cell line MCF-7/MG was originally obtained from Dr. Herbert Soule (Michigan Cancer Foundation), and has been maintained in the inventors' laboratory for the last 14 years.
  • MDA-MB-231 cells were obtained from the American Type Tissue Culture Collection (Rockville, MD).
  • MCF-7/MG, MDA-MB- 231, and COS cells were maintained as described in (Oelabor et al, 1996a).
  • the media was not supplemented with insulin or gentamicin sulfate.
  • T47DE and ZR-75 human breast cancer cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco-BRL), Grand Island, New York) supplemented with 1% (v/v) L-glutamine, 1% (v/v) glucose, 1% (v/v) penicillin/streptomycin and 5% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • Cells were plated at 2 x 10 5 cells/well in triplicate in six-well plates. The next day cells were transfected using Lipofectamine (Life Technologies, Grand Island, NY) and OPTTMEMTM (Gibco-BRL). Cells were transfected with 0.5 ⁇ g CF (hsp27 promoter fragment; see below) and with 100 ng HET (MDA-MB-231 and HeLa) or 250 ng HET (T47D, MCF-7/MG and COS cells), respectively. Cells were exposed for 16 h, and then incubated in MEM plus 10% FBS for a further 24 h.
  • Lipofectamine Life Technologies, Grand Island, NY
  • OPTTMEMTM Gibco-BRL
  • RLU relative luciferase units
  • the N-terminal cysteine residue is not part of the sequence of HET, but was added for purposes of coupling to KLH.
  • the resulting antiserum was purified by affinity chromatography.
  • the immunogenic peptide was cross-linked through the
  • N-terminal cysteine onto an activated Sepharose column (Sulfolink, Pierce) according to the manufacturer's protocol. After loading with anti-HET antibody, the column was washed with low and high-salt buffer. Antibody bound to the cross-linked peptide was eluted from the column with 100 mM glycine, pH 2.5. Purified antibody was dialyzed overnight against PBS (phosphate buffered saline) with 0.02%) NaN 3 .
  • Monoclonal antibodies against HET protein were generated against the same synthetic peptide sequence (SEQ ID NO: 9), using standard techniques as described above.
  • Protein extracts were resolved on polyacrylamide gels as previously described
  • PCRTM polymerase chain reaction
  • Reaction conditions were 200 ng template DNA, 0.01 mg of each primer, 2.0 mM MgCl 2 , 200 mM dNTPs, 50 mM KC1, and 1 U of Taq Gold polymerase.
  • An initial denaturation step of 15 min at 94°C was followed by 30 cycles of 1 min at 94°C,
  • PCRTM products were electrophoresed through 2.0%> agarose gel and visualized by staining with ethidium bromide.
  • a recombinant PI library was obtained from DuPont (Shepherd et al, 1994) and screened using the PCRTM primers listed above (SEQ ID NO: 5 and SEQ ID NO: 6). The library was screened using the pooling strategies first described by Green and Olsen (1990). DNA was isolated from positive clones using a preparation protocol obtained from DuPont Biosciences.
  • the chromosomes were counterstained with DAPI and examined using a Zeiss Axioscope fluorescent microscope equipped with FITC, DAPI and triple band pass filter sets. Digital images were captured by computer using Applied Imaging Probevision software (Pittsburg) and photographs printed on a Kodak XL 7700 color image printer.
  • Samples for LOH analysis were prepared by thin sectioning of tissue samples. Alternating 3 ⁇ m and 10 ⁇ m histologic sections. were cut from selected formalin-fixed, paraffin-embedded tissue blocks and float mounted on glass slides. Areas of interest on the H&E (hemotoxylin and eosin)-stained 3 ⁇ m slides were outlined with a felt-tip pen, and used as a template to guide the independent microdissection of corresponding regions on the unstained 10 ⁇ m slides. Microdissection was performed on a light box viewed under a dissecting microscope, and was precise enough to insure that > 75% of the cellularity of each sample was derived from the targeted tissue. Cellular enrichment was about 90% in the majority of samples.
  • DNA was liberated from samples by a modification of the method of Wright and Manos (1990). Paraffin and lipids were first extracted by adding 0.4 ml of octane to 1.5 ml microcentrifuge tubes containing the samples. Cell debris was then digested for 3 h at room temperature with 50 ⁇ l of 0.01 M Tris-HCl (pH 8.5), 0.001 M EDTA, 0.045% NP-40, and 0.045% Tween-20 containing 1.0 mg/ml proteinase K. The digest was heated to 95° C for 15 min to inactivate the protease, and the lysate was stored at -80° C until use.
  • Loss of heterozygosity was analyzed using primers for the D19S216 marker, located on human chromosome 19pter.
  • the marker has 5 alleles ranging from 179-191 base pairs and a heterozygosity of 76%.
  • the antisense primer was 5'-labeled in a standard polynucleotide kinase reaction with 3000 Ci/mmole [ ⁇ 32 P]-ATP (NEN) at a molar ratio of 18:1.
  • the locus-specific PCRTM assay was performed in a total volume of 15 ⁇ l in an assay containing approximately 1.5 mM MgCl 2 , 1 mM spermidine base, 0.75 U of Taq polymerase, 100 ⁇ M of each dNTP, 100 nM of each primer, and 5 ⁇ l of 1:15 diluted DNA template. Thirty PCRTM cycles consisting of 30 sec denaturing at 94°C, 1 min annealing at approximately 55-60°C and 1 min extension at 72°C were be carried out in a thermocycler. Amplified DNA was diluted 1:1 with stop solution (97%o formamide, 1% EDTA, 0.1% bromphenol blue, 0.1% xylene cyanol), and denatured at 90°C for 2 min.
  • AFM 164zb8a 5'-TCTTGTCACTCTAACTCCGC-3' (SEQ _D NO:7)
  • AFM 164zb8m 5'-GGCCCATGTCTTTTTTAGGT-3' (SEQ _ NO:8)
  • DNAs relative to the tumor DNA may be collected with a PhosphorlmagerTM laser scanning device, and analyzed with the Phosphorlmager ImageQuantTM software package.
  • HET cDNA Full-length HET cDNA was excised by EcoRI digestion from the Bluescript clone and ligated into the eukaryotic expression vector pcDNAl. This plasmid was then used to transfect NIH 3T3 cells. Thus, 1.5 x 10 6 cells were plated onto 10 cm petri dishes and co-transfected with HET-pcDNAl and the G418 selectable expression vector pSV-neo (molar ratio 25:1). Control transfection was performed using the pcDNAl vector alone plus pSV-neo. The lipofectamine method was used for transfection. After 16 h in OPTIMEMTM the cells were washed. One day later the cells were split into four 15 cm-dishes in media containing 1 mg/ml G418. Clones were picked 2-3 wk later.
  • cells were plated at 2500 cells/well into a 96- well plate. The next day cell number was assessed by MTT assay (Day 0). Cells were then incubated in serum-free medium, or 10% serum, and cell number calculated at days 2, 4 and 6 by MTT assay.
  • HET/pcDNAl antisense (0.02, 0,2 and 2 ⁇ g).
  • the inventors transfected pcDNAl plasmids only. Cells were kept overnight in serum-free OPTIMEMTM, and than allowed to recover for 24 h in 10%> serum containing media.
  • the number of transfected 293 cells in S-phase was determined by incorporating H-thymidine into DNA as a measurement of cell proliferation. After the recovery phase the cells were incubated in media containing H-thymidine (1 ⁇ Ci/ml) for 1 h. After washing the cells with cold PBS and with 5% cold TCA, cells were fixed with 5% cold TCA for 30 min on ice. Cells were than washed four times with cold water, and lysed in 1 ml 0.5 M NaOH. 750 ⁇ l lysate was added to scintillation fluid, and cpm were counted. In parallel, 50 ⁇ l of lysate was used to determine protein concentration (BCA method). Each transfection was set up in triplicate, and the measurements for each point were done in duplicates. The 3H-thymidine incorporation was corrected for protein content.
  • HET expression on MCF-7 colony formation was assayed by transfection of MCF-7 cells with 20 ⁇ g of pcDNAl control vector or pcDNAl -HET sense strand, along with 1 ⁇ g of pSVneo. Cells were incubated in 400 ⁇ g/ml G418S for 3 wk, after which colonies were stained with 1% crystal violet.
  • EXAMPLE 6 Localization of the HET Gene to Human Chromosome 19 Band pl3 and LOH Assays for Breast Cancer Diagnosis
  • the chromosomal locus of the HET gene was identified by somatic cell hybrid analysis and FISH, as described above. Fluorescent in situ hybridization analysis (FISH) of a human metaphase chromosomal spread with an HET PI probe showed labeling of chromosome 19 (FIG. 7). DAPI counterstaining of the metaphase chromosomes allowed further localization of the HET gene to the locus 19pl3.2-13.3.
  • FISH Fluorescent in situ hybridization analysis
  • LOH assays were performed as described above by comparative PCRTM analysis of DNA from tumor and normal tissue samples, using primers specific for microsatellite marker D19S216 (Genethon).
  • D19S216 marker was chosen because it is located near the 19pl3.2-13.3 chromosomal locus of HET and it shows a high endogenous level of heterozygosity. Loss of heterozygosity is indicated by the disappearance of an allelic band in the tumor DNA sample which is present in a normal DNA sample from the same individual.
  • analysis of PCRTM products of D 19S216 indicates that LOH occurs at 19p 13.
  • HET expression may be utilized to control the phenotype of tumor cells.
  • Transfection of tumor cells with expression vectors containing an HET gene is expected to lead to loss of the transformed phenotype and slower tumor growth rate.
  • transfected NIH 3T3 cells were transfected with the eukaryotic expression vector pcDNAl, into which a full-length HET cDNA was incorporated.
  • many positive clones were originally obtained (by DNA- PCRTM and Western blot analysis), they could not be kept in culture, indicating that HET is growth inhibiting/toxic for the cells. Only one clone overexpressing HET survived, and growth analysis confirmed significant growth inhibition as compared to control clones.
  • FIG. 10A and FIG. 10B Cell growth rate was determined by MTT assay at 0, 2, 4 and 6 days post-transfection. As shown in FIG. 10A and FIG. 10B, transfection with the HET cDNA resulted in a significant inhibition of tumor cell growth rate. Each point on FIG. 10A and FIG. 10B represents the mean and standard deviation for quadruplicate samples of control and HET-transfected cells.
  • FIG. 10A and FIG. 10B The inhibition of tumor cell growth rate was particularly striking in serum-free medium.
  • Cell growth in serum-free medium is characteristic of the transformed phenotype.
  • transfection of tumor cells with HET resulted in at least a partial reversal of the transformed phenotype.
  • HET histone deacetylase
  • Increasing amounts (0.02, 0.2 and 2.0 ⁇ g) of HET antisense construct were transiently transfected into 293 cancer cells.
  • Control cells were transfected with vector alone.
  • the number of cells in S-phase was determined by incorporation of [3H]-thymidine, as described above.
  • transfection with increasing amounts of antisense HET was directly correlated with an increased proportion of 293 cells in S-phase, determined by incorporation of [3H]- thymidine into DNA.
  • FIG. 12 A The inverse correlation between HET levels and cell proliferation is further demonstrated in FIG. 12.
  • FIG. 12 A total cellular levels of HET decreased progressively when MCF-7/MG cells were stimulated to proliferate with 5% and 10% serum, respectively, compared to cells in serum free medium (SFM).
  • SFM serum free medium
  • Treatment of MCF-7 cells with the phorbol ester TPA has been shown to cause growth inhibition.
  • FIG. 12B when MCF-7 cells were exposed to 50 nM TPA, HET levels increased within 4 h.
  • FIG. 12B A similar effect was observed when MCF-7 cells were transfected with an HET expression vector (FIG. 12B).
  • Expression of HET resulted in an inhibition of MCF-7 colony formation (HET sense), compared with MCF-7 cells transfected with control vector alone (PCDNA1) or with an antisense expression vector (HET antisense).
  • HET is a tumor suppressor whose absence predicts a more aggressive type of breast cancer.
  • This example concerns the prediction of anti-estrogen drug resistance in cancer patients and their tissues based upon the evaluation of the relative abundance of HET/SAF-B in tissue samples. Cancer tissues displaying low levels of HET/SAF-B in comparison to tissues of known anti-estrogen drug resistance are predicted to be or likely to become drug resistant.
  • HET/SAF-B levels may be assessed by obtaining a sample of cancerous tissues from patients. Samples may be obtained through biopsy, fine needle aspiration, surgery, filtration, centrifugation, or other means. The relative abundance of HET/SAF-B in those tissues may be assessed. In one embodiment, tissues are subjected to SDS extraction and Western blot analysis using HET/SAF-B-specific antibodies. Relative abundances of HET/SAF-B may be obtained through densitometric measurements of the resulting stains. Preferably, one or more tissue samples of known drug resistance are included in the analysis as standards for comparison. Even more preferably, at least one sample of drug resistant and at least one sample of drug susceptible tissues are included.
  • Comparison of the HET/SAF-B levels will indicate those tissues in which drug resistance has occurred or is likely to occur. Those samples displaying levels of HET/SAF-B below the drug susceptible standards or similar to drug resistant standards are drug resistant or likely to become so. Those samples displaying levels of HET/SAF-B above the drug susceptible standards or similar to drug susceptible standards are susceptible. The susceptible or resistant status of each tissue sample is expected to correlate with the period of time the source patient may expect to remain disease free.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • a steroid receptor coactivator, SRA functions as an RNA and is present in an SRC- 1 complex.
  • the steroid receptor coactivator- 1 contains multiple receptor interacting and activation domains that cooperatively enhance the activation function 1 (AFl) and AF2 domains of steroid receptors. J. Biol.
  • Steroid receptor coactivator- 1 is a histone acetyltransferase. Nature 389: 194-198

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Abstract

Mots-clés : récepteur d'oestrogène ; protéine de matrice nucléaire HET/SAF-B ; transcription, répression; anti-oestrogène; tamoxifène L'invention concerne des procédés de détection de cellules tumorales, en particulier de cellules du cancer du sein humain. Elle concerne en outre des sondes génétiques et des sondes d'anticorps ainsi que des procédés servant à déterminer la présence d'une prolifération de cellules tumorales et des surveiller celle-ci. Ces procédés consistent à mesurer l'expression du polypeptide HET, les taux d'ARNm ou la perte du caractère hétérozygote dans le locus chromosomique 19p13, afin de déterminer le degré de malignité des cellules tumorales. Ces procédés permettent en outre de distinguer les cancers du sein résistants aux antagonistes de l'oestrogène tels que le tamoxifène, des tumeurs sensibles aux antagonistes de l'oestrogène. L'invention concerne en outre des procédures consistant à transformer des cellules avec des vecteurs contenant un gène HET exprimant le polypeptide HET. Ces procédures peuvent être utiles pour convertir les tumeurs résistantes au tamoxifène en tumeurs sensibles au tamoxifène.
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Cited By (5)

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WO2003042238A2 (fr) * 2001-11-12 2003-05-22 The University Of Bristol Modification de l'activite oestrogenique
WO2004008137A2 (fr) * 2002-07-12 2004-01-22 Max-Delbrück-Centrum für Molekulare Medizin Methode de diagnostic et de traitement de tumeurs du sein resistant au traitement anti-estrogene
US8703810B2 (en) 2010-06-10 2014-04-22 Seragon Pharmaceuticals, Inc. Estrogen receptor modulators and uses thereof
US9187460B2 (en) 2011-12-14 2015-11-17 Seragon Pharmaceuticals, Inc. Estrogen receptor modulators and uses thereof
WO2019018537A1 (fr) * 2017-07-18 2019-01-24 Exosome Diagnostics, Inc. Séquençage d'acides nucléiques associés à l'isolement exosomal chez des patients atteints de glioblastome multiforme

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CN107368709A (zh) * 2017-08-14 2017-11-21 江南大学 基于原子力显微镜的启动子体外强度表征的新方法

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OESTERREICHER S ET AL: "Novel nuclear matrix protein HET binds to and influences activity of the HSP27 promoter in human breast cancer cells" JOURNAL OF CELLULAR BIOCHEMISTRY, WILEY-LISS INC, US, vol. 67, no. 2, 1 November 1997 (1997-11-01), pages 275-286, XP002097726 ISSN: 0730-2312 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003042238A2 (fr) * 2001-11-12 2003-05-22 The University Of Bristol Modification de l'activite oestrogenique
WO2003042238A3 (fr) * 2001-11-12 2003-10-16 Univ Bristol Modification de l'activite oestrogenique
WO2004008137A2 (fr) * 2002-07-12 2004-01-22 Max-Delbrück-Centrum für Molekulare Medizin Methode de diagnostic et de traitement de tumeurs du sein resistant au traitement anti-estrogene
WO2004008137A3 (fr) * 2002-07-12 2004-09-02 Max Delbrueck Centrum Methode de diagnostic et de traitement de tumeurs du sein resistant au traitement anti-estrogene
US8703810B2 (en) 2010-06-10 2014-04-22 Seragon Pharmaceuticals, Inc. Estrogen receptor modulators and uses thereof
US9078871B2 (en) 2010-06-10 2015-07-14 Seragon Pharmaceuticals, Inc. Estrogen receptor modulators and uses thereof
US9187460B2 (en) 2011-12-14 2015-11-17 Seragon Pharmaceuticals, Inc. Estrogen receptor modulators and uses thereof
US9193714B2 (en) 2011-12-14 2015-11-24 Seragon Pharmaceuticals, Inc. Fluorinated estrogen receptor modulators and uses thereof
WO2019018537A1 (fr) * 2017-07-18 2019-01-24 Exosome Diagnostics, Inc. Séquençage d'acides nucléiques associés à l'isolement exosomal chez des patients atteints de glioblastome multiforme
US11345957B2 (en) 2017-07-18 2022-05-31 Exosome Diagnostics, Inc. Methods of treating glioblastoma in a subject informed by exosomal RNA signatures

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