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United States Patent | 5,691,198 |
Jin ,   et al. | November 25, 1997 |
A variety of genetic constructs are disclosed that will find both in vitro and in vivo use in the area of tumor biology and cancer therapy. In particular, expression constructs are provided that contain a p16 encoding region and other regulatory elements necessary for the expression of a p16 transcript. One version of the expression construct is a replication-deficient adenoviral vector. Also provided are methods for the transformation of cell lines and the inhibition of cancer cell proliferation.
Inventors: | Jin; Xiaomei (Houston, TX); Roth; Jack A. (Houston, TX) |
Assignee: | Board of Regents, The University of Texas System (Austin, TX) |
Appl. No.: | 502881 |
Filed: | July 17, 1995 |
Intern'l Class: | A61K 048/00; C12N 005/00; C12N 015/00 |
Field of Search: | 514/44,8,2 536/23.1,24.1,172.3,240.25,320.1 424/93.21 |
5399346 | Mar., 1995 | Anderson et al. | 424/93. |
Foreign Patent Documents | |||
0273085 | Jul., 1988 | EP | . |
9428152 | Dec., 1994 | WO. |
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TABLE 1 ______________________________________ Amino Acids Codons ______________________________________ Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine VaI V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU ______________________________________
TABLE 2 ______________________________________ ENHANCER ______________________________________ Immunoglobulin Heavy Chain Immunoglobulin Light Chain T-Cell Receptor HLA DQ .alpha. and DQ .beta. .beta.-Interferon Interleukin-2 Interleukin-2 Receptor MHC Class II 5.sub..alpha. .sup.k MHC Class II HLA-DR.alpha. .beta.-Actin Muscle Creatine Kinase Prealbumin (Transthyretin) Elastase I Metallothionein Collagenase Albumin Gene .alpha.-Fetoprotein .tau.-Globin .beta.-Globin c-fos c-HA-ras Insulin Neural Cell Adhesion Molecule (NCAM) .alpha..sub.1-Antitrypsin H2B (TH2B) Histone Mouse or Type I Collagen Glucose-Regulated Proteins (GRP94 and GRP78) Rat Growth Hormone Human Serum Amyloid A (SAA) Troponin I (TN I) Platelet-Derived Growth Factor Duchenne Muscular Dystrophy SV40 Polyoma Retroviruses Papilloma Virus Hepatitis B Virus Human Immunodeficiency Virus Cytomegalovirus Gibbon Ape Leukemia Virus ______________________________________
TABLE 3 ______________________________________ Element Inducer ______________________________________ MT II Phorbol Ester (TFA) Heavy metals MMTV (mouse mammary Glucocorticoids tumor virus) .beta.-Interferon poly(rI)X poly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H.sub.2 O.sub.2 Collagenase Phorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1 SV40 Phorbol Ester (TFA) Murine MX Gene Interferon, Newcastle Disease Virus GRP78 Gene A23187 .alpha.-2-MacroglobuIin IL-6 Vimentin Serum MHC Class I Gerie H-2kB Interferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol Ester-TPA Tumor Necrosis Factor FMA Thyroid Stiniulating Hormone Thyroid Hormone .alpha. Gene ______________________________________
______________________________________ A/B/A B/A/B B/B/A A/A/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B ______________________________________
TABLE 4 ______________________________________ Flow Cytometry Analysis of Cell-Cycle Effects of p16.sup.INK4 Mediated by Ad-p16 Percent of Cells in cell types & infected viruses G.sub.0 /G.sub.1 S G.sub.2 /M ______________________________________ HBL100/Ad-p16 36 42 22 HBL100/Ad5CMV-lacZ 31 44 25 HBL100/medium 31 42 27 H460/Ad-p16 88 9 3 H460/Ad5CMV-lacZ 41 32 27 H460/medium 39 35 26 H322/Ad-p16 80 6 14 H322/Ad5CMV-lacZ 30 36 34 H322/medium 30 36 34 H226Br/Ad-p16 80 11 9 H226Br/Ad5CMV-lacZ 20 53 27 H226Br/medium 26 44 30 ______________________________________ Values are shown for a representative assay. Cells were infected the same way as in the cell growth rate determination assay. 24 hours after infection, cells were treated with 1% Tween20 and stained with propidium iodide and then analyzed by flow cytornetry for DNA synthesis and cell cycle status. Flow cytometric assay was performed with a FACScan (Becton Dickinson, San Jose, CA) equipped with an aircooled 15mW 488 nm argon laser. Red fluorescence was measured through a long pass filter with cutoff wavelength at 650 nm.
TABLE 5 ______________________________________ Effect of p16.sup.INK4 on Tumorigenicity of H460 Cell Line in Nude Mice No. of tumors Mean Volume Treatment No. of mice (%) (mm.sup.3 .+-. SD) ______________________________________ Experinient 1 Medium 4/4 (100%) 1047.3 .+-. 104.7 Ad5/CMV-LacZ 4/4 (100%) 740.5 .+-. 205.5 Ad-p16 0/4 (0%) -- Experirnent 2 Medium 4/4 (100%) 1158.6 .+-. 200.2 Ad5/CMV-LacZ 3/4 (75%) 658.3 .+-. 144.3 Ad-p16 4/4 (100%) 71.2 .+-. 19.6 ______________________________________ The H460 cells were infected with viruses at a MOI of 50 PFU/ce11 for 24 hours and injected subcutaneously at 5 .times. 10.sup.6 cells/mouse. Tumo sizes were determined at the end of a threeweek period.
__________________________________________________________________________ SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 7 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 987 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CGGAGAGGGGGAGAACAGACAACGGGCGGCGGGGAGCAGCATGGAGCCGGCGGCGGGGAG60 CAGCATGGAGCCTTCGGCTGACTGGCTGGCCACGGCCGCGGCCCGGGGTCGGGTAGAGGA120 GGTGCGGGCGCTGCTGGAGGCGGGGGCGCTGCCCAACGCACCGAATAGTTACGGTCGGAG180 GCCGATCCAGGTCATGATGATGGGCAGCGCCCGAGTGGCGGAGCTGCTGCTGCTCCACGG240 CGCGGAGCCCAACTGCGCCGACCCCGCCACTCTCACCCGACCCGTGCACGACGCTGCCCG300 GGAGGGCTTCCTGGACACGCTGGTGGTGCTGCACCGGGCCGGGGCGCGGCTGGACGTGCG360 CGATGCCTGGGGCCGTCTGCCCGTGGACCTGGCTGAGGAGCTGGGCCATCGCGATGTCGC420 ACGGTACCTGCGCGCGGCTGCGGGGGGCACCAGAGGCAGTAACCATGCCCGCATAGATGC480 CGCGGAAGGTCCCTCAGACATCCCCGATTGAAAGAACCAGAGAGGCTCTGAGAAACCTCG540 GGAAACTTAGATCATCAGTCACCGAAGGTCCTACAGGGCCACAACTGCCCCCGCCACAAC600 CCACCCCGCTTTCGTAGTTTTCATTTAGAAAATAGAGCTTTTAAAAATGTCCTGCCTTTT660 AACGTAGATATAAGCCTTCCCCCACTACCGTAAATGTCCATTTATATCATTTTTTATATA720 TTCTTATAAAAATGTAAAAAAGAAAAACACCGCTTCTGCCTTTTCACTGTGTTGGAGTTT780 TCTGGAGTGAGCACTCACGCCCTAAGCGCACATTCATGTGGGCATTTCTTGCGAGCCTCG840 CAGCCTCCGGAAGCTGTCGACTTCATGACAAGCATTTTGTGAACTAGGGAAGCTCAGGGG900 GGTTACTGGCTTCTCTTGAGTCACACTGCTAGCAAATGGCAGAACCAAAGCTCAAATAAA960 AATAAAATAATTTTCATTCATTCACTC987 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 156 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: MetGluProAlaAlaGlySerSerMetGluProSerAlaAspTrpLeu 151015 AlaThrAlaAlaAlaArgGlyArgValGluGluValArgAlaLeuLeu 202530 GluAlaGlyAlaLeuProAsnAlaProAsnSerTyrGlyArgArgPro 354045 IleGlnValMetMetMetGlySerAlaArgValAlaGluLeuLeuLeu 505560 LeuHisGlyAlaGluProAsnCysAlaAspProAlaThrLeuThrArg 65707580 ProValHisAspAlaAlaArgGluGlyPheLeuAspThrLeuValVal 859095 LeuHisArgAlaGlyAlaArgLeuAspValArgAspAlaTrpGlyArg 100105110 LeuProValAspLeuAlaGluGluLeuGlyHisArgAspValAlaArg 115120125 TyrLeuArgAlaAlaAlaGlyGlyThrArgGlySerAsnHisAlaArg 130135140 IleAspAlaAlaGluGlyProSerAspIleProAsp 145150155 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGGAGCCTTCGGCTGACTGG21 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CCTGTAGGACCTTCGGTGACT21 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GATCCGGCGGCGGGGAGCAGCATGGAGCCTTCGGCTGACTGG42 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GCCTCTCTGGTTCTTTCA18 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "DNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CGGGCGGGGAGCAGCATGGAGCCGGCGGCGGGGAGC36 __________________________________________________________________________