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United States Patent | 6,046,032 |
Sherr ,   et al. | April 4, 2000 |
The INK4A (MTS1, CDKN2) gene encodes a specific inhibitor (InK4a-p16) of the cyclin D-dependent kinases CDK4 and CDK6. InK4a-p16 can block these kinase from phosphorylating the retinoblastoma protein (pRb), preventing exit from the G1 phase of the cell cycle. Deletions and mutations involving the gene encoding InK4a-p16, INK4A, occur frequently in cancer cells, implying that INK4a-p16, like pRb, suppresses tumor formulation. However, a completely unrelated protein (ARF-p19) arises in major part from an alternative reading frame of the mouse INK4A gene. Expression of an ARF-p19 cDNA (SEQ ID NO:1) in rodent fibroblasts induces both G1 and G2 phase arrest. Economical reutilization of protein coding sequences in this manner is without precedent in mammalian genomes, and the unitary inheritance of INK4a-p16 and ARF-p19 may reflect a dual requirement for both proteins in cell cycle control.
Inventors: | Sherr; Charles J. (Memphis, TN); Quelle; Dawn (Coralville, IA); Roussel; Martine F. (Memphis, TN); Zindy; Frederique (Memphis, TN) |
Assignee: | St. Jude Children's Research Hospital (Memphis, TN) |
Appl. No.: | 129855 |
Filed: | August 6, 1998 |
Intern'l Class: | C12P 021/06; C07K 014/00; C07H 017/00 |
Field of Search: | 514/12,2,44 530/350 435/69.1 |
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5174986 | Dec., 1992 | Berns. | |
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5739027 | Apr., 1998 | Kamb. | |
5876965 | Mar., 1999 | Sherr et al. | 435/69. |
Foreign Patent Documents | |||
WO 92/20796 | Nov., 1992 | WO. | |
WO 95/25429 | Sep., 1995 | WO. | |
WO 95/28483 | Oct., 1995 | WO. |
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______________________________________ A = Ala C = Cys D = Asp E = Glu F = Phe G = Gly H = His I = Ile K = Lys L = Leu M = Met N = Asn P = Pro Q = Gln R = Arg S = Ser T = Thr V = Val W = Trp Y = Tyr ______________________________________ ABBREVIATIONS CDK = cyclindependent kinase (protein); cdk = gene cDNA = complementary deoxyribonucleic acid DNA = deoxyribonucleic acid DMEM = Dulbecco's modified Eagle's medium EDTA = ethylenediamine tetraacetic acid ES = embryonic stem FISH = fluorescent in situ hybridization InK = inhibitor of CDK (protein); INK = gene kb = kilobase(s) kDa = kilodaton(s) MEL = mouse erythroleukemia (cell line) nt = nucleotide(s) PBS = phosphatebuffered saline PCR = polymerase chain reaction pRB = retinoblastoma protein RT = reverse transcriptase SDS = sodium dodecyl sulfate Sf9 = Spodoptera frugiperda (cell line) Tg = transgenic TK = thymidine kinase
TABLE 1 ______________________________________ Cell Cycle Arrest by ARF-p19 Vector Cell Cycle Distribution Cell Line cDNA (48 hrs post-infection) (No. Expts) Insert % G.sub.0 /G1 % S % G2/M ______________________________________ NIH-3T3 (7) None 42.4 .+-. 3.3 42.0 .+-. 2.3 15.6 .+-. 3.3 NIH-3T3 (8) p19.sup.ARF 66.6 .+-. 5.2 12.5 .+-. 2.6 20.9 .+-. 5.1 3T3-D1 (8) None 41.1 .+-. 6.6 39.1 .+-. 4.4 19.8 .+-. 2.7 3T3-D1 (9) p19.sup.ARF 53.0 .+-. 4.3 11.6 .+-. 2.8 35.4 .+-. 4.2 BALB-3T3 (3) None 36.5 .+-. 8.9 49.0 .+-. 8.8 14.5 .+-. 1.2 BALB-3T3 (3) p19.sup.ARF 57.1 .+-. 4.9 26.0 .+-. 2.8 16.9 .+-. 1.4 ______________________________________
TABLE 1 ______________________________________ Age p16 p16 Mouse Sex (weeks) Treatment Histology PCR Blot ______________________________________ K5 M 18 None Fibrosarcoma +* + K11 M 8 None Metastic salivary +* + gland carcinoma K17 F 21 None Thymoma ND ND K75 F 11 None Malignant fibrous ND ND histiocytoma K116 M 18 None Lymphoma (brain) ND ND K199 M 16 None Fibrosarcoma ND ND K86 F 12 DMBA Epidermal ND + papilloma (4 sites) K88 F 20 DMBA Epidermal ND ND papilloma K90 F 14 DMBA Lymphoma and + + epidermal papilloma K98 F 13 DMBA Mestatic epidermoid ND + carcinoma K106 M 9 DMBA Invasive epidermoid +* + carcinoma K149 M 12 DMBA Fibrosarcoma, ND ND malignant adenexal tumor K150 M 14 DMBA Epidermal ND ND papilloma (2 sites) K151 F 13 DMBA Fibrosarcoma and ND ND epidermal papilloma K163 M 13 DMBA Epidermal ND ND papilloma (4 sites) K173 M 12 Irradiation Fibrosarcoma ND ND K175 M 12 Irradiation Lymphoma ND ND K178 F 13 Irradiation Fibrosarcoma ND ND K185 M 19 Irradiation Lymphoma ND ND ______________________________________ Age refers to date of tumor detection or death of the animal. Where indicated, mice received ionizing radiation (4 Gy) [Kemp et al., Nat. Genetics, 8:66-69 (1994)] or were treated with DMBA 5-7 days after birth [Serrano et al., Cell, 85:27-37 (1996)]. Nine of 11 DMBAtreated animals, of 6 animals that received sublethal .gamma. irradiation, and 6 of 18 untreated mice developed tumors by 5 months of age. Fibrosarcomas arose subcutaneously and were all highly # invasive to skeletal muscle and bone. Skin tumors in DMBAtreated animals exhibited variable degrees of anaplasia, with lower grade papillomatous growths sometimes arising at multiple independent sites, and with higher grade carcinomas presenting a either locally invasive or frankly metastatic varants. Lymphomas were anaplastic largecell type with T cell markers. Detection of p16.sup.INK4a transcripts (PCR) or protein (immunoblotting) in primary tumor tissues is noted by # "+"; N.D. = not done; (*) RTPCR products taken for nucleotide sequencing.
__________________________________________________________________________ # SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES:12 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 713 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown - - (ii) MOLECULE TYPE: DNA (genomic) - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 43..548 - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 43..551 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - GTCACAGTGA GGCCGCCGCT GAGGGAGTAC AGCAGCGGGA GC ATG GGT - # CGC AGG 54 - # - # Met Gly Arg Arg - # - # 1 - - TTC TTG GTG ACT GTG AGG ATT CAG CGC GCG GG - #C CGC CCA CTC CAA GAG 102 Phe Leu Val Thr Val Arg Ile Gln Arg Ala Gl - #y Arg Pro Leu Gln Glu 5 - # 10 - # 15 - # 20 - - AGG GTT TTC TTG GTG AAG TTC GTG CGA TCC CG - #G AGA CCC AGG ACA GCG 150 Arg Val Phe Leu Val Lys Phe Val Arg Ser Ar - #g Arg Pro Arg Thr Ala 25 - # 30 - # 35 - - AGC TGC GCT CTG GCT TTC GTG AAC ATG TTG TT - #G AGG CTA GAG AGG ATC 198 Ser Cys Ala Leu Ala Phe Val Asn Met Leu Le - #u Arg Leu Glu Arg Ile 40 - # 45 - # 50 - - TTG AGA AGA GGG CCG CAC CGG AAT CCT GGA CC - #A GGT GAT GAT GAT GGG 246 Leu Arg Arg Gly Pro His Arg Asn Pro Gly Pr - #o Gly Asp Asp Asp Gly 55 - # 60 - # 65 - - CAA CGT TCA CGT AGC AGC TCT TCT GCT CAA CT - #A CGG TGC AGA TTC GAA 294 Gln Arg Ser Arg Ser Ser Ser Ser Ala Gln Le - #u Arg Cys Arg Phe Glu 70 - # 75 - # 80 - - CTG CGA GGA CCC CAC TAC CTT CTC CCG CCC GG - #T GCA CGA CGC AGC GCG 342 Leu Arg Gly Pro His Tyr Leu Leu Pro Pro Gl - #y Ala Arg Arg Ser Ala 85 - # 90 - # 95 - #100 - - GGA AGG CTT CCT GGA CAC GCT GGT GGT GCT GC - #A CGG GTC AGG GGC TCG 390 Gly Arg Leu Pro Gly His Ala Gly Gly Ala Al - #a Arg Val Arg Gly Ser 105 - # 110 - # 115 - - GCT GGA TGT GCG CGA TGC CTG GGG TCG CCT GC - #C GCT CGA CTT GGC CCA 438 Ala Gly Cys Ala Arg Cys Leu Gly Ser Pro Al - #a Ala Arg Leu Gly Pro 120 - # 125 - # 130 - - AGA GCG GGG ACA TCA AGA CAT CGT GCG ATA TT - #T GCG TTC CGC TGG GTG 486 Arg Ala Gly Thr Ser Arg His Arg Ala Ile Ph - #e Ala Phe Arg Trp Val 135 - # 140 - # 145 - - CTC TTT GTG TTC CGC TGG GTG GTC TTT GTG TA - #C CGC TGG GAA CGT CGC 534 Leu Phe Val Phe Arg Trp Val Val Phe Val Ty - #r Arg Trp Glu Arg Arg 150 - # 155 - # 160 - - CCA GAC CGA CGG GCA TAG CTTCAGCTC AAGCACGCCC AGG - #GCCCTGG 581 Pro Asp Arg Arg Ala * 165 - - AACTTCGCGG CCAATCCCAA GAGCAGAGCT AAATCCGGCC TCAGCCCGCC TT - #TTTCTTCT 641 - - TAGCTTCACT TCTAGCGATG CTAGCGTGTC TAGCATGTGG CTTTAAAAAA TA - #CATAATAA 701 - - TGCTTTTTTT TT - # - # - # 713 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 169 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - Met Gly Arg Arg Phe Leu Val Thr Val Arg Il - #e Gln Arg Ala Gly Arg 1 5 - # 10 - # 15 - - Pro Leu Gln Glu Arg Val Phe Leu Val Lys Ph - #e Val Arg Ser Arg Arg 20 - # 25 - # 30 - - Pro Arg Thr Ala Ser Cys Ala Leu Ala Phe Va - #l Asn Met Leu Leu Arg 35 - # 40 - # 45 - - Leu Glu Arg Ile Leu Arg Arg Gly Pro His Ar - #g Asn Pro Gly Pro Gly 50 - # 55 - # 60 - - Asp Asp Asp Gly Gln Arg Ser Arg Ser Ser Se - #r Ser Ala Gln Leu Arg 65 - # 70 - # 75 - # 80 - - Cys Arg Phe Glu Leu Arg Gly Pro His Tyr Le - #u Leu Pro Pro Gly Asp 85 - # 90 - # 95 - - Arg Arg Ser Ala Gly Arg Leu Pro Gly His Al - #a Gly Gly Ala Ala Arg 100 - # 105 - # 110 - - Val Arg Gly Ser Ala Gly Cys Ala Arg Cys Le - #u Gly Ser Pro Ala Ala 115 - # 120 - # 125 - - Arg Leu Gly Pro Arg Ala Gly Thr Ser Arg Hi - #s Arg Ala Ile Phe Ala 130 - # 135 - # 140 - - Phe Arg Trp Val Leu Phe Val Phe Arg Trp Va - #l Val Phe Val Tyr Arg 145 1 - #50 1 - #55 1 - #60 - - Trp Glu Arg Arg Pro Asp Arg Arg Ala 165 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 540 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: DNA (genomic) - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 142..540 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - CGCGCCTGCG GGGCGGAGAT GGGCAGGGGG CGGTGCGTGG GTCCCAGTCT GC - #AGTTAAGG 60 - - GGGCAGGAGT GGCGCTGCTC ACCTCTGGTG CCAAAGGGCG GCGCAGCGGC TG - #CCGAGCTC 120 - - GGCCCTGGAG GCGGCGAGAA C ATG GTG CGC AGG TTC TTG - #GTG ACC CTC CGG 171 - # Met Val Arg Arg Phe Leu Val - #Thr Leu Arg - # 1 - # 5 - # 10 - - ATT CGG CGC GCG TGC GGC CCG CCG CGA GTG AG - #G GTT TTC GTG GTT CAC 219 Ile Arg Arg Ala Cys Gly Pro Pro Arg Val Ar - #g Val Phe Val Val His 15 - # 20 - # 25 - - ATC CCG CGG CTC ACG GGG GAG TGG GCA GCG CC - #A GGG GCG CCC GCC GCT 267 Ile Pro Arg Leu Thr Gly Glu Trp Ala Ala Pr - #o Gly Ala Pro Ala Ala 30 - # 35 - # 40 - - GTG GCC CTC GTG CTG ATG CTA CTG AGG AGC CA - #G CGT CTA GGG CAG CAG 315 Val Ala Leu Val Leu Met Leu Leu Arg Ser Gl - #n Arg Leu Gly Gln Gln 45 - # 50 - # 55 - - CCG CTT CCT AGA AGA CCA GGT CAT GAT GAT GG - #G CAG CGC CCG AGT GGC 363 Pro Leu Pro Arg Arg Pro Gly His Asp Asp Gl - #y Gln Arg Pro Ser Gly 60 - # 65 - # 70 - - GGA GCT GCT GCT GCT CCA CGG CGC GGA GCC CA - #A CTG CGC CGA CCC CGC 411 Gly Ala Ala Ala Ala Pro Arg Arg Gly Ala Gl - #n Leu Arg Arg Pro Arg 75 - # 80 - # 85 - # 90 - - CAC TCT CAC CCG ACC CGT GCA CGA CGC TGC CC - #G GGA GGG CTT CCT GGA 459 His Ser His Pro Thr Arg Ala Arg Arg Cys Pr - #o Gly Gly Leu Pro Gly 95 - # 100 - # 105 - - CAC GCT GGT GGT GCT GCA CCG GGC CGG GGC GC - #G GCT GGA CGT GCG CGA 507 His Ala Gly Gly Ala Ala Pro Gly Arg Gly Al - #a Ala Gly Arg Ala Arg 110 - # 115 - # 120 - - TGC CTG GGG CCG TCT GCC CGT GGA CCT GGC TG - #A - # 540 Cys Leu Gly Pro Ser Ala Arg Gly Pro Gly - #* 125 - # 130 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 132 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - Met Val Arg Arg Phe Leu Val Thr Leu Arg Il - #e Arg Arg Ala Cys Gly 1 5 - # 10 - # 15 - - Pro Pro Arg Val Arg Val Phe Val Val His Il - #e Pro Arg Leu Thr Gly 20 - # 25 - # 30 - - Glu Trp Ala Ala Pro Gly Ala Pro Ala Ala Va - #l Ala Leu Val Leu Met 35 - # 40 - # 45 - - Leu Leu Arg Ser Gln Arg Leu Gly Gln Gln Pr - #o Leu Pro Arg Arg Pro 50 - # 55 - # 60 - - Gly His Asp Asp Gly Gln Arg Pro Ser Gly Gl - #y Ala Ala Ala Ala Pro 65 - # 70 - # 75 - # 80 - - Arg Arg Gly Ala Gln Leu Arg Arg Pro Arg Hi - #s Ser His Pro Thr Arg 85 - # 90 - # 95 - - Ala Arg Arg Cys Pro Gly Gly Leu Pro Gly Hi - #s Ala Gly Gly Ala Ala 100 - # 105 - # 110 - - Pro Gly Arg Gly Ala Ala Gly Arg Ala Arg Cy - #s Leu Gly Pro Ser Ala 115 - # 120 - # 125 - - Arg Gly Pro Gly 130 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 125 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - Met Met Met Gly Asn Val His Val Ala Ala Le - #u Leu Leu Asn Tyr Gly 1 5 - # 10 - # 15 - - Ala Asp Ser Asn Cys Glu Asp Pro Thr Thr Ph - #e Ser Arg Pro Val His 20 - # 25 - # 30 - - Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Le - #u Val Val Leu His Gly 35 - # 40 - # 45 - - Ser Gly Ala Arg Leu Asp Val Arg Asp Ala Tr - #p Gly Arg Leu Pro Leu 50 - # 55 - # 60 - - Asp Leu Ala Gln Glu Arg Gly His Gln Asp Il - #e Val Arg Tyr Leu Arg 65 - # 70 - # 75 - # 80 - - Ser Ala Gly Cys Ser Leu Cys Ser Ala Gly Tr - #p Ser Leu Cys Thr Ala 85 - # 90 - # 95 - - Gly Asn Val Ala Gln Thr Asp Gly His Ser Ph - #e Ser Ser Ser Thr Pro 100 - # 105 - # 110 - - Arg Ala Leu Glu Leu Arg Gly Gln Ser Gln Gl - #u Gln Ser 115 - # 120 - # 125 - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - GCAAAGCTTG AGGCCGGATT TAGCTCTGCT C - # - # 31 - - - - (2) INFORMATION FOR SEQ ID NO:7: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - - AGGGATCCTT GGTCACTGTG AGGATTC - # - # 27 - - - - (2) INFORMATION FOR SEQ ID NO:8: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: - - CGGGATCCGC TGCAGACAGA CTGGCCAG - # - # 28 - - - - (2) INFORMATION FOR SEQ ID NO:9: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: - - CGTCTAGAGC GTGTCCAGGA AGCCTTCC - # - # 28 - - - - (2) INFORMATION FOR SEQ ID NO:10: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino - #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: Not Relev - #ant - - (ii) MOLECULE TYPE: peptide - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: - - Val Phe Val Tyr Arg Trp Glu Arg Arg Pro As - #p Arg Arg Ala
1 5 - # 10 - - - - (2) INFORMATION FOR SEQ ID NO:11: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 74 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: - - CGGGATCCGA ATTCAGCCAT GGGTTACCCA TACGACGTCC CAGACTACGC TA - #CCGGTCGC 60 - - AGGTTCTTGG TCAC - # - # - # 74 - - - - (2) INFORMATION FOR SEQ ID NO:12: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc - #= "Synthetic DNA" - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: - - GCCCGCGCGC TGAATCCTCA - # - # - # 20 __________________________________________________________________________