KR20010022741A - ISOLATION OF A NOVEL SENESCENCE-FACTOR GENE, p23 - Google Patents

Abstract

The present invention provides isolated senescence-related nucleic acid molecules (p23) encoding polypeptides of 23 kilodaltons, methods of expressing 23 kilodalton polypeptides in culture cells, recombinant p23 polypeptides, expression vectors of p23 and host cells, and antibodies against p23. To provide. The present invention also provides methods for regulating senescence using p23 and measuring expression of p23 in biological samples.

Description

Isolation of a New Aging-Factor Gene, ｐ23 {ISOLATION OF A NOVEL SENESCENCE-FACTOR GENE, p23}

<110> UNIVERSITY OF WASHINGTON

<120> ISOLATION OF A NOVEL SENESCENCE-FACTOR GENE, p23

<130> 1

<150> US 08 / 908,873

<151> 1997-08-08

<160> 19

<170> KOPATIN 1.5

<210> 1

<211> 3443

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (221) .. (853)

<400> 1

gagcaacctc agcttctagt atccagactc cagcgccgcc ccgggcgcgg accccaaccc 60

cgacccagag cttctccagc ggcggcgcag cgagcagggc tccccgcctt aacttcctcc 120

gcggggccca gccaccttcg ggagtccggg ttgcccacct gcaaactctc cgccttctgc 180

acctgccacc cctgagccag cgcgggcgcc cgagcgagtc atg gcc aac gcg ggg 235

Met Ala Asn Ala Gly

1 5

ctg cag ctg ttg ggc ttc att ctc gcc ttc ctg gga tgg atc ggc gcc 283

Leu Gln Leu Leu Gly Phe Ile Leu Ala Phe Leu Gly Trp Ile Gly Ala

10 15 20

atc gtc agc act gcc ctg ccc cag tgg agg att tac tcc tat gcc ggc 331

Ile Val Ser Thr Ala Leu Pro Gln Trp Arg Ile Tyr Ser Tyr Ala Gly

25 30 35

gac aac atc gtg acc gcc cag gcc atg tac gag ggg ctg tgg atg tcc 379

Asp Asn Ile Val Thr Ala Gln Ala Met Tyr Glu Gly Leu Trp Met Ser

40 45 50

tgc gtg tcg cag agc acc ggg cag atc cag tgc aaa gtc ttt gac tcc 427

Cys Val Ser Gln Ser Thr Gly Gln Ile Gln Cys Lys Val Phe Asp Ser

55 60 65

ttg ctg aat ctg agc agc aca ttg caa gca acc cgt gcc ttg atg gtg 475

Leu Leu Asn Leu Ser Ser Thr Leu Gln Ala Thr Arg Ala Leu Met Val

70 75 80 85

gtt ggc atc ctc ctg gga gtg ata gca atc ttt gtg gcc acc gtt ggc 523

Val Gly Ile Leu Leu Gly Val Ile Ala Ile Phe Val Ala Thr Val Gly

90 95 100

atg aag tgt atg aag tgc ttg gaa gac gat gag gtg cag aag atg agg 571

Met Lys Cys Met Lys Cys Leu Glu Asp Asp Glu Val Gln Lys Met Arg

105 110 115

atg gct gtc att ggg ggt gcg ata ttt ctt ctt gca ggt ctg gct att 619

Met Ala Val Ile Gly Gly Ala Ile Phe Leu Leu Ala Gly Leu Ala Ile

120 125 130

tta gtt gcc aca gca tgg tat ggc aat aga atc gtt caa gaa ttc tat 667

Leu Val Ala Thr Ala Trp Tyr Gly Asn Arg Ile Val Gln Glu Phe Tyr

135 140 145

gac cct atg acc cca gtc aat gcc agg tac gaa ttt ggt cag gct ctc 715

Asp Pro Met Thr Pro Val Asn Ala Arg Tyr Glu Phe Gly Gln Ala Leu

150 155 160 165

ttc act ggc tgg gct gct gct tct ctc tgc ctt ctg gga ggt gcc cta 763

Phe Thr Gly Trp Ala Ala Ala Ser Leu Cys Leu Leu Gly Gly Ala Leu

170 175 180

ctt tgc tgt tcc tgt ccc cga aaa aca acc tct tac cca aca cca agg 811

Leu Cys Cys Ser Cys Pro Arg Lys Thr Thr Ser Tyr Pro Thr Pro Arg

185 190 195

ccc tat cca aaa cct gca cct tcc agc ggg aaa gac tac gtg tgacaca 860

Pro Tyr Pro Lys Pro Ala Pro Ser Ser Gly Lys Asp Tyr Val

200 205 210

gaggcaaaag gagaaaatca tgttgaaaca aaccgaaaat ggacattgag atactatcat 920

taacattagg accttagaat tttgggtatt gtaatctgaa gtatggtatt acaaaacaaa 980

caaacaaaca aaaaacccat gtgttaaaat actcagtgct aaacatggct taatcttatt 1040

ttatcttctt tcctcaatat aggagggaag attttaccat ttgtattact gcttcccatt 1100

gagtaatcat actcaaatgg gggaaggggt gctccttaaa tatatataga tatgtatata 1160

tacatgtttt tctattaaaa atagacagta aaatactatt ctcattatgt tgatactagc 1220

atacttaaaa tatctctaaa ataggtaaat gtatttaatt ccatattgat gaagatgttt 1280

attggtatat tttctttttc gtccttatat acatatgtaa cagtcaaata tcatttactc 1340

ttcttcatta gctttgggtg cctttgccac aagacctagc ctaatttacc aaggatgaat 1400

tctttcaatt cttcatgcgt gcccttttca tatacttatt ttatttttta ccataatctt 1460

atagcacttg catcgttatt aagcccttat ttgttttgtg tttcattggt ctctatctcc 1520

tgaatctaac acatttcata gcctacattt tagtttctaa agccaagaag aatttattac 1580

aaatcagaac tttggaggca aatctttctg catgaccaaa gtgataaatt cctgttgacc 1640

ttcccacaca atccctgtac tctgacccat agcactcttg tttgctttga aaatatttgt 1700

ccaattgagt agctgcatgc tgttccccca ggtgttgtaa cacaacttta ttgattgaat 1760

ttttaagcta cttattcata gttttatatc cccctaaact acctttttgt tccccattcc 1820

ttaattgtat tgttttccca agtgtaatta tcatgcgttt tatatcttcc taataaggtg 1880

tggtctgttt gtctgaacaa agtgctagac tttctggagt gataatctgg tgacaaatat 1940

tctctctgta gctgtaagca agtcacttaa tctttctacc tcttttttct atctgccaaa 2000

ttgagataat gatacttaac cagttagaag aggtagtgtg aatattaatt agtttatatt 2060

actctcattc tttgaacatg aactatgcct atgtagtgtc tttatttgct cagctggctg 2120

agacactgaa gaagtcactg aacaaaacct acacacgtac cttcatgtga ttcactgcct 2180

tcctctctct accagtctat ttccactgaa caaaacctac acacatacct tcatgtggtt 2240

cagtgccttc ctctctctac cagtctattt ccactgaaca aaacctacgc acataccttc 2300

atgtggctca gtgccttcct ctctctacca gtctatttcc attctttcag ctgtgtctga 2360

catgtttgtg ctctgttcca ttttaacaac tgctcttact tttccagtct gtacagaatg 2420

ctatttcact tgagcaagat gatgtatgga aagggtgttg gcactggtgt ctggagacct 2480

ggatttgagt cttggtgcta tcaatcaccg tctgtgtttg agcaaggcat ttggctgctg 2540

taagcttatt gcttcatctg taagcggtgg tttgtaattc ctgatcttcc cacctcacag 2600

tgatgttgtg gggatccagt gagatagaat acatgtaagt gtggttttgt aatttgaaaa 2660

gtgctatact aagggaaaga attgaggaat taactgcata cgttttggtg ttgcttttca 2720

aatgtttgaa aataaaaaaa tgttaagaaa tgggtttctt gccttaacca gtctctcaag 2780

tgatgagaca gtgaagtaaa attgagtgca ctaaacgaat aagattctga ggaagtctta 2840

tcttctgcag tgagtatggc ccaatgcttt ctgtggctaa acagatgtaa tgggaagaaa 2900

taaaagccta cgtgttggta aatccaacag caagggagat ttttgaatca taataactca 2960

taaggtgcta tctgttcagt gatgccctca gagctcttgc tgttagctgg cagctgacgc 3020

tgctaggata gttagtttgg aaatggtact tcataataaa ctacacaagg aaagtcagcc 3080

accgtgtctt atgaggaatt ggacctaata aattttagtg tgccttccaa acctgagaat 3140

atatgctttt ggaagttaaa atttaaatgg cttttgccac atacatagat cttcatgatg 3200

tgtgagtgta attccatgtg gatatcagtt accaaacatt acaaaaaaat tttatggccc 3260

aaaatgacca acgaaattgt tacaatagaa tttatccaat tttgatcttt ttatattctt 3320

ctaccacacc tggaaacaga ccaatagaca ttttggggtt ttataatggg aatttgtata 3380

aagcattact ctttttcaat aaattgtttt ttaatttaaa aaaaggaaaa aaaaaaaaaa 3440

aaa 3443

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<213> Homo sapiens

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Met Ala Asn Ala Gly Leu Gln Leu Leu Gly Phe Ile Leu Ala Phe Leu

1 5 10 15

Gly Trp Ile Gly Ala Ile Val Ser Thr Ala Leu Pro Gln Trp Arg Ile

20 25 30

Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Ala Gln Ala Met Tyr Glu

35 40 45

Gly Leu Trp Met Ser Cys Val Ser Gln Ser Thr Gly Gln Ile Gln Cys

50 55 60

Lys Val Phe Asp Ser Leu Leu Asn Leu Ser Ser Thr Leu Gln Ala Thr

65 70 75 80

Arg Ala Leu Met Val Val Gly Ile Leu Leu Gly Val Ile Ala Ile Phe

85 90 95

Val Ala Thr Val Gly Met Lys Cys Met Lys Cys Leu Glu Asp Asp Glu

100 105 110

Val Gln Lys Met Arg Met Ala Val Ile Gly Gly Ala Ile Phe Leu Leu

115 120 125

Ala Gly Leu Ala Ile Leu Val Ala Thr Ala Trp Tyr Gly Asn Arg Ile

130 135 140

Val Gln Glu Phe Tyr Asp Pro Met Thr Pro Val Asn Ala Arg Tyr Glu

145 150 155 160

Phe Gly Gln Ala Leu Phe Thr Gly Trp Ala Ala Ala Ser Leu Cys Leu

165 170 175

Leu Gly Gly Ala Leu Leu Cys Cys Ser Cys Pro Arg Lys Thr Thr Ser

180 185 190

Tyr Pro Thr Pro Arg Pro Tyr Pro Lys Pro Ala Pro Ser Ser Gly Lys

195 200 205

Asp Tyr Val

210

<210> 3

<211> 247

<212> PRT

<213> Rattus norvegicus

<400> 3

Met Ser Met Ser Leu Glu Ile Thr Gly Thr Ser Leu Ala Val Leu Gly

1 5 10 15

Trp Leu Cys Thr Ile Val Cys Cys Ala Leu Pro Met Trp Arg Val Ser

20 25 30

Ala Phe Ile Gly Ser Ser Ile Thr Ala Gln Ile Thr Trp Glu Gly

35 40 45

Leu Trp Met Asn Cys Val Gln Ser Thr Gly Gln Met Gln Cys Lys Met

50 55 60

Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala Arg Ala

65 70 75 80

Leu Ile Val Val Ser Ile Leu Leu Ala Ala Phe Gly Leu Leu Val Ala

85 90 95

Leu Val Gly Ala Gln Cys Thr Asn Cys Val Gln Asp Glu Thr Ala Lys

100 105 110

Ala Lys Ile Thr Ile Val Ala Gly Val Leu Phe Leu Leu Ala Ala Val

115 120 125

Leu Thr Leu Val Pro Val Ser Trp Ser Ala Asn Thr Ile Ile Arg Asp

130 135 140

Phe Tyr Asn Pro Leu Val Pro Glu Ala Gln Lys Arg Glu Met Gly Thr

145 150 155 160

Gly Leu Tyr Val Gly Trp Ala Ala Ala Ala Leu Gln Leu Leu Gly Gly

165 170 175

Ala Leu Leu Cys Cys Ser Cys Pro Pro Arg Glu Lys Tyr Ala Pro Thr

180 185 190

Lys Ile Leu Tyr Ser Ala Pro Arg Ser Thr Gly Pro Gly Thr Gly Thr

195 200 205

Gly Thr Ala Tyr Asp Arg Lys Thr Thr Ser Glu Arg Pro Gly Ala Arg

210 215 220

Thr Pro His His His His Tyr Gln Pro Ser Met Tyr Pro Thr Arg Pro

225 230 235 240

Ala Cys Ser Leu Ala Ser Glu

245

<210> 4

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 4

aagctttttt tttttg 16

<210> 5

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 5

aagctttttt ttttta 16

<210> 6

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 6

aagctttttt tttttc 16

<210> 7

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 7

tttttttttt ttaa 14

<210> 8

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 8

tttttttttt ttga 14

<210> 9

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 9

tttttttttt ttca 14

<210> 10

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 10

tttttttttt ttag 14

<210> 11

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 11

tttttttttt ttgg 14

<210> 12

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 12

tttttttttt ttcg 14

<210> 13

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 13

tttttttttt ttac 14

<210> 14

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 14

tttttttttt ttgc 14

<210> 15

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 15

tttttttttt ttcc 14

<210> 16

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 16

tttttttttt ttat 14

<210> 17

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 17

tttttttttt ttgt 14

<210> 18

<211> 14

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 18

tttttttttt ttct 14

<210> 19

<211> 10

<212> DNA

<213> Artificial Sequence

<220>

<223> oligonucleotide

<400> 19

ggagggtgtt 10

Replication aging, ie the inability to divide cells in response to mitogen, was first described in cultured normal human fibroblasts (Expt. Cell Res. 37: 614-635, 1965). Reference). Since then, several types of other human cells have been observed to age after repeated passage cultures (see Smith and Pereira-Smith, Science 273: 63-67, 1996). Aging cells, even if they should though maintaining the metabolic activity for an extended incubation with a DNA content of the G Step _1, whose growth is prevented in the G phase ₁ does not enter the S phase, against the death by apoptosis (apoptosis) do.

Although senescent cells in culture can be identified by their inability to divide in response to mitogen, until recently it was not possible to distinguish senescent cells from cells that possess dividing capacity in vivo. However, enzymes with β-galactosidase activity, a biological marker that clearly identifies senescent human cells in culture, have recently been described (Dimri et al., Proc. Natl. Acad. Sci. USA 92: 9363-). 9367, 1995). This study demonstrated an inverse relationship between the ability to incorporate ³ H-thymidine into newly synthesized DNA in cultured cells and the expression of β-galactosidase. The presence of this enzyme can be easily detected by providing the cells with a substrate that produces a blue product upon cleavage by the enzyme. The researchers also observed that the aging-binding β-galactosidase increases with age in human skin, indicating that the accumulation of the enzyme is a marker for aging in fibroblasts and keratinocytes of the skin and possibly other epithelial tissues. Implying to provide

The aging phenotype seems to be regulated by one or more genes. In one study, cell fusion experiments were performed using 40 different immortal human cell lines to determine if the aging phenotype could be recovered. Based on the results, the cell lines were assigned to four different complementation groups, indicating that at least four genes or genetic pathways contribute to aging (Smience and Pereira-Smith, Science 273: 63-67, 1996). Other experiments have shown that genes located on human chromosomes 1, 4, 6, 7, 11, 18 and X are involved in aging (see above). Recently, a specific gene (mac25) overexpressed in senescent epithelial cells has been isolated and mapped to the long arm of chromosome 4 (Swisshelm et al., Proc. Natl. Acad. Sci. USA 92 4472-4476, 1995).

Since senescent cells are likely to be blocked at the G ₁ stage, one approach to identifying aging-related genes is to compare quiescent cells that are post-sera deficient and transcripts expressed during G ₁ in senescent cells. It was. Using this approach, a number of differences in gene expression between senescent and senescent cells have been demonstrated (Smith and Pereira-Smith, 1996; WO 96/13601, 1996). Aging fibroblasts have also been observed to express transcription factor binding activity at reduced levels (see supra). However, there have been no reports of inducing senescent cells into the cell cycle by introducing a single gene product, which is usually downregulated in senescent cells.

Some experiments suggest that aging can evolve into tumor suppression mechanisms and that aging is an indirect effect of this situation. Since there is no bond to growth regulation in tumor cells, these cells do not seem to express genes that produce products that promote or maintain the aging state. For example, in vitro studies have shown that aging can be partially arrested by inactivation of tumor suppressor proteins such as retinoblastoma tumor suppressor gene RB1 (Weinberg, Cell 81: 323-330 (1995)). This suggests the possibility of cells gaining replication benefits and eventually immortalizing by loss of functional tumor suppressor genes in vivo.

It has also been observed that a decrease in immune response coupled with increasing age results from a decrease in the proliferative response of T-lymphocytes exposed to antigens (Smith and Pereira-Smith, 1996). This decrease in the response of T cells to antigens is associated with a decrease in the responsiveness to mitogen in senescent culture cells, meaning that a decrease in the immune response of aging can result from the same or similar mechanism. Thus, once the genes causing aging are known, their correlation to loss of immune response with aging can be revealed, and it may be feasible to manipulate these genes to therapeutically boost the immune system.

To directly investigate the role of aging in tumor suppression, experiments were carried out to infect immortalized and non immortalized human fibroblasts with plasmids expressing either the SV40 T antigen or Ki-ras bearing RNA tumor virus, or both. It was done. Only immortalized fibroblasts became tumorigenic by this method, suggesting that unique molecular mechanisms govern immortalization and tumorigenesis, and also indicate that the former may be a prerequisite for the latter (Sager, See Environ. Health Perspect. 93: 59-62, 1991). Several studies have shown that escape from aging, ie immortality, results from alterations in the expression or loss of one or more aging genes. If such genes can be identified and isolated, their role in cancer may be further revealed and it may be possible to manipulate their expression so that controlled growth in malignant tumor tissue is restored.

Summary of the Invention

In the present invention, a new gene expressed at high levels in senescent cells was identified. The cDNA corresponding to this new gene was isolated and sequenced, and it was found that this cDNA contained an open reading frame encoding a protein with an estimated molecular weight of 23 kDa (SEQ ID NO: 1). Therefore, this gene was named "p23". mRNA transcribed from p23 can be detected reproducibly at higher levels in senescent cells than in proliferating cultured normal human breast epithelial cells. The function of p23 is unknown, but analysis of its putative amino acid sequence (SEQ ID NO: 2) suggests that p23 belongs to the transmembrane protein family known as the "PMP 22" family, or the "epithelial membrane protein" (EMP) family. Taylor et al., J. Biol. Chem. 270: 28824-28833, 1995; Lobsiger et al. Genomics 36: 379-387, 1996; by Taylor and Suter Gene 175: 115-120, 1996 ] Reference).

p23 is expressed in a number of human tissues such as adult and fetal liver, pancreas, placenta, adrenal gland, prostate, and ovary, all of which consist mainly of epithelial cells with endocrine or secretory function. It has also been shown that p23 RNA is significantly reduced or absent in many human breast cancer cell lines, suggesting that p23 polypeptides may play a role in inhibiting malignant phenotypes in normal breast tissue. p23-positive normal human breast epithelial cells (HMEC) expresses reduced levels of p23 when incubated with retinic acid, indicating that the p23 gene is regulated transcriptionally through the retinic acid receptor pathway.

Accordingly, the present invention provides an isolated p23 nucleic acid molecule involved in aging of human epithelial cells, and a recombinant p23 polypeptide encoded by the nucleic acid molecule. According to another aspect, the present invention provides a vector comprising a nucleic acid molecule and a host cell, an antibody specific for a polypeptide, a method for regulating aging, and a method for measuring p23 levels in a biological sample.

The present invention specifically relates to nucleic acid sequences from genes associated with aging of human epithelial cells (herein referred to as p23), recombinant polypeptides encoded by these sequences, expression vectors and host cells comprising the sequences, and p23 polypeptides. The present invention relates to a binding antibody, a method for regulating aging in a biological sample, and a method for measuring the amount of the polypeptide.

These and many additional advantages of the present invention will be more readily understood in conjunction with the following appended drawings, as well as better understood by reference to the following detailed description:

1 illustrates a comparison of the coding sequence of the p23 polypeptide (its putative amino acid sequence is shown in SEQ ID NO: 2) with the apoptosis-related mouse ventral prostate gene 1 (RVP1) shown in SEQ ID NO: 3.

2 illustrates the structural properties of the p23 polypeptide in which the putative amino acid sequence is shown in SEQ ID NO: 2. These properties were determined by analysis with the Motifs subroutine of the Genetics Computer Group (GCG). O indicates a hydrophobic region, while o indicates a hydrophilic region. “O” shown below the line between residues 50 and 100 indicates the location of putative glycosylation site at amino acid 72.

Detailed Description of the Preferred Embodiments

The identification of genes involved in inducing and maintaining the aging state deepens the purpose of controlling diseases such as cancer, chronic inflammation, and many proliferative and degenerative diseases. The present invention provides a nucleic acid molecule encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, which is representative of the p23 polypeptide. Representative examples of nucleic acid molecules encoding a p23 polypeptide include the nucleotide sequence shown in SEQ ID NO: 1 corresponding to the cDNA encoding the polypeptide whose amino acid sequence is shown in SEQ ID NO: 2. The open reading frame of SEQ ID NO: 1 is located between nucleotides 221 and 853, most of which is not translated. Representative examples of p23 polypeptides are provided by the amino acid sequences set forth in SEQ ID NO: 2.

Many uses of the nucleic acids set forth in SEQ ID NO: 1 depend on the ability of the double-stranded formation (ie to hybridize with each other) of the complementary nucleic acid strands. "Strict hybridization conditions" usually means that the nucleic acid double strands formed under these conditions are a perfect match or a near perfect match (see Sambrook et al., Molecular Cloning, 2nd ed., 1989). Is incorporated by reference). Thus, under stringent conditions, complementary nucleic acid molecules derived from different allelic forms of a gene are expected to form stable hybrids, since the allelic forms of the genes usually differ only in very few nucleotide positions. Similarly, probes derived from certain cDNAs are expected to form stable hybrids with cDNAs or genes corresponding to allelic or mutant forms of the same gene under stringent conditions.

Stringent hybridization conditions for polynucleotide molecules of> 200 nucleotides in length involve hybridization at temperatures of 15-25 ° C., most preferably 25 ° C., which are usually below the expected double-stranded melting point (Tm). For probes (<30 nucleotides) hybridize at 5-10 ° C. below Tm (see, eg, Sambrook et al., 1989, section 11.45). The Tm of a nucleic acid double strand can be calculated using the following formula based on the percentage of G + C contained in the nucleic acid, where chain length is considered (Sambrook et al., 1989; section 11.46):

Tm = 81.5-16.6 (log [Na ⁺ ]) + 0.41 (% G + C)-(600 / N)

Wherein N is the chain length.

From the above formula, it is clear that the effect of chain length on Tm is only significant when hybridizing rather short nucleic acids, and this chain length effect can also be neglected for nucleic acids of several hundred bases or more. Thus, those skilled in the art can make suitable p23 probes from substantially any portion or fragment of SEQ ID NO: 1. As long as the length of the selected probe molecule exceeds about 15 nucleotides, stringent hybridization conditions can be calculated using the above equation or using some similar equation. For a given probe, Tm can be confirmed experimentally by hybridizing the probe with the p23 nucleic acid molecule cloned, for example SEQ ID NO: 1, and then increasing the temperature until the double strand is dissolved. The optimal hybridization temperature for a given probe may be confirmed experimentally by testing the hybrid double strand formation at different temperatures. In addition, probes with lengths greater than 15 nucleotides are expected to hybridize specifically because sequences beyond this length are unlikely to occur extremely more than once in the mammalian genome (Sambrook et al., 1989, 11.7). part).

The choice of hybridization conditions is obvious to those skilled in the art and is generally based on the purpose of hybridization, the type of hybridization (DNA-DNA or DNA-RNA), and the level of relevance between the desired sequences. As noted above, hybridization methods and representative buffer compositions for high or low stringent hybridization are well established and are provided in the published literature (see, eg, Sambrook et al., 1989; Nucleic Acid Hybridization by Hames and Higgins). , A Practical Approach, IRL Press, 1985, Washington, DC; Edited by Berger and Kimmel, Methods in Enzymology, Volume 52, Guide to Molecular Cloning Techniques, Academic Press Inc, 1987, New York, NY; and Bothwell, Methods for Cloning and Analysis of Eukaryotic Genes, Yancopoulos and Alt Edit, Jones and Bartlett Publishers, 1990, Boston, Mass., Which are hereby incorporated by reference in their entirety).

Those skilled in the art will appreciate that the stability of nucleic acid double strands decreases with increasing number of mismatched bases. Thus, the stringency of hybridization can be engineered to maximize or minimize the stability of such double strands. Hybridization stringency controls the hybridization temperature; Adjusting the percentage of helical destabilizing agent (eg formamide) in the hybridization mixture; It can be altered by adjusting the temperature and / or salt concentration of the wash liquor. In general, the stringency of hybridization is controlled by changing the salt concentration and / or temperature during the post-hybridization washes. Hybridization stringency can be lowered by reducing the percent of formamide in the hybridization solution or by reducing the temperature of the wash liquor. Conditions of high stringency are in 65-68 ° C., or 50% formamide in an aqueous solution comprising hybridization at high temperature (eg 4-6 times SSC (1 × SSC = 0.15 M NaCl, 0.015 M sodium citrate) 42 ° C.) may be associated with washing at high temperature (eg 5-25 ° C. below Tm) in a low salt solution (eg 0.1 × SSC). Conditions of low stringency are combined with low hybridization temperatures (eg 35-42 ° C. in 20-50% formamide) and medium temperatures (eg 35-35) in wash solutions (2-6 X SSC) with relatively high salt concentrations. 60 ° C.). Mild stringency conditions that may include hybridization in 0.2-0.3 M NaCl at a temperature of 50-65 ° C. and washing in 0.1 × SSC, 0.1% SDS at 50-55 ° C. are performed jointly with polynucleotide molecules published as probes. Can be used to identify genomic or cDNA clones encoding related proteins (eg, other members of the EMP family).

Nucleic acid molecules comprising the sequence set forth in SEQ ID NO: 1 provide tools that can be used to identify and isolate p23 and variants of the p23 gene, eg, complete genes encoding allelic variants or mutants of the gene. . Under stringent conditions, DNA molecules corresponding to all or part of the p23 gene can be identified by hybridizing p23 probes, ie, probes derived from all or part of SEQ ID NO: 1, with phage or cosmid libraries of the human genome.

The invention also includes variant forms such as allelic variants and mutants of the p23 protein, gene, and cDNA. Genes encoding the variants of p23 and cDNA are readily identified by using probes based on SEQ ID NO: 1 as a tool to screen cDNAs or genomic libraries made from cells of interest, ie, cells that may contain variant forms of the p23 gene or mRNA. And then isolated. In order to maximize the specificity of the screening, hybridization is performed under stringent conditions. The identification that the clones thus isolated are p23 variants is accomplished by determining the nucleotide sequence of the cloned DNA and comparing this sequence, particularly the sequence of the coding region, with the p23 sequence shown in SEQ ID NO: 1. A variant of p23 is expected to share at least 90-95% of its nucleotide sequence.

In some cases, the cells may express nonfunctional p23 protein or contain no p23 protein due to genetic mutations or somatic mutations. Thus, such cells, which may include genetically defective cells or cancer cells, may escape the aging state. For p23-deficient cancer cells, the cancer cells can be treated in such a way that they overexpress wild-type p23 to allow the cells to stabilize at G ₁ . Accordingly, the present invention provides a method of inducing an aging phenotype in a cell by introducing a nucleic acid molecule encoding p23 into the cell, eg, the representative p23 sequence shown in SEQ ID NO: 1.

This method of inducing senescence in cells involves the deaging of a nucleic acid molecule encoding a protein of the amino acid sequence shown in SEQ ID NO: 1 or 2, or an allelic form of the protein having substantially the same biological activity in vivo or ex vivo. Introduction to a cell. In addition, the untranslated region of p23 cDNA shown in SEQ ID NO: 1 may provide important regulatory functions that may affect mRNA stability or processing, or other aspects of mRNA function.

The present invention relates to recombinant expression vectors expressing p23 in eukaryotic cells such as yeast cells (e.g., retroviruses, Herpes simplex virus, plasmids, vaccinia virus, adenoviruses, defective parvoviruses, CMV, etc.), and plasmid or cosmid vectors for expressing p23 in prokaryotic cells. Recombinant expression vectors of the invention are constructed by, for example, operably binding a nucleic acid molecule capable of encoding the p23 protein of SEQ ID NO: 2 to a suitable regulatory sequence. Nucleotides 221 to 853 of SEQ ID NO: 1 provide representative nucleotide sequences with essential coding ability. As used herein, "operably binding" refers to a regulatory element necessary for transcription and translation of p23, preferably a regulatory sequence in an expression vector (ie, expression, overexpression, or constitutive expression of the p23 gene). Ligation of the p23 nucleic acid molecule to the expression vector nucleic acid in such a way that it is correctly positioned under predetermined positive (or negative) control exerted by a regulatory sequence capable of proceeding, e.g., a promoter, enhancer, operator sequence, etc. Used to. Vectors can include inducing promoters, such as directing transcription only in the presence of certain hormones, ions (eg zinc), growth factors, cofactors, or metabolic substrates. Selectable markers may also be present in the expression vector. Representative examples of such selectable markers include enzymes, antigens, tolerable markers, or markers that meet the needs of cell growth. Regulatory elements that exert regulation in eukaryotic or prokaryotic cells, or both, may be present in a single vector.

The expression vectors are useful for transfecting or transducing cells to express transgenic p23 polypeptides, mutant p23 polypeptides, and antisense nucleic acids capable of double stranding with endogenous p23 mRNAs. Cells induced to express exogenous p23 are called "transgenic cells." The present invention therefore provides a cell line transformed with a vector expressing a transgenic p23 polypeptide in a transgenic cell. Transgenic cells of the invention can be used to produce large amounts of p23 polypeptides. To facilitate the harvesting of the p23 polypeptide from the transgenic cell, a transgene, ie, a DNA fragment encoding p23, is framed in a region encoding an amino acid that provides a signal for secreting the transgenic polypeptide into the culture medium. Can be connected accordingly. Transgenic cells that express p23 may be eukaryotic or prokaryotic.

Another embodiment of the invention provides a method of inducing an aging phenotype in eukaryotic cells. In this method, a p23 expression vector is introduced into the cell that is constitutively, conditionally or temporarily overexpressing p23. As a result of subsequent expression of p23 in the transduced cells, these cells proliferated at a slower rate than their parent cells, or all stopped proliferating (ie, the cells acquire an aging phenotype). For example, human diploid cells transduced with p23 are arrested at G ₁ . The cells taken from the culture cells or live host can be target cells of the p23 expression vector. Thus, when the cultured cells are targets, the present invention provides cell lines capable of expressing the transgenic p23 polypeptide. When cells taken from the live host are transduced, these cells can be returned to the host during culture or further studied.

One skilled in the art can also remove cells from a patient and transfect or transduce cells with an expression vector that expresses p23 or an antisense RNA that can inhibit the expression of endogenous p23. It will be understood that the benefits of gene therapy to return back to the patient (ie ex vivo gene manipulation). It will also be understood that transgenic animals (eg experimental animals and domestic animals) expressing p23 under the control of a tissue specific promoter or inducible promoter, or expressing antisense RNA for inhibiting endogenous p23 expression, can be constructed. will be.

In addition, p23 can be temporarily expressed in cells by introducing a cloned p23 nucleic acid using a nucleotide sequence encoding a p23 nucleotide into a cell such as electroporation, calcium phosphate precipitation, or the like into liposomes. Transient expression occurs when mRNA is transcribed from the initial introduction vector DNA prior to vector integration.

Antisense p23 nucleotide sequences, ie nucleotide sequences complementary to the transcriptional or nontranscribed strand of the p23 gene, can be used to block normal or mutant p23 expression in cancer cells or other proliferative cells. The use and use of antisense oligonucleotides is outlined in the literature (see, eg, Antisense Nucleic Acids and Proteins Fundamentals and Applications, edited by Mol and Van der Krul, New York, New York, 1992; Cited for reference). Suitable antisense oligonucleotides are 11 or more nucleotides in length and may include untranslated (upstream or intron) and binding coding sequences. As will be apparent to those skilled in the art, the optimal length of an antisense oligonucleotide is determined by the intensity of the interaction between the antisense oligonucleotide and its complementary target sequence, the temperature and ionic environment in which translation takes place, the base sequence of the antisense oligonucleotide, and the target mRNA and And / or the presence of secondary and tertiary structures in antisense oligonucleotides. Suitable target sequences of antisense oligonucleotides are intron-exon junctions, regions in which the DNA / RNA hybrid prevents the migration of mRNA from the nucleus to the cytoplasm, initiation factor binding sites, ribosomal binding sites, and sites that interfere with ribosomal progression. ) To prevent proper splicing. Particularly preferred target regions of antisense oligonucleotides are the 5 'untranslated (promoter / enhancer) regions of the gene of interest. Antisense oligonucleotides can be constructed by inserting a DNA molecule comprising a target DNA sequence into a suitable vector so that the DNA molecule is inserted downstream of the promoter in reverse configuration relative to the gene itself. Antisense oligonucleotides can then be expressed by transducing, transforming or transfecting suitable cells with the expression vector. Alternatively, antisense oligonucleotides can be synthesized using standard manual or automated synthesis techniques. Synthetic oligonucleotides may be introduced into suitable cells by electroporation, calcium phosphate precipitation, liposomes, microinjection, or other methods. Stability of antisense oligonucleotide-mRNA hybrids can be determined by, for example, adding a stabilizer to the oligonucleotide, for example by adding an intercalating agent attached to one end of the oligonucleotide, or by phosphoester, phosphor Nate, phosphorothioate, phosphorosenoate, phosphoramidate, or phosphorodithioate can be increased by incorporation into the phosphodiester backbone.

Proteins harvested from transgenic cells expressing p23 can be used for a number of purposes, for example to produce antiserum against p23. Accordingly, the present invention provides immunological binding partners for p23 polypeptides that can specifically bind p23 polypeptides, such as those of the amino acid sequence shown in SEQ ID NO: 2, such as polyclonal and monoclonal antibody molecules, and several thereof. Provide an antigen binding fragment. Antibodies can be raised against whole p23, or a fragment of this polypeptide. The p23 used as the antigen may be denatured before injection or in its native form. Antibodies to denatured proteins can often react with natural or denatured proteins and are often useful for western blotting. Antibodies can also be used for identification of senescent cells in culture or in tissue fragments using standard immunostaining protocols.

Immunospecific reactants capable of specific binding to p23 can be purified by hybridomas or in combination with suitable adjuvants (e.g. Freund's, ISCOMs, etc.) to repeat the selected proteins derived from purified proteins or p23 for suitable animals ( For example rabbits, sheep, or goats). Antibodies to p23 are useful for treatment, purification, and diagnosis. Therapeutic uses include binding p23 to a partner that normally binds to and inhibits binding to a ligand that promotes cell proliferation in the treated cell. Representative examples of purification uses include immunoaffinity chromatography and immunochemical methods using antibodies as affinity agents for purifying p23 from tissues that naturally produce p23, or from transgenic cultured cells expressing p23. Representative examples of diagnostic applications include enzyme binding and radioisotope immunoassays (ie, ELISA and RIA), immunofluorescence, time-based fluorescence immunoassays, etc., to measure the level of p23 protein in tissue samples, such as tumor cells. do.

Antibodies to p23 can also be used to selectively kill senescent cells in a cell population. Since p23 appears to be a transmembrane protein, antibodies to p23 can be used to selectively lyse cells in which the protein is present in the membrane. Thus, cell culture aging can be rejuvenated by exposing the antibody to conditions under which the antibody lyses cells expressing this protein, or by using fixed anti-p23 to pick out p23 expressing cells from the cell suspension. . This same method can also be used to single out or enrich the senescent cells from tissue samples removed from the patient. Combustion cells from this selected cell population can be returned to the cells or, if necessary, to return the senescent cells to the body instead.

p23-specific immunological binding pairs are also diagnostic assays that detect p23 in cells such as cultured cells and quantify their levels (eg proteins or antigens) to indicate the remaining number of cell divisions that can be expected in such cell lines. Commonly used in In this type of assay, the measured levels of p23 in the cell line are compared with previously measured levels in early, middle, and late passage cultures of the same cell type. For example, the expected lifespan of cultured epithelial cells can be predicted by comparing the levels of p23 in test cultures with those of standard p23 measured after several passages in a representative epithelial cell line, such as HMEC. Alternatively, the remaining number of passages that can be expected as a function of the percentage of cells in a culture expressing p23 detected by immunostaining, in situ hybridization, or Norton blot hybridization, or by other convenient methods to detect mRNA It can be estimated. In this assay, if more than 90% of the cells express high levels of p23 compared to the levels expressed in the cells during the initial passage, it may be considered that this culture will not substantially increase upon aging and replating.

Those skilled in the art also provide an opportunity to screen for recombinant p23 nucleic acids disclosed herein, cells expressing exogenous p23, and compounds where ex vivo assays modulate or completely alter the functional activity of p23 protein or p23 nucleic acid in cells. Will understand. In this regard, "modulation" is intended to indicate that the compound increases or decreases one or more functional activities of the p23 protein or nucleic acid, while "altering" means that the compound is functionally different from that of the p23 protein or nucleic acid. It is intended to indicate a complete change to activity. In this regard, examples of compounds that "regulate" the activity of p23 protein include inhibitors that can reduce p23 expression levels after administration of the compound to p23 expressing cells. Because p23 inhibited cells are expected to be susceptible to mitogen stimulation, the functional activity of p23 added to cells can be analyzed by measuring the responsiveness of the recipient cells to mitogen. Retinic acid is an example of a compound that reduces p23 expression.

Compounds that can modulate p23 activity include synthetic p23 polypeptides, organic chemical analogues, and the like. Such compounds have broad utility as selective inhibitors of p23. Such inhibitors can be used to restore the ability of cells to proliferate in response to agents such as growth factors, mitogens, cytokines and the like by providing competitive inhibition of p23. A “synthetic p23 polypeptide” is understood to include fragments of the p23 polypeptide that can be generated from full length p23 by using physical or enzymatic fragmentation, or recombinant DNA techniques to express a portion of the p23 polypeptide.

The p23 polypeptides of the invention include normal p23 polypeptides (i.e., polypeptides found in normal cells), mutant p23 polypeptides (e.g., those resulting from mutation or found in tumor cells), and chemically modified p23 polypeptides (e.g., For example, one or more chemically modified amino acids, in which case the indicated amino acids are converted, chemically substituted or derived from other amino acids). Functional sites in p23 polypeptides are identified by testing for expression products with altered functional properties, for example, by constructing mutants of the p23 nucleic acid and introducing the construct into actively growing cultured cells, which do not induce aging. .

It will also be appreciated that a mutant p23 nucleotide sequence can be constructed from the nucleotide sequence shown in SEQ ID NO: 1. One skilled in the art can mutate the sequence of SEQ ID NO: 1 (e.g., using chemicals or radiation, or using recombinant DNA technology), and identify and / or select cell clones comprising the mutated p23 nucleotide sequence. You will know a variety of ways. Mutant p23 nucleotide sequences of the invention are useful for modulating or altering the activity of p23 in cells. Mutant p23 nucleotide sequences of the invention can be introduced into cells using retroviral vectors, adenovirus vectors, or bacteriophage or plasmid vectors.

The present invention

a) detecting the absolute level and activity of p23 expression in a non-synchronized cell population (eg in tissue samples such as tumor tissue fragments);

b) comparing the level and activity of p23 polypeptide or mRNA in a synchronized or non-synchronized cell population in several passaged cultures;

c) measuring the level and activity of p23 expression products in biological fluids (ie blood, serum, plasma, mucus secretions, CNS fluid, cell extracts, etc.)

It includes an assay for the following. The absolute level and activity of p23 expressed in malignant biological fluids (eg tumor cell extracts, serum from cancer patients, etc.), and the level and activity of p23 expressed in cell extracts prepared from multiple passaged tumor cells It is possible to provide information on the aggressiveness of or to explain the possibility that tumor cells may be arrested at G ₁ by the recovery of p23. In this regard, the levels and activity of p23 analyzed can serve as diagnostic markers for:

a) staging of the tumor (because at least some types of metastatic malignant cells express little or no p23);

b) prognostic measurements, ie prediction of patient viability and tumor recurrence time (fast growing, malignant cells that can cause metastasis can generally express less p23 than differentiated cells); And / or

c) predicting the success of treatment, i.e., predicting a specific therapeutic regimen (more slowly growing cells may express higher levels of p23 or have more active p23 (ie, than well growing metastatic cells), and are also less strong and Because they have greater responsiveness to longer therapeutic regimens).

One of ordinary skill in the art would be useful to physicians in determining how the diagnostic assays of the present invention become tumors, how to select a suitable treatment regimen, how to evaluate the success of treatment, and how to assess a patient's risk or viability. Can provide results.

The present invention also provides a method of measuring p23 expression level in a biological sample. The sample may be cultured cells, biological fluids, patient tissue pieces, tumor tissue pieces, or other samples. Expression levels are, for example, hybridized with RNA of a biological sample and nucleic acid probes corresponding to at least 15 nucleotide regions of the nucleotide sequence of SEQ ID NO: 1 and using RNA standards from burned cultured epithelial cells and aged cultured epithelial cells. Can be measured by comparing Probes are generally labeled with ³² P or biotin in conventional protocols using enzymes such as, for example, polynucleotide kinases, Klenow or complete DNA polymerases (Sambrook et al., 1989). In one method commonly used to detect p23 expression in a sample, ie Norton blotting, the extracted RNA is immobilized on a membrane filter, hybridized with labeled denaturing probes, and the hybrid is radiographed or chromatographic. Is detected. Compared with RNA standards, eg, combustion (ie, low passage counts) and RNA from aging cultured epithelial cells, whether the amount of p23 RNA in the test sample is “low” or “high” (ie, selected standard). The level in the burning standard cell from the cell line is defined as "low" while the level in the aging standard cell is defined as "many"). Alternatively, p23 expression levels can be determined by measuring the amount of p23 polypeptide using an antibody against p23. Again, the amount of p23 polypeptide in burned and aged epithelial cells is provided as a standard for comparison. Thus, assays for p23 levels provide useful tools that enable the use of rare or rare cell lines, for example cell lines established from unique tissue samples, or maximize the efficient use of non-immortalized cell lines of unknown passage history. Provide useful tools. Such assays can also be used to characterize biotissue samples from normal tissues or diseased tissues, such as tissue fragments from tumor tissues or degenerative tissues.

According to another embodiment, the present invention provides an assay for detecting chromosomal rearrangements on chromosome 3 of human cells. The chromosome location of p23 was mapped by the computer analysis (Unigene program; Boguski et al. [Nature Genet. 10: 369-371, 1995]) between the q28 and q29 bands of chromosome 3 at the ends of the long arms. Thus, the cDNA sequence of the cloned p23 allows for the detection of translocations associated with this region of chromosome 3 by providing a hybridization probe that can be used for in situ hybridization to visualize the p23 gene in the mitotic mid-chromosome. Translocation of the p23 gene, ie from its normal position to another chromosome, can result in a phenotype of unregulated cell growth by transferring p23 from regulatory regulatory elements that ensure expression of p23 and subsequent cellular senescence. Thus, rearrangement of the p23 gene in cells can have dramatic consequences. If the rearrangement induces downexpression, the cells may acquire a malignant (ie, unregulated) growth phenotype, and if the rearrangement induces overexpression, the cells may prematurely age. Screening cell samples from individuals for chromosomal rearrangements associated with p23 may provide information regarding the relative risk of the patient developing a particular type of cancer or other disease such as autosomal dominant optic nerve atrophy (see below). . Rearrangements in the long arms of chromosome 3, which also include the q28 band, are associated with one or more types of tumors, ie, liposarcoma [Nature Genetics Special Issue, April 1997, p. 433].

The position in chromosome 3 of the p23 gene is the same as measured for OPA1, an autosomal dominant genetic disease in retinal ganglion cells or optic nerve degeneration (Lunkes, A. Am. J. Hum. Genet., 1995 Oct.] or Eiberg et al., Human Mol. Genetics 3: 977-980, 1994). p23 and OPA1 map to the long arm between q28 and q29 of chromosome 3, suggesting the possibility that optic nerve atrophy can result from mutations in p23 itself. Given the association with cellular senescence, p23 can be mutated to cause premature or overexpression of this gene, and thus entry of premature aging or abnormal state of the affected cells, resulting in neuronal degeneration. Since the interband region of chromosome 3 is large enough to accommodate several genes, p23 is likely to be closely linked to the gene that causes OPA1 rather than directly to OPA1, thus its proximity to the actual OPA1 gene. Provides genetic markers of disease location due to.

If rearrangement of the p23 gene causes loss of growth regulation, i.e. cancer, or improper atrophy, e.g. OPA1, normal growth provides a regulatory element lacking the translocated gene, thereby altering the malignant phenotype, or Can be recovered by inhibiting inappropriate overexpression of (eg, upon treatment of OPA1). Thus, the p23 gene and its regulatory elements are targets of gene therapy vectors designed to reactivate or inactivate rearranged genes using in situ directed recombination / mutation, or targeted integration to disrupt translocated genes. Can act as

Example 1

Cloning of Upregulated Genes in Aging Breast Epithelial Cells

Differential display (DD) techniques of mRNA (see Liang and Pardee, Science 257: 967-971, 1992; Liang et al. Nucl. Ac. Res. 22: 5763-5764, 1994). Were used to identify genes whose expression levels are associated with cell aging. In this technique, mRNAs of two populations were compared by making partial cDNA sequences from subgroups of mRNA populations using reverse transcription, followed by amplification of cDNA using polymerase chain reaction (PCR). For initial reverse transcription, different primers can be used. The primer used to transcribe the first DNA strand always hybridizes to a portion of the poly (A) terminus of the mRNA template and to one or more residues other than (A) at the 3 'end of the mRNA of the poly (A) junction. In the second strand synthesis, primers with random sequences that were complementary to the internal sequence at any point in the upstream (5 'direction) of the first primer were used. Different subsets of mRNA were targeted by varying the identity of base (s) complementary to residues other than (A) of the first primer. When whole cell RNA is used as a template to synthesize cDNA and then amplified by PCR, each primer pair usually produces about 50 to 100 bands ranging in size from 50 to 500 base pairs. After amplification by PCR in the presence of nucleotides labeled with ³⁵ S, these mixtures of short cDNA sequences were displayed for comparison on polyacrylamide sequencing gels. By comparing the products obtained using the same primers for mRNA from two types of different cells, one can identify bands that exist in one cell type but not in the other. Different cDNAs between the two populations can be recovered from the dry gel, reamplified by PCR and then cloned to further characterize. This method has been used successfully to identify many aging-related ESTs from fibroblasts (WO 96/13610).

The cell sources and culture conditions used in the experiment were as follows: Normal human breast epithelial cells (HMEC), strains AG11132 and AG11134 were obtained from the Coriell Institute (National Institutes of Aging Cell Repository, Camden, NY). HMEC contains 0.4% bovine pituitary extract (Clonetics), 10 mM HEPES (Sigma), 10 ng / ml human recombinant epidermal growth factor (EGF) (Upstate Biotechnology, Lake Placid, NY), 5 μg / serum-free breast basal medium (MEBM) supplemented with ml of human recombinant insulin (UBI), 0.5 μg / ml hydrocortisone (Sigma, H4001), and 10-5 M of isoproterenol (Sigma, I5627); Clonetics, San Diego, California, USA. Breast tumor cells were obtained from the American Type Culture Collection, Rockville, MD, 5% fetal bovine serum (Hyclone), 10 mM HEPES (Sigma), 1 mM sodium pyruvate (Sigma), 1 x non-essential Alpha MEM (BRL / Gibco) supplemented with amino acids (Sigma), 12.5 ng / ml EGF (Sigma), 1 μg / ml insulin (Sigma) and 1 μg / ml hydrocortisone (Sigma) Maintained.

Differential display was performed by comparing cDNAs from burning and aging AG11134 cells. AG11134 is a normal HMEC strain that has already been passaged 6-8 serially at the time point obtained from the Coriell Institute. Cells were expanded weekly at 1: 4 to 1: 2 dilution and cells were harvested for RNA preparation after 18 and 26 passages (p18 and p26). Whole cell RNA was purified as described above (Swisshelm et al., Cell Growth Differentiation 5: 133-141, 1994). The cells still proliferated rapidly at about 60-65 doublings at p18, but at p26 corresponding to about 85 doublings, 80-90% of the cells failed to replicate upon resizing. The aging phenotype was confirmed by analysis for the presence of pH-dependent β-galactosidase differentially expressed in senescent cells (Dimre et al., 1995). About 12% of the cells were positively stained for β-galactosidase in the proliferating population (p18), while 99.4% of the cells were positively stained for the enzyme in the p26 population.

Transcription in p18 and p26 HMEC was compared by extracting RNA from burned and aged AG11134 cells and performing differential display. Differential displays were performed according to published methods (Liang and Pardee, 1992; Liang et al., Methods Enzymol. 254: 304-321, 1995; Swisshelm et al., Proc. Natl. Acad. Sci. USA 92: 4472-4476, 1995).

Primers that hybridize to the poly (A) terminus of mRNA (“anchor” primers) included three different primers with a 5 ′ HindIII site to aid in cloning of the amplified fragment (Liang et al., 1994; United States). GenHunter Corp, Brooklyn, Mass.). These three "HT ₁₁ " primers have the following sequence:

In addition to the HT ₁₁ primers, the following T ₁₂ fixed primers (obtained from Operon Technology, Alameda, CA) were used:

The primers were coupled in PCR reactions with 30 different random sequence primers obtained from Operon Technology or GenHunter, each with 60-70% G + C and no autocomplementary ends.

PCR reactions were carried out as follows: denaturation: 94 ° C., 30 seconds; Annealing: 40 ° C., 2 minutes; Extension: 72 ° C., 30 seconds. This step was repeated a total of 40 times and ended with the extension step for 5 minutes. For individual PCR reactions, each fixed primer was paired with each random primer in a separate reaction tube, except that the same bases were present at the end of the T12 primers combined together in a single reaction.

CDNA band patterns corresponding to initial passages and senescent cells were displayed and compared on DD gels, and a number of bands were cut out of the dry gels which appeared to be more abundant or less abundant in senescent cells compared to combustion cells. First, about 50 candidate cDNA fragments were cut out of the gel and re-amplified by PCR. Label each of these amplified cDNA fragments with ³² P and use it as a probe to analyze RNA from burned and aged AG11134 cells in a Norton blot containing 5-10 μg / lane total cell RNA from each type of cell. It was. Norton blots were hybridized at 37 ° C. in a buffer containing 0.25M NaPO ₄ , 0.25M NaCl, 7% SDS, 1mM EDTA, 5% dextran sulfate, 100 mg / ml salmon sperm DNA, and 50% formamide. . Norton blots included whole cell RNA from each of the cell cultures. The filter was washed at 37 ° C. in buffer containing 2 × SSC and 0.1% SDS.

A probe corresponding to one of the cut DNA fragments (named “DD19”) was found to hybridize to about 4 kb in size mRNA present at much higher levels in senescent cells than in rapidly dividing cells. The primer pair flanked by the specific cDNA consists of 5 'GGAGGGTGTT 3' (SEQ ID NO: 19, random primer OPB15 from Kit B of Operon) and 5 'AAGCTTTTTTTTTTTC 3' (SEQ ID NO: 6, ie fixed primer HT ₁₁ C) It became. The DD19 probe was labeled with ³² P-dCTP using a random primer kit (manufactured by Boehringer-Mannheim).

To confirm that DD19 corresponds to increased mRNA in senescent cells, the Norton blot analysis was repeated using whole cell RNA from AG11132 and AG11134 cells (the former also being normal HMEC strains). The experimental results showed that transcripts hybridizing with DD19 probes were present at higher levels in senescent cells than in combustion cells in both HMEC cell lines. The most prominent band hybridizing with the probe was about 4.0 kb in size, but there were also less abundant transcripts that were about 3.0 kb in size. 3.0 kb mRNA may have arisen from differential splicing of the first p23 transcript.

Amplified DD19 DNA fragments of 326 bp in size were cloned into the pCR plasmid vector (InVitrogen, Carlsbad, Calif.) Using the TA cloning kit. Inserts of cloned DD19 were sequenced manually using SEQUENASE (manufactured by USB) and simultaneously by fluorescence using an ABI377 automated sequencer (Murdock Laboratories, University of Montana). Sequencing was performed using primers defined by the vectors, namely T7 and SP6.

DD19 cloned into the pCR vector was labeled and hybridized with an RNA panel as described above to confirm the initial observation that 4.0 kb mRNA was increased in senescent cells. The RNA sources in the panel were burned, aged and stopped AG11134 cells and burned, aged and stopped AG11132 cells. Stop cells are cells that have ceased dividing but have the ability to divide when placed under suitable conditions, for example when exposed to, or diluted and replated with, mitogen. Stop cells were prepared from early passage cells by densifying the cells and then keeping them in culture while feeding occasionally for an additional two weeks without further passage. RNA was isolated from quiescent cells about 48 hours after the final addition of fresh medium. As an internal control, p23 was stripped from the filter, and the labeling probe made from a cloned cDNA corresponding to 36B4, a phosphoprotein present in ribosomes, with corresponding mRNAs of 1.5 kb in size and relatively constant in a wide range of cell types. The filter was rehybridized (Masiakowski et al., Nucl. Ac. Res, 10: 7895-7903, 1982; Rio et al., Proc. Natl. Acad. Sci. USA 84: 9243-9247, 1987; Laborda, J. Nucl. Acids Res. 19: 3998, 1991).

Analysis of Norton blots comprising whole cell RNA from burned, aged, and quiescent cells by hybridization as described above showed that cloned DD19 DNA was expressed at elevated levels in senescent cells in both HMEC cell lines. It confirmed that it corresponds to a transcript.

As measured by analysis with a photographic densitometer, the expression level of 4.0 kb transcript in AG11132 cells was about 7 times higher in senescent cells than in burned cells. DD19-related transcripts were not elevated in quiescent cells of any of the HMEC cell lines.

Additional hybridization experiments were performed to confirm the expression level of 4.0 kb transcripts in a panel of breast tumor cell lines. The cells were Hs578T, MCF7A, MDA-MD-435, MDA-MB-231, SKBR3, and T47D cells. The hybridization was performed using two different probes, one of which was a cloned DD19 DNA fragment and the other a cDNA corresponding to DD19.5 DNA (most of the 4.0 kb transcript, or both of these transcripts). Clone) (see below). Hybridization conditions were as described above except that the filters hybridizing with the DD19.5 probe were washed at 50 ° C instead of 37 ° C. The same result was obtained for both probes. Transcripts hybridizing with labeled DD19 were not observed at all in these cells except for T47D, which was comparable to senescent cells in T47D. Interestingly, no 3.0 kb RNA was detected in any of the six breast cancer cell lines including T47D. This situation suggests that the absence of 3 kb mRNA may provide a marker for breast cancer cells.

Using the cloned DD19 DNA as a probe, cDNA clones corresponding to most or all of the 4.0 kb transcript were obtained as follows. Cloned DD19 DNA fragments were labeled and used as hybridization probes to screen cDNA libraries already prepared in lambda ZapII vectors using aged 76N cells as templates for reverse transcription of long cDNAs (Swisshelm et al., 1994). The cell is a normal human breast epithelial cell line. Hybridization buffers included 50% formamide, 5 X SSC, 100 mg / ml carrier DNA, 0.1% SDS, 0.1% BSA, 0.1% polyvinylpyrrolidone, and 0.1% picol. Hybridization was performed at 37 ° C. and filters were washed at 37 ° C. in 2 × SSC and 0.1% SDS. Approximately 1.25 × 10 ⁶ plaques were screened. Three positive clones were selected for further characterization.

Inserts from three positive clones were sequenced manually and simultaneously with an ABI automated sequencer. The longest of the three cDNA clones (named "DD19.5") included inserts from the remaining two selection clones. The entire cDNA cloned into DD19.5 was sequenced using a walking primer and the cloned insert was found to be 3443 nucleotides in length. The cloned DD19.5 was deposited in the American Type Culture Collection (Rockville Parkron Drive 12301, 20852, USA, USA) on August 4, 1997, and was assigned an access number under the Budapest Treaty. The nucleotide sequence of DD19.5 was analyzed using Wisconsin Package Version 9.0 (Genetics Computer Group, University Research Park, Madison, Wisconsin, USA), referred to below as the "GCG" package or program.

DD19.5 nucleotide sequence analysis showed that the nucleotide sequence included an unread reading frame (ORF) capable of encoding a protein having 211 amino acid residues with an estimated molecular weight of 23 kDa. Therefore, this gene was named "p23". The length of the open reading frame indicated that a large proportion of 4.0 kb mRNA was not translated. The long untranslated region is consistent with the assumption that p23 will belong to the EMP family of transmembrane proteins because the family often has mRNAs that have large untranslated regions (eg Chem et al., Genomics 41: 40-48). , 1997; Lobsiger et al., Genomics 36: 379-387, 1996).

Using FASTA homology studies, p23 contains several unknown partial cDNAs (ie, expressed sequence tags or “EST”), including unknown cDNAs from pancreatic islet cDNA libraries (GenBank Accession Number W51940), and It was measured to be similar or identical to six or more other ESTs. The latter has 45.3% identity in the overlapping 214 amino acids when GenBank accession number is AC000088; If the accession number is AC000005, it has 46.5% identity in the overlapping 198 amino acids; The access number is U19582 and has 34.6% identity at the overlapping 188 amino acids; With access number X94770 it has 24.3% identity in the overlapping 136 amino acids; With access number X15436 it has 52.0% identity in the 25 overlapping amino acids; Accession number M97881 has 43.2% identity in the overlapping 37 amino acids. No significant homology was detected between p23 and the aging related ESTs disclosed in WO 96/13610.

Computer analysis has also shown that the gene known as "RVP1" cloned from the rat ventral prostate-androgen withdrawal cDNA library (GenBank Accession Number A39484; Briehl and Miesfeld, Molec. Endocrinol. 5: 1381-1388, 1991). p23, when aligned to maximize its similarity, its amino acids were 48% identical to RVP1 and 69% similar (ie, amino acids were identical or exhibited 69% conservative substitutions). A comparison of the two protein sequences is illustrated in FIG. 1. The size of the most abundant transcript of the two genes is very different, the size of the transcript of the mouse gene is about 1.2 kb, the transcript size of the p23 gene was 4.0 kb. However, the transient protein products of the murine and p23 genes were more similar in size than their transcripts, with 280 amino acids of the RVP1 protein and 211 amino acids of the p23 protein. The degree of homology observed between p23 and the rat protein suggests that even though RVP1 does not rise in senescent cells but rises in apoptosis cells, these two proteins are far from relevant.

Functional motifs in the unread reading frame of p23 were identified based on amino acid sequence and consensus sequence domain using the Motifs tool (which is a subroutine of the GCG computer program package for sequence analysis). A single putative asparagine N-glycosylation site was identified at residue 72 in consensus sequence “NLSS”. The cAMP / cGMP phosphorylation site was found at residue 192 included in consensus sequence “RKTTS”. Two potential protein kinase C substrates, threonine and serine residues were identified at amino acids 193 and 206, respectively. Several features of the secondary structure expected in the p23 protein are shown in Figure 2, where the hydrophobic region is indicated by ◇ and the hydrophilic region is indicated by ○. The O-glycosylation site at residue 72 is also shown. The analysis further revealed that the isoelectric point of p23 was pH 8.02.

Based on Engelman et al. (Ann. Rev. Biophys. Biochem. 15: 321-353, 1986), and Kyte-Doolittle hydrophobic plots (Kyte and Doolittle, J. Mol. Biol. 157: 105-132, 1982) Two, possibly four, domains in the p23 amino acid sequence of No. 2 have been shown to contain an integral transmembrane region. This is noteworthy because the properties of several EMP family proteins, a family of which p23 is temporarily assigned, include putative transmembrane regions (see, eg, Schiemann et al., Anticancer Res. 17: 13-20, 1997). ] Reference). EMP proteins are also associated with cell growth arrest and degeneration, although they have been suggested to play a dual role in development and differentiation. For example, PMP22, the prototype gene of this family, may be involved in both growth inhibition and differentiation in Schwann cells (Taylar et al., 1995; Taylor and Sutor, Gene 175: 115-120, 1996). The putative transmembrane regions identified at p23 are amino acid residues 82-98 (76-108), 119-135 (115-141), 8-24 (3-28), and 170-186 (165-187) (in parentheses). Number represents another possibility of duplication). Features shared with the EMP protein support the suggestion that p23 resembles EMP protein and has a function of inhibiting cell division.

In addition to RVP1, two other proteins have been identified that appear to be associated with the p23 polypeptide. One of these is the product of the "TMVCF" gene, a gene associated with human autosomal dominant hereditary diseases involving a number of body malformations. The TMVCF gene encodes a protein of 219 amino acids, according to Kyle-Doolittle analysis, which has four putative transmembrane regions (Sirotkin et al., Genomics 42: 245-251, 1997). Another p23 related protein was isolated from monkey cells, which encodes a receptor for toxins produced by Clostidium perfringins and is called the "CPE-R" gene (Katahina et al. Cell Biol. 136: 1239-1247, 1997). The CPE-R protein encodes a 209 amino acid polypeptide and was expected to include four transmembrane regions. Compared to p23 using the GCG program, TMVCF protein had about 46% identity and 55% similarity to p23, while CPE-R protein had 46% identity and 57% similarity. The CPE-R and TMVCF genes produce 1.8 kb and 1.4 kb of mRNA, respectively. Since no transcript of this size was detected on the Norton blot using p23, the coding sequence of CPE-R and TMVCF diverges from the p23 coding sequence to the extent that it does not support mutual hybridization with the p23 probe under the hybridization conditions used in the Norton blot. Seems to have done.

Preliminary sutton blots revealed that the human genome contains only a single gene that can hybridize with the probe corresponding to p23. For this analysis, genomic DNA from five types of cells was digested with Bam HI, which had a single cleavage site in the p23 cDNA sequence. As expected, two fragments were observed when Sutton blot analysis was performed on cleaved DNA, and the blot was probed with labeled DD19.5. The blots hybridized at 50 ° C. in 2 × SSC, 0.1% SDS.

The location of the p23 gene on the human chromosome was mapped by computer analysis using the Unigene program (see Boguski et al. Nature Genet. 10: 369-371, 1995). This gene was found to be located between the q28 and q29 bands of the long arm end of chromosome 3. This position is consistent with the strongly associated chromosome position by pedigree analysis of OPA1 disease. The conventional map site suggests that although the interband region is large enough to accommodate several genes, mutations in the p23 gene may be a potential pathogen of OPA1. It may be important that the breakpoint of the chromosomal location is associated with one or more cancer types, ie liposarcoma (see Nature Genetics 15 (supplemented version): 417-474, 1997).

Example 2

Expression of p23 in Various Tissue Forms

The expression of p23 was further studied by analyzing several different pre-made Norton blots (manufactured by ClonTech, Palo Alto, Calif.) Which included various panels of poly (A) ⁺ mRNA. The hybridization probe used in this assay was a cloned 326 bp DD19 fragment. The hybridization showed that this gene is expressed at different levels in a wide range of tissues. p23 expression was observed in the heart, placenta, liver, fetal liver, lung, skeletal muscle, kidney, spleen, thymus, prostate, ovary, and small intestine. The human endocrine tissue panel Norton blot showed abundant expression in the pancreas and adrenal glands, and somewhat lower levels in the thyroid, testes and thymus. The human brain panel Norton blot showed expression at the laryngeal pole, lower expression in the medulla, and very low expression in the other brain. Human immune system Norton blot showed expression in the spleen, lymph nodes, thymus, and cecum. Of all the tissues analyzed, the greatest expression level was observed in liver, pancreas, and fetal liver. Levels expressed in other organs were about 10-50% lower than those observed in the liver and pancreas.

It was not possible to directly compare the p23 RNA observed in the ClonTech Norton blot panel with p23 transcript levels in senescent cells because the ClonTech blot did not contain senescent cell RNA. Aging cell RNA analysis described in Example 1 used whole RNA, while the ClonTech blot included polyadenylation RNA, making a meaningful comparison more impossible.

HL-60 (promyelocytic leukemia), HeLa S3 (cervical cancer), K-562 (chronic myeloid leukemia), MOLT-4 (lymphocytic leukemia), Raji (bucket lymphoma), SW480 (colorectal adenocarcinoma), A549 (Lung cancer), and a panel of human cancer cell lines made by ClonTech, including G361 (melanoma) cells, were also analyzed for the p23 transcript. Norton blot showed that p23 is expressed at low levels in SW480 and A549 cells but not at all in the other cell lines of the panel. It may be important that SW480 and A549 are epithelial cells. These results support the hypothesis that low levels of p23 expression are associated with unregulated cell growth.

Example 3

Expression of p23 in the Presence of Retinic Acid

In aging and early passage AG11132 cells, early passage AG11134 cells, MCF7 tumor cells (which do not express detectable p23), and T47D tumor cells (which express comparable levels of p23 to senescent epithelial cells) The response of the expression of p23 to retinic acid was tested. Cells were incubated for 48 hours as in Example 1 by adding 1 μM retinic acid to the culture medium. No retinic acid was added to the control culture. Whole cell RNA was extracted from the exposed and control cells and Norton blot analysis was performed. The probe used was cloned DD19 and hybridization conditions for the initial Norton blot test using RNA from combustion and aging AG11134 cells were as described in Example 1. Signals on the resulting autoradiograms were quantified by densitometry using AGFA flatbed scanner and NIH Image program. The results showed a 25-50% reduction in the amount of p23 mRNA in all cells exposed to retinic acid.

Example 4

Inhibition of Transformation Phenotype in Cultured Breast Cancer Cells

In this experiment, MDA-MD-231, Hs578T, MDA-MD-43J, SKBR3, and MCF7 cells were cultured as described in Example 1. The cloned p23 gene was introduced into cells as follows. The coding region of p23 cDNA was ligated to LXSN retroviral vectors as described in Seewaldt et al., Cell Growth and Differentiation 6: 1077-1088, 1995, which is incorporated herein by reference.

The vector carries a gene that confers resistance to the G418 drug, providing a basis for selection of effectively transduced cultured cells. In G418 selection, G418 (manufactured by GIBCO) was added to the culture medium at a concentration of 1 mg / ml, which is a concentration that is toxic to untransduced cells. In the presence of 4 mg / ml POLYBRENE (manufactured by Sigma), dividing cells were transduced by adding a p23 coding vector with an infection degree of 1: 1. Cell selection is as described above (Seewaldt et al., 1995). As a control, part of the cell culture was transduced with an "empty" vector, i.e., a vector that does not contain a p23 coding sequence.

Expression of p23 in transduced cells resistant to G418 was confirmed by Norton blot. Whole cell RNA was extracted with guanidinium hydrochloride, analyzed by electrophoresis on agarose gel after formaldehyde denaturation, hybridized to a probe prepared by transfer to nylon membrane and labeling DD19 or DD19.5. In contrast, suitable synthetic probes are based on the nucleotide sequence of SEQ ID NO: 1, and the specificity of this synthetic probe hybridizes to 4.0 kb and 3.0 kb mRNAs, which are expressed at higher levels in senescent HMECs than under strict conditions under combustion conditions. It was confirmed by proving that it did not hybridize with other mRNAs in the same cell.

Breast cancer cells transduced with a p23 expression vector are expected to divide less rapidly than control cells and enter an aging state arrested at G ₁ . To determine whether the cells actively divide, the cells were replated and ³ H-thymidine was added to the medium at 1-10 μCi / ml. After 1-6 hours cells were harvested, DNA extracted and the amount of ³ H incorporated into the DNA was assessed. Aging cells are arrested at G1 so that ³ H-thymidine is not incorporated into their DNA.

The effect of transducing p23 is further assessed by assessing cell multiplication time in p23 transduction and mimic-transduction (ie, cells infected with empty vectors) breast cancer cells. Cells were plated at 5 × 10 ⁴ per petri dish for 35 mm tissue culture and cultured in standard medium containing 1 mg / ml G418. Individual plates were trypsinized at 24-48 hour intervals and the number of harvested cells was duplicated and counted. Doubling time was obtained by plotting the logarithmic scale cell number against the linear scale time.

Cells expressing transduced p23 and increased doubling time were further evaluated to determine whether they had reduced tumorigenicity than before transduction. Tumorogenicity was assessed by injecting transgenic and mimic-infected cells subcutaneously into hairless mice using 10 ⁶ cells per injection into the intradermal, intraperitoneal, or breast fat pads, depending on the type of cells to be injected. For example, transduced breast tumor cells were injected into breast fat pads. Tumors were measured at weekly intervals after inoculation. The reduced tumor growth rate in p23 transduced cells compared to mimic-transduced cells indicates that induction of p23 expression can provide treatment for other forms of cancer, including breast cancer or epithelial cells.

While preferred embodiments of the invention have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims (13)

An isolated nucleic acid molecule encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2. The nucleic acid molecule of claim 1 comprising the nucleotide sequence set forth in SEQ ID NO: 1. An isolated polypeptide comprising the amino acid sequence of claim 2. A method of inducing an aging phenotype in a cell, comprising introducing the nucleic acid molecule of claim 1 into a cell. A recombinant expression vector comprising the nucleic acid molecule of claim 1 operably linked to a regulatory element of the vector. The recombinant expression vector of claim 5, wherein the regulatory element is provided for expression in eukaryotic cells. A method of inducing an aging phenotype in eukaryotic cells, comprising introducing the vector of claim 6 into a cell. A cell line transformed with the expression vector of claim 5 capable of expressing a transgenic p23 polypeptide. The cell line of claim 8, wherein said cell line secretes a transgenic p23 polypeptide. An immunospecific agent capable of specifically binding to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. The formulation of claim 10 comprising a monoclonal antibody, or a specific binding fragment thereof. The formulation of claim 10 comprising a polyclonal antibody, or specific binding fragment thereof. A method of measuring p23 expression level in a biological sample, comprising the following steps: Hybridizing RNA from cells with nucleic acid probes corresponding to at least 15 nucleotide regions of the nucleotide sequence of SEQ ID NO: 1 under stringent hybridization conditions; And Determining whether the level of p23 in the cell is high or low compared to a standard comprising RNA from cultured burned and aged epithelial cells.