REGULATOR OF CELL PROLIFERATION

Abstract: The invention relates to a novel regulator of cell proliferation, termed p24<INK>. This protein is involved in the regulation of the cell cycle and more specifically the biological clock that measures the finite life span of a cell involves an accumulation of p24<INK>. The isolation of this protein therefore provides a means for regulating the cell cycle both for investigative and clinical purposes.

Description: REGULATOR OF CELL PROLIFERATION The invention relates to a novel regulator of cell proliferation, hereinafter termed p24 INK ; a method for the isolation of p24 INK ; and uses thereof including, but not limited to, uses designed to affect the role of p24 INK in the cell cycle. Normal mammalian fibroblasts cultured in vitro undergo a limited number of divisions before entering a senescent phase in which they can be maintained for long periods but cannot be induced to divide. Even though the mechanism that regulates the finite proliferative potential is not known, it has been suggested that it is limited either by random accumulation of cell damage or by a genetic program. The cell damage hypothesis suggests that as cells divide they randomly accumulate mutations, karyotypic changes and other forms of cell damage which lead to changes in expression of positive and negative regulators of cell growth or to a predisposition to karyotypic instability resulting in the loss of proliferative potential. Recently the progressive loss of telomeric DNA and other essential sequences from the ends of chromosomes has been proposed to determine the finite proliferative potential; however, the processive loss of telomeric sequences has not been unequivocally demonstrated in rodent cells. The genetic program hypothesis suggests that the finite mitotic potential is regulated by a biological clock that measures the finite life span so that upon its completion cells cease dividing and enter into the post mitotic state of senescence. The mitotic cell cycle is considered largely to be regulated by cyclin- dependant kinases (Cdks) which were initially identified in promoting meiosis is Xenopus and in yeast as genes whose deletion would impair the cell cycle. Activation of Cdks is extremely complex, involving phosphorylation/dephosphorylation of cdks themselves, binding to cyclins, originally identified as a family of proteins which oscillate through the cell cycle and inhibition of their kinase activity by association with a family of molecules known as the Cdk inhibitors. One of these inhibitors, p27 κipl , inhibits cyclin E/cdk2 and cyclin A/cdk2 kinase activities and is induced in response to TGF-β and by contact inhibition. p2l Wafl/CiP,/sdil , another Cdk inhibitor, was identified as a gene that was transcriptionally upregulated by the tumour suppressor protein p53 and by virtue of its interaction with cdk2 in a yeast two hybrid system. Since transfection of p21 into cells inhibits DNA synthesis, it has been suggested that the growth inhibitory function of p53 may be mediated by p21. P21 and p27 proteins share homology in the region involved in binding to cyclin/cdk complexes. P21 also binds the proliferating cell nuclear antigen (PCNA); since PCNA is a component of DNA polymerse δ, it has been suggested that this interaction may be involved in promoting DNA repair. P21 mRNA was also isolated as a gene that was upregulated when human diploid fibroblasts undergo senescence and proposed to be a component of the mechanism that limits the finite proliferative potential. Thus our initial goal was to investigate whether p21 was similarly involved in the senescence of rodent embryonic fibroblasts. However, our investigations have led us surprisingly to identify a novel cell cycle regulator which we have termed p24 INK . Our investigations lead us to conclude that an accumulation of p24 INK has a part to play in regulating the cell cycle and more specifically that the biological clock that measures the finite life span of a cell involves an accumulation of p24 INK . Moreover, we also present results which indicate that the growth inhibitory effects of p24 INK can be overcome by its association with T antigen. Clearly our results are of considerable technical value because they provide a basis for the regulation of the cell cycle and more specifically they provide a basis for selectively inhibiting or promoting the cell cycle by providing and/or enhancing the levels of p24 INK , or, removing or reducing the levels of p24 INK , respectfully. We therefore also present hereinafter a method for the isolation and/or purification of p24 INK in order to provide for a supply of the novel protein which upon purification may be used to regulate the cell cycle as herein described; or alternatively it may be used to identify agents capable of interacting there with so as to block the effects of the p24 INK protein. In addition, notwithstanding the fact that an antibody that binds to p24 1NK is described herein, the isolation of p24 INK also provides for the production of potentially more specific antibodies thereto, especially monoclonal antibodies. It will be understood that antibodies, monoclonal or otherwise, will be manufactured against the isolated and purified p24 INK using conventional techniques. Clearly the invention is of particular importance in disorders characterised by aberrant cell growth. For instance, since the finite lifespan of a cell involves an accumulation of p24 INK , p24 INK can be used to selectively limit the lifespan of diseased tissue such as, but not limited to, cancer tissue, lymphoid hyperplasias, inflammatory regeneration, Keloid scarring of the skin and benign tumours. Alternatively, or in addition, agents that bind p24 1NK , such as T antigen, can be identified and the interaction between p24 INK and such agents can be blocked in diseased tissue in order to ensure that p24 INK activity is manifest. Moreover the expression of p24 INK could be manipulated by binding agents in situations where one might want to limit the number of times cells divide. For example, in stem cell transplantation or other cell transplantations, one may want to put back cells into individuals that are prevented from dividing too many times and consequently becoming cancerous. Alternatively, in bone marrow transplantations, it has been observed that transplanted cells do not exist for a long time and therefore repeated transplantations have been necessary. If one could manipulate the cells to be transplanted via, manipulation of p24 INK , to have a greater number of divisions before senescence then fewer transplantations might be necessary. In addition, in instances where one would like to provide for growth of tissue, and in particular sustained growth, for example in wound healing or tissue repair, agents such as antagonists may be used to selectively bind to p24 INK and thus mask the effects of same. In this invention reference made to p24 INK is intended to include homologues or analogues thereof and in particular agonists which behave in the same way as p24 κ . In addition further still, since it is known that p24 INK has at least a part to play in preventing cells dividing, the invention has application in the manipulation of the promoter relating thereto so that enhanced levels of p24 INK may be expressed. Having regard to the above it can be seen that it is an object of the invention to identify a novel regulator of cell proliferation and to provide a method for the isolation and/or purification of same. It is a further object of the invention to provide a method for regulating the cell cycle which involves the use of the novel regulator of cell proliferation of the invention, or agents that interact therewith. It is yet a further object of the invention to provide agents which are capable of interacting with the novel regulator of cell proliferation of the invention ideally with a view to blocking or enhancing the effects of same. According to a first aspect of the invention there is therefore provided a novel, isolated regulator of cell proliferation, p24 INK , having a molecular weight of 24 kilodaltons and a pattern of expression which is approximately maximal at the onset of cell senescence. Reference herein to a novel regulator of cell proliferation, p24 INK , is intended to include reference to any mammalian form of the protein given the high degree of inter-species homology and indeed the functional identity, and inter- changeability of the protein between species. This is demonstrated having regard to a review entitled Inhibitors of mammalian G, cyclin-dependent kinases (Genes & Development 9: 1149-1163, 1995 - Cold Spring Harbor Laboratory Press). For example, it is stated in this review that a recently identified cyclin-dependent kinase inhibitor, termed p57 kip2 , and more particularly in vitro translated mouse p57 Wp2 , bound to cdk2, cdk3, cdk4 and cdkό in a cyclin-dependent manner. Moreover, this inhibitor was shown to inhibit the kinase activity of cyclin D-cdk4, D-cdk6, E-cdk2, E-cdk3, and A- cdk2 complexes in vitro; and its transfection into mink lung cells or into human Saos-2 osteosarcoma cells induced G] arrest. Similarly, other cyclin- dependent kinase inhibitors have been shown to have a high degree of inter species homology. For example, p!5 INK4 which has been known to be induced in human epithelial (HaCaT) cells by growth factor has also been shown to be produced in many mouse tissues. Similarly, plo^ 1 40 and pl9 INK4d are also expressed ubiquitously in proliferating culture cells and in normal mouse tissues. Then the invention relates to any mammalian p24 INK . Ideally, said regulator is a cyclin-dependant kinase inhibitor. More preferably still, the expression of said inhibitor is independent of T antigen and/or independent of p53. More preferably further still, said regulator associates with T antigen. We consider that this interaction is the mechanism by which the inhibitory growth effects of p24 INK are overcome in cells expressing T antigen. According to a second aspect of the invention, there is provided a method for the purification of a novel regulator of cell proliferation, p24 INK , comprising: lysing cells expressing p24 INK ; and exposing at least protein extracts from said lysis to an antibody that is at least specific for p24 INK . In a preferred embodiment of the invention, in the instance where said antibody may recognise an antigen other than p24 INK , the method includes a further step of size-fractionation. Ideally the method of the invention involves the use of an immunoaffinity column to which the said antibody is attached and exposure of at least said extract thereto under conditions which facilitate binding of p24 mκ to said antibody; and then elution of said bound agent or antigen followed, optionally, depending upon the nature of the antibody, by a further separation technique which ideally involves a size-fractionation method. Alternatively, or in addition, the method for purification of the novel cell cycle regulator, p24 κ may involve genetic techniques. These techniques involve screening an expression library such as an E.coli library prepared with mRNA extracted from a p24 INK expressing cell such as a tsa cell line using a p24 INK specific antibody such as the 471 antibody. Once a clone is identified it is used to screen a full length cDNA library. This full length clone is then over expressed in order to purify the protein in a large quantity. In order to check that the correct gene has been cloned antisera to the protein will be used. Alternatively, or in addition, a PCR library is used. Since the sequence of the peptide that was used as an immunogen is known an oligonucleotide was designed which is used in combination with a vector specific primer to carry PCR on a cDNA library. This is done advantageously under fairly nonstringent conditions. As before, when a PCR fragment is generated it is used for screening a full length cDNA library. The isolated cDNA is then over expressed and used to generate antisera for determining the correct nature of the gene. According to a yet further aspect of the invention there is provided the use of a novel regulator of cell proliferation, p24 INK , to affect the cell cycle. According to a further aspect of the invention there is provided the use of agents that selectively bind with p24 ,NK to affect the cell cycle. According to a yet further aspect of the invention there is provided the use of p24 κ to identify agents that selectively bind therewith so as to inhibit the effects thereof. According to a yet further aspect of the invention there is provided antibodies, other than antibody 471 , with selectively bind with p24 INK , ideally, with a view to determining the level of expression thereof. The invention will now be described by way of example only and in particular a preferred method for purifying and/or isolating the novel protein of the invention will be described by way of example only and with reference to the accompanying figures wherein. Figure 1 shows P21 expression: (A) Northern blot analysis. 5μg of total RNA prepared from tsa 129, SV4 and REF cells was loaded onto agarose gel, transferred on nitrocellulose membrane and probed with rat p21 cDNA probe labelled with 32p by random priming. Comparison of p21 mRNA expression in tsal29 cell line when shifted up from 33° C to 39.5° C for 3 days (lane 1, 2) and in normal Rat Embryo Fibroblasts (REFs) in early (lane 3) and late passages (lane 4). SV4 cell lines were used to verify that p21 mRNA expression is not dependant on temperature. The Blot was stripped and reprobed with a human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe for control of equal RNA loading. (B) Western blot analysis, (a) tsa 14, 129, AP393 and SV4 total protein extracts were loaded at 30 μg lane and examinated by CP74, a p21 -specific monoclonal antibody. GST-p21 was loaded as positive control, (b) comparison of two peptide antibodies affinity purified. 471 and 472 were raised respectively against the C-terminus of murine and N-terminus of human p21 protein. Figure 2 shows passaging experiments at 33° C in normal Rat Embryo Fibroblasts from 13 day old Sprague-Dawley embryos in two separate experiments: same total cell extracts were analysed by Western blotting at 30 μg/lane with a variety of antisera against p21 (CP74, 472, 397), p24 INK (471) and p27 (528). REFs stop dividing at passage 4 in experiment on the left panel and passage 3 in experiment on the right panel (to corresponds to cells before plating). Cells were called senescent when the number of cells counted after 4 days was lower than number of cells plated to extracts were prepared from cells before plating. Figure 3 shows the expression of p21 and p24 INK in passaging experiments using transgenic mouse embryo fibroblasts expressing thermolabile tsa58 T Ag (A). The expression levels of p21 and p24 INK in normal (NMEFs) or transgenic (TMEFs) mouse embryo fibroblasts were compared. TMEFs and NMEFs were serially cultivated every 4 days in presence of 10 units per ml of recombinant mouse γ-INF obtained from Gibco-BRL at 33° C until the NMEFs stopped proliferating. Total cell extracts were prepared 2 days after replating. In this experiment, cells stop dividing at passage 6. The same extracts were assayed by immunoblotting in duplicate for p24 INK (upper panel) and p21 (lower panel) using 30 g of protein. (B) TMEFs were serially cultivated at 33° C + γ-INF or 39.5° C without γ-INF. NMEFs were passaged at 39.5° C without γ-INF. (C) Extracts were prepared from passage 12 TMEFs cultured at either 33° C + γ-INF or after shift up to 39.5° C for 3 days. 30μg total protein was analysed for p21 and p24 INK by immunoblotting with antibodies SX118 and 471. SX118 is another mouse monoclonal antibody specific for p21 and was provided Xin Liu. (D) p21 and p24 INK protein expression in p21-/- cell extracts compared to cell extracts TMEFs and NMEFs. Cell protein extracts (50 μg) were analysed first with a specific anti-p21 antibody (SX118) which detects only p21 and then reprobed with 471 for detection of p24 INK . Auto radiograms were then cut to compare p21 and p24 κ in each lane. Arrowheads indicate position of p21 and p24 INK . Figure 4 shows coprecipitation of T antigen. (A) An equal amount RIPA cell lysate prepared from tsa 129 cells cultured at 33° C was immunoprecipitated using antibodies specific for p21(SX118, 397), p27 (528) or p53 (421). Monoclonal antibody M73 specific for the Adeno virus EIA protein was used a negative control(). The immunoprecipitated proteins were fractionated and analysed for the presence of T antigen by immunoblotting using a mixture of monoclonal antibodies 419 and 416. (B) RIPA cell lysate prepared from tsal29 cells at 33° C was immunoprecipitated with 471 in the presence of increasing amounts of cold peptide used to raise this antiserum. IX corresponds to an equal amount of peptide whereas 10X is ten-fold higher amount of peptide. The immunoprecipitates were first analysed for the presence T antigen and then reprobed for p21 using SX118. Experiments to Show p21 Expression in Response to an Irreversible Growth Arrest In agreement with the data from human fibroblasts, p21 mRNA was upregulated both in senescent Rat Embryo Fibroblasts (REFs) and in a tsa cell line (tsal29) when it undergoes an irreversible growth arrest (Figure 1 A), tsa cell lines such as tsa 129 were derived by immortalising REFs with the thermolabile large T antigen encoded by the SV40 early region mutant tsA58 and thus grow continuously at the permissive temperature (33° ) but rapidly undergo growth arrest upon inactivation of T antigen at the nonpermissive temperature (39.5°). Isolation and characterisation of these cell lines has been previously described (Jat and Sharp 1989). The increase in p21 mRNA was greater in the tsal29 line (5-10 fold) in comparison to REFs. Similar increases in expression of p21 mRNA were observed in other tsa cell lines (tsa8, 14) upon inactivation of T antigen (Gonos et al). Experiments to Show Correlation Between p21 mRNA Expression and p21 Protein Expression in Response to an Irreversible Growth Arrest A number of p21 specific antibodies were used for analysis by immunoblotting: CP74 (provided by Ed Harlow) and antibody 472 (purchased from Santa Cruz) show a similar pattern of enhanced expression of p21 upon inactivation of T antigen. Even though the increase in protein was less than the increase in mRNA, a 2-5 fold increase was consistently observed (compare Figure IA with IB). However, antibody 471 (also purchased from Santa Cruz) revealed an unexpected pattern of expression (Figure IB lane 1, 2). It detected a protein that migrates slower than p21, at approximately 24 Kd (designated p24 INK ) and there was no alteration in the protein level upon shift up to 39.5° C. Nevertheless, 471 can detect a fusion protein of glutathione-S-transferase-p21 (GST-p21) which was used as the positive control in all immunoblotting experiments and 471 is also capable of immunoprecipitating p21 (Figure 4), thus confirming its specificity. Experiments to Show Levels of p21. p24 INK and p27 Expression in Rat Embryo Fibroblasts Upon Serial Passaging 13 days-old embryos from pregnant rat females were dissected and the head and tail removed. The remaining tissues were disaggregated and the cell population (designated tO) plated in Dulbecco's Modified Eagles Medium (DMEM) medium supplemented with 10% foetal calf serum. Cells were then passaged at 10° cells/ 10 cm plate every four days at 33° C until they stopped dividing ie became senescent; identical results were obtained at 37° C where the cultures senesce slower than those at 33° C. We reproducibly observed an upregulation of p24 INK upon passaging using antibody 471 (Figure 2); it was undetectable in extracts prepared from cells before plating (tO) and reaches a maximal level corresponding to onset of senescence (determined by counting the cell number). Expression of authentic p21 was also increased upon passaging; however the increase occurs earlier and reached a steady state well before the cells stopped dividing (compare CP74, 472,397 with 471 in Figure 2). In contrast analysis of p27 Kφl gave an inverse pattern of expression (Figure 2, right panel); it declined from a high level in tO cells to a low level in senescent fibroblasts and thus demonstrated that p24 INK was not p27. The observation that the level of p24 INK increases in fibroblasts upon serial passaging when taken in conjunction with the finding that the p24 INK level was independent of the presence of T antigen raised the possibility that p24 INK detected by 471 might be an excellent candidate for a component of the biological clock that limits the mitotic potential of rodent embryo fibroblasts. Experiments to Show p24 INK Expression in the Presence and Absence of T antigen in TMEFs The observation that increased expression of p24 INK was correlated with serial passaging in REFs (Figure 2) and its expression was not altered by inactivation of T antigen in tsa cell lines (Figure 1), prompted us to test the possibility that ρ24 INK exhibits the same pattern of expression both in the presence and absence of T antigen and compared the build up of p24 ,NK in these fibroblasts with embryonic fibroblasts prepared from nontransgenic control mice. We used embryonic fibroblasts prepared from the H-2K b tsA58 mice (Jat et al, 1991). These mice harbour the thermolabile f_?A58 T antigen under the control of the γ -interferon (γ-INF) inducible H-2K b promoter and thus allow the preparation of fibroblast populations in which the expression of T antigen can be modulated in every cell by manipulating the growth conditions. Previously, it has been shown that embryonic fibroblasts prepared from the H-2K b tsA58 mice became dependent upon the T antigen for growth only when the normal mitotic lifespan has elapsed and that the measurement of the lifespan continues normally in cells expressing this immortalising gene (Ikram et al, 1994). Fibroblast cultures were prepared from 12/13-day old mouse embryos from transgenic homozygous males x normal females (TMEFs) and normal males x normal females (NMEFs) and serially cultivated in the presence of 10 units per ml γ -interferon at 33° C (γ-INF) until the NMEFs ceased dividing. Under these permissive growth conditions TMEFs yield immortal cultures that can be passaged indefinitely whereas NMEFs stop dividing; γ-INF was included in both cultures to ensure that the cultures were directly comparable. At the indicated passages protein extracts were prepared and analysed. As the cultures were passaged there was a gradual accumulation of p24 ,NK with a peak around P6 when the NMEFs ceased dividing (Figure 3A). The kinetics of p24 INK accumulation were very similar in NMEFs and TMEFs (Figure 3 A upper panel). As for the REF cultures the increase in expression of p21 occurred more rapidly and reached a steady state earlier. Moreover, the maximum level of p21 in TMEFs was lower that in NMEFs (Figure 3 A lower panel). Identical results were obtained in several experiments. Since we have previously shown that TMEFs and NMEFs grown at 39.5° C in absence of γ-INF exhibit the same finite proliferative potential, we passaged TMEFs at 33° C in the presence of γ-INF and at 39.5° C in the absence of γ- INF and NMEFs at 39.5° C in the absence of γ-INF and examined the accumulation of p24 INK . p24 INK accumulates with the same kinetics in both cultures at 39.5° C (Figure 3B). Analysis of p24 INK in TMEF cultures serially cultivated at 33°C+γ-INF once the NMEFs had ceased dividing around passage 3 showed a reduction in the level of p24 INK to a steady state level which did not alter upon further passaging and also upon inactivation of T antigen. This profile of p24 INK expression correlates with the growth profile of TMEFs cultured at 33° C in the presence of γ-INF where there is always a small but reproducible decline in the growth rate after the rapid initial growth period and after which the growth rate remains relatively constant (data not shown and Ikram et al). Interestingly the decline in growth rate occurs at about the same time as NMEFs cease dividing. Moreover, it corresponds also to the maximum level of p24 INK . In other experiments where NMEFs ceased dividing later (P6), the peak of p24 ,NK occurred later at P6 and the steady state level of p24 INK was not achieved until passage 11 (data not shown). Determination of the level of T antigen in these cultures showed that a steady state level of T antigen was attained by passage 4. After 12 passages if these TMEFs were shifted to 39.5° C for 3 days, we reproducibly did not detect any further increase in the level of p24 INK but p21 was upregulated (Figure 3C). Thus these results show that p24 INK accumulates in rodent fibroblasts upon serial passaging in vitro and that this accumulation occurs irrespective of T antigen. Experiments to Show p21 and p24 INK are derived from different genes Protein extracts prepared from p21-/- fibroblasts were analysed. Results presented in Figure 3D show that although p21 level was undetectable in p21- /- extracts, approximately the same amount of p24 INK protein was present in these cells compared to TMEFs. Thus the p24 INK protein is not a product of the p21 Waf, Cip, SdU gene. Experiments to Show Interaction of T antigen with p24 INK Our results showed that in contrast to p21 whose expression was clearly influenced by the presence of T antigen, p24 INK expression was unaffected. Since p24 INK is probably related to p21 protein, it is possible that p24 INK may also be a cdk inhibitor and therefore by analogy an inhibitor of cell division and thus the high level of p24 INK in proliferating cells seems to be a paradox. Since sequestration of growth suppressors is one potential mechanism of removing an inhibitor, we investigated the possibility that T antigen might interact with p24 INK . Therefore we carried out immunoprecipitations using 35-S methionine labelled cell extracts and found that this antibody immunoprecipitates a number of proteins. Nevertheless two of these proteins migrated at 21 and 24 KD respectively suggesting that it might be precipitating both p21 and p24 INK . Therefore we immunoprecipitated unlabelled extracts prepared from tsa 129 cells cultured at the permissive temperature and analysed by immunoblotting with a mixture of T antigen specific monoclonal antibodies or a mouse monoclonal antibody specific for p21. Since T antigen is known to be present complexed to p53, extracts were immunoprecipitated by 421, a p53 specific monoclonal antibody, as a positive control; two antibodies that immunopreciptate authentic p21 and a p27 specific antibody were also included. The results in Figure 4 show that immunoprecipitation with 471 results in the precipitation of T antigen whereas antibodies that precipitate p21 or p27 do not bring down T antigen. Moreover, this precipitation was competed by an excess of the peptide that had been as the immunogen for the 471 antibody. Since there is no homology between the 471 peptide and T antigen, it suggests that T antigen was precipitate and as a consequence of a complex with a cellular protein recognised by 471. The most likely candidate for this cellular protein is p24 INK . Experiments for Isolating and/or Purifying p24 INK p24 INK was essentially extracted using immunoaffmity purification. Techniques relating to immunoaffinity purification are described in the laboratory text book "Antibodies: A Laboratory Manual" by Ed Harlow and David Lane, Cold Spring Harbour Laboratories, 1988, and in particular page 449 describing cell lysis and pages 511-552 describing large scale immunoaffinity purification. Essentially, the method involves three basic steps. 1. Preparation of an antibody -matrix. 2. Binding the antigen to the antibody-matrix. 3. Elution of the antigen. In the first step monoclonal antibodies or affinity-purified polyclonal antibodies are used. In our preferred method of the invention we have used the 471 antibody and covalently attached it to a solid-phase matrix that is a sepharose column. Any contaminating macro-molecules were removed by washing. Using standard cell lysis techniques, cells expressing p24 INK , and ideally cells maximally expressing p24 INK and thus approaching the onset of senescence were lysed and the cell lysis extracts were passed over the affinity column. The nature of the antibody used determined the amount of cross-binding. For example, 471 antibody may bind p21 as well as p24 INK and therefore following use of the immunoaffinity column, and elution of the bound antigen, p21 and p24 INK , using conventional techniques a further separation technique is required. Given the difference in size between p21 and p24 INK a sizing column was used to separate the two proteins such as an FPLC superose 12 or superose 75 column to size-fractionate the proteins. In the instance where a different antibody, ie a more specific antibody is used the immunoaffinity column alone is sufficient to purify p24 INK because of the reduction in cross-binding. Summary Here we have presented identification of p24 INK , a protein whose expression increases when rodent embryonic fibroblasts are serially passaged in vitro. This protein clearly shares some homology to p2l Wafl/c ' P1/sdil since it was identified using a peptide affinity purified antibody prepared against a p21 peptide. The p21 afl ' ciP1 ' sdil gene was originally isolated as a gene that was overexpressed during cellular senescence of human fibroblasts and transcriptionally regulated by ρ53. We have found that the p 21 af,/Ci P 1 ' Sdil gene was also upregulated during serial passaging of rodent embryonic fibroblasts but the pattern of accumulation does not correlate with the loss of proliferative potential; it also does not accumulate to the maximal level in the presence of T antigen which was not surprising since T antigen complexes with p53 and p53 is known to upregulate p21. Here we have shown that p24 INK was upregulated when rodent embryo fibroblasts are serially passaged in vitro and undergo senescence; the upregulation occurs at the same rate in the presence or absence of T antigen and is thus independent of p53. Moreover, the timing and pattem of accumulation of p24 INK correlates exactly with the biological counting mechanism that limits the finite proliferative potential of rodent embryo fibroblasts. We have also found that this protein may be present in a complex with T antigen. By analogy to p2i W fl ' CiP1 ' Sdil ) p24 INK is an inhibitor of growth, and so our results suggests that the biological clock that measures the finite life span involves the accumulation of ρ24 INK and that its growth inhibitory effects can be overcome by association with T antigen.

Claims: CLAIMS 1. An isolated regulator of cell proliferation, p24, having a molecular weight of 24 kilodaltons and a pattern of expression which is approximately maximal at the onset of cell senescence. 2. A regulator according to claim 1 wherein said regulator is a cyclin- dependent kinase inhibitor. 3. A regulator according to claim 1 or 2 wherein the expression of said regulator is independent of T antigen. 4. A regulator according to any preceding claim wherein the expression of said regulator is independent of p53. 5. A regulator according to any preceding claim wherein said regulator associates with T antigen. 6. A method for the purification of a novel regulator of cell proliferation, p24, comprising: lysing cells expressing p24; and exposing at least protein extracts from said lysis to an antibody that is at least specific for p24; and isolating said p24-antibody complex from said extract. 7. A method according to claim 6 wherein said method further includes a size-fractionation step in order to isolate the p24-antibody complex on the basis of the molecular weight of p24. 8. A method according to claims 6 or 7 wherein the method comprises the use of an immuno affinity column to which the said antibody is attached and exposure of said extract thereto, under conditions which facilitate binding of p24 to said antibody; and then elution of said bound antigen. 9. A method according to claim 8 wherein said method involves a further separation technique which comprises a size-fractionation step in order to isolate the p24-antibody complex on the basis of the molecular weight for p24. 10. A method for the purification of a novel regulator of cell proliferation, p24, comprising screening an expression library prepared with mRNA extracted from p24 expressing cells, using a p24 antibody in order to identify a p24 clone and then over expressing this clone in order to purify the protein in a suitable quantity. 11. A method for the purification of a novel regulator of cell proliferation, p24, comprising preparing a PCR fragment having regard to the nature of the antibody used to bind to p24, then using this PCR fragment for screening a full length cDNA library in order to detect p24 cDNA and then overexpressing this cDNA. 12. The use of an isolated regulator of cell proliferation, p24, to affect the cell cycle. 13. The use of agents that selectively bind with p24 to affect the cell cycle. 14. The use of p24 to identify agents that selectively bind therewith so as o inhibit the effects thereof. 15. Antibodies, other than antibody 471, which selectively bind with p24.

Inventors: JAT PARMJIT, MAZARS GEORGE-RAOUL, JAT, PARMJIT, MAZARS, GEORGE-RAOUL
Applicants: JAT PARMJIT, LUDWIG INST CANCER RES, MAZARS GEORGE RAOUL, JAT, PARMJIT, LUDWIG INSTITUTE FOR CANCER RESEARCH, MAZARS, GEORGE-RAOUL
Application year: 1997
Main classification: C07K14
IPC codes: A61K38, C07K14