Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
1999-03-23
2001-03-27
Kemmerer, Elizabeth (Department: 1646)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C530S387900, C530S388100, C530S388800, C530S389700, C530S387700, C435S331000, C435S346000
Reexamination Certificate
active
06207452
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of cell physiology, and more particularly to tumorigenesis and to apoptosis, i.e. programmed cell death. The novel peptides and nucleotides of the invention are useful in molecular screening of human tumors for the presence of mutations that allow the proliferation of cells that were otherwise marked for apoptosis. The novel peptides and nucleotides are also useful to screen for proteins that play a role in the modulation of cellular proliferation.
BACKGROUND OF THE INVENTION
The bcl-2 gene was discovered as typically involved in the t(14;18) chromosomal translocations observed in human follicular lymphoma (1-3). This chromosomal rearrangement results in deregulated high-level expression of the bcl-2 gene. In addition, Bcl-2 is also expressed at elevated levels in a variety of other tumors (4-6). The Bcl-2 protein suppresses apoptosis induced by a multitude of stimuli (7,8). Suppression of apoptosis by Bcl-2, while allowing cell survival, is characterized by growth arrest associated with Bcl-2 activity (40). Although bcl-2 was discovered as a candidate oncogene, conventional transformation assays indicate that it does not possess dominant oncogenic activity (9). It is therefore believed that unlike other oncogenes, bcl-2 contributes to oncogenesis primarily by extending cell viability, thereby perturbing the homeostatic mechanisms that control cell number and by providing an environment for other genetic changes (10).
In spite of a lack of detectable autonomous transforming activity, bcl-2 has been shown to synergize with c-myc in the generation of malignant cells (11). Since constitutive expression of c-myc induces apoptosis under certain conditions (12-14) that can be suppressed by Bcl-2 (14-16), it appears that the c-myc-cooperating oncogenic activity of bcl-2 may be related to its anti-apoptosis activity. In addition, Bcl-2 can also efficiently suppress apoptosis induced by tumor suppressor proteins such as p53 (17-21). This suggests that Bcl-2 may contribute to oncogenesis by suppressing apoptosis induced by oncogenes and tumor suppressor genes.
Although mutations within the Bcl-2 protein that permit proliferation of cells that would otherwise undergo total apoptosis could play a more direct role (as opposed to deregulated expression) in oncogenesis, thus far no such mutants have been identified in naturally arising tumors or under experimental conditions.
SUMMARY OF THE INVENTION
The present inventor here describes the identification and characterization of a hitherto unrecognized domain within human Bcl-2, which the inventor has designated the “anti-proliferation (AP) domain”, that is required for the proliferation-restraining activity of Bcl-2. Mutants in this domain of Bcl-2 are described that retain the ability to suppress apoptosis induced by the p53 tumor suppressor protein and Myc onco-protein, while allowing concomitant cell proliferation.
More specifically, the present inventor has identified a deletion mutant of Bcl-2 that has a novel activity. The deletion mutant, designated Bcl2&Dgr;51-85, not only suppresses apoptosis induced by the tumor suppressor protein p53 and the Myc onco-protein, but unlike wt Bcl-2, permits continued cell proliferation. These results may have important implications for oncogenesis involving Bcl-2. Unlike other oncogenes, the bcl-2 proto-oncogene promotes cell survival without significant cell proliferation. These results suggest that certain mutations can inactivate a proliferation-restraining activity. Further, the observed effect against oncogene/anti-oncogene-induced apoptosis may potentially prove to be of considerable significance in oncogenic events involving Bcl-2. Such inactivating mutations within the non-conserved region of Bcl-2 may enhance tumorigenesis by antagonizing the apoptotic activities of p53 and Myc as well as by permitting continued cell proliferation.
The molecular basis for the loss of proliferation-restraining activity in the Bcl-2 mutant has been partially elucidated as described in Example 3. The results suggest that the loss of activity does not correlate with the ability of Bcl-2 to interact with several proteins. However, the interaction between the Bcl-2 mutant and the death-promoting protein Bax appears to be enhanced compared to the interaction of Bax with wild type Bcl-2. It is not clear whether this enhancement is due to an increased affinity of the Bcl-2 mutant for Bax or increased stability of the Bcl-2/Bax complex. The importance of the Bcl-2/Bax interaction to the proliferation-restraining function of Bcl-2 is unknown.
Also, the region deleted in Bcl2&Dgr;51-85 contains several Ser and Thr residues. It has been reported that Bcl-2 activity can be modulated by phosphorylation (34, 46, 47, and 49). Analysis of the activity of several Bcl-2 mutants containing amino acid substitutions at Ser or Thr residues, as described in Example 4, suggests that modulation of the proliferation-restraining activity by phosphorylation is possible. Alternative explanations to account for the mutant phenotype are also possible. The deleted region is rich in Ala and Pro residues. Substitution of Pro residues in two positions within the AP domain resulted in Bcl-2 mutants that permit enhanced cell proliferation. The possibility that these residues play some negative regulatory role in Bcl-2 activity remains to be investigated.
In one aspect then, the invention provides isolated oligonucleotides that encode the Bcl-2 AP domain or fragments of the domain. The oligonucleotides and short segments thereof are useful for screening for mutations in the Bcl-2 AP domain by methods known in the art, such as single strand conformational polymorphism (SSCP) and PCR mismatch analysis.
In another aspect, the present invention is directed to identifying protein/protein interactions between the Bcl-2 AP domain and known or as yet unidentified cellular proteins. The Bcl-2 AP domain is also useful in the identification and cloning of genes whose protein products interact with this domain in Bcl-2. The interacting proteins may play a role in modulation of cellular proliferation.
The present invention also relates to an isolated polypeptide that is the Bcl-2 AP domain and fragments of the domain. The domain may be a target for allosteric regulators of Bcl-2 function, such as protein kinases and/or phosphatases. Accordingly, peptides derived from this domain, prepared synthetically or as bacterially expressed fusion proteins, can be used as substrates to identify and characterize potential regulatory kinases and/or phosphatases.
The invention further provides screening methods to identify molecules that modulate the proliferation-restraining activity of the AP domain. In one aspect, such screening methods involve the effect of a putative modulating molecule on the short term or long term proliferation of cells in culture expressing the AP domain. In another aspect, putative modulating molecules can be identified by screening for agents that disrupt necessary protein/protein interactions mediated by the AP domain, using in vitro binding assays.
In yet another aspect, the invention provides for expression vectors containing genetic sequences, hosts transformed with such expression vectors, and methods for producing the AP domain and fragments of the domain that hinder or completely block proliferation.
In additional aspects, the present invention relates to antibodies that specifically bind to the AP domain and fragments of the domain that hinder or completely block cell proliferation. Peptides comprising the domain are useful for producing antibodies thereto. Such antibodies are useful for detecting and isolating proteins comprising the AP domain in biological specimens including, for example, cells from all human tissues including heart tissue, lung tissue, tumor cells, brain tissue, placenta, liver, skeletal muscle, kidney, and pancreas, as well as for modulating the proliferation-restraining activity of proteins comprising the AP domain, in and from such biologi
Kaufman Claire M.
Kemmerer Elizabeth
St. Louis University Health Sciences Center
Sughrue Mion Zinn Macpeak & Seas, PLLC
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