Beclin, a novel bcl-2 interacting gene near BRCA1 on chromosome 17q21 and methods of use

This invention provides for an isolated nucleic acid which encodes a wildtype human Beclin and a mutant human Beclin. This invention also provides a vector containing the isolated nucleic acid which encodes a wildtype human Beclin. This invention also provides for a method of producing a wildtype human Beclin. This invention also provides for a purified, wildtype human Beclin. This invention also provides for a method for determining whether a subject has a predisposition for cancer. This invention also provides a method for determining whether a subject has cancer. This invention also provides for a method for inhibiting cell proliferation in cells unable to regulate themselves. This invention also provides for a method for treating a subject who has cancer. This invention also provides a pharmaceutical composition composed of the wildtype human Beclin. This invention also provides a method for detecting a mutant human Beclin in a subject. This invention also provides a method for treating a subject unable to control apoptosis in the cells of the subject.

BACKGROUND 
Throughout this application, various publications are referenced by author 
and date. Full citations for these publications may be found listed 
alphabetically at the end of the specification immediately preceding 
Sequence Listing and the claims. The disclosures of these publications in 
their entireties are hereby incorporated by reference into this 
application in order to more fully describe the state of the art as known 
to those skilled therein as of the date of the invention described and 
claimed herein. 
A. Regulation of Apoptosis 
1. Apoptosis is important in diverse physiologic processes; the abnormal 
regulation of apoptosis is important in diverse pathologic processes, 
including turmorigenesis. 
Apoptosis is a highly conserved innate mechanism by which mammalian cells 
commit suicide. This mechanism allows an organism to eliminate unwanted or 
defective cells by an orderly process of cellular disintegration, and is 
characterized by certain stereotypic biochemical (e.g. endonucleosomal 
cleavage into 180-200 bp multimers) and morphologic features (e.g. 
chromatin condensation, cytoplasmic blebbing, etc.). Apoptosis plays a 
role in physiologic processes such as differentiation during 
embryogenesis, establishment of immune self-tolerance, and killing of 
cytotoxic immune cells, and apoptosis can be induced in response to a 
variety of stimuli including DNA damage, growth factor withdrawal, 
Ca.sup.2+ influx, ischemia, and viral infection. The unwanted occurrence 
of apoptosis may play a role in neurodegenerative diseases and aging, and 
the diminution of apoptotic death may play a role in cancer and 
chemoresistance. 
2. Apoptosis and the cell cycle may share common pathways. 
In recent years, the concept that the cell cycle and apoptosis are 
inextricably linked has gained widespread support in the cell death field. 
Several different lines of evidence support this concept. For example, in 
response to different death signals, normally quiescent cells express 
elevated levels of cell cycle genes (Buttyan, 1991; Freeman, 1994). 
Oncogenes such as c-myc (Evan, et al., 1996), ras (Wyllie, et al., 1987; 
Tanaka, et al., 1994) and adenovirus ELA (White, 1991), that promote cell 
proliferation, also act as triggers of apoptosis. Loss of normal 
restraints at the Gl checkpoint, such as inactivation of the 
retinoblastoma gene product, p105Rb (Clarke, et al, 1992; Lee, et al 1992; 
Jacks, et al, 1992), or deregulated expression of the Gl-specific E2F 
transcription factors (Shan, et al., 1994; Qin, et al., 1994; Wu, et al., 
1994) results in uncontrolled proliferation and apoptosis. Loss of the p53 
tumor suppressor gene results in resistance to certain apoptotic triggers, 
and p53 overexpression induces some types of apoptosis (reviewed in Evan, 
1995). The morphologic features of apoptosis resemble those of mitotic 
catastrophe (reviewed in King and Cidlowski, 1995), and premature 
activation of cyclin dependent kinases is required for some forms of 
apoptosis (Shi, et al., 1994). Furthermore, several agents that block cell 
cycle progression also protect neuronal cells from apoptosis induced by 
withdrawal of trophic factor support (Farinelli and Greene, 1996) and T 
lymphocytes form apoptosis induced by T-cell receptor ligation (Boehme and 
Lenardo, 1993). These observations all support the notion of a link 
between the cell cycle and apoptosis. 
3. An evolutionarily conserved set of cellular genes regulate apoptosis. 
Several mammalian genes have been identified that function as either 
inducers (e.g. faslapo-1, bax, ICE-like cysteine proteases, p53) or 
repressors (e.g. bcl-2, bcl-x.sub.s, bcl-x.sub.L) of an evolutionarily 
conserved apoptotic death pathway. Prevailing hypotheses in the cell death 
field are that a family of ICE-like cysteine proteases (CED-3, ICE, 
Nedd-2/ICH-1, CPP32) constitute the pivotal triggers of both nematode and 
mammalian cell suicide program and that a family of bcl-2 related genes 
constitute the final downstream negative regulators of cell death. Despite 
the identification of several effectors and repressors of cell death, the 
precise molecular mechanisms underlining the action of each of these genes 
remains poorly defined. 
4. Bcl-2, the proto-oncogene, inhibits a variety of types of apoptosis. 
Bcl-2 (for B cell lymphoma 2) is the prototypic anti-apoptotic gene. It was 
first discovered by virtue of its involvement in the t(14:18) chromosomal 
translocations found in the majority of non-Hodgkin's B cell lymphomas 
(Tsujimoto and Croce, 1985). Bcl-2 can prevent or delay apoptosis induced 
by a wide variety of stimuli (reviewed in Park and Hockenbery, 1996), 
including growth factor deprivation, alterations in Ca.sup.2+, free 
radicals, cytoxic lymphokines. some types of viruses, radiation and most 
chemotherapeutic drugs. The ability of Bcl-2 to inhibit apoptosis induced 
by such diverse stimuli suggests that this oncoprotein controls a common 
final pathway involved in cell death regulation. 
5. Dysregulated Bcl-2 expression occurs in a wide variety of human cancers 
and contributes to neoplastic cell expansion. 
While the bcl-2 gene was first discovered because of its involvement in 
t(14:18) translocations found frequently in non-Hodgkin's lymphomas, high 
levels and aberrant patterns of bcl-2 gene expression have been reported 
in a wide variety of human cancers, including colorectal, gastric, 
prostate, non-small cell lung, neuroblastomas, breast and ovarian cancer 
(reviewed in Reed, et al., 1996). In these tumors, it is thought that 
Bcl-2 contributes to neoplastic cell expansion by preventing cell turnover 
caused by physiological cell death mechanisms. In addition to its role in 
the development of human tumors, high levels of Bcl-2 expression are 
thought to play an important role in the resistance of tumor cells to 
cytotoxic anticancer drugs and radiation. 
6. The mechanism by which Bcl-2 inhibits apoptosis is still poorly 
understood. 
Several potential mechanisms of action have been proposed for Bcl-2, 
including protection against oxidative stress (Hockenbery et al., 1993; 
Kane et al., 1993), regulation of intracellular Ca.sup.2+ homeostasis 
(Lam, et al., 1993), antagonism of cell death proteases (e.g. ICE-like 
family of cysteine proteases) (Miura, et al., 1993) and other cell death 
effectors (e.g. bax) (Yin, et al., 1994), and association with the signal 
transducing proteins, R-ras and Faf-1 (Fernandez-Sarbia and Bischoff, 
1993; Wang, et al., 1994). In addition, two recent reports have suggested 
that Bcl-2 may exert anti-apoptotic effects by delaying cell cycle 
progression (Mazel, et al., 1996; Borner, 1996). Despite these numerous 
proposed mechanisms, there is considerable contradictory evidence and no 
universal agreement in the cell death field as to how Bcl-2 actually 
works. Further elucidation of the precise mechanism(s) of action of Bcl-2 
is a high research priority in the field. 
7. No functional links have been identified between inhibitors of apoptosis 
and inhibitors of cell cycle. 
According to the concept that the cell cycle is linked to apoptosis, one 
would predict that cellular genes that inhibit apoptosis would be 
functionally linked to genes that exert effects on the cell cycle. Along 
these lines, Bcl-2 has been shown to delay cell cycle progression (as 
stated above), and Bcl-2 has also been postulated to function as a nuclear 
"gatekeeper" that regulates nuclear access of cyclin-dependent kinases. 
However, to date, Bcl-2 has not been shown to directly interact with any 
proteins that affect the cell cycle. 
8. Further investigation of the mechanism(s) underlaying the death 
repressor activity of Bcl-2, including the characterization of novel Bcl-2 
interacting proteins, will provide new insights into apoptosis and 
diseases in which apoptosis plays a pathogenetic role. 
Understanding how Bcl-2 inhibits cell death is a critical question that has 
important implications for an understanding of all physiologic processes 
that involve cell death. 
B. Molecular Pathogenesis of Breast and Ovarian Cancer 
1. Several genes are responsible for inherited breast and ovarian cancer. 
The existence of one gene predisposing to breast and ovarian cancer on 
chromosome 17q21, BRCA1, was proven by linkage analysis several years ago 
(Hall, et al., 1990), and isolated in 1994 by positional cloning (Miki, et 
al., 1994; Futreal, et al., 1994). BRCA1 is mutated in the germline and 
the normal allele is lost in tumor tissue from approximately 50% of cases 
of hereditary breast and ovarian cancer (reviewed in Szabo and King, 
1996). BRCA2, a second breast cancer susceptibility gene, has been mapped 
to chromosome 13q21 and is presently implicated in 20% of hereditary cases 
(reviewed in Szabo and King, 1996). At least two other genes, p53 and the 
androgen receptor are also responsible for inherited predisposition to 
breast cancer in families. Other epidemiologic studies have suggested that 
carriers of mutations in the ataxia telangieclasia gene and HRAS1 
minisatellite locus are also at increased risk of breast cancer. 
2. Molecular genetic evidence suggests chromosome 17q21 may contain a 
second tumor suppressor tumor suppressor gene (in addition to BRCA1) that 
is important in sporadic breast and ovarian cancer. 
Allelic deletions of chromosome 17q21 (loss of heterozygosity LOH! that 
include that BRCA1 region are found to occur in 50-70% of breast 
carcinomas (Futreal, et al., 1992; Cropp, et al., 1993; Saito, et al., 
1993) and in up to 75% of ovarian carcinomas (Russell, et al., 1991; Sato, 
et al., 1991; Eccles, et al., 1991; Yang-Feng, et al., 1993). However, 
while several studies have confirmed the role of germline BRCA1 mutations 
in hereditary breast and ovarian cancers (Miki, et al., 1994; Futreal, et 
al., 1994), somatic mutations in BRCA1 have been found in very few cases 
of sporadic cancers (Futreal, et al., 1994; Takahashi, et al., 1995; 
Merajver, et al., 1995; Hosking, et al. 1995). This raises the strong 
possibility that the frequent allelic loss on chromosome 17q21 in sporadic 
breast and ovarian cancer reflects the involvement of an additional tumor 
suppressor gene. In further support of this hypothesis, more detailed 
deletion mapping of sporadic epithelial ovarian carcinomas has revealed a 
common deletion unit, located on chromosome 17q21 that is located 
approximately 60 kb centromeric to BRCA1 (Tangir, et al., 1996). Thus, the 
presence of LOH is sporadic ovarian cancer cases of a region of chromosome 
17q21 that does not encompass BRCA1 may reflect the presence of an 
additional tumor suppressor gene. 
SUMMARY OF THE INVENTION 
This invention provides for an isolated nucleic acid which encodes a 
wildtype human Beclin. This invention also provides for a mutant human 
Beclin. 
This invention also provides for a vector comprising the isolated nucleic 
acid which encodes a wildtype human Beclin operatively linked to a 
promoter of RNA transcription, specifically, the plasmid pSG5/beclin. 
This invention also provides a method of obtaining a polypeptide in 
purified form, specifically a wildtype human Beclin. This invention also 
provides for purified wildtype human Beclin. 
This invention also provides for an oligonucleotide of at least 15 
nucleotides capable of specifically hybridizing with a unique sequence of 
nucleotides within a nucleic acid which encodes a wildtype Beclin without 
hybridizing to any sequence of nucleotides within a nucleic acid which 
encodes a mutant human Beclin. This invention also provides for an 
oligonucleotide of at least 15 nucleotides capable of specifically 
hybridizing with a unique sequence of nucleotides within a nucleic acid 
which encodes a mutant Beclin without hybridizing to any sequence of 
nucleotides within a nucleic acid which encodes a wildtype human Beclin. 
This invention also provides for a method for detecting a mutant human 
Beclin in a subject. This invention also provides for a method for 
determining whether a subject has a predisposition for cancer. Further, 
this invention also provides a method for determining whether a subject 
has cancer. 
This invention also provides a method for inhibiting cell proliferation in 
cells unable to regulate themselves. 
This invention also provides a pharmaceutical composition comprising a 
wildtype human Beclin and a pharmaceutically acceptable carrier. 
This invention also provides for a method for treating a subject who has 
cancer. 
This invention also provides for a method for detecting the presence of 
human chromosomal region 17q21 in a sample of genomic DNA. 
This invention also provides for a method for treating a subject unable to 
control apoptosis in the cells of the subject.

DETAILED DESCRIPTION OF THE INVENTION 
In order to facilitate an understanding of the Experimental Details section 
which follows, certain frequently occurring methods and/or terms are best 
described in Sambrook, et al.(1989). 
Throughout this application, references to specific nucleotides are to 
nucleotides present on the coding strand of the nucleic acid. The 
following standard abbreviations are used throughout the specification to 
indicate specific nucleotides: 
______________________________________ 
C = cytosine A = adenosine 
T = thymidine G = guanosine 
______________________________________ 
A "gene" means a nucleic acid molecule, the sequence of which includes all 
the information required for the normal regulated production of a 
particular protein, including the structural coding sequence, promoters 
and enhancers. 
As used herein a wildtype human Beclin means a polypeptide which has an 
amino acid sequence identical to that present in a naturally-occurring 
form of human Beclin. As used here a mutant human Beclin means a 
polypeptide having an amino acid sequence which differs by one or more 
amino residues from, any naturally occurring form, including deletions 
mutants containing less than all of the residues present in the wildtype 
polypeptide, substitution homologs wherein one or more residues are 
replaced by other residues, and addition homologs wherein on or more amino 
acid residues are added to a terminal or medial portion of the 
polypeptide. 
The nucleic acids and oligonucleotides described and claimed herein are 
useful for the information which they claimed herein are useful for the 
information which they provide concerning the amino acid sequence of the 
polypeptide and as products for the large scale synthesis of the 
polypeptide by a variety of recombinant techniques. The molecule is useful 
for generating new cloning and expression vectors, transformed and 
transfected prokaryotic and eukaryotic host cells, and new and useful 
methods for cultured growth of such host cells capable of expression of 
the polypeptide and related products. 
The present invention provides for an isolated nucleic acid which encodes a 
wildtype human Beclin. This invention further provides an isolated nucleic 
acid which encodes a mutant human Beclin. The above-described isolated 
nucleic acids can be DNA, specifically cDNA or genomic DNA, and RNA. In a 
preferred embodiment, the wildtype Beclin has an amino acid sequence 
substantially identical to the amino acid sequence designated Seq. I.D. 
No.: 1. In another preferred embodiment, the isolated nucleic acid 
comprises a nucleic acid having a sequence substantially the same as the 
sequence designated Seq. I.D. No.: 2. 
As used herein, "mutant human Beclin" means polypeptides whose nucleic acid 
sequence or amino acid seqeunce differs from that of the 
naturally-occuring wildtype human Beclin. For example, due to a point 
mutation, the translated polypeptide differs from the naturally-occuring 
wildtype protein. Further, a subject may have low expression of the 
naturally-occurring protein so that the cells with this low-expressing 
protein cannot inhibit cell proliferation. 
This invention also provides for a vector comprising the above-described 
nucleic acid operatively linked to a promoter of RNA transcription. 
Numerous vector backbones are known in the art and are useful for 
expressing proteins. Such vectors include plasmid vectors, cosmid vectors, 
yeast artificial chromosome (YAC), bacteriophage or eukaryotic viral DNA. 
For example, one such class of vectors comprises DNA elements derived from 
viruses such as bovine papilloma virus, polyoma virus, adenovirus, 
vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MoMLV), Semliki 
Forest virus or SV40 virus. Such vectors may be obtained commercially or 
assembled from the sequences described by methods well-known in the art. 
This invention specifically provides a plasmid designated pSG5/beclin. 
Plasmid pSG5/beclin was made by cleaving DNA which encodes a wildtype 
human Beclin and inserting the DNA into the Eco RI site of pSG5 (FIG. 5). 
pSG5/beclin was deposited on Jul. 18, 1996 with the American Type Culture 
Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., 
under the provisions of the Budapest Treaty For The International 
Recognition Of The Deposit Of Microorganisms For The Purposes Of Patent 
Procedure. pSG5/beclin has been accorded ATCC Accession Number 97664. 
These vectors may be introduced into a suitable host cell to form a host 
vector system for producing the inventive proteins. Methods of making host 
vector systems are well known to those skilled in the art. 
Suitable host cells include, but are not limited to, bacterial cells 
(including gram positive cells), yeast cells, fungal cells, insect cells 
and animal cells. Suitable animal cells include, but are not limited to 
HeLa cells, Cos cells, CV1 cells and various primary mammalian cells. 
Numerous mammalian cells may be used as hosts, including, but not limited 
to, the mouse fibroblast cell NIH-3T3 cells, CHO cells, HeLa cells, 
Ltk.sup.- cells and COS cells. Mammalian cells may be transfected by 
methods well known in the art such as calcium phosphate precipitation, 
electroporation and microinjection. 
This invention also provides a host vector system for the production of a 
polypeptide which comprises the above-described vector in a suitable host. 
This invention also provides a method of producing a polypeptide which 
comprises growing the above-described host vector system, under suitable 
conditions permitting production of the polypeptide and recovering the 
polypeptide so produced. Further, this invention also provides a method of 
obtaining a polypeptide in purified form which comprises (a) introducing 
the above-described vector into a suitable host cell, (b) culturing the 
resulting host cell so as to produce the polypeptide, (c) recovering the 
polypeptide produced into step (b); and (d) purifying the polypeptide so 
recovered. In the above-described method, the vector comprises a plasmid, 
cosmid, yeast artificial chromosome (YAC), bacteriophage or eukaryotic 
viral DNA and the suitable host cell comprises a bacterial, insect, plant 
or mammalian cell. 
This invention also provides a purified, wildtype human Beclin. Wildtype 
human Beclin means a polypeptide which has an amino acid sequence 
identical to that present in a naturally-occurring form of human Beclin. 
This invention also provides an oligonucleotide of at least 15 nucleotides 
capable of specifically hybridizing with a unique sequence of nucleotides 
within a nucleic acid which encodes a wildtype Beclin without hybridizing 
to any sequence of nucleotides within a nucleic acid which encodes a 
mutant human Beclin. Further, this invention also provides an 
oligonucleotide of at least 15 nucleotides capable of specifically 
hybridizing with a unique sequence of nucleotides within a nucleic acid 
which encodes a mutant Beclin without hybridizing to any sequence of 
nucleotides within a nucleic acid which encodes a wildtype human Beclin. 
The above-described oligonucleotides may DNA or RNA. Methods of 
manufacturing such oligonucleotides and using the oligonucleotides are 
well-known in the art. 
This invention also provides a method for determining whether a subject has 
a predisposition for cancer which comprises (a) obtaining an appropriate 
nucleic acid sample from the subject; and (b) determining whether the 
nucleic acid sample from step (a) is, or is derived from, a nucleic acid 
which encodes a mutant human Beclin so as to thereby determine whether a 
subject has a predisposition for cancer. Various methods of determining 
whether the nucleic acid sample is, or is derived from, a nucleic acid 
which encodes a mutant human Beclin exist. 
In one example, the nucleic acid sample in step (a) comprises mRNA 
corresponding to the transcript of DNA encoding a mutant Beclin, and 
wherein the determining of step (b) comprises (i) contacting the mRNA with 
the above-described oligonucleotide, which is capable of specifically 
hybridizing with a unique sequence of nucleotides within a nucleic acid 
which encodes a mutant Beclin without hybridizing to any sequence of 
nucleotides within a nucleic acid which encodes a wildtype human Beclin, 
under conditions permitting binding of the mRNA to the oligonucleotide so 
as to form a complex, (ii) isolating the complex so formed, and (iii) 
identifying the MRNA in the isolated complex so as to thereby determine 
whether the MRNA is, or is derived from, a nucleic acid which encodes a 
mutant human Beclin. 
In another example, the determining of step (b) comprises (i) contacting 
the nucleic acid sample of step (a), and the isolated nucleic acid which 
encodes a wildtype human Beclin with restriction enzymes under conditions 
permitting the digestion of the nucleic acid sample, and the isolated 
nucleic acid into distinct, distinguishable pieces of nucleic acid, (ii) 
isolating the pieces of nucleic acid; and (iii) comparing the pieces of 
nucleic acid derived from the nucleic acid sample with the pieces of 
nucleic acid derived from the isolated nucleic acid so as to thereby 
determine whether the nucleic acid sample is, or is derived from, a 
nucleic acid which encodes a mutant human Beclin. 
In another example, the determining of step (b) comprises (i) sequencing 
the nucleic acid sample of step (a); and (ii) comparing the nucleic acid 
sequence of step (i) with the above-described isolated nucleic acid having 
a sequence substantially the same as the sequence designated Seq. I.D. 
No.: 2, so as to thereby determine whether the nucleic acid sample is, or 
is derived from, a nucleic acid which encodes a mutant human Beclin. 
In another example, the nucleic acid sample in step (a) comprises mRNA 
corresponding to the transcript of DNA encoding mutant Beclin, and wherein 
the determining of step (b) comprises (i) translating the mRNA under 
suitable conditions to obtain an amino acid sequence; and (ii) comparing 
the amino acid sequence of step (i) with the above-described isolated 
nucleic acid which as an amino acid sequence designated Seq. I.D. No.: 1 
so as to thereby determine whether the nucleic acid sample is, or is 
derived from, a nucleic acid which encodes a mutant human Beclin. 
In another example, the determining of step (b) comprises (i) amplifying 
the nucleic acid present in the sample of step (a); and (ii) detecting the 
presence of the mutant human Beclin in the resulting amplified nucleic 
acid. 
The above-described methods of determining are well-known to those skilled 
in the art. In a preferred embodiment, the isolated nucleic acid or the 
oligonucleotide is labeled with a detectable marker, wherein the 
detectable marker is a radioactive isotope, a fluorophor or an enzyme. 
Further, in a specific embodiment, the sample comprises blood, tissue or 
sera. 
This invention also provides a method for determining whether a subject has 
cancer, which comprises (a) obtaining an appropriate nucleic acid sample 
from the subject; and (b) determining whether the nucleic acid sample from 
step (a) is, or is derived from, a nucleic acid which encodes a mutant 
human Beclin so as to thereby determine whether a subject has cancer. 
Various methods of determining whether the nucleic acid sample is, or is 
derived from, a nucleic acid which encodes a mutant human Beclin exist. 
In one example, the nucleic acid sample in step (a) comprises mRNA 
corresponding to the transcript of DNA encoding a mutant Beclin, and 
wherein the determining of step (b) comprises (i) contacting the mRNA with 
the above-described oligonucleotide, which is capable of specifically 
hybridizing with a unique sequence of nucleotides within a nucleic acid 
which encodes a mutant Beclin without hybridizing to any sequence of 
nucleotides within a nucleic acid which encodes a wildtype human Beclin, 
under conditions permitting binding of the mRNA to the oligonucleotide so 
as to form a complex, (ii) isolating the complex so formed, and (iii) 
identifying the mRNA in the isolated complex so as to thereby determine 
whether the mRNA is, or is derived from, a nucleic acid which encodes a 
mutant human Beclin. 
In another example, the determining of step (b) comprises (i) contacting 
the nucleic acid sample of step (a), and the isolated nucleic acid which 
encodes a wildtype human Beclin with restriction enzymes under conditions 
permitting the digestion of the nucleic acid sample, and the isolated 
nucleic acid into distinct, distinguishable pieces of nucleic acid, (ii) 
isolating the pieces of nucleic acid; and (iii) comparing the pieces of 
nucleic acid derived from the nucleic acid sample with the pieces of 
nucleic acid derived from the isolated nucleic acid so as to thereby 
determine whether the nucleic acid sample is, or is derived from, a 
nucleic acid which encodes a mutant human Beclin. 
In another example, the determining of step (b) comprises (i) sequencing 
the nucleic acid sample of step (a); and (ii) comparing the nucleic acid 
sequence of step (i) with the above-described isolated nucleic acid having 
a sequence substantially the same as the sequence designated Seq. I.D. 
No.: 2, so as to thereby determine whether the nucleic acid sample is, or 
is derived from, a nucleic acid which encodes a mutant human Beclin. 
In another example, the nucleic acid sample in step (a) comprises mRNA 
corresponding to the transcript of DNA encoding mutant Beclin, and wherein 
the determining of step (b) comprises (i) translating the mRNA under 
suitable conditions to obtain an amino acid sequence; and (ii) comparing 
the amino acid sequence of step (i) with the above-described isolated 
nucleic acid which as an amino acid sequence designated Seq. I.D. No.: 1 
so as to thereby determine whether the nucleic acid sample is, or is 
derived from, a nucleic acid which encodes a mutant human Beclin. 
In another example, the determining of step (b) comprises (i) amplifying 
the nucleic acid present in the sample of step (a), and (ii) detecting the 
presence of the mutant human Beclin in the resulting amplified nucleic 
acid. 
The above-described methods of determining are well-known to those skilled 
in the art. In a preferred embodiment, the isolated nucleic acid or the 
oligonucleotide is labeled with a detectable marker, wherein the 
detectable marker is a radioactive isotope, a fluorophor or an enzyme. 
Further, in a specific embodiment, the sample comprises blood, tissue or 
sera. 
This invention also provides for a method for inhibiting cell proliferation 
in cells unable to regulate themselves by introducing the isolated nucleic 
acid which encodes a wildtype human Beclin into the cells, specifically 
wherein the cells are cancerous. Various methods of introducing nucleic 
acids into cells are well-known to those skilled in the art. 
This invention also provides a method for treating a subject who has cancer 
which comprises introducing the isolated nucleic acid which encodes a 
wildtype human Beclin, into the subject so as to thereby treat the cancer. 
Various methods of introducing nucleic acids into cells are well-kwown to 
those skilled in the art. In one example, one can introduce the isolated 
nucleic acid by (a) recovering cancer cells from the subject, (b) 
introducing the isolated nucleic acid of claim 1 into the cells; and (c) 
reintroducing the cells of step (b) into the subject so as to treat the 
subject who has cancer. Many types of cancer cells exist and are 
well-known in the art, specifically, breast, ovarian, skeletal, cervical, 
colon, prostate or lung cells. 
This invention also provides a pharmaceutical composition comprising a 
purified wildtype human Beclin and a pharmaceutically acceptable carrier. 
This invention further provides a pharmaceutical composition comprising 
the polypeptide obtained from using the above-described method of 
obtaining a polypeptide in a purified form and a pharmaceutically 
acceptable carrier. 
This invention also provides a method for treating a subject who has cancer 
comprising administration of an effective amount of the above-described 
pharmaceutical compositions to the subject who has cancer. The 
administration of the pharmaceutical compositions may be by topical, oral, 
aerosol, subcutaneous administration, infusion, intralesional, 
intramuscular, intraperitoneal, intratumoral, intratracheal, intravenous 
injection, or liposome-mediate delivery. 
This invention also provides a method for detecting the presence of human 
chromosomal region 17q21 in a sample of genomic DNA which comprises (a) 
contacting the sample with the isolated nucleic acid which encodes a 
wildtype human Beclin, under conditions permitting formation of a complex 
between any genomic DNA present in the sample that is complementary to 
such nucleic acid, and (b) detecting the presence of any complex formed in 
step (a), the presence of such a complex indicating the human chromosomal 
region 17q21 is present in the sample. Further, one may contacting the 
sample with an oligonucleotide, which is capable of specifically 
hybridizing with a unique sequence of nucleotides within a nucleic acid 
which encodes a mutant Beclin without hybridizing to any sequence of 
nucleotides within a nucleic acid which encodes a wildtype human Beclin, 
under conditions permitting formation of a complex between any genomic DNA 
present in the sample that is complementary to such oligonucleotide, and 
(b) detecting the presence of any complex formed in step (a), the presence 
of such a complex indicating the human chromosomal region 17q21 is present 
in the sample. The nucleic acid may be labeled with a detectable marker, 
wherein the detectable marker is a radioactive isotope, a fluophor or an 
enzyme. 
This invention also provides a method for detecting a mutant human Beclin 
in a subject which comprises (a) obtaining an appropriate nucleic acid 
sample from the subject, and (b) determining whether the nucleic acid 
sample from step (a) is, or is derived from, a nucleic acid which encodes 
mutant human Beclin so as to thereby detect a mutant human Beclin in the 
subject. 
Various methods of determining whether the nucleic acid sample is, or is 
derived from, a nucleic acid which encodes a mutant human Beclin exist. 
In one example, the nucleic acid sample in step (a) comprises mRNA 
corresponding to the transcript of DNA encoding a mutant Beclin, and 
wherein the determining of step (b) comprises (i) contacting the mRNA with 
the above-described oligonucleotide, which is capable of specifically 
hybridizing with a unique sequence of nucleotides within a nucleic acid 
which encodes a mutant Beclin without hybridizing to any sequence of 
nucleotides within a nucleic acid which encodes a wildtype human Beclin, 
under conditions permitting binding of the mRNA to the oligonucleotide so 
as to form a complex, (ii) isolating the complex so formed, and (iii) 
identifying the mRNA in the isolated complex so as to thereby determine 
whether the mRNA is, or is derived from, a nucleic acid which encodes a 
mutant human Beclin. 
In another example, the determining of step (b) comprises (i) contacting 
the nucleic acid sample of step (a), and the isolated nucleic acid which 
encodes a wildtype human Beclin with restriction enzymes under conditions 
permitting the digestion of the nucleic acid sample, and the isolated 
nucleic acid into distinct, distinguishable pieces of nucleic acid, (ii) 
isolating the pieces of nucleic acid; and (iii) comparing the pieces of 
nucleic acid derived from the nucleic acid sample with the pieces of 
nucleic acid derived from the isolated nucleic acid so as to thereby 
determine whether the nucleic acid sample is, or is derived from, a 
nucleic acid which encodes a mutant human Beclin. 
In another example, the determining of step (b) comprises (i) sequencing 
the nucleic acid sample of step (a); and (ii) comparing the nucleic acid 
sequence of step (i) with the above-described isolated nucleic acid having 
a sequence substantially the same as the sequence designated Seq. I.D. 
No,: 2, so as to thereby determine whether the nucleic acid sample is, or 
is derived from, a nucleic acid which encodes a mutant human Beclin. 
In another example, the nucleic acid sample in step (a) comprises mRNA 
corresponding to the transcript of DNA encoding mutant Beclin, and wherein 
the determining of step (b) comprises (i) translating the mRNA under 
suitable conditions to obtain an amino acid sequence; and (ii) comparing 
the amino acid sequence of step (i) with the above-described isolated 
nucleic acid which as an amino acid sequence designated Seq. I.D. No.: 1 
so as to thereby determine whether the nucleic acid sample is, or is 
derived from, a nucleic acid which encodes a mutant human Beclin. 
In another example, the determining of step (b) comprises (i) amplifying 
the nucleic acid present in the sample of step (a); and (ii) detecting the 
presence of the mutant human Beclin in the resulting amplified nucleic 
acid. 
The above-described methods of determining are well-known to those skilled 
in the art. In a preferred embodiment, the isolated nucleic acid or the 
oligonucleotide is labeled with a detectable marker, wherein the 
detectable marker is a radioactive isotope, a fluorophor or an enzyme. 
Further, in a specific embodiment, the sample comprises blood, tissue or 
sera. 
This invention also provides a method for treating a subject unable to 
control apoptosis in the cells of the subject which comprises introducing 
the isolated nucleic acid of claim 1, into the subject so as to treat the 
subject unable to control apoptosis in the cells of the subject. In a 
specific embodiment, the cells are cancerous. Various method of 
introducing isolated nucleic acids into cells exist and are well-known in 
the art. In one example, one can introduce the isolated nucleic acid by 
(a) recovering cancer cells from the subject, (b) introducing the isolated 
nucleic acid of claim 1 into the cells; and (c) reintroducing the cells of 
step (b) into the subject so as to treat the subject who has cancer. 
This invention also provides a method of treating a subject unable to 
control apoptosis in the cells of the subject comprising administration of 
an effective amount of the above-described pharmaceutical compositions to 
the subject, wherein the administration comprises, topical, oral, aerosol, 
subcutaneous administration, infusion, intralesional, intramuscular, 
intraperitoneal, intratumoral, intratracheal, intravenous injection, or 
liposome-mediate delivery. 
This invention is illustrated in the Experimental Details section which 
follows. These sections are set forth to aid in an understanding of the 
invention but are not intended to, and should not be construed to, limit 
in any way the invention as set forth in the claims which follow 
thereafter. 
EXPERIMENTAL DETAILS 
Cell proliferation and apoptosis may share common pathways. Yet no 
functional links have been identified between cellular genes that inhibit 
apoptosis and cellular genes that inhibit proliferation. To investigate 
the mechanism by which anti-apoptotic genes function, the yeast two hybrid 
system was used to screen an adult mouse brain cDNA library for genes 
encoding proteins that interact with Bcl-2. Both Bcl-2 and its related 
family member, Bcl-x.sub.L, interact with a novel 60 kd protein, Beclin, 
encoded by a gene on a specific region of chromosome 17q21 that is 
postulated to contain a tumor suppressor gene important in sporadic breast 
and ovarian cancer. Mutations in the conserved BH1 domains of Bcl-2 and 
Bcl-x.sub.L that block anti-apoptotic function also disrupt binding with 
Beclin, suggesting that Bcl-2-Beclin and Bcl-x.sub.L -Beclin interactions 
may be important for inhibition of apoptosis. Using Sindbis virus as a 
vector and inducer of apoptosis in mammalian cells, antisense beclin RNA 
partially blocks the death-repressor activity of Bcl-2 and overexpression 
of Beclin induces a proliferative arrest. Thus, Bcl-2 may inhibit 
apoptosis by interacting with a gene that has effects on cellular 
proliferative machinery. 
The common deletion unit, located approximately 60 kb centromeric to BRCA1 
that is postulated to contain an additional tumor suppressor gene 
important for ovarian and possibly breast cancer, contains 12 previously 
identified genes (Friedman, et al., 1995). Six of them are known genes or 
human homologs of other species, gamma tubulin, homolog of D. melanogaster 
enhancer of zeste, pseudogene of HMG17, homolog of Pacific electric ray 
VAT1, glucose-6phosphatase and Ki antigen. The remaining six genes are 
novel genes, one of which is the gene referred to as beclin that is 
described in this invention. 
The mapping of beclin to this common deletion unit on chromosome 17q21, 
coupled with data that Beclin interacts with Bcl-2 and has 
anti-proliferative effects, raises the possibility that Beclin may 
function as a tumor suppressor gene important in ovarian and breast 
cancer. 
EXAMPLE 1 
Yeast Two Hybrid CDNA Library Screen To Isolate Bcl-2-interacting Proteins 
To further understand the mechanism by which bcl-2 protects against 
apoptosis, the yeast two hybrid system was used to screen a mouse brain 
library for complementary cDNAs encoding proteins that bind to Bcl-2. A 
bait plasmid (pGBT9/bcl-2) was constructed by fusing human bcl-2 (lacking 
the C' terminal signal-anchor sequence to ensure translocation to the 
nucleus) to the GAL4 DNA-binding domain, which was cotransformed with an 
oligo(dT) and random hexamer primed adult mouse brain cDNA fusion library 
in a GAL4-activating domain vector, pGAD10. pGBT9/bcl-2 was co-transformed 
with 1.times.10.sup.6 cDNAs from a mouse brain library fused to a GAL-4 
activation domain vector (Clontech), plated onto SD medium lacking 
tryptophan and leucine, and after incubation at 30.degree. C. for 4 days, 
screened for LacZ activity using a colony lift filter assay. Putative 
interacting clones were isolated by manipulation in leuB E. coli, and 
further tested against pGBT9 and control plasmids. Of one million 
transformants, one true positive colony (F1) was identified by the X-Gal 
filter assay. A positive .beta.-gal reaction between pGBT9/bcl-2 and clone 
F1 was obtained within 15-20 minutes. The sequence of the Eco RI insert in 
clone F1 was obtained using Sequenase(.TM.) and by automated dideoxy 
sequencing. Sequencing analysis of the cDNA plasmid rescued from this 
colony revealed a termination codon 42 base pairs downstream from the GAL 
4 activation domain, several predicted short open reading frames between 
nucleotides 124 and 1843, and a longer predicted open reading frame 
spanning from nucleotide 1855 to the 3' end of the insert, suggesting that 
either the 14 amino acid fusion protein was interacting with Bcl-2, or one 
of the downstream open reading frames encoded a protein that contains its 
own activation domain and interacts with Bcl-2. To identify the 
Bcl-2-interacting region of F1, nucleotides 1-1854 and 1855-2500 were 
fused to the GAL4 activation domain in pGAD424 and tested for interactions 
with Bcl-2. Nucleotides 1855-2500, but not 1-1800, encoded a protein that 
specifically interacts with Bcl-2 (Table 1). 
A database search revealed that the sequence of F1:1855-2500 overlapped 
with several clones isolated from a normalized infant human brain cDNA 
library in the Merck EST database as well as clones from human breast 
(GT197) (Rommens, 1995) and human fibroblast cells (B32) (Friedman, 1994). 
Clones GT197 and B32 were both isolated in the generation of transcription 
maps of the breast cancer susceptibility locus on chromosome 17q21 and are 
mapped to a region located approximately 100 kilobases centromeric to the 
gene BRCA1. These clones contain only partial open reading frames of a 
novel gene that encodes a protein with coiled coils. The gene was assigned 
the name beclin, because of the interaction of its encoded protein with 
bcl-2 (becl) and the predicted coiled coil structure of its encoded 
protein (in suffix). The overlapping partial clones in Genbank were 
aligned with the mouse beclin sequence to obtain a predicted sequence of 
the full-length open reading frame for human beclin. Human beclin was 
isolated from a normalized human brain infant cDNA library (Soares, 1994). 
TABLE 1 
__________________________________________________________________________ 
Summary of yeast two-hybrid assay results 
GAL4 BD 
GAL4 AD Empty 
Bcl-2 
Bcl-X.sub.L 
Bcl-X.sub.S 
Bax Lamin 
p53 
__________________________________________________________________________ 
Empty -- -- -- + -- -- -- 
F1 -- + + ND -- -- -- 
F1:1-1855 -- -- -- ND -- -- -- 
F1:1856-2563 
-- + + ND -- -- -- 
(Mus Beclin 1-708) 
Hu Beclin 1-708 
-- + + ND -- -- -- 
Hu Beclin 1-450 
-- + + ND -- -- -- 
Hu Beclin 1-258 
-- -- -- ND -- -- -- 
Hu Beclin 262-450 
-- + + ND -- -- -- 
Hu Beclin 451-708 
-- -- -- ND -- -- -- 
Hu Beclin 1-1383 
-- -- -- ND -- -- -- 
__________________________________________________________________________ 
Additional yeast two hybrid studies were performed to confirm that human 
beclin, like mouse beclin, encodes a protein that interacts with human 
Bcl-2, and to further define the Bcl-2-interacting region of human Beclin 
(see Table 1). Additional clones containing fragments of F1 or human 
beclin fused to the GAL4-activiation domain were constructed using PCR 
primers which incorporated Eco Ri and Sal I restriction sites into the 
forward and reverse primers, respectively. The ability of human beclin to 
bind to Bcl-2 in the yeast two hybrid system maps to amino acids 88-150. 
Interestingly, the coding sequence for this region of Beclin is deleted in 
some human infant brain cDNA clones in the Merck EST database, suggesting 
that Beclin exists in at least two forms--one form that contains a Bcl-2 
binding domain and one form that lacks a Bcl-2 binding domain. 
Sequencing Of Human Beclin. Primers immediately upstream and downstream of 
the predicted open reading frame were used to amplify the coding sequence 
of human beclin from a normalized human infant brain cDNA library (Soares, 
1994). The resulting PCR products from several independent reactions were 
cloned into pCR.sup.TMII and sequenced in both directions using Sequenase 
(US Biochemicals) as well as automated sequencing. The resulting 
nucleotide sequence (FIG. 1B, Seq. I.D. No.: 2) and deduced amino acid 
sequence (FIG. 1A, Sequ. I.D. No.: 1) were used to scan various data banks 
(Genbank, EMBL, SwissProt, PIR) for homologous sequences using the BLAST 
algorithms (Altschul, 1990). The amino acid sequence was also analyzed by 
the PROSITE program to identify functional motifs and by the COILS program 
to identify coiled coil regions (Lupas, 1991). 
Yeast Two Hybrid Analyses of Beclin-Bcl-2 Family Member Interactions. To 
investigate whether Beclin interacts with other Bcl-2 family members that 
positively or negatively regulate apoptosis, bax, bcl-x.sub.S and 
bcl-x.sub.L cDNAs were fused into the GAL4 binding domain vector and 
tested for interactions with Beclin in the yeast two hybrid system. (See 
Table 1). 
The sequences encoding amino acids 1-218 of human bcl-2, 1-212 of 
bcl-X.sub.L, 1-149 of bcl-x.sub.S, and 1-171 of bax were cloned into pGBT9 
in frame with the GAL4-binding domain. To avoid problems with targeting of 
proteins to the nucleus, the sequences encoding C'terminal transmembrane 
domains were omitted. To construct pGBT9/bcl-2, human bcl-2 was amplified 
by PCR from the plasmid pZIP/bcl-2, subcloned into pCR.sup.TMII, and the 
correct sequence of bcl-2 was confirmed prior to cloning an Eco RI-Sal I 
fragment into pGBT9. To construct pGBT9/bcl-x.sub.L, pGBT9/bcl-x.sub.s, 
and pGBT6/Bax, the Eco RI--Xho I fragments were excised from pGEG202 
plasmids previously described (Sato, et al., 1994) and cloned into the Eco 
RI--Sal I sites of pGBT9. Control pGBT9 plasmids containing lamin (pLAM5') 
and p53 (pVA3) inserts were obtained from Clontech. 
The Bcl-x.sub.s GAL4 DB construct activated transcription by itself, and 
therefore could not be tested for interactions with Beclin. The same 
region of Beclin (aa 88-150) that interacted with Bcl-2, also interacted 
with Bcl-x.sub.L (Boise, 1993), a related Bcl-2 family member that 
inhibits apoptosis. In contrast, Beclin did not react with Bax (Oltvai, 
1993), a family member that promotes apoptosis. The selective interaction 
of Beclin with Bcl-2 family members that have death repressor activity 
suggests a possible functional role of Beclin in anti-apoptotic pathways. 
Full-length human Beclin does not interact with Bcl-2 in the yeast 
two-hybrid system. This most likely reflects lack of translocation to the 
nucleus in yeast secondary to association with yeast intracellular 
membranes since full-length human Beclin expressed in mammalian cells is 
associated with the insoluble membrane fraction after cell lysis. 
To evaluate whether Bcl-2-Beclin and Bcl-x.sub.L -Beclin interactions are 
related to the ability of Bcl-2 and Bcl-X.sub.L to inhibit apoptosis, 
pGBT9 vectors were constructed containing bcl-2 and bcl-x.sub.L constructs 
with mutations in the conserved BH1 domain that are known to block death 
repressor activity. A G.fwdarw.A mutation at amino acid position 145 of 
Bcl-2 completely abrogates Bcl-2 death-repressor activity in interleukin-3 
deprivation, .gamma.-irradiation and glucocorticoid-induced apoptosis 
(Yin, 1994), and also blocks Bcl-2 binding to beclin in the yeast two 
hybrid system (Table 2). Similarly, substitutions of amino acids 136-138 
of Bcl-x.sub.L (VNW.fwdarw.AIL) completely abolishes death repressor 
activity in Sindbis virus-induced apoptosis (Cheng, 1996), and also blocks 
Bcl-x.sub.L binding to Beclin. Thus, mutations that block anti-death 
activity of bcl-2 and bcl-x.sub.L also block binding to beclin. 
TABLE 2 
__________________________________________________________________________ 
Effect of BH1 domain mutations on the ability of 
Bcl-2 and Bcl-x.sub.L to bind to beclin in the yeast two-hybrid assay 
Inhibition 
Beclin 
of Apoptosis 
Binding 
__________________________________________________________________________ 
WT BCL-2 
(SEQ. I.D. NO.: 3) 
ELFRDGVNWGRIVAFFEFGG 
+ + 
WT BCL-X.sub.L 
(SEQ. I.D. NO.: 4) 
ELFRDGVNWGRIVAFFSFGG 
+ + 
MT BCL-2 
(SEQ. I.D. NO.: 5) 
A---------- 
- - 
MT BCL-X.sub.L 
(SEQ. I.D. NO.: 6) 
AIL----------- 
- - 
__________________________________________________________________________ 
Oligonucleotide-directed mutagenesis of bcl-2 and bcl-x.sub.L was 
accomplished by a two-step polymerase chain reaction. Mutants were cloned 
into pCR.sup.TM11 and mutations were confirmed by dideoxy sequencing prior 
to cloning into pGBT9 pGBT9/bcl-2 and pGBT9/bcl-x.sub.L mutants were 
cotransformed with fragments of human beclin (1-450, 262-450, 1-708) fused 
to the GAL4-activation domain. Transformants wee screened by 
.beta.-galactosidase assays to determine whether mutations affected Bcl-2 
binding. 
Analysis of Beclin Expression in Mammalian Cells 
Human beclin is predicted to encode a novel 450 amino acid protein, 
containing a coiled coil region with 25-28% homology with myosin-like 
proteins (FIG. 1A). Western blot analysis of lysates prepared from BHK 
cells infected with a Sindbis virus vector that expresses flag 
epitope-tagged Beclin confirms that human beclin encodes a 60 kd protein 
(FIG. 2A). 
To construct the plasmid SIN/flag-beclin, human beclin was amplified by PCR 
from a human brain cDNA library, using primers that incorporated upstream 
and downstream Bst EII sites and an upstream sequence encoding the flag 
epitope. The Bst EII flag-beclin fragment was ligated into the Bst EII 
restriction site of the previously described double subgenomic SIN vector, 
ds633. Recombinant virus stocks were generated from SIN/flag-beclin 
plasmid as described. BHK cells were infected with SIN/flag-beclin or 
control constructs at a multiplicity of infection (MOI) of 1 
plaque-forming unit per cell and harvested 15 hours after infection. 
PROSITE analysis of human beclin identified several potential 
phosphorylation and myristoylation sites, but no other functional sequence 
motifs. RNA blot analysis revealed that expression of beclin mRNA is 
widespread in both mouse and human adult tissues. A beclin-specific probe 
hybridized to a 2.3 kb transcript present at highest levels in human 
skeletal muscle, but at detectable levels in all tissues examined (FIG. 
2B). In some tissues, additional 1.7 and 1.4 kb transcripts were observed, 
suggesting the presence of alternatively spliced transcripts. 
Multiple tissue Northern blots were probed according to manufacturer's 
instructions (Clontech) with a .sup.32 P-labeled 485 base pair probe 
corresponding to nucleotides 1-485 of human or mouse beclin. Equal amount 
of loading (2 .mu.g of polyA) was confirmed by hybridization to a B-actin 
probe. 
To examine the subcellular localization of Beclin in mammalian cells and to 
determine whether Beclin colocalizes with Bcl-2, baby hamster kidney cells 
were transmitted with the plasmids, pSG5/bcl-2 and pSG5/beclin, that 
express Bcl-2 and a flag-epitope tagged Beclin, respectively. 
Immunofluorescence staining with an anti-flag epitope antibody and an 
anti-Bcl-2 antibody revealed that both proteins were expressed in the 
perinuclear membrane/endoplasmic reticulum region (FIG. 2C). Confocal 
laser microscopy confirmed an identical pattern of immunostaining for 
Bcl-2 and Beclin in all cotransfected cells. Thus, Bcl-2 and Beclin 
colocalize in transfected mammalian cells. 
Role of Beclin in Virus-Induced Apoptosis 
Overexpression of many Bcl-2 family members (Boise, 1993; Oltvai, 1993) or 
Bcl-2 interacting proteins (Farrow, Takayama) results in either the 
acceleration or inhibition of apoptosis. The Sindbis virus vector system 
was employed, which has been previously used to study the anti-apoptotic 
function of several Bcl-2 family members (Cheng, 1996), to evaluate the 
effects of beclin overexpression on virus-induced apoptosis. While Bcl-2 
overexpression results in a significant delay in SIN-induced cell death of 
BHK cells (FIG. 3A), neither antisense beclin RNA nor beclin 
overexpression delays or accelerates virus-induced death. Therefore, 
rather than acting as an independent regulator of apoptosis, beclin may be 
a functional component of a pathway that is mechanistically involved in 
the death repressor activity of Bcl-2. 
To test this hypothesis, Beclin was tested to see if it plays a role in the 
ability of Bcl-2 to inhibit virus-induced apoptosis in mammalian cells. A 
bcl-2-transfected rat prostate adenocarcinoma cell line (AT3/bcl-2 cells) 
that is resistant to Sindbis virus-induced apoptosis (Levine, 1996) was 
infected with chimeric Sindbis viruses containing beclin in either the 
sense or antisense orientation. At 72 hours after infection, 77% of cells 
infected with SIN/beclin and 66% of cells infected with a control chimeric 
virus, SIN/CAT were still alive (FIG. 3B). In contrast, only 35% of cells 
infected with SIN/antisense beclin were still alive. The magnitude of this 
increase in cell death is similar to that seen after infection with a 
virus containing bcl-2 antisense RNA. The ability of antisense beclin, 
like antisense bcl-2, to partially inhibit bcl-2 protection against 
Sindbis virus-induced apoptosis demonstrates a functional role for Beclin 
in the death repressor activity of Bcl-2. 
Role of Beclin in Cellular Proliferation 
In the course of the above experiments, an apparent inhibition of cellular 
proliferation in both BHK cells and AT3/bcl-2 cells infected with 
SIN/beclin was observed. The number of AT3/bcl-2 cells 24 hours after 
infection with SIN/beclin was reduced by more than 50% as compared to the 
number of AT3/bcl-2 cells that were mock-infected or infected with SIN/CAT 
(FIG. 4A), whereas no significant differences were observed in AT3/bcl-2 
cell viability among the three groups (FIG. 4B). 
In summary, the yeast two hybrid system was used to isolate a cDNA that 
encodes a predicted coiled coil protein, Beclin, that interacts with 
members of the Bcl-2 family that negatively regulate apoptosis. A 
functional role for Beclin in anti-apoptotic pathways is suggested both by 
Bcl-2 and Bcl-x.sub.L mutational studies showing a correlation between 
disruption of anti-apoptotic function and binding to Beclin, and by 
studies in which beclin antisense RNA partially blocks Bcl-2-mediated 
protection against virus-induced apoptosis. While the function of beclin, 
when expressed at normal levels in mammalian cells, is still unknown, its 
overexpression can inhibit cellular proliferation. These observations are 
consistent with the hypothesis that Bcl-2 may inhibit apoptosis by 
interacting with a gene product that exerts effects on cellular 
proliferative machinery. Furthermore, these findings, coupled with 
previous studies that have mapped beclin transcripts to a breast and 
ovarian cancer susceptibility locus on chromosome 17q21 (Rommens, 1995; 
Friedman, 1994; Friedman, 1995), warrant additional investigation to 
determine whether beclin, and its interactions with Bcl-2, play a role in 
human cancer. 
REFERENCES 
1. Altschul, S. F., et al. (1990) "Basic local alignment search tool." J. 
Mol. Biol. 215: 403-410; 
2. Boehme, S. A. and Lenardo, M. J. (1993) "Propriocidal apoptosis of 
mature T lymphomcytes occurs at S phase of the cell cycle." Eur. J. 
Immunol 23: 1552-1560; 
3. Boise, L. H., et al. (1993) "Bcl-xL, a bcl-2-related gene that functions 
as a dominant regulator of apoptotic cell death." Cell 74: 597-608; 
4. Borner, C. (1996) "Diminished cell proliferation associated with the 
death-protective activity of Bcl-2." J. Biol. Chem. 271: 12695-12698; 
5. Boyd, J., et al. (1994) "Adenovirus E1B 19kDa and Bcl-2 proteins 
interact with a common set of cellular proteins."Cell 79: 341-351; 
6. Buttyan, R. (1991) "Genetic response of prostate cells to androgen 
deprivation: insights into the cellular mechanisms of apoptosis." In Tomei 
L D, Cope F O (eds): "Apoptosis: The Molecular Basis of Cell Death." 
Plainview, N.Y.: Cold Spring Harbor Laboratory Press 157-173; 
7. Cheng, E. H., et al. (1996) "Bax-independent inhibition of apoptosis by 
Bcl-x.sub.L." Nature 379: 554-556. 
8. Clarke, A., et al. (1992) "Requirement for a functional Rb-1 gene in 
murine development." Nature 359: 328-330 
9. Chittenden, T. (1995) "Induction of apoptosis by the Bcl-2 homologue 
Bak." Nature 374: 733-736; 
10. Cropp, C. S, et al. (1993) "Identification of three regions on 
chromosome 17q in primary human breast carcinomas which are frequently 
deleted." Cancer Res. 53: 3382-3385; 
11. Eccles, D. M., et al. (1992) "Early loss heterozygosity on 17q in 
ovarian cancer." Oncogene 7: 2069-2072; 
12. Evan, G. I., et al. (1995) "Apoptosis and the cell cycle." Curr. Opin. 
Cell Biol. 7: 825-834; 
13. Evan, G. I., et al. (1996) "Induction of apoptosis in fibroblasts by 
c-myc protein." Cell 69: 119-128; 
14. Evan, G. I., et al. (1995) "Apoptosis and the cell cycle." Curr. Opin. 
Cell Biol. 7: 825-834; 
15. Farinelli, S. E. and L. A. Greene (1996) "Cell blockers mimosine, 
ciclopirox, and deferoxamine prevent the death of PC12 cells and 
postmitotic sympathetic neurons after removal of trophic support." J. 
Neurosci. 16: 1150-1162; 
16. Farrow, S. N., et al. (1995) "Cloning of a bcl-2 homologue by 
interaction with adenovirus E1B 19K." Nature 374: 731-733; 
17. Fernandez-Sarabia, M. J., et al. (1993) "Bcl-2 associates with the 
ras-related protein R-ras p23." Nature 366: 274-275; 
18. Freeman, R. S., et al. (1994) "Analysis of cell cycle related gene 
expression in post-mitotic neurons: selective induction of cyclin D1 
during prrammed cell death." Neuron 12: 343-355; 
19. Friedman, L. S., et al. (1995) "Twenty-two genes from chromosome 17q21: 
cloning, sequencing and characterization of mutations in breast cancer 
families and tumors." Genomics 25: 256-263; 
20. Friedman, L. S., et al. (1994) "The search for BRCA1." Cancer Res. 54: 
6374-6382; 
21. Futreal, P. A., et al. (1994) "BRCA1 mutations in primary breast and 
ovarian carcinomas." Science 266: 120-122; 
22. Futreal, P. A., et al. "Detection of frequent allelic loss on proximal 
chromosome 17q in sporadic breast carcinoma using microsatellite length 
polymorphisms." Cancer Res. 52: 2624-2627; 
23. Hall, J. M., et al. (1990) "Linkage of early-onset breast cancer to 
chromosome 17q21." Science 250: 1684-1689; 
24. Hanada, M., et al. (1995) "Structure-function analysis of the Bcl-2 
protein." J. Biol. Chem. 270: 11962-11969; 
25. Hockenbery, D., et al. (1993) "Bcl-2 functions in an antioxidant 
pathway to prevent apoptosis." Cell. 75:241-251; 
26. Hosking, L., et al. (1995) "A somatic BRCA1 mutation in an ovarian 
tumour." Nature Genet 9: 343-344; 
27. Jacks, T., et al. (1992) "Effects of an Rb mutation in the mouse." 
Nature 359: 295-300; 
28. Kane, D. J., et al. (1993) "Bcl-2 inhibition of neural cell death: 
Decreased generation of reactive oxygen species." Science. 262:1274-1276. 
29. Kiefer, M. C., et al. (1995) "Modulation of apoptosis by the widely 
distributed Bcl-2 homologue Bak." Nature 374: 736-739; 
30. King, K. L. and Cidlowski, J. A. (1995) "Cell cycle and apoptosis: 
common pathways to life and death." J. Cell. Biochem. 58: 175-180; 
31. Lam, M. et al. (1994) "Evidence that Bcl-2 represses apoptosis by 
regulating endoplasmic reticulum-associated Ca.sup.2+ fluxes." Proc Natl 
Acad Sci USA 91:6569-6573. 
32. Lee, E-H, et al. (1992) "Mice deficient for Rb are nonviable and show 
defects in neurogenesis and haematopoiesis." Nature 359: 288-294; 
33. Levine, B., et al. (1993) "Conversion of lytic to persistent alphavirus 
infection by the bcl-2 cellular oncogene." Nature 361: 739-742; 
34. Lupas, A., et al. (1991) "Predicting Coiled Coils from Protein 
Sequences." Science 252: 1162-1164; 
35. Matzel, S., et al. (1996) "Regulation of cell division cycle 
progression by bcl-2 expression; a potential mechanism for inhibition of 
programmed cell death." J. Exp. Med. 183: 2219-2226; 
36. Miura, M., et al. (1993) Induction of apoptosis in fibroblasts by IL-1B 
converting enzyme, a mammalian homolog of the C. elegans cell death gene 
ced-2. Cell 75:653-660; 
37. Merajver, S. D., et al. (1995) "Somatic mutations in the BRCA1 gene in 
sporadic ovarian tumors." Nature Genet. 9: 439-443; 
38. Miki, Y., et al. (1994) "A strong candidate gene for the breast and 
ovarian cancer susceptibility gene BRCA1." Science 266: 66-71; 
39. Oltvai, Z., et al. (1993) "Bcl-2 heterodimerizes in vivo with a 
conserved homolog, Bax, that accelerates programed cell death." Cell 74: 
609-619; 
40. Park, J. R., and Hockenberry, D. M. (1996) "Bcl-2, a novel regulator of 
apoptosis." J. Cell. Biochem. 60: 12-17; 
41. Qin, X., et al. (1994) "Deregulated transcription factor E2F-1 
expression leads to S-phase entry and p53-mediated apoptosis." Proc Natl 
Acad Sci USA 91: 10918-10922; 
42. Reed, J. C., et al. (1990) "Bcl-2 family proteins; regulators of cell 
death involved in the pathogenesis of cancer and resistance to therapy." 
J. Cell. Biochem. 60: 23-32; 
43. Rommens, J. M., et al. (1995) "Generation of a transcription map at the 
HSD17B locus centromeric to BRCA1 at 17q21." Genomics 28: 530-542; 
44. Russell, S. E. H., et al. (1990) "Allele loss from chromosome 17 in 
ovarian cancer." Oncogene 5: 1581-1583; 
45. Sambrook, et al. (1989) Molecular Cloning--A Laboratory Manual, 2nd 
Edition, Cold Spring Harbor Laboratory Press. 
46. Saito, H., et al. (1993) "Detailed deletion mapping of chromosome 17q 
in ovarian and breast cancers: 2-cM region on 17q21.1 often and commonly 
deleted in tumors." Cancer Res. 53: 3382-3385; 
47. Sato, T., et al. (1994) "Interactions among members of the Bcl-2 
protein family analyzed with a yeast two-hybrid system." Proc. Natl. Acad. 
Sci USA 91: 9238-9242; 
48. Sedlak, T. W., et al. (1995) "Multiple Bcl-2 family members demonstrate 
selective dimerizations with Bax." Proc. Natl. Acad. Sci. USA 92: 
7834-7838; 
49. Soares, M. B., et. al. (1994) "Construction and characterization of a 
normalized cDNA library." Proc. Natl. Acad. Sci USA 91: 9228-9232; 
50. Shan, B. and Lee, W. H. (1994) "Deregulated expression of E2F-1 induces 
S-phase entry and leads to apoptosis." Mol Cell Biol 14: 8166-8173; 
51. Shi, L., et al. (1994) "Premature p34.sup.cdc2 activation required for 
apoptosis." Science 263: 1143-1145; 
52. Szabo, C. I. and King, M. C. (1996) "Inherited breast cancer and 
ovarian cancer." Hum. Mol. Genet. 4 review: 1811-1817; 
53. Takhashi, H., et al. (1995) "Mutation analysis of the BRCA1 gene in 
ovarian cancers." Cancer Res. 55: 2998-3002; 
54. Takayama, S., et al. (1995) "Cloning and functional analysis of BAG-1: 
a novel Bcl-2-binding protein with anti-cell death activity." Cell 80: 
279-284; 
55. Tanaka, N., et al. (1994) "Cellular commitment to oncogene-induced 
transformation or apoptosis is dependent on the transcription factor 
IRF-1." Cell 77: 829-839; 
56. Tangir, J., et al. (1996) "A 400 kb novel deletion unit centromeric to 
the BRCA1 gene in sporadic epithelial ovarian cancer." Oncogene 12: 
735-740; 
57. Tsujimoto, Y., et al. (1985) "Involvement of the bcl-2 gene in human 
follicular lymphoma." Science 228: 1440-1443; 
58. Wang, H-G., et al. (1994) Apoptosis regulation by interaction of Bcl-2 
protein and Raf-1 kinase. Oncoqene 90: 2751-2756. 
59. White, E., et al. (1991) "Adenovirus E1B 19-kilodalton protein 
overcomes the cytotoxicity of E1A proteins." J. Virol. 65: 2968-2978; 
60. Wyllie, A. H., et al. (1987) "Rodent fibroblast tumors expressing human 
myc and ras genes: Growth, metastasis and endogenous oncogene expression." 
Br. J. Cancer 56: 251-259; 
61. Wu, X., and Levine, A. J. (1994) "p53 and E2F-1 cooperate to mediate 
apoptosis." Proc Natl. Acad Sci USA 91: 3602-3606; 
62. Yan-Feng, et al. (1993) "Allelic loss in ovarian cancer. " Int. J. 
Cancer 54: 546-551; 
63. Yang, E., et al. (1995) "Bad, a heterodimeric partner for Bclxl and 
Bcl-2, displaces Bax and promotes cell death." Cell 80: 285-291; 
64. Yin, X. M., et al. (1994) "BH1 and BH2 domains of Bcl-2 are required 
for inhibition of apoptosis and heterodimerization with BAX." Nature 369: 
321-323. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 450 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
MetGluGlySerLysThrSerAsnAsnSerThrMetGlnValSerPhe 
151015 
ValCysGlnArgCysSerGlnProLeuLysLeuAspThrSerPheLys 
202530 
IleLeuAspArgValThrIleGlnGluLeuThrAlaProLeuLeuThr 
354045 
ThrAlaGlnAlaLysProGlyGluThrGlnGluGluGluThrAsnSer 
505560 
GlyGluGluProPheIleGluThrProArgGlnAspGlyValSerArg 
65707580 
ArgPheIleProProAlaArgMetMetSerThrGluSerAlaAsnSer 
859095 
PheThrLeuIleGlyGluValSerAspGlyGlyThrMetGluAsnLeu 
100105110 
SerArgArgLeuLysValThrGlyAspLeuPheAspIleMetSerGly 
115120125 
GlnThrAspValAspHisProLeuCysGluGluCysThrAspThrLeu 
130135140 
LeuAspGlnLeuAspThrGlnLeuAsnValThrGluAsnGluCysGln 
145150155160 
AsnTyrLysArgCysLeuGluIleLeuGluGlnMetAsnGluAspAsp 
165170175 
SerGluGlnLeuGlnMetGluLeuLysGluLeuAlaLeuGluGluGlu 
180185190 
ArgLeuIleGlnGluLeuGluAspValGluLysAsnArgLysIleVal 
195200205 
AlaGluAsnLeuGluLysValGlnAlaGluAlaGluArgLeuAspGln 
210215220 
GluGluAlaGlnTyrGlnArgGluTyrSerGluPheLysArgGlnGln 
225230235240 
LeuGluLeuAspAspGluLeuLysSerValGluAsnGlnMetArgTyr 
245250255 
AlaGlnThrGlnLeuAspLysLeuLysLysThrAsnValPheAsnAla 
260265270 
ThrPheHisIleTrpHisSerGlyGlnPheGlyThrIleAsnAsnPhe 
275280285 
ArgLeuGlyArgLeuProSerValProValGluTrpAsnGluIleAsn 
290295300 
AlaAlaTrpGlyGlnThrValLeuLeuLeuHisAlaLeuAlaAsnLys 
305310315320 
MetGlyLeuLysPheGlnArgTyrArgLeuValProTyrGlyAsnHis 
325330335 
SerTyrLeuGluSerLeuThrAspLysSerLysGluLeuProLeuTyr 
340345350 
CysSerGlyGlyLeuArgPhePheTrpAspAsnLysPheAspHisAla 
355360365 
MetValAlaPheLeuAspCysValGlnGlnPheLysGluGluValGlu 
370375380 
LysGlyGluThrArgPheCysLeuProTyrArgMetAspValGluLys 
385390395400 
GlyLysIleGluAspThrGlyGlySerGlyGlySerTyrSerIleLys 
405410415 
ThrGlnPheAsnSerGluGluGlnTrpThrLysAlaLeuLysPheMet 
420425430 
LeuThrAsnLeuLysTrpGlyLeuAlaTrpValSerSerGlnPheTyr 
435440445 
AsnLys 
450 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1353 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ATGGAAGGGTCTAAGACGTCCAACAACAGCACCATGCAGGTGAGCTTCGTGTGCCAGCGC60 
TGCAGCCAGCCCCTGAAACTGGACACGAGTTTCAAGATCCTGGACCGTGTCACCATCCAG120 
GAACTCACAGCTCCATTACTTACCACAGCCCAGGCGAAACCAGGAGAGACCCAGGAGGAA180 
GAGACTAACTCAGGAGAGGAGCCATTTATTGAAACTCCTCGCCAGGATGGTGTCTCTCGC240 
AGATTCATCCCCCCAGCCAGGATGATGTCCACAGAAAGTGCCAACAGCTTCACTCTGATT300 
GGGGAGGTATCTGATGGCGGCACCATGGAGAACCTCAGCCGAAGACTGAAGGTCACTGGG360 
GACCTTTTTGACATCATGTCGGGCCAGACAGATGTGGATCACCCACTCTGTGAGGAATGC420 
ACAGATACTCTTTTAGACCAGCTGGACACTCAGCTCAACGTCACTGAAAATGAGTGTCAG480 
AACTACAAACGCTGTTTGGAGATCTTAGAGCAAATGAATGAGGATGACAGTGAACAGTTA540 
CAGATGGAGCTAAAGGAGCTGGCACTAGAGGAGGAGAGGCTGATCCAGGAGCTGGAAGAC600 
GTGGAAAAGAACCGCAAGATAGTGGCAGAAAATCTCGAGAAGGTCCAGGCTGAGGCTGAG660 
AGACTGGATCAGGAGGAAGCTCAGTATCAGAGAGAATACAGTGAATTTAAACGACAGCAG720 
CTGGAGCTGGATGATGAGCTGAAGAGTGTTGAAAACCAGATGCGTTATGCCCAGACGCAG780 
CTGGATAAGCTGAAGAAAACCAACGTCTTTAATGCAACCTTCCACATCTGGCACAGTGGA840 
CAGTTTGGCACAATCAATAACTTCAGGCTGGGTCGCCTGCCCAGTGTTCCCGTGGAATGG900 
AATGAGATTAATGCTGCTTGGGGCCAGACTGTGTTGCTGCTCCATGCTCTGGCCAATAAG960 
ATGGGTCTGAAATTTCAGAGATACCGACTTGTTCCTTACGGAAACCATTCATATCTGGAG1020 
TCTCTGACAGACAAATCTAAGGAGCTGCCGTTATACTGTTCTGGGGGGTTGCGGTTTTTC1080 
TGGGACAACAAGTTTGACCATGCAATGGTGGCTTTCCTGGACTGTGTGCAGCAGTTCAAA1140 
GAAGAGGTTGAGAAAGGCGAGACACGTTTTTGTCTTCCCTACAGGATGGATGTGGAGAAA1200 
GGCAAGATTGAAGACACAGGAGGCAGTGGCGGCTCCTATTCCATCAAAACCCAGTTTAAC1260 
TCTGAGGAGCAGTGGACAAAAGCTCTCAAGTTCATGCTGACGAATCTTAAGTGGGGTCTT1320 
GCTTGGGTGTCCTCACAATTTTATAACAAATGA1353 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
GluLeuPheArgAspGlyValAsnTrpGlyArgIleValAlaPhePhe 
151015 
GluPheGlyGly 
20 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GluLeuPheArgAspGlyValAsnTrpGlyArgIleValAlaPhePhe 
151015 
SerPheGlyGly 
20 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
GluLeuPheArgAspGlyValAsnTrpAlaArgIleValAlaPhePhe 
151015 
GluPheGlyGly 
20 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(iii) HYPOTHETICAL: NO 
(iv) ANTI-SENSE: NO 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GluLeuPheArgAspGlyAlaIleLeuGlyArgIleValAlaPhePhe 
151015 
SerPheGlyGly 
20 
__________________________________________________________________________