The present invention relates to a novel BCSG1 protein. In particular,
isolated nucleic acid molecules are provided encoding the human
BCSG1 protein. BCSG1 polypeptides are also provided as are vectors,
host cells and recombinant methods for producing the same. Also
provided are diagnostic methods for detecting breast cancer.
What is claimed is:
1. An isolated polynucleotide comprising a nucleic acid encoding
amino acids 2 to 127 of SEQ ID NO:2.
2. The isolated polynucleotide of claim 1, comprising nucleotide
15 to 392 of SEQ ID NO:1.
3. The isolated polynucleotide of claim 1, comprising a nucleic
acid encoding amino acids 1 to 127 of SEQ ID NO:2.
4. The isolated polynucleotide of claim 3, comprising nucleotide
12 to 392 of SEQ ID NO:1.
5. The isolated polynucleotide of claim 1, which is DNA.
6. The isolated polynucleotide of claim 1, which is RNA.
7. The isolated polynucleotide of claim 1, further comprising a
8. The isolated polynucleotide of claim 7, wherein said heterologous
polynucleotide encodes a heterologous polypeptide.
9. A method of producing a vector that comprises inserting the
isolated polynucleotide of claim 1 into a vector.
10. A vector comprising the isolated polynucleotide of claim 1.
11. The vector of claim 10, wherein said polynucleotide is operably
associated with a heterologous regulatory sequence.
12. An isolated host cell comprising the isolated polynucleotide
of claim 1.
13. The host cell of claim 12, wherein said isolated polynucleotide
is operably associated with a heterologous regulatory sequence.
14. A method of producing a polypeptide that comprises culturing
the host cell of claim 13 under conditions such that said polypeptide
is expressed, and recovering said polypeptide.
15. A composition comprising the isolated polynucleotide of claim
1 and a pharmaceutically acceptable carrier.
16. An isolated polynucleotide comprising a nucleic acid encoding
the complete amino acid sequence encoded by the cDNA clone of ATCC
Deposit No. 97856.
17. The isolated polynucleotide of claim 16, which is DNA.
18. The isolated polynucleotide of claim 16, which is RNA.
19. The isolated polynucleotide of claim 16, further comprising
a heterologous polynucleotide.
20. The isolated polynucleotide of claim 19, wherein said heterologous
polynucleotide encodes a heterologous polypeptide.
21. A method of producing a vector that comprises inserting the
isolated polynucleotide of claim 16 into a vector.
22. A vector comprising the isolated polynucleotide of claim 16.
23. The vector of claim 22, wherein said polynucleotide is operably
associated with a heterologous regulatory sequence.
24. An isolated host cell comprising the isolated polynucleotide
of claim 16.
25. The host cell of claim 24, wherein said isolated polynucleotide
is operably associated with a heterologous regulatory sequence.
26. A method of producing a polypeptide that comprises culturing
the host cell of claim 25 under conditions such that said polypeptide
is expressed, and recovering said polypeptide.
27. A composition comprising the isolated polypeptide of claim
16 and a pharmaceutically acceptable carrier.
28. An isolated polynucleotide fragment of SEQ ID NO:1 consisting
of at least 100 contiguous nucleotides of the coding region of SEQ
ID NO:1 or the complete complement thereof.
29. The isolated polynucleotide fragment of claim 28, consisting
of at least 250 contiguous nucleotides of the coding region of SEQ
ID NO:1 or the complement thereof.
30. The isolated polynucleotide of claim 28, which is DNA.
31. The isolated polynucleotide of claim 28, which is RNA.
32. A method of producing a vector that comprises inserting the
isolated polynucleotide of claim 28 into a vector.
33. A vector comprising the isolated polynucleotide of claim 28.
34. An isolated host cell comprising the isolated polynucleotide
of claim 28.
35. An isolated polynucleotide, encoding a fragment of SEQ ID NO:2
selected from the group consisting of: (a) a polypeptide consisting
of at least amino acids 94 to 107 of SEQ ID NO:2; and (b) a polypeptide
consisting of at least amino acids 120 to 127 of SEQ ID NO:2.
36. The isolated polynucleotide of claim 35, wherein said polypeptide
37. The isolated polynucleotide of claim 35, wherein said polypeptide
38. The isolated polynucleotide of claim 35, which is DNA.
39. The isolated polynucleotide of claim 35, which is RNA.
40. The isolated polynucleotide of claim 35, further comprising
a heterologous polynucleotide.
41. The isolated polynucleotide of claim 40, wherein said heterologous
polynucleotide encodes a heterologous polypeptide.
42. A method of producing a vector that comprises inserting the
isolated polynucleotide of claim 35 into a vector.
43. A vector comprising the isolated polynucleotide of claim 35.
44. The vector of claim 43, wherein said polynucleotide is operably
associated with a heterologous regulatory sequence.
45. An isolated host cell comprising the isolated polynucleotide
of claim 33.
46. The host cell of claim 45, wherein said isolated polynucleotide
is operably associated with a heterologous regulatory sequence.
47. A method of producing a polypeptide that comprises culturing
the host cell of claim 46 under conditions such that said polypeptide
is expressed, and recovering said polypeptide.
48. A composition comprising the isolated polynucleotide of claim
35 and a pharmaceutically acceptable carrier.
FIELD OF THE INVENTION
The present invention relates to a novel breast cancer specific
marker. More specifically, isolated nucleic acid molecules are provided
encoding a human breast cancer specific gene 1 (BCSG1). BCSG1 polypeptides
are also provided, as are vectors, host cells and recombinant methods
for producing the same. Also provided are diagnostic methods for
detecting breast cancer. The invention further provides an isolated
BCSG1 polypeptide having an amino acid sequence encoded by a polynucleotide
More than 190,000 new cases of breast cancer are diagnosed in the
United States every year, with incidence increasing by approximately
1% annually (Goldhirsch, A., JNCI97:1141 1145 (1995); Emster, V.
L., et al., JAMA 275:913 918 (1996)). Studies linked to the discovery
of new genetic markers and additional risk factors could provide
new information that fits into the complex patient management issues
surrounding breast cancer. Many new prognostic and predictive factors
have been proposed and studied for breast cancer. HER 2/neu positive
tumors respond poorly to endocrine treatment (Allred D. C., et al.,
J. Clin Oncol. 10:599 605 (1992); Gusterson B. A., et al., J. Clin
Oncol. 10:1049 56 (1992)). p53 alteration has an indication of poorer
prognosis and poor response to tamoxifen (Bergh J., et al., Nature
Medicine 10: 1029 34 (1995); Elledge R. M., et al., Breast Cancer
Res Treat 27:95 102 (1993)). The lack of Nm23 expression has an
indicative value of metastatic potential and poor prognosis in invasive
ductal carcinoma (Steeg P. S., et al., Breast Cancer Res Treat 25:175
87 (1993)). Cathepsin D, a protease suggested to have a role in
breast cancer, appears to affect the potential for invasive growth
(Velculescu, V. E., et al., Science 270:484 7 (1995); Schena, M.,
et al., Science 270:467 70 (1995); M. L. Angerer & R. C. Angerer,
In: In situ hybridization, D. Rickwood and B. D. Hames (ed.). London:
LRL Press., (1992), pp. 15 32; Ferno M., et al., Eur J. Cancer 30A:2042
8 (1994)). Positive immunostaining of tumor sections with Factor
VIII antibodies seems to be a marker for angiogenesis (Klijn J.
G. M., et al., Breast Cancer 18:165 98 (1993); Harris A. L., et
al., Eur J. Cancer 31A:831 2 (1995); Gasparini G., et al., JNCI
85:1206 19 (1993) (errata JNCI 85:1605 (1993))). It has been postulated
that these tumors are targets for anti-angiogenesis drug treatment.
Expression of the mdr-1 gene is proposed to be an indicator of multidrug
resistance (Harris A. L., et al., Eur J. Cancer 31A:831 2 (1995);
Gasparini G., et al., JNCI 85:1206 19 (1993) (errata JNCI 85:1605
(1993))). Poor response to endocrine therapy has been indicated
for uPA/PAI-1, a plasminogen activator/inhibitor (Foekens J. A.,
et al., JNCI 87:751 6 (1995)). Also receiving major attention are
the familial breast cancer related genes, BRCA1 and BRCA2 (Miki,
Y., et al., Science 266:66 71 (1994); Wooster, R., et al., Science
265:2088 2090 (1994); Futreal, P. A., et al., Science 266:120 122
Thus, the onset and progression of breast cancer is accompanied
by multiple genetic changes that result in qualitative and quantitative
alterations in individual gene expression (Porter-Jordan, K. &
Lippman, M. E., Hematol. Oncol. Clin. N. Am. 8:73 100 (1994)). Many
of these quantitative genetic changes may manifest themselves as
alterations in the cellular complement of novel transcribed mRNAs.
Identification of these mRNAs could provide clinically useful information
for patient management and prognosis while enhancing our understanding
of breast cancer pathogenesis.
Identification of quantitative changes in gene expression that
occur in the malignant mammary gland may yield novel molecular markers
which may be useful in the diagnosis and treatment of human breast
cancer. Several differential cloning methods, such as differential
display polymerase chain reaction and subtractive hybridization,
have been used to identify the genes differentially expressed in
breast cancer biopsies, as compared to normal breast tissue controls
(Watson, M. A. & Fleming, T. P., Cancer Res. 54:4598 4602 (1994);
Sager, R., et al., FASEB J. 7:964 970 (1993); Chen, Z. & Sager,
R., Mol. Med. 1:153 160 (1995); Zhang, M., et al., Cancer Res. 55:2537
2541 (1995); Zou, Z., et al., Science 263:526 529)). However, these
investigations have involved the relatively time- and labor-intensive
steps of subcloning, library screening, and cDNA sequencing of individual
genes (Sager, R., et al., FASEB J. 7:964 970 (1993); Liang, P.,
et al., Cancer Res. 52:6966 6968 (1992)).
Although pathological endpoints such as tumor size, lymph node
status and status of estrogen receptor and progesterone receptor
remain the most useful guides in prognosis and selecting treatment
strategies for breast cancer (Manning, D. L., et al., Acta Oncol.
34:641 646 (1995)), there is still a need to further investigate
the molecular mechanisms that determine the properties of an individual
tumor e.g., probability of metastasis. While numerous prognostic
factors have been identified, few have contributed to defining clinical
response to therapy.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules
comprising a polynucleotide encoding the BCSG1 polypeptide having
the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino
acid sequence encoded by the cDNA clones deposited in a bacterial
host as ATCC Deposit Number 97175 on Jun. 2, 1995 or as ATCC Deposit
Number 97856 on Jan. 23, 1997.
The present invention also relates to recombinant vectors, which
include the isolated nucleic acid molecules of the present invention,
and to host cells containing the recombinant vectors, as well as
to methods of making such vectors and host cells and for using them
for production of BCSG1 polypeptides or peptides by recombinant
In accordance with another aspect of the present invention, there
is provided a method of and products for diagnosing breast cancer
metastases by detecting an altered level of a polypeptide corresponding
to the breast specific genes of the present invention in a sample
derived from a host, whereby an elevated level of the polypeptide
indicates a breast cancer diagnosis.
The present invention further relates to antibodies specific to
the polypeptides of the present invention, which may be employed
to detect breast cancer cells or breast cancer metastasis.
The polynucleotides and polypeptides described herein are useful
as markers for breast cancer.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid
(SEQ ID NO:2) sequences of BCSG1. The protein has a deduced molecular
weight of about 14.2 kDa. The predicted amino acid sequence of the
BCSG1 protein is also shown.
FIG. 2 shows the differential cDNA sequencing approach. Messenger
RNAs from normal and diseased tissues were extracted and used for
making the cDNA libraries. These libraries are searched by EST method
involving automated DNA sequence analysis of randomly selected cDNA
clones. The ESTs with overlapping sequences were grouped into unique
EST groups. Each unique EST group, which does not overlap to each
other in sequence, was analyzed for its relative expression by examining
the number of expressed individual EST in the libraries of normal
vs diseased tissues. Three EST groups are listed. Blue EST group
represents gene that is equally expressed in both libraries. Green
EST group represents gene that is more expressed in normal library
compared to diseased library. Red EST group represent gene that
is more expressed in diseased library compared to normal library.
FIG. 3 shows a schematic representation of the pHE4-5 expression
vector (SEQ ID NO:10) and the subcloned BSCG-1 cDNA coding sequence.
The locations of the kanamycin resistance marker gene, the BSCG-1
coding sequence, the oriC sequence, and the lacIq coding sequence
FIG. 4 shows the nucleotide sequence of the regulatory elements
of the pHE promoter (SEQ ID NO:11). The two lac operator sequences,
the Shine-Delgarno sequence (S/D), and the terminal HindIII and
NdeI restriction sites (italicized) are indicated.
The present invention provides isolated nucleic acid molecules
comprising a polynucleotide encoding a BCSG1 polypeptide having
the amino acid sequence shown in FIG. 1 (SEQ ID NO:2), which was
determined by sequencing a cloned cDNA. The BCSG1 protein of the
present invention shares sequence homology with human AD amyloid.
The nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) was obtained
by sequencing the 184,497 clone, which as deposited on Jan. 23,
1997 at the American Type Culture Collection, Patent Depository,
10801 University Boulevard, Manassas, Va. 20110-2209; and given
accession number 97856. The deposited clone is contained in the
pBluescript SK(-) plasmid (Stratagene, La Jolla, Calif.). The BSCG-1
gene was also deposited on Jun. 2, 1995 at the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Md. 20852, and given
accession number 97175.
Nucleic Acid Molecules
Unless otherwise indicated, all nucleotide sequences determined
by sequencing a DNA molecule herein were determined using an automated
DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.),
and all amino acid sequences of polypeptides encoded by DNA molecules
determined herein were predicted by translation of a DNA sequence
determined as above. Therefore, as is known in the art for any DNA
sequence determined by this automated approach, any nucleotide sequence
determined herein may contain some errors. Nucleotide sequences
determined by automation are typically at least about 90% identical,
more typically at least about 95% to at least about 99.9% identical
to the actual nucleotide sequence of the sequenced DNA molecule.
The actual sequence can be more precisely determined by other approaches
including manual DNA sequencing methods well known in the art. As
is also known in the art, a single insertion or deletion in a determined
nucleotide sequence compared to the actual sequence will cause a
frame shift in translation of the nucleotide sequence such that
the predicted amino acid sequence encoded by a determined nucleotide
sequence will be completely different from the amino acid sequence
actually encoded by the sequenced DNA molecule, beginning at the
point of such an insertion or deletion.
Using the information provided herein, such as the nucleotide sequence
in FIG. 1, a nucleic acid molecule of the present invention encoding
a BCSG1 polypeptide may be obtained using standard cloning and screening
procedures, such as those for cloning cDNAs using mRNA as starting
material. Illustrative of the invention, the nucleic acid molecule
described in FIG. 1 (SEQ ID NO:1) was discovered in a cDNA library
derived from breast cancer. The gene was also identified in cDNA
libraries from brain tissue. The determined nucleotide sequence
of the BCSG1 cDNA of FIG. 1 (SEQ ID NO:1) contains an open reading
frame encoding a protein of 127 amino acid residues, with an initiation
codon at positions 12 14 of the nucleotide sequence in FIG. 1 (SEQ
ID NO:1), and a deduced molecular weight of about 14.2 kDa. The
BCSG1 protein shown in FIG. 1 (SEQ ID NO:2) is about 54% identical
to non-A.beta. fragment of human Alzheimer's disease (AD) amyloid
As one of ordinary skill would appreciate, due to the possibilities
of sequencing errors, the predicted BCSG1 polypeptide encoded by
the deposited cDNA comprises about 127 amino acids, but may be anywhere
in the range of 110 140 amino acids.
As indicated, nucleic acid molecules of the present invention may
be in the form of RNA, such as mRNA, or in the form of DNA, including,
for instance, cDNA and genomic DNA obtained by cloning or produced
synthetically. The DNA may be double-stranded or single-stranded.
Single-stranded DNA or RNA may be the coding strand, also known
as the sense strand, or it may be the non-coding strand, also referred
to as the anti-sense strand.
By "isolated" nucleic acid molecule(s) is intended a
nucleic acid molecule, DNA or RNA, which has been removed from its
native environment. For example, recombinant DNA molecules contained
in a vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include recombinant
DNA molecules maintained in heterologous host cells or purified
(partially or substantially) DNA molecules in solution. Isolated
RNA molecules include in vivo or in vitro RNA transcripts of the
DNA molecules of the present invention. Isolated nucleic acid molecules
according to the present invention further include such molecules
Isolated nucleic acid molecules of the present invention include
DNA molecules comprising an open reading frame (ORF) shown in FIG.
1 (SEQ ID NO:1) and DNA molecules which comprise a sequence substantially
different from those described above but which, due to the degeneracy
of the genetic code, still encode the BCSG1 protein. Of course,
the genetic code is well known in the art. Thus, it would be routine
for one skilled in the art to generate such degenerate variants.
SEQ ID NO:12 is a full length cDNA sequence of breast specific gene
1 of the present invention.
In another aspect, the invention provides isolated nucleic acid
molecules encoding the BCSG1 polypeptide having an amino acid sequence
encoded by the cDNA clone contained in the plasmid deposited as
ATCC Deposit No. 97856 on Jan. 23, 1997 or contained in the plasmid
deposited as ATCC Deposit No. 97175 on Jun. 2, 1995. The invention
further provides an isolated nucleic acid molecule having the nucleotide
sequence shown in FIG. 1 (SEQ ID NO:1) or the nucleotide sequence
of the BCSG1 cDNA contained in the above-described deposited clone,
the full-length BCSG1 polypeptide lacking the N-terminal methionine
or a nucleic acid molecule having a sequence complementary to one
of the above sequences. Such isolated molecules, particularly DNA
molecules, are useful as probes for gene mapping, by in situ hybridization
with chromosomes, and for detecting expression of the BCSG1 gene
in human tissue, for instance, by Northern blot analysis.
The present invention is further directed to fragments of the isolated
nucleic acid molecules described herein. By a fragment of an isolated
nucleic acid molecule having the nucleotide sequence of the deposited
cDNA or the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) is
intended fragments at least about 15 nt, and more preferably at
least about 20 nt, still more preferably at least about 30 nt, and
even more preferably, at least about 40 nt in length which are useful
as diagnostic probes and primers as discussed herein. Of course,
larger fragments 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,
300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550 nt in length
are also useful according to the present invention as are fragments
corresponding to most, if not all, of the nucleotide sequence of
the deposited cDNA or as shown in FIG. 1 (SEQ ID NO:1). By a fragment
at least 20 nt in length, for example, is intended fragments which
include 20 or more contiguous bases from the nucleotide sequence
of the deposited cDNA or the nucleotide sequence as shown in FIG.
1 (SEQ ID NO:1).
Preferred nucleic acid fragments of the present invention include
nucleic acid molecules encoding epitope-bearing portions of the
BCSG1 protein. In particular, such nucleic acid fragments of the
present invention include nucleic acid molecules encoding: a polypeptide
comprising amino acid residues from about 94 to about 107 in FIG.
1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from
about 120 to about 127 in FIG. 1 (SEQ ID NO:2). The inventors have
determined that the above polypeptide fragments are antigenic regions
of the BCSG1 protein. Methods for determining other such epitope-bearing
portions of the BCSG1 protein are described in detail below.
In another aspect, the invention provides an isolated nucleic acid
molecule comprising a polynucleotide which hybridizes under stringent
hybridization conditions to a portion of the polynucleotide in a
nucleic acid molecule of the invention described above, for instance,
the cDNA clones contained in ATCC Deposits 97856 or 97175. By "stringent
hybridization conditions" is intended overnight incubation
at 42.degree. C. in a solution comprising: 50% formamide, 5.times.SSC
(750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6), 5.times. Denhardt's solution, 10% dextran sulfate, and 20
mg/ml denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C.
By a polynucleotide which hybridizes to a "portion" of
a polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more preferably
at least about 20 nt, still more preferably at least about 30 nt,
and even more preferably about 30 70 nt of the reference polynucleotide.
These are useful as diagnostic probes and primers as discussed above
and in more detail below.
By a portion of a polynucleotide of "at least 20 nt in length,"
for example, is intended 20 or more contiguous nucleotides from
the nucleotide sequence of the reference polynucleotide (e.g., the
deposited cDNA or the nucleotide sequence as shown in FIG. 1 (SEQ
ID NO:1)). Of course, a polynucleotide which hybridizes only to
a poly A sequence (such as the 3' terminal poly(A) tract of the
BCSG1 cDNA shown in FIG. 1 (SEQ ID NO:1)), or to a complementary
stretch of T (or U) resides, would not be included in a polynucleotide
of the invention used to hybridize to a portion of a nucleic acid
of the invention, since such a polynucleotide would hybridize to
any nucleic acid molecule containing a poly (A) stretch or the complement
thereof (e.g., practically any double-stranded cDNA clone).
As indicated, nucleic acid molecules of the present invention which
encode a BCSG1 polypeptide may include those encoding the amino
acid sequence of the polypeptide, by itself; the coding sequence
for the polypeptide and additional sequences, such as those encoding
an amino acid leader or secretory sequence, such as a pre-, or pro-
or prepro-protein sequence; the coding sequence of the polypeptide,
with or without the aforementioned additional coding sequences,
together with additional, non-coding sequences, including for example,
but not limited to introns and non-coding 5' and 3' sequences, such
as the transcribed, non-translated sequences that play a role in
transcription, mRNA processing, including splicing and polyadenylation
signals, for example--ribosome binding and stability of mRNA; an
additional coding sequence which codes for additional amino acids,
such as those which provide additional functionalities. Thus, the
sequence encoding the polypeptide may be fused to a marker sequence,
such as a sequence encoding a peptide which facilitates purification
of the fused polypeptide. In certain preferred embodiments of this
aspect of the invention, the marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (Qiagen, Inc.),
among others, many of which are commercially available. As described
in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821 824 (1989), for
instance, hexa-histidine provides for convenient purification of
the fusion protein. The "HA" tag is another peptide useful
for purification which corresponds to an epitope derived from the
influenza hemagglutinin protein, which has been described by Wilson
et al., Cell 37: 767 (1984). As discussed below, other such fusion
proteins include the BCSG1 fused to Fc at the N- or C-terminus.
The present invention further relates to variants of the nucleic
acid molecules of the present invention, which encode portions,
analogs or derivatives of the BCSG1 protein. Variants may occur
naturally, such as a natural allelic variant. By an "allelic
variant" is intended one of several alternate forms of a gene
occupying a given locus on a chromosome of an organism. Genes II,
Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally
occurring variants may be produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions,
deletions or additions, which may involve one or more nucleotides.
The variants may be altered in coding regions, non-coding regions,
or both. Alterations in the coding regions may produce conservative
or non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions, additions
and deletions, which do not alter the properties and activities
of the BCSG1 protein or portions thereof. Also especially preferred
in this regard are conservative substitutions.
Further embodiments of the invention include isolated nucleic acid
molecules comprising a polynucleotide having a nucleotide sequence
at least 90% identical, and more preferably at least 95%, 96%, 97%,
98% or 99% identical to (a) a nucleotide sequence encoding the BCSG1
polypeptide having the amino acid sequence in FIG. 1 (SEQ ID NO:2);
(b) a nucleotide sequence encoding the polypeptide having the amino
acid sequence in SEQ ID NO:2, but lacking the N-terminal methionine;
(c) a nucleotide sequence encoding the BCSG1 polypeptide having
the amino acid sequence encoded by the cDNA clones contained in
ATCC Deposit Nos. 97856 or 97175; or (d) a nucleotide sequence complementary
to any of the nucleotide sequences in (a), (b) or (c).
By a polynucleotide having a nucleotide sequence at least, for
example, 95% "identical" to a reference nucleotide sequence
encoding a BCSG1 polypeptide is intended that the nucleotide sequence
of the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to five point mutations
per each 100 nucleotides of the reference nucleotide sequence encoding
the BCSG1 polypeptide. In other words, to obtain a polynucleotide
having a nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide,
or a number of nucleotides up to 5% of the total nucleotides in
the reference sequence may be inserted into the reference sequence.
These mutations of the reference sequence may occur at the 5' or
3' terminal positions of the reference nucleotide sequence or anywhere
between those terminal positions, interspersed either individually
among nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule
is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) or to the
nucleotides sequence of the deposited cDNA clone can be determined
conventionally using known computer programs such as the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science Drive,
Madison, Wis. 53711. Bestfit uses the local homology algorithm of
Smith and Waterman, Advances in Applied Mathematics 2: 482 489 (1981),
to find the best segment of homology between two sequences. When
using Bestfit or any other sequence alignment program to determine
whether a particular sequence is, for instance, 95% identical to
a reference sequence according to the present invention, the parameters
are set, of course, such that the percentage of identity is calculated
over the full length of the reference nucleotide sequence and that
gaps in homology of up to 5% of the total number of nucleotides
in the reference sequence are allowed.
The present application is directed to nucleic acid molecules at
least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid
sequence shown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid sequence
of the deposited cDNA, irrespective of whether they encode a polypeptide
having BCSG1 activity. This is because even where a particular nucleic
acid molecule does not encode a polypeptide having BCSG1 activity,
one of skill in the art would still know how to use the nucleic
acid molecule, for instance, as a hybridization probe or a polymerase
chain reaction (PCR) primer. Uses of the nucleic acid molecules
of the present invention that do not encode a polypeptide having
BCSG1 activity include, inter alia, (1) isolating the BCSG1 gene
or allelic variants thereof in a cDNA library; (2) in situ hybridization
(e.g., "FISH") to metaphase chromosomal spreads to provide
precise chromosomal location of the BCSG1 gene, as described in
Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon
Press, New York (1988); and Northern Blot analysis for detecting
BCSG1 mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having sequences
at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic
acid sequence shown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid
sequence of the deposited cDNA which do, in fact, encode a polypeptide
having BCSG1 protein activity. By "a polypeptide having BCSG1
activity" is intended polypeptides exhibiting activity similar,
but not necessarily identical, to an activity of the BCSG1 protein
of the invention, as measured in a particular biological assay.
BCSG1 protein is believed to be involved with apoptosis. BCSG1 protein
activity can be measured using assays that measure apoptosis. For
example, human breast cancer cells cultured on Lab-Tek chamber slides
(Nunc, Inc.) are treated with or without recombinant BCSG1 protein
or a candidate BCSG1 protein. The cells are then treated with several
concentrations of an apoptotic inducer, such as adriamycin. Apoptosis
is compared between the treated and control cells where DNA fragmentation
is the criteria for apoptotic death using the following assay. At
various time points after the adriamycin treatment, adherent cells
are stained with DNA-specific fluorochrome diamino-2 phenylindole
(Boehringer Mannheim) in a 1 .mu.g/ml methanol solution. Cells are
counted within 20 minutes of staining on a Zeiss Axiophot epiflouresence
microscope. Experiments are performed in triplicate with at least
150 cells scored at each point. Fragmented or condensed nuclei are
scored as apoptotic. Intact or mitotic nuclei are scored as normal.
Of course, due to the degeneracy of the genetic code, one of ordinary
skill in the art will immediately recognize that a large number
of the nucleic acid molecules having a sequence at least 90%, 95%,
96%, 97%, 98%, or 99% identical to the nucleic acid sequence of
the deposited cDNA or the nucleic acid sequence shown in FIG. 1
(SEQ ID NO:1) will encode a polypeptide "having BCSG1 protein
activity." In fact, since degenerate variants of these nucleotide
sequences all encode the same polypeptide, this will be clear to
the skilled artisan even without performing the above described
comparison assay. It will be further recognized in the art that,
for such nucleic acid molecules that are not degenerate variants,
a reasonable number will also encode a polypeptide having BCSG1
protein activity. This is because the skilled artisan is fully aware
of amino acid substitutions that are either less likely or not likely
to significantly effect protein function (e.g., replacing one aliphatic
amino acid with a second aliphatic amino acid).
For example, guidance concerning how to make phenotypically silent
amino acid substitutions is provided in Bowie, J. U. et al., "Deciphering
the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,"
Science 247:1306 1310 (1990), wherein the authors indicate that
proteins are surprisingly tolerant of amino acid substitutions.
Vectors and Host Cells
The present invention also relates to vectors which include the
isolated DNA molecules of the present invention, host cells which
are genetically engineered with the recombinant vectors, and the
production of BCSG1 polypeptides or fragments thereof by recombinant
The polynucleotides may be joined to a vector containing a selectable
marker for propagation in a host. Generally, a plasmid vector is
introduced in a precipitate, such as a calcium phosphate precipitate,
or in a complex with a charged lipid. If the vector is a virus,
it may be packaged in vitro using an appropriate packaging cell
line and then transduced into host cells.
The DNA insert should be operatively linked to an appropriate promoter,
such as the phage lambda PL promoter, the E. coli lac, trp and tac
promoters, the SV40 early and late promoters and promoters of retroviral
LTRs, to name a few. Other suitable promoters will be known to the
skilled artisan. The expression constructs will further contain
sites for transcription initiation, termination and, in the transcribed
region, a ribosome binding site for translation. The coding portion
of the mature transcripts expressed by the constructs will preferably
include a translation initiating at the beginning and a termination
codon (UAA, UGA or UAG) appropriately positioned at the end of the
polypeptide to be translated.
As indicated, the expression vectors will preferably include at
least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include, but are not limited to, bacterial cells, such as
E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and Spodoptera
Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells;
and plant cells. Appropriate culture mediums and conditions for
the above-described host cells are known in the art.
In addition to the use of expression vectors in the practice of
the present invention, the present invention further includes novel
expression vectors comprising operator and promoter elements operatively
linked to nucleotide sequences encoding a protein of interest. One
example of such a vector is pHE4-5 which is described in detail
As summarized in FIGS. 3 and 4, components of the pHE4-5 vector
(SEQ ID NO:10) include: 1) a neomycinphosphotransferase gene as
a selection marker, 2) an E. coli origin of replication, 3) a T5
phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno
sequence, 6) the lactose operon repressor gene (lacIq). The origin
of replication (oriC) is derived from pUC19 (LTI, Gaithersburg,
Md.). The promoter sequence and operator sequences were made synthetically.
Synthetic production of nucleic acid sequences is well known in
the art. CLONTECH 95/96 Catalog, pages 215 216, CLONTECH, 1020 East
Meadow Circle, Palo Alto, Calif. 94303. A nucleotide sequence encoding
BSCG-1 (SEQ ID NO:1), is operatively linked to the promoter and
operator by inserting the nucleotide sequence between the NdeI and
Asp718 sites of the pHE4-5 vector.
As noted above, the pHE4-5 vector contains a lacIq gene. LacIq
is an allele of the lacI gene which confers tight regulation of
the lac operator. Amann, E. et al., Gene 69:301 315 (1988); Stark,
M., Gene 51:255 267 (1987). The lacIq gene encodes a repressor protein
which binds to lac operator sequences and blocks transcription of
down-stream (i.e., 3') sequences. However, the lacIq gene product
dissociates from the lac operator in the presence of either lactose
or certain lactose analogs, e.g., isopropyl B-D-thiogalactopyranoside
(IPTG). BSCG-1 thus is not produced in appreciable quantities in
uninduced host cells containing the pHE4-5 vector. Induction of
these host cells by the addition of an agent such as IPTG, however,
results in the expression of the BSCG-1 coding sequence.
The promoter/operator sequences of the pHE4-5 vector (SEQ ID NO:11)
comprise a T5 phage promoter and two lac operator sequences. One
operator is located 5' to the transcriptional start site and the
other is located 3' to the same site. These operators, when present
in combination with the lacIq gene product, confer tight repression
of down-stream sequences in the absence of a lac operon inducer,
e.g., IPTG. Expression of operatively linked sequences located down-stream
from the lac operators may be induced by the addition of a lac operon
inducer, such as IPTG. Binding of a lac inducer to the lacIq proteins
results in their release from the lac operator sequences and the
initiation of transcription of operatively linked sequences. Lac
operon regulation of gene expression is reviewed in Devlin, T.,
TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL CORRELATIONS, 4th Edition-(1997),
pages 802 807.
The pHE4 series of vectors contain all of the components of the
pHE4-5 vector except for the BSCG-1 coding sequence. Features of
the pHE4 vectors include optimized synthetic T5 phage promoter,
lac operator, and Shine-Delagarno sequences. Further, these sequences
are also optimally spaced so that expression of an inserted gene
may be tightly regulated and high level of expression occurs upon
Among known bacterial promoters suitable for use in the production
of proteins of the present invention include the E. coli lacI and
lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda
PR and PL promoters and the trp promoter. Suitable eukaryotic promoters
include the CMV immediate early promoter, the HSV thymidine kinase
promoter, the early and late SV40 promoters, the promoters of retroviral
LTRs, such as those of the Rous Sarcoma Virus (RSV), and metallothionein
promoters, such as the mouse metallothionein-I promoter.
The pHE4-5 vector also contains a Shine-Delgarno sequence 5' to
the AUG initiation codon. Shine-Delgarno sequences are short sequences
generally located about 10 nucleotides up-stream (i.e., 5') from
the AUG initiation codon. These sequences essentially direct prokaryotic
ribosomes to the AUG initiation codon.
Thus, the present invention is also directed to expression vector
useful for the production of the proteins of the present invention.
This aspect of the invention is exemplified by the pHE4-5 vector
(SEQ ID NO:10).
Among vectors preferred for use in bacteria include pQE70, pQE60
and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,
Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from
Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available
from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,
pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,
pMSG and pSVL available from Pharmacia. Other suitable vectors will
be readily apparent to the skilled artisan.
Introduction of the construct into the host cell can be effected
by calcium phosphate transfection, DEAE-dextran mediated transfection,
cationic lipid-mediated transfection, electroporation, transduction,
infection or other methods. Such methods are described in many standard
laboratory manuals, such as Davis et al., Basic Methods In Molecular
The polypeptide may be expressed in a modified form, such as a
fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve stability
and persistence in the host cell, during purification, or during
subsequent handling and storage. Also, peptide moieties may be added
to the polypeptide to facilitate purification. Such regions may
be removed prior to final preparation of the polypeptide. The addition
of peptide moieties to polypeptides to engender secretion or excretion,
to improve stability and to facilitate purification, among others,
are familiar and routine techniques in the art. A preferred fusion
protein comprises a heterologous region from immunoglobulin that
is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobin molecules together with
another human protein or part thereof. In many cases, the Fc part
in a fusion protein is thoroughly advantageous for use in therapy
and diagnosis and thus results, for example, in improved pharmacokinetic
properties (EP-A 0232 262). On the other hand, for some uses it
would be desirable to be able to delete the Fc part after the fusion
protein has been expressed, detected and purified in the advantageous
manner described. This is the case when Fc portion proves to be
a hindrance to use in therapy and diagnosis, for example when the
fusion protein is to be used as antigen for immunizations. In drug
discovery, for example, human proteins, such as, hIL5-receptor has
been fused with Fc portions for the purpose of high-throughput screening
assays to identify antagonists of hIL-5. See, D. Bennett et al.,
Journal of Molecular Recognition, Vol. 8:52 58 (1995) and K. Johanson
et al., The Journal of Biological Chemistry, Vol. 270, No. 16:9459
The BCSG1 protein can be recovered and purified from recombinant
cell cultures by well-known methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite chromatography
and lectin chromatography. Most preferably, high performance liquid
chromatography ("HPLC") is employed for purification.
Polypeptides of the present invention include naturally purified
products, products of chemical synthetic procedures, and products
produced by recombinant techniques from a prokaryotic or eukaryotic
host, including, for example, bacterial, yeast, higher plant, insect
and mammalian cells. Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention
may be glycosylated or may be non-glycosylated. In addition, polypeptides
of the invention may also include an initial modified methionine
residue, in some cases as a result of host-mediated processes.
BCSG1 Polypeptides and Fragments
The invention further provides an isolated BCSG1 polypeptide having
the amino acid sequence encoded by the deposited cDNA clones, or
the amino acid sequence in FIG. 1 (SEQ ID NO:2), or a peptide or
polypeptide comprising a portion of the above polypeptides.
It will be recognized in the art that some amino acid sequences
of the BCSG1 polypeptide can be varied without significant effect
of the structure or function of the protein. If such differences
in sequence are contemplated, it should be remembered that there
will be critical areas on the protein which determine activity.
Thus, the invention further includes variations of the BCSG1 polypeptide
which show substantial BCSG1 polypeptide activity or which include
regions of BCSG1 protein such as the protein portions discussed
below. Such mutants include deletions, insertions, inversions, repeats,
and type substitutions. As indicated above, guidance concerning
which amino acid changes are likely to be phenotypically silent
can be found in Bowie, J. U., et al., "Deciphering the Message
in Protein Sequences: Tolerance to Amino Acid Substitutions,"
Science 247:1306 1310 (1990).
Thus, the fragment, derivative or analog of the polypeptide of
FIG. 1 (SEQ ID NO:2), or that encoded by the deposited cDNA, may
be (i) one in which one or more of the amino acid residues are substituted
with a conserved or non-conserved amino acid residue (preferably
a conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a substituent
group, or (iii) one in which the polypeptide is fused with another
compound, such as a compound to increase the half-life of the polypeptide
(for example, polyethylene glycol), or (iv) one in which the additional
amino acids are fused to the polypeptide, such as an IgG Fc fusion
region peptide or leader or secretory sequence or a sequence which
is employed for purification of the polypeptide or a proprotein
sequence. Such fragments, derivatives and analogs are deemed to
be within the scope of those skilled in the art from the teachings
Of particular interest are substitutions of charged amino acids
with another charged amino acid and with neutral or negatively charged
amino acids. The latter results in proteins with reduced positive
charge to improve the characteristics of the BCSG1 protein. The
prevention of aggregation is highly desirable. Aggregation of proteins
not only results in a loss of activity but can also be problematic
when preparing pharmaceutical formulations, because they can be
immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331 340 (1967);
Robbins et al., Diabetes 36:838 845 (1987); Cleland et al. Crit.
Rev. Therapeutic Drug Carrier Systems 10:307 377 (1993)).
As indicated, changes are preferably of a minor nature, such as
conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 1).
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions. Aromatic
Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine
Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine
Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine
Of course, the number of amino acid substitutions a skilled artisan
would make depends on-many factors, including those described above.
Generally speaking, the number of substitutions for any given AIM-II
polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5
Amino acids in the BCSG1 protein of the present invention that
are essential for function can be identified by methods known in
the art, such as site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham and Wells, Science 244:1081 1085 (1989)). The latter
procedure introduces single alanine mutations at every residue in
the molecule. The resulting mutant molecules are then tested for
biological activity such as receptor binding or in vitro, or in
vitro proliferative activity. Sites that are critical for ligand-receptor
binding can also be determined by structural analysis such as crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith et al.,
J. Mol. Biol. 224:899 904 (1992) and de Vos et al. Science 255:306
The polypeptides of the present invention are preferably provided
in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a polypeptide
produced and/or contained within a recombinant host cell is considered
isolated for purposes of the present invention. Also intended as
an "isolated polypeptide" are polypeptides that have been
purified, partially or substantially, from a recombinant host cell
or from a native source. For example, a recombinantly produced version
of the BCSG1 polypeptide can be substantially purified by the one-step
method described in Smith and Johnson, Gene 67:31 40 (1988).
The polypeptides of the present invention include the polypeptide
encoded by the deposited cDNA; a polypeptide comprising amino acids
about 1 to about 127 in SEQ ID NO:2 (FIG. 1); a polypeptide comprising
amino acids about 2 to about 127 in SEQ ID NO:2; as well as polypeptides
which are at least 80% identical, more preferably at least 90% or
95% identical, still more preferably at least 96%, 97%, 98% or 99%
identical to the polypeptide encoded by the deposited cDNA, to the
polypeptide of FIG. 1 (SEQ ID NO:2), and also include portions of
such polypeptides with at least 30 amino acids and more preferably
at least 50 amino acids.
By a polypeptide having an amino acid sequence at least, for example,
95% "identical" to a reference amino acid sequence of
a BCSG1 polypeptide is intended that the amino acid sequence of
the polypeptide is identical to the reference sequence except that
the polypeptide sequence may include up to five amino acid alterations
per each 100 amino acids of the reference amino acid of the BCSG1
polypeptide. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a reference amino acid sequence,
up to 5% of the amino acid residues in the reference sequence may
be deleted or substituted with another amino acid, or a number of
amino acids up to 5% of the total amino acid residues in the reference
sequence may be inserted into the reference sequence. These alterations
of the reference sequence may occur at the amino or carboxy terminal
positions of the reference amino acid sequence or anywhere between
those terminal positions, interspersed either individually among
residues in the reference sequence or in one or more contiguous
groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at
least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or to the
amino acid sequence encoded by deposited cDNA clone can be determined
conventionally using known computer programs such the Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, 575 Science Drive, Madison,
Wis. 53711. When using Bestfit or any other sequence alignment program
to determine whether a particular sequence is, for instance, 95%
identical to a reference sequence according to the present invention,
the parameters are set, of course, such that the percentage of identity
is calculated over the full length of the reference amino acid sequence
and that gaps in homology of up to 5% of the total number of amino
acid residues in the reference sequence are allowed.
The polypeptide of the present invention could be used as a molecular
weight marker on SDS-PAGE gels or on molecular sieve gel filtration
columns using methods well known to those of skill in the art.
In another aspect, the invention provides a peptide or polypeptide
comprising an epitope-bearing portion of a polypeptide of the invention.
The epitope of this polypeptide portion is an immunogenic or antigenic
epitope of a polypeptide described herein. An "immunogenic
epitope" is defined as a part of a protein that elicits an
antibody response when the whole protein is the immunogen. On the
other hand, a region of a protein molecule to which an antibody
can bind is defined as an "antigenic epitope." The number
of immunogenic epitopes of a protein generally is less than the
number of antigenic epitopes. See, for instance, Geysen et al.,
Proc. Natl. Acad. Sci. USA 81:3998 4002 (1983).
As to the selection of peptides or polypeptides bearing an antigenic
epitope (i.e., that contain a region of a protein molecule to which
an antibody can bind), it is well known in that art that relatively
short synthetic peptides that mimic part of a protein sequence are
routinely capable of eliciting an antiserum that reacts with the
partially mimicked protein. See, for instance, Sutcliffe, J. G.,
Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies
that react with predetermined sites on proteins. Science 219:660
666. Peptides capable of eliciting protein-reactive sera are frequently
represented in the primary sequence of a protein, can be characterized
by a set of simple chemical rules, and are confined neither to immunodominant
regions of intact proteins (i.e., immunogenic epitopes) nor to the
amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention
are therefore useful to raise antibodies, including monoclonal antibodies,
that bind specifically to a polypeptide of the invention. See, for
instance, Wilson et al., Cell 37:767 778 (1984) at 777.
Antigenic epitope-bearing peptides and polypeptides of the invention
preferably contain a sequence of at least seven, more preferably
at least nine and most preferably between about at least about 15
to about 30 amino acids contained within the amino acid sequence
of a polypeptide of the invention.
Non-limiting examples of antigenic polypeptides or peptides that
can be used to generate BCSG1-specific antibodies include: a polypeptide
comprising amino acid residues from about 94 to about 107 in FIG.
1 (SEQ ID NO:2); a polypeptide comprising amino acid residues from
about 120 to about 127 in FIG. 1 (SEQ ID NO:2). As indicated above,
the inventors have determined that the above polypeptide fragments
are antigenic regions of the BCSG1 protein.
The epitope-bearing peptides and polypeptides of the invention
may be produced by any conventional means. Houghten, R. A. (1985)
General method for the rapid solid-phase synthesis of large numbers
of peptides: specificity of antigen-antibody interaction at the
level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131
5135. This "Simultaneous Multiple Peptide Synthesis (SMPS)"
process is further described in U.S. Pat. No. 4,631,211 to Houghten
et al. (1986).
As one of skill in the art will appreciate, BCSG1 polypeptides
of the present invention and the epitope-bearing fragments thereof
described above can be combined with parts of the constant domain
of immunoglobulins (IgG), resulting in chimeric polypeptides. These
fusion proteins facilitate purification and show an increased half-life
in vivo. This has been shown, e.g., for chimeric proteins consisting
of the first two domains of the human CD4-polypeptide and various
domains of the constant regions of the heavy or light chains of
mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature
331:84 86 (1988)). Fusion proteins that have a disulfide-linked
dimeric structure due to the IgG part can also be more efficient
in binding and neutralizing other molecules than the monomeric BCSG1
protein or protein fragment alone (Fountoulakis et al., J. Biochem
270:3958 3964 (1995)).
Cancer Diagnosis and Prognosis
There are two classes of genes affecting tumor development. Genes
influencing the cancer phenotype that act directly as a result of
changes (e.g., mutation) at the DNA level, such as BRCA1, BRCA2,
and p53, are called Class I genes. The Class II genes affect the
phenotype by modulation at the expression level. Development of
breast cancer and subsequent malignant progression is associated
with alterations of a variety of genes of both classes. Identification
of quantitative changes in gene expression that occur in the malignant
mammary gland, if sufficiently characterized, may yield novel molecular
markers which may be useful in the diagnosis and treatment of human
The present inventors have identified a new breast cancer marker
that is overexpressed in advanced infiltrating breast cancer cells.
The lack of expression of BCSG1 in normal or benign breast epithelial
cells and a weak expression in low grade in situ carcinomas suggest
that overexpression of BCSG1 indicates breast cancer malignant progression.
(See, Examples 6 and 7). It is unlikely that BCSG1 is overexpressed
as a secondary effect of cellular proliferation because no detectable
BCSG1 expression is evident in rapidly proliferating nonmalignant
breast lesions. (See, Example 7).
BCSG1 may be useful in clinical management and treatment of breast
cancer. In this regard, the expression of BCSG1 transcripts was
observed in the neoplastic epithelial cells of infiltrating breast
carcinoma but not in epithelial cells of normal and benign breast
tissue. (See, Example 7). The overexpression of BCSG1 in malignant
infiltrating breast epithelial cells compared to the low level expression
in the low grade in situ carcinoma suggests that up-regulation of
BCSG1 expression is associated with breast malignant progression
and may signal the more advanced invasive/metastatic phenotype of
human breast cancer. This implication is further supported by detection
of BCSG1 expression in 4/4 breast cancer cell lines derived from
ductal infiltrating carcinomas but not (0/3) in breast cancer cell
lines derived from primary solid carcinoma (See, Example 6). BCSG1
overexpression in ductal carcinoma in situ (DCIS) may indicate a
malignant progression leading to metastasis. There was a marked
increase in DCIS incidence beginning in the early 1980s (Emster,
V. L., et al., JAMA 275:913 918 (1996)). The total estimated number
of DCIS cases in the United States in 1992 was 200% higher than
expected based on 1983 rates and trends between 1973 and 1983 (Emster,
V. L., et al., JAMA 275:913 918 (1996)). While early detection of
invasive breast cancer is beneficial, the value of DCIS detection
is currently unknown. There is cause for concern about the large
number of DCIS cases that are being diagnosed as a consequence of
screening mammography, most of which are treated by some form of
surgery. In addition, the proportion of cases treated by mastectomy
may be inappropriately high (Emster, V. L., et al., JAMA 275:913
918 (1996)). BCSG1 expression may provide some prognostic information
on distinguishing the DCIS which is not likely to become invasive
from the DCIS which is most likely to become invasive, which will
help to reduce some inappropriate or unnecessary mastectomies. In
addition, the use of BCSG1 gene could be of great importance in
differentiating atypical proliferative breast lesions from cancer
and may be useful in screening of breast biopsies for potential
It is interesting to note that the predicted amino acid sequence
of BCSG1 gene shares high sequence homology with the recently cloned
non-A.beta. component of Alzheimer's disease (AD) amyloid precursor
protein (Ueda, K., et al., Proc. Natl. Acad. Sci. USA. 90 (23):11282
6 (1993)). A neuropathological hallmark of AD is a widespread amyloid
deposition resulting from beta-amyloid precursor proteins (beta
APPS). Beta APPs are large membrane-spanning proteins that either
give rise to the beta A4 peptide (AB fragment) (Masters, C. L.,
et al., Proc. Natl. Acad. Sci. USA 82:4245 4249 (1985)) or a non-A.beta.
component of AD amyloid (Ueda, K., et al., Proc. Natl. Acad. Sci.
USA. 90 (23):11282 6 (1993)) that is either deposited in AD amyloid
plaques or yielding soluble forms. While the insoluble membrane-bound
AD amyloid destabilizes calcium homeostasis and thus renders cell
vulnerable to excitotoxic conditions of calcium influx resulting
from energy deprivation or overexcitation (Mattson, M. P., et al.,
Ann. N.Y. Acad. Sci. 679:121 (1993)), the soluble AD amyloid proteins
are neuroprotective against glucose deprivation and glutamate toxicity,
perhaps through their ability to lower the intraneuronal calcium
concentration (Barger, S. W., J. Neurochem. 64:2087 96 (1995)).
It is possible that BCSG1, like soluble AD amyloid, may be potentially
involved in tissue damage resulting from tissue remodeling due to
the local cancer invasion. Nevertheless, Examples 6 and 7 demonstrate
a stage-specific BCSG1 expression and an association of BCSG1 overexpression
with clinical aggressiveness of breast cancers. BCSG1 overexpression
may indicate breast cancer malignant progression from benign breast
or low grade in situ carcinoma to the highly infiltrating carcinoma.
The Examples demonstrate that certain tissues in mammals with cancer
express significantly enhanced levels of the BCSG1 protein and mRNA
encoding the BCSG1 protein when compared to a corresponding "standard"
mammal, i.e., a mammal of the same species not having the cancer.
Further, it is believed that enhanced levels of the BCSG1 protein
can be detected in certain body fluids (e.g., sera, plasma, urine,
and spinal fluid) from mammals with cancer when compared to sera
from mammals of the same species not having the cancer. Thus, the
invention provides a diagnostic method useful during tumor diagnosis,
which involves assaying the expression level of the gene encoding
the BCSG1 protein in mammalian cells or body fluid and comparing
the gene expression level with a standard BCSG1 gene expression
level, whereby an increase in the gene expression level over the
standard is indicative of certain tumors.
Where a tumor diagnosis has already been made according to conventional
methods, the present invention is useful as a prognostic indicator,
whereby patients exhibiting enhanced BCSG1 gene expression will
experience a worse clinical outcome relative to patients expressing
the gene at a lower level.
By "assaying the expression level of the gene encoding the
BCSG1 protein" is intended qualitatively or quantitatively
measuring or estimating the level of the BCSG1 protein or the level
of the mRNA encoding the BCSG1 protein in a first biological sample
either directly (e.g., by determining or estimating absolute protein
level or mRNA level) or relatively (e.g., by comparing to the BCSG1
protein level or mRNA level in a second biological sample).
Preferably, the BCSG1 protein level or mRNA level in the first
biological sample is measured or estimated and compared to a standard
BCSG1 protein level or mRNA level, the standard being taken from
a second biological sample obtained from an individual not having
the cancer. As will be appreciated in the art, once a standard BCSG1
protein level or mRNA level is known, it can be used repeatedly
as a standard for comparison.
By "biological sample" is intended any biological sample
obtained from an individual, cell line, tissue culture, or other
source which contains BCSG1 protein or mRNA. Biological samples
include mammalian body fluids (such as sera, plasma, urine, synovial
fluid and spinal fluid) which contain secreted mature BCSG1 protein,
and ovarian, prostate, heart, placenta, pancreas liver, spleen,
lung, breast and umbilical tissue.
The present invention is useful for detecting cancer in mammals.
In particular the invention is useful during diagnosis of the of
following types of cancers in mammals: breast, ovarian, prostate,
bone, liver, lung, pancreatic, and spleenic. Preferred mammals include
monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans.
Particularly preferred are humans.
Total cellular RNA can be isolated from a biological sample using
the single-step guanidinium-thiocyanate-phenol-chloroform method
described in Chomczynski and Sacchi, Anal. Biochem. 162:156 159
(1987). Levels of mRNA encoding the BCSG1 protein are then assayed
using any appropriate method. These include Northern blot analysis
(Harada et al., Cell 63:303 312 (1990)), S1 nuclease mapping (Fujita
et al., Cell 49:357 367 (1987)), the polymerase chain reaction (PCR),
reverse transcription in combination with the polymerase chain reaction
(RT-PCR) (Makino et al., Technique 2:295 301 (1990)), and reverse
transcription in combination with the ligase chain reaction (RT-LCR).
Assaying BCSG1 protein levels in a biological sample can occur
using antibody-based techniques. For example, BCSG1 protein expression
in tissues can be studied with classical immunohistological methods
(Jalkanen, M., et al., J. Cell. Biol. 101:976 985 (1985); Jalkanen,
M., et al., J. Cell. Biol. 105:3087 3096 (1987)).
Other antibody-based methods useful for detecting BCSG1 protein
gene expression include immunoassays, such as the enzyme linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable labels are known in the art and include enzyme labels,
such as, Glucose oxidase, and radioisotopes, such as iodine (.sup.125I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium (.sup.3H),
indium (.sup.112In), and technetium (.sup.99mTc), and fluorescent
labels, such as fluorescein and rhodamine, and biotin.
The nucleic acid molecules of the present invention are also valuable
for chromosome identification. The sequence is specifically targeted
to and can hybridize with a particular location on an individual
human chromosome. The mapping of DNAs to chromosomes according to
the present invention is an important first step in correlating
those sequences with genes associated with disease.
In certain preferred embodiments in this regard, the cDNA herein
disclosed is used to clone genomic DNA of a BCSG1 protein gene.
This can be accomplished using a variety of well known techniques
and libraries, which generally are available commercially. The genomic
DNA then is used for in situ chromosome mapping using well known
techniques for this purpose.
In addition, in some cases, sequences can be mapped to chromosomes
by preparing PCR primers (preferably 15 25 bp) from the cDNA. Computer
analysis of the 3' untranslated region of the gene is used to rapidly
select primers that do not span more than one exon in the genomic
DNA, thus complicating the amplification process. These primers
are then used for PCR screening of somatic cell hybrids containing
individual human chromosomes.
Fluorescence in situ hybridization ("FISH") of a cDNA
clone to a metaphase chromosomal spread can be used to provide a
precise chromosomal location in one step. This technique can be
used with probes from the cDNA as short as 50 or 60 bp. For a review
of this technique, see Verma et al., Human Chromosomes: A Manual
Of Basic Techniques, Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location,
the physical position of the sequence on the chromosome can be correlated
with genetic map data. Such data are found, for example, in V. McKusick,
Mendelian Inheritance In Man, available on-line through Johns Hopkins
University, Welch Medical Library. The relationship between genes
and diseases that have been mapped to the same chromosomal region
are then identified through linkage analysis (coinheritance of physically
Next, it is necessary to determine the differences in the cDNA
or genomic sequence between affected and unaffected individuals.
If a mutation is observed in some or all of the affected individuals
but not in any normal individuals, then the mutation is likely to
be the causative agent of the disease.
Having generally described the invention, the same will be more
readily understood by reference to the following examples, which
are provided by way of illustration and are not intended as limiting.
Expression and Purification of BCSG1 in E. coli
The bacterial expression vector pQE9 (pD10) is used for bacterial
expression in this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311). pQE9 encodes ampicillin antibiotic resistance ("Amp.sup.r")
and contains a bacterial origin of replication ("ori"),
an IPTG inducible promoter, a ribosome binding site ("RBS"),
six codons encoding histidine residues that allow affinity purification
using nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity
resin sold by QIAGEN, Inc., supra, and suitable single restriction
enzyme cleavage sites. These elements are arranged such that an
inserted DNA fragment encoding a polypeptide expresses that polypeptide
with the six His residues (i.e., a "6.times.His tag"))
covalently linked to the amino terminus of that polypeptide.
The DNA sequence encoding the desired portion BCSG1 protein sequence
is amplified from the deposited cDNA clone using PCR oligonucleotide
primers which anneal to the amino terminal sequences of the desired
portion of the BCSG1 protein and to sequences in the deposited construct
3' to the cDNA coding sequence. Additional nucleotides containing
restriction sites to facilitate cloning in the pQE9 vector are added
to the 5' and 3' primer sequences, respectively.
For cloning the mature protein, the 5' primer has the sequence
5' GGGGATCCATGTTTTCAAGAAGG 3' (SEQ ID NO:3) containing the underlined
BamHI restriction site followed by 16 nucleotides complementary
to the amino terminal coding sequence of the BCSG1 sequence in FIG.
1. One of ordinary skill in the art would appreciate, of course,
that the point in the protein coding sequence where the 5' primer
begins may be varied to amplify a DNA segment encoding any desired
portion of the complete BCSG1 protein shorter or longer than the
protein. The 3' primer has the sequence 5' GGAAGCTTCTAGTCTCCCCCACTCTGG
3' (SEQ ID NO:4) containing the underlined HindIII restriction site
followed by 19 nucleotides complementary to the non-coding sequence
of the BCSG1 DNA sequence in FIG. 1.
The amplified BCSG1 DNA fragment and the vector pQE9 are digested
with BamHI/HindIII and the digested DNAs are then ligated together.
Insertion of the BCSG1 DNA into the restricted pQE9 vector places
the BCSG1 protein coding region downstream from the IPTG-inducible
promoter and in-frame with an initiating AUG and the six histidine
The ligation mixture is transformed into competent E. coli cells
using standard procedures such as those described in Sambrook et
al., Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli
strain M15/rep4, containing multiple copies of the plasmid pREP4,
which expresses the lac repressor and confers kanamycin resistance
("Kan.sup.r"), is used in carrying out the illustrative
example described herein. This strain, which is only one of many
that are suitable for expressing BCSG1 protein, is available commercially
from QIAGEN, Inc., supra. Transformants are identified by their
ability to grow on LB plates in the presence of ampicillin and kanamycin.
Plasmid DNA is isolated from resistant colonies and the identity
of the cloned DNA confirmed by restriction analysis, PCR and DNA
Clones containing the desired constructs are grown overnight ("O/N")
in liquid culture in LB media supplemented with both ampicillin
(100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N culture is used
to inoculate a large culture, at a dilution of approximately 1:25
to 1:250. The cells are grown to an optical density at 600 nm ("OD600")
of between 0.4 and 0.6. Isopropyl-b-D-thiogalactopyranoside ("IPTG")
is then added to a final concentration of 1 mM to induce transcription
from the lac repressor sensitive promoter, by inactivating the lacI
repressor. Cells subsequently are incubated further for 3 to 4 hours.
Cells then are harvested by centrifugation.
The cells are then stirred for 3 4 hours at 4.degree. C. in 6 M
guanidine-HCl, pH 8. The cell debris is removed by centrifugation,
and the supernatant containing the BCSG1 is loaded onto a nickel-nitrilo-tri-acetic
acid ("NiNTA") affinity resin column (available from QIAGEN,
Inc., supra). Proteins with a 6.times.His tag bind to the NI-NTA
resin with high affinity and can be purified in a simple one-step
procedure (for details see: The QIAexpressionist, 1995, QIAGEN,
Inc., supra). Briefly the supernatant is loaded onto the column
in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes
of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl
pH 6, and finally the BCSG1 is eluted with 6 M guanidine-HCl, pH
The purified protein is then renatured by dialyzing it against
phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer
plus 200 mM NaCl. Alternatively, the protein can be successfully
refolded while immobilized on the Ni-NTA column. The recommended
conditions are as follows: renature using a linear 6M 1M urea gradient
in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing
protease inhibitors. The renaturation should be performed over a
period of 1.5 hours or more. After renaturation the proteins can
be eluted by the addition of 250 mM immidazole. Immidazole is removed
by a final dialyzing step against PBS or 50 mM sodium acetate pH
6 buffer plus 200 mM NaCl. The purified protein is stored at 4.degree.
C. or frozen at -80.degree. C.
Cloning and Expression of BCSG1 Protein in a Baculovirus Expression
In this illustrative example, the plasmid shuttle vector pA2 GP
is used to insert the cloned DNA encoding the protein into a baculovirus
to express the BCSG1 protein, using a baculovirus leader and standard
methods as described in Summers et al., A Manual of Methods for
Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural
Experimental Station Bulletin No. 1555 (1987). This expression vector
contains the strong polyhedrin promoter of the Autographa californica
nuclear polyhedrosis virus (AcMNPV) followed by the secretory signal
peptide (leader) of the baculovirus gp67 protein and convenient
restriction sites such as BamHI, Xba I and Asp718. The polyadenylation
site of the simian virus 40 ("SV40") is used for efficient
polyadenylation. For easy selection of recombinant virus, the plasmid
contains the beta-galactosidase gene from E. coli under control
of a weak Drosophila promoter in the same orientation, followed
by the polyadenylation signal of the polyhedrin gene. The inserted
genes are flanked on both sides by viral sequences for cell-mediated
homologous recombination with wild-type viral DNA to generate viable
virus that expresses the cloned polynucleotide.
Many other baculovirus vectors could be used in place of the vector
above, such as pAc373, pVL941 and pAcIMI, as one skilled in the
art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation, secretion
and the like, including a signal peptide and an in-frame AUG as
required. Such vectors are described, for instance, in Luckow et
al., Virology 170:31 39.
The cDNA sequence encoding the BCSG1 protein in the deposited clone
shown in FIG. 1 (SEQ ID NO:2), is amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' sequences of the gene.
The 5' primer has the sequence 5' GGGGATCCcGATGTTTTCAAGAAGG 3'
(SEQ ID NO:5) (the lowercase "c" is a nucleotide included
to preserve the coding frame) containing the underlined BamHI restriction
enzyme site followed by 16 bases of the sequence of the BCSG1 protein
shown in FIG. 1, beginning with the N-terminus of the protein. The
3' primer has the sequence 5'GGGGTACCCTAGTCTCCCCCACTCTGG 3' (SEQ
ID NO:6) containing the underlined Asp718 restriction site followed
by 18 nucleotides complementary to the 3' noncoding sequence in
The amplified fragment is isolated from a 1% agarose gel using
a commercially available kit ("Geneclean," BIO 101 Inc.,
La Jolla, Calif.). The fragment then is digested with BamHI/Asp718
and again is purified on a 1% agarose gel. This fragment is designated
The plasmid is digested with the restriction enzymes BamHI/Asp718
and optionally, can be dephosphorylated using calf intestinal phosphatase,
using routine procedures known in the art. The DNA is then isolated
from a 1% agarose gel using a commercially available kit ("Geneclean"
BIO 101 Inc., La Jolla, Calif.). This vector DNA is designated herein
Fragment F1 and the dephosphorylated plasmid V1 are ligated together
with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts
such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)
cells are transformed with the ligation mixture and spread on culture
plates. Bacteria are identified that contain the plasmid with the
human BCSG1 gene using the PCR method, in which one of the primers
that is used to amplify the gene and the second primer is from well
within the vector so that only those bacterial colonies containing
the BCSG1 gene fragment will show amplification of the DNA. The
sequence of the cloned fragment is confirmed by DNA sequencing.
This plasmid is designated herein pBac BCSG1.
Five .mu.g of the plasmid pBacBCSG1 is co-transfected with 1.0
.mu.g of a commercially available linearized baculovirus DNA ("BaculoGold.TM.
baculovirus DNA", Pharmingen, San Diego, Calif.), using the
lipofection method described by Felgner et al., Proc. Natl. Acad.
Sci. USA 84:7413 7417 (1987). 1 .mu.g of BaculoGold.TM. virus DNA
and 5 .mu.g of the plasmid pBac BCSG1 are mixed in a sterile well
of a microtiter plate containing 50 .mu.l of serum-free Grace's
medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards,
10 .mu.l Lipofectin plus 90 .mu.l Grace's medium are added, mixed
and incubated for 15 minutes at room temperature. Then the transfection
mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded
in a 35 mm tissue culture plate with 1 ml Grace's medium without
serum. The plate is rocked back and forth to mix the newly added
solution. The plate is then incubated for 5 hours at 27.degree.
C. After 5 hours the transfection solution is removed from the plate
and 1 ml of Grace's insect medium supplemented with 10% fetal calf
serum is added. The plate is put back into an incubator and cultivation
is continued at 27.degree. C. for four days.
After four days the supernatant is collected and a plaque assay
is performed, as described by Summers and Smith, supra. An agarose
gel with "Blue Gal" (Life Technologies Inc., Gaithersburg)
is used to allow easy identification and isolation of gal-expressing
clones, which produce blue-stained plaques. (A detailed description
of a "plaque assay" of this type can also be found in
the user's guide for insect cell culture and baculovirology distributed
by Life Technologies Inc., Gaithersburg, page 9 10). After appropriate
incubation, blue stained plaques are picked with the tip of a micropipettor
(e.g., Eppendorf). The agar containing the recombinant viruses is
then resuspended in a microcentrifuge tube containing 200 .mu.l
of Grace's medium and the suspension containing the recombinant
baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
Four days later the supernatants of these culture dishes are harvested
and then they are stored at 4.degree. C. The recombinant virus is
To verify the expression of the BCSG1 gene, Sf9 cells are grown
in Grace's medium supplemented with 10% heat inactivated FBS. The
cells are infected with the recombinant baculovirus V-BCSG1 at a
multiplicity of infection ("MOI") of about 2. Six hours
later the medium is removed and is replaced with SF900 II medium
minus methionine and cysteine (available from Life Technologies
Inc., Rockville, Md.). If radiolabeled proteins are desired, 42
hours later, 5 .mu.Ci of .sup.35S-methionine and 5 .mu.Ci .sup.35S-cysteine
(available from Amersham) are added. The cells are further incubated
for 16 hours and then they are harvested by centrifugation. The
proteins in the supernatant as well as the intracellular proteins
are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus
of purified protein may be used to determine the amino terminal
sequence of the mature protein and thus the cleavage point and length
of the secretory signal peptide.
Cloning and Expression of BCSG1 in Mammalian Cells
A typical mammalian expression vector contains the promoter element,
which mediates the initiation of transcription of mRNA, the protein
coding sequence, and signals required for the termination of transcription
and polyadenylation of the transcript. Additional elements include
enhancers, Kozak sequences and intervening sequences flanked by
donor and acceptor sites for RNA splicing. Highly efficient transcription
can be achieved with the early and late promoters from SV40, the
long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLV
I, HIV I and the early promoter of the cytomegalovirus (CMV). However,
cellular elements can also be used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention
include, for example, vectors such as PSVL and PMSG (Pharmacia,
Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and
pBC12MI (ATCC 67109). Mammalian host cells that could be used include,
human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells,
Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese
hamster ovary (CHO) cells.
Alternatively, the gene can be expressed in stable cell lines that
contain the gene integrated into a chromosome. The co-transfection
with a selectable marker such as dhfr, gpt, neomycin, or hygromycin
allows the identification and isolation of the transfected cells.
The transfected gene can also be amplified to express large amounts
of the encoded protein. The DHFR (dihydrofolate reductase) marker
is useful to develop cell lines that carry several hundred or even
several thousand copies of the gene of interest. Another useful
selection marker is the enzyme glutamine synthase (GS) (Murphy et
al., Biochem J. 227:277 279 (1991); Bebbington et al., Bio/Technology
10:169 175 (1992)). Using these markers, the mammalian cells are
grown in selective medium and the cells with the highest resistance
are selected. These cell lines contain the amplified gene(s) integrated
into a chromosome. Chinese hamster ovary (CHO) and NSO cells are
often used for the production of proteins.
The expression vectors pC1 and pC4 contain the strong promoter
(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular
Biology, 438 447 (March, 1985)) plus a fragment of the CMV-enhancer
(Boshart et al., Cell 41:521 530 (1985)). Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites BamHI, XbaI and
Asp718, facilitate the cloning of the gene of interest. The vectors
contain in addition the 3' intron, the polyadenylation and termination
signal of the rat preproinsulin gene.
Cloning and Expression in COS Cells
The expression plasmid, pBCSG1 HA, is made by cloning a cDNA encoding
BCSG1 into the expression vector pcDNAI/Amp or pcDNAIII (which can
be obtained from Invitrogen, Inc.).
The expression vector pcDNAI/amp contains: (1) an E. coli origin
of replication effective for propagation in E. coli and other prokaryotic
cells; (2) an ampicillin resistance gene for selection of plasmid-containing
prokaryotic cells; (3) an SV40 origin of replication for propagation
in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron;
(5) several codons encoding a hemagglutinin fragment (i.e., an "HA"
tag to facilitate purification) followed by a termination codon
and polyadenylation signal arranged so that a cDNA can be conveniently
placed under expression control of the CMV promoter and operably
linked to the SV40 intron and the polyadenylation signal by means
of restriction sites in the polylinker. The HA tag corresponds to
an epitope derived from the influenza hemagglutinin protein described
by Wilson et al., Cell 37:767 (1984). The fusion of the HA tag to
the target protein allows easy detection and recovery of the recombinant
protein with an antibody that recognizes the HA epitope. pcDNAIII
contains, in addition, the selectable neomycin marker.
A DNA fragment encoding the BCSG1 is cloned into the polylinker
region of the vector so that recombinant protein expression is directed
by the CMV promoter. The plasmid construction strategy is as follows.
The BCSG1 cDNA of the deposited clone is amplified using primers
that contain convenient restriction sites, much as described above
for construction of vectors for expression of BCSG1 in E. coli.
Suitable primers include the following, which are used in this example.
The 5' primer, containing the underlined BamHI site, a Kozak sequence,
an AUG start codon and 4 codons of the 5' coding region of the complete
BCSG1 has the following sequence: 5' GGGGATccgccaccATGTTTTCAAGAAGG
3' (SEQ ID NO:7) (Kozak sequence is represented by the lowercase
letters). The 3' primer, containing the underlined BamHI site, a
stop codon, and 19 bp of 3' coding sequence has the following sequence
(at the 3' end): 5' GGGGATCCTCAgaaagcgtagtctgggacgtcgtatgggtaCTAGTCTCCCCCACTCT
GG 3' (SEQ ID NO:8) (the HA tag is represented by the lowercase
The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are
digested with BamHI and then ligated. The ligation mixture is transformed
into E. coli strain SURE (available from Stratagene Cloning Systems,
11099 North Torrey Pines Road, La Jolla, Calif. 92037), and the
transformed culture is plated on ampicillin media plates which then
are incubated to allow growth of ampicillin resistant colonies.
Plasmid DNA is isolated from resistant colonies and examined by
restriction analysis or other means for the presence of the BCSG1-encoding
For expression of recombinant BCSG1, COS cells are transfected
with an expression vector, as described above, using DEAE-DEXTRAN,
as described, for instance, in Sambrook et al., Molecular Cloning:
a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor,
N.Y. (1989). Cells are incubated under conditions for expression
of BCSG1 by the vector.
Expression of the BCSG1-HA fusion protein is detected by radiolabeling
and immunoprecipitation, using methods described in, for example
Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To this
end, two days after transfection, the cells are labeled by incubation
in media containing .sup.35S-cysteine for 8 hours. The cells and
the media are collected, and the cells are washed and lysed with
detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS,
0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al. cited
above. Proteins are precipitated from the cell lysate and from the
culture media using an HA-specific monoclonal antibody. The precipitated
proteins then are analyzed by SDS-PAGE and autoradiography. An expression
product of the expected size is seen in the cell lysate, which is
not seen in negative controls.
Cloning and Expression in CHO Cells
The vector pC4 is used for the expression of BCSG1 protein. Plasmid
pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.
37146). The plasmid contains the mouse DHFR gene under control of
the SV40 early promoter. Chinese hamster ovary- or other cells lacking
dihydrofolate activity that are transfected with these plasmids
can be selected by growing the cells in a selective medium (alpha
minus MEM, Life Technologies) supplemented with the chemotherapeutic
agent methotrexate. The amplification of the DHFR genes in cells
resistant to methotrexate (MTX) has been well documented (see, e.g.,
Alt, F. W., Kellems, R. M., Bertino, J. R., and Schimke, R. T.,
1978, J. Biol. Chem. 253:1357 1370, Hamlin, J. L. and Ma, C. 1990,
Biochem. et Biophys. Acta, 1097:107 143, Page, M. J. and Sydenham,
M. A. 1991, Biotechnology 9:64 68). Cells grown in increasing concentrations
of MTX develop resistance to the drug by overproducing the target
enzyme, DHFR, as a result of amplification of the DHFR gene. If
a second gene is linked to the DHFR gene, it is usually co-amplified
and over-expressed. It is known in the art that this approach may
be used to develop cell lines carrying more than 1,000 copies of
the amplified gene(s). Subsequently, when the methotrexate is withdrawn,
cell lines are obtained which contain the amplified gene integrated
into one or more chromosome(s) of the host cell.
Plasmid pC4 contains for expressing the gene of interest the strong
promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus
(Cullen, et al., Molecular and Cellular Biology, March 1985:438
447) plus a fragment isolated from the enhancer of the immediate
early gene of human cytomegalovirus (CMV) (Boshart et al., Cell
41:521 530 (1985)). Downstream of the promoter are BamHI, XbaI,
and Asp718 restriction enzyme cleavage sites that allow integration
of the genes. Behind these cloning sites the plasmid contains the
3' intron and polyadenylation site of the rat preproinsulin gene.
Other high efficiency promoters can also be used for the expression,
e.g., the human .beta.-actin promoter, the SV40 early or late promoters
or the long terminal repeats from other retroviruses, e.g., HIV
and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems
and similar systems can be used to express the BCSG1 in a regulated
way in mammalian cells (Gossen, M., & Bujard, H. 1992, Proc.
Natl. Acad. Sci. USA 89: 5547 5551). For the polyadenylation of
the mRNA other signals, e.g., from the human growth hormone or globin
genes can be used as well. Stable cell lines carrying a gene of
interest integrated into the chromosomes can also be selected upon
co-transfection with a selectable marker such as gpt, G418 or hygromycin.
It is advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
The plasmid pC4 is digested with the restriction enzymes BamHI/Asp718
and then dephosphorylated using calf intestinal phosphatase by procedures
known in the art. The vector is then isolated from a 1% agarose
The DNA sequence encoding the BCSG1 protein sequence is amplified
using PCR oligonucleotide primers corresponding to the 5' and 3'
sequences of the gene. The 5' primer has the sequence 5' GGGGATccgccaccATGTTTTCAAGAAGG
3' (SEQ ID NO:7) (Kozak sequence is represented by the lowercase
letters) containing the underlined BamHI restriction enzyme site
followed by an efficient signal for initiation of translation in
eukaryotes, as described by Kozak, M., J. Mol. Biol. 196:947 950
(1987), and 15 bases of the coding sequence of BCSG1 shown in FIG.
1 (SEQ ID NO:1). The 3' primer has the sequence 5' GGGGTACCTCACTAGTCTCCCCCACTCTGG
3' (SEQ ID NO:9) containing the underlined Asp718 restriction site
followed by 22 nucleotides complementary to the non-translated region
of the BCSG1 gene shown in FIG. 1 (SEQ ID NO:1).
The amplified fragment is digested with the endonucleases BamHI/Asp718
and then purified again on a 1% agarose gel. The isolated fragment
and the dephosphorylated vector are then ligated with T4 DNA ligase.
E. coli HB101 or XL-1 Blue cells are then transformed and bacteria
are identified that contain the fragment inserted into plasmid pC4
using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene are used
for transfection. 5 .mu.g of the expression plasmid pC4 is cotransfected
with 0.5 .mu.g of the plasmid pSV2-neo using lipofectin (Felgner
et al., supra). The plasmid pSV2neo contains a dominant selectable
marker, the neo gene from Tn5 encoding an enzyme that confers resistance
to a group of antibiotics including G418. The cells are seeded in
alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the
cells are trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml
of metothrexate plus 1 mg/ml G418. After about 10 14 days single
clones are trypsinized and then seeded in 6-well petri dishes or
10 ml flasks using different concentrations of methotrexate (50
nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest
concentrations of methotrexate are then transferred to new 6-well
plates containing even higher concentrations of methotrexate (1
.mu.M, 2 .mu.M, 5 .mu.M, 10 mM, 20 mM). The same procedure is repeated
until clones are obtained which grow at a concentration of 100 200
.mu.M. Expression of the desired gene product is analyzed, for instance,
by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
Tissue Distribution of BCSG1 mRNA Expression
Northern blot analysis was carried out to examine BCSG1 gene expression
in human tissues as follows. Total RNA was extracted from tissues
according to the method of Chomcznski and Sacchi (Chomczynski, P.
& Sacchi, N., Anal. Biochem. 162:156 159 (1987)). Equal aliquots
of RNA were electrophoresed in a 1.2% agarose gel containing formaldehyde
and transferred to nylon membrane (Boehringer Mannheim). The membrane
was pre-hybridized with ExpressHyb hybridization solution (Clontech,
Inc.) at 68.degree. C. for 30 min. The hybridization was carried
out in the same solution with .sup.32P-labeled BCSG1 probe (1.5.times.10.sup.6
cpm/ml) for 1 hour at 68.degree. C. The membrane was then rinsed
in 2.times.SSC containing 0.05% SDS three times for 30 min at room
temperature, followed by two washes with 0.1.times.SSC containing
0.1% SDS for 40 min at 50.degree. C. The full-length BCSG1 cDNA
(SEQ ID NO:1) was isolated from the Bluescript vector, following
EcoRI and Xhol digestion, and used as a template for preparation
of a random-labelled cDNA probe. Random primer DNA labeling kit
was obtained from Boehringer Mannheim, Indianapolis. .sup.32P-dATP
was purchased from Amersham.
The northern blot showed that BCSG1 was abundantly expressed as
the expected 1 kb transcript in brain which is a rich source for
AD amyloid family genes. A much less intense band of similar size
was also seen in the following tissues: ovary, testis, colon, and
Cloning of BCSG1 from cDNA Libraries
EST analysis was used to search for new genes differentially expressed
in breast cancer versus normal breast. A data base containing approximately
500,000 human partial cDNA sequences (expressed sequence tags) has
been established in a collaborative effort between the Institute
for Genomic Research and Human Genome Science Inc., using high throughput
automated DNA sequence analysis of randomly selected human cDNA
clones (Adams, M. D., et al., Science 252:1651 6 (1991)). RNAs from
a stage III breast carcinoma and patient-matched normal breast were
isolated and subjected to preparation of cDNA libraries. EST automated
DNA sequence analysis was performed on randomly selected cDNA clones.
Both libraries had about 60% novel gene sequences which did not
match exactly to published human genes. A total of 3048 ESTs from
breast cancer cDNA library and 2886 ESTs from normal breast cDNA
library were randomly picked and sequence analyzed. The ESTs with
overlapping sequences were grouped into unique EST groups; and each
EST group may represent a gene or a family of sequence-related genes.
There were more than 2,200 EST groups that were analyzed for quantitative
comparison of EST hits in the pair of cDNA libraries from normal
breast versus breast cancer by examining the expression of individual
EST sequences. The numbers of EST hits in the libraries reflect
the relative expression or mRNA transcript copy numbers of the EST.
This direct differential cDNA sequence, as illustrated in FIG. 2,
utilizing the direct EST sequencing analysis simultaneously on a
pair of cDNA libraries made from normal breast and breast cancer,
was used to study expression profile of individual genes and patterns
of genes in normal breast versus breast cancer.
cDNA libraries were generated from breast cancer biopsy specimen
and patient-matched normal breast and were analyzed by EST sequencing.
Approximately 6,000 ESTs were analyzed and grouped to different
groups based on sequence overlapping, and 2,200 unique EST groups
were first analyzed for relative expression in the cDNA libraries
from normal breast versus breast cancer and then subjected to tissue-specific
expression by examining tissue origins of individual EST sequences
against a large population of ESTs derived from a variety of different
tissue types. Three classes of EST groups were identified that were
differentially expressed in normal breast versus breast cancer.
As a demonstration of this approach, Table 1 shows a partial list
of three classes of genes that are differentially expressed in normal
breast versus breast cancer. Class I represents the genes more abundant
in breast cancer than in normal breast and includes cathepsin D,
a well-studied steroid regulated extracellular matrix-degrading
proteinase (Rochefort, H., et al., J. Cell. Biochem. 35:17 29 (1987);
Cavailles, V., et al., Biochem. Biophys. Res. Commun. 174:816 24
(1991); Capony, F. et al., Biochem. Biophys. Res. Commun. 171:972
80 (1990)). Cathepsin D is thought to play a role in breast cancer
metastasis (Rochefort, H., et al., J. Cell. Biochem. 35:17 29 (1987);
Cavailles, V., et al., Biochem. Biophys. Res. Commun. 174:816 24
(1991); Capony, F. et al., Biochem. Biophys. Res. Commun. 171:972
80 (1990)) and has been proposed as a prognostic marker in breast
cancer progression (Brouillet, J. P., et al., Eur. J. Cancer 26:437
41 (1990); Spyratos, F., et al., Lancet, 11:1115 8 (1989); Rochefort,
H., et al., J. steroid. Biochem. 34:177 82 (1989); Foekens J. A.,
et al., JNCI 87:751 6 (1995)). As listed, there were 5 cathepsin
D ESTs sequenced in the breast cancer cDNA library and only 1 EST
in the normal breast cDNA library. Another proposed breast cancer
metastasis-related gene and a prognostic marker for breast cancer,
67 kDa laminin receptor (Horan-Hand, P., et al., Cancer Res. 45:833
40 (1986); Hunt, G. Exp. Cell Biol 57 (3):165 76 (1989); Castronovo,
V., et al., Am. J. Pathol. 137 (6):1373 81 (1990); Marques, L. A.,
et al., Cancer Res. 50 (5):1479 83 (1990); Gasparini, G., et al.,
Int. J. Cancer. 60 (5):604 10 (1995)), was also picked up in this
class by the Differential cDNA Sequencing approach. Class II represents
genes that are more abundant in normal breast than in breast cancer.
Although the genes in classes I and II are differentially expressed
in normal breast versus breast cancer, none of these genes are unique
to breast tissues. Class III is a special group of genes that are
selectively expressed in breast relative to other tissue types.
The tissue-specific expression of the unique gene was searched against
approximately 500,000 ESTs using the BLAST program (Altschul, S.
F., et al., J. Mol. Biol. 215 (3):403 10 (1990)). None of these
breast cancer specific genes (BCSG) except the first one matched
with any sequences in public gene sequence databases. BCSG1 was
chosen for analysis as a first putative breast cancer maker gene
because 1) its sequence has been matched with the sequence in public
gene sequence database; and 2) most of the individual EST sequences
in BCSG1 derived from a breast tumor cDNA library. Of the eight
distinctive EST clones in BCSG1, seven of them were discovered in
breast cDNA libraries and only one in a brain library. Of the seven
EST clones discovered in the breast cDNA libraries, six of them
were identified in the breast tumor library and only one in the
normal breast library. After complete sequencing of all 6 EST clones,
one EST clone was found to have a complete full-length sequence.
The open reading frame of the resulting full-length gene is predicted
to encode a 127 amino acid polypeptide. After optimal alignment,
the putative BCSG1-encoded protein shows 54% sequence identity with
the recently cloned non-A.beta. fragment of human Alzheimer's disease
(AD) amyloid protein (Ueda, K., et al., Proc. Natl. Acad. Sci. USA.
90 (23):11282 6 (1993)).
TABLE-US-00002 TABLE 1 PARTIAL LIST OF DIFFERENTIAL EXPRESSED GENES
IN NORMAL VERSUS CANCEROUS BREAST IDENTIFIED BY DIFFERENTIAL cDNA
SEQUENCING EST Genes Cancer Normal Genes more abundant in breast
cancer Breast basic conserved gene 33 9 Cathepsin D 5 1 67 kDa laminin
Receptor 4 0 Elongation factor 1 13 5 Genes More Abundant in Normal
Breast Matrix Gla protein 0 8 23 kDa Highly basic Protein 3 11 EST
Genes NB 1 BC.sup.2 All Tissues Genes as Breast-Specific and Differentially
Expressed BCSG1 1 6 8 BCSG2 0 7 7 BCSG3 0 5 5 BCSG4 4 0 4 BCSG5
0 4 4 1 normal breast; .sup.2breast cancer
Table 1. Complementary DNA libraries were established from a stage
III breast carcinoma and patient-matched normal breast. A total
of 5,934 ESTs were randomly picked and sequence analyzed. More than
2,200 distinctive EST groups were analyzed for quantitative comparison
of EST hits in the pair of cDNA libraries from breast cancer versus
normal breast as described in "Materials and Methods".
The same EST groups were also analyzed by examining the tissue-specific
expression against the total of 500,000 ESTs from a variety of different
cDNA libraries. Only a unique EST group with more than 3 breast-specific
EST hits was listed and the rest of the several dozens EST groups
with fewer than 4 breast-specific EST hits were omitted in this
Expression of BCSG1 in Human Breast Cancer Cells
In an attempt to evaluate the potential biological significance
of BCSG1 on human breast cancer development and progression, BCSG1
gene expression in human breast biopsy samples was examined using
Northern blot analysis.
The RNA from human breast cancer cells was prepared using the RNA
isolation kit RNAzol B (Tel-Test, Inc) based on the manufacturer's
instruction. Equal aliquots of RNA were electrophoresed in a 1.2%
agarose gel containing formaldehyde and transferred to nylon membrane
(Boehringer Mannheim). The membrane was pre-hybridized with ExpressHyb
hybridization solution (Clontech, Inc.) at 68.degree. C. for 30
min. The hybridization was carried out in the same solution with
.sup.32P-labeled BCSG1 probe (1.5.times.10.sup.6 cpm/ml) for 1 hour
at 68.degree. C. The membrane was then rinsed in 2.times.SSC containing
0.05% SDS three times for 30 min at room temperature, followed by
two washes with 0.1.times.SSC containing 0.1% SDS for 40 min at
50.degree. C. The full-length BCSG1 cDNA (SEQ ID NO:1) was isolated
from the Bluescript vector, following EcoRI and Xhol digestion,
and used as a template for preparation of a random-labelled cDNA
probe. Random primer DNA labeling kit was obtained from Boehringer
Mannheim, Indianapolis. .sup.32P-dATP was purchased from Amersham.
The expression of BCSG1 in metastatic breast carcinoma and benign
breast tissue were analyzed by Northern blotting. Overexpression
of the BCSG1 transcript in breast carcinoma. In contrast, the BCSG1
transcript was undetectable in benign breast tissue. The presence
of BCSG1 transcript in human breast tissue and its overexpression
in breast carcinomas are consistent with the differential cDNA sequencing
cloning strategy which suggests a possible role or a biomarker of
up-regulation of BCSG1 in the development of breast cancer.
The expression of BCSG1 was also examined in a variety of human
breast cancer cell lines, namely, primary solid tumor derived cell
lines H3477, H3630, H3680B; pleural effusion derived cell lines
H3396, MCF7, SKBR-3 MDAMB231; infiltrating ductal carcinoma derived
cell lines H3914, H3922, ZR-75-1, T47D. Cell lines of T47D, ZR-75-1,
SKBR-3, MCF-7 and MDA-MB-231 are from ATCC; all other lines were
initially isolated at Bristol-Myers Squibb Pharmaceutical Research
Institute (Liu, J., Cancer Res.).
Northern blot detected the 1 Kb BCSG1 transcript in 2 of the 4
cell lines derived from pleural effusion (i.e., SKBR-3 MDAMB231)
and all 4 cell lines detected from ductal infiltrating carcinomas.
Interestingly, none of the cell lines derived from primary solid
breast carcinoma expressed BCSG1 mRNA. Among these lines, H3922
expressed the highest level of BCSG1 mRNA. The absence of BCSG1
mRNA in some pleural effusion derived cell lines suggest that the
expression of BCSG1 gene may require specific in vivo conditions,
or that it is induced by interactions between the tumor cells and
In Situ Hybridization of BCSG1 in Breast Cancer Cells
In order to localize the cellular source of the BCSG1 expression
and to further assess the biological relevance of the overexpression
of BCSG1 in breast cancers, in situ hybridization was performed
on fixed breast sections from 20 infiltrating carcinomas, 15 in
situ carcinomas, and 15 benign breast lesions (breast hyperplasia
In situ hybridization was carried out as described (M. L. Angerer
& R. C. Angerer, In: In situ hybridization, D. Rickwood and
B. D. Hames (ed.). London: LRL Press., (1992), pp. 15 32). Briefly,
deparaffinized and acid-treated sections (5-um thick) were treated
with proteinase K, pre-hybridized, and hybridized overnight with
digoxigenin labeled anti-sense transcripts from a BCSG1 cDNA insert.
The BCSG1 antisense probe is a 550 bp full-length fragment. The
probe was generated by PstI cut of BCSG1 cDNA plasmid and followed
by T7 polymerase. Hybridization was followed by RNase treatment
and three stringent washings. Sections were incubated with mouse
anti-digoxigenin antibodies (Boehringer) followed by the incubation
with biotin-conjugated secondary rabbit anti-mouse antibodies (DAKO).
The calorimetric detection were performed using a standard indirect
streptavidin-biotin immunoreaction method by DAKO's Universal LSAB
Kit according to manufacturer's instructions.
In these experiments, two aspects of BCSG1 expression were examined:
1) the tissue localization (stromal versus epithelial); and 2) the
correlation of BCSG1 expression and breast cancer malignant phenotype.
A strongly positive BCSG1 hybridization in neoplastic epithelial
cells of highly infiltrating breast carcinomas was observed. The
expression of BCSG1 mRNA was detectable in the neoplastic epithelial
cells in 18 of 20 infiltrating breast carcinomas. No expression
of BCSG1 was detected in the stromal cells. In contrast, expression
of BCSG1 was absent in all 15 cases of normal or benign breast lesions.
A representative negative staining of BCSG1 in an atypical proliferative
breast lesion, a benign fibroadenoma, and normal ductal breast epithelial
cells are presented. Furthermore, in a highly invasive breast carcinoma,
no detectable signal of BCSG1 expression was evident in the residual
normal lobular breast epithelial cells although the surrounding
invasive breast carcinoma cells were stained positive for BCSG1
expression. These in situ hybridization results are consistent with
the Northern blot analysis which showed a strong expression of BCSG1
transcript in breast carcinoma but not in normal or benign breast
It is interesting to note that although a strong BCSG1 signal was
easily detected in the malignant breast epithelial cells of infiltrating
breast carcinoma, the low grade in situ carcinomas showed a sparse
and a light staining. Among 15 in situ carcinomas, 9 specimens were
stained negatively and 6 specimens were partially stained. Both
the intensity of staining and the number of positive cells were
significantly reduced in the in situ breast carcinomas compared
to the strong expression in the metastatic breast carcinomas. These
results, which demonstrated a gradient and stage-specific BCSG1
expression from virtually no detectable expression in normal or
benign breast to the low level and partial expression in the low
grade in situ breast carcinoma and to the high expression in the
infiltrating malignant breast carcinomas, suggest an association
of BCSG1expression with breast cancer malignant progression. Based
on this BCSG1 expression pattern, BCSG1 is useful as a breast cancer
It will be clear that the invention may be practiced otherwise
than as particularly described in the foregoing description and
Numerous modifications and variations of the present invention
are possible in light of the above teachings and, therefore, are
within the scope of the appended claims.
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