A method for detection and prognosis of breast cancer and other
types of cancer. The method comprises detecting expression, if any,
for both an Id-1 and an Id-2 genes, or the ratio thereof, of gene
products in samples of breast tissue obtained from a patient. When
expressed, Id-1 gene is a prognostic indicator that breast cancer
cells are invasive and metastatic, whereas Id-2 gene is a prognostic
indicator that breast cancer cells are localized and noninvasive
in the breast tissue.
It is claimed:
1. A diagnostic and prognostic method useful for detection of aggressive,
metastatic and invasive cells in breast, cervical, ovarian, endometrium
and squamous, prostate and melanoma cells cancer tissue, comprising
steps: a) detecting expression for an Id-1 or Id-2 gene product
in breast tissue obtained from a patient; b) evaluating results
obtained in step (a) wherein Id-1 gene product expression is a prognostic
indicator that cancer cells in the cancer tissue are aggressive
and metastatic and Id-2 gene expression product is a prognostic
indicator that cancer cells in the cancer tissue are non-invasive.
2. The method of claim 1 wherein the expression of Id-1 gene product
to the expression of Id-2 product is defined as a ratio of Id-1
product to Id-2 product, wherein a high ratio indicates aggressive
and metastatic cancer and a low ratio indicates non-invasive localized
3. The method of claim 2 wherein the expression of Id-1 or Id-2
gene is detected as MRNA or Id-1 or Id-2 protein.
4. The method of claim 3 wherein the mRNA is detected using oligonucleotide
probes, primers or antisense sequences.
5. The method of claim 4 wherein the mRNA is detected with a Northern
6. The method of claim 3 wherein the Id-1 or Id-2 protein is detected
with Id-1 or Id-2 antibodies, immunohistochemically or radiographically.
7. The method of claim 3 wherein the proteins are detected with
a Western analysis.
8. The method of claim 3 wherein the Id-1 or Id-2 mRNA, Id-1 or
Id-2 protein is detected as a marker for breast cancer prognosis.
9. The method of claim 1 further detecting aggressive metastatic
and invasive prostate cancer and melanoma.
10. The method of claim 9 wherein the prostate cancer aggressiveness
and invasiveness is detected as high Id-2 expression product and
low Id-1 expression product in aggressive prostate cancer and melanoma.
11. A method for using Id-1 or Id-2 antibodies for detection and
prognosis of breast cancer, comprising steps: a) providing antibodies
specific for Id-1 protein and antibodies specific for Id-2 protein;
b) contacting the Id-1 antibodies with a first sample of breast
tissue and contacting the Id-2 antibodies with a second sample of
breast tissue under conditions allowing the antibodies to bind to
protein, if present, and c) comparing the amount of bound antibody
in each of the first and second samples.
12. The method as in claim 11 wherein the presence of bound antibodies
is determined by visual examination.
13. A kit useful for the diagnosis or prognosis of breast cancer,
comprising: a) a first quantity of antibodies specific for Id-1
protein; b) a second quantity of antibodies specific for Id-2 protein;
and, c) a means for performing an assay wherein the first quantity
of Id-1 antibodies is contacted with Id-1 protein in a breast tissue,
and the second quantity of Id-2 antibodies is adapted with Id-2
protein in a breast tissue, and wherein, if present, the Id-1 antibodies
form an Id-1 protein/antibody complex and the Id-2 antibodies form
an Id-2 protein/antibody complex and wherein the Id-1 protein/antibody
complex and the Id-2 protein/antibody complex quantities are in
a determinable ratio effective for predicting either invasiveness
or non-invasiveness of the breast cancer.
14. A method for treatment and amelioration of a breast, cervical,
ovarian, endometrial, squamous cells, prostate cancer and melanoma
in a patient comprising a step of: a) targeting Id-1 or Id-2 gene
expression with a delivery vehicle comprising a product which affects
positively or negatively Id-1 or Id-2 expression.
15. The method of claim 14 wherein said cancer is breast, cervical,
ovarian, endometrium or squamous cells cancer.
16. The method of claim 15 wherein the product is an antisense
transcript, ribozyme, a molecule that disrupts Id-1 interaction
with a transcription factor or enhances Id-2 interaction with a
transcription factor, RNAi, ITF-2 gene or protein.
17. The method of claim 16 wherein the molecule that disrupts Id-1
or enhances Id-2 interaction is a peptide, pharmaceutical agent
or an organic compound.
18. The method of claim 17 wherein the delivery vehicle is an adenoviral,
adeno-associated viral, lentis viral or retroviral vector, a cationic
liposome, polycationic polymer or polyplex, a pharmaceutically acceptable
composition, or a device which facilitates a delivery of such delivery
19. The method of claim 14 wherein said cancer is prostate cancer
20. The method of claim 19 wherein the product is an antisense
transcript, ribozyme, a molecule that enhances Id-1 interaction
with a transcription factor or disrupts Id-2 interaction with a
transcription factor, RNAi.
21. The method of claim 20 wherein the molecule that enhances Id-1
or disrupts Id-2 interaction is a peptide, pharmaceutical agent
or an organic compound.
22. The method of claim 21 wherein the delivery vehicle is adenoviral,
adeno-associated viral, lentisviral or retroviral, vector, cationic
liposome, polycationic polymer or polyplex, pharmaceutically acceptable
composition, or a device which facilitates a delivery of such delivery
23. The method of claim 22 wherein said cancer is prostate cancer
 This application is based on and claims priority of the
Provisional application Ser. Nos.: 60/232,529 and 60/232,558, both
filed on Sep. 14, 2000.
 This invention was made with government support under Contract
No. DE-AC03-76SF00098, awarded by the United States Department of
Energy, and under NIH Grant for NCI R01CA82548. The Government has
certain rights in this invention.
BACKGROUND OF THE INVENTION
 1. Field of the Invention
 The present invention concerns a diagnosis, prognosis and
treatment of breast, endometrium, cervical, ovarian, squamous cell,
prostate and melanoma cancer. Particularly, the invention concerns
the use of Id-1 and/or Id-2 genes or Id-1 and/or Id-2 products as
diagnostic markers for cancer cells metastatic aggressivity and
use of detection of the Id-1 or Id-2 genes, or a ratio thereof,
or use of detection of the Id-1 or Id-2 products, or a ratio thereof,
for diagnosis and prognosis of breast cancer. The invention further
concerns a method for treatment of breast cancer by targeting Id-1
or Id-2 genes, or a combination thereof, through delivery of antisense
transcripts, ribozymes, small therapeutically active molecules,
drugs, peptides or organic compounds that disrupt Id-1 protein interaction
with bHLH transcription factor or enhance Id-2 action with bHLH
transcription factor and vice versa, RNA, anti-Id-1 RNAi causing
degradation of homologous Id-1 mRNAs, Id-2 as a gene or a protein,
or ITF-2 as a gene or protein, or targeting Id-1 or Id-2 proteins
with antibodies or with compounds which either enhance or impair
their expression thereby affecting the feedback of the gene expression.
The invention further concerns the detection of Id-1 or Id-2 products
or genes or their ratio with a kit comprising anti Id-1 and/or Id-2
antibodies or Id-1 or Id-2 probes.
 2. Description of Related Art
 Breast cancer is one of the most common malignancy among
women and shares, together with lung carcinoma, the highest fatality
rate of all cancers affecting females.
 There are very few diagnostic markers available for breast
cancer detection and those which are available have a predictive
accuracy only about twenty percent. There is no marker available
that can detect or determine cancer cells metastatic aggressivity.
 The current treatment of the breast cancer is limited to
a very invasive, total or partial mastectomy, radiation therapy,
or chemotherapy, later two resulting in serious undesirable side
 It would thus be desirable to have available additional
new diagnostic methods which would detect the presence of cancer
with greater accuracy and which would permit determination of distinction
of highly aggressive breast cancer cells having a tendency to metastasize
from the cancer cells which remain localized and have low probability
of metastatic spread. It would also be desirable to have available
methods for less invasive treatment of the breast or other cancers.
 The mammary gland is one of the few organs that undergo
striking morphological and functional changes during adult life,
particularly during pregnancy, lactation, and involution.
 When normal epithelial breast cells become transformed,
a number of genetic alteration occur which lead to tumorigenesis
and metastasis. These alteration affect growth control, maintenance
of differentiated epithelial functions and invasiveness. Identifying
the genes involved in these processes is essential for understanding
how breast cancer develops, and for deriving better methods for
prognosis and treatment.
 In both humans and mice, fetal virgin adult, and pregnant
mammary glands undergo extensive temporal, structural and spatial
remodeling, which entails invasion, migration, and relocation of
cells to generate the ductal and alveolar structures of the gland.
Once lactation is terminated, there is additional and extensive
tissue remodeling as the gland returns to its resting state.
 During each menstrual cycle, and especially during pregnancy,
lactation and involution, mammary epithelial cells go through cycles
of proliferation, invasion, differentiation and apoptotic cell-death.
The mechanisms that regulate these complex and developmentally coordinated
cell phenotypes are only poorly understood. However, at least some
of the downstream genes that are regulated during these different
stages of mammary development have been identified.
 In recent years, some progress has been also made in elucidating
the mechanisms that regulate mammary gland-specific gene expression
and the transformation of mammary epithelial cells to malignancy.
However, the practical use of these findings for detection, prognosis
and treatment of cancer and its malignant propensities has not been
 It is, therefore, a primary objective of this invention
to provide a method and means for detection and prognosis of breast
cancer, for determination of the malignant aggressivity of cancer
cells and for providing therapeutically effective agents for suppression
and therapy of breast, endometrium, cervical, ovarian, squamous
cells and prostate cancer and melanoma.
 All patents, patent applications and publications cited
herein are hereby incorporated by reference.
SUMMARY OF THE INVENTION
 One aspect of the current invention is a method for diagnosis,
prognosis and treatment of breast, cervical, ovarian, endometrium,
squamous, prostate and melanoma cancer.
 Another aspect fo the current invention is the use of Id-1
and/or Id-2 genes as diagnostic markers for metastatic aggressivity
of breast, cervical, ovarian, endometrium and squamous cancer cells.
 Yet another aspect fo the current invention is the use of
Id-1 and/or Id-2 proteins as diagnostic markers for metastatic aggressivity
of prostate and melanoma cancer cells.
 Still another aspect of the current invention is a method
for detection of the Id-1 or Id-2 genes, or a ratio thereof, or
for detection of the Id-1 or Id-2 products, or a ratio thereof,
as the markers for diagnosis and prognosis of breast cancer.
 Still yet another aspect of the current invention is a method
for treatment of breast cancer and other types of cancer by targeting
Id-1 and/or Id-2 genes, or a combination thereof, through a delivery
of antisense transcripts, ribozymes, small therapeutically active
molecules, drugs, peptides or organic compounds that disrupt Id-1
interaction with a bHLH transcription factor or enhance Id-2 protein
action with a bHLH transcription factor, RNA, anti-Id-1 RNAi causing
degradation of homologous Id-1 mRNAs, Id-2 as a gene or a protein,
or ITF-2 gene or protein.
 Yet another aspect of the current invention is a kit for
detection of Id-1 or Id-2 genes or Id-1 or Id-2 products, or their
ratio, said kit comprising anti Id-1 and/or Id-2 antibodies or anti
Id-1 and/or Id-1 probes.
BRIEF DESCRIPTION OF THE DRAWINGS
 FIG. 1 is a Northern blot showing a pattern of Id-1 and
Id-2 expression in the mouse mammary gland.
 FIG. 2 is a Northern MRNA blot of Id-1 expression in cultured
breast cancer cells that were either growing in 10% serum (G) or
incubated in serum-free medium (SF).
 FIG. 3 is a Western analysis using a polyclonal antibody
against human Id-1 protein with cross-reactive bands around M.sub.R
40,000 (40 Kda) indicating loading and transfer efficiency with
nine cell clones of T47D-Id-1.
 FIG. 4 is a graphical representation of Boyden Chamber invasion
assay for T47D-Id-1 cell clones.
 FIG. 5 is an autoradiogram of T47D cells incubated with
 FIG. 6A is a Western analysis of an Id-1 protein expression
probed with the Id-1 antibody in non-invasive cancer T47D (lane
1) and metastatic cancer MDA-MB-231 (lane 2) cells. The position
of Id-1 protein is indicated. FIG. 6B is an immunohistogram wherein
panels (a), (b), and (c) are representative sections from ductal
carcinomas in situ (DCIS), and panels (d), (e), and (f) are Grade
3 invasive carcinomas analyzed by immunohistochemistry with anti
 FIG. 7 is a Northern blot showing Id-2 mRNA expression in
human breast cancer cell lines cultured in serum-free medium for
 FIG. 8 is a Western analysis showing inverse correlation
between Id-1 and Id-2 protein expression in growing (G) and differentiated
(Diff) mouse mammary SCp2 epithelial cells in culture.
 FIG. 9 is a Northern mRNA analysis showing an inverse correlation
between Id-1 and Id-2 mRNA expression in growing (G), serum starved
(SSt) and laminin-treated mouse mammary SCp2 cells in culture for
24 and 48 hours.
 FIG. 10A shows reduction of .beta.-casein expression in
mammary epithelial cells treated with Id-2 antisense oligonucleotides.
FIG. 10B shows increase of .beta.-casein expression in mammary epithelial
cells infected with a LXSN-Id-2-sense and Id-2-antisense expression
 FIG. 11 is a Northern mRNA analysis displaying a different
pattern of expression in the mouse mammary gland in vivo at different
stages of development wherein V indicates virgin, P indicates pregnant
and L indicates lactation stage. Northern analysis was performed
using cDNA probes for mouse .beta.-casein, Id-1 and Id-2.
 FIG. 12A is a Northern analysis of Id-1 and Id-2 mRNA expression
in human breast cancer cell lines. Cell were cultured in serum-free
medium for 48 hours before RNA was extracted. Lane 1: T47D; lane
2: MCF-7; lane 3: MDA-MB-231 and lane 4: MDA-MB-436 cell lines.
FIG. 12B shows Id-1 and Id-2 expression in MCF-7 growing in 10%
FBS (lane 1) and MCF-7 cultured in serum-free medium for 24 hours
 FIG. 13 is a Western blot showing Id-1 protein expression.
Lane 1 shows MDA-MB436 controls, lane 2 shows MDA-MB436 Id-1 sense
infected with an amphotropic retrovirus and lane 3 shows MDA-MB436
Id-1 antisense infected with an amphotropic retrovirus.
 FIG. 14 is graphical illustration of FIG. 13 showing a conversion
of aggressive MDA436 cells into non-aggressive cells when treated
with Id-1 antisense amphotropic retrovirus in an in vitro invasion
 FIG. 15 is a graph showing decrease in tumor number in 4T1/BalbC
mice treated with various constructs in vivo.
 As used herein:
 "Id" means inhibition of differentiation or DNA
 "Id proteins" means proteins which are inhibitors
of differentiation or DNA binding. Since Id proteins function by
binding basic helix-loop-helix (bHLH) transcription factors, Id-1-
or Id-2-interacting proteins are important transcriptional regulators
of mammary epithelial cell properties.
 "Id-1" means protein expressed by Id-1 gene. High
levels of Id-1 protein are expressed by aggressive and metastatic
breast, cervical, ovarian, endometrium and squamous cancer cells.
High levels of Id-1 protein are expressed in noninvasive prostate
cancer and melanoma.
 "Id-2" means protein expressed by Id-2 gene. Increased
levels of Id-2 protein are crucial for normal breast development.
Breast, cervical, endometrium and squamous cancer cells producing
high level of Id-2 protein are less invasive. Increased levels of
Id-2 protein are expressed by highly invasive and metastatic prostate
 "Id-1-interacting proteins" are proteins which
interact with Id-1 protein. These proteins are, therefore, important
transcriptional regulators of mammary epithelial cell properties.
 "ITF-2" is a bHLH transcription factor which interacts
with Id-1 and is, therefore, an example of Id-1 interacting protein.
ITF-2 appears to be constitutively expressed in SCp2 epithelial
cells. Although Id-1 expression fluctuates during mammary epithelial
cell growth and differentiation, the expression of ITF-2, determined
by ITF-2 mRNA, in such proliferating and differentiating SCp2 cells,
does not fluctuate. The mouse ITF-2 (mITF-2) insert was found to
contain a 950 bp open reading frame encoding the bHLH and C-terminal
domains of ITF-2, but missing the N-terminal region.
 "HLH" means helix-loop-helix.
 "bHLH" means basic helix-loop-helix.
 "GAPDH" means glyceraldehyde-3-phosphate dehydrogenase.
 "DAPI" means 4', 6-diamidino-2-phenylindole.
 "DCIS" means ductal carcinoma in situ.
 "EGR" means early growth.
 "Gene product" means a protein or mRNA.
 "RNAi" means RNA interference process for a sequence-specific
post-transcriptional gene silencing of a gene by providing a double-stranded
RNA (dsRNA) that is homologous in sequence to the silenced gene.
Small interfering RNAs (siRNAs) generated by ribonuclease III cleavage
from longer dsRNA are the mediators of sequence-specific mRNA degradation.
DETAILED DESCRIPTION OF THE INVENTION
 The current invention is based on findings that Id-1 and
Id-2 genes are involved in tumor progression of breast, cervical,
ovarian, endometrium, squamous cells and prostate carcinoma and
melanoma and that Id-1 and Id-2 genes are involved in the development
of breast cancer and are, therefore, suitable to serve as diagnostic
markers and therapeutic targets for these types of cancer.
 Specifically, it has been discovered that Id-1 gene is involved
in and plays a critical role in the development of a proliferative
and invasive phenotype in breast, cervical, endometrium and squamous
epithelial cells and that it is constitutively expressed in the
least differentiated and highly aggressive human cancer cells and
that Id-2 gene is involved in development of a less aggressive or
non-aggressive phenotype in these cancer cells.
 The Id-2 gene, on the other hand is involved in the development
of a proliferative and invasive phenotype in prostate cancer cells,
where Id-1 gene seems to play just the opposite role, that is, it
is involved in the development of a less aggressive or nonaggressive
phenotype and prostate and melanoma cancer cells.
 It has been further discovered that both Id-1 and Id-2 genes,
Id-1 and Id-2 proteins, and their respective ratios, may be conveniently
 Additionally, the invention is based on findings that both
Id-1 and Id-2 genes expression may be effectively suppressed or
at least decreased by the targeted conversion with amphotropic retrovirus
carrying Id-1 or Id-2 antisense.
 Consequently, the invention concerns, in its broadest scope,
a diagnosis, prognosis and treatment of breast, endometrial, cervical,
ovarian, squamous cells or prostate carcinoma or melanoma.
 I. Function of Id-1 and Id-2 Genes and Breast Cancer
 Aggressive breast cancer cells that are metastasizing to
other parts of the body have been known to loose a specific regulation
of the gene involved in normal breast cell development. By contrast,
normally developing breast cells maintain this regulation. Little
is known, however, about the transcriptional regulators that control
the expression of these developmental stage-specific genes.
 Basic helix-loop-helix (bHLH) transcription factors are
key regulators of lineage- and tissue-specific gene expression in
a number of mammalian and non-mammalian organisms. These transcription
factors bind DNA as homo- or heterodimers, and activate the transcription
of target genes containing E-boxes or E-box-like sequences in their
promoters. Dimerization occurs through the HLH domains, whereas
DNA binding occurs through the two basic domains.
 Id proteins, which are inhibitors of differentiation or
DNA binding, are helix-loop-helix (HLH) proteins that lack a basic
domain. Id proteins act as dominant inhibitors of bHLH transcription
factors by forming transcriptionally inactive heterodimers.
 So far, four Id genes (Id-1 through Id-4) have been identified.
These genes, although similar in their organization and HLH sequences,
localize to different chromosomes and show differences in their
pattern of expression and function. For example, the cytogenetic
location of Id-1 protein is 20q11, whereas location of Id-2 is 2p25,
location of Id-3 is 1p36.13-p36.12 and location of Id-4 is 6p22-p21.
 The helix-loop-helix protein Id-1 has been shown to inhibit
the activity of basic helix-loop-helix transcription factors, and
is an important regulator of cell growth and tissue-specific differentiation.
 These findings led inventors to investigate a possible correlation
between the levels of Id-1 protein and the aggressiveness of human
breast cancer cells leading to the current discovery.
 A. Id-1 and Id-2 Gene DNA Sequences
 Nucleotide sequences of human Id-1 and Id-2 genes are known
and have been deposited at GenBank under Accession numbers D13891
and X77956, respectively. Nucleotide sequence which is a source
for Id-1 gene comprises of 926 nucleotides with an Id-1 gene encoding
region starting at nucleotide 36 and ending at nucleotide 500. Nucleotide
sequence which is a source for Id-2 gene comprises of 1049 nucleotides
with Id-2 gene coding region starting at nucleotide 97 and ending
at nucleotide 501.
 B. Function of Id-1 and Id-2 Genes
 It has been now discovered that Id-1 and Id-2 genes function
as negative regulators of helix-loop-helix (bHLH) transcription
factors playing a critical role in the development of a proliferative
and invasive phenotype. Such function of Id-1 and Id-2 genes was
not previously known.
 During the development of the current invention the ectopic
expression of the Id-1 gene has been found to inhibit differentiation
and stimulate the proliferation and invasiveness of mammary epithelial
 The expression of Id-2 gene, on the other hand, has been
found to be up-regulated during differentiation of mammary epithelial
cells and its expression increased in the differentiated human breast
cancer cells. Such up-regulation of Id-2 expression was found to
be a necessary step toward a fully differentiated phenotype in breast
 Compared to expression of Id-1, expression of Id-2 was found
to be much higher in the differentiated human breast cells than
the expression of the very aggressive and metastatic cells leading
to conclusion that there may be a correlation between the levels
of Id-1 or Id-2 proteins and the aggressiveness or non-aggressiveness
in human breast cancer cells.
 The Id-1 and Id-2 protein levels change dramatically at
different stages of breast development. An increase in the level
of Id-2 protein is crucial for normal breast development. In breast
cancer cells, the cancer cells producing high levels of Id-2 protein
are less invasive. By contrast, aggressive and metastatic breast
cancer cells express high level of Id-1 mRNA and Id-1 protein.
 C. Experimental Evidence and Studies
 The evidence supporting the above described findings is
based on studies performed on murine epithelial cell lines, on normal
mouse mammary glands in vivo, on human breast cancer cells and on
human breast cancer biopsies.
 1. Effect of Manipulating Id-1 Expression on Differentiation
of Murine Mammary Epithelial SCp2 Cell Phenotypes
 SCp2 cells, a cell line developed from murine mammary gland,
are a useful model system for studying mammary epithelial cell growth
and differentiation in cell culture.
 A role of Id genes in the normal differentiation of SCp2
cells was first suggested by inventors prior findings that Id-1
expression declined rapidly to undetectable levels when the cells
differentiated in response to lactogenic hormones, such as insulin,
prolactin and hydrocortisone and upon contact with basement membrane
(Mol. Cell Biol., 15:3398-3404 (1995).
 To directly test the role of Id-1 in these cells, the cells
were transfected with an expressible murine Id-1 gene, in either
the sense or antisense orientation.
 In monolayer culture and low serum medium, Id-1 sense cells
grew faster than control cells transfected with the vector lacking
a cDNA insert. By contrast, Id-1 antisense cells grew more slowly
than controls. Both Id-1 sense and Id-1 antisense cells ceased growth
and formed aggregates or spheres when provided with basement membrane
and lactogenic hormones. However, Id-1 sense cells formed spheres
that were less compact than spheres formed by controls or antisense
expressing cells, and failed to express the milk protein .beta.-casein.
Under the same conditions, Id-1 antisense cells expressed .beta.-casein
at a higher level than control cells.
 Despite differences in .beta.-casein expression, control,
Id-1 sense and Id-1 antisense cells exposed to hormones and basement
membrane remained a growth arrested for 5 to 6 days. After 8-10
days, however, spheres of Id-1 sense cells began to disintegrate
as individual cells dissociated from the sphere, began to invade
the basement membrane and resumed growth. In the Boyden Chamber
invasion assay, Id-1 sense cells were much more invasive than normal
SCp2 or Id-1 antisense cells. The Id-1 sense cells, unlike control
or Id-1 antisense cells, expressed a gelatinase of approximately
120 kDa. The activity of this gelatinase was specifically inhibited
by inhibitors of matrix-metalloproteinases.
 Id-1 protein expression in the nontransformed SCp2 cells
resulted in a loss of cell-cell interaction, loss of ability to
express markers of differentiation and in an increased ability to
invade a basement membrane, migrate and proliferate.
 All these propensities make the cells expressing constitutively
high levels of Id-1 protein most highly aggressive and metastatic.
 2. The Role of Id-1 in Normal Mammary Gland Development
 The role of Id-1 in normal mammary gland was determined
by following the expression of Id-1 during normal mouse mammary
gland development in vivo, using Northern analysis of total RNA
from virgin (V), pregnant (P; days 2 to 18), and lactating (L) mice.
Result are shown in FIG. 1.
 FIG. 1 is a Northern analysis of total RNA extracted from
mouse mammary gland at different stages of development. Northern
analysis utilized cDNA probes for mouse .beta.-casein, Id-1 and
Id-2 gene expression.
 As seen in FIG. 1, .beta.-casein mRNA was evident only during
mid and late pregnancy and lactation. When the blot was reprobed
with Id-1 cDNA, Id-1 expression was found to be inversely correlated
with .beta.-casein expression, confirming the role of Id-1 gene
in vivo observed in the SCp2 cells.
 These results clearly show that Id-1 expression declines
when the mammary gland proceeds toward full differentiation during
pregnancy and at the lactation stage. Id-1 thus is expressed primarily
in cells which are nondifferentiated or begin to differentiate.
 3. Analysis of Id-1 Expression in Human Breast Cancer Cell
lines and Breast Biopsies
 Findings that ectopic Id-1 expression induced an invasive
phenotype in mouse mammary epithelial cells suggested that Id-1
gene could contribute to human breast cancer progression.
 To begin to explore this possibility, human breast cancer
cell lines exhibiting varying degrees of invasiveness in culture
and in vivo, using metastatic tumor formation in nude mice, was
examined. Results of these studies show that highly aggressive human
breast cells have lost their serum regulation of Id-1 gene expression.
Results are shown in FIGS. 2-6.
 The regulation of Id-1 gene expression in the presence of
serum was examined in non-invasive cancer T47D and MCF-7 cell lines
and in aggressive and invasive cancer MDA-MB-231 and MDA-MB-435
cell lines. The first two are noninvasive human breast cancer cell
lines, the latter two are highly invasive metastatic cells which
were selected for a highly aggressive phenotype by passage in immunodeficient
mice. All cells were purchased from the American Tissue Culture
 In some cells, Id-1 gene expression is known to be induced
by certain mitogens, such as, for example, serum. Consequently,
the effect of the presence or absence of serum on expression of
Id-1 gene in these two types of cells was investigated. RNA was
isolated from both types of cells that were grown on either 10%
serum (G) or incubated in serum-free medium (SF). RNA was then analyzed
by Northern analysis according to Example 3. Results are seen in
 FIG. 2 illustrates a loss of serum-regulated Id-1 expression
in aggressive breast cancer cells. Upper panel shows a position
of Id-1 mRNA (1.2 kb) . Lower panel shows a position of the ribosomal
28S RNA used as control for RNA integrity and quantitating.
 As seen in FIG. 2, T47D and MCF-7 non-invasive cancer cells
expressed high levels of Id-1 mRNA only when cultured in serum.
When cultured in serum-free medium for two days, such expression
levels were undetectable. In contrast, highly aggressive and metastatic
MDA-MB-231 and MDA-MB-435 cells constitutively expressed Id-1 mRNA,
regardless of the presence or absence of serum.
 These results show that in non-invasive breast cancer cell,
the expression of Id-1 gene could be induced by culturing these
cells in the presence of serum. However, in these non-invasive breast
cancer cells, this gene was not expressed and the expression could
not be induced in serum-free medium. On the contrary, the invasive
metastatic cancer cells expressed Id-1 gene in both the serum containing
and serum-free medium. Consequently, the invasive metastatic breast
cancer cells do not need Id-1 expression induction by serum but
it is in their cellular make-up to express Id-1 gene constitutively.
 4. Constitutive Id-1 Expression Converts a Nonaggressive
into a More Aggressive Breast Cancer Cell Line
 To test whether the unregulated Id-1 expression contributes
to aggressive phenotype of human breast cancer cells and to determine
if the induced constitutive Id-1 expression would convert a nonaggressive
cells into an aggressive metastatic cells, constitutive Id-1 expression
 For this purpose, the human Id-1 cDNA was expressed in nonaggressive
T47D cells using amphotropic retrovirus (pBabe-Id-1 ). Production
of pBabe-Id-1 retroviral vector and virus are described in Example
1. Retroviral infection is described in Example 2. Puromycin was
used to select virus-expressing cells.
 Briefly, approximately eight RT-units of either pBabe-puro
or pBabe-Id-1 retrovirus were mixed with 5 ml of medium containing
4 .mu.g/ml polybrene and were added to T47D cells in 100-mm dishes.
Cells expressing the retroviral genes were selected in 0.6 .mu.g/ml
puromycin, which killed all of the mock-infected cells within three
days, whereas 80 or 30% of the pBabe-puro or pBabe-Id-1-infected
cells, respectively, survived. The puromycin-resistant cells are
referred to as T47D-pBO or T47D-Id-1. To establish single-cell clones,
the T47D-Id-1 population was plated at 1-2 cells/well in 24-well
tissue LXSN retroviral vector was prepared in the same way except
neomycin was used to select virus expressing cells culture plates.
Clones that grew in the wells were expanded. Results are seen in
 FIG. 3 illustrates Id-1 protein levels obtained in nine
clones. When T47D cells were infected with pBabe-Id-1 retrovirus,
nine single-cell-derived clones (clone 1-clone 9) were obtained.
The clones were cultured in serum-free medium for two days before
protein extraction and Western analysis using a polyclonal antibody
against human Id-1. Positions of Id-1 protein and molecular weight
markers in each clone are indicated. Cross-reactive bands around
M.sub.r 40,000 (4OkDa) indicate loading and transfer efficiency.
 From nine single-cell-derived clones isolated from the T47D-Id-1
population, the clone 6 was lost during processing. Each of the
eight surviving clones expressed a different level of Id-1 protein,
as determined by Western analysis. Clones 1, 2, and 8 expressed
relatively high levels of Id-1 protein in serum-free medium, whereas
clones 4 and 9 expressed very low levels of Id-1 under these conditions.
The other clones expressed Id-1 at intermediate levels.
 Five T47D-Id-1 clones, expressing either high or low levels
of Id-1 in serum-free medium, were then examined for invasiveness
using the Boyden Chamber invasion assay. Conditions of the Boyden
Chamber invasion assay are described in Example 5. Results are shown
in FIG. 4.
 FIG. 4 illustrates Boyden Chamber invasion assay for T47D
clones. Cells were cultured in serum-free medium for 2 days before
they were placed in the upper chamber of Matrigel-coated trans-well
filters. The invasion assay was carried out for 20 hours in serum-free
medium and cells that migrated through the filter were stained and
counted. Results were averaged and SDs were calculated.
 As seen in FIG. 4, the invasive activity of each clone was
approximately proportional to the level of Id-1 protein expression.
Thus, clones with constitutively high levels of Id-1 (clones 1,
2, and 8) were more invasive then clones expressing low levels of
Id-1 protein (clones 4 and 9). The invasive activity of the low-expressing
clones resembled that of the uninfected parental T47D cells (not
 Ectopic Id-1 expression also conferred a growth advantage
in serum-free medium, as measured by the percentage of cells incorporating
[.sup.3H]-thymidine. Conditions of the [.sup.3H]-thymidine labeling
are described in Example 6. Results are seen in FIG. 5.
 FIG. 5 shows percentage of labeled nuclei of cells cultured
in serum-free medium for 32 hours before incubation with [.sup.3H]-thymidine
for additional 16 hours and processed by autoradiography. Cell that
incorporated [.sup.3H]-thymidine were calculated as a percentage
of total DAPI-stained nuclei.
 As seen in FIG. 5, the three T47D-Id-1 clones that expressed
Id-1 proteins at higher levels had a greater [.sup.3H]-thymidine-labeling
index than two clones in which Id-1 expression was lower. The three
T47D-Id-1 clones that expressed Id-1 protein at higher levels had
a greater thymidine-labeling index than two clones in which expression
of Id-1 protein was lower. Thymidine-labeling index for clones 1,
2 and 8 was 59%/average, for clones 4 and 9 it was 36%/average.
 These results show that when normal Id-1 regulation is lost
and Id-1 is constitutively expressed, human breast cancer cell lines
acquire increased invasiveness and a proliferative advantage in
a growth factor-deficient media. Ectopic Id-1 expression converted
a relatively nonaggressive breast cancer cell line into a relatively
 These results show that by determining a level of Id-1 protein
expression, evaluation of the breast cells aggressivity can be made.
 Since the above findings indicated that Id-1 expression
may serve as a prognostic marker for certain subset of aggressive
breast cancers, breast cancer biopsies for Id-1 expression were
further examined by immunohistochemistry.
 5. Id-1 Expression in Breast Cancer Biopsies
 To determine whether the above obtained observations are
applicable to humans, a large number of breast cancer biopsies were
obtained from patients and immunohistochemical reactions as well
as Western analyses were performed.
 Immunohistochemical determination of the expression of Id-1
protein was carried out on a total of eighty-three breast cancer
biopsies obtained from patients treated at California Pacific Medical
 Twenty-three of the biopsies were ductal carcinoma in situ
(DCIS), sixty biopsies were infiltrating carcinomas of which twelve
were Grade 1, seven were Grade 2 and forty-one were of Grade 3 carcinoma.
 Out of twenty-three ductal carcinomas in situ (DCIS), 18
were found negative (78%), three were weakly positive (13%), and
two were strongly positive (9%). Infiltrating carcinomas Grade 1,
which is the least aggressive amongst the invasive tumors, displayed
a pattern of Id-1 protein expression similar to the DCIS. Out of
twelve Grade 1 carcinoma, 10 were negative (83%), 1 was weakly positive
(8.5%), and 1 was strongly positive (8.5%). On the other hand, the
majority of the infiltrating Grade 2 and Grade 3 carcinomas, the
most aggressive tumors, were weakly or strongly Id-1 positive. Out
of seven Grade 2 carcinomas, 3 were negative, 1 was weakly positive,
and 3 were strongly positive. Out of forty-one Grade 3 carcinomas,
16 were negative (39%), 4 were weakly positive (10%), and 21 were
strongly positive (51%).
 Results are seen in Table 1.
1TABLE 1 Id-1 Protein Expression Determined By Immunohistochemistry
in 83 Breast Cancer Biopsies Id-1 Weakly Id-1 Strongly Tumor Type
Id-1 Negative Positive Positive Ductal 78% (18/23) 13% (3/23) 9%
(2/23) Carcinoma in Situ Infiltrating Carcinoma Grade 1 83% (10/12)
8.5% (1/12) 8.5% (1/12) Grade 2 43% (3/7) 14% (1/7) 43% (3/7) Grade
3 39% (16/41) 10% (4/41) 51% (21/41)
 Numbers in parenthesis indicate the actual number of biopsies
out of the total number of biopsies examined.
 Results of six selected representative samples in this assay
are seen in FIGS. 6A and 6B which show expression of Id-1 in human
breast cancer biopsies. Immunohistochemistry was carried out using
a specific batch of anti-Id-1 antibody, confirmed by Western analysis
to show no cross-reactive bands. Immunohistochemical procedure is
described in Example 8.
 FIG. 6A is a Western analysis showing the specificity of
the Id-1 antibody used for immunohistochemistry. Lane 1 shows non-invasive
T47D cancer cells, lane 2 shows invasive and metastatic MDA-MB-231
cancer cells. All cells were cultured in serum-free medium for 48
hours. Position of Id-1 protein is indicated. No cross-reactive
band is seen. Results shown in FIG. 6A clearly confirm high expression
of Id-1 protein in the cancer cells when compared to Id-1 expression
in T47D cells.
 FIG. 6B shows representative section from DCIS (panels a,
b, and c) and Grade 3 invasive carcinoma (panels d, e and f) which
were analyzed by immunohistochemistry using antiserum against Id-1
protein. The majority of DCIS were negative (panels a and b), one
showed strong positivity in its large ductal structure (panel c)
. The majority of infiltrating carcinoma, on the other hand, showed
strong Id-1 immunoreactivity (panel d and e) . Minority of the invasive
tumors were negative (panel f). In panel d, a differentiated glandular
section, the structure with the lumen was negative whereas infiltrating
cells showed strong immunoreactivity.
 These results show that almost all examined ductal carcinomas
in situ (DCIS) were negative for Id-1 staining. However, the majority
(51%) of infiltrating Grade 3 carcinomas of ductal origin were strongly
Id-1 positive. These results confirm that Id-1 is a reliable prognostic
marker for breast cancer invasiveness and metastatic propensity.
 6. Expression of Id-2 in Human Breast Cancer Cells
 To determine if the expression of Id-1 protein was specific
to aggressive malignant cancer cells or if this was common property
of Id proteins, the expression of the second Id protein, namely
Id-2 protein, in human breast cancer cells was examined.
 Id-2 expression in human cancer cells was determined by
Northern analysis. The same four types of cells were used as used
previously in studies with Id-1. These cells were cultured in serum-free
medium for two days before RNA was extracted. The blot was hybridized
with a human Id-2 cDNA probe. Results are shown in FIG. 7.
 FIG. 7 is a Northern analysis of Id-2 transcripts. Upper
panel shows expression of Id-2 mRNA in non-invasive T47D (lane 1)
and MCF7 (lane 2) cancer cells and in highly aggressive, metastatic
and invasive MDA-MB-231 (lane 3) and MDA-MB436 (lane 4) cancer cells.
Lower two panels show a positions of two ribosomal 28S and 18S RNA
used as control for RNA integrity.
 FIG. 7 shows that under the same experimental conditions
as those described for Id-1, Id-2 mRNA was found to be expressed
in lanes 1 and 2, which correlate with non-invasive T47D and the
MCF7 human breast cancer cell lines. As seen in FIG. 7, lanes 3
and 4, there was no detectable Id-2 mRNA in lanes 3 or 4, which
represent highly invasive MDA-MB-231 and MDA-MB-436 human breast
cancer cell lines.
 Thus, in contrast to Id-1, the expression of Id-2 gene products,
such as the protein and mRNA, correlates with non-aggressive or
 These results show that both Id-1 and Id-2 are fair indicators
of breast cancer presence and aggressivity and that each indicates
and is found in a different type of cancer cells. Detection of Id-1
expression indicates presence of highly aggressive, metastatic and
invasive cancer cells. Detection of Id-2 expression indicates presence
of noninvasive cancer cells.
 7. Inverse Correlation between Id-1 and Id-2 Expression
 A direct regulatory link has been found to exist between
Id-1 and Id-2 genes in breast cells. Id-2 protein expression is
generally high when Id-1 protein expression is low, both in vitro
and in vivo, confirming an existence of a negative correlation in
 a. Id-2 Expression In Vitro
 To determine the pattern of Id-2 expression during mammary
cell growth and differentiation, expression of Id-2 protein during
mammary epithelial cell differentiation in vitro and in vivo was
 For this purpose, the yeast two-hybrid system and the basic
helix-loop-helix protein ITF-2 as a bait were used to isolate Id-2
from a library derived from differentiated, milk-producing mammary
epithelial cells. First, Id-2 protein expression in SCp2 cells during
proliferation or differentiation was investigated, using Western
analysis. Results are shown in FIG. 8.
 FIG. 8 is a Western analysis showing inverse correlation
between Id-1 and Id-2 protein expression in growing (G) and differentiated
(Diff) SCp2 mammary epithelial cells treated with Matrigel and lactogenic
hormones for 48 and 72 hours. Protein was extracted and analyzed
using antibodies specific for Id-1, Id-2 and .beta.-casein milk
protein, which is the marker for mammary epithelial cells differentiation.
 As shown in FIG. 8, differentiated cells expressed high
levels of the Id-2 (16 kDa) protein, similarly to expression of
.beta.-casein, at both 48 and 72 hours. In comparison, Id-1 protein
was detectable only in proliferating cells (lane G). No expression
of Id-1 protein was detected in differentiated cells. These results
clearly show that there is an inverse correlation between Id-1 and
 To confirm this inverse correlation between Id-1 and Id-2
expression, Northern analysis of SCp2 cells proliferating or treated
with laminin for 24 and 48 hrs was performed. Laminin is an important
component of extracellular matrix and can trigger differentiation.
Results are seen in FIG. 9.
 FIG. 9 is a Northern analysis of inverse correlation between
Id-1 and Id-2 mRNA expression in growing (G), serum starved (SSt),
and Laminin-treated SCp2 mammary epithelial cells for 24 and 48
hours. Total RNA was extracted and analyzed using probes specific
for Id-1, Id-2 and .beta.-casein.
 Results seen in FIG. 9 confirm results seen in FIG. 8. There
was expression of both Id-2 and .beta.-casein in differentiated
cells, but there was no expression of Id-1 in these cells. Id-1
was expressed only in growing (G) cells confirming that the inverse
correlation exists between expression of Id-1 and Id-2 mRNA.
 In order to determine if Id-2 up-regulation was a crucial
event for mammary epithelial cell differentiation and milk production,
two sets of experiments were performed. In the first set, SCp2 cells
were treated with Laminin and lactogenic hormones for 48 hrs in
the presence of either control oligonucleotides or Id-2 antisense
oligonucleotides. Results are seen in FIG. 10.
 FIG. 1OA illustrates reduction of .beta.-casein expression
in mammary epithelial cells treated with Id-2 antisense oligonucleotides.
Lane 1 shows SCp2 cells treated with Laminin for 48 hours and control
oligonucleotide. Lane 2 shows Scp2 cells treated with Laminin for
48 hours and with Id-2 oligonucleotide. FIG. 10B illustrates increase
of .beta.-casein expression in mammary epithelial cells infected
with a LXSN-Id-2 sense expression vector (Lane 2) and inhibition
of .beta.-casein expression in cells infected with a LXSN-Id-2 antisense
expression vector (Lane3) . Lane 1 corresponds to cells infected
with a LXSN-control vector.
 As seen in FIG. 10A, a dramatic reduction of .beta.-casein
expression was observed in Id-2 antisense oligonucleotide treated
cells. In the second set of experiments, SCp2 cells were infected
with either LXSN-control, LXSN-Id2-sense or LXSN-Id2-antisense constructs,
selected with neomycin and treated with laminin for 48 hrs. As shown
in FIG. 10B, .beta.-casein expression was increased in SCp2-LXSN-Id2-sense
cells in comparison to control. Most dramatically, .beta.-casein
expression was almost undetectable in SCp2-LXSN-Id2-antisense cells.
 The results seen in FIGS. 1OA and lOB show that Id-2 is
involved and necessary in and its up-regulation occurs during mammary
cell differentiation. However, the results in FIG. 10B also shows
that such up-regulating can be effectively negated with Id-2 antisense
 b. Id-2 Expression In Vivo
 To determine Id-2 protein expression in vivo and to compare
it to the expression of Id-1 protein, another set of experiments
 In these studies, the level of Id-1 and Id-2 mRNA during
mammary gland development in vivo, using Northern analyses of total
RNA from virgins, pregnant and lactating mice were determined. Results
are seen in FIG. 11.
 FIG. 11 shows a different pattern of Id-1 and Id-2 protein
expression in the mouse mammary gland in vivo. Total RNA was extracted
from mouse mammary glands at different stages of development. Northern
analyses using sDNA probes for mouse .beta.-casein, Id-1 and Id-2
were performed. V indicates virgin; P indicates pregnant at days
2, 5, 12 and 18 and L indicates lactation mammary gland.
 As seen in FIG. 11, .beta.-casein mRNA was evident only
during mid and late pregnancy and during lactation. When the blot
was then reprobed with a mouse Id-1 cDNA, Id-1 mRNA expression resulted.
Such Id-1 expression was inversely correlated with .beta.-casein
expression, suggesting a similar role for Id-1 gene in vivo to that
observed in the SCp2 cells, that is, Id-1 expression declines when
the mammary gland proceeds towards full differentiation as, for
example, in lactation stage. On the other hand, expression of Id-2
mRNA was barely detectable in virgin gland and at the beginning
of pregnancy. Its expression increased at day 12 of pregnancy, when
epithelial cells start producing the milk protein .beta.-casein.
Id-2 expression was at its highest level toward the end of pregnancy
(day 18) and lactation, when the epithelial cells were fully differentiated.
 The above results show that the expression pattern of Id-2
mRNA or gene expression is different from that of Id-1 MRNA. Id-2
expression level is opposite to that of Id-1 expression during periods
of cell growth and differentiation. This further indicates a differentiating
role for Id-2, in contrast to Id-1, during mammary gland development.
 The terminal development of the mammary gland involves the
contribution of proliferative as well as differentiative events.
These events must be tightly coordinated. Id-2 as well as Id-1 were
shown to play a central role in this regulation by negatively regulating
different sets of bHLH proteins. Moreover, the expression of these
two genes was found to be tightly coordinated.
 c. Analysis of Id-2 Expression in Breast Cancer Cells
 To confirm that similar findings to those found in murine
mammary epithelial cells in vitro and in vivo, Id-2 expression was
investigated in human breast cancer cell lines in culture using
the same mouse Id-2 cDNA probe.
 For this purpose, the two T47D and MCF7 cancer cell lines
which display non-aggressive and differentiated characteristics
in culture (in absence of estrogen), and the two highly aggressive
and metastatic MDA-MB-231 and MDA-MB-436 cell lines were used. The
cells lines were described above. Results are seen in FIG. 12.
 FIG. 12A is a Northern analysis of Id-1 and Id-2 mRNA expression
in human breast cancer cell lines. Cells were cultured in serum-free
medium for 48 hours before RNA was extracted and subjected to blotting.
Lane 1 shows T47D cancer cell line; lane 2 shown MCF-7 cancer cell
line; lane 3 shows MDA-MB-231 cancer cell line and lane 4 shows
MDA-MB-436 cancer cell line. FIG. 12B shows Id-1 and Id-2 expression
in MCF-7 growing in 10% FBS (lane 1) and MCF-7 cultured in serum-free
medium for 24 hours (lane 2).
 As seen if FIG. 12A, when cultured in serum-free conditions
for 48 hrs, MCF-7 cells, and to a lesser extent T47D cells, expressed
high levels of Id-2 mRNA. However, Id-2 expression was undetectable
in the two aggressive cell lines MDA-MB-231 and MDA-MB-436 where,
as expected, Id-1 was highly expressed. Id-1 expression was not
detected in non-aggressive T47D and MCF-7 cancer cells.
 These results again confirm, this time in human breast cancer
cells, the inverse correlation between the expression of the two
HLH proteins that was previously determined to exist in mammary
epithelial cells and imply a different role for Id-2 from Id-1 in
breast cancer cell phenotypes. This is seen especially clearly in
FIG. 12B, where, upon serum-withdrawal, the levels of Id-2 mRNA
were found to be increased in MCF-7 cells whereas the levels of
Id-1 MRNA were decreased.
 All the data presented above clearly show the role of the
two helix-loop-helix proteins, Id-1 and Id-2, as molecular switches
not only between growth/invasion and differentiation in mammary
epithelial cells, but also during breast cancer progression.
 8. Targeting Id-1 Reduces Breast Cancer Cell Invasion In
 To determine whether the Id-1 is a key gene which regulates
the aggressive phenotype of human breast cancer cells, studies were
performed to determine whether Id-1 antisense expression converts
a very aggressive and metastatic breast cancer cell into a non-aggressive
 For this purpose, the human Id-1 cDNA was expressed in a
sense as well as an antisense orientation in human metastatic MDA-MB436
breast cancer cells using an amphotropic LXSN-Id-1 sense and antisense
retrovirus. Neomycin was used to select for virus-expressing cells.
Results are shown in FIG. 13.
 FIG. 13 is a Western analysis of Id-1 expression of highly
aggressive and invasive MDA-MB436 cancer cells. Actin was used as
control. Lane 1 shows MDA-MB436 cells as control against MDA-MB436
treated with Id-1 sense retrovirus (lane 2) or MDA-MB436 treated
with Id-1 antisense infected with retrovirus (lane 3).
 As seen in FIG. 13, cells infected with a control virus
(empty plasmid, lane 1) expressed detectable levels of Id-1 protein
in serum-free medium. The LXSN-ld-1 sense infected population (lane
2) expressed even higher levels of Id-1 protein whereas the LXSN-Id-1
antisense infected cells (lane 3) expressed very low levels of Id-1
under these conditions.
 The same three populations of cells were then tested in
a Boyden Chamber invasion assay to compare their ability to migrate
and invade a reconstituted basement membrane. Results are seen in
 FIG. 14 shows results of the invasion assay where the assays
were performed in modified Boyden Chambers assay described in Example
5 with 8 .mu.m pore filter inserts for 24-well plates obtained from
Collaborative Research. Filters were coated with 10-12 .mu.l of
ice-cold Matrigel (7.3 mg/ml protein) obtained from Collaborative
Research. Cells (100,000 per well) were added to the upper chamber
in 200 .mu.l of the appropriate medium containing 0.1% bovine serum
albumin (BSA). In general, cells were assayed in triplicate or quadruplicate,
and the results averaged. The lower chamber was filled with 300
.mu.l of NIH-3T3 cell-conditioned medium according to Cancer Res.,
47:3239-3245 (1987). After a 20 hours incubation, cells were fixed
with 2.5% glutaraldehyde in PBS and stained with 0.5% toluidine
blue in 2% Na.sub.2CO.sub.3. Cells that remained in the Matrigel
or attached to the upper side of the filter were removed with cotton
tips. Cells on the lower side of the filter were counted using light
 The invasive activity of each cell population was proportional
to the level of Id-1 protein expression as seen in Western blot
shown in FIG. 13. The population with high levels of Id-1 (LXSN-Id-1
sense cells, lane 2) was much more invasive than the population
expressing low levels of Id-1 (LXSN-Id-I antisense cells, lane 3).
The invasive activity of the control population expressing intermediate
levels of Id-1 protein was also intermediate (lane 1).
 These results further confirm that the aggressivity and
invasiveness of the human breast cancer cells can be attributed
to the high expression of Id-1 gene and also show that aggressivity
of cells expressing Id-1 protein can be reduced or eliminated by
treatment with an Id-1 antisense constructs. Consequently, the expression
of Id-1 in human breast cancer cells is a good prognostic and diagnostic
tool for detection of aggressive breast cancer and for distinguishing
such aggressive and invasive cancer from the non-invasive cancer
cells attributable to their expressing Id-2 protein.
 9. Targeting Id-1 Reduces Breast Cancer Cell Metastasis
 Following the finding that targeting Id-1 with an antisense
comprising construct reduces aggressivity of breast cancer cells
in vitro, further studies were undertaken to determine if the same
would be valid for breast cancer cells in vivo, and if the metastatic
propensity of cancer cells expressing Id-1 could be changed to nonaggressive
 In order to determine the role of Id-1 in the metastatic
process in vivo, the 4T1 murine metastatic breast cancer cell line
which express, like human MDA-MB231 and MDA-MB436 cells, high levels
of Id-1 mRNA and protein and which metastasize to the lungs were
used. In order to deliver the Id-1 antisense constructs, the technique
of cationic liposome-DNA complex (CLDC)-based intravenous gene delivery
according to J. Biol. Chem., 274:13338-13344(1999) was utilized.
This CLDC-based intravenous (iv) delivery (tail vein injections)
of Id-1 antisense construct, such as plasmid, significantly reduced
the metastatic spread of 4T1 breast cancer cells in 4T1BalbC mice.
Results are seen in FIG. 15.
 FIG. 15 is a graph illustrating a tumor reduction in 4T1/BalbC
mice treated with various constructs. Specifically, the mice were
treated with luciferase (lane 1), with irrelevant gene serving as
another control (lane 2) and with Id-1 antisense (lane 3).
 Results shown in FIG. 15 clearly show that the number of
highly aggressive and metastatic tumor decreases significantly when
the tumor cells are targeted with Id-1 antisense construct.
 Specifically, a single injection of CLDC containing Id-1
antisense, three days after iv injection of 50,000 4T1 cells, dramatically
reduced the total number of lung metastases (lane 3), when compared
to tumor-bearing mice treated with CLDC containing control genes
(luciferase as well as an irrelevant gene, lanes 1 and 2).
 These results show first that the aggressive tumor growth
and metastasis can be treated with antisense Id-1 construct and,
second, that CLDC-based plasmid antisense delivery, which is a novel
delivery approach, is a practical way of achieving such delivery.
 10. Cumulative Evidence for Id-1 and Id-2 Function in Breast
Cancer Aggressivity and Diagnosis and Treatment Thereof
 Invention described herein showed that aggressive metastatic
breast cancer cells express high levels of Id-1 mRNA because of
a loss of serum-dependent relation that is mediated by the 2.2-kb
region of the human Id-1 promoter. This suggests that unregulated
Id-1 gene expression may be an important regulator of the aggressive
phenotype of a subset of human breast cancer cells. The results
disclosed herein further implicated Id-1 gene as a critical downstream
target of steroid hormones and critical mediator of the aggressive
phenotype in a subset of human breast cancer cells.
 Specific findings are as follows:
 The Id-1 gene is highly expressed during proliferation,
and is down-regulated when mammary epithelial cells differentiate.
The Id-2 gene is not expressed in growing mammary epithelial cells,
and is up-regulated during differentiation.
 Id-1 expression declines when the mammary gland proceeds
toward full differentiation during pregnancy and at the lactation
stage. Id-1 thus is expressed primarily in cells which are nondifferentiated
or begin to differentiate.
 In non-invasive breast cancer cell, the expression of Id-1
gene can be induced by culturing these cells in the presence of
serum. However, in these non-invasive breast cancer cells, this
gene is not expressed and the expression cannot be induced in serum-free
medium. To the contrary, the invasive metastatic cancer cells express
Id-1 gene in both the serum containing and serum-free medium. Consequently,
the invasive metastatic breast cancer cells do not need Id-1 expression
induction by serum but it is in their cellular make-up to express
Id-1 gene constitutively.
 The constitutive expression of Id-1 inhibits differentiation
of mammary epithelial cells, and induces proliferation and invasion.
 Certain aggressive breast cancer cells constitutively express
high levels of Id-1 protein, apparently due to the loss of serum-dependent
 The expression of Id-1 directly correlates with the level
of aggressiveness in breast cancer cell lines and evaluation of
the breast cells aggressivity can be made in breast cancer biopsies
by determining a level of Id-1 protein expression. Almost all examined
ductal carcinomas in situ (DCIS) were negative for Id-1 staining.
However, the majority (51%) of infiltrating Grade 3 carcinomas of
ductal origin were strongly Id-1 positive. These results confirm
that Id-1 is a reliable prognostic marker for breast cancer invasiveness
and metastatic propensity.
 The expression of Id-2 directly correlates with the level
of differentiation and non-aggressiveness breast cancer cells. Id-2
is involved in and its up-regulation occurs during mammary cell
differentiation. Such up-regulating can be effectively negated with
Id-2 antisense carrying construct.
 Id-1 and Id-2 are fair indicators of breast cancer presence
and aggressivity and each indicates and is found in a different
type of cancer cells. Detection of Id-1 expression indicates presence
of highly aggressive, metastatic and invasive cancer cells. Detection
of Id-2 expression indicates presence of noninvasive cancer cells.
The expression pattern of Id-2 protein is different from that of
Id-1 protein. Id-2 expression level is opposite to that of Id-1
expression during periods of cell growth and differentiation.
 The expression of Id-1 in human breast cancer cells is a
good prognostic and diagnostic tool for detection of aggressive
breast cancer and for distinguishing such aggressive and invasive
cancer from the non-invasive cancer cells attributable to their
expressing Id-2 protein.
 The aggressive tumor growth can be treated with antisense
Id-1 construct and CLDC-based plasmid antisense delivery is a practical
way of achieving such delivery.
 The Id-2 protein level changes dramatically at different
stages of breast development in the opposite direction of the Id-1
protein level. The increase in the level of Id-2 protein is crucial
for normal breast development, and breast cancer cells that produce
high levels of Id-2 protein do not, or are less likely to, migrate
and invade. They will remain localized in the breast, will not metastasize
and are therefore easier to treat.
 II. Method for Detection, Diagnosis and Prognosis of Breast
 A method for detection of the aggressive and invasive cancer
cells or noninvasive cancer cells comprises detection of Id-1 and/or
Id-2 genes, or their ratio, or Id-1 and/or Id-2 products, or their
ratio, as diagnostic markers for detection of metastatic aggressivity
of carcinoma. Such detection is useful both for diagnostic and particularly
for prognostic purposes in patients.
 As earlier noted, Id-I protein is expressed at elevated
levels in aggressive breast cancer cell lines. These highly aggressive
breast cancer cells have lost serum-dependent regulation of the
Id-1 gene expression, which results in constitutively high levels
of Id-1 protein. Indeed, it appears that the Id-1 protein plays
a key role in the malignant progression of a subset of aggressive
and invasive human breast cancers.
 While Id-1 represents a marker of poor prognosis for invasive
and metastatic breast cancer, in contrast Id-2 represents a marker
of good prognosis for breast cancer since the breast cancer cells
expressing Id-2 will tend to be localized and not metastasized.
 A patient found to have breast cancer, but breast cancer
in which Id-2 is being expressed, is one for whom the prospect of
recovery by simpler and less invasive techniques, such as lumpectomy,
is suggested. Such a patient, therefore, likely does not need the
more radical treatments, such as mastectomy, radiation or chemotherapy,
that would otherwise be recommended for invasive breast cancer when
the high expression of Id-1 protein is detected.
 A. Methods Suitable For Detection of Id-1/Id-2 Expression
 In a therapeutic method of this invention described below,
the treating physician who has, for example, found tumors/lumps
will typically send a breast tissue sample, as a biopsy, to a pathologist
for examination and diagnosis.
 The examination and classification of the tissue is typically
based on a visual inspection of tissue morphology. For example,
the pathologist can decide whether the biopsied tissue is an infiltrating
or invasive carcinoma or whether it is ductal carcinoma in situ
(DCIS). Within each of these classifications the pathologist attempts
to assign grades of aggressiveness, such as infiltrating Grade 1
carcinoma, which is not overly aggressive, or infiltrating Grade
3 carcinoma that is very aggressive.
 The development of a DCIS into a highly aggressive and metastatic
breast tumor involves a series of sequential steps; breast epithelial
cells must lose the ability to interact with other cells, acquire
the ability to digest the surrounding basement membrane, migrate
toward the blood stream, and survive and proliferate in ectopic
sites. Invasiveness marks the onset of metastasis, which is a hallmark
of often final malignant progression.
 For detection of Id-1/Id-2 proteins, the immunohistochemistry
analysis using Id-1 antibodies can be used together with Id-2 antibodies,
since a determination of both Id-2 and Id-1 expression, or lack
of expression for one with respect to the other, will help the treating
physician and pathologist determine the type or grade of breast
cancer. Thus, determination of Id-1 or Id-2 expression ratio, or
the ratio of Id-1 to Id-2 gene product such as proteins or mRNA,
can be performed by various detection methods known to the art such
as immunohistochemistry or in situ hybridization.
 Where the gene products to be determined are proteins, then
the Id-1 and Id-2 proteins can be detected and analyzed, for example,
by immunohistochemistry as described in Examples 8 and 10, where
anti-serum is directed against the gene product of interest.
 Additionally, the presence or absence of a gene product,
mRNA, can be detected in accordance with this invention through
the use of probes, primers or anti-sense molecules. Such detection
utilizes, for example, probes for detecting and/or analyzing Id-1
and Id-2 expression, such as in in situ hybridization to detect
 Where the Id-1 and Id-2 gene products to be detected are,
for example, mRNA, then the detection can be accomplished, for example,
with nucleic acid probes. Other means for detecting the presence
or absence of the mRNA gene product that are known and useful can
utilize primers and anti-sense molecules.
 The DNA of the invention encoding the Id-1 or Id-2 gene
or homologues, analogues, or fragments thereof may be used in accordance
with the invention to diagnose disease states which are phenotypic
of an aberrant Id-1 or Id-2 genotype or of aberrant Id-1 or Id-2
 By way of another example, but not by way of limitation,
many tumors may be characterized by a lack of, or excess of, Id-1
or Id-2 activity which may stem from mutations in the Id-1 or Id-2
coding or regulatory sequence.
 In both of the examples above, afflicted cells, tissue sections
or biopsy specimens may be screened with the Id-1 or Id-2 DNA sequences
of the invention and isolated Id-1 or Id-2 sequenced to determine
which mutations in Id-1 or Id-2 are associated with the diseases.
The DNAs of the invention may also be used to determine whether
an individual carries an aberrant Id-1 or Id-2 gene.
 The detection of the aberrant Id-1 or Id-2 DNA is conducted
by PCR amplification, from a small tissue sample. Detection of Id-1
or Id-2 product may also be via in situ hybridization or immunocytochemistry
of pathology or biopsy specimens.
 The best mode contemplated for practicing the invention
for detection of breast cancer cell aggressivity is to perform assays
from biopsied breast tissue for both Id-1 and Id-2 proteins or mRNAs.
In practice, one or more of the sections made from an embedded biopsy
are tested for Id-1 and for Id-2. The results are then compared
for ratios of Id-1 and Id-2, since it appears that Id-1 and Id-2
are inversely correlated. The importance of determining the ratios
of Id-1 and Id-2 will be specific for breast tissue and breast cancers,
by contrast to other tissues and other cancers, where different
ratios may be found.
 B. Antibodies
 In addition, Id-I and Id-2 antibodies can be used in a number
of other detection methods, since many of the detection methods
known in the art that will be useful in detecting Id-1 and Id-2
gene products utilize antibodies.
 One aspect of this invention is a method for using Id-1
and Id-2 antibodies where the antibodies will bind to Id-1 and Id-2
proteins, respectively, if present, in a breast, cervical, ovarian,
endometrium, squamous cells, prostate or melanoma tissue sample.
The presence of bound antibodies can be determined by simple visual
examination, or can be detected by other known methods, such as
radioactivity or fluorescence.
 For the production of antibodies, various host animals may
be immunized by injection with the Id-2 or Id-1 gene product, or
a portion thereof including but not limited to, portions of the
Id-1 or Id-2 gene product in a recombinant protein. Such host animals
may include but are not limited to rabbits, mice, and rats, to name
but a few.
 Various adjuvants may be used to increase the immunological
response, depending on the host species, including but not limited
to Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolccithin, pluronic
polyols, polyanions, peptides, oil emulsions, kehole limpet hemocyanin,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum.
 Id-1 and Id-2 antibodies are commercially available. The
commercially available antibodies are typically polyclonal, and
bind to both the mouse and human proteins.
 Monoclonal antibodies may be prepared by using any technique
which provides for the production of antibody molecules by continuous
cell lines in culture. These include but are not limited to the
hybridoma technique originally described in Nature, 256:495-497
(1975), the human B-cell hybridoma technique, Immunology Today,
4:72 (1983), Proc. Natl. Acad. Sci., 80:2026-2030 (1983) and the
EBV-hybridoma technique, Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc., pp. 77-96 (1985).
 In addition, techniques developed for the production of
"chimeric antibodies", Proc. Natl. Acad. Sci., 81:6851-6855
(1984), Nature, 312:604-608 (1984), Nature, 314:452-454 (1985) by
splicing the genes from a mouse antibody molecule of appropriate
antigen specificity together with genes from a human antibody molecule
of appropriate biological activity can be used. Alternatively, techniques
described, for example, the production of single chain antibodies
(U.S. Pat. No. 4,946,778) can be adapted to produce single chain
antibodies specific to one of the binding partners.
 Antibody fragments which recognize specific epitopes may
be generated by know techniques. For example, such fragments include
but are not limited to: the F(ab.sup.1).sub.2 fragments which can
be produced by pepsin digestion of the antibody molecule and the
Fab fragments which can be generated by reducing the disulfide bridges
of the F(ab.sup.1).sub.2 fragments. Alternatively, Fab expression
libraries may be constructed according to Science, 246:1275-1281
(1989) to allow rapid and easy identification of monoclonal Fab
fragments with the desired specificity.
 C. Id-1 and Id-2 Genes, Id-1 and Id-2 Protein--Markers for
Detection of and Targets for Treatment
 Because of their negative correlation and different function
in the breast tissue, Id-1 or Id-2 genes, Id-1 or Id-1 mRNAs, or
Id-1 or Id-2 proteins may each individually be used as a marker
for detection and/or prognosis of malignant aggressivity or as a
target for gene therapy.
 D. Combination of Id-1 and Id-2 Genes--Marker and Target
 Similarly, a ratio of both genes and/expressed proteins
may be advantageously used for diagnosis and/or prognosis of breast
cancer cells aggressivity.
 E. Prognosis of Breast Cancer
 In one aspect of the present invention, a method is provided
that is useful in the prognosis of breast cancer.
 The method for prognosis comprises detecting expression
for an Id gene product in breast tissue obtained from a patient,
and more preferably by seeking to detect gene products, that is
Id-1 and Id-2 proteins or mRNAs. For example, the presence of Id-2
gene product (protein or MRNA) and the absence of Id-1 gene product,
or a relatively larger amount of Id-2 with respect to Id-1, is a
prognostic indicator that breast cancer cells in the breast tissue
will remain localized.
 III. A Diagnostic Kit for Detection of Breast and Other
Types of Cancer Aggressivity
 The invention further concerns the detection of Id-1, Id-2
or their ratio with a kit comprising anti Id-1 and/or Id-2 antibodies
or Id-1 and/or Id-2 probes.
 The kit for detection of breast cancer aggressivity is based
on a method of using Id-1 and Id-2 antibodies or probes.
 The kit typically comprises a detection means for detecting
either the Id-1 and/or Id-2 expression product mRNA, or Id-1 and/or
Id-2 product. For detection of Id-1 or Id-2 protein, antibodies
for Id-1 protein are contacted with breast tissue under conditions
allowing the Id-1 antibodies to bind to Id-1 protein, if present.
Another sample of the same breast tissue is similarly contacted
with antibodies for Id-2 protein under conditions allowing the Id-2
antibodies to bind to Id-2 protein, if present. The presence of
bound Id-2 antibodies with the absence of bound Id-1 antibodies
is a prognostic indicator that breast cancer cells in the breast
tissue are noninvasive and remain localized. The presence of Id-1
antibodies with the absence of Id-2 binding is a prognostic indicator
of the presence of aggressive cancer. Quantitating both responses
derives a ratio of Id-1/Id-2. The ratio above 1 indicates aggressive
cancer. The ratio lower than 1 indicates less aggressive or non-aggressive
 IV. Method for Treatment of Breast Cancer
 A method for treatment of breast, endometrial, cervical,
ovarian, squamous cells or prostate carcinoma or melanoma comprises
targeting of Id-1, or Id-2 genes, or a combination thereof, through
delivery of antisense transcripts, ribozymes, cationic liposomes,
small therapeutically active molecules, drugs, peptides or organic
compounds that disrupt Id-1 interaction with a bHLH transcription
factor or enhance Id-2 gene interaction with a bHLH transcription
factor and vice versa, RNA, anti-Id-1 RNAI causing degradation of
homologous Id-1 mRNAs, Id-2 as a gene or a protein, ITF-2 as a gene
or protein, or targeting Id-1 or Id-2 proteins with antibodies or
with compounds which either enhance or inhibit their production.
 A. Gene Therapy for Treatment
 Gene therapy provides a way to manipulate genetic make-up
of the cell. There are two general approaches to gene therapy.
 The first approach utilizes the introduction into a patient
of a vector that inserts into the genetic code a sequence in the
case of breast cancer, Id-2 sequence, that replaces the more aggressive
Id-1 gene, with the less aggressive Id-2 gene.
 The second approach utilizes the genetic code of Id-1 or
Id-2 to deliver to the breast cells Id-1 or Id-2 antisense molecules
that enter the breast cells and by sequence recognition, selectively
inhibit the gene, Id-1 gene in this case, expression.
 Both approaches are intended to be within the scope of this
 B. Gene Therapy Approaches
 A variety of gene therapy approaches may be used in accordance
with the invention to modulate expression of the Id-1 or Id-2 gene
in vivo. For example, antisense DNA molecules may be engineered
and used to block translation of mRNA in vivo.
 Alternatively, ribozyme molecules may be designed to cleave
and destroy the Id-1 or Id-2 mRNAs in vivo.
 In another alternative, oligonucleotides designed to hybridize
to the 5' region of the Id-1 or Id-2 gene (including the region
upstream of the coding sequence) and form triple helix structures
may be used to block or reduce transcription of the Id-1 or Id-2
 In yet another alternative, nucleic acid encoding the full
length wild-type Id-1 or Id-2 message may be introduced in vivo
into cells which otherwise would be unable to produce the wild-type
Id-1 or Id-2 gene product in sufficient quantities or at all.
 In a preferred embodiment, the antisense, ribozyme and triple
helix nucleotides are designed to inhibit the translation or transcription
of Id-1 with minimal effects on the expression of Id-2. In a preferred
embodiment, the antisense, ribozyme and triple helix nucleotides
are designed to inhibit the translation or transcription of Id-2
with minimal effects on the expression of Id-1. To accomplish this,
the oligonucleotides used are designed on the basis of relevant
sequences unique to Id-1 or Id-2, i.e., those sequences found in
Id-1 but not in Id-2 or Id-2 and not Id-1.
 For example, and not be way of limitation, the oligonucleotides
should not fall within those regions where the nucleotide sequence
of both Id genes is most homologous.
 Moreover, the aggressive propensity of Id-1 gene in breast
cancer cells may be effectively targeted with Id-1-antisense construct
and the aggressive breast cancer cells may be converted to non-aggressive
non-invasive cancer cells.
 B. Targeting Delivery Vehicles and Products
 The current gene delivery methods can be divided to two
classes: viral and non-viral.
 a. Viral Vectors
 The viral vectors currently used both for target validation
and gene therapy are mainly of the following types:
 1. Adenoviral vectors, mostly Ad2 and Ad5-based recombinant
vectors which may or may not contain targeting elements, either
via genetic modification or chemical modification of the viral capsid.
It can either be a replication-defective virus or a selectively
replicating competent virus.
 2. Lentis viral vectors with the same modifications as stated
for adenoviral vectors.
 3. Adeno-associated viral vectors (AAV).
 4. Retroviral vectors.
 Among these four, the first two are most commonly used for
 b. Non-viral Gene Delivery Vehicles
 There are several non-viral based gene delivery systems.
 1. One class includes physical devices to facilitate uptake
including direct injection of plasmid DNA, gene guns, electroporation,
microinjection, electrical pulses, and ultrasound.
 2. The other class of non-viral based methods more relevant
to systemic delivery are the synthetic gene delivery systems that
are defined by their use of:
 i) cationic lipids, also called cationic liposomes or lipoplexes;
Cationic lipids enter the cell by endocytosis and traverse the cytoplasm
through various endocytic compartments. In this process, these complexes
are either targeted to lysosomes for degradation, or are released
into the cytoplasm. One way to deliver gene to its target is by
forming cationic liposome-DNA complex which targets gene expression
to vascular endothelial cells, macrophages and tumor cells.
 In practice, for example, cationic liposome-Id-2-DNA complex
is prepared and targeted to carcinoma cells to replace a highly
aggressive Id-1 gene with less aggressive Id-2 gene.
 ii) polycationic polymers or polyplexes.
 3. Another delivery vehicle for targeting of the Id-1 gene
is RNA interference (RNAi) process. The RNAi process utilizes a
sequence-specific post-transcriptional gene silencing of Id-1 gene
by providing a double-stranded RNA (Id-1-dsRNA) that is homologous
in sequence to the Id-1 gene. Small interfering RNAs (siRNAs) generated
by ribonuclease III cleavage from longer Id-1-dsRNA are the mediators
of sequence-specific Id-1-mRNA degradation.
 4. Another type of targeting delivery vehicles are recently
newly developed nanotechnologies. There are currently two nanotechnologies
developed and available for gene transfers and drug delivery, namely
dendritic polymers and micellar nanoparticles. Dendritic polymers,
also called dendrimers are polymers suitable and useful for the
design and assembly of nanoscale materials. Micellar nanoparticles
are unique synthetic lipid vesicles that fuse with cell membrane.
 Non-viral based gene delivery systems offer ease of preparation,
enhanced DNA packaging capacity and low immunogenecity.
 In terms of the type of molecules the gene delivery vehicles
can deliver, they include plasmids expressing cDNA of the therapeutic
genes (ITF-2 or Id-2, for example in the breast) or the actual therapeutic
molecules. Additionally, anti-sense expressing plasmids (Id-1 antisense,
for example) or the anti-sense oligonucleotides themselves may be
used as a delivery vehicle to target cancer genes. Small molecule
inhibitors of Id-1-interacting proteins are also suitable.
 The use of antisense DNA and DNA vectors is described, for
example, in Clinical Trails of Genetic Therapy with Antisense DNA
and DNA Vectors, Ed. Eric Wickstrom, Marcel Decker, Inc. (1998),
incorporated by reference.
 In conclusion, there are different ways to develop cancer
therapeutics using helix-loop-helix proteins as targets. These different
ways include, but are not limited to, the ones previously described.
 VI. Pharmaceutical Formulations and Compositions
 Any of the identified compounds, antisense DNA molecules,
antibodies, delivery vehicles, etc., can be administered to a mammal,
including a human patient, directly, or in pharmaceutical compositions
comprising its admixture with suitable carriers or excipient(s)
at doses therapeutically effective to treat or ameliorate a breast,
cervical, ovarian, endometrium, squamous cells and prostate cancer
 A therapeutically effective dose refers to that amount of
the composition sufficient to result in treatment or amelioration
of symptoms associated with aggressive cancer cells. Various techniques
for formulation and administration of the compositions of the instant
application may be found in "Remington's Pharmaceutical Sciences,"
Mack Publishing Co., Easton, Pa., latest edition.
 The products of the invention may be designed or administered
for tissue specificity. If the compound comprises a nucleic acid
molecule, including those comprising an expression vector, it may
be linked to a regulatory sequence which is specific for the target
tissue, such as the breast tissue, cervix, ovarian, endometrium,
squamous cells, prostate or skin, etc., by methods which are know
in the art including those set forth in Ann. Oncol., 5 Suppl 4:59-65
(1994); Gene, 145:305-310 (1994); Surgery, 116:205213 ((1994); Cancer
Res., 54:4266-4269; Cancer, 74 (Suppl. 3):1021-1025 (1994); Proc.
Nat'l. Acad. Sci. USA, 91:1460-1464; Exp. Hematol., 22:223-230;
Prog. Clin. Biol. Res., 388:361-365 (1994). The compounds of the
invention may be targeted to specific sites by direct injection
to those sites, such as breast, in the case of breast cancer.
 Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
More specifically, a therapeutically effective amount means an amount
effective to stop aggressive metastatic cancer growth and to alleviate
the existing symptoms of the subject being treated. Determination
of the effective amounts is well within the capability of those
skilled in the art, especially in light of the detailed disclosure
 For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. For example, a dose can be formulated in animal
models to achieve a circulating concentration range that includes
the IC50 (the dose where 50% of the cells show the desired effects)
as determined in cell culture. Such information can be used to more
accurately determine useful doses in humans.
 A therapeutically effective dose refers to that amount of
the compound that results in amelioration of symptoms or a prolongation
of survival in a patient. Toxicity and therapeutic efficacy of such
compounds can be determined by standard pharmaceutical procedures
in cell cultures or experimental animals, e.g., for determining
the LD50 (the dose lethal to 50% of the population) and the ED50
(the dose therapeutically effective in 50% of the population).
 The dose ratio between toxic and therapeutic effects is
the therapeutic index and it can be expressed as the ratio between
LD50 and ED50. Compounds which exhibit high therapeutic indices
are preferred. The data obtained from the cell culture assays described
above and animal studies can be used in formulating a range of dosage
for use in humans. The dosage of such compounds lies preferably
within a range of circulating concentrations that include the ED50
with little or no toxicity.
 The dosage may vary within this range depending upon the
dosage from employed and the route of administration utilized. The
exact formulation, route of administration and dosage can be chosen
by the individual physician in view of the patent's condition. Dosage
amount and interval may be adjusted individually to provide plasma
levels of the active moiety which are sufficient to maintain the
 In cases of local administration or selective uptake, the
effective local concentration of the drug may not be related to
 The amount of composition administered will, of course,
be dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician.
 The pharmaceutical compositions of the present invention
may be manufactured in a manner that is itself known, e.g., by means
of conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
 Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the active
compounds into preparations which can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
 For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible buffers
such as Hank's solution, Ringer's solution, or physiological saline
buffer. For transmucosal administration, penetrants appropriate
to the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
 For oral administration, the compounds can be formulated
readily by combining the active compounds with pharmaceutically
acceptable carriers well known in the art. Such carriers enable
the compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions
and the like, for oral ingestion by a patient to be treated. Pharmaceutical
preparations for oral use can be obtained solid excipient, optionally
grinding a resulting mixture, and processing the mixture of granules
after adding suitable auxiliaries, if desired, to obtain tablets
or dragee cores. Suitable excipients are, in particular, fillers
such as sugars, including lactosse, sucrose, mannitol, or sorbitol;
cellulose preparations such as, for example, maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents
may be added, such as the cross-linked polyvinyl pyrrolidone, agar,
or alginic acid or a salt thereof such as sodium alginate.
 Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may optionally
contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene
glycol, and/or titanium dioxide, lacquer solutions and a suitable
organic solvent or solvent mixture. Dye stuffs or pigments may be
added to the tablets or dragee coatings for identification or to
characterize different combinations of active compound doses.
 Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticiser, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in admixture
with filler such as lactose, binders such as starches, and/or lubricants
such as talc or magnesium stearate and, optionally, stabilizers.
In soft capsules, the active compounds may be dissolved or suspended
in suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethlene glycols. In addition, stabilizers may be added. All
formulations for oral administration should be in dosages suitable
for such administration.
 For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
 For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs
or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluorethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g. gelatin for use
in an inhaler or insufflator may be formulated containing a powder
mix of the compound and a suitable powder base such as lactose or
 The compounds may be formulated for parenteral administration
by injection, e.g., by bolus injection or continuous infusion. Formulations
for injection may be presented in unit dosage form, e.g., in ampoules
or in multidose containers, with the added preservative. The compositions
may take such forms as suspensions, solutions or emulsions in oily
or aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispersing agents.
 Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions to the active compounds may be prepared
as appropriate oily injection suspensions. Suitable lipophilic solvents
or vehicles include fatty oils such as a sesame oil, or synthetic
fatty acid esters, such as ethyl oleate or triglycerides, liposomes
or cationic liposomes. Aqueous injection suspensions may contain
substances which increase the viscosity of the suspension, such
as sodium carboxymethyl cellulose, sorbitol, or destran. Optionally,
the suspension may also contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the preparation
of highly concentrated solutions.
 Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use.
 The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing conventional
suppository bases such as cocoa butter or other glycerides.
 In addition to the formulation described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for example
subcutaneously or intramuscularly) or by intramuscular injection.
Thus, for example, the compounds may be formulated with suitable
polymeric or hydrophobic materials (for example, as an emulsion
in an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
 A pharmaceutical carrier for the hydrophobic compounds of
the invention is a cosolvent system comprising benzyl alcohol, a
nonpolar surfactant, a water-miscible organic polymer, and an aqueous
phase. Naturally, the proportions of a co-solvent system may be
varied considerably without destroying its solubility and toxicity
characteristics. Furthermore, the identify of the co-solvent components
may be varied.
 Alternatively, other delivery systems for hydrophobic pharmaceutical
compounds may be employed. Liposomes, particularly cationic liposomes,
and emulsions are well known examples of delivery vehicles or carriers
for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide
also may be employed although usually at the cost of greater toxicity.
Additionally, the compounds may be delivered using a sustained-release
system, such as semipermeable matrices of solid hydrophobic polymers
containing the therapeutic agent. Various sustained-release materials
have been established and are well known by those skilled in the
art. Sustained-release capsules may, depending on their chemical
nature, release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability of
the therapeutic reagent, additional strategies for protein stabilization
may be employed.
 The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such carriers
or excipients include but are not limited to, calcium carbonate,
calcium phosphate, various sugars, starches, cellulose derivatives,
gelatin, and polymers such as polyethylene glycols.
 Many of the compounds of the invention may be provided as
salts with pharmaceutically compatible counterions. Pharmaceutically
compatible salts may be formed with many acids, including but not
limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic,
succinic, etc. Salts tend to be more soluble in aqueous or other
protonic solvents that are the corresponding free base forms.
 Routes of Administration
 Suitable routes of administration may, for example, include
oral, rectal, transmucosal, transdermal, or intestinal administration,
parenteral delivery, including intramuscular, subcutaneous, intravenous,
intraperitoneal or intranasal.
 Alternatively, one may administer the compound in a local
rather than systemic manner, for example, via injection of the compound
directly into an affected area, often in a depot or sustained release
 Furthermore, one may administer the drug in a targeted drug
delivery system, for example, in a liposome coated with an antibody
specific for affected cells. The liposomes will be targeted to and
taken up selectively by the cells.
 It is to be understood that while the invention has been
described above in conjunction with preferred specific embodiments,
the description and examples are intended to illustrate and not
limit the scope of the invention.
Production of pBabe- Id-1 Retroviral Vector and Virus
 This example describes production of pBabe-Id-1 retroviral
vector and virus.
 The full-length human Id-1 cDNA was excised from CMV-Id-1
and cloned into pBabe-puro, a gift from Dr. Hartmut Land, ICRF,
London, United Kingdom. Clones in which the Id-1 cDNA was inserted
in the sense orientation (pBabe-Id-1) were selected for use.
 pBabe-Id-1 was transfected into the TSA54 packaging cell
line (Cell Genesis; Foster City, Calif.) using calcium phosphate.
Twenty-four hours after transfection, culture medium containing
infectious virus was harvested twice at 4 hour intervals and was
frozen at -80.degree. C. Viral titers were determined by reverse-transcriptase
activity. Briefly, thawed aliquots of harvested media were incubated
with poly(A) (20 ng/.mu.l), oligo dT (10 ng/.mu.l), and [.sup.3H]TTP
(0.1.mu.Ci/.mu.l) in reaction buffer (50 mM Tris-HCl, 75 mM Kcl,
0.5 mM EGTA, and 5 mM MgCl.sub.2) for 30 minutes at 37.degree. C.
The reaction mixture was spotted on Whatman DE81 paper, which was
washed with 2.times. SSC and counted in a scintillation counter.
One unit of MMLV reverse transcriptase (Life Technologies, Inc.)
was subjected to the same reaction, and the amount of incorporated
[.sup.3H]TTP was defined as 1 RT unit. The retroviral titer (RT
units/ml) was determined by comparing the amount of [.sup.3]TTP
incorporated by the virus-containing medium with that incorporated
by MMLV reverse transcriptase.
Cell Culture and Retroviral Infection
 This example describes cell lines, cell culture conditions
and retroviral infection.
 Human breast cancer cell lines MCF7, T47D, and MDA-MB-231
were purchased from the American Tissue Culture Collection (ATCC).
Metastatic MDA-MB-435 cells from ATCC were selected for a highly
aggressive phenotype by passage in immunodeficient mice. Briefly,
cells were injected into nude mice and fast growing tumors were
harvested 3-4 weeks later and processed for in vitro cultivation.
Fibroblasts were eliminated from the culture by differential trypsinization,
and the tumor cells were expanded and cryopreserved for future use.
 Breast cancer cell lines were grown in DMEM or RPMI 1640
obtained from University of California, San Francisco, containing
10% fetal bovine serum and insulin (5 .mu.g/ml, Sigma) . For experiments
using serum-free medium, fetal bovine serum was omitted.
 Approximately eight RT-units of either pBabe-puro or pBabe-Id-1
retrovirus were mixed with 5 ml of medium containing 4 .mu.g/ml
polybrene and were added to T47D cells in 100-mm dishes. Cells expressing
the retroviral genes were selected in 0.6 .mu.g/ml puromycin, which
killed all of the mock-infected cells within three days, whereas
80 or 30% of the pBabe-puro- or pBabe-Id-1-infected cells, respectively,
survived. These puromycin-resistant cells are referred to as T47D-pBO
or T47D-Id-1. To establish single-cell clones, the T47D-Id-1 population
was plated at 1-2 cells/well in 24-well tissue culture plates. Clones
that grew in the wells were expanded.
RNA Isolation and Northern Analysis
 This example describes conditions used for RNA isolation
and Northern analysis.
 Total cellular RNA was isolated and purified as described
in Anal. Biochem., 162:156-159 (1987). Twenty .mu.g were separated
by electrophoresis through formaldehyde-agarose gels and transferred
to a nylon membrane (Hybond N; Amersham). The membrane was hybridized
to a .sup.32P-labeled human Id-1 cDNA or Id-2 or .beta.-casein probe
according to J. Biol. Chem., 269:2139-2145 (1994) and was washed
and exposed to XAR-5 film for autoradiography. The same blot was
hybridized to a 28S rRNA probe to control for RNA integrity and
 This example describes conditions used for Western analysis
of breast cancer cells.
 Cells were lysed in 2.times. Laemmli buffer and stored at
-70.degree. C. Protein concentration was determined by the DC protein
assay (Bio-Rad, Hercules, Calif.). Samples (20-30 .mu.g) were separated
by SDS-PAGE and were transferred to a Immobilin-P filter (Millipore)
by standard methods. The membrane was blocked for 1 hour at room
temperature with TBST (20 mM Tris Base, 137 mM NaCl, 3.8 mM, HCl,
and 0.1% Tween 20) containing 5% nonfat milk, and incubated with
a rabbit polyclonal antibody against human Id-1 or Id-2 (C-20; Santa
Cruz Biotechnology) or with a rabbit polyclonal antibody specific
for the PR-A and PR-B forms of the Pg receptor (C-20; Santa Cruz
Biotechnology) for 1.5 hours. The membrane was washed, incubated
with secondary antibody (goat antirabbit IgG-horseradish peroxidase;
Santa Cruz Biotechnology), washed again, and developed for enhanced
chemiluminescence using the Amersham ECL kit, according to the supplier's
Boyden Chamber Invasion Assays
 This example illustrates conditions used for Boyden Chamber
 Invasion assays were performed in modified Boyden chambers
with 8 .mu.m pore filter inserts for 24-well plates (Collaborative
Research). Filters were coated with 10-12 .mu.l of ice-cold Matrigel
(8 mg/ml protein; Collaborative Research). Cells (80,000 per well)
were added to the upper chamber in 200 .mu.l of the appropriate
medium containing 0.1% BSA. Cells were assayed in triplicate or
quadruplicate, and the results were averaged. The lower chamber
was filled with 300 .mu.l of NIH-3T3 cell-conditioned medium. After
a 20 hour incubation, cells were fixed with 2.5% glutaraldehyde
in PBS and were stained with 0.5% toluidine blue in 2% Na.sub.2CO.sub.3.
Cells that remained in the Matrigel or attached to the upper side
of the filter were removed with cotton tips. Cells on the lower
side of the filter were counted using light microscopy.
 This example describes conditions used for labeling cells
 Cells cultured on coverslips were given [.sup.3H]-thymidine
(10 .mu.Ci/ml; 60-80 Ci/mmol; Amersham) for the last 16 hours of
the experiments, unless otherwise indicated, whereupon they were
fixed with methanol/acetone (1:1) and stained with DAPI. [.sup.3H]-thymidine-labelin-
g was developed as described previously in Mol. Cell Biol., 18:4577-4588
(1988). The percentage of labeled nuclei was calculated by comparing
the number of [.sup.3H]-thymidine-labeled nuclei with the number
of DAPI-stained nuclei in a given field, using phase contrast and
Antisense Oligonucleotide Treatment
 This example describes conditions used for antisense oligonucleotide
treatment of T47D cells.
 Phosphorothiolated oligonucleotides were made by Life Technologies,
Inc. The Id-1 antisense oligonucleotide and nonspecific control
oligonucleotide were described in J. Biol. Cheml, 269:2139-2145
(1994). T47D cells were cultured on coverslips in serum-free medium
for 2 days. On days 3 and 4, the medium was changed in the morning
to serum-free medium containing either E2 (10 nM), or E2 and the
oligonucleotides (10 .mu.M). On the evening of day 4, protein was
extracted from one set of dishes, whereas [.sup.3H]-thymidine was
added to the other set for an additional 16 hours. Cells were fixed
on day 5 and assessed for labeled nuclei as described above.
 This example describes conditions used for immunohistochemical
treatment of tumor tissue sections.
 Formalin-fixed paraffin-embedded tumor tissue sections obtained
from the CPMC patient protein expression in both DCIS and infiltrating
Grades 1, 2 and 3 ductal carcinomas.
 Slides were de-waxed, re-hydrated, and placed in a container
containing 1 liter of 0.01 M citrate buffer (pH 6.0); they were
then microwaved at 700 W for 20 minutes, allowed to remain in the
hot citrate buffer for 15 minutes, and cooled down in running cold
water. The slides were washed in deionized water and incubated in
10% nonfat dry milk for 30 minutes at room temperature, washed in
TBS, and incubated with 1 .mu.g/ml of anti Id-1 antibody overnight
at 40.degree. C. Control slides were incubated with rabbit immunoglobulins.
The slides were washed in TBS and incubated with biotinylated swine
antirabbit F(ab').sub.2 fragments (I:400) for 30 minutes. After
washing in TBS, endogenous peroxidase was visualized by incubating
in 0.5 mg/ml diaminobenzidine-4-HCl and 0.03% hydrogen peroxide
in TBS for three minutes. The slides were washed in TBS and water
Manipulation of Id-2 Expression in Breast Cells
 This example describes methods used for manipulation of
Id-2 expression in breast cells.
 Id-2 cDNA was digested with XbaI and HindIII to isolate
a 1.2 kb fragment. The viral LXSN vector that was used for the mouse
Id-2 cDNA has already been digested with EcoRI, blunted with T4
DNA polymerase and dephosphorylated with CIAP. The Id-2 fragment
was similarly blunted with T4 DNA polymerase, was inserted inside
the dephosphorylated vector, and the ligation product transformed
into Top-10 cells. To identify the clones with sense or anti-sense
orientation, digestion of the recovered plasmids was performed with
either NcoI or BstEII enzymes, and the size of the expected fragments
determined on ethidium bromide agarose gels. The viral vectors was
then packaged in TSA-54 cells (Cell Genesis; Foster City, Calif.).
Mammary epithelial cells were infected with control, Id-2 sense
or Id-2 antisense vectors and selected with neomycin. One to two
weeks after infection, resistant colonies were pooled and expanded.
Id-2 Protein Expression in Tumor Biopsies
 This example describes studies performed to demonstrate
Id-protein expression in tumor biopsies.
 Breast samples have been obtained from patients undergoing
tumorectomies. In order to maintain the integrity of the tissue,
paraffin embedded sections were used instead of frozen sections.
Tissues were fixed overnight at 4.degree. C. in PBS, pH 7.2, containing
4% paraformaldehyde, dehydrated by graded alcohol and finally embedded
 Id-2 expression is studied in a representative number of
in situ and invasive breast tumors. As for Id-1, a sample size of
30 ductal carcinomas in situ as well as 30 invasive Grade I and
30 invasive Grade 3 tumor tissues are used.
 A specific rabbit anti-Id-2 antibody obtained from Santa
Cruz Biotechnology (C-20) is used for immunohistochemistry experiments.
Slides are dewaxed, re-hydrated and placed in a container containing
citrate buffer (pH 6.0), microwaved, allowed to remain in the hot
citrate buffer for 15 min, and cooled down in running cold water.
The slides are washed in deionized water and incubated in 10% non
fat dry milk, washed in TBS and incubated with 1 .mu.g/ml of anti
Id-2 antibody overnight at 4.degree. C. Control slides are incubated
with rabbit immunoglobulin, washed in TBS and incubated with biotinylated
swine anti-rabbit F(ab)'2 (1:400). The slides are then washed in
TBS and incubated with 1:500 streptavidin-horse radish peroxidase.
Peroxidase is visualized by incubating in 0.5 mg/ml diaminobenzidine-4HCl
and 0.03% hydrogen peroxide.