U.S. patent application number 10/252850 was filed with the patent office on 2003-05-08 for tumor-specific p450 protein.
This patent application is currently assigned to University of Aberdeen, a Scotland corporation. Invention is credited to Burke, Michael Danny, Greenlee, William Frank, Melvin, William Thomas, Murray, Graeme Ian.
Application Number | 20030087408 10/252850 |
Document ID | / |
Family ID | 10781203 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030087408 |
Kind Code |
A1 |
Melvin, William Thomas ; et
al. |
May 8, 2003 |
Tumor-specific P450 protein
Abstract
The discovery that CYP1B1 protein is detectable in a wide range
of human cancers of different histogenetic types, but is not
detectable in non-cancerous tissues, gives rise to diagnostic
methods for detecting tumors based on this protein as a marker, and
to the possibility of tumor therapies involving the protein. A
diagnostic method may include the steps of (a) obtaining from a
patient a tissue sample to be tested for the presence of cancer
cells; (b) producing a prepared sample in a sample preparation
process; (c) contacting the prepared sample with an antibody that
reacts with human CYP1B1 protein; and (d) detecting binding of the
antibody to CYP1B1 protein in the prepared sample.
Inventors: |
Melvin, William Thomas;
(Aberdeen, GB) ; Murray, Graeme Ian; (Aberdeen,
GB) ; Burke, Michael Danny; (Leicester, GB) ;
Greenlee, William Frank; (Worcester, MA) |
Correspondence
Address: |
J. PETER FASSE
Fish & Richardson P.C.
225 Franklin Street
Boston
MA
02110-2804
US
|
Assignee: |
University of Aberdeen, a Scotland
corporation
|
Family ID: |
10781203 |
Appl. No.: |
10/252850 |
Filed: |
September 23, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10252850 |
Sep 23, 2002 |
|
|
|
09874166 |
Jun 4, 2001 |
|
|
|
09874166 |
Jun 4, 2001 |
|
|
|
09043814 |
Jan 22, 1999 |
|
|
|
6242203 |
|
|
|
|
09043814 |
Jan 22, 1999 |
|
|
|
PCT/GB96/02368 |
Sep 25, 1996 |
|
|
|
Current U.S.
Class: |
435/184 ;
514/1 |
Current CPC
Class: |
G01N 33/57492 20130101;
A61P 35/00 20180101; C12Q 1/26 20130101; G01N 2333/90245 20130101;
G01N 2500/00 20130101; C12Y 114/14001 20130101; A61K 38/44
20130101; A61P 37/02 20180101; C12N 9/0077 20130101 |
Class at
Publication: |
435/184 ;
514/1 |
International
Class: |
A61K 031/00; C12N
009/99; C12Q 001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 25, 1995 |
GB |
9519490.8 |
Claims
What is claimed is:
1. A composition comprising a compound that is selectively
metabolized by CYP1B1 to generate a cytotoxic substance that kills
or inhibits the growth of a tumor cell.
2. The composition of claim 1, wherein the compound is non-toxic
prior to its metabolism by CYP1B1.
3. An anti-cancer drug comprising an amount of the composition of
claim 1 effective to kill or inhibit the growth of a tumor cell
when administered to an individual having cancer.
4. The anti-cancer drug of claim 3, wherein the tumor cell is a
tumor cell of the bladder, brain, breast, colon, connective tissue,
kidney, lung, lymph node, esophagus, ovary, skin, stomach, testis,
or uterus.
5. A composition comprising a compound that is selectively
metabolized by CYP1B1 to generate a substance that renders a tumor
cell susceptible to a cytotoxic agent.
6. An anti-cancer drug comprising an amount of the composition of
claim 5 effective to render a tumor cell susceptible to a cytotoxic
agent when administered to an individual having cancer.
7. The anti-cancer drug of claim 6, wherein the tumor cell is a
tumor cell of the bladder, brain, breast, colon, connective tissue,
kidney, lung, lymph node, esophagus, ovary, skin, stomach, testis,
or uterus.
8. A method of killing or inhibiting the growth of a tumor cell,
the method comprising contacting a tumor cell expressing CYP1B1
with the composition of claim 1, wherein the CYP1B1 expressed by
the tumor cell metabolizes the compound to generate the cytotoxic
substance and thereby kill or inhibit the growth of the tumor
cell.
9. The method of claim 8, wherein the tumor cell is a tumor cell of
the bladder, brain, breast, colon, connective tissue, kidney, lung,
lymph node, esophagus, ovary, skin, stomach, testis, or uterus.
10. A method of rendering a tumor cell susceptible to a cytotoxic
agent, the method comprising contacting a tumor cell expressing
CYP1B1 with the composition of claim 5, wherein the CYP1B1
expressed by the tumor cell metabolizes the compound to generate
the substance and thereby render the tumor cell susceptible to the
cytotoxic agent.
11. The method of claim 10, further comprising contacting the tumor
cell with an amount of the cytotoxic agent effective to kill or
inhibit the growth of the tumor cell.
12. The method of claim 11, wherein the tumor cell is a tumor cell
of the bladder, brain, breast, colon, connective tissue, kidney,
lung, lymph node, esophagus, ovary, skin, stomach, testis, or
uterus.
13. A method of treating cancer, the method comprising
administering to an individual diagnosed as having a tumor an
amount of the composition of claim 1 effective to kill or inhibit
the growth of a tumor cell in the individual.
14. The method of claim 13, further comprising detecting expression
of CYP1B 1 in the tumor prior to the administration of the
composition.
15. The method of claim 13, wherein the administration of the
composition does not result in liver toxicity in the
individual.
16. The method of claim 13, wherein the administration of the
composition does not result in systemic toxicity in the
individual.
17. The method of claim 13, wherein the tumor is a tumor of the
bladder, brain, breast, colon, connective tissue, kidney, lung,
lymph node, esophagus, ovary, skin, stomach, testis, or uterus.
18. A method of treating cancer, the method comprising:
administering to an individual diagnosed as having a tumor an
amount of the composition of claim 5 effective to render a tumor
cell in the individual susceptible to the cytotoxic agent; and
administering to the individual an amount of the cytotoxic agent
effective to kill or inhibit the growth of the tumor cell.
19. The method of claim 18, further comprising detecting expression
of CYP1B1 in the tumor prior to the administration of the
composition.
20. The method of claim 18, wherein the administration of the
composition does not result in liver toxicity in the
individual.
21. The method of claim 18, wherein the administration of the
composition does not result in systemic toxicity in the
individual.
22. The method of claim 18, wherein the tumor is a tumor of the
bladder, brain, breast, colon, connective tissue, kidney, lung,
lymph node, esophagus, ovary, skin, stomach, testis, or uterus.
23. A method of preparing an anti-cancer compound, the method
comprising: selecting a compound that is metabolized by CYP1B1; and
preparing the compound as a non-toxic substance that is metabolized
by CYP1B1 to generate a cytotoxic substance that kills or inhibits
the growth of a tumor cell.
24. The method of claim 23, wherein the compound is selected by
screening to identify compositions that are metabolized by CYP1B
1.
25. The method of claim 23, wherein the compound is selected by
designing compositions that are metabolized by CYP1B1.
26. A method of preparing an anti-cancer compound, the method
comprising: selecting a compound that is metabolized by CYP1B1; and
preparing the compound as a non-toxic substance that is metabolized
by CYP1B1 to generate a moiety that renders a tumor cell
susceptible to a cytotoxic agent.
27. The method of claim 26, wherein the compound is selected by
screening to identify compositions that are metabolized by
CYP1B1.
28. The method of claim 26, wherein the compound is selected by
designing compositions that are metabolized by CYP1B1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/874,166, filed on Jun. 4, 2001, which is a divisional of
U.S. application Ser. No. 09/043,814, filed on Jan. 22, 1999,
issued as U.S. Pat. No. 6,242,203 on Jun. 5, 2001, which is a
continuation under 35 USC .sctn.371 of PCT Application Number
PCT/GB96/02368, filed on Sep. 25, 1996, which claims priority from
Great Britain Application Number 9519490.8, filed on Sep. 25, 1995,
all of which are incorporated herein by reference in their
entirety.
TECHNICAL FIELD
[0002] This invention relates to tumor diagnosis and therapy, and
to materials and methods for use therein.
[0003] More particularly, the invention is based on the
identification of a cytochrome P450 form, specifically CYP1B1, in a
wide range of tumors, with a high frequency of expression in each
type, and proposes the use of this enzyme as a tumor marker, and as
the basis of a selective therapeutic approach involving the design
of drugs, e.g., which are activated to a cytotoxic form by the
action of CYP1B1.
BACKGROUND
[0004] The major goal of cancer chemotherapy is the development of
anti-cancer drugs that are effective in a wide range of cancers and
produce no toxic effects in normal tissues. The target of such
drugs should be expressed only in tumor cells and not normal cells.
However, to date, no such tumor specific target, general to all
types of cancers, has been identified.
[0005] The cytochromes P450 are a multi-gene family of constitutive
and inducible enzymes, which have a central role in the oxidative
metabolic activation and detoxification of both a wide range of
xenobiotics (2-4) and several groups of endogenous compounds active
in cell regulation and cell signaling including arachidonic acid
(5), steroid hormones (6) and fatty acids (7). The major families
of P450 involved in xenobiotic metabolism each consist of several
individual forms with different regulatory mechanisms and substrate
specificities (2). The majority of P450s are primarily expressed in
liver (2) although individual P450 forms are also expressed in
specific extra-hepatic tissues (8) including small intestine,
kidney and lung.
[0006] The human CYP1 gene family (individual P450 forms are
identified by the prefix CYP in accordance with the current P450
nomenclature (3)), which is one of the major P450 families involved
in the metabolism of xenobiotics, is now known to consist of three
individual forms classified into two sub-families. The CYP1A
subfamily contains two highly homologous and well characterized but
distinct members, CYP1A1 (9) and CYP1A2 (10). CYP1A1 is an
inducible P450 expressed primarily in extrahepatic tissues (11)
while CYP1A2 is a major form of P450 that is constitutively
expressed in liver (12). Recently a second human CYP1 subfamily has
been identified which to date contains one member, CYP1B1 (1). This
P450 is dioxin-inducible, and sequence analysis of CYP1B1 shows 40%
homology with both CYP1A1 and CYP1A2. Although CYP1B1 is assigned
to the CYP1 family on the basis of its sequence, it appears to be
structurally distinct from both CYP1A1 and CYP1A2.
[0007] Several forms of P450 are considered to have an important
role in tumor development since they can metabolize many potential
carcinogens and mutagens (13). Moreover, P450 activity may
influence the response of established tumors to anti-cancer drugs;
several cancer chemotherapeutic agents can be either activated or
detoxified by this enzyme system (14). The presence of individual
forms of P450 had previously been investigated in different types
of cancer including breast cancer (15), lung cancer (16), colon
cancer (17) and head and neck cancer (18) to determine if
intra-tumor metabolism of anti-cancer agents by P450 could occur
and thus influence the response of tumors to these agents. These
studies have generally shown that the level of the P450 forms
investigated is significantly reduced or absent in tumors when
compared with the adjacent normal tissue in which the tumors have
developed. However, our recent studies of several different types
of cancer (19) including breast cancer, esophageal cancer and soft
tissue sarcomas have shown that there may be tumor-specific
expression of a CYP1 form of P450.
[0008] Although CYP1B1 mRNA had previously been identified by
Northern blotting in several normal human tissues (1), the presence
of CYP1B1 protein itself had not been demonstrated.
SUMMARY
[0009] The present invention is based on the discovery that CYP1B1
is a tumor-specific form of P450, present in a wide range of
malignant tumors and not detected in normal tissues.
[0010] Accordingly, a first aspect of the present invention
provides a method for the identification of tumor cells, which
method comprises the use of a recognition agent, for example an
antibody, recognizing CYP1B1 protein to contact a sample of
tissues, cells, blood or body product, or samples derived
therefrom, and screening for a positive response. The positive
response may, for example, be indicated by an agglutination
reaction or by a visualizable change such as a color change or
fluorescence, e.g. immunostaining, or by a quantitative method such
as in the use of radio-immunological methods or enzyme-linked
antibody methods.
[0011] The method therefore typically includes the steps of (a)
obtaining from a patient a tissue sample to be tested for the
presence of cancer cells; (b) producing a prepared sample in a
sample preparation process; (c) contacting the prepared sample with
a recognition agent, such as an antibody, that reacts with human
CYP1B1 protein; and (d) detecting binding of the recognition agent
to CYP1B1 protein, if present, in the prepared sample. The human
tissue sample can be from for example the bladder, brain, breast,
colon, connective tissue, kidney, lung, lymph node, esophagus,
ovary, skin, stomach, testis, and uterus.
[0012] A preferred sample preparation process includes tissue
fixation and production of a thin section. The thin section can
then be subjected to immunohistochemical analysis to detect binding
of the recognition agent to CYP1B1 protein. Preferably, the
immunohistochemical analysis includes a conjugated enzyme labeling
technique. A preferred thin section preparation method includes
formalin fixation and wax embedding. Alternative sample preparation
processes include tissue homogenization, and preferably, microsome
isolation. When sample preparation includes tissue homogenization,
a preferred method for detecting binding of the antibody to CYP1B1
protein is Western blot analysis. Alternatively, an immunoassay can
be used to detect binding of the antibody to CYP1B1 protein.
Examples of immunoassays are antibody capture assays, two-antibody
sandwich assays, and antigen capture assays. Preferably, the
immunoassay is a solid support-based immunoassay. When Western blot
analysis or an immunoassay is used, preferably it includes a
conjugated enzyme labeling technique.
[0013] Although the recognition agent will conveniently be an
antibody, other recognition agents are known or may become
available, and can be used in the present invention. For example,
antigen binding domain fragments of antibodies, such as Fab
fragments, can be used. Also, so-called RNA aptomers may be used
(36, 37). Therefore, unless the context specifically indicates
otherwise, the term "antibody" as used herein is intended to
include other recognition agents. Where antibodies are used, they
may be polyclonal or monoclonal. Optionally, the antibody can
produced by a method so that it recognizes a preselected epitope of
said CYP1B1 protein.
[0014] A second aspect of the invention lies in the presence of
CYP1B1 protein selectively in tumors, e.g. in kidney tumors and not
normal renal tissue, combined with the absence of CYP1B1 protein
expression in normal liver, which provides a mechanism for the
selective targeting of anti-cancer drugs based on CYP1B1 metabolism
in tumors. Drugs can be designed for, or screened for, specific
metabolism by CYP1B1 in tumors whereby this metabolism converts a
non-toxic moiety into a toxic one, which kills or inhibits the
tumor or makes it more susceptible to other agents.
[0015] A third aspect of the invention provides for the targeting
of cytotoxic drugs or other therapeutic agents, or the targeting of
imaging agents, by virtue of their recognition of CYP1B1 epitopes
on the surface of a tumor cell, whether as part of the complete
CYP1B1 protein itself or in some degraded form such as in the
presentation on the surface of a cell bound to a MHC protein.
[0016] Another aspect of the invention provides stimulation of the
immune system of cancer patients, for example by activating
cytotoxic or helper T-cells which recognize CYP1B1 epitopes so as
to implement a cell-mediated or humoral immune response against the
tumor. The activation of the immune system can be achieved by
immunization with CYP1B1 sequences.
[0017] Because the expression of CYP1B1 is very common in tumors of
many different types, it is likely that this enzyme performs an
essential function for the tumor cells, for example by inactivating
endogenous anti-tumor compounds such as 2-methoxyestradiol.
[0018] Consequently, another aspect of the invention is the
reduction of CYP1B1 levels in tumor cells, for example, by the use
of suicide inhibitors or by using antisense RNA methods to decrease
the synthesis of the protein. Down-regulation of the CYP1B1
promoter could also achieve the reduction in CYP1B1 levels.
DESCRIPTION OF DRAWINGS
[0019] FIG. 1 shows SDS-PAGE and immunoblotting procedures using an
anti-CYP1B1 antibody to look for CYP1B1 protein in normal and
tumorous kidney tissue and in normal liver tissue.
[0020] FIG. 2 shows SDS-PAGE and immunoblotting to detect CYP1B1
protein in breast tumor and in normal liver tissue.
[0021] FIG. 3 shows an immunoblot of CYP1B1 in different types of
tumors and normal tissues.
[0022] FIG. 4 shows CYP1B1 and .beta.-actin mRNA in normal (A and
B) and corresponding tumor (C and D) samples which have been
detected by RT-PCR.
DETAILED DESCRIPTION
[0023] Expression of CYP1B1 was investigated in different types of
cancers that had developed in a broad range of different anatomical
sites (bladder, breast, colon, kidney, lung, esophagus, ovary,
skin, stomach, uterus, bone and connective tissue, lymph node,
brain and testis). Primary malignant tumors of these tissues
constitute different histogenetic types (carcinomas, lymphomas,
sarcomas, neuro-epithelial tumors and germ cell tumors) each with a
different biological behavior. These tumors also represent a range
of both common and less common types of cancer. The presence of
CYP1B1 was also investigated in a wide variety of normal
tissues.
[0024] Immunohistochemistry for CYP1B1 showed that in all the
different types of tumor there was strong immunoreactivity for
CYP1B1. CYP1B1 immunoreactivity was localized specifically to tumor
cells. Non-tumor cells including stromal cells, inflammatory cells,
and endothelial cells present in the sections of tumor showed no
immunoreactivity for CYP1B1. There was no significant intra-tumor
heterogeneity of CYP1B1 immunoreactivity and only in five out of
133 tumors was CYP1B1 not detected. There was no immunoreactivity
for CYP1B1 in any of the normal tissues studied which included
liver, kidney, small intestine and lung.
[0025] The absence or low level of individual forms of P450 in most
studies of human cancer (15-18), combined with extrapolation from
studies of rodent hepatic carcinogenesis (25), had led to the
general belief that tumor cells do not significantly express P450.
However, we have now shown that CYP1B1 is expressed in a wide
variety of malignant tumors of different histogenetic types and is
not present in normal tissues, indicating that this P450 is a tumor
specific form of P450. Tumors are composed of a variable proportion
of tumor cells and non-tumor cells. To identify that a protein is
tumor specific, it is important to demonstrate that the protein is
localized only to tumor cells. Immunohistochemistry allows the
direct visualization of tumor cells and has the spatial resolution
to separate tumor cells from non-tumor cells. Furthermore, it is
important to show there has been no differential degradation of
proteins in normal tissue samples compared with tumor samples, and
immunoblotting for .beta.-actin (as a positive control protein)
showed it to be present in every normal and tumor sample indicating
there was no protein degradation. In addition, Coomassie blue
staining of the polyacrylamide gels showed no evidence of protein
degradation. Moreover, immunohistochemistry of the tumor samples
provides its own internal control as sections of tumor contain
non-tumor cells.
[0026] The presence of CYP1B1 in many types of tumor suggests that
this P450 may have a crucial endogenous function in tumor cells and
CYP1B1 may contribute to drug resistance that is observed in many
types of tumor. CYP1B1 is also likely to be important in tumor
development and progression. Its identification in a diverse range
of cancers of different histogenetic types and its absence from
normal tissues appears to make CYP1B1 one of the common changes of
a gene product in malignancy (26).
[0027] A previous investigation (1) found mRNA in normal tissues.
In some tumors, increased (2-4.times.) mRNA was found compared with
normal. This might be due to increased transcription mediated by
hypoxia inducible factor. This is a novel heterodimeric
transcription factor which is induced by hypoxia, and the stimulus
in this case may be the hypoxic micro-environment that can exist in
tumors, and this factor can have as one of its components the Ah
receptor nuclear translocator (27). However, regulation of other
forms of P450 is complex (2, 28) and the regulation of CYP1B1 in
tumors is likely to be complex also, with multiple mechanisms
including transcriptional and post-transcriptional factors
involved.
[0028] The tumor-specific expression of CYP1B1 has important
consequences for both the diagnosis and treatment of cancer. New
diagnostic procedures based on the presence of CYP1B1 in cancer
cells can be developed, while the expression of CYP1B1 in tumor
cells provides a molecular target for the development of new
anti-cancer drugs that are selectively activated by CYP1B1 in tumor
cells. Since CYP1B1 is found in a wide range of tumors it would be
expected that such drugs would be effective in treating many
different types of cancer. An important feature is that it would be
anticipated these drugs would not be associated with the systemic
toxicity that limits the use of current anti-cancer drugs as CYP1B1
is not present in normal tissues especially liver, small intestine
and kidney that are the main tissues involved in drug metabolism.
Thus, a major problem to targeting anticancer drugs at tumors based
on their selective activation by P450 has been marked hepatic P450
metabolism of drugs resulting in decreased bioavailability and/or
undue toxicity. The absence of CYP1B1 protein in liver overcomes
this problem.
[0029] As regards tumor diagnosis, numerous methods for using
antibodies to detect a specific protein, including CYP1B1, in a
biological sample are known and can be used in the present
invention. Any of the various antibody methods can be used alone in
practicing the present invention. If desired, two or more methods
can be used to complement one another.
[0030] A preferred method for use in the present invention is
immunohistochemical analysis. Immunohistochemical analysis
advantageously avoids a dilution effect when relatively few cancer
cells are in the midst of normal cells. An early step in
immunohistochemical analysis is tissue fixation, which preserves
proteins in place within cells. This prevents substantial mixing of
proteins from different cells. As a result, surrounding normal
cells do not diminish the detectability of CYP1B1-containing cancer
cells. This is in contrast to assay methods that involve tissue
homogenization. Upon tissue homogenization, CYP1B1 protein from
cancer cells is mixed with proteins from any surrounding normal
cells present in the tissue sample. The concentration of CYP1B1
protein is thus reduced in the prepared sample, and it can fall
below detectable limits. Immunohistochemical analysis has at least
three other advantages. First, it requires less tissue than is
required by alternative methods such as Western blot analysis or
immunoassay. Second, it provides information on the intracellular
localization and distribution of immunoreactive material. Third,
information on cell morphology can be obtained from the same thin
section used to test for the presence of CYP1B1 protein.
Preferably, when immunohistochemical analysis is employed in the
practice of this invention, several thin sections from each tissue
sample are prepared and analyzed. This increases the chances of
finding small tumors.
[0031] Another preferred antibody method for use in the present
invention is Western blot analysis, i.e., sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by
immunoblotting. Sample preparation for Western blot analysis
includes tissue homogenization, and optionally isolation of
microsomes. Western blot analysis has the advantage of detecting
immunoreactivity on proteins that have been separated with high
resolution, according to (apparent) molecular weight.
[0032] Immunoassays such as antibody capture assays, two-antibody
sandwich assays, and antigen capture assays can also be used in the
present invention. Sample preparation for immunoassays includes
tissue homogenization, and optionally isolation of microsomes.
Immunoassays have the advantage of enabling large numbers of
samples to be tested relatively quickly, and they offer
quantitative precision.
[0033] Principles and practice of immunohistochemistry, Western
blot analysis, and immunoassays are well known. One of ordinary
skill in the art can select suitable protocols and carry out
immunohistochemical analysis, Western blot analysis, or an
immunoassay, in the practice of the present invention (30).
[0034] Experimental Details
[0035] 1. Preparation of Antibodies
[0036] As already noted, the diagnostic aspects of the present
invention can conveniently use an antibody that recognizes human
CYP1B1. Antibody specificity for CYP1B1 protein is preferable, but
not required. Preferably, any non-CYP1B1 protein recognized by the
antibody is readily distinguished from CYP1B1, e.g., according to
apparent molecular weight on a Western blot. With selection of an
appropriate assay-protocol, which is within ordinary skill in the
art, the invention can be practiced with a polyclonal antibody or a
monoclonal antibody.
[0037] A polyclonal antibody or monoclonal antibody suitable for
use in the present invention can be obtained according to
conventional procedures (30). Preparation of antibodies that react
with CYP1B1 protein is known (31). Procedures for obtaining
antibodies that react with human CYP1B1 protein can be carried out
using a preparation of non-human CYP1B1 protein, e.g., murine
CYP1B1 protein. A CYP1B1 protein preparation suitable for eliciting
antibodies useful in the present invention can be obtained
according to various procedures, including those described
(31).
[0038] Antibodies useful in the present invention can be obtained
by immunizing an animal with a preparation containing intact CYP1B1
protein. Alternatively, useful antibodies can be obtained by
immunizing an animal with a polypeptide or oligopeptide
corresponding to one or more epitopes on the CYP1B1 protein.
[0039] Preparation
[0040] To prepare an antibody according to the latter approach, two
15-mer peptides corresponding to epitopes on the human CYP1B1
protein were synthesized. Each corresponded to a different putative
surface loop region of the CYP1B1 enzyme. The first peptide
(designated 217A) consisted of 14 amino acids, i.e., ESLRPGAAPR
DMMD (SEQ ID NO: 1). Peptide 217A represented amino acid positions
312-325 of the deduced amino acid sequence. A carboxy terminal
cysteine was included for use in a conjugation reaction. The second
peptide (designated 218A) consisted of 14 amino acids, i.e.,
EKKAAGDSHG GGAR (SEQ ID NO: 2). Peptide 218A represented positions
332-345 of the deduced amino acid sequence. A carboxy terminal
cysteine was added for use in a conjugation reaction. Each of these
peptides was conjugated directly to KLH.
[0041] Male New Zealand rabbits were immunized at several
anatomical sites using 100 .mu.g of the 217A peptide conjugate or
the 218A peptide conjugate. The conjugates were dissolved in 300
.mu.l of PBS mixed with 300 .mu.l of Freund 's Complete Adjuvant.
Three weeks after the initial immunization, the rabbits were
boosted with 50 .mu.g of one of each of the conjugates (contained
in 300 .mu.l PBS mixed with 300 .mu.l of Freund 's Incomplete
Adjuvant, injected in several sites). One week later (four weeks
after the initial injection), the rabbits were boosted again, using
the same protocol. One week after this second boost, the first
serum sample was collected. Rabbits were subsequently boosted, and
serum samples were collected, weekly. Serum samples were screened
for anti-CYP1B1 titer and specificity by Western blotting against a
human CYP1B1-maltose binding fusion protein expressed in E. coli,
and human CYP1B1 protein expressed in COS-1 cells.
[0042] Anti-CYP1B1 IgG was purified by immunoafftinity
chromatography. The chromatography was carried out using the
appropriate CYP1B1 peptide linked directly to a commercial
N-hydroxysuccinamide ester of a derivatized, cross-linked agarose
gel bead support (AffiGel 10; Biorad, Richmond, Calif.).
Conjugation and chromatography were performed according to the
vendor 's recommended protocols.
[0043] 2. Detection of CYP1B1 Protein and its mRNA
[0044] In general in the experiments that follow, samples of normal
tissue were obtained from tissue specimens that were removed from
patients undergoing surgery for malignant disease. Normal liver,
stomach and small intestine were also obtained from organ
transplant donors. All the tissue samples were processed
immediately after excision to prevent any degradation of protein or
mRNA and ensure no deterioration in tissue morphology. We have
previously shown that human liver obtained in this way shows no
loss or degradation of individual forms of hepatic P450 (20).
Tissue blocks for immunohistochemistry were fixed in 10% neutral
buffered formalin for 24 hours and then embedded in wax, while
tissue samples for immunoblotting and mRNA analyses were rapidly
frozen in liquid nitrogen and stored at -80.degree. C. prior to
use.
[0045] The presence of CYP1B1 protein in tissue samples was
investigated using immunohistochemistry (21) and sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-PAGE) combined
with immunoblotting (22). Immunohistochemistry ensures the
identification of specific types of cells containing CYP1B1 and is
an ideal technique for investigating the presence of CYP1B1 in
tumor cells as tumors are composed of a variable proportion of
tumor cells and non-tumor cells.
[0046] Immunohistochemistry was used to determine the cellular
localization and distribution of CYP1B1 and was performed on
formalin fixed wax embedded sections using two antibodies which
recognize CYP1B1. Sites of immunoreactivity were detected using an
alkaline phosphatase anti-alkaline phosphatase (APAAP) technique
(21). Samples of tumor and normal tissue were fixed in 10% neutral
buffered formalin for 24 hour and then embedded in wax. Sections
were cut on to glass slides and for immunohistochemistry sections
of tumors and normal tissues were dewaxed in xylene, rehydrated in
alcohol and then washed sequentially in cold water and 0.05 M
Tris-HCl (pH 7.6) containing 0.15 M sodium chloride (TBS). The
sections were then immunostained with the CYP1B1 antibodies.
Subsequently, monoclonal mouse anti-rabbit immunoglobulin (1/100,
Dako Ltd, High Wycombe, Bucks; UK), mouse anti-rabbit
immunoglobulin (1/100, Dako) and mouse monoclonal APAAP (1/100,
Dako) were sequentially applied to the tissue sections for 30
minutes each. Between antibody applications the sections were
washed with TBS to remove unbound antibody. Sites of bound alkaline
phosphatase were identified using bromo-chloro-indolyl phosphate
and nitro blue tetrazolium as the enzyme substrate. After
incubating the sections for 30 minutes at room temperature, the
reaction was stopped by washing the sections in cold tap water. The
slides were then air-dried and mounted in glycerine jelly. The
sections were examined using bright field light microscopy in order
to establish the presence or absence of immunostaining, and its
distribution. SDS-PAGE and immunoblotting was followed by enhanced
chemihuuinescence (ECL) technique as described below.
Immunoblotting was also performed with a monoclonal antibody to
.beta.-actin (clone no. AC-15, Sigma, Poole, Dorset, UK) to show
the presence of a positive control protein in tumor and normal
samples and indicate that there was no evidence of protein
degradation in any of the tissue samples.
[0047] The presence of CYP1B1 in each tumor that showed CYP1B1 by
immunohistochemistry, and the absence of detectable CYP1B1 in
normal tissues, was confirmed by Western blot analysis. The
proteins subjected to Western blot analysis were from isolated
microsome preparations.
[0048] Microsomes were prepared essentially as described (32).
Tissue samples were thawed in 25 ml 0.01 M Tris-HCl buffer, pH 7.4,
containing 1.15% KCl before being homogenized in 0.01 M Tris-HCl
buffer, pH 7.4, containing 0.25 M sucrose, 15% glycerol, using an
Ultra-Turrax homogenizer (type TP 18/2; Janlke and Kunkel A G,
Staufen Breisgau, Germany). After centrifugation at 15,000.times. g
for 20 min, the supernatant was removed and recentrifuged at
116,000.times. g for 50 min. The pellet was resuspended a first
time in 0.1 M Tris-HCl buffer, pH 7.4, containing 15% glycerol, 1
mM EDTA, and recentrifuged at 116,000.times. g for 50 min. The
pellet was resuspended a second time in Tris-HCl-glycerol-EDTA
buffer. Microsomal protein concentration was determined (34).
[0049] A discontinuous polyacrylamide gel system, as described (33)
with modifications (32), was employed for separation of proteins in
the microsomes. 20 .mu.l of a 1 mg/ml preparation of normal
samples, and 40 .mu.l of a 0.5 mg/ml preparation of tumor samples,
in 0.125 M Tris-HCl, pH 6.8, containing 2.35% (w/v) sodium dodecyl
sulfate, 5% (v/v) 2-mercaptoethanol, and 0.005% bromophenol blue
tracking dye were loaded onto the gel. 10 .mu.l of a 1 mg/ml
preparation of human liver microsomes were used as positive
controls. Samples were run on a 10% non-gradient gel at 30 mA.
[0050] Following SDS-PAGE, resolved proteins were blotted onto a
nitrocellulose membrane (Schleicher & Schuell; Dassel, Germany)
overnight as described (35). Nonspecific binding site were blocked
with PBS containing 2% (w/v) nonfat milk, 0.05% (v/v) TWEEN 20TM
for 30 minutes at room temperature, with continuous shaking. This
buffer was also used for washing stages. The nitrocellulose
membrane was then incubated with CYP1B1-specific antibody (1:1000)
for 90 minutes and goat anti-rabbit immunoglobulin horseradish
peroxidase conjugate (1:2000 Bio-Rad Laboratories, Hemel Hempstead,
Herts, UK) for 60 minutes. The membrane was washed for three
successive 15-minute periods and one 60-minute period after each
incubation to remove unbound antibody. Bound horseradish peroxidase
was then visualized with an Enhanced Chemiluminescence (ECL) kit
(Amersham International, Aylesbury, Bucks, UK). Detection was
carried out as described in the ECL protocol, with the X-ray film
(Hyperfilm-ECL; Amersham) being exposed for 30 seconds.
EXAMPLE 1
[0051] Expression of CYP1B1 in Normal Kidney and Kidney Tumors was
Investigated.
[0052] Nephrectomy specimens (n=10) excised from primary renal cell
carcinoma were used. Samples or normal kidney were taken at least
several centimeters distant from the edge of each tumor, and only
macroscopically viable tumor was sampled. Normal human livers (n=5)
were obtained from renal transplant donors and stored at
-80.degree. C. prior to use.
[0053] Microsomes of normal kidney, kidney tumors and normal liver
were prepared and subjected to the SDS-PAGE and immunoblotting
procedures in an enhanced chemiluminescence technique (21, 22)
using an anti-CYP1 polyclonal antibody. Recognition of human CYP1B1
was demonstrated using a maltose-binding recombinant CYP1B1 fusion
protein expressed in E coli. Expressed CYP1A1 and CYP142 were
supplied by Dr C L Crespi, Gentest Corp, Ma, USA. The results are
shown in FIG. 1: lane 1 human liver, lane 2 expressed recombinant
CYP1B1 protein, lanes 3, 5, 7, 9, 11 normal kidney samples, lanes
4, 6, 8, 10, 12 corresponding kidney tumors. The same amount of
microsomal protein (30 .mu.g) was loaded into each lane, thus
allowing direct comparison between the kidney and liver
samples.
[0054] As shown in FIG. 1, the kidney tumors and expressed CYP1B1
show a single immunoreactive band at 60 kDa corresponding to the
molecular weight of expressed CYP1B1. In normal kidney none of the
samples showed an immunoreactive band at 60 kDa. In addition, none
of the kidney tumors or normal kidney samples showed the presence
CYP1A1.
[0055] Immunoblotting of liver samples showed an immunoreactive
band at 54 kDa corresponding to the molecular weight of CYP1A2. The
intensity of the band at 54 kDa showed liver-to-liver variation,
whereas there was no CYP1B1 immunoreactive band at 60 kDa in any of
the liver samples.
EXAMPLE 2
[0056] The expression of CYP1B1 was also investigated in breast
cancer using immunoblotting.
[0057] Samples of breast tissue were obtained from patients
undergoing surgery either for primary breast cancer or
non-neoplastic breast disease. Immunoblotting was performed on
breast cancers obtained from six patients (age range 45-67; three
non-smokers, information not available for three patients), and
histologically all these tumors were carcinomas of no special type.
The tissue samples were frozen in liquid nitrogen and stored at
-80.degree. C. prior to analysis.
[0058] SDS-PAGE and immunoblotting were carried out as described
previously. CYP1B1 was detected using the anti-CYP1 polyclonal
antibody referred to above. The results are shown in FIG. 2: lane 1
human liver, lane 2 expressed CYP1B1, lanes 3-8 breast tumors. As
can be seen, a single protein band of molecular weight 60 kDa
corresponding to the molecular weight of the expressed CYP1B1
protein was identified. As previously, CYP1B1 was not detectable in
the liver sample, but CYP1A2 was detected.
EXAMPLE 3
[0059] Immunohistochemistry was used to demonstrate the presence of
CYP1B1 specifically in a variety of normal and tumor tissues. The
results are shown in Table 1 and in FIG. 3.
[0060] Immunohistological localization of CYP1B1 was investigated
in tumors and normal tissues from invasive ductal carcinoma of the
breast, endometrial adenocarcinoma, transitional cell carcinoma of
the bladder, diffuse high grade malignant lymphoma, high grade
astrocytoma of the brain, soft tissue sarcoma (malignant fibrous
histiocytoma), normal liver, normal kidney, normal small intestine.
The antibody used was the 218A anti-CYP1B1 polyclonal antibody
described above.
[0061] FIG. 3 shows an immunoblot of CYP1B1 in different types of
tumors and normal tissues. Lane 1 normal colon, lane 2 colon
adenocarcinoma, lane 3 normal kidney, lane 4 carcinoma of kidney,
lane 5 normal breast, lane 6 breast cancer, lane 7 normal jejunum,
lane 8 normal stomach, lane 9 normal liver, lane 10 malignant mixed
Mullerian tumor, lane 11 endometrial adenocarcinoma, lane 12
ovarian carcinoma, lane 13 diffuse B cell lymphoma, lane 14
transitional cell carcinoma, lane 15 lung carcinoma, lane 16
positive control (dioxin-induced ACHN kidney tumor cells (panel A
only). In panel B, the same series of tissue samples have been
immunoblotted for .beta.-actin, which is present in all normal and
tumor samples. A Coomassie blue stained polyacrylamide gel of the
same series of tissue samples displayed no evidence of protein
degradation. The results demonstrated that this P450 is
specifically localized to tumor cells, and that there is no CYP1B1
immunoreactivity in normal tissues.
1TABLE 1 CYP1B1 Expression in Tumor Tissues and Normal Tissues.
Normal Tumor no pos./ no pos./ Histopathological Tissue no tested
no tested diagnosis Bladder 0/8 8/8 transitional cell carcinoma
Brain 0/12 11/12 astrocytoma Breast 0/10 12/12 invasive ductal
carcinoma Colon 0/10 11/12 adenocarcinoma Connective tissue 0/9 8/9
sarcoma Kidney 0/11 11/11 clear cell carcinoma n = 10; transitional
cell carcinoma n = 1 Liver 0/8 not tested not tested Lung 0/8 7/8
squamous carcinoma Lymph node 0/5 9/9 non-Hodgkin's lymphoma
Esophagus 0/8 8/8 squamous carcinoma Ovary not tested 7/7
Adenocarcinoma Skin 0/6 6/6 squamous carcinoma Small Intestine 0/5
not tested not tested Stomach 0/10 9/10 Adenocarcinoma Testis 0/8
14/14 malignant germ cell tumors Uterus 0/5 7/7 Adenocarcinoma n =
5; Malignant mixed Mullerian tumor n = 2 Total 0/123 128/133
EXAMPLE 4
[0062] Experiments were conducted to detect CYP1B1 RNA in various
tumor and normal tissues.
[0063] Reverse transcription polymerase chain reaction (RT-PCR)
experiments to detect CYP1B1 mRNA were carried out as described in
McKay et al (23). RNA was extracted from tissue samples and cDNA
was synthesized from the isolated RNA using oligo (dT). The CYP1B1
primers had the following sequences: Forward 5'-AAC TCT CCA TCA GGT
GAG GT-3' (SEQ ID NO:3) (nt 2104-2123); Reverse 5'-TAA GGA AGT ATA
CCA GAA GGC-3' (SEQ ID NO:4) (nt 2573-3593) giving a PCR product of
489 bp. .beta.-actin was used as a positive control to confirm the
presence and integrity of mRNA in each sample and the .beta.-actin
primers which were bought from Stratagene (Cambridge, UK) had the
following sequences: Forward 5'-TGA CGG GGT CAC CCA CAC TGT GCC CAT
CTA-3' (SEQ ID NO:5) (nt 1067-1105); Reverse 5'-CTA GAA GCA TTT GCG
GTG GAC GAT GGA GGG-3' (SEQ ID NO:6) (nt 1876-1905). PCR with 35
cycles of amplification for both CYP1B1 and .beta.-actin was
performed as described (23). The positive control for CYP1B1 was a
2.78 kb CYP1B1 cDNA and the negative control was sterile water in
place of cDNA. After PCR 10 .mu.l of the PCR product was
electrophoresed on a 1.5% agarose gel which incorporated 0.007% w/v
ethidium bromide and visualized by UV illumination. The CYP1B1 PCR
product was sequenced, after purification, by the direct dideoxy
sequencing technique with a T7 sequencing kit (Pharmacia, Milton
Keynes, UK) used according to the manufacturer 's protocol. To
further investigate the relative amount of CYP1B1 mRNA in normal
and tumor tissues, semi-quantitative RT-PCR of normal and tumor
kidney samples was performed using serial dilution cDNA of (24).
.beta.-actin mRNA was used as an internal control (29).
[0064] FIG. 4 shows CYP1B1 and .beta.-actin mRNA in normal (A and
B) and corresponding tumor (C and D) samples which have been
detected by RT-PCR. Lane 1 kidney, lane 2 colon, lane 3 skin, lane
4 esophagus, lane 5 stomach, lane 6 lymph node, lane 7 breast.
[0065] Analysis of the tumors by RT-PCR showed that all tumor
samples in which CYP1B1 had been identified contained CYP1B1 mRNA.
The PCR product was of the expected molecular size when analyzed by
agarose gel electrophoresis. Sequencing of the PCR product
confirmed identity with CYP1B1.
CONCLUDING REMARKS
[0066] The absence or low level of individual for P450 in most
studies of human cancer (15-18), combined with extrapolation from
studies of rodent hepatic carcinogenesis (25), had led to the
general belief that tumor cells do not significantly express P450.
However, we have now shown that CYP1B1 is expressed in a wide
variety of malignant tumors of different histogenetic types and is
not present in normal tissues, indicating that this P450 is a tumor
specific form of P450. Tumors are composed of a variable proportion
of tumor cells and non-tumor cells. To identify that a protein is
tumor specific, it is important to demonstrate that the protein is
localized only to tumor cells. Immunohistochemistry allows the
direct visualization of tumor cells and has the spatial resolution
to separate tumor cells from non-tumor cells. Furthermore, it is
important to show there has been no differential degradation of
proteins in normal tissue samples compared with tumor samples and
immunoblotting for .beta.-actin (as a positive control protein)
showed it to be present in every normal and tumor sample indicating
there was no protein degradation. In addition, Coomassie blue
staining of the polyacrylamide gels showed no evidence of protein
degradation. Moreover, immunohistochemistry of the tumor samples
provides its own internal control as sections of tumor contain
non-tumor cells.
REFERENCES
[0067] 1. T. R. Sutter et al., J. Biol. Chem. 269, 10392 (1994)
[0068] 2. S. A. Wrighton and J. C. Stevens, Crit. Rev. Toxicol. 22,
1 (1992)
[0069] 3. D. R. Nelson et al, Pharmacogenetics 6, 1 (1996)
[0070] 4. T. Shimada and F. P. Guengerich, Chem. Res. Toxicol. 4,
391 (1991); V. Nedelcheva and I. Gut, Xenobiotica 24, 1151 (1994);
B. K. Park, M. Pirmohamed, N. R. Kitteringham, Pharmac. Ther. 58,
385 (1995).
[0071] 5. J. H. Capdevila, J. R. Falck, R. W. Estabrook, FASEB J.
6, 731 (1992)
[0072] 6. W. L. Miller, Endocrine Rev. 9, 295 (1988)
[0073] 7. E. H. Oliw, Prog. Lipid Res. 33, 329 (1994)
[0074] 8. G. I. Murray and M. D. Burke, Biochem. Pharmacol. 50, 895
(1995); L. S. Kaminsky and M. J. Fasco, Crit. Rev. Toxicol. 21, 407
(1992); E. G. Scheutz et al., Arch. Biochem. Biophys. 294, 206
(1992)
[0075] 9. A. K. Jaiswal, F. J. Gonzalez, D. W. Nebert, Science 228,
80 (1985)
[0076] 10. A. K. Jaiswal, D. W. Nebert, F. J. Gonzalez, Nucl. Acid
Res. 14, 6773 (1986)
[0077] 11. D. Sesardic, M. Pasanen, O. Pelkonen, Carcinogenesis 11,
1183 (1990)
[0078] 12. H. Schweikl et al., Pharmacogenetics 3, 239 (1993)
[0079] 13. F. J. Gonzalez and H. V. Gelboin, Drug Metab. Rev. 26,
165 (1994); F. P. Guengerich, Cancer Res. 48, 2946 (1988); K.
Kawajiri and Y. Fujii-Kuriyama, Jpan. J. Cancer Res. 82, 1325
(1991); F. P. Guengerich, Pharmac. Ther. 54, 17 (1992)
[0080] 14. G. A. Le Blanc and D. J. Waxman, Drug Metab. Rev. 20,
395 (1989); K. T. Kivisto, H. K. Kroemer, M. Eichelbaum, Brit. J.
Clin. Pharmacol. 40, 523 (1995)
[0081] 15. N. Albin et al., Cancer Res. 53, 3541 (1993)
[0082] 16. C. Toussaint et al., Cancer Res. 53, 4606 (1993); M.
Czerwinski et al., Cancer Res. 54, 1085 (1994)
[0083] 17. W. H. M. Peters et al., Gastroenterology 103, 448
(1992); I. de Waziers et al., Carcinogenesis 12, 905 (1991); L.
Massad et al., Cancer Res. 52, 6567 (1992); K. Mekhail-Ishak et
al., Cancer Res. 49, 4866 (1989)
[0084] 18. F. Janot et al., Carcinogenesis 14, 1279 (1993)
[0085] 19. G. I. Murray et al., Br. J. Cancer 63, 1021 (1991); G.
I. Murray et al., J. Pathol. 171, 49 (1993); G. I. Murray et al.,
Gut 35, 599 (1994)
[0086] 20. R. J. WeaveR et al., Biochem. Pharmacol. 47, 763
(1994)
[0087] 21. J. A. McKay et al., J. Histochem. Cytochem. 43, 615
(1995)
[0088] 22. D. C. Spink et al., Arch. Biochem. Biophys. 293, 242-348
(1992)
[0089] 23. J. A. McKay et al., FEBS Letts. 374, 270 (1995)
[0090] 24. M. J. Dallman and A. C. G. Porter, In PCR, A practical
approach, eds. J. McPherson, P. Quirke and G. R. Taylor (IRL Press,
Oxford) p 215-224.
[0091] 25. A. Buchmann et al., Carcinogenesis 6, 513 (1985); M. W.
Roomi, R. K. Ho, D. S. R. Sarma, E. Farber, Cancer Res. 45, 564
(1985); A. Buchmann et al., Cancer Res. 47,2911(1987)
[0092] 26. T. G. Krontiris, N. Engl. J. Med. 333, 303 (1995); L. M.
Ponz de Leon, Recent Res. Cancer Res. 136, 35 (1994); S. K. Bahra,
B. K. A. Rasheed, S. H. Bigner, D. D. Bigner, Lab. Invest. 71, 621
(1994)
[0093] 27. G. L. Wang, B-H, Jiang, E. A. Rue, G. L. Semenza, Proc.
Natl. Acad. Sci. USA 92, 5510 (1995)
[0094] 28. J. P. Whitlock et al., FASEB. J. 10, 809 (1996)
[0095] 29. T. Horishika et al, Cancer Res. 52,108 (1992)
[0096] 30. Harlow et al., Antibodies--A Laboratory Manual, Cold
Spring Harbor, Cold Spring Harbor, N.Y. (1988)
[0097] 31. Otto et al., Endocrinology 129:970-982 (1991)
[0098] 32. Weaver et al. Biochem. Pharmacol. 46:1183-1197
(1993)
[0099] 33. Laemmli, Nature 227:68 (1970)
[0100] 34. Lowry, J. Biol. Chem. 193:265 (1951)
[0101] 35. Towbin et al. Proc. Natl. Acad. Sci. USA 76:4350
(1979)
[0102] 36. J. R. Willimson, Nature 382:112-113 (1996)
[0103] 37. A. D. Ellington et al, Nature 346:818-822 (1990)
Sequence CWU 1
1
6 1 14 PRT Homo sapiens 1 Glu Ser Leu Arg Pro Gly Ala Ala Pro Arg
Asp Met Met Asp 1 5 10 2 14 PRT Homo sapiens 2 Glu Lys Lys Ala Ala
Gly Asp Ser His Gly Gly Gly Ala Arg 1 5 10 3 20 DNA Artificial
Sequence Synthetically generated primer 3 aactctccat caggtgaggt 20
4 21 DNA Artificial Sequence Synthetically generated primer 4
taaggaagta taccagaagg c 21 5 30 DNA Artificial Sequence
Synthetically generated primer 5 tgacggggtc acccacactg tgcccatcta
30 6 30 DNA Artificial Sequence Synthetically generated primer 6
ctagaagcat ttgcggtgga cgatggaggg 30
* * * * *