U.S. patent application number 09/874166 was filed with the patent office on 2002-05-02 for tumour-specific p450 protein.
Invention is credited to Burke, Michael Danny, Greenlee, William Frank, Melvin, William Thomas, Murray, Graeme Ian.
Application Number | 20020052013 09/874166 |
Document ID | / |
Family ID | 10781203 |
Filed Date | 2002-05-02 |
United States Patent
Application |
20020052013 |
Kind Code |
A1 |
Melvin, William Thomas ; et
al. |
May 2, 2002 |
Tumour-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
|
Family ID: |
10781203 |
Appl. No.: |
09/874166 |
Filed: |
June 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09874166 |
Jun 4, 2001 |
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09043814 |
Jan 22, 1999 |
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6242203 |
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09043814 |
Jan 22, 1999 |
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PCT/GB96/02368 |
Sep 25, 1996 |
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Current U.S.
Class: |
435/7.23 ;
424/94.4 |
Current CPC
Class: |
G01N 2500/00 20130101;
C12Q 1/26 20130101; C12N 9/0077 20130101; A61P 37/02 20180101; G01N
2333/90245 20130101; G01N 33/57492 20130101; A61K 38/44 20130101;
C12Y 114/14001 20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/7.23 ;
424/94.4 |
International
Class: |
G01N 033/574; A61K
038/44 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 25, 1995 |
GB |
9519490.8 |
Claims
1. A method for detecting cancer cells, if present, in a tissue
sample from a human patient, the method comprising contacting a
tissue sample from a patent with a recognition agent for human
cytochrome P450 CYP1B1, and detecting binding of the recognition
agent to CYP1B1 protein in the prepared sample as an indication of
the presence of cancer cells in the tissue sample.
2. A method according to claim 1 which includes the steps of
obtaining from a patient a tissue sample to be tested for the
presence of cancer cells; and producing a prepared sample in a
sample preparation process; prior to contacting the prepared sample
with a recognition agent that reacts with human CYP1B1 protein.
3. The method of claim 2 wherein binding of said recognition agent
to CYP1B1 protein in sample is detected by immunohistochemical
analysis.
4. The method of claim 3, wherein the sample preparation process
comprises contacting the tissue with fixative and producing a thin
section suitable for immunohistochemical analysis.
5. The method of claim 4, wherein the fixative is formalin.
6. The method of claim 4 or claim 5, wherein the thin section is
wax-embedded.
7. The method of any one of claims 3 to 6, wherein the
immunohistochemical analysis comprises a conjugated enzyme
labelling technique.
8. The method of claim 2, wherein the sample preparation process
comprises tissue homogenization.
9. The method of claim 8, wherein the sample preparation process
further comprises isolating microsomes.
10. The method of claim 8 or claim 9, wherein the binding of said
recognition agent to the CYP1B1 protein in the prepared sample is
detected by Western blot analysis.
11. The method of claim 10, wherein the Western blot analysis
comprises a conjugated enzyme labelling technique.
12. The method of claim 8, wherein the binding of said recognition
agent to the CYP1B1 protein in the prepared is sample is detected
by an immunoassay.
13. The method of claim 12, wherein the immunoassay is selected
from antibody capture assay, two-antibody sandwich assay and
antigen capture assay.
14. The method of claim 12, wherein the immunoassay is a solid
support-based immunoassay.
15. The method of any one of claims 12-14, wherein the immunoassay
comprises a conjugated enzyme labelling technique.
16. The method of any one of the preceding claims, wherein the
recognition agent is a polyclonal antibody.
17. The method of any one of claims 1 to 15, wherein the
recognition agent is a monoclonal antibody.
18. The method of any one of the preceding claims, wherein the
recognition agent recognizes a preselected epitope of the CYP1B1
protein.
19. The method of any one of claims 1 to 17, wherein the
recognition agent is specific for CYP1B1 protein.
20. The method of any one of the preceding claims, wherein said
tissue sample is selected from bladder, brain, breast, colon,
connective tissue, kidney, lung, lymph node, oesophagus, ovary,
skin, stomach, testis, and uterus.
21. A method of obtaining drugs of potential use in cancer therapy,
which comprises screening for or selecting a substance which is
susceptible to specific metabolism by human cytochrome P450 CYP1B1,
and using that substance as a basis for a non-toxic moiety which
can be converted by the CYP1B1 metabolism into a toxic one, which
kills or inhibits a tumour cell expressing CYP1B1 or makes it more
susceptible to other agents.
22. A method of providing for the targeting of cytotoxic drugs or
other therapeutic agents, or the targeting of imaging agents, in
tumour diagnosis or therapy, which comprises providing the drug,
therapeutic agent or imaging agent with means for the recognition
of a human cytochrome P450 CYP1B1 epitope on the surface of a
tumour 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.
23. Use of a biologically active substance which is capable of
stimulation of the human immune system by activating cytotoxic or
helper T-cells which recognise human cytochrome P450 CYP1B1
epitopes in the preparation of a medicament to implement a
cell-mediated or humoral immune response against a cell in which
CYP1B1 is expressed.
24. The use of a biologically active substance according to claim
23, which activates the immune system by immunisation with at least
one CYP1B1 amino acid sequence.
25. Use of a substance in the reduction of human cytochrome P450
CYP1B1 levels in tumour cells in the preparation of a medicament,
wherein said substance comprising an inhibitor or means for
producing antisense RNA to decrease the synthesis of CYP1B1.
26. The use of a substance according to claim 25, which functions
by down-regulation of the CYP1B1 promoter.
Description
FIELD OF THE INVENTION
[0001] This invention relates to tumour diagnosis and therapy, and
to materials and methods for use therein.
[0002] More particularly, the invention is based on the
identification of a cytochrome P450 form, specifically CYP1B1, in a
wide range of tumours, with a high frequency of expression in each
type, and proposes the use of this enzyme as a tumour marker, and
as the basis of a selective therapeutic approach involving the
design of drugs, eg which are activated to a cytotoxic form by the
action of CYP1B1.
BACKGROUND OF THE INVENTION
[0003] 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 tumour cells and not normal
cells. However, to date, no such tumour specific target, general to
all types of cancers, has been identified.
[0004] 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 signalling 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.
[0005] 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 characterised 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.
[0006] Several forms of P450 are considered to have an important
role in tumour development since they can metabolise many potential
carcinogens and mutagens (13). Moreover, P450 activity may
influence the response of established tumours 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-tumour metabolism of anti-cancer agents by P450 could occur
and thus influence the response of tumours to these agents. These
studies have generally shown that the level of the P450 forms
investigated is significantly reduced or absent in tumours when
compared with the adjacent normal tissue in which the tumours have
developed. However, our recent studies of several different types
of cancer (19) including breast cancer, oesophageal cancer and soft
tissue sarcomas have shown that there may be tumour-specific
expression of a CYP1 form of P450.
[0007] 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 OF THE INVENTION
[0008] The present invention is based on the discovery that CYP1B1
is a tumour-specific form of P450, present in a wide range of
malignant tumours and not detected in normal tissues. Accordingly,
a first aspect of the present invention provides a method for the
identification of tumour cells, which method comprises the use of a
recognition agent, for example an antibody, recognising 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 visualisable change such as a colour
change or fluorescence, eg immunostaining, or by a quantitative
method such as in the use of radio-immunological methods or
enzyme-linked antibody methods.
[0009] 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, oesophagus,
ovary, skin, stomach, testis, and uterus.
[0010] 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 labelling
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
immnunoassay is a solid support-based immunoassay. When Western
blot analysis or an immunoassay is used, preferably it includes a
conjugated enzyme labelling technique.
[0011] 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.
[0012] A second aspect of the invention lies in the presence of
CYP1B1 protein selectively in tumours, eg in kidney tumours 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 tumours. Drugs can be designed for, or screened for, specific
metabolism by CYP1B1 in tumours whereby this metabolism converts a
non-toxic moiety into a toxic one, which kills or inhibits the
tumour or makes it more susceptible to other agents.
[0013] 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 tumour 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.
[0014] Another aspect of the invention provides stimulation of the
immune system of cancer patients, for example by activating
cytotoxic or helper T-cells which recognise CYP1B1 epitopes so as
to implement a cell-mediated or humoral immune response against the
tumour. The activation of the immune system can be achieved by
immunization with CYP1B1 sequences.
[0015] Because the expression of CYP1B1 is very common in tumours
of many different types, it is likely that this enzyme performs an
essential function for the tumour cells, for example by
inactivating endogenous anti-tumour compounds such as
2-methoxyestradiol. Consequently, another aspect of the invention
is the reduction of CYP1B1 levels in tumour 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] 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.
[0017] FIG. 2 shows SDS-PAGE and immunoblotting to detect CYP1B1
protein in breast tumour and in normal liver tissue.
[0018] FIG. 3 shows an immunoblot of CYP1B1 in different types of
tumours and normal tissues.
[0019] FIG. 4 shows CYP1B1 and .beta.-actin mRNA in normal (A and
B) and corresponding tumour (C and D) samples which have been
detected by RT-PCR.
DETAILED DESCRIPTION OF THE INVENTION
[0020] 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, oesophagus, ovary,
skin, stomach, uterus, bone and connective tissue, lymph node,
brain and testis). Primary malignant tumours of these tissues
constitute different histogenetic types (carcinomas, lymphomas,
sarcomas, neuro-epithelial tumours and germ cell tumours) each with
a different biological behaviour. These tumours 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.
[0021] Immunohistochemistry for CYP1B1 showed that in all the
different types of tumour there was strong immunoreactivity for
CYP1B1. CYP1B1 immunoreactivity was localised specifically to
tumour cells. Non-tumour cells including stromal cells,
inflammatory cells, and endothelial cells present in the sections
of tumour showed no immunoreactivity for CYP1B 1. There was no
significant intra-tumour heterogeneity of CYP1B1 immunoreactivity
and only in five out of 133 tumours 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.
[0022] 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 tumour cells do not significantly express P450.
However, we have now shown that CYP1B1 is expressed in a wide
variety of malignant tumours of different histogenetic types and is
not present in normal tissues, indicating that this P450 is a
tumour specific form of P450. Tumours are composed of a variable
proportion of tumour cells and non-tumour cells. To identify that a
protein is tumour specific, it is important to demonstrate that the
protein is localised only to tumour cells. Immunohistochemistry
allows the direct visualisation of tumour cells and has the spatial
resolution to separate tumour cells from non-tumour cells.
Furthermore, it is important to show there has been no differential
degradation of proteins in normal tissue samples compared with
tumour samples, and immunoblotting for .beta.-actin (as a positive
control protein) showed it to be present in every normal and tumour
sample indicating there was no protein degradation. In addition,
Coomassie blue staining of the polyacrylamide gels showed no
evidence of protein degradation. Moreover, immuno-histochemistry of
the tumour samples provides its own internal control as sections of
tumour contain non-tumour cells.
[0023] The presence of CYP1B1 in many types of tumour suggests that
this P450 may have a crucial endogenous function in tumour cells
and CYP1B1 may contribute to drug resistance that is observed in
many types of tumour. CYP1B1 is also likely to be important in
tumour 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).
[0024] A previous investigation (1) found mRNA in normal tissues.
In some tumours, 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
tumours, 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
tumours is likely to be complex also, with multiple mechanisms
including transcriptional and post-transcriptional factors
involved. The tumour-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 tumour
cells provides a molecular target for the development of new
anti-cancer drugs that are selectively activated by CYP1B1 in
tumour cells. Since CYP1B1 is found in a wide range of tumours 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 anti-cancer drugs at
tumours 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.
[0025] As regards tumour 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
practising the present invention. If desired, two or more methods
can be used to complement one another.
[0026] 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 analysed. This increases the chances of
finding small tumours.
[0027] Another preferred antibody method for use in the present
invention is Western blot analysis, ie 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.
[0028] Inmunoassays 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.
[0029] 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).
[0030] Experimental Details
[0031] 1. Preparation of Antibodies
[0032] 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, eg, 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 practised with a polyclonal antibody or a
monoclonal antibody.
[0033] 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, eg, 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).
[0034] 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.
[0035] Preparation
[0036] 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, ie, 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, ie,
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.
[0037] 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 titre 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.
[0038] Anti-CYP1B1 IgG was purified by immunoaffinity
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, Richmnond, Calif.).
Conjugation and chromatography were performed according to the
vendor's recommended protocols.
[0039] 2. Detection of CYP1B1 Protein and its mRNA
[0040] 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 immuno-histochemistry 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.
[0041] 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
tumour cells as tumours are composed of a variable proportion of
tumour cells and non-tumour cells.
[0042] Immunohistochemistry was used to determine the cellular
localisation and distribution of CYP1B1 and was performed on
formalin fixed wax embedded sections using two antibodies which
recognise CYP1B1. Sites of immunoreactivity were detected using an
alkaline phosphatase anti-alkaline phosphatase (APAAP) technique
(21). Samples of tumour 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 tumours and normal tissues were dewaxed in xylene, rehydrated in
alcohol and then washed sequentially in cold water and 0.05M
Tris-HCl (pH 7.6) containing 0.15M sodium chloride (TBS). The
sections were then immnunostained with the CYP1B1 antibodies.
Subsequently, monoclonal mouse anti-rabbit immunoglobulin
({fraction (1/100)}, Dako Ltd, High Wycombe, Bucks; UK), rabbit
anti-mouse immunoglobulin ({fraction (1/100)}, Dako) and mouse
monoclonal APAAP ({fraction (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 allcaline 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.
[0043] SDS-PAGE and immunoblotting was followed by enhanced
chemiluminescence (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 tumour and normal
samples and indicate that there was no evidence of protein
degradation in any of the tissue samples. The presence of CYP1B1 in
each tumour 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.
[0044] 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; Janke and Kunkel AG,
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 tune
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).
[0045] 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 tumour 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.
[0046] Following SDS-PAGE, resolved proteins were blotted onto a
nitrocellulose membrane (Schleicher & Schuell; Dassel, Germany)
overnight as described (35). Nonspecific binding sites were blocked
with PBS containing 2% (w/v) nonfat milk, 0.05% (v/v) TWEEN 20.TM.
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
[0047] Expression of CYP1B1 in normal kidney and kidney tumours was
investigated.
[0048] 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 tumour, and only
macroscopically viable tumour was sampled. Normal human livers
(n=5) were obtained from renal transplant donors and stored at
-80.degree. C. prior to use.
[0049] Microsomes of normal kidney, kidney tumours 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 CYP1A2 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 tumours. The same amount of
microsomal protein (30 .mu.g) was loaded into each lane, thus
allowing direct comparison between the kidney and liver
samples.
[0050] As shown in FIG. 1, the kidney tumours and expressed CYP1B1
show a single immuno-reactive 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 tumours or normal kidney samples showed the presence
of CYP1A1.
[0051] 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
[0052] The expression of CYP1B1 was also investigated in breast
cancer using immunoblotting.
[0053] 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 tumours were carcinomas of no special
type. The tissue samples were frozen in liquid nitrogen and stored
at -80.degree. C. prior to analysis.
[0054] 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 tumours. 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
[0055] Immunohistochemistry was used to demonstrate the presence of
CYP1B1 specifically in a variety of normal and tumour tissues. The
results are shown in Table 1 and in FIG. 3.
[0056] Immunohistological localisation of CYP1B1 was investigated
in tumours 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.
[0057] FIG. 3 shows an immunoblot of CYP1B1 in different types of
tumours 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 tumour, lane 11 eodometrial 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 tumour 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
tumour 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 localised to tumour cells, and that there is no CYP1B1
immunoreactivity in normal tissues.
1TABLE 1 CYP1B1 Expression in Tumour Tissues and Normal Tissues.
Normal Tumor no pos./ no pos./ Tissue no tested no tested
Histopathological 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 0/9 8/9
sarcoma tissue 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 Oesophagus 0/8 8/8 squamous carcinoma Ovary not tested 7/7
adenocarcinoma Skin 0/6 6/6 squamous carcinoma Small 0/5 not tested
not tested Intestine Stomach 0/10 9/10 adenocarcinoma Testis 0/8
14/14 malignant germ cell tumours Uterus 0/5 7/7 adenocarcinoma n =
5; malignant mixed Mullerian tumour n = 2 Total 0/123 128/133
EXAMPLE 4
[0058] Experiments were conducted to defect CYP1B1 RNA in various
tumour and normal tissues.
[0059] 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 synthesised from the isolated RNA using oligo (dT). The CYP1B1
primers had the following sequences: Forward 5'-AAC TCT CCA TCA GGT
GAG GT-3' (nt 2104-2123); Reverse 5'-TAA GGA AGT ATA CCA GAA GGC
-3' (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' (nt
1067-1105); Reverse 5'-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3'
(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 visualised 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 tumour tissues, semi-quantitative
RT-PCR of normal and tumour kidney samples was performed using
serial dilution of cDNA (24). .beta.-actin mRNA was used as an
internal control (29).
[0060] FIG. 4 shows CYP1B1 and .beta.-actin mRNA in normal (A and
B) and corresponding tumour (C and D) samples which have been
detected by RT-PCR. Lane 1 kidney, lane 2 colon, lane 3 skin, lane
4 oesophagus, lane 5 stomach, lane 6 lymph node, lane 7 breast.
[0061] Analysis of the tumours by RT-PCR showed that all tumour
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.
[0062] Concluding Remarks
[0063] 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 tumour cells do not significantly express P450.
However, we have now shown that CYP1B1 is expressed in a wide
variety of malignant turnouts of different histogenetic types and
is not present in normal tissues, indicating that this P450 is a
tumour specific form of P450. Tumours are composed of a variable
proportion of tumour cells and non-tumour cells. To identify that a
protein is tumour specific, it is important to demonstrate that the
protein is localised only to tumour cells. Immunohistochemistry
allows the direct visualisation of tumour cells and has the spatial
resolution to separate tumour cells from non-tumour cells.
Furthermore, it is important to show there has been no differential
degradation of proteins in normal tissue samples compared with
tumour samples and immunoblotting for .beta.-actin (as a positive
control protein) showed it to be present in every normal and tumour
sample indicating there was no protein degradation. In addition,
Coomassie blue staining of the polyacrylamide gels showed no
evidence of protein degradation. Moreover, immuno-histochemistry of
the tumour samples provides its own internal control as sections of
tumour contain non-tumour cells.
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