U.S. patent application number 10/137473 was filed with the patent office on 2003-06-05 for method of classifying a thyroid carcinoma using differential gene expression.
This patent application is currently assigned to CuraGen Corporation. Invention is credited to Gould-Rothberg, Bonnie E., Rastelli, Luca.
Application Number | 20030104419 10/137473 |
Document ID | / |
Family ID | 27383971 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030104419 |
Kind Code |
A1 |
Gould-Rothberg, Bonnie E. ;
et al. |
June 5, 2003 |
Method of classifying a thyroid carcinoma using differential gene
expression
Abstract
Disclosed are methods of diagnosing and treating carcinomas,
including metastatic thyroid carcinomas using differential gene
expression. Also disclosed are novel nucleic acid sequences whose
expression is differentially regulated in metastatic and
non-metastatic thyroid carcinomas.
Inventors: |
Gould-Rothberg, Bonnie E.;
(Guilford, CT) ; Rastelli, Luca; (Guilford,
CT) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS,
GLOVSKY AND POPEO, P.C.
One Financial Center
Boston
MA
02111
US
|
Assignee: |
CuraGen Corporation
New Haven
CT
|
Family ID: |
27383971 |
Appl. No.: |
10/137473 |
Filed: |
April 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10137473 |
Apr 30, 2002 |
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09552322 |
Apr 19, 2000 |
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6436642 |
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60130123 |
Apr 20, 1999 |
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60193203 |
Mar 30, 2000 |
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Current U.S.
Class: |
435/6.18 ;
435/7.23 |
Current CPC
Class: |
A61P 35/04 20180101;
C12Q 1/6886 20130101; C12Q 2600/136 20130101; A61P 5/14 20180101;
G01N 33/57407 20130101; C12Q 2600/158 20130101; A61P 35/00
20180101 |
Class at
Publication: |
435/6 ;
435/7.23 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Claims
What is claimed is:
1. A method of categorizing a thyroid carcinoma in a subject, the
method comprising: a) providing a test cell population from said
subject, wherein at least one cell in said test cell population is
capable of expressing one or more nucleic acid sequences selected
from the group consisting of MTCs: 8-51; b) measuring the
expression of one or more said nucleic acid sequences in said test
cell population; c) comparing the expression of said nucleic acid
sequences to the expression of said nucleic acid sequences in a
reference cell population comprising at least one cell whose
thyroid carcinoma stage is known; and d) identifying a difference
in expression levels of the MTC sequence, if present, in the test
cell population and reference cell population, thereby categorizing
said thyroid carcinoma in said subject.
2. The method of claim 1, wherein said method comprises measuring
the expression of one or more nucleic acid sequences selected from
the group consisting of MTCs: 8-18.
3. The method of claim 1, wherein said method comprises measuring
the expression of one or more nucleic acid sequences selected from
the group consisting of MTCs: 19-47.
4. The method of claim 1, wherein said method comprises measuring
the expression of one or more nucleic acid sequences selected from
the group consisting of MTCs:48-51.
5. The method of claim 1, wherein said method further comprises
measuring the expression of one or more nucleic acid sequences
selected from the group consisting of MTCs: 1-7.
6. The method of claim 2, wherein said method further comprises
measuring the expression of one or more nucleic acid sequences
selected from the group consisting of MTCs: 19-47.
7. The method of claim 6, wherein said method further comprises
measuring the expression of one or more nucleic acid sequences
selected from the group consisting of MTCs:48-51.
8. The method of claim 3, wherein said method further comprises
measuring the expression of one or more nucleic acid sequences
selected from the group consisting of MTCs:48-51.
9. The method of claim 1, wherein said carcinoma is a metastatic
papillary thyroid carcinoma.
10. The method of claim 1, wherein an alteration of the expression
of said nucleic acids in said test cell population as compared to
said reference cell population indicates that the test cell
population has a different thyroid carcinoma stage than the cells
in said reference cell population.
11. The method of claim 1, wherein a similar expression pattern of
said nucleic acids in said test cell population as compared to said
reference cell population indicates that the test cell population
has the same thyroid carcinoma stage as the cells in said reference
cell population.
12. The method of claim 1, wherein said subject is a human.
13. The method of claim 1, wherein said reference cell population
comprises a plurality of cells or a database.
14. A method of diagnosing a thyroid carcinoma in a subject, the
method comprising: a) providing a test cell population from said
subject, wherein at least one cell in said test cell population is
capable of expressing one or more nucleic acid sequences selected
from the group consisting of MTCs 8-51; b) measuring the expression
of one or more said nucleic acid sequences in said test cell
population; c) comparing the expression of said nucleic acid
sequences to the expression of said nucleic acid sequences to a
reference cell population comprising at least one cell whose
thyroid carcinoma stage is known, and d) identifying a difference
in expression levels of the MTC sequence, if present, in the test
cell population and reference cell population, thereby diagnosing a
thyroid carcinoma, if present, in said subject.
15. The method of claim 2, wherein said thyroid carcinoma is a
metastatic thyroid carcinoma.
16. A method of assessing the efficacy of a treatment of a thyroid
carcinoma in a subject, the method comprising: a) providing a test
cell population from said subject, wherein at least one cell in
said test cell population is capable of expressing one or more
nucleic acid sequences selected from the group consisting of MTCs
8-51; b) detecting the expression of one or more said nucleic acid
sequences in said test cell population; c) comparing the expression
of said nucleic acid sequences to the expression of said nucleic
acid sequences to a reference cell population comprising at least
one cell whose thyroid carcinoma stage is known; and d) identifying
a difference in expression levels of the MTC sequence, if present,
in the test cell population and reference cell population, thereby
assessing the efficacy of treatment of said thyroid carcinoma in
said subject.
17. A method for identifying a therapeutic agent individualized for
treating a thyroid carcinoma in a subject, the method comprising:
a) providing a test cell population from said subject, wherein at
least one cell in said test cell population is capable of
expressing one or more nucleic acid sequences selected from the
group consisting of MTCs: 8-51; b) contacting said test cell
population with a therapeutic agent; c) measuring the expression of
said nucleic acid sequence in said test cell population; d)
comparing the expression of said nucleic acid sequences to the
expression of said nucleic acid sequences in a reference cell
population comprising at least one cell whose thyroid carcinoma
stage is known; and e) identifying a difference in expression
levels of the MTC sequence, if present, in the test cell population
and reference cell population, thereby identifying a therapeutic
agent individualized for said subject.
18. The method of claim 17, wherein said thyroid carcinoma is a
metastatic thyroid carcinoma.
19. A method of identifying a candidate therapeutic agent for
treating a thyroid carcinoma, the method comprising: a) providing a
test cell population, wherein at least one cell in said test cell
population is capable of expressing one or more nucleic acid
sequences selected from the group consisting of MTCs:8-51; b)
contacting said test cell population with said candidate
therapeutic agent; c) measuring the expression of said nucleic acid
sequences in the test cell population; d) comparing the expression
of said nucleic acid sequences to the expression of said nucleic
acid sequences in a reference cell population comprising at least
one cell whose thyroid carcinoma stage is known; and e) identifying
a difference in expression levels of the MTC sequence, if present,
in the test cell population and reference cell population, thereby
identifying a therapeutic agent for treating a thyroid
carcinoma.
20. A method of assessing the prognosis of a subject with a thyroid
carcinoma, the method comprising: a) providing a test cell
population from said subject, wherein at least one cell in said
test cell population is capable of expressing one or more of said
nucleic acid sequences selected from the group consisting of MTCs:
8-51; b) measuring the expression or one or more said nucleic acid
sequences in said cell; and c) comparing the expression of said
nucleic acid sequences to the expression of said nucleic acid
sequences in a reference cell population comprising at least one
cell whose thyroid carcinoma stage is known; and d) identifying a
difference in expression levels of the MTC sequence, if present, in
the test cell population and reference cell population, thereby
assessing the prognosis of said subject.
21. A method of treating metastatic carcinoma, the method
comprising administering to a patient suffering from or at risk for
developing metastatic carcinoma, an agent that increases the
expression or activity of one or more nucleic acid sequences
selected from the group consisting of MTCs: 17, 24, 28, 32, 36, 38,
40, 43, and 46.
22. The method of claim 21, wherein said carcinoma is thyroid
carcinoma.
23. A method of treating metastatic carcinoma, the method
comprising administering to a patient suffering from or at risk for
developing metastatic carcinoma, an agent that decreases the
expression or activity of one or more nucleic acid sequences
selected from the group consisting of MTCs: 8-16, 18-23, 25-27,
29-31, 33-35, 37, 39, 41-42, 44-45, 48, and 50.
24. The method of claim 23, wherein said carcinoma is thyroid
carcinoma.
25. The method of claim 23, wherein said agent is an antibody to a
polypeptide encoded by said MTC nucleic acid sequence.
26. An isolated polynucleotide selected from the group consisting
of: a) a nucleotide sequence comprising one or more polymorphic
sequences of Table 2; b) a fragment of said nucleotide sequence,
provided that the fragment includes a polymorphic site in said
polymorphic sequence; c) a complementary nucleotide sequence
comprising a sequence complementary to one or more of said
polymorphic sequences; and d) a fragment of said complementary
nucleotide sequence, provided that the fragment includes a
polymorphic site in said polymorphic sequence.
27. The polynucleotide of claim 26, wherein said polynucleotide
sequence is DNA.
28. The polynucleotide of claim 26, wherein said polynucleotide
sequence is RNA.
29. The polynucleotide of claim 26, wherein said polynucleotide
sequence is between about 10 and about 100 nucleotides in
length.
30. The polynucleotide of claim 26, wherein said polynucleotide
sequence is between about 10 and about 90 nucleotides in
length.
31. The polynucleotide of claim 26, wherein said polynucleotide
sequence is between about 10 and about 75 nucleotides in
length.
32. The polynucleotide of claim 26, wherein said polynucleotide is
between about 10 and about 50 bas es in length.
33. The polynucleotide of claim 26, wherein said polynucleotide is
between about 10 and about 40 bases in length.
34. An isolated allele-specific oligonucleotide that hybridizes to
a first polynucleotide at a polymorphic site encompassed therein,
wherein the first polynucleotide is chosen from the group
consisting of: a) a nucleotide sequence comprising one or more
polymorphic sequences provided that the polymorphic sequence
includes a nucleotide other than the nucleotide recited in Table 2
for said polymorphic sequence; b) a nucleotide sequence that is a
fragment of said polymorphic sequence, provided that the fragment
includes a polymorphic site in said polymorphic sequence; c) a
complementary nucleotide sequence comprising a sequence
complementary to one or more polymorphic sequences recited in Table
2, provided that the complementary nucleotide sequence includes a
nucleotide other than the complement of the nucleotide recited in
non polymorphic sequence Table 2; and d) a nucleotide sequence that
is a fragment of said complementary sequence, provided that the
fragment includes a polymorphic site in said polymorphic
sequence.
35. The nucleic acid molecule of claim 34, wherein said nucleic
acid molecule is selected from the group consisting of SEQ ID
NOS:15-63.
36. A method of identifying a base occupying a polymorphic site in
a nucleic acid molecule, the method comprising: (a) obtaining a
nucleic acid molecule from a subject; (b) contacting said nucleic
acid molecule with one or more sequences selected from the group
consisting of SEQ ID NOS:15-63; (c) detecting hybridization; and
(d) identifying the sequences that hybridize with said nucleic acid
molecules wherein the nucleic sequence of the hybridizing sequences
indicates a polymorphic site in the nucleic acid molecule.
37. The method of claim 36, wherein the subject suffers from or is
at risk for metastatic thyroid carcinoma.
38. The method of claim 36, wherein the presence of the polymorphic
site is correlated with the presence of a metastatic thyroid
carcinoma.
39. The method of claim 36, wherein the nucleic acid molecule is
genomic DNA.
40. The method of claim 36, wherein the nucleic acid molecule is
cDNA.
41. A nucleic acid sequence 20-100 nucleotides in length comprising
the polymorphic site 10 identified in the method of claim 36.
42. The method of claim 36, wherein the nucleic acid molecule is
obtained from a plurality of subjects, and a base occupying one of
the polymorphic sites is determined in each of the subjects.
41. The method of claim 36, wherein the subject is a human or a
rodent.
42. A kit comprising one or more reagents for detecting two or more
nucleic acid sequences selected from the group consisting of MTCs:
8-51.
43. An array of probe nucleic acids, wherein said probe nucleic
acids detect two or more nucleic acid sequences selected from the
group consisting of MTCs: 8-51.
44. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of MTCs: 48-51, or
fragments thereof.
45. A vector comprising the nucleic acid of claim 44.
46. A cell comprising the vector of claim 45.
47. A pharmaceutical composition comprising the nucleic acid of
claim 44.
48. A polypeptide encoded by the nucleic acid of claim 44.
49. An antibody which specifically binds to the polypeptide of
claim 48.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Ser. No.
60/130,123, filed Apr. 20, 1999, and U.S. Ser. No. ______, filed
Mar. 30, 2000. Both of these applications are incorporated herein
by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates in general to nucleic acids and more
particularly to the use of genes differentially expressed in
metastatic thyroid carcinomas and non-metastatic thyroid
carcinomas.
BACKGROUND OF THE INVENTION
[0003] The thyroid is an endocrine gland involved in the
homeostatic regulation of bodily functions. Its basic morphologic
unit is the follicle, which contains two major parenchymal cell
types: the follicular cells and the parafollicular cells.
[0004] The diseases affecting the thyroid gland can be classified
as benign and malignant. Benign diseases can include diffuse
goiter, such as Graves' disease, and nodular goiter, such as
nodular hyperplastic goiter. Other benign thyroid disorders include
thyroiditis and benign neoplasms, such as adenomas.
[0005] Thyroid cancer is a common endocrine tumor. The reported
incidence of thyroid cancer is about 4 per 100,000, with the number
of afflicted males and females occurring at a relative ratio of
2:1. It is possible that the actual incidence of thyroid cancer is
higher, as thyroid cancers, like prostate malignancies, are most
typically found as occult tumors during autopsy.
[0006] Thyroid cancers are typically classified according to their
cellular origin and level of differentiation. For example,
follicular cells produce well-differentiated carcinomas and
anaplastic carcinomas, while parafollicular cells produce medullary
thyroid carcinomas. Other cancers associated with the thyroid gland
include sarcomas, originating from stromal cells of the thyroid,
and lymphomas, which originate from immune cells associated with
the thyroid.
[0007] A majority of thyroid malignancies are well-differentiated,
slow-growing, and remain local to the thyroid gland. Removal of the
thyroid, followed by .sup.125I gamma irradiation of any remaining
thyroid tissue, typically elicits a good response and prognosis for
patients whose cancer has not spread beyond the thyroid gland.
Extension of tumors into adjacent neck structures or more distant
sites from the thyroid is associated with a significantly worse
prognosis. The prognostic significance of local metastases to lymph
nodes in the region of the lymph node is less clear.
[0008] The mechanisms by which thyroid cancers metastasize to
distant sites in the body are not completely understood. For
example, not all the genes whose expression levels change in
response to a thyroid metastasis have been identified. In addition
to providing potential targets in metastatic tissue that could be
used for treat metastasized genes, such genes could also be used to
diagnose metastatic tissues.
SUMMARY OF THE INVENTION
[0009] The invention is based in part on the discovery of genes
whose expression levels can be correlated to one or more metastatic
cancerous states in a thyroid cell. Measuring expression levels of
these genes in a sample cell population allows for the type and
tumor stage of the cells in the sample to be determined.
[0010] Accordingly, in one aspect the invention relates to genes
that are differentially expressed in metastatic versus
non-metastatic thyroid cancer. These differentially expressed genes
are collectively referred to herein as "Metastatic Thyroid Cancer"
genes ("MTC genes"). The corresponding gene products are referred
to as "MTC products" and/or "MTC proteins". The MTC genes include
E-cadherin, alpha-1-antitrypsin, manganese superoxide,
thyroglobulin, fibronectin, CD18, calpain, clusterin, cathepsin E,
cystatin B, RIG-E, p8=candidate of metastasis 1, periplakin,
neuropilin, proteasome subunit HC5, NET-1, ras
GTPase-activating-like protein (IQGAP1), DUSP6 dual specificity MAP
kinase phosphatase, SET binding factor (SBF), 5-lipoxygenase,
lipocortin I, lipocortin II, annexin II, calgizzarin,
spermidine/spermine N1-acetyltransferase (SSAT),
daunorubicin-binding protein=aldehyde dehydrogenase 2, gelsolin,
integrin alpha-3, Type IV collagenase, antileukoprotease,
STE20-like protein kinase 3 (STK3), peflin, and kinectin.
[0011] In various aspects, the invention includes methods of
categorizing thyroid neoplasms, diagnosing thyroid carcinomas,
assessing the efficacy of a treatment of a thyroid carcinoma in a
subject, and assessing the prognosis of a subject with a thyroid
carcinoma. Each of these methods involves providing a test cell
population from a subject capable of expressing one or more
metastatic thyroid carcinoma nucleic acid sequences, termed MTC
sequences, measuring the expression of these MTC sequences in the
test cell population, and comparing the levels of expression in the
test cell population with the expression levels in a reference cell
population whose thyroid carcinoma stage is known.
[0012] In a further aspect, the invention includes a method of
selecting an individualized therapeutic agent appropriate for a
particular subject. This method includes providing from the
subject, a test cell population comprising a cell capable of
expressing one or more MTC sequences, contacting the test cell
population with the therapeutic agent, and comparing the expression
of the MTC sequences in the test cell population to the expression
levels in a reference cell population whose thyroid carcinoma stage
is known.
[0013] In another aspect, the invention provides a method of
identifying a candidate therapeutic agent for treating thyroid
carcinomas. This method includes providing a test cell population
capable of expressing one or more MTC sequences, contacting the
test cell population with a candidate therapeutic agent, and
comparing the expression of the MTC sequences to the expression in
a reference cell population whose thyroid carcinoma stage is
known.
[0014] The invention further provides a method of treating
metastatic cancer. In one embodiment, this method includes
administering to a patient suffering from or at risk for developing
metastatic cancer, an agent that increases the expression of one or
more MTC sequences that are down-regulated in metastatic versus
non-metastatic cancer. In another embodiment, this method involves
administering an agent that decreases the expression of one or more
MTC sequences that are up-regulated in metastatic versus
non-metastatic cancer.
[0015] Further provided is a kit comprising one or more reagents
for detecting the presence of two or more MTC nucleic acid
sequences and an array of probe nucleic acids capable of detecting
two or more MTC nucleic acid sequences. Also provided are isolated
nucleic acid molecules that are differentially expressed in
metastatic vs. non-metastatic thyroid cancer, as well as single
nucleotide polymorphisms in MTC sequences, and methods of using the
MTC single nucleotide polymorphisms.
[0016] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and are not intended to be
limiting.
[0017] Other features and advantages of the invention will be
apparent from the following detailed description and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a representation of the sequence homology between
a mouse MTC48 amino acid sequence according to the invention and a
human MTC48 amino acid sequence according to the invention.
[0019] FIG. 2 is a representation of a hydrophobicity plot for a
MTC48 nucleic acid according to the invention.
[0020] FIG. 3 is a representation of the sequence homology between
mouse MTC49 amino acid sequence according to the invention and a
human MTC49 amino acid sequence according to the invention.
[0021] FIG. 4 is an illustration of the sequence homology between a
nucleic acid encoding protein Q9Y3Z0 and a MTC50 nucleic acid
according to the invention.
[0022] FIG. 5 is an illustration of the sequence homology between
the rat gene E3-3 (AAB54063) and a MTC50 nucleic acid according to
the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention is based in part on the discovery of
changes in expression patterns of multiple nucleic acid sequences
in metastatic papillary thyroid carcinomas versus in non-metastatic
thyroid follicular adenomas. Changes in expression have been
observed in both heretofore undescribed nucleic acid sequences as
well as in previously identified nucleic acid sequences. By
measuring the expression of one or more of these nucleic acid
sequences in Response to various agents, agents for treating cancer
can be identified. In addition, the measurement of the expression
profiles of one or more of these sequences can be used, for
example, to diagnose a neoplasm, to categorize a neoplasm, to
assess prognosis, and to monitor the efficacy of neoplasm
treatment.
[0024] To identify differentially expressed genes according to the
invention, fresh frozen surgical specimens were obtained from three
thyroid follicular adenomas and three metastatic papillary thyroid
carcinomas. These samples were processed through GENECALLING.TM.
differential expression analysis, as described in U.S. Pat. No.
5,871,697 and in Shimkets et al., Nature Biotechnology 17:798-803
(1999). GENECALLING.TM. technology relies on Quantitative
Expression Analysis to generate a gene expression profile of a
given sample and then generates differential expression analysis of
pairwise comparison of these profiles to controls containing no
additions. Polynucleotides exhibiting differential expression are
confirmed by conducting a PCR reaction according to the
GENECALLING.TM. protocol with the addition of a competing unlabeled
primer that prevents the amplification from being detected.
[0025] Comparing any two samples with each other (either between or
across treatment groups) revealed a 10% difference rate between
samples. This difference highlights both the highly variable nature
of neoplastic growths and the high level of individual variation.
To identify those genes that are differentially expressed between
the hyperplastic adenamatous state and the metastatic carcinomous
state, all three follicular adenomas were compared as a group to
all three papillary carcinomas as a group. Differences were
confirmed if they exhibited consistent behavior across all adenomas
and were significantly different from all carcinomas. This strategy
reduced the number to significant difference to 80 (0.5%).
Fifty-one genes were confirmed as being differentially expressed.
For convenience and ease of reference, these genes have been
designated as MTC: 1-51.
[0026] A summary of the differentially expressed sequences is
presented in Table 1. The first three columns of this Table provide
descriptive information for these sequences. Specifically, Column 1
provides the common names of each of the genes, Column 2 lists the
sequence database reference number for previously known sequences,
and Column 3 provides the SEQ ID NOs for the newly discovered
nucleic acid sequences. Column 4 displays the change in expression
level of each sequence that was observed in metastatic thyroid
carcinomas as compared to nonmetastatic thyroid carcinomas. Column
5 provides the MTCX number assigned to each of the differentially
expressed genes for ease of reference.
[0027] The differentially expressed genes are subdivided into
groups in Table 1 as follows: genes whose expression is previously
correlated with metastatic thyroid cancer; genes whose expression
is newly correlated with metastatic thyroid cancer, and nucleic
acid sequences newly described herein.
[0028] One of ordinary skill in this art will recognize that the
information in Table 1 can be used to identify a particular MTC
sequence. For example, where the sequence database reference number
of a particular nucleic acid sequence is known, this reference
number can be used to identify a particular MTC sequence. For a
given MTC sequence, its expression can be measured by using any
methods commonly known in the art. Based on all of this, one of
ordinary skill in the art will be able to deduce the information
necessary for detecting and measuring the expression of each MTC
nucleic acid sequence, as required by each of the methods described
herein.
1 Sequence Change in Expression Description Database Level in
Metastic vs. MTCX of Sequence Reference SEQ ID NO: Non-Metastic
Cells Assignment Genes Whose Expression Is Previously Correlated
with Metastatic Thyroid Cancer Fibronectin X02761 K00055 K00799
+19.8 MTC1 K02273 X00307 X00739 Thyroglobulin X05615 -4.6 MTC2
Alpha-1-antitrypsin M11465 +7.1 MTC3 E-cadherin CAA79356, Z18923
-4.2 MTC4 Manganese superoxide X65965 +2.8 MTC5 dismutase (MnSOD)
Integrin beta-2, M15395 +2.5 MTC6 CD18 (ITGB2) (LFA-1/CR3/P150,95)
Nebulin X83957 +7.1 MTC7 Genes Whose Expression Is Newly Correlated
with Metastatic Thyroid Cancer Type IV collagenase J03210 +3.7 MTC8
Neuropilin 1 NM_003873 +11.8 MTC9 Ca2-activated M23254 +2.5 MTC10
neutral protease large subunit Clusterin NP_001822 +5.4 MTC11
(apolipoprotein J) Cathepsin E J05036 +3.1 MTC12 Cystatin B U46692
+1.6 MTC13 Lipocortin I X05908 +3.1 MTC14 Lipocortin II D00017 +4.5
MTC15 Retinoic acid induced U42376 +3.7 MTC16 RIG-E precursor
(RIG-E) p8 (candidate of AF069073 -2.3 MTC17 metastasis 1) NM_12385
protein phosphatase-1 L07395 +1.5 MTC18 gamma 1 Tetraspan NET-I
AF065388 +7.3 MTC19 Periplakin AF013717 +2.7 MTC20 Proteasome
subunit HC5 D00761 +2.1 MTC21 ras GTPase-activating- NP_003861 +2.7
MTC22 like protein L33075 (IQGAP1) DUSP6 dual specificity AB013382
+3.2 MTC23 MAP kinase phosphatase SET binding factor U93181 -4.5
MTC24 (SBF1) 5-lipoxygenase J03571 +3.7 MTC25 Calgizzarin D38583
+2.2 MTC26 Spermidine/spermine N1- M77693 +2.1 MTC27
acetyltransferase (SSAT) Aldehyde dehydrogenase 2 K03001 -2.7 MTC28
(ALDH-2) Gelsolin X04412 +2.7 MTC29 Integrin alpha-3 AAA3612 +2.9
MTC30 KIAA0937 AB023154 +3.5 MTC31 KIAA1131 AB032957 -2 MTC32
Antileukoprotease X04470 +11.1 MTC33 DAP12 AF019562 +3.5 MTC34
brain-specific STE20- AAD42039 +3.5 MTC35 like protein kinase 3
(MST-3b) Peflin BAA85163 -1.8 MTC36 Kinectin NM_004986 +3.5 MTC37
Prostaglandin transporter U70867 -4.9 MTC38 hPGT Ribophorin II
Y00282 +2.5 MTC39 PRO302 X25445 -2 MTC40 X52258 (Patent Database)
Stimulated trans- X82200 +3.6 MTC41 acting factor (50 kDa)
Iduronate 2-sulfatase M58342 +1.7 MTC42 Mitochondrial proteolipid
AF054175 -1.7 MTC43 68 MP Growth arrest specific AF141346 +2 MTC44
transcript 5 gene lactate dehydrogenase-A X02152 +1.5 MTC45 26-kDa
surface protein M33680 -2.9 MTC46 TAPA-1 mRNA, complete cds; CD81
Sodium dependent AF111856 +11.8 MTC47 phosphate transporter isoform
NaPi-3b Nucleic Acid Sequences Newly Described Herein Novel human
d010-145.3 = 88098062 SEQ ID NO:1 +6.6 MTC48 Cghs m1n0274.6_2 SEQ
ID NO:5 -2.1 MTC49 Human E3-3 = 95199195 SEQ ID NO:10 +2.7 MTC50
Cghs 10n0102.3_1 SEQ ID NO:12 -2 MTC51
[0029] Below follows a brief description of some sequences whose
expression level changes between metastatic and non-metastatic
thyroid carcinomas. For some sequences, a summary of SAGE
expression analysis is also presented below. Using this
information, one of ordinary skill in the art will recognize that
MTC sequences can be used to detect nucleic acid sequence
expression in cells or tissues in which they are expressed.
[0030] Genes Whose Expression is Previously Correlated with
Metastatic Thyroid Cancer
[0031] Genes identified as being differentially expressed in
studies leading to the invention include fibronectin, E-cadherin,
alpha-1-antitrypsin, manganese superoxide, thyroglobulin, and CD18.
These genes have been previously described as being differentially
expressed in metastatic and non-metastatic thyroid cancers. These
genes are briefly discussed below.
[0032] Fibronectin (MTC 1)
[0033] Fibronectin (genBank #X02761 K00055 K00799
K02273X00307X00739) is a 430,000 dalton dimeric glycoprotein that
exists in two forms, which are named cellular and plasma
fibronectin. Cellular fibronectin is the major cell surface
glycoprotein of many cells. A major function of the fibronectins is
to facilitate the adhesion of cells to extracellular materials,
such as solid substrata and matrices. While there is agreement that
fibronectin is up-regulated in metastatic thyroid cancer, it is not
yet completely understood whether this is due to the tumor cells or
to the infiltrating fibroblast (ee Takano et al., J Clin Endocrinol
Metab 85(2):765-8) (2000). GENECALLING.TM. analysis reveals that
fibronectin is up-regulated in metastatic vs. non-metastatic
thyroid cancer.
[0034] Thyroglobulin (MTC2)
[0035] Thyroglobulin (genBank #X05615) is the glycoprotein
precursor of the thyroid hormones thyroxine (T4) and
triiodothyronine (T3). Newly synthesized thyroglobulin is folded
and homodimerized in the endoplasmic reticulum (ER) prior to its
export to the site of iodination, where it serves as the precursor
for thyroid hormone synthesis. Thyroglobulin is a reliable tumor
marker in patients with well-differentiated thyroid cancer
("WDTC"). Low levels of thyroglobulin correlate with clinical stage
and metastatic potential. GENECALLING.TM. analysis reveals that
thyroglobulin is down-regulated in metastatic vs. non-metastatic
thyroid cancer.
[0036] Alpha-1-antitrypsin (MTC3)
[0037] Alpha-1-antitrypsin (GenBank #M11465) is a major plasma
serine protease inhibitor. Previous reports indicate that
alpha-1-antitrypsin is present in 9 out of 10 thyroid papillary
carcinomas while the normal thyroid tissue present in the vicinity
of each tumor showed no staining (ee Poblete et al., Am J Surg
Pathol 20(8):956-63 (1996)). GENECALLING.TM. analysis reveals that
alpha-1-antitrypsin is up-regulated in metastatic vs.
non-metastatic thyroid cancer.
[0038] E-cadherin (MTC4)
[0039] Loss of E-cadherin (uvomorulin) (genBank #CAA79356, Z18923),
a Ca(2+)-dependent cell adhesion molecule that is required for
normal epithelial function has been attributed a pathogenetic role
in tumor invasion. Previous reports indicate that steady-state
E-cadherin mRNA levels and immunostaining were either reduced or
lost in thyroid cancers. E-cadherin was greatly reduced upon
progression to primary tumor stage 4 (pT4) tumors, and this
reduction correlates with metastatic potential. (See Scheumman et
al., J Clin Endocrinol Metab 80(7):2168-72 (1995)). GENECALLING.TM.
analysis reveals that E-cadherin is down-regulated in metastatic
vs. non-metastatic thyroid cancer.
[0040] Manganese Superoxide Dismutase (MnSOD) (MTC5)
[0041] Manganese superoxide dismutase (GenBank #X65965) is a member
of a family of metalloenzymes that catalyze the dismutation of the
superoxide anion to H.sub.2O.sub.2. MnSOD is encoded by nuclear
chromatin, synthesized in the cytosol, and imported
posttranslationally into the mitochondrial matrix. The MnSOD
concentration is increased in papillary carcinoma or
papillary-growing cells. (See Iwase et al., Acta Endocrinol
(Copenh) 129(6):573-8 (1993)). GENECALLING.TM. analysis reveals
that MnSOD is up-regulated in metastatic vs. non-metastatic thyroid
cancer.
[0042] Integrin Beta-2, CD18 (ITGB2) (MTC6)
[0043] Integrin beta-2 is also known as human cell surface adhesion
glycoprotein LFA-1/CR3/P150,95 beta-subunit (genBank# M15395). This
leukocyte cell adhesion molecule belongs to the class of cell
membrane glycoproteins known as integrins, which are alpha-beta
heterodimers. The alpha subunits vary in size from 120 to 180 kD,
and each is noncovalently associated with a beta subunit (90 to 110
kD). Expression is increased in infiltrating lymphocytes and
vascular endothelium in thyroid glands from patients with
autoimmune thyroid disorders, Graves' disease (GD), and Hashimoto's
thyroiditis (HT). GENECALLING.TM. analysis reveals that ITGB2 is
up-regulated in metastatic vs. non-metastatic thyroid cancer. These
cancers have high levels of infiltrating lymphocytes.
[0044] Nebulin (MTC7)
[0045] Nebulin (genBank#X83957) is a giant protein component of the
cytoskeletal matrix that coexists with the thick and thin filaments
within the sarcomeres of skeletal muscle. In most vertebrates,
nebulin accounts for 3 to 4% of the total myofibrillar protein. Its
size varies from 600 to 800 kD in a manner that is tissue-,
species-, and developmental stage-specific. GENECALLING.TM.
analysis reveals that nebulin is up-regulated in metastatic vs.
non-metastatic thyroid cancer.
[0046] Nebulin is one of the autoantigens present in the peripheral
blood of patients with Graves' disease. (ee Kiljanski et al., Clin
Exp Rheumatol 14 Suppl 15:S69-76 (1996)). It is possible that the
overexpression of nebulin is a general feature of thyroid diseases
associated with immune responses.
[0047] Genes Whose Expression is Newly Correlated with Metastatic
Thyroid Cancer
[0048] Additional genes identified as being differentially
expressed in studies leading to the invention include Type IV
collagenase, neuropilin 1, calpain, clusterin, cathepsin E,
cystatin B, lipocortin I, RIG-E, p8=candidate of metastasis 1, and
protein phosphatase-1 gamma 1.
[0049] Their identification with the correct modulation indicates
that metastatic tumor cells from different tissues or sites in the
body might have an underlying common molecular pathway of gene
expression. The discovery of such a common pathway has important
implications for the diagnosis and treatment of metastatic
carcinomas of all origins. The diagnostic, screening, and
therapeutic applications are discussed in detail below. Moreover,
these genes and treatments developed based on this list, including
recombinant protein drugs, antibody drugs, and small molecule drugs
may have a diagnostic role in metastatic carcinomas other than
thyroid cancer.
[0050] Type IV Collagenase (MTC8)
[0051] Type IV collagenase (genBank# J03210) is a metalloproteinase
that specifically cleaves type IV collagen, the major structural
component of basement membranes. It is also known as matrix
metalloproteinase-2 and gelatinase A. Degradation of type IV
collagen in the basement membrane is an essential step in the
invasive/metastatic behavior of tumor cells. Therefore the enhanced
expression of the type IV collagenases, MMP-2 and MMP-9, or the
lack of their inhibitors, TIMP-1 and TIMP-2, has been associated
with tumor invasion and metastatic potential in several
experimental models as well as in human tumors. Inhibitors of MMP-2
and MMP-9 prevent tumor growth and invasion in animal models. (See
Koivunen et al., Nat Biotechnol 17(8):768-74 (1999)).
GENECALLING.TM. analysis reveals that type IV collagenase is
up-regulated in metastatic vs. non-metastatic thyroid cancer.
[0052] Neuropilin I (MTC9)
[0053] Neuropilin 1 (genBank #NM.sub.--003873) is a receptor for
the axonal chemorepellent Semaphorin III in neurons. In endothelial
and tumor cells, it is an isoform-specific receptor for vascular
endothelial growth factor ("VEGF"). It acts by modulating VEGF
binding to its receptor KDR and subsequent bioactivity. (ee Soker
et al., Int J Oncol 16(2):253-60 (2000)). A naturally-occurring
soluble form acts as a powerful VEGF antagonist and has anti-tumor
activity. GENECALLING.TM. analysis reveals that Neuropilin I is
up-regulated in metastatic vs. non-metastatic thyroid cancer.
[0054] Ca2-Activated Neutral Protease Large Subunit (CANP) (MTC
10)
[0055] Ca2-activated neutral protease large subunit is also known
as calpain (genBank #M23254). It is an intracellular cytoplasmatic
non-lysosomal cysteine endopeptidase that requires calcium ions for
activity. Many substrates of the calpain isoenzymes, such as the
transcription factors c-Fos and c-Jun, the tumor supressor protein
p53, protein kinase C, pp60c-src, and the adhesion molecule
integrin, have been implicated in the pathogenesis of different
human tumors. This suggests an important role for the calpains in
malignant diseases. In renal cell carcinomas, there is a
correlation of higher calpain I expression with increased
malignancy. Within the clear cell carcinoma subset, tumor samples
with advanced nodal status (N1 and N2) showed a significantly
higher level of calpain I expression than did tumors without
metastasis to regional lymph nodes. (See Braun et al., Int J Cancer
84(1):6-9 (1999)). An inhibitor of calpain was selectively
cytotoxic to human tumor cells from chronic myeloid leukemia
tissues, in a dose-dependent manner, and was also cytotoxic to
Walker rat tumor cells. GENECALLING.TM. analysis reveals that
calpain is up-regulated in metastatic vs. non-metastatic thyroid
cancer.
[0056] Clusterin (MTC11)
[0057] Clusterin is also known as complement lysis inhibitor,
SP40,40, sulfated glycoprotein 2, testosterone-repressed prostate
message 2, and apolipoprotein J (genBank#NP.sub.--001822). Sulfated
glycoprotein-2 (SGP-2) is the major secreted product of Sertoli
cells. It probably plays a critical role in spermatogenesis.
Clusterin acts as a control mechanism of the complement cascade.
Specifically, it prevents the binding of a C5b-C7 complex to the
membrane of the target cell and, in this way, inhibits
complement-mediated cytolysis. Apolipoprotein J is synthesized at
high levels in degenerating hippocampus from individuals with
Alzheimer's disease or Pick disease.
[0058] Clusterin may be a suicide gene active in programmed cell
death. Transforming growth factor beta-1 up-regulates clusterin
synthesis in thyroid epithelial cells and may be a marker of
TGFbeta-mediated thyrocyte dedifferentiation. (See Wegrowski et
al., Exp Cell Res 247(2):475-83 (1999)). Clusterin expression
correlates with prostate cancer malignancy. Specifically, cells
expressing clusterin are more resistant to androgen ablation and
apoptotic stimuli.
[0059] GENECALLING.TM. analysis reveals that clusterin is
up-regulated in metastatic vs. non-metastatic thyroid cancer.
[0060] Cathepsin E (MTC12)
[0061] Cathepsin E (genBank# J05036) is an immunologically discrete
aspartic protease found in the gastrointestinal tract. It is
overexpressed in pancreatic cancer and is associated with cellular
dedifferentiation in cervical intraepithelial neoplasia
Cathepsin-positive inflammatory cells are infiltrated in and around
the carcinoma tissue. Intensely stained inflammatory cells were
often located in the stroma at the border of the carcinoma tissue.
The incidence of this peculiar localization of intensely stained
carcinoma cells significantly correlated with the progression of
the carcinoma tissue. (ee Matsuo et al., Hum Pathol 27(2):184-90
(1996)).
[0062] GENECALLING.TM. analysis reveals that cathepsin E is
up-regulated in metastatic vs. non-metastatic thyroid cancer.
[0063] Cystatin B (MTC 13)
[0064] Cystatin B, also known as Stefin B, (genBank# U46692) is a
member of the superfamily of cysteine protease inhibitors, mainly
inhibiting cathepsin L. An imbalance between the cathepsins and the
cystatins has been observed at various levels in malignant human
tumor tissue as compared to normal and benign tissue counterparts.
These changes are highly predictive for the length of survival and
may be used for the assessment of risk of relapse and death for
breast, lung, brain, head and neck, ovarian, uterine, melanoma and
colorecta cancers. Specifically, overexpression of cystatin B in
colorectal cancer correlates significantly with Dukes' stage. Its
level is the highest in stage D. (ee Kos et al., Clin Cancer Res
6(2):505-11 (2000)). GENECALLING.TM. analysis reveals that cystatin
B is up-regulated in metastatic vs. non-metastatic thyroid
cancer.
[0065] Lipocortin I (MTC14)
[0066] Lipocortins are a family of calcium-dependent
phospholipid-binding proteins with phospholipase A2 inhibitory
activity. They are also known as annexins because they undergo
Ca(2+)-dependent binding to phospholipids that are preferentially
located on the cytosolic face of the plasma membrane. Lipocortin I
(genBank# x05908) is overexpressed in human hepatocellular
carcinoma (HCC). (See Masaki et al., Hepatology 24(1):72-81
(1997)). It is also overexpressed in breast tumors, where it might
correlate with malignancy. (See Ahn et al., Clin Exp Metastasis
15(2):151-6 (1997)). GENECALLING.TM. analysis reveals that
Lipocortin I is up-regulated in metastatic vs. non-metastatic
thyroid cancer.
[0067] Lipocortin II (MTC15)
[0068] Lipocortins are a family of calcium-dependent
phospholipid-binding proteins with phospholipase A2 inhibitory
activity. They are also known as annexins because they undergo
Ca(2+)-dependent binding to phospholipids that are preferentially
located on the cytosolic face of the plasma membrane. Annexin II
(genBank# d00017) is a major cellular substrate of the tyrosine
kinase encoded by the SRC oncogene. When it is exposed on the
membrane, it is the receptor for tissue-type plasminogen activator
and tenascin in endothelial cells where it mediates mitogenesis,
cell migration, and loss of focal adhesions. (See Chung et al., Mol
Biol Cell 7(6):883-92 (1996)). Increased levels of annexin II are
observed in various cancer cells and tissues, and the molecule has
been proposed as a marker of malignancy in vivo. GENECALLING.TM.
analysis reveals that Lipocortin II is up-regulated in metastatic
vs. non-metastatic thyroid cancer.
[0069] Retinoic acid Induced RIG-E Precursor (RIG-E) (MTC16)
[0070] Retinoic acid induced RIG-E precursor (genBank# U42376). The
Ly-6 Ag family consists of glycosyl-phosphatidylinositol-anchored
surface proteins with a molecular mass of about 15 kDa. Expression
of RIG-E is not restricted to myeloid differentiation, as with
other Ly-6 family member, but it is also present in thymocytes and
in a number of other tissues at different levels. Low expression of
RIG-E was correlated with the malignant potential human
hepatocellular carcinoma (See Kondoh et al., Cancer Res
9(19):4990-6 (1999)). GENECALLING.TM. analysis reveals that while
RIG-E is up-regulated in metastatic vs. non-metastatic thyroid
cancer when samples are compared group by group, it is
down-regulated when samples are compared one by one in the
poisoning reaction.
[0071] P8 (Candidate of Metastasis 1) (MTC17)
[0072] P8 (genBank# AF069073 or NM.sub.--012385) encodes a putative
DNA-binding protein of the helix-turn-helix type. It is
up-regulated in an animal model of breast tumor metastasis. In this
model, constitutive expression of candidate of metastasis 1 seemed
to distinguish breast cancer cells with metastatic potential from
cells without metastatic potential. (See Ree et al., Cancer Res
59(18):4675-80 (1999)). It is induced by serum starvation and is
mitogenic. (See Vasseur et al., Eur J Biochem 259(3):670-5
(1999)).
[0073] GENECALLING.TM. analysis reveals that P8 is down-regulated
in metastatic vs. non-metastatic thyroid cancer.
[0074] Protein Phosphatase-1 Gamma 1 (MTC18)
[0075] Protein phosphatase-1 gamma 1 (genBank# L07395) was
increased in about 50% in rat ascites hepatoma. It is significantly
higher in malignant liposarcoma than in lipoma and in tumorous
regions than in non-tumorous regions of malignant fibrous
histiocytomas. (See Sogawa et al, Res Commun Mol Pathol Pharmacol
93(1):3342 (1996)). The percentage of cells that stained positively
with antiserum against PP1 catalytic subunit isoform PP1 gamma 1,
was significantly higher in malignant osteogenic tumors
(chondrosarcoma, osteosarcoma, and Ewing's sarcoma) and in
malignant soft tissue tumors (liposarcoma and malignant fibrous
histiocytoma (MFH)) than in benign tumors (osteochondroma,
osteoblastoma, ossifying fibroma, enchondroma and lipoma). It is
down-regulated by GA3P an extracellular polysaccharide that induces
apoptosis in lymphoid and myeloid cell lines and it has a potential
role in survival. GENECALLING.TM. analysis reveals that protein
phosphatase-1 gamma I is up-regulated in metastatic vs.
non-metastatic thyroid cancer.
[0076] In addition to the preceding genes, genes whose expression
is newly correlated with metastatic thyroid cancer and which have
not been previously associated with any type of cancer metastasis,
were also differentially expressed in metastatic vs. non-metastatic
thyroid cancer. These genes include tetraspan NET-1, periplakin,
proteasome subunit HC5, ras GTPase-activating-like protein
(IQGAP1), DUSP6 dual specficity MAP kinase phosphatase, SET binding
factor (SBF1), 5-lipoxygenase, calgizzarin, spermidine/spermine
N1-acetyltransferase (SSAT), aldehyde dehydrogenase 2, gelsolin,
integrin alpha-3, KIAA0937, KIAA1131, antileukoprotease, DAP12,
brain-specific STE20-like protein kinase 3 (MST-3b), peflin,
kinectin, prostaglandin transporter hPCT, ribophorin II, PRO302,
stimulated trans-acting factor, iduronate 2-sulfatase,
mitochondrial proteolipid 68 MP, growth arrest specific transcript
gene, lactate dehydrogenase-A, 26-kDa surface protein TAPA-1 mRNA,
and sodium dependent phosphate transporter isoform NaPi-3b.
[0077] These genes have been shown to be differentially expressed
in tumor tissues, in animal models of human tumors, or in cell
lines derived from human tumors, but the available literature does
not describe a correlation between this expression and cancer
progression and/or metastatic potential. It is likely that these
genes contribute to the molecular phenotype of metastatic thyroid
cancer and, as discussed above, to the phenotype of metastatic
carcinomas in general. Genes in this group may have a diagnostic
role in all metastatic carcinomas. Treatments may be useful in the
treatment of all metastatic carcinomas.
[0078] tetraspan NET-1 (MTC19)
[0079] Tetraspanins such as tetraspan NET-1, (genBank# AF065388)
encode cell-surface proteins that span the membrane four times,
forming two extracellular loops. They act as "molecular
facilitators" by grouping specific cell-surface proteins and thus
increasing the formation and stability of functional signaling
complexes. NET-I has been described by Serru et al., Biochim
Biophys Acta 1478(1):159-163 (2000). This sequence is patented and
the patent describes that it is expressed by prostate cancer and
immunogenic in these patients. GENECALLING.TM. analysis reveals
that tetraspan NET-1 is up-regulated in metastatic vs.
non-metastatic thyroid cancer.
[0080] Periplakin (MTC 20)
[0081] The cornified envelope is a layer of transglutaminase
cross-linked protein that is assembled under the plasma membrane of
keratinocytes in the outermost layers of the epidermis. The
intermediate filament cytoskeleton of keratinocytes, composed of
keratins that are expressed in specific expression pairs according
to tissue and differentiation state, are interconnected through
transmembrane protein complexes called desmosomes. They connect
with the basement membrane via hemidesmosomes. The proteins that
are thought to make contact with the intermediate filaments of
keratinocytes belong to a family of proteins known as plakins.
Paraneoplastic pemphigus is an rare autoimmune bullous skin disease
and sera from these patients recognize periplakin (genBank#
AF013717) and envoplakin. Cancers associated with paraneoplastic
pemphigus are hematologic diseases such as non-Hodgkin's lymphomas
and chronic lymphoid leukemia. GENECALLING.TM. analysis reveals
that periplakin is up-regulated in metastatic vs. non-metastatic
thyroid cancer.
[0082] Proteasome subunit HC5 (MTC 21)
[0083] Proteasome subunit HC5 (genBank# D00761) is part of the 20S
proteasome. Proteasomes are multicatalytic proteinase complexes
consisting of a set of non-identical polypeptide components. The
20S proteasome is increased in skeletal muscle from patients with
cancer. (See Williams et al., Surgery 126(4):744-9 (1999)).
GENECALLING.TM. analysis reveals that proteasome subunit HC5 is
up-regulated in metastatic vs. non-metastatic thyroid cancer.
[0084] Ras GTPase-Activating-Like Protein (IQGAP1) (MTC 22)
[0085] RasGTPase-activating-like protein (IQ GAP 1) (genBank#
NP.sub.--003861) inhibited the GTPase activity of cdc42Hs and rac,
whereas no interaction with ras was detected. It interacts with the
cytoskeleton and binds to actin, to members of the Rho family, and
to E-cadherin. Calmodulin binds to IQGAP1 and regulates its
association with Cdc42 and actin. Disruption of the binding of
calmodulin to IQGAP1 enhances the association of IQGAP1 with
components of the cadherin-catenin complex at cell-cell junctions,
resulting in impaired E-cadherin function.
[0086] Mutant IQGAP1 mice exhibit a significant (P<0.0001)
increase in late-onset gastric hyperplasia relative to wild-type
animals of the same genetic background. GENECALLING.TM. analysis
reveals that IQ GAP I is up-regulated in metastatic vs.
non-metastatic thyroid cancer.
[0087] DUSP6 Dual Specificity MAP Kinase Phosphatase (MTC 23)
[0088] DUSP6 is also known as PYST1 (genBank# AB013382). Members of
the mitogen-activated protein (MAP) kinase family play a pivotal
role in cellular signal transduction. The dual-specificity
phosphatases can reverse MAP kinase activation by dephosphorylating
critical phosphotyrosine and phosphothreonine residues. DUSP6 is
localized on 12q21, one of the regions of frequent allelic loss in
pancreatic cancer. Reduced expression of the full-length
transcripts was observed in some pancreatic cancer cell lines. (ee
Furukawa et al., Cytogenet Cell Genet 82(34):156-9 (1998)).
GENECALLING.TM. analysis reveals that DUSP6 is up-regulated in
metastatic vs. non-metastatic thyroid cancer. While this result is
not in agreement with the reduced expression in pancreatic cell
lines, there is evidence that another MAP kinase phosphatase,
MKP-1, is overexpressed in prostate cancer and blocks Fas ligand
(FasL)-induced apoptosis. (ee Srikanth et al., Mol Cell Biochem
199(1-2):169-78 (1999)). It is possible that DUSP6 might have a
similar activity in thyroid cancer.
[0089] SET Binding Factor 1 (SBF1) (MTC24)
[0090] SET binding factor 1 (genBank# U93181) was originally
discovered by virtue of its interaction with a highly conserved
motif, the SET domain common to protein like Suvar3-9,
Enhancer-of-zeste, Trithorax, involved in epigenetic mechanisms of
gene regulation. (ee Cui et al., Nat Genet 18(4):331-7 (1998)). SET
domains mediate highly conserved interactions with a specific
family of proteins that display similarity with dual-specificity
phosphatases (dsPTPases). SBF1 is a homolog of the dsPTPase
myotubularin, but it lacks a functional catalytic domain which
dephosphorylates phosphotyrosine and serine-containing peptides in
vitro. It is possible that SBF1 acts by competing against dsPTPase
for their target kinases. There is evidence that SBF1 behaves as an
oncogene because overexpression of SBF1 can induce transformation
or growth advantage. GENECALLING.TM. analysis reveals that SBF1 is
down-regulated in metastatic vs non-metastatic thyroid cancer.
While this result is not in agreement with the activities described
above, in the context of the thyroid, SBF1 function might be to
suppress the 1 activity of DUSP6 and other dsPTPases that are
important for the switch to metastatic tumor, where up-regulation
of the dsPTPase DUSP6 is observed.
[0091] 5-Lipoxygenase (MTC25)
[0092] The enzyme 5-lipoxygenase (genBank# J03571) catalyzes 2
reactions in the formation of leukotrienes.The leukotrienes
constitute a group of arachidonic acid-derived compounds with
biologic activities in inflammation and immediate hypersensitivity.
Recently the 5-lipoxygenase product, 5-HETE has been shown to be
important for the survival of prostate cancer cells. (See Ghosh et
al., Proc Natl Acad Sci U S A 95(22):13182-7 (1998)).
GENECALLING.TM. analysis reveals that 5-lipoxygenase is
up-regulated in metastatic vs. non-metastatic thyroid cancer.
[0093] Calgizzarin (MTC26)
[0094] Calgizzarin (genBank# d38583) is a Ca2+-binding protein of
the S100 family that has been implicated in the regulation of
cytoskeletal function through its Ca2+-dependent interaction with
annexin I. Calgizzarin expression was remarkably elevated in
colorectal cancers, compared with expression in normal colorectal
mucosa. (See Tanaka et al., Cancer Lett 89(2):195-200 (1995)).
Calgizzarin is part of the cornified envelope (CE) like periplakin
and annexin I, two other genes described in this invention.
GENECALLING.TM. analysis reveals that calgizzarin is up-regulated
in metastatic vs. non-metastatic thyroid cancer, as are periplakin
and annexin I. These results indicate that they might be part of a
common pathway activated in metastatic cells.
[0095] Spermidine/Spermine N1-Acetyltransferase (SSAT) (MTC27)
[0096] Also known as diamine acetyltransferase and putrescine
acetyltransferase (genBank# m77693). It is a rate-limiting enzyme
in the catabolic pathway of polyamine metabolism. It catalyzes the
N(1)-acetylation of spermidine and spermine, and, by the successive
activity of polyamine oxidase, spermine can be converted to
spermidine and spermidine to putrescine. It is up-regulated in
prostate cancer, and this up-regulation correlates with malignancy.
(Saverio et al., Cancer Res 60(1):28-3400 (2000)). GENECALLING.TM.
analysis reveals that SSAT is up-regulated in metastatic vs.
non-metastatic thyroid cancer.
[0097] Interestingly, for therapy considerations, increased
transcription and ultimate superinduction of SSAT has been
associated with the antineoplastic activity of several new
antitumor polyamine analogues. (ee Alhonen et al., Mol Pharmacol
55(4):693-8 (1999)). Potentially, polyamine analogues could have
specific applications in the treatment of metastatic thyroid
cancer.
[0098] Aldehyde Dehydrogenase 2 (ALDH-2) (MTC28)
[0099] Aldehyde dehydrogenase 2 (genBank# k03001) encodes the
mitochondrial form of this alcohol metabolism enzyme. Inactive
ALDH2 is a risk factor for multiple carcinoma of the esophagus in
alcoholics due to the higher amounts of acetaldehyde produced by
these individuals. It was also shown that ALDH2 is down-regulated
or absent in tumor cell lines and in urethane-induced mouse liver
tumors. (See Banfi et al., Mol Pharmacol 46(5):896-900(1994)).
GENECALLING.TM. analysis reveals that ALDH-2 is down-regulated in
metastatic vs. non-metastatic thyroid cancer.
[0100] Gelsolin (MTC29)
[0101] Gelsolin (genBank# X04412) severs actin filaments in the
presence of submicromolar calcium concentrations. Circulating forms
of gelsolin act to dissolve and then clear actin from the
circulation. Plasma and cytoplasmic gelsolins are encoded by a
single gene. Gelsolin is an obligate downstream effector of Rac for
motility in dermal fibroblasts. It regulates phosphoinositide
signaling pathways and ion channel function in vivo and acts as
both a regulator and an effector of apoptosis. It is greatly
decreased in many transformed cell lines and tumor tissues. This
decrease correlates with malignancy in breast tumors.
GENECALLING.TM. analysis reveals that gelsolin is up-regulated in
metastatic vs. non-metastatic thyroid cancer. While this is in
disagreement with the above data, at the same time, there is
evidence that, in Tsc2 heterozygotic mice, renal cystadenomas
develop from intercalated cells of the cortical collecting duct
expressing gelsolin at high levels. (ee Onda et al., J Clin Invest
104(6):687-95 (1999)).
[0102] Integrin Alpha-3 (MTC30)
[0103] Integrins encompass a family of cell-surface molecules,
which play a crucial role in cell-cell and cell-extracellular
matrix interactions. They are usually organized as heterodimeric
transmembrane glycoproteins consisting of an alpha and beta chain.
Integrin alpha-3 (genBank# AAA3612) together with beta 1 (VLA-3)
functions as a receptor for fibronectin, laminin, and collagen.
GENECALLING.TM. analysis reveals that integrin alpha-3 is
up-regulated in metastatic vs. non-metastatic thyroid cancer, as is
fibronectin. It is possible that the up-regulation of fibronectin
and its receptor, integrin alpha-3, in metastatic thyroid cancer is
important for the ability of these tumors to migrate in the
extracellular matrix.
[0104] KIAA0937 (MTC31)
[0105] KIAA0937 (genBank# AB023154) is a homologue of human Deltex
(DTX1), a member of the Notch signaling pathway. It contains a RING
finger, which is a domain involved in protein-protein interactions
or in DNA binding. Sbase indicates that it has homology to several
transcription factors and to retinoic acid receptors and to serine
proteases. It is possible that this protein binds a receptor-like
notch, that is activated by processing and then translocated to the
nucleus to activate trasncription. The putative chicken orthologue
is expressed in many neural and sensory structures, such as neural
tube, migrating neural crest cells, epidermal placodes, cranial
ganglia, and the optic and otic vesicles. (See Frolova et al., Mech
Dev 92(2):285-289 (2000)). GENECALLING.TM. analysis reveals that
KIAA0937 is up-regulated in metastatic vs. non-metastatic thyroid
cancer. SAGE analysis reveals that this gene is also expressed in
colon cancer cell lines and tumors, in glioblastoma multiforme,
breast cancers, and ovarian tumor cell lines. This indicates that
overexpression of this gene may have a general role in
tumorogenesis.
[0106] KIAA1131 (MTC32)
[0107] KIAA1131 (genBank# AB032957) is described in Hirosawa et
al., DNA Res. 6:329-336 (1999). This gene has homology to
suppressor of Deltex and other ubiquitin ligase enzymes. This
homology is extremely relevant to its potential role in thyroid and
other metastatic cancers. In mammalian cells, notch has been shown
to be important for survival and tumorogeneis. Deltex and
suppressor of Deltex antagonize each other in their interaction
with Notch. It is therefore likely that KIAA0937 and KIAA1131 are
functional equivalent to Deltex and suppressor of Deltex. They
identify a new Notch signalling pathway important for cell survival
and metastatic potential. GENECALLING.TM. analysis reveals that
KIAA1131 is down-regulated in metastatic vs. non-metastatic thyroid
cancer. SAGE analysis reveals that this gene is down-regulated in
prostate tumors compared with normal prostate, indicating a more
general role of KIAA1131 downregulation in cancer.
[0108] Antileukoprotease (MTC33)
[0109] Antileukoprotease is also known as secretory leukocyte
protease inhibitor (genBank# X04470). It is an acid-stable
proteinase inhibitor with strong affinity for trypsin and
chymotrypsin as well as for neutrophil lysosomal elastase and
cathepsin-G. It is present in mucous fluids such as seminal plasma,
cervical mucus, bronchial and nasal secretions, and tears. It was
found to be expressed in lung, breast, oropharyngeal, bladder,
endometrial, ovarian, and colorectal carcinomas. (See Garver et
al., Gene Ther (1):46-50 (1994)). GENECALLING.TM. analysis reveals
that antileukoprotease is up-regulated in metastatic vs.
non-metastatic thyroid cancer. SAGE analysis indicate that
antileukoprotease is upregulated in ovarian tumors.
[0110] DAP12 (MTC34)
[0111] DAP12 (genBank# AF019562) is an Natural killer (NK)-cell
receptor that lacks ITIM (immunoreceptor tyrosine-based inhibitory
motifs) sequences. It has been proposed that DAP12 may activate,
rather than inhibit, NK cells. DAP12 non-covalently associates with
membrane glycoproteins of the killer-cell inhibitory receptor (KIR)
family without an ITIM in their cytoplasmic domain. Crosslinking of
KIR-DAP12 complexes results in cellular activation. Phosphorylated
DAP12 peptides bind ZAP-70 and Syk protein tyrosine kinases,
suggesting that the activation pathway is similar to that of the T-
and B-cell antigen receptors. GENECALLING.TM. analysis reveals that
DAP12 is up-regulated in metastatic vs. non-metastatic thyroid
cancer. This up-regulation is likely due to lymphocyte infiltration
of the metastatic thyroid cancers. SAGE analysis shows that its
up-regulation is common to other cancers, like glioblastoma
multiforme and ovarian tumors.
[0112] Brain-Specific STE20-Like Protein Kinase 3 (MST-3b)
(MTC35)
[0113] The Sterile-20 (Ste20) family of serine-threonine kinases
has been implicated in the activation of the stress-activated
protein kinase pathways. MST3b (genBank# AAD42039) mRNA is
restricted to the brain. MST3b, but not MST3, was effectively
phosphorylated by the activation of cyclic AMP-dependent protein
kinase (PKA) in both in vivo and in vitro assays. (See Zhou et al.,
J Biol Chem 275(4):2513-9 (2000)). GENECALLING.TM. analysis reveals
that MST-3b is up-regulated in metastatic vs. non-metastatic
thyroid cancer. SAGE analysis indicates that MST-3b is also
up-regulated in ovarian tumors.
[0114] Peflin (MTC36)
[0115] Peflin (genBank# BAA85163) is a member of the five-EF-hand,
Ca(2+)-binding protein family. It is similar to the
apoptosis-linked gene 2 (ALG-2) (ee Kitaura et al., Biochem.
Biophys. Res. Commun. 263(1): 68-75 (1999)). ALG-2 is required for
apoptosis, and ALG-2 depletion protects the mouse T cell hybridoma
3DO from programmed cell death induced by several stimuli, such as
synthetic glucocorticoids, TCR, and Fas triggering. (ee Lacana et
al., J Immunol 158(11):5129-35 (1997)). GENECALLING.TM. analysis
reveals that Peflin is down-regulated in metastatic vs.
non-metastatic thyroid cancer. This suggests that tumor
down-regulation of peflin might increase their resistance to
apoptosis. SAGE analysis reveals that this down-regulation is also
observed in breast, ductal carcinoma in situ vs. normal mammary
epithelium and in colon cancer vs. normal colon.
[0116] Kinectin (MTC37)
[0117] Kinectin (genBank# NM.sub.--004986) is a receptor for the
molecular motor kinesin, which is critically involved in
microtubule-based vesicle transport and membrane trafficking.
GENECALLING.TM. analysis reveals that kinectin is up-regulated in
metastatic vs. non-metastatic thyroid cancer. It is cleaved by
caspase 7 in cells subject to apoptotic stimuli. Therefore, its
up-regulation might be related to survival. This hypothesis is
confirmed by SAGE analysis. It is up-regulated by VEGF, a survival
factor in human microvessel endothelial cells and by hypoxia. It is
highly up-regulated in MDA453, from a human breast carcinoma with
malignant effusion, estrogen receptor negative, obtained from a
patient treated with radiation and chemotherapy. This evidence
together with kinectin's up-regulation in thyroid cancer indicates
a general role in promoting survival and metastatic potential.
[0118] Prostaglandin Transporter hPGT (MTC38)
[0119] Prostaglandins (PGs) play diverse and important roles in
human health and disease. Specifically, elevated prostaglandin
levels are associated with tumors and may promote tumor
progression. PGT (genBank# u70867) is likely to have a role in the
transport and/or metabolic clearance of PGs in diverse human
tissues. GENECALLING.TM. analysis reveals that hPTG is
down-regulated in metastatic vs. non-metastatic thyroid cancer. It
is possible that the down-regulation of hPTG by metastatic thyroid
cancer cells results in higher local concentration of
prostaglandins that will favor tumor progression.
[0120] Ribophorin II (MTC39)
[0121] Ribophorin II (genBank# y00282) is part, together with
ribophorin I (RI) and OST48, of the oligosaccharyltransferase
(OST), which has its active site exposed on the luminal face of the
endoplasmic reticulum (ER) and catalyzes the transfer of
preassembled high mannose oligosaccharides onto certain asparagine
residues of nascent polypeptides. Recently DAD1, the defender
against apoptotic cell death was shown to be a member of this
complex. DAD1 was initially identified as a negative regulator of
programmed cell death indicating the possibility that OST and
therefore ribophorin II have a role in cell survival.
GENECALLING.TM. reveals that ribophorin II is up-regulated in
metastatic vs. non-metastatic thyroid cancer. This result, together
with the evidence that ribophorin II is overexpressed in primary
colorectal cancer (ee Hufton, FEBS Lett 463(1-2):77-82 (1999)),
indicate that overexpression of ribophorin II promote a metastatic
phenotype by increasing the ability of cells to survive.
[0122] Pro302 (MTC40)
[0123] This gene encodes a vitellogenic carboxypeptidase that is
secreted protein. The pretin has anti-angiogenic activities that
are useful for the treatment of cancer among other diseases.
GENECALLING.TM. analysis reveals that Pro302 is down-regulated in
metastatic vs. non-metastatic thyroid cancer. The down-regulation
is in agreement with an anti-angiogenic and anti-tumor roles.
Therefore, these studies indicate a specific use for Pro302 in the
treatment of metastatic thyroid cancer.
[0124] Stimulated Trans-Acting Factor (50 kDa) (MTC41)
[0125] Stimulated trans-acting factor (50 kDa) (Staf50) (genBank#
X82200) is a member of the Ring finger family. It contains all the
features of a transcriptional regulator able to initiate a second
cascade of gene induction. It is induced by both type I and type II
IFN in various cell lines (See Tissot et al., J Biol Chem
270(25):14891-8 (1995)). GENECALLING.TM. analysis reveals that
Staf50 is up-regulated in metastatic vs. non-metastatic thyroid
cancer. This might be a reflection of the high level of lymphocyte
invasion of metastatic papillary carcinomas.
[0126] Iduronate 2-Sulfatase (MTC42)
[0127] GENECALLING.TM. analysis reveals that iduronate 2-sulfatase
(genBank #M583420) is up-regulated in metastatic vs. non-metastatic
thyroid cancer.
[0128] Mitochondrial Proteolipid 68 MP (MTC43)
[0129] Mitochondrial proteolipid 68 MP (genBank# AF054175) is
expressed by human CD34(+) hematopoietic stem/progenitor cells.
(See Mao et al., Proc. Natl. Acad. Sci. U.S.A. 95(14): 8175-8180
(1998)). GENECALLING.TM. analysis reveals that 68 MP is
down-regulated in metastatic vs. non-metastatic thyroid cancer. It
is likely that down-regulation of this gene indicates that cells in
the metastatic tumors are losing their differentiation state and
becoming similar to stem/progenitor cells.
[0130] Growth Arrest Specific Transcript 5 Gene (MTC43)
[0131] Growth arrest-specific transcript 5 (GAS5) (genBank#
AF141346) was initially discovered in a screen for potential tumor
suppressor genes expressed at high levels during growth arrest.
GAS5 is a non-protein-coding multiple small nucleolar RNA (snoRNA)
hostgene similar to UHG (U22 host gene). Encoded within the 11
introns of the mouse GAS5 gene are nine (10 in human) box
C/DsnoRNAs predicted to function in the 2'-O-methylation of rRNA.
In growing cells, active translation leads to rapid degradation of
the spliced GAS5 RNA, whereas inhibition of translation causes the
level of GAS5 transcript to rise. Likewise in growth-arrested
cells, the spliced GAS5 RNA accumulates, apparently because it is
sequestered in mRNP particles and is not translated.
GENECALLING.TM. analysis reveals that GAS5 is up-regulated in
metastatic vs. non-metastatic thyroid cancer. The differential
expression of GAS5 in metastatic thyroid cancer may be related to
an increase in the ability of these cells to survive stresses.
[0132] Lactate Dehydrogenase-A (MTC45)
[0133] GENECALLING.TM. analysis reveals that lactate
dehydrogenase-A (genBank #X02152) is up-regulated in metastatic vs.
non-metastatic thyroid cancer.
[0134] 26-kDa Cell Surface Protein TAPA-1 mRNA, complete cds.; CD81
(MTC46)
[0135] GENECALLING.TM. analysis reveals that CD81 (genBank #M33680)
is down-regulated in metastatic vs. non-metastatic thyroid
cancer.
[0136] sodium Dependent Phosphate Transporter Isoform NaPi-3b
AF111856 (hs640.sub.--0) (MTC47)
[0137] GENECALLING.TM. analysis reveals that sodium dependent
phosphate transporter isoform NaPi-3b (genBank #AF111856) is
up-regulated in metatstatic vs. non-metastatic thyroid cancer.
[0138] Nucleic Acid Sequences Newly Described Herein
[0139] MTC48
[0140] An MTC48 nucleic acid sequence according to the invention
includes the assembled sequence d010-145.3, or 88098062.
GENECALLING.TM. analysis reveals that it is up-regulated in
metastatic vs. non-metastatic thyroid cancer.
[0141] A human MTC48 nucleic acid sequence according to the
invention (SEQ ID NO:1), along with its encoded amino acid sequence
(SEQ ID NO:2), is provided below:
2 GGCGCCCGGGCGGTGCTGCGCTGCCAGAGCCCGCGCA (SEQ ID NO:1)
TGGTGTGGACCCAGGACCGGCTGCACGACCGCCAGCG
CGTGCTCCACTGGGACCTGCGCGGCCCCGGGGGTGGC
CCCGCGCGGCGCCTGCTGGACTTGTACTCGGCGGGCG
AGCAGCGCGTGTACGAGGCGCGGGACCGCGGCCGCCT
GGAGCTCTCGGCCTCGGCCTTCGACGACGGCAACTTC
TCGCTGCTCATCCGCGCGGTGGAGGAGACGGACGCGG
GGCTGTACACCTGCAACCTGCACCATCACTACTGCCA
CCTCTACGAGAGCCTGGCCGTCCGCCTGGAGGTCACC
GACGGCCCCCCGGCCACCCCCGCCTACTGGGACGGCG
AGAAGGAGGTGCTGGCGGTGGCGCGCGGCGCACCCGC
GCTTCTGACCTGCGTGAACCGCGGGCACGTGTGGACC
GACCGGCACGTGGAGGAGGCTCAACAGGTGGTGCACT
GGGACCGGCAGCCGCCCGGGGTCCCGCACGACCGCGC
GGACCGCCTGCTGGACGTCTACGCGTCGGGCGAGCGC
CGCGCCTACGGGCCCCTTTTTCTGCGCGACCGCGTGG
CTGTGGGCGCGGATGCCTTTGAGCGCGGTGACTTCTC
ACTGCGTATCGAGCCGCTGGAGGTCGCCGACGAGGGC
ACCTACTCCTGCCACCTGCACCACCATTACTGTGGCC
TGCACGAACGCCGCGTCTTCCACCTGACGGTCGCCGA
ACCCCACGCGGAGCCGCCCCCCCGGGGCTCTCCGGGC
AACGGCTCCAGCCACAGCGGCGCCCCAGGCCCAGACC
CCACACTGGCGCGCGGCCACAACGTCATCAATGTCAT
CGTCCCCGAGAGCCGAGCCCACTTCTTCCAGCAGCTG
GGCTACGTGCTGGCCACGCTGCTGCTCTTCATCCTGC
TACTGGTCACTGTCCTCCTGGCCGCCCGCAGGCGCCG
CGGAGGCTACGAATACTCGGACCAGAAGTCGGGAAAG
TCAAAGGGGAAGGATGTTAACTTGGCGGAGTTCGCTG
TGGCTGCAGGGGACCAGATGCTTTACAGGAGTGAGGA
CATCCAGCTAGATTACAAAAACAACATCCTGAAGGAG
AGGGCGGAGCTGGCCCACAGCCCCCTGCCTGCCAAGT
ACATCGACCTAGACAAAGGGTTCCGGAAGGAGAACTG
CAAATAGGGAGGCCCTGGGCTCCTGGCTGGGCCAGCA
GCTGCACCTCTCCTGTCTGTGCTCCTCGGGGCATCTC
CTGATGCTCCGGGGCTCACCCCCCTTCCAGCGGCTGG
TCCCGCTTTCCTGGAATTTGGCCTGGGCGTATGCAGA
GGCCGCCTCCACACCCCTCCCCCAGGGGCTTGGTGGC
AGCATAGCCCCCACCCCTGCGGCCTTTGCTCACGGGT
GGGCCCTGCCCACCCCTGGGCACAACCAAAATCCCAC
TGATGCCCATCATGCCCTCAGACCCTTCTGGGCTCTG
CCCGCTGGGGGCCTGAAGACATTCCTGGAGGACACTC
CCATCAGAACCTGGCAGCCCCAAAACTGGGGTCAGCC
TCAGGGCAGGAGTCCCACTCCTCCAGGGCTCTGCTCG
TCCGGGGCTGGGAGATGTTCCTGGAGGAGGACACTCC
CATCAGAACTTGGCAGCCTTGAAGTTGGGGTCAGCCT
CGGCAGGAGTCCCACTCCTCCTGGGGTGCTGCCTGCC
ACCAAGAGCTCCCCCACCTGTACCACCATGTGGGACT
CCAGGCACCATCTGTTCTCCCCAGGGACCTGCTGACT
TGAATGCCAGCCCTTGCTCCTCTGTGTTGCTTTGGGC
CACCTGGGGCTGCACCCCCTGCCCTTTCTCTGCCCCA
TCCCTACCCTAGCCTTGCTCTCAGCCACCTTGATAGT
CACTGGGCTCCCTGTGACTTCTGACCCTGACACCCCT
CCCTTGGACTCTGCCTGGGCTGGAGTCTAGGGCTGGG
GCTACATTTGGCTTCTGTACTGGCTGAGGACAGGGGA
GGGAGTGAAGTTGGTTTGGGGTGGCCTGTGTTGCCAC
TCTCAGCACCCCACATTTGCATCTGCTGGTGGACCTG
CCACCATCACAATAAAGTCCCCATCTGATTTTTAAAA AAAAAAAAAAAAAAAAAAAAAAA
[0142] Amino Acid Sequence: (SEQ ID NO:2)
3 MVWTQDRLHDRQRVLHWDLRGPGGGPARRLLDLYSAG (SEQ ID NO:2)
EQRVYEARDRGRLELSASAFDDGNFSLLIRAVEETDA
GLYTCNLHHHYCHLYESLAVRLEVTDGPPATPAYWDG
EKEVLAVARGAPALLTCVNRGHVWTDRHVEEAQQVVH
WDRQPPGVPHDRADRLLDLYASGERRAYGPLFLRDRV
AVGADAFERGDFSLRIEPLEVADEGTYSCHLHHHYCG
LHERRVFHLTVAEPHAEPPPRGSPGNGSSHSGAPGPD
PTLARGHNVINVIVPESRAHFFQQLGYVLATLLLFIL
LLVTVLLAARRRRGGYEYSDQKSGKSKGKDVNLAEFA
VAAGDQMLYRSEDIQLDYKNNILKERAELAHSPLPAK YIDLDKGFRKENCK
[0143] A mouse MTC48 nucleic acid sequence according to the
invention (SEQ ID NO:3), along with its encoded amino acid sequence
(SEQ ID NO:4), is provided below:
[0144] (SEQ ID NO:3)
4 TTCGGCACAGGACCTGCACCATCACTACTGCCACCTC (SEQ ID NO:3)
GATGAGAGCATGGCTGTGCGCCTCGAGGTTACAGAGG
ATCCCCTATTAAGTCGCGCATACTGGGACGGTGAGAA
GGAAGTGTTGGAGGTGGCCCATGGCGCGCCGGCACTG
ATGACCTGCATCAACCGTGCGCACGTGTGGACTGACC
GCCATTTAGAGGAGGCGCAACAGGATAGACAATTGGG
ACGACAGCTACCTGGGGTGTCACACGACCGCGCCGAC
CGCCTGCATGACCTGTATGCATCTGGCGAGCGCCGCG
CCTATGGGCCACCCTTCCTGCGTGATCGCGTGTCAGT
GAACACCAACGCTTTTGCACGCGGTGACTTCTCCCTA
CGCATCGATGAGCTGGAGCGAGCTGATGAGGGCATCT
ATTCCTGCCACCTGCACCATCACTACTGTGGCCTCCA
CGAGCGCCGAGTCTTCCACCTACAGGTCACAGAGCCT
GCCTTTGAGCCACCAGCTCGTGCTTCTCCTGGCAATG
GGTCTGGTCACAGCAGTGCTCCTAGCCCAGATCCCAC
CCTGACCCGAGGCCACAGCATCATCAATGTCATTTGT
CCCAGAGGACCACACACATTTCTTCCAGCAACTGGGC
TATGTGTTGGCCACGCTGCTGCTCTTCATCTTGCTGC
TCATCACTGTAGTCCTGGCTACACGATATCGTCACAG
CGGAGGATGCAAGACGTCGGACAAAAAAGCTGGGAAG
TCAAAGGGGAAGGAATGTCGACACGATGGTGGAGTTT
GCTGTAGCCACAAGGGATCAGGCTCCATATAGGACTG
AGGACATCCAGCTAGATTACAAAAACAACATCCTGCG
GTATTCCTGGCTCTTCTCAGCGGCTGGTCCGACTTAC
CTAGAAACTTGGCAGAGCAGCTGCCTGTACTTTGCCC
TTCCTAGAATCGCCACCCCTCATCTTGGTGAGCAACT
GTGGGTTCCCTAGAGACTCTGGTATAGTACGATTGCT
GCCCTTCACCTGTGCCCACTGATGGTTGTACCCCCAA
CTTAAACACAACAAAGATCCCTTGTTAATATCCACCA
AATGCAAAGTCCCTCGTGGCCTCTTACTGCTAGGGTC
AGGAAGACACTTAAAAATTCCAGTTAAGACTCCCTAG
CCACCAGTTAAACACATTAGCCATTGTCCTGGGGGGT
CTCCTGAGCTGCATTGTGCCTGTGTACTGTTCAG
[0145] Mouse Amino Acid Sequence: (SEQ ID NO: 4)
5 SAQDLHHHYCHLDESMAVRLEVTEDPLLSRAYWDGEK (SEQ ID NO:4)
EVLEVAHGAPALMTCINRAHVWTDRHLEEAQQDRQLG
RQLPGVSHDRADRLHDLYASGERRAYGPPFLRDRVSV
NTNAFARGDFSLRIDELERADEGIYSCHLHHHYCGLH
ERRVFHLQVTEPAFEPPARASPGNGSGHSSAPSPDPT
LTRGHSIINVICPRGPHTFLPATGLCVGHAAALHLAA
HHCSPGYTISSQRRMQDVGQKSWEVKGEGMSTRWWSL L
[0146] FIG. 1 shows the sequence homology between a human MTC48
amino acid sequence and a mouse MTC48 amino acid sequence according
to the invention. The conserved regions in each amino acid sequence
are highlighted in black.
[0147] Psort analysis indicates that this is a transmembrane
protein. The values are mitochondrial inner membrane P=0.8207 and
plasma membrane P=0.7000. Because the values are so similar, a
definitive cellular localization cannot yet be predicted.
[0148] Pfam analysis indicates the presence of two
immunoglobulin-like domains in the protein. One extends from amino
acid 23 to amino acid 81, and the includes amino acid 121 to amino
acid 216. These regions are also identified using Sbase analysis.
Sbase analysis also indicates that the encoded MET48 polypeptide
has homology to the COOH region of the HB-EGF-like family. Blocks
analysis finds homology to metallo-proteins involved in oxygen
carrying or electron transfer and to G-protein coupled
receptors.
[0149] FIG. 2 presents a hydrophobicity analysis of a human MTC48
polypeptide. A transmembrane domain is present between amino acids
260 to 300 When exposed on the plasma membrane, this region
presents a desirable target for immunotherapy. A MMTC48 is a GPCR
or a protein involved in enzymatic activity, it is potentially a
target for small molecule therapy.
[0150] MTC49
[0151] A MTC49 nucleic acid sequence according to the invention was
assembled using sequences from AC024267, which is derived from
human chromosome 17, as well as sequences expressed in thyroid
glands. The assembled sequence is downregulated in metastatic vs.
non-metastatic cancer cells. The presence of sequences from thyroid
glands supports the hypothesis that this gene is normally expressed
in the thyroid gland and that it is down-regulated in metastatic
thyroid tumors.
[0152] A human MTC49 nucleic acid sequence according to the
invention (SEQ ID NO:5), along with its encoded amino acid sequence
(SEQ ID NO:6), is provided below:
[0153] Human MTC49 cDNA (SEQ ID NO: 5)
6 CCAGCGCCATCATCAGATGGCAAGNTCAGCCCCGGCA (SEQ ID NO:5)
CGTTATCCATAGGAAGCGCTTTAACCGTACCCTCTTT
CCCAACCAACTCTACTGCCATGGTGGACCTCACCAAC
TCACTTCGAGCATTTATGGATGTCAATGGAGAAATCG
AGATAAATATGCTGGACGAGAAGCTGATCAAGTTTCT
GGCCTTGCAGAGAATACATCAGCTTTTCCCCTCCCGG
GTCCAACCTTCACCGGGCAGTGTCGGGACACATCAGC
TGGCTTCTGGAGGGCACCACATAGAAGNNNNNNNNNN
NNNTGTACAGGCCCGAGCTGTGTTCTACCCCCTCTTA
GGGTTGGGAGGAGCTGTGAACATGTCCTATCGAACCC
TCTACATCGGGACAGGAGCTGACATGGATGTGTGCCT
TACAAACTATGGTCACTGTAACTACGTGTCCGGGAAA
CATGCCTGCATATTCTACGATGAGAATACCAAACATT
ATGAGCTGTTAAACTACAGTGAGCATGGGACAACGGT
GGACAATGTGCTGTATTCATGTGACTTCTCGGAGAAG
ACCCCGCCAACCCCCCCAAGCAGTATTGTTGCCAAAG
TGCAGAGTGTCATCAGGCGCCGCCGGCACCAGAAACA
GGACGAAGAGCCAAGTGAGGAGGCAGCCATGATGAGT
TCCCAGGCCCAGGGGCCGCAGCGGAGACCCTGCAATT
GCAAAGCCAGCAGCTCGAGCTTGATTGGGGGCAGTGG
GGCCGGCTGGGAGGGCACAGCCTTACTGCACCATGGC
AGCTACATCAAGCTGGGCTGCCTGCAGTTTGTCTTCA
GCATCACTGAGTTTGCGACCAAACAGCCCAAAGGCGA
TGCCAGCCTGCTGCAGGATGGGGTCTTGGCCGAGAAG
CTCTCTCTCAAGCCCCACCAGGGCCCTGTGCTGCGCT
CCAACTCTGTTCCTTAGGACTGGCGGCTACCCCGCCA
CTGGCCTGTACACCCACCCAAGACTCCTGCAATGCAA
AAATGTACACAAACCAAGCCCGGGTGTTTTCTATACT
CTACCAGAAACCCTTCAACTACAATCTTTGCATGAAA
TGAAGAAAACCTTTTGACTGTTTTTTAAAAATCCTTT
TTCTTTTCTCAAGTTCTAGGGGGCATTTGCACATATA
TTTGTACTCAACATTTCATGGGAAAGCGGCAGACCTG
AGCTGAGGAACAGCGTGGNGCAGGGAGGGAAAGACCC
AGGGTCTGGACANTTTCTCCAACACAAAANCCTTTCC
CACCCANCTTCCTGCTTCCTTCCCCTTCGNGCNCCCC
ATTGTAAAATAATCAGGAAACTTGTTCTATTTTGTGG
CAGTGACAATAGTTTTATATTAAAAGAAAAAATACAG
TTTTCATAACCACAAATCTATTCAATATCATTGTTTT
ATTTAATATAAAGATCGCTACCCACCTTCCTTCCATG
GTTCCCACCCTCCACGTTATTTTCCCTTTCTGCAGCG
GTTGCACTACAGGTAGCTACTGTGTATTATGGACAAA
TGAGAAATGAATTCTTTTTCTGGCTGTCCATCTATTT
TATTTCAAATAAGGAAAAGTGTATTTGGATTTTGTGT
AAATACATCTAGTGATGACATTTTTTCAATGTTTTTA
AAAACCGTGTACAGTACTACATGTGGTAGAGCGTTTT
CTCAAATTGTCTATTGTAGCAAAAATGTTTTTGTCGT
AAACCTGTTTTGTCTCCTTTTTTTGTTCTCTTGCCAC
TTCTCTCCTCTTCCTCCTGCCCCTGGTTCCCTCCTCT
CCTCCCACCCCCACAACCAGTACCAATGTACATAGTA
ATTGTAATGTTTTAGACTTTACAGAAACTTTCCTGTA
TTCTGTATATAAAAAACAAAAATACTTCAAAAAAAAA AAAAAAAAA
[0154] Human MET49 Polypeptide (SEQ ID NO:6)
7 MVDLTNSLRAFMDVNGEIEINMLDEKLIKFLALQRIH (SEQ ID NO:6)
QLFPSRVQPSPGSVGTHQLASGGHHIEXXXXXVQARA
VFYPLLGLGGAVNMSYRTLYIGTGADMDVCLTNYGHC
NYVSGKHACIFYDENTKHYELLNYSEHGTTVDNVLYS
CDFSEKTPPTPPSSIVAKVQSVIRRRRHQKQDEEPSE
EAAMMSSQAQGPQRRPCNCKASSSSLIGGSGAGWEGT
ALLHHGSYIKLGCLQFVFSITEFATKQPKGDASLLQD GVLAEKLSLKPHQGPVLRSNSVP
[0155] A mouse MTC49 nucleic acid sequence according to the
invention (SEQ ID NO:7), along with its encoded amino acid sequence
(SEQ ID NO:8), is provided below:
8 Mouse cDNA CCCAAATGCCACCCTGGGTGCTCACTCCCCCCCAGGC (SEQ ID NO:7)
TGCAGGAGACAGTATCTTGGCCACAGGTGCCAACCAA
CGATTCTGCTCACCAGCGCCATCATCAGCTCTAGGAG
TTCCAGAGCCTGTGGGTTGAGAATCAAGAAGGGAACA
TCACCTTGGGTCCGAAATCCAGAACTGTCTCAACAAT
GGGGGATTGGACCGGTGGGTTTCCCTCAGGCGACCCA
ACTCTACTGCCATGGTGGACCTCACCAACTCACTTCG
AGCATTTATGGATGTCAACGGAGAAATCGAGATAAAT
ATGTTGGATGAGAAGCTGATCAAGTTTCTGGCCTTGC
AGAGAGTACATCAGCTTTTCCCTTCCCGGGTCCAAGC
TTCACCGGGCAATGTTGGGACACATCCGCTGGCTTCT
GGAGGGCACCACCCAGAAGTGCAAAGAAAGGAGGTAC
AGGCCCGAGCTGTGTTCTGCCCCCTCTTAGGGTTGGG
AGGAGCTGTGAACATGTGCTATCGAACCCTCTACATC
GGGACAGGAGCTGACATGGATGTGTGCCTTACAAACT
ATGGTCACTGTAACTACGTGTCCGGGAAACATGCCTG
CATATTCTACGATGAGAATACCAAACATTATGAGCTG
TTAAACTACAGTGAGCATGGGACAACGGTGGACAATG
TGCTGTATTCATGTGACTTCTCTGAGAAGACCCCGCC
AACCCCCCCAAGCAGTATTGTTGCCAAAGTACAGAGT
GTCATCAGGCGCCGCGAGCACCAGAAACAGGATGAAG
AGCCAAGTGAGGAGGCAGCCATGATGAGTTCCCAGGC
CCAGGGgcCACAGCGGAGACCCTGCAATTGCAAAGCC
AGCAGCTCAAGCTTGATTGGGGGCAGTGGGGCCGGCT
GGGAGGGCACAGCATTACTCCACCATGGCAGCTACAT
CAAGCTGGGCTGCCTGCAGTTTGTCTTCAGCATCACT
GAGTTTGCGACCAAACAGCCCAAAGGCGATGCCAGCC
TGCTGCAGGATGGGGTCTTGGCTGAGAAACTCTCTCT
CAAGCCCCATCAGGGCCCTGTGCTGCGCTCCAACTCC
GTTCCCTAGGCCATTGGCCTGGACGCCCACCCAAGAC
TCCTGCAATGCAAAAATGTACACGAACCAAGCCTGGG
TGTTTTCTATACCAGAAACCCTCAACTACAATCTTTG
CATGAAATGAAGAAAACCTTTTGACTGTTTTTTAAGA
CTTTTTTTCTTTTCTCAAGTTCTAGGGGGCATTTGCA
CATATATTTGTACTCAACATTTCATGGGAAAGCGGCA
GATCCGCGCTGAGGAGCAGCGAGGGCAGGGACAGGAG
GCCCTGGTCTGGACACTTCCTCCAGCACAATCCCTTC
CCCCGCCTCCTGCTCCTCCCCCTCGACCGCCTGCCCA
CTGTTGTAAAATAATCAGAAACTTGTTCTATTTTGTG
GCAGTGACAATAGTTTTATATTAAAAGAAAAAAATAC
AGTTTTCATACAGCAAAATCTATACAATATCATTGTT
TTATTTAATATAAAGATCGCTACCCACTCCTTTCCAT
GGTTCCCACCCTACAAGGACTTCCCCTCTCTGCAGCA
GTTGCACTACAGGTAGCTACTGTGTATATGGACAAAT
GAGAAATGAATCCTTTTTTCTGGCTGTCCATCTATTT
TATTTCAAATAAGGAAAAGTGTATTTGGATTTTGTGT
AAATACATCTAGTGATGGCATTTTTTCAATGTTTTTA
AAAGCTGTGTACAGTACATGTGGTAGAGTGTTTCTCA
AATTGTCTATTGTAGCAAAGGCGTTTTTGTCGTAAAC
CTGTTCTGTGTCCTTTTTTGTTCTTACCACTTCTCTT
CCTCCTCACCCCAGATACTTCCTCTTCCCCACAACCA
ATGTACATAGTAATTGTAATGTTTTAGACTTGACAGA
AACTTTCCTGTATTCTGTATATAAAAACCAAAAATAC TTCAAATT
[0156]
9 Mouse peptide MVDLTNSLRAFMDVNGEIEINMLDEKLIKFLALQRVH (SEQ ID NO:8)
QLFPSRVQASPGNVGTHPLASGGHHPEVQRKEVQARA
VFCPLLGLGGAVNMCYRTLYIGTGADMDVCLTNYGHC
NYVSGKHACIFYDENTKHYELLNYSEHGTTVDNVLYS
CDFSEKTPPTPPSSIVAKVQSVIRREHQKQDEEPSEE
AAMMSSQAQGPQRRPCNCKASSSSLIGGSGAGWEGTA
LLHHGSYIKLGCLQFVFSITEFATKQPKGDASLLQDG VLAEKLSLKPHQGPVLRSNSVP
[0157] FIG. 3 shows the sequence homology between a human MTC49
amino acid sequence 25; and a mouse MTC49 amino acid sequence
according to the invention. The conserved regions in each amino
acid sequence are highlighted in black.
[0158] MTC49 most likely encodes a new transcription factor. This
conclusion is supported by Psort analysis suggesting a nuclear
location (nucleus P=0.6000). A nuclear location for the encoded
MTC49 polypeptide is also likely in view of the homology of the
encoded polypeptide to O60129 FORK HEAD PROTEIN TYPE TRANSCRIPTION
FACTOR (46% ID in the region between 96 to 191aa) and weaker
homology to homeobox proteins.
[0159] Also included in the invention is a nucleic acid encoding a
polypeptide having an FHA domain. The FHA domain is a putative
nuclear signaling domain found in protein kinases and transcription
factors. This sequence is up-regulated in the GENECALLING.TM.
analysis described herein. Its nucleotide sequence is provided in
SEQ ID NO:9.
10 AATTTATAAAGAAAAGACATTTATTTTGGCTCACAAT (SEQ ID NO:9)
TCTGCAGGCTGTACTGGCATGGCACCAACATTTGCTC
AGCTTCTGGTGAGGGCCTCAGGAAGCTTACAGTAAAG
GCGGAAGGTGAAGGGGGAGCAGGCATATCACATGGCG
AGAAAGAGGGGAGAGGTCTCAGACTCTTTTAAACAAC
CATATCTATGTGAATTGAGTGAGAACTCACTCATCAC
CAAGGAGATGGTGCTGAGCCATTCATGAAGGATCCTC
TCTCATGATCCAAATACTTCCCACCAGGCTCCACTTC
CAACACTGGGAATTACATTTCAACATGAGATTTGGAG
GGGACGAGCATCCAAACCATATCAGATGGTGAGACAG
GAGAACTTTGTGTGTCCCAGCTGCACTGGTCTGAAGA
TATAACTAAGTCCCTGGACTTTTTCTCCCTTAATTGG
AGAATTCCTAATGTTCCATGATCAGCCTGATTGACCA
GTGGCTGACTGGTCCTGAGAGGGGAGATAAAAACAGA
CACACAGCTTTCTCCATAGACAAATCTCAACACTTTC
[0160] MTC50
[0161] An MTC50 nucleic acid according to the invention was
assembled and named 95199195. GENECALLING.TM. analysis reveals that
this gene is up-regulated in metastatic vs. non-metastatic thyroid
cancer. The assembled sequence is part of the genomic region
assigned to inosine-5'-monophosphate dehydrogenase type II, but it
represents a different gene as discussed by Zimmermann et al. See
Zimmermann et al., J Biol Chem 272(36):22913-23 (1997)). Zimmerman
et al. showed that there is a 2-kb gene that is oriented
tail-to-tail with respect to the IMPDH type II gene and terminates
I kb 3 to it.
[0162] An MTC50 nucleotide sequence according to the invention that
corrresponds to the reverse complement (SEQ ID NO: 10) of 95199195
is provided below. The amino acid sequence (SEQ ID NO:11) of an MTC
polypeptide is also provided
11 GGGGCTAGAAGTCTGGCACCCACCGCCTGGCCAGGT (SEQ ID NO:10)
GTTCGGGACGCGACCAGGTGGGCGGTCGCCGCCCCG
GGAGCGCGGCTTAATAGCTGAGAGCCCGGGGGCCAG
GCCGCGGCTGCGGCCCAGGCAACGCCCTGAGGGTGG
CCACGCTGCCAGGTGTTCCACTCCCCCGGGACTATG
GGCAAGGGCCGGGGCGGGGAGGGCGGCAGGTGCTGA
CACTGGAGCTGCCCGGAGTCGGGGAACTCGGCCTCC
TAAGACTGAGGACACTCGCCTGCTGGGCCGGTCGAG
CTGTGCGGTGCCCTCCGGACGCAGGGGGCGCTGCAG
CCACGCTGGGTCAGGCTCCGCAGGCCCTCCCAACCC
GGGGACTAACGGCGCCGGTGACGACTTCGCCGCGCG
TTGGTCAGCCATGGCCACCGCTCTCGCGCTACGTAG
CTTGTACCGAGCGCGACCCTCGCTGCGCTGTCCGCC
CGTTGAGCTTCCCTGGGCCCCGCGGCGAGGGCATCG
GCTCTCGCCGGCGGATGACGAGCTGTATCAGCGGAC
GCGCATCTCTCTGCTGCAACGCGAGGCCGCTCAGGC
AATGTACATCGACAGCTACAACAGCCGCGGCTTCAT
GATAAACGGAAACCGCGTGCTCGGCCCCTGCGCTCT
GCTCCCGCACTCGGTGGTGCAGTGGAACGTGGGATC
CCACCAGGACATCACCGAAGACAGCTTTTCCCTCTT
CTGGTTGCTGGAGCCCCGGATAGAGATCGTGGTGGT
GGGGACTGGAGACCGGACCGAGAGGCTGCAGTCCCA
GGTGCTTCAAGCCATGAGGCAGCGGGGCATTGCTGT
GGAAGTGCAGGACACGCCCAATGCCTGTGCCACCTT
CAACTTCCTGTGTCATGAAGGCCGAGTAACTGGAGC
TGCTCTCATCCCTCCACCAGGAGGGACTTCACTTAC
ATCTTTGGGCCAAGCTGCTCAATGAACCGCCAGGAA
CTGACCTGCTGACTGCACTCTGCCAGGCTTCCCAAT
GCTTTCACTCTTATCTACCCTTTGGCACTTATCTTG
CTTATCAACATAATAATTTATACACTTCTCCCATTT
TGTATCAGGTGTGTTGCTGGCCAGGAGCTGATGGCT
CACTGGGCTCTTGGAGGGGAATGTGAAGAAACCAAG
GAGTCACTTTTTCATCTAGATTACTTAGGATTCCTT
GACTTTTCAGAAGTCGGGAAGCAGTATGTTTGCCTG
TTGTAGACCTACTTGCTCACATGCAGATTTGAGAGG
ACCTCAACGGCTTTTCTCACAAAAAAAAA
[0163]
12 Amino Acid Sequence (SEQ ID NO:11)
MATALALRSLYRARPSLRCPPVELPWAPRRGHRLSPADDELYQRTRISLL
QREAAQAMYIDSYNSRGFMINGNRVLGPCALLPHSVVQWNVGSHQDITED
SFSLFWLLEPRIEIVVVGTGDRTERLQSQVLQAMRQRGIAVEVQDTPNAC
ATFNFLCHEGRVTGAALIPPPGGTSLTSLGQAAQ
[0164] The encoded MTC50 amino acid polypeptide is similar to a
hypothetical protein present in public databases that is named
Q9Y3Z0. Alignment of these two sequences reveals that they are
identical in their COOH terminus but diverge in the first 50 amino
acids (out of 190 amino acids).
[0165] FIG. 4 shows the sequence homology between a human MTC50
amino acid sequence and the hypothetical protein Q9Y2Z0. The
conserved regions in each amino acid sequence are highlighted in
black.
[0166] This sequence is similar to the rat gene E3-3 (AAB54063), an
entry in the database that is annotated as a novel nuclear protein
that is predominantly expressed in testis. Psort puts it in the
mitochondrial matrix space P=0.8414. BlastP against the database
finds homology to several eukaryotic hypothetical proteins and
bacterial membrane proteins. Blocks analysis shows the polypeptide
has homology to acid phosphatase in the NH terminus and to
CYCLOPHILIN-TYPE PPIASE FAMILY, in the COOH terminus. Sbase and
Prodom finds that the NH terminus is similar to the transit peptide
of FUMARATE HYDRATASE, another genomically encoded protein that is
localized to the mitochondrial membrane. Sbase also shows
similarity with the rat serotonin (5-HT6) receptor, a G-protein
coupled receptor (48% over the length of the protein).
[0167] FIG. 5 shows the sequence homology between a human MTC50
amino acid sequence and the rat gene E3-3 (AAB54063). The conserved
regions in each amino acid sequence are highlighted in black.
[0168] Cyclophilins are known to be involved in stress-related
responses, and they are up-regulated in cancer. This sequence might
represent a new member of this family that localizes to the
mitochondrial membrane. Inhibition of the Ppiase activity of this
protein may be useful to treat cancer.
[0169] MTC51
[0170] A nucleotide sequence of an MTC51 nucleic acid is provided
below (SEQ ID NO: 12). GENECALLING.TM. analysis reveals that it is
up-regulated in metastatic vs. non-metastatic thyroid cancer. This
sequence finds no match or similarity with sequences in any
database.
[0171] Nucleic Acid Sequence (SEQ ID NO: 12)
[0172]
tgtacagcagccgcttgaagtcctttaagangaaatctcttttcttgncgngttganccttccacgg-
ngtanttgacgtcccctggcntggtttt tgatccgga
[0173] Conceptual translation of this nucleic acid sequence reveals
that it can code for two open reading frames ("ORFs"). This
indicates the possibility that it can represent a fragment of the
coding region of a novel gene.
13 ORF1, Frame-1 SGSKTXPGDVXYXVEGSTRQEKRFXLKGLQAAAV (SEQ ID NO:13)
ORF2, Frame-2 PDQKPXQGTSXTPWKXQXXKKRDFXL- KDFKRLLY (SEQ ID
NO:14)
[0174] General Screening and Diagnostic Methods Using MTC
sequences
[0175] Expression of each MTC nucleic acid sequence can be
identified using the information provided above. In some
embodiments, the MTC nucleic acids correspond to nucleic acids
which include the various sequences (referenced above by SEQ ID
NOs) that have been disclosed for each MTC sequence.
[0176] In its various aspects and embodiments, the invention
includes providing a test cell population which includes at least
one cell that is capable of expressing one or more of the sequences
MTC 1-51. By "capable of expressing" is meant that the nucleic acid
sequence is present in an intact form in the cell and can be
expressed. Expression of one, some, or all of the MTC sequences is
detected, if present, and, preferably, measured. Using the sequence
information provided by the database entries for the known
sequences, or the sequence information provided herein for the
previously unknown sequences, the expression of the MTC sequences
can be detected, if present, and measured using techniques well
known to one of ordinary skill in the art. For example, sequences
within public sequence database entries for the MTC sequences or
within the novel sequences disclosed herein can be used to
construct probes for detecting MTC RNA sequences in, for example,
northern blot hybridization analyses. Alternatively, the sequences
can be used to construct primers for specifically amplifying the
MTC sequences in, for example, amplification-based detection
methods such as reverse transcription-based polymerase chain
reaction (PCR).
[0177] The expression level(s) of one or more of the MTC sequences
in the test cell population is then compared to expression levels
of the sequences in one or more cells from a reference cell
population. A reference cell population includes one or more cells
for which the compared parameter or condition is known. The
composition of the reference cell population will determine whether
the comparison of gene expression profile indicates the presence or
absence of the measured parameter or condition.
[0178] An alteration of the expression in the test cell population,
as compared to the reference cell population, indicates that the
measured parameter or condition in the test cell population is
different than that of the reference cell. The absence of the
alteration of expression in the test cell population, as compared
to the reference cell population, indicates that the measured
parameter or condition in the test cell population is the same as
that of the reference cell. As an example, if the reference cell
population contains noncancerous cells, a similar gene expression
profile in the test cell population indicates that the test cells
are also non-cancerous, whereas a different profile indicates that
the test cells are cancerous. Likewise, if the reference cell
population is made up of cancerous cells, a similar expression
profile in the test cell population indicates that the test cell
population also includes cancerous cells, and a different
expression profile indicates that test cells are noncancerous.
[0179] In some embodiments, the test cell population is compared to
multiple reference cell populations. Each of the multiple reference
cell populations might differ in the known. parameter. For example,
a test cell population may be compared to a reference cell
population containing nonmetastatic cancerous cells as well as a
second reference cell population known to contain metastatic
cancerous cells.
[0180] The test cell population may be known to contain or be
suspected of containing a neoplasm. In some embodiments, the test
cell will be included in a cell sample known to contain or
suspected of containing a thyroid follicular adenoma. In other
embodiments, the test cell sample will be derived from a region
known to contain, or suspected of containing a metastatic papillary
carcinoma. In other embodiments, the test cell population may be
known to contain or be suspected of containing thyroid follicular
adenomas and metastatic papillary carcinomas.
[0181] The test cell can be taken from a known or suspected
tumor-containing sample, or it may be taken from a bodily fluid or
biological fluid including blood, serum, urine, saliva, milk,
ductal fluid, or tears. For many applications, e.g., categorizing a
neoplasm, assessing the efficacy of treatment, or in diagnosing a
neoplasm in a subject, cells present in bodily fluids can be
examined instead of those from the primary lesion. Accordingly, the
need for taking a biopsy from a known or suspected neoplasm may be
obviated.
[0182] Preferably, cells in the reference cell population are
derived from a tissue type that is as similar to the test cell
population as possible. For example, the reference cell population
may be derived from thyroid tissue. In some embodiments, the
reference cell is derived from a region proximal to the region of
origin of the test cell population.
[0183] The subject is preferably a mammal. For example, the mammal
can be a human, non-human primate, mouse, rat, dog, cat, horse, or
cow.
[0184] In some embodiments, the reference cell population is
derived from a plurality of cells. The reference cell population
can be a database of expression patterns from previously tested
cells for which one of the assayed parameters or conditions is
known.
[0185] In various embodiments, the expression of 1-51 or more of
the sequences represented by MTC1-51 are measured. If desired,
expression of these sequences can be measured along with other
sequences whose expression is known to be altered according to one
of the herein described parameters or conditions. By "altered" is
meant that the expression of one or more nucleic acid sequences is
either increased or decreased as compared to the expression levels
in the reference cell population. Alternatively, the expression
profile of the test cell population may be the same as that of the
reference cell population.
[0186] The expression of the sequences disclosed herein can be
measured at the RNA by any method known in the art. For example,
northern blot hybridization analysis using probes that specifically
recognize one or more of these MTC sequences can be used to
determine gene expression. Alternatively, nucleic acid sequence
expression can be measured using reverse transcription-based PCR
assays that, for example, use primers specific for the
differentially expressed sequences designated as MTCs: 1-51.
[0187] Expression can also be measured at the protein level by, for
example, measuring the levels of polypeptides encoded by the gene
products described herein. Methods for measuring the levels of
polypeptides are well known in the art. For example, immunoassays
can be designed based on antibodies to proteins encoded by the
nucleic acid sequences.
[0188] When alterations in nucleic acid sequence expression are
associated with gene amplification or deletion, sequence
comparisons in the test and reference cell populations can be made
by comparing the relative levels of examined nucleic acid sequences
in both the test and reference cell populations.
[0189] Categorizing Thyroid Cancer Stage
[0190] In one aspect, the invention provides a method of
categorizing the stage of thyroid cancer in a subject. By
"categorizing thyroid cancer stage" is meant the determination of
the metastatic stage of the thyroid cancer. In other words,
determining whether a subject's thyroid cancer is metastatic as
opposed to non-metastatic.
[0191] The method includes providing a cell from the subject and
detecting the expression level of one or more of the nucleic acid
sequences MTC: 1-51 in the cell.
[0192] The expression of the nucleic acid sequences is then
compared to the level of expression in a reference cell population.
In general, any reference cell population may be used as long as
the thyroid cancer stage is known. In some embodiments, the
reference cell population is made up of non-metastatic thyroid
cancer cells. Test cell expression profiles that are similar to
those of such a reference cell population indicate that the test
cell population is also made up of non-metastatic thyroid cancer
cells. Conversely, test cell expression profiles that are different
from a non-metastatic thyroid cancer population are indicative of a
metastatic thyroid cancer test cell population. In other
embodiments, the reference cell population comprises metastatic
thyroid cancer cells. In such an embodiment, a test cell expression
profile similar to the reference cell would be indicative of
metastatic thyroid cancer whereas a different expression pattern is
indicative of nonmetastatic thyroid cancer.
[0193] If desired, relative expression levels within the test and
reference cell populations can be normalized by reference to the
expression level of a nucleic acid sequence that does not vary
according to thyroid cancer stage in a subject.
[0194] Diagnosing a Neoplasm
[0195] The invention further provides a method for diagnosing a
neoplasm, e.g., a thyroid carcinoma. A neoplasm is diagnosed by
examining the expression of one or more MTC nucleic acid sequences
from a test cell population that contain a suspected tumor. The
population of test cells may contain the primary tumor, e.g.,
thyroid tissue, or, alternatively, may contain cells into which the
primary tumor has disseminated, e.g., blood or lymphatic fluid.
[0196] The expression of one or more of the MTC sequences (MTCs:
1-51) is measured in the test cell and compared to the expression
of the sequences in the reference cell population. The reference
cell population must contain at least one cell whose neoplastic
state is known. For example, the thyroid cancer stage of the
reference cell population must be known. If the reference cell
contains no neoplastic cells, than a similarity in MTC sequence
expression between the test cell population and the reference cell
populations indicates that the test cell population likewise does
not contain any neoplastic cells. On the other hand, a difference
in expression of MTC sequence between the test and reference cell
population indicates that the test cell population contains a
neoplastic cell.
[0197] Conversely, when the reference cell population contains at
least one neoplastic cell, a similarity in MTC expression pattern
indicates that the test cell population also includes a neoplastic
cell. Alternatively, a differential expression pattern indicates
that the test cell population contains non-neoplastic cells.
[0198] Assessing the Efficacy of a Treatment of a Neoplasm in a
Subject
[0199] The differentially expressed MTC sequences identified herein
also allow the course of treatment of a neoplasm, such as a thyroid
carcinoma, to be monitored. In this method, a test cell population
is provided from a subject who is undergoing treatment for a
neoplasm. If desired, the test cell population can be taken from
the subject at various times before, during, and after treatment.
The expression of one or more of MTCs: 1-51 in the test cell
population is then measured and compared to a reference cell
population which includes cells whose neoplastic, i.e., thyroid
carcinoma, stage is known. Preferably, the reference cells have not
been exposed to the treatment.
[0200] If the reference cell population contains no neoplastic
cells, a similarity in expression between the test and reference
cell populations indicates that the treatment is efficacious.
However, a difference in expression patterns indicates that the
treatment is not efficacious.
[0201] By "efficacious" is meant that the treatment leads to a
decrease in size or metastatic potential of a neoplasm in a
subject, or a shift in a tumor stage to a less advanced stage. When
the treatment is applied prophylactically, "efficacious" means that
the treatment retards or prevents the formation of the neoplasm in
a subject.
[0202] When the reference cell population contains neoplastic
cells, a similar expression pattern indicates that the treatment is
not efficacious, whereas a dissimilar expression pattern indicates
that the treatment is efficacious.
[0203] Efficacy can be determined in association with any method
for treating a particular neoplasm.
[0204] Identifying a Therapeutic Agent Individualized for Treating
a Neoplasm
[0205] Genetic differences in individual subjects can result in
different abilities to metabolize various drugs. An agent that is
metabolized in a subject to act as an anti-neoplastic agent can
manifest itself by inducing a change in gene expression pattern in
the subject's cells from the pattern characteristic of the
non-neoplastic state. Thus, the differentially expressed MTC
sequences disclosed herein allow for a putative therapeutic or
prophylactic anti-neoplastic agent to be tested in a cell
population to determine if the agent is a suitable anti-neoplastic
agent in the subject.
[0206] According to this method of the invention, a test cell
population from the subject is exposed to a therapeutic agent. The
expression of one or more MTC sequences is then measured.
[0207] In some embodiments, the test cell population contains the
primary tumor, e.g. a thyroid carcinoma, or a bodily fluid, such as
blood or lymph, into which the tumor cell has disseminated. In
other embodiments, the agent is first mixed with a cell extract,
for example, a liver cell extract, which contains enzymes that
metabolize drugs into an active form. The activated form of the
drug is then mixed with the test cell population so that gene
expression can be measured. Preferably, the cell population is
contacted ex vivo with the agent or its activated form.
[0208] By "individualized" is meant that the particular therapeutic
agent selected takes the differences in genetic makeup of
individuals into account by insuring that the selected agent is
therapeutic in a particular subject.
[0209] Expression of the MTC sequences in the test cell population
is then compared with the expression patterns in the reference cell
population. Again, the reference cell population contains at least
one cell whose neoplastic, i.e., thyroid carcinoma, stage is known.
If the reference cell is non-cancerous, similar gene expression
patterns indicate that the agent is suitable for treating the
neoplasm in that subject. If the patterns are different, then the
particular agent is not suitable for treating the neoplasm in a
particular subject.
[0210] On the other hand, if the reference cell is cancerous,
similar sequence expression patterns indicate that the agent is not
suitable for the treatment of that subject. Conversely,
differential MTC expression indicates that the agent is suitable
for the treatment of that subject.
[0211] The test agent may be any compound or composition. In some
embodiments, the agent may be a compound or composition known to be
an anti-cancer agent. In other embodiments, the agent may be a
compound or composition not previously known to be an anti-cancer
agent.
[0212] Screening Assays for Identifying a Candidate Therapeutic
Agent for Treating or Preventing a Neoplasm
[0213] The differentially expressed MTC sequences disclosed herein
can also be used to identify candidate therapeutic agents for
treating a neoplasm, for example, a thyroid carcinoma. This method
is based on the screening of a candidate therapeutic agent to
determine if it converts an expression profile of MTCs: 1-51 that
is characteristic of a cancerous state to a pattern indicative of a
noncancerous state.
[0214] In this method, a test cell population is exposed to a test
agent or a combination of test agents, either sequentially or
simultaneously. The expression of one or more MTC sequence is
measured. Next, the expression of the MTC sequences in the test
cell population is compared to the expression level of the MTC
sequences in a reference cell population that has not been exposed
to the test agent.
[0215] An appropriate test agent candidate will increase the
expression of MTC sequences that are downregulated in cancerous
cells and/or will decrease the expression of those MTC sequences
that are upregulated in cancerous cells.
[0216] In some embodiments, the reference cell population includes
cancerous cells. When such a reference cell population is used, an
alteration in expression of the nucleic acid sequences in the
presence of the test agent from the expression pattern of the
reference cell population in the absence of the reagent indicates
that the agent is a candidate therapeutic agent for the treatment
of a neoplasm.
[0217] The test agent or agents used in this method can be a
compound(s) not previously described or can be a previously known
compound that has not been shown to be an anti-neoplastic
agent.
[0218] An agent that is effective in stimulating the expression of
underexpressed genes, or in suppressing the expression of
overexpressed genes can be further tested for its ability to
prevent tumor growth. Such an agent is also potentially useful for
the treatment of tumors. Further analysis of the clinical
usefulness of a given compound can be performed using standard
methods of evaluating toxicity and clinical effectiveness of
anti-cancer agents.
[0219] Categorizing a Neoplasm
[0220] Comparison of MTC expression patterns in test cell
populations and reference cell populations can be used to
categorize neoplasms in a subject. For example, such a comparison
can be used to categorize thyroid carcinoma in a subject.
[0221] This method includes providing a test cell population
containing at least one neoplastic cell from a subject and
measuring the expression of one or more MTC sequences in this test
cell. The expression of the nucleic acid sequences in the test cell
population is compared to the expression of the nucleic acid
sequences in a reference cell population comprising at least one
cell whose neoplastic state and category is known. A similarity in
expression patterns indicates that the cancerous cell in the test
cell population has the same neoplastic category as does the
reference cell population.
[0222] By "category" is meant the neoplastic state of a given
neoplasm. In other words, whether the neoplasm is metastatic or
nonmetastatic. In the case of metastatic neoplasms, "categorizing a
neoplasm" can mean determining the extent of the metastasis.
[0223] Assessing the Prognosis of a Subject with a Neoplasm
[0224] Also provided is a method of assessing the prognosis of a
subject having a neoplasm, such as a thyroid carcinoma, by
comparing the expression of one or more MTC sequences in a test
cell population, which contains at least one cancerous cell, to the
expression of the sequences in a reference cell population. By
comparing the gene expression profiles of one or more MTC
sequences, the prognosis of the subject can be assessed.
[0225] In alternative embodiments, the reference cell population
includes primarily noncancerous or cancerous cells. When the
reference cell contains primarily noncancerous cells, an increase
in the expression of an MTC sequence that is overexpressed in the
metastatic cancer state, or a decrease in the expression of an MTC
sequence that is underexpressed in the metastatic state, suggests a
less favorable prognosis. Specifically, an increase in any one or
more of MTCs: 1, 3, 5-16,18-23, 25-27, 29-31, 33-35, 37, 39, 41-42,
44-45, 48 or 50, or a decrease in any one or more of MTCs: 2, 4,
17, 24, 28, 32, 36, 38, 40, 43, or 46, compared to a noncancerous
reference cell is indicative of a less favorable prognosis.
[0226] When the reference cell population contains primarily
cancerous cells, a decrease in the expression of a MTC sequence
that is overexpressed in the metastatic state, or an increase in
the expression of a MTC sequence that is underexpressed in the
metastatic state, suggests a favorable prognosis. Thus, a decrease
in the expression of any one or more of MTCs: 1, 3, 5-16,
18-23,25-27, 29-31, 33-35, 37, 39, 41-42, 44-45,48 or 50, or an
increase in the expression of any one or more of MTCs: 2, 4, 17,
24, 28, 32, 36, 38, 40, 43, or 46, compared to a cancerous
reference cell population is indicative of a favorable prognosis
for the subject.
[0227] Treating Metastatic Cancer
[0228] Also provided is a method of treating metastatic cancer, for
example, metastatic thyroid carcinomas, in a patent suffering from
or at risk for developing metastatic cancer. By "at risk for
developing" is meant that the subject's prognosis is less favorable
and that the subject has an increased likelihood of developing
metastatic cancer. This method involves the administration of an
agent that modulates the expression of one or more MTC sequences to
a subject in need of treatment. Administration can be prophylactic
or therapeutic.
[0229] In one embodiment, this method comprises administering to a
subject, an agent that increases the expression of one or more
nucleic acid sequences selected from the group consisting of MTCs:
2, 4, 17, 24, 28, 32, 36, 38, 40, 43, and 46. These MTC sequences
are underexpressed in the metastatic state as compared to the
nonmetastatic state. The subject is treated with an effective
amount of a compound that increases the amount the underexpressed
nucleic acid sequences in the subject. Administration can be
systemic or local, e.g., in the immediate vicinity of the subject's
cancerous cells. This agent could be, for example, the polypeptide
product of the underexpressed gene or a biologically active
fragment thereof, a nucleic acid encoding the underexpressed gene
and having expression control elements permitting expression in the
carcinoma cells, or an agent which increases the endogenous level
of expression of the gene.
[0230] In another embodiment, this method comprises administering
an agent that decreases the expression of one or more nucleic acid
sequences selected from the group consisting of MTCs: 1, 3, 5-16,
18-23, 25-27, 29-31, 33-35, 37, 39, 41-42, 44-45, 48, and 50. These
MTC sequences are overexpressed in the metastatic cancerous state.
Again, the subject is treated with an effective amount of a
compound that decreases the amount of the overexpressed nucleic
acid sequences in the subject. As discussed above, administration
can be systemic or local. Expression can be inhibited in any of
several ways known in the art. For example, expression can be
inhibited by administering to the subject a nucleic acid that
inhibits, or antagonizes, the expression of the overexpressed gene
or genes. In one embodiment, an antisense oligonucleotide can be
administered to disrupt expression of the endogenous gene or
genes.
[0231] In an alternative embodiment, the patient may be treated
with one or more agents which decrease the expression of those MTC
sequences that are overexpressed in the metastatic state alone or
in combination with one or more agents which increase the
expression of those MTC sequences that are underexpressed in the
metastatic state.
[0232] Administration of a prophylactic agent can occur prior to
the manifestation of symptoms characteristic of aberrant gene
expression, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. Depending on the type of
aberrant expression detected, the agent can be used for treating
the subject. The appropriate agent can be determined based on
screening assays described herein. Determination of an effective
amount of a compound is within the ordinary skill of one in this
art.
[0233] In some embodiments, a subject, e.g., a human, is treated
with genes encoding secreted or membrane bound polypeptides, or
with the encoded polypeptide. These genes include, e.g.,
alpha-1-antitrypsin, Neuropilin, NET-1, Lipocortin II integrin
alpha-3 Type IV collagenase Antileukoprotease Periplakin Clusterin
DAP12. These secreted or membrane bound proteins are up-regulated
in metastatic thyroid cancers. Accordingly, they are also desirable
candidates for antibody screening and antibody-binding therapy.
[0234] In other embodiments, the patient is treated with a gene
encoding a membrane or secreted protein that is down-regulated in
metastatic thyroid cancer, or its encoded polypeptide. These genes
include, e.g., RIG-E, PRO302. These secreted or membrane protein
that can be easily delivered back to the affected patients as
recombinant soluble proteins for the treatment of metastatic
thyroid cancer.
[0235] Pharmaceutical Compositions for Treating Neoplasms
[0236] In another aspect, the invention includes pharmaceutical or
therapeutic compositions containing one or more therapeutic
compounds described herein. Pharmaceutical formulations may include
those suitable for oral, rectal, nasal, topical (including buccal
and sub-lingual), vaginal or parenteral (including intramuscular,
sub-cutaneous and intravenous) administration, or for
administration by inhalation or insufflation. The formulations may,
where appropriate, be conveniently presented in discrete dosage
units and may be prepared by any of the methods well known in the
art of pharmacy. All such pharmacy methods include the steps of
bringing into association the active compound with liquid carriers
or finely divided solid carriers or both as needed and then, if
necessary, shaping the product into the desired formulation.
[0237] Pharmaceutical formulations suitable for oral administration
may conveniently be presented as discrete units, such as capsules,
cachets or tablets, each containing a predetermined amount of the
active ingredient; as a powder or granules; or as a solution, a
suspension or as an emulsion. The active ingredient may also be
presented as a bolus electuary or paste, and be in a pure form,
i.e., without a carrier. Tablets and capsules for oral
administration may contain conventional excipients such as binding
agents, fillers, lubricants, disintegrant or wetting agents. A
tablet may be made by compression or molding, optionally with one
or more formulational ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active
ingredients in a free-flowing form such as a powder or granules,
optionally mixed with a binder, lubricant, inert diluent,
lubricating, surface active or dispersing agent. Molded tablets may
be made by molding in a suitable machine a mixture of the powdered
compound moistened with an inert liquid diluent. The tablets may be
coated according to methods well known in the art. Oral fluid
preparations may be in the form of, for example, aqueous or oily
suspensions, solutions, emulsions, syrups or elixirs, or may be
presented as a dry product for constitution with water or other
suitable vehicle before use. Such liquid preparations may contain
conventional additives such as suspending agents, emulsifying
agents, non-aqueous vehicles (which may include edible oils), or
preservatives. The tablets may optionally be formulated so as to
provide slow or controlled release of the active ingredient
therein.
[0238] Formulations for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include
suspending agents and thickening agents. The formulations may be
presented in unit dose or multi-dose containers, for example sealed
ampoules and vials, and may be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, saline, water-for-injection,
immediately prior to use. Alternatively, the formulations may be
presented for continuous infusion. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets of the kind previously described.
[0239] Formulations for rectal administration may be presented as a
suppository with the usual carriers such as cocoa butter or
polyethylene glycol. Formulations for topical administration in the
mouth, for example buccally or sublingually, include lozenges,
comprising the active ingredient in a flavored base such as sucrose
and acacia or tragacanth, and pastilles comprising the active
ingredient in a base such as gelatin and glycerin or sucrose and
acacia. For intra-nasal administration the compounds of the
invention may be used as a liquid spray or dispersible powder or in
the form of drops. Drops may be formulated with an aqueous or
non-aqueous base also comprising one or more dispersing agents,
solubilizing agents or suspending agents. Liquid sprays are
conveniently delivered from pressurized packs.
[0240] For administration by inhalation the compounds are
conveniently delivered from an insufflator, nebulizer, pressurized
packs or other convenient means of delivering an aerosol spray.
Pressurized packs may comprise-a suitable propellant such as
dichlorodifluoromethane, trichlorofluoromethane,
dichiorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount.
[0241] Alternatively, for administration by inhalation or
insufflation, the compounds may take the form of a dry powder
composition, for example a powder mix of the compound and a
suitable powder base such as lactose or starch. The powder
composition may be presented in unit dosage form, in for example,
capsules, cartridges, gelatin or blister packs from which the
powder may be administered with the aid of an inhalator or
insuffiator.
[0242] When desired, the above-described formulations, adapted to
give sustained release of the active ingredient, may be employed.
The pharmaceutical compositions may also contain other active
ingredients such as antimicrobial agents, immunosuppressants or
preservatives.
[0243] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention
may include other agents conventional in the art having regard to
the type of formulation in question, for example, those suitable
for oral administration may include flavoring agents.
[0244] Preferred unit dosage formulations are those containing an
effective dose, as recited below, or an appropriate fraction
thereof, of the active ingredient.
[0245] For each of the aforementioned conditions, the compositions
may be administered orally or via injection at a dose of from about
0.1 to about 250 mg/kg per day. The dose range for adult humans is
generally from about 5 mg to about 17.5 g/day, preferably about 5
mg to about 10 g/day, and most preferably about 100 mg to about 3
g/day. Tablets or other unit dosage forms of presentation provided
in discrete units may conveniently contain an amount which is
effective at such dosage or as a multiple of the same, for
instance, units containing about 5 mg to about 500 mg, usually from
about 100 mg to about 500 mg.
[0246] The pharmaceutical composition preferably is administered
orally or by injection (intravenous or subcutaneous), and the
precise amount administered to a subject will be the responsibility
of the attendant physician. However, the dose employed will depend
upon a number of factors, including the age and sex of the subject,
the precise disorder being treated, and its severity. Also the
route of administration may vary depending upon the condition and
its severity. Determination of the proper dose and route of
administration is within the ordinary skill of those familiar with
this art.
[0247] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having the nucleotide sequence of any of
MTCS:48-51, or a complement of any of these nucleotide sequences,
can be isolated using standard molecular biology techniques and the
sequence information provided herein. Using all or a portion of
these nucleic acid sequences as a hybridization probe, MTC nucleic
acid sequences can be isolated using standard hybridization and
cloning techniques (e.g., as described in Sambrook et al., eds.,
MOLECULAR CLONING: A LABORATORY MANUAL 2.sup.nd Ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and
Ausubel, et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John
Wiley & Sons, New York, N.Y., 1993.).
[0248] A nucleic acid of the invention can be amplified using cDNA,
mRNA or alternatively, genomic DNA, as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to MTC nucleotide
sequences can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0249] As used herein, the term "oligonucleotide" refers to a
series of linked nucleotide residues, which oligonucleotide has a
sufficient number of nucleotide bases to be used in a PCR reaction.
A short oligonucleotide sequence may be based on, or designed from,
a genomic or cDNA sequence and is used to amplify, confirm, or
reveal the presence of an identical, similar or complementary DNA
or RNA in a particular cell or tissue. Oligonucleotides comprise
portions of a nucleic acid sequence having at least about 10 nt and
as many as 50 nt, preferably about 15 nt to 30 nt. They may be
chemically synthesized and may be used as probes.
[0250] In another embodiment, an isolated nucleic acid molecule of
the invention comprises a nucleic acid molecule that is a
complement of the nucleotide sequence shown in MTCs: 48-51. In
another embodiment, an isolated nucleic acid molecule of the
invention comprises a nucleic acid molecule that is a complement of
the nucleotide sequence shown in any of these sequences, or a
portion of any of these nucleotide sequences. A nucleic acid
molecule that is complementary to the nucleotide sequence shown in
MTCs:48-51 is one that is sufficiently complementary to the
nucleotide sequence shown, such that it can hydrogen bond with
little or no mismatches to the nucleotide sequences shown, thereby
forming a stable duplex.
[0251] As used herein, the term "complementary" refers to
Watson-Crick or Hoogsteen base pairing between nucleotides units of
a nucleic acid molecule, and the term "binding" means the physical
or chemical interaction between two polypeptides or compounds or
associated polypeptides or compounds or combinations thereof.
Binding includes ionic, non-ionic, Von der Waals, hydrophobic
interactions, etc. A physical interaction can be either direct or
indirect. Indirect interactions may be through or due to the
effects of another polypeptide or compound. Direct binding refers
to interactions that do not take place through, or due to, the
effect of another polypeptide or compound, but instead are without
other substantial chemical intermediates.
[0252] Moreover, the nucleic acid molecule of the invention can
comprise only a portion of the nucleic acid sequence of MTCs:48-51
e.g., a fragment that can be used as a probe or primer or a
fragment encoding a biologically active portion of MTC. Fragments
provided herein are defined as sequences of at least 6 (contiguous)
nucleic acids or at least 4 (contiguous) amino acids, a length
sufficient to allow for specific hybridization in the case of
nucleic acids or for specific recognition of an epitope in the case
of amino acids, respectively, and are at most some portion less
than a full length sequence. Fragments may be derived from any
contiguous portion of a nucleic acid or amino acid sequence of
choice. Derivatives are nucleic acid sequences or amino acid
sequences formed from the native compounds either directly or by
modification or partial substitution. Analogs are nucleic acid
sequences or amino acid sequences that have a structure similar to,
but not identical to, the native compound but differs from it in
respect to certain components or side chains. Analogs may be
synthetic or from a different evolutionary origin and may have a
similar or opposite metabolic activity compared to wild type.
[0253] Derivatives and analogs may be full length or other than
full length, if the derivative or analog contains a modified
nucleic acid or amino acid, as described below. Derivatives or
analogs of the nucleic acids or proteins of the invention include,
but are not limited to, molecules comprising regions that are
substantially homologous to the nucleic acids or proteins of the
invention, in various embodiments, by at least about 45%, 50%, 70%,
80%, 95%, 98%, or even 99% identity (with a preferred identity of
80-99%) over a nucleic acid or amino acid sequence of identical
size or when compared to an aligned sequence in which the alignment
is done by a computer homology program known in the art, or whose
encoding nucleic acid is capable of hybridizing to the complement
of a sequence encoding the aforementioned proteins under stringent,
moderately stringent, or low stringent conditions. See e.g.
Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley
& Sons, New York, N.Y., 1993, and below. An exemplary program
is the Gap program (Wisconsin Sequence Analysis Package, Version 8
for UNIX, Genetics Computer Group, University Research Park,
Madison, Wis.) using the default settings, which uses the algorithm
of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482-489, which in
incorporated herein by reference in its entirety).
[0254] A "homologous nucleic acid sequence" or "homologous amino
acid sequence," or variations thereof, refer to sequences
characterized by a homology at the nucleotide level or amino acid
level as discussed above. Homologous nucleotide sequences encode
those sequences coding for isoforms of a MTC polypeptide. Isoforms
can be expressed in different tissues of the same organism as a
result of, for example, alternative splicing of RNA. Alternatively,
isoforms can be encoded by different genes. In the present
invention, homologous nucleotide sequences include nucleotide
sequences encoding for a MTC polypeptide of species other than
humans, including, but not limited to, mammals, and thus can
include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other
organisms. Homologous nucleotide sequences also include, but are
not limited to, naturally occurring allelic variations and
mutations of the nucleotide sequences set forth herein. A
homologous nucleotide sequence does not, however, include the
nucleotide sequence encoding a human MTC protein. Homologous
nucleic acid sequences include those nucleic acid sequences that
encode conservative amino acid substitutions (see below) in a MTC
polypeptide, as well as a polypeptide having a MTC activity. A
homologous amino acid sequence does not encode the amino acid
sequence of a human MTC polypeptide.
[0255] The nucleotide sequence determined from the cloning of human
MTC genes allows for the generation of probes and primers designed
for use in identifying and/or cloning MTC homologues in other cell
types, e.g., from other tissues, as well as MTC homologues from
other mammals. The probe/primer typically comprises a substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300,
350 or 400 consecutive sense strand nucleotide sequence of a
nucleic acid comprising a MTC sequence, or an anti-sense strand
nucleotide sequence of a nucleic acid comprising a MTC sequence, or
of a naturally occurring mutant of these sequences.
[0256] Probes based on human MTC nucleotide sequences can be used
to detect transcripts or genomic sequences encoding the same or
homologous proteins. In various embodiments, the probe further
comprises a label group attached thereto, e.g., the label group can
be a radioisotope, a fluorescent compound, an enzyme, or an enzyme
co-factor. Such probes can be used as a part of a diagnostic test
kit for identifying cells or tissue which misexpress a MTC protein,
such as by measuring a level of a MTC-encoding nucleic acid in a
sample of cells from a subject e.g., detecting MTC mRNA levels or
determining whether a genomic MTC gene has been mutated or
deleted.
[0257] "A polypeptide having a biologically active portion of MTC"
refers to polypeptides exhibiting activity similar, but not
necessarily identical to, an activity of a polypeptide of the
present invention, including mature forms, as measured in a
particular biological assay, with or without dose dependency. A
nucleic acid fragment encoding a "biologically active portion of
MTC" can be prepared by isolating a portion of MTCs:48-51, that
encodes a polypeptide having a MTC biological activity, expressing
the encoded portion of MTC protein (e.g., by recombinant expression
in vitro) and assessing the activity of the encoded portion of MTC.
For example, a nucleic acid fragment encoding a biologically active
portion of a MTC polypeptide can optionally include an ATP-binding
domain. In another embodiment, a nucleic acid fragment encoding a
biologically active portion of MTC includes one or more
regions.
[0258] MTC Variants
[0259] The invention further encompasses nucleic acid molecules
that differ from the disclosed or referenced MTC nucleotide
sequences due to degeneracy of the genetic code. These nucleic
acids thus encode the same MTC protein as that encoded by
nucleotide sequence comprising a MTC nucleic acid as shown in,
e.g., MTC48-51.
[0260] In addition to the MTC nucleotide sequences disclosed for
MTCs:48-51, it will be appreciated by those skilled in the art that
DNA sequence polymorphisms that lead to changes in the amino acid
sequences of a MTC polypeptide may exist within a population (e.g.,
the human population). Such genetic polymorphism in the MTC gene
may exist among individuals within a population due to natural
allelic variation. As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules comprising an
open reading frame encoding a MTC protein, preferably a mammalian
MTC protein. Such natural allelic variations can typically result
in 1-5% variance in the nucleotide sequence of the MTC gene. Any
and all such nucleotide variations and resulting amino acid
polymorphisms in MTC that are the result of natural allelic
variation and that do not alter the functional activity of MTC are
intended to be within the scope of the invention.
[0261] Moreover, nucleic acid molecules encoding MTC proteins from
other species, and thus that have a nucleotide sequence that
differs from the human sequence of MTC48-51, are intended to be
within the scope of the invention. Nucleic acid molecules
corresponding to natural allelic variants and homologues of the MTC
DNAs of the invention can be isolated based on their homology to
the human MTC nucleic acids disclosed herein using the human cDNAs,
or a portion thereof, as a hybridization probe according to
standard hybridization techniques under stringent hybridization
conditions. For example, a soluble human MTC DNA can be isolated
based on its homology to human membrane-bound MTC. Likewise, a
membrane-bound human MTC DNA can be isolated based on its homology
to soluble human MTC.
[0262] Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 6 nucleotides in length and
hybridizes under stringent conditions to the nucleic acid molecule
comprising the nucleotide sequence of MTCs:48-51. In another
embodiment, the nucleic acid is at least 10, 25, 50, 100, 250 or
500 nucleotides in length. In another embodiment, an isolated
nucleic acid molecule of the invention hybridizes to the coding
region. As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences at least 60%
homologous to each other typically remain hybridized to each
other.
[0263] Homologs (i.e., nucleic acids encoding MTC proteins derived
from species other than human) or other related sequences (e.g.,
paralogs) can be obtained by low, moderate or high stringency
hybridization with all or a portion of the particular human
sequence as a probe using methods well known in the art for nucleic
acid hybridization and cloning.
[0264] As used herein, the phrase "stringent hybridization
conditions" refers to conditions under which a probe, primer or
oligonucleotide will hybridize to its target sequence, but to no
other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures than shorter
sequences. Generally, stringent conditions are selected to be about
5.degree. C. lower than the thermal melting point (Tm) for the
specific sequence at a defined ionic strength and pH. The Tm is the
temperature (under defined ionic strength, pH and nucleic acid
concentration) at which 50% of the probes complementary to the
target sequence hybridize to the target sequence at equilibrium.
Since the target sequences are generally present at excess, at Tm,
50% of the probes are occupied at equilibrium. Typically, stringent
conditions will be those in which the salt concentration is less
than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium
ion (or other salts) at pH 7.0 to 8.3 and the temperature is at
least about 30.degree. C. for short probes, primers or
oligonucleotides (e.g., 10 nt to 50 nt) and at least about
60.degree. C. for longer probes, primers and oligonucleotides.
Stringent conditions may also be achieved with the addition of
destabilizing agents, such as formamide.
[0265] Stringent conditions are known to those skilled in the art
and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Preferably, the
conditions are such that sequences at least about 65%, 70%, 75%,
85%, 90%, 95%, 98%, or 99% homologous to each other typically
remain hybridized to each other. A non-limiting example of
stringent hybridization conditions is hybridization in a high salt
buffer comprising 6.times. SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA,
0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon
sperm DNA at 65.degree. C. This hybridization is followed by one or
more washes in 0.2.times. SSC, 0.01% BSA at 50.degree. C. An
isolated nucleic acid molecule of the invention that hybridizes
under stringent conditions to the sequence of MTCs:48-51
corresponds to a naturally occurring nucleic acid molecule. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein).
[0266] In a second embodiment, a nucleic acid sequence that is
hybridizable to the nucleic acid molecule comprising the nucleotide
sequence of MTCs:48-51 or fragments, analogs or derivatives
thereof, under conditions of moderate stringency is provided. A
non-limiting example of moderate stringency hybridization
conditions are hybridization in 6.times. SSC, 5.times. Denhardt's
solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at
55.degree. C., followed by one or more washes in 1.times. SSC, 0.1%
SDS at 37.degree. C. Other conditions of moderate stringency that
may be used are well known in the art. See, e.g., Ausubel et al.
(eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley
& Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A
LABORATORY MANUAL, Stockton Press, NY.
[0267] In a third embodiment, a nucleic acid that is hybridizable
to the nucleic acid molecule comprising the nucleotide sequence of
MTCs:48-51 or fragments, analogs or derivatives thereof, under
conditions of low stringency, is provided. A non-limiting example
of low stringency hybridization conditions are hybridization in 35%
formamide, 5.times. SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02%
PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA,
10% (wt/vol) dextran sulfate at 40.degree. C., followed by one or
more washes in 2.times. SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA,
and 0.1% SDS at 50.degree. C. Other conditions of low stringency
that may be used are well known in the art (e.g., as employed for
cross-species hybridizations). See, e.g., Ausubel et al. (eds.),
1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A
LABORATORY MANUAL, Stockton Press, NY; Shilo et al., 1981, Proc
Natl Acad Sci USA 78: 6789-6792.
[0268] Conservative Mutations
[0269] In addition to naturally-occurring allelic variants of the
MTC sequence that may exist in the population, the skilled artisan
will further appreciate that changes can be introduced into an MTC
nucleic acid or directly into an MTC polypeptide sequence without
altering the functional ability of the MTC protein. In some
embodiments, the nucleotide sequence of MTCs:48-51 will be altered,
thereby leading to changes in the amino acid sequence of the
encoded MTC protein. For example, nucleotide substitutions that
result in amino acid substitutions at various "non-essential" amino
acid residues can be made in the sequence of MTCs:48-51. A
"non-essential" amino acid residue is a residue that can be altered
from the wild-type sequence of MTC without altering the biological
activity, whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are
conserved among the MTC proteins of the present invention, are
predicted to be particularly unamenable to alteration.
[0270] In addition, amino acid residues that are conserved among
family members of the MTC proteins of the present invention, are
also predicted to be particularly unamenable to alteration. As
such, these conserved domains are not likely to be amenable to
mutation. Other amino acid residues, however, (e.g., those that are
not conserved or only semi-conserved among members of the MTC
proteins) may not be essential for activity and thus are likely to
be amenable to alteration.
[0271] Another aspect of the invention pertains to nucleic acid
molecules encoding MTC proteins that contain changes in amino acid
residues that are not essential for activity. Such MTC proteins
differ in amino acid sequence from the amino acid sequences of
polypeptides encoded by nucleic acids containing MTCs:48-51, yet
retain biological activity. In one embodiment, the isolated nucleic
acid molecule comprises a nucleotide sequence encoding a protein,
wherein the protein comprises an amino acid sequence at least about
45% homologous, more preferably 60%, and still more preferably at
least about 70%, 80%, 90%, 95%, 98%, and most preferably at least
about 99% homologous to the amino acid sequence of the amino acid
sequences of polypeptides encoded by nucleic acids comprising
MTCs:48-51.
[0272] An isolated nucleic acid molecule encoding a MTC protein
homologous to can be created by introducing one or more nucleotide
substitutions, additions or deletions into the nucleotide sequence
of a nucleic acid comprising MTCs:48-51, such that one or more
amino acid substitutions, additions or deletions are introduced
into the encoded protein.
[0273] Mutations can be introduced into a nucleic acid comprising
MTCs:48-51 by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Preferably, conservative
amino acid substitutions are made at one or more predicted
non-essential amino acid residues. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted nonessential amino acid residue in
MTC is replaced with another amino acid residue from the same side
chain family. Alternatively, in another embodiment, mutations can
be introduced randomly along all or part of a MTC coding sequence,
such as by saturation mutagenesis, and the resultant mutants can be
screened for MTC biological activity to identify mutants that
retain activity. Following mutagenesis of the nucleic acid, the
encoded protein can be expressed by any recombinant technology
known in the art and the activity of the protein can be
determined.
[0274] In one embodiment, a mutant MTC protein can be assayed for
(1) the ability to form protein:protein interactions with other MTC
proteins, other cell-surface proteins, or biologically active
portions thereof, (2) complex formation between a mutant MTC
protein and a MTC ligand; (3) the ability of a mutant MTC protein
to bind to an intracellular target protein or biologically active
portion thereof; (e.g., avidin proteins); (4) the ability to bind
ATP; or (5) the ability to specifically bind a MTC protein
antibody.
[0275] In other specific embodiments, the nucleic acid is RNA or
DNA.
[0276] Antisense
[0277] Another aspect of the invention pertains to isolated
antisense nucleic acid molecules that are hybridizable to or
complementary to the nucleic acid molecule comprising the
nucleotide sequence of a MTC sequence or fragments, analogs or
derivatives thereof. An "antisense" nucleic acid comprises a
nucleotide sequence that is complementary to a "sense" nucleic acid
encoding a protein, e.g., complementary to the coding strand of a
double-stranded cDNA molecule or complementary to an mRNA sequence.
In specific aspects, antisense nucleic acid molecules are provided
that comprise a sequence complementary to at least about 10, 25,
50, 100, 250 or 500 nucleotides or an entire MTC coding strand, or
to only a portion thereof Nucleic acid molecules encoding
fragments, homologs, derivatives and analogs of a MTC protein, or
antisense nucleic acids complementary to a nucleic acid comprising
a MTC nucleic acid sequence are additionally provided.
[0278] In one embodiment, an antisense nucleic acid molecule is
antisense to a "coding region" of the coding strand of a nucleotide
sequence encoding MTC. The term "coding region" refers to the
region of the nucleotide sequence comprising codons which are
translated into amino acid residues. In another embodiment, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding MTC.
The term "noncoding region" refers to 5' and 3' sequences which
flank the coding region that are not translated into amino acids
(i.e., also referred to as 5' and 3' untranslated regions).
[0279] Given the coding strand sequences encoding MTC disclosed
herein, antisense nucleic acids of the invention can be designed
according to the rules of Watson and Crick or Hoogsteen base
pairing. The antisense nucleic acid molecule can be complementary
to the entire coding region of MTC mRNA, but more preferably is an
oligonucleotide that is antisense to only a portion of the coding
or noncoding region of MTC mRNA. For example, the antisense
oligonucleotide can be complementary to the region surrounding the
translation start site of MTC mRNA. An antisense oligonucleotide
can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50
nucleotides in length. An antisense nucleic acid of the invention
can be constructed using chemical synthesis or enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed between the antisense and sense nucleic acids,
e.g., phosphorothioate derivatives and acridine substituted
nucleotides can be used.
[0280] Examples of modified nucleotides that can be used to
generate the antisense nucleic acid include: 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0281] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind to cellular mRNA and/or genomic DNA
encoding a MTC protein to thereby inhibit expression of the
protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by conventional nucleotide complementarity to
form a stable duplex, or, for example, in the case of an antisense
nucleic acid molecule that binds to DNA duplexes, through specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention includes direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind to receptors or antigens
expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies that
bind to cell surface receptors or antigens. The antisense nucleic
acid molecules can also be delivered to cells using the vectors
described herein. To achieve sufficient intracellular
concentrations of antisense molecules, vector constructs in which
the antisense nucleic acid molecule is placed under the control of
a strong pol II or pol III promoter are preferred.
[0282] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual P-units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res
15: 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett 215: 327-330).
[0283] Ribozymes and PNA Moieties
[0284] In still another embodiment, an antisense nucleic acid of
the invention is a ribozyme. Ribozymes are catalytic RNA molecules
with ribonuclease activity that are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave MTC mRNA transcripts to thereby
inhibit translation of MTC mRNA. A ribozyme having specificity for
a MTC-encoding nucleic acid can be designed based upon the
nucleotide sequence of a MTC DNA disclosed herein. For example, a
derivative of a Tetrahymena L-19 IVS RNA can be constructed in
which the nucleotide sequence of the active site is complementary
to the nucleotide sequence to be cleaved in a MTC-encoding mRNA.
See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al.
U.S. Pat. No. 5,116,742. Alternatively, MTC mRNA can be used to
select a catalytic RNA having a specific ribonuclease activity from
a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science
261:1411-1418.
[0285] Alternatively, MTC gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory
region of a MTC nucleic acid (e.g., the MTC promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the MTC gene in target cells. See generally,
Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al.
(1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays
14: 807-15.
[0286] In various embodiments, the nucleic acids of MTC can be
modified at the base moiety, sugar moiety or phosphate backbone to
improve, e.g., the stability, hybridization, or solubility of the
molecule. For example, the deoxyribose phosphate backbone of the
nucleic acids can be modified to generate peptide nucleic acids
(see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein,
the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid
mimics, e.g., DNA mimics, in which the deoxyribose phosphate
backbone is replaced by a pseudopeptide backbone and only the four
natural nucleobases are retained. The neutral backbone of PNAs has
been shown to allow for specific hybridization to DNA and RNA under
conditions of low ionic strength. The synthesis of PNA oligomers
can be performed using standard solid phase peptide synthesis
protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe
et al. (1996) PNAS 93: 14670-675.
[0287] PNAs of MTC can be used in therapeutic and diagnostic
applications. For example, PNAs can be used as antisense or
antigene agents for sequence-specific modulation of gene expression
by, e.g., inducing transcription or translation arrest or
inhibiting replication. PNAs of MTC can also be used, e.g., in the
analysis of single base pair mutations in a gene by, e.g., PNA
directed PCR clamping; as artificial restriction enzymes when used
in combination with other enzymes, e.g., S1 nucleases (Hyrup B.
(1996) above); or as probes or primers for DNA sequence and
hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996),
above).
[0288] In another embodiment, PNAs of MTC can be modified, e.g., to
enhance their stability or cellular uptake, by attaching lipophilic
or other helper groups to PNA, by the formation of PNA-DNA
chimeras, or by the use of liposomes or other techniques of drug
delivery known in the art. For example, PNA-DNA chimeras of MTC can
be generated that may combine the advantageous properties of PNA
and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H
and DNA polymerases, to interact with the DNA portion while the PNA
portion would provide high binding affinity and specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds between the
nucleobases, and orientation (Hyrup (1996) above). The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996)
above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For
example, a DNA chain can be synthesized on a solid support using
standard phosphoramidite coupling chemistry, and modified
nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can
be used between the PNA and the 5' end of DNA (Mag et al. (1989)
Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a
stepwise manner to produce a chimeric molecule with a 5' PNA
segment and a 3' DNA segment (Finn et al. (1996) above).
Alternatively, chimeric molecules can be synthesized with a 5' DNA
segment and a 3' PNA segment See, Petersen et al. (1975) Bioorg Med
Chem Lett 5: 1119-11124.
[0289] In other embodiments, the oligonucleotide may include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents facilitating transport across the
cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad.
Sci. USA. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad.
Sci. 84:648-652; PCT Publication No. WO88/09810) or the blood-brain
barrier (see, e.g., PCT Publication No. WO89/10134). In addition,
oligonucleotides can be modified with hybridization triggered
cleavage agents (See, e.g., Krol et al., 1988, BioTechniques
6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm.
Res. 5: 539-549). To this end, the oligonucleotide may be
conjugated to another molecule, e.g., a peptide, a hybridization
triggered cross-linking agent, a transport agent, a
hybridization-triggered cleavage agent, etc.
[0290] MTC Polypeptides
[0291] One aspect of the invention pertains to isolated MTC
polypeptides or proteins (these terms are used interchangeably
herein), and biologically active portions thereof, or derivatives,
fragments, analogs or homologs thereof. Also provided are
polypeptide fragments suitable for use as immunogens to raise
anti-MTC antibodies. In one embodiment, native MTC proteins can be
isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques.
In another embodiment, MTC proteins are produced by recombinant DNA
techniques. Alternative to recombinant expression, a MTC protein or
polypeptide can be synthesized chemically using standard peptide
synthesis techniques.
[0292] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the MTC protein is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of MTC protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. In one embodiment, the language
"substantially free of cellular material" includes preparations of
MTC protein having less than about 30% (by dry weight) of non-MTC
protein (also referred to herein as a "contaminating protein"),
more preferably less than about 20% of non-MTC protein, still more
preferably less than about 10% of non-MTC protein, and most
preferably less than about 5% non-MTC protein. When the MTC protein
or biologically active portion thereof is recombinantly produced,
it is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less
than about 10%, and most preferably less than about 5% of the
volume of the protein preparation.
[0293] The language "substantially free of chemical precursors or
other chemicals" includes preparations of MTC protein in which the
protein is separated from chemical precursors or other chemicals
that are involved in the synthesis of the protein. In one
embodiment, the language "substantially free of chemical precursors
or other chemicals" includes preparations of MTC protein having
less than about 30% (by dry weight) of chemical precursors or
non-MTC chemicals, more preferably less than about 20% chemical
precursors or non-MTC chemicals, still more preferably less than
about 10% chemical precursors or non-MTC chemicals, and most
preferably less than about 5% chemical precursors or non-MTC
chemicals.
[0294] Biologically active portions of a MTC protein include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequence of the MTC protein, e.g.,
the amino acid sequence encoded by a nucleic acid comprising MTC
1-20 that include fewer amino acids than the full length MTC
proteins, and exhibit at least one activity of a MTC protein.
Typically, biologically active portions comprise a domain or motif
with at least one activity of the MTC protein. A biologically
active portion of a MTC protein can be a polypeptide which is, for
example, 10, 25, 50, 100 or more amino acids in length.
[0295] A biologically active portion of a MTC protein of the
present invention may contain at least one of the above-identified
domains conserved between the MTC proteins. An alternative
biologically active portion of a MTC protein may contain at least
two of the above-identified domains. Another biologically active
portion of a MTC protein may contain at least three of the
above-identified domains. Yet another biologically active portion
of a MTC protein of the present invention may contain at least four
of the above-identified domains.
[0296] Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of a native MTC protein.
[0297] In some embodiments, the MTC protein is substantially
homologous to one of these MTC proteins and retains its the
functional activity, yet differs in amino acid sequence due to
natural allelic variation or mutagenesis, as described in detail
below.
[0298] In specific embodiments, the invention includes an isolated
polypeptide comprising an amino acid sequence that is 80% or more
identical to the sequence of a polypeptide whose expression is
modulated in a mammal to which PPAR.gamma. ligand is
administered.
[0299] Determining Homology Between Two or More Sequences
[0300] To determine the percent homology of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are homologous at that position (i.e., as used
herein amino acid or nucleic acid "homology" is equivalent to amino
acid or nucleic acid "identity").
[0301] The nucleic acid sequence homology may be determined as the
degree of identity between two sequences. The homology may be
determined using computer programs known in the art, such as GAP
software provided in the GCG program package. See Needleman and
Wunsch 1970 J Mol Biol 48: 443-453. Using GCG GAP software with the
following settings for nucleic acid sequence comparison: GAP
creation penalty of 5.0 and GAP extension penalty of 0.3, the
coding region of the analogous nucleic acid sequences referred to
above exhibits a degree of identity preferably of at least 70%,
75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part
of a DNA sequence comprising MTCS: 48-51.
[0302] The term "sequence identity" refers to the degree to which
two polynucleotide or polypeptide sequences are identical on a
residue-by-residue basis over a particular region of comparison.
The term "percentage of sequence identity" is calculated by
comparing two optimally aligned sequences over that region of
comparison, determining the number of positions at which the
identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case
of nucleic acids) occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the region of comparison (i.e., the
window size), and multiplying the result by 100 to yield the
percentage of sequence identity. The term "substantial identity" as
used herein denotes a characteristic of a polynucleotide sequence,
wherein the polynucleotide comprises a sequence that has at least
80 percent sequence identity, preferably at least 85 percent
identity and often 90 to 95 percent sequence identity, more usually
at least 99 percent sequence identity as compared to a reference
sequence over a comparison region.
[0303] Chimeric and Fusion Proteins
[0304] The invention also provides MTC chimeric or fusion proteins.
As used herein, an MTC "chimeric protein" or "fusion protein"
comprises an MTC polypeptide operatively linked to a non-MTC
polypeptide. A "MTC polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to MTC, whereas a "non-MTC
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein that is not substantially homologous to
the MTC protein, e.g., a protein that is different from the MTC
protein and that is derived from the same or a different organism.
Within an MTC fusion protein the MTC polypeptide can correspond to
all or a portion of an MTC protein. In one embodiment, an MTC
fusion protein comprises at least one biologically active portion
of an MTC protein. In another embodiment, an MTC fusion protein
comprises at least two biologically active portions of an MTC
protein. In yet another embodiment, an MTC fusion protein comprises
at least three biologically active portions of an MTC protein.
Within the fusion protein, the term "operatively linked" is
intended to indicate that the MTC polypeptide and the non-MTC
polypeptide are fused in-frame to each other. The non-MTC
polypeptide can be fused to the N-terminus or C-terminus of the MTC
polypeptide.
[0305] For example, in one embodiment an MTC fusion protein
comprises an MTC domain operably linked to the extracellular domain
of a second protein. Such fusion proteins can be further utilized
in screening assays for compounds which modulate MTC activity (such
assays are described in detail below).
[0306] In yet another embodiment, the fusion protein is a GST-MTC
fusion protein in which the MTC sequences are fused to the
C-terminus of the GST (i.e., glutathione S-transferase) sequences.
Such fusion proteins can facilitate the purification of recombinant
MTC.
[0307] In another embodiment, the fusion protein is an MTC protein
containing a heterologous signal sequence at its N-terminus. For
example, a native MTC signal sequence can be removed and replaced
with a signal sequence from another protein. In certain host cells
(e.g., mammalian host cells), expression and/or secretion of MTC
can be increased through use of a heterologous signal sequence.
[0308] In yet another embodiment, the fusion protein is an
MTC-immunoglobulin fusion protein in which the MTC sequences
comprising one or more domains are fused to sequences derived from
a member of the immunoglobulin protein family. The
MTC-immunoglobulin fusion proteins of the invention can be
incorporated into pharmaceutical compositions and administered to a
subject to inhibit an interaction between a MTC ligand and a MTC
protein on the surface of a cell, to thereby suppress MTC-mediated
signal transduction in vivo. The MTC-immunoglobulin fusion proteins
can be used to affect the bioavailability of an MTC cognate ligand.
Inhibition of the MTC ligand/MTC interaction may be useful
therapeutically for both the treatments of proliferative and
differentiative disorders, as well as modulating (e.g. promoting or
inhibiting) cell survival. Moreover, the MTC-immunoglobulin fusion
proteins of the invention can be used as immunogens to produce
anti-MTC antibodies in a subject, to purify MTC ligands, and in
screening assays to identify molecules that inhibit the interaction
of MTC with a MTC ligand.
[0309] An MTC chimeric or fusion protein of the invention can be
produced by standard recombinant DNA techniques. For example, DNA
fragments coding for the different polypeptide sequences are
ligated together in-frame in accordance with conventional
techniques, e.g. by employing blunt-ended or stagger-ended termini
for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers that give rise to
complementary overhangs between two consecutive gene fragments that
can subsequently be annealed and reamplified to generate a chimeric
gene sequence (see, for example, Ausubel et al. (eds.) CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
Moreover, many expression vectors are commercially available that
already encode a fusion moiety (e.g., a GST polypeptide). An
MTC-encoding nucleic acid can be cloned into such an expression
vector such that the fusion moiety is linked in-frame to the MTC
protein.
[0310] MTC Agonists and Antagonists
[0311] The present invention also pertains to variants of the MTC
proteins, e.g., MTC1-51, that function as either MTC agonists
(mimetics) or as MTC antagonists. Variants of the MTC protein can
be generated by mutagenesis, e.g., discrete point mutation or
truncation of the MTC protein. An agonist of the MTC protein can
retain substantially the same, or a subset of, the biological
activities of the naturally occurring form of the MTC protein. An
antagonist of the MTC protein can inhibit one or more of the
activities of the naturally occurring form of the MTC protein by,
for example, competitively binding to a downstream or upstream
member of a cellular signaling cascade which includes the MTC
protein. Thus, specific biological effects can be elicited by
treatment with a variant of limited function. In one embodiment,
treatment of a subject with a variant having a subset of the
biological activities of the naturally occurring form of the
protein has fewer side effects in a subject relative to treatment
with the naturally occurring form of the MTC proteins.
[0312] Variants of the MTC protein that function as either MTC
agonists (mimetics) or as MTC antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the MTC protein for MTC protein agonist or antagonist
activity. In one embodiment, a variegated library of MTC variants
is generated by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. A variegated library
of MTC variants can be produced by, for example, enzymatically
ligating a mixture of synthetic oligonucleotides into gene
sequences such that a degenerate set of potential MTC sequences is
expressible as individual polypeptides, or alternatively, as a set
of larger fusion proteins (e.g., for phage display) containing the
set of MTC sequences therein. There are a variety of methods which
can be used to produce libraries of potential MTC variants from a
degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an
appropriate expression vector. Use of a degenerate set of genes
allows for the provision, in one mixture, of all of the sequences
encoding the desired set of potential MTC sequences. Methods for
synthesizing degenerate oligonucleotides are known in the art (see,
e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu
Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et
al. (1983) Nucl Acid Res 11:477.
[0313] In one embodiment, proteins having enzymatic activities are
used for small molecule screening and small molecule drug therapy.
These proteins include, e.g., the DUSP6 dual specificity MAP kinase
phosphatase, ras GTPase-activating-like protein (IQGAP1),
Ca2-activated neutral protease large subunit calpain, Cathepsin E,
5-lipoxygenase, Spermidine/spermine N1-acetyltransferase (SSAT),
and STE20-like protein kinase 3 (STK3) protein phosphatase-1 gamma
1.
[0314] Polypeptide Libraries
[0315] In addition, libraries of fragments of the MTC protein
coding sequence can be used to generate a variegated population of
MTC fragments for screening and subsequent selection of variants of
an MTC protein. In one embodiment, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of a MTC coding sequence with a nuclease under conditions
wherein nicking occurs only about once per molecule, denaturing the
double stranded DNA, renaturing the DNA to form double stranded DNA
that can include sense/antisense pairs from different nicked
products, removing single stranded portions from reformed duplexes
by treatment with S I nuclease, and ligating the resulting fragment
library into an expression vector. By this method, an expression
library can be derived which encodes N-terminal and internal
fragments of various sizes of the MTC protein.
[0316] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of MTC proteins. The most widely used techniques, which
are amenable to high throughput analysis, for screening large gene
libraries typically include cloning the gene library into
replicable expression vectors, transforming appropriate cells with
the resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene whose product
was detected. Recursive ensemble mutagenesis (REM), a new technique
that enhances the frequency of functional mutants in the libraries,
can be used in combination with the screening assays to identify
MTC variants (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave
et al. (1993) Protein Engineering 6:327-331).
[0317] Anti-MTC Antibodies
[0318] An isolated MTC protein, or a portion or fragment thereof,
can be used as an immunogen to generate antibodies that bind MTC
using standard techniques for polyclonal and monoclonal antibody
preparation. The full-length MTC protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of MTC for use as immunogens. The antigenic peptide of MTC
comprises at least 8 amino acid residues of the amino acid sequence
encoded by a nucleic acid comprising the nucleic acid sequence
shown in MTC:1-51, e.g., MTC48-51 and encompasses an epitope of MTC
such that an antibody raised against the peptide forms a specific
immune complex with MTC. Preferably, the antigenic peptide
comprises at least 10 amino acid residues, more preferably at least
15 amino acid residues, even more preferably at least 20 amino acid
residues, and most preferably at least 30 amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions
of MTC that are located on the surface of the protein, e.g.,
hydrophilic regions. As a means for targeting antibody production,
hydropathy plots showing regions of hydrophilicity and
hydrophobicity may be generated by any method well known in the
art, including, for example, the Kyte Doolittle or the Hopp Woods
methods, either with or without Fourier transformation. See, e.g.,
Hopp and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte
and Doolittle 1982, J. Mol. Biol. 157: 105-142, each incorporated
herein by reference in their entirety.
[0319] MTC polypeptides or derivatives, fragments, analogs or
homologs thereof, may be utilized as immunogens in the generation
of antibodies that immunospecifically-bind these protein
components. The term "antibody" as used herein refers to
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site that specifically binds (immunoreacts with) an
antigen. Such antibodies include, but are not limited to,
polyclonal, monoclonal, chimeric, single chain, F.sub.ab and
F.sub.(ab')2 fragments, and an F.sub.ab expression library. Various
procedures known within the art may be used for the production of
polyclonal or monoclonal antibodies to an MTC protein sequence, or
derivatives, fragments, analogs or homologs thereof. Some of these
proteins are discussed below.
[0320] For the production of polyclonal antibodies, various
suitable host animals (e.g., rabbit, goat, mouse or other mammal)
may be immunized by injection with the native protein, or a
synthetic variant thereof, or a derivative of the foregoing. An
appropriate immunogenic preparation can contain, for example,
recombinantly expressed MTC protein or a chemically synthesized MTC
polypeptide. The preparation can further include an adjuvant.
Various adjuvants used to increase the immunological response
include, but are not limited to, Freund's (complete and
incomplete), mineral gels (e.g., aluminum hydroxide), surface
active substances (e.g., lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, dinitrophenol, etc.), human
adjuvants such as Bacille Calmette-Guerin and Corynebacterium
parvum, or similar immunostimulatory agents. If desired, the
antibody molecules directed against MTC can be isolated from the
mammal (e g., from the blood) and further purified by well known
techniques, such as protein A chromatography to obtain the IgG
fraction.
[0321] The term "monoclonal antibody" or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one species of an antigen binding site
capable of immunoreacting with a particular epitope of MTC. A
monoclonal antibody composition thus typically displays a single
binding affinity for a particular MTC protein with which it
immunoreacts. For preparation of monoclonal antibodies directed
towards a particular MTC protein, or derivatives, fragments,
analogs or homologs thereof, any technique that provides for the
production of antibody molecules by continuous cell line culture
may be utilized. Such techniques include, but are not limited to,
the hybridoma technique (see Kohler & Milstein, 1975 Nature
256: 495-497); the trioma technique; the human B-cell hybridoma
technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the
EBV hybridoma technique to produce human monoclonal antibodies (see
Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY,
Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be
utilized in the practice of the present invention and may be
produced by using human hybridomas (see Cote, et al., 1983. Proc
Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells
with Epstein Barr Virus in vitro (see Cole, et al., 1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.
77-96).
[0322] According to the invention, techniques can be adapted for
the production of single-chain antibodies specific to a MTC protein
(see e.g., U.S. Pat. No. 4,946,778). In addition, methods can be
adapted for the construction of F.sub.ab expression libraries (see
e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and
effective identification of monoclonal F.sub.ab fragments with the
desired specificity for a MTC protein or derivatives, fragments,
analogs or homologs thereof. Non-human antibodies can be
"humanized" by techniques well known in the art. See e.g., U.S.
Pat. No. 5,225,539. Antibody fragments that contain the idiotypes
to a MTC protein may be produced by techniques known in the art
including, but not limited to: (i) an F.sub.(ab')2 fragment
produced by pepsin digestion of an antibody molecule; (ii) an
F.sub.ab fragment generated by reducing the disulfide bridges of an
F.sub.(ab')2 fragment; (iii) an F.sub.ab fragment generated by the
treatment of the antibody molecule with papain and a reducing agent
and (iv) F.sub.v fragments.
[0323] Additionally, recombinant anti-MTC antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in PCT International Application No.
PCT/US86/02269; European Patent Application No. 184,187; European
Patent Application No. 171,496; European Patent Application No.
173,494; PCT International Publication No. WO 86/01533; U.S. Pat.
No. 4,816,567; European Patent Application No. 125,023; Better et
al.(1988) Science 240:1041-1043; Liu et al. (1987) PNAS
84:3439-3443; Liu et al. (1987)J Immunol. 139:3521-3526; Sun et al.
(1987) PNAS 84:214-218; Nishimura et al. (1987) Cancer Res
47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al.
(1988) J Nail Cancer Inst. 80:1553-1559); Morrison (1985) Science
229:1202-1207; Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J Immunol
141:4053-4060.
[0324] In one embodiment, methods for the screening of antibodies
that possess the desired specificity include, but are not limited
to, enzyme-linked immunosorbent assay (ELISA) and other
immunologically-mediated techniques known within the art. In a
specific embodiment, selection of antibodies that are specific to a
particular domain of a MTC protein is facilitated by generation of
hybridomas that bind to the fragment of a MTC protein possessing
such a domain. Antibodies that are specific for one or more domains
within a MTC protein, e.g. domains spanning the above-identified
conserved regions of MTC family proteins, or derivatives,
fragments, analogs or homologs thereof, are also provided
herein.
[0325] Anti-MTC antibodies may be used in methods known within the
art relating to the localization and/or quantitation of a MTC
protein (e.g., for use in measuring levels of the MTC protein
within appropriate physiological samples, for use in diagnostic
methods, for use in imaging the protein, and the like). In a given
embodiment, antibodies for MTC proteins, or derivatives, fragments,
analogs or homologs thereof, that contain the antibody derived
binding domain, are utilized as pharmacologically-active compounds
[hereinafter "Therapeutics"].
[0326] An anti-MTC antibody (e.g., monoclonal antibody) can be used
to isolate MTC by standard techniques, such as affinity
chromatography or immunoprecipitation. An anti-MTC antibody can
facilitate the purification of natural MTC from cells and of
recombinantly produced MTC expressed in host cells. Moreover, an
anti-MTC antibody can be used to detect MTC protein (e.g., in a
cellular lysate or cell supernatant) in order to evaluate the
abundance and pattern of expression of the MTC protein. Anti-MTC
antibodies can be used diagnostically to monitor protein levels in
tissue as part of a clinical testing procedure, e.g., to, for
example, determine the efficacy of a given treatment regimen.
Detection can be facilitated by coupling (i.e., physically linking)
the antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, -galactosidase,
oracetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0327] MTC Recombinant Expression Vectors and Host Cells
[0328] Another aspect of the invention pertains to vectors,
preferably expression vectors, containing a nucleic acid encoding
MTC protein, or derivatives, fragments, analogs or homologs
thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable of transporting another nucleic acid to which it
has been linked. One type of vector is a "plasmid", which refers to
a linear or circular double stranded DNA loop into which additional
DNA segments can be ligated. Another type of vector is a viral
vector, wherein additional DNA segments can be ligated into the
viral genome. Certain vectors are capable of autonomous replication
in a host cell into which they are introduced (e.g., bacterial
vectors having a bacterial origin of replication and episomal
mammalian vectors). Other vectors (e.g., non-episomal mammalian
vectors) are integrated into the genome of a host cell upon
introduction into the host cell, and thereby are replicated along
with the host genome. Moreover, certain vectors are capable of
directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "expression
vectors". In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" can be used interchangeably
as the plasmid is the most commonly used form of vector. However,
the invention is intended to include such other forms of expression
vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses and adeno-associated viruses), which
serve equivalent functions.
[0329] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell, which means that the
recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for
expression, that is operatively linked to the nucleic acid sequence
to be expressed. Within a recombinant expression vector, "operably
linked" is intended to mean that the nucleotide sequence of
interest is linked to the regulatory sequence(s) in a manner that
allows for expression of the nucleotide sequence (e g., in an in
vitro transcription/translation system or in a host cell when the
vector is introduced into the host cell). The term "regulatory
sequence" is intended to includes promoters, enhancers and other
expression control elements (e.g., polyadenylation signals). Such
regulatory sequences are described, for example, in Goeddel; GENE
EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,
San Diego, Calif. (1990). Regulatory sequences include those that
direct constitutive expression of a nucleotide sequence in many
types of host cell and those that direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by
those skilled in the art that the design of the expression vector
can depend on such factors as the choice of the host cell to be
transformed, the level of expression of protein desired, etc. The
expression vectors of the invention can be introduced into host
cells to thereby produce proteins or peptides, including fusion
proteins or peptides, encoded by nucleic acids as described herein
(e.g., MTC proteins, mutant forms of MTC, fusion proteins,
etc.).
[0330] Recombinant expression vectors of the invention can be
designed for expression of MTC in prokaryotic or eukaryotic cells.
For example, MTC can be expressed in bacterial cells such as E.
coli, insect cells (using baculovirus expression vectors) yeast
cells or mammalian cells. Suitable host cells are discussed further
in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,
Academic Press, San Diego, Calif. (1990). Alternatively, the
recombinant expression vector can be transcribed and translated in
vitro, for example using T7 promoter regulatory sequences and T7
polymerase.
[0331] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: (1) to
increase expression of recombinant protein; (2) to increase the
solubility of the recombinant protein; and (3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) that fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0332] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and
pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990)
60-89).
[0333] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant
protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128.
Another strategy is to alter the nucleic acid sequence of the
nucleic acid to be inserted into an expression vector so that the
individual codons for each amino acid are those preferentially
utilized in E. coli (Wada et al., (1992) Nucleic Acids Res.
20:2111-2118). Such alteration of nucleic acid sequences of the
invention can be carried out by standard DNA synthesis
techniques.
[0334] In another embodiment, the MTC expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J
6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San
Diego, Calif.).
[0335] Alternatively, MTC can be expressed in insect cells using
baculovirus expression vectors. Baculovirus vectors available for
expression of proteins in cultured insect cells (e.g., SF9 cells)
include the pAc series (Smith et al. (1983) Mol Cell Biol
3:2156-2165) and the pVL series (Lucklow and Summers (1989)
Virology 170:31-39).
[0336] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO
J. 6: 187-195). When used in mammalian cells, the expression
vector's control functions are often provided by viral regulatory
elements. For example, commonly used promoters are derived from
polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For
other suitable expression systems for both prokaryotic and
eukaryotic cells. See, e.g., Chapters 16 and 17 of Sambrook et al.,
MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0337] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv Immunol 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) PNAS
86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)
Science 230:912-916), and mammary gland-specific promoters (e.g.,
milk whey promoter; U.S. Pat. No. 4,873,316 and European
Application Publication No. 264,166). Developmentally-regulated
promoters are also encompassed, e.g., the murine hox promoters
(Kessel and Gruss (1990) Science 249:374-379) and the
.alpha.-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev
3:537-546).
[0338] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. That is, the DNA
molecule is operatively linked to a regulatory sequence in a manner
that allows for expression (by transcription of the DNA molecule)
of an RNA molecule that is antisense to MTC mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the
antisense orientation can be chosen that direct the continuous
expression of the antisense RNA molecule in a variety of cell
types, for instance viral promoters and/or enhancers, or regulatory
sequences can be chosen that direct constitutive, tissue specific
or cell type specific expression of antisense RNA. The antisense
expression vector can be in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense nucleic acids are
produced under the control of a high efficiency regulatory region,
the activity of which can be determined by the cell type into which
the vector is introduced. For a discussion of the regulation of
gene expression using antisense genes see Weintraub et al.,
"Antisense RNA as a molecular tool for genetic analysis,"
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0339] Another aspect of the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to the particular subject cell but also to the progeny or
potential progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0340] A host cell can be any prokaryotic or eukaryotic cell. For
example, MTC protein can be expressed in bacterial cells such as E.
coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[0341] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A
LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0342] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Various selectable markers
include those that confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding MTC or can be introduced on a separate vector. Cells
stably transfected with the introduced nucleic acid can be
identified by drug selection (e.g., cells that have incorporated
the selectable marker gene will survive, while the other cells
die).
[0343] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce (i.e.,
express) an MTC protein. Accordingly, the invention further
provides methods for producing MTC protein using the host cells of
the invention. In one embodiment, the method comprises culturing
the host cell of invention (into which a recombinant expression
vector encoding MTC has been introduced) in a suitable medium such
that MTC protein is produced. In another embodiment, the method
further comprises isolating MTC from the medium or the host
cell.
[0344] Kits, Arrays, and Pluralities
[0345] The invention provides for a kit comprising one or more
reagents for detecting two or more nucleic acid sequences selected
from the group consisting of MTCs: 1-51. In various embodiments,
the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,
40, 45, 50, or 51 or more of the sequences represented by MTCs:
1-51 are measured. The kit can identify the enumerated nucleic
acids by, e.g., having homologous nucleic acid sequences, such as
oligonucleotide sequences, complementary to a portion of the
recited nucleic acids, or antibodies to proteins encoded by the
genes.
[0346] The invention also includes an array of probe nucleic acids.
These probe nucleic acid sequences detect two or more nucleic acid
sequences selected from the group consisting of MTCs: 1-51. In
various embodiments, the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25, 30, 35, 40, 45, 50, or 51 or more of the sequences
represented by MTCs: 1-51 are identified.
[0347] The probe nucleic acids in the array can detect the
enumerated nucleic acids by, e.g., having homologous nucleic acid
sequences, such as oligonucleotide sequences, complementary to a
portion of the recited nucleic acids. The substrate array can be
on, e.g., a solid substrate, e.g., a "chip", as described in U.S.
Pat. No. 5,744,305.
[0348] The invention also includes an isolated plurality of nucleic
acid sequences. The plurality typically includes two or more of the
nucleic acid sequences represented by MTCs: 1-51. In various
embodiments, the plurality includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 50, or 51 or more of the sequences
represented by MTCs: 1-51.
[0349] An "isolated" nucleic acid molecule is one that is separated
from other nucleic acid molecules, which are present in the natural
source of the nucleic acid. Examples of isolated nucleic acid
molecules include, but are not limited to, recombinant DNA
molecules contained in a vector, recombinant DNA molecules
maintained in a heterologous host cell, partially or substantially
purified nucleic acid molecules, and synthetic DNA or RNA
molecules. Preferably, and "isolated" nucleic acid is free of
sequences which naturally flank the nucleic acid (i.e., sequences
located at the 5' and 3' ends of the nucleic acid) in the genomic
DNA of the organism from which the nucleic acid is derived. In
various embodiments, the isolated nucleic acid molecule can contain
less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb,
or 0.1 kb of nucleotide sequences which naturally flank the nucleic
acid molecule in genomic DNA of the cell from which the nucleic
acid is derived. Moreover, an "isolated" nucleic acid molecule,
such as a cDNA molecule, can be substantially free of other
cellular material or cultural medium when produced by recombinant
techniques, or of chemical precursors or other chemicals when
chemically synthesized.
[0350] Compositions Including Novel Nucleic Acids Differentially
Expressed in Metastatic and Nonmetastatic Cancer
[0351] The invention also provides compositions of novel nucleic
acid sequences that are differentially expressed in metastatic and
nonmetastic thyroid cancer. Thus, the invention includes isolated
nucleic acid molecules comprising a nucleotide sequence selected
from the group consisting of MTCs:48-51, or fragments thereof.
[0352] Single Nucleotide Polymorphisms in MTCX Genes
[0353] Also provided in the invention are nucleic acids
corresponding to polymorphisms in MTCX genes. MTCX Single
Nucleotide Polymorphisms ("SNPs") according to the invention
include those shown in Table 2.
14TABLE 2 SNPs SNP's found in CSNP's coding region that cSNP's
found in Confirmed found in 5`/3` do not change coding region that
Gene MTCX UTR's amino acids change amino acids KIAA1131 MTC32
G4944T; T4947C; A5060G; T5081C Periplakin MTC20 T5738C; C6107A
A3521G; C4118T G1777A (Arg-Gln); C4728G (Gln-Glu) Mitochondrial
MTC33 A264G; G283A A85G (Ile-Val); proteolipid T158C (Phe-Ser)
Phosphatase-1 MTC18 A1680G; A1951G gamma 1 Prostaglandin MTC38
A3163G; A3247G A1269G (Thr-Ala) transporter hPGT E-cadherin MTC4
C2076T Kinectin MTC37 T42G Staf50 MTC41 C2290T; T2458C Proteasome
MTC21 G534C (Gln-His) subunit HC5 Proteasome C331A (Pro-His);
activator hPA28 G374T (Val-Phe) subunit beta STE20-like protein
MTC35 C1509T; A2216G kinase 3 DUSP6 dual MTC23 G666A (Trp-STOP);
specificity MAP 1691G (Leu-Val); kinase phosphatase 781G (Ser-Ala)
Peflin MTC36 T905C P8-Candidate of MTC17 C110T (Thr-Ile) metastasis
1 Lipocortin MTC14 A1267T G179A; A401G C300T (Gin-STOP) MTC15
5-lipoxygenase MTC25 C2328T Type IV MTC8 A2196C C1102T; A1333G;
collagenase C1759T LFA-1/CR3/P150, 95 MTC6 G2505A; G2553C A891G
A638G (Asn-Ser) beta-subunit Integrin alpha-3 MTC30 C3773T G2228A
(Ala-Thr)
[0354] Table 2, columns 1 and 2 provide the gene name and
corresponding MTC reference number, respectively. Columns 3-5
provide the nucleotide position in which the polymorphism occurs,
with the nucleotide present in the polymorphic allele following the
number, and the nucleotide it replaces preceding the number. Column
3 lists polymorphisms occurring in the 5' or 3' non-translated
regions of genes, while column 4 lists polymorphisms which occur in
coding regions but which do not result in an altered encoded
polypeptide. Column 5 lists polymorphisms which do result in a
change of the encoded amino acid sequence.
[0355] In general, polymorphisms according to the invention can be
identified using any nucleic acid probe which specifically
identifies the polymorphic allele.
[0356] Examples of nucleic acid probes which recognize SNPs
according to the invention are provided below. Listed are the gene
from which the SNP is identified, with the polymorphic site
indicated with an ">". The frequency with which the polymorphic
nucleotide appeared in a sampled population is given in
parentheseses.
15 KIAA1131 (gbeh.sub.--aa570716) CATAAGCCCTTGTAAAGTAAC
A>G(2/214) CAGTGTTCTGTGCTATATACTTGCT (SEQ ID NO:15)
CAGTGTTCTGTGCTATATAC T>C(3/214) TGCTGGCTGGGTAGT (SEQ ID NO:16)
GTGTCATTAGCATGGTG G>A(31/190) ATCATATACTTCTCTGCACACAAACAC (SEQ
ID NO:17) AGCATGGTGGAT C>A(2/190) ATATACTTCTCTGCACACAAACA
Periplakin (af013717) TTCATGCTATAAATAAA T<G(6/13)
TTCCCTATTAGTTCCC (SEQ ID NO:19) GGTTTTAAGCCAGAA
G<A(4/13)TCTGGAGAGATGTCATGCCAG (SEQ ID NO:20) CTCCAGCTGCAGGTTTT
G>C(4/9) CCTCTCCAGCTGTAATTTGT (SEQ ID NO:21) GCTCTCGGCAAAGGC
A>G(3/6) CTCGCCTCGGCCC (SEQ ID NO:22) ACGATCTCCCGCACCTTCTCCTG
C>T(3/8) ACCACCACTTTGGCGTTCTCCT (SEQ ID NO:23)
TGCTCGTATTTCCGGTTGGTGTCCTCCACC C>T(3/6) (SEQ ID NO:24)
GGGTCCTCAGCAGGGGTGTGGTG (SEQ ID NO:25) Mitochondrial proteolipid
(af054175) GGCTTGCTGAAATTTA C>T(4/190) AGGCAGACTGACGTTTT (SEQ ID
NO:26) ACTGACGTTTTC T>C(3/190) TTCACATGTACTCC (SEQ ID NO:27)
ATTTTATAAACGATG A>G(3/170) AGCCCATCAGCCCCATT (SEQ ID NO:28)
TTCATGGGGATCCATA T>C(8/44) GTTTTTAATAATACTTT (SEQ ID NO:29)
Phosphatase-1 gamma 1 (107395) TTTTAACTTATAAGCC T>C(2/130)
CAACTTCACCGCAGAATAAAGAATGTAG (SEQ ID NO:30) AGAAGGCAGCATGTGTA
T>C(13/45) ACAACCATACC (SEQ ID NO:31) Prostaglandin transporter
hPGT (u70867) GCGCTTTGTTTTCTCTCTACAA G>A(5/11) CCATTCCCCGC (SEQ
ID NO:32) TGCCTCCAGAGAGGTGG G>A(3/7) TGCCTGGGTTGAGAGACACAGCT
(SEQ ID NO:33) GAGGTCATTCATCAACAAATAT A>G(4/8)
TTTATTGGAGACCGACTTT (SEQ ID NO:34) E-cadherin (z18923)
GTGTGTGACTGTGAAGGGGCCGC C>T(2/12) GGCGTCTGTAGGAA (SEQ ID NO:35)
Kinectin (z22551) CAATGTGATCCTATAA A>C(3/8) ACCCTGTGCGGCCGGGAAAG
(SEQ ID NO:36) Staf50 (x82200) TAATTCCTTTTCTTTTCTTC T>C(3/12)
TTATTTCCTCTGCCCCTT (SEQ ID NO:37) AAAATTAAAGCAAGAAGTCCA
T>C(3/14) AGTAATTTATTTGC (SEQ ID NO:38) Proteasome subunit HC5
(d00761) CAAGTGCCATGCTACA G>C(5/240) CCCCTGCTTGACAACCAGGTT (SEQ
ID NO:39) Proteasome activator hPA28 subunit beta (d45248)
TCCAGACTTCTGGCTTAA C>A(3/95) CAGGGCAAGCAGGGACAGGACTTT (SEQ ID
NO:40) AAATCCACACTTA G>T(2/55) GGACTTCTTTCTTCTCC (SEQ ID NO:41)
STE20-like protein kinase 3 (AF083420) GAAAAAGGAAATCAACCTC
T>C(11/62) AGGTGTACCAAAAGGGGC (SEQ ID NO:42)
GGAGGAGGATGAAGAAGGAAAAAA G>A(3/9) GAAAAACAAAACCCCAAATGCC (SEQ ID
NO:43) DUSP6 dual specificity MAP kinase phosphatase (AB013382)
CTCGCAATGCAGGG A>C(4/32) GAACTCGGCTTGGAA (SEQ ID NO:44)
CTTGAGCAGCAGCCCGAGCA A>C (20/38) CGACTCGCCGCCCGTATTCTCGT (SEQ ID
NO:45) CGCCGCCCGTATTCTCGTT C>T(2/38) CAGTCGCTGCTGCTCTCGTCGT (SEQ
ID NO:46) Peflin (gbeh.sub.--h72140) CTCTAAGAAGCCAGGAA A>G(2/32)
GGTCCCTGGTGCACTCCACTCT (SEQ ID NO:47) P8-Candidate of metastasis 1
(AF069073) GGTTGCTGGTGGGAAG G>A(2/80) TGGCCATCGTGCCTGGC (SEQ ID
NO:48) Lipocortin (x05908) AAAGGTGGTCCCGGATCAGC G>A(2/60)
GTGAGCCCCTATCCTACC (SEQ ID NO:49) TTCTAACTAAGCGAAACAATGCA
C>T(2/90) AGCGTCAACAGATCAAAGCAGCATAT (SEQ ID NO:50)
TTGAGGAGGTTGTTTT A>G(11/60) GCTCTGCTAAAAACTCCAGCG (SEQ ID NO:51)
AAATCATTTTTATATTATA A>T(2/60) CTCTGTATAATAGAGATAAGT (SEQ ID
NO:52) 5-lipoxygenase (j03571) AGACATCTATCAGGGTC
G>A(3/34)TGATTTGCTGTTGCTGCTT (SEQ ID NO:53) TGAAAATATCAAAG
T>G(3/9) ATCTCTTTAGGGG (SEQ ID NO:54) CTGCGATGAGCTTGGG
A>G(9/18) AAGCCAGGATCCATTTTCTT (SEQ ID NO:55) AGTGACAGGGCCCAG
T>C(21/63) GTGGGGGTGGGGCCGGTGCCAA (SEQ ID NO:56) GCCCCACTTGCGGTC
G>A(8/80) TCATCGTAGTTGGCTGTGGT (SEQ ID NO:57) Cell surface
adhesion glycoproteins LFA-1/CR3/P150,95 beta-subunit (m15395)
GGAGCTGTCCCCC C>G(6/18) GACGAGCCCCCAG (SEQ ID NO:58) CATTTTGAGGG
C>T(4/28) GGAAAATAACTG (SEQ ID NO:59) TGGCGCCCAGCTT C>T(4/12)
CCGTCGCCCGCGAA (SEQ ID NO:60) CCTTGTTGGGGCATGGG T>C(2/16)
TTCGCAGCTTATC (SEQ ID NO:61) Integrin alpha-3 (m59911)
GCCTTTTCCTCCC G>A(3/13) GCTCTGGTGGGAGG (SEQ ID NO:62)
ATGACCTCAAAGG C>T(3/33) GATGAGCAGCT (SEQ ID NO:63)
[0357] The invention also includes nucleic acid sequences that
include one or more polymorphic MTC sequences. Also included are
methods of identifying a base occupying a polymorphic in an MTC
sequence, as well as methods of identifying an individualized
therapeutic agent for treating pathologies (e.g., carcinomas,
including metastatic carcinomas such as metastatic thyroid
carcinomas) based on MTC sequence polymorphisms.
[0358] The nucleotide polymorphism can be a single nucleotide
polymorphism (SNP). A SNP occurs at a polymorphic site occupied by
a single nucleotide, which is the site of variation between allelic
sequences. The site is usually preceded by and followed by highly
conserved sequences of the allele (e.g., sequences that vary in
less than {fraction (1/100)} or {fraction (1/1000)} members of the
populations). A single nucleotide polymorphism usually arises due
to substitution of one nucleotide for another at the polymorphic
site. A transition is the replacement of one purine by another
purine or one pyrimidine by another pyrimidine. A transversion is
the replacement of a purine by a pyrimidine or vice versa. Single
nucleotide polymorphisms can also arise from a deletion of a
nucleotide or an insertion of a nucleotide relative to a reference
allele.
[0359] Polymorphic sequences according to the present invention can
include those shown in Table 2. Table 2 describes nine MTC
sequences for which polymorphisms have been identified. The first
column of the table lists the names assigned to the sequences in
which the polymorphisms occur. The second and third columns list
the rat and human GenBank Accession numbers for the respective
sequences. The forth column lists the position in the sequence in
which the polymorphic site has been found. The fifth column lists
the base occupying the polymorphic site in the sequence in the
database, i.e., the wildtype. The sixth column lists the
alternative base at the polymorphic site. The seventh column lists
any amino acid change that occurs due to the polymorphism.
[0360] The polymorphic sequence can include one or more of the
following sequences: (1) a sequence having the nucleotide denoted
in Table 2, column 5 at the polymorphic site in the polymorphic
sequence, and (2) a sequence having a nucleotide other than the
nucleotide denoted in Table 2, column 5. An example of the latter
sequence is a polymorphic sequence having the nucleotide denoted in
Table 2, column 6 at the polymorphic site in the polymorphic
sequence.
[0361] For example, a polymorphism according to the invention
includes a sequence polymorphism in the ATP citrate lyase gene
having the nucleotide sequence of GenBank Accession No. x64330, in
which the cytosine at nucleotide 609 is replaced by adenosine. In
some embodiments the polymorphic sequence includes a nucleotide
sequence of ATP citrate gene having the GenBank Accession No.
x64330, wherein the nucleotide at 609 is any nucleotide other that
cytosine.
[0362] In some embodiments, the polymorphic sequence includes the
full length of any one of the nine genes in Table2. In other
embodiments, the polymorphic sequence includes a polynucleotide
that is between 10 and 100 nucleotides, 10 and 75 nucleotides, 10
and 50 nucleotides, or 10 and 25 nucleotides in length.
[0363] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims. The following examples illustrate the identification
and characterization of genes differentially expressed in thyroid
follicular adenomas and metastatic papillary carcinomas.
EXAMPLE 1
RNA Preparation
[0364] Following treatment, total cellular RNA was isolated with
Trizol (GIBCO-BRL, Baltimore Md.), using one-tenth volume of
bromochloropropane (Molecular Research Corp., Cincinnati Ohio) for
phase separation. Contaminating DNA was removed by treatment with
DNAse I (Promega, Milwaukee Wis.) in the presence of 0.01M DTT
(GIBCO-BRL, Baltimore Md.) and 1 unit/.mu.l Rnasin (Promega,
Milwaukee Wis.). Following phenol/chloroform extraction, RNA
quality was evaluated by spectrophotomety and formaldehyde agarose
gel electrophoresis, and RNA yield was estimated by fluorometry
with OliGreen (Molecular Probes, Eugene Oreg.). Poly(A)+ RNA was
prepared from 100 .mu.g total RNA using oligo(dT) paramagnetic
beads (PerSeptive Biosynthesis, Boston Mass.), and quantitated with
fluorometry.
EXAMPLE 2
cDNA Synthesis
[0365] First strand cDNA was prepared from 1.0 .mu.g of poly(A)+
RNA with 200 pmoles oligo(dT).sub.25V (V=A, C or G) (PerSeptive
Biosynthesis, Boston Mass.) using 40 units of Superscript II
(GIBCO-BRL, Baltimore Md.). Second strand synthesis was performed
at 16.degree. C. for 2 h following the addition of 10 units of E.
coli DNA ligase (GIBCO-BRL, Baltimore Md.), 40 units of E. coli DNA
polymerase (GIBCO-BRL, Baltimore Md.), and 3.5 units of E. coli
RNase H (GIBCO-BRL, Baltimore Md.). 1 .mu.l of T4 DNA polymerase
(GIBCO-BRL, Baltimore Md.) was then added, and incubation at
16.degree. C. was continued for 5 min. The reaction was then
treated with 5 units of arctic shrimp alkaline phosphatase (USB,
Chicago Ill.) at 37.degree. C. for 30 min., and cDNA purified by
phenol/chloroform extraction. The yield of cDNA was estimated using
fluorometry.
EXAMPLE 3
GeneCalling.TM. Chemistry
[0366] For all samples, triplicate GeneCalling chemistry reactions
were executed in parallel for each of 96 subsequence pairs.
Restriction endonuclease digestion is performed in a reaction mix
containing 2.6 .mu.l H.sub.2O, 2 .mu.l 5M betaine (Sigma, St. Louis
Mo.), 1 .mu.l 10.times. restriction endonuclease buffer, 0.8 .mu.l
10 mM ATP (Pharmacia, Newark N.J.), 1 .mu.l 25% PEG (Fluka, St.
Louis Mo.), 0.2 .mu..mu.l Restriction enzyme 1 (NEB, Beverly Mass.,
or Fermentas, Amherst N.J.), 0.2 .mu.l Restriction enzyme 2, 1
.mu.l cDNA (1 ng/.mu.l).
[0367] Digestion of cDNA is performed with the following
thermocycler program: 30 mm@37.degree. C., 22 min ramp to
16.degree. C., 1 hour@16.degree. C., 15 min@37.degree. C., and 20
min@72.degree. C. Following digestion 0.2 .mu.l Ligase (BRL,
Baltimore Md.) with 1 .mu.l Primer set 1 (Genosys, The Woodlands
Tex., or Amitof, Boston Mass.), 1 .mu.l Primer set 2 are added to
the mix. The reactions are then maintained at 16.degree. C. for
ligation of PCR primers. For PCR, the following reagents are added:
2.mu.l 10 mM dNTP (USB, Chicago Ill.), 51 .mu.l 10.times. TB buffer
(500 mM Tris pH 9.15, 160 mM (NH4).sub.2SO.sub.4, 20 mM
MgCl.sub.2), 0.25 .mu.l Klentaq (Invitrogen, Carlsbad Calif.): PFU
(Stratagene, Los Angeles Calif.) (16:1), 32.75 .mu.l H.sub.2O. 20
cycles of amplification (30 sec@96.degree. C., 1 sec@57.degree. C.,
2 min@72.degree. C.) were followed with 10 min@72.degree. C. PCR
product purification was performed using MPG streptavidin beads
(CPG, Lincoln Park N.J.). After washing the beads twice with buffer
1 (3M NaCl, 10 mM TRIS, pH 7.5, 1 mM EDTA), 20 .mu.l were mixed
with the PCR product for 10 min at room temperature. separated with
a magnet, and washed once with buffer 2 (10 mM TRIS, pH 8.0, 1 mM
EDTA). The beads were then dried and resuspended in 3 .mu.l of
buffer 3 (80% formamide, 4 mM EDTA, 5% ROX-tagged molecular size
standard (ABI, San Fransisco Calif.)). In addition, every other
lane received 5% TAMRA (ABI, San Fransisco Calif.) as interlane
bleed control. Following denaturation (96.degree. C. for 3 min),
samples were loaded onto 5% polyacrylamide, 6M urea, 1.times. BE
ultrathin gels (Long-Ranger, FMC, Philadelphia Pa.), and
electrophoresed for 60 min at 3500 V on a Niagara.TM.
instrument.
EXAMPLE 4
Open Genome Initiative.TM. Software Gel Interpretation
[0368] The output of the electrophoresis instruments are processed
using the internet-based Open Genome Initiative.TM. (OGI.TM.)
software suite. Gel images are visually inspected for overall
quality and each lane tracked to delineanate the path of best fit.
Each lane contains a GeneCalling sample plus two sizing ladders
(labeled with ROX and TAMRA fluorochrome) spanning the range from
50 to 500 bp. The ladder peaks provide a relationship between
camera frames (typically collected at 1 Hz) and base pairs. After
tracking, lanes are extracted and the peaks in the sizing ladder
are resolved. Linear interpolation between the ladder peaks
converts the GeneCalling sample traces from frames to base pairs.
Each trace is evaluated and ruled out for low signal-to-noise, poor
peak resolution, absent ladder peaks, and lane-to-lane bleed. Lanes
that pass all criteria are submitted as point-by-point length vs.
amplitude addresses to the GeneScape Oracle 8 database. Submitted
traces are then organized by treatment group and fragmentation
primers. The 9 traces corresponding to each treatment
group/fragmentation pattern are superimposed and are manually
evaluated for intertrace alignment fidelity. Misaligned traces are
rejected and excluded from subsequent analyses.
EXAMPLE 5
Gene Isolation
[0369] 1 .mu.l of the GENECALLING.TM. chemistry reaction containing
the peak of interest is added to 3 .mu.l of 1.times. TAE buffer
(Sigma, St Louis Mo.) and 1 .mu.l of gel loading dye (Elchrom
Scientific, Lake Park Fla.) and electrophoresed on an Elchrom Mini
Gel (Elchrom Scientific, Lake Park Fla.) at 55.degree. C. 120 V for
30'-150' depending upon the size of the selected fragment Following
15' of ethidium bromide staining, the desired band length is
excised from gel lane, placed into 10 mM MgCl2, centrifuged at 3000
RPM for 5' and heated to 65.degree. C. for 30'. Eluted fragments
are PCR-amplified using J23 & R23 PCR primers (Amitof, Boston
Mass.) and cDNA polymerase (Clontech, Palo Alto Calif.) for 25
cycles of 30"@96.degree. C., 60"@57.degree. C., 2'@72.degree. C. 3
.mu.l aliquot is ligated to pCR2.1 cloning vector (Invitrogen,
Carlsbad Calif.) using the Fast-Link DNA ligation kit (Epicenter,
Madison Wis.). Vectors are electroporated into DH10B E. coli with
1.8 mV pulses and cells are then plated on LB plates with
ampicillin, kanamycin, and x-gal (Northeast Laboratories,
Waterville Mass.). Colonies with inserts are selected for PCR
amplification using 5M betaine (Sigma, St. Louis Mo.), DYN-A &
DYN-RE primers (Amitof, Boston Mass.) and polymerase (Clontech,
Palo Alto Calif.) for 29 cycles of 1'@96.degree. C., 1'@72.degree.
C. PCR products are submitted to sequencing for clone
identification.
EXAMPLE 6
Clone Sequencing
[0370] 30 .mu.l of clone template are added to 6 .mu.l of SPRI
beads (Bangs Laboratories, Inc., Fishers Ind.) in 0.5 M EDTA pH 8.0
(Amresco, Solon Ohio) and 30 .mu.l hybridization buffer (2.5 M
NaCl, 20% PEG 8000 (Sigma, St. Louis Mo.)) in 96-well plate format.
Plates are shaken for 5' at 600 rpm and settled for 2' on a magnet.
The beads are washed 4.times. with 200 .mu.l of 70% EtOH(AAPER,
Louisville Ky.) and air dried for 2 min. 36 .mu.l of Nanopure
H.sub.2O is added to the beads. Plates are again shaken for 5' at
600 rpm and the supernatant is collected for sequencing.
[0371] 3 .mu.l of purified product is transferred to each of A
(JOE-fluor). G (TAMRA-fluor), C (FAM-fluor) and T (ROX-fluor)
reaction mixes (2 .mu.l DYEnamic Direct Cycle sequencing kit:
DYEnamic-M13-40ET primers, premixed dGTP, Taq polymerase(Amersharn
Life Sciences, Piscataway N.J.) and 1.8 ml dNTP (Amersham Life
Sciences, Piscataway N.J.)) in 384-well format. Plates are placed
in a thermocycler for 15 cycles of 5"96.degree. C., 10"@52.degree.
C., 60"@72.degree. C. Reactions are quenched at 4.degree. C. For
each template, the four reactions are pooled into one well of a
96-well plate and 65 .mu.l of 100% EtOH (AAPER, Louisville Ky.) are
added. Plates are chilled at 4.degree. C. for 60 mm and centrifuged
at 4.degree. C. for 30 min at 2000 rpm. The supernatant is removed
and the plates are air dried to completion at 25.degree. C. 3 .mu.l
of formamide loading dye (Amersham, Piscataway N.J.) is added to
each well. In addition, 960 .mu.l of TAMRA-spiked loading dye (10:1
formamide (Amersham, Piscataway N.J.): 55mer TAMRA (Amitof, Boston
Mass.)) is added to select wells for electrophoresis quality
control. Samples are electrophoresed in a 1.times. TBE Long Ranger
(FMC, Philadelphia Pa.) polyacrylamide gel on the AB1377 (ABI, San
Fransisco Calif.) platform for 2.5 hrs at 3000 volts.
[0372] Gel images are resolved and interpreted by the OGI software
interface. Images are quality controlled for overall image fidelity
arid sequence quality of individual lanes. Lanes with truncated
sequences, absent signals in one or more channels, bleed and primer
dimer are failed and removed from further analysis. The sequences
are BaseCalled and imported into GeneScape assigned to the
difference peak corresponding to the sequence.
EXAMPLE 7
Oligonucleotide Poisoning
[0373] Restriction fragments which map in end sequence and length
to known rat genes are used as templates for the design of
unlabeled oligonucleotide primers. An unlabeled oligonucleotide
designed against one end of the restriction fragment is added in
excess to the original reaction. and is re-amplified for an
additional 15 cycles. This reaction is then electrophoresed and
compared to a control reaction reamplified without the unlabeled
oligonucleotide to evaluate the selective diminution of the peak of
interest.
EXAMPLE 8
Northern Blot Analysis
[0374] 1 .mu.g of Poly-A+ RNA prepared as described above was
transferred to Hybond plus membranes (Amersham. Piscataway N.J.)
and hybridized using standard techniques. Probes for cloned
fragments were reamplified from isolated E.coli colonies containing
the appropriate insert as described above and subcloned into the
pCR2.1 vector (Invitrogen). Probes for GeneCalled fragments were
obtained following PCR amplification of the fragment from sample
cDNA prepared as described above using primers designed from the
predicted database sequence designed to overlap the restriction
enzyme sites of the GeneCalled gene fragment and allow subcloning
into the pCR2.1 vector. 1 ng of plasmid was combined with 0.2 uM of
M13FSP6 forward and M13RT3 reverse primers and 200 uM of each dNTP
in 1.times. PCR buffer with 0.5 ul cDNA Taq polymerase (Clontech).
The mixture was subject 5'@94.degree. C. followed by 5.times. of
5"@94.degree. C./3'@72.degree. C., 5.times. of 5"@94.degree.
C./3'@70.degree. C., and 15.times. of 5"@94.degree.
C./3'@68.degree. C. PCR products were electrophoresed through a 1%
low melting agarose gel and purified using the Qiaex II gel
extraction kit (Qiagen). The RNA probe was transcribed using the
Stip-EZ RNA probe synthesis kit (Ambion). 100 ng of purified probe
was labelled using 25 uCi of 33P-UTP (Amersham) using either SP6 or
T3 polymerase to capture the noncoding strand of the probe.
Following transcription, 1 ul of DNAse I was added and incubated at
37.degree. C. for 15. Unincorporated nucleotides were removed using
ProbeQuant G-50 micro columns (Pharmacia Biotech) and the probe was
quantitated using a Bioscan QC-4000 (Bioscan).
[0375] RNA probes were hybridized to the Northern blots at
65.degree. C. in a Robbins Scientific Model 400 hybridization
incubator. The blots were prehybridized at 65.degree. C. in 10 ml
of Zip-Hyb (Ambion) for 30' and then 10.sup.6 dpm/ml of RNA probe
in 1 ml of Zip-Hyb was added to the Northern for a 2 hr incubation.
Following hybridization, the buffer was removed and the blots were
washed first in 2.times. SSC. 0.1% SDS.times.15'@65.degree. C. then
in 0.1.times. SSC. 0.1% SDS.times.15, @65.degree. C. The blots were
wrapped in Saran Wrap (Dow) and exposed to phosphor screens
(Molecular Dynamics) overnight. The screens were scanned on a Storm
840 (Molecular Dynamics) at 50 urn resolution.
EXAMPLE 9
Database Query for Sage Expression Analysis
[0376] Serial Analysis of Gene Expression, or SAGE, is an
experimental technique designed to gain a quantitative measure of
gene expression. The SAGE technique itself includes several steps
tilizing molecular biological, DNA sequencing and bioinformatics
techniques. These steps (reviewed in Adams Md., "Serial analysis of
gene expression: ESTs get smaller." Bioessays. 18(4):261-2 (1996))
have been used to produce 9 or 10 base "tags", which are then, in
some manner, assigned gene descriptions. For experimental reasons,
these tags are immediately adjacent to the 3' end of the 3'-most
NlaIII restriction site in cDNA sequences. The Cancer Genome
Anatomy Project, or CGAP, is an NCI-initiated and sponsored
project, which hopes to delineate the molecular fingerprint of the
cancer cell. It has created a database of those cancer-related
projects that used SAGE analysis in order to gain insight into the
initiation and development of cancer in the human body. The SAGE
expression profiles reported in this invention are generated by
first identifying the Unigene accession ID associated with the
given MTC gene by querying the Unigene database at
[0377] http://www.ncbi.nlm.nih.gov/UniGene/. This page has then a
link to the SAGE: Gene to Tag mapping
(http://www.ncbi.nlm.nih.gov/SAGE/SAGEcid.c-
gi?cid="unigeneID").
[0378] This generated the reports that are included in this
application, which list the number of tags found for the given gene
in a given sample along with the relative expression. This
information is then used to understand whether the gene has a more
general role in tumorogenesis and/or tumor progression. A list of
the SAGE libraries generated by CGAP and used in the analysis can
be found at http://www.ncbi.nlm.nih.gov/SAGE- /sagelb.cgi.
OTHER EMBODIMENTS
[0379] It is to be understood that, while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims. For example, the neoplasm
described herein can be a thyroid carcinoma, a breast carcinoma, a
colorectal carcinoma, and/or an ovarian carcinoma. Similarly, the
methods described herein can also be used for metastatic neoplasms
from non-thyroid tumors, e.g., carcinomas such as ovarian, breast,
and colorectal carcinomas.
* * * * *
References