MN and Hypoxia
The MN/CA IX protein is identified herein as a hypoxia marker. The MN/CA9 gene promoter is characterized, and the location of the HIF-1 binding site within the MN/CA9 promoter is identified. Further, the hypoxia inducibility of the MN/CA9 gene and the uses of such inducibility are disclosed. In one aspect, the invention provides diagnostic/prognostic tools for determining the presence of hypoxia in a tissue in a vertebrate, preferably a human, and for measuring the relative degree of hypoxia in said vertebrate. In another aspect, the invention provides methods using tumor biopsies to predict the radioresistance of a preneoplastic/neoplastic tissue in a vertebrate subject, preferably a human patient, for diseases in which MN/CA IX levels can be used to indicate radiobiologically relevant tumor hypoxia. Such predictive methods can be used as an aid in patient therapy selection.
1 . A method for determining the degree of hypoxia in a tissue in a subject vertebrate, comprising:
(a) isolating a sample from said tissue from said vertebrate; and
(b) immunologically detecting and quantifying the level of MN/CA IX protein/polypeptide in said tissue;
wherein the level of said MN/CA IX protein/polypeptide found in said tissue in step (b), relative to the level of MN/CA IX protein/polypeptide found at 0.1% O 2 in comparable cells isolated from an organism of the same taxonomic classification as the subject vertebrate, indicates the degree of hypoxia in said tissue.
2 . The method of claim 1 , wherein said tissue is a is preneoplastic/neoplastic tissue.
3 . A method to predict the degree of radioresistance of an affected tissue in a subject vertebrate with a preneoplastic/neoplastic disease, wherein MN/CA IX protein/polypeptide levels in said preneoplastic/neoplastic tissue can be used to indicate radiobiologically relevant tumor hypoxia in said tissue, comprising:
(a) performing an in vitro test of comparable preneoplastic/neoplastic cells, correlating the MN/CA IX protein/polypeptide levels in said cells with degrees of cellular radioresistance, wherein said cells are isolated from an organism of the same taxonomic classification as the subject vertebrate;
(b) isolating a sample from the affected tissue in said subject vertebrate;
(c) immunologically detecting and quantitating the MN/CA IX protein/polypeptide level in said vertebrate sample; and
(d) predicting the degree of radioresistance of the subject vertebrate tissue by comparing the MN/CA IX protein/polypeptide level found in step (c) with the MN/CA IX protein/polypeptide levels of step (a), and extrapolating therefrom a predicted degree of radioresistance of the subject vertebrate tissue.
4 . The method of claim 3 , wherein said comparable preneoplastic/neoplastic cells are isolated from the subject vertebrate.
5 . The method of claim 3 , wherein said method is used as an aid in the selection of treatment for said preneoplastic/neoplastic disease afflicting said vertebrate.
6 . The method of claim 5 , wherein the predicted degree of radioresistance of the affected vertebrate tissue is high, and the decision is made not to use radiation therapy, or wherein the predicted degree of radioresistance of the affected vertebrate tissue is low, and the decision is made to use radiation therapy.
7 . The method of claim 3 , wherein said preneoplastic/neoplastic is disease afflicting said subject vertebrate is selected from the group consisting of preneoplastic/neoplastic diseases of head and neck, mammary, urinary tract, kidney, bladder, ovarian, uterine, cervical, endometrial, vaginal, vulvar, prostate, liver, lung, skin, thyroid, pancreatic, testicular, brain, gastrointestinal, colon, colorectal and mesodermal tissues.
8 . The method of claim 3 wherein said preneoplastic/neoplastic disease is head and neck preneoplastic/neoplastic disease.
9 . The method of claim 3 wherein said subject vertebrate is a human patient.
10 . The method of claim 9 wherein said neoplastic disease is a tumor, and said sample is taken from said tumor and/or from a metastatic lesion derived from said tumor.
11 . The method of claim 3 , wherein said sample is a formalin-fixed, paraffin-embedded tissue sample.
12 . The method of claim 3 , wherein said correlating step (a), and said detecting and quantitating step (c), comprise the use of an assay selected from the group consisting of Western blots, enzyme-inked immunosorbent assays, radioimmunoassays, competition immunoassays, dual antibody sandwich assays, immunohistochemical staining assays, agglutination assays, and fluorescent immunoassays.
13 . The method according to claim 3 , wherein said correlating step (a), and said detecting and quantitating step (c), comprise the use of the M75 monoclonal antibody secreted by the hybridoma VU-M75 which has Accession No. ATCC HB 11128.
14 . The method of claim 3 , wherein said detecting and quantitating step (c) comprises the use of immunohistochemical staining with MN/CA IX-specific antibody to detect the levels of MN/CA IX protein in the sample.
15 . The method according to claim 14 , wherein said detecting and quantitating step (c) further comprises determining the percentage of MN/CA IX immunoreactive cells and/or the intensity of immunostaining of immunoreactive cells.
16 . The method of claim 3 , wherein said correlating step (a) comprises determining the oxygen enhancement ratio (OER) or modified oxygen enhancement ratio (OER′) in said comparable preneoplastic/neoplastic cells.
17 . The method of claim 9 , wherein said preneoplastic/neoplastic disease is head and neck cancer, and wherein said comparable preneoplastic/neoplastic cells are FaDu human pharyngeal carcinoma cells.
18 . The method of claim 9 , wherein said preneoplastic/neoplastic disease is head and neck cancer, and wherein said comparable preneoplastic/neoplastic cells are isolated from the subject human.
19 . A method to detect cells that are both hypoxic and metabolically active in a prenoplastic/neoplastic tissue derived from a subject vertebrate, comprising:
(a) isolating a sample from the affected tissue in said subject vertebrate; and
(b) immunologically detecting and quantitating the MN/CA IX protein/polypeptide level in said vertebrate sample;
wherein if detectable MN/CA IX protein/polypeptide is found in step (b), concluding that the cells in the vertebrate sample are both hypoxic and metabolically active; or if no detectable MN/CA IX protein/polypeptide is found in step (b), concluding that the cells in the vertebrate sample are not hypoxic and/or not metabolically active.
20 . The method of claim 19 , wherein if no detectable MN/CA IX protein/polypeptide is found in step (b), further comprising screening said vertebrate sample for a second hypoxic marker;
wherein if said second hypoxic marker indicates that the cells are hypoxic, further concluding that said cells in the vertebrate sample are hypoxic but not metabolically active.
21 . The method of claim 19 , wherein said method is used as an aid in the selection of treatment for said preneoplastic/neoplastic disease afflicting said vertebrate.