Human mitogenic oxidase

Details Human mitogenic oxidase Human mitogenic oxidase

1999-11-10

2003-09-16

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S192000, C436S023000

Reexamination Certificate

active

06620603

ABSTRACT:

TECHNICAL FIELD
The present invention relates to the field of normal and abnormal cell growth, in particular mitogenic regulation. The present invention provides the following: nucleotide sequences encoding for the production of enzymes that are mitogenic regulators; amino acid sequences of these enzymes; vectors containing these nucleotide sequences; methods for transfecting cells with vectors that produce these enzymes; transfected cells; methods for administering these transfected cells to animals to induce tumor formation; and antibodies to these enzymes that are useful for detecting and measuring levels of these enzymes, and for binding to cells possessing extracellular epitopes of these enzymes.
BACKGROUND OF THE INVENTION
Reactive oxygen intermediates (ROI) are partial reduction products of oxygen: 1 electron reduces O
2
to form superoxide (O
2
−
), and 2 electrons reduce O
2
to form hydrogen peroxide (H
2
O
2
). ROI are generated as a byproduct of aerobic metabolism and by toxicological mechanisms. There is growing evidence for regulated enzymatic generation of O
2
−
and its conversion to H
2
O
2
in a variety of cells. The conversion of O
2
−
to H
2
O
2
occurs spontaneously, but is markedly accelerated by superoxide dismutase (SOD). High levels of ROI are associated with damage to biomolecules such as DNA, biomembranes and proteins. Recent evidence indicates generation of ROI under normal cellular conditions and points to signaling roles for O
2
−
and H
2
O
2
.
Several biological systems generate reactive oxygen. Phagocytic cells such as neutrophils generate large quantities of ROI as part of their battery of bactericidal mechanisms. Exposure of neutrophils to bacteria or to various soluble mediators such as formyl-Met-Leu-Phe or phorbol esters activates a massive consumption of oxygen, termed the respiratory burst, to initially generate superoxide, with secondary generation of H
2
O
2
, HOCl and hydroxyl radical. The enzyme responsible for this oxygen consumption is the respiratory burst oxidase (nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) oxidase).
There is growing evidence for the generation of ROI by non-phagocytic cells, particularly in situations related to cell proliferation. Significant generation of H
2
O
2
, O
2
−
or both have been noted in some cell types. Fibroblasts and human endothelial cells show increased release of superoxide in response to cytokines such as interleukin-1 or tumor necrosis factor (TNF) (Meier et al. (1989)
Biochem J.
263, 539-545.; Matsubara et al. (1986)
J. Immun.
137, 3295-3298). Ras-transformed fibroblasts show increased superoxide release compared with control fibroblasts (Irani, et al. (1997)
Science
275, 1649-1652). Rat vascular smooth muscle cells show increased H
2
O
2
release in response to PDGF (Sundaresan et al. (1995)
Science
270, 296-299) and angiotensin II (Griendling et al. (1994)
Circ. Res.
74, 1141-1148; Fukui et al. (1997)
Circ. Res.
80, 45-51; Ushio-Fukai et al. (1996)
J. Biol. Chem.
271, 23317-23321), and H
2
O
2
in these cells is associated with increased proliferation rate. The occurrence of ROI in a variety of cell types is summarized in Table 1 (adapted from Burdon, R. (1995)
Free Radical Biol. Med.
18, 775-794).
TABLE 1
Superoxide
Hydrogen Peroxide
human fibroblasts
BaIb/3T3 cells
human endothelial cells
rat pancreatic islet cells
human/rat smooth muscle cells
murine keratinocytes
human fat cells
rabbit chondrocytes
human osteocytes
human tumor cells
BHK-21 cells
fat cells, 3T3 L1 cells
human colonic epithelial cells
ROI generated by the neutrophil have a cytotoxic function. While ROI are normally directed at the invading microbe, ROI can also induce tissue damage (e.g., in inflammatory conditions such as arthritis, shock, lung disease, and inflammatory bowel disease) or may be involved in tumor initiation or promotion, due to damaging effects on DNA. Nathan (Szatrowski et al. (1991)
Canc. Res.
51, 794-798) proposed that the generation of ROI in tumor cells may contribute to the hypermutability seen in tumors, and may therefore contribute to tumor heterogeneity, invasion and metastasis.
In addition to cytotoxic and mutagenic roles, ROI have ideal properties as signal molecules: 1) they are generated in a controlled manner in response to upstream signals; 2) the signal can be terminated by rapid metabolism of O
2
−
and H
2
O
2
by SOD and catalase/peroxidases; 3) they elicit downstream effects on target molecules, e.g., redox-sensitive regulatory proteins such as NF kappa B and AP-1 (Schreck et al. (1991)
EMBO J.
10, 2247-2258; Schmidt et al. (1995)
Chemistry
&
Biology
2, 13-22). Oxidants such as O
2
−
and H
2
O
2
have a relatively well defined signaling role in bacteria, operating via the SoxI/II regulon to regulate transcription.
ROI appear to have a direct role in regulating cell division, and may function as mitogenic signals in pathological conditions related to growth. These conditions include cancer and cardiovascular disease. O
2
−
is generated in endothelial cells in response to cytokines, and might play a role in angiogenesis (Matsubara et al. (1986)
J. Immun.
137, 3295-3298). O
2
−
and H
2
O
2
are also proposed to function as “life-signals”, preventing cells from undergoing apoptosis (Matsubara et al. (1986)
J. Immun.
137, 3295-3298). As discussed above, many cells respond to growth factors (e.g., platelet derived growth factor (PDGF), epidermal derived growth factor (EGF), angiotensin II, and various cytokines) with both increased production of O
2
−
/H
2
O
2
and increased proliferation. Inhibition of ROI generation prevents the mitogenic response. Exposure to exogenously generated O
2
−
and H
2
O
2
results in an increase in cell proliferation. A partial list of responsive cell types is shown below in Table 2 (adapted from Burdon, R. (1995)
Free Radical Biol. Med.
18, 775-794).
TABLE 2
Superoxide
Hydrogen peroxide
human, hamster fibroblasts
mouse osteoblastic cells
Balb/3T3 cells
Balb/3T3 cells
human histiocytic leukemia
rat, hamster fibroblasts
mouse epidermal cells
human smooth muscle cells
rat colonic epithelial cells
rat vascular smooth muscle
cells
rat vascular smooth muscle cells
While non-transformed cells can respond to growth factors and cytokines with the production of ROI, tumor cells appear to produce ROI in an uncontrolled manner. A series of human tumor cells produced large amounts of hydrogen peroxide compared with non-tumor cells (Szatrowski et al. (1991)
Canc. Res.
51, 794-798). Ras-transformed NIH 3T3 cells generated elevated amounts of superoxide, and inhibition of superoxide generation by several mechanisms resulted in a reversion to a “normal” growth phenotype.
O
2
−
has been implicated in maintenance of the transformed phenotype in cancer cells including melanoma, breast carcinoma, fibrosarcoma, and virally transformed tumor cells. Decreased levels of the manganese form of SOD (MnSOD) have been measured in cancer cells and in vitro-transformed cell lines, predicting increased O
2
−
levels (Burdon, R. (1995)
Free Radical Biol. Med.
18, 775-794). MnSOD is encoded on chromosome 6q25 which is very often lost in melanoma. Overexpression of MnSOD in melanoma and other cancer cells (Church et al. (1993)
Proc. of Natl. Acad. Sci.
90, 3113-3117; Femandez-Pol et al. (1982)
Canc. Res.
42, 609-617; Yan et al. (1996)
Canc. Res.
56, 2864-2871) resulted in suppression of the transformed phenotype.
ROI are implicated in growth of vascular smooth muscle associated with hypertension, atherosclerosis, and restenosis after angioplasty. O
2
−
generation is seen in rabbit aortic adventitia (Pagano et al. (1997)
Proc. Natl. Acad. Sci.
94, 14483-14488). Vascular endothelial cells release O
2
−
in response to cytokines (Matsubara et al. (1986)
J. Immun.
137, 3295-3298). O
2
−
is generated by aortic smooth muscle cells in culture, and increased O
2
−
generation is stimulated by angiotensin II which also induces cel

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