Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving luciferase
Reexamination Certificate
2002-04-26
2004-04-06
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving luciferase
C435S004000, C435S968000
Reexamination Certificate
active
06716595
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to non-radioactive enzymatic methods for detecting Sphingosine-1-Phosphate (S1P) in biological fluids and the correlation at a measurement of S1P to disease. The present invention further relates to a method of detecting the presence of cancer in a patient by the use of these and other methods of detecting S1P in biological samples from a patient.
BACKGROUND OF THE INVENTION
Sphingolipids are a diverse group of molecules that are found in the membranes of all eukaryotic cells (Merrill et al.,
Toxicol. Appl. Pharmacol.
142:208-225 (1997)). There are three classes of sphingolipids: sphingomyelins, cerebrosides and gangliosides. Sphingolipids include sphingomyelin, sphinganine, sphingosine, glycosphingolipids and ceramide. Sphingosine-1-phosphate (S1P) is a lysosphingolipid that is generated by the metabolism of sphingomyelin. (Spiegel and Merrill,
FASEB J.
10:1388-1397 (1996)). Sphingomyelin, the most abundant type of sphingolipid is first converted to ceramide (sphingosine+palmitate residues) by the removal of the head group. Ceramide is then cleaved to form sphingosine by the action of ceramidases. S1P is then generated from sphingosine by the action of sphingosine kinase. S1P may then be further metabolized into ethanolamine-phosphate and long-chain aldehyde (e.g. hexadecanal) by S1P lyase (Zhou and Saba,
Biochemical and Biophysical Research Communications,
242:502-507 (1998)). The web of enzymatic events governing the metabolism of Sphingolipids is diagrammed below:
In addition to their role as components of biological membranes, some of the metabolites of sphingolipids, such as ceramide, sphingosine and S1P, are potent chemical messengers (Spiegel and Merrill, supra, Meyer et al.,
FEBS Lett.
410:34-38 (1997); and Gomez-Munoz et al.,
J. Biol. Chem.
270:26318-26325 (1995)). S1P has been shown to act as an intracellular second messenger that is generated in cells that have been activated by various mitogens (Spiegel et al.,
Breast Cancer Res. Treat.
31:337-348 (1994); Sadahira et al.,
Proc. Natl. Acad. Sci. USA
89:9686-9690 (1992); and Spiegel et al.,
Ann. N.Y. Acad. Sci.
845:11-18 (1998)), and to act as an extracellular ligand for a group of cell surface receptors (Hecht et al.,
J. Cell. Biol.
135:1071-1083 (1996); An et al.,
Biochem. Biophys. Res. Commun.
231:619-622 (1997); Goetzl and An,
FASEB J.
12:1589-1598 (1998); van Koppen et al.,
J. Biol. Chem.
274:18997-19002 (1999); Yatomi et al.,
J. Biol. Chem.
272:5291-5297 (1997); and Ancellin and Hla,
J. Biol. Chem.
274:18997-19002 (1999)). S1P is the principal ligand for three G-protein coupled receptors known as endothelial cell differentiation gene-1, -3 and -5 (Edg-1, Edg-3 and Edg-5). Edg-1, -3 and -5 are expressed in a variety of human tissues. Edg-1 is ubiquitously expressed (Goetzl and An, supra). Edg-3 is abundant in cardiovascular tissue and leukocytes, and is also expressed widely (Id.). Edg-5 is found most abundantly in cardiovascular tissue, the central nervous system, gonadal tissue and the placenta (Id.). The presence of multiple, high affinity receptors for S1P and their wide distribution, may provide redundancy in the control of the biological processes mediated by S1P. Another possibility is that specific S1P-directed biological processes are achieved by differential coupling between the various receptors and G-proteins (Ancellin and Hla, supra). However, the full role of S1P in normal and abnormal cell function has not yet been elucidated.
Previously, S1P levels have been measured quantitatively in biological samples by various methods utilizing the addition of radioactive isotopic moieties to the molecule. One such method is the determination of the amount of radioactive acetic anhydride that is incorporated in S1P by acylation (Yatomi et al.,
Anal. Biochem.
230:315-320 (1995)). In this assay, the S1P is first extracted from cells into an upper aqueous phase under alkaline conditions, and then re-extracted into the lower chloroform phase under acidic conditions. The phosphorylated sphingoid base is then quantitatively converted to N-[
3
H]-acetylated S1P by N-acylation with [
3
H] acetic anhydride, forming C2-Cer-1-P (tritiated C2-ceramide 1-phosphate). The C2-Cer-1-P is resolved with thin-layer chromatography, detected with autoradiography, and quantified with a scintillation counter. The assay permits quantification of S1P over a range of at least 100 pmol to 10 nmol. Yatomi used this assay to measure the distribution of S1P in various organs in rats. The largest concentration of S1P was in the testis and intestine (~100 nmol S1P/gram of tissue), followed by the spleen and brain (~50 nmol S1P/gram of tissue) (Yatomi et al.,
FEBS Lett.
404:173-174 (1997)). The kidney, heart and lung contained between 10 and 20 nmol S1P/gram of tissue, with the liver and muscle containing the lowest concentration of S1P (<5 nmol S1P/gram of tissue).
Using their assay, Yatomi and coworkers measured the level of S1P in some human bodily fluids (Yatomi et al.,
J. Biochem
. (
Tokyo
) 121:969-973 (1997)). They determined the concentration of S1P in normal human plasma and serum, but were unable to detect S1P using their assay in urine, ascites, pleural effusion or cerebrospinal fluid. They determined that tritiated sphingosine was rapidly taken into platelets in platelet-rich serum, while S1P was stable, demonstrating that the enzymes required for S1P degradation are not present in serum (Yatomi et al.,
J. Biochem
. (
Tokyo
), supra). The authors suggested that the release of S1P from activated platelets might be involved in processes such as thrombosis, hemostasis, atherosclerosis and wound healing.
S1P has also been detected using an enzymatic method employing an alkaline lipid extraction to separate S1P from other phospholipids and sphingolipids (Edsall and Spiegel,
Anal. Biochem.
272:80-86 (1999)). The extracted S1P was converted to sphingosine by alkaline phosphatase treatment. The sphingosine was then quantitatively phosphorylated using recombinant sphingosine kinase and [&ggr;
32
P]ATP. The authors reported that levels of S1P varied in rat tissues between 0.5 and 6 pmol/mg wet wt. The lowest levels were found in heart and testes, while the brain contained the highest levels.
These prior known methods for measuring S1P focus on the incorporation of a radioactive isotopic moiety into the molecule, and require considerable processing time in the laboratory. These limitations make the current assays unsuitable for many clinical or laboratory settings where complex bench processes are not practical. Development of a rapid and sensitive non-radioactive assay for S1P would facilitate use of this compound as a marker for various cellular activities such as cell growth, cell migration, apoptosis, platelet activation, changes in cellular morphology and for detecting conditions associated with altered levels of S1P.
Cancers such as ovarian cancer, lung cancer, colon cancer, and breast cancer are among the most frequent causes of cancer death in the United States and Europe. Despite decades of cancer research, mortality rates among persons who contract cancer remain high. However, when a cancer is detected at an early stage, survivability increases dramatically. For example, when ovarian cancer is diagnosed at an early stage, the cure rate approaches 90%. In contrast, the 5 year outlook for women with advanced disease remains poor with no more than a 15% survival rate. Thus, early diagnosis is one of the most effective means of improving the prognosis for cancer.
Frequently, however, detection of cancer depends upon the detection and inspection of a tumor mass which has reached sufficient size to be detected by physical examination. The detection of molecular markers of carcinogenesis and tumor growth can solve many of the problems which the physical examination of tumors have encountered. Samples taken from the patient for screening by molecular techniques are typically blood or urine, and thus require mini
Johnson Jodi L.
Parrott Jeff A.
Skinner Michael K.
Baker & Hostetler LLP
Leary Louise N.
LPL Technologies, Inc.
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