Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-09-14
2001-07-10
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200
Reexamination Certificate
active
06258541
ABSTRACT:
S
TATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable
REFERENCE TO A “MICROFICHE APPENDIX”
Not applicable
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for the noninvasive detection of colonic biomarkers using fecal messenger RNA (mRNA). More particularly, the present invention relates to methods for the isolation of poly A+ RNA from feces, and includes the subsequent detection of, and quantitation of, particular mRNAs that correlate with a patient's diagnosis and/or prognosis of colon cancer thereby providing methods for noninvasively diagnosing and/or prognosticating colon cancer in a patient. One embodiment of the present invention relates to the detection of, and quantitation of, mRNA from sloughed colon cells in feces encoding particular isozymes of protein kinase C (PKC) whose levels are correlative with and predictive of colon cancer in a patient. Methods including semi-quantitative RT-PCR and biochip microarray technology may be made to assay and evaluate the fecal poly A+RNA.
2. General Background
Since colon cancer is the second most common cause of U.S. cancer deaths and since early detection can result in a high cure rate, an accurate screening method for colon cancer is imperative. Current detection methods have many drawbacks. For example, fecal occult blood screening can produce false positive results due to meat consumption, iron supplement intake and other common behaviors. The other routine screening technique, sigmoidoscopy, is an invasive expensive procedure which has inherent risks of perforation, reaction to sedative, or bleeding. In addition, the efficacy of sigmoidoscopy screening remains unproven (Levin, 1996). Because of these limitations, colon cancer cure rates have not improved in the past 30 year (Silverberg, 1988, WFR/AICR, 1997). Therefore, an accurate technique to detect early changes associated with the tumorigenic process is imperative in order to decrease the mortality from colon cancer.
Screening of colorectal cancer is recommended for all persons aged 50 and older with annual fecal occult blood testing or sigmoidoscopy, or both (Levin, 1996). However, each of these tests has limitations related to sensitivity and specificity (Levin, 1996). The presence of colorectal and pancreatic tumors has been detected in the stool and colonic effluent of patients by noninvasive methods based on the molecular pathogenesis of the disease (Sidransky, 1992: Tobi, 1994; Caldas, 1994). These protocols utilize DNA extraction procedures and the detection of oncogene mutations using PCR. The major disadvantage of this methodology is that it will not detect alterations in gene expression. Our methodology can quantitate the expression of any relevant gene by isolating and amplifying mRNA derived from fecal material containing sloughed colonocytes.
A sensitive molecular technique for the detection of colon cancer is important since early diagnosis can substantially reduce mortality (Levin, 1996). Our method is noninvasive, highly sensitive and specific. Our protocol is unique because it will determine colonic expression of any gene (e.g., tumor suppressor gene, oncogene), and provides early sensitive prognostic information and greatly enhances current methods of dietary and pharmacologic risk assessment.
SUMMARY OF THE PRESENT INVENTION
The present invention relates to a novel non-invasive technology to detect changes in colonocyte gene expression associated with early stages of colon tumorigenesis. This invention also covers the first known methods to isolate poly A+ RNA from feces. This methodology has the advantage of utilizing a fecal sample, which contains sloughed colon cells. Therefore, it does not require anesthesia or cause any discomfort to the patient. In addition, the invention utilizes a novel mRNA isolation process that results in an unexpectedly high yield and stability of isolated fecal mRNA, and utilizes an exquisitely sensitive technique, rapid competitive polymerase chain reaction (Jiang, 1996), developed by the inventors, to detect and quantify mRNA markers of the tumorigenic process. Thousands of gene markers for the tumorigenic process are assayable in the practice of the present invention. These markers include, but are not limited to, PKC isozymes such as, for example, PKC &bgr;II (PKC beta II) and PKC &zgr; (PKC zeta), where, for example, levels of these particular isozymes in feces are correlative of and predictive of the presence of, and development of colon cancer in a rat colon cancer model (Davidson, 1998). We have also successfully isolated poly A+ RNA from rectal vault eluate isolated at the initiation of colonoscopy. Yields from fecal eluate are generally in the range of 0.3-1.5 &mgr;g poly A+ RNA isolated per subject.
The pathogenesis of colon cancer is a multi-step process, in which tumor suppressor genes, oncogenes and other molecules involved in signal transduction are affected (Fearon, 1997). It is now clear the signals mediated via select isozymes of protein kinase C (PKC) are involved in colonic tumor development (Sakanoue et al., 1991; Kopp et al. 1991; Baum et al., 1990). PKCs are a family of serine-threonine kinases thought to regulate colonic cell proliferation, differentiation and programmed cell death. PKCs can be divided into three different sub-categories based on the cofactors needed for activation: classical PKCs (&agr;, &bgr;I, &bgr;II and &ggr;) require diacylglycerol (DAG) and Ca
2+
for activation; novel PKCs (&dgr;, &thgr;, &eegr; and &egr;) are Ca
2+
independent, but activated by DAG; and atypical PKCs (&lgr;, &igr; and &zgr;) are Ca
2+
and DAG independent. Although these isozymes are enzymatically similar, in vivo, they have different expression patterns depending on tissue and cell type (Blobe et al., 1996).
PKC &bgr;II protein is generally found in very low levels in normal rat colonic mucosa (Davidson et al., 1994). However, &bgr;II protein levels increase in colonic tumors as compared with normal colonic mucosa (Craven et al., 1992; Wali et al., 1995). In contrast, PKC &zgr; mRNA levels are significantly lower in human colorectal tumors than in normal colonic mucosa (Kuranami et al., 1995). PKC &zgr; protein levels also are significantly lower in preneoplastic colonic epithelium from rats injected with azoxymethane (AOM) as compared with saline-injected control rats (Wali et al., 1995; Roy et al., 1995; Jiang et al., 1997). Therefore PKC &bgr;II and &zgr; may serve as biomarkers to monitor the development of colon cancer.
In summary, no one has reported the isolation of intact poly A+ RNA from fecal material or rectal eluates obtained at colonoscopy. Utilization of this noninvasive procedure combined with either RT-PCR analyses or genechip microarrays is novel.
REFERENCES:
Ambion Inc. from Ambion.com website: description of poly(A) pure kits, and protocol, 1995.*
Barbee et al, Gene, vol. 132, pp 305-306, 1993.*
Greenham et al., Human Genetics, vol. 103, pp 483-487, 1998.*
Jiang et al.: “Rapid Competitive PCR Determination of Relative Gene Expression in Limiting Tissue Samples” Clin. Chemistry, vol. 42, No. 2, 1996, pp. 227-331, XP002075087.
Davidson et al.: “Noninvasive Detection of Putative Biomarkers for Colon Cancer Using Fecal Messenger RNA” Canc. Epidem. Biomarkers and Prevention, vol. 4, 1995, pp. 643-647, XP002075091.
Jiang et al.: “Dietary Fish Oil Blocks Carcinogen-Induced Down-Regulation of Colonic Protein Kinase C Isozymes” Carcinogenesis, vol. 18, No. 2, Feb. 1997, pp. 351-357, XP002075088.
Hardie and Hanks: “The Protein Kinase Factsbook; vol. 1: Protein-Serin Kinases” 1995, Academic, London, GB XP002075310, see pp. 80-88.
Blobe et al.: “Protein Kinase C Isozymes: Regulation and Function” Cancer Surveys, vol. 27, 1996, pp. 213-248, XP002075089.
Davidson et al.: “Protein Kinase C Isoforms in Human and Rat Colonic Mucosa” Arch. of Biochem. and Biophys., vol. 312, No. 2, 1994, pp. 547-553, XP002075090.
Davidson et al.: “Non-Invasive Detection of Fecal Protein Kinase C Be
Chapkin Robert S.
Davidson Laurie A.
Lupton Joanne R.
Arthur Lisa B.
Fulbright & Jaworski
Souaya Jehanne
Texas A&M University
LandOfFree
Noninvasive detection of colonic biomarkers using fecal... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Noninvasive detection of colonic biomarkers using fecal..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Noninvasive detection of colonic biomarkers using fecal... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2532059