WO1998045480A1 - Noninvasive detection of colonic biomarkers using fecal messenger rna - Google Patents

Noninvasive detection of colonic biomarkers using fecal messenger rna Download PDF

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WO1998045480A1
WO1998045480A1 PCT/US1998/006698 US9806698W WO9845480A1 WO 1998045480 A1 WO1998045480 A1 WO 1998045480A1 US 9806698 W US9806698 W US 9806698W WO 9845480 A1 WO9845480 A1 WO 9845480A1
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pkc
expression
rna
determined
colon cancer
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PCT/US1998/006698
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French (fr)
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WO1998045480A9 (en
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Robert S. Chapkin
Laurie A. Davidson
Joanne R. Lupton
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The Texas A & M University System
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Priority to EP98915287A priority Critical patent/EP0972083A1/en
Priority to CA002286328A priority patent/CA2286328A1/en
Priority to AU69510/98A priority patent/AU6951098A/en
Priority to US09/381,086 priority patent/US6258541B1/en
Publication of WO1998045480A1 publication Critical patent/WO1998045480A1/en
Publication of WO1998045480A9 publication Critical patent/WO1998045480A9/en
Priority to US09/854,880 priority patent/US20010044113A1/en

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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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  • LAURIE A.S. citizen of College Station, Texas.
  • 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 mRNA from feces and 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.
  • PKC protein kinase C
  • 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.
  • the efficacy of sigmoidoscopy screening remains unproven (Levine, 1996) . Because of these limitations, colon cancer cure rates have not improved in the past 30 years (Silverberg, 1988) . Therefore, an accurate technique to detect early changes associated with the tumorigenic process is imperative in order to decrease the mortality from colon cancer.
  • 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 of importance 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 gene expression, and provides early sensitive prognostic information and greatly enhances current methods of dietary and pharmacologic risk assessment .
  • the present invention relates to a novel non-invasive technology to detect changes in colonocytes associated with early stages of colon tumorigenesis .
  • 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.
  • the invention utilizes a novel mRNA isolation process that results in an unexpectedly high yield and stability of isolated fecal mRNA, and utilizes an extraordinarly sensitive technique, rapid competitive polymerase chain reaction
  • markers for the tumorigenic process are assayable in the practice of the present invention. These markers include, but are not limited to, PKC isozy ⁇ nes such as, for example, PKC ⁇ ll (PKC-betall) and PKC ⁇ (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 patient. (Davidson, 1998) .
  • PKC protein kinase C
  • DAG DAG
  • novel PKCs ⁇ , ⁇ , ⁇ and ⁇
  • atypical PKCs ⁇ , ⁇ and ⁇
  • DAG DAG
  • isozymes are enzymatically similar, in vivo, they have different expression patterns depending on tissue and cell type (Blobe et al . , 1996) .
  • PKC ⁇ n protein levels are generally found in very low levels in normal rat colonic mucosa (Davidson et al . , 1994) . However, ⁇ protein levels increase in colonic tumors as compared with normal colonic mucosa (Craven et al . , 1992; Wali et al . , 1995). In contrast, PKC ⁇ mRNA levels are significantly lower in human colorectal tumors than in normal colonic mucosa (Kuranami et al . , 1995). PKC ⁇ protein levels also are significantly lower in preneoplastic colonic epithelium from rats injected with azoxymethane (AOM) as compared with saline- injected control rats
  • AOM azoxymethane
  • PKC ⁇ ZI and ⁇ may serve as biomarkers to monitor the development of colon cancer.
  • FIG. 1 shows expression of protein kinase C (PKC) beta II ( ⁇ ll) in fecal poly A+ RNA as assessed by semi -quantitative rapid competitive polymerase chain reaction (RC-PCR) , wherein “E” is “expression”, “T” is “tumor”, and “NT” is “no tumor”.
  • Figure 2 shows representative competitive PCR products for determination of Liver-Fatty Acid Binding Protein (L-FABP) expression in fecal poly A+ RNA.
  • PIC protein kinase C
  • RC-PCR semi -quantitative rapid competitive polymerase chain reaction
  • Figure 3 shows representative gel showing rapid competitive- RT/PCR of PKC ⁇ .
  • Lane I marker; lanes 2-5 fecal poly (A) + samples.
  • Upper band is the amplified sample band (419 bp)
  • the lower band is the amplified internal standard (361 bp) .
  • Figure 4 shows representative rapid competitive RT-PCR showing expression of PKC ⁇ x and PKC ⁇ in brain but not in feces containing colonocytes.
  • Lane 1 marker; lane 2, PCK ⁇ x in brain
  • Figure 5 shows expression of PKC ⁇ : ⁇ . Rats were fed diets containing corn oil or fish oil and cellulose or pectin and injected with AOM or saline twice in a 2x2x2 factorial design.
  • the development of noninvasive techniques provides early sensitive prognostic information and will greatly enhance the current methods of dietary, pharmacologic, and cancer risk assessment .
  • the present invention describes a noninvasive method utilizing feces containing sloughed colonocytes as a sensitive technique for detecting diagnostic biomarkers in the colon.
  • a novel method of isolating fecal mRNA and by utilizing the extraordinar sensitivity of quantitative reverse transcriptase polymerase chain reaction (RC-PCR) , the method is capable of isolating and quantitating specific messenger RNAs (mRNAs) as candidate biomarkers in feces.
  • mRNAs messenger RNAs
  • the present invention has recognized a correlation between levels of particular biomarkers and the presence of and development of colon cancer.
  • the present invention recognizes that PKC beta II expression in fecal poly A+ RNA is positively correlated with colon tumor incidence, while the expression in fecal poly A+ RNA of PKC zeta is negatively correlated with tumor incidence (Davidson, 1998) . Further, the present invention also recognizes that, inter alia , the expression of these PKC tumor markers is also affected by diet. Specifically and for example, the present invention recognizes that there is a main effect of fat on protein kinase C isotype expression with dietary fish oil versus corn oil having an impact on the relative expression of protein kinase C beta II and zeta isozymes in feces isolated from carcinogen fed animals .
  • the method of the present invention involves a novel technique of isolating mRNA from feces that results in, inter alia , substantial improvement in yield, and stability of isolated poly A+ RNA from exfoliated colonocytes in feces, in a substantially reduced amount of time compared with known techniques in the art (for example, the technique of Davidson, 1995) .
  • the original method (Davidson, 1995) involved the isolation of total RNA from feces followed by poly A+ RNA isolation, which could subsequently be utilized for assessment of colon cancer biomarkers.
  • This older methodology resulted in a relatively low yield of poly A+ RNA, thereby limiting the diagnostic tests which could be performed.
  • the modifications, detailed below, result in approximately 10-fold increase in poly A+ yield, allowing for extensive screening of various colon cancer biomarkers.
  • the method is straight-forward and could be performed by a trained technician. Several samples (up to 12 or more) can be processed at once.
  • RNA isolation technology of the present invention has been validated using the rat chemical carcinogen model . Specifically, we have demonstrated that protein kinase C(PKC) ⁇ and PKC zeta in exfoliated colonocytes may serve as noninvasive markers for development of colon tumors (Davidson, 1998) .
  • the improved method is an improvement on the basic method set forth by Laurie A. Davidson, Yi-Hai Jiang, Joanne R. Lupton, and Robert S. Chapkin in Noninvasive Detection of Putative Biomarkers for Colon Cancer Using Fecal Messenger RNA, published in Cancer Epidemiology, Biomarkers & Prevention, Vol. 4, 643-647, September, 1995 -- this paper is hereby incorporated by reference in its entirety.
  • Instant improvements include, for example, immediately after defecation, poly(A) + RNA is directly isolated from feces using oligo dT cellulose based methodology.
  • the previous published report (Davidson, 1995) involved total RNA isolation from feces followed by poly (A) + isolation from the total RNA preparation.
  • the improved method shortens the mRNA isolation to 5 h (from 2 d with the previous methodology) and significantly increases yield by ⁇ 5- 10 fold.
  • the present invention is suitable for the detection, and quantitation of specific biomarkers whose expression in colon cells and thus, in polyA.
  • RNA isolated from feces correlates with and is predictive of states of colon cancer in a patient.
  • the present invention shows that PKC ⁇ and PKC ⁇ are suitable as biomarkers for monitoring the development of colon cancer.
  • Weanling rats were fed diets containing corn oil or fish oil at 15% by weight and injected with saline (control) or carcinogen (azoxymethane) in a 2 x 2 factorial design.
  • PKC isozyme expression was altered by the presence of tumors (P ⁇ 0.05), with tumor bearing animals having a 3-fold higher ⁇ expression and 6-fold lower ⁇ expression in exfoliated colonocytes than non-tumor bearing animals.
  • PKC ⁇ is more highly expressed in tumors compared with normal colonocytes
  • reduced PKC ⁇ mRNA levels in exfoliated cells from fish oil vs corn oil fed rats indicates a protective effect of fish oil with regard to carcinogen-induced tumor incidence.
  • expression of PKC ⁇ and ⁇ in exfoliated colonocytes may serve as a noninvasive marker for development of colon tumors .
  • At least the following features of the present invention are novel over the prior art: (1) Direct isolation of poly A+ RNA from feces; (2) Ten- fold increase in poly A+ yield with decrease in processing time by more than 50%; (3) Identification of protein kinase C (PKC) beta II as a marker for colon cancer; (4) Identification of PKC zeta as a marker for colon cancer; (5) Use of the novel relative competitive (RC) -PCR method to detect and quantify markers of colon cancer in feces containing exfoliated colon cells; and (6)
  • biomarkers such as:
  • ACBP Acyl CoA Binding Protein
  • NCA Antigen
  • EGFR Epidermal Growth Factor Receptor
  • Interleukin 1 and 2 expression Liver and Intestinal Fatty Acid Binding Protein expression
  • MAP kinase Mitogen-activated protein kinase
  • PCNA Proliferating cell nuclear antigen
  • SCP-2 Sterol Carrier Protein-2
  • Tumor suppressor gene APC mutations Tumor suppressor gene p53 mutations and expression
  • 13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase expression The present invention is suitable for noninvasive detection of PKC isozymes as predictive risk markers for human colon cancer.
  • Rat colon cancer model relates to the determination of the temporal effects of carcinogen on select PKC isozyme fecal mRNAs, and the elucidation of the chemopreventive effects of dietary n-3 polyunsaturated fatty acids (PUFAs) on select PKC isozyme fecal mRNAs .
  • PUFAs polyunsaturated fatty acids
  • EXPERIMENT 1 UTILIZATION OF ISOLATED FECAL POLY A+ RNA TO DETECT COLON CANCER MARKERS
  • Isolation of poly A+ RNA from feces 1. Collect 0.3-2.0 g of rat or human feces. Within 30 min of defecation, add 10 volumes of Lysis Solution (from Poly A+ Pure Kit, Ambion, Austin, TX) . Homogenize feces with a pestle. This homogenate can be stored at -80°C for several months before further processing . 2. Transfer homogenate to sterile 50 ml conical Falcon tube and measure the volume. Add 2 vol Dilution Buffer (Ambion Poly A+ Pure Kit) . Mix by inversion for 10 sec. Centrifuge at 4,000 x g, 15 min, 4°C. Transfer supernatant to a new sterile 50 ml Falcon tube.
  • oligo dT cellulose (Ambion kit) , an amount equal to 10% of the starting fecal weight. Mix by inversion to resuspend the oligo dT resin. 4. Rock the tube on a horizontal shaker at 100-150 rpm at room temperature for 1 h.
  • EXPERIMENT 2 UTILIZATION OF ISOLATED FECAL POLY A+ RNA TO DETECT COLON CANCER MARKERS II
  • L-FABP liver fatty acid binding protein
  • I-FABP intestinal fatty acid binding protein
  • EXPERIMENT 3 DETECTION OF COLON CANCER BIOMARKERS IN EXFOLIATED COLONOCYTRS
  • PKC colonic protein kinase C
  • a sample of liquid feces (20-30 g) is collected during colonscopy as distally as possible, added to 10 volumes of Lysis solution (from Poly A+ Pure Kit, Ambion, Austin, TX) , homogenized with pestle, and stored at -80°C before transport to College Station on dry ice.
  • the resin is then pelletized, supernatant discarded and resulting poly A+ RNA eluted from the resin and used to determine biomarker prevalence (Davidson et al., 1995) .
  • the biomarkers chosen for analysis are PKC-beta II and PKC- zeta, based on our previous research indicating the beta II isoform is positively correlated with colon tumor incidence ( Figure 1) , and the zeta isoform is negatively correlated with tumor incidence.
  • RC-PCR developed in our lab (Jiang et al . , 1996) , to detect the level of expression or each of the biomarkers .
  • Data is analyzed using the GLM models of SAS (SAS, 1985) . Differences between groups are determined by orthogonal contrasts. Data from healthy individuals are compared with those having either polyps or cancer to determine if the presence of the pathologies affect the relative mRNA expression for the two isozymes of PKC. In addition, a contrast of the individuals with polyps vs those with cancer is performed to determine if the expression changes with stage of the tumorigenic process.
  • rats were given two s.c. injections of AOM (Sigma Chemical Co., St. Louis, MO) at a dose of 15 mg/kg body weight or an equal volume of saline (one injection/week) (Chang et al . , 1997) . Animals were killed by C0 2 asphyxiation 36 weeks after the second injection. The colon was subsequently removed and the most distal fecal pellet collected. The pellet was immediately placed in denaturation solution for RNA isolation (Ambion Totally RNA kit, Austin, TX) . The colon was then visually inspected for tumors and tumor typing was determined
  • RNA Isolation (Chang et al . , 1997) . Briefly, tissue sections were fixed in 4% buffered formalin, embedded in paraffin, and stained with eosin and hematoxylin. Slides were then microscopically evaluated for tumors as we have previously described (Chang et al . 1997) . Following removal of suspected tumors for histological evaluation, the remaining colonic sections were gently scraped with a microscopic slide and the mucosa used for determination of stedy-state levels of PKC isozyme mRNA. Histological evaluation of this method indicated that epithelial cells and limina basement down to the muscularis mucosa were removed (Lee et al . , 1995) .
  • Fecal and mucosal total RNA were isolated using Ambion Totally RNA kit.
  • Fecal poly (A) + RNA was subsequently isolated using BioTecx isolation buffers (Houston, TX) and oligio dT cellulose spin columns (Collaborative Biomedical Products, Bedford, MA) as previously described (Davidson et al . , 1995) .
  • the fecal poly (A) + RNA pellet was resuspended in 20 ⁇ l of H 2 O/0.1 mM EDTA and stored at -80°C. Quantification of fecal poly (A) + RNA was performed as previously described (Davidson et al . , 1995).
  • Rat brain cDNA was run as a positive control.
  • PCR was performed using a Perkin-Elmer 2400 thermal cycler (Perkin-Elmer, Foster City, CA) with the following amplification program: 15 s denaturation (94°C) , 15 s annealing (59°C) , and 45 s extension (74°C) for 40 cycles.
  • PCR products were analyzed on a 4% agarose gel followed by ethidium bromide staining. All PCR products were sequenced to ensure the fidelity of amplification (Davidson et al . , 1994).
  • the primer pair for PKC Y was as follows (347 bp) ; forward, 5 ' TTGATGGGGAAGATGAGGAGG-3 ' , Sequence ID No.
  • Rapid competitive RT-PCR was performed in order to semi- quantitatively determine the PKC ⁇ and ⁇ ⁇ I fecal and mucosal mRNA levels as previously described (Jiang et al . , 1996) .
  • relative gene expression was determined by co-amplifying an exogenous DNA target ('internal standard') with a different size than the sample cDNA but with identical 5' and 3' ends. This allows for competition between the sample cDNA and the internal standard for primers (Jiang et al . , 1996) . Internal standards were prepared as described previously (Davidson et al . , 1995). Fecal poly (A) + RNA was processed as decribed above.
  • RNA was reverse transcribed in a 50 ⁇ l reaction and 10 ⁇ l was amplified in the presence of either 140 fg of PKC ⁇ internal standard or 31.2 fg PKC ⁇ IX internal standard.
  • the primer pair for the PKC ⁇ internal standard was (561 bp) : forward, 5'CGATGGGGTGGATGGGATCAAAA-3 ' , Sequence ID No. 5; reverse, 5' GTATTCATGTCAGGGTTGTCTGGATTTCGGGGGCG-3 ', Sequence ID No. 6, and for PKC ⁇ was (680 bp) : forward, 5 ' CGATGGGGTGGATGGGATCAAAA-3 ' , Sequence ID No.
  • the primer pair for PKC ⁇ ZI internal standard was (361 bp) : forward, 5 ' -TATCTGGGATGGGGTGACAACCGAGATCATTGCTTA-3 ' , Sequence ID No. 9; reverse, 5 ' -CGGTCGAAGTTTTCAGCGTTTC-3 ' , Sequence ID No. 10.
  • the primer pair for PKC l ⁇ was (419 bp) : forward, 5'- TATCTGGGATGGGGTGACAACC-3' , Sequence ID No. 11; reverse, 5'- CGGTCGAAGTTTTCAGCGTTTC-3 ' , Sequence ID No. 12.
  • RT-PCR was performed as stated above for PKC ⁇ with the exception that 161 fg of PKC ⁇ internal standard or 29.9 pg of PKC ⁇ : ⁇ internal standard was added to each PCR reaction.
  • PCR products were separated on a 4% agarose gel and stained with ethidium bromide. A representative gel is shown in Figure 3.
  • Gels were scanned and band intensities quantitated with Bioimage software version 2.1 (Ann Arbor, MI) .
  • the relative amount of sample mRNA was calculated by dividing the sample band intensity by the internal standard band intensity.
  • Specific amplification of mRNA was monitored by running PCR negative controls consisting of tubes containing either sample RNA without reverse transcription, reverse transcribed sample without mimic, or mimic only. To ensure reproducibility of results, selected sample were amplified in duplicate. In addition, the fidelity of all PCR reactions was confirmed by DNA sequencing (Jiang et al . , 1996) .
  • Statistical Analysis

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Abstract

A noninvasive method utilizing feces, containing sloughed colonocytes, as a sensitive technique for detecting diagnostic colonic biomarkers. By incorporating the sensitivity of semi-quantitative RT-PCR, this novel method is capable of isolating and quantitating specific eukaryotic mRNAs as candidate biomarkers in feces. In particular, measurement of the relative expression of fecal PKC βII and z serves as a biomarker for development of colon tumors.

Description

TITLE OF THE INVENTION "NONINVASIVE DETECTION OF COLONIC BIOMARKERS USING FECAL
MESSENGER RNA " INVENTORS: ROBERT S. CHAPKIN, a Canadian citizen of College Station, Texas;
LAURIE A. DAVIDSON, a U.S. citizen of College Station, Texas; and
JOANNE R. LUPTON, a U.S. citizen of College Station, Texas . CROSS-REFERENCE TO RELATED APPLICATIONS
Priority of U.S. Provisional Patent Application Serial No. 60/043,048, filed 4 April 1997, incorporated herein by reference, is hereby claimed. STATEMENT 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 mRNA from feces and 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.
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. In addition, the efficacy of sigmoidoscopy screening remains unproven (Levine, 1996) . Because of these limitations, colon cancer cure rates have not improved in the past 30 years (Silverberg, 1988) . 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 method 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 of importance 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 gene expression, 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 colonocytes associated with early stages of colon tumorigenesis . 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 . Several markers for the tumorigenic process are assayable in the practice of the present invention. These markers include, but are not limited to, PKC isozyτnes such as, for example, PKC βll (PKC-betall) and PKCζ (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 patient. (Davidson, 1998) .
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, 1995) . 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 and differentiation. PKCs can be divided into three different sub-categories based on the cofactors needed for activation: classical PKCs ( , βx, βΣI and y) require diacylglycerol
(DAG) and Ca2+ for activation; novel PKCs (δ, θ, η and ε ) are Ca+ independent, but activated by DAG; and atypical PKCs (λ, ι and ζ) are Ca2+ 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 βn protein levels are generally found in very low levels in normal rat colonic mucosa (Davidson et al . , 1994) . However, β protein levels increase in colonic tumors as compared with normal colonic mucosa (Craven et al . , 1992; Wali et al . , 1995). In contrast, PKC ζ mRNA levels are significantly lower in human colorectal tumors than in normal colonic mucosa (Kuranami et al . , 1995). PKC ζ 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 βZI and ζ may serve as biomarkers to monitor the development of colon cancer.
BRIEF DESCRIPTION OF THE DRAWINGS:
For a further understanding of the nature and objects of the present invention, reference should be had to the following detailed description taken in conjunction with the accompanying drawings, in which like parts are given like reference numerals, and wherein:
Figure 1 shows expression of protein kinase C (PKC) beta II (βll) in fecal poly A+ RNA as assessed by semi -quantitative rapid competitive polymerase chain reaction (RC-PCR) , wherein "E" is "expression", "T" is "tumor", and "NT" is "no tumor". Figure 2 shows representative competitive PCR products for determination of Liver-Fatty Acid Binding Protein (L-FABP) expression in fecal poly A+ RNA.
Figure 3 shows representative gel showing rapid competitive- RT/PCR of PKC β . Lane I, marker; lanes 2-5 fecal poly (A) + samples. Upper band is the amplified sample band (419 bp) , the lower band is the amplified internal standard (361 bp) .
Figure 4 shows representative rapid competitive RT-PCR showing expression of PKC βx and PKC γ in brain but not in feces containing colonocytes. Lane 1, marker; lane 2, PCK βx in brain
(639 bp) ; lanes 3 and 4, PKC βx in fecal RNA; lane 5, PKC γ in brain (347 bp) ; lanes 6 and 7, PKC γ in fecal RNA.
Figure 5 shows expression of PKC β:ι . Rats were fed diets containing corn oil or fish oil and cellulose or pectin and injected with AOM or saline twice in a 2x2x2 factorial design.
Feces were collected 36 weeks after the second injection and poly (A) + RNA was isolated. Colonic mucosa was scraped and total
RNA was isolated. Quantitative RT-PCR was performed using primers specific for PKC βXI. PCR products were separated on 4% agarose gels, stained with ethidium bromide, photographed and scanned on a densitometer to quantitate. Y-axis represents band intensities
(ODxmm2) . (A) Expression of PKC ττ in fecal mRNA from rats with or without tumors (mean ± SEM; P = 0.026; n = 12-29) . (B) Expression of PKC βι: in mucosal RNA from rats injected with azoxymethane (AOM) or saline (mean ± SEM; P = 0.036; n = 16-20). "BI" is "band intensity", "T" is "tumor", "NT" is "no tumor", "I" is "injection", and "S" is "saline".
Figure 6 show expression of PKC ζ in fecal mRNA from rats injected with azoxymethane (AOM) or saline. See Figure 5 legend for further details (mean ± SEM; P = 0.017; n = 21-22).
Figure 7 shows expression of PKC βXI/PKC ζ ratio in fecal mRNA from rats with or without tumors. See Figure 5 legend for further details (mean ± SEM; P = 0.025; n = 9-26).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT:
The development of noninvasive techniques, as shown in this invention, provides early sensitive prognostic information and will greatly enhance the current methods of dietary, pharmacologic, and cancer risk assessment . The present invention describes a noninvasive method utilizing feces containing sloughed colonocytes as a sensitive technique for detecting diagnostic biomarkers in the colon. By incorporating a novel method of isolating fecal mRNA and by utilizing the exquisite sensitivity of quantitative reverse transcriptase polymerase chain reaction (RC-PCR) , the method is capable of isolating and quantitating specific messenger RNAs (mRNAs) as candidate biomarkers in feces. Further, the present invention has recognized a correlation between levels of particular biomarkers and the presence of and development of colon cancer.
For example, but not in a limiting sense, the present invention recognizes that PKC beta II expression in fecal poly A+ RNA is positively correlated with colon tumor incidence, while the expression in fecal poly A+ RNA of PKC zeta is negatively correlated with tumor incidence (Davidson, 1998) . Further, the present invention also recognizes that, inter alia , the expression of these PKC tumor markers is also affected by diet. Specifically and for example, the present invention recognizes that there is a main effect of fat on protein kinase C isotype expression with dietary fish oil versus corn oil having an impact on the relative expression of protein kinase C beta II and zeta isozymes in feces isolated from carcinogen fed animals .
The method of the present invention involves a novel technique of isolating mRNA from feces that results in, inter alia , substantial improvement in yield, and stability of isolated poly A+ RNA from exfoliated colonocytes in feces, in a substantially reduced amount of time compared with known techniques in the art (for example, the technique of Davidson, 1995) .
Approximately one-sixth to one-third of normal adult colonic epithelial cells are shed daily (Potten, 1979) . Isolation of colonocytes from feces has been reported by another group (Albaugh, 1992) . This method is very time consuming and results in an extremely low yield such that useful diagnostic tests on the isolated cells are limited and very labor intensive. We therefore designed a technique to directly isolate poly A+ RNA from feces containing exfoliated colonocytes. The poly A+ isolated can be used to probe for early markers for colon cancer or other colorectal diseases .
Specifically, we have redesigned the protocol of the prior art (for example, Davidson, 1995) to significantly simplify and enhance the process, resulting in a greatly enhanced yield. In addition, we have combined the improved isolation protocol with an extremely sensitive detection technique, called rapid competitive polymerase chain reaction (RC-PCR) , a technology developed in our laboratory.
The original method (Davidson, 1995) involved the isolation of total RNA from feces followed by poly A+ RNA isolation, which could subsequently be utilized for assessment of colon cancer biomarkers. This older methodology resulted in a relatively low yield of poly A+ RNA, thereby limiting the diagnostic tests which could be performed. The modifications, detailed below, result in approximately 10-fold increase in poly A+ yield, allowing for extensive screening of various colon cancer biomarkers. In addition, the method is straight-forward and could be performed by a trained technician. Several samples (up to 12 or more) can be processed at once.
The refined RNA isolation technology of the present invention has been validated using the rat chemical carcinogen model . Specifically, we have demonstrated that protein kinase C(PKC) βπ and PKC zeta in exfoliated colonocytes may serve as noninvasive markers for development of colon tumors (Davidson, 1998) .
The improved method is an improvement on the basic method set forth by Laurie A. Davidson, Yi-Hai Jiang, Joanne R. Lupton, and Robert S. Chapkin in Noninvasive Detection of Putative Biomarkers for Colon Cancer Using Fecal Messenger RNA, published in Cancer Epidemiology, Biomarkers & Prevention, Vol. 4, 643-647, September, 1995 -- this paper is hereby incorporated by reference in its entirety. Instant improvements include, for example, immediately after defecation, poly(A)+RNA is directly isolated from feces using oligo dT cellulose based methodology. The previous published report (Davidson, 1995) involved total RNA isolation from feces followed by poly (A) + isolation from the total RNA preparation. The improved method shortens the mRNA isolation to 5 h (from 2 d with the previous methodology) and significantly increases yield by ~5- 10 fold.
In still another feature of the present invention, and an improvement over the prior art, the present invention is suitable for the detection, and quantitation of specific biomarkers whose expression in colon cells and thus, in polyA. RNA isolated from feces, correlates with and is predictive of states of colon cancer in a patient.
For example, the present invention shows that PKCβπ and PKC ζ are suitable as biomarkers for monitoring the development of colon cancer. The modulation of these putative biomarkers -- affected by both the presence or absence of colon tumor and by dietary factors -- is shown herein. Weanling rats were fed diets containing corn oil or fish oil at 15% by weight and injected with saline (control) or carcinogen (azoxymethane) in a 2 x 2 factorial design. Fresh fecal samples (n=6 per diet) were collected 36 weeks post injection, poly A+ RNA was isolated and quantitative RC-PCR performed using primers to PKCβπ and ζ . PKC isozyme expression was altered by the presence of tumors (P<0.05), with tumor bearing animals having a 3-fold higher βπ expression and 6-fold lower ζ expression in exfoliated colonocytes than non-tumor bearing animals. In addition, there was a main effect of fat with rats fed fish oil and injected with carcinogen having 5-fold lower βπ levels than animals consuming corn oil injected with carcinogen (P<0.05) . Since PKCβπ is more highly expressed in tumors compared with normal colonocytes, reduced PKCβπ mRNA levels in exfoliated cells from fish oil vs corn oil fed rats indicates a protective effect of fish oil with regard to carcinogen-induced tumor incidence. We propose that expression of PKCβπ and ζ in exfoliated colonocytes may serve as a noninvasive marker for development of colon tumors .
Also novel is the use of the rapid competitive PCR method (as first disclosed in Jang, 1996) to sensitively quantify biomarker expression in fecal poly A+ RNA. This method is described in detail in Rapid competitive PCR determination of relative gene expression in limiting tissue samples. Yi-Hai Jiang, Laurie A. Davidson, Joanne R. Lupton, and Robert S. Chapkin, Clinical Chemistry, 42:2, 227-231 (1996), which is hereby incorporated by reference in its entirety. This method is ideal for limiting RNA samples, since it requires only a single PCR reaction in order to determine relative gene expression. In contrast, the more traditional mimic reverse transcriptase (RT) -PCR technique requires a series of 5 to 7 PCR reactions in order to quantitate gene expression.
For example and for illustrative purposes only, at least the following features of the present invention are novel over the prior art: (1) Direct isolation of poly A+ RNA from feces; (2) Ten- fold increase in poly A+ yield with decrease in processing time by more than 50%; (3) Identification of protein kinase C (PKC) beta II as a marker for colon cancer; (4) Identification of PKC zeta as a marker for colon cancer; (5) Use of the novel relative competitive (RC) -PCR method to detect and quantify markers of colon cancer in feces containing exfoliated colon cells; and (6)
Validation of fecal homogenate stability after processing and storage prior to poly A+ isolation. The methods of the present invention can be utilized to detect predictive risk markers for colon cancer including, but not limited to, biomarkers such as:
Acyl CoA Binding Protein (ACBP) expression
Arginase expression bax expression bcl-2 expression
Bcl-XL expression
Bcl-Xs expression c-myc expression Carcinoembryonic Antigen (CEA) and Nonspecific Crossreacting
Antigen (NCA) expression
CD44 Glycoprotein expression
Cyclin-dependent kinase inhibitors (p27, pl6ink4) expression
Cyclin-dependent kinase cdk2/cdc2, cyclin Dl, and cdk4 expression Decay Activating Factor expression
E-Cadherin cell adhesion molecule expression
Epidermal Growth Factor Receptor (EGFR) expression
Fatty Acid Synthase expression
Fecal alpha- 1 Antitrypsin expression GDP-L- fucose:beta-D-galactoside-alpha-2-L-fucosyltranferase expression
Glutathione S-Transferase and Glutathione S-Transferase expression
Histone H3 expression
Interleukin 1 and 2 expression Liver and Intestinal Fatty Acid Binding Protein expression
Mitogen-activated protein kinase (MAP kinase) expression
MAP kinase phosphatase-1 expression NO synthase, inducible expression
Ornithine Decarboxylase expression p21 waf 1/cip 1 expression
P-glycoprotein, the mdr gene product expression Plasminogen Activator expression
Proliferating cell nuclear antigen (PCNA) expression
Prostaglandin Synthase Type II (COX II) expression
Protein Kinase A, Type I and II Isozyme expression
Protein Kinase C , δ, e, λ, i, μ expression Ras oncogene expression
Ras oncogene mutations
Stearoyl-CoA desaturase expression
Sterol Carrier Protein-2 (SCP-2) expression
Telomerase expression Transforming Growth Factor-beta I and II expression
Transforming Growth Factor-beta type II Receptor expression and mutations
Tumor Necrosis Factor Alpha expression
Tumor suppressor gene APC mutations Tumor suppressor gene p53 mutations and expression
Tumor suppressor gene retinoblastoma (Rb) protein expression
Villin expression
1, 25-dihydroxyvitamin D3 Receptor expression, and
13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase expression. The present invention is suitable for noninvasive detection of PKC isozymes as predictive risk markers for human colon cancer.
We have already validated the use of select PKC isozymes as predictive risk markers using the rat experimental colon cancer model (Davidson, 1998) . In addition, we have isolated human poly A+ RNA from feces and detected the presence of PKC isozymes.
Additionally, the present invention, using, for example the
Rat colon cancer model, relates to the determination of the temporal effects of carcinogen on select PKC isozyme fecal mRNAs, and the elucidation of the chemopreventive effects of dietary n-3 polyunsaturated fatty acids (PUFAs) on select PKC isozyme fecal mRNAs . The development of noninvasive techniques, as shown in this proposal, provide early sensitive prognostic information and greatly enhance our current methods of dietary and pharmacologic risk assessment . The method reported herein is novel since we are the first to report that poly A+ RNA from exfoliated colonocytes can be isolated directly from feces and can be used to probe for markers of colon cancer. We have also identified several markers present in fecal poly A+RNA that predict for colon cancer and are affected by, inter alia , dietary factors. EXPERIMENT 1: UTILIZATION OF ISOLATED FECAL POLY A+ RNA TO DETECT COLON CANCER MARKERS
Further details related to this method may be found in the article by Lauria A. Davidson, Christin M. Ayτnond, Yi-Hai Jiang, Nancy D. Turner, Joanne R. Lupton and Robert S. Chapkin, entitled "Non- invasive detection of fecal protein kinase C βIX and ζ messenger RNA: putative biomarkers for colon cancer", published in Carcinogenesis, vol. 19, no. 2, pp. 253-257, 1998, which is hereby incorporated by reference in its entirety.
EXPERIMENTAL METHODS:
Isolation of poly A+ RNA from feces: 1. Collect 0.3-2.0 g of rat or human feces. Within 30 min of defecation, add 10 volumes of Lysis Solution (from Poly A+ Pure Kit, Ambion, Austin, TX) . Homogenize feces with a pestle. This homogenate can be stored at -80°C for several months before further processing . 2. Transfer homogenate to sterile 50 ml conical Falcon tube and measure the volume. Add 2 vol Dilution Buffer (Ambion Poly A+ Pure Kit) . Mix by inversion for 10 sec. Centrifuge at 4,000 x g, 15 min, 4°C. Transfer supernatant to a new sterile 50 ml Falcon tube. 3. Add oligo dT cellulose (Ambion kit) , an amount equal to 10% of the starting fecal weight. Mix by inversion to resuspend the oligo dT resin. 4. Rock the tube on a horizontal shaker at 100-150 rpm at room temperature for 1 h.
5. Pellet the oligo dT resin by centrifuging at 4,000 x g, 3 min, 4°C. Remove and discard the supernatant.
6. Resuspend the resin with 6-10 ml Binding Buffer (Ambion kit) and mix well. Pellet resin as described in step 5 and discard. Repeat this step two more times.
7. Resuspend resin with 6-10 ml Wash Buffer (Ambion kit) and mix well. Centrifuge as described in step 5 and discard supernatant. Repeat this wash two more times. 8. Resuspend the resin in 1-2 ml wash buffer and transfer to a spin column in a 1.5 ml microfuge tube (Ambion kit) . Centrifuge at 5,000 x g, 10 sec, room temperature to remove the supernatant. Place spin column into a new microfuge tube. 9. Add 300 μl Elution Buffer (Ambion kit) which has been pre-warmed to 65°C. Immediately centrifuge at 5,000 x g, room temperature, 30 sec and save the eluate. Add another 300 μl pre-warmed Elution buffer and centrifuge at 5,000 x g, room temperature, 30 sec. Combine eluate with previous eluate. Discard the spin column. 10. Precipitate the poly A+ RNA by adding 60 μl 5M ammonium acetate, 10 μg glycogen and 2.5 vol 100% ethanol . Place at -80°C for 1 h. Recover poly A+ RNA by centrifugation at 12,000 x g, 20 min, 4°C. Remove and discard supernatant, add 0.5 ml chilled 80% ethanol to the tube, invert tube gently. Centrifuge at 12,000 x g, 5 min. Remove and discard the ethanol. Resuspend the poly A+ pellet in 60-200 μl water/0.1 mM EDTA. Vortex gently to resuspend.
This purified A+ RNA is used for colon cancer biomarker studies such as those detailed below. RESULTS :
Using the method described above, fecal poly A+RNA from rats injected with carcinogen or saline (control) was examined for colon cancer biomarkers. We determined that protein kinase C beta II expression in fecal poly A+ RNA is positively correlated with colon tumor incidence (Figure 1A) , while protein kinase C zeta is negatively correlated with tumor incidence (Figure IB) .
The ratio of PKC beta II to zeta is also strongly correlated with tumor presence (Table 1) .
Table 1. Relationship between PKC beta II: zeta ratio and tumor incidence .
PKC βll: ζ ratio Animals with tumors 4.27 ± 2.37 p=0.02 Animals without tumors 0.71 ± 0.14
EXPERIMENT 2 : UTILIZATION OF ISOLATED FECAL POLY A+ RNA TO DETECT COLON CANCER MARKERS II
We are actively pursuing liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP) as additional colon cancer biomarkers. Preliminary data indicates that expression of L-FABP and I-FABP are significantly depressed in carcinogen treated animals. Figure 2 (shown below) documents a typical gel containing rapid competitive PCR products for L-FABP. The upper band (sample, 390 base pairs) , the lower band (internal standard, 336 base pairs) .
EXPERIMENT 3 : DETECTION OF COLON CANCER BIOMARKERS IN EXFOLIATED COLONOCYTRS There is strong evidence that select dietary factors play a key role in the development of colon cancer. We have recently shown that dietary fat and fiber can alter colonic protein kinase C (PKC) activity and isozyme expression, which may influence the malignant transformation process (Chapkin, 1993; Jiang, 1997). In this study we examined the role of diet on the expression of colonic PKC βπ and ζ as markers for the development of colon cancer using a non- invasive technique. Weanling rats were fed diets containing corn oil or fish oil at 15% by weight and injected with saline or azoxymethane (AOM; 15mg/kg body weight) feces were collected 36 weeks after the second injection, poly A+ RNA was isolated and quantitative RC-PCR performed using primers to PKC βπ and ζ . PKC isozyme expression was altered by the presence of tumors (p<0.05), with tumor-bearing animals having 3-fold higher βπ expression and 6-fold lower ζ expression in exfoliated colonocytes than non-tumor bearing animals. In addition, there was a main effect of fat with rats fed fish oil and injected with AOM having 5- fold lower βπ levels than animals consuming corn oil injected with AOM (p,0.05) . Since PKC βπ is more highly expressed in tumors compared with normal colonocytes, reduced βπ mRNA levels in exfoliated cells from fish oil vs corn oil fed rats indicates a protective effect with regard to carcinogen- induced tumor incidence. We propose that expression of PKC βπ or ζ in exfoliated coloncytes may serve as a non-invasive marker for development of colon tumors. A sensitive technique for the detection of colon cancer is important since early diagnosis can substantially reduce mortality. EXPERIMENT 4: HUMAN CLINICAL TRIALS METHODOLOGY : Clinical . Patients presenting for colonscopy are individually typed as: 1) being free of colon cancer, 2) having adenomatous polyps (considered preneoplastic), or 3) having colon cancer. Thirty subjects for each group are recruited in order to reduce the effect of individual variation on the analysis. The sample size is based upon testing equality of means at α=0.05 and detecting a difference of size σ with a probability of 0.95 (Pearson and Hartley, 1966) . To achieve this level of statistical power requires 26 individuals. Thus 30 patients protect against loss of power if a sample becomes damaged during storage or analysis. Because patients randomly present for treatment, and the disease state will not be a controlled factor, we assume the data will be randomly distributed among the potentital population. Because patients with cancer are the limiting factor in sample collection, the first 30 individuals with cancer are those selected for inclusion in the study. In order to adjust for variation related to patient age, individuals free of colon cancer and those with polyps are age-matched to patients with colon cancer. Further, patients with polyps or free of pathology are selected after a sample is collected from a cancer patient.
A sample of liquid feces (20-30 g) is collected during colonscopy as distally as possible, added to 10 volumes of Lysis solution (from Poly A+ Pure Kit, Ambion, Austin, TX) , homogenized with pestle, and stored at -80°C before transport to College Station on dry ice.
Laboratory. Samples are stored at -80°C until being thawed on ice, and the homogenate transferred to sterile tubes and the volume measured. Dilution buffer (Ambion Poly A+ Pure Kit) is added and the contents mixed by inversion, and then centrifuged at 4,000 x g for 15 min at 4°C. Oligio dT resin is added to the sample and the supernatant is then mixed by inversion to resuspend the oligo dT resin prior to rocking the tube on a horizontal shaker. Following centrifugation, the resin pellet is resuspended with binding buffer (Ambion Kit) . The resin is then pelletized, supernatant discarded and resulting poly A+ RNA eluted from the resin and used to determine biomarker prevalence (Davidson et al., 1995) . The biomarkers chosen for analysis are PKC-beta II and PKC- zeta, based on our previous research indicating the beta II isoform is positively correlated with colon tumor incidence (Figure 1) , and the zeta isoform is negatively correlated with tumor incidence. We use RC-PCR, developed in our lab (Jiang et al . , 1996) , to detect the level of expression or each of the biomarkers .
Data is analyzed using the GLM models of SAS (SAS, 1985) . Differences between groups are determined by orthogonal contrasts. Data from healthy individuals are compared with those having either polyps or cancer to determine if the presence of the pathologies affect the relative mRNA expression for the two isozymes of PKC. In addition, a contrast of the individuals with polyps vs those with cancer is performed to determine if the expression changes with stage of the tumorigenic process.
Experiment 5: Detection of Fecal Protein Kinase C βIX and . Messenger RNA Colon Cancer Biomarkers
The animal use protocol conformed to NIH guidelines and was approved by the University Animal Care Committee of Texas A&M University. Forty-eight male weanling Sprague-Dawley rats (Harlan Sprague-Dawley, Houston, TX) were randomly divided into eight groups (six rats/treatment) as previously described (Chang et al . , 1997) . Briefly, after a 1-week acclimatization period of consuming standard rat chow, rats were stratified by body weight and assigned to one of eight treatments in a 2x2x2 factorial design with two types of fat (corn oil or fish oil) , two types of fiber (pectin or cellulose) and two types of injection (carcinogen or saline) . Animals received the assigned diet until the conclusion of the study (36 weeks after carcinogen/saline second injection) . Animals were housed individually in suspended cages in a temperature and humidity controlled animal facility with a 12 h light/dark cycle. Food and distilled water were provided ad libi tum . Forty-eight h food intakes and fecal outputs were measured during the study.
Body weights were recorded weekly.
Carcinogen Administration and Fecal Collection:
After 1 week of consuming semipurified diets, rats were given two s.c. injections of AOM (Sigma Chemical Co., St. Louis, MO) at a dose of 15 mg/kg body weight or an equal volume of saline (one injection/week) (Chang et al . , 1997) . Animals were killed by C02 asphyxiation 36 weeks after the second injection. The colon was subsequently removed and the most distal fecal pellet collected. The pellet was immediately placed in denaturation solution for RNA isolation (Ambion Totally RNA kit, Austin, TX) . The colon was then visually inspected for tumors and tumor typing was determined
(Chang et al . , 1997) . Briefly, tissue sections were fixed in 4% buffered formalin, embedded in paraffin, and stained with eosin and hematoxylin. Slides were then microscopically evaluated for tumors as we have previously described (Chang et al . 1997) . Following removal of suspected tumors for histological evaluation, the remaining colonic sections were gently scraped with a microscopic slide and the mucosa used for determination of stedy-state levels of PKC isozyme mRNA. Histological evaluation of this method indicated that epithelial cells and limina propria down to the muscularis mucosa were removed (Lee et al . , 1995) . RNA Isolation:
Fecal and mucosal total RNA were isolated using Ambion Totally RNA kit. Fecal poly (A) + RNA was subsequently isolated using BioTecx isolation buffers (Houston, TX) and oligio dT cellulose spin columns (Collaborative Biomedical Products, Bedford, MA) as previously described (Davidson et al . , 1995) . The fecal poly (A) + RNA pellet was resuspended in 20 μl of H2O/0.1 mM EDTA and stored at -80°C. Quantification of fecal poly (A) + RNA was performed as previously described (Davidson et al . , 1995). Briefly, samples were quantitated by blotting fecal poly (A) + onto a positively charged nylon membrain (Boehringer Mannheim, Indianapolis, IN) . A biotinylated oligo (dT) probe (Promega, Madison, WI) was hybridized to the RNA followed by detection with streptavidin- alkaline phosphatase . Dilutions of colonic mucosal total RNA of known concentration (as determined from absorbance at 260 nm) were also blotted to generate a standard curve. For concentration calculations, it was assumed that poly (A) + RNA constitutes 3% of total RNA. Mucosal total RNA was quantitated by absorbance at 260nm. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Assay for Negative Controls (PKC y and PKC βτ)
Aliquots of 40 ng fecal poly (A) + RNA in a 50 μl reaction were reverse transcribed to generate first strand cDNA using Superscript II reverse transcriptase (Gibco-BRL, Gaithersburg, MD) as previously described (Davidson et al . , 1995) . PCR was performed using Expand High Fidelity polymerase (Boehringer-Mannheim, Indianapolis, IN) . The 50 μl PCR reaction consisted of lx PCR buffer, 2% DMSO, 0.05 mM dNTPs, 1.5 mM MgCl2, 20 pmol each of forward and reverse primer, 2.6 U Expand High Fidelity polymerase and 5-10 μl of RT reaction. Rat brain cDNA was run as a positive control. PCR was performed using a Perkin-Elmer 2400 thermal cycler (Perkin-Elmer, Foster City, CA) with the following amplification program: 15 s denaturation (94°C) , 15 s annealing (59°C) , and 45 s extension (74°C) for 40 cycles. PCR products were analyzed on a 4% agarose gel followed by ethidium bromide staining. All PCR products were sequenced to ensure the fidelity of amplification (Davidson et al . , 1994). The primer pair for PKC Y was as follows (347 bp) ; forward, 5 ' TTGATGGGGAAGATGAGGAGG-3 ' , Sequence ID No. 1; reverse, 5 ' -GAAATCAGCTTGGTCGATGCTG-3 ' , Sequence ID No. 2. The primer pair for PKC βx was as follows (639 bp) : forward, 5 ' TGTGATGGAGTATGTGAACGGGGG- 3 ', Sequence ID No. 3; reverse, TCGAAGTTGGAGGTGTCTCGCTTG-3 ' , Sequence ID No . 4. Rapid Competitive Reverse Transcription-Polymerase Chain Reaction Assay for Fecal and Mucosal PKC and βTI
Rapid competitive RT-PCR was performed in order to semi- quantitatively determine the PKC ζ and βΣI fecal and mucosal mRNA levels as previously described (Jiang et al . , 1996) . Using this method, relative gene expression was determined by co-amplifying an exogenous DNA target ('internal standard') with a different size than the sample cDNA but with identical 5' and 3' ends. This allows for competition between the sample cDNA and the internal standard for primers (Jiang et al . , 1996) . Internal standards were prepared as described previously (Davidson et al . , 1995). Fecal poly (A) + RNA was processed as decribed above. In addition, 6 μg of mucosal total RNA was reverse transcribed in a 50 μl reaction and 10 μl was amplified in the presence of either 140 fg of PKC ζ internal standard or 31.2 fg PKC βIX internal standard. The primer pair for the PKC ζ internal standard was (561 bp) : forward, 5'CGATGGGGTGGATGGGATCAAAA-3 ' , Sequence ID No. 5; reverse, 5' GTATTCATGTCAGGGTTGTCTGGATTTCGGGGGCG-3 ', Sequence ID No. 6, and for PKC ζ was (680 bp) : forward, 5 ' CGATGGGGTGGATGGGATCAAAA-3 ' , Sequence ID No. 7; reverse, 5 ' -GTATTCATGTCAGGGTTGTCTG-3 ' , Sequence ID No. 8. The primer pair for PKC βZI internal standard was (361 bp) : forward, 5 ' -TATCTGGGATGGGGTGACAACCGAGATCATTGCTTA-3 ' , Sequence ID No. 9; reverse, 5 ' -CGGTCGAAGTTTTCAGCGTTTC-3 ' , Sequence ID No. 10. The primer pair for PKC lτ was (419 bp) : forward, 5'- TATCTGGGATGGGGTGACAACC-3' , Sequence ID No. 11; reverse, 5'- CGGTCGAAGTTTTCAGCGTTTC-3 ' , Sequence ID No. 12. RT-PCR was performed as stated above for PKC γ with the exception that 161 fg of PKC ζ internal standard or 29.9 pg of PKC β:ι internal standard was added to each PCR reaction. PCR products were separated on a 4% agarose gel and stained with ethidium bromide. A representative gel is shown in Figure 3. Gels were scanned and band intensities quantitated with Bioimage software version 2.1 (Ann Arbor, MI) . The relative amount of sample mRNA was calculated by dividing the sample band intensity by the internal standard band intensity. Specific amplification of mRNA was monitored by running PCR negative controls consisting of tubes containing either sample RNA without reverse transcription, reverse transcribed sample without mimic, or mimic only. To ensure reproducibility of results, selected sample were amplified in duplicate. In addition, the fidelity of all PCR reactions was confirmed by DNA sequencing (Jiang et al . , 1996) . Statistical Analysis:
Data were analyzed to determine the effects of carcinogen and presence of tumor using one-way ANOVA. When P-values were <0.05 for the effects of tumor or carcinogen, total means were separated using Duncan's multiple range test. RESULTS :
Colon Carcinoma Incidence :
There was no evidence of carcinoma in any saline injected animal, whereas 64% of carcinogen injected rats had carcinomas at the time of death. Effect of Carcinogen and Presence of Tumor on Fecal and Mucosal PKC Isozyme mRNA Levels:
To determine the specificity of this non-invasive procedure, PKC β- and γ primers were used as negative controls (Davidson et al . , 1995; Davidson et al . , 1994). No amplified products were detected after 40 cycles in any fecal poly (A) + or scraped colonic mucosa total RNA samples (Figure 4). However, both isozymes were detected using brain total RNA (positive control) .
PCR products for PCK βXI were detected in all fecal and mucosal samples. Samples processed without reverse transcriptase were used as negative controls and yielded no detectable amplified products (data not shown) . Using semiquantitative mimic PCR, it was determined that fecal PKC βXI mRNA levels were altered by the presence of a tumor with tumor-bearing animals having 3 -fold higher (P<0.05) PKC βIX expression as compared with animals without tumors, as seen in Figure 5A. In contrast, there was no effect of tumor incidence on mucosal PKC β expression. However, there was a significant effect (P<0.05) of injection on mucosal PKC βIT expression. Specifically, carcinogen (AOM) -injection increased mucosal PKC βIΣ mRNA expression compared with saline controls (Figure 5B) .
There were no treatment effects on colonic mucosal PKC ζ mRNA levels (data not shown) (Jiang, 1997) . In contrast, fecal PKC ζ expression in rats injected with AOM was less than half (P < 0.05) that of saline control, as shown in Figure 6. Since tumor incidence exerts a reciprocal effect on fecal PKC ζ on PKC βu mRNA expression, data were also expressed as the ratio between PKC βI]: and PKC ζ. The isozyme ratio was strongly relate to tumor incidence, i.e. ratio for animals with tumors was 2.18 ± 1.25
(n=9) , animals without tumors was 0.50 ± 0.16 [n = 26), P = 0.025
(Figure 7) . These data demonstrate that PKC β„ and PKC ζ may serve as non-invasive markers for developmment of colon tumors.
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36170005 . pac/david

Claims

CLAIMS :
1. A method for non- invasively determining the expression of PKC isozymes in colonocytes of a patient comprising: directly isolating from a patient poly (A) + RNA from feces, containing sloughed colonocytes; semi-quantitatively assaying the isolated poly (A) + RNA and determining the level, in the isolated poly (A) + RNA, of mRNA encoding at least one PKC isozyme.
2. The method of claim 1, wherein at least one PKC isozyme is selected from the group consisting of PKC ╬╢, PKC ╬▓I]:, PKC ╬│, and PKC ╬▓x.
3. The method of claim 1, wherein the level of expression of PKC isozymes PKC ╬╢, and PKC ╬▓n is determined.
4. The method of claim 3, wherein the ratio of expression of PKC ζ to PKC βIΣ is determined.
5. The method of claim 4, further comprising the step of comparing the ratio of expression of PKC ╬╢ to PKC ╬▓╬╣:r in said patient with similarly determined ratios of PKC ╬╢ to PKC ╬▓r╬╣ in other patients with known conditions.
6. The method of claim 5, wherein the ratio of expression of PKC ╬╢ to PKC ╬▓╬╣: in said patient is compared with similarly determined ratios of PKC ╬╢ to PKC ╬▓ΓÇ₧ in at least two other patients, one with colon cancer and one without colon cancer.
7. The method of claim 4, wherein the level of PKC ╬╢ is determined using the primer pair having sequence nos. 7 and 8, and the level of PKC ╬▓IX is determined using the primer pair selected from the group consisting of the primers having Sequence ID Numbers 11, and 12.
8. A method for non-invasively detecting colonic biomarkers in a patient using fecal messenger RNA comprising: directly isolating, from said patient, poly (A) + RNA from feces containing sloughed colonocytes; semi-quantitatively assaying the isolated poly (A) + RNA and determining the level, in the isolated poly (A) + RNA, of mRNA encoding at least one colonic biomarker.
9. The method of claim 8, wherein said at least one colonic biomarker is a specific isozyme of PKC.
10. The method of claim 9, wherein the specific isozyme of PKC is selected from the group consisting of PKC ╬╢, PKC ╬▓ΓÇ₧, PKC ╬│, and PKC ╬▓z.
11. The method of claim 10, wherein the level of expression of PKC isozymes PKC ╬╢, and PKC ╬▓XI is determined.
12. The method of claim 11, wherein the ratio of expression of PKC ╬╢ to PKC ╬▓ΓÇ₧ is determined.
13. The method of claim 12, further comprising the step of comparing the ratio of expression of PKC ╬╢ to PKC ╬▓XI in said patient with similarly determined ratios of PKC ╬╢ to PKC ╬▓IX in at least two other patients, one with colon cancer and one without colon cancer.
14. The method of claim 11, wherein the level of PKC ╬╢ is determined using at least one primer selected from the group consisting of the primers having Sequence ID Numbers 5, 6, 7, and 8, and the level of PKC ╬▓IX is determined using at least one primer selected from the group consisting of the primers having Sequence ID Numbers 9, 10, 11, and 12.
15. A method for non-invasively screening for colon cancer in a patient comprising: detecting the expression of at least one specific isozyme in sloughed colonocytes in said patient's feces; correlating the expression of said at least one specific isozyme with the presence or absence of colon cancer in said patient .
16. The method of claim 15, wherein said at least one specific isozyme is selected from the group consisting of PKC ╬╢, PKC ╬▓IX, PKC Y, and PKC ╬▓x.
17. The method of claim 16, wherein the level of expression of PKC isozymes PKC ζ, and PKC βIΣ is determined.
18. The method of claim 17, wherein the ratio of expression of PKC ╬╢ to PKC ╬▓XI is determined.
19. The method of claim 4, further comprising the step of comparing the ratio of expression of PKC ╬▓XI to PKC ╬╢ in said patient with similarly determined ratios of PKC ╬▓IX to PKC ╬╢ in at least two other patients, one with colon cancer and one without colon cancer.
20. The method of claim 17, wherein the level of PKC ╬╢ is determined using at least one primer selected from the group consisting of the primers having Sequence ID Numbers 5, 6, 7, and 8, and the level of PKC ╬▓XI is determined using at least one primer selected from the group consisting of the primers having Sequence ID Numbers 9, 10, 11, and 12.
PCT/US1998/006698 1997-04-04 1998-04-03 Noninvasive detection of colonic biomarkers using fecal messenger rna WO1998045480A1 (en)

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