WO1988001301A1

WO1988001301A1 - Detection marker for colonic lesions

Info

Publication number: WO1988001301A1
Application number: PCT/US1987/001889
Authority: WO
Inventors: Sebastiano Gattoni-Celli; Kurt J. Isselbacher
Original assignee: The General Hospital Corporation
Priority date: 1986-08-13
Filing date: 1987-08-06
Publication date: 1988-02-25
Also published as: JPH02500323A; EP0318502A1

Abstract

A method for the detection of neoplastic disease of the colon in an RNA containing colonic sample, which comprises contacting the RNA of the sample with a detectably labeled H-LTR or H-env nucleotide probe; and detecting the amount of hybridization between the probe and the RNA, relative to the amount in a known, non-cancerous, colonic sample.

Description

TITLE OF THE INVENTION

DETECTION MARKER FOR COLONIC LESIONS

This invention was made using U.S. Government funds. The U.S. Government has rights in the inven¬ tion.

Field of the Invention

This invention relates to molecular biological approaches to the diagnosis of pre alignant and malig¬ nant colonic tissues, especially by nucleic ,acid hy- bridization procedures.

Description o-fi the Background Art

Im unological approaches to the diagnosis of pre¬ malignant and malignant colonic tissues have been re¬ ported by Schlom et al. at the National Institutes of Health of the United States. Thor et al.. Nature 311:562 (1984) and Schlom et al., J. Cell. Biochem. Supp. ^Aj36 (1985) disclose the detection of specific oncogene products in human colonic tissue. These reports show the use of monoclonal antibody (Mab) to define differential ras gene expression of the p21 protein in benign and malignant colonic diseases. Immunohistochemical analyses of individual cells with¬ in tissue sections revealed differences in ras p21 expression in colon carcinomas, compared with normal colonic epithelium, benign colon tumors, and inflam¬ matory or dysplastic colon lesions. The authors con¬ cluded that ras p21 is probably a relatively late event in colon carcinogenesis.

In Thor et al.. Lab. Invest. :68A (1985); Hand et al., Proc. Nat. Acad. Sci. USA £1.:5227 (1984); and Hand et al., Hybridoma 68A (1985), the research group examined; a variety of cancer tissues for ras p21 expression using Mab to p21. Ras gene expression was found in 49% of colonic adenosarcoma, 63% of ductal carcinomas, 10% of benign breast tumors, and in 0% of benign colon lesions and normal colons. The majority of human colonic and mammary gland adenocarcinomas exhibited ras gene expression, whereas the majority of abnormal ducts and lobules from fibroadenoma and fibrocystic disease ^" patients were negative. The authors maintained that these findings form the basis of a quantitative RIA for a ras translational product, and provide a means to evaluate ras p21 expression within normal cells, as well as within benign, pre- malignant, and malignant lesions.

Type C-related human endogenous retroviral se¬ quences have recently been discovered and characteriz¬ ed. They appear to play a role in controlling trans¬ cription of linked genes, especially during cell transformation.

Several - publications have appeared discussing human endogenous retroviral sequences. For example, Callahan et al.. Science 228:1208 (1985), disclose a human recombinant DNA clone (HLM-2) that comprises a mosaic of retroviral-related sequences with the organ¬ ization and length of known endogenous retroviral genomes. A particular HLM-2 long terminal repeat (LTR) hybridized with LTR of a type-D retrovirus. The HLM-2 gag and pol genes share extensive homology with a type-A related retrovirus, a type-B retrovirus, and a type-C retrovirus. .There were regions in the HLM-2 pol gene that were up to 70% identical to a mammary tumor virus pol gene. A portion of the putative HLM-2 env gene hybridized with the corresponding region of a type-A retroviral genome. Martin et al. , PNAS 75:4892 (1982) disclosed the detection and cloning of type-C retroviral sequences in human brain, baboon skin fibroblasts, and rhesus monkey liver.

Rabson et al. , Nature 306:604 (1983) disclosed the characterization of a full-length human retroviral clone containing 2 LTR elements 8.4 kb apart, as well as gag, pol, and putative env regions. Hybridization experiments revealed that the human cells (placenta) contained species of poly(A) RNA that annealed to segments of the full-length retroviral clone that con¬ tains only 4.1 kb of gag-pol sequences, bounded by a tandem array of imperfect repeats 72 to 76 base pairs in length, and lacking LTR's.

Repaske et al., J. Virol. j> :764 (1985) disclosed the complete nucleotide sequence of a full-length (8.8 kb) endogenous C-type human retroviral DNA (clone 4-1) cloned from a human genomic DNA library; colinearity and 40% amino acid homology were found in comparison with Moloney murine leukemia viral DNA.

Rabson et al., J. Virol. 6:176 (1985) disclosed the structure of human endogenous retroviral env RNA transcripts by Northern blot hybridization and cDNA cloning. Poly(A) 3.0- and 1.7-kb env RNA's were identified in placenta, colon carcinoma, and breast carcinoma- cells. No accurate nucleic acid - based marker, however, has yet been disclosed to be useful for colonic cancer lesions.

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SUMMARY OF THE INVENTION

The present invention is based on the analysis of the pattern of expression of type C-related retroviral sequences in the pol (A) RNA extracted from a number of human primary colon cancers, from adjacent colon mucosa and from two colon cancer cell lines (HCT and Caco2), using as probes labeled LTR and envelope se¬ quences (designated H-LTR and H-env) . In most of the tumor samples examined, the inventors observed a striking decrease in the amount of a 3.6kb LTR-related transcript, which is very prominent and abundant in normal colon mucosa (NCM) ? in contrast, either or both of two env-related transcripts of 3.0 and 1.7kb, especially the 1.7kb one, increased in colonic tumors versus NCM.

The present invention thus provides a method for the detection of neoplastic disease of the colon in an RNA containing colonic sample, comprising:

(1) contacting the RNA of the sample with a de¬ tectably labeled H-LTR or H-env nucleotide probe; and

(2) detecting the amount of hybridization between the probe and the RNA, relative to the amount in a known, non-cancerous colonic sample.

In a specific preferred embodiment, the invention is directed to a method for analyzing colonic tissues for the transcriptional pattern of type C-related human endogenous retroviral sequences, comprising ex¬ tracting total RNA from the tissue, recovering poly (A) RNA through oligo-dT cellulose chromatography, size-separating this RNA by denaturing gel electro- phoresis, hybridizing with labeled H-LTR or H-env human proviral probes, detecting the species hybridiz¬ ed, and quantifying changes in the LTR-specific or env-related transcripts.

DESCRIPTION OF THE FIGURES

Figure 1 shows the complete sequence of the env- probe.

Figure 2 shows the complete sequence of the H-LTR probe.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Two nucleic acid probes are used in the method of the ^ invention. The first probe is a sequence iden¬ tical to or homologous to^" a sequence from endogenous C-type human retroviral nucleic acid, hereinafter the "envelope" or "env" probe. The complete sequence of the envelope type C retroviral gene in question is given in Repaske et al. , J. Virol. 5_4:764 (June 1985). The preferred segment is a Bam HI-Hind III fragment thereof, which is shown in Figure 1, or subfragments thereof capable of hybridizing a target nucleic acid.

The second probe is named "H-LTR" and the complete sequence thereof is shown in Figure 2. The H-LTR probe is also derived from the complete sequence of the envelope type C retroviral gene shown in the Repaske et al. publication (supra) . The H-LTR probe is a sequence identical to, part of, or homologous to the sequence shown in Figure 2.

The desired nucleotide probe sequence may include flanking naturally occurring nucleotides as well, with the proviso that these flanking nucleotides may not be present in such numbers as to alter the hybridization specificity of the DNA or RNA sequence. Typically, the probe sequence will contain at least 18 nucleo¬ tides. Thus, further intended within the scope ~-f this invention are any and all polynucleotides con¬ taining, as a minimum, 18 members that are part of or homologous to the env or H-LTR probes.

These probes can be either in DNA or in RNA form. They can be obtained by known and published isolation and digestion procedures (supra) or synthesized by standard methods. The probe may be obtained from mes¬ senger RNA, from cDNA obtained by reverse transcrip¬ tion of messenger RNA with reverse transcriptase or by cleavage of the genome, conveniently by endonuclease digestion, followed by cloning of the gene or gene fragment . in accordance with known techniques. See, for example, Kornberg, DNA Replication,' W. H. Freeman & Co., San Francisco, 1980, pp. 670-679. Alternative- ^* ly, the probe may be synthesized according to the technique described by Merrifield, J. M. Che . Soc. , 85_:2149 (1962). After isolation of the DNA fragment, the fragment may be used for preparation of the probe.

The probe can be by itself or may be part of a plasmid. For example, the H-LTR probe is flanked by Taq I sites. It can thus be subcloned into the Taq I site of plasmid pBR322, for example, and cloned in E. coli.

The probe is detectably labelled, the labels of most utility being radioactive atoms, enzymes, chromo- phores, biotin/avidin, or the like. A more complete discussion of nucleic acid hybridization technology may be found in Huang, E. S. et al., Vol. 6, pp. 457-497 (1977), incorporated by reference herein. Oligonucleotide probe technology is also disclosed by Szostak, J. . et al. , Meth. Enzymol., 68:419-428 (1979), also incorporated by reference herein. The polynucleotide or oligonucleotide probe may be labelled with an atom or inorganic radical, most com¬ monly using radionuclides, but also perhaps heavy metals. In some situations, it may also be possible to employ an antibody which will bind specifically to the probe hybridized to the target DNA.

Most commonly, a radioactive label is employed, suitable radioactive labels including P, H, C

125 I, or the like. Any radioactive label may be employed which provides for an adequate signal and has sufficient half-life. Other labels include ligands, fluorescers, chemiluminescers, enzymes, antibodies, and the like.

In one technique of labelling, E^ coli DNA poly- merase I may be utilized to add nucleotide residues to the 3'-hydroxy- terminus that is created when one strand of a double-stranded DNA molecule is nicked. In addition, the enzyme, by virtue of its 5¹ to 3¹ exonucleolytic activity, may remove nucleotides from the 5' side of the nick. The elimination of nucleo¬ tides from the 5' side and the sequential addition of nucleotides to the 3' side results in the formation of the nick (nick translation) along the DNA (Kelley et al. , J. Biol. Chem. , 245: 39 (1970)). By replacing the preexisting nucleotides with highly radioactive nucleotides, it is possible to prepare labelled probe

Q with a specific activity well in excess of 10 cpm/ug (Rigby, P. W. J. et ____, J. Mol. Biol., 113: 237 (1977)).

Probes maybe labelled to high specific activity using either 3H-thymidme tπ.phosphate or alpha-32P- deoxynucleotide triphosphates by such nick translation (Rigby et al. , supra) . In testing a colonic sample for the marker(s), RNA can be isolated from tissue by sectioning on a cryostat and lysing the sections with a detergent such as SDS and a chelating* agent such as EDTA, optionally with overnight digestion with proteinase K (50 ug/ l). Protein is removed by phenol and chloroform extrac¬ tions, and nucleic acids are precipitated with ethanol. RNA is isolated by chromotography on an oligo dT column and then eluted therefrom. Further fractionation can also be carried out.

A number of techniques for molecular hybridization are used for the detection of retroviral RNA sequences in colonic tissues? each has certain advantages and disadvantages. When large amounts of tis'sue are available, analysis of hybridization kinetics provides the opportunity to accurately quantitate the amount of retroviral RNA present, as well as to- 'distinguish se¬ quences that are closely related but not identical to the probe, and determine the percent homology.

Reactions are run under conditions of hybridiza¬ tion (Tm-25°C) in which the rate of reassociation of the probe is optimal (Wet ur, J. G. et al. , J. Mol. Biol., 31: 349-370 (1968)). The kinetics of the reac¬ tion are second- order when the sequences in the tis¬ sue are identical to those of the probe; however, the reaction exhibits complex kinetics when probe se¬ quences have partial homology to those in the tissue (Sharp, P. A. et al. , J. Mol. Biol._r 86: 709-726 (1974)).

The concentration of probe to- cell RNA is deter¬ mined by the sensitivity desired. To detect one re¬ troviral transcript per cell would require about 100 pg of probe per ug of total cellular RNA. The nucleic acids are mixed, denatured, brought to the appropriate salt concentration and temperature, and allowed to hybridize for various periods of time. The rate of reassociation can be 'determined by quantitating the amount of probe hybridized either by hydroxy apatite chro atography (Britten, R. J. et al. , Science, 161: 529-540 (1968)) or SI nuclease digestion (Sutton, W. D., Biochim. Biophys. Acta, 240: 522-531 (1971)).

A more flexible method of hybridization is the Northern blot technique. This technique offers variability in the stringency of the hybridization reaction, as well as determination of the state of the retroviral sequences in the specimen under analysis. Cell RNA is denatured jlri situ with alkali, neutralized and transferred to a nitrocellulose membrane.

After washing, the membrane is baked under vacuum and prehybridized in 10X Denhardts solution (0.2% each of Ficoll, bovine serum albumin, polyvinylpyrollidβne) in 4X SSC (SSC = 0.15M NaCl, 0.05M sodium citrate) containing 50 ug/ml sonicated and denatured salmon sperm DNA for four hours at 60°C. Stringent hybridiza¬ tion or non-stringent hybridization can be carried out. Membranes are washed extensively in 4X SSC at 52°C, air dried and detected.

A major consideration associated with hybridiza¬ tion analysis of retroviral RNA sequences is the de¬ gree of relatedness the probe has with the sequences present in the specimen under study. This is impor¬ tant with the blotting technique, since a moderate degree of sequence homology under nonstringent condi¬ tions of hybridization can yield a strong signal even though the probe and sequences in the sample represent different retroviral types. The particular hybridization technique is not es¬ sential to the invention, any technique commonly used in the art being within the scope of the present in¬ vention. Typical probe technology is described in United States Patent 4,358,535 to Falkow et al. , in¬ corporated by reference herein.

The labelled probes, as described above, provide a general, diagnostic method for detection of colonic lesions:.. The method is reasonably rapid, has a simple protocol, has reagents which can be standardized and provided as commercial kits, and allows for rapid screening of large numbers of samples.

In one method for carrying out the procedure, a clinical isolate containing RNA transcripts is fixed to a support. The affixed nucleic acid is contacted with a labelled polynucleotide having a base sequence complementary or homologous to the coding strand of the retroviral gene.

The hybridization assays of the present invention are particularly well suited for preparation and com¬ mercialization in kit form, the kit comprising a car¬ rier means compartmentalized to receive one or more container means (vial, test tube, etc.) in close con¬ finement, each of said container means comprising one of the separate elements to be used in the hybridiza¬ tion assay.

For example, one vial may contain soluble, detect¬ ably labelled HLR or env probe, while one or more dif¬ ferent vials may contain different, predetermined amounts of env or HLR RNA transcripts. The latter con¬ tainers may be used to construct a standard curve for interpolating data obtained from the unknown sample. The presence of colonic lesions is determined by the variation in the appearance and/or quantity of probe-related RNA transcripts in tested tissue. Two LTR specific transcripts and three env-related trans¬ cripts are observed.

Table 1 below shows the pattern of transcript dis¬ tribution in normal colonic mucosa (NCM) versus can¬ cerous colonic tissue.

TABLE I

NCM Cancerous Tissue

LTR related transcripts

3.6 kb ft

2.1 kb

Env - - related transcripts

3.0 kb t

1.7 kb if

0.6 kb t

The 1.7 kb env-related transcript clearly in¬ creases, and the 3.6 kb LTR-related transcript clearly decreases in cancerous colonic tissue.

Generally a significant increase or decrease, re¬ spectively, of these transcripts should be observed for an accurate diagnosis. Preferably a 3-fold or higher for the 1.7 kb transcript, and a 60% or more decrease for the 3.6 kb transcript, respectively should be observed.

Certain examples are now provided for illustration purposes only. EXAMPLES Materials and Methods Cell Cultures, Chemicals and Tissue Specimens.

HCT and Caco2 colon cancer cell lines were cultur- ed in Dulbecco's modified Eagle's medium (DMEM) sup¬ plemented with 20% fetal calf serum (GIBCO). Cell cultures were grown in a humidified incubator at 37°C with 5% C0₂ lh air and fed three times a week. For DNA and RNA extraction, colon tissue surgical speci¬ mens were used, which were not needed for routine pathology and in accordance with the NIH guidelines on human studies.

Probes and Nucleic Acid Hybridizations.

In the initial studies the H-LTR and H-env, cloned in pBR322, were [ 32P] labeled by nick-translation. In later experiments, the H-LTR segment was subcloned in the pSP64 vector system (Melton, D.A. et al. , Nuc. Ac. . Res. 1,2:7035 (1984)) to generate a [³²P] labeled ribo-' probe. Chromosomal DNA was extracted from cell cul¬ tures or tissue specimens following a previously described procedure (Gattoni, S. , Mol. Cell. Biol. 42-51 (1982)), subjected to restriction endonuclease (New England Biolabs), digestion, and to Southern blot hybridization. Total RNA was extracted using the method described by Cathala et al. (DNA _,2^:_!_.9-335 (1983))? poly(A)⁺ RNA was selected through oligo-dT cellulose chromatography (Aviv, H. et al., PNAS §9_ι1408-1412 (1972)), size separated by denaturing gel electrophoresis (Thomas, P.A., PNAS 77:5201-5208 (1980)) and blot hybridized (Melton, supra, Wahl, G.M. et al. PNAS 76_:3683-3687 (1979)). DNA and RNA blots were subsequently rinsed and exposed for autoradio- graphy using X-ray films (KODAK) at-70°C. EXAMPLE 1 Northern Blot Analyses

The findings of 'Rabson, Martin and coworkers (Nature 305:604-607 (1983)), that H-LTR and H-env probes detect specific transcripts in a series of human tissues and cell lines was confirmed. The at¬ tention was then focused on normal colon mucosa CNCM), a number of primary colon tumors (T) and two colon cancer cell lines - HCT and Caco2 - from which RNA was extracted and poly(A) RNA was selected and size- separated on denaturing agarose gels for Northern blot analysis. In all samples, the H-env probe identified three transcripts of 3.0, 1.7 and 0.6 kb, while the H-LTR probe gave rise to marked differences both in the quality and intensity of hybridization. The H-LTR probe identified a major 3.6 kb species which was very prominent in NCM and Tl? in comparison, T3 and T4 showed hardly any hybridization (comparable amounts of RNA were loaded in each lane) and HCT showed much less intense hybridization than the normal colon mucosa. Moreover, the 3.6 kb species did not appear to cross hybridize with H-env, nor with a gag-pol probe.

It was subsequently confirmed that every colon RNA sample analyzed thus far (whether from normal mucosa or tumor), exhibited the three env-specific tran¬ scripts, although there was some difference in the intensity of hybridization (tumors seemed to contain consistently more of the 1.7 kb and sometimes more of the 3.0 and 0.6 kb species). In addition, of the four colon cancer cell lines tested (HCT, Caco2, SW480 and Colo320) , only the HCT and Caco2 showed the above pat- tern and have been studied further. In this respect, it should be noted that HCT and Caco2 exhibit a dis¬ tinct epithelial morphology which has not been observ¬ ed in stocks of SW480 and Colo 320. The H-env probe, therefore, appears to identify a rather specific pat¬ tern of hybridization which has not been detected in a number of human fibroblastic and cancer cell lines; moreover it provides a useful marker for checking RNA integrity. Such a control is important for validating the dramatic decrease of the 3.6 kb LTR transcript in a majority of the colon tumors and in all the colon cancer cell lines examined to date.

A substantially improved resolution of RNA tran¬ scripts as well as the sensitivity of the H-LTR probe were also achieved. The LTR insert was subcloned in the pSP6 vector (Melton, supra) and the corresponding riboprobe was^" used for reprobing filters previously hybridized with a nick-translated H-env probe. An example of such an experiment is one wherein five dif¬ ferent poly(A)⁺ RNAs - two NCM controls, two malignant tumors (T5, T6) and one villoglandular polyp (Pi) - were probed with H-env and H-LTR riboprobe. H-env gave the usual pattern (3.0, 1.7 and 0.6 kb tran¬ scripts), although the intensity of the signal for the 1.7 and 0.6 kb species was stronger in the tumors. In PI all three H-env transcripts were clearly increased.

Reprobing the same filters with H-LTR confirmed the previous findings but also provided evidence to suggest that alteration in the transcription of endo¬ genous retroviral sequences may constitute an early event in the process of carcinogenesis. Specifically, a consistent number of differences emerged in both premalignant and malignant tumors, compared to every sample of normal colon mucosa (NCM) studied thus far. There was a decrease, often very striking, in the amount of the LTR specific 3.6 kb RNA and an increase in the 1.7 kb transcript, sometimes accompanied by a parallel increase in the 3.0 and 0.6 kb species.

In this regard, it is of interest that in the one case of dysplasia in chronic ulcerative colitis (Dl) examined thus far, the 1.7 kb transcript was clearly increased (see Table 2 below). (A total colectomy was later performed on this patient and a carcinoma "in situ" was identified in the colon specimen. )

The 3.0 and 1.7 kb RNAs appeared to cross-hybrid¬ ize with both env and LTR probes. The reason for their being virtually undetectable in NCM using the LTR probe may derive from differential homology be¬ tween the probe and the LTR portion of the 3.6 kb RNA compared to the 3.0 and 1.7 kb RNAs. Therefore, only when the latter two transcripts were increased above a given threshold did they become detectable. Alterna¬ tively, each probe may have identified different, but comigrating transcripts.

The summary of the analyses is shown in Table 2.

TABLE 2 Summary of Northern blot analyses of human colon tissue and colon cancer cell lines

Surgical LTR LTR-env env Specimens Transcripts Transcripts Transcript 3.6 kb 2.1 kb 3.0 kb 1.7 kb 0.6 kb

NCM* ++++ + + ₊S§ +

** Tl ++++ + + ++ ++

T2 + + — + ++

T3 + + + + +

T4 + + + + +

T5 + + + + ++

T6 +++ + + + ++

T7 + + + + ++

T8 ++ + - + +

T9 +++ + + + ++

T10 + + + + ++ il . + + + + +

T12 + + + ++ ND

T13 •+>. ^" + .+ ^■ ND

T14 ++ + + + ND

T15 + + + + +++

. ***

Pi + + + + ++

Dl§ +++ + - + +

Cell lines

HCT + + ++ ++ ++

CA-I + + — ++ +

CaCo2 CA-II - + + ++ +

CA-III *^~~* *+ + ++ +

Normal Colon Mucosa: a sample of normal mucosa (NCM) adjacent to each tumor specimen was analyzed and no detectable differences in the transcription pattern of such controls were found.

**

Tl-14 were colon carcinomas_? T15 was a rectal car¬ cinoma.

***

PI villoglandular polyp § Dl dysplasia in chronic ulcerative colitis.

§ § The + symbol = barely detectable; ND = not done. The remarkable abundance of the 3,6 kb H-LTR spe¬ cific transcript and its striking decrease in most of the primary human colon cancers tested suggests that the regulation of the corresponding sequence is alter¬ ed in tumors in contrast to normal colon mucosa. Co¬ lon tumors, on the other hand, appear to express high¬ er levels of the env-related transcripts, especially the= -L. kb species, including the one case of dyspla- si examined thus far. This observation and the find¬ ing of a "cancer-like" pattern in a non-metastatic, non-malignant tumor (PI) indicates that type C-related endogenous retroviral sequences provide early detec¬ tion markers of colonic lesions progressing to neo- plasia. In particular, the increase of the 1.7 kb RNA and the often dramatic decrease of the 3.6 kb species represent the best indices of an altered transcription pattern corresponding to an altered phenotype.

COMPARATIVE EXAMPLE 1 Examination of Retroviral Sequences in Genomic DNA

Southern blot hybridization of the genomic pattern of type C-related human endogenous retroviral se¬ quences was analyzed in a number of DNA samples deriv¬ ed either from human colon carcinoma cell lines or from human colon surgical specimens.

H-LTR was used as a probe to compare the pattern of integration of homologous genomic sequences in seven different chromosomal DNA samples extracted from a human fibroblastic cell line (HF), four colon cancer cell lines (Caco2, SW480, Colo320 and HCT), a specimen of normal colon mucosa (NCM) and a colon tumor (Tl). The Southern blot hybridization of these PstI di¬ gested chromosomal DNAs indicated that the H-LTR probe identified multiple homologous sequences in the human genome, as was previously shown with a gag-env probe (Steele, P.E., Science 225:943-847 (1984)).

Although the complexity of the hybridization pat¬ tern did not allow detection of minor variations, there appeared to be no obvious difference among the DNAs examined.

EXAMPLE 2 Studies on Human Colon Cancer Cell Lines

In parallel, the HCT and Caco2 cell lines were also characterized, since they share several markers and morphological features with intestinal epithelia. In addition, confluent Caco2 cells have been shown to express a ^"large increase in the levels of intestinal alkaline phosphatase as well as markers of enterocytic differentiation. In this respect Caco2 cells provide an inducible system, susceptible to in vitro manipula¬ tion.

Therefore, Caco2 cells were cultured and extracted for RNA at different times after seeding - namely, in exponential growth (CA-I) at subsconfluence (CA-II) and at confluence (CA-III). The corresponding poly(A)⁺ RNAs were subjected to Northern blot hybridi¬ zation with the H-LTR riboprobe and compared with the normal mucosa control (NCM), the HCT cell line, and two primary tumors (T7 and T8).

The 3.6 kb transcript was again very prominent in NCM and was decreased in both primary tumors and HCT cells. However, the Caco2 cells appeared to produce the 3.6 kb transcript only when growing exponentially. The transcriptional pattern of these sequences in HCT and Caco2 cells is remarkable similar to the pat¬ tern observed in some primary tumors (see Table 2). T7 exhibits both env-related transcripts (3.0 and 1.7 kb) and is therefore comparable to the HCT cells in which these RNAs are rather prominent? T8 instead, shows only the smaller transcript as is the case in Caco2 cells, although in subconfluent and confluent cultures very small amounts of the 3.0 kb transcript are detectable.

DISCUSSION OF EXAMPLES

The first consistent finding was the increase in the LTR-env 1.7 kb transcript in all primary tumors and in the two non-malignant lesions tested (i.e. polyp and dysplasia).

The second relevant finding is the decrease, often very striking, of the 3.6 kb LTR-related transcript. The "in vitro" studies with the Caco2 cell line showed that the expression of such a transcript can be modu¬ lated, suggesting that the corresponding gene product may be involved in the process of differentiation of colon mucosal cells.

The variability of the transcription pattern in tumors and tumor cell lines as compared to the remark¬ able consistent pattern in 15 normal colon mucosal specimens is also compatible with the concept of tumor heterogeneity, which encompasses morphological as well as biochemical features of cancer, especially primary ones.

The present results indicate that an altered pat¬ tern of expression of retroviral transcripts provides markers for the detection of the neoplastic disease at its initial stages.

Claims

WHAT IS CLAIMED IS:

1. A method for the detection of neoplastic disease of the colon> in an RNA containing colonic sample, which comprises:

(1) contacting the RNA of said sample with a detectably labeled H-LTR or H-env nucleotide probe; and

(2) detecting the amount of hybridization between the probe and the RNA, relative to the amount in a known, non-cancerous, colonic sample.

2. The method of claim 1 wherein said probe is H-env.

3. The method of claim 1 wherein said probe is

H-LTR. <

4. The method of claim 2 wherein said H-env probe has at least a portion, or is homologous to a portion, of the sequence of Figure 1.

5. The method of claim 3 wherein said H-LTR probe has at least a portion, or is homologous to a portion, of the sequence of Figure 2.

6. The method of any of claims 4 or 5 wherein said portion is at least 18 nucleotides long.

7. The method of claim 2 which comprises detect¬ ing a significant increase in a 1.7 Kb retroviral RNA transcript present in said sample relative to said non-cancerous sample.

8. The method of claim 3 which comprises detec¬ ting a significant decrease in a 3.6 Kb retroviral RNA transcript present in said sample relative to said non-cancerous sample.

9. The method of claim 1 which comprises, prior to said step- (1), purifying RNA from said sample.

10. The method of claim 9 wherein said RNA is purified by oligo-dT chromatography.

11. The method of claim 1 wherein said probe is labeled with a radioactive atom, an enzyme, or a biotin or avidin label.