Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

• This invention relates generally to human laminin binding protein and, more specifically, to a full length cDNA encoding a human laminin binding protein derived from human colon carcinoma and to the molecular cloning thereof.
• Colon carcinoma is one of the leading causes for cancer death in humans. Over 140,000 new cases of colon carcinoma are expected in 1988 in the United
• CEA carcinoembryonic antigen
• Laminin has been implicated in a wide variety of biological processes. Most relevant to colon cancer, laminin has been linked to cell adhesion, motphogenesis, mitogenesis, differentiation and metastasis. Laminin is a major constituent of basement membrane. Laminin is a large glycoprotein consisting of three polypeptide chains, A chain (M r 400,000), B1 chain (M r 210,000) and B2 chain (M r 200,000) in a cross-shaped structure. It mediates the attachment of both normal and neoplastic cells to the basement membrane.
• a proteolytic fragment of laminin and a pentapeptide of Tyr-Ile-Gly-Ser-Arg from the B1 chain with cell attachment activity have been identified.
• This invention is directed to a full length human laminin binding protein cDNA sequence derived from human colon carcinoma cells comprising substantially the sequence as set forth in Figure 3. It also concerns a subsequence corresponding substantially to the sequence starting with nucleotide number 162 and ending with nucleotide number 829 as set forth in Figure 3.
• this invention also relates to recombinant clones encoding a human laminin binding protein.
• this invention relates to a laminin binding protein encoded by either sequence and to substantially isolated DNA encoding the laminin binding protein of the above-described cDNAs.
• Also of concern is a method for detecting the presence of carcinoma in a patient and a reagent for detecting the presence of carcinoma in a patient.
• Figure 1 Blot-hybridization of RNAs from a human colon carcinoma and adjacent normal epithelium obtained from surgery with 32 p-labeled cDNA inserts from three plasmids. Lanes 1,3,5 used total RNA from normal human colonic epithelium; Lanes 2,4,6 from colon
• Lanes 5,6 with 1-4E Arrows indicated the major species of mRNA hybridized, 1.2 kb, 1.7 kb, and 0.7 kb, respectively.
• FIG. 1 Blot-hybridization of RNAs from twenty human colon carcinoma cell lines with 32 p-labeled cDNA insert from 8-2V plasmid.
• Lanes 1 used RNAs from HT29; 2 from CX-1; 3 from RCA; 4 from CCL222; 5 from CCL220.1; 6 from CCL228; 7 from HCT116b; 8 from HTB39; 9 from Clone A; 10 from Clone D; 11 from MIP101; 12 from CCL229; 13 from Gly; 14 from Moser; 15 from CCL233; 16 from CCL224; 17 from CCL237; 18 from CL187, 19 from CCL227, and 20 from CCL234.
• FIG. 3 Nucleotide sequence and deduced amino acid sequence of a full-length cDNA insert from J-9 lambda gt10 phage. All of the sequence of 8-2V insert is located substantially in the sequence shown here (nucleotide #162 to #829). Sequences of interest are underlined.
• Figure 4 is a hydropathy plot of the deduced amino acid sequences shown in Figure 3.
• Figures 5 and 6 are Northern blots of total RNA isolated from various human carcinoma cell lines which show significant levels of expression of the
• FIG. 7 shows that the in vitro translated products of laminin binding gene from rabbit
• reticulocyte binds to a laminim sepharose column and the molecular weight of laminin binding protein in vitro translated products migrate on SDS PAGE as a 45 kd protein.
• the recombinant clone designated J-9 was deposited in the American Type Culture Collection (ATCC) under the Budapest Treaty on August 30, 1988 and was accorded ATCC accession number 40489.
• the recorr_oir_ant clone designated 8-2V was deposited in the ATCC under the Budapest Treaty on August 30 , 1988 and was accorded ATCC accession number 40490 .
• sequence encodes the same protein or a functional equivalent with conservative substitutions, additions or deletions.
• substantially purified means synthesized or, if naturally occurring, isolated free of other cellular components with which it is normally associated in a particular genome.
• cDNA libraries were constructed from human colon carcinoma cell lines. These libraries were screened for specific clones that hybridized with mRNAs more abundant in colon carcinomas. A 1.2 kb mRNA was identified by its hybridization with the cDNA insert in recombinant clone 8-2V which is discussed below. This mRNA was about 9-fold more abundant in human colon carcinoma than that of adjacent normal colonic
• the cDNA of this 1.2 kb mRNA appears to be the first full length human laminin binding protein cDNA sequence derived from human colon carcinoma cells comprising substantially the sequence as set forth in Figure 3.
• nucleotide triplet can code for the same amino acid and, due to the existence of degenerate codes, variations in the
• nucleotide sequence can arise. Thus, variations of the nucleotide sequence set forth in Figure 3 can occur and fall within the scope of this invention.
• Identification of 8-2V Plasmid To search for cellular mRNAs that are more abundant in human colon carcinoma than that of normal counterparts, a cDNA library was constructed in plasmid pBR322 using
• Initial screenings were made by hybridizing 1,500 plasmids with cDNAs generated from mRNAs of Clone A as well as CX-1, a well differentiated human colon carcinoma cell line. Whereas the great majority of plasmids hybridized equally between the two or more with that of CX-1, 10 plasmids were studied because there was greater hybridization with cDNAs from Clone A than that of CX-1, cDNA inserts in these plasmids were then used to compare the levels of mRNA between normal human colonic epithelial tissues and colon carcinoma of the same patient by blot
• RNA. gels One of the plasmids, 8-2V, hybridized with 1.2 kb mRNA that was more abundant in carcinoma than that of adjacent normal epithelium (Fig. 1) . Equal amounts of total RNA from carcinoma and normal tissue were compared. Densitometric analysis suggested that the level of this mRNA might be
• RNAs were hybridized with cDNA inserts from two other plasmids. Plasmid 1-4E hybridized with a 0.7 kb mRNA far more abundant in normal colonic epithelium than does carcinoma, whereas plasmid 3-4W hybridized with a 1.7 kb mRNA equally abundant between the two. These results suggested that 1.2 kb mRNA which hybridized with the cDNA insert in 8-2V has the ability to encode for a polypeptide which is useful for distinguishing colon carcinoma from normal colonic epithelium.
• Fig. 2 shows that essentially all human colon carcinoma cell lines tested express significant levels of this mRNA except CCL234, a mucin-producing, well-differentiated, rectal adenocarcinoma cell line. Correlation between the level of this 1.2 kb mRNA and the degree of
• the predicted polypeptide contains the twenty amino acid sequence (Pro-Thr-Glu-Asp-Trp-Ser-Ala-Gln-Pro-Ala-Thr-Glu-Asp-Trp-Ser-Ala-Ala-Pro-Thr-Ala) recognized by the polyclonal antibodies that inhibit laminin-mediated cell attachment.
• the product of the product of 1.2 kB mRNA which hybridiized with 8-2V cDNA also binds laminin.
• nucleotide sequence from three different sources, human umbilical vein endothelial cells. Clone A and CX-1.
• Computer-assisted analysis revealed an open-reading frame (ORF) starting from the first ATG and terminating at TAA as indicated. This ORF is preceded by an inframe stop codon at position -18, and ANN immediately before the first ATG.
• polyadenylation are also present.
• This ORF can encode for a 32,817 Da polypeptide of about 295 amino acid residues.
• the deduced amino acid sequence is a 32,817 Da polypeptide of about 295 amino acid residues.
• Unusual sequences include two repeats of Thr-Glu-Asp-Trp-Ser-Ala-X-Pro (264-271 and 273-280(*)
• Figures 5 and 6 show there is a significant level of expression of the 1.2 kB mRNA in human
• Figure 5 is a Northern blot of total RNA isolated from various human carcinoma cell lines.
• Lanes 1-6 contained RNA isolated from the following carcinoma cell lines: 1, CE81T; 2,
• CE48T 3, HID (1, 2 & 3, human esophageal carcinoma
• Figure 6 is also a Northern blot of total RNA isolated from various human carcinoma cell lines.
• Total RNA (15 ⁇ g/lane) was separated on a 1.0% agarose/formaldehyde gel, transferred to nitrocellulose filters, and probed with 32 P-iabeled cDNA insert from clone 8-2V which encodes a laminin binding protein.
• Lanes 1-6 contained RNA isolated from the following carcinoma cell lines: 1, T24; 2, EJ (1 & 2, human bladder adenocarcinoma); 3, CX-1; 4, CCL218WiDr (3 & 4, human colon adenocarcinoma); 5, HTB40HuTu80, human duodenum adenocarcinoma; 6, CCL185A549; 7, 0AT4; 8, Calu (6, 7 & 8, human lung carcinoma); 9, A2058 (human melanoma);
• CRL1435PC-3 human prostate adenocarcinoma
• Arrowhead indicates the hybridized species of 1.2 Kb RNA.
• Figure 7 shows that the in vitro translated pro ⁇ ucts of laminin binding protein gene appeared as a 45 kd protein on SDS PAGE which bound to laminin sepharose column.
• the in vitro translated pro ⁇ ucts of laminin binding protein gene appeared as a 45 kd protein on SDS PAGE which bound to laminin sepharose column.
• Lane 1 is the background control of in vitro translation of this particular batch rabbit reticulocyte lysate.
• Laminin receptor has been suggested to be involved in the initial interactions of tumor cells via laminin with the vascular basement membrane to
• differentiated human carcinomas including that of colon have been shown to express high levels of cytoplasmic laminin receptor by immunoperoxidase stainings than that of well-differentiated. Moreover, inhibition of
• metastasis of melanoma in animal model systems has been demonstrated by laminin-derived pentapeptide recognized by laminin receptor. It is possible that intervening interactions between laminin and its receptor prevent the metastasis of colon carcinomas.
• laminin receptor the product of 1.2 kb mRNA, if not the 67 kDa laminin receptor, is probably a laminin binding protein. That the product of this 1.2 kb mRNA might be a laminin binding protein is strongly supported by the fact that the coding region for an epitope in 67 kDa laminin receptor recognized by the monoclonal antibody, 2H5, which blocks the binding of laminin with its receptor is present entirely in the sequence
• Asp(Glu)-X-X-X-Glu(Asp)-X-Tyr-X-Tyr-Lys(Arg)-X-X-X-Asp(Glu) in positions 31-44 and 196-209 was a notable repeat. If the 140 amino acid stretch flanked by the two cysteines is considered a linking domain, the two flanking domains would each have one of this repeat sequence. There is another repeat, Ala-Ala-Ala-X-X-Ala-X(-)-Thr, one each in these two flanking domains.
• the C-terminal domain after residue 225 contains a 70-amino acid segment with 13 aspartic plus glutamic residues and without any lysine or arginine residues.
• This trypsin-resistant, highly acidic domain might mark laminin binding proteins.
• this invention concerns a reagent and method for detecting carcinoma in a patient which comprises providing a labelled cDNA probe having one of the cDNA sequences mentioned above, exposing the probe to a sample of tissue or body fluid from the patient, and monitoring the reaction for hybridization.
• Hybridization selection techniques are described in Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1982).
• Useful labels include any moiety detectable subsequent to hybridization such as radionuclides, e.g., 32 P, 3 H, or 14 C, enzymes, fluorophores, chemiluminescent compounds, chromogens and chromophores. These labels can be attached directly or indirectly to the probe using techniques commonly known to those skilled in the art. It is also within the skill of the art to identify the probe using specific labelled antibodies.
• DLD-1 subclones of DLD-1, were from D. Dexter (Du Pont Co.), MIP101 from N. Zamcheck (Mallory Institute), CX-1 from S. Bernal (Dana-Farber Cancer Institute), RCA, HCT116b, Gly and Moser from M. Brattain (Baylor College of DLD-1).
• RNA Dulbecco's modified Eagles' medium (GIBCO) and 50% RPMI 1640 MEDIUM (GIBCO) supplemented with 10% fetal calf serum (MA Bioproducts) and 1% Nutridoma NS (Boehringer) at 37°C with 5% CO 2 and 100% humidity.
• RNA Total cellular RNAs from cultured cells were isolated by the methods of Cox (Cox, R. A. (1986) Methods Enzym. 12, 120-129) and Strohman et al. (Strohman, R. C, Moss, P. S., Eastwood, J. M. & Spector, D. (1977) Cell 10, 265-273).
• Total cellular RNAs from surgical specimens were prepared according to Chirgwin et al . (Chirgwin, J . M . , Pryzbyla, A . E . ,
• RNAs from Clone A cells were purified by a poly(U)-Sephadex column twice as described by Haff et al. (Haff, L. A. & Bogorad, L. (1976) Biochem. 15, 4110-4115).
• the first strands of cDNAs were synthesized by AMV reverse transcriptase (Molecular Genetics Resources) using oligo-dT as a primer, the second strands with DNA polymefase I according to Wickens et al. (Wickens, M. P., Buell, G. N. & Schimke, R. T. (1978) J. Biol. Chem.
• cDNAs were treated with S1 nuclease, tailed with dCTP, and fractionated by Bio-Gel A-50 column. Only dC-tailed cDNAs larger than 400 bp were pooled and annealed to Pst I-digested poly(G)-tailed pBR322 plasmids. Transformation in E. coli HB101 was carried out according to Dagert et al. (Dagert, M. & Ehrlich, S. D. (1979) Gene 6, 23-28). The complexity of this library was approximately 5X10 ⁇ clones/ ⁇ g cDNA.
• RNA Gels Blot-Hybridization Total RNAs (15 ⁇ g) extracted from tissues and cultured cells were electrophoresed in agarose gels containing formaldehyde, and transferred to GeneScreenPlus (Du Pont) according to Seed (Seed, B. (1982) Genet. Eng. 4, 91-102).
• Recombinant plasmids were prepared by alkaline lysis and CsCl gradient centrifugation (Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A
• Plasmid DNA was purified and digested by Pst I. After electrophoresis, cDNA insert was eluted with LID/X filter syringes (Xydex), extracted with
• RNAs were isolated by extraction of cultured human colon carcinoma cell line, CX-1, with guanidinium thiocyanate. Poly(A)+RNAs were selected on oligo(dT)-cellulose affinity columns.
• oligo(dt) 12-18 100 ⁇ /ml of RNAs in 10 ⁇ g of poly(A)+RNA, and 1000 ⁇ /mi of avian reverse transcriptase for 1.5 h at 42°C.
• the main reaction for the first strand synthesis was not radiolabelled. Labelling was done only, in a separate tube, to monitor the incorporation reaction containing the same
• Second Strand Synthesis The cDNA pellet from first strand reaction was dissolved in water and the second strand synthesis conducted in a reaction (400 ⁇ l volume) containing 0.5-1.0 ⁇ g of first strand cDNA, 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 5 mM MgCl 2 , 10 mM DTT, 10 mM (NH 4 ) 2 SO 4 , 50 ⁇ g/ml BSA (nuclease-free), 150 ⁇ M B-NAD ⁇ /ml RNAse H, 50 ⁇ /ml E. Coli DNA ligase, 40 ⁇ M dNTPs, 20 uCi [ 32 P]dCTP (specific activity: 3000
• the fill-in reaction was performed in a reaction (80 ⁇ l volume) consisting of 30 mM
• the cDNA was recovered by EtOH precipitation and
• Sepharose resin was prepared in 5 ml pipet and
• oligonucleotides were kindly provided by P . H. Sayre in the laboratory of E. Reinherz (Dana-Farber Cancer

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