WO1995007708A2 - Utilisation therapeutique d'un produit genique contre la predisposition au retinoblastome - Google Patents

Utilisation therapeutique d'un produit genique contre la predisposition au retinoblastome Download PDF

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WO1995007708A2
WO1995007708A2 PCT/US1994/010357 US9410357W WO9507708A2 WO 1995007708 A2 WO1995007708 A2 WO 1995007708A2 US 9410357 W US9410357 W US 9410357W WO 9507708 A2 WO9507708 A2 WO 9507708A2
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protein
cell
cells
retinoblastoma
gene
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PCT/US1994/010357
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WO1995007708A3 (fr
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Wen-Hwa Lee
Eva Y-H. P. Lee
H. Michael Shepard
Duane Johnson
David W. Goodrich
Nan Ping Wang
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The Regents Of The University Of California
Canji, Inc.
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Priority to AU78349/94A priority Critical patent/AU7834994A/en
Publication of WO1995007708A2 publication Critical patent/WO1995007708A2/fr
Publication of WO1995007708A3 publication Critical patent/WO1995007708A3/fr

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4736—Retinoblastoma protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • This invention relates generally to a method of cell therapy to prevent proliferation of pathologically proliferating cells, i.e., to accomplish tumor suppression by administration of either a prophylactically or therapeutically effective amount of a cancer suppressor protein such at the RB protein or polypeptide.
  • Wilms tumor a childhood cancer of the kidney, is thought to arise by inactivation of a gene on chromosome 11.
  • chromosome 11 a normal chromosome 11 into Wilm's tumor cells suppressed the tumorigenicity .
  • the introduction of chromosomes X and 13 did not have this effect.
  • Retinoblastoma is a malignant tumor of the sensory layer of the retina.
  • the neoplastic tumor is composed of primitive retinal cells, occurring often bilaterally, usually before the third year of life. It exhibits a familiar tendency.
  • Retinoblastomas are characterized by small round cells with deeply stained nuclei, and elongated cells forming rosettes. They usually cause death by local invasion, especially along the optic nerves.
  • the retinoblastoma may be hereditary but also acquired. It is the most common intraocular tumor and represents one of the prototypes of inheritable cancers.
  • the hereditary form is characterized by early age of onset and multiple tumor foci. Acquired form occurs later in life with single unilateral tumor (Proc. Natl. Acad. Sci. (1971) 68:820-823; Hum. Genet. (1979) 52:1-54; Science (1984) 223:1028-1033.
  • hereditary retinoblastoma might arise from a precursor retinoblast cell, carrying one inherited defective allele which suffers an additional somatic mutation, while nonhereditary cases would require two somatic mutations in the same cell.
  • Recent circumstantial evidence supports the existence of such cancer suppressor genes in retinoblastoma as well as nephroblastoma also known as Wilm's tumor, neuroblastoma, and osteosarcoma.
  • the RB locus was implicated in non-hereditary retinoblastoma by observing frequent abnormalities of chromosome 13 in tumor karyotypes and reduced esterase D activity in tumors (Cancer Genet. Cytogenet. (1983) 10:311-333; Cancer Genet. Cytogenet. (1982) 6:213-221. It has been proposed that inactivation of both alleles of the RB gene located in region 13ql4 resulted in retinoblastoma. Such proposal was based in part on a case of hereditary retinoblastoma in which both RB alleles were inferred to be absent (Science (1983) 219:973-975).
  • the human retinoblastoma gene has been successfully cloned, identified and sequence (Science
  • the retinoblastoma gene was located in the chromosome 13 region 13ql4:ll in the close proximity of the esterase D gene, which also has been identified, cloned and sequenced (Proc. Natl. Sci. (1986) 83:6790-6794; Proc. Natl. Acad. Sci. (1986) 83:6337-6341; Proc. Natl.
  • RB gene was identified on the basis of chromosomal location, homozygous deletion and tumor-specific alterations in expression.
  • RB gene was shown to have 4723 nucleotides and encodes a messenger RNA (mRNA) of 4.8 kilobases (kb).
  • mRNA messenger RNA
  • This invention provides methods for preventing or inhibiting the proliferation of a pathologically proliferating cell, wherein the pathological proliferation of the cell is the result of the absence of a functional retinoblastoma protein or polypeptide in the cell.
  • the methods require contacting the cell with an effective amount of retinoblastoma protein or polypeptide. These methods also are useful to prevent or treat retinoblastoma or a cancer secondary to retinoblastoma, by administering to a patient a functional retinoblastoma protein or polypeptide.
  • Figure 1 is the chromatogram illustrating the identification of RB proteins by immunoprecipitation with rabbit anti-RB IgG in various cell lines.
  • Human cells such as neuroblastoma LAN-1 (Lanes 1 and 2), Alexander hepatoma
  • retinoblastomas (Lanes 6 to 10) were labeled with 35 S-methionine and immunoprecipitated with preimmune rabbit IgG (Lane 1) or rabbit anti-RB IgG (Lanes 2-10). The immunoprecipitates were analyzed by 7.5% SDS-polyacrylamide gel electrophoresis and autoradiographed.
  • Figure 2 is the complete RB cDNA nucleotide sequence and predicted amino acid sequence of the RB protein.
  • This protein is designated ppRB 110 or p110 RB .
  • the most 5' ⁇ 240 nucleotides were obtained from a cDNA clone from retinoblastoma cell line Y79. Nucleotide sequences from this clone and the original normal RB clones were aligned by sequence overlap. The first and second initiation sites are boxed, and alanine and proline clusters underlined.
  • FIG 3 is the chromatogram illustrating the modifications of the RB protein.
  • LAN-1 cells were labeled with 35 S-methionine (lanes 1-3) or 32 P-phosphoric acid (0.5 mci/ml) (lanes 4 and 5) for three hours.
  • Cellular lysates were immunoprecipitated with preimmune rabbit IgG (lane 1 and 4) or anti-RB IgG (lanes 2, 3 and 5) .
  • Aliquots of 35 S-methionine-labeled RB proteins were digested with endoglycosidase H (ICN ImmunoBiologicals) overnight (Lane 3). These immunoprecipitates were then analyzed by 7.5% SDS-polyacrylamide gel as described in Figure 1.
  • FIG. 4 is the chromatogram illustrating a conservation of the RB gene product in different vertebrate species.
  • Cell lines of human neuroblastoma, LAN-5 (Lanes 1 and 2), monkey cos, (Lanes 3 and 4), quail fibroblast, QT6, (Lanes 5 and 6), mouse fibroblast, NIH/3T3), (Lanes 7 and 8), and rat fibroblast, rat-2, (Lanes 9 and 10) were labeled with 32 P-phosphoric acid and immunoprecipitated with preimmune IgG (odd numbered lanes) or anti-RB IgG (even numbered lanes) and analyzed as described in Figure 1.
  • RB proteins of similar but distinguishable sizes were found among different vertebrate pieces .
  • Figure 5A is the chromatogram showing localization of the RB protein.
  • 35 S-methionine labeled LAN-1 cells (Lane 4) were fractionated into membrane (Lane 1) , cytoplasm (Lane 2) and nucleus (Lane 3) and protein was immunoprecipitated with anti-pRB 110 IgG. The immunoprecipitates were then analyzed by SDS-PAGE as described for Figure 1.
  • Figure 5B is the chromatogram showing the results of the immunofluorescence studies of RB protein localization within the osteosarcoma cell line U20S. Cells reacted with (i) anti-RB IgG and (ii) preimmune rabbit IgG.
  • Figure 6A is the picture of the column chromatography of the RB gene phosphoprotein in single stranded DNA cellulose.
  • Lane 1 shows the whole cell lysate immunoprecipitated with anti-RB IgG.
  • Figure 6B is the picture of the column chromatography of the RB gene phosphoprotein in double stranded DNA cellulose.
  • Line 1 shows the whole cell lysate immunoprecipitated with anti-RB IgG.
  • Figure 7A is the drawing illustrating the production of the TRP E-RB fusion protein. EcoRl-EcoRl cDNA RB fragment (0.7 kb) was fused in-frame into the EcoRl site of pATH3 vector. Orientation was confirmed by detailed restriction enzyme mapping. The recombinant plasmid was then transformed into E. coli mm294.
  • Figure 7B is the picture of the polyacrylamide gel electrophoresis of the recombinant TRP E-RB fusion protein.
  • the recombinant plasmid was then transformed into E. coli mm294, and grown in M9 minimal medium supplemented with 20 mg/ml of tryptophan. The culture was diluted to
  • Figure 8A is the chromatogram showing immunoprecipitation of RB protein/anti-RB protein IgG from the various human cell lines.
  • 35 S-methionine-labeled cells extracts prepared from a human hepatoma Alexander cell line (Lane 1), human osteosarcoma cell line, U20S (Lane 2), normal human fibroblast (Lane 3), human neuroblastoma cell line, LAN-5 (Lane 4), and from neuroblastoma lysates precipitated by preimmune rabbit IgG (Lane 5) were immunoprecipitated with purified rabbit anti-RB IgG. Doublet bands with apparent molecular weight about 110-114 kD were observed in Lanes 1-4.
  • Figure 8B is the chromatogram showing immunoprecipitation of RB protein/anti-RB protein IgG from the several retinoblastoma cells.
  • Figure 9 is the chromatogram showing a biochemical fractionation to demonstrate the localization of the RB protein.
  • 35 S-methionine labeled whole cells of LAN 5 (Lane 4) was fractionated into membrane (Lane 1), cytoplasm (Lane 2) and nucleus (Lane 3) and were subsequently immunoprecipitated with rabbit anti-RB IgG.
  • the majority of the RB 110-114 protein was found in the nucleus with minor portions in membrane or cytoplasm.
  • Figure 10A and 10B depict Soiled DNA-binding assays. Six ⁇ g of purified TRPE-RB fusion proteins, as well as purified baculovirus-expressed pp110 RB , were applied to 10% SDS-PAGE.
  • Figure 10A depicts a Southwestern DNA binding assay of fusion proteins and baculovirus-expressed p110 RB applied to 10% SDS-PAGE, Coomassie brilliant blue staining. Coomassie brilliant blue staining was used and incubated with 32 P-labeled DNA fragments and analyzed by autoradiography. The following are shown: lane 1: RB19-22; lane 2: RB23-27; lane 3: RB19-27; lane 4: purified RB protein from AcNPV-Y4 RB infected insect cells.
  • Figure 10B is an autoradiograph of a blot from a parallel gel to the gel used to produce Figure 10A. It has been demonstrated that fusion protein RB19-27, which contains the major domain for interacting with DNA, has a 20-fold higher affinity for DNA than either of two subregions, RB19-22 and RB23-27. In this regard, lane 3 of Figure 10B can be compared with lanes 1 and 2, while the purified full-length RB protein exhibited ( Figure 10B; lane 4) . DNA-binding activity of the purified RB protein from insect cells was also demonstrated by retention of the protein by DNA-cellulose and its subsequent elution from the column, at approximately 400 mM NaCl.
  • Figure 11 is a chromatograph showing complex formation of baculovirus-expressed RB protein (p110 RB ) with SV40 T-antigen.
  • the purified full length RB protein was mixed with purified T-antigen in a test tube, i.e., in vitro. Identical aliquots of the mixtures were then immunoprecipitated with PAB419 (lane 2) or anti-RBO.47
  • Lane 3 shows purified SV40 T-antigen immunoprecipitated with PAB419, and purified baculovirus-expressed RB protein immunoprecipitated with anti-RBO.47 antibody, respectively.
  • Mixing of RB protein with SV40 T antigen in vitro resulted in the co-immunoprecipitation of the RB protein with PAB419 (lane 2 ) , as well as the co- immunoprecipitation of T with anti-RBO.47 antibody (lane 3).
  • Figure 12 is a photograph depicting nuclear translocation of purified protein after microinjection into the cytoplasm of Soas-2 cells. The protein preparations used for microinjection were analyzed by SDS-PAGE.
  • Lane 1 p110 RB from insect cells infected with recombinant RB baculovirus; lane 2: biotinylated rabbit anti-goat antibody; lane 3: anti-RB .495 antibody; lane 4: anti-RB R2 antibody; lane 5: anti-RB .47 antibody; lane 6: histone H1; lane 7: p56 RB from E. coli.
  • One microliter of each sample was loaded on a 15% acrylamide gel. The gel was stained with Coomassie brilliant blue. The positions of molecular weight standards, in kiloDaltons, are indicated.
  • Figure 13 depicts SDS-PAGE analysis of the concentration and purity of protein preparations used for microinj ection.
  • Figures 14A through 14F are photomicrographs depicting microinjection and immunostaining of labeled Saos-2 cells.
  • Figure 15 graphically depicts the percentage of labeled cells up to four days after injection.
  • Figure 16 graphically depicts the percentage of labeled cells as a function of p56 concentration.
  • Figure 17 graphically depicts the effect on cells when p56 RB is injected in S phase.
  • Figures 18A through 18C graphically depict the dependence of cell cycle arrest on the time period of injection for the truncated protein.
  • Figure 19 shows p110 RB inhibits the growth of both small cell and non-small cell lung cancer cell lines which are defective in Rb protein expression.
  • the results of this experiment demonstrate that p110 RB can inhibit proliferation of NCL-H596 (NSCLC; Rb-negative).
  • p110 RB has less activity on the A549 (NSCLC; Rb-positive) tumor cell line.
  • the p110 RB used in this experiment was derived from either baculovirus (B-Rb; purified as described in Cell Growth (1990) supra) or from E. coli (E-Rb).
  • the control protein employed was bovine serum albumin (BSA). All proteins were present in the growth media at 25 ⁇ g/ml.
  • Figure 20 is the results of a time course study of RB-mediated growth inhibition. 3 H-thymidine incorporation is expressed as the percent of a buffer control +/- S.D.
  • Figures 21A through 21D show cellular uptake and nuclear localization of 125 I-p110 RB .
  • Figure 21A shows time-dependent localization of
  • Figure 21B shows immunoprecipitation and identification of 125 I -p110 RB in NCl-H596 nuclear fractions. Molecular size standards are indicated. Lane 1: 0 hours; Lane 2: 1 hour; Lane 3: 48 hours; Lane 4: 125 I-p110 RB protein standard.
  • Figure 21C shows immunoprecipitation and identification of 125 I-p110 RB in nuclear fractions of Lung cell lines.
  • Lane 1 NCl-H596 NSCLC (RB neg );
  • Lane 2 A549 NSCLC (RB pos );
  • Lane 3 MRC-9 normal lung epithelium (RB pos );
  • Lane 4 125 I-p110 RB protein standard.
  • Figure 21D shows quantification of nucleus-localized 125 I in NCl-H596 NSCLC cells. Cells were harvested at 0, 8, 24, 48 and 72 hours and nuclear 125 I-p110 RB (I) or 125 I-p56 RB ( ⁇ ) was quantitated.
  • Figure 22 shows the effect of peritumoral injection of RB protein on the growth of subcutaneous lung tumors in nude mice.
  • Figure 22A indicates the NCl-H596 (RB neg ) tumor sizes shown represent the mean value of each treatment group of 3 animals ⁇ S.D.
  • Tumor sizes at day 87 (mm 2 ) were: p110 RB -treated: 169, 130, 100; p56 RB -treated: 400, 360, 252; buffer treated: 858, 420, 770 ( ⁇ )-p110 RB , ( ⁇ )-p56 RB ,
  • Figure 22B indicates the A549 (RB 1308 ) tumor sizes shown represent the mean value of each treatment group of 3 animals ⁇ S.D. Tumor sizes of ' individual animals at day 87 (mm 2 ) : p110 RB treated: 120, 136, 118; p56 RB -treated: 118, 132, 121; buffer treated: 110, 139, 126. ( ⁇ )-p110 RB , ( ⁇ )-p56 RB ( ⁇ ) -buffer control.
  • Figure 23 shows the effect of intravenous administration of p110 RB on the growth of human lung tumor cells in nude mice.
  • Active p110 RB 50 ⁇ g/dose ( ⁇ ); active p110 RB 200 ⁇ g/dose ( ⁇ ); or inactive p110 RB 200 ⁇ g/dose (O). Values shown are Mean ( ⁇ SEM). Arrows indicate days of treatment.
  • Figure 24 shows p110 RB can inhibit the growth of non-small cell lung cancer cells in vitro while having no effect on the proliferation of normal cell lines.
  • p110 RB was prepared from E. coli strain BL21, containing a T7 promoter driving expression of full length p110 RB (described in S. Huang et al., infra.). The p110 RB was purified according to the protocol described in Cell Growth and Diff. (1990) 1:429-437.
  • cells were incubated with the indicated concentrations of p110 RB added every 8 hours for the duration of the experiment. 3 H-Thymidine was added overnight for the last 8 hours to measure cellular DNA synthesis.
  • Cell types used for these experiments were purchased from the American Type Culture Collection.
  • H596 Rb-negative; Non-small cell lung cancer
  • MRC-9 Rb-positive; normal lung epithelial cell line
  • WI- 38 Rb-positive; normal lung epithelial cell line
  • CCD 597 SK Rb-positive; normal foreskin epithelial cell line
  • FSH 738 BL Rb-positive; normal bladder epithelial cell line
  • NIH 3T3 Rb-positive; non-transformed murine cell line
  • Figure 25 shows p110 RB inhibits the growth of both small cell and non-small cell lung cancer cell lines which are defective in Rb protein expression.
  • the results of this experiment demonstrate that p110 RB can inhibit proliferation of H128 and H69 cell lines (both Rb-negative), and H596 (NSCLC; Rb-negative).
  • p110 RB has less activity on the A549 (Rb-positive) tumor cell line.
  • the p110 RB used in this experiment was derived from either baculovirus (B-Rb; purified as described herein) or from E. coli (E-Rb) .
  • the control protein employed was bovine serum albumin (BSA) . All proteins were present in the growth media at 25 ⁇ g/ml .
  • the assay was otherwise performed as described in the legend to Figure 24.
  • Figure 26 shows that p110 RB can enter cells and localize to the nucleus.
  • I 125 -labeled p110 RB is shown to enter cells in a time-dependent manner and localize in the nucleus. Cells were fractionated as described in Nature
  • Figure 27 shows that intact p110 RB can be detected in the nuclei of H596 tumor cells treated with I 125 -labeled p110 RB .
  • This figure shows that when the nuclear fraction (prepared as described (Ibid)), is examined by denaturing SDS gel electrophoresis and autoradiography, that full length p110 RB is seen in the nucleus following 48 hours of incubation. An iodinated p110 RB was used as marker for the autoradiography.
  • Figure 28 shows both p110 RB and p56 RB have biological activity for subcutaneous therapy of non-small cell lung cancer in an animal model.
  • Both proteins were prepared from E. coli BL21, as described above, using the purification methodology as described by herein, 100 ⁇ g of p110 RB or p56 RB was administered daily/subcutaneously in the region of the tumor. Both proteins had demonstrable biological activity as shown in the Figure. Although p56 RB had less activity than p110 RB , this experiment does demonstrate that fragments of p110 RB do have biological activity.
  • Figure 29 shows parenteral therapy of lung cancer using p110 RB in a relevant animal model.
  • 200 ⁇ g of p110 RB was injected three times weekly via the tail vein of Balb/c nude mice which had established and rapidly growing subcutaneous H596 human NSCLC tumors. This dose corresponds to a human dose of approximately 1 mg per kg.
  • the experiment was carried out over a 1 month period, as shown. During therapy, the untreated tumors grew rapidly and the treated tumors either did not grow, or grew at a much reduced rate.
  • Figure 30 shows the amino acid sequence of retinoblastoma protein produced in E. coli. As compared to Figure 2, when the protein is produced in E. coli, the second amino acid (proline) is changed to alanine, for cloning convenience.
  • Figure 31 is a diagrammatic representation of the construction of the baculovirus expression vector for pp110 RB synthesis.
  • Figures 32A and 32B are western blots of ppRB infected insect cells ( Figure 32A) and a blot identifying cellular extracts from infected cells at up to 72 hours post-infection.
  • Figure 33 is an autoradiograph depicting phosphorylation of RB protein in insect cells and the results of dephosphorylation analysis.
  • the present invention comprises a method for cell therapy wherein a specific cancer suppressor gene protein product is delivered to the affected cell to accomplish tumor suppression.
  • the present invention provides a method for treating cancer which reduces the need for conventional radiation and chemotherapy.
  • the inventive technique may be employed at a very early stage, after a genetic predisposition to cancer has been discovered, but before the onset of tumorigenesis.
  • a significant advantage of the present invention is that it uses a cancer suppressor gene protein product in a convenient, and relatively inexpensive manner to accomplish cancer suppression at the cellular level.
  • the cellular introduction of the gene protein product is a novel and advantageous approach to the treatment of malignancy.
  • a further advantage of the present is that, unlike conventional, cytotoxic cancer therapies, the cell therapy herein disclosed accomplishes beneficial changes at the cellular levels, while minimizing trauma to the organism.
  • the present invention is a method of cell therapy wherein a specific cancer suppressor gene protein product is delivered to the affected cell to accomplish tumor, suppression.
  • a given cell may be defective in that it has a missing or defective gene thereby leading to a deficiency in protein expression in the cell.
  • the inventive method relates to using a gene protein product related to the defective gene.
  • the purified protein product is delivered to the affected cell to accomplish, for example, tumor suppression.
  • the product is delivered in a pharmacologically suitable carrier, thereby enabling the protein product to function at the cellular, or subcellular level.
  • an RB gene protein has been delivered to cells having a missing or defective RB gene, which is a cancer suppressor gene.
  • retinoblastoma a rare childhood cancer of the developing retina
  • retinoblastoma susceptibility gene was cloned. This gene contains 27 exons dispersed within 200 kilobases of genomic DNA and expresses a 4.7 kilobase mRNA transcript in all normal tissue examined. Sequence analysis of the complementary DNA clones revealed a long open reading frame that could encode a hypothetical protein of 928 amino acids.
  • the RB gene product has been identified as a nuclear phosphoprotein with relative molecular mass (Mr) of 110,000-114,000, and was named pp110 RB .
  • Mr relative molecular mass
  • pp110 RB relative molecular mass
  • the loss of RB gene function has also been implicated in the development of several other tumor types, including breast cancer, osteosarcoma, prostate cancer and small cell lung carcinoma.
  • a cell therapy method has been invented for the delivery of specific gene protein product to cells having defective or absent genes. Utilizing the present invention, appropriate amounts of substantially purified, intact and biochemically active gene product proteins can be delivered to defective cells in therapeutically effective dosages.
  • the cell therapeutic methods of the present invention has broad applications.
  • the protein product has utility, not only in the treatment of defective cells but in the elucidation of gene functions as the genes interact with one another at the cellular level.
  • retinoblastoma gene protein product pp110 RB
  • pp110 RB has broad application for treatment of eucaryotic cells having a defective, or missing, RB gene.
  • the purified protein can bind DNA and form a specific complex with SV40 T antigen in the same way as the authentic human pp110 RB .
  • the prompt nuclear translocation of the protein after microinjection further suggests the active nature, and therapeutic applications for the purified gene product protein.
  • the present invention includes a method and compositions for controlling cell cycle progression, by introducing into a cell to be controlled, during the interphase of the cell, a cycle regulating composition.
  • the composition is selected from the group consisting of a gene protein product and a fragment of the protein, to alter reversibly the cell cycle progression of the cell while maintaining its viability.
  • the protein fragments have been found to have the unexpected and surprising characteristic of being soluble in low concentration of glycerol, thereby enhancing their value in pharmaceutical applications.
  • An advantage of the present invention is that cell cycle progression can be reversibly arrested in a convenient and safe manner, without insult to the organism. Thus, tumorigenesis may, for example, be controlled.
  • a further advantage of the present invention is that the composition utilized for cell cycle progression control is relatively inexpensive and readably obtainable.
  • a still further advantage of the present invention is the fact that the composition therein utilized possesses little or no toxic effects on healthy cells, and may be used in conjunction with other methods of cancer treatment .
  • compositions and techniques are compatible for use with the regulatory regimens and are physiologically compatible with other methods and devices for regulating certain physiological processes of the body such as blood cell production and gamete production.
  • this invention provides a method of preventing or inhibiting the proliferation of a pathologically proliferating cell, wherein the pathological proliferation of the cell is the result of an absence of a functional retinoblastoma polypeptide or protein in the cell, comprising contacting the cell with an effective amount of retinoblastoma polypeptide or protein, thereby preventing or inhibiting the proliferation of the cell.
  • the term "inhibiting the proliferation of” is intended to be a reduction in the rate of replication of the target cell. In one embodiment, this reduction is characterized by apoptosis or death of the target cell. In a separate embodiment, this reduction is characterized by the target cell being transformed from a malignant phenotype to a differentiated, mature or benign phenotype. The loss of the functional protein or polypeptide can occur by mutation (as defined below) of the retinoblastoma gene or allele or by mutation affecting the expression of the gene, such that a subnecessary concentration is expressed.
  • mutated is defined as altered forms that are commonly found in nature or the one arbitrarily designated as normal. Genetic mutation can be gross deletions, point deletions, additions, substitutions and translocations . All of these genetic alterations at the 13ql4 locus have been implicated in retinoblastoma and pathologies secondary to retinoblastoma.
  • This invention also provides therapeutic and prophylactic regimens for patients having non-ocular malignancies, cancers, or tumors, whose presence has been associated with a mutated RB protein.
  • patients having non-ocular malignancies, cancers, or tumors whose presence has been associated with a mutated RB protein.
  • individuals with hereditary susceptibility to retinoblastoma who have never contacted the disease or been cured are at a higher than normal risk of contacting certain other non-ocular primary cancers.
  • These non-ocular primary cancers for the purposes of this invention are termed “secondary retinoblastoma-linked cancer” or a pathology "secondary to retinoblastoma.”
  • Secondary retinoblastoma-linked cancer includes sporadic bilateral cases wherein two independent mutational events occur on each allele of 13ql4 resulting in a pathologically mutated RB protein.
  • secondary retinoblastoma-linked cancers or tumors include, but are not limited to, osteosarcoma, bone cancer, synovial sarcoma, breast cancer, small cell and non-small cell lung carcinomas, bladder carcinoma, renal cell carcinoma, gastric cancer, prostate carcinoma, leukemia, cervical carcinoma, fibrosarcoma, glioblastoma, acoustic neuroma, chronic lymphocytic leukemia, and acute myelogenous leukemia.
  • this invention is intended to provide a prophylaxis and/or treatment for cancers, tumors or malignancies, the presence of which is the result of an absent or pathologically mutated RB protein in cells of the tumor or cancerous tissue.
  • cancer is defined as a class of diseases of animals characterized by uncontrolled cellular growth, e.g., leukemia, lymphoma, sarcoma, carcinoma, teratoma, metastasis and neoplasm.
  • a tumor is a clump of cells due to abnormal proliferation.
  • this invention provides a therapy to stop the uncontrolled cellular growth in the patient thereby alleviating the symptoms of the disease or cachexia present in the patient.
  • the effect of this treatment and/or prophylaxis includes prolonged survival of the patient, reduction in tumor mass or burden or the reduction of the number of circulating tumor cells.
  • patient and “subject” are synonymous and are intended to include human patients as well as vertebrate subjects, such as mammals.
  • this invention provides a method of preventing or inhibiting uncontrolled cellular growth related to the absence of a normal or wild-type RB protein in the cell.
  • the RB protein is pathologically mutated resulting in the loss of functional retinoblastoma protein in the cell .
  • this invention provides a method of preventing or inhibiting the proliferation of a pathologically proliferating cell, wherein the pathological proliferation of the cell is the result of an absence of a functional retinoblastoma polypeptide or protein in the cell, by contacting the cell with an effective amount of functional or biologically active retinoblastoma polypeptide or protein, thereby preventing or inhibiting the proliferation of the cell.
  • the method prevents uncontrolled cell growth or pathological proliferation of the cell .
  • a tumor or cancer to be treated contains a large number of cells (sometimes of different cell types) at various stages of growth and division, certain cells within a tumor contacted with the RB protein are prevented from uncontrolled growth while simultaneously, others are inhibited from uncontrolled growth.
  • the term "pathologically proliferating cell” is intended to include but is not limited to cells having the capacity for autonomous growth, i.e., existing and reproducing independently of normal regulatory mechanisms . These cells are pathologic because they deviate from normal cells, whether or not associated with a diseased state.
  • Such cells include, but are not limited to, a retinal cell, a prostate cell, a psoriatic cell, a thyroid cell, a breast cell, a colon cell, a lung cell, a sarcoma cell, a leukemia cell, or a lymphoma cell.
  • tumor cells characteristic of cancers such as retinoblastoma, osteosarcoma, fibrosarcoma, glioblastoma, breast cancer, lung cancer, transitional cell carcinoma of bladder, small cell lung carcinoma, non-small cell lung carcinoma, renal cell carcinoma, acoustic neuroma, for example.
  • the method of this invention requires contacting the cell with an effective amount of functional retinoblastoma protein or polypeptide.
  • the contacting may be in vitro or in vivo. When the contacting is in vitro, it is done by removing a sample of cells from a subject and mixing the retinoblastoma protein or polypeptide with the cells.
  • the RB protein may be microinjected into the cells.
  • a culture of cells of a known type also are useful, especially for screening proteins believed to be a functional equivalent of RB protein, such as an RB protein mimetic.
  • the polypeptide or protein can be added to cell culture medium which in turn is then added to the sample of cells.
  • the in vitro method is useful as an assay to determine if the protein therapy of this invention is useful to treat a subject's uncontrolled cellular growth or tumor. If the cells are inhibited in vitro, the examples provided below show that there is a direct correlation between in vitro efficacy of the method and in vivo efficacy of the method.
  • a sample of the suspected tissue can be administered to a mouse or rat and allowed to form a tumor in the animal.
  • the RB protein or polypeptide can be administered to the animal to determine if this protein therapy will be effective in the patient alone, or in conjunction with traditional anti-cancer therapies. If effective, the same ' or similar course of treatment can be utilized with the patient.
  • the successful use of the therapy at the cellular or tissue level is predictive of the therapeutic utility of the method in a patient having hyperproliferative cells present, for example, in a tumor or cancer.
  • telophase telophase
  • the cell enters into an interphase which, depending on a variety of factors, may be of short duration or last for a long period of time.
  • the nerve cells may have a very long interphase.
  • cellular interphase may be regarded as having three stages: G1 in which cell growth occurs without DNA replication, S phase, in which DNA replication occurs, and G2, in which DNA replication has been completed and the cell prepares for division.
  • G1 in which cell growth occurs without DNA replication
  • S phase in which DNA replication occurs
  • G2 in which DNA replication has been completed and the cell prepares for division.
  • certain gene protein products, or fragments thereof have the capacity for controlling progression through the cell cycle by stopping reversibly the progression at G1.
  • cell cyle controlling compositions were introduced into cells during the interphase portion of their cell cycles, to cause a reversible alteration of the cell cycle progression, while maintaining cell viability. After a certain time, when the compositions degraded sufficiently within the cell, the cell cycle has been observed to be reinstated with the cell progressing toward subsequent stages of interphase.
  • cancer suppressor gene protein products such as the RB protein, or a fragment thereof, were utilized to arrest Saos-2 osteosarcoma cells in the G1 stage of interphase. It has been found that the administration of the protein to the cells had no toxic effect on the cells, and was reversible.
  • the method of this invention is useful for treating a patient having, or susceptible to, a retinoblastoma-related pathology or malignancy by administering an effective amount of the RB protein or polypeptide to the patient.
  • this method is useful to treat a pathology characterized by a cell's inability to express a functional retinoblastoma polypeptide or protein by contacting the cell with a functional retinoblastoma polypeptide.
  • the animal having a retinoblastoma- related pathology can be utilized to test new drugs or therapies (an assay system) to be used in conjunction with the protein therapy described herein.
  • the polypeptide or protein is first mixed with a pharmaceutically acceptable carrier for administration.
  • a pharmaceutically acceptable carrier is intended to include, but not be limited to any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, glycerol, mannitol, sucrose human serum albumin, Tween 80, Tris, sodium carbonate and emulsions, such as oil/water emulsions and various types of wetting agents.
  • administering for in vivo purposes means providing the subject with an effective amount of the RB polypeptide or protein, effective to prevent or inhibit proliferation of the target cell or growth of the tumor.
  • Methods of administering pharmaceutical compositions are well known to those of skill in the art and include, but are not limited to, intratumoral injection, oral administration, intravenous administration or parenteral administration. Administration can be effected continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage are well known to those of skill in the art and will vary with the protein or polypeptide used for therapy, the purpose of the therapy, the cell or tumor being treated, and the subject being treated. As an example, a suitable dosage range is from about 0.1 mg/kg/body weight to about 10 mg/kg/body weight .
  • a "functional retinoblastoma protein” is intended to encompass naturally occurring retinoblastoma protein isolated from human cell lines as shown in the examples below or recombinantly produced retinoblastoma protein, for example, encoded by the cDNA shown in Figure 2.
  • the retinoblastoma protein is the protein designated pRB 110 .
  • the amino acid sequence of pRB 110 is shown in Figure 2. Also included within this definition are functional equivalents of purified retinoblastoma protein or recombinantly produced protein pRB 110 .
  • a functional equivalent is a protein having an amino acid sequence different from purified retinoblastoma or pRB 110 but having the ability to produce the same phenotypic effect. These equivalent proteins can have additions, deletions or substitutions that do not substantially affect the ability of the protein to inhibit cell proliferation.
  • a method of determining equivalent proteins is by the in vitro assay described above.
  • Equivalent proteins or polypeptides include fragments of purified retinoblastoma protein or pRB 110 . Examples of a biologically active fragments include, but are not limited to, p56 RB , and the fragments depicted in Figure 2 from amino aicds 393-772, 393 to 870 and 871 to 928.
  • a suitable cell for the practice of this invention is an animal cell; for example, a mammalian cell such as a human or mouse cell. Suitable subjects are mammals, such as mice or humans.
  • the above method is useful not only to inhibit the proliferation of a pathologically proliferating cell, but also is useful to prevent a cell susceptible to such proliferation.
  • patients with hereditary retinoblastoma are at risk for development of a secondary cancer, as defined above.
  • an effective amount or dosage for prophylactic use can differ from a therapeutic amount or dosage, but can be determined by those of skill in the art.
  • This invention also provides a method for treating a pathology in a subject caused by the absence of a functional retinoblastoma protein or polypeptide, or the absence of or the presence of a mutated retinoblastoma gene.
  • the method requires administering to the subject an effective amount of a functional retinoblastoma protein or polypeptide, as described above.
  • the RB gene protein product or the RB protein is an alternatively phosphorylated protein having an apparent molecular weight of about HOkDa to 116 kDa on SDS-PAGE. It is present in normal, i.e., non-transformed, cells in a variety of vertebrate species . In some cases, a pathologically or absent RB protein is the result of a mutation at the chromosomal locus of the RB gene. In a normal cell, the RB protein is the transcription product of a gene located at the 13ql4 region.
  • retinoblastoma In familial cases of retinoblastoma, ("familial bilateral retinoblastoma") a mutation in one allele of this gene in the embryonic retina eventually leads to a retinoblastoma tumor in the developing child. For the unilateral or bilateral form of the disease that has no familial disposition, somatic mutations affecting both alleles must occur in the same retinoblast. See Proc. Natl. Acad. Sci. USA (1971) 68:820-823. However, this invention provides therapeutic and prophylactic regimens for unilateral and bilateral retinoblastomas caused by these mutations.
  • the RB protein is a alternatively phosphorylated protein that can be affinity-purified from cellular extracts. Due to variations in the phosphorylation of the protein, it migrates as a diffuse band of molecular weight 110 to 114 kDa on SDS-PAGE. Phosphorylation of the RB protein occurs on serine and threonine. (Cell (1989) 56:57-65 and Oncogene Res. 1:205-214); this property oscillates during the cell division cycle. The underphosphorylated forms predominate in the G 0 and G 1 phases of the cell cycle while the more highly phosphorylated forms predominate in the G 2 , M, and S phases.
  • RB protein is intended to encompass all variously or alternatively phosphorylated forms of the protein. In certain embodiments, more than one form will be used in the methods of this invention and alternative forms can be administered simultaneously.
  • a "functional retinoblastoma protein", "RB protein” or “RB polypeptide” also is intended to encompass naturally occurring retinoblastoma protein isolated from human or vertebrate cell lines as shown in the examples below or recombinantly produced retinoblastoma protein, for example, encoded by the cDNA shown in Figure 2 in procaryotic and eucaryotic expression systems.
  • the retinoblastoma protein is the protein designated ppRB 110 .
  • the amino acid sequence of ppRB 110 is shown in Figures 2 and 30.
  • the term “purified” or “substantially purified” refers to the approximate level of purity obtained by the experimental procedures described below. Purity can be measured by any known method in the art, such as by gel imaging or using a densitometer.
  • a functional equivalent is a protein having an amino acid sequence different from purified retinoblastoma (ppRB 110 but having the ability to produce the same phenotypic effect.
  • These equivalent proteins can have additions, deletions or substitutions that do not substantially affect the ability of the protein to inhibit uncontrolled cell proliferation.
  • a method of determining equivalent proteins is by the in vitro assay described above.
  • Equivalent proteins or polypeptides include fragments of purified retinoblastoma protein or p110 RB .
  • recombinant RB protein or polypeptide is defined as the product of recombinant expression of a cDNA (as shown in Figure 2) or fragment thereof, in recombinant expression system.
  • a recombinant expression system is a recombinant expression vector stably transformed into a suitable host cell for the recombinant production of RB protein.
  • Recombinant expression vector includes vectors which are capable of expressing DNA sequences contained therein, where such sequences are operatively linked to other sequences capable of effecting their expression. Exemplified below are several expression vectors. It is implied, although not always explicitly stated, that these expression vectors must be replicable in the host organisms either as episomes, as an integral part of the chromosomal DNA, or as a separate chromosome. In sum, “vector” is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified DNA sequence disposed therein is included in this term as it is applied to the specified sequence.
  • “Host-vector system” refers to host cells which have been transfected with vectors constructed using recombinant DNA techniques. Insertion of the vector or DNA can be accomplished by microcell transfer, retrovirus- mediated gene transfer, transfection, cell fusion, etc.
  • the vectors and methods disclosed herein are suitable for use in host cells over a wide range of procaryotic and eucaryotic organisms.
  • recombinant DNA methods currently used by those skilled in the art include the polymerase chain reaction (PCR) which, combined with the synthesis of oligonucleotides, allows easy reproduction of DNA sequences.
  • PCR polymerase chain reaction
  • a DNA segment of up to approximately 6000 base pairs in length can be amplified exponentially starting from as little as a single gene copy by means of PCR.
  • a denatured DNA sample is incubated with two oligonucleotide primers that direct the DNA polymerase-dependent synthesis each afford an approximate doubling of the amount of target sequence.
  • Each cycle is controlled by varying the temperature to permit denaturation of the DNA strands, annealing the primers, and synthesizing new DNA strands.
  • the use of a thermostable DNA polymerase eliminates the necessity of adding new enzyme for each cycle, thus permitting fully automated DNA amplification. Twenty-five amplification cycles increase the amount of target sequence by approximately 10 6 -fold.
  • the PCR technology is the subject matter of United States Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202.
  • nucleic acid molecule shown in Figure
  • a suitable cell for the practice of this invention is an animal cell; for example, a mammalian cell such as a human, rat, or mouse cell. Suitable subjects are therefore animals and mammals, such as rats, simian, mice or humans.
  • the above method is useful not only to inhibit the proliferation of a pathologically proliferating cell, but also is useful to prevent a cell susceptible to such proliferation from uncontrolled cellular growth.
  • patients with hereditary retinoblastoma are at risk for development of a secondary cancer, as defined above. This method is particularly applicable to use in these patients.
  • an effective amount or dosage for prophylactic use can differ from a therapeutic amount or dosage, but it easily can be determined by those of skill in the art.
  • This invention also provides a method for treating a pathology in a subject caused by the absence of a functional retinoblastoma protein or polypeptide, or the absence of or the presence of a mutated retinoblastoma gene.
  • the method requires administering to the subject an effective amount of a functional retinoblastoma protein or polypeptide, as described above.
  • retinoblastoma gene and encoding of the amino acid sequence was identified at 13 chromosome, 13q14:11 region.
  • esterase D cDNA clones and by screening the genomic and cDNA libraries, several clones were obtained. From these clones, two cDNA overlapping clones RB-1 (about nucleotide 100 to about nucleotide 1,700 as shown in Figure 2) and RB-2 (about nucleotide 100 to about nucleotide 1,000 as shown in Figure 2) of 1.6 kb and 0.9 kb, respectively, were identified in human cDNA libraries. Later on, another clone RB-5 (about nucleotide 1,250 to about nucleotide 4,757 as shown in Figure 2) also was identified.
  • the RB-1 clone was hybridized with 4.8 kb mRNA transcript in human fetal retina and placenta. In retinoblastoma samples, RB-1 clone either detected an abnormal mRNA transcript or the mRNA transcripts were not observed at all. Subsequently identified RB-5 clone, with a 3.5 kb insert, gave identical results as RB-1 in mRNA hybridization. Restriction enzyme analysis suggested that RB-5 and RB-1 clones overlapped in a 0.4 kb region and both together defined a DNA segment of about 4.6 kb, a size close to that of the normal RB mRNA transcript.
  • Nucleotide sequence analysis of clones RB-1 and RB-5 was performed by the dideoxy-terminator method described in Proc. Natl. Acad. Sci. (1977) 74:5463-5467 and yielded the reconstructed complete cDNA sequence.
  • Different deletion templates were generated by the "cyclone" method (Ibid) in single stranded M13 phage clones, which yielded greater than 95% of the sequence. The remaining gaps were sequenced by primer extension in both strands. The complete sequence identified in this way contained 4,523 nucleotides.
  • the predicted protein sequence included ten potential glycosylation sites (CRC Crit. Rev. Biochem. (1981) 10:307-366) but a candidate transmembrane domain (at least 20 consecutive hydrophobic residues) was not found.
  • the amino acid hydropathy plot showed a mildly hydrophobic region near the putative amino terminus and a hydrophilic region at the carboxyl terminus. Two pairs of short amino acid sequences were identified that were bracketed by cysteine and histidine residues in the manner of a metal-binding domains found in nucleic acid-binding proteins
  • the RB gene product may be a nucleic acid-binding protein.
  • nucleic acid-binding protein a nucleic acid-binding protein
  • RB protein antigen was prepared by expressing the fusion protein in E. coli. For that purpose, a 20 kb polypeptide fragment of the RB gene was fused with TRP E protein and the fusion protein has been expressed in E. coli.
  • Plasmid pATH3-0.9 RB was constructed from the fragment 5' 0.9 kb inserted into
  • Plasmid pATH3-0.7 RB was constructed by inserting middle 0.7 kb fragment of RB-1 clone into EcoRI-EcoRI site of pATH3, and the plasmid pATH3-1.8 RB was constructed by inserting 3' 1.8 kb fragment into BglII-BglII site of pATH3 vector.
  • the recombinant plasmids pATH3 - 0.9RB, pATH3 - 0.7RB and pATH3-1.8RB were then transformed into E. coli mm 294 and grown in M9 minimal medium which was supplemented with tryptophan preferably of concentration of about 20 mg/ml.
  • the culture mixture was diluted from 1:10 to 1:150, preferably 1:100, with M9 medium, with casamino acids and ampicillin added.
  • the procedure for recombinant plasmid construction is described in J. Virol. (1984) 49:132-141.
  • the fusion of the fragments into pATH vector frames at the site of restriction enzymes is described in Proc. Natl. Acad. Sci. (1986) 83:4685-4689.
  • the obtained fusion protein had a molecular weight of 57 kD. Since the molecular weight of TRYP E is known to be 37 kD, 20 kD protein portion of the fusion protein was derived from the RB.
  • Purified fusion protein was used as an antigen in generating specific anti-RB protein antibody.
  • the specific rabbit polyclonal antibody against RB protein were prepared by the procedure described generally in Proc. Natl. Acad. Sci. (1986) 83:6790-6794.
  • Complete Freund's adjuvant generally consists of an emulsion of the antigen, in this case the fusion protein, in saline and a mixture of an emulsifying agent, such as for example, Arlacel A, in mineral oil and killed mycobacteria. Incomplete Freund's adjuvant is the same except that it does not have the mycobacteria.
  • the purified anti-RB IgG antibody was used for immunoprecipitation or immunostaining, for localization of RB protein and will be equally useful for diagnostic identification of RB protein in human tissue samples.
  • To identify the RB protein several human cell lines known to have either normal or altered RB gene expression were selected.
  • LAN-1 neuroblastoma cell line, normal human fibroblasts, human hepatoma Alexander cell line and osteosarcoma U20S cell line were used as positive controls containing normal RB mRNA. All these cells were obtained from the American Type Culture Collection (ATCC) depository.
  • ATCC American Type Culture Collection
  • RB mRNA lines with expected shortened or absent RB mRNA such as retinoblastomas cell lines Y79 (ATCC), RB355 (gifted from Robert Philips, Toronto, Canada), WERI-1, WERI-24, and WERI-27 (gifted from T. Sery Wills' Eye Hospital, Philadelphia) were used as negative controls.
  • Cells from all human cell lines were metabolically labeled with 35 S-methionine according to procedure described in J. Virol. (1981) 38:1064-1076.
  • Labeled cell mixtures were immunoprecipitated with 1-20 ⁇ l, preferably 10 ⁇ l, of from 50 ⁇ g/ml - 200 ⁇ g/ml, preferably 100 ⁇ g/ml of anti-RB antibody IgG using the procedure described in J. Virol. (1981) 38:1064-1076.
  • the RB proteins immunoprecipitated with rabbit anti-RB IgG were analyzed by SDS/polyacrylamide gel electrophoresis and into auto-radiographed. The results are shown in Figure 1.
  • the RB protein presence is visible at approximately 110 kD region in lanes 2-5 which illustrate the immunoprecipitation of the normal positive, i.e., RB protein containing cell lines labeled with 35 S-methionine.
  • Lane 1 is the control line of neuroblastoma cell immunnoprecipitated with the preimmune, hence without anti-RB protein antibody, rabbit IgG.
  • Lanes 6-10 are obtained by immunoprecipitation of labeled 35 S-methionine cell lines from five retinoblastomas. There is no RB protein present in any of these cell lines.
  • the hypothetical protein predicted from the nucleotide sequence was expected to have MW about 98 kD.
  • the immunoprecipitated protein has a MW about 110-114 kD.
  • Complete RB amino acid sequence is illustrated in Figure 2. This complete sequence is obtained from the newly reconstructed clone which contains the most 5' end stretch missing in the original cDNA clone (Science (1987) 235:1394-1399).
  • the first and second initiation methionines are boxed and alanine and proline clusters are underlined.
  • the amino acid sequence (Figure 2) is written in the single-letter abbreviation code recognized in the art.
  • the RB cDNA sequence (Science (1987) 235:1394- 1399) contained a long open reading frame from nucleotide 1 through 2688, which is translated from the first methionine condon yields a hypothetical protein of 816 amino acids and molecular weight 98 kD. Another RB cDNA clone was isolated which contains an additional 234 base pairs on the 5' end.
  • the revised RB cDNA sequence ( Figure 2) still maintains the same open reading frame as in the original clones, and an additional methionine condon was found at nucleotide 139. When this methionine was used as an initiation condon, the predicted RB protein had 928 amino acids and a molecular weight of 110 kD -- identical to the apparent M.W. determined by SDS-PAGE.
  • the additional 5' sequence contains a GC-rich region that translation into an unusual cluster of alanine and proline residues ( Figure 2).
  • the RB gene was detected in other vertebrates at the DNA level (also described in Science (1987) 235:1394-1399 and Figure 4) suggesting that the RB gene is present in many species during the evolution and further suggesting an important physiological role.
  • the cells from several vertebrate species, such as QT6 (quail), NIH/3T3 (mouse), Rat-2 (Rat) and cos (monkey) were labeled with 32 P-phosphoric acid as described previously and proteins were immunoprecipitated with anti-RB IgG using the same procedure as used for human cells.
  • antigenically related proteins were detected in all cells with apparent similar molecular weights of 108 kD in quail, 120 kD in mouse, 128 kD in rat and 108-110 kD in monkey, as compared to 110-114 kD in human cells.
  • Antigenically related proteins with varied molecular weights observed in different vertebrate species such as quails, mice, rats and monkeys suggest that the RB protein is conserved through the evaluation, most probably in proportion to evolutionary relatedness . Since both antigens and molecular weights are simultaneously conserved in these vertebrate species, it is likely that the RB gene product is present and functionally similar in other species as well.
  • the predicted whole amino acid sequence of the ppRB 110 protein has several characteristics similar to those appearing in other oncogenes. Therefore, the subcellular localization of the ppRB 110 was investigated by cellular fractionization.
  • the osteosarcoma cell line U20S known to have an advantageous cell morphology for immunohistochemical staining were used.
  • the U20S cells were immunoprecipitated with anti- ppRB 110 IgG.
  • the U20S cells were immunoprecipitated with preimmune IgG. Both groups were then incubated with rhodamine conjugated goat anti-rabbit IgG obtained commercially from Sigma. Immunofluorescence was observed in cells reacted with anti-ppRB 110 IgG, namely in the cell nucleus ( Figure 5B). Cells reacted with preimmune control did not show any fluorescence ( Figure 5Bii) .
  • ppRB 110 plays an important regulatory function in regulating other genes and has a DNA - binding activity.
  • Certain cell lines, particularly those from tumors other than retinoblastoma, such as neuroblastoma LAN-1 cells were radioactively labeled with 32 P-phosphoric acid. Cellular lysates of these labeled cell mixtures were separated by single or double stranded calf thymus DNA - cellulose columns according to the method described in Mol. Cell. Biol. (1986) 6:4450-4457.
  • ppRB 110 binds only to a limited number of DNA sites that are easily saturated. It has been previously shown that other protooncogenes such as c-myc, n-myc, c-myb and c-fos are nuclear phosphoproteins with DNA binding activity (Mol. Cell. Biol. (1986) 6:4450-4457; Nature (1982) 296:262-266). Oncogenic activation of these proto-oncogenes occurs by deregulation of gene expression or by structural modification, and the gene product is essential for oncogenicity.
  • protooncogenes such as c-myc, n-myc, c-myb and c-fos are nuclear phosphoproteins with DNA binding activity (Mol. Cell. Biol. (1986) 6:4450-4457; Nature (1982) 296:262-266). Oncogenic activation of these proto-oncogenes occurs by deregulation of gene expression or by structural modification, and the gene product is essential for oncogenicity.
  • the ppRB 110 is therefore an important regulatory protein which can prevent and inhibit, by its presence, and trigger, by its absence, the malignant growth. Thus, the ppRB 110 's importance is in regulating other genes.
  • the absence or loss of ppRB 110 mediates oncogenicity.
  • the presence or absence of the ppRB 110 shall serve as a diagnostic tool in determination of presence or predisposition to retinoblastoma and other retinoblastoma-related non-ocular pathologies or tumors of the human and animal fetus, embryo or newborn babies .
  • Such early diagnosis will allow an early warning and treatment of retinoblastoma and other tumors with the possibility of preventing development of the secondary tumor.
  • the diagnostic method disclosed also is particularly for useful screening families with the history of hereditary retinoblastoma.
  • the diagnostic method is also intended to be used for prophylactic prenatal and postnatal screening.
  • the diagnostic method also will be used for prediction of the development of secondary cancer, for example, osteosarcoma, fibrosarcoma, glioblastoma, breast cancer, etc., whether or not occurring in a patient whp previously had a retinoblastoma tumor.
  • Recombinant fusion protein was prepared for use as an antigen for immunization.
  • the conserved 5' 0.9 kb, middle 0.7 kb and 3' 1.8 kb regions of RB cDNA were subcloned into an inducible, high-level TRP E expression vector, pATH-3 (University of California, San Diego) using a standard procedure described in Maniatis et al. Molecular Cloning: A Laboratory Manual (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • Three RB cDNA subfragments containing the coding sequence namely 5' 0.9 kb (EcoRI-EcoRI), middle 0.7 kb (EcoRI-EcoRI of RB-1) and 3' 1.8 kb (BglII-BglII) were fused in frame with pATH3 vectors, respectively.
  • the TRP E - RB gene product of pATH3-0.7 plasmid was expressed in E. coli using a method described in J. Virol. (1984) 49:132-141.
  • pATH3-0.7 RB was constructed as illustrated in drawing 7A.
  • cDNA RB fragment was inserted in-frame into the EcoRI endonuclease site of pATH-3 plasmid.
  • the recombinant plasmid was transformed into E. coli mm294 and grown in M9 minimal medium supplemented with 20 mg/ml of tryptophan.
  • the culture was diluted to 1:100 in M9 plus casamino acids and ampicillin.
  • a 1:1000 dilution of a 10 mg/ml stock of indoleacrylic acid in 100% ethanol was added to induce the expression of the TRP E promoter.
  • Bacteria cells were pelleted and boiled in Laemmli gel sample buffer for 15 minutes and analyzed by polyacrylamide gel electrophoresis . Gel was then stained with Coomassie blue. Large quantities of fusion protein were prepared and purified through preparative polyacrylamide gel electrophoresis and eluted by overnight extraction. SDS and soluble acrylamide were removed by dialysis. The proteins were then concentrated and mixed with adjuvant for immunization of rabbits. About 6 mg protein was recovered which was subsequently used for immunization of rabbits.
  • the obtained fusion protein had a molecular weight of 57 kD. Since the molecular weight of TRP E is known to be 37 kD, 20 kD protein portion of the fusion protein was derived from the RB.
  • E. coli produced, after induction, a 57 kD fusion protein comprising 20% of total E. coli protein.
  • TRP E-RB fusion protein Production of the TRP E-RB fusion protein can be seen in Figure 7A and the results of the PAGE/gel electrophoresis in 7B.
  • a 58 kD protein was found in induction culture (Lane 2) but not control culture (Lane
  • Purified fusion protein was used as an antigen in generating specific anti-RB protein antibody.
  • the specific rabbit polyclonal antibody against RB protein were prepared by the procedure described generally in Proc. Natl. Acad. Sci. (1986) 83:6790-6794. New Zealand Red rabbits were immunized with the
  • TRP E-RB fusion protein obtained, as described above, following a standard protocol. Specifically, rabbits were repeatedly injected, preferably at 14 day intervals with 1-20 ⁇ g, preferable 10 ⁇ g, of purified fusion protein mixed with complete Freund's adjuvant (initial injection) and then given booster injections of the same amount of the fusion protein in incomplete Freund's adjuvant (repeated injections).
  • Complete Freund's adjuvant generally consists of an emulsion of the antigen, in this case the fusion protein, in saline and a mixture of an emulsifying agent, such as for example, Arlacel A, in mineral oil and killed mycobacteria. Incomplete Freund's adjuvant is the same except that it does not have the mycobacteria.
  • both rabbits produced high titers of antibodies that reacted with both TRP E and the fusion protein.
  • the rabbits were bled and the blood was collected into plastic containers and clotted. The serum was obtained by centrifugation at 1000g for 10 minutes.
  • Rabbit anti-ppRB 110 immunoglobulin (IgG) was purified by passing the antisera through the two affinity columns. To enrich for antibodies recognizing only RB determinants, two affinity columns were prepared, one with TRP E protein and the other with the fusion protein. The antisera was passed through the fusion protein-Sepharose column and eluted with 0.1M glycine HCl buffer (pH 2.3).
  • LAN-1 neuroblastoma cell line normal human fibroblasts, human hepatoma Alexander cell line and osteosarcoma (U20S) cell line which all contain normal RB mRNA were used as positive controls. All these cells were obtained from the American Type Culture Collection (ATCC) depository. Cell lines with expected shortened or absent RB mRNA, such as retinoblastomas cell lines Y79, RB355, WERI-1, WERI-24, and WERI-27 were used as negative controls. These cell lines were obtained as described above.
  • Immunoprecipitation was carried out with 5 ⁇ l of preimmune rabbit antisera, followed by absorption to formalin-fixed Staphylococcus aureus obtained from the Enzyme Center, Inc. To supernatant of each experimental sample was added 10 ⁇ l of 100 ⁇ g/ml of anti-ppRB 110 IgG and to supernatant of each control sample was added 10 ⁇ l of the preimmune sera for control. Protein A sepharose beads (Sigma) were then added. Immunoprecipitates were subsequently washed with 1) lysis buffer, 2) 1 M NaCl in lysis buffer, 3) 0.15 M NaCl in lysis buffer, and 4) lysis buffer to remove nonspecifically bound proteins.
  • the immunoprecipitated proteins were analyzed by 7.5% SDS- polyacrylamide gel electrophoresis and autoradiographed. Gels of 35 S-labeled proteins were fluorographed at -70°C after impregnation with acetic acid-based 2.5-diphenyloxazole.
  • the cells were labeled with 32 P-phosphoric acid or with 14 C or 3 H-glucosamine and subsequently digested with Endoglycosidase H.
  • LAN-1 cells were metabolically labeled with 32 P-phosphoric acid.
  • 32 P To label LAN-1 cells with 32 P, around 1.5 ⁇ 10 6 cells in 60-mm petri dishes were starved by incubation at 37°C for 80 minutes in phosphate-free medium and then incubated for 1 to 2 hours in 2 ml of phosphate-free medium supplemented with 32 PO 4 3 - (1m Ci/ml) medium.
  • Cell extracts were prepared in lysis buffer containing 25 mM Tris-hydrochloride of pH 7.4, 50 mM NaCl, 0.2% Nonideb P-40, 0.5% deoxycholate and 200 units/ml of kallikrein inactivator obtained from Calbiochem. The lysate was clarified at 4°C at 20,000 ⁇ g for 20 minutes.
  • Immunoprecipitation was carried out with anti-ppRB 110 IgG according to standard procedure. Immunoprecipitate was absorbed to formalin-fixed Staphylococcus aureus obtained from the Enzyme Center and subsequently washed with 1) lysis buffer; 2) 1 M NaCl, lOmM Tris-hydrochloride (pH 7.4) and 0.1% Nonidet P-40; 3) 0.15 mM NaCl, 10 mM Tris-hydrochloride (pH 7.4), 0.1 Nonidet P-40; and 4) lysis buffer.
  • the immunoprecipitated proteins were prepared for electrophoresis following the procedure described in J.
  • Human neuroblastoma cells LAN-1 were labeled with 32 S-methionine as described above, and fractionated into membrane, cytoplasm and nucleus. The labeled protein was subsequently immunoprecipitated with anti-RB IgG.
  • Cell fractionation protocol is essentially adapted from that described in J. Cell. Biol. (1983) 97:1601-1611.
  • Two to five 100-mm plates containing a total of 2.0 ⁇ 10 7 to 7.5 ⁇ 10 7 LAN-1 cells were metabolically labeled with 35 S-methionine for 2-3 hours prior to use. All subsequent procedures were performed at 4°C.
  • Cells were rinsed twice with phosphate-buffered saline (PBS), scraped into PBS, and pelleted for 5 minutes at 375 ⁇ g in a table top centrifuge. Half the cells were resuspended in lysis buffer for the whole cell lysate.
  • PBS phosphate-buffered saline
  • the remaining cells were rinsed once in hypotonic RSB buffer (10 mM HEPES pH 6.2, 10 mM NaCl, 1.5 mM MgCl 2 , 200 units/ml Aprotinin) and then resuspended in RSB.
  • the cell suspension was homogenized by 20 strokes in a tight fitting Dounce homogenizer, and volume was adjusted to exactly 3 ml with RSB buffer.
  • the homogenate was centrifuged at 1,500 rpm in a Sorvall HB4 rotor at 375 ⁇ g for 10 minutes, and the pellet was resuspended in lysis buffer to generate the nuclear fraction.
  • the supernatant was centrifuged in thick-walled polyallomer tubes in a Beckman SW50.1 rotor at 35,000 rpm
  • the following fractionation method also can be used. All procedures were performed at 0 to 4°C. Two to five 100-mm plates containing a total of 2.0 ⁇ 10 7 to 7.5 ⁇ 10 7 LAN-1 cells were rinsed twice with phosphate-buffered saline (PBS), scraped into PBS, and pelleted for 1 minute at 1,000 ⁇ g in a clinical centrifuge. After the pellet was rinsed once with hypotonic TKM buffer (20 mm Tris pH 7.1, 5 mM KCl, 1 mM MgCl 2 , 1% Aprotinin), the cells were dispersed and swollen in TKM for 15 minutes. The cell suspension was homogenized by 20 strokes in a tight fitting Dounce homogenizer. The volume was adjusted to exactly 3 ml with TKM buffer, and samples were removed for analysis by immunoprecipitation.
  • PBS phosphate-buffered saline
  • Nuclear pellet was generated by low-speed centrifugation, and the supernatant from this initial centrifugation was subjected to high-speed centrifugation to obtain a precipitate and a soluble fraction.
  • the homogenate was centrifuged at 1,500 rpm in a Sorvall HB4 rotor (375 ⁇ g) for 10 minutes at 0°C, and the crude nuclear pellet was suspended in 1 ml of TKM. This pellet was then homogenized five times in the Dounce homogenizer and aspirated three times through a 1 ml syringe fitted with a 25-gauge needle.
  • the suspension was then pelleted as described above, suspended in TKM buffer and aspirated again five times through the same syringe. After a final centrifugation, the nuclear pellet was suspended in TKM buffer and analyzed for RB protein content and for subcellular markers.
  • the original postnuclear supernatant (PNS) and the supernatants from the nuclear pellet washes were pooled and centrifuged in thick-walled polyallomer tubes in a Beckman SW50.1 rotor at 38,000 rpm (150,000 ⁇ g) for 90 minutes at 0°C to generate particulate (P 150 ) and soluble (S 150 ) fractions. The fractions were then adjusted to equal volumes with TKM buffer and assayed directly for RB protein and subcellular markers.
  • Plasma membrane content was determined by measuring 5' nucleotidase.
  • the samples were taken up in TKM buffer and incubated in an assay mixture containing 10 mM MgCl 2 , 0.1 mM AMP, 100 mM glycine (pH 9.0), and 2 ⁇ Ci of 3 H-adenosine in the supernatant determined by liquid scintillation counting (Ibid).
  • the soluble protein in each fraction was determined assaying for lactate dehydrogenase activity according to Proc. Natl. Acad. Sci. (1962) 48:2123-2130 and the endoplasmic reticulum content was measured with an assay for NADH diaphorase according to procedure described in Biochem. Biophys. Acta. (1971) 233:334-347. The results are illustrated in Figure 5B. Using methods of biochemical fractionation and immunofluorescence
  • the RB protein was determined to be localized primarily in the nucleus. (The fluorescence was present mainly within the nucleus and the preimmune control was negative.)
  • a majority (about 85%) of 35 S-labeled protein was located in the nuclear fraction while a minor portion (less than 10%) was associated with membrane. There was no detectable RB protein within the cytoplasmic fraction, or secreted into the medium.
  • the human osteosarcoma cell line U20S obtained from American Type Culture Collection, was used for immunofluorescent staining. About 10 4 U20S cells were seeded onto 12-mm glass cover slips and used 18 hours later for immunofluorescent staining. The cells were washed once with PBS buffer and fixed with cold acetone for 10 minutes at room temperature. Fixed and permeabilized cells were hydrated in PBS for 1 to 2 minutes. Each cover slip was incubated with 200 ⁇ l of rabbit anti-RB IgG (1:20 dilution) or preimmune serum in a moist chamber for 45 minutes at room temperature.
  • the cover slips were incubated with 200 ⁇ l or rhodamine-conjugated goat anti-rabbit IgG (25 ⁇ g/ml) obtained from Sigma for 45 minutes at room temperature. The cover slips were again washed extensively in PBS and viewed with the Zeiss photomicroscope III.
  • immunofluorescent staining of LAN- 1 neuroblastoma cell lines was carried out as follows. IO 4 LAN-1 cells were seeded onto 12-mm glass cover slips and used 18 hours later for immunofluorescent staining.
  • the cells were washed once with PHEM buffer consisting of 0.06 M Pipes, 0.025 M HEPES, 0.01 M EGTA, 0.002 M MgCl 2 , pH 6.9 and fixed with 2% paraformaldehyde in PHEM buffer for 20 minutes at room temperature. Fixed and permeabilized cells were washed once in PHEM buffer and three times in PBS. Each cover slip was incubated with 12 ⁇ l of a 1:80 dilution of a rabbit anti-RB IgG, or preimmune serum in a moist chamber for 45 minutes at room temperature. After three washes in PBS, the cover slips were incubated with 12 ⁇ l
  • the supernatant was diluted 10-fold with loading buffer (1 mM DTT, 0.5% NP40, 10 mM potassium phosphate, 10% glycerol pH 6.2, Aprotinin 200 units/ml).
  • the diluted extract was then applied to calf-thymus DNA-cellulose columns (Pharmacia) Mol. Cell Biol. (1986) 6:4450-4457, equilibrated with loading buffer containing 50 mM NaCl. After allowing binding to occur for 40 minutes, the column was washed with 5 ml of loading buffer and then eluted with buffer containing 1 mM DTT, 10 mM Tris-HCl, pH 8.0 with increasing NaCl concentration from 0.05 to 1.0 M.
  • Human tumor cells disassociated from biopsy tissue taken from a patient was labeled with 35 S methionine or 32 P-phosphoric acid and immunoprecipitated with anti- ppRB 110 IgG according to the procedure set forth above.
  • protein lysates extracted from bioptic tissue can be directly diagnosed using the western blotting analysis probed with either radioactive labeled or non-radioactive labeled anti-RB specific antibody.
  • the presence or absence of immunoprecipitated proteins serves as a diagnostic tool in determination of retinoblastoma or other diseases controlled by the retinoblastoma gene.
  • Wild-type or normal RB protein is known to possess two biochemical properties of DNA binding proteins. One is the ability to bind DNA intrinsically (Nature (1987) 329:642-645) and the second is the ability to form specific complexes with oncoproteins of several DNA tumor viruses
  • RB protein purified from a baculovirus-infected insect cell system and RB truncated proteins purified from an E. coli expression system were tested for these two known biochemical properties.
  • TRP E-RB fusion proteins described above also were used in this assay.
  • a baculovirus expression system was utilized for the recombinant production of a full-length RB protein.
  • the protein was designated p110 RB .
  • the expression vector was constructed as described in Cell Growth and Diff. (1990) 1:429-437. Recombinant baculovirus construction
  • the baculovirus Autograph californica nuclear polyhedrosis virus (AcNPV) is known to be suitable as a helper-independent viral expression vector for the high-level production of recombinant proteins in cultured insect cells.
  • This virus propagates in cultured Fall Army worm Spodoptera frugiperda (Sf9) cells and has a strong temporally regulated promoter of the polyhedrin gene, whose product represents 50% or more of total cellular proteins during a lytic infection.
  • the coding sequence of a foreign gene can be placed under the transcriptional control of the polyhedrin promoter, resulting in a high level of protein expression.
  • such proteins may be correctly folded and contain appropriate post-translational modifications like those proteins in the original higher eucaryotes.
  • cloned human RB cDNA containing the complete coding sequence of the RB gene, was introduced into the AcNPV expression vector and the recombinant viruses were propagated in insect cells.
  • Successful expression of human p110 RB at high level, by the host-vector system was achieved.
  • the protein produced is phosphorylated and correctly targeted to the nuclei of infected cells.
  • methods for the purification of RB protein were developed. It was found that the purified protein can bind DNA and form a specific complex with SV40 T antigen in the same way as the authentic human pp110 RB .
  • the prompt nuclear translocation of the protein after microinjection further suggests the active nature of the purified RB protein.
  • recombinant transfer vectors were constructed with deletion of most of the 5' non-coding sequence of the RB gene.
  • site-specific mutagenesis two BamHI sites were introduced into the RB cDNA at nucleotides 116 and 2935 to facilitate construction of the recombinant transfer vector.
  • the resulting vector encodes an mRNA that contains the entire (60 base pairs) polyhedrin 5' non-coding sequence fused to 23 base pairs of the 5' untranslated region of the RB cDNA, followed by the complete coding sequence.
  • This recombinant gene contains no ATG codons upstream of the authentic RB initiation site at nucleotide 139.
  • the recombinant gene encodes a non-fusion, full-length RB protein.
  • FIG. 31 there is depicted the transfer vector pAcYMl, which has all the upstream sequences of the polyhedrin gene, including the A of the initiating codon, followed by a unique BamHI site.
  • the transfer vector was designated pAcYMl, which has all the upstream sequences of the polyhedrin gene, including the A of the initiating ATG codon, followed by a unique BamHI site.
  • the transfer vector has been described by Matsura et al. J. Gen. Virol. (1987) 68:1233-1250.
  • pRB44-2 contains the complete RB cDNA coding sequence from nucleotides 116 to 2935 subcloned into the BamHI site of plasmic pGEMl
  • the recombinant baculovirus vector, pAcYMl/RB2.8 was constructed by inserting the 2.8 kb BamHI fragment from pRB44-2 into the BamHI site of pAcYMl in a proper orientation so that the transcription of the RB gene would be under the direct control of the polyhedrin promoter.
  • p"44-2 consists of the complete RB cDNA coding sequence from nucleotide 116 to 2935 subcloned into the BamHI site of pGEMl.
  • pAcYMl contains the approximately 7 kb EcoRI fragment of the viral DNA sequence flanking the polyhedrin gene in which the leader sequence remains intact, but all of the polyhedrin coding sequences except the first A of the ATG are replaced by a BamHI linker.
  • the recombinant baculovirus vector, pAcYMl/RB2.8, containing polyhedrin promoter-RB cDNA fusion was constructed by inserting the RB2.8 BamHI fragment into the BamHI site of pAcYMl so that the transcription of the RB gene would be under the direct control of the polyhedrin promoter.
  • the sequence at the junction of the fusion is shown at the bottom of Figure 31 with the lower case symbol representing the polyhedrin promoter, and the upper case representing the RB cDNA sequence, while the BamHI linker is underlined.
  • (+1) of Figure 31 represents the first A of the translation start codon ATG of the polyhedrin gene.
  • Transfer of RB cDNA from the recombinant plasmid to the viral genome was achieved by cotransforming pAcYMl/RB2.8 DNA with wild-type Autographa californica nuclear polyhedrosis virus DNA by lipofection (BRL).
  • the recombinant viruses in which the polyhedrin gene had been inactivated by allelic replacement with the RB gene through homologous recombination, were identified by their distinct plaque morphology as they showed no polyhedrin occlusion bodies in infected cells.
  • the viruses were subjected to three rounds of plaque purification to obtain a pure stock of RB-containing baculovirus, which was designated as AcNPV-Y4 RB.
  • Sf9 cells were prepared.
  • Sf9 a clonal isolate of Spodoptera frugiperda IPLB-Sf21-AE In vi tro, 13:213-217 (1977) was grown as a monolayer or suspension cultures at 27°C in Grace's insect medium supplemented with 3.33 gm/l of yeastolate, lactalbumin hydrolysate (GIBCO), and 10% heat-inactivated fetal bovine serum (GIMINI) Bull. 1555 (1987), (Texas Agricultural Experiment Station, College Station, TX). In large-scale preparation of cellular lysates, spinner cultures of Sf9 cells were grown in EX-CELL 400 serum-free defined medium
  • Molt-4 cells a human T cell leukemia line, were cultured in suspension in RPMI 1640 supplemented with 20% calf serum.
  • Saos-2 cells an osteosarcoma cell line, were grown in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal bovine serum.
  • Nonidet P-40 0.2% Nonidet P-40; 1 mM EDTA; 100 mM NaCl; 50 mM NaF and 1 mM PMSF) , and the lysates were clarified by centrifugation (4°C, 20,000 ⁇ g) for 5 minutes. Lysates were then incubated with anti-RBO.47 antibody, and immunoprecipitates were separated by 7.5% SDS-PAGE. Proteins were then transferred to nitrocellulose paper, following conventional techniques. After overnight blocking, the nitrocellulose paper was incubated with pMG3-245 anti-fRB monoclonal antibody for 3 hours, followed by alkaline phosphatase- conjugated goat anti-mouse IgG and colorigenic substrates, as described in Cell (1988) 54:275-283.
  • the RB gene encodes a nuclear phosphoprotein of about Mr 110,000.
  • AcNPV-Y4 RB-infected Sf9 cells were immunostained with anti-RBP.47 antibody 40 hours after infection. Such a condition is characteristic of the cytopathic effect of baculovirus infection.
  • Phosphorylation of RB protein occurs at multiple serine and threonine residues and accounts for the molecular weight heterogeneity of RB protein in the SDS-PAGE see Figure 33 and Oncogene Res. (1989) 1:205-214 and Cell (1989) 56:57-65.
  • AcNPV-Y4 RB-infected Sf9 cells were metabolically labeled with 35 S-methionine or 32 P-orthophosphate for 3 hours at 40 hours after infection.
  • Cell extracts were subjected to immunoprecipitation and analyzed by SDS-PAGE followed by autoradiography.
  • Cell extracts were then prepared in lysis buffer (50 mM Tris-HCl, pH 7.4; 0.2% Nonidet P-40; 1 mM EDTA; 100 mM NaCl; 50 mM NaF and 1 mM PMSF) , and immunoprecipitation with anti-RBO.47 antibody was performed.
  • lysis buffer 50 mM Tris-HCl, pH 7.4; 0.2% Nonidet P-40; 1 mM EDTA; 100 mM NaCl; 50 mM NaF and 1 mM PMSF
  • the cellular lysate washed, and resuspended in an extraction buffer containing 50 mM Tris-HCl, pH 7.4; 0.2% NP-40; 1 mM EDTA; 100 mM NaCl; 10% (v/v) glycerol; ImM DTT; ImM PMSF; 25 ⁇ g/ml leupeptin and 50 units/ml aprotinin.
  • an extraction buffer containing 50 mM Tris-HCl, pH 7.4; 0.2% NP-40; 1 mM EDTA; 100 mM NaCl; 10% (v/v) glycerol; ImM DTT; ImM PMSF; 25 ⁇ g/ml leupeptin and 50 units/ml aprotinin.
  • Immunoaffinity chromatography of the RB protein was carried out on a two milliliter volume column containing an anti-fRB monoclonal antibody (pMG3-245) linked to protein G-Agarose. After passing the supernatant through the column four times, the column was washed sequentially with 200 bed-volumes of each of the following: lysis buffer, lysis buffer containing 500 mM NaCl, and washing solution (200 mM NaCl; ImM EDTA; ImM DTT; ImM PMSF; 10% glycerol).
  • Bound proteins were then eluted from the column by alkaline elution buffer containing 20 mM triethylamine, pH 10.8; 200 mM NaCl; ImM EDTA; 1mM DTT; ImM PMSF and 10% glycerol.
  • alkaline elution buffer containing 20 mM triethylamine, pH 10.8; 200 mM NaCl; ImM EDTA; 1mM DTT; ImM PMSF and 10% glycerol.
  • One-ml fractions were collected, immediately neutralized with one-twentieth volume of 1M Tris-HCl (pH 7.5) and stored at -70°C in 10% glycerol.
  • the amount of total protein was determined and subsequently, Soiled DNA-binding assays and SV40 T-antigen binding assays were performed as described below.
  • the amount of total protein in the elution fraction of the immunoaffinity column was determined by Micro-BCA assay (PIERCE).
  • the total eluted protein sample was then analyzed by SDS-PAGE, and the amount of RB protein in the eluate was estimated by Coomassie brilliant blue staining followed by densitometry.
  • the amount of total protein in the cellular extract was measured by the method of Bradford (Bio-Rad) Anal. Biochem. (1976) 72:248-254.
  • Western blotting was performed using serially diluted purified RB protein as standard followed by densitometric comparison of the band intensity.
  • TRP E-RB fusion proteins were included as controls. Each TRP E-RB fusion protein was named according to the exons of the RB gene that the protein contains .
  • RB19-22, RB23-27, and RB19-27 spanned the regions of the recombinant protein from exon 19 to 22 (amino acids 612-775), exon 23 to 27 (amino acid 776-928) and exon 19 to 27 (amino acid 612-928), respectively.
  • SV40 T-antigen was purified by immunoaffinity chromatography from Ad-SV XI-infected 293 cells, see J. Virol. (1985) 53:1001-1004 and "Eucaryotic Viral Vectors" Cold Spring Harbor Press. (1982) Cold Spring Harbor, NY pp. 187-192, and an anti-T monoclonal antibody (PAB419 antibody) was obtained from Oncogene Inc. A known complex formation assay was performed as described in Cell (1988) 54:275-283, with minor modification.
  • baculovirus-expressed RB protein were mixed with 1 ml of EBC buffer (50 mM Tris-HCL, pH 8.0; 120 mM NaCl; 0.5% Nonidet P-40) containing 1 mM PMSF 25 ⁇ g/ml leupeptid 50 units/ aprotinin.
  • EBC buffer 50 mM Tris-HCL, pH 8.0; 120 mM NaCl; 0.5% Nonidet P-40
  • PMSF 25 ⁇ g/ml leupeptid 50 units/ aprotinin.
  • 8 hundred (800) ng of purified T antigen was added to the mix; it was incubated on ice for 90 minutes. Aliquots of the mixture were immunoprecipitated with either anti-RBO.47 or PAB419 monoclonal antibody and subjected to western blotting analysis. Blots were sequentially reacted with pMG3-245 followed by PAB419. After incubating with alkaline phosphatase-conjugated goat anti
  • FIGS 10A and 10B depict Sumble DNA-binding assays.
  • Coomassie brilliant blue staining was utilized while in the assay of Figure 10B, a parallel gel was electrotransferred onto nitrocellulose paper; it was then incubated with 32 P-labeled DNA fragments and analyzed by autoradiography.
  • Sf9 cells were infected with AcNPV-Y4 at a MOI of 1.0, and cultured in suspension (1 ⁇ 16 6 cells/ml, 1000 ml). After 40 hours of infection, the cells were pelleted by low-speed centrifugation, washed, and resuspended in an extraction buffer containing 50 mM Tris-HCl, pH 7.4; 0.2% NP-40; 1 mM EDTA; 100 mM NaCl; 10% (v/v) glycerol; ImM DTT; ImM PMSF; 25 ⁇ g/ml leupeptin and 50 units/ml aprotinin.
  • lysis buffer lysis buffer containing 500 mM NaCl, and washing solution (200 mM NaCl; ImM EDTA; ImM DTT; ImM PMSF; 10% glycerol).
  • Bound proteins were then eluted from the column by alkaline elution buffer containing 20 mM triethylamine, pH 10.8; 200 mM NaCl; ImM EDTA; 1mM DTT; ImM PMSF and 10% glycerol.
  • alkaline elution buffer containing 20 mM triethylamine, pH 10.8; 200 mM NaCl; ImM EDTA; 1mM DTT; ImM PMSF and 10% glycerol.
  • One-ml fractions were collected, immediately neutralized with one-twentieth volume of 1M Tris-HCl (pH 7.5) and stored at -70°C in 10% glycerol.
  • the amount of total protein was determined and, subsequently Soiled DNA-binding assays and SV40 T antigen binding assays were performed.
  • the amount of total protein in the elution fraction of the immunoaffinity column was determined by Micro-BCA Assay (PIERCE).
  • the eluted protein sample was then analyzed by SDS-PAGE, and the amount of RB protein in the eluates was estimated by Coomassie brillian blue staining followed by densitometry.
  • the amount of total protein in the cellular extract was measured by the method of Bradford (Bio-Rad) Anal. Biochem. (1976) 72:248-254.
  • Western blotting was performed using serially diluted purified RB protein as standard followed by densitometric comparison of the band intensity. In this regard, see the Table above.
  • Protein blotting was performed, utilizing conventional techniques. Incubation of blots with radiolabeled DNA followed the protocols described by Nucleic Acids Res. (1980) 1-21. The procedure was carried out at room temperature. Blots were rinsed briefly with water and then washed three times with 6M urea; 0.2% NP-40 (20 minutes each), followed by four washes (30 minutes each) with DNA-binding buffer (10 mM Tris-HCl, pH 7.0; 1 mM EDTA; 50 mM NaCl; 0.2 % BSA; 0.2% Ficoll 400 and 0.2% polyvinyl pyrolidone). The blots were then incubated for 30 minutes in DNA-binding buffer containing 32 P-labeled DNA.
  • TRPE-RB fusion proteins were included as controls. Each TRPE-RB fusion protein was named according to the exons that the protein contains.
  • RB19-22, RB23-27, and RB19-27 spanned the regions of pp110 RB from exon 19 to 22 (amino acids 612-775), exon 23 to 27 (amino acid 776-928) and exon 19 to 27 (amino acid 612-928), respectively.
  • SV40 T antigen was purified by immunoaffinity chromatography from Ad-SV Xl-infected 293 cells J. Vir. (1985) 53:1001-1004; Cold Spring Harbor Press. Cold Spring Harbor, NY pp. 187-192 (1982) and anti-T monoclonal PAB419 antibody was obtained from Oncogene Inc.
  • a known complex formation assay was performed, with minor modification, in which 800 ng of baculovirus expressed RB protein was mixed with 1 ml of EBC buffer (50 mM Tris-HCl, pH 8.0 , 120 mM NaCl and 0.5% Nonidet P-40) containing 1 mM PMSF, 25 ⁇ g/ml leupeptid and 50 units/ml aprotinin. 800 ng of purified T was added to the mix and mixture was incubated on ice for 90 minutes. Aliquots of the mixture were immunoprecipitate with either anti-RBO.47 or PAB 419 antibody and subjected to western blotting analysis. Blots were sequentially reacted with pMG3-245 followed by PAB419. After incubating with alkaline phosphatase-conjugated goat anti-mouse IgG, the blots were developed with colorigenic substrates.
  • EBC buffer 50 mM Tris-HCl, pH 8.0 , 120 mM NaCl
  • Figure 11 depicts complex formation of baculovirus-expressed RB protein (p110 RB ) with SV40 T-antigen.
  • the purified full length RB protein was mixed with purified T-antigen in a test tube, i.e., in vi tro .
  • Identical aliquots of the mixtures were then immunoprecipitated with PAB419 (lane 2) or anti-RBO.47 (lane 3) and analyzed by western blotting.
  • Lanes 1 and 4 show purified SV40 T-antigen immunoprecipitated with
  • PAB419 PAB419, and purified baculovirus-expressed RB protein immunoprecipitated with anti-RBO.47 antibody, respectively.
  • RB protein Purified RB protein, p110 RB , was injected into the cytoplasm of Saos-2 cells, an osteosarcoma cell line which contains a defective RB gene with deletion of exons 21-27 and encodes a C-terminal truncated RB protein (p95), Proc. Natl. Acad. Sci. USA (1990) 87:6-10.
  • the p95 protein is located in the cytoplasm in such minute amounts that it is not recognized by the anti-RBO.47 antibody used herein, even though the antibody is directed against the C-terminus of RB protein.
  • cells were fixed and subjected to immunostaining analysis.
  • purified RB protein was dialyzed into injection buffer containing 20 mM Tris-HCl, pH 7.4; 10 mM KCl ; 0.1 mM EDTA; 0.1 mM DTT and 2% glycerol to a final concentration of 0.5 mg/ml.
  • Saos-2 cells growing on glass chamber slides were microinjected according to conventional techniques, using glass capillary needles (Eppendorf).
  • An Eppendorf micromanipulator, equipped with a vacuum and pressure device, and an inverted phase-contrast microscope (Nikon) were employed for micromanipulation of the capillary and visualization of the microinjection process.
  • the cells were immediately fixed by 4% formaldehyde in 0.04 M phosphate buffer (pH 7.4) and subjected to immunostaining analysis.
  • Figure 12 shows nuclear translocation of the recombinant RB protein p110 RB after microinjection into the cytoplasm of Saos-2 cells.
  • the cells were injected with purified RB protein and subjected to immunostaining analysis.
  • the arrow in Figure 12 indicates the intense staining of the nucleus after microinjection, as compared to that of uninfected cells.
  • the intense staining of the nucleus of the injected cell as compared to that of the uninfected control indicates the rapid transport of the injected protein into the nuclei. Since RB protein has been known as a nuclear protein, the prompt and accurate nuclear translocation of purified protein across the nuclear membrane after microinjection, further suggests that the recombinant RB protein is active in vivo.
  • RB protein produced in a baculovirus expression system has been shown to be accurately targeted to the nuclei of insect cells implying that mammalian nuclear translocation signals are also recognized by insect cells.
  • glycosylation of recombinant proteins in the baculovirus expression system seems limited to the O-linked and N-linked oligosaccharides of the high mannose-type, appropriate phosphorylation of foreign proteins has been reported for the expression of c-myc and HTLV-I p40 x .
  • RB protein has previously been shown to be phosphorylated but not glycosylated, making the baculovirus expression system suitable for the production of functional protein.
  • the RB p110" protein produced in infected insect cells is post-translationally phosphorylated, and multiple bands can be differentiated by western blotting analysis, just as in the case of authentic mammalian RB protein.
  • band intensity un- and hypophosphorylated forms are predominant when compared to the hyperphosphorylated RB protein. It is not known whether this phenomenon is a reflection of the cell cycle status of the population during a viral lytic infection, or is simply due to the insufficient phosphorylation of the protein by insect kinases because of the massive amount of exogenous RB present in the cells . Precise mapping of phosphorylation sites in the RB protein will determine whether the phosphorylation patterns are truly identical to that of mammalian protein.
  • the total level of recombinant RB protein expressed in the baculovirus system is about 17-18 mg per liter of infected insect cell culture ( ⁇ 10 9 cells). This level of expression is comparable to other mammalian proteins produced by this system, such as 10-20 mg/l for interleukin 2 The Banbury Report. Fields, B., et al. (ed.)
  • RB protein may be enhanced by using a recombinant transfer vector containing the intact polyhedrin 5' untranslated region, fused with the RB cDNA deprived of most of its 5' non-coding region.
  • This sequence of the RB mRNA is highly G+C rich, a factor which may favor the formation of stable secondary structures. These structures, when present in front of an initiation codon, are thought to decrease the translational efficiency of the corresponding mRNA.
  • Saos-2 An osteosarcoma cell line, Saos-2, was used for these studies. It was obtained from the American Type Culture Collection. This cell line lacks expression of wild-type RB protein, but contains a cytoplasmic, carboxytruncated 95 kDa protein which cannot bind SV40 T-antigen. Saos-2 cells respond to expression of exogenously added gene encoding RB, introduced by retrovirus mediated gene transfer, by an initial enlargement of cell size and loss of tumorigenicity of cells transferred to nude mice. Hence, it was determined that these cells might be particularly sensitive to injection of RB protein.
  • the cell line SR-40 which stably expresses exogenous RB gene to RB protein, was derived from Saos-2 by single cell cloning after infection with a retrovirus carrying the RB gene as described in Science (1988) 242:1563-1566.
  • African Green monkey kidney cell lines CV-1 and COS-7 were also obtained from American Type Culture Collection. COS-7 was derived from CV-1 by transformation with an origin-defective SV-40 as described in Cell (1981) 23:175-182. All the cells were cultured in Dulbecco's modified Eagle's medium plus 10% fetal calf serum as recommended.
  • RB protein Two forms of RB protein were prepared for the microinjection experiments. Recombinant full length RB protein, (p110 RB both hypophosphorylated and unphosphorylated forms) was purified to near homogeneity from baculovirus infected insect cells. At concentrations approaching 1 mg/ml however, the protein aggregated into a form which could not be injected. To partially alleviate this problem, RB protein was purified, stored, and injected in buffers containing 10% glycerol. An unphosphorylated, amino-truncated 56 kDa RB protein (P56 RB or truncated RB protein), containing an intact T-antigen binding domain, was expressed in E. coli and purified to near homogeneity.
  • RB truncated protein is the C-terminal half of the RB protein and contains both regions essential for SV40 T-antigen binding. It is produced in E. coli from a T7 RNA polymerase expression system as disclosed in Nature (1991) 350:160-162. p110 RB is produced in insect cells by recombinant means as disclosed above and in Cell Growth and Diff . (1990) 1:429-437. Both p110"* and p56" proteins were purified to homogeneity by conventional chromatography.
  • Histone HI and rabbit anti-goat IgG were purchased from Boehringer Mannheim and Vector laboratories, respectively.
  • Antibodies .495 (against the TRP E-RB fusion protein consisting of sequences from exons 19-22), .47 (against the TRP E-RB fusion protein containing sequences from exons 23-27 of RB) and R2 were concentrated in microinjection buffer to an approximate concentration of 1 mg/ml.
  • the T peptide and p53 peptides were the gift of Nicholas Lin.
  • the T peptide comprises amino acids 101-118 and was dissolved in microinjection buffer at 1 mM or 5 mM as described in Cell (1989) 56:57-65.
  • the mutant T peptide contains a lysine to aspartic acid substitution and was used at 5 mM, Cell (1989) supra.
  • the p53 peptide was dissolved in microinjection buffer at 5 mM.
  • Protein preparations except the full-length RB protein, were concentrated in an injection buffer containing 20 mM Tris, pH 7.4; 0.1 mM EDTA; 10 mM KCl; 1 mM 2-mercaptoethanol and 2% glycerol using the Centricon 30 micro-concentrator (Amicon).
  • p110 RB was kept in a buffer containing 10% glycerol to reduce aggregation.
  • the retinoblastoma gene when referred to, it is intended to mean the gene having the nucleotide sequence depicted in Figure 2.
  • the protein having the amino acid sequence also depicted in Figure 2 is also intended.
  • the truncated protein fragment, p56 RB is illustrated.
  • the p56"k, fragment is depicted in Figure 2 as commencing at arrow A and terminating at arrow B.
  • the C terminal peptide is illustrated, commencing at amino acid 917 (arrow C) and terminating at amino acid 928 (arrow B). Protein preparations were analyzed by SDS-PAGE using standard techniques.
  • Figure 12 shows the concentration and purity of protein preparations used in the microinjection techniques discussed below.
  • the protein preparations used for microinjection were analyzed by SDS- PAGE.
  • Lane 1 p110 RB from insect cells infected with recombinant RB baculovirus
  • lane 2 biotinylated rabbit anti-goat antibody
  • lane 3 anti-RB .495 antibody
  • lane 4 anti-RB R2 antibody
  • lane 5 anti-RB .47 antibody
  • lane 6 histone HI
  • lane 7 p56 RB from E. coli.
  • One microliter of each sample was loaded on a 15% acrylamide gel. The gel was stained with Coomassie brilliant blue.
  • a solution composed of equal volumes of glycerol and PBS was added and the specimens were covered with a glass coverslip. Specimens were examined under a fluorescent microscope with Texas Red and Fluorescein filters.
  • the following examples demonstrate the experimental results achieved after introduction of RB proteins into tumor cell lines. Specifically, the examples show the capacity of the RB proteins, or fragments thereof, to arrest progression of a cell infected with exogenously added RB protein, through the cell cycle. In addition, the examples demonstrate that the effect of the blocking can be relieved by SV40 T antigen.
  • the RB gene product was used to arrest the cell cycle progression of an osteosarcoma cell line, Saos-2, treated with full length RB protein or a fragment thereof. It was discovered that cell cycle progression was arrested in G1 and that this progression is reversible.
  • Figures 14A through 14F there is depicted the results of microinjection and immunostaining of Saos-2 cells labeled with BrdU. Saos-2 cells were injected as described herein.
  • Figure 14A shows cells injected with p56 RB , immediately fixed, and indirectly immunostained for RB protein with Texas Red.
  • Figure 14B contains uninf ected cells labeled with BrdU for 4 hours, and then fixed and immunostained with fluorescein conjugated anti-BrdU antibody.
  • Figures 14C and 14D are a single field of synchronized cells co-injected in early G1 with p56 ⁇ and R ⁇ G, incubated with BrdU for 24 hours, then fixed and stained. The cells stained with Texas Red mark injected cells, while the cells stained with fluorescein indicate cells which have incorporated BrdU.
  • Figures 14E and 14F are a single field of cells also injected in early G1 with R ⁇ G alone.
  • asynchronously growing Saos-2 cells were cytoplasmically microinjected with p56 RB or p110 RB and fixed 5 to 15 minutes later. The cells were then immunostained with rabbit anti-RB antibody .47 and a Texas Red conjugated anti-rabbit antibody. Staining was mainly observed in the nucleus ( Figure 14), although there was some staining observed in the cytoplasm of some injected cells. Both p110 RB and p56 RB proteins were capable of being transported to the nucleus within 15 minutes.
  • RB protein a biotinylated, polyclonal rabbit anti-goat antibody (R ⁇ G) that served as a cytoplasmic marker for injected cells. It was estimated that 5-50 million molecules of RB protein were injected per cell. The number of endogenous RB protein molecules per cell was estimated to be approximately 1 million. Hence the injected protein represented at least a 5 to 50-fold excess over endogenous levels.
  • the cells were incubated in growth media containing bromodeoxyuridine (BrdU). Cells progressing through S phase during the labeling period will incorporate BrdU into their DNA.
  • PrdU bromodeoxyuridine
  • RB was injected into synchronized cells.
  • Cells were synchronized by treatment with nocodazole, which arrest cells in mitosis, and were then released and incubated for an additional 6 hours at which time non-adherent cells were removed and the remaining cells injected.
  • nocodazole which arrest cells in mitosis, and were then released and incubated for an additional 6 hours at which time non-adherent cells were removed and the remaining cells injected.
  • cells were fixed and stained for BrdU and R ⁇ G. At least 80-90% of uninfected cells could be stained for BrdU.
  • truncated RB protein was injected into cell line SR-40 which was derived from Saos- 2; it stably expresses full length RB protein.
  • the effect of RB protein injection at early G1 phase on synchronized SR-40 cells was identical to the effect on Saos-2 cells (Table 2); very few cells injected with the RB truncated protein entered S phase over the 24 hour labeling period.
  • the presence of endogenous wild-type full length RB protein did not interfere with the effect of the RB truncated protein on cell cycle progression.
  • Figure 15 graphically shows the number of injected Saos-2 cells which incorporated BrdU during the labeling period.
  • Saos-2 cells were co-injected with 56 kD RB protein and R ⁇ .G 6-7 hours after release from nocodazole treatment. After injection, cells were incubated in media supplemented with BrdU for the indicated number of days before fixing and staining. The percentage value indicates the number of injected cells which had incorporated BrdU during the labeling period. The values represent at least 100 injected cells.
  • FIG. 16 graphically shows the dependence of cell cycle arrest on the dose of p56 RB .
  • Saos-2 cells were co-injected 6-8 hours after release from nocodazole treatment with the indicated concentrations of the truncated protein and 1 mg/ml of R ⁇ G. After 24 hours of incubation in growth medium with BrdU, cells were fixed and stained for BrdU and R ⁇ G. The histogram indicates the percentage of injected cells which incorporated BrdU. Each value represents at least 150 injected cells.
  • the block was alleviated with reagents that bound specifically to the truncated protein.
  • the RB protein (p56 RB ) was mixed with 1 mg/ml solutions of rabbit polyclonal antibodies .495, .47, or R2. These antibodies were raised against unique RB fusion proteins, and recognized the RB truncated protein on Western blots (31, 49, 56). Injection of the mixture of the RB truncated protein and .495 resulted in BrdU incorporation in about 30% of injected cells, compared to an almost total lack of incorporation in cells injected with this protein alone (Table 2).
  • the protein preparations used for injection are indicated.
  • the percentage of injected cells that stained for BrdU after the respective labeling period is shown. After injection, cells were released from aphidicolin treatment and incubated with BrdU for 4-6 hours, fixed, and immunostained for BrdU and R ⁇ G as described. In contrast to cells injected in early G1, approximately 60% of injected, truncated RB protein, aphidicolin arrested cells stained positively for BrdU incorporation. The percentage of cells entering S phase and the intensity of BrdU staining was similar to cells injected with histone and R ⁇ G, or R ⁇ G alone.
  • aphidicolin arrested cells injected with the RB protein were incubated for an additional 6 hours prior to release and labeling with BrdU.
  • BrdU staining was probably caused by the prolonged incubation with aphidicolin itself, the percentage comparable to that of cells injected with R ⁇ G alone.
  • FIGS 18A through 18C graphicaphically depict the dependence of cell cycle arrest on the time period of injection for the truncated protein.
  • Saos-2 cells were arrested in mitosis by treatment with nocodazole. After release from arrest, the cells were incubated for the indicated number of hours in the presence of BrdU, then fixed and stained with an anti-BrdU antibody. The histogram indicates the percentage of counted cells that stained for BrdU. Each value represents at least 200 cells.
  • Figure 18B depicts the time of onset of S phase in Saos-2 cells, in hours, after release from nocodazole arrest in mitosis.
  • the length of G1 phase is about 22 to 24 hours.
  • the length of S phase is about 7 to 8 hours.
  • the length of the G2 phase has not been precisely determined.
  • the arrows indicate the time points of injection that are used for the experiment described in the legend to Figure 18C.
  • Saos-2 cells were co-injected with 1 mg/ml the truncated RB protein and R ⁇ G at the indicated times, in hours, after release from nocodazole treatment. Incubation was continued after injection in growth medium with BrdU until 30 hours after the original release from nocodazole. After BrdU labeling, cells were fixed and stained for BrdU and R ⁇ G. The histogram shows the percentage of the protein injected cells which incorporated BrdU. Each value represents at least 200 injected cells. Very few cells entered S phase at 30 hours after release if injected with the protein 5-10 hours after release from nocodazole ( Figure 20C).
  • SV40 T antigen relieves the blocked cell cycle progression by the truncated RB protein
  • the effect of the truncated RB protein injection on G1 progression in the presence or absence of SV40 T antigen was compared.
  • a 1 mM T peptide solution which was capable of binding to the p56 RB was mixed with an equal volume of the truncated RB protein and injected into synchronized cells in early G1 phase. At this concentration, the peptide was at a 100 to 200-fold molar excess over the RB protein.
  • COS-7 cells were chosen since they were derived from CV-1 cells by transformation with an origin-defective from CV-1 cells by transformation with an origin-defective SV-40 mutant, and they expressed a high level of T-antigen. Therefore, they can serve as a good system for testing the effect, if any, of the presence of endogenous T-antigen on the activity of the injected RB protein.
  • the percentage of synchronized CV-1 cells injected with the truncated RB protein in early G1 phase that incorporated BrdU was at least 5-fold lower than cells injected with R ⁇ G alone (Table 3).
  • Synchronized COS-7 cells were not inhibited at all from progressing into S phase. These results indicated that the presence of endogenous T-antigen, or co-injection with a T-antigen peptide, could neutralize the inhibitory activity or RB protein. Thus, those skilled in the art can utilize this system to screen RB protein fragments to identify those fragments capable of blocking cell cycle progression by comparing the percentage of CV-1 to COS-7 cells entering S phase in the presence of the RB protein fragment.
  • RB protein inhibits progression to S phase at a critical point in G1 phase.
  • the 6-10 hour time window prior to DNA synthesis may be analogous to the time period following the G1 restriction point, a point of irreversible commitment to S phase in mammalian cell lines.
  • the time window defined may not correspond precisely to the time point when RB protein functions under normal physiological conditions.
  • the time it takes for injected full length RB protein or the truncated RB protein to appear in an active configuration and/or location is not clear from these experiments.
  • RB proteins are transported to the nucleus within 15 minutes after cytoplasmic injection, and continued incubation of the cells injected with the truncated RB protein with aphidicolin for several hours prior to release and labeling does not decrease the percentage of cells which incorporate BrdU.
  • RB may be involved in a regulatory decision which the cell makes at a point about 6-10 hours prior to the G1/S transition.
  • the RB protein can be phosphorylated at multiple sites both in vivo and in vi tro, yet it is not yet clear which phosphorylation sites are important for RB function. Subtle increases in phosphorylation of during G1 phase may be responsible for the cells' transition from an RB-responsive to a non-responsive state. Some phosphorylation of the recombinant, full length RB protein has been observed in the G1 phase, although this may have been due to incompletely synchronized cells.
  • the carboxy-terminal 56 kilodaltons of RB is sufficient to inhibit progression through the G1 phase. This indicates that the carboxy terminal half of the recombinantly produced, full length protein is, in fact, a functional domain with respect to its effect on the cell cycle.
  • Two biochemical activities have been ascribed to the carboxy-terminal half of this protein, DNA binding and protein binding. Based on findings to date, the sequence specificity of RB binding to DNA is low; although RB binds with slightly higher affinity to DNA with high G/C content, no particular sequence is strongly preferred. On the other hand, specificity has been observed in binding of RB to proteins.
  • RB protein The transforming proteins of several DNA transforming viruses as well as a subset of cellular proteins bind to the same domain of the recombinantly produced, full length RB protein therefore can compete with one another for RB protein binding. This region is where the majority of naturally occurring inactivating mutations of RB are located. It seems likely, then, that the block to progression of G1 phase by RB protein is dependent on specific protein-protein interactions.
  • the transforming proteins of some DNA tumor viruses may promote cell growth, at least in part, by binding and inactivating underphosphorylated recombinantly produced full length RB protein.
  • the immortalization of cells and the induced escape from quiescence upon expression of these transforming proteins are phenotypes consistent with deregulation of the cell cycle.
  • the carboxy-terminal half of RB protein is biologically active, the question remains as to the function of the amino terminal half of the protein. Sequences within this region may be required for the proper phosphorylation of the protein. In murine cells, polypeptides similar to the truncated RB protein are not hyperphosphorylated. Also, several consensus sites for the cdc2/MPF kinase are present within this region. It follows that the amino terminal half of the RB protein may contain a regulatory domain which can modulate RB function.
  • RB protein segregates other proteins in an inactive form.
  • RB protein segregates with cellular replication proteins to sites of Herpes virus DNA replication upon infection, a situation where normal regulation of the cell cycle is subverted.
  • the underphosphorylated form of recombinant, full length RB is tightly associated with a particular nuclear locale.
  • This purified protein also tends to polymerize, perhaps explaining the difficulty in maintaining its solubility at high concentrations, and it has limited homology to a certain class of intermediate filament proteins.
  • the model predicts that regulation of RB activity could be accomplished by specifying its nuclear location and/or the cellular proteins to which it binds.
  • the biological consequence of complete loss of RB gene and/or protein function is the generation of retinoblastoma tumors and some secondary tumors.
  • Involvement of RB in the cell cycle provides a means to explain this phenomenon.
  • RB may act to halt progression through G1 phase until the cell receives proper signals for commitment to continuation of the cell cycle.
  • loss of RB function may contribute to tumor formation in different tissues by permitting unscheduled cell proliferation.
  • the RB gene product is a nuclear phosphoprotein which undergoes changes in the phosphorylation status in synchrony with the cell cycle.
  • RB protein p110 RB was purified from E. coli BL21 (DE3)/pLyS/pETRBc using a modification of the method described in Nature (1991) 350:160-162. Briefly, 120 g of wet cell paste was resuspended in lysis buffer, (10 mM Tris-Cl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 1 mM benzamidine, 1 ⁇ g/mL leupeptin, pH 8.5) processed through a microfluidizer at 13,000 psi and the soluble fraction was brought to 50% ammonium sulfate.
  • lysis buffer (10 mM Tris-Cl, 10% glycerol, 1 mM EDTA, 1 mM DTT, 1 mM benzamidine, 1 ⁇ g/mL leupeptin, pH 8.5
  • Precipitated protein was recovered by centrifugation at 10,000 ⁇ g and was solubilized in lysis buffer, clarified by centrifugation and dialyzed against lysis buffer. The dialysate was loaded on a phosphocellulose P-11 (Whatman) column in 10 mM Tris-Cl, 10% glycerol, 1 mM EDTA, 1 mM DTT, pH 8.5 and was washed and eluted with a gradient of 0-0.7 M NaCl. Fractions containing p56 RB and p110 RB (as judged by SDS-gel electrophoresis) were pooled and protein was precipitated with 70% ammonium sulfate.
  • the precipitate was recovered by centrifugation and was dialyzed into 20 mM sodium phosphate, 0.2 M NaCl, 10% glycerol, 1 mM EDTA, pH 7.5, and was loaded onto a Sephacryl-200 (Pharmacia) sizing column.
  • p56 RB was separated from p110 RB during this purification step. Pooled fractions of p110 RB were then loaded onto a DEAE column (Whatman) and were washed with 10 mM Tris-Cl, 10% glycerol, 1 mM EDTA, pH 8.8 and eluted with a gradient of 0-0.25 M NaCl.
  • the p110 RB and p56 RB used in these studies were >95% pure as judged by Coomassie blue stained SDS-polyacrylamide gels. Purified p110 RB and p56 RB was dispensed into aliquots and stored frozen at -80°C in 20 mM sodium phosphate, 0.2 M NaCl, 10% glycerol, pH 7.5.
  • 125 I-p110 RB and 125 I-p56 RB were prepared from purified p110 RB or p56 RB protein and 125 I (ICN Biomedical, Irvine, CA) using the chloramine-T method (Nature (1962) 194:495). Cell lines
  • WI-38 normal human lung
  • CCD 976Sk normal human foreskin fibroblast
  • RB pos denotes cell lines in which there is expression of wild-type p110 RB .
  • RB neg denotes cell lines that produce mutant, nonfunctional RB or cell lines in which RB protein expression is not detectable by immunostaining or western blotting with the ⁇ -RB MAb 3C8 (J. Immunol. Meth. (1994) 169:231-240).
  • p110 RB -mediated growth inhibition 2 X 10 4 cells were seeded in 96-well plates, allowed to adhere overnight and were incubated with 0-25 ⁇ g/mL of RB protein in RPMI-1640, 10% fetal bovine serum (heat inactivated). Assays were performed in triplicate. p110 RB -containing medium was changed every 6 hours for 72 hours. One ⁇ Cl of 3 H-thymidine was added to each well 12-18 hours before harvest. Cells were harvested and assayed for 3 H-thymidine uptake as described in Proc.
  • NCl-H596 RB neg , NSCLC
  • Nuclear localization of p110 RB The time-dependent nuclear localization of p110 RB was assessed as follows: NCl-H596 tumor cells were seeded at 10 6 cells/well (triplicate wells for each time point) in a 6-well plate in RPMI-1640 (10% FBS) and allowed to adhere overnight. 0.75 ⁇ Ci of 125 I-p110 RB was added to the medium in 8 hour intervals for a total of 72 hours. Medium was replaced every 24 hours. A 0, 8, 24, 48 and 72 hours, cells were cooled to 4°C and residual 125 I-p110 RB was removed by sequentially rinsing the cells with 1 ml of PBS three times.
  • the homogenate was centrifuged at 500 ⁇ g for 5 minutes at 4°C and the pellet (nuclear fraction) was resuspended in 1 ml ice cold hypotonic buffer.
  • the supernatant, containing the membrane and cytoplasmic fractions was further centrifuged at 10,400 ⁇ g for 45 minutes at 4°C.
  • the resulting supernatant cytoplasmic fraction was removed and the pellet (membrane fraction) was resuspended in 1 ml ice cold hypotonic buffer.
  • One-tenth (0.1 ml) of each fraction was subjected to liquid scintillation counting (Cytoscint fluid).
  • NCl-H596 non-small cell lung carcinoma (NSCLC) cells Three (3) X 10 6 NCl-H596 non-small cell lung carcinoma (NSCLC) cells were seeded in 10 ml RPMI-1640 media (10% fetal bovine serum (heat inactivated)) in 100 mm tissue culture dishes (Costar) and were allowed to adhere overnight. Medium was removed and 10 ml of fresh RPMI-1640 containing 10% fetal bovine serum (heat inactivated) and 0.8 ⁇ Ci 125 I-p110 RB (specific activity 0.008 ⁇ Ci/ ⁇ g) was added to each dish.
  • RPMI-1640 media 10% fetal bovine serum (heat inactivated)
  • tissue culture dishes Costar
  • RPMI-1640 containing 10% fetal bovine serum (heat inactivated) and either 230 pCi 125 I-p110 RB (specific activity 9.23 pCi/ ⁇ g) or 991 pCi 125 I-p56 RB (specific activity 39.6 pCi/ ⁇ g) was added to each of 3 wells. At the indicated time points, cells from 3 wells were washed twice with 1 ml of HBSS, harvested, pooled and the nuclear fraction was isolated as described above. Nucleus-specific radioactivity was quantitated in a ⁇ -scintillation counter. Peritumoral treatment of subcutaneous tumors
  • mice Groups of 3 nude mice (Balb/C NCR nu/nu female, Simonsen, Gilroy, CA) were each subcutaneously inoculated with either 10 7 NCl-H596 (RB neg ) or 10 7 A549 (RB pos ) NSCLC cells.
  • Tumor bearing mice were randomly assigned to different cages. When tumor size was approximately 9 mm 2 , subcutaneous injections of RB protein were made around the tumor. Each tumor was divided into 4 quadrants and 25 ⁇ g of RB protein was subcutaneously injected into alternating quadrants every six hours for ten days. Each animal received a total of 100 ⁇ g of RB protein per day for a total of 1 mg during the 10 day delivery period. Tumor measurements were made in two dimensions using vernier calipers twice a week.
  • mice received a single subcutaneous injection of -20 X 10 6 NCl-H596 (NSCLC, RB neg ) tumor cells. Tumors were permitted to grow for 21 days and external tumor sizes were measured with calipers. Mice were randomized by tumor size into one of four groups each containing 9-10 animals. Mice received single daily dozes (5 doses/week) for a period of 3 weeks of either active p110 RB , 50 ⁇ g/dose; active p110 RB 200 ⁇ g/dose; or inactive p110 RB 200 ⁇ g/dose.
  • Biological activity of p110 RB was determined by its ability to suppress growth of NCl-H596 tumor cells in the 3 H-thymidine uptake assay described in Figure 22.
  • Tumor dimension (l,w,h) and body weights were measured twice per week by two independent investigators and animals were observed daily for signs of morbidity.
  • Tumor volumes were estimated for each animal assuming a spherical geometry of radius equal to one-half the average of the measured tumor dimensions. Comparison of average tumor sizes between each group was performed using the Mann-Whitney U-test as implemented by StatView (Abacus Software, Berkeley, CA).
  • NCl-H596 The ability of RB protein to selectively inhibit growth of the RB neg non-small cell lung carcinoma line NCl-H596 is shown in Figure 19A).
  • the results demonstrate that NCl-H596 cells are growth inhibited by p110 RB in a dose dependent manner.
  • the IC 50 for p110 RB in this assay is approximately 5 ⁇ g/ml [ ⁇ 50 nM].
  • p56 RB a C-terminal derivative of p110 RB (amino acids 379-928) containing the "pocket" region necessary for growth suppression (Mol. Cell Biol. (1993) 13:3384-3391; Genes and Develop .
  • p110 RB is a nuclear phosphoprotein and its interaction with key transcription elements such as E2F underscores its normal regulatory role in the cell cycle (for a review see TIBS (1992) 17:312-315 and Science (1992) 258:424-429). Thus, if p110 RB is entering cells and affecting their growth by its normal pathway, then it must also localize to the nucleus of target cells. Accordingly, 125 I-p110 RB was added to cultures of NCl-H596 tumor cells. Cells were harvested and fractionated into membrane, cytoplasm and nuclear enriched components as described in herein. Figure 21A shows the time-dependent internalization of 125 I-p110 RB into various subcellular fractions.
  • a 110 kD radiolabelled band appeared in a time-dependent manner, indicating the presence of intact 125 I -p110 RB in the nucleus.
  • the time-dependent nuclear accumulation of acid precipitable 125 I cpm and immunoprecipitable 125 I-p110 RB was also examined in the RB pos lung tumor cell lines MRC-9 and
  • Tumor cells disassociated from biopsy tissue from the subject was labeled with 35 s methionine or 32 P-phosphoric acid an immunoprecipitated with anti-ppRB 110 IgG according to the procedure described above.
  • protein lysates extracted from bioptic tissue can be directly diagnosed using western blotting analysis probed with either radioactive labeled or non-radioactive labeled anti- RB specific antibody.
  • the presence or absence of immunoprecipitated proteins serves as a diagnostic tool in determination of retinoblastoma or other diseases controlled by the retinoblastoma gene.
  • Tumor suppressor genes achieve their oncogenic effect following mutation inactivation of both normal alleles . Recognition of the importance of tumor suppressor genes for human cancer first emerged with studies of retinoblastoma. In hereditary retinoblastoma, one inactivated copy of the retinoblastoma (RB) gene is inherited, and the second gene copy is inactivated during growth and differentiation of the offspring. Characterization of the RB gene has shown that it encodesa nuclear phosphoprotein (p110 RB ). p110 RB appears to regulate cell cycle progression, at least in part, through its association with several target proteins, including the transcription factor E2F.
  • E2F nuclear phosphoprotein
  • RB gene structure and expression have now been associated with many human malignancies, including cancers of the lung, prostate, mammary glands, urogenital system and hematologic malignancies.
  • Evidence supporting an important clinical role for altered RB gene expression in tumorigenesis is two-fold. First, introduction of a functioning copy of a normal RB gene into tumor cells characterized by RB gene mutations will suppress the malignant phenotype or the target cell. Second, decreased expression of p110 RB has recently been shown to indicate a worsened prognosis in acute myelogenous leukemia and early stage bladder cancer patients (reviewed in Annual Rev. Biochem. (1993) 62:623-651.
  • p110 RB protein replacement therapy
  • p110 RB or a functional derivative of it, is introduced into tumor cells characterized by loss of expression of the wild type tumor suppressor protein.
  • p110 RB A truncated version of p110 RB , called p56 RB also has limited activity in an in vivo model. These results provide further support for the application of tumor suppressor protein therapy for human lung cancers characterized by defective RB expression. p110 RB inhibits tumor formation in vivo
  • A549 NSCLC tumors were established in nude mice and used an identical treatment protocol to that described in Figure 22A.
  • the data demonstrate that p110 RB and p56 RB protein therapy has no effect on the growth of the A549 tumors, indicating that tumor growth is reduced only for RB neg tumors.
  • Average tumor size on day 49 was significantly smaller for mice treated with 200 ⁇ g/dose of active p110 RB than for animals treated with the same dose of inactive p110 RB (p ⁇ 0.01) or the lower dose (50 ⁇ g/dose) of p110 RB (p ⁇ 0.05).
  • the highest rate of tumor growth was observed in mice that received inactive preparations of RB protein (as judged by lack of activity in the in vitro 3 H-thymidine incorporation assay) or a buffer control. Mice treated with active p110 RB showed no signs of toxicity or morbidity during the study.
  • NCl-H596 does not express detectable levels of p110 RB as judged by immunoblotting and ELISA whereas A549 produces normal amounts of p110 RB (see J. Immunol. Meth.
  • the breast carcinoma line MDA-MB-468 harbors a partial RB gene deletion as well as a point mutation in it p53 gene (see Mol. Cell Biol. (1990) 9:1628-1634 and Oncogene (1993) 8:279-288).
  • Introduction of a single-copy of the RB gene into MDA-MB-468 using retroviral vectors results in decreased tumorigenicity in nude mice and reduced ability to grow in soft agar, but the growth rate in culture was not affected.
  • exogenous p110 RB suppressed the growth of MDA-MB- 468.
  • the extent of growth suppression of MDA-MB-468 in vi tro may be a function of the higher intracellular level of p110 RB that was achieved in these experiments.
  • NCI H-69 produces an aberrantly migrating species of RB when analyzed by SDS-polyacrylamide gel electrophoresis (Proc. Natl. Acad. Sci. USA (1990) 87:2775-2779).
  • a genomic point mutation in NCI H-69 causes abnormal precursor mRNA splicing, resulting in mRNA in which exon 21 is fused in frame to exon 23, eliminating 38 amino acids of the exon 22 coding sequence.
  • the RB protein produced in these cells migrates with an apparent molecular weight of ⁇ 4 kD less than p110 RB and is defective for E1A and SV-40 T-antigen binding.
  • the RB-mediated growth suppression in these experiments is specific for RB neg cell lines.
  • RB pos NSCLC line are not growth inhibited by p110 RB .
  • overexpression of p110 RB does not affect the growth rate of NIH-3T3 fibroblast cells (Exp. Cell Res. (1993) 207:99-106). These data are in direct contrast to another report that overexpression of wild-type p110 RB leads to growth arrest in normal cells (Oncogene
  • lung epithelial lines such as WI-38 were growth arrested by transfection with a plasmid overexpressing RB cDNA and subsequent selection of stable transfectants.
  • the inability to see growth suppression of normal cell lines is most likely due to limitations on the intracellular concentration of p110 RB that can be achieved by our method.
  • the uptake of radiolabeled p110 RB revealed that only a small fraction (0.5-1% of cpm added) of the 125 I-p110 RB was taken up by the cells.
  • the work presented herein presents a practical way of restoring normal RB function to RB neg tumors, without the need for genetic modification. This is significant, because loss of RB function occurs in a wide variety of tumor types.
  • the growth suppression of a variety of RB neg tumor cells in vitro demonstrates that exogenous RB protein can functionally substitute for endogenous wild-type RB. While normal cells can take up p110 RB , they are not growth inhibited, demonstrating that p110 RB has selective anti-proliferative activity against RB neg tumor cells. Since reduced tumorigenicity in vivo is a more sensitive indicator of the restoration of tumor suppression function, the experiments included nude mouse xenograft models. Recent observations indicate that reintroduction of the RB gene into RB neg SCLC cells carrying additional multiple genetic alterations suppresses their tumorigenicity in nude mice (Oncogene (1993) 8:2175-2181).

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Abstract

Procédé de prévention ou d'inhibition de la prolifération pathologique d'une cellule, ladite prolifération pathologique de la cellule étant le résultat de l'absence d'une protéine ou d'un polypeptide fonctionnels de rétinoblastome dans la cellule. Ledit procédé consiste à mettre en contact la cellule avec une quantité efficace de protéine ou de polypeptide de rétinoblastome. Ledit procédé est également utile pour prévenir ou traiter le rétinoblastome ou un cancer secondaire du rétinoblastome en administrant à un patient une protéine ou un polypeptide fonctionnels de rétinoblastome.
PCT/US1994/010357 1993-09-13 1994-09-13 Utilisation therapeutique d'un produit genique contre la predisposition au retinoblastome WO1995007708A2 (fr)

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US7223842B1 (en) 1986-08-11 2007-05-29 Massachusetts Eye And Ear Infirmary Detection of proteins whose absence is associated with a neoplasm
US7384735B1 (en) 1986-08-11 2008-06-10 Massachusetts Eye And Ear Infirmary Retinoblastoma nucleic acids
WO1997047745A1 (fr) * 1996-06-13 1997-12-18 Consejo Superior De Investigaciones Cientificas Proteines vegetales associees au retinoblastome
US6384299B1 (en) 1996-06-13 2002-05-07 Consejo Superior De Investigaciones Cientificas Plant retinoblastoma-associated gene
WO1998021228A1 (fr) * 1996-11-15 1998-05-22 Canji, Inc. Expression specifique tissulaire de la proteine du retinoblastome
US6379927B1 (en) 1996-11-15 2002-04-30 Canji, Inc. Retinoblastoma fusion proteins
US6902731B1 (en) 1996-11-15 2005-06-07 Canji, Inc. Methods of treating hyperproliferative disorders using retinoblastoma fusion proteins
CN100338219C (zh) * 1996-11-15 2007-09-19 坎吉有限公司 成视网膜细胞瘤蛋白的组织特异性表达
WO1998037091A2 (fr) * 1997-02-20 1998-08-27 Board Of Regents, The University Of Texas System Proteines modifiees de suppression des retinoblastomes
WO1998037091A3 (fr) * 1997-02-20 1998-11-05 Univ Texas Proteines modifiees de suppression des retinoblastomes
WO2004094996A2 (fr) * 2001-07-12 2004-11-04 Duke University Systeme et technique de determination d'expression de proteine differentielle, systeme de decouverte de biomarqueur de diagnostic et technique d'utilisation de ce systeme et biomarqueur de proteine et utilisation therapeutique et diagnostique de ceux-ci
WO2004094996A3 (fr) * 2001-07-12 2005-03-10 Univ Duke Systeme et technique de determination d'expression de proteine differentielle, systeme de decouverte de biomarqueur de diagnostic et technique d'utilisation de ce systeme et biomarqueur de proteine et utilisation therapeutique et diagnostique de ceux-ci

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ZA947065B (en) 1995-05-03
IL110954A0 (en) 1994-11-28
AU7834994A (en) 1995-04-03
WO1995007708A3 (fr) 1995-06-08

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