WO1992022640A1 - Rb TRANSFERRED CELLS AND METHOD - Google Patents

Rb TRANSFERRED CELLS AND METHOD Download PDF

Info

Publication number
WO1992022640A1
WO1992022640A1 PCT/US1992/004934 US9204934W WO9222640A1 WO 1992022640 A1 WO1992022640 A1 WO 1992022640A1 US 9204934 W US9204934 W US 9204934W WO 9222640 A1 WO9222640 A1 WO 9222640A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell line
human
cdna
cells
gene
Prior art date
Application number
PCT/US1992/004934
Other languages
French (fr)
Inventor
Rei Takahashi
William F. Benedict
Original Assignee
Research Development Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Research Development Foundation filed Critical Research Development Foundation
Publication of WO1992022640A1 publication Critical patent/WO1992022640A1/en

Links

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • Rb retinoblastoma gene
  • the lack of the retinoblastoma gene (Rb) has been linked not only to retinoblastoma, the most common malignant ocular tumor in children, but also to osteosarcoma, fibrosarcomas, other soft tissue sarcomas, as well as some of the most common human tumors including lung, prostate, bladder and breast tumors.
  • the Rb gene was the first human tumor suppressor gene isolated.
  • the Rb gene is on chromosome 14, region 13q14.
  • the cDNA of the Rb gene has been seguenced. Gene probes identifying the presence of certain Rb gene DNA sequences have been developed.
  • the loss of Rb gene function can occur through mutation or total loss of the gene. In some cases the mutation of the gene leads to the production of a truncated Rb protein rather than total loss of the protein.
  • the normal Rb protein includes both under phosphorylated and phosphorylated forms of the Rb protein recognized as a 110-kDa band and a smeary region between 110 and 116 kDa, respectively.
  • Cell lines have been isolated that lack Rb function or produce an abnormal truncated Rb protein.
  • Highly specific antibodies have been developed to detect the presence or absence of the functional Rb gene. "Purified, Highly Specific Antibodies against Gene Products Including Retinoblastoma and Method" PCT International Application No. PCT US90/02017, International Publication No. WO 90/12807, November 1, 1990.
  • a method for producing human cancer lines with a transferred Rb gene expressing Rb protein from a cell line lacking Rb expression The human cancer cell lines with the transferred Rb gene are stable and express the normal Rb protein indefinitely without reversion to lack of expressed Rb gene product like the parent line prior to transformation.
  • the pair of cell lines, one with and one without the Rb gene, can serve as a model system for research.
  • the Rb+ celf line is produced by selecting an
  • Rb negative human cancer cell line as a recipient.
  • the cell line is transfected with the Rb gene-containing plasmid to produce a stable transformed cell line expressing Rb protein.
  • the stable cell lines have been produced with human cancer cell lines including human bladder carcinoma, fibrosarcoma, and prostate cancer.
  • the expression vector used with successful transfection includes the human ⁇ -actin promoter with the Rb cDNA.
  • the Rb positive cells are selected after transfection by methods known in the art. From the mass culture, individual cells can be isolated and cloned to produce cell lines.
  • the Rb+ and Rb- cells comprise a system for studying the differential sensitivity to anticancer agents. Also, the Rb gene has been implicated in cell growth and regulation. The cell lines can be used for studying the effects of cell growth factors.
  • Figure 1 is a schematic drawing of the Rb expression plasmid, pBARB.
  • Figure 2 is part of the Rb-cDNA showing the 5' integration site by the closed arrow.
  • Rb expression plasmid indicated as pBARB, shown in schematic form in Figure 1 was constructed.
  • the complete Rb gene coding sequence with the 5' and 3' untranslated regions was isolated from a cDNA library derived from mRNAs of human embryo fibroblasts. Miki, T., et al., 83 Gene 137-146 (1989).
  • a 3.8 kb Rb-cDNA probe was used to screen the library.
  • a 4.74 kb cDNA with the 5' and 3' untranslated regions and complete coding sequence was isolated.
  • In vitro expression of full length Rb protein 110 kDa was achieved. From the 4.74 kb cDNA, a 3.8 kb Rb-cDNA insert was prepared.
  • FIG. 1 is a schematic of the Rb-cDNA.
  • Figure 2 also contains the DNA sequence for a portion of the 5' untranslated region and a short length of the coding sequence after the first ATG is shown.
  • the integration site in the 5' translation region is noted with the closed arrow.
  • the 3' termination point at +3684 bp is indicated with an open arrow.
  • the Rb expression vector, pBARB was constructed by ligating the Rb-cDNA fragment shown in
  • the pBARB plasmid contains 3 kb of human ⁇ -actin 5' flanking sequence, 78 bp of 5' untranslated region and intervening sequence I as described in Gunning, et al. (supra).
  • the Rb gene sequence was inserted in the pH ⁇ APr-1-neo plasmid shown in Figure 2 of Gunning, et al., at the Bam Hl site. Integration points in the pH ⁇ APr-1-neo plasmid are indicated by the closed arrow
  • the Rb cDNA is followed by an SV40 Poly A sequence.
  • the expression vector included a dominant selection marker gene neo under the control of the SV40 early promoter which is indicated in Figure 1 as SV-neo.
  • the neo gene is neortycin-resistant.
  • an ampicillin-resistent gene indicated as AmpR in Figure 1 was included in the expression plasmid.
  • These marker genes were used to select cells that were successfully transfected with the plasmid containing pBARB. However, other marker genes or selection methods may be used.
  • the mammalian expression plasmid pBARB in E. coli is deposited as DHpBARB with the American Type Culture Collection ATCC number 68569.
  • Rb gene containing plasmid, pBARB was transfected by the Ca ++ PO 4 using the Stratagene Mammalian Transfection Kit by the method described in Chen, C. and Okayama, H. , "High efficiency transformations of mammalian cells by plasmid DNA," 7 J. Mol. Cell. Biol. 2745-2752 (1987). See also, Stragene Protocol, Mammalian Transfection Kit, Instruction Manual (Catalog #200285) February 5, 1989, 11099 North Torrey Pines Road, La Jolla, California 92037.
  • Exponentially growing cells were incubated with 1.37 pmole (10 ⁇ g or 13.6 ⁇ g) of pBARB for 12 hours in 3% CO 2 atmosphere, treated with 20% dimethylsulfoxide (DMSO) for 5 minutes, washed twice with DMEM media, and cultured in 5% CO 2 atmosphere for 48 hours. The cells were subcultured into five 100-mm dishes and cultured for 24 hours. The medium was then changed to G418-containing DMEM in the active concentration of 200 ⁇ g/ml for selection of the neomycin-resistant transfected cells.
  • DMSO dimethylsulfoxide
  • the culture was handled in two ways.
  • a mass culture of the G418-resistant cells was grown after transfection with pBARB. The cells were then diluted and seeded at a concentration to yield an average of less than a single cell/well in microliter plates. Clones are selected after analysis for the Rb protein expression. Another method was also employed. After two weeks, each G418- resistant colony was transferred separately to wells of 24-well plates using a cloning ring. The isolated colony was cloned from an independent G418-mass culture. Other methods of culture isolation know to those skilled in the art may be used.
  • Rb- clones for comparison as a control were made by transfecting the cells with pH ⁇ APr-1-neo (plasmid without the Rb cDNA).
  • the Rb+ cells produced normal Rb proteins which were identical in size and phosphorylation pattern to the Rb+ control cells normal fetal lung fibroblast cells WI- 38 from the ATCC.
  • the Rb protein source was further confirmed from pBARB plasmid by RNA blotting analysis.
  • the Rb-cDNA used for the pBARB construction predicted a transcript size of 3.8 kb as compared to a normal 4.7 kb endogenous Rb transcript.
  • the 3.8 kb transcript was detected in the Rb+ transfectants with the less than normal sized Rb transcript.
  • the presence of the RNA was isolated by guanidium thiocyanate method as described in Deeley, R. G., et al., 252 J. Biol. Chem. 8810-8819.
  • RNA was denatured in glyoxal and DMSO at 50°C for 1 hour, electrophoresed in 1% agarose gel in 10mM phosphate buffer (pH 6.7), and transferred to nylon membranes (Hybond-N, Amersham) as described in Thomas, P.S., 77 Proc. Nat'l. Acad. Sci. 5201-5205 (1980).
  • Membranes were hybridized with a 32 P-labeled 3.8 kb Rb probe. After washing, the membranes were exposed to Kodak XAR-5 film at -80°C. The presence of the RNA Rb transcript was confirmed over time for each of the Rb+ transfectants.
  • the presence of nuclear Rb+ protein staining was also confirmed by immunohistochemical analysis.
  • Cells were grown overnight on sterile coverslips in culture dishes. Cells on the coverslips were then fixed in 45%..acetone, 10% formaldehyde and 0.1 M phosphate buffer for 5 minutes. After washing six times with PBS, the cells were pre-incubated in 5% BSA and 4% normal goat serum in phosphate buffer for 4 hours at room temperature.
  • the MAb-1 anti-Rb monoclonal antibody (Triton Biosciences, Inc.) was diluted 1:25 in 3% BSA, 1.5% normal horse serum, 0.02% Triton X-100 and phosphate buffer and was incubated with the cells overnight.
  • the Rb+ colonies transfectants did not form colonies in soft agar. Cell proliferation under standard culture conditions was not affected. Media with greater than 3% serum should be used to culture Rb+ cells.
  • a stable transfected cell line was prepared by the method described above using the human bladder carcinoma cell line, HTB9.
  • the cell line lacks detectable Rb expression as previously reported by
  • HTB9 cells were transfected with the pBARB plasmid as described above. Clones were isolated independently from a mass culture and also by seeding from growth of a mass culture as previously described. A clone isolated from the mass culture, H/Rb-M-CL5, is deposited as H/Rb-M with the ATCC
  • the Rb+ transfectants were used for investigation of tumorigenicity in immunodeficient nude mice.
  • Rb- parental HTB9 cells and Rb- control plasmid -transfected HTB9 were used as controls.
  • a variable number of Rb+ cells (1 ⁇ 10 6 , 2 ⁇ 10 6 , or 1 ⁇ 10 7 ) were injected subcutaneously in 0.2 ml PBS into the right flank of nude mice.
  • the Rb + clones were confirmed by Western blotting, RNA blotting, and immunohistochemical staining.
  • the Rb- parental HTB9 and a parental line transfected with a plasmid without the Rb cDNA, HTB9-PL, were used as controls.
  • the parental cell line, HTB9 and the control plasmid-transfected HTB9-PL lack Rb protein, Rb transcript and exhibited no nuclear staining.
  • Control Rb- HTB9 or HTB9-PL cells were injected at an identical concentration into the left flank of the same mice.
  • the Rb-parental HTB9 cells and Rb- HTB9-PL cells formed large progressively growing tumors following inoculation. Tumors were measured every one to two weeks.
  • tumors averaged 207.2 mm 3 and 218.3 mm 3 , respectively, when 1 ⁇ 10 7 cells were injected.
  • the Rb+ cells induced much smaller tumors and nonprogressive tumors.
  • two Rb+ clones produced tumors with an average size of 6.1 mm 3 and 5.1 mm 3 .
  • the Rb+ cell line was re-established from the small tumors in the nude mice.
  • the small tumors were minced under sterile conditions and transferred to dishes in the presence of G418-containing complete medium.
  • the re-established cell lines expressed normal Rb protein and contained the 3.8 kb RNA transcript after passage in nude mice and did not revert to Rb-.
  • a stable Rb+ cell line was also established by transfection of a fibrosarcoma cell line Hs913T from the ATCC with the pBARB plasmid. Rb+ stability has been confirmed several months after transfection.
  • a stable Rb+ cell line was also established by transfection of a prostate cancer cell line DU145 with the pBARB plasmid. Rb+ stability has been confirmed several months after transfection.

Abstract

A method has been developed to transfer the Rb gene to human cancer cells that do not express Rb protein thus producing a pair of cell lines with and without Rb protein expression. A plasmid containing the Rb cDNA effects the transfer. The transformed Rb positive cell exhibits normal and stable Rb protein production.

Description

Rb Transferred Cells and Method
Origin of the Invention
The invention described herein was made using federal funds and may be manufactured or used by or for the Government of the United States of America for governmental purposes without payment of any royalties thereon or therefor.
Background of the Invention
Lack of the retinoblastoma gene (Rb) normally present in humans has been linked to the development of tumors. The lack of the retinoblastoma gene (Rb) has been linked not only to retinoblastoma, the most common malignant ocular tumor in children, but also to osteosarcoma, fibrosarcomas, other soft tissue sarcomas, as well as some of the most common human tumors including lung, prostate, bladder and breast tumors. The Rb gene was the first human tumor suppressor gene isolated.
The Rb gene is on chromosome 14, region 13q14. The cDNA of the Rb gene has been seguenced. Gene probes identifying the presence of certain Rb gene DNA sequences have been developed.
The loss of Rb gene function can occur through mutation or total loss of the gene. In some cases the mutation of the gene leads to the production of a truncated Rb protein rather than total loss of the protein. The normal Rb protein includes both under phosphorylated and phosphorylated forms of the Rb protein recognized as a 110-kDa band and a smeary region between 110 and 116 kDa, respectively. Cell lines have been isolated that lack Rb function or produce an abnormal truncated Rb protein. Highly specific antibodies have been developed to detect the presence or absence of the functional Rb gene. "Purified, Highly Specific Antibodies Against Gene Products Including Retinoblastoma and Method" PCT International Application No. PCT US90/02017, International Publication No. WO 90/12807, November 1, 1990.
Attempts have been made to take known Rb negative cell lines and introduce the Rb gene with a retroviral vector. In one study, the loss of Rb gene expression reoccurred after temporary Rb gene expression in the cell culture of two known Rb negative cell lines. Huang, et al., "Suppression of the Neoplastic Phenotype by Replacement of the Rb Gene in Human Cancer Cells," 242 Science (December 16, 1988). This study was reproduced with similar results and failure to produce a stable transformed Rb positive cell line from an Rb negative parent culture. Sumegi, et al., "Expression of the Rb Gene under the Control of the MuLV-LTR Suppresses Tumorigenicity of WERI-Rb27 Retinoblastoma Cells in Immunodefective Mice," 1 Cell Growth and Differentiation 247-250 (1990). In another study using prostate carcinoma cell line, DU145, which contains a partial deletion in the Rb gene, incomplete suppression of tumorigenicity in nude mice, as well as lack of exogenous Rb proteins in tumor tissue were reported. Bookstein, et al., "Suppression of Tumorigenicity of Human Prostate Carcinoma Cells by Replacing a Mutated Rb Gene," 247 Science (February 9, 1990).
Summary of the Invention
A method has been developed for producing human cancer lines with a transferred Rb gene expressing Rb protein from a cell line lacking Rb expression. The human cancer cell lines with the transferred Rb gene are stable and express the normal Rb protein indefinitely without reversion to lack of expressed Rb gene product like the parent line prior to transformation. The pair of cell lines, one with and one without the Rb gene, can serve as a model system for research.
The Rb+ celf line is produced by selecting an
Rb negative human cancer cell line as a recipient. The cell line is transfected with the Rb gene-containing plasmid to produce a stable transformed cell line expressing Rb protein. The stable cell lines have been produced with human cancer cell lines including human bladder carcinoma, fibrosarcoma, and prostate cancer.
The expression vector used with successful transfection includes the human β-actin promoter with the Rb cDNA. The Rb positive cells are selected after transfection by methods known in the art. From the mass culture, individual cells can be isolated and cloned to produce cell lines.
Two cell lines virtually identical except for the presence or absence of a normal Rb expressing gene have been developed. The Rb+ and Rb- cells comprise a system for studying the differential sensitivity to anticancer agents. Also, the Rb gene has been implicated in cell growth and regulation. The cell lines can be used for studying the effects of cell growth factors. Brief Description of the Drawings
Figure 1 is a schematic drawing of the Rb expression plasmid, pBARB.
Figure 2 is part of the Rb-cDNA showing the 5' integration site by the closed arrow.
Detailed Description of the Invention
An Rb expression plasmid, indicated as pBARB, shown in schematic form in Figure 1 was constructed. The complete Rb gene coding sequence with the 5' and 3' untranslated regions was isolated from a cDNA library derived from mRNAs of human embryo fibroblasts. Miki, T., et al., 83 Gene 137-146 (1989). A 3.8 kb Rb-cDNA probe was used to screen the library. A 4.74 kb cDNA with the 5' and 3' untranslated regions and complete coding sequence was isolated. In vitro expression of full length Rb protein (110 kDa) was achieved. From the 4.74 kb cDNA, a 3.8 kb Rb-cDNA insert was prepared.
The 5' end of the Rb-cDNA insert was at the position -57 base pairs from the first start codon (ATG) of translation. The 3' end of the insert was at the +3684 base pair from the first ATG. Figure 2 is a schematic of the Rb-cDNA. Figure 2 also contains the DNA sequence for a portion of the 5' untranslated region and a short length of the coding sequence after the first ATG is shown. The integration site in the 5' translation region is noted with the closed arrow. The 3' termination point at +3684 bp is indicated with an open arrow.
The Rb expression vector, pBARB, was constructed by ligating the Rb-cDNA fragment shown in
Figure 2 into a mammalian expression vector, pHβAPr-1-neo. The plasmid pHβAPr-1-neo is described in detail in
Gunning, et al., "A human β-actin expression vector system directs high-level accumulation of antisense transcripts," 84 Proc. Nat'l. Acad. Sci. U.S.A. 4831-4835
(July 1987), which is incorporated herein by reference.
The pBARB plasmid contains 3 kb of human β-actin 5' flanking sequence, 78 bp of 5' untranslated region and intervening sequence I as described in Gunning, et al. (supra). The Rb gene sequence was inserted in the pHβAPr-1-neo plasmid shown in Figure 2 of Gunning, et al., at the Bam Hl site. Integration points in the pHβAPr-1-neo plasmid are indicated by the closed arrow
Eco Rl and the open arrow Bam Hl. The Rb cDNA is followed by an SV40 Poly A sequence.
The expression vector included a dominant selection marker gene neo under the control of the SV40 early promoter which is indicated in Figure 1 as SV-neo. The neo gene is neortycin-resistant. In addition, an ampicillin-resistent gene indicated as AmpR in Figure 1 was included in the expression plasmid. These marker genes were used to select cells that were successfully transfected with the plasmid containing pBARB. However, other marker genes or selection methods may be used.
The mammalian expression plasmid pBARB in E. coli is deposited as DHpBARB with the American Type Culture Collection ATCC number 68569.
Cell lines selected for transfection did not have detectable Rb expression. The Rb gene containing plasmid, pBARB, was transfected by the Ca++PO4 using the Stratagene Mammalian Transfection Kit by the method described in Chen, C. and Okayama, H. , "High efficiency transformations of mammalian cells by plasmid DNA," 7 J. Mol. Cell. Biol. 2745-2752 (1987). See also, Stragene Protocol, Mammalian Transfection Kit, Instruction Manual (Catalog #200285) February 5, 1989, 11099 North Torrey Pines Road, La Jolla, California 92037. Exponentially growing cells were incubated with 1.37 pmole (10 μg or 13.6 μg) of pBARB for 12 hours in 3% CO2 atmosphere, treated with 20% dimethylsulfoxide (DMSO) for 5 minutes, washed twice with DMEM media, and cultured in 5% CO2 atmosphere for 48 hours. The cells were subcultured into five 100-mm dishes and cultured for 24 hours. The medium was then changed to G418-containing DMEM in the active concentration of 200 μg/ml for selection of the neomycin-resistant transfected cells.
After selection of the transfected cells, the culture was handled in two ways. A mass culture of the G418-resistant cells was grown after transfection with pBARB. The cells were then diluted and seeded at a concentration to yield an average of less than a single cell/well in microliter plates. Clones are selected after analysis for the Rb protein expression. Another method was also employed. After two weeks, each G418- resistant colony was transferred separately to wells of 24-well plates using a cloning ring. The isolated colony was cloned from an independent G418-mass culture. Other methods of culture isolation know to those skilled in the art may be used.
Rb- clones for comparison as a control were made by transfecting the cells with pHβAPr-1-neo (plasmid without the Rb cDNA).
Some of the clones selected as Rb+ by the drug marker have been confirmed as Rb positive by several methods over a year's time to determine stability of the transfected Rb gene in the culture. Western blotting was used to confirm the presence of the Rb protein. Cellular protein was extracted by lysing 5 × 106 cells in 1 ml of 100 mM Na CI, 0.2% sodium deoxycholate, 0.1% sodium dodecylsulfate, and 50 mM Tris.HCl, pH 8.0. Cellular lysates were clarified by centrifugation and normalized by total cellular protein as determined by the..Bradford protein assay (BioRad). Fifty μg of cellular protein was electrophoresed in an 8% SDS-polyacrylamide gel and electroblotted to Immobilon PVDF membranes . After blocking with 4% bovine serum albumin (BSA) and 1% normal goat sera in Tris-buffered saline, the membranes were incubated overnight with a highly specific Rb antibody at a final concentration of 0.4 μg/ml. The blot was then probed by the Protiblot Western Blot AP System (Promega).
The Rb+ cells produced normal Rb proteins which were identical in size and phosphorylation pattern to the Rb+ control cells normal fetal lung fibroblast cells WI- 38 from the ATCC.
The Rb protein source was further confirmed from pBARB plasmid by RNA blotting analysis. The Rb-cDNA used for the pBARB construction predicted a transcript size of 3.8 kb as compared to a normal 4.7 kb endogenous Rb transcript. The 3.8 kb transcript was detected in the Rb+ transfectants with the less than normal sized Rb transcript. The presence of the RNA was isolated by guanidium thiocyanate method as described in Deeley, R. G., et al., 252 J. Biol. Chem. 8810-8819. The RNA was denatured in glyoxal and DMSO at 50°C for 1 hour, electrophoresed in 1% agarose gel in 10mM phosphate buffer (pH 6.7), and transferred to nylon membranes (Hybond-N, Amersham) as described in Thomas, P.S., 77 Proc. Nat'l. Acad. Sci. 5201-5205 (1980). Membranes were hybridized with a 32P-labeled 3.8 kb Rb probe. After washing, the membranes were exposed to Kodak XAR-5 film at -80°C. The presence of the RNA Rb transcript was confirmed over time for each of the Rb+ transfectants. The presence of nuclear Rb+ protein staining was also confirmed by immunohistochemical analysis. Cells were grown overnight on sterile coverslips in culture dishes. Cells on the coverslips were then fixed in 45%..acetone, 10% formaldehyde and 0.1 M phosphate buffer for 5 minutes. After washing six times with PBS, the cells were pre-incubated in 5% BSA and 4% normal goat serum in phosphate buffer for 4 hours at room temperature. The MAb-1 anti-Rb monoclonal antibody (Triton Biosciences, Inc.) was diluted 1:25 in 3% BSA, 1.5% normal horse serum, 0.02% Triton X-100 and phosphate buffer and was incubated with the cells overnight. After washing, the coverslips were processed for immunostaining with avidin biotinylated peroxidase complex (ABC) method according to the technical manual (Vector Labs). Each Rb+ cell line confirmed by Western blotting showed positive nuclear Rb protein staining.
The Rb+ colonies transfectants did not form colonies in soft agar. Cell proliferation under standard culture conditions was not affected. Media with greater than 3% serum should be used to culture Rb+ cells.
Example l
A stable transfected cell line was prepared by the method described above using the human bladder carcinoma cell line, HTB9. The cell line lacks detectable Rb expression as previously reported by
Horowitz, et al., 87 Proc. Nat'l. Acad. Sci. 2775-2779
(1990), which absence was further confirmed using the protein and RNA assays described above. The HTB9 cells were transfected with the pBARB plasmid as described above. Clones were isolated independently from a mass culture and also by seeding from growth of a mass culture as previously described. A clone isolated from the mass culture, H/Rb-M-CL5, is deposited as H/Rb-M with the ATCC
No. CRL 10707. A second clone, H/RB-CL2, from an independent G418-resistant mass culture is deposited with the ATCC No. CRL 10708.
The Rb+ transfectants were used for investigation of tumorigenicity in immunodeficient nude mice. Rb- parental HTB9 cells and Rb- control plasmid -transfected HTB9 were used as controls. After trypsinization, a variable number of Rb+ cells (1 × 106, 2 × 106, or 1 × 107) were injected subcutaneously in 0.2 ml PBS into the right flank of nude mice.
The Rb+ clones were confirmed by Western blotting, RNA blotting, and immunohistochemical staining. The Rb- parental HTB9 and a parental line transfected with a plasmid without the Rb cDNA, HTB9-PL, were used as controls. The parental cell line, HTB9 and the control plasmid-transfected HTB9-PL lack Rb protein, Rb transcript and exhibited no nuclear staining. Control Rb- HTB9 or HTB9-PL cells were injected at an identical concentration into the left flank of the same mice. The Rb-parental HTB9 cells and Rb- HTB9-PL cells formed large progressively growing tumors following inoculation. Tumors were measured every one to two weeks. At one month, tumors averaged 207.2 mm3 and 218.3 mm3, respectively, when 1 × 107 cells were injected. The Rb+ cells induced much smaller tumors and nonprogressive tumors. For instance, two Rb+ clones produced tumors with an average size of 6.1 mm3 and 5.1 mm3.
The Rb+ cell line was re-established from the small tumors in the nude mice. The small tumors were minced under sterile conditions and transferred to dishes in the presence of G418-containing complete medium. The re-established cell lines expressed normal Rb protein and contained the 3.8 kb RNA transcript after passage in nude mice and did not revert to Rb-.
Example 2
A stable Rb+ cell line was also established by transfection of a fibrosarcoma cell line Hs913T from the ATCC with the pBARB plasmid. Rb+ stability has been confirmed several months after transfection.
Example 3
A stable Rb+ cell line was also established by transfection of a prostate cancer cell line DU145 with the pBARB plasmid. Rb+ stability has been confirmed several months after transfection.
The examples are illustrative of the invention. Other embodiments will be understood by those skilled in the art and are considered part of the invention. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Takahashi, Rei
Benedict, W. F.
(ii) TITLE OF INVENTION: Rb Transferred Cells and Method
(iii) NUMBER OF SEQUENCES: 1
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: James F. Weiler, Attorney-at-Law
(B) STREET: One Riverway, Suite 1560
(C) CITY: Houston
(D) STATE: Texas
(E) COUNTRY: USA
(F) ZIP: 77056
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 Mb
storage
(B) COMPUTER: IBM compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WordPerfect 5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/712,470
(B) FILING DATE: 10-June-1991
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Weiler, James F.
(B) REGISTRATION NUMBER: 16,040
(C) REFERENCE/DOCKET NUMBER: D-5446 PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (713) 626-8646
(B) TELEFAX: (713) 622-1981 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 161
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
(vi) ORIGINAL SOURCE:
(A) ORGANISM: human gene
(G) CELL TYPE: human embryo fibroblasts
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: cDNA
(vii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT: 13 - human retinoblastoma gene
(B) MAP POSITION: Region 13ql4
(C) UNITS: Portion of 5' untranslated region and
5' integration site
(ix) FEATURE:
(A) NAME/KEY: 5' untranslated region Rb-cDNA
(B) LOCATION: 5' untranslated regions and 25bp
after ATG site
(C) IDENTIFICATION METHOD: cDNA probe
(D) OTHER INFORMATION: Particle cDNA sequence from human retinoblastoma gene
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GGGCGCGCCG GTTTTTCTCA GGGGACGTTG AAATTATTTT TGTAACGGGA GTCGGGAGAG 60 GACGGGGCGT GCCCCGCGTG CGCGCGCGTC GTCCTCCCCG GCGCTCCTCC ACAGCTCGCT 120 GGCTCCCGCC GCGGAAAGGC GTC ATG CCG CCG AAA ACC CCC 161
Met Pro Pro Lys Thr Pro
5
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001

Claims

Claims
1. A human cancer cell line comprising,
a human cancer cell line with the human Rb gene;
said human cancer cell line transformed from a human cancer cell line that did not express the normal human Rb gene product; and said Rb gene transfected in the human cancer cell line to provide a stable Rb positive human cancer cell line expressing Rb protein.
2. A human cancer cell line of Claim 1 wherein the human cancer cell line is selected from the group consisting of human bladder carcinoma, fibrosarcoma and prostate cancer.
3. A method of making a human cancer cell line expressing the normal Rb gene product comprising the steps of,
isolating the human Rb cDNA;
incorporating the human Rb cDNA into an expression vector;
transfecting a human cancer cell line that does not express Rb protein with the Rb expression vector;
selecting the transfected cells; and culturing the transfected cells.
4. A method of claim 3 including the step of selecting the Rb positive cells by identifying the cells with Rb protein expression.
5. A method of Claim 3 wherein the expression vector includes the human β-actin promoter.
6. A method of Claim 3 wherein in the 5' end the Rb cDNA started at -57 bp from the first ATG.
7. A method of Claim 3 wherein the 3' end of the Rb cDNA is +3684 bp from the first ATG.
8. A method of Claim 3 wherein the expression vector is pBARB.
9. A method of Claim 3 wherein the transfected cell line is selected from the group consisting of human bladder carcinoma, fibrosarcoma and prostate cancer.
10. A method of Claim 3 wherein the Rb positive cell line is cultured in media containing greater than 3% serum.
11. A method of Claim 3 wherein one of said transfected cells is isolated and comprising the additional steps of isolating the Rb positive cell and culturing a cloned Rb positive cell line.
12. A cell line produced by the process of
Claim 3.
13. A cell line produced by the process of Claim 11.
14. An Rb expression vector for transfecting human cancer cells comprising,
a plasmid;
a promoter; and Rb cDNA .
15. An Rb expression vector of Claim 14 wherein said promoter is human β-actin.
16. An Rb expression vector of Claim 14 wherein said 5' end of the Rb cDNA starts at -57 bp from the first ATG.
17. An Rb expression vector of Claim 14 wherein the 3' end of the Rb cDNA is +3684 bp from the first ATG.
18. An Rb expression vector of Claim 14 wherein the Rb cDNA is inserted in the plasmid pHβAPr-1-neo.
PCT/US1992/004934 1991-06-10 1992-06-09 Rb TRANSFERRED CELLS AND METHOD WO1992022640A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US71247091A 1991-06-10 1991-06-10
US712,470 1991-06-10
US88507392A 1992-05-18 1992-05-18
US885,073 1992-05-18

Publications (1)

Publication Number Publication Date
WO1992022640A1 true WO1992022640A1 (en) 1992-12-23

Family

ID=27108842

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/004934 WO1992022640A1 (en) 1991-06-10 1992-06-09 Rb TRANSFERRED CELLS AND METHOD

Country Status (4)

Country Link
AU (1) AU2336592A (en)
IE (1) IE921876A1 (en)
IL (1) IL102165A0 (en)
WO (1) WO1992022640A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021115A1 (en) * 1993-03-25 1994-09-29 Baylor College Of Medicine Broad spectrum tumor suppressor genes, gene products and methods for tumor suppression gene therapy
WO1994027426A1 (en) * 1993-05-26 1994-12-08 La Jolla Cancer Research Foundation Methods of using bcl-2 for the therapeutic treatment and prevention of diseases
WO1995007708A2 (en) * 1993-09-13 1995-03-23 The Regents Of The University Of California Therapeutic use of the retinoblastoma susceptibility gene product

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Vol. 84, issued July 1987, P. GUNNING et al., "A Human beta-Actin Expression Vector System Directs High-Level Accumulation of Antisense Transcripts", pages 4831-4835. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Vol. 87, issued April 1990, J.M. HOROWITZ et al., "Frequent Inactivation of the Retinoblastoma Anti-Oncogene is Restricted to a Subset of human Tumor Cells", pages 2775-2779. *
SCIENCE, Vol. 235, issued 13 March 1987, W.H. LEE et al., "Human Retinoblastoma Susceptibility Gene: Cloning, Identification, and Sequence", pages 1394-1399. *
SCIENCE, Vol. 247, issued 09 February 1990, R. BOOKSTEIN et al., "Suppression of Tumorigenicity of Human Prostate Carcinoma Cells by Replacing a Mutated RB Gene", pages 712-715. *
SCIENCE, Volume 242, issued 16 December 1988, H.J.S. HUANG et al., "Suppression of the Neoplastic phenotype by Replacement of the RB Gene in Human Cancer Cells", pages 1563-1566. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994021115A1 (en) * 1993-03-25 1994-09-29 Baylor College Of Medicine Broad spectrum tumor suppressor genes, gene products and methods for tumor suppression gene therapy
WO1994027426A1 (en) * 1993-05-26 1994-12-08 La Jolla Cancer Research Foundation Methods of using bcl-2 for the therapeutic treatment and prevention of diseases
US5550019A (en) * 1993-05-26 1996-08-27 La Jolla Cancer Research Foundation Methods of identifying compounds which alter apoptosis
US6514761B1 (en) 1993-05-26 2003-02-04 The Burnham Institute Methods of using BCL-2 for the therapeutic treatment and prevention of diseases
US7427495B2 (en) 1993-05-26 2008-09-23 La Jolla Cancer Research Foundation Methods of using Bcl-2 for the therapeutic treatment and prevention of diseases
US7910370B2 (en) 1993-05-26 2011-03-22 Sanford-Burnham Medical Research Institute Methods of using BCL-2 for the therapeutic treatment and prevention of diseases
WO1995007708A2 (en) * 1993-09-13 1995-03-23 The Regents Of The University Of California Therapeutic use of the retinoblastoma susceptibility gene product
WO1995007708A3 (en) * 1993-09-13 1995-06-08 Univ California Therapeutic use of the retinoblastoma susceptibility gene product

Also Published As

Publication number Publication date
AU2336592A (en) 1993-01-12
IE921876A1 (en) 1992-12-16
IL102165A0 (en) 1993-01-14

Similar Documents

Publication Publication Date Title
Sarria et al. Regulated expression of vimentin cDNA in cells in the presence and absence of a preexisting vimentin filament network.
EP1164195B1 (en) Adeno-associated virus with inverted terminal repeat sequences as promoter
US4663281A (en) Enhanced production of proteinaceous materials in eucaryotic cells
CA2005016C (en) Dna fragment containing promoter region for human polypeptide chain elongation factor-1.alpha. and expression plasmid containing the dna fragment
HU208711B (en) Method for preparing gm-csf and transformation vector containing csf/cdna, also a methold for purifying the csf protein
CA2260365C (en) Differentiation-suppressive polypeptide
Chang et al. Transformation of human cultured fibroblasts with plasmids carrying dominant selection markers and immortalizing potential
Clement et al. SV40T-immortalized lung alveolar epithelial cells display posttranscriptional regulation of proliferation-related genes
JPH09501568A (en) Tumor suppressor protein PRB2, related gene product and DNA encoding the same
WO1992022640A1 (en) Rb TRANSFERRED CELLS AND METHOD
Bauer et al. Cellular mutation mediates T-antigen-positive revertant cells resistant to simian virus 40 transformation but not to retransformation by polyomavirus and adenovirus type 2
Bloemendal Lens research: from protein to gene
KR100237953B1 (en) Mutant aox2 promoter, microorganism carrying same, method of preparation thereof and production of heterologous protein using such microorganism
EP0529160A1 (en) Gene protein products and methods of cell therapy
Yang et al. Intermediate filaments in higher plant cells and their assembly in a cell-free system
EP0162319B1 (en) Method of joining heterologous genes
EP0204302A2 (en) Laminin and the production thereof
Affara et al. Analysis of chromatin changes associated with the expression of globin and non-globin genes in cell hybrids between erythroid and other cells
US7141389B1 (en) Chromosome DNA coding for human hepatocyte growth factor
Kay Expression of types I and IV collagen genes in normal and in modulated corneal endothelial cells.
AU2038283A (en) The use of eucaryotic promoter sequences in the production ofproteinaceous materials
DE69534246T2 (en) MN GENE AND PROTEIN
KR100441202B1 (en) Fertilized egg of mouse expressing HPV E6
EP0681028B9 (en) A gene expression vector using the gene expression regulating region of the ADP ribosylation factor
CA2000094A1 (en) H. saimiri-htlv-x region vector

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA FI JP KR NO RU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase