IE921876A1

IE921876A1 - Rb Transferred Cells and Method

Info

Publication number: IE921876A1
Application number: IE187692A
Authority: IE
Inventors: Rei Takahashi; William F Benedict
Original assignee: Res Dev Foundation
Priority date: 1991-06-10
Filing date: 1992-07-01
Publication date: 1992-12-16
Also published as: WO1992022640A1; IL102165A0; AU2336592A

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

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 13ql4.

The cDNA of the Rb gene has been sequenced. 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 -2been 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 -3without 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+ cell 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 -4derived 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, pH/?APr-lneo. The plasmid pH/?APr-l-neo is described in detail in Gunning, et al., A human 0-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 0-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 pH0APr-l-neo plasmid shown in Figure 2 of Gunning, et al., at the Bam Hl site. Integration points in the pH0APr-l-neo plasmid are indicated by the closed arrow Eco Rl and the open arrow Bam Hl. The Rb cDNA is -5followed 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 neomycin-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 15 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 /zg or 13.6 μ

Claims

1. A human cancer cell line comprising, a human cancer cell line with the human Rb gene; 5 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 10 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 15 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 20 steps of, isolating the human Rb cDNA; incorporating the human Rb cDNA into an expression vector; transfecting a human cancer cell line 25 that does not express Rb protein with the Rb expression vector; selecting the transfected cells; and culturing the transfected cells. 30

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 -13vector includes the human 0-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 10 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. 20

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 -14Rb CDNA.

15. An Rb expression vector of Claim 14 wherein said promoter is human 0-actin.

16. An Rb expression vector of Claim 14 wherfein said 5' end of the Rb cDNA starts at -57 bp from the first ATG. 10

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 15 wherein the Rb >DNA is inserted in the plasmid pH0APr-lneo.

19. A human cancer cell line according to Claim 1, substantially as hereinbefore described and exemplified.

20. A method according to Claim 3 of making a human cancer cell line expressing the normal Rb gene product, substantially as hereinbefore described and exemplified.

21. . A human cancer cell line expressing the normal Rb gene product, whenever made by a method claimed in a preceding claim.

22. An Rb expression vector according to Claim 14, substantially as hereinbefore described and exemplified.