Methods and artificial genes for antagonizing the function of an oncogene

A method of antagonizing the effects of an oncogene by constructing an artificial gene which antagonizes the function of the oncogene is described herein. In one embodiment, the artificial gene comprises a transcriptional promoter segment, an inverted oncogene segment, and a polyadenylation segment.

TECHNICAL FIELD 
This invention is in the field of molecular biology and relates to 
oncogenes. 
BACKGROUND ART 
Although the molecular basis for malignant transformation leading to cancer 
is not yet fully understood, much information has been developed recently 
using molecular biology techniques. For example, while it has long been 
thought that transformation involved the alteration of critical genes, 
referred to as "oncogenes", such discrete oncogenes have only recently 
been isolated and shown to cause transformation. 
For example, isolated human sequences from the c-K-ras oncogene present in 
certain human lung tumors have been described. See Nakano, H., Yamamoto, 
F., Neville, C., Evans, D., Mizuno, T., and Perucho, M., "Isolation of 
Transforming Sequences of Two Human Lung Carcinomas: Structural and 
Functional Analysis of the Activated c-K-ras Oncogenes", Proc. Acad. Sci. 
USA, 81, 71-75, January, 1984; Santos, E., Martin-Zanca, M., Reddy, P., 
Pierotti, M. A., Della Porta, G., Barbacid, M., "Malignant Activation of A 
K-ras Oncogene and Lung Carcinoma But Not in Normal Tissue of the Same 
Patient", Science, 223, pp. 661-4, Feb. 17, 1984. Additionally, an 
oncogene isolated from the EJ bladder carcinoma cell line has been found 
to cause transformation when it was transfected into NIH 3T3 cells. See 
Shih, C., and Weinberg, R., Cell, 29, 161-9 (1982). 
DISCLOSURE OF THE INVENTION 
This invention relates to Applicants' discovery of a method for 
antagonizing the function of an oncogene. In this method, an artificial 
gene is constructed so that, upon introduction into a cell containing the 
oncogene, the artificial gene produces an RNA transcript which antagonizes 
the function of the oncogene. More specifically, the antagonizing gene is 
constructed to include: (1) a transcriptional promoter segment; (2) an 
oncogene segment that is inverted with regard to the transcriptional 
promoter; and (3) a polyadenylation segment. 
Transcription of the inverted oncogene segment occurs along the 
complementary strand of DNA, which is not transcribed in the oncogene 
itself to produce an RNA transcript capable of antagonizing the function 
of the oncogene. Thus, the artificially-constructed gene can be introduced 
into a cell containing an oncogene to antagonize the function of the 
oncogene. 
In a further embodiment of the invention, the function of the oncogene is 
antagonized and proto-oncogene function is restored to a cell. In this 
embodiment, a first artificially-constructed gene, as described above, is 
introduced into cells containing an oncogene to antagonize the function of 
the oncogene. In addition, a second artificially-constructed gene is 
introduced into the same cells and the second gene is constructed to 
restore proto-oncogene function to the cell; its function is not 
antagonized by the first gene. 
By employing this invention, the function of an oncogene can be antagonized 
in intact cells without the necessity of disrupting the cells' structure. 
Further, the artificially constructed genes can be designed to be 
selective for antagonism of a gene of choice within cells, leaving other 
genes to function normally. Additionally, this invention provides a method 
by which the function of an oncogene can be antagonized while 
simultaneously restoring proto-oncogene function to cells containing the 
oncogene.

BEST MODE FOR CARRYING OUT THE INVENTION 
As used herein, the term "oncogene" is used to mean a genetic sequence 
whose expression within a cell provides a function, including one of 
several functions, in the steps leading from a normal cell into a tumor 
cell. Similarly, the term "proto-oncogene" is used herein to mean a 
genetic sequence, residing in the normal genome of a normal, non-tumor 
cell, which has the potential, when altered in the appropriate manner, of 
becoming an oncogene. 
The experimental work described herein employed the k-ras oncogene. 
Additional oncogenes are known to those skilled in the art. These include, 
for example, the h-ras and n-ras oncogenes which have been implicated in 
many human tumors. See Tabin et al., Nature, 300, p. 143 (1982); and 
Shimizu et al., PNAS 80, p. 2112 (1983). Additionally, the myc gene has 
reported to be aberrantly expressed in Burkitt's lymphomas, and n-myc has 
been reported as being expressed at high levels in neuroblastomas. See 
Taub et al., PNAS, 79, p. 7837 (1982) and Schwab et al., Nature, 305, p. 
245 (1983). 
HTLV, a human retrovirus, is also believed to contain genes important to 
tumorigenesis whose function can be antagonized by the methods of this 
invention. See Wong-Staal et al., Nature, 302, p. 626 (1983). 
Additionally, many oncogenes have been isolated in mammalian and avian 
retroviruses and are suspected of being important in human tumors. These 
include src, erb, yes, fes, raf, myb, fos, rel, ski, etc. It is believed 
that oncogene function of these genes could be antagonized by the methods 
of this invention. 
An additional description of oncogenes is contained in Weinberg, R. A., 
Science, (1984), the teachings of which are hereby incorporated by 
reference. 
Oncogenes may be present in cells because they are introduced by 
experimental manipulation, such as gene transfer. Alternatively, oncogenes 
can be introduced into cells by an applied viral vector capable of 
carrying the oncogene into cells or may be introduced by a naturally 
occurring virus whose genome carries the oncogene. Additionally, an 
oncogene may be present in a tumor cell because of mutation occurring in a 
cell that was ancestral to the tumor cell, the mutation creating an 
oncogene from a pre-existing normal cellular gene, i.e., proto-oncogene. 
FIG. 1 illustrates transcription of an oncogene. Therein, an oncogene is 
illustrated having two strands of DNA complementary to each other, of 
opposite polarity, that constitute a single DNA molecule. The 
complementarity is indicated by lines connecting the two strands of DNA. 
Thus, sequence A is complementary to sequence A' on the opposite strand of 
DNA, sequence B is complementary to sequence B' on the opposite strand of 
DNA, and so forth. 
Transcription of the DNA molecule directs synthesis of one strand of RNA 
having sequences complementary to the sequences on the strand of DNA 
transcribed. Thus, the RNA transcript has sequences A-F which are 
complementary to the sequence of the transcribed strand A'-F' of DNA. Such 
an RNA transcript is sometimes referred to as sense RNA. 
FIG. 2 illustrates the construction of a tripartite gene designed to 
antagonize the oncogene shown in FIG. 1. The dotted lines indicate where 
the DNA molecule is cut, employing restriction enzymes, to create the 
tripartite construction. The two strands of DNA containing the oncogene 
segment are cleaved and inverted 180.degree. with respect to the promoter 
and polyadenylation signal. The polarity of the inverted strand is the 
same as the polarity of the sequence into which it is inserted. Such 
inversion results in the F-A sequence of DNA being transcribed. As a 
result, the RNA transcript of the gene illustrated in FIG. 2 is 
complementary to the F-A sequence. 
The promoter serves as a signal conferring expression on the sequences that 
lie on one side of it. It may be construed as all the signals necessary 
for successful initiation of transcription in a given cell type, including 
sequences such as enhancers, TATA boxes, and other signals. Its operation 
is taken to be strictly polar, leading to transcription of one of the two 
strands of DNA in a 3' to 5' direction on the DNA, leading to a 5' to 3' 
formation of an RNA molecule. 
The polyadenylation signal may be construed as such signals that provide an 
accurate and efficient termination of a transcript, whether by cessation 
of transcription or a post-transcriptional cleavage event. Polyadenylation 
itself is a post-transcription processing event in which a cleaved RNA has 
ribose adenosines attached to its 3' end which may confer stability to the 
RNA or be involved in processing and transport. 
FIG. 3 illustrates how the tripartite artificially-constructed gene of FIG. 
2 may antagonize the function of the oncogene of FIG. 1. In FIG. 3, an 
interaction is illustrated that might occur between the RNA transcripts 
produced by the genes of FIGS. 1 and 2. A complementary interaction 
involves two strands of opposite polarity to each other, so that the RNA 
transcript from FIG. 2 is rotated in FIG. 3 so that its 5' end lies to the 
right rather than the left. The region that was inverted in the tripartite 
construction of FIG. 2 allows a complementary region to form between the 
two RNA transcripts. Regions outside the inverted region do not bear a 
complementary relationship to each other. 
The transcriptional promoter can, in principle, be of any origin as long as 
it specifies a rate of transcription that is compatible with antagonizing 
the oncogene. To achieve such antagonism, it is desirable to employ a very 
efficient transcriptional promoter, which eliminates many promoters 
naturally associated with proto-oncogenes because they are weak promoters. 
Viral promoters, such as a retrovirus promoter, are potent promoters and 
therefore function well in the tripartite genes according to this 
invention. Other sources of potent promoters include cellular genes that 
are expressed at a high level. These may be genes that are actively and 
powerfully transcribed in most cell types, such as tubulin genes, or genes 
that are optimally transcribed only in one or a few cell types, such as 
globin genes in pre-erythrocytes or muscle actin in myoblasts or 
myofibrils. Promoters from genes transcribed in only a few cell types 
would be most useful in the same type of cell. 
The promoter segment may be a constitutive promoter or a regulatable 
promoter. Regulatable promoters provide selectivity to the antagonism of 
oncogene function. One example of a regulatable promoter is described in: 
Mayo, K. E., Warren, R. and Palmiter, R. D., "The Mouse Metallothionein-1 
Gene Is Transcriptionally Regulated By Cadmium Following Transfection Into 
Human or Mouse Cells", Cell, 29, 99-108 (May 1982), the teaching of which 
are hereby incorporated by reference. Regulatable promoters may include 
those that can be regulated externally such as the metallothionein I gene 
which can be regulated by administration of heavy metals, and those that 
are regulated internally, such as histone genes that are regulated in the 
cell cycle. 
The oncogene segment which is inverted in the tripartite gene described 
herein must have a close sequence relationship to the oncogene segment it 
is constructed to antagonize. Although the precise reason for this is not 
known, it is likely that this is due to the fact that the two kinds of RNA 
molecules recognize one another which only happens when they share a close 
or identical sequence relationship. For this reason, it is often preferred 
to employ oncogene segments from the same species of origin as the 
oncogene which is to be antagonized. Despite this, it is believed that 
oncogene segments from different species of origin than the oncogene to be 
antagonized will be sufficient in some cases. This is particularly true 
where the two genes are decended from the same gene carried by a common 
progenitor organism but have evolved separately during independent 
evolutions of the two lineages, causing the two genes to have diverged 
somewhat in consequence; such genes are no longer completely analogous but 
are closely related and are known as homologs. Indeed, there may be some 
advantages in some applications to use oncogene segments from species of 
origin different from the oncogene to be antagonized in such applications. 
The polyadenylation segment is employed in the tripartite construction 
because production of a stable RNA molecule requires that the molecule go 
through discrete processing or maturation events. These events are 
facilitated by the presence in a gene of a precise cleavage/processing 
signal of this nature; the absence of such a signal causes a decreased 
level of functional, stable RNA molecules in the cytoplasm of the cell. 
Such a signal could be obtained from a retrovirus, as in this instance, or 
another virus, or a cellular gene. In each case, it is necessary that the 
poly A site so provided prove an efficient site of cleavage/termination in 
the organism and cell in which the antagonism is desired. This can best be 
determined empirically. 
The construction of a gene capable of antagonizing the function of k-ras 
oncogene will now be described and is illustrated schematically in FIGS. 4 
and 5. This gene is constructed employing a promoter region from the left 
(5' proximal) end of the Moloney leukemia virus provirus. This 5' proximal 
proviral segment includes the left (5' proximal) long terminal repeat 
(LTR) of the provirus; a middle portion containing a cDNA segment of the 
human cellular cKi-ras2 gene; and a portion that includes the right (3' 
proximal) LTR of the Moloney leukemia virus provirus, including an 
associated polyadenylation site. 
This particular construct will be referred to as a "6-segment construct" 
and serves as the basis for constructing tripartite genes as described 
herein. It can be described as follows, moving from left to right. 
Segment 1. This is a derivative of the pZIP plasmid (See Hoffmann et al., 
J. Virol 44: 144-157), whose essential portion is 1 kb (kilobase) long, 
beginning with 0.6 kb of the left LTR of Moloney murine leukemia virus 
(MLV) and proceeding another 0.4 kb to the first Pst I site usually 
encountered in the MLV viral genome (termed the MLV provirus). The Pst I 
site has been replaced by a Bam HI endonuclease site. Segment 1 contains 
the transcriptional promoter and, therefore, the first of the 3 elements 
of a tripartite gene. 
Segment 2. This is a 0.4 kb segment derived from the pZIP clone of the MLV 
provirus, encompassing a "splice-acceptor" site, and originating from the 
provirus region defined by map units 5.9 to 6.3 and the endonuclease sites 
BglII and XbaI within the MLV provirus, these two sites having been 
modified to BamHI and XhoI endonuclease sites, respectively. 
Segment 3. This is a 1.4 kb segment that was derived originally from the 
tn5 neomycin resistance gene was isolated from pBRneo [See Southern and 
Berg, J. Mol. Appl. Genet. 1: 327 (1982)]. This segment was inserted 
between HindIII and BamHI endonuclease sites at nucleotide positions 29 
and 375 within pBR322; in this construction these sites have been replaced 
by XhoI and a null cleavage site, respectively. 
Segment 4. This is a 0.23 kb segment derived from the portion of simian 
virus 40 (SV40) genome containing the viral origin of replication and 
mapping from nucleotides 160 to 5154 of the SV40 genome, these two sites 
representing RII and HindIII endonuclease sites, these sites being 
replaced by a null cleavage site and an EcoRI cleavage site, respectively, 
in the construct. 
Segment 5. This is a 0.58 kb segment of pBR322 containing the plasmid 
origin of replication and originating from a ThaI endonuclease-generated 
fragment extending from nucleotide 2521 to nucleotide 3102 of the pBR322 
plasmid. These two ThaI sites being replaced by an EcoRI site on the left 
and an XhoI site on the right. 
Segment 6. This is a 1.05 kb segment deriving from the pZIP plasmid and 
containing an MLV proviral segment originating at a site at 7.75 map units 
within the MLV provirus, defined by a HpaI endonuclease site in the 
naturally occurring provirus and continuing for 0.45 kb until the right 
LTR of the MLV provirus, and continuing farther for 0.6 kb of the 
provirus. The naturally occurring Hpa I site has been replaced by an XhoI 
site in this construct. Thus segment 6 contains the polyadenylation site 
and therefore constitutes the rightmost segment of a tripartite gene. 
The second or middle portion of a tripartite gene is not present in the 
above 6-segment construction. A site existing between segment 1 and 
segment 2, and corresponding to a BamHI endonuclease site, exists as an 
empty cassette into which various versions of the desired middle portion 
of a tripartite construction can be inserted. 
This BamHI cleavage site has been used as a point of entry for the 
introduction of DNA segments leading to 3 tripartite constructs. These 
are: 
a. A DNA segment homologous to the cKi-ras2 gene of the human genome (See 
McCoy et al., Nature, 302: 79-81, 1983) and being specifically a 1.2 kb 
DNA segment deriving from reverse transcription of the cKi-ras2 mRNA, 
being termed therefore a cDNA. This gene was retrieved from a library 
prepared by Okayama and Berg (See Mol. Cell. Biol. 3: 280, 1983). This 1.2 
kb cKi-ras2 segment acquired BamHI sites as a consequence of its presence 
in the Okayama and Berg cloning vector and could be retrieved from said 
vector by cleavage with endonuclease BamHI. This segment could be 
introduced into the 6-segment construct at its BamHI site in a fashion 
such that the promoter of the 6-segment construct will cause synthesis of 
the anti-sense RNA strand from the cKI-ras2 DNA template. 
b. The identical 1.2 kb cKi-ras2 cDNA can be introduced into the same site 
of the 6-segment construct in the opposite orientation, so that the 
transcriptional promoter of the 6-segment construct will cause synthesis 
of "sense" RNA from the cKi-ras2 DNA template. 
c. The BamHI site can be left unoccupied, such that no additional DNA is 
introduced into this site. 
These three separately constructed tripartite genes were introduced 
separately via transfection into NIH3T3 mouse fibroblasts that had 
previously acquired, via transfection, copies of the oncogene of the human 
SW480 colon carcinoma cell line. See Murray et al., Cell, August, 1981. 
Cells from each of the three resulting cultures were placed under neomycin 
selection following transfection and neomycin-resistant colonies were 
observed and studied in detail. This selection assured outgrowth of only 
those cells that had acquired a copy or copies of the segment construct 
and associated inserted segments. Such outgrowth was assured because the 
6-segment construct carried a gene ("segment 3" above) conferring 
resistance to killing by neomycin. 
Five out of six colonies carrying the cKi-ras DNA gene inserted in the 
reverse orientation into the 6-segment construct, thus, carrying an 
antagonizing gene that served as template for synthesis of "anti-sense 
RNA," lost their transformed morphology and therefore reverted to normal 
growth patterns. 
Four out of seven colonies that carried the cKi-ras DNA gene inserted in 
the normal orientation into the 6-segment construct, thus carrying a gene 
that served as template for synthesis of "sense RNA," retained their 
transformed morphology and became even "hyper-transformed" in their 
morphology. 
Four out of five colonies that carried no cKi-ras segment inserted into the 
6-segment construct showed no effect whatsoever on morphology or growth 
behavior. 
The antagonizing gene can be introduced into cells containing oncogenes by 
a number of different techniques. One technique is transfection, which can 
be done by several different methods. One method of transfection involves 
the addition of DEAE-dextran to increase the uptake of the naked DNA 
molecules by a recipient cell. See McCutchin, J. H. and Pagano, J. S. , J. 
Natl. Cancer Inst., 41, pp. 351-7 (1968). Another method of transfection 
is the calcium phosphate precipitation technique which depends upon the 
addition of Ca.sup.++ to a phosphate-containing DNA solution. The 
resulting precipitate apparently includes DNA in associate with calcium 
phosphate crystals. These crystals settle onto a cell monolayer, the 
resulting apposition of crystals and cell surface appears to lead to 
uptake of the DNA. A small proportion of the DNA taken up becomes 
expressed in a transfectant, as well as in its clonal descendants. See 
Graham, F. L. and van der Eb, A. J., Virology 52, pp. 456-467 (1973) and 
Graham, F. L. an van der Eb, A. J., Virology 54, pp. 536-539 (1973). 
Alternatively, the reverting gene can be introduced into cells, in vitro or 
in vivo, via a transducing viral vector. See Tabin, C. J., Hoffmann, J. 
W., Goff, S. P., and Weinberg, R. A. (1982) "Adaption of a Retrovirus as a 
Eucaryotic Vector Transmitting the Herpes Simplex Virus Thymidine Kinase 
Gene", Mol. Cel. Biol. 2: 426-436. Use of retrovirus, for example, will 
infect a variety of cells and cause the antagonizing gene to be inserted 
into the genome of infected cells. Such infection could either be done 
with the aid of helper retrovirus, which would allow the virus to spread 
through the organism, or the antisense retrovirus could be produced in a 
helper-free system, such as .psi.2-like cells (See Mann et al., Cell, 
1983) that package amphotropic viruses. A helper-free virus might be 
employed to minimize spread throughout the organism. Viral vectors in 
addition to retroviruses can also be employed, such as paporaviruses, 
SV40-like viruses, or papilloma viruses. 
The use of viruses to transfer genes into cells has recently been described 
in Science, 223, 1376, Mar. 30, 1984, and the teachings of this article 
are hereby incorporated by reference. 
Vesicle fusion could also be employed to deliver the antagonizing gene. 
Vesicle fusion may be physically targeted to the tumor tissue if the 
vesicle were approximately designed to be taken up by the cells containing 
the oncogene. For example, vesicles containing asialoglycoproteins would 
be preferentially taken up by liver cells or other cells containing 
asialoglycoprotein receptor. Such a delivery system would be expected to 
have a lower efficiency of integration and expression of the antagonizing 
gene delivered, but would have a higher specificity than a retroviral 
vector. A combination strategy of targeted vesicles containing papilloma 
virus or retrovirus DNA molecules might provide a method for increasing 
the efficiency of expression of targeted molecules. 
Still another alternative is to introduce the antagonizing gene via 
micro-injection. See for example, Laski et al., Cell, 1982. 
The precise mechanisms of antagonism provided by introduction of an 
antagonizing gene are not well understood. It is clear, however, that the 
introduction of the antagonizing gene prevents the target oncogene from 
serving as a template for the synthesis of protein whose structure is 
translated from the RNA transcript of the oncogene. While not wishing to 
be bound by this explanation, it is possible that the antagonism stems 
from the ability of the anti-sense RNA transcript of the antagonizing gene 
to bond non-covalently to a sense RNA transcript of opposite polarity 
produced by transcription of the oncogene. 
The construction and functioning of two artificial genes, one of which is 
intended to antagonize oncogene function, and the other of which is 
intended to restore proto-oncogene function, will now be described with 
reference to FIGS. 6-8. 
An antagonizing gene which will antagonize the function of a gene whose 
initial transcript is an unspliced RNA molecule is constructed. Such an 
antagonizing gene will not antagonize the function of a related gene whose 
initial transcript is already spliced. Thus, the antagonizing gene will be 
introduced into a tumor or transformed cell and antagonize the function of 
an unspliced oncogene residing in that cell. At the same time, a second 
gene is constructed and introduced into this cell. This second 
cointroduced gene is a cDNA constructed to express functions of the normal 
cellular counterpart of the oncogene and will therefore be termed a 
"proto-oncogene." Because this cointroduced proto-oncogene will express 
only an already spliced RNA transcript, its functioning will not be 
antagonized by the reverting gene. Consequently, by introducing the two 
genes into a tumor or transformed cell, oncogene function can be 
antagonized while simultaneously replacing proto-oncogene function. 
The transcription of most cellular genes results in synthesis of RNA 
molecules having 2 types of RNA segments termed exons and introns. These 
exons and introns reside together in the same initial transcript. During 
subsequent maturation of the RNA molecule, the introns are removed in a 
process termed "splicing." The resulting spliced RNA molecule is then 
exported from its site of synthesis and maturation (the cellular nucleus) 
into its site of utilization (the cytoplasm). Artificial versions of the 
gene can be constructed that contain only those sequences of the gene 
which are represented in the spliced version of the gene. These artificial 
versions can be made, for example, by the reverse transcription of spliced 
RNA molecules, creating a complementary DNA copy of the spliced RNA 
molecule termed a cDNA copy. 
The replacement method involves constructing a tripartite gene whose middle 
segment contains, in reverse orientation, an intron sequence which resides 
normally in the unspliced versions of the oncogene and related 
proto-oncogene. When this tripartite reverting gene is introduced into a 
tumor or transformed cell, it will antagonize function of the oncogene 
residing in that cell. A cointroduced gene carrying in normal orientation 
a cDNA version of the proto-oncogene will not be affected by the presence 
of the reverting gene, because this cDNA proto-oncogene does not carry the 
intron sequence whose transcription creates an RNA molecule that is 
antagonized by the reverting gene. 
An alternative technique for antagonizing oncogene function and restoring 
proto-oncogene function to cells is illustrated in FIG. 9. 
An antagonizing gene which will antagonize the functioning of an oncogene 
by generating anti-sense RNAs which are complementary to the untranslated 
portions of the messenger RNA of the oncogene is illustrated. This 
tripartite construction will serve as template for the synthesis of RNA 
segments that are complementary to (a) sequences of the oncogene mRNA 
which lie between the 5' (beginning) of the RNA and the site on the mRNA 
at which translation of the encoded oncogene protein is begun; or (b) 
sequences of the oncogene mRNA which lie between the 3' (end) of the 
protein-encoding protion of the mRNA at which translation is terminated 
and the 3' site of the mRNA at which polyadenylation is initiated; or (c) 
a combination of (a) and (b). 
Into the same cell that acquires the above construction, a version of the 
normal proto-oncogene is introduced to serve as template for synthesis of 
a "sense" mRNA encoding a normal protein. This normal gene is constructed 
so that the mRNA synthesized on its DNA template will lack that portion or 
portions of the mRNA which are complementary to anti-sense RNAs that are 
synthesized on the reverting gene template. Thus, this normal construct 
will escape antagonism by the anti-sense RNAs. 
Although the discussion herein, including the experimental work, has been 
directed to antagonizing oncogenes, it is believed that the methods of 
this invention can also be employed to antagonize the function of other 
functioning genes contained within the DNA of cells. Such genes might 
include, for example, genes responsible for such auto-immune diseases as 
arthritis, Huntington's Chorea, etc. 
This invention will now be further and more specifically illustrated by the 
following example. 
EXEMPLIFICATION 
One .mu. g of pZIPNeo DNA (the 6-segment construct of FIG. 4) obtained from 
C. Cepko and R. Mulligan at the Massachusetts Institute of Technology was 
cut to completion with BamHI (New England Biolabs) and treated with calf 
intestinal phosphatase (Boehringer-Mannheim Biochemicals). The backbone 
plasmid pZIP is related to a plasmid described in J. Virol., 44, 144-57, 
the teachings of which are incorporated by reference. The human c-Kirsten 
cDNA was isolated from an SV40-transformed human fibroblast library (see 
Okayama and Berg, Mol. Cel. Biol., 3, p. 280, 1983). The 1.2 kb cDNA was 
isolated from the pcD vector by digestion with BamHI and subsequent gel 
purification. The cDNA and pZipNeo were ligated using T.sub.4 DNA ligase 
(Collaborative Research) under standard conditions (50 mM Tris pH 7.6, 10 
mM MgCl.sub.2, 6.6 mM .beta.-mercaptoethanol, 1 mM ATP). Recombinants were 
identified by restriction mapping. 
PZipNeo, ZNCK (cDNA in sense orientation with respect to left LTR), and 
ZNKC (cDNA antisense orientation with respect to left LTR) were 
transfected by the method of Graham and van der Eb, Virology 52: 456 
(1973) as modified by Anderson, Goldfarb and Weinberg, Cell 16: 63 (1979). 
Briefly, 50 ng of each DNA was mixed with 37.5 .mu.g of sheared NIH3T3 
carrier DNA, ethanol precipitated, and resuspended in 1.25 ml of 8 g/l 
NaCl, 5 g/l HEPES pH 6.95, and 0.15 g/l Na.sub.2 HPO.sub.4 .times.7H.sub.2 
O. To this was added with vortexing 50 .mu.l of 2.5 M CaCl.sub.2, and a 
precipitate was allowed to form for 30' at room temperature. SW-2-3 cells 
[McCoy et al., Nature 302: 79 (1983)] were fed with 5 mls of 10 percent 
calf serum/DME and the DNA precipitate. Four hours later the precipitate 
was removed and the cells refed with 10 mls of 10 percent calf serum/DME. 
Sixteen hours after transfection the cells were trypsinized and split 1 to 
4. Twenty-four hours after transfection the cells were refed with medium 
containing 10 percent calf serum/DME/1 mg/ml G418 (Gibco). Cells were 
refed with this medium every three days. Sixteen days after transfection, 
G418-resistant colonies were picked using plexiglass cloning cylinders and 
grown up to make the cell lines described. 
Cellular morphology was observed when the cells were at various stages of 
confluence in 10 cm dishes. Cells scored as transformed were refractile 
and splindly, and piled on top of each other. Cells scored as 
nontransformed were nonrefractile and strongly inhibited from overgrowing 
a monolayer. Of the ZipNeo lines, 4/5 scored transformed; of the ZNCK and 
4/7 scored as transformed, and of the ZNKC lines, 5/6 scored as 
nontransformed. 
Subsequently, ZNKC lines were analysed to insure that the transforming 
Kirsten-ras oncogene was still present. Total cellular DNA was isolated by 
lysis in 100 mM NaCl/10 mM Tris pH 7.6/10 mM EDTA/0.5% SDS and 0.2 
.mu.g/ml Prolinase K (Boehringer-Mannhein Biochemicals). Lysates were 
extracted twice with phenol, twice with chloroform, and ethanol 
precipitated. 10 .mu.g of DNA were digested with XbaI (New England 
Biolabs) in 50 mM NaCl/10 mM Tris pH7.6/10 mM MgCl.sub.2 and separated on 
a 1.1% agarose gel. DNA was transferred to nitrocellulose (Schliecher and 
Schuell) by the method of Southern [JMB 98:503 (1975)] and probed with 
10.sup.7 cpm of human c-Kirsten-ras DNA nick translated to a specific 
activity of 6.times.10.sup.8 cpm/.mu.g. Hybridization was carried out in 
50% formamide /S X SSCPE/1.times.Denhardt's/100 .mu.g/ml salmon sperm DNA 
at 42.degree. for 16 hours. Filters were washed with 2.times.SSCPE/0.1% 
SDS at 68.degree. for 4 hours and exposed next to X-ray film overnight. 
The phenotypically nontransformed ZNKC lines were shown to contain two 
sets of Kirstein-ras sequences by this analysis: (1) the SW480-derived 
Kirsten-ras oncogene sequence; and (2) the transfected ZNKC sequence. 
Mouse c-Kirsten sequences should not hybridize at this stringency. This 
demonstrated that these cells were not transformed even though the 
oncogene segments were present. 
In addition, cell lines were analysed for growth in soft agar, plating 
10,000 cells into 0.35% nobel agar (Gibco) in 10% calf serum/DME and 
growing at 37.degree. C. for two weeks. Phenotypically untransformed ZNKC 
lines showed up to 100-fold inhibition of colony-forming ability compared 
to parental SW-2-3 cells or Zip Neo-transfected cells. 
Industrial Applicability 
The invention described herein is useful in antagonizing transformation of 
cells caused by oncogenes within the cells, and in a particularly 
preferred embodiment, in restoring proto-oncogene function to the cells in 
addition to reversing the effects of the oncogene. In addition, treatment, 
in vivo, of other diseases, such as auto-immune diseases caused by 
malfunctioning genes can be treated by the methods of this invention. In 
addition, there are certain in vitro applications, such as antagonizing 
genes to prevent undesired gene products in commercially important 
products of cell lines. Such products might be proteases in a line used 
for purifying a protein, e.g., interferon or IL-2 producing lines, or gene 
products that contaminate otherwise pure preparations. 
Equivalents 
Those skilled in the art will recognize or be able to ascertain, using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims.