The work described herein was supported by grants from the National Institutes of Health, the National Cancer Institute and the American Business Cancer Research Foundation.
This invention is in the field of molecular biology and more specifically relates to isolating discrete genes from mammalian DNA and to defining differences between mutant alleles and their corresponding wild type alleles, particularly oncogenes and proto-oncogenes, and to assays which take advantage of such differences.
Previous work relating to chemical carcinogenesis has demonstrated that carcinogenic potency of a compound often correlates with its mutagenic power. See McCann, J., Choi, E., Yamasaki, E. and Ames, B. N. Proc. Natl. Acad. Sci. USA 72: 5135-5139 (1975); McCann, J. and Ames, B. N. Proc. Natl. Acad. Sci. USA 73: 950-954 (1976); Bridges, B. A. Nature 261: 195-200 (1976); and, Bouck, N. and diMayorca, G. Nature 264: 722-727 (1976). This suggests that DNA is the ultimate target of carcinogenic activation. Because of this, researchers have attempted to identify and study DNA segments in tumor cells, often referred to as xe2x80x9concogenes,xe2x80x9d whose alteration is critically important for oncogenic conversion.
The molecular basis of malignant transformation leading to cancer is poorly understood. It is generally thought that transformation begins with damage to DNA, although the exact nature of the damage is in dispute. Presumably, the alteration of critical genes, sometimes called oncogenes, which are carried in the DNA is an essential step in a conversion of a normal cell to one that is capable of causing cancer. Such oncogenes could be activated by carcinogens of various kinds known to cause cancer.
There has been a tremendous amount of research effort directed toward identifying, and presumably subsequently isolating, such oncogenes. Despite the amount of effort which has been undertaken, little is understood about activation and activity of oncogenes.
This invention relates to a method for isolating a discrete, transmissible gene from DNA of mammalian origin and to an investigation of the differences between oncogenes of mammalian origin and their corresponding proto-oncogenes. In particular, it relates to definition of differences between the EJ oncogene, previously shown to cause human bladder cancer, and its proto-oncogene and to assessment of cellular DNAs to determine whether they include lesions or alterations in a neu gene which result in activation of the proto-oncogene and conversion of the proto-oncogene to an oncogene. The procedures involved in defining the differences in these proto-oncogene/oncogene pairs can be used to define differences between any mutant allele and its corresponding wild type allele; the procedures are particularly useful in defining the differences between oncogenes and their corresponding proto-oncogenes.
Identifying and isolating oncogenes has many desirable consequences. The oncogene isolated can be compared with closely related sequences in normal DNA and such comparison should lead to an understanding of what alterations occur to lead to the creation of an active oncogene; in fact, such comparison has made it possible to demonstrate that a proto-oncogene can be converted to an oncogene by a single nucleotide alteration or point mutation. This has made it possible to produce oligonucleotide probes which are specific for nucleotide sequences of the region in which the activation mutation(s) resides. Such probes can be used to determine the presence or absence of an oncogene which differs from its proto-oncogene. Methods of detecting an oncogene using such probes are described herein. As described in detail in the following sections, investigation of the activity and characteristics of oncogenes and their corresponding proto-oncogenes entailed discovery of a method for isolating a discrete, transmissible gene of mammalian origin, initial assessment of the basis of functional differences between the two (oncogene and proto-oncogene) and subsequent determination of the means by which a proto-oncogene is converted or activated to its oncogene form.
As described in application Ser. No. 379,721, filed May 19, 1982, now abandoned, the concept of a discrete, definable oncogene has been directly demonstrated by molecular isolation of discrete transforming genes from the EJ human bladder carcinoma cell line. That such a discrete, definable oncogene exists was demonstrated using a method described in the co-pending application.
In the method described, mammalian DNA from a donor is fragmented into a multiplicity of fragments, at least one of which contains a discrete, transmissible gene of interest. A xe2x80x9cmarkerxe2x80x9d is provided on the fragment containing the gene of interest, if such a marker is not already present. The multiplicity of fragments are then transmitted into recipient cells which are capable of phenotypically expressing the presence of the discrete transmissible gene, i.e., the phenotype of the gene can be scored by cells carried in culture. These recipient cells are then cultured under conditions which allow phenotypic expression by the gene of interest, and cells showing such phenotypic expression are selected. Because of the phenotypic expression, the selected recipient cells are known to contain the gene of interest; they may also contain additional DNA sequences on the donor fragment transmitted into the recipient cells, as well as their own endogenous DNA. The selected recipient cells"" DNA is then recovered and used in the aforementioned series of steps in place of the original donor DNA. These steps are repeated until the recipient cells selected in the last step have acquired essentially only the discrete, transmissible, mammalian gene and its associated marker. At this point, the discrete, transmissible gene is recovered from the cells selected employing its associated marker.
In one embodiment of this invention which has actually been experimentally performed, an oncogene for human bladder cancer has been isolated from DNA obtained from a human bladder cancer cell line. The original DNA was serially passed by transfection into mouse fibroblast cells using the above method until a mouse fibroblast cell containing essentially only the human bladder cancer oncogene and a marker was selected. In this case, the marker was an Alu DNA sequence which is repeated about 300,000 times in a human DNA molecule but is not present in mouse fibroblast cells. The interspecies transfection thus resulted in the ultimate selection of a cell containing the oncogene of interest and its associated marker. All of the DNA from the transfected cell was employed in the creation of a genomic library in a lambdaphage and the appropriate chimeric lambdaphage was then selected using a probe specific for the human Alu marker.
A sub-cloned insert of 6.6 kb which carried transforming activity was amplified in the plasmid vector pBR322. The sub-cloned oncogene has been used as a sequence probe in Southern blot analyses. The oncogene appears to derive from sequences present in normal cellular DNA. Structural analysis has so far failed to reveal differences between the oncogene and its normal cellular homolog. The oncogene is unrelated to transforming sequences detected in a variety of other types of human cell lines of colonic, lung, and neuroblastoma origin. In contrast, the human bladder oncogene isolated from one cell line appears closely related to oncogenes active in other human bladder carcinoma cell lines.
Initially, experiments were performed to determine whether the dramatic functional difference between the EJ oncogene and its proto-oncogene were due to a regulation mechanism or to one of sequence differences. These experiments provide data indicating that upregulation of this gene was not responsible for cellular transformation. Thus, it was concluded that the dramatic functional differences must be due to changes in the DNA sequence of said genes.
Subsequent to the determination that functional differences between an oncogene and it corresponding proto-oncogene must be due to differences in the DNA sequences of the two gene types, assessment of the two demonstrated what that difference is: a single base substitution, or point mutation, in the nucleotide sequence of the proto-oncogene, which resulted in its activation or conversion to an oncogene, which was accompanied by a difference in the amino acid sequence of the encoded protein.
In the case of the EJ oncogene and its corresponding proto-oncogene, the area of the 6.6 kb pEJ responsible for cellular transformation in NIH3T3 fibroblasts was narrowed to a 350 kb segment by a series of in vitro recombinations. This 350 kb segment was then sequenced for the oncogene and proto-oncogene, and it was found that single base substitutions accounted for the difference at the 60th nucleotide from the XmaI restriction site. This substitution in the codon for glycine (Gly12), normally occurring as GGC, was changed to the sequence GTC, which codon expresses valine. Thus, the specific difference in cellular DNA from the EJ and its proto-oncogene was located, and the difference in the amino acid sequence of the corresponding p21 proteins was also determined.
Assays for detecting such changes in DNA sequences were then developed. In one type of assay, restriction enzymes specific for a site on either the oncogene or the proto-oncogen, but not both, were employed to detect differences in such DNA sequences. In another type of assay, polynucleotide probes specific for a nucleotide site on either the oncogene or the proto-oncogene, but not both, are employed to detect differences in such DNA sequences or in RNA transcribed from such DNA sequences.
Since the p21 proteins encoded by these genes are also different, serological reagents, such as polyclonal or monoclonal antibodies, can also be developed which are specific for the altered or normal sequence domains in p21 proteins, or for an amino acid sequence not involved in the alteration which occurs during carcinogenesis. Such serological reagents can then be employed in various protocol to provide a very sensitive test for human bladder carcinogenesis. Similarly, these assays could be employed to detect changes in other wild type alleles causing mutant alleles.
In the case of the neu proto-oncogene, it was determined that conversion into an oncogene also occurs as a result of a single nucleotide alteration, or a point mutation. This point mutation was initially seen in a rat neuroblastoma induced by transplacental expsoure to a carcinogen (e.g., ethylnitrosourea) and found to affect the amino acid sequence of the transmembrane region of the p185 encoded by the DNA. That is, a valine present in the normal protein is replaced by a glutamic acid residue.
Oligonucleotide probes used to assay for similar point mutations suspected to be present in seven additional neu oncogenes, each of which arose in a separate, independently induced tumor, demonstrated the presence of the same activating mutations in all seven neu oncogenes. The same amino acid substitution (glutamic acid replacing valine) resulted in these cells. Further assays with oligonucleotide probes homologous to the neu gene demonstrates that the mutagen methylnitrosourea induces formation of nervous system tumors and activation of neu genes at the same position as shown to occur in the ethylnitrosurea-induced tumors.
The human homolog of the neu gene (also known as c-erbB2 or HER2) is thought to achieve an oncogenic state through the action of a similar mechanism: alteration of a single nucleotide in the normal cellular DNA sequence (the proto-oncogene), resulting in activation of the oncogene. The activating lesion found can be identified in the DNA of a variety of spontaneously arising human tumors through the use of oligonucleotide probes constructed to be specifically reactive with the region of the human neu gene corresponding to the region in the rat neu oncogene known to contain the activating mutations. Identification in human tumor cells of the activating point mutation responsible for conversion of the proto-oncogene into the neu oncogene can serve as the basis for construction of oligonucleotide hybridization probes useful in testing human tumor DNAs for the presence or absence of point mutations responsible for activation of neu oncogenes. These hybridization probes can be used in detecting the occurrence of the neu proto-oncogene and of the neu oncogene in cells and in determining the profile of oncogene activations in human tumor specimens. Such oligonucleotide probes are described, as are methods for their use in detecting the presence or absence of neu oncogenes in tumor cells. Antibodies specific for the p185 protein encoded by the neu oncogene, which can be used to detect the occurrence of the neu oncogene, are also described.
The protein coded for by the oncogene can be produced in significant quantities so that it can be studied to understand the metabolic alterations that occur in the cell during tumorigenesis. This may also lead to insights into methods by which one could antagonize or inhibit its functioning. In addition, it is expected to lead to sensitive tests for the presence of this protein which will be useful in the early detection of the onset of cancer.