Chapter 4: Cellular Oncogenes
4.1: Transfection of DNA provides a strategy for detecting non-viral oncogenes
In the 1970s, scientists speculated that chemically transformed cells carried mutated cellular genes and that these genes were responsible for programming the aberrant growth of these cells.
This was a daunting task to try to prove experimentally, though, because if cancer-causing genes were really present in the genomes of chemically transformed cells, how could they be found?
If these genes were mutant versions of normal cellular genes, then they were embedded in cancer cell genomes together with hundreds of thousands of other genes, each present in at least one copy per cell genome.
To determine whether non-viral oncogenes existed in chemically transformed cells, a novel experimental strategy was devised.
It involved introducing DNA of cancer cells into normal cells, and then determining whether the recipient cells became transformed in response to the introduced tumor cell DNA.
In 1972, a highly effective gene transfer procedure termed transfection was developed, making it possible to introduce naked DNA molecules from donor cells directly into mammalian cells serving as recipients.
Researchers chose cells that had been treated repeatedly with the potent carcinogen 3-MC, a known component of coal tars.
Importantly, these transformed cells bore no traces of either tumor virus infection or activated endogenous retroviral genes.
Hence, any transforming oncogenes detected in the genome of these donor cells would, with great likelihood, be of cellular origin.
In 1978-1979, the cells plucked from the resulting foci were found to be both anchorage-independent and tumorigenic. This proved that the donor tumor DNA carried one or several genetic elements that were able to convert a non-tumorigenic recipient cell into a cell that was strongly tumorigenic.
At first, it seemed difficult to determine whether the tumor cells carried a single oncogene in their genomes or several distinct oncogenes that acted in concert to transform the recipient cells.
However, they discovered that only about 0.1% of a cell genome’s worth of donor DNA became established in the genome of each transfected recipient cell.
The probability of two independent, genetically unlinked donor genes both being introduced into a single recipient cell was highly unlikely.
Scientists could infer that only a single gene was responsible for the transformation of the recipient cells following the transfection.
These transfection experiments proved strong indication that oncogenes can arise in the genomes of cells through mechanisms that have no apparent connection with viral infection.
Most importantly, this work demonstrated directly that chemically transformed cells as well as certain human tumor cells contained mutant genes that were capable of driving such cells into a neoplastic growth state.
4.2: Oncogenes discovered in human tumor cell lines are related to those carried by transforming retroviruses
The oncogenes detected by transfection in the genomes of various human tumor cells were ostensibly derived from preexisting normal cellular genes that lacked oncogenic function.
This seemed parallel to the process that led to the appearance of transforming retroviruses, where preexisting normal cellular genes (proto-oncogenes) became associated with viral genomes and were activated into potent oncogenes.
Could the same group of cellular proto-oncogenes become activated into oncogenes by marauting retroviruses in one context and by non-viral mutagnes in another?
Or, did the retrovirus-associated oncogenes and those activated by non-viral mechanisms arise from two very distinct groups of cellular proto-oncogenes?
Scientists discovered that the retrovirus-associated oncogenes were present in increased copy number in human tumor cell genomes.
The Myc oncogene was found to be present in multiple copies in the human promyelocytic leukemia cell line due to gene amplification, which favored proliferation of cancer cells.
The erbB gene was discovered to be present in an increased copy number in the DNAs of human stomach, breast, and brain tumor cells. Elevated expression of the homolog of the erbB gene is now thought to be present in the majority of human carcinomas.
Amplification of the erbB-related gene known as erbB2/HER2 was reported in many breast cancers.
Patients whose tumors expressed elevated levels of its encoded protein had a median survival of only three years. This provided a strong indication that this gene, in amplified form, was causally involved in driving the malignant growth of the breast cancer cells.
The lesson taught by these numerous cross connections was simple and clear:
Many of the oncogenes originally discovered through their association with avian and mammalian retroviruses could be found in a mutated, activated state in human tumor cell genomes.
This meant that a common set of cellular proto-oncogenes might be activated either by retroviruses (in animas) or, alternatively, by non-viral mutational mechanisms operating during the formation of human cancers.
4.3: Proto-oncogenes can be activated by genetic changes affecting either protein expression level or structure
A number of proto-oncogenes were found in activated, oncogenic form in human tumor genomes. The precise genetic alterations that led to many of these activations remained unclear.
With retrovirus-associated oncogenes, one mechanism became clear once the organization of the transforming retrovirus genomes was known.
After insertion into the retrovirus genome, the expression of this captured gene was now controlled by a retroviral transcriptional promoter instead of the regular promoter.
This drove the gene’s expression unceasingly and at high levels, rather than at the regular levels. Transcription of this virus associated gene, now an oncogene, was therefore no longer responsive to the cellular signals that had previously regulated its expression.
It was difficult to find what converted a normal human Ras proto-oncogene to the potent oncogene that was detected by transfection of human bladder carcinoma.
It was found that a single point mutation at glycine 12. Ras is a GTPase that is inactive when bound to GDP. It is active when bound to GTP, and then a GAP will hydrolyze the GTP to GDP to inactivate it again.
From this point mutation, Ras can bind to GTP, but the replaced amino acid causes steric interference at the protein active site. This means that GAP cannot hydrolyze GTP anymore, leaving Ras constitutively active.
A large number of human tumors were found that carried point mutations in one of the three Ras genes present in the mammalian genomes.