Chapter 3: Cancer as an infectious disease
3.1: Peyton Rous discovers a chicken sarcoma virus
Cancer was considered a candidate infectious disease.
Peyton Rous began his study of a sarcoma that had appeared in the breast muscle of a hen.
He ground up a sarcoma fragment in sand and filtered the resulting homogenate under conditions in which only subcellular particles could pass through the filter.
When he injected the resulting filtrate into young birds, they too developed tumors, sometimes within several weeks.
He subsequently found that these induced tumors could also be homogenized to yield an infectious agent that could be transmitted, following filtration, to yet other birds, which also developed sarcomas at the sites of injection.
The carcinogenic agent was a virus that could cause the development of a sarcoma in an injected chicken, doing so on a predictable timetable.
This came to be called the Rous sarcoma virus.
3.2: Rous sarcoma virus is discovered to transform infected cells in culture
A group of researchers found that when stocks of RSV were introduced to petri dishes carrying cultures of chicken embryo fibroblasts, the RSV infected cells survived, apparently indefinitely.
It seemed that RSV parasitized these cells, forcing them to produce a steady stream of progeny virus particles for many days, weeks, even months.
Most importantly, the RSV infected cells in these cultures displayed many of the traits associated with cancer cells.
Normal cells form confluent cultures
When first introduced into a petri dish, cells formed islands scattered across the bottom, and the proliferated to fill up all the space in the bottom of the dish.
Once they reached the confluence, however, these cells stopped proliferating, resulting in a one-cell-thick layer of cells.
The cessation of growth of these normal cells after forming confluent monolayers was the result of a process that came to be contact inhibition.
RSV-infected foci lost contact inhibition
These cells continued to proliferate, piling on top of one another and creating multi layer cell foci
3.3: The continued presence of RSV is needed to maintain transformation
The descendants of an RSV-infected cell continued to harbor copies of the RSV genome.
A mutant of RSV was developed that was capable of transforming chicken cells when these cells were cultured at 37 degrees but not at 41 degrees.
Temperature sensitive mutants like this were known to encode partially defective proteins, which retain their normal structure and function at one temperature and lose their function at another temperature.
After the chicken embryo fibroblasts were infected with the mutant of RSV, these cells became transformed if they were subsequently cultured at the lower temperature.
These cells could be propagated for many cell generations at this lower temperature and continued to grow and divide just like cancer cells.
But, weeks later, if the temperature of these infected cultures was raised to 41 degrees, these cells lost their transformed shape and quickly reverted to the shape and growth pattern of cells that never had experienced the infection.
If these cells were again returned to the permissive temperature, they regained their transformed appearance.
Since the cells that descended from a temperature sensitive RSV infected cell continued to show temperature sensitive growth trait, it was obvious that the copies of the genome of the infecting virus persisted in these cells for weeks after the initial infection.
Most importantly, the continuing actions of some temperature sensitive protein were required in order to maintain the transformed growth phenotype of the RSV infected cells.
The viral transforming gene was required to both initiate and maintain the transformed phenotype.
3.7: Retroviral genomes become integrated into the chromosomes of infected cells
The genome of RSV is made of single stranded RNA, which clearly cannot be integrated directly into the chromosomal DNA of an infected cell.
Still, RSV succeeded in transmitting its genetic information through many successive cycles of cell growth and division.
It was hypothesized that after RSV particles infected a cell, they made double stranded DNA (dsDNA) copies of their RNA genomes.
It was these dsDNA versions of the viral genome that became established in the chromosomal DNA of the host cell.
Once established, the DNA version of the viral genome (called a provirus) then assumed the molecular configuration of a cellular gene and would be replicated each time the cell replicated its chromosomal DNA.
In addition, the proviral DNA could them serve as a template for transcription by cellular RNA polymerase, thereby yielding RNA molecules that could be incorporated into progeny virus particles, or alternatively, could function as mRNA that was used for the synthesis of viral proteins.
This was supported by the discovery that RSV and related virus particles carried reverse transcriptase.
3.8: A version of the Src gene carried by RSV is also present in uninfected cells
Geneticists speculated that all of the viral transforming functions of RSV resided in a single gene, which they termed Src, to indicate its role in triggering the formation of sarcomas in infected chickens.
In 1975, a research group using an Src-specific probe found that Src sequences were clearly present among the DNA sequences of uninfected chicken cells.
3.9: RSV exploits a kidnapped cellular gene to transform cells
The presence of a highly conserved Src gene in the genome of a normal organism implied that this cellular version of Src, sometimes termed c-Src (cellular Src), played some role in the life of regular cells.
This viral transforming gene (v-Src) was closely related to the c-Src, yet the two genes had drastically different effects and apparent functions.
The v-Src gene borne by the RSV genome acted as a potent oncogene—a gene capable, following its introduction into a cell by an infecting virus particle, of transforming the previously normal cell into a tumor cell.
One solution to this puzzle came from considering the possibility that perhaps the Src gene of RSV was not naturally present in the retrovirus ancestral to RSV.
This suggested that during the course of infecting a chicken cell, an ancestral virus somehow acquired sequences from the genome of a cell that it had infected.
Once present in the genome of RSV, the kidnapped Src gene could then be altered and exploited by this virus to transform and subsequently infect cells.
C-Src is a precursor to an active oncogene, called a proto-oncogene.
Genomes of normal vertebrate cells carry a gene that has the potential, under certain circumstances, to induce cell transformation and thus cancer.