In the early 1980s, evidence indicated that the oncogenic transformation of primary cells involved at least two stages: establishment (the immortalization of cells) and cellular transformation. With this in mind, Hartmut Land, Luis F. Parada and Robert Weinberg, working at the same time as Earl Ruley, investigated how oncogenes cooperate to induce tumour development.
Weinberg and colleagues examined the effect of expressing two recently identified oncogenes — an activated variant of Harvey RAS1, EJ RAS (see Milestone 17), and either a viral or a mammalian clone of Myc — in primary rat embryonic fibroblasts (REFs). They found that, despite its capacity to transform rodent cell lines, EJ RAS could not transform REFs. These cells initially proliferated, but then underwent crisis and arrest. EJ RAS-expressing REFs were also unable to form tumours in immunocompromised mice. However, REFs expressing Myc and RAS grew rapidly as foci that were able to establish long-term cultures when passaged in vitro, and could form tumours in mice. These tumours were not metastatic, implying that beyond MYC and RAS cooperation, further oncogenic events might be required to produce an invasive tumour (although we now know that tumours seeded subcutaneously in mice often do not metastasize). Similar results were found by Ruley using adenoviral E1A, polyoma virus middle T antigen and T24 Harvey RAS expressed in baby rat kidney cells.
Oncogene cooperation was also studied in avian erythroid progenitor cells by Thomas Graf and colleagues. In 1986, they showed that the non-transforming viral gene v-erbA could cooperate both in vitro and in vivo with v-src, v-Ha-ras and v-erbB
The ability of different oncogenes to cooperate in producing cellular transformation has been a cornerstone of cancer research during the intervening years. With the development of further molecular techniques, it has become possible to piece together how and why specific oncogenes cooperate so effectively.
Land and colleagues went on to show that high expression levels of RAS, like those used in the experiments described above, induce G1 arrest in primary cells owing to the expression of the cell-cycle inhibitor p21. By contrast, expression of MYC was found to induce both proliferation and apoptosis (see Milestone 12). MYC and RAS cooperate because MYC can act in numerous ways to circumvent the RAS-induced G1 growth arrest, and RAS prevents MYC-induced apoptosis by activation of the anti-apoptotic kinase AKT.
B-cell lymphoma 2 (BCL2) and MYC also cooperate effectively (see Milestone 12). BCL2 is an unusual oncogene product in that, unlike RAS, it cannot transform rodent cell lines and, unlike MYC, it does not induce proliferation. Nevertheless, in 1990, Andreas Strasser and colleagues showed that BCL2 synergizes with MYC to rapidly produce tumours in mice. This is because, as shown later by several groups, MYC-induced apoptosis is suppressed by the expression of BCL2, leaving the proliferative capacity of MYC unchecked.
Identification of the growth-restrictive aspects of oncogene activation, such as cell-cycle arrest and apoptosis, allowed cancer biologists to appreciate the complexities of the molecular cross-talk in tumour cells, and to begin to understand the pathways that have evolved to limit cellular transformation (see Milestone 20).
