Half a century ago, epidemiologists proposed that cancers result from multiple 'hits' (see Milestone 9). Initially, the focus was on dominantly acting viral oncogenes and activating mutations in the RAS oncogene. Later, cell fusion and genetic experiments showed that recessive mutations cause defects in tumour suppression (see Milestone 11). Bert Vogelstein reconciled the oncogene and tumour-suppressor camps by describing how both events are necessary for colorectal carcinogenesis (see Milestone 14). Arnold Levine, David Lane and colleagues discovered the first tumour-suppressor gene, p53 (also known as TP3), although it was initially described as an oncogene. Levine showed that p53 suppresses transformation, while Vogelstein reported that both p53 alleles are mutated in colorectal cancer, a finding subsequently extended to most common human tumour types, with over 20,000 p53 mutations now on record. The second tumour suppressor to emerge was the retinoblastoma protein RB (see Milestones in Cell Division Milestone 15). Both RB and p53 have been on the citation bestseller lists ever since it became apparent that the main DNA tumour viruses transform cells by inactivating both Rb and p53. The RB pathway is now firmly enshrined in cell-cycle regulation, and defects in this pathway are a universal feature of cancer.

In 1989, David Livingston and Ed Harlow published an early milestone: they found that RB is phosphorylated in a cell cycle-dependent manner, as synchronized primary and immortalized cells enter the DNA-replication phase (S phase). They reported, separately, that SV40 T antigen, which can drive G1-arrested cells into the cycle, only binds unphosphorylated RB — the first indication that this is the growth-suppressive form of RB. Therefore, they surmised that unphosphorylated RB acts to block exit from G1.

p53 has emerged as a crucial guardian of the genome, and several exceptional papers first described its role in the DNA damage-checkpoint response. It was known that both p53 and DNA damage inhibit DNA replication, and cause G1 cell-cycle arrest. Michael Kastan and colleagues connected these findings in haematopoietic cells by showing that the G1-checkpoint arrest correlates with p53 protein induction, and that this response is sensitive to caffeine — later shown to block ATM kinase — and cycloheximide. Importantly, cells with mutant or no p53 did not arrest in G1 after γ-irradiation (IR), while maintaining a second checkpoint arrest in G2. In a second paper, Kastan generalized these findings and showed that re-expression of p53 in p53-null cells rescued the IR-induced G1-checkpoint arrest. Conversely, a p53 mutant was able to abrogate the G1 checkpoint in p53 wild-type cells in a dominant–negative fashion. A third paper by Kastan placed p53 in a checkpoint-signalling pathway; he noted that cells from ataxia telangiectasia (AT) patients also lacked the G1 DNA-damage checkpoint and, proposing that the defects in AT and p53 are functionally linked, he documented a decreased p53 induction in AT cells after IR. Importantly, this paper used primary embryonic fibroblasts from p53-null mice, rather than transformed cell lines. Just previously, p53 had been shown to be a sequence-specific DNA-binding protein capable of transcriptional activation. Furthermore, it was known that the radiation sensitive GADD45 gene was not induced in AT and several tumour cell lines. Kastan showed that GADD45 induction requires p53, and that wild-type p53 bound to a p53 consensus site in the gene promoter. Therefore, this paper not only uncovered upstream and downstream events in the p53-dependent DNA damage-signalling pathway, but also described one of the first p53 target genes. The importance of these papers is threefold: they explain how the cell cycle is arrested after DNA damage, and how p53 loss might contribute to genetic instability and tumour formation, and they show that DNA damage elicits a signal-transduction response involving the gene mutated in AT (now known to be the ATM kinase), p53 and p53 target genes.

By the mid-1990s, it became clear that apoptosis was a key tumour-suppressive pathway (see Milestone 12), and that p53 induces apoptosis and is required for DNA damage and oncogene-induced apoptosis. To investigate the role of p53-dependent apoptosis in brain tumour progression, Holly Symonds and colleagues used transgenic mice expressing a SV40 T-antigen mutant that inhibits RB, but not p53. Tumour growth relative to wild-type T antigen slowed in p53-wild type, but not in p53-null, mice; p53-heterozygous mice exhibited stochastic emergence of p53-null tumours, and this correlated with decreased apoptosis. At the same time, Sharon Morgenbesser and colleagues reported increased proliferation and apoptosis in the developing ocular lens of RB-null mice; apoptosis was suppressed in RB/p53 double-null mice, indicating p53 dependence. These papers, together, are the first to describe that inappropriate S-phase entry owing to loss of RB results in p53-dependent apoptosis, thereby linking the two central tumour-suppressor pathways in the cell.

These studies represent only a couple of the milestones in our understanding of RB and p53, and their role in cell-cycle and DNA-damage checkpoints, which have dominated cancer research for the past decade.