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The retinoblastoma tumour suppressor protein (Rb) has come a long way since its initial discovery in 1986. Encoded by the first candidate tumour suppressor gene it has emerged a versatile and context-dependent modulator of cell behaviour. Its activity is managed by signalling networks sensing intra- and extracellular cues. These cues are relayed to hold or permit inactivation of Rb by phosphorylation. Loss or mutation of the retinoblastoma gene is rare in sporadic cancers but defects in the pathways that license inactivation of Rb are found in the majority of them, suggesting that loss of Rb control is central to tumour development and arguing that its reinstatement might reverse tumour formation. Furthermore, mouse models with engineered defects in the Rb-phosphorylating kinases provide evidence that moderation of Rb inactivation may be a strategy for the prevention of tumour formation. The rationale behind these arguments, their underlying molecular concepts and strategies towards therapeutic application will be discussed.

The tumor suppressor protein, pRb, regulates progression through the G1 phase of the cell cycle by its ability to bind to and regulate the activity of a variety of transcription factors. This function of pRb is disabled through its phosphorylation by the cyclin-dependent kinase (CDK) family of serine/threonine kinases. In many human cancers, genetic alteration such as loss of CDK inhibitor function and deregulated G1 cyclin expression leads to inappropriate phosphorylation and hence inactivation of this tumor suppressor. Identification of cell-permeable small molecules that block pRb phosphorylation in these tumors could therefore lead to development of an effective anticancer treatment. As a result, we have developed a high-throughput assay to detect changes in the level of pRb phosphorylation in cells. Signal detection is by a time-resolved fluorescence-based cellular immunosorbant assay on a fixed monolayer of cells. This comprises a mouse monoclonal antibody that recognizes the phosphorylated form of serine 608 on pRb, a known site of CDK phosphorylation, and a Europium-labeled secondary antibody for signal detection. The assay is reproducible and amenable to automation and has been used to screen 2000 compounds in a search for cell-permeable small molecules that will block pRb phosphorylation.

Two opposing enzymatic reactions control the activity of the retinoblastoma tumour suppressor protein, pRB. Phosphorylation inactivates pRB's ability to sequester miscellaneous cellular proteins, mostly involved in regulating gene transcription, whereas pRB dephosphorylation restores this ability. For some time now it has been suspected that members of the cyclin/cyclin-dependent kinase (cyclin/cdk) family mediate pRB inactivation. Recent results indicate that pRB phosphorylation is not executed by single kinase but by a combination of cyclin/cdks, each one phosphorylating a subset of pRB's phosphorylation sites. The different kinases appear to be activated by growth factors through distinct signal transduction pathways. This lends itself to an attractive model whereby pRB phosphorylation may constitute an integration point for these signalling pathways, perhaps allowing cell cycle progression only when concurrent activation of these signalling pathways has been achieved.

Cells made ischemic rapidly manifest many distinct structural and functional alterations as a consequence of the depletion of their energy stores. In attempting to determine which of these are causally related to the eventual cell death, the authors have emphasized the relationship to the reversibility of the ischemic injury. Two phenomena have consistently characterized irreversibly in contrast to reversibly injured ischemic cells: the inability to restore mitochondrial function and evidence of plasma membrane damage. Studies in the authors' laboratory are reviewed that have focused on the pathogenesis, biochemical nature, and the relationship to irreversible cell injury of both of these alterations. A number of mitochondrial abnormalities are related to changes in long-chain acyl-CoA metabolism with inhibition of adenine nucleotide translocation and potentiation of a Ca2+-dependent increase in the permeability of the inner mitochondrial membrane. These changes are reversible upon reoxygenation only when the large increase in intracellular Ca2+ content that accompanies the phospholipid depletion from other cellular membranes is prevented. This disorder in phospholipid metabolism is felt to be the critical lesion that produces irreversible cell injury in ischemia. It affects the endoplasmic and sarcoplasmic reticular membranes of liver and myocardial cells, respectively, and probably the plasma membranes of both. It is prevented by pretreatment with chlorpromazine. An activation of endogenous phospholipases by an elevated, cytosolic free Ca2+ ion concentration is suggested as the mechanism underlying this phospholipid disturbance. The central role of intracellular Ca2+ in the initiation and functional consequences of ischemic cell injury are emphasized.