The existence and molecular basis of an amino acid sensitive cell cycle control mechanism in human cells is described for the first time. Withdrawal of a single amino acid (arginine) from normal human fibroblast cultures caused a rapid cessation of proliferation characterised with a loss of accumulation of cells with G1 DNA content, consistent with a loss of cyclin D1-associated kinase activity and the predominance of hypo-phosphorylated pRb. Restoration of amino acid caused a synchronous reentry to cycle after a delay in excess of that for M to S transit in freely cycling populations, indicating exit from a quiescent-like state. The cellular response was thus consistent with Pardee's concept of a pivotal cell cycle control mechanism in G1, sensitive to extracellular conditions (<I>ie</I> the R-point) (Pardee, 1974). Inhibition of expression of the pRb phosphorylating kinase, cdk4, was identified as the key regulatory element in the response. A hypothetical cellular communication pathway coupling amino acid shortage to translational suppression of cdk4 (the '-Arg/cdk4 response pathway') has been synthesised from the known biochemical effects of deprivation and the recognised determinants of this suppression, including a 5'UTR mediated wild-type p53 dependency. A strategy for analysis and interrogation of this translational control mechanism, based upon the synthesis of epitope-tagged protein from full length or 5'URT truncated cdk4 cDNAs, and attempts to confirm the primacy of cdk4 suppression to the antiproliferative response by its enforced expression, are described. A highly deranged human tumour cell line (HeLa) was found to be deficient in amino acid sensitive cell cycle control. These cells continued in cycle after withdrawal but this was accompanied by a rapid loss of viability and cell disintegration. Simultaneous cell cycle blocks conferred partial protection from arginine deprivation induced cell death. The possibility that inappropriate cell cycle progression was the cause of cell death is discussed. Not all human tumour lines were vulnerable to arginine deprivation. This responsivity was found to be predicted by the status of the functional determinants identified or inferred (<I>ie</I> wild-type pRb, with cdk4 as the predominant phosphorylating kinase, intact'-Arg'cdk4 response pathway). This work describes a novel cellular response mechanism, complementing recent similar findings from elsewhere, to connect cellular biosynthetic capacity with control of cell cycle progression, with significance to the maintenance of normal cell growth regulation and suppression of the malignant phenotype, and providing a broader understanding of 'physiological' cell cycle control.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:245976 |
| Date | January 1998 |
| Creators | Lamb, Justin |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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