ABSTRACT
Recently, we demonstrated that downregulation of inosine-5'-monophosphate dehydrogenase (IMPD; IMP:NAD oxidoreductase, EC 1.2.1.14), the rate-limiting enzyme for guanine nucleotide biosynthesis, is required for p53-dependent growth suppression. These studies were performed with cell lines derived from immortal, nontumorigenic fibroblasts that express wild-type p53 conditionally by virtue of a metal-responsive promoter. Here, the p53-dependent properties of the original "p53-inducible" fibroblasts are presented in detail and compared to related properties of epithelial cells that also express wild-type p53 conditionally, but by virtue of a temperature-responsive promoter. Both types of p53-inducible cells were designed to approximate normal physiologic relationships between the host cell and the regulated p53 protein. Together, they were used to investigate expression relationships between IMPD and other p53-responsive genes proposed as mediators of p53-dependent growth suppression. In both types of cells, IMPD activity, protein, and mRNA were consistently coordinately reduced in response to p53 expression. In contrast, mRNAs for waf1, bax, and mdm2 showed disparate patterns of expression, being induced in one conditional cell type, but not the other. This distinction in regulation pattern suggests that under normal growth conditions, unlike IMPD downregulation, bax and waf1 induction is not a rate-determining event for p53-dependent growth suppression.
Subject(s)
Cyclins/metabolism , IMP Dehydrogenase/metabolism , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/biosynthesis , Animals , Cell Division , Cell Line , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Enzyme Activation , Fibroblasts , Gene Expression Regulation , IMP Dehydrogenase/genetics , Mice , Proto-Oncogene Proteins/genetics , RNA, Messenger/antagonists & inhibitors , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/physiology , Zinc/physiology , bcl-2-Associated X ProteinABSTRACT
The application of the exponential growth equation is the standard method employed in the quantitative analyses of mammalian cell proliferation in culture. This method is based on the implicit assumption that, within a cell population under study, all division events give rise to daughter cells that always divide. When a cell population does not adhere to this assumption, use of the exponential growth equation leads to errors in the determination of both population doubling time and cell generation time. We have derived a more general growth equation that defines cell growth in terms of the dividing fraction of daughter cells. This equation can account for population growth kinetics that derive from the generation of both dividing and non-dividing cells. As such, it provides a sensitive method for detecting non-exponential division dynamics. In addition, this equation can be used to determine when it is appropriate to use the standard exponential growth equation for the estimation of doubling time and generation time.
Subject(s)
Cell Division/physiology , Models, Biological , Cell Count , Cells, Cultured , Humans , Tumor Suppressor Protein p53/biosynthesis , Tumor Suppressor Protein p53/geneticsABSTRACT
The predominant type of cell division in adult mammals is renewal growth. Renewing stem cells in somatic tissues undergo continuous asymmetric divisions. One new daughter cell retains the division potential of the original stem cell, while the other differentiates into a functional constituent of the tissue. Disruptions of this process lead to the development of human cancers. We show that through a guanine nucleotide-dependent mechanism, the p53 antioncogene can induce exponentially dividing cells to switch to an asymmetric stem cell growth pattern. This finding suggests that the observed high frequency of p53 mutations in human cancers reflects a critical function in the regulation of somatic renewal growth.
Subject(s)
Gene Expression , Genes, p53 , Guanine Nucleotides/physiology , Stem Cells/cytology , Tumor Suppressor Protein p53/biosynthesis , Animals , Cell Cycle , Cell Division/physiology , Cell Line , Humans , Kinetics , Mammals , Neoplasms/genetics , Neoplasms/pathology , Regression Analysis , Stem Cells/metabolism , Time FactorsABSTRACT
We have examined properties of IMPD activity in soluble extracts from immortalized murine epithelial and fibroblastic cells. The absence of significant xanthine oxidase activity in these extracts allowed the use of a spectrophotometric assay to study the enzyme activity. The observed enzymatic activity had subcellular localization and kinetic properties similar to those of previously described mammalian IMPD from other sources. Analysis of IMPD activity in extracts from cells in different states of growth related to serum concentration gave a surprising result. Extracts from exponentially growing cells exhibited a level of IMPD activity similar to that of extracts from quiescent cells arrested by serum-deprivation. In previous studies, the cellular variable designated to account for changes in IMPD activity was proliferative rate. Our findings suggest that either proliferative rate is not the functionally significant variable related to IMPD regulation or that there are other factors that can supersede it in certain contexts. Given the role of the enzyme in regulating the synthesis of guanine nucleotides, which are key regulatory molecules for many cellular processes, this may indeed be the case. Using immortalized cell lines growth-arrested by serum deprivation, we have experimentally isolated the enzyme activity from the previously assigned variable of growth rate. Based on our findings we propose that regulation of IMPD activity is more appropriately related to proliferative capacity as opposed to proliferative rate.
