Decreased H3K27 and H3K4 trimethylation on mortal chromosomes in distributed stem cells.

The role of immortal DNA strands that co-segregate during mitosis of asymmetrically self-renewing distributed stem cells (DSCs) is unknown. Previously, investigation of immortal DNA strand function and molecular mechanisms responsible for their nonrandom co-segregation was precluded by difficulty in identifying DSCs and immortal DNA strands. Here, we report the use of two technological innovations, selective DSC expansion and establishment of H2A.Z chromosomal asymmetry as a specific marker of 'immortal chromosomes,' to investigate molecular properties of immortal chromosomes and opposing 'mortal chromosomes' in cultured mouse hair follicle DSCs. Although detection of the respective suppressive and activating H3K27me3 and H3K4me3 epigenetic marks on immortal chromosomes was similar to randomly segregated chromosomes, detection of both was lower on mortal chromosomes destined for lineage-committed sister cells. This global epigenomic feature of nonrandom co-segregation may reveal a mechanism that maintains an epigenome-wide 'poised' transcription state, which preserves DSC identity, while simultaneously activating sister chromosomes for differentiation.

The importance of valid disclosures in the human embryonic stem cell research debate.

Misinformation erodes the legitimacy of any public debate. Since the start of human embryonic stem cell research deliberations in the USA, misinformation concerning the nature of human embryos, their availability for research, and the potential for using them to develop new medical therapies have been widespread and persistent. Basic facts, well understood by physicians and biologists, have been so misstated and misrepresented in the news media and political speeches that the general public has been put in a state of constant uncertainty. The solution to the present troubling condition is better education in the form of diligent, honest, and complete scientific disclosure by responsible scientists and physicians; and more care given to accurate reporting by news media. Several key aspects of newly emerging embryonic and non-embryonic stem cell technologies are defined and discussed as they relate to the debate over the use of human embryos for medical research. An important topic for consideration is how to disclose with clarity the scientific basis for human embryonic life. Thereafter, failings in proposed technologies for developing new therapies with human embryonic stem cells, that have been grossly under-reported, are examined. Finally, properties of adult stem cells are presented in contradistinction to embryonic stem cells, both in terms of adult stem cells as a scientifically better alternative to embryonic stem cells and in terms of the technological challenges that must be overcome to realize the potential of adult stem cells for new medical therapies.

Comparison of bax, waf1, and IMP dehydrogenase regulation in response to wild-type p53 expression under normal growth conditions.

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.

Inosine-5'-monophosphate dehydrogenase is a rate-determining factor for p53-dependent growth regulation.

We have proposed that reduced activity of inosine-5'-monophosphate dehydrogenase (IMPD; IMP:NAD oxidoreductase, EC 1.2.1.14), the rate-limiting enzyme for guanine nucleotide biosynthesis, in response to wild-type p53 expression, is essential for p53-dependent growth suppression. A gene transfer strategy was used to demonstrate that under physiological conditions constitutive IMPD expression prevents p53-dependent growth suppression. In these studies, expression of bax and waf1, genes implicated in p53-dependent growth suppression in response to DNA damage, remains elevated in response to p53. These findings indicate that under physiological conditions IMPD is a rate-determining factor for p53-dependent growth regulation. In addition, they suggest that the impd gene may be epistatic to bax and waf1 in growth suppression. Because of the role of IMPD in the production and balance of GTP and ATP, essential nucleotides for signal transduction, these results suggest that p53 controls cell division signals by regulating purine ribonucleotide metabolism.

Evidence for E-selectin complement regulatory domain mRNA splice variants in the rat.

The adhesion protein E-selectin is one mediator of endothelial cell-leukocyte interaction during acute inflammation. To investigate the molecular regulation of E-selectin function, we have examined the expression of E-selectin mRNA in target rat tissues after administration of lipopolysaccharide, a potent inducer of acute inflammation. In the course of these studies we isolated two unique rat E-selectin cDNA fragments. Both cDNA fragments show extensive nucleotide sequence homology to previously isolated mouse and human E-selectin cDNAs. However, they differ for the presence of sequences that encode complement regulatory domain-5 (CR5). Previous studies have shown that different animal species express E-selectin mRNAs that encode different numbers of CR domains. The isolation of these two rat E-selectin cDNA fragments, which differ only for the presence of CR5, represents the first direct evidence for the existence of E-selectin CR-variant mRNAs in the same species. Moreover, the sequence of the CR5(-) cDNA is consistent with its origin from an mRNA splice variant of a CR5(+) mRNA. We have demonstrated the presence of the two predicted mRNA species in rat heart tissue and have investigated their expression in response to lipopolysaccharide. Although both mRNA variants were greatly induced by lipopolysaccharide, the CR5(-) form was more abundant in both treated and control tissues. This difference in mRNA abundance may indicate different levels of CR5 variant proteins that perform functionally distinct tasks in E-selectin dependent inflammatory processes.

Expression of E-selectin mRNA during ischemia/reperfusion injury.

To better understand molecular and cellular processes involved in tissue inflammation, we have examined expression of endothelial leukocyte adhesion molecule 1 (E-selectin) mRNA in adult male rats after ischemia/reperfusion (I/R) injury and after intravenous injection of lipopolysaccharide (LPS). The polymerase chain reaction was used to generate a rat E-selectin cDNA fragment by using heart total RNA from rats exposed to LPS. This partial cDNA fragment spanned sequences from complement repeat region-5 to the second cytoplasmic tail domain. Comparison of the predicted amino acid sequence from the rat cDNA fragment to mouse and human E-selectin protein sequences showed significant conservation. The rat E-selectin cDNA fragment was used as a probe to examine the regulation of E-selectin mRNA expression by Northern blot analysis. As previously described in other animal species, E-selectin mRNA expression was induced after intravenous injection of LPS. In contrast, ischemia did not induce E-selectin mRNA expression, except in the setting of I/R injury. I/R injury triggered expression of E-selectin mRNA in the kidney. These experiments represent a first in vivo examination of E-selectin mRNA expression after I/R injury and constitute an initial step in characterizing a model system for investigating inflammation in the setting of acute ischemic injury.

A quantitative method for the analysis of mammalian cell proliferation in culture in terms of dividing and non-dividing cells.

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.

Expression of the wild-type p53 antioncogene induces guanine nucleotide-dependent stem cell division kinetics.

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.

Inosine-5'-monophosphate dehydrogenase activity is maintained in immortalized murine cells growth-arrested by serum deprivation.

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.

Guanine nucleotide biosynthesis is regulated by the cellular p53 concentration.

As an approach to defining the role of p53 in cellular proliferation, murine cell lines were derived which contain a stably transfected temperature-inducible p53 expression system. Cell lines derived with the system exhibited a 3-6-fold physiologic elevation in the cellular p53 concentration when grown at 32.5 degrees C. A p53 induction phenotype was defined by examination of the growth properties of these lines at 32.5 degrees C. The induction phenotype had three main features: 1) a 2-4-fold increase in doubling time and biphasic growth kinetics; 2) delayed early S phase transit; and 3) complete reversibility either by growth at 37 degrees C or by growth in the presence of added hypoxanthine or xanthosine. The reversal of the induction phenotype by these purine salvage precursors implicated the purine nucleotide biosynthetic pathway as the cellular target for the antiproliferative action of p53. Subsequent genetic and biochemical analyses identified a p53 induction-related purine pathway defect which was localized to the step of inosine 5'-monophosphate conversion to xanthosine 5'-monophosphate. This enzymatic step catalyzed by inosine 5'-monophosphate dehydrogenase (EC 1.2.1.14) is the rate-limiting step for GTP synthesis. Extracts from p53-inducible cells growing at the induction temperature show a specific reduction in inosine 5'-monophosphate dehydrogenase enzymatic activity. These findings define p53 as a cellular regulator of the synthesis of GTP, a key regulatory nucleotide for many important cellular processes. Moreover, observations of the growth behavior of p53-inducible cells suggest that by regulating the production of GTP, p53 can control cellular quiescence.

Regulation of human thymidine kinase during the cell cycle.

As an approach to defining the molecular basis for the periodic expression of thymidine kinase activity during the cell cycle, we have examined properties of the cytosolic enzyme in cycling HeLa cells synchronized by centrifugal elutriation and mitotic selection. By immunoblot analyses with a specific antiserum raised against the purified HeLa enzyme, we have demonstrated that changes in the levels of thymidine kinase activity reflect similar changes in the levels of thymidine kinase polypeptide. In contrast, the steady state levels of thymidine kinase mRNA show relatively small changes during the cell cycle. Using pulse labeling methods, we have shown that the synthetic rate of thymidine kinase protein is about 10-fold greater in S phase than in G1 phase, indicating that the efficiency of translation of thymidine kinase mRNA increases as cells begin DNA replication. In addition, the stability of thymidine kinase protein dramatically decreases upon cell division, resulting in the rapid clearance of the enzyme from newly divided G1 cells. Thus, two different post-transcriptional mechanisms largely account for the periodic behavior of the enzyme activity during the cell cycle.

Human cytosolic thymidine kinase. Purification and physical characterization of the enzyme from HeLa cells.

The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, we have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic Rm of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of Mr = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity. 1) The Mr = 24,000 polypeptide co-migrates with thymidine kinase activity in electrophoretic and sedimentation analyses. 2) A subunit Mr = 25,504 is predicted by the nucleotide sequence of a recently isolated cDNA clone that encodes HeLa thymidine kinase. 3) Mouse LTK- cells transformed with this clone express a cytosolic thymidine kinase activity, as well as a novel Mr = 24,000 polypeptide detectable with immune serum raised against the purified human enzyme.