Maarten H.K. Linskens

Human Neural Precursor Cells Express Low Levels of Telomerase in Vitro and Show Diminishing Cell Proliferation with Extensive Axonal Outgrowth following Transplantation

Worldwideattention is presently focused on proliferating populations of neural precursor cells as an in vitro source of tissue for neural transplantation and brain repair. However, successful neuroreconstruction is contingent upon their capacity to integrate within the host CNS and the absence of tumorigenesis. Here we show that human neural precursor cells express very low levels of telomerase at early passages (less than 20 population doublings), but that this decreases to undetectable levels at later passages. In contrast, rodent neural precursors express high levels of telomerase at both early and late passages. The human neural precursors also have telomeres (approximately 12 kbp) significantly shorter than those of their rodent counterparts (approximately 40 kbp). Human neural precursors were then expanded 100-fold prior to intrastriatal transplantation in a rodent model of Parkinsons disease. To establish the effects of implanted cell number on survival and integration, precursors were transplanted at either 200,000, 1 million, or 2 million cells per animal. Interestingly, the smaller transplants were more likely to extend neuronal fibers and less likely to provoke immune rejection than the largest transplants in this xenograft model. Cellular proliferation continued immediately post-transplantation, but by 20 weeks there were virtually no dividing cells within any of the grafts. In contrast, fiber outgrowth increased gradually over time and often occupied the entire striatum at 20 weeks postgrafting. Transient expression of tyrosine hydroxylase-positive cells within the grafts was found in some animals, but this was not sustained at 20 weeks and had no functional effects. For Parkinsons disease, the principal aim now is to induce the dopaminergic phenotype in these cells prior to transplantation. However, given the relative safety profile for these human cells and their capacity to extend fibers into the adult rodent brain, they may provide the ideal basis for the repair of other lesions of the CNS where extensive axonal outgrowth is required.

p34Cdc28-mediated control of Cln3 cyclin degradation.

Cln3 cyclin of the budding yeast Saccharomyces cerevisiae is a key regulator of Start, a cell cycle event in G1 phase at which cells become committed to division. The time of Start is sensitive to Cln3 levels, which in turn depend on the balance between synthesis and rapid degradation. Here we report that the breakdown of Cln3 is ubiquitin dependent and involves the ubiquitin-conjugating enzyme Cdc34 (Ubc3). The C-terminal tail of Cln3 functions as a transferable signal for degradation. Sequences important for Cln3 degradation are spread throughout the tail and consist largely of PEST elements, which have been previously suggested to target certain proteins for rapid turnover. The Cln3 tail also appears to contain multiple phosphorylation sites, and both phosphorylation and degradation of Cln3 are deficient in a cdc28ts mutant at the nonpermissive temperature. A point mutation at Ser-468, which lies within a Cdc28 kinase consensus site, causes approximately fivefold stabilization of a Cln3-beta-galactosidase fusion protein that contains a portion of the Cln3 tail and strongly reduces the phosphorylation of this protein. These data indicate that the degradation of Cln3 involves CDC28-dependent phosphorylation events.

Organization of Replication of Ribosomal DNA in Saccharomyces cerevisiae

Using recently developed replicon mapping techniques, we have analyzed the replication of the ribosomal DNA in Saccharomyces cerevisiae. The results show that (i) the functional origin of replication colocalizes with an autonomously replicating sequence element previously mapped to the nontranscribed spacer region, (ii) only a fraction of the potential origins are utilized in a single S phase, and (iii) the replication forks moving counter to the direction of transcription of the 37S precursor RNA stop at or near the termination site of transcription. Consequently, most ribosomal DNA is replicated unidirectionally by forks moving in the direction of transcription and most replicons are larger than the repeat unit. The significance of this finding for the replication of abundantly transcribed genes is discussed.

Telomere shortening and survival in free-living corvids

Evidence accumulates that telomere shortening reflects lifestyle and predicts remaining lifespan, but little is known of telomere dynamics and their relation to survival under natural conditions. We present longitudinal telomere data in free-living jackdaws (Corvus monedula) and test hypotheses on telomere shortening and survival. Telomeres in erythrocytes were measured using pulsed-field gel electrophoresis. Telomere shortening rates within individuals were twice as high as the population level slope, demonstrating that individuals with short telomeres are less likely to survive. Further analysis showed that shortening rate in particular predicted survival, because telomere shortening was much accelerated during a birds last year in the colony. Telomere shortening was also faster early in life, even after growth was completed. It was previously shown that the lengths of the shortest telomeres best predict cellular senescence, suggesting that shorter telomeres should be better protected. We test the latter hypothesis and show that, within individuals, long telomeres shorten faster than short telomeres in adults and nestlings, a result not previously shown in vivo. Moreover, survival selection in adults was most conspicuous on relatively long telomeres. In conclusion, our longitudinal data indicate that the shortening rate of long telomeres may be a measure of ‘life stress’ and hence holds promise as a biomarker of remaining lifespan.

Altered expression of plasminogen activator and plasminogen activator inhibitor during cellular senescence

Fibroblast senescence is associated with a loss of proliferative potential and an alteration in extracellular gene expression. Because the expression of extracellular gene products are frequently growth state dependent, we undertook a comparative study of the regulation of the components of the plasminogen activation system in young and senescent cells under controlled conditions of growth. Young and senescent cells were compared in quiescent and activated growth conditions for the secretion of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2). Whereas young cells showed decreased levels of PAI-1 in the secreted and extracellular matrix pools upon serum deprivation, senescent cells showed a more constitutive pattern of gene expression, with no noticeable decrease of the levels in a low concentration of serum. RNA analysis revealed that senescent lung and skin cells, independent of the growth state, constitutively express levels of u-PA and PAI-1 comparable to the expression levels in young mitotically growing cells. These expression levels are down-regulated in quiescent young cells. In contrast, both t-PA and PAI-2 were markedly overexpressed in senescent skin lung cells under all growth conditions. Total plasminogen activator activity in conditioned medium was 50-fold higher in senescent-cell medium compared to young when cultured in 0.5% fetal calf serum (FCS) for five days, with the majority of the activity co-migrating on zymograms with u-PA. Increases in PAI-1 was also observed in senescent human umbilical vein endothelial cells. In summary, cells of various types display alterations in plasminogen activator activity during replicative senescence. The inappropriate over-expression of plasminogen activator activity in vivo may be expected to lead to a progressive disruption of extracellular matrix maintenance. Thus, our observations suggest that cellular replicative senescence is associated with an altered expression of several genes regulating tissue maintenance which, in turn, could lead to degenerative changes in tissue in age-related disease(s).

New beginnings in studies of eukaryotic DNA replication origins

DNA fiber autoradiographic studies carried out over 20 years ago first suggested that eukaryotic DNA replication begins at discrete sites spaced at irregular intervals along chromosomal DNA molecules [1]. The discrete initiation sites were called origins. Later on, the DNA replicated from a single origin was termed a replicon (reviewed iri Ref. 2). At the time, it was impossible to determine whether these origins and replicons corresponded to specific nucleotide sequences or were randomly located (with different origins being used in different cells). In the absence of accurate

Efficient control of gene expression by a tetracycline-dependent transactivator in single Dictyostelium discoideum cells.

We established a tetracycline-regulated gene expression system that tightly controls expression of genes in Dictyostelium discoideum. The control elements are contained in two plasmid vectors, one being an integrated plasmid encoding a chimeric tetracycline-controlled transcriptional activator protein (tTA(s)(*)). The second component is an extrachromosomal plasmid harboring the gene of interest preceded by an inducible promoter. This promoter contains a tetracycline-responsive element, which is the binding site for tTA(s)(*). Tetracycline prevents tTA(s)(*) from binding to the tetracycline-responsive element, rendering the promoter virtually silent. In the absence of tetracycline, tTA(s)(*) binds to its target sequence and strongly induces gene expression. The kinetics of activation and repression of the system were monitored using luciferase as a reporter. The results reveal efficient inhibition of gene expression by low concentrations of tetracycline and an induction of gene expression by several orders of magnitude within a few hours after removal of tetracycline. Green fluorescent protein (GFP) provided information about the effects of modulation of the tetracycline concentration on gene expression, at the single cell level, using fluorescence activated cell sorting (FACS). We also report that not all cells in a clonal population express the reporter gene.

The two faces of higher eukaryotic DNA replication origins

Maarten H. K. Linskens and Joel A. Huberman Department of Molecular and Cellular Biology Roswell Park Cancer Institute Buffalo, New York 14263 Earlier this year, Vaughn et al. (1990) described convinc- ing evidence for the surprising conclusion that the DNA replication origin located downstream of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells consists of a broad initiation zone extending over 26 kb. However, the data presented by Burhans et al. (1990) in this issue suggest the contradictory conclusion that replication forks must emanate bidirectionally from a site that is no larger than 450 nucleotides. In this review, we hope to show that these apparently conflicting data are not necessarily ir- reconcilable. Instead, the two sets of data may illuminate two different faces of the higher eukaryotic chromosomal replication origin. The existence of replication origins in mammalian cells was first suggested by DNA fiber autoradiography, which revealed that replication forks move bidirectionally out- ward from sites (origins) irregularly spaced along chro- mosomal DNA molecules. However, the resolution of fiber autoradiography (15-30 kb) is insufficient to provide infor- mation about events during initiation of replication or to determine whether origins correspond to specific nucleo- tide sequences. The subsequent mapping of replication origins in E. coli and prokaryotic and eukaryotic viruses (most notably SV40) to a few hundred nucleotides or less suggested that eukaryotic chromosomal origins might correspond to short stretches of defined nucleotide se- quence. Indeed, chromosomal replication origins in the yeast S. cerevisiae correspond to specific DNA segments of a few hundred nucleotides or less (Linskens and Huber- man, 1966). The identification of replication origins in higher eukary- otes, however, has proven to be more difficult than in yeast (reviewed in Umek et al., 1969). The complexity of the higher eukaryotic genome has been a major obstacle. To overcome this difficulty, Hamlin’ s laboratory developed a strain of Chinese hamster ovary cells containing 500- 1000 copies of 240 kb surrounding the DHFR gene. By de- termining which restriction fragments of the amplified do- main are replicated earliest in S phase, Heintz and Hamlin (1982) identified a region downstream of the DHFR gene that appeared to contain a replication origin. This region is still the only stretch of mammalian chromosomal DNA has been proven to contain a replication origin. High resolution, early S phase labeling (Burhans et al., 1986; Leu and Hamlin, 1989) and replication polarity anal- ysis (Handeli et al., 1989) have suggested the existence of two separate origins about 22 kb apart within the DHFR downstream region. Figure 1 summarizes these data. Bur- hans et al. (1990) measured Okazaki fragment polarity by an independent technique to map the left-hand origin (Figure 1) to even higher resolution. Their data suggest

Reconstitution of active telomerase in primary human foreskin fibroblasts: effects on proliferative characteristics and response to ionizing radiation.

Purpose: Telomere shortening has been proposed to trigger senescence, and since most primary cells do not express active telomerase, reactivation of telomerase activity was proposed as a safe and non‐transforming way of immortalizing cells. However, to study radiation responses, it is as yet unclear whether cells immortalized by telomerase reactivation behave in a similar manner as their parental primary cells. Materials and methods: Primary human foreskin fibroblasts were transfected with the human catalytic subunit of telomerase, the reverse transcriptase (hTERT), and their growth characteristics and response to DNA damage were characterized. Results: The sole expression of the human hTERT was sufficient to immortalize the human foreskin fibroblasts. With time in culture, the immortalized cells almost doubled their average telomeric length and the clonal population contained almost no post‐mitotic fibroblasts anymore. Up to 300 population doublings, no alterations compared with the parental primary cells were seen in terms of clonogenic radiosensitivity, DNA double‐strand break repair, radiation‐induced increases in p53 and p21WAF‐1,CIP‐1 expression, and the G1/S and G2/M cell cycle checkpoints. Moreover, mitogen‐induced mitotic arrest of fibroblasts was still possible in the hTERT‐immortalized clones. Conclusions: Immortalizing fibroblasts by reconstitution of active telomerase seems a good, reliable manner to generate a large source of cells with a radiation damage response similar to the primary cells.

Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in Dictyostelium discoideum

The soil amoeba Dictyostelium discoideum is a host cell that provides simple genetics in combination with complex protein synthesis. We show that the complex human heterodimeric gonadotropins can be produced and secreted by this organism. Furthermore, both follicle stimulation hormone and choriogonadotropin produced by D. dictyostelium bind to their human receptors and elicit a biological response comparable to the wild‐type hormones. We also show that structure‐function analysis using random mutagenesis and screening of recombinant glycoprotein hormones is feasible. Thus, expression of gonadotropins in D. dictyostelium opens the way to the engineering of potential new therapeutic analogues. Linskens, M. H. K., Grootenhuis, P. D. J., Blaauw, M., Huisman‐de Winkel, B., van Ravestein, A., van Haastert, P. J. M., and Heikoop, J. C. Random mutagenesis and screening of complex glycoproteins: expression of human gonadotropins in Dictyostelium discoideum. FASEB J. 13, 639–645 (1999)